Glucose in vertebrate skeletal muscle proteins.

Neutral sugar was found in proteins extracted with acid chloroform/methanol from skeletal muscle of frog, normal and dystrophic chicken as well as in conventional preparations of frog tropomyosin and rabbit myosin. Whenever tested, the sugar content remained unaffected by two extractions with 5% trichloroacetic acid and by treatment with acid (0.1 N hydrochloric acid) or alkali (0.1 N sodium hydroxide, 60--90 min, 100 degrees C). These and other chemical properties favor the notion that sugar is present in covalent linkage to protein rather than as an attending contaminant. In every protein studied sugar profiles consisted of glucose only except rabbit myosin where it was accompanied by another sugar, presumably ribose.

Inhibition and stimulation of rat luteal protein phosphorylation by protein kinase effectors.

Estradiol-17 beta (E2) predetermined protein phosphorylation systems have been identified recently in midpregnant rat corpus luteum. Major type protein kinase activities in these systems were explored here using as probes protein kinase inhibitors. Luteal nuclear, mitochondrial, microsomal and cytosolic fractions were obtained from rats hysterectomized and hypophysectomized on day 12 of pregnancy and then treated for 72 h with E2. In vitro phosphate transfer from [gamma-32P]ATP was monitored by SDS-PAGE followed by autoradiography. Polymyxin B (PMB), 1-200 microM, a PKC inhibitor, completely blocked, in a dose dependent manner, the Ca2+ phospholipid (PL) stimulated radiolabeling of nuclear fraction Mr 79,000 substrate(s) as expected. Similarly, the calmodulin (CaM) antagonist compound 48/80, 1-20 micrograms/ml, inhibited the Ca2+/CaM-dependent phosphorylation of the microsomal fraction Mr 60,000 and Mr 56,000 proteins. The Ca2+ PL-enhanced labeling of mitochondrial fraction Mr 76,000 substrate(s) was only partially susceptible to inhibition by PMB or compound 48/80. Studies of microsomal fraction phosphoprotein bands not stimulated by added cofactors indicated that the radiolabeling of Mr 75,000 protein(s) was partially blocked by compound 48/80 but not by PMB. Phosphate transfer to Mr 41,000 protein(s) was inhibited by the cAMP-dependent kinase protein inhibitor (PKI), while the phosphorylation of Mr 31,000 protein(s) was refractory to all inhibitors employed here. Surprisingly, regardless of hormonal pretreatment, PMB and compound 48/80 activated in every subcellular fraction the cofactor independent appearance of at least one phosphoprotein band, between Mr 87,000-99,000. This novel observation should be instrumental in understanding the actions of these compounds towards living cells.

Isolation of phosphorylated acid chloroform/methanol-soluble proteins from live frog muscle.

About 6-7% of the total proteins from trichloroacetic acid-washed and freeze-dried frog muscle could be extracted with acid chloroform/methanol. Three of these proteins were found to be phosphorylated in the live frog. They were purified to apparent homogeneity by gel chromatography and preparative gel electrophoresis. The apparent molecular weights, determined by sodium dodecyl sulfate gel electrophoresis, were 34 000, 19 000 and 10 000. Each phosphorylated protein contained 3 mol of a covalently bound neutral sugar but they did not contain any tightly bound lipids. All three proteins incorporated 32P into serine phosphate. The 10 000 dalton protein, which had the highest specific radioactivity contained an unusually high proportion of serine, 14% of the total amino acids. It also did not stain with Coomassie Blue.

Hydrogen bonds in the tripeptide Pro-Leu-Gly-NH2. 17O and 1H studies.

17O-NMR measurements of labeled Pro-Leu-Gly-NH2 were carried out at different pH levels and in mixed solvents of water/acetonitrile. Complementary studies of the amide protons were carried out in acetonitrile-d3. Only the prolyl C = 17O group was sensitive to the pH level. Protonation of the amine group resulted in an upfield chemical shift of 18 ppm. The chemical shifts of each of the three oxygen sites was sensitive to the ratio water:acetonitrile. Solvent composition dependence of the chemical shift and linewidth suggests that the prolyl C = 17O is involved in intramolecular hydrogen bond formation when Pro-Leu-Gly-NH2 is dissolved in acetonitrile, while in water there is no intramolecular H bond.

