The influence of cadmium (CdCl2) on the canine plasma levels of gastrin,cholecystokinin (CCK), and pancreatic polypeptide (PP)

The influence of cadmium on basal and stimulated plasma levels of gastrin, cholecystokinin (CCK), and pancreatic polypeptide (PP) was investigated in conscious dogs using three doses of cadmium (0.15, 0.5, and 0.75 mg Cd/kg-h). Levels of gastrointestinal (GI) hormones were stimulated with bombesin (BBS), a peptide known to stimulate GI hormone release. Plasma cadmium was measured employing atomic absorption spectrophotometry and GI hormone levels were measured with specific radioimmunoassays (RIA). Basal plasma levels of hormones (pg/mL) in the dogs were in the range (mean ± SE): 38±5 to 44±6 for gastrin, 80±25 to 107±17, for CCK and 120±5 to 142±5 for PP; these levels did not change with cadmium. Significant increases above basal levels in all three hormones were found with infusions of BBS and with BBS plus cadmium. Gastrin levels remained steady during Cd and saline after BBS; however, CCK and PP levels dropped to values that were 68 and 73% less than their stimulated peak levels. With reinfusion of BBS, gastrin, CCK, and PP were significantly elevated above basal; however, the peak values for CCK and PP, but not gastrin, were less than those found during the first BBS infusion. The data suggest that in response to bombesin, cadmium has little or no effect on the release of gastrin, but that is exerts a latent effect on the release of both CCK and PP.     

Seasonal and diurnal melatonin production in exercising sled dogs

Melatonin is a hormone that is released from the pineal gland into the blood stream and is controlled by nerve impulses from the suprachiasmatic nuclei. Melatonin synthesis, which is inhibited by light on the mammalian retina, peaks in plasma concentrations during the night. Though still a subject of intense research, melatonin in mammals is known to effect the reproductive system, thyroid function, and adaptations to seasonal changes. Sled dogs in Fairbanks, Alaska (65 degrees N) can be exposed to anywhere from 21 h of daylight in the summer to 4 h in the winter. While light may be the primary factor influencing melatonin production, we hypothesized that exercise may also affect melatonin production. In the current study, sled dogs were used to study seasonal and diurnal variation in melatonin production. Sled dogs by nature are elite athletes and therefore exercise was a focus in the study. Both exercise and non exercise dogs from 2 distinct latitudes were used. The peak in melatonin production was prolonged in high latitude dogs (65 degrees N), compared with lower latitude dogs (45 degrees N). Dogs at both latitudes show a reduction in peak melatonin levels with exercise, and winter melatonin levels in both locations were higher than the summer. Surprisingly, sled dogs in Alaska had lower melatonin levels than sled dogs in New York.     

Energy metabolism of Inuit sled dogs

We explored how seasonal changes in temperature, exercise and food supply affected energy metabolism and heart rate of Inuit dogs in Greenland. Using open flow respirometry, doubly labeled water, and heart rate recording, we measured metabolic rates of the same dogs at two different locations: at one location the dogs were fed with high energy food throughout the year while at the other location they were fed with low energy food during summer. Our key questions were: is resting metabolic rate (RMR) increased during the winter season when dogs are working? Does feeding regime affect RMR during summer? What is the proportion of metabolic rate (MR) devoted to specific dynamic action (SDA), and what is the metabolic scope of working Inuit sled dogs? The Inuit dogs had an extremely wide thermoneutral zone extending down to ?25°C. Temperature changes between summer and winter did not affect RMR, thus summer fasting periods were defined as baseline RMR. Relative to this baseline, summer MR was upregulated in the group of dogs receiving low energy food, whereas heart rate was downregulated. However, during food digestion, both MR and HR were twice their respective baseline values. A continuously elevated MR was observed during winter. Because temperature effects were excluded and because there were also no effects of training, we attribute winter elevated MR to SDA because of the continuous food supply. Working MR during winter was 7.9 times the MR of resting dogs in winter, or 12.2 times baseline MR.     

