Prokaryotic expression and functional analysis of molecular chaperone Acr2 protein of Mycobacterium tuberculosis / 中国生物制品学杂志

@#Objective To express the molecular chaperone Acr2 protein of Mycobacterium tuberculosis(Mtb)in E.coli and analyze the function. Methods The recombinant plasmid pET-28a-Acr2 was transformed into competent E. coli BL21(DE3),and induced by IPTG. The expressed His-Acr2 protein was purified by Ni-NTA chromatography and SuperdexTM200 10/300 GL gel filtration chromatography to obtain Acr2 protein. The Acr2 protein was refolded by spontaneous refolding and reassembly after thermal denaturation(100 ℃ for 15 min)and chemical denaturation(8 mol/L urea,37 ℃ for 4 h).The secondary structure of Acr2 protein before and after denaturation-renaturation was detected by circular dichroism spectroscopy and non-denaturing SDS-PAGE,and the molecular chaperone function of Acr2 protein in vitro was detected by substrate binding assay. Results The purified Acr2 protein had the relative molecular mass of about 232 000,the purity of over 90%,and the concentration of about 2 mg/mL,which recovered its natural secondary structure after denaturationrenaturation,and formed stable complexes with the denatured malate dehydrogenase(MDH)at 48 ℃. Conclusion The Acr2protein can restore its natural molecular conformation with molecular chaperone activity in vitro after denaturation-renaturation treatment,providing a new strategy for the preparation of Mtb protein antigen with natural activity.

Design of small molecules targeting molecular chaperone system: review and perspective / 药学学报

Molecular chaperone system, which mainly consist of heat shock proteins family and their cochaperones, is crucial for maintaining proteostasis in life. It assists in folding, maturation and ubiquitin-proteasome-mediated degradation of proteins, thus to play a key role in cell proliferation and apoptosis. Functional disorder of molecular chaperone system is highly relevant to occurrence and development of multiple diseases including cancers, autoimmune disease/inflammatory, infective diseases, neurodegenerative disease, etc. Therefore, molecular chaperone system has long been regarded as potential drug targets. In this review, we outline the progress in the design of small molecules targeting molecular chaperone system and analyze the features of small molecules with different mechanisms. Finally, we put forward expects about potential development directions for future drug design in this field.

Effect of Modified Shengjiangsan on Gd-IgA1 in IgA Nephropathy Rats： Based on C1GALT1/C1GALT1C1 Signaling Pathway / 中国实验方剂学杂志

