RESUMEN

Background: Chronic HIV is associated with increased inflammation and tissue fibrosis despite suppressive antiretroviral therapy (ART). Monocytes and macrophages have been implicated in the pathogenesis of fibrosis, facilitated by chemokine receptor interactions.Methods: We assessed systemic fibrotic biomarkers (transforming growth factor beta-1 [TGF-ß1], thrombospondin-1 [TSP-1], C-terminal pro-peptide of collagen type I [CICP], and IL-11) in banked plasma from a previously published 24-week open-label trial of cenicriviroc (CVC), a dual CCR2/CCR5 antagonist, among persons living with HIV (PLWH) on stable ART with undetectable plasma HIV RNA (<50 copies/mL). Fibrotic markers were assessed by ELISA and Luminex. Untreated HIV-seronegative individuals (n = 6) of similar age and demographics served as a comparator group.Results: Median age of PLWH was 55 years. At baseline, PLWH had higher median TGF-ß1 (2.11 vs 1.62 ng/mL, p = 0.01), TSP-1 (236.74 vs 83.29 ng/mL, p < 0.0001), and CICP (200.46 vs 111.28 ng/mL, p = 0.01), but lower IL-11 (36.00 vs 53.74 pg/mL, p = 0.01) compared to HIV-uninfected individuals. Over 24 weeks, median TGF-ß1 (-0.74 ng/mL, p = 0.006), TSP-1 (-52.12 ng/mL, p < 0.0001), and CICP (-28.12 ng/mL, p < 0.0001) decreased and IL-11 (28.98 pg/mL, p < 0.0001) increased in PLWH. At week 24, TGF-ß1, CICP, and IL-11 were similar between the two groups (p > 0.05), while TSP-1 remained elevated in PLWH (p = 0.009) compared to controls.Conclusions: PLWH had higher levels of the plasma fibrotic markers TGF-ß1, TSP-1, and CICP. After 24 weeks of CVC, fibrotic markers generally returned to levels comparable to HIV-uninfected controls. Dual CCR2 and CCR5 blockade may ameliorate the detrimental fibrotic events that persist in treated HIV.

Asunto(s)

RESUMEN

BACKGROUND: An elevated lactate level reflects impaired tissue oxygenation and is a predictor of mortality. Studies have shown that the anion gap is inadequate as a screen for hyperlactataemia, particularly in critically ill and trauma patients. A proposed explanation for the anion gap's poor sensitivity and specificity in detecting hyperlactataemia is that the serum albumin is frequently low. This study therefore, sought to compare the predictive values of the anion gap and the anion gap corrected for albumin (cAG) as an indicator of hyperlactataemia as defined by a lactate > or =2.5 mmol/l. METHODS: A retrospective review of 639 sets of laboratory values from a tertiary care hospital. Patients' laboratory results were included in the study if serum chemistries and lactate were drawn consecutively. The sensitivity, specificity, and predictive values were obtained. A receiver operator characteristics curve (ROC) was drawn and the area under the curve (AUC) was calculated. RESULTS: An anion gap > or =12 provided a sensitivity, specificity, positive predictive value, and negative predictive value of 39%, 89%, 79%, and 58%, respectively, and a cAG > or =12 provided a sensitivity, specificity, positive predictive value, and negative predictive value of 75%, 59%, 66%, and 69%, respectively. The ROC curves between anion gap and cAG as a predictor of hyperlactataemia were almost identical. The AUC was 0.757 and 0.750, respectively. CONCLUSIONS: The sensitivities, specificities, and predictive values of the anion gap and cAG were inadequate in predicting the presence of hyperlactataemia. The cAG provides no additional advantage over the anion gap in the detection of hyperlactataemia.

