Role of PHB2 in mitochondrial calcium dynamics and mitophagy / 中国药理学与毒理学杂志

OBJECTIVE Mitochondria plays a crucial role in cellular homeostasis by regulating various pro-cesses,including calcium signaling and mitophagy.This study aimed to explore the involvement of prohibitin 2(PHB2),an inner mitochondrial membrane protein,in the modulation of mitochondrial calcium dynamics and mitoph-agy.METHODS HEK293T cells were used as the experi-mental cells and were divided into control,PHB2 knock-down,and PHB2 overexpression groups.To evaluate mitochondrial calcium dynamics,Rhod-2 AM and Mito-Tracker Green fluorescence dyesrhod-2 staining and laser confocal microscopy were employed to visualize mito-chondrial calcium imaging.Additionally,Green-5N was utilized to measure the rate of mitochondrial calcium uptake.The mitochondrial membrane potential was assessed using JC-10 staining and laser confocal micros-copy,while cellular ATP levels were determined using ATP assay kits.Furthermore,mitochondrial autophagy was induced by treatment with CCCP,and the expression lev-els of TOM20,LC3,and PARKIN,key mitophagy-related proteins,were analyzed using Western blotting.RESULTS The results demonstrated that compared to the control group,the overexpression of PHB2 increased mitochon-drial calcium concentration,mitochondrial calcium uptake rate,ATP level and expression levels of LC3 and PAR-KIN,but decreased mitochondrial membrane potential and TOM20 expression.In contrast,PHB2 knockdown reduced mitochondrial calcium concentration,ATP level and expression levels of LC3 and PARKIN,but elevated mitochondrial membrane potential,and TOM20 expres-sion.CONCLUSION This study provides evidence that PHB2 plays a vital role in regulating mitochondrial calci-um dynamics,which in turn influences mitochondrial func-tion and modulates mitochondrial autophagy.These find-ings contribute to our understanding of the molecular mechanisms underlying the interplay between PHB2,mitochondrial calcium signaling,and mitophagy.

Respiration in Azotobacter vinelandii and its relationship with the synthesis of biopolymers

Azotobacter vinelandii is a gram-negative soil bacterium that produces two biopolymers of biotechnological interest, alginate and poly(3-hydroxybutyrate), and it has been widely studied because of its capability to fix nitrogen even in the presence of oxygen. This bacterium is characterized by its high respiration rates, which are almost 10-fold higher than those of Escherichia coli and are a disadvantage for fermentation processes. On the other hand, several works have demonstrated that adequate control of the oxygen supply in A. vinelandii cultivations determines the yields and physicochemical characteristics of alginate and poly(3-hydroxybutyrate). Here, we summarize a review of the characteristics of A. vinelandii related to its respiration systems, as well as some of the most important findings on the oxygen consumption rates as a function of the cultivation parameters and biopolymer production.

Improvement of polyhydroxybutyrate production by deletion of csrA in Escherichia coli

BACKGROUND: Poly-3-hydroxybutyrate (PHB) can be efficiently produced in recombinant Escherichia coli by the overexpression of an operon (NphaCAB) encoding PHB synthetase. Strain improvement is considered to be one of critical factors to lower the production cost of PHB in recombinant system. In this study, one of key regulators that affect the cell growth and PHB content was confirmed and analyzed. RESULT: S17-3, a mutant E. coli strain derived from S17-1, was found to be able to achieve high cell density when expressing NphaCAB with the plasmid pBhya-CAB. Whole genome sequencing of S17-3 revealed genetic alternations on the upstream regions of csrA, encoding a global regulator cross-talking between stress response, catabolite repression and other metabolic activities. Deletion of csrA or expression of mutant csrA resulted in improved cell density and PHB content. CONCLUSION: The impact of gene deletion of csrA was determined, dysfunction of the regulators improved the cell density of recombinant E. coli and PHB production, however, the detail mechanism needs to be further clarified.

