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At least 865 records · Page 48Linked to original sources
PubMed2006

CEACAMs: their role in physiology and pathophysiology.

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) belong to a group of mammalian immunoglobulin-related glycoproteins. They are involved in cell-cell recognition and modulate cellular processes that range from the shaping of tissue architecture and neovascularization to the regulation of insulin homeostasis and T-cell proliferation. CEACAMs have also been identified as receptors for host-specific viruses and bacteria in mice and humans, respectively, making these proteins an interesting example of pathogen-host co-evolution. Forward and reverse genetics in the mouse now provide powerful novel models to elucidate the action of CEACAM family members in vivo.

Animals↗
PubMed2006

Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation.

Higher eukaryotes sense microbes through the perception of pathogen-associated molecular patterns (PAMPs). Arabidopsis plants detect a variety of PAMPs including conserved domains of bacterial flagellin and of bacterial EF-Tu. Here, we show that flagellin and EF-Tu activate a common set of signaling events and defense responses but without clear synergistic effects. Treatment with either PAMP results in increased binding sites for both PAMPs. We used this finding in a targeted reverse-genetic approach to identify a receptor kinase essential for EF-Tu perception, which we called EFR. Nicotiana benthamiana, a plant unable to perceive EF-Tu, acquires EF-Tu binding sites and responsiveness upon transient expression of EFR. Arabidopsis efr mutants show enhanced susceptibility to the bacterium Agrobacterium tumefaciens, as revealed by a higher efficiency of T-DNA transformation. These results demonstrate that EFR is the EF-Tu receptor and that plant defense responses induced by PAMPs such as EF-Tu reduce transformation by Agrobacterium.

Agrobacterium tumefaciens↗
PubMed2006

Role of PI3K and AKT specific isoforms in ovarian cancer cell migration, invasion and proliferation through the p70S6K1 pathway.

Ovarian cancer is the leading cause of death from gynecological malignancy for women. The amplification of the PI3K catalytic subunit (p110alpha) and the lost function of PTEN are frequently detected in ovarian cancer cells. PI3K plays an important role in tumorigenesis. To specifically inhibit PI3K activity in ovarian cancer cells, we constructed small interfering RNA (siRNA) against p110alpha. The expression of p110alpha siRNA significantly decreased cell migration, invasion, and proliferation compared to the siSCR control cells. The expression of p110alpha siRNA induced CDK inhibitor p27(KIP1) levels, and decreased levels of cyclin D1, CDK4, and phosphorylated retinoblastoma protein. PI3K transmits the mytogenic signal through AKT. AKT has three isoforms in the cells: AKT1, AKT2 and AKT3. We found that inhibition of AKT1 is sufficient to affect cell migration, invasion, and proliferation. Expression of AKT1 siRNA had a similar effect as p110alpha siRNA in the cells. We showed the roles of specific PI3K and AKT isoforms in the cells, which are important to understanding the mechanism of PI3K/AKT signaling in ovarian cancer cells. Both p110alpha and AKT1 siRNA-expressing cells decreased the activation of p70S6K1. Inhibition of p70S6K1 activity by its siRNA also decreased cell migration, invasion, and proliferation associated with the induction of p27(KIP1) levels, and with the inhibition of cell cycle-associated proteins including cyclin D1, CDK2, and phosphorylated retinoblastoma protein. This study demonstrates the important role of the PI3K/AKT/mTOR/p70S6K1 pathway in cell proliferation, migration, and invasion in ovarian cancer cells by using siRNA-mediated gene silencing as a reverse genetic method.

Catalytic Domain↗
PubMedDate not supplied

How do we get fat? An epidemiologic and metabolic approach.

Obesity results when the energy intake exceeds expenditure for a long period. The first law of thermodynamics, which describes this relationship, does not provide insight into the failures to couple energy intake and expenditure. Research over the past two decades has provided an unprecedented expansion of knowledge about the physiological and molecular mechanisms regulating body fat. Perhaps the greatest impact has resulted from the cloning of genes corresponding to the five mouse monogenic obesity syndromes and the subsequent characterization of the human counterparts to these syndromes. Extensive molecular and reverse genetic studies (mouse knockouts) have helped establish other critical pathways that regulate body fat and food intake, and also have either validated or refuted the importance of previously identified pathways.

Adipocytes↗
PubMed2006

Ablation of ghrelin improves the diabetic but not obese phenotype of ob/ob mice.

