Calcium and lithium ion production for laser ion source.

Calcium and lithium ion beams are required by NASA Space Radiation Laboratory at Brookhaven National Laboratory to simulate the effects of cosmic radiation. To identify the difficulties in providing such highly reactive materials as laser targets, both species were experimentally tested. Plate shaped lithium and calcium targets were fabricated to create ablation plasmas with a 6 ns 1064 nm neodymium-doped yttrium aluminum garnet laser. We found significant oxygen contamination in both the Ca and Li high charge state beams due to the rapid oxidation of the surfaces. A large spot size, low power density laser was used to create low charge state beams without scanning the targets. The low charge state Ca beam did not have any apparent oxygen contamination, showing the potential to clean the target entirely of oxide with a low power beam once in the chamber. The Li target was clearly still oxidizing in the chamber after each low power shot. To measure the rate of oxidation, we shot the low power laser at the target repeatedly at 10 s, 30 s, 60 s, and 120 s interval lengths, showing a linear relation between the interval time and the amount of oxygen in the beam.

Proton beam production by a laser ion source with hydride target.

We studied proton beam production from a laser ion source using hydrogen rich target materials. In general, gas based species are not suitable for laser ion sources since formation of a dense laser target is difficult. In order to achieve reliable operation, we tested hydride targets using a sub nanosecond Q-switched Nd-YAG laser, which may help suppress target material consumption. We detected enough yields of protons from a titanium hydride target without degradation of beam current during the experiment. The combination of a sub nanosecond laser and compressed hydride target may provide stable proton beam.

Transcriptional feedback and definition of the circadian pacemaker in Drosophila and animals.

The modern era of Drosophila circadian rhythms began with the landmark Benzer and Konopka paper and its definition of the period gene. The recombinant DNA revolution then led to the cloning and sequencing of this gene. This work did not result in a coherent view of circadian rhythm biochemistry, but experiments eventually gave rise to a transcription-centric view of circadian rhythm generation. Although these circadian transcription-translation feedback loops are still important, their contribution to core timekeeping is under challenge. Indeed, kinases and posttranslational regulation may be more important, based in part on recent in vitro work from cyanobacteria. In addition, kinase mutants or suspected kinase substrate mutants have unusually large period effects in Drosophila. This chapter discusses our recent experiments, which indicate that circadian transcription does indeed contribute to period determination in this system. We propose that cyanobacteria and animal clocks reflect two independent origins of circadian rhythms.

Enhanced proliferation and differentiation of rat hepatocytes cultured with bone marrow stromal cells.

Liver transplantation is the only clinically effective method of treating acute liver failure. However, wider application of this therapeutic modality is restricted primarily by shortage of donor organs. In the search for alternative methods of liver replacement therapy, investigators have focused on transplantation of normal allogeneic hepatocytes and on the development of liver support systems utilizing isolated hepatocytes. Since all human livers suitable for cell harvest are being used for transplantation, hepatocyte therapy using human tissue would require growing of cells in vitro. Unfortunately, although hepatocytes have tremendous capacity to proliferate in vivo, their ability to grow in culture is severely limited. Stromal cells from bone marrow and other blood-forming organs have been found to support hematopoiesis. In this paper, we show that bone marrow-derived stromal cells (BMSCs) enhance proliferation and support differentiation of rat hepatocytes in culture. Further, we demonstrate that in hepatocyte/BMSC co-cultures, clonal expansion of small hepatocytes (SH) is increased. Using semipermeable membrane cultures, we established that direct cell-cell contact is necessary for stimulation of cell proliferation. We also show that BMSCs which are in direct contact with hepatocytes and SH colonies express Jagged1. This suggests a potential role for Notch signaling in the observed effects. Finally, we present evidence that the expression and activity of liver specific transcription factors, CCAAT/enhancer binding proteins and liver specific key enzymes such as tryptophan 2,3-dioxygenase, are improved in hepatocyte/BMSC co-cultures. In conclusion, results of this study indicate that BMSCs could facilitate proliferation and differentiation of primary rat hepatocytes and their progenitors (SH) in vitro.

Caco-2 monolayers in experimental and theoretical predictions of drug transport.

