Cerebral microbleeds predict infectious intracranial aneurysm in infective endocarditis.

BACKGROUND AND PURPOSE: Magnetic resonance imaging (MRI) features such as cerebral microbleeds and sulcal susceptibility-weighted imaging (SWI) or gradient-echo T2* lesions in infective endocarditis (IE) have been associated with the presence of infectious intracranial aneurysm (IIA). Our aim was to validate these MRI predictors for IIA in order to better assist in assessing the appropriate indications for digital subtraction angiography (DSA). METHODS: The derivation cohort comprised IE patients with neurological evaluation, MRI and DSA at a single tertiary referral center from January 2015 to July 2016. Validation was performed in a cohort of IE patients who underwent MRI and DSA at the same center from 2010 to 2014. RESULTS: Of 62 patients in the derivation cohort, 10 (16%) had IIAs. Of 129 in the validation cohort, 19 (15%) IIAs were identified. The MRI predictors for IIA consist of (i) contrast enhancement with microbleeds, (ii) cerebral microbleeds >5 mm or sulcal SWI lesions and (iii) any MRI hemorrhages. The sensitivity for the presence of IIA in each group of the derivation cohort was 90%, 80% and 100%, respectively. The sensitivity in the validation cohort was 47%, 68% and 94% respectively. The specificity in the derivation cohort was 87%, 85% and 18%. In the validation cohort, the specificity was similar at 87%, 75% and 27%. CONCLUSIONS: The absence of MRI hemorrhages may not necessitate the need for DSA.

A 39-kD DNA-binding protein from mouse brain stimulates transcription of myelin basic protein gene in oligodendrocytic cells.

The MB1 regulatory sequence of the myelin basic protein (MBP) gene spanning between nucleotides -14 to -50 with respect to the transcription start site is critical for cell type-specific transcription of the MBP gene, which encodes the major protein component of myelin sheath in cells derived from the central nervous system (CNS). This regulatory sequence has the ability to interact with a developmentally controlled DNA-binding protein from mouse brain that stimulates transcription of MBP promoter in an in vitro system (Haas, S., J. Gordon, and K. Khalili. 1993. Mol. Cell. Biol. 13:3103-3112). Here, we report the purification of a 39-kD protein from mouse brain tissue at the peak of myelination and MBP production that binds to the MB1 regulatory motif. Following partial amino acid sequence analysis, we have identified a complementary DNA encoding a 39-kD DNA-binding protein called pur alpha. Expression of pur alpha cDNA in the prokaryotic and eukaryotic cells resulted in the synthesis of a protein with characteristics similar to the purified brain-derived 39-kD protein in band shift competition assays. Cotransfection of the recombinant pur alpha expressor plasmid with MBP promoter construct indicated that Pur alpha stimulates transcription of the MBP promoter in oligodendrocytic cells, and that the nucleotide sequence required for binding of the 39-kD Pur alpha to DNA within the MB1 region is crucial for this activity. Moreover, transient expression of Pur alpha caused elevation in the level of endogenous MBP RNA in oligodendrocytic cells. Thus, Pur alpha, a sequence-specific DNA-binding protein upon binding to MB1 regulatory region may play a significant role in determining the cell type-specific expression of MBP in brain.

The transcription factor E2F-1 modulates TGF-beta1 RNA expression in glial cells.

The cell type specificity of the regulation of expression of the potent growth inhibitory cytokine transforming growth factor-beta (TGF-beta), prompted our analyses of the regulation of TGF-beta1 gene expression in glial cells by viral and cellular oncoproteins. We have shown that SV40 T-antigen diminished TGF-beta1 expression in glial cells and this repression was dependent on the ability of T-antigen to interact with the tumor suppressor protein, pRb, and two structurally related proteins, p107 and p130. The cellular transcription factor E2F-1, which is a downstream effector of T-antigen, was unable to influence expression from the TGF-beta1 promoter by itself. Interestingly, E2F-1 could overcome viral T-antigen-mediated repression of the TGF-beta1 promoter, suggesting potential feedback loop between TGF-beta and E2F in virally transformed glial cells. Using deletion analyses, we have mapped two E2F-1-responsive regions on the TGF-beta1 promoter: a T-antigen-dependent negative regulatory sequence (TdNRS) between -323 and -175, and a T-antigen-independent positive regulatory sequence (TiPRS) between -34 and +10 on the TGF-beta1 promoter. Further examination of TiPRS revealed the presence of a functional E2F binding site. Interestingly, the amino terminus of E2F-1 was required for its activation of TGF-beta1 expression, as mutations in that domain abolished the ability of E2F-1 to increase TGF-beta1 expression. These data suggest that yet-uncharacterized interaction between the amino terminus of E2F-1 and cellular proteins regulates TGF-beta1 expression. The mechanism for E2F-1-mediated T-antigen-dependent regulation of TGF-beta1 expression from TdNRS awaits further characterization.

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.

Role of nitric oxide in sympathetic neurotransmission in opossum internal anal sphincter.

