Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica最新文献

To identify molecular features of neoplasms associated with EB virus, human peripheral blood lymphocytes (huPBL) were isolated from healthy volunteer donors and were transplanted intraperitoneally into SCID mice, and then huPBL/SCID mice were infected with EB virus. Serum levels of human IgG were measured by unidirectional immunodiffusion assay. Human Alu sequence and EBER-1 in tumor tissues were detected with PCR and in situ hybridization. Immunohistochemical staining was used to examine leukocyte differentiation antigens (LCA, L26, UCHL1, PS1), viral gene products (LMP1, EBNA2, BZLF1) and cellular oncoproteins (p53, C-myc, Bcl-2 and Bax). The experiments showed that tumors developed in 24 of 34 surviving huPBL/SCID mice by EBV infection. Histopathological and immunohistochemical observations demonstrated that all of the induced tumors in SCID mice were malignant lymphomas derived from human B-lymphocytes. In situ hybridization showed that tumor cells had EBV-encoded small RNA-1 (i.e. EBER-1). Alu sequence could be amplified by PCR from human genome of tumor tissues. Immunohistochemistry detected positive staining of BZLF1-encoded protein in a small population of tumor cells of almost all cases, and positive staining of LMP1 and EBNA2 only in small number of tumor cells. Human IgG could be found in the serum of 12 SCID mice on the 15th day after huPBL engraftment, and then increased with time and with the development of induced tumors in 6 mice. Positive rates of p53, C-myc, Bcl-2 and Bax expression were 83.33%, 100%, 95.83%, 91.67%, respectively, in 24 cases of the EBV-induced lymphomas. The results indicate that molecular lesions associated with the induced B-cell lymphoma involved EBV infection, expression of oncogenic viral genes, and abnormal expression of cellular oncogenes in human xenografts. Human IgG level in the serum of huPBL/SCID mice can be considered as a useful index for tumor development.

The interaction of bovine serum albumin (BSA) with a new palladium(II) complex [Pd(bpy)(Oct-Gly)]NO(3) (bpy, 2,2 -bipyridine; Oct-Gly, octyl-glycine) was studied by isothermal titration UV-visible spectrophotometry and microcalorimetry in 30 mmol/L Tris buffer, pH 7.0. There is a set of 18 binding sites for this complex on BSA at 300 and 310 K with positive cooperativity in the binding process. The Hill coefficients at 300 and 310 K are 2.2 and 2.4, respectively. The binding of this palladium complex on BSA is endothermic with mean association binding constant of 21.0 and 16.4 (mmol/L) (-1) at 300 and 310 K, respectively. The complex can denature the protein as surfactants. The stability of BSA in the interaction study with the complex is 84 and 58 kJ/mol at 300 and 310 K, respectively. Also, the enthalpy of BSA denaturation due to the interaction with the complex is 842 kJ/mol.

The cellulase genes of some animals, most coding for endo-beta-1,4-glucanases, were found and cloned. There has been no reports about genes encoding exo-beta-1,4-glucanase or endo- -1,4-xylanase from animal. Here we cloned the cDNA of a cellulase designated as EGX from mollusc, Ampullaria crossean, and expressed it in Pichia pastoris for the first time. The cellulase EGX is a multi-functional beta cellulase with the activities of exo-beta-1,4-glucanase, endo-beta-1,4-glucanase and endo-beta-1,4-xylanase. The opening reading frame of EGX cDNA is 1185 bp and encodes 395 amino acids. The EGX gene can also be amplificated from the genomic DNA by PCR, which verified the endogenous origin of this gene. This EGX gene was the first multi-functional cellulase gene that was directly isolated from animals.

To study the genetic variation in 5'-regulatory region of aldose reductase (AR) gene that might influence expression and the relationship between variations and diabetic complication (DC), PCR-single stranded conformational polymorphism (SSCP) was used to screen the 5'-regulatory region of AR gene in Chinese patients with type 2 diabetes mellitus. A novel mutation, C(-167) --> A substitution which created a new CCAAT box was found only in two diabetic patients. These two patients have no retinopathy, and the AR activity of their erythrocytes was within low range in patients without DC. The DNA segments of AR wild type and mutant were subcloned into pCAT reporter vector, and CAT assays were performed to assess promoter activity. The interaction between the DNA segments and nuclear proteins was determined by using competitive gel electrophoretic mobility shift assay (EMSA). The transcriptional activity of mutant (5.7% +/-2.9%) was lower than that of wild type (15.7% +/- 4.1%) (P<0.01), and the mobility shift of mutant was also slower than that of wild type. The results indicated the mutation C(-167) -->A in AR gene might prevent or delay the development of DC by repressing the expression of AR gene.

