Immunofluorescence and Histochemical Methods for Neural M1 Pyruvate Kinase Localization

Abstract: The distribution of pyruvate kinase (ATP pyruvate phosphotransferase, EC 2.7.1.40) in the nervous system has been studied by both immunofluorescence and a histochemical procedure using nitro blue tetrazolium. The localization in various parts of rat central nervous system in situ , cerebellar and cerebral cortex, was compared to that found in vitro in cultures of cerebellum, spinal ganglia, cerebral astrocytes, and skin fibroblasts. (1) Pyruvate kinase was found predominantly in the cytoplasm of neuronal cell bodies. (2) Large neurons were better visualized than small ones. (3) No glial localization was clearly demonstrated in situ , although this does not rule out the presence of some M₁ pyruvate kinase. (4) Regions expected to be rich in nerve terminals, such as the cerebellar glomeruli or the cerebellar molecular layer, showed intense staining even when the cell bodies themselves were negative. This was expected, owing to the previous demonstration of the presence of M₁ pyruvate kinase in nerve ending by subcellular fractionation methods. (5) The localization was similar in situ and in tissue culture, except that nerve processes were better seen in the latter and astrocytes were sometimes stained in vitro. (6) Variation in intensity of staining was observed in similar cell types in the same section or in the same culture. This could represent different metabolic or functional or maturational states.     

Microanalysis of plant mitochondrial protein synthesis products:

Summary A mitochondrial fraction obtained from 0.5 g of leaves was purified on a 0.75 ml Percoll gradient and used for an in vitro mitochondrial protein synthesis assay in the presence of [³⁵S] methionine. A set of 15 to 20 labeled polypeptides were revealed by autoradiography after sodium dodecylsulfate polyacrylamide gel electrophoresis. This could be applied at an early growth stage by using a few leaves from individual seedlings. It revealed the presence of variant mitochondrially translated polypeptides in green leaves of cytoplasmic male sterile lines from various cultivated plants of large economic importance: maize, wheat, sugar beet, tobacco and faba bean. This non-destructive microanalysis is thus of general use and opens new possibilities for rapid and large mass screening of mitochondrial parameters such as male sterility.     

A yeast strain with mutated beta-subunits of mitochondrial ATPase-ATPsynthase: high azide and bicarbonate sensitivity of the ATPase activity

A phenotypic revertant with modified beta-subunits of mitochondrial ATPase-ATP synthase has been obtained for the first time by selection from a beta-less mutant of the yeast Schizosaccharomyces pombe. Contrary to the parental mutant, the phenotypic revertant grows on glycerol, has normal respiratory activity and shows immunodetectable beta-subunits. However the kinetic properties of its submitochondrial particles ATPase activity differ markedly from those of the wild strain. The optimal pH is increased by about one unit. The maximal rate of the revertant ATPase activity at pH 8.5 is 4 to 5-fold lower than that of the wild strain, but it can be greatly increased upon addition of bicarbonate whereas the wild strain is completely insensitive to this anion. Furthermore the revertant ATPase activity is much more sensitive to azide inhibition. The results suggest that ADP dissociation is the rate-limiting step of ATP hydrolysis by the revertant.     

Organization and function of the plant pleiotropic drug resistance ABC transporter family

Among the ABC transporters, the pleiotropic drug resistance (PDR) family is particular in that its members are found only in fungi and plants and have a reverse domain organization, i.e., the nucleotide binding domain precedes the transmembrane domain. In Arabidopsis and rice, for which the full genome has been sequenced, the family of plant ABC transporters contains 15 and 23 PDR genes, respectively, which can be tentatively organized using the sequence data into five subfamilies. Most of the plant PDR genes so far characterized belong to subfamily I and have been shown to be involved in responses to abiotic and biotic stress, in the latter case, probably by transporting antimicrobial secondary metabolites to the cell surface. Only a single subfamily II member has been characterized. Induction of its expression by iron deficiency suggests its involvement in iron deficiency stress, thus, enlightening a new physiological role for a PDR gene.     

DLG1/SAP97 modulates transforming growth factor alpha bioavailability

TGFalpha and its receptor EGFR participate in the development of a wide range of tumors including gliomas, the main adult primary brain tumors. TGFalpha soluble form results from the cleavage by the metalloprotease TACE/ADAM17 of the extracellular part of its transmembrane precursor, pro-TGFalpha. To gain insights into the mechanisms underlying TGFalpha bioavailability, a yeast two-hybrid screen was performed to identify proteins interacting with pro-TGFalpha intracellular domain (ICD). DLG1/SAP97 (Discs Large Gene 1 or Synapse Associated Protein 97) was found to interact with both pro-TGFalpha and TACE ICDs through distinct PDZ domains. An in vivo pro-TGFalpha-DLG1-TACE complex was detected in U251 glioma cells and in gliomas-derived tumor initiating cells. Interaction between DLG1 and TACE diminished in response to stimulations promoting pro-TGFalpha shedding. Manipulation of DLG1 levels revealed dual actions of DLG1 on pro-TGFalpha shedding, favoring approximation of pro-TGFalpha and TACE, while limiting TACE full shedding activity. These results show that DLG1 participates in the control of TGFalpha bioavailability through its dynamic interaction with the growth factor precursor and TACE.