Endothelial cell leptin receptor mutant mice have hyperleptinemia and reduced tissue uptake

Hyperleptinemia is usually associated with obesity and leptin resistance. Endothelial cell leptin receptor knockout (ELKO) mice without a signaling membrane‐bound leptin receptor in endothelia, however, have profound hyperleptinemia without signs of leptin resistance. Leptin mRNA in adipose tissue was unchanged. To test the hypothesis that the ELKO mutation results in delayed degradation and slowed excretion, we determined the kinetics of leptin transfer in groups of ELKO and wildtype mice after intravenous bolus injection of ¹²⁵I‐leptin and the reference substance ¹³¹I‐albumin. The degradation pattern of ¹²⁵I‐leptin in serum and brain homogenates at different time points between 10 and 60 min was measured by HPLC and acid precipitation. Although ELKO mice had reduced uptake of ¹²⁵I‐leptin uptake by the brain and several peripheral organs, leptin was more stable in blood and tissue. There was no change in the rate of renal excretion. ELISA showed that serum soluble leptin receptor, known to antagonize leptin transport, had a 400‐fold increase, probably contributing to the hyperleptinemia and reduced tissue uptake. Thus, the ELKO mutation unexpectedly increased the stability of leptin but suppressed its tissue uptake. These changes probably contribute to the known partial resistance of the ELKO mice to diet‐induced obesity. J. Cell. Physiol. 228: 1610–1616, 2013. © 2013 Wiley Periodicals, Inc.     

Identification of the active site tyrosine of Flp recombinase. Possible relevance of its location to the mechanism of recombination

A combination of site-directed mutagenesis and amino acid sequence analysis identifies Tyr-343 of Flp recombinase as the residue that covalently attaches to DNA during the strand-cleavage step of recombination. This residue is part of the invariant His-Arg-Tyr triad of the Int family of recombinases. Tyr-343 is located in a highly protease-accessible (and hence "open") region of Flp. This placement may provide the conformational flexibility required for the dual role of Tyr-343 in recombination: nicking of the DNA strands to initiate recombination and joining of the nicked strands across partner substrates to complete recombination. In-frame insertion of a few amino acids close to Tyr-343 (and to its amino-terminal side) does not affect substrate recognition by Flp but abolishes its catalytic function.     

Free energy simulations: Applications to the study of liquid water,hydrophobic interactions and solvent effects on conformational stability

Free energy simulations using the Metropolis Monte Carlo method and the coupling parameter approach with umbrella sampling are described for several problems of interest in structural biochemistry; the liquid water, the hydrophobic interaction of alkyl and phenyl groups in water and solvent effects on the conformational stability of the alanine dipeptide and the dimethyl phosphate anion in water. Proximity analysis of results is employed to identify stabilizing factors. Implications of result with respect to the structural chemistry of proteins and nucleic acids is considered.     

Preformed messengers inMicrosporum canis macroconidia

Macroconidia ofMicrosporum canis, when placed in a nutrient medium produce germ tubes within 4–6 h. Precursor incorporation studies showed that protein synthesis occurred prior to RNA synthesis. Sucrose density gradient analysis of wet and dry spore extracts revealed the presence of 16 % and 11 % polysomes respectively. The polysomal content increased to about 50% within 15 min of germination. Synthesis of RNA occurred only after 2 h of germination. Pool equilibration of the radioactive precursors was not limiting to these measurements. Polyadenylated RNA was isolated from macroconidia and was found to comprise 2–2.5 % of the total RNA. The poly(A)⁺ RNAs were heterodisperse and translatable in a wheat germ cell free translating system. It was concluded that macroconidia ofMicrosporum canis contain pre-formed mRNA which is translated early in germination     

Mating type-like conversion promoted by the 2 micrograms circle site-specific recombinase: implications for the double-strand-gap repair model.

Double-strand breaks in DNA are known to promote recombination in Saccharomyces cerevisiae. Yeast mating type switching, which is a highly efficient gene conversion event, is apparently initiated by a site-specific double-strand break. The 2 micrograms circle site-specific recombinase, FLP, has been shown to make double-strand breaks in its substrate DNA. By using a hybrid 2 micrograms circle::Tn5 plasmid, a portion of which resembles, in its DNA organization, the active (MAT) and the silent (HML) yeast mating type loci, it is shown that FLP mediates a conversion event analogous to mating type switching. Whereas the FLP site-specific recombination is not dependent on the RAD52 gene product, the FLP-induced conversion is abolished in a rad52 background. The FLP-promoted conversion in vivo can be faithfully reproduced by making a double-stranded gap in vitro in the vicinity of the FLP site and allowing the gap to be repaired in vivo.     

The yeast plasmid 2μ circle encodes components required for its high copy propagation

The yeast plasmid 2μ and certain hybrid plasmids constructed from it are maintained stably and at high copy number in yeast cells. By examining various mutant hybrid 2μ plasmids, we show that these properties require the integrity of four plasmid loci. Two of these, designated REPI and REP2, are active in trans and correspond to two open coding regions of 2μ. The other two loci are active only in cis and correspond to the origin of replication and to a region, designated REP3, located several hundred bp away from the origin and consisting of direct repeats of a 62 bp sequence. We propose that the REP loci constitute a copy control system that overrides normal cellular restriction on plasmid replication and amplifies the plasmid when copy number is low.     

Effect of vitamin A deprivation on the cholesterol side-chain cleavage enzyme activity of testes and ovaries of rats (Short Communication)

The cholesterol side-chain cleavage enzyme activity is decreased considerably at the mild stage of vitamin A deficiency in rat testes and ovaries and the decrease in activity becomes more pronounced with progress of deficiency. Supplementation of the deficient rats with retinyl acetate, but not retinoic acid, restores the enzyme activity to normal values. The cholesterol side-chain cleavage enzyme of adrenals is not affected by any of the above treatments.     

DNA-protein complexes during attachment-site synapsis in Mu DNA transposition.

Initial events in Mu DNA transposition involve specific recognition of Mu DNA ends (att sites) and an internal enhancer site by the Mu transposase (A protein). This interaction between A protein and Mu DNA sequences present on a supercoiled DNA substrate leads to the formation of a stable synaptic complex in which the att ends are nicked, prior to DNA strand transfer. This study examines the properties of a synaptic complex proficient for DNA transposition. We show that the A protein binds as a monomer to its binding sites, and causes the DNA to bend through approximately 90 degrees at each site. All six att binding sites (three at each Mu end) are occupied by A within the synaptic complex. Three of these sites are loosely held and can be emptied of A upon challenge with heparin. A synaptic complex with only three sites occupied is stable and is fully competent in the subsequent strand-transfer step of transposition.     

Acid-fast bacilli in aspiration smears from tuberculous lymph nodes. An analysis of 255 cases

An analysis of fine needle aspiration (FNA) smears from 255 patients with tuberculous lymphadenopathy was done. The aspirates were either purulent, cheesy or mixed with blood. A total of 56.4% of all cases aspirated showed acid-fast bacilli. Of the cases in which purulent material was aspirated, 66% were positive for acid-fast bacilli. These findings stress the importance of doing Ziehl-Neelsen staining in smears of all cases suspected of being tuberculous in etiology, particularly when purulent material is aspirated.