Mechanisms of modulation of neuronal nicotinic receptors by substance P and OAG

Substance P is known to modulate neuronal nicotinicacetylcholine receptors (nAChRs) in the sympathetic nervous system.There are two conflicting proposals for the mechanism of this effect, an indirect action mediated by protein kinase C (PKC) and a direct interaction with receptor subunits. We studied the mechanisms of thiseffect in PC-12 cells. Substance P enhanced the decay of thenicotine-induced whole cell current. This effect was fast in its onsetand was not antagonized by guanosine5'-O-(2-thiodiphosphate), a G protein blocker, orstaurosporine, a nonselective PKC blocker. Staurosporine failed toreverse the inhibition by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a synthetic diacylglycerol analog known to activate PKC. Theinhibitory effects of the peptide and OAG were preserved in excisedpatches, but substance P applied to the extra patch membrane wasineffective in the cell-attached patch configuration. We conclude thatsubstance P modulates neuronal nAChRs most likely by direct interactions with the receptors but independently from activation ofPKC or G proteins and that PKC does not participate in modulation by OAG.

     

Filipin-sterol complexes in golden hamster sperm membranes with special reference to epididymal maturation

Summary The distribution of membrane filipin-sterol complexes (FSCs) was qualitatively surveyed on freeze-fracture replicas of spermatozoa from the male reproductive tract and ejaculates of golden hamster. In the head, the acrosomal plasma membrane showed the strongest filipin labeling on the principal segment, but it was absent in the quilt-like pattern areas. These latter were observed in both caput and corpus epididymal spermatozoa, but were absent in mature spermatozoa. The postacrosomal plasma membrane had few FSCs and both the outer and inner acrosomal membranes were always negative to filipin. The nuclear membrane of the principal segment was constantly filipinpositive. The nuclear membrane of the postacrosomal region had more FSCs than that of the principal segment, particularly in mature spermatozoa. Many linear, rod-like FSCs were observed on the postacrosomal nuclear membrane of mature spermatozoa, especially in the uterine spermatozoan samples. In the neck, the plasma membrane had only a few FSCs. The redundant nuclear membrane was slightly filipin-positive, while the membrane scroll of mature spermatozoa was heavily labeled. In the tail, the plasma membrane of both the middle and principal piece was moderately labeled.     

Species-dependent regulation of monocyte/macrophage Ia antigen expression and antigen presentation by prostaglandin E

The expression of Ia antigen by various murine and human macrophage populations and the ability of prostaglandins of the E series to regulate Ia antigen expression were explored. Monocytes and macrophages from human and murine populations demonstrated a dichotomy in the expression of Ia antigen. Both human monocytes and macrophages expressed elevated levels of Ia antigen compared to their murine counterpart. Murine macrophages appear to express elevated levels of Ia antigen only when actively interacting with T lymphocytes in vivo or with lymphokines in vitro. Prostaglandins of the E series can suppress murine macrophage Ia antigen expression, but have little effect on the expression of Ia antigen by human monocytes and macrophages. Also, prostaglandins of the E series do not modulate the ability of human monocytes to present antigen to autologous lymphocytes when studied over a broad concentration range. These data suggest that prostaglandin E compounds do not profoundly affect human monocyte/macrophage Ia antigen expression or human monocyte antigen presenting activity.     

The structures of the carbohydrate moieties of mouse kidney gamma-glutamyltranspeptidase: occurrence of X-antigenic determinants and bisecting N-acetylglucosamine residues

Paper electrophoresis and Bio-Gel P-4 column chromatography of the oligosaccharides released from mouse kidney gamma-glutamyltranspeptidase by hydrazinolysis gave fractionation patterns quite distinct from those of the bovine and rat kidney enzymes. Structural studies of the fractionated oligosaccharides by sequential exoglycosidase digestion in combination with methylation analysis showed that mouse kidney gamma-glutamyltranspeptidase contains a series of bisected complex-type asparagine-linked sugar chains with the following oligosaccharides as their outer chain moieties: GlcNAc beta 1----, Sia alpha 2----Gal beta 1----4GlcNAc beta 1----, Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----, and sialylated N-acetyllactosamine repeating sugar chains. Some of these sugar chains were found for the first time in glycoproteins.     

Expression in Escherichia coli of chemically synthesized gene for the human immune interferon.

A 454 base pair fragment of double stranded DNA consisting of a gene for a human immune interferon (hIFN-gamma), initiation and termination signals plus appropriate restriction endonuclease sites, was totally synthesized. The synthesis involved preparation of 62 oligodeoxyribonucleotides by rapid, solid phase procedures, and enzymatic ligation of the oligonucleotides. This synthetic gene was expressed in E. coli under the control of the lac UV5 promoter. The product has antiviral activity which was acid labile and completely neutralized by antiserum to hIFN-gamma but not by antiserum to hIFN-alpha or hIFN-beta. Molecular weight of hIFN-gamma produced by E. coli was estimated to be about 32,000 and 17,000 by gel filtration and SDS-polyacrylamide gel electrophoresis respectively.     