Inhibition of protein kinase CK2 expression and activity blocks tumor cell growth

Protein kinase CK2 (CK2) is a highly conserved and ubiquitous serine/threonine kinase. It is a multifunctional and pleiotropic protein kinase implicated in the regulation of cell proliferation, survival, and differentiation. Deregulation of CK2 is observed in a wide variety of tumors. It has been the focus of intensive research efforts to establish the cause–effect relationship between CK2 and neoplastic growth. Here, we further validate the role of CK2 in cancer cell growth using siRNA approach. We also screened a library of more than 200,000 compounds and identified several molecules, which inhibit CK2 with IC₅₀ < 1 μM. The binding mode of a representative compound with maize CK2 was determined. In addition, the cellular activity of the compounds was demonstrated by their inhibition of phosphorylation of PTEN Ser370 in HCT116 cells. Treatment of a variety of cancer cell lines with the newly identified CK2 inhibitor significantly blocked cell growth with IC₅₀s as low as 300 nM.     

Seasonal changes in adenine phosphoribosyltransferase and adenosine kinase activities as markers of the dormancy and the growth of Fragaria × ananassa

Because of the potential involvement of adenosine in the winter re-acquisition of nucleotide synthesis capability of strawberry plants (Fragaria × ananassa Duch., Elsanta), the properties and the time-course activity of an adenosine kinase (EC 2.7.1.20) and an adenine phosphoribosyltransferase (APRTase, EC 2.4.2.7) of the adenosine recycling pathway were characterized. The results showed an increase in APRTase activity during winter re-acquisition of nucleotide synthesis capability and an increase in adenosine kinase activity during spring growth of strawberry plants. Western blot analysis, performed with polyclonal rabbit antibodies raised against peach bud adenosine kinase, showed a concomitant rise in the strawberry enzyme. These results suggest that APRTase activity could be a good marker of the break of strawberry plant dormancy and adenosine kinase a marker of subsequent spring growth.     

Nonspecific, Reversible Inhibition of Voltage-Gated Calcium Channels by CaMKII Inhibitor CK59

Investigation of kinase-related processes often uses pharmacological inhibition to reveal pathways in which kinases are involved. However, one concern about using such kinase inhibitors is their potential lack of specificity. Here, we report that the calcium–calmodulin-dependent kinase II (CaMKII) inhibitor CK59 inhibited multiple voltage-gated calcium channels, including the L-type channel during depolarization in a dose-dependent manner. The use of another CaMKII inhibitor, cell-permeable autocamtide-2 related inhibitory peptide II (Ant-AIP-II), failed to similarly decrease calcium current or entry in hippocampal cultures, as shown by ratiometric calcium imaging and whole-cell patch clamp electrophysiology. Notably, inhibition due to CK59 was reversible; washout of the drug brought calcium levels back to control values upon depolarization. Furthermore, the IC₅₀ for CK59 was approximately 50 μM, which is only fivefold larger than the reported IC₅₀ values for CaMKII inhibition. Similar nonspecific actions of other CaMKII inhibitors KN93 and KN62 have previously been reported. In the case of all three kinase inhibitors, the IC₅₀ for calcium current inhibition falls near that of CaMKII inhibition. Our findings demonstrate that CK59 attenuates activity of voltage-gated calcium channels, and thus provide more evidence for caution when relying on pharmacological inhibition to examine kinase-dependent phenomena.     

CHICK BRAIN CREATINE KINASE ONTOGENY BEFORE THE ADVENT OF CREATINE AND INSULIN

The ontogeny of brain creatine kinase (CK) was studied during chick embryo development. The cytosolic activity increased 270% in 10 h from the 2nd to the 3rd days of incubation; this was followed by a plateau phase throughout development and at the end of incubation there appeared to be another increase of cytosolic and mitochondrial CK activities. Therefore, early embryonic chick brain CK is another‘constitutive’enzyme like the early embryonic chick heart CK since creatine has not been enzymatically detected in the embryo until day 4 of incubation. Insulin does not appear to stimulate the early increase of brain CK activity since the hormone is not present in the embryo until day 5 of incubation. It is likely that CK increase is associated with neuronal multiplication at early stages and possibly to neuronal maturation before hatching.     

