Studies on the interactions between phospholipids and membrane-bound enzymes in microsomes. Effects of phospholipases C on the glucose-6-phosphatase system of rat liver microsomes

The role of phospholipids in the glucose-6-phosphatase system, including glucose-6-P phosphohydrolase and glucose-6-P translocase, was studied in rat liver microsomes by using phospholipases C and detergents. In the time course experiments on detergent exposure, the maximal activation of glucose-6-P phosphohydrolase varied according to the nature of the detergent used. On treatment of microsomes with phospholipase C of C. perfringens, the activity of glucose-6-P phosphohydrolase without detergent (i.e. without rupture of translocase activity) was gradually decreased with the progressive hydrolysis of phosphatidylcholine and phosphatidylethanolamine on the microsomal membrane, and was restored by incubation of these microsomes with egg yolk phospholipids. The extent of decrease in this phosphohydrolase activity in the detergent-exposed microsomes (with rupture of translocase activity) also varied depending on the detergent used (Triton X-114 or taurocholate). When 66% of the phosphatidylinositol on the membrane was hydrolyzed by phosphatidylinositol-specific phospholipase C of B. thuringiensis, the inhibition of glucose-6-P phosphohydrolase activity without detergent was very small. Although the inhibition of enzyme activity with detergent was apparently greater than that without detergent, the enzyme activity was stimulated by the breakdown of phosphatidylinositol when the enzyme activity was measured at lower concentration (0.5 mM) of substrate, glucose-6-P. The latency of mannose-6-P phosphohydrolase, a plausible index of microsomal integrity, remained above 70% after the hydrolysis of phosphatidylcholine, phosphatidylethanolamine, or phosphatidylinositol. The results show that the glucose-6-phosphatase system requires microsomal phospholipids for its integrity, suggesting that there exists a close relation between phosphatidylinositol and glucose-6-P translocase.     

Cloning and sequence analysis of cDNA encoding aspartate aminotransferase isozymes from Panicum miliaceum L., a C4 plant.

The cytosolic and mitochondrial isozymes of aspartate aminotransferase (AspAT) function in the C4 dicarboxylate cycle of photosynthesis. We constructed a cDNA library from leaf tissues of Panicum miliaceum, an NAD-malic-enzyme-type C4 plant and screened the library for AspAT isozymes. A full-length cDNA clone for cytosolic AspAT was isolated. This clone contains an open reading frame that encodes 409 amino acids. We also isolated two cDNA clones for different precursors of mitochondrial AspAT. Comparing these two sequences in the coding regions, we found 12 amino acid substitutions out of 28 base substitutions. The encoded amino acid sequences predict that mitochondrial AspAT are synthesized as precursor proteins of 428 amino acid residues, which each consist of a mature enzyme of 400 amino acid residues and a 28-amino-acid presequence. This prediction coincides with the observation that the in vitro translation product of the mRNA for mitochondrial AspAT was substantially larger than the mature form. A comparison of the amino acid sequences of the AspAT isozymes from P. miliaceum with the published sequences for the enzymes from various animals and microorganisms reveals that functionally and/or structurally important residues are almost entirely conserved in all AspAT species.     

Pathology of sternal deformity in the Sprague-Dawley rat

Pathological examination of two 12-week-old male Sprague-Dawley rats revealed bending of the sternebrae. Grossly, the sternebrae distal to the third sternebra were bent towards the underside, and the episternal extremity was turned towards the head. Microscopically, the 4th sternebra was observed to have slipped behind the third sternebra, and fractures of the episternal cortex and slanting of the episternal cartilage were also observed.     

Target site specificity of the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon-rich regions of the genome

Because retrotransposons are the major component of plant genomes, analysis of the target site selection of retrotransposons is important for understanding the structure and evolution of plant genomes. Here, we examined the target site specificity of the rice retrotransposon Tos17, which can be activated by tissue culture. We have produced 47,196 Tos17-induced insertion mutants of rice. This mutant population carries approximately 500,000 insertions. We analyzed >42,000 flanking sequences of newly transposed Tos17 copies from 4316 mutant lines. More than 20,000 unique loci were assigned on the rice genomic sequence. Analysis of these sequences showed that insertion events are three times more frequent in genic regions than in intergenic regions. Consistent with this result, Tos17 was shown to prefer gene-dense regions over centromeric heterochromatin regions. Analysis of insertion target sequences revealed a palindromic consensus sequence, ANGTT-TSD-AACNT, flanking the 5-bp target site duplication. Although insertion targets are distributed throughout the chromosomes, they tend to cluster, and 76% of the clusters are located in genic regions. The mechanisms of target site selection by Tos17, the utility of the mutant lines, and the knockout gene database are discussed. --The nucleotide sequence data were uploaded to the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession numbers AG020727 to AG025611 and AG205093 to AG215049.     

