OBJECTIVETo compare the transcription difference of the mannitol PTS genes between epidemic and non-epidemic strains of Vibrio cholerae El Tor in mannitol ferment tests.

METHODSGrowth curves of 10 epidemic strains (slow-ferment) and 10 non-epidemic strains (rapid-ferment) of Vibrio cholerae were detected in the process of fermentation test, and the transcriptional level of mannitol PTS operon of these strains were determined with quantitative reverse-transcriptional PCR.

RESULTSAfter 4 hours of test, the non-epidemic strains became positive and the average growth density of the non-epidemic strains was higher than that of the epidemic strains; however, some were still lower than the epidemic strains. In contrast, at the eighth hour of test, when epidemic strains got positive, they showed higher average growth density. Compared to the epidemic strains, the transcription of mannitol PTS genes of the non-epidemic strains were much more active at the 1st and 2nd hour and were lower at the 4th and 8th hour.

CONCLUSIONSThe difference of mannitol PTS operon transcription level should be an important feature to identify the epidemic and non-epidemic strains of Vibrio cholerae, which directly influences the mannitol fermentation rate during the test. The growth rate is not a key factor that affect such difference.

Bacterial Proteins ; genetics ; Gene Expression Regulation, Bacterial ; Mannitol ; metabolism ; Operon ; Phosphoenolpyruvate ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sugar Alcohol Dehydrogenases ; metabolism ; Transcription, Genetic ; Vibrio cholerae ; classification ; genetics ; metabolism

There are over 200 secretive proteins in the epididymis. Spermatozoa are generally considered to become mature and full-functional after interacting with secretive proteins in the epididymis. This review is aimed at summarizing some aspects of the biochemical, molecular and functional characterization of some new proteins recently detected in human epididymis, and is expected to contribute to further researches on the mechanism of epididymal reproduction and contraception.
Epididymis ; secretion ; Humans ; Male ; Proteins ; metabolism ; Spermatozoa ; metabolism ; Sugar Alcohol Dehydrogenases ; metabolism

OBJECTIVETo acquire purified recombinant human epididymal sperm protein P34H for basic and clinical studies.

METHODSOn the basis of cloning of P34H coding region, P34H fragment was subcloned into the pQE-30 expression vector. The recombinant expression vector designated pQE-30/P34H was transformed into E. coli to induce the expression of the recombinant protein P34H on the reduction of IPTG. After sonication, the recombinant protein P34H was purified from the supernatant with Ni-NTA resin under native conditions. It was identified by SDS-PAGE analysis and DNA sequencing.

RESULTSRecombinant expression vector pQE-30/P34H was correctly constructed, identified with PCR and double-enzyme digestion. And the results of SDS-PAGE analysis and DNA sequencing showed that the protein was what we had hoped to acquire.

CONCLUSIONPurified recombinant P34H can be acquired successfully with the above mentioned prokaryotic expression method.

Cloning, Molecular ; Gene Expression ; Genetic Vectors ; Humans ; Male ; Recombinant Proteins ; biosynthesis ; Sugar Alcohol Dehydrogenases ; biosynthesis ; genetics

1,3-Dihydroxyacetone is widely used in cosmetics, medicines and food products. We reviewed the recent progress in metabolic pathways, key enzymes, as well as metabolic engineering for microbial production of 1,3-dihydroxyacetone. We addressed the research trend to increase yield of 1,3-dihydroxyacetone by improving the activity of glycerol dehydrogenase with genetic engineering, and regulating of fermentation process based on metabolic characteristic of the strain.
Dihydroxyacetone ; biosynthesis ; Fermentation ; Genetic Engineering ; methods ; Gluconobacter oxydans ; genetics ; metabolism ; Industrial Microbiology ; methods ; Metabolic Engineering ; methods ; Sugar Alcohol Dehydrogenases ; metabolism

During epididymal transit, mammalian spermatozoa acquire new surface proteins that are necessary for gamete interaction. P34H, a member of the short-chain dehydrogenase/reductase(SDR) superfamily, is acquired on the acrosomal cap of human spermatozoon during its maturation arising within epididymis. P34H has been shown to be involved in sperm-zona pellucida interaction. Research revealed that the occurrence of low concentration of sperm protein P34H were significant amongst the idiopathic infertile male population and P34H protein could also be considered as a marker of epididymal sperm maturation in human. Therefore the level of sperm protein P34H is proposed to be a auxiliary diagnostic tool for male infertility. This paper reviews the molecular properties and regulation of the expression of P34H and its association with male reproduction.
Acyl-CoA Dehydrogenase ; Fatty Acid Desaturases ; chemistry ; Gene Expression ; Humans ; Male ; Proteins ; genetics ; physiology ; Sperm Maturation ; physiology ; Sugar Alcohol Dehydrogenases

