Phosphorylation of smg p21, a ras p21-like GTP-binding protein, by cyclic AMP-dependent protein kinase in a cell-free system and in response to prostaglandin E1 in intact human platelets

We have separated multiple small Mr GTP-binding proteins (G proteins) from bovine brain membranes by several column chromatographies and purified to near homogeneity four of them, including a novel Mr 24,000 G protein (smg p25A), a novel Mr 22,000 G protein (smg p21), the rho protein (rho p20), and the c-Ki-ras protein (c-Ki-ras p21). Among these small Mr G proteins, only smg p21 is phosphorylated stoichiometrically by cAMP-dependent protein kinase (protein kinase A), and c-Ki-ras p21 is phosphorylated to a small extent by protein kinase A in a cell-free system. None of smg p25A, rho p20, and other partially purified small Mr G proteins is phosphorylated by protein kinase A. Neither smg p21 nor other small Mr G proteins are phosphorylated by protein kinase C. About 1 mol of phosphate is maximally incorporated into 1 mol of smg p21 by protein kinase A. Only serine residue(s) are phosphorylated. The guanosine 5'-3-O-(thio) triphosphate (GTP gamma S)-bound and GDP-bound forms of smg p21 are phosphorylated with the same reaction velocity. The phosphorylation of smg p21 affects neither its GTP gamma S-binding nor GTPase activity. smg p21 is found in human platelets, and this human platelet smg p21 is also phosphorylated by protein kinase A at the same site(s) as bovine brain smg p21 in a cell-free system. When intact human platelets are stimulated by prostaglandin E1 known to elevate the cAMP level, four proteins with apparent Mr values of 240,000, 50,000, 24,000, and 22,000 are phosphorylated. These four proteins are also phosphorylated by the action of dibutyryl cAMP but not by the action of thrombin, Ca2+ ionophore A23187, or 12-O-tetradecanoylphorbol-13-acetate. Among the four proteins, the Mr 22,000 protein is identified as smg p21. The site(s) of phosphorylation of smg p21 by protein kinase A in a cell-free system are identical to that phosphorylated in response to prostaglandin E1 in intact platelets. These results indicate that among many small Mr G proteins, smg p21 is selectively phosphorylated by protein kinase A and that this G protein is also phosphorylated by this protein kinase in response to prostaglandin E1 in intact human platelets.     

Soil-transmitted helminth infection,anemia, and malnutrition among preschool-age children in Nangapanda subdistrict,Indonesia

BackgroundSoil-transmitted helminth (STH) infections are still prevalent in Indonesia, with roughly one-third of infected population being preschool-age children (PSC), which are generally at higher risk of morbidity such as malnutrition and anemia. This study aimed to investigate the association of STH infections with nutritional status and anemia among PSC in Nangapanda subdistrict, Ende, East Nusa Tenggara.MethodsA cross-sectional survey involving PSC ranging from 12 to 59 months old from Nangapanda subdistrict, Ende district, East Nusa Tenggara was performed. Socio-demographic, breastfeeding, and complementary feeding information was obtained from structured questionnaires, while nutritional and anemia status was determined from anthropometry and hemoglobin measurements, respectively. Anthropometric z-scores were calculated based on the World Health Organization 2006 standards and stool samples were examined using Kato-Katz method.ResultsA total of 393 PSC randomly selected from 22 villages were examined. The prevalence of underweight, stunting, wasting, and anemia were 33.1%, 40.2%, 17.1%, and 60.3%, respectively. STH infection, predominated by Ascaris lumbricoides, was found in 160 (58.8%) PSC. Single STH infection, but not multiple infection, was independently associated with a lower risk of anemia (odds ratio [OR] 0.320, 95% confidence interval [CI]: 0.126–0.809, p = 0.016). Similar association with anemia was also found on mild STH infection (OR 0.318 [95% CI: 0.114–0.887], p = 0.029). On the other hand, younger children were found to have a higher risk of anemia and stunting. None of the examined variables were independently associated with underweight and wasting.ConclusionSTH infection as well as anemia and malnutrition were prevalent in this region. However in this study, current STH infections seemed to have minimal negative impact on children’s nutritional status.     