BACKGROUND: The role of nitric oxide in the gut in response to sympathetic nerve stimulation has not been examined. The present study examined the influence of the NO synthase inhibitor L-NG-nitro-arginine (L-NNA) on responses to hypogastric nerve stimulation (HGNS) in the opossum internal anal sphincter (IAS). METHODS: Resting pressures in the IAS (IASP) were monitored using low-compliance continuously perfused catheters. RESULTS: The predominant response to HGNS was an elevation of the resting tone in the IAS. The other responses of infrequent nature were a decrease in the IASP and a biphasic response (an initial increase followed by a decrease in the IASP). beta-Adrenoceptor antagonist propranolol had no significant effect on the increase in the IASP by HGNS but almost abolished the decrease in the IASP caused by HGNS and unmasked the excitatory responses. The IAS responses to HGNS were frequency dependent and abolished by guanethidine (adrenergic blocker). L-NNA caused significant and stereoselective suppression of all IAS responses to HGNS. The suppressed HGNS responses were completely reversed by the NO precursor L-arginine stereoselectively. The NO synthase inhibitor and guanethidine had no effect on the increase in IASP by phenylephrine. CONCLUSIONS: NO may play a significant role in the facilitatory modulation of sympathetic responses in the IAS.

Nitric oxide synthase inhibitor inhibits catecholamines release caused by hypogastric sympathetic nerve stimulation.

Recent studies suggest that nitric oxide (NO) plays a significant role in the parasympathetic inhibitory neurotransmission to the internal and sphincter (IAS). The role of NO in the sympathetic neurotransmission in the gut is not known. The resting intraluminal pressures of anesthetized opossum IAS (IASP) were monitored using low-compliance continuously perfused catheters. The plasma levels of catecholamines norepinephrine, epinephrine and dopamine were measured using a radioenzymatic assay. Parallel studies to determine the effects of hypogastric nerve stimulation (HGNS) on the resting IASP and catecholamine release were performed. The influence of NO in the sympathetic neurotransmission was determined by examining the effect of the NO synthase inhibitor L-NG-nitro-arginine (L-NNA) and the reversal of the effect by L-arginine. HGNS caused a frequency-dependent rise in the IASP accompanied by rises in norepinephrine, epinephrine and dopamine levels. The increases in the IASP and norepinephrine, epinephrine and dopamine levels were stereoselectively and significantly attenuated by L-NNA. Furthermore, the L-NNA-induced attenuation of rises in the resting IASP and catecholamine levels in response to HGNS was reversed stereoselectively by L-arginine. These studies for the first time (to the authors' knowledge) show that the NO synthase inhibitor causes specific suppression of sympathetic neurotransmitter release in the gut smooth muscle. It was concluded that, in the anorectum, NO has a facilitory role in the release of sympathetic neurotransmitters.

Human immunodeficiency virus tat gene transfer to the murine central nervous system using a replication-defective herpes simplex virus vector stimulates transforming growth factor beta 1 gene expression.

The high incidence of neurological disorders in patients afflicted with acquired immunodeficiency syndrome (AIDS) may result from human immunodeficiency virus type 1 (HIV-1) induction of chemotactic signals and cytokines within the brain by virus-encoded gene products. Transforming growth factor beta1 (TGF-beta1) is an immunomodulator and potent chemotactic molecule present at elevated levels in HIV-1-infected patients, and its expression may thus be induced by viral trans-activating proteins such as Tat. In this report, a replication-defective herpes simplex virus (HSV)-1 tat gene transfer vector, dSTat, was used to transiently express HIV-1 Tat in glial cells in culture and following intracerebral inoculation in mouse brain in order to directly determine whether Tat can increase TGF-beta1 mRNA expression. dSTat infection of Vero cells transiently transfected by a panel of HIV-1 long terminal repeat deletion mutants linked to the bacterial chloramphenicol acetyltransferase reporter gene demonstrated that vector-expressed Tat activated the long terminal repeat in a trans-activation response element-dependent fashion independent of the HSV-mediated induction of the HIV-1 enhancer, or NF-kappaB domain. Northern blot analysis of human astrocytic glial U87-MG cells transfected by dSTat vector DNA resulted in a substantial increase in steady-state levels of TGF-beta1 mRNA. Furthermore, intracerebral inoculation of dSTat followed by Northern blot analysis of whole mouse brain RNA revealed an increase in levels of TGF-beta1 mRNA similar to that observed in cultured glial cells transfected by dSTat DNA. These results provided direct in vivo evidence for the involvement of HIV-1 Tat in activation of TGF-beta1 gene expression in brain. Tat-mediated stimulation of TGF-beta1 expression suggests a novel pathway by which HIV-1 may alter the expression of cytokines in the central nervous system, potentially contributing to the development of AIDS-associated neurological disease.

Reciprocal Id expression and myelin gene regulation in Schwann cells.

Id proteins are thought to act as dominant negative antagonists of basic helix-loop-helix (bHLH) transcription factors that direct differentiation in various cell types. We found that Schwann cells express all four Id-family genes and that their transcript levels were reciprocally regulated in pairs during nerve maturation in vivo and cAMP-mediated differentiation in vitro. The rapid induction as part of the early response to axonal membranes and cytokines suggested that Id3 is involved in myelin gene repression. An inverse relationship between Id1/3 and myelin P0 expression was consistent with a role for these two Id proteins as inhibitors of differentiation, and Id1/3 proteins strongly repressed myelin gene promoter activity. Nuclear factors isolated from Schwann cells and intact sciatic nerves were found to bind three different HLH recognition sequences (E boxes) in the proximal region of the P0 promoter, and production of these DNA binding complexes was altered during differentiation. These data support the concept that Id proteins regulate myelin gene expression by controlling the formation of specific bHLH DNA binding complexes with different E-box preferences.

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.