Separation of hepatocyte plasma membrane domains by free flow electrophoresis

We have applied free flow electrophoresis to separate the canalicular and basolateral (sinusoidal and lateral) domains of rat hepatocyte plasma membranes. Hepatocyte plasma membranes were prepurified by rat zonal and discontinous sucrose gradient centrifugation. In electrophoretic separation, the canalicular membranes were more deflected toward the anode than the basolateral membranes. Na+-dependent taurocholate uptake could be measured in both membrane fractions, transport activity being highest in fractions containing the highest specific activity in the basolateral marker enzyme Na+-K+-ATPase. Thus, differences in electrophoretic mobility permit the separation of functional intact plasma membrane vesicles derived from basolateral and canalicular plasma membrane domains of rat hepatocyte.     

Macromolecular organization of collagen fibres in natural and tanned basement membrane

The elastic constants and ultrastructure of natural and tanned basement membrane of the crystalline lens of the adult rat have been investigated. Sonicated and negatively stained specimens of both membranes show parallel filaments that have similar spacing of 3.5(+/- 0.1) nm and a different periodicity. In natural membrane the periodicity is 3.7(+/- 0.13) nm, whilst in tanned basement membrane the periodicity is 3.2(+/- 0.15) nm. The periodicity ratio of tanned membrane to natural membrane was 0.86 +/- 0.04, whilst the elongation ratio of tanned membrane compared with natural membrane was 0.88 +/- 0.05. In contrast to this, the thickness ratio of tanned to natural membrane was 1.098 +/- 0.045. Tanned basement membrane showed a shrinkage of 12% in length but an increase in thickness of about 10%. These data suggest, firstly, that the degree of extension of the superhelices of the filaments follows closely the degree of extension of the intact membrane and, secondly, that the coiled superhelices of tanned membrane have an angle of tilt of about 42 degrees compared with those of natural membrane, where the angle is about 50 degrees. The Young's modulus of elasticity and ultimate stress of tanned basement membrane are, respectively, eight times greater and one-third as great as natural membrane. The entropy change in basement membrane was calculated from the external work necessary to extend the tanned membrane, and was estimated to be -13.5(+/- 2.4) J K-1 mol-1. An estimate of the change in entropy from thermodynamic measurements made on a suspension of collagen tanned with glutaraldehyde was found to be -30.1(+/- 9.5) J K-1 mol-1. The two different estimates of the change in entropy of collagen following tanning suggest that in basement membrane only about 45% of the collagenous protein has an extensile helical structure.     

Reticular meshwork of the spleen in rats studied by electron microscopy

The reticular meshwork of the rat spleen, which consists of both fibrous and cellular reticula, was investigated by transmission electron microscopy. The fibrous reticulum of the splenic pulp is composed of reticular fibers and basement membranes of the sinuses. These reticular fibers and basement membranes are continuous with each other. The reticular fibers are enfolded by reticular cells and are composed of two basic elements: 1) peripheral basal laminae of the reticular cells, and 2) central connective tissue spaces in which microfibrils, collagenous fibrils, elastic fibers, and unmyelinated adrenergic nerve fibers are present. The basement membranes of the sinuses are sandwiched between reticular cells and sinus endothelial cells and are composed of lamina-densalike material, microfibrils, collagenous fibrils, and elastic fibers. The presence of these connective tissue fibrous components indicates that there are connective tissue spaces in these basement membranes. The basement membrane is divided into three parts: the basal lamina of the reticular cell, the connective tissue space, and the basal lamina of the sinus endothelial cell. When the connective tissue space is very small or absent, the two basal laminae may fuse to form a single, thick basement membrane of the splenic sinus wall. The fibrous reticulum having these structures is responsible for support (collagenous fibrils) and rebounding (elastic fibers). The cells of the cellular reticulum--reticular cells and their cytoplasmic processes, which possess abundant contractile microfilaments, dense bodies, hemidesmosomes, basal laminae, and a well-developed, rough-surfaced endoplasmic reticulum, and Golgi complexes, which are characteristic of both fibroblasts and smooth muscle cells--are considered to be myofibroblasts. They may play roles in splenic contraction and in fibrogenesis of the fibrous reticulum. The contractile ability may be influenced by the unmyelinated adrenergic nerve fibers that pass through the reticular fibers. The three-dimensional reticular meshwork of the spleen consists of sustentacular fibrous reticulum and contractile myofibroblastic cellular reticulum. This meshwork not only supports the organ but also contributes to a contractile mechanism in circulation regulation, in collaboration with major contractile elements in the capsulo-trabecular system.     

