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Catecholamine-induced polymorphic ventricular tachycardia (CPVT) is a familial disorder caused by cardiac ryanodine receptor type 2 (RyR2) or calsequestrin 2 (CASQ2) gene mutations. To define how CASQ2 mutations cause CPVT, we produced and studied mice carrying a human D307H missense mutation (CASQ(307/307)) or a CASQ2-null mutation (CASQ(DeltaE9/DeltaE9)). Both CASQ2 mutations caused identical consequences. Young mutant mice had structurally normal hearts but stress-induced ventricular arrhythmias; aging produced cardiac hypertrophy and reduced contractile function. Mutant myocytes had reduced CASQ2 and increased calreticulin and RyR2 (with normal phosphorylated proportions) but unchanged calstabin levels, as well as reduced total sarcoplasmic reticulum (SR) Ca(2+), prolonged Ca(2+) release, and delayed Ca(2+) reuptake. Stress further diminished Ca(2+) transients, elevated cytosolic Ca(2+), and triggered frequent, spontaneous SR Ca(2+) release. Treatment with Mg(2+), a RyR2 inhibitor, normalized myocyte Ca(2+) cycling and decreased CPVT in mutant mice, indicating RyR2 dysfunction was critical to mutant CASQ2 pathophysiology. We conclude that CPVT-causing CASQ2 missense mutations function as null alleles. In the absence of CASQ2, calreticulin, a fetal Ca(2+)-binding protein normally downregulated at birth, remains a prominent SR component. Adaptive changes to CASQ2 deficiency (increased posttranscriptional expression of calreticulin and RyR2) maintained electrical-mechanical coupling, but increased RyR2 leakiness, a paradoxical response further exacerbated by stress. The central role of RyR2 dysfunction in CASQ2 deficiency unifies the pathophysiologic mechanism underlying CPVT due to RyR2 or CASQ2 mutations and suggests a therapeutic approach for these inherited cardiac arrhythmias.

The cardiac conduction system is an anatomically discrete segment of specialized myocardium that initiates and propagates electrical impulses to coordinate myocardial contraction. To define the molecular composition of the mouse ventricular conduction system we used microdissection and transcriptional profiling by serial analysis of gene expression (SAGE). Conduction-system-specific expression for Id2, a member of the Id gene family of transcriptional repressors, was identified. Analyses of Id2-deficient mice demonstrated structural and functional conduction system abnormalities, including left bundle branch block. A 1.2 kb fragment of the Id2 promoter proved sufficient for cooperative regulation by Nkx2-5 and Tbx5 in vitro and for conduction-system-specific gene expression in vivo. Furthermore, compound haploinsufficiency of Tbx5 and Nkx2-5 or Tbx5 and Id2 prevented embryonic specification of the ventricular conduction system. We conclude that a molecular pathway including Tbx5, Nkx2-5, and Id2 coordinates specification of ventricular myocytes into the ventricular conduction system lineage.

A 5-year-old, neutered male, Shar Pei dog was presented with weight loss, anorexia, lethargy, stranguria, and distal limb edema. Clinicopathologic abnormalities included anemia, an inflammatory leukogram, azotemia, icterus, urinary tract infection, and hepatomegaly with a markedly hypoechoic liver. Cytologic findings in a fine-needle aspirate of the liver included large amounts of amorphous, pink, extracellular matrix between hepatocytes. The amorphous material was congophilic using Congo red stain on a hepatic cytology specimen and green birefringent areas were observed under polarized light, confirming the presence of amyloid. The dog was euthanized and a necropsy was done. Histopathologic evaluation using H&E and Congo red staining confirmed amyloid deposits within the liver, kidneys, intestinal vessels, pancreas, and mesenteric ganglia. Immunohistochemical staining of liver and kidney sections using anti-AA amyloid and anti-P component antibodies confirmed the presence of AA amyloid. In this case, we demonstrated that Congo red staining and polarized light microscopy are a useful diagnostic technique in cytologic specimens of suitable thickness for confirming the presence of amyloid.

