PCDHB14- and GABRB1-like nervous system developmental genes are altered during early neuronal differentiation of NCCIT cells treated with ethanol.

Ethanol (EtOH) exposure during embryonic development causes dysfunction of the central nervous system (CNS). Here, we examined the effects of chronic EtOH on gene expression during early stages of neuronal differentiation. Human embryonic carcinoma (NCCIT) cells were differentiated into neuronal precursors/lineages in the presence or absence of EtOH and folic acid. Gene expression profiling and pathway analysis demonstrated that EtOH deregulates many genes and pathways that are involved in early brain development. EtOH exposure downregulated several important genes, such as PCDHB14, GABRB1, CTNND2, NAV3, RALDH1, and OPN5, which are involved in CNS development, synapse assembly, synaptic transmission, and neurotransmitter receptor activity. GeneGo pathway analysis revealed that the deregulated genes mapped to disease pathways that were relevant to fetal alcohol spectrum disorders (FASD, such as neurotic disorders, epilepsy, and alcohol-related disorders). In conclusion, these findings suggest that the impairment of the neurological system or suboptimal synapse formation resulting from EtOH exposure could underlie the neurodevelopmental disorders in individuals with FASD.

Comparative analysis of virulence factors secreted by Bacillus anthracis Sterne at host body temperature.

AIMS: For the analysis of virulence factors produced and secreted by Bacillus anthracis vegetative cells during mammalian host infection, we evaluated the secretome of B. anthracis Sterne exposed to host-specific factors specifically to host body temperature. METHODS AND RESULTS: We employed a comparative proteomics-based approach to analyse the proteins secreted by B. anthracis Sterne under host-specific body temperature conditions. A total of 17 proteins encoded on a single chromosome and the pXO1 plasmid were identified by peptide mass fingerprinting. Multiple algorithms were used to predict the secretion mechanisms of the detected proteins in B. anthracis. CONCLUSIONS: Several putative virulence factors and known factors responsible for sporulation were differentially regulated, including CodY, pXO1-130 and BA1952, revealing insights into temperature cues in the B. anthracis secretome. SIGNIFICANCE AND IMPACT OF THE STUDY: This study identified temperature-regulated proteins. Further studies aimed at understanding the physical and functional roles of these proteins in infection and control by elevated temperatures will contribute to detection, diagnostics and prophylaxis.

Time-lapse, single cell based confocal imaging analysis of caspase activation and phosphatidylserine flipping during cellular apoptosis.

Apoptosis is an important phenomenon for investigating the efficacy of anti-cancer drug candidates. The conventional assays for cellular apoptosis, such as enzyme-linked immunosorbent assay, absorbance monitoring for the activity of caspase, and flow cytometric assay, have focused only on biochemical events. We investigated the staurosporine (STS)-induced apoptosis of the murine macrophage RAW-264.7 cell using a cell based bioimaging technique. Using time-lapse confocal microscopy, we monitored caspase-3 activation during apoptosis by imaging the translocation of green fluorescent protein from the cytosol to the nuclei. Five hours after 1 µM STS treatment, caspase-3 was observed to be activated and membrane blebbing was observed simultaneously. Also, the loss of phosphatidylserine (PS) asymmetry in the phospholipid bilayer of plasma membrane during early apoptosis was monitored by imaging annexin-V labeled with fluorescein isocyanate binding to the externalized PS at various concentrations of STS. Moreover, disintegration of the plasma membrane during late apoptosis was confirmed using a nuclear dye, propidium iodide. The single cell based bioimaging data agreed well with those of the biochemical assays for caspase activation and morphological observation for membrane integrity.

Studies on the interaction of REST4 with the cholinergic repressor element-1/neuron restrictive silencer element.