Stretch-shortening cycle characteristics during vertical jumps carried out with small and large range of motion

In the present study we investigated kinematical characteristics of the knee and ankle extensors to estimate the length change properties of the contractile and the passive elements in countermovement jumps (CMJ) and drop jumps (DJ) performed with small (40°) and large (80°) range of joint motion (SRM and LRM). At SRM the accelerations at maximal muscle lengths compared with the last phase of joint flexion were greater for the gastrocnemius and the soleus (124.9% and 79.4%) and also were greater than at the beginning of joint extension, while no difference was measured at LRM. The differences suggest that at LRM the length change of the serial passive elements from the end of joint flexion to the beginning of joint extension is minimal and simultaneously the length change of the contractile elements is significant, but at SRM – especially in the plantar flexors – the length change of the contractile elements is minimal while in the passive elements significant. It can be presumed that for SRM at the end of joint flexion significant elastic energy is stored and at the beginning of joint extension reused, while for LRM elastic energy storage is not dominant.     

Influence of preparative procedures on the membrane viscoelasticity of human red cell ghosts

The effects of systematic variations in the preparative procedures on the membrane viscoelastic properties of resealed human red blood cell ghosts have been investigated. Ghosts, prepared by hypotonic lysis at 0 degrees C and resealing at 37 degrees C, were subjected to: measurement of the time constant for extensional recovery (tc); measurement of the membrane shear elastic modulus (mu) via three separate techniques; determination of the membrane viscosity (eta m) via a cone-plate Rheoscope. Membrane viscosity was also determined as eta m = mu X tc. Compared to intact cells, ghosts had shorter tc, regardless of their residual hemoglobin concentration (up to 21.6 g/dl). However, prolonged exposure to hypotonic media did increase their recovery time toward the intact cell value. The shear elastic modulus, as judged by micropipette aspiration of membrane tongues (mu p), was similar for all ghosts and intact cells. This result, taken with the tc data, indicates that ghosts have reduced membrane viscosity. Rheoscopic analysis also showed that eta m was reduced for ghosts, with the degree of reduction (approx. 50%) agreeing well with that estimated by the product mu p X tc. However, flow channel and pipette elongation estimates indicated that the ghost membrane elastic modulus was somewhat elevated compared to intact cells. We conclude that: ghosts have reduced membrane viscosity; ghosts have membrane rigidities close to intact cells, except possibly when the membrane is subjected to very large strains; the reduction in eta m is not directly related to the loss of hemoglobin; prolonged exposure of ghosts to low-ionic strength media increases the membrane viscosity toward its initial cellular level. These data indicate that the mechanical characteristics of ghost membranes can be varied by changing the methods of preparation and thus have potential application to further studies of the structural determinants of red cell membrane viscoelasticity.     

Abnormal extracellular matrix and excessive growth of human adult polycystic kidney disease epithelia.