Regulation of energy flux through the creatine kinase reaction in vitro and in perfused rat heart. 31P-NMR studies.

Fluxes catalyzed by soluble creatine kinase (MM) in equilibrium in vitro and by the creatine kinase system in perfused rat hearts were studied by 31P-NMR saturation transfer method. It was found that in vitro both forward and reverse fluxes through creatine kinase at equilibrium were almost equal and very stable to changes in phosphocreatine/creatine ratio (from 0.2 to 3.0) as well as to changes in pH (from 7.4 to 6.5 or 8.1), free Mg2+ concentration and 2-fold decrease of total adenine nucleotides and creatine pools (from 8.0 to 4.0 mM and from 30 to 14 mM, respectively). In the rat hearts perfused by the Langendorff method the creatine kinase-catalyzed flux from phosphocreatine to ATP was increased by 50% when oxygen consumption grew from 8 to 55 mumol/min per g of dry wt. due to transition from rest to high workload. These changes could not be exclusively explained on the basis of the equilibrium model by activation of heart creatine kinase due to some decrease in [phosphocreatine]/[creatine] ratio (from 1.8 to 0.8) observed during transition from rest to high workload. Analysis of our data showed that an increase in the flux via creatine kinase is correlated with an increase in the rate of ATP synthesis with a linearity coefficient higher than 1.0. These data are more consistent with the concept of energy channeling by phosphocreatine shuttle than with that of the creatine kinase equilibrium in the heart.

Identification and characterization of an abundant phosphoprotein specific to the large luteal cell.

An abundant protein with a relative mol wt of 32K present specifically in the large cells of the pregnant rat corpus luteum has been identified. Separation of large and small luteal cells by elutriation, followed by protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), have revealed that the 32K protein was present as a major protein in the large luteal cells but was practically absent in the small cell population. This protein appears to be highly tissue and cell specific and resolves into three protein species by two-dimensional SDS-PAGE with the major protein having an isoelectric point (pI) greater than or equal to 8.5. It was not detected in preantral follicles or placentas of the same pregnant rats, or in any other tissue examined. After subcellular fractionation, the 32K protein(s) was found in the particulate fraction and was localized principally in the microsomal compartment. Autoradiographic analysis of 35S-amino acid-labeled tissue demonstrated that the 32K protein(s) is synthesized in the corpus luteum. When particulate fractions from small and large cells were incubated in the presence of [gamma-32P]ATP followed by SDS-PAGE, phosphorylation of the 32K protein was apparent. Phosphorylation of this protein was not enhanced by the addition of cofactors for cAMP, Ca2(+)-calmodulin- or Ca2(+)-phospholipid-dependent kinases. Experimental inhibition of steroidogenesis with amino-glutethimide caused a remarkable reduction in the luteal content of this 32K protein whereas estradiol and human CG treatment increased its content. In summary, we have discovered and partially characterized a unique 32K protein(s) which is expressed and phosphorylated only in large luteal cells of the corpus luteum. This protein(s), which is regulated by estradiol formed locally, may serve as a powerful marker for both the large luteal cell and estrogen action in the corpus luteum.

Protein kinase C catalyzed phosphorylation of sterol carrier protein 2.

