Evidence that Ser775 in the alpha subunit of the Na,K-ATPase is a residue in the cation binding pocket

Substitution of alanine for Ser775 in a ouabain-resistant alpha1 sheep isoform causes a 30-fold decrease in apparent affinity for K+ as an activator of the Na,K-ATPase, as well as an increase in apparent affinity for ATP (Arguello, J. M., and Lingrel, J. B (1995) J. Biol. Chem. 270, 22764-22771). This study was carried out to determine whether Ser775 is a direct cation-ligating residue or whether the change in apparent affinity for K+ is secondary to a conformational alteration as evidenced in the change in ATP affinity, with the following results. Kinetics of K+(Rb+) influx into intact cells show that the change is due to a change in K+ interaction at the extracellular surface. The K+ dependence of formation of K+-occluded enzyme (E2(K)) and of the rate of formation of deoccluded enzyme from E2(K) indicate that the Ser775 --> Ala mutation results in a marked increase (>/=30-fold) in rate of release of K+ from E2(K). The high affinity Na+-like competitive antagonist 1,3-dibromo2,4,6-tris-(methylisothiouronium)benzene (Br2TITU), which interacts with the E1 conformation and blocks cytoplasmic cation binding (Hoving, S., Bar-Shimon, M., Tijmes, J. J. , Tal, D. M., and Karlish, S. J. D. (1995) J. Biol. Chem. 270, 29788-29793), inhibits Na+-ATPase of the mutant less than the control enzyme. With intact cells, Br2TITU acts as a competitive inhibitor of extracellular K+ activation of both the mutant and control enzymes. In this case, the mutant was more sensitive to inhibition. With vanadate as a probe of conformation, a difference in conformational equilibrium between the mutant and control enzymes could not be detected under turnover conditions (Na+- ATPase) in the absence of K+. These results indicate that the increase in apparent affinity for ATP effected by the Ser775 --> Ala mutation is secondary to a change in intrinsic cation affinity/selectivity. The large change in affinity for extracellular K+ compared with cytoplasmic Na+ and to Br2TITU binding supports the conclusion that the serine hydroxyl is either part of the K+-gate structure or a direct cation-ligating residue that is shared by at least one Na+ ion, albeit with less consequence on rate constants for Na+ binding or release compared with K+.     

Effect of tidal volume and frequency on airway responsiveness in mechanically ventilated rabbits

We evaluated the effects of the rate and volume of tidal ventilation on airway resistance (Raw) during intravenous methacholine (MCh) challenge in mechanically ventilated rabbits. Five rabbits were challenged at tidal volumes of 5, 10, and 20 ml/kg at a frequency of 15 breaths/min and also under static conditions (0 ml/kg tidal volume). Four rabbits were subjected to MCh challenge at frequencies of 6 and 30 breaths/min with a tidal volume of 10 ml/kg and also under static conditions. In both groups, the increase in Raw with MCh challenge was significantly greater under static conditions than during tidal ventilation at any frequency or volume. Increases in the volume or frequency of tidal ventilation resulted in significant decreases in Raw in response to MCh. We conclude that tidal breathing suppresses airway responsiveness in rabbits in vivo. The suppression of narrowing in response to MCh increases as the magnitude of the volume or the frequency of the tidal oscillations is increased. Our findings suggest that the effect of lung volume changes on airway responsiveness in vivo is primarily related to the stretch of airway smooth muscle.     

C-C chemokines, pivotal in protection against HIV type 1 infection

Exposure to HIV type 1 (HIV-1) does not usually lead to infection. Although this could be because of insufficient virus titer, there is now abundant evidence that some individuals resist infection even when directly exposed to a high titer of HIV. This protection recently has been correlated with homozygous mutations of an HIV-1 coreceptor, namely CCR5, the receptor for the beta-chemokines. Moreover, earlier results already had shown that the same chemokines markedly suppress the nonsyncitial inducing variants of HIV-1, the chief virus type transmitted from person to person. CCR5 mutation, as a unique mechanism of protection, is, however, suspect because HIV-1 variants can use other chemokine receptors as their coreceptor. Moreover, recent results have established that infection can indeed sometimes occur with such mutations. Here, we report on transient natural resistance over time of most of 128 hemophiliacs who were inoculated repeatedly with HIV-1-contaminated Factor VIII concentrate from plasma during 1980-1985 before the development of the HIV blood test. Furthermore, and remarkably, 14 subjects remain uninfected to this date, and in these subjects we found homozygous CCR5 mutations in none but in most of them overproduction of beta chemokines. In vitro experiments confirmed the potent anti-HIV suppressive effect of these chemokines.     

