Evaluation of endometrial tissue specific complement activation in women with endometriosis. To determine if endometrial tissue specific complement (C) activation occurs in patients with endometriosis. Prospective. University of Oklahoma Health Sciences Center, a tertiary care referral center. Twenty-six women, 9 with endometriosis and 17 without endometriosis. None. Uterine and ectopic endometria were evaluated by the immunofluorescence assay for the presence of activated products of the initial (C3d) and terminal (C5b-9) C pathway, C3 regulatory proteins (decay-accelerating factor and membrane cofactor protein), and terminal C regulatory proteins (clusterin and vitronectin). The initial (C3d) and terminal (C5b-9) C components were deposited on blood vessel walls in uterine and/or ectopic endometria of women with and without endometriosis. In the stromal compartment at both sites, C deposition was colocalized with terminal C inhibitors/cell-cell attachment factors, clusterin and vitronectin on elastic fibers. No specific staining for C proteins was detected on the glandular epithelium of uterine and ectopic endometrial tissues examined. Furthermore, intense staining of endometrial epithelium for C3 regulatory proteins, decay-accelerating factor, and membrane cofactor protein was noted on endometrial glands from women with and without endometriosis. Our findings demonstrate a lack of specific deposition of C in the glandular epithelial cells of endometria of women with endometriosis. The localization of C3 regulatory proteins, decay-accelerating factor, and membrane cofactor protein on glandular epithelium may suggest the involvement of intrinsic protective mechanisms on these cells from autologous C attack in vivo.


Detection of Leptospiraceae by amplification of 16S ribosomal DNA. The polymerase chain reaction (PCR) was developed to detect Leptospiraceae. Primers were used to amplify 1 631 base-pair (bp) 5'-region of 16S rDNA. Representative strains from the species, Leptospira interrogans sensu stricto, L. borgpetersenii, L. noguchii, L. santarosai, L. weilii, L. inadai, L. meyeri and the single member strain of Leptonema were amplified. In contrast, strains representing the saprophytic species. L. biflexa, L. wolbachii and L. parva were not amplified. There was no PCR product from 23 phylogenetically unrelated species of bacteria. As little as 10-1 pg of purified DNA and as few as 10-1 leptospires could be detected using the PCR analysis. Isolates of leptospires from clinical sources gave a positive PCR band, but those from surface waters did not.


Purification and characterization of LPS-free porins isolated from Salmonella minnesota. Porins of Salmonella minnesota, R595, were purified by anion exchange chromatography and subsequently isolated in their monomeric form by chromatofocusing. Two forms of porin could be isolated, both with an apparent molecular mass of 37,000, but of differing isoelectric points (pI 4.6 versus pI of 4.9). Porins with pI 4.9 did not contain any detectable LPS, but porins with pI 4.6 were found to contain trace amounts of LPS (1.3 x 10(-4) micrograms LPS/1 microgram porin) as measured using a highly sensitive limulus assay. Unlike the LPS-associated porins the monomeric porins were biologically inert with regard to pore formation, but they were still able to bind C1q, a subcomponent of the first component of complement.


The serum resistance of gonococci in the majority of urethral exudates is due to sialylated lipopolysaccharide seen as a surface coat. Examined before subculture, gonococci in 18 urethral exudates collected from different patients were serum-resistant. For 15 exudates, the resistance was drastically reduced by treatment with neuraminidase and by one subculture on laboratory media. It was restored by incubation with cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA). Electron microscopic examination of gonococci in eight exudates showed a surface structure stained by Ruthenium red which disappeared in most samples when they were treated with neuraminidase. These results were identical with those of previous studies on in vitro grown gonococci which had shown that serum resistance is due to sialylation of a 4.5-kDa conserved component of gonococcal lipopolysaccharide (LPS) by host CMP-NANA, which masks the target site for bactericidal IgM and renders surface LPS stainable by Ruthenium red. The serum resistance of gonococci in the remaining three exudates was not reduced by neuraminidase nor by subculture. The mechanism of this stable resistance is unknown.


Percutaneous absorption of benzyl acetate through rat skin in vitro. 2. Effect of vehicle and occlusion. The effect of vehicle and occlusion on the in vitro percutaneous absorption of [methylene-14C]-benzyl acetate (1.7-16.6 mg/cm2) has been studied in diffusion cells using full thickness skin from male Fischer 344 rats. Absorption of neat benzyl acetate through rat skin occluded with parafilm was 49.3 +/- 2.0% (mean +/- SD, n = 4) after 48 hr. When benzyl acetate in ethanol was applied to the skin and the skin was occluded with parafilm, the extent of absorption at 48 hr was not significantly different from that after neat application. However at 6 hr, as the ethanol content of the application mixture was increased, the absorption of benzyl acetate through occluded skin was enhanced proportionally (r = 0.999). When phenylethanol was used as a vehicle, the extent of the benzyl acetate absorption through occluded skin at 48 hr was enhanced (P less than 0.05) compared with that after application neat; with 50% (v/v) phenyl-ethanol, absorption at 48 hr was 56.3 +/- 4.9%. However, this enhanced absorption did not correlate with the proportion of phenylethanol in the application mixture. When dimethylsulphoxide was used as a vehicle, the extent of absorption of benzyl acetate through occluded skin at 48 hr was enhanced (P less than 0.05) compared with that after application neat (absorption was 59.3 +/- 3.7% of the applied dose when 50% (v/v) dimethylsulphoxide was used). As the dimethylsulphoxide content of the application mixture was increased, the absorption of benzyl acetate was enhanced proportionally. Occlusion of the skin surface with parafilm often significantly enhanced absorption (P less than 0.05), although the effect varied with time and vehicle. In general, the degree of any enhanced absorption caused by the use of a vehicle or occlusion of the skin was small, and, in most cases, would be unlikely to be toxicologically significant.


Is yohimbine-induced increase in salivary secretion a kinin-dependent mechanism? We have previously reported that the alpha 2-adrenoceptor antagonist yohimbine induced a significant increase in both salivary flow rate and kallikrein output. In order to assess the possible role of the kinin-kallikrein system in the increase in salivary secretion elicited by yohimbine, the effects of aprotinin, an inhibitor of kallikrein activity, were investigated in yohimbine-treated conscious dogs. Aprotinin (at a dose, 5000 IU/kg iv, which reduced both resting and yohimbine-induced increase in kininogenase and amidolytic activities of saliva) which remained inactive alone, failed to modify the increase in salivary volume elicited by yohimbine (0.5 mg/kg iv). These results show that the rise in salivary flow rate observed under alpha 2-adrenoceptor antagonist is not induced by the kinin-kallikrein system. The release of kallikrein into saliva observed after yohimbine is rather the consequence than the cause of the increase in salivary secretion.


Early detection of prostate cancer.

Early detection of prostate cancer. Prostate cancer is unique among the potentially lethal human malignancies in the wide discrepancy between the high prevalence of histologic changes recognizable as cancer and the much lower prevalence of the clinical disease. Despite the availability of effective tests for early detection and of effective treatment for cancers so detected, the diagnosis usually is not established until the tumor is locally advanced or metastatic. Yet, physicians hesitate to use these tests for fear that many cancers found would be latent, of little threat to the life or health of the host, and treatment could introduce inappropriate morbidity. Latent or "clinically unimportant" cancers can be distinguished from those that are clinically important by the larger volume, higher grade, and greater invasiveness of the latter. The available tests can detect only those cancers large enough to be palpable, visible on ultrasound, or capable of elevating the serum level of prostate-specific antigen. Such cancers are clinically important and should be treated for cure if the life expectancy of the patient is sufficiently long and the morbidity rate of therapy is low. Early detection of prostate cancer using the tests that are available today may widen the window of opportunity so that treatment indeed becomes possible in those for whom it is necessary.


Prostatic intraepithelial neoplasia: a premalignant lesion. Putative premalignant changes in the prostate have been recognized for a number of years. A variety of synonyms have been given to the most commonly described lesion, one characterized by proliferation and dysplasia of the normal two cell layers lining prostatic acini and ductules. Currently, "prostatic intraepithelial neoplasia" (PIN) is the term most commonly used to describe this entity. In this review, the requirements for a premalignant lesion, the reason why PIN fulfills the majority of these requirements, and a detailed description of the morphologic and phenotypic similarities between PIN and carcinoma will be outlined. Finally, the clinical significance of PIN will be reviewed.


Histochemistry of the prostate.

Histochemistry of the prostate. Histochemistry, including immunohistochemistry, is helpful to the practicing pathologist in the diagnosis of prostatic carcinoma. Of equal importance, histochemistry is being increasingly used to study the pathobiology of the prostate. This article reviews these histochemical techniques and their applications.


Elevated expression of ICAM1 (CD54) and minimal expression of LFA3 (CD58) in Epstein-Barr-virus-positive nasopharyngeal carcinoma cells. Undifferentiated nasopharyngeal carcinoma (NPC) is a remarkable entity among human tumors because of its constant association with the Epstein-Barr virus (EBV). Malignant epithelial cells harbor the EBV genome and often express at least 2 species of latent EBV protein (EBNA1 and LMP1). Despite the massive presence of tumor-infiltrating lymphocytes, NPC cells obviously escape immune surveillance directed to EBV antigens. Previous investigations carried out on EBV-positive Burkitt lymphoma (BL) cells have shown that this fact may be partially accounted for by a lack of expression of ICAM1 (CD54) and LFA3 (CD58). ICAM1 and LFA3 have therefore been investigated in fresh NPC biopsies and transplanted NPCs. With only 1 exception out of 9 cases, NPC cells appear to express high levels of ICAM1 and low levels of LFA3. This is a complete inversion of the pattern observed in normal epithelial cells in vivo. Additional investigations will be required to determine to what extent these characteristics affect T-cell interactions with NPC cells, specially in the process of EBV-antigen recognition.


Recombinant interferon alpha-2A combined with prednisone in metastatic renal-cell carcinoma: treatment results, serum interferon levels and the development of antibodies. Five partial responses were seen in 23 patients with metastatic renal-cell carcinoma (MRCC) receiving interferon-alpha 2a (IFN)+prednisone (P). Four of 24 subsequent patients responded to IFN+P (combined response rate 19%). The median response duration was 8 months (3 to 30 months). The one-year survival for all eligible patients was 52%. Eight of 26 evaluable patients developed antibodies detected by an enzyme immunoassay. In 2 patients, high levels of neutralizing antibodies were also found, together with particularly low IFN levels. In one patient the development of neutralizing antibodies coincided with the loss of initial response. The treatment was well tolerated by most patients. Premature discontinuation of treatment was necessary in only 2 patients. In MRCC, combination treatment with IFN+P is as effective as IFN monotherapy (response rate 19%), with significantly reduced subjective toxicity. The clinical relevance of the development of antibodies against IFN requires further investigation.


