PubMed Health⌕ Search

Biomedical subjects

J Shiloach

Publications and source records attributed to J Shiloach.

At least 91 records · Page 5Linked to original sources
PubMed1985

Bacillus subtilis contains multiple forms of somatostatin-like material.

Extracts of B. subtilis contain somatostatin-like immunoactivity (1-20 pg per g wet weight cells). Two major forms were detected, one with reactivity in both N- and C-terminal immunoassays similar to somatostatin-28 and a second form reactive only in the C-terminal specific immunoassay similar to somatostatin-14. Both forms were active in a bioassay and the bioactivity was neutralized in the presence of antibody to the central, biologically active part of somatostatin-14. Preconditioned medium contained no detectable somatostatin whereas conditioned medium had 80-380 pg per liter.

Animals↗
PubMed1985

Evidence for multiple molecular weight forms of somatostatin-like material in Escherichia coli.

Extracts of Escherichia coli grown in defined medium contain somatostatin-related material (1-10 pg/g wet weight of cells). Preconditioned medium had no immunoactive somatostatin whereas, conditioned medium had 110-150 pg/l. Following purification of the extracted material on Sep-pak C18, Bio-Gel P-6 and HPLC, multiple molecular weight forms of somatostatin- (SRIF-) related material were identified. The material in one peak reacted in both the N-terminal and C-terminal SRIF immunoassay and coeluted on HPLC with SRIF-28, whereas that in a second peak eluted near SRIF-14 and was reactive only in the C-terminal SRIF assay. The two peaks are thus similar to SRIF-28 and SRIF-14 of vertebrates. These findings add support to the suggestion that vertebrate-type peptide hormones and neuropeptides have early evolutionary origins.

Chromatography, Gel↗
PubMed1985

Salmon calcitonin-like immunoactivity in extracts of Tetrahymena pyriformis.

Acid extracts of Tetrahymena pyriformis, a ciliated protozoan grown in defined medium revealed the presence of materials with salmon-type calcitonin immunoactivity. These findings add support to earlier reports of the presence of materials closely resembling vertebrate peptide hormones in unicellular microbes.

Animals↗
PubMed1985

Insulin-related material in microbes: similarities and differences from mammalian insulins.

We have reported that extracts of Escherichia coli and Tetrahymena grown in synthetic media contained material that reacted specifically in the immunoassay and bioassay for insulin. One additional strain of Tetrahymena and four of E. coli yielded amounts of material similar to those reported previously. In addition to their behavior on Sephadex G-50, the immunoactive insulin-related materials from the microbial sources behaved like authentic vertebrate insulins in their ability to be adsorbed to and eluted from disposable octadecasilylsilica cartridges, DEAE-Sephadex, DEAE-cellulose, and one system of high-pressure liquid chromatography (HPLC). As with less purified microbial material, the "insulin" that had been purified on DEAE and HPLC, when tested for its bioactivity, had an immunoactivity:bioactivity ratio of approximately unity and the bioactivity was largely neutralized by anti-insulin antibody. Because the material from the microbes was so similar to authentic insulins, studies were undertaken to demonstrate that inadvertent contamination with vertebrate insulins was highly unlikely. Blanks carried through the entire procedure were always negative. Tetrahymena grown and extracted in another laboratory gave the same results. Tetrahymena that had been grown but then allowed to stand in the fermenter under adverse conditions and then carried through the entire procedure were devoid of insulin. Tetrahymena that were homogenized and subjected to acid hydrolysis were devoid of insulin. Further substantiation that exogenous contamination was highly unlikely was provided by two other types of experiments. In one of these, it was shown that the subcellular distribution of exogenously added porcine insulin or porcine 125I-labeled insulin was different from the distribution of endogenous insulin. In the second type of experiment, it was shown that during the log phase of growth of Tetrahymena or of E. coli the insulin content of the system increased multifold in a fashion that might be expected for living organisms but quite unexpected for exogenous contamination. (Interestingly, the insulin content of the E. coli medium far exceeded that which might be contributed by death of cells, estimated by the content in the medium of an intracellular enzyme.) When E. coli was grown and processed in four other laboratories having no contact with our own, similar levels of insulin-related material were recovered.

Animals↗
PubMed1984

Agonist and antagonist properties of calmodulin fragments.

