l-Asparaginase from Proteus vulgaris

To produce an immunologically and enzymologically new type of l-asparaginase, 108 strains of bacteria were screened for enzyme production. As a result, 13 bacteria belonging to the genera Alcaligenes, Bacterium, and Proteus were found to produce l-asparaginases in high levels. Among these l-asparaginases, partially purified l-asparaginases from B. cadaveris and P. vulgaris showed antitumor activity. A partially purified l-asparaginase preparation of P. vulgaris did not react with the antibody of Escherichia colil-asparaginase on the Ouchterlony agar plate. Culture conditions for the production of l-asparaginase by P. vulgaris were investigated in detail. The enzyme was produced in high yields when cells were grown aerobically in a medium containing sodium fumarate and corn steep liquor. The addition of glucose or ammonium ion to the medium, however, resulted in depressed production of l-asparaginase. Under the optimum conditions, 3,700 international units of l-asparaginase was obtained from 1 liter of culture medium.     

Detection of lactate dehydrogenase B4 in human hemolysate of patients deficient in lactate dehydrogenase B subunit activity using enzyme immunoassay

We developed a sensitive enzyme immunoassay system specific for human lactate dehydrogenase (LDH)- B₄ with antiacetylated LDH-B₄ Fab-horse-radish peroxidase conjugate. The enzyme immunoassay system was not interfered with by up to 0.3 mg/tube of hemoglobin. Thus, we measured LDH-B₄ concentrations in the hemolysate of seven heterozygous individuals deficient in LDH-B subunit activity and eight normal individuals. We could not find a significant difference between the LDH-B₄ concentrations in heterozygous and those in normal individuals. These results demonstrate that heterozygous individuals deficient in LDH-B subunit activity produce enzymatically inactive B subunits.This work was supported in part by grants in aid for Scientific Research from the Ministry of Education, Japan (59570998), and from the Clinical Pathology Research Foundation of Japan.     

Structural polymorphism of murine factor B controlled by a locus closely linked to the H-2 complex and demonstration of multiple alleles

New alleles of murine factor B (Bf) protein were demonstrated. When ethylenediaminetetraacetic acid (EDTA)-plasmas from inbred and wild mice were analyzed by isoelectro-focusing (IEF) and immunofixation, murine Bf proteins were visualized as distinct protein bands in all mice tested. Four variants of murine Bf could be demonstrated in a large number of tested mice: Bf 1 (isoelectro-focusing point (P.I.) range of 5.8–6.1) exemplified by B10 and B10.BR, Bf 2 (P.I. range of 5.8–6.0) exemplified by B10.MOL (OHM), Bf 3 (P.I. range of 5.6–5.9) exemplified by B10.MOL (TEN2) and Mus musculus (Mus m.) subspecies Chc, Bf4 (P.I. range of 6.0–6.3) exemplified by Mus m. subspecies Shh. The genetic linkage between S locus and Bf locus was studied with two backcross progenies — [B 10.BR × (B10.BR × Mus m. subspecies Chc)F₁] and [B 10.BR × (B10.BR × Mus m. subspecies Shh)F₁]. Totally, 256 backcross progenies were typed for Bf type and for Ss type (plasma level of the fourth complement protein regulated by S locus). The results indicated that murine Bf was controlled by a single codominant locus located close to the H-2 complex because no mouse showing recombination between Bf locus and S locus was found.     

Genotypic analysis of families with lactate dehydrogenase A(M) deficiency by selective DNA amplification

Summary Genomic DNA prepared from LDH-A-deficient whole blood was amplified by the polymerase chain reaction technique using two primers specific for the active human LDH-A gene. The amplified fragment was examined by direct agarose gel electrophoresis, and a deletion of 20 base pairs (bp) in exon 6 of the LDH-A gene was found. The results permitted a clear distinction between the homozygous mutant, the heterozygous mutant, and wild-type genotypes. Moreover, HinfI digestion and direct sequencing of the amplified product confirmed the results from direct agarose gel electrophoresis. Four families, including 18 individuals, were shown to contain the same mutation, that is a 20-bp deletion in exon 6. All genotypes were consistent with their biochemical phenotypes as evaluated by the ratio of LDH-B to LDH-A subunits in erythrocytes. Thus, all four known affected families in Japan have been shown to carry the same mutant gene, which may have been derived from a single mutational event.     

Light and electron microscopical demonstration of c-erbB-2 gene product-like immunoreactivity in human malignant tumors

C-erbB-2 is a human protooncogene homologous with the well-known c-erbB. Genes and gene products of the EGF receptor and c-erbB are known to be closely related and to be closely homologous in their intracellular domain. Inspection of the deduced amino acid sequence suggested that the c-erbB-2 gene encodes a receptor for a yet unidentified growth factor. An immunohistological study was performed by introducing an antibody raised in the rabbit by immunization with a synthetic peptide corresponding to a part of the intracytoplasmic domain of predicted gene product. Specimens from 13 normal human organs, fresh frozen tissue from 41 surgically excised human malignant tumors and eight cell lines maintained in nude mice were studied. Positive staining was found in 4 of the 41 (9.8%) malignant tumors. All of the positive tumors were adenocarcinomas and two adenocarcinoma cell lines were also positive. Amongst the normal human tissues, epithelial cells in stomach, small and large intestine were faintly stained. When the positively stained cell lines were studied by immunoelectronmicroscopy, the reaction was most prominent in the membrane of microvilli, but part of the nuclear membrane, the endoplasmic reticulum and the outer cell membrane were also stained. DNA and mRNA blot assays, as well as our immunoprecipitation test, revealed that immunohistologically positive cell lines bore amplified c-erbB-2 DNA, c-erbB-2 mRNA and 185 kD protein which is supposed to be the gene product, while negative cell lines did not.     

