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R Beavis

Publications and source records attributed to R Beavis.

10 recordsLinked to original sources
PubMed1998

Structural studies of alpha-N-acetylgalactosaminidase: effect of glycosylation on the level of expression, secretion efficiency, and enzyme activity.

alpha-N-Acetylgalactosaminidase (alphaNAGAL, EC 3.2.1.49) is an exoglycosidase specific for the hydrolysis of terminal alpha-linked N-acetylgalactosamine from oligosaccharide chains. After cloning of its cDNA, the recombinant alphaNAGAL (ralphaNAGAL) was produced in Pichia pastoris, a methylotrophic yeast strain. The enzyme was hyperglycosylated by the host cells, resulting in a protein with a molecular mass of approximately 50 kDa, which was 7 kDa larger than that of its native counterpart. When deglycosylated with endoglycosidase H under nondenaturing conditions, ralphaNAGAL remained fully active, suggesting that the glycosylation is not required for enzyme activity. Data derived from mass spectrometry indicated that all three putative N-glycosylation sites [Asn residues at positions 161 (N1), 185 (N2), and 369 (N3)] in the enzyme were glycosylated, and a high-mannose structure, which was possibly phosphorylated, was attached to the sites N1 and N2. In order to examine the effect of individual N-linked oligosaccharide chains on the expression of ralphaNAGAL in P. pastoris, we mutated each of the N-glycosylation sites, as well as all three sites in the same protein molecule, by substituting the Asn with a Gln residue. The results indicate that ralphaNAGAL mutations in any of the three glycosylation sites, N2 being the most profound, impaired the expression level, altered subcellular distribution, and decreased the efficiency of secretion. Our data suggest that the N-glycosylation of ralphaNAGAL expressed in P. pastoris may be important in protein folding and resistance to protease degradation during protein synthesis, although it is apparently not required for enzyme activity.

Animals↗
PubMed1997

Bacterial interference caused by autoinducing peptide variants.

The synthesis of virulence factors and other extracellular proteins by Staphylococcus aureus is globally controlled by the agr locus, which encodes a two-component signaling pathway whose activating ligand is an agr-encoded autoinducing peptide. The cognate peptides produced by some strains inhibit the expression of agr in other strains, and the amino acid sequences of peptide and receptor are markedly different between such strains, suggesting a hypervariability-generating mechanism. Cross-inhibition of gene expression represents a type of bacterial interference that could be correlated with the ability of one strain to exclude others from infection or colonization sites, or both.

Amino Acid Sequence↗
PubMed1997

Alzheimer's soluble amyloid beta is a normal component of human urine.

Soluble A beta (Sa beta) is normally present at a low concentration in human plasma and cerebrospinal fluid. Although the factors involved in the regulation of Sa beta plasma levels are still unknown, we have explored its excretion in the urine as one of the possible homeostatic mechanisms. The presence of Sa beta in the urine was investigated via immunoprecipitation experiments with anti-A beta antibodies followed by detection and identification by immunoblot, MALDI mass spectrometry and sequence analysis. Soluble A beta (4.3 kDa) immunoreactivity was present in the urine of normal donors, Down's syndrome individuals as well as in patients with renal disorders exhibiting glomerular or mixed proteinuria. Edman degradation of the immunoprecipitated material yielded the intact A beta N-terminus and mass spectra analysis indicated the existence of a major component at mlz 4327, corresponding to the molecular mass of A beta1-40. Semiquantitative data obtained from the immunoprecipitation experiments indicate that under normal conditions the daily excretion of intact Sa beta in the urine represents less than 1% of the circulating pool.

Amyloid beta-Peptides↗
PubMed1997

alpha-Latrotoxin stimulates exocytosis by the interaction with a neuronal G-protein-coupled receptor.

alpha-Latrotoxin is a potent stimulator of neurosecretion. Its action requires extracellular binding to high affinity presynaptic receptors. Neurexin I alpha was previously described as a high affinity alpha-latrotoxin receptor that binds the toxin only in the presence of calcium ions. Therefore, the interaction of alpha-latrotoxin with neurexin I alpha cannot explain how alpha-latrotoxin stimulates neurotransmitter release in the absence of calcium. We describe molecular cloning and functional expression of the calcium-independent receptor of alpha-latrotoxin (CIRL), which is a second high affinity alpha-latrotoxin receptor that may be the major mediator of alpha-latrotoxin's effects. CIRL appears to be a novel orphan G-protein-coupled receptor, a member of the secretin receptor family. In contrast with other known serpentine receptors, CIRL has two subunits of the 120 and 85 kDa that are the result of endogenous proteolytic cleavage of a precursor polypeptide. CIRL is found in brain where it is enriched in the striatum and cortex. Expression of CIRL in chromaffin cells increases the sensitivity of the cells to the effects of alpha-latrotoxin, demonstrating that this protein is functional in coupling to secretion. Syntaxin, a component of the fusion complex, copurifies with CIRL on an alpha-latrotoxin affinity column and forms stable complexes with this receptor in vitro. Interaction of CIRL with a specific presynaptic neurotoxin and with a component of the docking-fusion machinery suggests its role in regulation of neurosecretion.

