Glutamate-Dependent Translational Control of Glutamine Synthetase in Bergmann Glia Cells.

Glutamate is the major excitatory transmitter of the vertebrate brain. It exerts its actions through the activation of specific plasma membrane receptors expressed both in neurons and in glial cells. Recent evidence has shown that glutamate uptake systems, particularly enriched in glia cells, trigger biochemical cascades in a similar fashion as receptors.

Nuclear localization of the dystrophin-associated protein α-dystrobrevin through importin α2/β1 is critical for interaction with the nuclear lamina/maintenance of nuclear integrity.

Although α-dystrobrevin (DB) is assembled into the dystrophin-associated protein complex, which is central to cytoskeletal organization, it has also been found in the nucleus. Here we delineate the nuclear import pathway responsible for nuclear targeting of α-DB for the first time, together with the importance of nuclear α-DB in determining nuclear morphology. We map key residues of the nuclear localization signal of α-DB within the zinc finger domain (ZZ) using various truncated versions of the protein, and site-directed mutagenesis.

The P1/P2 protein heterodimers assemble to the ribosomal stalk at the moment when the ribosome is committed to translation but not to the native 60S ribosomal subunit in Saccharomyces cerevisiae.

The four structural acidic ribosomal proteins that dissociate from P1A/P2B and P1B/P2A heterodimers of Saccharomyces cerevisiae were searched in the 60S ribosomal subunit, the 80S monosome, and the polysomal fractions after ribosome profile centrifugation in sucrose gradients in TMN buffer, and after dissociation of monosomes and polysomes to small and large ribosomal subunits in LMS buffer. Analysis by isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting of these fractions or the purified acidic protein samples showed eight bands that correspond to the acidic ribosomal proteins in the 60S dissociated subunits of the 80S monosome and polysomes. After samples had been radiolabeled with (32)P, four bands were shown to correspond to the phosphorylated form of the acidic ribosomal proteins located in the 80S monosome and the polysomes.

Glutamate regulates eEF1A phosphorylation and ribosomal transit time in Bergmann glial cells.

Glutamate, the major excitatory transmitter in the vertebrate brain, is involved in neuronal development and synaptic plasticity. Glutamatergic stimulation leads to differential gene expression patterns in neuronal and glial cells. A glutamate-dependent transcriptional control has been established for several genes.

Heterologous expression and biochemical characterization of an alpha1,2-mannosidase encoded by the Candida albicans MNS1 gene.

Protein glycosylation pathways, commonly found in fungal pathogens, offer an attractive new area of study for the discovery of antifungal targets. In particular, these post-translational modifications are required for virulence and proper cell wall assembly in Candida albicans, an opportunistic human pathogen. The C.

Kex2 protease converts the endoplasmic reticulum alpha1,2-mannosidase of Candida albicans into a soluble cytosolic form.

Cytosolic alpha-mannosidases are glycosyl hydrolases that participate in the catabolism of cytosolic free N-oligosaccharides. Two soluble alpha-mannosidases (E-I and E-II) belonging to glycosyl hydrolases family 47 have been described in Candida albicans. We demonstrate that addition of pepstatin A during the preparation of cell homogenates enriched alpha-mannosidase E-I at the expense of E-II, indicating that the latter is generated by proteolysis during cell disruption.

Endoplasmic reticulum alpha-glycosidases of Candida albicans are required for N glycosylation, cell wall integrity, and normal host-fungus interaction.

The cell surface of Candida albicans is enriched in highly glycosylated mannoproteins that are involved in the interaction with the host tissues. N glycosylation is a posttranslational modification that is initiated in the endoplasmic reticulum (ER), where the Glc(3)Man(9)GlcNAc(2) N-glycan is processed by alpha-glucosidases I and II and alpha1,2-mannosidase to generate Man(8)GlcNAc(2). This N-oligosaccharide is then elaborated in the Golgi to form N-glycans with highly branched outer chains rich in mannose.

