Quantitative autoradiographic analysis of [I]-human CGRP binding sites in the dorsal horn of rat following chronic constriction injury or dorsal rhizotomy

Quantitative receptor autoradiography was used to examine the binding of [¹²⁵I]-human CGRP in the dorsal horn of the L4 spinal segment of rats with a chronic constriction injury (CCI) of the sciatic nerve or unilateral dorsal rhizotomies of spinal segments L1–L6. At the times selected for study, we found no change in the amount of CGRP binding in any areas examined following CCI. In contrast, our results showed a temporally related increase in the amount of CGRP binding in areas within laminae I–II and in lateral lamina V of the dorsal horn ipsilateral to the rhizotomies. These results indicate that CGRP binding sites are regulated, most likely, by changes in the release of CGRP. Further, our results suggest that the release of CGRP from primary afferent neurons is unchanged in animals with a CCI.     

Calcitonin gene-related peptide and its mRNA in pulmonary neuroendocrine cells and ganglia.

The occurrence of calcitonin gene-related peptide (CGRP) and it's mRNA was studied in lungs of rats and piglets using in situ hybridization with two synthetic oligonucleotide probes followed by immunocytochemistry (ICC). CGRP mRNA was present in pulmonary neuroendocrine cells (PNEC) of both the solitary type and cluster type (neuroepithelial body; NEB) at all levels of the airway epithelium from bronchi to alveoli. The distribution of labelled cells was similar to that previously described with ICC. The 44-mer probe provided stronger hybridization signal than the 34-mer and the two combined increased labelling slightly. Formalin fixation reduced labelling and tended to increase background. Labelling for CGRP mRNA was evenly distributed over the cytoplasm, whereas CGRP-like immunoreactivity (LI) usually was of highest intensity toward the base of the PNEC, suggesting basal accumulation of synthesized peptide. CGRP-LI was also observed in occasional rat ganglia and in some, but not all, piglet ganglia. These local neurons may contribute to the CGRP fibers of airways and vasculature, and could theoretically bridge their dendrites and axons between NEB and the effector organ (e.g. artery or arteriole) thus accomplishing a function similar to the postulated axon reflex.     

O-ribosyl-phosphate purine as a constant modified nucleotide located at position 64 in cytoplasmic initiator tRNAs(Met) of yeasts.

The unknown modified nucleotide G*, isolated from both Schizosaccharomyces pombe and Torulopsis utilis initiator tRNAs(Met), has been identified as an O-ribosyl-(1"----2')-guanosine-5"-phosphate, called Gr(p), by means of HPLC, UV-absorption, mass spectrometry and periodate oxidation procedures. By comparison with the previously published structure of Ar(p) isolated from Saccharomyces cerevisiae initiator tRNA(Met), the (1"----2')-glycosidic bond in Gr(p) has been postulated to have a beta-spatial conformation. The modified nucleotide Gr(p) is located at position 64 in the tRNA(Met) molecules, i.e. at the same position as Ar(p). Since we have also characterized Gr(p) in Candida albicans initiator tRNA(Met), the phosphoribosylation of purine 64 can be considered as a constant nucleotide modification in the cytoplasmic initiator tRNAs(Met) of all yeast species so far sequenced. Precise evidence for the presence of Gr(p) in initiator tRNAs(Met) of several plants is also reported.     

Inhibition of the cellular response to interferons by products of the adenovirus type 5 E1A oncogene.

Expression of the E1A oncogene of adenovirus type 5 inhibits the response of interferon (IFN)-inducible constructs to Type I (alpha,beta) and II (gamma) IFNs in transient transfection assays. In human cell lines stably expressing E1A mRNA and protein acquisition of an antiviral state and the induction of a number of genes in response to alpha- and gamma-IFNs is inhibited. A short IFN-stimulable response element (ISRE) present in the 5' flanking region of a number of genes mediates induction by alpha- and gamma-IFNs. In cells expressing E1A there is a substantial reduction in the levels of the ISRE-binding factors E and M, inducible by alpha-IFN, and of factor G, inducible by gamma-IFN. In E1A-expressing cells the E alpha subunit of factor E is activated normally in response to alpha-IFN; the defect is in the production or activation of the E gamma subunit. The inhibitory activity of E1A is lost upon deletion of the CR1 domain. The induction of HLA class II genes by gamma-IFN, which involves a different DNA response element(s), and of beta-IFN mRNA in response to double-stranded RNA are also inhibited by E1A. An essential component(s) of a number of signalling pathways must, therefore, be subject, directly or indirectly, to inhibition by E1A.     

Immunological identification of uncoupling protein in interscapular "brown" adipose tissue of suckling and adult pipistrelle bats (Pipistrellus pipistrellus).

1. Interscapular adipose tissue of suckling and adult pipistrelle bats was examined for the presence of the 32,000 Mr "uncoupling protein" diagnostic of brown adipose tissue. 2. Following separation by SDS-polyacrylamide gel electrophoresis, mitochondrial proteins were blotted onto nitrocellulose and probed for uncoupling protein with an anti-(ground squirrel uncoupling protein) serum. 3. Immunoreactivity consistent with the presence of uncoupling protein was found in all samples of adipose tissue mitochondria from both suckling and adult bats. 4. It is concluded that interscapular adipose tissue in pipistrelle bats exhibits the critical biochemical criterion for being designated functionally "brown".     

