Long-term treatment with glibenclamide increases susceptibility of streptozotocin-induced diabetic rat heart to reperfusion-induced ventricular tachycardia

This study investigated the effects of long-term treatment with glibenclamide (GLIB) on the susceptibility of streptozotocin (STZ)-induced diabetic heart to ischemia/reperfusion insults. Starting 4 weeks after the injection of STZ, rats were treated with GLIB (0.1 mg/kg, ip, three times a week, STZ-GLIB group) or vehicle (STZ-VEH group) for 8 weeks. The recovery of cardiac performance, released creatine kinase (CK), and incidence of ventricular arrhythmias were studied during the reperfusion period in isolated hearts from rats in STZ-GLIB (n = 14) and in STZ-VEH groups (n = 13) and from age-matched control rats (CNT group, n = 14). Each heart was subjected to 5 min of global low-flow ischemia followed by 25 min of no-flow ischemia, with a subsequent 30 min of reperfusion. Plasma glucose level was not significantly different between the STZ-GLIB and STZ-VEH groups. The recovery of cardiac performance and the released CK during reperfusion period were significantly lower in both STZ-VEH and STZ-GLIB groups than in the CNT group (P < 0.01 and P < 0.05, respectively). Reperfusion resulted in an incidence of ventricular fibrillation in 23% and 21% in STZ-VEH and STZ-GLIB groups, respectively (P = ns). These values were significantly lower than that of the CNT group (100%, P < 0.001 for both). More importantly, the incidence of ventricular tachycardia in the STZ-GLIB group was significantly higher than that in the STZ-VEH group (93% vs 54%, P < 0.05) and was not significantly different from that in the CNT group (93% vs 100%, P = ns). The results suggest that STZ-induced protection against reperfusion-induced ventricular arrhythmias in diabetic heart may be partially abrogated by long-term treatment with GLIB.     

Establishment of a monoclonal antibody for human LXRα: Detection of LXRα protein expression in human macrophages

Liver X activated receptor alpha (LXRalpha) forms a functional dimeric nuclear receptor with RXR that regulates the metabolism of several important lipids, including cholesterol and bile acids. As compared with RXR, the LXRalpha protein level in the cell is low and the LXRalpha protein itself is very hard to detect. We have previously reported that the mRNA for LXRalpha is highly expressed in human cultured macrophages. In order to confirm the presence of the LXRalpha protein in the human macrophage, we have established a monoclonal antibody against LXRalpha, K-8607. The binding of mAb K-8607 to the human LXRalpha protein was confirmed by a wide variety of different techniques, including immunoblotting, immunohistochemistry, and electrophoretic mobility shift assay (EMSA). By immunoblotting with this antibody, the presence of native LXR protein in primary cultured human macrophage was demonstrated, as was its absence in human monocytes. This monoclonal anti-LXRalpha antibody should prove to be a useful tool in the analysis of the human LXRalpha protein.     

Expression of the HB9 homeobox gene concomitant with proliferation accompanying epidermal stratification during development of chick embryonic tarsometatarsal skin

A homeobox gene, HB9, has been isolated from the tarsometatarsal skin of 13-day-old chick embryos using a degenerate RT-PCR-based screening method. In situ hybridization analysis revealed that, during development of chick embryonic skin, the HB9 gene was expressed in epidermal basal cells of the placodes, but not in those of interplacodes, and in the dermal cells under the placodes at 9 days before addition of an intermediate layer by proliferation of the basal cells in the placodes. With the onset of epidermal stratification, the direction of the basal cell mitosis changed, with the axis becoming vertical to the epidermal surface. Placodes and interplacodes form outer and inner scales, respectively, after they have elongated distally (Tanaka S, Kato Y (1983b) J Exp Zool 225: 271–283). During scale ridge elongation at 12–15 days, HB9 was strongly expressed in the epidermis of the outer scale face, where the cell proliferation is more active than in the epidermis of the inner scale face; hence, stratification of the outer scale face is more prominent than that of the inner scale face. After 16 days, when mitotic activity in the epidermal basal cells decreases and the thickness of the epidermis is maintained at a constant level, the HB9 expression decreases with the onset of epidermal keratinization. These results suggest that HB9 may be involved in the proliferation of the epidermal basal cells that accompanies epidermal stratification.     

