Isolation of pulmonary interstitial fluid in rabbits by a modified wick technique

Interstitial fluid protein concentration (C(protein)) values in perivascular and peribronchial lung tissues were never simultaneously measured in mammals; in this study, perivascular and peribronchial interstitial fluids were collected from rabbits under control conditions and rabbits with hydraulic edema or lesional edema. Postmortem dry wicks were implanted in the perivascular and peribronchial tissues; after 20 min, the wicks were withdrawn and the interstitial fluid was collected to measure C(protein) and colloid osmotic pressure. Plasma, perivascular, and peribronchial C(protein) values averaged 6.4 +/- 0.7 (SD), 3.7 +/- 0.5, and 2.4 +/- 0.7 g/dl, respectively, in control rabbits; 4.8 +/- 0.7, 2.5 +/- 0.6, and 2.4 +/- 0.4 g/dl, respectively, in rabbits with hydraulic edema; and 5.1 +/- 0.3, 4.3 +/- 0.4 and 3.3 +/- 0.6 g/dl, respectively, in rabbits with lesional edema. Contamination of plasma proteins from microvascular lesions during wick insertion was 14% of plasma C(protein). In control animals, pulmonary interstitial C(protein) was lower than previous estimates from pre- and postnodal pulmonary lymph; furthermore, although the interstitium constitutes a continuum within the lung parenchyma, regional differences in tissue content seem to exist in the rabbit lung.     

Isolation of interstitial fluid from rat mammary tumors by a centrifugation method

Access to interstitial fluid is of fundamental importance to understand tumor transcapillary fluid balance, including the distribution of probes and therapeutic agents. Tumors were induced by gavage of 9,10-dimethyl-1,2-benzanthracene to rats, and fluid was isolated after anesthesia by exposing tissue to consecutive centrifugations from 27 to 6,800 g. The observed (51)Cr-EDTA (extracellular tracer) tissue fluid-to-plasma ratio obtained from whole tumor or from superficial tumor tissue by centrifugation at 27-424 g was not significantly different from 1.0 (0.92-0.99), suggesting an extracellular origin only. However, fluid collected from excised central tumor parts had a significantly lower ratio (0.66-0.77) for all imposed G forces, suggesting dilution by fluid deriving from a space unavailable for (51)Cr-EDTA. The colloid osmotic pressure in tumor fluid was generally higher than in fluid isolated from the subcutis, attributable to less selective capillaries and impaired lymphatic drainage in tumors. HPLC analysis of tumor fluid showed that low-molecular-weight macromolecules not present in arterial plasma were present in tumor fluid obtained by centrifugation and in venous blood draining the tumor, most likely representing proteins derived from tumor cells. We conclude that low-speed centrifugation may be a simple and reliable method to isolate interstitial fluid from tumors.     

A novel angiogenesis model using interstitial cells derived from skeletal muscle

A novel angiogenesis model using co-culture of endothelial and interstitial cells was developed, in which bovine capillary endothelial cells (BCEC) formed capillary-like structure on the monolayer of interstitial cells (like smooth muscle cells) isolated from rat skeletal muscle. The capillary formation of BCEC occurred even under serum-free conditions. Insulin stimulated the capillary growth under serum-free condition, but not BCEC growth in the single culture system. These results suggested that the insulin effect on the capillary growth was brought about indirectly through the interstitial cells, and that this co-culture system may be useful for the study of angiogenesis (especially in skeletal muscle).     

