Cloning and expression of endo-β-glucanase II gene in Humicola insolens H31-3

Endo-β-glucanase Ⅱ (EG Ⅱ) gene cDNA was isolated from the fungus Humicola insolens H31-3 by RT-PCR.It was cloned into the expression vector pGAPZαA.The resultant recombinant plasmid was introduced into Pichia pastoris GS115 by electroporation after being lin-earized by BspHI digestion.The recombinant Pichia pas-toris strain was obtained and SDS-PAGE showed that the molecular weight of the expression protein was about 55 kD.The cultivation condition and the characteristics of the recombinant EG Ⅱ were also explored.     

NMR spectroscopy of 14-3-3ζ reveals a flexible C-terminal extension: differentiation of the chaperone and phosphoserine-binding activities of 14-3-3ζ

Intracellular 14-3-3 proteins bind to many proteins, via a specific phosphoserine motif, regulating diverse cellular tasks including cell signalling and disease progression. The 14-3-3ζ isoform is a molecular chaperone, preventing the stress-induced aggregation of target proteins in a manner comparable with that of the unrelated sHsps (small heat-shock proteins). 1H-NMR spectroscopy revealed the presence of a flexible and unstructured C-terminal extension, 12 amino acids in length, which protrudes from the domain core of 14-3-3ζ and is similar in structure and length to the C-terminal extension of mammalian sHsps. The extension stabilizes 14-3-3ζ, but has no direct role in chaperone action. Lys(49) is an important functional residue within the ligand-binding groove of 14-3-3ζ with K49E 14-3-3ζ exhibiting markedly reduced binding to phosphorylated and non-phosphorylated ligands. The R18 peptide binds to the binding groove of 14-3-3ζ with high affinity and also reduces the interaction of 14-3-3ζ ligands. However, neither the K49E mutation nor the presence of the R18 peptide affected the chaperone activity of 14-3-3ζ, implying that the C-terminal extension and binding groove of 14-3-3ζ do not mediate interaction with target proteins during chaperone action. Other region(s) in 14-3-3ζ are most likely to be involved, i.e. the protein's chaperone and phosphoserine-binding activities are functionally and structurally separated.     

Characterization of the 3′ untranslated region of mouse DNA topoisomerase IIα mRNA

Expression of DNA topoisomerase IIα protein varies through the cell cycle with its peak in G₂/M. This cell-cycle-dependent expression depends on changes in topoisomerase IIα mRNA stability as well as promoter activity. We isolated the 3′ genomic region of the mouse topoisomerase IIα gene and investigated whether or not the 3′ untranslated region (UTR) of the topoisomerase IIα mRNA participates in the cell-cycle-dependent mRNA stability. Interestingly, genomic- and RT-PCR analyses revealed that the topoisomerase IIα 3′ UTR is formed via splicing in mouse, but not in human and hamster. Comparison of the mouse 3′ region with the human and hamster regions suggests that this mouse-specific splicing has resulted from an accidental acquisition of the consensus 5′ splice site. The minority of the non-spliced topoisomerase IIα 3′ UTR in mouse was confirmed by Northern blot analysis. We performed transient expression assays using luciferase constructs with the mouse topoisomerase IIα 3′ genomic region, or the major spliced form of the 3′ UTR. However, neither construct affected the cell-cycle-dependent expression of the reporter gene driven by the topoisomerase IIα promoter. Our results strongly suggest that the mouse topoisomerase IIα 3′ UTR by itself is not involved in the cell-cycle-dependent mRNA stability.     

