Cross-linking of elongation factor 2 to rat-liver ribosomal proteins by 2-iminothiolane

Complexes containing rat liver 80S ribosomes treated with puromycin and high concentrations of KCl, elongation factor 2 (EF-2) from pig liver, and guanosine 5'-[beta, gamma-methylene]triphosphate were prepared. Neighboring proteins in the complexes were cross-linked with the bifunctional reagent 2-iminothiolane. Proteins were extracted and then separated into 22 fractions by chromatography on carboxymethylcellulose of which seven fractions were used for further analyses. Each protein fraction was subjected to diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Nine cross-linked protein pairs between EF-2 and ribosomal proteins were shifted from the line formed with monomeric proteins. The spots of ribosomal proteins cross-linked to EF-2 were cut out from the gel plate and labelled with 125I. The labelled protein was extracted from the gel and identified by three kinds of two-dimensional gel electrophoresis, followed by autoradiography. The following proteins of both large and small subunits were identified: L9, L12, L23, LA33 (acidic protein of Mr 33000), P2, S6 and S23/S24, and L3 and L4 in lower yields. The results are discussed in relation to the topographies of ribosomal proteins in large and small subunits. Furthermore we found new neighboring protein pairs in large subunits, LA33-L11 and LA33-L12.     

Partial two-dimensional gel electrophoresis (2-DE) maps of Streptococcus iniae ATCC29178 and Lactococcus garvieae KG9408

To examine the proteomes of 2 important causative agents of fish streptococcosis, Streptococcus iniae ATCC29178 and Lactococcus garvieae KG9408, we used 2-dimensional gel electrophoresis (2-DE) followed by mass spectrometry to generate 2-DE maps of these type strains. Silver-stained 2-DE gels of S. iniae ATCC29178 and L. garvieae KG9408 revealed approximately 320 and 300 spots, respectively, and immobilized pH gradient strips (13 cm, pH 4 to 7) revealed that the majority of the detected spots were concentrated in the pH range of 4.5 to 5.5. The spots were randomly selected from the 2-DE profiles and identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time of flight mass spectrometry. The majority of the identified proteins were functionally related to energy and carbohydrate metabolism (e.g. enolase ATPase, glyceraldehyde-3-phosphate dehydrogenase) or translation and translocation (e.g. elongation factor G, elongation factor Tu, DNA-directed RNA polymerase alpha chain). These data, along with our partial 2-DE maps of S. iniae ATCC29178 and L. garvieae KG9408, may help suggest antigenic proteins for the development of effective diagnostic tools and vaccines against S. iniae and L. garvieae.     

Characterisation of the ribosomal binding site for eukaryotic elongation factor 2 by chemical cross-linking

Ribosomal complexes containing elongation factor 2 (EF-2) were formed by incubation of 80 S ribosomes in the presence of EF-2 and the non-hydrolysable GTP analogue GuoPP[CH2]P. The factor was covalently coupled to the ribosomal proteins located at the factor binding site, by treatment with bifunctional reagents. After isolation of the covalent EF-2.ribosomal protein complexes, the proteins were labelled with 125I and the introduced covalent links cleaved. The ribosomal proteins were identified by electrophoresis in two independent two-dimensional gel systems, followed by autoradiography. After cross-linking with bis(hydroxysuccinimidyl) tartrate (4 A between the reactive groups), protein S3/S3a, S7 and S11 were found as the major ribosomal proteins covalently linked to EF-2. The longer reagent, dimethyl 3,8-diaza-4,7-dioxo-5,6-dihydroxydecanbisimidate (11 A between the reactive groups), covalently coupled proteins S7, S11, L5, L13, L21, L23, L26, L27a and L32 to EF-2. After cross-linking with dimethyl suberimidate (9 A between the reactive groups) proteins S3/3a, S7, S11, L5, L8, L13, L21, L23, L26, L27a, L31 and L32 were identified as belonging to the EF-2-binding site. The results indicate that the ribosomal domain interacting with EF-2 is located on both the small and the large ribosomal subunit close to the subunit interface.     