Nanoscale Distribution of Ryanodine Receptors and Caveolin-3 in Mouse Ventricular Myocytes: Dilation of T-Tubules near Junctions

We conducted super-resolution light microscopy (LM) imaging of the distribution of ryanodine receptors (RyRs) and caveolin-3 (CAV3) in mouse ventricular myocytes. Quantitative analysis of data at the surface sarcolemma showed that 4.8% of RyR labeling colocalized with CAV3 whereas 3.5% of CAV3 was in areas with RyR labeling. These values increased to 9.2 and 9.0%, respectively, in the interior of myocytes where CAV3 was widely expressed in the t-system but reduced in regions associated with junctional couplings. Electron microscopic (EM) tomography independently showed only few couplings with caveolae and little evidence for caveolar shapes on the t-system. Unexpectedly, both super-resolution LM and three-dimensional EM data (including serial block-face scanning EM) revealed significant increases in local t-system diameters in many regions associated with junctions. We suggest that this regional specialization helps reduce ionic accumulation and depletion in t-system lumen during excitation-contraction coupling to ensure effective local Ca²⁺ release. Our data demonstrate that super-resolution LM and volume EM techniques complementarily enhance information on subcellular structure at the nanoscale.The contraction of cardiac ventricular myocytes depends on the rapid cell-wide transient increase in intracellular [Ca²⁺] upon depolarization of the cell-membrane potential. The cardiac ryanodine receptor (RyR) (1), which is the intracellular Ca²⁺ release channel in the sarcoplasmic reticulum (SR), plays a central role in shaping Ca²⁺ transients. RyRs form clusters of various sizes (2,3) with the majority located within junctions between the SR and the surface membrane and its cytoplasmic extension, the transverse tubular (t-) system. It has been suggested that some RyR clusters are associated with caveolae, a specialized signaling microdomain of the surface membrane. Previous studies were complicated by the limited resolution of optical imaging methods of ∼250 nm, much larger than the nanometer scale of RyRs and caveolae. Accordingly, these studies report varying colocalization between RyRs and caveolin-3 (CAV3), a caveolar marker also expressed in the t-system (4,5).In this work, we investigated the relative distribution of CAV3 and RyRs in mouse ventricular myocytes both in the cytosol and near the cell surface with super-resolution fluorescence microscopy that achieves a resolution approaching 30 nm. Our data revealed unexpected local t-system swellings near junctional couplings, which was supported by two different three-dimensional electron microscopy (EM) modalities with <10-nm resolution: EM tomography and serial block-face scanning EM (SBFSEM).Super-resolution images of CAV3 and RyR labeling at the surface sarcolemma of mouse myocytes showed little overlap, suggesting that few RyRs were in couplings with caveolae (Fig. 1 A, for detailed methods, see the Supporting Material). Only ∼4.8% of RyR labeling was associated with CAV3 positive areas and ∼3.5% of CAV3 associated with RyR positive areas (n = 6 cells from three animals, Fig. 1 B, see also Table S1 in the Supporting Material), broadly consistent with previous data in rats (6). To support this finding, EM tomography was applied to mouse ventricular tissue that included a part of the surface sarcolemma, to our knowledge for the first time. Segmentation of peripheral couplings (containing RyR foot structures) and surface caveolae (∼60 nm in diameter and often interconnected) confirmed that the great majority of peripheral couplings were in regions devoid of caveolae (Fig. 1 C). A few junctional couplings containing feet were between caveolae and subsarcolemmal SR (Fig. 1 D, see also Fig. S1 and Movie S1 in the Supporting Material). We conducted a similar analysis in the cytosol where CAV3 expression occurs in the t-system (5) and RyRs are abundant in dyadic junctions between the t-system and SR terminal cisterns.Open in a separate windowFigure 1Colocalization of CAV3 and RyRs at the surface sarcolemma. (A) Super-resolution micrograph of the distribution of CAV3 (green) and RyRs (red) at the surface of a mouse cardiac myocyte. (B) Analysis of the association of CAV3 with RyRs. The fraction of RyR labeling within CAV3 positive areas was ∼4.8% (front data) whereas ∼3.5% of CAV3 was found in RyR-positive membrane areas. (C) Segmented EM tomogram containing a patch of surface sarcolemma (light blue) and associated caveolae (green) as well as peripheral couplings (red). (D) Detailed view of a region with abundant caveolae. (Arrows) Couplings with caveolae.As shown in Fig. 2 A, the spatial distribution of CAV3 and RyR clusters in super-resolution micrographs taken several microns below the surface sarcolemma is consistent with this view. The association of the two labels is slightly increased (as compared to the surface), according to distance analysis with 9% of CAV3 and 9.2% of RyR labeling