Biomedical subjects

Michael H Gelb

Publications and source records attributed to Michael H Gelb.

At least 19 recordsLinked to original sources

PubMed2025

Operationalizing the Wilson-Jungner principles for the genomics era: Consensus recommendations from the International Consortium on Newborn Sequencing.

PURPOSE: For decades, the selection of disorders included in newborn screening (NBS) programs has been guided by principles published by Wilson and Jungner in 1968. As research explores the expansion of conditions included in NBS through genomic sequencing, there is a critical need for updated recommendations to address the opportunities and complexities of genomic data. METHODS: The International Consortium on Newborn Sequencing includes leaders from over 16 research projects investigating genomic NBS across the United Kingdom, Europe, United States, and Oceania. Consortium members were invited to participate in a modified Delphi study, aggregating opinion on the selection of conditions for genomic NBS through 3 rounds of online questionnaires, with feedback provided to participants between rounds. RESULTS: In round 1, 94 participants completed the questionnaire, and 10 of 43 statements reached consensus. In round 2, 81 participants completed the questionnaire, and 14 of 27 statements reached consensus. In round 3, 68 participants completed the questionnaire, and all 10 statements reached 72% or more consensus. CONCLUSION: The 10 consensus recommendations developed in this study can guide future research and public health programs performing genomic NBS. This process also identified key areas of participant discordance, highlighting important topics for future research.

Humans↗

PubMed2006

Resistance mutations at the lipid substrate binding site of Plasmodium falciparum protein farnesyltransferase.

The post-translational farnesylation of proteins serves to anchor a subset of intracellular proteins to membranes in eukaryotic organisms and also promotes protein-protein interactions. This enzymatic reaction is carried out by protein farnesyltransferase (PFT), which catalyzes the transfer of a 15-carbon isoprenoid lipid unit, a farnesyl group, from farnesyl pyrophosphate to the C-termini of proteins containing a CaaX motif. Inhibition of PFT is lethal to the pathogenic protozoa Plasmodium falciparum. Previously, we have shown that parasites resistant to a tetrahydroquinoline (THQ)-based PFT inhibitor BMS-388891 have mutations leading to amino acid substitutions in PFT that map to the peptide substrate binding domain. We now report the selection of parasites resistant to another THQ PFT inhibitor BMS-339941. In whole cell assays sensitivity to BMS-339941 was reduced by 33-fold in a resistant clone, and biochemical analysis demonstrated a corresponding 33-fold increase in the BMS-339941 K(i) for the mutant PFT enzyme. More detailed kinetic analysis revealed that the mutant enzyme required higher concentration of peptide and farnesyl pyrophosphate substrates for optimum catalysis. Unlike previously characterized parasites resistant to BMS-388891, the resistant parasites have a mutation which is predicted to be in a distinct location of the enzymatic pocket, near the farnesyl pyrophosphate binding pocket. This is the first description of a mutation from any species affecting the farnesyl pyrophosphate binding pocket with reduced efficacy of PFT inhibitors. These data provide further support that PFT is the target of THQ inhibitors in P. falciparum and suggest that PFT inhibitors should be combined with other antimalarial agents to minimize the development of resistant parasites.

Alkyl and Aryl Transferases↗

PubMed2006

Sustained activation of proton channels and NADPH oxidase in human eosinophils and murine granulocytes requires PKC but not cPLA2 alpha activity.

The prevailing hypothesis that a signalling pathway involving cPLA(2)alpha is required to enhance the gating of the voltage-gated proton channel associated with NADPH oxidase was tested in human eosinophils and murine granulocytes. This hypothesis invokes arachidonic acid (AA) liberated by cPLA(2)alpha as a final activator of proton channels. In human eosinophils studied in the perforated-patch configuration, phorbol myristate acetate (PMA) stimulation elicited NADPH oxidase-generated electron current (I(e)) and enhanced proton channel gating identically in the presence or absence of three specific cPLA(2)alpha inhibitors, Wyeth-1, pyrrolidine-2 and AACOCF(3) (arachidonyl trifluoromethyl ketone). In contrast, PKC inhibitors GFX (GF109203X) or staurosporine prevented the activation of either proton channels or NADPH oxidase. PKC inhibition during the respiratory burst reversed the activation of both molecules, suggesting that ongoing phosphorylation is required. This effect of GFX was inhibited by okadaic acid, implicating phosphatases in proton channel deactivation. Proton channel activation by AA was partially reversed by GFX or staurosporine, indicating that AA effects are due in part to activation of PKC. In granulocytes from mice with the cPLA(2)alpha gene disrupted (knockout mice), PMA or fMetLeuPhe activated NADPH oxidase and proton channels in a manner indistinguishable from the responses of control cells. Thus, cPLA(2)alpha is not essential to activate the proton conductance or for a normal respiratory burst. Instead, phosphorylation of the proton channel or an activating molecule converts the channel to its activated gating mode. The existing paradigm for regulation of the concerted activity of proton channels and NADPH oxidase must be revised.

