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Biomedical subjects

I M Verma

Publications and source records attributed to I M Verma.

At least 73 records · Page 4Linked to original sources
PubMed1997

BRCA1 is a cell cycle-regulated nuclear phosphoprotein.

We have characterized the BRCA1 gene product by using four polyclonal antibodies raised against peptides from four different regions of the protein. The antibodies specifically recognize an approximately 220-kDa BRCA1 protein that is predominantly expressed in the nucleus of both normal and neoplastic breast cancer cells. It is a serine phosphoprotein that undergoes hyperphosphorylation during late G1 and S phases of the cell cycle and is transiently dephosphorylated early after M phase. We propose that BRCA1 is a phosphoprotein that alters in a qualitative and quantitative manner during cell cycle progression.

Antibodies↗
PubMed1997

Immunological defects in mice with a targeted disruption in Bcl-3.

The proto-oncogene bcl-3 is a member of the IkappaB family. The Bcl-3 protein is known to interact specifically with the p50 and p52 subunits of NFkappaB. However, the function of this interaction is not well understood. To determine the in vivo role of Bcl-3, mice were generated that lack the bcl-3 gene, Bcl 3(-/-). Here we report that Bcl 3(-/-) mice appear developmentally normal, but exhibit severe defects in humoral immune responses and protection from in vivo pathogenic challenges. Relative to wild-type mice, Bcl 3(-/-) mice are unable to clear L. monocytogenes and are more susceptible to infection with S. pneumoniae. This phenotype is similar to that observed in the p50(-/-) mice and the cross between the Bcl-3(-/-) and p50(-/-) mice generates animals with an enhanced phenotype. In accordance with the observed defects in their immune response, the Bcl 3(-/-) mice have normal immunoglobulin levels before and after immunization, but fail to produce antigen-specific antibodies. Additionally, spleens from Bcl-3(-/-) mice are abnormal and void of germinal centers. In contrast, the p50(-/-) mice have normal germinal centers. We propose that in in vivo, Bcl-3 can function independently of p50.

Animals↗
PubMed1997

Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors.

Successful gene therapy approaches will require efficient gene delivery and sustained expression of the transgene in recipients. A variety of methods, ranging from direct DNA delivery to infection with recombinant viruses containing foreign genes, have been developed, but they all have some major limitations that restrict their utility. We have described a human lentiviral (HIV)-based vector that can transduce non-dividing cells in vitro and deliver genes in vivo. With this vector, expression of transgenes in the brain has been detected for more than six months--the longest period tested so far. Because lentiviral vectors are pseudotyped with vesicular stomatitis virus G glycoprotein (VSVG; ref. 8), they can transduce a broad range of tissues and cell types. We now describe the ability of lentiviral vectors to introduce genes directly into liver and muscle. Sustained expression of green fluorescent protein (GFP), used as a surrogate for therapeutic protein, can be observed for more than 22 weeks in the liver. Similar long-term expression (more than eight weeks) was observed in transduced muscle. In contrast, little or no GFP could be detected in liver or muscle transduced with the Moloney murine leukaemia virus (M-MLV), a prototypic retroviral based vector. At a minimum, 3-4% of the total liver tissue was transduced by a single injection of 1-3 x 10(7) infectious units (I.U.) of recombinant HIV vector. Furthermore, no inflammation of recruitment of lymphocytes could be detected at the site of injection. Animals previously transduced with a lentiviral vector can be efficiently re-infected with lentiviral vectors. Additionally, we show that the requirement for lentiviral accessory proteins to establish efficient transduction in vivo is tissue dependent.

Animals↗
PubMed1997

Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector.

The identification of monogenic and complex genes responsible for neurological disorders requires new approaches for delivering therapeutic protein genes to significant numbers of cells in the central nervous system. A lentivirus-based vector capable of infecting dividing and quiescent cells was investigated in vivo by injecting highly concentrated viral vector stock into the striatum and hippocampus of adult rats. Control brains were injected with a Moloney murine leukemia virus, adenovirus, or adeno-associated virus vector. The volumes of the areas containing transduced cells and the transduced-cell densities were stereologically determined to provide a basis for comparison among different viral vectors and variants of the viral vector stocks. The efficiency of infection by the lentivirus vector was improved by deoxynucleoside triphosphate pretreatment of the vector and was reduced following mutation of integrase and the Vpr-matrix protein complex involved in the nuclear translocation of the preintegration complex. The lentivirus vector system was able to efficiently and stably infect quiescent cells in the primary injection site with transgene expression for over 6 months. Triple labeling showed that 88.7% of striatal cells transduced by the lentivirus vector were terminally differentiated neurons.

Adult↗
PubMed1996

Suppression of TNF-alpha-induced apoptosis by NF-kappaB.