Initiation factor and ribosome levels are coordinately controlled in Escherichia coli growing at different rates

The levels of initiation factors and other translational components were compared in crude lysates of Escherichia coli grown at different rates. Cells were grown in media containing [35S]sulfate and different carbon sources, and harvested during mid-exponential phase after about 10 generations of balanced growth. Initiation factors (IF), elongation factors (EF), and a number of ribosomal proteins were identified unambiguously in gel patterns following two-dimensional polyacrylamide gel electrophoresis. The molar concentration of each protein was calculated from measurements of the radioactivity in excised gel spots and knowledge of the sulfur content of each protein. We found that the ribosomal proteins and elongation factors were present in equimolar amounts except for L7/L12 and EF-Tu which were 4-fold and 5-fold more abundant, respectively, and that the levels of each protein increased in proportion to growth rate. These results are similar to ones obtained previously by other methods, and serve to confirm the validity of our method. We found that the levels of IF2a and IF3 also were approximately proportional to growth rate. We also measured the levels of all three initiation factors using a radioimmune assay, showed that the factors are present in equimolar amounts, and confirmed that their abundance increased in parallel with ribosomes. We conclude that initiation factor levels are coordinately regulated with one another and with ribosome and elongation factor levels.     

苜蓿青贮用乳酸菌复合系Al2的组成多样性

苜蓿是高蛋白的饲料作物,青贮是保存青草过冬的主要手段,但苜蓿是难青贮的作物,添加乳酸菌制剂是解决苜蓿青贮难的有效措施。本研究室通过连续的限制性培养筛选到苜蓿青贮用乳酸菌复合系Al2,用变性梯度凝胶电泳(DGGE)、平板分离及建立16S rDNA克隆文库3种手段相结合分析Al2的组成多样性,确认Al2复合菌系由7株菌组成,全部属于Lactobacillus。其中3株菌Al2-1i,Al2-2i,Al2-3i通过平板分离得到,分别属于L.plantarum(99.9%)、L.kimchii(99.4%)、L.farciminis(100%)。在Al2复合菌系的16S rDNA克隆文库中,上述7种菌的组成比例分别为55.21%、19.79%、14.58%、3.13%、3.13%、3.13%、1.03%。     

Genotypic analyses of lactobacilli with a range of tannase activities isolated from human feces and fermented foods

A total of 77 tannase producing lactobacilli strains isolated from human feces or fermented foods were examined for their genotypic profiles and intensities of tannase production. With a PCR-based assay targeting recA gene, all strains except one isolate were assigned to either Lactobacillus plantarum, L. paraplantarum, or L. pentosus whereas a 16/23S rDNA targeted PCR-based assay identified all except 6 isolates (inclusive of the above one isolate) as one of the closely related species. Subsequent DNA/DNA hybridization assays revealed that these 6 exceptional isolates showed low homology (between 1.2% and 55.8% relative DNA binding) against type strains of the three species. Supplemental carbohydrate fermentation profiles on the 6 isolates indicated that two of them were identified as L. acidophilus, one as Pediococcus acidilactici, one as P. pentosaceus, and two remained unidentifiable. The evidence suggests that the 16/23S rDNA targeted PCR assay can be used as a reliable identification tool for the closely related lactobacilli, and that the tannase gene is widely distributed within members of the Lactobacillaceae family. Meanwhile, a randomly amplified polymorphism DNA (RAPD) analysis revealed that all except 8 isolates were well allocated in 4 major RAPD clusters, though not species specific, consisting of two L. plantarum predominant clusters, one L. paraplantarum predominant, and one L. pentosus predominant. The RAPD patterns of the 8 non-clustered isolates, which consisted of the 6 unidentifiable isolates and 2 isolates identified as L. pentosus, were <40% similarity to those belonging to the 4 clusters. A quantitative assay of the tannase activities showed that there was a marked variation in the activities among the strains, which did not correlate with either species identification or clustering by RAPD.     

