The role of glutathione biosynthesis in heavy metal resistance in the fission yeast Schizosaccharomyces pombe

Abstract: Plants and the fission yeast Schizosaccharomyces pombe synthesize small cadmium-binding peptides, called phytochelatins, in response to cadmium. Derived from glutathione (GSH: λ-Glu-Cys-Gly), they have the general structure (λ-Glu-Cys) n Gly, where n is 2–11. In order to study the biosynthesis of phytochelatins, we used the mutagen N -methyl- N '-nitro- N nitrosoguanidine (MNNG) to select mutants with a lowered GSH content. GSH-deficient mutants show a Cd-sensitive phenotype, whereas resistance to Cu is only slightly influenced. These Cd-sensitive mutants contain 2–15% of the wild-type GSH level. For three mutants a lowered activity of λ-glutamylcysteine synthetase was measured. One of the mutants was transformed to Cd-resistance and the complementing fragment was analyzed further. The complementing fragment hybridized with chromosome III. In the transformants, GSH content was restored up to wild-type levels, whereas the activity of λ-glutamylcysteine synthetase was significantly increased compared with the wild-type. Possible mechanisms for Cd-resistance in the transformants are discussed.     

The induction of chromosomal damage and cell killing in mouse spermatogonial stem cells following combined treatments with hydroxyurea, 3-aminobenzamide and X-rays

To analyze in more detail the relation between the sensitivity of spermatogonial stem cells to killing and the induction of genetic damage, mature male mice received combined treatments with hydroxyurea (HU), 3-aminobenzamide (3-AB) and X-rays. Stem cell killing was determined using the repopulation index method and translocations were studied via spermatocyte analysis. HU was administered at 16 or at 48 h before further treatment in order to create stem cell populations with different sensitivities in whic the translocation induction and stem cell killing could be studied and compared. The sensitivities for cell death and genetic damage appeared to be strongly correlated: at 16 h after HU significantly higher values were found than at 48 h or in controls without HU pretreatment.By using 3-AB in the treatment schedules we were able to investigate whether the sensitization of stem cells towards cell death and genetic damage is the outcome of a radiation- or drug-induced G₁ delay. The effect of 3-AB was most pronounced at 16 h after HU. This confirms that at this interval a large fraction of stem cells is in G₁. Our data therefore indicate that all treatments that induce an enrichment of G₁ cells also result in a sensitization of stem cells to cell killing or the induction of mutagenic damage.     

TGGE and HIEF: a comparison of two methods in the detection of carriers of the Z mutation of the alpha-1-antitrypsin gene

Alpha-1-antitrypsin (-1-AT) deficiency can lead to juvenile liver cirrhosis and lung emphysema in adulthood. The deficiency Z allele is caused by a base transition. Temperature gradient gel electrophoresis (TGGE) and hybrid isoelectric focusing (HIEF) were used to detect carriers of the Z mutation of the -1-AT gene. The resulting data were compared. To verify carriers at the sequence level, a manual nonradioactive sequencing strategy was established. Among our sample of carriers of the Z mutation, two were not detected by HIEF that could be identified by TGGE. DNA of all TGGE identified individuals harboring the Z mutation of the -1-AT gene were sequenced nonradioactively. All carriers harbored a G to A transition at position 11.940. This mutation is described to cause the altered protein.     

Bioprocess development to produce a hyperthermostable S-methyl-5′-thioadenosine phosphorylase in Escherichia coli

Nucleoside phosphorylases are important biocatalysts for the chemo-enzymatic synthesis of nucleosides and their analogs which are, among others, used for the treatment of viral infections or cancer. S-methyl-5′-thioadenosine phosphorylases (MTAP) are a group of nucleoside phosphorylases and the thermostable MTAP of Aeropyrum pernix (ApMTAP) was described to accept a wide range of modified nucleosides as substrates. Therefore, it is an interesting biocatalyst for the synthesis of nucleoside analogs for industrial and therapeutic applications. To date, thermostable nucleoside phosphorylases were produced in shake flask cultivations using complex media. The drawback of this approach is low volumetric protein yields which hamper the wide-spread application of the thermostable nucleoside phosphorylases in large scale. High cell density (HCD) cultivations allow the production of recombinant proteins with high volumetric yields, as final optical densities >100 can be achieved. Therefore, in this study, we developed a suitable protocol for HCD cultivations of ApMTAP. Initially, optimum expression conditions were determined in 24-well plates using a fed-batch medium. Subsequently, HCD cultivations were performed using E. coli BL21-Gold cells, by employing a glucose-limited fed-batch strategy. Comparing different growth rates in stirred-tank bioreactors, cultivations revealed that growth at maximum growth rates until induction resulted in the highest yields of ApMTAP. On a 500-mL scale, final cell dry weights of 87.1–90.1 g L⁻¹ were observed together with an overproduction of ApMTAP in a 1.9%–3.8% ratio of total protein. Compared to initially applied shake flask cultivations with terrific broth (TB) medium the volumetric yield increased by a factor of 136. After the purification of ApMTAP via heat treatment and affinity chromatography, a purity of more than 90% was determined. Activity testing revealed specific activities in the range of 0.21 ± 0.11 (low growth rate) to 3.99 ± 1.02 U mg⁻¹ (growth at maximum growth rate). Hence, growth at maximum growth rate led to both an increased expression of the target protein and an increased specific enzyme activity. This study paves the way towards the application of thermostable nucleoside phosphorylases in industrial applications due to an improved heterologous expression in Escherichia coli.     

