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At least 937 records · Page 52Linked to original sources
PubMed2004

Oncolysis of multifocal hepatocellular carcinoma in the rat liver by hepatic artery infusion of vesicular stomatitis virus.

Hepatocellular carcinoma (HCC) is a lethal malignancy with poor prognosis and few effective treatments, as well as ever-increasing frequencies in the Western world. Viruses that replicate selectively in cancer cells hold considerable promise as novel therapeutic agents for the treatment of malignancy. Vesicular stomatitis virus (VSV) is a negative-strand RNA virus with intrinsic oncolytic specificity due to significantly attenuated antiviral responses in many tumor cells. The aim of this study was to evaluate the potential of VSV, administered via the hepatic artery, as an effective and safe therapeutic agent for treating "multifocal" HCC in the rat liver. Recombinant VSV vector expressing beta-galactosidase (rVSV-beta-gal) was generated by reverse genetics and infused into the hepatic artery of Buffalo rats bearing orthotopically implanted multifocal HCC. Access by the virus to multifocal HCC lesions in the liver, as well as the kinetic profiles of intratumoral viral replication and spread, was established by X-gal staining of liver and tumor sections. Plaque assays were also performed to determine the infectious viral yields in tumor and normal liver tissues. Pharmacotoxicology studies, including serum chemistries and proinflammatory cytokine production, as well as organ histopathology, were performed. Buffer- or vector-treated tumor-bearing rats were followed for survival and the results were analyzed by the Kaplan-Meier method and the log-rank test. Hepatic arterial infusion of rVSV-beta-gal at the maximum tolerated dose in tumor-bearing rats resulted in efficient viral transduction of multifocal HCC lesions in their livers, tumor-selective viral replication, and extensive oncolysis. Importantly, no significant vector-associated toxicities were noted and, in particular, no damage to the hepatic parenchyma was seen. Finally, survival of vector-treated rats was substantially prolonged over that of animals in the control treatment group (p < 0.028). Thus, hepatic arterial administration of VSV is both effective and safe in an orthotopic animal model of multifocal HCC. The results suggest that oncolytic VSV can be developed into an effective and safe therapeutic modality for patients with multifocal HCC in the future.

Animals↗
PubMed2004

Inhibition of Staphylococcus aureus gene expression and growth using antisense peptide nucleic acids.

Gene function studies in bacteria lag behind progress in genome sequencing, in part because current reverse genetics technology based on genome disruption does not allow subtle control of gene expression for all genes in a range of species. Essential genes and clustered regions are particularly problematic. Antisense technology offers an attractive alternative for microbial genomics. Unfortunately, bacteria lack RNAi mechanisms and conventional oligonucleotides are not taken up efficiently. However, in Escherichia coli, efficient and gene-specific antisense knock down is possible using antisense peptide nucleic acids (PNAs) attached to carrier peptides (KFFKFFKFFK). Carrier peptides can enter a range of microbial species, and in this study we asked whether peptide-PNAs could mediate antisense effects in Staphylococcus aureus. Using low micromolar concentrations we observed dose- and sequence-dependent inhibition of the reporter gene gfp and endogenous gene phoB. Also, antisense peptide-PNAs targeted to the essential genes fmhB, gyrA, and hmrB were growth inhibitory. Control peptide-PNAs were much less effective, and sequence alterations within the PNA and target mRNA sequences reduced or eliminated inhibition. Further development is needed to raise the antibacterial potential of PNAs, but the present results show that the approach can be used to study gene function and requirement in this important pathogen.

Anti-Bacterial Agents↗
PubMed1999

Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry.

