Cysteine protects Na,K-ATPase and isolated human lymphocytes from silver toxicity. Metal-binding proteins are important components of retroviruses such as human immunodeficiency virus (HIV). Therefore, metals could be used as antiviral agents. However, most metals are toxic for humans with the exception of silver which is toxic only to prokaryotic cells and viruses. In addition, HIV infection causes a decrease in body cysteine. We formed a complex of silver and cysteine, named silver-cysteine. Healthy human lymphocytes were incubated with silver-nitrate or silver-cysteine. Negligible cell survival was seen at 50 microM silver-nitrate. However, in presence of 1 mM cysteine, the viability remained unaffected up to 1 mM of silver. Further, silver inhibition of isolated Na,K-ATPase was easily reversed by cysteine. Thus, non-toxic silver-cysteine could be used as an anti-viral and cysteine-replenishing agent.


1 beta, 25 (OH)2-vitamin D3 is an antagonist of 1 alpha,25 (OH)2-vitamin D3 stimulated transcaltachia (the rapid hormonal stimulation of intestinal calcium transport). The steroid hormone 1 alpha,25-dihydroxyvitamin-D3 [1 alpha,25(OH)2D3] stimulates biological responses via both genomic mechanisms and nongenomic mechanisms (opening of voltage-gated Ca2+ channels). We report here that 1 beta, 25(OH)2-vitamin-D3 (a) is devoid of activity as an agonist for transcaltachia, (b) is a potent stereospecific antagonist of 1 alpha,25 (OH)2D3 stimulation of the nongenomic transcaltachia response and also (c) has less than 1% the ability of 1 alpha,25(OH)2D3 to bind to the chick intestinal nuclear 1 beta,25(OH)2D3 receptor. We conclude that the membrane response element(s) which generates the nongenomic response of transcaltachia has a different ligand specificity than the classic nuclear 1 alpha, 25(OH)2D3 receptor.


Insulin receptor tyrosine kinase domain auto-dephosphorylation. We have observed dephosphorylation of the soluble, 48 kDa insulin receptor tyrosine kinase domain following its tyrosine autophosphorylation. Dephosphorylation was associated with generation of inorganic phosphate, thereby making catalysis by reversal of the kinase reaction unlikely. The kinase domain preparations could not be shown to contain detectable, contaminating protein tyrosine phosphatase activity. In addition, dephosphorylation was insensitive to protein phosphatase inhibitors. However, it was blocked by the kinase inhibitor staurosporine. These results are consistent with insulin receptor kinase domain auto-dephosphorylation via catalysis involving the kinase itself. These findings raise the possibility of a novel mechanism for termination of the insulin receptor signal.


Valinomycin modifies phosphorescence quenching in cytochrome c oxidase. The spectrum of resting cytochrome c oxidase is modulated by valinomycin addition, which induces a red shift of the Soret band. The enzyme is known both to fluoresce and phosphorescence, effects which can be modulated by certain protein reagents and quenchers. Valinomycin had little effect upon fluorescence at 335 nm, whether excited at 295 nm or 280 nm (tryptophans or both tryptophans and tyrosines, respectively). Phosphorescence at 445 nm was slightly enhanced upon the binding of valinomycin to the enzyme, suggesting a small conformational change accompanying the spectral shift of the heme groups. The quenching by nitrite of the phosphorescence excited at 260 nm, but not that excited at 295 nm, was diminished by valinomycin. This suggests that the valinomycin-induced conformational change may involve (i) a change in the accessibility of tyrosines to the quencher and/or (ii) a change in distribution of distances and/or orientations between populations of tyrosines and tryptophans.


Heme O biosynthesis in Escherichia coli: the cyoE gene in the cytochrome bo operon encodes a protoheme IX farnesyltransferase. The cytochrome bo complex of Escherichia coli is encoded by the cyoABCDE operon and functions as a redox-coupled proton pump. In this study, we have constructed eight cyoE deletion mutants and found that all the mutants were nonfunctional. Spectroscopic and heme analyses of the mutant oxidases revealed that the mutations specifically substituted protoheme IX for heme O present in the high-spin heme binding site. We found also that the overexpression of the cyoE gene in a cyo operon deletion strain resulted in a conversion of protoheme IX to heme O. Since the CyoE protein contains the putative allylic polyprenyldiphosphate binding domain, we concluded that the cyoE gene encodes a novel enzyme, protoheme IX farnesyltransferase, essential for heme O biosynthesis.


Agonist-induced association of the p21ras GTPase-activating protein with phosphatidylinositol 3-kinase. The signal transduction properties of the 21-kDa GTP-binding proteins, encoded by the ras genes, are only partly known. In a recent report, we demonstrated that the signaling pathway of p21ras, like that of several growth factors, is closely associated with phosphatidylinositol 3-kinase (PtdIns 3-kinase) activity. We showed that insulin-like growth factor-1 (IGF-1) and insulin increased the phosphatidylinositol 3-kinase activity in immunoprecipitates obtained with anti-phosphotyrosine and anti-ras antibodies in Ha-ras-transformed epithelial cells. Several findings in this previous study suggested that an additional protein was likely to be associated with the PtdIns 3-kinase. The suggestion that p21ras GTPase-activating protein (GAP) acts not only as a regulator of p21ras activity but also as a direct downstream target in the signaling pathway of p21ras led us to investigate the possible association of PtdIns 3-kinase with GAP. The stimulation of Ha-ras-transformed epithelial cells with IGF-1 caused an increased association of PtdIns 3-kinase activity with GAP, as seen by immunoprecipitation with anti-p21ras and anti-GAP antibodies. The 85-kDa regulatory subunit of PtdIns 3-kinase was present in immunoprecipitates obtained with antibodies against GAP and p21ras of IGF-1 stimulated cells. These data suggest that GAP acts as a downstream target for p21ras via its association with PtdIns 3-kinase.


Vitamin K-dependent gamma-carboxylation of the 1,25-dihydroxyvitamin D3 receptor. It has been reported that vitamin K deficiency in the rat markedly increases the 1,25-dihydroxyvitamin D3 receptor (VDR) binding to DNA and that vitamin K-dependent gamma-carboxylation of endogenous substrates of the intestinal and renal cytosol, also containing VDR, sharply reduced that binding (Sergeev, I.N., and Spirichev, V.B. (1989) Nutr. Res. 9, 725-733). In the present study we have evaluated vitamin K-dependent 14CO2 incorporation to VDR quantitated by immunoprecipitation with anti-VDR monoclonal antibodies. The results obtained strongly suggest that VDR in vitro can undergo gamma-carboxylation in the presence of vitamin K1 and that 15-25% of Glu residues in the VDR are carboxylated in vivo. Taking into account our earlier findings, it is likely that the VDR gamma-carboxylation modulates its binding to DNA.


5' sequence of vesicular stomatitis virus N-gene confers selective translation of mRNA. The infection of cells by vesicular stomatitis virus results in the rapid inhibition of host-cell protein synthesis, but not of viral protein synthesis. To determine if this translational selectivity might be conferred by the viral mRNA, we constructed a plasmid (pUCLN beta-4) containing the 5' end of the viral nucleocapsid (N)-gene, including the ribosome binding site, fused in frame with the gene encoding beta-galactosidase, and compared it to a control plasmid (pMC1924) containing the cellular rabbit beta-globin gene 5' end fused with the beta-galactosidase encoding gene. Both plasmids contained identical promoter and 3' nontranslated regions and expressed similar levels of beta-galactosidase in the indicator cell line 293. In cells transfected with either plasmid, viral infection resulted in a approximately 70% decrease in protein synthesis by five hours. The level of beta-galactosidase from cells transfected with pMC1924 also decreased concomitantly with the decrease in total protein synthesis. However, the level of beta-galactosidase from cells transfected with pUCLN beta-4 was not affected by viral infection. Our data suggest that sequences in the 5' end of the viral mRNA allow for the selective translation of the viral message in the presence of an inhibited translational machinery.


Analysis of the ACP1 gene product: classification as an FMN phosphatase. The relationship between the ACP1 gene product, an 18kDa acid phosphatase (E.C. 3.1.3.2) postulated to function as a protein tyrosyl phosphatase, and the cellular flavin mononucleotide (FMN) phosphatase has been examined in vitro and by using cultured Chinese hamster ovary (CHO) cells. Kinetic analysis indicated that at pH 6 the acid phosphatase utilized a variety of phosphate monoesters as substrates. While small molecules such as FMN were effectively utilized as substrates (kcat/Km = 7.3 x 10(3) s-1M-1), the tyrosyl phosphorylated form of the adipocyte lipid binding protein was a relatively poor substrate (kcat/Km = 1.7 x 10(-1) s-1M-1) suggesting a role for the phosphatase in flavin metabolism. Fractionation of CHO cell extracts revealed that 90% of the FMN phosphatase activity was soluble and that all of the soluble activity eluted from a Sephadex G-75 column with the acid phosphatase. All of the soluble FMN phosphatase activity was inhibited by immunospecific antibodies directed against the bovine heart ACP1 gene product. These results suggest that the ACP1 gene product functions cellularly not as a protein tyrosyl phosphatase but as a soluble FMN phosphatase.


Effect of eosinophil peroxidase on beta-adrenergic receptor density on guinea pig lung membrane. We studied whether eosinophil peroxidase (EPO), an eosinophil granule basic protein, can alter beta-adrenergic receptor (BAR) density on the guinea pig lung membrane. Lung membrane was first preincubated with 1-10 U/ml EPO and then incubated with 10(-4) M NaI and 10(-4) or 10(-6) M H2O2 for 2 hours. BAR density was determined using (-)125I-cyanopindolol. EPO combined with 10(-4) M H2O2 and I decreased the BAR density in a concentration-dependent manner. When only 10(-4) M H2O2 was used, the decrease in BAR density was small but significant. When compared to I, bromide was less effective and chloride alone was not effective. These results suggest that EPO is one of the factors responsible for beta-adrenergic blockade in asthma.


Tyrosine kinase inhibitors block calcium channel currents in vascular smooth muscle cells. Selective inhibitors of tyrosine kinases, tyrphostin 23 and genistein, produced concentration-dependent inhibition of voltage-operated calcium channel currents in vascular smooth muscle cells isolated from rabbit ear artery. The potency of these two structurally dissimilar inhibitors was similar to that reported for their action as inhibitors of tyrosine kinases. Daidzein, an inactive analogue of genistein, had little inhibitory effect on calcium channel currents at concentrations below 300 microM consistent with an action of these agents at a tyrosine kinase. However, tyrphostin 1, a reportedly less active tyrphostin derivative, also inhibited calcium channel currents with a potency similar to tyrphostin 23. These findings suggest that voltage-operated calcium channels in vascular smooth muscle may be modulated by endogenous tyrosine kinase(s) which display different sensitivities to inhibitors compared with the epidermal growth factor (EGF) receptor. Alternatively the possibility of direct blocking actions of these inhibitors at voltage-operated calcium channels cannot be excluded.