Population dynamics, growth and reproduction of Corophium insidiosum (Crustacea: Amphipoda) at low salinities in Monolimni lagoon (Evros Delta, North Aegean Sea)

Distribution, population dynamics, growth and aspects of reproductive biology of Corophium insidiosum were investigated in Monolimni lagoon. Samples were collected in July 1997 (at 30 psu S) and during February 1998–May 1999 (at 0.1–5.7 psu S). Corophium insidiosumwas almost exclusively found in the outer part of the lagoon, which showed a higher water renewal rate. Population density gradually decreased during winter and spring, when salinity was lower than 1 psu and the amphipod finally vanished from the lagoon. Salinity increase during summer (1.2–5.7 psu) was followed by the re-occurrence of C. insidiosum with a time lag of 2–3 months. Population density increased in autumn and peaked in early winter at salinities 1.6–4.2 psu. Three cohorts appeared in the population during September 1998–March 1999. Breeding activity peaked in early autumn (14–21?°C, 4 psu S) and ceased after December (2–6.5?°C, ¡1.5 psu S). The preponderance of females in the large size classes resulted in a female- biased sex ratio in the whole population. The population showed a growth rate of 7.5–11.2 μm d^?1 being faster in autumn (9–21?°C, 3–4 psu S) than in winter (2–12?°C, 0.2–3 psu). An exponential relation existed between body length and cephalic length or dry body weight, while brood size was directly related to body length. Mean brood size was small (4.96 early embryos) and egg loss during development high (53%), possibly as a consequence of low salinities.     

Casein kinase II activity in the brain of an insect,Acheta domesticus: Characterization and hormonal regulation

This study documented casein kinase II (CK II) activity in Acheta domesticus brain using specific antibodies and its regulation by polyamines. In control animals a transient decrease in CK II activity at day 3 after imaginal moult was observed in the brain but not in the fat body. If deprived of ecdysone by ovariectomy a different pattern was observed, with CK II activity being significantly higher on days 3 and 4 after emergence. After ecdysone injection in ovariectomized females, CK II activity decreased to levels similar to those in controls. The implications of ecdysone regulation of brain CK II activity are discussed. © 1997 Wiley-Liss, Inc.     

Combination of Omega-3 Fatty Acids,Lithium, and Aripiprazole Reduces Oxidative Stress in Brain of Mice with Mania

Manic episode in bipolar disorder (BD) was evaluated in the present study with supplementation of omega-3 fatty acids in combination with aripiprazole and lithium on methylphenidate (MPD)-induced manic mice model. Administration of MPD 5 mg/kg bw intraperitoneally (i.p.) caused increase in oxidative stress in mice brain. To retract this effect, supplementation of omega-3 fatty acids 1.5 ml/kg (p.o.), aripiprazole 1.5 mg/kg bw (i.p.), and lithium 50 mg/kg bw (p.o) were given to mice. Omega-3 fatty acids alone and in combination with aripiprazole- and lithium-treated groups significantly reduced the levels of superoxide dismutase (SOD), catalase (CAT), and lipid peroxidation products (thiobarbituric acid reactive substances) in the brain. MPD treatment significantly decreased the reduced glutathione (GSH) level and glutathione peroxidase (GPx) activity, and they were restored by supplementation of omega-3 fatty acids with aripiprazole and lithium. There is no remarkable difference in the effect of creatine kinase (CK) activity between MPD-induced manic model and the treatment groups. Therefore, our results demonstrate that oxidative stress imbalance and mild insignificant CK alterations induced by administration of MPD can be restored back to normal physiological levels through omega-3 fatty acids combined with lithium and aripiprazole that attributes to effective prevention against mania in adult male Swiss albino mice.     

Architectonics of the hair of sled dogs of Chukotka

Architectonics of guard hairs from dogs of recent breeds, mongrel sled dogs, and fossil dogs from ancient settlements of Chukotka have been investigated using scanning electron microscopy. Distinct features of hair structure important for adaptation, including the adaptation to harness in sled dogs, were identified. Hairs of Chukchi sled dogs were most similar to those of the fossil dogs.     

