Comparative study on the in vitro replication and genomic variability of Argentinean field isolates of bovine herpesvirus type 4 (BoHV-4).

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus, belonging to the Rhadinovirus genus, which is increasingly associated with various problems of the reproductive tract of cattle. In Argentina, analysis of BoHV-4 strains isolated from cervico-vaginal mucus of aborted cows revealed a high genetic divergence among strains, which could be classified in three different groups: Genotype 1 comprises Movar-like strains (European prototype), Genotype 2 includes DN599-like strains (American prototype) and Genotype 3 corresponds to a novel genotype group. Understanding the replication behavior in cell cultures and the molecular characteristics of this pathogen of cattle is critical for the rational design of in vitro experiments. The aim of this work was to quantitatively evaluate the replication properties of different Argentinean BoHV-4 strains and to characterize their phylogenetic relationships. Significant differences were evident among the virus titers of the different BoHV-4 isolates in vitro. The most conserved gene was the major capsid protein (ORF25). The glycoprotein B (gB), glycoprotein H (gH), and thymidine kinsase (TK) genes displayed both synonymous and non-synonymous substitutions, with the highest diversity observed for gB, which displayed amino acid substitutions in 24 out of the 178 positions examined. Strains 09/759, 12/512, and 07/568 presented a deletion encompassing amino acid position 27 to 35, whereas strains 07/435 and 09/227 had a deletion from position 28 to 35. Two strains, 07/435 and 09/227, also displayed the highest divergence compared to the other strains analyzed. This study provides information about the in vitro replication and behavior of nine field isolates of BoHV-4. These findings are relevant since available information on the in vitro growth characteristics of BoHV-4 strains is scarce. The results from this study may also be useful for establishing comparisons with other related viruses.

Systematic review of cardiovascular disease and cardiovascular death in patients with a small abdominal aortic aneurysm.

BACKGROUND: Screening for abdominal aortic aneurysm (AAA) has reduced the rate of AAA rupture. However, cardiovascular disease is still a major cause of death in men with an AAA. The aim of this study was to assess cardiovascular risk in patients with a small AAA. METHODS: Standard PRISMA guidelines were followed. Analysis was performed of studies reporting cardiovascular outcomes in patients with a small AAA (30-54 mm). Weighted metaregression was performed for cardiovascular death in patients with a small AAA, and the prevalence of cardiovascular disease was reviewed. RESULTS: Twenty-one articles were identified describing patients with an AAA, and the prevalence of, and death from, cardiovascular disease. Ten of these reported cardiovascular death rates in patients with a small AAA. Some 2323 patients with a small AAA were identified; 335 cardiovascular deaths occurred, of which 37 were due to AAA rupture. Metaregression demonstrated that the risk of cardiovascular death was 3·0 (95 per cent c.i. 1·7 to 4·3) per cent per year in patients with a small AAA (R(2) = 0·902, P < 0·001). The prevalence of ischaemic heart disease (44·9 per cent), myocardial infarction (26·8 per cent), heart failure (4·4 per cent) and stroke (14·0 per cent) was also high in these patients. CONCLUSION: The risk of cardiovascular death in patients with a small AAA is high and increases by approximately 3 per cent each year after diagnosis. Patients with a small AAA have a high prevalence of cardiovascular disease. Patients a small AAA should be considered for lifestyle modifications and secondary cardiovascular protection.

Genetic data generated from virus-host complexes obtained by membrane co-immobilization are equivalent to data obtained from tangential filtrate virus concentrates and virus cultures.

The sieving and immobilization of virus-host complexes using impact filtration (aka membrane co-immobilization or MCI) is a novel approach to the study of plankton viruses. One of the most interesting characteristics of the method is the possibility of generating data on potential viral hosts without the need of culturing hosts cells. MCI has demonstrated to be useful for studying viruses of picoalgae, but studies comparing data generated by MCI to data obtained by other techniques are lacking. In this work, Ostreococcus virus (OV) and Ostreococcus sp. sequences generated from virus-host complexes obtained by MCI were compared to sequences obtained from tangential filtration (TF) concentrates and virus cultures (VC). Statistical parsimony, phylogenetic analyses, pairwise distance comparisons, and analysis of molecular variance showed that the viral and host sequences obtained by the three methods were highly related to each other, indicating that MCI, TF, and VC produce equivalent results. Minor differences were observed among viral sequences obtained from VC and TF concentrates as well as among host sequences generated from VC and MCI. As discussed in the body of the paper, the divergence observed for cultured cells could be due to selective pressures exerted by culture conditions, whereas the correlate observed for the corresponding viral sequences could obey to a hitchhiking effect.