Repression of the viral latent promoter BC-R2 in Epstein-Barr virus negative cell lines. The regulation of the latent promoter BC-R2 of Epstein-Barr virus (EBV) was examined using the chloramphenicol acetyl transferase (CAT) gene reporter system. A 5' deletion analysis of BC-R2 promoter sequences has been used to characterize a region, described previously as a transcriptional enhancer in EBV positive cell lines, which can repress the BC-R2 transcriptional activity in EBV negative cell lines.


Role of cysteine residues in the extracellular domain and exoplasmic loops of the transmembrane domain of the TSH receptor: effect of mutation to serine on TSH receptor activity and response to thyroid stimulating autoantibodies. The extracellular domain of the thyrotropin (TSH) receptor is the primary site with which TSH and receptor autoantibodies interact. Cysteines 494 or 569 in the 1st and 2nd exoplasmic loops, respectively, of the transmembrane domain of the TSH receptor are important in this process or in coupling ligand binding to signal generation. Thus, when either is mutated to serine, a receptor results which has no detectable TSH binding and no cAMP response to TSH or thyroid stimulating autoantibodies after transfection, despite the fact the mutant receptor is normally synthesized, processed, and integrated in the membrane, as evidenced by Western blotting using a TSH receptor-specific antibody. Additional site directed mutagenesis studies are performed in order to identify cysteine residues in the extracellular domain of the receptor which, with cysteines 494 and 569, are important for tertiary structure and receptor bioactivity.


Modification of arginyl and lysyl residues of flavokinase from rat small intestine. The amino acid composition of flavokinase has been determined to contain five arginyl and four lysyl residues per mole. Flavokinase is inactivated by arginine-specific reagents. The substrates riboflavin and especially ATP impede inactivation, whereas neither of the products, ADP or FMN, protect. Among lysine-modifying reagents, only 2,4,6-trinitrobenzene sulfonic acid caused inactivation of the enzyme, especially under conditions for denaturation. In this case it was noted that the activity was enhanced at the early stage of the reaction but this enhancement was repressed in the presence of ATP along with the significant protection of activity. Results with modification of arginyl residues and incorporation of 14C-phenylglyoxal suggest that one such residue is involved in the substrate-binding site. The involvement of lysyl residues in catalytic function remains unclear, but seems less critical.


Phosphoinositide fatty acid composition of peripheral blood lymphocytes from aging humans. The interaction of the T-cell receptor complex with the ligands is associated with early molecular events involved in the process of signal transduction implicating phosphoinositide breakdown. In elderly people, abnormalities in membrane signal transduction pathways are the basis of the immune deficiency associated with aging. Peripheral blood lymphocytes from aging humans and young subjects were stimulated with anti-CD3 monoclonal antibody and the phosphoinositide fractions were analyzed in order to determine the fatty acid composition in resting and stimulated conditions. In aging humans, in resting conditions, the all three phosphoinositide fractions appeared more saturated than the corresponding fractions in young subjects. Following anti-CD3 stimulation a decrease in arachidonic acid relative molar content was detected in both young and old subjects, but the arachidonic acid content in resting conditions greatly differed between the two groups, suggesting a different modulation of the microenvironment of the T-cell receptor complex in elderly people, so determining alterations in the early activation steps of lymphocytes.


Modulation of fish liver fructose-1, 6-bis phosphatase by ammonia stress. Hepatic fructose 1, 6 bisphosphatase (Fru-P2ase) from Channa punctatus exhibits 100% and 60% elevations of its maximal catalytic rates (Vmax) at pH 7.5 and 9.5 respectively in summer over those in winter. Both the neutral and alkaline activities of the enzyme are enhanced remarkably (100%) in the winter-adapted fish due to exposure to 10 mg N/1 of unionized ammonia for three weeks. While the neutral activity of the purified enzyme is increased in vitro by NH+4 ions in dose-dependent manner upto about 150 mM NH4, the alkaline activity is found to be activated at a very low concentration of NH4+ (25 mM) and inhibited at 100-150 mM NH4+. The enzyme also shows a characteristics potentiation of 5'-AMP inhibition by NH4+ (125 mM).


The conformation of glucagon in dilute aqueous solution as studied by 1H NMR. The structure of monomeric glucagon in dilute aqueous solution was studied by 500 MHz NMR. Hydrogen-deuterium exchange experiments monitored by NMR showed that the backbone amide NHs form intrastrand hydrogen bonds suggesting the existence of some degree of compact structure. The temperature dependent shift of several amide NH resonances supported the above conclusion. The small J coupling constants arising from the interactions between 6 amide NHs and C alpha Hs (less than 6 Hz) imply a helical structure.


A novel low molecular weight factor detected in the cytosol of guinea pig neutrophils to enhance superoxide anion production. Cytosolic low molecular components in guinea pig neutrophils were examined for the activity to enhance superoxide anion (O2-)-generating NADPH oxidase activity. A component was separated by Sephadex G-25 gel filtration from high molecular weight components, the latter revealed NADPH oxidase activity in a cell-free system in combination with the membrane fraction and arachidonic acid. Addition of this cytosolic low molecular weight component to the cell-free system significantly enhanced NADPH oxidase activity, though this component did not substitute the high molecular weight components in constituting the system. The low molecular weight NADPH oxidase activation factor (LMWAF) found here was not of protein nature, since protease treatment failed to reduce its activity. This factor did not contain phosphate, and was neither flavin nor guanine nucleotide. Though LMWAF was extractable with chloroform-methanol, it was not identical with diacylglycerol.


Hormonal regulation of vitamin D-binding protein production by a human hepatoma cell line. Regulation of vitamin D-binding protein production by various hormones was studied in an established human hepatoma cell line, HuH-7. Among all the hormones studied, the maximal production of the binding protein was obtained by an extra-cellular addition of triamcinolone or epidermal growth factor. Cell numbers were not so changed except for the addition of insulin or glucagon. Our data indicate that the protein production may be regulated by insulin, estradiol, triamcinolone, dihydrotestosterone or epidermal growth factor.


Involvement of thiol groups in the reaction mechanism of Mn(2+)-activated alkaline p-nitrophenylphosphate phosphatase of the extreme halophilic archaebacterium Halobacterium halobium. Halobacterium halobium contains a cytosolic alkaline p-nitrophenylphosphate phosphatase which is selectively activated by Mn2+ ions (Bonet et al., 1991: Int. J. Biochem. 12, 1445-1451). We investigated the reaction mechanism of the enzyme in the presence and the absence of Mn2+ by means of pH studies and the application of some group-specific reagents. A pKes 8.4 of a group in the Mn(2+)-enzyme-substrate complex involved in catalysis could not be detected when catalysis in the absence of the cation was analysed. Only the enzyme with bound Mn2+ was inhibited by p-hydroxymercuribenzoate. Reducing agents stimulated the Mn(2+)-dependent activity but had no effect on the native enzyme activity. Our results indicate that the reaction mechanism is different whether the activating cation is present or not, and that the reaction mechanism in the presence of Mn2+ involves thiol groups.


[Effects of fertilization on yield of Asparagus cochinchinensis (Lour.) Merr]. The experiment revealed that the use of various kinds of fertilizers has apparent effect on the yield of Asparagus cochinchinensis during its growth period. Nitrogen fertilizer and compound fertilizer are suitable for the seeding period, while compound fertilizer, phosphorus and potassium fertilizers are suitable for the period of root forming. The quantity of fertilizer to be used depends on the growing and soil conditions.


[Application of the regression orthogonal design to the fertilization of Psoralea corylifolia L. for optimum yield]. This paper probes into the determination of the optimum amount of fertilization by applying regression orthogonal design. After a significance test by regression equation, a regression equation can be set up, i. e. Y = 25.21 + 9.18 Xn + 15.68 Xp - 0.4 Xn2 - 0.98 Xp2 + 0.062 Xn Xp. This equation can be used to describe the relationship between yield and amount of fertilization.


Postnatal administration of two peptide solutions affects passive avoidance behaviour of young rats. The effects of two subcutaneously injected peptide solutions CERE (100 mg/kg b. wt.) and E021 (1 mg/kg b. wt.) and of 0.9% saline on passive avoidance reaction (PAR) of young rats were examined. Animals were trained and tested in a step-through avoidance task using a footshock of 0.5 mA or 1 mA. Step-through latencies were observed up to 200 s and from these data the percentage of good learners (latency = 200 s) and bad learners (latency < 200 s) was calculated. Two experimental schedules were performed (n > 6). In Expt. 1 rat pups were chronically treated with the substances within the first 7 days after birth. In Expt. 2 the 7 days of treatment started in the 4th postnatal week. In both experiments PAR acquisition was trained on the 28th day after birth (learning trial), PAR extinction testing started on the 29th day (retention trials). After applying a 0.5-mA footshock, rat pups treated with E021 within the first 7 days of life (Expt. 1) displayed significantly slower PAR extinction when compared to saline- and CERE-treated rats. In the 1 mA groups, significant differences in step-through latencies were measured between 0.9% saline- and E021-pretreated animals on retention day 11 and between saline and CERE on retention days 9 and 13. E021-treated rats of Expt. 2, receiving a footshock intensity of 0.5 mA, showed significant lower step-through latencies when compared to E021-treated rats of Expt. 1. In Expt. 2 no significant differences between treatment groups were observed.(ABSTRACT TRUNCATED AT 250 WORDS)


A downstream sequence of the rpL32 promoter competes with the glucocorticoid responsive element for a protein factor. The murine ribosomal protein (rp) L32 gene contains essential promoter sequences located both upstream and downstream of the cap site. A combination of gel mobility shift, UV cross-linking, and cell-free transcription assays were used to analyze the interaction of factors binding to a downstream element (located at position +25 to +37). The rpL32 downstream element identified polypeptides (transcription factors) ranging in size from 45 to 25 kilodaltons (kDa). Four base pair changes in the wild-type sequence of the downstream element eliminated binding. An oligonucleotide containing the glucocorticoid responsive element sequence competed specifically for the 45-kDa protein in both the gel mobility shift assay and in the UV cross-linking studies. Our data also indicate that the downstream binding factors contribute to cell-free transcription of the rpL32 gene.


EBV infection patterns in Hodgkin's disease and normal lymphoid tissue: expression and cellular localization of EBV gene products. The present study was performed to clarify the reported inconsistencies regarding the frequency of the association of Epstein-Barr virus (EBV) and Hodgkin's disease (HD). Biopsies from 102 patients with HD were screened for the presence of EBV-encoded small nuclear RNA (EBER) and latent membrane protein (LMP) by using a non-isotopic in situ hybridization (ISH) and immunohistology (IH), respectively. The results were additionally compared with those obtained by polymerase chain reaction (PCR) for EBV-DNA detection. EBV was detected by EBER-ISH in 67% of the HD cases and in 25% of the control group cases consisting of normal lymph nodes. The results of PCR performed on cases with amplifiable DNA were overall congruent with those obtained by EBER-ISH. With respect to the cellular localization of EBV, four categories of HD could be established: (a) cases with EBV-infected tumour cells (42/102), (b) cases with additional infection of bystander cells (4/102); (c) cases with EBV infection restricted to non-malignant bystander cells (23/102); and (d) cases with neither EBV-infected tumour cells nor bystander cells (33/102). LMP expression was detectable only in the neoplastic cell population of those cases with EBER-positive tumour cells, suggesting a frequent involvement of EBV in the pathogenesis of HD.


