Recombinant protein production via fed-batch culture of the yeast Saccharomyces cerevisiae

Protein production with the recombinant yeast Saccharomyces cerevisiae in fed-batch culture is investigated in this work using beta-galactosidase as a model protein. Segregational instability was negligible during the observed culture periods. The final volumetric productivity, as determined by both cell concentration and gene expression, was strongly affected by the time course of the glucose levels in the bioreactor. It was found that an average glucose feed rate of 1.31 g glucose h-1 resulted in both the maximum beta-galactosidase production rate of 831-950 units ml-1 h-1 and the maximum cell production rate of 0.520-0.524 mg ml-1 h-1.     

Glutathione depletion in the yeast Saccharomyces cerevisiae

The effect of chosen compounds on the total glutathione (GSH) level in stationary cultures of S. cerevisiae was compared. 1-Chloro-2,4-dinitrobenzene, 1-fluoro-2,4-dinitrobenzene, maleimide, iodacetamide and allyl alcohol (1 mM), and menadione (0.5 mM) caused an almost complete GSH depletion during several minutes. Bromobenzoic acid and chloramine T (I mM), and daunomycin (60 mu M) induced a slower GSH decrease, down to 30-70% after 60 min. Paraquat (1 mM), CuSO(4) (0.5 mM) and cadmium acetate (1 mM) decreased glutathione level down to ca 70%. Diamide (0.5 mM), phenazine methosulphate, phenylhydrazine, acetylphenylhydrazine and H(2)O(2) (1 mM), and t-butyl hydroperoxide (2 mM) did not affect total GSH during 60-min exposure. There was no clear-cut dependence between the ability of various chemicals to deplete cellular GSH and their increased toxicity to a glutathione-poor mutant.     

Synthesis and intracellular transport of aminoglycerophospholipids in permeabilized cells of the yeast, Saccharomyces cerevisiae

The sequence of biosynthetic steps from phosphatidylserine to phosphatidylethanolamine (via decarboxylation) and then phosphatidylcholine (via methylation) is linked to the intracellular transport of these aminoglycerophospholipids. Using a [3H]serine precursor and permeabilized yeast cells, it is possible to follow the synthesis of each of the aminoglycerophospholipids and examine the requirements for their interorganelle transport. This experimental approach reveals that in permeabilized cells newly synthesized phosphatidyl-serine is readily translocated to the locus of phosphatidylserine decarboxylase 1 in the mitochondria but not to the locus of phosphatidylserine decarboxylase 2 in the Golgi and vacuoles. Phosphatidylserine transport to the mitochondria is ATP independent and exhibits no requirements for cytosolic factors. The phosphatidylethanolamine formed in the mitochondria is exported to the locus of the methyltransferases (principally the endoplasmic reticulum) and converted to phosphatidylcholine. The export of phosphatidylethanolamine requires ATP but not any other cytosolic factors and is not obligately coupled to methyltransferase activity. The above described lipid transport reactions also occur in permeabilized cells that have been disrupted by homogenization, indicating that the processes are extremely efficient and may be dependent upon stable structural elements between organelles.     

Cobalt-mediated generation of reactive oxygen species and its possible mechanism

Electron spin resonance spin trapping was utilized to investigate free radical generation from cobalt (Co) mediated reactions using 5,5-dimethyl-1-pyrroline (DMPO) as a spin trap. A mixture of Co with water in the presence of DMPO generated 5,5-dimethylpyrroline-(2)-oxy(1) DMPOX, indicating the production of strong oxidants. Addition of superoxide dismutase (SOD) to the mixture produced hydroxyl radical (.OH). Catalase eliminated the generation of this radical and metal chelators, such as desferoxamine, diethylenetriaminepentaacetic acid or 1,10-phenanthroline, decreased it. Addition of Fe(II) resulted in a several fold increase in the .OH generation. UV and O2 consumption measurements showed that the reaction of Co with water consumed molecular oxygen and generated Co(II). Since reaction of Co(II) with H2O2 did not generate any significant amount of .OH radicals, a Co(I) mediated Fenton-like reaction [Co(I) + H2O2-->Co(II) + .OH + OH-] seems responsible for .OH generation. H2O2 is produced from O2.- via dismutation, O2.- is produced by one-electron reduction of molecular oxygen catalyzed by Co. Chelation of Co(II) by biological chelators, such as glutathione or beta-ananyl-3-methyl-L-histidine alters, its oxidation-reduction potential and makes Co(II) capable of generating .OH via a Co(II)-mediated Fenton-like reaction [Co(II) + H2O2-->Co(III) + .OH + OH-]. Thus, the reaction of Co with water, especially in the presence of biological chelators, glutathione, glycylglycylhistidine and beta-ananyl-3-methyl-L-histidine, is capable of generating a whole spectrum of reactive oxygen species, which may be responsible for Co-induced cell injury.     