Asunto(s)

RESUMEN

Human bone marrow (BM) is a tissue of complex architectural organization, which includes granulopoietic loci, erythroblastic islets, and lymphocytic nodules. Oxygen tension (pO(2)) is an important determinant of hematopoietic stem and progenitor cell proliferation and differentiation. Thus, understanding the impact of the BM architectural organization on pO(2) levels in extravascular hematopoietic tissue is an important biophysical problem. However, currently it is impossible to measure pO(2) levels and their spatial variations in the BM. Homogeneous Kroghian models were used to estimate pO(2) distribution in the BM hematopoietic compartment (BMHC) and to conservatively simulate pO(2)-limited cellular architectures. Based on biophysical data of hematopoietic cells and characteristics of BM physiology, we constructed a tissue cylinder solely occupied by granulocytic progenitors (the most metabolically active stage of the most abundant cell type) to provide a physiologically relevant limiting case. Although the number of possible cellular architectures is large, all simulated pO(2) profiles fall between two extreme cases: those of homogeneous tissues with adipocytes and granulocytic progenitors, respectively. This was illustrated by results obtained from a parametric criterion derived for pO(2) depletion in the extravascular tissue. Modeling results suggest that stem and progenitor cells experience a low pO(2) environment in the BMHC.

Asunto(s)

RESUMEN

Hematopoietic cells of various lineages are organized in distinct cellular architectures in the bone marrow hematopoietic compartment (BMHC). The homogeneous Kroghian model, which deals only with a single cell type, may not be sufficient to accurately describe oxygen transfer in the BMHC. Thus, for cellular architectures of physiological significance, more complex biophysical-transport models were considered and compared against simulations using the homogeneous Kroghian model. The effects of the heterogeneity of model parameters on the oxygen tension (pO(2)) distribution were examined using the multilayer Kroghian model. We have also developed two-dimensional Kroghian models to simulate several cellular architectures in which a cell cluster (erythroid cluster) or an individual cell (megakaryocyte or adipocyte) is located in the BMHC predominantly occupied by mature granulocytes. pO(2) distributions in colony-type cellular arrangements (erythroblastic islets, granulopoietic loci, and lymphocytic nodules) in the BMHC were also evaluated by modifying the multilayer Kroghian model. The simulated results indicate that most hematopoietic progenitors experience low pO(2) values, which agrees with the finding that low pO(2) promotes the expansion of various hematopoietic progenitors. These results suggest that the most primitive stem cells, which are located even further away from BM sinuses, are likely located in a very low pO(2) environment.

Asunto(s)

RESUMEN

The assembly of the beta-subunit of the gastric H-K-ATPase (HKbeta) with the alpha-subunit of the H-K-ATPase or the Na-K-ATPase (NaKalpha) was characterized with two anti-HKbeta monoclonal antibodies (MAbs). In fixed gastric oxyntic cells, in H-K-ATPase in vitro, and in Madin-Darby canine kidney (MDCK) cells transfected with HKbeta, MAb 2/2E6 was observed to bind to HKbeta only when interactions between alpha- and beta-subunits were disrupted by various denaturants. The epitope for MAb 2/2E6 was mapped to the tetrapeptide S(226)LHY(229) of the extracellular domain of HKbeta. The epitope for MAb 2G11 was mapped to the eight NH(2)-terminal amino acids of the cytoplasmic domain of HKbeta. In transfected MDCK cells, MAb 2G11 could immunoprecipitate HKbeta with alpha-subunits of the endogenous cell surface NaKalpha, as well as that from early in the biosynthetic pathway, whereas MAb 2/2E6 immunoprecipitated only a cohort of unassembled endoglycosidase H-sensitive HKbeta. In HKbeta-transfected LLC-PK(1) cells, significant immunofluorescent labeling of HKbeta at the cell surface could be detected without postfixation denaturation or in live cells, although a fraction of transfected HKbeta could also be coimmunoprecipitated with NaKalpha. Thus assembly of HKbeta with NaKalpha does not appear to be a stringent requirement for cell surface delivery of HKbeta in LLC-PK(1) cells but may be required in MDCK cells. In addition, endogenous posttranslational regulatory mechanisms to prevent hybrid alpha-beta heterodimer assembly appear to be compromised in transfected cultured renal epithelial cells. Finally, the extracellular epitope for assembly-sensitive MAb 2/2E6 may represent a region of HKbeta that is associated with alpha-beta interaction.

