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.

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.

Evolutionary conservation, developmental expression, and genomic mapping of mammalian Twisted gastrulation.

The twisted gastrulation gene (tsg) encodes a secreted protein required for the correct specification of dorsal midline cell fate during gastrulation in Drosophila. We report that tsg homologs from human, mouse, zebrafish, and Xenopus share 72-98% identity at the amino acid level and retain all 24 cysteine residues from Drosophila. In contrast to Drosophila where tsg expression is limited to early embryos, expression is found throughout mouse and human development. In Drosophila, tsg acts in synergy with decapentaplegic (dpp), a member of the TGF-beta family of secreted proteins. The vertebrate orthologs of dpp, BMP-2 and -4, are crucial for gastrulation and neural induction, and aberrant signaling by BMPs and other TGF-beta family members results in developmental defects including holoprosencephaly (HPE). Interestingly, human TSG maps to the HPE4 locus on Chromosome 18p11.3, and our analysis places the gene within 5 Mbp of TG-interacting factor (TGIF).

A physical map of human chromosome 14.

We report the construction of a tiling path of around 650 clones covering more than 99% of human chromosome 14. Clone overlap information to assemble the map was derived by comparing fully sequenced clones with a database of clone end sequences (sequence tag connector strategy). We selected homogeneously distributed seed points using an auxiliary high-resolution radiation hybrid map comprising 1,895 distinct positions. The high long-range continuity and low redundancy of the tiling path indicates that the sequence tag connector approach compares favourably with alternative mapping strategies.

A first high-density map of 981 biallelic markers on human chromosome 14.

As the largest set of sequence variants, single-nucleotide polymorphisms (SNPs) constitute powerful assets for mapping genes and mutations related to common diseases and for pharmacogenetic studies. A major goal in human genetics is to establish a high-density map of the genome containing several hundred thousand SNPs. Here we assayed 3.7 Mb (154,397 bp in 24 alleles) of chromosome 14 expressed sequence tags (ESTs) and sequence-tagged sites, for sequence variation in DNA samples from 12 African individuals. We identified and mapped 480 biallelic markers (459 SNPs and 21 small insertions and deletions), equally distributed between EST and non-EST classes. Extensive research in public databases also yielded 604 chromosome 14 SNPs (dbSNPs), 520 of which could be mapped and 19 of which are common between CNG (i.e., identified at the Centre National de Génotypage) and dbSNP polymorphisms. We present a dense map of SNP variation of human chromosome 14 based on 981 nonredundant biallelic markers present among 1345 radiation hybrid mapped sequence objects. Next, bioinformatic tools allowed 945 significant sequence alignments to chromosome 14 contigs, giving the precise chromosome sequence position for 70% of the mapped sequences and SNPs. In addition, these tools also permitted the identification and mapping of 273 SNPs in 159 known genes. The availability of this SNP map will permit a wide range of genetic studies on a complete chromosome. The recognition of 45 genes with multiple SNPs, by allowing the construction of haplotypes, should facilitate pharmacogenetic studies in the corresponding regions.

Transactivation of EGF receptor induced by angiotensin II regulates fibronectin and TGF-beta gene expression via transcriptional and post-transcriptional mechanisms.

The signaling cascade elicited by angiotensin II (Ang II) resembles that characteristic of growth factor, and recent evidence indicates transactivation of epidermal growth factor receptor (EGF-R) by G protein-coupled receptors. Here, we report the involvement of EGF-R in Ang II-induced synthesis of fibronectin and TGF-beta in cardiac fibroblasts. Ang II stimulated fibronectin mRNA levels dose-dependently with a maximal increase (approximately 5-fold) observed after 12 h of incubation. Ang II-, or calcium ionophore-induced fibronectin synthesis was completely abolished by tyrosine kinase inhibitors and intracellular Ca2+ chelating agents. Ang II-induced fibronectin mRNA was not affected by PKC inhibitors or PKC depletion, whereas specific inhibition of EGF-R function by a dominant negative EGF-R mutant and tyrphostin AG1478 abolished induction of fibronectin mRNA. We isolated the rat fibronectin gene including the 5'-flanking region and found that the AP-1 binding site present in the promoter region was responsible for the Ang II responsiveness of this gene. Gel retardation assay revealed the binding of nuclear protein to the AP-1 site, which was supershifted with anti-c-fos and anti-c-jun but not anti-ATF-2 antibodies. Conditioned medium from Ang II-treated cells contained TGF-beta bioactivity and addition of neutralizing TGF-beta antibody modestly (46%) inhibited induction of fibronectin. Ang II-induced synthesis of TGF-beta was also abolished by inhibition of EGF-R function. The effect of TGF-beta was exerted by stabilizing fibronectin mRNA without affecting the promoter activity and required de novo protein synthesis. We concluded that Ang II-induced expression of fibronectin and TGF-beta is mediated by downstream signaling of EGF-R transactivated by Ca2+-dependent tyrosine kinase, and that Ang II-induced fibronectin mRNA expression is regulated by two different mechanisms; transcriptional control by binding of c-fos/c-jun complex to the AP-1 site, and post-transcriptional control by mRNA stabilization due to autocrine and/or paracrine effects of TGF-beta. Thus, this study suggested that the action of Ang II on extracellular matrix formation should be interpreted in association with the EGF-R signaling cascade.