Isolation and properties of myoglobin from murine skeletal muscle

Myoglobin (Mb) was isolated from skeletal muscle of JCL-ICR mice by heat denaturation-gel filtration combined with ion exchange chromatography or chromatofocusing by which isoelectric point of the main component was estimated as 7.63 +/- 0.09 (20 degrees C). The Mb was homogeneous by gel electrophoretic and ultracentrifugal analysis. The molecular weight by sedimentation equilibrium was 1.80 X 10(4) and essentially identical with the values by the iron analysis (1.82 X 10(4)) and amino acid composition (1.78 X 10(4)) in which one residue of cysteine was found per molecule. The spectroscopic properties of deoxy-, oxy-, carboxy- and ferri-derivatives of the protein were determined in ultraviolet, Soret and visible regions. The pK' of acid-alkaline transition of the ferri-form was estimated as 8.57 +/- 0.30 (30 degrees C) from the pH-dependent spectral changes. The oxygen equilibrium studies revealed complete absence of such allosteric properties as heme-heme interaction, anion effect and Bohr effect. Oxygen tension for the half-oxygenation (P50) was 0.69 +/- 0.06 Torr (20 degrees C) and its temperature-dependent change gave the delta H degrees of -14.1 kcal/mole.     

Isolation and characterization of plasma membranes from rabbit skeletal muscle

An improved procedure was developed for the isolation of skeletal muscle plasma membranes. This method includes a DNAse treatment of the homogenate prior to the isolation of membranes by differential and sucrose gradient centrifugation techniques. We obtained two light fractions which were highly enriched in many biochemical and chemical plasma membrane markers. These fractions were shown to be mostly inside-out vesicles containing a Ca2+-ATPase activity. These results suggested that this enzyme could participate in the extrusion of calcium ions from the muscle cells.     

Isolation of skeletal muscle collagen

A new method for the isolation of a high yield of collagen from human skeletal muscle is described. This technique employs rapid stirring of the homogenate of skeletal muscle with a magnetic stirrer. Fibrous material entangled during rapid stirring was recovered by passing the homogenate through a sieve and then sequentially extracted with Hasselbach-Schneider solution and 0.6 m KI-0.06 m Na₂S₂O₃. The insoluble residue obtained after these extractions was shown to be highly purified collagen by amino acid analysis, and the recovery of collagen by this method was found to be more than 90% of the total collagen in skeletal muscle. The isolated collagen from 5 years of age was mostly composed of Type I collagen, and small amounts of Type III collagen and an unidentified collagenous protein migrating in a position near Type V collagen (αB chain) were also found on a sodium dodecyl sulfate-polyacrylamide gel.     

Isolation of skeletal muscle nuclei

A method for isolation of nuclei from rat skeletal muscle is described. The tissue is homogenized in the presence of Triton X-100, resulting in release of the nuclei from the muscle fibers. A crude nuclear pellet is prepared by differential centrifugation, and further purification is accomplished by centrifugation through dense sucrose. Bovine serum albumin is used to stabilize the nuclei during the isolation procedure. The isolated nuclei have been characterized microscopically and by chemical and enzymatic assay procedures.     

Isolation and composition of thick filaments from rabbit skeletal muscle

A method has been developed for the isolation of thick filaments from rabbit skeletal muscle. We found that the thick filaments of this muscle are readily dispersed in the presence of a relaxing medium if the M and Z-line structures are first extracted in a low-salt solvent system. Thick filaments were separated from thin filaments by zone sedimentation in a 10% to 30% glycerol density gradient. The isolated filaments are homogeneous in length (1.5 to 1.6 μm) and retain the physical characteristics of these structures observed in sectioned muscle. Gel electrophoresis of thick filaments in the presence of sodium dodecyl sulfate showed a band of C-protein as well as bands with mobilities characteristic of the heavy and light chains of myosin. No other protein species was detected in these experiments. Thus our results provide evidence against the presence of a special protein component which would serve as the core of the skeletal thick filament structure. From the relative stain density of bands, the molar ratio of C-protein to myosin was estimated to be 1 to 5.8.     

Isolation and characterization of basement membrane from frog skeletal muscle

Isolation of basement membrane from frog skeletal muscle has been described. The membrane preparation contained 35 micrograms hexoses, 1.72 micrograms sialic acid, 6.8 micrograms phospholipids, 0.21 micrograms cholesterol/mg protein. Na + K-ATPase and 5'-nucleotidase could not be detected in the membrane preparation. Glycine accounted for about 20% of the total amino acids. On SDS-PAGE, the membrane resolved into 20-22 polypeptide bands.     

