Diversity of aquatic parasites in pristine spring waters in Tehsil Babuzai, Swat, Pakistan.

Global access to clean and safe drinking water remains a formidable challenge, contributing to a myriad of health issues. This research exposes the existence of waterborne parasites in seemingly pristine spring waters, indicating potential contamination. Daily extensive sampling of Seventeen water sources was conducted in the untarnished freshwater streams of Tehsil Babuzai, District Swat, Khyber Pakhtunkhwa, Pakistan, from February to September 2021. Employing a stringent filtration process, the collected samples were effectively concentrated to detect any waterborne parasites. Subsequent application of the wet mount technique, combined with the capabilities of a compound microscope, revealed a disconcerting reality: all examined samples tested positive for various parasites. Identified parasites included Schistosoma species, Ascaris lumbricoides, Trichiuria trichiuria, Taenia saginata, Entamoeba histolytica, Amoeba, Lacrymana olor, Tintinnids, Paramecium, Dileptus, Euglena, Loxodes striatus, Acanthocyclops lynceus, Spondylosium, Oscillatoria, Cyanobacteria, Cilindros, Cilindros cerro, Commensal amoeba mature cysts,, Filliform larva of Strongyloides, Cercaria larva, Larva of Taenia solium, Egg of Enterobius vermiculais, Egg of Isospora belli, Egg of Tapeworm, Egg of Schistosoma species, Egg of Toxocara, and Egg of Diphyllobothrium latum. These findings clearly demonstrate the presence of a diverse array of parasites in the freshwater springs of Tehsil Babuzai, Swat, Pakistan. Implementing robust water treatment protocols, conducting regular monitoring and testing, and raising awareness about the risks of waterborne parasites are crucial steps to safeguard public health in the region.

Etoposide Model-Induced Changes in Antioxidant Studies Histological and Ultrastructural Evaluations of Hepatic, Renal, and Cardiac Tissues of Male Rats / Cambios Inducidos por el Modelo de Etopósido en Estudios de Antioxidantes, Evaluaciones Histológicas y Ultraestructurales de Tejidos Hepáticos, Renales y Cardíacos de Ratas Macho

SUMMARY: Etoposide is an effective antimitotic and antineoplastic agent used to treat various human malignancies. In the present study, Etoposide was injected intraperitoneally into the rats at 1 mg/kg/day for 52 days (52 doses). The control animals received physiological saline (0.5 ml) intraperitoneally daily for 52 doses. The body weight of etoposide-treated rats was significantly reduced compared to control rats. Lipid peroxidation demonstrated an insignificant rise in hepatic tissue, a non-significant decline in renal tissue, and a significant reduction in cardiac tissue. The levels of GSH in hepatic and renal tissue were found to be non-significantly increased but significantly increased in cardiac tissue compared to controls. GR activity was found to be considerably decreased in the treated group. G-S-T levels increased significantly in all treated group. Etoposide injections caused a non-significant change in the GPX level of hepatic tissue, whereas renal and cardiac tissues showed a significant increase. The activity of CAT in hepatic tissue was significantly increased, while CAT activity in renal tissue showed a non-significant decrease, whereas in cardiac tissue, significantly lower levels were observed than in control group. The level of CYTp450 in hepatic and cardiac tissues showed a significant increase; however, renal tissue showed non-significant depletion, whereas CYTb5 in hepatic, renal, and cardiac tissues was significantly lower than controls. The protein content in the hepatic tissue was not significantly increased, whereas the total protein in the renal and cardiac tissues was increased significantly. The research finding is indicative of detoxification activity in the etoposide model.

