Gene Expression Profiling of Preovulatory Follicle in the Buffalo Cow: Effects of Increased IGF-I Concentration on Periovulatory Events

The preovulatory follicle in response to gonadotropin surge undergoes dramatic biochemical, and morphological changes orchestrated by expression changes in hundreds of genes. Employing well characterized bovine preovulatory follicle model, granulosa cells (GCs) and follicle wall were collected from the preovulatory follicle before, 1, 10 and 22 h post peak LH surge. Microarray analysis performed on GCs revealed that 450 and 111 genes were differentially expressed at 1 and 22 h post peak LH surge, respectively. For validation, qPCR and immunocytochemistry analyses were carried out for some of the differentially expressed genes. Expression analysis of many of these genes showed distinct expression patterns in GCs and the follicle wall. To study molecular functions and genetic networks, microarray data was analyzed using Ingenuity Pathway Analysis which revealed majority of the differentially expressed genes to cluster within processes like steroidogenesis, cell survival and cell differentiation. In the ovarian follicle, IGF-I is established to be an important regulator of the above mentioned molecular functions. Thus, further experiments were conducted to verify the effects of increased intrafollicular IGF-I levels on the expression of genes associated with the above mentioned processes. For this purpose, buffalo cows were administered with exogenous bGH to transiently increase circulating and intrafollicular concentrations of IGF-I. The results indicated that increased intrafollicular concentrations of IGF-I caused changes in expression of genes associated with steroidogenesis (StAR, SRF) and apoptosis (BCL-2, FKHR, PAWR). These results taken together suggest that onset of gonadotropin surge triggers activation of various biological pathways and that the effects of growth factors and peptides on gonadotropin actions could be examined during preovulatory follicle development.

Phenobarbitone-mediated translocation of the cytosolic proteins interacting with the 5 '-proximal region of rat liver CYP2B1/B2 gene into the nucleus

The positive element (PE) (-69 to -98 bp) within the 5'-proximal region of the CYP2B1B2 gene (+1 to -179 bp) of rat liver is essential for phenobarbitone (PB) response and gives a single major complex with the rat liver cytosol in gel shift analysis. This complex corresponds to complex I (top) of the three complexes given by the nuclear extracts. PB treatment of rats leads to a decrease in complex I formation with the cytosol and PE and an increase in the same with the nuclear extract in gel shift analysis. Both the changes are counteracted by simultaneous okadaic acid administration. The nuclear protein giving rise to complex I has been isolated and has an M-r of 26 kDa. The cytosolic counterpart consists of two species, 26 and 28 kDa, as revealed by Southwestern blot analysis using labeled PE. It is concluded that PB treatment leads to the translocation accompanied by processing of the cytosolic protein species into the nucleus that requires protein dephosphorylation. It is suggested that PB may exert a global regulation on the transcription of many genes by modulating the phosphorylation status of different protein factors involved in transcriptional regulation. (C) 2002 Elsevier Science (USA).

Scheduling expression trees for delayed-load architectures

In this paper we consider the problem of scheduling expression trees on delayed-load architectures. The problem tackled here takes root from the one considered in [Proceedings of the ACM SIGPLAN '91 Conf. on Programming Language Design and Implementation, 1991. p. 256] in which the leaves of the expression trees all refer to memory locations. A generalization of this involves the situation in which the trees may contain register variables, with the registers being used only at the leaves. Solutions to this generalization are given in [ACM Trans. Prog. Lang. Syst. 17 (1995) 740, Microproc. Microprog. 40 (1994) 577]. This paper considers the most general case in which the registers are reusable. This problem is tackled in [Comput. Lang, 21 (1995) 49] which gives an approximate solution to the problem under certain assumptions about the contiguity of the evaluation order: Here we propose an optimal solution (which may involve even a non-contiguous evaluation of the tree). The schedule generated by the algorithm given in this paper is optimal in the sense that it is an interlock-free schedule which uses the minimum number of registers required. An extension to the algorithm incorporates spilling. The problem as stated in this paper is an instruction scheduling problem. However, the problem could also be rephrased as an operations research problem with a difference in terminology. (C) 2002 Elsevier Science B.V. All rights reserved.

