Cloning and expression of a gene encoding an integrin-like protein in Candida albicans.

The existence of integrin-like proteins in Candida albicans has been postulated because monoclonal antibodies to the leukocyte integrins alpha M and alpha X bind to blastospores and germ tubes, recognize a candidal surface protein of approximately 185 kDa, and inhibit candidal adhesion to human epithelium. The gene alpha INT1 was isolated from a library of C. albicans genomic DNA by screening with a cDNA probe from the transmembrane domain of human alpha M. The predicted polypeptide (alpha Int1p) of 188 kDa contains several motifs common to alpha M and alpha X: a putative I domain, two EF-hand divalent cation-binding sites, a transmembrane domain, and a cytoplasmic tail with a single tyrosine residue. An internal RGD tripeptide is also present. Binding of anti-peptide antibodies raised to potential extracellular domains of alpha Int1p confirms surface localization in C. albicans blastopores. By Southern blotting, alpha INT1 is unique to C. albicans. Expression of alpha INT1 under control of a galactose-inducible promoter led to the production of germ tubes in haploid Saccharomyces cerevisiae and in the corresponding ste12 mutant. Germ tubes were not observed in haploid yeast transformed with vector alone, in transformants expressing a galactose-inducible gene from Chlamydomonas, or in transformants grown in the presence of glucose or raffinose. Transformants producing alpha Int1p bound an anti-alpha M monoclonal antibody and exhibited enhanced aggregation. Studies of alpha Int1p reveal novel roles for primitive integrin-like proteins in adhesion and in STE12-independent morphogenesis.

Grb-IR: a SH2-domain-containing protein that binds to the insulin receptor and inhibits its function.

To identify potential signaling molecules involved in mediating insulin-induced biological responses, a yeast two-hybrid screen was performed with the cytoplasmic domain of the human insulin receptor (IR) as bait to trap high-affinity interacting proteins encoded by human liver or HeLa cDNA libraries. A SH2-domain-containing protein was identified that binds with high affinity in vitro to the autophosphorylated IR. The mRNA for this protein was found by Northern blot analyses to be highest in skeletal muscle and was also detected in fat by PCR. To study the role of this protein in insulin signaling, a full-length cDNA encoding this protein (called Grb-IR) was isolated and stably expressed in Chinese hamster ovary cells overexpressing the human IR. Insulin treatment of these cells resulted in the in situ formation of a complex of the IR and the 60-kDa Grb-IR. Although almost 75% of the Grb-IR protein was bound to the IR, it was only weakly tyrosine-phosphorylated. The formation of this complex appeared to inhibit the insulin-induced increase in tyrosine phosphorylation of two endogenous substrates, a 60-kDa GTPase-activating-protein-associated protein and, to a lesser extent, IR substrate 1. The subsequent association of this latter protein with phosphatidylinositol 3-kinase also appeared to be inhibited. These findings raise the possibility that Grb-IR is a SH2-domain-containing protein that directly complexes with the IR and serves to inhibit signaling or redirect the IR signaling pathway.

A model for the transcriptional regulation of the CYP2B1/B2 gene in rat liver.

The phenobarbitone-responsive minimal promoter has been shown to lie between nt -179 and nt + 1 in the 5' (upstream) region of the CYP2B1/B2 gene in rat liver, on the basis of the drug responsiveness of the sequence linked to human growth hormone gene as reporter and targeted to liver as an asialoglycoprotein-DNA complex in vivo. Competition analyses of the nuclear protein-DNA complexes formed in gel shift assays with the positive (nt -69 to -98) and negative (nt -126 to -160) cis elements (PE and NE, respectively) identified within this region earlier indicate that the same protein may be binding to both the elements. The protein species purified on PE and NE affinity columns appear to be identical based on SDS/PAGE analysis, where it migrates as a protein of 26-28 kDa. Traces of a high molecular weight protein (94-100 kDa) are also seen in the preparation obtained after one round of affinity chromatography. The purified protein stimulates transcription of a minigene construct containing the 179 nt on the 5' side of the CYP2B1/B2 gene linked to the I exon in a cell-free system from liver nuclei. The purified protein can give rise to all the three complexes (I, II, and III) with the PE, just as the crude nuclear extract, under appropriate conditions. Manipulations in vitro indicate that the NE has a significantly higher affinity for the dephosphorylated form than for the phosphorylated form of the protein. The PE binds both forms. Phenobarbitone treatment of the animal leads to a significant increase in the phosphorylation of the 26- to 28-kDa and 94-kDa proteins in nuclear labeling experiments followed by isolation on a PE affinity column. We propose that the protein binding predominantly to the NE in the dephosphorylated state characterizes the basal level of transcription of the CYP2B1/B2 gene. Phenobarbitone treatment leads to phosphorylation of the protein, shifting the equilibrium toward binding to the PE. This can promote interaction with an upstream enhancer through other proteins such as the 94-kDa protein and leads to a significant activation of transcription.