Effect of nocturnal melatonin intake on cellular damage and recovery from repeated sprint performance during an intensive training schedule

ABSTRACT

An optimal recovery between training sessions is of similar if not greater importance as the training content and program of the training, itself. One of the most used strategies for improving recovery is the ingestion of supplements. The present study aimed to evaluate the effect of 5 mg oral melatonin supplementation on the recovery from repeated sprint (RSA) of performance and biochemical responses (i.e. oxidative stress, leukocytosis cellular damage) after an intensive training camp (TC). Twenty soccer players performed an RSA test before and after an intensive six-day TC associated with nocturnal melatonin (n = 10) or placebo (n = 10) ingestion. Resting and post-RSA test blood samples were obtained before and after the TC. Compared to placebo, melatonin intake decreased resting oxidative stress markers (i.e, advanced oxidation protein products), leukocytosis (i.e. white blood cells (WBC), neutrophils (NE)) and biomarkers of cellular damage (i.e. creatine kinase (CK)). It also lowered post-exercise leukocytosis (i.e. WBC, NE, lymphocytes (LY), monocytes (MO)) and biomarkers of cellular damage (i.e. CK, aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT)) and raised the activity of the main antioxidant enzymes (i.e. glutathione peroxidase (GPx), glutathione reductase (GR)). In addition, compared to placebo, melatonin reduced the deterioration of the best and total time during the RSA test after the TC. In conclusion, nocturnal melatonin supplementation during an intensive TC alleviated oxidative stress, leukocytosis and cellular damage and improved recovery of RSA performance in soccer players.     

Seasonal activity patterns and movements of the raccoon dog, a vector of diseases and parasites, in southern Finland

Activity patterns and movements of raccoon dogs (Nyctereutes procyonoides) were studied in Virolahti, southeast Finland, in 2000–2003. Activity data were compared to those collected from Evo, south-central Finland, in 1990–1993. Activity in winter was compared to weather (temperature and snow depth), day length and moon. Also circadian activity rhythm was studied in Evo. Raccoon dogs moved fastest in late winter after winter dormancy and slowest in autumn before settling in their winter dens. In March, males were moving more often than females. Raccoon dogs stayed usually in their dens in mid-winter (December–February) but were sometimes wandering around also during the harshest months of the year and changed their winter den on average three times. Both day length and weather affected the activity of raccoon dogs in winter. Animals usually stayed in their dens, when temperature was below –10 °C, snow depth >35 cm and day length <7 h and were moving around, when temperature was >0 °C, there was no snow and day length was >10 h. Day length and snow depth together predicted rather well the probability of animals being active during winter. Although raccoon dogs were more often active at night than during the light hours, they also showed rather much diurnal activity.     

Preclinical evaluation of the Aurora kinase inhibitors AMG 900, AZD1152-HQPA,and MK-5108 on SW-872 and 93T449 human liposarcoma cells

