Regucalcin increases Ca2+-ATPase activity and ATP-dependent calcium uptake in the microsomes of rat kidney cortex

The effect of regucalcin, a Ca²⁺-binding protein, on Ca²⁺ transport system in rat renal cortex microsomes was investigated. The presence of regucalcin (10^-8 to 10^-6 M) in the reaction mixture caused a significant increase in Ca²⁺-ATPase activity and ATP-dependent⁴⁵ Ca²⁺ uptake in the microsomes. Regucalcin (10^-7 M) increased Ca²⁺-ATPase activity independently of increasing concentrations of CaCl_{_2}. The microsomal Ca²⁺-ATPase activity and⁴⁵ Ca²⁺ uptake were markedly decreased by the presence of vanadate (0.1 mM) or N-ethylmaleimide (NEM; 5 mM) in the absence or presence of regucalcin. Dithiothreitol (DTT; 5 mM) markedly elevated Ca²⁺-ATPase activity and ⁴⁵Ca²⁺ uptake in the microsomes. The DTT effects were not further enhanced by regucalcin (10^-7 M). Meanwhile, the microsomal Ca²⁺-ATPase activity and ⁴⁵Ca²⁺ uptake were significantly decreased by the presence of dibutyryl cyclic AMP (DcAMP; 10^-5 and 10^-3 M) or inositol 1,4, 5-trisphosphate (IP3; 10^-7 and 10^-5 M). The effect of regucalcin (10^-7 M) on Ca²⁺ ATPase activity and ⁴⁵Ca²⁺ uptake was weakened in the presence of DcAMP or IP₃. The present results demonstrate that regucalcin has a stimulatory effect on ATP-dependent Ca²⁺ uptake in the microsomes of rat renal cortex due to acting on the thiol groups of Ca²⁺-ATPase.     

Geographical variation in photoperiodic induction of larval diapause in the bruchid beetle, Bruchidius dorsalis: polymorphism in overwintering stages

The bruchid beetle, Bruchidius dorsalis Fahraeus (Coleoptera: Bruchidae), has a multivoltine life cycle and shows geographical variation of overwintering stages in Japan. Our previous study found that B. dorsalis enters larval diapause in the final instar under short photoperiods. In cooler areas, we observed that most individuals overwinter in the final larval stage in diapause, whereas beetles at different developmental stages (non‐diapausing young instars, diapausing instars, and adults) were overwintering in warmer areas. In this study, we investigated geographical variation in the photoperiodic response for induction of larval diapause at 20 °C (three populations) and 24 °C (two populations) to clarify the overwintering strategy of B. dorsalis. We observed that (1) diapause incidence at 20 °C changed sharply from ca. 100% to 0% with a change in photoperiod in all the populations, (2) critical photoperiod was longer at 20 °C in populations from cooler areas, and (3) critical photoperiod at 24 °C was shorter than at 20 °C and a fraction of the larvae did not enter diapause, even under short photoperiods. Overwintering stages estimated from these results were consistent with those actually observed in the field. This study indicates that the geographical variation of overwintering stages is likely to reflect adaptive diapause induction in each local environment.     

Suppressed expression of calcium-binding protein regucalcin mRNA in the renal cortex of rats with chemically induced kidney damage

The alteration of Ca²⁺-binding protein regucalcin mRNA expression in the kidney cortex of rats administered cisplatin and cephaloridine, which can induce kidney damage, was investigated. Cisplatin (0.25, 0.5 and 1.0 mg/100 g body weight) or cephaloridine (25, 50 and 100 mg/100 g) was intraperitoneally administered in rats, and 1, 2 and 3 days later they were sacrificed. The alteration in serum findings after the administration of cisplatin (1.0 mg/100 g) or cephaloridine (50 and 100 mg/100 g) demonstrated chemically induced kidney damage; blood urea nitrogen (BUN) concentration increased markedly and serum inorganic phosphorus or calcium concentration decreased significantly. Moreover, the administration of cisplatin (1.0 mg/100 g) or cephaloridine (100 mg/100 g) caused a remarkable increase of calcium content in the kidney cortex of rats, indicating kidney damage. The expression of regucalcin mRNA in the kidney cortex was markedly reduced by the administration of cisplatin or cephaloridine in rats, when the mRNA levels were analyzed by Northern blotting using rat liver regucalcin cDNA (0.9 kb). The mRNA decreases were seen with the used lowest dose of cisplatin or cephaloridine. The present study clearly demonstrates that the mRNA expression of Ca²⁺-binding protein regucalcin in the kidney cortex of rats is decreased by chemically induced kidney damage.     

