Biomarkers to evaluate the effects of temperature and methanol on recombinant Pichia pastoris

Pichia pastoris is methylotrophic yeast used as an efficient expression system for heterologous protein production. In order to evaluate the effects of temperature (10 and 30 °C) and methanol (1 and 3% (v/v)) on genetically-modified Pichia pastoris, different biomarkers were evaluated: Heat stress (HSF-1 and Hsp70), oxidative stress (OGG1 and TBARS) and antioxidant (GLR). Three yeast cultures were performed: 3X = 3% methanol-10 °C, 4X = 3% methanol-30 °C, and 5X = 1% methanol-10°C. The expression level of HIF-1α, HSF-1, HSP-70 and HSP-90 biomarkers were measured by Western blot and in situ detection was performed by immunocytochemistry. Ours results show that at 3% methanol -30 °C there is an increase of mitochondrial OGG1 (mtOGG1), Glutathione Reductase (GLR) and TBARS. In addition, there was a cytosolic expression of HSF-1 and HSP-70, which indicates a deprotection against nucleolar fragmentation (apoptosis). On the other hand, at 3% methanol -10 °C and 1% and at methanol -10 °C conditions there was nuclear expression of OGG1, lower levels of TBARS and lower expression of GLR, cytosolic expression of HSF-1 and nuclear expression HSP-70. In conclusion, our results suggest that 3% methanol-30 °C is a condition that induces a strong oxidative stress and risk factors of apoptosis in modified-genetically P. pastoris.

HSF-1, HIF-1and HSP90 expression on recombinant Pichia pastoris under fed-batch fermentation

Pichia pastoris is a methylotrophic yeast used as an efficient expression system for heterologous protein production as compared to other expression systems. Considering that every cell must respond to environmental changes to survive and differentiate, determination of endogenous protein related to heat stress responses and hypoxia, it would necessary to establish the temperature and methanol concentration conditions for optimal growth. The aim of this study is characterize the culture conditions through the putative biomarkers in different conditions of temperature and methanol concentration. Three yeast cultures were performed: 3X = 3% methanol -10 °C, 4X = 3% methanol -30 °C, and 5X = 1% methanol -10 °C. The expression level of HIF-1α, HSF-1, HSP-70 and HSP-90 biomarkers were measured by Western blot and in situ detection was performed by immunocytochemistry. The western blot results of HIF-1α and HSP-90 did not indicate statistically significant in the culture conditions studied. Respect to biomarkers location, HIF-1α and HSP-90 presented differences between cultures. In conclusion, the results suggest the cultures in a hypoxic condition produce a high density and yeast cells smaller. Beside the high density would not necessary related with a high production of recombinant proteins in modified-genetically P. pastoris.

Anti-apoptotic effect of NGF on H9 c2 cardiac myocytes in a hypoxia/reoxygenation injury model / 中国药理学通报

Aim To investigate the anti-apoptotic effect of NGF on H9 c2 cardiac myocytes in a hypoxia / reox-ygenation injury model and its mechanism. Methods The H9 c2 cardiac myocytes were randomly divided into five groups:control group ( C group) , hypoxia/reoxy-genation group ( H/R group) , NGF group ( N group) , NGF+LY294002 group ( N+L group) and LY294002 group( L group) . Each group received the correspond-ing treatment. Cell survival rate was tested by cell counter kit-8 methods. Apoptotic rate was evaluated by propidium iodide ( PI ) staining and flow cytometry (FCM). The levels of Caspase-12, p-Akt/Akt were e-valuated by Western blot. Results The NGF group could significantly protect the H9 c2 cardiac myocytes under the hypoxia / reoxygenation injury with increased cell survival rate. It also decreased the apoptotic per-centage, upregulated the level of p-Akt/Akt and inhib-ited the expression of Caspase-12 . As the specific in-hibitor of PI3k receptor, LY294002 decreased the level of p-Akt. Conclusion NGF has the effect of anti-ap-optosis on H9 c2 cardiac myocytes exposed to hypoxia /reoxygenation injury via PI3k-Akt signal pathway.

Expression of Heat Stress Protein 70 mRNA in Patients with Chronic Ob-structive Pulmonary Disease and Its Significance / 华中科技大学学报（医学）（英德文版）

The effects of cigarette smoke extract (CSE) on the expression of heat stress protein 70(Hsp70) in human bronchi smooth muscle cells were investigated in vitro, and the changes in Hsp70 mRNA in the patients with chronic obstructive pulmonary disease and their significance were explored. Human bronchi smooth muscle cells were cultured with CSE at the different concentrations. The expression of Hsp70 mRNA and Hsp70 was detected by reverse translation-polymerase chain reaction (RT-PCR) and Western blotting respectively. Levels of Hsp70 mRNA and Hsp70 in lymphocytes from 20 patients with COPD and 20 healthy smoking control subjects were measured by RT-PCR and Western blotting. The results showed the expression of both Hsp70 mRNA and Hsp70 was decreased conformably in human bronchi smooth muscle cells treated with CSE at certain concentration in vitro. The A values of the Hsp70 mRNA expression were 0. 24±0.11 and 0.42±0.13 respectively in COPD patients and healthy smoking controls with the difference being significant (P＜0.01). There was also significant difference in the A values of the Hsp70 expression between COPD patients and healthy smoking controls (20.9±9.9 vs 44.8±15.3, P＜0.01). The levels of Hsp70 mRNA had strongly positive correlation with Hsp70 protein (r = 0. 85, P＜0.01). It was suggested that the expression of Hsp70 mRNA was in concordance with the expression of Hsp70, which could provide a basis on the study of Hsp70 gene regulation and Hsp70 gene in the development of COPD.