ObjectiveThe effect of modified Shengjiangsan on immunoglobulin A （IgA） nephropathy was observed. The microRNA-148b （miRNA-148b）， interleukin 6 （IL-6）， core 1 beta 1，3-galactosyltransferase （C1GALT1）， molecular chaperone Cosmc （core1β3-Gal-T-specific molecular chaperone C1GALT1C1）， and galactose-deficient IgA1 （Gd-IGA1） in serum and kidney tissues of IgA nephropathy rats were detected to explore the underlying mechanism. The result is expected to lay a scientific basis for clinical application of modified Shengjiangsan in the treatment of IgA nephropathy. MethodA total of 42 SPF male SD rats were randomized into the normal group （8rats） and modeling group （34 rats） with the random number table method. After one week of adaptive feeding， rats for modeling were given bovine serum albumin （BSA， gavage）， lipopolysaccharide （LPS， injection into tail vein）， carbon tetrachloride （CCl4， subcutaneous injection）， and castor oil to induce IgA nephropathy. After modeling， two rats were randomly selected to test the modeling outcome. Then the model rats were classified into the model group， low-dose Chinese medicine group （modified Shengjiangsan，6.27 g·kg-1）， high-dose Chinese medicine group （modified Shengjiangsan，12.54 g·kg-1）， and benazepril group （10 mg·kg-1） with the random number table method， 8 in each group. The administration （gavage， once a day） lasted 4 weeks. The 24-h urinary total protein （24 h-UTP） was detected at the end of the 1st， 9th， and 13th week of the experiment. At the 14th week， after anesthesia， femoral artery blood was collected and centrifugated. The supernatant was collected to detect albumin （ALB）， aspartate aminotransferase （AST）， alanine aminotransferase （ALT）， serum creatinine （SCr）， and blood urea nitrogen （BUN）. The expression levels of IL-6 and Gd-IGA1 were determined by enzyme-linked immunosorbent assay （ELISA）. Based on hematoxylin-eosin （HE）/Masson/periodic Schiff-methenamine silver （PASM） staining， the pathological changes of renal tissues were observed. Ultrastructural changes of glomeruli were observed by transmission electron microscopy. The expression of miRNA-148b， IL-6， C1GALT1， and C1GALT1C1 was detected by immunohistochemistry. The mesangial area of the glomeruli was observed by immunofluorescence. Real-time polymerase chain reaction （Real-time PCR） was employed to determine the mRNA levels of mirNA-148b， IL-6， C1GALT1， and C1GALT1C1， and Western blot was used to detect the protein levels of IL-6， C1GALT1， and C1GALT1C1. ResultCompared with normal group， the model group showed increase in the content of 24 h-UTP， SCr， ALT， IL-6， and GD-IGA1 （P<0.05）， decrease in ALB content （P<0.05）. Moreover， rats in the model group demonstrated hyperplasia of glomerular mesangial cells， thickening of mesangial area， podocyte foot process effacement， and a large number of granular IgA immune complex in the mesangial area. In addition， the model group showed increase in the expression of IL-6 in mesangial area and podocytes， decrease in the expression of C1GALT1 and C1GALT1C1 in mesangial area and podocytes， enhanced expression of IL-6 mRNA and miRNA-148b （P<0.01）， weakened expression of C1GALT1 mRNA and C1GALT1C1 mRNA （P<0.01）， rise of IL-6 protein expression （P<0.01）， and reduction in the protein expression of C1GALT1 and C1GALT1C1 （P<0.01）. Compared with the model group， modified Shengjiangsan decreased the content of 24 h-UTP， SCr， ALT， IL-6， and Gd-IGA1 （P<0.05） and increased the content of ALB （P<0.05， P<0.01）. Moreover， with the treatment of this Chinese medicine， the pathological damage was significantly alleviated and the deposition of IgA immune complex in basement membrane was reduced. The expression of IL-6 in the mesangial area and podocytes of rats was decreased， and the expression of C1GALT1 and C1GALT1C1 in the mesangial area and podocytes of rats was increased. Moreover， the expression of IL-6 mRNA and miRNA-148b was decreased （P<0.01）， and the expression of C1GALT1 mRNA and C1GALT1C1 mRNA was increased （P<0.01）. The protein expression of IL-6 was decreased （P<0.05， P<0.01）， and the protein expression of C1GALT1 and C1GALT1C1 was enhanced （P<0.05， P<0.01）. The Chinese medicine group showed obvious dose-effect trend. ConclusionModified Shengjiangsan may reduce the expression of miRNA-148b and IL-6 in serum and kidney tissue of IgA nephropathy rats， restore the expression of C1GALT1 and C1GALT1C1， and decrease the generation of Gd-IGA1， so as to reduce renal pathological damage and proteinuria， protect the kidney protection， and finally delay the disease progression. Moreover， the effect is enhanced with the rise of dose.

Changes in molecular chaperone-mediated autophagy expression of developmental rats after recurrent-status seizures / 中华实用儿科临床杂志

Objective:To observe the expression of molecular chaperone-mediated autophagy in hippocampal neurons and its relationship with brain injury after recurrent-status seizures.Methods:Seven-day-old SD rats were divided into two groups according to simple randomization: the control group (NS group, 6 rats) and the recurrent-seizure group (RS group, 39 rats). Rats in the RS group were subjected to recurrent seizures after repeated inhalation of flurothyl, with 30 minutes once each day for consecutive 7 days.A total of 30 convulsive models were successfully established (9 rats that failed to establish models were discarded), and they were further divided into 0 h, 1.5 h, 3 h, 12 h and 24 h after the last seizure according to simple randomization, with 6 rats in each group.Western blot and reverse transcription-polymerase chain reaction (RT-PCR) were adopted for the observation of the expression of molecular chaperone-mediated autophagy markers [heat shock cognate protein 70 (Hsc70), lysosome-associated membrane protein type 2a (LAMP-2a), heat shock protein 40(HSP40) and heat shock protein 90(HSP90)] in hip-pocampal neurons, and apoptosis was detected by TdT-mediated dUTP nick-end labeling (TUNEL).Results:(1) RT-PCR and Western blot showed that, compared with the NS group, the expression of Hsc70, as a molecular chape-rone, started to increase at 1.5 h and continued until 24 h after the last seizure in the RS group ( P<0.05). HSP90 increased immediately after the last seizure and lasted until 24 h after the seizure ( P<0.01); the expression of HSP40 and LAMP-2a also showed high expression after the last seizure episode ( P<0.05). (2) The TUNEL method showed that the number of apoptotic cells in the hippocampal CA1 region increased significantly at 3 h (36.33±5.16)/40 field, 12 h (44.83±4.83)/40 field and 24 h (54.83±7.16)/40 field after the last seizure compared with NS group(15.16±2.48)/40 field ( P<0.01). (3) Pearson correlation analysis showed that the level of apoptosis in hippocampal CA1 region of rats after recurrent seizures was positively correlated with the expression of molecular chaperone marker molecules (Hsc70: r=0.734, P=0.001; LAMP2a: r=0.790, P<0.001). Conclusions:After recurrent seizures in developmental rats, the presence of increased expression of multiple molecular chaperone-mediated autophagy, which may positively correlate with apoptosis, may be involved in the process of brain injury.

