Salivary immunoglobulin A antibodies to gp41 in human immunodeficiency virus-seropositive patients: lack of correlation with disease progression.

Mucous membranes are the main route of transmission of human immunodeficiency virus (HIV). Interestingly, some viral inhibitory activities have been found in saliva. The purpose of this study was to determine the level of salivary immunoglobulin A (IgA) antibodies to gp41 in HIV+ patients at various disease stages to identify whether gp41 was able to induce vigorous humoral responses. Unstimulated saliva samples were obtained from three groups of subjects (n=37): group A (HIV-), group B (HIV+, CD4+ <200/mm3), and group C (HIV+, CD4+ >200/mm3). IgA antibody levels to purified gp41 were determined by enzyme-linked immunosorbent assay (ELISA). Western blot analyses were performed using HIV+ saliva to confirm IgA reactivity to gp41. ELISA demonstrated that HIV+ subjects had higher IgA antibody to gp41 than HIV- individuals. No significant differences were noted between HIV+, CD4+ <200/mm3 and CD4+ >200/mm3 subjects. High (81.25%) IgA reactivity to gp41 was demonstrated by Western blotting of saliva from all HIV+ individuals. In conclusion, gp41 responses are important in the HIV disease process, as indicated by the high IgA levels and gp41 reactivity in saliva of HIV+ patients.

Lovastatin induces apoptosis of spontaneously immortalized rat brain neuroblasts: involvement of nonsterol isoprenoid biosynthesis inhibition.

We have examined the effects of lovastatin and pravastatin (competitive HMG-CoA reductase inhibitors) on the growth and survival of rat brain neuroblasts. Lovastatin, but not pravastatin, suppressed cell growth by inducing apoptosis of neuroblasts in a dose- and time-dependent manner. Apoptosis was accompanied by a decrease in both Bcl-2 and Bcl-xL protein levels, suggesting that changes in the expression of these genes may contribute to apoptosis following lovastatin treatment. Lovastatin treatment was also associated with decreased prenylation of both Ras and Rho A proteins whereas Rac 1 geranylgeranylation was not affected. Lovastatin effects were fully prevented by mevalonate. The present data suggest that lovastatin induces apoptosis of rat brain neuroblasts by its capacity to decrease the prenylation of specific proteins involved in signal transduction pathways that control growth and survival of neuronal cells.

In vitro fluoride dose-response study of sterilized enamel lesions.

Currently our intra-oral model uses enamel specimens that have been disinfected by soaking in buffered formalin (pH 6.8). However, because of increasing emphasis on infection control, it is important to identify a way to sterilize these specimens. The aim of this study was to determine if autoclaved, or gas sterilized, lesioned enamel responds to fluoride (F) in the same way alcohol-disinfected enamel lesions do. Seventy-two formalin-disinfected, human enamel specimens (3 mm) were lesioned in demineralizing solution for 96 h and were then divided into three groups. One group was autoclaved; one group was gas sterilized (ethylene oxide), and the remaining 24 specimens were further disinfected in 70% ethanol for 10 min. Specimens in each group were then treated 4 times/day for 4 weeks with 0, 250 or 1,100 ppm F dentifrice slurries in an in vitro cycling, remin/demin model. Following treatment, fluoride uptake was analyzed by microdrill biopsy, and lesion depth and mineral content changes (DeltaM) were determined by transverse microradiography. Data were analyzed by one-way ANOVA analysis. In all three groups of specimens there were significant (p<0.05) differences in fluoride uptake in response to different fluoride treatments. Autoclaved lesions failed to provide dose- response data with regard to changes in lesion mineral content. Because formalin and 70% alcohol are only disinfectants, and autoclaving altered the responsiveness of enamel lesions, results from this study suggest that, of the methods tested, gas sterilization is the preferred method for sterilizing enamel specimens that will be used in intra-oral studies.

