Heterogeneity in the myoglobin content of chicken heart Purkinje fibers

Summary The myoglobin content of chicken myocardial cells was studied using indirect-immunoperoxidase histochemistry. While ordinary myocardial cells exhibited a homogeneous reaction pattern, the reactions for ventricular Purkinje fibers were remarkably heterogeneous. On the basis of the degree of staining, three types of cells, i.e., dark, intermediate, and clear, were distinguishable. In addition, the cytological heterogeneity of Purkinje cells was confirmed using conventional and immunological electron microscopy. The dark cells contained more myofibrils, mitochondria, and other organelles (e.g., ribosomes) than the clear cells.A part of this study was supported by a scientific research grant (59440037) from the Ministry of Education, Japan     

The influence of reduced glutathione in fertilization medium on the fertility of in vitro–matured C57BL/6 mouse oocytes

It is well known that IVM oocytes show a decreased potential for fertility and development compared with in vivo–matured oocytes. In this study, we added reduced glutathione (GSH) to the fertilization medium during IVF to investigate its effect on the fertility and early embryo development of IVM oocytes. The fertilization rate for IVM oocytes and fresh sperm increased with the addition of GSH (0, 1.0, and 2.0 mM: 51%, 76%, and 70%). Moreover, the addition of GSH to the fertilization medium also improved the developmental potential compared with the control sample (0 mM). In addition, we performed IVF using IVM oocytes and frozen/thawed sperm that had been cryopreserved in a mouse bank. Results indicated a marked increase in the fertilization rate when 1.0 mM GSH was added to the fertilization medium compared with when no GSM was used (0.0 mM GSH: 2% (3/195); 1.0 mM GSH: 33% (156/468)). Furthermore, the fertilization rate improved dramatically via zona drilling using laser equipment (52%: 267/516), whereas normal offspring were obtainsed after transferring embryos created via IVF using IVM oocytes and frozen/thawed sperm. This is the first report in which offspring have been obtained via IVF using IVM oocytes and frozen/thawed sperm.     

Increased expression of SLC2A9 decreases urate excretion from the kidney

Urate is the final metabolite of purine in humans. Renal urate handling is clinically important because under-reabsorption or underexcretion causes hypouricemia or hyperuricemia, respectively. We have identified a urate-anion exchanger, URAT1, localized at the apical side and a voltage-driven urate efflux transporter, URATv1, expressed at the basolateral side of the renal proximal tubules. URAT1 and URATv1 are vital to renal urate reabsorption because the experimental data have illustrated that functional loss of these transporter proteins affords hypouricemia. While mutations affording enhanced function via these transporter proteins on urate handling is unknown, we have constructed kidney-specific transgenic (Tg) mice for URAT1 or URATv1 to investigate this problem. In our study, each transgene was under the control of the mouse URAT1 promoter so that transgene expression was directed to the kidney. Plasma urate concentrations in URAT1 and URATv1 Tg mice were not significantly different from that in wild-type (WT) mice. Urate excretion in URAT1 Tg mice was similar to that in WT mice, while URATv1 Tg mice excreted more urate compared with WT. Our results suggest that hyperfunctioning URATv1 in the kidney can lead to increased urate reabsorption and may contribute to the development of hyperuricemia.     

Heterogeneous responses of recombinant human thyrotropin receptor to immunoglobulins from patients with Graves' disease.

Non-thyroid mammalian cells, CHO-K1 cells, stably expressing human thyrotropin receptor (CHO-TSH-R cells) were used for the assay of thyroid stimulating antibody (TSAb) activities of IgGs from 24 patients with Graves' disease and we compared them with the values obtained in porcine thyroid cells. A significant positive correlation was observed between the results given by CHO-TSH-R cells (hTSAb) and porcine thyrocytes (pTSAb) (r = 0.94, p less than 0.001). However, we found that hTSAb values of IgGs from 5 patients were extremely different from their hTSAb values. Four out of these 5 IgGs showed strong pTSAb activity but exhibited a weak or negative hTSAb activity. Conversely, one out of 5 autoantibodies was very strong for hTSAb but its pTSAb was low. These heterogeneous responses of recombinant hTSH-R to Graves' IgGs suggest that there exist different types of TSAb and also that the epitope(s) for TSAb may be different from case to case.     

Comparison of stable nitroxide, 3-substituted 2,2,5,5-tetramethylpyrrolidine-N-oxyls, with respect to protection from radiation, prevention of DNA damage, and distribution in mice

