Sarcolemma-enriched fractions were isolated from skeletal muscles of normal and dystrophic mice. Comparison between the two muscles was possible because almost equivalent subcellular fractions were obtained with respect to the distribution of marker enzymes. Unusual findings in the dystrophic sarcolemma include a marked decrease in the specific activity of 5’-nucleotidase, and an alteration in immunoreactivity of NaKATPase. Also, quantitative changes in some glycoproteins were seen in the dystrophic sarcolemma; disappearance of a glycoprotein with an apparent molecular mass of 30 kDa was the most noteworthy finding.
The effect of environmental tonicity on the interaction between arginine vasopressin (AVP) and prostaglandin (PG) E in the production of adenosine-3’,5’-cyclic monophosphate (cAMP) was studied in cultured rat renal papillary collecting tubule cells. Both AVP and PGE2 stimulated intracellular cAMP accumulation in a dose-dependent manner in an isotonic medium of 300 mOsm/kg H2O in the presence of a phosphodiesterase inhibitor. Environmental tonicity modulated the effect of AVP, PGE, or of both, on cellular cAMP production. The hypertonic medium with 1,800 mOsm/kg H2O enhanced the effect of 1×10-9 M AVP on the cellular cAMP production 1.6-fold, but markedly reduced the effect of 2×10-8 MPGE1 or PGE2 to one-sixth or one-third of that in the isotonic medium. The inhibitory effect of PGE1 and PGE2 on the action of AVP was not shown in the isotonic medium, but was seen in the hypertonic medium of 1,800 mOsm/kg H2O. Synthesis of[3H]PGE2 was enhanced by hypertonicity, but not by AVP, when [3H]arachidonic acid was incorporated in the medium. The results indicate that an environmental tonicity is an important factor in modulating the effect of PGE1 or PGE2 on AVP-mediated cAMP production in renal papillary collecting tubule cells.
Ca2+ storage by vesicles in human lymphocytes was studied using saponin-permeabilized cells. Saponin-treated lymphocytes could accumulate Ca2+ in the presence of ATP. In the presence of NaN3 (10 mM), the maximal storage of Ca2+ (0.87 nmol/4X106 cells) was obtained at free Ca2+ concentration of 10-5 M, and the apparent affinity constant for Ca2+ was approximately 1.4×106 M-1. Stored Ca2+ was rapidly released by A23187. Ca2+ accumulation was markedly enhanced by oxalate, and precipitates of Ca-oxalate were electron-microscopically observed inside the endoplasmic reticulum-like structures. In the absence of NaN3, the maximal amount of intracellular Ca2+ was about 30 nmol/4×106 cells, while the affinity for Ca2+ was much lower. Application of inositol 1,4,5-trisphosphate to saponin-permeabilized cells which had accumulated Ca2+ by ATP in the presence of NaN3 rapidly released 35% of stored Ca2+ within 1 min. However, when the concentration of free Ca2+ was higher than 1.5×10-6 M, Ca2+ release by this agent was inhibited. The results suggest that inositol 1,4,5-trisphosphate may function as a mediator in lymphocytes, linking activation of surface membrane receptors with mobilization of internal Ca2+, which plays an important role in lymphocyte responses involved in proliferation.
An in vitro model of connective tissue was reconstituted by introducing fibroblasts into a three-dimensional space of the hydrated collagen lattice. The model was maintained in culture to investigate morphological and biochemical changes in collagen fibrils. Evidence was obtained that the collagen fibrils in the model were stabilized on one hand and were metabolized on the other hand. Collagen fibrils were contracted and were reorganized by the cells in an early stage of culture. Collagen fibrils in the freshly prepared model were easily hydrolyzed by bacterial collagenase. The fibrils became resistant to the enzyme digestion during culture. The acquired resistance seemed to be partly due to the formation of a complex of collagen with polysaccharides secreted by the cells. The amount ofcollagen in the model decreased gradually during culture and a hydroxyproline-containing substance or substances accumulated in the medium, indicating that fibroblasts catabolized the collagen fibrils. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis demonstrated that the collagen was degraded to a peptide or peptides with molecular weights less than 20 K. The electron microscopical examinations of the model revealed some bizzare bodies with a striated banding pattern, which resembled the zebra-bodies reported in vivo. The origin of this striated structure is discussed.