Developing a management procedure robust to uncertainty for southern bluefin tuna: a somewhat frustrating struggle to bridge the gap between ideals and reality

Fisheries management is conducted to achieve sustainable use of fishery resources, mainly through regulation of fishing activities. For almost a decade, the Commission for the Conservation of Southern Bluefin Tuna (CCSBT) struggled to reach agreement on a total allowable catch (TAC) for southern bluefin tuna (SBT) because of stock assessment uncertainties. To address this, in 2002 the CCSBT commenced development of a management procedure (MP), a pre-agreed set of rules to determine how the TAC will be adjusted as new monitoring data become available. The CCSBT Scientific Committee tested various candidate MPs using operating models which simulate fish population and fishery dynamics as well as incorporate process, observation, and model uncertainties. Candidate MPs were evaluated using performance measures related to the following management objectives: maximize catches, avoid stock collapse, and minimize interannual catch variation. Of the MPs explored, some relied solely on empirical data [i.e., adjusted TAC based on catch per unit effort (CPUE) trends], whereas others were more complicated, based on population models. In 2005, the CCSBT adopted a model-based MP that realized a moderate catch with low variability and avoided stock collapse. This MP struck a compromise between the risk-prone and risk-averse standpoints of the different stakeholders. However, despite this concerted scientific effort, the MP was not implemented because, shortly after its adoption, it became evident that historical catches may have been substantially underreported. This complication necessitates returning to near the beginning of the development process. MP approaches have various advantages and challenges to be explored further. However, it is essential to lessen human-introduced uncertainty (such as catch misreporting) by enhanced enforcement, and to increase management robustness to biological uncertainties by implementing MPs.     

Calcium-binding protein regucalcin mRNA expression in the kidney cortex is suppressed by saline ingestion in rats

The effect of adrenalectomy (ADX) or saline ingestion, which is a hypertensive factor, on the expression of calcium-binding protein regucalcin mRNA in the kidney cortex of rats was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using rat liver regucalcin complementary DNA (0.9 kb of open-reading frame). Regucalcin mRNA was expressed in the kidney cortex but not the medulla. Rats were adrenalectomized, and 48 h later they were sacrificed. ADX caused a reduction of regucalcin mRNA levels in the kidney cortex, suggesting that adrenal glands participate in the regulation of the mRNA expression. This reduction was not restored by the subcutaneous administration of dexamethasone with an effective dose (1 mg/kg body weight), which can stimulate kidney regucalcin mRNA expression. Regucalcin mRNA levels in the kidney cortex of rats were markedly suppressed by the ingestion of saline for 7 days. The ADX-induced decrease of renal cortex regucalcin mRNA levels was not appreciably restored by saline ingestion. Moreover, regucalcin mRNA levels in the kidney cortex of spontaneous hypertensive rats (SHR) were clearly decreased as compared with that of control (Wistar-Kyoto) rats. Meanwhile, calcium content in the kidney cortex was not significantly decreased by ADX or saline ingestion. The present study suggests that the expression of regucalcin mRNA in the kidney cortex of rats is suppressed by saline administration.     

