Proteinase-sensitive release of enzymes from pancreatic microsomal fraction.

Suspensions of rat pancreatic microsomal fraction release alpha-amylase and ribonuclease on incubation at 37 degrees C, but not at 2 degrees C. The release is abolished by proteolytic enzymes. Ribonuclease associated with the microsomal fraction is protected from subtilisin BPN' attack, but is sensitive after release.     

Receptor-operated calcium-channels in isolated rabbit jejunum.

Calcium channels were studied in isolated spontaneously rhythmic rabbit jejunum using the muscarinic agonist carbachol as stimulant. Carbachol failed to produce the characteristic phasic and tonic components of smooth muscle contractions. A variety of chemically distinct calcium antagonists, viz. bepridil, diltiazem, isradipine (PN 200-110), nifedipine, and verapamil, non-competitively inhibited the contractions. Diltiazem was most potent (-logIC50 = 8.30) and bepridil least potent (-logIC50 = 6.19) in inhibiting the contractions. The findings conclude with the presence of pharmacologically distinct receptor-operated calcium-channels, besides the potential-dependent calcium-channels, in the rabbit jejunum.     

Characterization of autoantigenic sites on isolated dog heart mitochondria.

1. Anti-heart mitochondria autoantibodies were developed in serum from dogs following experimental myocardial infarction. 2. Heart mitochondria frozen and thawed repeatedly in a sucrose/Tris-chloride buffer retained both their functional integrity as measured by the respiratory control ratio and their ability to serve as an antigen in a complement fixation test. Mitochondria frozen and thawed in a potassium chloride/Tris-chloride buffer lost both their functional integrity and their autoantigenic activity after one freeze-thaw cycle. 3. Extraction of the heart mitochondria with acetone/water mixtures to remove phospholipids from the membrane led to a complete loss of the ability of the mitochondria to react in the complement fixation test but did not affect the ability of the membranes to bind autoantibody in absorption experiments. 4. Treatment of the mitochondrial membranes with increasing concentrations of trypsin caused a loss of up to approximately 50% of the membrane protein with a gradual decrease in the autoantigenic activity of the membrane without impairment of the ability of the membrane to bind autoantibody. 5. Removal of up to 90% of the sialic acid of the mitochondrial membrane with neuraminidase resulted in a considerable increase in the complement-fixing autoantigenic activity of the membrane without changing the apparent ability of the membrane to bind autoantibody in absorption experiments. 6. Exposure of mitochondrial membranes to autoantibody and complement caused an inhibition of both an inner mitochondrial membrane enzyme, i.e. cytochrome oxidase (48%) and an outer mitochondrial membrane enzyme, i.e. NADH cytochrome c reductase (rotenone insensitive) (37%).     

Polymorphisms of three new microsatellite sites of the dystrophin gene

To look for novel microsatellites in the dystrophin gene for the diagnosis of Duchenne muscular dystrophy, candidate microsatellite sites in the dystrophin gene were analyzed with the SSRHunter software and were also genotyped. Among the 15 candidate microsatellite sites, three novel microsatellite sites in the 60th, 30th, and 2nd intron were found to have a high degree of polymorphism. We submitted these three new loci to the European Molecular Biology Laboratory, under accession Nos. FN547040, FN547041 and FN557526, which were called DXSDMD-in60, DXSDMD-in30 and DXSDMD-in2, respectively. In these three loci, we found 9, 6 and 11 alleles, respectively, in the 205 individuals. In addition, we also detected 20, 19 and 20 genotypes for the three loci in female samples, with a polymorphism information content of more than 0.600. In conclusion, the three microsatellite sites in the intron region of the dystrophin gene have a high degree of polymorphism, and they can be used in population genetics, as well as to provide a theoretical basis for genetic diagnosis and elucidation of molecular mechanisms in Duchenne muscular dystrophy.     

Transcobalamin II--cobalamin binding sites are present on rabbit germ cells.

Specific binding sites for rabbit transcobalamin II have been found on isolated adult rabbit germ cells. Scatchard analysis revealed a single class of binding sites for [57Co]cyanocobalamin-transcobalamin II with an association constant (Ka) of 1.3 x 10(10) M-1 and 700 sites per cell. Binding was reversible, saturable and calcium dependent. Electron microscope radioautography following incubation with iodinated transcobalamin II at 4 degrees C led to a detectable labeling mainly restricted to the plasma membrane.     

