Expression of gap-junctional protein (connexin 43 or alpha 1 gap junction) is down-regulated at the transcriptional level during adipocyte differentiation of H-1/A marrow stromal cells. Bone marrow stromal cells are requisite for the proliferation of hematopoietic cells and communicate with each other via gap junctions. Marrow stromal cells expressed connexin 43, but not connexin 32. H-1/A, a murine marrow stromal cell line, underwent adipocyte differentiation at confluence, and expressed the 3.0 kilobase mRNA species of connexin 43 before differentiation. H-1/A cells studied with the dye-transfer method showed gap-junctional communication with adjacent cells but lost this communication during differentiation. The connexin 43 transcripts in H-1/A cells were down-regulated before the expression of glycero-monophosphate dehydrogenase was induced. Loss of gap-junctional communication was regulated at the mRNA level of connexin 43. Connexin 43 expression was down-regulated at the transcriptional level.


Immortalization of fetal mouse brain glial cells by human papillomavirus type 16 E7 genes. Fetal mouse brain glial cells in primary cultures were immortalized by recombinant retroviruses containing human papillomavirus type 16 E7 genes, and named VR-2g cells. The presence and expression of E7 genes in VR-2g cells were demonstrated by the Southern and the Northern blot analyses. VR-2g cells did not form colonies in soft agar culture, indicating that VR-2g cells have no transforming phenotypes. By the karyotype analysis, VR-2g cells consisted of two cell populations, the pseudo-diploid and the pseudo-tetraploid. VR-2g cells were positive in immunostaining with mono- and polyclonal antibodies against glial fibrillary acidic proteins (glial-specific intermediate filaments). In addition, VR-2g cells secreted neurotrophic factors as assayed with primary cultures of fetal rat striatum neurons, although molecular characterization of the factor(s) was not yet determined. These results indicate that the present method for cell immortalization will be useful for establishing untransformed cell lines from primary cultures of fetal brain cells.


Antitumor activity and cross-resistance of carmethizole hydrochloride in preclinical models in mice. Carmethizole hydrochloride [1-methyl-2-methylthio-4,5-bis(hydroxymethyl)imidazole-4', 5'-bis(N-methylcarbamate)hydrochloride, NSC 602,668; hereafter called carmethizole] is a new antitumor drug that has shown relatively broad activity in initial evaluations against several murine tumors and human tumor xenografts in vivo. The present studies were designed to address questions about carmethizole's activity against established disease, its activity on different treatment schedules, and the extent of its cross-resistance with established drugs. Human MX-1 mammary carcinoma, human NCI-H82 small-cell lung carcinoma, and human LOX amelanotic melanoma xenografts in athymic mice were used to determine the drug's activity against established disease; the NCI-H82 lung-tumor xenograft in athymic mice was used to explore its schedule dependence; and a series of drug-resistant murine leukemias provided an in vivo cross-resistance profile. When injected i.p., carmethizole exhibited antitumor activity against advanced-stage s.c. MX-1 mammary, s.c. NCI-H82 lung, and i.p. LOX melanoma xenografts and was as effective against established disease (MX-1 and LOX) as it was against early-stage disease (no data are available for early-stage NCI-H82). The therapeutic effect of carmethizole was not route-dependent, as was evidenced by the similar delays observed in tumor growth following i.p. and i.v. administration. The use of a split-dose schedule on a single day instead of one bolus injection yielded an increase in the total dose delivered, resulting in an increased delay in tumor growth. Murine leukemias resistant to vincristine (VCR), amsacrine (AMSA), or methotrexate (MTX) were not cross-resistant to carmethizole. However, murine leukemias resistant to doxorubicin (ADR), melphalan (L-PAM), cisplatin (DDPt), 1-beta-D-ara-binofuranosylcytosine (ara-C), and 5-fluorouracil (5-FU) were cross-resistant to carmethizole, suggesting that patients who have previously been treated with any of these agents might be less likely to respond to carmethizole than those who have had no opportunity to develop resistance to any of these compounds. We anticipate that the information derived from these studies may be useful in the design of clinical trials of carmethizole and may stimulate additional basic research on the mechanism of action of this new agent.


Nutrition and cancer.

Nutrition and cancer. Diet can play a key role in the pathogenesis of cancer. Diets high in fat and low in fiber predispose individuals to colon cancer. A high-fat diet is also implicated in breast cancer and prostate cancer. The dietary fat-cancer linkage is supported by epidemiological evidence, animal studies, and prospective trials. The antioxidants vitamin E, ascorbic acid, and beta-carotene have a protective effect and act as antipromoters of carcinogenesis. A diet of less than or equal to 10% of calories from fat and less than or equal to 40 g of fiber daily that includes fruits and vegetables will prevent up to 35% of cancers.


Induction of neutral proteinase and prostanoid production in bovine nasal chondrocytes by interleukin-1 and tumor necrosis factor alpha: modulation of these cellular responses by interleukin-6 and platelet-derived growth factor. We have previously reported that recombinant human interleukin-1 (IL-1) stimulates matrix erosion in bovine nasal cartilage explants (R. J. Smith et al., Inflammation 13, 367-382, 1989). This action of IL-1 is believed to be caused by matrix-degrading neutral proteinases produced by activated chrondrocytes. Accordingly, we investigated the effects of recombinant human interleukin-1 alpha (IL-1 alpha), recombinant human interleukin-1 beta (IL-1 beta), and recombinant human tumor necrosis factor alpha (TNF alpha) on bovine nasal chondrocyte (BNC) responsiveness. IL-1 alpha and IL-1 beta stimulated a time (0-72 hr) and concentration-dependent (0.01-10 ng/ml) production of collagenase, gelatinase, caseinase, and prostaglandin E2 (PGE2) in BNC monolayer cultures. Neutral proteinase and PGE2 production by BNC was also induced by TNF alpha (0.2-200 ng/ml) in a time-dependent (0-72 hr) manner. Recombinant human interleukin-6 (IL-6) caused a concentration-dependent (6-200 ng/ml) potentiation of IL-1-stimulated neutral proteinase and PGE2 production by BNC. However, recombinant human platelet-derived growth factor homodimer BB suppressed BNC responsiveness to IL-1. A recombinant human IL-1 receptor antagonist protein inhibited BNC activation by IL-1 but not TNF alpha.


Entry of lomefloxacin and temafloxacin into human neutrophils, peritoneal macrophages, and tissue culture cells. The uptake of lomefloxacin (difluoroquinolone) and temafloxacin (trifluoroquinolone) by human polymorphonuclear leukocytes (PMNs), peritoneal macrophages (PM phis), and two tissue culture cells (McCoy and Vero) was measured by a fluorometric assay. Both antimicrobials reached high intracellular concentrations in PMNs [cellular to extracellular ratio (C/E) greater than 4], in PM phis (C/E greater than 3) and lower in tissue culture cells (C/E greater than 1) at an extracellular concentration of 5 mg/L. Lomefloxacin uptake by PMNs was more rapid than that of temafloxacin. Entry of both quinolones into PMNs was environmental temperature-dependent, but not affected by cell viability. Ingestion of opsonized Staphylococcus aureus did not affect the ability of PMNs to concentrate these antimicrobials. Ingestion of opsonized zymosan or stimulation with phorbol myristate acetate significantly increased the PMN association of both quinolones, this effect being particularly marked with temafloxacin. It is concluded that both lomefloxacin and temafloxacin are markedly concentrated within human phagocytes and tissue culture cells, although this phenomenon is not dependent on the degree of fluorination of the molecule.


Latex agglutination test for cytomegalovirus antibody screening of transplant donors. Important aspects. In a retrospective study, false negative readings of CMV Scan results were detected. In a following prospective survey of 72 transplant donor sera, CMV Scan had a sensitivity and a negative predictive value of greater than 95%. Although CMV Scan is a rapid and useful test, a higher reliability can be achieved if results are controlled by another sensitive test or, at the least, CMV Scan is read by two different persons.


Pretreatment staging evaluation in small cell lung carcinoma. A new approach to medical decision making. The real need for extensive staging at the time of diagnosis is discussed in regard to small cell lung carcinoma. We performed a decisional retrospective analysis on a series of 182 patients, based on three staging steps: the first step included physical examination and routine biologic tests. The second step consisted of liver ultrasonography and needle aspiration of any clinically detectable tumor mass, and the third step included bone marrow examination, radionuclide bone scan, thoracic, abdominal, and brain CT scan. A stepwise multivariate logistic regression performed on 11 variables considered in the first step shows that a four-parameter model can predict the spread of the disease (limited or extensive): weight loss, performance status, and elevated LDH or alkaline phosphatase levels. Limited disease can be predicted in two ways: (1) elevated LDH with normal alkaline phosphatases, no weight loss, and good performance status, or (2) normal LDH and alkaline phosphatases. In this series, 28 percent of patients can be predicted as having extensive disease and can be treated with chemotherapy alone without chest irradiation. After the second step, the probability of disease being extensive is only 25 percent, and only 84 (46.15 percent) patients would need to undergo the third step of staging procedures (brain CT scan, bone marrow aspiration and biopsy, radionuclide bone scan) with this method. We conclude that a multistep approach represents a simple staging method and offers the advantage of harmlessness and lower costs for patients not to be evaluated in prospective clinical trials.


Spontaneous pneumothorax in small cell lung cancer. Two patients with extensive small cell lung cancer developed unilateral, spontaneous pneumothoraces while receiving chemotherapy. Both pneumothoraces wee asymptomatic, required no special procedure, and resolved with continued chemotherapy. Development of spontaneous pneumothorax during chemotherapy in patients with known small cell lung cancer may represent a response to treatment.


Pulmonary blastoma presenting with massive hemothorax. Pulmonary blastoma, a rare primary lung neoplasm occurring in both children and adults, is histologically distinct from other lung tumors. A patient is described who presented with massive and persistent hemothorax. Based on experience, it is possible that in advanced cases of this tumor, more vigorous chemotherapy is needed against both histologic elements.


[Prevention of thromboembolism in surgery--results of a survey in West German hospitals]. In 1990 a questionnaire on methods for prevention of deep venous thrombosis (DVT) and pulmonary embolism (LE) was mailed to 940 surgical centers in West Germany (FRG). The return rate was 60% or 564 answers, covering about 1,200,000 operations/year. The results are as follows: (1) Physical therapeutic measures (early mobilisation, elastic stockings) and drug administration are routinely used in all centers. The duration of prophylaxis is 3-8 days in 36% of centers, up to mobilisation in 31%, 9-16 days after operation in 17% and until demission in 16%. (2) A single drug regime is employed in 60% of centers (49% standard heparin, 9% low molecular heparin in combination with DHE) 40% of centers use all three drugs without clear cut guidelines concerning the indications. (3) The reported rates of thromboembolic complications diagnosed by clinical criteria are 0.55 +/- 0.62% for DVT and 0.22 +/- 0.29% for fatal or nonfatal LE. There is no evidence from the analysed data that the drug regimes influences the clinical outcome. The need for administration of drugs prevent DVT is widely accepted. A polypragmatic approach seems to be effective. However, standardized regimes for defined clinical conditions are desirable.


