Kinetics of DNA replication in the Indian muntjac chromosomes as studied by quantitative autoradiography

DNA replication patterns of individual chromosomes and their various euchromatic and heterochromatic regions were analyzed by means of quantitative autoradiography. The cultured cells of the skin fibroblast of a male Indian muntjac were pulse labeled with ³H-thymidine and chromosome samples were prepared for the next 32 h at 1–2 h intervals. A typical late replication pattern widely observed in heterochromatin was not found in the muntjac chromosomes. The following points make the DNA replication of the muntjac chromosomes characteristics: (1) Heterochromatin replicated its DNA in a shorter period with a higher rate than euchromatin. (2) Two small euchromatic regions adjacent to centromeric heterochromatin behaved differently from other portions of euchromatin, possessing shorter Ts, higher DNA synthetic rates and starting much later and ending earlier their DNA replication. (3) Segmental replication patterns were observed in the chromosomes 2 and 3 during the entire S phase. (4) Both homologues of the chromosome 3 showed a synchronous DNA replication pattern throughout the S phase except in the distal portion of the long arms during the mid-S phase.     

Induction of differentiation of cultured human promyelocytic leukemia cells by retinoids

Human promyelocytic leukemia cells (HL-60) were induced to phagocytize, reduce NBT dye(nitroblue tetrazolium), and change into forms that were morphologically similar to mature granulocytes by retinoic acid and related retinoids, but not by the pyridyl analog of retinoic acid. Induction of differentiation could be detected after 4 days of treatment of the cells with retinoic acid at as low a dose as 4 × 10^?8 M. Thus, retinoids may be used in studies on the control of cell differentiation and malignancy of human myeloid leukemia cells.     

Ly-m19: The Lyb-2 region of mouse chromosome 4 controls a new surface alloantigen

Spleen cells from an SJL mouse immunized with 70'/3 cells, an established pre-B cell line, were fused with cells of the nonsecretor myeloma line NS.1. One established hybridoma cell line (clone K10.6) continuously secreted antibody that recognized a new antigenic specificity tentatively named Ly-m19. This newly found antigen is detectable on both T and B cells. Cytotoxicity assays reveal that 75 percent of the spleen and lymph-node cells, 35 percent of bone-marrow cells, and 15 percent of thymus cells reacted with antibody of clone K10.6. Strains expressing the specificity Ly-m19.1 are characterized by negative reactions and include the strains AKR, CE/J, RF/J, GR/A, SJL, P/J, BDP/J, and LG/J. All other strains so far tested are Ly-m19.2. This strain distribution pattern distinguishes Ly-m19 from any known murine lymphocyte alloantigen, but it parallels the Lyb-2 ^c haplotype. Linkage test of a set of AKXL recombinant inbred strains revealed close linkage of Ly-m19 and Lyb-2 loci on mouse chromosome 4.Abbreviations used in this paper LPS lipopolysaccharide - B6 C57BL/6 - Con-A concanavalin A - MLC mixed-lymphocyte culture The prefix m (monoclonal) is used following a suggestion by Klein and co-workers (1979).     

Stereoselective hydrolysis of dl-menthyl succinate by gel-entrapped Rhodotorula minuta var. texensis cells in organic solvent

Summary dl-Menthyl succinate was successfully hydrolyzed stereoselectively by Rhodotorula minuta var. texensis cells entrapped within photo-crosslinked or polyurethane resin gels in water-saturated n-heptane. The hydrolyzed product was found to be pure l-menthol. The catalytic activity of the immobilized cells, especially those entrapped in urethane polymers, was far more stable than that of the free cells. The half-life of the polyurethaneentrapped cells was estimated to be 55–63 days in the organic solvent.Dedicated to the 65th birthday od Professor Dr. G. Manecke     

Reduction by a model of NAD(P)H: XI. Stereochemistry of a lactate dehydrogenase-model reaction

Stereochemistry of the biomimetic reduction of α-keto esters with NAD(P)H-model compounds has been investigated. The model compound with the R-configuration reduces the α-keto esters to the (R)-α-hydroxy esters, whereas (S)-α-hydroxy esters are afforded by the reduction with the S-configurational model compounds. It has been concluded that pro-R and -S hydrogens of the model compounds with R- and S-configuration, respectively, contribute predominantly to the reduction.     

Studies on the oligomeric structure of yeast aldehyde dehydrogenase by cross-linking with bifunctional reagents.

The molecular w:ight of yeast aldehyde dehydrogenase determined by sucrose density gradient centrifugation was 207,000 +/- 13,000. The enzyme activity was proportional to the enzyme concentration in the range of 2 X 10(-11) M to 1 X 10(-7) M. Cross-linking patterns obtained with yeast aldehyde dehydrogenase after treatment with a series of diimidoesters of increasing chain lengths with different reaction times resulted in the appearance of tetramers as the largest cross-linked product of the enzyme subunits. The molecular weights of its monomer, dimer, trimer, and tetramer were, 57,000, 114,000, 171,000, and 228,000, respectively, as estimated from their mobilities on SDS-electrophoresis. In tetramers monomers are probably assembled in a heterologous square arrangement.     