Molecular characterization of hepatitis C virus genotype 4 sequences in HIV-coinfected patients from Argentina.

The prevalence of hepatitis C virus genotype 4 (HCV-4) is increasing in different parts of the World but in Latin America the data are still scarce. We aimed to characterize HCV-4 isolates from 383 HIV-coinfected patients in Argentina. Sequence analyses were based on the non-structural 5B region of HCV. Results from 18 patients indicated a genetic heterogeneity that involved three genotype 4 subtypes. Sequences were ascribed to subtype 4d (67%), 4a (22%), and 4m (11%). In spite of different sources of transmission were defined among patients, no statistical association was found with the genotype 4 subtype. The scenario is also compatible with multiple importation of the epidemic and there is no evidence for transmission-specific clusters or network-like transmission of HCV-4. This HCV-4 does not represent a recent introduction in Argentina, it circulates in all transmission groups and its presence is increasing among HIV-infected patients.

Evaluation of multiplex ligation-dependent probe amplification as a method for the detection of copy number abnormalities in B-cell precursor acute lymphoblastic leukemia.

Recent genomic studies have shown that copy number abnormalities (CNA) of genes involved in lymphoid differentiation and cell cycle control are common in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). We have evaluated Multiplex Ligation-dependent Probe Amplification (MLPA) on 43 BCP-ALL patients for the detection of the most common deletions among these genes and compared the results to those obtained by fluorescence in situ hybridization (FISH) and genomic quantitative PCR (qPCR). There was good correlation between methods for CDKN2A/B, IKZF1, and PAX5 deletions in the majority of cases and MLPA confirmed the presence of deletions within the PAR1 region in two of three cases identified by FISH. Small intragenic aberrations detected by MLPA, which were below the resolution of FISH for CDKN2A/B (n = 7), IKZF1 (n = 3), and PAX5 (n = 3) were confirmed by qPCR. MLPA and qPCR were unable to detect populations present at a low level (<20%) by FISH. In addition, although MLPA identified the presence of a deletion, it was unable to discern the presence of mixed cell populations which had been identified by FISH: CDKN2A/B (n = 3), IKZF1 (n = 1), PAX5 (n = 2), and PAR1 deletion (n = 1). Nevertheless, this study has demonstrated that MLPA is a robust technique for the reliable detection of CNA involving multiple targets in a single test and thus is ideal for rapid high throughput testing of large cohorts with a view to establishing incidence and prognostic significance.

Robotic Pancreatoduodenectomy: Patient Selection, Volume Criteria, and Training Programs.

INTRODUCTION: There has been a rapid development in minimally invasive pancreas surgery in recent years. The most recent innovation is robotic pancreatoduodenectomy. Several studies have suggested benefits as compared to the open or laparoscopic approach. This review provides an overview of studies concerning patient selection, volume criteria, and training programs for robotic pancreatoduodenectomy and identified knowledge gaps regarding barriers for safe implementation of robotic pancreatoduodenectomy. MATERIALS AND METHODS: A Pubmed search was conducted concerning patient selection, volume criteria, and training programs in robotic pancreatoduodenectomy. RESULTS: A total of 20 studies were included. No contraindications were found in patient selection for robotic pancreatoduodenectomy. The consensus and the Miami guidelines advice is a minimum annual volume of 20 robotic pancreatoduodenectomy procedures per center, per year. One training program was identified which describes superior outcomes after the training program and shortening of the learning curve in robotic pancreatoduodenectomy. CONCLUSION: Robotic pancreatoduodenectomy is safe and feasable for all indications when performed by specifically trained surgeons working in centers who can maintain a minimum volume of 20 robotic pancreatoduodenectomy procedures per year. Large proficiency-based training program for robotic pancreatoduodenectomy seem essential to facilitate a safe implementation and future research on robotic pancreatoduodenectomy.

