Molecular cloning,nucleotide sequence and expression of a cDNA encoding an intracellular protein tyrosine phosphatase,PTPase-2, from mouse testis and T-cells

The PTP-2 cDNA encoding an intracellular protein tyrosine phosphatase (PTPase-2) was isolated and sequenced from mouse testis and T-cell cDNA libraries. This PTP-2 cDNA was found to be homologous to human PTP-TC and rat PTP-S, and contained 1,551 nucleotides, including 1,146 nucleotides encoding 382 amino acids as well as 5 (61 nucleotides) and 3 (344 nucleotides) non-coding regions. Northern blot analysis indicated that PTP-2 mRNA of 1.9 Kb was most abundant in testis and kidney, although it was also present in spleen, muscle, liver, heart and brain.Abbreviations PTPase Protein Tyrosine Phosphatase (EC3.1.3.48) - PTKase Protein Tyrosine Kinase (EC2.7.1.112)     

Glucosylation of Salicyl Alcohol by Gardenia jasminoides Cell Cultures

Cultured cells of Gardenia jasminoides produced both salicinand isosalicin from exogenously supplied salicyl alcohol. Theglucosylation activity of the cells was highest in the exponentialphase of growth and ca. 70% of the added substrate was convertedto the glucosides within 4 days. The rate of glucosylation wasalso dependent on the medium composition such as auxin and sucroseconcentrations. The ratio of salicin to isosalicin formed fromsalicyl alcohol was influenced by the growth stage of the culturedcells. Salicin was converted to isosalicin when exogenouslyadded to the culture. (Received October 11, 1985; Accepted March 10, 1986)     

Two classes of continuous cell lines established from Syrian hamster 9 day gestation embryos: Preneoplastic cells and progenitor cells

Summary Primary cultures of 9-d-gestation Syrian hamster embryo (E9) cells are distinct from primary cultures of later gestational age in terms of their growth and differentiation. First, primary E9 cell cultures express multiple mesenchymal differentiation lineages (e.g., adipocyte, myoblast) only rarely seen in cultures of 13-d-gestation fetal (F13) cells. Second, although most primary E9 cultures have a limited in vitro proliferative life span and exhibit cellular senescence similar to primary cultures of F13 cells, E9 cultures seem to have higher frequency of escape from senescence and conversion to continuous cell lines compared to F13 cells. Moreover, this frequency can be further increased 4- to 5-fold by continuous exposure of the E9 cells to tumor promoters or epidermal growth factor. Eleven continuous cell lines have been isolated from unreated, promoter-treated, or epidermal growth factor-treated primary E9 cultures. Seven of these are neoplastic or preneoplastic. However, the remaining four do not show any evidence of being in neoplastic progression and three of these continue to express the same differentiated phenotype observed in ther parental primary cell cultures. These studies were supported in part by grants from the National Institutes of Health (AG 01998), Bethesda, MD, and the U.S. Department of Energy (DE-A-C02-76-EVO-3280), Washington, DC.     

Determination of non-protein-bound iron in human synovial fluid by high-performance liquid chromatography with electrochemical detection

Non-protein-bound iron in human synovial fluid was determined using high-performance liquid chromatography with electrochemical detection. The procedure was based on the separation of the iron—diethylenetriaminepentaacetic acid (DPTA) complex formed directly on a chromatographic column containing an anion-exchange resin followed by electrochemical detection. The method enabled more than 0.1 μM Fe(III) to be determined with an injection volume of 10 μl. A mixture of synovial fluid, 20 μM DTPA and acetate buffer was incubated in the presence and absence of superoxide (O⁻₂) generated by a xanthine—xanthine oxidase system and was ultrafiltered through a 30 000 molecular mass cut-off filter. No iron was detected in the ultrafiltrate at physiological pH. However, the presence of iron was observed in the ultrafiltrate at low pH, and O⁻₂ and decreased pH, iron may be released into the synovial fluid.     

Effect of pancreastatin on pancreatic endocrine function in the conscious rat

We have studied the effects of pancreastatin on insulin and glucagon secretions in vivo in the conscious rat. Rats were prepared with a gastric fistula and with both external jugular veins cannulated. We found that an i.v. infusion of pancreastatin (1 and 10 nmol/kg/h) inhibited the plasma insulin response and increased the plasma glucose response to the intragastric infusion of glucose in a dose-dependent manner. Furthermore, the infusion of pancreastatin increased the plasma glucagon response to the i.v. infusion of arginine in a dose-dependent manner, and it inhibited the plasma insulin response. However, such an infusion of pancreastatin had no effect on the basal plasma glucose level, nor did it have any effect on plasma insulin and glucagon concentrations. Thus, it is suggested that in the rat, the newly discovered pancreastatin is a regulator of islet cell function.     