Tissue sections from aggressive human intraocular (uveal) and metastatic cutaneous melanomas generally lack evidence of significant necrosis and contain patterned networks of interconnected loops of extracellular matrix. The matrix that forms these loops or networks may be solid or hollow. Red blood cells have been detected within the hollow channel components of this patterned matrix histologically, and these vascular channel networks have been detected in human tumors angiographically. Endothelial cells were not identified within these matrix-embedded channels by light microscopy, by transmission electron microscopy, or by using an immunohistochemical panel of endothelial cell markers (Factor VIII-related antigen, Ulex, CD31, CD34, and KDR[Flk-1]). Highly invasive primary and metastatic human melanoma cells formed patterned solid and hollow matrix channels (seen in tissue sections of aggressive primary and metastatic human melanomas) in three-dimensional cultures containing Matrigel or dilute Type I collagen, without endothelial cells or fibroblasts. These tumor cell-generated patterned channels conducted dye, highlighting looping patterns visualized angiographically in human tumors. Neither normal melanocytes nor poorly invasive melanoma cells generated these patterned channels in vitro under identical culture conditions, even after the addition of conditioned medium from metastatic pattern-forming melanoma cells, soluble growth factors, or regimes of hypoxia. Highly invasive and metastatic human melanoma cells, but not poorly invasive melanoma cells, contracted and remodeled floating hydrated gels, providing a biomechanical explanation for the generation of microvessels in vitro. cDNA microarray analysis of highly invasive versus poorly invasive melanoma tumor cells confirmed a genetic reversion to a pluripotent embryonic-like genotype in the highly aggressive melanoma cells. These observations strongly suggest that aggressive melanoma cells may generate vascular channels that facilitate tumor perfusion independent of tumor angiogenesis.

Biomarkers, Tumor↗
PubMed2001

Proteins of the cyanobacterial photosystem I.

Cyanobacterial photosystem (PS) I is remarkably similar to its counterpart in the chloroplast of plants and algae. Therefore, it has served as a prototype for the type I reaction centers of photosynthesis. Cyanobacterial PS I contains 11-12 proteins. Some of the cyanobacterial proteins are modified post-translationally. Reverse genetics has been used to generate subunit-deficient cyanobacterial mutants, phenotypes of which have revealed the functions of the missing proteins. The cyanobacterial PS I proteins bind cofactors, provide docking sites for electron transfer proteins, participate in tertiary and quaternary organization of the complex and protect the electron transfer centers. Many of these mutants are now being used in sophisticated structure-function analyses. Yet, the roles of some proteins of the cyanobacterial PS I are unknown. It is necessary to examine functions of these proteins on a global scale of cell physiology, biogenesis and evolution.

Cyanobacteria↗
PubMed2001

An arabidopsis T-DNA mutant affected in Nrt2 genes is impaired in nitrate uptake.

Expression analyses of Nrt2 plant genes have shown a strict correlation with root nitrate influx mediated by the high-affinity transport system (HATS). The precise assignment of NRT2 protein function has not yet been possible due to the absence of heterologous expression studies as well as loss of function mutants in higher plants. Using a reverse genetic approach, we isolated an Arabidopsis thaliana knock-out mutant where the T-DNA insertion led to the complete deletion of the AtNrt2.1 gene together with the deletion of the 3' region of the AtNrt2.2 gene. This mutant is impaired in the HATS, without being modified in the low-affinity system. Moreover, the de-regulated expression of a Nicotiana plumbaginifolia Nrt2 gene restored the mutant nitrate influx to that of the wild-type. These results demonstrate that plant NRT2 proteins do have a role in HATS.

Anion Transport Proteins↗
PubMed2002

Ccz1p/Aut11p/Cvt16p is essential for autophagy and the cvt pathway.

In a reverse genetics screen, we here identify Ccz1p as an essential component of the cvt pathway and autophagy. Ccz1p is identical with the so far unknown Cvt16p. GFP-Aut7p, a specific cargo of autophagosomes, accumulates in ccz1 Delta cells at punctate, vesicular structures in the cytosol, suggesting a block in the autophagic pathway prior to vacuolar fusion of autophagosomes. Proteinase protection experiments using hypotonically lysed ccz1 Delta spheroplasts demonstrate that proaminopeptidase I, another specific cargo of autophagy and the cvt pathway, is trapped inside membrane-enclosed vesicles. Taken together our findings are compatible with a function of Ccz1p in vacuolar fusion of cvt vesicles and autophagosomes.

