VEGF receptor 2/‐3 heterodimers detected in situ by proximity ligation on angiogenic sprouts

The vascular endothelial growth factors VEGFA and VEGFC are crucial regulators of vascular development. They exert their effects by dimerization and activation of the cognate receptors VEGFR2 and VEGFR3. Here, we have used in situ proximity ligation to detect receptor complexes in intact endothelial cells. We show that both VEGFA and VEGFC potently induce formation of VEGFR2/‐3 heterodimers. Receptor heterodimers were found in both developing blood vessels and immature lymphatic structures in embryoid bodies. We present evidence that heterodimers frequently localize to tip cell filopodia. Interestingly, in the presence of VEGFC, heterodimers were enriched in the leading tip cells as compared with trailing stalk cells of growing sprouts. Neutralization of VEGFR3 to prevent heterodimer formation in response to VEGFA decreased the extent of angiogenic sprouting. We conclude that VEGFR2/‐3 heterodimers on angiogenic sprouts induced by VEGFA or VEGFC may serve to positively regulate angiogenic sprouting.     

Redox-sensitive mechanism of no scavenging by nitronyl nitroxides

Nitronyl nitroxides, NN., have been increasingly used in the field of NO-related studies as specific antagonists of NO. . We employed a combination of EPR and NMR spin trapping to study the mechanisms of the reaction of NN. with NO. in reducing environments. EPR allowed observation of NO-induced transformation of the paramagnetic trap, NN., to the corresponding iminonitroxide, IN. . In a complementary way, corresponding EPR-invisible diamagnetic products (the hydroxylamines NN-H and IN-H) were detected by 19F-NMR using newly synthesized fluorinated traps. Addition of reducing agents to a solution of NN. resulted in fast disappearance of its EPR spectra and appearance of a 19F-NMR peak of the corresponding hydroxylamine, NN-H. Addition of NO. as a bolus, or NO. generated on sodium nitroprusside photolysis, resulted in 19F-NMR-detectable accumulation of the hydroxylamine, IN-H. Upon high rates of NO. generation in ascorbate-containing solutions, partial recovery of NN. was observed, which undergoes further reactions with NO. and ascorbate in a competitive manner. Using 19F-NMR and a fluorinated trap, NO-induced conversion of NN-H into IN-H was also observed in vivo in hypertensive ISIAH rats compared with normotensive WAG rats. The results provide insight into a new potential redox-sensitive mechanism of the antagonistic action of NN. against NO., which may provide insight into previously unexplained behavior of this category of NO-reacting compounds.     

Impact of drug resistance on fitness of Mycobacterium tuberculosis strains of the W-Beijing genotype

Mycobacterium tuberculosis strains of the W-Beijing genotype became a common cause of tuberculosis during the past years and they are often associated with drug resistance. The biological factors facilitating the selection and wide dissemination of these strains are not known. To determine how acquisition of drug resistance affected growth of strains of the W-Beijing genotype, the growth of 55 M. tuberculosis isolates were studied using the BBL MGIT Mycobacteria Growth Indicator Tube and the BACTEC MGIT 960 System. Susceptible strains of non-Beijing genotypes were found to be the most fit strains. Drug-resistant strains of non-Beijing genotypes were more likely to grow slower than susceptible strains (P=0.001). Drug-resistant strains of the W-Beijing genotype had two tendencies of growth: some of them showed reduced growth compared to susceptible strains, while others did not show loss of fitness measured as growth.     

Genomic organization and analysis of the <Emphasis Type="Italic">hairless</Emphasis> gene in four hypotrichotic rat strains

More than 25 different hypotrichotic mutations have been described in laboratory rats, yet the molecular basis for these mutations has not been determined for most of these phenotypes. Their similarity to the hairless (hr) mutations described in mice suggests a possible role for the hairless gene in the formation of rat hypotrichotic phenotypes, though whether hr is responsible for these rat phenotypes has yet to be determined. Therefore, in order to understand the basis for the rat hypotrichotic phenotypes and their relationship to the hr gene, we determined the genomic organization of the hr gene and subsequently analyzed the coding sequence in four hypotrichotic rat strains. Analysis revealed that the first two exons of the mouse, monkey, and human hr gene were fused in the rat gene, while the rest of the gene showed strong evolutionary conservation. Despite their designation as hairless, no mutations within the coding sequences were identified, indicating that the hairless phenotype in all four hypotrichotic rat strains are not allelic with hr.     

Synthesis of [26,27-2H6]brassinosteroids from 23,24-bisnorcholenic acid methyl ester

A number of hexadeuterated brassinosteroids (BS) containing a hydroxy group at C-22 or a 22R,23R-diol function were prepared starting from 23,24-bisnorcholenic acid methyl ester for biosynthetic studies. Synthesis of the cyclic part was accomplished via the initial hydroboration-oxidation of Delta(5)-double bond. The key step in the synthesis of the side chain involved addition of (2S)-[3,4-(2)H(6)]2,3-dimethylbutylphenyl sulfone to the corresponding C-22 aldehydes.     

Novel role of phospholipase C-delta1: regulation of liver mitochondrial Ca2+ uptake

Mitochondrial Ca2+ (mCa2+) handling is an important regulator of liver cell function that controls events ranging from cellular respiration and signal transduction to apoptosis. Cytosolic Ca2+ enters mitochondria through the ruthenium red-sensitive mCa2+ uniporter, but the mechanisms governing uniporter activity are unknown. Activation of many Ca2+ channels in the cell membrane requires PLC. This activation commonly occurs through phosphitidylinositol-4,5-biphosphate (PIP2) hydrolysis and the production of the second messengers inositol 1,4,5-trisphosphate [I(1,4,5)P3] and 1,2-diacylglycerol (DAG). PIP2 was recently identified in mitochondria. We hypothesized that PLC exists in liver mitochondria and regulates mCa2+ uptake through the uniporter. Western blot analysis with anti-PLC antibodies demonstrated the presence of PLC-delta1 in pure preparations of mitochondrial membranes isolated from rat liver. In addition, the selective PLC inhibitor U-73122 dose-dependently blocked mCa2+ uptake when whole mitochondria were incubated at 37 degrees C with 45Ca2+. Increasing extra mCa2+ concentration significantly stimulated mCa2+ uptake, and U-73122 inhibited this effect. Spermine, a uniporter agonist, significantly increased mCa2+ uptake, whereas U-73122 dose-dependently blocked this effect. The inactive analog of U-73122, U-73343, did not affect mCa2+ uptake in any experimental condition. Membrane-permeable I(1,4,5)P3 receptor antagonists 2-aminoethoxydiphenylborate and xestospongin C also inhibited mCa2+ uptake. Although extra mitochondrial I(1,4,5)P3 had no effect on mCa2+ uptake, membrane-permeable DAG analogs 1-oleoyl-2-acetyl-sn-glycerol and DAG-lactone, which inhibit PLC activity, dose-dependently inhibited mCa2+ uptake. These data indicate that PLC-delta1 exists in liver mitochondria and is involved in regulating mCa2+ uptake through the uniporter.