An open-type microdevice to improve the quality of fluorescence labeling for axonal transport analysis in neurons.

Abnormal axonal transport of vesicles as well as organelles in a particular set of neurons is implicated in the pathogenesis of many neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease. Although various types of microfluidic multicompartmental devices with closed microchannels have been recently developed and widely used for axonal transport analysis, most of the existing devices are troublesome and time-consuming to handle, such as culture maintenances, sample collections, and immunocytochemistry. In this study, we overcome such inherent shortcomings by developing a novel open-type device that enables easy cell maintenance and sample collections. In our device, microgrooves instead of microchannels were directly fabricated on a glass substrate, thereby making possible a high-resolution optical observation. Compared with the conventional closed-type devices, our newly designed device allowed us to efficiently and precisely label the axonal acidic vesicles by fluorescent dyes, facilitating a high-throughput analysis of axonal vesicular transport. The present novel device, as a user-friendly and powerful tool, can be implemented in molecular and cellular pathogenesis studies on neurological diseases.

A gene encoding a putative GTPase regulator is mutated in familial amyotrophic lateral sclerosis 2.

Amyotrophic lateral sclerosis 2 (ALS2) is an autosomal recessive form of juvenile ALS and has been mapped to human chromosome 2q33. Here we report the identification of two independent deletion mutations linked to ALS2 in the coding exons of the new gene ALS2. These deletion mutations result in frameshifts that generate premature stop codons. ALS2 is expressed in various tissues and cells, including neurons throughout the brain and spinal cord, and encodes a protein containing multiple domains that have homology to RanGEF as well as RhoGEF. Deletion mutations are predicted to cause a loss of protein function, providing strong evidence that ALS2 is the causative gene underlying this form of ALS.

Conversion of ammonia to dinitrogen in wastewater by Nitrosomonas europaea.

Because Nitrosomonas europaea contains ammonia-oxidizing enzyme, nitrite reductase, and nitrous oxide reductase, the conversion of ammonia to dinitrogen was tried with different reaction conditions. In aerobic reaction conditions, ammonium was converted to nitrite (NO2-), while under oxygen-limiting or oxygen-free conditions, NO2(-)-N formed from ammonia oxidation by N. europaea was reduced to N2O and dinitrogen with 22% conversion. During denitrification, optimal pH for the production of N2O and dinitrogen was found to be 7.0-8.0. Dinitrogen was not produced in acidic pH <7.0. A low partial oxygen pressure as well as oxygen-free conditions are favorable for high production of dinitrogen.

Cloning and characterization of three novel genes, ALS2CR1, ALS2CR2, and ALS2CR3, in the juvenile amyotrophic lateral sclerosis (ALS2) critical region at chromosome 2q33-q34: candidate genes for ALS2.

Amyotrophic lateral sclerosis is a progressive neurodegenerative disease that manifests as selective upper and lower motor neuron degeneration. The autosomal recessive form of juvenile amyotrophic lateral sclerosis (ALS2) has previously been mapped to the 1.7-cM interval flanked by D2S116 and D2S2237 on human chromosome 2q33-q34. We identified three novel full-length transcripts encoded by three distinct genes (HGMW-approved symbols ALS2CR1, ALS2CR2, and ALS2CR3) within the ALS2 critical region. The intron-exon organizations of these genes as well as those of CFLAR, CASP10, and CASP8, which were previously mapped to this region, were defined. These genes were evaluated for mutations in ALS2 patients, and no disease-associated sequence alterations in either exons or intron-exon boundaries were observed. Sequence analysis of overlapping RT-PCR products covering the whole coding sequence for each transcript revealed no aberrant mRNA sequences. These data strongly indicate that ALS2CR1, ALS2CR2, ALS2CR3, CFLAR, CASP10, and CASP8 are not causative genes for ALS2.

Identification and characterization of the miniature pig Huntington's disease gene homolog: evidence for conservation and polymorphism in the CAG triplet repeat.