Tumorigenicity, mucin production and AM-3 epitope expression in clones selected from the HT-29 colon carcinoma cell line. Two mucin-producing cell clones (16.2 and 12.2) and a mucin-deficient clone (15.2) were selected from the established human adenocarcinoma cell line HT-29 by limiting dilution and Alcian blue staining. The amounts of the mucin antigen detectable on the cell surface with the monoclonal antibody (MAb) AM-3 decreased in the order HT-29 greater than 16.2 greater than 12.2 greater than 15.2 = 0. The binding avidity of AM-3 antibody to cells as well as to mucin extracts from each cell line decreased in the same order, indicating that the epitope density on the cell-bound mucins was highest in HT-29 and lowest in 12.2 cells. The parental line and the mucin-producing cell clones 16.2 and 12.2 showed no contact inhibition and grew as aggregates, while the 15.2 cells were well spread and formed a regular monolayer. The mucin-producing cell lines injected into nude mice yielded solid tumors with different growth rates (HT-29 greater than 16.2 greater than 12.2), while the 15.2 cell clone was not tumorigenic at all. The relative amounts of total mucin-bound hexoses and of the mucin epitope AM-3 decreased in the xenografts in the order HT-29 greater than 16.2 greater than 12.2. The present system is suitable for investigating the role of mucins in growth of colon carcinoma cells and indicates that increased tumorigenicity in nude mice coincides with the increase in total mucin expression and the expression of the AM-3 mucin epitope in tumor tissue.


Patterns of angiogenic response to mast cell granule constituents. Agents derived from mast cell granule constituents, and compound 48/80 which stimulates release of mast cell granules, have been used by us to develop new methods for quantitating angiogenesis in the chick chorioallantoic membrane. Two of these methods provide different insights, demonstrating different patterns of response to dosage and over time, produced by different agents. Counting mesenchymal blood vessels is convenient for obtaining dose-response data. Histamine and compound 48/80 have been shown previously to give a sigmoid dose-response curve resulting in a plateau before the lethal dose. This contrasts with the effect of porcine sodium heparin (Evans Biologicals) which results in a minor increase then a relative decline in vessel number due to a failure of growth. Here, the ability to produce angiogenesis or antiangiogenesis appears to be dose-dependent. Measurement of the changes in DNA synthesis, leading to visible angiogenesis, may be performed once the optimal angiogenic dose is known, and again distinctive patterns of response with different agents have been found. Histamine results in a fall then rise to a peak at 36 hr. We now show that two types of heparin each produce a peak at 12 hr. Compound 48/80 results in a distinctive pattern that looks like a composite of the histamine and especially the heparin effects, and this suggests that both are relevant to induction of angiogenesis by mast cells. The elicitation of this pattern of response also provides a method, additional to electron microscopy, for discovering whether or not an angiogenic substance is likely to operate via mast cell stimulation. Such characteristic patterns offer a new way of classifying angiogenic substances.


Clinical experience with patient administered subcutaneous dihydroergotamine mesylate in refractory headaches. This study examines the practicality and efficacy of dihydroergotamine mesylate (DHE) when self-administered subcutaneously in a population of refractory headache patients. Forty-three patients with chronic daily headache or migraine headache without aura, who had been taught self-injection of DHE either through the Raskin Protocol or in an outpatient headache clinic, were contacted by telephone and administered a questionnaire regarding usage and results from DHE injection. Ninety-two percent of patients could successfully administer DHE. Forty-six percent of patients experienced 90% or greater relief of pain and the majority of patients (77%) had greater than 50% relief. Emergency room use was decreased in 83% and 80% preferred DHE to their previous therapy. While side effects were common (79%), only four patients (9%) stopped DHE for this reason. No convincing evidence for the development of rebound headaches due to DHE was found in this sample.


Production of multiple growth factors by a newly established human thyroid carcinoma cell line. A multiple growth factor-producing tumor cell line (NIM-1) was newly established from a patient with thyroid cancer and remarkable neutrophilia. NIM-1 cells also caused severe neutrophilia in nude mice bearing tumors. NIM-1-conditioned medium (NIM-1CM) contained activities that supported not only granulocyte, macrophage and eosinophil colony formation of human bone marrow cells but also the growth of colony-stimulating factor (CSF)-dependent cell lines, NFS60-KX and TF-1. Northern blot hybridization analysis revealed the constitutive expression of granulocyte-CSF (G-CSF), granulocyte/macrophage-CSF (GM-CSF) and interleukin(IL)-6 mRNAs in NIM-1 cells. Enzyme-linked immunosorbent assays (ELISA) using NIM-1CM also confirmed the production of IL-1 alpha and a small amount of IL-1 beta besides G-CSF, GM-CSF and IL-6 in NIM-1 cells. In addition, unexpected production of IL-11 in NIM-1 cells was detected by northern blot hybridization analysis and by bioassay using an IL-11-dependent cell line. Therefore, NIM-1 cell line is shown to produce multiple cytokines including potentially megakaryopoietic growth factors such as GM-CSF, IL-6 and IL-11.


Site-dependent development of complete and incomplete intestinal metaplasia types in the human stomach. The topographical distribution of complete and incomplete types of intestinal metaplasia in human stomach samples was investigated in order to elucidate their mutual histogenetic relationship and significance in carcinogenesis. Subgross stereomicroscopic examination of alcian blue and hematoxylin-stained gastric mucosae allowed clear distinction of complete and incomplete intestinal metaplasia types as white (with or without purple hue) and purple foci, respectively, against the background magenta areas of non-intestinalized mucosa. Intestinal metaplasias which developed in the fundic area were predominantly of the complete type whereas those of the antrum were a mixture of both with a distinct predilection for expression of the incomplete type. Although there was some variation among foci regarding the hue of white or purple, the color feature was principally homogeneous within each individual intestinal metaplasia focus. Thus phenotypic analysis indicated intestinal metaplasia expression to be clearly influenced by intragastric topography. The study did not provide any evidence that a shift from incomplete to complete type intestinal metaplasia may occur with time or that the incomplete type may be more intimately associated with development of well-differentiated carcinomas.


Tumor-specific T cell lines: capacity to proliferate and produce interleukin 2 in response to various forms of tumor antigens. Anti-tumor proliferative T cell lines were established from cultures of lymph node cells from BALB/c mice immunized to syngeneic CSA1M fibrosarcoma with the CSA1M tumor cell membrane. The cultures were maintained throughout in the absence of exogenous interleukin 2 (IL2). Cell surface phenotypes of all T cell lines established were Thy-1+, Ig-, L3T4+ and Lyt-2-. Their proliferation was induced in a tumor antigen dose-dependent fashion and a tumor antigen-specific way. Such proliferative responses were inhibited by the addition to cultures of anti-class II H-2d (anti-I-Ad) or anti-L3T4 but not of anti-class I H-2d or anti-Lyt-2 monoclonal antibody. None of the T cell lines exhibited any cytotoxic T lymphocyte activity but they all produced IL2 upon stimulation with CSA1M tumor antigens, indicating that they represent helper-type T cell (Th) lines. The activation of these tumor-specific Th lines was induced with either CSA1M tumor cells themselves, or their membrane or detergent-solubilized fraction depending on the presence of antigen-presenting cells (APC). Most importantly, activation was also inducible by membranous tumor antigen-pulsed APC, which were capable of producing potent anti-tumor protective immunity when administered in vivo into syngeneic BALB/c mice. These results indicate that the tumor-specific Th lines established here can be activated with various forms of tumor antigens for their expression of helper function. Since Th lines of this type have not been described previously, our Th lines provide an intriguing tool for investigating the cellular and molecular mechanisms by which tumor-specific Th recognize tumor antigens.


Sandwich capture ELISA by a murine monoclonal antibody against a genus-specific LPS epitope for the detection of different common serotypes of salmonellas. A sandwich capture ELISA based on a murine monoclonal antibody against a genus-specific epitope in the outer core region of the Salmonella lipopolysaccharide is described for the detection of different common serotypes of salmonellas. Four h broth cultures of seven standard and 24 wild strains of salmonellas were all detected by the capture ELISA while overnight broth cultures of 21 non-salmonella standard strains were all negative. The capture ELISA detected 1 ng/ml of Ra lipopolysaccharide, 10(6)/ml of a smooth wild strain of Salm. typhimurium, and 1120 cells of Salm. heidelberg after enrichment culture for 4 h.


The Tn10-encoded tetracycline resistance mRNA contains a translational silencer in the 5' nontranslated region. We performed a mutational analysis of the left half of Tn10-encoded tet operator O2, located in the 5' nontranslated region of the mRNA for the resistance protein TetA, and determined the importance of that region for translation efficiency and mRNA stability. Transcriptional fusions of 17 mutants to lacZ expressed the same amounts of beta-galactosidase, while translational fusions varied 35-fold in expression efficiency. The mRNA half-lives varied 24-fold, with 9.6 min for the most highly expressed mRNA and 0.4 min for the least efficiently expressed mRNA. Toeprint experiments were performed to distinguish whether these mutations define a determinant of mRNA stability or influence translation initiation. The highly expressed mRNA was 24-fold more efficient in forming the initiation complex in vitro than the low-expression mutant. It was concluded that this sequence, albeit located upstream of the ribosome-binding sequence, is an important determinant for efficient initiation of translation. Secondary-structure calculations of the mRNAs revealed no correlation of the potential to form double strands masking the ribosome-binding sequence with expression efficiency.


The cystic fibrosis transmembrane conductance regulator. Effects of the most common cystic fibrosis-causing mutation on the secondary structure and stability of a synthetic peptide. Deletion of phenylalanine 508 (delta Phe-508) in the cystic fibrosis transmembrane conductance regulator (CFTR) protein causes approximately 70% of all cases of cystic fibrosis. This residue lies in a region of the protein that we have synthesized chemically and shown to bind adenine nucleotides (Thomas, P. J., Shenbagamurthi, P., Ysern, X., and Pedersen, P. L. (1991) Science 251, 555-557). A peptide lacking this critical residue, but otherwise corresponding to this crucial part of the protein, now also has been chemically synthesized and purified. This mutant peptide (P-66) exhibits a significant loss of beta-sheet structure as compared with the wild type peptide (P-67). Furthermore, urea denaturation of peptide structure reveals that P-66 is less stable than P-67. Although under non-denaturing conditions both peptides bind adenine nucleotides with high affinity, the loss of structural stability is reflected in the binding function of the peptides. Thus, P-67, in contrast to P-66, retains a significant capacity for nucleotide binding in 4 M urea. These results suggest a model for impaired delta Phe-508 CFTR function.