Molecular chaperones facilitate soluble expression of recombinant non-toxic mutant CRM197 of diphtheria toxin in Escherichia coli / 生物工程学报

Diphtheria toxin is an ADP-ribosyltransferase toxic to human cells. Mutation of the active site in its catalytic domain eliminates the toxicity, but retains its immunogenicity. A non-toxic mutant of diphtheria toxin known as CRM197 protein has become an ideal carrier protein for conjugate vaccines. CRM197 can further improve its immunogenicity by cross-linking with other antigens, so it has good potential to find broad applications. Unfortunately, inclusion bodies are easily formed during the expression of recombinant CRM197 protein in Escherichia coli, which greatly reduces its yield. In order to address this problem, pG-KJE8 vector carrying molecular chaperones and plasmid pET28a-CRM197, were co-expressed in Escherichia coli. The results showed that the recombinant CRM197 protein was successfully expressed and appeared largely in inclusion bodies. The molecular chaperones DnaK, DnaJ, GrpE, GroES and GroEL5 expressed can facilitate correct and rapid folding of CRM197. Furthermore, it can also improve the recovery rate of soluble CRM197 protein. The soluble expression of CRM197 was maximized upon addition of 1.0 mmol/L IPTG, 0.5 mg L-arabinose, 5.0 ng/mL tetracycline and induction at 20oC for 16 h. The soluble CRM197 protein shows good immunoreactivity, demonstrating the molecular chaperones expressed from pG-KJE8 facilitated the soluble expression of CRM197 protein in E. coli.

Advances in research progress on acid tolerance mechanism of Gram- negative bacteria mediated by molecular chaperone protein / 中国药科大学学报

@#The tolerance of microorganism under acid stress is of significant importance to the growth and industrial production of bacterial strain. When the bacterial cells are exposed to the external acid environment, proteins in periplasmic space are under higher acid stress and are thusmore susceptible to severe damage from acid than the intracellular protein, and these are also more susceptible to severe damage from acid than the intracellular protein. During the acid-resisting process of Gram-negative bacteria, in addition to the intracellular decarboxylase system, the molecular chaperone can also participate in the identification and protection of the space structure of protein as an important "correcting" mechanism. This paper, reviews the current researches on the function, structure, acid-resisting mechanism of variousmolecular chaperones, including HdeA, HdeB, DnaK and GroEL. Finally, the research progress of the acid-resisting mechanism among Gram-negative bacteria is summarized. Through in-depth investigation and analysis of the physiological adaptation strategy of molecular chaperone to the acid stress environment, this study can help to conduct physiological property modification of target strain and improve their viability and tolerance in acid stress environment, which has its important theoretical and practical significance.

Research progress of the relationship between heat shock protein 27 and related intestinal diseases / 解放军医学杂志

Heat shock proteins (HSP) are highly conserved proteins that protect cells and enhance the body's resistance to stress. Heat shock protein 27 (HSP27) also known as heat shock protein beta-1 (HSPB1) is a small molecular weight HSP that in humans is encoded by the HSPB1 gene. HSP27 exerts its effect mainly in the form of phosphorylation activity, and has a variety of important biological functions, such as chaperone activity, thermotolerance, anti-oxidation and inhibition of apoptosis. HSP27 was found to be highly expressed in a variety of intestinal diseases. This article reviews the structure, function, mechanism of action of HSP27 and the relationship between HSP27 and intestinal diseases, and explores the clinical value and application prospect of HSP27 in intestinal diseases.