Interaction with the nuclear matrix of a chimeric construct containing a replication origin and a transcription unit. We have studied the interaction of a chimeric construct containing an origin of replication (from bovine papilloma virus) and a hormonally regulated transcription unit (long terminal repeat from the mouse mammary tumor virus, driving the v-Ha-ras gene) with the nuclear scaffold and matrix from mouse fibroblasts. We used two experimental approaches because the nuclear matrix protein composition depends largely on the isolation conditions, making its definition mostly operational. In situ studies and in vitro experiments performed in 1361.5 cells, a cell line in which multiple copies of the construct have been established, indicate that two interesting regions of the construct interact with the nuclear matrix. The first region is located in the v-Ha-ras gene 5'-flanking sequences. These sequences come from the Harvey virus and contain a piece of the virus like 30S (VL30) sequences in which the v-Ha-ras gene is embedded. This DNA fragment was coupled to the thymidine kinase (TK) promoter driving the reporter luciferase gene and assayed in transient transfection experiments. Its insertion, in the sense orientation, upstream of the TK promoter resulted in a moderate enhancement (2-3-fold) of the luciferase activity. The second region is the most interesting from a physiological point of view. It contains the plasmid maintenance sequence 1 (PMS-1) and the core origin of replication of the bovine papilloma virus. Differences in the results from in situ (nuclear scaffold) and in vitro (nuclear matrix) experiments suggest that the components involved in the interaction with PMS-1 and the viral origin of replication are different. This may be of importance in the context of the recently proposed view that PMS-1 could be part of a composite origin of replication and provide information at a distance.


Molecular cloning and characterization of a mouse alpha 2C2 adrenoceptor subtype gene. A clone MA2C2 encoding the murine homologue of adrenoceptor alpha 2C2 was isolated from a mouse genomic library using a subtype specific probe. The nucleotide and the deduced amino acid sequences derived from an ApaI fragment (2 kb) of the clone reveal a single open reading frame encoding a putative receptor protein of 455 amino acids. The 5' untranslated region (0.5 kb) sequenced is characterized by high GC content and CpG island count.


Protein phosphatase X has been highly conserved during mammalian evolution. Complementary DNA encoding human protein phosphatase X (PPX, also designated PPP4 in the human genome nomenclature) was isolated from a teratocarcinoma library. The deduced amino acid sequence of human PPX differs from that of rabbit PPX in only two in 307 amino acids, demonstrating that the structure of PPX has been highly conserved during the course of mammalian evolution.


Caffeine promotes survival of cultured sympathetic neurons deprived of nerve growth factor through a cAMP-dependent mechanism. The effects of caffeine on neuronal survival independent of trophic factor support were examined in developing superior cervical ganglion in vitro. We found that caffeine promoted neuronal survival in the absence of nerve growth-factor (NGF) in a dose-dependent manner (EC50 = 6 mM). Pulse treatment with caffeine or high K+ (40 mM), which caused only a transient increase in intracellular free Ca2+ levels ([Ca2+]i), did not promote survival. In contrast, caffeine potentiated the saving effect of various phosphodiesterase inhibitors including theophylline (EC50 = 3 mM) and 3-isobutyl-1-methylxanthine (EC50 = 0.4 mM). Non-xanthine phosphodiesterase inhibitor Ro 20-1724 potentiated the survival promoting effect of caffeine or IBMX. Indeed, administration of 20 mM caffeine rapidly restored the cAMP level of NGF-deprived neurons to normal (0.34 pmol/well) within 10 min; the level reached a plateau level (0.69 pmol/well) at 10 h. Even after 1 day, the sustained level was maintained in the presence of caffeine. In contrast, noradrenaline and isoproterenol, which cause only a transient increase in cAMP levels, did not support survival. These data, in conjunction with others, suggest that sustained levels of second messengers, including not only the [Ca2+]i but also the cAMP level, would support the survival of superior cervical ganglion cells independent of trophic factor support.


Effects of androgen on alpha- and beta-adrenergic receptors in membranes from the rat seminal vesicle. The effects of castration and androgen-replacement on adrenergic receptors in membranes from the rat seminal vesicle were studied. Membranes from seminal vesicles showed saturable and high-affinity binding sites for the beta-adrenergic receptor antagonist, [3H]dihydroalprenolol ([3H]DHA), and the alpha 1-adrenergic receptor antagonist, [3H]prazosin. Castration markedly reduced beta-adrenergic receptors with decreasing the effect of GTP modulating the receptor-ligand affinity, suggesting defects in both the receptor per se and the guanine-nucleotides-regulating mechanism after castration. In contrast, castration increased alpha 1-adrenergic receptors and androgen-replacement reversed this change. The effects of GTP decreasing the alpha 1-receptor binding affinity to the radioligand were observed to a similar extent in the castrated and control membranes. These results demonstrate an inverse regulation by androgen on beta- and alpha 1-adrenergic receptors in membranes of the rat seminal vesicle.


Transcobalamin II--cobalamin binding sites are present on rabbit germ cells. Specific binding sites for rabbit transcobalamin II have been found on isolated adult rabbit germ cells. Scatchard analysis revealed a single class of binding sites for [57Co]cyanocobalamin-transcobalamin II with an association constant (Ka) of 1.3 x 10(10) M-1 and 700 sites per cell. Binding was reversible, saturable and calcium dependent. Electron microscope radioautography following incubation with iodinated transcobalamin II at 4 degrees C led to a detectable labeling mainly restricted to the plasma membrane.


A site-directed antibody that inhibits phosphorylation of the rat-brain sodium channel by cyclic-AMP-dependent protein kinase. Antibodies were raised against three peptides corresponding to the potential protein phosphorylation sites of rat-brain sodium channels by the cAMP-dependent protein kinase (PKA). One of the antibody against sequence (C561-575) reacted to the channel molecule. This immunoreaction occurred in a sequence-specific manner, as it was inhibited by the antigen peptide itself but not inhibited by two other peptides. Although PKA phosphorylates two synthetic peptides, C561-575 and C681-689, of the three, anti-(C561-575) antibody can only inhibit the phosphorylation of peptide (C561-575). PKA catalyzed the incorporation of 3.1-3.5 mol of phosphates into the alpha subunit of the purified sodium channel. The anti-(C561-575) antibody inhibited the channel phosphorylation by 40%. Digestion of the phosphorylated sodium channel with lysyl endoproteinase yielded four major phosphorylated fragments of 3.5, 5.0, 7.0, and 10 kDa. However, similar digestion of the channel that was phosphorylated in the presence of anti-(C561-575) antibody did not yield the phosphorylated fragment of 3.5 kDa and gave the 7.0 kDa fragment in reducing yield. Inspection of these phosphorylated fragments by the predicted sizes of the peptide fragments containing the five potential phosphorylation sites gives a conclusion that anti-(C561-575) antibody inhibits the phosphorylation on Ser-573 completely, and on either Ser-610 or Ser-623 partially, probably due to their proximity orientation in the tertiary structure.


Dictyostelium myosin II heavy-chain kinase A is activated by heparin, DNA and acidic phospholipids and inhibited by polylysine, polyarginine and histones. Dictyostelium myosin II heavy-chain kinase A (MHCK A) is activated by autophosphorylation. Heparin and DNA, as well as vesicles composed of phosphatidylserine or phosphatidylinositol, were found to increase the initial rate of MHCK A autophosphorylation 5-10-fold in a Ca(2+)-independent manner. The negatively charged molecules also increased the activity of the autophosphorylated MHCK A by about 2-fold. In contrast, positively charged polypeptides such as poly(D-lysine), poly(L-lysine), poly(L-arginine) and histones strongly inhibited (IC50 of 0.5 micrograms/ml) the activity of the active, autophosphorylated MHCK A. Similar levels of inhibition, on a weight basis, were observed for poly(L-lysine) fractions with molecular weights from 3800 to 150,000-300,000. The inhibition was competitive with respect to peptide substrate and mixed with respect to ATP. At much higher concentrations poly(L-lysine) also inhibited the ability of MHCK A to autophosphorylate. It is proposed that negatively charged compounds and autophosphorylation increase the activity of MHCK A by weakening the interaction between the catalytic domain and a positively charged autoinhibitory domain.


The roles of cell Ca2+, protein kinase C and the Na(+)-H+ antiport in the development of hypertension and insulin resistance. There is evidence that the cytosolic free Ca2+, protein kinase C, and the Na(+)-H+ antiport cross-communicate with one another through positive and negative feedback mechanisms, thereby maintaining cellular Ca2+ and pH homeostasis. This triumvirate may play a role in the development of insulin resistance--a common characteristic of both essential hypertension and non-insulin-dependent diabetes mellitus. Circulating cells from patients with essential hypertension and non-insulin-dependent diabetes mellitus demonstrate elevated cytosolic free Ca2+, increased protein kinase C activity, or both, and these perturbations are associated with augmented activity of the Na(+)-H+ antiport. If present in other cells (e.g., striated muscle cells and adipocytes), these alterations could underlie insulin resistance in essential hypertension and non-insulin-dependent diabetes mellitus.


