Interactions between mycoplasma lipid-associated membrane proteins and the host cells

Mycoplamas are a group of wall-less prokaryotes widely distributed in nature, some of which are pathogenic for humans and animals. There are many lipoproteins anchored on the outer face of the plasma membrane, called lipid-associated membrane proteins (LAMPs). LAMPs are highly antigenic and could undergo phase and size variation, and are recognized by the innate immune system through Toll-like receptors (TLR) 2 and 6. LAMPs can modulate the immune system, and could induce immune cells apoptosis or death. In addition, they may associate with malignant transformation of host cells and are also con-sidered to be cofactors in the progression of AIDS.

Expression of inducible nitric oxide synthase induced by lipid-associated membrane proteins of Ureaplasma urealyticum is regulated by nuclear factor κB-mediated mechanism in murine macrophages

The aim was to investigate the molecular mechanisms responsible for the inducible nitric oxide synthase （iNOS） gene expression stimulated by lipid associated membrane proteins （LAMPs） of Ureaplasma urealytictan （ U. urealyticum ）. Detection of NO, the expression of iNOS and the activation of nuclear factor κB （NF-κB） in direct response to U. urealyticum LAMPs in a murine macrophages, the effects of pyrrolidine dithiocarbamate （PDTC）, an inhibitor of NF-κB and of cycloheximide （CHX）, a protein synthase inhibitor were available. The results indicated that U. urealyticum LAMPs stimulated mouse macrophages to express iNOS and thus produce NO in dose- and time-dependent manner by activating NF-κB. The expression of iNOS, NO production and the activation of NF-κB were inhibited by U. urealyticum LAMPs combination with PDTC or CHX. In conclusion, our findings suggest that U. urealyticum may be an etiological factor to certain diseases due to its ability to stimulate the expression of iNOS, which is probably mediated through the activation of NF-κB.

Lipid-associated membrane proteins of Mycoplasma penetrans induce production of proinflammatory cytokines in human monocytic cells

The aim of this study is to explore potential pathogenicity of Mycoplasma penetrans, and to investigate whether M. penetrans lipid-associated membrane proteins （LAMPs） could induce human monocytic cell line （THP- 1 ） to produce some proinflammatory cytokines in vitro, including interleukin- 1β （ IL- 1β）, tumor necrosis factor alpha （TNF-α）, and IL-8. THP-1 was stimulated with different concentrations of M.penetrans LAMPs and at different time to analyze the production of human IL-1β, TNF-α and IL-8. The protein levels of human IL-1β, TNF-α and IL-8 were measured by enzyme-linked immunoadsorbent assay （ELISA） and the mRNA levels of these proinflammatory cytokines were detected by reverse transcriptase-PCR （RT-PCR）. It was demonstrated in the present study that the production of IL-1β, TNF-α and IL-8 increased in dose- and time-dependent manner after stimulation with M. penetrans LAMPs in THP-1 cells. M. penetrans LAMPs also induced the expression of IL-1β, TNF-α and IL-8 mRNA. The production of IL-1β, TNF-α and IL-8 and the expression of mRNA were down-regulated by pyrrolidine dithiocarbamate （PDTC）. This study demonstrated that M. penetrans LAMPs can induce the production of proinflammatory cytokines in human monocytic cells in vitro, thus suggesting that it may be an important etiological factor.

Ureaplasma urealyticum-derived lipid-associated membrane proteins introduce IL-6, IL-8, and TNF-α cytokines into human amniotic epithelial cells via Toll-like receptor 2

Objective： The purpose of this study was to determine the role of Ureaplasma urealyticum-derived lipidassociated membrane proteins （LAMPs） in the host innate immune system, specifically their effect on Toll-like receptors （TLRs）. Methods： LAMPs were derived from U. urea/yticum strains, and human amniotic epithelial cells （HAECs） were isolated from healthy full-term placentas. Cytokine concentrations were determined by enzyme-linked immunosorbent assay （ELISA） and TLR2 mRNA by real-time PCR. Expression of TLR2 was confirmed by Western blotting and immunohistochemistry. Results： LAMPs induced HAECs to produce inflammatory cytokines interleukin （IL）-6, IL-8, and tumor necrosis factor （TNF）-α. Cytokine production was reduced after blocking TLR2 using TLR2 inhibitor （anti-hTLR2-IgA）. Conclusions： LAMPs isolated from U. urealyticum induced TLR2-dependent up-regulation of inflammatory genes and cytokines in HAECs.