Somatostatin binding sites in cytosolic fraction isolated from rabbit antral and fundic gastric mucosa

Specific binding sites for somatostatin have been identified and characterized in cytosolic fraction of rabbit gastric mucosa at both antrum and fundus levels. The binding depended on time, temperature and pH, and was reversible and saturable. The stoichiometric data suggested the presence of two classes of binding sites: a class with high affinity (Kd = 26.7 and 37.0 nM in antrum and fundus, respectively) and low capacity (2.1 and 4.1 pmol somatostatin/mg protein in antrum and fundus, respectively), and a class with low affinity (Kd = 246.4 and 162.5 nM in antrum and fundus, respectively) and high capacity (134.1 and 110.9 pmol somatostatin/mg protein in antrum and fundus, respectively) at 25 degrees C and pH 7.4. The binding sites were shown to be highly specific for somatostatin since neuropeptides such as Leu-enkephalin, neurotensin and substance P behaved as ligands with very low affinity.     

Binding sites involved in the interaction of actin with the N-terminal region of dystrophin.

Two actin-binding sites have been identified on human dystrophin by proton NMR spectroscopy of synthetic peptides corresponding to defined regions of the polypeptide sequence. These are Actin-Binding Site 1 (ABS1) located at residues 17-26 and Actin-Binding Site 2 (ABS2) in the region of residues 128-156. Using defined fragments of the actin amino acid sequence, ABS1 has been shown to bind to actin in the region represented by residues 83-117 and ABS2 to the C-terminal region represented by residues 350-375. These dystrophin-binding sites lie on the exposed domain in the actin filament.     

Effects of mercaptans upon dihydropyridine binding sites on transverse tubules isolated from triads of rabbit skeletal muscle

The binding of nitrendipine to transverse (T) tubules isolated from skeletal muscle triads is inhibited by dithiothreitol (KI approximately 0.05 mM) and glutathione (KI approximately 3 mM). The t 1/2's of inhibition (18.3 and 11.5 min, respectively) suggest that these hydrophylic reagents act upon the exposed surface of the vesicles. Dithiothreitol shifts the apparent KD for nitrendipine from 8.5 nM to 30 nM without altering the Bmax extrapolated by Scatchard analysis. That T-tubules isolated by disruption of triad junctions are constrained to have the protoplasmic (P) face uniformly exposed was experimentally confirmed. These studies show that a sulfhydryl residue on the P-face of the T-tubule influences the affinity of the receptor for dihydropyridines.     

Oxytocin binding sites in lactating rabbit mammary gland.

Material which specifically binds oxytocin was prepared from a crude preparation of lactating rabbit mammary gland by purification on a sucrose density gradient. On examination of activities of enzyme markers and the molar ratio of cholesterol to phospholipid, this material was considered to be a highly purified plasma membrane fraction. For the determination of specificity and time course of oxytocin binding, a Scatchard plot analysis was carried out for the crude and purified fractions. Dissociation constant (Kd) and binding capacity values were found to be as follows: crude, Kd equals 1.83 X 10(-9) M, capacity equals 670 fmol/mg protein; purified, Kd equals 2.8 X 10(-9) M, capacity equals 1700 fmol/mg protein. Treatment of the purified material with different detergents resulted in loss of all [3H]oxytocin binding capacity. However, preincubation of this material with [3H]oxytocin prior to detergent treatment resulted in solubilization of a receptor-hormone complex. This complex remained in the supernatant even after centrifugation at 210 000 X g for 30 min. Using oxytocin analogs, we have shown this solubilized complex to be oxytocin specific.     

Identification of osteopontin in isolated rabbit osteoclasts.

Bone remodeling is a complex process coupling bone formation and resorption. Osteoblasts, the bone-forming cells, are known to produce various bone matrix proteins and cytokines; however, little is known about protein factors produced by osteoclasts or bone-resorbing cells. A method utilizing the high affinity of osteoclasts for tissue culture dishes was developed to isolate a large number of pure osteoclasts from rabbit long bones. A cDNA library was then constructed from these isolated osteoclasts, and differential cDNA screening was performed between osteoclasts and spleen cells. Two clones representing osteoclast-specific clones, named OC-1 and OC-2, were isolated. By Northern blot analysis, OC-1 was expressed in osteoclasts and in kidneys, whereas OC-2 was specific for osteoclasts. OC-1 was found to encode osteopontin from its nucleotide sequence, and therefore, osteopontin may have other functions for osteoclastic bone resorption besides osteoclast attachment to bone.     