Sarcomere protein gene mutations cause hypertrophic cardiomyopathy (HCM), a disease with distinctive histopathology and increased susceptibility to cardiac arrhythmias and risk for sudden death. Myocyte disarray (disorganized cell-cell contact) and cardiac fibrosis, the prototypic but protean features of HCM histopathology, are presumed triggers for ventricular arrhythmias that precipitate sudden death events. To assess relationships between arrhythmias and HCM pathology without confounding human variables, such as genetic heterogeneity of disease-causing mutations, background genotypes, and lifestyles, we studied cardiac electrophysiology, hypertrophy, and histopathology in mice engineered to carry an HCM mutation. Both genetically outbred and inbred HCM mice had variable susceptibility to arrhythmias, differences in ventricular hypertrophy, and variable amounts and distribution of histopathology. Among inbred HCM mice, neither the extent nor location of myocyte disarray or cardiac fibrosis correlated with ex vivo signal conduction properties or in vivo electrophysiologically stimulated arrhythmias. In contrast, the amount of ventricular hypertrophy was significantly associated with increased arrhythmia susceptibility. These data demonstrate that distinct somatic events contribute to variable HCM pathology and that cardiac hypertrophy, more than fibrosis or disarray, correlates with arrhythmic risk. We suggest that a shared pathway triggered by sarcomere gene mutations links cardiac hypertrophy and arrhythmias in HCM.

Cisplatin is one of the mostly used antineoplastic drugs in the treatment of cancer, but its clastogenic potential has become of great interest. In patients treated with long-term cisplatin, genetic damage can be observed during chemotherapy or many years later. The aim of this study was to investigate the possible anticlastogenic effect of pretreatment with olive, extra virgin olive, canola or corn oils on cisplatin-induced chromosomal aberrations in Wistar rat bone marrow cells. The animals received pretreatment with a single dose of vegetable oils (5 ml/kg b.w.) by gavage before cisplatin i.p. (5 mg/kg b.w.), and were sacrificed 24 h after cisplatin injection. The pretreatment with a single dose of olive, extra virgin olive and canola oils caused a statistically significant decrease in the total of chromosomal aberrations and abnormal metaphases induced by cisplatin when compared with the groups treated with cisplatin alone. The possible explanation for the anticlastogenic effects observed in the pretreatment with olive, extra virgin olive and canola oils is ascribed to the oil contents. In conclusion, from the findings we suggest that these oils have some antioxidant effect, and the anticlastogenesis mechanisms of these oils need to be explored further before their use during cisplatin chemotherapy.