Intracellular calcium during chemotaxis of Dictyostelium discoideum: a new fura-2 derivative avoids sequestration of the indicator and allows long-term calcium measurements. During stimulation of Dictyostelium discoideum amoebae with the chemoattractant cAMP, extracellular calcium is taken up by the cells. The aim of this study was to determine the cytosolic free calcium concentration ([Ca++]i) during chemotaxis of Dictyostelium cells. In contrast to most vertebrate cells, three major drawbacks were encountered: 1) the indicator fura-2 could not be introduced into the cells by incubation with the ester form, 2) once loaded, the dye was rapidly sequestered into vesicles, 3) the organic anion transport blocker probenecid was not suitable to block sequestration. These problems were met by introducing the indicator into the cells with the scrape-loading technique adapted for use with Dictyostelium and the construction of a new fura-2 derivative, fura-2-dextran. Scrape-loading of Dictyostelium yielded up to 40% of labeled, vital cells. Fura-2-dextran fulfilled the following criteria: 1) it remained homogeneously distributed in the cytoplasm of motile Dictyostelium cells, 2) it retained the fluorescence intensity of fura-2 and the affinity for calcium binding, 3) it was very well suitable to demonstrate changes of [Ca++]i in serum-stimulated fibroblasts. [Ca++]i-measurements with fura-2-dextran in chemotactically active D. discoideum amoebae revealed that the large decrease in the extracellular calcium concentration is not accompanied by an overall change in [Ca++]i. Chemotaxis in this organism occurs in the absence of global changes in [Ca++]i. However, we cannot exclude either short-lived or local changes just beneath the plasma membrane.


Diversity of B2 bradykinin receptors with nanomolar affinity expressed in passaged IMR90 human lung fibroblasts. IMR90 human fetal lung fibroblasts express bradykinin receptors activating the pathway for biosynthesis of PGE2. A receptor of the B2 subtype stimulates half-maximal PGE2 production at 4.8 nM bradykinin, and maximal output takes place at 25 nM bradykinin. Radioligand binding studies reveal a population of [3H]bradykinin binding sites whose affinity correlates with this B2 receptor's biologic activity, with a KD of 2.5 nM. As IMR90 cells reach 60% of their defined life span in culture, they spontaneously induce expression of a second site of lower affinity, with half-maximal binding of [3H]bradykinin at 44 nM. This second site displays a characteristic primary B2 receptor recognition profile, but differs from the 2.5 nM site on a secondary level in recognition among different B2 ligands. Bradykinin is the most potent ligand at both sites; they each preferentially recognize an N-terminal extended bradykinin peptide construct having selectivity for the rat myometrial B2 receptor, suggesting that both sites have structural features in common. However, they display diversity in their order of preference for Met-Lys-bradykinin versus Lys-Lys-bradykinin; at the 44 nM site this order is completely reversed from the order of potency exhibited at the 2.5 nM site. Expression of the second site changes the manner in which these fibroblasts control their PGE2 production; it affords a graded response of PGE2 production at bradykinin levels beyond those which would normally saturate the 2.5 nM site. The inducibility of the 44 nM site in cultured fibroblasts addresses in vivo conditions in an inflammatory environment where continuing generation of bradykinin-related peptides takes place and presents a possible mechanism for overriding constraints that would otherwise limit the progression of inflammation.


Molecular cloning of mouse connexins26 and -32: similar genomic organization but distinct promoter sequences of two gap junction genes. Connexins26 and -32 are subunit proteins of gap junctions that are coexpressed in hepatocytes and several tissues but individually expressed in other cells. Molecular cloning of both corresponding mouse genes revealed similar genomic organization, i.e., each gene consists of two exons with the complete coding region located in the second exon. The first exon of each gene is preceded by a TATA-less promoter region. The promoter of the mouse Cx26 gene has at least two transcription start sites and is located in a very GC-rich region which is reminiscent of promoters of house-keeping genes. Putative consensus sequences for a metal response element, the transcription factor NFkappaB, and several GC-boxes were found within 600 bp upstream of the Cx26 transcription start sites. The promoter region of the mouse Cx32 gene contains two putative binding sites for the transcription factor HNF-1 and consensus motifs for NF-1 as well as NFkappaB within 680 bp upstream of the main transcription start site. Thus the sequence comparison of mouse Cx26 and Cx32 promoter regions provides hints for possible consensus elements that could control individual expression as well as common regulation of these gap junction genes in various tissues. Cx26 mRNA is much more abundant in adult mouse skin than in adult kidney and liver where Cx32 transcripts are relatively strongly expressed.


Alterations of choline acetyltransferase, phosphoinositide hydrolysis, and cytoskeletal proteins in rat brain in response to colchicine administration. Colchicine, a microtubule disrupting toxin, was administered intracerebroventricularly to rats, followed by measurements of (i) the activity of choline acetyltransferase, a biochemical marker of cholinergic neurons, (ii) cytoskeletal protein concentrations, including tau, MAP-2, spectrin, and tubulin, and (iii) the activity of the second messenger-generating system, receptor-coupled phosphoinositide hydrolysis. One week after colchicine treatment there was a 60% decrease in choline acetyltransferase activity in the hippocampus, which was followed by a gradual increase in only a 29% deficit after 12 weeks. In the striatum and cerebral cortex, choline acetyltransferase activity was slightly reduced (by 13% and 19%, respectively) 1 week after colchicine treatment followed by increases to control values. The concentrations of tau and tubulin in the hippocampus were unaltered by colchicine treatment, and MAP-2 and spectrin were only slightly reduced 4 weeks after colchicine. Hippocampal phosphoinositide hydrolysis induced by norepinephrine was elevated approximately 28% 1 and 2 weeks after colchicine treatment and that induced by ibotenate was increased by 53% 2 weeks after colchicine. These results demonstrate that colchicine causes a severe depletion of choline acetyltransferase 1 week after administration. There was not a significant reduction of the concentration of any of the cytoskeletal proteins after 1 week, possibly due to the cell-selectivity of the toxic effect of colchicine, but there was a delayed, and temporary, decline of MAP-2 and spectrin concentrations. Associated with the decreased choline acetyltransferase activity after 1 week was an enhanced phosphoinositide hydrolysis in response to norepinephrine, and after 2 weeks there were enhanced responses to norepinephrine and to ibotenate.(ABSTRACT TRUNCATED AT 250 WORDS)


Incidence of gestational trophoblastic disease in Stockholm County, 1975-1988. An epidemiological study with the aim of establishing the incidence of hydatidiform mole, persistent trophoblastic disease and choriocarcinoma in Stockholm County was performed. Based on the regional cancer registry and hospital registers, the incidence for 1975-1988 was calculated for the number of deliveries as well as the total number of pregnancies. Of the molar pregnancies, 6% were treated with chemotherapy because of invasive mole or choriocarcinoma. Non-molar choriocarcinoma occurred in 1/33,717 deliveries. Difficulties in assessing the incidence of gestational trophoblastic disease are discussed.


Absence of antibodies to human herpesvirus-6 in patients with slowly-progressive human immunodeficiency virus type 1 infection. To evaluate a possible role for Human Herpesvirus-type 6 (HHV-6) coinfection as a co-factor in the progression of HIV-1 disease, we investigated the prevalence of seropositivity for HHV-6 in a cohort of HIV-1 infected patients. These patients were retrospectively divided into two groups according to the decline of CD4+ T cells during the follow up: 11 were classified as rapid decliners (less than 400 CD4+/cmm within 1 year), and 38 as slow decliners (greater than 400 CD4+/cmm after at least 4 years' follow up). HHV-6 antibodies were detected by a commercial immunofluorescence assay and by a Western blotting assay developed in our laboratory. Our results show that Western blot appears to provide results satisfactorily free of false positivities. We found that the frequency of HHV-6 seropositivity was significantly lower in the group of slow decliners, compared both to rapid decliners and to the general population. These data suggest a role for HHV-6 co-infection in the progression of HIV-1 disease.


Evolution of hepatitis A antibodies prevalence in young French military recruits. Hepatitis A antibodies (anti-HAV) were surveyed in 1000 French recruits during 1990. The prevalence of anti-HAV in this group was 21.35%. Compared to a 1985 survey a 9% fall in the anti-HAV prevalence rate was observed. Living in a coastal area, low educational level, stay overseas were the main risk factors, as already noted in 1985.


High-affinity kainate and domoate receptors in rat brain. Mammalian brain expresses receptors which bind the potent neurotoxins, kainate and domoate, with high affinity, and which form a subclass of ionotropic glutamate receptors. A new member of these receptors, expressed in both adult and embryonic CNS is compared in its ligand binding properties to its closely sequence-related homologs.


Identification of a paramagnetic species as an early intermediate in the coenzyme B12-dependent glutamate mutase reaction. A cob(II)amide? Highly active and cobamide-free glutamate mutase was obtained from Clostridium cochlearium by a modification of the original purification procedure. After incubation of the enzyme with dithiothreitol, adenosylcobalamin (coenzyme B12) and the substrate (S)-glutamate, a paramagnetic species was observed by EPR-spectroscopy. The signal was maximal within 15 ms after mixing with glutamate. Different signals were detected after incubating the system with the competitive inhibitors (2S,4S)-4-fluoroglutamate or 2-methyleneglutarate instead of the substrate. The former developed with an at least 100-fold lower rate then the signal with glutamate. All three signals are probably due to low-spin cob(II)amide species with an extraordinary low gxy value as compared with cob(II)alamin.


Ubiquitin metabolism in HeLa cells starved of amino acids. Radio-iodinated ubiquitin (Ub) was introduced into HeLa cells by red blood cell-mediated microinjection. The half-life and solubility of Ub, as well as the molecular weight distributions of Ub conjugates, were then measured in HeLa cells grown in complete medium or in medium lacking amino acids and fetal calf serum. Ub metabolism was similar in the two sets of cells. Thus, the dramatic changes in Ub metabolism induced by thermal stress are not observed upon amino acid deprivation.


Serine residues responsible for tetracycline transport are on a vertical stripe including Asp-84 on one side of transmembrane helix 3 in transposon Tn10-encoded tetracycline/H+ antiporter of Escherichia coli. Putative transmembrane helix 3 of the tetracycline/H+ antiporter encoded by a transposon, Tn10, contains four serine residues, Ser-77, Ser-82, Ser-91 and Ser-92. Each of these serine residues was replaced by site-directed mutagenesis. Of these four serine residues, Ser-77 was important for the transport function, and a bulky side chain at position 91 hindered substrate translocation, whereas Ser-82 and Ser-92 did not play any role. Ser-77 and Ser-91 are on the same vertical stripe, that includes the essential Asp-84, on the hydrophilic side of putative helix 3. These observations suggest that helix 3 is part of the tetracycline translocation channel across the membrane.


