Beneficial effect of vitamin E administration on nitric oxide function in subjects with hypercholesterolaemia

Study of the solubilization of commercial grades of soya phosphatidylcholine (SPC) with different purities by bile salts (BS) indicated that only highly pure grades of SPC are suitable for the preparation of clear solutions of BS/SPC-mixed micelles (BS/SPC-MM). The solubilizing capacity of different BS towards SPC increased in the following order; Sodium cholate (SC) < sodium deoxycholate (SDC) < sodium glycocholate (SGC). Moreover, egg phosphatidylcholine (EPC) was solubilized to a higher extent than SPC. Furthermore, the solubility study of different drugs in the prepared MM showed substantial enhancement of solubility, the extent of which is essentially affected by the chemical nature of the drug and the composition of MM. Benzodiazepine drugs such as clonazepam, tetrazepam, diazepam, and lorazepam displayed higher affinity for MM compared with BS alone, whereas steroidal drugs, such as estradiol, prednisolone and progesterone, compared with benzodiazepines, displayed relatively higher affinity for BS alone. The solubilizing capacity of MM for the different drugs was increased to different degrees by the addition of benzyl alcohol which was comparable to the solubility of the drug in pure benzyl alcohol. The interaction between benzyl alcohol and the drug in MM could be proved by NMR.     

Layer-specific dendritic regression of pyramidal cells with ageing in the human prefrontal cortex

Huntingtin is the protein product of the gene for Huntington's disease (HD) and carries a polyglutamine repeat that is expanded in HD (>36 units). Huntingtin-associated protein (HAP1) is a neuronal protein and binds to huntingtin in association with the polyglutamine repeat. Like huntingtin, HAP1 has been found to be a cytoplasmic protein associated with membranous organelles, suggesting the existence of a protein complex including HAP1, huntingtin, and other proteins. Using the yeast two-hybrid system, we found that HAP1 also binds to dynactin P150(Glued) (P150), an accessory protein for cytoplasmic dynein that participates in microtubule-dependent retrograde transport of membranous organelles. An in vitro binding assay showed that both huntingtin and P150 selectively bound to a glutathione transferase (GST)-HAP1 fusion protein. An immunoprecipitation assay demonstrated that P150 and huntingtin coprecipitated with HAP1 from rat brain cytosol. Western blot analysis revealed that HAP1 was enriched in rat brain microtubules and comigrated with P150 and huntingtin in sucrose gradients. Immunofluorescence showed that transfected HAP1 colocalized with P150 and huntingtin in human embryonic kidney (HEK) 293 cells. We propose that HAP1, P150, and huntingtin are present in a protein complex that may participate in dynein-dynactin-associated intracellular transport.     

Intracellular diadenosine polyphosphates: a novel second messenger in stimulus-secretion coupling

In pancreatic beta-cells, stimulatory glucose concentrations increase cytosolic diadenosine polyphosphates ([ApnA]i) to concentrations sufficient to block ATP-sensitive K+ (KATP) channels. High-performance liquid chromatography and patch clamp techniques were used to study the metabolic pathways by which pancreatic beta-cells synthesize ApnA and the mechanism through which ApnA inhibit KATP channels. ApnA show a glucose- and time-dependent cytosolic concentration increase parallel, though 30- to 50-fold higher, to changes observed in adenine nucleotides. Other fuel secretagogues, leucine and 2-ketoisocaproate, raise [ApnA]i as efficiently as 22 mM glucose. Blockade of glycolysis or Krebs cycle decreases glucose-induced [ApnA]i. No significant increase in cytosolic ApnA concentrations is induced by nonnutrient secretagogues or nonmetabolizable nutrient secretagogues. Inorganic pyrophosphatase inhibition with sodium fluoride blocks 22 mM glucose-induced [ApnA]i increase. ApnA inhibition of KATP channel resembles that of ATP in efficacy, but shows clear functional differences. Unlike ATP, Ap4A does not restore channel activity after rundown. Furthermore, these compounds do not compete with each other for the same site. These features suggest a prominent role for Ap4A in beta-cell function, comparable to ATP. We conclude that nutrient metabolism through pyrophosphatase activation is necessary to induce ApnA synthesis, which in turn constitutes a new, ATP-independent, metabolic regulator of KATP channel activity.     

