Dystrophin predominantly localizes to the transverse tubule/Z-line regions of single ventricular myocytes and exhibits distinct associations with the membrane

Dystrophin is a high molecular weight protein present at low abundance in skeletal, cardiac and smooth muscle and in trace amounts in brain. In skeletal muscle, dystrophin is uniformly distributed along the inner surface of the plasma membrane. Biochemical fractionation studies have shown that all detectable skeletal muscle dystrophin is tightly associated with a complex of wheat germ agglutinin (WGA)-binding and concanavalin A (Con A) binding sarcolemmal glycoproteins. Absence of dystrophin is the primary biochemical defect in patients with Duchenne muscular dystrophy and leads to segmental necrosis of their skeletal myofibers. Although present in similar amounts in normal cardiac and skeletal muscle, the absence of dystrophin from cardiac muscle has less severe effects on the survival of cardiac cells. We have therefore examined whether there are differences in the properties of cardiac and skeletal dystrophin. We report that in contrast to skeletal muscle, cardiac dystrophin is distributed between distinct pools: a soluble cytoplasmic pool, a membrane-bound pool not associated with WGA-binding glycoproteins and a membrane-bound pool associated with WGA-binding glycoproteins. Cardiac dystrophin was not associated with any Con A binding glycoproteins. Immunohistochemical localization studies in isolated ventricular myocytes reveal a distinct punctate staining pattern for dystrophin, approximating to the level of the transverse tubule/Z-line and contrasting with the uniform sarcolemmal staining reported for skeletal muscle fibers. The distinct properties of cardiac dystrophin suggest unique roles for this protein in cardiac versus skeletal muscle function.Abbreviations Dys Dystrophin - T-tubule Transverse tubule - SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis - WGA Wheat Germ Agglutinin - Con A Concanavalin A - DHP Dihydropyridine receptor - FITC Fluorescein Isothiocyanate Conjugate - NAG N-Acetyl-D-Glucosamine - NP-40 NONIDET P-40 - PBS Phosphate-Buffered Saline - TBST Tris Buffered Saline-Tween     

Photoaffinity cross-linking of F1ATPase from spinach chloroplasts by 3''-arylazido-β-alanyl-8-azido ATP

UV irradiation of the ATPase (CF₁) from spinach chloroplasts in the presence of 3'-arylazido-β-alanyl-8-azido ATP (8,3'-DiN₃ATP) results in a nucleotide-dependent inactivation of the enzyme and in a nucleotide-dependent formation of -β cross-links. The results demonstrate an interfacial localization of the nucleotide binding sites on CF₁.     

Molecular cloning of a chloroplastic proteinassociated with both the envelope and thylakoid membranes

Chloroplasts consist of six morphologically distinct compartments. Each compartment has a specific set of polypeptides that perform distinct biochemical functions. We report here the identification of a membrane-associated protein with a novel localization. This protein was synthesized as a 37 kDa precursor and was processed to a mature protein of 30 kDa after being imported into isolated pea chloroplasts. Fractionation of chloroplasts showed that the 30 kDa mature protein was associated with both of the envelope membranes as well as with thylakoid membranes. Immunocyto-chemical localization of the 30 kDa protein revealed that the protein occurred in clusters in the vicinity of both the envelope and the thylakoid. Possible functions of this 30 kDa protein, inferred from its novel localization pattern, are discussed.Abbreviations CAB light-harvesting chlorophyll a/b-binding protein of photosystem II - prCAB precursor protein to CAB - SS small subunit of ribulose-1,5-bisphosphate carboxylase - prSS precursor protein to SS - RCF relative centrifugation force     

Distribution of unesterified and esterified pectins in cell walls of pollen tubes of flowering plants

Immunocytochemical localization of polygalacturonic acid (pectin) and methyl-esterified pectin in the walls of pollen tubes of 20 species of flowering plants grown in vitro was investigated by using monoclonal antibodies (MAbs) JIM5 and JIM7 and by means of confocal laser scanning microscopy (CLSM). In general, periodic annular deposits of pectins were found coating the tube wall in species possessing solid styles, and a more uniform pectin sheath in tube walls in species having hollow styles or no styles. We hypothesize that the periodic ring-like structure of the pectin sheath reinforces pollen tubes for passing through the transmitting tract in the style. Esterified pectin which prevents Ca²⁺-induced gelification of pectate is located predominantly at the apex. This implies that pectin esterification is related to tip wall loosening that is required for cell wall expansion during tip growth of pollen tubes. The occurrence of unesterified pectins in other areas of pollen tube walls suggests that de-esterification of pectin following tip expansion leads to a more rigid form of pectin that contributes to the construction of the pollen tube wall.     

The cortical actin cytoskeleton in a Dipteran embryo: Analysis of the spatial reorganization of F-actin aggregates during the early nuclear division cycles

Rhodamine phalloidin-staining was used to study the organization of the cortical actin cytoskeleton of the early Ceratitis capitata embryo. The dynamics of the actin aggregates and their changes in distribution during the formation of the syncytial blastoderm, were followed in detail. It was found that these aggregates formed a shell-like cluster around the interphase nuclei, and concentrated toward the poles of the mitotic apparatus when the nuclei divided. Laser scanning confocal microscopy revealed that aggregates not clustered at the poles of the mitotic apparatus were closely associated with fine fibers of a dense cytoplasmic network of actin filaments.     

