Phosphorylation-regulated axonal dependent transport of syntaxin 1 is mediated by a Kinesin-1 adapter

Presynaptic nerve terminals are formed from preassembled vesicles that are delivered to the prospective synapse by kinesin-mediated axonal transport. However, precisely how the various cargoes are linked to the motor proteins remains unclear. Here, we report a transport complex linking syntaxin 1a (Stx) and Munc18, two proteins functioning in synaptic vesicle exocytosis at the presynaptic plasma membrane, to the motor protein Kinesin-1 via the kinesin adaptor FEZ1. Mutation of the FEZ1 ortholog UNC-76 in Caenorhabditis elegans causes defects in the axonal transport of Stx. We also show that binding of FEZ1 to Kinesin-1 and Munc18 is regulated by phosphorylation, with a conserved site (serine 58) being essential for binding. When expressed in C. elegans, wild-type but not phosphorylation-deficient FEZ1 (S58A) restored axonal transport of Stx. We conclude that FEZ1 operates as a kinesin adaptor for the transport of Stx, with cargo loading and unloading being regulated by protein kinases.     

A vaccine directed to B cells and produced by cell-free protein synthesis generates potent antilymphoma immunity

Clinical studies of idiotype (Id) vaccination in patients with lymphoma have established a correlation between the induced anti-Id antibody responses and favorable clinical outcomes. To streamline the production of an Id vaccine, we engineered a small diabody (Db) molecule containing both a B-cell-targeting moiety (anti-CD19) and a lymphoma Id. This molecule (αCD19-Id) was designed to penetrate lymph nodes and bind to noncognate B cells to form an antigen presentation array. Indeed, the αCD19-Id molecule accumulated on B cells in vivo after s.c. administration. These noncognate B cells, decorated with the diabody, could then stimulate the more rare Id-specific B cells. Peptide epitopes present in the diabody linker augmented the response by activating CD4(+) helper T cells. Consequently, the αCD19-Id molecule induced a robust Id-specific antibody response and protected animals from tumor challenge. Such diabodies are produced in a cell-free protein expression system within hours of amplification of the specific Ig genes from the B-cell tumor. This customized product can now be available to vaccinate patients before they receive other, potentially immunosuppressive, therapies.     

Central diabetes insipidus associated with impaired renal aquaporin-1 expression in mice lacking liver X receptor β

The present study demonstrates a key role for the oxysterol receptor liver X receptor β (LXRβ) in the etiology of diabetes insipidus (DI). Given free access to water, LXRβ(-/-) but not LXRα(-/-) mice exhibited polyuria (abnormal daily excretion of highly diluted urine) and polydipsia (increased water intake), both features of diabetes insipidus. LXRβ(-/-) mice responded to 24-h dehydration with a decreased urine volume and increased urine osmolality. To determine whether the DI was of central or nephrogenic origin, we examined the responsiveness of the kidney to arginine vasopressin (AVP). An i.p. injection of AVP to LXRβ(-/-) mice revealed a partial kidney response: There was no effect on urine volume, but there was a significant increase of urine osmolality, suggesting that DI may be caused by a defect in central production of AVP. In the brain of WT mice LXRβ was expressed in the nuclei of magnocellular neurons in the supraoptic and paraventricular nuclei of the hypothalamus. In LXRβ(-/-) mice the expression of AVP was markedly decreased in the magnocellular neurons as well as in urine collected over a 24-h period. The persistent high urine volume after AVP administration was traced to a reduction in aquaporin-1 expression in the kidney of LXRβ(-/-) mice. The LXR agonist (GW3965) in WT mice elicited an increase in urine osmolality, suggesting that LXRβ is a key receptor in controlling water balance with targets in both the brain and kidney, and it could be a therapeutic target in disorders of water balance.     