Steady-state force–velocity relation of ATP-dependent sliding between slime mold myosin, arranged on paramyosin filaments, and algal cell actin cables

To investigate characteristics of ATP-dependent sliding of a non-muscle cell myosin, obtained from a cellular slime mold Dictyostelium discoideum, on actin filament, we prepared hybrid thick filaments, in which Dictyostelium myosin was regularly arranged around paramyosin filaments obtained from a molluscan smooth muscle. A single to a few hybrid filaments were attached to a polystyrene bead (diameter, 4.5 μm; specific gravity, 1.5), and the filaments were made to slide on actin filament arrays (actin cables) in the internodal cell of an alga Chara corallina, mounted on the rotor of a centrifuge microscope. The filament-attached bead was observed to move with a constant velocity under a constant external load for many seconds. The steady-state force–velocity relation of Dictyostelium myosin sliding on actin cables was hyperbolic in shape except for large loads ≤0.7–0.8 P₀, being qualitatively similar to that of skeletal muscle fibres, despite a considerable variation in the number of myosin molecules interacting with actin cables. Comparison of the P–V curves between Dictyostelium myosin and muscle myosins sliding on actin cables suggests that the time of attachment to actin in a single attachment–detachment cycle is much longer in Dictyostelium myosin than in muscle myosins.     

Quantitative determination of disopyramide, verapamil and flecainide enantiomers in rat plasma and tissues by high-performance liquid chromatography

Enantiomers of disopyramide (DP), flecainide (FLC) and verapamil (VP) were extracted from rat plasma and tissues (brain, lung, heart, liver, kidney and muscle), followed by quantitative determination using enantioselective high-performance liquid chromatography with chiral stationary-phase columns. The recoveries of S-(+)- and R-(−)-DP from tissues were higher than 69%, and the within- and between-day coefficients of variation were very low (0.5 – 5.7%). The lower limits of detection in each tissue were less than 289 ng/g tissue. The recoveries of S-(+)- and R-(−)-FLC from tissues were higher than 88%, and the within- and between-day coefficients of variation were 1.2–6.0%. The lower limits of detection in each tissue were less than 37 ng/g tissue. The recoveries of S-(−)- and R-(+)-VP from tissues were higher than 80%, and the within- and between-day coefficients of variation were 0.5–6.2%. The lower limits of detection in each tissue were less than 51 ng/g tissue. The analytical methods established in this study will be suitable for determining the concentrations of the enantiomers of these anti-arrhythmic agents in rat plasma and tissues.     

Integrated Copy Number and Expression Analysis Identifies Profiles of Whole-Arm Chromosomal Alterations and Subgroups with Favorable Outcome in Ovarian Clear Cell Carcinomas

Ovarian clear cell carcinoma (CCC) is generally associated with chemoresistance and poor clinical outcome, even with early diagnosis; whereas high-grade serous carcinomas (SCs) and endometrioid carcinomas (ECs) are commonly chemosensitive at advanced stages. Although an integrated genomic analysis of SC has been performed, conclusive views on copy number and expression profiles for CCC are still limited. In this study, we performed single nucleotide polymorphism analysis with 57 epithelial ovarian cancers (31 CCCs, 14 SCs, and 12 ECs) and microarray expression analysis with 55 cancers (25 CCCs, 16 SCs, and 14 ECs). We then evaluated PIK3CA mutations and ARID1A expression in CCCs. SNP array analysis classified 13% of CCCs into a cluster with high frequency and focal range of copy number alterations (CNAs), significantly lower than for SCs (93%, P < 0.01) and ECs (50%, P = 0.017). The ratio of whole-arm to all CNAs was higher in CCCs (46.9%) than SCs (21.7%; P < 0.0001). SCs with loss of heterozygosity (LOH) of BRCA1 (85%) also had LOH of NF1 and TP53, and LOH of BRCA2 (62%) coexisted with LOH of RB1 and TP53. Microarray analysis classified CCCs into three clusters. One cluster (CCC-2, n = 10) showed more favorable prognosis than the CCC-1 and CCC-3 clusters (P = 0.041). Coexistent alterations of PIK3CA and ARID1A were more common in CCC-1 and CCC-3 (7/11, 64%) than in CCC-2 (0/10, 0%; P < 0.01). Being in cluster CCC-2 was an independent favorable prognostic factor in CCC. In conclusion, CCC was characterized by a high ratio of whole-arm CNAs; whereas CNAs in SC were mainly focal, but preferentially caused LOH of well-known tumor suppressor genes. As such, expression profiles might be useful for sub-classification of CCC, and might provide useful information on prognosis.     

The Investigation of Posttraumatic Pseudoaneurysms in Patients Treated with Nonoperative Management for Blunt Abdominal Solid Organ Injuries

Background

Posttraumatic pseudoaneurysms (PAs) have been recognized as the cause of delayed hemorrhage complicated with nonoperative management (NOM), although the need for intervention in patients with small-sized PAs and the relationship between the occurrence of PAs and bed-rest has been also unclear.

Objectives

The purpose of this study was to investigate the clinical history of small-sized PAs (less than 10 mm in diameter) which occurred in abdominal solid organs, and to analyze the relationship between the occurrence of PAs and early mobilization from bed.

