OBJECTIVETo obtain the Escherichia coli strains expressing N-Acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase).

METHODSThe gene (nanA) coding Neu5Ac aldolase was cloned from Escherichia coli C600, and the recombinant plasmid was sequenced and expressed in Escherichia coli.

RESULTSSequencing data revealed that the open reading frame was 894 bp and predicted to encode a protein consisting of 298 amino acids. The patterns of SDS-PAGE showed that the purified enzyme protein as a single protein band with a molecular weight of 33 kD, which was consistent with those reported in the reference. In the recombinant plasmid pRY1, the expression of nanA gene was controlled by the lac promoter with the induction of IPTG or lactose. The plasmid pRY3 was constructed, in which the nanA gene ws controlled by the tac promoter. The protein of Neu5Ac aldolase was constitutively expressed using the recombinant strain, E.coli DH5 alpha/pRY3 without induction of IPTG or lactose. The crystal was finally obtained with the efficiency of 90.2% of Neu5Ac. The HPLC indicated that the Neu5Ac crystal prepared in this experiment was same as Simga product.

CONCLUSIONThe protein products expressed by two recombinant strains E.coli BL21(DE3)/pRY1 and DH5 alpha/pRY3 has the characteristics of Neu5Ac.

Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Open Reading Frames ; Oxo-Acid-Lyases ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Recombination, Genetic

A N-acetylneuraminate lyase gene (shnal) from Staphylococcus hominis was cloned into pET-28a and expressed in Escherichia coli BL21 (DE3) host cells. The recombinant enzyme was purified and characterized. It is a homotetrameric enzyme with the optimum pH at 8.0 for the cleavage direction and the optimum pH and temperature were 7.5 and 45 degrees C for the synthetic direction. The activity of ShNAL is stable when incubated at 45 degrees C for 2 h but decreased rapidly over 50 degrees C. ShNAL showed high stability in a wide range pH from 5.0 to 10.0 with the residual activity being > 70% when the enzyme was incubated in different buffers at 4 degrees C for 24 h. Its K(m) towards N-acetylneuraminic acid, pyruvate and ManNAc were (4.0 +/- 0.2) mmol/L, (35.1 +/- 3.2) mmol/L and (131.7 +/- 12.1) mmol/L, respectively. The k(cat)/K(m) value of Neu5Ac, ManNAc, and Pyr for ShNAL were 1.9 L/(mmol x s), 0.08 L/(mmol x s) and 0.08 L/(mmol x s), respectively.
Bacterial Proteins ; genetics ; metabolism ; Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Hydrogen-Ion Concentration ; Oxo-Acid-Lyases ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Staphylococcus hominis ; enzymology ; Temperature

Amino Acid Metabolism, Inborn Errors ; diagnosis ; genetics ; therapy ; Death, Sudden ; etiology ; Hereditary Central Nervous System Demyelinating Diseases ; diagnosis ; etiology ; Humans ; Hydroxymethylglutaryl-CoA Synthase ; deficiency ; Infant, Newborn ; Male ; Mutation ; Oxo-Acid-Lyases ; genetics ; Spectrometry, Mass, Electrospray Ionization ; Tandem Mass Spectrometry

BACKGROUND: Enhanced lipogenesis plays a critical role in cell senescence via induction of expression of the mature form of sterol regulatory element binding protein 1 (SREBP1), which contributes to an increase in organellar mass, one of the indicators of senescence. We investigated the molecular mechanisms by which signaling molecules control SREBP1-mediated lipogenesis and senescence. METHODS: We developed cellular models for stress-induced senescence, by exposing Chang cells, which are immortalized human liver cells, to subcytotoxic concentrations (200 microM) of deferoxamine (DFO) and H2O2. RESULTS: In this model of stress-induced cell senescence using DFO and H2O2, the phosphorylation profile of glycogen synthase kinase 3alpha (GSK3alpha) and beta corresponded closely to the expression profile of the mature form of SREBP-1 protein. Inhibition of GSK3 with a subcytotoxic concentration of the selective GSK3 inhibitor SB415286 significantly increased mature SREBP1 expression, as well as lipogenesis and organellar mass. In addition, GSK3 inhibition was sufficient to induce senescence in Chang cells. Suppression of GSK3 expression with siRNAs specific to GSK3alpha and beta also increased mature SREBP1 expression and induced senescence. Finally, blocking lipogenesis with fatty acid synthase inhibitors (cerulenin and C75) and siRNA-mediated silencing of SREBP1 and ATP citrate lyase (ACL) significantly attenuated GSK3 inhibition-induced senescence. CONCLUSION: GSK3 inactivation is an important upstream event that induces SREBP1-mediated lipogenesis and consequent cell senescence.
Aging* ; Aminophenols ; ATP Citrate (pro-S)-Lyase ; Carrier Proteins* ; Cell Aging ; Deferoxamine ; Fatty Acid Synthetase Complex ; Glycogen Synthase Kinase 3* ; Glycogen Synthase Kinases* ; Glycogen Synthase* ; Glycogen* ; Humans ; Lipogenesis* ; Liver ; Maleimides ; Multienzyme Complexes ; Oxo-Acid-Lyases ; Phosphorylation ; RNA, Small Interfering ; Sterol Regulatory Element Binding Protein 1

