Gamma/delta T cell antigen receptors expressed on tumor-infiltrating lymphocytes from patients with solid tumors.

The expression of gamma/delta T cell antigen receptors (TcR) in T cell lines or clones derived from tumor-infiltrating lymphocytes (TIL) from patients with solid tumors was investigated. gamma/delta TcR T cell lines were derived from TIL from patients with Wilms tumor, sarcoma or metastatic melanoma by stimulation with autologous tumor cells alone and recombinant interleukin 2 and they exhibited nonspecific cytotoxicity against autologous and allogeneic tumor cells, or cells of the K562 or the MEL21 tumor cell lines. Two T cell lines were derived from a patient with Wilms tumor. One of them expressed a non-disulfide-linked gamma/delta TcR using the 60-kDa gamma chain, whereas, the other expressed a disulfide-linked gamma/delta TcR. A T cell line was derived from a patient with sarcoma and expressed a disulfide-linked gamma/delta TcR, whereas, a T cell line derived from a patient with melanoma expressed a non-disulfide-linked gamma chain of 62 kDa. Several T cell clones were developed from patients with metastatic melanoma or Wilms tumor and expressed either disulfide- or non-disulfide-linked gamma/delta TcR. Northern analysis of RNA from certain of these clones revealed a full-length gamma chain transcript, whereas, the alpha or beta chain transcripts were either absent or truncated. These T cell clones exhibited nonspecific cytotoxicity. Both disulfide- and non-disulfide-linked TIL T cell lines and clones expressed the delta TCS1 determinant. gamma/delta TcR+ cells in freshly prepared TIL from these patients were present in low proportions (less than 5%) and their delta TCS1/delta 1 ratios were within the range observed in the peripheral blood of normal donors. These results demonstrate that both disulfide- and non-disulfide-linked gamma/delta TcR are expressed on T cell lines and clones derived from TIL from solid tumors. Non-disulfide-linked gamma/delta TcR using the 56-66-kDa gamma chain are frequently found on TIL-derived T cell lines and clones. These 56-66-kDa gamma chains are rarely expressed on T cell lines or clones derived from peripheral blood lymphocytes of normal donors.


Immunization with soluble simian virus 40 large T antigen induces a specific response of CD3+ CD4- CD8+ cytotoxic T lymphocytes in mice.

C57BL/6 (B6) mice (H-2b) were immunized with the large tumor antigen (T Ag) of simian virus 40 (SV40). Intraperitoneal or subcutaneous sensitization with soluble T Ag specifically primed cytotoxic lymphocyte precursors (CTLp). T Ag-specific cytotoxic T lymphocytes (CTL) were detected in a cytotoxicity assay after specific in vitro restimulation of effector cell populations from mice immunized with 2-10 micrograms purified, soluble T Ag and boosted with an injection of 2 micrograms T Ag 2-4 weeks after priming. Cells used for in vitro restimulation and as targets in cytotoxicity assays were syngeneic (B6-derived) RBL5 lymphoma cells expressing SV40 T Ag after transfection with a T Ag-encoding expression vector. Effector cells of this response were H-2 class I-restricted CD3+ CD4-CD8+ CTL. The magnitude of the anti-T Ag CTL response of B6 mice stimulated by soluble virus protein was comparable to the anti-T Ag CTL response of SV40-infected B6 mice. Injections of denatured or native T Ag protein primed CTLp equally well, but immunization with an equal dose of antigen emulsified in incomplete Freund's adjuvants inefficiently stimulated CTLp.


Expression of the murine interleukin 6 receptor in hepatoma cells: the intracytoplasmic domain is not required for interleukin 6 signal transduction.

This paper reports on cDNA coding for the 80-kDa murine IL6 receptor (mIL6R) that was cloned from a mouse liver cDNA library. Human hepatoma Hep3B cells transfected transiently or stably with an expression vector carrying the entire coding region for mIL6R become responsive to mouse IL6 (mIL6). We monitored response to the cytokine through the transcriptional activation of a co-transfected IL6-inducible human C-reactive protein (CRP) promoter; response to mIL6 is lost upon treatment of the cells with increasing amounts of a monoclonal antibody to mIL6R. mIL6R mutants have been generated in the carboxy-terminal portion of the molecule. Their functional analysis in hepatoma cells shows that the intracytoplasmic domain of the receptor is not absolutely essential to IL6 signal transduction (i.e. CRP promoter activation), but that the last 40 amino acids contribute to maximal IL6 response in these cells.


Immunoglobulin production in severe combined immunodeficient (SCID) mice reconstituted with human peripheral blood mononuclear cells.

Immunodeficient C.B-17 scid/scid (SCID) mice were reconstituted with human peripheral blood mononuclear cells and analyzed for humoral immunity. The majority of adoptively transferred animals had serum levels of 1-4 mg/ml of human IgG 8-12 weeks after i.p. reconstitution with 20 x 10(6) PBMC, whereas the IgM and especially IgA concentrations were considerably lower. The half-lives of human IgG, IgM, and IgA in SCID mice were 12 days, 36, and 23 hours, respectively. Furthermore, IgA was rapidly secreted into bile indicating that the low IgA concentration was mainly caused by a high turnover rate. IgG subclass distribution in mouse serum was similar to that found in the donor serum. Irradiation with 3 Gy prior to adoptive transfer resulted in increased levels of human IgG early after reconstitution, whereas both IgM and IgA concentrations were impaired. A polyclonal serum Ig pattern was found 4 weeks after transfer of human cells later frequently followed by a predominance of oligoclonal bands. Unexpectedly, oligoclonal bands were also found using donors with a negative Epstein-Barr virus serology. Human cells were found to reside in the peritoneal cavity for several months. Within 2 weeks of reconstitution, human cells were also identified in lymphoid structures in the vicinity of the liver hilus with a later spread to other lymphoid organs. Homing of human cells to skin and gut was not seen.


Biochemical analysis of the mouse mammary tumor virus long terminal repeat product. Evidence for the molecular structure of an endogenous superantigen.

Recent reports have shown that both exogenous and endogenous mouse mammary tumor viruses (MMTV) can encode superantigens. Transfection and transgenic studies have identified the open reading frame (ORF) present in the 3' long terminal repeat (LTR) as encoding superantigen function. In this study, we have used an in vitro translation system in an attempt to characterize the molecular nature of the protein encoded by the 3' ORF of Mtv-8. Using various constructs encoding full-length and truncated versions of the ORF product, we report that the hydrophobic region close to the amino terminus of the 36-kDa protein can function as a transmembrane domain. Protease digestion experiments also demonstrate that the protein has a type-II transmembrane conformation with an extra-cytoplasmic carboxy terminus. Since this hydrophobic region is conserved between all known MMTV, we speculate that LTR ORF, including those proposed to encode the minor lymphocyte stimulatory antigens, are also capable of encoding type-II transmembrane glycoproteins. The polymorphism between MMTV LTR ORF products, which correlates with deletion phenotypes, is predominantly in the carboxy-terminal extracellular region, consistent with a major role in interaction with the T cell receptor.


Epidemiology and aetiology of acute bronchiolitis in Hong Kong infants.

The epidemiological, clinical and virological features of 1220 children with acute bronchiolitis admitted to the Prince of Wales Hospital, Hong Kong, from 1985 to 1988 are reported. They accounted for 6.6% of total paediatric admissions and provided a case incidence of bronchiolitis requiring admission to hospital of approximately 21 per 1000 children 0-24 months of age. The clinical course and outcome was in general benign. The average hospital stay was 5 days and there were no deaths. Ten per cent of patients were repeatedly admitted to hospital with recurrent wheezing after discharge. Two infants developed bronchiolitis obliterans. Respiratory syncytial virus (RSV) was shown by direct immunofluorescence, virus culture and serology to be the commonest cause of acute bronchiolitis in Hong Kong. Other aetiological agents included parainfluenza and influenza viruses, adenoviruses, and Mycoplasma pneumoniae. In contrast to western countries, a seasonal variation of bronchiolitis was found with a peak incidence in the summer months. The significance of these observations is discussed.


A 2-year survey of the prevalence of enteric viral infections in children compared with contamination in locally-harvested oysters.

We studied, for two years, the prevalence of indigenous human enteric viruses in wild oysters gathered each month from the bottom of Mikawa Bay, Aichi Prefecture, Japan. Viruses were detected periodically in 9 out of 54 oyster pools prepared by the acid or polyethylene glycol precipitation method although all these 9 pools met current national bacteriological safety standards. Since most of the serotypes of the enteric viruses detected in the oysters were identical with those of viruses isolated from sick children living in the area, it is suggested that contamination of enteric viruses in the oysters would depend on the prevalence of enteric viral infections in the local inhabitants.


Antibody-capture enzyme-linked immunosorbent assays that use enzyme-labelled antigen for detection of virus-specific immunoglobulin M, A and G in patients with varicella or herpes zoster.

Antibody-capture enzyme-linked immunosorbent assays (AC-ELISA) which use enzyme-labelled antigen were developed for detection of varicella-zoster virus-(VZV) specific IgM, IgA and IgG antibody in patients with varicella or herpes zoster and in sera from healthy individuals. All 18 patients with varicella developed a VZV-IgM and a VZV-IgG response, 17 also a VZV-IgA response. In contrast, all 19 patients with herpes zoster were shown to be positive for VZV-IgA whereas only 13 of these reacted positively for VZV-IgM. A VZV-IgM response was detected in only two sera from 100 healthy individuals and an IgA response in only one. The presence of virus-specific IgA and IgG in the cerebrospinal fluid as determined by AC-ELISA was a useful indicator of VZV infection of the central nervous system. By AC-ELISA, VZV-IgG was detected predominantly in sera from patients with acute or recent VZV infection. Only 14 sera from 100 healthy individuals were positive for VZV-IgG by AC-ELISA, whereas all were positive by an indirect ELISA. These results indicate that AC-ELISA's may be useful assays for determination for acute or recurrent VZV infection, but are not suitable for determination of past infection with this virus.


Rotavirus detection by dot blot hybridization assay using a non-radioactive synthetic oligodeoxynucleotide probe.

A synthetic oligodeoxynucleotide of 40 nucleotides corresponding to nucleotides 33-72 of the gene coding for the viral protein VP7 of rotavirus, was used as a nucleic acid probe to develop a non-radioactive hybridization method for rotavirus detection. The probe was labelled at the 3' end with biotin-7-dATP. The sensitivity and specificity of the dot blot hybridization assay for rotavirus detection was evaluated with 303 stool specimens. The results indicate that the hybridization assay has a higher sensitivity than both PAGE and EIA. Among the rotavirus strains tested 37 different electropherotypes were found. The results suggest that rotavirus diagnosis by dot hybridization using a non-radioactive probe may become routine laboratory procedure because it is simple, highly specific and very sensitive.


Evolution of Ig- and T-cell receptor gene configuration in a Ph1+ hybrid leukemia patient.

