Biomedical subjects

I Verma

Publications and source records attributed to I Verma.

10 recordsLinked to original sources

PubMed1997

Overexpression of the FosB2 gene in thymocytes causes aberrant development of T cells and thymic epithelial cells.

We have examined the role of the AP-1 transcription factor on thymocyte maturation and thymus architecture by overexpressing FosB2 in transgenic mice. FosB2 is a naturally occurring splice variant of the FosB2 gene, encoding a truncated protein which lacks two domains necessary for transcriptional activation. The expression of FosB2 in the thymocytes severely affected their maturation and the structure of the whole thymus: the phenotype developed slowly during the first months of life, resulting in a progressive expansion of the medulla and concomitant reduction of the cortex. CD4+ thymocytes represented the major thymocyte population, whereas the CD4+ 8+ thymocytes were virtually absent. This phenotype appeared to be an intrinsic property of bone marrow derived cells, as it could be reproduced in bone marrow chimaeric mice. This pathology was very reminiscent to that observed in mice overexpressing c-Fos in thymic epithelium: also in that case the thymus underwent with age a progressive expansion of the epithelium and major changes in the ratio of thymocyte subsets, but the phenotype appeared to be an intrinsic property of the epithelial cells since it could not be reproduced by transgenic bone marrow transplantation. We speculate that both overexpression of FosB2 in thymocytes and overexpression of c-Fos in thymic epithelium results in aberrant signaling between thymocytes and stroma, that ultimately alters the thymic micromilieu, leading to this severe pathology.

Age Factors↗

PubMed1997

Cellular immune responses to cell wall peptidoglycan associated protein antigens in tuberculosis patients and healthy subjects.

We have isolated cell wall peptidoglycan associated proteins (CW-Pr) of Mycobacterium tuberculosis H37Ra by chemical treatment with trifluoromethanesulfonic acid:anisole (2:1), which further resolved into 71, 60 and 45 kDa proteins on SDS-PAGE. A study was carried out to investigate the immunoreactivity of these proteins with blood samples from 4 categories, including 15 tuberculous patients (TB), 5 tuberculous patients on ATT (TBT), 10 PPD non-reactive healthy controls (HPPD-) and 11 PPD reactive healthy controls (HPPD+). Comparing the proliferative responses to cell wall protein antigens, it was observed that the 71 kDa protein gave maximum stimulation with PBMCs from the TB and HPPD+ groups. The adherent PBMCs from the TB group also demonstrated enhanced phagocytosis, particularly in the presence of 71 and 45 kDa proteins, and the phagocytic index was significantly higher (P < 0.05) than the TBT group. However, PBMCs from of the groups recognized the 60 kDa cell wall antigen. Our results suggest that the 71 kDa protein from the cell wall of M. tuberculosis is highly immunogenic.

Antigens, Bacterial↗

PubMed1997

Immunoreactivity of mycobacterial 70 kDa protein in different physico-chemical forms.

The immunoreactivity of 70 kDa culture filtrate protein of Mycobacterium tuberculosis H37 Ra has been examined as such and by increasing its hydrophobicity through its conjugation to stearic acid ester (70 kDa-FAester). The cell mediated immune responses produced by 70 kDa-FAester encapsulated in phosphatidylcholine (PC) liposomes were significantly higher particularly at the 3rd week post immunization (wk p. im.) than that produced by the 70 kDa protein in PC liposomes, whereas humoral immune responses were non-significant. Further, these immune responses were comparable to that elicited by 70 kDa protein complexed with Freund's incomplete adjuvant (70 kDa-FIA). Results of this study suggest that changes in physicochemical nature of 70 kDa protein influences both the humoral and cellular immunoreactivity.

Adjuvants, Immunologic↗

PubMed1994

Memory. A model with good CREdentials.

The sea snail Aplysia provides a relatively simple model system for studying both short-term and long-term memory; a known transcriptional mechanism is implicated in establishment of the latter.

Animals↗

PubMed1994

Strategy for reliable prenatal detection of normal male carriers of the fragile X syndrome.

Prenatal diagnosis of fragile X syndrome identifying full mutations has been described. Here we report on a case of a prenatal test concerning a normal male carrier of the fragile X syndrome. Southern blot analysis of the fragile X gene resulted in the identification of a premutation in DNA isolated from the chorionic villus sample. Using a polymerase chain reaction (PCR)-based assay the CGG repeat length was determined to be 82 CGG repeat units. Confirmation of this premutation in the chorionic villus sample was based on the cytogenetic analysis of the expression of the fragile site at Xq27 applied on a cordocentesis-derived blood sample.

Chorionic Villi Sampling↗

PubMed1992

Purified I kappa B-beta is inactivated upon dephosphorylation.

In uninduced cells, the NF-kappa B transcription factor resides in the cytoplasm in complex with an inhibitory protein, I kappa B. I kappa B is a specific inhibitor of DNA binding and apparently prevents nuclear uptake of NF-kappa B. Stimulation of cells, for instance with the cytokine tumor necrosis factor, releases I kappa B and allows nuclear translocation and DNA binding of NF-kappa B to regulatory DNA sequences in many genes. We recently reported on the purification of a major form of I kappa B, referred to as I kappa B-alpha, with a molecular size of 37 kDa. Here, we purified and characterized I kappa B-beta, a chromatographically distinct second form of I kappa B. I kappa B-beta has a size of 43 kDa and, as I kappa B-alpha, an acidic isoelectric point between 4.8 and 5.0. Both forms of I kappa B were inactivated by a treatment with protein kinases A and C in vitro. In contrast to I kappa B-alpha, I kappa B-beta lost its inhibiting activity upon a treatment with phosphatase. Phosphatase treatment also released active NF-kappa B from its inactive complex with I kappa B-beta suggesting that the activation of NF-kappa B in intact cells might not only rely on phosphate transfer onto I kappa B but also on phosphate removal from one form of I kappa B.