Screening of lactic-acid bacteria from South African barley beer for the production of bacteriocin-like compounds

Strains of Lactobacillus paracasei subsp. paracasei (strain ST11BR), L. pentosus (strain ST151BR), L. plantarum (strain ST13BR), and Lactococcus lactis subsp. lactis (strain ST34BR) producing bacteriocin-like peptides were isolated from barley beer produced in the Western, Northern and Eastern provinces of South Africa. The peptides (bacST11BR, bacST151BR, bacST13BR and bacST34BR) lost their activity after treatment with proteinase K, a proteinase, papain, chymotrypsin, trypsin, pepsin and pronase, but not when they were treated with alpha-amylase, suggesting that the peptides are not glycosylated. The peptides inhibited the growth of Lactobacillus casei, L. sakei, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis, but not Enterobacter cloacae, Lactobacillus bulgaricus subsp. delbrueckii, L. plantarum, L. salivarius, Listeria innocua, Staphylococcus aureus, Streptococcus uberis, S. agalactiae, S. caprinus and S. pneumoniae. Peptides bacST11BR and bacST13BR differed from the other 2 peptides by failing to kill Klebsiella pneumoniae and one of the E. coli strains. Peptides were stable after 2 h of incubation at pH 2.0-12.0, and after 90 min at 100 degrees C. When autoclaved (121 degrees C, 20 min), only bacST13BR lost its activity. The bacteriocin-like peptides were produced at a growth temperature of 30 degrees C, but not at 37 degrees C.     

Systematic search for zinc-binding proteins in Escherichia coli.

A systematic search for Escherichia coli proteins with the zinc-binding activity was performed using the assay of radioactive Zn(II) binding to total E. coli proteins fractionated by two methods of two-dimensional gel electrophoresis. A total of 30-40 radioactive spots were identified, of which 14 have been assigned from N-terminal sequencing. In addition to five known zinc-binding proteins, nine zinc-binding proteins were newly identified including: acetate kinase (AckA), DnaK, serine hydroxymethyltransferase (GlyA), transketolase isozymes (TktA/TktB), translation elongation factor Ts (Tsf), ribosomal proteins L2 (RplB), L13 (RplM) and one of S15 (RpsO), S16 (RpsP) or S17 (RpsQ). Together with about 20 known zinc-binding proteins, the total number of zinc-binding proteins in E. coli increased up to more than 30 species (or more than 3% of about 1000 proteins expressed under laboratory culture conditions). The specificity and affinity of zinc-binding were analysed for some of the zinc-binding proteins.     

Heat shock response in Lactobacillus plantarum

Heat stress resistance and response were studied in strains of Lactobacillus plantarum. Stationary-phase cells of L. plantarum DPC2739 had decimal reduction times (D values) (D value was the time that it took to reduce the number of cells by 1 log cycle) in sterile milk of 32.9, 14.7, and 7.14 s at 60, 72, and 75 degrees C, respectively. When mid-exponential-phase cells were used, the D values decreased. The temperature increases which caused a 10-fold reduction in the D value ranged from 9 to 20 degrees C, depending on the strain. Part of the cell population treated at 72 degrees C for 90 s recovered viability during incubation at 7 degrees C in sterile milk for 20 days. When mid-exponential- or stationary-phase cells of L. plantarum DPC2739 were adapted to 42 degrees C for 1 h, the heat resistance at 72 degrees C for 90 s increased ca. 3 and 2 log cycles, respectively. Heat-adapted cells also showed increased growth at pH 5 and in the presence of 6% NaCl. Two-dimensional gel electrophoresis of proteins expressed by control and heat-adapted cells revealed changes in the levels of expression of 31 and 18 proteins in mid-exponential- and stationary-phase cells, respectively. Twelve proteins were commonly induced. Nine proteins induced in the heat-adapted mid-exponential- and/or stationary-phase cells of L. plantarum DPC2739 were subjected to N-terminal sequencing. These proteins were identified as DnaK, GroEL, trigger factor, ribosomal proteins L1, L11, L31, and S6, DNA-binding protein II HlbA, and CspC. All of these proteins have been found to play a role in the mechanisms of stress adaptation in other bacteria. Antibodies against GroES detected a protein which was induced moderately, while antibodies against DnaJ and GrpE reacted with proteins whose level of expression did not vary after heat adaptation. This study showed that the heat resistance of L. plantarum is a complex process involving proteins with various roles in cell physiology, including chaperone activity, ribosome stability, stringent response mediation, temperature sensing, and control of ribosomal function. The physiological mechanisms of response to pasteurization in L. plantarum are fundamental for survival in cheese during manufacture.     