In a longitudinal study of a 32-year-old male with Ph1+ hybrid leukemia we have followed the immunophenotype and configuration of Ig- and TCR genes during the course of different chemotherapy regimens directed first against the myeloid and later against the lymphoid components of the disease. We identified changes in all parameters, interpretable as an evolution of the malignant clone resulting in a leukemic switch towards a more lymphoid character. Thus, while the expression of the myeloid antigens CD13 and CD33 decreased, that of CD10 (CALLA) and CD20 (B1) increased. Moreover, while the configuration of the Ig heavy and light chain lambda genes remained constant during the whole period of treatment, that of the Ig light chain kappa gene and TCR beta gene displayed extensive rearrangements after initiation of ALL therapy. Since this patient represents a de novo acute leukemia as evaluated by location of the translocation-breakpoint on chromosome 22, our data clearly indicate that Ig- and TCR gene rearrangements might prove a valuable addition in monitoring Ph1+ hybrid leukemias, providing guidelines for optimizing chemotherapy.


Fluorescence study on cardiac glycoside binding to the Na,K-pump. Ouabain binding is associated with movement of electrical charge.

Recently we have presented evidence that the fluorescence probe RH 421 can be used to detect binding and release of ions at the extracellular face of the pump since these processes are associated with translocation of electrical charge. Applying this method to experiments with cardiac glycosides we found that: (1) ouabain induced fluorescence changes of the electrochromic dye, RH 421, were caused by the change of charges bound to the enzyme; (2) independent of the sodium concentration, the final fluorescence amplitude indicated that approximately 2 Na+ ions were bound to the pump; (3) the sodium release to the extracellular side involved two distinct electrogenic steps; (4) the kinetics of inhibition depended on the Na(+)-concentration. Experiments with hydrophobic ions indicated that the kinetics of ouabain binding to the Na-ATPase is voltage dependent; and (5) the applied technique is a convenient tool to characterize binding of cardiac glycosides to the Na,K-pump.


Action of Escherichia coli and human 5'----3' exonuclease functions at incised apurinic/apyrimidinic sites in DNA.

The 5'----3' exonuclease activity of E. coli DNA polymerase I and a related enzyme activity in mammalian cell nuclei, DNase IV, are unable to catalyse the excision of free deoxyribose-phosphate from apurinic/apyrimidinic (AP) sites incised by an AP endonuclease. Instead, the sugar phosphate residue is slowly released as part of a short oligonucleotide. These products have been characterised as dimers and trimers by comparison of their retention time on reverse-phase HPLC with reference compounds prepared by acid depurination of a dinucleotide, trinucleotide and tetranucleotide containing a 5'-terminal dAMP residue. The similar mode of action of these enzymes at 5'-incised AP sites provides an explanation for the minority of repair patches larger than one nucleotide observed when AP sites are repaired by E. coli and mammalian cell extracts in vitro and strengthens the functional analogy between the two activities.


Identification and characterization of urokinase receptors in natural killer cells and T-cell-derived lymphokine activated killer cells.

Fluorescence-activated cell scanning analysis of human blood cells revealed novel urokinase receptors in large granular lymphocytes and a small subset of T-cells (CD3+). Culturing of T-cells with interleukin-2 to generate CD3+ lymphokine-activated killer cells caused a large increase in urokinase binding, suggesting that the urokinase receptor is an activation antigen. The receptor in lymphocytes was similar to that in monocytes with regard to size, affinity and ligand specificity, but did not mediate degradation of urokinase-inhibitor complexes. It is suggested that lymphocyte-bound pro-urokinase is activated, e.g. by the human T-cell-specific serine proteinase, HuTSP-1, and thereby starts a cascade of plasminogen activation important for extravasation of the cells.


Inhibition of neutrophil superoxide production by human plasma alpha 1-antitrypsin.

We report here that human plasma alpha 1-antitrypsin (alpha 1-AT) inhibited human neutrophil O2.- release elicited by a variety of stimulants. In comparison, the inhibitory capacities of two serine protease inhibitors, L-1-tosylamide 2-phenylethyl chloromethyl ketone (TPCK) and soybean trypsin inhibitor (SBTI), and the human recombinant alpha 1-AT mutant, alpha 1-AT-Arg358 were in the order: alpha 1-AT = TPCK much greater than alpha 1-AT-Arg358 greater than SBTI when cells were stimulated with concanavalin A plus cytochalasin E. These data suggest that, in human inflammatory fluids containing relatively high concentrations of alpha 1-AT (such as rheumatoid arthritis synovial fluid), (i) alpha 1-AT may down-regulate the inflammatory process by inhibiting the neutrophil respiratory burst and (ii) serpin oxidation by neutrophil-released reactive oxygen species is unlikely to occur.


Detection of elsamicin-DNA binding specificity by restriction enzyme cleavage.

The sequence specificity of elsamicin A, an anti-tumour antibiotic, binding to DNA was elucidated considering the inhibition of the rate of digestion of linearised pBR322 DNA by AatII, ClaI, EcoRI, HindIII and NruI restriction enzymes. Elsamicin A inhibits the rate of digestion by NruI (recognition sequence TCG/CGA) to a greater extent than it does for the other enzymes, thus evidencing the sequence-selective binding of elsamicin to CGC regions in DNA. Our results also show the important role of the neighbouring sequences in the elsamicin A-DNA interactions and their effects on the cleavage by restriction enzymes.


Identification of a component separated on Mono Q purification of Escherichia coli RNA polymerase as an NTPase.

Standard preparations of Escherichia coli RNA polymerase (RNAP) contain NTPase activity. High-performance anion-exchange chromatography on Mono Q has recently been used by Hager et al. [1990, Biochemistry 29, 7890-7894] to fractionate RNAP into holoenzyme (alpha 2 beta beta' sigma) and core (alpha 2 beta beta') forms, plus other protein components. We found that one of these components, of protomer size slightly larger than the sigma 70 subunit, has NTPase activity; it is efficiently separated on Mono Q, leaving transcriptionally active holoenzyme and core apparently free of NTPase activity. Because of the similarity in size with sigma 70, the NTPase component may escape detection by routine gel electrophoresis.


The use of the amplifiable high-expression vector pEE14 to study the interactions of autoantibodies with recombinant human thyrotrophin receptor.

In order to produce significant quantities of the human thyrotrophin (TSH) receptor we have investigated the use of two eukaryotic high expression systems. DNA encoding the receptor was obtained by the polymerase chain reaction (PCR) applied to thyroid cDNA. Receptor DNA was inserted into the baculovirus system; despite high mRNA levels there was little or no demonstrable protein production. However, using a novel amplifiable glutamine synthetase system, clones of transfected Chinese hamster ovary (CHO) cells expressed a high affinity TSH receptor (KD 0.225 +/- 0.046 nM, Bmax 20,000-45,000 sites/cell for individual clones). This was coupled to adenylate cyclase as measured by a TSH-stimulatable increase in extracellular cyclic AMP (cAMP), a detectable response being noted at 1 microU/ml TSH with half-maximal at around 25-50 microU/ml. The high expression allowed detection of both TSH binding inhibition and adenylate cyclase stimulation by autoantibodies in unfractionated sera from patients with Graves' disease.


TPA decreases 1,25(OH)2D3 binding and calbindin D-28K in renal (MDBK) cells.

The effect of the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) on vitamin D receptors (VDRs) was studied in MDBK cells, a normal bovine renal epithelial cell line. 24 h treatment of MDBK cells with TPA resulted in down-regulation of VDR number, with no change in the binding affinity for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or approximate molecular weight determined by fast protein liquid chromatography (FPLC). TPA treatment also reduced the level of calbindin D-28K, a vitamin D-dependent renal protein. 4 alpha-Phorbol 12,13-didecanoate (4 alpha-PDD), an inactive phorbol ester, did not affect either 1,25(OH)2D3 binding or calbindin D-28K levels. TPA elicited a significant decrease in membrane-associated protein kinase C (PKC) activity which coincided with the reduction in VDR number and calbindin D-28K. These data support a link between TPA, PKC activity and vitamin D actions in kidney.


Osmolytes improve the reconstitution of luteinizing hormone/human chorionic gonadotropin receptors into proteoliposomes.

Rat ovarian membrane luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor was reconstituted into proteoliposomes. The ability of sodium cholate to extract and reconstitute hCG binding activity was dependent on the protein/detergent ratio. Trypsinization of the LH/hCG receptor containing proteoliposomes indicated that approximately 57% of hCG binding sites were oriented extravesicularly. The presence of 20% glycerol or other osmolytes during reconstitution increased the accessibility of LH/hCG receptors but not the activity of adenylate cyclase in proteoliposomes. This beneficial effect was independent of any specific detergent or its presence during detergent solubilization of proteins. Dynamic properties of membranes were monitored by electron spin resonance of 16-, 12-, and 5-doxyl stearic acid. Reconstituted proteoliposomes contain less ordered membrane lipids than do native membranes. Addition of glycerol before reconstitution increased the order of lipid bilayer and shifted it to the physical state of the native membrane. These findings are consistent with the hypothesis that a rise in membrane ordering increases the accessibility of membrane-bound LH/hCG receptors.


In vivo proliferation of rat vascular smooth muscle in relation to diabetes mellitus insulin-like growth factor I and insulin.

The roles of diabetes mellitus, insulin-like growth factor I and insulin in vascular smooth muscle proliferation in vivo were studied. Proliferation was induced by endothelial injury (balloon catheterization) of rat aorta, and was measured as 3H-thymidine incorporation into DNA. Levels of insulin-like growth factor I mRNA and insulin-like growth factor I receptor mRNA were measured with a solution hybridization assay. The increase in DNA synthesis was most pronounced 2 days after injury in both normal and diabetic rats and declined thereafter, but DNA synthesis in aortas from diabetic rats was lower throughout the time period studied. Levels of insulin-like growth factor I mRNA and the receptor mRNA were both increased in balloon catheterized aortas, and time-course studies showed an increase in receptor mRNA prior to the increase in insulin-like growth factor I mRNA. Diabetic rats were treated with equimolar concentrations of insulin (35 nmol/day) or insulin-like growth factor I (31 nmol/day) for 5 days. Insulin-like growth factor I increased DNA synthesis in injured aortas 2 days after injury without improving blood glucose, whereas the effect of insulin was associated with a decrease in blood glucose levels. In conclusion, vascular smooth muscle proliferation is impaired by diabetes and stimulated by insulin treatment. Insulin-like growth factor I infusion stimulates vascular smooth muscle proliferation without affecting blood glucose, and gene expressions of insulin-like growth factor I and its receptor are increased in proliferating vascular smooth muscle, indicating that insulin-like growth factor I and involved in vascular smooth muscle proliferation in vivo.


Effect of phospholipase treatment on insulin receptor signal transduction.

To study the role of membrane lipids in signal transduction by the insulin receptor, we have studied the effect of phospholipase C (Clostridium perfringens) and a phosphatidylinositol-specific phospholipase (Staphylococcus aureus) on insulin binding, a function of the alpha-subunit, and tyrosine kinase activity, a function of the beta-subunit in IM-9 lymphocytes and NIH 3T3 fibroblasts transfected with the human insulin receptor. Treatment of the cells with phospholipase C at concentrations up to 3.4 U/ml did not affect specific insulin binding, but reduced insulin-stimulated receptor phosphorylation by 50%. This effect of phospholipase C was observed within 10 min of treatment and occurred with no change in the basal level of phosphorylation. Pre-treatment of cells with insulin for 5 min prior to enzyme addition prevented any change in kinase activity. Insulin-stimulated phosphorylation of pp 185, the presumed endogenous substrate for the insulin receptor kinase, was also reduced following phospholipase C treatment, with an almost complete loss of insulin stimulation after exposure of cells to enzyme at concentrations as low as 0.6 U/ml. In contrast to these effects of phospholipase C on intact cells, receptor autophosphorylation was not affected in insulin receptors purified on wheat germ agglutinin-agarose from phospholipase C treated cells. Likewise, the phospholipase C effect was reduced by the addition of phosphatidylcholine, but not by the addition of the protease inhibitors, aprotinin and phenylmethylsulfonyl fluoride, to the incubation indicating its dependence on phospholipid hydrolysis. Treatment of cells with the phosphatidylinositol-specific phospholipase C did not affect any of the parameters studied.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of islet amyloid polypeptide on hepatic insulin resistance and glucose production in the isolated perfused rat liver.