The role of epigenetic inactivation of 14-3-3σ in human cancer

Cancer cells show characteristic alterations in DNA methylation patterns. Aberrant CpG methylation of specific promoters results in inactivation of tumor suppressor genes and therefore plays an important role in carcinogenesis. The p53-regulated gene 14-3-3σ undergoes frequent epigenetic silencing in several types of cancer, including carcinoma of the breast, prostate, and skin, suggesting that the loss of 14-3-3σ expression may be causally involved in tumor progression. Functional studies demonstrated that 14-3-3σ is involved in cell-cycle control and prevents the accumulation of chromosomal damage. The recent identification of novel 14-3-3if-associated proteins by a targeted proteomics approach implies that 14-3-3σ regulates diverse cellular processes, which may become deregulated after silencing of 14-3-3σ expression in cancer cells.     

Function and structure of microvirid phage α3 genome: II. Isolation and properties of various mutants of α3

Various mutants were isolated from a microvirid (isometric single-stranded DNA) phage α3, by mutagenesis with hydroxylamine or nitrous acid. They were divided into eight complementation groups, and mainly by genetic crosses the gene alignment was determined as -A-B-C′-D-J′-F-G-H-. Except for groups C′ and J′, each defective gene product was clearly discerned in electropherograms of proteins extracted from the phage-infected suppressor-negative (Su^?) Escherichia coli. Only gene A mutants abolished synthesis of the progeny replicative-form DNA (RF), whereas mutants belonging to groups B, C′, D, E, F and J′ affected RF replication at late stage, as well as synthesis of the single-stranded DNA (SS). Additional properties of several mutants are also discussed.     

Cloning and expression of endo-β-glucanase II gene in Humicola insolens H31-3

Endo-β-glucanase II (EG II) gene cDNA was isolated from the fungus Humicola insolens H31-3 by RT-PCR. It was cloned into the expression vector pGAPZαA. The resultant recombinant plasmid was introduced into Pichia pastoris GS115 by electroporation after being linearized by BspHI digestion. The recombinant Pichia pastoris strain was obtained and SDS-PAGE showed that the molecular weight of the expression protein was about 55 kD.The cultivation condition and the characteristics of the recombinant EG II were also explored. __________ Translated from Microbiology, 2006, 33(6): 68273 [译自: 微生物学 通报]     

Small-molecule modulators of 14-3-3 protein–protein interactions

14-3-3 Proteins are eukaryotic adapter proteins that regulate a plethora of physiological processes by binding to several hundred partner proteins. They play a role in biological activities as diverse as signal transduction, cell cycle regulation, apoptosis, host-pathogen interactions and metabolic control. As such, 14-3-3s are implicated in disease areas like cancer, neurodegeneration, diabetes, pulmonary disease, and obesity. Targeted modulation of 14-3-3 protein–protein interactions (PPIs) by small molecules is therefore an attractive concept for disease intervention. In recent years a number of examples of inhibitors and stabilizers of 14-3-3 PPIs have been reported promising a vivid future in chemical biology and drug development for this remarkable class of proteins.     

Effect of phosphorylation on interaction of human tau protein with 14-3-3ζ

Interaction of the shortest isoform of tau protein (τ3) with human 14-3-3ζ was analyzed by means of native gel electrophoresis, chemical crosslinking and size-exclusion chromatography. Phosphorylation by cAMP-dependent protein kinase (up to 2 mole of phosphate per mole of τ3) strongly enhanced interaction of τ3 with 14-3-3. Apparent K_D of the complexes formed by phosphorylated τ3 and 14-3-3 was close to 2 μM, whereas the corresponding constant for unphosphorylated τ3 was at least 10 times higher. The stoichiometry of the complexes formed by phosphorylated τ3 and 14-3-3 was variable and was different from 1:1. 14-3-3 decreased the probability of formation of chemically crosslinked large homooligomers of phosphorylated τ3 and at the same time induced formation of crosslinked heterooligomeric complexes of τ3 and 14-3-3 with an apparent molecular mass of 120–140 kDa.     

