Expression and refolding of OLA Ⅰ protein with peptides derived from sheeppox virus / 生物工程学报

The aim of this study was to refold the OvisAries leukocyte antigen (OLA) class Ⅰ protein with peptides derived from sheeppox virus (SPPV) to identify SPPV T cell epitopes. Two pairs of primers were designed based on the published sequence of a sheep major histocompatibility complex Ⅰ to amplify the heavy chain gene of OLA Ⅰ α-BSP and the light chain gene of OLA Ⅰ-β2m. Both genes were cloned into a pET-28a(+) expression vector, respectively, and induced with ITPG for protein expression. After purification, the heavy chain and light chain proteins as well as peptides derived from SPPV were refolded at a ratio of 1:1:1 using a gradual dilution method. Molecular exclusion chromatography was used to test whether these peptides bind to the OLA Ⅰ complex. T-cell responses were assessed using freshly isolated PBMCs from immunized sheep through IFN-γ ELISPOT with peptides derived from SPPV protein. The results showed that the cloned heavy chain and light chain expressed sufficiently, with a molecular weight of 36.3 kDa and 16.7 kDa, respectively. The protein separated via a SuperdexTM 200 increase 10/300 GL column was collected and verified by SDS-PAGE after refolding. One SPPV CTL epitope was identified after combined refolding and functional studies based on T-cell epitopes derived from SPPV. An OLA Ⅰ/peptide complex was refolded correctly, which is necessary for the structural characterization. This study may contribute to the development of sheep vaccine based on peptides.

Búsqueda de biomarcadores a partir de péptidos expresados en biopsias de cáncer de mama / Search for biomarkers from peptides expressed in breast cancer biopsies / Busca de biomarcadores a partir de peptídeos expressos em biópsias câncer de mama

El cáncer de mama (CM) es una de las principales causas de muerte en México. Se ha observado un incremento en la incidencia de éste en mujeres de 15-29 años. A fin de comprender las causas en el desarrollo del CM, se pretendió buscar la asociación entre los genes/enfermedad empleando técnicas de Biología Molecular. Se analizaron, por genómica funcional, 50 biopsias frescas de pacientes con CM (BFCM), 50 biopsias embebidas en parafina de CM (BEPCM) y 10 biopsias frescas de pacientes con sospecha de CM (BFSC), obtenidas de mujeres que residen en Coahuila, México. Las muestras proteicas se cuantificaron y se resolvieron en geles de poliacrilamida dodecil sulfato de sodio (SDS-PAGE) y en dos dimensiones (2-DE). El perfil proteico de las BFCM, BEPCM versus BFSC mostró diferencias entre las bandas peptídicas observadas en los geles. Aquellos péptidos que se diferenciaron por su expresión fueron analizados por cromatografía líquida acoplada a masas en tándem (LC/ MS/MS). Las huellas peptídicas obtenidas, a su vez, se analizaron por medio del banco de genes (PubMed). Se encontraron, en las muestras de cáncer, proteínas asociadas a migración celular, supresión de tumores, estrés oxidativo y choque térmico. Por último, estos hallazgos se confirmaron empleando inmuno-electro transferencia o Western blot (WB) con anticuerpos contra vimentina.

Breast cancer (BC) is one of the leading causes of death in Mexico. Moreover, BC is the main cause of death in women between 15-29 years old in northern Mexico. Proteomic techniques have been used in order to achieve a better understanding of the genes involved in the development of BC. The proteins in BC extracted from 50 fresh breast cancer tissues (FBCT), 50 paraffin embedded breast cancer tissues (PEBCT) and 10 biopsies from women suspected of cancer (SC), residing in Coahuila, Mexico were analyzed in this paper. The quantity of protein extracted was similar in both samples FBCT and PEBCT. However, protein quality was lower in PEBCT than FBCT. Subsequently, these proteins were resolved in SDS-PAGE and 2DE. Differences were noticed in protein profile and all those suspect proteins were analyzed by LC/MS/MS. Amino acidic fingerprint allowed for the identification of peptides associated with a) cell migration, b) tumor suppression, c) oxidative stress or heat shock.