Sp1 functions in a chromatin-dependent manner to augment human alpha-globin promoter activity.

The 5' flanking region of the human alpha-globin gene is highly G + C rich and contains multiple copies of the consensus sequence for the Sp1 binding site. We investigated the role of this G + C-rich region in augmenting alpha-globin promoter activity in the presence of the far-upstream alpha-globin enhancer, HS-40. We show that in transiently transfected erythroid cells, deletion of the alpha-globin G + C-rich 5' flanking region has no effect on alpha-globin promoter activity. However, upon stable integration into chromatin, deletion of this region causes a nearly 90% decrease in promoter activity compared with expression from an alpha-globin promoter retaining this region. These results suggest that the alpha-globin G + C-rich 5' flanking region augments alpha-globin promoter activity in a chromatin-dependent manner. We further show that this G + C-rich region is required for the activation of alpha-globin gene expression during erythroid differentiation. Finally, we show by both footprint analysis and functional assays that the ability of the G + C-rich region to increase alpha-globin promoter activity from a stably integrated alpha-globin gene is mediated by its multiple binding sites for the transcription factor Sp1.

Identification of human granzyme B promoter regulatory elements interacting with activated T-cell-specific proteins: implication of Ikaros and CBF binding sites in promoter activation.

Granzyme B serine protease is found in the granules of activated cytotoxic T cells and in natural and lymphokine-activated killer cells. This protease plays a critical role in the rapid induction of target cell DNA fragmentation. The DNA regulatory elements that are responsible for the specificity of granzyme B gene transcription in activated T-cells reside between nt -148 and +60 (relative to the transcription start point at +1) of the human granzyme B gene promoter. This region contains binding sites for the transcription factors Ikaros, CBF, Ets, and AP-1. Mutational analysis of the human granzyme B promoter reveals that the Ikaros binding site (-143 to -114) and the AP-1/CBF binding site (-103 to -77) are essential for the activation of transcription in phytohemagglutinin-activated peripheral blood lymphocytes, whereas mutation of the Ets binding site does not affect promoter activity in these cells.

Three-dimensional structure of a cysteine-rich repeat from the low-density lipoprotein receptor.

The low-density lipoprotein (LDL) receptor plays a central role in mammalian cholesterol metabolism, clearing lipoproteins which bear apolipoproteins E and B-100 from plasma. Mutations in this molecule are associated with familial hypercholesterolemia, a condition which leads to an elevated plasma cholesterol concentration and accelerated atherosclerosis. The N-terminal segment of the LDL receptor contains a heptad of cysteine-rich repeats that bind the lipoproteins. Similar repeats are present in related receptors, including the very low-density lipoprotein receptor and the LDL receptor-related protein/alpha 2-macroglobulin receptor, and in proteins which are functionally unrelated, such as the C9 component of complement. The first repeat of the human LDL receptor has been expressed in Escherichia coli as a glutathione S-transferase fusion protein, and the cleaved and purified receptor module has been shown to fold to a single, fully oxidized form that is recognized by the monoclonal antibody IgG-C7 in the presence of calcium ions. The three-dimensional structure of this module has been determined by two-dimensional NMR spectroscopy and shown to consist of a beta-hairpin structure, followed by a series of beta turns. Many of the side chains of the acidic residues, including the highly conserved Ser-Asp-Glu triad, are clustered on one face of the module. To our knowledge, this structure has not previously been described in any other protein and may represent a structural paradigm both for the other modules in the LDL receptor and for the homologous domains of several other proteins. Calcium ions had only minor effects on the CD spectrum and no effect on the 1H NMR spectrum of the repeat, suggesting that they induce no significant conformational change.

