ABSTRACT
The site of synthesis of mollusc lipoproteins is hitherto unknown and was investigated for perivitellin 2 (PV2), an egg lipoprotein found in the freshwater snail Pomacea canaliculata. Tissues (albumen gland, gonad-digestive gland complex, and muscle) from vitellogenic females were incubated in vitro with 14C-leucine at 25 degrees C for 12 hr. At the end of incubation, soluble proteins from tissue homogenates and medium were analyzed for de novo protein synthesis by electrophoresis and HPLC, and radiolabeled proteins were quantified by liquid scintillation. Two albumen gland radiolabeled proteins (67 and 31 kDa) co-migrated with the subunits of PV2, and they represented 6.0% of the total labeled protein in that tissue. Western blot analysis confirmed the presence of PV2 only in the albumen gland. In vivo experiments where adult females were injected with 3H-leucine revealed that PV2 was not present in hemolymph. ELISA analysis in all tissues of the snail confirmed the PV2 presence only in the albumen gland and developing eggs with levels of 26 and 98 mg/g protein, respectively. Therefore, the albumen gland is the only site for PV2 synthesis, and no extra-gland synthesis, circulation, or accumulation could be found. PV2 subunits were further characterized analyzing N-terminal sequences which showed no homology with other proteins.
Subject(s)
Egg Proteins/biosynthesis , Snails/growth & development , Vitellogenesis/physiology , Animals , Blotting, Western , Chromatography, High Pressure Liquid , Egg Proteins/analysis , Eggs , Endocrine Glands/chemistry , Enzyme-Linked Immunosorbent Assay , Female , Hemolymph/chemistryABSTRACT
From a crude extract of the sinus glands of the shrimp Penaeus (litopenaeus) schmitti a peptide with hyperglycemic activity in a homologous bioassay was isolated and characterized by a combination of automatic Edman degradation, enzymatic digestions, TLC of dansyl-amino acids, and mass spectrometry. Its M(r) is 8359.4 Da by MS, which coincides with the deduced sequence. Its N-terminus is free and its C-terminus is amidated. It has 6 Cys residues in conserved positions compared with other known CHHs. This is the first sinus gland hormone from an Atlantic Ocean shrimp characterized to date. It has a remarkable 90% sequence similarity to the Indo-Pacific shrimp P. (marsupenaeus) japonicus Pej-VII hyperglycemic hormone.
Subject(s)
Endocrine Glands/chemistry , Glucose/metabolism , Invertebrate Hormones/chemistry , Invertebrate Hormones/pharmacology , Penaeidae , Amino Acid Sequence , Animals , Biological Assay , Endopeptidases , Hemolymph/drug effects , Hemolymph/metabolism , Invertebrate Hormones/isolation & purification , Mass Spectrometry , Molecular Sequence Data , Molecular Weight , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
Glucocorticoids are the most effective anti-inflammatory agents currently available, but a variety of adverse effects limit their clinical usefulness. This work explores further two facets of their interaction between glucocorticoids and the skin, with the aim of identifying means of reducing glucocorticoid toxicity. (a) Metabolism of glucocorticoids by skin: Human skin is active in the terminal metabolism of corticol to cortisone, but the biological implications of this process in skin are uncertain. BEcause there are technical difficulties in dealing with human skin, an animal model, the nude mouse, has been evaluated for its suitability to the study of the metabolism of corticosterone to IIB-dehydrocorticosterone (the homologous reaction in rodents of cortisol to cortisone conversion in man); a process mediated by IIB-hydroxysteroid dehydrogenase. (b) Skin vasoconstrictor response (blanching) to topical glucocorticoids: Glucocorticoids applied topically to human skin produce vasoconstriction in dermal vessels, the degree of which correlates closely with the potency and clinically efficacy of these compounds (AU)