Response at three months is a good predictive factor for newly diagnosed chronic myeloid leukemia patients treated by recombinant interferon-alpha

In a single institution, we have used recombinant interferon- (IFN-) to treat 116 newly diagnosed Philadelphia-positive (Ph+) chronic myeloid leukemia (CML) patients and analyzed the predictive factors for response and survival. The patients whose median age was 50.3 years (range, 9 to 70) were administered IFN- (5 million units/m2/d) subcutaneously. The IFN- dose was subsequently adjusted to maintain the white blood cell and platelet counts between 1.5 and 5 x 10(9)/L, 50 and 100 x 10(9)/L, respectively. At diagnosis, the Sokal score was used to classify the patients into three groups: low (n = 57), intermediate (n = 42), and high risk (n = 16). A complete hematological response (CHR) was achieved in 93 cases (80.2%). Of the 116 patients, 113 were available for cytogenetic evaluation. Fifty patients (43%) achieved a major cytogenetic response (MCR) (=65% marrow Ph- cells), 37 of them having a complete cytogenetic response (CCR). The estimated 5-year survival of the 116 patients was 68% +/- 11% (95% confidence interval [CI]) with a median follow-up of 42 months (range, 3 to 114) and 85% +/- 11% (95% CI) with a median follow-up of 30.9 (range, 3 to 111) when patients were censored at the time of transplantation. Event-free survival at 5 years (adding death and transplant as event) was 46% +/- 11% (95% CI). Using proportional hazards regression to study time-dependent variables, we confirmed that the most significant factor associated with survival was the cytogenetic response (MCR or CCR) (P <.0001). This factor was independent compared with the Sokal score and baseline variables used to calculate the Sokal score. Moreover, using either univariate or multivariate analysis, the achievement of CHR within 3 months was strongly correlated with MCR (P <.0001). Minimum cytogenetic response (mCR, ie, at least 5% of Ph- metaphases) at 3 months was also a significant predictive factor for MCR (P <.0001). These results show that IFN- can induce a high rate of hematological and cytogenetic response when administered in doses leading to myelosuppression. Factors such as the achievement of CHR and mCR within 3 months could be useful to identify early those patients who will not respond to IFN- and who need alternative treatments such as stem cell transplantation.     

Risks and benefits of diagnostic angiography after aneurysm surgery: a retrospective analysis of 597 studies

INTRODUCTION: Cerebral angiography performed after aneurysm surgery can identify causes of morbidity and mortality that may be corrected. The risks and benefits of angiography that is performed after aneurysm surgery, however, have not been clearly defined. We therefore reviewed our experience with postoperative angiography to determine its dangers and benefits. METHODS: During 10 years, 543 consecutive patients received treatment for cerebral aneurysms. A retrospective analysis of 597 diagnostic angiograms obtained after aneurysm surgery for 494 of these patients was performed. RESULTS: Catheter-induced vessel spasm and dissection, occurring most frequently in the internal carotid artery, were observed in seven (1.2%) and six (1%) studies, respectively. No angiography-associated strokes were identified. No association between age, smoking, hypertension, blood pressure, atherosclerosis, or severe vasospasm and angiographic complications was observed. Aneurysm remnants were identified in 36 (5.7%) of the 637 aneurysms that were surgically treated. Atherosclerosis (P < 0.01) or multiple clip applications (P < 0.01) were significantly associated with aneurysm remnants. Angiographic vessel occlusion was observed in 28 (5.7%) patients and resulted in stroke in 14 of these patients. Vessel occlusion was significantly associated with increasing aneurysm size (P < 0.001), atherosclerosis (P < 0.001), temporary clips (P < 0.001), multiple clips (P=0.03), multiple clip applications (P=0.001), and a new postoperative neurological deficit (P=0.002). Severe vasospasm and newly identified aneurysms were observed in 51 and 16 patients, respectively. CONCLUSION: Angiography after aneurysm surgery is safe and can be routinely performed. Angiography after aneurysm surgery should be particularly considered for patients with large aneurysms or cerebrovascular atherosclerosis and for those who develop new postoperative neurological deficits.     