This review examines the use of Caco-2 monolayers in the prediction of intestinal drug absorption. First, the different routes of drug transport in Caco-2 monolayers are compared with those seen in vivo. Second, the prediction of drug absorption in vivo from transport experiments in cell monolayers is discussed for different classes of drugs. Finally, the use of Caco-2 monolayers as a reference model in physico-chemical and theoretical predictions of drug absorption is discussed. We conclude that Caco-2 monolayers can be used to identify drugs with potential absorption problems, and possibly also to select drugs with optimal passive absorption characteristics from series of pharmacologically active molecules generated in drug discovery programs.

Fetal and adult human CNS stem cells have similar molecular characteristics and developmental potential.

The mammalian central nervous system (CNS) contains multipotent stem cells that develop into neurons, astrocytes and oligodendrocytes. Our current data show that fetal and adult human CNS stem cell isolates display similar proliferation kinetics, differentiate into three major cell types of the nervous system and express similar sets of regulatory genes. However, each individual CNS stem cell isolate could be distinguished by its specific gene expression and developmental potential.

Prediction of maternal and fetal complications in preeclampsia.

INTRODUCTION: Preeclampsia is associated with an increased risk for maternal and fetal morbidity. The aim of this study was to identify factors predicting for maternal or fetal complications. MATERIAL AND METHODS: One hundred and eleven patients with preeclampsia were included. Endpoint variables were maternal and fetal complications. Any of the diagnosis eclampsia, hemolysis elevated liver low platelet (HELLP) syndrome, oliguria or placental abruption was considered a maternal complication. Fetal complications were: small for gestational age (SGA) infant, umbilical artery pH<7.10 and admittance of the infant to a neonatal intensive care unit (NICU). Independent variables were maternal age, parity, gestational age, renal function, platelet count, liver enzymes and blood pressure. Logistic regression analysis was used for statistical evaluation. RESULTS: The following independent significant predictors were identified: Maternal complication (n=10) was predicted only by diastolic blood pressure; odds ratio (OR) 1.13 (95% confidence interval 1.01-1.25). SGA (n=21) was associated with maternal prepregnancy weight, OR 0.94 (0.89-0.99) and gestational age at debut of preeclampsia, OR 0.97 (0.94-0.99). NICU admittance (n=31) was only predicted by gestational age at delivery, OR 0.80 (0.67-0.96). No association was found between any of the independent variables and a low umbilical artery pH (n= 10). CONCLUSIONS: In the surveillance of patients with established preeclampsia, the value of serial blood and urine sampling, which is common practice in many obstetrical units, might be questioned.

Neuron-specific splicing of zinc finger transcription factor REST/NRSF/XBR is frequent in neuroblastomas and conserved in human, mouse and rat.

Neuron-restrictive silencer factor (NRSF), also known as repressor element RE1 binding transcription factor (REST) or repressor binding to the X2 box (XBR) (REST/NRSF/XBR), is a zinc finger transcription factor that during early embryogenesis is required to repress a subset of neuron-specific genes in non-neural tissues and undifferentiated neural precursors. We have previously shown that splicing within the coding region of rat REST/NRSF/XBR (rREST) generates several different transcripts all of which are expressed in the adult nervous system. rREST transcripts with short neuron-specific exons (exon N) have in-frame stop codons and encode truncated proteins which have an N-terminal repressor domain and weakened DNA binding activity. The aim of this study was to analyze the regulatory mechanisms underlying REST/NRSF/XBR activity in human and mouse as compared to rat. We show that the structure of REST/NRSF/XBR gene and its regulation by neuron-specific splicing is conserved in human, mouse and rat. Expression levels of REST/NRSF/XBR transcripts with the insertion of exon N are increased during the neuronal differentiation of mouse teratocarcinoma PCC7 and rat pheocromocytoma PC12 cells and are high in several human and mouse neuroblastoma cells as compared to the relatively low levels in the developing and adult nervous system. The exclusive expression of the neuronal forms of REST/NRSF/XBR mRNAs in mouse neuroblastoma Neuro-2A cells is not caused by rearrangement of the REST/NRSF/XBR gene nor by mutations in the sequence of the splice sites flanking exon N. These data suggest that changes in REST/NRSF/XBR splicing pattern may result from altered levels of splicing factors reflecting the formation and/or progression of neuroblastoma tumors.

Effect of molecular charge on intestinal epithelial drug transport: pH-dependent transport of cationic drugs.

