Biomedical subjects

Brian M Martin

Publications and source records attributed to Brian M Martin.

26 records · Page 2Linked to original sources

PubMed2003

Cystatin C as a cerebrospinal fluid biomarker for pain in humans.

Through a process of subtraction cloning and differential hybridization, we previously identified several new genes whose expression was induced by peripheral inflammation. One of these coded for cystatin C, a secreted cysteine protease inhibitor in the cystatin superfamily. We hypothesized that, concurrent with increased expression in dorsal horn, increased secretion would elevate the cystatin C content in cerebrospinal fluid (CSF) during active pain states. Alterations were assessed by immunoassay and by surface enhanced laser desorption ionization (SELDI) mass spectrometry with either reverse phase or immobilized anti-cystatin C antibody surfaces using CSF from ten age-matched obstetrical patients at term. Five control subjects were scheduled for an elective caesarian section and were not in pain. Another five subjects were in labor for 8.9+/-1h and were in severe pain as assessed with a visual analog scale and the McGill short form questionnaire. The level of cystatin C as measured by immunoassay in the non-pain patients was 2.77+/-0.75 microg/ml and in the pain patients 5.36+/-0.92 microg/ml (P<0.02). The elevation occurred without significant change in total CSF protein or beta-endorphin content. The cystatin C increase also was detectable by SELDI with either raw CSF or after antibody capture. These data are consistent with our previous animal study and the idea that persistent pain induces the synthesis and release of cystatin C in dorsal spinal cord, the surplus of which overflows into the CSF.

Adult↗

PubMed2003

Myoclonic epilepsy in Gaucher disease: genotype-phenotype insights from a rare patient subgroup.

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, presents with a wide spectrum of manifestations. Although Gaucher disease has been divided into three clinical types, patients with atypical presentations continue to be recognized. A careful phenotypic and genotypic assessment of patients with unusual symptoms may help define factors that modify phenotype in this disorder. One such example is a rare subgroup of patients with type 3 Gaucher disease who develop progressive myoclonic epilepsy. We evaluated 16 patients with myoclonic epilepsy, nine of whom were diagnosed by age 4 y with severe visceral involvement and myoclonus, and seven with a more chronic course, who were studied between ages 22 and 40. All of the patients had abnormal horizontal saccadic eye movements. Fourteen different genotypes were encountered, yet there were several shared alleles, including V394L (seen on two alleles), G377S (seen on three alleles), and L444P, N188S, and recombinant alleles (each found on four alleles). V394L, G377S, and N188S are mutations that have previously been associated with non-neuronopathic Gaucher disease. The spectrum of genotypes differed significantly from other patients with type 3 Gaucher disease, where genotypes L444P/L444P and R463C/null allele predominated. Northern blot studies revealed a normal glucocerebrosidase transcript, whereas Western studies showed that the patients studied lacked the processed 56 kD isoform of the enzyme, consistent with neuronopathic Gaucher disease. Brain autopsy samples from two patients demonstrated elevated levels of glucosylsphingosine, a toxic glycolipid, which could contribute to the development of myoclonus. Thus, although there were certain shared mutant alleles found in these patients, both the lack of a shared genotype and the variability in clinical presentations suggest that other modifiers must contribute to this rare phenotype.

Adolescent↗

PubMed2002

The Aes protein and the monomeric alpha-galactosidase from Escherichia coli form a non-covalent complex. Implications for the regulation of carbohydrate metabolism.

Aes, a 36-kDa acetylesterase from Escherichia coli, belongs to the hormone-sensitive lipase family, and it is involved in the regulation of MalT, the transcriptional activator of the maltose regulon. The activity of MalT is depressed through a direct protein-protein interaction with Aes. Although the effect is clear-cut, the meaning of this interaction and the conditions that trigger it still remain elusive. To perform a comparative thermodynamic study between the mesophilic Aes protein and two homologous thermostable enzymes, Aes was overexpressed in E. coli and purified. At the last step of the purification procedure the enzyme was eluted from a Mono Q HR 5/5 column as a major form migrating, anomalously, at 56 kDa on a calibrated Superdex 75 column. A minor peak that contains the Aes protein and a polypeptide of 50 kDa was also detected. By a combined analysis of size-exclusion chromatography and surface-enhanced laser desorption ionization-time of flight mass spectrometry, it was possible to demonstrate the presence in this peak of a stable 87-kDa complex, containing the Aes protein itself and the 50-kDa polypeptide in a 1:1 ratio. The homodimeric molecular species of Aes and of the 50-kDa polypeptide were also detected. The esterase activity associated with the 87-kDa complex, when assayed with p-nitrophenyl butanoate as substrate, proved 6-fold higher than the activity of the major Aes form of 56 kDa. Amino-terminal sequencing highlighted that the 50-kDa partner of Aes in the complex was the alpha-galactosidase from E. coli. The E. coli cells harboring plasmid pT7-SCII-aes and, therefore, expressing Aes were hampered in their growth on a minimal medium containing raffinose as a sole carbon source. Because alpha-galactosidase is involved in the metabolism of raffinose, the above findings suggest a potential role of Aes in the regulation of carbohydrate metabolism in E. coli.

Acetylesterase↗

PubMed2002

The YbgC protein encoded by the ybgC gene of the tol-pal gene cluster of Haemophilus influenzae catalyzes acyl-coenzyme A thioester hydrolysis.

