Differences in the abilities of human tau isoforms to promote microtubule assembly.

Three isoforms of human tau protein were compared for their abilities to induce microtubule assembly. The three isoforms, tau 3 (tau containing three microtubule-binding domains), tau 4 (tau containing four microtubule-binding domains) and tau 4L (tau containing four microtubule binding domains plus a 58-amino-acid insert near the N-terminus) were expressed in E. coli and purified using ammonium sulfate precipitation, ion exchange, and size exclusion chromatography. All three isoforms induced microtubule assembly at micromolar concentrations and showed similar critical concentrations for assembly of 0.4-0.45 microM. However, tau 4 induced microtubule formation at a rate five- to tenfold faster than either tau 3 or tau 4L. The rate of microtubule elongation seen with tau 4 was twofold greater than with tau 3 or tau 4L, suggesting that the faster rate of microtubule assembly seen with tau 4 was due, at least in part, to faster elongation. Tau 4 induced a greater number of microtubules to form at steady state than did tau 3 or tau 4L. The microtubules generated with each tau isoform had similar steady-state length distributions and were equally susceptible to cold-induced disassembly. These results indicate that the additional microtubule-binding domain in tau 4 enhances microtubule assembly, while the 58-amino-acid insert negates the stimulatory effect of the fourth microtubule-binding domain.     

Clinical review: Vasculitis on the intensive care unit – part 2: treatment and prognosis

The second part of this review addresses the treatment and prognosis of the vasculitides Wegener's granulomatosis, microscopic polyangiitis, Churg–Strauss syndrome and polyarteritis nodosa. Treatment regimens consist of an initial remission phase with aggressive immunosuppression, followed by a more prolonged maintenance phase using less toxic agents and doses. This review focuses on the initial treatment of fulminant vasculitis, the mainstay of which remains immunosuppression with steroids and cyclophosphamide. For Wegener's granulomatosis and microscopic polyangiitis plasma exchange can be considered for first-line therapy in patients with acute renal failure and/or pulmonary haemorrhage. Refractory disease is rare and is usually due to inadequate treatment. The vasculitides provide a particular challenge for the critical care team. Particular aspects of major organ support related to these conditions are discussed. Effective treatment has revolutionized the prognosis of these conditions. However, mortality is still approximately 50% for those requiring admission to intensive care unit. Furthermore, there is a high morbidity associated with both the diseases themselves and the treatment.     

Coupling of lactose molecules to the carrier protein hinders the spleen and bone marrow uptake of doxorubicin conjugated with human albumin.

Several attempts have been made to enhance doxorubicin (DOXO) concentrations in tumour cells by drug conjugation with human albumin (HSA). HSA-DOXO has the drawback of causing DOXO accumulation in spleen and bone marrow, with a consequent leucopoenia not produced when lactose molecules are coupled to the carrier protein. In the present experiments we demonstrated that the effect of HSA lactosamination is not a consequence of a more rapid disappearance from the bloodstream of the lactosaminated conjugate (L-HSA-DOXO), which is rapidly internalized by the liver through the asialoglycoprotein receptor, but is due to a hindered uptake by spleen and bone marrow cells caused by the coupled lactose molecules. Experiments in vitro showed that HSA-DOXO produced an inhibition of murine macrophage proliferation not caused by L-HSA-DOXO. This result can be explained by higher amounts of the former conjugate entering in these cells and suggests macrophages as the cell type responsible for the spleen and bone marrow internalization of HSA-DOXO hindered by lactose coupling. Importantly, lactosamination of HSA did not reduce the marked uptake of HSA-DOXO by chemically induced rat hepatocellular carcinoma. L-HSA-DOXO, by avoiding DOXO accumulation in bone marrow is an attractive candidate for clinical trials against tumors which were found to actively internalize this conjugate in laboratory animals, such as hepatocellular carcinoma.     

Tau protein induces bundling of microtubules in vitro: comparison of different tau isoforms and a tau protein fragment.

