Multi-decadal survival of an Antarctic nematode, Plectus murrayi, in a -20°C stored moss sample.

It is not clear for how long Antarctic soil nematodes might tolerate freezing. Samples of the Antarctic moss, Bryum argenteum, were collected on 1 October 1983 at Langhovde, Soya coast, eastern Antarctica and were stored at -20°C. After 25.5 years of storage, living nematodes were recovered from the samples and were identified as Plectus murrayi by morphological examination and nucleotide sequencing of ribosomal RNA loci. The nematodes can grow and reproduce in a water agar plate with bacteria (mainly Pseudomonas sp.) cultured from the moss extract. They showed freezing tolerance at -20°C and -80°C and their survival rate after exposure to -20°C, but not -80°C, was increased if they were initially frozen slowly at a high sub-zero temperature. They also showed some ability to tolerate desiccation stress.

Refined human artificial chromosome vectors for gene therapy and animal transgenesis.

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis.

DNA sequence and comparative analysis of chimpanzee chromosome 22.

Human-chimpanzee comparative genome research is essential for narrowing down genetic changes involved in the acquisition of unique human features, such as highly developed cognitive functions, bipedalism or the use of complex language. Here, we report the high-quality DNA sequence of 33.3 megabases of chimpanzee chromosome 22. By comparing the whole sequence with the human counterpart, chromosome 21, we found that 1.44% of the chromosome consists of single-base substitutions in addition to nearly 68,000 insertions or deletions. These differences are sufficient to generate changes in most of the proteins. Indeed, 83% of the 231 coding sequences, including functionally important genes, show differences at the amino acid sequence level. Furthermore, we demonstrate different expansion of particular subfamilies of retrotransposons between the lineages, suggesting different impacts of retrotranspositions on human and chimpanzee evolution. The genomic changes after speciation and their biological consequences seem more complex than originally hypothesized.

Three-body nonmesonic weak decay of the (Lambda)12C hypernucleus.

We have measured the branching ratio of the three-body process in the nonmesonic weak decay of Lambda12C to be 0.29+/-0.13. This result was obtained by reproducing the nucleon and the nucleon pair yields introducing a measured final state interaction. At the same time, we have determined the absolute decay widths, Gamma(n) and Gamma(p), along with Gamma2N, whose relative ratio has been a long-standing puzzle. Including the three-body process, we have successfully reproduced the nucleon energy distribution, the coincidence two-nucleon angular correlation, and the momentum sum distribution simultaneously.

Binding of neuroligins to PSD-95.

PSD-95 is a component of postsynaptic densities in central synapses. It contains three PDZ domains that localize N-methyl-D-aspartate receptor subunit 2 (NMDA2 receptor) and K+ channels to synapses. In mouse forebrain, PSD-95 bound to the cytoplasmic COOH-termini of neuroligins, which are neuronal cell adhesion molecules that interact with beta-neurexins and form intercellular junctions. Neuroligins bind to the third PDZ domain of PSD-95, whereas NMDA2 receptors and K+ channels interact with the first and second PDZ domains. Thus different PDZ domains of PSD-95 are specialized for distinct functions. PSD-95 may recruit ion channels and neurotransmitter receptors to intercellular junctions formed between neurons by neuroligins and beta-neurexins.

Unexpected serum level of vancomycin after oral administration in a patient with severe colitis and renal insufficiency.

OBJECTIVE: To report a case in which the serum concentration of vancomycin (VCM) reached the supratherapeutic range following oral administration in a patient with severe pseudomembranous colitis and renal insufficiency. CASE SUMMARY: A 65-year-old, 70 kg weighing man with severe acute pancreatitis and acute renal failure was subjected to continuous hemodiafiltration (CHDF). CHDF could only be performed intermittently because of the unstable circulation dynamic of this patient. After admission, intravenous VCM therapy was initiated. Thereafter, oral VCM administration was begun (0.5 g every 6 h). Despite the discontinuation of intravenous VCM after the first 2 days of oral VCM, the serum VCM concentration increased gradually to 49.8 mg/l over a period of 2 weeks from the initiation of oral administration (34.4 mg/l). Based on pharmacokinetic analysis, the bioavailability of VCM was estimated to over 33%. Autopsy findings indicated broadly distributed necrosis on the lamina propria of the mucosa throughout all parts of the intestine below the duodenum. DISCUSSION: This case indicates necessity of the careful monitoring after oral high-dose VCM administration in a patient with a broadly distributed necrosis and renal insufficiency. CONCLUSIONS: TDM should be considered according to renal function, the severity of enteritis and the total dosage of oral VCM administration.