Acute unilateral nephrectomy elicits a specific increase in plasma of peptides derived from the N-terminal region of proopiomelanocortin. Acute unilateral nephrectomy (AUN) causes natriuresis from the contralateral kidney through neurohumoral reflex pathways that involve an increase in the plasma of peptides derived from the N-terminal region of the adrenocorticotropic hormone (ACTH)/beta-endorphin precursor proopiomelanocortin (POMC). To determine the specificity of these humoral changes, the concentrations in plasma of ACTH and two peptides arising from the N-terminal fragment (NTF) of POMC, NTF32-49 and gamma-melanocyte-stimulating hormone (gamma-MSH), and of another natriuretic peptide, atrial natriuretic peptide (ANP), were measured by RIA with highly specific antisera to these epitopes. Group I experiments followed the course of sodium excretion (UNaV) for 120 min after AUN or sham nephrectomy. UNaV more than doubled within 60 min of AUN, and this natriuresis was maintained for the remainder of the experiment, whereas UNaV in sham rats did not change. There was no difference in plasma immunoreactive (ir) ACTH or ir-ANP concentrations between sham and AUN rats 120 min after the procedure, but plasma ir-NTF concentration was double in AUN rats compared with sham (P < 0.03). In Group II experiments, animals were killed 30, 60, 90, or 120 min after AUN and the urinary response related to peptide concentrations in plasma. UNaV rose rapidly after AUN, reaching a maximum value within 45 min that again was double the control value and remained stable for the duration of the experiment, up to 120 min after AUN. There was no significant change in ir-ACTH or ir-ANP at any point after AUN compared with values in sham AUN rats. However, plasma concentrations of both ir-NTF and ir-gamma-MSH were elevated 30 min after AUN and reached values at 120 min that were again double the values in sham rats (P < 0.05 for both).(ABSTRACT TRUNCATED AT 250 WORDS)


Stimulation of Na+/H+ exchange is not required for induction of hypertrophy of renal cells in vitro. Hypertrophy of renal proximal tubular cells is associated with an early increase in Na+/H+ antiport activity both in vivo and in vitro. The purpose of the study presented here was to determine whether functioning Na+/H+ antiport activity is required for hypertrophy to occur. LLC-PK1 cells deficient in Na+/H+ antiport activity were prepared by the "proton-suicide" method. Mutant cells had 28 to 40% of the normal Na+/H+ antiport activity. The addition of 50 nM methylisobutylamiloride to these cells decreased the antiport activity to less than 5% of the control value. In the mutant cells, steady-state intracellular pH was normal as was the protein content. After exposure of the wild-type cells for 72 h to 10(-6) M insulin and 10(-9) M insulin-like growth factor 1, cell protein content increased significantly. The increase in protein content induced by these growth factors in the mutant cells did not differ significantly from the response of the wild-type cells. Lowering the Na+/H+ exchange further by the addition of methylisobutylamiloride (50 nM) to less than 5% of the control value did not blunt the hypertrophic response in the mutant cells. These studies indicate that hypertrophy can be induced in LLC-PK1 cells by growth factors when basal Na+/H+ antiport activity is reduced to low levels by selective mutation or by competitive inhibition. The results suggest that stimulation of the Na+/H+ antiporter is not an essential prerequisite for the induction of hypertrophy in renal cells.


Vasoactive substances induce cytoskeletal changes in cultured rat glomerular epithelial cells. Angiotensin II (ANG II), atrial natriuretic peptide III (ANP), and sodium nitroprusside (SNP) alter capillary hydraulic conductivity in isolated glomeruli. These agents also affect cyclic nucleotide levels of glomerular epithelial cells (GEC). ANG II increases cAMP, whereas ANP and SNP increase cGMP. The effects of these vasoactive substances on GEC cytoskeleton were tested by incubating cells from primary cultures or an established cell line with each agent. Changes in the cytoskeleton were assessed by staining for F-actin with Bodipy phallacidin and for tubulin with NBD-colcemid. Control cells exhibited short bundles of F-actin or stress fibers near the base of the cells. These were frequently arranged in parallel and occasionally appeared to radiate from the center of the cell. Microtubules were arranged in a fine network throughout the cell with increased density adjacent to the nucleus and within the nucleolus. Incubation of GEC with 10(-7) M ANG II, cholera toxin, or 8Br-cAMP for 2 at 37 degrees C resulted in rearrangement of F-actin into distinct stellate patterns with a decrease in the relative intensity of the peripheral staining, all concurrent with a fivefold increase in intracellular cAMP. The incubation of GEC with 10(-6) M ANP or 10(-7) M SNP for 2 h at 37 degrees C resulted in apparent disassembly of stress fibers, sparse and more diffuse fluorescence, and some increase of fluorescence at the periphery of the cells, all concurrent with a 10-fold increase in intracellular cGMP. Cytochalasin D incubation led to complete disassembly of actin filaments.(ABSTRACT TRUNCATED AT 250 WORDS)


Chronology of cellular events leading to derangements in function of pancreatic islets in chronic renal failure. In chronic renal failure (CRF), a multitude of metabolic derangements occur in the pancreatic islets, resulting in impaired glucose-induced insulin secretion. These abnormalities include a rise in the basal level of cytosolic calcium ([Ca2+]i) in the islet, a decrease in their basal and stimulated ATP and ATP/ADP ratio, a reduction in the Vmax of Ca2+ATPase and Na(+)-K+ATPase, and an impaired glucose-induced calcium signal. The sequence of events that lead to these derangements and to the impairment in insulin secretion during the evolution of CRF are not defined. The study presented here examined this issue by measuring the metabolic profile of pancreatic islets weekly during the evolution of CRF over a period of 6 wk. The results show that serum levels of parathyroid hormone (PTH) begin to rise during the first week of CRF. The Vmax of Ca2+ATPase and Na(+)-K+ATPase increased during weeks 1 to 3 of CRF but fell to low levels thereafter. At week 3 of CRF, the basal level of [Ca2+]i began to rise, whereas basal and the stimulated ATP content and ATP/ADP ratio started to fall. Glucose-induced calcium signal, delta[Ca2+]i/basal [Ca2+]i, and insulin secretion became abnormally low between weeks 3 and 6 of CRF. The data allow the following formulation: as serum levels of PTH begins to rise, calcium entry into islets is augmented; this in turn will stimulate the activity of Ca2+ATPase and the Na(+)-Ca2+ exchanger, and hence, calcium extrusion out of the islets is increased. As a result, [Ca2+] remains normal during the first 2 wk of CRF.(ABSTRACT TRUNCATED AT 250 WORDS)


Intratumoral immunotherapy with interferon-alpha and interleukin-2 in glioblastoma. The combination of the cytokines interferon-alpha 2b and rIL-2, at doses of 10 x 10(6) IU and 9 x 10(6) IU, respectively, in a 1.5 cc solution, can be safely injected into cystic glioblastoma without any evidence of side effects or increase in surrounding tumoral oedema. Nevertheless, an immunotherapeutic protocol in ten glioblastoma patients with tumour growth, that consisted of one intralesional injection per week, over 4 weeks of this combination of cytokines, had no effect on tumour progression.


Pharmacologic action of Escherichia coli heat-stable (STa) enterotoxin. Escherichia coli produces a heat-stable (STa) enterotoxin that belongs to a family of peptides that mediate several diarrheal diseases, including traveler's diarrhea and epidemic diarrhea in infants and newborns. The STa enterotoxin consists of 18 or 19 amino acids and is encoded by genes specified on a transposon. Intestinal secretion is induced by specific binding to high affinity receptors that reside on the brush border cell membrane of the small intestine. Receptor activation by STa enterotoxin induces a sequence of events that culminate in the release of fluid and electrolytes into the intestinal lumen. These events include the stimulation of particulate guanylate cyclae and subsequent increase of intracellular cyclic GMP, involvement of particulate protein kinase, elevation of intracellular calcium, and activation of the phosphatidylinositol pathway. The release of archidonic acid and production of prostaglandins and/or leukotrienes have also been implicated in the action of STa. Evidence indicates that the STa enterotoxin receptor may be a single multifunctional membrane protein.


A method for the simultaneous and continuous monitoring of vascular tone and protein leakage in the synovium. A technique has been developed to monitor changes in synovial vascular tone and leakage of macromolecules, two of the cardinal features of inflammation. The technique permits not only quantitative sequential measurements of the leakage of 125I-albumin from the circulation into the synovium, but also semiquantitative continuous monitoring of the leakage changes. It allows simultaneous continuous monitoring of changes in synovial vascular tone as well. Vascular protein leakage was monitored semiquantitatively by measuring the radioactivity of 125I-albumin in synovial perfusate when passed through a flow cell consisting of a plastic tubing coil situated inside the well of a gamma detector. Quantitative measurements were provided by collecting and counting the synovial perfusate for 125I-albumin. Synovial vascular tone was monitored continuously by measurement of the change in joint radiation emitted by in vivo-labelled 99m-Tc erythrocytes. The in vivo labelling procedure yielded an 86% +/- 9% labelling efficiency. The degree of erythrocyte labelling was stable throughout the course of the experiment. Using this technique, the abilities of histamine to induce changes in synovial vascular tone and protein leakage were examined. Intraarticular infusion of 1 ng/mL histamine produced a 61% +/- 24% increase in synovial vascular blood volume, but did not significantly increase 125I-albumin leakage above that produced by Krebs solution alone. Higher concentrations produced a concentration-related increase in the leakage of 125I-albumin, which was not accompanied by appreciable increases in synovial vascular blood volume above the basal line. The simultaneous and continuous monitoring provided by this technique has revealed an apparent dissociation between vascular tone and vascular protein leakage in response to intraarticular histamine.


Oxygen toxicity.

Oxygen toxicity. The objective of this article is to provide an overview of the biochemistry of oxygen metabolism, including the formation of free radicals and the role of endogenous antioxidants. Pathophysiologic correlates underlying the clinical manifestations of oxygen toxicity are reviewed and management strategies are outlined. References from basic science and clinical journals were selected from the authors' files and from a search of a computerized database of the biomedical literature. Articles selected for review included both historical and current literature concerning the biochemistry and pathophysiology of oxygen toxicity in animals and humans. The benefits of oxygen therapy have been known for many years; however, its potential toxicity has not been recognized until the last two decades. The lungs, the eyes, and, under certain conditions, the central nervous system are the organs most affected by prolonged exposure to hyperoxic environments. Free radical formation during cellular metabolism under hyperoxic conditions is recognized as the biochemical basis of oxygen injury to cells and organs. Endogenous antioxidants are a primary means of detoxifying reactive oxygen species and preventing hyperoxia-induced cellular damage. When this defense fails or is overwhelmed by the excessive production of hyperoxia-induced free-radical species, distinctive morphologic changes occur at the cellular level. The amount of hyperoxia required to cause cellular damage and the time course of these changes vary from species to species and from individual to individual within the same species. Age, nutritional status, presence of underlying diseases, and certain drugs may influence the development of oxygen toxicity. There is currently no reliably effective drug for preventing or delaying the development of oxygen toxicity in humans. Use of the lowest effective oxygen concentration, the avoidance of certain drugs, and attention to nutritional and metabolic factors remain the best means currently available to avoid or minimize oxygen toxicity. Research is continuing into more effective ways to prevent, diagnose, and treat this disorder.


A path to integration in an academic health science center. This article describes a networking and integration strategy in use at the University of Michigan Medical Center. This strategy builds upon the existing technology base and is designed to provide a roadmap that will direct short-term development along a productive, long-term path. It offers a way to permit the short-term development of incremental solutions to current problems while at the same time maximizing the likelihood that these incremental efforts can be recycled into a more comprehensive approach.