This paper examines the catalytic function of the protein YbgC, encoded by the ybgC gene of the tol-pal gene cluster in Haemophilus influenzae. The YbgC protein, a homologue of the Pseudomonas sp. strain CBS3 4-hydroxybenzoyl-coenzyme A thioesterase, conserves the active site Asp residue associated with thioesterase activity. The H. influenzae ybgC gene was cloned and overexpressed in Escherichia coli. The recombinant protein was purified and tested for thioesterase activity towards acyl-CoA and acyl-N-acetylcysteamine thioesters. The YbgC protein catalyzes the hydrolysis of short chain aliphatic acyl-CoA thioesters, while the D18N YbgC mutant protein (prepared to serve as a control) does not.

Acyl Coenzyme A↗

PubMed2002

Glucosylsphingosine accumulation in tissues from patients with Gaucher disease: correlation with phenotype and genotype.

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, presents with a wide spectrum of clinical manifestations including neuronopathic and non-neuronopathic forms. While the lipid glucosylceramide is stored in both patients with Gaucher disease and in a null allele mouse model of Gaucher disease, elevated levels of a second potentially toxic substrate, glucosylsphingosine, are also found. Using high performance liquid chromatography, glucosylsphingosine levels were measured in tissues from patients with type 1, 2, and 3 Gaucher disease. Glucosylsphingosine was measured in 16 spleen samples (8 type 1; 4 type 2; and 4, type 3) and levels ranged from 54 to 728 ng/mg protein in the patients with type 1 disease, 133 to 1200 ng/mg protein in the patients with type 2, and 109 to 1298 ng/mg protein in the type 3 samples. The levels of splenic glucosylsphingosine bore no relation to the type of Gaucher disease, the age of the patient, the genotype, nor the clinical course. In the same patients, hepatic glucosylsphingosine levels were lower than in spleen. Glucosylsphingosine was also measured in brains from 13 patients (1 type 1; 8 type 2; and 4 type 3). While the glucosylsphingosine level in the brain from the type 1 patient, 1.0 ng/mg protein, was in the normal range, the levels in the type 3 samples ranged from 14 to 32 ng/mg protein, and in the type 2 samples from 24 to 437 ng/mg protein, with the highest values detected in two fetuses with hydrops fetalis. The elevated levels found in brains from patients with neuronopathic Gaucher disease support the hypothesis that glucosylsphingosine may contribute to the nervous system involvement in these patients.

Adolescent↗

PubMed2002

The characterization and purification of a human transcription factor modulating the glutathione peroxidase gene in response to oxygen tension.

An oxygen responsive transcription factor regulating human glutathione peroxidase gene (GPx) through two oxygen responsive elements (ORE I and ORE2) has been purified and characterized by sequence-specific DNA affinity chromatography. The DNA binding activity, termed Oxygen Responsive Element Binding Protein (OREBP), was partially represented by a 77 kD polypeptide (p70) possessing a blocked N-terminus. The p70 subunit co-eluted with an 86 kD subunit (p80) from affinity columns. N-terminal sequencing analysis of the 86 kD component revealed that this protein represented the larger member of the Ku antigen complex. The identity of the purified 77 kD subunit was determined by Western blot analysis using an antibody directed against the p70 protein. In addition to binding the GPx-ORE, the OREBP was itself regulated by oxygen tension. It was found that the abundance of the ORE binding activity was decreased in cells maintained at low oxygen tension (40 mm Hg). Anti-Ku-antibodies specifically supershifted the OREBP-ORE DNA complex. These observations further add to the numerous nuclear roles of the Ku-transcription factor.

Antibodies↗

PubMed2002

The 2-aminoethylphosphonate-specific transaminase of the 2-aminoethylphosphonate degradation pathway.

The 2-aminoethylphosphonate transaminase (AEPT; the phnW gene product) of the Salmonella enterica serovar Typhimurium 2-aminoethylphosphonate (AEP) degradation pathway catalyzes the reversible reaction of AEP and pyruvate to form phosphonoacetaldehyde (P-Ald) and L-alanine (L-Ala). Here, we describe the purification and characterization of recombinant AEPT. pH rate profiles (log V(m) and log V(m)/K(m) versus pH) revealed a pH optimum of 8.5. At pH 8.5, K(eq) is equal to 0.5 and the k(cat) values of the forward and reverse reactions are 7 and 9 s(-1), respectively. The K(m) for AEP is 1.11 +/- 0.03 mM; for pyruvate it is 0.15 +/- 0.02 mM, for P-Ald it is 0.09 +/- 0.01 mM, and for L-Ala it is 1.4 +/- 0.03 mM. Substrate specificity tests revealed a high degree of discrimination, indicating a singular physiological role for the transaminase in AEP degradation. The 40-kDa subunit of the homodimeric enzyme is homologous to other members of the pyridoxalphosphate-dependent amino acid transaminase superfamily. Catalytic residues conserved within well-characterized members are also conserved within the seven known AEPT sequences. Site-directed mutagenesis demonstrated the importance of three selected residues (Asp168, Lys194, and Arg340) in AEPT catalysis.

Aminoethylphosphonic Acid↗

PubMedDate not supplied

Advancing cancer biotherapy with proteomics.

Proteomics is becoming increasingly important for cancer biotherapy. The development of high-throughput platforms now allows the analysis of multiple proteins from small quantities of material. While these techniques are being used to discover new biomarkers, they are particularly important for assessing complex biologic processes such as immunotherapy for cancer. Recent advances in this field are reviewed, as well as the use of proteomics to assess the effectiveness and toxicities of high-dose IL-2 cancer therapy. Proteomics is becoming useful in assessing cancer biotherapies and in unraveling their mechanisms of action. High-throughput proteomic technologies have now advanced to a stage where they have the potential to become effective discovery tools for biomarkers/predictors of disease, disease recurrence, and response to therapy.

Humans↗