Characterization of the gene encoding the envelope fusion glycoprotein GP64 from Bombyx mori nucleopolyhedrovirus

We describe here the characterization of the gene gp64 encoding the envelope fusion protein GP64 (open reading frame) ORF 105 from Bombyx mori nucleopolyhedrovirus (BmNPV). gp64 was transcribed from the early to late stages of infection and the transcripts were seen from 6 to 72 h post infection (hpi). The early transcripts initiated from a consensus CAGT motif while the late transcripts arose from three conserved TAAG motifs, all of which were located in the near upstream region of the coding sequence. Both early and late transcripts terminated at a run of T residues following the second polyadenylation signal located 31 nt downstream of the translation termination codon. BmGP64 protein was detectable from 6 hpi and was present in larger quantities throughout the infection process from 12 hpi, in BmNPV-infected BmN cells. The persistent presence of GP64 in BmN cells differed from the protein expression pattern of GP64 in Autographa californica multinucleocapsid nucleopolyhedrovirus infection, where the protein levels decreased significantly by late times (48 hpi). BmGP64 was located in the membrane and cytoplasm of the infected host cells and as a component of the budded virions. The production of infectious budded virus and the fusion activity were reduced when glycosylation of GP64 was inhibited. (C) 2003 Elsevier Science B.V. All rights reserved.

Identification of a core promoter and a novel isoform of the human TSCI gene transcript and structural comparison with mouse homolog

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder with loci on chromosome 9q34.12 (TSC1) and chromosome 16p13.3 (TSC2). Genes for both loci have been isolated and characterized. The promoters of both genes have not been characterized so far and little is known about the regulation of these genes. This study reports the characterization of the human TSC1 promoter region for the first time. We have identified a novel alternative isoform in the 5' untranslated region (UTR) of the TSC1 gene transcript involving exon 1. Alternative isoforms in the 5' UTR of the mouse Tsc1 gene transcript involving exon I and exon 2 have also been identified. We have identified three upstream open reading frames (uORFs) in the 5' UTR of the TSC1/Tsc1 gene. A comparative study of the 5' UTR of TSC1/Tsc1 gene has revealed that there is a high degree of similarity not only in the sequence but also in the splicing pattern of both human and mouse TSC1 genes. We have used PCR methodology to isolate approximately 1.6 kb genomic DNA 5' to the TSC1 cDNA. This sequence has directed a high level of expression of luciferase activity in both HeLa and HepG2 cells. Successive 5' and 3' deletion analysis has suggested that a -587 bp region, from position +77 to -510 from the transcription start site (TSS), contains the promoter activity. Interestingly, this region contains no consensus TATA box or CAAT box. However, a 521-bp fragment surrounding the TSS exhibits the characteristics of a CpG island which overlaps with the promoter region. The identification of the TSC1 promoter region will help in designing a suitable strategy to identify mutations in this region in patients who do not show any mutations in the coding regions. It will also help to study the regulation of the TSC1 gene and its role in tumorigenesis. (C) 2003 Elsevier B.V. All rights reserved.

Recombination Drives Genetic Diversification of Streptococcus dysgalactiae Subspecies equisimilis in a Region of Streptococcal Endemicity

Infection of the skin or throat by Streptococcus dysgalactiae subspecies equisimilis (SDSE) may result in a number of human diseases. To understand mechanisms that give rise to new genetic variants in this species, we used multi-locus sequence typing (MLST) to characterise relationships in the SDSE population from India, a country where streptococcal disease is endemic. The study revealed Indian SDSE isolates have sequence types (STs) predominantly different to those reported from other regions of the world. Emm-ST combinations in India are also largely unique. Split decomposition analysis, the presence of emm-types in unrelated clonal complexes, and analysis of phylogenetic trees based on concatenated sequences all reveal an extensive history of recombination within the population. The ratio of recombination to mutation (r/m) events (11:1) and per site r/m ratio (41:1) in this population is twice as high as reported for SDSE from non-endemic regions. Recombination involving the emm-gene is also more frequent than recombination involving housekeeping genes, consistent with diversification of M proteins offering selective advantages to the pathogen. Our data demonstrate that genetic recombination in endemic regions is more frequent than non-endemic regions, and gives rise to novel local SDSE variants, some of which may have increased fitness or pathogenic potential.

NETGEM: Network Embedded Temporal GEnerative Model for gene expression data

Background: Temporal analysis of gene expression data has been limited to identifying genes whose expression varies with time and/or correlation between genes that have similar temporal profiles. Often, the methods do not consider the underlying network constraints that connect the genes. It is becoming increasingly evident that interactions change substantially with time. Thus far, there is no systematic method to relate the temporal changes in gene expression to the dynamics of interactions between them. Information on interaction dynamics would open up possibilities for discovering new mechanisms of regulation by providing valuable insight into identifying time-sensitive interactions as well as permit studies on the effect of a genetic perturbation. Results: We present NETGEM, a tractable model rooted in Markov dynamics, for analyzing the dynamics of the interactions between proteins based on the dynamics of the expression changes of the genes that encode them. The model treats the interaction strengths as random variables which are modulated by suitable priors. This approach is necessitated by the extremely small sample size of the datasets, relative to the number of interactions. The model is amenable to a linear time algorithm for efficient inference. Using temporal gene expression data, NETGEM was successful in identifying (i) temporal interactions and determining their strength, (ii) functional categories of the actively interacting partners and (iii) dynamics of interactions in perturbed networks. Conclusions: NETGEM represents an optimal trade-off between model complexity and data requirement. It was able to deduce actively interacting genes and functional categories from temporal gene expression data. It permits inference by incorporating the information available in perturbed networks. Given that the inputs to NETGEM are only the network and the temporal variation of the nodes, this algorithm promises to have widespread applications, beyond biological systems. The source code for NETGEM is available from https://github.com/vjethava/NETGEM