Characterization of the coexisting multiple mechanisms of methotrexate resistance in mouse 3T6 R50 fibroblasts. We have studied the discrepancy in the degree of methotrexate (MTX) resistance that exists between two clonal cell lines, mouse 3T6 R50 cells and Chinese hamster ovary B11 0.5 cells that overexpress comparable levels of dihydrofolate reductase, yet exhibit a 100-fold difference in MTX resistance while maintaining similar sensitivity to the lipophilic antifolates trimetrexate and piritrexim. These data suggested that R50 cells may possess additional mechanism(s) of antifolate resistance, such as MTX transport alteration. Flow cytometric analysis using fluorescein methotrexate revealed comparable levels of fluorescein MTX displacement with lipophilic antifolates in viable R50 and B11 0.5 cells, but marked insensitivity of R50 cells to MTX competition, thus suggesting a poor uptake of MTX into R50 cells. Analysis of the kinetic parameters of dihydrofolate reductase from R50 cells neither showed alterations in enzyme affinities for various antifolates nor in the Michaelis constant for folic acid and NADPH nor a change in the pH activity optimum. R50 cell-free extracts contained wild-type levels of folylpoly-gamma-glutamyl synthetase activity. However, following metabolic labeling with [3H]MTX, no MTX polyglutamates could be detected in R50 cells. We conclude that the high level of MTX resistance in R50 cells is multifactorial, including overexpression of dihydrofolate reductase, reduced MTX transport, and possibly altered formation of MTX polyglutamates. The potential interactions between the different modalities of MTX resistance in R50 cells are being discussed.


Local anesthetics induce fast Ca2+ efflux through a nonenergized state of the sarcoplasmic reticulum Ca(2+)-ATPase. The effect of the local anesthetics SKF 525-A, dibucaine, tetracaine, procaine, and benzocaine on sarcoplasmic reticulum vesicles was studied. All the anesthetics tested inhibited the phosphorylation of the Ca(2+)-ATPase by Pi in a competitive manner. Tertiary amine and positively charged anesthetics, in addition to competing with Pi, also decreased the apparent affinity of the ATPase for Mg2+. There was a good correlation between the octanol/water partition coefficients and the inhibitory activity of the different anesthetics. All the anesthetics tested induced a 5- to 10-fold increase in the rate of Ca2+ efflux. This was promoted by the same drug concentration that inhibited the phosphorylation of the ATPase by Pi. The effect on Ca2+ efflux was antagonized by the ligands of the ATPase (Mg2+, K+, Ca2+, MgATP, and ADP) and by the organic polyamines ruthenium red, spermine, spermidine, and putrescine. The natural anion heparin was found to potentiate the effect of the positively charged anesthetics on the rate of Ca2+ efflux. It is concluded that the local anesthetics increase the Ca2+ efflux through a nonenergized state of the Ca(2+)-ATPase, rather than promoting a nonspecific Ca2+ leakage through the membrane.


Structure of an mdr-like gene from Arabidopsis thaliana. Evolutionary implications. Multidrug resistance of mammalian tumor cells is caused by the enhanced expression of P-glycoproteins. These proteins are encoded by mdr genes and mediate the energy-dependent efflux of a variety of lipophilic drugs from cells. To test whether in plants mdr-like genes might be involved in certain cases of cross-resistance to different herbicides, we have cloned and characterized a gene from Arabidopsis thaliana, atpgp1, encoding a putative P-glycoprotein homologue. Like the mammalian P-glycoproteins, with which it shares extensive sequence homology and a similar organization in structural domains, this protein is internally duplicated. Seven of the nine introns in the atpgp1 gene match introns in the mammalian mdr genes to within a few nucleotides, and the positions of these suggest that P-glycoprotein genes evolved by duplication and subsequent fusion of an intron-containing primordial gene prior to the evolutionary separation of plants and mammals. The atpgp1 gene gives rise to transcripts present in all plant parts but particularly abundant in inflorescence axes.


Identification of the fourth member of the mammalian endoprotease family homologous to the yeast Kex2 protease. Its testis-specific expression. We used the polymerase chain reaction to identify a mouse testis cDNA that represented another member of a growing class of mammalian endoproteases involved in the processing of precursor proteins. This cDNA encoded a 655-residue protein, designated PC4, containing a bacterial subtilisin-like catalytic domain closely related to those of the recently characterized precursor-processing endoproteases, furin, PC1/PC3, PC2, and Kex2. Within this domain, the amino acid sequence of PC4 was 70, 58, 55, and 45% identical with those of mouse furin, mouse PC1/PC3, mouse PC2, and yeast Kex2, respectively. Northern blot analysis indicated that the PC4 mRNA was detectable only in the testes after the 20th day of postnatal development. Moreover, this message was mainly expressed in the round spermatids. These data suggest that PC4 represents a prime candidate for a precursor-processing endoprotease in the testicular germ cells and that its gene expression is regulated during spermatogenesis.


Use of a multiple S1 nuclease protection assay to monitor changes in RNA levels for type 1 phosphatase and several proto-oncogenes in response to insulin. Changes in insulin-regulated gene expression occur in a time- and tissue-dependent fashion. To monitor these changes we have adapted the S1 nuclease protection assay to allow simultaneous estimation of multiple RNA species in a single sample by using synthetic oligonucleotides of various lengths as probes for specific RNA species, which can then be resolved by electrophoresis. The multiple S1 nuclease protection assay was used to assess the influence of insulin on the RNA concentrations of 12 different genes in human skeletal muscle. Estimates obtained by this assay were comparable with those obtained by Northern analysis. RNA levels for proto-oncogene c-src displayed a transient 4-fold increase, whereas RNA levels for type 1 protein phosphatase were suppressed by 50% during the same time period. RNAs corresponding to known insulin-responsive genes such as c-fos, c-myc, c-Ha-ras, and c-src displayed rapid and transient 2-4-fold increases between 30 and 60 min as detected by either Northern analysis or the multiple S1 nuclease protection assay. In addition, RNA levels for the insulin receptor, Glut-4, Glut-3, and c-jun were apparently unaffected by exposure of the cells to insulin.


Fatty acid regulation of gene expression. Transcriptional and post-transcriptional mechanisms. Fatty acids are important metabolic substrates and may also be involved in pathological syndromes such as the insulin resistance of diabetes and obesity. We demonstrate here that fatty acids can regulate specific gene expression; mRNAs encoding the fatty acid binding protein adipocyte P2 (aP2) and the Fos-related transcription factor Fra1 are specifically induced at least 20-fold upon treatment of preadipocytes with oleate. For aP2, the effect requires long chain fatty acids and occurs without a generalized activation of the genes linked to adipocyte differentiation. Other fibroblastic cells without preadipocyte characteristics do not induce aP2 mRNA in response to fatty acids. Unlike aP2, Fra1 induction by fatty acids also can be detected in NIH 3T3 and 3T3-C2 fibroblasts. Nuclear transcription assays in 3T3-F442A preadipocytes demonstrate that fatty acids elicit no transcriptional increase in the aP2 gene. Fra1, on the other hand, shows a 3-4-fold increase in transcription. These results demonstrate at least two distinct mechanisms by which fatty acids may influence gene expression.


The Drosophila G protein gamma subunit gene (D-G gamma 1) produces three developmentally regulated transcripts and is predominantly expressed in the central nervous system. A genomic clone, 536, located at the 44CD region of polytene chromosomes of Drosophila melanogaster, has been characterized for its neurobiological importance. We found that this clone contains a gene which produces 2.6-, 1.3- and 1.1-kilobase (kb) RNAs. While the 2.6-kb RNA is expressed only in the head, the 1.3-kb RNA is present exclusively in the body. The 1.1-kb RNA, however, is found in both the head and body, but in much higher concentration in the head. DNA sequence analysis of a 2.6-kb RNA-specific cDNA showed that this gene encodes a 70-amino acid polypeptide which is the putative Drosophila homologue to the gamma subunit of the bovine G-protein. The Drosophila protein, named D-G gamma 1, shares 46, 43, and 28% identity, and 59, 52, and 60% similarity, with the gamma 2, gamma 3, and gamma t proteins of bovine G proteins, respectively. Sequencing of the 1.1-kb RNA-specific cDNA clone revealed that the 1.1-kb RNA is produced from the 2.6-kb transcription unit by usage of an alternative polyadenylation site, and has a coding region identical to that of the 2.6-kb RNA. Genomic Southern blot hybridization indicated that the Drosophila genome has only one D-G gamma 1 gene. Throughout development the 1.1-kb RNA is found to be the most prevalent species; its level peaks between 9 and 12 h of embryogenesis. As is the case for the other G protein genes of Drosophila, the D-G gamma 1 gene is predominantly expressed in the central nervous system of the fly.


Structure and regulation of the glpFK operon encoding glycerol diffusion facilitator and glycerol kinase of Escherichia coli K-12. The glpFK operon maps near minute 88 on the linkage map of Escherichia coli K-12 with glpF promoter proximal. The glpF gene encodes a cytoplasmic membrane protein which facilitates the diffusion of glycerol into the cell. The glpK gene encodes glycerol kinase. In the present work, the nucleotide sequence of the 5'-end of the operon, including the control region, the glpF gene, and part of the glpK gene, was determined. The facilitator was predicted to contain 281 amino acids with a calculated molecular weight of 29,780. It is a highly hydrophobic protein with a minimum of six potential transmembrane alpha helices. The transcription start site for the glpFK operon was located 71 base pairs upstream from the proposed translation start codon for glpF. Preceding the transcription start site were sequences similar to the -10 and -35 consensus sequences for bacterial promoters. Binding sites for the cAMP-cAMP receptor protein (CRP) complex and the glp repressor were identified by DNase I footprinting. The region protected by the cAMP.CRP complex contained tandem sequences resembling the consensus sequence for CRP binding. The CRP sites were centered at 37.5 and 60.5 base pairs upstream of the start of transcription. The glp repressor protected an extensive area (-89 to -7 relative to the start point of transcription), sufficient for the binding of four repressor tetramers. Two additional binding sites for the repressor were identified within the glpK coding region. The DNA containing these two operators synergistically increased the apparent affinity of glp repressor for DNA fragments containing the four operators in the promoter region of the glpFK operon. With this study, a total of 13 operators for the glp regulon have been characterized. Comparison of these operators revealed the consensus 5'-WATGTTCGWT-3' for the operator half-site (W = A or T). The relative affinity of the glp repressor for the various glp operators was assessed in vivo using a promoter-probe vector. The relative apparent affinity of the control regions for glp repressor was glpFK greater than glpD greater than glpACB greater than glpTQ. The degree of catabolite repression for each of the operons was assessed using a similar system. In this case, the relative sensitivity of the glp operons to catabolite repression was glpTQ greater than glpFK greater than glpACB greater than glpD.