O câncer da mama (CM) é uma das principais causas de morte no México. Observou-se um aumento na incidência desse câncer em mulheres entre os 15-29 anos de idade. Para compreender as causas do desenvolvimento de CM, visou-se encontrar a associação entre os genes/doença utilizando técnicas de Biologia molecular. Analisaram-se por genômica funcional, 50 biópsias frescas de pacientes com CM (BFCM), 50 biópsias embebidas em parafina (BEPCM) e 10 biópsias frescas de pacientes com suspeita de CM (BFSC), obtidas de mulheres residentes em Coahuila, México. As amostras de proteínas foram quantificadas e separadas em géis de poliacrilamida dodecil sulfato de sódio (SDS-PAGE) e em duas dimensões (2-DE). O perfil proteico das BFCM, BEPCM comparado com BFSC mostrou diferenças entre as bandas peptídicas observadas nos géis. Esses peptídeos que diferem em sua expressão foram analisados por cromatografia líquida acoplada a massas em tandem (LC/MS/MS). As pegadas peptídicas obtidas, por sua vez, foram analisadas utilizando o banco de genes (PubMed). Verificaram-se nas amostras de câncer, proteínas associadas à migração celular, supressão de tumores, estresse oxidativo e choque térmico. Finalmente, estes achados foram confirmados utilizando a imuno-eletro transferência ou Western Blot (WB) com anticorpos contra vimentina.

A novel M2e-multiple antigenic peptide providing heterologous protection in mice

Swine influenza viruses (SwIVs) cause considerable morbidity and mortality in domestic pigs, resulting in a significant economic burden. Moreover, pigs have been considered to be a possible mixing vessel in which novel strains loom. Here, we developed and evaluated a novel M2e-multiple antigenic peptide (M2e-MAP) as a supplemental antigen for inactivated H3N2 vaccine to provide cross-protection against two main subtypes of SwIVs, H1N1 and H3N2. The novel tetra-branched MAP was constructed by fusing four copies of M2e to one copy of foreign T helper cell epitopes. A high-yield reassortant H3N2 virus was generated by plasmid based reverse genetics. The efficacy of the novel H3N2 inactivated vaccines with or without M2e-MAP supplementation was evaluated in a mouse model. M2e-MAP conjugated vaccine induced strong antibody responses in mice. Complete protection against the heterologous swine H1N1 virus was observed in mice vaccinated with M2e-MAP combined vaccine. Moreover, this novel peptide confers protection against lethal challenge of A/Puerto Rico/8/34 (H1N1). Taken together, our results suggest the combined immunization of reassortant inactivated H3N2 vaccine and the novel M2e-MAP provided cross-protection against swine and human viruses and may serve as a promising approach for influenza vaccine development.

Association between overweight and characteristics of young adult students: support for nursing care / Associação entre excesso de peso e características de adultos jovens escolares: subsídio ao cuidado de enfermagem / Asociación entre exceso de peso y características de adultos jóvenes escolares: contribución para el cuidado de enfermería

OBJECTIVE: to verify associations between overweight and the characteristics of young adult students to support nursing care. METHOD: case-control study conducted with young adults from public schools. The sample was composed of 441 participants (147 cases and 294 controls, with and without excess weight, respectively). Sociodemographic and clinical characteristics were collected together with exposure factors and anthropometrics. Multiple logistic regression was used. The study received Institutional Review Board approval. RESULTS: statistically significant association with overweight: non-Caucasian, having a partner; weight gain during adolescence, mother's excess weight, the use of obesogenic medication, augmented diastolic blood pressure, of abdominal circumference and waist/hip ratio. In addition to these, schooling and weight gain during childhood were also included in the multivariate analysis. After adjustment, the final model included: having a partner, weight gain during adolescence, augmented diastolic blood pressure and abdominal circumference. CONCLUSION: the analysis of predictor variables for excess weight among young adult students supports nurses in planning and developing educational practices aimed to prevent this clinical condition, which is a risk factor for other chronic comorbidities, such as cardiovascular diseases. .