Immunoelectron microscopical localization of phospholamban in adult canine ventricular muscle.

The subcellular distribution of phospholamban in adult canine ventricular myocardial cells was determined by the indirect immunogold-labeling technique. The results presented suggest that phospholamban, like the Ca2+-ATPase, is uniformly distributed in the network sarcoplasmic reticulum but absent from the junctional portion of the junctional sarcoplasmic reticulum. Unlike the Ca2+-ATPase, but like cardiac calsequestrin, phospholamban also appears to be present in the corbular sarcoplasmic reticulum. Comparison of the relative distribution of phospholamban immunolabeling in the sarcoplasmic reticulum with that of the sarcolemma showed that the density of phospholamban in the network sarcoplasmic reticulum was approximately 35-fold higher than that of the cytoplasmic side of the sarcolemma, which in turn was found to be three- to fourfold higher than the density of the background labeling. However, a majority of the specific phospholamban labeling within 30 nm of the cytoplasmic side of the sarcolemma was clustered and present over the sarcoplasmic reticulum in the subsarcolemmal region of the myocardial cells, suggesting that phospholamban is confined to the junctional regions between the sarcolemma and the sarcoplasmic reticulum, but absent from the nonjunctional portion of the sarcolemma. Although the resolution of the immunogold-labeling technique used (60 nm) does not permit one to determine whether the specific labeling within 30 nm of the cytoplasmic side of the sarcolemma is associated with the sarcolemma and/or the junctional sarcoplasmic reticulum, it is likely that the low amount of labeling in this region represents phospholamban associated with sarcoplasmic reticulum. These results suggest that phospholamban is absent from the sarcolemma and confined to the sarcoplasmic reticulum in cardiac muscle.

Single-molecule motions of oligoarginine transporter conjugates on the plasma membrane of Chinese hamster ovary cells.

To explore the real-time dynamic behavior of molecular transporters of the cell-penetrating-peptide (CPP) type on a biological membrane, single fluorescently labeled oligoarginine conjugates were imaged interacting with the plasma membrane of Chinese hamster ovary (CHO) cells. The diffusional motion on the membrane, characterized by single-molecule diffusion coefficient and residence time (tau R), defined as the time from the initial appearance of a single-molecule spot on the membrane (from the solution) to the time the single molecule disappears from the imaging focal plane, was observed for a fluorophore-labeled octaarginine (a model guanidinium-rich CPP) and compared with the corresponding values observed for a tetraarginine conjugate (negative control), a lipid analogue, and a fluorescently labeled protein conjugate (transferrin-Alexa594) known to enter the cell through endocytosis. Imaging of the oligoarginine conjugates was enabled by the use of a new high-contrast fluorophore in the dicyanomethylenedihydrofuran family, which brightens upon interaction with the membrane at normal oxygen concentrations. Taken as a whole, the motions of the octaarginine conjugate single molecules are highly heterogeneous and cannot be described as Brownian motion with a single diffusion coefficient. The observed behavior is also different from that of lipids, known to penetrate cellular membranes through passive diffusion, conventionally involving lateral diffusion followed by membrane bilayer flip-flop. Furthermore, while the octaarginine conjugate behavior shares some common features with transferrin uptake (endocytotic) processes, the two systems also exhibit dissimilar traits when diffusional motions and residence times of single constructs are compared. Additionally, pretreatment of cells with cytochalasin D, a known actin filament disruptor, produces no significant effect, which further rules out unimodal endocytosis as the mechanism of uptake. Also, the involvement of membrane potential in octaarginine-membrane interaction is supported by significant changes in the motion with high [K(+)] treatment. In sum, this first study of single transporter motion on the membrane of a living cell indicates that the mode by which the octaarginine transporter penetrates the cell membrane appears to either be a multimechanism uptake process or a mechanism different from unimodal passive diffusion or endocytosis.

A prospective analysis of dietary energy density at age 5 and 7 years and fatness at 9 years among UK children.

