Biomedical subjects

Malgorzata Simm

Publications and source records attributed to Malgorzata Simm.

7 recordsLinked to original sources

PubMed2026

Cloning of human DING: Developmental expression and downregulation by EtOH in-utero.

INTRODUCTION: An estimated 15-20% of women consume alcohol (EtOH) during pregnancy. Women with alcohol use in early pregnancy are likely to have a child with fetal alcohol spectrum disorders (FASD). Recently, we reported neuroprotective effects of human DING (a member of the DING family of phosphatases) against EtOH-mediated toxicity in rats and in human fetal cortical neurons in vitro. Now, we report the sequencing and developmental expression patterns of endogenous DING in human fetal brain. METHODS: DING cDNA was cloned from human U87MG astrocytoma cells with primers specific to the plant DING gene and known prokaryotic DING genes. This cDNA was used to prepare antibodies. The full-length human DING gene p38hu (1095 nucleotide bases) is flanked by the first initiating codon, ATG, and the last, stop codon, TAA. Post-mortem fetal tissues and maternal blood were collected during pregnancy between 8 and 37 weeks' gestation. The developmental, spatial, and temporal expression of DING protein in fetal brain tissue was analyzed by immunohistochemistry. Developmental expression of DING in fetal brain and placenta was quantified by qWestern blots. DING promoter expression was assayed by ddPCR. Statistical analysis included ANOVA. RESULTS: Sequencing revealed different-sized genomic DNA clones. The anti-DING antibody detected proteins ranging in size from 35 to 40 kDa, and high molecular weight precursor protein in fetal brain and placenta. DING protein was present in fetal brain at early stages and its level was increased at later gestational ages. The DING promoter was expressed in fetal brain, neurospheres, and fetal brain-derived exosomes. DING levels were reduced in samples exposed to maternally consumed alcohol. CONCLUSIONS: Because DING is neuroprotective, its reduced expression in fetuses exposed to alcohol may suggest a mechanism that contributes to the pathogenesis of FASD, which could lead to the development of therapeutic tools aimed at preventing, ameliorating or reversing this prevalent group of syndromes that are implicated in as many as 5% of births world-wide.

DING gene cloning↗

PubMed2005

A soluble factor secreted by an HIV-1-resistant cell line blocks transcription through inactivating the DNA-binding capacity of the NF-kappa B p65/p50 dimer.

The identity and activity of several anti-HIV soluble factor(s) secreted by CD8 and CD4 T lymphocytes have been determined; however, some of them still await definition. We have established an HIV-1-resistant, transformed CD4 T cell line that secretes HIV-1 resistance protein(s). Our studies indicate that this protein(s), called HIV-1 resistance factor (HRF), inhibits transcription of the virus by interfering with the activity of NF-kappaB. In the present report we identified the site at which HRF exerts this inhibition by evaluating a set of discrete events in NF-kappaB action. We tested the kappaB oligonucleotide binding activity in nuclei of resistant cells, nuclear translocation and binding to the HIV-1 long terminal repeat of p65 and p50 proteins from susceptible cells after exposure to HRF, and the binding of recombinant p50 to the kappaB oligonucleotide in vitro as affected by prior or simultaneous exposure to HRF. The results of this experimental schema indicate that HRF interacts with p50 after it enters the nucleus, but before its binding to DNA and that this interaction impedes the formation of an NF-kappaB-DNA complex required for the promotion of transcription. These findings suggest that HRF mediates a novel innate immune response to virus infection.

Active Transport, Cell Nucleus↗

PubMed2004

Monoubiquitinated histone H1B is required for antiviral protection in CD4(+)T cells resistant to HIV-1.

Linker histone H1B (H1B) coeluted with an antiviral activity during the purification of HIV-1 resistance factor (HRF) from supernatants of HRF(+) cells. Western blot analysis of the supernatant using alpha-H1 and alpha-ubiquitin antibodies detected the same band of roughly 46 kDa; this band was absent from the control supernatant. Depletion of histone from biologically active material did not affect its potential, suggesting that ubiquitinated H1B is not required for the HRF-mediated antiviral protection in HIV-1 susceptible target cells; however, specific silencing of histone H1B via RNAi in HRF(+) cells reduced the biological activity of cell culture supernatants by 96% and reversed the HIV-1 resistance phenotype of HRF(+) cells. Exposure to HRF induced ubiquitination and secretion of H1B from target HIV-1 susceptible cells, suggesting that ubiquitinated H1B is a cofactor of HRF, possibly regulating its expression and secretion from CD4(+)T cells induced to resist HIV-1 infection.

CD4-Positive T-Lymphocytes↗

PubMed2004

Detection of hepatitis C virus in the nasal secretions of an intranasal drug-user.