OBJETIVO: verificar a associação entre excesso de peso e características de adultos jovens escolares, como subsídio ao cuidado de enfermagem. MÉTODO: estudo caso-controle, realizado com adultos jovens de escolas públicas. Amostra composta por 441 participantes (147 casos e 294 controles, com e sem excesso de peso, respectivamente). Coletaram-se informações sociodemográficas, clínicas, fatores de exposição e antropometria. Utilizou-se regressão logística múltipla. O estudo foi aprovado em comitê de ética. RESULTADOS: detectou-se associação estatística significativa com excesso de peso em: não brancos, ter companheiro(a), ganho ponderal na adolescência, excesso de peso materno, uso de fármacos obesogênicos, pressão arterial diastólica aumentada, circunferência abdominal e relação cintura quadril. Além destas, entraram na análise multivariada as variáveis escolaridade e ganho ponderal na infância. Após etapa de ajuste permaneceram no modelo final: estado civil com companheiro(a), ganho ponderal na adolescência, pressão arterial diastólica aumentada e circunferência abdominal aumentada. CONCLUSÃO: a análise das variáveis preditoras para o excesso de peso em adultos jovens escolares possibilita ao enfermeiro bases para elaboração e planejamento de práticas educativas que visem à prevenção desta condição clínica, visualizada como fator de risco para outras comorbidades de caráter crônico, como as doenças cardiovasculares. .

OBJETIVO: verificar la asociación entre exceso de peso y características de adultos jóvenes escolares como contribución para el cuidado de enfermería. MÉTODO: estudio de caso control realizado con adultos jóvenes de escuelas públicas. Muestra compuesta por 441 participantes (147 casos y 294 controles, con y sin exceso de peso, respectivamente). Se recolectaron características sociodemográficas, clínicas, factores de exposición y antropometría. Se utilizó la regresión logística múltiple. El estudio fue aprobado por comité de ética. RESULTADOS: se detectó asociación estadística significativa con exceso de peso: no blancos, tener compañero, aumento de peso en la adolescencia, exceso de peso materno, uso de medicamentos obesogénicos, presión arterial diastólica aumentada, circunferencia abdominal aumentada y relación cintura-cadera. Además de estas, entraron en el análisis multivariado las variables escolaridad y aumento de peso en la infancia. Después de la etapa de ajuste permanecieron en el modelo final: estado civil con compañero, aumento de peso en la adolescencia, presión arterial diastólica aumentada y circunferencia abdominal aumentada. CONCLUSIÓN: el análisis de las variables de predicción para el exceso de peso en adultos jóvenes escolares suministra al enfermero bases para la elaboración y planificación de prácticas educativas que objetiven la prevención de esta condición clínica, visualizada como factor de riesgo para otras enfermedades concomitantes de carácter crónico, como las enfermedades cardiovasculares. .

Association between Promoter Polymorphisms of TFF1, TFF2, and TFF3 and the Risk of Gastric and Diffuse Gastric Cancers in a Korean Population

Gastric cancer is one of the most common cancers in the world. The aims of this study were to evaluate the association between polymorphisms in TFF gene family, TFF1, TFF2, and TFF3 and the risk of gastric cancer (GC) and GC subgroups in a Korean population via a case-control study. The eight polymorphisms in TFF gene family were identified by sequencing and genotyped with 377 GC patients and 396 controls by using TaqMan genotyping assay. The rs184432 TT genotype of TFF1 was significantly associated with a reduced risk of GC (odds ratio, [OR) = 0.45; 95% confidence interval, [CI] = 0.25-0.82; P = 0.009), more protective against diffuse-type GC (OR = 0.20; 95% CI = 0.05-0.89; P = 0.035) than GC (OR = 0.34; 95% CI = 0.14-0.82; P = 0.017) in subjects aged < 60 yr, and correlated with lymph node metastasis negative GC and diffuse-type GC (OR = 0.44; 95% CI = 0.23-0.86; P = 0.016 and OR = 0.20; 95% CI = 0.05-0.87; P = 0.031, respectively). In addition, a decreased risk of lymph node metastasis negative GC and diffuse-type GC was observed for rs225359 TT genotype of TFF1 (OR = 0.46, 95% CI = 0.24-0.88; P = 0.020 and OR = 0.21, 95% CI = 0.05-0.88; P = 0.033, respectively). These findings suggest that the rs184432 and rs225359 polymorphisms in TFF1 have protective effects for GC and contribute to the development of GC in Korean individuals.

