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Biomedical subjects

M Hackett

Publications and source records attributed to M Hackett.

At least 19 recordsLinked to original sources

Complete genome sequence of the genetically tractable hydrogenotrophic methanogen Methanococcus maripaludis.

The genome sequence of the genetically tractable, mesophilic, hydrogenotrophic methanogen Methanococcus maripaludis contains 1,722 protein-coding genes in a single circular chromosome of 1,661,137 bp. Of the protein-coding genes (open reading frames [ORFs]), 44% were assigned a function, 48% were conserved but had unknown or uncertain functions, and 7.5% (129 ORFs) were unique to M. maripaludis. Of the unique ORFs, 27 were confirmed to encode proteins by the mass spectrometric identification of unique peptides. Genes for most known functions and pathways were identified. For example, a full complement of hydrogenases and methanogenesis enzymes was identified, including eight selenocysteine-containing proteins, with each being paralogous to a cysteine-containing counterpart. At least 59 proteins were predicted to contain iron-sulfur centers, including ferredoxins, polyferredoxins, and subunits of enzymes with various redox functions. Unusual features included the absence of a Cdc6 homolog, implying a variation in replication initiation, and the presence of a bacterial-like RNase HI as well as an RNase HII typical of the Archaea. The presence of alanine dehydrogenase and alanine racemase, which are uniquely present among the Archaea, explained the ability of the organism to use L- and D-alanine as nitrogen sources. Features that contrasted with the related organism Methanocaldococcus jannaschii included the absence of inteins, even though close homologs of most intein-containing proteins were encoded. Although two-thirds of the ORFs had their highest Blastp hits in Methanocaldococcus jannaschii, lateral gene transfer or gene loss has apparently resulted in genes, which are often clustered, with top Blastp hits in more distantly related groups.

Archaeal Proteins↗

Related IgA1 and IgG producing cells in blood and diseased mucosa in ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory bowel disease in which the colonic mucosa is infiltrated with plasma cells producing IgG autoantibodies. It is not known whether this represents a local mucosal response which has switched to IgG or a peripheral response which may have been initiated by peripheral antigen which homed to the colonic mucosa. The clonal distribution of IgG secreting cells and isotype switched variants in UC is not known. AIMS: To investigate the clonal distribution of mucosal IgG in UC and to search for related IgG and IgA secreting cells in normal and diseased mucosa and blood in UC. To investigate characteristics which may discriminate between the mucosal and peripheral repertoire in the normal mucosa and in UC. PATIENTS: Blood and normal and diseased mucosa from two patients with UC were studied. METHODS: Immunoglobulin gene analysis and clone specific polymerase chain reaction were used to study the clonal distribution and characteristics of IgG and related IgA in the mucosa and blood of patients with UC. RESULTS: The IgG response in the mucosa of UC patients included widespread clones of cells that were present in both the diseased mucosa and blood but that were scarce in normal mucosa. Clonally related IgA class switch variants, all IgA1, were detected but also only in the diseased mucosa and blood. This suggests that these clones home preferentially to the diseased mucosa. We showed that J(H)1 usage was characteristic of the peripheral repertoire, and that examples of J(H)1 usage were observed in mucosal IgG in UC. CONCLUSIONS: Overall, these data are consistent with a model of UC in which a peripheral response is expressed and expanded in the colonic mucosa.

Antibody-Producing Cells↗

Acylation of lysine 983 is sufficient for toxin activity of Bordetella pertussis adenylate cyclase. Substitutions of alanine 140 modulate acylation site selectivity of the toxin acyltransferase CyaC.

The capacity of adenylate cyclase toxin (ACT) to penetrate into target cells depends on post-translational fatty-acylation by the acyltransferase CyaC, which can palmitoylate the conserved lysines 983 and 860 of ACT. Here, the in vivo acylating capacity of a set of mutated CyaC acyltransferases was characterized by two-dimensional gel electrophoresis and mass spectrometric analyses of the ACT product. Substitutions of the potentially catalytic serine 20 and histidine 33 residues ablated acylating activity of CyaC. Conservative replacements of alanine 140 by glycine (A140G) and valine (A140V) residues, however, affected selectivity of CyaC for the two acylation sites on ACT. Activation by the A140G variant of CyaC generated a mixture of bi- and monoacylated ACT molecules, modified either at both Lys-860 and Lys-983, or only at Lys-860, respectively. In contrast, the A140V CyaC produced a nearly 1:1 mixture of nonacylated pro-ACT with ACT monoacylated almost exclusively at Lys-983. The respective proportion of toxin molecules acylated at Lys-983 correlated well with the cell-invasive activity of both ACT mixtures, which was about half of that of ACT fully acylated on Lys-983 by intact CyaC. These results show that acylation of Lys-860 alone does not confer cell-invasive activity on ACT, whereas acylation of Lys-983 is necessary and sufficient.

