Optimization of glutathione production in batch and fed-batch cultures by the wild-type and recombinant strains of the methylotrophic yeast Hansenula polymorpha DL-1

<p>Abstract</p> <p>Background</p> <p>Tripeptide glutathione (gamma-glutamyl-L-cysteinyl-glycine) is the most abundant non-protein thiol that protects cells from metabolic and oxidative stresses and is widely used as medicine, food additives and in cosmetic industry. The methylotrophic yeast <it>Hansenula polymorpha </it>is regarded as a rich source of glutathione due to the role of this thiol in detoxifications of key intermediates of methanol metabolism. Cellular and extracellular glutathione production of <it>H. polymorpha </it>DL-1 in the wild type and recombinant strains which overexpress genes of glutathione biosynthesis (<it>GSH2</it>) and its precursor cysteine (<it>MET4</it>) was studied.</p> <p>Results</p> <p>Glutathione producing capacity of <it>H. polymorpha </it>DL-1 depending on parameters of cultivation (dissolved oxygen tension, pH, stirrer speed), carbon substrate (glucose, methanol) and type of overexpressed genes of glutathione and its precursor biosynthesis during batch and fed-batch fermentations were studied. Under optimized conditions of glucose fed-batch cultivation, the glutathione productivity of the engineered strains was increased from ~900 up to ~ 2300 mg of Total Intracellular Glutathione (TIG) or GSH+GSSG_in, per liter of culture medium. Meantime, methanol fed-batch cultivation of one of the recombinant strains allowed achieving the extracellular glutathione productivity up to 250 mg of Total Extracellular Glutathione (TEG) or GSH+GSSG_ex, per liter of the culture medium.</p> <p>Conclusions</p> <p><it>H. polymorpha </it>is an competitive glutathione producer as compared to other known yeast and bacteria strains (<it>Saccharomyces cerevisiae, Candida utilis, Escherichia coli, Lactococcus lactis </it>etc.) with good perspectives for further improvement especially for production of extracellular form of glutathione.</p

Crystal structure of Hop2-Mnd1 and mechanistic insights into its role in meiotic recombination

In meiotic DNA recombination, the Hop2−Mnd1 complex promotes Dmc1-mediated single-stranded DNA (ssDNA) invasion into homologous chromosomes to form a synaptic complex by a yet-unclear mechanism. Here, the crystal structure of Hop2−Mnd1 reveals that it forms a curved rod-like structure consisting of three leucine zippers and two kinked junctions. One end of the rod is linked to two juxtaposed winged-helix domains, and the other end is capped by extra α-helices to form a helical bundle-like structure. Deletion analysis shows that the helical bundle-like structure is sufficient for interacting with the Dmc1-ssDNA nucleofilament, and molecular modeling suggests that the curved rod could be accommodated into the helical groove of the nucleofilament. Remarkably, the winged-helix domains are juxtaposed at fixed relative orientation, and their binding to DNA is likely to perturb the base pairing according to molecular simulations. These findings allow us to propose a model explaining how Hop2−Mnd1 juxtaposes Dmc1-bound ssDNA with distorted recipient double-stranded DNA and thus facilitates strand invasio

Artificial External Glottic Device for Passive Lung Insufflation

PURPOSE: For patients with neuromuscular disease, air stacking, which inflates the lungs to deep volumes, is important for many reasons. However, neuromuscular patients with severe glottic dysfunction or indwelling tracheostomy tubes cannot air stack effectively. For these patients, we developed a device that permits deep lung insufflations substituting for glottic function. MATERIALS AND METHODS: Thirty- seven patients with bulbar-innervated muscle weakness and/or tracheostomies were recruited. Twenty-three had amyotrophic lateral sclerosis, and 14 were tetraplegic patients due to cervical spinal cord injury. An artificial external glottic device (AEGD) was used to permit passive deep lung insufflation. In order to confirm the utility of AEGD, vital capacity, maximum insufflation capacity (MIC), and lung insufflation capacity (LIC) with AEGD (LICA) were measured. RESULTS: For 30 patients, MICs were initially zero. However, with the use of the AEGD, LICA was measurable for all patients. The mean LICA was 1,622.7±526.8 mL. Although MIC was measurable for the remaining 7 patients without utilizing the AEGD, it was significantly less than LICA, which was 1,084.3±259.9 mL and 1,862.9±248 mL, respectively (p<0.05). CONCLUSION: The AEGD permits lung insufflation by providing deeper lung volumes than possible by air stacking.ope

Protective Mechanism of KIOM-4 in Streptozotocin-Induced Pancreatic β-Cells Damage Is Involved in the Inhibition of Endoplasmic Reticulum Stress

