Functional domains of the alpha1 catalytic subunit of the AMP-activated protein kinase

The AMP-activated protein kinase is a heterotrimeric enzyme, important in cellular adaptation to the stress of nutrient starvation, hypoxia, increased ATP utilization, or heat shock. This mammalian enzyme is composed of a catalytic alpha subunit and noncatalytic beta and gamma subunits and is a member of a larger protein kinase family that includes the SNF1 kinase of Saccharomyces cerevisiae. In the present study, we have identified by truncation and site-directed mutagenesis several functional domains of the alpha1 catalytic subunit, which modulate its activity, subunit association, and protein turnover. C-terminal truncation of the 548-amino acid (aa) wild-type alpha1 protein to aa 312 or 392 abolishes the binding of the beta/gamma subunits and dramatically increases protein expression. The full-length wild-type alpha1 subunit is only minimally active in the absence of co-expressed beta/gamma, and alpha1(1-392) likewise has little activity. Further truncation to aa 312, however, is associated with a large increase in enzyme specific activity, thus revealing an autoinhibitory sequence between aa 313 and 392. alpha-1(1-312) still requires the phosphorylation of the activation loop Thr-172 for enzyme activity, yet is now independent of the allosteric activator, AMP. The increased levels of protein expression on transient transfection of either truncated alpha subunit cDNA are because of a decrease in enzyme turnover by pulse-chase analysis. Taken together, these data indicate that the alpha1 subunit of AMP-activated protein kinase contains several features that determine enzyme activity and stability. A constitutively active form of the kinase that does not require participation by the noncatalytic subunits provides a unique reagent for exploring the functions of AMP-activated protein kinase.     

Features of the parasitic system of Ixodid ticks--Borrelia--small mammals in the Russian Northwest

BACKGROUND AND AIMS: Long-term treatment with H(+)-K(+)-adenotriphosphatase (ATPase) inhibitors, such as omeprazole or lansoprazole, for severe gastroesophageal reflux disease is now widely used. Whether such treatment will result in vitamin B12 deficiency is controversial. We studied whether long-term treatment with omeprazole alters serum vitamin B12 levels in patients with Zollinger-Ellison syndrome. METHODS: In 131 consecutive patients treated with either omeprazole (n = 111) or histamine H2-receptor antagonists (n = 20), serum vitamin B12 and folate levels and complete blood counts were determined after acid secretion had been controlled for at least 6 months. These studies were repeated yearly. Serum vitamin B12 and folate levels were correlated with the type of antisecretory drug and the extent of inhibition of acid secretion. RESULTS: The mean duration of omeprazole treatment was 4.5 years, and for H2-receptor antagonists 10 years. Vitamin B12 levels, but not serum folate levels or any hematological parameter, were significantly (P = 0.03) lower in patients treated with omeprazole, especially those with omeprazole-induced sustained hyposecretion (P = 0.0014) or complete achlorhydria (P < 0.0001). In 68 patients with two determinations at least 5 years apart, vitamin B12 levels decreased significantly (30%; P = 0.001) only in patients rendered achlorhydric. The duration of omeprazole treatment was inversely correlated with vitamin B12 levels (P = 0.013), but not folate levels. Eight patients (6%) developed subnormal B12 levels during follow-up. CONCLUSIONS: Long-term omeprazole treatment leads to significant decreases in serum vitamin B12 but not folate levels. These results suggest patients with Zollinger-Ellison syndrome treated with H(+)-K(+)-ATPase inhibitors should have serum vitamin B12 levels monitored. Furthermore, these results raise the possibility that other patients treated chronically with H(+)-K(+)-ATPase inhibitors may develop B12 deficiency.     

Diagnostic accuracy of HIV-associated central nervous system toxoplasmosis

Our objective was to examine the accuracy of diagnosis of HIV-associated central nervous system (CNS) toxoplasmosis. Individuals diagnosed with HIV-associated CNS toxoplasmosis and controls were ascertained from a population-based database. Diagnosis was confirmed by response to therapy or by histology. Symptoms, results of anti-Toxoplasma serology and use of Pneumocystis carinii pneumonia (PCP) prophylaxis were recorded. Central nervous system toxoplasmosis was confirmed in 54 (76%) of 75 patients. Reactive anti-Toxoplasma serology was associated with CNS toxoplasmosis (OR=20.4, 95% CI 3.1-175.8). Adjusting for CD4 and use of dapsone or aerosolized pentamidine, trimethoprim-sulphamethoxazole (TMP-SMX) for PCP prophylaxis was associated with lower likelihood of CNS toxoplasmosis (OR 0.3, 95% CI 0.1-0.7). Diagnosis of CNS toxoplasmosis is often incorrect. Another diagnosis is most likely in patients who are anti-Toxoplasma seronegative or who are receiving prophylactic TMP-SMX.     

Comparative analysis of semiotic shifts, established by LCS of blood plasma from random samples of studied subjects from the zone of the Chernobyl accident, "Ural Radiation Trace", and collaborators from St. Petersburg Institute of Nuclear Physics of the Russian Academy of Sciences

The effects of an alternative chelation program in thalassemic patients with severe iron overload are investigated. The schedule of treatment, feasible at home, consists in the administration of deferoxamine intravenously (100 mg/kg/die 8 hours 10 days a month), followed by 50 mg/kg/die subcutaneously in the remaining 20 days of the month. The results in 34 patients who underwent this program over 8 months are reported. After intensive chelation therapy serum ferritin and transaminase levels were significantly lower, and daily urinary iron excretion values were significantly higher when compared to the levels observed before the treatment. After the period of study, echocardiography revealed an ejection fraction (EF) significantly higher in 15 out of 34 cardiopathic patients. In conclusion, the alternative chelation program is effective in reducing iron overload of thalassemic patients, protecting them also against cardiac disease.     

Severing of F-actin by the amino-terminal half of gelsolin suggests internal cooperativity in gelsolin

Gelsolin is a Ca2+-regulated actin-binding protein that can sever, cap, and nucleate growth from the pointed ends of actin filaments. In this study we have measured the binding of the amino-terminal half of gelsolin, G1-3, to pyrene-labeled F-actin as a function of Ca2+ concentration. The rate of binding is shown to be dependent on micromolar concentrations of Ca2+. Independent experiments demonstrate that conformational changes in G1-3 are induced by micromolar concentrations of Ca2+. Titrations of pyrene-F-actin with G1-3 and gelsolin show that the quenching of pyrene fluorescence is identical in extent and stoichiometry for both G1-3 and gelsolin. In contrast, severing of F-actin by G1-3 is found to be much less efficient than is severing by gelsolin. In experiments in which F-actin severing is quantitatively measured, the filament number is found to be proportional to the 1.35 power of the G1-3 concentration. This deviation from linearity may be explained by cooperativity; the binding of two G1-3 molecules in close proximity may lead to cooperative severing of the polymer, thus increasing the severing efficiency. This model is supported by experiments that show that the efficiency of G1-3 severing of F-actin increases with increasing G1-3:F-actin ratios. Extrapolating from these results, we conclude that G4-6, the carboxyl-terminal half of gelsolin, has an active role in the severing of F-actin by intact gelsolin. Whereas F-actin severing by G1-3 is enhanced by cooperative binding of two separate G1-3 molecules, cooperativity is inherent to intact gelsolin because the cooperative partners are covalently linked.