The impact of (pancreatic) islet amyloid polypeptide on glucose metabolism and insulin sensitivity was examined in isolated rat livers perfused in a non-recirculating system. Continuous infusion of 10(-7) mol/l islet amyloid polypeptide affected neither basal nor glucagon (10(-9) mol/l)-stimulated glucose output by livers from fed rats, but it did increase the hepatic cyclic AMP release within 44 min (7.91 +/- 12.07 vs control: 0.07 +/- 0.03 pmol x 100 g body weight-1). The effect of the peptide on the ability of insulin to inhibit glucagon-induced hepatic glycogenolysis was measured in three experimental groups (n = 6). As expected glucagon (7 x 10(-11) mol/l) increased integral hepatic glucose release within 84 min (763.4 +/- 161.7 vs -25.7 +/- 73.2 mumol x 100 g body weight-1 in the control group, p less than 0.001), while insulin (100 mU/l) decreased the glucagon-stimulated glucose production (395.2 +/- 180.0 mumol x 100 g body weight-1, p less than 0.01). Simultaneous infusion of 10(-7) mol/l islet amyloid polypeptide however, was not able to reverse insulin-dependent inhibition of glucagon-stimulated hepatic glucose output (370.0 +/- 102.5 mumol x 100 g body weight-1, NS) or to enhance lactate-induced gluconeogenesis of livers from 24 h fasted rats (n = 8). The glucose production stimulated by 10(-9) mol/l glucagon was slightly greater in islet amyloid polypeptide-pre-treated livers than in a control group without addition of islet amyloid polypeptide (5 min: 3.60 +/- 3.36 vs 1.67 +/- 1.28 mumol.min-1 x 100 g body weight-1).(ABSTRACT TRUNCATED AT 250 WORDS)


[Granular cell tumor misdiagnosed as scirrhous breast cancer].

The granular cell tumour, a rare benign tumour of the breast, is described. Its appearance is highly suspicious for malignancy on clinical, mammographic and ultrasonographic investigations. Only histological examination of the tumour, which mimics a scirrhous carcinoma, leads to the definite diagnosis.


[Postmenopausal virilization in thecosis of the ovaries].

We describe the case of a 74-year-old woman with a fifteen-year old history of virilism and with a Type-II-diabetes mellitus treated with insulin. After the removal of the ovaries, the formerly elevated serum-testosterone levels normalised and the signs of virilism gradually abated. The histological finding of stromal thecosis in the ovaries is discussed in relation to hyperinsulinaemia and other potential stimuli for ovarian stromal hyperplasia.


Characterization of three RXR genes that mediate the action of 9-cis retinoic acid.

An understanding of the differences and similarities of the retinoid X receptor (RXR) and retinoic acid receptor (RAR) systems requires knowledge of the diversity of their family members, their patterns of expression, and their pharmacological response to ligands. In this paper we report the isolation of a family of mouse RXR genes encoding three distinct receptors (RXR alpha, beta, and gamma). They are closely related to each other in their DNA- and ligand-binding domains but are quite divergent from the RAR subfamily in both structure and ligand specificity. Recently, we demonstrated that all-trans retinoic acid (RA) serves as a "pro-hormone" to the isomer 9-cis RA, which is a high-affinity ligand for the human RXR alpha. We extend those findings to show that 9-cis RA is also "retinoid X" for mouse RXR alpha, beta, and gamma. Trans-activation analyses show that although all three RXRs respond to a variety of endogenous retinoids, 9-cis RA is their most potent ligand and is up to 40-fold more active than all-trans RA. Northern blot and in situ hybridization analyses define a broad spectrum of expression for the RXRs, which display unique patterns and only partially overlap themselves and the RARs. This study suggests that the RXR family plays critical roles in diverse aspects of development, from embryo implantation to organogenesis and central nervous system differentiation, as well as in adult physiology.


New tools for the physical and genetic mapping of Lactococcus strains.

Tools for the genetic and physical analysis of the Lactococcus lactis subsp. lactis genome were developed. Plasmid pRC1 does not replicate in Gram+ bacteria; it contains unique ApaI, NotI and SmaI restriction sites and an erythromycin-resistance (ErR) encoding gene, ermAM, functional in L. lactis subsp. lactis. When a chromosomal L. lactis subsp. lactis DNA fragment was cloned into this vector, the resulting plasmid became integrated, after transformation, into the bacterial chromosome by homologous recombination in a Campbell-like manner. The integration lead to the generation of new rare restriction sites near to the host fragment. This procedure allows precise mapping of cloned genes onto the chromosomal restriction map. The mapping of the his operon of L. lactis subsp. lactis provides an illustration. The cloning into pRC1 of an IS element able to transpose into the chromosome of the target cell, gave rise to an integration plasmid able to insert randomly rare restriction sites onto the bacterial chromosome. The L. lactis IS element, ISS1RS, was cloned into pRC1, yielding pRL1. Pulsed-field gel electrophoresis analysis of ErR clones obtained after transformation with pRL1, showed that this plasmid was stably integrated at a number of different sites in the L. lactis subsp. lactis chromosome, via transposition. Plasmids pRC1 and pRL1 can greatly facilitate the construction of the physical and genetic map of the chromosome of lactococcal strains.


Complete nucleotide sequence of Tn1721: gene organization and a novel gene product with features of a chemotaxis protein.

The complete 11,139-nucleotide sequence of transposon Tn1721 has been determined. It contains three 38-bp inverted repeats, and (in this order) a new orfI, a resolution site (res), genes encoding resolvase (tnpR), transposase (tnpA), tetracycline-resistance (TcR) repressor (tetR), TcR (tetA) and a truncated transposase gene (tnpA'). The modulator origin of Tn1721 from at least three separate sources is supported by the distinctive codon usages of orfI, tnpR/tnpA and tetR/tetA, and by sequence similarities with Tn501 (tnpR/tnpA) and RP1 (tetR/tetA). The ORFI-encoded 56-kDa polypeptide exhibits features of a methyl-accepting chemotaxis protein (MCP) with a conserved signal domain and a potential transmembrane domain; this polypeptide cross-reacts with anti-MCP antiserum. Like chemotaxis genes, orfI is transcribed from a sigma 28-like promoter. The overexpressed orfI gene product interferes with MCP-dependent chemotaxis suggesting that it completes for soluble transducer protein(s) in the cell. The potential selective advantage of this novel transposon-borne gene is discussed.


Physical mapping of the Saccharomyces cerevisiae Ap4A phosphorylase I-encoding gene by the Achilles' cleavage method.

LacI-mediated Achilles' cleavage (AC) is a method for selective fragmentation of chromosomes at special lac operator sites introduced by gene targeting methods [Koob and Szybalski, Science 250 (1990) 271-273]. The Saccharomyces cerevisiae APA1 gene, coding for diadenosine 5', 5"'-P1, P4-tetraphosphate phosphorylase I, has previously been shown to be located on chromosome III [Kaushal et al., Gene 95 (1990) 79-84]. We have now used the AC method to map APA1 gene to a site 44 kb from the left terminus of the chromosome, between the HIS4 and HML genes. This location was confirmed by the comparison of restriction maps of the APA1 gene region to published restriction maps of chromosome III.


TnblaM: a transposon for directly tagging bacterial genes encoding cell envelope and secreted proteins.

A transposon, TnblaM, designed for the direct selection of bacterial mutants with insertions in genes encoding cell envelope and secreted proteins, was constructed and subcloned into plasmid and bacteriophage lambda delivery vectors. TnblaM is a spectinomycin-resistant derivative of Tn5 with an unexpressed open reading frame encoding mature beta-lactamase (BlaM) at its left end. Therefore, when it inserts into genes in the correct orientation and reading frame, gene fusions encoding hybrid proteins are generated. By introducing TnblaM into bacterial cells and selecting ampicillin-resistant (ApR) colonies, the subset of isolates producing extracytoplasmic BlaM, and hence containing TnblaM inserted in genes encoding secreted proteins and cell envelope proteins, can be directly selected. TnblaM, like TnphoA, can therefore be used to preferentially mutagenise genes encoding extracytoplasmic proteins, but it has the advantage over TnphoA that the desired mutants can be isolated by direct selection (as ApR colonies) rather than by phenotypic screening. Isolates in which TnblaM occupies sites in the chromosome from which it can transpose at high frequency are readily identifiable, and constitute TnblaM donors, with which to simply and efficiently generate rare types of insertion mutants. Moreover, the ApR selection that is used with TnblaM can be fine-tuned to obtain blaM fusions to poorly or well-expressed genes.


A two-component T7 system for the overexpression of genes in Pseudomonas aeruginosa.

A two-component T7 expression system was developed for efficient expression of genes in the nonenteric bacterium, Pseudomonas aeruginosa. The first component of the expression system is a bacteriophage-based transposable element that contains a lacUV5/lacIq-regulated T7 RNA polymerase gene and a selectable antibiotic-resistance determinant. This element, designated miniD-180, was stably integrated into the P. aeruginosa PAO1 chromosome. The second component of this system includes several improved broad-host-range expression vectors containing the T7 gene 10 promoter and multiple cloning site (MCS). These vectors (pEB8, pEB11, and pEB12) contain transcriptional terminators (T1(4)) upstream from the T7 promoter, and T7 terminators downstream from the MCS. Because the T7 promoter is somewhat leaky in these vectors, pEB14 was constructed to decrease transcription of target genes by basal levels of T7 RNA polymerase. This vector contains a core sequence of the lac operator located 19 bp downstream from the transcriptional start point of the T7 promoter, thereby providing a dually regulated system. The utility of this system was demonstrated by placing a promoterless chloramphenicol acetyltransferase (CAT) cassette under control of the T7 promoter and monitoring the isopropyl-beta-D-thiogalactopyranoside-dependent accumulation of CAT in cell-free extracts of P. aeruginosa. We observed up to nearly a 60-fold increase in CAT levels 4 h post-induction, at which time this polypeptide represented up to 20% of the total soluble protein.


Localization of the exonuclease and polymerase domains of Bacillus subtilis DNA polymerase III.

