Demonstration of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes by monoclonal antibodies

Calmodulin-dependent cyclic nucleotide phosphodiesterase from bovine brain is found to be composed of two distinct subunits, 60,000- and 63,000-dalton polypeptides. Peptide mapping of the subunits by partial proteolysis demonstrated that the 60-kDa polypeptide is not derived from the 63-kDa species. The interaction of the enzyme with three monoclonal antibodies, A6, C1, and A2, and the analysis of immunocomplexes by sucrose density gradient centrifugation revealed that calmodulin-dependent cyclic nucleotide phosphodiesterase exists in three different forms, i.e. (a) homodiamer of 60-kDa, (b) heterodimer of 60- and 63-kDa, and (c) homodimer of 63-kDa. A6 antibody reacts with both 60- and 63-kDa polypeptides indicating that they are immunologically related. C1 and A2 antibodies react with only 60-kDa polypeptide species. By using C1 Sepharose 4B affinity column chromatography, the 63-kDa homodimer which did not bind to the column (Fraction I) was separated from the 60-kDa polypeptide containing isozymes (the heterodimer and the 60-kDa homodimer) which were retained on the column and later eluted as a mixture (Fraction II). Fraction I, the 63-kDa homodimer enzyme, has higher Vmax toward cGMP as substrate than cAMP whereas the opposite was found with Fraction II. The specific activity of Fraction II enzyme toward cAMP was approximately 500 mumol/min/mg, the highest value ever reported for brain calmodulin-dependent cyclic nucleotide phosphodiesterase preparations.     

Identification of the native form of chicken gizzard myosin light chain kinase with the aid of monoclonal antibodies

Examination, by immunoblotting, of myosin light chain kinase-containing fractions obtained during purification of the enzyme from chicken gizzard has shown that a single species (Mr = 136,000) exists in the muscle and that this enzyme is degraded, primarily to a 130,000-dalton fragment, during purification. These conclusions were confirmed by phosphorylation of the different species of myosin light chain kinase by the isolated catalytic subunit of cyclic AMP-dependent protein kinase.     

Dielectric constant of sickle cell hemoglobin. Dielectric properties of sickle cell hemoglobin in solution and gel

The dielectric constants of sickle cell hemoglobin were determined before and after gelation. The dielectric properties of oxy and deoxy sickle cell hemoglobin in solution are nearly identical to those of oxy and deoxy hemoglobin A. Only in the gel state did deoxy sickle cell hemoglobin display dielectric behavior different from that in solution. Upon gelation of deoxy sickle cell hemoglobin, the dielectric constant showed a marked decrease, and the relaxation frequency shifted towards higher frequencies. This result suggests that dielectric constant measurement can be used for the investigation of the kinetics of polymerization of sickle cell hemoglobin molecules. Despite the marked decrease in the dielectric constant, deoxy sickle cell hemoglobin still showed a well-defined dielectric dispersion even in the gel state. This indicates that individual molecules have considerable freedom of rotation in gels. It was observed that the dielectric properties of gelled deoxy sickle cell hemoglobin were affected by electrical fields at the level of 10 to 20 V/cm. This observation suggests that electrical fields of moderate strengths are able to perturb the gel structure if the system is near the transition region. The non-linear electrical behavior of gelled sickle cell hemoglobin will be discussed further in subsequent papers.     

Fluctuation in the development of various skeletal muscles in the chick embryo,with special reference to AChE activity and the formation of neuromuscular junctions

Acetylcholinesterase (AChE)-rich cytoplasmic granules in the developing myofibers increased remarkably until the establishment of neuromuscular junctions and thereafter decreased rapidly, whereas junctional AChE activities continued to increase (K. Wake, 1976, Cell Tissue Res. 173, 383–400). In the present paper, during the developmental course of the chick embryo, the temporal and regional gradients in differentiation of skeletal muscles at various sites were examined with special reference to the fluctuation of intracellular AChE activity. AChE-rich granules in each muscle throughout the whole body of chick embryos were observed. Since the distribution pattern of these granules changed regularly in the course of the muscle fiber development, advances of muscle differentiation in various sites of the body were compared. (1) The process of muscle development is more advanced in the trunk muscles than in the limb muscles. (2) The dorsal trunk muscles differentiate one day earlier than the ventral ones. (3) Within the same limb, proximal muscles differentiate approximately 24 hr ahead of distal ones. (4) The development of posterior limb muscles advances faster than that of anterior limb muscles. (5) Within the thigh muscles, the flexor muscles tend to differentiate earlier than the extensor muscles.     

