Salt bridge impact on global rigidity and thermostability in thermophilic citrate synthase.

It has been suggested that structural rigidity is connected to thermostability, e.g. in enzymes from thermophilic microorganisms. We examine the importance of correctly handling salt bridges, and interactions which we term 'strong polars', when constructing the constraint network for global rigidity analysis in these systems. Through a comparison of rigidity in citrate synthases, we clarify the relationship between rigidity and thermostability. In particular, with our corrected handling of strong polar interactions, the difference in rigidity between mesophilic and thermophilic structures is detected more clearly than in previous studies. The increase in rigidity did not detract from the functional flexibility of the active site in all systems once their respective temperature range had been reached. We then examine the distribution of salt bridges in thermophiles that were previously unaccounted for in flexibility studies. We show that in hyperthermophiles these have stabilising roles in the active site; occuring in close proximity to key residues involved in catalysis and binding of the protein.

Ouabain-insensitive salt and water movements in duck red cells. I. Kinetics of cation transport under hypertonic conditions.

Duck red cells in hypertonic media experience rapid osmotic shrinkage followed by gradual reswelling back toward their original volume. This uptake of salt and water is self limiting and demands a specific ionic composition of the external solution. Although ouabain (10(-4)M) alters the pattern of cation accumulation from predominantly potassium to sodium, it does not affect the rate of the reaction, or the total amount of salt or water taken up. To study the response without the complications of active Na-K transport, ouabain was added to most incubations. All water accumulated by the cells can be accounted for by net salt uptake. Specific external cation requirements for reswelling include: sufficient sodium (more than 23 mM), and elevated potassium (more than 7 mM). In the absence of external potassium cells lose potassium without gaining sodium and continue to shrink instead of reswelling. Adding rubidium to the potassium- free solution promotes an even greater loss of cell potassium, yet causes swelling due to a net uptake of sodium and rubidium followed by chloride. The diuretic furosemide (10(-3)M) inhibits net sodium uptake which depends on potassium (or rubidium), as well as inhibits net sodium uptake which depends on sodium. As a result, cell volume is stabilized in the presence of this drug by inhibition of shrinkage, at low, and of swelling at high external potassium. The response has a high apparent energy of activation (15-20 kcal/mol). We propose that net salt and water movements in hypertonic solutions containing ouabain are mediated by direct coupling or cis-interaction, between sodium and potassium so that the uphill movement of one is driven by the downhill movement of the other in the same direction.

Ouabain-insensitive salt and water movements in duck red cells. II. Norepinephrine stimulation of sodium plus potassium cotransport.

Catecholamines induce net salt and water movements in duck red cells incubated in isotonic solutions. The rate of this response is approximately three times greater than a comparable effect observed in 400 mosmol hypertonic solutions in the absence of hormone (W.F. Schmidt and T. J. McManus. 1977 a.J. Gen. Physiol. 70:59-79. Otherwise, these two systems share a great many similarities. In both cases, net water and salt movements have a marked dependence on external cation concentrations, are sensitive to furosemide and insensitive to ouabain, and allow the substitution of rubidium for external potassium. In the presence of ouabain, but the absence of external potassium (or rubidium), a furosemide-sensitive net extrusion of sodium against a large electrochemical gradient can be demonstrated. When norepinephrine-treated cells are incubated with ouabain and sufficient external sodium, the furosemide-sensitive, unidirectional influxes of both sodium and rubidium are half- maximally saturated at similar rubidium concentrations; with saturating external rubidium, the same fluxes are half-maximal at comparable levels of external sodium. In the absence of sodium, a catecholamine-stimulated, furosemide-sensitive influx of rubidium persists. In the absence of rubidium, a similar but smaller component of sodium influx can be seen. We interpret these results in terms of a cotransport model for sodium plus potassium which is activated by hypertonicity or norepinephrine. When either ion is absent from the incubation medium, the system promotes an exchange-diffusion type of movement of the co-ion into the cells. In the absence of external potassium, net movement of potassium out of the cell leads to a coupled extrusion of sodium against its electrochemical gradient.

Ouabain-insensitive salt and water movements in duck red cells. III. The role of chloride in the volume response.