Objective: Previous reports have shown that pain is managed inadequately in newborn infants. Ironically, clinicians believe that infants can experience pain much like adults, that infants are exposed daily to painful procedures, and that pain protection should be provided. In adults, a close relationship has been shown in how adults behave in response to pain, how painful they sense the stimulus to be, and physical measurements of the intensity of the stimulus. Whether similar parallels exist in newborn infants has not been examined. If these parallels do not exist in infants, it may help explain why clinicians fail to manage procedural pain in infants more effectively. The objective of this study was to determine whether the magnitude of infants' responses to nursing/medical procedures: 1) differs as a function of the invasiveness or intensity of the procedure; 2) differs as a function of intrauterine (gestational age at birth) and/or extrauterine (conceptional age) development; and 3) parallels the subjective pain ratings of clinicians for those procedures., Methods: A broad developmental and clinical range of newborn infants was studied shortly before (baseline and preparatory periods), throughout, and shortly after (recovery period) required nursing/medical procedures during hospitalization. Heart rate, oxygen saturation, mean arterial pressure, and behavioral state (percentage of time spent in sleep or in agitation) were measured, and the magnitude of change in each in response to procedures was calculated. Procedures were categorized as mildly, moderately, and highly invasive to examine differences in response magnitude as a function of procedural invasiveness. Responses were compared as a function of prematurity and postnatal age. Clinicians' procedural pain ratings were compared with the magnitude of infants' responses., Results: Of the original 152 infants, 135 were studied at least two times (range 2-27). Significant changes occurred in physiologic and behavioral measures in response to procedures indicative of pain responses. The magnitude of response generally increased with increased procedural invasiveness although there was considerable overlap of magnitude with invasiveness. Both premature and full-term infants differentiated procedural invasiveness. Very premature infants (<28 weeks' gestational age) exhibited increased increments in response magnitude with increasing postnatal age. Clinician's ratings of procedural painfulness were correlated with and predicted the magnitude of heart rate response to individual procedures., Conclusions: Similar to what has been shown in adults, newborn and developing infants show increased magnitude physiologic and behavioral responses to increasingly invasive procedures, demonstrating that even very prematurely born infants respond to pain and differentiate stimulus intensity. However, the considerable overlap of magnitude with invasiveness suggests that there is not a physiologic or behavioral threshold that clearly marks the presence of pain. Inconsistencies in physiologic and behavioral responses make reliance on a pain index difficult. The best approach may be one of universal precaution to provide pain management systematically to reduce the acute and long-term impact of early procedural pain. development, stimulus intensity, pain response.

Apoptosis is mediated by members of the caspase family of proteases which can be activated by release of mitochondrial cytochrome c. Additional members of the caspase family are activated at the cell surface in response to direct stimulus from the external environment such as by activation of the Fas receptor. It has been suggested that these upstream caspases directly activate the downstream caspases which would obviate a role for cytochrome c in apoptosis induced by the Fas receptor. We demonstrate that cytochrome c is released from mitochondria of Jurkat cells in response to both staurosporine and an agonistic anti-Fas antibody and that only the latter is inhibited by the caspase inhibitor z-VAD-FMK. This suggests that an upstream caspase such as caspase-8 is required for the Fas-mediated release of mitochondrial cytochrome c. The protein phosphatase inhibitor calyculin A prevented cytochrome c release and apoptosis induced by both agents, suggesting that release of cytochrome c is required in both models. Zinc, once thought of as an endonuclease inhibitor, has previously been shown to prevent the activation of caspase-3. We show that zinc prevents the activation of downstream caspases and apoptosis induced by both insults, yet does not prevent release of mitochondrial cytochrome c. The ability of calyculin A and zinc to prevent DNA digestion implies that the mitochondrial pathway is important for induction of apoptosis by both agents. These results do not support an alternative pathway in which caspase-8 directly activates caspase-3. These results also demonstrate that a critical protein phosphatase regulates the release of cytochrome c and apoptosis induced by both insults., (Copyright 1999 Academic Press.)

During apoptosis, DNA fragmentation and intracellular acidification occur concurrently. Previous results have shown that intracellular acidification is not required for DNA fragmentation, while the alternative, that acidification is a consequence of DNA fragmentation was analyzed here. To obviate the requirement of any nuclear function in acidification, apoptosis was induced by staurosporine in cytoplasts made from the breast tumor cell line MDA-MB-468. Both cells and cytoplasts demonstrated externalization of phosphatidylserine that was prevented by the pan-caspase inhibitor zVAD-fluoromethylketone or by expression of Bcl-2. Intracellular acidification was observed in both cells and cytoplasts and this was also inhibited by both zVAD-fluoromethylketone and Bcl-2. These results show that intracellular acidification and DNA fragmentation are independent consequences of caspase action during apoptosis., (Copyright 1999 Academic Press.)

