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)     

The effect of PGP on β-hexosaminidase and histamine secretion in rat peritoneal mast cells in vitro

The investigation of the effect of peptide prolyl-glycyl-proline (PGP) on β-hexosaminidase and histamine secretion by mast cells in primary culture has shown that incubation of mast cells with PGP (6 × 10⁻⁵ M) before their activation by synacten significantly decreased the amount of secreted histamine and β-hexosaminidase in comparison with the action of synacten only. The peptide in investigated concentration had no influence on the level of spontaneous secretion. Incubation of cells with PGP did not prevent their activation by compound 48/80. Therefore, PGP can have a direct effect on isolated rat mast cells in vitro and diminish their secretory activity under activation by synacten.     

Human phosphoglycolate phosphatase (PGP) E.C.3.1.3.18: Linkage analysis

Summary Linkage data on phosphoglycolate phosphatase (PGP) E.C.3.1.3.18 and 26 other human genetic markers are presented. One hundred and one families from the southwestern area of Germany were tested. Close linkage between PGP and the following markers could be ruled out: AB0, acP, ADA, GPT, PGM₁, GLO, HLA, and PGM₃. There is some evidence for possible linkage with MNSs, Rh, Gm and EsD. Family segregation data confirm the hypothesis formerly established by Barker and Hopkinson: three common alleles PGP¹, PGP² and PGP³ at an autosomal locus PGP.Supported by the Deutsche ForschungsgemeinschaftSupported by DAAD and Portuguese Inst. for Scientific Research (INIC)     

Immunohistochemical Localization of Nerve Fibers in the Pseudocapsule of Fibroids

The pseudocapsule surrounding fibroids consists of compressed myometrium containing nerves and blood vessels that continue into adjacent myometrium. Oxytocin (OXT) is thought to affect wound healing after myomectomy. We determined the presence of OXT and protein gene product 9.5 (PGP9.5) immunoreactive nerve fibers in pseudocapsule compared to adjacent myometrium. Samples (N=106) of pseudocapsule and adjacent myometrium were collected from 57 women with uterine fibroids undergoing myomectomy, and stained with anti-OXT and PGP 9.5 antibodies to demonstrate the presence of nerve fibers. Nerve fibers in the pseudocapsule stained positively with OXT (89/106, 84.0%) and PGP 9.5 (94/106, 88.7%). The densities of nerve fibers staining with PGP 9.5 and OXT in the pseudocapsule were highest in the isthmus (23.68±22.45/mm² and 43.35±40.74/mm², respectively). There were no significant differences in the density of nerve fibers, stained with either OXT or PGP 9.5, between the pseudocapsule, and adjacent normal myometrium regardless of the fibroid location in the uterus (P>0.05). These results suggest that the pseudocapsule should avoid to be damaged during the myomectomy procedure.Key words: fibroid pseudocapsule, nerve fibers, oxytocin, myomectomy, protein gene product 9.5, immunohistochemistry     

Protein kinase C-independent correlation between P-glycoprotein expression and volume sensitivity of Cl− channel

The possible correlation between P-glycoprotein (PGP) and volume-sensitive Cl⁻ channel was examined in a pair of cell lines: a subline of the human epidermoid KB cell (KB-3-1) and the corresponding MDR1-transfected cell line (KB-G2). Western blot analysis and indirect immunofluorescence studies indicated that KB-G2, but not KB-3-1, exhibits the PGP expression. Patch-clamp whole-cell recordings showed that osmotic swelling activates Cl⁻ currents not only in PGP-expressing but also in PGP-lacking cells. The amplitude of the maximal current was indistinguishable between both cells. Activation of protein kinase C (PKC) or loading with a PKC inhibitor failed to affect the swelling-induced activation of the Cl⁻ currents in both cells. The relation between whole-cell Cl⁻ currents and cell size measured simultaneously showed that volume sensitivity of the Cl⁻ channel was augmented by the PGP expression irrespective of the activity of PKC on the plasma membrane. A similar increase in volume sensitivity of the Cl⁻ channel was also induced by the expression of the ATP hydrolysis-deficient PGP mutant, K433M. We conclude that P-glycoprotein does not represent the volume-sensitive Cl⁻ channel but that its expression modulates volume sensitivity of the Cl⁻ channel in a manner independent of its ATPase activity or of the protein kinase C activity. Received: 25 September 1996/Revised: 12 December 1996     

Role of multidrug resistance P-glycoprotein in the secretion of aldosterone by human adrenal NCI-H295 cells