Somatostatin interferes with thyrotropin-induced G1-S transition mediated by cAMP-dependent protein kinase and phosphatidylinositol 3-kinase. Involvement of RhoA and cyclin E x cyclin-dependent kinase 2 complexes.

cAMP-mediated cell proliferation is a complex process that involves multiple pathways. Using a cAMP-dependent cell system, FRTL-5 thyroid cells, we have previously demonstrated the existence of a precise autocrine loop in the control of cell proliferation that involves the positive effector thyrotropin (TSH) and the general inhibitor somatostatin. In search of the regulatory mechanisms responsible for the TSH and somatostatin control of cell proliferation, we analyzed the cell cycle regulatory proteins and the cellular pathways involved in the action of both signals. The results show that specific inhibition of cAMP-dependent protein kinase (PKA) and phosphatidylinositol (PI) 3-kinase blocks independently TSH-induced FRTL-5 cell proliferation and that somatostatin interferes with both signals. Each pathway activates different proteins required for G(1)/S progression. Thus, PKA is responsible for the TSH-induction of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA levels, RhoA activation, and down-regulation of p27(kip1). These correlated events are necessary for FRTL-5 cell proliferation after TSH stimulation. Moreover, TSH through PKA pathway increases cyclin-dependent kinase 2 levels, whereas PI 3-kinase signaling increases cyclin E levels. Together, both pathways finally converge, increasing the formation and activation of cyclin E x cyclin-dependent kinase 2 complexes and the phosphorylation of the retinoblastoma protein, two important steps in the transition from G(1) to S phase in growth-stimulated cells. Somatostatin exerts its antiproliferative effect inhibiting more upstream the TSH stimulation of PKA and PI 3-kinase, interfering with the TSH-mediated increases of intracellular cAMP levels by inactivation of adenylyl cyclase activity. Together, these results suggest the existence of a PKA-dependent pathway and a new PKA-independent PI 3-kinase pathway in the TSH/cAMP-mediated proliferation of FRTL-5 thyroid cells.

Mutagenesis in the switch IV of the helical domain of the human Gsalpha reduces its GDP/GTP exchange rate.

The Galpha subunits of heterotrimeric G proteins are constituted by a conserved GTPase "Ras-like" domain (RasD) and by a unique alpha-helical domain (HD). Upon GTP binding, four regions, called switch I, II, III, and IV, have been identified as undergoing structural changes. Switch I, II, and III are located in RasD and switch IV in HD. All Galpha known functions, such as GTPase activity and receptor, effector, and Gbetagamma interaction sites have been found to be localized in RasD, but little is known about the role of HD and its switch IV region. Through the construction of chimeras between human and Xenopus Gsalpha we have previously identified a HD region, encompassing helices alphaA, alphaB, and alphaC, that was responsible for the observed functional differences in their capacity to activate adenylyl cyclase (Antonelli et al. [1994]: FEBS Lett 340:249-254). Since switch IV is located within this region and contains most of the nonconservative amino acid differences between both Gsalpha proteins, in the present work we constructed two human Gsalpha mutant proteins in which we have changed four and five switch IV residues for the ones present in the Xenopus protein. Mutants M15 (hGsalphaalphaS133N, M135P, P138K, P143S) and M17 (hGsalphaalphaS133N, M135P, V137Y, P138K, P143S) were expressed in Escherichia coli, purified, and characterized by their ability to bind GTPgammaS, dissociate GDP, hydrolyze GTP, and activate adenylyl cyclase. A decreased rate of GDP release, GTPgammaS binding, and GTP hydrolysis was observed for both mutants, M17 having considerably slower kinetics than M15 for all functions tested. Reconstituted adenylyl cyclase activity with both mutants showed normal activation in the presence of AlF(4)(-), but a decreased activation with GTPgammaS, which is consistent with the lower GDP dissociating rate they displayed. These data provide new evidence on the role that HD is playing in modulating the GDP/GTP exchange of the Gsalpha subunit.

Denervation-induced supersensitivity to forskolin and pinacidil is not related to changes in the adenylate cyclase transduction pathway in the rabbit femoral artery.