Acylation↗

Mass spectrometric analysis of recombinant adenylate cyclase toxin from Bordetella pertussis strain 18323/pHSP9.

The adenylate cyclase toxin-hemolysin (ACT) is a key virulence factor of the whooping cough agent Bordetella pertussis (Bp). The major cytotoxic activity of this 1706-residue protein consists of its capacity to invade a variety of eukaryotic cells directly across their cytoplasmic membrane and to deliver into cells a catalytic adenylate cyclase domain. This causes impairment of immune effector cells and apoptosis of lung macrophages by uncontrolled conversion of ATP to cAMP. The adenylate cyclase toxin-hemolysin acquires biological activity upon post-translational amide-linked palmitoylation of the epsilon-amino group of lysine 983 (K983) by the accessory fatty acyltransferase, CyaC. However, an additional conserved acylation site can be identified in ACT at lysine 860 (K860) and this residue is palmitoylated when recombinant ACT is produced in Escherichia coli (r-Ec-ACT). In this paper we report the double acylation of r-Bp-ACT secreted by a recombinant Bp strain 18323/pHSP9. This strain overproduces ACT from an oligocopy plasmid carrying the entire cya locus of Bordetella pertussis 18323. Palmitoylation of both conserved lysines (K860 and K983) of r-Bp-ACT expressed by this Bp strain was found. In addition, an error in the deduced protein sequence was identified, with Leu being the real residue at position 1001 and not the Val residue given in the published gene sequence. We also discuss these results in comparison with those from recombinant ACT expressed in E. coli strain K12 XL1-Blue. The analytical approach for characterization of the fatty acylation of ACT from strain 18323/pHSP9 consisted of multiple proteolytic digestion procedures (trypsin, Asp-N), microcapillary liquid chromatography/tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Adenylate Cyclase Toxin↗

Searching the Porphyromonas gingivalis genome with peptide fragmentation mass spectra.

An approach is described for genomic database searching based on experimentally observed proteolytic fragments, e.g., isolated from 1D or 2D gels or analyzed directly, that can be applied to unfinished prokaryotic genomic data in the absence of annotations or previously assigned open reading frames (ORFs). This variation on the database search is in contrast to the more familiar use of peptide mass spectral fragmentation data to search fully annotated inferred protein databases, e.g., OWL or SWISS-PROT. We compared the SEQUEST search results from a six reading frame translation of the Porphyromonas gingivalis genome DNA sequence with those from computationally derived ORFs created using publicly available genomics software tools. The ORF approach eliminated many of the artifacts present in output from the six reading frame search. The method was applied to uninterpreted tandem mass spectrometric data derived from proteins secreted by the periodontal pathogen Porphyromonas gingivalis in response to the gingival epithelial cell environment, a model system for the study of host-pathogen interactions relevant to human periodontal disease.

Bacterial Proteins↗

Escherichia coli alpha-hemolysin (HlyA) is heterogeneously acylated in vivo with 14-, 15-, and 17-carbon fatty acids.

alpha-Hemolysin (HlyA) is a secreted protein virulence factor observed in certain uropathogenic strains of Escherichia coli. The active, mature form of HlyA is produced by posttranslational modification of the protoxin that is mediated by acyl carrier protein and an acyltransferase, HlyC. We have now shown using mass spectrometry that these modifications, when observed in protein isolated in vivo, consist of acylation at the epsilon-amino groups of two internal lysine residues, at positions 564 and 690, with saturated 14- (68%), 15- (26%), and 17- (6%) carbon amide-linked side chains. Thus, HlyA activated in vivo consists of a heterogeneous family of up to nine different covalent structures, and the substrate specificity of the HlyC acyltransferase appears to differ from that of the closely related CyaC acyltransferase expressed by Bordetella pertussis.