We determinedthe role of the multidrug resistance (MDR1) gene product,P-glycoprotein (PGP), in the secretion of aldosterone by the adrenalcell line NCI-H295. Aldosterone secretion is significantly decreased bythe PGP inhibitors verapamil, cyclosporin A (CSA), PSC-833, andvinblastine. Aldosterone inhibits the efflux of the PGP substraterhodamine 123 from NCI-H295 cells and from human mesangial cells(expressing PGP). CSA, verapamil, and the monoclonal antibody UIC2significantly decreased the efflux of fluorescein-labeled (FL)-aldosterone microinjected into NCI-H295 cells. In MCF-7/VP cells,expressing multidrug resistance-associated protein (MRP) but not PGP,and in the parental cell line MCF7 (expressing no MRP andno PGP), the efflux of microinjected FL-aldosterone was slow. In BC19/3cells (MCF7 cells transfected with MDR1), the efflux of FL-aldosteronewas rapid and it was inhibited by verapamil, indicating thattransfection with MDR1 cDNA confers the ability to transportFL-aldosterone. These results strongly indicate that PGP plays a rolein the secretion of aldosterone by NCI-H295 cells and in other cellsexpressing MDR1, including normal adrenal cells.

     

Phosphatidylglycerol biosynthesis in Bacillus licheniformis Resolution of membrane-bound enzymes by affinity chromatography on cytidinediphospho-sn-1,2-diacylglycerol Sepharose.

Cytidinediphospho-sn-1,2-diaclglycerol (CDP-diglyceride) has been covalently linked to Sephrose 4B via adipic acid dihydrazide spacer arm forming an effective affinity chromatography column. This liponucleo-tide ligand and sn-glycero-3-phosphate are subtracts for the formation of 3-sn-phoshatidyl-1'-sn-glycero-3'-phosphate (PGP) catalyzed in both eukaryotic and prokaryotic organisms by sn-glycero-3-phosphate: CMP phosphatidlytranferase (PGP synthetase). Using this CDP-diglyceride Sephrose affinity column we were able to resolve the membrane associated 3-sn-phosphatidyl'1-sn-glycerol (PG) synthesizing system present in Bacillus licheniformis into two activities. A PGP synthetase activity was adsorbed to the affinity column and was eluted using buffer containg CDP-diglyceride; a PGP phosphatease acactivity had no affinity for the column. Both PGP synthase and PGP phosphatase of B. licheniformis were associated with a membrane component of the cell as evidenced by sucrose gradient centrifugation, differential centrifugation, and solubilization by buffers containing detergent...     

Phosphoglycolate phosphatase in several population groups in Israel

The genetic polymorphism of phosphoglycolate phosphatase (PGP) found in red blood cells has been investigated in several population groups in Israel: Ashkenazi Jews, non-Ashkenazi Jews from Iraq, Yemen, Turkey, Iran, Balkan, North Africa and Arabs. The distribution of the PGP genes was not homogeneous (chi 2 = 40.545; d.f. = 20; p less than 0.005). The PGP2 gene frequency varied between 0.0185 in the Yemenite and 0.0688 in the Iranian Jews. PGP3 gene frequency ranged between 0.0062 in the Iranian and 0.0547 in the Moroccan Jews. Depsite this heterogeneity all the Israeli population groups showed some unifying characteristics which differentiated them from a random European population sample, namely higher frequencies of PGP1 gene (92-97% as opposed to 82% in th European sample) and lower frequencies of PGP2 gene (1.8-6.8% compared to 12.9% among Europeans).     

Luteibacter rhizovicinus MIMR1 promotes root development in barley (Hordeum vulgare L.) under laboratory conditions

In order to preserve environmental quality, alternative strategies to chemical-intensive agriculture are strongly needed. In this study, we characterized in vitro the potential plant growth promoting (PGP) properties of a gamma-proteobacterium, named MIMR1, originally isolated from apple shoots in micropropagation. The analysis of the 16S rRNA gene sequence allowed the taxonomic identification of MIMR1 as Luteibacter rhizovicinus. The PGP properties of MIMR1 were compared to Pseudomonas chlororaphis subsp. aurantiaca DSM 19603^T, which was selected as a reference PGP bacterium. By means of in vitro experiments, we showed that L. rhizovicinus MIMR1 and P. chlororaphis DSM 19603^T have the ability to produce molecules able to chelate ferric ions and solubilize monocalcium phosphate. On the contrary, both strains were apparently unable to solubilize tricalcium phosphate. Furthermore, the ability to produce 3-indol acetic acid by MIMR1 was approximately three times higher than that of DSM 19603^T. By using fluorescent recombinants of strains MIMR1 and DSM 19603^T, we also demonstrated that both bacteria are able to abundantly proliferate and colonize the barley rhizosphere, preferentially localizing on root tips and in the rhizoplane. Finally, we observed a negative effect of DSM 19603^T on barley seed germination and plant growth, whereas MIMR1, compared to the control, determined a significant increase of the weight of aerial part (+22 %), and the weight and length of roots (+53 and +32 %, respectively). The results obtained in this work make L. rhizovicinus MIMR1 a good candidate for possible use in the formulation of bio-fertilizers.