Construction of DNA-probes and their immunochemical detection using nonradioactive hybridization tests

Simple and efficient chemical approaches to preparation of DNA probes carrying 2,4-dinitrophenyl, dansyl or biotin residues were developed. The residues were introduced using following DNA derivatization procedures: a) transamination of cytidine residues with O-(4-aminobutyl)hydroxylamine; b) mercuration of pyrimidine residues followed by beta-mercaptoethanol modification. It was shown that 2,4-dinitrophenyl-containing DNA probes can be used for nonradioactive hybridization detection of nucleic acids. DNP-DNA: DNA complexes were detected using mouse antibodies specific to 2,4-dinitrophenyl groups, which were developed with peroxidase-conjugated antimouse immunoglobulins. Peroxidase-catalyzed chemoluminescent reaction of luminol oxidation with hydrogen peroxide allowed to detect 10 picograms of the dinitrophenylated single-stranded DNA probe.     

Functionally important lysine residues in inorganic pyrophosphatase from E. coli. II. Isolation and characteristics of modified tryptic peptide and analysis of the functional role of lysine residues controlling enzyme activity

Inactivation of inorganic pyrophosphatase from E. coli by pyridoxal-5'-phosphate is due to the modification of a lysine residue located in the tryptic peptide with the Asp-Leu-Pro-Glu N-terminal sequence. In course of the enzymatic process this lysine-residue appears to be in the protonated state and either operators as the proton donor for the product of the enzymatic reaction or is involved in stabilization of the transition state.     

Characterization of the purified multifunctional cellulase component of Penicillium funiculosum

Summary The endoglucanase component (CMCase I) ofPenicillium funiculosum cellulase was purified to apparent homogeneity by ultrafiltration and gel chromatography. It consists of a single polypeptide chain with a molecular weight of 56000 and is a glycoprotein. Viscometric and end-product analysis revealed the randomness of enzyme action. Multifunctional characteristic of CMCase I was studied with various carbohydrate substrates.NCL Communication No.: 4307     

Structure of a protein catalyzing the formation of 11 cis-retinal in the visual cycle of invertebrate eyes

A pigment made up of a protein able to bind retinal as well as retinol is described. The molecule consists of a dimer with a molecular weight of 50,000 which binds one molecule of retinal. The binding site for retinal is a Schiff base buried in the interior of the protein. Retinol is probably bound to the protein in the same site as for retinal, although not covalently, as suggested by the absorbance spectra. The protein, extracted from honeybee retina, is involved in visual pigment metabolism, and its structure may elucidate the mechanism of the stereospecific photoisomerization of all trans-retinal to 11-cis-retinal.     

Temperature regulation in a burrow-nesting tropical seabird,the Wedge-tailed Shearwater (Puffinus pacificus)

Summary At low air temperatures (2.3–13.9°C), Wedge-tailed Shearwaters (Puffinus pacificus) shivered and their oxygen consumption increased to as much as 283% of the mean value (0.77 ml O₂/g·h) within the thermoneutral zone of air temperature (23–34°C). The minimal thermal conductance of the tissues and plumage was similar to the value predicted from the body mass (320.5 g). The oxygen consumption of the birds within their thermoneutral zone was lower than predictions based on body mass. At elevated air temperatures, the shearwaters panted at respiratory frequencies as high as 260 respirations/min; maximal respiratory frequencies were not invoked until the birds had become hyperthermic. During exposure to a hot environment, the oxygen consumption of the birds increased and in most instances the shearwaters were not able to lose heat equivalent to their concurrent metabolic heat production.Symbols and abbreviations BMR basal metabolic rate - C _total total thermal conductance - f respiratory frequency - TEWL total evaporative water loss - T _st stomach temperature - T _re rectal temperature     

Isolation of antiserum against rat transcortin and characteristics of specific antibodies

Repeated chromatography of rat plasma protein on DEAE-cellulose, hydroxylapatite and subsequent gel-filtration through Sephadex G-200 were used to obtain a pure rat transcortin homogeneous upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of transcortin was about 66 kDa as determined by SDS-polyacrylamide gel electrophoresis. Immunization of a rabbit with the homogeneous preparation of rat plasma transcortin caused development of antibodies to transcortin. It was shown that the antibodies of rabbit antisera in the experiments made in vitro and in vivo neutralized 60 and 65% of 3H-corticosterone-transcortin complexes, respectively. Specific antibodies to the transcortin were isolated from the homogeneous fraction of IgG by affinity chromatography on transcortin-sepharose 4B. 125J-labelled antibodies were adsorbed by protein A-sepharose; IgG can be eluted by IM acetic acid as a sharp peak. The SDS-polyacrylamide gel electrophoresis demonstrated that affinity-eluted material contains 25 and 50 kDa polypeptides.     