Acylation↗

Characteristics of human IgA and IgM genes used by plasma cells in the salivary gland resemble those used in duodenum but not those used in the spleen.

Immunologically, the parotid salivary gland is an effector site that secretes large quantities of polyspecific Abs into the saliva, mainly of the IgA isotype. It is considered to be part of the common mucosal immune system but the inductive site for the Ab-producing cells of the salivary gland has not yet been clearly identified. The origin and diversity of cells of B lineage can be investigated by analyzing their Ig heavy chain genes (IgH). We have obtained sequences of IgM and IgA VH4-34 genes from plasma cells in human salivary gland, duodenal lamina propria, and splenic red pulp. Related sequences were found in different areas sampled within each tissue studied, indicating that the plasma cells carrying these genes are widespread with limited diversity. Examples of related IgH genes that are isotype switched were also seen in the salivary gland. The genes from plasma cells of the salivary gland were highly mutated, as were duodenal plasma cell sequences. The level of mutation was significantly higher than that seen in splenic plasma cell sequences. Analysis of CDR3 regions showed that the sequences from salivary gland had significantly smaller CDR3 regions than sequences from spleen, due to differences in number and type of DH regions used. Sequences from duodenum also had smaller CDR3 regions. Therefore, plasma cells from human duodenum and salivary gland showed characteristics that differed from those of human splenic plasma cells.

Base Sequence↗

Rapid isolation method for lipopolysaccharide and lipid A from gram-negative bacteria.

A fast, convenient extraction method for lipopolysaccharide (LPS), using a commercial RNA isolating reagent, allows the isolation of LPS or lipid A from low milligram (dry weight) quantities of bacterial cells. The method avoids the use of specialized equipment and has been used for processing relatively large numbers of samples. The major components of the commercial RNA isolating reagent, Tri-Reagent, are phenol and guanidinium thiocyanate in aqueous solution. The bacterial cell membranes are disrupted with guanidinium thiocyanate, which eliminates the need for mechanical cell disruption (e.g. French press) or heating. LPS and its degradation products, with particular attention paid to its bioactive lipid A portion, were measured and compared with those from the most common conventional extraction method, hot phenol-water. Negative ion quadrupole ion trap and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, fatty acid composition analysis by capillary gas chromatography, total and free phosphate by UV spectrophotometry and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that LPS and lipid A isolated using the Tri-Reagent approach were cleaner and suffered less degradation through loss of phosphate and (or) fatty acyl side chains from lipid A. The Tri-Reagent extraction method generated low free phosphate contamination, 11% of the total phosphate concentration, whereas the hot phenol-water extraction method gave approximately 58% as free, inorganic phosphate. Similar results were observed for the degradation of fatty acyl side chains. The time required by the new method is considerably shorter (two or three days) than that required by conventional hot phenol-water extraction (about two weeks).

Gram-Negative Bacteria↗

A remote flow cell for UV absorbance detection with capillary HPLC based on a single strand fiber optic.

A remote flow cell based on a single strand of fused-silica fiber optic was built for UV absorbance detection with a packed capillary HPLC system, using commercially available pumps, detection electronics (Shimadzu) and fittings. This 'off-column' flow cell design is applicable to both pressure and electro-osmotically driven systems. The goals were to minimize the linearity and light leakage problems that often limit the performance of UV absorbance detection with capillary chromatography. A linear dynamic range of 10(3) (reserpine, lambda = 220 nm), and a concentration detection limit of 5.1 x 10(-8) mol l-1 were observed. Baseline noise was measured at 3.5 x 10(-5) absorbance units (AU), with a standard deviation of 1.7 x 10(-5) AU. The illuminated volume of approximately 3 nl was optimized for capillaries with inner diameters in the range 50-100 microns, and flow rates from 100 nl min-1 to 1 microliter min-1. These modifications of readily available instrumentation have allowed the construction of a practical system for fractionating complex mixtures of peptides in small amounts, prior to mass spectrometry or additional wet chemistry steps.

Chromatography, High Pressure Liquid↗

A PhoP-regulated outer membrane protease of Salmonella enterica serovar typhimurium promotes resistance to alpha-helical antimicrobial peptides.