Changes of mitochondrial respiratory function and phb1 expression in rat skeletal muscle after moderate-intensity training / 中国组织工程研究

BACKGROUND: Studies have shown that long-term moderate-intensity regular exercise can Improve the activity of mitochondrial respiratory chain complex enzymes In skeletal muscle cells, thereby Improving their power capability as well as the ability to resist fatigue. OBJECTIVE: To explore the effect of moderate-intensity training on prohibitin (PHB) expression and mitochondrial respiratory function in rat skeletal muscle. METHODS: Thirty-two healthy male Sprague-Dawley rats were randomly divided into two groups: Quiet control group and moderate-intensity training group, each of 16 rats. In the moderate-intensity training group, the treadmill training was performed at a slope of 10°: In the 1st week, running at 10 m/min, 10 minutes per day, 6 days per week; in the 2nd week, running at 15 m/min, increased from 10 minutes per day to 60 minutes per day with an increase of 10 minute per day, 6 days per week; in the 3rd to 8th week, running at 15 m/min, 60 minutes per day, 6 days per week for a total of 8 weeks. Rats were sacrificed at 48 hours after the final experiment, and mitochondria were extracted from skeletal muscle samples. Mitochondrial respiration control rate, adenosine triphosphate content, reactive oxygen species level, complex V activity and PHB1 protein expression were measured. RESULTS AND CONCLUSION: Compared with the quiet control group, in the moderate-intensity training group, mitochondrial respiration control rate in the skeletal muscle increased significantly (P < 0.001), adenosine triphosphate content increased significantly (P < 0.05), reactive oxygen species level decreased significantly (P < 0.001), the activity of complex V was significantly increased (P < 0.05) and PHB1 expression was significantly increased (P < 0.01). The correlation analysis showed that after 8 weeks of moderate-intensity training, the expression of PHB1 in the skeletal muscle of rats was positively correlated with adenosine triphosphate content and complex V activity, and negatively correlated with reactive oxygen species level. Therefore, moderate-intensity training improves mitochondrial oxidative phosphorylation, effectively maintains mitochondrial membrane structure and enhances mitochondrial respiratory function by increasing the expression of PHB1.

Prohibitin (PHB) interacts with AKT in mitochondria to coordinately modulate sperm motility / 亚洲男科学杂志(英文版)

Prohibitin (PHB), an evolutionarily conserved mitochondrial inner membrane protein, is highly expressed in cells that require strong mitochondrial function. Recently, we demonstrated that the deletion of Phb in spermatocytes results in impaired mitochondrial function. In addition, PHB expression in the mitochondrial sheath of human sperm has a significantly negative correlation with mitochondrial reactive oxygen species levels, but a positive one with mitochondrial membrane potential and sperm motility. These results suggest that mitochondrial PHB expression plays a role in sperm motility. However, the mechanism of PHB-mediated regulation of sperm motility remains unknown. Here, we demonstrate for the first time that PHB interacts with protein kinase B (AKT) and exists in a complex with phospho-PHB (pT258) and phospho-AKT in the mitochondrial sheath of murine sperm, as determined using colocalization and coimmunoprecipitation assays. After blocking AKT activity using wortmannin (a phosphatidylinositol 3-kinase [PI3K] inhibitor), murine sperm have significantly ( P < 0.05) decreased levels of phospho-PHB (pT258) and the total and progressive motility. Furthermore, significantly ( P < 0.05) lower levels of phospho-PI3K P85 subunit α+γ (pY199 and pY467) and phospho-AKT (pS473; pT308) are found in sperm from infertile asthenospermic and oligoasthenospermic men compared with normospermic subjects, which suggest a reduced activity of the PI3K/AKT pathway in these infertile subjects. Importantly, these sperm from infertile subjects also have a significantly ( P < 0.05) lower level of phospho-PHB (pT258). Collectively, our findings suggest that the interaction of PHB with AKT in the mitochondrial sheath is critical for sperm motility, where PHB phosphorylation (pT258) level and PI3K/AKT activity are key regulatory factors.

