[Preliminary exploration of the metabolic profile and metabolic pathways in newly diagnosed multiple myeloma].

Objective: To explore the metabolite profile and metabolic pathways of newly diagnosed multiple myeloma (MM). Methods: Gas chromatography-mass spectrometry (GC-MS) was employed for the high-throughput detection and identification of serum samples from 55 patients with MM and 37 healthy controls matched for age and sex from 2016 to 2017 collected at the First Affiliated Hospital of Soochow University. The relative standard deviation (RSD) of quality control (QC) samples was employed to validate the reproducibility of GC-MS approach. The differential metabolites between patients with MM and healthy controls were detected by partial least squares discrimination analysis (PLS-DA), and t-test with false discovery rate (FDR) correction. Metabolomics pathway analysis (MetPA) was employed to construct metabolic pathways. Results: There were 55 MM patients, including 34 males and 21 females. The median age was 60 years old (42-73 years old). There were 30 cases of IgG type, 9 cases of IgA type, 1 case of IgM type, 2 cases of non-secreted type, 1 case of double clone type and 12 cases of light chain type, including 3 cases of kappa light chain type and 9 cases of lambda light chain type. The result of QC sample test showed that the proportion of compounds with the RSD of the relative content of metabolites < 15% was 70.21% obtained by the reproducibility of GC-MS experimental data, which implied that the experimental data were reliable. A total of 17 metabolites were screened differently with the healthy control group, including myristic acid, hydroxyproline, cysteine, palmitic acid, L-leucine, stearic acid, methionine, phenylalanine, glycerin, serine, isoleucine, tyrosine, valine, citric acid, inositol, threonine, and oxalic acid (VIP>1, P<0.05). Metabolic pathway analysis suggested that metabolic disorders in MM patients comprised mainly phenylalanine metabolism, glyoxylic acid and dicarboxylic acid metabolism, phosphoinositide metabolism, cysteine and methionine metabolism, glycerolipid metabolism, glycine, serine, and threonine metabolism. Conclusion: Compared with normal people, patients with newly diagnosed MM have obvious differences in metabolic profiles and metabolic pathways.

[New discovery and short-term effect analysis of tensor tympani muscle tenotomy for Meniere's disease under otoscope].

Objective: To explore the feasibility and short-term effect of tensor tympani muscle Tenotomy in the treatment of Meniere's disease under otoscope. The possible pathogenesis was discussed and our views were put forward. Methods: The clinical data of 9 cases of Meniere's disease treated by otoscopic Tenotomy were analyzed retrospectively, including 2 males, 7 females, 5 right ones, 2 left ones and 2 bilateral ones. The average age was (56.33± 10.56) years, ranging from 38 to 75 years. We evaluated intraoperative findings and short-term postoperative efficacy, respectively evaluated postoperative aural fullness, tinnitus and hearing recovery, and evaluated postoperative vertigo attack in a short time. Results: Nine patients were completed the operation under general anaesthesia and otoscopy, and no serious complications occurred. We found new pathological changes in tympanic cavity in some cases during operation. There were rupture of round window membrane in 1 case, severe fibrous hyperplasia near the round window membrane and vestibular window and adhesion with ossicular chain in 1 case, fibrous cord and membranous hyperplasia near vestibular window and round window membrane in 1 case, fibrous hyperplasia and adhesion near the round window membrane in 2 cases, membranous hyperplasia and adhesion around vestibular window in 1 case. No fibrous hyperplasia was found in 3 cases in the tympanic cavity. The round window membrane can be exposed in 4 cases and failed in 5 cases. After 3 months of follow-up, we found that we found that 5/5 cases of aural fullness disappeared, 2/2 cases of earache disappeared, 3/8 cases of tinnitus improved, 5/8 cases presented with improvement and no aggravation, 3/3 cases of hearing allergy improved, 4/9 cases of hearing improved, and 5/9 cases showed no improvement or decrease. 9 patients were followed up for 3 months, of whom 8 patients had no vertigo, one patient suffered from vertigo twice within 3 months after operation, and the patient suffered from rupture of round window membrane. Conclusions: Endoscopic Tenotomy for Meniere's disease has obvious curative effect and quick recovery after operation. During the operation, we find that most of Meniere's patients have fibrous cord hyperplasia near the inner ear window membrane, which may be the pathological manifestation after repeated rupture and repair of the inner ear window membrane. The vertigo of Meniere's disease may be related to the destruction and repair of inner ear membrane structure caused by improper contraction or spasm of tympanic tensor muscle.