Phenotypic and genotypic characteristics of lactic acid bacteria isolated from sour congee in Inner Mongolia of China

Sour congee is a popular food in the western regions of Inner Mongolia in China. It has a complex microbial population, which contributes to its unique flavor and functional properties. In this study, we chose 28 sour congee samples that were collected from different areas in Inner Mongolia for analysis of the microbial community of lactic acid bacteria (LAB) by classical biochemical tests, 16S rRNA gene sequencing, multiplex PCR assay of recA gene and restriction fragment length polymorphism (RFLP) analysis of the tuf gene (encoding elongation factor Tu). The results revealed that all the isolates were identified as Lactobacillus (L.) paracasei (38 strains), L. fermentum (28 strains), L. plantarum (7 strains), L. brevis (4 strains), L. reuteri (2 strains), L. amylolyticus (1 strain), Enterococcus (E.) faecalis (3 strains), E. italicus (2 strains) or Lactococcus lactis subsp. lactis (1 strain). The predominant LAB were L. casei and L. fermentum in sour congee samples. The diversity of LAB derived from sour congee could offer useful information for further research on sour congee, and the results demonstrated that the combination of tuf gene and RFLP patterns can be considered as a useful tool for differentiation of the L. casei group.     

Identification and characterization of lactic acid bacteria isolated from traditional pickles in Sichuan, China

The pickle, a traditional fermented vegetable product, is popular in Sichuan Province of China. The objective of this study was to investigate the diversity of dominant lactic acid bacteria (LAB) in pickles by analyzing 36 samples collected from 6 different regions in Sichuan Province. The LAB counts in these samples varied from 3.90 to 8.40 log cfu ml^-1. In total, 185 presumptive LAB with Gram-positive and catalase-negative properties were obtained from these samples using MRS agar, and those strains were identified at the species level by physiological tests, 16S rRNA gene sequencing and multiplex PCR assay. The results revealed that all isolates were accurately identified as Enterococcus thailandicus (2 strains), Lactobacillus alimentarius (16 strains), L. brevis (24 strains), L. paracasei (9 strains), L. plantarum (81 strains), L. pentosus (38 strains), L. sakei (8 strains), L. spicheri (1 strain), Leuconostoc lactis (1 strain) and Pediococcus ethanolidurans (5 strains). The predominant LAB in Sichuan pickle was L. plantarum, which were isolated from most samples. The results also demonstrated that different regions in Sichuan Province have complex compositions of LAB species, and such a rich resource of LAB strains provides raw data for further studies involving probiotic strain selection.     

应用rep-PCR分型技术筛选潜在治疗性乳杆菌

分离鉴定阴道弯曲乳酸杆菌并对其进行基因分型分析和产H2O2能力测定,初步筛选具有潜在防治女性生殖道感染的弯曲乳酸杆菌菌株。将健康妇女阴道分泌物接种到de Man,Rogosa and Sharpe (MRS) 培养基,分离培养乳酸杆菌。通过16S rRNA序列进行乳酸杆菌分类鉴定,重复序列片段PCR扩增方法进行弯曲乳酸杆菌的基因分型,并进一步采用pH直接测酸法和辣根过氧化物酶催化四甲基联苯胺与过氧化氢反应显色法检测了10株弯曲乳酸杆菌产酸和产H2O2能力。经过序列比对鉴定,共得到65株乳酸杆菌。其中,弯曲乳杆菌Lactobacillus crispatus 19株,詹氏乳杆菌Lactobacillus jensenii 17株,发酵乳杆菌Lactobacillus fermentum 12株;rep-PCR分型发现不同种类的乳酸杆菌和同一种弯曲乳酸杆菌均表现为不同的带型指纹图;10株弯曲乳酸杆菌均产酸,其中T22-3和T29-5两株弯曲乳酸杆菌产H2O2量最高。结果表明个体阴道内乳酸杆菌分布具有差异,弯曲乳酸杆菌具有种内多样性,产H2O2丰富的T22-3和T29-5两株弯曲乳酸杆菌有可能作为防治女性生殖道感染的有益菌株。     

