Essential role for gamma-tubulin in the acentriolar female meiotic spindle of Drosophila.

Microtubule nucleation in vivo requires gamma-tubulin, a highly conserved component of microtubule-organizing centers. In Drosophila melanogaster there are two gamma-tubulin genes, gammaTUB23C and gammaTUB37C. Here we report the cytological and molecular characterization of the 37C isoform. By Western blotting, this protein can only be detected in ovaries and embryos. Antibodies against this isoform predominantly label the centrosomes in embryos from early cleavage divisions until cycle 15, but fail to reveal any particular localization of gamma-tubulin in the developing egg chambers. The loss of function of this gene results in female sterility and has no effect on viability or male fertility. Early stages of oogenesis are unaffected by mutations in this gene, as judged both by morphological criteria and by localization of reporter genes, but the female meiotic spindle is extremely disrupted. Nuclear proliferation within the eggs laid by mutant females is also impaired. We conclude that the expression of the 37C gamma-tubulin isoform of D. melanogaster is under strict developmental regulation and that the organization of the female meiotic spindle requires gamma-tubulin.     

Differential expression of cyclooxygenase-1 and cyclooxygenase-2 in the cornea during wound healing

Cyclooxygenases (COXs) are the key enzymes in the production of prostaglandins (PGs) and exist in two isoforms. Isoform 1 (COX-1) is constitutively expressed in most tissues, whereas cyclooxygenase-2 (COX-2) is rapidly induced by a variety of different stimuli. In this study, we have quantitatively analyzed mRNA expression of COX-1 and COX-2 and protein distribution during corneal reparative processes after wound. Total RNA was isolated from cornea samples of New Zealand rabbits that had been subjected to corneal wound by mechanical brush scraping. Quantification of RT-PCR results was made by using a DNA mimic approach. The localization and expression of the enzymes was studied by immunocytochemistry and Western blotting. In normal corneas COX-1 is expressed throughout the cornea in the whole tissue, while COX-2 is strongly expressed in stromal keratocytes. Following injury, COX-2 levels drastically increase and, at least in the epithelium, COX-2 becomes the predominant isoform of cyclooxygenases at an early stage of healing. Moreover, in the epithelium COX-2 is expressed predominantly by those cells close to the wound. These cells become migratory and move toward the injured area. In contrast, COX-1 levels remain unaffected in all corneal tissues. The system returns to the pre-injury state in about 24h. Thus, the expression of COX-2 in the corneal epithelium during wound repair is tightly regulated both temporally and spatially.     

Identification of the full-length AE2 (AE2a) isoform as the Golgi-associated anion exchanger in fibroblasts.

Na(+)-independent Cl(-)/HCO(3)(-) exchangers (AE1, AE2, AE3) are generally known as ubiquitous, multispanning plasma membrane proteins that regulate intracellular pH and transepithelial acid-base balance in animal tissues. However, previous immunological evidence has suggested that anion exchanger (AE) proteins may also be present in intracellular membranes, including membranes of the Golgi complex and mitochondria. Here we provide several lines of evidence to show that an AE protein is indeed a resident of the Golgi membranes and that this protein corresponds to the full-length AE2a isoform in fibroblasts. First, both the N- and C-terminal antibodies to AE2 (but not to AE1) detected an AE protein in the Golgi membranes. Golgi localization of this AE2 antigen was evident also in cycloheximide-treated cells, indicating that it is a true Golgi-resident protein. Second, our Northern blotting and RT-PCR analyses demonstrated the presence of only the full-length AE2a mRNA in cells that show prominent Golgi staining with antibodies to AE2. Third, antisense oligonucleotides directed against the translational initiation site of the AE2a mRNA markedly inhibited the expression of the endogenous AE2 protein in the Golgi. Finally, transient expression of the GFP-tagged full-length AE2a protein resulted in predominant accumulation of the fusion protein in the Golgi membranes in COS-7 and CHO-K1 cells. Golgi localization of the AE2a probably involves its oligomerization and/or association with the recently identified Golgi membrane skeleton, because a substantial portion of both the endogenous AE2a and the GFP-tagged fusion protein resisted detergent extraction in cold. (J Histochem Cytochem 49:259-269, 2001)     

Immunocytochemical localization of ribonuclease in yolk granules of adult Rana cttesbeiana oocytes

To determine the localization of the pyrimidine-guanine sequence-specific ribonuclease in Rana catesbeiana (bullfrog) oocytes, the RNase was first isolated and used to prepare a specific rabbit antiserum. Only one protein of similar molecular size to the RNase was immunoprecipitated from ovary homogenate by the antiserum, but two bands were observed by Western blotting analysis. These two proteins were shown by further purification of antibody and Western blotting analysis to have similar antigenicity. Immunoprecipitation and Western blotting of tissue homogenates showed that the RNase was found predominantly in the ovary, but not in other tissues. The specific localization of the RNase was determined by immuno-electron microscopy of oocyte sections incubated with the specific antiserum; the yolk granules, but not other organelles, were found to contain the RNase. Most of the RNase was evenly distributed in the lateral amorphous area of the yolk granule but not in the central yolk crystal area which contains stored vitellogenin proteins. Our results indicate that the RNase is compartmentalized in the yolk granules of oocytes, which might prevent damage to cellular RNAs.     

