NRT1 family proteins play an important roles for absorbing and transporting of nitrate in different plants. In order to identify the NRT1 family genes of Rehmannia glutinosa, this study used 11 NRT1 homologous proteins of Arabidopsis as probe sequences and aligned with the transcriptome data of R. glutinosa by using NCBI BLASTN software. Resulting there were 18 NRT1 proteins were identified in R. glutinosa. On basis of this, a series of the molecular characteristics of R. glutinosa NRT1 proteins including the conserved domains, the transmembrane structure, the subcellular location and phylogenetic features were in detail analyzed. At same time, it were systematically analyzed that the temporal and spatial expression patterns and characteristics of R. glutinosa NRT1 family genes in response to different stress factors. The results indicated that 18 R. glutinosa NRT1 family genes with the length of coding region from 1 260 bp to 1 806 bp, encoded proteins ranging from 419 to 601 amino acids, and all of they owned the domains of typical peptide transporter with 7 to 12 transmembrane domains. These R. glutinosa NRT1 family proteins mostly were found to locate on cellular plasma membrane, and belonged to the hydrophobic proteins. Furthermore, the evolutionary analysis found that the 18 R. glutinosa NRT1 protein family could be divided into two subfamilies, of which 14 NRT1 family genes might occur the positive selection, and 4 genes occur the passivation selection during the evolution process of R. glutinosa. In addition the expression analysis showed that 18 R. glutinosa NRT1 family genes have the distinct expression patterns in different tissues of R. glutinosa, and their expression levels were also obvious difference in response to various stress. These findings infield that 18 R. glutinosa NRT1 family proteins might have obviously different functional roles in nitrate transport of R. glutinosa. In conclusion, this study lays a solid theoretical foundation for clarifying the absorption and transport molecular mechanism of N element during R. glutinosa growth and development, and at same time for deeply studying the molecular function of R. glutinosa NRT1 proteins in absorption and transport of nitrate.
Anion Transport Proteins ; Membrane Transport Proteins ; Nitrates ; Phylogeny ; Plant Proteins/metabolism* ; Rehmannia/genetics* ; Transcriptome

In Escherichia coli, uptake of L-tryptophan is done by three distinct permeases, encoded by mtr, tnaB, and aroP. Based on the mtr single-gene knockout, we constructed the mtr.tnaB and mtr.aroP double-gene knockout mutants and the mtr.tnaB.aroP triple-gene knockout mutant. The fermentation results showed that the mtr.tnaB and mtr.aroP knockout mutants produced 1.38 g/L and 1.27 g/L L-tryptophan, respectively, which was 17% and 9% higher than that of the mtr knockout mutant. However, the mtr.tnaB.aroP knockout mutant was significantly affected on cell growth and only produced 0.63 g/L L-tryptophan. During the fed-batch fermentation in a 3-L fermentor, the mtr.tnaB knockout mutant produced 12.2 g/L L-tryptophan, which was 27% higher than that of the mtr knockout mutant. This study demonstrates the effect of multi-gene knockouts of L-tryptophan transport system of Escherichia coli on the biosynthesis of L-tryptophan.
Amino Acid Transport Systems ; genetics ; Biological Transport ; Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; Fermentation ; Gene Knockout Techniques ; Membrane Transport Proteins ; genetics ; metabolism ; Mutant Proteins ; metabolism ; Tryptophan ; biosynthesis ; genetics ; metabolism

LR11, also known as SorLA or SORL1, is a type-I membrane protein from which a large extracellular part, soluble LR11 (sLR11), is released by proteolytic shedding on cleavage with a disintegrin and metalloproteinase 17 (ADAM17). A shedding mechanism is presumed to have a key role in the functions of LR11, but the evidence for this has not yet been demonstrated. Tetraspanin CD9 has been recently shown to regulate the ADAM17-mediated shedding of tumor necrosis factor-alpha and intercellular adhesion molecule-1 on the cell surface. Here, we investigated the role of CD9 on the shedding of LR11 in leukocytes. LR11 was not expressed in THP-1 monocytes, but it was expressed and released in phorbol 12-myristate 13-acetate (PMA)-induced THP-1 macrophages (PMA/THP-1). Confocal microscopy showed colocalization of LR11 and CD9 proteins on the cell surface of PMA/THP-1. Ectopic neo-expression of CD9 in CCRF-SB cells, which are LR11-positive and CD9-negative, reduced the amount of sLR11 released from the cells. In contrast, incubation of LR11-transfected THP-1 cells with neutralizing anti-CD9 monoclonal antibodies increased the amount of sLR11 released from the cells. Likewise, the PMA-stimulated release of sLR11 increased in THP-1 cells transfected with CD9-targeted shRNAs, which was negated by treatment with the metalloproteinase inhibitor GM6001. These results suggest that the tetraspanin CD9 modulates the ADAM17-mediated shedding of LR11 in various leukemia cell lines and that the association between LR11 and CD9 on the cell surface has an important role in the ADAM17-mediated shedding mechanism.
ADAM Proteins/*metabolism ; Antigens, CD9/genetics/*metabolism ; Cell Line, Tumor ; Humans ; LDL-Receptor Related Proteins/genetics/*metabolism ; Leukocytes/*metabolism ; Macrophages/metabolism ; Membrane Transport Proteins/genetics/*metabolism ; Proteolysis