Liposarcoma is a malignant soft tissue tumor that originates from adipose tissue and is one of the most frequently diagnosed soft tissue sarcomas in humans. There is great interest in identifying novel chemotherapeutic options for treating liposarcoma based upon molecular alterations in the cancer cells. The Aurora kinases have been identified as promising chemotherapeutic targets based on their altered expression in many human cancers and cellular roles in mitosis and cytokinesis. In this study, we investigated the effects of an Aurora kinase A inhibitor (MK-5108), an Aurora kinase B inhibitor (AZD1152-HQPA), and a pan-Aurora kinase inhibitor (AMG 900) on undifferentiated SW-872 and well-differentiated 93T449 human liposarcoma cells. Treatment of the SW-872 and 93T449 cells with MK-5108 (0–1000 nM), AZD1152-HQPA (0–1000 nM), and AMG 900 (0–1000 nM) for 72 h resulted in a dose-dependent decrease in the total viable cell number. Based upon the EC₅₀ values, the potency of the three Aurora kinase inhibitors in the SW-872 cells was as follows: AMG 900 (EC₅₀ = 3.7 nM) > AZD1152-HQPA (EC₅₀ = 43.4 nM) > MK-5108 (EC₅₀ = 309.0 nM), while the potency in the 93T449 cells was as follows: AMG 900 (EC₅₀ = 6.5 nM) > AZD1152-HQPA (EC₅₀ = 74.5 nM) > MK-5108 (EC₅₀ = 283.6 nM). The percentage of polyploidy after 72 h of drug treatment (0–1000 nM) was determined by propidium iodide staining and flow cytometric analysis. AMG 900 caused a significant increase in polyploidy starting at 25 nM in the SW-872 and 93T449 cells, and AZD1152-HQPA caused a significant increase starting at 100 nM in the SW-872 cells and 250 nM in the 93T449 cells. The Aurora kinase A inhibitor MK-5108 did not significantly increase the percentage of polyploid cells at any of the doses tested in either cell line. The expression of Aurora kinase A and B was evaluated in the SW-872 cells versus differentiated adipocytes and human mesenchymal stem cells by real-time RT-PCR and Western blot analysis. Aurora kinase A and B mRNA expression was significantly increased in the SW-872 cells versus the differentiated adipocytes and human mesenchymal stem cells. Western blot analysis revealed a ~ 48 kDa immunoreactive band for Aurora kinase A that was not present in the differentiated adipocytes or the human mesenchymal stem cells. A ~ 39 kDa immunoreactive band for Aurora kinase B was detected in the SW-872 cells, differentiated adipocytes, and human mesenchymal stem cells. A smaller immunoreactive band for Aurora kinase B was detected in the SW-872 cells but not in the differentiated adipocytes and human mesenchymal stem cells, and this may reflect the expression of a truncated splice variant of Aurora kinase B that has been associated with poor patient prognosis. The 93T449 cells demonstrated decreased expression of Aurora kinase A and B mRNA and protein compared to the SW-872 cells, and also expressed the truncated form of Aurora kinase B. The results of these in vitro studies indicate that Aurora kinase inhibitors should be further investigated as possible chemotherapeutic agents for human liposarcoma.     

Effects of fasting and refeeding on antral, duodenal, and serum gastrin levels and on colonic thymidine kinase activity in rats

Antral, duodenal, and serum gastrin levels and colonic thymidine kinase activity were determined in 1- to 4-day-fasted rats and after refeeding of 4-day-fasted rats for 3-24 h. The effect of pentagastrin on colonic thymidine kinase activity was also determined. Total deprivation of food caused a drastic reduction in gastrin concentrations in serum and tissues. After 4 days of fasting, serum gastrin levels in most animals fell below the present detection limit of the assay (10-15 pg/ml), and antral and duodenal gastrin levels decreased to 15 and 50% of the respective initial fed control. After 9 and 24 h of refeeding, gastrin concentration in serum and antrum had increased to about 35% of the initial fed level. On the other hand, refeeding for 3-24 h produced no significant change in duodenal gastrin concentration. Fasting for 1-4 days resulted in a 60-70% reduction in colonic thymidine kinase activity, compared to the initial fed control. Refeeding caused a prompt stimulation in the enzyme activity, which after 6 h was found to be 72% above the 4-day-fasted group. Daily injection of pentagastrin, at doses between 125 and 500 micrograms/kg, during a 4-day fasting period resulted in a significant stimulation in colonic thymidine kinase activity, compared to the saline-treated control. The maximal stimulation of an enzyme activity 90% higher than in the saline control was attained with a pentagastrin dose of 125 micrograms/kg. Higher doses decreased the maximal stimulatory effect of pentagastrin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of single and repeated heat stress on chemical signals of heat shock response cascade in the rat’s heart