Arachidonic Acid Binds 14-3-3ζ, Releases 14-3-3ζ from Phosphorylated BAD and Induces Aggregation of 14-3-3ζ

Polyunsaturated fatty acids, like arachidonic acid, can bind proteins and affect their function. The 14-3-3 proteins bind phosphorylated sites on a diverse array of client proteins and, in this way, are involved in many intracellular signaling pathways. In this study, we used a novel approach to discover that 14-3-3ζ is able to directly bind arachidonic acid. Furthermore, arachidonic acid, at physiological concentrations, reduced the binding of 14-3-3ζ to phosphorylated BAD, an interaction that is important in regulating apoptosis. In addition, high concentrations of arachidonic acid caused the polymerization of 14-3-3ζ, an event observed in neurodegenerative disorders. Taken together, these results indicate that arachidonic acid directly interacts with 14-3-3ζ and that this interaction may be important in both normal and pathological cellular events. If so, then factors that mediate the release, metabolism and reacylation of arachidonic acid into membranes represent key points of regulation.     

The down-regulated expression of 14-3-3σ may activate the other six 14-3-3 isoforms and they may take over the role of 14-3-3σ in the tumor genesis

Seven isoforms of 14-3-3 protein family have different functions in the cancer genesis and progress. It is found that six isoforms were up-regulated expression and inclined to sustain the cancer survival. Conversely, 14-3-3σ strongly promotes cancer apoptosis. Its down-regulated expression was found in many cancer tissues and thought to be an early event in the tumor genesis. Interestingly, no suggestions are made about the possible effect that the down-regulated expression of 14-3-3σ activated the other six 14-3-3 isoforms and they take over the role of 14-3-3σ in the tumor genesis. The inactivation of 14-3-3σ in the early stage of tumor genesis is a clue to trigger the other six 14-3-3 isoforms activation.     

14-3-3 η inhibits chondrogenic differentiation of ATDC5 cell

It was previously shown that 14-3-3η is overexpressed in the synovial fluid of patients with joint inflammation, which is often associated with growth failure. In this study, we investigated the role of 14-3-3η in chondrogenesis using ATDC5 cells. Upon treatment with TNF-α, cells overexpressed 14-3-3η with inhibition of chondrogenesis. Chondrogenesis was also inhibited by overexpression of 14-3-3η without TNF-α treatment, whereas silencing of 14-3-3η promoted chondrogenic differentiation. Further, G1 phase arrest was inhibited by overexpression of 14-3-3η. In summary, we suggest that 14-3-3η plays a regulatory role in chondrogenic differentiation.     

Targeted proteomic analysis of 14-3-3σ in nasopharyngeal carcinoma

14-3-3σ is a potential tumor suppressor, and loss of 14-3-3σ expression plays an important role in carcinogenesis and metastasis. To explore the possible mechanism of 14-3-3σ in nasopharyngeal carcinoma (NPC) invasion and metastasis, targeted proteomic analysis was performed on 14-3-3σ-associated proteins from NPC cells. As the results, 112 proteins associated with 14-3-3σ were identified, and four 14-3-3σ-interacted proteins: keratin 8, epidermal growth factor receptor (EGFR), small GTP-binding protein RAB7, and p53 were confirmed by coimmunoprecipitation and Western blot analysis. The 14-3-3σ-associated proteins could be grouped into eight clusters based on their molecule functions. Protein–protein interaction (PPI) analysis indicated that 14-3-3σ/EGFR/keratin 8 interactions may be involved in the invasion and metastasis of NPC. 14-3-3σ/EGFR/keratin 8 could form complexes in NPC cells. 14-3-3σ downregulation in NPC may lead to the overexpression of EGFR and keratin 8, which increases the invasion ability of NPC cells possibly by activating the downstream signal molecules and reorganizing cytoskeleton. The data suggest that the biological functions of 14-3-3σ in NPC are diversified, and 14-3-3σ could inhibit the in vitro invasive ability of NPC cells possibly through 14-3-3σ/EGFR/keratin 8 interaction.     