Glycosaminoglycans and glycoproteins isolated from rabbit femur.

1. Complex carbohydrate fractions were extracted successively with 40% aqueous EDTA (pH 7.4) and 6M urea (PH 7.8) FROM ACETONE-DRIED bone powder of rabbit femur. 2. The carbohydrate fraction extracted with EDTA (E=Fr) was separated into five fractions,D1approximatelyD5 by DEAE-Dephadex A-50 column chromatography. Chemical and infrared spectral analyses, and enzymatic digestion indicate that D2 contained lessacidic glycoprotein, D3 contained sialoglycoprotein, D4 contained a low sulfated proteokeratan sulfate-like substance, and d5 contained glycoprotein-bound chondroitin sulfate A plus protein-free chondroitin sulfate A. 3. Two fractions, HU-D1 and HU-D2, were isolated from the carbohydrate fraction extracted with urea (HU-Fr) by successive digestion with collagenase [EC 3.4.99.5] and pronase, followed by gel-filtration on Sephadex G-100 and then DEAE-Sephadex A-50 column chromatography. HU-D1 and HU-D2 contained a low sulfated keratan sulfate-like substance linked to peptide and glycopeptide-bound chondroitin sulfated keratan sulfate-like substance linked to peptide and glycopeptide-bound chondroitin sulfate A, respectively. 4. The present findings indicate that rabbit femur contains low sulfated proteokeratan sulfate-like substances with varying sulfate contents and glycoprotein-bound chondroitin sulfate A as the principal glycosaminoglycans. The macromolecules bound more tightly to the tissue contain much more sulfate than the corresponding loosely bound ones.     

Molecular shape of dystrophin.

The molecular shape of dystrophin has been reported to be a 175 nm flexible rod [Pons, F. et al. (1990) Proc. Natl. Acad. Sci. USA 87, 7851-7855] or a 120 nm dumbbell [Murayama, T. et al. (1990) Proc. Jpn. Acad. 66B, 96-99]. The present work revealed that 100 nm flexible rods with or without spheres were predominant in highly purified dystrophin preparations. When the sample was subjected to gel filtration, dystrophin oligomers were isolated just after the void volume and the fraction largely consisted of dumbbell-shaped molecules. From various rotary-shadowed images, it was suggested that dystrophin is a rod with spheres at both ends, approximately 110 nm long and 2 nm wide. It appeared that this monomer binds to another monomer in a staggered way, forming a dimer, and the dimers associate with each other side-by-side, forming a dumbbell-shaped tetramer, 130 nm long and 5 nm wide. The tetramers form an end-to-end aggregate. It seemed that the dumbbell structure was not affected by alkaline (pH 11) treatment to dissociate dystrophin associated glycoproteins, but was deteriorated by detergent, NP-40, Triton X-100, or CHAPS, used for solubilization of membrane-bound dystrophin.     

Effect of copper on isolated rabbit blastocysts

The effect of copper on the electrical membrane properties of the isolated-perfused 6-day rabbit blastocyst was studied to understand changes in the intrauterine environment caused by the copper IUD. Blastocysts were perfused in an environmental chamber containing Krebs-Ringer bicarbonate with 1 mg bovine serum albumin/ml. Electrical measurements made included short-circuit current (SCC) (the net result of currents produced by all net active ionic transport processes when there is no electrochemical gradient), transmural potential difference (p.d.), and conductance (computed from the ratio of open circuit p.d. to SCC). Control values were obtained and 9 experiments were performed in which 10 mcl aliquots of ?cuCl2 was added to the bathing solution. Electrical parameters of solutions containing 10-5M concentration CuCl2 remained essentially unchanged. 2.5 x 10-5 M reduced average p.d. 25% and average SCC 12%, WHILE 5 X 10-4C-5 M further reduced p.d. 48% and SCC 38% after 30 minutes. At 7.5 x 10-5 M p.d. was depressed 89% after 10 minutes with 1/3 of the values being positive, and SCC values decreased to 71% at 10 minutes and then increased to 77% of control values at 30 minutes. The subsequent changes in p.d. and SCC caused a 6-fold increase in membrane conductance. 9 experiments were performed on a 2nd group of blastocysts in which the effects of a single addition of CuCl2 at 10-4 M were studied. Average p.d. decreased reversing to positive values at 30 minutes. There was a biphasic response to SCC decreasing to 46% after 20 minutes then increasing to 1.7 times control values. Single additions of copper ions collapsed all blastocysts after a return to copper-free solutions. Serial additions showed only 3 out of 9 collapsing under similar conditions. Further experiments involving simultaneous SCC-isotope flux are necessary to determine which specific actively transported ions are affected by copper and to determine the effect on conductance. It is suggested that the action of copper in these experiments might have some bearing on the effectiveness of the copper IUD.     