Domains of transcription factor Sp1 required for synergistic activation with sterol regulatory element binding protein 1 of low density lipoprotein receptor promoter.

Feedback regulation of transcription from the low density lipoprotein (LDL) receptor gene is fundamentally important in the maintenance of intracellular sterol balance. The region of the LDL receptor promoter responsible for normal sterol regulation contains adjacent binding sites for the ubiquitous transcription factor Sp1 and the cholesterol-sensitive sterol regulatory element-binding proteins (SREBPs). Interestingly, both are essential for normal sterolmediated regulation of the promoter. The cooperation by Sp1 and SREBP-1 occurs at two steps in the activation process. SREBP-1 stimulates the binding of Sp1 to its adjacent recognition site in the promoter followed by enhanced stimulation of transcription after both proteins are bound to DNA. In the present report, we have defined the protein domains of Sp1 that are required for both synergistic DNA binding and transcriptional activation. The major activation domains of Sp1 that have previously been shown to be essential to activation of promoters containing multiple Sp1 sites are required for activation of the LDL receptor promoter. Additionally, the C domain is also crucial. This slightly acidic approximately 120-amino acid region is not required for efficient synergistic activation by multiple Sp1 sites or in combination with other recently characterized transcriptional regulators. We also show that Sp1 domain C is essential for full, enhanced DNA binding by SREBP-1. Taken together with other recent studies on the role of Sp1 in promoter activation, the current experiments suggest a unique combinatorial mechanism for promoter activation by two distinct transcription factors that are both essential to intracellular cholesterol homeostasis.

Prostate cancer in a transgenic mouse.

Progress toward understanding the biology of prostate cancer has been slow due to the few animal research models available to study the spectrum of this uniquely human disease. To develop an animal model for prostate cancer, several lines of transgenic mice were generated by using the prostate-specific rat probasin promoter to derive expression of the simian virus 40 large tumor antigen-coding region. Mice expressing high levels of the transgene display progressive forms of prostatic disease that histologically resemble human prostate cancer, ranging from mild intraepithelial hyperplasia to large multinodular malignant neoplasia. Prostate tumors have been detected specifically in the prostate as early as 10 weeks of age. Immunohistochemical analysis of tumor tissue has demonstrated that dorsolateral prostate-specific secretory proteins were confined to well-differentiated ductal epithelial cells adjacent to, or within, the poorly differentiated tumor mass. Prostate tumors in the mice also display elevated levels of nuclear p53 and a decreased heterogeneous pattern of androgen-receptor expression, as observed in advanced human prostate cancer. The establishment of breeding lines of transgenic mice that reproducibly develop prostate cancer provides an animal model system to study the molecular basis of transformation of normal prostatic cells and the factors influencing the progression to metastatic prostate cancer.