Conversion of alpha1,2-mannosidase E-I from Candida albicans to alpha1,2-mannosidase E-II by limited proteolysis.

Previous studies demonstrated the presence in Candida albicans ATCC 26555 of two soluble alpha1,2-mannosidases: E-I and E-II. In contrast, in the C. albicans CAI-4 mutant only E-I was detected and it could be processed by a membrane-bound proteolytic activity from the ATCC 26555 strain, generating an active 43 kDa polypeptide.

Loss of DNA: a plausible molecular level explanation for crustacean neuropeptide gene evolution.

Alignment of nucleotides of APGWamide, RPCH and AKH genes gives region stretches (common regions) present in all family member variants. Common regions were separated by gap sections in the larger variants of family members. Consensus sequences for single polynucleotides from virtual hybrid molecules of DNA were obtained by joining the common regions of DNA and deleting the extra DNA nucleotides.

Purification of soluble alpha1,2-mannosidase from Candida albicans CAI-4.

A soluble alpha-mannosidase from Candida albicans CAI-4 was purified by conventional methods of protein isolation. Analytical electrophoresis of the purified preparation revealed two polypeptides of 52 and 27 kDa, the former being responsible for enzyme activity. The purified, 52 kDa enzyme trimmed Man9GlcNAc2, producing Man8GlcNAc2 isomer B and mannose, and was inhibited preferentially by 1-deoxymannojirimycin.

Circadian oscillations of RPCH gene expression in the eyestalk of the crayfish Cherax quadricarinatus.

The RPCH and beta-actin cDNAs from the crayfish Cherax quadricarinatus were amplified, cloned and sequenced. The primary structure sequences of these cDNAs were compared to other members of the AKH/RPCH family. Fluctuations in the amount of the C.

Hydrolysis of Man9GlcNAc2 and Man8GlcNAc2 oligosaccharides by a purified alpha-mannosidase from Candida albicans.

A soluble alpha-mannosidase from Candida albicans was purified to homogeneity by sequential size exclusion, ion exchange, and affinity chromatographies in columns of Sepharose CL6B, DEAE Bio-Gel A, and Concanavalin A Sepharose 4B, respectively. Analytical electrophoresis of the purified preparation in 10% SDS-polyacrylamide gels stained with Coomassie blue revealed a single polypeptide of 43 kDa that was responsible for enzyme activity. The purified enzyme primarily trimmed Man(9)GlcNAc(2) to produce Man(8)GlcNAc(2) isomer B and mannose as a function of time of incubation up to 12 h at 37 degrees C.

A possible molecular ancestor for mollusk APGWamide, insect adipokinetic hormone, and crustacean red pigment concentrating hormone.

Precursor structures of various members of the neuropeptide family adipokinetic hormone/red pigment concentrating hormone (AKH/RPCH) of mandibular arthropods and the APGWamide family of mollusks were compared. Amino acid alignments showed a common overall architecture (signal peptide, active peptide, related peptide), with a similar alpha helix-random coil secondary structure. DNA sequence alignments revealed close similarities between the genes encoding for the peptides of the two families.

The genomic organization of the open reading frame of the red pigment concentrating hormone gene in the blue crab Callinectes sapidus.

The open reading frame (ORF) of the gene for the precursor of the octapeptide Red Pigment Concentrating Hormone (RPCH) from the blue crab Callinectes sapidus was cloned by PCR with oligonucleotides targeted to the initiation and the end of the translation coding sequences. A 272 bp intron was characterized between nucleotides 343 and 344 of the reported cDNA, present in the region coding for the last amino acids of the precursor related peptide of RPCH. The intron genomic structure here described is similar to that reported for the gene coding for the Adipokinetic Hormone (AKH) of the grasshopper Schistocerca nitans.

Isolation and analysis of a new developmentally regulated gene from amastigotes of Leishmania mexicana mexicana.

Leishmania differentiates from the promastigote to the amastigote stage during its digenetic life cycle. Characterization of the developmentally regulated genes during that process would help to elucidate the mechanisms of gene regulation. In this study, specific fragments of mRNAs from the amastigote stage of L.