The kinetic mechanisms of two key enzymes in the biotransformation of glycerol to 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae, glycerol dehydrogenase (GDH) and 1,3-propanediol oxidoreductase (PDOR), was characterized. Kinetics on initial velocity and product inhibition revealed that GDH and PDOR follow an ordered Bi-Bi sequential mechanism. Kinetic models for GDH and PDOR showed that the oxidation reaction catalyzed by GDH was the rate-limiting step in coupled enzymatic reaction when the GDH/PDOR was 1:1, and the NAD+ was the main form of coenzyme in the reaction. Knowledge about the kinetic mechanisms will be helpful to understand how these enzymes is regulated, which will be useful for further enzyme catalysis and metabolic engineering studies.
Alcohol Dehydrogenase ; metabolism ; Bacterial Proteins ; metabolism ; Glycerol ; metabolism ; Kinetics ; Klebsiella pneumoniae ; enzymology ; Models, Theoretical ; Propylene Glycols ; metabolism ; Substrate Specificity ; Sugar Alcohol Dehydrogenases ; metabolism

OBJECTIVESTo identify, clone and sequence human sperm protein P34H gene and perform semi-quantitative analysis of its expression in testis and epididymidis.

METHODSTotal RNA was prepared from human epididymidis tissue. cDNA fragment encoding human P34H was amplified by RT-PCR using specific primers, and then was cloned into pGEM-T vector. Inserted P34H gene was sequenced by ABI 377 DNA Sequencer. Meanwhile, semi-quantitative analysis of P34H expression in testis and caput, corpus, cauda epididymidis was done by RT-PCR based on beta-actin as an inner control.

RESULTSHuman sperm protein P34H gene was successfully cloned and its cDNA sequence was submitted to GenBank(Accession No. AF515625).

CONCLUSIONSThe cloning of P34H gene may be useful for the further basic and clinical studies on P34H.

Base Sequence ; Cloning, Molecular ; DNA, Complementary ; analysis ; Epididymis ; metabolism ; Humans ; Male ; Molecular Sequence Data ; Proteins ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sugar Alcohol Dehydrogenases ; Testis ; metabolism

The gldA gene coding glycerol dehydrogenase (GDH) was amplified by PCR with the genomic DNA of Klebsiella pneumoniae as the template. The gldA were inserted in pMD-18T to construct the recombinant cloning vector pMD-gldA. After the DNA sequence was determined, the gldA was subcloned into expression vector pET-32a (+) to construct the recombinant expression vector pET-32gldA. Upon lactose induction, soluble GDH was over-produced by E. coli BL21 (DE3) harboring the expression construct. Recombinant GDH purified by Ni-NTA affinity chromatography showed a single band about 54 kD on SDS-PAGE gel, and the specified activity was about 188 u/mg, the purification fold is 3 times and the activity recovery is 67.5%.
Chromatography, Affinity ; Cloning, Molecular ; DNA, Bacterial ; genetics ; Escherichia coli ; genetics ; isolation & purification ; metabolism ; Klebsiella pneumoniae ; enzymology ; genetics ; Sugar Alcohol Dehydrogenases ; biosynthesis ; genetics ; isolation & purification

1,3-propanediol production by microbial fermentation has become the research hot spot for its amiability with the environment. Here the molecular mechanism of glycerol bioconversion to 1,3-propanediol was outlined by elucidating the fermentation strains, metabolic pathways, regulon and key enzymes. Of enzymes, glycerol dehydrogenase, the velocity-limiting enzyme in glycerol reductive pathway, was emphatically discussed with regard to its molecular structure and reactivating factors. This paper aims to provide the basis for genetic modification of fermentation strains.
Bacteria ; enzymology ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Biosynthetic Pathways ; Fermentation ; Gene Order ; Glycerol ; metabolism ; Hydro-Lyases ; genetics ; metabolism ; Industrial Microbiology ; methods ; trends ; Propylene Glycols ; metabolism ; Sugar Alcohol Dehydrogenases ; genetics ; metabolism

OBJECTIVETo prepare and identify anti-human epididymal sperm protein P34H monoclonal antibody (McAb).

METHODSThe previously purified recombinant P34H protein was used as an antigen to immunize BALB/c mouse. Cell strains secreting anti-P34H McAb were established by hybridoma technique and then ascitic fluid-type McAb prepared. The sensitivity and specificity of McAb were detected by ELISA and Western blot, respectively.

RESULTSOne strain (2C4) of IgG1 Kappa anti-P34H McAb was harvested. The titer detected by ELISA technique was 1:10(3) - 1:10(5). Western blot of healthy human sperm samples and purified recombinant P34H antigen probed with the prepared McAb were immunoreactive at 34,000 and 27,000, respectively.

CONCLUSIONAnti-P34H McAb has been prepared successfully by the above methods, which may provide a powerful tool for exploring the relationship between P34H and male fertility.

Animals ; Antibodies, Monoclonal ; analysis ; biosynthesis ; Blotting, Western ; Cell Line, Tumor ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Hybridomas ; secretion ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; immunology ; Sensitivity and Specificity ; Sugar Alcohol Dehydrogenases ; immunology