Huntington's disease (HD) is associated with a significant expansion of a CAG trinucleotide repeat, which results in a lengthened polyglutamine tract in the single gene product, huntingtin, on human 4p16.3. We isolated cDNA clones that encompassed the entire coding sequence of the miniature pig HD gene (Sus HD) from two porcine testis cDNA libraries. The cDNA contig revealed a 12,749-nucleotide transcript coding for a 345-kDa protein (3139 amino acid residues), which exhibited 96% peptide sequence homology to human huntingtin. Northern blot analysis revealed that the Sus HD gene was ubiquitously expressed as two large transcripts of approximately 11 and 13 kb in size in all the tested tissues, much like the human HD gene. The CAG trinucleotide repeat was found to be interrupted by CAA triplets and to encode 17 or 18 consecutive glutamine residues. In our laboratory stock of miniature pig, three allotypes in the triplet repeat sequence were found. Thus, the Sus HD gene closely resembles its human counterpart in terms of sequence and expression pattern. In particular, human-miniature pig similarities in the normal length of the CAG triplet repeat as well as its repeat-number polymorphism may indicate that miniature pig would provide a good animal model for Huntington's disease.

The RS447 human megasatellite tandem repetitive sequence encodes a novel deubiquitinating enzyme with a functional promoter.

We have recently identified a tandem repetitive DNA sequence that we designated the RS447 megasatellite. In this study, we describe a functional novel deubiquitinating enzyme (USP17, 60 kDa) gene that is intronless and encoded by the RS447 repeating unit. Northern blot analysis in conjunction with 5' and 3' rapid amplification of cDNA ends confirmed the presence of poly(A)(+) containing RS447 RNA in normal cells. We also identified a functional promoter sequence as well as an open reading frame within every RS447 repeat. When USP17 was expressed in Escherichia coli, it exhibited deubiquitinating activity in vivo. An antibody against USP17 detected USP17 protein in human cells. Our results indicate that the RS447 repeating unit on this megasatellite repeat codes for and actively expresses a functional deubiquitinating enzyme. Although it is expressed ubiquitously in human tissues, USP17 exhibited a unique expression pattern in that its complementary strand is transcribed as an antisense transcript that may modulate the level of USP17 expression in the human brain.

Complicating risk factors for pyelonephritis after extracorporeal shock wave lithotripsy.

PURPOSE: The score to predict the risk of post-extracorporeal shock wave lithotripsy (ESWL) pyelonephritis was evaluated. The score was based on the multivariate analysis of risk factors available pre-operatively. Stone size, pyuria, bacteriuria, previous pyelonephritis and other adjunctive procedures had been selected and scored. METHODS: Three-hundred and forty-eight adult patients without active urinary infection undergoing ESWL therapy were studied. One of three regimens were selected by either doctor or patient: (i) no antimicrobial treatment; (ii) one dose of levofloxacin; or (iii) 1 week course of levofloxacin. Who and why selected it were described. Post-ESWL fever over 38 degrees C was defined as the unfavorable event. RESULTS/CONCLUSION: With increasing score, doctors recommend taking an antimicrobial. There were 11 bacteriuric patients and post-ESWL pyelonephritis developed in one of them. Bacteria within the stone and post-ESWL ureteral obstruction caused by the stone fragments were considered to be important in developing pyelonephritis. However, multiple factors were related with it. Although their decision was not based simply on the score, the score was confirmed to be useful in identifying the high-risk patients and, therefore, to implement cost-effective antimicrobial use.

Identification of two distinct transcripts for the neuronal apoptosis inhibitory protein gene.

The spinal muscular atrophies (SMA), characterized by motor neuron loss and progressive paralysis, are among the most common autosomal recessive disorders. Recently, two SMA candidate genes, NAIP (neuronal apoptosis inhibitory protein) and survival motor neuron (SMN), were reported and a 131-kb genomic sequence of 5q13.1 encompassing these two genes was determined. Based upon this genomic sequence, the original NAIP cDNA sequence published in 1995 was shown to contain foreign fragments. We therefore conducted an extensive cDNA cloning of NAIP from a human fetal brain library. Our studies confirmed that the cDNA sequence deduced from the 131-kb genomic sequence was the major transcript in the human fetal brain. In addition, a shorter and minor transcript was also newly identified. We thus designated the longer and shorter transcripts as NAIPl and NAIPs, respectively. The cDNA clones for NAIPl and NAIPs should facilitate the functional analysis of the NAIP gene and its association with neuronal apoptosis and SMA.

A yeast artificial chromosome-based physical map of the juvenile amyotrophic lateral sclerosis (ALS2) critical region on human chromosome 2q33-q34.

The autosomal recessive form of juvenile amyotrophic lateral sclerosis (ALS2; RFALS Type 3) has previously been mapped to the 8-cM interval flanked by D2S115 and D2S155 on human chromosome 2q33-q34. We have established a yeast artificial chromosome (YAC) contig spanning an approximately 8-Mb region of the ALS2 candidate region and mapped 52 transcribed DNA sequences including 13 known genes and 39 expressed sequenced tags within this YAC contig. The establishment of a YAC contig and transcript map that spans the region containing the ALS2 mutation is an essential step in the identification of the ALS2 gene.