Amino Acid Sequence↗
PubMed1996

The length of amyloid-beta in hereditary cerebral hemorrhage with amyloidosis, Dutch type. Implications for the role of amyloid-beta 1-42 in Alzheimer's disease.

In hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D), a genetic variant (E22Q) of amyloid beta (Abeta) accumulates predominantly in the small vessels of leptomeninges and cerebral cortex, leading to fatal strokes in the fifth or sixth decade of life. Abeta deposition in the neuropil occurs mainly in the form of preamyloid, Congo red negative deposits, while mature neuritic plaques and neurofibrillary tangles, hallmark lesions in Alzheimer's disease (AD), are characteristically absent. A recent hypothesis regarding the pathogenesis of AD states that Abeta extending to residues 42-43 (as opposed to shorter species) can seed amyloid formation and trigger the development of neuritic plaques followed by neuronal damage in AD. We characterized biochemically and immunohistochemically Abeta from three cases of HCHWA-D to determine its length in vascular and parenchymal deposits. Mass spectrometry of formic acid-soluble amyloid, purified by size-exclusion gel chromatography, showed that Abeta 1-40 and its carboxyl-terminal truncated derivatives were the predominant forms in leptomeningeal and cortical vessels. Abeta 1-42 was a minor component in these amyloid extracts. Immunohistochemistry with antibodies S40 and S42, specific for Abeta ending at Val-40 or Ala-42, respectively, were consistent with the biochemical data from vascular amyloid. In addition, parenchymal preamyloid lesions were specifically stained with S42 and were not labeled by S40, in agreement with the pattern reported for AD, Down's syndrome, and aged dogs. Our results suggest that in HCHWA-D the carboxyl-terminal Abeta heterogeneity is due to limited proteolysis in vivo. Moreover, they suggest that Abeta species ending at Ala-42 may not be critical for the seeding of amyloid formation and the development of AD-like neuritic changes.

Alzheimer Disease↗
PubMed1996

The calcium-independent receptor of alpha-latrotoxin is not a neurexin.

alpha-Latrotoxin (alpha-LTx), a vertebrate neurotoxin isolated from Black Widow Spider venom, causes massive spontaneous neurotransmitter release. The molecular mechanism(s) by which the toxin exerts its effect is largely unknown. Here we report identification and purification of a novel membrane receptor with high affinity for alpha-LTx. Unlike neurexin Ia, a previously described high affinity alpha-LTx receptor, this novel protein binds alpha-LTx independently of Ca2+ presence and therefore may be a mediator of the calcium-independent stimulation of neurotransmitter release by alpha-latrotoxin. The major protein component of calcium-independent alpha-LTx receptors is a novel M(r) 120,000 protein which does not belong to the neurexin family. Among several tissues tested, the M(r) 120,000 protein was found only in brain.

Amino Acid Sequence↗
PubMed1991

Hemoglobin degradation in the human malaria pathogen Plasmodium falciparum: a catabolic pathway initiated by a specific aspartic protease.

Hemoglobin is an important nutrient source for intraerythrocytic malaria organisms. Its catabolism occurs in an acidic digestive vacuole. Our previous studies suggested that an aspartic protease plays a key role in the degradative process. We have now isolated this enzyme and defined its role in the hemoglobinolytic pathway. Laser desorption mass spectrometry was used to analyze the proteolytic action of the purified protease. The enzyme has a remarkably stringent specificity towards native hemoglobin, making a single cleavage between alpha 33Phe and 34Leu. This scission is in the hemoglobin hinge region, unraveling the molecule and exposing other sites for proteolysis. The protease is inhibited by pepstatin and has NH2-terminal homology to mammalian aspartic proteases. Isolated digestive vacuoles make a pepstatin-inhibitable cleavage identical to that of the purified enzyme. The pivotal role of this aspartic hemoglobinase in initiating hemoglobin degradation in the malaria parasite digestive vacuoles is demonstrated.

Amino Acid Sequence↗
PubMed1987

Redox reactions occurring during secondary ion mass spectrometry of some heteroanthracycline antitumor antibiotic precursors.

Positive and negative secondary ion mass spectra of the title compounds were produced by bombardment of solid deposits by 13 and 23 KeV cesium ions, respectively, and analyzed with a time-of-flight mass spectrometer. In addition to familiar protonation and alkali (mainly sodium) ion addition processes observed in the positive ion spectra and proton abstractions in the negative ion spectra (which enable determination of molecular weight) there is evidence from both spectral modes for oxidation-reduction processes in which species such as [M - H]+, [M - H + Na]+. and [M + 1]- are formed. It is argued that these may be formed rapidly from pre-associated molecules during bombardment/ionization rather than as a result of radiation damage. Sodium ion addition is apparently facilitated by the presence of electron pair donor groups, especially if chelation is possible.

Antibiotics, Antineoplastic↗
PubMed1983

Boyle's machine.

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Anesthesiology↗