A novel cell surface antigen involved in thymocyte and thymic epithelial cell adhesion.

A murine mAb, 7D3, was produced by fusion of spleen cells obtained from mice immunized with a rat thymic epithelial cell line, Tu-D3 and NS/1 myeloma cells. 7D3 antibody reacted with approximately 95% thymocytes, 17% spleen cells, less than 9% of mesenteric lymph node cells and 32% of bone marrow cells of rat origin. 7D3 also reacted with two rat thymic epithelial cell lines but not with a rat fibroblastic cell line. Immunochemical analysis demonstrated that 7D3 antibody recognized a single polypeptide with molecular weight of 80,000 in FTE cells and 80,000 to 96,000 in thymocytes. 7D3 antibody strongly inhibited the thymocyte binding to thymic epithelial cells. In addition, 7D3 antibody inhibited TPA-induced thymocyte aggregation. 7D3 negative rat thymic lymphoma cells bound to 7D3 positive thymic epithelial cells and this binding was inhibited by 7D3 antibody, indicating that a part of thymocyte-thymic epithelial cell binding was mediated by the interaction of 7D3 Ag and undefined ligand to 7D3.     

Stimulation by growth hormone of MAP kinase activity in 3T3-F442A fibroblasts.

We have previously demonstrated that growth hormone (GH) promotes an increase in tyrosine kinase activity associated with the GH receptor. To gain insight into the role of GH-dependent tyrosine kinase activity in signaling by GH, we investigated the possibility that GH might stimulate MAP kinase, a serine/threonine/tyrosine kinase thought to be a common element in tyrosine kinase-initiated response cascades. Treatment of 3T3-F442A fibroblasts with 100 ng/ml GH results in a 3-6-fold increase in the ability of cell-free extracts to phosphorylate MAP-2 and myelin basic protein. GH-stimulated kinase activity is unaffected by heparin, H7, or cAMP-dependent protein kinase inhibitor peptide, partially reduced by staurosporin and inhibited by fluoride and calcium ions, indicating that the kinase is not protein kinase C or A, casein kinase, or a calcium/calmodulin-dependent protein kinase. Based on gel permeation chromatography, the molecular mass of the GH-stimulated MAP kinase is approximately kDa. Furthermore, anti-phosphotyrosine antibodies revealed the GH-dependent appearance of two phosphotyrosine-containing proteins in cell-free lysates of GH-treated cells that co-migrate with proteins recognized by anti-MAP kinase antibodies. The GH-dependent increase in MAP kinase activity displays a biphasic time course and is dependent on the concentration of GH applied to the cells. GH-dependent MAP kinase activity, partially purified by Mono-Q chromatography, is inactivated by treatment with alkaline phosphatase. Addition of H7 to the cells prior to the addition of GH has no effect, whereas addition of H8 increases MAP kinase activity in control cells with no effect in GH-treated cells, indicating that protein kinase C is unlikely to be an intermediary in the GH-dependent stimulation of MAP kinase activity. These findings indicate that signaling by GH in 3T3-F443A cells may, at least in part, utilize a kinase cascade similar to those that have been proposed for other membrane receptors with associated tyrosine kinase activity.     

Age-related alterations in the size of human hepatocytes

Age-related alterations in the size of human hepatocytes (both mononuclear and binucleate forms), were studied in histological sections and in separated cells and nuclei using cytophotometrical and microspectrophotometrical methods. The following results were obtained: 1. The volume of nuclear DNA increased in proportion to nuclear size. The increase occurred in a group pattern reflecting nuclear polyploidization. 2. Cell size increased in proportion to nuclear size. Tetraploid cells (4C) were roughly two times greater than diploid cells (2C). 3. In most of the binucleate cells examined, the ploidy class of the two nuclei in a binucleate cell was observed to be equal. Heterogeneity of the ploidy class among the nuclei of a binucleate cell was present in less than 1% of total binucleate cells examined. The nuclear DNA volume of individual nuclei in binucleate cells appeared to be the same as that of mononuclear cells. 4. The cell size of binucleate cells corresponded with that of mononuclear cells whose ploidy class was the same as the sum of the ploidy classes of two nuclei of a binucleate cell. 5. The incidence of binucleate cells in the lobular periphery was about 4 to 6% in the third decade, and increased slightly with age up to 5 to 7% in the tenth decade. 6. The incidence of binucleate cells in the liver at different ages followed a similar pattern to that observed in mononuclear cells whose ploidy class was half of the sum of ploidy classes of the two nuclei of the binucleate cell.