Artificial feeding and successful reproduction inOrnithodoros moubata moubata (Murray, 1877) (Acarina: Argasidae)

The increasing demand for laboratory-reared argasid and ixodid ticks for research and control purposes makes it necessary to develop effective and standardized tick feeding methods without using live animals as hosts. The in vitro maintenance technique, described in this paper, has been used successfully for rearingOrnithodoros moubata moubata by feeding all nymphal and adult instars through Parafilm^R M sealing film on heparinized bovine blood (fresh standard). The technique is based on a specially designed tick feeding apparatus with a capacity to feed 2000 first nymphal instars (N 1) or up to 200 adults at one time. For different 1-month-old instars feeding rates were between 80–100%. Using this feeding technique the subsequent egg production of female ticks was remarkably high, producing an average of 210.2 eggs per tick with a hatch rate of 96.72%. There was no overall difference in the reproductive capacity of 1-month-old femaleO. m. moubata fed on heparinized and haemolyzed bovine blood (kept deep-frozen), heparinized rat blood and defibrinated ovine blood when compared with those fed on heparinized bovine blood (fresh standard).     

Biosynthesis of betagranin in pancreatic beta-cells. Identification of a chromogranin A-like precursor and its parallel processing with proinsulin.

The biosynthesis of insulin and betagranin, a 20-21 kDa co-secreted chromogranin A-related protein, were investigated in isolated insulinoma cells and islets. The insulinoma tissue processed proinsulin to insulin with kinetics similar to those reported in islet tissue. Unlike islets, however, the insulinoma released almost one-quarter of the newly synthesized proinsulin into the medium 10-40 min after its formation. Betagranin was initially immunoprecipitated as a 100 kDa precursor form, which was indistinguishable from chromogranin A in size and immunoreactivity and by peptide mapping. After an initial lag of 10-20 min, the precursor was converted progressively into betagranin, which appeared to be a stable end product. Formation of betagranin and insulin from their respective precursors followed a parallel course and could likewise be inhibited by NH4+, chloroquine and monensin, added either before labelling or at any point of time up to 15 min after labelling. As with proinsulin, approximately one-quarter of the betagranin precursor was released 10-40 min after synthesis. It is concluded that betagranin is produced by limited proteolysis from a chromogranin A precursor in pancreatic beta-cells by a cellular pathway indistinguishable from that of insulin from proinsulin. Chromogranin A is highly conserved in the N-terminal region represented by betagranin, further suggesting that the biological activity of chromogranin A may reside in a derived peptide rather than in the parent molecule.     

Proteolytic processing of chromogranin A in purified insulin granules. Formation of a 20 kDa N-terminal fragment (betagranin) by the concerted action of a Ca2+-dependent endopeptidase and carboxypeptidase H (EC 3.4.17.10).

The nature and subcellular localization of the enzymic activities responsible for the production of the 20 kDa protein betagranin from its 100 kDa chromogranin-A-like precursor was investigated in transplantable insulinoma tissue. [35S]Methionine-labelled precursor was converted by lysed insulin-secretory granules into betagranin and one or more proteins of 47 kDa, via intermediates in the 60-65 kDa range. Lysosome-enriched fractions also processed the precursor, but not into the peptides found in vivo; other fractions, including those enriched in Golgi, were inactive. Conversion of the precursor by granules was quantitative and the products were stable. Inhibitor studies showed that processing occurred by initial endoproteolytic cleavage at sites marked by pairs of basic amino acids, followed by removal of these by carboxypeptidase H. The endopeptidase activity appeared to be a novel metalloenzyme, with a markedly acidic pH optimum (4.8-5). It was inhibited by alanyl-L-lysyl-L-arginyl chloromethane (K0.5 = 1.3 microM), but to a much lesser extent by inhibitor analogues of processing sites defined by single or unpaired basic amino acid residues, e.g. alanyl-L-norleucyl-L-arginylchloromethane (K0.5 greater than 100 microM), leupeptin (K0.5 = 150 microM) and antipain (K0.5 = 40 microM). p-Chloromercuribenzoate (K0.5 = 13 microM), Hg2+ (K0.5 = 16 microM), Zn2+ (K0.5 = 0.8 mM) and vanadate (K0.5 = 7 microM) also abolished activity, as did various anions (SCN- greater than I- greater than Cl- greater than SO4(2-). Group-specific inhibitors of serine, thiol and acidic endopeptidases were without effect. EDTA and CDTA (1,2-cyclohexanediaminetetra-acetic acid), but not 1,10-phenanthroline, abolished endoproteolytic activity. Several bivalent cations could restore activity after EDTA or CDTA inhibition, including Ca2+, Zn2+, Mn2+ and Sr2+; however, the ion of physiological importance appeared to be Ca2+ (K0.5 = 8 microM). The properties of the granule endopeptidase and its subcellular localization suggested that it is of importance in processing chromogranin A in the pancreatic beta-cell.     