The primary structure and genomic organization of five novel transcripts located close to the Huntington's disease gene on human chromosome 4p16.3.

Five distinct novel transcripts (RES4-22, -23, -24, -25 and -26) that mapped to the 1-Mb interval between D4S180 and D4S183 on human chromosome 4p16.3 close to the Huntington's disease (HD) gene were isolated, and the structure and exon/intron organization of each gene were thoroughly analyzed. The transcripts of the RES4-22, -23 and -24 genes each have several isoforms by alternative splicing and these have also been defined. Two transcripts, RES4-24 and RES4-25, reside in the same genomic region with opposite polarities and they also clearly overlap. Among these transcripts, RES4-26 was found to encode a novel zinc finger protein. The transcript map based upon our current level of analysis combined with data from previous studies reveals the gene-rich nature and the intricate organization of the genes in the HD locus.

Cell death attenuation by 'Usurpin', a mammalian DED-caspase homologue that precludes caspase-8 recruitment and activation by the CD-95 (Fas, APO-1) receptor complex.

Apoptotic cell suicide initiated by ligation of CD95 (Fas/APO-1) occurs through recruitment, oligomerization and autocatalytic activation of the cysteine protease, caspase-8 (MACH, FLICE, Mch5). An endogenous mammalian regulator of this process, named Usurpin, has been identified (aliases for Usurpin include CASH, Casper, CLARP, FLAME-1, FLIP, I-FLICE and MRIT). This protein is ubiquitously expressed and exists as at least three isoforms arising by alternative mRNA splicing. The Usurpin gene is comprised of 13 exons and is clustered within approximately 200 Kb with the caspase-8 and -10 genes on human chromosome 2q33-34. The Usurpin polypeptide has features in common with pro-caspase-8 and -10, including tandem 'death effector domains' on the N-terminus of a large subunit/small subunit caspase-like domain, but it lacks key residues that are necessary for caspase proteolytic activity, including the His and Cys which form the catalytic substrates diad, and residues that stabilize the P1 aspartic acid in substrates. Retro-mutation of these residues to functional caspase counterparts failed to restore proteolytic activity, indicating that other determinants also ensure the absence of catalytic potential. Usurpin heterodimerized with pro-caspase-8 in vitro and precluded pro-caspase-8 recruitment by the FADD/MORT1 adapter protein. Cell death induced by CD95 (Fas/APO-1) ligation was attenuated in cells transfected with Usurpin. In vivo, a Usurpin deficit was found in cardiac infarcts where TUNEL-positive myocytes and active caspase-3 expression were prominent following ischemia/reperfusion injury. In contrast, abundant Usurpin expression (and a caspase-3 deficit) occurred in surrounding unaffected cardiac tissue, suggesting reciprocal regulation of these pro- and anti-apoptotic molecules in vivo. Usurpin thus appears to be an endogenous modulator of apoptosis sensitivity in mammalian cells, including the susceptibility of cardiac myocytes to apoptotic death following ischemia/ reperfusion injury.

A novel tandem repeat sequence located on human chromosome 4p: isolation and characterization.

In an effort to analyze the genomic region of the distal half of human chromosome 4p, to where Huntington disease and other diseases have been mapped, we have isolated the cosmid clone (CRS447) that was likely to contain a region with specific repeat sequences. Clone CRS447 was subjected to detailed analysis, including chromosome mapping, restriction mapping, and DNA sequencing. Chromosome mapping by both a human-CHO hybrid cell panel and FISH revealed that CRS447 was predominantly located in the 4p15.1-15.3 region. CRS447 was shown to consist of tandem repeats of 4.7-kb units present on chromosome 4p. A single EcoRI unit was subcloned (pRS447), and the complete sequence was determined as 4752 nucleotides. When pRS447 was used as a probe, the number of copies of this repeat per haploid genome was estimated to be 50-70. Sequence analysis revealed that it contained two internal CA repeats and one putative ORF. Database search established that this sequence was unreported. However, two homologous STS markers were found in the database. We concluded that CRS447/pRS447 is a novel tandem repeat sequence that is mainly specific to human chromosome 4p.

Transcript map of the human chromosome 4p16.3 consisting of 627 cDNA clones derived from 1 Mb of the Huntington's disease locus.

