Roles of dopa decarboxylase and phenoloxidase in the melanization of the tobacco hornworm and their control by 20-hydroxyecdysone

Summary WhenManduca sexta larvae are allatectomized 5 h before head capsule slippage (HCS) in the final larval molt, the new larval cuticle contains granules that melanize 3 h before ecdysis when the ecdysteroid titer falls (Curtis et al. 1984). In both the epidermis and hemolymph of these allatectomized larvae dopamine was higher than dopa prior to and at the time of melanization. Dopamine also increased in the new cuticle as melanization began. Dopa decarboxylase (DDC) activity increased in the epidermis, cuticle, and fat body beginning 16 h after HCS, with a two-fold greater increase in the epidermis of allatectomized larvae. Both -MDH and -fluoromethyl-dopa inhibited epidermal DDC activity and inhibited melanization in vitro when dopa was used as a precursor. Addition of dopamine to the medium allowed melanization in the presence of the inhibitors. All these results indicate that dopamine is likely the primary precursor of cuticular melanin. The diphenoloxidase in the premelanin granules was activated in vivo between 19 and 21 h after HCS and was found to prefer dopamine to dopa and not to convert tyrosine to melanin. The activation of the prophenoloxidase was inhibited by 20-hydroxyecdysone (20-HE), both in vivo and in vitro, if hormone was given by 16 h after HCS. Infusion of 1.2 g/ml 20-HE into allatectomized larvae for 24 h from HCS prevented both the increase in DDC activity and the activation of the premelanin granules. Although the larvae ecdysed after a 15 h delay, melanization never occurred.Abbreviations -MDH L-3-(3,4 dihydroxyphenyl)-2-hydrazine-methylpropionic acid - -FM-dopa R-S--fluoromethyl-dopa - DCC dopa decarboxylase - 20-HE 20-hydroxyecdysone - JH juvenile hormone - HCS head capsule slippage     

Plasma levels of 13,14-dihydro-15-keto prostaglandin F2 alpha, estrogens, and progesterone during stretch-induced labor at term

The uterus of six healthy multiparous women at term was mechanically stretched by a rubber catheter and balloon. Apparent labor was inaugurated in all cases within 5 hours and increased progressively with time. Advanced cervical softening and dilatation were also evident after the stretch treatment. Significant increases in the levels of 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) were observed with the progress of treatment (P less than 0.01). Plasma estrogens and progesterone levels did not change significantly during the treatment (P greater than 0.05). Stretching and/or resulting uterine contractions appear to induce the secretion of prostaglandin F2 alpha (PGF) from the organ, which in turn seems to be involved in both cervical softening, and the onset and progress of labor, under stable conditions of plasma estrogens and progesterone.     

General kinetic model for protein-mediated phospholipid transfer between membranes

Phospholipid transfer protein catalyzes the transfer of phospholipids between bilayer membranes. A general model is developed for describing the kinetics of this process. While previous models derive detailed expressions only for the initial rate of transfer from donor to acceptor membranes, this model takes into account donor-to-donor, acceptor-to-acceptor, and acceptor-to-donor transfers, in addition to the usual donor-to-acceptor transfer. The apparent rate of transfer along any of these specific routes is given as the product of the total rate of transfer (the sum of the rates of transfer along all four routes) and a probability function uniquely defined for each route. The model explains adequately the effects of membrane concentration on phospholipid transfer activity as well as the consequences of varying membrane surface charge and size. Using bovine liver phosphatidylcholine transfer protein, the model is applied to the kinetic analysis of phosphatidylcholine transfer between two populations of small unilamellar vesicles. Rates of protein-catalyzed phosphatidylcholine transfer between vesicles with identical phosphatidic acid content (2 or 6 mol%) are determined experimentally as a function of total vesicle concentration to calculate apparent dissociation constants and maximum rates of transfer; apparent rates of transfer between various combinations of vesicles containing 2 or 6 mol% phosphatidic acid are then deduced from the derived velocity expression. Reasonably good agreement is seen between theoretical apparent rate-vesicle concentration relationships and those measured experimentally. The results support the general treatment of the kinetics of protein-mediated phospholipid transfer and permit an estimation of useful kinetic parameters.     

Oxygenation mechanism in the oxidation of xenobiotic aldehyde to carboxylic acid by mouse hepatic microsomes

11-Oxo-delta 8-tetrahydrocannabinol was oxidized to delta 8-tetrahydrocannabinol-11-oic acid by mouse hepatic microsomes. The oxygenation mechanism in the reaction was confirmed by the incorporation of oxygen-18 from molecular oxygen into delta 8-tetrahydrocannabinol-11-oic acid. The oxygenation of aldehyde to carboxylic acid represents a novel mechanism in biological oxidation of aldehyde to carboxylic acid.     

Evidence for Disulfide Bonds in Membrane-Bound and Solubilized Opioid Receptors

The effects of pretreatment with dithiothreitol (DTT) on opioid binding activities of membrane-bound and digitonin-solubilized opioid receptors from bovine adrenal medulla were studied. Pretreatment of membranes with DTT or mercaptoethanol inhibited [3H]diprenorphine binding by reducing the number of binding sites. The inhibitory action of DTT was time and dose dependent. The binding of [3H]D-Ala2-D-Leu5-enkephalin was also inhibited by DTT pretreatment. Pretreatment of digitonin-solubilized binding sites with DTT also reduced the number of [3H]diprenorphine binding sites. The action of DTT was diminished by preincubating the DTT solution with H2O2. [3H]Diprenorphine protected the opioid binding sites from the inhibitory action of DTT. The present results provide evidence that disulfide bonds are implicated in opioid binding activity of the opioid receptor system.     