Male golden hamsters were exposed gradually to low temperatures and short photoperiods in a climatic chamber, and kept finally in an environment of 5°C and 3 h light/21 h darkness (3L/21D) for more than 5 weeks. Control animals were maintained at 20°C and 14L/10D or 12L/12D. The following changes were induced in animals kept under the artificial winter conditions. 1) Pinealocytes showed proliferation of the smooth- and rough-surfaced endoplasmic reticulum (sER and rER) and marked development of the Golgi apparatus, indicating an enhanced cellular function. Secretory granules were increased in cell processes, and lipid droplets and myeloid bodies were frequently observed. 2) LH-RH-positive neurons in the brain were immunostained more numerously than in controls, in agreement with a significant increase in hypothalamic LH-RH demonstrated by radioimmunoassay. 3) Pituitary gonadotrophs showed signs of suppressed secretory activity, such as accumulation of granules, whereas mammotrophs showed signs of activation including a marked development of the rER and Golgi apparatus. 4) Testes and accessory sex organs exhibited significant atrophy, whose weight was decreased to 13% and to 39% of that in the respective controls. It was assumed that pineal hormones secreted excessively under the experimental winter condition inhibited the discharge of hypothalamic LH-RH, resulting in a suppression of gonadotropin release and subsequent gonadal atrophy.
Chromosome condensation and nuclear membrane breakdown were induced in cultured chick myotubes by exposure to a solution containing 50 mM β-glycerophosphate. These changes were effectively induced only when the Ca2+ concentration in the solution was reduced below several micromolar level. The chromosome condensation proceeded in the presence of cycloheximide (1 mg/ml), but was partially inhibited by ethidium bromide (10 μg/ml). Mononucleated cells in the myogenic culture also underwent the chromosome condensation when exposed to the solution containing β-glycerophosphate. Thus, the chromosome condensation in multinucleated skeletal muscle cells was chemically induced for the first time. The results suggest that the chromosomecondensing mechanism is preserved, but is not functioning in myotubes, which never enter the mitotic state under the physiological condition. A possible mechanism of the β-glycerophosphate-induced chromosome codensation was discussed.
Messenger RNA (mRNA) for Ca2+-activated neutral protease (CANP) in chicken tissues and its translation product were identified. CANP mRNA was 3.5 kilobase-long, and the molecular weight of the primary translation product was about 80,000 which was indistinguishable from that of mature CANP. The lung had the highest content of CANP mRNA, whereas its content in the liver was low. Ubiquitous occurrence of CANP mRNA in chicken tissues indicates its essential role in cellular functions.
Spot 35 protein (S 35) is a soluble protein which has been shown to occur specifically in Purkinje cells. The present study deals with immunohistochemical demonstration of S 35 in the sensory organs of guinea pigs. The S 35-like immunoreactivity was recognized in gustatory cells, hair cells in the organ of Corti and olfactory receptor cells. In the retina, horizontal neurons stained positively, but rods and cones were negative in reaction. Since we have demonstrated an extensive distribution of S 35 in peptidic endocrine cells, it is suggested that many, if not all, paraneurons of both endocrine and sensory nature share this protein with cerebellar Purkinje cells.
Glucose tolerance in alloxan-diabetic rats was significantly improved by intraperitoneal administration of 2-aminomethyl-4-tert-butyl-6-propionylphenol (ONO-3144), a new free radical scavenger. Also, hyperglycemia and glucosuria which usually develop in 80% of female non-obese diabetic mice in adulthood were not observed when ONO-3144 had been orally given to mice after the age of 5 weeks.
Perikarya of bovine spinal ganglion cells were dissected out from freeze-dried sections and their protein composition was examined by two-dimensional gel electrophoresis and immunoblotting. New components which reacted with an antibody against the axonal 220 K neurofrlament protein were found as two skewed bands on the gel. They showed high pI and low molecular weight compared with the axonal counterpart. In the previous work (14), we found the following neurofilament proteins in perikarya: a 150 K protein and a group of proteins ranging from 150 K to 170 K, in addition to a trace amount of the 170 K component. Thus two of the three neurofilament proteins, high and intermediate molecular weight components, are different between axons and perikarya. Post-translational modifications of neurofilament proteins may explain the differences.
The stoichiometric concentration of cytochalasin D is known to enhance the initial rate of actin polymerization. At the initial stage of polymerization, the number of actin filaments formed in the presence of cytochalasin D was larger and the average length was shorter than in its absence. The length distribution of F-actin formed under the influence of stoichiometric concentrations of cytochalasin D was somewhat of the Poisson type in contrast to the exponential distribution seen in the absence of this agent. These observations support the view that cytochalasin D causes a rapid formation of nuclei at the initial stage of actin polymerization.
OK-432, a streptococcal preparation, induced a marked increase in the number of rodcored vesicles in pit cells (natural killer cells) of the rat liver. OK-432 enhanced also the liver-associated natural killer activity. OK-432 injected intravenously was incorporated into lysosomes of Kupffer cells. Kupffer cells and pit cells were increased in their number and were in wide contact with each other. Prominent Golgi complex and many rodcored vesicles characterized the actively secreting pit cells. No obvious morphological changes were detected in dense granules in pit cells. Itissuggested that the increment of rod-cored vesicles induced by OK-432 is responsible for the augmentation of the liverassociated natural killer activity.