This paper describes the effect of external chloride on the typical swelling response induced in duck red cells by hypertonicity or norepinephrine. Lowering chloride inhibits swelling and produces concomitant changes in net movements of sodium and potassium in ouabain-treated cells, which resemble the effect of lowering external sodium or potassium. Inhibition is the same whether chloride is replaced with gluconate or with an osmotic equivalent of sucrose. Since changes in external chloride also cause predictable changes in cell chloride, pH, and water, these variables were systematically investigated by varying external pH along with chloride. Lowering pH to 6.60 does not abolish the response if external chloride levels are normal, although the cells are initially swollen due to the increased acidity. Cells deliberately preswollen in hypotonic solutions with appropriate ionic composition can also respond to norepinephrine by further swelling. These results rule out initial values of cell water, chloride, and pH as significant variables affecting the response. Initial values of the chloride equilibrium potential do have marked effect on the direction and rate of net water movement. If chloride is lowered by replacement with the permeant anion, acetate, E(Cl) is unchanged and a normal response to norepinephrine, which is inhibited by furosemide, is observed. Increasing internal sodium by the nystatin technique also inhibits the response. A theory is developed which depicts that the cotransport carrier proposed in the previous paper (W.F. Schmidt and T.J. McManus. 1977b. J. Gen. Physiol. 70:81-97) moves in response to the net electrochemical potential difference driving sodium and potassium across the membrane. Predictions of this theory fit the data for both cations and anions.

Effect of norepinephrine on swelling-induced potassium transport in duck red cells. Evidence against a volume-regulatory decrease under physiological conditions.

Duck red cells exhibit specific volume-sensitive ion transport processes that are inhibited by furosemide, but not by ouabain. Swelling cells in a hypotonic synthetic medium activates a chloride-dependent, but sodium-independent, potassium transport. Shrinking cells in a hypertonic synthetic medium stimulates an electrically neutral co-transport of [Na + K + 2 Cl] with an associated 1:1 K/K (or K/Rb) exchange. These shrinkage-induced modes can also be activated in both hypo- and hypertonic solutions by beta-adrenergic catecholamines (e.g., norepinephrine). Freshly drawn cells spontaneously shrink approximately 4-5% when removed from the influence of endogenous plasma catecholamines, either by incubation in a catecholamine-free, plasma-like synthetic medium, or in plasma to which a beta-receptor blocking dose of propranolol has been added. This spontaneous shrinkage resembles the response of hypotonically swollen cells in that it is due to a net loss of KCl with no change in cell sodium. Norepinephrine abolishes the net potassium transport seen in both fresh and hypotonically swollen cells. Moreover, cells swollen in diluted plasma, at physiological pH and extracellular potassium, show no net loss of KCl and water ("volume-regulatory decrease") unless propranolol is added. Examination of the individual cation fluxes in the presence of catecholamines demonstrates that activation of [Na + K + 2Cl] co-transport with its associated K/Rb exchange prevents, or overrides, swelling-induced [K + Cl] co-transport. These results, therefore, cast doubt on whether the swelling-induced [K + Cl] system can serve a volume-regulatory function under in vivo conditions.

Catecholamine-stimulated ion transport in duck red cells. Gradient effects in electrically neutral [Na + K + 2Cl] Co-transport.

The transient increase in cation permeability observed in duck red cells incubated with norepinephrine has been shown to be a linked, bidirectional, co-transport of sodium plus potassium. This pathway, sensitive to loop diuretics such as furosemide, was found to have a [Na + K] stoichiometry of 1:1 under all conditions tested. Net sodium efflux was inhibited by increasing external potassium, and net potassium efflux was inhibited by increasing external sodium. Thus, the movement of either cation is coupled to, and can be driven by, the gradient of its co-ion. There is no evidence of trans stimulation of co-transport by either cation. The system also has a specific anion requirement satisfied only by chloride or bromide. Shifting the membrane potential by varying either external chloride (at constant internal chloride) or external potassium (at constant internal potassium in the presence of valinomycin and DIDs [4,4'-diisothiocyano-2,2'-disulfonic acid stilbene]), has no effect on nor-epinephrine-stimulated net sodium transport. Thus, this co-transport system is unaffected by membrane potential and is therefore electrically neutral. Finally, under the latter conditions-when Em was held constant near EK and chloride was not at equilibrium-net sodium extrusion against a substantial electrochemical gradient could be produced by lowering external chloride at high internal concentrations, thereby demonstrating that the anion gradient can also drive co-transport. We conclude, therefore, that chloride participates directly in the co-transport of [Na + K + 2Cl].