Spectroscopic identification of the haem ligands of cellobiose oxidase. A spectroscopic study of the flavocytochrome b enzyme, cellobiose oxidase, employing optical, NMR, EPR and near infra-red MCD techniques, has identified the axial ligands of the b-type haem. These are a histidine and a methionine, and this ligation set is discussed in relation to the functional role of the haem group.


Engineering firefly luciferase as an indicator of cyclic AMP-dependent protein kinase in living cells. A bioluminescent indicator for protein kinase A has been developed by mutating V217 in firefly (Photinus pyralis) luciferase to R, and the C-terminal peroxisomal signal removed by PCR. The cDNA for normal and the RRFS mutant luciferase were inserted into pSV7d and expressed in COS-7 cells. Transient expression in approximately 5% of cells was confirmed by extraction of active luciferase, light emission from cells in the presence of luciferin, and immuno-localisation. The cyclic-AMP analogue, 8-(4-chlorophenylthio)-cyclic AMP caused a 5-10% decrease in light emission within 4 min in COS cells expressing the RRFS mutant, but not in cells expressing normal luciferase. This provides for the first time an indicator for detecting and quantifying protein kinase A activation in living cells.


CDC25-dependent induction of inositol 1,4,5-trisphosphate and diacylglycerol in Saccharomyces cerevisiae by nitrogen. The addition of ammonium sulfate to starved yeast cells leads to a 3- to 4-fold rapid increase of the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), the products of phosphoinositide-specific phospholipase C (PI-PLC). This response is reduced by dissecting the RAS-activating Cdc25 protein, and is completely abolished by the cdc25-1 mutation even at permissive temperature. Starved cdc25-1 mutant cells have a strongly reduced IP3 content, but an at least 10-fold increased DAG level compared to the isogenic wild-type strain. NH4 does not stimulate cAMP synthesis, and glucose does not induce IP3 and DAG. Our data suggest that the Cdc25 protein controls a nitrogen-specific signalling pathway involving the effector PI-PLC, in addition to the glucose-induced activation of adenylyl cyclase (AC).


Membrane interactions of mastoparan analogues related to their differential effects on protein kinase C, Na, K-ATPase and HL60 cells. Membrane interactions of tetradecapeptide toxin mastoparan (MP) and analogues (MP-3, MP-X and polistes MP), as indicated by inhibition of various enzymatic and cellular activities, were investigated. MP-3 was found to be the least active in inhibiting protein kinase C (PKC; activated by phosphatidylserine vesicles, synaptosomal membranes or phorbol ester), synaptosomal membrane Na,K-ATPase and proliferation and viability of leukemia HL60 cells. MP-3, however, was as active as others in inhibiting PKC activated by arachidonate monomers and phorbol ester binding. The unique properties of MP-3, the [des-Ile1-Asn2]-analogue of MP, might be related to its low functional amphiphilicity compared to others and useful in further delineating biological activities associated with or regulated by membranes.


Stoichiometric binding of 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate to bovine heart cytochrome c oxidase. The binding of 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) to isolated bovine heart cytochrome c oxidase (COX) was studied by following its specific spectral change at 510 nm. The quantitative titration revealed two binding sites for TNP-ATP per monomer COX with a Kd of 1.6 microM.


Abolishment of bradykinin-induced calcium oscillations in ras-transformed fibroblasts by the expression of 80 kDa diacylglycerol kinase. Our previous study showed bradykinin-induced periodic Ca2+ changes (Ca2+ oscillations) in v-Ki-ras-transformed NIH/3T3 (DT) cells in which protein kinase C (PKC) activity is partially down-regulated by a sustained high level of 1,2-diacylglycerol (DAG) [FEBS Lett. (1991) 281, 263-266]. In the present study, DAG kinase with 80 kDa mass (80K DGK) has been successfully transfected in DT cells, which exhibited enhanced cellular DAG kinase activities, decreased cellular DAG contents, and increased PKC activities compared to the control vector-transfected cells. Furthermore, these DGK-transfectants showed strong inhibition in bradykinin-induced Ca2+ oscillations. The results suggest that the sustained DAG increase down-regulates the PKC activity, thereby leading to the induction of Ca2+ oscillations in DT cells.


Isoproterenol-induced Mg2+ uptake in liver. Isoproterenol increased the Mg2+ content of hepatocytes after injection into rats or after addition to collagenase-dispersed hepatocytes. cAMP also the increased cellular Mg2+ content of isolated hepatocytes. This effect was prevented by staurosporine. Phorbol ester had no effect on the Mg2+ content of isolated hepatocytes, and after injection of isoproterenol into rats, protein kinase C of liver was not affected. It was concluded that isoproterenol induced long-term Mg2+ influx via the activation of protein kinase A which can be inhibited by staurosporine.


Effects of glycosylation on protein conformation and amide proton exchange rates in RNase B. Assignment of most of the proton NMR resonances of bovine pancreatic RNase B has been achieved using standard NMR techniques and by comparison with the published assignments for RNase A. A comparison of the NMR spectra of RNase B with RNase A shows that glycosylation of the enzyme has little overall effect on the conformation of the protein in solution. Comparisons of hydrogen-deuterium solvent exchange rates for the NH protons of RNase A and RNase B were made using two-dimensional 1H correlation spectroscopy. In the case of the glycosylated enzyme the exchange rates decreased for the NH protons of residues 9-14, 23-24, 32, 34-35, 39-40, 43-44, 48-49, 60, 71, 75-76, 80, 83-85, 100-101, 107, 111 and 122, relative to the unglycosylated RNase A. These results are consistent with the presence of the oligosaccharide inducing enhanced global dynamic stability and consequent changes to the unfolding equilibrium of the enzyme. The enhanced stability is observed not only for residues in the vicinity of the glycosylation site, asparagine-34, but also at residues remote from this site, as much as 30 A away.


Effects of bradykinin on cell volume and intracellular pH in NIH 3T3 fibroblasts expressing the ras oncogene. BCECF fluorescence has been applied to determine intracellular pH (pHi) in NIH 3T3 fibroblasts expressing the Ha-ras oncogene (+ras) and otherwise identical cells not expressing the oncogene (-ras). In +ras cells, pHi is significantly more alkaline (6.79 +/- 0.03 n = 12) than in -ras cells (6.64 +/- 0.02, n = 8). Bradykinin (100 nmol/l) leads to intracellular alkalinization in both +ras (to 6.96 +/- 0.04, n = 12) and -ras cells (to 6.85 +/- 0.02, n = 8). The effect of bradykinin is completely abolished in the presence of dimethylamiloride (100 mumol/l), which does not modify pHi in the absence of bradykinin. Similar to bradykinin, cell shrinkage by addition of 15 mmol/l NaCl to the extracellular fluid leads to intracellular alkalinization (by 0.08 +/- 0.01, n = 15). Cell volume is significantly greater in +ras cells (2.7 +/- 0.4 pl, n = 15) than in -ras cells (2.2 +/- 0.4 pl, n = 15). Bradykinin leads to cell shrinkage in both +ras cells (by 7 +/- 1%, n = 17) and -ras cells (by 5 +/- 1%, n = 15). The effect of bradykinin on cell volume can be reversed by the reduction of extracellular NaCl concentration by 15 mmol/l NaCl in +ras cells and by 7 mmol/l NaCl in -ras cells. This maneuver completely abolishes (in -ras cells) or blunts (in +ras cells) the alkalinizing effect of bradykinin. In conclusion, +ras cells are more alkaline than -ras cells. Bradykinin leads to further intracellular alkalinization by activation of the Na+/H(+)-exchanger, at least in part secondary to hormone-induced cell shrinkage.


Expression and purification of mouse TIMP-1 from E. coli. Tissue inhibitors of metalloproteinases (TIMPs) constitute a family of secreted glycoproteins involved in regulating extracellular matrix degradation in both normal and malignant tissues. We have expressed a cDNA clone of mouse TIMP-1 as a 22-kDa protein with 12 cysteine residues in E. coli and purified protein that shows inhibitory activity against collagenase following renaturation by chemical means. The low specific activity and circular dichroism measurements suggest, however, that the renaturation of the mouse recombinant (non-glycosylated) protein is not efficient under the conditions we have used, indicative of either thermodynamic instability or the transition to kinetic intermediates which have very low in vitro refolding rates.


Ubiquitin of Entamoeba histolytica deviates in six amino acid residues from the consensus of all other known ubiquitins. The amino acid sequence of ubiquitin from Entamoeba histolytica, as deduced from a cDNA nucleotide sequence, deviated at six positions from the consensus of all other known ubiquitins (ranging from Trypanosoma cruzi to Homo sapiens). The corresponding residues were scattered over the primary sequence, but came close together on the surface of the folded protein structure. We conclude that (i) E. histolytica branched off very early from the main eukaryotic line, and (ii) this organism may yield clues as to the evolutionary development of the ubiquitin system.


Cooperative proton and calcium binding by sarcoplasmic reticulum ATPase. The classic work on binding of calcium to CaATPase is analyzed by an objective non-linear least squares procedure of 74 data points over six pH values. Binding of two calciums to the basic form of the sites occurs with an equilibrium stability constant product of log K1K2 = 13.2. Owing to competition from protons, this value drops in acidic and neutral solutions, becoming, for example, 11.9 at pH 6.8. Binding of the two calciums is so strongly cooperative that its extent is difficult to estimate reliably; there is very little of the one calcium species. Two protons are also bound cooperatively to the calcium sites. In solutions of calcium free protein, at pH less than 7.6 the predominant species holds two protons at the calcium sites, while at greater pH the dominant species bears no protons; there is very little of the intermediate one proton species. The analysis also reveals the likely presence of a small, less than statistical, amount of a ternary complex bearing one calcium and one proton.


Tyrosine-89 is important for enzymatic activity of S. cerevisiae inorganic pyrophosphatase. 7-Chloro-4-nitro-benzofurazan selectively modifies one PPase Tyr residue per subunit and lowers the enzyme activity. Hydrolysis of the modified protein by trypsin and then by chymotrypsin produces the 82-89 peptide which possesses modified Tyr-89. Substrate analog (CaPPi) and the product of the enzyme reaction, MgPi, protect the enzyme against inactivation. Ions of metal-activators (Mg2+, Zn2+) exert no influence on the inactivation rate. On the contrary, the Ca(2+)-inhibitor of the enzyme accelerates the reaction by binding to the high-affinity site, and effectively decreases it when Ca2+ binds to both sites. Mg2+ competes with Ca2+ for one binding site, which is the low affinity site for Mg2+ and the high-affinity site for Ca2+. The Ca2+ saturation of the high-affinity site decreases the pK2 of Tyr-89, probably due to direct coordination between Tyr and Ca2+. The observed properties of Tyr-89 modification enable us to propose that Tyr-89 serves as a proton donor for phosphate releasing during enzymatic hydrolysis of pyrophosphate. The Ca2+ inhibitory effect on the enzyme activity may be due to the existence of a Tyr-89 bond in the Ca2+ pyrophosphatase complex.


