Evidence for an interferon-related inflammatory reaction in the trisomy 16 mouse brain leading to caspase-1-mediated neuronal apoptosis.

The trisomy of human chromosome 21 (Down syndrome) is the leading genetic cause of learning difficulties in children, and predisposes this population to the early onset of the neurodegeneration of Alzheimer's disease. Down syndrome is associated with increased interferon (IFN) sensitivity resulting in unexpectedly high levels of IFN inducible gene products including Fas, complement factor C3, and neuronal HLA I which could result in a damaging inflammatory reaction in the brain. Consistent with this possibility, we report here that the trisomy 16 mouse fetus has significantly increased whole brain IFN-gamma and Fas receptor immunoreactivity and that cultured whole brain trisomy 16 mouse neurons have increased basal levels of caspase 1 activity and altered homeostasis of intracellular calcium and pH.

Spacing constraints on reinitiation of paramyxovirus transcription: the gene end U tract acts as a spacer to separate gene end from gene start sites.

The paramyxovirus gene end U tracts are thought to serve as templates for the addition of a 3' polyA tail to viral mRNAs. The goal of the work described here was to determine the function in transcription of the naturally occurring variability in length of the gene end U tracts of the paramyxovirus simian virus 5 (SV5). An anchored RT-PCR assay was developed to test the hypothesis that the variable U tracts template the addition of variable lengths of polyA tails to mRNAs.

Regulation of the Epstein-Barr virus C promoter by AUF1 and the cyclic AMP/protein kinase A signaling pathway.

EBNA2 is an Epstein-Barr virus (EBV)-encoded protein that regulates the expression of viral and cellular genes required for EBV-driven B-cell immortalization. Elucidating the mechanisms by which EBNA2 regulates viral and cellular gene expression is necessary to understand EBV-induced B-cell immortalization and viral latency in humans. EBNA2 targets to the latency C promoter (Cp) through an interaction with the cellular DNA binding protein CBF1 (RBPJk).

Regulation of c-myc mRNA decay in vitro by a phorbol ester-inducible, ribosome-associated component in differentiating megakaryoblasts.

The K562 leukemia cell line is bipotential for erythroid and megakaryoblastic differentiation. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) activates a genetic program of gene expression in these cells leading to their differentiation into megakaryoblasts, a platelet precursor. Thus, K562 cells offer a means to examine early changes in gene expression necessary for megakaryoblastic commitment and differentiation.

Regulation of cyclooxygenase 2 mRNA stability by the mitogen-activated protein kinase p38 signaling cascade.

A tetracycline-regulated reporter system was used to investigate the regulation of cyclooxygenase 2 (Cox-2) mRNA stability by the mitogen-activated protein kinase (MAPK) p38 signaling cascade. The stable beta-globin mRNA was rendered unstable by insertion of the 2, 500-nucleotide Cox-2 3' untranslated region (3' UTR). The chimeric transcript was stabilized by a constitutively active form of MAPK kinase 6, an activator of p38.

Protective effect of the energy precursor creatine against toxicity of glutamate and beta-amyloid in rat hippocampal neurons.

The loss of ATP, which is needed for ionic homeostasis, is an early event in the neurotoxicity of glutamate and beta-amyloid (A(beta)). We hypothesize that cells supplemented with the precursor creatine make more phosphocreatine (PCr) and create larger energy reserves with consequent neuroprotection against stressors. In serum-free cultures, glutamate at 0.

A fluorescence-based assay for 3' --> 5' exoribonucleases: potential applications to the study of mRNA decay.

A cell-free mRNA decay assay has been adapted to permit the kinetics of 3' --> 5' exoribonuclease activities to be monitored in real time. RNA probes containing 5' caps and 3' poly(A) tails generated by transcription in vitro are 3' labeled using fluorescein-N6-ATP and poly(A) polymerase. Release of fluorescein-conjugated adenosine residues from the 3' end of the RNA substrate is monitored by a time-dependent decrease in fluorescence anisotropy in the presence of cytosolic proteins.

