Partial purification and characterization of a factor which stimulates an increase in ornithine decarboxylase activity in the liver from tumor cell-free ascites fluid

A factor responsible for stimulating an increase in ornithine decarboxylase activity in the liver of mice was found in tumor cell-free ascites fluid of mice 3 days after inoculation of tumor cells. The factor was purified about 70-fold in 25% yield from tumor cell-free ascites fluid. As little as 1 μg of protein of purified fraction, injected intraperitoneally into normal mice, significantly increased the activity of ornithine decarboxylase in the liver. The most active preparation of the factor formed two major protein bands on analytical polyacrylamide gel electrophoresis and both these bands stained with periodic acid-Schiff's reagent. The factor was a heat-labile, alkaline-stable, acidic protein with a molecular weight of more than 300 000. It was inactivated by treatment with 10 mM dithiothreitol, 5M urea, pronase or mixed glycosidase, but was stable on treatment with DNAase, RNAase or neuraminidase.     

Tyrosine Hydroxylase Is Activated and Phosphorylated on Different Sites in Rat Pheochromocytoma PC12 Cells Treated with Phorbol Ester and Forskolin

Abstract: Incubation of rat pheochromocytoma PC12 cells with 4β-phorbol-12β-myristate-13α-acetate (PMA), an activator of Ca²⁺/phospholipid-dependent protein kinase (protein kinase C), or forskolin, an activator of adenylate cyclase, is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase. Neither the activation nor increased phosphorylation of tyrosine hydroxylase produced by PMA is dependent on extracellular Ca²⁺. Both activation and phosphorylation of the enzyme by PMA are inhibited by pretreatment of the cells with trifluo-perazine (TFP). Treatment of PC 12 cells with l-oleoyl-2-acetylglycerol also leads to increases in the phosphorylation and enzymatic activity of tyrosine hydroxylase; 1, 2-diolein and 1, 3-diolein are ineffective. The effects of forskolin on the activation and phosphorylation of the enzyme are independent of Ca²⁺ and are not inhibited by TIT⁵. Forskolin elicits an increase in cyclic AMP levels in PC 12 cells. The increases in both cyclic AMP content and the enzymatic activity and phosphorylation of tyrosine hydroxylase following exposure of PC 12 cells to different concentrations of forskolin are closely correlated. In contrast, cyclic AMP levels do not increase in cells treated with PMA. Tryptic digestion of the phosphorylated enzyme isolated from untreated cells yields four phosphopeptides separable by HPLC. Incubation of the cells in the presence of the Ca²⁺ ionophore ionomycin increases the phosphorylation of three of these tryptic peptides. However, in cells treated with either PMA or forskolin, there is an increase in the phosphorylation of only one of these peptides derived from tyrosine hydroxylase. The peptide phosphorylated in PMA-treated cells is different from that phosphorylated in forskolin-treated cells. The latter peptide is identical to the peptide phosphorylated in dibutyryl cyclic AMP-treated cells. These results indicate that tyrosine hydroxylase is activated and phosphorylated on different sites in PC 12 cells exposed to PMA and forskolin and that phosphorylation of either of these sites is associated with activation of tyrosine hydroxylase. The results further suggest that cyclic AMP-dependent and Ca²⁺/ phospholipid-dependent protein kinases may play a role in the regulation of tyrosine hydroxylase in PC 12 cells.     

Lack of association and linkage between HLA and familial polyposis coli

Summary We investigated possible association of and linkage between HLA and familial polyposis coli (FPC). In 182 individuals from 66 pedigrees of FPC and 108 individuals from a normal population, HLA-A,-B, and-C antigens were determined. When the frequencies of HLA antigens in 66 unrelated patients and in normal controls were compared, no association of FPC with HLA was observed. For the linkage analysis, HLA haplotypes of 17 affected sib pairs were investigated by the affected sib pair method. The number of pairs which shared two, one, and no haplotypes identical by descent was not significantly different from the number expected with random occurrence (P>0.95). Finally, seven families were analyzed using Morton's sequential test. A maximum lod score of-0.056 at a recombination fraction of 0.4, and a lod of-3.089 at a recombination fraction of 0.05 were obtained. Therefore, there is neither an association of nor linkage between FPC and HLA.     