BACKGROUND: Enhanced lipogenesis plays a critical role in cell senescence via induction of expression of the mature form of sterol regulatory element binding protein 1 (SREBP1), which contributes to an increase in organellar mass, one of the indicators of senescence. We investigated the molecular mechanisms by which signaling molecules control SREBP1-mediated lipogenesis and senescence. METHODS: We developed cellular models for stress-induced senescence, by exposing Chang cells, which are immortalized human liver cells, to subcytotoxic concentrations (200 microM) of deferoxamine (DFO) and H2O2. RESULTS: In this model of stress-induced cell senescence using DFO and H2O2, the phosphorylation profile of glycogen synthase kinase 3alpha (GSK3alpha) and beta corresponded closely to the expression profile of the mature form of SREBP-1 protein. Inhibition of GSK3 with a subcytotoxic concentration of the selective GSK3 inhibitor SB415286 significantly increased mature SREBP1 expression, as well as lipogenesis and organellar mass. In addition, GSK3 inhibition was sufficient to induce senescence in Chang cells. Suppression of GSK3 expression with siRNAs specific to GSK3alpha and beta also increased mature SREBP1 expression and induced senescence. Finally, blocking lipogenesis with fatty acid synthase inhibitors (cerulenin and C75) and siRNA-mediated silencing of SREBP1 and ATP citrate lyase (ACL) significantly attenuated GSK3 inhibition-induced senescence. CONCLUSION: GSK3 inactivation is an important upstream event that induces SREBP1-mediated lipogenesis and consequent cell senescence.
Aging* ; Aminophenols ; ATP Citrate (pro-S)-Lyase ; Carrier Proteins* ; Cell Aging ; Deferoxamine ; Fatty Acid Synthetase Complex ; Glycogen Synthase Kinase 3* ; Glycogen Synthase Kinases* ; Glycogen Synthase* ; Glycogen* ; Humans ; Lipogenesis* ; Liver ; Maleimides ; Multienzyme Complexes ; Oxo-Acid-Lyases ; Phosphorylation ; RNA, Small Interfering ; Sterol Regulatory Element Binding Protein 1

OBJECTIVETo explore the role of BM2 protein in the life cycle of influenza B virus.

METHODSThe authors screened human kidney MATCHMAKER cDNA library for new binding partners of BM2 of influenza B virus by using the yeast two hybrid system with truncated BM2 (26-109 aa) as the bait.

RESULTSSix positive plasmids encoding N-acetylneuraminate pyruvate lyase, angiopoietin 3, zinc finger protein 251, ribosomal protein S20, protein arginine N-methyltransferase 1 variant 1 (PRMT) and transcription factor-like 1 (TCFL1) were obtained.

CONCLUSIONThe results suggest that BM2 may play an important role in the life cycle of influenza B virus.

Angiopoietin-like Proteins ; Angiopoietins ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Gene Library ; Humans ; Influenza B virus ; genetics ; metabolism ; Kidney ; metabolism ; Oxo-Acid-Lyases ; genetics ; metabolism ; Plasmids ; genetics ; Protein Binding ; Protein-Arginine N-Methyltransferases ; genetics ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Ribosomal Proteins ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism ; Two-Hybrid System Techniques ; Viral Proteins ; genetics ; metabolism ; Zinc Fingers ; genetics