BACKGROUND: One controversial source of infection for hepatitis C virus (HCV) involves the sharing of contaminated implements, such as straws or spoons, used to nasally inhale cocaine and other powdered drugs. An essential precondition for this mode of transmission is the presence of HCV in the nasal secretions of intranasal drug users. METHODS: Blood and nasal secretion samples were collected from five plasma-positive chronic intranasal drug users and tested for HCV RNA using RT-PCR. RESULTS: HCV was detected in all five blood samples and in the nasal secretions of the subject with the highest serum viral load. CONCLUSIONS: This study is the first to demonstrate the presence of HCV in nasal secretions. This finding has implications for potential transmission of HCV through contact with contaminated nasal secretions.

Journal Article↗

PubMed2004

Microarray analysis of differentially expressed genes in cells resistant to HIV-1.

We have previously isolated two matched transformed human T cell clones: one of which is resistant to HIV-1 replication and secretes an HIV-1 resistance factor(s) (HRF) and the second which retains the susceptibility of the parental cell line to HIV-1 infection. We employed cDNA arrays to investigate the spectrum of changes in cellular gene expression that correlate with the acquisition of HIV-1 resistance and the secretion of HRF. Using a tissue based immunology/hematology array, we identified 29 transcripts that were differentially expressed by HRF(+) and HRF(-) cells. HRF(+) cells showed a selective down-regulation of 11 genes involved in transcription, several of which are implicated in either susceptibility of cells to HIV-1 or the promotion of HIV-1 transcription itself. In the group of the up-regulated genes, three were linked directly to the cellular resistance to HIV-1. One of the cDNAs placed on the array, representing the hypothetical protein KIAA0117 hybridized only with poly A+ RNA probes derived from HRF(+) cells. The specific up-regulation of two genes, the transcription repressor (CTCF) and hypothetical protein KIAA0117 was confirmed by RT-PCR and Northern blot. The role of KIAA0117 transcript in the resistance to HIV-1 replication needs to be determined.

Blotting, Northern↗

PubMed2003

Customized rapid subtraction hybridization (RaSH) gene microarrays identify overlapping expression changes in human fetal astrocytes resulting from human immunodeficiency virus-1 infection or tumor necrosis factor-alpha treatment.

Genes displaying altered expression as a function of human immunodeficiency virus (HIV)-1 infection of cultured primary human fetal astrocytes (PHFA) were previously identified using a rapid subtraction hybridization (RaSH) method. This scheme identified both known and novel genes displaying elevated expression, astrocyte elevated genes (AEG), and decreased expression, astrocyte suppressed genes (ASG), in PHFA as a consequence of infection with HIV-1 or treatment with HIV-1 envelope glycoprotein (gp120). RaSH also identified both known and novel genes displaying enhanced (HR) or reduced (HS) expression in HIV-1 resistant versus HIV-1 susceptible human T-cell clones. In the present study, a customized microarray approach employing these RaSH-derived genes was used to distinguish overlapping gene expression changes occurring in PHFA as a function of treatment with HIV-1 and the neurotoxic agent tumor necrosis factor (TNF)-alpha. RaSH cDNAs were spotted (microarrayed) on nylon membranes and probed with temporally isolated reverse transcribed cDNAs from HIV-1-infected and TNF-alpha-treated PHFA. This strategy identified genes displaying parallel changes after TNF-alpha treatment as observed following HIV-1 infection. Confirmation of genuine differential expression was achieved by Northern blotting. These studies document that TNF-alpha can induce a set of corresponding changes in specific AEGs and ASGs as does HIV-1 infection in PHFA. Furthermore, this customized microarray approach with RaSH-derived clones represents an efficient and sensitive methodology for elucidating molecular changes in PHFA occurring as a consequence of treatment with pharmacological agents affecting astrocyte physiology.

Astrocytes↗

PubMed2002

Induction of secreted human immunodeficiency virus type 1 (HIV-1) resistance factors in CD4-positive T lymphocytes by attenuated HIV-1 infection.

This report describes induction of HIV-1 resistance and synthesis of resistance factors in immortal CD4-positive T lymphocytes. SupT1 cells were infected by NL4-3 attenuated by a defect in the vif gene through coculture with infected primary lymphocytes. Cell lines from this infection, termed R1, expressed CD4 and CXCR4, carried low levels of HIV-1 DNA, but expressed no other detectable viral products and were resistant to infection by wild-type HIV-1. Investigation of challenge infection in resistant R1 lines demonstrated entry, reverse transcription, and integration by incoming HIV-1 but no synthesis of viral RNA. By assay of marker gene expression, we found that Tat was unable to activate LTR-driven transcription in R1 lines. HIV-1-resistant R1 lines secreted soluble factors that inhibited productive infection of primary lymphocytes by several strains of HIV-1 and blocked viral RNA synthesis in newly infected cells. Resistance factors also blocked the induction of HIV-1 transcription in ACH-2 cells as assayed by viral antigen expression and Northern blot of viral RNA. Soluble factors produced by HIV-1-resistant, immortal R1 cells may form the basis of new approaches to control HIV-1 infection.

CD4-Positive T-Lymphocytes↗