[New drugs in endocrine treatment of breast cancer]. Therapeutic access in the treatment of breast cancer with the antiestrogen tamoxifen has been established by world-wide clinical trials since the drug was introduced by Cole et al, in 1971. In recent years, however, a new series of antiestrogens (the derivatives of tamoxifen) such as trioxifene, toremifene and droloxifene have been studied with regard to clinical efficacy as a first-line treatment for postmenopausal patients with breast cancer and occasionally even for patients who previously responded to tamoxifen and then relapsed. Luteinizing hormone releasing hormone (LHRH) agonists are now available for premenopausal patients that will produce a medical castration, when given continuously, by down-regulation of the pituitary LHRH receptors. Four compounds, leuprolide, buserelin, tryptorelin and goserelin have been available for clinical use, but goserelin (Zoladex) is now widely used by long-acting depot preparations, which are given subcutaneously once every 4 weeks. Another series of drugs which inhibit estrogen synthesis in postmenopausal patients and are termed "aromatase inhibitors" have been developed. The pure aromatase inhibitors newly developed include two types of both a steroidal compound (4-hydroxyandrostenedione) and a non-steroidal one which is a tetrahydroimidazopyrimidine derivative (CGS 16949A). This review describes the pharmacological and clinical aspects of these new agents.


Plant fructose-1,6-bisphosphatases: characteristics and properties. In this minireview the properties and characteristics of plant fructose-1,6-bisphosphatases (D-fructose-1,6-bisphosphatase 1-phosphohydrolase, EC 3.1.3.11) are discussed. The properties and characteristics of the chloroplastic and cytoplasmic forms of the enzyme are reviewed. For purposes of comparison some reference is made to fructose-1,6-bisphosphatases from other species.


Modulation of dihydropyridine-sensitive gastric mucosal calcium channels by GM1-ganglioside. 1. A dihydropyridine-sensitive calcium channel complex was solubilized from gastric mucosal cell membranes and purified by affinity chromatography on wheat germ agglutinin. 2. The calcium channel complex labeled with [3H]PN200-110, when reconstituted into phosphatidylcholine vesicles, exhibited active 45Ca2+ uptake into intravesicular space as evidenced by La3+ displacement and osmolarity studies. The channel complex responded in a dose-dependent manner to dihydropyridine calcium antagonist, PN200-110, which at 0.5 microM exerted maximal inhibitory effect of 66% in 45Ca2+ uptake. 3. The uptake of 45Ca2+ into vesicle-reconstituted gastric mucosal calcium channel complex was inhibited by GM1-ganglioside. Maximum inhibitory effect was achieved at 10-15 nM GM1, at which point a 74% decrease in 45Ca2+ uptake occurred. Furthermore, GM1 also inhibited dihydropyridine binding to gastric mucosal membranes, indicating the extracellular orientation of calcium channel domains for GM1. 4. The ability of GM1 to modulate the intracellular calcium levels may be an important feature in gastric mucosal protection by this ganglioside.


Direct evidence for the alterations in protein structure and conformation upon in vitro nonenzymatic glycosylation. The formation of nonenzymatic glycosylation products appears to be a link between chronic hyperglycaemia and long-term diabetic complications. However, little is known concerning the glycation-induced modifications in the structure and conformation of proteins, which possibly underlie their altered functional characteristics. This study conveys a direct evidence for and compares the glucose-induced modifications in the conformation of three proteins with various half-lives: bovine serum albumin, human haemoglobin and bovine tendon collagen. These proteins incubated in vitro with glucose in various media containing optionally EDTA and Fe2+ ions contained up to 4-10 times as much attached glucose as did their relevant controls, and the extent of glycation was the highest in the samples incubated under air or in the absence of EDTA. The fluorescence and ESR data indicate that the Trp in albumin molecule, given albumin glycation-induced structural modifications, became more exposed to water surrounding solution whereas the Trp residues of haemoglobin remained shielded from water; also collagen fluorescence derived from the supposedly newly formed covalent crosslinks is vastly increased, and particularly when collagen was glycated under air or in the presence of Fe2+ ions. Possible mechanisms underlying the increased mobility of selected protein domains and glycation-mediated alterations in protein conformation are considered and discussed.


Position-dependence of retinoic acid receptor-beta gene expression in the chick limb bud. Retinoic acid and 3,4-didehydroretinoic acid are metabolites of vitamin A that can induce duplications and other malformations when locally applied to the anterior margin of the chick limb bud. There is evidence that they may be natural signaling substances in the limb bud. Both compounds are thought to act by binding to ligand-dependent transcription factors that belong to the steroid/thyroid hormone nuclear receptor superfamily. In situ hybridization analyses show that in the mesenchyme of the chick wing bud between embryonic stages 20 and 27, retinoic acid receptor-beta (RAR-beta) transcripts are restricted to the proximal region of the bud and are present at highest levels in the region of the limb bud mesenchyme that contributes to the shoulder. We have performed grafting experiments in order to examine whether RAR-beta gene expression in limb bud mesenchyme cells is cell-autonomous or whether it is dependent upon the cell's position within the limb bud. When tissue from the proximal region of the stage 22 wing bud, which contains high levels of RAR-beta transcripts, was grafted to the distal tip of the bud, RAR-beta transcripts were undetectable in the graft 6 hr later. When tissue from the distal tip of the bud was grafted to a proximal site, most of the grafts exhibited a slight increase in the level of RAR-beta transcripts, which was detectable 6 hr after grafting. However, the levels of RAR-beta transcripts in these grafts never approached those found in the proximal core of the bud. These data indicate that RAR-beta gene expression in the chick wing bud is position-dependent in that it is repressed at the distal tip of the bud and partially activated by grafting distal tissue to a proximal site. However, accumulation of RAR-beta transcripts to high levels appears to be a characteristic of mesenchyme that was initially specified to form proximal structures.


Developmental expression of voltage-dependent calcium currents in identified mouse motoneurons. Previous work has shown that during chick embryonic development, large changes occur in the density of specific, motoneuronal calcium currents just prior to the period of naturally occurring motoneuron cell death. Here we report on calcium currents in mouse motoneurons isolated from embryos at the time of peak cell death and also during a subsequent developmental stage when supernumerary synapses are being eliminated. In mouse motoneurons, the density of high-voltage-activated calcium current increases significantly after the phase of cell death, during the period of synapse elimination.


Fibroblast growth factor receptors contain a conserved HAV region common to cadherins and influenza strain A hemagglutinins: a role in protein-protein interactions? The first extracellular domain of the cadherins has been shown to exhibit extensive sequence homology with the amino termini of the HA1 chains of influenza strain A hemagglutinins. These regions of homology are known to be functionally important in both the cadherins and the hemagglutinins. The homologous regions harbor the tripeptide HAV, which has been identified as being the cadherin cell adhesion recognition sequence. Here we report that members of the rapidly expanding family of fibroblast growth factor receptors also possess HAV-containing regions. These regions are homologous to the HAV-containing regions present within both the hemagglutinins and the cadherins and appear to be involved in regulating the function of the fibroblast growth factor receptors. We speculate that the HAV motif may represent an evolutionarily conserved amino acid sequence that will prove to be functionally important in a wide variety of proteins.


Functional alterations of G-proteins in diabetic rat retina: a possible explanation for the early visual abnormalities in diabetes mellitus. We examined changes in guanosine triphosphate-dependent signal transduction mechanisms in the retina from the early stages of the streptozotocin-diabetic rat, a model for Type 1 (insulin-dependent) diabetes mellitus. Guanosine triphosphate binding, guanosine triphosphatase activity, and binding of (azido) guanosine triphosphate decreased significantly in the retina as early as 2 weeks after the induction of diabetes. The ability of guanosine triphosphate to inhibit forskolin-stimulatable adenyl cyclase was also abolished. These data suggest functional deterioration of G-proteins, especially Gi, in diabetic retina. Further studies using retinal rod outer segments revealed deterioration in light-sensitive, guanosine triphosphate-dependent functions of transducin in diabetic rats. Pertussis toxin-catalysed ADP ribosylation of the alpha subunit of transducin, a heterotrimeric G-protein of rod outer segments, was also reduced in diabetes. No functional effects were seen in purified subunits of transducin subjected to non-enzymatic glycation in vitro. On the other hand, incubation of non-diabetic rod outer segments with (12-0-tetradeconyl) phorbol-13-acetate, a protein kinase C agonist, in the presence of magnesium and adenosine triphosphate resulted in the reduction of guanosine triphosphate-binding and hydrolysis, thus indicating that protein kinase C may be involved in the regulation of these activities. The significance of these observations in the early visual abnormalities associated with diabetes is discussed.


Effect of insulin on human adipose tissue metabolism in situ. Interactions with beta-adrenoceptors. The effects of insulin, and its interactions with catecholamines through beta-adrenoceptors, on human adipose tissue glucose utilization and lipolysis were investigated in vivo. Microdialysis of the extracellular compartment of abdominal subcutaneous adipose tissue was performed in healthy subjects of normal weight, before and during a 2-h hyperinsulinaemic (61 +/- 3 mU/l), euglycaemic clamp. The tissue was perfused with or without the beta-adrenergic agonist isoproterenol (10(-6) mol/l), and the tissue dialysate concentrations of glucose, glycerol (lipolysis index) lactate and pyruvate were determined. During the insulin infusion, glucose in adipose tissue decreased by 20% (p less than 0.001), despite arterial steady-state normoglycaemia. The concentrations of lactate and pyruvate increased gradually to a steady-state plateau of twice the basal level in adipose tissue and arterial blood. Insulin-induced suppression of glycerol (lipolysis index) was, if anything, more marked in adipose tissue than in plasma (65% vs 50% decrease from baseline levels, p less than 0.05). In situ perfusion of adipose tissue with isoproterenol, starting either at the beginning of the study period or at 45 min after initiation of the insulin infusion, resulted in marked and rapid elevations of all the investigated metabolites in the adipose tissue extracellular compartment (p less than 0.05-0.005). It is concluded that insulin action on glucose uptake and lipolysis in human adipose tissue in vivo is counteracted by beta-adrenoceptor stimulation. In contrast, insulin and beta-adrenoceptors have synergistic effects on non-oxidative glucose metabolism in human adipose tissue in situ.


