Henry S. Slayter

Autocrine Release of Angiotensin II Mediates Stretch-Induced Hypertrophy of Cardiac Myocytes In Vitro

Hypertrophy is a fundamental adaptive process employed by postmitotic cardiac and skeletal muscle in response to mechanical load. How muscle cells convert mechanical stimuli into growth signals has been a long-standing question. Using an in vitro model of load (stretch)-induced cardiac hypertrophy, we demonstrate that mechanical stretch causes release of angiotensin II (Ang II) from cardiac myocytes and that Ang II acts as an initial mediator of the stretch-induced hypertrophic response. The results not only provide direct evidence for the autocrine mechanism in load-induced growth of cardiac muscle cells, but also define the pathophysiological role of the local (cardiac) renin-angiotensin system.

The release of heparin binding peptides from platelet thrombospondin by proteolytic action of thrombin, plasmin and trypsin

Abstract When purified human platelet thrombospondin is exposed to thrombin (4 Units/ml) or plasmin (2 Units/ml) for prolonged periods of time proteolytic cleavages occur which result in a decrease in the molecular weight of the 145,000 dalton thrombospondin chains to 115,000 daltons. Low molecular weight fragments produced by thrombin were observed in reduced samples at 30,000, 25,000, 18,500 and 12,500 daltons. Heparin-Sepharose affinity chromatography of thrombin, plasmin or trypsin-treated thrombospondin indicates that the 30,000 dalton polypeptide contains the heparin binding site. The heparin-binding polypeptides separate into multiple bands on 10% discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis and during isoelectric focusing. The results of thrombin treatment of thrombospondin in the supernatant from thrombin-treated platelets suggests that some of the proteolytic cleavages are contingent upon the presence of calcium and a nondialyzable component of the native supernatant.

High-resolution metal replication of macromolecules.

By adjustment of various parameters affecting crystallite size in thin metal replica films used for contrasting biological macromolecules for electron microscopy, improvements in the level of image information retrieved are demonstrated. Mass thickness, replica metal type, substrate temperature and composition are found to be important determinants of replica quality and conditions are discussed and tabulated for optimizing these. Typical correction curves for replicas are given for platinum and tungsten, from which approximate true dimensions may be calculated from measurements of unknown replicas. Deviation of evaporant sources from ideality are quantitated for both heated filament and electron gun sources. Metal replicas exposed to 80 kV electron beams long enough receive 10(6)e/nm2 show no observable changes other than those attributable to contamination as a function of time after the first few seconds. A variety of artifacts occasionally produced in replicas are discussed in relation to image interpretation.

Chemical and physical properties of human bronchial mucus glycoproteins

Human bronchial mucus glycoproteins of different chemical types were isolated by Ecteola and gel exclusion chromatography. Chemical analysis indicated polydispersity with regard to content of sulfate and sialic acid. No blood group A, B or H activity was found in these glycoproteins. Compositions are reported for amino acid and sugar residues for several fractions obtained from both cystic fibrotic and chronic bronchitic mucus. It is noteworthy that glycoproteins extracted from a single subject contain molecules with different acid groups as well as significant differences in carbohydrate chain length.

ELECTRON MICROSCOPIC STUDIES OF FIBRINOGEN STRUCTURE: HISTORICAL PERSPECTIVES AND RECENT EXPERIMENTS*

Early attempts to visualize macromolecules in the electron microscope were fraught with difficulties of several types. Thirty years ago, instrumental resolving powers were still quite restricted; although 20 A resolution was being approached, 50 A was more typical during routine operation. The convenience of modern instruments was as yet undreamed of. Technologies for specimen preparation were inadequate. Although the shadow casting method had been introduced during the 1 9 4 0 ~ , ~ l # G 2 making it possible to enhance the contrast of small particles, the technique remained rudimentary. In particular, irregularities of supporting films tended to be as large, and therefore as subject to contrast enhancement, as details of the specimens themselves. The negative contrast, or “negative staining” method, on the other hand, had yet to be proposed. Finally, many electronmicroscopists at that time were physicists, with little interest or training in the methods of preparative biochemistry. In the face of these difficulties, prospects for visualizing particles in the molecular weight range from 30,000 to 500,000 d seemed problematic at best. Fibrinogen, however, was readily available at reasonably high purity and, as a subject of intense interest with respect to its role in the clotting process, was well characterized in physicochemical terms. It was to become one of the first subjects of macromolecular electron microscopy. The first electron micrographs of fibrinogen were published in 1949 by 4 2 who suggested that the structure of the molecule resembled a string of beads. Such was the level of background “noise,” however, that neither the number of subunits nor the length of the molecule could be determined. Shortly afterwards, Porter and Hawn 4 3 suggested quite a different model: that of an oblate ellipsoid. Subsequently, Mitchel 6,1 found spherical particles in fibrinogen preparations, while Siegel, Mernan and Scheraga 45 returned to Hall’s original string-of-beads suggestion. In 1956 an important advance in the technology of specimen preparation appeared: the development, by Hall, of the so-called “mica method.” Until that time, shadowing techniques had inevitably contrasted irregularities in the substrate film along with the surface contours of particles to be studied. Even the most carefully prepared plastic or carbon film substrates were rough enough

Immunoelectron-microscopic studies of human platelet thrombospondin, von Willebrand factor, and fibrinogen redistribution during clot formation.