The aim of this study was to investigate the effect of ionization on drug transport across the intestinal epithelium in order to include this effect in structure-absorption relationships. The pH-dependent permeation of one rapidly (alfentanil) and one slowly (cimetidine) transported basic model drug across Caco-2 cell monolayers was investigated. Both drugs had pK(a)values in the physiological pH range. The permeability coefficients (P(c)) of the model drugs were obtained at varying apical buffer pHs, thus varying the degree of drug ionization (from 5 to 95%). The relationship between P(c) and the fraction of the drug in un-ionized form (f(u)) was analyzed to delineate the permeability coefficients of the un-ionized (P(c,u)) and ionized (P(c,i)) forms of the drugs. Theoretical estimates of the pK(a) values were also calculated from ionization energies for each model compound. For both drugs, a linear increase in P(c) was observed with increasing f(u). Transport of the un-ionized form was 150- and 30-fold more rapid than transport of the ionized form for alfentanil and cimetidine, respectively. However, when f(u) <0.1, the contribution of the ionized form was significant. Because f(u) is <0.1 over the entire physiological pH range for a large number of drugs, these results will have implications on predictions of in vivo intestinal drug absorption both from in vitro studies in cell cultures and from computed structural properties of drug molecules.

Brain-derived neurotrophic factor expression in vivo is under the control of neuron-restrictive silencer element.

Neuron-restrictive silencer element (NRSE) has been identified in multiple neuron-specific genes. This element has been shown to mediate repression of neuronal gene transcription in nonneuronal cells. A palindromic NRSE (NRSEBDNF) is present in the proximal region of brain-derived neurotrophic factor (BDNF) promoter II. Using in vitro binding assays, we establish that the upper half-site is largely responsible for the NRSEBDNF activity. To delineate the in vivo role of NRSE in the regulation of rat BDNF gene, promoter constructs with intact and mutated NRSEBDNF were introduced into transgenic mice. Our data show that NRSEBDNF is controlling the activity of BDNF promoters I and II in the brain, thymus, and lung, i.e. in the tissues in which the intact reporter gene and endogenous BDNF mRNAs are expressed. Mutation of NRSEBDNF did not lead to the ectopic activation of the reporter gene in any other nonneural tissues. In the brain, NRSEBDNF is involved in the repression of basal and kainic acid-induced expression from BDNF promoters I and II in neurons. However, NRSEBDNF does not control the activity of the BDNF gene in nonneuronal cells of brain.

Neuronal expression of zinc finger transcription factor REST/NRSF/XBR gene.

The identification of a common cis-acting silencer element, a neuron-restrictive silencer element (NRSE), in multiple neuron-specific genes, together with the finding that zinc finger transcription factor REST/NRSF/XBR could confer NRSE-mediated silencing in non-neuronal cells, suggested that REST/NRSF/XBR is a master negative regulator of neurogenesis. Here we show that, although REST/NRSF/XBR expression decreases during neuronal development, it proceeds in the adult nervous system. In situ hybridization analysis revealed neuronal expression of rat REST/NRSF/XBR mRNA in adult brain, with the highest levels in the neurons of hippocampus, pons/medulla, and midbrain. The glutamate analog kainic acid increased REST/NRSF/XBR mRNA levels in various hippocampal and cortical neurons in vivo, suggesting that REST/NRSF/XBR has a role in neuronal activity-implied processes. Several alternatively spliced REST/NRSF/XBR mRNAs encoding proteins with nine, five, or four zinc finger motifs are transcribed from REST/NRSF/XBR gene. Two of these transcripts are generated by neuron-specific splicing of a 28-bp-long exon. Rat REST/NRSF/XBR protein isoforms differ in their DNA binding specificities; however, all mediate repression in transient expression assays. Our data suggest that REST/NRSF/XBR is a negative regulator rather than a transcriptional silencer of neuronal gene expression and counteracts with positive regulators to modulate target gene expression quantitatively in different cell types, including neurons.

Evaluation of dynamic polar molecular surface area as predictor of drug absorption: comparison with other computational and experimental predictors.

The relationship between various molecular descriptors and transport of drugs across the intestinal epithelium was evaluated. The monolayer permeability (Pc) of human intestinal Caco-2 cells to a series of nine beta-receptor-blocking agents was investigated in vitro. The dynamic polar molecular surface area (PSAd) of the compounds was calculated from all low-energy conformations identified in molecular mechanics calculations in vacuum and in simulated chloroform and water environments. For most of the investigated drugs, the effects of the different environments on PSAd were small. The exception was H 216/44, which is a large flexible compound containing several functional groups capable of hydrogen bonding (PSAd,chloroform = 70.8 A2 and PSAd,water = 116.6 A2). The relationship between Pc and PSAd was stronger than those between Pc and the calculated octanol/water distribution coefficients (log Dcalc) or the experimentally determined immobilized liposome chromatography (ILC) retention. Pc values for two new practolol analogues and H 216/44 were predicted from the structure-permeability relationships of a subset of the nine compounds and compared with experimental values. The Pc values of the two practolol analogues were predicted well from both PSAd calculations and ILC retention studies. The Pc value of H 216/44 was reasonably well-predicted only from the PSAd of conformations preferred in vacuum and in water. The other descriptors overestimated the Pc of H 216/44 100-500-fold.