Human autosomal dominant polycystic kidney disease (ADPKD) epithelia were grown in primary monolayer cultures and their properties compared with intact kidney epithelial cultures derived from individually microdissected normal human kidney proximal convoluted tubules (PCT), proximal straight tubules (PST), and cortical collecting tubules (CCT). In vivo, ADPKD cyst epithelia exhibited a thickened basement membrane, and immunofluorescence demonstrated the presence of laminin, fibronectin, type IV collagen, and heparan sulfate proteoglycan in basement membranes and type I collagen in the interstitium. ADPKD epithelia grown in culture synthesized and secreted basally a unique, extracellular matrix that took the form of proteinaceous spheroids when the cells were grown on dried, type I collagen. Incorporation of H2[S35O4] into basement membrane extracts was increased more than ten-fold in ADPKD epithelia by comparison to normal PST and CCT. In addition to incorporation into the normal tubular basement membrane 220 kD band, radioactivity was also seen at 175 kD and 150 kD in ADPKD extracts. Growth in culture of cyst-lining ADPKD epithelia was more rapid than normal tubules, and was abnormal since there was no absolute requirement for added extracellular matrix. However, when ADPKD epithelia were grown on different, exogenous matrix protein components, a profound influence on both structure and epithelial cell proliferation was seen. Growth on a complete basement membrane three-dimensional gel derived from the Engelbreth-Holm-Swarm (EHS) sarcoma led to a reduction in the numbers of spheroids and increase in amorphous filaments. Incorporation of [3H]-thymidine into ADPKD epithelia was greater than into normal PCT, PST, and CCT and was also greatly modified by the type of extracellular matrix components provided. In studies using single matrix components, the strongest proliferative response was seen when ADPKD epithelia were plated on type I collagen greater than type IV collagen greater than fibronectin greater than laminin. These findings suggest that the excessive growth of cyst-lining epithelia may be, at least in part, a result of abnormal basement membrane and extracellular matrix production by ADPKD cells.     

Passive resistance of the human knee: The effect of remobilization

Changes of circumferential dimensions and passive resistance of the human knee caused by immobilization, were studied during remobilization. Patients immobilized with a long leg cast after tibial fractures or ligamentous injuries were studied immediately after removal of the cast and after mean periods of 18, 36 and 81 days of remobilization. Immobilization resulted in a decrease of circumferential dimensions. The difference in mid-thigh circumference between the immobilized and the unaffected leg was still present after 81 days of remobilization both for the patients with tibial fractures and for the patients with ligamentous lesions. An increase of midpatellar circumference was present exclusively in the patients with ligamentous lesions at all four testing dates, indicating that this is an effect of the ligamentous lesion and not of immobilization per se. Variables of the hysteresis diagrams, resulting from sinusoidal movement of the knee at a range of knee angles, were used to quantify passive resistance of the knee in the flexion-extension plane (the muscles crossing the knee are inactive). Variables related to the elastic storage and release of energy, and variables related to energy dissipation were discerned. During remobilization the increased resistance to flexion (shown by the variables related to the elastic storage of energy), as found immediately after removal of the cast, disappears and the resistance becomes identical to the resistance of the unaffected leg. This may indicate a rapid readaptation of the length of ventral structures (shortened due to immobilization in a shortened position) to almost normal values. The variables related to energy dissipation, which do not differ between the two legs immediately after immobilization, are low in comparison with the unaffected knee during the whole remobilization period studied, indicating a remaining atrophy (as is also shown by the still decreased aircumference of the lower limb). All resistance variables are still lower at the last date of testing when the knee is extended. The remobilization period studied was not long enough to achieve complete restoration of the dimensions of the lower limb and full return of normal passive resistance of the knee.     

Repopulation of a human alveolar matrix by adult rat type II pneumocytes in vitro : A novel system for type II pneumocyte culture

This paper describes the preparation of lung acellular alveolar matrix fragments and culture of rat type II pneumocytes directly on the alveolar epithelial basement membrane, thereby permitting study of the effect of lung basement membrane on the morphology and function of type II cells. Collagen types I, III, IV and V, laminin and fibronectin were located by immunofluorescence in the lung matrix with the same patterns as those described for the normal human lung. Transmission electron microscopy (TEM) of the fragments revealed intact epithelial and endothelial basement membranes. The matrix maintained the normal three-dimensional alveolar architecture. Glycosaminoglycans were still present by Alcian Blue staining. Isolated adult rat type II pneumocytes cultured on 150 micron thick fragments of acellular human alveolar extracellular matrix undergo gradual cytoplasmic flattening, with loss of lamellar bodies, mitochondria, and surface microvilli. These changes are similar to the in vivo differentiation of type II pneumocytes into type I pneumocytes. The type II pneumocyte behaviour on the lung epithelial basement membrane contrasted sharply with that of the same cell type cultured on a human amnionic basement membrane. On the latter surface the cells retained their cuboidal shape, lamellar bodies and surface microvilli for up to 8 days. These observations suggest that the basement membranes from different organ systems exert differing influences on the morphology and function of type II pneumocytes and that the alveolar and amnionic basement membranes may have differing three-dimensional organizations. The technique of direct culture of type II cells on the lung basement membrane provides a useful tool for studying the modulating effect of the basement membrane on alveolar epithelial cells.     

