Selective eosinophil leukocyte recruitment by transendothelial migration and not by leukocyte-endothelial cell adhesion. Eosinophil infiltration is the hallmark of allergic inflammatory events. However, the mechanisms governing the influx of eosinophils into the tissue at a site of an allergic reaction remains unclear. We have examined the interactions of eosinophils and neutrophils isolated from the same atopic donor with cultured human umbilical vein endothelial cell (EC) monolayers in the search for a mechanism for this selective eosinophil recruitment. First, the adherence of eosinophils and neutrophils to ECs stimulated with lipopolysaccharide, interleukin (IL)-1 alpha, and tumor necrosis factor-alpha were compared. Each mediator induced a similar dose-dependent enhancement of eosinophil adhesiveness for both eosinophils and neutrophils. Thus, although cytokine activation of ECs in the vasculature adjacent to an inflammatory site probably serves as an important focusing mechanism for the extravasation of inflammatory cells at this site, there does not appear to be any selective EC-dependent mechanism for eosinophil recruitment. Little or no effect on eosinophil and neutrophil adherence was observed with IL-3, IL-5, granulocyte/macrophage colony-stimulating factor, platelet-activating factor (PAF), leukotriene B4, or histamine. Second, the migration of eosinophils and neutrophils through an EC monolayer in response to chemoattractants was examined. PAF was found to selectively enhance eosinophil transendothelial migration at doses of 10(-7) to 10(-10) M, with optimal effect at 10(-8) M. This effect was gradient dependent and could be inhibited by WEB 2086, a specific PAF inhibitor. These results suggest that localized production of PAF may be a prime factor in the events leading to eosinophil accumulation at allergic inflammatory sites, and that selectivity for eosinophil recruitment occurs at the stage of transendothelial cell migration under the influence of cell-specific chemoattractants.


Cell surface antigens on human marrow-derived mesenchymal cells are detected by monoclonal antibodies. Human bone marrow has been shown to contain mesenchymal cells, which fabricate the connective tissue network of the marrow called the stroma. A subset of these marrow-derived mesenchymal cells can be isolated, expanded in culture, and then induced to differentiate into bone-producing osteoblasts and ultimately osteocytes when placed in the proper environment. At present, there are no methods for definitively identifying these cells in human marrow tissue or following their differentiation into osteogenic phenotypes. Therefore, we culture-expanded, marrow-derived mesenchymal cells from human donors and used these cells to immunize cells from human donors and used these cells to immunize mice whose spleens were used to generate hybridoma cell lines, which secrete antibodies to antigens on the cell surface of these culture-expanded mesenchymal cells. Hybridoma culture supernatants were successively screened against highly enriched samples of culture-expanded, marrow-derived mesenchymal cells in cryosections and live cell cultures to identify unique cell surface antigens. Positive clones were then screened against cell suspensions of whole and fractionated marrow to identify hybridomas whose supernatants were nonreactive with marrow hemopoietic cells. Three hybridoma cell lines, SH2, SH3, and SH4, were identified; these hybridomas secrete antibodies that recognize antigens on the cell surface of marrow-derived mesenchymal cells, but fail to react with marrow-derived hemopoietic cells. Additional tissue screening reveals unique tissue distributions for each of the recognized antigens, which suggests different antigen recognition for each antibody. However, all three antibodies fail to react with the cell surface of osteoblasts or osteocytes, suggesting that the antigens recognized by these antibodies are developmentally regulated and specific for primitive or early-stage cells of the osteogenic lineage.


Chromosomal heterogeneity of various Mycoplasma hyopneumoniae field strains. Restriction enzyme digestion and field inversion gel electrophoresis were used to analyze the chromosomes of strains of Mycoplasma hyopneumoniae and the related organism Mycoplasma flocculare. The chromosome size for the M. hyopneumoniae type strain was calculated from individual fragments to be 1,011.3 +/- 32.9 kbp. The chromosomes of M. hyopneumoniae field strains were approximately the same size. The restriction patterns obtained for the chromosomes of phenotypically similar M. hyopneumoniae strains were quite different. Therefore, the species M. hyopneumoniae seems to be very heterogeneous. A field inversion gel electrophoresis analysis of the entire chromosomes allowed us to distinguish M. hyopneumoniae strains easily and hence to characterize further the species M. hyopneumoniae. The chromosome size for M. flocculare was calculated to be 988.3 +/- 39.5 kbp. Restriction enzyme XhoI, which statistically should cut the M. hyopneumoniae chromosome frequently, did not cut the DNA of any of the M. hyopneumoniae strains but did digest M. flocculare DNA, indicating that there is a site-specific modification at CTCGAG which probably belongs to a restriction modification system in M. hyopneumoniae and is absent in M. flocculare.


In vitro toxicity of 3'-azido-3'-deoxythymidine, carbovir and 2',3'-didehydro-2',3'-dideoxythymidine to human and murine haematopoietic progenitor cells. The myelotoxicities of three antiretroviral agents, 3'-azido-3'-deoxythymidine (AZT), carbovir (CBV) and 2',3'-didehydro-2',3'-dideoxythymidine (d4T), were evaluated in vitro with normal human and murine haematopoietic progenitor cells. These studies demonstrated that continuous AZT exposure was more inhibitory to human and murine colony formation than 1 h exposure, with murine and human progenitors similarly inhibited by continuous AZT exposure. These in vitro results on AZT's myelotoxicity correlate with both human and murine in vivo studies. CBV was only moderately toxic to human and murine cells following either 1 h or continuous exposure, with human and murine progenitors similarly suppressed by continuous CBV exposure. 1 h d4T exposure was less toxic to both human and murine marrow cells than continuous exposure and both species were equivalently inhibited when continuously exposed to d4T. In general, CBV was the least toxic agent to human and murine haematopoietic cells and AZT the most toxic. The study establishes CBV and d4T as less myelotoxic agents to human and murine haematopoietic progenitor cells in vitro than AZT which therefore could be considered as alternatives to AZT for the treatment of HIV infection.


[The clinical usefulness of the serological study of IgG antibodies and IgA against the antigens associated with the Epstein-Barr virus in patients with nasopharyngeal carcinoma]. The relationship between nasopharyngeal carcinoma (NPC) and Epstein-Barr virus (EBV) has been already well established. Titulation of the antibodies IgG and IgA against EBV-related antigens has been proved reliable but always suitable having tested and age-matched control population. We present here the preliminary results from the 14 patients bearing NPC and 12 patients with other carcinoma of the head and neck and healthy donors.


Unique genotypic features of infant acute lymphoblastic leukaemia at presentation and at relapse. Acute lymphoblastic leukaemia (ALL) of infants aged less than 1 year represents a group of patients with peculiar biological features, poor response to therapy and unfavourable prognosis. In order better to characterize this type of leukaemia, we have investigated the immunoglobulin (Ig) and T-cell receptor (TCR) genes configuration of 21 infants with ALL, and compared the genotypic features with the phenotypic and karyotypic data, as well as with the clinical outcome. All cases had a pre-B phenotype; 12 (57%) of them were pre-pre-B ALL (CD10-, CD19+). Six of the 16 cases evaluated (38%) displayed chromosomal abnormalities; five had the typical translocation t(4;11)(q21;23). Eleven cases presented with a white blood cell count greater than 100 x 10(9)/l. The clinical course was unfavourable in 14 patients. The genotype of this group of ALL revealed several peculiarities. (1) Of the 21 cases, six (29%) displayed a multiple rearrangement pattern at the IgH locus. (2) In three cases (15%), the light chain genes were rearranged. (3) The TCR beta and gamma genes were rearranged in only one case (one case at the TCR beta and one at the TCR gamma locus). (4) The TCR delta chain was rearranged in eight cases (40%) and rarely deleted; the rearrangements observed were those most frequently observed in B cell-precursor ALL. Two cases were evaluated both at presentation and at relapse. While the immunophenotype had remained unmodified, comparison of Ig heavy chain gene rearrangements revealed clonal variations in both cases. Taken together, these findings further underline the biological peculiarities of infant ALL compared to ALL which occurs in older children and in adults, and stress the need of differentiated and aggressive therapeutic approach for these patients.


A prospective study of conization of the cervix in the management of cervical intraepithelial glandular neoplasia (CIGN)--a preliminary report. To assess the efficacy of cervical conization as primary management of cervical intraepithelial glandular neoplasia (CIGN). A multicentre prospective cohort study. CRC Clinical Trials Unit, Birmingham. 84 women registered with the Unit between May 1986 and January 1989. After excluding 33 women, 51 who had been managed in accordance with the described protocol and had the presence of CIGN confirmed by central review of diagnostic histopathological material were included in the study. INTERVENTION/PROTOCOL: Women with CIGN diagnosed on a cervical cone specimen were managed in accordance with a specific protocol: (a) women with negative cone margins were managed conservatively and followed up with regular cervical cytological and colposcopic examinations; (b) women with involved cone margins were managed by hysterectomy. Presence or absence of CIGN at cone margins, results of cervical cytological examinations following conization, results of histopathological assessment of any surgical specimens taken after initial cone biopsy. Of the 51 women with confirmed CIGN, managed by conization, 14 (27%) were aged 30 or less and 15 (29%) were nulliparous. Thirty five women who had a cone biopsy showing margins free of CIGN have been managed by conization alone. After a median follow-up period of 12 months there is no apparent residual CIGN or invasive disease in this group. Thirteen women have had further surgical procedures (according to protocol) and two have had a hysterectomy for benign gynaecological disorders. Eight further procedures were carried out because the original cone biopsy had margins involved with CIGN, and only one of them was found to have residual CIGN. The other five procedures were carried out solely because of abnormal cytology, only one of them had a diagnosis of CIN 1. A total of 10 women had cytological abnormality following cone biopsy, one had CIGN, one had CIN 1 and a third had CIN 3. Our preliminary data suggests that when a diagnosis of CIGN is made upon a cone biopsy, further surgery is unnecessary in those women in whom the margins of the cone specimen are free of disease. Cytological and colposcopic follow up, including cytological sampling of the endocervical canal, is recommended for these women.


Expression of human foamy virus is differentially regulated during development in transgenic mice. The human foamy virus (HFV) is a recently characterized member of the spumavirus family. Although no diseases have been unequivocally associated with HFV infection, expression of HFV regulatory genes in transgenic mice induces a characteristic acute neurodegenerative disease and a myopathy. To better characterize the sequence of events leading to disease, and to gain a better understanding of the underlying pathogenetic mechanisms, we have analyzed in detail the transgene expression pattern during development. Transcription of a construct containing all regulatory elements and ancillary genes of HFV was analyzed by in situ hybridization and was shown to occur in two distinct phases. At midgestation, low but widespread expression was first detected in cells of extraembryonic tissues. Later, various tissues originating from embryonic mesoderm, neuroectoderm, and neural crest transcribed the transgene at moderate levels. However, expression decreased dramatically during late gestation and was suppressed shortly after birth. After a latency period of up to 5 weeks, transcription of the transgene resumed in single cells distributed irregularly in the central nervous system and in the skeletal muscle. By the age of 8 weeks, an increasing number of cells displayed much higher expression levels than in embryonic life and eventually underwent severe degenerative changes. These findings demonstrate that HFV transgene expression is differentially regulated in development and that HFV cytotoxicity may be dose-dependent. Such biphasic pattern of expression differs from that of murine retroviruses and may be explained by the specificity of HFV regulatory elements in combination with cellular factors. Future studies of this model system should, therefore, provide novel insights in the mechanisms controlling retroviral latency.