Autologous perfusion of an isolated rabbit gastrointestinal tract.

Most perfusion techniques rely on mechanical means to provide blood flow to the isolated organ for maintaining its physiological conditions. The approach usually requires a complicated mechanical system with the associated problems of blood type matching and prevention of blood cell damage. This paper describes a gastrointestinal tract perfusion technique that uses the rabbit's own cardiopulmonary system as the autologous blood supply source. The technique allows for the removal of the complete intestinal loop from the abdominal cavity of the rabbit, and maintains its blood circulation through silastic tubing connections of the catheterized portal vein and cranial and caudal mesenteric arteries. An alternative perfusion site that uses the aorta as the arterial blood supply and the vena cava as the venous return also is described. The isolated perfused GI tract may then be placed in a separate test environment for controlled experiments. For an acute animal test, the approach was found to be a convenient alternative to conventional approaches.     

Perfusate cytochrome c reduction in isolated rabbit lungs.

The reduction of ferricytochrome c within the perfusate in isolated lung perfusion systems has been demonstrated previously. We carried out the present study 1) to determine what reducing agents might be responsible for this reduction and 2) to determine whether the cytochrome c (cyto c) reduction within the recirculating perfusion system can be accounted for by relatively stable reducing agents released into the perfusate or whether some of the reduction is dependent on short-lived agents and/or proximity to the source of the agents within the lungs. Experiments were carried out with the use of isolated rabbit lungs perfused for 1 h in a recirculating system. In one group of experiments, ferricytochrome c was included in the recirculating perfusion system. In another group, the cyto c was added to produce the same concentration in samples after they were removed from a cyto c-free recirculating system. The recirculating cyto c was reduced at a rate of approximately 1.76 mumol/h, and approximately 22% was inhibitable by superoxide dismutase. Most of the rest could be inhibited by ascorbate oxidase within the recirculating perfusate. When the ferricytochrome c was added to the samples removed from the cyto c-free perfusion system, virtually the entire cyto c reducing capacity was inhibitable by ascorbate oxidase. Although reduced glutathione did accumulate in the recirculating perfusate, the quantity was not sufficient to have an important role in the cyto c reduction. We conclude that most of the cyto c reducing capacity within the lung perfusate could be accounted for by ascorbate released from the lungs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ventilation-perfusion relationships in isolated blood-free perfused rabbit lungs.

The multiple inert gas elimination technique (MIGET) was applied to blood-free perfused isolated rabbit lungs. Commonly accepted criteria for reliability of the method were found to be fulfilled in this model. Ventilation-perfusion (VA/Q) distributions in isolated control lungs corresponded to those repeatedly detected under physiological conditions. In particular, a narrow unimodal dispersion of perfusate flow was observed: perfusion of low-VA/Q areas ranged below 1% and shunt flow approximately 2-3%; perfusion of high-VA/Q regions was not detected. Gas flow was characterized by narrow dispersion in the midrange-VA/Q areas. Application of a low level of PEEP (1 cmH2O) reduced shunt flow to less than 1%, and low-VA/Q areas were no longer noted. By using this PEEP-level, stable gas exchange conditions were maintained for greater than 5 h of extracorporeal perfusion. Graded embolization with small air bubbles caused a typical rightward shift (to higher VA/Q ratios) of mean ventilation, associated with the appearance of high-VA/Q regions and an increase in dead space ventilation. Mean perfusion was shifted leftward, and shunt flow was approximately doubled. Whole lung lavage with saline for washout of surfactant evoked a progressive manifold increase in shunt flow, accompanied by a moderate rise of perfusate flow to low-VA/Q areas. We conclude that the MIGET can be applied to isolated blood-free perfused rabbit lungs for assessment of gas exchange and that typical patterns of VA/Q mismatch are reproduced in this model.