Characteristics of settling matter and its role in nutrient cycles in a deep oligotrophic lake

The settling flux of seston (dry weight, DW), chlorophyll a (Chl a), particulate organic carbon (POC), particulate organic nitrogen (PON), and particulate phosphorus (PP) was measured monthly in 1981–1983 at 10 different depths in Lake Chuzenji, Japan; an oligotrophic lake with a maximum depth of 163 m. The Ti concentration in entrapped matter was used to separate the sedimentation flux into allochthonous and autochthonous components. Inflow loads of dissolved nutrients (DN: 4.5, DP: 0.48 g m^-2a^-1) were almost sufficient to supply the autochthonous fluxes at 30 m (PON: 2.9, PP: 0.51 g m^-2a^-1 ), and this flux of POC (26.6 g m^-2a ^-1) was about one-third of primary production (84 g C M^-2a^-1). Sedimentation of particulate matter was the main path of losing nutrients from lake water, explaining more than 80% removal of inflow loads (TN, TP). Decomposition rates during sedimentation which were calculated from the vertical difference in the autochthonous flux agreed very closely with the results obtained by laboratory experiments of a 100-day incubation (content ratios from field observations were: POC 0.67, PON 0.65, PP 0.85; and from laboratory experiments they were: POC 0.68, PON 0.70, PP 0.94). These decomposition rates and those near the sediment interface were used to explain dissolved oxygen depletion and nitrate increase in the hypolimnion during stratification. The average sinking velocities were 1.82m d^-1 for seston and 1.16 m d^-1 for Chl a at 30m, they were influenced by Chl a content of seston.     

Cloning of genes for a hemolytic factor of Leptospira interrogans serovar autumnalis strain Congo 21-543

A DNA fragment encoding a hemolytic factor was cloned from the parasitic spirochete Leptospira interrogans serovar autumnalis strain Congo 21-543. Initial clones were isolated by screening a genomic library in pBR322 in Escherichia coli for hemolytic activity. Hemolytic activity was coded by a 4.5 kilobase BamHI-HindIII fragment. Southern hybridization with DNAs from other strains of Leptospira using this gene as a probe showed that DNAs from non-parasitic strains failed to hybridize with the probe, whereas those from all parasitic strains tested had the sequence which hybridize to the probe.     

Localization of follicle regulatory protein in the porcine ovary

The granulosa cell secretes a protein (follicle regulatory protein: FRP) that affects the responsiveness of other follicles to gonadotropin stimulation. This protein was purified, partially characterized, and rabbit antisera as well as monoclonal antibodies were prepared against FRP. Fixed sections of porcine ovaries were prepared on slides and then incubated with the monoclonal antibody or polyclonal antisera and then incubated with either biotinylated mouse IgM or rabbit IgG antisera, respectively. These sections were then incubated with avidin conjugated to horseradish peroxidase, followed by substrate. Staining with both the monoclonal antibody and the antisera was present in the cytoplasm of granulosa cells of small- or medium-sized antral follicles. Staining distribution was localized preferentially to cells near the basal lamina; the antral granulosa cells of viable follicles did not stain. Neither primordial follicles nor pre-antral follicles (less than 300 microns in diameter) showed any positive staining. Thecal cells were not stained in follicles less than 5 mm in diameter, whereas some large follicles (greater than 5 mm) contained staining in the theca. In the latter, specific granulosa staining was only weakly positive with the polyclonal antibody and negative with the monoclonal antibody. Atretic follicles contained significant staining of all epithelial cells adjacent to the basal lamina by both the monoclonal and polyclonal antibody preparations. Staining of the luteal ovary by the monoclonal antibody was limited to the large luteal cells. These findings suggest that FRP is produced by the granulosa cells of porcine follicles at the stage of maturation corresponding to 0.5 mm in diameter. As the viable follicle increases in size, production of FRP in the granulosa is reduced below the detectable level when the follicle exceeds 5 mm in diameter. The main source of FRP during the luteal phase is the large cell of the corpus luteum.     