Endotoxaemia in rats: role of leukocyte sequestration in rapid pulmonary nitric oxide synthase-2 expression.

Nitric oxide (NO), depending on the amount, time and source of generation may exert both, protective and deleterious actions during endotoxic acute lung injury (ALI). Evaluation of the expression and localization of NOS isoforms in the lung of lipopolysaccharide (LPS)-treated rats may contribute to understanding the role of NO in pathogenesis of ALI. Tissue samples (lung, heart, liver, kidney and spleen) as well as peripheral blood polymorphonuclear cells (PMNs) were collected from control male Wistar rats and LPS - treated animals, 15, 30, 60, 120 and 180 min after LPS injection (2 mg kg(-1) min(-1) for 10 minutes, i.v.). Levels of NOS-2 and NOS-3 mRNA and protein in tissues and PMNs were estimated by RT-PCR, Northern blotting and Western blotting. Additionally, myeloperoxidase (MPO) activity in tissue samples was assayed. NOS-3 mRNA as well as protein were detected in lungs of control animals; pulmonary NOS-3 expression was not influenced by LPS. The induction of NOS-2 mRNA in rat lungs and in PMNs isolated from peripheral blood was observed 15 minutes after LPS challenge. In contrast, increase of NOS-2 mRNA in the heart, kidneys, liver and spleen was observed 2-3 hours after LPS injection. In all tissues rise in NOS-2 mRNA was followed after 1-2 hours by increase of NOS-2 protein. Importantly, progressive leukocyte sequestration in the lung parenchyma that started as early as 15 min after LPS injection was revealed only in the lungs; in other organs no significant changes in MPO activity were detected up to 180 min after LPS injection. In conclusion, infusion of LPS caused much more rapid expression of NOS-2 in lungs as compared to the heart, kidneys, liver and spleen. Early induction of NOS-2 may depend on the LPS-stimulated rapid neutrophil sequestration within lung vasculature and fast induction of NOS-2 in sequestrated neutrophils.     

Characterization of a long isoform of IL-1R8 (TIR8/SIGIRR)

IL-1R8, also known as SIGIRR or TIR8, is a trans-membrane protein belonging to the IL-1 receptor family. The human gene includes ten exons, and alternative splicing can result in different isoforms. We, herein, characterized a longer isoform of IL-1R8 containing an in-frame additional sequence between the TIR domain and the C-terminal portion of the protein. IL-1R8 Long (IL-1R8L1) mRNA was specifically expressed and regulated in distinct cell lines, in a manner similar to the classic isoform. Overexpression of IL-1R8L1 resulted in the production of a corresponding protein that showed a pattern of cell localization similar to the classic isoform. An antibody directed against an IL-1R8L1 specific peptide, detected this novel isoform in different cell lines and tissues where this protein may complement the anti-inflammatory functions of classic IL-1R8.     

Cloning and functional characterization of the human GLUT7 isoform SLC2A7 from the small intestine

Facilitated glucose transporters (GLUTs) mediate transport of sugars across cell membranes by using the chemical gradient of sugars as the driving force. Improved cloning techniques and database analyses have expanded this family of proteins to a total of 14 putative members. In this work a novel hexose transporter isoform, GLUT7, has been cloned from a human intestinal cDNA library by using a PCR-based strategy (GenBank accession no. AY571960). The encoded protein is comprised of 524 amino acid residues and shares 68% similarity and 53% identity with GLUT5, its most closely related isoform. When GLUT7 was expressed in Xenopus oocytes, it showed high-affinity transport for glucose (K(m) = 0.3 mM) and fructose (IC(50) = 0.060 mM). Galactose, 2-deoxy-d-glucose, and xylose were not transported. Uptake of 100 microM d-glucose was not inhibited by 200 microM phloretin or 100 microM cytochalasin B. Northern blotting indicated that the mRNA for GLUT7 is present in the human small intestine, colon, testis, and prostate. Western blotting and immunohistochemistry of rat tissues with an antibody raised against the predicted COOH-terminal sequence confirmed expression of the protein in the small intestine and indicated that the transporter is predominantly expressed in the enterocytes' brush-border membrane. The unusual substrate specificity and close sequence identity with GLUT5 suggest that GLUT7 represents an intermediate between class II GLUTs and the class I member GLUT2. Comparison between these proteins may provide key information as to the structural determinants for the recognition of fructose as a substrate.     

