The effects of nucleotides and potassium channel openers on the SUR2A/Kir6.2 complex K+ channel expressed in a mammalian cell line, HEK293T cells

 The effects of potassium channel opening drugs and intracellular nucleotides on the ATP-sensitive K+ (K_ATP) channel composed of SUR2A and Kir6.2 in HEK293T cells were examined using the patch-clamp technique. The SUR2A/Kir6.2 channel was activated effectively by pinacidil, marginally by nicorandil but not by diazoxide. The pinacidil-activated channel currents were inhibited by glibenclamide with a K _i value of 160 nM. Upon formation of inside-out (I-O) patches, spontaneous openings of the channels appeared, which were inhibited by intracellular ATP (ATP_i) equipotently in the presence and in the absence of intracellular Mg²⁺ (Mg²⁺ _i). The channel activity ran-down gradually in I-O patches. The run-down channels could be reactivated by ATP_i only in the presence of Mg²⁺ _i. Uridine 5’-diphosphate (UDP) antagonized the ATP_i-mediated inhibition of the channel activity before run-down. After run-down, UDP activated the channel without antagonizing ATP_i-mediated channel inhibition. Thus, the SUR2A/Kir6.2 reproduced the major properties of the native cardiac K_ATP channel well in terms of nucleotide regulation and pharmacology, and therefore can be a useful tool with which to elucidate the molecular mechanisms characterizing the K_ATP channel. Received: 24 October 1997 / Received after revision and accepted: 4 December 1997     

Renal primitive neuroectodermal tumor: An immunohistochemical and cytogenetic analysis

The cytogenetic and morphologic characteristics of a case with a primitive neuroectodermal tumor (PNET) arising from the left kidney in a 22 year old man are presented. The patient was detected as having a left renal mass with a tumor embolus In the inferior vena cava and multiple pulmonary metastases. A radical nephrectomy with tumor embolectomy of the Inferior vena cava, along with a resection of the pulmonary nodules were performed. Histologic examination revealed a dense proliferation of small round cells with many Homer-Wright type rosettes and perlvascular pseudo-rosettes. Immunohlstochemically, the tumor cells stained strongly positive for HBA71(p30/32^{M IC2}), a surface glycopro-teln specific to PNET and Ewlng's sarcoma. In addition, the tumor cells expressed several neural markers (neuron specific enolase, neurofilament, synaptophysin, and Leu-7) and vimentin, while the epithelial, muscular, and lymphocytlc markers were negative in the tumor cells. Cytogenetic analysis of cultured tumor cells showed a reciprocal translocation t(11; 22)(q24; q12) that is considered to be specific to PNET and Ewing's sarcoma. In conclusion, this case suggested that a karyotyping analysis is a useful diagnostic tool for renal PNET, and it may therefore be utilized to help distinguish between difficult cases of small round cell tumors and Wilms' tumor of the kidney.     

Hippocampal input to preoptic thermosensitive neurons in the rat

The effects of electrical stimulation of ventral subiculum (VSB) of the hippocampus of the thermosensitive neurons in the preoptic area were studied in urethane-anesthetized rats. VSB stimulation affected thermosensitive neurons more frequently (92.1%, 58 of 63) than thermally insensitive neurons (71.4%, 55 of 77). The majority of the VSB-responsive thermosensitive neurons (33 of 44 warm-units and 11 of 14 cold-units) were initially inhibited following stimulation. The result provides further support for the involvement of hippocampus in the central control of thermoregulation.     

Expression of insulin-like growth factor 1 receptor in primary breast cancer: immunohistochemical analysis

Insulin-like growth factor-1 receptor (IGF-1R) has been implicated in regulation in tumor growth. The results of previous studies performed by radioimmunoassay are conflicting, and the prognostic significance of IGF-1R expression in primary breast cancer is still controversial. IGF-1R expression was evaluated in formalin-fixed, paraffin-embedded tissue of 210 primary breast cancer patients by using anti-IGF-1R antibody. The clinicopathologic variables and 5-year disease-free survival were studied, and their correlations between IGF-1R expressions were investigated. IGF-1R overexpression was observed in 43.8% of tumors. IGF-1R overexpression had no correlation with prognosis or with other clinicopathologic parameters, such as age, tumor size, nodal status, histologic grade, hormone receptor status, and human epidermal growth factor 2 status. Though its prognostic value in breast cancer is limited, immunohistochemical evaluation of IGF-1R by using this monoclonal antibody may be useful in translational research using archived material.     

