Gene Expression Profiles Stratified according to Type 1 Diabetes Mellitus Susceptibility Regions

The MHC region (6p21) aggregates the major genes that contribute to susceptibility to type 1 diabetes (T1D). Three additional relevant susceptibility regions mapped on chromosomes 1p13 (PTPN22), 2q33 (CTLA-4), and 11p15 (insulin) have also been described by linkage studies. To evaluate the contribution of these susceptibility regions and the chromosomes that house these regions, we performed a large-scale differential gene expression on lymphomononuclear cells of recently diagnosed T1D patients, pinpointing relevant modulated genes clustered in these regions and their respective chromosomes. A total of 4608 cDNAs from the IMAGE library were spotted onto glass slides using robotic technology. Statistical analysis was carried out using the SAM program, and data regarding gene location and biological function were obtained at the SOURCE, NCBI, and FATIGO programs. Three induced genes were observed spanning around the MHC region (6p21-6p23), and seven modulated genes (5 repressed and 2 repressed) were seen spanning around the 6q21-24 region. Additional modulated genes were observed in and around the 1p13, 2q33, and 11p15 regions. Overall, modulated genes in these regions were primarily associated with cellular metabolism, transcription factors and signaling transduction. The differential gene expression characterization may identify new genes potentially involved with diabetes pathogenesis.

Expression and characterization of HPV-16 L1 capsid protein in Pichia pastoris

Human papillomaviruses (HPVs) are responsible for the most common human sexually transmitted viral infections. Infection with high-risk HPVs, particularly HPV16, is associated with the development of cervical cancer. The papillomavirus L1 major capsid protein, the basis of the currently marketed vaccines, self-assembles into virus-like particles (VLPs). Here, we describe the expression, purification and characterization of recombinant HPV16 L1 produced by a methylotrophic yeast. A codon-optimized HPV16 L1 gene was cloned into a non-integrative expression vector under the regulation of a methanol-inducible promoter and used to transform competent Pichia pastoris cells. Purification of L1 protein from yeast extracts was performed using heparin-sepharose chromatography, followed by a disassembly/reassembly step. VLPs could be assembled from the purified L1 protein, as demonstrated by electron microscopy. The display of conformational epitopes on the VLPs surface was confirmed by hemagglutination and hemagglutination inhibition assays and by immuno-electron microscopy. This study has implications for the development of an alternative platform for the production of a papillomavirus vaccine that could be provided by public health programs, especially in resource-poor areas, where there is a great demand for low-cost vaccines.

Growth factors and cytokines modulate gene expression of cell-surface proteoglycans in human periodontal ligament cells

Cell-surface proteoglycans have been known to be involved in many functions including interactions with components of the extracellular microenvironment, and act as co-receptors which bind and modify the action of various growth factors and cytokines. The purpose of this study was to determine the regulation by growth factors and cytokines on cell-surface proteoglycan gene expression in cultured human periodontal ligament (PDL) cells. Subconfluent, quiescent PDL cells were treated with various concentrations of serum, bFGF, PDGF-BB, TGF-beta1, IL-1 beta, and IFN-gamma. RT-PCR technique was used, complemented with Northern blot for syndecan-1, to examine the effects of these agents on the mRNA expression of five cell-surface proteoglycans (syndecan-1, syndecan-2, syndecan-4, glypican and betaglycan). Syndecan-1 mRNA levels increased in response to serum, bFGF and PDGF-BB, but decreased in response to TGF-beta1, IL-1 beta and IFN-gamma. In contrast, syndecan-2 mRNA levels were upregulated by TCF-beta1 and IL-1 beta stimulation, but remained unchanged with the other agents. Betaglycan gene expression decreased in response to serum, but was upregulated by TCF-beta1 and unchanged by the other stimulants. Additionally, syndecan-4 and glypican were not significantly altered in response to the regulator molecules studied, with the exception that glypican is decreased in response to IFN-gamma. These data demonstrate that the gene expression of the five cell-surface proteoglycans studied is differentially regulated in PDL cells lending support to the nation of distinct functions for these cell-surface proteoglycans. (C) 2001 Wiley-Liss, inc.

Cell-surface proteoglycan expression by lymphocytes from peripheral blood and Gingiva in health and periodontal disease

Cell-surface proteoglycans are involved in lymphocyte migration and activation. This study investigated the expression of syndecan-1, syndecan-4, and glypican in peripheral blood lymphocytes and by lymphocytes in variously inflamed periodontal tissues. Gingival specimens from healthy, gingivitis, or chronic periodontitis sites were stained by means of antibodies against B- and T-lymphocytes and also syndecan-1, syndecan-4, and glypican. Syndecan-1 expression by peripheral blood mononuclear cells (PBMC) from healthy, gingivitis, and chronic periodontitis subjects was assessed by flow cytometry. Syndecan-1 was expressed by B-cells/plasma cells but not T-cells in both gingivitis and chronic periodontitis lesions, Both B-cells/plasma cells and T-cells in gingivitis and chronic periodontitis expressed syndecan-4. Glypican was expressed only by macrophages. Stimulation of PBMC with mitogens and growth factors modulated syndecan-1 expression in both the T- and B-cells. Thus, cell-surface proteoglycan expression by lymphocytes in periodontal inflammation is cell-type-specific and may be modulated by inflammation.