Coordinated regulation of shrinkage-induced Na/H exchange and swelling-induced [K-Cl] cotransport in dog red cells. Further evidence from activation kinetics and phosphatase inhibition.

Hypertonic shrinkage of dog red cells caused rapid activation of Na/H exchange and rapid deactivation of [K-Cl] cotransport. Hypotonic swelling caused delayed deactivation of Na/H exchange and delayed activation of [K-Cl] cotransport. Okadaic acid stimulated shrinkage-induced Na/H exchange and inhibited swelling-induced [K-Cl] cotransport. The data are compatible with the kinetic model of Jennings and Al-Rohil (1990. J. Gen. Physiol. 95:1021-1040) for volume regulation of [K-Cl] cotransport in rabbit red cells and suggest that in dog red cells Na/H exchange and [K-Cl] cotransport are controlled by a common regulatory system. The proposal of Jennings and Schulz (1991. J. Gen. Physiol. 96:799-817) that activation/deactivation of volume-sensitive transport involves phosphorylation/dephosphorylation of a regulatory protein is supported by these observations.

Coordinated regulation of Na/H exchange and [K-Cl] cotransport in dog red cells.

Swelling-activated [K-Cl] cotransport and shrinkage-activated Na/H exchange were studied in dog red cells with altered internal Mg or Li content. The two pathways responded in a coordinated fashion. When cells were depleted of Mg, [K-Cl] cotransport was stimulated and Na/H exchange was inhibited. Raising internal Mg had the opposite effect: [K-Cl] cotransport was inhibited and Na/H exchange was stimulated. Li loading, previously shown to stimulate Na/H exchange, inhibited [K-Cl] cotransport. From these reciprocal effects and from other evidence, we surmise that the regulation of Na/H exchange and [K-Cl] cotransport is conducted and coordinated by a discrete mechanism that responds to changes in cell volume and is sensitive to cytoplasmic Mg and Li concentrations.

Sexual partners, penetrative sexual partners and HIV risk.

This paper argues that the notion of sexual partners per se is insufficient for estimating levels of HIV risk behaviour or changes in HIV risk over time, even though it is a crucial element of most epidemiological models of HIV. The concept of a penetrative sexual partner (PSP) is introduced as a considerably more accurate measure of HIV risk. Using data from a longitudinal study of 930 homosexually active men in England and Wales, this paper demonstrates that variation in numbers of PSPs (and thus HIV risk) is not related to variation in the gross numbers of sexual partners.

No connection between alcohol use and unsafe sex among gay and bisexual men.

OBJECTIVE: To investigate the relationship between alcohol use and unsafe sexual behaviour. METHODS: The paper discusses data collected from 461 gay and bisexual men interviewed in England and Wales by Project SIGMA during 1991-1992. These data were collected during face-to-face interviews using retrospective weekly diary techniques and include details of all sexual sessions and alcohol use. The 819 reported sexual sessions with other men are divided into those that involved alcohol use (30.6%) and those that did not. RESULTS: Differences in the incidence of HIV risk behaviours between sexual sessions that involved alcohol use and those that did not are small, and none are statistically significant. Furthermore, for those men who engaged in sexual behaviour whilst under the influence of alcohol, the quantity of alcohol consumed had no effect on sexual behaviour. CONCLUSIONS: Among gay and bisexual men, sex under the influence of alcohol is no more likely to be unsafe than sex among men who have not consumed alcohol.

Sex role separation in sexual diaries of homosexual men.

OBJECTIVE: To measure types of sex role prevalence in common and risk-related behaviours among gay men for modelling HIV transmission. DESIGN: Cohort study of 385 homosexually active men recording sexual diaries over 1-month periods. METHODS: Measures of incidence of behavioural sex roles for masturbation, fellatio, anal intercourse and anilingus by relationship type, derived from 1-month sexual diary data. RESULTS: Low behavioural role rigidity for masturbation and fellatio, but higher rigidity for anal intercourse and anilingus. Participants with no regular partner showed a relatively low frequency of anal intercourse, whereas those in closed relationships showed a high frequency. CONCLUSION: Although anal intercourse shows a certain degree of behavioural role rigidity, this rigidity is not large enough to conclude that gay men exclusively engage in either an active or a passive role. Typical rates for exclusive active and passive roles for anal intercourse during the month the diaries were recorded were in the range of 12-15%; the dual role was significantly higher.