Progress in Molecular Chaperone Regulation of Heat Shock Protein 90 and Cancer / 分析化学

Heat shock protein 90 (HSP90) is a member of genetically conserved heat shock protein family. As an important molecular chaperone in eukaryotic cells, HSP90 plays a key regulatory role in maintaining cellular protein homeostasis. HSP90 clients encompass a wide range of proteins, thus HSP90 is involved in diverse biological process. With the deeper study, it is found that HSP90 takes an important part in the development and metastasis of cancer,and has become a promising target for the study of anticancer biology. We review the progress of HSP90 as molecular chaperone and its relationship with cancer.

Heat Shock Protein Hspa5 Interacts with and Protects Tyrosinase Activity

In this study, heat shock protein, Hspa5 was cloned, expressed and purified subsequently confirmed that it interacted with the tyrosinase (TYR) in vitro. Then, using the crystal structure of the homologous protein from the bacteria as a template, a homology model of human TYR was constructed. This model was further applied to investigate the molecular docking with Hspa5. The model showed that the interaction between the TYR and Hspa5 was mainly maintained by some hydrogen bonds in a quite low energy state. The results indicated that TYR was protected in different denaturation conditions by Hspa5. It was concluded that Hspa5 served as a molecular chaperone of TYR, which could help to better understand the molecule regulation mechanism of TRY in many kinds of diseases.

GRP78 Protein Expression as Prognostic Values in Neoadjuvant Chemoradiotherapy and Laparoscopic Surgery for Locally Advanced Rectal Cancer / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: We investigated the relationships between biomarkers related to endoplasmic reticulum stress proteins (glucose-regulated protein of molecular mass 78 [GRP78] and Cripto-1 [teratocarcinoma-derived growth factor 1 protein]), pathologic response, and prognosis in locally advanced rectal cancer. MATERIALS AND METHODS: All clinical stage II and III rectal cancer patients received 50.4 Gy over 5.5 weeks, plus 5-fluorouracil (400 mg/m2/day) and leucovorin (20 mg/m2/day) bolus on days 1 to 5 and 29 to 33, and surgery was performed at 7 to 10 weeks after completion of all therapies. Expression of GRP78 and Cripto-1 proteins was determined by immunohistochemistry and was assessed in 101 patients with rectal cancer treated with neoadjuvant chemoradiotherapy (CRT). RESULTS: High expression of GRP78 and Cripto-1 proteins was observed in 86 patients (85.1%) and 49 patients (48.5%), respectively. Low expression of GRP78 protein was associated with a significantly high rate of down staging (80.0% vs. 52.3%, respectively; p=0.046) and a significantly low rate of recurrence (0% vs. 33.7%, respectively; p=0.008) compared with high expression of GRP78 protein. Mean recurrence-free survival according to GRP78 expression could not be estimated because the low expression group did not develop recurrence events but showed a significant correlation with time to recurrence, based on the log rank method (p=0.007). GRP78 also showed correlation with overall survival, based on the log rank method (p=0.045). CONCLUSION: GRP78 expression is a predictive and prognostic factor for down staging, recurrence, and survival in rectal cancer patients treated with 5-fluorouracil and leucovorin neoadjuvant CRT.

Therapeutic Approaches for Inhibition of Protein Aggregation in Huntington's Disease

Huntington's disease (HD) is a late-onset and progressive neurodegenerative disorder that is caused by aggregation of mutant huntingtin protein which contains expanded-polyglutamine. The molecular chaperones modulate the aggregation in early stage and known for the most potent protector of neurodegeneration in animal models of HD. Over the past decades, a number of studies have demonstrated molecular chaperones alleviate the pathogenic symptoms by polyQ-mediated toxicity. Moreover, chaperone-inducible drugs and anti-aggregation drugs have beneficial effects on symptoms of disease. Here, we focus on the function of molecular chaperone in animal models of HD, and review the recent therapeutic approaches to modulate expression and turn-over of molecular chaperone and to develop anti-aggregation drugs.

