Identification and extraction of proteins that compose the triad junction of skeletal muscle.

Treatment of both transverse tubules and terminal cisternae with a combination of Triton X-100 and hypertonic K cacodylate causes dissolution of nonjunctional proteins and selective retention of membrane fragments which are capable of junction formation. Treatment of vesicles with Triton X-100 and either KCl or K gluconate causes complete dissolution of all components. Therefore K cacodylate exerts a specific preservative action on the junctional material. The membrane fragment from treatment of transverse tubules with Triton X-100 + cacodylate contains a protein of Mr = 80,000 in SDS gel electrophoresis as the predominant protein while lipid composition is enriched in cholesterol. The membrane fragment retains in electron microscopy the trilaminar appearance of the intact vesicles. Freeze fracture of transverse tubule fragments reveals a high density of low-profile, intercalated particles, which frequently form strings or occasional small arrays. The fragments from Triton X-100 plus cacodylate treatment of terminal cisternae include the protein of Mr = 80,000 as well as the spanning protein of the triad, calsequestrin, and some minor proteins. The fragments are almost devoid of lipid and display an amorphous morphology suggesting membrane disruption. The ability of the transverse tubular fragment, which contains predominantly the Mr = 80,000 protein, to form junctions with terminal cisternae fragments suggests that it plays a role in anchoring the membrane to the junctional processes of the triad. The junctional proteins may be solubilized in a combination of nonionic detergent and hypertonic NaCl. Subsequent molecular sieve chromatography gives an enriched preparation of the spanning protein. This protein has subunits of Mr = 300,000, 270,000 and 140,000 and migrates in the gel as a protein of Mr = 1.2 X 10(6) indicating a polymeric structure.

The use of millipore filters in ultrastructural studies of cell cultures and viruses.

A technique is described for embedding tissue culture cells that have been adsorbed or grown on Millipore filters. The acetone used during the embedding process rendered the filters transparent so that specific areas or cells could be chosen with the aid of the light microscope. Lymphoblastoid cells processed on the filters possessed well-defined plasma membranes and microvilli which were rarely present in cells from parallel cultures that were prepared by pelleting in the centrifuge. Fibroblast cells grown on filters retained their elongated appearance, in contrast to the rounded cells in pelleted preparations. Millipore filters were also used as a means of embedding virus pellets for sectioning. Preparations containing as few as 4 x 10(8) virus particles were suitable for study by the filter technique. Crude tissue-culture harvests of vaccinia virus and purified preparations of Rauscher murine leukemia and adeno-satellite viruses were successfully examined.

Ultrastructural observations of isolated intact and fragmented junctions of skeletal muscle by use of tannic acid mordanting.

Tannic acid mordanting during fixation of isolated vesicles from skeletal muscle enhanced the resolution of the images. Isolated triadic junctions displayed two characteristic features not previously described: (a) a clear gap separated terminal cisternae from transverse tubules; (b) this gap was bridged by a separating array of structures which resembled the "feet" of intact muscle. When the triad was broken in a French press and subsequently reassembled by joining the two organelles, a similar gap was seen but the structure of the feet was less well defined. When the membrane of the triad was extracted by Triton X-100, the junctional region was retained and a similar gap between the two organelles could be discerned. The terminal cisternae characteristically displayed a thickening of the cytoplasmic leaflet of the membrane in select areas in which electron-dense material was apposed on the luminal leaflet. This thickened membrane was not observed in longitudinal reticulum or in terminal cisternae regions distal to the electron-dense matter. This thickened leaflet was not invariably associated with the junction, and some junctional regions did not display discernible thickening of the membrane. When the triad was treated with KCl, the electron-dense aggregate was dispersed and the thickened leaflet of the terminal cisternae dissipated, whereas the triadic junctional region with its feet remained unchanged. KCl treatment caused dissolution of three proteins of Mr = 77,000, 43,000, and 38,000. Treatment of Triton-resistant vesicles with KCl caused the loss of electron-dense aggregate but did not otherwise influence the appearance of the junction. A good degree of correlation both qualitatively and in quantitative parameters between the isolated vesicles and the intact muscle was observed.

Isolation, characterization, and localization of the spanning protein from skeletal muscle triads.

