Biomedical subjects

G Pietrini

Publications and source records attributed to G Pietrini.

6 recordsLinked to original sources

PubMed1992

Isoforms of the Na,K-ATPase are present in both axons and dendrites of hippocampal neurons in culture.

The distributions of isoforms of the Na,K-ATPase alpha subunit were determined in mature cultured hippocampal neurons and in a polarized epithelial cell line. We find that hippocampal neurons express the alpha 1 and alpha 3 isoforms in the membranes of both axons and dendrites. In contrast the alpha 1 and alpha 3 proteins are exclusively basolateral when expressed endogenously or by stable transfection in renal epithelial cells. These data suggest that epithelial cells and hippocampal neurons localize these proteins by different mechanisms. These observations contrast with those made for the vesicular stomatitis virus and the influenza glycoproteins, which are polarized in both epithelial and neuronal cells.

Animals↗

PubMed1992

A single mRNA, transcribed from an alternative, erythroid-specific, promoter, codes for two non-myristylated forms of NADH-cytochrome b5 reductase.

Two forms of NADH-cytochrome b5 reductase are produced from one gene: a myristylated membrane-bound enzyme, expressed in all tissues, and a soluble, erythrocyte-specific, isoform. The two forms are identical in a large cytoplasmic domain (Mr approximately 30,000) and differ at the NH2-terminus, which, in the membrane form, is responsible for binding to the bilayer, and which contains the myristylation consensus sequence and an additional 14 uncharged amino acids. To investigate how the two differently targeted forms of the reductase are produced, we cloned a reductase transcript from reticulocytes, and studied its relationship to the previously cloned liver cDNA. The reticulocyte transcript differs from the liver transcript in the 5' non-coding portion and at the beginning of the coding portion, where the seven codons specifying the myristoylation consensus are replaced by a reticulocyte-specific sequence which codes for 13 non-charged amino acids. Analysis of genomic reductase clones indicated that the ubiquitous transcript is generated from an upstream "housekeeping" type promoter, while the reticulocyte transcript originates from a downstream, erythroid-specific, promoter. In vitro translation of the reticulocyte-specific mRNA generated two products: a minor one originating from the first AUG, and a major one starting from a downstream AUG, as indicated by mutational analysis. Both the AUGs used as initiation codons were in an unfavorable sequence context. The major, lower relative molecular mass product behaved as a soluble protein, while the NH2-terminally extended minor product interacted with microsomes in vitro. The generation of soluble reductase from a downstream AUG was confirmed in vivo, in Xenopus oocytes. Thus, differently localized products, with respect both to tissues and to subcellular compartments, are generated from the same gene by a combination of transcriptional and translational mechanisms.

Amino Acid Sequence↗

PubMed1988

Two transcripts encode rat cytochrome b5 reductase.

A cDNA expression library in lambda gt11 was screened with affinity-purified polyclonal anti-rat cytochrome b5 reductase antibodies. One positive clone out of 450,000 clones was isolated and found to be incomplete. This clone was used to rescreen the library, and a second, overlapping clone that contained the entire coding sequence was isolated. RNA gel blots showed that the two overlapping clones contained approximately 90% of the reductase mRNA sequence. Sequencing data showed (i) that rat reductase has a 93% sequence similarity with bovine and human reductase and (ii) that reductase is not synthesized as a high molecular weight precursor. Results of Southern blot analysis were consistent with the hypothesis that a single gene codes for the soluble and membrane-bound (microsomal and mitochondrial) forms of the reductase, present in erythrocytes and liver, respectively. The cloned cDNA was used to study reductase transcripts in liver and reticulocytes. Two antisense RNA probes that together covered the entire coding region and part of the noncoding region of reductase mRNA were used in RNase A protection experiments. These probes detected only one transcript in liver, suggesting that endoplasmic reticulum and mitochondrial reductase are translated from the same mRNA. In contrast, two transcripts were detected in reticulocytes, one of which mismatched the liver probe approximately 30 nucleotides downstream from the initiation codon. Since the soluble and membrane form of the reductase are known to differ at the N terminus, we suggest that this second transcript encodes soluble reductase.

Amino Acid Sequence↗

PubMed1987

Three translationally regulated mRNAs are stored in the cytoplasm of clam oocytes.

In situ hybridization was used to examine the spatial distributions of three translationally controlled maternal RNAs in oocytes and two-cell embryos of the clam Spisula. 3H-labeled single-stranded RNA probes were generated from SP6 recombinant clones containing DNA inserts encoding portions of histone H3 (the DNA sequence which is presented here), cyclin A, and the small subunit of ribonucleotide reductase. Hybridization of these probes to oocytes, in which the mRNAs are translationally inactive, shows that these mRNAs are stored in the cytoplasm. There is no evidence for sequestration of any of the RNAs within the nucleus or any other discrete structure. Instead they appear to be evenly distributed throughout the cytoplasm.

Amino Acid Sequence↗

PubMed1987

Concentration of NADH-cytochrome b5 reductase in erythrocytes of normal and methemoglobinemic individuals measured with a quantitative radioimmunoblotting assay.

The activity of NADH-cytochrome b5 reductase (NADH-methemoglobin reductase) is generally reduced in red cells of patients with recessive hereditary methemoglobinemia. To determine whether this lower activity is due to reduced concentration of an enzyme with normal catalytic properties or to reduced activity of an enzyme present at normal concentration, we measured erythrocyte reductase concentrations with a quantitative radioimmunoblotting method, using affinity-purified polyclonal antibodies against rat liver microsomal reductase as probe. In five patients with the "mild" form of recessive hereditary methemoglobinemia, in which the activity of erythrocyte reductase was 4-13% of controls, concentrations of the enzyme, measured as antigen, were also reduced to 7-20% of the control values. The concentration of membrane-bound reductase antigen, measured in the ghost fraction, was similarly reduced. Thus, in these patients, the reductase deficit is caused mainly by a reduction in NADH-cytochrome b5 reductase concentration, although altered catalytic properties of the enzyme may also contribute to the reduced enzyme activity.

Animals↗

PubMed1986

Distribution of the integral membrane protein NADH-cytochrome b5 reductase in rat liver cells, studied with a quantitative radioimmunoblotting assay.

The intracellular localization of the post-translationally inserted integral membrane protein, NADH-cytochrome b5 reductase, was investigated, using a quantitative radioimmunoblotting method to determine its concentration in rat liver subcellular fractions. Subcellular fractions enriched in rough or smooth microsomes, Golgi, lysosomes, plasma membrane and mitochondrial inner or outer membranes were characterized by marker enzyme analysis and electron microscopy. Reductase levels were determined both with the NADH-cytochrome c reductase activity assay, and by radioimmunoblotting, and the results of the two methods were compared. When measured as antigen, the reductase was relatively less concentrated in microsomal subfractions, and more concentrated in fractions containing outer mitochondrial membranes, lysosomes and plasma membrane than when measured as enzyme activity. Rough and smooth microsomes had 4-5-fold lower concentrations, on a phospholipid basis than did mitochondrial outer membranes. Fractions containing Golgi, lysosomes and plasma membrane had approximately 14-, approximately 16, and approximately 9-fold lower concentrations of antigen than did mitochondrial outer membranes, respectively, and much of the antigen in these fractions could be accounted for by cross-contamination. No enzyme activity or antigen was detected in mitochondrial inner membranes. Our results indicate that the enzyme activity data do not precisely reflect the true enzyme localization, and show an extremely uneven distribution of reductase among different cellular membranes.

Animals↗