Background: Previous reports have suggested that healthy, full-term newborn infants who are in more aroused behavioral states tend to respond more robustly to painful events. Others have shown that acutely ill premature and full-term infants who undergo significant handling and immobilization as part of required nursery procedures respond less robustly to concurrent painful events., Purpose: To investigate, using an experimental manipulation, the effect of arousal associated with handling and immobilization on response to acute pain in generally healthy, premature and full-term newborn infants., Methods: Infants were randomly assigned to a group that underwent a series of handling and immobilization procedures before a heelstick or to a group that underwent the heelstick without previous handling and immobilization. Heart rate, behavioral state, and facial activity were compared between the handled (n = 21) and nonhandled (n = 27) infants during an undisturbed baseline, a preparatory, and a standard heelstick procedure., Results: In the handled group, heart rate increased over baseline levels in response to the handling but promptly returned to prehandling levels. There were no significant differences between handled and nonhandled groups in mean heart rate, behavioral state, or facial activity during the baseline or preparatory periods before the heelstick. However, in response to the heelstick, handled infants had a higher mean heart rate, greater behavioral arousal, and displayed more facial activity as compared with nonhandled infants., Conclusions: Healthy premature and full-term newborn infants who undergo common nursery experiences such as handling and immobilization as part of their routine care can exhibit greater physiologic and behavioral reactivity to subsequent painful procedures. The effects of the previous handling may be undetectable, using conventional indices of reactivity, until the painful event. These findings emphasize the importance of identifying reliable markers of previous stress, particularly for newborn infants who may become clinically compromised as a result of the physiologic instability associated with response to pain.

Background: Despite an increased awareness among clinicians regarding pain and pain management for infants undergoing surgery, pain associated with procedures performed outside the operating room may not be adequately managed., Purpose: To examine the beliefs and self-described behavior of physicians and nurses regarding the management of procedural pain in newborn infants., Methods: A survey was distributed to 467 clinicians (nurses and physicians) working in 11 level II and 4 level III nurseries in a large metropolitan area. Respondents were asked to rate the painfulness of 12 common bedside nursery procedures and how often pharmacologic and nonpharmacologic (comfort) measures are currently used and should be used for those procedures. Demographic data were also collected., Results: Surveys were completed by 374 clinicians (80% response rate). Physicians and nurses believe infants feel as much pain as adults and that 9 of the 12 listed procedures are moderately to very painful. Neither pharmacologic nor comfort measures are believed to be used frequently, even for the most painful procedures. Physicians and nurses believe both pharmacologic and comfort measures should be used more frequently, but nurses believe comfort measures should be used more frequently than do physicians. Beliefs about infant pain and procedural pain were related to pain management preferences. Physicians' but not nurses' ratings were associated with significant personal pain., Conclusions: Despite their beliefs that infants experience significant procedure-related pain, clinicians believe pain management for infants remains below optimal levels. Barriers to more consistent and effective pain management need to be identified and surmounted.

Apoptosis is commonly associated with DNA digestion, but it remains controversial as to which endonuclease is involved. The ability of zinc to inhibit DNA digestion in intact cells, and inhibit a Ca2+/Mg2+-dependent endonuclease in cell lysates, has been used frequently to suggest this is the endonuclease involved. However, zinc has many other effects on cells, and here it is shown that zinc also prevents many upstream events in apoptosis. These studies were performed in human ML-1 cells following incubation with etoposide. During apoptosis, these cells undergo intracellular acidification, increased accumulation of Hoechst 33342, DNA digestion and chromatin condensation. Zinc inhibited all of these events. An upstream event in apoptosis is activation of ICE/CED-3 proteases which is commonly observed as proteolysis of a substrate protein, poly(ADP-ribose) polymerase (PARP). The ICE/CED-3 proteases are themselves activated by proteolysis, and this was detected here by cleavage of one family member CPP32. Zinc prevented cleavage of both CPP32 and PARP. We recently demonstrated that dephosphorylation of the retinoblastoma susceptibility protein Rb was a marker of an event even further upstream in apoptosis; zinc was also found to inhibit Rb dephosphorylation. Therefore, zinc must protect cells at a very early step in the apoptotic pathway, and not as a direct inhibitor of an endonuclease.

