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Biomedical subjects

M Zamboni

Publications and source records attributed to M Zamboni.

At least 91 records · Page 5Linked to original sources
PubMed1990

Isolation of an inhibitor of cell-free protein synthesis from Salmonella enteritidis.

Sonic extracts of Salmonella enteritidis, serotype enteritidis, contain a factor which inhibits eukaryotic cell-free protein synthesis tested on a rabbit reticulocyte lysate system and on a poly(U)-dependent system from Artemia salina embryos. The factor is heat-labile, does not diffuse through dialysis bags and is precipitated by ammonium sulphate. A 78% recovery of the factor was obtained in the 60-80 per cent ammonium sulphate saturation range, with a 3.3-fold purification. The factor binds to DEAE-cellulose. In the fraction eluted at 0.55 M NaCl, SDS-polyacrylamide gel electrophoresis shows only two protein bands (Mr 55,000 and 29,000). While the inhibitory activity on protein synthesis of the crude factor is fairly stable at 4 degrees C, it becomes very labile after DEAE-cellulose chromatography. The ID50 of the partially purified factor was less than 0.55 microgram (20-fold purification).

Ammonium Sulfate↗
PubMed1989

High-pressure-liquid-chromatographic and fluorimetric methods for the determination of adenine released from ribosomes by ricin and gelonin.

The high fluorescence of adenine-containing compounds after reaction with chloroacetaldehyde was used to measure the adenine released from rat liver and Artemia salina ribosomes by the action of ricin A chain and gelonin, two ribosome-inactivating proteins (RIPs) that share the same mechanism of action, consisting in the hydrolysis of the N-glycosidic bond of A-4324 of 28 S rRNA. Two methods were employed: (i) h.p.l.c. of the chloroacetaldehyde-reactive material released by RIPs; h.p.l.c. associated with a fluorescence detector allows the identification of adenine and its dosage at quantities as low as 2 ng; (ii) the direct fluorimetric measurement of the material that had reacted with chloroacetaldehyde. The amount of adenine released increases when ribosomes are pretreated in conditions that lead to their dissociation into subunits. Adenine protects ribosomes from the inhibition by ricin A-chain. When ribosomes were incubated with ricin A-chain in the presence of [14C]adenine no incorporation of radioisotope in ribosomes was observed, indicating that neither exchange nor reversal reactions occurred. A binding of [14C]adenine to ricin A chain was not detected by equilibrium dialysis.

Adenine↗
PubMed1989

Alpha-sarcin impairs the N-glycosidase activity of ricin on ribosomes.

In a recently described method the RNA N-glycosidase activity of ricin is measured by h.p.l.c. determination of the adenine released from ribosomes after conversion of the base into its fluorescent 1-N6-etheno derivative [Zamboni et al. (1989) Biochem. J., 259, in press]. Unlike previously available methods, based on the separation of RNA fragments by gel electrophoresis, the new method allows one to investigate the activity of ricin on ribosomes pretreated with alpha-sarcin, a cytotoxin which cleaves 28S rRNA at one nucleotide distance from the site of attack of ricin. alpha-Sarcin makes ribosomes a poor substrate for ricin, the release of adenine requiring concentrations of ricin 50-times higher than those effective on untreated ribosomes.

Adenine↗
PubMed1989

Effect of the antibiotic purpuromycin on cell-free protein-synthesizing systems.

Purpuromycin, an antibiotic isolated from the culture broth of Actinoplanes ianthinogenes, which is very active against Gram-positive bacteria and fungi, inhibits protein synthesis in both prokaryotic and eukaryotic cell-free systems. The ID50 was 9 microM with the endogenous mRNA-directed rabbit reticulocyte lysate, 17 microM with a poly(U)-directed system from Escherichia coli and 69 microM with a poly(U)-directed system from Artemia salina cysts. Of the three steps of elongation, purpuromycin does not affect the peptidyl-transferase reaction, inhibits the elongation factor 1 (EF-1) dependent binding of phenylalanyl-tRNA and stimulates the GTP-dependent binding of EF-2. When protein synthesis is stopped by the addition of purpuromycin, the nascent peptide chains are found in the puromycin-reactive P site. The results suggest that the mechanism of action of purpuromycin is similar to that of fusidic acid. Both antibiotics would seem to produce a stable guanine nucleotide-ribosome-EF-2 complex which allows one round of translocation but prevents, because of a common or overlapping ribosomal binding site for the two elongation factors, the subsequent EF-1-dependent binding of aminoacyl-tRNA.

