Developmental changes in the effects of carbachol and morphine on cGMP contents of plasma, heart, and lung of mice

It is considered that carbachol increases plasma cGMP levels by acting on muscarinic receptors and morphine increases these levels by acting on opioid receptors, followed by stimulation of muscarinic receptors. We investigated the ability of carbachol and morphine to increase cGMP contents of plasma, heart, and lung and the guanylate cyclase activity of heart and lung homogenate in 1-, 2-, 3-, and 7-week-old mice. The increase in plasma cGMP levels induced by carbachol showed a peak at 2 and 3 weeks of age. The basal cGMP contents in heart and lung and their rise induced by carbachol, as well as the guanylate cyclase activity of these organs, were decreased in 7-week-old mice. The effects of morphine on the cGMP contents showed a similar developmental change, except for no effect in 1-week-old mice. These changes in the effects of carbachol and morphine may be the result of developmental changes of the muscarinic receptor--guanylate cyclase system and opioid receptors.     

Evidence for age-related change in plasma 19-hydroxyandrostenedione

The steroid, 19-hydroxyandrost-4-ene-3, 17-dione (19-hydroxyandrostene-dione, 19-OH-A-dione) has been known to enhance the mineralocorticoid action of aldosterone. To investigate the age-related change in the plasma 19-OH-A-dione concentration, plasma 19-OH-A-dione, androst-4-ene-3, 17-dione (A-dione), aldosterone and cortisol of 38 non-hypertensive healthy subjects (18 young men and 20 aged men) measured by specific radioimmunoassays. The basal plasma 19-OH-A-dione and A-dione concentration in aged men was significantly lower than in young men (P less than 0.01). Moreover, there was found to be a positive correlation between plasma 19-OH-A-dione and A-dione (P less than 0.01). On the other hand, plasma aldosterone and cortisol in aged men showed a tendency to decrease, but no statistical significance compared to young men was observed. This study demonstrated that there was an apparent age-related decrease not only in plasma A-dione, but also in plasma 19-OH-A-dione, an amplifier or aldosterone action.     

Calyculin A and okadaic acid: inhibitors of protein phosphatase activity

Calyculin A and okadaic acid induce contraction in smooth muscle fibers. Okadaic acid is an inhibitor of phosphatase activity and the aims of this study were to determine if calyculin A also inhibits phosphatase and to screen effects of both compounds on various phosphatases. Neither compound inhibited acid or alkaline phosphatases, nor the phosphotyrosine protein phosphatase. Both compounds were potent inhibitors of the catalytic subunit of type-2A phosphatase, with IC50 values of 0.5 to 1 nM. With the catalytic subunit of protein phosphatase type-1, calyculin A was a more effective inhibitor than okadaic acid, IC50 values for calyculin A were about 2 nM and for okadaic acid between 60 and 500 nM. The endogenous phosphatase of smooth muscle myosin B was inhibited by both compounds with IC50 values of 0.3 to 0.7 nM and 15 to 70 nM, for calyculin A and okadaic acid, respectively. The partially purified catalytic subunit from myosin B had IC50 values of 0.7 and 200 nM for calyculin A and okadaic acid, respectively. The pattern of inhibition for the phosphatase in myosin B therefore is similar to that of the type-1 enzyme.     

Cytochrome P450 in livers of diabetic rats: regulation by growth hormone and insulin

The effects of pituitary and pancreatic hormones on the change in hepatic cytochrome P450s were studied in alloxan- or streptozotocin-induced male rats. In two major sex-specific forms, P450-male and P450(6 beta-1), the former was decreased in chronic (5 week) diabetes to only less than one-third of controls and the latter was also reduced in early (1 week) diabetes. In contrast, a main phenobarbital-inducible form, P450b, was enhanced 25- to 30-fold in these diabetic rats. 3-Methylcholanthrene-inducible P448H was also elevated 3-fold in alloxan-induced diabetes. These changes in hepatic contents of P450-male, P450-6 beta-1, and P450b, which are under the regulation of pituitary growth hormone, associated well with the reported results of time-dependent changes in growth hormone levels in diabetes (G.S. Tannenbaum (1981) Endocrinology 108, 76-82), suggesting that the change in growth hormone level is a factor responsible for alterations in hepatic cytochrome P450s. Normalizing effects of insulin on these forms were also studied. Treatment of diabetic rats with insulin reversed the decreased amounts of both P450-male protein and mRNA. Insulin also normalized hepatic contents of P450b, P4506 beta-1, and P448H. However, the treatment of hypophysectomized rats with insulin had no effect, and treatment of diabetic rats with growth hormone or a suppressing agent of somatostatin, cysteamine, showed trivial effects on P450-male and P450b. These results suggest that insulin does not act directly as a substitute of growth hormone, but exerts its effect indirectly through the normalization of a growth hormone-mediated process(es) in diabetic rats.     

