Genome architecture catalyzes nonrecurrent chromosomal rearrangements

To investigate the potential involvement of genome architecture in nonrecurrent chromosome rearrangements, we analyzed the breakpoints of eight translocations and 18 unusual-sized deletions involving human proximal 17p. Surprisingly, we found that many deletion breakpoints occurred in low-copy repeats (LCRs); 13 were associated with novel large LCR17p structures, and 2 mapped within an LCR sequence (middle SMS-REP) within the Smith-Magenis syndrome (SMS) common deletion. Three translocation breakpoints involving 17p11 were found to be located within the centromeric alpha-satellite sequence D17Z1, three within a pericentromeric segment, and one at the distal SMS-REP. Remarkably, our analysis reveals that LCRs constitute >23% of the analyzed genome sequence in proximal 17p--an experimental observation two- to fourfold higher than predictions based on virtual analysis of the genome. Our data demonstrate that higher-order genomic architecture involving LCRs plays a significant role not only in recurrent chromosome rearrangements but also in translocations and unusual-sized deletions involving 17p.     

Potassium and tetraphenylphosphonium ion-selective electrodes for monitoring changes in the permeability of bacterial outer and cytoplasmic membranes

A tetraphenylphosphonium ion (TPP(+))-selective electrode, originally developed as a membrane potential indicator, is useful for measuring increases in the permeability of bacterial outer membranes induced by antimicrobial agents. The combination of this electrode with a potassium ion-selective electrode enabled us to determine changes in the permeability of bacterial outer and cytoplasmic membranes simultaneously. Outer membrane permeabilization induced by antimicrobial agents, chlorhexidine and polyhexamethylene biguanide (PHMB), as monitored with the TPP(+) electrode, correlated closely with the ability of the agents to release lipopolysaccharide (LPS) from the outer membrane.     

A mutant receptor tyrosine phosphatase,CD148, causes defects in vascular development

Vascularization defects in genetic recombinant mice have defined critical roles for a number of specific receptor tyrosine kinases. Here we evaluated whether an endothelium-expressed receptor tyrosine phosphatase, CD148 (DEP-1/PTPeta), participates in developmental vascularization. A mutant allele, CD148(DeltaCyGFP), was constructed to eliminate CD148 phosphatase activity by in-frame replacement of cytoplasmic sequences with enhanced green fluorescent protein sequences. Homozygous mutant mice died at midgestation, before embryonic day 11.5 (E11.5), with vascularization failure marked by growth retardation and disorganized vascular structures. Structural abnormalities were observed as early as E8.25 in the yolk sac, prior to the appearance of intraembryonic defects. Homozygous mutant mice displayed enlarged vessels comprised of endothelial cells expressing markers of early differentiation, including VEGFR2 (Flk1), Tal1/SCL, CD31, ephrin-B2, and Tie2, with notable lack of endoglin expression. Increased endothelial cell numbers and mitotic activity indices were demonstrated. At E9.5, homozygous mutant embryos showed homogeneously enlarged primitive vessels defective in vascular remodeling and branching, with impaired pericyte investment adjacent to endothelial structures, in similarity to endoglin-deficient embryos. Developing cardiac tissues showed expanded endocardial projections accompanied by defective endocardial cushion formation. These findings implicate a member of the receptor tyrosine phosphatase family, CD148, in developmental vascular organization and provide evidence that it regulates endothelial proliferation and endothelium-pericyte interactions.     

Intron length variation of the Adh gene in Brachyscome (Asteraceae)

Eighteen polymerase chain reaction (PCR) products of the partial sequence of the Adh (alcohol dehydrogenase) gene from 10 Brachyscome species were sequenced and compared. These products contained the 5 three fourths of exon 4 and whole sequences of intron 3. They varied extensively in length due to the differences in length of intron 3. A total of 10 long insertions were flanked by direct repeats of 5 to 12 bp sequences, indicating inserted elements. These inserted elements were classified into the following five categories based on nucleotide sequence characteristics and length; (1) a region homologous to that of 5S RNA genes (5S DNA), (2) A-rich structure at the 3 end-like short interspersed elements (SINEs) in animals, (3) a sequence of 280 bp with no characteristic features, (4) a sequence of 125 bp with no characteristic features, (5) termini of 11 bp inverted repeats flanked by 5 bp sequence of direct repeats characteristics of a transposon.     