The transport of cholesterol to the inner mitochondrial membrane, a key step in steroidogenesis, is subject to hormonal modulation that, at least in part, could be mediated by protein phosphorylation. This step is stimulated by sterol carrier protein 2 (SCP2) and Ca2+. To explore whether SCP2 itself is a potential control point for regulation by Ca2+-dependent phosphorylation we investigated whether highly purified SCP2 could serve as a substrate for major type Ca2+ and non-Ca2+-dependent protein kinases. Phosphorylation by calmodulin protein kinase II (CaM-PK II), myosin light chain kinase (MLCK), cAMP-dependent kinase (PKA) and protein kinase C (PKC) was monitored under optimal conditions for each enzyme. PKA, CaM-PK II and MLCK catalyzed the radiolabeling of histone 2A, synapsin I and myosin light chain (MLC), known substrates for these kinases, respectively, yet no phosphate transfer to SCP2 was observed. In contrast, PKC from two different sources (rat and calf brain) effectively catalyzed the phosphorylation of the highly purified SCP2. The phosphorylation of SCP2 depended on the addition of Ca2+ and phospholipids and was completely blocked by Polymyxin B, a PKC inhibitor. PKC catalyzed phosphorylation of SCP2 displayed a similar dependence on the concentration of ATP. Lineweaver Burk plots of the data indicate Km values for ATP of approximately 6 microM for the phosphorylation of SCP2. Our results, which have revealed for the first time that SCP2 is a substrate for PKC, are consistent with the possibilities that the control of steroidogenesis by tropic hormones and by PKC activation are mediated, at least in part, by the phosphorylation/dephosphorylation of SCP2.

Calcium-calmodulin and calcium-phospholipid dependent phosphorylation of membranous fraction proteins related to the tropic regulation by estradiol in the corpus luteum.

Estradiol assumes a major role in the regulation of growth, vascularization, and progesterone synthesis in the midpregnant rat corpus luteum. To explore whether molecular events triggered by estradiol could be mediated, at least in part, by protein phosphorylation, we investigated whether estradiol treatment in vivo affects endogenous luteal protein phosphorylation systems detectable in vitro. Luteal nuclear, mitochondrial, and microsomal fractions were obtained by differential centrifugation from rats hypophysectomized and hysterectomized on day 12 of pregnancy and treated with or without estradiol for 72 h. Using [gamma-32P]ATP as phosphate donor, proteins were phosphorylated in the presence or absence of either calcium (Ca), Ca plus calmodulin, or Ca plus phospholipid. Phosphoproteins were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography. The Coomassie blue stained proteins and phosphoprotein profiles were markedly different in the various fractions. Estradiol treatment in vivo caused an increase in the basal endogenous phosphorylation of several proteins in vitro. It also substantially enhanced the protein kinase C (PKC) and Ca-calmodulin kinase-dependent phosphorylation of selected proteins in subcellular fractions. The Ca-calmodulin kinase catalyzed phosphorylation of microsomal 56 and 60 kilodalton (kDa) proteins was remarkably increased by estradiol. Proteins (56 and 60 kDa) were also phosphorylated when Ca-calmodulin was added to the nuclear fraction, however, this phosphorylation did not appear to be affected by estradiol treatment. A major PKC substrate in the nuclear fraction was an 80 kDa protein whose phosphorylation was increased remarkably by estradiol treatment. In the mitochondrial fraction the most striking effect of estradiol was a marked increase in PKC-mediated phosphate transfer into a 76 kDa substrate. To determine whether estradiol action on protein phosphorylation was related to its tropic effect in the corpus luteum, the hormone was administered to day 10 hypophysectomized and hysterectomized pregnant rats. In this rat model, where estradiol has no stimulatory effect on either luteal steroidogenesis or growth, neither endogenous nor kinase-mediated phosphorylation was affected by this steroid. In summary, the present investigation has revealed that in vivo treatment with estradiol affects the PKC and the Ca-calmodulin dependent in vitro phosphorylation of selected proteins localized in different subcellular compartments and further suggests that phosphorylation systems are potential control points for estradiol regulation of rat corpus luteum function.

ESR spin trapping and NMR spectroscopy of the same heart shows correlation between energy depression and radical formation during postischemic reperfusion.

The relevance of radical formation in disturbances of energy metabolism in the postischemic heart is not clear. This study provides the first evidence of a significant correlation between the amount of oxy-radicals trapped in the effluent of isolated hearts upon reperfusion and the decreased myocardial content of phosphocreatine and ATP. This suggests that the loss of high-energy compounds might contribute to oxy-radical production during reperfusion. The application of ESR spin trapping and of NMR technique to the same heart is a new approach to investigate the pathobiochemical relevance of free radicals for the heart muscle.

