Conversion of proparathyroid hormone to parathyroid hormone: studies in vitro with trypsin

The conversion of proparathyroid hormone to parathyroid hormone (PTH) was studied in vitro employing pancreatic trypsin as a prototype converting enzyme. Digestion of intact radiolabeled bovine prohormone with trypsin (0.1%) (w/w) resulted in release of a peptide comigrating with intact hormone marker in systems resolving both on the basis of charge (urea polyacrylamide gels, pH 4.4) and size (sodium dodecyl sulfate-urea polyacrylamide gels, pH 7.2). Tryptic digestions of a synthetic analogue of bovine prohormone, ProPTH-(-6 + 34), consisting of the prohormone hexapeptide covalently bonded to the NIH2 terminus of the active fragment of the hormone, released in high yield the hexapeptide and the intact active hormone fragment before any other smaller fragments. Analyses of digestions were by: (i) thin-layer chromatography and amino acid analysis of digestion products; (ii) comparison of the biological activity of the prophormone substrate and the products of digestion; and (iii) peptide end-group analysis by the Edman method during progressive tryptic hydrolysis over 22 h. The latter experiments demonstrated cleavage of more than 75% of the hexapeptide-hormone peptide bond before cleavage of other trypsin-sensitive sites within the molecule. It is concluded that the specificity of cleavage at the hexapeptide-hormone bond in the process of intracellular hydrolysis of proparathyroid hormone resides primarily in the sequence and/or conformation of the precursor molecule; inasmuch as conversion of prohormone to hormone can be efficiently accomplished by pancreatic trypsin in vitro, there is, therefore, no need to postulate the existence of an intracellular converting enzyme within the parathyroid cell that possesses unique hydrolytic specificity.     

Pyogenic arthritis of the sacro-iliac joint. Long-term follow-up

Nine cases of sacro-iliac pyarthrosis are presented. The difficulty in localizing the infection is attributable mostly to failure to appreciate the posteriorly situated physical findings. This, and the difficulty with early roentgenographic demonstration of the lesion, may lead to unnecessary abdominal exploration (as in two of our patients) or to prolonged delay in diagnosis and hence spread of the infection. Awareness of the usual physical findings and prompt use of radioisotope scanning to localize the infection led to earlier diagnosis and avoidance of surgery in three patients seen recently. Antibiotic therapy, with or without surgery, led to cure in all patients, with minimum sequelae.     

Influence of inhibitors of cellular function on chemotactic factor-induced neutrophil aggregation

Chemotactic factors which induce polymorphonuclear neutrophils (PMN) to migrate directionally and release granular enzyme constituents also induce these cells to aggregate. The potency of these factors in inducing aggregation closely parallels their chemotactic and enzyme-releasing potencies. Several reagents known to influence the migratory and degranulatory response of PMN to chemotactins have been examined for their influence on chemotactic factor-induced aggregation of PMN. We have found that ambient temperatures below 37 degrees C, deoxyglucose, and iodoacetic acid inhibit PMN aggregation, whereas sodium cyanide and dinitrophenol have no effect. Inhibitors of microtubules (colchicine and vinca alkaloids) and of protein synthesis (cyclohexamide) had no effect. Cytochalasin B markedly enhanced aggregation. We conclude that chemotactin-induced aggregation is similar to the other chemotactin-induced PMN functions in the requirements for proper temperature and intact glycolytic pathways; in contrast, however, and intact cytoskeletal microtubular system appears unessential for this response. This may be explained by assuming that the chemotactic factor-induced aggregation of PMN is predominantly a surface membrane-dependent phenomenon.     

Diagnosis of prostatic carcinoma: value of random transrectal sonographically guided biopsies

OBJECTIVE: We studied the efficacy of random, transrectal sonographically guided biopsies in the diagnosis of prostatic carcinoma in a high-risk population. SUBJECTS AND METHODS: During a 2-year period, 570 transrectal sonographically guided prostatic biopsies were done because of clinical findings suggestive of prostatic carcinoma. Biopsies of hypoechoic lesions that were suggestive of carcinoma and segmental random biopsies of normal-appearing lobes of the prostate were performed. Transrectal sonographic findings were correlated with results of pathologic examination of the biopsy specimen and with surgical results, when available. RESULTS: Of the 202 patients found to have carcinoma, the carcinoma was detected with directed biopsy in 145 patients (72%). One hundred twenty (71%) of 169 carcinomas were detected with random biopsy when that procedure was performed. Random biopsies were the only method of diagnosing 57 (28%) of the 202 carcinomas, increasing the yield by 39%. CONCLUSION: Yield of carcinoma on transrectal sonographically guided biopsies increases significantly when segmental random biopsies are performed. Transrectal sonographically guided biopsies should include cores through hypoechoic lesions that are suggestive of carcinoma and bilateral segmental random biopsies.     

Hyperosmolarity: effects on nerves and smooth muscle of cutaneous veins

The technique consists in employing successively an extraction method using the dithizone-carbon tetrachloride system, at 4 different pH values, then thin-layer chromatography on silica gel, to identify and separate Ag, Cd, Co, Cu, Hg, Ni, Pb, and Zn in the form of their dithizonates. Sensitivity is of the order of 10(-7) g ion/l. This method is directly applicable in hydrology; after destruction of organic matter in the case of biological samples (blood, urine, excrement). We have applied it in toxicological analysis together with other methods for the detection of copper, lead, mercury and zinc in cases of poisoning.     

ExoY, an adenylate cyclase secreted by the Pseudomonas aeruginosa type III system

The exoenzyme S regulon is a set of coordinately regulated virulence genes of Pseudomonas aeruginosa. Proteins encoded by the regulon include a type III secretion and translocation apparatus, regulators of gene expression, and effector proteins. The effector proteins include two enzymes with ADP-ribosyltransferase activity (ExoS and ExoT) and an acute cytotoxin (ExoU). In this study, we identified ExoY as a fourth effector protein of the regulon. ExoY is homologous to the extracellular adenylate cyclases of Bordetella pertussis (CyaA) and Bacillus anthracis (EF). The homology among the three adenylate cyclases is limited to two short regions, one of which possesses an ATP-binding motif. In assays for adenylate cyclase activity, recombinant ExoY (rExoY) catalyzed the formation of cAMP with a specific activity similar to the basal activity of CyaA. In contrast to CyaA and EF, rExoY activity was not stimulated or activated by calmodulin. A 500-fold stimulation of activity was detected following the addition of a cytosolic extract from Chinese hamster ovary (CHO) cells. These results indicate that a eukaryotic factor, distinct from calmodulin, enhances rExoY catalysis. Site-directed mutagenesis of residues within the putative active site of ExoY abolished adenylate cyclase activity. Infection of CHO cells with ExoY-producing strains of P. aeruginosa resulted in the intracellular accumulation of cAMP. cAMP accumulation within CHO cells depended on an intact type III translocation apparatus, demonstrating that ExoY is directly translocated into the eukaryotic cytosol.