Mutations away from splice site recognition sequences might cis-modulate alternative splicing of goat alpha s1-casein transcripts. Structural organization of the relevant gene. alpha s1-Casein variants F and D, synthesized in goat milk at lower levels than variant A, essentially differ from it by internal deletions of 37 and 11 amino acid residues, respectively. Northern blot analysis of mRNAs encoding alpha s1-casein F and A and sequencing of the relevant cloned cDNAs, as well as sequencing of in vitro amplified genomic fragments, revealed multiple alternatively processed transcripts, from the F allele. Although correctly spliced messengers were identified, most of the FmRNAs lacked three exons. These exons, further identified as exons 9, 10, and 11, together encode the 37 amino acid residues present in alpha s1-casein variant A but missing in variant F. Exon 9 codes for the sequence present in variant A but deleted in variant D. A single nucleotide deletion in exon 9 and two insertions, 11 and 3 base pairs in length, in the downstream intron, were identified as mutations potentially responsible for the alternative skipping of these 3 exons. From a computer-predicted secondary structure it appeared that the 11-base pair insertion might be involved in base-pairing interactions with the intron 5' splice site which might consequently be less accessible to U1 snRNA. We also report here the complete structural organization of the goat alpha s1-casein transcription unit, deduced from polymerase chain reaction experiments. It contains 19 exons scattered within a nucleotide stretch nearly 17-kilobase pairs long.


Interleukin-1-inducible tumor growth arrest is characterized by activation of cell type-specific "early" gene expression programs. Human melanoma cells, A375-C6, were "committed" to growth arrest within a few hours of exposure to interleukin-1 (IL-1). Co-treatment with actinomycin D rescued the cells from the "commitment," suggesting that "early" gene activation events may be crucial for growth arrest. To understand the mechanism of IL-1 action, we are studying early genes whose expression is induced by the cytokine. Five early genes associated with IL-1 action in the melanoma cells were isolated by differential screening of a cDNA library, which was enriched for sequences representing IL-1 responsive genes (IRGs). Nucleotide sequencing identified four of the genes as gro-alpha, gro-beta, c-jun and nur77/NGF1-B/NAK1, respectively, while the fifth was judged as novel by GenBank search and designated IRG-9. None of the early genes was uniquely associated with the antiproliferative action of IL-1: other growth-inhibitory as well as growth-stimulatory signals induced these genes in diverse cell types. However, analysis of the induction patterns of the IRGs and other well known early genes revealed that IL-1 action in the melanoma cells is characterized by activation of a unique primary gene expression program. This program was defined by the magnitude and temporal pattern of induction of the five IRGs, feeble induction of c-fos, and lack of induction of Egr-1 and c-myc. We present evidence that this program is growth arrest-specific in the melanoma cells and that distinct cell type-specific programs are associated with IL-1 growth-regulatory actions in other tumor cells. Based on these data, we propose that early genes may play multifunctional roles in tumor growth control, but specificity for the growth arrest action of IL-1 is determined by the composite early gene induction program.


Primary structure and chromosomal localization of human and mouse rod photoreceptor cGMP-gated cation channel. We have determined the primary structures of the human and mouse retinal rod cGMP-gated cation channel by analysis of cDNA clones and amplified DNA. The open reading frames predicted polypeptides of 690 and 683 residues exhibiting 88% sequence similarity. Sequence comparison indicated that the rod channels consist of a variable 90-residue N-terminal region, a short highly charged segment rich in lysine and glutamate, and a 540-residue C-terminal portion that is well conserved in three mammalian species. Significant sequence similarity (59%) of the visual cGMP-gated channel to the olfactory cAMP-gated channel established the existence of a family of cyclic nucleotide-gated ion channel genes. RNA blot analysis revealed transcripts of 3.2 kilobases (kb) in human, mouse, and dog, 3.2, 4.6, and 5.2 kb in bovine, and 3.6 kb in fish. The human channel gene was mapped by polymerase chain reaction of somatic cell hybrid DNAs to chromosome 4 (p14-q13) near the centromere. The mouse channel gene locus (Cncg) was mapped by interspecific backcross haplotype analysis 0.9 centimorgan proximal of the Kit locus on chromosome 5.


Epitope mapping of anti-recA protein IgGs by region specified polymerase chain reaction mutagenesis. Monoclonal IgGs were shown to be useful for the specific inhibition of a set of activities of the recA protein, a key protein in homologous genetic recombination. The mapping of the epitopes for these IgGs and site-directed mutagenesis based on the mapping will facilitate location of the functionally active sites on the tertiary structure of the protein, which is being solved by means of physicochemical techniques. We developed a novel technique for region-specified mutagenesis and applied the technique to epitope mapping. Using the polymerase chain reaction in the presence of deoxyinosine triphosphate, we introduced random base substitutions specifically into a region of the recA gene defined by a pair of primers. RecA mutants exhibiting altered antigenicity were selected, in plaque-immunoblotting experiments, from libraries of mutagenized recA genes constructed on the lambda gt11 expression vector. Mutant recA genes were obtained at the frequency of about 10(-2) among the plaques expressing fused recA genes and then each one was expressed as a whole protein, which was characterized by enzyme-linked immunosorbent assay. Analyzing the DNA sequences of the mutant recA genes, we located at the amino acid sequence level the epitopes for two anti-recA IgGs which could not be located in previous studies. One of the antibodies was shown to prevent self-assembly of the recA protein and the other was suggested to inhibit the binding of double-stranded DNA. Thus, the active sites involved in these functions would be located in the space around or near the relevant epitope.


Cloning of the cDNa for a Na+/myo-inositol cotransporter, a hypertonicity stress protein. Kidney medullary cells in situ, as well as kidney-derived Madin-Darby canine kidney (MDCK) cells accumulate nonperturbing, small organic solutes (osmolytes), including myo-inositol, when bathed in hypertonic media. Accumulation of osmolytes balances the osmolality of extracellular fluid without raising intracellular salts that would perturb cellular functions. In hypertonic media, increased myo-inositol accumulation is the result of increased activity of a Na+/myo-inositol cotransporter. We have isolated a cDNA encoding a Na+/myo-inositol cotransporter from MDCK cells using expression in Xenopus oocytes. The cDNA sequence predicts a protein of 718 amino acids with a significant amino acid sequence similarity to the Na+/D-glucose cotransporters of absorbing epithelia. Transporter mRNA is present in kidney and brain and is markedly induced in MDCK cells by medium hypertonicity, demonstrating that adaptation to hypertonic stress involves up-regulation of transporter mRNA accumulation.


Design and immunological properties of topographic immunogenic determinants of a protein antigen (LDH-C4) as vaccines. Antibodies elicited by immunization with short peptides containing antigenic determinants have been shown, in general, to bind with greatly reduced affinity to the corresponding region in the native proteins. Thus, contiguous linear peptides have not proven to be effective immunogens in generating high affinity neutralizing or protective antibodies and consequently appear to be poor prospects for vaccines. The molecular basis for such reduced reactivity is clear from the crystal structure determination of antibody Fabs bound to protein antigens, which showed the complementarity between interfaces to be lock-and-key-like and extending over a large area (750 A2) involving discontinuous segments of the polypeptide chain. Thus, small perturbations in the secondary and tertiary structure of the antigen have profound effects on the fit of the antigen and its corresponding antibody. Because short peptides are unlikely to assume any particular conformation in solution, the fit is likely to be poor. New strategies are therefore required to produce conformationally stable peptides that mimic the critical structural features of the protein antigenic site. Here we show that a putative topographic determinant of the testis-specific isozyme of lactate dehydrogenase C4 (LDH-C4), designed and synthesized to adopt a well defined alpha-helical secondary and tertiary structure (four-helix bundle motif) in aqueous solutions, is highly immunogenic in both rabbits and mice, inducing IgG antibodies that bind to native LDH-C4. This engineered conformational 40-residue peptide is considerably more effective in inducing antibodies, as compared with the corresponding linear peptide. The antibody response is obtained without coupling the peptide to a carrier protein, suggesting that the peptide contains a T-cell antigenic determinant. The strategy described here to produce a conformationally stable peptide that mimics the native structure may have general applications in vaccine design.


Identification of alpha 2-macroglobulin conformational intermediates by electron microscopy and image analysis. Comparison of alpha 2-macroglobulin-thrombin and alpha 2-macroglobulin reacted with cis-dichlorodiammineplatinum(II) and trypsin. Human alpha 2-macroglobulin (alpha 2M) exists in two well defined, highly distinct conformations and in less well described intermediate conformations. In this study, previously characterized reactions were used to partially or completely transform the conformation of alpha 2M. Electron micrographs of each preparation were subjected to image analysis. Ternary alpha 2M-trypsin (2 mol of trypsin/mol of alpha 2M) was analyzed as a control for the fully transformed state. Correspondence analysis (CORAN) and hierarchical ascendant classification (HAC) generated five image clusters from 330 aligned alpha 2M-trypsin complexes. Average images of each cluster resembled the letter "H" with four nearly equivalent lateral arms. Abnormally shaped lateral arms were not demonstrated by HAC, using a variety of factor sets. In a native polyacrylamide gel electrophoresis system, alpha 2M-thrombin migrated in a diffuse band partially behind alpha 2M-trypsin, suggesting conformational heterogeneity. CORAN and HAC of 733 alpha 2M-thrombin complexes identified two neighboring clusters, the average images of which showed an H-like structure in which one arm was replaced by a globular stain-excluding body. The two alpha 2M-thrombin clusters included 125 images (17.1% of image population). The complete absence of atypical lateral arm structure in the alpha 2M-trypsin clusters suggests that this variation is not the result of orientation or staining artifact. Native alpha 2M was reacted with cis-dichlorodiammineplatinum(II) and then with trypsin to form alpha 2M-Pt-trypsin, a preparation that includes partially transformed alpha 2M structures. CORAN and HAC of 580 alpha 2M-Pt-trypsin complexes generated five clusters, the average images of which showed atypical lateral arm structure equivalent to that demonstrated with alpha 2M-thrombin. The five alpha 2M-Pt-trypsin clusters accounted for 15.2% of the image population. These studies suggest that alpha 2M conformational change intermediates demonstrate common structural characteristics, permitting an elucidation of the steps involved in this complex transformation.


Molecular cloning and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line. Macrophages activated by exposure to cytokines and/or to endotoxin produce nitric oxide (NO.), a free radical that is a mediator of the host response to infection. Activation induces the expression of nitric oxide synthase, the enzyme that catalyzes formation of NO. from L-arginine and molecular oxygen. We report the cloning of a cDNA encoding the inducible nitric oxide synthase from a murine macrophage cell line, RAW264.7, exposed to interferon-gamma and lipopolysaccharide. Oocytes injected with mRNA transcribed from this cDNA demonstrate arginine-dependent production of nitrite, a stable metabolite of NO.. Nitric production is blocked by the enzyme inhibitor, NG-monomethylarginine, and is independent of calcium/calmodulin. RAW264.7 cells demonstrate rapid accumulation of the nitric oxide synthase-encoding mRNAs upon activation. Comparison of the deduced amino acid sequence to the calcium/calmodulin-dependent nitric oxide synthase previously purified (Bredt, D. S., and Synder, S.H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 682-685) and cloned (Bredt, D. S., Hwang, P. M., Glatt, C. E., Lowenstein, C., Reed, R. R., and Synder, S. H. (1991) nature 351, 714-718) from rat brain identifies shared binding sites for the cofactors NADPH and flavins in the C-terminal half of both proteins and an additional conserved region near the N terminus that may recognize L-arginine and/or contribute to the active site.