Autophagy-Related Proteins↗
PubMed2000

Upper thermal tolerances of the beachflea Orchestia gammarellus (Pallas) (Crustacea: Amphipoda: Talitridae) associated with hot springs in Iceland.

The upper thermal tolerance (CT(max)) of beachfleas Orchestia gammarellus (Pallas) collected from a number of different locations in Iceland was determined. Differences were recorded between field populations associated with thermal springs and those from non-thermal sites. A number of reciprocal acclimation experiments (where animals from thermal and non-thermal sites were acclimated to the measured ambient temperatures of thermal (17 and 22 degrees C) and non-thermal (11 degrees C) sites) were performed. Differences between at least one thermal population and a non-thermal population were maintained following this reciprocal acclimation, supporting the hypothesis that population differences were due to non-reversible genetic differences and not local acclimatisation. Animals from one thermal site (Reykjanes) had a mean CT(max)=37.1+/-0.5 degrees C when acclimated at 11 degrees C and 38.6+/-0.3 degrees C when acclimated at 22 degrees C, whereas animals from a non-thermal site (Hvassahraun) had CT(max) values of 35.9+/-0.5 and 37.9+/-0.3 degrees C, respectively. In other cases, differences are best explained by local acclimatisation. Results are discussed in relation to ambient local conditions and the degree of isolation of the different populations.

Journal Article↗
PubMed1990

Characterization of a G-protein alpha-subunit gene from the nematode Caenorhabditis elegans.

A gene encoding the alpha-subunit of a guanine nucleotide binding regulatory protein (G-protein) was isolated from a library of genomic Caenorhabditis elegans DNA. The predicted coding region is colinear to related genes from mammals and the 356 amino acid residues show 63% sequence identity to e.g. rat Gi alpha 2. Three of the eight introns within the coding sequence are at exactly the same positions as those in a Drosophila G-protein alpha-subunit gene, and two of these are also conserved in the mammalian homologues. The nematode gene does not encode the cysteine residue that forms the substrate site for pertussis toxin-catalyzed ADP-ribosylation in several G-proteins. In spite of the similarity to mammalian G-protein alpha-subunit genes the gene can not unambiguously be categorized in one of the classes of G-proteins recognized in mammals (G alpha i, o, z, etc.). The position of the gene on the physical map of the animal was determined (chromosome V). The cloning and sequencing of this gene can be the starting point of reverse genetics experiments aimed at the isolation of animals mutated in a G-protein alpha-subunit gene.

Amino Acid Sequence↗
PubMed2003

A 2-oxoglutarate-dependent dioxygenase is integrated in DIMBOA-biosynthesis.

Benzoxazinoids are secondary metabolites of grasses that function as natural pesticides. While many steps of DIMBOA biosynthesis have been elucidated, the mechanism of the introduction of OCH(3)-group at the C-7 position was unknown. Inhibitor experiments in Triticum aestivum and Zea mays suggest that a 2-oxoglutarate-dependent dioxygenase catalyses the hydroxylation reaction at C-7. Cloning and reverse genetics analysis have identified the Bx6 gene that encodes this enzyme. Bx6 is located in the Bx-gene cluster of maize.

Amino Acid Sequence↗
PubMed2003

Multiple amino acid residues confer temperature sensitivity to human influenza virus vaccine strains (FluMist) derived from cold-adapted A/Ann Arbor/6/60.

FluMist influenza A vaccine strains contain the PB1, PB2, PA, NP, M, and NS gene segments of ca A/AA/6/60, the master donor virus-A strain. These gene segments impart the characteristic cold-adapted (ca), attenuated (att), and temperature-sensitive (ts) phenotypes to the vaccine strains. A plasmid-based reverse genetics system was used to create a series of recombinant hybrids between the isogenic non-ts wt A/Ann Arbor/6/60 and MDV-A strains to characterize the genetic basis of the ts phenotype, a critical, genetically stable, biological trait that contributes to the attenuation and safety of FluMist vaccines. PB1, PB2, and NP derived from MDV-A each expressed determinants of temperature sensitivity and the combination of all three gene segments was synergistic, resulting in expression of the characteristic MDV-A ts phenotype. Site-directed mutagenesis analysis mapped the MDV-A ts phenotype to the following four major loci: PB1(1195) (K391E), PB1(1766) (E581G), PB2(821) (N265S), and NP(146) (D34G). In addition, PB1(2005) (A661T) also contributed to the ts phenotype. The identification of multiple genetic loci that control the MDV-A ts phenotype provides a molecular basis for the observed genetic stability of FluMist vaccines.