Exposure to sublethal heat stress activates a complex cascade of signaling events, such as activators (NO), signal molecules (PKCε), and mediators (HSP70 and COX-2), leading to implementation of heat preconditioning, an adaptive mechanism which makes the organism more tolerant to additional stress. We investigated the time frame in which these chemical signals are triggered after heat stress (41?±?0.5°С/45 min), single or repeated (24 or 72 h after the first one) in heart tissue of male Wistar rats. The animals were allowed to recover 24, 48 or 72 h at room temperature. Single heat stress caused a significant increase of the concentration of HSP70, NO, and PKC level and decrease of COX-2 level 24 h after the heat stress, which in the next course of recovery gradually normalized. The second heat stress, 24 h after the first one, caused a significant reduction of the HSP70 levels, concentration of NO and PKC?, and significant increase of COX-2 concentration. The second exposure, 72 h after the first heat stress, caused more expressive changes of HSP70 and NO in the 24 h-recovery groups. The level of PKC? was not significantly changed, but there was significantly increased COX-2 concentration during recovery. Serum activity of AST, ALT, and CK was reduced after single exposure and increased after repeated exposure to heat stress, in both time intervals. In conclusion, a longer period of recovery (72 h) between two consecutive sessions of heat stress is necessary to achieve more expressive changes in mediators (HSP70) and triggers (NO) of heat preconditioning.     

Cardiac depression and cellular injury in hemorrhagic shock and reinfusion: Role of free radicals

We investigated the effects of hemorrhagic shock and reinfusion on the cardiac function and contractility, plasma CK and CK-MB activity and lactate concentration, oxyradical-producing activity of polymorphonuclear leukocytes (PMNL-CL), cardiac chemiluminescence (LV-CL), antioxidant enzyme activity [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-P_X)] and malondialdehyde (MDA) concentration in anesthetized dogs to determine the role of oxyradicals in cardiac depression and cellular injury in hemorrhagic shock and reinfusion. The dogs were assigned into three groups: I (sham), 4 h duration; II (S + R), 2 h of shock followed by reinfusion for 2 h; III (SOD + S + R), as II but pretreated with PEG-SOD. Hemorrhagic shock was produced by withdrawal of blood to maintain the mean arterial pressure at 50 ± 5 mm Hg. Cardiac function and contractility were depressed during hemorrhagic shock. Plasma CK, CK-MB and lactate increased during shock. Following reinfusion after 2 h of shock hemodynamic parameters and plasma lactate tended to return towards control values. Plasma CK and CK-MB, PMNL-CL and cardiac MDA, total-, Mn- and CuZn-SOD activity increased while LV-CL decreased. In spite of the increase in the antioxidant reserve, there was oxidative damage. Pretreatment with SOD attenuated the deleterious effects of shock and reinfusion on the cardiovascular function, plasma CK, and CK-MB, PMNL-CL, cardiac MDA, SOD, and LV-CL. Protection was incomplete for cardiovascular function and plasma CK and CK-MB. These results suggest that oxyradicals may partly be involved in the deterioration of cardiovascular function and cellular injury during hemorrhagic shock and reinfusion.     

Induction and regulation of casein kinase II during B lymphocyte activation.

This study evaluates the regulation of casein kinase II (CK II) activity in resting B cells induced to enter the cell cycle. The induction of B cell cycle progression PMA and ionomycin results in an oscillatory expression of CK II. This kinase activity is also elicited after direct physical interaction between B cells and activated, fixed Th cells, indicating that the increase seen in CK II activity is probably associated with the delivery of the competence-inducing signal to resting B cells. The selective inhibition of ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine biosynthesis, during PMA and ionomycin-induction of B cell cycle progression, inhibits the expression of CK II activity. The addition of polyamines to cytosolic preparations recovered from cells in which ODC is inhibited results in the appearance of CK II activity, showing that the ODC inhibitor does not directly inhibit the kinase. The treatment of B cells with cycloheximide results in the appearance of CK II activity within 15 min, and this induction is partially explainable by a cycloheximide-elicited increase in cellular levels of polyamines. The artificial elevation of cellular levels of cAMP simultaneous with the addition of PMA and ionomycin results in a 150 to 200% increase in detectable CK II levels, suggesting that the cAMP-dependent signaling cascade may participate during the early regulation of CK II. In contrast, the inhibition of protein kinase C does not adversely influence the early expression of CK II, while actually enhancing kinase activity by 18 h poststimulation.     