OBJECTIVE: To analyse whether high dietary energy density (DED) is associated with increased fat mass and risk of excess adiposity in free-living children. DESIGN: Longitudinal, observational cohort study. SUBJECTS: Six hundred and eighty-two healthy children from the Avon Longitudinal Study of Parents and Children. MEASUREMENTS: Diet was assessed at age 5 and 7 years using 3-day diet diaries, and DED (kJ g(-1)) was calculated excluding drinks. Fat mass was estimated at age 9 years using Dual-Energy X-ray Absorptiometry. To adjust for body size, fat mass index (FMI) was calculated by dividing fat mass (kg) by height (m(5.8)). Excess adiposity was defined as the top quintile of logFMI. RESULTS: Mean DED at age 5 years was higher among children with excess adiposity at age 9 years compared to the remaining sample (8.8+/-0.16 vs 8.5+/-0.07 kJ g(-1)), but there was no evidence of an association with excess adiposity at age 9 years (odds ratio (OR)=1.14, 95% confidence interval (CI) 0.90-1.44) after controlling for potential confounders. Mean DED at age 7 years was higher among children with excess adiposity compared to the remaining sample (9.1+/-0.12 vs 8.8+/-0.06 kJ g(-1)) and a 1 kJ g(-1) rise in DED increased the odds of excess adiposity at 9 years by 36% (OR=1.36, 95% CI 1.09-1.69) after controlling for potential confounders. CONCLUSION: Higher DED at age 7 years, but not age 5 years, is a risk factor for excess adiposity at age 9 years, perhaps reflecting deterioration in the ability to compensate for extra calories in an energy-dense diet. DED tracks strongly from age 5 to 7 years suggesting intervention to alter dietary habits need to commence at younger ages to prevent the formation of preferences for energy dense foods.

Lifestyle modification in the treatment of obesity: an educational challenge and opportunity.

As many as two-thirds of adults in developed nations are overweight (body mass index (BMI)=25.0-29.9 kg/m2) or obese (BMI>or=30 kg/m2), and many of these individuals suffer from weight-related comorbidities such as hypertension, hyperlipidemia, and type II diabetes. On a more positive note, recent studies have demonstrated that losses as small as 5-10% of initial weight can improve these health complications. For example, the Diabetes Prevention Program demonstrated that a 7% reduction in initial weight, coupled with 150 min/week of physical activity, reduced the risk of developing type II diabetes by 58% compared with placebo. Behavioral treatment consistently induces weight losses in this range. This paper describes the behavioral treatment of obesity, including its short- and long-term results as well as approaches for improving the maintenance of lost weight. The terms "behavioral treatment," "lifestyle modification," and "behavioral weight control" are often used interchangeably, as they are in this paper. Lifestyle modification includes three principal components: diet, physical activity, and behavior therapy. The latter term, as applied to weight control, refers to a set of principles and techniques to help patients adopt new diet and exercise habits that can be sustained long term to promote health.

Enhanced myocardial relaxation in vivo in transgenic mice overexpressing the beta2-adrenergic receptor is associated with reduced phospholamban protein.

To assess the effect of targeted myocardial beta-adrenergic receptor (AR) stimulation on relaxation and phospholamban regulation, we studied the physiological and biochemical alterations associated with overexpression of the human beta2-AR gene in transgenic mice. These mice have an approximately 200-fold increase in beta-AR density and a 2-fold increase in basal adenylyl cyclase activity relative to negative littermate controls. Mice were catheterized with a high fidelity micromanometer and hemodynamic recordings were obtained in vivo. Overexpression of the beta2-AR altered parameters of relaxation. At baseline, LV dP/dt(min) and the time constant of LV pressure isovolumic decay (Tau) in the transgenic mice were significantly shorter compared with controls, indicating markedly enhanced myocardial relaxation. Isoproterenol stimulation resulted in shortening of relaxation velocity in control mice but not in the transgenic mice, indicating maximal relaxation in these animals. Immunoblotting analysis revealed a selective decrease in the amount of phospholamban protein, without a significant change in the content for either sarcoplasmic reticulum Ca2+ ATPase or calsequestrin, in the transgenic hearts compared with controls. This study indicates that myocardial relaxation is both markedly enhanced and maximal in these mice and that conditions associated with chronic beta-AR stimulation can result in a selective reduction of phospholamban protein.

Regulation of Ca2+ signaling in transgenic mouse cardiac myocytes overexpressing calsequestrin.