Synthesis of ribonucleic acid in normal bone in vitro

1. The incorporation of [2-(14)C]uridine into nucleic acids of bone cells was studied in rat and pig trabecular-bone fragments surviving in vitro. 2. The rapid uptake of uridine into trichloroacetic acid-soluble material, and its subsequent incorporation into a crude nucleic acid fraction of bone or purified RNA extracted from isolated bone cells, was proportional to uridine concentration in the incubation medium over a range 0.5-20.0mum. 3. During continued exposure to radioactive uridine, bulk RNA became labelled in a curvilinear fashion. Radioactivity rapidly entered nuclear RNA, which approached its maximum specific activity by 2hr. of incubation; cytoplasmic RNA, and particularly microsomal RNA, was more slowly labelled. The kinetics of labelling and rapid decline of the nuclear/microsomal specific activity ratio were consistent with a precursor-product relationship. 4. Bulk RNA preparations were resolved by zonal centrifugation in sucrose density gradients into components with approximate sedimentation coefficients 28s, 18s and 4s. 5. Rapidly labelled RNA, predominantly nuclear in location, demonstrated a polydisperse sedimentation pattern that did not conform to the major types of stable cellular RNA. Material of highest specific activity, sedimenting in the 4-18s region and insoluble in 10% (w/v) sodium chloride, rapidly achieved its maximum activity during continued exposure to radioactive precursor and decayed equally rapidly during ;chase' incubation, exhibiting an average half-life of 4.3hr. 6. Ribosomal 28s and 18s RNA were of lower specific activity, which increased linearly for at least 6hr. in the continued presence of radioactive uridine. There was persistent but variable incorporation into ribosomal RNA during ;chase' incubation despite rapid decline in total radioactivity of the acid-soluble pool containing RNA precursors.     

Acid hydrolases of the rat uterus in relation to pregnancy, post-partum involution and collagen breakdown

1. The rat uterus contains acid cathepsin, beta-glucuronidase, beta-galactosidase, acid phosphatase and deoxyribonuclease II at concentrations comparable with those found in liver. Two non-hydrolytic uterine enzymes, cytochrome c oxidase and aspartate aminotransferase, display only 2-6% of the activity found in liver. 2. The concentrations of acid cathepsin and beta-glucuronidase are significantly decreased in pregnancy and increase 3-4-fold during post-partum involution. 3. The concentrations of beta-galactosidase and acid phosphatase are not decreased in pregnancy and increase only 2-3-fold during involution. 4. The concentrations of these four acid hydrolases increase linearly during the first 4 days post partum and reach their peak values at the same time that wet weight and collagen content fall to their lowest point. 5. The concentration of deoxyribonuclease is depressed in pregnancy but does not rise above normal in the post-partum period. 6. Only a small proportion of each hydrolytic activity can be isolated in the mitochondrial-lysosomal fraction of sucrose homogenates of the rat uterus. This proportion increases during involution. However, the extensive mitochondrial rupture occurring during homogenization indicates that the technique is probably too harsh to obtain a true measure of the proportion of lysosomes present in the intact tissue. 7. There are no significant changes in either the concentration or subcellular distribution of the five acid hydrolases in the livers of the experimental rats during pregnancy or involution. In each case the largest proportion of the activity is found in the mitochondrial-lysosomal fraction of liver homogenates. 8. The results are interpreted in terms of the lysosomal theory of intracellular digestion.     

