Ultrastructural localization of the calcium-binding protein parvalbumin in neurons of the song system of the zebra finch,Poephila guttata

Summary The distribution of parvalbumin (PV) within neurons of the vocal motor nucleus hyperstriatum ventralepars caudalis (HVc) was investigated in the forebrain of adult male zebra finches by means of light and electron microscopy using the indirect immunoperoxidase technique. Parvalbumin-reaction product was located in the amorphous material of perikarya, dendrites and nuclei, and associated to microtubuli, postsynaptic densities and intracellular membranes; it was found in some axons and Gray type-2 boutons, but rarely in type-1 boutons and never in the Golgi apparatus. These observations suggest that parvalbumin may regulate calcium-dependent processes at the postsynaptic membrane and in the cytosol. Furthermore, the partial association of parvalbumin to microtubuli points to an involvement in calcium-dependent tubular functions. Calcium currents and microtubular assembly or transport may be relevant for the known functions of HVc in song learning.     

Neuropeptides in the gastrointestinal canal of Necturus maculosus

Summary The presence and distribution of regulatory peptides in nerves and endocrine cells of the stomach, intestine and rectum of a urodele amphibian, the mudpuppy, Necturus maculosus, was studied immunohistochemically in sections or whole-mount preparations of the gut wall. The effect of the occurring peptides on gut motility was studied in isolated strip preparations of circular and longitudinal smooth muscle from different parts of the gut.Bombesin-, neurotensin-, substance P- and VIP-like immunoreactivity was present in abundant nerve fibres in the myenteric plexus of both stomach, intestine and rectum. Single fibres or bundles were present in the circular muscle layer and in a well-developed deep muscular plexus in the intestine and rectum. Immunoreactive nerve cells were found in the myenteric plexus of the stomach, intestine (neurotensin only) and rectum. Gastrin/CCK-like immunoreactivity was observed only in a few fibres in stomach and rectum.Endocrine cells containing bombesin-, met-enkephalin-, gastrin/CCK-, neurotensin-, somatostatin- or substance P- like immunoreactivity were present in the mucosa.The effect of bombesin was an inhibition of the rhythmic activity in circular muscle preparations and in longitudinal muscle from the rectum, while longitudinal muscle from the stomach usually responded with a weak increase in tonus. Neurotensin, like bombesin, was inhibitory on the spontaneous rhythmic activity of circular muscle throughout the gut, while the effect on longitudinal muscle was an increase in tonus. Met-enkephalin and substance P increased the tonus of all types of preparations, and often, in addition, initiated a rhythmic activity superimposed on this maintained tonus. VIP had a general inhibitory effect on the preparations, decreasing tonus and/or abolishing rhythmic activity.It is concluded that bombesin-, neurotensin-, substance P- and VIP-like peptides are present in nerves throughout the urodele gut and may have physiological functions in regulating the motility of the gut. The gastrin/CCK-like peptide present in nerves of the stomach and rectum may affect the function of these parts of the gut. The regulatory peptides present in endocrine cells may, perhaps with the exception of the somatostatin-like peptide, affect the motility humorally.     

Immunohistochemical localization of cyclic AMP and ultrastructural demonstration of adenylate cyclase activity in the testis of Esox lucius at time of spermiation

Summary In the testis of Esox lucius at the time of spermiation, activity of cyclic adenosine 3,5-monophosphate (cAMP) was immunocytochemically localized at the level of the Sertoli cells. In these cells adenylate cyclase activity was also ultracytochemically demonstrated by using adenylyl imidodiphosphate as a substrate. Reaction products of adenylate cyclase were primarily detectable on the basal and adluminal plasma membranes and on the surface of protrusions of the cell body into the lumen.     

Multicellular antennal sensilla containing a sensory cell with a short dendrite and dense-core granules in the insect,Hypogastrura socialis (Collembola): intermolt and molting stages

Summary At the antennal tip of the collembolan insect Hypogastrura socialis two terminal-pore sensilla are located, which, in addition to normally structured and most probably chemosensitive sensory cells, also contain aberrant sensory cells. Portions of these cells resemble chemoreceptors but also shown are features that, as a rule, occur in mechanoreceptors. One cell in each sensillum is remarkable in two characteristics: (1) Its dendritic outer segment does not reach the cuticular outer structures of the sensillum; (2) it contains dense-core granules (diameter 60–110 nm) within its perikaryon, its dendritic inner segment and its axon. Additionally, these two cells do not show lengthening of their dendritic outer segment during molt as do all other sensory cells. Among the fibers of one major branch of the antennal nerve within the head capsule a single axon was observed to contain dense-core granules. This axon was traced to its termination where normal synaptical contacts were found. Based on the assumption that the axon belongs to one of the granule-containing sensory cells two alternative hypotheses are proposed: (1) an individual sensory cell of a sensillum may synthesize a transmitter that is different from that of the other sensory cells of this sensillum; (2) the aberrant cells have lost exteroceptive functions but act as neuromodulatorsSupported by the Deutsche Forschungsgemeinschaft (SFB 4/G1)     