Cofactor residues lysine 165 and 166 are critical for protein substrate recognition by the tissue factor-factor VIIa protease complex. High affinity binding of factor VIIa (VIIa) to its cellular receptor tissue factor (TF), as well as association of factor X with phospholipid are required for optimal assembly of the extrinsic activation complex. In addition to the interactions of substrate with phospholipid and enzyme, we here provide evidence that cofactor residues Lys-165 and Lys-166 specifically contribute to the recognition of macromolecular substrate. Ala for Lys replacement in TFA165A166 was compatible with high affinity binding of VIIa when analyzed on cell surfaces as well as in the absence of phospholipid. Dissociation of TFA165A166.VIIa did not occur with a faster rate compared to TF.VIIa, further supporting unaltered VIIa binding function of TFA165A166. Cleavage of chromogenic peptidyl substrate by TFA165A166.VIIa complexes was not diminished, demonstrating that TFA165A166 supported enhancement of catalytic function of the VIIa protease domain. In contrast, factor X activation was reduced in the presence and absence of phospholipid. Further, TFA165A166 effectively competed with wild-type TF in the cleavage of factor X at limited VIIa concentrations. Selective reduction in macromolecular substrate hydrolysis combined with normal VIIa binding by TFA165A166 indicates that the cofactor TF does contribute, either directly or indirectly via specific interactions with VIIa, to factor X recognition.


Role of integrin alpha 4 beta 7/alpha 4 beta P in lymphocyte adherence to fibronectin and VCAM-1 and in homotypic cell clustering. Integrins are heterodimeric cell surface proteins that mediate both cell-cell and cell-extracellular matrix interactions. We and others recently identified cDNAs encoding a novel integrin beta subunit, beta 7, in lymphocytes. We have now detected beta 7 mRNA in mouse TK-1 T lymphoma cells, which are known to express the putative Peyer's patch homing receptor alpha 4 beta P. We used an anti-peptide antiserum and a novel mAb against the beta 7 subunit to show that TK-1 cells express beta 7 as the only subunit associated with alpha 4. We conclude that beta 7 and beta P are identical. We also show that activated peripheral blood T cells express alpha 4 beta 7. We studied the function of alpha 4 beta 7/alpha 4 beta P in TK-1 cells, which do not express very late antigen (VLA)-4 (alpha 4 beta 1). Cells adhered to intact fibronectin and to a fibronectin fragment containing the CS-1 region, but not to a fragment containing the RGD sequence. Adhesion to fibronectin was inhibited by antibodies to alpha 4, suggesting that alpha 4 beta 7 is a fibronectin receptor. We confirmed that alpha 4 beta 7 binds to the CS-1 region of fibronectin using affinity chromatography. TK-1 cell adhesion to the vascular cell adhesion molecule VCAM-1 was also inhibited by antibodies to alpha 4, implying that alpha 4 beta 7 also plays a role in the adherence of lymphocytes to endothelial cells. TK-1 cell binding to fibronectin and VCAM-1 is markedly increased by brief PMA stimulation. We also found that mAbs against alpha 4 and beta 7 induce homotypic clustering of TK-1 cells. Taken together these results suggest that alpha 4 beta 7/alpha 4 beta P recognizes some or all of the same widely distributed ligands recognized by VLA-4 (alpha 4 beta 1) and that the role of alpha 4 beta 7/alpha 4 beta P may not be restricted to lymphocyte homing.


Characterisation of visna virus reverse transcriptase: a micro scale reverse transcriptase assay adapted for use with an automated cell harvester. The reverse transcriptase of the sheep lentivirus visna/maedi virus has been characterised. Optima for magnesium ion concentration (5-10 mM), potassium ion concentration (150 mM) and pH (8.25) for this enzyme are very similar to those previously described for the human immunodeficiency viruses. The assay used for this work makes use of a cell harvester to speed up the processing of multiple samples. It is small scale, requiring 15 microliters of sample, is rapid, and is able to detect virus at titres below 10(3)/ml. Harvesting the assay onto either DEAE paper or using TCA and glass fibre mats make it suitable for use with either tissue culture media or infected cell lysates, but not with body fluids. It has been used to detect cell-associated reverse transcriptase in choroid plexus cells within 36 h of visna infection.


Detection of HIV-1 RNA in heparinized plasma of HIV-1 seropositive individuals. The interference of reverse transcription by heparin was removed by heparinase. When the HIV-1 RNA in the presence of heparin was detected by a combination of reverse transcription and the polymerase chain reaction (PCR), heparinase treatment followed by removal of Ca2+ before the reverse transcription step permitted the efficient detection of HIV-1 RNA. Prior treatment with heparinase revealed HIV-1 RNA in 68% (13/19) of heparinized plasma samples from HIV-1 carriers, whereas only 26% (5/19) of the same specimens were positive without the heparinase step. Heparinase removed the inhibition of reverse transcription by heparin and is highly recommended when detecting low levels of viral RNA in heparinized plasma.


Immunoelectron microscopic epitope locations of titin in rabbit heart muscle. The location of cardiac titin epitopes in the sarcomere of rabbit cardiac, atrial and ventricular muscle was studied by using polyclonal antibodies against skeletal muscle titin. The results show that incubation with the antibody leads to the appearance of four electron-dense stripes in the A band of both atrial and ventricular cardiac muscle. The location and intensity of these stripes were identical to those observed in skeletal muscle. In conclusion we demonstrate that titins from skeletal and cardiac muscles share some common antigenic determinants.


Protein C inhibitor in human body fluids. Seminal plasma is rich in inhibitor antigen deriving from cells throughout the male reproductive system. An assay was developed for the measurement of human protein C inhibitor antigen (PCI) in blood plasma and other biological fluids. Both native PCI, modified inhibitor, and complexes of inhibitor with activated protein C or plasma kallikrein could be measured with the assay. Inhibitor antigen concentrations were found to be very high in seminal plasma (greater than 200 mg/liter), more than 40 times the concentration of PCI found in blood plasma. The inhibitor in seminal plasma was unable to form complexes with activated protein C. Gel filtration and immunoblotting findings indicated that the inhibitor in seminal plasma is present in a high molecular mass complex or cleaved to its modified form. As PCI antigen was absent from seminal plasma of patients with dysfunctional seminal vesicles, the seminal vesicle glands would appear to be the major source of seminal plasma PCI, a conclusion supported by immunohistochemical demonstration of the presence of PCI epitopes in the secretory epithelium of the seminal vesicles. Specific PCI immunoreactivity was also shown to be present in the testes, the epididymis glands, and the prostate, suggesting the inhibitor to have a complex or multiple function in the male reproductive system. Conclusive evidence of a local synthesis of PCI in the four male sex glands was provided by Northern blot analysis of RNA from these organs.


Decreased serum insulin-like growth factor-I associated with growth failure in newborn lambs with experimental cyanotic heart disease. To determine whether chronic hypoxemia results in alterations in endocrine function that may contribute to growth failure, we measured growth hormone (GH), somatomedins (insulin-like growth factors I and II, IGF-I and IGF-2), hepatic growth hormone receptors, and circulating IGF-binding proteins IGFBP-3 and IGFBP-2 in 12 newborn lambs with surgically created pulmonic stenosis and atrial septal defect, and in 10 controls. During chronic hypoxemia (oxygen saturation of 60-74% for 2 wk), weight gain was 60% of control (hypoxemic, 135 +/- 20 vs. control, 216 +/- 26 g/d, P less than 0.02). IGF-I was decreased by 43% (hypoxemic 253.6 +/- 29.3 SE vs. control 448.0 +/- 75.5 ng/ml, P = 0.01), whereas GH was unchanged (19.9 +/- 5.1 vs. 11.9 +/- 3.0 ng/ml, NS). The increase in IGF-1 was associated with a decrease in IGFBP-3 (hypoxemic, 5.09 +/- 1.25 vs. control, 11.2 +/- 1.08 arbitrary absorbency units per mm (Au.mm), P less than 0.01), and increase in IGFBP-2 (0.47 +/- 0.03 vs. 0.19 +/- 0.13 Au.mm, P less than 0.05), but no significant downregulation of hepatic GH receptors (hypoxemic, 106.1 +/- 20.1 vs. control, 147.3 +/- 25.9 fmol/mg, NS). Thus, chronic hypoxemia in the newborn is associated with a decrease in IGF-I and IGFBP-3 in the face of normal GH. This suggests peripheral GH unresponsiveness, similar to protein-calorie malnutrition or GH receptor deficiency dwarfism, but mediated at a level distal to the hepatic GH receptor.


Configuration of immunoglobulin and T cell receptor beta and gamma genes in acute myeloid leukaemia: pitfalls in the analysis of 40 cases. To evaluate the overall incidence of immunoglobulin (Ig) and T cell receptor (TCR) beta and gamma gene rearrangements in a series of 40 cases of acute myeloid leukaemia (AML) and to determine whether structural modifications of these genes could be correlated with the abnormal expression of lymphoid markers in malignant cells. All cases were classified according to the criteria of the FAB group and immunophenotyped with a panel of monoclonal antibodies reactive with myeloid and lymphoid differentiation antigens. DNA analysis was performed by the method of Southern using probes for the Ig JH, TCR-C beta 1, and TCR-J tau 1 regions. Phenotypic analysis showed that in addition to myeloid markers, 10 cases expressed lymphoid antigens: CD7 in seven (of which three were TdT positive, one CD2 positive, and one CD19 positive) and CD19 in three. Southern blot analysis showed that bands with sizes different from the germ line control were present in the TCR beta genes in 11 cases: in six of 30 with pure myeloid phenotype and in five of 10 of those expressing lymphoid markers. A close observation of the size and patterns of those bands, however, showed that they could be artefactual. Indeed, further analysis showed that they were either due to resistant Eco RI/Hind III sites at the beta locus or to plasmid contamination. Rearranged genes were eventually found in only two of the 40 cases: at the Ig JH region in one of the 30 with pure myeloid phenotype (3.3%) and at the TCR gamma genes in one of 10 with lymphoid markers (10%). These observations showed that Ig/TCR gene rearrangements were rare in this AML series (overall incidence of 5%) and that they were not significantly more common in cases with aberrant expression of lymphoid markers. The size and pattern of the potential non-germline bands that can be found in these loci must be carefully evaluated.