Cation-anion cotransport.

Two methods have been described for the study of cation-chloride cotransport systems. The zero-trans efflux method is designed to determine stoichiometric relationships between cotransported ions under conditions where ion exchanges cannot occur. These exchanges (e.g., Na+/Na+, K+/K+) may occur as partial or incomplete reactions of a cotransport process and can lead to erroneous determinations of the stoichiometry of the cotransport process. The zero-trans efflux method can also be used to study the effects of cell volume, pH, and intracellular ion concentrations on cotransport processes. The valinomycin method is used to determine the electrogenicity or electroneutrality of transport, and in this regard can be used in conjunction with other methods such as those employing potential-sensitive dyes or microelectrodes. Other, more recently developed ionophores with specificity for lithium rather than potassium have now been used to study the effect of Em on the ATP-dependent Na+/K+ pump. It may be possible to use such ionophores to confirm the suspected electroneutrality of (K+ + Cl-) cotransport systems, as well as for other studies of specific potassium transport processes in which valinomycin obviously cannot be used. Both methods discussed in detail in this chapter, and particularly the valinomycin method, were originally devised for use in red blood cells in order to take advantage of (or circumvent) properties of the red cell membrane, such as its low permeability to sodium and potassium and relatively high permeability to chloride. However, valinomycin has been used successfully to demonstrate the electroneutrality of (Na+ + K+ + 2Cl-) cotransport in MDCK cells, and the zero-trans efflux method should be applicable to the study of transport processes in other types of cells in suspension, so long as the transport system being studied can be accurately defined (e.g., as an inhibitor-sensitive or chloride-dependent cation flux) and comprises a significant fraction of the total salt efflux.

A study of the relationship between ABO blood groups, secretor status and infection with Neisseria gonorrhoeae.

The distribution of ABO blood groups and secretor status among 216 male caucasian patients with gonorrhoea was not significantly different from that in 2043 male caucasians who formed a comparison population.

Trends in sexual behaviour in a cohort of homosexual men: a 7 year prospective study.

A cohort of homosexual men at risk for human immunodeficiency virus (HIV) infection were studied prospectively over a 7-year period (1982/88) to assess trends in sexual behaviour and amyl nitrite intake. During the period, there were dramatic declines in the proportion of HIV seropositive and seronegative subjects reporting multiple casual partners for anal intercourse, unprotected anal intercourse and recreational use of amyl nitrite. Reported rates of orogenital intercourse remained the same during the period, whilst the total number of seroconversions fell from 17 for the period 1982-84 to 8 for 1985-88. High-risk sexual and related social behaviour among homosexual men, as assessed by patterns of anal intercourse behaviour and nitrite intake, changed over the 7-year period, with the greatest changes apparent before the widespread availability of HIV antibody testing and public education campaigns. This highlights the effectiveness of peer-group and community-based programmes in modifying the sexual behaviour of their members.

Eight year prospective study of HIV infection in a cohort of homosexual men--clinical progression, immunological and virological markers.

Three hundred and fourteen homosexual/bisexual men at risk for human immunodeficiency virus (HIV) infection (170 seroprevalent HIV-positive, 144 seronegative) were prospectively studied over 8 years to assess rates of HIV infection and disease progression, in conjunction with cellular and HIV serological markers. In HIV-positive subjects, CD4+ lymphocyte counts rose strikingly during the period surrounding seroconversion, then fell progressively over the intervening period to a mean level of 300 cells/mm3 when AIDS developed. Changes in CD8+ lymphocyte counts were less consistent. The trend for HIV serological markers over the study period was of progressive decline in the proportion of subjects with anti-p24 antibody, associated with an increase in the proportion of subjects with detectable HIV antigenaemia. However, only 45% of subjects tested had lost anti-p24 antibody by the time of AIDS diagnosis, and HIV antigen was detectable up to 4 years before this. Different HIV serological patterns were also observed in subjects presenting either with Kaposi's sarcoma or opportunist infections. Our data support the continued use of cellular and virological markers in the evaluation of HIV disease; however, the variability observed in this study highlights their limited ability in predicting specific clinical events. Care should therefore be taken to encompass both clinical and laboratory information in the medical assessment of the HIV-infected individual.