Roles of receptor tyrosine kinases in Drosophila development. Communication between cells is a fundamental component of development and morphogenesis. Identification of the molecules mediating cell-cell communication is crucial for elucidation of the molecular basis of these processes. Receptor tyrosine kinases (RTKs) appear to play a central role in this context by transmitting into cells information dictating their fate. The functions of RTKs in Drosophila are extremely diverse, and include maternal determination of embryonic polarity (torso and torpedo), determination of neuroblast identity (faint little ball), and guidance of tracheal cell migration in the embryo (breathless). During compound eye development, RTKs affect the number of photoreceptor clusters (Ellipse) and the determination of photoreceptor R7 identity (sevenless). The phenotypes of mutations in RTK loci serve as a starting point for understanding processes dictating cell identity at the level of the whole organism. Recently, they have also begun to provide a basis for selection of second-site suppressor mutations, encoding additional elements in their signal transduction pathway. Common themes between the functions, regulation, and signal transduction pathways of Drosophila RTKs are drawn.


Poliovirus induces indoleamine-2,3-dioxygenase and quinolinic acid synthesis in macaque brain. Accumulation of the neurotoxin quinolinic acid within the brain occurs in a broad spectrum of patients with inflammatory neurologic disease and may be of neuropathologic significance. The production of quinolinic acid was postulated to reflect local induction of indoleamine 2,3-dioxygenase by cytokines in reactive cells and inflammatory cell infiltrates within the central nervous system. To test this hypothesis, macaques received an intraspinal injection of poliovirus as a model of localized inflammatory neurologic disease. Seventeen days later, spinal cord indoleamine 2,3-dioxygenase activity and quinolinic acid concentrations in spinal cord and cerebrospinal fluid were both increased in proportion to the degree of inflammatory responses and neurologic damage in the spinal cord, as well as the severity of motor paralysis. The absolute concentrations of quinolinic acid achieved in spinal cord and cerebrospinal fluid exceeded levels reported to kill spinal cord neurons in vitro. Smaller increases in indoleamine 2,3-dioxygenase activity and quinolinic acid concentrations also occurred in parietal cortex, a poliovirus target area. In frontal cortex, which is not a target for poliovirus, indoleamine 2,3-dioxygenase was not affected. A monoclonal antibody to human indoleamine 2,3-dioxygenase was used to visualize indoleamine 2,3-dioxygenase predominantly in grey matter of poliovirus-infected spinal cord, in conjunction with local inflammatory lesions. Macrophage/monocytes in vitro synthesized [13C6]quinolinic acid from [13C6]L-tryptophan, particularly when stimulated by interferon-gamma. Spinal cord slices from poliovirus-inoculated macaques in vitro also converted [13C6]L-tryptophan to [13C6]quinolinic acid. We conclude that local synthesis of quinolinic acid from L-tryptophan within the central nervous system follows the induction of indoleamine-2,3-dioxygenase, particularly within macrophage/microglia. In view of this link between immune stimulation and the synthesis of neurotoxic amounts of quinolinic acid, we propose that attenuation of local inflammation, strategies to reduce the synthesis of neuroactive kynurenine pathway metabolites, or drugs that interfere with the neurotoxicity of quinolinic acid offer new approaches to therapy in inflammatory neurologic disease.


Germinal and somatic activity of the maize element Activator (Ac) in Arabidopsis. We have investigated the germinal and somatic activity of the maize Activator (Ac) element in Arabidopsis with the objective of developing an efficient transposon-based system for gene isolation in that plant. Transposition activity was assayed with a chimeric marker that consists of the cauliflower mosaic virus 35S promoter and a bacterial streptomycin phosphotransferase gene (SPT). Somatic activity was detected in seedlings germinated on plates containing streptomycin as green-resistant sectors against a background of white-sensitive cells. Germinal excisions resulted in fully green seedlings. The transposition frequency was extremely low when a single copy of the transposon was present, but appeared to increase with an increase in Ac copy number. Plants that were selected as variegated produced an increased number of green progeny. The methylation state of the Ac elements in lines with either low or high levels of excision was assessed by restriction analysis. No difference was found between these lines, indicating that the degree of methylation did not contribute to the level of Ac activity. Germinal excision events were analyzed molecularly and shown to carry reinserted transposons in about 50% of the cases. In several instances, streptomycin-resistant siblings carried the same transposed Ac element, indicating that excision had occurred prior to meiosis in the parent. We discuss parameters that need to be considered to optimize the use of Ac as a transposon tag in Arabidopsis.


The mechanism of somatic inhibition of Drosophila P-element pre-mRNA splicing: multiprotein complexes at an exon pseudo-5' splice site control U1 snRNP binding. Somatic inhibition restricts splicing of the Drosophila P-element third intron (IVS3) to the germ line. We have exploited this simple system to provide a model for a mechanism of alternative pre-mRNA splicing. Biochemical complementation experiments revealed that Drosophila somatic extracts inhibited U1 snRNP binding to the 5' splice site. Using sensitive RNase protection and modification-interference assays, we found that U1 snRNP bound to a pseudo-5' splice site in the 5' exon and that multiprotein complexes bound to an adjacent site. Binding of these factors appeared to mediate the inhibitory effect, because mutations in the pseudo-5' splice sites blocked binding and activated splicing in vitro. Likewise, wild-type, but not mutant, 5' exon RNA titrated inhibitory factors away from the pre-mRNA and activated splicing. Thus, we have defined the pseudo-5' splice sites as crucial components of the regulatory element, correlated the inhibitory activity with specific RNA binding factors from Drosophila somatic cells, and provided a mechanistic description of somatic inhibition. Because the inhibitory activity involves general splicing functions such as protein recognition of 5' splice site sequences and changes in the distribution of bound U1 snRNP, our data may also provide insights into how splice sites are selected.


[Studies on angiotensin I converting enzyme (ACE) inhibitory effect of imidapril. (I). Inhibition of various tissue ACEs in vitro]. Imidapril is a newly synthesized non-sulfhydryl-containing angiotensin I converting enzyme (ACE) inhibitor. The present study describes the inhibitory effects of imidapril and its active metabolite 6366A on ACEs from various tissues and compares its effects to those of captopril, enalapril and enalaprilat in vitro. 6366A inhibited swine renal and human serum ACEs with an inhibition constant (Ki) of 0.067 nM and 0.04 nM, respectively. These values were 3 to 18 times more potent than those of the other inhibitors. The kinetic study showed that 6366A exerted competitive type inhibition. The ACE inhibition (IC50 values) of 6366A, enalaprilat and the structurally related compounds (6366DM and 6366PY) were compared in homogenates of lung, aorta, heart, brain and kidney from spontaneously hypertensive rats (SHRs) and Wistar Kyoto rats (WKYs). The inhibitory effects of 6366A on all tissue ACEs from SHRs and WKYs were the most potent among these compounds. And the inhibitory potencies of these compounds were correlated with their chemical structure. The present results suggest that 6366A may show a strong inhibitory effect on ACEs from several tissues and species due to its chemical characteristics.


[Affinity of Z-105 to the 1,4-dihydropyridine type calcium channel and several other receptor bindings in the central nervous system]. The affinity of a 1,4-dihydropyridine (DHP) type calcium channel blocker, NZ-105 ((+/-)-2-[benzyl (phenyl) amino] ethyl 1,4-dihydro-2, 6-dimethyl-5- (5,5-dimethyl-2-oxo-1,3,2-dioxaphosphorinan- 2-yl)-4-(3-nitrophenyl)-3-pyridinecarboxylate hydrochloride ethanol), on the DHP-binding site in the central nervous system and various receptor sites were compared with nicardipine and diltiazem by the use of a receptor binding assay technique. NZ-105 exhibited a displacement effect against [3H]nimodipine in the rat brain DHP-binding site with a potency similar to that of nicardipine. Nicardipine also inhibited the specific binding of several other [3H]-labelled ligands to their receptor such as adrenergic alpha 1, alpha 2, beta, dopamine D1, D2, opioid mu, delta, and kappa-type receptors. Diltiazem also showed a similar inhibitory property. However, NZ-105 showed only weak inhibition against the binding to these receptors. These results suggest that Z-105 has strong affinity to the DHP-binding site in voltage-dependent calcium channels with higher specificity.


Mucoid cytoplasmic inclusions in urothelial carcinomas. To date, mucoid cytoplasmic inclusions in urothelial carcinomas have rarely been noted. However, we were impressed by the fact that these corpuscles are readily detectable in numerous urothelial neoplasms. Therefore, a histologic analysis of 100 cases of urothelial carcinomas was performed. Overall, 37 cases revealed periodic acid-Schiff-positive cytoplasmic inclusions. These were observed in 14% of grade 1, 49% of grade 2, and 63% of grade 3 carcinomas. The inclusions were histochemically, immunohistochemically, and ultrastructurally identified as cytoplasmic deposits of mucoid materials. Two types of deposits, condensed and noncondensed, could be distinguished. The demonstration of mucoid deposits in otherwise poorly differentiated metastatic carcinomas may be of some differential diagnostic importance insofar as urothelial carcinoma has to be considered as the possible primary tumor.


Parathyroid hormone-like peptide in pancreatic endocrine carcinoma and adenocarcinoma associated with hypercalcemia. Parathyroid hormone-like peptide (PLP) is produced by a number of tumors commonly associated with hypercalcemia as well as by nontumorous tissue, including some endocrine organs. We applied immunohistochemistry using the avidin-biotin-peroxidase technique to localize PLP in formalin-fixed, paraffin-embedded tissues of two human pancreatic carcinomas associated with hypercalcemia and normal blood parathyroid hormone levels. One tumor was endocrine and one was exocrine in differentiation. There was no evidence of bone metastasis in either case. We documented normalization of serum calcium level after removal of the pancreatic endocrine tumor. These observations support the suggestion that PLP production by pancreatic carcinoma may play a role in the development of hypercalcemia. However, the presence of PLP in tumors not associated with hypercalcemia indicates that other factors in addition to PLP are necessary for the manifestation of hypercalcemia. Hypercalcemia associated with exocrine pancreatic tumor has rarely been reported. Our exocrine pancreatic tumor appears to be the first reported case in which immunohistochemistry localized PLP. Since PLP has been localized to cells of exocrine ducts and ductules of normal pancreas, our results provide insight into the cell of origin of this tumor type.


Sinonasal small cell neoplasm developing after radiation therapy for retinoblastoma: an immunohistologic, ultrastructural, and cytogenetic study. Patients with retinoblastoma have an increased risk of developing second primary tumors. Only a few examples of sinonasal small cell neoplasms developing after radiation therapy for retinoblastoma have been reported. We report one such case that developed 18 years after treatment for retinoblastoma. Histologic examination revealed a small, blue, round cell tumor without rosettes or cytoplasmic glycogen. Immunohistochemically, the tumor cells were positive for neuron-specific enolase, synaptophysin, and S-100 protein, but negative for epithelial and mesenchymal markers, suggesting that this was a primitive neuroectodermal tumor. Cytogenetic studies of this tumor failed to reveal the chromosome 13 abnormality typical of retinoblastoma and the t(11:22) translocation typical of the group of peripheral neuroepitheliomas.