Two-colour immunoenzymatic technique using sequential staining by APAAP to evaluate two cell antigens. To extend the alkaline phosphatase-antialkaline phosphatase (APAAP) immunoenzyme single stain method to a more generally applicable double stain technique. This will allow two primary antibodies of the same isotype of IgG and specifically the nuclear antigen bromodeoxyuridine (BRdU) to be evaluated with a cell surface antigen identifier. Sequential applications of the APAAP method showed two antigen sites by different dye couplings to a common alkaline phosphatase substrate, producing blue and red reaction products on the same slide. Antigens on different cell populations as well as those in different compartments of the same cell were analysed. The method allowed a surface antigen monoclonal to be revealed first, using an optimal fixative, before alcohol/gluteraldehyde fixation was used to start the second (BRdU) staining sequence. An analysis of double staining of T lymphocyte subsets (CD4 and CD8) showed no significant difference in the order of application of the primaries (n = 10) and no significant difference from their corresponding single stain results (n = 50), confirming the validity of the technique where antigens are exclusively distributed. Other examples, including antigens distributed in different compartments of the same cell, displayed discrete staining which implied validity. Double staining by APAAP with this technique seems to be applicable to those cases where antigens are exclusively distributed and includes cases where different compartments of the same cell are stained. It is especially useful in revealing antigens that require different fixation and preparation--that is DNA incorporated BRdU with a surface antigen. But it does seem to have a limited ability to produce a dual colour at a common site.


New screening system for simultaneous determination of two marker proteins by homogeneous enzyme immunoassay. To save time and labour in mass screening, by detecting two marker proteins on one specimen using only one test. alpha Fetoprotein and ferritin were chosen to demonstrate the principal of this system. The assay reagents were horseradish peroxidase (HRP)-labelled anti-alpha fetoprotein and HRP-labelled anti-ferritin antibodies. After the serum sample had been incubated with these reagents the substrate for HRP was added and the absorbance measured. An absorbance value below the cutoff point indicated that both parameters were within normal limits; a value above the cutoff point indicated that at least one of the two parameters was abnormally high. Fifty sera from healthy Japanese subjects were assayed by the simultaneous assay method. All samples gave absorbancy values below the cutoff point. Fifty serum samples from patients with high alpha fetoprotein concentrations (over 20 ng/ml) and 50 samples with high ferritin concentrations (over 200 ng/ml) were also assayed. The absorbancy values of all samples with high alpha fetoprotein concentrations, and all but one sample with high ferritin concentrations gave values above the cutoff point. Although this homogeneous enzyme assay method was applied to the combination of alpha fetoprotein and ferritin, it could be used in mass screening for any other combination of two markers.


Hepatocholangioadenoma in a pig.

Hepatocholangioadenoma in a pig. A Landrace sow had several neoplastic nodules in the liver. Histopathologically, the individual nodules consisted of two types of closely associated neoplastic cells; one was of hepatocellular type, the other of cholangiocellular type. Based on this finding, the term "hepatocholangioadenoma" is considered to be appropriate for this tumour. Previously reported in man, the dog and the duck, a mixed type of primary hepatic neoplasm has now been found in the pig.


Poorly differentiated squamous cell carcinomas of the skin can express vimentin. Thirty cases of poorly differentiated carcinomas of the skin were examined for the expression of vimentin. All cases expressed cytokeratins; in addition, 12 cases were positive for vimentin. These were all non-reactive with antibodies to S100 protein, HMB45 and desmin. The finding of vimentin in poorly differentiated squamous cell carcinomas underscores the need for caution in the use of immunohistochemical stains for tumor typing. Cutaneous squamous cell carcinomas are an addition to the list of epithelial tumors which are known to coexpress vimentin intermediate filaments. Other carcinomas which consistently express vimentin include those of renal, endometrial, thyroid, pulmonary, ovarian, salivary gland, adrenal and more recently, those of breast and prostatic origin.


Neutrophil elastase and its inhibitors in human gingival crevicular fluid during experimental gingivitis. The relative concentrations and absolute amounts of neutrophil elastase and its two inhibitors, alpha 2-macroglobulin (alpha 2-M) and alpha 1-antitrypsin (alpha 1-AT), were determined in gingival crevicular fluid (GCF) collected from six dental students who refrained from brushing the upper left or right quadrant during three weeks. Plaque and gingival indices and flow of GCF were measured before, during, and after the three weeks of no brushing. Functional elastase, representing the enzyme complexed with alpha 2-M, was measured by use of a low-molecular-weight fluorogenic substrate. Elastolytic activity in GCF was also assayed by use of elastin as substrate. Antigenic elastase, representing the enzyme complexed with alpha 1-AT, as well as the inhibitors alpha 2-M and alpha 1-AT were measured by ELISA. After three weeks of plaque accumulation, the concentrations of both functional and antigenic elastase increased by a factor of about 3, whereas the concentrations of the inhibitors increased in a much higher proportion. No free elastase could be detected in GCF, as evidenced by the Sephadex G-75 elution profile of GCF, by the negative results obtained when elastin was used as substrate, and by the demonstration that pure enzyme kept its activity against the low-molecular-weight substrate after being saturated by alpha 2-M.


Postprandial cholecystokinin secretion in elderly with protein-energy undernutrition. Malnutrition is currently observed in aged people, and cholecystokinin is an important peripheral satiety signal. The aim of this study was to examine the effect of aging and protein-energy malnutrition on postprandial cholecystokinin (CCK) release. Non-randomized, cross-sectional comparison by age group. Gastroenterology section of a teaching hospital. Twenty-one human volunteers divided into three groups: young healthy subjects (Group 1: mean 29 years, n = 7), aged healthy subjects (Group 2, mean 80 years, n = 7), and aged subjects with an important degree of malnutrition (Group 3, mean 84.6 years, n = 7). Each subject ingested a standardized liquid meal after an overnight fast. Plasma cholecystokinin was measured using a sensitive bioassay before and after the ingestion of the liquid meal. Basal cholecystokinin levels were similar (0.9 to 1 pM equivalent CCK-8) in the three groups. Postprandial levels were significantly increased over basal (P less than 0.05). The maximal cholecystokinin value was lower in Group 1 (3.5 +/- 0.8 pM equivalent CCK-8) and Group 2 (3.3 +/- 0.77 pM equivalent CCK-8) than in Group 3 (8.3 +/- 2 pM equivalent CCK-8) (P less than 0.05). Integrated plasma cholecystokinin was also similar in Group 1 (171 +/- 38 pM.60 min), (P less than 0.05). The increase of postprandial maximal levels of cholecystokinin is more related to malnutrition than to aging.


Effects of dibutyrylcyclic adenosine monophosphate on bleomycin-induced lung toxicity in hamsters. Cyclic nucleotides play an important role in the regulation of fibroblast proliferation and collagen metabolism. In the present study, the antifibrotic potential of dibutyrylcAMP (Bt2cAMP) was evaluated in the bleomycin (BLM)-hamster model of pulmonary fibrosis. Bt2cAMP (10 mg kg-1, s.c.) or saline (SA, s.c.) was given daily two days prior to the first intratracheal (i.t.) dose of BLM or SA and thereafter throughout the study. BLM or SA was instilled i.t. in three consecutive doses (2.5, 2.0 and 1.5 U 5ml-1 kg-1) at weekly intervals. Hamsters were killed at 7, 14 and 20 days after the third i.t. instillation. Bt2cAMP significantly reduced the contents of lung hydroxyproline and lung thiobarbituric acid reactive substance equivalents in BLM-treated animals at 7 and 14 days. Bt2cAMP significantly elevated lung superoxide dismutase activity in BLM-treated animals at 7 days. Lung prolyl hydroxylase activity was significantly elevated at 14 and 20 days in SABLM- and Bt2cAMPBLM-treated animals. The ratio of cAMP/cGMP was significantly reduced at all time points in SABLM-treated animals but only at 7 and 14 days in Bt2cAMPBLM-treated animals. Bt2cAMP caused no significant changes in lung calcium and calmodulin levels and protein content of the bronchoalveolar lavage. BLM significantly increased various inflammatory cell counts in the lavage at all three time points. The cell counts in the Bt2cAMPBLM groups were generally lower at 7 days and higher at 20 days than those of the SABLM groups. Histological evaluation showed that the lungs of Bt2cAMPBLM-treated hamsters progressed from an inflammatory cell lesion to a fibrotic lesion at a slower rate than the SABLM groups. It was concluded that Bt2cAMP attenuated BLM-induced pulmonary fibrosis in hamsters in part by delaying the acute phase of the inflammatory reaction.


Large volume paracentesis and intravenous dextran to treat tense ascites. Forty patients with cirrhosis of the liver and tense ascites were randomized to receive either aldactone 400 mg/day and furosemide 80 mg/day (n = 20) or repeated large volume paracentesis (LVP) and infusion of low molecular weight dextran (n = 20). Both treatment groups were similar in clinical and laboratory parameters. Complete mobilization of the ascitic fluid was achieved in all receiving LVP and dextran therapy within 1 week of the treatment, in contrast to the minimal mobilization of the ascitic fluid in patients receiving diuretics even after 2 weeks of therapy. Renal function, the clinical parameters of systemic hemodynamics, serum electrolytes, and hepatic function remained stable in patients receiving LVP and dextran and were similar to those in the diuretic-treated patients. We found no deterioration of these functions in the nonedematous patients treated by LVP and dextran even though the protective effect of edema against LVP was lacking in them. Plasma volume estimation in six nonedematous cirrhotic patients treated by LVP and dextran did not reveal any hypovolemia after complete mobilization of ascites. The frequency of complications and death were similar in the two groups. Dextran infusion is a safe, effective, and low-cost replacement therapy in patients with cirrhotic ascites treated by LVP.


AIDS-like disproportion of minor T-cell subsets in Japanese patients with Wegener's granulomatosis. Fifteen Japanese patients with Wegener's granulomatosis (WG) were evaluated according to their lymphocyte subset abnormalities. Two colour immunofluorescent flow cytometry was used to distinguish the lymphocyte subset alterations. WG group showed a decrease in the percentage of CD4+ cells and the increase of CD8+ cells. Within the NK cell family, the functionally unidentified CD8+57+ cells were markedly elevated. The disproportion of the lymphocyte subsets (CD4+ decreases CD8+57+ increases) were similar to those of Acquired Immunodeficiency Syndrome (AIDS) and AIDS relating complexes (ARC). To assess silent infection of Human immunodeficiency virus (HIV), the polymerase chain reaction (PCR) was done for all patients to selectively amplify specific HIV proviral DNA sequences in peripheral blood mononuclear cells, HIV-1 proviral DNA was not found in any patients but these changes of WG might suggest a possible adaptive response to unknown viral infection.