Animals↗

PubMed2006

Secreted phospholipase A2 inhibitors are also potent blockers of binding to the M-type receptor.

Mammalian secreted phospholipases A(2) (sPLA(2)s) constitute a family of structurally related enzymes that are likely to play numerous biological roles because of their phospholipid hydrolyzing activity and binding to soluble and membrane-bound proteins, including the M-type receptor. Over the past decade, a number of competitive inhibitors have been developed against the inflammatory-type human group IIA (hGIIA) sPLA(2) with the aim of specifically blocking its catalytic activity and pathophysiological functions. The fact that many of these inhibitors, including the indole analogue Me-Indoxam, inhibit several other sPLA(2)s that bind to the M-type receptor prompted us to investigate the impact of Me-Indoxam and other inhibitors on the sPLA(2)-receptor interaction. By using a Ca(2+) loop mutant derived from a venom sPLA(2) which is insensitive to hGIIA inhibitors but still binds to the M-type receptor, we demonstrate that Me-Indoxam dramatically decreases the affinity of various sPLA(2)s for the receptor, yet an sPLA(2)-Me-Indoxam-receptor complex can form at very high sPLA(2) concentrations. Me-Indoxam inhibits the binding of iodinated mouse sPLA(2)s to the mouse M-type receptor expressed on live cells but also enhances binding of sPLA(2) to phospholipids. Because Me-Indoxam and other competitive inhibitors protrude out of the sPLA(2) catalytic groove, it is likely that the inhibitors interfere with the sPLA(2)-receptor interaction by steric hindrance and to different extents that depend on the type of sPLA(2) and inhibitor. Our finding suggests that the various anti-inflammatory therapeutic effects of sPLA(2) inhibitors may be due not only to inhibition of enzymatic activity but also to modulation of binding of sPLA(2) to the M-type receptor or other as yet unknown protein targets.

Animals↗

PubMed2006

Tandem mass spectrometry for the direct assay of enzymes in dried blood spots: application to newborn screening for mucopolysaccharidosis II (Hunter disease).

BACKGROUND: A treatment for mucopolysaccharidosis II (Hunter syndrome) has recently become available. Therefore, we developed a high-throughput assay method appropriate for newborn screening for the relevant enzyme, iduronate 2-sulfatase. METHODS: We synthesized a new iduronate 2-sulfatase substrate that can be used to assay the enzyme by use of tandem mass spectrometry together with an internal standard. The assay uses a dried blood spot on a newborn screening card as the enzyme source. RESULTS: When the assay was tested on dried blood spots, the iduronate 2-sulfatase activity measured for 13 patients with Hunter syndrome was well below the interval found for 57 randomly chosen newborns. The assay was more sensitive than previously reported iduronate 2-sulfatase assays. CONCLUSIONS: This newly developed tandem mass spectrometry assay has the potential to be adopted for newborn screening of Hunter syndrome. This method also has the potential to be carried out in multiplex fashion to assay several different enzymes relevant to lysosomal storage diseases that are assayed in a single infusion into the mass spectrometer.

Blood Specimen Collection↗

PubMed2006

Metal affinity capture tandem mass spectrometry for the selective detection of phosphopeptides.