Quantitation of cytomegalovirus DNA in lung tissue of bone marrow transplant recipients. Five bone marrow transplant recipients who died of respiratory failure were retrospectively analyzed with polymerase chain reaction (PCR) assay for pulmonary cytomegalovirus (CMV) infection. Two patients had CMV interstitial pneumonitis according to the virus isolation and the histologic and immunofluorescent examinations of the lungs, while the other three patients had non-CMV diseases (ie, idiopathic interstitial pneumonitis, pulmonary aspergillosis, or Streptococcus mitis septicemia). Cytomegalovirus DNA was amplified from the postmortem lung tissue with PCR. The PCR assay showed apparent PCR signals specific to CMV DNA in the two patients with CMV pneumonitis. In contrast, CMV DNA was hardly detectable or undetectable in the three patients without CMV disease. With quantitative PCR assay the initial CMV copy number in the lung tissue of the two patients with CMV pneumonitis was more than 10(4) copies/micrograms DNA and was over 1,000-fold more than that of the three patients without CMV pneumonitis. These results show that quantitative PCR assay could be useful as a diagnostic measure for pulmonary CMV infection.


T-cell receptor gamma/delta: comparison of gene configurations and function between humans and chimpanzees. The human and chimpanzee T-cell receptor gamma-delta (TCR gamma delta) bearing cells represent a minor subset (3-8%) of T lymphocytes. In the periphery, the TCR gamma delta population has a restricted combinatorial repertoire. The TCRD-V1 and -V2 gene products are expressed in a mutually exclusive fashion, whereas, the TCRD-V2 and the TCRG-V9 encoded proteins show, in general, a coordinated expression. Restriction fragment length polymorphism analysis showed conservation of the restriction sites that identify the TCRG-V9 and TCRD-V2 rearrangements. The human TCRG-V9 locus has two alleles, TCRG-V9A1 and TCRG-V9A2 differing at codon position 31. The chimpanzee TCRG-V9 gene product differs from the products of the human TCRG-V9A1 and TCRG-V9A2 allele by two and three amino acid replacements, respectively. The human and the chimpanzee TCRG-V9-TCRD-V2 lymphocytes show a similar specific proliferative and cytolytic response to human Daudi Burkitt's lymphoma cells. Therefore, the amino acid replacements found in the chimpanzee TCRG-V9 gene product do not change the superantigen specificity across this species barrier.


Tcrb-V3+ T-cell deletion and a mouse mammary tumor provirus, Mtv-27. Genes encoding superantigens which delete Tcrb-V3+ T cells co-segregate with mouse mammary tumor proviruses (Mtv), Mtv-1, Mtv-3, Mtv-6, Mtv-13, and Mtv-44. We have examined percentages of Tcrb-V3+ T cells and Mtv integrations in [(B10 x NZB)F1 x B10.BR] mice, and show that Mtv-27 as well as Mtv-3 from NZB mice co-segregate with genes encoding deletion ligands for Tcrb-V3+ T cells without recombination.


Beneficial health effects of modest weight loss. The medical effects of modest weight reduction (approximately 10% or less) in patients with obesity-associated medical complications were reviewed. The National Library of Medicine MEDLINE database and the Derwent RINGDOC database were searched to identify English language studies that examined the effects of weight loss in obese patients with serious medical complications commonly associated with obesity (non-insulin dependent diabetes mellitus (NIDDM or type II), hypertension, hyperlipidemia, hypercholesterolemia, and cardiovascular disease). Studies in which patients experienced approximately 10% or less weight reduction were selected for review. Studies indicated that, for obese patients with NIDDM, hypertension or hyperlipidemia, modest weight reduction appeared to improve glycemic control, reduce blood pressure, and reduce cholesterol levels, respectively. Modest weight reduction also appeared to increase longevity in obese individuals. In conclusion, a large proportion of obese individuals with NIDDM, hypertension, and hyperlipidemia experienced positive health benefits with modest weight loss. For patients who are unable to attain and maintain substantial weight reduction, modest weight loss should be recommended; even a small amount of weight loss appears to benefit a substantial subset of obese patients.


Waist/hip ratio, body mass index and premature cardiovascular disease mortality in US Army veterans during a twenty-three year follow-up study. A retrospective longitudinal analysis of fat distribution and cause-specific mortality was performed on data from 105,062 men discharged from the United States Army in 1946-47. Baseline height, weight, waist and hip girth, and 23-year follow-up mortality data were available for 84,910 white men. Proportional hazard survival analysis was performed by 5-year age group for waist/hip ratio (WHR) and for body mass index (BMI) in prediction of time to death from ischaemic heart disease (IHD) and stroke. Relative risk of IHD fatality per standard deviation (s.d.) of WHR ranged from 1.11 to 1.17, the higher values appearing in younger age groups. Relative risk due to BMI was not significant in the group aged 16-20 years at time of discharge from service, but ranged from 1.22 to 1.25 per s.d. among the 21-30 year olds. WHR was predictive of cerebrovascular disease mortality among 16-25 year olds, carrying a relative risk of 1.24 to 1.35 per s.d. BMI was not predictive of cerebrovascular disease mortality in any age group. Multivariate models indicated that WHR and BMI were related to subsequent IHD independently of each other in most age groups. WHR and BMI both contribute to risk of premature IHD mortality and WHR to risk of cerebrovascular disease mortality in an initially relatively healthy population of young men, although the effects are not equivalent in all age groups.


Effects of increasing brain GABA on the meal patterns of genetically obese vs. lean Zucker rats. To explore recent suggestions that genetically obese Zucker rats show less anorexia when brain gamma-aminobutyric acid (GABA) is elevated, obese vs. lean littermates received 100, 50 and 0 micrograms of the GABA-transaminase inhibitor, ethanolamine-O-sulfate (EOS), intra-cisternally in a longitudinal design where their feeding patterns were monitored 24 h daily. Obese rats were refractory to EOS-induced anorexia as evidenced by less suppression of daily food intake and fewer alterations to both meal size and meal frequency, particularly in the night. This effect was not due to an inability of EOS to increase brain GABA since equivalent, specific dose-dependent increments were seen in the brains of separate obese vs. lean rats after analysis of endogenous GABA and seven other amino acids. An unexpected finding was elevated levels of brain taurine for obese rats regardless of EOS dosage, implying a hitherto unknown neurochemical trait whose potential significance is unclear. The primary data obtained provide further support for recent hypotheses that obese Zucker rats possess altered brain GABAergic mechanisms that may serve as one contributor to their over-eating.


Laboratory diagnosis of congenital human cytomegalovirus infection using polymerase chain reaction and shell vial culture. Congenital HCMV infection was diagnosed at the 22th week of gestation. The infection was suspected because HCMV IgM was detected in a serum sample obtained from the woman's husband. HCMV infection was detected in the amniotic fluid by polymerase chain reaction, shell vial culture (immunoperoxidase assay) and conventional virus isolation. Serologic testing in paired sera of the woman and in umbilical cord blood for specific IgM and IgA remained negative. As serological data (preconceptional HCMV serostatus) were incomplete, a clear differentiation between primary and secondary infection could not be achieved; consequently, risk quantification could not be determined. Viruria was detected in the offspring during the 1st week post partum. No clinical signs of cytomegalic inclusion disease were diagnosed up to six weeks post partum. Our case report indicates that for pregnancy surveillance, serologic testing for HCMV antibody should also be performed in the spouse.


In situ hybridization study of obesity-associated alteration in growth hormone mRNA levels. In order to investigate whether the impaired GH secretion associated with obesity is due to a pituitary disorder we studied GH mRNA levels by in situ hybridization in genetically obese and lean Zucker rats. The levels of GH mRNA were at least two fold lower in obese rats in comparison to that in lean controls as quantified by both the scanning of autoradiographs of tissue sections and Northern blot analysis. Quantification of somatotrophs revealed no significant difference in their number between lean and obese rat pituitaries. It is therefore likely that the attenuated GH mRNA levels in genetically obese Zucker rats are due to a decrease in GH transcripts per somatotroph rather than a result of a pituitary defect involving a preferential decrease in somatotroph population.


Body fat distribution in pubertal girls quantified by magnetic resonance imaging. We examined body fat distribution in relation to anthropometrically derived variables in 24 girls in early and late stages of puberty. The amounts of subcutaneous and intra-abdominal body fat were derived from transverse slices at the levels of the waist, hip and trochanter using magnetic resonance imaging (MRI), and were compared to the related circumferences, the circumference ratios and the trunk-to-extremity skinfold ratios. Waist, hip and trochanter circumferences were highly correlated to the respective related MRI total fat surface area both in early and late pubertal girls (r = 0.79-0.97), while waist circumference, and waist-hip, waist-thigh or skinfold ratios were not significantly correlated to intra-abdominal fat areas. Late pubertal girls (n = 11) were significantly taller, heavier and fatter compared to early pubertal girls (n = 13), yet their anthropometric waist-to-hip or waist-to-trochanter circumference ratios were significantly lower. The intra-abdominal fat area measured in a transverse MRI section at the level of the waist was 24.1 +/- 4.1 cm2 in early pubertal girls and 25.7 +/- 4.1 cm2 in late pubertal girls (mean +/- s.e.m.). As compared to early pubertal girls, the MRI derived amount of subcutaneous fat in late pubertal girls was significantly higher at the trochanter level (142.1 +/- 12.7 vs. 201.3 +/- 26.3 cm2; P less than 0.05). We conclude that circumferences at the trunk are good measures for the related amounts of fat in pubertal girls. In contrast conventional anthropometric measurements, such as trunk-to-extremity skinfold ratio or waist-to-hip circumference ratio, cannot be used to predict the amount of intra-abdominal fat in pubertal girls.(ABSTRACT TRUNCATED AT 250 WORDS)