Ischemic preconditioning in the protection of global cerebral ischemia in rats by reducing endoplasmic reticulum stress / 国际脑血管病杂志

Objective To investigate the effect of inhibition of endoplasmic reticulum stress (ERS) in ischemic preconditioning-induced cerebral ischemia tolerance.Methods A total of 120 adult male SpragueDawley rats were randomly allocated into three groups:sham operation,global cerebral ischemic and ischemic preconditioning groups (ischemic preconditioning for 3 minutes,and global cerebral ischemia for 15 minutes after 2 days).Three time points (day 1,day 3 and day 7) were set.Sugawara method was used to observe the changes of neurological behavior in rats.TUNEL staining was used to observe the conditions of cortical neuronal apoptosis.Immunofluorescence staining and Western blot analysis were used to detect the expression levels of ERS-related protein CHOP,GRP78,and caspase-12.Results The neurological behavior score showed that the sham operation group did not have neurological deficits.Both the global cerebral ischemic group and the ischemic preconditioning group had obvious neurological deficits,and they improved gradually with the passage of time,but after modeling,the neurological scores at each time point in the global cerebral ischemic were significantly lower than those in the ischemic preconditioning group:at day 1∶11.00 ±0.63 vs.14.33 ±0.33 (t =21.74,P＝0.001); at day 3∶ 12.17±0.31vs.15.17±0.48 (t=27.93,P =0.000); at day 7:14.67±0.49 vs.16.33 ±0.33 (t =7.81,P=0.020).TUNEL staining showed that at day 7 after ischemia,the positive cell count per mm2 in the sham operation,global cerebral ischemic and ischemic preconditioning groups were 4.83 ±1.85vs.395.67± 43.43 and 146.17± 27.38 respectively (F=23.62,P=0.001).The ischemic preconditioning group was significantly lower than that in the global cerebral ischemic group (P =0.001).Immunofluorescence staining showed that at day 7 after ischemia,the numbers of positive cells of CHOP (26.50±3.89vs.82.33±4.25; P=0.000),GRP78 (15.00±2.02vs.35.67±2.99; t=0.000),and caspase-12 (22.33 ± 2.76 vs.66.50± 7.25; P=0.000) in the ischemic preconditioning group were significantly less than those in the global cerebral ischemic group.Western blotting showed that at day 7 after ischemia,the expression levels of CHOP (1.22 ± 0.38 vs.3.22 ± 0.51; t =24.50,P =0.001),GRP78 (1.78 ± 0.45 vs.3.16 ± 0.76; t =14.29,P =0.005),and caspase-12 (2.89 ± 0.53 vs.5.96 ± 0.67; t =77.73; P =0.000) in the ischemic preconditioning group were significantly lower than those in the global cerebral ischemic group.Conclusions Ischemic preconditioning demonstrated a neuroprotective effect for the second lethal ischemia,its mechanism may be associated with the relief of ERS and downregulation of ERS-related protein.

Endoplasmic reticulum molecular chaperone involved in the impairment of inner ear consistented with the mimetic aging rats / 临床耳鼻咽喉头颈外科杂志

Objective:To explore the involvement of endoplasmic reticulum molecular chaperone GRP78 in the impairment of inner ear consistented with the mimetic aging model.Method:Twenty-four Wistar rats were randomly divided into two groups. model group was in duced by daily hypodermic injection of 10% D-galactose (800 mg·kg~(-1)·d~(-1)) for 8 weeks and the control group was given saline accordingly. Spatial learning and memory was measured by Morris-Water-Maze. Colorimetry was used to analyze superoxide dismutase (SOD) and malondialdehyde (MDA) extracted from inner ear tissue. Hearing threshold of rats were detected with Auditory brainstem response (ABR).In addition, expression of GRP78 in the inner ear was detected by immunohistochemistry,RT-PCR and Western blot. The control group was studied parallel.Result:The escape latency in the model group injected with D-galactose was markedly longer than that in the control group.accordingly ,the changes of SOD and MDA were more significant in the model group, the difference between two groups was significant(t-test,P0.05). The expression of GRP78 was significantly different between two groups ,which is increased in the inner ear tissue of model group(P<0.01).Conclusion:The impairment of inner ear tissue partly dued to the oxidative stress in the model, which was induced by D-galactose.and endoplasmic reticulum molecular chaperone was thought to contribute to the impairment mechanism of inner ear in mimetic aging model.