Asunto(s)

RESUMEN

alpha-Toxin-permeabilized gastric glands represent a functional model in which acid secretion can be elicited by either adenosine 3',5'-cyclic monophosphate (cAMP) or ATP, with proven morphological and functional transition between resting and secretory states [X. Yao, S. M. Karam, M. Ramilo, Q. Rong, A. Thibodeau, and J. G. Forte. Am. J. Physiol. 271 (Cell Physiol. 40): C61-C73, 1996.] In this study we use alpha-toxin-permeabilized rabbit gastric glands to study energy metabolism and the interplay between nucleotides to support acid secretion, as indicated by the accumulation of aminopyrine (AP). When permeabilized glands were treated with a phosphodiesterase inhibitor, the secretory response to cAMP was inhibited, whereas the secretory response to ATP was potentiated. This implied that 1) ATP provided support not only as an energy source but also as substrate for adenylate cyclase, 2) activation of acid secretion by cAMP needed ATP, and 3) ATP and cAMP exchanged rapidly inside parietal cells. To address these issues, we tested the action of adenine nucleotides in the presence and absence of oxidizable substrates. All adenine nucleotides, including AMP, ADP, ATP, and cAMP, could individually enhance the glandular AP accumulation in the presence of substrates, whereas only a high concentration of ATP (5 mM) was able to support secretory activity in substrate-free buffer. Moreover, ATP could maintain 75-80% of maximal secretory activity in phosphate-free buffer; cAMP alone could not support secretion in phosphate-free buffer. In glands and in H(+)-K(+)-adenosinetriphosphatase-rich gastric microsomes, we showed the operation of adenylate kinase, creatine kinase, and ATP/ADP exchange activities. These enzymes, together with endogenous adenylate cyclase and phosphodiesterase, provide the recycling of nucleotides essential for the viability of alpha-toxin-permeabilized gastric glands and imply the importance of nucleotide recycling for energy metabolism in intact parietal cells.

Asunto(s)

Asunto(s)

RESUMEN

H+/K(+)-ATPase is the proton pump in the gastric parietal cell that is responsible for gastric acid secretion. Stimulation of acid secretion is associated with a reorganization of the parietal cells resulting in the incorporation of H+/K(+)-ATPase from a cytoplasmic membrane pool, the tubulovesicle compartment, into the apical canalicular membrane. To better characterize the role of membrane trafficking events in the morphological and physiological changes associated with acid secretion from parietal cells, we have characterized the expression and localization of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in these cells. Each of the six different SNARE proteins examined [syntaxins 1 through 4 of 25-kDa synaptosome-associated protein, and vesicle-associated membrane protein] were found to be expressed in parietal cells. Furthermore, two of these SNAREs, vesicle-associated membrane protein and syntaxin 3, were associated with H+/K(+)-ATPase-containing tubulovesicles while the remainder were excluded from this compartment. The expression of syntaxin 1 and synaptosome-associated protein of 25 kDa in parietal cells, two SNAREs previously thought to be restricted to neuroendocrine tissues, suggests that parietal cells may utilize membrane trafficking machinery that is similar to that utilized for regulated exocytosis in neurons. Furthermore, the localization of syntaxin 3, a putative target membrane SNARE, to the tubulovesicle compartment indicates that syntaxin 3 may have an alternative function. These observations support a role for intracellular membrane trafficking events in the regulated recruitment of H+/K(+)-ATPase to the plasma membrane after parietal cell stimulation.

Asunto(s)

RESUMEN

Jejunoileal diverticula are estimated to occur in 1-5% of the population. The incidence increases with age, peaking at the sixth and seventh decades. The pathogenesis is believed to involve an acquired defect of the intestinal smooth muscle or myenteric plexus. Eighty percent of jejunoileal diverticula are localized to the jejunum, 15% to the ileum, and 5% to both. Diverticula in the jejunum tend to be large and multiple, whereas those in the ileum are small and solitary. Symptoms of intermittent abdominal pain, flatulence, diarrhea, and constipation are reported in 10-30% of patients with jejunoileal diverticula. The radiographic diagnosis of these diverticula is difficult to establish. Enteroclysis should be reserved for patients who have persistent abdominal pain despite nonrevealing endoscopic and contrast enhanced studies of the upper and lower gastrointestinal tracts. Asymptomatic jejunoileal diverticula should be managed conservatively. Complications occur in 6-10% of patients and include obstruction, diverticulitis, hemorrhage, perforation, malabsorption, and chronic debilitating abdominal pain. When surgical therapy is indicated, intestinal resection with primary anastomosis is the preferred treatment.