Expression of tau protein in non-neuronal cells can result in a redistribution of the microtubule cytoskeleton into thick bundles of tau-containing microtubules (Lewis et al.: Nature 342:498-505, 1989; Kanai et al.: J Cell Biol 109:1173-1184, 1989). We reconstituted microtubule bundles using purified tubulin and tau in order to study the assembly of these structures. Taxol-stabilized tubulin polymers were incubated with various concentrations of recombinant human tau and examined by electron microscopy. With increasing concentrations of tau 3 (tau isoform containing three microtubule binding domains) or tau 4 (isoform containing four microtubule binding domains) the microtubules changed orientation from a random distribution to loosely and tightly packed parallel arrays and then to thick cables. In contrast, tau 4L, the tau isoform containing four microtubule binding domains plus a 58-amino acid insert near the N-terminus, showed minimal bundling activity. tau 4-induced bundling could be inhibited by the addition of 0.5 M NaCl or 0.4 mM estramustine phosphate, conditions which are known to inhibit tau binding to microtubules. A tau construct that contained only the microtubule binding domains plus 19 amino acids to the C-terminus was fully capable of bundling microtubules. Phosphorylation of tau 3 with cAMP-dependent protein kinase had no effect on its ability to induce microtubule bundling. These results indicate that tau protein is directly capable of bundling microtubules in vitro, and suggests that different tau isoforms differ in their ability to bundle microtubule filaments.     

Prevalence, clinical characteristics, quality of life, and prognosis of patients with congestive heart failure and isolated left ventricular diastolic dysfunction.

Prevalence of isolated left ventricular (LV) diastolic dysfunction has been reported to be as high as one-third of all heart failure (HF) cases, with an increasing prevalence in the elderly population. However, there is a paucity of prospective data about the prevalence and prognosis of isolated LV diastolic dysfunction in an unselected population of patients hospitalized with HF. Therefore, we prospectively evaluated 179 consecutive patients discharged from our hospital with HF to assess the prevalence of systolic versus diastolic LV dysfunction among patients hospitalized with HF and to compare their demographics, clinical features, self-perceived quality of life (QOL), and 6-month readmission rate and mortality. Among them, 133 (59% men, median age 74 years) showed in sinus rhythm and had no significant primary valvular disease. LV diastolic dysfunction was diagnosed on the basis of the European Study Group on Diastolic HF echocardiographic criteria. QOL was assessed at hospital discharge and 6-month follow-up visit using the Minnesota Living with HF questionnaire. Survival of patients with HF was compared with that of age- and sex-matched general population. In all, 29 patients (22%) had isolated LV diastolic dysfunction and 102 (78%) had prevalent LV systolic dysfunction (ie, LV ejection fraction 相似文献   

Stable preference for high ethanol concentrations after ethanol deprivation in Sardinian alcohol-preferring (sP) rats.

Results of a recent study have demonstrated that exposure to multiple ethanol concentrations and repeated ethanol deprivation periods in Indiana ethanol-preferring (P) rats resulted in the development of an alcohol deprivation effect (ADE; the temporary increase in voluntary ethanol intake after a period of deprivation from ethanol) characterized by consumption of intoxicating amounts of ethanol. The current study was designed to possibly extend these results to Sardinian alcohol-preferring (sP) rats, generated with the same selective program previously used for P rats. To this aim, ethanol-naive sP rats were exposed initially to the home cage four-bottle choice [10%, 20%, and 30% (vol./vol.) ethanol solutions and water] for eight consecutive weeks. Subsequently, rats were divided into two groups: The first group had continuous access to the four-bottle regimen (nondeprived rats), and the second group was exposed to five cycles of 14-day periods of deprivation from ethanol and 14-day periods of reexposure to the four-bottle regimen. An ADE developed after each deprivation period. However, the extra intake of ethanol was limited to the first hour of each reaccess period. Magnitude of ADE did not change with repeated periods of deprivation. However, a shift in preference toward the two highest concentrations of ethanol solutions was evident from the first reexposure to ethanol and was maintained throughout the study. These results provide further evidence on the heterogeneity of ethanol-drinking behavior among rat lines selectively bred for high ethanol preference and consumption.     