Intracarotid glucose induced norepinephrine response and the development of diet induced obesity. Intracarotid glucose infusions cause increased plasma norepinephrine (NE) levels in some rats. This is used as an index of sympathetic activation. Similar increases in plasma NE levels are produced by intravenous glucose injections and these levels correlate positively with the amount of weight gained by adult rats when they are subsequently fed a diet enriched in calories, sucrose and fat (condensed milk (CM) diet) for three months. Thus, rats prone to develop diet induced obesity (DIO) on the CM diet have greater intravenous glucose induced NE responses than those which are diet resistant (DR). To test the hypothesis that this relationship is mediated by the brain, 17 chow fed, adult male Sprague-Dawley rats were infused for 60 min with intracarotid glucose at 4 mg/kg/min and blood samples were obtained for plasma catecholamines, insulin and glucose. They were then placed on the CM diet for three months. After three months on the CM diet, there was a wide variability in body weight gain and the weights of retroperitoneal fat pads, an indirect measure of carcass adiposity. For all 17 rats, there was a significant correlation between both body weight gain (r = 0.685, P = 0.002) and retroperitoneal fat pad weights (r = 0.590, P = 0.013) with the levels of plasma NE reached 45-60 min into the preceding intracarotid glucose infusions. For the six lowest and six highest glucose induced NE responders, the correlation between NE response and body weight gain on CM diet was r = 0.944 (P = 0.0001). Plasma epinephrine, insulin and glucose levels were unchanged during such infusions.(ABSTRACT TRUNCATED AT 250 WORDS)


Reduction of plasma insulin levels does not restore integrated concentration of growth hormone to normal in obese children. Obesity in childhood is characterized by subnormal integrated concentrations of growth hormone (IC-GH) and elevated integrated concentrations of insulin (IC-I). We tested whether a reduction of IC-I induced by a low calorie diet would lead to a rise of IC-GH into the normal range for age. Six obese children (body mass index (BMI) 39.1 +/- 9.2 kg/m2) underwent integrated concentration (IC) studies by continuous withdrawal before and again 5-8 weeks after being on a low calorie diet. In response to the diet BMI was lower 34.7 +/- 9.4 kg/m2 (P less than 0.003), and IC-I was considerably reduced, 479 +/- 255 pM initially vs. 109 +/- 109 pM on the diet, P less than 0.0008. IC-GH increased modestly from 1.6 +/- 0.6 micrograms/l initially to 2.4 +/- 0.6 micrograms/l, P less than 0.01 on the diet. None of the patients had repeat IC-GH levels which were above the lower limit of normal for lean children of normal stature (3.2 micrograms/l). Single sample insulin-like growth factor 1 (IGF-1) levels were unchanged: 40.9 +/- 23.1 nM initially vs. 49.7 +/- 25.7 nM (314.6 +/- 197.7 vs. 382.5 +/- 217.0 ng/ml, n.s.). Thus reduction of high insulin concentrations during 5-8 weeks of a low calorie diet has only a small effect on IC-GH in obese children. Factors other than circulating insulin levels are likely to play the major role in mediating the reduced levels of GH observed in obesity.


Swedish obese subjects (SOS). Recruitment for an intervention study and a selected description of the obese state. SOS (Swedish obese subjects) is an on-going intervention trial designed to determine whether the mortality and morbidity rates among obese individuals who lose weight by surgical means (gastric banding, vertical banded gastroplasty and gastric by-pass) differ from the rates associated with conventional treatment. For this purpose, the study is recruiting a sample of obese men and women who constitute a registry of potential subjects from which the participants are drawn. Eligibility criteria for participation in the registry were: age at application 37-57 years and BMI greater than or equal to 34 kg/m2 for men and greater than or equal to 38 kg/m2 for women. Before receiving a health examination, all patients complete extensive questionnaires on current and past health status, utilization of medical care and medications, socio-economic status, psychological profiles, dietary habits, physical activity, weight history, and familial disposition to obesity. Each surgical case is matched to its optimal control in the registry, to ensure that the two groups do not differ systematically with respect to any of 18 matching variables that may affect prognosis. The first 1006 subjects included in the registry have been studied with respect to morbidity and compared with on-going population studies of men and women in Göteborg, Sweden. The relative risks of prevalent disease and symptoms associated with obesity in 50-year-old males and females respectively were 4.3 and 4.7 (dyspnoea), 14.7 and 11.8 (angina), 6.3 (myocardial infarction, males only), 2.1 and 4.5 (hypertension), 5.2 and 6.6 (diabetes), 4.6 and 26.1 (claudication) and 1.7 and 1.8 (gall bladder disease). Correspondingly, obese males and females display elevations of systolic and diastolic blood pressure, fasting glucose, insulin, triglyceride, and uric acid levels. However, total cholesterol was not increased in obese males and was in fact significantly lower in obese compared with reference women. HDL-cholesterol was lower in obese than reference men (data were not available in reference women). The rate of taking sick pensions was over twice as high in SOS obese patients than in population controls. Finally, comparison of measurements with self-reported prevalence estimates revealed a considerable amount of previously undiagnosed hypertension and diabetes in the obese subjects. These data suggest that the excess health risks associated with obesity may not be fully appreciated.


A comment on the 1990 recommendations of the ICRP. ICRP Publication 60 recommends a change relating to the numerical assessment of radiation quality in radiation protection. The quality factor, Q, is to be replaced by "radiation weighting factors," WR, and the quantity "effective dose equivalent" is to be supplanted by "effective dose." Reasons are given why it is virtually impossible to measure this quantity and why it appears unavoidable that practical measurements will continue to be based on the current system. No sensible justification was provided for the proposed change, which is likely to cause confusion.


Leaching of uranium from glass and ceramic foodware and decorative items. Beginning as early as the first century A. D. and continuing until at least the 1970s, uranium was used as a coloring agent in glass and in ceramic glazes. The leaching of uranium from such items is of interest as some were designed for food storage or serving. Thirty-three glass items and two ceramic items were leached sequentially with deionized water, dilute acetic acid, and 1 M nitric acid to assess realistic and worst-case scenario leaching by foods and beverages. The maximum quantity of uranium leached from the uranium-bearing glasses was about 30 micrograms L-1, while that from the ceramic-glazed items was about 300,000 micrograms L-1.


[Gracilis muscle repair in perineal and genital soft tissue problems]. The transposition of the gracilis muscle is shown as a reliable modality of treatment in chronic soft tissue problems and fistulas of the anogenital region. A short summary of the specific anatomy is given. The clinical application is illustrated with 2 case reports.


Variations of response of cattle to experimentally induced viral papillomatosis. The common bovine papilloma virus type 1 has been widely used to stimulate basic research on papilloma viruses involved in some cancers of mankind. The usually benign neoplasms of cattle caused by bovine papilloma viruses are frequent clinical problems for veterinarians. Approximately 240 experimentally induced cutaneous fibropapillomas on 8 susceptible calves had a uniform appearance of initial growth. Their size and duration ranged from about 2 months to nearly 3 years but were similar for the multiple papillomas of each calf. Sequential biopsies were done to examine the histologic changes and existence of viral antigen. Veterinarians in practice may encounter the common fibropapillomas caused by bovine papilloma virus 1 and 2 as well as papillomas with no fibromatous element. These types may develop on teats of cows in some herds. Interdigital papillomas cause a problem in some dairy herds and a virus suspected but not yet found. Prophylactic vaccination with a formalin-killed vaccine will protect against infection with bovine papilloma virus 1 and 2.


Feeding value of frost-damaged soybeans for lambs. Three experiments were conducted to determine the relative feeding value of frost-damaged soybeans (FDS) for ruminants. Frost-damaged soybean ether-extract content was variable and differed (P less than .05) from mature soybeans. Isonitrogenous supplementation of corn silage diets with soybean meal (SBM), SBM+soybean oil (SBO), mature raw soybeans (MSB), and FDS was compared in sheep. Acid detergent fiber and apparent N digestion were lower (P less than .001) for supplements containing oil. Nitrogen retention was reduced (P less than .07) only for raw soybean supplements. Ruminal NH3 N and branched-chain VFA concentrations differed (P less than .01) between SBM and supplements containing oil. Maximum tolerable inclusion level of FDS in corn silage diets was tested in wethers using diets containing 0, 7, 14, or 21% FDS. Dry matter and ADF digestibility declined linearly (P less than .01) with increasing dietary FDS. Ether extract digestibility was unchanged due to treatment, but GE digestibility decreased quadratically (P less than .01). The most pronounced decline in GE digestibility occurred when FDS increased from 14 to 21% of the diet. The effects of FDS on corn silage utilization were similar to MSB effects. Oil content and antinutritional factors contributed to detrimental effects. Frost-damaged soybeans should not exceed 14% of corn silage diets fed to growing wethers.


Effects of protein and energy supplementation of wheat straw-based diets on site of nutrient digestion and nitrogen metabolism of lambs. A 4 x 4 Latin square metabolism trial with 2 x 2 factorial arrangement of treatments was conducted with lambs to determine effects of energy and CP supplementation of wheat straw-based (WS) diets on apparent N digestion, retention, and flow to the abomasum. Four wether lambs (average weight, 32 kg) fitted with ruminal and abomasal cannulas were fed 70 vs 42% WS (remainder of the diet was concentrate) and 9.5 vs 12.5% CP. Ruminal and total tract DM and OM digestion was 41 and 33% greater (P less than .03) for high-energy than for low-energy diets. Apparent N digestibility was greater (P less than .05) for 12.5% CP than for 9.5% CP diets (69.2 vs 62.0%, respectively) and also greater (P less than .03) for high-energy than for low-energy diets (67.4 vs 63.7%, respectively). High-energy diets resulted in a 23% greater (P less than .03) N retention (percentage of N intake) than did low-energy diets; 12.5% CP diets resulted in a 9% greater N retention (P less than .10) than did 9.5% CP diets. Nitrogen retention (percentage of N digested) was 15% higher (P less than .03) for high-energy than for low-energy diets. Protein level had no effect (P greater than .10). Nitrogen retention (grams/day) was 5.65, 6.97, 5.28, and 7.43 for low-energy, high-energy, low-protein, and high-protein diets, respectively; there were responses to energy level (P less than .03) and protein level (P less than .05). Total N flow (grams/day) to the abomasum did not differ (P greater than .10) due to treatment.(ABSTRACT TRUNCATED AT 250 WORDS)


Differentiation between thermophilic Campylobacter species by species-specific antibodies. The four species of thermophilic campylobacters, Campylobacter jejuni, C. coli, C. upsaliensis and C. lari, are difficult to distinguish from each other because of their lack of reactivity in many conventional biochemical and physiological tests. Those tests which do discriminate sometimes give discordant results. Species-specific antibody preparations (APs), capable of discriminating between the thermophilic campylobacter species by dot-ELISA, were raised by inoculation of mice with partially purified membrane protein. The APs produced were absorbed with cells of cross-reactive species and tested by dot-ELISA against reference and natural strains, the identities of which were confirmed by DNA/DNA hybridization. The results showed that such APs could be useful as an alternative to DNA/DNA hybridization for rapid species identification, for example in epidemiological surveys. Western blotting experiments with the APs showed that the specificity of the antibodies was not due to a single antigen.