A biologically motivated partitioning of mortality

For over a century, actuaries and biologists working independently of each other have presented arguments for why total mortality needs to be partitioned into biologically meaningful subcomponents. These mortality partitions tended to overlook genetic diseases that are inherited because the partitions were motivated by a paradigm focused on aging. In this article, we combine and extend the concepts from these disciplines to develop a conceptual partitioning of total mortality into extrinsic and intrinsic causes of death. An extrinsic death is either caused or initiated by something that orginates outside the body of an individual, while an intrinsic death is either caused or initiated by processes that originate within the body. It is argued that extrinsic mortality has been a driving force in determining why we die when we do from intrinsic causes of death. This biologically motivated partitioning of mortality provides a useful perspective for researchers interested in comparative mortality analyses, the consequences of population aging, limits to human life expectancy, the progress made by the biomedical sciences against lethal diseases, and demographic models that predict the life expectancy of future populations.     

Heteronuclear NMR studies of the combined Src homology domains 2 and 3 of pp60 c-Src: effects of phosphopeptide binding

The results of heteronuclear NMR studies on the combined Src homology domains 2 and 3 (SH3-SH2) of pp60 c-Src are presented. Resonance assignments were obtained using heteronuclear triple-resonance experiments in conjunction with 15N-separated nuclear Overhauser effect spectroscopy (NOESY) data. A modified three-dimensional 13CO-15N-1H spectral correlation experiment [(HACA)CO(CA)-NH] with improved sensitivity is presented that provided additional sequential information and resolved several ambiguities. Chemical shifts and sequential- and medium-range NOE cross peaks indicate that the structures of both the SH3 and SH2 portions of the polypeptide are very similar to those of the isolated SH3 and SH2 domains. Binding of a high-affinity phosphopeptide, EPQpYEEIPIYL, induces large chemical shift changes at several locations in the SH2 domain. Comparison with known results for peptide binding to SH2 domains shows that the residues displaying the largest effects are all involved in peptide binding or undergo significant conformational changes upon binding. However, subtle changes of both 1H and 15N chemical shifts are observed for residues within the SH3 domain and the connecting linker region, indicating possible cross-domain communication.     

The impact of mucositis on alpha-hemolytic streptococcal infection in patients undergoing autologous bone marrow transplantation for hematologic malignancies

BACKGROUND: Antibacterial prophylaxis with quinolone antibiotics has resulted in an increase in streptococcal infections among bone marrow transplantation (BMT) recipients with myelosuppression. Oral ulceration (mucositis), which frequently occurs as a consequence of chemotherapy, has been implicated as a significant portal of entry for streptococci. The objectives of this study were to confirm the correlation between mucositis and streptococcal bacteremia, determine the risk associated with this correlation, and evaluate the impact of mucositis and streptococcal bacteremia on hospital course and costs associated with autologous BMT. METHODS: This was a retrospective, case-control study in which the charts of autologous BMT recipients treated for hematologic malignancies between 1990 and 1996 were reviewed. Twenty-four patients were identified who met the criteria of autologous BMT; their blood cultures confirmed (x2) alpha-hemolytic streptococcal sepsis. A control group of 45 without positive cultures was matched by gender, age, diagnosis, and treatment to the study group. RESULTS: The results confirm that ulcerative mucositis is a significant risk factor for alpha-hemolytic streptococcal bacteremia among autologous BMT patients. Of the 24 patients with bacteremia, 15 of 24 (62%) had ulcerative mucositis, compared with 16 of 45 (36%) of patients in the control population (P < 0.05). Patients with ulcerative mucositis were found to be three times as likely to develop alpha-hemolytic streptococcal bacteremia as those without ulcerative mucositis (odds ratio=3.02). Both independently and as a cofactor associated with bacteremia, mucositis adversely affected the length of hospital stay (LOS). Of all the patients studied, those with oral ulcerations had a LOS of 34 days, compared with 29 days for patients without oral ulcerations (P < 0.05). Of patients in the study group, those with oral ulcerations stayed in the hospital 6 days longer than patients without oral ulcerations (40 days vs. 34 days, P < 0.05). CONCLUSIONS: Oral ulcerative mucositis is a significant, common, and important risk factor for alpha-hemolytic streptococcal bacteremia in BMT recipients with myelosuppression; it results in longer hospital stay and increased costs.     

