Evidence for pyroglutamyl peptidase I and prolyl endopeptidase activities in the rat insulinoma cell line RINm 5F: lack of relationship with TRH metabolism

Thyrotropin-R eleasing hormone (TRH)-degrading pyroglutamyl peptidase I(PGP I) and prolyl endopeptidase (PE) activities have been demonstrated in rat insulinoma RINm 5F cell line. These two enzymes catalyze the conversion of TRH to Histydyl-Proline-Diketopiperazine and to acid TRH respectively.After cell fractionation, we found all the PGP I and PE activities in the cytosolic fraction. The membranebound PGP II activity is not detectable in the RINm 5F cells. Further investigations on these two cytosolic enzymes show that pyroglutamyl- and proline-containing peptides are inhibitors of each TRH-degrading enzyme.Gelfiltration chromatography on Sephadex G100 shows that PGP I and PE activity have an apparent molecular mass of about 18 kDa and 57 kDa, respectively. Kinetic analysis with TRH as substrate, gives a Km of 44 µM and 235 µM, and a Vmax of 1.49 and 8.80 pmoUmin/µg protein for PGP I and PE, respectively. Immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH levels in the cell line extracts are 2.2 ± 0.9, 22.5 ± 11.1 and 28.7 ± 14.6pg/10⁶ cells, respectively. When cells have been incubated for 2 to 72 hours with a P. E. inhibitor (Z-Gly-Pro-CHN₂) at 5 × 10^–7M, both cell PGP I and PE activities are inhibited. No change in the cellular content of immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH have been observed in treated cells.These data suggest that TRH is not degraded by cytosolic, unspecific PGP I and PE enzymes in RINm 5F. The finding that these cells contain 10 and 13 times more His-Pro-Diketopiperazine and acid TRH than TRH may be an indirect evidence for the existence of another precursor than TRH for these two peptides or of the possibility that TRH can be degraded by other peptidases.Abbreviations TRH Thyrotropin-Releasing Hormone or Thyroliberin - His-Pro-DKP Histidyl-ProlineDiketopiperazine - TRH-OH acid TRH or deamidated TRH - LH-RH Luteinizing Hormone-Releasing Hormone - Z-Gly-Pro-CHN₂ N-benzyloxycarboxyl-Gly-Pro-diazomethylketone - PGP Pyroglutamyl Peptidase, PGP I (EC 3.4.19.3) and PGP II (EC 3.4.19.-) - PE Prolyl Endopeptidase or post-proline cleaving enzyme (EC 3.4.21.26)     

Characterization of a frequent polymorphism in the coding sequence of the Tp53 gene in colonic cancer patients and a control population

Summary We describe a simple method for characterizing a frequent polymorphism (that subsitutes an arginine for a proline) in the coding sequence of the Tp53 gene in patients with colonic cancer and in a control population. We could find no evidence that this polymorphism is associated with a marked predisposition to colorectal cancer.     

The localisation of an Mr 74,000 major chromatin antigen on native salivary chromosomes of Drosophila melanogaster

An antigen making a major contribution to the immune response to Drosophila melanogaster chromatin resides primarily on a nonhistone charge-class family of proteins of Mr 74,000. Immunofluorescence detects this antigen at interbands, puffs and diffuse bands of D. melanogaster salivary chromosomes isolated without exposure to acid fixatives, and on nucleoplasmic ribonucleoprotein droplets. In the electron microscope, gold labelling reveals the binding of monoclonal antibodies specific for the antigen at chromosomal loci generally bearing putative ribonucleoprotein (RNP) particles. However, the locus 3C 11–12 is remarkable in that it bears putative RNP particles but is virtually unlabelled, suggesting protein specificity at different active loci.     

Anaerobic C1 Metabolism of the O-Methyl-14C-Labeled Substituent of Vanillate

The O-methyl substituents of aromatic compounds constitute a C₁ growth substrate for a number of taxonomically diverse anaerobic acetogens. In this study, strain TH-001, an O-demethylating obligate anaerobe, was chosen to represent this physiological group, and the carbon flow when cells were grown on O-methyl substituents as a C₁ substrate was determined by ¹⁴C radiotracer techniques. O-[methyl-¹⁴C]vanillate (4-hydroxy-3-methoxy-benzoate) was used as the labeled C₁ substrate. The data showed that for every O-methyl carbon converted to [¹⁴C]acetate, two were oxidized to ¹⁴CO₂. Quantitation of the carbon recovered in the two products, acetate and CO₂, indicated that acetate was formed in part by the fixation of unlabeled CO₂. The specific activity of ¹⁴C in acetate was 70% of that in the O-methyl substrate, suggesting that only one carbon of acetate was derived from the O-methyl group. Thus, it is postulated that the carboxyl carbon of the product acetate is derived from CO₂ and the methyl carbon is derived from the O-methyl substituent of vanillate. The metabolism of O-[methyl-¹⁴C]vanillate by strain TH-001 can be described as follows: 3¹⁴CH₃OC₇H₅O₃ + CO₂ + 4H₂O → ¹⁴CH₃COOH + 2¹⁴CO₂ + 10H⁺ + 10e^- + 3HOC₇H₅O₃.     