Patch-clamp analysis of the effects of the insecticide deltamethrin on insect neurones. 1. The mode of action of the pyrethroid insecticide deltamethrin on inexcitable embryonic cultured cockroach neurones has been investigated using the patch-clamp technique. 2. Whole-cell recordings of the current induced by step depolarizations of the cell membrane showed that concentrations of deltamethrin ranging from 10(-8) to 5 x 10(-6) mol l-1 induced a small tetrodotoxin (TTX)-sensitive inward current that peaked at around +10 mV and reversed at around +60 mV. The activation and inactivation kinetics of this current were much slower than those of the axonal sodium current in this same species and were relatively insensitive to membrane potential. Steady-state inactivation was almost absent. 3. Single-channel activity associated with the action of the insecticide was analyzed using the cell-attached configuration. Three distinct patterns of activity were found: (1) discrete single-channel events of relatively short duration, (2) long events of comparatively small amplitude and (3) complex bursts made up of a succession of openings and closings to several levels. These three patterns were analyzed quantitatively using specially designed programs. 4. The first pattern of activity could be seen in most patches. It consisted of short (1-10 ms) rectangular events of comparatively small amplitude (1.5 pA at rest) and very low open time probability (around 0.001). The current-voltage relationship of these small events was linear over the voltage range studied and the (extrapolated) reversal potential approximated ENa. 5. The second pattern of activity was observed less frequently. The channels could stay open for very long periods (up to several seconds) and occasionally flickered between two or more levels. 6. The third pattern of activity was observed in many patches. During the burst, which could last from a few milliseconds to a few hundred milliseconds, the single-channel current jumped almost continuously between several levels (up to 7 or 8).


Prevalence and geographical distribution of major LDL receptor gene rearrangements in Finland. In order to determine the prevalence of major rearrangements of the low density lipoprotein (LDL) receptor gene in Finland, DNA samples of 199 unrelated Finnish patients with the heterozygous form of familial hypercholesterolaemia (FH) were examined by Southern blot analysis. The FH-Helsinki mutation, characterized by a 9.5-kb deletion in the 3'-end of the LDL receptor gene, was found in 75 (38%) of the patients. The prevalence of this mutation ranged from 26-58% in different areas of Finland. A striking exception was the North Karelia region, where only one out of 26 (4%) FH patients was found to carry the FH-Helsinki allele. Two patients were found to carry other types of large nucleotide rearrangements of the LDL receptor gene. One mutation was a 7.5-kb deletion eliminating exons 7 to 10, and the other was a 13-kb deletion covering exons 11 to 16 of the LDL receptor gene. Serum lipoprotein levels were very similar in each category of mutation, i.e. in patients with the FH-Helsinki gene, those with the two other types of deletion, and the remaining patients with as yet unknown types of LDL receptor gene defects. These results show that, even in genetically uniform populations, FH may be heterogeneous at the DNA level. DNA techniques enable an unequivocal diagnosis for almost 40% of the Finnish patients with the heterozygous form of FH.


Localization of conserved B-cell epitopes among encapsulated and non-encapsulated Haemophilus influenzae P2 porin proteins using synthetic peptides. The P2 outer membrane protein of Haemophilus influenzae belongs to a class of apparently ubiquitous proteins in Gram-negative bacteria that function as porins. Murine hybridomas raised to the P2 protein and synthetic peptides were used to investigate the structural and antigenic relationships among P2 proteins of encapsulated and non-encapsulated H. influenzae. Three monoclonal antibodies (mAbs), P2-17, P2-18 and P2-19, recognizing epitopes on the P2 protein, as shown by Western immunoblotting of outer membrane preparations, and purified and recombinant P2 proteins are described. The epitopes reactive with the mAbs were widely distributed among H. influenzae strains since 70-100% of strains of encapsulated and non-encapsulated isolates collected worldwide were recognized by individual mAbs. None of the mAbs reacted with H. parainfluenzae or other bacterial species. The peptide composition of P2 epitopes was determined by analysis of mAb reactivity with a series of overlapping synthetic peptides that covered the amino acid sequences of H. influenzae type b. The domains recognized by these mAbs were completely distinct. mAb P2-18, reactive with an epitope conserved among all H. influenzae P2 porin molecules which were screened, recognized a peptide corresponding to the N-terminal segment (residues 1-14). The P2-17- and P2-19-specific epitopes were located between residues 28 and 55, and 101 and 129, respectively. None of the epitopes were exposed on the cell surface since no mAbs bound to intact live bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)


Diversity of cleavage patterns of Salmonella 23S rRNA. The recent discovery of the phenomenon that some prokaryotes fragment their 23S rRNA during post-transcriptional processing of precursor rRNA has been shown to be particularly prevalent among strains and species of Salmonella. Some strains of Salmonella cleaved 23S rRNA at multiple sites producing several fragments. The cleavage patterns of 23S rRNA differed among Salmonella strains and sometimes among the rRNA operons in the same strain. Fragmentation of 23S rRNA was not observed in strains of the closely related species Escherichia coli. Fragmentation of 23S rRNA occurred in Brucella and Agrobacterium but the cleavage pattern was not as diverse as that demonstrated in Salmonella. Introduction of cleavage sites into precursor 23S rRNA of Salmonella is probably a recent evolutionary event.


Identification of indigo-related pigments produced by Escherichia coli containing a cloned Rhodococcus gene. Pigments produced by Escherichia coli containing a cloned piece of DNA from Rhodococcus sp. ATCC 21145 were extracted in chloroform and separated into blue and pink components. Evidence from TLC, NMR spectroscopy, absorption spectrum analysis and solubility behaviour suggested that the blue pigment was indigo and the pink pigment was indirubin, a structural isomer of indigo. The proposed pathway for pigment production on LB agar involves the conversion of tryptophan to indole by tryptophanase of E. coli and the oxidation of indole to indigo by the product of the cloned Rhodococcus DNA insert.


Visceral targets specify calcitonin gene-related peptide and substance P enrichment in trigeminal afferent projections. Rat trigeminal ganglion projections to a visceral target (intracranial blood vessels) are enriched in calcitonin gene-related peptide (CGRP) and substance P (Sub P) compared to trigeminal ganglion projections to a cutaneous target (the forehead skin). We asked if transplants of a novel visceral target (fetal stomach antrum tissue) into the path of the neonatal rat trigeminal frontal nerve projection to forehead skin would induce neuronal CGRP and Sub P enrichment. By postnatal day (P) 25, the percentage of nerves containing CGRP increased from 14-15% in the control trigeminal projection to forehead skin to 20-31% (in different experiments) in the trigeminal projection to transplanted stomach antrum. The percentage of Sub P-containing neurons increased from 10% in the control forehead skin projection to 22% in the trigeminal projection to stomach transplants over the same time period. The number of neurons in the trigeminal frontal nerve projection to stomach antrum transplants was not significantly different from the number of frontal neurons projecting to control forehead skin. We suggest that respecification of trigeminal neurons to the CGRP and Sub P phenotype, not selective survival of CGRP- and Sub P-positive afferents, is the mechanism by which stomach antrum induces enrichment of CGRP and Sub P. A subpopulation of rat trigeminal neurons with cutaneous forehead skin projections also sends a transient axon collateral projection to a visceral target (the cerebral arteries) during early postnatal development. Postnatal maintenance of an axonal projection to a cutaneous target (forehead skin) may be incompatible with a neuron also maintaining a visceral collateral to the cerebral arteries.(ABSTRACT TRUNCATED AT 250 WORDS)


Asymmetric modulation of cytosolic cAMP activity induces growth cone turning. The possible role of cyclic nucleotides as second messengers mediating growth cone turning was studied by producing an asymmetric distribution of cyclic nucleotides across the growth cone. A repetitive pulse application method was developed to produce microscopic chemical gradients near the growth cone of embryonic Xenopus neurons in cell culture. When picoliters of a solution containing 20 mM dibutyryl cAMP (dB-cAMP), a membrane-permeable analog of cAMP, were repetitively ejected from a micropipette near the growth cone, neurite growth was consistently directed toward the pipette. Theoretical analysis of the diffusion gradient showed that the neurite is capable of detecting a 10% difference in dB-cAMP concentration across the growth cone. Similar responses were also observed using gradients of the phosphodiesterase inhibitor isobutylmethylxanthine, or of forskolin, which activates adenylate cyclase. Dibutyryl cGMP, however, produced no significant turning. These results suggest that a cytoplasmic gradient of cAMP across the growth cone is sufficient to initiate its turning response, and that cAMP in the growth cone could serve as a second messenger in mediating the action of extracellular guidance cues.


Effect of select media supplements on motility and development of Eimeria nieschulzi in vitro. A technique was developed to examine the effects of exogenous substances on apicomplexan sporozoite motility in vitro. Sporozoites of Eimeria nieschulzi were placed in the top compartments of blind well chemotactic chambers and separated from potential chemoattractant/chemokinetic agents by 8.0-microns pore size Millipore filters. After 3-hr incubations, the number of sporozoites migrating through the filters was assessed using a hemacytometer. Results revealed that several substances, especially albumin and fetuin, enhanced motility of coccidial sporozoites. Cell culture assays supported these data, with higher numbers of parasites found in cultures supplemented with albumin and/or fetuin.


Distribution of cytokeratins in oral cytological smears of HIV-infected patients. Cytological smears (CS), taken from the lateral border of the tongue of HIV-seropositive patients (HIV+) (n = 34) and of seronegative controls (HIV-) (n = 16), were examined by means of immunocytochemistry (APAAP) for the distribution patterns of different cytokeratins and MHC class II antigens. Compared with HIV- patients in CS of HIV-infected patients cornification associated cytokeratins 10/11 were increased, while the number of keratinocytes positive for cytokeratins 13/16 was comparable in both groups. Expression of simple epithelial cytokeratins 19, rarely observed in CS of HIV- patients, was a frequent findings in CS of HIV+ patients. Keratinocytes positive for MHC class II antigens were observed in CS of 12/34 HIV+, while all control CS were negative. In the group of HIV+ patients no correlation was found between the clinical presence of HL and the expression of cytokeratins or class II antigens. The altered distribution of cytokeratins may reflect local responses to proliferative stimuli or local inflammation due to the presence of microbial antigens or may occur as a general unspecific reaction in the setting of systemic viral infection. This non-invasive technique seems to be a valuable tool to determine the proliferation rate of oral epithelial cells.