Knockdown of 14-3-3zeta enhances radiosensitivity and radio-induced apoptosis in CD133+ liver cancer stem cells

14-3-3zeta is related to many cancer survival cellular processes. In a previous study, we showed that silencing 14-3-3zeta decreases the resistance of hepatocellular carcinoma (HCC) to chemotherapy. In this study, we investigated whether silencing 14-3-3zeta affects the radioresistance of cancer stem-like cells (CSCs) in HCC. Knockdown of 14-3-3zeta decreased cell viability and the number of spheres by reducing radioresistance in CSCs after gamma-irradiation (IR). Furthermore, the levels of pro-apoptotic proteins were upregulated in CSCs via silencing 14-3-3zeta after IR. These results suggest that 14-3-3zeta knockdown enhances radio-induced apoptosis by reducing radioresistance in liver CSCs.

Optimal electroporation condition for small interfering RNA transfection into MDA-MB-468 cell line

Electroporation is a valuable tool for small interfering RNA [siRNA] delivery into cells because it efficiently transforms a wide variety of cell types. Since electroporation condition for each cell type must be determined experimentally, this study presents an optimal electroporation strategy to reproducibly and efficiently transfect MDA-MB 468 human breast cancer cell with siRNA. To identify the best condition, the cells were firstly electroporated without siRNA and cell viability was determined by trypan blue and MTT assays. Then siRNA transfection in the best condition was performed. Western blot analysis was used for monitoring successful siRNA transfection. Results: The best condition for electroporation of this cell line was 220 volt and 975 microF in exponential decay using the Gene Pulser X cell electroporation system. Our data demonstrated that by using proper electroporation condition, DNA methyl transferase mRNA was silenced by 10 nmol DNMT1 siRNA in MDA-MB 468 cells when compared with negative control siRNA electroporation. Analysis of cell viability demonstrated that optimal electroporation condition resulted in 74% and 78% cell viability by trypan blue staining and MTT assay, respectively. Transfection of the MDA-MB-468 breast cancer cell line with siRNA in the obtained electroporation condition was successful and resulted in effective gene silencing and high cellular viability

Prolonged expression of MHC class I - peptide expression in bone marrow derived retrovirus transfected matured dendritic cells by continuous centrifugation in the presence of IL-4.

Background & objectives: Dendritic cells (DCs) are potent antigen presenting cells which proceed from immature to a mature stage during their differentiation. There are several methods of obtaining long lasting mature antigen expressing DCs and different methods show different levels of antigen expressions. We investigated bone marrow derived DCs for the degree of maturation and genetically engineered antigen presentation in the presence of interleukin-4 (IL-4) as a maturity enhancer. Methods: DCs and transfected retrovirus were cultured together in the presence of granulocyte-macrophage colony stimulating factor (GMCSF)-IL4, GMCSF +IL4, lipopolysaccharide (LPS). B 7.1, B7.2 and CD11c were measured by the degree of immune fluorescence using enhanced green fluorescent protein (EGFP) shuttled retrovirus transfected antigen. Degree of MHC class I molecule with antigen presentation of antigen was also evaluated by fluorescence activated cell sorting. The antigen presenting capacity of transfected DCs was investigated. Bone marrow DCs were generated in the presence of GMCSF and IL-4 in vitro. Dividing bone marrow cells were infected with EGFP shuttled retrovirus expressing SSP2 by prolonged centrifugation for three consecutive days from day 5, 6 and 7 and continued to culture in the presence of GMSCF and IL-4 until day 8. Results: IL-4 as a cytokine increased the maturation of retrovirus transfected DCs by high expression of B 7-1 and B 7-2. Also, IL-4 induced DC enhanced by the prolonged centrifugation and it was shown by increased antigen presentation of these dendric cells as antigen presenting cell (APC). Cytolytic effects were significantly higher in cytotoxic T cell response (CTLs) mixed with transfected DCs than CTLs mixed with pulsed DCs. Interpretation & conclusions: There was an enhanced antigen presentation by prolonged expression of antigen loaded MHC class I receptors in DCs in the presence of IL-4 by prolonged centrifugation.