OBJECTIVETo introduce a new method for detecting mitochondrial permeability transition pore (PTP) opening with flow cytometry using the resveratrol-inducing PTP opening model.

METHODSMitochondria were isolated from rat livers and selectively labeled with nonyl acridine orange. The mitochondrial membrane potential was detected using flow cytometry with TMRE (tetramethylrhodamine, ethyl ester) labeling. PTP opening induced by resveratrol was represented by the changes of mitochondrial side-scattering (SSC) detected by flow cytometry.

RESULTSFlow cytometry was capable of defining the purity of the mitochondria isolated. The fluorescence intensities and SSC of the mitochondria were decreased after resveratrol treatment, indicating that resveratrol could induce PTP opening. Ciclosporin A inhibited resveratrol-induced PTP opening.

CONCLUSIONFlow cytometric analysis allows accurate and convenient detection of mitochondrial membrane potential, mitochondrial swelling and PTP opening.

Animals ; Apoptosis ; Flow Cytometry ; Membrane Potential, Mitochondrial ; genetics ; Mitochondria, Liver ; metabolism ; Mitochondrial Membrane Transport Proteins ; metabolism ; Rats ; Rhodamines

AIMTo determine the feasibility of establishing the heterologous expression model of human- serotonin transporter(hSERT or 5-HTT).

METHODScRNA of SERT was transcribed from cDNA, which was cloned in the pOTV vector. Each oocyte of mature xenopus laevis was injected with transcribed cRNA in vivo and incubated at room temperature for 4-9 days. Recording the current induced by 5-HT with voltage clamp technique tested the function of the expressed 5-HT transporter.

RESULTSThe transporter current could be observed in Ringer's solution containing 5-HT, and the 5-HT induced current were concentration-dependent. Norepinephrine and dopamine could not induce the transporter current while the 5-HT induced current could be specifically inhibited by 5-HTT blocker, desipramine.

CONCLUSIONThe results demonstrate that the heterologous expression product in xenopus laevis oocytes is human 5-HT transporter.

Animals ; Carrier Proteins ; genetics ; DNA, Complementary ; genetics ; Female ; Gene Expression ; Models, Animal ; Oocytes ; metabolism ; RNA, Messenger ; genetics ; Serotonin ; metabolism ; Serotonin Plasma Membrane Transport Proteins ; biosynthesis ; genetics ; Xenopus laevis

Selection of suitable signal peptides is an important factor for efficient secretion of heterologous proteins. We defined structural fusion degree (SFD) as the compatibility degree of target proteins and signal peptides by a bioinformatics approach. We mathematically analyzed the interaction of fused signal peptides and adjacent residues of proteins, and proposed a mathematical model of extended signal region and the protein. SFD Features was extracted from this model to characterize the secretability of heterologous proteins. Simulation tests showed that SFD features can effectively discriminate high secretory proteins from poor ones in the host Bacillus subtilis. Results from this research will be useful in signal peptide selection and have a better guiding significance for the optimization of heterologous protein secretion.
Amino Acid Sequence ; Bacillus subtilis ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Biotechnology ; methods ; Membrane Transport Proteins ; genetics ; metabolism ; Metalloendopeptidases ; genetics ; metabolism ; Molecular Sequence Data ; Protein Sorting Signals ; genetics ; Proteins ; secretion ; Recombinant Fusion Proteins ; genetics ; metabolism

OBJECTIVETo investigate the role of AdeABC efflux pump in carbapenems resistance of Acinetobacter baumannii in light of the phenotype and genetype of the efflux pump.

METHODSThe phenotype of the efflux pump was detected in 138 clinical isolates of A.baumannii using the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The mRNA expression of pump-encoding gene adeB in the strains was detected using quantitative real-time RT-PCR.

