Clonal nature of murine hemopoietic blast cell colonies

Murine hemopoietic blast cell colonies obtained from spleen cells of 5-fluorouracil (5-FU)-treated mice give rise to many multilineage colonies including granulocyte - erythrocyte - macrophage - megakaryocyte (GEMM) colonies in secondary cultures. Progenitor cells for blast cell colonies are considered to be more primitive than colony forming units (CFU)-GEMM. To determine whether they are clonal, we examined the phosphoglycerate kinase-1 (PGK-1) isozyme type of colonies originally grown from spleen cells of 5-FU-treated mice which had PGK-1 isozyme mosaicism. PGK assays of whole secondary colonies derived from one blast cell colony showed that they were either of type A or type B but not both. These results suggest that murine hemopoietic blast cell colonies are clonal.     

Morphology,physics, chemistry and biology of Lake Rara in West Nepal

A survey of oligotrophic Lake Rara, the biggest lake in Nepal, was carried out from 1982 till 1984. Mean depth is 100 m, and maximum depth is 167 m. The surface area covers 9.8 km², and the lake contains 0.98 km³ volume of water. Transparency was about 16 m, photoquantum yield decreased exponentially with depth below 5 m, and the extinction coefficient was 8.3 × 10⁻². The concentration of Chl.-a was in the range of 0.06–0.46 mg m⁻³, and total nitrogen was 18–30 μg 1⁻¹. The whole water column was well oxygenated. Primary productivity was extremely low. It has more than 30 inflowing brooks and one outlet. The water quality of the brooks changes drastically with their location. The pH, electrical conductivity, and EDTA hardness in the waters from a landslide area were high. In the waters from a rich pine forest they were extremely low. The zooplankton consisted of two species of protozoa, five species of rotifers, two species of Cladocera, and two species of Copepoda. The zooplankton density range was 6200–16200 individuals m⁻³. The minimum was on November 11th, 1983 and the maximum on August 19th, 1983.     

The pyrogallol related compounds reduce UV-induced mutations in Escherichia coli B/r WP2

Plant components with bio-antimutagenic activity were screened on UVC (254 nm)-induced mutagenesis using E. coli B/r WP2. The components with a pyrogallol moiety including gallic acid, (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG) reduced the mutation induction, but other components such as caffeic acid, chlorogenic acid and quercetin did not. The above compounds with a pyrogallol moiety were also effective on UVAB (295-400 nm)-induced mutagenesis, while they showed little effect on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutagenesis. As this bio-antimutagenic effect was not seen in the DNA excision-repair-deficient strains WP2s and ZA159, the activity by the above plant components might be based on the promotion of the excision-repair system in E. coli B/r WP2.     

Early development of fin-supports ani fin-rays in the milkfishChanos chanos

Development of fin-supports and fin-rays was observed in larval and juvenileChanos chanos, Chondrification of the caudal complex started at 4.70 mm SL. Ossification of the caudal elements started at 7.80 mm SL and was nearly completed at about 30 mm SL. Cartilaginous fusion of caudal elements, which occurs in hypurals of higher teleostean fishes but is not seen in lower teleosts, was observed between the neural arch of the preural centrum 1 and that of the ural centrum 1 via a small cartilage bridging the distal tips of the two arches. Caudal finrays began to develop at 6.60 mm SL, and an adult complement of principal rays was attained at 7.35 mm SL. Dorsal and anal pterygiophore elements were first evident at 6.70 mm and 6.65 mm SL, respectively. All proximal radiais were formed at 8.15 mm SL in both fins. Formation of dorsal and anal fin-rays started simultaneously at 8.60 mm SL, and adult fin-ray complements were attained at 10,00 mm and 10.70 mm SL, respectively. In the pectoral fin, the cleithrum, coraco-scapular cartilage and blade-like cartilage (fin plate) had already been formed at 4.65 mm SL. The mesocoracoid was observed to originate from the coraco-scapular cartilage and become detached from it in the course of ossification. Pectoral fin-ray formation started at 13.80 mm SL and was completed in number of rays at 20.00 mm SL. In the pelvic fin, the basipterygium was first evident at 13.00 mm SL. Pelvic fin-rays appeared at 13.80 mm SL and attained their adult count at 17.15 mm SL.     

Purification and physicochemical characterization of murine T cell replacing factor (TRF)

Murine T cell replacing factor (TRF) was purified from a cellfree supernatant of a T cell hybridoma (B151K12) that constitutively produces TRF. Two assay systems for TRF activity were employed: 1) induction of anti-DNP IgG PFC responses in cultures of splenic B cells from DNP-KLH-primed BALB/c mice, and 2) induction of IgM PFC in chronic B cell leukemic cells (BCL1). The purification scheme consisted of ammonium sulfate precipitation, DEAE-cellulose chromatography, Blue-Sepharose chromatography, hydroxylapatite chromatography, gel permeation with fast protein liquid chromatography (FPLC), and disc polyacrylamide gel electrophoresis. Overall, TRF was purified approximately 34,000-fold with a maximum 3.8% recovery of activity, and the specific activity of the purified TRF was approximately 9.6 X 10(4) U/mg. The TRF that is active in these systems is distinct from the other lymphokines such as IL 1, IL 2, BCGFI (now known as BSFp1), and gamma-interferon. The TRF is extremely hydrophobic, with an apparent m.w. of 50,000 to 60,000 on gel permeation chromatography and 18,000 on SDS-PAGE under reducing conditions. Highly purified B151-TRF abrogated the activity by treatment with trypsin but not with RNase. Moreover, it bound to lima bean agglutinin-Sepharose specific for N-acetylgalactosamine residues, indicating that B151-TRF is a glycosylated glycoprotein containing N-acetylgalactosamine residues. The role of N-acetylgalactosamine residues on TRF activity was additionally substantiated by the fact that the addition of appropriate amounts of N-acetylgalactosamine in the assay systems for TRF preferentially induced a profound suppression for TRF-mediated PFC responses.     