Prolonged sleep apnea and respiratory instability: a discriminative study.

This report is based on a study of 53 infants, 28 of whom had clinically observed prolonged apnea (greater than or equal to 20 seconds) during sleep whereas the remaining did not. In addition to the clinical observations, each infant was studied in a sleep laboratory during a complete nap, and a continuous recording was made of respiratory activity and rapid eye movements. Measurements were made of all apneic pauses (greater than or equal to 2 seconds) observed in the laboratory and the two groups of infants were compared in terms of the frequency and average duration of apneic pauses, the longest apneic pause, the amount of periodic apnea, and the relative amount of apnea. The infants with prolonged sleep apnea had, during a single nap, more frequent and longer apneic pauses and more periodic apnea. This supports the hypothesis that respiratory instability during sleep and prolonged apnea have a common etiology. By employing a multiple linear regression model and including all laboratory apnea measures in a single analysis, a composite laboratory score was developed to differentiate members of the two groups. It would thus appear that the study of infants during a single nap could assist in the identification of infants at risk for prolonged sleep apnea and provide an indirect method for determining the influence of a number of variables on the occurrence of prolonged sleep apnea. Pediatrics, 59:962-970, 1977, SLEEP, APNEA, RESPIRATORY INSTABILITY.

Nasopharyngitis and the sudden infant death syndrome.

Infants who died of SIDS in Onondaga County (New York) were classified into a Nasopharyngitis Group (N = 59) or a No Nasopharyngitis Group (N = 57) on the basis of symptoms, within one week prior to death, referable to the upper respiratory tract. Examination of the postnatal age of death revealed that infants in the Nasopharyngitis Group tended to be older than those in the No Nasopharyngitis Group. These results are consistent with a prediction derived from the hypothesis that prolonged sleep apnea is part of a pathophysiologic process resulting in SIDS.

Nasopharyngitis and prolonged sleep Apnea.

The effect of nasopharyngitis on the simultaneous occurrence of prolonged sleep apnea (greater than or equal to 20 seconds in duration) was studied in 26 infants managed at home on an apnea monitor. During the observation period, these infants had a total of 69 illnesses which appeared to represent an upper respiratory tract inflammatory process. In general, the daily frequency of prolonged apneic episodes was significantly greater during nasopharyngitis when compared to comparable time intervals immediately prior to and following the illness. In addition, there was a decrease in the frequency of apneic episodes with increasing postnatal age until the episodes finally ceased to occur during the illness-related intervals. Apneic episodes ceased to occur at an earlier age for the before- and after-illness intervals than for the time interval during which there were clinical symptoms. Thus, it would appear that infants go through an age-related phase wherein prolonged apnea occurs during nasopharyngitis but not when free of illness. The implications of these results for the management of infants having prolonged sleep apnea are discussed. In view of the hypothesis that prolonged sleep apnea is part of the physiological process resulting in the sudden infant death syndrome, these results also provide for the prediction that infants who suddenly die in association with nasopharyngitis would do so, in general, at a later age than those who succumb when free of an upper respiratory tract inflammatory process.

Parental reports of apnea and bradycardia: temporal characteristics and accuracy.

A prospective examination was made of the temporal course of parental observations in response to a monitor alarm of apnea (apnea setting of 20 seconds) or bradycardia (bradycardia setting of 80 beats per minute). Data were obtained from 155 subsequent sudden infant death syndrome siblings followed up at home, during the first 20 weeks of life, on an apnea/bradycardia monitor with an attached event recorder. In addition, parental reports were compared to an objective recording of the pattern of cardiorespiratory activity surrounding each monitor alarm. Only those parental observations were considered which reported the infant to be asleep with no apparent equipment malfunction following an apnea alarm (with or without pallor, cyanosis, or the provision of external stimulation) or a low heart rate alarm associated with pallor, cyanosis, or stimulation. Observations were analyzed within each of five age periods (less than 29, 29 through 56, 57 through 84, 85 through 112, 113 through 140 days). The percentage of infants reported to have prolonged apnea, prolonged apnea with stimulation, or bradycardia with stimulation was found to decrease with age. An examination of the linked event recordings failed to document an episode of apnea as long as 15 seconds for any of the reported episodes of apnea. Furthermore, bradycardia as long as 5 seconds in duration could be documented in only 3 of 422 reported episodes of bradycardia. These results indicate the potential for considerable error when total reliance is placed on parental observations and point to the necessity for objective event recordings when using home monitors in the clinical management of at-risk infants.