Chromatography, Liquid↗

PubMed1991

Muscle-specific transcriptional activation by MyoD.

We focus on the mechanism by which MyoD activates transcription. Previous experiments showed that when the 13-amino-acid basic region of E12 replaced the corresponding basic region of MyoD, the resulting MyoD-E12Basic chimeric protein could bind specifically to muscle-specific enhancers in vitro and form dimers with E12, but could not activate a cotransfected reporter gene or convert 10T1/2 cells to muscle. Here we show that back mutation of this chimeric protein (with the corresponding residues in MyoD) re-establishes activation, and we identify a specific alanine involved in increasing DNA binding and a specific threonine required for activation. Using a reporter gene containing MyoD-binding sites located downstream from the transcription start site, we show that MyoD-E12Basic can bind in vivo and thereby inhibit expression of the reporter. In vivo binding is also supported by the fact that the addition of the "constitutive" VP16 activation domain to MyoD-E12Basic restores full trans-activation potential. The normal MyoD-activation domain maps within the amino-terminal 53 residues and can be functionally replaced by the activation domain of VP16. The activity of the MyoD-activation domain is dramatically elevated when deletions are made almost anywhere in the rest of the MyoD molecule, suggesting that the activation domain in MyoD is usually masked. Surprisingly, MyoD-E12Basic can activate transcription in CV1 and B78 cells (but not in 10T1/2 or 3T3 cells), suggesting that the activation function of the basic domain requires a specific factor present in CV1 and B78 cells. We propose that to function, the masked MyoD-activation domain requires the participation of a second factor that recognizes the basic region. We refer to such a factor as a recognition factor.

Amino Acid Sequence↗

PubMed1987

Gene therapy for thioguanine-resistant human leukemia.

The feasibility of using retroviral gene therapy to overcome drug resistance was assessed by determining the efficiency by which a retrovirus containing the human HGPRT gene could sensitize hypoxanthine-guanine phosphoribosyltransferase (HGPRT) negative human promyelocytic leukemia cells to 6-thioguanine. A single three-hour exposure at a virus to cell ratio of 6 X 2:1 restored sensitivity to 70(+/- 18)% of the clonogenic cells. The efficacy varied as a function of virus concentration and duration of viral exposure; the time allowed for integration and expression between one and five days post-infection had little effect. Cells successfully sensitized contained a proviral insert and expressed HGPRT activity that ranged from 1 to 92% of that in the wild-type cells. The mutation rate of the inserted gene varied from the same as that of the endogenous HGPRT gene to 200-fold greater in different clones. Failure of sensitization following viral exposure was associated with absence of an integrated provirus, and clonogenic cells failing to be sensitized by one virus exposure were sensitized with approximately the same efficiency by a second viral exposure. These results demonstrate the feasibility of transferring a drug sensitivity gene to a human leukemia cell line.

Cell Line↗

PubMed1981

Detection and elimination of cellular nucleic acids in biologicals produced on continuous cell lines.

Experiments have been conducted to determine the extent to which currently available purification techniques can remove contaminating substrate cellular DNA from inactivated poliovirus vaccine produced on continuous cell lines rising highly [32P]-labeled, nick-translated cellular DNA added to poliovirus suspensions, we found that purification procedures were capable, in small-scale experiments, of reducing contaminating DNA by factors of 10(3) (DNAse treatment followed by gel filtration) and 10(3)-3X10(5) (ion exchange chromatography). Sequential application of these purification steps should reduce contaminating cellular DNA to acceptable levels. We also examined the potential usefulness of immobilized nucleic acid hybridization techniques for the routine direct testing of residual cellular nucleic acids in final production lots of inactivated poliovirus vaccine and other biologicals. A filter hybridization test, using [32P]-labeled, nick-translated cellular DNA as a probe, was capable of detecting 40 pg of homologous cellular DNA. Using probes of higher specific activity the assay should be sensitive enough for use in routine quality control.

Animals↗

PubMedDate not supplied

Immunobiological properties of a 30 kDa secretory protein of Mycobacterium tuberculosis H37Ra.

Six different secretory proteins of molecular weights (15, 26, 30, 41, 55 and 70 kDa) were isolated from 8-day-old culture filtrate of Mycobacterium tuberculosis H37Ra using different column chromatography techniques. These proteins were further examined for their ability to induce cell mediated (T-cell proliferation assay) and humoral immune response (ELISA) in mice immunized with total culture filtrate proteins. Out of six proteins, three proteins showed good reactivity. However, the activity was at a maximum with 30 kDa antigen. The immune response induced by 30 kDa antigen emulsified in Freund's incomplete adjuvant (FIA) was investigated and was found to be dose dependent. The T-cell response induced by this protein was skewed towards T-helper (Th1) cells as determined by the pronounced secretion of interleukin-2 (IL-2) and gamma-interferon (IFN-gamma). The protective activity of the 30 kDa protein was also evaluated and compared with reference to Bacillus Calmette Guerin (BCG) vaccine in the mice challenged with virulent M. tuberculosis H37Rv. The degree of protection afforded by the 30 kDa antigen on the basis of mortality and the significant decrease in c.f.u.'s recovered from different organs (lung, liver, spleen) after 30 days of challenge with LD50 of M. tuberculosis H37Rv was significantly higher in comparison to BCG vaccinated animals. However, the degree of immunity induced by this antigen decreased with time (when challenged 8 and 12 weeks post-immunization) but it was still comparable with BCG. These findings suggest that 30 kDa secretory protein of M. tuberculosis is the key immunoprotective antigen and may be a suitable candidate for the development of an alternative subunit vaccine against tuberculosis.

Animals↗