The outer membrane protein contents of Salmonella enterica serovar Typhimurium strains with PhoP/PhoQ regulon mutations were compared by two-dimensional gel electrophoresis. At least 26 species of outer membrane proteins (OMPs) were identified as being regulated by PhoP/PhoQ activation. One PhoP/PhoQ-activated OMP was identified by semiautomated tandem mass spectrometry coupled with electronic database searching as PgtE, a member of the Escherichia coli OmpT and Yersinia pestis Pla family of outer membrane proteases. Salmonella PgtE expression promoted resistance to alpha-helical cationic antimicrobial peptides (alpha-CAMPs). Strains expressing PgtE cleaved C18G, an 18-residue alpha-CAMP present in culture medium, indicating that protease activity is likely to be the mechanism of OmpT-mediated resistance to alpha-CAMPs. PhoP/PhoQ did not regulate the transcription or export of PgtE, indicating that another PhoP/PhoQ-dependent mechanism is required for PgtE outer membrane localization. PgtE is a posttranscriptionally regulated component of the PhoP/PhoQ regulon that contributes to Salmonella resistance to innate immunity.

Amino Acid Sequence↗

Specific lipopolysaccharide found in cystic fibrosis airway Pseudomonas aeruginosa.

Cystic fibrosis (CF) patients develop chronic airway infections with Pseudomonas aeruginosa (PA). Pseudomonas aeruginosa synthesized lipopolysaccharide (LPS) with a variety of penta- and hexa-acylated lipid A structures under different environmental conditions. CF patient PA synthesized LPS with specific lipid A structures indicating unique recognition of the CF airway environment. CF-specific lipid A forms containing palmitate and aminoarabinose were associated with resistance to cationic antimicrobial peptides and increased inflammatory responses, indicating that they are likely to be involved in airway disease.

Acylation↗

The conserved lysine 860 in the additional fatty-acylation site of Bordetella pertussis adenylate cyclase is crucial for toxin function independently of its acylation status.

The Bordetella pertussis RTX (repeat in toxin family protein) adenylate cyclase toxin-hemolysin (ACT) acquires biological activity upon a single amide-linked palmitoylation of the epsilon-amino group of lysine 983 (Lys983) by the accessory fatty-acyltransferase CyaC. However, an additional conserved RTX acylation site can be identified in ACT at lysine 860 (Lys860), and this residue becomes palmitoylated when recombinant ACT (r-Ec-ACT) is produced together with CyaC in Escherichia coli K12. We have eliminated this additional acylation site by replacing Lys860 of ACT with arginine, leucine, and cysteine residues. Two-dimensional gel electrophoresis and microcapillary high performance liquid chromatography/tandem mass spectrometric analyses of mutant proteins confirmed that the two sites are acylated independently in vivo and that mutations of Lys860 did not affect the quantitative acylation of Lys983 by palmitoyl (C16:0) and palmitoleil (cis Delta9 C16:1) fatty-acyl groups. Nevertheless, even the most conservative substitution of lysine 860 by an arginine residue caused a 10-fold decrease of toxin activity. This resulted from a 5-fold reduction of cell association capacity and a further 2-fold reduction in cell penetration efficiency of the membrane-bound K860R toxin. These results suggest that lysine 860 plays by itself a crucial structural role in membrane insertion and translocation of the toxin, independently of its acylation status.

Acylation↗

Fragmentation pathways of selectively labeled uropranolol using electrospray ionization on an ion trap mass spectrometer and comparison with ions formed by electron impact.

Propranolol, deuterium- and 18O-labeled propranolol and related compounds were analyzed using an ion trap mass spectrometer equipped with a modified Finnigan API electrospray interface. Sequential product ion (MSn) experiments were used to elucidate fragmentation pathways for these compounds. The observed ions were compared to those observed under electron impact (EI) conditions. The electrospray ionization (ESI) ion trap spectra, as well as the EI spectra, afford useful information to allow assignments of most product ions, many of which retain portions of the aliphatic three-carbon side chain.

Adrenergic beta-Antagonists↗

Mass spectral fragmentation pathways of propranolol related beta-fluorinated amines studied by electrospray and electron impact ionization.