To probe the physiological role of calsequestrin in excitation-contraction coupling, transgenic mice overexpressing cardiac calsequestrin were developed. Transgenic mice exhibited 10-fold higher levels of calsequestrin in myocardium and survived into adulthood, but had severe cardiac hypertrophy, with a twofold increase in heart mass and cell size. In whole cell-clamped transgenic myocytes, Ca2+ channel- gated Ca2+ release from the sarcoplasmic reticulum was strongly suppressed, the frequency of occurrence of spontaneous or Ca2+ current-triggered "Ca2+ sparks" was reduced, and the spark perimeter was less defined. In sharp contrast, caffeine-induced Ca2+ transients and the resultant Na+-Ca2+ exchanger currents were increased 10-fold in transgenic myocytes, directly implicating calsequestrin as the source of the contractile-dependent pool of Ca2+. Interestingly, the proteins involved in the Ca2+-release cascade (ryanodine receptor, junctin, and triadin) were downregulated, whereas Ca2+-uptake proteins (Ca2+-ATPase and phospholamban) were unchanged or slightly increased. The parallel increase in the pool of releasable Ca2+ with overexpression of calsequestrin and subsequent impairment of physiological Ca2+ release mechanism show for the first time that calsequestrin is both a storage and a regulatory protein in the cardiac muscle Ca2+-signaling cascade. Cardiac hypertrophy in these mice may provide a novel model to investigate the molecular determinants of heart failure.

Quality of food eaten in English primary schools: school dinners vs packed lunches.

OBJECTIVE: To compare the food and nutrient intakes of primary school children eating school dinners and packed lunches. SUBJECTS: Six-hundred and twenty-one 7-year-old children participating in the Avon Longitudinal Study of Parents and Children in South West England. METHODS: Diet was assessed by 3-day unweighed food record. RESULTS: The composition of both types of school meals compared unfavourably with dietary guidelines. Intakes of energy, non-starch polysaccharides (NSP), calcium, iron, folate, retinol equivalents, zinc, copper, magnesium, iodine and riboflavin were too low, and intakes of total and saturated fat were too high. However, children who ate school dinners had higher lunchtime intakes of protein, starch, NSP and most vitamins and minerals and lower intakes of sugar (14.2 and 20.9% of energy in school dinners and packed lunches, respectively, P<0.001) and saturated fat (12.0 and 16.2%, P<0.001). Only around half of the recommended amount of fruit and vegetables was eaten by children having either type of school meal. There were also differences in the whole day's nutrient intake according to school meal type. Children eating packed lunches had lower daily intakes of potassium and zinc, and higher intakes of sugar and saturated fat. Differences in nutrient intake were independent of maternal education. CONCLUSIONS: The food and nutrient content of both school dinners and packed lunches needed improvement. However, the standard of food brought from home by children was, if anything, worse than that served at school. Recent moves to improve school dinners will need to be complemented by education about what constitutes a healthy packed lunch.

Molecular characterization of bovine rotavirus circulating in beef and dairy herds in Argentina during a 10-year period (1994-2003).

Group A bovine rotavirus (BRV) is one of the main causes of neonatal calf diarrhea. The present study reports the incidence of rotavirus diarrhea and the genotypes of BRV strains circulating in beef and dairy herds from Argentina, during a 10-year period (1994-2003). Group A BRV was detected in 62.5% (250/400) of the total studied cases of diarrhea. Positive cases were analyzed by heminested multiplex RT-PCR for P and G genotypes identification. Sixty percent of them were typed as P[5]G6, 4.4% P[11]G10, 4.4% P[11]G6 and 2.4% P[5]G10. Additionally, 9.2% of the cases were initially typed as G8 combined with P[5] or P[11], but sequence analysis revealed they belonged to genotype G6, lineage Hun4-like. Partial typing was assessed in 12.0% of the cases. One of the partially typed samples was closely related to genotype G15. BRV was detected in 71% and 58% of the outbreaks registered in beef and dairy farms, respectively. A clear differential distribution of G/P types was found according to the herd type. P[5]G6 was the prevalent strain in beef herds, while P[11] was the prevalent P-type in dairy herds (71%), associated in similar proportions with G6 and G10, These findings indicate that BRV genotypes included in the current commercially available rotavirus vaccines (G6, G10, P[5] and P[11]) should protect calves from most Argentinean field strains. Nevertheless, continuous surveillance is necessary to detect the emergence of new variants.