Type II p21-activated kinases (PAKs) are regulated by an autoinhibitory pseudosubstrate

The type II p21-activated kinases (PAKs) are key effectors of RHO-family GTPases involved in cell motility, survival, and proliferation. Using a structure-guided approach, we discovered that type II PAKs are regulated by an N-terminal autoinhibitory pseudosubstrate motif centered on a critical proline residue, and that this regulation occurs independently of activation loop phosphorylation. We determined six X-ray crystal structures of either full-length PAK4 or its catalytic domain, that demonstrate the molecular basis for pseudosubstrate binding to the active state with phosphorylated activation loop. We show that full-length PAK4 is constitutively autoinhibited, but mutation of the pseudosubstrate releases this inhibition and causes increased phosphorylation of the apoptotic regulation protein Bcl-2/Bcl-X_L antagonist causing cell death and cellular morphological changes. We also find that PAK6 is regulated by the pseudosubstrate region, indicating a common type II PAK autoregulatory mechanism. Finally, we find Src SH3, but not β-PIX SH3, can activate PAK4. We provide a unique understanding for type II PAK regulation.     

Elevated admission microalbuminuria predicts poor myocardial blood flow and 6-month mortality in ST-segment elevation myocardial infarction patients undergoing primary percutaneous coronary intervention

Background:

Microalbuminuria (MA) is considered a major risk factor predisposing to cardiovascular morbidity and mortality. Outcomes after percutaneous coronary intervention (PCI) for patients with acute myocardial infarction (AMI) complicated by MA have been well described. However, data regarding admission MA and coronary and myocardial flow are scant. The aims of this study were to evaluate the effects of admission MA on coronary blood flow and prognosis in ST‐segment elevation myocardial infarction (STEMI) patients undergoing primary PCI.

Hypothesis:

Did elevated admission microalbuminuria predict poor myocardial blood flow and 6‐month mortality in ST‐segment elevation myocardial infarction patients undergoing primary percutaneous coronary intervention?

Methods:

A total of 247 patients undergoing primary PCI for STEMI within 12 hours after symptom onset were studied. Patients were divided into 2 groups according to admission urinary albumin extraction rate (UAER): (1) an MA group (UAER 20–200 µg/min), and (2) a normoalbuminuria (NA) group (UAER < 20 µg/min).

Results:

Microalbuminuria was observed in 108 patients. Univariate analyses showed statistical differences between the NA and MA groups in serum creatine level, plasma glucose level, and peak creatine kinase level on presentation. Thrombolysis In Myocardial Infarction (TIMI) flow grades (TFGs) 0–2 in the MA group were more frequent (9.4% vs 21.2%, P < 0.05) than in the NA group, and corrected TIMI frame count was higher (23.9 ± 18.5 vs 29.8 ± 23.5, P < 0.05). Admission MA was an independent predictor of poor myocardial perfusion (adjusted relative risk: 3.14, 95% confidence interval: 0.99–6.78) and a higher rate of 6‐month mortality in STEMI patients undergoing primary PCI (adjusted relative risk: 1.58, 95% confidence interval: 0.74–3.39).

Conclusions:

Admission MA levels are associated with impaired myocardial flow and poor prognosis in STEMI patients undergoing primary PCI. © 2012 Wiley Periodicals, Inc. The authors have no funding, financial relationships, or conflicts of interest to disclose.

     

An Integrated Alcohol Abuse and Medical Treatment Model for Patients with Hepatitis C

Background  

Patients with chronic hepatitis C virus (HCV) infection have high rates of alcohol consumption, which is associated with progression of fibrosis and lower response rates to HCV treatment.     

白色假丝酵母菌pACT1-GFP质粒的构建及表达

目的构建能够稳定表达绿色荧光蛋白（GFP）的白色假丝酵母菌菌株,以便对目的基因进行示踪。方法构建pACT1-GFP质粒,以白色假丝酵母菌CAI4菌株作为感受态进行转化培养后,分别观察绿色荧光蛋白在两相型下的表达情况。结果含有pACT1-GFP的白色假丝酵母菌菌株,99%在两相型下均有较高水平的荧光蛋白表达,且荧光强度没有明显差别。结论pACT1-GFP能够在白色假丝酵母菌体内稳定表达。