Direct gene transfer to the liver with herpes simplex virus type 1 vectors: transient production of physiologically relevant levels of circulating factor IX. We have used gene transfer vectors derived from a replication-defective mutant of herpes simplex virus type 1 (HSV-1) expressing the hepatitis B virus surface antigen (HBsAg), Escherichia coli beta-galactosidase (beta-gal), or canine factor IX (cFIX) from the immediate early promoter of human cytomegalovirus (hCMV) to infect mouse liver by direct injection or through the portal vein. By either route, high levels of transgene expression were demonstrated by the detection of immunoreactive HBsAg or cFIX in the circulation and by histochemical detection of beta-gal activity in situ. The results were striking in that the serum level of cFIX reached 10% of the normal murine levels. Although the level of transgene expression from the hCMV promoter was transient, a significant number of persistent vectors could be rescued from the livers of recipient mice up to 2 months after inoculation. Replacement of the hCMV promoter with the HSV-1 latency-associated transcript (LAT) promoter resulted in reduced but prolonged expression of both HBsAg and cFIX. The very high level of factor IX expression suggests that clinically useful gene transfer may eventually be feasible through direct vector delivery to the liver.


Backbone dynamics of the Bacillus subtilis glucose permease IIA domain determined from 15N NMR relaxation measurements. The backbone dynamics of the uniformly 15N-labeled IIA domain of the glucose permease of Bacillus subtilis have been characterized using inverse-detected two-dimensional 1H-15N NMR spectroscopy. Longitudinal (T1) and transverse (T2) 15N relaxation time constants and steady-state (1H)-15N NOEs were measured, at a spectrometer proton frequency of 500 MHz, for 137 (91%) of the 151 protonated backbone nitrogens. These data were analyzed by using a model-free dynamics formalism to determine the generalized order parameter (S2), the effective correlation time for internal motions (tau e), and 15N exchange broadening contributions (Rex) for each residue, as well as the overall molecular rotational correlation time (tau m). The T1 and T2 values for most residues were in the ranges 0.45-0.55 and 0.11-0.15 s, respectively; however, a small number of residues exhibited significantly slower relaxation. Similarly, (1H)-15N NOE values for most residues were in the range 0.72-0.80, but a few residues had much smaller positive NOEs and some exhibited negative NOEs. The molecular rotational correlation time was 6.24 +/- 0.01 ns; most residues had order parameters in the range 0.75-0.90 and tau e values of less than ca. 25 ps. Residues found to be more mobile than the average were concentrated in three areas: the N-terminal residues (1-13), which were observed to be highly disordered; the loop from P25 to D41, the apex of which is situated adjacent to the active site and may have a role in binding to other proteins; and the region from A146 to S149. All mobile residues occurred in regions close to termini, in loops, or in irregular secondary structure.


Highly efficient photoaffinity labeling of the hormone binding domain of atrial natriuretic factor receptor. A high-efficiency photoaffinity derivative of atrial natriuretic factor (ANF) was developed for studying the peptide binding domain of the receptor protein and for better characterization of this receptor in tissues with a low density of binding sites. The position of the photosensitive residue was chosen on the basis of a molecular conformational model and on structure-activity relationship studies which both indicate that the carboxy-terminal end of the peptide is part of a hydrophobic pole likely to interact deeply within the ANF binding pocket of the receptor. Selection of the photoreactive residue p-benzoylphenylalanine (BPA) as a substitute for arginine in position 125 of the peptide sequence led to a photoaffinity derivative with a high (63%) efficiency of covalent incorporation to the receptor protein. This derivative (BPA-ANF) has a 10-fold lower affinity when compared with ANF, but it is a full agonist in stimulating cGMP production and inhibiting aldosterone secretion in bovine adrenal zona glomerulosa. Photoaffinity labeling with BPA-ANF specifically identifies ANF-R1 and ANF-R2 receptor proteins with a 10-fold higher efficiency than with azido derivatives of ANF or with cross-linking agents. This new ANF derivative therefore appears to be useful for studying ANF receptors in tissues with low levels of expression, for locating receptor following cellular internalization, and for tagging proteolytic fragments of the receptor amenable to amino acid microsequencing.


Signal transmission by epidermal growth factor receptor: coincidence of activation and dimerization. Dimerization of epidermal growth factor receptor dissolved in a solution of nonionic detergent was followed with a resolution of 1 min by quantitative cross-linking with glutaraldehyde. Upon addition of epidermal growth factor to the solution, the initially monomeric protein dimerized in a reaction that was second-order in the concentration of receptor. A second-order rate constant, on the basis of enzymatic activity as a measure of the concentration of functional receptor, was calculated from time courses of dimerization at various initial concentrations of receptor. The activation of the protein tyrosine kinase of the receptor was monitored directly under the same conditions with an exogenous substrate. The increase in tyrosine kinase activity displayed kinetics that were also second-order in the concentration of receptor. A second-order rate constant for the activation of the tyrosine kinase could be calculated from the time courses. The second-order rate constant for the activation of the tyrosine kinase by epidermal growth factor was indistinguishable from the second-order rate constant for the dimerization induced by epidermal growth factor. Therefore, dimerization of epidermal growth factor receptor and activation of its tyrosine kinase are coincident events, both initiated by the binding of epidermal growth factor.


Spectroscopic studies of lipids and biological membranes: carbon-13 and proton magic-angle sample-spinning nuclear magnetic resonance study of glycolipid-water systems. We have obtained 1H and 13C magic-angle sample-spinning (MAS) nuclear magnetic resonance (NMR) spectra of three glycosyldiacylglycerol-water (1:1, weight ratio) mesophases, at 11.7 T, as a function of temperature, in order to probe lipid headgroup, backbone, and acyl chain dynamics by using natural-abundance NMR probes. The systems investigated were monogalactosyldiacyldiglyceride [MGDG; primarily 1,2-di[(9Z,12Z,15Z)octadec-9,12,15-trienoyl++ +]-3-beta-D-galactopyranosyl- sn-glycerol]; digalactosyldiacyldiglyceride [DGDG; primarily 1,2-di[(9Z,12Z,15Z)octadec-9,12,15-trienoyl++ +]-3- (alpha-D-galactopyranosyl-1-6-beta-D-glactopyranosyl)-sn-glycerol] ; and sulfoquinovosyldiacyldiglyceride [SQDG; primarily 1-[(9Z,12Z,15Z)octadec-9,12,15-trienoyl]-2 -hexadecanoyl-3-(6-deoxyl-6- sulfono-alpha-D-glucopyranosyl)-sn-glycerol]. At approximately 22 degrees C, all three lipid-water systems give well-resoled 13C and 1H MAS NMR spectra, characteristic of fluid, liquid-crystalline mesophases. 13C spin-lattice relaxation times of the headgroup and glycerol backbone carbons of all three materials give, within experimental error, the same NT1 values (approximately 400 ms), implying similar high-frequency motions, independent of headgroup size and charge. Upon cooling, pronounced line broadenings are observed, due to an increase in slow motional behavior. For each lipid, the onset of line broadening is seen with the glycosyl headgroup, glycerol backbone, and the first two or three carbons of the acyl chains. By approximately -20 degrees, all headgroup carbon resonances are broadened beyond detection. Both galactose moieties in DGDG "freeze out" together, implying a rigid-body motion of the disaccharide unit. Upon further cooling, the bulk polymethylene chain resonances in all three systems (in both 13C and 1H MAS) broaden greatly, followed by the olefinic and allylic carbon resonances.(ABSTRACT TRUNCATED AT 250 WORDS)


Investigation of the origin of the "S3" EPR signal from the oxygen-evolving complex of photosystem 2: the role of tyrosine Z. The origin of the "S3" EPR signal from calcium-depleted photosystem 2 samples has been investigated. This signal is observed after freezing samples under illumination and has been assigned to an interaction between the manganese cluster and an oxidized histidine radical [Boussac et al. (1990) Nature 347; 303-306]. In calcium-depleted samples prepared by three different methods, we observed the trapping of the tyrosine radical YZ+ under conditions which also formed the "S3" signal. An "S3"-type signal and YZ+ were also formed in PS2 samples treated with the water analogue ammonia. Following illumination at 277 K, the "S3" and YZ+ signals decayed at the same rate at 273 K in the dark. Both the YZ+ and "S3" signals decayed on storage at 77 K and could be subsequently regenerated by illumination at 8-77 K. No evidence to support histidine oxidation was found. The effects of DCMU, chelators, and alkaline pH on the dark-stable multiline S2 and the "S3" signals from calcium-depleted samples were determined. Both signals required the presence of EGTA or citrate for maximum yield. The addition of DCMU caused a reduction in the yield of "S3" generated by freezing under illumination. Incubation at pH 7.5 resulted in the loss of both signals. We propose that a variety of treatments which affect calcium and chloride binding cause a stabilization of the S2 state and slow the reduction of YZ+. This allows the trapping of YZ+, the interaction with the manganese cluster (probably in the S2 state) resulting in the "S3" signal. The data allow the position of the manganese cluster to be estimated as within 10 A of tyrosine Z (D1-161).