Identification of membrane anchoring site of human renal dipeptidase and construction and expression of a cDNA for its secretory form

The chemical properties of human renal dipeptidase (hrDP) purified from the membrane fraction of kidney have been characterized. When treated with phosphatidylinositol-specific phospholipase C, hrDP was released from renal membrane fractions. After digestion with trypsin, carboxyl-terminal peptide was isolated employing anhydrotrypsin-agarose column chromatography and reversed-phase high performance liquid chromatography. The amino acid sequence of the peptide was identified at positions 363-369 in the primary structure deduced from the cDNA sequence (Adachi, H., Tawaragi, Y., Inuzuka, C., Kubota, I., Tsujimoto, M., Nishihara, T., And Nakazato, H. (1990) J. Biol. Chem. 265, 3992-3995). Further examination of the chemical composion of the peptide showed that it contained, respectively, 2, 1, 5, 1, and 1 mol of ethanolamine, glucosamine, mannose, inositol, and phosphate in addition to amino acids. These results suggest that the mature hrDP molecule lacks the carboxyl-terminal hydrophobic peptide extension predicted from the cDNA sequence and is anchored at Ser369 via glycosylphosphatidylinositol to the membrane. To characterize further the action of the enzyme, we have established expression systems for both secretory and membrane anchored forms of hrDP using COS-1 cells and found that both recombinant forms were as active as natural enzyme. Our expression system made it possible to prepare large amounts of soluble enzyme, and will contribute toward elucidation of the physiological roles of the enzyme.     

Regulation of Histamine Turnover via Muscarinic and Nicotinic Receptors in the Brain

To clarify the regulation of central histaminergic (HAergic) activity by cholinergic receptors, the effects of drugs that stimulate the cholinergic system on brain histamine (HA) turnover were examined, in vivo, in mice and rats. The HA turnover was estimated from the accumulation of tele-methylhistamine (t-MH) during the 90-min period after administration of pargyline (65 mg/kg, i.p.). In the whole brain of mice, oxotremorine, at doses higher than 0.05 mg/kg, s.c., significantly inhibited the HA turnover, this effect being completely antagonized by atropine but not by methylatropine. A large dose of nicotine (10 mg/kg, s.c.) also significantly inhibited the HA turnover. This inhibitory effect was antagonized by mecamylamine but not by atropine or hexamethonium. A cholinesterase inhibitor, physostigmine, at doses higher than 0.1 mg/kg, s.c., significantly inhibited the HA turnover. This effect was antagonized by atropine but not at all by mecamylamine. None of these cholinergic antagonists used affected the steady-state t-MH level or HA turnover by themselves. In the rat brain, physostigmine (0.1 and 0.3 mg/kg, s.c.) also decreased the HA turnover. This inhibitory effect of physostigmine was especially marked in the striatum and cerebral cortex where muscarinic receptors are present in high density. Oxotremorine (0.2 mg/kg, s.c.) and nicotine (1 mg/kg, s.c.) also decreased the HA turnover in the rat brain. However, these effects showed no marked regional differences. These results suggest that the stimulation of central muscarinic receptors potently inhibits the HAergic activity in the brain and that strong stimulation of central nicotinic receptors can also induce a similar effect.     

Functional comparison of transactivation by human retrovirus rev and rex genes.

The effect of rev-responsive element deletion on human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) gene expression was examined. The phenotypes of HIV-1 and HIV-2 provirus DNAs lacking the rev-responsive element, as determined by transfection experiments, were indistinguishable from those of virus DNAs carrying rev gene mutations. By using rev-response elements derived from these two viruses, we developed two monitoring systems to evaluate the functionality of HIV-1 rev, HIV-2 rev, and human T-lymphotropic virus type I rex. In both systems, HIV-1 rev and human T-lymphotropic virus type I rex transactivated HIV-2 very efficiently. On the contrary, HIV-2 rev and human T-lymphotropic virus type I rex were poor activators of HIV-1. No functional replacement of rex by HIV-2 rev was observed.