Emotional and health impact of home monitoring on mothers: a controlled prospective study.

The psychologic and health effects of home monitoring were evaluated in mothers, whose infants (epidemiologically not at high risk for sudden infant death syndrome) were placed on electronic surveillance because of results obtained from a laboratory sleep study conducted at 4 weeks of age. Mothers of these infants were studied prospectively at several periods following the infants' births: 6 weeks, 12 weeks, and 1 year. The initial sample consisted of 56 mothers. Evaluation procedures included the Neonatal Perception Inventories, Anxiety Inventory (State and Trait), Depression Inventory, Brief Symptom Inventory, and a Recent Life Changes Questionnaire. The results obtained from these women were compared with those of women who had delivered at about the same time but whose infants were not placed on a home monitoring program. A total of 57 women started in the control group. Very few statistically significant group differences were observed between the two subject groups: 2 weeks after initiating the home monitoring program, monitor mothers (when compared to nonmonitor mothers) perceived their infant's behavior differently (although not more bothersome) and had an increased degree of situational anxiety (although not to an abnormal degree). There were no statistically significant differences between the two groups 12 weeks or 1 year after giving birth. These results suggest that a home monitoring program, which includes an aggressive and readily available support system, does not impose a marked health hazard to mothers.

Cost implications of event recordings in apnea/bradycardia home monitoring: a theoretical analysis.

OBJECTIVES: To evaluate the financial impact of incorporating event recordings as an integral component of home apnea/bradycardia monitoring. STUDY DESIGN: This theoretical analysis examines the cost of home monitoring when medical decisions are based on an evaluation of the cardiorespiratory waveforms surrounding each apnea/bradycardia monitor alarm (documented monitoring) compared to those based on parental observations. Data for both approaches were obtained from 155 infants referred within the first 10 days of life, because a sibling died of sudden infant death syndrome. All were followed on an impedance type apnea/bradycardia monitor with an attached event recorder. The monitor settings were 20 seconds for apnea and 80 beats per minute (bpm) for bradycardia. Parents were taught how to use the equipment, resuscitative techniques, and to complete an alarm log. The clinical protocol provided for home monitoring until there were no "episodes" (prolonged apnea or prolonged bradycardia) for 16 consecutive weeks. A polysomnogram would be obtained if an "episode" occurred. For each infant two independent approaches were used to judge the occurrence of an "episode": (1) parental report of an apnea alarm occurring during sleep or a physiologic alarm associated with skin color change or resuscitative intervention and (2) apnea > or = 20 seconds long or bradycardia > or = 10 seconds. The cost was calculated assuming a 4-week monitor rental fee of $350, a 4-week waveform interpretation fee of $180, and a $600 fee for performing and interpreting a polysomnogram. RESULTS: Episodes defined from an interpretation of the cardiorespiratory waveforms resulted in fewer diagnostic studies, a shorter period of home monitoring, and lower per patient treatment costs. CONCLUSION: Despite the increased monthly cost, incorporating event recordings as an integral component of home monitoring resulted in a lower average per patient cost.

The sudden infant death syndrome and apnea/obstruction during neonatal sleep and feeding.