Temperature-dependent abnormalities of the erythrocyte membrane in porcine malignant hyperthermia

The temperature dependence of ATPase activities and stearic acid spin label motion in red blood cells of normal and MH-susceptible pigs have been examined. Arrhenius plots of red blood cell ghost Ca-ATPase and calmodulin-stimulable Ca-ATPase activities were identical for both normal and MH erythrocyte ghosts. Arrhenius plots of Mg-ATPase activity exhibited a break (defined as a change in slope) at 24 degrees C in both MH and normal erythrocyte ghosts. However, below 24 degrees C the apparent activation energy for this activity was less in MH than normal ghosts. To determine whether breaks in ATPase Arrhenius plots could be correlated with changes in the physical state of the red blood cell membrane, the spin label 16-doxyl-stearate was introduced into the bilayer of both erythrocyte ghosts and red blood cells. With both ghosts and intact cells, at each temperature examined, the mobility of the probe in the lipid bilayer, as measured by electron paramagnetic resonance, was greater in normal than in MH membranes. While there were no breaks in Arrhenius plots for probe motion in the erythrocyte ghosts, the apparent activation energy for probe motion was significantly greater in normal than in MH ghost membranes. While there was no break in the Arrhenius plot of probe motion in normal intact red blood cell membranes, there were breaks in the Arrhenius plot of probe motion at both 24 and 33 degrees C in intact MH red blood cell membranes. Based on the altered temperature dependence of Mg-ATPase activity and spin probe motion in membranes derived from MH red blood cells, we conclude that there may be a generalized membrane defect in MH pigs which is reflected in the red blood cell as an altered membrane composition or organization.     

A simple, versatile, nondisruptive method for the isolation of morphologically and chemicaly pure basement membranes from several tissues.

A simple procedure has been developed for the isolation of ultrastructurally pure, intact basement membranes from bovine retinal and brain blood vessels, rabbit renal tubules and rat renal glomeruli. By this procedure, cell membranes and intracellular materials are selectively solubilized with 4% sodium deoxycholate to yield morphologically and chemically intact basement membrane preparations. Therefore, this method appears to be a versatile, nondisruptive procedure for the isolation and characterization of basement membranes from a variety of tissues. Its applicability has been demonstrated by the preparation for the first time of isolated basement membranes from non-renal mammalian blood vessels.     

Activation of zeaxanthin is an obligatory event in the regulation of photosynthetic light harvesting.

By dynamic changes in protein structure and function, the photosynthetic membranes of plants are able to regulate the partitioning of absorbed light energy between utilization in photosynthesis and photoprotective non-radiative dissipation of the excess energy. This process is controlled by features of the intact membrane, the transmembrane pH gradient, the organization of the photosystem II antenna proteins and the reversible binding of a specific carotenoid, zeaxanthin. Resonance Raman spectroscopy has been applied for the first time to wild type and mutant Arabidopsis leaves and to intact thylakoid membranes to investigate the nature of the absorption changes obligatorily associated with the energy dissipation process. The observed changes in the carotenoid Resonance Raman spectrum proved that zeaxanthin was involved and indicated a dramatic change in zeaxanthin environment that specifically alters the pigment configuration and red-shifts the absorption spectrum. This activation of zeaxanthin is a key event in the regulation of light harvesting.     

Characterization of plasma-membrane glycoproteins from functional domains of the rat hepatocyte.