A monoclonal antibody has been developed against the putative junctional protein or spanning protein (SP) from skeletal muscle triads. By immuno-affinity chromatography, we have purified this protein. The native protein has a molecular mass of 630-800 kD, as determined by gel filtration and rate zonal centrifugation. Within the limits of the methods used, the basic unit of the SP appears to be a dimer. In electron micrographs, it is shown to exhibit a circular profile with a diameter of approximately 100 A. In thin section analysis, the protein is frequently observed as parallel tracks of electron-dense particles bordering a translucent core. We suggest that the basic unit of the junctional structure is a dimer of 300-kD subunits and that four such entities constitute the intact SP. The purified protein has been used to develop polyclonal antibodies. By immunoelectron microscopy using immunogold probes, the SP has been localized to the junctional gap of the triad. By attaching the SP to an affinity resin, three proteins have been identified as forming associations with the SP. The Mrs of the proteins are 150, 62, and 38 kD; the 62-kD protein is calsequestrin.

Tissue culture reduces Ia antigen-bearing cells in rat islets and prolongs islet allograft survival.

Prolongation of rat pancreatic islet allograft survival by a prior 7-day period of tissue culture was demonstrated, confirming previous reports by others. We then sought to identity those cells in islets capable of stimulating allograft rejection (Ia antigen-bearing cells) and to determine whether such cells and/or their Ia antigens might be reduced by tissue culture. Freshly isolated and 7-day-cultured Wistar-Furth rat islets were incubated with a mouse anti-rat Ia nonpolymorphic monoclonal antibody, then with peroxidase-conjugated goat anti-mouse antibody, and processed for electron microscopy. Peroxidase (Ia)-positive lymphocytes, macrophages, and capillary endothelial cells were identified in fresh but not in cultured islets. A radioligand assay, using 125I-protein A, revealed a 45% decrease in binding of Ia antibody to cultured compared with fresh islet cells. We conclude that Ia antigen-bearing lymphocytes, macrophages, and capillary endothelial cells in rat islets are reduced by tissue culture and that this may account, at least in part, for the decreased immunogenicity of cultured islet allografts.

Cerebral blood flow restoration and reperfusion injury after ultraviolet laser-facilitated middle cerebral artery recanalization in rat thrombotic stroke.

BACKGROUND AND PURPOSE: A reversible model of focal thrombotic stroke was developed in the rat and examined for histological evidence of reperfusion injury after clinically relevant times of recanalization. METHODS: The distal middle cerebral artery of 28 male Sprague-Dawley rats was occluded by 562-nm laser-driven photothrombosis for 0.5, 2, and 3 hours or permanently (each n=7) and was recanalized by 355-nm UV laser irradiation. Occlusive material was examined by transmission electron microscopy. Cortical cerebral blood flow was monitored by laser-Doppler flowmetry. Brain infarcts were examined histologically at 3 days. RESULTS: After occlusion, cortical cerebral blood flow was reduced to 33+/-4% of baseline for all groups and was restored to 82+/-9%, 75+/-3%, and 93+/-7% of baseline for the 0.5-, 2-, and 3-hour groups, respectively, following recanalization after 29+/-8, 38+/-20, and 70+/-33 minutes of UV laser irradiation. The thrombotic occlusion contained compactly aggregated platelets but no fibrin, with length (1.2 to 1.8 mm) proportional to the ischemic period. During recanalization, microchannels containing erythrocytes and scattered leukocytes and bordered by intact disaggregated platelets infiltrated the thrombus. Infarct volumes (mm3) at 3 days were 12+/-3 for the permanent case and 8+/-4, 24+/-3, and 30+/-9 for the 0.5-, 2-, and 3-hour cases, respectively, thus demonstrating reperfusion injury histologically in the latter 2 groups. No hemorrhage was seen. CONCLUSIONS: UV laser-facilitated dissolution of a conventionally refractory platelet thrombus provides a novel and effective method for restoring blood flow without hemorrhagic complications during thrombotic stroke. This was the first observation of histologically confirmed reperfusion injury in such a model.

Effects of anti-triadin antibody on Ca2+ release from sarcoplasmic reticulum.

The monoclonal antibody, mAb GE 4.90, raised against triadin, a 95 kDa protein of sarcoplasmic reticulum (SR), inhibits the slow phase of Ca2+ release from SR following depolarization of the T-tubule moiety of the triad. The antibody has virtually no effect on the fast phase of depolarization-induced Ca2+ release nor on caffeine-induced Ca2+ release. Since the slow phase of depolarization-induced Ca2+ release is also inhibited by dihydropyridines (DHP), these results suggest that triadin may be involved in the functional coupling between the DHP receptor and the SR Ca2+ channel.