The protective effects of aspirin on the α-crystalline molecular chaperone-like activity in naphthalene-induced cataract / 眼科研究

Background Age-related cataract is the leading cause of visual impairment worldwide.To seek the effective prevention and drugs for management of cataract is important.Naphthalene-induced cataract of rat is an ideal animal model for the research of human age-related cataract,and aspirin has been proven to inhibit the development of human age-related cataract.ObjectiveThe present study is to investigate the role of aspirin on naphthalene-induced cataract.Methods Forty-five 150-160 g female SD rats were divided into three groups randomly.Naphthalene was orally taken with 0.5mg/kg per day for 3 days and then 1mg/kg per day for 70 days,and then 100mg/kg of aspirin was given per day for 70 days following four-day washout period in group A.In group B,the animals was given orally only naphthalene at the same way.No any intervention was used in group C.Naphthalene-induced cataract was examined under the slim lamp every week.The experimental animals were sacrificed and lenses were obtained in 70 days.α-Crystalline was extracted from lens homogenate and purified and identified using High Performance Liquid Chromatography(HPLC),2-dimentional electrophoresis gel and Western blot.Different abilities of α-crystalline to protect β low crystalline from aggregation were observed using ultraviolet spectrophotometer.Results Naphthalene-induced cataract formed at the third week in only naphthalene group but at the sixth week in naphthalene+aspirin group under the slim lamp.No significant difference was found in the degree of lenses opacity in the second week among these three groups(F=0.032,P=0.969).However,a statistically significant difference was seen in the degree of lenses opacity in the fourth,sixth,eighth and tenth week among these three groups(F= 5031.130,P=0.000;F=115964.000,P=0.000;F=169846.500,P=0.000;F= 195431.200,P=0.000).Themolecular chaperone-like activity was significantly higher than that of the naphthalene-induced group.Conclusion Aspirin delays the progression of lens opacification through protecting α-crystalline molecular chaperone-like activity.

Influencing factors in the refolding process of artificial molecular chaperone assisting chicken IL-18 recombination protein / 中国兽医学报

The recombinant plasmid of mChlL-18 prokaryotic expression was transformed into E.coli BL21(DE3) strain and then induced by IPTG at 37℃.After crushed and washed,the expressing inclusion bodies were thoroughly denatured with 6 mol/L guanidine hydrochloride.Then according to experiment design,the effects of rChlL-18 protein refolding yield at different densities were investigated by the systems of artificial chapercne at different densities.Experiment results indicate that there is a optimal condition on assiting rChIL-18 protein by using the artificial chaperone technique.The optimal condition can improve the refolding yield of rChIL-18 protein,and then the expressed product of fusion chicken IL-18 gene in E.coli has a relativity high bioactivity.

Expression of αB-crystallin in X-ray irradiated rat lenses / 中华放射医学与防护杂志

Objective To detect the changes of soluble αB-crystallin in X-ray irradiated rat lens,and to explore the potential role of αB-crystallin in the pathogenesis of radiation-induced cataract.Methods The radiation cataract model was established with the accelerator linear in male Sprague-Dawley(SD)rats.The rats were divided into normal control group,experimental control group and X-irradiated group(the doses were 5,15,and 25 Gy,respectively).The rats were killed at 3 morlths post-irradiation,and the lenses were carefully isolated and homogenized.The protein changes of αB-crystallin in lens supernatant were measured by Western blotting.Results Typical radiation-induced cataract was observed in 15 and 25 Gy groups,while the lenses of the normal control group,experimental control group and the 5 Gy irradiation group remained transparent.The αB-crystallin protein level was significandy decreased in a dose-dependent manner(5 Gy group:0.871±0.085;15 Gy group:0.643±0.096;25 Gy group:0.338±0.160;F=40.764,P＜0.05).Conclusions The decreased expression of the molecular chaperone αB-crystallin in X-irradiated rat lens indicates its important role in the pathogenesis of irradiation cataract.