Desoxyepothilone B: an efficacious microtubule-targeted antitumor agent with a promising in vivo profile relative to epothilone B

A new class of 16-membered macrolides, the epothilones (Epos), has been synthesized and evaluated for antitumor potential in vitro and in vivo. Recent studies in these and other laboratories showed that epothilones and paclitaxel (paclitaxel) share similar mechanisms of action in stabilizing microtubule arrays as indicated by binding-displacement studies, substitution for paclitaxel in paclitaxel-dependent cell growth, and electron microscopic examinations. The present study examined cell growth-inhibitory effects in two rodent and three human tumor cell lines and their drug-resistant sublines. Although paclitaxel showed as much as 1, 970-fold cross-resistance to the sublines resistant to paclitaxel, adriamycin, vinblastine, or actinomycin D, most epothilones exhibit little or no cross-resistance. In multidrug-resistant CCRF-CEM/VBL100 cells, IC50 values for EpoA (1), EpoB (2), desoxyEpoA (3) (dEpoA), desoxyEpoB (4) (dEpoB), and paclitaxel were 0.02, 0.002, 0.012, 0.017, and 4.14 microM, respectively. In vivo studies, using i.p. administration, indicated that the parent, EpoB, was highly toxic to mice and showed little therapeutic effect when compared with a lead compound, dEpoB. More significantly, dEpoB (25-40 mg/kg, Q2Dx5, i.p.) showed far superior therapeutic effects and lower toxicity than paclitaxel, doxorubicin, camptothecin, or vinblastine (at maximal tolerated doses) in parallel experiments. For mammary adenocarcinoma xenografts resistant to adriamycin, MCF-7/Adr, superior therapeutic effects were obtained with dEpoB compared with paclitaxel when i.p. regimens were used. For ovarian adenocarcinoma xenografts, SK-OV-3, dEpoB (i.p.), and paclitaxel (i. v.) gave similar therapeutic effects. In nude mice bearing a human mammary carcinoma xenograft (MX-1), marked tumor regression and cures were obtained with dEpoB.     

Report of the first workshop on the genetic map of bovine chromosome 1

Allergic bronchopulmonary aspergillosis (ABPA), occurring primarily in patients with asthma or cystic fibrosis (CF), is a hypersensitivity reaction to Aspergillus fumigatus (Af), and is characterized by increased serum IgE levels and peripheral blood and pulmonary eosinophilia. We evaluated the IgE and cytokine profile in ABPA through enzyme-linked immunosorbent assay (ELISA), and evaluated eosinophil activity with the eosinophil peroxidase (EPO) assay. IgE and cytokines were measured in supernatants from cultures of peripheral blood mononuclear cells (PBMC) from three subject groups: ABPA patients, patients with asthma, and healthy individuals. All cultures for the three subject groups were studied in the presence and absence of two purified Af antigens (the 35-kD antigen and heat shock protein 1). We found that increased in vitro levels of IgE in unstimulated PBMC culture supernatants correlated significantly with serum IgE concentrations in ABPA patients. We measured a decrease in IgE levels of up to 75% of baseline values in supernatants from PBMC cultured with Af antigens. Interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) concentrations in cultures with Af were increased in ABPA, whereas concentrations of IL-4 did not differ in the three subject groups. An inverse relation was noted between the changes in IgE and IFN-gamma measured in 4 of 5 ABPA patients. The PBMC supernatants also promoted EPO activity in purified eosinophils from ABPA patients, and to a lesser extent in purified eosinophils from healthy subjects. These results show that the 35-kD antigen and HSP1 from Af downregulate IgE in vitro but are capable of inducing eosinophilia in ABPA. Further studies could result in the characterization of epitopes leading to these disparate effects. An identification of the IgE-down-regulating epitopes in Af antigens might have therapeutic significance.     