Network information security in a phase III Integrated Academic Information Management System (IAIMS). The developing Integrated Academic Information System (IAIMS) at Columbia-Presbyterian Medical Center provides data sharing links between two separate corporate entities, namely Columbia University Medical School and The Presbyterian Hospital, using a network-based architecture. Multiple database servers with heterogeneous user authentication protocols are linked to this network. "One-stop information shopping" implies one log-on procedure per session, not separate log-on and log-off procedures for each server or application used during a session. These circumstances provide challenges at the policy and technical levels to data security at the network level and insuring smooth information access for end users of these network-based services. Five activities being conducted as part of our security project are described: (1) policy development; (2) an authentication server for the network; (3) Kerberos as a tool for providing mutual authentication, encryption, and time stamping of authentication messages; (4) a prototype interface using Kerberos services to authenticate users accessing a network database server; and (5) a Kerberized electronic signature.


A fast track to IAIMS: the Vanderbilt University strategy. In July 1991, Vanderbilt University Medical Center (VUMC) initiated a fast track approach to the implementation of an Integrated Academic Information Management System (IAIMS). The fast track approach has four elements: 1) an integrated organizational structure combining various operational information management units and the academic informatics program into a single entity to enhance efficiency; 2) technology transfer and network access to remote resources in preference to de novo development; 3) parallel IAIMS planning and infrastructure construction; 4) restriction of the scope of the initial IAIMS to permit a manageable implementation project. The fast track approach is intended to provide a truly functional IAIMS within a time period (7 years) associated with other major construction projects such as the building of a replacement hospital.


Online bibliographic information: integration into an emerging IAIMS environment. The Medical Library at Yale University has developed an online free-text database containing Current Contents citations. The database was designed to be integrated into an emerging campus-wide information environment. To this end Current Contents at Yale was designed with a user interface familiar to the Yale community, an alerting service based on electronic mail, and search expansion using the National Library of Medicine's Meta-1 metathesaurus.


Analysis of the leukotriene D4 receptor in the granulation tissue of allergic inflammation in rats. Leukotriene (LT) D4 receptor in the granulation tissue formed in the air pouch-type allergic inflammation model in rats was analyzed. Membrane preparation of the granulation tissue obtained 3-9 days after the antigen challenge has specific binding sites of [3H]LTD4. Scatchard analysis showed that the affinity (Kd) and the density (Bmax) were not changed among the granulation tissue obtained 3-9 days after the antigen challenge. The Kd value in the granulation tissue (0.90 +/- 0.12 nM) was close to that in the rat lung (1.00 +/- 0.24 nM) and the guinea pig lung (0.86 nM). On the other hand, Bmax (62 +/- 8 fmol/mg protein) in the granulation tissue was higher than that in the rat lung (21 +/- 4 fmol/mg protein) but was far less than that in the guinea pig lung (405 fmol/mg protein). LTC4 and LTE4 inhibited the binding of [3H]LTD4 to the membrane preparation of the granulation tissue in a concentration-dependent manner. IC50 of LTC4 and LTE4 were 1 x 10(-7) and 2 x 10(-7) M, respectively. A guanine nucleotide, guanyl-5'-yl-imido-diphosphate (GppNHp), reduced [3H]LTD4 binding to the membrane preparation of the granulation tissue suggesting that LTD4 receptors in the granulation tissue are associated with G proteins. These results indicate that LTD4 binding sites in the granulation tissue are high affinity receptors for LTD4. A possible role of LTD4 in the recurrence of allergic inflammation in the chronic phase is discussed.


Human foamy virus antigens in thyroid tissue of Graves' disease patients. Human foamy virus (HFV) is a recently characterized member of the retrovirus subfamily Spumaretrovirinae. HFV has a complex structure: it encodes the classical three retroviral structural genes gag, pol and env, but also possesses additional regulatory, so-called bel sequences. Foamy viruses have been discussed occasionally as being possibly involved in the pathogenesis of autoimmune thyroiditis. By indirect immunofluorescence we were able to demonstrate the expression of HFV gag proteins on the epithelial cells of 7/7 thyroid glands of patients with Graves' disease, but not in those of 9 patients with struma parenchymatosa, 3 with follicular carcinoma and 2 normal thyroids. The thyroids of 5 patients with Hashimoto's disease were also negative with the exception of a single small focus in 1 case. These observations may have importance for our understanding of the development of Graves' disease.


An automated sequential sampling system for particulate acid aerosols: description, characterization, and field sampling results. An automated sequential sampling system for the measurement of particulate acid aerosols was developed in order to allow the routine collection of daily samples with only once-per-week site visits. The system's design, operation, and associated quality assurance (QA), quality control (QC), and data validation procedures are described. Results from a multi-year field application of this system in the Buffalo, Albany, and New York City metropolitan areas are reported, as well as from external audits and interlab analytical intercomparisons. It is found that the system successfully collected 94% of all possible samples in these three cities between May 1988 and December 1989. Weekly QA field blanks and positive control filters indicate that these data were collected with negligible acid contamination or neutralization. Peak aerosol acidity levels at these sites were found to be highest during the summer months, with peak 24-hr average levels in the 300-400 nmol/m3 range. It is concluded that this system represents a more convenient means of collecting extended daily records of valid particulate acid aerosol concentrations than has been possible in the past.


Functional characterization of human IL-10. Rapid progress has been made over the past two years in the characterization of the biological activities of interleukin-10. Interleukin-10, produced by T cells, B cells, macrophages/monocytes and keratinocytes, alters profoundly the morphology, the expression of MHC class II antigens and the production of cytokines by monocytes which in turn can affect a variety of immunological responses including antigen specific proliferation and cytokine production of both soluble and allo-antigens by T cells, cytokine production by natural killer cells and immunoglobulin production by B cells. IL-10 also directly affects the function and growth of T cells, B cells and mast cells. These characteristics indicate that IL-10 has strong anti-inflammatory activities and may act as a general suppressor factor of immune reactions with consequences for transplantation, tolerance, cancer therapy and infectious diseases.


A multi-year study of air pollution and respiratory hospital admissions in three New York State metropolitan areas: results for 1988 and 1989 summers. As part of a multi-year study of air pollution and respiratory hospital admissions in the Buffalo, Albany, and New York City, New York, metropolitan areas, filter samples were collected daily at suburban air monitoring sites and analyzed for their content of particulate phase aerosol strong acidity (i.e., hydrogen ion, H+) and sulfate (SO4 = ). In addition, daily hospital admissions for respiratory causes, other community air pollutant measurements (e.g., ozone, O3), and meteorological data (e.g., temperature) were also obtained for these metropolitan areas. The summer months (June-August) were selected for analysis because that is when the highest H+ (and O3) are usually experienced at these sites, and because these months are rarely complicated by other major influences (e.g., high pollen counts). Thus, any pollution-admissions relationships were expected to be most clearly discernible in this season. Prior to the health effects analysis, the summer admissions and environmental data were first detrended to eliminate long-wave autocorrelations, and day-of-week effects were removed via regression. Cross-correlations of the filtered 1988 and 1989 admissions and environmental data revealed strong associations between elevated summer haze pollution (i.e., H+, SO4 =, and O3) and increased total respiratory and asthma admissions on the same day and/or on subsequent days in Buffalo and New York City, especially during the summer of 1988 (when pollution levels were more extreme). Regression analyses indicated that the pollution-admissions associations remained significant (p < 0.05) even after the simultaneous inclusion of lagged daily maximum temperature. Mean effects calculations for these cities indicated that summertime haze can play a significant role in the occurrence of respiratory admissions in that season: accounting for an average 6 to 24% of 1988 Buffalo and NYC asthma admissions (depending on the pollutant index employed). O3 consistently had the highest mean effects estimates. Relative risk (RR) calculations indicated that the risk of admission for asthma was increased by a factor of 1.19 to 1.43 in these cities on maximum 1988 summertime pollution days, with H+ consistently having the highest RR estimates. These results are consistent with the hypothesis that ambient acid aerosol peaks (e.g., H+ > or = 100 nmol/m3) can potentiate the respiratory disease effects of O3. Associations were weaker in the less urbanized Albany metropolitan area and in the New York City (NYC) suburbs, even though the NYC suburban O3 exposures were similar to (and the H+ concentrations may even be somewhat higher than) those in the center city.(ABSTRACT TRUNCATED AT 400 WORDS)


Alginate--its role in neutrophil responses and signal transduction towards mucoid Pseudomonas aeruginosa bacteria. Mucoid Pseudomonas aeruginosa bacteria impaired neutrophil functions, e.g. chemiluminescence response, and leukotriene formation to a significantly higher degree as compared to nonmucoid P. aeruginosa bacteria. To study the cell biological requirements for the different cellular response pattern by mucoid and nonmucoid (NM) P. aeruginosa bacteria, further experiments were performed with purified alginate, the mucoid exopolysaccharide of P. aeruginosa (MEP). In this regard the MEP (alginate) significantly reduced the zymosan-induced leukotriene B4 (LTB4) formation (from 40 +/- 7 to 2 +/- 4 ng). The chemiluminescence response induced by NM bacteria was abolished when the bacteria were precoated with the MEP. Mucoid and NM P. aeruginosa bacteria interacted with components of the cellular signal transduction pathway to a different degree. Mucoid bacteria induced a 2-fold enhanced GTPase activity but activated the protein kinase C (PKC) to a lesser degree than NM P. aeruginosa bacteria. Prior exposure of neutrophils to the MEP increased the sodium fluoride (NaF)-induced GTPase activity and guanylylimidodiphosphate binding [Gpp(NH)p] by approximately 60 and 30%, respectively. The phorbol myristic acid-induced PKC activation was inhibited by 30-40% in the presence of the MEP. However, the MEP by itself was inactive in all assay systems. Our results indicate that the MEP represents an important component which modulates neutrophil responses of mucoid as compared to NM P. aeruginosa bacteria, e.g. the chemiluminescence response, LTB4 generation, and the interaction with components (G proteins, PKC) of the signal transduction pathway.


Metabolic characterization of neurological diseases by proton localized NMR spectroscopy of the human brain. Proton localized Magnetic Resonance Spectroscopy (MRS) of the brain allows the non invasive detection of intracellular cerebral metabolites. Localized MRS has been performed using short stimulated-echo times in various neurological diseases including stroke, multiple sclerosis, and AIDS-related leukoencephalopathies. Principal component analysis (PCA) was used to determine the critical parameters defining the metabolic profile of normal and diseased brain. PCA clearly differentiates the demyelinating processes from ischaemic lesions and leukoencephalopathies. Localized MRS of the brain appears growingly as a tool of choice to discriminate, quantitate and assess cerebral metabolic damage in patients with neurological disorders.


Rous sarcoma virus does not induce sarcoma in early chick embryos by lack of the v-src gene expression. The Schmidt-Ruppin or the B77 strain of Rous sarcoma virus (RSV) was inoculated into limb buds of 4.5-days-old avian embryos. No sarcoma but blister formation was observed in those RSV-inoculated embryos. Protein kinase activity of pp60v-src in RSV-inoculated embryos, even in the site of virus inoculation, was the same as that in mock-infected embryos. This indicated that the expression of the v-src gene did not attain superiority over that of the c-src gene in RSV-inoculated embryos. The v-src gene was detected in every DNA from tissues of RSV-inoculated embryos but not in DNAs from tissues of RSV-inoculated chicken except for the DNA from Rous sarcoma. Those results confirmed that the lack of sarcoma induction in early avian embryos by RSV was due to the lack of the expression of the v-src gene which was present in the target cells.