Spatial epidemiological characteristics and exponential smoothing model application of tuberculosis in Qinghai Plateau, China.

The epidemic of tuberculosis has posed a serious burden in Qinghai province, it is necessary to clarify the epidemiological characteristics and spatial-temporal distribution of TB for future prevention and control measures. We used descriptive epidemiological methods and spatial statistical analysis including spatial correlation and spatial-temporal analysis in this study. Furthermore, we applied an exponential smoothing model for TB epidemiological trend forecasting. Of 43 859 TB cases, the sex ratio was 1.27:1 (M:F), and the average annual TB registered incidence was 70.00/100 000 of 2009-2019. More cases were reported in March and April, and the worst TB stricken regions were the prefectures of Golog and Yushu. High TB registered incidences were seen in males, farmers and herdsmen, Tibetans, or elderly people. 7132 cases were intractable, which were recurrent, drug resistant, or co-infected with other infections. Three likely cases clusters with significant high risk were found by spatial-temporal scan on data of 2009-2019. The exponential smoothing winters' additive model was selected as the best-fitting model to forecast monthly TB cases in the future. This research indicated that TB in Qinghai is still a serious threaten to the local residents' health. Multi-departmental collaboration and funds special for TB treatments and control are still needed, and the exponential smoothing model is promising which could be applied for forecasting of TB epidemic trend in this high-altitude province.

CT-based staging and prognosis of novel coronavirus (COVID-19) pneumonia: correlation with blood glucose levels.

OBJECTIVE: Based on the latest epidemic situation and field experience, this study aims to explore the correlation of computed tomography (CT) stages and blood glucose level in patients with novel coronavirus pneumonia (COVID-19). PATIENTS AND METHODS: The clinical data of first and multiple CT imaging re-examination and blood glucose levels from 62 confirmed cases of COVID-19 were collected for a retrospective analysis to determine the correlation between glucose level and CT-based staging. RESULTS: Of the 62 COVID-19 patients, 48 cases of early stage and 14 cases of advanced stage were found in the CT data of the first diagnosis. These 62 cases were currently under follow-up (17-32 days): 18 cases in early stage-resorption stage, 25 cases in early stage-advanced stage-resorption stage, 12 cases in advanced stage-resorption stage, 5 cases in early stage -advanced stage-severe stage-resorption stage, and 2 cases in advanced stage-severe stage-resorption stage. Among them, the CT of 14 patients with advanced stage at the first diagnosis showed multiple stage lesions (advanced stage + early stage) at the same time. Patients presented with statistically significant changes in blood glucose at early stage-resorption stage, early stage-advanced stage-resorption stage, advanced stage-resorption stage, and early stage-advanced stage-severe stage-resorption stage (p<0.05). However, no statistically significant alterations were observed in the glucose level of patients with advanced stage-severe stage-resorption stage (p>0.05). CONCLUSIONS: Alteration of blood glucose is positively correlated with CT-based staging of COVID-19. Blood glucose is of great value in clinical diagnosis of COVID-19 and in determining the stage and prognosis of this disease.

[Years of potential life lost due to premature death of cardiovascular diseases among residents in Suzhou from 1987 to 2017].