Limited proteolysis of calmodulin with trypsin in the presence of ethylene glycol bis(beta-aminoethyl ether)-N, N,N',N'-tetracetic acid (EGTA) or Ca2+ was performed according to a modification of the method of Drabikowski et al. (Drabikowski, W., Kuznicki, J., and Grabarek, Z. (1977) Biochim. Biophys. Acta 485, 124-133). The resulting peptides were purified by reverse-phase high performance liquid chromatography. Tryptic digests in EGTA yielded peptides 1-106, 1-90, and 107-148 with yields of 9, 47, and 61%, respectively. The digests performed with Ca2+ yielded peptides 1-77 and 78-148 in 35 and 45% yield. Analysis by high performance liquid chromatography indicated that the purified fragments contained less than 0.1% contamination by calmodulin, thus allowing a definitive study of the ability of these fragments to activate, or interact with, calmodulin-regulated enzymes and anti-calmodulin drugs. Each of the fragments, except 107-148, bound to a phenothiazine affinity column in a Ca2+-dependent manner. Thus, calmodulin contains two interaction sites for phenothiazines: one on the NH2-terminal half (fragment 1-77) and one on the COOH-terminal half (fragment 78-148). None of the fragments activates the protein phosphatase, calcineurin, or prevents its stimulation by calmodulin, nor does any of the fragments stimulate Ca2+-dependent cAMP phosphodiesterase. A single cleavage in the middle of the calmodulin molecule results in the rapid dissociation of the two resultant fragments and a loss of ability to activate cAMP phosphodiesterase. One fragment, 78-148, interacts with phosphodiesterase and prevents its activation by calmodulin (Ki: 1.5 +/- 0.4 X 10(-6) M). The same fragment, 78-148, can fully activate phosphorylase kinase but with a lower affinity than calmodulin (Kuznicki, J., Grabarek, Z., Brzeska, H., Drabikowski, W., and Cohen, P. (1981) FEBS Lett. 130, 141-145). Thus, peptide 78-148 behaves as a calmodulin agonist or antagonist or as neither, depending on the enzyme under study.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1984

Serum antibody responses of juvenile and infant rhesus monkeys injected with Haemophilus influenzae type b and pneumococcus type 6A capsular polysaccharide-protein conjugates.

Juvenile and infant rhesus monkeys were injected subcutaneously with saline solutions of Haemophilus influenzae type b (Hib) and pneumococcus type 6A (Pn6A) capsular polysaccharides conjugated to either tetanus toxoid (TT), horseshoe crab hemocyanin, or cholera toxin (CT), and the antibody responses of the monkeys to both bacterial components were measured. All three Hib conjugates were immunogenic and elicited booster responses; their comparative immunogenicity was Hib-CT greater than Hib-TT greater than Hib-horseshoe crab hemocyanin. Hib alone did not elicit antibodies in the juveniles. Juveniles responded earlier and with higher levels of antibodies than did infants. TT, as well as diphtheria-tetanus toxoids-pertussis vaccine adsorbed injected concurrently at a separate site, increased both Hib and TT antibody responses in juveniles (P less than 0.05). Concurrent injection of 5 Lf of fluid TT with a nonimmunogenic 5-micrograms dose in infants elicited levels of Hib antibodies comparable to those elicited by 50 micrograms of Hib-TT. Hib antibodies elicited by the conjugates remained at protective levels in both juveniles and infants 2 months after the last injection, were bactericidal, and conferred passive immunity against bacteremia in infant rats. Passive immunization of juveniles with tetanus immune globulin before each injection of Hib-TT did not suppress Hib antibodies. Hib-TT and Hib-CT elicited increases of Hib antibodies of the immunoglobulin M and G isotypes in the infants. The Pn6A-TT conjugate was considerably less immunogenic than the Hib-TT conjugate; only a few of the juveniles or infants responded with protective levels of Pn6A antibodies. Pn6A antibodies from responders conferred protection in mice against intraperitoneal challenge with Pn6A organisms. TT antibodies were elicited in both juvenile and infant animals after one injection of 50 micrograms of Hib-TT and in the infants injected with 5 micrograms of Hib-TT plus 5 Lf of TT; 5 micrograms of Hib-TT and Pn6A-TT in combination alone did not elicit TT antibodies. Hib-CT elicited CT antibodies in both juveniles and infants.

Adjuvants, Immunologic↗
PubMed1983

Are messenger molecules in microbes the ancestors of the vertebrate hormones and tissue factors?

Peptides very similar to hormones and other messenger molecules of vertebrates have been detected in extracts of unicellular eukaryotes (and prokaryotes). We present arguments to suggest the possibility that these molecules 1) originated evolutionarily in unicellular microbes, 2) serve as intercellular messenger molecules in these organisms, and 3) represent the phylogenetic ancestors of the hormones and neurotransmitters as well as paracrine and other tissue factors of the vertebrates. We suggest that the biochemical elements of intercellular communication arose very early in evolution and are highly conserved; evolution largely changed the anatomy, i.e., the nature of the secretory cell, the target cell, and the fluid compartment that carries the messenger molecule from one to the other. Such an approach suggests a more rational relationship between different modes of intercellular chemical signaling in vertebrates.