1. The present study investigates whether chemical sympathectomy compromises the relaxation of the rabbit femoral artery precontracted with serotonin. The vasodilating agents promoted cyclic adenosine monophosphate (cAMP) accumulation or opening of the potassium channels. The effect of denervation on the adenylyl cyclase transduction pathway was also studied. 2. 6-Hydroxydopamine treatment did not impair the relaxation to adenosine, 8-bromoadenosine-cAMP (a membrane-permeable analog of cAMP) and 3-isobutyl-1-methylxanthine (a cAMP phosphodiesterase inhibitor). Moreover, denervation enhanced the relaxation to forskolin (a direct Gs-type protein activator) and pinacidil (a potassium channel opener). 3. Denervation modified neither adenosine diphosphate ribosylation of Gs- and Gi-proteins nor adenylyl cyclase activity.

Lovastatin decreases prolactin and growth hormone gene expression in GH4C1 cells through a cAMP dependent mechanism.

The heterotrimeric G protein Gs couples several surface ligand receptors to cAMP production, as well as to both growth hormone (GH) and prolactin (PRL) gene expression in pituitary and GH cells. It has been shown that constitutively active alpha s stimulates transient expression of both PRL- and GH- chloramphenicol acetyl transferase (CAT) constructions, which indicates that both the PRL and GH promoter regions are under the influence of signal pathways mediated by alpha s. We have previously shown that the cholesterol lowering drug lovastatin decreases both the amount of G alpha s subunit in the membrane and the adenylyl cyclase activity in GH4C1 cells. Thus, we tried to verify whether that decrease in alpha s levels could affect PRL and GH secretion, as well as the expression of PRL- and GH-CAT constructions. Since the regulation of these two genes is dependent on the pituitary specific transcription factor Pit-1, the effect of lovastatin on the expression of Pit-1-CAT constructions was also studied. Our results show that lovastatin decreased the basal expression of these three cAMP-responsive genes in GH4C1 cells, being partially reversed by the addition of mevalonate to the culture medium. This effect of lovastatin on the promoter activities of the transfected constructions was also observed in PRL and GH secretion to the medium, suggesting that this drug produces similar changes in the endogenous promoters of both hormones. Moreover, the presence of lovastatin did not prevent the response to the cAMP activator forskolin, indicating that the main effect of this drug could be exerted through upstream adenylyl cyclase. In conclusion, our data indicate that lovastatin decreases the basal expression of Pit-1 and consequently of both GH and PRL genes through a mechanism probably mediated by the decrease of G alpha s levels in the cell membrane. Taken together, these results suggest that the activity of membrane heterotrimeric G proteins regulates the basal transcription of specific cellular genes in GH4C1 cells. Moreover the effects of lovastatin may be taken into account in the study of constitutively endocrine disorders associated with an increased secretion of either PRL or GH.

Effect of mevalonate availability on the association of G-protein alpha-subunits with the plasma membrane in GH4C1 cells.

We show that the levels and activity of the alpha-subunits of Gs and Gi proteins in plasma membrane of GH4C1 cells are regulated by the availability of mevalonate (MVA), and not by changes in cholesterol cell content. Changes in the levels of MVA, induced by modulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, determine the amount of both membrane-bound G alpha-subunits, which correlated with the activity of their effector adenylyl cyclase. Lipoprotein deficient serum (LPDS) decreases cholesterol content and increases both HMG-CoA reductase activity and G alpha-subunits in the membrane. Cholesterol and 25-hydroxycholesterol (25-HC) each repress HMG-CoA reductase and diminish G alpha-subunit levels. However, while cholesterol cell content is also decreased by 25-HC, exogenous cholesterol increases it. In addition, the decrease of both G alpha-subunits is reversed by the presence of MVA. This regulation appears to be mediated by nonsterol products generated from MVA. We assume that the first is the prenylation of the gamma-subunits, since the attachment of G alpha-subunits to the membrane is dependent on this modification. However, as neither of our treatments completely abolished protein prenylation, we conclude that another MVA derivative is required in addition to prenyl residues to the presence and activity of alpha-subunits in the membrane.