Altered mucin expression in the gastrointestinal tract: a review

Early studies of changes in mucin expression in disorders of the gastrointestinal tract focused on alterations in the carbohydrate chain. This review briefly considers the various mechanisms by which such alterations may come about: (a) normal variation, (b) sialic acid alterations, (c) defective assembly of carbohydrate side-chains, (d) changed expression of core proteins and (e) epithelial metaplasia. The availability of monoclonal antibodies to mucin core proteins adds a new dimension to mucin histochemistry. It is now possible to offer explanations for traditional mucin histochemical findings on the basis of lineage-specific patterns of mucin core protein expression. Changes in core protein expression are described in inflammatory, metaplastic and neoplastic disorders of the gastrointestinal tract. The possibility that mucin change could be important in the aetiology of some diseases such as ulcerative colitis and H. pylori gastritis is considered. It is more probable, however, that changes in mucin expression are secondary to reprogramming of cellular differentiation and altered cell turnover. As such they may serve as markers to explain pathogenesis and provide novel diagnostic and prognostic information.

Transcriptional response of murine macrophages upon infection with opsonized Paracoccidioides brasiliensis yeast cells

Paracoccidioides brasiliensis is the etiologic agent of the Paracoccidioidomycosis the most common systemic mycosis in Latin America. Little is known about the regulation of genes involved in the innate immune host response to P. brasiliensis. We therefore examined the kinetic profile of gene expression of peritoneal macrophage infected with P. brasiliensis. Total RNA from macrophages at 6, 24 and 48 h was extracted, hybridized onto nylon membranes and analyzed. An increase in the transcription of a number of pro-inflammatory molecules encoding membrane proteins, metalloproteases, involved in adhesion and phagocytosis, are described. We observed also the differential expression of genes whose products may cause apoptotic events induced at 24 h. In addition, considering the simultaneous analyses of differential gene expression for the pathogen reported before by our group, at six hours post infection, we propose a model at molecular level for the P. brasiliensis-macrophage early interaction. In this regard, P. brasiliensis regulates genes specially related to stress and macrophages, at the same time point, up-regulate genes related to inflammation and phagocytosis, probably as an effort to counteract infection by the fungus. (c) 2007 Elsevier Masson SAS. All fights reserved.

Interrelationship between Epstein-Barr virus infection in gastric carcinomas and the expression of apoptosis-associated proteins

Aims: Epstein-Barr virus (EBV) and its associated proteins may be protective against the occurrence of apoptosis that would normally inhibit cancer development and progression. Alternatively, the viral infection may cause altered or mutated expression of oncogenes or tumour suppressor genes that are necessary for tumour development. an action that may also involve apoptosis, In this study, a relationship was sought between occurrence of EBV infection, expression of apoptosis-associated proteins (tumour suppressor gene p53 and oncogenes c-myc and bcl-2) and levels of cell death (apoptosis or necrosis) in 119 cases of gastric carcinoma. Methods and results: The EBV status of the gastric carcinomas (using the EBV-encoded small RNA I (EBER-1) and in-situ hybridization), stage and grade of tumour and sex of patients were compared for bcl-2, p53 and c-myc expression patterns. EBER-1 was detected in approximately 20% of cases studied. There was no significant correlation between levels of cell death in the tumour tissue and EBV status. In the protein analyses, development and progression of gastric carcinoma, with or without EBV infection. was independent of bcl-2 expression. However, in gastric cancers with EBV infection, p53 overexpression was inhibited and c-myc expression was increased in early stage cancers, in comparison with decreased c-myc expression in late stage cancers. Conclusions: The p53 and c-myc expression patterns indicate that EBV-infected gastric carcinomas are less likely to have a natural regression via apoptosis at an early stage and explain, in part, the resistance to treatment of late stage of gastric cancers.

