Occurrence of a new enzyme catalyzing the direct conversion of NG,NG-dimethyl-L-arginine to L-citrulline in rats

An enzyme concerned with the degradation of NG,NG-dimethyl-L-arginine to L-citrulline was investigated in rats. The enzyme purified from rat kidney catalyzed the direct conversion of NG,NG-dimethyl-L-arginine to L-citrulline with liberation of dimethylamine from the methylated guanidino moiety. The reaction required no co-factor and the maximum activity was obtained at pH 6.5. The enzyme was highly specific for NG,NG-dimethyl-L-arginine.     

Bone-derived and recombinant transforming growth factor beta's are potent inhibitors of tumor cell growth

Two naturally occurring chrondogenesis inducing peptides have been purified to homogeneity from demineralized bovine bone. Cartilage-inducing factors A and B are the bone-derived equivalents of transforming growth factor-beta types I and II. Both peptides exhibit identical biological activities in chondrogenesis assays and stimulate anchorage independent cell growth. In this study we show that both bone-derived factors are potent (ng/ml) inhibitors of both DNA synthesis and the anchorage independent growth of a variety of human and non-human tumor cells. Unique in this study is also a comparison of the activities of these polypeptide growth factors with recombinant transforming growth factor type I expressed in mammalian cells.     

A monoclonal antibody that recognizes a cluster of a disaccharide, NeuAc alpha(2----6)GalNAc, in mucin-type glycoproteins

The structure of an epitopic carbohydrate recognized by a monoclonal antibody, MLS 102, was determined. A disaccharide, NeuAc alpha (2----6)GalNAc, the major prosthetic group of ovine submaxillary mucin (OSM) and related synthetic glycosides, NeuAc alpha(2----6)GalNAc alpha----Ser, NeuAc alpha(2----6)GalNAc beta----Ser, and NeuAc alpha (2----6)GalNAc beta----propyl, reacted with MLS 102 to similar extents, but the reaction was considerably weaker compared to that of OSM. This difference in reactivity could be ascribed to the occurrence of a cluster of the disaccharide on OSM. Purification of MLS 102-reactive antigens from a Triton X-100 extract of LS 180 cells by means of immunoaffinity chromatography gave mucin fractions (cMLS 102 antigen) with an OSM-like domain. Correlation between the content of the disaccharide, NeuAc alpha(2----6)GalNAc, in mucins and their reactivity with MLS 102 was observed.     

Decrease in the level of poly(ADP-ribose) synthetase during nerve growth factor-promoted neurite outgrowth in rat pheochromocytoma PC12 cells

We have studied the changes in the levels of the enzyme molecule and mRNA for poly(ADP-ribose) synthetase during nerve growth factor-promoted neurite outgrowth in rat pheochromocytoma PC12 cells. When the PC12 cells were cultured in the presence of nerve growth factor, the content of enzyme molecules decreased along with neurite outgrowth to 50% of the original amounts in 2 days and the content of mRNA for the enzyme also decreased to approximately 50% in 2 days. These results suggest that the decrease of the enzyme molecule may be due to depression of expression of the gene for synthetase during the process. Taken together with previous observations, the decrease of the synthetase seems to be required for some cellular differentiation.     

Effect of ovarian steroids on cyclic adenosine 3':5'-monophosphate production stimulated by arginine vasopressin in rat renal monolayer cultured cells

Renal resistance to antidiuretic hormone (ADH) has been speculated to be a mechanism of transient nephrogenic diabetes insipidus occurring during late pregnancy. In order to study possible involvement of ovarian steroids in this mechanism, their effect on cyclic adenosine 3':5'-monophosphate (cAMP) response to arginine vasopressin (AVP) was examined utilizing rat and human renal medullary cells in monolayer culture. In both rat and human cells, estradiol significantly reduced cAMP response to AVP; estradiol at 1.84 x 10(-8) M, 1.84 x 10(-7) M and 1.84 x 10(-6) M decreased cAMP production stimulated by 10(-8) M AVP to 78 +/- 5%, 67 +/- 2% (P less than 0.05) and 52 +/- 1% (P less than 0.001) of the control in rat renal cells, respectively, and in human renal cells the effect of estradiol was comparable to that in rat cells. In rat renal cells, progesterone also reduced cAMP response to AVP dose-dependently; progesterone at 1.59 x 10(-7) M, 1.59 x 10(-6) M and 1.59 x 10(-5) M decreased cAMP production stimulated by 10(-8) M AVP to 87 +/- 1%, 72 +/- 5% (P less than 0.001) and 37 +/- 5% (P less than 0.001) of the control, respectively. On the other hand, corticosterone and dexamethasone at concentrations ranging from 10(-8) M to 10(-5) M and aldosterone at concentrations ranging from 10(-9) M to 10(-5) M did not alter cAMP response to AVP significantly. The suppressive effect of estradiol increased with time until six hours and thereafter it reached a plateau.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and some properties of a novel ethylene-forming enzyme produced by Penicillium digitatum

We purified heat-labile enterotoxins (LThs) from YT3, H-10407 and YT240 strains isolated from human diarrheal patients. These LThs were immunologically identical to each other. The molecular weights of their A and B subunits were also the same by means of SDS-polyacrylamide gel electrophoresis. However, the ionic charges of the molecular surfaces of these LThs were different as shown by polyacrylamide gel isoelectric focusing. Though the pI points of B subunits of the LThs were identical to each other, the pI points of A subunits were found to be different. These data suggest that the ionic charge differences among A subunits cause differences in holo LThs in their charge, and that there is heterogeneity among A subunits produced by strains of human enterotoxigenic Escherichia coli.     

