The identification and structure-activity relations of a cardioactive FMRFamide-related peptide from the blue crab Callinectes sapidus.

The pericardial organs and thoracic ganglia of the blue crab Callinectes sapidus were resected and extracted. The extracts were fractionated by HPLC and the fractions analyzed by a radioimmunoassay (RIA) to FMRFamide. Multiple peaks of immunoreactivity were present and one of these, upon fast atom bombardment mass spectrometry (FAB-ms) and microsequencing, yielded the sequence GYNRSFLRFamide. The amount of this peptide in each crab is between 7 and 13 pmol. Several incomplete sequences were also characterized, suggesting a precursor with multiple copies of peptides related to GYNRSFLRFamide might occur. The peptide caused a dose-dependent increase in heart rate; threshold was 10 to 30 nM, and the EC₅₀ was 323±62 nM. A structure-activity study of GYNRSFLRFamide on the crab heart suggests that, for full potency, a peptide should be at least a heptapeptide with the sequence XXZFLRFamide, where X is any amino acid and Z is either asparagine or serine.     

Acute exposure to offshore produced water has an effect on stress- and secondary stress responses in three-spined stickleback Gasterosteus aculeatus

Pollution is one of today's greatest problems, and the release of contaminants into the environment can cause adverse changes in vitally important biological pathways. In this study, we exposed three-spined stickleback Gasterosteus aculeatus to produced water (PW), i.e. wastewater from offshore petroleum production. PW contains substances such as alkylphenols (APs) and aromatic hydrocarbons (PAHs) known to induce toxicant stress and endocrine disruption in a variety of organisms. Following exposure to PW, a standardized confinement treatment was applied as a second stressor (PW-stress), testing how fish already under stress from the pollutant would respond to an additional stressor. The endpoint for analysis was a combination of blood levels of cortisol and glucose, in addition to transcribed levels of a set of genes related to toxicant stress, endocrine disruption and general stress. The findings of this study indicate that low doses of PW do not induce vitellogenin in immature female stickleback, but do cause an upregulation of cytochrome (CYP1A) and UDP-glucuronsyltransferase (UDP-GT), two biomarkers related to toxicant stress. However, when the second stressor was applied, both genes were downregulated, indicating that the confinement exposure had a suppressive effect on the expression of toxicant biomarkers (CYP1A and UDP-GT). Further, two of the stress related genes, heat shock protein 90 (HSP90) and stress-induced phosphoprotein (STIP), were upregulated in both PW- and PW-stress-treatment, but not in the water control confinement treatment, indicating that PW posed as a larger stress-factor than confinement for these genes. The confinement stressor caused an increased level of glucose in both control and PW-treated fish, indicating hyperglycemia, a commonly reported stress response in fish.     

Polyamine synthesis in plants: isolation and characterization of spermidine synthase from soybean (Glycine max) axes

Spermidine synthase (EC 2.5.1.16) was purified to homogeneity for the cytosol of soybean (Glycine max) axes using ammonium sulfate fractionation and chromatography on DEAE-Sephacel, Sephacryl S-300, ω-aminooctyl-Sepharose and ATPA-Sepharose. The molecular mass of the enzyme estimated by gel filtration and SDS–PAGE is 74 kDa. Cadaverin and 1,6-diaminohexane could not replace putrescine as the aminopropyl acceptor. Kinetic behaviors of the substrate are consistent with a ping pong mechanism. The kinetic mechanism is further supported by direct evidence confirming the presence of an aminopropylated enzyme and identification of product, 5′-deoxy-5′-methylthioadenosine, prior to adding putrescine. The K_m values for decarboxylated S-adenosylmethionine and putrescine are 0.43 μM and 32.45 μM, respectively. Optimum pH and temperature for the enzyme reaction are 8.5 and 37°C, respectively. The enzyme activity is inhibited by N-ethylmaleimide and DTNB, but stimulated by Co²⁺, Cu²⁺ and Ca²⁺ significantly, suggesting that these metal ions could be the cellular regulators in polyamine biosynthesis.     

HPLC/RIA analysis of bioactive methionine enkephalin content in the seizure-susceptible El mouse brain

We previously reported a deficit of methionine enkephalin-like immunoreactivity (ME-LI), in the cerebral cortex, septal area, hippocampus, and striatum and the abnormal metabolism of opioid peptides in the hippocampus and striatum of seizure-susceptible El mice, which are involved in the pathogenesis of seizures. However, these findings suggest that the ME-LI does not necessarily reflect the bioactive methionine enkephalin (ME). Herein, we measured the biologically active peptide, ME excluding cross-reactive substances by using HPLC coupled with radioimmunoassay to clarify the abnormal function of enkephalinergic neurons in the El mouse brain. The ME content in 25-day-old El mice that had no seizures was significantly decreased in the hippocampus and septal area, as compared with corresponding regions in ddY mice (seizure-nonsusceptible; the mother strain of El). At the age of 50 days when El mice displayed abortive seizures, this content in both stimulated El[s] and nonstimulated El[ns] was significantly reduced in the septal area and cerebral cortex. At the age of 150 days when El mice exhibit tonic-clonic seizures, this content in both El[s] and El[ns] was significantly reduced in the septal area, cerebral cortex and striatum. These findings were generally compatible with our previous findings. This study further supports our hypothesis that a deficit of anticonvulsant endogenous ME, in the cerebral cortex, septal area, and hippocampus of seizuresusceptible El mice play an important role in the pathogenesis of seizures.     

Insulin-related material in prokaryotes

Abstract We have reported previously on the presence of vertebrate-type peptide hormones in Tetrahymena pyriformis and Escherichia coli . We now have examined other prokaryotes for immunologically detectable insulin-like material. The bacteria studied were E. coli, Acinetobacter calcoaceticus RAG-1, Bordetella pertussis and Halobacteria solinarium; they were grown in defined minimal salt media under varying growth conditions. All these bacteria contain insulin-related material (1–12 pg per g cell wet wt). No insulin immunoactivity was detected in the media prior to inoculation. The content of insulin-related material was not affected by the carbon source used for cell growth. These data are in good agreement with data published previously, and suggest that prokaryotes and unicellular eukaryotes are capable of producing hormone-like material; the function of these peptides, if any, is as yet unknown.