Expression of matrix metalloproteinases and their tissue inhibitors in the serum and cerebrospinal fluid of patients with HIV-1 infection and syphilis or neurosyphilis

The potential mechanisms for altered matrix metalloproteinase (MMP) or tissue inhibitors of matrix metalloproteinase (TIMP) function in patients with syphilis and HIV-1 co-infection (HIV-S) was unclear. To determine the expression of MMP-2, 9 and TIMP-1, 2, 4 in the serum and cerebrospinal fluid (CSF) of HIV-S patients, a total of 20 HIV-S patients and 8 controls were enrolled in a HIV-1 clinical cohort for diagnosis of neurosyphilis in Taiwan. Serum and CSF concentrations of MMP-2, 9, and TIMP-1, 2, 4 were determined by ELISA. Gelatin zymography was used to detect the expression of MMP-2 and MMP-9 in the CSF. Neurosyphilis was defined as a CSF white blood cell count ≥ 20 cells/μL or a reactive CSF Venereal Disease Research Laboratory (VDRL). All the patients with HIV-S were males. Most (85%) had sex with men (MSM) and serum rapid plasma reagin (RPR) titers of ≥ 1:32. The median age was 35 years (IQR 30-43). The median CD4 T cell counts at the time of the diagnosis of syphilis were 270 cells/μL (IQR 96-484). Ten patients (50%) had neurosyphilis based on a reactive CSF VDRL test (n=8) or increased CSF white cell counts ≥ 20/μL (n=2). The concentrations of CSF MMP-9, TIMP-1, and TIMP-2 were significantly higher in patients with HIV-S than the controls (P<0.05). The CSF TIMP-4 concentrations were significantly lower in those with HIV-S (452 pg/ml) than controls (3101 pg/ml), P<0001. There were no significant differences in serum concentrations between the groups. The only finding that distinguished HIV-1 patients with from those without neurosyphilis is a significant higher expression of CSF MMP-9. In conclusion, the MMP/TIMP system was found to be dysregulated in patients with HIV-S regardless of whether they met the laboratory definition of neurosyphilis. The CSF level of MMP-9 was the only measure that distinguished those with or without neurosyphilis.     

Ku80 is differentially expressed in human lung carcinomas and upregulated in response to irradiation in mice

Based on the role of Ku80 in mediating radiation-induced DNA repair, we investigated Ku80 expression in human lung cancers of different pathological types and evaluated the effect of radiotherapy on Ku80 expression levels in a mouse model. We used immunohistochemistry and real-time PCR to determine Ku80 protein and mRNA levels, respectively. We inoculated nude mice with A549 cells and subjected the tumor-bearing mice to varying doses of irradiation. Lung carcinoma tissue exhibited higher Ku80 mRNA and protein levels when compared with normal tissue. Among the tumor subtypes, lung adenocarcinoma and lung squamous carcinoma showed higher levels of Ku80 protein and mRNA, compared with small-cell lung carcinoma. There was a dose-dependent and time-dependent increase in Ku80 mRNA levels in nude mice that were inoculated with A549 cells and exposed to varying doses of irradiation. Ku80 may play an important role in the DNA damage response pathway. Higher Ku80 levels in lung squamous carcinoma and adenocarcinoma may explain their lower radiosensitivity when compared with small-cell lung carcinoma. Ku80 expression levels could be useful in predicting radiosensitivity of lung tumors and inhibition of Ku80 may be an interesting target to improve radiosensitivity in lung cancer patients.     

Construction and characterization of a cDNA library from shark regenerated hepatic tissue

Sharks are a type of fish with a full cartilaginous skeleton and have big livers. To better understand liver regeneration in sharks and screening for the important genes participated in disease-defense, in this study, a cDNA library of regenerated liver tissues of shark, Chiloscyllium plagiosum, was constructed. A total of 2103 expressed sequence tags (ESTs), which represents 997 unique genes, were sequenced. Among these genes, 434 (43.53%) of them showed significant similarity (E-values < 10^?5) to the sequences in NCBI Nt database, 685 (68.71%) of these unique genes showed significant similarity (E-values < 10^?5) to the sequences in NCBI Nr database, and 662 (66.40%) of these unique genes showed significant similarity (E-values < 10^?5) to the Swiss-Prot database. Preliminary analysis of unique genes according to COG database showed that unigenes were further grouped into 21 functional categories including inorganic ion transport and metabolism, energy production and conversion, posttranslational modification, protein turnover and chaperones, general function prediction only, translation, and ribosomal structure and biogenesis. Several possible candidate genes involved in liver regeneration were selected to analyze their expression with relative quantification real-time PCR. This study may contribute to our better understanding of the molecular mechanism of regeneration in shark liver. Furthermore, the EST cataloguing and profiling of shark will be also benefited to the functional genomic research in this marine species.     