Characterization of the antimicrobial peptide attacin loci from Glossina morsitans.

The antimicrobial peptide Attacin is an immune effector molecule that can inhibit the growth of gram-negative bacteria. In Glossina morsitans morsitans, which serves as the sole vectors of African trypanosomes, Attacins also play a role in trypanosome resistance, and in maintaining parasite numbers at homeostatic levels in infected individuals. We characterized the attacin encoding loci from a Bacterial Artificial Chromosome (BAC) library. The attacin genes are organized into three clusters. Cluster 1 contains two attacin (attA) genes located in head-to-head orientation, cluster 2 contains two closely related genes (attA and attB) located in a similar transcriptional orientation, and cluster 3 contains a single attacin gene (attD). Coding and transcription regulatory sequences of attA and attB are nearly identical, but differ significantly from attD. Putative AttA and AttB have signal peptide sequences, but lack the pro domain typically present in insect Attacins. Putative AttD lacks both domains. Analysis of attacin cDNA sequences shows polymorphisms that could arise either from allelic variations or from the presence of additional attacin genomic loci. Real time-PCR analysis reveals that attA and attB expression is induced in the fat body of flies per os challenged with Escherichia coli and parasitized with trypanosomes. In the midgut, expression of these attacins is similarly induced following microbial challenge, but reduced in response to parasite infections. Transcription of AttD is significantly less relative to the other two genes, and is preferentially induced in the fat body of parasitized flies. These results indicate that the different attacin genes may be differentially regulated.

Preparation of ortho-para ratio controlled D2 gas for muon-catalyzed fusion.

A negative muon in hydrogen targets, e.g., D2 or D-T mixture, can catalyze nuclear fusions following a series of atomic processes involving muonic hydrogen molecular formation (muon-catalyzed fusion, muCF). The ortho-para state of D2 is a crucial parameter not only for enhancing the fusion rate but also to precisely investigate various muonic atom processes. We have developed a system for controlling and measuring the ortho-para ratio of D2 gas for muCF experiments. We successfully collected para-enriched D2 without using liquid-hydrogen coolant. Ortho-enriched D2 was also obtained by using a catalytic conversion method with a mixture of chromium oxide and alumina. The ortho-para ratio of D2 gas was measured with a compact Raman spectroscopy system. We produced large volume (5-30 l at STP), high-purity (less than ppm high-Z contaminant) D2 targets with a wide range of ortho-para ratios (ortho 20%-99%). By using the ortho-para controlled D2 in muCF experiments, we observed the dependence of muCF phenomena on the ortho-para ratio.

FLT3 is fused to ETV6 in a myeloproliferative disorder with hypereosinophilia and a t(12;13)(p13;q12) translocation.

The FMS-like tyrosine kinase 3 (FLT3) gene, belonging to the receptor tyrosine kinase (TK) subclass III family, plays an important role in normal hematopoiesis and is one of the most frequently mutated genes in hematologic malignancies as well as an attractive target for directed inhibition. Activating mutations of this gene, including internal tandem duplication in the juxtamembrane (JM) domain and point mutations in the TK domain, are found in approximately one-third of patients with acute myeloid leukemia and in a smaller subset of patients with acute lymphoblastic leukemia. We report here that FLT3 may contribute to leukemogenesis in a patient with myeloproliferative disorder and a t(12;13)(p13;q12) translocation through generating a fusion gene with the ETS variant gene 6 (ETV6) gene. ETV6 has been reported to fuse to various partner genes, including TK and transcription factors. Both ETV6/FLT3 and reciprocal FLT3/ETV6 transcripts were detected in the patient mRNA by reverse transcriptase-polymerase chain reaction. At the protein level, however, only ETV6/FLT3 products were expressed. Among them, one retains the helix-loop-helix (HLH) oligomerization domain of ETV6 and the JM as well as TK domain of FLT3. FLT3 receptor in leukemic cells might be inappropriately activated through dimerization by HLH domain of ETV6, which consequently interfered with proliferation and differentiation of hematopoietic cells.

Identification and characterization of a 500-kb homozygously deleted region at 1p36.2-p36.3 in a neuroblastoma cell line.

Loss of heterozygosity of the distal region of chromosome 1p where tumor suppressor gene(s) might harbor is frequently observed in many human cancers including neuroblastoma (NBL) with MYCN amplification and poor prognosis. We have identified for the first time a homozygously deleted region at the marker D1S244 within the smallest region of overlap at 1p36.2-p36.3 in two NBL cell lines, NB-1 and NB-C201 (MASS-NB-SCH1), although our genotyping has suggested the possibility that both lines are derived from the same origin. The 800-kb PAC contig covering the entire region of homozygous deletion was made and partially sequenced (about 60%). The estimated length of the deleted region was 500 kb. We have, thus far, identified six genes within the region which include three known genes (DFF45, PGD, and CORT) as well as three other genes which have been reported during processing our present project for the last 3(1/2) years (HDNB1/UFD2, KIAA0591F/KIF1B-beta, and PEX14). They include the genes related to apoptosis, glucose metabolism, ubiquitin-proteasome pathway, a neuronal microtubule-associated motor molecule and biogenesis of peroxisome. At least three genes (HDNB1/UFD2, KIAA0591F/KIF1B-beta, and PEX14) were differentially expressed at high levels in favorable and at low levels in unfavorable subsets of primary neuroblastoma. Since the 1p distal region is reported to be imprinted, those differentially expressed genes could be the new members of the candidate NBL suppressor, although RT-PCR-SSCP analysis has demonstrated infrequent mutation of the genes so far identified. Full-sequencing and gene prediction for the region of homozygous deletion would elucidate more detailed structure of this region and might lead to discovery of additional candidate genes. Oncogene (2000) 19, 4302 - 4307

A clonal study of hematopoiesis using the M27 beta probe: aberrant band patterns caused by incomplete digestion of a methyl-sensitive enzyme in the the inactive X-chromosome.

The M27 beta probe has been used to determine the clonality of human tumors, based upon X-chromosome inactivation. However, it occasionally gives rise to aberrant results. In this study, the M27 beta probe was used for clonal analysis in Japanese women with clonal stem cell disorders and in those with normal hematopoiesis. Restriction digestion with PstI indicated heterozygosity for the DXS255 locus in 41 out of 50 individuals (82%). Further digestion with HpaII in heterozygous women led to four distinct band patterns: I, both fragments were partially digested; II, either one of the two fragments was completely digested; III, a three-band pattern; and IV, neither fragment was digested. Of 21 hematologically normal females, 17 (81%) and four (19%) had patterns I and III, respectively. In some subjects with pattern I, imbalanced HpaII digestion in the two alleles was seen. Fifteen (65%) of the 23 patients with clonal stem cell disorders had pattern II, while the remainder (35%) had pattern IV. The normal tissues of three acute myeloid leukemia patients with pattern IV all revealed pattern I. It is possible that the aberrant band patterns could be caused by incomplete HpaII digestion in inactive X-chromosomes. In this study, we propose a hypothesis whereby, in normal tissues, aberrant cells, the DXS255 locus of which is not digested with HpaII despite their inactive status, would be mixed with cells demonstrating the usual methylation pattern. In normal tissues, complex of proportion of aberrant cells and skewed Lyonization could produce a variety of band patterns. If a cell with the usual methylation pattern proliferated monoclonally, pattern II would be seen: whereas if an aberrant cell proliferated, pattern IV would be demonstrated.

Incidence and characteristics of clonal hematopoiesis in remission of acute myeloid leukemia in relation to morphological dysplasia.

We studied 34 patients in remission of acute myeloid leukemia (AML) by performing clonal analysis of peripheral blood polymorphonuclear (PMN) cells and mononuclear (MN) cells, using X-linked DNA polymorphisms, in conjunction with the assessment of morphological myelodysplastic changes, performed by a scoring method. Nine patients demonstrated a non-random or skewed X-chromosome inactivation pattern in PMN cells. Three of these nine patients had an apparently random pattern in MN cells (group A), whereas the remaining six patients demonstrated no difference between the inactivation patterns of PMN and MN cells (group B). The PMN cells of the other 25 patients showed a random X-chromosome inactivation pattern, and the patterns of the PMN cells did not differ from those of the MN cells (group C). The scores for myelodysplasia were high (> or = 4) in all three patients in group A, intermediate (2-3) in two patients and low (score < 2) in four patients in group B, and intermediate in five patients and low in 20 patients in group C. The duration of remission in patients with a myelodysplasia score of > or = 2 was significantly shorter than that of patients with a score of < 2 (P < 0.01). We conclude that clonal remission actually occurs with myelodysplastic features in some patients with AML (around 10%, group A). It is possible that this clonal analysis may not be sensitive enough to detect the preleukemic clone with myelodysplastic features when this clone constitutes only a minor population of remission hematopoiesis. To further elucidate the biology of such preleukemic clones it is essential to develop more sensitive molecular methods for the detection of genetic abnormalities specific to preleukemic hematopoiesis.

Arrangement of high-energy neutron irradiation field and shielding experiment using 4 m concrete at KENS.

An irradiation field of high-energy neutrons produced in the forward direction from a thick tungsten target bombarded by 500 MeV protons was arranged at the KENS spallation neutron source facility. In this facility, shielding experiment was performed with an ordinary concrete shield of 4 m thickness assembled in the irradiation room, 2.5 m downstream from the target centre. Activation detectors of bismuth, aluminium, indium and gold were inserted into eight slots inside the shield and attenuations of neutron reaction rates were obtained by measurements of gamma-rays from the activation detectors. A MARS14 Monte Carlo simulation was also performed down to thermal energy, and comparisons between the calculations and measurements show agreements within a factor of 3. This neutron field is useful for studies of shielding, activation and radiation damage of materials for high-energy neutrons, and experimental data are useful to check the accuracies of the transmission and activation calculation codes.

Isolation and characterization of a novel zinc finger gene, ZNF219, and mapping to the human chromosome 14q11 region.

We have isolated and characterized a novel zinc finger gene by screening a human testis cDNA library. The isolated cDNA, termed ZNF219, contains an open reading frame of 2169 nucleotides encoding 723 amino acids. Sequence analysis revealed 9 sets of Kruppel-related zinc finger structures and proline-rich regions in several parts. ZNF219 exhibited ubiquitous expression in all fetal and adult tissues examined. The transcript size was 5.5 kb in adult tissues, while the main transcript size in the embryo stage was 3.5 kb. The transcript size is developmentally regulated. When the plasmid cloned with green fluorescent protein (GFP)-tagged ZNF219 was expressed in HeLa and COS7 cells, strong fluorescence intensities were observed only in the nucleus of both cells by fluorescence confocal microscopy. These data suggest that ZNF219 may be related to the regulation of transcription and developmental regulation. Genomic structure analysis mapped ZNF219 to chromosome 14q11 between markers D14S72 and D14S990, because a sequence tagged site mapped to the locus was found in the intron region of the ZNF219 gene.

Isolation and analysis of a novel gene, HXC-26, adjacent to the rab GDP dissociation inhibitor gene located at human chromosome Xq28 region.

We screened potential promoter regions from NotI-linking cosmid clones mapped on human chromosome Xq28 region with our constructed trapping vector and isolated six fragments containing transcription activity. Using one of the obtained fragments as a probe, a novel gene was isolated by screening a human skeletal muscle cDNA library. The isolated cDNA, termed HXC-26, contained an open reading frame of 975 nucleotides encoding 325 amino acids (38,848 Da). The HXC-26 gene was composed of 13 exons that span approximately 8 kb. Several potential GC boxes were found in the putative promoter region, but no typical TATA box. The HXC-26 gene associated with a CpG island was located adjacent to the rab GDP dissociation inhibitor (GDI) gene.

Mode of activation of the GC box/Sp1-dependent promoter of the human NADH-cytochrome b5 reductase-encoding gene.

Many eukaryote promoters, particularly those for so-called housekeeping genes, have multiple GC boxes which are the binding sites of the transcription factor, Sp1. It has been proposed that Sp1 binds to the multiple GC boxes, and then the GC box-bound Sp1 interact with each other to synergistically stimulate transcription. Here, we describe a Sp1-dependent promoter which does not necessarily fit the synergistic activation mechanism. The promoter of the human NADH-cytochrome b5 reductase-encoding gene (CYTB5R) possesses five potential GC box sequences. Deletion and mutagenesis studies coupled with CAT assays revealed that three out of five GC box-like sequences were functionally active and activated transcription additively (rather than synergistically). Our results suggested that Sp1-mediated activation of transcription occurs in a promoter context-dependent manner.

Cloning and chromosomal localization of a paralog and a mouse homolog of the human transaldolase gene.

