In vitro dynamics of chromatin organization and migration

The organization of eukaryotic chromatin is not static but changes as a function of cell status during processes such as proliferation, differentiation, and migration. DNA quantification has not been used extensively to investigate chromatin dynamics in combination with cellular migration. In this context, an optimized DNA-specific, nonperturbant method has been developed for studying chromatin organization, using the fluorescent vital bisbenzimidazole probe Hoechst 33342: this property has been described by Hamori et al. (1980). Computer-assisted image analysis was used to follow migratory activity and chromatin organization of L929 fibroblasts during in vitro wound healing. Cell movements were analyzed using an optical flow technique, which consists in the calculation of the velocity field of cells and nuclear movements in the frame. This system allows the correlation of cell migration and position in the cell cycle. It makes it possible to study chromatin dynamics using a quantitative analysis of nuclear differentiation reorganization (nuclear texture) and to correlate this with migration characteristics. The present system would be of interest for studying cell-extracellular matrix interactions using differing substrates, and also the migratory response to chemotactic factors. Such a model is a prerequisite for gaining better understanding of drug action.     

The complete mitochondrial DNA sequence of the greater Indian rhinoceros, Rhinoceros unicornis, and the Phylogenetic relationship among Carnivora, Perissodactyla, and Artiodactyla (+ Cetacea)

The sequence (16,829 nt) of the complete mitochondrial genome of the greater Indian rhinoceros, Rhinoceros unicornis, was determined. Like other perissodactyls studied (horse and donkey) the rhinoceros demonstrates length variation (heteroplasmy) associated with different numbers of repetitive motifs in the control region. The 16,829-nt variety of the molecule includes 36 identical control region motifs. The evolution of individual peptide-coding genes was examined by comparison with a distantly related perissodactyl, the horse, and the relationships among the orders Carnivora, Perissodactyla, and Artiodactyla (+ Cetacea) were examined on the basis of concatenated sequences of 12 mitochondrial peptide-coding genes. The phylogenetic analyses grouped Carnivora, Perissodactyla, and Artiodactyla (+ Cetacea) into a superordinal clade and within this clade a sister group relationship was recognized between Carnivora and Perissodactyla to the exclusion of Artiodactyla (+ Cetacea) . On the basis of the molecular difference between the rhinoceros and the horse and by applying as a reference to Artiodactyl/Cetacean divergence set at 60 million years ago (MYA), the evolutionary divergence between the families Rhinocerotidae and Equidae was dated to approximately 50 MYA.      

Cloning, nucleotide sequences, and enzymatic properties of glucose dehydrogenase isozymes from Bacillus megaterium IAM1030.

Bacillus megaterium is known to have several genes that code for isozymes of glucose dehydrogenase. Two of them, gdhI and gdhII, were cloned from B. megaterium IAM1030 in our previous work (T. Mitamura, R. V. Evora, T. Nakai, Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Ferment. Bioeng. 70:363-369, 1990). In the present study, two new genes, gdhIII and gdhIV, were isolated from the same strain and their nucleotide sequences were identified. Each gene has an open reading frame of 783 bp available to encode a peptide of 261 amino acids. Thus, a total of four glucose dehydrogenase genes have been cloned from B. megaterium IAM1030. In addition, this strain does not seem to have other glucose dehydrogenase genes that can be distinguished from the four cloned genes so far examined by Southern hybridization analysis. The two newly cloned genes were expressed in Escherichia coli cells, and the products, GlcDH-III and GlcDH-IV, were purified and characterized and compared with the other isozymes, GlcDH-I and GlcDH-II, encoded by gdhI and gdhII, respectively. These isozymes showed different mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (GlcDH-I greater than GlcDH-III = GlcDH-IV greater than GlcDH-II), although they have the same number of amino acid residues. Double-immunodiffusion tests showed that GlcDH-I is immunologically different from the other isozymes and that GlcDH-III and GlcDH-IV are identical to one another but a little different from GlcDH-II. These glucose dehydrogenases were stabilized in the presence of 2 M NaCl. The effect of NaCl was especially large for GlcDH-III, which is most unstable enzyme. Kinetic studies showed that these isozymes are divided into two groups with respect to coenzyme specificity, although they can utilize both NAD and NADP: GlcDH-III and GlcDH-IV prefer NAD, and GlcDH-I and GlcDH-II prefer NADP. The phylogenic relationship of these glucose dehydrogenase genes is also discussed.     

The tryptic activation pathway of monomeric procarboxypeptidase A

Procarboxypeptidases are the remaining major digestive zymogens the activation process of which remains unsolved. Here it is shown that in the tryptic activation of monomeric procarboxypeptidase A from porcine pancreas, the generation of carboxypeptidase A (CPA) activity parallels the limited proteolysis of the 94-residue activation segment. This degradation proceeds from the COOH-terminal end of the molecule, and CPA itself makes an important and unexpected contribution by excising the COOH-terminal arginine residue of the released primary activation fragment. Successive cleavages at some of the peptide bonds of the activation segment nearest to the COOH terminus were found to be of prime importance in eliciting CPA activity, particularly those involving the carbonyl groups of Arg94 and Gly93 which were first cleaved. It is also shown that the rate of activation does not depend directly upon the generation of CPA-alpha and its conversion to CPA-beta.     

Short-term handling of the slimming agent oleoyl-estrone in liposomes (Merlin-2) by the rat

Female adult rats were injected in the jugular vein with oleoyl-³H-estrone incorporated into liposomes. The label rapidly disappeared from the blood, being taken up by the tissues, mainly liver, spleen and lung, which filtered most of the label. However, many other tissues, such as the heart, brown adipose tissue, adrenals and visceral fat incorporated significant amounts of oleoyl-estrone. The analysis of the form in which the label remained 10 min after the injection showed that it was hydrolysed in a large proportion even in liver and lungs. However, in most tissues (brain, brown and white - periovaric - adipose tissues and ovaries), intact oleoyl-estrone accounted for less than one quarter of all tissue label, and less than 10% in the case of subcutaneous adipose tissue and uterus. This rapid destruction of oleoyl-estrone is in agreement with the active role of this compound in the control of body weight.     

Glucose influenced degradation of α-santonin in pseudomonas sp. strain S (ATCC 43388)

Pseudomonas sp., strain S ATCC 43 388 utilizes α-santonin by inducible enzyme system measurable by oxygen uptake. Cells grown on acetate or benzoate show negligible oxygen consumption with α-santonin. However, glucose grown cells show evidence of a rapid induction of santonin utilizing enzyme system indicating the implication of glucose or its metabolites in the regulation of degradation of santonin. As a consequence, growth of strain S on mixtures of glucose and α-santonin occurs at rates higher than on either of the substrates alone. Mutants with lesion in the glucose metabolism, independent of α-santonin degradation, fail to exhibit higher growth rates with the binary substrates. The results infer simultaneous metabolism of substrates.