Phosphatidic acid regulates tyrosine phosphorylating activity in human neutrophils: enhancement of Fgr activity

In human neutrophils, the activation of phospholipase D and the Tyr phosphorylation of proteins are early signaling events upon cell stimulation. We found that the pretreatment of neutrophils with ethanol (0.8%) or 1-butanol (0.3%), which results in the accumulation of phosphatidylalcohol at the expense of phosphatidic acid (PA), decreased the phorbol myristate acetate-stimulated Tyr phosphorylation of endogenous proteins (42, 115 kDa). When neutrophil cytosol was incubated in the presence or absence of PA, these and other endogenous proteins became Tyr-phosphorylated in a PA-dependent manner. In contrast, phosphatidylalcohols exhibited only 25% (phosphatidylethanol) or 5% (phosphatidylbutanol) of the ability of PA to stimulate Tyr phosphorylation in the cell-free assay. Similarly, other phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, polyphosphoinositides, and sphingosine 1-phosphate) showed little ability to stimulate Tyr phosphorylation. These data suggest that PA can function as an intracellular regulator of Tyr phosphorylating activity. Gel filtration chromatography of leukocyte cytosol revealed a peak of PA-dependent Tyr phosphorylating activity distinct from a previously described PA-dependent phosphorylating activity (Waite, K. A., Wallin, R., Qualliotine-Mann, D., and McPhail, L. C. (1997) J. Biol. Chem. 272, 15569-15578). Among the protein Tyr kinases expressed in neutrophils, only Fgr eluted exclusively in the peak of PA-dependent Tyr phosphorylating activity. Importantly, Fgr isolated from unstimulated neutrophil lysates showed increased activity in the presence of PA but not phosphatidylbutanol. Moreover, the pretreatment of neutrophils with 1-butanol decreased Fgr activity in cells stimulated with formyl-methionyl-leucyl phenylalanine plus dihydrocytochalasin B. Together, these results suggest a new second messenger role for PA in the regulation of Tyr phosphorylation.     

Enhancement of synaptic plasticity through chronically reduced Ca2+ flux during uncorrelated activity

The plasticity of synapses within neural circuits is regulated by activity, but the underlying mechanisms remain elusive. Using the dye FM1-43 to directly image presynaptic function, we found that large numbers of presynaptic terminals in hippocampal cultures have a low release probability. While these terminals were not readily modifiable, a transient but not permanent long-term reduction of network activity or Ca2+ influx could increase their modifiability. This modulation of plasticity was mediated by Ca2+ flux through NMDA and voltage-gated calcium channels and was lost within 48 hr. A more permanent enhancement of synaptic plasticity was achieved by selectively reducing the Ca2+ flux associated with uncorrelated activity via adjustment of the voltage-dependent Mg2+ block of the NMDAR. Upregulation of NR2B-containing NMDARs induced by this treatment is an important but not sole contributor to the enhancement of plasticity. Thus, quantity and quality of activity have differential effects on the intrinsic plasticity of neurons.     

Synthesis of OSW saponin analogs with modified sugar residues and their antiproliferative activities

Eight monosaccharide analogs of the potent antitumor OSW saponins (2-9) were synthesized. One analog, 2-O-acetyl-alpha-l-arabinopyranoside 3, showed antiproliferative activity against the Jurkat cells (IC(50)=0.078microM) comparable to that of the disaccharide derivative (1).     

The lost correlation between heat shock protein 70 (HSPA1A) and plasminogen activator inhibitor-1 in patients with type 2 diabetes and albuminuria

We aimed to study the relation between plasma levels of stress-induced heat shock protein 70 (HSPA1A) with plasminogen activator inhibitor-1 (PAI-1) and high-density lipoprotein cholesterol (HDL-C), apolipoprotein A1 (Apo-A1), and HDL-C/Apo-A1 ratio. In a matched case-control study on patients with diabetes (40 patients with albuminuria and 40 without albuminuria matched for age, sex, and body mass index), we observed that plasma levels of HSPA1A and PAI-1 are increased in patients with albuminuria (0.55 ± 0.02 vs. 0.77 ± 0.04 ng/ml, p value <0.001 for HSPA1A; 25.9 ± 2 vs. 31.8 ± 2.4 ng/ml, p value <0.05 for PAI-1). There was a significant correlation between HSPA1A and PAI-1 in diabetic patients without albuminuria (r = 0.28; p value = 0.04), but not in those with albuminuria (r = 0.07; p value = 0.63). No association was found between HSPA1A and HDL-C, between HSPA1A and Apo-A1, or between HSPA1A and HDL-C/Apo-A1 ratio. We concluded that there is a direct correlation between plasma HSPA1A and PAI-1 levels in patients with diabetes, which is lost when they develop albuminuria.     

3D QSAR studies,pharmacophore modeling,and virtual screening of diarylpyrazole–benzenesulfonamide derivatives as a template to obtain new inhibitors,using human carbonic anhydrase II as a model protein

A 3D-QSAR modeling was performed on a series of diarylpyrazole-benzenesulfonamide derivatives acting as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The compounds were collected from two datasets with the same scaffold, and utilized as a template for a new pharmacophore model to screen the ZINC database of commercially available derivatives. The datasets were divided into training, test, and validation sets. As the first step, comparative molecular field analysis (CoMFA), CoMFA region focusing and comparative molecular similarity indices analysis (CoMSIA) in parallel with docking studies were applied to a set of 41 human (h) CA II inhibitors. The validity and the prediction capacity of the resulting models were evaluated by leave-one-out (LOO) cross-validation approach. The reliability of the model for the prediction of possibly new CA inhibitors was also tested.     

The 2008 Anatomy Ceremony: Voices, Letter, Poems

Yale University medical and PA students express their gratitude in a compilation of reflections on learning human anatomy.     

Transacylase formation of bis(monoacylglycerol)phosphate

Recent work within our laboratory has focused on the enzymes we hypothesize are involved in the biosynthesis of bis(monoacylglycerol)phosphate from phosphatidylglycerol. Here we describe a transacylase, active at acidic pH values, isolated from a macrophage-like cell line, RAW 264.7. This enzyme acylates the head group glycerol of sn-3:sn-1' lysophosphatidylglycerol to form sn-3:sn-1' bis(monoacylglycerol)phosphate. Here we demonstrate that this enzyme uses two lysophosphatidylglycerol molecules, one as an acyl donor and another as an acyl acceptor, and that the acyl contributions from all other lipids tested are comparatively minor. This enzyme prefers saturated acyl chains to monounsaturates, 16 and 18 carbon fatty acids over 14 carbon fatty acids, and saturated acyl chains at the sn-1 position to monounsaturated acyl chains on the sn-2 carbon of lysophosphatidylglycerol. We present data which show the transacylase activity depends on the presence of a lipid-water interface and the lipid polymorphic state.