Activation of nuclear factor κB and induction of inducible nitric oxide synthase by lipid-associated membrane proteins isolated from Mycoplasma penetrans

Background This study was designed to investigate the potential pathogenicity of Mycoplasma penetrans (M. penetrans) and its molecular mechanisms responsible for the induction of iNOS gene expression in mouse macrophages stimulated by lipid-associated membrane proteins (LAMPs) prepared from M. penetrans.Methods Mouse macrophages were stimulated with M. penetrans LAMPs to assay the production of nitric oxide (NO). The expression of inducible nitric oxide synthase (iNOS) was detected by RT-PCR and Western blotting. The activity of nuclear factor κB (NF-κB) and the effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, on the production of nitric oxide and the expression of iNOS were also assessed in mouse macrophages treated with M. penetrans LAMPs by indirect immunofluorescence and Western blotting.Results M. penetrans LAMPs stimulated mouse macrophages to produce nitric oxide in a dose- and time-dependent manner. The mRNA and protein levels of iNOS were also upregulated in response to LAMP stimulation and inhibited by PDTC treatment. M. penetrans LAMPs were found to trigger NF-κB activation, a possible mechanism for the induction of iNOS expression.Conclusion This study demonstrated that M. penetrans may be an important etiological factor of certain diseases due to the ability of M. penetrans LAMPs to stimulate the expression of iNOS, which is probably mediated through the activation of NF-κB.

Functional characterization of sensory neuron membrane protein 1a involved in sex pheromone detection of Apolygus lucorum(Hemiptera:Miridae)

The mirid bug Apolygus lucorum(Hemiptera:Miridae)is a polyphagous pest that affects a wide range of host plants.Its control remains challenging mainly due to its rapid reproduction,necessitating an understanding of sex pheromone communication.The recognition of sex pheromones is vital for courtship and mating behaviors,and is mediated by various chemosensory-associated proteins.Among these,sensory neuron membrane protein(SNMP),a CD36-related protein,is suggested to play crucial roles in detecting sex pheromones.In this study,we employed transcriptomic and genomic data from A.lucorum and phylogenetic approaches,and identified four putative SNMP genes(AlucSNMP1a,AlucSNMP1b,AlucSNMP2a,and AlucSNMP2b)with full open reading frames.Expression analysis revealed the ubiquitous presence of AlucSNMP transcripts in multiple tissues,with only AlucSNMP1a exhibiting male-biased expression in the antennae,suggesting its potential role in male chemosensation.Functional analysis using the Xenopus oocyte expression system,coupled with two-electrode voltage clamp recording,demonstrated that the co-expression of AlucSNMP1a with specific pheromone receptors(PRs)and the Odorant receptor co-receptor(Orco)significantly enhanced electrophysiological responses to sex pheromones compared to the co-expression of PRs and Orco alone.Moreover,the results indicated that the presence of AlucSNMP1a not only affected the responsiveness to sex pheromones but also influenced the kinetics(activation and inactivation)of the induced signals.In contrast,the co-expression of AlucSNMP1b with AlucPR/Orco complexes had no impact on the inward currents induced by two pheromone compounds.An examination of the selective pressures on SNMP1 genes across 20 species indicated strong purifying selection,implying potential functional conservation in various insects.These findings highlight the crucial role of AlucSNMP1a in the response to sex pheromones.