Topology of innervation of labial salivary glands by protein gene product 9.5 and synaptophysin immunoreactive nerves in patients with Sjögren's syndrome. Glandular secretion and integrity, local blood flow, salivary secretion, pain perception and neurogenic inflammation can all be controlled by the nervous system. Therefore, the pattern of innervation of labial salivary glands (LSG) was studied in 10 patients with Sjögren's syndrome using neuronal markers: protein gene product 9.5 (PGP 9.5), a cytoplasmic, noncytoskeletal epitope and synaptophysin, a glycoprotein present in presynaptic vesicles. PGP 9.5 immunoreactive nerve fibers were found surrounding the acini, salivary ducts and blood vessels. The rich innervation of LSG was even more evident in immunofluorescence stained sections analyzed using confocal laser scanning imaging. Synaptophysin immunoreactive nerve endings and preterminal varicosities also demarcated the LSG acini. Furthermore, in the small foci, PGP 9.5 and synaptophysin immunoreactive nerve fibers were found amid inflammatory mononuclear cells and in extensive inflammatory areas nerve fibers were found in the peripheral parts of such infiltrates. This suggests a possible neurogenic influence on the local cellular inflammation. When LSG patient samples were compared to unaffected glands from 7 controls, acinar atrophy was found in the areas devoid of a local delivery system of neurogenic trophic stimuli, suggesting this as a possible cause of glandular degeneration. It may become necessary to incorporate this neglected, but existing system into our current view on the local but possibly centrally controlled or influenced pathogenetic mechanisms of Sjögren's syndrome.


Cytoplasmic chloride regulates cation channels in the vacuolar membrane of plant cells. This study is concerned with the characterization of the ionic currents in the vacuolar membrane (tonoplast) of plant cells. Voltage patch-clamp experiments at the whole vacuole and single channel levels were employed to study the effects of cytoplasmic chloride on the tonoplast inward rectifying currents of sugar beet cultured cells. Whole vacuole experiments showed that removal of cytoplasmic chloride induced a decrease in the level of the inward currents, an effect that was reversed upon returning to control levels of cytoplasmic chloride. Substitution of cytoplasmic chloride by any other anion (organic or inorganic) resulted in a reduction in the level of the inward currents. At a given negative tonoplast potential, the inward currents showed a linear relationship with the concentration of cytoplasmic chloride between 10 and 100 mM, with the slope of these relationships increasing as the potential was made more negative. Single channel experiments showed that reduction of cytoplasmic chloride changed the gating mechanism of the channels without affecting the single channel conductance. Reduction of cytoplasmic chloride caused a decrease in the open probability of the tonoplast cation channels by reducing their mean open time and by inducing the appearance of an additional closed state.


Ion selectivity of colicin E1: modulation by pH and membrane composition. Colicin E1 is a plasmid-encoded bacteriocidal protein which, though water soluble when secreted by its host bacterium, spontaneously interacts with planar lipid bilayers to form voltage-gated ion channels. In asolectin bilayers, the preference for anions over cations exhibited by these channels at low pH can be reversed by raising the pH on either side of the membrane. When incorporated into membranes composed of either of the two zwitterionic lipids, bacterial phosphatidylethanolamine and diphytanoyl phosphatidylcholine, colicin E1 channels were nearly ideally anion selective in the limit of low pH and moderately cation selective at the high pH limit. In phosphatidylcholine membranes, however, the response of these channels to changes in pH exhibited a pattern of behavior peculiar to this lipid. If the side of the membrane on which the protein had been introduced (the cis side) was exposed to pH 4.0, all the channels in the bilayer, whether opened or closed, became refractory to further changes in pH. This irreversibility has been interpreted as evidence that the selectivity of colicin E1 is under the control of a pH-sensitive conformational change. Protonation of groups on the cis side of the membrane appear to be essential to the conversion to the anion-selective state. These groups are rendered kinetically inaccessible to the aqueous phase when the transition takes place in phosphatidylcholine membranes.


Complementary DNA-DNA hybridization in Drosophila. We have performed DNA-DNA hybridization experiments among several species of Drosophila using the evolutionarily conserved portion of the genome representing sequences coding for amino acids of proteins. This was done by using as tracer, radioactively labeled complementary DNA that was reverse transcribed from adult mRNA. We show that this procedure extends phylogenetically the distance over which the technique can be applied to fast-evolving groups such as Drosophila. The major phylogenetic conclusions are (1) the subgenus Sophophora is a monophyletic lineage; (2) within Sophophora the melanogaster subgroup is closer to the obscura group than either group is to the willistoni group; (3) the subgenus Drosophila is complex with most major lineages originating deep in the phylogeny; the subgenus may not be monophyletic; (4) as with most groups classically placed in Drosophila, the Hawaiian Drosophila originate early, supporting the notion that this lineage is older than the extant islands; and (5) the virilis/repleta lineage is monophyletic within Drosophila.


Expression of insulinlike growth factor (IGF) and IGF-binding protein genes in human lung tumor cell lines. The presence of multiple, low-molecular-weight, insulinlike growth factor (IGF)-binding proteins in lung tumor cell-conditioned medium and lung cancer patient serum has been recently reported. To begin to elucidate the genetic basis for these observations, the present study examines the expression by lung tumor cell lines of three IGF-binding protein genes, namely, IGFBP-1, IGFBP-2, and IGFBP-3. Since IGF-binding proteins are thought to modulate the biologic action of the IGFs, the relationship between the expression of IGF-binding protein genes and the genes encoding IGF-I and IGF-II also has been investigated. Gene expression was studied in four small-cell lung cancer (SCLC) and three non-small-cell lung cancer (NSCLC) cell lines using Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR) for IGFBP-1. IGFBP-1 gene expression was detected by Northern blot analysis in one NSCLC cell line only. However, RT-PCR revealed that the IGFBP-1 gene was expressed in all four SCLC cell lines and in two of the three NSCLC lines. Northern blot analysis of IGFBP-2 gene expression demonstrated that all lung tumor cell lines expressed this gene. A low level of IGFBP-3 gene expression was detected in one SCLC cell line and in all three NSCLC cell lines. All lung tumor cell lines expressed the IGF-II gene as determined by Northern blot analysis. In marked contrast, none of the lines showed evidence of IGF-I gene expression using this method. However, RT-PCR revealed a low level of IGF-I gene expression in one SCLC and one NSCLC cell line only. These observations indicate 1) that IGF-binding proteins secreted by lung tumors are encoded by at least three different genes; 2) that there may be a close association between IGF-II and IGFBP-2 gene expression, such that, where there is production of IGF-II, IGFBP-2 is the principal BP; and 3) that the IGF-II gene is more widely expressed than the IGF-I gene in human lung tumor cell lines.


Longitudinal evaluation of prostate-specific antigen levels in men with and without prostate disease. To evaluate longitudinal changes in prostate-specific antigen (PSA) levels in men with and without prostate disease. Case-control study of men with and without prostate disease who were participants in a prospective aging study. Gerontology Research Center of the National Institute on Aging; the Baltimore (Md) Longitudinal Study of Aging. Sixteen men with no prostate disease (control group), 20 men with a histologic diagnosis of benign prostatic hyperplasia (BPH), and 18 men with a histologic diagnosis of prostate cancer. Multiple PSA and androgen determinations on serum samples obtained from 7 to 25 years prior to histologic diagnosis or exclusion of prostate disease. Changes in androgen levels with age did not differ between groups. Control subjects did not show a significant change in PSA levels with age. There was a significant difference in the age-adjusted rate of change in PSA levels between groups (prostate cancer greater than BPH greater than control; P less than .01). At 5 years before diagnosis when PSA levels did not differ between subjects with BPH and prostate cancer, rate of change in PSA levels (0.75 micrograms/L per year) was significantly greater in subjects with prostate cancer compared with control subjects and subjects with BPH. Also, rate of change in PSA levels distinguished subjects with prostate cancer from subjects with BPH and control subjects with a specificity of 90% and 100%, respectively. The most significant factor affecting serum PSA levels with age is the development of prostate disease. Rate of change in PSA levels may be a sensitive and specific early clinical marker for the development of prostate cancer.


[Congenital pelvic arteriovenous fistula in a male. A case report]. A congenital arteriovenous fistula (AV fistula) in the true pelvis is extremely rare, especially in males. We present a case of this disease with pollakiuria and intrarectal discomfort. Diagnosis was made by computed tomography and magnetic resonance imaging and confirmed by cine angiography. Because the AV fistula involved the bladder, prostate and rectum, ligation of the main feeding arteries was performed. He also had benign prostatic hypertrophy and was treated by conservative therapy because of a high risk of massive bleeding in surgical treatment.


The effect of digital rectal examination on prostate-specific antigen levels. OBJECTIVE--To identify the effect of digital rectal examination (DRE) on serum prostate-specific antigen (PSA) levels. DESIGN--A prospective trial before and after DRE. SETTING--Multicenter outpatient screening program. PATIENTS--A total of 2754 healthy men aged 40 years and older who presented to a prostate cancer screening program and consented to two phlebotomies. MAIN OUTCOME MEASURE--Changes in serum PSA levels after DRE. RESULTS--Patients were divided into four groups based on their initial serum PSA levels. The levels were chosen based on previous studies that showed different incidences of prostate cancer within these groups. The two groups with the lowest initial PSA values (0.1 through 4 micrograms/L and 4.1 through 10 micrograms/L) were found to have statistically insignificant changes in the serum PSA levels after DRE. The group with initial PSA levels of 10.1 through 20 micrograms/L had increases in serum PSA values that showed a trend toward statistical significance. The group with initial PSA levels of greater than 20 micrograms/L had statistically significant increases in serum PSA values after DRE. The alterations in serum PSA levels in the two groups with the highest PSA values were not clinically important as the patients' clinical treatment was not altered. CONCLUSIONS--No clinically important effects on serum PSA levels were noted after DRE.


Gastrointestinal function in obesity: motility, secretion, and absorption following a liquid test meal. Digestive responses to a 300-mL liquid fat-rich meal (432 kcal) in a group of massively obese patients were compared with those observed in a group of healthy lean subjects of variable body weight. Gastric and intestinal propulsion, digestive secretions, and absorption in the proximal 70 cm of intestine were measured using a multiple-marker dilution method. The average gastric emptying of energy, acid, volumes, and meal marker were similar in the two groups 80 minutes after intake, justifying a comparison of intestinal processing of the meal. Compared with lean subjects, the obese subjects responded with less pancreatic secretion (P less than .05) and gallbladder emptying, but absorbed a larger proportion of the emptied energy in the test segment (P less than .01) during a similar or shorter transit time. In addition, when the entire meals were compared, the obese group generally absorbed the test meal more effectively and rapidly in the upper part of the intestine. As a consequence, the flow volumes at the exit of the test segment were lower (P less than .05), and less of the test meal was propulsed to distal parts of the intestine. In the lean subjects, the body weight or height correlated positively with the gastric emptying rate, peak gastric acid output, and pancreatic responses, and negatively with (P less than .05) the segment transit time. The taller the subject, the greater the proportion of the meal which was rapidly propulsed unabsorbed to lower parts of the intestine, indicating that a large intestinal area was exposed for rapid energy uptake. No such correlations were observed in the obese group.