Apoptosis-inducing factor (AIF) is a phylogenetically conserved mitochondrial intermembrane flavoprotein which has the ability to induce apoptosis via a caspase-independent pathway. AIF plays an important role in inducing nuclear chromatin condensation as well as large-scale DNA fragmentation (approximately 50 kb), and is essential for programmed cell death during cavitation of embryoid bodies. Two homologous proteins, AIF-homologous mitochondrion-associated inducer of death (AMID) and p53-responsive gene 3 (PRG3), also have apoptosis-inducing effects. Recent studies on mechanisms of AIF-mediated apoptotic DNA degradation in Caenorhabditis elegans reveal that WAH-1(an AIF homolog in C. elegans) induced apoptosis is CED-3-dependent. AIF also interacts with cytochrome c and caspases during mammalian apoptosis processes, indicating that different apoptotic pathways may be mutually cross-regulated to activate an apoptotic program.

In previous study, we demonstrated that the specific activity of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) in skeletal muscle of induced hibernating jerboa (hibernating GAPDH) was 3 4 folds lower than that of the one in the skeletal muscle of the euthermic jerboa (euthermic GAPDH). A significant decrease in both GAPDH protein and GapC mRNA levels occurs when hibernating, but the purified hibernating GAPDH is less active than the euthermic GAPDH. To investigate the physico-chemical basis of this lower activity, the behaviour during thermal inactivation of skeletal muscle GAPDH from hibernating and euthermic tissues was examined by a variety of spectroscopic techniques, including fluorescence emission, circular dichroism and ultraviolet absorption. A clear resistance to thermal denaturation was observed in the hibernating GAPDH compared with the euthermic GAPDH. The different temperature of denaturation found in these proteins by both fluorimetry and circular dichroism indicates that there might exist conformational changes of GAPDH upon hibernation that could affect the stability of this enzyme.

Twenty-two DnaA boxes were identified in the chromosome replication origin (oriC) of Streptoverticillum caespitosus ATCC27422 based upon the characteristics of consensus sequences. The 21st and 22nd DnaA boxes overlapped 8 base pairs each other reversely. Compared with the oriC database of actinomycetes, similar overlapping DnaA boxes were recognized in several species of Streptomyces and Mycobacterium. These overlapping DnaA boxes were composed of the last two DnaA box (21st and 22nd) in the Streptomyces species, but the of 1st and 2nd ones in the Mycobacterium species. The consensus sequence of the overlapping DnaA box is CTGTGCACAA, one base longer than the normal DnaA box sequence presumably due to the overlapping structure. Although the DnaA boxes exist in the 189 792 bp region only, the 1 188 bp and 793 939 bp regions are also important to the DNA replication. Deletion of the 1 188 bp region may cause absolute loss of DNA replication initiation activity measured by the transformation efficiency of plasmids with truncated oriC. When the 793 939 bp region was truncated, the transformation efficiency reduced about 40%. If the oriC was cloned into a vector with partial flanking region sequences (partial dnaA and dnaN gene sequences), the transformation rate was about 4.3-fold lower than that of the construct containing the oriC region only. However, the transformants were much more similar to the host with respect to the morphology of colony and mycelium. The cis-regulatory functions of the flanking sequences, which may influence the initiation efficiency of the chromosome replication and/or the stability of replicon, are thus suggested.

To investigate the effects of lentivirus-mediated transfection of GDNF on Parkinson s disease (PD). The pNL-gdnf plasmid was constructed by replacing the LacZ-coding region present in pNL-lacZ/CMV. Vector particles involved a three-plasmid lentivirus expression system were co-transferred into 293T cells through calcium phosphate method. High-titer virus was collected from infected 293T cells and injected into lesion-side striatum of PD rats, and their apomorphine-induced rotations were assayed at day 14, 30 and 60, respectively. GDNF protein was detected by Western blot analysis, and the expression of lacZ and TH were detected by immunochemistry. Results showed that behavioral recovery gradually appeared after transplantation, and a significant reduction in the rotational response was observed at the 14th day. Meanwhile, gdnf expression maintained for at least 60 d, which had dopaminergic trophism to a certain degree, indicating that lentivirus-mediated transfection gdnf could effectively improve the clinical function of PD rats, which provide a potential attractive tool of gene therapy for PD.

The phycobilisomes were isolated from blue-green alga Spirulina platensis, and could form monolayer film at air/water interface. The monolayer film of phycobilisomes was transferred to newly cleaved mica, and coated with gold. Scanning tunneling microscope was used to investigate the structure of the Langmuir-Blodgett film of phycobilisomes. It was shown that phycobilisomes in the monolayer arrayed in rows with core attaching on the substrate surface and rods radiating towards the air phase, this phenomenon was similar to the arrangement of phycobilisomes on cytoplasmic surface of thylakoid membrane in vivo. The possible applications of the Langmuir-Blodgett film of phycobilisomes were also discussed.

Ribonuclease inhibitor (RI) is an acidic 50 kD protein with a high content of leucine and cysteine residues. RI inhibits RNases of the pancreatic type. A variant of RI was cloned from human fetal liver cDNA library by polymerase chain reaction (PCR). Compared with the reported RI, only two variations Arg(359)Ala and Leu(365)Pro were found in RIv amino acid sequence. Recombinant RIv has been expressed both in Escherichia coli and silkworm insect cells (Bombyx mori). The recombinant RIv exhibited inhibition activity on ribonucleolytic activity of RNase A in vitro system.