Peptidolytic microbial community of methanogenic reactors from two modified UASBs of brewery industries

We studied the peptide-degrading anaerobic communities of methanogenic reactors from two mesophilic full-scale modified upflow anaerobic sludge blanket (UASB) reactors treating brewery wastewater in Colombia. Most probable number (MPN) counts varied between 7.1 x 10(8) and 6.6 x 10(9) bacteria/g volatile suspended solids VSS (Methanogenic Reactor 1) and 7.2 x 10(6) and 6.4 x 10(7) bacteria/g (VSS) (Methanogenic Reactor 2). Metabolites detected in the highest positive MPN dilutions in both reactors were mostly acetate, propionate, isovalerate and, in some cases, negligible concentrations of butyrate. Using the highest positive dilutions of MPN counts, 50 dominant strains were isolated from both reactors, and 12 strains were selected for sequencing their 16S rRNA gene based on their phenotypic characteristics. The small-subunit rRNA gene sequences indicated that these strains were affiliated to the families Propionibacteriaceae, Clostridiaceae and Syntrophomonadaceae in the low G + C gram-positive group and Desulfovibrio spp. in the class d-Proteobacteria. The main metabolites detected in the highest positive dilutions of MPN and the presence of Syntrophomonadaceae indicate the effect of the syntrophic associations on the bioconversion of these substrates in methanogenic reactors. Additionally, the potential utilization of external electron acceptors for the complete degradation of amino acids by Clostridium strains confirms the relevance of these acceptors in the transformation of peptides and amino acids in these systems.

A Case of Congenital Central Hypoventilation Syndrome with PHOX2B Gene Mutation in a Korean Neonate

Congenital central hypoventilation syndrome (CCHS) is a life-threatening disorder with apnea and cyanosis during sleep requiring immediate endotracheal intubation during the first day of life. The PHOX2B gene has been identified as the major gene involved in CCHS. This is the first report of a Korean neonate with CCHS confirmed to have a PHOX2B mutation with expanded alleles containing 20 polyalanine repeats that is a relatively small number compared to previous cases. The patient required intermittent ventilator support during sleep only and did not suffer from any other disorders of the autonomic nerve system. He consistently needs ventilator support during sleep and remains alive. Analysis of PHOX2B gene is useful for diagnosis and appropriate therapeutic intervention of CCHS patients.

DNA Methylation Patterns of Ulcer-Healing Genes Associated with the Normal Gastric Mucosa of Gastric Cancers

Recent evidence suggests that gastric mucosal injury induces adaptive changes in DNA methylation. In this study, the methylation status of the key tissue-specific genes in normal gastric mucosa of healthy individuals and cancer patients was evaluated. The methylation-variable sites of 14 genes, including ulcer-healing genes (TFF1, TFF2, CDH1, and PPARG), were chosen from the CpG-island margins or non-island CpGs near the transcription start sites. The healthy individuals as well as the normal gastric mucosa of 23 ulcer, 21 non-invasive cancer, and 53 cancer patients were examined by semiquantitative methylation-specific polymerase chain reaction (PCR) analysis. The ulcer-healing genes were concurrently methylated with other genes depending on the presence or absence of CpG-islands in the normal mucosa of healthy individuals. Both the TFF2 and PPARG genes were frequently undermethylated in ulcer patients. The over- or intermediate-methylated TFF2 and undermethylated PPARG genes was more common in stage-1 cancer patients (71%) than in healthy individuals (10%; odds ratio [OR], 21.9) and non-invasive cancer patients (21%; OR, 8.9). The TFF2-PPARG methylation pattern of cancer patients was stronger in the older-age group (> or =55 yr; OR, 43.6). These results suggest that the combined methylation pattern of ulcer-healing genes serves as a sensitive marker for predicting cancer-prone gastric mucosa.