Changes in milk fat globule membrane proteins along lactation stage of Laoshan dairy goat

The milk fat globule membrane(MFGM)is a complex structure with numerous functions,and its composition is affected by many factors.There have been few systematic investigations on goat MFGM proteome profiling during lactation.Individual milk samples from 15 healthy dairy goats were obtained at six lactation time points for investigation of the MFGM proteome using both data-independent acquisition(DIA)and data-dependent acquisition(DDA)proteomics techniques combined with multivariate statistical analysis.Using the DIA method,890 variably abundant MFGM proteins were discovered throughout the lactation cycle.From 1 to 240 d,butyrophilin subfamily 1 member A1,lipoprotein lipase,perilipin-2,and adipose triglyceride lipase were upregulated,while APOE,complement C3,clusterin,and IgG were downregulated.Furthermore,from 1 to 90 d,annexin A1,annexin A2,and antithrombin-ll were downregulated,then upregulated by d 240.Albumin had a high degree of connectedness,indicating that it was a key protein,according to protein-protein interaction research.Overall,our findings gave new insights into the biological features of MFGM protein in goat milk throughout lactation,which may aid in the creation of specialized MFGM products and infant formula.

Study on Outer Membrane Protein Patterns of Escherichia coli O38,O53 and O75 Isolated from Chickens

[Objective] This study aimed to investigate the outer membrane protein （OMP） patterns of Escherichia coli 038, 053 and 075 isolates from chickens. [Method] Eight pathogenic E. coil isolates with various serotypes were used as experimental materials to extract OMP by using supersonic schizolysis method and Sarcosyl. After SDS-PAGE electrophoresis, OMP patterns of the extracted products were determined based on the OMP model diagram. [Result] OMP of eight E. coil isolates with three serotypes were divided into three patterns, to be specific, 2 075 isolates respectively belonged to OMP-I and OMP-II pattern, 1 053 isolate belonged to OMP-II pattern, and 5 038 isolates belonged to OMP-I and OMP-III pattern. [Conclusion] Experimental results showed that E. coli isolates with the same serotype may belong to completely different OMP patterns, while serologically unrelated isolates may belong to the same OMP pattern. OMP of E. coil isolates with the same serotype may generate genetic differentiation; in addition, OMP of E. coli isolates with different serotypes may have different genetic correlation.

Fluorescence Study on The Interaction Between Cisplatinand Erythrocyte Membrane Proteins

The interaction between cisplatin and erythrocyte membrane proteins was studied based on the quenching effect of cisplatin on the intrinsic fluorescence of proteins.A concentration-dependent quenching effect was observed.The presence of chloride and sulphate weakens the effect significantly.A pH-dependence was also noted with a stronger effect in acidic solution. The nature of the interaction is considered to be platinum-thiol group binding according to the effect of cisplatin on the fluorescence of FMA labeled membrane. The mechanism of the cisplatin-protein interactions was discussed based on the effect of coexisting anion

Prokaryotic Expression and Identification of Outer Membrane Protein 2 of Chlamydia trachomatis

Objective: To construct a recombinant plasmid containing the outer membrane protein 2 (Omp2) gene of Chlamydia trachomatis and express Omp2 in E.coli. Methods: The omp2 gene of C. trachomatis serovar D was cloned into pQE30 vector following PCR amplification from genomic DNA. E. coli M15 transformants were induced to express the fusion protein by IPTG and the product was identified by SDS-PAGE and Western blot. Results: Confirmed by enzyme cleavage analysis and DNA sequencing, a correct recombinant plasmid pQE30/omp2 was constructed. The fusion protein from the transformants was approximately 60 kDa in size in SDS-PAGE analysis, which could specially react with anti-6 X His mouse monoclonal IgG antibodies. Conclusion: We successfully expressed Omp2 in E. coli M15, providing an efficient and simple system for assaying the immunological properties of Omp2.

Epithelial membrane protein 1 negatively regulates cell growth and metastasis in colorectal carcinoma

AIM: To determine the expression and function of epithelial membrane protein 1 (EMP1) in colorectal carcinoma.