Transforming potential of a myc-containing variant of feline leukemia virus in vitro in early-passage feline cells.

We studied a naturally occurring variant of feline leukemia virus (FeLV) in which the oncogene myc has substituted for a portion of the viral structural genes (myc-FeLV). myc-FeLV was rescued by replication in the presence of FeLV as helper, and its biological activity was examined in early-passage feline cells in vitro. Infection of leukocytes from peripheral blood, spleen, or thymus, or of kitten fibroblasts did not immortalize these cells or alter them morphologically. Northern blot (RNA blot) analysis of virion RNA prepared from the supernatant of infected cells demonstrated the 8.2-kilobase genome of FeLV, but did not demonstrate the 5.0-kilobase genome of myc-FeLV. Apparently, the myc-FeLV genome was lost in the absence of the selective pressure of transformation. In contrast, infection of embryonic fibroblasts with myc-FeLV(FeLV) rendered these cells capable of greatly increased, if not infinite, proliferative potential. The cells were morphologically altered compared with controls and were only loosely adherent to the substrate. The cells failed to proliferate in semisolid medium and did not form tumors when inoculated subcutaneously into athymic mice. Blot analyses demonstrated the presence and expression of integrated proviral DNAs of both FeLV and myc-FeLV in these cells. They appear, then, to represent cells partially transformed by infection with myc-FeLV(FeLV). The action of feline v-myc in early-passage cells in vitro was compared to that of avian v-myc.     

The association between spontaneous pyelonephritis and maturity-onset diabetes mellitus in male MM mice

Blood glucose and glucose tolerance tests demonstrated that many male MM mice are diabetic. Serial urine sampling showed that the diabetes occurred only in mature MM males and consisted of a single self-limiting episode. Histological examination of the pancreas, together with measurements of body weight, glycosylated haemoglobin and plasma insulin, revealed that the diabetes was of the maturity-onset insulin-resistant type. Bacteriological examination of the urine samples showed that urinary tract infection, a known feature of male MM mice, occurred in the diabetics but only after the onset of hyperglucosuria. It was concluded that the high urinary glucose levels of diabetic MM males are of prime importance in the aetiology of the renal infection which occurs rarely in non-diabetic MM males or in other strains in the colony. An infectious aetiology for the diabetes per se was excluded by the existence of diabetes in germfree MM males.     

Antitrypanosomal action of cis-diamminedichloroplatinum (II) analogs

The present report deals with the alterations produced by cis-diamminedichloroplatinum (II) (DDP), and 2 of its analogs: cis-Pt(II)(tranylcypromine)2Cl2 and cis-Pt(II)(benzothiazole)2Cl2 in cultured epimastigote forms of Trypanosoma cruzi. Studies have been performed at the ultrastructural level and the inhibitory effect of these complexes on macromolecule synthesis, evaluated by 3H-thymidine, 3H-uridine, and 3H-leucine incorporation, has been investigated. DDP at concentrations of 50 and 100 micrograms/ml does not inhibit significantly the incorporation of radioactive precursors, but a clear decrease was observed with the 2 analogs. Eight hours of treatment at a concentration of 10 micrograms/ml rendered in all 3 cases an increase in autophagic vacuoles and lipids as well as an abnormal condensation of the nucleus chromatin.     

Influence of adrenergic receptors on ovarian progesterone secretion in the pseudopregnant cat and oestradiol secretion in the oestrous cat

The infusion of isoprenaline or propranolol into the abdominal aorta of the pseudopregnant cat caused an increase or decrease respectively in the ovarian progesterone secretion rate. These observations suggest that the sympathetic innervation of the ovary has a physiological influence on normal progesterone secretion, and this mechanism may explain stress-related increases in progesterone concentrations. The infusion of isoprenaline or propranolol after the stimulation of follicular growth had no consistent or convincing effect on oestradiol secretion.