Effects of lovastatin on adenylyl cyclase activity and G proteins in GH4C1 cells.

We studied the effect of lovastatin, a cholesterol lowering drug, on the basal state of G-proteins in GH4C1 cells. Our data show that the addition of lovastatin markedly decreased the amount of the alpha-subunits of the Gs and Gi-proteins in the plasma membrane. The decrease of alpha s was correlated with a decrease in adenylyl cyclase activity, and both effects were reverted by the presence of mevalonate. As the attachment of G protein subunits to the membrane is dependent on gamma-subunit prenylation, we assume that the mechanism through which lovastatin exerts its effects on G-proteins is the lack of mevalonate for the synthesis of prenyl residues. In conclusion, our data indicate that some of the effects of lovastatin are mediated through changes in the basal state of G-protein in the membrane and consequently on adenylyl cyclase activity.

Information transfer in high dependency environments: an ergonomic analysis.

We have studied the information flow in HDE (with special focus on the information transfer process) using data provided by a group of experienced health care professionals. A model of the information flow in HDE was built up. It postulates the existence of quanta of information (due to the artificial fragmentation of the information flow produced by the clinical working processes: organization in shifts, demand of simultaneous activities from different staff members, etc.). This fragmentation is described by using the so-called Clinical Information Process Units (CIPUs), which correspond to patient care activities going on in parallely and serially linked blocks, performed by the staff in the specific environments. Due to a transfer in responsibility over the patient the CIPUs are linked by information transfer events which are described using transfer modules (TraMs). We exemplified 32 CIPUs related to the clinical environments (PreOp, Surgery, Recovery Intensive Care, Ward, Diagnostics, Outpatient) and the health care professional groups (Anesthesiologist/Intensivist, Surgeon, Nurse, Physician, Diagnostic Physician, Physical Therapist). A matrix was established providing the transfer situations among the CIPUs enabling a systematic classification of the TraMs. The contents of the TraMs are built up of information link elements, which are assembled according to the specific settings of the transfer situation given by the emitter, receiver and purpose. In summary we modelled the process of information transfer in HDE through CIPUs, TraMs and information links in a way, which may be useful to design information technology applications or to reorganize the information management in HDE.

Cholesterol cell content affects prolactin but not growth hormone release in GH4C1 cells.

It is generally accepted that cholesterol affects dynamic membrane properties and the function of membrane bound proteins involved in secretion processes. In the present study we employed GH4C1 cells treated with human lipoprotein-deficient serum (h-LPDS), exogenous cholesterol and high density lipoprotein (HDL3) to investigate the role of cholesterol cell content on PRL and GH basal release. Incubation of GH4C1 cells with h-LPDS decreased free cholesterol content and cholesterol added to the media increased it. HDL3 did not act as a cholesterol acceptor in either cholesterol-depleted or cholesterol-loaded cells; however, in depleted cells HDL3 was a net donor, significantly increasing cell cholesterol. Control or cholesterol loaded cells incubated in media with h-LPDS increased their secretion of PRL in parallel with the loss of cell cholesterol. Conversely, the addition of either cholesterol or HDL3 to cholesterol depleted cells inhibited PRL release. However, GH secretion was not modified by changes in free cholesterol in any of these situations. In the experiments in which HDL3 was present, a highly positive correlation was found between cholesterol cell content at the end of the experiment and PRL secretion, no effect could be related to the amount of added HDL3, suggesting that the HDL3 had no specific effect on the secretion of PRL or GH. Our results indicate that cholesterol cell content is an important factor in the release of PRL but not of GH, and emphasize the differences in the basal regulation of the secretion of both hormones.

Regulation by butyrate of the cAMP response to cholera toxin and forskolin in pituitary GH1 cells.