Lactic acid bacteria isolated from fermented flour of finger millet,its probiotic attributes and bioactive properties

This study aims to isolate and identify lactic acid bacteria from fermented flour of selected finger millet varieties grown in Sri Lanka and to evaluate their probiotic attributes and bioactive properties in vitro. Fifteen lactic acid bacteria were isolated from three varieties of fermented finger millet flour namely ravi, raavana and oshadha. These isolates were screened for phenotypical and biochemical characteristics. The selected isolates were identified by 16 S rRNA sequencing as Bacillus cereus (five strains), Streptococcus lutetiensis, Lactobacillus plantarum, Lactobacillus fermentum (two strains), Brevibacillus borstelensis, Paenibacillus species, Lactococcus lactis subspecies lactis, Enterococcus faecium, Pediococcus acidilactici, and Enterococcus lactis, and their partial sequences were deposited in GenBank. Among them, five isolates including two isolates, L. plantarum MF405176.1 and L. fermentum MF033346.1 isolated from ravi; two isolates, L. lactis MF480428.1 and E. faecium MF480431.1 isolated from raavana; and P. acidilactici MF480434.1 isolated from oshadha varieties respectively, exhibited in vitro safety attributes and could tolerate acid, gastric juice, bile, salt, phenol, and temperature under simulated gastric conditions, and also were susceptible to antibiotics tested. Further, they demonstrated bactericidal activity against both drug-sensitive and multidrug-resistant pathogens. Among the selected isolates, L. plantarum MF405176.1 demonstrated highest hydrophobicity and adhesion to both colon colorectal adenocarcinoma and colon colorectal carcinoma cell lines. L. lactis subspecies lactis MF480428.1 exhibited the highest auto-aggregation and 2, 2, diphenyl-1-pricrylhydrazyl free radical scavenging activity. P. acidilactici MF480434.1 demonstrated the lowest IC50 values against HCT-116 and HT-29 cells. None of the LAB isolates could assimilate > 10% cholesterol in vitro.     

Comparative study between prokaryotes and eukaryotes by chemical iodination of ribosomal proteins.

Escherichia coli and Saccharomyces cerevisiae ribosomal proteins were chemically iodinated with 125I by chloramine T under conditions in which the proteins were denatured. The labelled proteins were subsequently separated by two-dimensional gel electrophoresis with an excess of untreated ribosomal proteins from the same species. The iodination did not change the electrophoretic mobility of the proteins as shown by the pattern of spots in the stained gel slabs and their autoradiography. The 125I radioactivity incorporated in the proteins was estimated by cutting out the gel spots from the two-dimensional electrophoresis gel slabs. The highest content of 125I was found in the ribosomal proteins L2, L11, L13, L20/S12, S4 and S9 from E. coli, and L2/L3, L4/L6/S7, L5, L19/L20, L22/S17, L29/S27, L35/L37 and S14/S15 from S. cerevisiae. Comparisons between the electrophoretic patterns of E. coli and S. cerevisiae ribosomal proteins were carried out by coelectrophoresis of labelled and unlabelled proteins from both species. E. coli ribosomal proteins L5, L11, L20, S2, S3 and S15/S16 were found to overlap with L15, L11/L16, L36/L37, S3, S10 and S33 from S. cerevisiae, respectively. Similar coelectrophoresis of E. coli 125I-labelled proteins with unlabelled rat liver and wheat germ ribosomal proteins showed the former to overlap with proteins L1, L11, L14, L16, L19, L20 and the latter with L2, L5, L6, L15, L17 from E. coli.     