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum.

ATP and the divalent cations Mg2+ and Ca2+ regulated K+ stimulation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K+-stimulated ATPase activity of the Ca2+ pump by two mechanisms. First, ATP chelated free Mg2+ and, at low ionized Mg2+ concentrations, K+ was shown to be a potent activator of ATP hydrolysis. In the absence of K+ ionized Mg2+ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K+ the concentration of ionized Mg2+ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K+-stimulation. With a saturating concentration of ionized Mg2+, stimulation by K+ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K+ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold. Activation of K+-stimulated ATPase activity by Ca2+ was maximal at an ionized Ca2+ concentration of approx. 1 microM. At very high concentrations of either Ca2+ or Mg2+, basal Ca2+-dependent ATPase activity persisted, but the enzymic response to K+ was completely inhibited. The results provide further evidence that the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme.

Evidence for the presence of phospholamban in the endoplasmic reticulum of smooth muscle.

In microsomal vesicles isolated from several smooth muscles many polypeptides were phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. In pig stomach and in rabbit and dog aorta components of Mr 22,000 and 11,000 were identified as forms of phospholamban. These polypeptides were, however, not observed in pig aorta. These phospholamban-like polypeptides presented the same electrophoretic mobility in sodium dodecyl sulphate gels as cardiac phospholamban, and the 22,000 Mr form showed a similar reaction to heat treatment in sodium dodecyl sulphate. Antibodies against purified canine cardiac phospholamban cross-reacted with the 22,000 and 11,000 Mr phosphorylatable polypeptides from smooth muscle membranes. Subcellular fractionation of porcine stomach smooth muscle indicated that phospholamban was present in the membranes of the endoplasmic reticulum and not in the plasma membranes. Phospholamban was also phosphorylated by an endogenous calcium-calmodulin-dependent protein kinase and by an endogenous cyclic AMP-dependent kinase. It is concluded that the endoplasmic reticulum of many, but possibly not all, smooth muscles contains phospholamban. However, the physiological role of phospholamban in smooth muscle remains to be established.

Smooth-muscle endoplasmic reticulum contains a cardiac-like form of calsequestrin.

It is proposed that smooth-muscle endoplasmic reticulum contains calsequestrin and that this protein in smooth muscle resembles cardiac calsequestrin more than the skeletal-muscle form. This proposal is based on seven similarities between the smooth-muscle protein and cardiac calsequestrin. Proteins with an Mr of 55,000 can be extracted from the membranes of smooth muscle and of cardiac muscle using 100 mM Na2CO3. The protein from smooth muscle binds to phenyl-Sepharose in the absence of Ca2+ and is released by 10 mM CaCl2, as has been observed for cardiac calsequestrin. The protein from smooth muscle comigrates with the cardiac calsequestrin on Laemmli-type SDS-polyacrylamide gel electrophoresis. The protein of Mr 55,000 from smooth muscle and cardiac calsequestrin both strain blue with the carbocyanine dye Stains-all. Both proteins present similar one-dimensional Cleveland peptide maps although minor differences might exist. From an analysis of subcellular membranes separated by sucrose gradient centrifugation it is concluded that the protein with Mr 55,000 from the smooth muscle is confined to the endoplasmic reticulum, the same subcellular structure from which, in heart muscle, calsequestrin can be isolated. Antibodies raised against canine cardiac calsequestrin bind to a protein of similar Mr in smooth-muscle endoplasmic reticulum. In addition to the calsequestrin, three other extrinsic proteins with an Mr of 130,000, 100,000 and 63,000, stain blue with Stains-all and occur in the endoplasmic reticulum of smooth muscle.

Structural and functional comparison of a 22 kDa protein from internal human platelet membranes with cardiac phospholamban.