We report a new method called metal affinity capture that when coupled with tandem mass spectrometry (MAC-MSMS) allows for the selective detection and identification of phosphopeptides in complex mixtures. Phosphopeptides are captured as ternary complexes with Ga(III) or Fe(III) and N(alpha),N(alpha)-bis(carboxymethyl)lysine (LysNTA) in solution and electrosprayed as doubly or triply charged positive ions. The gas-phase complexes uniformly dissociate to produce a common (LysNTA + H)+ ion that is used as a specific marker in precursor ion scans. The advantages of MAC-MSMS over the current methods of phosphopeptide detection are as follows. (1) MAC-MSMS uses metal complexes that self-assemble in solution at pH <5, which is favorable for the production of positive ions by electrospray. (2) Phosphorylation at tyrosine, serine, and threonine is detected by MAC-MSMS. (3) The phosphopeptide peaks in the mass spectra are encoded with the 69Ga-71Ga isotope pattern for selective recognition in mixtures. Detection by MAC-MSMS of singly and multiply phosphorylated peptides in tryptic digests is demonstrated at low-nanomolar protein concentrations.

Amino Acid Sequence↗

PubMed2006

The first potent inhibitor of mammalian group X secreted phospholipase A2: elucidation of sites for enhanced binding.

Using the X-ray structure of human group X secreted phospholipase A(2) (hGX), we carried out structure-based design of indole-based inhibitors and prepared the compounds using a new synthetic route. The most potent compound inhibited hGX and the mouse orthologue with an IC(50) of 75 nM. This compound is the most potent hGX inhibitor reported to date and was also found to inhibit a subset of the other mouse and human sPLA(2)s.

Animals↗

PubMed2006

Neurotoxicity and other pharmacological activities of the snake venom phospholipase A2 OS2: the N-terminal region is more important than enzymatic activity.

Several snake venom secreted phospholipases A2 (sPLA2s) including OS2 exert a variety of pharmacological effects ranging from central neurotoxicity to anti-HIV activity by mechanisms that are not yet fully understood. To conclusively address the role of enzymatic activity and map the key structural elements of OS2 responsible for its pharmacological properties, we have prepared single point OS2 mutants at the catalytic site and large chimeras between OS2 and OS1, a homologous but nontoxic sPLA2. Most importantly, we found that the enzymatic activity of the active site mutant H48Q is 500-fold lower than that of the wild-type protein, while central neurotoxicity is only 16-fold lower, providing convincing evidence that catalytic activity is at most a minor factor that determines central neurotoxicity. The chimera approach has identified the N-terminal region (residues 1-22) of OS2, but not the central one (residues 58-89), as crucial for both enzymatic activity and pharmacological effects. The C-terminal region of OS2 (residues 102-119) was found to be critical for enzymatic activity, but not for central neurotoxicity and anti-HIV activity, allowing us to further dissociate enzymatic activity and pharmacological effects. Finally, direct binding studies with the C-terminal chimera, which poorly binds to phospholipids while it is still neurotoxic, led to the identification of a subset of brain N-type receptors which may be directly involved in central neurotoxicity.

Amino Acid Sequence↗

PubMed2006

Analysis of expression of secreted phospholipases A2 in mouse tissues at protein and mRNA levels.

Secreted phospholipases A(2) (sPLA(2)) form a group of low-molecular weight enzymes that catalyze the hydrolysis of phospholipids. Some sPLA(2)s are likely to play a role in inflammation, cancer, and as antibacterial enzymes in innate immunity. We developed specific and sensitive time-resolved fluroimmunoassays (TR-FIA) for mouse group (G) IB, GIIA, GIID, GIIE, GIIF, GV and GX sPLA(2)s and measured their concentrations in mouse serum and tissues obtained from both Balb/c and C57BL/6J mice. We also analyzed the mRNA expression of the sPLA(2)s by quantitative real-time reverse transcriptase PCR (qPCR). In most tissues, the concentrations of sPLA(2) proteins corresponded to the expression of sPLA(2)s at the mRNA level. With a few exceptions, the sPLA(2) proteins were found in the gastrointestinal tract. The qPCR results showed that GIB sPLA(2) is synthesized widely in the gastrointestinal tract, including esophagus and colon, in addition to stomach and pancreas. Our results also suggest that the loss of GIIA sPLA(2) in the intestine of GIIA sPLA(2)-deficient C57BL/6J mice is not compensated by other sPLA(2)s under normal conditions. Outside the gastrointestinal tract, sPLA(2)s were expressed occasionally in a number of tissues. The TR-FIAs developed in the current study may serve as useful tools to measure the levels of sPLA(2) proteins in mouse serum and tissues in various experimental settings.

