Cysteine substitutions reveal dual functions of the amino-terminal tail in cystic fibrosis transmembrane conductance regulator channel gating

Previously, we observed that the cystic fibrosis transmembrane conductance regulator (CFTR) channel openings are destabilized by replacing several acidic residues in the amino-terminal tail with alanines (Naren, A. P., Cormet-Boyaka, E., Fu, J., Villain, M., Blalock, J. E., Quick, M. W., and Kirk, K. L. (1999) Science 286, 544-548). Here we determined whether this effect is due to the loss of negative charge at these sites and whether the amino-terminal tail also modulates other aspects of channel gating. We introduced cysteines at two of these positions (E54C/D58C) and tested a series of methanethiosulfonate (MTS) reagents for their effects on the gating properties of these cysteine mutants in intact Xenopus oocytes and excised membrane patches. Covalent modification of these sites with either neutral (MMTS) or charged (2-carboxyethylmethanethiosulfonate (MTSCE) and 2-(trimethylammonium)ethylmethanethiosulfonate (MTSET)) reagents markedly inhibited channel open probability primarily by reducing the rate of channel opening. The MTS reagents had negligible effects on the gating of the wild type channel or a corresponding double alanine mutant (E54A/D58A) under the same conditions. The inhibition of the opening rate of the E54C/D58C mutant channel by MMTS could be reversed by the reducing agent dithiothreitol (200 microm) or by elevating the bath ATP concentration above that required to activate maximally the wild type channel (>1 mm). Interestingly, the three MTS reagents had qualitatively different effects on the duration of channel openings (i.e. channel closing rate), namely the duration of openings was negligibly changed by the neutral MMTS, decreased by the positively charged MTSET, and increased by the negatively charged MTSCE. Our results indicate that the CFTR amino tail modulates both the rates of channel opening and channel closing and that the negative charges at residues 54 and 58 are important for controlling the duration of channel openings.     

Hydrogen-deuterium exchange as a probe of folding and assembly in viral capsids

The dynamics of proteins within large cellular assemblies are important in the molecular transformations that are required for macromolecular synthesis, transport, and metabolism. The capsid expansion (maturation) accompanying DNA packaging in the dsDNA bacteriophage P22 represents an experimentally accessible case of such a transformation. A novel method, based on hydrogen-deuterium exchange was devised to investigate the dynamics of capsid expansion. Mass spectrometric detection of deuterium incorporation allows for a sensitive and quantitative determination of hydrogen-deuterium exchange dynamics irrespective of the size of the assembly. Partial digestion of the exchanged protein with pepsin allows for region-specific assignment of the exchange. Procapsids and mature capsids were probed under native and slightly denaturing conditions. These experiments revealed regions that exhibit different degrees of flexibility in the procapsid and in the mature capsid. In addition, exchange and deuterium trapping during the process of expansion itself was observed and allowed for the identification of segments of the protein subunit that become buried or stabilized as a result of expansion. This approach may help to identify residues participating in macromolecular transformations and uncover novel patterns and hierarchies of interactions that determine functional movements within molecular machines.     

Age-sensitive and -insensitive pathways leading to JNK activation in mouse CD4(+) T-cells

The c-Jun N-terminal kinase (JNK) can be activated in T-cells either by the combination of TCR and CD28 costimulation or by a variety of stress-related stimuli including UV light, H(2)O(2), and hyperosmolar sorbitol solutions. In T-lymphocytes, TCR/CD28 stimulation of JNK leads to induction of new gene expression via c-Jun, ATF-2, and Elk-1. Phosphorylation of c-Jun in CD4(+) T-cells stimulated by CD3/CD4/CD28 cross-linking declines with age, due to diminished activation of JNK. Here we show that the age-related decline in TCR/CD28 activation of JNK reflects two effects of age: the accumulation of memory cells (in which JNK stimulation is poor regardless of donor age) and age-dependent declines in JNK activation within the naive subset. Cyclosporin A inhibits induction of JNK function by TCR/CD28, PMA/ionomycin, ceramide, or H(2)O(2), but not induction by UV light or hyperosmolar sorbitol. Although aging impairs JNK induction by UV light, it has no effect on JNK activation by ceramide, H(2)O(2), or sorbitol. The data as a whole indicate that there are at least four pathways that activate JNK in CD4(+) T-cells, of which two are age-sensitive and two others unaffected by aging. Two of the pathways (UV and hyperosmolar sorbitol) are insensitive to cyclosporin inhibition. Finally, we show that the alterations in JNK function are not due to changes in the expression of MKK4, an upstream activator of JNK, and that another JNK kinase, MKK7, is not expressed in splenic T-cells.     

