Obesity-inducing lesions of the central nervous system alter leptin uptake by the blood-brain barrier.

Leptin regulates body adiposity by decreasing feeding and increasing thermogenesis. Obese humans and some obese rodents are resistant to peripherally administered leptin, suggesting a defect in the transport of leptin across the blood-brain barrier (BBB). Defective transport of exogenous leptin occurs in some models of obesity, but in other models transport is normal. This shows that factors other than obesity are associated with impairment of leptin transport across the BBB. In order to further investigate these factors, we determined leptin transport in rats made obese by lesioning of the ventromedial hypothalamus (VMH), paraventricular nucleus (PVN), or posterodorsal amygdala (PDA). These regions all contain leptin receptors and lesions there induce obesity and hyperleptinemia and alter the levels of many feeding hormones which might participate in leptin transporter regulation. We measured the uptake of radioactively labeled leptin by the BBB by multiple-time regression analysis which divides uptake into a reversible phase (Vi, e.g., receptor/transporter binding to the brain endothelial cell) and an irreversible phase (Ki, complete transport across the BBB). Leptin uptake was not affected in rats with VMH lesions. No significant change occurred in the entry rate (Ki) for any group, although Ki declined by over 35% in rats with PVN lesions. Decreased uptake was observed in rats with PVN lesions and with PDA lesions. This was primarily due to a reduced Vi (about 21% for the PDA). This decreased uptake is most likely explained by decreased binding of leptin to the brain endothelial cell, which could be because of decreased binding by either receptors or transporters. This suggests that some of the feeding hormones controlled by the PVN and PDA may participate in regulating leptin uptake by the BBB.     

DIATOM SILICA BIOMINERALIZATION: AT NANOSCALE LEVEL A CHEMICALLY UNIFORM PROCESS

Using a high-brilliance synchrotron X-ray source, combined small- and wide-angle X-ray scattering (SAXS and WAXS) was applied to study nanoscale characteristics, in particular pore size in the range of 3 to 65 nm, of a variety of unialgal cultures of centric and pennate diatoms, and of mixed diatom populations sampled in the field. Results of scattering analysis were compared with details of pore size, structure and orientation visible at the electron microscopic level. WAXS patterns did not reveal any crystalline phase or features of microcrystallinity (resolution 0.07 to 0.51 nm), which implies a totally amorphous character of the SiO₂ matrix of the frustule material. SAXS data (resolution 3 to 65 nm) provided information on geometry, size, and distribution of pores in the silica. Overall, two pore regions were recognized that were common to the silica of all samples: the smallest (d less than 10 nm) regularly spaced and shaped spherically, the larger (up to 65 nm) being cylinders or slits. Apparently, at a nanoscale level diatomaceous silica is quite homologous among species, in agreement with the chemical principles of silica polymerization under the conditions of pH and precursor concentrations inside the silicon deposition vesicle. The final frustule "macro"-morphology is of course species-specific, being determined genetically. Synthetically-derived MCM-type silicas have a similarly organized pore distribution in an amorphous silica matrix as we found in all diatom species studied. We therefore suggest that organic molecules of a kind used as structure-directing agents to produce these artificial silicas play a role in the nucleation of the silica polymerization reaction and the shaping of pore morphology inside the silicon deposition vesicle of diatoms. Structure-directing molecules now await isolation from the SDV, followed by identification and characterisation by molecular techniques.     