Avaliação do efeito de polimorfismos genéticos com a dependência à nicotina

Introdução: A identificação de variantes genéticas que predispõem a maior susceptibilidade à dependência à nicotina pode ser importante para a prevenção e o tratamento do tabagismo. No contexto de medicina personalizada, os principais objetivos do presente estudo foram avaliar se polimorfismos nos genes CHRNA2, CHRNA3, CHRNA5 e CHRNB3 estão associados com o nível de dependência em indivíduos fumantes e com o resultado do tratamento antitabágico. Métodos: Estudo de coorte com 1049 pacientes fumantes que receberam tratamento farmacológico (vareniclina, vareniclina e bupropiona, bupropiona e/ou terapia de reposição nicotínica). O sucesso na cessação tabágica foi considerado para os pacientes que completaram 6 meses de abstinência contínua. O teste de Fagerström para a dependência à nicotina (FTND) e o escore de consumo situacional Issa foram utilizados para avaliar a dependência à nicotina. A escala de conforto PAF foi utilizada para avaliar o conforto do paciente durante o tratamento. Os polimorfismos CHRNA2 rs2472553, CHRNA3 rs1051730, CHRNA5 rs16969968, CHRNA5 rs2036527 e CHRNB3 rs6474413 foram genotipados pela análise da curva de melting. Resultados: As mulheres portadoras dos genótipos GA e AA para os polimorfismos CHRNA5 rs16969968 e rs2036527 obtiveram maior taxa de sucesso no tratamento antitabagismo: 44,0% e 56,3% (rs16969968), 41,5% e 56,5% (rs2036527), respectivamente; em comparação com as mulheres portadoras do genótipo GG: 35,7% (rs16969968) e 34,8% (rs2036527), (P=0,03; n=389; P=0,01; n=391). Os genótipos GA ou AA para os rs16969968 e rs2036527 foram associados com maior OR para o sucesso em mulheres (OR=1,63; IC 95%=1,04-2,54; P=0,03 e OR=1,59; IC 95%=1,02-2,48; P=0,04; respectivamente), em um modelo multivariado. Não foi encontrada associação dos polimorfismos no gene CHRNA5 com o escore de FTND. Para os polimorfismos CHRNA2 rs2472553, CHRNA3 rs1051730 e CHRNB3 rs6474413 não foram encontradas associações significativas com os fenótipos estudados. Conclusão: Os polimorfismos rs16969968 e rs2036527 no gene CHRNA5 foram associados com maior taxa de sucesso no tratamento antitabagismo em mulheres. Estes resultados podem contribuir com avanços na terapêutica baseada em medicina personalizada

Interactive effects of MC1R and OCA2 on melanoma risk phenotypes

The relationships between MC1R gene variants and red hair, skin reflectance, degree of freckling and nevus count were investigated in 2331 adolescent twins, their sibs and parents in 645 twin families. Penetrance of each MC1R variant allele was consistent with an allelic model where effects were multiplicative for red hair but additive for skin reflectance. Of nine MC1R variant alleles assayed, four common alleles were strongly associated with red hair and fair skin (Asp84Glu, Arg151Cys, Arg160Trp and Asp294His), with a further three alleles having low penetrance (Val60Leu, Val92Met and Arg163Gln). These variants were separately combined for the purposes of this analysis and designated as strong 'R' (OR=63.3; 95% CI 31.9-139.6) and weak 'r ' (OR=5.1; 95% CI 2.5-11.3) red hair alleles. Red-haired individuals are predominantly seen in the R/R and R/r groups with 67.1 and 10.8%, respectively. To assess the interaction of the brown eye color gene OCA2 on the phenotypic effects of variant MC1R alleles we included eye color as a covariate, and also genotyped two OCA2 SNPs (Arg305Trp and Arg419Gln), which were confirmed as modifying eye color. MC1R genotype effects on constitutive skin color, freckling and mole count were modified by eye color, but not genotype for these two OCA2 SNPs. This is probably due to the association of these OCA2 SNPs with brown/green not blue eye color. Amongst individuals with a R/R genotype (but not R/r), those who also had brown eyes had a mole count twice that of those with blue eyes. This suggests that other OCA2 polymorphisms influence mole count and remain to be described.

The policy and ethical implications of genetic research on attention deficit hyperactivity disorder

Objective: To review the policy and ethical implications of recent research on the molecular genetics of attention deficit hyperactivity disorder (ADHD). Method: MEDLINE and psycINFO database searches were used to identify studies on the genetics of ADHD. The implications of replicated candidate genes are discussed. Results: The findings for most genes have been inconsistent but several studies have implicated the genes in the dopaminergic pathway in the aetiology of ADHD. Conclusions: The current evidence on the genetics of ADHD is insufficient to justify genetic screening tests but it will provide important clues as to the aetiology of ADHD. Genetic information on susceptibility to ADHD has the potential to be abused and to stigmatize individuals. Researchers and clinicians need to be mindful of these issues in interpreting and disseminating the results of genetic studies of ADHD.