Water relations and an expression analysis of plasma membrane intrinsic proteins in sensitive and tolerant rice during chilling and recovery

A symptom of chilling injury is development of water deficit in shoots, resulting from an imbalance of water transport and transpiration. In this work, two rice varieties （Oryza sativa L. var. Wasetoitsu and Somewake） seedlings were chilled at 7 ℃, followed by recovery at 28 ℃. Based on the growth phenotype and electrolyte leakage tests, Somewake was shown to be a chilling-tolerant variety, and Wasetoitsu a chilling-sensitive one. The chilling stress reduced markedly the relative water content （RWC） of leaves, accumulative transpiration and osmotic root hydraulic conductivity （Lp） in both varieties. But when retumed to 28 ℃, the water relation balance of Somewake recovered better. The mRNA expression profile of all the 11 plasma membrane intrinsic proteins （PIPs）, a subgroup of aquaporins, was subsequently determined by real-time reverse transcription （RT）-PCR with TaqMan-minor grove binder （MGB） probes derived from rice var. Nipponbare during chilling treatment and recovery. Most of the PIP genes was down-regulated at the low temperature, and recovered at the warm temperature. The relative expression of some PIPs in both Somewake and Wasetoitsu decreased in parallel during the chilling. However during the recovery, the relative expression of OsPIP1;1, OsPIP2;1, OsPIP2;7 in shoots and OsPIP1：1, OsPIP2：1 in roots were significantly higher in Somewake than Wasetoitsu. This supports the role of PIPs in re-establishing water balance after chilling conditions. We discuss the diversified roles played by members of the aquaporin PIP subfamily in plant chilling tolerance depending on aquaporin isoforms, plant tissue and the stage of chilling duration.

An Inner Membrane Protein(Imp) of Xanthomonas oryzae pv. oryzicola Functions in Carbon Acquisition, EPS Production, Bacterial Motility and Virulence in Rice

Xanthomonas oryzae pv. oryzicola （Xoc） causes bacterial leaf streak, a devastating disease in rice-growing regions worldwide. A Tn5-insertion mutant in Xoc_3248, encoding an inner membrane protein （Imp）, showed reduced virulence in rice. To explore the potential function of this gene in virulence, a deletion mutant R？imp was constructed in the wild-type RS105. The R？imp mutant was signiifcantly impaired for bacterial virulence and growth in planta. The mutation in imp made the pathogen insufifciently utilize glucose, fructose, mannose or pyruvate as a sole carbon source, leading to less extracellular polysaccharide （EPS） production and reduced motility. The deifciencies noted for the mutant were restored to wild-type levels when imp was introduced in trans. Transcription of imp was signiifcantly declined when hrpG and hrpX was mutated and the expression of hrpG and hrpX was also signiifcantly declined when imp was deleted. Cell sublocalization in planta showed Imp membrane-binding feature. These results suggest that Imp is a virulence factor with roles in the catabolism of sugars, EPS production, and bacterial motility.

Effects of cell membrane phospholipid level and protein kinase C isoenzyme expression on hepatic metastasis of colorectal carcinoma

BACKGROUND: The molecular mechanism of hepaticmetastasis of colorectal cancer is not well understood. Theaim of this study was to assess the relations between phos-pholipid contents of cellular membrane and isoenzyme ex-pression of protein kinase C (PKC) and their effects on he-patic metastasis of colorectal cancer.METHODS: High performance liquid chromatography wasused to detect contents of cell membrane phospholipids:phosphatidylinosital (PI), phosphatidylserine (PS), phos-phatidylethanolamine (PE) and phosphatidylcholine (PC)in primary foci, paratumor mucosa and hepatic metastaticfoci in patients with colorectal carcinoma. The mRNA ex-pression levels of PKC-α, -δ, -ε, -λ, -ξ isoenzymeswere detected with the QRT-PCR technique.RESULTS: The levels of PI, PC and PE in primary foci andhepatic metastatic foci were higher than those in paratumormucosa. The level of PE in hepatic metastatic foci wasmuch higher than that in primary foci (t =98.88, P <0.01);but the levels of PI and PC were not significantly differentbetween primary foci and hepatic metastatic foci (t =1.73 ,1.36, P>0.05). The expression levels of -δ, -ε,-λ, -ξ were enhanced in primary foci and hepatic metasta-tic foci, but the level of PKC-α in primary foci was de-creased as compared with that in paratumor mucosa. Thelevels of PKC-δ, -ε, -λ, -ξ in hepatic metastatic foci werehigher than those in primary foci. A positive correlationwas observed between the expression levels of PI, PC andand also between those of PE and PKC-δ, -ε, -λ,-ξ. However, there was a close negative correlation be-tween PE and PKC-α.CONCLUSION: Increased levels of PI and PC and de-creased ratio of PKC-α to are related to colorectalcancer genesis. Increased levels of PE, increased expressionof PKC-δ, -ε, -λ, -ξ isoenzymes and decreased level ofPKC-α are related to hepatic metastasis in colorectal carci-noma.