The Expression of a Novel 90 kDa Stress Protein in Human Malignant Neoplasms / 대한암학회지

PURPOSE: When cells are subjected to stressful stimuli such as, heat shock, toxic metal, nutrient deprivation, and metabolic disruption, they increase production of specific stress proteins that buffer them from harm. We reported that the expression of a navel 90 kDa cellular protein was increased by the infection of a fish rhabdovirus and heat shock in a fish cell. This new 90 kDa protein is not expressed in normal animal tissues but is highly induced in progressively transforming tissues or cells. That gives us some ideas tl at it is possible for this stress protein to be expressed in specific human cancer tissues. MATERIALS AND METHODS: Commercialized checkerboard multi-tumor block (DAKO Co. Carpinteria, CA) was used for immunohistochemical analysis. The samples of human gastric cancer, colon cancer and breast cancer tissues were evaluated by Western blot and Northern blot for overexpression of the novel 90 kDa stress protein. Sera of those patients were analyzed by ELISA for the presence of antibody against the novel 90 kDa stress protein. RESULTS: Immunohistochemical staining of human tumor tissue blocks showed significant immunostaining of novel 90 kDa stress protein in carcinomas such as colon cancer, breast cancer and stomach cancer but no apparent immunostaining in sarcomas. Coinciding with the immunohistochemical result, Western blotting and Northern blotting analyses indicate that the expression of the novel 90 kDa stress protein was increased in carcinomas. In addition, the antibody titer against the novel 90 kDa stress protein was found to be elevated in the sera of cancer patients. CONCLUSIONS: The novel 90 kDa stress protein gene expression was elevated in carcinomas such as gastric cancer, breast cancer and colon cancer. These findings suggest that this new stress protein can be used as a tumor marker and may function as a chaperone in tumor growth.

Fiber type specific distribution of stress proteins in rat skeletal muscle / 体力科学

To determine whether fiber type-specific expression of heat shock protein (HSP, or stress protein) occurs in unstressed rat skeletal muscle, the medial gastrocnemius of adult female Sprague-Dawley rats was subjected to immunohistochemical analysis. Antibodies against 5 types of anti-myosin heavy chain (MHC) were used to classify the type of fibers, and 2 types of anti-HSP antibodies were employed to analyze the fiber type-specific expression.<BR>Serial cross-sections of 10 μm thick cut by a cryostat were incubated with primary anti-MHC or anti-HSP 60 and 72 antibodies, followed by biotinylated secondary anti-mouse antibodies, and avidin-biotin complex solution. A peroxidase DAB substrate kit (Vector SK-4100) or BCIP/NBT solution was used to visualize the immunoreaction of each fiber type.<BR>By using the 5 types of anti-MHC antibodies, fibers were classified into 4 types : slow-type I, fasttypes IIA, IIX, and IIB. Anti-HSP 72 antibody reacted with many, but not all, type I and IIA fibers, whereas anti-HSP 60 antibody reacted specifically with type I fibers. Neither type IIX nor IIB fibers showed immunoreactivity with anti-HSP 60 or 72 antibodies. These results suggest that the expression of HSP 60 protein is related to that of type I MHC, and that the expression of HSP 72 protein may be related to that of types I and ha MHC, in unstressed rat skeletal muscle.

Stress Protein Expression in Kainate-Induced Experimental Temporal Lobe Epilepsy in Rats

To investigate neuronal injury developed in an experimental model of temporal lobe epilepsy, the expression of c-FOS, c-JUN and HSP72 on the amygdala, hippocampus and temporal neocortex was studied. Epileptic seizure was induced in rats by microinjection of kainic acid(1microgrm/microl) dissolved in phosphate buffer(0.1 M, pH 7.4) into the left amygdala. Selective and delayed neuronal injuries appeared in the CA3 region of the hippocampus after 14 days and were characterized by swelling of cytoplasm and neurites, nuclear pyknosis and loss of neurons. Early induction of c-FOS and c-JUN was noted on the injection-side amygdala at 1-12h, and delayed expression developed at 7 days after the injection. HSP72 expression appeared continuously 3 hrs after the injection. Delayed induction of c-FOS, c-JUN and continuous expression of HSP72 were observed in the hippocampus and entorhinal cortex. In the hippocampus, c-FOS expression was relatively strong in the neurons of CA3 and dentate gyrus at 7~14 days after the injection. Similar findings were also noted in the neurons of the entorhinal cortex. Induction of HSP72 occurred slightly later than on that of c-FOS and c-JUN in the amygdala, with the prominent induction being noted in the neurons of amygdala, CA2, CA3, CA4 and dentate gyrus at 3 to 21 days after the injection. These results suggested that the delayed expressions of c-FOS and c-JUN in the hippocampus correlated well with impending clinical epileptic seizure.