Oxygen restriction of neonate rats elevates neuregulin-1alpha isoform levels: possible relationship to schizophrenia

Neuregulin-1 (NRG-1), a replicated gene in schizophrenia-association studies, exhibits six mRNA-types and two types of the EGF-like domain, alpha and beta. The beta-isoform was extensively studied, less is known about the extent and specific localization of adult brain NRG-1alpha. NRG-1alpha protein levels were reported reduced in postmortem prefrontal-cortex of schizophrenia patients. NRG-1 type I mRNA levels were found higher in postmortem brain in schizophrenia. In an attempt to decipher between a genetic or environmental involvement in the differences in NRG-1 levels in postmortem brain in schizophrenia, and since obstetric complications were suggested non-genetic risk-factors of schizophrenia, we studied the effect of perinatal hypoxia in rats on brain NRG-1alpha protein levels. Seven-day-old rats were exposed to hypoxia versus air. Frontal-cortex levels of NRG-1alpha isoform were quantified at adulthood by Western blotting. Frontal-cortex NRG-1alpha was 32% elevated in hypoxia-exposed rats. The data support the role of non-genetic factors, e.g. oxygen restriction, in the expression of genes associated with schizophrenia.     

Experimental testing of predicted myristoylation targets involved in asymmetric cell division and calcium-dependent signalling

Evolutionary conservation of N-terminal N-myristoylation within protein families indicates significant functional impact of this lipid posttranslational modification for function. In the MYRbase study (Maurer-Stroh et al. (2004) Genome Biology 5, R21), protein families with relevance to asymmetric cell division in animals and the group of plant calcium-dependent protein kinases (CPKs) have surfaced with many predicted myristoylated members. Here, we describe experimental in vitro verification of predicted myristoylation and explore its impact on subcellular localization for these targets in vivo. Our results confirm that, indeed, Numb isoform A, Neuralized isoforms C and D from Drosophila melanogaster and two Neuralized-like homologues from Mus musculus have the capability for N-terminal myristoylation in vitro and in vivo (in fly tissue and in mouse 3T3 cells respectively) whereas other isoforms such as Neuralized A and B have not. The latter two cases are an example of different potential of various isoforms for posttranslational modifications. Additionally, the Arabidopsis thaliana CDPKs CPK6, CPK9 and CPK13 are shown to be substrates for myristoylation in vitro, which also affects their subcellular localization (in Arabidopsis protoplasts and tobacco leaves). At the same time, CPK6 and CPK13 do not appear to be substrates of a NMT1-like enzyme; the reasons for differing substrate specificities of NMT homologues in plants are derived from the evolutionary divergence of their N-myristoyl transferase sequences. As a methodical advance, we describe a fast and very sensitive technique (compared to traditional autoradiography) for in vitro testing of myristoylation based on thin layer chromatography read-out of the incorporated radioactive myristoyl anchor with subsequent Western blotting detection for protein yield determination using the same membrane.     

MCT1 confirmed in rat striated muscle mitochondria.

We sought to test the hypothesis that monocarboxylate transporter isoform 1 (MCT1) is the inner mitochondrial membrane lactate/pyruvate transporter, and, as such, contributes to functioning of the intracellular lactate shuttle. However, presence of a mammalian mitochondrially localized MCT1 (mMCT1) has been contested. We sought to confirm by Western blotting the mitochondrial localization of MCT1 in rat cardiac, soleus, and extensor digitorum longus muscles utilizing three different cell fractionation methods and three different antibodies. We performed Western blotting using antibodies to cell membrane glucose transporter isoform GLUT1, inner mitochondrial constituent cytochrome oxidase, the monocarboxylate transporter protein chaperone CD147, as well as custom and commercially available MCT1 antibodies. Western blots demonstrated similar results with each MCT1 antibody and two of three methods of fractionation. MCT1 was found in the mitochondria, as well as in the sarcolemmal membrane and whole muscle homogenates. Probing with GLUT1 and CD147 demonstrated that mitochondrial fractions were not contaminated with sarcolemmal remnants. Probing with cytochrome oxidase showed mitochondrial localization of MCT1. Comparison of these results to the findings of others indicates that the most likely source of discrepancy is the cell fractionation procedure utilized.