Immunoproteomics of membrane proteins of Shigella flexneri 2a 2457T

AIM： To screen the immunogenic membrane proteins of Shigella Aexneri 2a 2457T. METHODS： The routine two-dimensional polyacrylamide gel electrophoresis （2-DE） and Western blotting were combined to screen immunogenic proteins of S. Aexneri 2a 2457T. Serum was gained from rabbits immunized with the same bacteria. Immunogenic spots were cut out from the polyacrylamide gel and digested by trypsin in-gel. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry （MALDI-TOF-MS） was performed to determine the molecular weight of peptides. Electrospray ionization （ESI-MS/MS） was performed to determine the sequences of the interesting peptides. RESULTS： A total of 20 spots were successfully identified from Coomassie brilliant blue stained gels representing 13 protein entries, 5 known antigens and 8 novel antigens. A hypothetical protein （YaeT） was detected, which might be a candidate target of vaccine. CONCLUSION： Membrane proteins of S. flexneri 2a 2457T were successfully observed by 2-DE. Several known and novel antigens were identified by mass spectrum.

Plasma membrane-anchored fluorescent tracker based on boron-dipyrromethene

The construction of a stable-membrane tracker has significant implications for the visualization of the membrane in live cells.However,most current plasma trackers are not suitable for tracking plasma membranes for a long time due to their limited retention time.Herein,Mem580-F-Sulfo is designed to target and anchor cell membranes and therefore track cell membranes for a longer time.This tracker is composed of a lipophilic boron-dipyrromethene(BODIPY)derivative and a hydrophilic zwitterion to form an amphiphilic structure,which enables its targeting ability toward cell membranes.Moreover,a reactive ester group is included to bind with proteins through covalent bonds in cell membranes nonspecifically,which extends retention time in cell membranes.Mem580-F-Sulfo shows intense brightness(94600),with a high molar absorption coefficient of up to about 100000 L·mol^(-1)·cm^(-1)and a fluorescence quantum yield of up to 0.97.It shows fast cell membrane targeting ability and long retention up to 90 min.In brief,this work has not only developed a tracker with good cell membrane targetability but also provided a new strategy for improving the targeting stability of cell membranes.

Development and Evaluation of a MAb-Based ELISA for Detection of Chlamy- dophila pneumoniae Infection with Variable Domain 2 and 3 of the Major Outer Membrane protein

Objective This paper aims to develop a monoclonal antibodies （MAbs）- based ELISA for detecting Chlamydophila pneumoniae （C. pneumonioe） antigens in humans with the variable domains （VD） 2 and 3 of the major outer membrane protein （MOMPvD2-VD~） and to assess its sensitivity and specificity by comparing with a widely used MAb that is able to recognize the elementary bodies of C. pneumoniae. Methods MOMPvo2-vo3were overexpressed in Escherichia coil and purified by affinity chromatography. Mice were immunized with the recombinant antigen, and hybridomas secreting MAbs were screened. Three stable hybridomas clones were selected and named 5D6, 7G3, and 8C9. The MAbs-based ELISA was scrutinized for species-specific recognition with a number of human throat swab samples from Group I （156 patients with typical respiratory illness clinically confirmed before） and Group II （57 healthy donors）. Results In Group I, 55 positive cases were detected by anti-EB MAb-based ELISA, 51 cases were positive by MAbs 5D6-based ELISA, and 33 and 38 cases were positive by MAb 8C9 and 7G3-based ELISA respectively. Of the 57 samples from Group II ＂healthy donors＂, 5 were positive and 52 were negative with both anti-EB and 5D6-based tests, while 2 and 3 positive cases were identified by the other two MAb-based ELISAs respectively. Conclusion The novel MOMPvD2.VD3 MAb-based assay may have higher specificity than the anti-EB MAb, which may possibly be used as an alternative tool for the diagnosis of C. pneumoniae infection.