Long-term maintenance of patterns of hippocampal pyramidal cells on substrates of polyethylene glycol and microstamped polylysine.

For neurons to attach and remain in precise micropatterns for weeks in culture, background molecules that remain nonpermissive for extended culture durations need to be identified. Nonpermissive background molecules of either polyethylene glycol (PEG) or the amino acid serine (C3H7NO3) were evaluated. The foreground regions were microstamped with 3-, 5-, or 10-micron lines of poly-D-lysine (PDL), which promotes neural attachment and growth.

Identification of AUF1 as a parathyroid hormone mRNA 3'-untranslated region-binding protein that determines parathyroid hormone mRNA stability.

Parathyroid hormone (PTH) mRNA levels are post-transcriptionally increased by hypocalcemia and decreased by hypophosphatemia, and this is mediated by cytosolic proteins binding to the PTH mRNA 3'-untranslated region (UTR). The same proteins are also present in other tissues, such as brain, but only in the parathyroid is their binding regulated by calcium and phosphate. The function of the PTH mRNA 3'-UTR-binding proteins was studied using an in vitro degradation assay.

Mutation causing von Willebrand's disease in Scottish Terriers.

Von Willebrand's Disease (vWD) in the Scottish Terrier breed is a serious, often fatal, hereditary bleeding disorder. Elimination of the mutated gene by selective breeding is an important goal for the health of this breed. Although the standard protein-based tests are accurate for identification of affected Scottish Terriers, they are not reliable for the identification of carriers of the mutant gene unless multiple replicate assays are performed.

Treatment of metastatic cancer with tetrathiomolybdate, an anticopper, antiangiogenic agent: Phase I study.

Preclinical and in vitro studies have determined that copper is an important cofactor for angiogenesis. Tetrathiomolybdate (TM) was developed as an effective anticopper therapy for the initial treatment of Wilson's disease, an autosomal recessive disorder that leads to abnormal copper accumulation. Given the potency and uniqueness of the anticopper action of TM and its lack of toxicity, we hypothesized that TM would be a suitable agent to achieve and maintain mild copper deficiency to impair neovascularization in metastatic solid tumors.

Treatment of Wilson's disease with zinc. XVII: treatment during pregnancy.

Therapy of Wilson's disease continues to evolve. In 1997, zinc acetate was added to the list of drugs approved by the Food and Drug Administration, which includes penicillamine and trientine. The mechanism of zinc's anticopper action is unique.

Recognition, diagnosis, and management of Wilson's disease.

Wilson's disease is a relatively rare inherited disorder of copper accumulation and toxicity, caused by a defect in an enzyme that is part of the pathway of biliary excretion of excess copper. Clinically, patients usually present as older children or young adults with hepatic, neurologic, or psychiatric manifestations, or some combination of these. Wilson's disease is unusual among genetic diseases in that it can be very effectively treated.

N-type Ca2+ channels in cultured rat sphenopalatine ganglion neurons: an immunohistochemical and electrophysiological study.

Results from pharmacological studies have suggested that presynaptic N-type Ca2+ channels play an important role in regulating neuronal Ca2+ influx and transmitter nitric oxide (NO) release in isolated cerebral arteries. However, the presence of N-type Ca2+ channels in cerebral perivascular nerves has not been directly demonstrated. As a major source of cerebral perivascular NOergic innervation is the sphenopalatine ganglion (SPG), adult rat SPGs were cultured and examined by whole-cell patch-clamp technique.

CTCF is essential for up-regulating expression from the amyloid precursor protein promoter during differentiation of primary hippocampal neurons.

The transcriptional mechanism underlying amyloid precursor protein (APP) regulation in primary neurons during development was investigated. We observed an approximately threefold elevation of APP mRNA levels in differentiating rat hippocampal neurons between day 1 and day 7 in culture and in rat brain hippocampi between embryonic day 18 and postnatal day 3. When an APP promoter construct extending to position -2,832 upstream from the main transcriptional start site was transfected into primary rat hippocampal neurons, promoter activity increased from day 1 until reaching a maximum on day 7 in culture.