Animals↗
PubMed1982

Corticotropin and beta-endorphin-like materials are native to unicellular organisms.

Multiple molecular forms of immunoreactive corticotropin (ACTH) and beta-endorphin were present in extracts of a unicellular eukaryote (Tetrahymena pyriformis). One form of immunoreactive ACTH reacted similarly with two different ACTH antisera (one specific for the 11-24 sequence and the other with determinants within sequences 1-14 and 17-39) and migrated with synthetic hACTH-(1-39) in a gel filtration system. This form also exhibited ACTH bioactivity in a dispersed rat adrenal cell bioassay system, with a mean immunoassay/bioassay ratio of 1.5. Gel filtration revealed multiple size classes of immunoreactive beta-endorphin; a major peak of radioreceptor activity was detected which exhibited a K(av) similar to that of authentic beta-endorphin. A major portion of immunoreactive beta-endorphin-sized material exhibited retention times similar to those of synthetic human and camel beta-endorphin upon reverse-phase high-pressure liquid chromatography. These distinctive properties and specificities would seem to exclude the presence of limited homologies with sequences present in other proteins. High molecular weight material containing both ACTH and beta-endorphin antigenic determinants was also demonstrated, suggesting, but not proving, the presence of a common precursor molecule.

Journal Article↗
PubMed1980

Evolutionary origins of vertebrate hormones: substances similar to mammalian insulins are native to unicellular eukaryotes.

Tetrahymena pyriformis, Neurospora crassa, and Aspergillus fumigatus that had been grown in simple defined media were extracted with acid ethanol by a classic method for recovering insulin from pancreas. After filtration of the extracts on Sephadex G-50, distinct peaks of insulin immunoreactivity were recovered in the region typical of insulin. The gel-filtered material from the Tetrahymena had reactivity in the pork insulin radioimmunoassay about equal to its reactivity in the insulin bioassay (stimulation of lipogenesis in isolated rat adipocytes), and the gel-filtered material from neurospora had an immunoreactivity-to-bioactivity ratio of about 1:3. The material that stimulated lipogenesis could be neturalized by anti-insulin sera (i.e., 75-95% of the Tetrahymena material and 60% of the Neurospora material). Bioactive and immunoactive insulin was found in the conditioned medium equal in amount to that in the cells. The findings suggest that insulin did not arise evolutionarily in the intestinal or neural tissues of primitive vertebrates or complex invertebrates but rather has its molecular origins at least as far back as the simplest unicellular eukaryotes.

Aspergillus↗
PubMed1977

Enzyme replacement therapy in Gaucher's disease: large-scale purification of glucocerebrosidase suitable for human administration.

Enzyme replacement therapy for the alleviation of Gaucher's disease has been impeded because of the difficulty in preparing large amounts of glucocerebrosidase, the enzyme that is deficient in patients with this disorder. A large-scale procedure for the purification of human placental glucocerebrosidase has been developed. The method uses cholate extraction, ammonium sulfate fractionation, acid precipitation, butanol extraction, and hydrophobic chromatography; the final enzyme preparation has a specific activity of more than 10(6) units/mg of protein with an overall recovery of 30%. In addition, the contamination of enzyme preparations, intended for human infusion, prepared by isolation procedures involving concanavalin A columns has been studied and is reported here.

Female↗
PubMed1975

The influence of the peptide chain length on the activity of peptidyl-tRNA hydrolase from E. coli.

The dependence of the Vmax and Km on the length of the peptide moiety in the peptidyl-tRNA series (Gly)n-Val tRNA, was measured in the system peptidyl-tRNA hydrolase-peptidyl-tRNA. It was found that the Km value decreases from 7.2 X 10-7 M for Gly-Val-tRNA to 4.6 X 10-7 M FOR (Gly)2-Val-tRNA and to 1.7 X 10-7M for (Gly)3-Val-tRNA; further increase of the peptide chain is not followed by decrease of the Km. The Vmax values are 5.7 pmole/min/EU for Gly-Val-tRNA and 42 pmole/min/EU for (Gly)3-Val-tRNA. The enzyme activity is inhibited competitively by uncharged tRNA with a KI value of about 10-5M. The significance of these results described in this paper, in relation to the fact that peptides and peptide esters do not inhibit the enzyme activity, and in relation to the proposed physiological role of the enzyme, is discussed.

Carboxylic Ester Hydrolases↗