Tumor necrosis factor alpha (TNF-alpha) signaling gives rise to a number of events, including activation of transcription factor NF-kappaB and programmed cell death (apoptosis). Previous studies of TNF-alpha signaling have suggested that these two events occur independently. The sensitivity and kinetics of TNF-alpha-induced apoptosis are shown to be enhanced in a number of cell types expressing a dominant-negative IkappaBalpha (IkappaBalphaM). These findings suggest that a negative feedback mechanism results from TNF-alpha signaling in which NF-kappaB activation suppresses the signals for cell death.

Animals↗
PubMed1996

Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector.

We describe the construction of a safe, replication-defective and efficient lentiviral vector suitable for in vivo gene delivery. The reverse transcription of the vector was found to be a rate-limiting step; therefore, promoting the reaction inside the vector particles before delivery significantly enhanced the efficiency of gene transfer. After injection into the brain of adult rats, sustained long-term expression of the transgene was obtained in the absence of detectable pathology. A high proportion of the neurons in the areas surrounding the injection sites of the vector expressed the transduced beta-galactosidase gene. This pattern was invariant in animals sacrificed several months after a single administration of the vector. Transduction occurs by integration of the vector genome, as it was abolished by a single amino acid substitution in the catalytic site of the integrase protein incorporated in the vector. Development of clinically acceptable derivatives of the lentiviral vector may thus enable the sustained delivery of significant amounts of a therapeutic gene product in a wide variety of somatic tissues.

Animals↗
PubMed1996

In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.

A retroviral vector system based on the human immunodeficiency virus (HIV) was developed that, in contrast to a murine leukemia virus-based counterpart, transduced heterologous sequences into HeLa cells and rat fibroblasts blocked in the cell cycle, as well as into human primary macrophages. Additionally, the HIV vector could mediate stable in vivo gene transfer into terminally differentiated neurons. The ability of HIV-based viral vectors to deliver genes in vivo into nondividing cells could increase the applicability of retroviral vectors in human gene therapy.

Amino Acid Sequence↗
PubMed1996

Applications of gene therapy to the CNS.

Gene therapy is a new method with potential for treating a broad range of acquired and inherited neurologic diseases, where the causative gene defect or deletion has been identified. In addition to gene replacement the application of gene products that reduce cellular dysfunction or death represent new therapeutic options. Gene transfer techniques to express novel proteins using different viral vectors in vitro and in vivo, as well as animal models and human trials will be reviewed in this article. We will focus on a new lentiviral vector as a recent gene transfer method and degenerative disorders of the CNS, and their related model systems.

Adenoviridae↗
PubMed1996

The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses.

We describe the construction and characterization of retroviral vectors and packaging plasmids that produce helper-free retrovirus with titers of 1 X 10(6) to 5 X 10(6) within 48 h. These vectors contain the immediate early region of the human cytomegalovirus enhancer-promoter fused to the Moloney murine leukemia virus long terminal repeat at the TATA box in the 5' U3 region, yielding the pCL promoter. By selecting vectors designed to express genes from one of four promoters (dihydrofolate reductase, Rous sarcoma virus, long terminal repeat, or cytomegalovirus), the pCL system permits the investigator to control the level of gene expression in target cells over a 100-fold range, while maintaining uniformly high titers of virus from transiently transfected producer cells. The pCL packaging plasmids lack a packaging signal (delta-psi) and include an added safety modification that renders them self-inactivating through the deletion of the 3' U3 enhancer. Ecotropic, amphotropic (4070A), and amphotropic-mink cell focus-forming hybrid (10A1) envelope constructions have been prepared and tested, permitting flexible selection of vector pseudotype in accordance with experimental needs. Vector supernatants are free of helper virus and are of sufficiently high titer within 2 days of transient transfection in 293 cells to permit infection of more than 50% of randomly cycling target cells in culture. We demonstrated the efficacy of these vectors by using them to transfer three potent cell cycle control genes (the p16(INK4A), p53, and Rb1 genes) into human glioblastoma cells.

DNA, Recombinant↗
PubMed1996

Signal-induced degradation of I(kappa)B(alpha): association with NF-kappaB and the PEST sequence in I(kappa)B(alpha) are not required.

Signal-induced degradation of I(kappa)B(alpha) via the ubiquitin-proteasome pathway requires phosphorylation on residues serine 32 and serine 36 followed by ubiquitination on lysines 21 and 22. We investigated the role of other regions of I(kappa)B(alpha) which may be involved in its degradation. Here we report that the carboxy-terminal PEST sequence is not required for I(kappa)B(alpha) signal-induced degradation. However, removal of the PEST sequence stabilizes free I(kappa)B(alpha) in unstimulated cells. We further report that a PEST deletion mutant does not associate well with NF-(kappa)B proteins but is degraded in response to signal. Therefore, we conclude that both association with NF-(kappa)B and a PEST sequence are not required for signal-induced I(kappa)B(alpha) degradation. Additionally, the PEST sequence may be required for constitutive turnover of free I(kappa)B(alpha).