El etopósido es un agente antimitótico y antineoplásico eficaz que se utiliza para tratar diversas neoplasias malignas humanas. En el presente estudio, se inyectó etopósido por vía intraperitoneal a las ratas a razón de 1 mg/kg/día durante 52 días (52 dosis). Los animales control recibieron solución salina fisiológica (0,5 ml) por vía intraperitoneal diariamente por 52 dosis. El peso corporal de las ratas tratadas con etopósido se redujo significativamente en comparación con las ratas del grupo control. La peroxidación lipídica demostró un aumento insignificante del tejido hepático, una disminución no significativa del tejido renal y una reducción significativa del tejido cardíaco. Se encontró que los niveles de GSH en el tejido hepático y renal no aumentaron significativamente, pero sí aumentaron significativamente en el tejido cardíaco en comparación con los controles. Se encontró que la actividad de GR disminuyó considerablemente en el grupo tratado. Los niveles de G-S-T aumentaron significativamente en todos los grupos tratados. Las inyecciones de etopósido provocaron un cambio no significativo en el nivel de GPX del tejido hepático, mientras que los tejidos renal y cardíaco mostraron un aumento significativo. La actividad de CAT en el tejido hepático aumentó significativamente, mientras que la actividad de CAT en el tejido renal mostró una disminución no significativa, mientras que en el tejido cardíaco se observaron niveles significativamente más bajos que en el grupo de control. El nivel de CYTp450 en los tejidos hepático y cardíaco mostró un aumento significativo; sin embargo, el tejido renal mostró un agotamiento no significativo, mientras que CYTb5 en los tejidos hepático, renal y cardíaco fue significativamente menor que los controles. El contenido de proteínas en el tejido hepático no aumentó significativamente, mientras que la proteína total en los tejidos renal y cardíaco aumentó significativamente. El hallazgo de la investigación es indicativo de la actividad de desintoxicación en el modelo de etopósido.

Assessment of Babesia spp. prevalence in various domestic animals across Southern Punjab, Pakistan.

Parasitic diseases, notably babesiosis, exert a substantial impact on the global cattle industry, posing challenges to commerce, economies, and human health. This study, conducted in Southern Punjab, Pakistan, aimed to assess the prevalence of Babesia spp. across various livestock species using microscopic and PCR methods. A total of 180 blood samples (60 from each district) were systematically collected from apparently healthy animals, with 36 samples obtained from each domestic animal species, including camel, cattle, buffalo, goat, and sheep, noting that 12 samples were collected from each district for each animal species. Overall prevalence was determined to be 32.8% (59/180), with varying rates among species: 25.0% in cattle, 41.66% in buffalo, 30.55% in goats, 33.3% in sheep, and 33.3% in camels. Microscopic examination revealed slightly varied infection rates among large and small domestic animals (22.2%), while PCR results indicated a 32.8% overall infection rate in both large and small domestic animals, with no statistical significance. District-wise analysis showed regional variations, with Muzaffargarh recording a prevalence rate of 23.33% through microscopic examination, while Lodhran and Bahawalpur recorded 21.67%. PCR results revealed higher rates (38.33%, 26.67%, and 33.33%, respectively), underlining the importance of employing PCR for accurate detection. Examining ruminant types, large ruminants exhibited a 32.4% infection rate, while small domestic animals showed 33.3%, with no significant difference (p=0.897). District-wise prevalence showcased significant variation, with Muzaffargarh demonstrating a 25% prevalence, Lodhran 22%, and Bahawalpur 22%, through microscopic examination. PCR results displayed 38.33%, 27%, and 33.3%, respectively, with no statistical significance. Detailed analysis of individual districts highlighted variations in infection rates among camels, cattle, buffalo, goats, and sheep. The binomial test indicated significant differences through microscopic analysis (P=0.011) but non-significant variations through PCR (P=0.065), emphasizing the precision of PCR. Regional variations in prevalence, notably with Punjab exhibiting the highest frequency (33.87%) and KPK the lowest (13.24%), suggest potential influences from varying veterinary practices and environmental factors. This study underscores the pivotal role of PCR alongside microscopy for accurate babesiosis diagnosis. These findings contribute to the broader understanding of babesiosis prevalence, emphasizing the necessity of advanced molecular techniques for informed control measures.

South-to-north migration preceded the advent of intensive farming in the Maya region.

The genetic prehistory of human populations in Central America is largely unexplored leaving an important gap in our knowledge of the global expansion of humans. We report genome-wide ancient DNA data for a transect of twenty individuals from two Belize rock-shelters dating between 9,600-3,700 calibrated radiocarbon years before present (cal. BP). The oldest individuals (9,600-7,300 cal. BP) descend from an Early Holocene Native American lineage with only distant relatedness to present-day Mesoamericans, including Mayan-speaking populations. After ~5,600 cal. BP a previously unknown human dispersal from the south made a major demographic impact on the region, contributing more than 50% of the ancestry of all later individuals. This new ancestry derived from a source related to present-day Chibchan speakers living from Costa Rica to Colombia. Its arrival corresponds to the first clear evidence for forest clearing and maize horticulture in what later became the Maya region.