Cryofiltration and oral corticosteroids provide successful treatment for an elderly patient with cryoglobulinemic glomerulonephritis associated with hepatitis C virus infection.

A 75-year-old man was admitted due to nephrotic syndrome, purpura on the legs, which was associated with hepatitis C virus (HCV), and type II mixed cryoglobulinemia. Renal biopsy revealed features of cryoglobulinemic glomerulonephritis. Since the patient was elderly and the HCV genotype was Ib, interferon-alpha for reducing HCV was not indicated. Four sessions of cryofiltration and the administration of corticosteroids improved the proteinuria and renal function strikingly without adverse effects. This case demonstrates that an elderly patient who has nephrotic syndrome caused by cryoglobulinemic glomerulonephritis associated with HCV can be treated safely by cryofiltration with low doses of oral corticosteroids.

Newly identified repeat sequences, derived from human chromosome 21qter, are also localized in the subtelomeric region of particular chromosomes and 2q13, and are conserved in the chimpanzee genome.

Subtelomeric regions have been a target of structural and functional studies of human chromosomes. Markers having a defined structure are especially useful to such studies. Here, we report 93 bp tandem repeat sequences found in the subtelomeric region of human chromosome 21q. They were also detected in the telomeric region of several other chromosomes. Interestingly, the repeat was also found in the 2q13 region which is known to be a position of chromosomal fusion, a major difference between the human and chimpanzee karyotypes. To the best of our knowledge, this repetitive sequence is a new member of human subtelomeric interspersed repeats.

Angiotensin II initiates tyrosine kinase Pyk2-dependent signalings leading to activation of Rac1-mediated c-Jun NH2-terminal kinase.

Ca(2+)-sensitive tyrosine kinase Pyk2 was shown to be involved in angiotensin (Ang) II-mediated activation of extracellular signal-regulated kinase (ERK) via transactivation of epidermal growth factor receptor (EGF-R). In this study, we tested the involvement of Pyk2 and EGF-R in Ang II-induced activation of JNK and c-Jun in cardiac fibroblasts. Ang II markedly stimulated JNK activities, which were abolished by genistein and intracellular Ca(2+) chelators but partially by protein kinase C depletion. Inhibition of EGF-R did not affect Pyk2 and JNK activation by Ang II. Stable transfection with a dominant negative (DN) mutant for Pyk2 (PKM) completely blocked JNK activation by Ang II. DN mutants of Rac1 (DN-Rac1) and MEK kinase (DN-MEKK1) also abolished it, whereas those of Cdc42, RhoA, and Ha-Ras had no effect. Induction of c-Jun gene transcription by Ang II was abolished in PKM, DN-Rac1, and DN-MEKK1, in which Ang II-induced binding of ATF2/c-Jun heterodimer to the activator protein-1 sequence at -190 played a key role. These results suggest that 1) in cardiac fibroblasts activation of JNK and c-Jun by Ang II is initiated by Pyk2-dependent signalings but not by downstream signals of EGF-R or Ras, 2) Rac1 but not Cdc42 is required for JNK activation by Ang II upstream of MEKK1, and 3) ATF-2/c-Jun binding to the activator protein-1 sequence at -190 plays a key role for induction of c-Jun gene by Ang II.

The DNA sequence of human chromosome 21.

Chromosome 21 is the smallest human autosome. An extra copy of chromosome 21 causes Down syndrome, the most frequent genetic cause of significant mental retardation, which affects up to 1 in 700 live births. Several anonymous loci for monogenic disorders and predispositions for common complex disorders have also been mapped to this chromosome, and loss of heterozygosity has been observed in regions associated with solid tumours. Here we report the sequence and gene catalogue of the long arm of chromosome 21. We have sequenced 33,546,361 base pairs (bp) of DNA with very high accuracy, the largest contig being 25,491,867 bp. Only three small clone gaps and seven sequencing gaps remain, comprising about 100 kilobases. Thus, we achieved 99.7% coverage of 21q. We also sequenced 281,116 bp from the short arm. The structural features identified include duplications that are probably involved in chromosomal abnormalities and repeat structures in the telomeric and pericentromeric regions. Analysis of the chromosome revealed 127 known genes, 98 predicted genes and 59 pseudogenes.

Angiotensin II type 2 receptor overexpression activates the vascular kinin system and causes vasodilation.

Angiotensin II (Ang II) is a potent vasopressor peptide that interacts with 2 major receptor isoforms - AT1 and AT2. Although blood pressure is increased in AT2 knockout mice, the underlying mechanisms remain undefined because of the low levels of expression of AT2 in the vasculature. Here we overexpressed AT2 in vascular smooth muscle (VSM) cells in transgenic (TG) mice. Aortic AT1 was not affected by overexpression of AT2. Chronic infusion of Ang II into AT2-TG mice completely abolished the AT1-mediated pressor effect, which was blocked by inhibitors of bradykinin type 2 receptor (icatibant) and nitric oxide (NO) synthase (L-NAME). Aortic explants from TG mice showed greatly increased cGMP production and diminished Ang II-induced vascular constriction. Removal of endothelium or treatment with icatibant and L-NAME abolished these AT2-mediated effects. AT2 blocked the amiloride-sensitive Na(+)/H(+) exchanger, promoting intracellular acidosis in VSM cells and activating kininogenases. The resulting enhancement of aortic kinin formation in TG mice was not affected by removal of endothelium. Our results suggest that AT2 in aortic VSM cells stimulates the production of bradykinin, which stimulates the NO/cGMP system in a paracrine manner to promote vasodilation. Selective stimulation of AT2 in the presence of AT1 antagonists is predicted to have a beneficial clinical effect in controlling blood pressure.

Multiple spontaneous small bowel perforations due to systemic cholesterol atheromatous embolism.

A-65-year-old man was admitted for coronary and peripheral angiography to evaluate angina pectoris and peripheral vascular disease. Following angiography, he suffered from blue toes, livedo reticularis and progressive renal failure. The patient's condition continued to deteriorate, including the development of malnutrition. Four months later he suddenly developed panperitonitis, went into shock and died. The autopsy verified multiple perforations of the small bowel with disseminated cholesterol atheromatous embolism. The other organs including kidney were also invaded by atheroembolism. This was a rare case of multiple spontaneous perforations of small bowel due to systemic cholesterol atheromatous embolism.

Ras-mediated signaling pathway regulates the expression of a low-molecular-weight heat-shock protein in fission yeast.

In fission yeast, Schizosaccharomyces pombe, deficiency of ras1 gene causes an abnormal cell shape and abolishes mating ability. However, target genes of this signaling pathway are largely unknown because of the lack of an appropriate analysis system. To overcome this problem, we have started a novel project to categorize entire genes based on their expression levels under different growth conditions. Using this strategy, we screened genes whose expression levels were affected in the presence or absence of the ras1 gene product. For this purpose, we utilized high-density arrays of clones covering the entire genome of the fission yeast, and probed with labelled cDNA derived from various strains and growth conditions. Here, we demonstrate the detection of a low-molecular-weight heat-shock protein gene, hsp16, whose expression is very likely to be regulated by a ras-mediated signaling pathway, but not by the heat-shock response.

[Aortic valvular vegetation in annuloaortic ectasia due to idiopathic cystic medial necrosis: a case report].

Annuloaortic ectasia is often accompanied by Marfan syndrome and associated with infective endocarditis usually involving the mitral valves. We treated a patient with annuloaortic ectasia due to idiopathic cystic medial necrosis who developed congestive heart failure with aortic valvular vegetation. A 56-year-old man had dyspnea on effort since the beginning of January, 1997 and was admitted to our hospital on April 6, 1997 because of orthopnea. The diagnosis was congestive heart failure due to severe aortic regurgitation with annuloaortic ectasia detected by echocardiography. Medication and rest after hospitalization relieved his symptoms but congestive heart failure deteriorated after he had a high fever. At this time, a vegetation attached to the noncoronary cusp of the aortic valve was found which had not been detected on admission. Blood culture yielded Streptococcus sanguis. The diagnosis was infective endocarditis involving the aortic valve. Surgical correction (Bentall method) improved congestive heart failure and he was discharged on August 4, 1997 without recurrence of endocarditis. Infective endocarditis involving the aortic valves is a possible cause of development or deterioration of congestive heart failure in patients with annuloaortic ectasia.

Identification of a brain-specific APC homologue, APCL, and its interaction with beta-catenin.

We isolated a novel gene, APCL, that showed significant homology to the adenomatous polyposis coli (APC) tumor suppressor gene. This novel gene, located on chromosome 19p13.3, encodes a protein of 2303 amino acids that is expressed specifically in the brain. The predicted protein of APCL contains five copies of a 20-amino-acid motif (FXVEXTPXCFSRXSSLSSLS). Like APC, this domain of APCL was able to bind to beta-catenin and deplete the intracellular beta-catenin pool. A reporter-gene assay revealed that APCL could also regulate interaction of beta-catenin with T cell-specific transcription factor, although less actively than APC. These results suggest that the APCL protein may be involved in the Wnt/Wingless signal pathway, and the identification of a novel relative of APC may provide new insights into the function of APC.