IL-1 rescues scleroderma myofibroblasts from serum-starvation-induced cell death

Normal senescent adult human fibroblasts are relatively insensitive to the effects of serum deprivation in culture. In contrast, the myofibroblast phenotype, which is predominant in scleroderma-affected skin and cultured fibroblasts from scleroderma-affected skin, shows signs of deterioration within 3 days of serum starvation. The addition of IL-1 (5 ng/ml) prevents this deterioration. We propose that IL-1 is a factor promoting myofibroblast longevity with consequent fibrosis in scleroderma skin.     

Indirect intracoronary delivery of adenovirus encoding adenylyl cyclase increases left ventricular contractile function in mice

We performed indirect intracoronary delivery of adenovirus vectors in mice and explored techniques including hypothermia and pharmacological means to increase cardiac gene transfer. Mice were maintained in a normothermic state or cooled to 25 degrees C. The aorta or both the pulmonary artery and aorta were clamped while a needle was advanced into the left ventricular cavity to deliver adenovirus vectors encoding enhanced green fluorescent protein (EGFP) or murine adenylyl cyclase type VI (AC(VI)) with saline, sodium nitroprusside, acetylcholine, or serotonin. Clamping was maintained for 30 s (normothermia) or 2 min (25 degrees C) after adenovirus administration. Mice were killed 7 or 21 days later, and hearts were examined for EGFP expression. Compared with clamping the aorta alone and with no cooling, gene transfer was increased as follows: 1) 1.3-fold with hypothermia to extend dwell time; 2) 4.5-fold by clamping the aorta and the pulmonary artery; 3) 11.4-fold with nitroprusside administration; 4) 11.8-fold with serotonin addition, and 5) 14.3-fold with acetylcholine delivery. Gene expression remained substantial at 21 days, and no significant inflammatory response was seen. Efficacy of the method was tested by performing gene transfer of adenovirus encoding AC(VI). Fourteen days after gene transfer, hearts isolated from mice that received adenovirus encoding AC(VI) showed increased contractile function. Indirect intracoronary delivery of adenovirus vectors in mice is associated with efficient cardiac gene transfer and increased left ventricular function after AC(VI) gene transfer.     

The race concept in six regions: variation without consensus

Race, once the central concept in physical anthropology worldwide, now varies in the degree of support it receives in different regions. We present the currently available information on the status of the concept in the United States, the Spanish language areas, Poland, Europe, Russia, and China. Rejection of race ranges from high to low with the highest rejection occurring among anthropologists in the United States (and Canada). Rejection of race is moderate in Europe, sizeable in Poland and Cuba, and lowest in Russia and China. A discussion on the scientific and contextual reasons influencing these variations is presented. The tension between scientific evidence and social influences varies from region to region. The methods used in the studies reported here included questionnaires and content analysis. Response rates to questionnaires were often around 50 percent (with exception of the Polish studies). We discuss reasons for the low rates. Although a uniform method of data gathering is desirable, it may not suit scientists working in different traditions of theory and research. We conclude that it is once again timely to discuss the race concept in international meetings where all scientific and political changes occurring throughout the world in recent past decades are taken into account.     