Polar molecular surface properties predict the intestinal absorption of drugs in humans.

PURPOSE: A theoretical method has been devised for prediction of drug absorption after oral administration to humans. METHODS: Twenty structurally diverse model drugs, ranging from 0.3 to 100% absorbed, were investigated. The compounds also displayed diversity in physicochemical properties such as lipophilicity, hydrogen bonding potential and molecular size. The dynamic molecular surface properties of the compounds were calculated, taking into account their three-dimensional shape and flexibility. RESULTS: An excellent sigmoidal relationship was established between the absorbed fraction after oral administration to humans (FA) and the dynamic polar molecular surface area (PSAd) (r2 = 0.94). The relationship was stronger than those obtained for more established predictors of drug absorption. Drugs that are completely absorbed (FA > 90%) had a PSAd < or = 60 A2 while drugs that are < 10% absorbed had a PSAd > or = 140 A2. CONCLUSIONS: The results indicate that PSAd can be used to differentiate poorly absorbed drugs at an early stage of the drug discovery process.

Solitary Langerhans cell histiocytosis lesion of the parieto-occipital lobe: a case report and review of the literature.

A 38 year-old woman with a solitary intracerebral Langerhans cell histocytosis (LCH) lesion is presented, in whom, cerebral magnetic resonance imaging (MRI) revealed a non-enhancing mass in the right parieto-occipital lobe. The surgical specimen consisted of a nodular polymorphic infiltrate of mononuclear histiocytic cells, macrophages, eosinophilic granulocytes, lymphocytes and Langerhans giant cells involving leptomeninges, cerebral cortex and white matter. The histiocytes displayed features of Langerhans cells such as CD1a and S-100 immunopositivity, and of reticulum cells such as Ki-M4P and X-12 immunopositivity. This case as well as ten other cases from the literature are reviewed.

Correlation of drug absorption with molecular surface properties.

The correlation between dynamic surface properties of drug molecules and drug absorption in two common in vitro models of the intestinal wall (Caco-2 monolayers and rat intestinal segments) has been investigated. A homologous series of beta-adrenoreceptor antagonists were used as model compounds. Dynamic molecular surface properties, considering all low-energy conformations, of the compounds were calculated. The flexibility of the molecules was studied by molecular mechanics calculations (MM2) and the van der Waals' (vdW), and water accessible surface areas were calculated and averaged according to a Boltzmann distribution. Excellent correlations were obtained between the dynamic polar vdW surface areas and cell permeabilities in Caco-2 cells and rat ileum (r2 = 0.99 and 0.92, respectively). These correlations were stronger than those between calculated octanol/buffer partition coefficients (log Doct,7.4) and permeability (r2 = 0.80 and 0.73, respectively). Moreover, the calculated log Doct,7.4 values failed to rank the permeability coefficients through Caco-2 monolayers and rat ileum in the correct order. The results indicate that dynamic polar surface area is a promising alternative model for the prediction of oral drug absorption.

Helix-loop-helix transcription factors mediate activation and repression of the p75LNGFR gene.

Sequence analysis of rat and human low-affinity nerve growth factor receptor p75LNGFR gene promoter regions revealed a single E-box cis-acting element, located upstream of the major transcription start sites. Deletion analysis of the E-box sequence demonstrated that it significantly contributes to p75LNGFR promoter activity. This E box has a dual function; it mediates either activation or repression of the p75LNGFR promoter activity, depending on the interacting transcription factors. We showed that the two isoforms of the class A basic helix-loop-helix (bHLH) transcription factor ME1 (ME1a and ME1b), the murine homolog of the human HEB transcription factor, specifically repress p75LNGFR promoter activity. This repression can be released by coexpression of the HLH Id2 transcriptional regulator. In vitro analyses demonstrated that ME1a forms a stable complex with the p75LNGFR E box and likely competes with activating E-box-binding proteins. By using ME1a-overexpressing PC12 cells, we showed that the endogenous p75LNGFR gene is a target of ME1a repression. Together, these data demonstrate that the p75LNGFR E box and the interacting bHLH transcription factors are involved in the regulation of p75LNGFR gene expression. These results also show that class A bHLH transcription factors can repress and Id-like negative regulators can stimulate gene expression.