In pituitary GH1 cells, a rat growth hormone-producing cell line, butyrate elicited a dose-dependent increase in cholera toxin receptors as measured by an increased binding of 125I-labeled cholera toxin to the intact cells. Butyrate did not alter the affinity of cholera toxin binding, the dissociation constant being 0.4 nM for both control and butyrate-treated cells. Despite the increased binding, the cAMP response to cholera toxin was strongly reduced after exposure to butyrate. This reduction was dose-dependent and with butyrate 1--5 mM, intracellular and extracellular (medium) cAMP levels were decreased by more than 70% in cells incubated for 24 h with 1 nM cholera toxin. Forskolin (30 microM) elicited a cAMP response similar to that found with the toxin, and a similar inhibition of cAMP was also found after incubation of GH1 cells with butyrate. Butyrate also affected basal cAMP levels which were reduced by 40--60% in cells cultured for 24--48 h with the fatty acid. In order to study whether butyrate influenced cAMP synthesis and/or cAMP degradation, adenylyl cyclase and phosphodiesterase activities were determined in control cells and in cells incubated for 24 h with cholera toxin or forskolin. Butyrate had a dual effect since, besides activating phosphodiesterase by more than twofold, it also inhibited the cyclase by 40--50% in all groups. The in vitro response of adenylyl cyclase to stimulatory (NaF) and inhibitory (carbachol and adenosine) effectors was also examined. The absolute activity of the cyclase was always 40--50% lower in the cells incubated with butyrate, but the percentage change of activity obtained in butyrate-treated and untreated cells was unaltered. In addition, ADP-ribosylation of the guanine nucleotide stimulatory component of the cyclase (Gs) was not affected in the cells incubated with butyrate. These results suggest that the catalytic (C) subunit of adenylyl cyclase and/or its interaction with the regulatory components might be altered in butyrate-treated GH1 cells. The inhibition of the cAMP response in GH1 cells was accompanied by an inhibition of a biological action of the nucleotide, namely growth hormone (somatotropin) production which is primarily controlled by thyroid hormones in these cells. Forskolin alone did not affect the somatotropin levels but potentiated the growth hormone response to triiodothyronine. Butyrate produced a dose-dependent inhibition of this response, which was totally abolished at concentrations of butyrate higher than 1 mM.

[Predictive value of the APACHE and TISS indices on the postoperative morbidity in vascular surgery]. / Valor predictivo de los índices APACHE y TISS sobre la morbilidad postoperatoria en cirugía vascular.

The predictive value of age, weight, APACHE score (preoperative and 48 hours after surgery), and TISS index (24 and 48 hours after surgery) on morbidity has been studied on a group of 101 patients undergoing diverse vascular surgical procedures, with a complication rate of 47%. The 48 hour after surgery TISS alone, could correctly predict the postoperative course in 78% of the cases. This index, combined with the preoperative APACHE score, adequately predicted the course in 79% of the patients. When the 48 hour postoperative APACHE score was added to these two, the prediction was correct in 80%. A discriminant function including both APACHE scores, the 24 hour postoperative TISS, and age allows a proper classification in 81% of the cases. When the same criteria were used in another group of 56 patients--with 38% of postoperative complications--the results obtained showed no statistically significant difference. We conclude that both, the bayesian criterium, and the discriminant function can successfully be used for the prediction of the postoperative morbidity in vascular surgery.

Mechanism of action of somatostatin.

The chain of events leading to the manifestation of the biological action of somatostatin are described. Internalization is mediated by cytoskeletal proteins in the presence of calmodulin. Transduction of the somatostatin message at the membrane level takes place through inhibition of cyclic AMP accumulation and blockade of cytosol calcium increases. The influence of central and peripheral factors upon these processes is discussed and the importance of the Ni/Ns components is stressed. Thus, somatostatin also suppresses phosphoinositide turnover and stimulates soluble phosphodiesterase, thus reinforcing its negative effect on cyclase generation.

Somatostatin stimulates phosphodiesterase in rat anterior pituitary and brain, and GH4C1 cells.

