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Biomedical subjects

L Denisova

Publications and source records attributed to L Denisova.

10 recordsLinked to original sources
PubMed2000

Plasmid DNA vaccines: tissue distribution and effects of DNA sequence, adjuvants and delivery method on integration into host DNA.

A variety of factors could affect the frequency of integration of plasmid DNA vaccines into host cellular DNA, including DNA sequences within the plasmid, the expressed gene product (antigen), the formulation, delivery method, route of administration, and the type of cells exposed to the plasmid. In this report, we examined the tissue distribution and potential integration of plasmid DNA vaccines following intramuscular administration in mice and guinea pigs. We compared needle versus Biojector (needleless jet) delivery, examined the effect of aluminum phosphate adjuvants, compared the results of different plasmid DNA vaccines, and tested a gene (the human papilloma virus E7 gene) whose protein product is known to increase integration frequency in vitro. Six weeks following intramuscular injection, the vast majority of the plasmid was detected in the muscle and skin near the injection site; lower levels of plasmid were also detected in the draining lymph nodes. At early time points (1-7 days) after injection, a low level of systemic exposure could be detected. Occasionally, plasmid was detected in gonads, but it dissipated rapidly and was extrachromosomal - indicating a low risk of germline transmission. Aluminum phosphate adjuvant had no effect on the tissue distribution and did not result in a detectable increase in integration frequency. Biojector delivery, compared with needle injection, greatly increased the uptake of plasmid (particularly in skin at the injection site), but did not result in a detectable increase in integration frequency. Finally, injection of a plasmid DNA vaccine containing the human papilloma virus type 16 E7 gene, known to increase integration in vitro, did not result in detectable integration in mice. These results suggest that the risk of integration following intramuscular injection of plasmid DNA is low under a variety of experimental conditions.

Adjuvants, Immunologic↗
PubMed1998

Cooperative actions of HIV-1 Vpr and p53 modulate viral gene transcription.

Transcription of the human immunodeficiency virus type-1 (HIV-1) genome is controlled by cooperative interaction of viral encoded proteins and host regulatory proteins. In this study, we have examined the capacity of the viral auxiliary protein, Vpr, to modulate transcriptional activity of the HIV-1 promoter sequence located within the long terminal repeat (LTR). We demonstrate that ectopic expression of Vpr in human astrocytic cells, U-87MG, enhances the basal activity of the viral promoter in transfected cells and that the GC-rich sequences, spanning nucleotides -80 to -43, are important for this activity. Since this region serves as the target for p53-induced suppression of LTR activity and interacts with the ubiquitous transcription factor, Sp1, we examined the cooperative activity of Vpr, p53, and Sp1 upon LTR transcription. Results from co-transfection studies indicated that overexpression of wild type p53, but not mutant p53, decreases the level of activation of the LTR by Vpr. Transcriptional activation of the LTR by Vpr required the presence of Sp1 since overexpression of Vpr in cells with no endogenous Sp1 failed to augment LTR activity. Results from protein-protein interaction studies indicated that Vpr is associated with both p53 and Sp1 in cells with ectopic expression of these proteins. Moreover, it was evident that p53 and Sp1 interact with each other in these cells. These functional and structural studies provided a working model on the cooperative interaction of Vpr with cellular proteins Sp1 and p53 and control of viral gene transcription at immediate early stage of infection prior to the participation of other viral regulatory proteins.

Astrocytes↗
PubMed1998

Regulation of TNFalpha and TGFbeta-1 gene transcription by HIV-1 Tat in CNS cells.