Polymorphism in Two Parasitoids Detected Using Random Amplified Polymorphic DNA Polymerase Chain Reaction

We used Chelex 100 chelating resin to prepare DNA for the polymerase chain reaction (PCR) from two species of Hymenopteran parasitoids, Trioxys pallidus and Diglyphus begini. Chelex 100 produces consistent DNA yields for both species, as measured with Hoescht dye fluorometry. Approximately twice as much DNA was obtained from individual D. begini wasps than from T. pallidus wasps, but there were no differences in yield between sexes. We used this DNA to perform random amplified polymorphic DNA (RAPD) analysis, a PCR technique that amplifies various regions of the genome using arbitrarily chosen 10-base primers. Of the 120 primers tested using T. pallidus, 92 produced a total of 342 scorable bands, 118 of which exhibited presence/absence polymorphism. Of the 25 primers tested using D. begini, 18 produced a total of 53 scorable bands, 30 of which exhibited presence/absence polymorphism. The level of genetic variation detected using this technique was greater than any found in Hymenoptera using allozymes. Scorable bands segregated as dominant Mendelian traits. Potential uses of RAPD-PCR in genetic analyses on parasitic Hymenoptera are discussed.     

New gene assignments using a complete, characterized sheep-hamster somatic cell hybrid panel

The generation and characterization of new sheep-hamster cell hybrids is reported from the fusion of sheep white blood cells with six different hamster auxotrophs. Selection from these and previously generated cell hybrids has led to the production of a panel of 30 hybrids covering the complete sheep genome of 28 chromosomes. Over half of the cell hybrids in this panel contain single sheep chromosomes. By complementation, the following new assignments have been made using the panel: phosphoribosyl N-formylglycinamide amidotransferase (PRFGA) to sheep chromosome (chr) 11; adenylosuccinate synthetase (ADSS) to sheep chr 12; adenylosuccinate lyase (ADSL) to sheep chr 3q; 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGCS) to sheep chr 16; dihydrofolate reductase (DHFR) to sheep chr 5; and adenine phosphoribosyltransferase (APRT) to sheep chr 14. The gene phosphoribosylaminoinidazole-carboxamide formyltransferase/Inosinicase (PRACFT) has now been regionally assigned to chr 2q. By isozyme analysis, phosphogluconate dehydrogenase (PGD) was assigned to sheep chr 12, anchoring the sheep syntenic group U1 to this chromosome, and mannose phosphate isomerase (MPI) was assigned to sheep chr 18. Furthermore, the chromosomal assignment of 110 microsatellites was confirmed using this cell panel.     

Temperature influences the expression of fimbriae and flagella in <Emphasis Type="Italic">Hafnia alvei</Emphasis> strains: an immunofluorescence study

Hafnia alvei, a Gram negative bacillus related to the Enterobacteriaceae family, is considered an opportunistic pathogen of several animal species and humans. In this communication, we describe fimbrial-like structures from different strains of H. alvei that cannot be easily ascribed to any of the previously reported fimbrial types in this species (type I or type III). Polymerase chain reaction (PCR) and immunofluorescence assays were carried out to study fimbriae and flagella in H. alvei strains isolated from different sources. No correlation between the results obtained by PCR and those obtained by phenotypic methods were found, and the antibodies used gave cross or different recognition patterns of the surface structures present in these strains. We report as well that strain and growth temperature influence fimbriation and expression of flagella in human and animal isolates of H. alvei. This study also indicates that the absence of fimbriae have a significant positive influence on the initial adhesion of H. alvei to human epithelial cells.     

Determination of deuterium-labeled tryptophan in proteins by means of high-performance liquid chromatography and thermospray mass spectrometry

In order to develop direct methods for determining the extent of metabolic incorporation of isotopically labeled amino acids into a protein, the determination of deuterated tryptophan in [2H5]tryptophan-bacteriorhodopsin was investigated. The isotopically modified protein was subjected to alkaline hydrolysis. After phenyl isothiocyanate derivatization of the hydrolysate, the mixture was separated by reversed-phase liquid chromatography. Field desorption mass spectrometry and thermospray mass spectrometry were investigated for their ability to determine the ratio between [2H5]tryptophan and total tryptophan in the collected fractions. In order to check the procedure a set of known tryptophan/[2H5]tryptophan mixtures were passed through the same derivatization, HPLC separation, and lyophilization procedure as used for the biological samples.