Topological orientation of mitochondrial GDPmannose: dolichyl-monophosphate mannosyltransferase in the outer membrane

Previous studies have shown the existence of an autonomous mitochondrial GDPmannose: dolichylmonophosphate mannosyltransferase, located in mitochondiral outer membrane of liver cells. As nothing is known about glycosylation sites in mitochondria, we have investigated the topological orientation of this enzyme in intact mitochondria, using controlled proteolysis with trypsin. Mitochondria were purified sequentially by mild ultrasonic treatment and sucrose density gradient. Purity and homogeneity of mitochondrial fraction were assessed by electron microscopy and specific marker enzymes measures. Our data provide evidence for a mitochondrial GDPmannose: dolichylmonophosphate mannosyltransferase facing the cytoplasmic side of the outer membrane. However, the exposure of this enzyme to the water phase has been shown to be dependent on the ionic strength of the environment.     

Atrazine and diuron resistant plants from photoautotrophic protoplast-derived cultures of Nicotiana plumbaginifolia

Atrazine and diuron resistant clones were isolated from diploid photoautotrophic protoplastderived colonies of Nicotiana plumbaginifolia. Protoplasts were mutagenised with 0.1 mM N-ethyl-N-nitrosourea and colonies were screened for resistance after plating. Selection of calli was carried out on their ability to grow and green on a selective medium containing either atrazine or diuron. Plants were regenerated from most tolerant calli. Herbicide spray showed that plants of 6 and 4 clones were resistant to atrazine and diuron, respectively.Abbreviations Atrazine 2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine - diuron 3-(3,4-dichlorophenyl)-1,1-dimethylurea - NEU N-ethyl-N-nitrosourea - PSII photosystem II     

Platelet-Activating Factor Antagonist BN52021 Decreases Accumulation of Free Polyunsaturated Fatty Acid in Mouse Brain During Ischemia and Electroconvulsive Shock

We have investigated the effects of the specific platelet-activating factor (PAF; 1-alkyl-2-acetyl-glycerophosphocholine) antagonist BN52021 on free fatty acid (FFA) and diacylglycerol (DG) accumulation and on the loss of fatty acids from phosphatidylinositol-4,5-bisphosphate (PIP2) in mouse brain. Mice were pretreated with BN52021 (10 mg/kg, i.p.) 30 min before electroconvulsive shock (ECS) or postdecapitation ischemia. These procedures cause rapid breakdown of PIP2 and accumulation of FFA and DG. Lipid extracts were prepared from microwave-fixed cerebrum and fractionated by TLC, and the fatty acid methyl esters were prepared by methanolysis and quantified by capillary GLC. In saline or vehicle (dimethyl sulfoxide)-treated mice, ECS caused marked accumulation of FFA and DG and loss of mainly stearic (18:0) and arachidonic (20:4) acids from PIP2. BN52021 pretreatment of ECS-treated mice decreased the accumulation of free palmitic (16:0), 18:0, 20:4, and docosahexaenoic (22:6) acids with no effect on the fatty acids in DG or the loss of PIP2. BN52021 had no effect on basal levels of FFA, DG, or PIP2. One minute of postdecapitation ischemia induced PIP2 loss and accumulation of FFA and DG. BN52021 attenuated the accumulation of free 20:4 and 22:6 acids, decreased the content of oleic (18:1), 20:4, and 22:6 acids in DG, but had no effect on PIP2 loss. These data indicate that BN52021 reduces the injury-induced activation of phospholipase A2 and lysophospholipase, which mediate the accumulation of FFA in brain, while having a negligible effect on phospholipase C-mediated degradation of PIP2.     

Sib pair linkage analysis of renin gene haplotypes in human essential hypertension