MAP kinase pathways in the yeast Saccharomyces cerevisiae

A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.     

Direct effects of intraperilymphatic reactive oxygen species generation on cochlear function

Reactive oxygen species (ROS) generation may play a role in ototoxicity, however, the specific effects of ROS generation upon cochlear function are unstudied. Therefore, guinea pig cochleas were instilled with artificial perilymph (AP), H2O2, or confirmed generating systems for the superoxide anion (O2-) or the hydroxyl radical (OH.), or with an ROS system plus its respective scavenger -catalase (CAT), superoxide dismutase (SOD) or deferoxamine (DEF). O2- generating system instillation led to significantly greater mean high frequency compound action potential (CAP) threshold shifts at 10 and 120 min post infusion than seen in AP control or SOD/O2- groups. H2O2 group CAP threshold shifts were significantly greater than control and CAT/H2O2 group values at 10 (16-30 kHz), and 120 min (above 12 kHz). OH generating system instillation led to significantly greater CAP threshold shifts at 10 (12-30 kHz) and 120 min (above 6 kHz) than seen in control or DEF/OH groups. No significant CAP differences were found between controls and scavenger/ROS groups. Mean 1.0 microV cochlear microphonic isopotential curve shift values did not systematically differ among groups. The rapid degradation of high frequency CAP threshold sensitivity seen here may provide insight into the portion of cochlear dysfunction which is ROS-mediated following noise, radiation or chemical exposures.     

Cascades of mammalian caspase activation in the yeast Saccharomyces cerevisiae

Caspases (aspartate-specific cysteine proteases) play a critical role in the execution of the mammalian apoptotic program. To address the regulation of human caspase activation, we used the yeast Saccharomyces cerevisiae, which is devoid of endogenous caspases. The apical procaspases, -8beta and -10, were efficiently processed and activated in yeast. Although protease activity, per se, was insufficient to drive cell death, caspase-10 activity had little effect on cell viability, whereas expression of caspase-8beta was cytotoxic. This lethal phenotype was abrogated by co-expression of the pan-caspase inhibitor, baculovirus p35, and by mutation of the active site cysteine of procaspase-8beta. In contrast, autoactivation of the executioner caspase-3 and -6 zymogens was not detected. Procaspase-3 activation required co-expression of procaspase-8 or -10. Surprisingly, activation of procaspase-6 required proteolytic activities other than caspase-8, -10, or -3. Caspase-8beta or -10 activity was insufficient to catalyze the maturation of procaspase-6. Moreover, a constitutively active caspase-3, although cytotoxic in its own right, was unable to induce the processing of wild-type procaspase-6 and vice versa. These results distinguish sequential modes of activation for different caspases in vivo and establish a yeast model system to examine the regulation of caspase cascades. Moreover, the distinct terminal phenotypes induced by various caspases attest to differences in the cellular targets of these apoptotic proteases, which may be defined using this system.     

Sensitivity of Ca2+ transport of mitochondria to reactive oxygen species

To determine the association between specific structural changes in the hemagglutinin gene and pathogenicity of avian influenza viruses (AIVs), groups of 4-week-old White Plymouth Rock chickens were inoculated intravenously or intranasally with AIVs of varying pathogenicities isolated from chickens in central Mexico during 1994-1995. Mildly pathogenic (MP) viruses had a common hemagglutinin-connecting peptide sequence of Pro-Gln-Arg-Glu-Thr-Arg decreases Gly and had restricted capability for replication and production of lesions in tissues. The principle targets for virus replication or lesion production were the lungs, lymphoid organs, and visceral organs containing epithelial cells, such as kidney and pancreas. Death was associated with respiratory and/or renal failure. By contrast, highly pathogenic (HP) AIVs had one substitution and the addition of two basic amino acids in the hemagglutinin connecting peptide, for a sequence of Pro-Gln-Arg-Lys-Arg-Lys-Thr-Arg decreases Gly. The HP AIVs were pantropic in virus replication and lesion production ability. However, the most severe histologic lesions were produced in the brain, heart, adrenal glands, and pancreas, and failure of multiple critical organs was responsible for disease pathogenesis and death. No differences in lesion distribution patterns or in sites of AIV replication were evident to explain the variation in mortality rates for different HP AIVs, but HP AIVs that produced the highest mortality rates had more severe necrosis in heart and pancreas. The ability of individual HP AIVs to produce low or high mortality rates could not be explained by changes in sequence of the hemagglutinin-connecting peptide alone, but probably required the addition of other undetermined genomic changes.     

