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.

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.

Precise switching of DNA replication timing in the GC content transition area in the human major histocompatibility complex.

The human genome is composed of long-range G+C% (GC%) mosaic structures thought to be related to chromosome bands. We previously reported a boundary of megabase-sized GC% mosaic domains at the junction area between major histocompatibility complex (MHC) classes II and III, proposing it as a possible chromosome band boundary. DNA replication timing during the S phase is known to be correlated cytogenetically with chromosome band zones, and thus the band boundaries have been predicted to contain a switch point for DNA replication timing. In this study, to identify to the nucleotide sequence level the replication switch point during the S phase, we determined the precise DNA replication timing for MHC classes II and III, focusing on the junction area. To do this, we used PCR-based quantitation of nascent DNA obtained from synchronized human myeloid leukemia HL60 cells. The replication timing changed precisely in the boundary region with a 2-h difference between the two sides, supporting the prediction that this region may be a chromosome band boundary. We supposed that replication fork movement terminates (pauses) or significantly slows in the switch region, which contains dense Alu clusters; polypurine/polypyrimidine tracts; di-, tri-, or tetranucleotide repeats; and medium-reiteration-frequency sequences. Because the nascent DNA in the switch region was recovered at low efficiency, we investigated whether this region is associated with the nuclear scaffold and found three scaffold-associated regions in and around the switch region.

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.

Identification and characterization of a novel trans-membrane protein gene, pdh1, from Schizosaccharomyces pombe.

We have cloned a new gene, pdh1, from genomic DNA of fission yeast Schizosaccharomyces pombe. pdh1 is actively transcribed as 1400-nucleotide mRNA in vegetatively growing cells and can code for a 226 amino acid polypeptide (pdh1p). Computational structural prediction has revealed that the pdh1p is a highly hydrophobic protein with seven transmembrane domains. The prediction has also detected a possible C-kinase phosphorylation site within the longest hydrophilic loop.

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.

Two mammalian cell systems for propagation of the hepatitis B virus genome in extrachromosomal and chromosomally integrated states: production of the surface and e antigens.

A recombinant plasmid consisting of (i) the entire genome of hepatitis B virus (HBV) DNA, (ii) the replication origin of SV40 virus, and (iii) a deletion derivative of pBR322 was introduced either into COS cells of monkey origin which constitutively express SV40 large T antigen, or into thymidine kinase(TK)-deficient mouse L cells together with the TK DNA of Herpes simplex virus. In the COS cell system, the transfecting recombinant DNA replicates via SV40 origin and is maintained in an autonomously replicating state. The cells carrying these extrachromosomal elements express the hepatitis B surface antigen gene at moderate rate, and release the products into the culture medium. However, neither core antigen nor e antigen expression was detected in this system. In the L cell system, the transformed L cells carry the recombinant DNA in a chromosomally integrated state. Such cells express the surface antigen gene at high rate, and release the products into the culture medium. This system also excretes the e antigen into the culture medium. The core antigen was not detected.

Chromosome assignment of aberrant NotI restriction DNA fragments in primary hepatocellular carcinoma.

DNA aberrations in human hepatocellular carcinoma (HCC) were studied by two-dimensional DNA electrophoresis analysis. Five intensified and 60 dwindling spots were detected recurrently in the two-dimensional profile which showed about 3000 restriction DNA fragments as distinctive spots. We assigned these aberrant spots to chromosomes, using the chromosome-assigned two-dimensional profile. Four of the five intensified, and 53 of the 60 dwindling spots were given chromosome assignments. Intensified spots were assigned to chromosomes 5, 6, 9 through 12, 16 and 18. Among the dwindling spots, the highest incidence of aberrations was found on chromosome 16, followed by 9 through 12 and chromosome 2. No aberrations were detected in chromosomes 7, 21, 22 or Y.

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.

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.

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.