Differential responsiveness to agents which stimulate cAMP production in normal versus neoplastic mouse lung epithelial cells. We examined the responsiveness of normal and neoplastic lung cells to agents which stimulate cAMP production. While their basal cAMP levels were similar, spontaneous in vitro transformant E9 cells and tumor-derived PCC4 cells produced much less cAMP in response to 1 microM isoproterenol compared to non-tumorigenic C10 cells derived from normal mouse lung epithelium. Iodocyanopindolol binding studies indicated that both neoplastic lines contained fewer beta-adrenergic receptors than normal C10 cells. When receptors were bypassed via treatment with 10 pM cholera toxin, the pattern of cAMP-responsiveness was reversed; both neoplastic cell lines produced more cAMP than C10 cells. Direct stimulation of adenylate cyclase with 100 microM forskolin greatly increased cAMP concentrations in all three cell lines. These anomalies at both the receptor and G-protein levels in neoplastic lung epithelial cells may contribute to their deregulated growth.


Methylation of DNA and its modulation by estrogen in the uterus of aging rats. The effect of estrogen on methylation of DNA from the uteri of young (20 weeks) and old (96 weeks) female Wistar rats has been examined by isoschizomeric restriction enzymes and HPLC analysis. In vitro methylation of DNA is significantly higher in the uteri of young rats as compared to old ones. This is reduced by estrogen to greater extent in young than in old age. Furthermore, the digestion of DNA with EcoRI+Msp I shows a distinct 1.2 kb band only in young control. Such band is absent in old control and estrogen-treated sets of both age groups. The HPLC data further reveal that the level of 5-methyl cytosine is high in young and decreases by nearly 18% in old. Estrogen lowers the level of 5-methyl cytosine by 8% in young but shows no effect in the old. Such age-dependent changes in the methylation of DNA brought by estrogen in the rat uterus attribute to alterations in gene expression during aging.


Detection of mutations in the p53 gene in human head and neck carcinomas by single strand conformation polymorphism analysis. Using the polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) analysis, we have examined the highly conserved regions of the p53 gene in 58 biopsy samples of head and neck tumors. Mutations were found in 13/58 (23%) tumor specimens, but not in 6 normal tissues. Ten of 13 mutations were due to single base changes and the remaining 3 were 1- or 8-base deletion mutants. These mutations were clustered in exons 5 and 7 and resulted in amino acid changes. Our results seem to indicate that mutations in the p53 gene contribute to a significant number of cases of the head and neck tumors including 20% of nasopharyngeal carcinoma biopsies. The relationship of Epstein-Barr virus or human papillomavirus and p53 gene mutations in this group of cancers was also analyzed and discussed.


Receptor mediated endocytosis of N-acetylglucosamine and mannose exposing molecules by cultured chick embryo hepatocytes. We studied the receptor mediated endocytosis of a modified glycoprotein (N-acetylglucosamine-BSA) and mannan in cultured hepatocytes isolated from 19-days-old embryos. The binding sites for molecules exposing terminal N-acetylglucosamine (GlcNac) and mannose residues were localized and quantified at the ultrastructural level by means of protein-gold complexes. The binding sites were found to be randomly distributed as single gold particles on cultured hepatocyte cell surfaces not restricted to specialized areas of the plasma membrane. The gold ligands were internalized following a receptor mediated pathway, which was studied at different interval times (15, 30 and 60 min.) after incubating the cells with the electron dense markers.


Resolution of acute inflammation and the role of apoptosis in the tissue fate of granulocytes. We have identified a pathway for granulocyte removal in tissues which is controlled by apoptosis. This process can be modulated by agents in the inflammatory microenvironment and leads to loss of neutrophil secretory function and its phagocytosis by macrophages which utilize a novel recognition mechanism such that the macrophage does not release pro-inflammatory mediators. It is hypothesized that this represents an alternative fate to necrosis, and one which would tend to limit tissue injury and promote inflammatory resolution. This is not to suggest that granulocyte necrosis does not occur; even at sites of 'beneficial inflammation' some necrotic neutrophils may be seen. However, since the development of inflammatory disease is currently thought to result from a multifactoral, quantitative imbalance between potentially injurious inflammatory influences and tissue defences, it is reasonable to suggest that the balance between neutrophil apoptosis and necrosis could represent one of several possible pivotal points in the control of inflammation. Finally, as the specific trigger and induction processes which permit closely related cells to undergo apoptosis at markedly different rates are uncovered, it may be possible to design new anti-inflammatory therapeutic strategies directed towards causing specific cell types to 'commit suicide' and to be cleared by the mechanisms which 'nature intended'. Thus we have begun to dissect the cellular events occurring in the resolution of acute inflammation. We believe that the rapid cessation of neutrophil emigration which occurs remarkably early in the evolution of the acute inflammatory response represents one of the earliest events in the resolution process. Clearly there is much work to be done on the underlying mechanisms. These are likely to be more accessible with the recent molecular characterization of chemotactic cytokines and surface molecules involved in neutrophil-endothelial adhesion transmigration events. It is also clear that apoptosis, which represents an alternative tissue injury-limiting fate to necrosis in situ, may be important in limiting tissue injury and determining whether inflammation persists or resolves. It is also possible that there are circumstances in which the macrophage recognition and clearance of apoptotic neutrophils is impaired. However, a second glance at the events which must occur during the resolution of even the simplest model of acute inflammation (Fig. 1) is sufficient reminder that the 'surface has barely been scratched'. There remain many obscure areas, not the least of which is the fate of the inflammatory macrophage after it has completed its scavenging functions.


Alpha-tocopherol, lipids and lipoproteins in knee-joint synovial fluid and serum from patients with inflammatory joint disease. 1. We have determined the antioxidant status of synovial fluid and serum of patients with inflammatory joint disease in terms of the biologically active lipid-soluble antioxidant, alpha-tocopherol. Synovial fluid concentrations of alpha-tocopherol were significantly lower relative to those of paired serum samples (P < 0.001). Serum levels of alpha-tocopherol in these patients did not differ significantly from those in control serum. 2. Lower concentrations of cholesterol, triacylglycerol and low-density lipoprotein were also observed in patients' synovial fluid compared with matched serum samples. However, multiple regression analysis of the data indicated that there remained a significant depletion of alpha-tocopherol, which was largely independent of these co-variables, in inflammatory synovial fluid. These findings are consistent with the consumption of alpha-tocopherol within the inflamed joint via its role in terminating the process of lipid peroxidation. 3. Nuclear magnetic resonance spectroscopic analysis of matched inflammatory synovial fluid and serum confirmed lower concentrations of triacylglycerol in synovial fluid together with evidence of a shortened mean triacylglycerol chain length. The latter metabolic difference suggests an increased utilization of triacylglycerols for energy within the inflamed joint.


Whole-body and site-specific bone remodelling in patients with previous femoral fractures: relationships between reduced physical activity, reduced bone mass and increased bone resorption. 1. A new tracer method is described for the non-invasive measurement of bone formation in the proximal femur. The method is based on our previously described whole-body method using 85Sr as the tracer (Reeve, J., Hesp, R. & Wootton, R. Calcif. Tissue Res. 1976; 22, 191-206). It allows correction to be made for long-term exchange processes within the skeleton. 2. The method has been applied in a study of regional and whole-body bone formation in 12 rehabilitated patients who had previously suffered a fracture of the proximal femur. Twelve healthy control subjects were studied, who were selected for their good health and continued physical activity. The aim was to explore the relationship between bone formation and physical activity. 3. Bone formation was similar in the two groups, both regionally and in the whole body. Based on analyses of four cadaver specimens, bone formation in the proximal femur was about one and two-thirds times that in the whole skeleton when related to mass of calcium in the region of interest. 4. Whole-body bone resorption, estimated from five measurements per subject of hydroxyproline excretion in relation to creatinine excretion, was significantly higher in the fracture patients (P < 0.01, Wilcoxon's test). 5. Estimates of current physical activity (and immediate pre-fracture physical activity) were made with a newly devised questionnaire. Historical levels of physical activity (at ages 15-45 years) were determined with Astrom's questionnaire. No bone formation index correlated with any index of physical activity. Urinary hydroxyproline excretion correlated inversely both with current physical activity and historical physical activity (for both regression coefficients P < 0.01). 6. The results are discussed in the light of our current understanding of the control of bone remodelling by the discrete basic multicellular units of bone. The opportunity to study regional bone resorption by the additional use of serial dual X-ray absorptiometry of the same region will in future allow the direct monitoring of the effects of therapeutic interventions which have been designed to prevent contralateral hip fracture.


Evidence for the indirect utilization of glucose for the synthesis of hepatic glycogen in man. 1. This study was designed to test the hypothesis that three-carbon intermediates can be used in the 'indirect' pathway of glycogen synthesis in human liver (i.e. a route additional to the use of glucose by the 'direct' pathway). 2. After an overnight fast, 13 patients were given an infusion of 20% (w/v) glucose before elective abdominal operation. All received a 2.5 g bolus of 2220 kBq of selectively 3H- and 14C-labelled glucose before removal of a 1 g biopsy of liver. 3H and 14C were determined in purified glycogen as well as in glucose and lactate from samples of peripheral blood. 3. The ratio and specific activities of 3H and 14C in glycogen were found to be significantly lower than those in administered glucose. By calculation, 7-74% of glycogen repletion occurred by indirect pathways and not all of this was from the glucose supplied. 4. This study suggests that the operation of a direct pathway in man is not exclusive and that significant repletion of hepatic glycogen occurs by an indirect route.


Glucose-6-phosphatase in normal adult human intestinal mucosa. 1. The existence of specific glucose-6-phosphatase activity in human intestinal mucosa has been somewhat controversial. 2. We have demonstrated the presence of low levels of specific glucose-6-phosphatase activity in normal human adult intestinal mucosa. Activity was found in oesophagus, stomach, duodenum and colon. 3. Immunoblot analysis using antibodies monospecific for the 36.5 kDa liver glucose-6-phosphatase catalytic subunit demonstrated that intestinal mucosa contains low levels of the glucose-6-phosphatase enzyme protein. 4. The low levels of activity together with problems of proteolysis make human intestinal biopsies unsuitable for use in the diagnosis of type 1 glycogen-storage disease.