A cromoglycate binding protein from rat mast cells of a leukemia line is a nucleoside diphosphate kinase. Recently, we have shown that a membrane-permeant derivative of the antiasthmatic drug cromoglycate (CG) effectively inhibits the Fc epsilon-receptor-mediated secretory response of rat mucosal mast cells (line RBL-2H3) at a stage preceding the transient rise in the cytoplasmic free calcium concentration [Hemmerich, S., Sijpkins, D., & Pecht, I. (1991) Biochemistry 30, 1523-1532]. In contrast to cromoglycate itself, which is membrane impermeant and ineffective in these cells, its bis-acetoxymethyl ester derivative (CG/AM) can diffuse across the plasma membrane into the cytosol, where it is hydrolyzed into the impermeant CG dianion, which presumably may interact with intracellular components involved in the Fc epsilon R signal transduction pathway. In order to identify cytosolic components involved in the stimulus-secretion coupling that interact with this drug, we coupled CG to an insoluble matrix. This matrix was indeed effective in the affinity isolation from RBL cells of a cytosolic protein that exhibits an apparent molecular mass of 18 kDa on reducing SDS gels. This protein was purified to homogeneity and then fragmented, and the amino acid sequence of three resultant peptides was determined. Using the corresponding synthetic oligonucleotides, we cloned and sequenced a cDNA that encodes the full-length 18-kDa polypeptide (p18). The protein sequence deduced from this cDNA is identical to that of rat nucleoside diphosphate kinase [Kimura, N., Shimada, N., Nomura, K., & Watanabe, K. (1990) J. Biol. Chem. 265, 15744-15749] and highly homologous (88%) to the human NM23 gene product whose expression is associated with reduced metastatic potential, as well as with the Drosophila awd gene product (77% sequence identity).(ABSTRACT TRUNCATED AT 250 WORDS)


Oligomeric structure and autophosphorylation of nucleoside diphosphate kinase from rat mucosal mast cells. Nucleoside diphosphate (NDP) kinases have been found to be involved in a wide range of fundamental biological processes ranging from developmental control to signal transduction and metastasis. We have recently cloned and sequenced a cDNA encoding an NDP-kinase of the rat mucosal mast cell line RBL-2H3 [Hemmerich, S., Yarden, Y., & Pecht, I. (1992) Biochemistry (preceding paper in this issue)]. The enzyme itself has been isolated by means of its affinity to the bischromone cromoglycate. Here we report several of its biochemical characteristics: A structural model for the native protein is proposed in which two disulfide-linked pairs of similar 18-kDa subunits (p18) associate to form a 72-kDa tetramer (p72). This is based on the migration properties of the purified enzyme on gel filtration columns, sodium dodecylsulfate gel electrophoresis, and two-dimensional electrophoresis, together with peptide mapping data. In the absence of NDP, both intact p72 and the dissociated 18-kDa subunits (p18) were shown to undergo Mg(2+)-dependent stoichiometric autophosphorylation utilizing adenosine and guanosine triphosphate or gamma-thiotriphosphate as phosphate donor. This autophosphorylation activity was found to be retained by the 18-kDa subunits even following fractionation by SDS-PAGE and electrophoretic transfer to nitrocellulose. The Michaelis constant of this autophosphorylation reaction with either ATP, ATP gamma S, GTP, or GTP gamma S was determined to be 6.5 +/- 1 microM, and maximally 2 mol of phosphate were found to be incorporated per p72 molecule, thus indicating that phosphorylation occurs at a single site on only two of the four 18-kDa subunits of the holoenzyme.(ABSTRACT TRUNCATED AT 250 WORDS)


Thiolate ligation of the active site Fe2+ of isopenicillin N synthase derives from substrate rather than endogenous cysteine: spectroscopic studies of site-specific Cys----Ser mutated enzymes. Isopenicillin N synthase (IPNS) catalyzes double ring closure of the tripeptide (L-alpha-amino-delta-adipoyl)-L-cysteinyl-D-valine (ACV) to form the beta-lactam and thiazolidine rings of penicillin-type antibiotics. Our previous spectroscopic study using IPNS from Cephalosporium acremonium expressed in Escherichia coli [Chen, V. J., Orville, A. M., Harpel, M. R., Frolik, C. A., Surerus, K. K., Münck, E., & Lipscomb, J. D. (1989) J. Biol. Chem. 264, 21677-21681] indicated that a thiolate enters the coordination of the essential active site Fe2+ when ACV binds to IPNS. The presence of an Fe-S bond in the IPNS.ACV complex is confirmed by EXAFS data presented in the preceding paper [Scott, R. A., Wang, S., Eidsness, M. K., Kriauciunas, A., Frolik, C. A. & Chen, V. J. (1992) Biochemistry (preceding paper in this issue)]. However, these studies leave unclear whether the coordinating thiolate derives from ACV or an endogenous cysteine. Here, we examine the spectroscopic properties of three genetically engineered variants of IPNS in which the only two endogenous cysteines are individually and collectively replaced by serine. The EPR, Mössbauer, and optical spectra of the mutant enzymes and their complexes with ACV, NO, or both ACV and NO are found to be essentially the same as those of wild-type IPNS, showing that the endogenous cysteines are not Fe2+ ligands in any of these complexes. Spectral quantitations show that the double Cys----Ser mutation decreases the affinity of the enzyme for ACV by about 6-fold, suggesting that the endogenous cysteines influence the structure of the substrate binding pocket remote from the iron. Thiolate complexation of the Fe2+ is also examined using ACV analogues. All ACV analogues examined in which the cysteinyl thiol moiety is unaltered are found to bind to the IPNS.NO complex to give optical and EPR spectra very similar to those of the ACV complex. In contrast, analogues in which the cysteinyl moiety of ACV is replaced with serine or cysteic acid fail to elicit the characteristic EPR and optical features despite the fact that they are bound with reasonable affinity to the enzyme. These results demonstrate that the thiolate of ACV coordinates the Fe2+. The EPR spectra of both the IPNS.NO and IPNS.ACV.NO complexes are broadened for samples prepared in 17O-enriched water, showing that water (or hydroxide) is also an iron ligand in each case. Thus, the Fe2+ coordination of the IPNS.ACV.NO complex accommodates at least three exogenous ligands.(ABSTRACT TRUNCATED AT 400 WORDS)


Characterization of the flavins and the iron-sulfur centers of glutamate synthase from Azospirillum brasilense by absorption, circular dichroism, and electron paramagnetic resonance spectroscopies. Azospirillum brasilense glutamate synthase has been studied by absorption, electron paramagnetic resonance, and circular dichroism spectroscopies in order to determine the type and number of iron-sulfur centers present in the enzyme alpha beta protomer and to gain information on the role of the flavin and iron-sulfur centers in the catalytic mechanism. The FMN and FAD prosthetic groups are demonstrated to be non-equivalent with respect to their reactivities with sulfite. Sulfite reacts with only one of the two flavins forming an N(5)-sulfite adduct with a Kd of approximately 1 mM. The enzyme-sulfite complex is reduced by NADPH, and the complexed sulfite is competitively displaced by 2-oxoglutarate, which suggests the reactive flavin to be at the imine-reducing site. These data are in agreement with the two-site model of the enzyme active center proposed on the basis of kinetic studies [Vanoni, M.A., Nuzzi, L., Rescigno, M., Zanetti, G., & Curti, B. (1991) Eur. J. Biochem. 202, 181-189]. Each enzyme protomer was found, by chemical analysis, to contain 12.1 +/- 0.5 mol of non-heme iron. Electron paramagnetic resonance spectroscopic studies on the oxidized and reduced forms of glutamate synthase demonstrated the presence of three distinct iron-sulfur centers per enzyme protomer. The oxidized enzyme exhibits an axial spectrum with g values at 2.03 and 1.97, which is highly temperature-dependent and integrates to 1.1 +/- 0.2 spin/protomer. This signal is assigned to a [3Fe-4S]1+ cluster (Fe-S)I. Reduction of the enzyme with an NADPH-regenerating system results in reduction of the [3Fe-4S]1+ center to a species with a g approximately 12 signal characteristic of the S = 2 spin state of a [3Fe-4S]0 cluster. The NADPH-reduced enzyme also exhibits an [Fe-S] signal at g values of 1.98, 1.95, and 1.88, which integrates to 0.9 spin/protomer and is due to a second cluster (Fe-S)II. Reduction of the enzyme with the light/deazaflavin method results in a signal characteristic of [Fe-S] clusters with g values of 2.03, 1.92, and 1.86 and an integrated intensity of 1.9 spin/protomer. This signal arises from reduction of the (Fe-S)II center and from that of the third, lower potential iron-sulfur center (Fe-S)III. Circular dichroism spectral data on the oxidized and reduced forms of the enzyme are more consistent with the assignment of (Fe-S)II and (Fe-S)III as [4Fe-4S] clusters rather than [2Fe-2S] centers.


Thermodynamics of ion binding to phosphatidic acid bilayers. Titration calorimetry of the heat of dissociation of DMPA. The heat of dissociation of the second proton of 1,2-dimyristoylphosphatidic acid (DMPA) was studied as a function of temperature using titration calorimetry. The dissociation of the second proton of DMPA was induced by addition of NaOH. From the calorimetric titration experiment, the intrinsic pK0 for the dissociation reaction could be determined by applying the Gouy-Chapman theory. pK0 decreases with temperature from ca. 6.2 at 11 degrees C to 5.4 at 54 degrees C. From the total heat of reaction, the dissociation enthalpy, delta Hdiss, was determined by subtracting the heat of neutralization of water and the heat of dilution of NaOH. In the temperature range between 2 and 23 degrees C, delta Hdiss is endothermic with an average value of ca. 2.5 kcal.mol-1 and shows no clear-cut temperature dependence. In the temperature range between 23 and 52 degrees C, delta Hdiss calculated after subtraction of the heat of neutralization and dilution is not the true dissociation enthalpy but includes contributions from the phase transition enthalpy, delta Htrans, as the pH jump induces a transition from the gel to the liquid-crystalline phase. The delta Cp for the reaction enthalpy observed in this temperature range is positive. Above 53 degrees C, the pH jump induces again only the dissociation of the second proton, and the bilayers stay in the liquid-crystalline phase. In this temperature range, delta Hdiss seems to decrease with temperature. The thermodynamic data from titration calorimetry and differential scanning calorimetry as a function of pH can be combined to construct a complete enthalpy-temperature diagram of DMPA in its two ionization states.


Molecular model of the cyclic GMP-binding domain of the cyclic GMP-gated ion channel. The structure of the cyclic GMP-binding domain of the cyclic GMP-gated ion channel from bovine retinal rod photoreceptors has been modeled by analogy to the crystal structure of the homologous cyclic AMP-binding domain of catabolite gene activator protein (CAP). The modeled cyclic GMP-binding domain has a three-residue deletion and a five-residue insertion between beta strands compared to CAP. The major interactions of the ion channel with cyclic GMP are similar to those observed for cyclic AMP bound to CAP and predicted for cGMP bound to the cGMP-dependent protein kinase: Gly 543 and Glu 544 make hydrogen-bond interactions with the ribose 2'-OH, Arg 559 forms an ion pair with the charged phosphate oxygen, and Thr 560 forms hydrogen-bond interactions with an exocyclic phosphate oxygen and with the 2-amino group of cGMP. Three additional potential interactions were predicted from the model structure. Ile 545 O and Ser 546 OH form hydrogen-bond interactions with an exocyclic phosphate oxygen, and Phe 533 may interact with the aromatic ring of cGMP. This model is in agreement with both the analogue binding experiments and the mutational analysis of Thr 560.


Structures of aspartic acid-96 in the L and N intermediates of bacteriorhodopsin: analysis by Fourier transform infrared spectroscopy. The light-induced difference Fourier transform infrared spectrum between the L or N intermediate minus light-adapted bacteriorhodopsin (BR) was measured in order to examine the protonated states and the changes in the interactions of carboxylic acids of Asp-96 and Asp-115 in these intermediates. Vibrational bands due to the protonated and unprotonated carboxylic acid were identified by isotope shift and band depletion upon substitution of Asp-96 or -115 by asparagine. While the signal due to the deprotonation of Asp-96 was clearly observed in the N intermediate, this residue remained protonated in L. Asp-115 was partially deprotonated in L. The C = O stretching vibration of protonated Asp-96 of L showed almost no shift upon 2H2O substitution, in contrast to the corresponding band of Asp-96 or Asp-115 of BR, which shifted by 9-12 cm-1 under the same conditions. In the model system of acetic acid in organic solvents, such an absence of the shift of the C = O stretching vibration of the protonated carboxylic acid upon 2H2O substitution was seen only when the O-H of acetic acid is hydrogen-bonded. The non-hydrogen-bonded monomer showed the 2H2O-dependent shift. Thus, the O-H bond of Asp-96 enters into hydrogen bonding upon conversion of BR to L. Its increased hydrogen bonding in L is consistent with the observed downshift of the O-H stretching vibration of the carboxylic acid of Asp-96.