RESULTSOf the 138 strains, 28 showed positivities for AdeABC efflux pump identified by Mueller-Hinton Broth with CCCP. Of the 39 strains resistant to meropenem, 15 (38.4%) showed positive results in CCCP assay, a rate significantly higher than that among the 99 sensitive strains (13.1%, 13/99) (X(2)=12.477(b), P=0.01). The mRNA expression of efflux pump-encoding gene adeB was detected by real-time RT-PCR at a level of 0.899∓∓1.172 in meropenem-sensitive strains, significantly lower than the level of 21.101∓∓21.443 in meropenem-resistant strains (t=4.403, P=0.000).

CONCLUSIONSEfflux plays a role in carbapenems resistance in the clinical isolates of A. baumannii. The AdeABC efflux pump may be an important factor in reducing carbapenems sensitivity in A. baumannii.

Acinetobacter baumannii ; drug effects ; isolation & purification ; Bacterial Proteins ; genetics ; metabolism ; Carbapenems ; pharmacology ; Humans ; Membrane Transport Proteins ; genetics ; metabolism ; beta-Lactam Resistance ; genetics

Cocaine analogue, CFT (2beta-carbomethoxy-3beta-(4-fluorophenyl) tropane) binding to dopamine transporter (DAT) in different species is quite heterogeneous. CFT is scarcely detected in bovine DAT whereas it is conspicuous in humans. To examine the structural basis for this functional discrepancy, we analyzed transporter chimeras of these two DATs. The CFT binding activities are avid in all of the chimeric DATs of which both of the 3rd and the 6-8th transmembrane domain (TM) are composed of human DAT sequences. On the contrary, CFT binding activities were scarcely detected if either or both of two regions are replaced with bovine sequences. These findings indicate that the CFT binding absolutely requires human DAT sequences, at least, in the regions encompassing the 3rd and 6-8th transmembrane domain (TM), and that these regions might contribute to form the 3-dimensional pocket for CFT binding.
Animal ; Cattle ; Chimeric Proteins/genetics/metabolism ; Cocaine/*analogs & derivatives/*metabolism ; Human ; Membrane Transport Proteins/*genetics/*metabolism ; Protein Binding ; Protein Structure, Tertiary

Drug transport is an important source of inter-individual variations in drug responses and is also a common site where drug-drug interactions happen. In recent years, more and more novel identified transporters have been added into the transporter super family, and this trend will continue in the future. Among the transporter members of this family, ATP-dependent efflux transporter P-glycoprotein (MDR1) and organic anion transporters (OATP) are the most important proteins involved in drug transport. MDR1 is the most well known transporter. Widely distributed in tissues such as the gastrointestinal tract, liver, kidney and so on, MDR1 plays an important role in drug absorption, distribution and excretion. Its functional genetic polymorphisms have significantly changed the pharmacokinetics of its substrate drugs, which has important clinical implications. OATP expressed in multiple tissues, and it mediated the drug excretion through the bile acid and kidney. Some genetic polymorphism of OATP genes is the cause of some abnormal drug responses.
ATP Binding Cassette Transporter, Subfamily B, Member 1 ; genetics ; Drug Interactions ; genetics ; Humans ; Membrane Transport Proteins ; genetics ; metabolism ; Organic Anion Transporters ; genetics ; Pharmaceutical Preparations ; metabolism ; Polymorphism, Genetic

Sugarcane molasses containing large amounts of sucrose is an economical substrate for succinic acid production. However, Escherichia coli AFP111 cannot metabolize sucrose although it is a promising candidate for succinic acid production. To achieve sucrose utilizing ability, we cloned and expressed cscBKA genes encoding sucrose permease, fructokinase and invertase of non-PTS sucrose-utilization system from E. coli W in E. coli AFP111 to generate a recombinant strain AFP111/pMD19T-cscBKA. After 72 h of anaerobic fermentation of the recombinant in serum bottles, 20 g/L sucrose was consumed and 12 g/L succinic acid was produced. During dual-phase fermentation comprised of initial aerobic growth phase followed by anaerobic fermentation phase, the concentration of succinic acid from sucrose and sugarcane molasses was 34 g/L and 30 g/L, respectively, at 30 h of anaerobic phase in a 3 L fermentor. The results show that the introduction of non-PTS sucrose-utilization system has sucrose-metabolizing capability for cell growth and succinic acid production, and can use cheap sugarcane molasses to produce succinic acid.
Bioreactors ; Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; Fermentation ; Membrane Transport Proteins ; genetics ; Metabolic Engineering ; Molasses ; Saccharum ; chemistry ; Succinic Acid ; chemistry ; Sucrose ; chemistry