Interaction between a cellular protein that binds to the C-terminal region of adenovirus E1A (CtBP) and a novel cellular protein is disrupted by E1A through a conserved PLDLS motif

Adenovirus E1A proteins immortalize primary animal cells and cooperate with several other oncogenes in oncogenic transformation. These activities are primarily determined by the N-terminal half (exon 1) of E1A. Although the C-terminal half (exon 2) is also essential for some of these activities, it is dispensable for cooperative transformation with the activated T24 ras oncogene. Exon 2 negatively modulates in vitro cooperative transformation with T24 ras as well as the tumorigenic and metastatic potentials of transformed cells. A short C-terminal sequence of E1A governs the oncogenesis-restraining activity of exon 2. This region of E1A binds with a cellular phosphoprotein, CtBP, through a 5-amino acid motif, PLDLS, conserved among the E1A proteins of human adenoviruses. To understand the mechanism by which interaction between E1A and CtBP results in tumorigenesis-restraining activity, we searched for cellular proteins that complex with CtBP. Here, we report the cloning and characterization of a 125-kDa protein, CtIP, that binds with CtBP through the PLDLS motif. E1A exon 2 peptides that contain the PLDLS motif disrupt the CtBP-CtIP complex. Our results suggest that the tumorigenesis-restraining activity of E1A exon 2 may be related to the disruption of the CtBP-CtIP complex through the PLDLS motif.     

2-Amino-4-methylpyridine as a potent inhibitor of inducible NO synthase activity in vitro and in vivo

1. The ability of 2-amino-4-methylpyridine to inhibit the catalytic activity of the inducible NO synthase (NOS II) enzyme was characterized in vitro and in vivo. 2. In vitro, 2-amino-4-methylpyridine inhibited NOS II activity derived from mouse RAW 264.7 cells with an IC50 of 6 nM. Enzyme kinetic studies indicated that inhibition is competitive with respect to arginine. 2-Amino-4-methylpyridine was less potent on human recombinant NOS II (IC50 = 40 nM) and was still less potent on human recombinant NOS I and NOS III (IC50 = 100 nM). NG-monomethyl-L-arginine (L-NMMA), N6-iminoethyl-L-lysine (L-NIL) and aminoguanidine were much weaker inhibitors of murine NOS II than 2-amino-4-methylpyridine but, unlike 2-amino-4-methylpyridine, retained similar activity on human recombinant NOS II. L-NMMA inhibited all three NOS isoforms with similar potency (IC50S 3-7 microM). In contrast, compared to activity on human recombinant NOS III, L-NIL displayed 10 x selectivity for murine NOS II and 11 x selectivity for human recombinant NOS II while aminoguanidine displayed 7.3 x selectivity for murine NOS II and 3.7 x selectivity for human recombinant NOS II. 3. Mouse RAW 264.7 macrophages produced high levels of nitrite when cultured overnight in the presence of lipopolysaccharide (LPS) and interferon-gamma. Addition of 2-amino-4-methylpyridine at the same time as the LPS and IFN-gamma, dose-dependently reduced the levels of nitrite (IC50 = 1.5 microM) without affecting the induction of NOS II protein. Increasing the extracellular concentration of arginine decreased the potency of 2-amino-4-methylpyridine but at concentrations up to 10 microM, 2-amino-4-methylpyridine did not inhibit the uptake of [3H]-arginine into the cell. Addition of 2-amino-4-methylpyridine after the enzyme was induced also dose-dependently inhibited nitrite production. Together, these data suggest that 2-amino-4-methylpyridine reduces cellular production of NO by competitive inhibition of the catalytic activity of NOS II, in agreement with results obtained from in vitro enzyme kinetic studies. 4. When infused i.v. in conscious unrestrained rats, 2-amino-4-methylpyridine inhibited the rise in plasma nitrate produced in response to intraperitoneal injection of LPS (ID50 = 0.009 mg kg-1 min-1). Larger doses of 2-amino-4-methylpyridine were required to raise mean arterial pressure in untreated conscious rats (ED50 = 0.060 mg kg-1 min-1) indicating 6.9 x selectivity for NOS II over NOS III in vivo. Under the same conditions, L-NMMA was nonselective while L-NIL and aminoguanidine displayed 5.2 x and 8.6 x selectivity respectively. All of these compounds caused significant increases in mean arterial pressure at doses above the ID50 for inhibition of NOS II activity in vivo. 5. 2-Amino-4-methylpyridine also inhibited LPS-induced elevation in plasma nitrate after either subcutaneous (ID50 = 0.3 mg kg-1) or oral (ID50 = 20.8 mg kg-1) administration. 6. These data indicate that 2-amino-4-methylpyridine is a potent inhibitor of NOS II activity in vitro and in vivo with a similar degree of isozyme selectivity to that of L-NIL and aminoguanidine in rodents.