Identification and validation of genes involved in gastric tumorigenesis

Background  

Gastric cancer is one of the common cancers seen in south India. Unfortunately more than 90% are advanced by the time they report to a tertiary centre in the country. There is an urgent need to characterize these cancers and try to identify potential biomarkers and novel therapeutic targets.     

Maternal periconceptional smoking and alcohol consumption and risk for select congenital anomalies

BACKGROUND: This study examined the association between maternal smoking and alcohol use (including binge drinking) during the periconceptional period (i.e., 2 months before through 2 months after conception) and the risk of orofacial clefts, NTDs, and conotruncal heart defects in offspring. METHODS: Data were drawn from a population‐based case‐control study of fetuses and live‐born infants among a cohort of California births between July 1999 and June 2003. The 1,355 cases comprised of 701 orofacial clefts, 337 NTDs, and 323 conotruncal heart defects. Information on smoking and alcohol consumption was obtained via telephone interviews with mothers of 1,355 (80% of eligibles) cases and 700 (77% of eligibles) nonmalformed, live‐born controls. RESULTS: Maternal smoking of five cigarettes or less per day was associated with reduced risks of NTDs (OR 0.7; 95% CI: 0.3, 1.4), whereas the risk associated with higher cigarette consumption was lower for conotruncal heart defects (OR 0.5; 95% CI: 0.2, 1.2). Maternal intake of alcohol less than 1 day per week was associated with a 1.6‐ to 2.1‐fold higher risk of NTDs (95% CI: 0.9, 2.6), d‐transposition of the great arteries (95% CI: 1.1, 3.2), and multiple cleft lip with or without cleft palate (CLP) (95% CI: 0.8, 4.5). Risks associated with more frequent alcohol intake were 2.1 for NTDs (95% CI: 1.1, 4.0) and 2.6 for multiple CLP (95% CI: 1.1, 6.1). CONCLUSIONS: This study observed that maternal alcohol intake increased the risk for d‐transposition of the great arteries, NTDs, and multiple CLP in infants. By contrast, smoking was associated with a lower risk of NTDs and conotruncal heart defects. Birth Defects Research (Part A), 2008. © 2008 Wiley‐Liss, Inc.     

Effects of smoking and alcohol consumption on vertebral deformity in the elderly--an epidemiological study

The aim of the study was to assess the role of smoking and alcohol consumption as possible risk factors for vertebral deformities in an elderly Croatian population sample. Data on smoking habit, alcohol consumption, body mass index, and overall life activity were collected in 425 randomly chosen community dwelling subjects. Radiographic morphometric method was used to assess vertebral deformities of thoracic and lumbar spine. Men smoked and drank significantly more than women. There was no association of either smoking status or number of cigarettes, or frequency of alcohol intake with prevalent vertebral deformities. There was a tendency of an increased risk of vertebral deformities in heavy drinkers (OR = 1.69; 95% CI = 0.98-2.91), and a reduced risk of these deformities in female regular drinkers (OR = 0.72; 95% CI = 0.14-3.66). Further studies in the Croatian population are needed to establish the association of smoking and alcohol consumption with vertebral deformities.     

Influence of paternal age, smoking, and alcohol consumption on congenital anomalies

The potential effects of paternal exposures on fetal development are of great public and scientific concern, yet few epidemiologic studies have examined this association. Single live births from 1959 to 1966 among 14,685 Kaiser Foundation Health Plan members who participated in the Child Health and Development Studies were analyzed to assess the impact of paternal age, cigarette smoking, and alcohol consumption on the occurrence of birth defects in the offspring. Prevalence odds ratios for anomalies identified by age 5 were analyzed, contrasting exposed to unexposed fathers with adjustment for maternal age, race, education, smoking, and alcohol use. Advanced paternal age was associated with increased risk of preauricular cyst, nasal aplasia, cleft palate, hydrocephalus, pulmonic stenosis, urethral stenosis, and hemangioma. Father's cigarette smoking was more common among children with cleft lip +/- cleft palate, hydrocephalus, ventricular septal defect, and urethral stenosis. Alcohol use by the father was most positively related to the offspring's risk of ventricular septal defect. For both smoking and alcohol use, inverse associations were more common than positive associations. These data generally do not indicate strong or widespread associations between paternal attributes and birth defects. However, because of this study's imprecision, limited ability to isolate defects most likely to be of paternal origin, and the identification of several suggestive associations with age and smoking, further study of this issue would be of value.     