Asunto(s)

Asunto(s)

RESUMEN

Protein metabolic labeling in vivo was used to determine a time course for trafficking of nascent H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) from endoplasmic reticulum (ER) to mature tubulovesicles in parietal cells. Stomachs of cimetidine-treated rabbits were taken 15-90 min after injection of [35S]methionine/cysteine, and mucosal microsomes were fractionated on sucrose gradients for analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, and autoradiography. After 15 min, labeled alpha-subunit peaked at approximately 1.14 g/ml, matching the distribution of the high-mannose beta-subunit precursor, "pre-beta." After 30 min, most labeled alpha-subunit was in a peak at approximately 1.10 g/ml, considered to be Golgi. By 90 min, most labeled alpha-subunit was in a light peak, at approximately 1.07 g/ml, aligned with the major peak of total H(+)-K(+)-ATPase previously characterized as mature tubulovesicles. From material enriched in pre-beta, alpha-subunit was coprecipitated with pre-beta by a terminal mannose-specific lectin, Galanthus nivalis agglutinin, in the same ratio as the mature alpha:beta ratio. Thus alpha- and beta-subunits associated early in the ER. This is the first use of protein metabolic labeling to study early trafficking of the H(+)-K(+)-ATPase in vivo. The techniques may be usefully applied to examining changes in H(+)-K(+)-ATPase synthetic rate in response to various pharmacological treatments and studying the divergent pathways for nascent H(+)-K(+)- and Na(+)-K(+)-ATPases.

Asunto(s)

RESUMEN

Structural and functional interactions between alpha- and beta-subunits of the H,K-ATPase were explored. The sensitivity to trypsinolysis of alpha-subunit was monitored by SDS-PAGE in control H,K-ATPase-enriched microsomes and in microsomes in which disulfide bonds of the beta-subunit were reduced using 2-mercaptoethanol (2-ME). Reduction of beta-subunit disulfide bonds increased the susceptibility of the alpha-subunit to tryptic digestion. Kinetics of trypsinolysis were also carried out in the presence of ligands known to bind with H,K-ATPase and favor a particular conformer state in the native enzyme. The time-course for release of tryptic peptides was monitored in protein stained gels and Western blots probed with monoclonal antibody alpha-H,K,12.18. In control preparations, where beta-subunit disulfides remained intact, trypsinolysis in the presence of ATP or K+ produced distinctive patterns of tryptic fragments, each characteristic of the conformational states induced by the respective ligand. For 2-ME-treated microsomes the altered alpha-subunit was unable to undergo ligand-induced conformational changes. The increased susceptibility of the alpha-subunit to trypsinization, the change in accessibility of tryptic cleavage sites and the inability of the alpha-subunit to undergo ligand-induced conformational changes after reduction of the beta-subunit disulfides suggest that the interactions between alpha- and beta-subunits are important for the conformational stability of the functional holoenzyme. A model localizing the most susceptible tryptic cleavage sites in control and 2-ME-reduced states is presented.

Asunto(s)