A halotolerant mutant of Saccharomyces cerevisiae.

FRD, a nuclear and dominant spontaneous mutant of Saccharomyces cerevisiae capable of growing in up to 2 M NaCl, was isolated. Compared with parental cells, the mutant cells have a lower intracellular Na+/K+ ratio, shorter generation times in the presence of 1 M NaCl, and alterations in gene expression.

Eukaryotic acidic phosphoproteins interact with the ribosome through their amino-terminal domain.

Variable-size fragments of the four yeast acidic ribosomal protein genes rpYP1 alpha, rpYP1 beta, rpYP2 alpha and rpYP2 beta were fused to the LacZ gene in the vector series YEp356-358. The constructs were used to transform wild-type Saccharomyces cerevisiae and several gene-disrupted strains lacking different acidic ribosomal protein genes. The distribution of the chimeric proteins between the cytoplasm and the ribosomes, tested as beta-galactosidase activity, was estimated.

Alterations in the cell wall of Saccharomyces cerevisiae induced by the alpha sex factor or a mutation in the cell cycle.

We performed experiments in parallel to study the rate of synthesis of cell wall polysaccharides and the activity of glycosyl transferases in Saccharomyces cerevisiae after arrest of a cdc 28 mutant in G1 phase by either addition of alpha-factor or transfer to the non-permissive temperature. Both effectors brought about similar time-dependent increases in the rate of synthesis and deposition of the cell wall polysaccharides chitin, glucan and mannan. These changes in cell wall composition were accompanied by an increase in the specific activities of glucan and chitin synthetases.

The yeast 5S rRNA binding ribosomal protein YL3 is phosphorylated in vivo.

The 5S rRNA-ribosomal protein YL3 ribonucleoprotein particle was isolated from yeast cells labeled with [32P]orthophosphate. The protein moiety was purified and found to be radioactive. Labeled phosphoserine was detected after partial hydrolysis of the protein.

The acidic ribosomal proteins as regulators of the eukaryotic ribosomal activity.

The acidic proteins, A-proteins, from the large ribosomal subunit of Saccharomyces cerevisiae grown under different conditions have been quantitatively estimated by ELISA tests using rabbit sera specific for these polypeptides. It has been found that the amount of A-protein present in the ribosome is not constant and depends on the metabolic state of the cell. Ribosomes from exponentially growing cultures have about 40% more of these proteins than those from stationary phase.

Isolation and partial characterization of 2-microns yeast plasmid as a transcriptionally active minichromosome.

Yeast cell extracts from 2-microns-containing strains (cir+) showed higher transcriptional activity than their corresponding isogenic sets (cir0). These extracts were used to purify transcriptionally active 2-microns minichromosomes in a sucrose gradient. Minichromosomes were transcribed in vitro and, employing hybridization techniques, the RNA synthesized was shown to present 2-microns-specific sequences.

P5/P5' the acidic ribosomal phosphoproteins from Saccharomyces cerevisiae.

P5/P5', the most acidic and exchangeable phosphoproteins from the large ribosomal subunit in the yeast Saccharomyces cerevisiae, were purified from the rest of the ribosomal proteins by means of polyacrylamide gel electrophoresis. Antibodies against the pure P5/P5' proteins cross-reacted with polypeptides from the cytoplasm suggesting that a cytoplasmic pool of P5/P5' exist. The cytoplasmic polypeptides immunologically related to P5/P5' are not phosphorylated.

The ribosomal proteins of Saccharomyces cerevisiae. Phosphorylated and exchangeable proteins.

Sixty-seven ribosomal proteins of the yeast Saccharomyces cerevisiae have been distinguished by two-dimensional polyacrylamide gel electrophoresis. Five of the ribosomal proteins are phosphorylated in vivo. One of the phosphorylated proteins appears to correspond to the phosphorylated ribosomal protein, S6, of rat liver.