Degradation of Mitochondrial Phospholipids During Experimental Cerebral Ischemia in Rats

Changes in content of brain mitochondrial phospholipids were examined in rats after 30 and 60 min of decapitation ischemia compared with controls, to explore the degradation of the mitochondrial membrane and its relation to dysfunction of mitochondria. Activities of respiratory functions and respiratory enzymes (cytochrome c oxidase; F0F1-ATPase) decreased significantly during ischemia. Considerable decreases in cardiolipin and phosphatidylinositol content were observed after 60 min, and other phospholipids showed similar but nonsignificant decreases in content. The amount of polyunsaturated fatty acids chains, such as arachidonic and docosahexaenoic acids, was reduced in each phospholipid, in some cases significantly, after 30 and 60 min of ischemia in time-dependent manners. Degradation of mitochondrial phospholipids during ischemia associated with the deterioration of mitochondrial respiratory functions suggested the significance of such changes in phospholipid content in disintegration of cellular energy metabolism during cerebral ischemia.     

Methylation status of ribosomal RNA gene clusters in the flow-sorted human acrocentric chromosomes

Southern blot analysis of the human acrocentric chromosomes that were flow-sorted from B-lymphoblastoid cell line GM130B revealed that the sensitivity of the ribosomal RNA (rDNA) gene clusters to the restriction enzyme NotI differs among these rDNA-containing chromosomes: the rDNA clusters of Chromosomes (Chr) 13, 14, and 15 are much more sensitive to NotI digestion than those of Chrs 21 and 22 in this particular cell line. Detailed analysis by use of methylation-sensitive enzymes HpaII and HhaI and methylation-insensitive enzyme MspI confirmed the significant variation in the methylation status of rDNA clusters among these chromosomes. Quantitative analysis by fluorescent in situ hybridization (FISH) indicated that copy number of rDNA varies among individual chromosomes, but the average copy number in the acrocentric Chrs 21 and 22 is significantly greater than that of the Chrs 13, 14, and 15 in GM130B cells. Similar analysis reveals that the methylation status of rDNA clusters in another B-lymphoblastoid cell line GM131 was different from that of GM130B. These data together indicate that the copy number and methylation patterns of rDNA clusters differ among individual acrocentric chromosomes in a given cell line, and they are different among cell lines.     

Dissolved DNA produced through a prey-predator relationship in a species-defined aquatic microcosm

Changes in concentration of dissolved DNA was studied in a species-defined microcosm consisting of bacteria Escherichia coli, protozoa Tetrahymena thermophila and algae Euglena gracilis. A marked increase in dissolved DNA was observed when T. thermophila grazed on E. coli and grew. This meant the prey–predatory relationship between E. coli and T. thermophila was responsible for the increase of dissolved DNA. This revised version was published online in September 2006 with corrections to the Cover Date.     

Dynamic Organization of Microtubules in Guard Cells of Viciafaba L. with Diurnal Cycle

Stomatal movement is regulated by changes in the volume of guardcells, thought to be mainly controlled by an osmo-regulatorysystem. In the present study, we examined the additional involvementof cytoskeletal events in the regulation of stomatal movement.Microtubules (MTs) in guard cells of Viciafaba L., grown undersunlight, were observed during the day and night by immunofluorescencemicroscopy. Cortical MTs began to be organized in a radial arrayat dawn and increased in numbers in the morning following theincrease in the stomatal aperture size. Thereafter, MTs becamelocalized near the nucleus and began to be destroyed from theevening to midnight, following the decrease in stomatal aperturesize. These diurnal changes in MT organization were observedeven two days after transfer from natural light condition tototal darkness, and were accompanied by corresponding changesin stomatal aperture. The increase in stomatal aperture sizein the early morning was inhibited by 50 µM propyzaraide,which destroys cortical MTs in guard cells, whereas the decreasein aperture size in the evening was suppressed by 10 µMtax-ol, which stabilizes cortical MTs. These results suggestthat radially-organized cortical MTs of guard cells may controldiurnal stomatal movement. (Received September 3, 1997; Accepted November 5, 1997)     

The Gene for Death Agonist BID Maps to the Region of Human 22q11.2 Duplicated in Cat Eye Syndrome Chromosomes and to Mouse Chromosome 6

Cat eye syndrome (CES) is associated with a duplication of a segment of human chromosome 22q11.2. Only one gene,ATP6E, has been previously mapped to this duplicated region. We now report the mapping of the human homologue of the apoptotic agonistBidto human chromosome 22 near locus D22S57 in the CES region. Dosage analysis demonstrated thatBIDis located just distal to the CES region critical for the majority of malformations associated with the syndrome (CESCR), as previously defined by a single patient with an unusual supernumerary chromosome. However,BIDremains a good candidate for involvement in CES-related mental impairment, and its overexpression may subtly add to the phenotype of CES patients. Our mapping of murineBidconfirms that the synteny of the CESCR and the 22q11 deletion syndrome critical region immediately telomeric on human chromosome 22 is not conserved in mice.Bidand adjacent geneAtp6ewere found to map to mousechromosome 6, while the region homologous to the DGSCR is known to map to mouse chromosome 16.     