Because of the concern about escape of antibiotic- or herbicide-resistant transgenes from transgenic crops, selectable marker genes from plant origin would be an alternative choice for plant transformation. In this study, a feedback-insensitive anthranilate synthase gene ( ASA2 ) cloned from a tobacco cell line was tested for Agrobacterium-mediated transformation of axis tissue of soybean mature embryo, with a tryptophan analogue 5-methyltryptophan (5-MT) as the selective agent. Southern blot analysis of the To transgenic lines confirmed the integration of the ASA2 gene into the soybean genome. Northern blot analysis showed the ASA2 gene was also expressed in the leave tissue, and the free tryptophan content in the leaf tissue of transgenic soybean was about 59% to 123% more than that in the wild type. PCR analysis of the T1 progeny showed that the transgene was inherited in a Mendelian fashion. All these results indicate that this feedback-insensitive ASA2 gene can be used as a selectable marker gene for plant transformation. This work also demonstrated that the ASA2 gene coding for the a-subunits from one plant (tobacco) can interact with the n-subunits of a heterologous plant (soybean) to form an active anthranilate synthase enzyme. The use of this feedback-insensitive gene as a novel selectable marker for plant transformation is also discussed.
Anthranilate Synthase ; genetics ; Feedback, Physiological ; Plants, Genetically Modified ; genetics ; Soybeans ; genetics ; Transformation, Genetic ; Tryptophan ; analogs & derivatives ; metabolism

Talaromyces marneffei is the only dimorphic species in its genus and causes a fatal systemic mycosis named talaromycosis. Our previous study indicated that knockdown of AcuD gene (encodes isocitrate lyase of glyoxylate bypass) of T. marneffei by RNA interference approach attenuated the virulence of T. marneffei, while the virulence of the AcuD knockout strains was not studied. In this study, T. marneffei-zebrafish infection model was successfully established through hindbrain microinjection with different amounts of T. marneffei yeast cells. After co-incubated at 28°C, the increasing T. marneffei inoculum doses result in greater larval mortality; and hyphae generation might be one virulence factor involved in T. marneffei-zebrafish infection. Moreover, the results demonstrated that the virulence of the ΔAcuD was significantly attenuated in this Zebrafish infection model.
Gene Knockout Techniques ; Hyphae ; Isocitrate Lyase ; Microinjections ; Mortality ; Rhombencephalon ; RNA Interference ; Talaromyces ; Virulence ; Yeasts ; Zebrafish

The glyoxylate cycle was hypothesed to be indispensable for glutamate overproduction in coryneform bacteria, for it was thought to fulfill anaplerotic functions and to supply energy during the growth phase. During glutamate overproduction phase, however, it has been noted that the high level of the cycle is detrimental to the glutamate production. In order to clarify the relationship between the glutamate production and the glyoxylate cycle, a chromosomal aceA-disrupted mutant of wild-type C. glutamicum ATCC 13032 was constructed. The isocitrate lyase (ICL) activity of the parental strain was 0.011 u/mg of protein and reached 1.980 u/mg of protein after acetate induction; the mutant strain WTdeltaA, however, had no detectable ICL activity and was no longer able to grow on minimal medium with acetate as the sole carbon source. Compared with the wild-type C. glutamicum WT, the mutant strain WTdeltaA, exhibited the same growth rate with glucose as the sole carbon source, indicating glyoxylate cycle is not required for its growth on glucose. On the contrary, the glutamate production in WTdeltaA was severely impaired and more residual glucose was found in the fermentation broth at the end of fermentation with the mutant strain than with the wild-type strain. Further investigations into the relationship between the glutamate production and the glyoxylate cycle are under the way, which may help to elucidate the mechanism of glutamate overproduction.
Corynebacterium glutamicum ; genetics ; growth & development ; metabolism ; Culture Media ; Fermentation ; Glucose ; metabolism ; Glutamic Acid ; biosynthesis ; Glyoxylates ; metabolism ; Isocitrate Lyase ; metabolism

OBJECTIVETo construct recombinant plasmid with isocitrate lyase (ICL) gene of Mycobacterium tuberculosis H37Rv for stable and high level expression of ICL in prokaryotic expression system.

METHODSThe recombinant plasmid with ICL gene (pET30 (a)-Rv0467) was constructed by polymerase chain reaction and cloning. The fusion protein was expressed in E. coli host strain BL21 (DE3). Activity of the fusion protein was studied after it was purified with metal chelating chromatography.

RESULTSWe constructed the plasmid which could highly express Mycobacterium tuberculosis H37Rv ICL. The plasmid was highly expressed in E. coli BL21 (DE3), in which the fusion protein accounted for 30% of total protein content. After having been purified by metal chelating chromatography, the purity of the soluble fusion protein was 90%. The fusion protein had activity of ICL.

CONCLUSIONUsing the prokaryotic expression system, the ICL gene of Mycobacterium tuberculosis H37Rv was successfully cloned and expressed, which build the basis for screening new anti-tuberculosis drugs with ICL as the target point.

Cloning, Molecular ; Escherichia coli ; genetics ; Gene Transfer Techniques ; Isocitrate Lyase ; biosynthesis ; genetics ; Mycobacterium tuberculosis ; classification ; enzymology ; genetics ; immunology ; Plasmids ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics