Light- and electron-microscopic immunocytochemistry of peptidergic neurons innervating thoracico-abdominal neurohaemal areas in the blowfly

Summary Ventral thoracic neurosecretory cells (VTNCs) of the blowflies, Calliphora erythrocephala and C. vomitoria, innervating thoracic neuropil and the dorsal neural sheath of the thoracico-abdominal ganglion have been shown to be immunoreactive to a variety of mammalian peptide antisera. In the neural sheath the VTNC terminals form an extensive neurohaemal network that is especially dense over the abdominal ganglia. The same areas are invaded by separate, ut overlapping serotonin-immunoreactive (5-HT-IR) projections derived from neuronal cell bodies in the suboesophageal ganglion. Immunocytochemical studies with different antisera, applied to adjacent sections at the lightmicroscopic level, combined with extensive cross-absorption tests, suggest that the perikarya of the VTNCs contain co-localized peptides related to gastrin/cholecystokinin (CCK), bovine pancreatic polypeptide (PP), Met- and Leuenkephalin and Met-enk-Arg⁶-Phe⁷ (Met-enk-RF). Electron-microscopic immunogold-labeling shows that some of the terminals in the dorsal sheath react with several of the individual peptide antisera, whilst others with similar cytology are non-immunoreactive. In the same region, separate terminals with different cytological characteristics contain 5-HT-IR. Both 5-HT-IR and peptidergic terminals are localized outside the cellular perineurium beneath the acellular permeable sheath adjacent to the haemocoel. Hence, we propose that various bioactive substances may be released from thoracic neurosecretory neurons into the circulating haemolymph to act on peripheral targets. The same neurons may also interact by synaptic or modulatory action in the CNS in different neuropil regions of the thoracic ganglion.     

Distribution of FMRFamide-like immunoreactivity in the nervous system of the slug Limax maximus

Summary The distribution of FMRFamide-like immunoreactive neurons in the nervous system of the slug Limax maximus was studied using immunohistochemical methods. Approximately one thousand FMRFamide-like immunoreactive cell bodies were found in the central nervous system. Ranging between 15 m and 200 m in diameter, they were found in all 11 ganglia of the central nervous system. FMRFamide-like immunoreactive cell bodies were also found at peripheral locations on buccal nerve roots. FMRFamide-like immunoreactive nerve fibres were present in peripheral nerve roots and were distributed extensively throughout the neuropil and cell body regions of the central ganglia. They were also present in the connective tissue of the perineurium, forming an extensive network of varicose fibres. The large number, extensive distribution and great range in size of FMRFamide-like immunoreactive cell bodies and the wide distribution of immunoreactive fibres suggest that FMRFamide-like peptides might serve several different functions in the nervous system of the slug.     

In vitro and ex vivo effects of antidepressants on rat brain membrane-bound phosphatidylinositol synthetase activity

The in vitro and ex vivo effects of antidepressant drugs on membrane-bound phosphatidylinositol (PI) synthetase and PI: myo-inositol exchange enzyme activities were examined. In rat brain subcellular fractions, PI synthetase occurred exclusively in the microsomes. In comparison, the activity of CDP-diglyceride independent PI: myo-inositol exchange enzyme was low (3%). Of the various CDP-diglycerides tested for the activation of PI synthetase, CDP-dipalmitin was the most active. Addition of 1 mM of desipramine, amitriptyline, imipramine, iprindole, clomipramine and mianserin in vitro significantly inhibited (30–60%) PI synthetase activity, whereas the same concentration of zimelidine and fluoxetine had no effect. At low liponucleotide concentrations, PI synthetase activity was significantly enhanced by imipramine (1 mM), whereas the enzyme activity was inhibited at higher liponucleotide concentrations (>0.3 mM). In contrast, imipramine had no effect on the PI: myo-inositol exchange enzyme activity. No significant alteration in the PI synthetase activity was found following either acute (2 h) or chronic (21 d) treatment of rats with imipramine. The above results indicate that the de novo synthesis of PI is inhibited in vitro but not ex vivo by some antidepressant drugs. However, in view of the high concentration of the drugs required, the pharmacological significance of this inhibitory action with respect to their therapeutic effects is doubtful.     

Analysis of the Escherichia coli glycogen gene cluster suggests that catabolic enzymes are encoded among the biosynthetic genes

The nucleotide sequences of the Escherichia coli genome between the glycogen biosynthetic genes glgB and glgC, and 1170 bp of DNA which follows glgA have been determined. The region between glgB and glgC contains an open reading frame (ORF) of 1521 bp which we call glgX. This ORF is capable of coding for an M_r 56 684 protein. The deduced amino acid (aa) sequence for the putative product shows significant similarity to the E. coli glycogen branching enzyme, and to several different glucan hydrolases and transferases. The regions of sequence similarity include residues which have been reported to be involved in substrate binding and catalysis by taka-amylase. This suggests that the proposed product may catalyze hydrolysis or glycosyltransferase reactions. The cloned region which follows glgA contains an incomplete ORF (1149 bp), glgY, which appears to encode 383 aa of the N terminus of glycogen phosphorylase, based upon sequence similarity with the enzyme from rabbit muscle (47% identical aa residues) and with maltodextrin phosphorylase from E. coli (37% identical aa residues). Results suggest that neither ORF is required for glycogen biosynthesis. The localization of glycogen biosynthetic and degradative genes together in a cluster may facilitate the regulation of these systems in vivo.     

意蜂和中蜂四种同工酶的研究

用聚丙烯酰胺凝胶等电聚焦电泳分析了意蜂和中蜂的酯酶(Est)、异柠檬酸脱氢酶(Idh)、苹果酸酶(Me)和苹果酸脱氢酶(Mdh)同工酶.两个蜂种的四种同工酶谱有不同程度的差别、意蜂酯酶Ⅳ和苹果酸脱氢酶Ⅲ是多态性的;中蜂的四种同工酶没有多态现象.     

An experiment in enzyme evolution studies withPseudomonas aeruginosa amidase

The regulation of amidase synthesis inP. aeruginosa is under positive control. This review describes the experimental evolution of amidase and its regulator protein for the hydrolysis of novel substrates and experiments to elucidate the mechanism of the control system.     

Synthesis of active Olisthodiscus luteus ribulose-1,5-bisphosphate carboxylase in Escherichia coli

The ribulose-1,5-bisphosphate carboxylase (Rubisco) large- and small-subunit genes are encoded on the chloroplast genome of the eukaryotic chromophytic alga Olisthodiscus luteus. Northern blot experiments indicate that both genes are co-transcribed into a single (>6 kb) mRNA molecule. Clones from the O. luteus rbc gene region were constructed with deleted 5 non-coding regions and placed under control of the lac promoter, resulting in the expression of high levels of O. luteus Rubisco large and small subunits in Escherichia coli. Sucrose gradient centrifugation of soluble extracts fractionated a minute amount of carboxylase activity that cosedimented with native hexadecameric O. luteus Rubisco. Most of the large subunit synthesized in E. coli appeared insoluble or formed an aggregate with the small subunit possessing an altered charge: mass ratio compared to the native holoenzyme. The presence in O. luteus of a polypeptide that has an identical molecular mass and cross reacts with antiserum generated against pea large-subunit binding protein may indicate that a protein of similar function is required for Rubisco assembly in O. luteus.     

Growth and activities of enzymes of primary metabolism in batch cultures of Catharanthus roseus cell suspension under different pCO2 conditions

In vitro enzyme activities of glycolysis, pentose-phosphate pathway and dark CO₂ fixation were assayed in batch cultures of heterotrophic Catharanthus roseus cells under various gassing rates and partial pressures of carbon dioxide. Detrimental effects of low pCO₂ culture conditions on the growth characteristics could be linked to marked changes in levels of enzymes of primary metabolism during growth. The enzyme levels observed during the early stages of growth were found to be more stable when a constant pCO₂ (20 mbar) was maintained and enabled exponential growth to be reached more rapidly.The importance of carbon dioxide as a conditioning factor of the culture medium is discussed.     

Nicotinic Agonists Regulate α-Bungarotoxin Binding Sites of TE671 Human Medulloblastoma Cells

The TE671 human medulloblastoma cell line expresses a variety of characteristics of human neurons. Among these characteristics is the expression of membrane-bound high-affinity binding sites for alpha-bungarotoxin, which is a potent antagonist of functional nicotinic acetylcholine receptors on these cells. These toxin binding sites represent a class of nicotinic receptor isotypes present in mammalian brain. Treatment of TE671 cells during proliferative growth phase with nicotine or carbamylcholine, but not with muscarine or d-tubocurarine, induced up to a five-fold increase in the density of radiolabeled toxin binding sites in crude membrane fractions. This effect was blocked by co-incubation with the nicotinic antagonists d-tubocurarine and decamethonium, but not by mecamylamine or by muscarinic antagonists. Following a 10-13 h lag phase upon removal of agonist, recovery of the up-regulated sites to control values occurred within an additional 10-20 h. These studies indicate that the expression of functional nicotinic acetylcholine receptors on TE671 cells is subject to regulation by nicotinic agonists. Studies of the murine CNS have consistently indicated nicotine-induced up-regulation of nicotinic acetylcholine receptors, thereby supporting the identification of the toxin binding site on these cells as the functional nicotinic receptor. Although a mechanism for this effect is not apparent, nicotine-induced receptor blockade does not appear to be involved.