Cervical spinal cord injury in the adult rat: assessment of forelimb dysfunction.

Traumatic injury to the adult human spinal cord most frequently occurs at the mid-to-low cervical segments and produces tetraplegia. To investigate treatments for improving upper extremity function after cervical spinal cord injury (SCI), three behavioral tests were examined for their potential usefulness in evaluating forelimb function in an adult rat model that mimics human low cervical SCI. Testing was conducted pre- and up to 4 weeks post-operation in adult female rats subjected to either contusion injury at the C7 spinal cord segment or sham-surgery. Modified Forelimb Tarlov scales revealed significant proximal and distal forelimb extension dysfunction in lesion rats at l-to-4 weeks post-cervical SCI. The Forelimb Grip Strength Test showed a significant decrease in forelimb grip strength of lesion rats throughout the 4 weeks post-cervical SCI. Significant deficits in reach and pellet retrieval by lesion rats were measured at l-to-4 weeks post-cervical SCI with the conditioned pellet retrieval Staircase Test. The results demonstrate that these qualitative and quantitative forelimb behavioral tests can be used to evaluate forelimb function following low cervical SCI and may be useful to investigate treatments for improving forelimb function in these lesions.

Age-related differences in the local cellular and molecular responses to injury in developing spinal cord of the opossum, Monodelphis domestica.

Immature spinal cord, unlike adult, has an ability to repair itself following injury. Evidence for regeneration, structural repair and development of substantially normal locomotor behaviour comes from studies of marsupials due to their immaturity at birth. We have compared morphological, cellular and molecular changes in spinal cords transected at postnatal day (P)7 or P14, from 3 h to 2 weeks post-injury, in South American opossums (Monodelphis domestica). A bridge between severed ends of cords was apparent 5 days post-injury in P7 cords, compared to 2 weeks in P14. The volume of neurofilament (axonal) material in the bridge 2 weeks after injury was 30% of control in P7- but < 10% in P14-injured cords. Granulocytes accumulated at the site of injury earlier (3 h) in P7 than in P14 (24 h)-injured animals. Monocytes accumulated 24 h post-injury and accumulation was greater in P14 cords. Accumulation of GFAP-positive astrocytes at the lesion occurred earlier in P14-injured cords. Neurites and growth cones were identified ultrastructurally in contact with astrocytes forming the bridge. Results using mouse inflammatory gene arrays showed differences in levels of expression of many TGF, TNF, cytokine, chemokine and interleukin gene families. Most of the genes identified were up-regulated to a greater extent following injury at P7. Some changes were validated and quantified by RT-PCR. Overall, the results suggest that at least some of the greater ability to recover from spinal cord transection at P7 compared to P14 in opossums is due to differences in inflammatory cellular and molecular responses.

Light and electron microscopic assessment of progressive atrophy following moderate traumatic brain injury in the rat.

The presence of progressive white matter atrophy following traumatic brain injury (TBI) has been reported in humans as well as in animal models. However, a quantitative analysis of progressive alterations in myelinated axons and other cellular responses to trauma has not been conducted. This study examined quantitative differences in myelinated axons from several white and gray matter structures between non-traumatized and traumatized areas at several time points up to 1 year. We hypothesize that axonal numbers decrease over time within the structures analyzed, based on our previous work demonstrating shrinkage of tissue in these vulnerable areas. Intubated, anesthetized male Sprague-Dawley rats were subjected to moderate (1.8-2.2 atm) parasagittal fluid-percussion brain injury, and perfused at various intervals after surgery. Sections from the fimbria, external capsule, thalamus and cerebral cortex from the ipsilateral hemisphere of traumatized and sham-operated animals were prepared and. estimated total numbers of myelinated axons were determined by systematic random sampling. Electron micrographs were obtained for ultrastructural analysis. A significant (P<0.05) reduction in the number of myelinated axons in the traumatized hemisphere compared to control in all structures was observed. In addition, thalamic and cortical axonal counts decreased significantly (P<0.05) over time. Swollen axons and macrophage/microglia infiltration were present as late as 6 months post-TBI in various structures. This study is the first to describe quantitatively chronic axonal changes in vulnerable brains regions after injury. Based on these data, a time-dependent decrease in the number of myelinated axons is seen to occur in vulnerable gray matter regions including the cerebral cortex and thalamus along with distinct morphological changes within white matter tracts after TBI. Although this progressive axonal response to TBI may include Wallerian degeneration, other potential mechanisms underlying this progressive pathological response within the white matter are discussed.