Cloning and sequencing of a cDNA encoding ascorbate peroxidase fromArabidopsis thaliana

A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage gt11 library of cDNA fromArabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced. The cDNA insert hybridized to a 1.1 kb poly(A)⁺ RNA from leaves ofA thaliana. Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene. The N-terminal amino acid sequence ofArabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach. The predicted amino acid sequence of the mature AP ofA. thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochromec peroxidase of yeast, but less homologous with other plant peroxidases. Amino acid residues at the active site of yeast cytochromec peroxidase are conserved in the amino acid sequence ofArabidopsis AP. The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3 untranslated region of the cDNA.     

By-product runaway evolution by adaptive mate choice: A behavioural aspect of sexual selection

Summary From a behavioural perspective on adaptive female choice, I developed a by-product runaway model of adaptive mate choice. The model illustrates the evolution of the tail size of peacocks. I consider the causal mechanisms of adaptive female choice: (1) why (ultimate reasons); (2) how (proximate mechanisms). Assumptions are developed based on these behavioural aspects. For (1) ultimate reasons, I assume that many male losers (low-fitness males) always occur due to genetic and environmental uncertainty (A-1). For (2) proximate mechanisms, I assume that losers tend to differ in the expression of a fitness-sensitive trait (an ultimate target, e.g. body size; A-2), that the fitness-sensitive trait correlates with a secondary sexual trait (a proximate cue, e.g. allometry in body size and tail size; (A-3), and that the cue trait has a genetic basis that is independent of the target trait (e.g. a genetic basis in tail ratio to body size; A-4). The model's results are: persistent female choice by means of a proximate cue (R-1); by-product selection on the independent genetic basis of the cue (R-2); and the non-adaptive or maladaptive runaway evolution of the male proximate cue (R-3). In this model, female mate preferences are non-arbitrary and adaptive, whereas the resulting evolution of male secondary sexual traits is non-adaptive in the sense of survival selection.     

In vitro transfection of fresh thymocytes and T cells shows subset-specific expression of viral promoters.

We describe conditions under which exogenous DNA templates can be introduced for transient expression into primary murine T lymphocytes. T cells at various stages of development, including concanavalin A-activated splenic T cells, immature pre-T cells, and even small cortical thymocytes, could be successfully transfected. A variety of model DNA constructs were compared in which different viral promoter regions were used to drive expression of the chloramphenicol acetyltransferase (CAT) reporter gene. All showed enhanced expression in cells that had been acutely stimulated with the Ca2+ ionophore A23187 and phorbol ester as chemical proxies for T-cell receptor-mediated signals. In addition, splenocytes but not thymocytes required prior treatment with a mitogen and interleukin-2 in order to express these constructs, implying that even postmitotic thymocytes may be held in a quasiactivated state. A most striking result was the finding that the viral regulatory sequences in the Rous sarcoma virus long terminal repeat and the simian virus 40 early region were subject to sharply differential regulation, with a rank order that changed depending on the developmental stage of the T cells. The most immature thymic blasts and several lymphoma cell lines expressed the pRSV-Cat and pSV2-Cat constructs similarly, but cortical thymocytes exhibited a strong preference for pSV2-Cat. Splenic concanavalin A-stimulated blasts, on the other hand, slightly preferred pRSV-Cat, a tendency which became exaggerated in factor-dependent T-cell lines. The ratio of pRSV-Cat to pSV2-Cat expression varied according to cell type by as much as 500-fold. These results argue against a trivial linkage of promoter preference to cell cycle status but instead provide evidence that activation of T cells at distinct stages of differentiation results in the expression of different ensembles of nuclear regulatory proteins. In contrast to the simian virus 40 and Rous sarcoma virus promoter regions, the long terminal repeats of the retroviruses mink cell focus-forming virus and Akv were expressed well in all primary T-lineage cells. Thus, they represent excellent model promoters for engineering developmental stage-independent expression of exogenous genes in murine T cells.     

A case of Graves' disease with false hyperthyrotropinemia who developed silent thyroiditis.

We encountered a patient who developed silent thyroiditis during the course of Graves' disease. The diagnosis of silent thyroiditis was made on the basis of a low thyroidal 131I uptake, no response to the thyrotropin releasing hormone (TRH) test, and subsequent hypothyroidism despite the presence of high titers of thyrotropin (TSH) receptor antibody (TRAb) and thyroid stimulating antibody (TSAb). The patient, in addition, had a discrepancy between serum TSH and thyroid hormone values. This was due to the presence of interfering substances that react to mouse IgG in the sera since serum TSH levels were decreased in a dose dependent manner by the addition of increasing amounts of mouse IgG to the sera. It should therefore be noted that silent thyroiditis can develop in patients with Graves' disease. Furthermore, clinicians should be aware that two-site immunoassay kits that use mouse monoclonal antibodies are subject to interference by some substances, possibly antibodies which react to mouse IgG.