associating with each other (Fig. 2 B, n = 6 cells from three animals). The similarity of manually traced t-system in EM tomograms (Fig. 2 C) and super-resolved CAV3 labeling suggested that CAV3 is widely distributed in the t-system except for regions where dyadic membrane junctions occur as CAV3 labeling was much weaker in regions with strong RyR labeling. It was notable that the t-system diameter appeared to increase at regions of strong RyR labeling (Fig. 2 D), broadly consistent with the behavior seen in tomograms (Fig. 2 C). This was confirmed by a quantitative analysis of t-tubule diameters in dyadic versus extradyadic regions on the basis of CAV3 and RyR labeling, with full-width at quarter-maximum mean diameters increasing from ∼150 nm distal to dyads, to ∼190 nm (using CAV3 signal only) or ∼280 nm (using CAV3 and RyR signal) near dyads (Fig. 2, G and H, see also Methods in the Supporting Material). The combined RyR and CAV3 signals seemed to be a better representation of the entire t-system lumen near junctions (see Fig. S2).Open in a separate windowFigure 2Distribution of CAV3 and RyRs in the cell interior. (A) Super-resolution micrograph of CAV3 (green) and RyR (red) distribution at t-system. (Arrow) Direction of longitudinal cell axis. (B) Distance analysis of the CAV3 and RyR association (N = 6 cells per group). (C) Segmented EM tomogram of a similar region with three-dimensional mesh models of t-system membrane (green) and dyadic couplings (red). (D) This image illustrates the tracing (white path) of t-tubules. The label distribution was extracted and linearized along the path (E) to calculate a mask that shows the full width at quarter-maximum diameter along tubules, CAV3 (green) and RyR (red) (F). (G) Histograms of local diameters extracted from traced t-tubules. (H) Mean diameters in junctional (dyad) and nonjunctional (ex-dyad) regions. See main text and the Supporting Material for details. ^**p < 0.01.Taken together, super-resolution imaging and EM tomography strongly support the presence of local t-system dilations in regions where the t-system opposes SR at dyads and such t-system bulges are connected by narrower tubule segments. Further support was provided by SBFSEM, another volume EM technique to study larger cell volumes (albeit at the expense of a slightly lower resolution). SBFSEM clearly showed local t-system dilations were regularly involved in the architecture of most (but not all) dyads (Fig. 3, see also Fig. S3 and Movie S2), as also observed in full three-dimensional super-resolution images (see Fig. S3 C).Open in a separate windowFigure 3Segmented SBFSEM data showing t-system dilations near dyadic junctions. (A) The overview shows t-system membranes (green) and jSR (red) in a mouse myocyte. (B, enlarged inset from panel A) Thin connecting tubules (arrows) and regular swellings in junctional regions at z-lines.Our data identify local dilations of the t-system associated with dyads in mouse cardiac myocytes. Frequent tubule distensions had been observed especially at the intersections of transverse and axial tubules (7), and constrictions were seen in rabbit myocytes although their relationship to dyads was unknown (8). The increased local t-system lumen near junctions may help reduce the predicted ionic accumulation/depletion during excitation-contraction coupling (9). Alternatively, it might simply be secondary to increasing local membrane area and allow the formation of large area junctions that harbor many RyRs. In connection with this point, it would be interesting to investigate the t-system near junctions in species that have larger average tubule diameters (e.g., human and rabbit (10)), or if this architecture changes in mouse heart failure models where t-tubule diameters are often increased.Most peripheral couplings were in regions void of surface caveolae, although a small number of RyR clusters were in junctional couplings between subsarcolemmal SR and caveolae as shown both by the low colocalization between CAV3 and RyRs as well as direct evidence from EM tomography. Similarly, a relatively small fraction of CAV3 colocalized with RyR clusters in the t-system although CAV3 was expressed widely in the t-system. A structural role of CAV3 in the t-system is still unclear—t-tubules in tomogram data did not reveal distinct caveolae shapes on the t-system membrane (see Fig. S4), although this might change in pathology (11). In any case, the t-system exhibits high curvature orthogonal to the tubule axis, which may be supported by CAV3 oligomerization. In addition, the presence of CAV3 in the t-system may be important for regulating other signaling systems (e.g., adrenergic signaling).Finally, our data demonstrate that complementary data from optical super-resolution and three-dimensional EM images assists data interpretation and reliability. We suggest that truly correlative optical and EM imaging approaches should provide further information and improve our knowledge of the basis of cardiac excitation-contraction coupling.     