We compared three 3-substituted 2,2,5,5-tetramethylpyrrolidine-N-oxyls (PROXYLs): carbamoyl-, methoxycarbonyl-, and hydroxymethyl-PROXYL (CM-, MC-, and HM-PROXYL, respectively) with respect to radioprotection, prevention of DNA damage, and in vivo distribution in mice. The PROXYLs provided protection to C3H mice against lethal X-irradiation (8 Gy) with the following order of magnitude, HM- > CM- approximately MC-PROXYL. In contrast, radioprotection at the cellular level assessed by the colony formation of leukemia cell line L5178Y showed no difference among them. The degree of protection from X ray-induced oxidation of DNA bases measured by the formation of 8-hydroxydeoxyguanosine in salmon DNA and the cleavage of DNA measured by electrophoresis of plasmid pBR322 DNA did not differ among the PROXYLs. Redox potentials were also similar for each. However, the blood concentration of the PROXYLs injected ip into the mice showed different maximum concentrations (HM- > CM- approximately MC-PROXYL), although all reached a maximum at around 5-10 min and gradually decreased thereafter. Their concentration in bone marrow showed a similar pattern, suggesting that the difference in in vivo radioprotection among the three PROXYLs is due to the difference in their distribution to bone marrow. In general, the radioprotection provided by stable nitroxides is affected not only by redox potential and reactivity in vitro but also by pharmacokinetics.     

Oat5 and NaDC1 Protein Abundance in Kidney and Urine After Renal Ischemic Reperfusion Injury

The aim of this study was to evaluate the abundance of the organic anion transporter 5 (Oat5) and the sodium-dicarboxylate cotransporter 1 (NaDC1) in kidney and urine after renal ischemic reperfusion injury. Renal injury was induced in male Wistar rats by occlusion of both renal pedicles for 0 (Group Sham), 5 (Group I5R60), or 60 (Group I60R60) min. The studies were performed after 60 min of reperfusion. The expression of Oat5 and NaDC1 was evaluated by IHC and Western blotting. Oat5 and NaDC1 abundance and alkaline phosphatase activity (AP) were assayed in urine. A decreased expression in renal homogenates and apical membranes and an increase in urinary excretion of Oat5 and NaDC1 were observed in I60R60 rats, as well as alterations of other widely used parameters for renal dysfunction and injury (plasma creatinine, urinary AP activity, kidney weight, histological lesions). In contrast, in the I5R60 group, only an increase in urinary excretion of Oat5 and mild histopathological damage was detected. This is the first study on Oat5 and NaDC1 detection in urine. These results suggest that urinary excretion of Oat5 might be an early indicator of renal dysfunction, which is useful for detection of even minor alterations in renal structural and functional integrity. (J Histochem Cytochem 57:17–27, 2009)     

Genetic polymorphism of the swine major histocompatibility complex (SLA) class I genes, SLA-1, -2 and -3

In order to identify and characterize genetic polymorphism of the swine major histocompatibility complex (Mhc: SLA) class I genes, RT-PCR products of the second and third exons of the three SLA classical class I genes, SLA-1, SLA-2 and SLA-3 were subjected to nucleotide determination. These analyses allowed the identification of four, eight and seven alleles at the SLA-1, SLA-2 and SLA-3 loci, respectively, from three different breeds of miniature swine and one mixed breed. Among them, 12 alleles were novel. Construction of a phylogenetic tree using the nucleotide sequences of those 19 alleles indicated that the SLA-1 and -2 genes are more closely related to each other than to SLA-3. Selective forces operating at single amino acid sites of the SLA class I molecules were analyzed by the Adaptsite Package program. Ten positive selection sites were found at the putative antigen recognition sites (ARSs). Among the 14 positively selected sites observed in the human MHC (HLA) classical class I molecules, eight corresponding positions in the SLA class I molecules were inferred as positively selected. On the other hand, four amino acids at the putative ARSs were identified as negatively selected in the SLA class I molecules. These results suggest that selective forces operating in the SLA class I molecules are almost similar to those of the HLA class I molecules, although several functional sites for antigen and cytotoxic T-lymphocyte recognition by the SLA class I molecules may be different from those of the HLA class I molecules.The DNA sequence data reported in this paper have been submitted to the DDBJ, EMBL and GenBank nucleotide databases and have been assigned the accession numbers, AB105379, AB105380, AB105381, AB105382, AB105383, AB105384, AB105385, AB105386, AB105388, AB105389, AB105390 and AB105391     

Pineal and retinal melatonin is involved in the control of circadian locomotor activity and body temperature rhythms in the pigeon

Summary Although pinealectomy or blinding resulted in loss of the clarity of the free-running rhythm of locomotor activity and body temperature and reduced the peak level of circulating melatonin rhythms to approximately a half in intact pigeons, neither pinealectomy nor blinding abolished any of these rhythms. However, when pinealectomy and blinding were combined, the rhythms of locomotor activity and body temperature disappeared in prolonged constant dim light, and melatonin concentration was reduced to the minimum level of detection. In order to examine the role of melatonin in the pigeon's circadian system, it was administered either daily or continuously to PX + EX-pigeons in LLdim. Daily administration of melatonin restored circadian rhythms of locomotor activity which entrained to melatonin injections, but continuous administration did not induce any remarkable change of locomotor activity. These results suggest that melatonin synthesized in the pineal body and the eye contributes to circulating melatonin and its rhythmicity is important for the control of circadian rhythms of locomotor activity and body temperature in the pigeon.Abbreviations LD Light-dark - LLdim constant dim light - LLbright constant bright light - PX pinealectomy - EX blinding - SCN suprachiasmatic nucleus