Inhibition of mouse acrosome reaction and sperm-zona pellucida binding by anti-human sperm membrane protein 1 antibody

Aim： To investigate the possible functions of human sperm membrane protein （hSMP-1） in the process of fertilization. Methods： A 576-bp cDNA fragment of HSD-1 gene coding for the extracellular domain of hSMP-1 was cloned and expressed. The localization of this protein on human and mouse sperm was determined by indirect immunofluorescent staining by using anti-recombinant hSMP-1 （anti-rhSMP-1） antibodies. Sperm acrosome reaction and spermzona pellucida （ZP） binding assay were carried out in 10-week-old BALB/c mice. Results： Recombinant hSMP-1 was successfully cloned and expressed. The expression of the native protein was limited on the acrosome of human and mouse sperm. Treatment of anti-rhSMP-1 antibodies significantly decreased the average number of sperms bound to each egg. Meanwhile, the percentage of acrosome reaction was decreased in comparison to pre-immune control after treatment with anti-rhSMP-1 （P 〈 0.05）. Conclusion： The results suggest that anti-rhSMP-1 antibody inhibited mouse acrosome reaction and sperm-ZP binding.

Analysis of Sperm Membrane Protein Relevant to Antisperm Antibody by Two-Dimensional Gel Electrophoresis and Western Blotting

Objective To identify the sperm membrane proteins that are associated with antisperm antibody Methods Using antisperm antibody positive serum through unidimensional polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis followed by Western blot analysis to determine the molecular weights (MW) and isoelectric points (pI) of sperm membrane proteins that are associated with antisperm antibody. Results Eight kinds of MW with more than ten sperm membrane proteins can be recognized by antisperm antibody positive serum, of which the MWs and pI were 23 kD, 31 kD, 32 kD, 34 kD, 41 kD, 51 kD, 60 kD, 78 kD and 5.3, 5.5,5.7, 5.0, 5.3, 5.8, 6.0, 5.5～6.2, 4.6,5.1,5.5～5.8 respectively. The identification ratios of the sperm membrane proteins on 78 kD (60.7%), 60 kD (71.4%), 51 kD (14.9%) and 23 kD (14.29%) were higher. Conclusion The sperm membrane proteins with MW of 78 kD, 60 kD, 51 kD and 23 kD were associated with antisperm antibody and immunological infertility. Two- dimensional gel electrophoresis and Western blotting can precisely identify the sperm membrane proteins that are associated with antisperm antibody.

Antimicrobial Susceptibility and Characterization of Outer Membrane Proteins of Aeromonas hydrophila Isolated in China

Aeromonas hydrophila isolates from clinical cases （n=43） were tested against 8 antimicrobial agents and typed by outer membrane protein （OMP） pattern by using sodium dodecyl sulfate gel electrophoresis. All isolates were resistant to ampicillin （MICs, ≥16 μg mL-1） and sulfamonomethoxine （MICs≥64 μg mLl）, but susceptible to norfloxacin （MICs,≤0.5 μg mL-1）. There was a high incidence of resistance to erythromycin （90.70%） and tylosin （93.02%）, while a low incidences of resistance to ciprofloxacin （2.33%）, enrofloxacin （2.33%） and florfenicol （4.65%）. Six different outer membrane protein patterns were found among 34 isolates by analyzing proteins in the range of 22 to 50 kDa, other than 9 isolates with their respective profiles. The strains with the similar OMP profiles had similar resistances. Compared with the other strains from the same OMP patterns, NB-1, A.Pun and MR-1 had lacked the proteins in the range of 30 to 45 kDa and their resistance to florfenicol substantially increased. It is speculated that the outer membrane protein changes might correlate with decreased susceptibility to florfenicol in the three strains. Some strains which showed completely identical OMP types had a little difference in their resistance to fluoroquinolones, indicating that there might be other factors that were involved in the antimicrobial resistance of A. hydrophila.