Dominant cultivable Lactobacillus species from the feces of healthy adults in northern Spain.

The aim of this study was to identify numerically dominant cultivable lactobacilli species in the feces of healthy adults. Ten individuals from Asturias, northern Spain, were chosen. Bacterial colonies grown under anoxic conditions on MRS with cysteine were microscopically examined for lactobacilli. Isolates were subsequently grouped based on the analysis of their carbohydrate fermentation profiles and then identified by partial amplification, sequencing, and comparison of their 16S rRNA gene sequences. Lactobacilli varied from undetectable levels in three subjects (10(5) CFU/g feces) to around 10(9) CFU/g feces. Among the 71 isolates obtained from seven individuals, 12 Lactobacillus species were identified. High interindividual variation was observed in terms of total numbers, number of species, and dominant species. Lactobacillus paracasei was found in four of the seven individuals; L. gasseri, L. delbrueckii, and L. plantarum in three. Phenotyping showed that only one strain per species was in the majority in each individual.     

植物乳杆菌LpT1和LpT2体外降胆固醇机制

【目的】初步探讨植物乳杆菌LpT1和LpT2的体外降胆固醇机制。【方法】以MRS、MRS+CH、MRS+CH+S和MRS+CH+N四种培养基为基础,接种植物乳杆菌LpT1和LpT2进行培养,通过分析比较培养基上清液、菌体沉淀和菌体细胞内部胆固醇含量以及接种和未接种两种情况上清液、沉淀和细胞内胆固醇总量变化,推测植物乳杆菌体外降胆固醇机制。【结果】乳酸菌体外降胆固醇存在非代谢和代谢降解两条途径,非代谢途径与共沉淀作用和菌体吸收有关。代谢降解是由于植物乳杆菌在生长过程中产生了特殊的酶系,从而将胆固醇降解成其他物质,导致其含量降低。【结论】研究结果为进一步研究植物乳杆菌体外降胆固醇的机制奠定了良好基础。     

Multi-locus sequence typing (MLST) of non-fermentative Gram-negative bacilli isolated from bloodstream infections in southern Poland

Non-fermentative Gram-negative bacilli are now one of the most important causes of severe infections in Polish hospitals. Acinetobacter species are serious concern because of the high prevalence of multi-drug resistance among strains. Resistance profiles for 53 Gram-negative non-fermentative blood isolates were done. MLST was carried out using 44 strains representing the most commonly isolated species: A. baumannii, P. aeruginosa, and S. maltophilia. MLST revealed that all 22 A. baumannii belonged to sequence type (ST) 2. The P. aeruginosa isolates belonged to 10 different STs. Four S. maltophilia isolates matched STs present in the database (ST4, ST15, ST116, ST142), seven isolates showing novel sequence types. Among P. aeruginosa and S. maltophilia PFGE confirmed the genetical variety of strains.     

Differential protein expression of murine macrophages upon interaction with Candida albicans

Numerous studies highlight the importance of macrophages for optimal host protection against systemic Candida albicans infections. We chose the murine macrophage cell line RAW 264.7 and the wild-type strain C. albicans SC5314 to study of the induced expression/repression of proteins in macrophages when they are in contact with C. albicans, based on 2-DE, comparison between different gels and protein identification. RAW 264.7 cells were allowed to interact with C. albicans cells for 45 min, and a significant differential protein expression was observed in these macrophages compared to controls. Gels were stained with SYPRO Ruby, allowing a better quantification of the intensity of the protein spots. Fifteen spots were up-regulated, whereas 32 were down-regulated; 60 spots appeared and 49 disappeared. Among them, we identified 11 proteins: annexin I, LyGDI (GDID4), Hspa5 (Grp78, Bip), tropomyosin 5 and L-plastin, that augment; and Eif3s5, Hsp60, Hspa9a, Grp58 (ER75), and Hspa8a (Hsc70), that decrease. The translation elongation factor (Eef2p) is modified in some of its different protein species. Many processes seem to be affected: cytoskeletal organisation, oxidative responses (superoxide and nitric oxide production) and protein biosynthesis and refolding.     