Characterization of the rat light neurofilament (NF-L) gene promoter and identification of NGF and cAMP responsive regions.

We have isolated a genomic DNA clone covering the coding and 14 kb upstream region of the rat light neurofilament (NF-L) gene and sequenced 2.3 kb of its promoter. DNase I hypersensitive sites have been mapped in PC12 cells. For functional analysis of the NF-L promoter, constructs carrying 38, 97, 407, 564, 650, 1,099, 1,660, 2,003 base pairs (bp) upstream region in front of the chloramphenicol acetyltransferase (CAT) reporter gene were tested for their capability to direct CAT expression after transient transfection into various cell lines. Similar CAT activities were recorded both in rat pheochromocytoma (PC12) and mouse neuroblastoma N115 cells and also in several nonneural cell lines (HeLa, C127, NIH 3T3). Regions responsible for the basic promoter activity were located between -407 and +75 bp from the transcription initiation site. The NGF-responsive element was located between -38 and +75 bp, and sequence -97 to -38 was found to contain a functional cAMP-responsive element. In PC12 cells in which nerve growth factor (NGF) induces neurite outgrowth and NF-L transcription, NF-L promoter-driven CAT expression was stimulated up to 12-fold within three days of NGF treatment, whereas epidermal growth factor (EGF) had no effect. Rat NF-L promoter contained Sp1, AP-2 and CGCCCCCGC elements. In PC12 cells, NGF transiently induced the binding of transcription factors to the deoxyoligonucleotide probes containing the binding sites of these elements. The role of these factors in NF-L gene transcriptional induction by NGF in PC12 cells is discussed.

Neurotrophins and their receptors in rat peripheral trigeminal system during maxillary nerve growth.

We examined the expression of the neurotrophins (NTFs) and their receptor mRNAs in the rat trigeminal ganglion and the first branchial arch before and at the time of maxillary nerve growth. The maxillary nerve appears first at embryonic day (E)10 and reaches the epithelium of the first branchial arch at E12, as revealed by anti-L1 immunohistochemistry. In situ hybridization demonstrates, that at E10-E11, neurotrophin-3 (NT-3) mRNA is expressed mainly in the mesenchyme, but neurotrophin-4 (NT-4) mRNA in the epithelium of the first branchial arch. NGF and brain-derived neurotrophic factor (BDNF) mRNAs start to be expressed in the distal part of the first brachial arch shortly before its innervation by the maxillary nerve. Trigeminal ganglia strongly express the mRNA of trkA at E10 and thereafter. The expression of mRNAs for low-affinity neurotrophin receptor (LANR), trkB, and trkC in trigeminal ganglia is weak at E10, but increases by E11-E12. NT-3, NT-4, and more prominently BDNF, induce neurite outgrowth from explant cultures of the E10 trigeminal ganglia but no neurites are induced by NGF, despite the expression of trkA. By E12, the neuritogenic potency of NGF also appears. The expression of NT-3 and NT-4 and their receptors in the trigeminal system prior to target field innervation suggests that these NTFs have also other functions than being the target-derived trophic factors.

Multiple promoters direct tissue-specific expression of the rat BDNF gene.

Brain-derived neurotrophic factor (BDNF) supports the survival of a specific set of neurons in the vertebrate nervous system. Here we show that the rat BDNF gene consists of four short 5' exons and one 3' exon encoding the mature BDNF protein. Eight different BDNF mRNAs with four different 5' ends and two alternative polyadenylation sites are transcribed from this gene. BDNF mRNAs containing exons I, II, and III are expressed predominantly in the brain, whereas exon IV transcripts predominate in the lung and heart. mRNAs containing exons I, II, and III increase markedly in the brain after kainic acid-induced seizures, whereas exon IV mRNA increases only slightly. Several transcription initiation sites were mapped upstream of the four 5' exons, and transfection of promoter-reporter gene constructs confirmed that these sequences act as promoters. Combined, the data demonstrate that alternative usage of four promoters within the BDNF gene and differential splicing control tissue-specific and seizure-induced expression of BDNF mRNA.