From 1987 to 2017, cardiovascular disease (CVD) had been ranking the first cause of death in Suzhou, and the mortality rate showed an upward trend annual percentage changes (APC=0.62%, P=0.001), while the standardized mortality rate showed a downward trend (APC=-2.65%, P<0.001). The probability of premature death of CVD declined consistently from 7.06% in 1987 to 2.00% in 2017 (APC=-4.45%, P<0.001). When the life expectancy was set at 70, the potential years of life lost rate (PYLLR) decreased from 6.35‰ in 1987 to 3.30‰ in 2017, and the standardized PYLLR decreased from 7.30‰ to 2.68‰. When the life expectancy was set at 75, the PYLLR decreased from 10.12‰ to 5.19‰, and the standardized PYLLR decreased from 11.44‰ to 3.88‰. With the increase of years, all PYLLR and standardized PYLLR showed a significantly downward trend (APC=-2.51%--3.89%, P<0.001).

The value of Gd-BOPTA- enhanced MRIs and DWI in the diagnosis of intrahepatic mass-forming cholangiocarcinoma.

The aim of this study is to explore the value of unenhanced magnetic resonance imaging (MRI), gadobenate dimeglumine injection (Gd-BOPTA)-enhanced MRI and diffusion-weighted imaging (DWI) in the diagnosis of intrahepatic mass-forming cholangiocarcinoma (IMCC). Totally 59 IMCC patients who underwent Gd-BOPTA-enhanced MRIs were recruited. The time-signal intensity curves and lesion periphery enhancement rates of the IMCC and liver parenchyma was drawn using apparent diffusion coefficient (ADC) values. The Gd-BOPTA-enhanced MRI showed that the peripheries of 30 lesions in the arterial phase exhibited irregular ring enhancement. However, lesions in the portal and delayed phases (which were gradually filled with a contrast agent), presented a patchy or latticed enhancement. Twenty-two lesions in the arterial and delayed phases exhibited uneven mild/moderate patchy enhancements with a progressive and centripetal lesion. Five lesions emerged from the arterial phase without any significant enhancement and had only gradual enhancement during the delayed phase. The remaining 2 lesions had a decreased mild enhancement, presented comparatively high signals and the lesion center had visible small spotted low signals. The DWI signals displayed a slightly high or high unevenness. Some lesion peripheries had a high signal but lesion centers displayed a relatively low or slightly low signal and irregular patches. There were significant differences between the ADC values of the lesion edge, lesion center and liver parenchyma. The IMCC detection rates of the Gd-BOPTA-enhanced MRI and DWI were higher than those of the unenhanced MRI. Our study demonstrated that both the Gd-BOPTA-enhanced MRI and DWI had higher accuracies rates than an unenhanced MRI. Furthermore, the hepatobiliary phase of IMCC plays an important role in the diagnosis and identification of IMCC constituents.

[Effect of CCR3 gene knockout on eosinophils in mice].

Objective:To investigate the effect of CCR3 gene knockout on the proliferation, maturation and apoptosis of eosinophils (EOS) in mice. Method:Bone marrow cells from CCR3 gene knockout mice (experimental group) and wild-type mice (control group) were cultured in vitro and induced differentiation into mature EOS. EOS proliferation was observed by cell counting.Expression of degranulation protein mRNA was detected by qRT-PCR. EOS apoptosis was detected by Annexin V-FITC/PI double staining method. Result:①The number of cells on the 0-14 day of EOS cultured in vitro showed that the number of cells in the experimental group was less than that in the control group at tenth, twelfth, fourteenth days, and the proliferation was slower, the difference was statistically significant (P<0.01). ②The test results of EOS cells apoptosis showed that, under the condition of containing 10 ng/ml IL-5 or no IL-5, the apoptosis rate of EOS cells in experimental group was higher than that in normal control group, the difference between both groups was statistically significant (P<0.01). ③The expression results of ECP, EPO, MBP mRNA in EOS showed that the expression levels of ECP, EPO, MBP mRNA in the experimental group compared with the normal control group were reduced in varying degrees, the difference between both groups was statistically significant (P<0.05). Conclusion:Knockout CCR3 gene can inhibit the proliferation and maturation of EOS and promote its apoptosis, which provides a theoretical basis for CCR3 as a target gene to treat allergic rhinitis.