RESUMEN

We have reviewed the structural and functional role of the beta-subunit in a subfamily of the P-ATPases known as the alpha/beta-heterodimeric, cation-exchange ATPases. The subfamily consists of the various isoforms of Na+/K(+)-ATPase and H+/K(+)-ATPase, both of which pump a cation out of the cell (Na+ or H+, respectively) in recycle exchange for K+. Much of the earlier work has emphasized the functional activities of the alpha-subunit, which shares many characteristics with the broader P-ATPase family. It is now clear that the glycosylated beta-subunit is an essential component of the cation-exchange ATPase subfamily. All beta-subunit isoforms have three highly conserved disulfide bonds within the extracellular domain that serve to stabilize the alpha-subunit, alpha/beta interaction and functional activity of the holoenzyme. Evidence strongly suggests that the beta-subunit is involved in the K(+)-dependent reactions of the enzymes, such as the E1-E2 transition and K+ occlusion, and that the extracellular domain of the beta-subunit plays an important role in determining the kinetics of K+ interaction. In most vertebrate cells, the unassociated alpha-subunit is restricted to the endoplasmic reticulum (ER), and assembly of the alpha/beta complex occurs within the ER. Signals for exiting the ER and directing the correct intracellular trafficking are primarily determined by the beta-subunit; Na+/K(+)-ATPase typically terminates in the plasma membrane facing the basolateral membrane, whereas all isoforms of H+/K(+)-ATPase terminate in the apical membrane. The C-terminal extracellular domain of the beta-subunit is important for proper interaction with the alpha-subunit and for correct intracellular trafficking. Oligosaccharides on the beta-subunit are not essential for enzyme function, but do serve to enhance the efficiency of alpha/beta association by increasing the lifetime of the unassociated beta-subunit and the stability of the alpha/beta complex to tryptic attack. We propose that highly specialized glycosylation on the beta-subunit of the gastric H+/K(+)-ATPase may help to protect that enzyme from the harsh extracellular environment of the stomach.

Asunto(s)

RESUMEN

Acquired Immunodeficiency Syndrome (AIDS) has become the biggest problem facing the health profession of Thailand today. The Ministry of Public Health reports that there are 400,000 individuals in Thailand already infected with the Human Immunodeficiency Virus (HIV) and is predicting that 4 million will be infected by the year 2000. This explosive epidemic first occurred among intravenous drug abusers (IVDAs) and subsequently spread to other high risk groups, especially prostitutes. The heterosexual population was next affected. The AIDS problem in Thailand was seen close-up by this writer, then a fourth year medical student, studying during an international health elective. At all three hospitals where I worked, I encountered large numbers of AIDS related admissions. Ten percent of medical beds at a Bangkok hospital were occupied by patients with AIDS related problems. In comparison, two hospitals located in the northern province of Chiang Mai had 15-20% and 30-40% of their beds occupied by patients with AIDS complications. Opportunistic infections were the primary reason for admissions. This paper describes the current AIDS epidemic in Thailand and the preventive measures being undertaken to combat it. Strategies to combat AIDS focus on preventive measures. The current program in Thailand emphasizes AIDS education and awareness, the promotion of condom usage, decreasing needle sharing, the screening of donated blood, and the development of the GP160 vaccine. The program, however, has been undermined by the country's well organized sex industry. Without a clear commitment from the Thai government, Thailand faces serious health and economic consequences from this epidemic in the coming decade.

PIP: Thailand's Ministry of Health estimates that 400,000 persons are infected with human immunodeficiency virus (HIV) and this statistic is projected to reach 4 million by the year 2000. From 10-40% of Thailand's hospital beds are occupied by patients with acquired immunodeficiency syndrome (AIDS)-related complications. HIV is believed to have been introduced by Thai homosexuals who had travelled abroad, and spread rapidly among intravenous drug users and prostitutes until establishing a hold in the heterosexual population. Among prostitutes in brothels, who serve a total of 450,000 clients each day, HIV seroprevalence is close to 25%. Current prevention strategies include AIDS education and awareness, promotion of condom use, decreases in needle sharing, and screening of donated blood. At present, 95% of rural and 98.8% of urban Thais are aware that AIDS is transmitted by sexual intercourse and that condom use can prevent its spread. Nonetheless, only 21.7% of urban and 23.3% of rural males routinely use condoms. Counseling programs have increased condom use among prostitutes to 38-54%, but client resistance (only 10% of men who have sex with prostitutes use condoms) remains an obstacle. The cost of caring for an AIDS patient, from diagnosis to death, is about US$40,000. Thailand has been selected as a site for trials of the GP160 vaccine, but given the length of time before the vaccine will be available to the general public, the emphasis must continue to be on other preventive measures.