Animals↗
PubMed2003

Rescue of Hantaan virus minigenomes.

Hantavirus infections are a major public health concern worldwide. Their widespread geographical distribution and their ability to produce serious, often fatal, human disease underline the need for a system that allows manipulation of these viruses. We describe here the first successful establishment of a reverse genetics technology for Hantaan virus, the prototype of the genus Hantavirus. The system offers a unique opportunity to study the biology of hantaviruses, the pathogenesis of the diseases, and the efficacy of antiviral and prophylactic measures against hantavirus infections.

Animals↗
PubMed2003

Production and release of infectious hepatitis C virus from human liver cell cultures in the three-dimensional radial-flow bioreactor.

Lack of efficient culture systems for hepatitis C virus (HCV) has been a major obstacle in HCV research. Human liver cells grown in a three-dimensional radial-flow bioreactor were successfully infected following inoculation with plasma from an HCV carrier. Subsequent detection of increased HCV RNA suggested viral replication. Furthermore, transfection of HCV RNA transcribed from full-length cDNA also resulted in the production and release of HCV virions into supernatant. Infectivity was shown by successful secondary passage to a new culture. Introduction of mutations in RNA helicase and polymerase regions of HCV cDNA abolished virus replication, indicating that reverse genetics of this system is possible. The ability to replicate and detect the extracellular release of HCV might provide clues with regard to the persistent nature of HCV infection. It will also accelerate research into the pathogenicity of HCV, as well as the development of prophylactic agents and new therapy.

Amino Acid Sequence↗
PubMed2006

The molecular biology of coronaviruses.

Coronaviruses are large, enveloped RNA viruses of both medical and veterinary importance. Interest in this viral family has intensified in the past few years as a result of the identification of a newly emerged coronavirus as the causative agent of severe acute respiratory syndrome (SARS). At the molecular level, coronaviruses employ a variety of unusual strategies to accomplish a complex program of gene expression. Coronavirus replication entails ribosome frameshifting during genome translation, the synthesis of both genomic and multiple subgenomic RNA species, and the assembly of progeny virions by a pathway that is unique among enveloped RNA viruses. Progress in the investigation of these processes has been enhanced by the development of reverse genetic systems, an advance that was heretofore obstructed by the enormous size of the coronavirus genome. This review summarizes both classical and contemporary discoveries in the study of the molecular biology of these infectious agents, with particular emphasis on the nature and recognition of viral receptors, viral RNA synthesis, and the molecular interactions governing virion assembly.

Animals↗
PubMed2006

RNAi therapy for neurodegenerative diseases.

RNA interference (RNAi) mediates gene silencing in a sequence-specific manner and has proven to be an exceptionally valuable discovery for bench scientists. In the laboratory, RNAi technologies provide efficient means for validating drug targets and for performing reverse genetics to study gene function (Friedman and Perrimon, 2004). Patients may also benefit from RNAi as applications extend to potential human therapies. RNAi-based treatments are being investigated and may provide hope for patients suffering from cancer, viral infections, or genetic diseases for which effective therapies are currently lacking. Notably, several independent studies have demonstrated that RNAi therapy can improve disease phenotypes in various mouse models of human disease. In this chapter, we focus on the potential of RNAi in treating neurologic diseases for which reduction of mutant or toxic gene expression may provide therapeutic benefit. We discuss approaches to achieving RNAi in vivo, progress in the field, and the potential pitfalls associated with RNAi-based therapies.