Identification of new genes involved in disaccharide fermentation in yeast

Summary Maltose non-fermenting mutants were obtained from strains carrying a MAL4 allele which permits constitutive synthesis of maltase. Cells carrying this allele are able to utilize sucrose in the absence of the classical sucrose genes. All maltose non-fermenting mutants were also sucrose non-fermenters. Eight mutants had become maltase negative; 19 mutants could still form maltase constitutively.In crosses with segregational maltose and sucrose non-fermenting strains, enzyme negative mutants gave diploids unable to ferment maltose and sucrose. Enzyme positive, non-fermenting mutants gave diploids which readily fermented maltose and sucrose. This latter type of mutants was designated dsf (disaccharide fermentation) mutants.The diploids derived from crossing non-fermenting mutants with segregational non-fermenters were subjected to tetrad analysis. Enzyme negative non-fermenters gave only non-fermenting progeny. The dsf mutants segregated both fermenting and non-fermenting progeny, some of which showed the dsf phenotype. This indicated that none of the dsf mutants had a defect in a gene closely linked to MAL4. Crosses between dsf mutants and strains carrying the maltose genes MAL2 and MAL3 showed that the mutations affected maltose fermentation in general. Sucrose fermentation in the presence of the classical sucrose gene SUC3 was not affected, nor were fermentation of glucose, fructose and galactose.The uptake of radioactivity from uniformly labeled maltose appeared to be blocked in mutants of at least four of the dsf genes. Only one non-leaky and a leaky mutant showed a significant uptake.These results suggest that there is an extremely complex transport system for maltose and sucrose or that the utilization of these disaccharides requires a complex series of metabolic reactions.     

Identification of genes involved in terbinafine resistance in Aspergillus nidulans

AIMS: To determine the pattern and the genetic basis of resistance to terbinafine, a drug extensively used for the treatment of fungal infections in humans. METHODS AND RESULTS: Four resistant mutants from Aspergillus nidulans isolated after irradiation with ultraviolet light were crossed with the master strain F (MSF). Genetic analysis revealed that a single gene, located on chromosome IV, is responsible for resistance to terbinafine and that the alleles responsible for this resistance in these mutants are of a codominant or dominant nature at high terbinafine concentrations. Furthermore, the interaction of this mutation with another one identified on chromosome II causes the double mutant to be highly resistant. CONCLUSIONS: Periodic surveillance of antimycotic susceptibility would be an important measure in detecting the emergence and spread of resistance. Mutation in a single gene could be responsible for resistance to terbinafine and a genic interaction may be responsible for a higher level of antimycotic resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: The understanding of the mechanisms that lead to changes in the sensitivity of a fungus to a given antifungal agent is important both in order to define strategies for the use of such agent and to guide the development of new antifungal agents.     

Identification of Listeria monocytogenes genes involved in salt and alkaline-pH tolerance

The capacity of Listeria monocytogenes to tolerate salt and alkaline stresses is of particular importance, as this pathogen is often exposed to such environments during food processing and food preservation. We screened a library of Tn917-lacZ insertional mutants in order to identify genes involved in salt and/or alkaline tolerance. We isolated six mutants sensitive to salt stress and 12 mutants sensitive to salt and alkaline stresses. The position of the insertion of the transposon was located in 15 of these mutants. In six mutants the transposon was inserted in intergenic regions, and in nine mutants it was inserted in genes. Most of the genes have unknown functions, but sequence comparisons indicated that they encode putative transporters.     

Identification of new genes involved in human adipogenesis and fat storage

Since the worldwide increase in obesity represents a growing challenge for health care systems, new approaches are needed to effectively treat obesity and its associated diseases. One prerequisite for advances in this field is the identification of genes involved in adipogenesis and/or lipid storage. To provide a systematic analysis of genes that regulate adipose tissue biology and to establish a target-oriented compound screening, we performed a high throughput siRNA screen with primary (pre)adipocytes, using a druggable siRNA library targeting 7,784 human genes. The primary screen showed that 459 genes affected adipogenesis and/or lipid accumulation after knock-down. Out of these hits, 333 could be validated in a secondary screen using independent siRNAs and 110 genes were further regulated on the gene expression level during adipogenesis. Assuming that these genes are involved in neutral lipid storage and/or adipocyte differentiation, we performed InCell-Western analysis for the most striking hits to distinguish between the two phenotypes. Beside well known regulators of adipogenesis and neutral lipid storage (i.e. PPARγ, RXR, Perilipin A) the screening revealed a large number of genes which have not been previously described in the context of fatty tissue biology such as axonemal dyneins. Five out of ten axonemal dyneins were identified in our screen and quantitative RT-PCR-analysis revealed that these genes are expressed in preadipocytes and/or maturing adipocytes. Finally, to show that the genes identified in our screen are per se druggable we performed a proof of principle experiment using an antagonist for HTR2B. The results showed a very similar phenotype compared to knock-down experiments proofing the "druggability". Thus, we identified new adipogenesis-associated genes and those involved in neutral lipid storage. Moreover, by using a druggable siRNA library the screen data provides a very attractive starting point to identify anti-obesity compounds targeting the adipose tissue.     

Identification of Bradyrhizobium nod genes involved in host-specific nodulation.

Three loci important for soybean nodulation by Bradyrhizobium japonicum were delimited by Tn5 mutagenesis on a 5.3-kilobase EcoRI fragment adjacent to the nodABC genes. Results of hybridization studies suggested that this region is conserved in Bradyrhizobium species but absent in all Rhizobium species. lacZ translational fusions of two of the loci contained in this region were found to be inducible by host-produced flavonoid chemicals via a mechanism requiring a functional nodD gene product. A mutation in one of the loci was found to result in an alteration of the host range of B. japonicum. This mutation appears to block nodulation at the step at which plant root cortical cell division is induced.     