Animals↗

PubMed2006

Heterologous expression of proteins from Plasmodium falciparum: results from 1000 genes.

As part of a structural genomics initiative, 1000 open reading frames from Plasmodium falciparum, the causative agent of the most deadly form of malaria, were tested in an E. coli protein expression system. Three hundred and thirty-seven of these targets were observed to express, although typically the protein was insoluble. Sixty-three of the targets provided soluble protein in yields ranging from 0.9 to 406.6 mg from one liter of rich media. Higher molecular weight, greater protein disorder (segmental analysis, SEG), more basic isoelectric point (pI), and a lack of homology to E. coli proteins were all highly and independently correlated with difficulties in expression. Surprisingly, codon usage and the percentage of adenosines and thymidines (%AT) did not appear to play a significant role. Of those proteins which expressed, high pI and a hypothetical annotation were both strongly and independently correlated with insolubility. The overwhelmingly important role of pI in both expression and solubility appears to be a surprising and fundamental issue in the heterologous expression of P. falciparum proteins in E. coli. Twelve targets which did not express in E. coli from the native gene sequence were codon-optimized through whole gene synthesis, resulting in the (insoluble) expression of three of these proteins. Seventeen targets which were expressed insolubly in E. coli were moved into a baculovirus/Sf-21 system, resulting in the soluble expression of one protein at a high level and six others at a low level. A variety of factors conspire to make the heterologous expression of P. falciparum proteins challenging, and these observations lay the groundwork for a rational approach to prioritizing and, ultimately, eliminating these impediments.

Animals↗

PubMed2006

Identification of the expressed form of human cytosolic phospholipase A2beta (cPLA2beta): cPLA2beta3 is a novel variant localized to mitochondria and early endosomes.

In this study, we identify the principal splice variant of human cytosolic phospholipase A(2)beta (cPLA(2)beta) (also known as Group IVB cPLA(2)) present in cells. In human lung, spleen, and ovary and in a lung epithelial cell line (BEAS-2B), cPLA(2)beta is expressed as a 100-kDa protein, not the 114-kDa form originally predicted. Using RNA interference, the 100-kDa protein in BEAS-2B cells was confirmed to be cPLA(2)beta. BEAS-2B cells contain three different RNA splice variants of cPLA(2)beta (beta1, beta2, and beta3). cPLA(2)beta1 is identical to the previously cloned cPLA(2)beta, predicted to encode a 114-kDa protein. However, cPLA(2)beta2 and cPLA(2)beta3 splice variants are smaller and contain internal deletions in the catalytic domain. The 100-kDa cPLA(2)beta in BEAS-2B cells is the translated product of cPLA(2)beta3. cPLA(2)beta3 exhibits calcium-dependent PLA(2) activity against palmitoyl-arachidonyl-phosphatidylethanolamine and low level lysophospholipase activity but no activity against phosphatidylcholine. Unlike Group IVA cPLA(2)alpha, cPLA(2)beta3 is constitutively bound to membrane in unstimulated cells, localizing to mitochondria and early endosomes. cPLA(2)beta3 is widely expressed in tissues, suggesting that it has a generalized function at these unique sites.

Alternative Splicing↗

PubMed2006

Intracellular actions of group IIA secreted phospholipase A2 and group IVA cytosolic phospholipase A2 contribute to arachidonic acid release and prostaglandin production in rat gastric mucosal cells and transfected human embryonic kidney cells.