The T to R transition in the copper(II)-substituted insulin hexamer. Anion complexes of the R-state species exhibiting type 1 and type 2 spectral characteristics. The R-state conformation of the Cu(II)-substituted insulin hexamer has been identified, and a number of its derivatives have been studied via 1H NMR, ESR, and UV-visible spectroscopy. This work establishes that the Cu(II)-substituted insulin hexamer undergoes an analogous T to R conformational transition in solution that has been identified previously for Zn(II)- and Co(II)-insulin hexamers [Roy, M., Brader, M.L., Lee, R. W.-K., Kaarsholm, N.C., Hansen, J., & Dunn, M.F. (1989) J. Biol. Chem. 264, 19081-19085]. The data indicate that each Cu(II) center of the R-state Cu(II)-insulin hexamer possesses a coordination site that is accessible to anions from solution. Both phenol and anionic ligands that coordinate to the Cu(II) ions are required to generate the necessary heterotropic interactions that stabilize the R-state structure. With phenylmethylthiolate (PMT), a Cu(II)-R6 adduct that displays the spectral features of blue (type 1) copper proteins is obtained. This complex is proposed to embody a pseudotetrahedral CuIIN3S(PMT) chromophore, in which N is HisB10 (imidazolyl). The remaining ligands examined gave rise to Cu(II)-R6 adducts that possessed the spectral characteristics of normal (type 2) Cu(II) proteins. Under reducing conditions, Cu(I)-T6 and Cu(I)-R6 hexamers have been identified.


Structure and organization of the heart isoform gene for bovine cytochrome c oxidase subunit VIIa. Mammalian cytochrome c oxidase (COX) is a 13-subunit polypeptide complex that contains 10 subunits coded by the nucleus and 3 by the mitochondria. The nuclear-encoded subunits, though of unknown function, are presumed to play a regulatory role. Three of these (subunits VIa, VIIa, and VIII) generally exist in one of two isoforms--a constitutive (L) isoform or a skeletal muscle/heart-specific (H) isoform. To study the regulation, and possibly function, of these isoforms, we have begun characterizing the genes. In this paper we describe the isolation and characterization of the gene for the bovine COX VIIa-H isoform. The gene consists of four exons spanning 1.58 kb and is associated with a CpG island. There are no canonical TATA or CCAAT boxes immediately upstream of the transcription start site. Putative DNA sequence elements associated with respiratory function, muscle gene activation, and housekeeping function are present both in the upstream regions and within introns.


Properties of oligomycin-induced occlusion of Na+ by detergent-solubilized Na,K-ATPase from pig kidney or shark rectal gland. Oligomycin induces occlusion of Na+ in membrane-bound Na,K-ATPase. Here it is shown that Na,K-ATPase from pig kidney or shark rectal gland solubilized in the nonionic detergent C12E8 is capable of occluding Na+ in the presence of oligomycin. The apparent affinity for Na+ is reduced for both enzymes upon solubilization, and there is an increase in the sigmoidicity of binding curves, which indicates a change in the cooperativity between the occluded ions. A high detergent/protein ratio leads to a decreased occlusion capacity. De-occlusion of Na+ by addition of K+ is slow for solubilized Na,K-ATPase, with a rate constant of about 0.1 s-1 at 6 degrees C. Stopped-flow fluorescence experiments with 6-carboxyeosin, which can be used to monitor the E1Na-form in detergent solution, show that the K(+)-induced de-occlusion of Na+ correlates well with the fluorescence decrease which follows the transition from the E1Na-form to the E2-form. There is a marked increase in the rate of fluorescence change at high detergent/protein ratios, indicating that the properties of solubilized enzyme are subject to modification by detergent in other respects than mere solubilization of the membrane-bound enzyme. The temperature dependence of the rate of de-occlusion in the range 2 degrees C to 12 degrees C is changed slightly upon solubilization, with activation energies in the range 20-23 kcal/mol for membrane-bound enzyme, increasing to 26-30 kcal/mol for solubilized enzyme. Titrations of the rate of transition from E1Na to E2K with oligomycin can be interpreted in a model with oligomycin having an apparent dissociation constant of about 2.5 microM for C12E8-solubilized shark Na,K-ATPase and 0.2 microM for solubilized pig kidney Na,K-ATPase.


Evidence for an internal pool of nucleoside transporters in mammalian reticulocytes. Comparisons of the site specific binding of nitrobenzylthioinosine (NBMPR) to intact and lysed red cells from various mammalian and avian species suggest the presence of a cytoplasmic pool of nucleoside transporters. In some species the cytoplasmic pool is about 50% of the total (mouse). On the average, the cytoplasmic pool is approx. 20% of the surface pool of NBMPR-binding sites. In sheep reticulocytes, both pools disappear in an energy-dependent manner during the maturation of the reticulocyte in vitro.


Characterization of Na+/H+ exchange in FRTL-5 thyroid cells. Evidence for dependence on activation of protein kinase C. Na+/H+ exchange activity was investigated in cultured rat thyroid follicular FRTL-5 cells using the pH sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Basal intracellular pH (pHi) was 7.13 +/- 0.10 in cells incubated in Hepes-buffered saline solution. The intracellular buffering capacity beta i was determined using the NH4Cl-pulse method, yielding a beta i value of 85 +/- 12 mM/pH unit. The relationship between extracellular Na+ and the initial rate of alkalinization of acid-loaded cells showed simple saturation kinetics, with an apparent Km value of 44 +/- 26 mM, and an Vmax value of 0.3 +/- 0.01 pH unit/min. The agonist-induced activation of Na+/H+ exchange was investigated in cells acidified with nigericin. Addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) or ATP induced rapid cytosolic alkalinization in acid-loaded cells. The action of both TPA and ATP was abolished by preincubating the cells with 100 microM amiloride, by substituting extracellular Na+ with equimolar concentrations of choline+, and by pretreating the cells with TPA for 24 h. Chelating extracellular Ca2+, or depleating intracellular Ca2+ pools did not affect the ATP-induced alkalinization. The results indicate, that FRTL-5 cells have a functional Na+/H+ exchange mechanism. Furthermore, stimulation of protein kinase C activity is of importance in activating the antiport.


Effect of pCMBS on anion transport in human red cell membranes. The kinetics of binding of the mercurial sulfhydryl reagent, pCMBS (p-chloromercuribenzene sulfonate), to the extracellular site(s) at which pCMBS inhibits water and urea transport across the human red cell membrane, have previously been characterized. To determine whether pCMBS binding alters Cl- transport, we measured Cl-/NO3- exchange by fluorescence enhancement, using the dye SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium). An essentially instantaneous extracellular phase of pCMBS inhibition is followed by a much slower intracellular phase, correlated with pCMBS permeation. We attribute the instantaneous phase to competitive inhibition of Cl- binding to band 3 by the pCMBS anion. The ID50 of 2.0 +/- 0.1 mM agrees with other organic sulfonates, but is very much greater than that of pCMBS inhibition of urea and water transport, showing that pCMBS reaction with water and urea transport inhibition sites has no effect on anion exchange. The intracellular inhibition by 1 mM pCMBS (1 h) is apparently non-competitive with Ki = 5.5 +/- 6.3 mM, presumably an allosteric effect of pCMBS binding to an intracellular band 3-related sulfhydryl group. After N-ethylmaleimide (NEM) treatment to block these band 3 sulfhydryl groups, there is apparent non-competitive inhibition with Ki = 2.1 +/- 1.2 mM, which suggests that pCMBS reacts with one of the NEM-insensitive sulfhydryl groups on a protein that links band 3 to the cytoskeleton, perhaps ankyrin or bands 4.1 and 4.2.


Assay of the Ca pump ATPase activity of intact red blood cells. An assay for the Ca pump ATPase of intact human red blood cells (RBCs) was developed. The assay utilized a small volume (typically 10 microliters) of packed RBCs in 1 ml of a buffer of known composition. The assay was based on the exposure of intact RBCs to the ionophore, A23187, in the presence of Ca. Such exposure caused a rapid degradation of ATP in RBCs. This degradation process is modeled in a numerical simulation in a companion paper (Vincenzi, F. F. and Hinds, T. R. (1992) Biochim. Biophys. Acta 1105, 63-70). The loss of ATP followed pseudo-first-order kinetics, and the rate constants for ATP degradation was taken as a measure of the capacity of the Ca pump ATPase. A number of variables were examined to optimize the activity of the ATPase. These variables included the concentrations of Ca and A23187. Because A23187 can promote loss of cellular Mg, it was necessary to include MgCl2 in the incubation medium to optimize ATPase activity. Likewise, it was determined that inclusion of iodoacetic acid optimized the rate of ATP loss, presumably by preventing the resynthesis of ATP from ADP and inorganic phosphate. Cobalt inhibited the ionophore-dependent loss of ATP by apparent competition with Ca for binding to A23187. Results of many assays demonstrated substantial differences in the rate constant for ATP loss in RBCs from different individuals. RBCs were selected according to density. Density associated loss of Ca pump ATPase activity was observed both by the intact RBC assay, and by assay of Ca pump ATPase activity in saponin lysates of RBCs. The correlation coefficient between the two assays was 0.93. It is suggested that the rate constant for ATP loss in intact RBCs exposed to A23187 and Ca can be taken as a measure of the Ca pump ATPase activity. This may be useful when isolated membrane ATPase assays fail (e.g., dog RBCs). The intact cell assay can also be carried out on very small volumes of cells and may be of particular value when RBC volumes are limited.


Numerical simulation of assay of the calcium pump of intact red blood cells. An assay of the Ca pump ATPase of intact human RBCs is described in a companion paper (Wu, L., Hinds, T. R. and Vincenzi, F. F. (1992) Biochim. Biophys. Acta 1106, 56-62). The assay is based on the rapid loss of ATP in RBCs that occurs when the cells are exposed to the ionophore, A23187, in the presence of Ca. An unexpected finding was that the initial loss of ATP follows pseudo-first-order kinetics. This was unexpected because the ATP content of RBCs is somewhat higher than the Km of the Ca pump for ATP. Thus, the initial loss of ATP would be expected to follow zero-order kinetics; at least if the Ca pump ATPase operated with Michaelis kinetics. We performed a series of computer simulations of the Ca pump ATPase to investigate the possible cause of the unexpected pseudo-first-order behavior. The results confirmed that the data can not be accounted for by Michaelis kinetics of the Ca pump ATPase. Possible effects of adenylate kinase were tested and were also not found to account for the pseudo-first-order behavior of an ATPase operating with Michaelis kinetics. The enzymatic properties of the Ca pump ATPase were re-examined. It was found that the Ca pump ATPase exhibits positive cooperativity toward ATP. The apparent cooperativity was 1.91. In simulations it was found that positive cooperativity of the Ca pump ATPase in the range of 1.5 to 2.0 could account for the pseudo-first-order behavior. Excellent fit of the simulation data to first-order behavior was true with or without any contribution from adenylate kinase. Rate constants of ATP loss were thus examined using cooperativity of 2.0. Over a wide range the rate constant of the loss of ATP was directly proportional to the assumed Vmax of the Ca pump ATPase, but only if the data were limited to loss of less than 67% of the initial ATP. It is suggested, therefore, that the rate constant for the initial loss of ATP in intact RBCs, as stimulated by the ionophore A23187, can be taken as a measure of the capacity of the Ca pump ATPase.