Isolation and characterization of a high molecular weight protein phosphatase from rabbit skeletal muscle

A high molecular weight protein phosphatase (phosphatase H-II) was isolated from rabbit skeletal muscle. The enzyme had a Mr = 260,000 as determined by gel filtration and possessed two types of subunit, of Mr = 70,000 and 35,000, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On ethanol treatment, the enzyme was dissociated to an active species of Mr = 35,000. The purified phosphatase dephosphorylated lysine-rich histone, phosphorylase a, glycogen synthase, and phosphorylase kinase. It dephosphorylated both the alpha- and beta-subunit phosphates of phosphorylase kinase, with a preference for the dephosphorylation of the alpha-subunit phosphate over the beta-subunit phosphate of phosphorylase kinase. The enzyme also dephosphorylated p-nitrophenyl phosphate at alkaline pH. Phosphatase H-II is distinct from the major phosphorylase phosphatase activities in the muscle extracts. Its enzymatic properties closely resemble that of a Mr = 33,500 protein phosphatase (protein phosphatase C-II) isolated from the same tissue. However, despite their similarity of enzymatic properties, the Mr = 35,000 subunit of phosphatase H-II is physically different from phosphatase C-II as revealed by their different sizes on sodium dodecyl sulfate-gel electrophoresis. On trypsin treatment of the enzyme, this subunit is converted to a form which is a similar size to phosphatase C-II.     

Isolation and characterization of four lipolytic preparations from rat skeletal muscle

Four different lipolytic preparations have been isolated from rat skeletal muscle. Two of these, provisionally designated monopalmitin lipase (MPL) and monomyristin lipase (MML), are associated with insoluble cellular particulate fractions. The other two enzymes, provisionally designated tricaproin lipase (TCL) and monolaurin lipase (MLL), are found in the high-speed supernatant fraction. Taken as a group, these enzymes are capable of hydrolyzing short-chain triglycerides (acyl moieties of C(3) to C(8)) and monoglycerides of lauric, myristic, palmitic, stearic, and oleic acids. All of these enzymes have a serine residue at or near the catalytic site as they are strongly inhibited by diisopropyl fluorophosphate. The two particulate preparations contain a sulfhydryl group and are sensitive to p-chloromercuribenzoate and N-ethylmaleimide, while the soluble preparations are not. The MLL, MML, and MPL preparations all have alkaline pH optima, while the TCL preparation has an acidic optimum. Buffer type is important: some buffer compounds completely inhibited one or more preparations. Of the soluble enzymes, MLL withstood heating to 60 degrees C, while TCL is completely inactivated at this temperature. Of the particulate preparations, only MML was stable to lyophilization. It is concluded that there are at least four lipolytic enzymes in rat skeletal muscle. The possible significance of the presence of these enzymes in muscle is discussed.     

Isolation and immunofluorescent localization of actin derived from rat skeletal muscle

Summary Rat skeletal muscle actin was extracted, purified and its homogeneity established according to the criteria of ultracentrifugation and electrophoresis. Immunofluorescence procedure using antisera prepared in rabbits against the purified rat skeletal muscle actin revealed localized staining reaction in the I band region of the skeletal muscle. Similar studies on rat embryo muscle cultures showed a diffuse cytoplasmic fluorescence in fibroblastlike cells and an intense fluorescence in the multi-nucleated myoblasts of the younger cultures. In the older cultures strong fluorescence was detectable in scattered parallel rows and in the presumptive I bands of mononucleated myoblasts an in the thread-like mitochondria of fibroblasts. The distribution of fluorescence in these cells is considered indicative of the association of actin with the contracile protein in general and with mitochondria which in cultured myoblasts assume enormous lengths and appear to be extremely motile.     

Isolation of two proteolipids from rabbit skeletal muscle sarcoplasmic reticulum

We have isolated two proteolipids from rabbit skeletal muscle sarcoplasmic reticulum by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. One, PL-II, is identical to the proteolipid previously obtained by others using organic solvent extraction. The other, PL-I, has an amino acid composition very similar to those of proteolipids we previously isolated from canine cardiac SR and lamb kidney (Na,K)-ATPase.