Propranolol, its 1"-mono-, di-, and trifluorinated analogs, and other related compounds were analyzed under electrospray ionization ion trap collision-induced dissociation (ESI-CID) and electron impact (EI) conditions. Interesting trends were observed in the fragment ions formed in both cases. Under ESI conditions, the abundances of product ions easily explained by protonation on the amine nitrogen decreased relative to the abundances of those formed from the ether-protonated species as the number of fluorines increased from zero to three. Under EI conditions, the distribution of fragment ions was shifted away from those arising from a nitrogen-centered cation radical and toward those arising from an ether oxygen-centered cation radical. The changes observed in apparent molecular sites of protonation and of ion radical formation in the mass spectra are consistent with the electron-withdrawing effects of the sequentially added fluorines. These effects are correlated with changes in solution phase pK( a)'s of the fluorinated amines.

Adrenergic beta-Antagonists↗

Potential utility of electronic drug compliance monitoring in measures of adverse outcomes associated with immunosuppressive agents.

Poor compliance with prescribed medications limits the effectiveness of many pharmacologic therapies and enhances their potential toxicities. Traditional methods of measuring drug-taking behavior, including direct observation, patient self-report, pill counts, and therapeutic drug level monitoring, all have well-described limitations in validity and interpretability. Electronic medication event monitoring has been used to assess compliance with therapies for hypertension, glaucoma, anemia, and epilepsy, overcoming many problems of traditional approaches. However, no published reports describe the use of electronic monitoring with immunosuppressive agents, despite their increasing use for non-life-threatening conditions and their many dose-dependent toxicities. Transplant recipients are thought to be at particular risk from noncompliance. Therefore, we undertook this study to assess the feasibility of electronically monitoring compliance with immunosuppressive drugs among renal allograft recipients. Twenty-five kidney transplant patients receiving immunosuppressive medications from a single pharmacy were enrolled. Each subject received electronic monitors with their immunosuppressive serum drug refills for cyclosporine and azathioprine. Each subject returned their monitors after the first month of this 2-month study for downloading data. The frequency distribution of interdose intervals were described. Two measures of average non-compliance were calculated for both drugs: the proportion of monitored days that had missed doses, and the proportion of missed doses. Once daily and twice daily regimens of cyclosporine were compared. Concordance in drug compliance between the two drugs was calculated for each subject and averaged over the study population. Twenty-two of 25 subjects missed one or more doses of cyclosporine or azathioprine. Seventeen (68%) subjects never missed four or more consecutive doses. Subjects were non-compliant with cyclosporine on 8.7% of monitored days, and non-compliance with azathioprine on 9.8% of monitored days. Subjects were non-compliant with 6.8% of their cyclosporine doses and 9.8% of their azathioprine doses. Patients were compliant with both drugs on 86.6% of days and were non-compliant with both drugs on 5.1% of days. Subjects were non-compliant with cyclosporine during 5% and 13.2% of monitored days for once and twice daily dosing regimens, respectively. Concordance analysis demonstrated that for 91.7% of days of monitoring, compliance information was identical for both drugs. This study demonstrated the feasibility of electronic medication event monitoring among kidney transplant patients. This methodology represents an important tool for monitoring compliance of immunosuppressive agents essential to their safe and effective use, and should be considered for use in future studies of these drugs and others with substantial dose-dependent toxicity.

Journal Article↗

A cardioactive peptide from the southern armyworm, Spodoptera eridania.

A cardioactive peptide was isolated from extracts of whole heads of the southern armyworm, Spodoptera eridania. This peptide has the sequence ENFAVGCTPGYQRTADGRCKPTF (Mr = 2516.8), determined from both Edman sequencing and tandem mass spectrometry in combination with off-line micropreparative capillary liquid chromatography. This peptide, termed Spoer-CAP23, has excitatory effects on a semi-isolated heart from larval Manduca sexta, causing an inotropic effect at low concentrations of peptide and chronotropic and inotropic effects at high doses. The threshold concentration for stimulatory effects of the synthetic peptide on the semi-isolated heart was about 1 nM, suggesting a physiological role as a neuropeptide.

Amino Acid Sequence↗

Clinical governance: culture, leadership and power--the key to changing attitudes and behaviours in trusts.

Clinical governance places for legal duty for quality on Trust chief executives. The article deals with how chief executives can overcome the cultural and behavioural obstacles to change which impede the journey to developing effective structures for clinical governance. To establish effective structures for clinical governance, chief executives will need to address these features of organisational life--culture, power and leadership. The article seeks to provide practical ways in which they can do this.

Governing Board↗