Heterogeneity of the Na(+)-H+ antiport systems in renal cells. This study analyzes the differential characteristics of the Na(+)-H+ antiport systems observed in several epithelial and non-epithelial renal cell lines. Confluent monolayers of LLC-PK1A cells have a Na(+)-H+ antiport system located in the apical membrane of the cell. This system, however, is not expressed during cell proliferation or after incubation in the presence of different mitogenic agents. In contrast, confluent monolayers of MDCK4 express minimal Na(+)-H+ antiport activity in the confluent monolayer state but reach maximal antiport activity during cell proliferation or after activation of the cells by different mitogenic agents. Similar results were obtained with the renal fibroblastic cell line BHK. The system present in MDCK4 cells is localized in the basolateral membrane of the epithelial cell. In LLC-PK1A cells, an increase in the extracellular Na+ concentration produces a hyperbolic increase in the activity of the Na(+)-H+ antiporter. In MDCK4 and BHK cells, however, an increase in external Na+ produces a sigmoid activation of the system. Maximal activation of the system occur at a pHo 7.5 in LLC-PK1A cells and pHo 7.0 in MDCK4 cells. The Na(+)-H+ antiporter of LLC-PK1A cells is more sensitive to the inhibitory effect of amiloride (Ki 1.8 x 10(-7) M) than is the antiporter of MDCK4 cells (Ki 7.0 x 10(-6) M). Moreover, 5-(N-methyl-N-isobutyl)amiloride is the most effective inhibitor of Na(+)-H+ exchange in LLC-PK1A cells, but the least effective inhibitor in MDCK4 cells. Conversely, the analog, 5-(N,N-dimethyl)amiloride, is the most effective inhibitor of Na(+)-H+ exchange in MDCK4 cells, but is the least effective inhibitor in LLC-PK1A cells. These results support the hypothesis that Na(+)-H+ exchange observed in LLC-PK1A and other cell lines may represent the activity of different Na(+)-H+ antiporters.


Incorporation and binding of estrogens into melanin: comparison of mushroom and mammalian tyrosinases. The activities of mushroom and melanoma tyrosinases towards the estrogens were compared. While the fungal enzyme is capable of hydroxylating estradiol to the 2-hydroxy compound and to oxidize the latter to the quinone, the mammalian enzyme does not have this ability. With dopa as substrate and an estrogen present in the reaction mixture, both enzyme reactions yield melanin with the steroid firmly incorporated into the pigment, although with the mammalian enzyme the incorporation is small. The steroid appears to be incorporated by covalent linkage. It is suggested that the incorporation of estrogens into melanin produced by mammalian tyrosinase is via their oxidation by oxidized intermediates of the dopa to melanin transformation. Melanin itself may function as oxidant for the estrogens. Whole melanoma cells are capable of binding estrogens and incorporating small amounts into melanosomes. Similarly, fresh melanosomes in isolation can incorporate estrogens into their structure, presumably by covalent bonding to their melanin.


Metal-ion-directed site-specificity of hydroxyl radical detection. A wide variety of .OH detectors are in use for determination of biological .OH production. The chemical generation of .OH is site-specific with respect to the metal-binding site, and thus .OH detectors with metal-binding properties may affect the biological damage and bias .OH detection. The present study shows that both salicylate and phenylalanine, added as low molecular weight .OH indicators, decreased Cu(II) binding to erythrocyte ghosts. In a cell-free system, Cu(II) complexed to both salicylate and phenylalanine. Phenylalanine is a stronger Cu(II) chelator than salicylate, both when competing for Cu(II) bound to ghosts and when competing directly with each other. When OH radicals were generated by ascorbate and Cu(II), the amount of .OH detected as dihydroxybenzoates was proportional to the amount of .OH produced. However, when phenylalanine was added to this system, the efficiency of .OH detection by salicylate strongly decreased, concomitant with the transfer of Cu(II) binding from salicylate to the amino acid. This decrease was larger than that predicted by calculations for random competition of the two detectors for .OH. Deoxyribose and mannitol, which do not bind copper appreciably, competed poorly with salicylate for the .OH. Hydroxylation of phenylalanine, on the other hand, was only slightly affected by the presence of salicylate and unaffected by deoxyribose and mannitol. These results suggest that the detection of .OH by low molecular weight .OH indicators was related to the relative affinity of the detectors for the catalyzing metal, and thus partially site-specific. Furthermore, glutamate, which does not contain an aromatic ring but binds Cu(II) with considerable affinity, competed strongly with salicylate for the .OH, indicating that metal-binding properties rather than the presence of an aromatic ring were the cause of the deviation from random competition. The results indicate that .OH indicators with metal-binding properties affect the distribution of catalytic metal ions in a biological system, causing a shift of free radical damage and localizing a site-specific reaction of .OH on these detectors, with a resulting positive bias in the apparent .OH production.


Detection of radicals produced by reaction of hydroperoxides with rat liver microsomal fractions. EPR spin trapping using the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 3,5-dibromo-4-nitrosobenzene sulphonic acid (DBNBS) has been employed to examine the generation of radicals produced on reaction of a number of primary, secondary and lipid hydroperoxides with rat liver microsomal fractions in both the presence and absence of reducing equivalents. Two major mechanisms of radical generation have been elucidated. In the absence of NADPH or NADH, oxidative degradation of the hydroperoxide occurs to give initially a peroxyl radical which in the majority of cases can be detected as a spin adduct to DMPO; these radicals can undergo further reactions which result in the generation of alkoxyl and carbon-centered radicals. In the presence of NADPH (and to a lesser extent NADH) alkoxyl radicals are generated directly via reductive cleavage of the hydroperoxide. These alkoxyl radicals undergo further fragmentation and rearrangement reactions to give carbon-centered species which can be identified by trapping with DBNBS. The type of transformation that occurs is highly dependent on the structure of the alkoxyl radical with species arising from beta-scission, 1,2-hydrogen shifts and ring closure reactions being identified; these processes are in accord with previous chemical studies and are characteristic of alkoxyl radicals present in free solution. Studies using specific enzyme inhibitors and metal-ion chelators suggest that most of the radical generation occurs via a catalytic process involving haem proteins and in particular cytochrome P-450. An unusual species (an acyl radical) is observed with lipid hydroperoxides; this is believed to arise via a cage reaction after beta-scission of an initial alkoxyl radical.


8-methoxycaffeine inhibition of Drosophila DNA topoisomerase II. We have investigated the effect of 8-methoxycaffeine on the interaction between Drosophila DNA topoisomerase II and DNA. We have shown that 8-methoxycaffeine affected the enzyme strand-passing activity by inhibiting decatenation of kinetoplast DNA, and that it interfered with the breakage-reunion reaction by stabilizing a cleavable complex. Treatment of the cleavable complex with protein denaturant resulted in DNA breaks. High resolution mapping of the cleavage sites in the central spacer region of Tetrahymena rDNA revealed that, contrary to what was observed with clinically important DNA topoisomerase II inhibitors, 8-methoxycaffeine did not modify the cleavage pattern observed without the drug.


cDNA cloning and membrane topology of the rabbit gastric H+/K(+)-ATPase alpha-subunit. We have cloned and sequenced a cDNA for the rabbit gastric proton-potassium pump (H+/K(+)-ATPase) alpha-subunit. The deduced peptide contains 1035 amino acids (Mr 114,201) and shows 97% sequence identity with the respective rat and hog proteins. A monoclonal antibody 146-14 has been shown previously to react with the extracytoplasmic side of the catalytic H+/K(+)-ATPase subunit and here we show that the epitope is in the region between amino acids 855 and 902 (the numbering of the H+/K(+)-ATPase catalytic subunit throughout the paper refers to the rabbit sequence). The localization of this epitope in conjunction with previously observed trypsin cleavage sites in the C-terminal one third of the enzyme and the hydrophobicity plot of the deduced peptide sequence are evidence for a structural model for the alpha-subunit of the H+/K(+)-ATPase which contains at least ten membrane spanning segments, similar to that deduced for the Ca(2+)-ATPase of sarcoplasmic reticulum.


Oxidation of hydroquinone by both cellular and extracellular grapevine peroxidase fractions. The oxidation of hydroquinone by two peroxidase (EC 1.11.1.7) fractions obtained from the cells and spent medium of cell cultures of grapevine (Vitis vinifera cv Monastrell) has been studied, and their comparative efficacy (kcat/KM ratio) studied in both the H2O2-consuming and hydroquinone-consuming reactions. While the efficacy in the H2O2-consuming reaction is practically identical for both enzyme fractions, the cellular peroxidase has five-fold more efficacy in the hydroquinone-consuming reaction than the peroxidase located in the spent medium. Screening of cellular peroxidases capable of oxidizing hydroquinone on polyacrylamide gels, by means of a staining reaction based on the nucleophilic attack of 4-aminoantipyrine on p-benzoquinone in acidic media, reveals that all the cellular peroxidase isoenzymes are capable of oxidizing hydroquinone, probably yielding a quinone-diimine as a product of the staining reaction. Since isoperoxidases found in cellular fractions are also present in the spent medium, the values found for the different efficacies in the hydroquinone-consuming reaction must be considered as the results of the different proportions in which each peroxidase isoenzyme was found in the two fractions. The localization of a benzoquinone-generating system of high efficacy inside the plant cell, and probably located in vacuoles, is discussed with respect to the harmful role which the quinone/semiquinone pair might play in cell death, as part of the hypersensitive response expressed within the mechanism of plant disease resistance.