Apoptosis occurs during development and tissue homeostasis, and under conditions of physical and chemical stress. During apoptosis, cells digest their DNA, decrease intracellular pH, shrink, exhibit protein phosphatase activity, and activate members of the ICE/CED-3 family of proteases. This protease activity is identified by cleavage of poly(ADP-ribose) polymerase (PARP). Phosphatase activity during apoptosis is observed as dephosphorylation of the retinoblastoma susceptibility protein (Rb). Serine/threonine phosphatase inhibitors can prevent dephosphorylation of Rb and apoptosis, suggesting that Rb dephosphorylation is an indication of a critical regulator of apoptosis. The experiments described here were designed to establish the temporal relationship between these events. Apoptosis was induced in human ML-1 cells by the topoisomerase inhibitor etoposide. An inhibitor of the ICE/CED-3 protease family, z-VAD-fluoromethylketone (FMK), showed concentration-dependent protection from PARP cleavage, intracellular acidification, DNA digestion, early changes in membrane permeability, and cell shrinkage, thereby placing all of these events downstream of the ICE/CED-3 protease action. However, z-VAD-FMK did not prevent the dephosphorylation of Rb, placing this change upstream of the protease. These results suggest that the imbalance between protein phosphatase and kinase that is responsible for the dephosphorylation of Rb is also responsible for the activation of ICE/CED-3 proteases, which in turn is responsible for all the other events associated with apoptosis.

To investigate the effect of dementia on response to pain, 51 community-dwelling, generally healthy, cognitively intact individuals > or = 65 years old and 44 community- or nursing home-dwelling persons > or = 65 years old with varying severity of dementia were studied. Cognitive status was assessed by standardized clinical evaluation and psychometric test performance. The following responses were measured before, during and after a standard venipuncture procedure: heart rate, the amplitude of respiratory sinus arrhythmia (RSA), self-reported anxiety and pain, and videotaped facial expressions. Although RSA did not differentiate procedural phases, in both samples, mean heart rate increased in the preparatory phase and decreased in the venipuncture phase. Independent of age, increasing severity of dementia was associated with blunting of physiologic response as measured by diminished heart rate increase in the preparatory phase and heart rate increase with venipuncture. Dementia significantly interfered with the subjects' ability to respond to direct questions about anxiety and pain. Those who were able to respond were relatively accurate self-assessors: higher anxiety was associated with greater magnitude heart rate responses. Facial expression was increased in demented individuals but it could not be classified by specific emotions. We conclude that dementia influences both the experience and reporting of pain in elderly individuals.

Many events in apoptosis have been identified but their temporal relationships remain obscure. Apoptosis in human ML-1 cells induced by etoposide is characterized by intracellular acidification, enhanced Hoechst 33342 fluorescence, DNA digestion, chromatin condensation, and proteolysis of poly(ADP-ribose) polymerase. This proteolysis is a marker for the action of ICE/CED-3 proteases, which are critical activators of apoptosis. We observed that three serine/threonine protein phosphatase inhibitors, okadaic acid, calyculin A, and cantharidin, prevented all of these apoptotic characteristics. To determine which protein phosphatase was involved, we investigated the dephosphorylation of the retinoblastoma susceptibility protein Rb, a substrate for protein phosphatase 1 but not protein phosphatase 2A. Rb was dephosphorylated during apoptosis, and each inhibitor prevented this dephosphorylation at the same concentrations that prevented apoptosis. No increase in protein phosphatase 1 activity was observed in apoptotic cells suggesting that dephosphorylation of Rb may result from loss of Rb kinase activity in the presence of a constant level of protein phosphatase activity. Long term inhibition of protein phosphatase 1 (>8 h) also led to the appearance of dephosphorylated Rb, cleavage of poly(ADP-ribose) polymerase and apoptosis, suggesting these events are not solely dependent upon protein phosphatase 1. Rb dephosphorylation was also observed in several other models of apoptosis. Hence, an imbalance between protein phosphatase 1 and Rb kinase may be a common means to activate ICE/CED-3 proteases resulting in the subsequent events of apoptosis.