SummaryThe relative distributions of the human platelet α-granule proteins fibrinogen, thrombospondin, and von Willebrand factor were mapped by immunoelectron microscopy in thin cryosections of activated platelets, platelet aggregates, and clots during the first 24 h ofin vitro clot formation. In early activated platelets, the results suggest that the canalicular system constitutes a significant component of the external platelet surface, and may act as a compartment for biochemical reactions occurring during granule relase. Further, detection of coagulation proteins by various non-morphological procedures may reflect protein contained within canalicular elements. Later in the release process, von Willebrand factor was detected as a major antigen on the platelet canalicular and plasma membranes; thrombospondin, on the other hand, showed minimal binding to platelets and only limited binding to the extensive fibrin network. Comparison of radioimmunoassays of supernatants of thrombin-stimulated platelets in plasma, clotted whole blood, and Triton X-100 platelet releasates indicated that virtually all of the platelet thrombospondin appears in serum. These data confirm the immunocytochemical results indicating that very little platelet thrombospondin binds to the platelet surface, compared with von Willebrand factor, studied here under the same conditions, which binds extensively to the platelet membrane following release and clot formation.

Thrombospondin expression in traumatized skeletal muscle

SummaryBiochemical and immuno-microscopic techniques were used to study temporal involvement of thrombospondin in relation to fibrinogen in muscle regeneration using a rat skeletal muscle-wound model. In undamaged control muscle, no fibrinogen and minimal thrombospondin antigen was found. Following crushing injury, fibrin networks appear immediately, followed by a gradual ordered accumulation of thrombospondin (within a few hours) in the vicinity of the vascular bed and adjacent endomysial connective tissue. Later, thrombospondin becomes associated with connective tissue and basal laminae around muscle fibers throughout the damaged muscle, maximal labelling occurring 3–6 days post-injury. Thrombospondin immunoreactivity decreased thereafter to near normal levels after 7 days post-injury, coincident with the appearance of regenerating muscle fibers. In contrast, little fibrin material remained by five days after injury. Quantitative radioimmunoassay of soluble thrombospondin antigen and radioimmune labelling of thick frozen sections reinforced the qualitative immuno-microscopic observations, with levels peaking at 3–4 days post-trauma, 10-fold over control levels. SDS-PAGE immunoblotting of non-reduced muscle extracts three days after a crush assault shows that the bulk of the thrombospondin incorporated into the injury site exists in a polymerized state (≤1000 kD). These results demonstrate that the temporal appearance and disappearance of thrombospondin in the healing of a crushing lesion in muscle is related more closely to the regeneration phase of muscle than to the coagulation phase.

Intestinal mucin of germ-free rats. Biochemical and electron-microscopic characterization.

Purified germ-free rat intestinal mucin was found by chemical analysis to contain 25% protein, enriched in serine, threonine, and proline, 75% carbohydrate, and no nucleic acid. It was analyzed by darkfield electron microscopy and found to consist of long filamentous molecules with a maximum length of approximately 740 nm, a mean length of 456 nm, and a mean width of 7 nm. Given reasonable assumptions derived from earlier work on other well-characterized mucins, the molecular weight of the peptide, calculated by the length from electron microscopy, was 200,000, and, given the chemical composition, the molecular weight of the entire mucin molecule was calculated to be approximately 800,000.

Electron microscopy of cardiac myosin: its shape and properties as determined by the regulatory light chain.

SummaryStructural properties of dog cardiac myosin and the influence of the regulatory light chain (LC2) on the shape of myosin heads were investigated by electron microscopy. LC2 was reversibly removed using a neutral protease from myopathic hamsters (Margossian,J. Biol. Chem.260 (1985) 13747–54). The distribution of myosin head length centred around 17 nm with the mean length being 18.9 nm. Statistical analysis suggested that myosin heads became more globular upon removal of LC2. No extensive aggregation of myosin could be detected after LC2 was dissociated, either by sedimentation velocity or by gels run under non-denaturing conditions. The centre-to-centre distance between heads remained constant at about 21 nm, regardless of the presence or absence of LC2. The distribution of length of the globular region reveals two peaks at 7.5 and 9.5 nm, suggesting an extended and a shorter configuration of this region. The decrease in mass at the head/tail junction upon LC2 removal suggests that it is the binding site for the regulatory light chains. A bend at 57 nm from the head/tail joint was sometimes noticed, corresponding to the myosin hinge region. In high resolution micrographs individual particles revealed invaginations along the contours of the head, possibly delineating the boundaries of structural domains within the head. The conformation of arrowheads in actin decorated with either subfragment 1 (S1) or heavy meromyosin (HMM) was investigated in the presence and absence of LC2.

Physico-chemical properties of rat and dog cardiac α-actinin

Abstract α-Actinin exists in several polymorphic forms which appear to be characteristic of the muscle type from which it is isolated. In order to determine the possible physiological role of this structural protein in cardiac muscle, we describe and compare here the physico-chemical properties of cardiac α-actinin from two different mammalian species, rat (fast contracting muscle) and dog (slow contracting muscle). Purification of cardiac α-actinin was achieved by chromatography on DEAE-cellulose and hydroxyapatite columns. The α-actinins isolated were different in their electrophoretic mobility (SDS-polyacrylamide gel electrophoresis), molecular size and α-helical content. However, their shape as revealed by electron microscopy and their activating effect on Mg2+-ATPase activity of actomyosin appear to be similar. These studies suggest that the rat and dog cardiac α-actinin are structurally different but functionally similar proteins.