A sequence homologous to the transaldolase gene (TALDO) was identified in a polymorphic cosmid DNA mapped on human chromosome 11p15 by exon trapping with pSPL3. Analysis of lambda clones contiguous to the cosmid clone showed that the related gene (TALDOR) consists of 8 exons spanning approximately 19kb from the translation start site to the polyadenylation signal. The exon sequence of TALDOR was almost identical with that of TALDO localized on 1p33-34. 1, but its exons corresponding to exons 4 and 5 of TALDO were found to be split by 4 introns in TALDOR. To examine the evolutionary conservation of two genes for transaldolase, we have isolated the cDNA for its mouse homolog and determined the nucleotide sequence covering the complete coding region. Fluorescence in situ hybridization using the cDNA as a probe showed that the mouse transaldolase gene (Taldo) is localized on chromosome 7 F3-F4 as a single copy gene. This chromosomal region is known to be syntenic to human chromosome 11p15 rather than to 1p33-p34.1, suggesting that TALDOR is the ancestral form. The existence of TALDOR implies a duplication of the mammalian transaldolase gene after divergence of rodent and primate.

[Clinical study on azithromycin in 10% fine granules and 100mg capsules in the field of pediatrics].

Azithromycin (AZM), a new oral macrolide antibiotic, in 10% fine granules or 100 mg capsules was given to pediatric patients to treat various infections. The following results were obtained in our studies of AZM for its antibacterial activities against clinical isolates, its pharmacokinetics, its efficacy, and its safety. 1. MICs of AZM, erythromycin (EM) and clarithromycin (CAM) were determined against a total of 57 strains all at 10(6) cfu/ml. Among Gram-positive cocci, MICs of AZM ranged from 0.78 to > 100 micrograms/ml against Staphylococcus aureus (20 strains), from 0.05 to 0.1 microgram/ml against Streptococcus pyogenes (11 strains), and from 0.0125 to 3.13 micrograms/ml against Streptococcus pneumoniae (10 strains). These MICs were similar to those of the other macrolides. Among Gram-negative bacilli, MICs of AZM were 0.05 micrograms/ml against Moraxella subgenus Branhamella catarrhalis (1 strain), from 0.78 to 3.13 micrograms/ml against Haemophilus influenzae (9 strains), 0.78 micrograms/ml against Haemophilus parainfluenzae (1 strain) and 6.25 micrograms/ml against salmonella sp. (1 strain). These values were similar to or lower than those of the other macrolides. Against Mycoplasma pneumoniae, MICs of AZM were < or = 0.0008 micrograms/ml in three strains. One strain of M. pneumoniae showed tolerance to AZM at MIC 25 micrograms/ml. The other agents exhibited higher MIC than AZM against this organism. 2. Plasma samples were collected from five patients receiving fine granules and four patients receiving capsules for drug level determination. The patients received AZM at 10.0 approximately 16.3 mg/kg body weight once daily for 3 days. Drug concentrations in plasma at two hours after Day 3 dosing were in a range between 0.02 and 0.19 micrograms/ml for fine granules and were in a range between 0.11 and 0.42 micrograms/ml for capsules. 3. Urine samples were collected from four patients receiving fine granules and four patients receiving capsules. Drug levels were determined to be 3 micrograms/ml at post-treatment 48 hours for fine granules and post-treatment 72 hours for capsules. Urinary excretion rates of AZM in three patients on capsules lied in a range between 4.69 and 10.17%. 4. Effectiveness of AZM in fine granules was evaluated in 128 patients having a total of 19 different infections. AZM was rated "excellent" in 51 patients, "good" in 63, "fair" in 8, "poor" in 6, resulting in an efficacy rate of 89.1%. Effectiveness of AZM in capsular form was evaluated in 23 patients with five different infections. AZM was found "excellent" in 13 patients and "good" in 10, resulting in an efficacy rate of 100%. 5. AZM in fine granules eradicated 45 strains of 54 in 8 different bacteria. AZM in capsules eradicated 9 strains of 10 strains in 6 different bacteria. 6. As for adverse reactions, one patient complained of eruption, one vomiting, one loose stool, five diarrhea, when administered with fine granular form of AZM. One patient on AZM capsules experienced urticaria and vomiting. 7. As for abnormal laboratory changes, three patients were found with decreased WBC, seven with increased eosinophil, two with increased GOT and GPT, one with increased GPT. They were all on fine granular form of AZM. As far as abnormalities found in patients administered with AZM in capsular form, two showed decreased WBC, one decreased WBC along with increased eosinophil, and three increased eosinophil.