Plasma-membrane glycoproteins from the three different functional domains of the rat hepatocyte were radioactively labelled by oxidation with NaIO4, followed by reduction with NaB3H4. Analysis of the radioactively labelled glycoproteins by polyacrylamide-gel electrophoresis revealed the presence of at least 12 major sialoglycoproteins in each different region of the hepatocyte surface. The Mr-110 000 component was homogeneously distributed over the plasma membrane, whereas the Mr-90 000 polypeptide was only located at the sinusoidal face. These radiolabelled glycoproteins were solubilized in 1% Triton X-100, and the soluble fraction was subjected to affinity chromatography on Sepharose-conjugated wheat-germ agglutinin (WGA). The labelled glycoproteins were poorly bound to WGA. Membrane glycoproteins were also labelled by the galactose oxidase/NaB3H4 method. The results show that the polypeptides with apparent Mr 170 000 from the sinusoidal, 230 000 from the canalicular and 170 000 from the lateral membranes were specifically labelled. When the membranes were treated with neuraminidase and galactose oxidase/NaB3H4, the electrophoretic patterns showed changes in the apparent Mr values of the glycoproteins, owing to loss of sialic acid, and a clear increase in labelling in the sinusoidal and canalicular membranes compared with the lateral membranes. When these labelled membranes were solubilized in 1% Triton X-100 and subjected to affinity chromatography on Sepharose-conjugated Ricinus communis agglutinin and/or Lens culinaris agglutinin, the results showed that the former columns efficiently bound the radiolabelled glycoproteins, whereas the latter columns bound poorly. The results show that there is a differential distribution of glycoproteins along the hepatocyte's surface.     

A model for passive elastic properties of rat vena cava

A two-dimensional model for the elastic properties of vena cava abdominalis under orthotropic deformation is introduced and tested against the experimental data obtained from six specimen of rat venae cavae by pressurization experiments. The model is based on membrane approximation and suited for vessels where most of the elastic elements are oriented axially, while circumferential contraction is exerted by redirecting axial stress by some network of oblique fibers. For the experimental data considered in this paper, the ratio between axial and circumferential stress depends almost exclusively on the circumferential extension ratio. As a consequence, the mechanical system can be formally decomposed in a kinematic system reacting by axial contraction on circumferential extension without any loss or storage of energy, serially connected to a hyperelastic system acting only in axial direction. Both systems are modeled separately by equations obtained by a purely phenomenological approach with two parameters for each system. This leads to reasonable reproduction of the experimental data. Introducing a correction parameter, which takes into account that the model assumption on the decomposition does not hold exactly, we get better reproduction of data. However, this is paid for by loss of physical rigor and in particular by departing from the assumption of hyperelasticity.     

Modulation of erythrocyte membrane proteins by membrane cholesterol and lipid fluidity.

Human erythrocyte membranes were enriched or depleted of cholesterol and effects on membrane proteins assessed with a membrane-impermeant sulfhydryl reagent, [35S]glutathione-maleimide. Reaction of the probe with intact cells quantifies exofacial sulfhydryl groups and reaction with leaky ghost membranes permits quantification of endofacial sulfhydryl groups. The mean endofacial sulfhydryl titer of cholesterol-enriched membranes exceeded that of cholesterol-depleted membrane by approximately 45 nmol/mg of protein or 64%. The corresponding exofacial titer of cholesterol-enriched cells was less than that of cholesterol-depleted cells by approximately 0.4 nmol/mg of protein, or 14%. Labeled membranes were examined by autoradiography of sodium dodecyl sulfate-polyacrylamide gel electropherograms to determine the labeling patterns of individual protein bands. Cholesterol enrichment enhanced the surface labeling of Coomassie brilliant blue stained bands 1,2,3, and 5, decreased the labeling of band 6, and did not change significantly that of band 4. The results demonstrate that changes in membrane cholesterol which influence lipid fluidity can alter the surface labeling of both intrinsic and extrinsic membrane proteins.     