In vitro α1-3 or α1-4 fucosylation of type I and II oligosaccharides with secreted forms of recombinant human fucosyltransferases III and VI

Transgalactosylation of chitobiose and chitotriose employing -galactosidase from bovine testes yielded mixtures with 1-3 linked galactose (type I) and 1-4 linked galactose (type II) in a final ratio of 1:1 for the tri- and 1:1.4 for the tetrasaccharide. After 24 h incubations of the two purified oligosaccharide mixtures with large amounts (20-fold increase compared with standard conditions) of human 1, 3/4-fucosyltransferase III (FucT III), the type I tri-/tetrasaccharides were completely converted to the Lewisa structure, whereas approximately 10% fucosylation of the type II isomers to the Lewisx oligosaccharides was observed in long-term incubations.Employing large amounts of human 1, 3-fucosyltransferase VI (FucT VI), the type I trisaccharide substrate was exclusively fucosylated at the proximal O-4 substituted N-acetylglucosamine (GlcNAc) (20%) whereas almost all of the type II isomers was converted to the corresponding Lewisx product. 45% of the type I tetrasaccharide was fucosylated at the second GlcNAc solely by FucT VI. The type II isomer was almost completely 1-3 fucosylated to yield the Lewisx derivative with traces of a structure that contained an additional fucose at the reducing GlcNAc. The results obtained in the present study employing high amounts of enzyme confirmed our previous results that FucT III acts preponderantly as a 1-4 fucosyltransferase onto GlcNAc in vitro. Human FucT VI attaches fucose exclusively in an 1-3 linkage to 4-substituted GlcNAc in vitro and does not modify any 3-substituted GlcNAc to yield Lewisa oligosaccharides. With 8-methoxycarbonyloctyl glycoside acceptors used under standard conditions, FucT III acts exclusively on the type I and FucT VI only on the type II derivative. With lacto-N-tetraose, lacto-N-fucopentraose I, or LS-tetrasaccharide as substrates, FucT III modified the 3-substituted GlcNAc and the reducing glucose; FucT VI recognized only lacto-N-neotetraose as a substrate.     

氨基糖苷-(3)-乙酰转移酶II的表达纯化及初步活性

用PCR扩增的氨基糖苷-(3)-乙酰转移酶II[aac(3)-II]基因构建了能表达较高活性AAC(3)-II的重组工程菌,并获取重组AAC(3)-II。含pET28a质粒的工程菌转入aac(3)-II基因前后对氨基糖苷类抗生素的最低抑菌浓度进行比较,重组菌超声裂解上清液及其纯化的AAC(3)-II进行SDS-PAGE和Western印迹电泳鉴定。最低抑菌浓度表明转入aac(3)-II基因的工程菌比未转入的工程菌对庆大霉素(gentamicin,GEN)的提高了256倍,对妥布霉素(tobramycin,TOB)及其奈替米星(netilmicin,NTL)提高了16倍,电泳鉴定表明纯化获取的是重组AAC(3)-II,其相对分子质量约为32kDa,纯度大于95%,纯化后的10μgAAC(3)-II,分别在10s内使80μgGEN、30s内使80μgTOB和NTL乙酰化而失去抑菌作用。研究为氨基糖苷类抗生素伴侣的开发研究初步打下基础。     

14-3-3ε Overexpression Contributes to Epithelial-Mesenchymal Transition of Hepatocellular Carcinoma

Background

14-3-3ε is implicated in regulating tumor progression, including hepatocellular carcinoma (HCC). Our earlier study indicated that elevated 14-3-3ε expression is significantly associated with higher risk of metastasis and lower survival rates of HCC patients. However, the molecular mechanisms of how 14-3-3ε regulates HCC tumor metastasis are still unclear.