Reconstructing the Deep Population History of Central and South America.

We report genome-wide ancient DNA from 49 individuals forming four parallel time transects in Belize, Brazil, the Central Andes, and the Southern Cone, each dating to at least ∼9,000 years ago. The common ancestral population radiated rapidly from just one of the two early branches that contributed to Native Americans today. We document two previously unappreciated streams of gene flow between North and South America. One affected the Central Andes by ∼4,200 years ago, while the other explains an affinity between the oldest North American genome associated with the Clovis culture and the oldest Central and South Americans from Chile, Brazil, and Belize. However, this was not the primary source for later South Americans, as the other ancient individuals derive from lineages without specific affinity to the Clovis-associated genome, suggesting a population replacement that began at least 9,000 years ago and was followed by substantial population continuity in multiple regions.

Early effects of Epac depend on the fine-tuning of the sarcoplasmic reticulum Ca2+ handling in cardiomyocytes.

In cardiac muscle, signaling through cAMP governs many fundamental cellular functions, including contractility, relaxation and automatism. cAMP cascade leads to the activation of the classic protein kinase A but also to the stimulation of the recently discovered exchange protein directly activated by cAMP (Epac). The role of Epac in the regulation of intracellular Ca2+ homeostasis and contractility in cardiac myocytes is still matter of debate. In this study we showed that the selective Epac activator, 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate (8-CPT), produced a positive inotropic effect when adult rat cardiac myocytes were stabilized at low [Ca2+]o (0.5mM), no changes at 1mM [Ca2+]o and a negative inotropic effect when [Ca2+]o was increased to 1.8mM. These effects were associated to parallel variations in sarcoplasmic reticulum (SR) Ca2+ content. At all [Ca2+]o studied, 8-CPT induced an increase in Ca2+ spark frequency and enhanced CaMKII autophosphorylation and the CaMKII-dependent phosphorylation of SR proteins: phospholamban (PLN, at Thr17 site) and ryanodine receptor (RyR2, at Ser2814 site). We used transgenic mice lacking PLN CaMKII phosphorylation site (PLN-DM) and knock-in mice with an inactivated CaMKII site S2814 on RyR2 (RyR2-S2814A) to investigate the involvement of these processes in the effects of Epac stimulation. In PLN-DM mice, 8-CPT failed to induce the positive inotropic effect at low [Ca2+]o and RyR2-S2814A mice showed no propensity to arrhythmic events when compared to wild type mice myocytes. We conclude that stimulation of Epac proteins could have either beneficial or deleterious effects depending on the steady-state Ca2+ levels at which the myocyte is functioning, favoring the prevailing mechanism of SR Ca2+ handling (uptake vs. leak) in the different situations.

A pediatric case of mammary analogue secretory carcinoma within the parotid.

Mammary analogue secretory carcinoma (MASC) is a recently described entity in the differential diagnosis of salivary gland tumors. It is notable for a characteristic t(12;15)(p13;q25) translocation that results in a unique fusion protein, ETV6-NTRK3. While several studies have retrospectively identified this translocation in cases previously diagnosed as a different salivary malignancy, there have been relatively few cases where this translocation was identified on initial pathology results, and fewer still in a pediatric population. We present a case of a 15 year old female with a slowly enlarging, painless, left facial mass. MRI demonstrated a cystic mass extending into the deep lobe of the parotid, and she underwent parotidectomy. The tumor cells stained positive for S100 and CK19. ETV6 translocation was present, confirming the diagnosis. Mammary analogue secretory carcinoma is a recently described tumor of the salivary glands, which often masquerades as more common primary salivary gland tumors and cysts. More research is needed to characterize the typical behavior of this neoplasm and the optimal treatment regimen. With identification of its characteristic translocation, mammary analogue secretory carcinoma can be easily differentiated from its more prevalent counterparts, and should therefore remain within the differential of the pathologist and head and neck surgeon.

Calcium-calmodulin dependent protein kinase II (CaMKII): a main signal responsible for early reperfusion arrhythmias.