Proteolytic conversion of proinsulin into insulin. Identification of a Ca2+-dependent acidic endopeptidase in isolated insulin-secretory granules.

The metabolism of cysteine and cysteinesulphinate was studied in freshly isolated rat hepatocytes. Over 80% of the 14CO2 formed from [1-14C]cysteinesulphinate could be accounted for by production of hypotaurine plus taurine in incubations of rat hepatocytes with either 1 mM- or 25 mM-cysteinesulphinate. In similar incubations with 1 mM- or 25 mM-cysteine, less than 10% of 14CO2 evolution from [1-14C]cysteine could be accounted for by production of hypotaurine plus taurine. In incubations with cysteine, but not with cysteinesulphinate, the production of urea and ammonia was substantially increased above that observed in incubations without substrate. Addition of unlabelled cysteinesulphinate did not affect 14CO2 production from [1-14C]cysteine. Addition of 2-oxoglutarate resulted in a marked increase in cysteinesulphinate catabolism via the transamination pathway, but addition of neither 2-oxoglutarate nor pyruvate to the incubation system had any effect on cysteine catabolism. Inhibition of cystathionase with propargylglycine decreased 14CO2 production from [1-14C]cysteine about 50% and markedly decreased production of ammonia plus urea N; cysteinesulphinate catabolism by cysteinesulphinate-independent pathways in the rat hepatocyte and, furthermore, that cleavage of cyst(e)ine by cystathionase may be an important physiological pathway for cysteine catabolism in rat liver.     

Co-secretion of carboxypeptidase H and insulin from isolated rat islets of Langerhans.

The release of carboxypeptidase H activity from isolated rat islets was determined and compared to the secretion of immunoreactive insulin. Analysis of pancreatic islet cells sorted into beta and non-beta types indicated that approx. 80% of islet carboxypeptidase H activity is present in the beta cell. The release of both insulin and carboxypeptidase H was stimulated markedly by increasing the glucose concentration in the medium from 2.8 to 28 mM. The fractional release was in accordance with the observed cellular distribution of both proteins. The secretory response was biphasic with time, with an initial rapid transient phase of release within 5 min, followed by a more sustained response. The concentration-dependencies of glucose stimulation of release of insulin and carboxypeptidase H were similar, with a threshold for stimulation around 5.6 mM-glucose and maximal stimulatory response at 16.7-28 mM-glucose. The release of both proteins was inhibited by 20 mM-mannoheptulose, removal of Ca2+ from the medium and addition of 1 microM-noradrenaline. The combination of 10 mM-4-methyl-2-oxopentanoate and 10 mM-glutamine stimulated the release of carboxypeptidase H and insulin, as did 3-isobutyl-1-methylxanthine and 350 microM-tolbutamide in the presence of glucose. It is evident that carboxypeptidase H is released from the pancreatic beta-cell by an exocytotic process from the same intracellular compartment as insulin. The release of carboxypeptidase H by a constitutive process was at best equivalent to 0.4%/h, or less than 2% of the maximal rate of release via the regulated pathway. It is concluded that carboxypeptidase H can be used as a sensitive index of beta-cell secretion and an alternative marker to the insulin-related peptides.     

Sequence of human adenosine deaminase cDNA including the coding region and a small intron

The nucleotide sequence for an unusual, cloned human adenosine deaminase cDNA has been determined. Contained within a sequence of 1535 nucleotides is a coding sequence of 1089 nucleotides that encodes a protein of 40,762 daltons. The coding sequence is interrupted by a non-coding region containing 76 nucleotides. Both the 3' and 5' ends of this region have consensus sequences generally associated with splice sites. The 3' untranslated sequence contained 308 nucleotides, including a polyadenylation signal sequence 20 nucleotides from the end. The cloned cDNA appears to correspond to a nuclear mRNA precursor which contains a small intron.     