Macrophage factors which enhance the mixed leukocyte reaction initiated by dendritic cells

Stimulator cells for the allogeneic MLR are dendritic cells but not macrophages (M phi). M phi, however, enhance the MLR initiated by relatively low doses of dendritic cells. The present report demonstrates that the enhancement of the MLR is mediated by two factors produced by M phi. One is IL-1, inasmuch as it has a Mr approximately 15 kDa, and both partially purified IL-1 and rIL-1 also enhance the MLR. The other has been identified as granulocyte-macrophage (GM)-CSF. It had a Mr approximately 25 kDa, and is reproduced by rGM-CSF. Moreover, the MLR-enhancing activity of both the 25-kDa molecule and rGM-CSF have been neutralized by anti-GM-CSF antiserum. Both IL-1 and GM-CSF have autonomous enhancing activity, but they collaborate with each other in enhancing the MLR. Both factors act on DC to augment their stimulatory activity for allogeneic T lymphocytes.     

Litter-fall and reproductive seasonalities in aLeucaena leucocephala forest at Chichijima,Ogasawara (Bonin) Islands

Seasonal changes in litter components were measured in a closedLeucaena leucocephala forest stand in Chichijima, Ogasawara (Bonin) Islands, situated in a wet subtropical climate. The phytomass of the stand was estimated as 5.62, 1.47 and 0.448 kg d.w. m⁻² for stems, branches and leaves, respectively. Litter from the canopy was collected semimonthly by ten 0.5 m² traps. Leaf-litter production rate was low in spring, medium in summer and autumn, and peaked in mid-winter. Annual production of leaf-litter was 498 g d.w. m⁻². Annual turnover rate of canopy leaves was 1.1. Flower-litter showed biannual production with peaks in spring and autumn, and the former peak largely exceeded the latter one in quantity. Seed maturation needed about 3 months. Annual production of reproductive organs was 89.2, 89.4, 19.1 and 99.4 g d.w. m⁻² for flowers, mature seeds, immature ones and pods, respectively. Number, of mature seeds disseminated exceeded 2,500 m⁻². Annual production of branch- and bark-litter was 345 g d.w.m⁻². Annual amount of plant litter from the canopy was as large as 1,150 g d.w. m⁻², showing higher productivity and higher rate of matter turnover in this forest than in temperate forests.     

Structure of Marek's disease virus DNA: detailed restriction enzyme map.

Purified virion DNA (120 X 10(6) molecular weight [MW]) of Marek's disease virus strain GA was cleaved with BamHI restriction endonuclease, and 27 out of the 29 fragments were cloned into bacterial plasmids. Restriction maps for BamHI, BglI, and SmaI endonucleases were constructed. The genomic structure of Marek's disease virus DNA was found to be similar to that of herpes simplex virus types 1 and 2. A long unique region (75 X 10(6) MW, located at 10 X 10(6) to 85 X 10(6) MW [10-85] from the left end of the genome), which was subdivided into segment 1 (22 X 10(6) MW, located at 10-32) and segment 2 (51 X 10(6) MW, located at 34-85) by direct repeats (32-34), was flanked by a long terminal region (10 X 10(6) MW, located at 0-10) and a long inverted region (10 X 10(6) MW, located at 85-95). A short unique region (8 X 10(6) MW, located at 103-111) was flanked by a short terminal region (8 X 10(6) MW, located at 111-119) and a short inverted region (8 X 10(6) MW, located at 95-103). The direct repeat fragments (0.9 X 10(6) could be isolated by cleavage with SmaI. The right terminal end was found to be heterogenous .     

Effect of concentration of compounds containing iron on the growth of Porphyromonas gingivalis

Abstract We examined the effect of the concentration of various types of iron molecules on the regulation of growth of Porphyromonas gingivalis . Bacterial growth was monitored spectrophotometrically. The hemin-depleted cells of P. gingivalis 381 were incubated in the basal medium plus test substrates such as hemoglobin, hemin, transferrin and various inorganic iron compounds. The relationship between the specific growth rate of organisms and the concentration of iron-containing compounds was determined. The value of K _s, a parameter analogous to the Michaelis-Menten constant, was estimated. P . gingivalis 381 showed a K _ss value of 3.85, 4.91 and 0.0017 μM for hemin, transferrin and hemoglobin, respectively. However, the inorganic iron compounds tested did not support growth of P. gingivalis . These findings suggest that P. gingivalis utilizes hemoglobin as an iron source much more effectively than other iron-containing compounds under an iron-limited environment.