Structural gene mutants were cloned and exploited to identify the major catalytic domains of Bacillus subtilis DNA polymerase III (BsPolIII), a 162.4-kDa [1437 amino acids (aa)] polymerase: 3'-5' exonuclease (Exo) required for replicative DNA synthesis. Analysis of the sequence, mutagenicity, and catalytic behavior of natural and site-directed point mutants of BsPolIII unequivocally located the domain involved in exonuclease catalysis within a 155-aa residue segment displaying homology with the Exo domain of Escherichia coli DNA polymerase I. Sequence analysis of four structural gene mutations which specifically alter then enzyme's reactivity to the inhibitory dGTP analog, 6-(p-hydroxyphenylhydrazino)uracil, and the inhibitory arabinonucleotide, araCTP, defined a domain (Pol) involved in dNTP binding. The Pol domain was in the C-terminal fourth of the enzyme within a 98-aa segment spanning aa 1175-1273. The primary structure of the domain was unique, displaying no obvious conservation in any other DNA polymerase, including the distantly related PolIIIs of the Gram- organisms, E. coli and Salmonella typhimurium.


The influence of different sera on the in vitro immobilisation of Percoll purified Treponema pallidum, Nichols strain.

Investigation of sera, especially rabbit serum, in preventing in vitro immobilisation of Percoll purified T. pallidum. The immobilisation of Percoll purified T. pallidum (Nichols) was studied after pre-incubations with basal reduced medium (BRM), heat-inactivated serum of seven different species of animals, heat-inactivated normal human serum (NHS) and rabbit sera containing a different level of antitreponemal antibodies. Also increasing percentages of heat-inactivated normal rabbit serum (NRS) were studied. The rapid immobilisation of purified treponemes by NHS is delayed by pre-incubation with NRS in a dose-dependent manner. The treponemes from 5-day infections were immobilised significantly more slowly than treponemes from 7- and 8-day infections. Compared with NRS, pre-incubations with a high-titred, low-titred and "autologous" serum resulted in significantly more rapid immobilisation of the treponemes. With most other animal sera resistance to immobilisation was slight compared with that produced by NRS. Immunofluorescent studies revealed that the treponemes were covered with a layer of the human third complement factor (C3b), within an hour of incubation. With two sequential pre-incubations, a delay of the immobilisation was only noted in those test mixtures in which NRS had been present in both preincubations. Rabbit serum delays the rapid in vitro immobilisation of Percoll purified treponemes by normal human serum. There was no evidence that this was caused by preventing access of antibodies (in vivo as well as in vitro) to, or preventing the activation of complement on, the treponemal surface. The evidence points to a mechanism in the fluid phase, suggesting participation of a third factor in the immobilisation process, for instance an enzyme, which can be partially inhibited by rabbit serum component(s).


Treatment of vaginal candidosis: a comparative study of the efficacy and acceptability of itraconazole and clotrimazole.

To compare the clinical and mycological efficacy and patient acceptability of the oral antifungal itraconazole with vaginal clotrimazole in the treatment of vaginal candidosis. A multicentre, single-blind, randomised, parallel group comparison of itraconazole and clotrimazole. 17 Genito Urinary Medicine clinics in UK hospitals. Women with symptomatic, culture positive vaginal candidosis. Patients were randomly allocated 2 x 100 mg itraconazole capsules to be taken twice in a 24 hour period, or a 500 mg clotrimazole vaginal tablet. Clinical and mycological assessments were made at entry and after approximately seven and 35 days. Cure rate was defined in terms of mycological results, and patients were questioned on their opinion of treatment. Of 214 patients, 109 received itraconazole and 105 clotrimazole with similar improvement in clinical signs and symptoms. Mycological cure rates one week after treatment were obtained in 72 of 97 patients (74%) in the itraconazole group and 64 of 89 patients (72%) in the clotrimazole group. Identical mycological cure rates six weeks after treatment were obtained with 40 of 79 patients (51%) receiving itraconazole and 39 of 78 patients (50%) receiving clotrimazole. Clotrimazole and itraconazole were found to be equally effective. A majority of patients receiving the latter preferred it to previous treatments.


G proteins in T cell signal transduction.

This review summarises recent data on G protein implication in receptor signalling in T cells. The data show that PPI-specific PLC in T cell membranes is under G protein control. Some evidence indicates that a G protein couples PLC to TCR. Differences are revealed between the effects induced by direct G protein activators, such as GTP gamma S or AlF4-, and TCR ligands, which imply that TCR ligands may trigger some G-protein-independent signals. An analysis of the conflicting results on the action of PTX and CTX, one of the main tools in studying G proteins, has shown that the toxins produce both G protein-dependent and independent effects. The G protein which couples PLC to TCR appears insensitive to both PTX and CTX. Some findings suggest G protein involvement in signalling induced by interleukins; however, in this case the effector molecules implicate often remain unknown. Scarce data on G protein involvement in signalling from differentiation antigens, on direct G protein regulation of ion channels, and on identification of G proteins in T cells, are also discussed.


The immune response against foot-and-mouth disease virus: influence of the T lymphocyte growth factors IL-1 and IL-2 on the murine humoral response in vivo.

Recombinant and pure "natural" IL-1 and IL-2 were compared with the muramyl dipeptide (MDP) component of Freund's adjuvant for their capacity to enhance the humoral immune response against foot-and-mouth disease (FMD) virus antigen. Using a dose of this antigen which alone did not give a detectable immune response, anti-FMD virus antibody was measured at 14 and 28 days post-vaccination. Although IL-1 could enhance the response against the virus antigen, in particular when administered 24 h before the vaccine, this was not as strong as that obtained when MDP was adjuvant. In contrast, IL-2 was at least as efficient as MDP when applied concomitantly with the antigen. If the IL-2 treatment preceded the vaccination by 24 h, a diminution in the magnitude of the response was seen; however, this was countered by the fact that 10 times less IL-2 was required, compared with concomitant cytokine/vaccine administration, in order to have the maximum effect. When both IL-1 and IL-2 were used together, an even greater enhancement of the immune response against FMD virus antigen was observed, but only when given concomitantly with the antigen. These results demonstrate the relevance of T lymphocyte growth factors to the immune response against FMD virus, and how current immunological and biotechnological knowledge could be applied to the improvement of adjuvant systems in a chemically and biologically defined manner.


Signal transduction during antibody-dependent cellular cytotoxicity mediated by U937 cells.

Stimulation of the human promonocytic cell line U937 with antibody-coated chicken red blood cells (Ab-CRBC), leads to inositol phosphate (IP) release in the effector cells. Neomycin (5 x 10(-4) M) completely inhibits activation of phosphoinositide breakdown, while ADCC is suppressed in a dose-dependent manner. Bordetella pertussis toxin (PT) (0.5 micrograms/ml), entirely inhibits IP release, while ADCC activity is markedly suppressed. The PKC inhibitors H-7 and propranolol also suppress ADCC. HA-1004, which has far lower PKC inhibitory activity than H-7, has a minimal effect on ADCC. The calmodulin antagonists W-7 and TFP are strongly inhibitory. These results indicate that stimulation of U937 cells for ADCC is associated to an increase in IP levels, which may provide positive transduction signals for the activation of this lytic mechanism.


Effect of angiotensin II on renin production by rat adrenal glomerulosa cells in culture.

Angiotensin II (Ang II) inhibits renin secretion and production from the kidney, but the effect of Ang II on adrenal renin is not clear. Nephrectomy, via elevated plasma adrenocorticotropic hormone (ACTH) and potassium, is a strong stimulator of adrenal renin production in the rat. This stimulation is inhibited by the infusion of Ang II, suggesting a negative feedback between Ang II and adrenal renin. In the present study, we examined the effect of Ang II on adrenal renin using a primary culture of rat glomerulosa cells. Cells were exposed to ACTH (10(-11) M), high potassium (8 and 12 mM), db-cyclic AMP (db-cAMP), (10(-3) M), or Ang II (10(-11) to 10(-5) M) for 24 hours, and active renin and inactive renin were measured. Active renin was predominant in the cells, whereas inactive renin predominated in the medium. Ang II stimulated renin production in a dose-dependent fashion (cell-active renin, 1.21 +/- 0.20 to 2.39 +/- 0.16; medium-inactive renin, 2.59 +/- 0.40 to 6.14 +/- 0.49 ng Ang I/10(6) cells). Both ACTH and db-cAMP significantly stimulated active renin in the cells (ACTH, 1.73 +/- 0.14 to 9.44 +/- 0.98; db-cAMP, 1.45 +/- 0.16 to 3.96 +/- 0.71 ng Ang I/10(6) cells) and inactive renin in the medium (ACTH, 4.98 +/- 0.38 to 43.7 +/- 5.63; db-cAMP, 3.80 +/- 0.32 to 33.55 +/- 5.62 ng Ang I/10(6) cells). The addition of Ang II (10(-7) M) blunted the stimulation of renin production by both ACTH and db-cAMP by 60%. High potassium-stimulated renin production was not inhibited by Ang II.(ABSTRACT TRUNCATED AT 250 WORDS)


Inhibition by converting enzyme inhibitors of pig kidney aminopeptidase P.

Several inhibitors of angiotensin converting enzyme were also found to inhibit aminopeptidase P, whereas inhibitors of other mammalian aminopeptidases were ineffective. Aminopeptidase P purified from pig kidney cortex was found to contain one atom of zinc per polypeptide chain, confirming its metalloenzyme nature. The concentrations of converting enzyme inhibitors required to cause 50% inhibition (I50) of aminopeptidase P were in the low micromolar range. The most potent converting enzyme inhibitors toward aminopeptidase P were the carboxylalkyl compounds, cilazaprilat, enalaprilat, and ramiprilat (I50 values of 3-12 microM). The sulfhydryl compounds captopril (I50 110 microM) and YS980 (I50 20 microM) were slightly less potent at inhibiting aminopeptidase P. In contrast, the carboxylalkyl compounds benazeprilat, lisinopril, and pentoprilat; the sulfhydryl compound rentiapril; and the phosphoryl compounds ceranopril and fosinoprilat had no inhibitory effect against aminopeptidase P. This compares with I50 values in the 1-6 nM range for these inhibitors with angiotensin converting enzyme. Inhibition of aminopeptidase P may account for some of the effects or side effects noted with the clinical use of converting enzyme inhibitors. These results may provide the basis for the design of more selective inhibitors of angiotensin converting enzyme or mixed inhibitors of aminopeptidase P and angiotensin converting enzyme, or both.


Influence of immunization on Porphyromonas gingivalis colonization and invasion in the mouse chamber model.

The effects of immunization with invasive or noninvasive Porphyromonas (Bacteroides) gingivalis strains on the pathogenesis of infection in a mouse chamber model were examined. BALB/c mice were immunized by a single injection of heat-killed P. gingivalis invasive strain A7436 or W83 or noninvasive strain 33277, HG405, or 381 directly into subcutaneous chambers. P. gingivalis-specific antibody was detected in chamber fluid 21 days postimmunization, and mice were subsequently challenged by injection of exponential-phase P. gingivalis into chambers. Immunization with A7436 or W83 followed by challenge with A7436 protected mice against secondary abscess formation and death; however, P. gingivalis persisted in chambers for up to 14 days postchallenge. Immunization with noninvasive strain 33277, HG405, or 381 followed by challenge with invasive strain A7436 or W83 protected mice against secondary lesion formation and death. P. gingivalis was cultured from 33277- or HG405-immunized and nonimmunized animals to day 14. All P. gingivalis strains induced an immunoglobulin G response, as measured by an enzyme-linked immunosorbent assay and Western immunoblotting of P. gingivalis whole-cell and outer membrane protein preparations. Western blot analyses indicated that sera from mice immunized with different invasive and noninvasive strains recognized common P. gingivalis antigens. In summary, immunization with invasive P. gingivalis A7436 and W83 or noninvasive P. gingivalis 33277, HG405, and 381 protected mice from secondary lesion formation and death after challenge with invasive P. gingivalis A7436 or W83. P. gingivalis-specific antibody did not, however, inhibit the colonization of P. gingivalis within chambers.


