The role of phospholipase in beta-agonist-induced down regulation in guinea pig lungs

It has been observed that repeated and prolonged beta-agonist treatment causes the impairment of beta-adrenergic function, so-called "desensitization" or "down regulation". To clarify the mechanism of down regulation, the following experiment was performed using guinea pig lungs. Animals were divided into four groups: In the metaproterenol groups, guinea pigs were treated with metaproterenol (10 mg/kg/day) by intraperitoneal injection once a day for 1 day or for 7 successive days In the control groups, guinea pigs were treated with saline by the same procedure as in the metaproterenol groups. In the group treated with metaproterenol for 7 days, there was a 45% reduction in the number of beta-adrenoceptors and a 62% reduction in adenylate cyclase activity, compared with those of the control group. However, there were no significant changes in the dissociation constant (Kd) of the receptors. On the other hand, no reduction in the number of beta-adrenoceptors and adenylate cyclase activity was observed in the group treated with metaproterenol once a day for 1 day, compared with those of the control group. Phospholipase (PLase) activity in the lung microsomes of guinea pigs injected with metaproterenol for 1 day and for 7 days was elevated by 14.4 and 33.1%, respectively, compared with that of the control groups. Phospholipid contents of lung membranes prepared from the animals treated with metaproterenol for 7 days were significantly decreased compared with those of the control group, though in the group treated with metaproterenol once a day for 1 day, phospholipid contents did not differ from those of the control. Lung membranes treated with PLase A2 revealed decreases both in the number of beta-adrenoceptors and adenylate cyclase activity, dose dependently. These results and the fact that membrane phospholipids are involved in the beta-adrenoceptor system suggest that down regulation observed during beta-agonist administration is, at least in part, attributed to degradation of phospholipids of lung membranes by the persistent activation of PLase in the tissue.     

A novel proenkephalin processing carboxypeptidase and its activation by cyclic AMP dependent protein kinase

Carboxypeptidase B-like enzymes cleaving Met-enkephalin-Arg from synaptosomes of the rat striatum purified using a DEAE-cellulose column and Met-Arg-CH-Sepharose 4B affinity column proved to be different from enkephalin-convertase, lysosomal carboxypeptidase B-like enzyme, pancreas carboxypeptidase B and carboxypeptidase Y, in effects of inhibitors and activators, pH optimum (7.5-8.5) and molecular size (50,000). This enzyme, named "Processin CP-E" was activated by cAMP dependent protein kinase, and the Vmax was increased from 4.3 to 13.3 microM/min/mg protein, while the Km (28.2 microM) was unchanged.     

A study of cytoplasmic and nuclear estrogen and progestin receptors in gynecologic neoplasms

A rapid method for simultaneous preparation of cytosol and nuclear estrogen (E) and progestin (P) receptors and their in vitro determination is described. The method was applied to several uterine or ovarian surgical specimens to evaluate their steroid hormone "dependence". The results suggest that low cytoplasmic E receptor levels (ERc) are associated with higher nuclear E receptor (ERn) levels but no apparent correlation was observed between PRc and ERn levels. The method appeared to be suitable for screening steroid hormone receptor content in tumor tissues and may provide better estimation of steroid dependence since both cytoplasmic and nuclear compartments can be studied simultaneously.     

Dissociation kinetics of 19 base paired oligonucleotide-DNA duplexes containing different single mismatched base pairs.

The dissociation kinetics of 19 base paired oligonucleotide-DNA duplex containing a various single mismatched base pair are studied on dried agarose gels. The kinetics of the dissociation are first order under our experimental conditions. The incorporation of a single mismatched base pair destabilizes the DNA duplexes to some extent, the amount depending on the nature of the mismatched base pair. G-T and G-A mismatches slightly destabilize a duplex, while A-A, T-T, C-T and C-A mismatches significantly destabilize it. The activation energy for the overall dissociation processes for these oligonucleotide-DNA duplexes containing 19 base pairs is 52 +/- 2 Kcal mol-1 as determined from the slope of Arrhenius plot.     

Cross-reactivity between human and canine helper and suppressor T cell antigens using monoclonal antibodies RPA-T4 and HuLy-m8

The murine monoclonal antibodies RPA-T4 and HuLy-m8, specific for a framework determinant of human helper/inducer and suppressor/cytotoxic T cell antigens, cross-reacted with canine cell membrane molecules recognizing a biomolecular complex (50,000 to 55,000 daltons) similar to that described in humans. We investigated the distribution of these helper and suppressor T cell-like antigens on canine peripheral blood lymphocytes. With complement-mediated lymphocytotoxicity, 34% and 35% of the canine lymphocytes expressed the helper T cell-like antigens and the suppressor-like T cell antigens, respectively. When the lymphocytes were treated with RPA-T4 and HuLy-m8, the respective helper and suppressor function was significantly inhibited.     

The development of porcine zona pellucida using monoclonal antibodies: I. Immunochemistry and light microscopy

We established three monoclonal antibodies (Mabs) against the zonae pellucidae (ZP) of porcine oocytes, named STA-1, STA-2, and STA-3, and eventually we determined that they all reacted with the isolated ZP. Based on Western blotting without 2-mercaptoethanol (2-ME), STA-1 reacted with the 80,000-110,000 Mr component, STA-2 with the 42,000-63,000 Mr component, and STA-3 with the 40,000-80,000 Mr component of ZP. We immunohistochemically specified the components of porcine ZP reactive with the three Mabs during the course of follicular development. Each Mab reacted with both the ZP and the interfollicular cell space (IFCS). One ZP component, reactive with STA-2 and STA-3, was first produced in the primordial follicle and was not found at the cumulus follicle stage, which corresponds to the stage of large antral follicles more than 5 mm in diameter. Another ZP component, reactive with STA-1, was not produced until the secondary follicle stage, and was never found at the antral follicle stage. These results suggest that each ZP component is produced and secreted at a specific stage or stages of folliculogenesis.     