Amino Acid Sequence↗
PubMed1996

Constitutive phosphorylation of IkappaBalpha by casein kinase II occurs preferentially at serine 293: requirement for degradation of free IkappaBalpha.

IkappaBalpha is a phosphoprotein that sequesters the NF-kappaB/Rel transcription factors in the cytoplasm by physical association. Following induction by a wide variety of agents, IkappaBalpha is further phosphorylated and degraded, allowing NF-kappaB/Rel proteins to translocate to the nucleus and induce transcription. We have previously reported that the constitutive phosphorylation site resides in the C-terminal PEST region of IkappaBalpha and is phosphorylated by casein kinase II (CKII). Here we show that serine 293 is the preferred CKII phosphorylation site. Additionally, we show compensatory phosphorylation by CKII at neighboring serine and threonine residues. Thus, only when all five of the serine and threonine residues in the C-terminal region of IkappaBalpha are converted to alanine (MutF), is constitutive phosphorylation abolished. Finally, we show that constitutive phosphorylation is required for efficient degradation of free IkappaBalpha, in that unassociated Mutf has a half-life two times longer than wild-type IkappaBalpha. A serine residue alone at position 293, as well as aspartic acid at this position, can revert the Mutf phenotype. Therefore, the constitutive CKII phosphorylation site is an integral part of the PEST region of IkappaBalpha, and this phosphorylation is required for rapid proteolysis of the unassociated protein.

Amino Acid Sequence↗
PubMed1995

Constitutive phosphorylation of I kappa B alpha by casein kinase II.

The NF-kappa B/Rel proteins are sequestered in the cytoplasm in association with the phosphorylated form of I kappa B alpha. Upon induction with a wide variety of agents, the activity of NF-kappa B/Rel proteins is preceded by the rapid degradation of I kappa B alpha protein. We report the identification and partial purification of a cellular kinase from unstimulated or stimulated murine cells, which specifically phosphorylates the C terminus of I kappa B alpha. There are several consensus sites for casein kinase II (CKII) in the C-terminal region of I kappa B alpha. Additionally, the activity of the cellular kinase is blocked by antibodies against the alpha subunit of CKII. No phosphorylation of the C-terminal region of I kappa B alpha can be detected if the five possible serine and threonine residues that can be phosphorylated by CKII are mutated to alanine. A two-dimensional tryptic phosphopeptide map of I kappa B alpha from unstimulated cells was identical to that obtained by in vitro phosphorylation of I kappa B alpha with the partially purified cellular kinase. We propose that constitutive phosphorylation of I kappa B alpha is carried out by CKII.

Amino Acid Sequence↗
PubMed1995

Generation of targeted retroviral vectors by using single-chain variable fragment: an approach to in vivo gene delivery.

We report the generation of a retroviral vector that infects human cells specifically through recognition of the low density lipoprotein receptor. The rationale for this targeted infection is to add onto the ecotropic envelope protein of Moloney murine leukemia virus, normally trophic for murine cells, a single-chain variable fragment derived from a monoclonal antibody recognizing the human low density lipoprotein receptor. This chimeric envelope protein was used to construct a packaging cell line producing a retroviral vector capable of high-efficiency transfer of the Escherichia coli beta-galactosidase gene to human cells expressing low density lipoprotein receptor. This approach offers a generalized plan to generate cell and tissue-specific retroviral vectors, an essential step toward in vivo gene therapy strategies.

Animals↗
PubMed1995

Cellular and humoral immune responses to adenoviral vectors containing factor IX gene: tolerization of factor IX and vector antigens allows for long-term expression.

Recombinant adenoviruses containing the canine factor IX (FIX) cDNA were directly introduced in the hind leg muscle of mice. We show that (i) in nude mice, high expression (1-5 micrograms/ml in plasma) of FIX protein can be detected for > 300 days; (ii) in contrast, expression of FIX protein was transient (7-10 days) in normal mice; (iii) CD8+ lymphocytes could be detected within 3 days in the infected muscle tissue; (iv) use of beta 2-microglobulin and immunoglobulin M heavy chain "knockout" mice showed that lack of sustained expression of FIX protein is due to cell-mediated and humoral immune responses; (v) normal mice, once infected with recombinant adenovirus, could not be reinfected efficiently for at least 30 days due to neutralizing viral antibodies; and, finally, (vi) using immunosuppressive drugs, some normal mice can be tolerized to produce and secrete FIX protein for > 5 months. We conclude that currently available adenoviral vectors have serious limitations for use for long-term gene therapy.

Adenoviridae↗