Interrelation between Ethylene and Carbon Dioxide in Relation to Respiration and Adenylate Content in the Pre-Germination Period of Cocklebur Seeds

Effects of C₂H₄ and CO₂ on respiration of pre-soaked upper cocklebur(Xanthium pennsylvanicum Wallr.) seeds during a pre-germinationperiod were examined in relation to effects of the two gaseson germination. At 33?C, cocklebur seed germination was greatlystimulated. This high temperature-stimulated germination wasseverely inhibited by C₂H₄, but not by CO₂, although both gasesstimulated germination at 23?C. C₂H₄ promoted seed respirationat 23?C, but its promotive effect decreases with increasingtemperature and disappeared at about 35?C, while CO₂ stimulatedrespiration regardless of temperature. CO₂ augmented the operationof the CN-sensitive, cytochrome path (CP) regardless of temperature,resulting in an increase in the ratio of the CP flux to a CN-resistant,alternative path (AP) flux. On the other hand, C₂H₄ augmentedthe operation of both paths, particularly of the AP, at 23?C,where it promoted germination. However, at 33?C where germinationis suppressed by C₂H₄, C₂H₄ preferentially stimulated respirationvia the AP, thus leading to an extremely high ratio of AP toCP. The inhibitory effect of C₂H₄ on germination at 33?C disappearedcompletely in enriched O₂, under which conditions CP is knownto be augmented. At 23?C, CO₂ and C₂H₄ acted independently incontrolling seed respiration, but they were antagonistic at33?C. The independent action appeared when the AP flux was verylow relative to the CP flux, while the antagonism appeared whenthe AP flux had risen. This differential action of the two gasesat different temperatures was also observed in the ATP level,adenylate pool size and energy charge of the axial tissues.These results suggest that the germination-controlling actionsof both CO₂ and C₂H₄ are fundamentally manifested through themodification of respiratory systems. However, the germination-inhibitingeffect of C₂H₄ at 33 ?C was not removed by inhibitors of AP,and there was little difference in the adenylate compounds betweenthe C₂H₄-treated and non-treated seeds at 33?C. Therefore, thephysiological action of C₂H₄ can not be explained only in termsof regulation of the respiratory system. (Received January 24, 1986; Accepted November 17, 1986)     

Steroid hormonal regulation of calcium-binding protein regucalcin mRNA expression in the kidney cortex of rats

Ethylenediamine tetraacetate ( EDTA ) inhibits lactoperoxidase (LPO)-catalyzed rate of iodide oxidation in concentration and pH-dependent manner. A plot of log Ki_app values against various pH yields a sigmoidal curve from which an ionisable group of pK_a value 6.0 could be ascertained for controlling the inhibition of catalytically active LPO by EDTA. Kinetic studies indicate that EDTA competitively inhibits iodide oxidation by acting as an electron donor. EDTA al so reduces LPO-compound-11 to the native ferric state by one-electron transfer as evidenced by the spectral shift from 428 to 412 nm. Optical difference spectroscopic studies indicate that EDTA binds to LPO with the apparent equilibrium dissociation constant (KD) of 12 ± 2 mM at pH 6.5. A plot of log K_D values against various pH produces a sigmoidal curve from which an ionisable group of LPO having pk_a = 5.47 could be calculated, deprotonation of which favours EDTA binding. EDTA also binds to LPO-CN- complex indicating its binding site away from heme iron centre. The K_D of LPO-EDTA complex is significantly increased (62 ± 5 mM) by iodide suggesting that EDTA binds close to the iodide binding site. EDTA also increases the K_D value of LPO-hydroquinone complex from 62 ± 5 mM to 200 ± 21 mM indicating that EDTA and aromatic donor binding sites are also close. We suggest that EDTA inhibits iodide oxidation competitively as an electron donor by interacting at or near the iodide binding site and these sites are close to the aromatic donor binding site.     