Quantitation and immunohistochemical localization of cathepsins E and D in rat tissues and blood cells

The distribution of cathepsins E and D in various rat tissues and blood cells was determined by immunoprecipitation and by immunohistochemistry with discriminative antibodies specific for each enzyme. While cathepsin D was detected in all of the tissues and blood cells tested (except for erythrocytes), cathepsin E had a relatively limited distribution. The cathepsin E content was highest in the stomach and was succeeded in the following order by the urinary bladder, thymus, spleen, cervical lymph node and bone marrow. Significant amounts of cathepsin E were also found in the colon, rectum, jejunum, skin, lung, kidney and submandibular gland. The other tissues tested had little or no detectable cathepsin E content. Of the blood cells tested, lymphocytes and peritoneal neutrophils contained high levels of cathepsin E. Erythrocytes had cathepsin E only as aspartic proteinases. When the subcellular localization of cathepsin E in the neutrophils was investigated by fractionation of the postnuclear supernatants, the enzyme behaved as a soluble cytosolic enzyme. In contrast, cathepsin D was mainly associated with the granular fraction. The immunohistochemical localization of cathepsins E and D was clearly different in the stomach, large intestines, kidney and urinary bladder, but was similar in the lymph node and spleen. The tissue-fixed macrophages, which were notable in the skeletal and cardiac muscle tissues, submucosal layers of the gastrointestinal tracts, salivary gland, lung and trachea, also exhibited similar intense immunoreactivities demonstrative of both cathepsins E and D.     

Radioimmunoassay for non-enzymatically glycated serum proteins

We have developed a radioimmunoassay (RIA) for nonenzymatically glycated serum proteins. The polyclonal antibodies prepared against reduced glycated human albumin were specific for the glucitollysine residues of serum proteins. Serum proteins from diabetic patients (n = 25) contained 5.3 +/- 2.8 nmoles of glucitollysine/mg protein, compared to 2.0 +/- 0.2 in controls (n = 20). The intra- and inter-assay variables were 3.2-6.2% and 4.4-8.6%, respectively. Results from this assay procedure correlated well with those from the boronate affinity chromatography procedure (r = 0.94; P less than 0.001). The data suggested that diabetic serum proteins contained at least 2.5 times as much immunochemically detectable glucitollysine residures as normal serum proteins after reduction of the proteins with sodium borohydride.     

Carboxyl-terminal deletion analysis of the Streptococcus mutans glucosyltransferase-I enzyme

Sequential deletion of the carboxyl-terminal amino acids (including the six direct repeating units) of the glucosyltransferase-I (GTF-I) enzyme of Streptococcus mutans revealed differential effects on sucrase and GTF activities. Removal of all but one repeating unit resulted in a truncated enzyme with significant sucrase activity but no detectable GTF activity. These results are compatible with the presence of two functional domains in the enzyme.     

Anomalous secondary vascular tissue inHelminthostachys zeylanica (Ophioglossaceae)

The vascular anatomy ofHelminthostachys zeylanica was examined with special reference to anomalous secondary tissue. Primary xylem development gradually takes place centrifugally. In branched rhizomes with destroyed apices, the vascular cylinder apical to the insertion of branch traces is generally composed of primary xylem, accessory xylem, inner parenchyma of radially arranged cells, outer parenchyma of irregularly arranged cells, and partly crushed phloem, listed in order going outwards. The accessory xylem as well as the inner parenchyma ofHelminthostachys zeylanica is probably secondarily produced, partly to contribute to the branch traces, in a position corresponding to that of secondary vascular tissue developed from a normal cambium inBotrychium sensu lato. It is suggested that although a cambium is lacking inHelminthostachys zeylanica, the secondary vascular tissues are comparable between the genera. The phylogenetic implication of this tissue is discussed.     

The polymerase chain reaction for Mycoplasma pulmonis

In vitro DNA amplification by polymerase chain reaction was examined to detect Mycoplasma pulmonis. A pair of synthetic oligonucleotide primers was constructed, and used to amplify a unique sequence of M. pulmonis DNA. Amplified products were detected by agarose gel electrophoresis and verified by blot hybridization with a synthetic oligonucleotide probe. This system detected cellular DNA of M. pulmonis but not M. arthritidis or M. neurolyticum, and thus appears to be useful for M. pulmonis diagnosis.     

Heterocyclic amine-DNA adducts analyzed by 32P-postlabeling method

DNA adducts formed by 12 heterocyclic amines were analyzed by 32P-postlabeling method. Several DNA adducts were detected in rat liver by administration of each heterocyclic amine. Total adduct levels ranged from 0.5 for 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to more than 250 for 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) per 10(7) nucleotides 24 hr after intragastric administration of these compounds. The N-hydroxy derivative of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was reactive toward DNA in vitro to form adducts. Addition of acetic anhydride to N-OH-MeIQx greatly enhanced its reactivity to DNA. 32P-Postlabeling analysis revealed that the MeIQx-DNA adducts formed in vivo and in vitro were identical. Thus, MeIQx would be metabolized in vivo to N-hydroxy form and further esterified to produce more reactive species, such as N-acetoxy form, which modify DNA to form adducts.