REST4 is a neuron specific truncated form of the transcription factor REST/NRSE derived by alternative splicing. REST4 was previously shown to block the repressor activity of REST/NRSF by forming a hetero-oligomer, Shimojo et al. [Mol. Cell. Biol. 19 (1999) 6788-6795]. A series of deletion mutants have now been used to characterize REST4 in terms of its structure and DNA binding. REST4 was found to be O-glycosylated between between residues 87 and 152. Binding of REST4 to the cholinergic RE-1/NRSE was approximately 1/10 to 1/20 as strong as full length REST/NRSF. DNA binding was enhanced by deletion of the first 86 residues and was found to require all four of the C-terminal zinc fingers as well as a twelve amino acid sequence preceding the first of these zinc fingers. REST4 can form homo-oligomers, however only the monomer was found to bind to DNA. REST4 binds to the 3' sequence of the cholinergic NRSE suggesting an anti-parallel orientation of the protein to the DNA.

Phosphorylation of the rat vesicular acetylcholine transporter.

Metabolic labeling of a mutant PC12 cell line, A123.7, expressing recombinant rat vesicular acetylcholine transporter (VAChT) with radiolabeled inorganic phosphate was used to demonstrate phosphorylation of the transporter on a serine residue. Mutational analysis was used to demonstrate that serine 480, which is located on the COOH-terminal cytoplasmic tail, is the sole phosphorylation site. Phosphorylation of serine 480 was attributable to the action of protein kinase C. Using a permanently dephosphorylated form of rat VAChT, S480A rVAChT, it was shown that this mutant displays the same kinetics for the transport of acetylcholine and the binding of the inhibitor vesamicol as does the wild type transporter. However, sucrose gradient density centrifugation showed that, unlike wild type VAChT, the S480A mutant did not localize to synaptic vesicles. These results suggest that phosphorylation of serine 480 of VAChT is involved in the trafficking of this transporter.

Expression patterns of mouse repressor element-1 silencing transcription factor 4 (REST4) and its possible function in neuroblastoma.

The expression pattern of the repressor element-1 silencing transcription factor (REST) also known as the neuron-restrictive silencer factor (NRSF) and its truncated forms have been analyzed in the neuroblastoma cell lines, NS20Y and NIE115 and in NIH3T3 cells. The neuroblastoma cell lines express transcripts of REST/NRSF and its neuron-specific truncated form REST4; with REST4 being the major transcript. NIH3T3 cells express predominantly REST/NRSF, with no detectable REST4. The cellular localization of REST4, determined using a REST4-GFP fusion protein, was shown to be nuclear. Mutational analysis implicates the zinc finger domains as the nuclear-targeting signal. Analysis of reporter-gene activities in the NS20Y cell line showed that the presence of four RE-1/NRSE sequences did not affect promoter activity. However, coexpression of exogenous REST4 produces a small increase in promoter activity of the reporter plasmid, whereas expression of exogenous REST/NRSF leads to repression. In the NIH3T3 cell line, the RE-1/NRSE sequence leads to repression of reporter-gene activity, whereas introduction of exogenous REST4 leads to de-repression. These data indicate that REST4 does not act as a transcriptional repressor. However, they support a mechanism where REST4 can block the repressor activity of REST/NRSF.

Mutational analysis of aspartate residues in the transmembrane regions and cytoplasmic loops of rat vesicular acetylcholine transporter.