Subcellular localization and glycoprotein nature of the invertase from the fission yeast Schizosaccharomyces pombe

The subcellular localization of the enzyme invertase in Schizosaccharomyces pombe cells, both repressed and derepressed for synthesis of the enzyme, was studied. Most of the invertase was found to be located outside the plasma membrane and only a small percentage was found to be associated to membranes. A substantial portion of the external enzyme remained firmly bound to cell-wall material.All of the invertase recovered in soluble form from cellular extracts reacted with concanavalin A and with the lectin from Bandeiraea simplicifolia seeds, indicating the presence in the enzyme of a carbohydrate moiety which probably contains terminal mannosyl (or structurally related) and galactosyl residues.The possibility of the presence of two different forms of invertase in S. pombe was considered. An intracellular, soluble form of invertase, devoid of carbohydrate, similar to the small invertase of the budding yeast Saccharomyces cerevisiae, was not found in S. pombe. However, the Michaelis constant for sucrose of the enzyme present in repressed cells was smaller than that of the invertase synthesized under derepressing conditions, although this difference could also be the result of a different pattern of glycosylation of the invertase synthesized under different growth conditions.     

Auxin action: the search for the receptor

Abstract. The molecular specificity of the substances which have auxin activity implies the existence of specific receptors. There have been many efforts to identify and isolate these receptors on the assumption that they should bind auxins with affinities coordinate to their activities in bioassays. However, the known complexity of auxin uptake and metabolism make this assumption seriously deficient. Although several such binding sites have, in fact, been identified, proof of a connection between these sites and auxin action has been lacking. Definite proof would include a requirement that the site be reconstituted, together with the appropriate macro-molecular machinery, to construct a model of an auxin response. At the moment, our ignorance of the biochemistry and molecular biology of auxin growth responses makes such a proof difficult. However, two avenues of research promise to accelerate the rate of progress. The increasingly potent tools of molecular biology should soon allow the dissection of auxin-regulated gene expression, while improved knowledge of plasma membrane proton pumps and the mechanism of cell wall biosynthesis should produce, in parallel, an understanding of the auxin regulation of acid growth.     

A method for efficient gene isolation from phage lambda gt11 libraries: use of antisera to denatured, acetone-precipitated proteins

Experience with cloning pseudorabies virus (PRV) DNA in the lambda gt11 phage vector has shown that there are special requirements for the antisera used in screening the libraries, in addition to the requirement that the antisera recognize proteins on a Western blot. Initial screening of a lambda gt11 library of sheared PRV DNA fragments in Escherichia coli for expression of PRV antigens using PRV hyperimmune antisera was unsuccessful. It was only after screening the library with antisera raised against PRV proteins eluted from sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels that positive results were obtained. These "gel-slice" antisera (GSA) were equivalent in potency to hyperimmune antisera in standard immunoassays (including ELISA, immunoprecipitation, Western blots, and neutralization of virus), but only the GSA could recognize PRV fusion proteins expressed by recombinant lambda gt11 phage. This difference was seen despite the fact that hyperimmune antisera performed satisfactorily on Western blots of denatured PRV-infected cell extracts. These results show that the efficiency of screening expression libraries in E. coli can be improved if antibodies are raised against denatured proteins.     

Restriction endonuclease EcoO109 from Escherichia coli H709c with heptanucleotide recognition site 5'-PuG/GNCCPy

A new restriction endonuclease, EcoO109, has been isolated from Escherichia coli H709c by polyethyleneimine (PEI) precipitation, DEAE-cellulose chromatography and heparin agarose chromatography. The yield was high, more than 3000 units/g of wet cells. The EcoO109 endonuclease recognizes and cleaves a nucleotide sequence of (formula: see text), in the presence of 10 mM Mg2+. The enzyme will be useful for structural analysis and molecular cloning of DNA because of the stability, high yield and easy handling of the producer strain.     

Development of a high-frequency transforming vector for Aspergillus nidulans

The pyr4 gene of Neurospora crassa, which codes for orotidine-5'-phosphate decarboxylase, is capable of transforming an Aspergillus nidulans pyrG mutant by chromosomal integration, despite low homology between the transforming DNA and the recipient genome. Integration of pFB6, a plasmid carrying pyr4 and capable of replication in Escherichia coli, was not observed at the pyrG locus. The efficiency of transformation was considerably enhanced (50-100 fold) by inclusion in the transforming vector of a 3.5-kb A.nidulans chromosomal sequence, ans1. Although this sequence was isolated on the basis of replicating activity in Saccharomyces cerevisiae, there was no evidence for such activity in A.nidulans. Part of the ans1 fragment appears to be reiterated in the A.nidulans genome, though it is not yet clear whether this is directly responsible for the high transformation frequency. The efficiency of transformation of A.nidulans by plasmids bearing ans1, using an improved protocol, was approx. 5 X 10(3) stable transformants per microgram of plasmid DNA.