Gastric epithelial cells liberate prostaglandin E(2) in response to cytokines as part of the process of healing of gastric lesions. Treatment of the rat gastric epithelial cell line RGM1 with transforming growth factor-alpha and interleukin-1beta leads to synergistic release of arachidonate and production of prostaglandin E(2). Results with highly specific and potent phospholipase A(2) inhibitors and with small interfering RNA show that cytosolic phospholipase A(2)-alpha and group IIA secreted phospholipase A(2) contribute to arachidonate release from cytokine-stimulated RGM1 cells. In the late phase of arachidonate release, group IIA secreted phospholipase A(2) is induced (detected at the mRNA and protein levels), and the action of cytosolic phospholipase A(2)-alpha is required for this induction. Results with RGM1 cells and group IIA secreted phospholipase A(2)-transfected HEK293 cells show that the group IIA phospholipase acts prior to externalization from the cells. RGM1 cells also express group XIIA secreted phospholipase A(2), but this enzyme is not regulated by cytokines nor does it contribute to arachidonate release. The other eight secreted phospholipases A(2) were not detected in RGM1 cells at the mRNA level. These results clearly show that cytosolic and group IIA secreted phospholipases A(2) work together to liberate arachidonate from RGM1 cell phospholipids in response to cytokines.

Animals↗

PubMed2006

Protein cross-linking analysis using mass spectrometry, isotope-coded cross-linkers, and integrated computational data processing.

Distance constraints in proteins and protein complexes provide invaluable information for calculation of 3D structures, identification of protein binding partners and localization of protein-protein contact sites. We have developed an integrative approach to identify and characterize such sites through the analysis of proteolytic products derived from proteins chemically cross-linked by isotopically coded cross-linkers using LC-MALDI tandem mass spectrometry and computer software. This method is specifically tailored toward the rapid analysis of low microgram amounts of proteins or multimeric protein complexes cross-linked with nonlabeled and deuterium-labeled bis-NHS ester cross-linking reagents (both commercially available and readily synthesized). Through labeling with [18O]water solvent and LC-MALDI analysis, the method further allows the possible distinction between Type 0 and Type 1 or Type 2 modified peptides (monolinks and looplinks or cross-links), although such a distinction is more readily made from analysis of tandem mass spectrometry data. When applied to the bacterial Colicin E7 DNAse/Im7 heterodimeric protein complex, 23 cross-links were identified including six intersubunit cross-links, all between residues that are close in space when examined in the context of the X-ray structure of the heterodimer. In addition, cross-links were successfully identified in five single subunit proteins, beta-lactoglobulin, cytochrome c, lysozyme, myoglobin, and ribonuclease A, establishing the generality of the approach.

Chromatography, Liquid↗

PubMed2006

Therapeutic intervention based on protein prenylation and associated modifications.

In eukaryotic cells, a specific set of proteins are modified by C-terminal attachment of 15-carbon farnesyl groups or 20-carbon geranylgeranyl groups that function both as anchors for fixing proteins to membranes and as molecular handles for facilitating binding of these lipidated proteins to other proteins. Additional modification of these prenylated proteins includes C-terminal proteolysis and methylation, and attachment of a 16-carbon palmitoyl group; these modifications augment membrane anchoring and alter the dynamics of movement of proteins between different cellular membrane compartments. The enzymes in the protein prenylation pathway have been isolated and characterized. Blocking protein prenylation is proving to be therapeutically useful for the treatment of certain cancers, infection by protozoan parasites and the rare genetic disease Hutchinson-Gilford progeria syndrome.

Animals↗

PubMed2006

Antibacterial effects of human group IIA and group XIIA phospholipase A2 against Helicobacter pylori in vitro.

Group IIA phospholipase A2 (PLA2-IIA) is an enzyme which has important roles in inflammation and infection. Recently, a novel human secretory PLA2 called group XIIA PLA2 (PLA2-XIIA) has been identified. Both PLA2-IIA and PLA2-XIIA are bactericidal against Gram-positive bacteria like many other secretory PLA2s. However, PLA2-XIIA is the only known PLA2 displaying significant bactericidal activity against the Gram-negative bacterium Escherichia coli. We examined the antibacterial properties of recombinant human PLA2-IIA and PLA2-XIIA against Helicobacter pylori, a Gram-negative bacterium, in vitro. PLA2-IIA was not bactericidal against H. pylori, whereas PLA2-XIIA effectively killed H. pylori at a concentration of 50 microg/ml but was not bactericidal at concentrations of 0.5 microg/ml and 5 microg/ml.

Anti-Bacterial Agents↗

PubMed2006

Cytosolic phospholipase A2 enzymes are not required by mouse bone marrow-derived macrophages for the control of Mycobacterium tuberculosis in vitro.