Animals↗
PubMed1989

Effect of alpha-sarcin and ribosome-inactivating proteins on the interaction of elongation factors with ribosomes.

alpha-Sarcin from Aspergillus giganteus and the ribosome-inactivating proteins (RIPs) from higher plants inactivate the 60 S ribosomal subunit. The former is an RNAase, whereas RIPs are N-glycosidases. The site of cleavage of RNA and that of N-glycosidic depurinization are at one nucleotide distance in 28 S rRNA [Endo & Tsurugi (1987) J. Biol. Chem. 262, 8128-8130]. The effect of alpha-sarcin and that of RIPs on the interaction of elongation factors with Artemia salina (brine shrimp) ribosomes have been investigated. alpha-Sarcin inhibits both the EF1 (elongation factor 1)-dependent binding of aminoacyl-tRNA and the GTP-dependent binding of EF2 (elongation factor 2) to ribosomes, whereas two of the RIPs tested, ricin from Ricinus communis (castor bean) and volkensin from Adenia volkensii (kilyambiti), inhibit only the latter reaction. EF2 protects ribosomes from inactivation by both alpha-sarcin and ricin. The EF1-binding site is affected only by alpha-sarcin. The sensitivity of this site to alpha-sarcin is increased by pretreatment of ribosomes with ricin. A. salina ribosomes were highly resistant to the third RIP tested, namely gelonin from Gelonium multiflorum. All four proteins tested have, however, a comparable activity on the rabbit reticulocyte-lysate system.

Aspergillus↗
PubMed1988

Captopril in patients with type II diabetes and renal insufficiency: systemic and renal hemodynamic alterations.

PURPOSE: To our knowledge, clinical studies on the long-term use of angiotensin converting enzyme inhibitors in patients with type II diabetes mellitus and nephropathy with incipient renal failure are nonexistent. We therefore assessed the effects of long-term treatment with captopril on systemic and renal hemodynamics and urinary protein excretion in patients with type II diabetes mellitus and the clinical syndrome of diabetic nephropathy. PATIENTS AND METHODS: Twelve patients, 10 men and two women, with an average age of 52 years (range, 40 to 66), participated in the study. After the basal hemodynamic evaluation, the patients received captopril in two daily doses. The dosage was adjusted at weekly intervals in order to obtain normalization of blood pressure without exceeding the maximum allowable dosage. Four patients also received furosemide (20 to 40 mg/day). RESULTS: After six months of treatment, the intra-arterial blood pressure fell (from 162 +/- 17/103 +/- 5 to 139 +/- 26/89 +/- 10 mm Hg) due to the reduction in total peripheral vascular resistance index (from 3,720 +/- 658 to 3,190 +/- 762 dynes/second/cm-5/m2), with no change in cardiac index (2.78 +/- 0.36 to 2.79 +/- 0.47 liters/minute/m2). No significant change was seen in renal vascular resistance (from 30,175 +/- 5,371 to 30,173 +/- 5,372 dynes/second/cm-5/1.73 m2) and in filtration fraction (from 26 +/- 8 to 27 +/- 10 percent). A slight, not significant, decrease in renal plasma flow (from 243 +/- 97 to 217 +/- 108 ml/minute/1.73 m2), in glomerular filtration rate (from 57 +/- 17 to 51 +/- 19 ml/minute/1.73 m2), and in proteinuria (from 4.50 +/- 3.10 to 3.40 +/- 2.31 g/day) was also observed. CONCLUSION: Our findings suggest that captopril is an effective antihypertensive agent in patients with diabetic nephropathy, but the renal beneficial effects seem to be limited when this syndrome is complicated by renal insufficiency.