A polymorphism in the agouti signalling protein (ASIP) is associated with decreased levels of mRNA

To date, a role for agouti signalling protein (ASIP) in human pigmentation has not been well characterized. It is known that agouti plays a pivotal role in the pigment switch from the dark eumelanin to the light pheomelanin in the mouse. However, because humans do not have an agouti banded hair pattern, its role in human pigmentation has been questioned. We previously identified a single polymorphism in the 3'-untranslated region (UTR) of ASIP that was found at a higher frequency in African-Americans compared with other population groups. To compare allele frequencies between European-Australians and indigenous Australians, the g.8818A -> G polymorphism was genotyped. Significant differences were seen in allele frequencies between these groups (P < 0.0001) with carriage of the G allele highest in Australian Aborigines. In the Caucasian sample set a strong association was observed between the G allele and dark hair colour (P = 0.004) (odds ratio 4.6; 95% CI 1.4-15.27). The functional consequences of this polymorphism are not known but it was postulated that it might result in message instability and premature degradation of the transcript. To test this hypothesis, ASIP mRNA levels were quantified in melanocytes carrying the variant and non-variant alleles. Using quantitative real-time polymerase chain reaction the mean ASIP mRNA ratio of the AA genotype to the AG genotype was 12 (P < 0.05). This study suggests that the 3'-UTR polymorphism results in decreased levels of ASIP and therefore less pheomelanin production.

Identification and molecular modeling of a novel, plant-like, human purple acid phosphatase

Purple acid phosphatases are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. Only one isoform of similar to 35 kDa has been isolated from animals, where it is associated with bone resorption and microbial killing through its phosphatase activity, and hydroxyl radical production, respectively. Using the sensitive PSI-BLAST search method, sequences representing new purple acid phosphatase-like proteins have been identified in mammals, insects and nematodes. These new putative isoforms are closely related to the similar to 55 kDa purple acid phosphatase characterized from plants. Secondary structure prediction of the new human isoform further confirms its similarity to a purple acid phosphatase from the red kidney bean. A structural model for the human enzyme was constructed based on the red kidney bean purple acid phosphatase structure. This model shows that the catalytic centre observed in other purple acid phosphatases is also present in this new isoform. These observations suggest that the sequences identified in this study represent a novel subfamily of plant-like purple acid phosphatases in animals and humans. (c) 2006 Elsevier B.V. All rights reserved.

Neoplastic progression occurs through mutator pathways in hyperplastic polyposis of the colorectum

Aim-Colorectal cancer has been described in association with hyperplastic polyposis but the mechanism underlying this observation is unknown. The aim of this study was to characterise foci of dysplasia developing in the polyps of subjects with hyperplastic polyposis on the basis of DNA microsatellite status and expression of the DNA mismatch repair proteins hMLH1, hMSH2, and hMSH6. Materials and methods-The material was derived from four patients with hyperplastic polyposis and between one and six synchronous colorectal cancers. Normal (four), hyperplastic (13), dysplastic (13), and malignant (11) samples were microdissected and a PCR based approach was used to identify mutations at 10 microsatellite loci, TGF beta IIR, IGF2R, BAX, MSH3, and MSH6. Microsatellite instability-high (MSI-H) was diagnosed when 40% or more of the microsatellite loci showed mutational bandshifts. Serial sections were stained for hMLH1, hMSH2, and hMSH6. Result-DNA microsatellite instability was found in 1/13 (8%) hyperplastic samples, in 7/13 (54%) dysplastic foci, and in 8/11 (73%) cancers. None of the MSI-low (MSI-L) samples (one hyperplastic, three dysplastic, two cancers) showed loss of hMLH1 expression. All four MSI-H dysplastic foci and six MSI-H cancers showed loss of hMLH1 expression. Loss of hMLH1 in MSI-H but not in MSI-L lesions showing dysplasia or cancer was significant (p< 0.001, Fisher's exact test). Loss of hMSH6 occurred in one MSI-H cancer and one MSS focus of dysplasia which also showed loss of hMLH1 staining. Conclusion-Neoplastic changes in hyperplastic polyposis may occur within a hyperplastic polyp. Neoplasia may be driven by DNA instability that is present to a low (MSI-L) or high (MSI-H) degree. MSI-H but not MSI-L dysplastic foci are associated with loss of hMLH1 expression. At least two mutator pathways drive neoplasia in hyperplastic polyposis. The role of the hyperplastic polyp in the histogenesis of sporadic DNA microsatellite unstable colorectal cancer should be examined.