Calcium ion influx during sporulation in the yeast Saccharomyces cerevisiae

The changes in intra- and (or) extra-cellular concentrations of Ca2+, Mg2+, K+, and Na+ during sporulation of a MATa/MAT alpha diploid yeast of Saccharomyces cerevisiae were examined in a nutrition-deprived medium with potassium acetate. Among these, Ca2+ in external medium was preferentially incorporated into cells, and sporulation was induced when the magnitude of free Ca2+ gradient between cytosol [Ca2+]i and external medium [Ca2+]o reached more than 3 x 10(3) ([Ca2+]i/[Ca2+]o = 3.5 x 10(3)). The result indicated that the meiosis and (or) sporulation signal of the yeast S. cerevisiae was generated through elevated Ca2+ influx rather than release from the internal Ca2+ stores.     

Lead exposure causes generation of reactive oxygen species and functional impairment in rat sperm

The relationships between blood lead, sperm lead, sperm reactive oxygen species (ROS) level, and sperm fertile capability were investigated to understand the effects of lead exposure on sperm function and the mechanism of these effects. Male Sprague-Dawley rats, 7 weeks old, were randomly divided into control group and lead-treated group. The controls and lead-treated animals received intraperitoneal injection of 10 mg sodium acetate and 10 mg lead acetate/kg body weight, respectively, weekly for 6 or 9 weeks. The blood lead and epididymal sperm lead were analyzed by graphite furnace atomic absorption spectrophotometer. Chemiluminescence was measured to evaluate the generation of sperm ROS. Sperm-oocyte penetration rate (SOPR) was measured to evaluate sperm function. After 6 weeks of lead exposure, the rats had average blood lead levels of 32 microg/dl, sperm lead levels of 0.67 +/- 0.11 microg/10(9) sperm, unchanged epididymal sperm counts, percent of motile sperms, and motile epididymal sperm counts compared with control animals. However, after 9 weeks of lead exposure, the rats had average blood lead levels of 48.0 +/- 4.3 microg/dl, sperm lead levels of 0.88 +/- 0.16 microg/10(9) sperm, statistically lower epididymal sperm counts, and lower motile epididymal sperm counts. There was a good correlation between the blood lead and sperm lead(r2 = 0.946, P < 0.001). The sperms of lead-exposed rats produced significantly higher counts ofchemiluminescence than did those from the control rats (P < 0.001). The chemiluminescence counts were positively associated with sperm lead level (r2 = 0.613, P < 0.001). Epididymal sperm counts, motility and motile epididymal sperm counts were negatively associated with sperm chemiluminescence (r2 = 0.255, 0.152, and 0.299; P < 0.01, 0.05, and 0.01, respectively). The SOPR were positively associated with epididymal sperm counts, motility and motile epididymal sperm counts (r2 = 0.136, 0.285, and 0.264; P < 0.05, 0.01, and 0.001, respectively). The sperm chemiluminescence was negatively associated with SOPR (r2 = 0.519, P < 0.001). It is concluded that lead exposure probably affected the sperm function by activating one of the pathways of ROS generation.     

Molecular cloning of the actin gene from yeast Saccharomyces cerevisiae

Two overlapping DNA fragments from yeast Saccharomyces cerevisiae containing the actin gene have been inserted into pBR322 and cloned in E.coli. Clones were identified by hybridization to complementary RNA from a plasmid containing a copy of Dictyostelium actin mRNA. One recombinant plasmid obtained (pYA102) contains a 3.93-kb Hindlll fragment, the other (pYA208) a 5.1-kb Pstl fragment, both share a common 2.2-kb fragment harboring part of the actin gene. Cloned yeast actin DNA was identified by R-loop formation and translation of the hybridized actin mRNA and by DNA sequence analysis. Cytoplasmic actin mRNA has been estimated to be about 1250 nucleotides long. There is only one type of the actin gene in S.cerevisiae.     