Coronary stent implantation throughout technical evolution: immediate and follow-up results

Coronary stenting (stent implantation) has evolved over the last 5 years with changes in stent design, stent material and the implantation technique. The use of high-pressure balloon inflation (HP), intravascular ultrasound (IVUS) and appropriate antiplatelet therapy have contributed to the abolishment of the need for subsequent anticoagulation, allowing extended stent applications. We compared results in three groups of patients having stent implantation throughout the period of evolution: group A: no IVUS, no HP, with subsequent anticoagulation treatment (n 3 434); group B: no IVUS, yes HP, without subsequent anticoagulation treatment (n 3 192); and group C: yes IVUS, yes HP, without subsequent anticoagulation treatment (n 3 588). The primary success rates were comparable in all groups. There was a clear change in indications for stenting in groups B and C compared with group A (elective stenting: group A 3 32%; group B 3 66%; group C 3 69%; P < 0.0001), in reference vessel size (group A 3 3.22 3 0.37 mm; group B 3 2.92 3 0.56 mm; group C 3 2.98 3 0.57 mm; P < 0.0001), and for presence of type B2 and C lesions (group A 3 57%; group B 3 72%; group C 3 74%; P < 0.001). The complication rate significantly decreased in group C (group A 3 3.6%; group B 3 4.1%; group C 3 1.2%; P < 0.001) and the mean patient hospital stay decreased to 2 days in groups B and C due to the abolition of the need for anticoagulant treatment. The angiographic restenosis rate increased in groups B and C (group A 3 20%; group B 3 34%; group C 3 32%; P < 0.001). The need for a repeat procedure increased as stenting of more complex lesions and smaller vessels was attempted: target lesion revascularization (TLR) was performed in 16% of patients in group A (73/434), in 18% of group B (35/192) and in 22% of group C (129/588) (P 3 0.04 for A versus C). Major cardiac events (MACE) occurred in 142 patients in group A (33%), 60 patients in group B (31%) and in 181 patients in group C (30%). The evolving technique of coronary stenting has expanded the spectrum of indications and range of coronary vessels attempted, and decreased the complication rates and hospital stay. However, in less-favorable subsets, additional improvements are needed to affect the long-term outcome.     

Overinitiation of chromosome replication in the Escherichia coli dnaAcos mutant depends on activation of oriC function by the dam gene product

The activity of DnaA protein, the initiator of chromosome replication in Escherichia coli , is regulated by adenine nucleotide binding; the ATP-bound form, not the ADP-bound form, is active. DnaAcos is a mutant protein that is insensitive to negative regulation by ADP. Initiation of chromosome replication occurs excessively in the dnaAcos mutant at 30°C, a restrictive temperature for growth. To determine the control factors that act independently of adenine nucleotide binding of DnaA, we analysed suppressors from the dnaAcos mutant isolated by Tn 5 insertion mutagenesis. Three of the suppressors carried Tn 5 in the aroK or aroB gene, the first two cistrons in the dam operon. Complementation tests revealed that the dam gene is responsible for the suppression. Over-replication of the chromosome was inhibited in the dnaAcos aroK ::Tn 5 double mutant, and initiation of chromosome replication in the dnaA ⁺ aroK ::Tn 5 mutant was partially inhibited. The aroK  (or B  )::Tn 5 cells contained DnaA molecules at a level similar to that in the parental aroBK  ⁺ strain. Moreover, dnaAcos suppression depended on the function of the seqA gene. Thus, Dam activity positively regulates initiation of chromosome replication in vivo . SeqA function seems to be distinguished from the control of DnaA protein by adenine nucleotide binding.     

Human ribosomal RNA gene cluster: identification of the proximal end containing a novel tandem repeat sequence

Human ribosomal RNA genes (rDNA) are arranged as tandem repeat clusters on the short arms of five pairs of acrocentric chromosomes. We have demonstrated that a majority of the rDNA clusters are detected as 3-Mb DNA fragments when released from human genomic DNA by EcoRV digestion. This indicated the absence of the EcoRV restriction site within the rDNA clusters. We then screened for rDNA-positive cosmid clones using a chromosome 22-specific cosmid library that was constructed from MboI partial digests of the flow-sorted chromosomes. Three hundred twenty rDNA-positive clones negative for the previously reported distal flanking sequence (pACR1) were chosen and subjected to EcoRV digestion. Seven clones susceptible to EcoRV were further characterized as candidate clones that might have been derived from the junctions of the 3-Mb rDNA cluster. We identified one clone containing part of the rDNA unit sequence and a novel flanking sequence. Detailed analysis of this unique clone revealed that the coding region of the last rRNA gene located at the proximal end of the cluster is interrupted with a novel sequence of 147 bp that is tandemly repeated and is connected with an intervening 68-bp unique sequence. This junction sequence was readily amplified from chromosomes 21 and 15 as well as 22 using the polymerase chain reaction. Fluorescence in situ hybridization further indicated that the 147-bp sequence repeat is commonly distributed among all the acrocentric short arms.