Body composition changes in patients with human immunodeficiency virus infection

Malnutrition characterized by weight loss and often extreme wasting generally develops when patients progress from infection with human immunodeficiency virus (HIV) to AIDS. There is evidence that before the development of AIDS, HIV-infected patients without weight loss show early signs of malnutrition, defined as an increase in the ratio of extracellular mass (ECM) to body cell mass (BCM). As part of a dietary intervention study, body composition measurement were obtained at baseline and after 6 wk in 18 patients with HIV infection and CD4 counts between 140 and 740 cells/mm3. Only one patient had a prior weight loss (3.7 kg); patients gained 2 pounds after 3 wk of dietary supplementation of 500 kcal daily. Bioelectrical impedance was used to measured body compartments. The average ECM/BCM ratio (0.77 +/- 0.13) was within the normal range (0.83 +/- 0.16) indicating the absence of malnutrition by this criterion. Most measurements of BCM (kg) approximated normal values, while several for BCM (kg) exceeded normal. BCM (kg) correlated poorly with the ECM/BCM ratio (r2 = 0.08; P = 0.11) in contrast to ECM (kg), which was well correlated (r2 = 0.82; P = 0.00). In addition, there was a significant correlation of body mass index (BMI) with the ECM/BCM ratio (r2 = 0.38; P = 0.00) and with ECM (r2 = 0.244; P = 0.003) indicating that overweight patients may be more likely to be considered malnourished than normal weight patients using this ratio. Without use of bioelectrical impedance, these subtle changes might be missed. Once significant weight loss has occurred coupled with decreases in BCM (kg), the ECM/BCM ratio may be more reflective of malnutrition. These conjectures will require prospective evaluation, but for now it seems reasonable to include bioelectrical impedance as a potentially useful tool in the evaluation of malnutrition in this population.     

Genomic instability and catalase gene amplification induced by chronic exposure to oxidative stress

Chronic exposure (>200 days) of HA1 fibroblasts to increasing concentrations of H2O2 or O2 results in the development of a stable oxidative stress-resistant phenotype characterized by increased cellular antioxidant levels, particularly catalase (D. R. Spitz et al, Arch. Biochem. Biophys., 279: 249-260, 1990; D. R. Spitz et al., Arch. Biochem. Biophys., 292: 221-227, 1992; S. J. Sullivan et al., Am. J. Physiol. (Lung Cell. Mol. Physiol.), 262: L748-L756, 1992). Acutely stressed cells failed to develop a stably resistant phenotype or increased catalase activity, suggesting that chronic exposure is required for the development of this phenotype. This study investigates the mechanism underlying increased catalase activity in the H2O2- and O2-resistant cell lines. In H2O2- and O2-resistant cells, catalase activity was found to be 20-30-fold higher than that in the parental HA1 cells and correlated with increased immunoreactive catalase protein and steady-state catalase mRNA levels. Resistant cell lines also demonstrated a 4-6-fold increase in catalase gene copy number by Southern blot analysis, which is indicative of gene amplification. Chromosome banding and in situ hybridization studies identified a single amplified catalase gene site located on a rearranged chromosome with banding similarities to Z-4 in the hamster fibroblast karyotype. Simultaneous in situ hybridization with a Z-4-specific adenine phosphoribosyltransferase (APRT) gene revealed that the amplified catalase genes were located proximate to APRT on the same chromosome in all resistant cells. In contrast, HA1 cells contained only single copies of the catalase gene that were not located on APRT-containing chromosomes, indicating that amplification is associated with a chromosomal rearrangement possibly involving Z-4. The fact that chronic exposure of HA1 cells to either HO2 or 95% O2 resulted in gene amplification suggests that gene amplification represents a generalized response to oxidative stress, contributing to the development of resistant phenotypes. These results support the hypothesis that chronic exposure to endogenous metabolic or exogenous environmental oxidative stress represents an important factor contributing to gene amplification and genomic instability.