Impaired fatty-meal-stimulated gallbladder contractility in patients with Crohn's disease. 1. The incidence of gallstones in patients with Crohn's disease is increased compared with that in healthy control subjects. This is in part due to reduced terminal ileal bile salt absorption and consequent increased cholesterol saturation in bile. The aim of this study was to evaluate gallbladder contractility, a second important factor in the pathogenesis of gallstones, in Crohn's disease. 2. Thirty patients with Crohn's disease and no known biliary tract disease and nine healthy control subjects were studied. After an overnight fast, gallbladder volume was determined by real-time ultrasonography before and 10, 20, 30, 40, and 50 min after ingestion of a standard liquid fatty meal. 3. Compared with healthy control subjects, patients with Crohn's disease had similar fasting gallbladder volumes (control, 18.7 +/- 2.3 ml; Crohn's disease, 18.2 +/- 2.3 ml). Percentage emptying was significantly impaired at 30, 40 and 50 min in patients with Crohn's disease compared with control subjects. Patients with Crohn's disease limited to the small bowel had gallbladder contractility that was comparable with that of control subjects, whereas in those with large-bowel disease, minimum residual gallbladder volume was significantly smaller than in control subjects. Patients with both large- and small-bowel Crohn's disease demonstrated the most marked abnormalities, with gallbladder volumes significantly larger than those of control subjects at 30, 40 and 50 min. Likewise, patients with Crohn's disease who had undergone previous bowel resection had impaired emptying at 30, 40 and 50 min.(ABSTRACT TRUNCATED AT 250 WORDS)


Relationship between hepatic lipid peroxidation and fibrogenesis in carbon tetrachloride-treated rats: effect of zinc administration. 1. Lipid peroxidation and hepatic fibrogenesis were investigated in 25 carbon tetrachloride-treated rats and in 25 control animals. Rats were further divided into two groups to receive either a standard diet or one supplemented with zinc. From each group, animals were killed at weeks 3 and 18 of the experiment for histological and biochemical assessments which included hepatic lipid peroxide and collagen concentrations and plasma zinc concentration as well as the hepatic activities of proline hydroxylase and collagenase. 2. Results indicated that oral zinc supplementation was associated with a decrease in lipid peroxidation (mean 51%; P < 0.05), collagen deposition (mean 32%; P < 0.001) and proline hydroxylase activity (mean 30%; P < 0.05) at week 18, together with an increase in collagenase activity (mean 208%; P < 0.01) at week 3, in carbon tetrachloride-treated rats. 3. There was a significant direct correlation between lipid peroxidation and proline hydroxylase activity in carbon tetrachloride-treated rats (r = 0.52; P < 0.01) and also a significant inverse correlation between lipid peroxidation and plasma zinc concentration in these animals (r = -0.62; P < 0.001). 4. These findings are consistent with the hypothesis that hepatic lipid peroxidation plays an important role in the aetiology of hepatic fibrogenesis and that zinc mitigates the process.


Glucocorticoid ameliorates altered gene expression of extracellular matrix components in kidneys of New Zealand black/white F1 mice. 1. We examined the effects of methylprednisolone on the levels of messenger RNA encoding for extracellular matrix components, including alpha 1(IV) collagen chain, laminin B1 and B2 chains, heparan sulphate proteoglycan and alpha 1(I) and alpha 1(III) collagen chains, and on the accumulation of these proteins in the renal cortex of New Zealand Black/White F1 mice. 2. At the onset of nephritis, at about 5 months of age, New Zealand Black/White F1 mice were divided in two groups that received either methylprednisolone or saline injections for 5 months. 3. The development of histological lesions and the glomerular deposition of IgG, IgM and C3 were suppressed by methylprednisolone treatment from 5 to 10 months of age. 4. The distribution and intensity of type IV collagen, laminin, heparan sulphate proteoglycan, type I collagen and type III collagen in renal cortex were decreased by the administration of methylprednisolone. 5. Levels of messenger RNA encoding for alpha 1(IV) collagen chain, laminin B1 and B2 chains, heparan sulphate proteoglycan and alpha 1(I) and alpha 1(III) collagen chains in the renal cortex of New Zealand Black/White F1 mice were significantly ameliorated at 8-10 months of age by methylprednisolone administration. 6. These results indicate that methylprednisolone treatment can serve as an effective therapeutic approach to abnormal extracellular matrix regulation in murine lupus nephritis.


Effects of parathyroid hormone and calcium and their interrelationship on urinary acidification in the rat. 1. The influence of thyroparathyroidectomy on renal function and specifically on acid excretion was studied in rats with or without oral supplementation of calcium. 2. Thyroparathyroidectomy caused a significant decrease in glomerular filtration rate, in the urinary/plasma inulin ratio and in overall acid excretion. These changes were not corrected by calcium supplementation. 3. Rates of proximal tubular acidification were studied by means of double-barrelled resin/reference microelectrodes. Acidification half-time was significantly increased in both thyroparathyroidectomized and calcium-supplemented thyroparathyroidectomized rats (8.38 s and 7.40 s, respectively) compared with control rats (5.44 s). 4. When 10(-6) mol/l A23187, a calcium ionophore, was added to the luminal bicarbonate solution, the acidification half-time returned to 3.97 s in the thyroparathyroidectomized rats, whereas no significant changes were detected in the properties of acidification in the control rats. 5. These data show that parathyroid hormone and cellular calcium are important factors involved in proximal tubular H+ secretion, which appears to be largely dependent on a well-defined concentration range of these agents.


Aortic compliance in young patients with heterozygous familial hypercholesterolaemia. 1. Aortic compliance and plasma lipid and lipoprotein levels were measured in 20 young patients with heterozygous familial hypercholesterolaemia and in 20 age- and sex-matched control subjects. 2. Patients with familial hypercholesterolaemia had significantly higher plasma cholesterol, low-density lipoprotein-cholesterol and triacylglycerol levels than control subjects (P < 0.001, P < 0.001 and P < 0.005, respectively). The patients with familial hypercholesterolemia also had significantly more compliant (distensible) aortas than the control subjects (P < 0.001), a significant inverse correlation being observed between compliance and age (r = 0.73, P < 0.001) and between compliance and mean blood pressure (r = -0.60, P < 0.005). 3. When the effects of age and sex on aortic compliance were corrected for, the blood pressure effect disappeared, significant correlations being observed between normalized compliance and cholesterol (r = 0.50, P < 0.03), low-density lipoprotein-cholesterol (r = 0.54, P < 0.01), high-density lipoprotein-cholesterol (r = -0.44, P < 0.05), low-density lipoprotein-/high-density lipoprotein-cholesterol ratio (r = 0.60, P < 0.0006) and duration of disease (r = 0.67, P < 0.002). Multivariate regression analysis showed that the low-density lipoprotein-/high-density lipoprotein-cholesterol ratio (P < 0.03) and duration of disease (P < 0.04) were the best predictors of normalized compliance. 4. We suggest that the measurement of aortic compliance in young patients with familial hypercholesterolaemia may potentially be a useful, non-invasive, research tool for assessing their susceptibility to atheroma.


Effect of chronic hypoxia on rat pulmonary resistance vessels: vasodilatation by atrial natriuretic peptide. 1. We have investigated the vasoreactivity of isolated pulmonary resistance vessels of rats after acclimatization to chronic hypoxia in a normobaric, hypoxic chamber. Vasoconstriction, in response to KCl and prostaglandin F2 alpha, and vasodilation, in response to atrial natriuretic peptide, were studied isometrically in a small-vessel myograph. Resting tensions were set to simulate transmural pressures of 17.5 mmHg or 35 mmHg. 2. There were no significant differences between intergroup internal vessel diameters or maximal contractile responses to either agonist. Both control and chronically hypoxic vessels generated significantly greater active contractions at 35 mmHg than at 17.5 mmHg. There were significant positive correlations between vessel diameter and maximum contractility for both control and chronically hypoxic vessels, but when contraction was expressed as equivalent transmural pressure no correlation existed. 3. There was a significant increase in potency (as measured by the concentration necessary to produce 50% of the maximum response) of KCl in chronically hypoxic vessels compared with control vessels at 35 mmHg, but not at 17.5 mmHg. In contrast, the potency of prostaglandin F2 alpha was significantly increased in chronically hypoxic vessels at 17.5 mmHg, but not at 35 mmHg. Thus the change in contractile responses of vessels from chronically hypoxic animals, in terms of maximal response and potency, is dependent on both resting pressure and agonist used. 4. After exposure to chronic hypoxia, atrial natriuretic peptide induced significantly greater maximal relaxation of pulmonary resistance vessels at both resting pressures, but its potency was unaffected.


[125I]calcitonin gene-related peptide binding in membranes of the ciliary body-iris block. Calcitonin gene-related peptide (CGRP) is a mediator of intraocular inflammatory responses, but it may also affect aqueous humour dynamics. The aim of the present work was to characterize CGRP binding sites in the eyes of various mammals. The binding of radiolabelled human CGRP to membranes from the ciliary body-iris (c+i) block of porcine eye showed characteristics expected of an interaction with a receptor site: it was reversible, saturable and displaced by rat CGRP and calcitonin. Studies with CGRP fragments demonstrated the importance of rather long carboxy-terminal sequences of the CGRP molecule for high-affinity binding to the receptor. Rat islet amyloid polypeptide (IAPP), which has about 50% structural similarity to CGRP, displaced radioligand binding nearly as efficiently as CGRP, while human IAPP was about twenty-fold less potent. No displaceable CGRP binding could be reliably demonstrated by the present method in c+i membranes from cat, rabbit and bovine eyes, thus indicating differences in the number or localization of CGRP receptors between different mammalian species.


Dityrosine formation in the proteins of the eye lens. The presence of dityrosine crosslinks in the proteins of the lens is a subject of some debate. We have investigated the formation of dityrosine in the lens proteins, the crystallins, through reactions mediated by reactive oxygen species, as well as through direct photolysis of proteins in the UVB region. Multiple methods were used to identify dityrosine. These include amino acid analysis of protein hydrolysates, quenching of the fluorescence at 400-410 nm by borate/boric acid solutions specific for dityrosine, and inhibition of formation of dityrosine in the presence of dithiothreitol. We find that reaction of the crystallins, alpha (alpha), beta (beta) and gamma (gamma), with singlet oxygen (1O2), hydroxyl radicals (.OH) and hydrogen peroxide (H2O2), does not result in the formation of either intermolecular or intramolecular dityrosine. It appears that the formation of dityrosine involves a radical mediated electron transfer reaction. Consistent with this, direct photolysis in the UVB region produces intramolecular dityrosine in all three proteins, with the efficiency varying in the order gamma - > beta - > alpha. Since the levels of electron transfer agents (e.g., Fe ions) in the lens are usually negligible, and since the amount of UVB radiation reaching the gamma--crystallin--rich lens nucleus is normally quite low, the changes of dityrosine formation in the crystallins in vivo appear remote.