Porphyromonas gingivalis virulence in mice: induction of immunity to bacterial components.

Selected cell envelope components of Porphyromonas gingivalis were tested in a BALB/c mouse model in an attempt to elucidate further the outer membrane components of this putative oral pathogen that might be considered as virulence factors in host tissue destruction. Lipopolysaccharide (LPS), outer membrane, and outer membrane vesicles of P. gingivalis W50, ATCC 53977, and ATCC 33277 were selected to examine an immunological approach for interference with progressing tissue destruction. Mice were actively immunized with heat-killed (H-K) or Formalin-killed (F-K) whole cells or with the outer membrane fraction, LPS, or outer membrane vesicles of the invasive strain P. gingivalis W50. The induction of invasive spreading lesions with tissue destruction and lethality were compared among different immunization groups in normal, dexamethasone-treated (dexamethasone alters neutrophil function at the inflammatory site), and galactosamine-sensitized (galactosamine sensitization increases endotoxin sensitivity) mice after challenge infection with the homologous strain (W50) and heterologous strains (ATCC 53977 and ATCC 33277). Enzyme-linked immunosorbent assay analyses revealed significantly elevated immunoglobulin G and M antibody responses after immunization with H-K or F-K cells or the outer membrane fraction compared with those of nonimmunized mice. The killed whole-cell vaccines provided significantly greater protection against challenge infection in normal mice (decreased lesion size and death) than did either the outer membrane fraction or LPS immunization. The lesion development observed in dexamethasone-pretreated mice was significantly enhanced compared with that of normal mice after challenge with P. gingivalis. Immunization with P. gingivalis W50 provided less protection against heterologous challenge infection with P. gingivalis ATCC 53977; however, some species-specific antigens were recognized and induced protective immunity. Only viable P. gingivalis induced a spreading lesion in normal, dexamethasone-treated, or galactosamine-sensitized mice; F-K or H-K bacteria did not induce lesions. The F-K and outer membrane vesicle immunization offered greater protection from lesion induction than did the H-K immunogen after challenge infection simultaneous with galactosamine sensitization. The H-K cell challenge with galactosamine sensitization produced 100% mortality without lesion induction, suggesting that LPS or LPS-associated outer membrane molecules were functioning like endotoxin. Likewise, P. gingivalis W50 LPS (1 micrograms per animal) administered intravenously produced 80% mortality in galactosamine-sensitized mice. In contrast to the effects of immunization on lesion development, immunization with H-K or F-K cells or LPS provided no protection against intravenous challenge with LPS; 100% of the mice died from acute endotoxin toxicity.(ABSTRACT TRUNCATED AT 400 WORDS)


Biochemical characterization of Porphyromonas (Bacteroides) gingivalis collagenase.

A protease was purified from Porphyromonas gingivalis 1101, a clinical isolate, by sequential sodium dodecyl sulfate-polyacrylamide gel electrophoresis, substrate diffusion gel electrophoresis, and electroelution. The enzyme cleaved radiolabeled human basement membrane type IV collagen and the synthetic collagen peptide substrate for eukaryotic collagenases. It was inactivated by the thiol protease inhibitor N-ethylmaleimide but not by EDTA or EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] and activated by reducing agents such as beta-mercaptoethanol. The enzyme exists as an active precursor protein of molecular mass 94 kDa and undergoes proteolytic cleavage to 75-, 56-, and 19-kDa forms. Biotin-labeled collagen bound specifically to the 94-kDa form of the protein and to its cleavage products in ligand blots, suggesting a role for this enzyme not only in collagen degradation but also in adhesion to collagenous substrata.


Adherence to respiratory epithelia by recombinant Escherichia coli expressing Klebsiella pneumoniae type 3 fimbrial gene products.

We examined the role of Klebsiella fimbrial types 1 and 3 in mediating adherence to human buccal and tracheal cells and to lung tissue sections. We found that clinical isolates of Klebsiella pneumoniae producing type 3 fimbriae and Escherichia coli HB101 containing a recombinant plasmid encoding expression of Klebsiella type 3 fimbriae (pFK10) demonstrated increased adherence to tracheal cells, trypsinized buccal cells, and lung tissue sections, in contrast to nonfimbriate and to type 1 fimbriate bacteria. Adherence by type 3 fimbriate bacteria was inhibited by purified type 3 fimbriae and Fab fragments derived from type 3 fimbrial-specific polyclonal immunoglobulin G. Type 3 fimbriae mediated attachment to the basolateral surface of tracheal cells and to the basal epithelial cells and the basement membrane regions of bronchial epithelia. Using an E. coli transformant (pDC17/pFK52), which expresses nonadherent P fimbrial filaments, along with the type 3 fimbrial adhesin (MrkD), we demonstrated that type 3 fimbrial attachment to respiratory cells was attributable to the MrkD adhesin subunit. Subsequent experiments demonstrated that the epithelial target of the type 3 fimbrial adhesin was most likely a peptide molecule rather than a carbohydrate. The results of this study demonstrate that, in vitro, the Klebsiella type 3 fimbrial adhesin mediates adherence to human respiratory tissue.


Age-dependent pulmonary clearance of Pseudomonas aeruginosa in a mouse model: diminished migration of polymorphonuclear leukocytes to N-formyl-methionyl-leucyl-phenylalanine.

Ten-, 20-, and 35-day-old mice were subjected to an aerosol containing Pseudomonas aeruginosa. The lung clearance of the organism was decreased in mice under 20 days of age. This deficiency was accompanied by decreased migration of polymorphonuclear leukocytes (PMN) to the airways in response to the P. aeruginosa challenge. Similar results were obtained in both outbred, C5-sufficient Swiss mice and inbred, C5-deficient DBA/2 mice. The diminished clearance of P. aeruginosa was related to a transient, age-related decrease in PMN chemotaxis to formyl-methionyl oligopeptides. PMN chemotaxis levels similar to those seen in adults were regained by day 35 after birth.


Apparent retrovirus-induced immunosuppression in a yearling llama.

Immunosuppression suspected to be associated with retrovirus infection was diagnosed in an 18-month-old female llama. The llama had a 6-month history of weight loss, intermittent lameness, and infections that were nonresponsive to treatment. Serial CBC indicated persistent nonregenerative anemia and leukopenia characterized by absolute neutropenia and lymphopenia. Functional hypoplasia of myeloid and erythroid cell lines was detected in serial bone marrow biopsy specimens. Notable pathologic findings included inadequate hematopoiesis, generalized lymphoid hypoplasia and plasma cell depletion, and pulmonary alveolar histiocytosis. Pneumocystis carinii cysts and viral particles of the size and morphologic features consistent with the retrovirus family were observed in lung sections examined by transmission electron microscopy. Antemortem macrophage and postmortem lymph node cultures were positive for reverse transcriptase activity.


WS-7338, new endothelin receptor antagonists isolated from Streptomyces sp. No. 7338. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activities.

WS-7338 A, B, C and D, novel endothelin receptor antagonists, have been isolated from fermentation broth of Streptomyces sp. No. 7338. These antagonists were purified from the culture mycelium by extraction with acetone, followed by carbon column chromatography and HPLC. Among them, WS-7338 B showed good activity in an endothelin receptor binding assay with an IC50 of 2.7 x 10(-7) M.


WS-7338, new endothelin receptor antagonists isolated from Streptomyces sp. No. 7338. II. Biological characterization and pharmacological characterization of WS-7338 B.

WS-7338, produced by Streptomyces sp. No. 7338, was found to be a competitive and specific antagonist against ET-1 receptors in in vitro studies and WS-7338 B is also active in vivo. Furthermore, WS-7338 B was a specific antagonist for vascular ET-1 receptors (ETA receptors) and significantly prevented the accumulation of intracellular inositol 1,4,5-triphosphate (IP3) in endothelin treated rat aorta tissues.


Supplementation of ammoniated wheat straw: performance and forage utilization characteristics in beef cattle receiving energy and protein supplements.

Two experiments were conducted to evaluate performance and forage utilization characteristics of beef cattle fed ammoniated wheat straw (AWS) unsupplemented except for minerals or supplemented with energy and protein. In Exp. 1, 194 crossbred beef cows (BW = 472 kg) in late gestation were allotted by weight, breed type, and age during two consecutive winters to 12 drylot pens (three pens/treatment) for a 60-d feeding trial. The AWS (3% NH3 wt/wt) was tubground through a 7.6-cm screen, and cows were allowed ad libitum access to the AWS. In Exp. 2, 16 ruminally fistulated Angus x Hereford steers (BW = 300 kg) were blocked by weight and randomized to treatments in a 35-d intake-digestion trial. Daily supplementation treatments in both experiments were Control, no supplemental energy or protein; LSG, 1.36 kg of sorghum grain (SG); HSG, 2.72 kg of SG; and SG + SBM, 1.02 kg of SG + .34 kg of soybean meal (SBM). All animals received .23 kg of mineral mixture formulated to meet a pregnant cow's mineral requirements. Supplements LSG and SG + SBM were fed to provide the same daily ME, and HSG and SG + SBM were fed to provide the same daily CP. Cows were managed as one group during and after calving. In Exp. 1, all supplements increased gain (P less than .01) vs Control, and cows fed SG + SBM had higher (P = .05) gains than those fed LSG. The SG + SBM supplement increased (P less than .01) change in cow body condition score compared with LSG.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of light intensity on circadian profiles of melatonin, prolactin, ACTH, and cortisol in pigs.

Eleven crossbred barrows were housed in environmentally controlled rooms with an 8-h photoperiod. Pigs in one room received control illumination of 113 1x (CON; n = 6), and pigs in the other room received intense illumination of 1,783 1x (INT; n = 5) fluorescent light. Pigs were given at least 20 d of exposure to the environment before blood samples were taken every 3 h for 48 h. Data were analyzed by split-plot analysis of variance. Except for prolactin, no treatment x time interactions were noted for the hormone profiles evaluated (P greater than .10). Pigs in INT had greater (P less than .05) concentrations of prolactin in serum than pigs in CON at every sampling time. Concentrations of ACTH and cortisol in plasma were similar for INT (33.9 +/- 3.2 pg/mL of ACTH and 24.4 +/- 3.4 ng/mL of cortisol, respectively) and CON (34.9 +/- 3.0 pg/mL of ACTH and 31.3 +/- 3.1 ng/mL of cortisol, respectively). Within the INT treatment, serum melatonin concentrations were more than doubled (P less than .05) during darkness (66.8 +/- 9.3 pg/mL) compared with during light (30.4 +/- 9.3 pg/mL); however, within the CON treatment, concentrations during light and darkness did not differ (38.4 +/- 9.3 pg/mL and 42.9 +/- 9.3 pg/mL, respectively). Results indicate that light of greater intensity is required to entrain circadian rhythms of melatonin in serum of pigs. Furthermore, pigs may respond to bright light with greater secretion of prolactin, even under constant duration of photoperiod.