Methods

Sixty-two patients who were successfully managed with NOM were investigated. Mobilization within three days post-injury was defined as “early mobilization” and bed-rest lasting over three days was defined as “late mobilization.” A comparison of the clinical factors, including the duration of bed-rest between patients with and without PAs detected by follow-up CT was performed. Furthermore, a multiple logistic regression model analysis on the occurrence of PAs was performed.

Results

PAs were detected in 7 of the 62 patients. The One patient with PAs measuring larger than 10 mm received trans-arterial embolization, and the remaining six patients with PAs smaller than 10 mm were managed conservatively. Consequently, no delayed hemorrhage occurred, and the PAs spontaneously disappeared in all of the six patients managed without intervention. The multiple regression model analysis revealed that early mobilization was not a significant factor predicting new-onset PAs.

Conclusions

Small PAs can be expected to disappear spontaneously. Moreover, early mobilization is not a significant risk factor for the occurrence of PAs.     

Laminar high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in endothelium. A quantitative analysis

Remodeling of endothelial basement membrane is important in atherogenesis. Since little is known about the actual relationship between type IV collagen and matrix metalloprotease−2 (MMP-2) in endothelial cells (ECs) under shear stress by blood flow, we performed quantitative analysis for type IV collagen and MMP-2 in ECs under high shear stress. The mRNA of type IV collagen from ECs exposed to high shear stress (10 and 30 dyn/cm²) had a higher expression compared to ECs exposed to a static condition or low shear stress (3 dyn/cm²) (P < 0.01). ³H-proline uptake analysis and fluorography revealed a remarkable increase of type IV collagen under high shear stress (P < 0.01). In contrast, zymography revealed that exposing to high shear stress, however similar positivity was leveled in the intracellular MMP-2 in the control and high shear stress-exposed ECs, reduced the secretion of MMP-2 in ECs. The results of Northern blotting, gelatin zymography and monitoring the intracellular trafficking of GFP-labeled MMP-2 revealed that MMP-2 secretion by ECs was completely suppressed by high shear stress, but the intracellular mRNA expression, protein synthesis, and transport of MMP-2 were not affected. In conclusion, we suggest that high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in ECs, which plays an important role in remodeling of the endothelial basement membrane and may suppress atherogenesis.     

Dynamic movements of Ro52 cytoplasmic bodies along microtubules

The RING-finger protein Ro52/TRIM21 is known as an autoantigen and is recognized by anti-Ro/SSA antibodies, which are commonly found in patients with Sjögren’s syndrome and systemic lupus erythematosus. Recently, Ro52 has been shown to localize to distinct structures called cytoplasmic bodies and function as an E3 ubiquitin ligase. However, the Ro52 cytoplasmic bodies have not been well characterized. In this study, we investigated the Ro52 cytoplasmic bodies using fluorescence microscopy. This analysis revealed that the Ro52 cytoplasmic bodies are diffusely located in the cytoplasm and exist independently of TRIM5α cytoplasmic bodies. Our results further showed that the Ro52 cytoplasmic bodies are not stained with MitoTracker dye and are not colocalized with the proteasome subunit Rpt5, the caveolae component caveolin-1, the endosome markers (EEA1, Rab5, and Rab7), and the lysosome marker LAMP2. These results indicate that the Ro52 cytoplasmic bodies are not mitochondria, proteasome-enriched structures, caveolae, endosomes, or lysosomes. Importantly, the Ro52 cytoplasmic bodies are highly motile and are located along the microtubule network. These results suggest that the Ro52 cytoplasmic bodies are unidentified structures that are transported along the microtubule network.     

Cytoplasmic relocation of Daxx induced by Ro52 and FLASH

The RING-finger protein Ro52/TRIM21 is known to be an autoantigen and is recognized by anti-Ro/SSA antibodies, which are commonly found in patients with Sjögren’s syndrome and systemic lupus erythematosus. We recently showed that Ro52 is an E3 ubiquitin ligase and localizes to cytoplasmic bodies that are highly motile along the microtubule network. To expand our knowledge of Ro52, we searched partners co-operating with Ro52. We performed a yeast two-hybrid screening of a human brain cDNA library with Ro52 as bait. This screening identified several genes encoding Ro52-interacting proteins, including the apoptosis-related proteins, Daxx and FLASH. Further yeast two-hybrid assays revealed that Daxx binds to the B30.2 domain of Ro52 and that FLASH binds to coiled-coil domains of Ro52 through its death-effector domain-recruiting domain. These results suggest that Ro52, Daxx, and FLASH form heteromeric protein complexes. Indeed, this was supported by results of immunoprecipitation experiments in which we found that Daxx is co-immunoprecipitated with Ro52 in the presence of overexpressed FLASH. Importantly, our fluorescence microscopy revealed that, although Daxx is predominantly located in the nucleus, overexpression of both Ro52 and FLASH leads to relocation of Daxx into the cytoplasm. Thus, Ro52 seems to co-operate with FLASH to induce cytoplasmic localization of Daxx in cells.