The vesicular acetylcholine transporter (VAChT) is responsible for the transport of the neurotransmitter acetylcholine (ACh) into synaptic vesicles using an electrochemical gradient to drive transport. Rat VAChT has a number of aspartate residues within its predicted transmembrane domains (TM) and cytoplasmic loops, which may play important structural or functional roles in acetylcholine transport. In order to identify functional charged residues, site-directed mutagenesis of rVAChT was undertaken. No effect on ACh transport was observed when any of the five aspartate residues in the cytoplasmic loop were converted to asparagine. Similarly, changing Asp-46 (D46N) in TM1 or Asp-255 (D255N) in TM6 had no effect on ACh transport or vesamicol binding. However, replacement of Asp-398 in TM10 with Asn completely eliminated both ACh transport and vesamicol binding. The conservative mutant D398E retained transport activity, but not vesamicol binding, suggesting this residue is critical for transport. Mutation of Asp-193 in TM4 did not affect ACh transport activity; however, vesamicol binding was dramatically reduced. With mutant D425N of TM11 transport activity for ACh was completely blocked, without an effect on vesamicol binding. Activity was not restored in the conservative mutant D425E, suggesting the side chain as well as the negative charge of Asp-425 is important for substrate binding. These mutants, as well as mutant D193N, clearly dissociated ACh binding and transport from vesamicol binding. These data suggest that Asp-398 in TM10 and Asp-425 in TM11 are important for ACh binding and transport, while Asp-193 and Asp-398 in TM4 and TM10, respectively, are involved in vesamicol binding.

Distribution of genotypes in the 5' untranslated region of hepatitis C virus in Korea.

Hepatitis C virus (HCV) is an important human pathogen that can cause acute and chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Recently, partial and entire sequence data from HCV isolates have been reported, suggesting various genotypes of HCV. The genotype may be correlated with the progression of hepatitis and maybe a prognostic marker of treatment. Thus, the availability of an assay for typing HCV RNA is important. This study developed a convenient method for genotyping HCV into six groups by PCR-RFLP with four restriction endonucleases (BstUI, HaeIII, NciI, RsaI) in the 5' untranslated region (UTR) of HCV. The HCV genotypes from 169 patients with HCV infections in Korea were analysed. Two genotypes, type 1b and type 2a, accounted for 47.3% and 42.6% of HCV infections, respectively.

Expression of DAZ (deleted in azoospermia), DAZL1 (DAZ-like) and protamine-2 in testis and its application for diagnosis of spermatogenesis in non-obstructive azoospermia.

Spermatogenesis is regulated by hormones, local regulatory factors in the testes and specific gene expression of spermatogenic cells in humans. In this study, we have detected the expression of the deleted in azoospermia (DAZ), the DAZ-like autosome (DAZL1), and the protamine-2 genes in spermatogenic cells. Spermatogenesis in 38 male infertility patients was evaluated by the semen analysis and histological examination. Patients were diagnosed as Sertoli cell-only syndrome (n = 20), maturation arrest (n = 6), hypospermatogenesis (n = 6), and obstructive azoospermic patients with normal spermatogenesis (n = 6). After microscopic observation of the wet preparation of the testis tissues, seminiferous tubule contents were used for reverse transcription-polymerase chain reaction (RT-PCR) analysis of DAZ, DAZL1 and protamine-2. In cases with Sertoli-cell only syndrome, we found spermatogenic cells in 30% of patients (6/20) by the wet preparation method. There was no difference between the histology and the wet preparation results in maturation arrest and obstructive azoospermia; however, in one case of hypospermatogenesis, spermatozoa were not detectable by the wet preparation method. Using in-situ hybridization with DAZ and protamine-2 ribonuclear probes, we confirmed spermatogenic cell-specific expression of DAZ (spermatogonia/early spermatocyte) and protamine-2 (spermatid/spermatozoon). DAZ and protamine-2 expression can therefore be considered spermatogenic cell markers and could be useful in molecular diagnosis of spermatogenesis. In 13 patients with spermatozoa under the wet preparation, the expression of DAZ, DAZL1 and protamine-2 was detected in all the preparations. In one wet preparation showing only spermatogonia/spermatocyte, only DAZ and DAZL1 RNA were detected. In 14 wet preparations showing no spermatogenic cells, DAZ, DAZL1 and protamine-2 were not detected except in one preparation where DAZL1 expression was detected. In 10 wet preparations representing spermatogonia/spermatocyte to spermatids, but showing no spermaozoa, DAZ and DAZL1 were detected in eight and nine preparations respectively, and protamine-2 was detected in six preparations. These results of gene expression were similar to the wet preparation results. RT-PCR for DAZ, DAZL1 and protamine-2 was informative for the existence of germ cells, germ cell physiology and differentiation. From these results, we suggest that the analysis of DAZ, DAZL1 and protamine-2 expression by RT-PCR and wet preparation might offer a better method for finding the spermatogenic cells compared to the histological method.