Yeast glycogenin (Glg2p) produced in Escherichia coli is simultaneously glucosylated at two vicinal tyrosine residues but results in a reduced bacterial glycogen accumulation.

Saccharomyces cerevisiae possesses two glycogenin isoforms (designated as Glg1p and Glg2p) that both contain a conserved tyrosine residue, Tyr232. However, Glg2p possesses an additional tyrosine residue, Tyr230 and therefore two potential autoglucosylation sites. Glucosylation of Glg2p was studied using both matrix-assisted laser desorption ionization and electrospray quadrupole time of flight mass spectrometry. Glg2p, carrying a C-terminal (His6) tag, was produced in Escherichia coli and purified. By tryptic digestion and reversed phase chromatography a peptide (residues 219-246 of the complete Glg2p sequence) was isolated that contained 4-25 glucosyl residues. Following incubation of Glg2p with UDPglucose, more than 36 glucosyl residues were covalently bound to this peptide. Using a combination of cyanogen bromide cleavage of the protein backbone, enzymatic hydrolysis of glycosidic bonds and reversed phase chromatography, mono- and diglucosylated peptides having the sequence PNYGYQSSPAM were generated. MS/MS spectra revealed that glucosyl residues were attached to both Tyr232 and Tyr230 within the same peptide. The formation of the highly glucosylated eukaryotic Glg2p did not favour the bacterial glycogen accumulation. Under various experimental conditions Glg2p-producing cells accumulated approximately 30% less glycogen than a control transformed with a Glg2p lacking plasmid. The size distribution of the glycogen and extractable activities of several glycogen-related enzymes were essentially unchanged. As revealed by high performance anion exchange chromatography, the intracellular maltooligosaccharide pattern of the bacterial cells expressing the functional eukaryotic transgene was significantly altered. Thus, the eukaryotic glycogenin appears to be incompatible with the bacterial initiation of glycogen biosynthesis.     

Phosphate uptake and polyphosphate metabolism of mycorrhizal and nonmycorrhizal roots of pine and of Suillus bovinus at varying external pH measured by in vivo 31P-NMR

 Comparative in vivo ³¹P-NMR analyses of mycorrhizal and nonmycorrhizal roots of Pinus sylvestris and the fungus of Suillus bovinus in pure culture were used to investigate alterations in phosphate metabolism due to changes in external pH in the range 3.5–8.5. All control samples maintained a constant pH in both cytoplasm and vacuole. Mycorrhizal roots and pure fungus, but not nonmycorrhizal roots, transformed accumulated inorganic phosphate into mobile polyphosphate with a medium chain length. Phosphate uptake rates and polyphosphate accumulation responded differently to external pH. In all cases, maximal phosphate uptake occurred at an external pH close to 5.5. At an external pH of 8.5, both roots and fungus showed a distinct lag in phosphate uptake, which was abolished when the external pH was lowered to 7.5. An irreversible effect on phosphate uptake as a consequence of variation in external pH was also observed. The central role of the fungus in regulating mycorrhizal phosphate metabolism is discussed. Accepted: 15 April 1997     

Neural and smooth muscle development in the chicken gizzard

The development of the autonomic ganglia of Auerbach's plexus and gizzard smooth muscle was studied in chicken embryos. Nervous system and smooth-muscle-specific antibodies were employed in immunofluorescence stainings on tissue sections to investigate the temporal and spatial frame of neural and muscular differentiation in relation to each other. Subserosal clusters of neural cells were clearly demonstrable at embryonic day 5 (ED5), the earliest stage analysed, with the monoclonal antibody El (SGIII-1). Fine nerve fibres (ED6) and, later, large axon bundles projecting from subserosal neuron clusters towards the lumen were followed and found to reach the luminal border by ED11. Already in early development the area of the future laminar tendons on the ventral and dorsal surface of the gizzard was devoid of neuroblasts, and nerve fibres were not extending to the muscle-tendon borderline until ED16. Double stainings with antibodies to smooth muscle myosin (SMM) and El revealed that SMM expression, taken as an indicator for muscle differentiation, followed neural growth. It was first detectable in close apposition to the differentiating neuroblasts in the caudal and cranial portion of the gizzard at ED6. With further development, myosin expression proceeded inward towards the lumen in a wave which followed the ingrowth of E1-positive nerve fibres from the prospective Auerbach plexus. Neuromuscular differentiation deviated from this pattern in the lateral tendon area where nerve growth was delayed and myosin expression preceeded the arrival of E1-positive nerve fibres. The findings suggest that the gizzard could serve as a model system for the analysis of potential early nervous system imprints on smooth premuscle mesenchyme differentiation.