A functional identification of cardiac junctional sarcoplasmic reticulum.

Junctional sarcoplasmic reticulum (SR) has been identified in microsomes from canine ventricular muscle by the presence of calsequestrin and ryanodine-sensitive Ca2+ release channels. These properties, however, are not common to cardiac cells from all species. Seiler et al (1) have recently described a high Mr polypeptide in canine junctional SR similar to the spanning protein subunits of skeletal muscle triads. We now report the existence of a polypeptide with the same mobility in SR from rabbit ventricular muscle and show that those cardiac membranes can associate with transverse (T-) tubules from rabbit skeletal muscle in K cacodylate medium. We propose that this polypeptide and the reaction with T-tubules be considered as criteria for the identification of cardiac junctional SR.

Localization by immunoelectron microscopy of spanning protein of triad junction in terminal cisternae/triad vesicles.

Polyclonal antibodies have been developed against the junctional feet or spanning protein from skeletal muscle triads. These probes in combination with immunogold labels have been used to localize the spanning protein by electron microscope of isolated vesicles from terminal cisternae/triads. The spanning protein antibodies specifically bind to the electron dense junctional feet. In vesicles permeabilized by hypotonic treatment or by saponin, some gold particles may be seen on the luminal side of the vesicle. Trypsin treatment of vesicles causes complete loss of the 300 K spanning protein from SDS gels while dot blots show that some but not all the antigenic activity is lost. This treatment is associated with the loss of the electron dense projections from the membrane surface and is coincident with the loss of immunogold staining when antibody is added to the intact vesicles. On the other hand, in experiments in which the luminal portions of the isolated vesicles have been made accessible to the polyclonal antibodies by sectioning lightly fixed vesicles before immunogold tagging, extensive gold labelling was found to occur in trypsin treated vesicles which have lost detectable projections from the cytoplasmic side of the membrane. These data support the view that the spanning protein projects from the sarcoplasmic reticulum towards the transverse tubules but further suggest that spanning protein extends into and probably through the sarcoplasmic reticulum membrane in accord with the proposition that it is a Ca2+ channel.

ATP-energized Ca2+ pump in isolated transverse tubules of skeletal muscle.

A modified protocol for isolation of transverse tubules incorporated an extra stage of purification. The existence of an ATP-energized Ca2+ pump in transverse tubules isolated from rabbit skeletal muscle has been demonstrated. Isolated transverse tubules had a Ca-ATPase activity of 0.78 mu mol/min . mg; this was 300% in excess of that activity attributable to sarcoplasmic reticulum contamination. The distribution of part of the CaATPase activity and ATP-energized Ca2+ uptake coincided with the distribution of transverse tubules in isopycnic sucrose gradients loaded with mechanically disrupted triad junctions. Transverse tubules accumulated over 70 nmol of Ca2+/mg of protein; this uptake was abolished by the Ca2+ ionophore A23187. Neither digitoxin nor monensin inhibited Ca2+ uptake, indicating that Ca2+ accumulation did not occur through a sodium/calcium exchange. Conditions for half-maximal Ca2+ uptake were 5 micro M free Ca2+ and 10 micro M ATP. The Ca2+ pump of isolated transverse tubules was distinguished from the Ca2+ pump of sarcoplasmic reticulum and sarcolemma in that the transverse tubule Ca2+ pump: 1) was not enhanced by oxalate; 2) was not energized by acetyl phosphate, p-nitrophenyl phosphate, or 3-O-methylfluorescein phosphate; and 3) did not hydrolyze p-nitrophenyl phosphate or 3-O-methyl-fluorescein phosphate. Using Ca2+-dependent 3-O-methylfluorescein phosphatase as a marker for sarcoplasmic reticulum, the contamination of the transverse tubule preparation was calculated to be 6%. This agreed with a contamination level of 5% estimated by freeze-fracture electron microscopy.

Biophysical studies of vesicular stomatitis virus.