Impact of the Distance from the Stent Edge to the Residual Plaque on Edge Restenosis following Everolimus-Eluting Stent Implantation

Objectives

This study aimed to assess the relation between stent edge restenosis (SER) and the distance from the stent edge to the residual plaque using quantitative intravascular ultrasound.

Background

Although percutaneous coronary intervention with drug-eluting stents has improved SER rates, determining an appropriate stent edge landing zone can be challenging in cases of diffuse plaque lesions. It is known that edge vascular response can occur within 2 mm from the edge of a bare metal stent, but the distance to the adjacent plaque has not been evaluated for drug-eluting stents.

Methods

A total of 97 proximal residual plaque lesions (plaque burden [PB] >40%) treated with everolimus-eluting stents were retrospectively evaluated to determine the distance from the stent edge to the residual plaque.

Results

The SER group had significantly higher PB (59.1 ± 6.1% vs. 51.9 ± 9.1% for non-SER; P = 0.04). Higher PB was associated with SER, with the cutoff value of 54.74% determined using receiver operating characteristic (ROC) curve analysis. At this cutoff value of PB, the distance from the stent edge to the lesion was significantly associated with SER (odds ratio = 2.05, P = 0.035). The corresponding area under the ROC curve was 0.725, and the cutoff distance value for predicting SER was 1.0 mm.

Conclusion

An interval less than 1 mm from the proximal stent edge to the nearest point with the determined PB cutoff value of 54.74% was significantly associated with SER in patients with residual plaque lesions.     

LARGE2 facilitates the maturation of alpha-dystroglycan more effectively than LARGE

The LARGE gene is thought to encode a putative glycosyltransferase because of its typical topology. However, no enzyme activity has been demonstrated yet, although the gene apparently supports the functional maturation of alpha-dystroglycan by glycosylation when it is transfected into cells. A novel homologous gene to LARGE was identified and named LARGE2. LARGE2 recombinant was co-expressed with alpha-dystroglycan in human embryonic kidney 293T cells to determine its activity to support the maturation of alpha-dystroglycan. The alpha-dystroglycan co-transfected with LARGE2 was more highly glycosylated than that co-transfected with LARGE. Pull-down experiments demonstrated binding activity of LARGE2 as well as LARGE toward alpha-dystroglycan. LARGE2 was found to support the maturation of alpha-dystroglycan more effectively than LARGE. Both of them are ubiquitously expressed in many tissues, except the brain where LARGE2 was not expressed at all. This compensatory function can explain the residual functionally glycosylated alpha-dystroglycan in a patient with MDC1D whose LARGE genes are congenitally null.     

Endothelin-1 promotes vascular structural remodeling during the progression of heart failure prevention of vascular remodeling using a specific endothelin-converting enzyme inhibitor.

To evaluate the effects of endothelin (ET)-converting enzyme (ECE) inhibitor on vascular remodeling in dogs with congestive heart failure (CHF), we chronically administered an ECE inhibitor, FR901533 (FR, iv. 0.3mg/kg/hr, n=6), to dogs with CHF induced by rapid ventricular pacing. Vehicle CHF dogs were given saline (n=7). In the vehicle CHF group after 3 weeks of pacing, the ET system was activated in the plasma and vasculature (3 and 5 times higher than normal, respectively). Inward remodeling occurred in the femoral artery; medial thickness (MT, 225+/-5 vs 193+/-4 microm, P<0.05) and deposition of collagen (DC, 22+/-2 vs 17+/-1%, P<0.01) significantly increased, while lumen diameter (LD, 1173+/-39 vs 1481+/-44 microm, P<0.05) decreased in the femoral artery with CHF compared with the normal femoral artery. There were significant correlations between the number of ET-1 positive cells and MT, DC, LD and systemic vascular resistance. FR significantly suppressed the changes in these vascular parameters compared with the changes in the vehicle CHF group despite the lack of an effect on blood pressure, and moreover FR caused decreases in ET-1 levels in both the plasma and femoral artery (reduced to 43% and 54%, respectively, of the levels in the vehicle CHF group, P<0.05). In conclusion, ET-1 plays a critical role in the structural deterioration of the vasculature during the progression of CHF, and ECE inhibitors can prevent the development of vascular remodeling.     

Effects of age and weaning on the immature rat uterotrophic assay using ethynylestradiol.

The purpose of this study was to evaluate the most appropriate rat age for the start of administration, and the effect of weaning, in the immature rat uterotrophic assay using ethynylestradiol (EE). Animals weaned on postnatal day (PND) 20 were administered subcutaneously EE at doses of 0.06-6 micrograms/kg/day for 3 days beginning on PND 21, 23 or 25. EE at the same doses was also administered to rats weaned on PND 17 or 20 from PND 21 for 3 days. Although uterine weight was significantly increased in the rats given 0.6-6 micrograms/kg EE in both of the studies, the percentage increase relative to the control in each group given EE from PND 21 and weaned on PND 20 was higher than in those groups given EE from PND 23 or 25, and the group weaned on PND 17.