Evaluation of short-chain-length polyhydroxyalkanoate accumulation in Bacillus aryabhattai

Abstract This study was focused on the polyhydroxybutyrate (PHB) accumulation property of Bacillus aryabhattai isolated from environment. Twenty-four polyhydroxyalkanoate (PHA) producers were screened out from sixty-two environmental bacterial isolates based on Sudan Black B colony staining. Based on their PHA accumulation property, six promising isolates were further screened out. The most productive isolate PHB10 was identified as B. aryabhattai PHB10. The polymer production maxima were 3.264 g/L, 2.181 g/L, 1.47 g/L, 1.742 g/L and 1.786 g/L in glucose, fructose, maltose, starch and glycerol respectively. The bacterial culture reached its stationary and declining phases at 18 h and 21 h respectively and indicated growth-associated PHB production. Nuclear Magnetic Resonance (NMR) spectra confirmed the material as PHB. The material has thermal stability between 30 and 140 °C, melting point at 170 °C and maximum thermal degradation at 287 °C. The molecular weight and poly dispersion index of the polymer were found as 199.7 kDa and 2.67 respectively. The bacterium B. aryabhattai accumulating PHB up to 75% of cell dry mass utilizing various carbon sources is a potential candidate for large scale production of bacterial polyhydroxybutyrate.

From waste to treasure: turning activated sludge into bioplastic poly-3-hydroxybutyrate / 生物工程学报

Large quantity of activated sludge is generated from wastewater treatment but without yet an appropriate deposition. High temperature can lyse the activate sludge so that nitrogen and phosphorus containing nutrients are released. Halomonas CJN was found to grow on the heat lysed activated sludge and glucose for production of bioplastic poly-3-hydroxybutyrate (PHB) in the absence of yeast extract, nitrogen and phosphorus sources as well as trace elements. This reduces the PHB production cost significantly. Furthermore, acetic acid formed from anaerobic fermentation of heat lysed activated sludge can be used to replace glucose for cell growth but not much for PHB production. After construction of an additional PHB synthesis pathway in Halomonas CJN, we can produce PHB entirely from heat lysed activated sludge, reducing production cost of PHB roughly from ¥ 30 000 Yuan/ton to ¥ 20 000 Yuan/ton, thus turning waste activated sludge to valuable raw material resource.

Study effects and mechanism of PHB on reactive oxygen species and apoptosis in cardiomyocytes under high glucose / 中国免疫学杂志

Objective:To investigate the effect and mechanism of PHB on the apoptosis of H 9C2 cardiomyocytes under high glucose.Methods:Select pCDNA3-PHB and pCDNA3-NC recombinant plasmid transfect into cardiomyocytes and effected by high glu-cose.The experiment were divided into four groups:control group,high glucose group(HG group),high glucose+empty vector group (HG+pCDNA3-NC group),high glucose+pCDNA3-PHB plasmid group(HG+pCDNA3-PHB group).The expression of PHB,Bax/Bcl-2,c-caspase3,AKt and p-AKt protein after transfected were detected by Western blot .ROS were detected under high glucose by DHE staining.The TNF-αmRNA and IL-6 mRNA were detected by Real-time quantitative PCR(qRT-PCR).Flow cytometry was used to detect the apoptotic rate .Results:The recombinant plasmid pCDNA 3-PHB could increase the expression of PHB .The apoptotic rate of HG group,HG+pCDNA3-NC group and HG+pCDNA3-PHB group were significantly higher than control group (P<0.05).Compared with HG group,the apoptotic rate of HG+pCDNA3-NC group was not significant(P>0.05),but that of HG+pCDNA3-PHB group was significantly lower(P<0.05).The levels of ROS,TNF-αmRNA,IL-6 mRNA,Bax/Bcl-2 and c-caspase3(P<0.05) protein in the HG group,HG+pCDNA3-NC group,HG+pCDNA3-PHB group were significantly higher than the control group .Compared with HG group , the level of ROS,TNF-αmRNA,IL-6 mRNA,Bax/Bcl-2 and c-caspase3 (P>0.05)protein in the HG+pCDNA3-NC group were no significant difference,but the ROS,TNF-αmRNA,IL-6 mRNA、Bax/Bcl-2 and c-caspase3 (P<0.05)protein in the HG+pCDNA3-PHB group were significantly decreased .Statistical analysis showed that there was no significant difference of AKt protein in each group (P>0.05).The relative expression of p-AKt protein in HG group,HG+pCDNA3-NC group and HG+pCDNA3-PHB group were signif-icantly lower than that in control group ( P<0.05 ) ,HG+pCDNA3-NC group was no significantly different than HG group ( P>0.05 ) , However ,HG+pCDNA3-PHB group was significantly higher than HG group ( P<0.05 ) .Conclusion: PHB overexpression can inhibit the apoptosis of H9C2 cardiomyocytes in high glucose environment ,andreduce the inflammatory response by effected the regulation of PI3K/Akt signaling pathway,reduced ROS,Bax/Bcl-2,c-caspase3 protein levels,that give us a new ideas and methods of DCM .