Asunto(s)

RESUMEN

Noninvasive prediction of the maximum axial load that a spinal bone screw will be able to withstand after anterior surgical placement would be highly useful. To investigate if this is feasible, we first performed preliminary experiments to distinguish the trabecular and cortical contributions to overall stiffness; the trabecular component was found to dominate. We then used a commercial computed tomography bone mineral package to determine the mineral density of the trabecular region of 41 porcine vertebrae in terms of equivalent K2HPO4 concentration; values ranged from 104 to 343 mg/cm3. A 6.5-mm diameter cancellous bone screw was then inserted laterally in each vertebra, and the ultimate tensile strength (UTS) of the screw/bone interface was measured using a tensile testing machine. The UTS values ranged from 589 to 2,620 Newtons. A superlinear relation was found between UTS and the projected K2HPO4 concentration in the direction of the screw axis, expressed in units of mg/cm2.

Asunto(s)

RESUMEN

The gastric proton pump, H(+)-K(+)-ATPase, is composed of alpha- and beta-subunits. The 95-kDa alpha-subunit has been referred to as the catalytic subunit containing sites for ATP binding and phosphorylation. The beta-subunit is a glycoprotein with a 34-kDa core peptide that has a single transmembrane segment, a small cytoplasmic NH2-terminal, and a large extracellular COOH-terminal domain with seven potential N-linked glycosylation sites. To further study the beta-subunit, we developed monoclonal antibodies that identified a 52-kDa mannose-rich glycoprotein that was deglycosylated by endoglycosidase H such that six transient intermediates were identified, as well as a 34-kDa beta-subunit core peptide. These observations suggest that the beta-subunit is synthesized as a 52-kDa glycoprotein with seven N-linked precursor high-mannose oligosaccharides that mature into complex oligosaccharides. One antibody, 2G11, inhibits the K(+)-stimulated ATP hydrolysis as well as K(+)-stimulated p-nitrophenyl phosphatase (pNPPase) activity of the H(+)-K(+)-ATPase. Kinetic studies revealed that 2G11 inhibited maximum velocity (Vmax) of ATP hydrolysis by approximately 50% with no change in the Km for K+, whereas, for pNPPase both Vmax and Km were altered. These studies demonstrate a functional role for the beta-subunit in the H(+)-K(+)-ATPase activity, especially the K(+)-induced conformational states.

Asunto(s)

RESUMEN

Amounts and fractional distributions of gastric H(+)-K(+)-adenosinetriphosphatase (ATPase) activity and H(+)-K(+)-ATPase protein as well as properties of H(+)-K(+)-ATPase-containing membranes were studied in rabbits injected with omeprazole (OM; 1 mg/kg sc twice daily for 5 days). Total H(+)-K(+)-ATPase activity decreased to 22 +/- 2% of control (n = 4). Densitometry of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots showed H(+)-K(+)-ATPase protein was decreased to 60-70% of control. In vitro reduction of the enzyme-OM disulfide bond with 0.1 M 2-mercaptoethanol increased microsomal H(+)-K(+)-ATPase activity to 56 +/- 7% of control (n = 3), consistent with a substantial decrease in enzyme protein. Incorporation of 35S-labeled methionine for 30 min before death resulted in 2.2-fold more label per unit of microsomal alpha-subunit protein (5 days OM vs. control). Thus the decrease in enzyme protein resulted from increased breakdown rather than decreased synthesis. A striking shift in distribution of H(+)-K(+)-ATPase-containing microsomes (tubulovesicles) on sucrose gradients reflected slow equilibration of most control vesicles with the gradient medium and faster equilibration after 5 days OM, indicating increased permeability. After 5 days OM, microsomal vesicle acidification (by acridine orange uptake assay) was negligible, even with 2-mercaptoethanol treatment, and H+ leakage on sudden delta pH was faster than control. We conclude that extended OM treatment not only inhibits H(+)-K(+)-ATPase but accelerates its breakdown and renders H(+)-K(+)-ATPase-containing membranes more permeable. It is thus possible that increased backward H+ flux contributes to profound inhibition of acid secretion during extended omeprazole treatment. In parallel experiments, H(+)-K(+)-ATPase activity and density gradient sedimentation of tubulovesicles returned to near normal 3 days after OM withdrawal.