Pancreatic B-cells secrete a range of novel peptides besides insulin

Insulin secretion by a transplantable rat islet B-cell tumour is accompanied by the release of two putative proinsulin cleavage intermediates, four peptides of M_r 9000–12 000 (excluding proinsulin) and peptides of M_r 21 000, 34 000 and 60 000. Granule-enriched subcellular preparations contain major peptides of identical M_r values. Of these peptides seven at least coincide in molecular weight with peptides secreted by isolated rat islets and thus may be constituents of the normal insulin secretory granule.     

Characterization of normal and mutant adenosine deaminase messenger RNAs by translation and hybridization to a cDNA probe

Summary Using both in vitro translation and hybridization to an adenosine deaminase (ADA) cDNA probe, ADA mRNA has been characterized in B lymphoblast lines established from seven ADA-deficient children, two parents of an ADA-deficient child, and three normal people. All ADA-deficient lines except GM-2825A, including those with less than 1% of normal catalytic activity, had normal or greater amounts of hybridizable, 1.6 kilobase in size, ADA mRNA. Immunoreactive ADA protein of normal size was produced by in vitro translation of the mRNAs. Deficiency of ADA activity in these lines appears secondary to synthesis of structurally altered proteins rather than to a quantitative deficiency in ADA mRNA. The GM-2825A line contains electrophoretically abnormal species of RNA which hybridize to the cDNA probe. Deficiency of ADA activity in this line appears at least in part secondary to a structural defect in the ADA mRNA or its precursors.     

Ca2+-sensitive phosphatidylinositol 4-phosphate metabolism in a rat beta-cell tumour.

Subcellular fractions were isolated from a rat beta-cell tumour by centrifugation of homogenates on Percoll and Urografin density gradients. Fractions were incubated with [gamma-32P]ATP, and labelling of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was used to measure phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase activities, respectively. The distribution of enzyme markers in density gradients indicated that phosphatidylinositol kinase was located in both the plasma membrane and the secretory-granule membrane. Phosphatidylinositol 4-phosphate kinase activity was low in all fractions. Phosphatidylinositol kinase activity of secretory granules and plasma membranes was decreased to 10-20% of its initial value by raising the free [Ca2+] from 1 microM to 5 microM. The enzyme had a Km (apparent) for ATP of 110 microM (secretory granule) or 120 microM (plasma membrane) and a Ka for Mg2+ of 7 mM (secretory granule) or 6 mM (plasma membrane). Ca2+-sensitivity of phosphatidylinositol kinase in calmodulin-depleted secretory granules and plasma membranes was not affected by addition of exogenous calmodulin, although activity was stimulated by trifluoperazine in the presence of 0.1 microM or 40 microM-Ca2+. Trifluoperazine oxide had no effect on the enzyme activity of secretory granules. Plasma membranes had a phosphatidylinositol 4-phosphate phosphatase activity which was stimulated by raising the free [Ca2+] from 0.1 to 40 microM. The secretory granule showed no phosphatidylinositol 4-phosphate-degrading activity. These results suggest the presence in the tumour beta-cell of Ca2+-sensitive mechanisms responsible for the metabolism of polyphosphoinositides in the secretory granule and plasma membrane.     

Phorbol ester stimulation of insulin release and secretory-granule protein phosphorylation in a transplantable rat insulinoma.

The effects of tumour-promoting phorbol esters on protein-phosphorylation reactions and secretion in rat insulinoma tissue were investigated with the objective of assessing the possible role of Ca2+- and phospholipid-dependent protein kinases (protein kinase C) in insulin release. 4 beta-Phorbol 12-myristate 13-acetate (TPA) was a potent secretagogue at concentrations above 0.1 microM. TPA-induced release was inhibited by adrenaline or omission of Ca2+ from the extracellular medium and was augmented by theophylline. These findings suggested that TPA activated an exocytotic process. TPA enhanced the Ca2+- and phospholipid-dependent phosphorylation of histone III-S by a soluble protein fraction of the tissue. Endogenous phosphorylation reactions involving soluble and secretory-granule membrane proteins were also stimulated by TPA in tissue homogenates and reconstituted subcellular fractions. Histone phosphorylation and the granule-protein phosphorylation reactions showed similar concentration-dependencies for activation by both Ca2+ and TPA, thus indicating that the same enzyme was involved. It is concluded that the phosphorylation of cytosolic and membrane protein substrates by protein kinase C may be important in the stimulus-secretion coupling mechanism of insulin release.