Cell Specificity of Molecular Changes During Memory Storage

Abstract: The aeolid nudibranch, Hermissenda crassicornis , exhibits Pavlovian conditioning to paired light and rotational stimuli and it has been suggested that protein kinase C (PKC) may play a critical role in the cellular mechanism for this conditioned behavioral response in the B-cell photoreceptor. The present study was designed to further examine learning-specific PKC involvement in identified cellular areas, particularly those in the visual-vestibular network, of the Hermissenda nervous system after Pavlovian conditioning. As used in previous vertebrate studies, the highly specific PKC radioligand, [³H]phorbol-12,13-dibutyrate ([³H]-PDBU), was used to determine the binding characteristics of the molluscan protein receptor considered to be PKC. The binding was specific, saturable, and could be displaced by a soluble diacylglycerol analogue. The binding activity was distributed evenly between the cytosol and the membrane. All of these analyses suggest that [³H]PDBU binds primarily to PKC in Hermissenda as it does in many other systems. Computerized grain image analysis was then used to determine the cellular localization of PKC as a function of Pavlovian conditioning. The medial and intermediate B photoreceptor and the optic ganglion showed significantly increased [³H]PDBU binding in conditioned animals. The present results provide the first report of an associative learning change of a key signal transduction component in identified neurons.     

Effect of dissolved inorganic carbon on the expression of carboxysomes,localization of Rubisco and the mode of inorganic carbon transport in cells of the cyanobacterium Synechococcus UTEX 625

In the cyanobacterium Synechococcus UTEX 625, the extent of expression of carboxysomes appeared dependent on the level of inorganic carbon (CO₂+HCO _inf3 ^sup- ) in the growth medium. In cells grown under 5% CO₂ and in those bubbled with air, carboxysomes were present in low numbers (<2 · longitudinal section^-1) and were distributed in an apparently random manner throughout the centroplasm. In contrast, cells grown in standing culture and those bubbled with 30 l CO₂ · 1^-1 possessed many carboxysomes (>8 · longitudinal section^-1). Moreover, carboxysomes in these cells were usually positioned near the cell periphery, aligned along the interface between the centroplasm and the photosynthetic thylakoids. This arrangement of carboxysomes coincided with the full induction of the HCO _inf3 ^sup- transport system that is involved in concentrating inorganic carbon within the cells for subsequent use in photosynthesis. Immunolocalization studies indicate that the Calvin cycle enzyme ribulose bisphosphate carboxylase was predominantly carboxysome-localized, regardless of the inorganic carbon concentration of the growth medium, while phosphoribulokinase was confined to the thylakoid region. It is postulated that the peripheral arrangement of carboxysomes may provide for more efficient photosynthetic utilization of the internal inorganic carbon pool in cells from cultures where carbon resources are limiting.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon (CO₂+HCO _inf3 ^sup- +CO _inf3 ^sup2- ) - PRK phosphoribulokinase - RuBP ribulose 1,5-bisphosphate - Rubisco LS large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase     

Enhancement of Antigenic Site Detection with Gold Labeled Secondary and Tertiary Antibodies Using the Immunogold-Silver Staining Method

We report a modification of the immunogold-silver staining method (IGSS) for localizing hepatic phosphoenolpyruvate carboxykinase (PEPCK) in tissue sections, and we compare the efficacy of localizing the primary antibody with either a 5 nm gold labeled secondary antibody or 5 nm gold labeled secondary and tertiary antibodies. Light microscope examination of 10 μm frozen sections demonstrated that the use of combined secondary and tertiary gold labeled antibodies was superior to using a secondary gold labeled antibody alone. The increased labeling density (number of colloidal gold particles/antigenic site/cell) achieved by combined gold labeled antibodies was confirmed by electron microscopy. The increased labeling density resulted in a two-thirds reduction in the time needed for the IGSS physical development of the silver shells and less background. We achieved intense specific staining of hepatocytes expressing PEPCK while minimizing background staining. The use of combined secondary and tertiary gold labeled antibodies enhances the signal-to-noise ratio, achieves high resolution and is a suitable method for use in both light and electron microscopy.     

Passive sound localization of prey by the pallid bat (Antrozous p. pallidus)

Summary The pallid bat (Antrozous p. pallidus) uses passive sound localization to capture terrestrial prey. This study of captive pallid bats examined the roles of echolocation and passive sound localization in prey capture, and focused on their spectral requirements for accurate passive sound localization.Crickets were used as prey throughout these studies. All tests were conducted in dim, red light in an effort to preclude the use of vision. Hunting performance did not differ significantly in red light and total darkness, nor did it differ when visual contrast between the terrestrial prey and the substrate was varied, demonstrating that the bats did not use vision to locate prey.Our bats apparently used echolocation for general orientation, but not to locate prey. They did not increase their pulse emission rate prior to prey capture, suggesting that they were not actively scanning prey. Instead, they required prey-generated sounds for localization. The bats attended to the sound of walking crickets for localization, and also attacked small, inanimate objects dragged across the floor. Stationary and/or anesthetized crickets were ignored, as were crickets walking on substrates that greatly attenuated walking sounds. Cricket communication sounds were not used in prey localization; the bats never captured stationary, calling crickets.The accuracy of their passive sound localization was tested with an open-loop passive sound localization task that required them to land upon an anesthetized cricket tossed on the floor. The impact of a cricket produced a single 10–20 ms duration sound, yet with this information, the bats were able to land within 7.6 cm of the cricket from a maximum distance of 4.9 m. This performance suggests a sound localization accuracy of approximately ±1° in the horizontal and vertical dimensions of auditory space. The lower frequency limit for accurate sound localization was between 3–8 kHz. A physiological survey of frequency representation in the pallid bat inferior colliculus suggests that this lower frequency limit is around 5 kHz.