Heterologous expression of P5CS gene in chickpea enhances salt tolerance without affecting yield

Vigna Delta(1)-pyrroline-5-carboxylate synthetase (P5CS) cDNA was transferred to chickpea (Cicer arietinum L.) cultivar Annigeri via Agrobacterium tumefaciens mediated transformation. Following selection on hygromycin and regeneration, 60 hygromycin-resistant plants were recovered. Southern blot analysis of five fertile independent lines of T0 and T1 generation revealed single and multiple insertions of the transgene. RT-PCR and Western blot analysis of T0 and T1 progeny demonstrated that the P5CS gene is expressed and produced functional protein in chickpea. T1 transgenic lines accumulated higher amount of proline under 250 mM NaCl compared to untransformed controls. Higher accumulation of Na(+) was noticed in the older leaves but negligible accumulation in seeds of T1 transgenic lines as compared to the controls. Chlorophyll stability and electrolyte leakage indicated that proline overproduction helps in alleviating salt stress in transgenic chickpea plants. The T1 transgenics lines were grown to maturity and set normal viable seeds under continuous salinity stress (250 mM) without any reduction in plant yield in terms of seed mass.

Direct detection of Salmonella without pre-enrichment in milk, ice-cream and fruit juice by PCR against hilA gene

A novel PCR based assay was devised to specifically detect contamination of any Salmonella serovar in milk, fruit juice and ice-cream without pre-enrichment. This method utilizes primers against hilA gene which is conserved in all Salmonella serovars and absent from the close relatives of Salmonella. An optimized protocol, in terms time and money, is provided for the reduction of PCR contaminants from milk, ice-cream and juice through the use of routine laboratory chemicals. The simplicity, efficiency (time taken 3-4 h) and sensitivity (to about 5-10 CFU/ml) of this technique confers a unique advantage over other previously used time consuming detection techniques. This technique does not involve pre-enrichment of the samples or extensive sample processing, which was a pre-requisite in most of the other reported studies. Hence, this assay can be ideal for adoption, after further fine tuning, by food quality control for timely detection of Salmonella contamination as well as other food-borne pathogens (with species specific primers) in food especially milk, ice-cream and fruit juice. (C) 2011 Elsevier Ltd. All rights reserved.

Why Is Less Cationic Lipid Required To Prepare Lipoplexes from Plasmid DNA than Linear DNA in Gene Therapy?

The most important objective of the present study was to explain why cationic lipid (CL)-mediated delivery of plasmid DNA (pDNA) is better than that of linear DNA in gene therapy, a question that, until now, has remained unanswered. Herein for the first time we experimentally show that for different types of CLs, pDNA, in contrast to linear DNA, is compacted with a large amount of its counterions, yielding a lower effective negative charge. This feature has been confirmed through a number of physicochemical and biochemical investigations. This is significant for both in vitro and in vivo transfection studies. For an effective DNA transfection, the lower the amount of the CL, the lower is the cytotoxicity. The study also points out that it is absolutely necessary to consider both effective charge ratios between CL and pDNA and effective pDNA charges, which can be determined from physicochemical experiments.

Loading of single-walled carbon nanotubes in cationic cholesterol suspensions significantly improves gene transfection efficiency in serum

We present here a series of cholesterol based cationic lipid suspensions that solubilize single-walled carbon nanotubes (SWCNT) efficiently in water. Each cationic lipid formulation was characterized in terms of their energy minimized molecular structures, bilayer widths of the aggregates based on X-ray diffraction. Then these aggregates were investigated pertaining to their DNA binding and release efficiency, effect of CNT inclusion on the stability of cationic cholesterol lipid-DNA complexes, Zeta potential values and changes in the chiro-optical property of DNA, effect on Raman spectral shift and changes in morphology by SEM and AFM. Each cationic lipid formulation was optimized for the amount of SWCNT solubilized in water, lipid-DNA ratio, amount of the plasmid DNA that can be transfected and the effect on the cellular toxicity. The resulting SWCNT-lipid formulations were then used for in vitro transfection of pEGFP-C3 in A549 (human alveolar basal epithelial) cells and HeLa (human cervical cancer) cells. Advantageously, the CNT-loaded formulations confer an excellent transfection efficiency even in high percentages of blood serum and showed significantly better gene transfer efficiency compared to one of the potent, well-known commercial transfection reagent, Lipofectamine2000.