Identification of the active-site arginine in rat neutral endopeptidase 24.11 (enkephalinase) as arginine 102 and analysis of a glutamine 102 mutant.

Neutral endopeptidase 24.11 contains an active-site arginine residue involved in binding the free carboxylate of substrate peptides and inhibitors. This arginine reacts rapidly with [14C]phenylglyoxal, and its reaction is selectively blocked by the presence of either the substrate Met5-enkephalin, the competitive inhibitor phenylalanylalanine, or the transition state analog phosphoramidon. The phenylglyoxal-modified peptide was isolated by a procedure involving limited digestion by trypsin, separation of the tryptic peptides by high pressure liquid chromatography (HPLC), further digestion of the modified peptide by pepsin, and a final purification by HPLC. By this procedure arginine 102 was identified as the active-site arginine. Verification of this finding came from the use of site-directed mutagenesis in which this arginine was replaced by glutamine. Both the mutant and wild-type enzyme reacted equally well with an amide containing substrate, glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide. However, reaction of the mutant enzyme with a substrate containing a free COOH-terminal carboxylate, 5-dimethylaminonaphthalene-1-sulfonyl-D-Ala-Gly-(NO2)Phe-Gly, was barely detectable with the mutant enzyme. Similarly the mutant enzyme showed a loss of selectivity in inhibition by D-Ala2-Met5-enkephalin compared to the corresponding amide but exhibited no difference in the maximal velocity for hydrolysis of D-Ala2-Met5-enkephalin and its amide.

A photoreversible phytochrome affinity column chromatography for putative phytochrome receptor studies.

Using a photoreversible phytochrome affinity column, we have isolated a small RNA molecule that binds to the affinity column. The RNA was identified by a A260/A280 ratio of 2.0, hypochromicity, orcinol, ribonuclease A and DNase tests. Agarose gel electrophoresis and circular dichroic spectral characteristics also indicate it to be a small RNA molecule. The successful preparation of a photoreversible phytochrome affinity column, the possible usage of the column and the significance of the RNA binding have been discussed.

A photoreversible conformational change in 124 kDa Avena phytochrome.

Tryptophan (Trp) fluorescence quenching of phytochrome has been studied using anionic, cationic and neutral quenchers, I-, Cs+ and acrylamide, respectively, in an effort to understand the molecular differences between the Pr and Pfr forms. The data have been analyzed using both Stern-Volmer and modified Stern-Volmer kinetic treatments. The anionic quencher, I-, was proven to be an ineffective quencher with Stern-Volmer constants, Ksv, of 0.60 and 0.63 M-1, respectively, for the Pr and Pfr forms of phytochrome. The cationic quencher, Cs+, showed about a 2-fold difference in the Ksv of Pr and Pfr, indicating a significant change in the fluorescent Trp environments during the Pr to Pfr phototransformation. However, only 25-37% of the fluorescent Trp residues were accessible to the cationic quencher. Most of the fluorescent Trp residues were accessible to acrylamide, but the quenching by acrylamide was indistinguishable for the Pr and Pfr forms. An additional quenching by acrylamide after a saturated quenching with Cs+ showed more than 40% increase in the Ksv of Pfr over Pr. These observations, along with the finding of two distinct components in the Trp fluorescence lifetime, indicate the existence of Trp residues in at least two different sets of environments in the phytochrome protein. The two components of the fluorescence had lifetimes of 1.1 ns (major) and 4.7 ns (minor) for Pr and 0.9 ns (major) and 4.6 ns (minor) for Pfr. Fluorescence quenching was found to be both static and dynamic as the Stern-Volmer constants for the steady-state fluorescence quenching were higher than for the dynamic fluorescence quenching. Based on the quenching results, in combination with the location of Trp residues in the primary structure, we conclude that the Pr to Pfr phototransformation involves a significant conformation change in the phytochrome molecule, preferentially in the 74 kDa chromophore-bearing domain.