Biochemical and immunofluorescence analysis of the constitutively expressed HSP27 stress protein in monkey CV-1 cells.

The α-crystallin-related stress protein HSP27, which promotes cellular resistance to different types of stress, is constitutively expressed during the growth of several primate tissue culture cells. Here, we report an analysis of the cellular localization of this protein in CV-1 monkey cells. Following cell lysis and fractionation in the absence of detergent about 2 5 % of the cellular content of HSP27 was recovered in the particulate fractions while the remaining of this protein was in the soluble cytoplasmic fraction. This association of HSP27 with particulate fractions was no more observed when cells were lysed in the presence of non-ionic detergent or when cells were pretreated with drugs, such as monensin and colcemid, that disrupt cytoskeletal architecture. Immunofluorescence analysis revealed that HSP27 is concentrated in a polarized perinuclear zone of CV-1 cells from where microtubules radiate. The particular locale of HSP27 was investigated in cells exposed to drugs or treatments, such as monensin, colcemid, cold stess and serum starvation, that disrupt the cellular architecture of microtubules. A correlation was observed between HSP27 cellular locale and microtubules integrity. Our results suggest a possible interaction of a fraction of HSP27 with cytoplasmic organelles or structures, different from the Golgi apparatus, whose distribution depends upon the organization of microtubules.

The Protective Effects of Various Stress Modalities on Ischemic / Reperfused Hearts of Rats

BACKGROUND: It has been found that sterss challenge with heat shock produces the acquisition of cellular resistance to ischemin injury in the hearts, which is associated with stress protein induction. The conventional heat shock(42degrees C of rectal temperature for 15min, anesthetized animal), however, is strong enough to endanger the animal life and then not suitable for practiocal application in human. The present study was performedd in an attempt to search the safely applicabel stress modalities to acquire the myocardial tolerance to ischemia-reperfusion in jury. METHODS: Male, Sprague-Ddawley rats(200-250g) were exposed to various stressful conditions, such as heat stimulation(environmental temperature of 42degrees C for 30min, live animal), swimming(20min), immobilization(60min), treadmill exercise(20M/min, 30min) and hyperbaric oxygenation(3atm, 60min) given once a day for 5 days. Twenty-four hours after the last application the hearts were isolated and perfused with oxygenated Krebs-Henseleit buffer solution by Langendorff method. Ischemia-reperfusion injury was produced by 20 min-global ischemia followed by 30 min-reperfusion. Cardiac mechanical function, lactate dehydrogenase release, the induction of stree proteins were assayed and compared dbetween the stressed dand the control animals. RESULTS: Upon reperfusion after ischemia the recovery of cardiac function was significantly improved in the stressed animals. The percentile recovery at 30min of reperfusion was in a range from 55.3%(swimming) to 89.3%(treadmill exercise), which was significantly higher than that of the control hearts(38%). The functional recovery of the conventional heat shocked heart was 57.7%. In stressed animals, lactate dehydrogenase release, which indicates myocardial cell injury, was significantly reduced by 20 to 30% compared to that for the control. The expression of an inducible form of 70 series stress protein, SP72, which was assayed by immunoblotting method, was markedly increased by heat stimulation while the other stress modalities failed to increase, it. There were no appreciable inductions of SP73(constitutive form) and GRR78 in the stressed animals. CONCLUSION: These results suggest that the cardiac protection from the ischemia-reperfusion injury could be induced by the repetitive non-fatal stress stimulations and that SP70 family proteins may be partly involved in the cardioprotective effect produced by heat stimulation, but not play the essential roles in anti-ischemic effects produced by other stress modalities.

The Effect of Dehydration after a Sauna and Intermittent Hypoxia Stimuli on Heat Stress Protein 70 in Human Leucocytes and Aerobic Capacity during Incremental Exercise / 中国运动医学杂志

The purpose of this study was to compare Heat Stress Protein70 (HSP70) in human leucocytes, VO2max and Blood Lactate (BLa) during incremental exercise under a thermoneutral condition without taking a sauna with those during the same workload and under same thermoneutral condition after a sauna and after intermittent hypoxia stimuli. Ten unacclimated men performed an incremental test to exhaustion on a treadmill under a thermoneutral condition without taking a sauna (N25℃, relative humidity 65%) and during the same workload and under same thermoneutral condition after a sauna (D25℃, relative humidity 65%), and after intermittent hypoxia stimuli (LN25℃, relative humidity 65%) . The results were as follows: (1) HSP70 levels before incremental exercise in D25℃ were significantly higher than that in N25℃(13701.87?5367.17vs 7517.57?1980.01,P