Immune responses of selected phagotopes from monoclonal antibodies of Burkholderia pseudomallei.

Random peptide libraries displayed by bacteriophage T7 and M13 were employed to identify mimotopes from 4 monoclonal antibodies (MAbs) specific to Burkholderia pseudomallei. Insert DNA sequences of bound phages selected from four rounds of panning with each MAb revealed peptide sequences corresponding to B. pseudomallei K96243 hypothetical protein BPSL2046, hypothetical protein BpseP_02000035, B. pseudomallei K96243 hypothetical protein BPSS0784, B. pseudomallei 1710b hypothetical protein BURPS1710b_1104, and B. cenocepacia H12424 TonB-dependent siderophore receptor, all located at the outer membrane. The immune responses from all selected phagotopes were significantly higher than that of lipopolysaccharide. The study demonstrates the feasibility of identifying mimotopes through screening of phage-displayed random peptide libraries with B. pseudomallei MAbs.

Inhibition of LPS-induced cyclooxygenase 2 and nitric oxide production by transduced PEP-1-PTEN fusion protein in Raw 264.7 macrophage cells

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor. Although it is well known to have various physiological roles in cancer, its inhibitory effect on inflammation remains poorly understood. In the present study, a human PTEN gene was fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-PTEN fusion protein. The expressed and purified PEP-1-PTEN fusion protein were transduced efficiently into macrophage Raw 264.7 cells in a time- and dose- dependent manner when added exogenously in culture media. Once inside the cells, the transduced PEP-1-PTEN protein was stable for 24 h. Transduced PEP-1-PTEN fusion protein inhibited the LPS-induced cyclooxygenase 2 (COX-2) and iNOS expression levels in a dose-dependent manner. Furthermore, transduced PEP-1-PTEN fusion protein inhibited the activation of NF-kappa B induced by LPS. These results suggest that the PEP-1-PTEN fusion protein can be used in protein therapy for inflammatory disorders.

Whole blood assays to identify Th1 cell antigens and peptides encoded by mycobacterium tuberculosis-specific RD1 genes

To identify Th1 cell-stimulating antigens/peptides encoded by the genes predicted in the Mycobacterium tuberculosis-specific genomic region of difference [RD]1, deleted in Mycobacterium bovis Bacille Calmette-Guerin[BCG], by using synthetic peptides and whole blood from tuberculosis [TB] patients. Heparinized peripheral blood was obtained from culture-proven pulmonary TB patients [n = 16] attending the Chest Disease Hospital, Kuwait. Whole blood was diluted with tissue culture medium RPMI-1640 and tested for Th1 cell stimulation using antigen-induced proliferation and interferon- [IFN-] secretion assays. The antigens included a peptide pool of 220 peptides covering the sequence of 12 open reading frames [ORFs] of RD1 [RD1mix], peptide pools of RD1 ORF5 [ORF5mix], ORF6 [ORF6mix] and ORF7 [ORF7mix], and individual peptides of ORF6 [P6.1-P6.6] and ORF7 [P7.1-P7.6]. M. tuberculosis culture filtrate, cell walls and whole-cell M. bovis BCG were used as complex mycobacterial antigens. The results obtained with different antigens and peptides were statistically analyzed for significant differences using Z test. The complex mycobacterial antigens [culture filtrate, cell walls and M.bovis BCG] and RD1mix induced comparable [p > 0.05] positive antigen-induced proliferation and IFN- responses with whole blood from TB patients. However, the positive IFN- responses induced by ORF6mix and ORF7mix were higher than ORF5mix. Among the individual peptides, P6.4 and P7.1 of ORF6 and ORF7, respectively, induced the highest IFN- responses, suggesting that these peptides represented the immunodominant Th1 cell epitopes of RD1 ORF6 and ORF7 in the patients tested. The whole blood assays with synthetic peptides are useful to identify Th1 cell antigens/peptides encoded by genes located in M. tuberculosis-specific genomic regions

Polyglutamine expansion in Drosophila: thermal stress and Hsp70 as selective agents.