Overt diabetes induced by overeating in neonatally STZ-treated impaired glucose tolerant mice: long-term follow up study

This study has investigated the effect of a long period of overeating on the glycemic control and pancreatic beta-cell function in neonatally streptozocin treated impaired glucose tolerant mice. Neonatally streptozocin (60 mg/kg) treated male ICR mice with 150-200 mg/dl of fed blood glucose levels were divided into two groups at 6 weeks of age. One group was maintained on a cafeteria diet (SZC) and the other on ordinary mouse chow (SZ) until 30 weeks of age. Normal male ICR mice were divided into a cafeteria diet group (CC) and an ordinary chow group (Cont). SZC and CC consumed 134-124% of the caloric intake in SZ and Cont throughout the study. Marked elevation of the fed blood glucose level was observed and the glucose tolerance was progressively impaired in SZC. On pancreas perfusion at 30 weeks of age, insulin secretion to 30 mM glucose in SZC was significantly decreased compared with that in SZ. That in CC was slightly decreased compared with that in Cont. The pancreatic insulin concentration in SZC was significantly less than that in SZ. We conclude that chronic hyperglycemia, induced by the long period of overeating, accelerated the selective loss of beta-cell sensitivity to glucose. Even in normal mice that did not have marked hyperglycemia, insulin secretion to glucose was suppressed, probably by chronic stimulation of the beta-cell due to the long period of dietary excess.     

An aberrant retinal pathway and visual centers in Xenopus tadpoles share a common cell surface molecule, A5 antigen

A monoclonal antibody A5 (MAb-A5), which was raised against Xenopus tadpole tectal cells, recognizes a cell surface-related protein molecule (A5 antigen) expressed on the visual centers of Xenopus tadpoles (S. Takagi, T. Tsuji, T. Amagai, T. Takamatsu, and H. Fujisawa, 1987, Dev. Biol. 122, 90-100). The present immunohistochemistry using MAb-A5 indicated that, in addition to the visual centers, A5 antigen was expressed on the general somatic sensory tract in the medulla and spinal cord of Xenopus tadpoles. As the general somatic sensory tract has been shown to be a pathway for ectopically transplanted retinal axons (M. Constantine-Paton and R. R. Capranica, 1976, J. Comp. Neurol. 170, 17-32; M. J. Katz and R. J. Lasek, 1979, J. Comp. Neurol. 183, 817-832), we examined whether retinal axons transplanted close to the spinal cord or medulla preferentially grow into the A5 antigen-positive general somatic sensory tract. We performed eye transplantation at embryonic stages and detected precise locations and trajectories of transplanted retinal axons within the medulla and spinal cord in tadpoles after filling retinal axons with horseradish peroxidase (HRP). HRP histochemistry in combination with MAb-A5 immunohistochemistry indicated that almost all HRP-filled transplanted retinal axons joined the A5 antigen-positive general somatic sensory tract. These findings suggest the involvement of A5 antigen in specific cell-cell recognition between retinal axons and their targets.     

Effects of Tween 80 and sodium fluoride on extracellular glucosyltransferase production and membrane lipids of Streptococcus mutans

1. Both Tween 80 and sodium fluoride significantly enhanced total extracellular glucosyltransferase activities of Streptococcus mutans. 2. Water-insoluble and water-soluble glucan formation were uniformly increased by Tween 80, whereas fluoride stimulated only water-soluble glucan formation. 3. Elevated glucan formation was due to an increase in enzymes secreted from bacterial cells. 4. Fatty acid composition and phospholipid content in bacterial membrane were changed by Tween 80, although sodium fluoride scarcely showed these changes. 5. Comparative results suggest that modulation of membrane lipids participates in mutansucrase production but not in dextransucrase production of S. mutans.     

Amino acid sequence of the calcium-binding light chain of myosin from the lower eukaryote, Physarum polycephalum

We have established a new method for preparing Physarum myosin whose actin-activated ATPase activity is inhibited by micromolar levels of Ca2+. This Ca2+-inhibition is mediated by the Ca2+ binding to the myosin rather than by the Ca2+-dependent modification of the phosphorylated state of the myosin (Kohama, K., and Kendrick-Jones, J. (1986) J. Biochem. (Tokyo) 99, 1433-1446). Ca2+-binding light chain (CaLC) has been suggested to be primary importance in this Ca2+ inhibition (Kohama, K., Takano-Ohmuro, H., Tanaka, T., Yamaguchi, T., and Kohama, T. (1986) J. Biol. Chem. 261, 8022-8027). The amino acid sequence of CaLC was determined; it was composed of 147 amino acid residues and the N terminus was acetylated. The molecular weight was calculated to be 16,131. The homology of CaLC in the amino acid sequence with 5,5'-dithiobis-(2-nitrobenzoic acid) light chain and alkali light chain of skeletal muscle myosin were rather low, i.e., 25% and 30%, respectively. Interestingly, however, the CaLC sequence was 40% homologous with brain calmodulin. This amino acid sequence was confirmed by sequencing the cloned phage DNA accommodating cDNA coding CaLC. Northern and Southern blot analysis indicated that 0.8-kilobase pair mRNA was transcribed from a single CaLC gene. This is the first report on the amino acid sequence of myosin light chain of lower eukaryotes and nucleotide sequence of its mRNA.