Effects of nitrogen fertilization and maturity of bromegrass on nitrogen and amino acid utilization by cows.

Four nonlactating Holstein cows (593 kg) with ruminal cannulas were fed bromegrass hay that was fertilized with 0 or 89 kg of N/ha and harvested in the late-boot or full-head stage of maturity. Total tract apparent digestibility of N was less for full-head than for late-boot bromegrass (48 vs 64%; P less than .01) and was greater for N-fertilized than for unfertilized bromegrass (60 vs 52%, P less than .01). True N digestibility was greater (P less than .01) by cows fed late-boot hay than by those fed full-head bromegrass (85 vs 81%). Maturity did not affect N retention. Nitrogen fertilization increased N retained, both grams per day (70 vs 20; P less than .01) and as a percentage of N intake (P less than .01). Nitrogen and amino acid disappearance kinetics were determined in situ. Rate constants for N disappearance within rapidly and slowly degraded pools were not different among treatments, but because late-boot had a larger proportion of N in the rapidly degraded pool, overall disappearance rate for N was faster (P less than .05) for late-boot than for full-head bromegrass (12.0 vs 7.1 %/h). Treatment effects observed for individual amino acids were similar to those found for N. Both composition and kinetic data were needed to explain differences in N digestion and balance. Distribution of N within the forage had profound effects on the N economy of the cows.


Forage systems for beef production from conception to slaughter: II. Stocker systems.

Fall weaned Angus calves grazed stockpiled 1) tall fescue (Festuca arundinacea Schreb.), 2) tall fescue-red clover (Trifolium pratense L.), or 3) tall fescue-alfalfa (Medicago sativa L.) or were barn-fed, 4) tall fescue hay, 5) orchardgrass (Dactylis glomerata L.)-alfalfa hay, or 6) tall fescue silage from late October to early April during each of 5 yr. Infection of the fescue with Acremonium coenophialum ranged from 0 to 55%. There were two replications each of steers and heifers for each forage system in a completely random design. Each replicate was grazed by three Angus stockers, except for System 1, which was grazed by six stockers, for a total of 420 stockers. Each pasture replicate contained .8 ha (except System 1, which was 1.6 ha), and the stocking rate was one stocker per .27 ha. Fescue hay and silage were harvested each spring for barn-fed systems from the area stockpiled for grazing by cattle in System 1. Nitrogen fertilizer (90 kg/ha) was applied in early spring and again in early August, before stockpiling; no N was applied to stockpiled fescue grown with legumes. Daily gains by calves grazing stockpiled fescue-alfalfa were greater (P less than .01) than by calves grazing stockpiled fescue-red clover or N-fertilized stockpiled fescue (.50, .33, and .34 kg/d, respectively), but fescue-alfalfa calves required more days (P less than .01) of supplemental hay feeding (105, 60, and 36, respectively). Calves fed fescue hay in the barn gained more (P less than .01) than those fed fescue silage.(ABSTRACT TRUNCATED AT 250 WORDS)


Phenotypic expression of the Pseudomonas syringae pv. syringae 61 hrp/hrm gene cluster in Escherichia coli MC4100 requires a functional porin.

Plants, in general, appear to be able to detect the presence of incompatible Pseudomonas syringae strains by a hypothetical cell-cell recognition process to initiate inducible defense mechanisms that contribute to disease resistance. A 25-kb hrp/hrm gene cluster isolated from P. syringae pv. syringae 61(pHIR11) enables Escherichia coli to elicit a hypersensitive response (HR), a plant response generally considered to be a manifestation of recognition and resistance. To identify the nature of the HR-eliciting signal produced by E. coli cells carrying pHIR11, bacterial surface features were surveyed by immunological and biochemical procedures. No immunoreactive epitopes or outer membrane proteins were detected that were associated with expression of the P. syringae pv. syringae 61 hrp/hrm cluster in E. coli MC4100. Phenotypic expression of the P. syringae pv. syringae 61 hrp/hrm cluster in E. coli MC4100, however, was found to be dependent upon ompC and ompF, which control outer membrane permeability to hydrophilic solutes. The results suggest that deployment of the HR-eliciting signal occurs via outer membrane porins and imply that a low-molecular-weight, hydrophilic factor mediates signal exchange between the bacterium and the responding plant cell.


Hyperhemolytic phenomena associated with insertions of Tn916 into the hemolysin determinant of Enterococcus faecalis plasmid pAD1.

Members of the Tn916 family of conjugative transposons are able to insert themselves into Enterococcus faecalis hemolysin/bacteriocin plasmid pAD1 (and related elements) in such a way as to generate hyperexpression of the hemolysin/bacteriocin. To examine this phenomenon in more detail, E. faecalis (pAD1::Tn916) derivatives defective or altered in hemolysin expression were isolated and characterized with respect to production of the L (lytic) or A (activator) component (also known as CylA) and the specific location of the transposon. The mutants fell into five classes. Class 1 strains were nonhemolytic, and the related insertions mapped in a location known to affect expression of the L component. The other four classes varied from an inability to express hemolysin (class 2) to different degrees of hyperhemolytic expression (classes 3 to 5); the insertions in these classes mapped in a similar place within cylA, near the 3' end of the determinant. A previous study provided evidence that CylA is also necessary for bacteriocin immunity; however, these insertions did not destroy this function. (A Tn917 insertion in the 5' half of the determinant eliminates immunity.) In mutant classes 3 to 5, the presence of tetracycline enhanced hemolysin expression. In late-exponential-phase broth cultures, hemolysin could not be detected in supernatants of classes 2 to 5, in contrast to a wild-type control strain; however, different amounts of the L component could be detected, with the lowest in class 2 and greater-than-normal amounts in classes 3 to 5. Although nucleotide sequencing showed that the Tn916 insertions in classes 2 to 5 were at identical sites, the transposon junction sequences differed in some cases. The data indicated that cylA translation into the transposon would result in different truncation sites, and these differences were probably related to phenotype differences.


Regulation of the pAD1-encoded sex pheromone response in Enterococcus faecalis: nucleotide sequence analysis of traA.

The Enterococcus faecalis plasmid pAD1 conjugatively transfers in response to a sex pheromone, cAD1, excreted by potential recipient cells. A key determinant responsible for regulation of pAD1 transfer is traA, which encodes a negative regulator also believed to function in signal sensing. In this study, we analyzed the nucleotide sequence and transcription of traA. A protein of 319 amino acids with a molecular weight of 37,856 was inferred and found to exhibit limited homology with several DNA-binding proteins. Analysis of Tn917-lac insertions resulting in transcriptional lacZ fusions within the 3' end of the traA transcript showed that it overlaps slightly with a convergently-transcribed C-region transcript. Insertional mutations affecting TraA repressor function and signal sensing functions were localized.


Characterization of stress-responsive behavior in Pseudomonas aeruginosa PAO: isolation of Tn3-lacZYA fusions with novel damage-inducible (din) promoters.

Although the pervasive soil and water microorganism Pseudomonas aeruginosa demonstrates heightened sensitivity to UV radiation, this species possesses a recA gene that, based on structural and functional properties, could mediate a DNA damage-responsive regulon similar to the SOS regulon of Escherichia coli. To determine whether P. aeruginosa encodes such stress-inducible genes, the response of P. aeruginosa to DNA-damaging agents including far-UV radiation (UVC) and the quinolone antimicrobial agent norfloxacin was investigated by monitoring the expression of fusions linking P. aeruginosa promoters to a beta-galactosidase reporter gene. These fusions were obtained by Tn3-HoHoI insertional mutagenesis of a P. aeruginosa genomic library. Eight different damage-inducible (din) gene fusions were isolated which lack homology to the P. aeruginosa recA gene. Expression of the three gene fusions studied, dinA::lacZYA, dinB::lacZYA, and dinC::lacZYA, increased following UVC and quinolone exposure but not following heat shock. Similar to E. coli SOS genes, the din genes were induced to different extents and with dissimilar kinetics following UVC irradiation.


Cloning and sequence of Bacillus subtilis purA and guaA, involved in the conversion of IMP to AMP and GMP.

Bacillus subtilis genes purA, encoding adenylosuccinate synthetase, and guaA, coding for GMP synthetase, appear to be lethal when cloned in multicopy plasmids in Escherichia coli. The nucleotide sequences of purA and guaA were determined from a series of gene fragments isolated by polymerase chain reaction amplification, library screening, and plasmid rescue techniques. Identifications were based on amino acid sequence alignments with enzymes from other organisms. Comparison of the 5'-flanking regions of purA and guaA with the pur operon suggests similarities in mechanisms for gene regulation. Nucleotide sequences are now available for all genes involved in the 14-step pathway for de novo purine nucleotide synthesis in B. subtilis.


Cloning and characterization of pathogenicity genes from Xanthomonas campestris pv. glycines.

Nonpathogenic mutants of Xanthomonas campestris pv. glycines 8ra were generated with N-methyl-N-nitro-N'-nitrosoguanidine to identify and characterize pathogenicity genes of the bacterium. A total of 16 nonpathogenic mutants were isolated from 2,000 colonies. One mutant, NP1, was chosen for further study. NP1 did not multiply in soybean cotyledons. A genomic library of strain 8ra was constructed in the cosmid pLAFR3, and the cosmids were tested for complementation in NP1. One cosmid clone, pIH1, which contained a 31-kb insert, complemented mutant NP1. A restriction map of pIH1 was constructed, and deletion analyses identified a 10-kb HindIII fragment that restored pathogenicity to NP1. Southern hybridization analysis indicated that DNA sequences in the 10-kb HindIII fragment are conserved among other X. campestris pathovars tested. Three regions responsible for restoring pathogenicity have been identified by Tn3-HoHo1 mutagenesis. A 2.7-kb ClaI fragment was sequenced, and two possible open reading frames (ORF1 and ORF2) were found. Results indicated that ORF2 but not ORF1 may be expressed in Escherichia coli and in X. campestris pv. glycines. The carboxy terminus of the potential polypeptide encoded by ORF2 has an amino acid sequence similar to that of the gamma subunit of oxaloacetate decarboxylase, which is involved in sodium ion transport in Klebsiella pneumoniae.


The dehalogenase gene dehI from Pseudomonas putida PP3 is carried on an unusual mobile genetic element designated DEH.

As a result of the production of two dehalogenases (DehI and DehII), Pseudomonas putida PP3 utilized halogenated alkanoic acids, such as 2-monochloropropionic acid (2MCPA), as sole sources of carbon and energy. The DehI gene (dehI) was carried on a mobile genetic element (DEH) located on the chromosome of strain PP3. DEH recombined with target plasmid DNAs at high frequencies (e.g. 3.8 x 10(-4) per RP4.5 plasmid transferred). The regulated expression of dehI was detected in P. putida, Pseudomonas aeruginosa, and Escherichia coli strains containing derivative plasmids of RP4.5 and pWW0 recombined with DEH. Movement of DEH from the unstable RP4 derivatives pNJ5000 and pMR5 resulted in the insertion of DEH into the chromosome of RecA+ strains of P. putida but not in RecA+ nor RecA- strains of E. coli. Rescue of DEH from the chromosome of P. putida KT2441 onto plasmid RP4 involved recombination at a frequency (2.7 x 10(-4) per RP4 plasmid transferred) comparable to that observed in strain PP3. The DEH element was not classified as a conventional transposon because it did not move as a discrete DNA fragment: dehI-containing inserts in plasmid DNA targets varied in size between 6 and 13 kb. In addition, DEH exhibited a marked preference for insertion into a specific site on the plasmid pWW0, but its transposition, independent of host recombinational systems, remains to be demonstrated. However, the transposonlike characteristics of DEH included the conservation of restriction endonuclease sites, high-frequency recombination with different target replicons (plasmid and chromosomal DNA), and promiscuous insertion into plasmid RP4-based replicons. Therefore, it is proposed that DEH is an unusual mobile genetic element.