A ubiquitin ligase-associated chaperone holdase maintains polypeptides in soluble states for proteasome degradation

Endoplasmic reticulum-associated degradation (ERAD) employs membrane-bound ubiquitin ligases and the translocation-driving ATPase p97 to retrotranslocate misfolded proteins for proteasomal degradation. How retrotranslocated polypeptides bearing exposed hydrophobic motifs or transmembrane domains (TMDs) avoid aggregation before reaching the proteasome is unclear. Here we identify a ubiquitin ligase-associated multiprotein complex comprising Bag6, Ubl4A, and Trc35, which chaperones retrotranslocated polypeptides en route to the proteasome to improve ERAD efficiency. In vitro, Bag6, the central component of the complex, contains a chaperone-like activity capable of maintaining an aggregation-prone substrate in an unfolded yet soluble state. The physiological importance of this holdase activity is underscored by observations that ERAD substrates accumulate in detergent-insoluble aggregates in cells depleted of Bag6, or of Trc35, a cofactor that keeps Bag6 outside the nucleus for engagement in ERAD. Our results reveal a ubiquitin ligase-associated holdase that maintains polypeptide solubility to enhance protein quality control in mammalian cells.     

Dicer's helicase domain discriminates dsRNA termini to promote an altered reaction mode

The role of Dicer's helicase domain is enigmatic, but in?vivo it is required for processing certain endogenous siRNA, but not miRNA. By using Caenorhabditis elegans extracts or purified Drosophila Dicer-2 we compared activities of wild-type enzymes and those containing mutations in the helicase domain. We found the helicase domain was essential for cleaving dsRNA with blunt or 5'-overhanging termini, but not those with 3' overhangs, as found on miRNA precursors. Further, blunt termini, but not 3' overhangs, led to increased siRNAs from internal regions of dsRNA; this activity required ATP and a functional helicase domain. Our data suggest that blunt or 5'-overhanging termini engage Dicer's helicase domain to facilitate accumulation of siRNAs from internal regions of a dsRNA, an activity suited for processing long siRNA precursors of low abundance, but not necessary for the single cleavage required for miRNA processing.     

Robust and responsive silk ionomer microcapsules

We demonstrate the assembly of extremely robust and pH-responsive thin shell LbL microcapsules from silk fibroin counterparts modified with poly(lysine) and poly(glutamic) acid, which are based on biocompatible silk ionomer materials in contrast with usually exploited synthetic polyelectrolytes. The microcapsules are extremely stable in an unusually wide pH range from 1.5 to 12.0 and show a remarkable degree of reversible swelling/deswelling response in dimensions, as exposed to extreme acidic and basic conditions. These changes are accompanied by reversible variations in shell permeability that can be utilized for pH-controlled loading and unloading of large macromolecules. Finally, we confirmed that these shells can be utilized to encapsulate yeast cells with a viability rate much higher than that for traditional synthetic polyelectrolytes.     

Biosynthesis and characterization of poly(3-hydroxydodecanoate) by β-oxidation inhibited mutant of Pseudomonas entomophila L48

A medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) producer Pseudomonas entomophila L48 was investigated for microbial production of 3-hydroxydodecanote homopolymer. Pseudomonas entomophila L48 was found to produce MCL PHA consisting of 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD), and 3-hydroxydodecanoate (3HDD) from related carbon sources fatty acids. In this study, some of the genes encoding key enzymes in β-oxidation cycle of P. entomophila such as 3-hydroxyacyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase, and acetyl-CoA acetyltransferase were deleted to study the relationship between β-oxidation and PHA synthesis in P. entomophila. Among the mutants constructed, P. entomophila LAC26 accumulated over 90 wt % PHA consisting of 99 mol % 3HDD. A fed-batch fermentation process carried out in a 6 L automatic fermentor produced 7.3 g L(-1) PHA consisting of over 97 mol % 3HDD fraction. Properties of MCL PHA were significantly improved along with increasing 3HDD contents. P(2.1 mol % 3HD-co-97.9 mol % 3HDD) produced by P. entomophila LAC25 had the widest temperature range between T(g) and T(m), which were -49.3 and 82.4 °C, respectively, in all MCL PHA reported so far. The new type of PHA also represented high crystallinity caused by side-chain crystallization compared with short side chain PHA. For the first time, P(3HDD) homopolymers were obtained.     

大兴安岭林区兴安落叶松人工林植被碳贮量

通过样地调查,研究了大兴安岭林区10、12、15、26和61年生兴安落叶松人工林中乔木、草本和植被总体碳储量,并以空间代替时间的方法,探讨落叶松人工林生长过程中植被碳库贮量变化.结果表明:随林龄的增加,兴安落叶松人工林植被碳库贮量逐渐增加,61 a时达105.69 t.hm-2,碳汇作用显著;15～26 a兴安落叶松人工林的碳汇能力最强.其中,树干碳库贮量占乔木碳库总贮量的54.3%～73.9%,且随林龄增加,其碳库比率和碳密度增加;其余器官碳库比率随林龄增加而减小,碳密度则逐渐增加,直至趋于平衡或末期略有减少.大兴安岭林区兴安落叶松人工林的轮伐期以≥60 a为宜.