Detrimental effects after dobutamine infusion on rat left ventricular function: mechanical work and energetics

We have previously reported that continuous infusion of dobutamine into the coronary artery induces positive inotropic effects but induces no detrimental effects in cross-circulated, excised normal rat hearts and even in Ca2+ overload-induced contractile failing rat hearts. However, we hypothesized that some detrimental effects on left ventricular (LV) function are induced after continuous dobutamine infusion and the following clearance of blood dobutamine, as is the case after beta-adrenergic receptor stimulation. To test this hypothesis, we investigated LV mechanical work and energetics in the same type of preparations that underwent continuous dobutamine infusion and clearance of blood dobutamine. We found that both mean end-systolic pressure and systolic pressure-volume area (PVA; a measure of total mechanical energy per beat) at midrange LV volume were significantly (P < 0.01) decreased. The mean myocardial oxygen consumption per beat intercept, which is composed of for the total Ca2+ handling in excitation-contraction coupling and basal metabolism, of the and PVA linear relation was also significantly (P < 0.05) decreased (n=8). The mean slope of the linear relation was unchanged in such hearts. Post-dobutamine basal metabolism was unchanged (n = 5 of the 8 hearts). The moderate proteolysis of a cytoskeleton protein, alpha-fodrin was identified (n = 7 of the 8 hearts with the decreased intercept), after clearance of blood dobutamine. In agreement with our hypothesis, the detrimental effect of the post-beta-adrenergic receptor stimulation was induced by a moderate concentration of dobutamine; we found systolic dysfunction due to the impairment of Ca2+ handling in excitation-contraction coupling in the rat LV and proteolysis of a cytoskeleton protein, alpha-fodrin.     

Role of ubiquitin carboxy terminal hydrolase-L1 in neural cell apoptosis induced by ischemic retinal injury in vivo

Ubiquitin is thought to be a stress protein that plays an important role in protecting cells under stress conditions; however, its precise role is unclear. Ubiquitin expression level is controlled by the balance of ubiquitinating and deubiquitinating enzymes. To investigate the function of deubiquitinating enzymes on ischemia-induced neural cell apoptosis in vivo, we analyzed gracile axonal dystrophy (gad) mice with an exon deletion for ubiquitin carboxy terminal hydrolase-L1 (UCH-L1), a neuron-specific deubiquitinating enzyme. In wild-type mouse retina, light stimuli and ischemic retinal injury induced strong ubiquitin expression in the inner retina, and its expression pattern was similar to that of UCH-L1. On the other hand, gad mice showed reduced ubiquitin induction after light stimuli and ischemia, whereas expression levels of antiapoptotic (Bcl-2 and XIAP) and prosurvival (brain-derived neurotrophic factor) proteins that are normally degraded by an ubiquitin-proteasome pathway were significantly higher. Consistently, ischemia-induced caspase activity and neural cell apoptosis were suppressed approximately 70% in gad mice. These results demonstrate that UCH-L1 is involved in ubiquitin expression after stress stimuli, but excessive ubiquitin induction following ischemic injury may rather lead to neural cell apoptosis in vivo.     

The critical role of ocular-infiltrating macrophages in the development of choroidal neovascularization

Choroidal neovascularization (CNV) is directly related to visual loss in some eye diseases, such as age-related macular degeneration. Although several human histological studies have suggested the participation of macrophages in CNV formation, the precise mechanisms are still not fully understood. In this study, we elucidated the role of ocular-infiltrating macrophages in experimental CNV using CCR2 knockout (KO) mice, wild-type mice, and C57BL/6 (B6) mice. CCR2 is the receptor of monocyte chemoattractant protein-1, and the number of infiltrating macrophage and the area of CNV were significantly reduced in CCR2 KO mice. Enriched ocular-infiltrating macrophages from B6 mice actually showed angiogenic ability in a dorsal air sac assay. Moreover, their expression of class II, CD40, B7-1 and B7-2 molecules, and the mRNA for potential angiogenic factors, such as vascular endothelial growth factor, basic fibroblast growth factor, and tumor necrosis factor alpha, was also observed. Collectively, we conclude that ocular-infiltrating macrophages play an important role in CNV generation.     

Cytogenetic studies of Hynobiidae (Urodela) XIX. Morphological variation of sex chromosomes pairing behavior of sex lampbrush chromosomes in <Emphasis Type="Italic">Hynobius quelpaertensis</Emphasis> (Mori) from Cheju Island,South Korea

Using Giemsa staining, C-banding and Ag-NOR staining techniques, we analyzed chromosomes in adult male and female Hynobius quelpaertensis and in embryos of this species in egg sacs collected from eight localities of Cheju Island, South Korea. Chromosome pair 21 was consistently homomorphic in male specimens, while it was heteromorphic in female specimens, suggesting the occurrence of ZZ/ZW sex chromosome constitution in this species. The W chromosome, being much larger than the Z chromosome, was of three morphologically distinct types: W^A, W^B and W^C. Lampbrush chromosomes examined in the oocytes of one female specimen having the W^A chromosome showed that the short arm of the W^A chromosome and the long arm of the Z chromosome paired closely and hence are genetically homologous. We also tried to analyze the structural relationship among the three types of W chromosomes based on their C-banding and Ag-NOR patterns.