Adult↗
PubMed1988

Intramuscular administration of salmon calcitonin impairs delayed cutaneous hypersensitivity.

The delayed cutaneous hypersensitivity to seven ubiquitous antigens (Multitest CMI, Istitute Mĕrieux) was evaluated in normal healthy volunteers before and at the time of intramuscular administration of 50 (12 subjects) or 50 + 50 (nine subjects) units of salmon calcitonin or 2 ml of saline (eight subjects). The delayed cutaneous hypersensitivity, in terms of number of positive tests or sum of positive tests, significantly fell in both groups given salmon calcitonin, but no changes were observed in the control group. These results indicate that salmon calcitonin, as well as inhibiting osteoclastic bone resorption, has important immunological effects.

Adult↗
PubMed1986

Interaction of alpha-sarcin and gelonin with cibacron blue.

Alpha-sarcin and gelonin, two proteins which inactivate the 60S ribosomal subunit, interact with Cibacron blue and bind to blue dextran-Sepharose, from which they are partially desorbed by nucleoside triphosphates and, more efficiently, by homopolynucleotides. It is further shown that the two proteins bind to poly(U)-Sepharose and that homopolynucleotides protect dilute solutions of both inhibitors from inactivation. It is suggested that the presence of a polynucleotide site on alpha-sarcin is related to its ribonuclease activity. The existence of a similar site on gelonin might be a clue to its yet unknown mechanism of action.

Binding, Competitive↗
PubMed1985

A controlled trial using ephedrine in the treatment of obesity.

A double-blind controlled study was performed in unselected obese outpatients to assess the effects of ephedrine on weight loss. Patients were treated for 3 months with placebo (group I), 25 mg t.i.d. or 50 mg t.i.d. of ephedrine hydrochloride orally administered (groups II and III, respectively). Dietary treatment consisted of 1000 kcal/day for females and 1200 kcal/day for males. The three groups were matched for age, sex, body mass index and pre-treatment spontaneous caloric intake. Weight loss was similar in all groups. Patients in group III (ephedrine 150 mg/day) showed significantly more side effects than the placebo group. These results do not seem to favour the hypothesis that ephedrine, a thermogenic agent, may be effective in the therapy of unselected simple obesity. On the other hand, it cannot be excluded that the drug may be useful in obese patients in whom defective thermogenesis may be clearly demonstrated.

Adolescent↗
PubMed1982

Biotin transport by a biotin-deficient strain of Escherichia coli.

Biotin uptake has been investigated using an Escherichia coli biotin requiring auxotroph grown under biotin-deficient conditions. This strain accumulated biotin in the free and bound form. In agreement with a previous report by O. Prakash and M.A. Eisenberg (J. Bacteriol. 120 (1974) 785-791), the biotin entry proved to be an active process which depended on an energy source and was inhibited in the presence of uncouplers. The kinetic parameters have been determined (KM = 0.05 microM, Vmax = 7 pmol/min per mg dry weight). The pool of free biotin could be readily exchanged with external biotin and decreased to a very low level in the absence of an energy source. The use of several biotin analogues revealed that this transport system was quite specific for biotin: slight modifications, for instance in the valeric chain, lowered drastically the affinity for the carrier.

Biological Transport, Active↗
PubMed1981

Ribosomal core-particles as the target of ricin.

Core-particles and split-proteins were prepared by treatment with ethanol and NH4Cl of control and ricin-treated Artemia salina ribosomes. No modifications of the ricin-treated split-proteins was detected by polyacrylamide-gel electrophoresis. Moreover, the split-proteins from ricin-treated ribosomes complemented control core-particles in poly(U)-directed phenylalanine polymerization. Conversely, ricin-treated core-particles remained totally inactive when supplemented with control split-proteins.