Ghrelin and leptin are suggested to regulate energy homeostasis as mutual antagonists on hypothalamic neurons that regulate feeding behavior. We employed reverse genetics to investigate the interplay between ghrelin and leptin. Leptin-deficient mice (ob/ob) are hyperphagic, obese, and hyperglycemic. Unexpectedly, ablation of ghrelin in ob/ob mice fails to rescue the obese hyperphagic phenotype, indicating that the ob/ob phenotype is not a consequence of ghrelin unopposed by leptin. Remarkably, deletion of ghrelin augments insulin secretion in response to glucose challenge and increases peripheral insulin sensitivity; indeed, the hyperglycemia exhibited by ob/ob mice is markedly reduced when ob/ob mice are bred onto the ghrelin(-/-) background. We further demonstrate that ablation of ghrelin reduces expression of Ucp2 mRNA in the pancreas, which contributes toward enhanced glucose-induced insulin secretion. Hence, chronically, ghrelin controls glucose homeostasis by regulating pancreatic Ucp2 expression and insulin sensitivity.

Animals↗
PubMed2004

OptiRNAi, an RNAi design tool.

RNA interference (RNAi), a recently developed reverse genetics tool, has many advantages compared to traditional gene knockout methods. Appropriate selection of double stranded RNAs identical to a specific region(s) of the target gene is critical for the successful implementation of this technology. Recently, Elbashir et al. [Methods 26 (2002) 199] has established empirical criteria for siRNA sequence selection that significantly improved the success rate for RNAi attempts. We have developed OptiRNAi, a computational tool, which uses the Elbashir et al. criteria to predict appropriate target sequences for siRNA production. Specificity of these siRNAs for the target of interest can then be assessed by the investigator using the embedded Blast search engine optimized for RNAi design. Thus, OptiRNAi is an efficient and user friendly tool for RNAi design based on criteria that are more stringent than other available tools.

Animals↗
PubMed2006

Vaccine development against malaria.

Malaria vaccine development aims to significantly reduce mortality and morbidity in the two high-risk groups: young children and pregnant women in sub-Saharan Africa. A pre-erythrocytic subunit vaccine, RTS,S, was recently evaluated in a first Phase IIb clinical trial in young children that live in a mesoendemic area of malaria transmission. The identification of a major parasite-encoded ligand that was found to be involved in pregnancy-associated malaria allows for the rational development of a tailor-made subunit vaccine for young women. The generation of transgenic yellow fever viruses and defined attenuated vaccine lines by reverse genetics are complementary approaches that might further boost the development of safe vaccines that elicit protective immune responses in this population.

Animals↗
PubMed2005

The temporal patterning microRNA let-7 regulates several transcription factors at the larval to adult transition in C. elegans.

The let-7 microRNA is phylogenetically conserved and temporally expressed in many animals. C. elegans let-7 controls terminal differentiation in a stem cell-like lineage in the hypodermis, while human let-7 has been implicated in lung cancer. To elucidate let-7's role in temporal control of nematode development, we used sequence analysis and reverse genetics to identify candidate let-7 target genes. We show that the nuclear hormone receptor daf-12 is a let-7 target in seam cells, while the forkhead transcription factor pha-4 is a target in the intestine. Additional likely targets are the zinc finger protein die-1 and the putative chromatin remodeling factor lss-4. Together with the previous identification of the hunchback ortholog hbl-1 as a let-7 target in the ventral nerve cord, our findings show that let-7 acts in at least three tissues to regulate different transcription factors, raising the possibility of let-7 as a master temporal regulator.

Animals↗
PubMed2005

Psoralen-sensitive mutant pso9-1 of Saccharomyces cerevisiae contains a mutant allele of the DNA damage checkpoint gene MEC3.

Complementation analysis of the pso9-1 yeast mutant strain sensitive to photoactivated mono- and bifunctional psoralens, UV-light 254 nm, and nitrosoguanidine, with pso1 to pso8 mutants, confirmed that it contains a novel pso mutation. Molecular cloning via the reverse genetics complementation approach using a yeast genomic library suggested pso9-1 to be a mutant allele of the DNA damage checkpoint control gene MEC3. Non-complementation of several sensitivity phenotypes in pso9-1/mec3Delta diploids confirmed allelism. The pso9-1 mutant allele contains a -1 frameshift mutation (deletion of one A) at nucleotide position 802 (802delA), resulting in nine different amino acid residues from that point and a premature termination. This mutation affected the binding properties of Pso9-1p, abolishing its interactions with both Rad17p and Ddc1p. Further interaction assays employing mec3 constructions lacking the last 25 and 75 amino acid carboxyl termini were also not able to maintain stable interactions. Moreover, the pso9-1 mutant strain could no longer sense DNA damage since it continued in the cell cycle after 8-MOP + UVA treatment. Taken together, these observations allowed us to propose a model for checkpoint activation generated by photo-induced adducts.

Alleles↗
PubMed2005

Characterization of outer membrane protein fractions of Bdellovibrionales.