Cardioprotective effects of ingliforib, a novel glycogen phosphorylase inhibitor

Interventions such as glycogen depletion, which limit myocardial anaerobic glycolysis and the associated proton production, can reduce myocardial ischemic injury; thus it follows that inhibition of glycogenolysis should also be cardioprotective. Therefore, we examined whether the novel glycogen phosphorylase inhibitor 5-Chloro-N-[(1S,2R)-3-[(3R,4S)-3,4-dihydroxy-1-pyrrolidinyl)]-2-hydroxy-3-oxo-1-(phenylmethyl)propyl]-1H-indole-2-carboxamide (ingliforib; CP-368,296) could reduce infarct size in both in vitro and in vivo rabbit models of ischemia-reperfusion injury (30 min of regional ischemia, followed by 120 min of reperfusion). In Langendorff-perfused hearts, constant perfusion of ingliforib started 30 min before regional ischemia and elicited a concentration-dependent reduction in infarct size; infarct size was reduced by 69% with 10 microM ingliforib. No significant drug-induced changes were observed in either cardiac function (heart rate, left ventricular developed pressure) or coronary flow. In open-chest anesthetized rabbits, a dose of ingliforib (15 mg/kg loading dose; 23 mg.kg(-1).h(-1) infusion) selected to achieve a free plasma concentration equivalent to an estimated EC(50) in the isolated hearts (1.2 microM, 0.55 microg/ml) significantly reduced infarct size by 52%, and reduced plasma glucose and lactate concentrations. Furthermore, myocardial glycogen phosphorylase a and total glycogen phosphorylase activity were reduced by 65% and 40%, respectively, and glycogen stores were preserved in ingliforib-treated hearts. No significant change was observed in mean arterial pressure or rate-pressure product in the ingliforib group, although heart rate was modestly decreased postischemia. In conclusion, glycogen phosphorylase inhibition with ingliforib markedly reduces myocardial ischemic injury in vitro and in vivo; this may represent a viable approach for both achieving clinical cardioprotection and treating diabetic patients at increased risk of cardiovascular disease.     

Induction of methyl tertiary butyl ether (MTBE)-oxidizing activity in Mycobacterium vaccae JOB5 by MTBE

Alkane-grown cells of Mycobacterium vaccae JOB5 cometabolically degrade the gasoline oxygenate methyl tertiary butyl ether (MTBE) through the activities of an alkane-inducible monooxygenase and other enzymes in the alkane oxidation pathway. In this study we examined the effects of MTBE on the MTBE-oxidizing activity of M. vaccae JOB5 grown on diverse nonalkane substrates. Carbon-limited cultures were grown on glycerol, lactate, several sugars, and tricarboxylic acid cycle intermediates, both in the presence and absence of MTBE. In all MTBE-containing cultures, MTBE consumption occurred and tertiary butyl alcohol (TBA) and tertiary butyl formate accumulated in the culture medium. Acetylene, a specific inactivator of alkane- and MTBE-oxidizing activities, fully inhibited MTBE consumption and product accumulation but had no other apparent effects on culture growth. The MTBE-dependent stimulation of MTBE-oxidizing activity in fructose- and glycerol-grown cells was saturable with respect to MTBE concentration (50% saturation level = 2.4 to 2.75 mM), and the onset of MTBE oxidation in glycerol-grown cells was inhibited by both rifampin and chloramphenicol. Other oxygenates (TBA and tertiary amyl methyl ether) also induced the enzyme activity required for their own degradation in glycerol-grown cells. Presence of MTBE also promoted MTBE oxidation in cells grown on organic acids, compounds that are often found in anaerobic, gasoline-contaminated environments. Experiments with acid-grown cells suggested induction of MTBE-oxidizing activity by MTBE is subject to catabolite repression. The results of this study are discussed in terms of their potential implications towards our understanding of the role of cometabolism in MTBE and TBA biodegradation in gasoline-contaminated environments.     

A role for immunology in invasion biology

Invasive species are of increasing conservation and economic concern, yet mechanisms underlying invasions remain poorly understood. We propose that variation in immune defences might help explain why only some introduced populations become invasive. Introduced species escape many of their native diseases, but also face novel pathogens that can induce costly, and sometimes deadly, immune responses in na?ve hosts. Therefore, favouring less resource-demanding and dangerous defence mechanisms and allocating a greater proportion of resources to growth and reproduction should favour invasion. Specifically, we argue that successful invaders should reduce costly systemic inflammatory responses, which are associated with fever and metabolic and behavioural changes, and rely more heavily on less expensive antibody-mediated immunity. Here we provide supporting arguments for this hypothesis and generate predictions that are testable using tools from the growing field of ecological immunology.