Animals↗
PubMed2003

From genes to photosynthesis in Arabidopsis thaliana.

Although photosynthesis in higher plants is of cyanobacterial descent, it differs strikingly in organization and regulation from the prokaryotic process. Genomics, proteomics, and comparative genome analysis are now providing powerful new tools for the molecular dissection of photosynthesis in higher plants. Mutant screens and reverse genetics identify an increasing number of gene-function relationships that have a bearing on photosynthesis, revealing a marked interdependency between photosynthesis and other cellular processes. Photosynthesis-related functions are mostly located in the chloroplast, but can also be located in other compartments of the plant cell. The analysis by DNA-array hybridization of mRNA expression patterns both in the chloroplast and the nucleus, under various environmental conditions and/or in different genetic backgrounds that affect the function of the plastid, is rapidly improving our understanding of how photosynthesis is regulated, and it reveals that plastid-to-nucleus signaling plays a central role in its control.

Arabidopsis↗
PubMed2001

Differential display analysis of gene expression altered by ras oncogene.

The goal of the signal transduction pathways, such as those controlled by Ras, is in large part to ensure highly stringent regulation of the target genes in the nucleus, which are collectively responsible for the signal output, or phenotypes, of the cell. Understanding of the Ras effect ultimately requires the identification of these downstream target genes. Reverse genetic approaches would trace back the pathways by which they are regulated by Ras. While newer methods such as DNA microarray are emerging, differential display has allowed the identification of a greater number of differentially expressed genes than have been cloned by all the other methods combined, based on Medline search. Much of this success has been attributed to its simplicity (RT-PCR and DNA-sequencing gel) and versatility (compare more than two RNAs for both up- and downregulated genes). It has become obvious that finding the genes by either differential display or DNA microarray is only the first step toward the understanding of biological problems under investigation. It is hoped that finding the right genes through careful experimental designs, such as outlined here, will narrow down the number of relevant genes and increase the odds for solving the puzzles of nature, such as ras.

Animals↗
PubMed2005

Ectopic targeting of substrates to the ubiquitin pathway.

Explanation of the physiological function of a cellular protein often requires targeted removal of that protein to reveal the associated biochemical and phenotypic alterations. A variety of technologies such as gene targeting and RNAi have been developed to abrogate the biosynthesis of the protein of interest. Recently, targeted protein degradation by harnessing the cellular ubiquitin-proteolytic machinery has emerged as a novel reverse genetic tool for loss-of-function studies. Targeted proteolysis operates at the posttranslational level to directly accelerate the turnover rate of the target protein and opens up new avenues for the dissection of complicated protein functions associated with posttranslational events, which are unattainable by a simple blocking of the biosynthesis of the target protein.

Base Sequence↗
PubMed1993

Storage and release of neurotransmitters.

Because synaptic vesicles and secretory granules are simple in composition and easy to purify, many of their protein components have been identified and often sequenced. Attempts are underway to link the small number of membrane proteins to the small number of functions the vesicles perform. The discovery of sequence homologies has helped greatly with this. In addition, techniques that have begun to prove successful involve microinjection, identification of proteins that bind synaptic vesicle proteins, DNA transfection into cells and oocytes, and more recently, in vitro reconstitution of exocytosis, endocytosis, and vesicle biogenesis. Advances in the latter areas have been strongly influenced by the breakthroughs in our knowledge of membrane traffic in nonneuronal cells. The budding reactions involved in making synaptic vesicles and secretory granules resemble in many ways the generation of carrier vesicles from the ER and the Golgi complex. Finally, exocytosis in neurons may closely resemble fusion of carrier vesicles with target organelles in nonneuronal cells, using complexes of peripheral membrane proteins, GTP hydrolysis, and integral membrane proteins with fusogenic domains. The usefulness of in vitro reconstitution, reverse genetics, and the parallels with better understood systems compensates in part for a major weakness in the field, namely the difficulty in obtaining viable mutants that are defective in the storage and release of secretory vesicle content.

Animals↗