Michael J. Keherly

Ciz1, Cip1 Interacting Zinc Finger Protein 1 Binds the Consensus DNA Sequence ARYSR(0–2)YYAC

The matrin 3 family of nuclear proteins consists of members with potentially diverse activities. Matrin 3 and NP220 share RNA-binding domains, and NP220 has been shown to recognize and bind to the DNA sequence, CCCCC (G/C). We have isolated and characterized another member of the matrin 3 family, designated NP94, from a medulloblastoma. This protein, also named Ciz1, has previously been characterized for its ability to interact with p21(Cip1/Waf1) and contains 3 zinc finger domains and a matrin 3-homologous domain 3. Our immunofluorescence and Northern blot analysis data indicate that Ciz1 is localized in the nucleus and is expressed in a wide range of tissues, especially the pancreas and the brain; within the brain, the highest message levels are found in the cerebellum. A modified selected and amplified binding (SAAB) sequence method was used to identify DNA sequences recognized by Ciz1. From the analysis of the retrieved SAAB sequences and verification using electrophoretic mobility shift assays, we formulated a consensus DNA sequence, ARYSR(0-2)YYAC, recognized by Ciz1. The potential activities of Ciz1, including those involved in brain tumorigenesis, are discussed.

Potential role for white matter lysosome expansion in HIV-associated dementia.

Expansion of the lysosomal apparatus occurs in subcortical white matter in brains from persons with AIDS. This study examined whether HIV-associated subcortical dementia (HAD) is significantly related to this lysosomal anomaly. Brain cortex and adjacent white matter from the middle frontal gyrus were obtained from the National NeuroAIDS Tissue Consortium. Lysosomal hydrolase activity was assayed in 57 subjects who underwent neuropsychological testing within 6 months prior to autopsy. Decedents were evaluated from 4 geographical sites in the United States: Galveston/Houston, Texas (n = 36), Los Angeles, California (n = 5), New York, New York (n = 5), and San Diego, California (n = 11). Increased β-glucuronidase activity, a representative lysosomal glycosidase, was correlated with the amount of neurocognitive impairment. Significant correlation was present in 5 of 7 functional testing domains, including some that draw upon frontal lobe output (r = 0.419; P < 0.002). The biochemical anomaly was negligible in cerebral cortex and cerebrospinal fluid and was not correlated with brain dysfunction in those compartments. Glycosidase activation was associated significantly with increased HIV RNA concentration in brain tissue (r = 0.469; P < 0.021) and possibly with HIV RNA in cerebrospinal fluid (r = 0.266; P < 0.067). HIV RNA in blood plasma was not correlated. These results support the suggestion that abnormal metabolism in white matter glial cells contributes to cognitive slowing in persons with HAD. Because membrane turnover is routed through the endosome-lysosome apparatus, these data are in agreement with brain spectroscopic data that have suggested that there is an increase in membrane turnover in white matter glia.

The thrombin peptide, TP508, enhances cytokine release and activates signaling events.

The thrombin peptide, TP508, accelerates tissue repair and initiates a cascade of cellular events. We have previously shown that alpha-thrombin induces cytokine expression in human mononuclear cells. We, therefore, investigated the possibility that TP508 might activate cytokine production and intracellular signaling pathways associated with cytokine activation. Our results show that TP508 induces cytokine expression in human mononuclear cells. TP508 treatment enhances extracellular signal-regulated kinase (Erk1/2) activities in U937 cells, as well as Erk1/2 and p38 activation in Jurkat T cells. These data support the hypothesis that TP508 may accelerate tissue repair through the activation of the inflammatory response.

Lamp-1 is upregulated in human glioblastoma cell lines induced to undergo apoptosis

Lysosome-associated membrane protein (LAMP)-1, one of the major protein components of the lysosomal membrane, is upregulated in the human glioblastoma cell lines, U-373 MG and LN-Z308, which undergo cisplatin-induced apoptosis. These human brain tumor cell lines demonstrated apoptosis in response to cisplatin/nifedipine treatment. Both cell lines demonstrated an apoptotic response by more than one criterion. Apoptosis was demonstrated by DNA fragmentation techniques such as DNA laddering, ApopTag® in situ labeling, and an ELISA-based method of detecting liberated oligosomes. These cells also had characteristic morphologic changes and upregulation of bax consistent with apoptosis. LAMP-1 expression at the protein and mRNA level was examined and found to increase with cisplatin/nifedipine treatment. LAMP-1 expression was examined using indirect immunofluorescent staining, Northern blot analysis and Western blot analysis. The finding of an augmentation of LAMP-1 in these cells induced to die is enigmatic. These findings raise the possibility of LAMP-1 involvement in the apoptotic process.

Characterization of somatic cell hybrids exhibiting extinction of AFP, Albumin and an AFP-HPRT Transgene

We utilized an AFP-HPRT transgene, i.e. the HPRT coding sequences under the regulation of AFP enhancer and promoter sequences, to localize the AFP extinguisher locus in intertypic somatic cell hybrids (hepatoma X fibroblast). This hybrid gene construct, which directly links AFP regulation to a reversibly selective gene, enabled the selection of stably transfected cells which express AFP, as well as cells showing extinction of AFP. Mouse hepatoma cells stably transfected with and expressing the transgene were fused to human fibroblasts, and the resulting somatic cell hybrids were characterized using Southern, Northern and karyotypic analyses. That several hybrids exhibited the proper extinction of AFP, AFP-HPRT and albumin suggests coregulation of these genes by an extinguisher. Segregant lines derived from these hybrids were selected for the loss of extinguisher activity and for reexpression of the transgene. Karyotypic analysis of hybrid and segregant lines, exhibiting proper AFP, albumin and AFP-HPRT phenotypes, revealed that the presence of human chromosome 7 was most closely associated with the AFP-extinguished state. The hybrids generated in these studies now make it possible to isolate the sequences responsible for AFP and albumin extinction.