Repetitive DNA sequences that encode polyglutamine tracts are prone to expansion and cause highly deleterious phenotypes of neurodegeneration. Despite this tendency,polyglutamine tracts ("polyQs") are conserved features of eukaryotic genomes. PolyQs are the most frequent protein-coding homotypic repeat in insect genomes, and are found predominantly in genes encoding transcription factors conserved from Drosophila through human. Although highly conserved across species, polyQ lengths vary widely within species. In D. melanogaster, polyQs in 25 genes have more alleles and higher heterozygosity than all other poly-amino acid tracts. The heat shock protein Hsp70 is a principal suppressor of polyQ expansions and may play a key role in modulating the phenotypes of the alleles that encode them. Hsp70 also promotes tolerance of natural thermal stress in Drosophila and diverse organisms,a role which may deplete the chaperone from buffering against polyQ toxicity. Thus in stressful environments, natural selection against long polyQ alleles more prone to expansion and deleterious phenotypes may be more effective. This hypothesis can be tested by measuring the phenotypic interactions between Hsp70 and polyQ transgenes in D. melanogaster undergoing natural thermal stress, an approach which integrates comparative genomics with experimental and ecological genetics.

In silico analysis identifies a C3HC4-RING finger domain of a putative E3 ubiquitin-protein ligase located at the C-terminus of a polyglutamine-containing protein

Almost identical polyglutamine-containing proteins with unknown structures have been found in human, mouse and rat genomes (GenBank AJ277365, AF525300, AY879229). We infer that an identical new gene (RING) finger domain of real interest is located in each C-terminal segment. A three-dimensional (3-D) model was generated by remote homology modeling and the functional implications are discussed. The model consists of 65 residues from terminal position 707 to 772 of the human protein with a total length of 796 residues. The 3-D model predicts a ubiquitin-protein ligase (E3) as a binding site for ubiquitin-conjugating enzyme (E2). Both enzymes are part of the ubiquitin pathway to label unwanted proteins for subsequent enzymatic degradation. The molecular contact specificities are suggested for both the substrate recognition and the residues at the possible E2-binding surface. The predicted structure, of a ubiquitin-protein ligase (E3, enzyme class number 6.3.2.19, CATH code 3.30.40.10.4) may contribute to explain the process of ubiquitination. The 3-D model supports the idea of a C3HC4-RING finger with a partially new pattern. The putative E2-binding site is formed by a shallow hydrophobic groove on the surface adjacent to the helix and one zinc finger (L722, C739, P740, P741, R744). Solvent-exposed hydrophobic amino acids lie around both zinc fingers (I717, L722, F738, or P765, L766, V767, V733, P734). The 3-D structure was deposited in the protein databank theoretical model repository (2B9G, RCSB Protein Data Bank, NJ).

Crystal structure of raw pure Mysore silk fibre based on (Ala-Gly)2-Ser-Gly peptide sequence using Linked-Atom-Least-Squares method.

We have carried out crystal structure analysis of raw pure Mysore silk fibers belonging to Bombyx mori on the basis of model parameters of Marsh et al using Linked-Atom-Least-Squares technique. The intensity of all the reflections were computed employing CCP13 software. We observe that the molecular modification is essentially same as b-pleated structure with antipolar-antiparallel arrangements formed by hydrogen bonds. The essential differences observed in the structure are highlighted and discussed.