Localization and functional analysis of structural and regulatory dehalogenase genes carried on DEH from Pseudomonas putida PP3.

Pseudomonas putida PP3 expressed two dehalogenases, DehI and DehII. The DehI gene (dehI) was located on a mobile DNA element (DEH) which inserted at high frequencies into target plasmids from its chromosomal location. From a recombinant TOL plasmid (pWW0) containing a 6.0-kb DEH element inserted into the plasmid's 5.6-kb EcoRI-G restriction endonuclease fragment, an 11.6-kb EcoRI fragment was cloned. Subcloning analysis and insertion mutagenesis produced a structural map of the DEH element and located the dehalogenase functions. The gene dehI was transcribed from a regulated promoter on DEH which was expressed in P. putida and Escherichia coli. The direction of transcription of dehI was determined, and it was also found to be under positive control, activated by an adjacent regulatory gene (dehRI). Expression of dehI in clones containing the intact DEH supported good growth on 2-monochloropropionate (2MCPA). Subclones lacking dehRI expressed dehI at levels which allowed only slow growth on 2MCPA, even when dehI expression was initiated from vector promoters. Expression of dehI in P. putida containing the intact DEH element required rpoN, suggesting that it was omega 54 dependent. The intact DEH element transferred to P. putida on a suicide plasmid donor pAWT34 (pBR325 replicon), and dehI was stably inherited, without vector DNA sequences, in transformants selected on 2MCPA. This indicated that the cloned DEH element contained functions associated with recombination.


Structural relatedness of enteric bacterial porins assessed with monoclonal antibodies to Salmonella typhimurium OmpD and OmpC.

The immunochemistry and structure of enteric bacterial porins are critical to the understanding of the immune response to bacterial infection. We raised 41 monoclonal antibodies (MAbs) to Salmonella typhimurium OmpD and OmpC porin trimers and monomers. Enzyme-linked immunosorbent assays, immunoprecipitations, and/or Western immunoblot techniques indicated that 39 MAbs (11 anti-trimer and 28 anti-monomer) in the panel are porin specific and one binds to the lipopolysaccharide; the specificity of the remaining MAb probably lies in the porin-lipopolysaccharide complex. Among the porin-specific MAbs, 10 bound cell-surface-exposed epitopes, one reacted with a periplasmic epitope, and the remaining 28 recognized determinants that are buried within the outer membrane bilayer. Many of the MAbs reacting with surface-exposed epitopes were highly specific, recognizing only the homologous porin trimers; this suggests that the cell-surface-exposed regions of porins tends to be quite different among S. typhimurium OmpF, OmpC, and OmpD porins. Immunological cross-reaction showed that S. typhimurium OmpD was very closely related to Escherichia coli NmpC and to the Lc porin of bacteriophage PA-2. Immunologically, E. coli OmpG and protein K also appear to belong to the family of closely related porins including E. coli OmpF, OmpC, PhoE, and NmpC and S. typhimurium OmpF, OmpC, and OmpD. It appears, however, that S. typhimurium "PhoE" is not closely related to this group. Finally, about one-third of the MAbs that presumably recognize buried epitopes reacted with porin domains that are widely conserved in 13 species of the family Enterobacteriaceae, but apparently not in the seven nonenterobacterial species tested. These data are evaluated in relation to host immune response to infection by gram-negative bacteria.


Surface presentation of Shigella flexneri invasion plasmid antigens requires the products of the spa locus.

An avirulent, invasion plasmid insertion mutant of Shigella flexneri 5 (pHS1059) was restored to the virulence phenotype by transformation with a partial HindIII library of the wild-type invasion plasmid constructed in pBR322. Western immunoblot analysis of pHS1059 whole-cell lysates revealed that the synthesis of the invasion plasmid antigens VirG, IpaA, IpaB, IpaC, and IpaD was similar to that seen in the corresponding isogenic S. flexneri 5 virulent strain, M90T. IpaB and IpaC, however, were not present on the surface of pHS1059 as was found in M90T, suggesting that the transport or presentation of the IpaB and IpaC proteins onto the bacterial surface was defective in the mutant. pHS1059 was complemented by pWR266, which carried contiguous 1.2- and 4.1-kb HindIII fragments of the invasion plasmid. pHS1059(pWR266) cells were positive in the HeLa cell invasion assay as well as colony immunoblot and enzyme-linked immunosorbent assays, using monoclonal antibodies to IpaB and IpaC. These studies established that the antigens were expressed on the surface of the transformed bacteria. In addition, water extraction of pHS1059 and pHS1059(pWR266) whole cells, which can be used to remove IpaB and IpaC antigens from the surface of wild-type M90T bacteria, yielded significant amounts of these antigens from pHS1059(pWR266) but not from pHS1059. Minicell and DNA sequence analysis indicated that several proteins were encoded by pWR266, comprising the spa loci, which were mapped to a region approximately 18 kb upstream of the ipaBCDAR gene cluster. Subcloning and deletion analysis revealed that more than one protein was involved in complementing the Spa- phenotype in pHS1059. One of these proteins, Spa47, showed striking homology to ORF4 of the Bacillus subtilis flaA locus and the fliI gene sequence of Salmonella typhimurium, both of which bear strong resemblance to the alpha and beta subunits of bacterial, mitochondrial, and chloroplast proton-translocating F0F1 ATPases.


Anaerobic induction of the alkylation-inducible Escherichia coli aidB gene involves genes of the cysteine biosynthetic pathway.

The Escherichia coli aidB gene is a component of the adaptive response to alkylation damage. This gene is subject to two different forms of induction: an ada-dependent alkylation induction and an ada-independent induction that occurs when cells are grown anaerobically (M. R. Volkert, L. I. Hajec, and D. C. Nguyen, J. Bacteriol. 171:1196-1198, 1989; M. R. Volkert, and D. C. Nguyen, Proc. Natl. Acad. Sci. USA 81:4110-4114, 1984). In this study, we isolated and characterized strains bearing mutations that specifically affect the anaerobic induction pathway. This pathway requires a functional cysA operon, which encodes sulfate permease. Mutations in cysA block this pathway of aidB induction. In contrast, mutations in either cysH, cysD, cysN, or cysC result in elevated levels of aidB expression during aerobic growth. These results indicate that the sulfate transport genes perform a role in anaerobic induction of the aidB gene and suggest that growth under anaerobic conditions may modify either the function or the expression of gene products encoded by the cysA operon.


Anabolic steroid use among substance abusers in treatment.

Increasing illicit use of anabolic steroids in adolescent and young adult populations has been reported. To determine the scope of this problem and its relationship to psychoactive substance abuse, we evaluated the prevalence of anabolic steroid use among individuals seeking inpatient treatment for substance abuse. A randomized mail survey of 175 inpatient substance abuse treatment directors elicited information regarding the prevalence of anabolic steroid use for inpatients treated in 1989 and the first half of 1990. Additionally, directors were surveyed for experience with DSM-III-R psychoactive substance dependence criteria for anabolic steroid use. Only 19% of centers responding had treated at least one individual using anabolic steroids. Facilities encountering anabolic steroid users reported a prevalence of less than 1% among all admissions. Anabolic steroid users were seen more commonly in privately funded facilities. Directors reported a majority of anabolic steroid users had at least three DSM-III-R psychoactive substance dependence criteria for anabolic steroid use. Treatment directors rarely found anabolic steroid use acknowledged as a problem by users and rarely found anabolic steroid use a primary reason for treatment. Users of illicit anabolic steroids may have significant clinical differences compared with users of other psychoactive substances of abuse and dependence.


The effect of glutamate receptor blockade on anoxic depolarization and cortical spreading depression.

We examined the effect of blockade of N-methyl-D-aspartate (NMDA) and non-NMDA subtype glutamate receptors on anoxic depolarization (AD) and cortical spreading depression (CSD). [K+]e and the direct current (DC) potential were measured with microelectrodes in the cerebral cortex of barbiturate-anesthetized rats. NMDA blockade was achieved by injection of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate [MK-801; 3 and 10 mg/kg] or amino-7-phosphonoheptanoate (APH; 4.5 and 10 mg/kg). Non-NMDA receptor blockade was achieved by injection of 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX; 10 and 20 mg/kg). MK-801 and APH blocked CSD, while NBQX did not. In control rats, the latency from circulatory arrest to AD was 2.1 +/- 0.1 min, while the amplitude of the DC shift was 21 +/- 1 mV, and [K+]e increased to 50 +/- 6 mM. All variables remained unchanged in animals treated with MK-801, APH, or NBQX. Finally, MK-801 (14 mg/kg) and NBQX (40 mg/kg) were given in combination to examine the effect of total glutamate receptor blockade on AD. This combination slightly accelerated the onset of AD, probably owing to circulatory failure. In conclusion, AD was unaffected by glutamate receptor blockade. In contrast, NMDA receptors play a crucial role for CSD.


Regional distribution and kinetics of three sites on the GABAA receptor: lack of effect of portacaval shunting.

The regional distribution of binding sites on the GABAA receptor and their kinetic parameters were measured by quantitative autoradiography in brains from normal rats and rats with a portacaval shunt, a model of portal systemic encephalopathy in which GABA neurotransmission may be altered. The ligands used were [3H]flunitrazepam (a benzodiazepine-site agonist), [3H]-Ro15-1788 (a benzodiazepine-site antagonist), [3H]muscimol (a GABA-site agonist), and [35S]t-butylbicyclophosphorothionate (35S-TBPS, a convulsant that binds to a site near the chloride channel). Some brains were analyzed by computerized image analysis and three-dimensional reconstruction. The regional distribution of binding of the benzodiazepines was very similar, but the patterns obtained with [3H]muscimol and [35S]TBPS were different in many areas, suggesting a heterogeneous distribution of several subtypes of the GABAA receptor. The kinetic parameters were determined in brain regions for [3H]flunitrazepam, [3H]Ro15-1788, and [3H]muscimol. For each ligand, the Kd showed a significant heterogeneity among brain regions (at least threefold), contrary to conclusions drawn from earlier studies. In portacaval shunted rats, binding of all four ligands was essentially unchanged from that in control rats, indicating that, if there was an abnormality in GABA neurotransmission during portal systemic shunting, it was not reflected by altered binding to the main sites on the GABAA receptor.


Mineralocorticoids and mineralocorticoid receptors in mononuclear leukocytes in patients with pregnancy-induced hypertension.