Somatostatin administration to female rats increased the activity of calmodulin-dependent soluble phosphodiesterase, both in pituitary and brain. This effect was also seen in homogenates of GH4C1 cells pretreated with the hormone. When assayed in the presence of EGTA no differences in rat brain and pituitary phosphodiesterase were observed between controls and somatostatin-treated, but when assayed in the presence of calcium or calcium plus calmodulin a clear increase in the activity of the enzyme was detected. In GH4C1 homogenates prepared from somatostatin-pretreated cells there was an increase in phosphodiesterase activity assayed in the presence of EGTA vs non-treated controls, which was more clear when assayed in the presence of calcium or calcium plus calmodulin. These observations suggest that somatostatin effects derive, at least in part, from increased cyclic nucleotide degradation.

G-protein distribution in canine cardiac sarcoplasmic reticulum and sarcolemma: comparison to rabbit skeletal muscle membranes and to brain and erythrocyte G-proteins.

Herein we describe the distribution of G-proteins in canine cardiac sarcolemma (SL) and sarcoplasmic reticulum (SR) and in rabbit skeletal muscle SL, T-tubules, and junctional and longitudinal SR in comparison to G-proteins of human erythrocyte and bovine brain. G-proteins were unequivocally present in cardiac SL and SR and in skeletal T-tubules. Both cardiac fractions had two substrates specifically ADP-ribosylated by cholera toxin migrating on a sodium dodecyl sulfate-polyacrylamide gel at about 42 and 45 kDa. In skeletal muscle membranes, cholera toxi-labeled substrates migrated at about 42 and 62 kDa. Three substrates for pertussis toxin were resolved by sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis in cardiac SL at about 38, 40, and 43 kDa. Only the two higher molecular weight substrates were detected in cardiac SR and in any of several skeletal muscle membrane fractions. Comparison of G-proteins in muscle membrane fractions with G-proteins isolated from bovine brain and human erythrocyte as well as their reaction with antisera to either a common sequence of alpha subunits of G-proteins (G alpha common antibody) or to a unique sequence of the alpha subunit of Go (G alpha o antibody) indicated that the two lower molecular weight bands in cardiac SL are Go or Go-like, and therefore the upper band is probably Gi. These data demonstrate that pertussis toxin substrates are more heterogeneous than previously described and have implications for studies attempting to attribute physiological functions to G-protein isolates.

Inhibitory regulation of adenylyl cyclases. Evidence inconsistent with beta gamma-complexes of Gi proteins mediating hormonal effects by interfering with activation of Gs.

The possible effect of cholera toxin (CTX) on hormonal inhibition of adenylyl cyclase in somatostatin (SST)-sensitive GH3 cells was quantitatively evaluated. The toxin treatment employed led to an essentially complete ADP ribosylation of all alpha s subunits of the stimulatory regulatory component (Gs) of the system and to ca. 5- to 7-fold increases in the activity measured, yet it failed to affect the inhibitory action of SST regardless of whether analyzed in terms of degree of inhibition (ca. 60%) that is attainable or in terms of the apparent Kact with which the inhibitory hormone elicits its action. In absolute terms the activity inhibited after CTX was ca. 6 times larger than that inhibited under control conditions, indicating that SST is equally effective in regulating control and CTX-stimulated adenylyl cyclase system and that interpretations are independent of possible intramembraneous compartmentalizations of adenylyl cyclase and its various regulatory components. Since CTX-mediated ADP ribosylation of the alpha-subunits of Gs has been demonstrated to result in an at least 10-fold decrease in the potency (i.e. EC50) with which the beta gamma-complexes of G proteins act to stabilize preactivated purified alpha-subunits of Gs and in an approximately 300-fold decrease in the potency with which exogenously added beta gamma-complexes act to prevent activation of Gs in intact membranes, the present data indicate that beta gamma-complexes cannot be mediating the inhibitory effects of hormones by interfering with activation of the Gs of adenylyl cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)