Reevaluation of Two Quantitative Trait Loci for Type II Resistance to Fusarium Head Blight in Wheat Germplasm PI 672538.

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, is a destructive disease in wheat. A population consisting of 229 F2 and F2:3 plants derived from the cross PI 672538 × L661 was used to evaluate the reactions to FHB. The FHB resistance data distribution in the F2 population indicates that some quantitative trait loci (QTLs) were controlling the FHB resistance in PI 672538. We further detected two major QTLs (Qfhs-2B, Qfhs-3B) from analysis of the resistance data and the PCR-amplified results using WinQTLCart 2.5 software. Qfhs-2B, flanked by Xbarc55-2B and Xbarc1155-2B, explained more than 11.6% of the phenotypic variation of the percentage of diseased spikelets (PDS), and Qfhs-3B, flanked by Xwmc54-3B and Xgwm566-3B, explained more than 10% of the PDS phenotypic variation in the F2:3 population. In addition, Qfhs-3B was different from Fhb1 in terms of the pedigree, inheritance, resistance response, chromosomal location, and marker diagnosis. We also detected QTLs for other disease resistance indices, including the percentage of damaged kernels and 1,000-grain weight, in similar chromosomal regions. Therefore, the FHB resistance of PI 672538 was mainly controlled by two major QTLs, mapped on 2B (FhbL693a) and 3B (FhbL693b). PI 672538 could be a useful germplasm for improving wheat FHB resistance.

[Efficacy of Bailemian capsule combined with self-help cognitive behavioral therapy in treatment of chronic insomnia].

Objective: To evaluate the efficacy of Bailemian capsule combined with self-help cognitive behavioral therapy (CBTI-SH) in treatment of chronic insomnia. Methods: Approved by the Ethics Committee of the hospital, 60 patients with chronic insomnia were randomly divided into two groups, the test group (Bailemian capsule combined with CBTI-SH) and the control group (CBTI-SH alone). Each group contained 30 cases. After 4 weeks for therapy, the sleep quality, mood and adverse reactions of treatment in patients were evaluated by sleep diary, sleep severity index scale (ISI), self-rating anxiety scale (SAS), self-rating depression scale (SDS) and treatment emergent symptom scale (TESS) respectively. The data were statistically analyzed. Results: The total effective rate in the test group was significantly higher than that of the control group (73.3% vs 46.7%, P<0.05). Compared to the control group, the sleep onset latency was significantly shorten [(38.3±13.1) vs (27.5±9.8) min, P<0.05], while the sleep efficiency were increased markedly [(76.6±5.7)% vs (80.5±6.6)%, P<0.05] in the test group. In the test group, the sleep onset latency, the total sleep time, the time in bed and sleep efficiency both improved significantly after treatment [(27.5±9.8) vs (56.2±19.4) min, (334.4±41.6) vs (310.8±31.7) min, (415.6±38.9) vs (446.9±39.9) min, (80.5±6.6)% vs (69.6±4.9)%, all P<0.05], while in the control group, the sleep onset latency, the time in bed and sleep efficiency also improved significantly after therapy [(38.3±13.1) vs (55.2±16.2) min, (430.4±32.6) vs (452.4±34.4) min, (76.6±5.7)% vs (69.9±5.2)%, all P<0.05]. After combined treatment, the SAS and SDS scores [(51.5±6.5) vs (55.0±5.8), (52.0±5.3) vs (55.3±4.4), both P<0.05] both decreased significantly than those of the control group, at the same time, the SAS and SDS scores decreased significantly after treatment in both the test group and the control group [(51.5±6.5) vs (61.5±4.8), (52.0±5.3) vs (60.2±4.5), (55.0±5.8) vs (62.5±3.7), (55.3±4.4) vs (62.2±3.7), all P<0.01]. Conclusion: The efficacy of Bailemian capsule combined with CBTI-SH in the treatment of chronic insomnia is more effective.