Small proline-rich protein 1A is a gp130 pathway- and stress-inducible cardioprotective protein

The interleukin-6 cytokines, acting via gp130 receptor pathways, play a pivotal role in the reduction of cardiac injury induced by mechanical stress or ischemia and in promoting subsequent adaptive remodeling of the heart. We have now identified the small proline-rich repeat proteins (SPRR) 1A and 2A as downstream targets of gp130 signaling that are strongly induced in cardiomyocytes responding to biomechanical/ischemic stress. Upregulation of SPRR1A and 2A was markedly reduced in the gp130 cardiomyocyte-restricted knockout mice. In cardiomyocytes, MEK1/2 inhibitors prevented SPRR1A upregulation by gp130 cytokines. Furthermore, binding of NF-IL6 (C/EBPbeta) and c-Jun to the SPRR1A promoter was observed after CT-1 stimulation. Histological analysis revealed that SPRR1A induction after mechanical stress of pressure overload was restricted to myocytes surrounding piecemeal necrotic lesions. A similar expression pattern was found in postinfarcted rat hearts. Both in vitro and in vivo ectopic overexpression of SPRR1A protected cardiomyocytes against ischemic injury. Thus, this study identifies SPRR1A as a novel stress-inducible downstream mediator of gp130 cytokines in cardiomyocytes and documents its cardioprotective effect against ischemic stress.     

The cardiac mechanical stretch sensor machinery involves a Z disc complex that is defective in a subset of human dilated cardiomyopathy

Muscle cells respond to mechanical stretch stimuli by triggering downstream signals for myocyte growth and survival. The molecular components of the muscle stretch sensor are unknown, and their role in muscle disease is unclear. Here, we present biophysical/biochemical studies in muscle LIM protein (MLP) deficient cardiac muscle that support a selective role for this Z disc protein in mechanical stretch sensing. MLP interacts with and colocalizes with telethonin (T-cap), a titin interacting protein. Further, a human MLP mutation (W4R) associated with dilated cardiomyopathy (DCM) results in a marked defect in T-cap interaction/localization. We propose that a Z disc MLP/T-cap complex is a key component of the in vivo cardiomyocyte stretch sensor machinery, and that defects in the complex can lead to human DCM and associated heart failure.     

ENH, containing PDZ and LIM domains, heart/skeletal muscle-specific protein, associates with cytoskeletal proteins through the PDZ domain

The Enigma homologue protein (ENH), containing an N-terminal PDZ domain and three C-terminal LIM domains, is a heart and skeletal muscle-specific protein that has been shown to preferentially interact with protein kinase C beta (PKCbeta) through the LIM domains (Kuroda et al., J. Biol. Chem. 271, 31029-31032, 1996). We here demonstrate that ENH is colocalized with a cytoskeletal protein alpha-actinin in the Z-disk region of rat neonatal cardiomyocytes. Pull-down assays using the glutathione-S-transferase-fusion system also showed the interaction of the PDZ domain of ENH with actin and alpha-actinin. Furthermore, by combined use of the in silico and conventional cDNA cloning methods, we have isolated three ENH-related clones from a mouse heart-derived cDNA library: mENH1 (591 amino acid residues) corresponding to rat ENH, mENH2 (337 residues), and mENH3 (239 residues); the latter two containing only a single PDZ domain. Deciphering their cDNA sequences, these mENH1-3 mRNAs appear to be generated from a single mENH gene by alternative splicing. Northern blot analyses using human cancer cells and mouse embryos have shown expression of each mENH mRNA to vary considerably among the cell types and during the developmental stage. Together with a recent finding that PKCbeta is markedly activated in the cardiac hypertrophic signaling, these results suggest that ENH1 plays an important role in the heart development by scaffolding PKCbeta to the Z-disk region and that ENH2 and ENH3 negatively modulate the scaffolding activity of ENH1.     