Respiratory electron transfer activity in an asolectin-isooctane reverse micellar system. Bovine heart submitochondrial particles (SMP) were solubilized in an asolectin isooctane reverse micellar system and the functionality of the respiratory chain was tested by spectroscopic and amperometric techniques. Electron transfer rate supported by NADH was very slow as evidenced by the low cytochrome reduction levels attained over long incubation periods. In the presence of KCN, NADH caused 34% and 12.5% reduction of the cytochromes aa3 and c, respectively, and negligible reduction of cytochrome b. Supplementation of the system with menadione rose the NADH-dependent reduction of all the cytochromes to levels that were close to the total content. However, no measurable O2 uptake activity took place in the presence of NADH plus menadione, or with ascorbate (or NADH) plus TMPD reducing systems. Therefore, it is suggested that in the organic medium, electron transfer from NADH to O2 is arrested at the terminal oxidase step. Cytochrome oxidase reduced by ascorbate (or NADH) plus TMPD seems to be trapped in its half reduced state (ie, a2+ a3(3+)). Although it is poorly reactive with O2, it can transfer electrons back to cytochrome c and TMPD. The electron transfer block to O2 was overcome when PMS was used instead of TMPD. This seems to be due to the recognized capacity of PMSH2 to carry out simultaneous reduction of both a CuA and a3 CuB redox centers of cytochrome oxidase. The cytochrome oxidase reaction in the organic solvent was highly sensitive to KCN (Ki 1.9 microM) and showed bell-shaped kinetics towards the PMS concentration and a sigmoidal response to water concentration, reaching its maximal turnover number (18 s-1) at 4 mM PMS and 1.1% (v/v) water.(ABSTRACT TRUNCATED AT 250 WORDS)


Differential migratory response of U-2 OS osteosarcoma cell to the various forms of platelet-derived growth factor. U-2 OS osteosarcoma cells are mesenchymal-derived transformed cells spontaneously expressing both platelet-derived growth factor (PDGF) A- and B-chain genes, and releasing PDGF AA dimers in culture. Using modified Boyden chemotactic chambers, platelet-purified PDGF was shown to be a chemoattractant for U-2 OS cells. More specifically, U-2 OS cells migrated in the presence of PDGF AB and BB dimers but not in the presence of PDGF AA dimers. This pattern of response was similar to that observed with human fibroblasts and this similarity is consistent with the fact that U-2 OS cells express PDGF receptor alpha- and beta-subunits in a similar fashion to human fibroblasts.


The measurement of nuclear topoisomerase II inhibition in vitro: a possible tool for detecting resistance on a subcellular level in haematopoietic malignancies. Anthracyclines, podophyllotoxines, N-[4-(9-acridinylamino)-3-methoxyphenyl]-methanesulphoneamide (amsacrine, INN) and mitoxantrone are cytostatic agents which exert several molecular effects in the cell. Among these, the inhibition of the nuclear enzyme topoisomerase II appears to be instrumental in cytotoxicity. Tumour cells can acquire resistance to these drugs by several molecular mechanisms. The alteration of target protein sensitivity is one of these. In the present study, we directly measured the inhibition of topoisomerase II by cytostatic drugs. The procedure included isolation of cell nuclei, extraction of nuclear proteins, fractionation of nuclear extracts by anion exchange chromatography and measurement of the catalytic activity in the presence of various concentrations of the drugs. All steps can be performed within one day and require a minimum sample of 10(7)-10(8) malignant cells. We used cell samples from a multidrug-resistant subclone of the human promyelocytic cell line HL-60 to study the feasibility of the approach. We found an increased resistance of topoisomerase II to etoposide and amsacrine, which correlates with the increased cellular resistance to these drugs as determined by exposure in short term liquid cultures.


Cathepsins B, H and L in human breast carcinoma. We have measured the activity of the cysteine proteinase cathepsin B, the inhibitory activity of cysteine proteinase inhibitors and the amounts of cathepsins B, H and L in normal sera and sera from patients with breast cancer, as well as in tissue homogenates of tumorous and non-tumorous samples. The amounts of cathepsin B, determined by ELISA in tumour sera (n = 17) were significantly higher than the amounts determined in normal sera (n = 20). On the other hand, the differences in the amounts measured in tumour sera were not significant when compared with the known histopathological characteristics. In cytosols of breast cancer tumour tissue in general, the level of cathepsin H was higher than those of cathepsins B and L in all samples tested. In the same samples, at least a 10-fold increase of cathepsin B (activity and quantity) was detected in matched pairs (n = 20) of carcinoma and normal tissue of the same breast (p less than 0.01). The amount of cathepsin B correlated with the degree of malignancy inside the histological subtypes of invasive ductal carcinoma (n = 90, p less than 0.01). In addition, a negative correlation of values for cathepsin B with the involvement of regional lymph nodes (n = 75, p less than 0.01) was found inside the same group. In contrast, the activities of cysteine proteinase inhibitors did not correlate with any of the known clinical data. Our data provide further indirect evidence for the involvement of cathepsin B in the processes of tumour growth and metastasis in breast carcinoma. The follow-up studies will verify the prognostic value of these findings.


A discrete-time model for the statistical analysis of infectious disease incidence data. A discrete-time model is devised for the per-time-unit distribution of infectious disease cases in a sample of households. Using the time at which an individual is identified (e.g., when illness symptoms appear) as a marker for being infected, the probabilities of becoming infected from the community or from a single infectious household member are estimated for various risk factor levels. Maximum likelihood procedures for estimating the model parameters are given. An individual may be classified with regard to level of susceptibility and level of infectiousness. The model is fitted to a combination of symptom and viral culture data from a rhinovirus epidemic in Tecumseh, Michigan. In general, it is observed that decreasing risk of infection is associated with increasing age.


Inositol trisphosphate, cyclic AMP, and cyclic GMP in rat brain regions after lithium and seizures. The mechanism of action of lithium, the primary treatment for bipolar affective disorder, is unknown but may involve inhibition of second messenger production in the brain. Therefore, the concentrations of three second messengers, inositol 1,4,5 trisphosphate (Ins 1,4,5P3), cyclic adenosine monophosphate (AMP), and cyclic guanosine monophosphate (GMP), were measured in rat cerebral cortex and hippocampus after acute or chronic lithium administration, as well as after treatment with the cholinergic agonist pilocarpine alone or in combination with lithium at a dose that induces seizures only in lithium pretreated rats. Neither acute nor chronic lithium treatment altered the hippocampal or cortical concentration of Ins 1,4,5P3, cyclic AMP, or cyclic GMP. Pilocarpine administered alone increased Ins 1,4,5P3 in both regions, did not alter cyclic AMP, and slightly increased cyclic GMP in the cortex. Coadministration of lithium plus pilocarpine caused large increases in the concentrations of all three second messengers and the production of each of them was uniquely attenuated: lithium reduced pilocarpine-induced increases of Ins 1,4,5P3 in the cortex at 60 min; chronic lithium administration reduced stimulated cyclic AMP production in the hippocampus; and chronic lithium treatment impaired stimulated cyclic GMP production in both regions. In summary, chronic lithium treatment appeared only to reduce Ins 1,4,5P3 and cyclic AMP concentrations after a long period of stimulation whereas cyclic GMP production was reduced by chronic lithium administration after both short and long periods of stimulation. Thus cyclic GMP was most sensitive to lithium and lithium attenuation of second messenger formation may be most important in excessively activated pathways.


N-estimation from retrospectively ascertained events with applications to AIDS. It is a common sampling scheme in retrospective studies that the data set includes only individuals who satisfy a certain sampling criterion. In this paper we consider the situation when the sampling criterion is a specified event, and assume that an earlier event can be retrospectively identified given the occurrence of the specified event. A semiparametric method, which is a compromise between nonparametric and parametric methods, is employed for the estimation of the expected number of the specified events (namely, the N-estimation) occurring in arbitrarily given intervals. A number of statistical properties of the estimates are developed. Due to the limitation of semiparametric models, our estimates should be regarded as conservative estimates since in general they underestimate the actual number of the specified events. This type of limitation, however, cannot be avoided with nonparametric or semiparametric models. Applications to acquired immunodeficiency syndrome (AIDS) cases are considered. The blood transfusion AIDS cases reported to the Centers for Disease Control are analyzed in detail.


Estimating the ventilation-perfusion distribution: an ill-posed integral equation problem. The distribution of ventilation-perfusion ratio over the lung is a useful indicator of the efficiency of lung function. Information about this distribution can be obtained by observing the retention in blood of inert gases passed through the lung. These retentions are related to the ventilation-perfusion distribution through an ill-posed integral equation. An unusual feature of this problem of estimating the ventilation-perfusion distribution is the small amount of data available; typically there are just six data points, as only six gases are used in the experiment. A nonparametric smoothing method is compared to a simpler method that models the distribution as a histogram with five classes. Results from the smoothing method are found to be very unstable. In contrast, the simpler method gives stable solutions with parameters that are physiologically meaningful. It is concluded that while such smoothing methods may be useful for solving some ill-posed integral equation problems, the simpler method is preferable when data are scarce.


Saturation transfer, continuous wave saturation, and saturation recovery electron spin resonance studies of chain-spin labeled phosphatidylcholines in the low temperature phases of dipalmitoyl phosphatidylcholine bilayers. Effects of rotational dynamics and spin-spin interactions. The saturation transfer electron spin resonance (STESR) spectra of 10 different positional isomers of phosphatidylcholine spin-labeled in the sn-2 chain have been investigated in the low temperature phases of dipalmitoyl phosphatidylcholine (DPPC) bilayers. The results of continuous wave saturation and of saturation recovery measurements on the conventional ESR spectra were used to define the saturation properties necessary for interpreting the STESR results in terms of the chain dynamics. Spin labels with the nitroxide group located in the center of the chain tended to segregate preferentially from the DPPC host lipids in the more ordered phases, causing spin-spin interactions which produced spectral broadening and had a very pronounced effect on the saturation characteristics of the labels. This was accompanied by a large decrease in the STESR spectral intensities and diagnostic line height ratios relative to those of spin labels that exhibited a higher degree of saturation at the same microwave power. The temperature dependence of the STESR spectra of the different spin label isomers revealed a sharp increase in the rate of rotation about the long axis of the lipid chains at approximately 25 degrees C, correlating with the pretransition of gel phase DPPC bilayers, and a progressive increase in the segmental motion towards the terminal methyl end of the chains in all phases. Prolonged incubation at low temperatures led to an increase in the diagnostic STESR line height ratios in all regions of the spectrum, reflecting the decrease in chain mobility accompanying formation of the subgel phase. Continuous recording of the central diagnostic peak height of the STESR spectra while scanning the temperature revealed a discontinuity at approximately 14-17 degrees C, corresponding to the DPPC subtransition which occurred only on the initial upward temperature scan, in addition to the discontinuity at 29-31 degrees C corresponding to the pretransition which displayed hysteresis on the downward temperature scan.


Cryo-EM of the native structure of the calcium release channel/ryanodine receptor from sarcoplasmic reticulum. The native structure of the calcium release channel (ryanodine receptor) from rabbit skeletal muscle has been analyzed in two dimensions from electron micrographs of frozen hydrated specimens. Within a resolution of 3.0 nm there is excellent agreement between the structure as seen in vitreous water and in negative stained specimens. Features seen in the three-dimensional reconstruction of the negatively stained channel can be identified in the projection of the unstained receptor.