Methodology and Principal Findings

In this study, we show that increased 14-3-3ε expression induces HCC cell migration and promotes epithelial-mesenchymal transition (EMT), which is determined by the reduction of E-cadherin expression and induction of N-cadherin and vimentin expression. Knockdown with specific siRNA abolished 14-3-3ε-induced cell migration and EMT. Furthermore, 14-3-3ε selectively induced Zeb-1 and Snail expression, and 14-3-3ε-induced cell migration was abrogated by Zeb-1 or Snail siRNA. In addition, the effect of 14-3-3ε-reduced E-cadherin was specifically restored by Zeb-1 siRNA. Positive 14-3-3ε expression was significantly correlated with negative E-cadherin expression, as determined by immunohistochemistry analysis in HCC tumors. Analysis of 14-3-3ε/E-cadherin expression associated with clinicopathological characteristics revealed that the combination of positive 14-3-3ε and negative E-cadherin expression is significantly correlated with higher incidence of HCC metastasis and poor 5-year overall survival. In contrast, patients with positive 14-3-3ε and positive E-cadherin expression had better prognostic outcomes than did those with negative E-cadherin expression.

Significance

Our findings show for the first time that E-cadherin is one of the downstream targets of 14-3-3ε in modulating HCC tumor progression. Thus, 14-3-3ε may act as an important regulator in modulating tumor metastasis by promoting EMT as well as cell migration, and it may serve as a novel prognostic biomarker or therapeutic target for HCC.     

Theoretical description of the magnetic properties of μ3-hydroxo bridged trinuclear copper(II) complexes

A theoretical study of the magnetic properties, using density functional theory, of a family of trinuclear μ₃-OH copper(II) complexes reported in the literature is presented. The reported X-ray crystal structures of [Cu₃(μ₃-OH)(aat)₃(H₂O)₃](NO₃)₂·H₂O (HUKDUM), where aat: 3-acetylamine-1,2,4-triazole; [Cu₃(μ₃-OH)(aaat)₃(H₂SO₄)(HSO₄)(H₂O)] (HUKDOG), where aaat: 3-acetylamine-5-amine-1,2,4-triazole; [Cu₃(μ₃-OH)(PhPyCNO)₃(tchlphac)₂] (HOHQUR), where PhPyCNO: phenyl 2-pyridyl-ketoxime and tchlphac: acid 2,4,5-trichlorophenoxyacetic; [Cu₃(μ₃-OH)(PhPyCNO)₃(NO₃)₂(CH₃OH)] (ILEGEM); [Cu₃(μ₃-OH)(pz)₃(Hpz)₃(ClO₄)₂] (QOPJIP), where Hpz?=?pyrazole; [Cu₃(μ₃-OH)(pz)₃(Hpz)(Me₃CCOO)₂]?2Me₃CCOOH (DEFSEN) and [Cu₃(μ₃-OH)(8-amino-4-methyl-5-azaoct-3-en-2-one)₃][CuI₃] (RITXUO), were used in the calculations. The magnetic exchange constants were calculated using the broken-symmetry approach. The calculated J values are for HUKDUM J₁?=??68.6 cm^?1, J₂?=??69.9 cm^?1, J₃?=??70.4 cm^?1; for HUKDOG, J₁?=??73.5 cm^?1, J₂?=??58.9 cm^?1, J₃?=??62.1 cm^?1; for HOHQUR J₁?=??128.3 cm^?1, J₂?=??134.1 cm^?1, J₃?=??120.4 cm^?1; for ILEGEM J₁?=??151.6 cm^?1, J₂?=??173.9 cm^?1, J₃?=??186.9 cm^?1; for QOPJIP J₁?=??118.3 cm^?1, J₂?=??106.0 cm^?1, J₃?=??120.6 cm^?1; for DEFSEN J₁?=??74.9 cm^?1, J₂?=??64.0 cm^?1, J₃?=??57.7 cm^?1 and for RITXUO J₁?=??10.9 cm^?1, J₂?=?+14.3 cm^?1, J₃?=??35.4 cm^?1. The Kahn-Briat model was used to correlate the calculated magnetic properties with the overlap of the magnetic orbitals. Spin density surfaces show that the delocalization mechanism is predominant in all the studied compounds.