Degradation of intact basement membranes by human and murine tumor enzymes

Homogenates from malignant tumors, obtained from surgery specimens or from transplants of Walker 256 carcinosarcoma in rats, contained an enzyme activity capable of degrading intact 3H-acetylated basement membranes from bovine lens. The enzyme activity from murine tumor was purified about 7500-fold by (NH4)2SO4 fractionation, ion exchange and gel chromatography. The apparent molecular weight of the purified enzyme was approximately 50,000. The rate of degradation of 3H-labelled basement membrane by the murine tumor enzyme was reduced by addition of excess type IV collagen, but not of excess type I, type III or type V collagen. These results suggested specificity of this enzyme for type IV collagen. Inhibitors of serine proteinases, thiol proteinases and soybean trypsin inhibitor were without effect on the enzyme activity. Chelators such as 1,10-phenanthroline or EDTA reduced the activity to control levels, indicating that the enzyme activity was due to a metalloproteinase. Chromatographic and electrophoretic separation of the enzymatic products from 3H-labelled basement membrane and type IV collagen indicated that the enzyme activity was due to a type IV collagenase. The use of basement membrane in the native physiological state as a substrate for the study of basement membrane-degrading activity by homogenates of solid malignant tumors offers an in vitro model for the investigation of the metastatic potential of these tumors.     

Vascular permeability and hyperpermeability in a murine adenocarcinoma after fractionated radiotherapy: an ultrastructural tracer study

Large radiation doses cause postradiation vascular hyperpermeability by disrupting endothelia. The cumulative sequences of small doses (fractionated radiotherapy) standard in clinical practice cause it too, but not by endothelial disruption: the mechanisms are unknown. In this study, correlated fluorescent and ultrastructural localisation of a tracer revealed the architecture, fine structure and function of microvessels in mouse AT17 tumours, before and after 42 Gy fractionated radiation. Before irradiation, tumour vascular permeability lay in the normophysiological range defined by the gut and cerebral cortex. A double barrier regulated permeability: vesicular transport through the endothelial wall required approximately 2 h and then the basement membrane charge barrier trapped tracer for 2 h longer. Irradiation abolished the double barrier: tracer passed instantly through both endothelial wall and underlying basement membrane, forming diffusion haloes around microvessels within 2-5 min. Structurally, irradiated tumour microvessels were lined by a continuous and vital endothelium with closed interendothelial junctions; endothelial basement membranes were intact, though loosened. Irradiated endothelia exhibited extremely active membrane motility and intracellular vesicle trafficking. Radiation treatment raised vascular permeability by enhancing transendothelial transcytosis, and by altering the passive filter properties of the subendothelial basement membrane. This type of vascular hyperpermeability should be susceptible to pharmacological modulation.     

Highly selective extraction of spiralin from the Spiroplasma citri cell membrane with alkyl-N-sulfobetaines

The extraction of proteins from the membrane of the mollicute (mycoplasma) Spiroplasma citri by sodium N-dodecyl-N,N-dimethyl-3-amino-1-propane sulfonate (SB12) and sodium N-tetradecyl-N,N-dimethyl-3-amino-1-propane sulfonate (SB14) was studied with electrophoretic methods. The membranes were prepared by osmotic lysis of the cells and depleted of the bulk of extrinsic proteins. It was possible to extract up to 35 and 45% of membrane proteins with SB12 and SB14, respectively. Maximal yield was obtained in both cases with detergent concentrations greater than or equal to 5 mumoles/mg of membrane protein. Spiralin, the major protein in the S. citri membrane, was highly selectively solubilized without the loss of antigenicity, with a yield of about 90% with SB12 and close to 100% with SB14, for a detergent concentration greater than or equal to 0.2 M. The degree of selectivity in favour of spiralin was higher with SB12 (purity approximately equal to 70%) than with SB14 (purity approximately equal to 50%). Treatment of the S. citri membrane with high concentrations of SB12 is a simple and fast procedure for partial purification of spiralin. This example shows that, in some cases, it should be possible to modulate the selectivity of the extraction of membrane proteins simply by varying the relative concentration of detergent.     