The Role of Src Kinase in the Caspase-1 Pathway After Hypoxia in the Brain of Newborn Piglets

Hypoxia induces a cerebral inflammatory response, which contributes to brain injury. Inflammasomes are complex intracellular molecular structures that initiate the inflammatory cascade. Caspase-1 and interleukin 1-β (IL-1β), have been established as markers of inflammasome activation. Src kinase, a cytosolic non-receptor protein tyrosine kinase, is linked to cell proliferation and differentiation and is up regulated during hypoxia. The role of Src kinase in the above pathway is not fully understood. The present study tests the hypothesis that inhibition of Src kinase, by a selective inhibitor, PP2, will prevent the activation of caspase-1 and production of IL-1β acutely, as well as at 1 and 15 days after hypoxia in the cerebral cortex of the newborn piglet. Piglets were divided into: Normoxia (Nx), Hypoxia acute (Hx), Hypoxia-day 1 (Hx-day 1), and Hypoxia day 15 (Hx-day 15). Piglets pretreated with Src kinase inhibitor, PP2, 1 mg/kg IV, 30 min prior to hypoxia were divided into: Hypoxia acute (Hx + PP2), 1 day (Hx + PP2-day 1), and day 15 (Hx + PP2-day 15). Hypoxia was induced by exposing the piglets to an FiO₂ of 0.07 for 1 hour. Caspase-1 activity and expression were determined with spectrophotometry and Western blot respectively, while IL-1β levels were measured by solid phase ELISA. Caspase-1 activation was achieved immediately (within 1 h) after hypoxia and persisted for 15 days. IL-1β level was also increased after hypoxia reaching a maximum level at 24 h following hypoxia and returned to baseline by 15 days. Administration of PP2 attenuated the activity acutely, but not the expression of the caspase-1. IL-1β level at 24 h after hypoxia returned to baseline in piglets that were pretreated with PP2. We provide evidence that inhibition of Src kinase in the acute phase after hypoxia involves changes in the production or processing of caspase-1 subunits. Our data suggest that Src kinase mediates hypoxia-induced caspase-1 activation in the cerebral cortex of newborn piglets. Inhibition of Src kinase may attenuate the neuroinflammatory response and could represent a potential target for neuroprotection after hypoxic injury.     

Stimulation of CK2-dependent Grp94 phosphorylation by the nuclear localization signal peptide

The nuclear localization signal sequence (NLS) of SV40 Large T antigen is essential and sufficient for the nuclear translocation of the protein. Phosphorylation often modulates the intracellular distribution of signaling proteins. In this study, we investigated effects of the NLS-peptide of Large T antigen on protein phosphorylation. When crude cell lysates were incubated with [γ-³²P]ATP, phosphorylation of several endogenous substrates with molecular masses of 100, 80, 50, and 45 kDa by an endogenous kinase was stimulated by the addition of the wild type NLS-peptide (CPKKKRKVEDP). The mutated NLS-peptide (CPKTKRKVEDP) and the reversed NLS-peptide (PDEVKRKKKPC) are weak in the nuclear localization activity, and they only weakly stimulated phosphorylation of these substrates. The mobility of the 100 kDa phosphoprotein was indistinguishable with that of an endoplasmic reticulum (ER)-resident molecular chaperone glucose-regulated protein 94 (Grp94) belonging to the Hsp90 family, and purified Grp94 was phosphorylated by a kinase in cell lysates in an NLS-dependent fashion. The 100 kDa protein was identified as Grp94 by immunoprecipitation and reconstitution experiments. Purification of the NLS-dependent Grp94 kinase by sequential biochemical column chromatography steps resulted in isolation of two polypeptides with molecular masses of 42 and 27 kDa, which were identified as α and β subunit of protein kinase CK2, respectively, by western blotting analysis and biochemical characterization. Moreover, effect of an excess amount of GTP and V8 peptide mapping showed that the NLS-dependent Grp94 kinase in the cell lysate is identical with CK2. Surprisingly purified CK2 did phosphorylate Grp94 even without the NLS-peptide, suggesting that an additional suppressive factor is required for NLS-dependent phosphorylation of Grp94 by CK2. We suggest a possible general role for CK2-catalyzed phosphorylation in the regulation of NLS-dependent protein nuclear translocation.