A photoreversible circular dichroism spectral change in oat phytochrome is suppressed by a monoclonal antibody that binds near its N-terminus and by chromophore modification.

Accompanying the phototransformation of native 124-kilodalton (kDa) oat phytochrome from red-absorbing form (Pr) to far-red-absorbing form (Pfr), there is a photoreversible change in circular dichroism (CD) in the far-UV region indicative of a 3% increase in alpha-helical folding of apoprotein. To elucidate the conformational change involved in the phytochrome phototransformation, several monoclonal antibodies have been used as epitope-specific probes. Monoclonal antibody oat-25 suppressed the photoreversible CD spectral change using phytochrome with an A666/A280 as Pr of 1.13. Monoclonal antibodies oat-22, oat-13, and oat-31 did not significantly affect the CD spectral change of phytochrome. Oat-25 requires an epitope near the N-terminus of phytochrome. Oat-22, oat-13, and oat-31 recognize epitopes on the N-terminus, chromophore-containing half of phytochrome, albeit further removed from the N-terminus than that recognized by oat-25. Interestingly, oat-13 and oat-31 did, however, induce a time-dependent decrease in the far-UV CD, apparently due to aggregation of phytochrome (both Pr and Pfr forms). Monoclonal antibodies oat-26 and oat-28, which recognize epitopes on the C-terminus half of phytochrome, also did not suppress the photoreversible CD change, although oat-26 and oat-28 slightly inhibited it. The photoreversible CD spectral change can also be inhibited by sodium borohydride, which bleaches the chromophore by reducing it, and by tetranitromethane, which oxidizes the chromophore of phytochrome. Although explanations of these results based on indirect interactions between the chromophore and the N-terminus segment are possible, we propose that an additional alpha-helical folding of the Pfr form of the phytochrome may result from a photoreversible interaction between the Pfr form of the chromophore and the N-terminus segment.

Purification and spectroscopic properties of 124-kDa oat phytochrome.

A simplified procedure for the isolation and purification of 124-kDa phytochrome from etiolated Avena seedlings has been developed using the method of ammonium sulfate back-extraction. After hydroxyapatite chromatography of seedling tissue extracts, the pooled phytochrome was subjected to ammonium sulfate back-extraction instead of the usual application to an Affi-Gel Blue column. The resulting phytochrome had specific absorbance ratios (SAR = A666/A280) ranging from 0.85 to 0.95. Subsequent Bio-Gel filtration chromatography yielded highly pure 124-kDa phytochrome with SAR values ranging from 0.99 to 1.13. The absorption maxima of 124-kDa phytochrome were at 280, 379, and 666 nm for the red absorbing form of phytochrome (Pr) and at 280, 400 and 730 nm for the far-red absorbing form (Pfr). The A730/A673 ratio in Pfr was found to be 1.5 to 1.6. The mole fraction of Pfr under red light photoequilibrium was 0.88. No dark reversion was detected within 5 h at 3 degrees C. A photoreversible far-uv-circular dichroism was observable with all phytochrome preparations examined. Fluorescence and phosphorescence lifetimes were measured to further characterize the differences between the phytochromes prepared under different conditions. The Trp fluorescence and phosphorescence lifetimes of Pr and Pfr with the chromophore "X", probably polyphenolic in nature, were significantly shorter than those of phytochrome without the contaminant X. The short lifetime of the fluorescence of the Pr chromophore is attributable to X in the former.