Characterization of a novel heparan sulfate proteoglycan found in the extracellular matrix of liver sinusoids and basement membranes

A novel heparan sulfate proteoglycan (HSPG) present in the extracellular matrix of rat liver has been partially characterized. Proteoglycans were purified from a high salt extract of total microsomes from rat liver and found to consist predominantly (approximately 90%) of HSPG. A polyclonal antiserum raised against this fraction specifically recognized HSPG by immunoprecipitation and immunoblotting. The intact, fully glycosylated HSPG migrated as a broad smear (150-300 kD) by SDS-PAGE, but after deglycosylation with trifluoromethanesulfonic acid only a single approximately 40-kD band was seen. By immunocytochemistry this HSPG was localized in the perisinusoidal space of Disse associated with irregular clumps of basement membrane-like extracellular matrix material, some of which was closely associated with the hepatocyte sinusoidal cell surface. It was also localized in biosynthetic compartments (rough ER and Golgi cisternae) of hepatocytes, suggesting that this HSPG is synthesized and deposited in the space of Disse by the hepatocyte. The anti-liver HSPG IgG also stained basement membranes of hepatic blood vessels and bile ducts as well as those of kidney and several other organs (heart, pancreas, and intestine). An antibody that recognizes the basement membrane HSPG found in the rat glomerular basement membrane did not precipitate the 150-300-kD rat liver HSPG. We conclude that the liver sinusoidal space of Disse contains a novel population of HSPG that differs in its overall size, its distribution and in the size of its core protein from other HSPG (i.e., membrane-intercalated HSPG) previously described in rat liver. It also differs in its core protein size from HSPG purified from other extracellular matrix sources. This population of HSPG appears to be a member of the basement membrane HSPG family.     

Effects of volatile anesthetics on stiffness of mammalian ventricular muscle.

To assess the effects of halothane, isoflurane, and sevoflurane on cross bridges in intact cardiac muscle, electrically stimulated (0.25 Hz, 25 degrees C) right ventricular ferret papillary muscles (n = 14) were subjected to sinusoidal load oscillations (37-182 Hz, 0.2-0.5 mN peak to peak) at the instantaneous self-resonant frequency of the muscle-lever system. At resonance, stiffness is proportional to m * omega(2) (where m is equivalent moving mass and omega is angular frequency). Dynamic stiffness was derived by relating total stiffness to values of passive stiffness at each length during shortening and lengthening. Shortening amplitude and dynamic stiffness were decreased by halothane > isoflurane > or = sevoflurane. At equal peak shortening, dynamic stiffness was higher in halothane or isoflurane in high extracellular Ca(2+) concentration than in control. Halothane and isoflurane increased passive stiffness. The decrease in dynamic stiffness and shortening results in part from direct effects of volatile anesthetics at the level of cross bridges. The increase in passive stiffness caused by halothane and isoflurane may reflect an effect on weakly bound cross bridges and/or an effect on passive elastic elements.     

Entactin, a novel basal lamina-associated sulfated glycoprotein

A sulfated glycoprotein, entactin, of apparent molecular weight 158,000 has been isolated from an extracellular basement membrane-like matrix. This matrix is elaborated in cell culture by a mouse endodermal cell line. Antibodies prepared in rabbits against this sulfated glycoprotein react with mouse and rat basement membranes from a variety of tissues. These antibodies also react in a specific manner with a discrete component of mouse and rat kidney glomeruli. The electrophoretic mobility of this component is identical to that of entactin. The mouse kidney antigen, as shown by immunoelectron microscopic studies, is predominantly localized at the surface of epithelial cells of tubules and glomeruli adjacent to the basement membrane. Some antigen is also present in the basal lamina adjacent to the epithelial cells. Entactin is distinct from the basement membrane-associated protein GP-2, a protein similar to laminin. Entactin differs from GP-2 in electrophoretic mobility, cyanogen bromide peptide fragmentation pattern, immunological cross-reactivity, and incorporation of H235SO4. Entactin is insensitive to treatment with chrondroitinase ABC. It is suggested that this molecule plays a role in the interaction of the extracellular matrix and the cell surface.