Asunto(s)

RESUMEN

BACKGROUND: Primary tracheal tumors are a rare malignancy. Before 1960, most patients had a biopsy, followed by external orthovoltage irradiation or radon seed implantation. Advances in surgery and in radiation therapy during the past three decades have allowed more patients to undergo definitive treatment. METHODS: Between 1957 and 1988, 22 patients with primary tracheal malignancy were treated with curative intent at The University of Texas M.D. Anderson Cancer Center. Five patients underwent primary surgical resection (Group 1), 5 patients had surgical resection and adjuvant irradiation (Group 2), and 12 patients had primary irradiation (Group 3). RESULTS: Median survival times were 26 months for all patients; 16 months for Group 1; 61 months for Group 2; and 26 months for Group 3. Local control was attained in 1 of 5 patients in Group 1, 4 of 5 patients in Group 2, and 4 of 12 patients in Group 3. Among those treated with primary radiation therapy, local control was attained by three of four patients who received 60 Gy or higher and one of eight patients who received less than 60 Gy. Results of chi-square test (P = 0.03) were statistically significant. Severe complications, including treatment-related deaths, occurred in 2 of 5 patients in Group 1, 2 of 5 patients in Group 2, and 3 of 12 patients in Group 3. CONCLUSION: Radiation therapy has a role in the treatment of patients with tracheal malignancy, either as postoperative adjuvant therapy or as sole therapy for those who refuse surgery or have medically inoperable disease. Alternative methods for increasing the local administration of radiation therapy, such as endotracheal brachytherapy, should be investigated for improvement in local control.

Asunto(s)

RESUMEN

H(+)-K(+)-ATPase activity of rabbit isolated gastric microsomes was irreversibly inactivated by reducing agents, such as 2-mercaptoethanol and dithiothreitol. Similar to what has been observed for Na(+)-K(+)-ATPase, high concentrations of reagents, at moderately elevated temperatures, were required to inactivate H(+)-K(+)-ATPase, suggesting relative inaccessibility of the responsible disulfide bonds. Resistance against inactivation was conferred by monovalent cation activators of K(+)-stimulated ATPase and p-nitro-phenylphosphatase. The effectiveness of K+ congeners in protecting the enzyme was similar in sequence (Tl+ greater than K+ greater than Rb+) and concentration to their respective affinities for stimulating enzymatic activity, suggesting that the K(+)-bound form of the enzyme is more resistant to reduction than the free enzyme. Furthermore, Na+ antagonized the protective effect of K+. Labeling studies using fluorescein-maleimide indicated that 60-70% of the cysteine residues in the beta-subunit are in the oxidized form. Coupled with primary sequence data, this suggests that three disulfide bonds are present in the native beta-subunit. In contrast, less than 10% of the cysteine residues in the alpha-subunit are in the oxidized form. Kinetic studies showed that the 2-mercaptoethanol-induced loss of H(+)-K(+)-ATPase activity was correlated with a reduction of disulfide groups in the beta-subunit, while there was no significant change in the alpha-subunit. We conclude that reduction of disulfide bonds irreversibly inhibits H(+)-K(+)-ATPase activity, binding of K+ to the enzyme confers a resistance to disulfide bond reduction, and the responsible disulfide bonds are present in the beta-subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

RESUMEN

Na,K-ATPase and H,K-ATPase are the only members of the P-type ATPases in which a glycosylated beta-subunit is part of the purified active enzyme. In this study, we have followed the synthesis and the posttranslational processing of the beta-subunit of H,K-ATPase (beta HK) in Xenopus oocytes injected with beta HK cRNA and have tested whether it can act as a surrogate for the beta-subunit of Na,K-ATPase (beta NaK) to support the functional expression of Na,K-pumps. In Xenopus oocytes, beta HK is processed from an Endo H-sensitive 51-kDa coreglycosylated form to an Endo H-resistant 71-kDa fully glycosylated form. Similar to beta NaK, beta HK can stabilize and increase the trypsin resistance of alpha-subunits of Na,K-ATPase (alpha NaK). Finally, expression of beta HK together with alpha NaK leads to an increased number of ouabain binding sites at the plasma membrane accompanied by an increased Rb+ uptake and Na,K-pump current. Our data suggest that beta HK, similar to beta NaK, can assemble to alpha NaK, support the structural maturation and the intracellular transport of catalytic alpha NaK, and ultimately form active alpha NaK-beta HK complexes with Na,K-pump transport properties.

Asunto(s)