Resistance in sunflower and interaction with Bacillus thuringiensis for control of banded sunflower moth (Lepidoptera: Tortricidae).

Four sunflower accessions were compared with a susceptible check, hybrid '894', in the greenhouse to determine their resistance to the banded sunflower moth, Cochylis hospes Walsingham, and their interaction with Bacillus thuringiensis Berliner variety kurstaki. Antibiosis, expressed as lower larval weight, was detected in all of the accessions. In addition to being antibiotic, sunflower accession Ames 3291 was antixenotic to banded sunflower moth oviposition and exhibited an additional impact on larval weight when B.

Developmental regulation of RNA transcript destabilization by A + U-rich elements is AUF1-dependent.

The developmental immaturity of neonatal phagocytic function is associated with decreased accumulation and half-life (t((1)/(2))) of granulocyte/macrophage colony-stimulating factor (GM-CSF) mRNA in mononuclear cells (MNC) from the neonatal umbilical cord compared with adult peripheral blood. The in vivo t((1)/(2)) of GM-CSF mRNA is 3-fold shorter in neonatal (30 min) than in adult (100 min) MNC. Turnover of mRNA containing a 3'-untranslated region (3'-UTR) A + U-rich element (ARE), which regulates GM-CSF mRNA stability, is accelerated in vitro by protein fractions enriched for AUF1, an ARE-specific binding factor.

Assembly of AUF1 oligomers on U-rich RNA targets by sequential dimer association.

Many labile mammalian mRNAs are targeted for rapid cytoplasmic turnover by the presence of A + U-rich elements (AREs) within their 3'-untranslated regions. These elements are selectively recognized by AUF1, a component of a multisubunit complex that may participate in the initiation of mRNA decay. In this study, we have investigated the recognition of AREs by AUF1 in vitro using oligoribonucleotide substrates.

Regeneration and proliferation of embryonic and adult rat hippocampal neurons in culture.

Adult mammalian CNS neurons appear to be terminally differentiated and postmitotic. However, this conclusion may be due to nonpermissive conditions in the brain or in culture media. If embryonic rat hippocampal neurons are cultured in Neurobasal/B27 with FGF2, nearly all neurons proliferated until a maximum density was reached.

Post-transcriptional regulation of the DNA damage-inducible gadd45 gene in human breast carcinoma cells exposed to a novel retinoid CD437.

The biologically active synthetic retinoid CD437 (6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene, AHPN) and different human breast carcinoma (HBC) cell lines were used to examine the possible mechanism(s) of gadd45 induction. Northern blot analysis of mRNA isolated from MCF-7, MDA-MB-468 and MDA-MB-231 HBC cell lines demonstrated a progressive increase in the 1.4 kb gadd45 transcript after exposure to 1 microM CD437.

Evidence for a 3'-5' decay pathway for c-myc mRNA in mammalian cells.

Many mRNAs in mammalian cells decay via a sequential pathway involving rapid conversion of polyadenylated molecules to a poly(A)-deficient state followed by rapid degradation of the poly(A)-deficient molecules. However, the rapidity of this latter step(s) has precluded further analyses of the decay pathways involved. Decay intermediates derived from degradation of poly(A)-deficient molecules could offer clues regarding decay pathways, but these intermediates have not been readily detected.

Regulation of AUF1 expression via conserved alternatively spliced elements in the 3' untranslated region.

The A+U-rich RNA-binding factor AUF1 exhibits characteristics of a trans-acting factor contributing to the rapid turnover of many cellular mRNAs. Structural mapping of the AUF1 gene and its transcribed mRNA has revealed alternative splicing events within the 3' untranslated region (3'-UTR). In K562 erythroleukemia cells, we have identified four alternatively spliced AUF1 3'-UTR variants, including a population of AUF1 mRNA containing a highly conserved 107-nucleotide (nt) 3'-UTR exon (exon 9) and the adjacent downstream intron (intron 9).