An intrinsic DNA curvature found in the cyanobacterium Microcystis aeruginosa K-81 affects the promoter activity of rpoD1 encoding a principal sigma factor.

The rpoD1 gene in the unicellular cyanobacterium Microcystis aeruginosa K-81 encodes a principal sigma factor of RNA polymerase and is transcribed under light and dark conditions to produce multiple monocistronic transcripts. In the 5'-upstream region from rpoD1 Promoter 2, which has a sequence of Escherichia coli type, we found a sequence-directed DNA curvature with an AT-rich sequence. Insertions of 2 to 21 base pairs introduced into the curved center changed a gross geometry of the original curved DNA structure. The rpoD1 promoter activities assayed in vivo by using transcriptional lacZ fusions were correlated with the change in the gross geometry in not only a cyanobacterium but also E. coli. In addition, RNA polymerase binding to the rpoD1 promoter region and the efficiency of the mRNA synthesis from the rpoD1 Promoter 2 were also affected in vitro by the change in the geometry. These results suggest that the tertiary structure of the curved DNA is important for the rpoD1 transcription. The deletion of the center region of the curvature resulted in a considerable reduction of the transcription from Promoter 2 in the cyanobacterium. This report demonstrates that a curved DNA plays a significant role in transcription in cyanobacteria, and that this functional curvature is located in the 5'-upstream region from the rpoD gene, which encodes a principal sigma factor in eubacteria.

A novel genetic organization: the leuA-rpoD1 locus in the cyanobacterium Microcystis aeruginosa K-81.

We cloned and sequenced the region upstream of rpoD1, which encodes a principal sigma factor in the cyanobacterium Microcystis aeruginosa K-81. An open reading frame (orf1, 1599 bp) was discovered, the deduced amino-acid sequence of which (533 aa, 58, 016 Da) exhibits homology to another bacterial leuA gene product, 2-isopropylmalate synthase. The leuA (orf1) gene specifically complemented an E. coli leuA mutant. The 5'-upstream region of leuA did not contain possible leader peptide or stem-loop structures for attenuation. These findings indicate that the genetic structure of the leuA-rpoD1 locus in M. aeruginosa K-81 significantly differs from those of known leuA and rpoD loci found in other bacteria.

Highly repetitive sequences and characteristics of genomic DNA in unicellular cyanobacterial strains.

Microcystis aeruginosa (Synechocystis) is a unicellular cyanobacterium that performs oxygenic photosynthesis. We found two novel sets of repetitive sequences, A (REP-A) and B (REP-B), on the M. aeruginosa K-81 genomic DNA, which consisted of distinct motifs of tandem repeated sequences located in the up- and downstream regions of the orf1 structural gene, respectively. Genomic Southern hybridization revealed multicopies of REP-A and -B on the genome. Furthermore, genomic Southern blots of cyanobacteria species with the REP-A and -B probes revealed that different hybridization signals appeared on the genomic DNAs of all 12 Microcystis strains, but no signal appeared on those of Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, and Anabaena sp. PCC 7120.

Effect of age on physiologic and mechanically stressed rat alveolar bone: a cytologic and histochemical study.

The effect of age on physiologic and mechanically stressed rat alveolar bone was assessed with special focus on bone formation and resorption in maxillary molar sections of 1-, 4-, 9-, 16-, and 24-month-old male Fischer rats. Tetracycline- and calcein-labeled undecalcified sections were used for observation by confocal laser scanning microscopy and contact microradiography. The localization of osteoclasts and the morphological change of osteoblasts were observed on decalcified sections. Mechanical stress was applied by an orthodontic elastic inserted between the first and second maxillary molars for 7 days. Results showed that bone-formative activity of osteoblasts and bone-resorptive activity of osteoclasts declined with age around the maxillary molar section of physiologic rat alveolar bone. However, results of mechanically stressed rat alveolar bone revealed no evidence of numerical difference in number, size, and activity of osteoclasts and osteoblasts among the ages, suggesting that mechanical stress provokes a higher activity of osteoblasts and osteoclasts similarly in both adult and younger rat alveolar bone.

Histological observations of dental tissues using the confocal laser scanning microscope.

To investigate the time course of mineralization in undecalcified dental tissues, calcein- and tetracycline-labeled rat maxillary molar sections were stained with Villanueva bone stain en bloc, embedded in methyl-methacrylate (MMA), ground to 50 microns thickness, and observed by confocal laser scanning microscopy (CLSM). This method allowed observation of dental structures including odontoblasts, pulp cells and periodontal ligament, and dentinal tubules and enamel rods at high resolution; labeled enamel, dentine, and cementum could be observed simultaneously regardless of section thickness. CLSM permitted simultaneous observation of both the components of calcified tissue and the cellular components of dental tissues, and assessment of the mineralization time course of hard tissues labeled by tetracycline or calcein. The technique is useful for both assessing the elements composing dental structure and observing the histological dynamics by which dental structure develops.

Successful surgical repair of left ventricular rupture after acute myocardial infarction.

A 61-year-old man with impending myocardial infarction was admitted and treated by percutaneous transluminal coronary recanalization (PTCR) therapy using Urokinase. Patient's symptoms subsided and his general condition maintained stable until the fifth hospital day, when he developed the signs of cardiac rupture. He was operated upon immediately, and the left ventricular rupture was confirmed and repaired successfully. Patient recovered from the surgery well. This report describes the rare successful surgical case of cardiac rupture secondary to acute myocardial infarction in Japan.

Significance of echocardiographic evaluation on surgery to mitral stenosis.

1) Diastolic descent rate (DDR) of the anterior mitral leaflet improved after the surgery. However, this improvement was not modified with a) atrial fibrillation b) damages of the subvalvular supporting tissue c) valvular calcification d) the method of the operation, namely the open or closed commissurotomy. 2) DDR was fairly well correlated with the mitral orifice area, the cardiac output and the pressure gradient across the mitral valve. Therefore, the echocardiographic diagnosis and the follow up evaluation of the mitral stenosis after the surgery were thought to be very reliable, considering the accuracy of this diagnosis as disclosed in this study. The contributory effect to DDR was discussed.