Characterization and developmental regulation of tyramine-beta-hydroxylase in the CNS of the moth, Manduca sexta.

Octopamine (OA) is present in insect nervous tissue, but little is known about its biosynthesis. In the CNS of Manduca sexta, OA levels increase markedly during postembryonic adult development. To study this increase, we developed an assay for tyramine-beta-hydroxylase, the putatively rate-limiting enzyme for OA biosynthesis. Tyramine-beta-hydroxylase activity in extracts of M. sexta CNS tissue: (1) was time- and protein-dependent, and with protein concentrations up to 2 microg/microl, was linear for 20 min; (2) had a pH optimum of 7.0 for conversion of tyramine to OA; (3) required ascorbate, copper, and catalase; and (4) had an apparent K(M, tyramine) of 0.22+/-0.04 mM. These characteristics resemble those of the mammalian enzyme dopamine-beta-hydroxylase, suggesting that these two enzymes are functionally related. During adult development, tyramine-beta-hydroxylase activity increased 11-fold in the brain and 9-fold in the abdominal ganglia, paralleling increases in OA levels in those CNS structures during metamorphosis. The apparent kinetic constants of tyramine-beta-hydroxylase suggested that the amount of this enzyme present in the tissues increases. The increase in OA levels during adult development thus appears to be due to an increase in the level of enzyme available for OA synthesis and may reflect an increase in the number of octopaminergic neurons.

Crustacean cardioactive peptide in the sphinx moth, Manduca sexta.

The isolation, identification, and actions of crustacean cardiactive peptide (CCAP) have been examined in the sphinx moth Manduca sexta. A sensitive and specific enzyme-linked immunosorbent assay (ELISA) was used to quantify CCAP-like immunoreactivity in the nervous system. The CCAP-like immunoreactivity from the abdominal CNS was then purified, and its sequence was ascertained by amino acid analysis, mass spectral analysis, and HPLC. These studies showed that the nervous system of M. sexta contains a peptide with the sequence Pro-Phe-Cys-Asn-Ala-Phe-Thr-Gly-Cys-NH2, identical to CCAP originally isolated and sequenced from the shore crab Carcinus maenas. The actions of CCAP on the isolated heart of M. sexta and the extensor-tibia muscle of Schistocerca americana were tested. Crustacean cardioactive peptide had excitatory actions on both preparations: a dose-dependent increase in the rate of contractions was observed on the heart, and an increase in the rate of the myogenic rhythm was observed in the leg muscle. Moreover, purified and synthetic CCAP had identical effects on the isolated heart. We conclude that CCAP occurs in M. sexta and exerts potent neurotransmitter or neurohormonal actions on a variety of muscles.

Optical metrology for two large highly aspheric telescope mirrors.

We describe a relatively simple, but highly effective, approach to the system design and alignment of an all-refractive Offner null corrector and phase-measuring Shack cube interferometer. In addition we outline procedures for fabricating and testing the optical components. Allowable errors for all parameters are determined by a tolerance analysis that separates axisymmetric and residual figure errors. An open construction optics frame provides a high degree of metering flexibility by incorporating simple kinematic mounts that provide adjustment of each lens while also allowing the lens to be removed and replaced with <2microm absolute repeatability. Nonaxisymmetric alignment errors are removed by rotating the optics on a high-precision bearing. Axial spacings are measured with contact transducers attached to both ends of an Invar metering rod. Two completed systems have guided the stressed-lap polishing of 1.8-m f/ 1.0 and 3.5-m f/ 1.5 aspheric mirrors.

Influence of sex and glucocorticoid hormones on 3H cholesterol uptake in the aortic wall and 3H adrenaline in the brain.

The mechanisms involved in the atherogenesis process and the connection of the latter with stress were studied, by trying to elucidate the difference in the response of the two sexes to the causative factors. To this purpose, the role of the sex hormones (testosterone, estradiol, progesterone) and glucocorticoids (hydrocortisone hemisuccinate) in 3H cholesterol uptake in the aortic wall and 3H adrenaline in the brain was investigated. In males, the results show that these hormones favoured the uptake of the two markers whose level was significantly raised (p greater than 0.05; p greater than 0.01) as against the controls all along the 20 days of treatment. In females, the level of 3H cholesterol in the aorta and 3H adrenaline in the brain had a statistical significance (p greater than 0.05) only in the first days of treatment but after the 6th day it began to decline.