Steady-state availability of sodium channels. Interactions between activation and slow inactivation. Changes in holding potential (Vh), affect both gating charge (the Q(Vh) curve) and peak ionic current (the F(Vh) curve) seen at positive test potentials. Careful comparison of the Q(Vh) and F(Vh) distributions indicates that these curves are similar, having two slopes (approximately 2.5e for Vh from -115 to -90 mV and approximately 4e for Vh from -90 to -65 mV) and very negative midpoints (approximately -86 mV). Thus, gating charge movement and channel availability appear closely coupled under fully-equilibrated conditions. The time course by which channels approach equilibration was explored using depolarizing prepulses of increasing duration. The high slope component seen in the F(Vh) and Q(Vh) curves is not evident following short depolarizing prepulses in which the prepulse duration approximately corresponds to the settling time for fast inactivation. Increasing the prepulse duration to 10 ms or longer reveals the high slope, and left-shifts the midpoint to more negative voltages, towards the F(Vh) and Q(Vh) distributions. These results indicate that a separate slow-moving voltage sensor affects the channels at prepulse durations greater than 10 ms. Charge movement and channel availability remain closely coupled as equilibrium is approached using depolarizing pulses of increasing durations. Both measures are 50% complete by 50 ms at a prepulse potential of -70 mV, with proportionately faster onset rates when the prepulse potential is more depolarized. By contrast, charge movement and channel availability dissociate during recovery from prolonged depolarizations. Recovery of gating charge is considerably faster than recovery of sodium ionic current after equilibration at depolarized potentials. Recovery of gating charge at -140 mV, is 65% complete within approximately 100 ms, whereas less than 30% of ionic current has recovered by this time. Thus, charge movement and channel availability appear to be uncoupled during recovery, although both rates remain voltage sensitive. These data suggest that channels remain inactivated due to a separate process operating in parallel with the fast gating charge. We demonstrate that this behavior can be simulated by a model in which the fast charge movement associated with channel activation is electrostatically-coupled to a separate slow voltage sensor responsible for the slow inactivation of channel conductance.


Solvent effects on myoglobin conformational substates as studied by electron paramagnetic resonance. Electronic paramagnetic resonance spectra of frozen horse myoglobin solutions at two different pH values and with different added organic solvents are analyzed by computer simulation in terms of Gaussian distributions of some ferric ion crystal field parameters. The mean values and the corresponding variances of these distributions, thought as arising from a distribution of the protein conformational substates, are found to be affected by both the pH and the addition of organic solvents. The significant narrowing of the conformational substate distribution, induced by large addition of glycerol, is discussed.


Protein phosphorylation driven by intracellular calcium oscillations: a kinetic analysis. Given the ubiquitous nature of signal-induced Ca2+ oscillations, the question arises as to how cellular responses are affected by repetitive Ca2+ spikes. Among these responses, we focus on those involving protein phosphorylation. We examine, by numerical simulations of a theoretical model, the situation where a protein is phosphorylated by a Ca(2+)-activated kinase and dephosphorylated by a phosphatase. This reversible phosphorylation system is coupled to a mechanism generating cytosolic Ca2+ oscillations; for definiteness, this oscillatory mechanism is based on the process of Ca(2+)-induced Ca2+ release. The analysis shows that the average fraction of phosphorylated protein increases with the frequency of repetitive Ca2+ spikes; the latter frequency generally rises with the extent of external stimulation. Protein phosphorylation therefore provides a mechanism for the encoding of the external stimulation in terms of the frequency of signal-induced Ca2+ oscillations. Such a frequency encoding requires precise kinetic conditions on the Michaelis-Menten constants of the kinase and phosphatase, their maximal rates, and the degree of cooperativity in kinase activation by Ca2+. In particular, the most efficient encoding of Ca2+ oscillations based on protein phosphorylation occurs in conditions of zero-order ultrasensitivity, when the kinase and phosphatase are saturated by their protein substrate. The kinetic analysis uncovers a wide variety of temporal patterns of phosphorylation that could be driven by signal-induced Ca2+ oscillations.


Multivalent ions are necessary for poly[d(AC).d(GT)] to assume the Z form: a CD study. In previous work, it was shown that poly[d(AC).d(GT)] could be forced into the Z form by strong dehydrating conditions, provided EDTA was not present. Presumably multivalent impurities were also necessary for the transition. In order to gain control over the B to Z transition for this DNA, we carefully removed all divalent contaminants from the sample and asked the obvious question: What ions are necessary for the transition under dehydrating conditions? We systemically investigated the effect of various multivalent ions. The common contaminants Ca2+, Mg2+, and Fe3+ will not cause the transition, but Co2+ and Ni2+ facilitate the transition, undoubtedly because of their well-known propensity to bind to purine N7. Since the transition also depends on the synergistic dehydrating action of sodium perchlorate and ethanol, we include CD spectra for the independent variations of these two factors. In addition, vacuum-uv CD spectra for the A form and various B forms of poly[d(AC).d(GT)] are presented for the first time.


Oxygen-derived free radical and active oxygen complex formation from cobalt(II) chelates in vitro. The electron paramagnetic resonance (EPR) spin trapping technique was used to study the generation of oxygen free radicals from the reaction of hydrogen peroxide with various Co(II) complexes in pH 7.4 phosphate buffer. The 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin trap was used in these experiments to detect superoxide and hydroxyl free radicals. Superoxide radical was generated from the reaction of H2O2 with Co(II), but was inhibited when Co(II) was chelated with adenosine 5'-diphosphate or citrate. Visible absorbance spectra revealed no change in the final oxidation state of the cobalt ion in these samples. The EDTA complex also prevented detectable free-radical formation when H2O2 was added, but visible absorbance data indicated oxidation of the Co(II) to Co(III) in this case. The amount of DMPO/.OOH adduct detected by EPR was greatly enhanced when H2O2 reacted with the nitrilotriacetate complex relative to Co(II) alone, and in addition, concurrent formation of the DMPO/.OH adduct due to slow oxidation of Co(II) was observed. The hydroxyl radical adduct formation was suppressed by ethanol, but not DMSO, indicating that free hydroxyl radical was not formed. The deferoxamine nitroxide radical was exclusively formed when H2O2 was added to the Co(II) complex of this ligand, most probably in a site-specific manner. In the presence of ethylenediamine, Co(II) bound molecular O2 and directly oxidized DMPO to its DMPO/.OH adduct without first forming free superoxide, hydroxyl radical, or hydrogen peroxide. An experiment using 17O-enriched water revealed that the Co(II)-ethylenediamine complex caused the DMPO to react with solvent water to form the DMPO/.OH adduct. The relevance of these results to toxicological studies of cobalt is discussed.


The group I intron of apocytochrome b gene from Chlamydomonas smithii encodes a site-specific endonuclease. The mitochondrial DNA molecules of two interfertile algal species, Chlamydomonas smithii and C. reinhardtii, are co-linear except for a 1075 bp intron (the alpha-insert) that is present in the cob gene of C. smithii. The alpha-insert, a group I intron (Cs cob.1) containing an open reading frame (ORF) which encodes a basic, hydrophilic protein of 237 amino acids, is unidirectionally transmitted to all diploid progeny during interspecific crosses. In this report, we show that the Cs cob.1-encoded protein is a site-specific endonuclease (I-Csm I) which could mediate the intron transfer via the gene conversion mechanism. The Cs cob.1 ORF was cloned into the vector pMALcr1 and over-expressed as a hybrid protein fused to maltose-binding protein (MBP). This fusion protein exhibited an in vivo endonuclease activity which specifically cleaved the intron homing site within the intronless cob gene.


Synthesis, structure, and stability of the zinc complex of the dimethyl ester of meso-2,3-dimercaptosuccinic acid. The dimethyl ester of meso-2,3-dimercaptosuccinic acid (meso-DiMeDMSA) reacts with Zn2+ ions to form protonated and polynuclear complexes. The species [Zn2L3H]- and [Zn2L3]2- are formed at pH values between 3 and 6.5 and have overall formation constants of 10(37.53) and 10(32.52), respectively, whereas the mononuclear complex [ZnL2]2- is formed at physiological pH and has a formation constant of 10(18.06). The mononuclear complex was synthesized and isolated as an ion association complex with the tetramethylammonium ion, [(Me)4N]2[Zn(DiMeDMSA)2], and its structure in solution was determined by low-temperature 1H and 13C NMR spectroscopy. In this structure the Zn2+ ion is tetrahedrally coordinated to four thiolate groups from two meso-DiMeDMSA molecules.


Purification and properties of DNA topoisomerase I from broccoli. We have purified a topoisomerase activity from broccoli (Brassica oleracea var. italica) to near homogeneity. The enzyme is an 80 kDa monomer as judged by gel filtration chromatography and SDS gel electrophoresis, though it may represent a proteolytic fragment of a larger protein. The enzyme is capable of removing both negative and positive supercoils in steps of one, does not absolutely require Mg2+, is only very weakly stimulated by NaCl, is inhibited by camptothecin, and cross-reacts with an antibody directed against human DNA topoisomerase I. These properties identify the enzyme as a eukaryotic type I topoisomerase.


Characterization of a Gy4 glycinin gene from soybean Glycine max cv. forrest. The glycinin gene family encoding the glycinin subunits in soybean plants is composed of at least five gene members. A genomic clone lambda S312 containing the Gy4 gene from a genomic library of cv. Forrest was isolated and partially characterized. The organization of this gene was found to be similar to that of a null allele from cv. Raiden, but different from the Gy4 gene from cv. Dare. The complete nucleotide sequence of this gene has been determined. It is 2599 bp long consisting of four exons and three introns. Comparing the DNA sequences between this gene and the gene from Dare and a null allele from Raiden, the difference found in the coding region was 5'-GCAGTGCAAG-3' (nt 824 to 833) in the former case versus 5'-TGGAGTTGCAATT-3' (nt 1314 to 1326) in the latter case in the exon 2 domain, resulting in three amino acid differences and one amino acid absence. Some other differences were also found in the non-coding region. The coding sequence and 5'-flanking region of the Gy4 gene, when compared with that of other legumin genes as well as group 1 glycinin subunit genes, revealed some interesting features: (1) a transposable element-like sequence was found in the hypervariable region (HVR) of the exon 3 domain, which was lacking in the legumin and the glycinin group 1 genes; (2) in the 5'-flanking region from nt -145 to -1, two high-homology sequences were found: one from nt -141 to nt -132, the other from nt -118 to nt -92 which includes the 'legumin box' and the RY repeat element.


Conversion of human fibroblasts to tissue macrophages by the Snyder-Theilen feline sarcoma virus (ST:FeSV): tumoricidal potential in monolayers and in agar suspensions. In earlier studies [1-3], we have demonstrated the conversion of human fibroblasts (HF) to tissue macrophages (TM) by the Snyder-Theilen feline sarcoma virus (ST:(FeSV)). The purpose of the present study is to determine the cytolytic potential of ST:FeSV(FeLV)-induced TM against tumorigenic target cells under defined conditions in vitro. The results show that ST:FeSV-induced TM, but not mock-infected HF, produced significant lysis of human colon adenocarcinoma cells (LS-180) after a 3-day preincubation period, followed by a 4-day coincubation period at an effector to target cell ratio of 5:1. The presence of IFN-gamma, or lipopolysaccharides (LPS), and especially of M-CSF, during the coincubation period generally yielded optimal lysis of the tumor cells. Addition of LS-180 specific antibody (NRCO-4) substantially increased the cytolytic potential of TM. Significantly, coincubation of TM with LS-180 tumor cells in an agar medium, where no direct contact between cells occurs, resulted in the inhibition of tumor cell proliferation. Addition of LPS has further accentuated this inhibition. The results indicate that ST:FeSV-induced macrophages are potent oncocytolytic agents of LS-180 tumor cells in the absence and in the presence of direct contact between effector and target cells.