Hyperthermic Cancer Therapy and Its Mechanism / 日本補完代替医療学会誌

We have reported that heat as well as X-rays induced <i>p53</i>-centred signal transduction. The p53 molecule determines the fate of cells, especially apoptosis. Wild-type (wt) <i>p53 </i>cells are resistant to heat as compared with the mutated-type (m) <i>p53 </i>cells. Apoptosis is efficiently induced in the wt<i>p53</i> cells by heat through the activation of Bax and Caspase-3, not but m<i>p53</i> cells. Therefore, we proposed that wt<i>p53</i> patients would be more suitable for hyperthermic therapy than m<i>p53</i> patients. To enhance apoptosis in m<i>p53</i> cells, however, we succeeded the establishing new cancer therapies against m<i>p53</i> cells using chemical chaperon therapy with glycerol and peptide therapy with p53 C-terminal peptide. In addition, we applied siRNA or gene therapy with <i>p53</i>-targeted genes to m<i>p53</i> and <i>p53</i>-deficient cells. It is our hope to show that these new therapies prove more effective as cancer therapies as soon as possible.<br>

Biochemical and Enzymatic Characterization of a Thermostable DNA Ligase Encoded by Thermophilic Acidophilic Archaebacterium Strain JP2 / 生物化学与生物物理进展

A thermostable DNA ligase gene was identified, and the biochemical and enzymatic properties of the ligase were characterized from JP2 strain which was enriched from geothermally active sites in Papua New Guinea. The nucleotide and amino acid sequences showed much high identities compared with that of archeabacterium species Sulfolobus solfataricus and Sulfolobus shibatae,especially in the six conserved motif sequences, which are known to be closely related to the key function of ligase. Recombinant JP2 ligase showed high activity in nick-joining reaction. It was the most active when Mn2+ present as divalent metal cofactor rather than Mg2+ and Ca2+ etc.. Assay of thermostability over a range of temperatures showed that at 50～80℃ the enzyme displayed relative high activity. Further thermostability experiment indicated that the activity of JP2 ligase could last for a long time at 80℃ and 85℃,however, at 90℃ and 95℃, it became unstable quickly. An investigation on the acquired thermotolerance of recombinant JP2 ligase was done by applying a chaperonin known as TF55 in thermophile on JP2 ligase reaction. Result showed that TF55 could not help in improving thermostability of ligase at 85℃. The possible reason might be that at 85℃ in vitro, the chaperonin itself was denatured.

Molecular chaperones in the Paracoccidioides brasiliensis transcriptome

Paracoccidioides brasiliensis is a thermally dimorphic and a human pathogenic fungus. Our group has partially sequenced its transcriptome and generated a database of mycelial and yeast PbAESTs (P. brasiliensis assembled expressed sequence tags). In the present review we describe the identification of PbAESTs encoding molecular chaperones. These proteins, involved in protein folding and renaturation, are also implicated in several other biological processes, where the dimorphic transition is of particular interest. Another important issue concerning these proteins refers to their participation in the immunopathogenicity of infectious diseases. We have found 438 ESTs (184 in mycelium and 253 in yeast) encoding P. brasiliensis molecular chaperones and their co-chaperones, which were clustered in 48 genes. These genes were classified in families, corresponding to three small chaperones, nine HSP40s, 10 HSP60s, seven HSP70s, five HSP90s, four HSP100s, and 10 other chaperones. These results greatly increase the knowledge on P. brasiliensis molecular chaperones, since only eight of such proteins had been previously characterized.

Effect of carnosine on steroid-induced modification of lens α-crystallin / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To explore whether carnosine can protect α -crys-tallin modification and decreased chaperone by a steroid,and whether carnosine could directly react with a steroid.METHODS: Bovine lens α L-crystallin was separated by size-exclusion chromatography on a Sephacyl S-300 HR column. α L-Crystallin was incubated with different concentrations of prednisolone-21-hemisuccinate (P-21-H)with or without carnosine for different times. The chaperone activity of α L-crystallin was monitored using the prevention thermal aggregation of α L-crystallin. The modified α L-crystallinwas examined by SDS-PAGE and fluorescence measurements. The absorbance spectra of solutions of carnosine and P-21-H were investigated.RESULTS: P-21-H decreased the chaperone activity ofα L-crystallin in a concentration- and time-dependent fashion. Carnosine only worsened this effect. The tryptophan fluorescence intensity of α L-crystallin modified by P-21-H was significantly decreased compared to unmodified crystallin, whereas its non-tryptophan fluorescence was increased with a shift to longer wavelengths in a time- and dose-dependent manner, suggesting that new fluorophores are possibly formed. Carnosine readily reacted with P-21-H thereby inhibiting steroid-mediated protein modification as revealed electrophoretically. The increased absorbance was time-dependent, suggesting adducts may be formed between carnosine and P-21-H.CONCLUSION: Carnosine reacts with P-21-H, which suggests carnosine's potential as a possible anti-seroid agent.