To explore whether CaMKII-dependent phosphorylation events mediate reperfusion arrhythmias, Langendorff perfused hearts were submitted to global ischemia/reperfusion. Epicardial monophasic or transmembrane action potentials and contractility were recorded. In rat hearts, reperfusion significantly increased the number of premature beats (PBs) relative to pre-ischemic values. This arrhythmic pattern was associated with a significant increase in CaMKII-dependent phosphorylation of Ser2814 on Ca(2+)-release channels (RyR2) and Thr17 on phospholamban (PLN) at the sarcoplasmic reticulum (SR). These phenomena could be prevented by the CaMKII-inhibitor KN-93. In transgenic mice with targeted inhibition of CaMKII at the SR membranes (SR-AIP), PBs were significantly decreased from 31±6 to 5±1 beats/3min with a virtually complete disappearance of early-afterdepolarizations (EADs). In mice with genetic mutation of the CaMKII phosphorylation site on RyR2 (RyR2-S2814A), PBs decreased by 51.0±14.7%. In contrast, the number of PBs upon reperfusion did not change in transgenic mice with ablation of both PLN phosphorylation sites (PLN-DM). The experiments in SR-AIP mice, in which the CaMKII inhibitor peptide is anchored in the SR membrane but also inhibits CaMKII regulation of L-type Ca(2+) channels, indicated a critical role of CaMKII-dependent phosphorylation of SR proteins and/or L-type Ca(2+) channels in reperfusion arrhythmias. The experiments in RyR2-S2814A further indicate that up to 60% of PBs related to CaMKII are dependent on the phosphorylation of RyR2-Ser2814 site and could be ascribed to delayed-afterdepolarizations (DADs). Moreover, phosphorylation of PLN-Thr17 and L-type Ca(2+) channels might contribute to reperfusion-induced PBs, by increasing SR Ca(2+) content and Ca(2+) influx.

Increased intracellular Ca2+ and SR Ca2+ load contribute to arrhythmias after acidosis in rat heart. Role of Ca2+/calmodulin-dependent protein kinase II.

Returning to normal pH after acidosis, similar to reperfusion after ischemia, is prone to arrhythmias. The type and mechanisms of these arrhythmias have never been explored and were the aim of the present work. Langendorff-perfused rat/mice hearts and rat-isolated myocytes were subjected to respiratory acidosis and then returned to normal pH. Monophasic action potentials and left ventricular developed pressure were recorded. The removal of acidosis provoked ectopic beats that were blunted by 1 muM of the CaMKII inhibitor KN-93, 1 muM thapsigargin, to inhibit sarcoplasmic reticulum (SR) Ca(2+) uptake, and 30 nM ryanodine or 45 muM dantrolene, to inhibit SR Ca(2+) release and were not observed in a transgenic mouse model with inhibition of CaMKII targeted to the SR. Acidosis increased the phosphorylation of Thr(17) site of phospholamban (PT-PLN) and SR Ca(2+) load. Both effects were precluded by KN-93. The return to normal pH was associated with an increase in SR Ca(2+) leak, when compared with that of control or with acidosis at the same SR Ca(2+) content. Ca(2+) leak occurred without changes in the phosphorylation of ryanodine receptors type 2 (RyR2) and was blunted by KN-93. Experiments in planar lipid bilayers confirmed the reversible inhibitory effect of acidosis on RyR2. Ectopic activity was triggered by membrane depolarizations (delayed afterdepolarizations), primarily occurring in epicardium and were prevented by KN-93. The results reveal that arrhythmias after acidosis are dependent on CaMKII activation and are associated with an increase in SR Ca(2+) load, which appears to be mainly due to the increase in PT-PLN.

The importance of the Thr17 residue of phospholamban as a phosphorylation site under physiological and pathological conditions.

The sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) is under the control of an SR protein named phospholamban (PLN). Dephosphorylated PLN inhibits SERCA2a, whereas phosphorylation of PLN at either the Ser16 site by PKA or the Thr17 site by CaMKII reverses this inhibition, thus increasing SERCA2a activity and the rate of Ca2+ uptake by the SR. This leads to an increase in the velocity of relaxation, SR Ca2+ load and myocardial contractility. In the intact heart, beta-adrenoceptor stimulation results in phosphorylation of PLN at both Ser16 and Thr17 residues. Phosphorylation of the Thr17 residue requires both stimulation of the CaMKII signaling pathways and inhibition of PP1, the major phosphatase that dephosphorylates PLN. These two prerequisites appear to be fulfilled by beta-adrenoceptor stimulation, which as a result of PKA activation, triggers the activation of CaMKII by increasing intracellular Ca2+, and inhibits PP1. Several pathological situations such as ischemia-reperfusion injury or hypercapnic acidosis provide the required conditions for the phosphorylation of the Thr17 residue of PLN, independently of the increase in PKA activity, i.e., increased intracellular Ca2+ and acidosis-induced phosphatase inhibition. Our results indicated that PLN was phosphorylated at Thr17 at the onset of reflow and immediately after hypercapnia was established, and that this phosphorylation contributes to the mechanical recovery after both the ischemic and acidic insults. Studies on transgenic mice with Thr17 mutated to Ala (PLN-T17A) are consistent with these results. Thus, phosphorylation of the Thr17 residue of PLN probably participates in a protective mechanism that favors Ca2+ handling and limits intracellular Ca2+ overload in pathological situations.

The importance of the Thr17 residue of phospholamban as a phosphorylation site under physiological and pathological conditions

The sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) is under the control of an SR protein named phospholamban (PLN). Dephosphorylated PLN inhibits SERCA2a, whereas phosphorylation of PLN at either the Ser16 site by PKA or the Thr17 site by CaMKII reverses this inhibition, thus increasing SERCA2a activity and the rate of Ca2+ uptake by the SR. This leads to an increase in the velocity of relaxation, SR Ca2+ load and myocardial contractility. In the intact heart, ß-adrenoceptor stimulation results in phosphorylation of PLN at both Ser16 and Thr17 residues. Phosphorylation of the Thr17 residue requires both stimulation of the CaMKII signaling pathways and inhibition of PP1, the major phosphatase that dephosphorylates PLN. These two prerequisites appear to be fulfilled by ß-adrenoceptor stimulation, which as a result of PKA activation, triggers the activation of CaMKII by increasing intracellular Ca2+, and inhibits PP1. Several pathological situations such as ischemia-reperfusion injury or hypercapnic acidosis provide the required conditions for the phosphorylation of the Thr17 residue of PLN, independently of the increase in PKA activity, i.e., increased intracellular Ca2+ and acidosis-induced phosphatase inhibition. Our results indicated that PLN was phosphorylated at Thr17 at the onset of reflow and immediately after hypercapnia was established, and that this phosphorylation contributes to the mechanical recovery after both the ischemic and acidic insults. Studies on transgenic mice with Thr17 mutated to Ala (PLN-T17A) are consistent with these results. Thus, phosphorylation of the Thr17 residue of PLN probably participates in a protective mechanism that favors Ca2+ handling and limits intracellular Ca2+ overload in pathological situations.

Phosphorylation of phospholamban in ischemia-reperfusion injury: functional role of Thr17 residue.

Phospholamban (PLB) is a sarcoplasmic reticulum (SR) protein that when phosphorylated at Ser16 by PKA and/or at Thr17 by CaMKII increases the affinity of the SR Ca2+ pump for Ca2+. PLB is therefore, a critical regulator of SR function, myocardial relaxation and myocardial contractility. The present study was undertaken to examine the status of PLB phosphorylation after ischemia and reperfusion and to provide evidence about the possible role of the phosphorylation of Thr17 PLB residue on the recovery of contractility and relaxation after a period of ischemia. Experiments were performed in Langendorff perfused hearts from Wistar rats. Hearts were submitted to a protocol of global normothermic ischemia and reperfusion. The results showed that (1) the phosphorylation of Ser16 and Thr17 residues of PLB increased at the end of the ischemia and the onset of reperfusion, respectively. The increase in Thr17 phosphorylation was associated with a recovery of relaxation to preischemic values. This recovery occurred in spite of the fact that contractility was depressed. (2) The reperfusion-induced increase in Thr17 phosphorylation was dependent on Ca2+ entry to the cardiac cell. This Ca2+ influx would mainly occur by the coupled activation of the Na+ / H+ exchanger and the Na+ / Ca2+ exchanger working in the reverse mode, since phosphorylation of Thr17 was decreased by inhibition of these exchangers and not affected by blockade of the L-type Ca2+ channels. (3) Specific inhibition of CaMKII by KN93 significantly decreased Thr17 phosphorylation. This decrease was associated with an impairment of myocardial relaxation. The present study suggests that the phosphorylation of Thr17 of PLB upon reflow, may favor the full recovery of relaxation after ischemia.