Murine B-cell stimulatory factor-1 (BSF-1)/interleukin-4 (IL-4) is a multilineage colony-stimulating factor that acts directly on primitive hemopoietic progenitors

Interleukin-4 (IL-4), which was originally identified as a B-cell growth factor, has been shown to produce diverse effects on hemopoietic progenitors. The present study investigated the effects of purified recombinant murine IL-4 on early hemopoetic progenitors in methylcellulose culture. IL-4 supported the formation of blast cell colonies and small granulocyte/macrophage (GM) colonies in cultures of marrow and spleen cells of normal mice as well as spleen cells of mice treated with 150 mg/kg 5-fluorouracil (5-FU) 4 days earlier. When the blast cell colonies were individually picked and replated in cultures containing WEHI-3 conditioned medium and erythropoietin (Ep), a variety of colonies were seen, including mixed erythroid colonies, indicating the multipotent nature of the blast cell colonies supported by IL-4. To test whether or not IL-4 affects multipotent progenitors directly, we replated pooled blast cells in cultures under varying conditions. In the presence of Ep, both IL-3 and IL-4 supported a similar number of granulocyte/erythrocyte/macrophage/megakaryocyte (GEMM) colonies. However, the number of GM colonies supported by IL-4 was significantly smaller than that supported by IL-3. When colony-supporting abilities of IL-4 and IL-3 were compared using day-4 post-5-FU spleen and day-2 post-5-FU marrow cells, IL-4 supported the formation of fewer blast cell colonies than did IL-3. IL-4 and IL-6 revealed synergy in support of colony formation from day 2 post-5-FU marrow cells. These results indicate that murine IL-4 is another direct-acting multilineage colony-stimulating factor (multi-CSF), similar to IL-3, that acts on primitive hemopoietic progenitors.     

Distribution of electric charges and concanavalin A binding sites on autophagic vacuoles and lysosomes in mouse hepatocytes

The distributions of electric charges and Concanavalin A binding sites in autophagic vacuoles and lysosomes in mouse hepatocytes were studied by utilizing a frozen ultrathin section labeling method with cationized ferritin (CF) or anionized ferritin and ferritin-conjugated Concanavalin A (Con A-F) as visual probes. Our observations revealed that the inner surface of the autophagic vacuole membrane has more anionic sites (CF binding) than other organelle membranes. This suggests that if the limiting membranes of autophagic vacuoles originate from preexisting membranes, such membranes must undergo structural and compositional alternation during the formation of the autophagic vacuoles. In contrast to CF, Con A-F showed no distinct binding to the membranes of autophagic vacuoles, but the contents of vacuoles displayed varying Con A-F binding, depending on the stage of the autophagic process. Increased binding was seen in more mature autophagic vacuoles. Since lysosomes showed a preferential accumulation of Con A-F particles, molecules with Con A-F binding sites in autophagic vacuoles may be of lysosomal origin. Con A-F distribution varied from lysosome to lysosome in the same cell, indicating heterogeneity of lysosomal contents. These results suggest that ferritin-conjugated lectin labeling methods applied to frozen, ultrathin section are a useful new approach in analyzing the natural history of autophagic vacuoles and the heterogeneity of lysosomes.     

Ca-release by phosphoinositides from sarcoplasmic reticulum of frog skeletal muscle

To clarify the biological role of phosphoinositides including inositol trisphosphate (IP3) in the skeletal muscle, we examined the Ca-releasing action on the heavy fraction of sarcoplasmic reticulum (HFSR) from bullfrog skeletal muscle of IP3, phosphatidylinositol monophosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), and glycerophosphoinositol 4,5-bisphosphate (GPIP2). Only PIP2 caused dose-dependent Ca release. IP3 (up to 55 microM), PIP (up to 37 microM), and GPIP2 (up to 33 microM) were ineffective. The PIP2-induced Ca release is due to the direct action of PIP2, but not its metabolite(s). The properties of the PIP2-induced Ca release are unique and cannot be accounted for by the Ca release mechanisms already reported, such as Ca2+-induced, ionic substitution-induced, or IP3-induced Ca release. The rate of the PIP2-induced Ca release, however, is so slow that it may have no physiological relevance unless stimulating factors or agents exist.