Random diffusion can account for topA-dependent suppression of partition defects in low-copy-number plasmids. The maintenance of partition-defective (Par-) mini-P1 and mini-F plasmids was studied in topA strains of Escherichia coli, which are defective in topoisomerase I activity. The partition defects were substantially but not completely suppressed in broth-grown cultures. This suppression was not due to a large increase in copy number. However, the absolute number of copies of Par- mini-P1 plasmids per average dividing cell is sufficiently high to account for the modest stability observed if a random distribution of the copies to daughter cells is assumed. The similar number of Par- plasmid copies in wild-type cells are distributed in a considerably worse-than-random fashion. Thus, it is unnecessary to propose, as was suggested previously, that an active, par-independent pathway operates in topA strains to ensure proper segregation of the plasmids to daughter cells. Rather, it seems likely that the lack of topoisomerase I activity aids the random distribution of the partition-defective plasmids, perhaps by facilitating their separation after replication. The results of studies carried out at reduced growth rates were consistent with this view; when topA cells containing Par- mini-P1 plasmids were cultured in minimal medium, in which the copy number of the plasmids per average cell is sharply reduced, very little suppression of the partition defect was observed.


Reevaluation, using intact cells, of the exclusion limit and role of porin OprF in Pseudomonas aeruginosa outer membrane permeability. Earlier studies that used model membrane reconstitution methods have come to different conclusions regarding the exclusion limit of the outer membrane of Pseudomonas aeruginosa and whether OprF is the major channel-forming protein in the outer membrane. In this study, a 6.2-kbp SalI fragment, encoding only two cytoplasmic enzymes, alpha-galactosidase and sucrose hydrolase, and the inner membrane raffinose permease, was cloned behind the m-toluate-inducible tol promoter of vector pNM185 to create plasmid pFB71. P. aeruginosa strains harboring pFB71, when grown with inducer, produced both enzymes encoded by the insert and had acquired the ability to grow on the disaccharide melibiose and the trisaccharide raffinose. The rate of growth was dependent on the concentration and size of the saccharide and was decreased three- to fivefold by the absence of OprF, as examined by measuring the growth on melibiose and raffinose of an isogenic OprF-deficient omega insertion derivative, H636(pFB71). At high concentrations, di-, tri-, and tetrasaccharides could pass across the outer membrane to plasmolyze P. aeruginosa, as measured by light scattering and confirmed by electron microscopy. The initial rate kinetics of light-scattering changes were dependent on the size of the saccharide being used. Furthermore, the rates of change in light scattering due to raffinose and stachyose uptake across the outer membrane for strain H636 were fivefold or more lower than for its OprF-sufficient parent H103. These data are consistent with model membrane studies showing that OprF is the most predominant porin for compounds larger than disaccharides in P. aeruginosa and suggest that the exclusion limit for this porin and the outer membrane is greater than the size of a tetrasaccharide. In addition, these data confirmed the existence of other porins with a predominant function in monosaccharide uptake and a more minor function in the uptake of larger saccharides.


Cloning, sequence analysis, and expression in Escherichia coli of a streptococcal plasmin receptor. Plasmin(ogen) receptors are expressed by many gram-positive and gram-negative bacteria. We previously isolated a plasmin receptor from a pathogenic group A streptococcal strain (C. C. Broder, R. Lottenberg, G. O. von Mering, K. H. Johnston, and M. D. P. Boyle, J. Biol. Chem. 266:4922-4928, 1991). The gene encoding this plasmin receptor, plr, was isolated from a lambda gt11 library of chromosomal DNA from group A streptococcal strain 64/14 by screening plaques with antibodies raised against the purified streptococcal plasmin receptor protein. The gene was subcloned by using a low-copy-number plasmid and stably expressed in Escherichia coli, resulting in the production of an immunoreactive and functional receptor protein. The DNA sequence of the gene contained an open reading frame encoding 335 amino acids with a predicted molecular weight of 35,787. Upstream of the open reading frame, putative promoter and ribosomal binding site sequences were identified. The experimentally derived amino acid sequences of the N terminus and three cyanogen bromide fragments of the purified streptococcal plasmin receptor protein corresponded to the predicted sequence encoded by plr. The deduced amino acid sequence for the plasmin receptor protein revealed significant similarity (39 to 54% identical amino acid residues) to glyceraldehyde 3-phosphate dehydrogenases.


NAD(P)H-dependent chromium (VI) reductase of Pseudomonas ambigua G-1: a Cr(V) intermediate is formed during the reduction of Cr(VI) to Cr(III). An NAD(P)H-dependent Cr(VI) reductase (molecular weight = 65,000) was purified from a Cr(VI)-resistant bacterium, Pseudomonas ambigua G-1. Stoichiometric analysis of the enzymatic reaction showed that the enzyme catalyzed the reduction of 1 mol of Cr(VI) to Cr(III) while consuming 3 mol of NADH as an electron donor. Chromium(VI) was reduced to Cr(V) by one equivalent NADH molecule in the absence of the enzyme. Electron spin resonance analysis showed that Cr(V) species (g = 1.979) was formed during the enzymatic reduction. The amount of Cr(V) species formed was about 10 times larger than that of the nonezymatic reduction. These findings show that the Cr(VI) reductase reduced Cr(VI) to Cr(III) with at least two reaction steps via Cr(V) as an intermediate.


Evidence that recBC-dependent degradation of duplex DNA in Escherichia coli recD mutants involves DNA unwinding. Infection of Escherichia coli with phage T4 gene 2am was used to transport 3H-labeled linear duplex DNA into cells to follow its degradation in relation to the cellular genotype. In wild-type cells, 49% of the DNA was made acid soluble within 60 min; in recB or recC cells, only about 5% of the DNA was made acid soluble. Remarkably, in recD cells about 25% of the DNA was rendered acid soluble. The DNA degradation in recD cells depended on intact recB and recC genes. The degradation in recD cells was largely decreased by mutations in recJ (which eliminates the 5' single-strand-specific exonuclease coded by this gene) or xonA (which abolishes the 3' single-strand-specific exonuclease I). In a recD recJ xonA triple mutant, the degradation of linear duplex DNA was roughly at the level of a recB mutant. Results similar to those with the set of recD strains were also obtained with a recC++ mutant (in which the RecD protein is intact but does not function) and its recJ, xonA, and recJ xonA derivatives. The observations provide evidence for a recBC-dependent DNA-unwinding activity that renders unwound DNA susceptible to exonucleolytic degradation. It is proposed that the DNA-unwinding activity causes the efficient recombination, DNA repair, and SOS induction (after application of nalidixic acid) in recD mutants. The RecBC helicase indirectly detected here may have a central function in Chi-dependent recombination and in the recombinational repair of double-strand breaks by the RecBCD pathway.


DNA binding specificity and sequence of Xanthomonas campestris catabolite gene activator protein-like protein. The Xanthomonas campestris catabolite gene activator protein-like protein (CLP) can substitute for the Escherichia coli catabolite gene activator protein (CAP) in transcription activation at the lac promoter (V. de Crecy-Lagard, P. Glaser, P. Lejeune, O. Sismeiro, C. Barber, M. Daniels, and A. Danchin, J. Bacteriol. 172:5877-5883, 1990). We show that CLP has the same DNA binding specificity as CAP at positions 5, 6, and 7 of the DNA half site. In addition, we show that the amino acids at positions 1 and 2 of the recognition helix of CLP are identical to the amino acids at positions 1 and 2 of the recognition helix of CAP:i.e., Arg at position 1 and Glu at position 2.


Mutations causing aminotriazole resistance and temperature sensitivity reside in gyrB, which encodes the B subunit of DNA gyrase. Certain mutations in gyrA and gyrB, the genes encoding the two subunits of DNA gyrase, are known to influence expression of the his operon (K. E. Rudd and R. Menzel, Proc. Natl. Acad. Sci. USA 84:517-521, 1987). Such mutations lead to a decrease in tRNA(His) levels and consequently to an attenuator-dependent increase in his operon expression. This effect presumably is due to the dependence of the hisR promoter (hisR encodes tRNA(His) on supercoiling for maximal activity. We used a relaxed (Rel-) strain of Escherichia coli to isolate gyrB mutants by selecting for resistance to the histidine antimetabolite 3-amino-1,2,4-triazole and then screening for temperature-sensitive growth on rich medium. Rel- mutants, which generally have lower basal levels of ppGpp (a positive regulator of his operon transcription), are more sensitive than wild-type E. coli to aminotriazole. The chance of isolating spoT mutants, which can be selected with a similar procedure, was decreased by selecting in the presence of a multicopy plasmid that carries the wild-type spoT gene. Under these conditions, gyrB mutants were isolated preferentially. This scheme selects for loss of function of DNA gyrase, rather than for its alteration due to resistance to specific gyrase inhibitors, and thus a greater variety of gyrase mutations might be obtainable.


Retroviral integrase functions as a multimer and can turn over catalytically. A number of studies have demonstrated that the retroviral protein integrase (IN) alone is sufficient to carry out two discrete steps required for retroviral integration: the endonucleolytic processing of viral DNA ends and the cleavage and joining of host DNA to the processed viral DNA termini. Little is known about the biochemical and biophysical mechanisms involved in these reactions. Here, we employ in vitro assays of Rous sarcoma virus IN to demonstrate for the first time that IN is capable of multiple turnover in both the processing and joining reactions. The turnover number calculated for the processing reaction is 0.26 cleavages/min/mol of IN. Our steady state kinetic studies indicate that both the processing and joining activities require a multimeric form of IN. Ultracentrifugation analyses reveal a substrate-independent reversible equilibrium among the monomeric, dimeric, and tetrameric forms of this protein. From these results we conclude that the minimal functional unit for both the processing and joining of each viral DNA end is an IN dimer.


Members of the Gq alpha subunit gene family activate phospholipase C beta isozymes. The relative specificities of members of the G alpha q family of GTP-binding proteins were tested for their ability to activate different phosphoinositide-specific phospholipase C (PI-PLC) beta isozymes. Cos-7 cells were transfected with cDNA corresponding to G alpha q, G alpha 11, G alpha 14, and G alpha 16. Most of the recombinant protein was bound to the cell membrane and these membranes were washed to elute endogenous PI-PLC activity. The membrane preparation was reconstituted with purified preparations of the PI-PLC beta isozymes and guanosine 5'-O-thiotriphosphate (GTP gamma S)-stimulated enzyme activity was measured. All four proteins of the G alpha q family were found to stimulate PI-PLC beta 1, with G alpha q and G alpha 11 being most efficient. On the other hand, G alpha 16 was found to most effectively activate PI-PLC beta 2, while G alpha q, G alpha 11, and G alpha 14 showed less stimulation. Specific anti- G alpha 16 antibody blocked the stimulation of both PI-PLC beta 1 and PI-PLC beta 2 in the enriched membrane fraction. We conclude that there is specificity in the interaction of different members of the Gq family with different PI-PLC beta effectors. This specificity may be important in generating tissue- or receptor-specific responses in vivo.