Effects of phospholipids on L-lactate dehydrogenase from membranes of Escherichia coli. Activation and stabilization of the enzyme with phospholipids.

Membrane-bound L-lactate dehydrogenase was freed from the detergent used during purification. The detergent-free enzyme had about one-half the specific activity of the enzyme in 1.0% Tween 80, and was only partially sensitive to the specific antibody. This enzyme was activated about 3-fold with phosphatidylglycerol, cardiolipin, or a mixture of phospholipids. The phospholipid-activated enzyme had a similar Km value for L-lactate to that of the membrane enzyme and was completely inhibited by the specific antibody. On heat treatment, the phospholipid-activated enzyme was more stable than detergent-free enzyme and was as stable as membrane-bound enzyme. The alpha helical content of the enzyme increased 1.7-fold during preincubation with these lipids and the alpha helix became more stable during heat treatment than that of the detergent-free enzyme. These results suggest that the enzyme showed monomolecular dispersion in the lipid bilayer and that its conformation, including its active site and secondary structure, was different from that of the detergent-free enzyme. Phosphatidylethanolamine, dilauroyl lecithin and lecithin from egg yolk had none of the above effects on the activity or the secondary structure of the enzyme. On the other hand, mixtures of each of these lipids and cholate had essentially similar effects to phosphatidylglycerol.     

Kinetics of substrate utilization in adenine-limited chemostat cultures of Bacillus subtilis KYA741.

The specific rates of limiting substrate utilization were investigated in adenine- or glucose-limited chemostat cultures of Bacillus subtilis KYA741, an adenine-requiring strain, at 37 degrees C. With the glucose-limited cultures, the specific rate of glucose consumption versus dilution rate gave a linear relationship from which the true growth yield and maintenance coefficient were determined to be 0.09 mg of bacteria per mg of glucose and 0.2 mg of glucose per mg of bacteria per h, respectively. With the adenine-limited cultures, adenine as the limiting substrate was not completely consumed at lower dilution rates (e.g., D less than 0.1), unlike in the glucose-limited cultures. When a linear relationship of specific rate of adenine consumption versus dilution rate was extrapolated to zero dilution rate, a negative value for the specific rate of adenine consumption, -0.01 mg of adenine per mg of bacteria per h, was obtained, giving a true growth yield for adenine of 5.2 mg of bacteria per mg of adenine. On the other hand, the maintenance coefficient of oxygen uptake gave a positive value of 8.1 x 10(-3) mmol/mg of bacteria per h. Based on previous results showing that adenine is resupplied by lysing cells, we developed kinetic models of adenine utilization and cell growth that gave a good estimation of the peculiar behavior of cell growth and adenine utilization in adenine-limited chemostat cultures.     

The gene and deduced protein sequences of the zymogen of Aspergillus niger acid proteinase A

Proteinase A obtained from the culture medium of Aspergillus niger var. macrosporus is a unique acid endopeptidase that is insensitive (or less sensitive) to specific inhibitors of ordinary acid or aspartic proteinases, such as pepstatin, diazoacetyl-DL-norleucine methyl ester, and 1,2-epoxy-3-(p-nitrophenoxy)-propane. In the preceding paper (Takahashi, K., Inoue, H., Sakai, K., Kohama, T., Kitahara, S., Takishima, K., Tanji, M., Athauda, S. B. P., Takahashi, T., Akanuma, H., Mamiya, G., Yamasaki, M. J. Biol. Chem. 266, 19480-19483), we reported the complete primary structure of the mature enzyme determined at the protein level. The enzyme has a unique two-chain structure with a 39-residue light (L) chain and a 173-residue heavy (H) chain linked noncovalently. As an extension of this study, we isolated genomic and cDNA clones encoding this proteinase and determined their nucleotide sequences. To isolate a genomic clone, the genomic DNA was selectively amplified by polymerase chain reaction using mixed oligonucleotide primers designed from the amino acid sequence of the H chain, and a specific probe thus generated was used for screening a lambda gt10 genomic library. A cDNA for the enzyme was also selectively amplified by polymerase chain reaction using primers synthesized based on the sequence of the genomic DNA. Sequencing of the cloned genomic DNA and cDNA revealed the nucleotide sequence of the structural gene for the enzyme of 846 base pairs without introns. It encodes the precursor form of proteinase A, including an NH2-terminal preprosequence of 59 residues, the L chain of 39 residues, an intervening sequence of 11 residues, and the H chain of 173 residues linked in that order. Thus, proteinase A is thought to be synthesized as a single peptide chain preproenzyme of 282 residues, which is processed to generate the mature two-chain form.