Markers of immune cell activation and disease progression. Cell activation in HIV disease.

Immune cell activation is a feature of infection with the human immunodeficiency virus (HIV). Here we report our studies on a cohort of over 400 patients with HIV infection studied cross-sectionally and longitudinally to examine the relationship between markers of immune cell activation and disease progression. To examine disease progression, 340 patients with HIV infection but without AIDS were followed for a total of 574 patient years, during which 56 developed AIDS. In our first study, 157 patients in CDC groups II-IV were examined cross-sectionally for in vivo expression of the activation markers HLA-DR and CD25 on CD3, CD4 and CD8 T cells. Levels of CD3+ HLA-DR+ T cells are high in HIV infection and show a significant negative correlation with CD4 counts (r = 0.52; p < 0.001). The appearance of HLA-DR+ CD3+ T cells is an early feature of asymptomatic HIV+ patients, with a greater proportion (82%) showing abnormally high levels of these than abnormally low levels of CD4 (52%; p < 0.001). Examining activation of the CD4 subset specifically is likely to be of greater interest, given that this cell is the viral target. Indeed, we found that in the cross-sectional study, levels of HLA-DR+ and CD25+ CD4 lymphocytes show a step-wise linear increase with increasing disease severity (significant test for linear trend; p < 0.001). In our previous studies, only declining CD4 count has shown such a significant linear trend. These data suggest that measuring activated CD4+ T cells in the periphery may be a powerful predictive tool. In our second study, we examined the expression of other markers acquired (CD45R0) and lost (CD45RA) following activation of naïve T cells. Examining expression of these on CD4 and CD8 cells cross-sectionally in 71 HIV+ patients, we found abnormalities in percentage levels of CD45RA+ and CD45R0+ populations, none of which showed any relationship to disease severity. Intriguingly, however, we noted that the surface density of both CD45RA and CD45R0 molecules on CD4 and CD8 cells was markedly and significantly reduced at all stages of HIV infection (eg relative specific fluorescence reduced by up to 50%; p < 0.001). This abnormality was confirmed in studies using antibodies to a common epitope on all CD45 isoforms (pan-CD45) and to the CD45RB isoform. Finally, returning to the question of immune cell markers of activation and disease progression, we have examined some of the best documented markers in our longitudinal study.(ABSTRACT TRUNCATED AT 400 WORDS)

Vibrio damsela infection in a stranded leatherback turtle (Dermochelys coriacea).

Necropsy of a stranded adult leatherback turtle (Dermochelys coriacea) determined that the animal died as a result of valvular endocarditis and septicemia. Vibrio damsela was isolated from the endocardial thrombus. The route of entry for infection probably was through the gastrointestinal tract.

Death in sheep following dosing with copper diethylamine oxyquinoline sulphonate as a commercial injectable copper preparation.

Death occurred in sheep following diethylamine oxyquinoline sulphonate (DOS) copper injections given at recommended dose rates. The copper content in unused portions of DOS copper packs was normal and free of bacterial contamination. Liver and blood copper levels in dead and sick sheep were not high. Sick sheep showed signs of hepatic encephalopathy and dead sheep were generally piled against fences and scrub. Deaths were associated with acute, severe, generalised, centrilobular, hepatocellular necrosis and live sheep had elevated circulating levels of liver enzymes consistent with liver damage. In recovered sheep there were no residual complications. It would appear that even at 0.5 mg/kg of DOS copper the safety threshold may sometimes be exceeded in some sheep.

Helicobacter pylori: an emerging infectious disease.

Helicobacter pylori is the most common chronic bacterial infection in the world, colonizing the stomachs of more than 50% of the human population. The discovery of this bacterium has changed the concept of care and management for peptic ulcer disease, mucosa-associated lymphomas, gastritis, and gastric carcinoma. Although the mode of transmission is not definitively known, person-to-person contact is suspected. This article discusses H. pylori, the associated clinical syndromes and diseases, risk factors, and current pharmacologic management.