To examine the role of mineralocorticoids in the pathophysiology of pregnancy-induced hypertension (PIH), we studied plasma aldosterone and 18-hydroxycorticosterone levels in 25 women with PIH and 25 normal pregnant women, as controls. Furthermore, we evaluated the mineralocorticoid receptor (MR) status in mononuclear leukocytes in the 2 groups. MR count was significantly (P less than 0.0005) decreased in the PIH group (148 +/- 9 binding sites/cell) compared with the control group (300 +/- 17 binding sites/cell; mean +/- SEM). Plasma aldosterone in women with PIH was 281 +/- 61 pmol/L; in normal pregnant women it was 697 +/- 172 pmol/L (P less than 0.025). Plasma 18-hydroxycorticosterone was also significantly (P less than 0.025) lower (PIH, 1071 +/- 149 pmol/L; controls, 1907 +/- 318 pmol/L). These values were determined at the onset of clinical symptoms of PIH. These results cannot be explained by receptor down-regulation due to higher levels of mineralocorticoids in PIH; a hitherto unknown mineralocorticoid may, thus, be responsible for the hypertension and altered MR status.


Ras mutations in human pituitary tumors.

The cellular basis for pituitary neoplasia is poorly understood. Mutations that activate the ras protooncogenes have been identified in a number of different types of human cancers and potentially represent one of the genetic alterations that occur in pituitary tumors. In this study we examined 19 pituitary tumors for the occurrence of ras mutations. The tumor types included 11 nonfunctioning adenomas, 6 somatotroph adenomas, and 2 prolactinomas. Each of the three ras genes (K-ras, N-ras, and H-ras) was amplified from pituitary tumor DNA using the polymerase chain reaction. Oligonucleotide-specific hybridization was used to screen for mutations that inhibit GTPase activity and cause activation of the ras oncogene. No ras mutations were observed in 18 of the pituitary adenomas. However, a mutation was identified in codon 12 of the H-ras gene (Gly to Val) in a recurrent prolactinoma that was highly invasive and ultimately proved to be fatal. We conclude that ras mutations are uncommon in pituitary adenomas, but may provide a marker for highly invasive tumors.


Mineralocorticoid effector mechanism in preeclampsia.

Mineralocorticoid effector mechanisms were evaluated in 29 patients with preeclampsia and in 25 uncomplicated pregnancies by measurement of plasma aldosterone, levels of mineralocorticoid receptor (MR) in mononuclear leucocytes, and subtraction potential difference (SPD; rectal minus oral values). Mean values for plasma aldosterone were not different between the two groups, but significant differences were observed for MR (preeclampsia, 81 +/- 44 receptors/cell; controls, 306 +/- 168) and SPD (preeclampsia, 65 +/- 7 mV; controls, 12 +/- 5 mV). In six cases we determined MR, plasma aldosterone, and SPD in patients with preeclampsia before and 3 months after delivery. MR were reduced before delivery (96 +/- 27 receptors/cell), and SPD increased (64 +/- 8 mV), with both parameters normalizing after delivery (MR, 242 +/- 79; SPD, 14.0 +/- 4 mV). Aldosterone levels returned to normal nonpregnant values after delivery. These data suggest an important role for abnormalities in mineralocorticoid effector mechanisms in the etiology of preeclampsia and could be an useful marker for diagnosis.


Increased level of atrial natriuretic peptide messenger RNA in the hypothalamus and brainstem of spontaneously hypertensive rats.

The aim of this study was to investigate atrial natriuretic peptide (ANP) gene expression in the central nervous system (CNS) during hypertension. We measured and compared immunoreactive atrial natriuretic peptide (irANP) and ANP messenger RNA (mRNA) in the hypothalamus and brainstem of 17-week-old spontaneously hypertensive rats (SHR) with those of age-matched Wistar-Kyoto (WKY) rats using ribonuclease (RNase) protection assay for ANP mRNA and a specific radioimmunoassay for irANP. RNase protection assay revealed that the concentrations of ANP mRNA in the hypothalamus and brainstem of SHR were higher than those of WKY rats. IrANP concentrations in the hypothalamus and brainstem of SHR were determined by a specific radioimmunoassay and found to be higher than those of WKY rats. Elevated mRNA levels in the hypothalamus and brainstem of SHR indicated that increased level of irANP in the CNS resulted from increased synthesis of ANP. We propose that increased synthesis of brain ANP in SHR may reflect a compensatory mechanism induced by hypertension.


Central interaction between endothelin and brain natriuretic peptide on vasopressin secretion.

To examine the interaction between i.c.v. administration of endothelin and brain natriuretic peptide (BNP) on vasopressin (AVP) secretion in unanesthetized, freely moving rats. I.c.v. cannulation and femoral artery catheterization were performed 7-8 days and 2 days before the experiment, respectively. Endothelin and BNP were injected into the third ventricle through the guide cannula. One millilitre of blood was collected for AVP measurement 30 min before and 10 min after i.c.v. injection. Central administration of endothelin (20 or 40 pmol/2 microliters) dose-dependently evoked the elevation of plasma AVP levels. Preinjection of BNP (0.2 or 1 nmol/3 microliters, i.c.v.) dose-dependently attenuated central endothelin (40 pmol/2 microliters)-induced plasma AVP secretion. We have already reported that BNP attenuated central endothelin-induced pressor response and plasma catecholamine secretion. Taken together, the results indicate that BNP attenuated central endothelin-induced pressor response, at least partially, by suppressing sympathetic nervous system activation and plasma AVP secretion.


The effect of chronic bilateral nephrectomy on plasma and brain angiotensin.

To prove the independence of local tissue renin-angiotensin system (RAS) in brain from classical kidney RAS, we measured local angiotensin levels in bilaterally nephrectomized rats which had been dialyzed with a dialysis technique to greatly prolong survival time. Two groups of animals were used: (1) bilaterally nephrectomized rats with intraperitoneal dialysis, where both kidneys were surgically removed; and (2) controls with intact kidneys and dialysis. By using this protocol, we were able to study plasma and brain angiotensins 5 days after nephrectomy (no longer periods have been attempted). Plasma sodium, potassium and pH were monitored while rats were dialyzed four times a day. Plasma samples and brain areas were obtained and angiotensin II measured by radioimmunoassay and high-pressure liquid chromatography (HPLC). Plasma angiotensin II was significantly diminished in the nephrectomized rats but was still detectable, the levels being above the minimal detectable value. The identity of angiotensin I and angiotensin II detected by radioimmunoassay was confirmed by HPLC. In the brain, angiotensin II content was significantly increased in all areas studied. The highest increments were in hypothalamus and brain stem. Our results demonstrate that: (1) brain angiotensin II is regulated independently of peripheral angiotensin II; and (2) a reduced plasma angiotensin II persists 5 days after bilateral nephrectomy. We conclude that the angiotensin II in the plasma was derived from non-renal tissue and the results support the conclusion that tissue RAS has paracrine and autocrine functions independent of the endocrine function of circulating plasma angiotensin.


Renal sites of action of physiological increases in plasma atrial natriuretic factor concentration in essential hypertension.

To investigate the renal, haemodynamic and neurohormonal responses to low-dose infusions of atrial natriuretic factor (ANF) in hypertensive humans. Ten patients with mild-to-moderate essential hypertension received incremental infusions of 3 and 6 ng/kg per min ANF or vehicle alone whilst on a constant dietary sodium intake. A 90-min basal clearance period was followed by two 2-h infusion periods, with urine collection in the last 90 min of each period. In each of the three clearance periods, glomerular filtration rate (GFR), renal tubular function, and the activity of the renin-angiotensin and sympathetic nervous systems were determined. The renal sites of ANF action were established by simultaneous measurements of 51Cr-ethylenediaminetetraacetate lithium and sodium clearances. Plasma concentrations of neurohormones were measured by radioimmunoassays. Plasma ANF concentrations increased by 1.6- and 2.5-fold during the lower and higher ANF infusion rates, respectively. Plasma cyclic guanosine monophosphate concentrations increased in parallel. ANF caused no changes in supine systolic and diastolic blood pressure or in heart rate. In contrast, haematocrit values increased progressively across the study. The renal effects of ANF administration were characterized by an unaltered GFR and significant increases in the renal clearances of lithium (a marker of end-proximal fluid delivery) and sodium when compared with vehicle infusions, whereas urine flow did not change. Estimated values of fractional proximal and distal tubular sodium reabsorption decreased significantly. Plasma concentration of active renin decreased during ANF infusions, but no significant changes in plasma levels of renin substrate, angiotensin I, angiotensin II or aldosterone were observed. A subtle activation of the sympathetic nervous system was indicated by a moderate increase in plasma noradrenaline during the ANF infusions. These results indicate that even small increases in plasma ANF, as can be found during physiological conditions, induce natriuresis in patients with essential hypertension by enhancing fluid delivery from the proximal tubules, in addition to impairing distal fractional sodium reabsorption. With minor exceptions, the ANF infusions caused qualitatively and quantitatively similar renal, haemodynamic and endocrine effects in the hypertensive patients as in a previously studied group of normotensive subjects.


Existence of renin in the endothelium of human artery.

The existence and localization of renin in situ was examined in human arteries under non-pathological conditions. Biochemical and immunohistochemical procedures were adopted to examine the existence of renin in human gastroepiploic arteries using an antibody raised against human recombinant renin or a specific renin inhibitor. (1) Renin activity (angiotensin I generating activity sensitive to antihuman recombinant renin antibody or a specific renin inhibitor) in the homogenate of human gastroepiploic arteries with and without endothelium was compared; (2) vascular renin was immunohistochemically stained using a modified avidin-biotin-peroxidase complex method. (1) Renin activity in human gastroepiploic arteries with endothelium was significantly higher than in those without endothelium; (2) immunoreactive staining selectively occurred in the endothelial cells of human gastroepiploic arteries. Renin is present in endothelial cells of human arteries under non-pathological conditions. Endothelial renin may play a role in the control of vascular tone through local production of angiotensin II.


The effect of angiotensin II on platelet intracellular free calcium concentration in human pregnancy.

The primary objective of this study was to determine the effect of i.v. angiotensin II infusion on platelet intracellular free calcium concentration in potentially hypertensive primigravid women. Patients at 28-32 weeks gestation were selected on the basis of a diastolic pressure less than or equal to 80 mmHg taken at the antenatal clinic visit. An angiotensin II infusion test (4-16 ng/kg per min) was performed in 13 women. Platelet intracellular free calcium concentration, platelet angiotensin II binding site density and plasma angiotensin II concentration were measured before and at the end of the infusion. Pregnancy outcome (normotension or hypertension) was recorded. Platelet intracellular free calcium concentration rose in 12 of 13 women during angiotensin II infusion. This rise was not directly correlated with initial or final platelet angiotensin II binding site density, plasma angiotensin II concentration or the evoked change in systolic or diastolic blood pressure. Five women subsequently developed pregnancy-induced hypertension. Although basal platelet intracellular free calcium concentration did not differ between the two groups, platelet intracellular free calcium concentration rose twice as much in response to angiotensin II in the five hypertensive pregnant women. There was a 12-fold increase in platelet angiotensin II binding in the future hypertensives, although basal angiotensin II was the same in the two groups. The enhanced rise in platelet intracellular free calcium concentration in response to angiotensin II administration in women who subsequently became hypertensive, together with their increased angiotensin II binding site density suggest a possible enhanced stimulus-effect coupling. Pregnancy-induced hypertension is a state of marked vasoconstriction, with an enhanced pressor response to angiotensin II. These data supply grounds for a hypothesis concerning the mechanism.