Bdellovibrio-and-like organisms (BALOs) are predatory bacteria that prey upon Gram-negative bacteria and are taxonomically subsumed in the order Bdellovibrionales. Despite their unique lifestyle, these bacteria show remarkable genotypic diversities. The outer membrane of the predators is likely to play an important role during the recognition and invasion stage, as well as in the intraperiplasmic growth phase. In this study, the outer membrane protein fractions of type strains of Bdellovibrio, Bacteriovorax and Peredibacter were investigated, revealing the presence of outer membrane proteins (Omps) similar to the major Omps of Bdellovibrio bacteriovorus. The primary structures of these Omps of Bdellovibrio sp. W, Bacteriovorax stolpii and Peredibacter starrii were elucidated by a combined mass spectrometric-reverse genetic approach. The similarity between the analyzed Omps of the investigated BALOs ranges from 32% to 89% showing conserved amino acid regions in their primary structure.

Amino Acid Sequence↗
PubMed2005

RNA silencing as a tool for exploring gene function in ascomycete fungi.

We have developed a pHANNIBAL-like silencing vector, pSilent-1, for ascomycete fungi, which carries a hygromycin resistance cassette and a transcriptional unit for hairpin RNA expression with a spacer of a cutinase gene intron from the rice blast fungus Magnaporthe oryzae. In M. oryzae, a silencing vector with the cutinase intron spacer (147 bp) showed a higher efficiency in silencing of the eGFP gene than did those with a spacer of a GUS gene fragment or a longer intron (850 bp) of a chitin binding protein gene. Application of pSilent-1 to two M. oryzae endogenous genes, MPG1 and polyketide synthase-like gene, resulted in various degrees of silencing of the genes in 70-90% of the resulting transformants. RNA silencing was also induced by a pSilent-1-based vector in Colletotrichum lagenarium at a slightly lower efficiency than in M. oryzae, indicating that this silencing vector should provide a useful reverse genetic tool in ascomycete fungi.

Base Sequence↗
PubMed2006

Baculovirus-mediated gene silencing in insect cells using intracellularly produced long double-stranded RNA.

Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetics tool to silence gene expression in multiple organisms, including plants, nematodes and insects. In this study, DNA vectors capable of promoting the synthesis of long hairpin dsRNAs in vivo from a DNA template to suppress gene expression in insect cells have been successfully constructed. The inhibition of the expression of a gene encoding enhanced green fluorescent protein (eGFP) in insect cells was demonstrated by using plasmid or baculovirus vectors. Both plasmid and baculovirus vectors were able to inhibit eGFP expression in a dose dependent manner. Complete inhibition was obtained when co-transfection ratios of target plasmid to inhibition plasmid were 1:1 and 1:0.1. Eighty percent suppression was still maintained even when the ratio of eGFP plasmid to 'hairpin' plasmid was as high as 1:0.01. When the hairpin dsRNAs were encoded in a baculovirus, the suppression was about 50% when the ratio of 'target' baculovirus to 'inhibition' baculovirus reached 1:10. Therefore, the designed plasmid and baculovirus vectors are useful to induce RNAi in insect cell systems.

Animals↗
PubMed2005

Lipoxygenases: occurrence, functions and catalysis.

Lipid peroxidation is common to all biological systems, both appearing in developmentally and environmentally regulated processes. Products are hydroperoxy polyunsaturated fatty acids and metabolites derived there from collectively named oxylipins. They may either originate from chemical oxidation or are synthesized by the action of various enzymes, such as lipoxygenases (LOXes). Signalling compounds such as jasmonates, antimicrobial and antifungal compounds such as leaf aldehydes or divinyl ethers, and a plant-specific blend of volatiles including leaf alcohols are among the numerous products. Cloning of many LOXes and other key enzymes metabolizing oxylipins, as well as analyses by reverse genetic approaches and metabolic profiling revealed new insights on oxylipin functions, new reactions and the first hints on enzyme mechanisms. These aspects are reviewed with respect to function of specific LOX forms and on the development of new models on their substrate and product specificity.

Catalysis↗
PubMed2007

An improved method for the recovery of recombinant paramyxovirus vaccine candidates suitable for use in human clinical trials.

We describe a method for the generation of clinical grade, live-attenuated vaccines in Vero cells entirely from cDNA plasmids. The entire electroporation procedure can be completed in less than 15 minutes and this is a significant improvement over previous lipid or electroporation based transfection techniques that also involve a heat-shock step. Importantly, the virus preparations can be generated with a minimal use of animal product derived materials, an important consideration for a vaccine candidate that is to be tested in humans. Since it is likely that all live-attenuated parainfluenza virus and pneumovirus vaccines in the future will be generated using reverse genetics, this simplified method provides guidance on how this can be achieved.

Animals↗
PubMed2005

Silencing viruses by RNA interference.

