Thermodynamic and kinetic study of thermal denaturation of Kunitz soybean trypsin inhibitor by differential scanning microcalorimetry

The thermal denaturation of soybean trypsin inhibitor (Kunitz inhibitor) has been studied in pH-region from 2.0 to 11.0 by differential scanning microcalorimetry. The thermodynamic characteristics have been determined. It has been established that the denaturation transition of protein may be described by a two-state model. It has been shown, that two side hydrogen bonds between carboxylate-ion and tyrosyl and carboxylate-ion and lysyl take part in the stabilization of the inhibitor's native structure. The activation of denaturation is accompanied by cleavage of one side hydrogen bond.     

Isolation and regional localization of a large collection (2,000) of single-copy DNA fragments on human chromosome 3 for mapping and cloning tumor suppressor genes

Summary A collection of 2,000 lambda phage-carrying human single-copy inserts (> 700 bp) were isolated from two chromosome-3 flow-sorted libraries. The single-copy DNA fragments were first sorted into 3p and 3q locations and about 700 3p fragments were regionally mapped using a deletion mapping panel comprised of two humanhamster and two-human-mouse cell hybrids, each containing a chromosome 3 with different deletions in the short arm. The hybrids were extensively mapped with a set of standard 3p markers physically localized or ordered by linkage. The deletion mapping panel divided the short arm into five distinct subregions (A-E). The 3p fragments were distributed on 3p regions as follows: region A, 26%; B, 31%; C, 4%; D, 4% and E, 35%. We screened 300 single-copy DNA fragments from the distal part of 3p (regions A and B) with ten restriction endonucleases for their ability to detect restriction fragment length polymorphisms (RFLPs). Of these fragments 110 (36%) were found to detect useful RFLPs: 35% detected polymorphisms with frequency of heterozygosity of 40% or higher, and 25% with frequency of 30% or higher. All polymorphisms originated from single loci and most of them were of the base pair substitution type. These RFLP markers make it possible to construct a fine linkage map that will span the distal part of chromosome 3p and encompasses the von Hippel-Lindau disease locus. The large number of single-copy fragments (2,000) spaced every 100–150 kb on chromosome 3 will make a significant contribution to mapping and sequencing the entire chromosome 3. The 300 conserved chromosome 3 probes will increase the existing knowledge of man-mouse homologies.     

Thiol-induced oligomerization of α-lactalbumin at high pressure

Denaturation and aggregation of-lactalbumin at high pressure (up to 10 kbar, 1000 MPa) were studied by means of circular dichroism, gel-permeation chromatography, sodium dodecyl sulfate and gel electrophoresis. It was found that the unfolding of-lactalbumin at high pressure is reversible even in basic pH and at a protein concentration as large as 10%. In these conditions only a negligible fraction of the protein is denatured irreversibly and aggregates. The rate of aggregation of-lactalbumin at high pressure increases significantly in the presence of low-molecular reducing agents such as cysteine, 2-mercaptoethanol, and dithiothreitol. Maximal yield of-lactalbumin oligomerization (over 90%) was achieved in the presence of cysteine at the molar cysteine/protein ratioq=2 and atpH 8.5. Apparent molecular weight of the obtained oligomers was over 500 kDa. It was shown that the size distribution of oligomers can be modulated by varyingpH and reducing agent. The size distribution shifts in the direction of very large, poorly soluble particles whenpH decreases. Maximal content of the insoluble fraction (about 30%) can be reached at pH 5.5 when cysteine (q=2) is used as reducing agent. The oligomers of-lactalbumin are stabilized mainly by nonnative interchain disulfide bridges. Circular dichroism measurements point to an additional mechanism of cohesion of polypeptide chains in the oligomers, which is formation of intermolecular-sheets.     

Regulation of N-Methyl-d-aspartate receptor-mediated calcium transport and norepinephrine release in rat hippocampus synaptosomes by polyamines

The role of polyamines (PA) synthesis in NMDA receptor-mediated⁴⁵Ca²⁺ fluxes and norepinephrine release was studied in rat hippocampal synaptosomes. NMDA (50M) caused a sharp (>2-fold) transient increase in PA synthesis regulating enzyme, ornithine decarboxylase (ODC) activity with concomitant elevation in PA levels in the order putrescine>spermidine>spermine. ODC inhibitor, -difluoromethylornithine (DFMO), and NMDA antagonist, 2-amino-5-phosphonovaleric acid (D-AP5), both blocked increases in ODC activity and PA levels. Activation of NMDA receptors induced a sharp (3 to 4-fold) and quick (15 seconds) increase in⁴⁵Ca²⁺ uptake by synaptosomes within 15 seconds of exposure at 37°C. The efflux of⁴⁵Ca²⁺ and³H-norepinephrine (NE) release at 22°C from pre-loaded synaptosomes was also significantly (2 to 4-fold) enhanced by NMDA within 15 seconds. These NMDA receptor-mediated effects on calcium fluxes and NE release were blocked by NMDA receptor-antagonists (DAP-5 and MK-801) and PA synthesis inhibitor, DFMO and the DFMO inhibition nullified by exogenous putrescine. These observations establish that ODC/PA cascade play an important role in transduction of excitatory amino acid mediated signals at NMDA receptors.Special issue dedicated to Dr. Sidney Ochs.     