To investigate the relations among popular measures of neonatal stress and their link to subsequent temperament, 50 full-term newborns from a normal care nursery were examined responding to a heelstick blood draw. Baseline and heelstick measures of behavioral state, heart period, vagal tone, and salivary cortisol were obtained. Recovery measures of behavioral and cardiac activity were also analyzed. Mothers completed Rothbart's Infant Behavior Questionnaire when their infants reached 6 months of age. Baseline vagal tone predicted cortisol in response to the heelstick, suggesting that baseline vagal tone reflects the infants' ability to react to stressors. Greater reactivity to the heelstick (more crying, shorter heart periods, lower vagal tone, and higher cortisol) was associated with lower scores on "Distress-to-Limitations" temperament at 6 months. This finding was consistent with the expectation that the capacity to react strongly to an aversive stimulus would reflect better neurobehavioral organization in the newborn. Recovery measures of cardiac activity approximated and were correlated with baseline measures indicating the strong self-righting properties of the healthy newborn. Finally, vagal tone and salivary cortisol measures were not significantly related, suggesting the importance of assessing both systems in studies of the ontogeny of stress-temperament relations.

Previous studies have shown that a number of different genotoxic carcinogens that induce different types of DNA damage preferentially alter the expression of inducible genes in vivo. To investigate further the mechanistic basis for these effects, we examined the effects of the human lung carcinogen chromium(VI) on expression of the hormone-inducible cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene in chick embryo liver. Chromium(VI) pretreatment had significant effects on both basal and glucocorticoid-inducible PEPCK expression in 14-d-old embryo liver. These effects were principally a result of changes in PEPCK transcription. In contrast, treatment with chromium(VI) 1 h after treatment with glucocorticoid had no effect on PEPCK induction, suggesting that an early event in the induction process is the target for carcinogen effects. In 16-d-old liver, in which PEPCK expression is no longer responsive to glucocorticoid induction, both basal and inducible PEPCK expression were also refractory to chromium(VI) effects, indicating that carcinogen responsiveness is a phenotypic rather than an inherent property of inducible genes and is related to their competence for induction. Chromium(VI) had no effect on cAMP induction of PEPCK expression, demonstrating that carcinogens target their effects to specific regulatory pathways. Comparison of the effects of chromium(VI) with those of cycloheximide suggests that chromium(VI) targets its effects to a labile, constitutively expressed repressor involved in PEPCK gene regulation.

In view of the importance of Ca2+-channels in controlling the entry of Ca2+ into the myocardium, this study was undertaken to examine the effects of oxygen free radicals on the binding of Ca2+-channel antagonists in rat heart by employing [3H]-nitrendipine as a ligand. Isolated heart membranes were incubated with xanthine + xanthine oxidase (a superoxide anion radicals generating system), hydrogen peroxide (an activated species of oxygen), or hydrogen peroxide + Fe2+ (a hydroxyl radicals generating system). The assay of the [3H]-nitrendipine binding activity revealed that the maximal number of binding sites (Bmax) were reduced in a time-dependent manner by superoxide radicals without any changes in the binding constant (Kd); a significant reduction of Bmax was seen after incubating membranes with xanthine + xanthine oxidase for a 10-min-period. Superoxide dismutase showed a protective effect on the superoxide radicals induced reduction in Bmax. Both hydrogen peroxide and hydroxyl radicals also depressed the Bmax for [3H]-nitrendipine binding without any significant change in Kd; catalase and mannitol showed protective effects on hydrogen peroxide or hydroxyl radicals induced depression in Bmax, respectively. These results indicate that oxygen free radicals may reduce the number of Ca2+-channels in the cell membrane and this change may contribute towards decreasing the voltage-dependent Ca2+ influx in the cardiac cell.