During the course of infection Mycobacterium tuberculosis predominantly resides within macrophages, where it encounters and is often able to resist the antibacterial mechanisms of the host. In this study, we assessed the role of macrophage phospholipases A2 (PLA2s) in defense against M. tuberculosis. Mouse bone marrow-derived macrophages (BMDMs) expressed cPLA2-IVA, cPLA2-IVB, iPLA2-VI, sPLA2-IIE, and sPLA2-XIIA. The expression of cPLA2-IVA was increased in response to M. tuberculosis, gamma interferon, or their combination, and cPLA2-IVA mediated the release of arachidonic acid, which was stimulated by M. tuberculosis in activated, but not unactivated, macrophages. We confirmed that arachidonic acid is highly mycobactericidal in a concentration- and pH-dependent manner in vitro. However, when M. tuberculosis-infected macrophages were treated with PLA2 inhibitors, intracellular survival of M. tuberculosis was not affected, even in inducible nitric oxide synthase-deficient macrophages, in which a major bactericidal mechanism is removed. Moreover, intracellular survival of M. tuberculosis was similar in cPLA2-IVA-deficient and wild-type macrophages. Our results demonstrate that the cytosolic PLA2s are not required by murine BMDMs to kill M. tuberculosis.

Animals↗

PubMed2005

Thematic review series: lipid posttranslational modifications. Fighting parasitic disease by blocking protein farnesylation.

Protein farnesylation is a form of posttranslational modification that occurs in most, if not all, eukaryotic cells. Inhibitors of protein farnesyltransferase (PFTIs) have been developed as anticancer chemotherapeutic agents. Using the knowledge gained from the development of PFTIs for the treatment of cancer, researchers are currently investigating the use of PFTIs for the treatment of eukaryotic pathogens. This "piggy-back" approach not only accelerates the development of a chemotherapeutic agent for protozoan pathogens but is also a means of mitigating the costs associated with de novo drug design. PFTIs have already been shown to be efficacious in the treatment of eukaryotic pathogens in animal models, including both Trypanosoma brucei, the causative agent of African sleeping sickness, and Plasmodium falciparum, one of the causative agents of malaria. Here, current evidence and progress are summarized that support the targeting of protein farnesyltransferase for the treatment of parasitic diseases.

Animals↗

PubMed2005

Polychlorinated biphenyls induce arachidonic acid release in human platelets in a tamoxifen sensitive manner via activation of group IVA cytosolic phospholipase A2-alpha.

Polychlorinated biphenyls (PCBs) are stable compounds commonly found in nature as environmental pollutants. PCBs can affect the endocrine function of hormones such as steroid-hormones. Also, PCBs are known to be inducers of arachidonic acid release in various cells. We report, here, the effects of PCBs on eicosanoid formation, arachidonic acid release and cytosolic phospholipase A2-alpha (cPLA2-alpha) activation in human platelets. Ortho-substituted PCBs induced a time and dose-dependent release of arachidonic acid and the concomitant formation of 12(S)-hydroxy-5,8-cis-10-trans-14-cis-eicosatetraenoic acid (12-HETE) and 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid (12-HHT) in human platelets. The release of arachidonic acid and the formation of 12-HETE was completely blocked by the cPLA2-alpha inhibitors AACOCF3 or pyrrolidine-1. PCB-treatment of platelets demonstrated that the cPLA2-alpha protein as well as PLA2 activity translocated to the membrane fraction, independent of a rise in intracellular Ca2+. Furthermore, electrophoretic gel mobility shift analysis of cPLA2-alpha on SDS-PAGE demonstrated a PCB-dependent phosphorylation of cPLA2-alpha. The effects of 17beta-estradiol and two structurally unrelated anti-estrogens, nafoxidin and tamoxifen on PCB-induced arachidonic acid release in platelets were also investigated. Both nafoxidin and tamoxifen inhibited PCB-induced arachidonic acid release as well as 12-HETE and 12-HHT formation. Interestingly, platelets incubated with PCBs did not aggregate despite the fact that robust release of arachidonic acid was observed. In summary, these results demonstrate that certain PCBs induce activation of cPLA2-alpha independent of a rise in intracellular calcium and a robust release of arachidonic acid release with resulting eicosanoid formation in human platelets.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