The isochore patterns of mammalian genomes and their phylogenetic implications

The compositional distributions of high molecular weight DNA fragments from 20 species belonging to 9 out of the 17 eutherian orders were investigated by analytical CsCl density gradient centrifugation and by preparative fractionation in Cs₂SO₄/BAMD density gradients followed by analysis of the fractions in CsCl. These compositional distributions reflect those of the isochores making up the corresponding genomes. A “general distribution” was found in species belonging to eight mammalian orders. A “myomorph distribution” was found in Myomorpha, but not in the other rodent infraorders Sciuromorpha and Histricomorpha, which share the general distribution. Two other distributions were found in a megachiropteran (but not in microchiropteran, which, again, shares the general distribution) and in pangolin (a species from the only genus of the order Pholidota), respectively. The main difference between the general distribution and all other distributions is that the former contains sizable amounts (6–10%) of GC-rich isochores (detected as DNA fragments equal to, or higher than, 1.710 g/cm³ in modal buoyant density), which are scarce, or absent, in the other distributions. This difference is remarkable because gene concentrations in mammalian genomes are paralleled by GC levels, the highest gene concentrations being present in the GC-richest isochores. The compositional distributions of mammalian genomes reported here shed light on mammalian phylogeny. Indeed, all orders investigated, with the exception of Pholidota, seem to share a common ancestor. The compositional patterns of the megachiropteran and of Myomorpha may be derived from the general pattern or have independent origins.     

Anopheles culicifacies: The occurrence of multiple insemination under laboratory conditions

Multiple insemination was detected under laboratory conditions in 38 of 399 females of Anopheles culicifacies Giles using a sex-linked, recessive eye-color marker system. Multiple mating occurred less frequently at low pair density and was not detected in a large cage. Remating after a 24-hr delay was detected in 5 of 131 families. The high incidence of multiple mating was attributed to interrupted copulations resulting in the transference of inadequate quantities of male accessory gland substance and/or by remating before insemination barriers were established after the initial insemination.

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Anopheles culicifacies giles: La presence d'inseminations multiples dans les conditions de laboratoire

Résumé Les inséminations multiples ont été mises en évidence dans les conditions de laboratoire chez 38 sur 399 femelles d'Anopheles culicifacies en utilisant un marqueur récessif colorant l'il, lié au sexe. Les copulations répétées sont moins fréquentes aux faibles densités et n'ont pas été observées dans de grandes cages. La recopulation après un délai de 24 h a été observée dans 5 familles sur 131. La haute fréquence de copulations multiples est envisagée comme conséquence de copulations interrompues, empéchant le transfert de quantités adéquates de sécrétions des glandes accessoires mâles et/ou par la recopulation avant que les barrières de non réceptivité aient été établies par l'insémination initiale.

     

Use of polysaccharides to remove lipids from the protein globulin fraction of baker's yeast

The precipitation of lipid-protein complexes from the baker's yeast protein globulin fraction by polysaccharides (gum arabic and arabinogalactan) was investigated. Lipid-protein complexes were precipitated more readily with the polysaccharides under study than with other globulin fractions components. A method for the removal of lipids from the globulin fraction of baker's yeast by precipitation of the lipid-protein complexes with polysaccharides is suggested.     

Anopheles subpictus Grassi: Observations on survivorship and population size using mark-release-recapture and dissection methods

A mark-release-recapture experiment to estimate population survivorship and absolute size was performed with wild-caught An. subpictus adults at the village of Khano-Harni, Lahore District, Punjab Province, Pakistan during September 1978, the end of the monsoon rainy season, when temporal population abundance was maximized. Daily survival rate estimated from the recapture sequence of marked adults was low, males=0.192 and females=0.343. Survivorship for females estimated by several vertical age-grading procedures ranged from 0.347 to 0.628. Both stage- and age-specific life tables were calculated from vertical age-grading data determined by the dilatation method. Female and male population size was estimated byBailey 's modification of theLincoln Index and was found to average 4478.4 and 6106.8, respectively. The bionomics, survivorship and population size of An. subpictus in the Lahore are indicated that this species was probably not important in the transmission of human malaria.     

Determination of nystatin component composition using HPLC and TLC with densitometry]

The component composition of nystatin produced by an improved strain of Streptomyces noursei was determined by HPLC on Milichrom chromatograph (USSR). It was shown that the antibiotic consisted of nystatins A1, A2, A3 and B and admixture substances. The data appeared to be in good agreement with the results of the complex TLC investigation, by using densitometry. The component composition of the samples was evidenced by SIEAP mass spectrometry. Physiochemical and biological characteristics of separate components are presented.