Phosphorylation of phospholamban in ischemia-reperfusion injury: Functional role of Thr(17) residue.

Phospholamban (PLB) is a sarcoplasmic reticulum (SR) protein that when phosphorylated at Ser(16) by PKA and/or at Thr(17) by CaMKII increases the affinity of the SR Ca(2+) pump for Ca(2+). PLB is therefore, a critical regulator of SR function, myocardial relaxation and myocardial contractility. The present study was undertaken to examine the status of PLB phosphorylation after ischemia and reperfusion and to provide evidence about the possible role of the phosphorylation of Thr(17) PLB residue on the recovery of contractility and relaxation after a period of ischemia. Experiments were performed in Langendorff perfused hearts from Wistar rats. Hearts were submitted to a protocol of global normothermic ischemia and reperfusion. The results showed that (1) the phosphorylation of Ser(16) and Thr(17) residues of PLB increased at the end of the ischemia and the onset of reperfusion, respectively. The increase in Thr(17) phosphorylation was associated with a recovery of relaxation to preischemic values. This recovery occurred in spite of the fact that contractility was depressed. (2) The reperfusion-induced increase in Thr(17) phosphorylation was dependent on Ca(2+) entry to the cardiac cell. This Ca(2+) influx would mainly occur by the coupled activation of the Na(+)/H(+) exchanger and the Na(+)/Ca(2+) exchanger working in the reverse mode, since phosphorylation of Thr(17) was decreased by inhibition of these exchangers and not affected by blockade of the L-type Ca(2+) channels. (3) Specific inhibition of CaMKII by KN93 significantly decreased Thr(17) phosphorylation. This decrease was associated with an impairment of myocardial relaxation. The present study suggests that the phosphorylation of Thr(17) of PLB upon reflow, may favor the full recovery of relaxation after ischemia. (Mol Cell Biochem 263: 131-136, 2004).

Role of dual-site phospholamban phosphorylation in the stunned heart: insights from phospholamban site-specific mutants.

Phosphorylation of phospholamban (PLB) at Ser16 (protein kinase A site) and at Thr17 [Ca2+/calmodulin kinase II (CaMKII) site] increases sarcoplasmic reticulum Ca2+ uptake and myocardial contractility and relaxation. In perfused rat hearts submitted to ischemia-reperfusion, we previously showed an ischemia-induced Ser16 phosphorylation that was dependent on beta-adrenergic stimulation and an ischemia and reperfusion-induced Thr17 phosphorylation that was dependent on Ca2+ influx. To elucidate the relationship between these two PLB phosphorylation sites and postischemic mechanical recovery, rat hearts were submitted to ischemia-reperfusion in the absence and presence of the CaMKII inhibitor KN-93 (1 microM) or the beta-adrenergic blocker dl-propranolol (1 microM). KN-93 diminished the reperfusion-induced Thr17 phosphorylation and depressed the recovery of contraction and relaxation after ischemia. dl-Propranolol decreased the ischemia-induced Ser16 phosphorylation but failed to modify the contractile recovery. To obtain further insights into the functional role of the two PLB phosphorylation sites in postischemic mechanical recovery, transgenic mice expressing wild-type PLB (PLB-WT) or PLB mutants in which either Thr17 or Ser16 were replaced by Ala (PLB-T17A and PLB-S16A, respectively) into the PLB-null background were used. Both PLB mutants showed a lower contractile recovery than PLB-WT. However, this recovery was significantly impaired all along reperfusion in PLB-T17A, whereas it was depressed only at the beginning of reperfusion in PLB-S16A. Moreover, the recovery of relaxation was delayed in PLB-T17A, whereas it did not change in PLB-S16A, compared with PLB-WT. These findings indicate that, although both PLB phosphorylation sites are involved in the mechanical recovery after ischemia, Thr17 appears to play a major role.

The highly selective cyclooxygenase-2 inhibitor DFU is neuroprotective when given several hours after transient cerebral ischemia in gerbils.