Hepatic calcium-binding protein regucalcin concentration is decreased by streptozotocin-diabetic state and ethanol ingestion in rats

The alteration in calcium-binding protein regucalcin in the liver and serum of rats with streptozotocin (STZ)-diabetic state or ethanol ingestion was investigated. STZ (6.0 mg/100 g body weight) was subcutaneously administered in rats, and 1 or 3 weeks later they were sacrificed by bleeding. Liver regucalcin mRNA levels were not clearly altered by the diabetic state, as evidenced by Northern blotting using regucalcin cDNA (0.9 kb of open reading frame). Based on enzyme-linked immunoadsorbent assay (ELISA) with rabbit-anti-regucalcin IgG, hepatic regucalcin concentration was decreased about 50% of control levels by STZ treatment. However, serum regucalcin concentration was not significantly altered by STZ treatment. Meanwhile, when rats ingested ethanol (10 and 30%) in the drinking water for 2 weeks, liver regucalcin mRNA levels were clearly increased, although hepatic regucalcin concentration was significantly decreased. Serum regucalcin concentration was not appreciably altered. Serum transaminases (GOT and GPT) activities were significantly increased at 1 or 3 weeks after STZ administration in rats, while their activities were not altered by ethanol ingestion. The present study demonstrates that hepatic regucalcin concentration is decreased independent of mRNA expression in the STZ-diabetes and during ethanol ingestion in rats.     

Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent protein kinase activity in rat renal cortex cytosol

The effect of regucalcin on Ca²⁺/calmodulin-dependent protein kinase activity in the cytosol of rat renal cortex was investigated. Regucalcin is a calcium-binding protein which exists in rat liver and renal cortex. Protein kinase activity in renal cortex cytosol was markedly increased by the addition of CaCl₂ (0.5 mM) plus calmodulin (10 µg/ml) in the enzyme reaction mixture. This increase was completely prevented by the addition of trifluoperazine (25 µM), an antagonist of calmodulin. The cytosolic Ca²⁺/calmodulin- dependent protein kinase activity was clearly inhibited by the addition of regucalcin; an appreciable effect of regucalcin was seen at 0.01 µM. The cytosolic Ca²⁺/calmodulin-dependent protein kinase activity was fairly increased by increasing concentrations of added Ca²⁺ (100-1000 µM). This increase was markedly blocked by the presence of regucalcin (0.1 µM). The inhibitory effect of regucalcin on the protein kinase activity was also seen with varying concentrations of calmodulin (2-20 µg/ml). These results demonstrate that regucalcin can regulate Ca²⁺/calmodulin-dependent protein kinase activity in renal cortex cells.     

Expression of calcium-binding protein regucalcin mRNA in the kidney cortex of rats: The stimulation by calcium administration

The expression of calcium-binding protein regucalcin mRNA in the kidney cortex of rats was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin complementary DNA (0.9 kb of open-reading frame). Regucalcin mRNA was expressed in the kidney cortex, and this expression was clearly increased by a single intraperitoneal administration of calcium chloride solution (5–15 mg Ca/100 g body weight) in rats; this increase was remarkable at 60–120 min after the administration. Thyroparathyroidectomy (TPTX) caused a slight decrease of regucalcin mRNA levels in the kidney cortex. However, the administration of calcium (10 mg/100 g) in TPTX rats produced a clear increase of regucalcin mRNA levels in the kidney cortex. The subcutaneous administration of calcitonin (10–100 MRC mU/100 g) or parathyroid hormone [1–34] (1–10 U/100 g) in TPTX rats which received calcium (10 mg/100 g) administration did not cause an appreciable alteration of regucalcin mRNA levels in the kidney cortex, suggesting that the mRNA expression is not stimulated by calcium-regulating hormones. The administration of trifluoperazine (TFP; 5 mg/100 g), an inhibitor of Ca²⁺/calmodulin action, completely blocked the expression of regucalcin mRNA stimulated by calcium administration. Now, calcium content in the kidney cortex was significantly elevated by a single intraperitpneal administration of calcium (10 mg/100 g) in rats. The present study clearly demonstrates that the expression of regucalcin mRNA in the kidney cortex is stimulated by calcium administration in rats. This expression may be mediated through Ca²⁺/calmodulin action in the kidney cortex.