Production and characterization of polyhydroxybutyrate (PHB) produced by Bacillus sp. isolated from Egypt.

Synthetic polymers are non-degradable and accumulated in the environment so, the efforts of scientists were forwarded to provide us with alternative environmentally biopolymers. Polyhydroxyalkanoates (PHAs) including polyhydroxybutyrate (PHB) are Group of the interesting biopolymers which have several medical applications such as drug delivery, suture, scaffold and heart valves. PHAs are biological macromolecules, thermoplastics, biodegradable and biocompatible. In this study, new bacterial isolates from Egypt were screened for their ability to produce PHB using Nile red dye. Out of 44 isolates, 19 bacterial isolates were selected according to strong of their fluorescence on mineral salt medium (MSM) agar plates supplemented with Nile red. The most potent strain was identified using biochemical tests as Bacillus sp. N-2. Production of PHB was carried out in limitation of nitrogen source using a minimal salt medium (MSM) supplemented with an excess of glucose as sole carbon source. PHB was accumulated in relation to cell dry weight about 20% (PHB/CDW). The obtained biopolymer was purified and analyzed using NMR, FT-IR, TGA and DSC thus; it was highly pure and identified as PHB. Optimization of PHB production from cheap sources appears to be a realistic goal in the future for reducing the costs and obtaining high yield.

Extraction and Characterization of Polyhydroxybutyrates (PHB) from Bacillus thuringiensisKSADL127 Isolated from Mangrove Environments of Saudi Arabia

ABSTRACTPolyhydroxybutyrate (PHB) is a renowned biodegradable plastic that do not release any toxins or residues in the environment like petroleum based plastics. In the present study, 50 bacteria isolated from mangrove niche, Saudi Arabia, were screened for maximum PHB production. All the 50 strains showed positive for PHB production, of which one strain showed maximum of 137 mgL-1. The most PHB accumulated bacterium was selected and identified asBacillus thuringiensis KSADL127, based on phenotypic characterization and 16S rRNA sequence analysis. Characterization of extracted PHB was carried out by FT-IR, NMR, UV spectroscopy, DSC, TGA, and LC-MS, which later confirmed the presence of intracellular accumulated polymer and substantiated as PHB.

Isolation and optimization of poly β hydroxybutyrate producing cyanobacterial strains.

Cyanobacteria have many unexploited potential for natural products with a huge variability in structure and biological activity. Under stress conditions they are reported to produce biopolymers like poly-β-hydroxybutyrate (PHB), which can be produced intracellularly.Cyanobacteria are capable of synthesizing small amount of poly-β-hydroxybutyrate (PHB) under nitrogen and phosphorous starvation conditions. High performance liquid chromatography (HPLC) analysis revealed that about percentage PHB of the cell dry weight (CDW) was accumulated under mixotrophic culture condition. However, Nile red stained cells showed the presence of large quantities of granules in the cell cytoplasm when viewed under fluorescent microscope. The qualitative observation was in contrast to the quantitative HPLC analysis which suggested that the fluorescent granules are PHB. Among the ten different isolates, three strains showed the accumulation of PHB. The amount of PHB produced after 10 days in the depleted conditions are 1.182 mg/l for spirulina, 2.322 mg/l for anabena and 3.741 mg/l. The maximum PHB producer was further studied in detail. The extracted polymer was compared with the authentic PHB and was confirmed to be PHB using FTIR analysis. The present study shows increased PHB accumulation in different species by nitrogen and phosphorous depleted condition and pH concentration in the growth media.