Syphilitic dementia and lipid metabolism.

BACKGROUND AND PURPOSE: Chronic syphilitic infection may lead to dementia. It is in general paresis (GP), which is the major late form of neurosyphilis, that cognitive impairment frequently occurs. The association between lipid metabolism and GP is unclear. METHODS: In this study, serum lipids were studied in 188 GP patients, in 241 syphilitic patients without neurosyphilis and in 539 healthy controls. The Mini-Mental State Examination (MMSE) was tested in all GP patients. Thirty-five GP patients had a follow-up evaluation 3 months after penicillin treatment. RESULTS: Significantly lower apolipoprotein A-I (apoA-I) levels were found in GP and in syphilitic patients without neurosyphilis compared to controls. In the 25-44-year-old groups, the male syphilitic patients without neurosyphilis had lower serum apoA-I levels and higher apolipoprotein B (apoB)/apoA-I ratios compared with female patients. A follow-up evaluation of 35 GP patients 3 months after penicillin treatment showed a significant positive correlation between increased apoA-I levels and MMSE scores. CONCLUSION: Abnormal apoA-I metabolism may be associated with the decline of cognitive performance. Long-term decrease of apoA-I level and higher apoB/apoA-I ratio may be contributing factors in syphilitic dementia. These results suggest a similar overlap between syphilitic dementia and lipid metabolism to that occurring in Alzheimer's disease.

Endothelial injury preceding intracranial aneurysm formation in rabbits.

OBJECTIVE: This study investigates the change of endothelial cell morphology and function at the rabbit basilar bifurcations in response to sustained high blood flow after bilateral common carotid artery ligation. METHODS: Fifteen adult female New Zealand white rabbits were divided into experimental and sham control groups. The experimental group was subjected to bilateral common carotid artery ligation to increase the compensatory basilar artery flow. Basilar artery flow was monitored by transcranial Doppler after surgery. The endothelial cells at the arterial bifurcations were studied morphologically by electron microscopy and immunohistochemistry using ß-catenin antibodies. Basilar artery flow increased significantly following common carotid artery ligation. RESULTS: Early-stage basilar artery bifurcation aneurysms were present in all rabbits at three months after ligation. The endothelial cells changed from a fusiform to column shape at the basilar artery bifurcation. Gaps between endothelial cells of the experimental group appeared wider in the electron microscopic photographs compared with those of the control group. The expression of endothelial ß-catenin at the arterial bifurcations also decreased. CONCLUSION: This study is the first to present endothelial cell changes of basilar artery bifurcation in response to sustained high blood flow in rabbits. Endothelial cell impairment possibly initiates aneurysm formation.

In vitro degradation of porous PLLA/pearl powder composite scaffolds.

The in vitro degradation behavior of poly-L-lactide (PLLA), PLLA/aragonite pearl powder and PLLA/vaterite pearl powder scaffolds was investigated. The scaffolds were soaked in phosphate buffer solution (PBS) up to 200 days. Scanning electron microscopy (SEM), gel permeation chromatography (GPC), and differential scanning calorimetry (DSC) were used to observe any degradation of the scaffolds. Degradation behaviors such as changes in pH, porosity, bulk density, water absorption, weight loss and mechanical properties were discussed. The results show that a gradual increase of the pH in composite scaffolds can decrease the rate of hydrolysis of PLLA. PLLA/vaterite and PLLA/aragonite scaffolds have a similar degradation behavior but a slower rate of degradation than PLLA.

