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.

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.

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.

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.

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.

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.

Demethylation of miR-34a upregulates expression of membrane palmitoylated proteins and promotes the apoptosis of liver cancer cells

BACKGROUND Liver cancer is a common cancer and the main cause of cancer-related deaths worldwide.Liver cancer is the sixth most common cancer in the world.Although miR-34a and palmitoyl membrane palmitoylated protein(MPP2)are reportedly involved in various cell processes,their precise roles in liver cancer are still unclear.AIM To investigate the expression of micro RNA 34a(miR-34a),methylation of the miR-34a promoter and the expression of MPP2 in liver cancer cells and their related mechanisms.METHODS Together,78 cases of liver cancer tissues and 78 cases of adjacent tissues were collected.The methylation degree of miR-34a promoter in liver cancer/paracancerous tissue and liver cancer cells/normal liver cells,and the expression levels of miR-34a and MPP2 in the above samples were detected.Demethylation of liver cancer cells or transfection of liver cancer cells with miR-34a mimetic was performed.The MPP2 overexpression vector was used to transfect liver cancer cells,and the changes in proliferation,invasion,apoptosis,migration,and other biological functions of liver cancer cells after the above interventions were observed.Double luciferase reporter genes were used to detect the targeting relationship between miR-34a and MPP2.RESULTS Clinical samples showed that the expression levels of miR-34a and MPP2 in liver cancer tissues were lower than those in the normal tissues.The methylation degree of miR-34a promoter region in liver cancer cells was higher than that in normal liver cells.After miR-34a demethylation/mimetic transfection/MPP2 overexpression,the apoptosis of liver cancer cells was increased;the proliferation,invasion and migration capabilities were decreased;the expression levels of caspase 3,caspase 9,E-cadherin,and B-cell lymphoma 2(Bcl-2)-associated X protein were increased;and the expression levels of Bcl-2,N-cadherin,andβ-catenin were decreased.Double luciferase reporter genes confirmed that MPP2 is targeted by miR-34a.Rescue experiments showed that small interfering MPP2 could counteract the promoting effect of miR-34a demethylation on apoptosis and the inhibitory effect on cell proliferation,invasion,and migration.CONCLUSION miR-34a demethylation upregulates the expression level of MPP2 in liver cancer cells and promotes the apoptosis of liver cancer cells.miR-34a demethylation is a potential method for liver cancer treatment.

Study on screening potential allergenic proteins from infant milk powders based on human mast cell membrane chromatography and histamine release assays

Cow's milk allergy is mainly observed in infants and young children. Most allergic reactions affect the skin, followed by the gastrointestinal and respiratory systems. Conventional diagnosis is based on positive allergy studies and evaluation of parameters including IgE and IgG1 levels, acute allergic skin response and anaphylactic shock reactions. We developed a cell membrane chromatographic(CMC)method based on human mast cells(HMC-1) for screening potential allergens in infant formula milk powders(IFMP). HMC-1 cell membranes were extracted and mixed with silica to prepare cell membrane chromatography columns(10 mm ? 2 mm i.d., 5 mm). Under the conditions of 0.2 mL/min flow rate and214 nm detection wavelength, human breast milk showed no retention. However, IFMP showed clear retention. The retained fractions were collected and analyzed through matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS). Four major milk proteins, i.e., α-casein, β-casein, α-lactalbumin, and β-lactoglobulin A, were identified. Furthermore, these proteins and β-lactoglobulin B showed clear retention on HMC-1/CMC columns. To test the degranulation effects of the five proteins, histamine and β-hexosaminidase release assays were carried out. All five proteins induced HMC-1 cells to release histamine and β-hexosaminidase. Also, we established a reversed phase liquid chromatographic(RPLC) method for the determination of the five proteins in IFMP and the results showed that 90% proteins in IFMP were α-casein and β-casein. We concluded that cow's milk proteins may be potential allergens and caseins cause more β-casein allergic risk than other proteins. This conclusion was consistent with other studies.

CLONING AND IDENTIFICATION OF A RABBIT SPERM MEMBRANE PROTEIN GENE

Antibodies raised against rabbit sperm membrane protein(rSMP-B)interacted with a rattestis cytosolic protein with an estimated molecular weight of 70 kD.The clone expressingrSMP-B was isolated by epitope selection from a rat testis,λgt11 vector cDNA library,An Eco Rl-eut 2.0 kb cDNA fragment encoding rSMP-B was identified.The cDNA fragment(RSD-1)was isolated,subcloned and restriction mapped.Dot hybridization analysis of mRNAprepared from human,rabbit and rat testes showed that the gene encoding rSMP-B is ex-pressed in the testes of all three mammals.

Effects of Monoclonal Antibody Against Adipocyte-Specific Membrane Protein on Lipid Metabolism in Pigs

This study was to investigate the regulation of monoclonal antibodies against adipocyte membrane proteins （McAb） on lipid metabolism in pigs. Forty Landrace x Saba pigs were randomly divided into eight groups; the control group was given 10 mL saline and the treat groups were given monoclonal antibody against adipocyte-specific membrane protein with 0.1, 0.5, and 1.0 mg kg-I body weight at 15 and 60 kg body weight, respectively, by intraperitoneal injection. The results showed that McAb could increase, significantly, serum lipoprotein lipase activity and reduce serum nonesterified fatty acid （NEFA） content. Meanwhile, McAb increased content of serum lipid, triglyceride （TG）, cholesterol （CHO）, high density lipoprotein （HDL）, and low density lipoprotein （LDL） both at 15 and 60 kg body weight. However, McAb affected more significantly the lipid metabolism at 15 kg body weight than at 60 kg body weight. Moreover, this effect of McAb on lipid metabolism exhibited dose-dependent effect. These results suggested that this monoclonal antibody increased lipase activity, promoted lipolysis, and utilization of lipid so that McAb could be applied to restrain excessive fat deposition in porcine production through the regulation of fat metabolism.