The sulphydryl groups of ox brain and liver glutamate dehydrogenase preparations and the effects of oxidation on their inhibitor sensitivities

Glutamate dehydrogenase preparations from several sources have been shown to have suffered limited proteolysis during purification. This proteolysis has been previously shown to involve removal of the N-terminal tetrapeptide and to result in changes in the regulatory properties of the enzyme. In the present work the previously unidentified N-terminal residue of the unproteolysed enzyme from ox brain and liver is shown to be cysteine. The thiol group of this residue is masked in the native enzyme but it becomes accessible after reduction. Exposure of solutions of the unproteolysed enzyme to air oxidation causes large changes in its sensitivity to inhibition by the antipsychotic drug perphenazine, GTP and by high concentrations of NADH. No such changes occurred in the behaviour of preparations of the enzyme that had suffered proteolysis during purification under these conditions.Special issue dedicated to Dr. Santiago Grisolia.     

Chrysoperla carnea predation on Heliothis virescens larvae parasitized by Microplitis croceipes

Predation by third instar larvae of Chrysoperla (=Chrysopa) carnea (Stephens) (Chrysopidae) did not alter the ratio of unparasitized Heliothis virescens (F.) (Noctuidae) larvae to H. virescens larvae parasitized by Microplitis croceipes (Cresson) (Braconidae) when these second instar larvae were exposed together to the predator on cotton (Gossypium hirsutum L., Malvaceae) in field cages. This indicates that C. carnea larvae did not prefer either parasitized or unparasitized larvae.

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Prédation par Chrysopa carnea des chenilles d'Heliothis virescens parasitées par Microplitis croceipes

Résumé Les prédations de chenilles d'Heliothis virescens, parasitées ou saines, élevées sur coton (Gossypium hirsutum L.), de la variété Stoneville 213, ont été comparées, dans des cages de 10 m² chacune placées dans la nature. Des chenilles du second stade ont été placées sur des pieds de coton dans 10 cages, à raison de 160 chenilles préalablement exposées à M. croceipes pendant leur premier stade et 40 chenilles saines par cage. Des larves du troisième stade de Chrysopa carnea ont été ajoutées dans 6 cages, à raison de 500/cage, et 4 cages ont servi de témoins pour évaluer les autres causes de mortalité. L'expérience a été répétée 2 fois. Les chenilles d'H. virescens ont été retirées au bout d'un jour dans une expérience et au bout de 2 jours dans l'autre. C. carnea n'a fait aucun choix entre chenilles parasitées ou non; la fréquence moyenne de chenilles parasitées n'a pas présenté de différence significative entre les cages avec ou sans C. carnea. Qui qu'il en soit, C. carnea a réduit significativement la survie des chenilles d'H. virescens parasitées ou non.

     

Construction of a dihydrofolate reductase-deficient mutant of Escherichia coli by gene replacement.

The dihydrofolate reductase (fol) gene in Escherichia coli has been deleted and replaced by a selectable marker. Verification of the delta fol::kan strain has been accomplished using genetic and biochemical criteria, including Southern analysis of the chromosomal DNA. The delta fol::kan mutation is stable in E. coli K549 [thyA polA12 (Ts)] and can be successfully transduced to other E. coli strains providing they have mutations in their thymidylate synthetase (thyA) genes. A preliminary investigation of the relationship between fol and thyA gene expression suggests that a Fol- cell (i.e., a dihydrofolate reductase deficiency phenotype) is not viable unless thymidylate synthetase activity is concurrently eliminated. This observation indicates that either the nonproductive accumulation of dihydrofolate or the depletion of tetrahydrofolate cofactor pools is lethal in a Fol- ThyA+ strain. Strains containing the thyA delta fol::kan lesions require the presence of Fol end products for growth, and these lesions typically increase the doubling time of the strain by a factor of 2.5 in rich medium.     

Distribution of isoaccepting tRNAs and codons for proline and glycine in collagenous and noncollagenous chicken tissues

The relation between codon usage and tRNA content for proline and glycine, the major constituents of collagen, was studied in two tissues: the magnum of laying hen oviduct and the leg tendons of chick embryo where collagen is produced. Although the relative contents of tRNA(GCCGly) and tRNA(IGGPro) in tendons, as compared to magnum indicate a specialization of the tRNA population for collagen synthesis, the distribution of the preponderant codons in collagen mRNA is correlated but at a lesser extent to that of their cognate tRNAs.     

The effect of polymerised fibrin on the catalytic activities of one-chain tissue-type plasminogen activator as revealed by an analogue resistant to plasmin cleavage

A one-chain recombinant tissue-type plasminogen activator (EC 2.4.31.-) (tPA) analogue was constructed in which Arg-275 of the activation site was changed to Gly by site-directed mutagenesis. This analogue, tPA-Gly275, was very resistant to plasmin (EC 2.4.21.5) cleavage. It has been used to gain information about the activity of the uncleaved one-chain tPA form, also when plasmin is generated as a result of a plasminogen activation reaction. The amidolytic activity of tPA-Gly275 with less than Glu-Gly-Arg-pNA was investigated and compared to that of one-chain and two-chain wild-type recombinant tPA. A small but significant intrinsic amidolytic activity was observed with the analogue as well as the wild-type one-chain tPA form. However, it was much lower than that of two-chain tPA. Polymerised fibrin enhanced the amidolytic activity of both one-chain tPA forms but not of two-chain tPA. Measurements of the plasminogen activation kinetics in the absence of fibrin revealed that tPA-Gly275 possessed a significant intrinsic activity. However, it was 30-fold lower than that of two-chain tPA. Addition of polymerised fibrin profoundly enhanced the plasminogen activation rate of both tPA-Gly275 and wild-type one- and two-chain tPA to approximately the same maximal level. The results were interpreted to mean that fibrin binding can induce an activated state of the intact tPA one-chain form.