Apoptosis and Expression of Bcl-2, Bcl-XL, and Bax in Renal Cell Carcinomas

There are at present disparate published results with regard to the relevance of the Bcl-2 gene family, levels of apoptosis, and cell proliferation in the development and progression of renal cell carcinoma (RCC). The present study v analyses the interrelationship between the expression of representatives of the anti-apoptotic (Bcl-2, Bcl-X-L) or pro-apoptotic (Bax) Bcl-2 proteins, incidence of apoptosis, and mitosis in a selected small group of 22 graded RCCs that had paired normal renal tissue, or non-neoplastic tissue in the renal biopsy specimen. The cases were chosen to determine the feasibility of measuring these parameters as potential surrogate markers of progression or treatment failure of the cancers. The results showed that in approximately 50% of the RCCs, where Bcl-2 and/or Bcl-X-L expression was high, apoptosis it-as not detected, and when expression of these proteins was low or not found, increased levels of apoptosis were seen. In most of the remaining 50% of samples, high levels of Bcl-X-L but not Bcl-2 were negatively correlated with low levels of apoptosis (Bcl-X-L: r = -0.437, P = 0.07 and Bcl-2: r = + 0.560, P = 0.02). For the same group of samples, high Bax expression was found in association with apoptosis (r = + 0.578, P = 0,02). A novel finding was an association between low expression of Bcl-2 an/or Bcl-X-L in normal tissue and the level of expression of these proteins in the RCCs, an intrinsic variation that may be an individual patient factor. The results indicate that, in RCCs with increased expression of Bcl-2 and/or Bcl-X-L, levels of apoptosis are minimal and these combined factors may assist in progression of the cancers and resistance to treatments.

Cdc25B activity is regulated by 14-3-3

In the G2 phase cell cycle checkpoint arrest, the cdc25-dependent activation of cyclin B/cdc2, a critical step in regulating entry into mitosis, is blocked. Studies in yeast have demonstrated that the inhibition of cdc25 function involves 14-3-3 binding to cdc25, In humans, two cdc25 isoforms have roles in G2/M progression, cdc25B and cdc25C, both bind 14-3-3, Abrogating 14-3-3 binding to cdc25C attenuates the G2 checkpoint arrest, but the contribution of 14-3-3 binding to the regulation of cdc25B function is unknown. Here we demonstrate that high level over-expression of cdc25B in G2 checkpoint arrested cells can activate cyclin B/cdc2 and overcome the checkpoint arrest. Mutation of the major 14-3-3 binding site, S323, or removal of the N-terminal regulatory domain are strong activating mutations, increasing the efficiency with which the mutant forms of cdc25B not only overcome the arrest, but also initiate aberrant mitosis, We also demonstrate that 14-3-3 binding to the S323 site on cdc25B blocks access of the substrate cyclin/cdks to the catalytic site of the enzyme, thereby directly inhibiting the activity of cdc25B, This provides direct mechanistic evidence that 14-3-3 binding to cdc25B can regulate its activity, thereby controlling progression into mitosis.

Insulin-like growth factor 2 cDNA cloning and ontogeny of gene expression in the liver of the marsupial brushtail possum (Trichosurus vulpecula)

The cDNA sequence for insulin-like growth factor 2 (IGF-2) was determined from the liver of the marsupial brushtail possum (Trichosurus vulpecula) using reverse transcription followed by polymerase chain reaction (RT-PCR) with gene-specific primers. The 359 bp of possum sequence encompassed the mature peptide, 27 bp of the signal peptide, and 125 bp of the E-peptide. Alignment of the deduced amino acid sequence with those from other species indicated that the mature peptide was 71 amino acids in length, 4 amino acids longer than most other mammals. At both the nucleotide and amino acid levels there was a high degree of sequence identity with IGF-2 from other mammalian and nonmammalian species. Amino acid identity ranged from 94.4% with a variant form of human IGF-2 to 80.3% with zebrafinch IGF-2. Northern analysis revealed that radiolabeled possum IGF-2, cDNA hybridized to multiple transcripts in the liver of both adult possums and 150-day-old pouch young and that the overall level of expression was greater in pouch young. Semiquantitative RT-PCR with total RNA from liver samples of pouch young aged 12 to 150 days postpartum and adults confirmed that IGF-2 gene expression was two to three times more abundant in pouch young than in adults but there was no significant change in the level of expression during pouch life. Unlike other mammalian species, in which there is a decline in levels of liver IGF-2 gene expression around the time of birth, levels in the marsupial brushtail possum remain elevated for at least 150 days after birth. This suggests that the decline in liver IGF-2 expression in marsupials and eutherians occurs at a similar stage of development and may reflect a role for this growth factor during the postnatal growth and development of the marsupial, (C) 2001 Academic Press.

Glyt-1 expression in cultured human Muller cells and intact retinae

In this study, we demonstrate that Muller cells cultured from human retinas are capable of strongly expressing the glycine transporter Glyt-1 as assessed by immunocytochemistry. By contrast, intact normal and pathological human retinas exhibit Glyt-1 immunoreactivity only in neurons. These data suggest that Glyt-1 expression in cultured Muller cells is an epiphenomenon associated with culturing in vitro, rather than a normal physiological or even pathophysiological phenomenon in vivo. (C) 2001 Wiley-Liss, Inc.