A new ECG measure (RSh) for detecting possible sodium channel blockade in vivo in rats. A new electrocardiographic (ECG) measure for detecting possible sodium channel blocking actions of drugs in anaesthetized rats is described. The conventional measures for sodium channel blockers are increased QRS width and/or P-R prolongation, however, these are limited in their sensitivity. This new measure, RSh, is the height from the peak of the R wave to the bottom of the S wave; it is more sensitive to known sodium channel blocking agents than conventional measures. This was shown by comparing the ECG effects of sodium channel blocking class I antiarrhythmic drugs from the three subclasses lidocaine (Ia), quinidine (Ib), and flecainide (Ic). In each case, RSh increased before changes could be detected in QRS or P-R. With tetrodotoxin and quinacainol, a new class I agent, changes in RSh correlated directly with previously reported changes in dV/dtmax of intracellular potentials recorded in vivo from epicardial cells. Representatives from antiarrhythmic classes II, III, and IV were also tested and only changed RSh when they had known sodium channel blocking properties at high doses. Other physiological maneuvers for altering heart rate, such as changing vagal activity, administration of catecholamines, or direct right atrial pacing, did not alter RSh. Thus RSh is a useful in vivo measure for the detection of possible class I antiarrhythmic actions. It has the advantages of being sensitive, selective, easy to measure, and involving minimal preparation.


Localization of genes and anonymous DNA probes on the short arm of chromosome 7. We have previously described the cytogenetic analysis of two patients with Greig cephalopolysyndactyly syndrome (GCPS) and various microdeletions on the short arm of Chromosome (Chr) 7. Using genes and anonymous DNA probes from 7p we analyzed the DNA of our patients for loss of heterozygosity, or we determined the copy number by semi-quantitative Southern hybridization. We have been able to show hemizygosity for the genes of INHBA, IGFBP1 and GLI3 in both patients and therefore can give the chromosomal assignment 7p12.3-p13. CRI-R944 and CRI-P137 map to the same region, whereas CRI-S207 can be assigned to 7p13-p14.2; TM102L, TS93, TS194, TM77 and TN177 showed no change and these probes map distal to 7p14.2.


Assignment of the rat genes coding for alpha 1-antitrypsin (PI), phosphoenolpyruvate carboxykinase (PEPCK), alcohol dehydrogenase (ADH), and fructose-1,6-bisphosphatase (FDP). The chromosomal localization of four genes which are expressed mainly in the liver has been undertaken for the rat. Using a panel of hybrid clones segregating rat chromosomes, and Southern blot analysis, alpha 1-AT (PI), PEPCK, ADH and FDP are assigned to rat Chromosomes (Chr) 6, 3, 2 and 17, respectively. Groups of synteny among rat, mouse and human species are discussed in relationship to the new assignments.


A new in vitro bioassay for human calcitonin: validation and comparison to the rat hypocalcemia bioassay. The human breast cancer cell line T 47 D expresses calcitonin (CT) receptors that are coupled to adenylate cyclase and which reveal a dose-dependent cyclic AMP response to CT. We used this model to establish an in vitro bioassay for synthetic human CT (hCT) preparations to overcome some of the obstacles of the standard rat hypocalcemia in vivo bioassay. The detection limit of the in vitro bioassay was 1 x 10(-10) M hCT (EC 50: 8.7 pM +/- 26%) compared to 7.3 x 10(-9) M (EC 50: 7.2 microM +/- 32%) for the in vivo bioassay. The relative potencies of test preparations revealed a good correlation (r = 0.89) and several hCT-related substances produced comparable results when tested by the two methods. The standard deviations of precision and accuracy, however, were significantly smaller (P less than 0.05) for the in vitro bioassay. According to these data the T 47 D in vitro bioassay is more sensitive, superior in precision and accuracy, and comparable in specificity to the rat hypocalcemia bioassay.


IgG and IgA antibody responses against porins in Yersinia-triggered reactive arthritis. Using an enzyme immunoassay, the development and persistence of serum IgG and IgA class antibody responses against porins, a class of the major outer membrane proteins, were compared between patients who developed reactive arthritis and subjects who did not after Yersinia infection. A significant difference was seen between the two patient groups in the beginning of the disease: those with reactive arthritis had higher levels of IgG and IgA class antibodies against the porins. A similar difference in the IgA class antibodies was also seen at follow-up. This supports the hypothesis of persistence of the pathogen within the arthritic host.


Phase II trial of high-dose 24-hour continuous intravenous 5-fluorouracil for advanced non-small cell lung cancer: a Cancer and Leukemia Group B study. Eighty-six eligible patients with non-small cell lung cancer were treated on a Phase II, CALGB study with high-dose, 24-h continuous intravenous 5-fluorouracil every 2 weeks. Objective responses were seen in 7 (8%) patients with 1 (1%) complete response and 6 (7%) partial responses. The median survival for these patients without prior chemotherapy was 3.8 months. Gastrointestinal and hematologic toxicity were acceptable for most patients. However, two patients experienced acute clinical deterioration characterized by worsening central nervous system and hemodynamic function beginning near the completion of chemotherapy treatment and resulting in death. Because of its potential for severe, unpredictable neurologic and cardiac toxicity, we do not recommend this dose and schedule of 5-FU for future trials.


The origins of DNA breaks: a consequence of DNA damage, DNA repair, or apoptosis? DNA breaks can arise from many sources after incubation of cells with toxic agents. Very few agents break DNA directly, rather most breaks occur as a result of metabolic participation by the cell, such as during attempts to repair the damage. It is now realized that many DNA breaks arise as a consequence of steps in the pathway of cell death. Upon reanalyzing the methodology commonly used to detect DNA breaks, it is evident that many studies would not have observed DNA breaks associated with cell death. Frequently experimental conditions have been used that are extremely toxic to cells with the justification that the cells were still viable as measured by their ability to exclude dyes such as trypan blue. However, the DNA digestion associated with cell death by apoptosis occurs prior to changes in membrane integrity. Because the possibility of endogenous endonuclease activity was not realized, many studies may have inaccurately assumed that DNA breaks arose during, for example, inhibition of DNA repair or as intermediates in recombination. In light of the new understanding of apoptosis and the formation of DNA breaks as an early event in cell death, it is important to both reevaluate past conclusions and to ensure that future studies fully consider the breaks derived from the cytotoxicity of every agent under investigation.


Metabolic control and B cell function in patients with diabetes mellitus secondary to chronic pancreatitis. To investigate the relationship between metabolic control and beta cell functions in chronic pancreatitis, 30 patients were selected for study, including 10 with diabetes mellitus in insulin-dependent state (group 1, Mean age 37.6), 10 with diabetes mellitus in non-insulin-dependent state (group 2, Mean age 47.8), and 10 with normal fasting glucose levels (group 3, Mean age 42.1). Each patient received urine routine, stool fat, renal function, biochemical study such as: serum lipid and glycosylated hemoglobin, eye fundi and X-ray examinations. Beta cell function was measured by C-peptide concentration six minutes after intravenous infusion of 1 mg glucagon. The results showed that the glycosylated hemoglobin concentrations were higher in group 1 than in group 2 or 3 patients (P less than 0.05), and were higher in group 2 than in group 3 patients (P less than 0.001) as well. The cholesterol and triglyceride levels were not significantly different among three groups. Furthermore, eight and two of group 1 and 2 patients manifested pancreatic calcification on abdomen X-ray examination (P less than 0.05). All and eight of group 1 and 2 patients received insulin injection respectively. In addition, group 1 patients were more likely to develop steatorrhea, other associated diseases and uncontrolled plasma glucose levels as compared with group 2 patients. In conclusion, insulin-dependent pancreatic diabetics had more advanced disease process and were therefore more likely to get other associated diseases than noninsulin-dependent pancreatic diabetics.


Comparison of effective renal plasma flow (ERPF) and endogenous creatinine clearance (Ccr) in evaluation of the differential kidney function: an in vivo study. Eleven patients with obstructive uropathy were inserted with a pigtail catheter either for urinary diversion, endopyelotomy or percutaneous nephrolithotomy. Differential kidney functions were obtained by single-sample technique with I-131 orthoiodohippurate (OIH) and separate endogenous creatinine clearance (Ccr). Good linear correlation was noted between effective renal plasma flow (ERPF) and Ccr (r = 0.84, p less than 0.005), but there was still some discrepancy between these two examinations, chiefly found in two severely obstructed kidneys. Determination of ERPF by I-131-OIH single-sample technique is a simple, rapid, inexpensive and relative accurate test to measure the differential kidney function. But if the ERPF is at extremely low level, further investigations such as sonography of kidney and separate endogenous creatinine clearance should be done to avoid unnecessary nephrectomy.


The effect of lasers on glycocalyx production by staphylococcus. The present study was undertaken to evaluate the effects of lasers on glycocalyx production by Staphylococcus epidermidis. Three strains of S. epidermidis were irradiated (He-Ne, Ga-As, CO2, and argon laser) at different energy densities ranging from 0.24 to 100 J/cm2. The viability of the bacteria was measured by colony forming units (CFU) and glycocalyx production was quantitated by toluidine blue staining and spectrophotometer. The He-Ne, Ga-As, and Co2 laser (2W; 90 J/cm2) had no effect on glycocalyx production and CFU, when compared to the control group (p greater than 0.05). However, glycocalyx production by the high-producer strains of S. epidermidis was significantly suppressed by agron laser when compared to the control group (p less than 0.05). It was concluded that the agron laser is a potentially advantageous mean of inhibiting the glycocalyx production by S. epidermidis.


Seventy-two patients' experience with an implantable drug delivery system for chemotherapy or total parenteral nutrition. An implantable drug delivery system will play an important role for patients who need chemotherapy or prolonged total parenteral nutrition in the future. We implanted 72 injection capsules in 74 patients who were undergoing aggressive chemotherapy or total parenteral nutrition programs. These capsules were implanted in different positions, including central access in 67 cases, peritoneal cavity in 1, hepatic artery in 2, portal vein in 1 and intrathecal space in 1. The actual median functional survival of the injection capsules was 12.3 months (range, 2 months to 4 years). The total cumulative puncture numbers were 1651 (range, 5 to 153; median 23). There was no unsuccessful instance in infusion or injection. To date, 27 patients still survive. The complication rate was about 7% including 1 sepsis, 1 skin infection, 1 catheter thrombosis, 1 dislodgement of reservoir and 1 catheter-reservoir disengagement, which were all corrected by a secondary operation without mortality. Patency of the Port-A-Cath system was maintained using a regular flushing schedule once every 30 days. The results of this study suggest that the implantable drug delivery system can provide a safe and reliable method for venous access or even arterious access in patients requiring intermittent or prolonged intravenous therapy.