GM1 improves neurofascin155 association with lipid rafts and prevents rat brain myelin injury after hypoxia-ischemia.

White matter injury characterized by damage to myelin is an important process in hypoxic-ischemic brain damage (HIBD). Because the oligodendrocyte-specific isoform of neurofascin, neurofascin 155 (NF155), and its association with lipid rafts are essential for the establishment and stabilization of the paranodal junction, which is required for tight interaction between myelin and axons, we analyzed the effect of monosialotetrahexosyl ganglioside (GM1) on NF155 expression and its association with lipid rafts after HIBD in Sprague-Dawley rats, weighing 12-15 g, on day 7 post-partum (P7; N = 20 per group). HIBD was induced on P7 and the rats were divided into two groups: one group received an intraperitoneal injection of 50 mg/kg GM1 three times and the other group an injection of saline. There was also a group of 20 sham-operated rats. After sacrifice, the brains of the rats were removed on P30 and studied by immunochemistry, SDS-PAGE, Western blot analysis, and electron microscopy. Staining showed that the saline group had definite rarefaction and fragmentation of brain myelin sheaths, whereas the GM1 group had no obvious structural changes. The GM1 group had 1.9-2.9-fold more GM1 in lipid rafts than the saline group (fraction 3-6; all P < 0.05) and 0.5-2.4-fold higher expression of NF155 in lipid rafts (fraction 3-5; all P < 0.05). Injection of GM1 increased the content of GM1 in lipid rafts as well as NF155 expression and its lipid raft association in HIBD rat brains. GM1 may repair the structure of lipid rafts, promote the association of NF155 (or other important proteins) with lipid rafts, stabilize the structure of paranodes, and eventually prevent myelin sheath damage, suggesting a novel mechanism for its neuroprotective properties.

GM1 improves neurofascin155 association with lipid rafts and prevents rat brain myelin injury after hypoxia-ischemia

White matter injury characterized by damage to myelin is an important process in hypoxic-ischemic brain damage (HIBD). Because the oligodendrocyte-specific isoform of neurofascin, neurofascin 155 (NF155), and its association with lipid rafts are essential for the establishment and stabilization of the paranodal junction, which is required for tight interaction between myelin and axons, we analyzed the effect of monosialotetrahexosyl ganglioside (GM1) on NF155 expression and its association with lipid rafts after HIBD in Sprague-Dawley rats, weighing 12-15 g, on day 7 post-partum (P7; N = 20 per group). HIBD was induced on P7 and the rats were divided into two groups: one group received an intraperitoneal injection of 50 mg/kg GM1 three times and the other group an injection of saline. There was also a group of 20 sham-operated rats. After sacrifice, the brains of the rats were removed on P30 and studied by immunochemistry, SDS-PAGE, Western blot analysis, and electron microscopy. Staining showed that the saline group had definite rarefaction and fragmentation of brain myelin sheaths, whereas the GM1 group had no obvious structural changes. The GM1 group had 1.9-2.9-fold more GM1 in lipid rafts than the saline group (fraction 3-6; all P < 0.05) and 0.5-2.4-fold higher expression of NF155 in lipid rafts (fraction 3-5; all P < 0.05). Injection of GM1 increased the content of GM1 in lipid rafts as well as NF155 expression and its lipid raft association in HIBD rat brains. GM1 may repair the structure of lipid rafts, promote the association of NF155 (or other important proteins) with lipid rafts, stabilize the structure of paranodes, and eventually prevent myelin sheath damage, suggesting a novel mechanism for its neuroprotective properties.

Dose estimation by chromosome aberration analysis and micronucleus assays in victims accidentally exposed to (60)Co radiation.