Altered membrane proteins and permeability correlate with cardiac dysfunction in cardiomyopathic hamsters

A mutation in the delta-sarcoglycan (SG) gene with absence of delta-SG protein in the heart has been identified in the BIO14.6 cardiomyopathic (CM) hamster, but how the defective gene leads to myocardial degeneration and dysfunction is unknown. We correlated left ventricular (LV) function with increased sarcolemmal membrane permeability and investigated the LV distribution of the dystrophin-dystroglycan complex in BIO14.6 CM hamsters. On echocardiography at 5 wk of age, the CM hamsters showed a mildly enlarged diastolic dimension (LVDD) with decreased LV percent fractional shortening (%FS), and at 9 wk further enlargement of LVDD with reduction of %FS was observed. The percent area of myocardium exhibiting increased membrane permeability or membrane rupture, assessed by Evans blue dye (EBD) staining and wheat germ agglutinin, was greater at 9 than at 5 wk. In areas not stained by EBD, immunostaining of dystrophin was detected in CM hamsters at sarcolemma and T tubules, as expected, but it was also abnormally expressed at the intercalated discs; in addition, the expression of beta-dystroglycan was significantly reduced compared with control hearts. As previously described, alpha-SG was completely deficient in CM hearts compared with control hearts. In myocardial areas showing increased sarcolemmal permeability, neither dystrophin nor beta-dystroglycan could be identified by immunolabeling. Thus, together with the known loss of delta-SG and other SGs, abnormal distribution of dystrophin and reduction of beta-dystroglycan are associated with increased sarcolemmal permeability followed by cell rupture, which correlates with early progressive cardiac dysfunction in the BIO14.6 CM hamster.     

Isozyme analysis and relationships among three species in Malaysian Trichoderma isolates

Isozyme and protein electrophoresis data from mycelial extracts of 27 isolates of Trichoderma harzianum, 10 isolates of T. aureoviride and 10 isolates of T. longibrachiatum from Southern Peninsular Malaysia were investigated. The eight enzyme and a single protein pattern systems were analyzed. Three isozyme and total protein patterns were shown to be useful for the detection of three Trichoderma species. The isozyme and protein data were analyzed using the Nei and Li Dice similarity coefficient for pairwise comparison between individual isolates, species isolate group, and for generating a distance matrix. The UPGMA cluster analysis showed a higher degree of relationship between T. harzianum and T. aureoviride than to T. longibrachiatum. These results suggested that the T. harzianum isolates had high levels of genetic variation compared to the other isolates of Trichoderma species.     

Erratum to: Characterization of a KCS-like KASII from<Emphasis Type="Italic"> Jessenia bataua</Emphasis> that Elongates Saturated and Monounsaturated Stearic Acids in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

As the world population grows, the demand for food increases. Although vegetable oils provide an affordable and rich source of energy, the supply of vegetable oils available for human consumption is limited by the “fuel vs food” debate. To increase the nutritional value of vegetable oil, metabolic engineering may be used to produce oil crops of desirable fatty acid composition. We have isolated and characterized β-ketoacyl ACP-synthase II (KASII) cDNA from a high-oleic acid palm, Jessenia bataua. Jessenia KASII (JbKASII) encodes a 488-amino acid polypeptide that possesses conserved domains that are necessary for condensing activities. When overexpressed in E. coli, recombinant His-tagged JbKASII was insoluble and non-functional. However, Arabidopsis plants expressing GFP-JbKASII fusions had elevated levels of arachidic acid (C20:0) and erucic acid (C22:1) at the expense of stearic acid (C18:0) and oleic acid (C18:1). Furthermore, JbKASII failed to complement the Arabidopsis KASII mutant, fab1-2. This suggests that the substrate specificity of JbKASII is similar to that of ketoacyl-CoA synthase (KCS), which preferentially elongates stearic and oleic acids, and not palmitic acid. Our results suggest that the KCS-like JbKASII may elongate C18:0 and C18:1 to yield C20:0 and C22:1, respectively. JbKASII may, therefore, be an interesting candidate gene for promoting the production of very long chain fatty acids in transgenic oil crops.