Six hundred and twenty-seven cDNA clones from human brain cDNA libraries were characterized and integrated into a transcript map of the 1-Mb region on human chromosome 4p16.3 containing the Huntington's disease (HD) gene. Six hundred and seventy-two cDNA clones were obtained by a direct screening of the cDNA libraries, probing with pools of single copy microclones generated from the HD region specific yeast artificial chromosome (YAC)-DNA. So far, 93% of the obtained clones (627 cDNA clones) have been mapped onto the 1-Mb HD gene region by hybridization with HD region-specific cosmid, P1 and YAC clones. DNA sequence and expression analyses revealed that several cDNA clones might encode novel genes, some of which are situated within or close to the IT15, IT11, and alpha-adducin (ADD1) gene region, suggesting the presence of the overlapping genes in this region. This collection of cDNA clones will greatly facilitate the construction of the complete map of the transcripts in the HD region.

Isolation and characterization of 21 novel expressed DNA sequences from the distal region of human chromosome 4p.

We have established an approach to the isolation of expressed DNA sequences from a defined region of the human chromosome. The method relies on the direct screening of cDNA libraries using pooled single-copy microclones generated by a laser chromosome micro-dissection in conjunction with a single unique primer polymerase chain reaction (SUP-PCR) procedure. We applied this method to the distal region of human chromosome 4p (4p15-4pter), which contains the Huntington disease (HD) and the Wolf-Hirschhorn syndrome (WHS) loci. Twenty-one nonoverlapping and region-specific cDNA clones encoding novel genes were isolated in this manner. Ten of 21 clones were subregionally assigned to 4p16.1-4pter, and the remainder mapped to the region proximal to 4p16.1. Northern blot and reverse transcription followed by the PCR (RT-PCR) analysis revealed that 16 of these 21 clones detected transcripts in total RNA from human tissues. Our method is applicable to other chromosomal regions and is a powerful approach to the isolation of region-specific cDNA clones.

Isolation of expressed sequences encoded by the human Xq terminal portion using microclone probes generated by laser microdissection.

The genes that cause a variety of neurologic and neuromuscular disorders have been mapped to the distal region of Xq. In an effort to isolate genes from this area, a regional genomic library of the distal 30% of Xq was constructed from a single metaphase spread by means of laser microdissection and single unique primer-polymerase chain reaction. Using pooled probes of 1000 clones from the genomic library, human brain cDNA libraries were screened for expressed sequences encoded by this region. From the 250,000 cDNA clones screened so far, 10 nonoverlapping sequences that mapped back to the target portion were isolated. The complete nucleotide sequences of these cDNA clones have been determined. Analysis of the sequences indicates that none has significant similarity to previously characterized primate genes. One sequence mapping to Xq27.3-qter contained an open reading frame of 281 amino acids and was expressed in every tissue tested. This gene, as well as others isolated in this manner, may prove to be a candidate gene for heritable disorders mapping to this region.

[Laser chromosome microdissection and cloning of the genetic disease loci].

We have developed an argon ion laser chromosome microdissection technique in conjunction with a single unique primer polymerase chain reaction (SUP-PCR) to directly amplify microdissected chromosomes. The 22-mer primer used in PCR, although unique in sequence, randomly primed and amplified any target DNA. These methods were applied to both the terminal region of the human chromosome 4p (4p 16) and Xq (Xq26-q28), and two chromosome region-specific DNA libraries were constructed. The resulting libraries contained approximately 1000 nonoverlapping DNA sequences with an average size of 230-350 bp, at an average spacing of 10-65 Kbp along the chromosomes of origin. Our new method is a simple and general approach for constructing a chromosome region-specific DNA library from a single metaphase spread.

Generation of high-density DNA markers from yeast artificial chromosome DNA by single unique primer-polymerase chain reaction.

We have developed a method for the whole sequence amplification of yeast artificial chromosome (YAC) DNA excised from preparative pulsed-field gel electrophoresis using single unique primer-polymerase chain reaction procedures. We used seven contiguous YAC clones, which span 2 Mbp of the Huntington disease gene region on 4p16.3, to amplify the YAC DNAs. The average size of the amplified DNA was approximately 300 bp long, and 12 DNA markers located on the YAC clones positively hybridized with these amplified products, implying that the sequences of the YAC clones were comprehensively amplified by our procedures. These amplified YAC DNAs greatly facilitate the characterization of YAC clones, leading to the detailed analysis of the defined chromosomal region.