A new assay for the analysis of X-chromosome inactivation based on methylation-specific PCR

The pattern of X-chromosome inactivation in females is currently evaluated by assays of differential methylation in the genes between the active and the inactive X chromosomes, with methylation-sensitive enzymes. We report a new assay in the human androgen receptor (HUMARA) locus involving a methylation-specific polymerase chain reaction (M-PCR) technique, independent of the use of restriction enzymes. The assay involves the chemical modification of DNA with sodium bisulfite and subsequent PCR. By using the assay with specific primers for the methylated allele, we obtained an X-inactivation pattern based on the ratio of the maternal inactive X to the paternal inactive X. These patterns were consistent with those obtained by conventional PCR assay at the same locus in 48 female cases. We also obtained another X-inactivation pattern based on the ratio of the maternal active X to the paternal active X by using specific primers for the unmethylated allele. The latter pattern was complementary to the former pattern, and a combination of these patterns produced a reliable X-inactivation pattern. The assay revealed that 12 (11%) of the 105 normal females had non-random inactivation patterns (>80:20 or <20:80). Four patients with an X; autosome translocation showed extremely non-random patterns, and these results were consistent with those obtained by previous molecular/cytogenetic studies. We conclude that M-PCR provides an accurate assay for X-inactivation and that it can be performed on various DNA samples unsuitable for restriction digestion. Received: 3 September 1998 / Accepted: 10 October 1998     

Suppression of 3-deoxyglucosone and heparin-binding epidermal growth factor-like growth factor mRNA expression by an aldose reductase inhibitor in rat vascular smooth muscle cells

Reactive carbonyl compounds and oxidative stress have been recently shown to up-regulate the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent mitogen for vascular smooth muscle cells (SMCs) produced by SMC themselves. Because the polyol pathway has been reported to influence the formation of carbonyl compounds and the oxidative stress in various cells, we conducted this study to investigate whether the polyol pathway affects HB-EGF expression along with the generation of carbonyl compounds and the oxidative stress in SMCs. We found that, compared with those cultured with 5.5mM glucose, SMCs cultured with 40 mM glucose showed the accelerated thymidine incorporation, elevated levels of intracellular sorbitol, 3-deoxyglucosone (3-DG), advanced glycation end products (AGEs), and thiobarbituric acid-reactive substances (TBARS) along with the enhanced expression of HB-EGF mRNA. An aldose reductase inhibitor (ARI), SNK-860, significantly inhibited all of these abnormalities, while aminoguanidine suppressed 3-DG levels and HB-EGF mRNA expression independent of sorbitol levels. The results suggest that the polyol pathway may play a substantial role in SMC hyperplasia under hyperglycemic condition in part by affecting HB-EGF mRNA expression via the production of carbonyl compounds and oxidative stress.     

Hepatocyte growth factor attenuates cerebral ischemia-induced learning dysfunction

Hepatocyte growth factor (HGF) acts as an organotropic factor for regeneration and protection in various organs and has the ability to attenuate cerebral ischemia-induced cell death. However, the effect of HGF on learning and memory function after a cerebral ischemic event is unknown. We demonstrate here that administration of human recombinant HGF (hrHGF) into the ventricle reduced the prolongation of the escape latency in the acquisition and retention tests in the water maze task on days 12-28 after microsphere embolism-induced cerebral ischemia. In addition, disruption of the blood-brain barrier at the early stage after microsphere embolism, which was determined by FITC-albumin leakage, was markedly reduced by treatment with hrHGF. We demonstrated that this effect of hrHGF on the blood-brain barrier was associated with protection against the apoptotic death of the cerebral endothelial cells at the early stage after the ischemia. These results suggest that hrHGF can prevent the learning and memory dysfunction soon after sustained cerebral ischemia by protecting against injury to the endothelial cells. The use of HGF may be a potent strategy for the treatment of cerebrovascular diseases, including cerebral infarct and vascular dementia.     

Induction of gicerin/CD146 in the rat carotid artery after balloon injury

Gicerin is a cell adhesion molecule belonging to the immunoglobulin superfamily. It is reported that the human homologous molecule, CD146, is expressed in the endothelial cells. Here, we found that the expression of gicerin was increased in the rat carotid arteries after balloon injury. Immunohistochemical analysis demonstrated that the expression of gicerin protein was increased in the medial smooth muscle cells prior to the formation of neointima one week after the injury and was also increased in the luminal edge of the neointima after two weeks. We employed A10 cells, a cell line derived from rat aortic smooth muscle cell, and examined the effect of growth factors on the expression of gicerin, such as IGF-1, PDGF-BB, and bFGF. We found that IGF-1, but not PDGF-BB and bFGF, significantly increases the expression of gicerin protein in A10 cells. These suggest gicerin might be involved in the arteriosclerotic neointima formation in the artery.