Protein phosphatases and cell division cycle control. Fission yeast has at least ten protein phosphatase genes that appear to play distinct roles in cell cycle control. Because of functional overlap, a clear lethal phenotype can be obtained only after multiple genetic alterations. Cells that have lost the protein phosphatase 1 (PP1)-like dis2/sds21 phosphatase activities prematurely enter mitosis and remain in a defective mitotic state with high H1 kinase activity and without sister chromatid disjunction. The same phenotype can be obtained in the presence of hydroxyurea. Overexpression of PP1-like phosphatase, on the other hand, delays the entry into mitosis. Cells that have lost PP2A-like ppa2 phosphatase activity also prematurely enter mitosis with a reduction in cell size. This semi-wee phenotype is enhanced in delta ppa2 mutants treated with the phosphatase inhibitor, okadaic acid. Genetic interactions between ppa2 and mitotic regulators suggest that ppa1/ppa2 phosphatase may directly or indirectly inhibit p34cdc2/cyclin kinase. Thus both PP1- and PP2A-like phosphatases in fission yeast may negatively regulate entry into mitosis. The major property of the dis2/sds21 mutant which is distinct from those of the ppa2/ppa1 mutant is its failure to inactivate the p34cdc2/cyclin complex after entry into mitosis. A novel phosphatase regulator encoded by sds22+ binds to dis2 phosphatase and controls the substrate specificity which appears to become essential in the progression from metaphase to anaphase.


DNA replication and the cell cycle.

DNA replication and the cell cycle. The replication of DNA in the eukaryotic cell cycle is one of the most highly regulated events in cell growth and division. Biochemical studies on the replication of the genome of the small DNA virus simian virus 40 (SV40) have resulted in the identification of a number of DNA replication proteins from human cells. One of these, Replication Protein A (RPA), was phosphorylated in a cell cycle-dependent manner, beginning at the onset of DNA replication. RPA was phosphorylated in vitro by the cell cycle-regulated cdc2 protein kinase. This kinase also stimulated the unwinding of the SV40 origin of DNA replication during initiation of DNA replication in vitro, suggesting a mechanism by which cdc2 kinase may regulate DNA replication. Functional homologues of the DNA replication factors have been identified in extracts from the yeast Saccharomyces cerevisiae, enabling a genetic characterization of the role of these proteins in the replication of cellular DNA. A cellular origin binding protein had not been characterized. To identify proteins that function like T antigen at cellular origins of DNA replication, we examined the structure of a yeast origin of DNA replication in detail. This origin consists of four separate functional elements, one of which is essential. A multiprotein complex that binds to the essential element has been identified and purified. This protein complex binds to all known cellular origins from S. cerevisiae and may function as an origin recognition complex.


A small protein in model membranes: a time-resolved fluorescence and ESR study on the interaction of M13 coat protein with lipid bilayers. Model membranes with unsaturated lipid chains containing various amounts of M13 coat protein in the alpha-helical form were studied using time-resolved fluorescence and ESR spectroscopy. The lipid-to-protein (L/P) ratios used were > 12 to avoid protein-protein contacts and irreversible aggregation leading to beta-polymeric coat protein. In the ESR spectra of the 12-SASL probe in dioleoyl phosphatidylcholine (DOPC) bilayers no second protein induced component is observed upon incorporation of M13 coat protein. However, strong effects are detected on the ESR lineshapes upon changing the protein concentration. The ESR lineshapes are simulated by assuming a fixed ratio between the parallel (D parallel) and perpendicular (D perpendicular) diffusion coefficients of 4, and an order parameter equal to zero. It is found that increasing the protein concentration from L/P infinity to L/P 15 results in a decrease of the rotational diffusion coefficient D perpendicular from 3.4 x 10(7) to 1.9 x 10(7) s-1. In the time-resolved fluorescence experiments with DPH-propionic acid as a probe, it is observed that increasing the M13 coat protein concentration causes an increase of the two fluorescent lifetimes, indicating an increase in bilayer order. Analysis of the time-resolved fluorescence anisotropy decay allows one to quantitatively determine the order parameters <P2> and <P4>, and the rotational diffusion coefficient D perpendicular of the fluorescent probe. The order parameters <P2> and <P4> increase from 0.34 to 0.55 and from 0.59 to 0.77, respectively, upon adding M13 coat protein to DOPC bilayers with an L/P ratio of 35.(ABSTRACT TRUNCATED AT 250 WORDS)


Motional narrowing of the 2H NMR spectra near the chain melting transition of phospholipid/D2O mixtures. The reduction in spectral splitting, or motional narrowing, of the deuterium spectra of D2O/phospholipid mixtures near the main chain melting phase transition was studied for palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and equimolar mixtures of the two at 10% hydration. For POPC the splitting was about 1700 Hz in both the fluid and gel phases, dropping to zero near the phase transition (as reported previously). For POPE the splitting remained approximately constant above the phase transition. Below the phase transition the spectrum showed a single broad line whose linewidth varied between 100 Hz and 800 Hz. This was interpreted as being due to small domains of water within a weakly hydrated crystal. POPC:POPE (1:1) samples exhibited motional narrowing behaviour similar to that for POPC except that the splitting above the phase transition was approximately twice that below the transition. The relatively broad temperature range (approximately 20 K) of the transition is explained using a simple physical model involving lipid fluctuations near the phase transition.


The effect of maximal exercise on the activity of neutrophil granulocytes in highly trained athletes in a moderate training period. Leucocyte cell counts and the phagocytic and chemotactic activities of neutrophil granulocytes were investigated in highly endurance-trained long-distance runners (n = 10) and triathletes (n = 10) during a moderate training period and compared with untrained subjects (n = 10) before and up to 24 h after a graded exercise to exhaustion on a treadmill. After exercise a leucocytosis was noted with a significant increase in lymphocyte (P < or = 0.01) and neutrophil (P < or = 0.01) counts in all groups. In neutrophils the number of ingested inert latex beads was significantly increased (P < or = 0.01) from 0.21 (SD 0.09) to 0.45 (SD 0.22) in controls, from 0.20 (SD 0.12) to 0.56 (SD 0.16) in long-distance runners and from 0.25 (SD 0.08) to 1.03 (SD 0.42) particles per cell in triathletes 24 h after exercise, compared with resting values. The capability of neutrophils to produce microbicidal reactive oxygen species fell (P < or = 0.05) immediately after exercise in all subjects and then increased by 36 (SD 8)%, 31 (SD 6)% and 19 (SD 9)% in controls, runners and triathletes respectively up to 24 h after exercise (P < or = 0.05) compared with pre-start values. With respect to the absolute number of neutrophils, ingestion capacity, production of superoxide anions and chemotactic activity, no significant differences were found between athletes and control subjects at rest and after exercise.(ABSTRACT TRUNCATED AT 250 WORDS)


Neuromuscular fatigue during repetitive stimulation in elderly and young adults. The purpose of this investigation was to examine the integrity of neuromuscular transmission and impulse propagation during fatigue by examining the muscle compound action potential (M wave) in elderly and young adults. The tibialis anterior muscle of nine elderly [mean = 67.7 (SE 1.7) years] and nine young [mean = 26.7 (SE 1.2) years] adults was maximally stimulated repetitively at frequencies of 20, 30 or 40 Hz for 60 s on separate occasions. There was a significantly smaller resting M wave amplitude [7.9 (SE 0.4) mV versus 9.9 (SE 0.6) mV] and M wave area [0.038 (SE 0.005) mV s versus 0.06 (SE 0.004) mV.s] in the elderly versus the young adults respectively. Measurement of the evoked muscle contractile properties revealed significantly (P < 0.05) longer twitch durations and a significantly (P < 0.05) greater peak twitch torque [4.6 (SE 0.4) Nm versus 3.2 (SE 0.5) Nm] in the elderly versus the young adults, respectively. The elderly adults had a significantly greater torque decline during the 20-Hz trial; however, the decline in torque during the 30-Hz and 40-Hz trials was similar in the elderly and the young adults (30 Hz: 40%; 40 Hz: 56%). Throughout each of the stimulation trials, the decline in torque was accompanied by a significant reduction in M wave amplitude (20 Hz: 14%; 30 Hz: 53%; 40 Hz: 67%); M wave area also declined significantly during the 30-Hz (31%) and 40-Hz (53%) trials. There was no significant difference between the elderly and the young adults in the reduction in the M wave amplitude or area during each trial.(ABSTRACT TRUNCATED AT 250 WORDS)


Purification and characterization of proteinase In, a trypsin-like proteinase, in Escherichia coli. We previously found a trypsin-like proteinase which momentarily appears immediately before DNA synthesis in the cell cycle of Escherichia coli synchronized by phosphate starvation and which is closely related to the initiation of DNA replication (Kato, M., Irisawa, T., Morimoto, Y. and Muramatu, M., unpublished results). The proteinase was named proteinase In. It was purified approximately 2880-fold with a recovery of 15%. The isolated enzyme appeared homogeneous by gel filtration and electrophoresis. Its molecular mass was estimated by analytical gel filtration and SDS/PAGE as approximately 66 kDa. The isoelectric point of proteinase In is 4.9 and its optimal pH is approximately 9. Although protein In hydrolyzes fluorogenic substrate for trypsin, its hydrolytic activity seems markedly affected by amino-acid sequence lying towards the N-terminal from the P1 (lysine, arginine) residue. The proteinase does not hydrolyze N2-benzoyl-D,L-arginine-4-nitronanilide and fluorogenic substrates for chymotrypsin and elastase. The proteinase activity is inhibited by leupeptin, antipain and 4-nitrophenyl 4-guanidinobenzoate, but the effects of tosyl-L-lysine chloromethane, diisopropylfluorophosphate, benzamidine and pentamidine isethionate on the proteinase activity are weak or not inhibitory. Its activity is strongly affected in the presence of NaCl and KCl, and at a concentration of 1.5 M, these increase the activity 14-fold and 13-fold, respectively, above that without salt. Proteinase In was strongly inhibited by various esters of trans-4-guanidinomethylcyclohexanecarboxylic acid, and their inhibitory effects were roughly correlated with those on growth of E. coli. Proteinase activity was found in the cytoplasmic fraction.


The control of protein phosphatase-1 by targetting subunits. The major myosin phosphatase in avian smooth muscle is a novel form of protein phosphatase-1. The major protein phosphatase that dephosphorylates smooth-muscle myosin was purified from chicken gizzard myofibrils and shown to be composed of three subunits with apparent molecular masses of 130, 37 and 20 kDa, the most likely structure being a heterotrimer. The 37-kDa component was the catalytic subunit, while the 130-kDa and 20-kDa components formed a regulatory complex that enhanced catalytic subunit activity towards heavy meromyosin or the isolated myosin P light chain from smooth muscle and suppressed its activity towards phosphorylase, phosphorylase kinase and glycogen synthase. The catalytic subunit was identified as the beta isoform of protein phosphatase-1 (PP1) and the 130-kDa subunit as the PP1-binding component. The distinctive properties of smooth and skeletal muscle myosin phosphatases are explained by interaction of PP1 beta with different proteins and (in conjunction with earlier analysis of the glycogen-associated phosphatase) establish that the specificity and subcellular location of PP1 is determined by its interaction with a number of specific targetting subunits.