Stabilization of microsatellite sequences by variant repeats in the yeast Saccharomyces cerevisiae

We examined the effect of a single variant repeat on the stability of a 51-base pair (bp) microsatellite (poly GT). We found that the insertion stabilizes the microsatellite about fivefold in wild-type strains. The stabilizing effect of the variant base was also observed in strains with mutations in the DNA mismatch repair genes pms1, msh2 and msh3, indicating that this effect does not require a functional DNA mismatch repair system. Most of the microsatellite alterations in the pms1, msh2 and msh3 strains were additions or deletions of single GT repeats, but about half of the alterations in the wild-type and msh6 strains were large (> 8 bp) deletions or additions.     

Precipitated immune complexes of IgM induce the generation of reactive oxygen species by rabbit polymorphonuclear leucocytes

We report that immune complexes of IgM (ICIgM) antibodies and ovalbumin in the form of a precipitate from the equivalence zone induce the generation of reactive oxygen species by rabbit blood polymorphonuclear leucocytes (PMN), as measured by the chemiluminescence (CL) production in the presence of luminol. The kinetics of CL generation induced by ICIgM is quite different from that induced by precipitated immune complexes of IgG (ICIgG): the maximum rate of CL production for ICIgM occurs around 14 min, whereas for ICIgG it occurs about 5 min after incubation with the cells. Also the triggering of the process requires a higher concentration of ICIgM than of ICIgG. Evidence is presented that these effects are not mediated by interaction of the antigen (ovalbumin) with the cell, since immune precipitates of ovalbumin and the F(ab')2 fragment had no effect. Our observations that precipitated ICIgM can also be an effective stimulus for CL generation and thus for O2- production reveal a new functional capability of PMN. These results may have implications for the understanding of the participation of ICIgM (as well as of ICIgG) in inflammatory reactions mediated by PMN in immune complex diseases, and in the mechanisms of defense against microbes and other non-self agents.     

Adenylosuccinate synthetase of the yeast Saccharomyces cerevisiae: purification and properties

Motor and sensory nerve conduction velocities (MNCV and SNCV) were reduced in the sciatic nerve of rats after 4 weeks of untreated streptozotocin-induced diabetes, and declined further during the following 4 weeks. Treating diabetic rats with the novel peptide HP228 had no effect on the decline of MNCV after the first 4 weeks of diabetes but attenuated the decline in SNCV. HP228 treatment also prevented any further decline in MNCV or SNCV between weeks 4 and 8 of diabetes. Consequently, at the conclusion of the study, the nerve conduction velocities (NCVs) in treated rats were significantly (both P < .001) higher than in untreated diabetic rats. Reduced nerve homogenate Na+,K+-adenosine triphosphatase (ATPase) activity in diabetic rats was significantly (P < .05) increased by HP228 but remained significantly (P < .05) lower than in untreated controls. HP228 treatment also reduced nerve Na+,K+-ATPase activity of control rats compared with untreated controls (P < .05). There was no effect of HP228 on the hyperglycemia, nerve polyol accumulation, myo-inositol depletion, reduced nerve laser Doppler blood flow, thermal hypoalgesia, or reduced mean axonal caliber in diabetic rats or on any of these parameters in control rats. These data demonstrate that a novel peptide may protect against the slowing of nerve conduction in prolonged diabetes and that the mechanism of action is unrelated to aldose reductase inhibition, prevention of nerve ischemia, or axonal atrophy. HP228 may prove a potential therapeutic agent for the treatment of prolonged diabetic neuropathy.     

Nuclear genes of human complex I of the mitochondrial electron transport chain: state of the art

Exposure to aromatic amines is considered a major risk factor for the development of bladder cancer. In this study, we have analysed the pattern of point mutations in several tumour genes in 21 cases of bladder cancer arising among western European workers exposed to aromatic amines in an attempt to determine whether this exposure may be associated with a unique spectrum of mutations. Of the four genes analysed (p53, p16MTS1, p21WAF1 and H-ras), only p53 showed a high frequency of mutations (in 8 out of 21 cases, 38%). Two mutations were found in p16, one in H-ras and none in p21 exon 3. All mutations were at G:C base pairs, mostly at non-CpG residues. This spectrum of mutations, which is highly suggestive of an involvement of exogenous carcinogens, is however identical to the spectrum of p53 mutations detected in bladder cancers of the general population. In exposed workers, p53 mutations were associated with tumour grade and with high occupational and tobacco exposure. Taken together, our data suggest that the same carcinogens may be responsible for the development of bladder cancers in workers exposed to aromatic amines and in the general population.