Pro domain peptide of HGCP-Iv cysteine proteinase inhibits nematode cysteine proteinases

Cysteine proteinases (CPs) are synthesized as zymogens and converted to mature proteinase forms by proteolytic cleavage and release of their pro domain peptides. A cDNA encoding a papain-like CP, called hgcp-Iv, was isolated from a Heterodera glycines J2 cDNA library, expressed and utilized to assess the ability of its propeptide to inhibit proteinase in its active form. The hgcp-Iv cDNA sequence encodes a polypeptide of 374 amino acids with the same domain organization as other cathepsin L-like CPs, including a hydrophobic signal sequence and a pro domain region. HGCP-Iv, produced in Escherichia coli as a fusion protein with thioredoxin, degrades the synthetic peptide benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin and is inhibited by E-64, a substrate and inhibitor commonly used for functional characterization of CPs. Recombinant propeptides of HGCP-Iv, expressed in E. coli, presented high inhibitory activity in vitro towards its cognate enzyme and proteinase activity of Meloidogyne incognita females, suggesting its usefulness in inhibiting nematode CPs in biological systems. Cysteine proteinases from other species produced no noticeable activity.

Intracellular trafficking and metabolic turnover of yeast prepro-alpha-factor-SRIF precursors in GH3 cells

Chimeric genes coding for prepro region of yeast alpha-factor and anglerfish SRIF were expressed in rat GH3 cells to determine whether yeast signals could regulate hormone processing in mammalian cells. We report that nascent hybrid polypeptides were efficiently targeted to ER, where cleavage of signal peptides and core glycosylation occurred, and were localized mainly in Golgi. These data indicate that prepro region of yeast alpha-factor functions in sorting molecules to secretory pathway in mammalian cells. A hybrid construct with a mutated signal peptide underwent similar ER translocation, whereas such a mutation resulted in defective translocation in yeast (Cheong et al., 1997). This difference may be due to the differences in ER translocation between yeast and mammalian cells, i.e., posttranslational versus cotranslational translocation. Processing and secretion of metabolically labeled hybrid propeptides to mature SRIF peptides were assessed by HPLC. When pulse-labeled cells were chased for up to 2 h, intracellular propeptides disappeared with a half-life of approximately 25 min, showing that -68% of initially synthesized propeptides were secreted constitutively. About 22% of SRIF-related products were proteolytically processed to mature SRIF, of which 38.7% were stored intracellularly with a half-life of - 2 h. In addition, immunocytochemical localization showed that a small proportion of SRIF molecules accumulated in secretory vesicles. All these results suggest that yeast prepropeptide could direct hybrid precursors to translocate into ER lumen and transit through secretory pathway to the distal elements of Golgi compartment, but could process and target it less efficiently to downstream in rat endocrine cells.

Intracellular trafficking and metabolic turnover of yeast prepro-alpha-factor-SRIF precursors in GH3 cells

Chimeric genes coding for prepro region of yeast alpha-factor and anglerfish SRIF were expressed in rat GH3 cells to determine whether yeast signals could regulate hormone processing in mammalian cells. We report that nascent hybrid polypeptides were efficiently targeted to ER, where cleavage of signal peptides and core glycosylation occurred, and were localized mainly in Golgi. These data indicate that prepro region of yeast alpha-factor functions in sorting molecules to secretory pathway in mammalian cells. A hybrid construct with a mutated signal peptide underwent similar ER translocation, whereas such a mutation resulted in defective translocation in yeast (Cheong et al., 1997). This difference may be due to the differences in ER translocation between yeast and mammalian cells, i.e., posttranslational versus cotranslational translocation. Processing and secretion of metabolically labeled hybrid propeptides to mature SRIF peptides were assessed by HPLC. When pulse-labeled cells were chased for up to 2 h, intracellular propeptides disappeared with a half-life of approximately 25 min, showing that -68% of initially synthesized propeptides were secreted constitutively. About 22% of SRIF-related products were proteolytically processed to mature SRIF, of which 38.7% were stored intracellularly with a half-life of - 2 h. In addition, immunocytochemical localization showed that a small proportion of SRIF molecules accumulated in secretory vesicles. All these results suggest that yeast prepropeptide could direct hybrid precursors to translocate into ER lumen and transit through secretory pathway to the distal elements of Golgi compartment, but could process and target it less efficiently to downstream in rat endocrine cells.