Respiratory observations made during nutritive feeding and a complete daytime nap within the first week of life on ten newborns who subsequently were victims of sudden infant death syndrome (SIDS) were compared with normative data based on 1,301 infants of comparable ages. Measurements were obtained of all apneic pauses and/or transient respiratory obstructive events at least two seconds in duration. A statistically significant increased number of future SIDS victims were found to have an unusual amount of apnea/obstruction episodes during nutritive feeding when compared with the normative group. Future SIDS victims, as a group, also had an increased frequency of apneic pauses during sleep. Furthermore, all SIDS victims demonstrated either an unusual amount of apnea/obstruction during nutritive feeding or apneic pauses during sleep. None of the airway obstruction measures during sleep differentiated the SIDS infants from the normative group. These results are consistent with the general hypothesis that prolonged apnea or airway obstruction is part of the pathophysiologic process resulting in SIDS and those hypotheses implicating unstable respiratory activity during sleep as well as apnea and pharyngeal/laryngeal dysfunction induced by liquid stimulation of the upper airway. They are also compatible with the growing body of evidence indicating that factors that predispose an infant to SIDS originate in the perinatal period.

Apnea and airway obstruction during feeding and sleep.

Recent awareness of the magnitude of sudden unexplained deaths in apparently healthy infants has lead to an increased interest in those circumstances that are associated with or can elicit prolonged and serious apneic episodes. In the present studies, attention was directed toward the study of physiologic activity during sleep and feeding. Apneic episodes of varying durations occur during sleep which, in some instances, can be of sufficient length to warrant resuscitative intervention. A number of infants also reveal transient upper airway obstruction following brief periods of sleep apnea. This functional airway obstruction produces sudden and severe bradycardia. Similarly, infant feeding can induce dangerously prolonged periods of apnea and, in some infants, transient airway obstruction. Few detailed studies have been performed to identify the anatomical level or characteristics of the obstruction. Available evidence suggests that this can take the form either of muscle hypotonicity or hypertonicity. Two infants observed by means of direct laryngoscopy revealed transient failure of vocal cord abduction. These results have provided for the development of two theoretical models that can result in the sudden infant death syndrome; furthermore, continuous recordings of respiratory and cardiac activity during feeding and sleep can be extremely valuable in elucidating the mechanisms responsible for the sudden development of apneic and cyanotic episodes in infants.

Sudden infant death syndrome prevention and an understanding of selected clinical issues.

The etiology and prevention of sudden infant death syndrome (SIDS) are among the more frustrating topics in pediatrics. This article addresses several clinically relevant issues, including the relationships between apnea and SIDS, the nature of the "terminal event," effectiveness of home monitoring, the role of gastroesophageal reflux in apparent life-threatening events, and the association between the prone sleeping position and SIDS. This article is intended to provide the clinician with an awareness of the issues and the scientific basis needed to understand their contribution to SIDS.

Sudden infant death syndrome (SIDS), apnea, and near miss for SIDS.

Sudden infant death syndrome (SIDS) probably represents a number of specific processes rather than one disease, the causes of which have so far eluded scientists. Various hypotheses as to cause are discussed, as is the role of the emergency physician. Also considered are apparently life-threatening events such as prolonged sleep apnea, laryngeal-induced apnea, gastroesophageal reflux-induced apnea, and seizure-associated apnea.

Clinical characteristics of an apparent life-threatening event (ALTE) and the subsequent occurrence of prolonged apnea or prolonged bradycardia.

The objective of this study was to test the hypothesis that the characteristics of an apparent life-threatening event (ALTE) influence the risk for a subsequent episode of prolonged apnea or bradycardia. Data were obtained from 182 infants referred because of an apparent life-threatening event who were followed up at home for 2 lunar months on an apnea/bradycardia monitor with recording capability. A prolonged episode was defined as apnea > or = 20 sec long or an episode of bradycardia > or = 10 sec in duration as validated and measured from the event recordings. Seventeen percent of infants had at least one episode of prolonged apnea or prolonged bradycardia. Change in skin color, degree of resuscitative intervention, and behavioral state were not statistically significant factors influencing the risk of a subsequent prolonged episode. Contrary to clinical expectation, a significantly greater percentage of infants subsequently had a prolonged episode whose muscle tone was described as normal during the ALTE (24.0%), when compared with those infants whose ALTE was associated with limpness or increased muscle tone (9.3%). The coexistence of gastroesophageal reflux did not influence the overall results. The observations in this study place in question the clinical approach of relying on the detailed characteristics of an ALTE in determining the need for follow-up studies and/or home monitoring.