McCombs, Robert M. (Baylor University College of Medicine, Houston, Tex.), Matilda Benyesh-Melnick, and Jean P. Brunschwig. Biophysical studies of vesicular stomatitis virus: J. Bacteriol. 91:803-812. 1966.-The infectivity and morphology of vesicular stomatitis virus (VSV) were studied after density gradient centrifugation in cesium chloride (CsCI), potassium tartrate (KT), and sucrose. Centrifugation in CsCl revealed two equally infectious bands corresponding to densities of 1.19 and 1.22 g/ml, and a third (density, 1.26 g/ml) band of low infectivity. Two bands (densities of 1.16 and 1.18 g/ml) were observed in the KT gradient, in which the lighter band contained most of the infectivity. Centrifugation in sucrose resulted in a single broad infectious band (density, 1.16 g/ml). The typical rod-shaped VSV particles were found mainly in the lighter bands obtained in CsCl (1.19 g/ml) and KT (1.16 g/ml) and in the single sucrose gradient band (1.16 g/ml). Bent particles equally as infectious as the rod-shaped particles were a constant finding in the CsCl preparations, and were observed mainly in the second band (density, 1.19 g). Numerous strands 15mmu wide were found in the third CsCl (density, 1.26 g/ml) and the second KT (1.18 g/ml) bands. Similar strands could be liberated from VSV particles after treatment with deoxycholate. Internal transverse striations were found to be a regular feature of VSV particles examined with the pseudoreplication negative-staining technique. For crude virus stocks, the physical particle-to-infectivity ratio ranged from 73 to 194. Several morphological similarities between VSV and myxoviruses were observed, including 10 mmu surface projections, pleomorphic morphological forms, and 15 mmu seemingly nucleoprotein strands.

Identification of a new subpopulation of triad junctions isolated from skeletal muscle; morphological correlations with intact muscle.

It has been previously recognized that a number of protocols may cause breakage of the triad junction and separation of the constituent organelles of skeletal muscle. We now describe a fraction of triad junctions which is refractory to the known protocols for disruption. Triads were passed through a French press and the dissociated organelles were separated on a sucrose density gradient, which was assayed for PN200-110, ouabain and ryanodine binding. Ryanodine binding showed a single peak at the density of heavy terminal cisternae. On the other hand, the PN200-110 and ouabain, which are external membrane ligands, bound in two peaks: one at the free transverse tubule region and the other at the light terminal cisternae. Similarly, a two peak pattern of PN200-110 and ouabain binding was observed when triad junctions were broken by the Ca2(+)-dependent protease, calpain, which selectively hydrolyzes the junctional foot protein. The light terminal cisternae vesicles were subjected to three different procedures of junctional breakage: French press, hypertonic salt treatment, and protease digestion using calpain or trypsin. The treated membranes were then centrifuged on density gradients. Only extensive trypsin digestion caused a partial shift of ouabain activity into the free transverse tubule region. These observations suggest that the triads are a composite mixture of breakage susceptible, "weak," and breakage resistant, "strong," triads. Scatchard analysis of PN200-110 suggests that the transverse tubules of strong triads contain a relatively high number of dihydropyridine receptors compared to those of weak triads. Thin section electron microscopic images of the strong triads comparable to those of intact muscle are presented.

Determinants of triad junction reformation: identification and isolation of an endogenous promotor for junction reformation in skeletal muscle.

The junction of isolated triads can be mechanically broken by passage through a French press and subsequently reformed by incubation of the isolated organelles with certain salts of weak acids (e.g., K cacodylate, K propionate, and K butyrate). In contrast, other salts (e.g., KCl, K phosphate, and K benzoate) are ineffective in promoting triad formation. An endogenous factor obtained from a muscle homogenate acts in the same manner as these artificial compounds. When rabbit skeletal muscle is homogenized in a KCl solution and centrifuged to remove large cellular components and membrane fractions, an endogenous factor is extracted into the high speed supernatant which promotes the reformation of mechanically broken triads. A three-stage purification of this factor has been achieved using: ammonium sulfate fractionation, adsorption chromatography, and molecular sieve chromatography. SDS-PAGE showed that the protein was purified to homogeneity and had a subunit Mr of 34,000 daltons. This protein has the following characteristics: it exists in 0.1 M KCl as a polymeric substance with an estimated Mr = 123,000 on molecular sieve chromatography and a Mr = 155,000 on sedimentation equilibrium; it promotes the formation of triadic vesicles from isolated organelles in a low ionic strength medium; Both this protein and cacodylate share the property of specifically catalyzing the association and aggregation of junctional proteins which had previously been dissolved by neutral detergent and salt; it appears to be identical to an extrinsic constituent of terminal cisternae, which has been described as a protein of Mr = 34K. It is not clear, however, whether this protein is a necessary and integral component of the junctional feet or whether it exerts predominantly a catalytic role in the formation of the triad junction.