The objective of this study was to assess the radiation exposure levels in victims of a (60)Co radiation accident using chromosome aberration analysis and the micronucleus assay. Peripheral blood samples were collected from three victims exposed to (60)Co 10 days after the accident and were used for the chromosome aberration and micronucleus assays. After in vitro culture of the lymphocytes, the frequencies of dicentric chromosomes and rings (dic+r) and the numbers of cytokinesis blocking micronuclei (CBMN) in the first mitotic division were determined and used to estimate radiation dosimetry. The Poisson distribution of the frequency of dic+r in lymphocytes was used to assess the uniformity of the exposure to (60)Co radiation. Based on the frequency of dic+r in lymphocytes, estimates of radiation exposure of the three victims were 5.61 Gy (A), 2.48 Gy (B) and 2.68 Gy (C). The values were estimated based on the frequencies of CBMN, which were 5.45 Gy (A), 2.78 Gy (B) and 2.84 Gy (C). The estimated radiation dosimetry demonstrated a critical role in estimating the radiation dose and facilitating an accurate clinical diagnosis. Furthermore, the frequencies of dir+r in victims A and B deviated significantly from a normal Poisson distribution. Chromosome aberration analysis offers a reliable means for estimating biological exposure to radiation. In the present study, the micronucleus assay demonstrated a high correlation with the chromosome aberration analysis in determining the radiation dosimetry 10 days after radiation exposure.

Development of tertiary hyperparathyroidism after phosphate supplementation in oncogenic osteomalacia.

Oncogenic osteomalacia is a rare paraneoplastic syndrome. It is characterized by bone pain, muscle weakness, gait disturbance, fractures and skeletal deformities. Hypophosphatemia, diminished renal phosphate reabsorption, decreased 1,25 dihydroxy Vitamin D and elevated alkaline phosphatase are the biochemical hallmarks of this disorder. Most tumors are of mesenchymal origin. We report the case of a 39-year-old woman with oncogenic osteomalacia caused by osteosarcoma of the right scapula which was unrecognized for several years. She subsequently developed tertiary hyperparathyroidism after treatment with oral phosphate and Vitamin D. This case illustrates that oncogenic osteomalacia may persist for many years before the tumor is discovered. This is because the tumors are frequently very small and are in obscure locations. The uniqueness of this case is the coexistence of hyperparathyroidism and oncogenic osteomalacia. Five other cases have been reported up to date. All patients had received phosphate supplement, ranging from 10 to 14 years prior to their diagnosis. Interestingly, our patient was on the treatment for only 2 years. The proposed mechanism is that exogenous phosphate stimulates parathyroid activity through sequestration of calcium.

Small heat-shock protein structures reveal a continuum from symmetric to variable assemblies.

The small heat-shock proteins (sHSPs) form a diverse family of proteins that are produced in all organisms. They function as chaperone-like proteins in that they bind unfolded polypeptides and prevent uncontrolled protein aggregation. Here, we present parallel cryo-electron microscopy studies of five different sHSP assemblies: Methanococcus jannaschii HSP16.5, human alphaB-crystallin, human HSP27, bovine native alpha-crystallin, and the complex of alphaB-crystallin and unfolded alpha-lactalbumin. Gel-filtration chromatography indicated that HSP16.5 is the most monodisperse, while HSP27 and the alpha-crystallin assemblies are more polydisperse. Particle images revealed a similar trend showing mostly regular and symmetric assemblies for HSP16.5 particles and the most irregular assemblies with a wide range of diameters for HSP27. A symmetry test on the particle images indicated stronger octahedral symmetry for HSP16.5 than for HSP27 or the alpha-crystallin assemblies. A single particle reconstruction of HSP16.5, based on 5772 particle images with imposed octahedral symmetry, resulted in a structure that closely matched the crystal structure. In addition, the cryo-EM reconstruction revealed internal density presumably corresponding to the flexible 32 N-terminal residues that were not observed in the crystal structure. The N termini were found to partially fill the central cavity making it unlikely that HSP16.5 sequesters denatured proteins in the cavity. A reconstruction calculated without imposed symmetry confirmed the presence of at least loose octahedral symmetry for HSP16.5 in contrast to the other sHSPs examined, which displayed no clear overall symmetry. Asymmetric reconstructions for the alpha-crystallin assemblies, with an additional mass selection step during image processing, resulted in lower resolution structures. We interpret the alpha-crystallin reconstructions to be average representations of variable assemblies and suggest that the resolutions achieved indicate the degree of variability. Quaternary structural information derived from cryo-electron microscopy is related to recent EPR studies of the alpha-crystallin domain fold and dimer interface of alphaA-crystallin.