Endoplasmic reticulum stress-mediated apoptosis plays an important role in the destruction of pancreatic β-cells and contributes to the development of type 1 diabetes. The present study examined the effect of KIOM-4, a mixture of four plant extracts, on streptozotocin- (STZ-) induced endoplasmic reticulum (ER) stress in rat pancreatic β-cells (RINm5F). KIOM-4 was found to inhibit STZ-induced apoptotic cell death, confirmed by formation of apoptotic bodies and DNA fragmentation. STZ was found to induce the characteristics of ER stress; mitochondrial Ca2+ overloading, enhanced ER staining, release of glucose-regulated protein 78 (GRP78), phosphorylation of RNA-dependent protein kinase (PKR) like ER kinase (PERK) and eukaryotic initiation factor-2α (eIF-2α), cleavage of activating transcription factor 6 (ATF6) and caspase 12, and upregulation of CCAAT/enhancer-binding protein-homologous protein (CHOP). However, KIOM-4 attenuated these changes induced by STZ. Furthermore, KIOM-4 suppressed apoptosis induced by STZ in CHOP downregulated cells using CHOP siRNA. These results suggest that KIOM-4 exhibits protective effects in STZ-induced pancreatic β-cell damage, by interrupting the ER stress-mediated pathway

How Respiratory Muscle Strength Correlates with Cough Capacity in Patients with Respiratory Muscle Weakness

PURPOSE: The purpose of this study is to investigate how respiratory muscle strength correlates to cough capacity in patients with respiratory muscle weakness. MATERIALS AND METHODS: Forty-five patients with amyotrophic lateral sclerosis (ALS), 43 with cervical spinal cord injury (SCI), and 42 with Duchenne muscular dystrophy (DMD) were recruited. Pulmonary function tests including forced vital capacity (FVC) and respiratory muscle strength (maximal expiratory pressure, MEP; maximal inspiratory pressure, MIP) were performed. The correlation between respiratory muscle strength and cough capacity was analyzed. RESULTS: In the SCI group, FVC in a supine position (2,597 +/- 648 mL) was significantly higher than FVC in a sitting position (2,304 +/- 564 mL, p < 0.01). Conversely, in the ALS group, FVC sitting (1,370 +/- 604 mL) was significantly higher than in supine (1,168 +/- 599 mL, p < 0.01). In the DMD group, there was no statistically significant difference between FVC while sitting (1,342 +/- 506 mL) and FVC while supine (1,304 +/- 500 mL). In addition, the MEP and MIP of all three groups showed a significant correlation with peak cough flow (PCF) (p < 0.01, Pearson's correlation analysis). In the SCI group, MIP was more closely correlated with PCF, while in the ALS and DMD groups, MEP was more closely correlated with PCF (p < 0.01, multiple regression analysis). CONCLUSION: To generate cough flow, inspiratory muscle strength is significantly more important for SCI patients, while expiratory muscle function is significantly more important for ALS and DMD patients.ope

KIOM-4 Protects against Oxidative Stress-Induced Mitochondrial Damage in Pancreatic β-cells via Its Antioxidant Effects

The protective effect of KIOM-4, a mixture of plant extracts, was examined against streptozotocin (STZ)-induced mitochondrial oxidative stress in rat pancreatic β-cells (RINm5F). KIOM-4 scavenged superoxide and hydroxyl radicals generated by xanthine/xanthine oxidase and Fenton reaction (FeSO4/H2O2), respectively, in a cell-free chemical system. In addition, a marked increase in mitochondrial reactive oxygen species (ROS) was observed in STZ-induced diabetic cells; this increase was attenuated by KIOM-4 treatment. Mitochondrial manganese superoxide dismutase (Mn SOD) activity and protein expression were down-regulated by STZ treatment and up-regulated by KIOM-4 treatment. In addition, NF-E2 related factor 2 (Nrf2), a transcription factor for Mn SOD, was up-regulated by KIOM-4. KIOM-4 prevented STZ-induced mitochondrial lipid peroxidation, protein carbonyl and DNA modification. Moreover, KIOM-4 treatment restored the loss of mitochondrial membrane potential (Δψ) that was induced by STZ treatment, and inhibited the translocation of cytochrome c from the mitochondria to the cytosol. In addition, KIOM-4 treatment elevated the level of ATP, succinate dehydrogenase activity and insulin level, which were reduced by STZ treatment. These results suggest that KIOM-4 exhibits a protective effect through its antioxidant effect and the attenuation of mitochondrial dysfunction in STZ-induced diabetic cells

Process development in Hansenula polymorpha and Arxula adeninivorans, a re-assessment

A range of industrial H. polymorpha-based processes exist, most of them for the production of pharmaceuticals. The established industrial processes lean on the use of promoters derived from MOX and FMD, genes of the methanol metabolism pathway. In Hansenula polymorpha these promoters are de-repressed upon depletion of a range of carbon sources like glucose and glycerol instead of being induced by methanol as reported for other methylotrophs. Due to these characteristics screening and fermentation modes have been defined for strains harbouring such expression control elements that lean on a limited supplementation of glycerol or glucose to a culture medium. For fermentation of H. polymorpha a synthetic minimal medium (SYN6) has been developed. No industrial processes have been developed so far based on Arxula adeninivorans and only a limited range of strong promoter elements exists, suitable for heterologous gene expression. SYN6 originally designed for H. polymorpha provided a suitable basis for the initial definition of fermentation conditions for this dimorphic yeast. Characteristics like osmo- and thermotolerance can be addressed for the definition of culture conditions