Discriminant analysis in diagnosing carcinoma of the pancreas and of the papilla of Vater

The clinical and biochemical presentation of carcinoma of the pancreas (PC) and of the papilla of Vater (CPV) are very similar, and, consequently, detailed investigations are required to correctly distinguish between them. The aim of the present study was to select the clinical and biochemical variables that would most efficiently discriminate the precise site of tumor origin. The study group consisted of 72 patients with PC and 22 patients with CPV consecutively hospitalized in our department. The following clinical parameters were considered: age, asthenia, anorexia, vomiting, weight loss, pain, fever, pruritis, and constipation; the biochemical parameters considered were total, direct, and indirect bilirubin, glucose, alkaline phosphatase, gamma glutamy transferase, transaminase, total protein, amylase, and occult blood in stools. The results indicated that in the initial phase of PC the most frequent clinical parameters were weight loss (P<0.0001), anorexia (P<0.02), constipation (P<0.001), and pruritus (P<0.01). In contrast, in CPV, fever (P<0.003) was most frequent in the same phase. There was a statistically significant difference in occult blood in stools (P<0.0001), total (P<0.03) and direct bilirubin (P<0.02), alkaline phosphatase (P<0.05), and transaminase (P<0.002) values in the two groups. On discriminant analysis, weight loss, constipation, pruritus, nausea, anorexia, and fever were the variables which best discriminated between the two types of tumors. In fact, the presence of weight loss, anorexia, asthenia, constipation, and pruritus correctly classified 87.5% of the patients with PC, while the presence of fever and nausea correctly classified 72.7% of the patients with CPV.     

Pyridoxol L,2-pyrrolidon-5 carboxylate prevents active fibroplasia in CCl4-treated rats.

In the present study we evaluated the protective activity of pyridoxol L,2-pyrrolidon-5 carboxylate (metadoxine) against CCl4 intoxication in rats, especially in relation to liver fibrosis. After 6 consecutive weeks of CCl4 treatment, the animals developed liver fibrosis and inflammation as revealed by histological analysis which also included semiquantitative scoring of these features. In addition the serum levels of the immunoreactive prolyl hydroxylase (SIRPH), an enzyme involved in the hydroxylation of the procollagen molecule, were significantly higher (44.2 +/- 16.3 micrograms/ml; P less than 0.005) in this group of animals than in controls (26.1 +/- 8.06). On the contrary, animals treated with CCl4 + metadoxine (200 mg/kg i.p.) had less severe liver fibrosis and normal SIRPH levels (21.5 +/- 14.6). These data suggest that metadoxine may be an effective pharmacological tool for preventing the progression of liver disease in rats exposed to CCl4 to cirrhosis.     

Metabolism of 7-(1,3-thiazolidin-2-ylmethyl)theophylline by rat liver microsomes. Evidence for a monooxygenase-dependent step in 1,3-thiazolidine ring cleavage.

Metabolic transformation of the mucoregulator and bronchodilator 7-(1,3-thiazolidin-2-ylmethyl)theophylline was studied in vitro with a rat liver microsomal preparation containing a NADPH-generating system. The only metabolite observed was 7-theophyllinacetaldehyde. In contrast to previous literature pointing out the chemical nature of 2-substituted thiazolidine ring cleavage, the formation of 7-theophyllinacetaldehyde was mediated by monooxygenase-dependent oxidation. Possibly an unstable sulfoxide was the first metabolic product, rapidly converted to 7-theophyllinacetaldehyde by hydrolysis. The sulfoxidation was apparently catalyzed mainly by flavin-containing monooxygenases, as selective thermal inactivation and methymazole significantly reduced the rate of formation of the metabolite. No N7-dealkylation pathway producing theophylline was detected, indicating a high regioselectivity in in vitro metabolism, due to the nucleophilicity of the sulfur atom.