Several studies suggest that cyclooxygenase-2 contributes to the delayed progression of ischemic brain damage. In this study we examined whether the highly selective cyclooxygenase-2 inhibitor DFU reduces neuronal damage when administered several hours after 5 min of transient forebrain ischemia in gerbils. The extent of ischemic injury was assessed behaviorally by measuring the increases in locomotor activity and by histopathological evaluation of the extent of CA1 hippocampal pyramidal cell injury 7 days after ischemia. DFU treatment (10 mg/kg, p.o.) significantly reduced hippocampal neuronal damage even if the treatment is delayed until 12 h after ischemia. These results suggest that selective cyclooxygenase-2 inhibitors may be a valuable therapeutic strategy for ischemic brain injury.

Endoplasmic reticulum contribution to the relaxant effect of cGMP- and cAMP-elevating agents in feline aorta.

The contribution of endoplasmic reticulum (ER) and phosphorylation of phospholamban (PLB) to the relaxant effect of cGMP- and cAMP-elevating agents was studied in feline aorta. Sodium nitroprusside (NP, 100 microM) completely relaxed contracture induced by 10 microM norepinephrine. This NP-induced relaxation was partially prevented by tetraethylammonium, suggesting that a fraction of NP-induced relaxation was mediated by activation of K(+) channels. In the absence and presence of tetraethylammonium, the relaxant effect of NP was associated with a significant increase in Ser(16) phosphorylation of PLB immunodetected by phosphorylation site-specific antibodies. The relaxant effect of NP on aortic strips precontracted with 80 mM KCl was significantly reduced by 1 microM thapsigargin. This decrease, which represents the ER contribution to the relaxant effect of NP, reached 23 +/- 9% at 100 microM NP and was closely associated with a dose-dependent increase in Ser(16) phosphorylation (128 +/- 49% over control at 100 microM NP). Effects of NP were associated with a significant increase in activity of protein kinase G and were mimicked by 8-bromo-cGMP. Forskolin produced a dose-dependent relaxant effect on KCl-induced contracture, which reached 64 +/- 8% at 50 microM and was associated with an increase in phosphorylation of Ser(16) residue of PLB (88 +/- 18% over control). Thapsigargin reduced this relaxant effect by 38 +/- 9%. 8-Bromo-cAMP mimicked effects of forskolin. The ER-mediated relaxant effect and the increase in Ser(16) phosphorylation produced by forskolin were partially blocked by the protein kinase A inhibitor H-89 (5 microM). The results indicate that ER partially contributes to the relaxant effect of NP and forskolin in feline aorta. This effect may be mediated by the associated increase in Ser(16) phosphorylation of PLB.

Mechanisms involved in the acidosis enhancement of the isoproterenol-induced phosphorylation of phospholamban in the intact heart.

Previous experiments have shown that acidosis enhances isoproterenol-induced phospholamban (PHL) phosphorylation (Mundiña-Weilenmann, C., Vittone, L., Cingolani, H. E., Orchard, C. H. (1996) Am. J. Physiol. 270, C107-C114). In the present experiments, performed in isolated Langendorff perfused rat hearts, phosphorylation site-specific antibodies to PHL combined with the quantitative measurement of 32P incorporation into PHL were used as experimental tools to gain further insight into the mechanism involved in this effect. At all isoproterenol concentrations tested (3-300 nM), phosphorylation of Thr17 of PHL was significantly higher at pHo 6.80 than at pHo 7.40, without significant changes in Ser16 phosphorylation. This increase in Thr17 phosphorylation was associated with an enhancement of the isoproterenol-induced relaxant effect. In the absence of isoproterenol, the increase in [Ca]o at pHo 6.80 (but not at pHo 7.40) evoked an increase in PHL phosphorylation that was exclusively due to an increase in Thr17 phosphorylation and that was also associated with a significant relaxant effect. This effect and the phosphorylation of Thr17 evoked by acidosis were both offset by the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62. In the presence of isoproterenol, either the increase in [Ca]o or the addition of a 1 microM concentration of the phosphatase inhibitor okadaic acid was able to mimic the increase in isoproterenol-induced Thr17 phosphorylation produced by acidosis. In contrast, these two interventions have opposite effects on phosphorylation of Ser16. Whereas the increase in [Ca]o significantly decreased phosphorylation of Ser16, the addition of okadaic acid significantly increased the phosphorylation of this residue. The results are consistent with the hypothesis that the increase in phospholamban phosphorylation produced by acidosis in the presence of isoproterenol is the consequence of two different mechanisms triggered by acidosis: an increase in [Ca2+]i and an inhibition of phosphatases.