Differences in adhesiveness among type 1 fimbriate strains of Enterobacteriaceae revealed by an in vitro HEp2 cell adhesion model

Ten type 1 fimbriate strains of Enterobacteriaceae were examined in an in vitro adhesion assay with HEp2 epithelial cells. The range of HEp2 cell adhesiveness, which was characteristic for each strain, was affected by motility, type 1 fimbriation and production of mannose sensitive haemagglutinin. Nevertheless, not all type 1 fimbriate strains adhered well in this model. The findings are discussed with regard to the possibility that different type 1 fimbriate enterobacteria, though all are mannose sensitive, recognize different mannose-containing receptors present or available on the surfaces of the HEp2 cells.     

Decreased Incorporation of [3H]Inositol and [3H]Glycerol into Glycerolipids of Sciatic Nerve from the Streptozotocin Diabetic Rat

The incorporation of [3H]myo-inositol into individual phosphoinositides and of [3H]glycerol into glycerolipids was determined in sciatic nerve obtained from normal and streptozotocin diabetic rats and incubated in vitro. The uptake of inositol into lipid was approximately linear with time. More than 80% of the label was present in phosphatidylinositol with the remainder divided about equally between phosphatidylinositol phosphate and phosphatidylinositol-4,5-bisphosphate. Labeling was unchanged 2 weeks after induction of diabetes, but was reduced by 32% after 20 weeks of the disease. Glycerol incorporation occurred primarily into phosphatidylcholine and triacylglycerol and was depressed up to 45% into major phosphoglycerides in nerves from both 2- and 20-week diabetic animals. Triacylglycerol labeling was also substantially decreased, and the reduction was comparable in intact and epineurium free nerve, suggesting that a metabolically active pool of this compound, which is sensitive to hyperglycemia and/or insulin deficiency, is located in or immediately adjacent to the nerve fibers. The considerable decline in incorporation of these lipid precursors in diabetic nerve may be related to impaired inositol transport and to decrease overall energy utilization by the tissue.     

A microassay for detection of DNA and RNA in small numbers of plant cells

Summary A microtechnique for the detection of DNA or RNA in small numbers of plant cells (1–50) has been developed using cauliflower mosaic virus (CaMV) infection of turnip as a model system. Both DNA and RNA extracted from 10 mesophyll protoplasts from CaMV-infected plants can be detected by hybridization using a radioactive probe made from cloned CaMV DNA (pCaMV10). No hybridization above background was detected in extracts of protoplasts from uninfected plants. At least 0.15 pg (11 000 molecules) of purified pCaMV10 DNA can be detected. This method is superior to existing macro techniques for nucleic acid detection as smaller amounts of tissue are required and the detection is approximately 100-fold more sensitive. re]19850326 rv]19850530 ac]19850611     

Occurrence of a microcanalicular system within the ossicles and dermal tissue of Asterias rubens and Marthasterias glacialis (Echinodermata: Asteroidea)

Summary A microcanalicular network is demonstrated within the ossicle stroma and the dermal tissue of two asteroid species. Microcanaliculi are presumed to be mesodermal structures. They consist of convoluted tubular ducts lined by epithelial cells associated with scattered basiepithelial nervous processes. Such a microcanalicular system has not been reported previously from any echinoderm species. Its discovery in asteroids entails some conceptual changes, especially considering the physiology of the body wall.Research assistants of the National Fund for Scientific Research (NFSR, Belgium)     

Influenza vaccination in the elderly: 2. The economics of sending reminder letters

Reminder letters and follow-up telephone calls were used to increase influenza vaccination acceptance by 273 well elderly registered at an urban community health centre. The net effect of the reminder letters was to increase overall coverage to 43%, from 17% in the previous year. Follow-up telephone calls to patients who had not responded to the letters increased coverage to only 55%. Calculation of costs per additional vaccination given revealed that the use of reminder letters alone was much more cost-effective than follow-up telephone calls in increasing coverage. However, with the current fee-for-service reimbursement by medical care insurance in Ontario, neither means of improving vaccination coverage would result in net practice earnings. The implications for an effective and efficient annual influenza program in Canada are discussed.