The Mutagenic Effect on PHB Accumulation of Acidiphilium cryptum DX1-1 / 微生物学通报

The strain Acidiphilium cryptum DX1-1 producing PHB was irradiated respectively by UV and Co60 to raise PHB production. The results indicated that the effect of UV better than using Co60. One strain of the UV mutagenized called UV60-3 has the highest PHB production yield, showing final PHB concentra- tion of 28.56 g/L, 1.45 times higher than that of original strain. FT-IR spectroscopy analysis shows that the polymers obtained from the strain DX1-1 have the same IR spectra of standard PHB. Further research about the best appropriate C/N ratio of the mutant was done. The optimum ratio of C/N was about 3.76, the final PHB concentration reaches to 30.57 g/L.

System of Intein-mediated PHB Purified Human Antimicrobial Peptide LL-37 / 中国生物工程杂志

The new intein-mediated PHB purify protein system is a high expression, automatic cutting, for purification, low-cost protein purification system,it is conducive to large-scale protein purification.Choose human antibacterial peptide LL-37 as the purification objects,which is poison to prokaryotic cell.We construct intein-mediated PHB purified human antimicrobial peptide LL-37 system through genetic engineering technology and use this system to purify LL-37. The results show that this system can highly express LL-37 fusion protein and purifiy the product as same size with expectations.

Polyhydroxybutyrate Synthesis in Recombinant Zymonomas mobilis Affected Ethanol Production / 中国生物工程杂志

Zymonomas mobilis was transformed with a polyhydroxybutyrate synthesis operon phbCAB equipped with a pdc promoter from Z. mobilis. For the first time,PHB was produced in recombinant Z.mobilis. Shake flask studies indicated that accumulation of PHB in Zymomonas mobilis increased approximately 10% ethanol productivity for the first 48h of anaerobic fermentation. After that,the PHB effect was observed as insignificant probably due to the exhaustion of the sugar.

Application of PHB in transplantation of adrenocortical cells among allorats / 中华器官移植杂志

Objective To investigate the effect of PHB on the proliferation and secretion function of adrenocortical cells and the efficiency of PHB as a cell carrier in transplantation of adrneocortical cells. Methods Adrenocortical cells from rat adrenal gland were separated and cultured in vitro. The effect of PHB on the proliferation and secrete function of adrenocortical cells were evaluated by MTT and RIA method. Adrenocortical cells were seeded into PHB. After cells were cultured in vitro for about 7 days, they were implanted into rats subject to bilateral adrenalectomy. The changes of blood corticosterone and aldosterone and local histological changes were observed. Results Adrenocortical cells could grow and passage on PHB. PHB had no effect on the proliferation and secretion function of adrenocortical cells. Adrenocortical cells from autograft, allograft and xenograft greatly suppressed the proliferation of spleen cells (P

STUDY ON THE POLY-?-HYDROXYBUTYRIC ACID PRODUCING STRAIN NS-82# / 微生物学通报

Poly-?-hydroxybutyric acid (PHB) produ c ing strain NS-82# which was isolated from the soil was identified to be Bacillus sp.The purified sample from fermentation was similar to t he standard sample produced by Aldric Chemical Company Inc after determinated wi th ultraviolet absorption,IR-absorption,gas chromatographiyc and nuclea r magnetic resonance analysis of polyesters.

Study of the PHB/PEO polymer combined with nerve growth factorin sciatic nerve injury / 重庆医科大学学报

Objective:To investigate the effect of PHB/PEO polymer combined with nerve growth factor in the peripheral nerve injury.Method:The male SD rats were treated with autogentic neural transplantation,xenogenic neural transplantation under the immunosuppression,transplantation of PHB/PEO polymer combined with nerve growth factor and PHB/PEO guidance transplantation,respectively.The nerve function and the nerve anatomic structure were evaluated by the sciatic function index(SFI),nerval electrophysiology test,nerve fiber number and electron microscope.Result:The SFI,nerve conduction velocity,nerve fiber number and the regeneration of nerve fiber and myeline sheath of the PHB/PEO polymer combined with nerve growth factor group is similar to the autogentic neural transplantation group,and is better than the xenogenic neural transplantation group and PHB/PEO guidance group significantly.Conclusion:The effect of PHB/PEO polymer combined with nerve growth factor is similar to the autogentic neural transplantation,and is better than the xenogenic neural transplantation and PHB/PEO guidance significantly in the repair of sciatic nerve injury.