Reduced IgG anti-small nuclear ribonucleoprotein autoantibody production in systemic lupus erythematosus patients with positive IgM anti-cytomegalovirus antibodies

Introduction

In rheumatoid arthritis (RA), synovial fluid (SF) contains a large number of neutrophils that contribute to the inflammation and destruction of the joints. The SF also contains granulocyte-macrophage colony-stimulating factor (GM-CSF), which sustains viability of neutrophils and activates their functions. Using proteomic surveillance, we here tried to elucidate the effects of GM-CSF on neutrophils.

Methods

Neutrophils stimulated by GM-CSF were divided into four subcellular fractions: cytosol, membrane/organelle, nuclei, and cytoskeleton. Then, proteins were extracted from each fraction and digested by trypsin. The produced peptides were detected using matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF MS).

Results

We detected 33 peptide peaks whose expression was upregulated by more than 2.5-fold in GM-CSF stimulated neutrophils and identified 11 proteins out of the 33 peptides using MALDI-TOF/TOF MS analysis and protein database searches. One of the identified proteins was neutrophil gelatinase-associated lipocalin (NGAL). We confirmed that the level of NGAL in SF was significantly higher in patients with RA than in those with osteoarthritis. We next addressed possible roles of the increased NGAL in RA. We analysed proteome alteration of synoviocytes from patients with RA by treatment with NGAL in vitro. We found that, out of the detected protein spots (approximately 3,600 protein spots), the intensity of 21 protein spots increased by more than 1.5-fold and the intensity of 10 protein spots decreased by less than 1 to 1.5-fold as a result of the NGAL treatment. Among the 21 increased protein spots, we identified 9 proteins including transitional endoplasmic reticulum ATPase (TERA), cathepsin D, and transglutaminase 2 (TG2), which increased to 4.8-fold, 1.5-fold and 1.6-fold, respectively. Two-dimensional electrophoresis followed by western blot analysis confirmed the upregulation of TERA by the NGAL treatment and, moreover, the western blot analysis showed that the NGAL treatment changed the protein spots caused by post-translational modification of TERA. Furthermore, NGAL cancelled out the proliferative effects of fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) on chondrocytes from a patient with RA and proliferative effect of FGF-2 on chondrosarcoma cells.

Conclusions

Our results indicate that GM-CSF contributes to the pathogenesis of RA through upregulation of NGAL in neutrophils, followed by induction of TERA, cathepsin D and TG2 in synoviocytes. NGAL and the upregulated enzymes may therefore play an important role in RA.     

Cross-linking study on localization of the binding site for elongation factor 1 alpha on rat liver ribosomes

Complexes containing rat liver 80 S ribosomes, poly(uridylic acid), phenylalanyl-tRNA, elongation factor 1 alpha, and guanylyl(beta, gamma-methylene)-diphosphonate were prepared. Neighboring proteins in the complexes were cross-linked with the bifunctional reagent 2-iminothiolane. Proteins were extracted and then separated into 26 fractions by chromatography on carboxymethylcellulose. Each protein fraction was subjected to diagonal polyacrylamide-sodium dodecyl sulfate gel electrophoresis. Four cross-linked pairs containing elongation factor 1 alpha were on the vertical line below the diagonal. The ribosomal protein spot of each pair was cut out from the gel plate and labeled with 125I. The labeled proteins were extracted from the gel and identified by two-dimensional gel electrophoresis, followed by autoradiography. The following proteins of both 60 S and 40 S subunits were identified: L12, L23, L39, S23/S24, and S26, three proteins of which had been found to be cross-linked also to elongation factor 2 (Uchiumi, T., Kikuchi, M., Terao, K., Iwasaki, K., and Ogata, K. (1986) Eur. J. Biochem. 156, 37-44). These results afford direct evidence that both elongation factors interact with partially overlapping sites on rat liver ribosomes.