Identification of Brucella spp. genes involved in intracellular trafficking

After uptake by host cells, the pathogen Brucella transits through early endosomes, evades phago–lysosome fusion and replicates in a compartment associated with the endoplasmic reticulum (ER). The molecular mechanisms underlying these processes are still poorly understood. To identify new bacterial factors involved in these processes, a library of 1800 Brucella melitensis 16M mini-Tn 5catkm mutants was screened for intracellular survival and multiplication in HeLa cells and J774A.1 macrophages. Thirteen mutants were identified as defective for their intracellular survival in both cell types. In 12 of them, the transposon had inserted in the virB operon, which encodes a type IV-related secretion system. The preponderance of virB mutants demonstrates the importance of this secretion apparatus in the intracellular multiplication of B. melitensis . We also examined the intracellular fate of three virB mutants ( virB2 , virB4 and virB9 ) in HeLa cells by immunofluorescence. The three VirB proteins are not necessary for penetration and the inhibition of phago–lysosomal fusion within non-professional phagocytes. Rather, the virB mutants are unable to reach the replicative niche and reside in a membrane-bound vacuole expressing the late endosomal marker, LAMP1, and the sec61β protein from the ER membrane, proteins that are present in autophagic vesicles originating from the ER.     

Identification of human genes involved in repair and tolerance of DNA damage

Summary An overview is presented on strategies of cloning mammalian DNA repair genes. Complementation of human and rodent repair defects and mutagen hypersensitivities by chromosome and DNA mediated gene transfer and mRNA microinjection is described, and the features of the cloned human DNA repair genes are summarized. It is shown that transfection of repair deficient cell lines with cloned bacterial and human genes may give rise to protection from the genotoxic effects of mutagens.Abbreviations MGMT O⁶-methylguanine-DNA methyltransferase - MNNG N-methyl-N-nitro-N-nitrosoguanidine - HeCNU N-hydroxyethyl-N-chloroethylnitrosourea - MMC mitomycin C - MPA mycophenolic acid - ERCC excision-repair cross-complementing rodent UV-complementation group - UV ultraviolet light - XP xeroderma pigmentosum - CHO Chinese hamster ovary Dedicated to Prof. Dr. U. Hagen on the occasion of his 65th birthdayExtended version of an oral presentation given at the workshop Molecular Radiation Biology. German Section of the DNA Repair Network, München-Neuherberg, 21.–23.3.90     

Identification of glucosyltransferase genes involved in sinapate metabolism and lignin synthesis in Arabidopsis

Sinapic acid is a major phenylpropanoid in Brassicaceae providing intermediates in two distinct metabolic pathways leading to sinapoyl esters and lignin synthesis. Glucosyltransferases play key roles in the formation of these intermediates, either through the production of the high energy compound 1-O-sinapoylglucose leading to sinapoylmalate and sinapoylcholine or through the production of sinapyl alcohol-4-O-glucoside, potentially leading to the syringyl units found in lignins. While the importance of these glucosyltransferases has been recognized for more than 20 years, their corresponding genes have not been identified. Combining sequence information in the Arabidopsis genomic data base with biochemical data from screening the activity of recombinant proteins in vitro, we have now identified five gene sequences encoding enzymes that can glucosylate sinapic acid, sinapyl alcohol, and their related phenylpropanoids. The data provide a foundation for future understanding and manipulation of sinapate metabolism and lignin biology in Arabidopsis.     

Identification of genes involved in ceramide-dependent neuronal apoptosis using cDNA arrays

Background

Ceramide is important in many cell responses, such as proliferation, differentiation, growth arrest and apoptosis. Elevated ceramide levels have been shown to induce apoptosis in primary neuronal cultures and neuronally differentiated PC 12 cells.

Results

To investigate gene expression during ceramide-dependent apoptosis, we carried out a global study of gene expression in neuronally differentiated PC 12 cells treated with C₂-ceramide using an array of 9,120 cDNA clones. Although the criteria adopted for differential hybridization were stringent, modulation of expression of 239 genes was identified during the effector phase of C₂-ceramide-induced cell death. We have made an attempt at classifying these genes on the basis of their putative functions, first with respect to known effects of ceramide or ceramide-mediated transduction systems, and then with respect to regulation of cell growth and apoptosis.

Conclusions

Our cell-culture model has enabled us to establish a profile of gene expression during the effector phase of ceramide-mediated cell death. Of the 239 genes that met the criteria for differential hybridization, 10 correspond to genes previously involved in C₂-ceramide or TNF-α signaling pathways and 20 in neuronal disorders, oncogenesis or more broadly in the regulation of proliferation. The remaining 209 genes, with or without known functions, constitute a pool of genes potentially implicated in the regulation of neuronal cell death.