Summary Although essential arterial hypertension is believed to have a strong genetic predisposition, the gene(s) responsible are unknown. The mechanisms underlying the regulation of blood pressure and experimental studies place the renin gene among the main candidate genes that need to be tested in humans. We tested the hypothesis of a linkage between the renin gene and essential hypertension using the affected sib pair method. Siblings (133 subjects, 52.1±10.9 years) from 57 families were selected for sustained hypertension (160.7 ± 22.9/99.5 ± 12.8 mmHg with 80% of patients under antihypertensive treatment), of early onset (40.7 ± 12.0 years), in the absence of obesity, diabetes mellitus, and secondary hypertension. Eight renin haplotypes were generated from three diallelic renin restriction fragment length polymorphisms (RFLPs) (TaqI, Hinfi, HindIII) located throughout the renin gene. The allelic concordance between the sib pairs was analyzed by identity by state relationships for 98 sib pairs (41 for 41 couples, 39 for 13 trios, 18 for 3 quartets). Allelic frequencies in the 57 hypertensive probands were similar to those observed among 102 hypertensive subjects studied previously. Six of eight possible haplotypes were observed, the informativity of the marker corresponded to 70% of heterozygosity. Allelic concordance for all sib pairs according to sibship size was not significantly different from that expected under the hypothesis of no linkage (t = 0.52, P = 0.15) reflecting only a small excess of renin alleles shared by the hypertensive sibs (1.44 ± 0.6 vs 1.36 ± 0.6). Likewise the linkage hypothesis was unsupported by weighted estimates to correct for possible bias due to large sibship size. Thus, the sib pair analysis suggests that the renin gene does not have a frequent role in the pathogenesis of essential hypertension; further more powerful linkage studies or other approaches will be needed to detect contributions at the renin locus to the heritability of essential hypertension.     

Improvement of four sigma analysis for the investigation of oxygen evolution by Photosystem II

We present here an improvement to the analysis of oxygen evolution with four sigma coefficients (4-S) by computing z, the sum of the S-state probabilities, which was introduced earlier (Delrieu and Rosengard 1987, Biochim Biophys Acta 892: 163–171). We demonstrate that z is equal to the ratio of two consecutive Mean Y (the estimation of the steady state oxygen production based on local properties) found by three sigma analysis. The quantity z is useful for computing double-hits, and for showing the inactivation/activation processes of PS II complexes. Three sigma analysis assumes z=1 exactly; since this is not verified, it is argued that four sigma analysis is closer to the real workings of the water oxidizing complex. Oxygen evolution can then be interpreted in the frame of a modified Kok's model where the sum of the probabilities equals z. We therefore suggest that the closer fitting of four sigma analysis to oxygen production data is not simply due to an extra, unnecessary variable, but to the fact that PS II complexes can be inactivated and reactivated under flashing light. Finally, in order to facilitate the use of four sigma analysis, a computer program is made available upon request.     

Sugars fermented byBifidobacterium infantis ATCC 27920 in relation to growth and α-galactosidase activity

Summary The ability of Bifidobacterium infantis ATCC 27 920 to ferment glucose, galactose, lactose, melibiose and raffinose was investigated with respect to -galactosidase (-d-galactoside galactohydrolase, E.C. 3.2.1.22). The sugars were tested at three concentrations: 0.5, 1.0 and 2.0%. The growth of B. infantis was slower on glucose compared with the other sugars. The highest specific growth rate was observed on melibiose followed by lactose. High cell numbers could be rapidly obtained on galactose-containing sugars. For each carbohydrate, enzyme activity was maximal at the end of the exponential phase and the highest specific -galactosidase activities were recorded on the two -1,6 galactosaccharides (melibiose and raffinose: 3.0 and 4.5 nkat · 10⁹ colony-forming units, respectively).Contribution no. 186 from the Food Research and Development Centre Offprint requests to: D. Roy     

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.     

Comparison of transforming growth factor β and a human tumour-derived suppressor factor

Summary Serum-free supernatants from the human melanoma cell line G361 contain a factor that can potently suppress the generation of tumouricidal lymphokine-activated killer (LAK) cells in response to interleukin-2. To characterise the suppressive factor of tumour origin we performed a number of physicochemical and functional comparisons with another immunosuppressive protein, transforming growth factor (TGF). The bioactivity of tumour-derived suppressor factor (TDSF), assayed by suppression of LAK cell generation, was unaffected by a reducing agent but lost when denatured with a chaotropic agent. In contrast, TGF was inactivated by reduction but not denaturation. TDSF lost bioactivity in conditions of pH less than 4, whereas TGF showed no loss of activity. The TDSF moiety has an estimated pI of 4.3 and a molecular mass of 69–87 kDa. This differs from published values of pI 9.5, and 25 kDa molecular mass for TGF. Anti-TGF antiserum reversed the effects of TGF but did not affect the suppression of LAK cell generation caused by TDSF. These findings provide compelling evidence that the TDSF moiety is not TGF, and may be a novel immunoregulatory cytokine.