[Studies on the effects of astrocyte during glial scar formation by recombinant antisense GFAP retrovirus].

The present study was to investigate the changes of macroglia and the effect of gonadal hormone on reactive gliosis and of antisense GFAP retrovirus on astrocyte, gliosis and glial scar formation after brain stab injury (BSI). The results indicate that astrocytes are the main cells of glial scar. GFAP plays an important role in the maintenance of structure and function of astrocytes. The oligodendrocyte is not an active cell during glial scar formation. The gonadal hormone can modify the reactivity of astrocyte after BSI, but has no significant effect on differentiation and proliferation of astrocyte. The recombined GFAP retrovirus can effectively inhibit the growth of astrocyte, and GFAP expression of injured astrocyte in vitro and glial scar forming in vivo.

Mutation of alpha B-crystallin: effects on chaperone-like activity.

A recent paper by Plater et al. [20], showed that the mutation of a single phenylalanine residue F27R in mouse alpha B completely abolished the chaperone-like property of alpha-crystallin when assayed with insulin at 25 degrees C or with gamma-crystallin at 66 degrees C. We have produced the same mutation as well as some additional mutations in human alpha B-crystallin. Our data suggest that the F27R mutation effected the thermal stability of alpha B-crystallin making it unstable at temperatures > or = 60 degrees C. In agreement with the published work, at these temperatures the F27R human recombinant alpha B-crystallin does not protect the target protein from aggregation. When assayed with insulin or alpha-lactalbumin at 25 or 37 degrees C, however, there were no differences in the protective abilities between the native alpha B-crystallin or the F27R mutated human alpha B-crystallin. Several other multiple mutations involving proline residues were also produced. These mutations did not effect the chaperone-like properties of human alpha B-crystallin, but some of them did effect the native molecular weight size as judged by gel filtration chromatography.

Overproduction, in Escherichia coli, of soluble taxadiene synthase, a key enzyme in the Taxol biosynthetic pathway.

Taxadiene synthase catalyzes the conversion of the universal precursor of diterpenoids, geranylgeranyl diphosphate, to taxadiene, a key intermediate in Taxol (paclitaxel) biosynthesis. The gene encoding taxadiene synthase was cloned recently. Here we report a method for the heterologous overexpression of cDNA encoding taxadiene synthase in Escherichia coli using a thioredoxin fusion expression system, which increases the solubility of expressed protein. Taxadiene synthase cDNA was amplified by polymerase chain reaction and then subcloned into pET3d and pET32a(+) to form pET3dTX and pET32TX, respectively. The expressed taxadiene synthase from E. coli BL21(DE3)/pET3dTX was present completely as inclusion bodies. The transformant E. coli BL21(DE3)/pET32TX produced a thioredoxin fusion taxadiene synthase (15-20% of total soluble protein) when induced with isopropyl beta-D-thiogalactopyranoside at low temperature (20 degrees C). The recombinant enzyme was purified by a single step with a His-binding metal affinity column. The maximal production attained was 13 mg of purified, active fusion protein per 500 ml culture of E. coli BL21(DE3)/pET32TX. The purified recombinant taxadiene synthase fusion protein was similar to native protein in steady-state kinetic parameters and mobility on sodium sulfate-polyacrylamide gel electrophoresis. The protein purified from E. coli BL21(DE3)/pET3dTX had the expected N-terminal (AQLSFNA) sequence.