Tat is a transcription transactivator produced by the human immunodeficiency virus type 1 (HIV-1) at the early phase of infection and plays a critical role in the expression and replication of the viral genome. This 86 amino acid protein, which can be secreted from the infected cells, has the ability to enter uninfected cells and exert its activity upon the responsive genes. Earlier results indicated that in addition to the HIV-1 promoter, Tat has the capacity to induce transcription of a variety of cellular genes. In this study, we demonstrate that exposure of cells from the central nervous system (U-87MG and SK-N-MC) and the lymphoid T cells (Jurkat) to highly purified Tat increases transcriptional activity of the reporter constructs containing the promoters from the transforming growth factor beta-1 (TGFbeta-1), the tumor necrosis factor alpha (TNFalpha), and the HIV-1 LTR. In addition, Tat treatment results in increased levels of TGFbeta-1 and TNFalpha mRNAs in these cells. Activation of the TGFbeta-1 and TNFalpha promoter constructs by Tat in U-87MG and SK-N-MC cells required amino acid residues 2 to 36 which spans the acidic and the cysteine-rich domains of Tat. In both CNS and lymphoid cells, the level of endogenous TGFbeta-1 mRNA was increased by mutant Tat protein containing amino acids 1 to 48 but not with a mutant Tat protein with a deletion between residues 2 to 36. TNFalpha mRNA level was increased by mutant Tat spanning residues 1 to 48 in U-87MG cells, but not in SK-N-MC and Jurkat cells. These observations suggest that activation of cellular and viral genes by Tat in various cells may be mediated by different pathways as evidenced by the requirements of the different regions of Tat. Activation of the TGFbeta-1 and TNFalpha promoters by wild-type Tat was severely affected by the mutant peptides spanning residues 2 to 36 and 1 to 48 suggesting that both truncated Tat peptides may function as dominant negative mutants over TNFalpha and TGFbeta-1 gene transcription. The importance of these findings in Tat-induced regulation of viral and cellular genes in various cell types is discussed.

Brain↗
PubMed1998

Cycloserine-induced decrease of cerebroside in myelin.

L-Cycloserine has been shown specifically to lower brain cerebroside levels in vivo, but the extent to which the decrease in whole brain cerebroside content reflects lower myelin cerebroside levels is not known since a substantial portion of cerebroside is found in nonmyelin membranes. The present report demonstrates that chronically administered cycloserine lowers the proportion of cerebroside in rat brain myelin. Cycloserine-induced decrease of myelin cerebroside should provide a useful tool in investigating the role of cerebroside in maintaining myelin stability.

Animals↗
PubMed1997

Identification of a cellular protein that binds to Tat-responsive element of TGF beta-1 promoter in glial cells.

Tat is a transcriptional transactivator produced by the human immunodeficiency virus type 1 (HIV-1) and plays a pivotal role in enhancing expression of the viral genome in the infected cells. Although initial studies have suggested that interaction of Tat with the transactivation responsive element (TAR); located within the LTR, is essential for Tat function, subsequent studies indicated that Tat has the ability to augment transcription of viral and cellular genes by a TAR-independent mechanism. In early studies we demonstrated that HIV-1 Tat stimulates transcription of the transforming growth factor, TGF beta-1, gene in glial cells. In this study, we have identified a cellular protein that interacts with the Tat-responsive region located between nucleotides -323 to -453 of the regulatory sequence of the TGF beta-1 promoter. Results from footprinting analysis revealed association of cellular proteins with the 130 nucleotide sequence located in the Tat-responsive region. Analysis of the associated protein by UV-crosslinking suggested the involvement of a protein between 40-45 kDa in size which preferentially interacts with the GC/GA rich sequence of the TGF beta-1 Tat-responsive sequence in a single-stranded configuration. The ability of the previously identified 40 kDa protein, named Pur alpha to bind to the GC/GA sequence in the single-stranded configuration, similar to those from TGF beta-1 promoter prompted us to investigate its binding capacity to the TGF beta-1 sequence and its transcriptional activity on the TGF beta-1 promoter. Results from band shift studies indicated the association of the bacterially produced Pur alpha to the TGF beta-1 DNA sequences positioned within the Tat-responsive region. Overexpression of Pur alpha in glial cells constitutively producing Tat augmented transcription of the TGF beta-1 gene. These results are consistent with previous reports on the cooperative action of Pur alpha and Tat in modulating other eukaryotic promoters. The importance of these findings with regard to deregulation of other cellular genes by HIV-1 Tat is discussed.

Astrocytes↗
PubMed1997

Synthesis and secretion of von Willebrand factor and fibronectin in megakaryocytes at different phases of maturation.