Preparation ofN-acetylneuraminic acid from delipidated egg yolk

Egg yolk, a large proportion of the egg, was studied for the preparation ofN-acetylneuraminic acid (Neu5Ac). The delipidated hen egg yolk (DEY; 500 kg containing 0.2% w/w, Neu5Ac) was hydrolysed with HCl (pH 1.4) at 80 °C and neutralized with NaOH (pH 6.0). The mixture was filtered and electrodialysed until the conductivity was 240 µS cm^–1. The filtrate was applied on a column of Dowex HCR-W2 (20–50 mesh), followed by a column of Dowex 1-X8 (200–400 mesh). The latter column was washed with water, and then eluted with a linear gradient of HCO₂H (0–2m). The eluates containing Neu5Ac were concentrated using a reverse osmosis membrane and, finally, rotary evaporated at 40 °C. The residue was then lyophilized to yield 500 g Neu5Ac. The purity of Neu5Ac was >98% (TBA method). HPLC, NMR spectroscopy and TLC chromatography of the product obtained from the DEY showed that Neu5Ac was the sole derivative present in egg yolk. The DEY, a byproduct from egg processing plants, was found to be an excellent source for the large-scale preparation of Neu5Ac.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Gc N-glycolylneuraminic acid - DEY delipidated egg yolk - HPLC high performance liquid chromatography - TLC thin layer chromatography - NMR nuclear magnetic resonance - IR infrared spectroscopy Presented at the 11th International Symposium on Glycoconjugates, Toronto, Canada.     

Enhanced Phosphorylation of Tyrosine Hydroxylase at More Than One Site Is Induced by 56 mM K+ in Rat Pheochromocytoma PC 12 Cells in Culture

Incubation of rat pheochromocytoma PC12 cells with dibutyryl cyclic AMP or 56 mM K+ is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase in situ. Following incubation of the PC12 cells with 32Pi, rapid isolation of the tyrosine hydroxylase, and tryptic digestion of the enzyme, two distinct 32P-peptides can be identified after paper electrophoresis. 56 mM K+ increases 32Pi incorporation into both of these peptides, whereas dibutyryl cyclic AMP increases 32Pi incorporation into only one of these peptides. The rate of increase in the incorporation of 32Pi into these two peptides in cells treated with 56 mM K+ is similar. The phosphorylation of tyrosine hydroxylase in PC12 cells occurs exclusively on serine residues. These results suggest that tyrosine hydroxylase in PC12 cells is phosphorylated on serine residues at two or more distinct sites after 56 mM K+ -induced depolarization. Since only one of these sites is phosphorylated by cyclic AMP-dependent protein kinase, activation of tyrosine hydroxylase by 56 mM K+ may involve phosphorylation by multiple protein kinases in rat pheochromocytoma PC12 cells.     

Serologic dissection of HLA-D specificities by the use of monoclonal antibodies

To study the gene products of the HLA complex, we produced two monoclonal antibodies, termed HU-18 and HU-23. They were active in complement-dependent cytotoxicity and detected B-cell alloantigens encoded by a locus (or loci) linked to HLA. When three types of HLA-DR4 homozygous B-cell lines with different HLA-D specificities were tested for reactivity with HU-18 and HU-23, they displayed distinct reaction patterns depending on the HLA-D specificities they possessed: EBV-Wa (HLA-DYT homozygous), negative for both HU-18 and HU-23; KT2 and KOB (HLA-DKT2 homozygous), positive only for HU-18; and ER (HLA-Dw4 homozygous), positive for both. These differential reaction patterns were further confirmed by testing against a panel of 17 HLA-DR4-positive peripheral blood lymphocytes with known HLA-D specificities. Thus, these monoclonal antibodies allow us to identify HLA-DYT, HLA-DKT2, and HLA-Dw4 solely by serologic methods. This is the first clearcut serologic identification of these three HLA-DR4-associated HLA-D specificities, which have been indistinguishable by conventional serology and identified only by cellular techniques. It is hoped that immunochemical investigations using HU-18 and HU-23 will advance our understanding of the HLA-D region on a molecular level.     

Kinetics of microbial cell growth

As a rate equation of microbial cell growth, the Monod equation is widely used. However, this equation cannot fully correspond to real courses of microbial cell growth in many batch cultivations. Especially, predicted values based on this equation do not agree with observed values in many continuous cultivations. In this paper, which introduces new concepts of critical concentration and coefficient of consumption activity, the growth rate equation which corresponds to the whole period including lag period is newly derived and characteristics of microbial cell growth in batch cultivation are clarified. Further, applying the new rate equation to continuous cultivation, a general equation with which to calculate cell concentration is derived and characteristics of microbial cell growth in continuous cultivation are clarified. The calculated values of cell concentration based on the new theory showed quite good agreement with the observed values in both batch and continuous cultivation.