Binding of penicillin to thiol-penicillin-binding protein 3 of Escherichia coli: identification of its active site

Summary In order to determine the active site of penicillin-binding protein 3 of Escherichia coli (PBP3), the serine residue at position 307 was replaced with alanine, threonine or cysteine by oligonucleotide-directed site-specific mutagenesis. Since a unique BanII site exists at the position corresponding to serine-307, BanII digestion of the plasmid DNA after mutagenesis resulted in significant enrichment of the mutant plasmids. For mutagenesis, the gene coding for PBP3 (ftsI) was inserted into the expression cloning vector pIN-IIB. The hybrid protein produced was able to bind penicillin while mutant PBP3 in which serine-307 was replaced with either alanine or threonine did not lead to any detectable binding. However, contrary to the report of Broome-Smith et al. (1985) thiol-penicillin-binding protein 3, in which serine-307 was replaced with cysteine, was still able to bind penicillin. Replacement of serine-445 with an alanine residue had no effect on penicillin binding to PBP3.     

Measurement of urinary steroid profile in patients with adrenal tumor as a screening method for carcinoma

Results of measurement of urinary steroid metabolite profile using gas chromatographic analysis in eight patients with adrenocortical tumors, i.e. 3 adenomas with Cushing's Syndrome, one adenoma with virilization, one adenoma without clinical manifestations, one carcinoma with Cushing's syndrome and virilization, one carcinoma with Cushing's syndrome and feminization, and one carcinoma without endocrinological symptoms, are reported. A unique pattern dominated by 5 beta and 11 beta-hydroxy steroid metabolites was confirmed in five patients with Cushing's syndrome consisting of three cases with adenomas and two with carcinomas. Excessive 3 alpha, 17 alpha, 21-trihydroxy-5 beta-pregnan-20-one (tetrahydro-11-deoxycortisol, THS) and delta 5-pregnene-3 beta, 11 alpha, 20 alpha-triol (delta 5-pregnenetriol) values were found in all three carcinomas including a nonfunctional carcinoma. These findings would strongly suggest the tumor to be a carcinoma, although excessive excretion of THS and delta 5-pregnenetriol was detected in one patient with a large adenoma associated with virilization. One patient with carcinoma was responsive to ACTH stimulation while the remainder show almost no response to exogenous ACTH. Urinary steroid profiling using gas chromatographic analysis, especially the values for THS and delta 5-pregnenetriol, appears to be a useful method to use in detecting these steroid metabolic characteristics in patients with adrenocortical carcinoma.     

Isolation and characterization of a monoclonal antibody against pig kidney sodium- and potassium-activated ATPase

A hybridoma cell line producing mouse monoclonal antibody against pig kidney Na,K-ATPase was established. The antibody, named 38 (mAb38, IgG1), was purified from mouse ascites fluid by chromatography on a protein A-Sepharose column. Antigens immobilized on microplate wells with p-benzoquinone were used for titer assays. mAb38 cross-reacted with both dodecyloctaethyleneglycol monoether (C12E8)-solubilized enzyme and membranous sodium dodecyl sulfate (SDS)-treated enzyme from kidney with high affinity (50% binding = 0.6 nM). However, the antibody bound to neither alpha- nor beta-subunit separated by preparative SDS-polyacrylamide gel electrophoresis (PAGE). The stoichiometry of antibody binding to the purified enzyme was estimated to be about 0.86 mol of IgG per mol of alpha beta-protomer. Na,K-ATPase proteins were recovered from a column of mAb38-coupled Affi-Gel by elution with pH 3 buffer when C12E8-solubilized kidney enzyme or detergent extracts of brain microsomes were applied to it, confirming that the mAb is directed to Na,K-ATPase. mAb38 at saturation level concentrations had no effect on kidney Na,K-ATPase activity or on ouabain-sensitive Rb uptake in erythrocytes. In an immunofluorescence study, the antibody bound to intact erythrocytes much more strongly than control IgG1 (mAb50c), but the extent of the antibody binding to inside-out vesicles under hypotonic conditions was lower than that of the control. Most of the antibody binding activity remained when the kidney enzyme was treated with sialidase. These results suggest that this mAb38 was raised against an intact conformation of a cell-surface-exposed site of Na,K-ATPase.     

Specificity of hydrogen transfer of mammalian and avian carbonyl and aldehyde reductases

Aldehyde reductases from several mammalian and avian tissues transferred the pro-4R hydrogen of NADPH to the substrate, whereas the stereospecificity of carbonyl reductases was not uniform being correlated with the ability to catalyze the oxidoreduction of hydroxysteroids.