Our goals have been to define the biochemical characteristics of megakaryocytes during maturation that are critical for platelet assembly and release into the circulation and to introduce biochemical markers for megakaryocytes. To achieve these goals, we have studied fibronectin (FN) and von Willebrand factor (vWF), which are large adhesive proteins that are synthesized by megakaryocytes, stored in alpha granules, and thought to have a fundamental role in hemostasis. The study demonstrated that vWF is primarily synthesized in mature megakaryocytes, which synthesized 7.5 times more vWF than immature megakaryocytes. Brefeldin A, which blocks the exit of proteins from the rough endoplasmic reticulum (RER), inhibited the formation of vWF multimers but did not affect the synthesis of monomers and dimers in mature megakaryocytes. These data are consistent with the formation of vWF dimers in the RER and the assembly of vWF multimers in the trans- and post-golgi. The synthesis of both the 260-kD and 275-kD pro-vWF was detected. However, the synthesis of 275-kD pro-vWF and 220-kD mature vWF was only evident after 2 hours, suggesting that the transit time of nascent vWF through the RER is about 2 hours. Constitutive secretion of vWF was demonstrated in megakaryocytes. About 14.5% and 4.6% of synthesized vWF was secreted by mature and immature megakaryocytes, respectively. In contrast, the synthesis of FN monomers and dimers was established in immature megakaryocytes, and their synthesis in mature megakaryocytes was very similar. Constitutive secretion of FN was not seen in megakaryocytes. Brefeldin A did not inhibit the synthesis of FN dimers; thus, formation of FN dimers occurs in the RER. The demonstration that vWF and FN are synthesized at different phases of megakaryocyte maturation and that only vWF is constitutively secreted by megakaryocytes provides new information relevant to alpha granule formation and possibly bone marrow matrix assembly.

Animals↗
PubMed1996

Fibronectin isoforms in megakaryocytes.

Our studies have shown that megakaryocytes (MK) can synthesize fibronectin (FN) and alternatively spliced fibronectin, FN EIIIB. FN EIIIB is primarily present in embryonic, proliferating and migrating cells, and thought to be important for cell maturation. MK, but not nonmegakaryocytic bone marrow cells, contain FN EIIIB and thus, MK and platelets are among a small number of adult cells and tissues that synthesize and contain FN EIIIB. Thrombin can induce the secretion of general FN, but does not cause the secretion of FN EIIIB into the medium. Analysis of immunostained cells by confocal microscopy revealed that both general FN and FN EIIIB accumulated on the MK surface following thrombin treatment. Thus, FN EIIIB can be released only to be bound to the MK surface. The expression of FN EIIIB on the MK surface may have a unique role in MK migration and maturation.

Animals↗
PubMedDate not supplied

[Magnetic resonance cholangiopancreatography and a problem in diagnosis of hepatopancreatoduodenal diseases].

Based on the findings of 54 magnetic resonance studies, the authors used 19 of them authors to study the types of normalcy. A role of the new noninvasive technique magnetic resonance cholangiopancreatography (MRCPG) in the diagnosis of hepatopancreatoduodenal diseases is assessed. The potentialities of MRCPG in the detection of most common diseases and malformations of the biliary system are demonstrated. Comparative studies of MRCPG and endoscopic retrograde cholangiopancreatography (ERCPG) were conducted in 18 cases. The paper shows a methodological approach to MRCPG and analyzes the studies by describing the MR semiotics of major diseases. Emphasis is laid on how it is important to combine routine MRI and MRCPG in certain diagnostic situations. The authors consider that MRCPG is able to replace X-ray endoscopic studies and primarily ERCPG in diagnostic terms, by reserving their therapeutical functions for itself. MRCPG has great potentialities that, require further investigations and analysis.

Adult↗
PubMedDate not supplied

[Role of magnetic resonance imaging in the complex radiation diagnosis of hormonally active adrenal tumors].

Based on the results of examination of 30 patients with hormonally active adrenal tumors, the authors consider the MRI sympatomatology of their different types and the potentialities of the technique in the complex radiation diagnosis of this pathology. The authors present their assessments of the relative intensity of a signal and the structure of each type of hormone-producing tumors of the glands by using different MRI pulse sequences that may be useful in establishing a presumptive morphological diagnosis. They identify MRI sequences that are of the greatest informative value for the diagnosis of each type of hormonally active adrenal tumors. There is evidence for that MRI is highly effective in detecting all types of hormonally active adrenal tumors and, in the context of their topographic and anatomic diagnosis, has an unquestionable advantage over ultrasonography and X-ray computed tomography in some cases.

Adenocarcinoma↗