We have shown that a platelet 22 kDa phosphoprotein is similar to the cardiac regulator phospholamban, in that both are phosphorylated by cAMP- and calmodulin-dependent protein kinases, and that both can be phosphorylated simultaneously by these two classes of protein kinases to yield an additive stimulation of the respective Ca2+ pumps (Adunyah, S.E. and Dean, W.L. (1987) Biochim. Biophys. Acta 930, 401-409). However, whereas phosphorylation of phospholamban increases the affinity of the cardiac Ca2+-ATPase for Ca2+, phosphorylation of the platelet 22 kDa protein increased the Vmax of the pump. In addition, antibodies raised against canine phospholamban did not crossreact with the human platelet 22 kDa protein. Finally, it was not possible to dissociate the platelet protein into lower-molecular-weight subunits by boiling in sodium dodecylsulfate which is characteristic of cardiac phospholamban. These results show that although phosphorylation of low-molecular-weight membrane-associated regulator proteins in cardiac muscle and platelets appears to stimulate the respective Ca2+ pumps, these proteins have different chemical and physical properties.

Secondary structure of detergent-solubilized phospholamban, a phosphorylatable, oligomeric protein of cardiac sarcoplasmic reticulum.

The structure of phospholamban, a 30-kDa oligomeric protein integral to cardiac sarcoplasmic reticulum, was probed using ultraviolet absorbance and circular dichroism spectroscopy. Purified phospholamban was examined in three detergents: octyl glucoside, n-dodecyloctaethylene glycol monoether (C12E8) and sodium dodecyl sulfate (SDS). Ultraviolet absorption spectra of phospholamban reflected its aromatic amino acid content: absorption peaks at 275-277 nm and 253, 259, 265 and 268 nm were attributed to phospholamban's one tyrosine and two phenylalanines, respectively. Phospholamban phosphorylated at serine 16 by the catalytic subunit of cAMP-dependent protein kinase exhibited no absorbance changes when examined in C12E8 or SDS. Circular dichroism spectroscopy at 250-190 nm demonstrated that phospholamban possesses a very high content of alpha-helix in all three detergents and is unusually resistant to denaturation. Dissociation of phospholamban subunits by boiling in SDS increased the helical content, suggesting that the highly ordered structure is not dependent upon oligomeric interactions. The purified COOH-terminal tryptic fragment of phospholamban, containing residues 26-52 and comprising the hydrophobic, putative membrane-spanning domain, also exhibited a circular dichroism spectrum characteristic of alpha-helix. Circular dichroism spectra of phosphorylated and dephosphorylated phospholamban were very similar, indicating that phosphorylation does not alter phospholamban secondary structure significantly. The results are consistent with a two-domain model of phospholamban in which each domain contains a helix and phosphorylation may act to rotate one domain relative to the other.

Membrane localization of myocardial type II cyclic AMP-dependent protein kinase activity.

Crude cardiac membrane vesicles were separated into subfractions of sarcolemma and sarcoplasmic reticulum. The subfractions were used to determine the origin and type of cyclic AMP-dependent protein kinase activity present in myocardial membranes. A cyclic AMP-binding protein of molecular weight 55,000 was covalently labeled with the photoaffinity probe 8-azido adenosine 3',5'-mono[32P]phosphate, and found to copurify with the (Na+ + K+)-ATPase activity of sarcolemma, and away from the (Ca2+ + K+)-ATPase activity of sarcoplasmic reticulum. Endogenous cyclic AMP-dependent protein kinase activity also copurified with sarcolemma. Protein substrates phosphorylated by cyclic AMP-dependent protein kinase activity had apparent molecular weights of 21,000 and 8000 and were present in both sarcolemma and sarcoplasmic reticulum. However, while addition of cyclic AMP alone resulted in phosphorylation of sarcolemma proteins, both cyclic AMP and exogenous, soluble cyclic AMP-dependent kinase were required for phosphorylation of sarcoplasmic reticulum proteins. Addition of the calcium-binding protein, calmodulin, to either sarcolemma or sarcoplasmic reticulum resulted in phosphorylation of the 21,000 and 8000-dalton proteins, as well. The results suggest that cardiac sarcolemma contains an intrinsic type II cyclic AMP-dependent protein kinase activity that is not present in sarcoplasmic reticulum. On the other hand, Ca2+- and calmodulin-dependent protein kinase activity is present in both sarcolemma and sarcoplasmic reticulum.