Animals↗
PubMed1980

Mechanism of the antibiotic action of alpha-dehydrobiotin.

alpha-Dehydrobiotin, a naturally occurring biotin analogue, exhibits antibiotic properties [Hanka, L. J., Reineke, L. M., & Martin, D. G.(1969) J. Bacteriol. 100, 42--46]. It is shown in this paper that in addition to its activity as corepressor of the transcription of the biotin locus [Guha, A., Saturen, Y., & Szybalski, W. (1971) J. Mol. Biol. 56, 53--62] alpha-dehydrobiotin acts at the enzyme level. The synthesis of specifically tritiated alpha-dehydrobiotin has been achieved. By use of this labeled compound and a biotin-department strain of Escherichia coli, it has been demonstrated that alpha-dehydrobiotin can be linked covalently to proteins without further transformation. The fixation of biotin to apocarboxylases is inhibited irreversibly after preincubation with alpha-dehydrobiotin. This strongly supports the hypothesis that alpha-dehydrobiotin can be specifically linked to apocarboxylases in place of biotin and leads to carboxylases that are inactive. Thus, the antibiotic properties of alpha-dehydrobiotin would be partly due to the fact that it compete with biotin for the fixation on the apocarboxylases, producing irreversibly inactive enzymes.

Biological Transport↗
PubMed1980

Inhibition of protein synthesis in vitro by proteins from the seeds of Momordica charantia (bitter pear melon).

1. A haemagglutinating lectin was purified from the seeds of Momordica charantia by affinity chromatography on Sepharose 4B and on acid-treated Sepharose 6B. It has mol.wt. 115 000 and consists of four subunits, of mol.wts. 30 500, 29 000, 28 500 and 27 000. 2. The lectin inhibits protein synthesis by a rabbit reticulocyte lysate with an ID50 (concentration giving 50% inhibition) of approx. 5 micrograms/ml. Protein synthesis by Yoshida ascites cells is partially inhibited by the lectin at a concentration of 100 micrograms/ml. 3. From the same seeds another protein was purified which has mol.wt. 23 000 and is a very potent inhibitor of protein synthesis in the lysate system, with an ID50 of 1.8 ng/ml. This inhibitor has no effect on protein synthesis by Yoshida cells, and has no haemagglutinating properties. 4. Artemia salina ribosomes preincubated with the lectin or with the inhibitor lose their capacity to perform protein synthesis. The proteins seem to act catalytically, since they inactivate a molar excess of ribosomes. 5. The lectin and the inhibitor are somewhat toxic to mice, the LD50 being 316 and 340 micrograms/100 g body wt. respectively.

Animals↗
PubMed1980

Purification and properties of different forms of modeccin, the toxin of Adenia digitata. Separation of subunits with inhibitory and lectin activity.

1. The subunits were isolated of modeccin (subsequently referred to as modeccin 4B), the toxin purified from the roots of Adenia digitata by affinity chromatography on Sepharose 4B [Gasperi-Campani, Barbieri, Lorenzoni, Montanaro, Sperti, Bonetti & Stirpe (1978) Biochem J. 174, 491-496]. They are an A subunit (mol.wt. 26 000), which inhibits protein synthesis, and a B subunit (mol.wt. 31 000), which binds to cells. Both sununits, as well as intact modeccin, gave single bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, but showed some heterogeneity on isoelectric focusing and on polyacrylamide-gel electrophoresis at pH 9.5. 2. A second form of modeccin, not retained by Sepharose 4B, was purified by affinity chromatography on acid-treated Sepharose 6B: this form is subsequently termed modeccin 6B 3. Modeccin 6B has a molecular weight indistinguishable from that of modeccin 4B, and consists of two subunits of mol.wts. 27 000 and 31 000, joined by a disulphide bond. The subunits were not isolated because of their high insolubility in the absence of sodium dodecyl sulphate. 4. As compared with modeccin 4B, modeccin 6B is slightly less toxic to animals, does not agglutinate erythrocytes, and is a more potent inhibitor of protein synthesis in a lysate of rabbit reticulocytes, giving 50% inhibition at the concentration of 0.31 microgram/ml.

Animals↗