Post-transcriptional gene silencing (PTGS) makes possible new approaches for studying the various steps of the viral cycle. Plus-strand RNA viruses appear to be attractive targets for small interfering RNAs (siRNAs), as their genome functions as both mRNA and replication template. PTGS creates an alternative to classic reverse genetics for viruses with either negative-strand or double-stranded RNA genomes and for those with a large genome. PTGS allows modification of the expression of a given cellular gene as a means to elucidate its role in the viral cycle and in virus-host cell interactions, and to investigate cellular pathways involved in viral pathogenesis. It also allows the creation of new animal models of human diseases. In addition, PTGS already appears to be a promising new therapeutic tool to fight viral multiplication and dissemination through the host and to prevent inflammation and virus-induced pathogenesis, including virus-induced tumorigenesis.

Animals↗
PubMed2005

Low growth ability of recent influenza clinical isolates in MDCK cells is due to their low receptor binding affinities.

Madin Darby canine kidney (MDCK) cells have generally been used to isolate influenza viruses from patients. However, in recent years, most fresh isolates of the H3N2 subtype have shown poor growth in MDCK cell cultures. Such low-growth viruses were often converted to high-growth viruses after several passages through MDCK cell cultures. In the present study, viruses were found to lose a potential glycosylation site near the receptor-binding pocket of hemagglutinin (HA), at the same time as they acquired the high-growth property. The growth curves of viruses in MDCK cell cultures revealed that multi-cycle replication did not function well in the low-growth viruses. However, the production of progeny viruses within a single cycle of growth did not differ much between the low- and high-growth viruses. The high-growth viruses showed higher infection efficiency in MDCK cell cultures than the low-growth viruses. The HA genes of both low- and high-growth viruses were separately cloned into the SV40 vector to compare their receptor binding affinities. The HA of high-growth viruses showed a much higher receptor binding affinity than that of low-growth viruses, when assayed by hemadsorption and the release kinetics of erythrocytes with bacterial neuraminidase. Reverse genetics studies demonstrated that HA was a crucial determinant for multi-cycle replication in MDCK cell cultures. Taken together, these results demonstrate that inefficient multi-cycle growth of fresh isolates is due to their low receptor binding affinities.

Animals↗
PubMed2004

Role of fusion protein cleavage site in the virulence of Newcastle disease virus.

Newcastle disease virus (NDV) causes a highly contagious and economically important disease in poultry. Viral determinants of NDV virulence are not completely understood. The amino acid sequence at the protease cleavage site of the fusion (F) protein has been postulated as a major determinant of NDV virulence. In this study, we have examined the role of F protein cleavage site sequence in NDV virulence using reverse genetics technology. The sequence G-R-Q-G-R present at the cleavage site of the F protein of avirulent strain LaSota was mutated to R-R-Q-K-R, which is present in the F cleavage site of neurovirulent strain Beaudette C (BC). The resultant mutated LaSota V.F. virus did not require exogenous protease for infectivity in cell culture, indicating that the F protein was cleaved by intracellular proteases. The virulence of the mutant and parental viruses was evaluated in vivo by intracerebral pathogenicity index (ICPI) and intravenous pathogenicity index (IVPI) tests in chickens. Our results showed that the modification of the F protein cleavage site resulted in a dramatic increase in virulence from an ICPI value of 0.00 for LaSota to a value of 1.12 for LaSota V.F. However, the ICPI value of LaSota V.F. was lower than that of BC, which had a value of 1.58. Interestingly, the IVPI tests showed values of 0.00 for both LaSota and LaSota V.F. viruses, compared to the IVPI value of 1.45 of BC. In vitro characteristics of the viruses were also studied. Our results demonstrate that the efficiency of cleavage of the F protein plays an important role if the NDV is delivered directly into the brains of chicks, but there could be other viral factors that probably affect peripheral replication, viremia, or entry into the central nervous system.

Amino Acid Sequence↗
PubMed2005

The use of in situ hybridization and immunohistochemistry to study the pathogenesis of various Newcastle disease virus strains and recombinants in embryonated chicken eggs.

Avian paramyxovirus type 1, commonly referred to as Newcastle disease virus (NDV), is a serious pathogen of significant economic importance to the industry. To investigate the role of the fusion (F), hemagglutinin-neuraminidase (HN), and (P) phosphoprotein gene sequences in virulence, six strains of Newcastle disease virus (NDV) representing all pathotypes and seven recombinant strains created by reverse genetics were inoculated into 9-day-old chicken embryos. Tissues and chorioallantoic membranes (CAM) were harvested at 24-hour intervals post-inoculation. Riboprobe in situ hybridization and immunohistochemistry highlighted distinct tissue tropisms among the viruses. Presence of F and/or HN from virulent viruses inserted into lentogenic backbones caused dissemination of virus in a manner similar to wild type virulent viruses. Disruption of P gene decreased dissemination of velogeinic infectious clones. It is concluded that each of these genes contributes to pathogenicity.

Animals↗