Monokine production by hypersensitivity (Schistosoma mansoni egg) and foreign body (Sephadex bead)-type granuloma macrophages. Evidence for sequential production of IL-1 and tumor necrosis factor

The present study examined the potential role of IL-1 and TNF in granuloma formation. Mice were given i.v. injections of Schistosoma mansoni eggs or Sephadex beads to induce synchronous immune T cell-mediated (hypersensitivity type) or nonimmune (foreign-body type) granulomas, respectively. Granuloma macrophages isolated at 2, 4, 8, 16, and 32 days of granuloma formation were evaluated for their capacity to produce IL-1 and TNF in response to 1 microgram/ml LPS. This was related to circulating levels of the acute phase protein, serum amyloid P (SAP) and expression of Ia Ag by monocytes and macrophages. Macrophages from nonimmune bead lesions were generally weak producers of IL-1 and TNF. In contrast, those from T cell-mediated egg lesions produced significant levels of both monokines. Moreover, there was a clear pattern of sequential monokine production such that IL-1 was produced in greatest amounts early (2 to 4 days), whereas TNF was produced later (8 to 16 days). Levels of SAP showed an initial sharp rise following particle embolization, then decreased rapidly in bead injected animals. However, mice with immune granulomas showed a prolonged elevation in SAP levels that corresponded to the period of maximal IL-1 production (2 to 4 days). Macrophage/monocyte Ia Ag expression was greatest at 8 to 16 days, corresponding to the period of TNF production. Bead injected animals showed low levels of Ia expression over the study period. These findings suggest that IL-1 may be important in the early recruitment stages of granuloma formation while TNF may take part in later maintenance or effector functions. The extent of production of both is likely influenced by the local or systemic milieu of lymphokines.     

Interactive effects of salinity, nitrogen and sulphur on the organic solutes in Spartina alterniflora leaf blades

Glycinebetaine, proline, asparagine, sucrose, glucose, and dimethylsulphoniopropionate(DMSP) were the major organic solutes in Spartina alternifloraleaf blades. To investigate the physiological role(s) of thesesolutes, the effects of salinity, nitrogen, and sulphur treatmentson leaf blade solute levels were examined. Glycinebetaine wasthe major organic solute accumulated in leaf blades grown at500 mol m^–3 NaCl, although asparagine and proline alsoaccumulated when the supply of nitrogen was sufficient. Thesesolutes may play a role in osmotic adjustment. In contrast,DMSP levels either did not change or were reduced in responseto the 500 mol m^–3 NaCl treatment. Furthermore, elevatednitrogen supply decreased leaf blade DMSP levels, which wasopposite to the response of glycinebetaine, proline, and asparagine.A 1000-fold increase in external sulphate concentration hadno effect on the leaf blade levels of DMSP, glycinebetaine,proline, or asparagine. These findings suggest that the majorphysiological role of DMSP in S. alterniflora leaf blades isnot for osmotic adjustment, even under conditions of nitrogendeficit and excess sulphur. Instead, DMSP which was presentat 45—130 µmol g^–1 dry weight, may play arole as a constitutive organic osmoticum. Key words: Spartina alterniflora, dimethylsulphoniopropionate, glycinebetaine, nitrogen, salinity     

Purification and properties of a novel enzyme,N-acylamino acid racemase,from Streptomyces atratus Y-53

A novel enzyme, N-acylamino acid racemase, was purified to homogeneity from Streptomyces atratus Y-53 and characterized. This enzyme catalyzes the interconversion of optically active N-acylamino acids. The relative molecular mass (M_r) of the enzyme was estimated to be about 41 000 and 244 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively, indicating that the enzyme is composed of six subunits with an equal M_r. The enzyme showed a broad substrate specificity toward N-acylamino acids, such as N-acetylmethionine, N-chloroacetylphenylalanine and N-chloroacetylvaline. The apparent Michaelis constant (K_m) values for N-acetyl-l-methionine and N-acetyl-d-methionine were calculated to be 15.2 and 5.6 mm, respectively. Enzyme activity was markedly enhanced by divalent metal ions, such as Co²⁺, Mg²⁺ and Mn²⁺, and was inhibited by metal-chelating reagent, indicating that the enzyme is a metalloenzyme. We propose to name the enzyme N-acylamino acid racemase (acylamino acid racemase). Correspondence to: S. Tokuyama