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Adrian J Lloyd

Publications and source records attributed to Adrian J Lloyd.

11 recordsLinked to original sources
PubMed2025

Transcriptomic and enzymological evidence for plastid peptidoglycan synthesis in the gymnosperm Picea abies.

It is understood that a cyanobacterium was the progenitor of plastids and that the biosynthesis of cell wall peptidoglycan was lost during chloroplast evolution. However, accumulated data, especially from the moss Physcomitrium patens, suggest that peptidoglycan remains essential for plastid division in some land plants. A fundamental set of peptidoglycan biosynthesis (Mur) genes has been identified in the genomes of these land plants, while many angiosperms no longer encode some core Mur genes, including a bifunctional penicillin-binding protein (PBP). Ten incomplete Mur genes were previously identified in the genome of the gymnosperm Picea abies but these could be pseudogenes or encode proteins that have been repurposed. For instance, mutant albino maize and Arabidopsis seedlings possess a defective UDP-N-acetylmuramoyl-l-alanyl-d-glutamate--2,6-diaminopimelate ligase (MurE), an intact MurE ligase being essential for peptidoglycan synthesis. In this study, we isolated a full set of cDNAs for peptidoglycan biosynthesis from P. abies. GFP fusion proteins with either P. abies (Pa)MurE or PaPBP were detected in chloroplasts. Cross-species complementation assays with PaMurE in Arabidopsis albino MurE mutants and Physcomitrium MurE chloroplast division mutants showed that the gymnosperm MurE completely rescued both mutant phenotypes. Enzymatic assay of recombinant PaMurE proteins revealed they catalyze the same reaction performed by their bacterial MurE homologs. Moreover, the expression of the PaPbp cDNA partially rescued the giant chloroplast phenotype in the moss Pbp knockout line. These results are consistent with the operation of a functional Mur gene set in the Norway spruce genome.

Peptidoglycan↗
PubMed2006

Fluorescent reagents for in vitro studies of lipid-linked steps of bacterial peptidoglycan biosynthesis: derivatives of UDPMurNAc-pentapeptide containing d-cysteine at position 4 or 5.

UDPMurNAc-L-Ala-gamma-D-Glu-X-D-Ala-DAla (X = L-Lys or m-DAP) is the cytoplasmic precursor for the lipid-linked cycle of bacterial peptidoglycan biosynthesis, consisting of at least four enzymatic reactions, which are targets for antibacterial agents. Fluorescent derivatives of the UDPMurNAc-pentapeptide labelled at the 3rd, 4th, and 5th position of the peptide chain were prepared chemoenzymatically, in order to study the reactions catalysed by enzymes in this cycle. Derivatives labelled on the epsilon-amino group of the 3rd amino acid (N-dansyl, N-fluorescamine and N-phthalaldehyde) were prepared by chemical modification. Two methods were developed for preparation of analogues of UDPMurNAc-pentapeptide containing D-cysteine at position 4 or 5: either by MurF-catalysed ligation of the UDPMurNAc-tripeptide to synthetic D-Ala-D-Cys or D-Cys-D-Ala dipeptides; or by enzymatic synthesis of D-Ala-D-Cys by ligase VanD. D-Cys-containing UDPMurNAc-pentapeptides were labelled with pyrene maleimide, to give 4-pyrene and 5-pyrene labelled derivatives. The fluorescent UDPMurNAc-pentapeptides were processed as substrates by Escherichia coli MraY or E. coli membranes, giving 1.5-150-fold changes in fluorescence upon transformation to lipid intermediate I. Subsequent processing to lipid intermediate II gave rise only to small changes in fluorescence. Pyrene-labelled lipid intermediates I and II can be generated using Micrococcus flavus membranes, enabling the study of the later lipid-linked steps.

Bacterial Proteins↗
PubMed2006

Use of a codon alteration strategy in a novel approach to cloning the Mycobacterium tuberculosis diaminopimelic acid epimerase.

Previous attempts to express the diaminopimelate epimerase gene dapF of Mycobacterium tuberculosis in Escherichia coli resulted in undetectable enzyme yields. We used silent mutation of the first 10 codons of the recombinant ORF in an attempt to reduce the formation of secondary structures that might occur near the 5' end of the mRNA and inhibit translation. This significantly increased the yield of the enzyme, which was purified and characterized biochemically. This strategy could be generally applied to other mycobacterial genes that are difficult to express hetero-specifically and here provided pure M. tuberculosis DapF, a good foundation for future research in antimycobacterial agents.

Amino Acid Isomerases↗
PubMed2006

Phospho-MurNAc-pentapeptide translocase (MraY) as a target for antibacterial agents and antibacterial proteins.

Phospho-MurNAc-pentapeptide translocase (MraY, translocase I) catalyses the first step of the lipid-linked cycle of reactions of bacterial peptidoglycan biosynthesis. MraY is the target for five families of nucleoside antibacterial natural products: the tunicamycins, the mureidomycins (also pacidamycins, napsamycins), the liposidomycins, the muraymycins, and the capuramycins. Recent structure-activity studies on these families have led to the identification of active pharmacophores, and insight into their mechanisms of action. This step of peptidoglycan biosynthesis is also the target for the bacteriolytic E protein from bacteriophage phiX174, and for cyclic peptides of the amphomycin family which complex the undecaprenyl phosphate co-substrate. The mechanisms of enzyme inhibition by these agents are discussed, and the state of knowledge regarding the transmembrane structure, active site, and catalytic mechanism of MraY. The availability of high throughput assays and prospects of MraY as an antibacterial target are also discussed.

Anti-Bacterial Agents↗
PubMed2004

Expression, purification, crystallization and preliminary characterization of uridine 5'-diphospho-N-acetylmuramoyl L-alanyl-D-glutamate:lysine ligase (MurE) from Streptococcus pneumoniae 110K/70.

An ORF designated sp1530 (murE) in the Streptococcus pneumoniae TIGR4 genome sequence, identified as uridine 5'-diphospho-N-acetylmuramoyl-L-alanyl-D-glutamate:L-lysine ligase (MurE; EC 6.3.2.7), was cloned into the high-expression plasmid pET21b and overexpressed in Escherichia coli BL21 (DE3) Star. The enzyme was purified in three steps to 99% purity. Crystals were obtained by the hanging-drop vapour-diffusion method at 291 K from solutions containing 25%(w/v) polyethylene glycol 2000 monomethylether, 0.2 M potassium thiocyanate, 0.1 M MES pH 6.5 in the presence of uridine 5'-diphospho-N-acetylmuramoyl alanyl glutamate (UDP-MurNAc-L-Ala-D-Glu) with and without 5'-adenylyl imidophosphate (AMP-PNP), a non-hydrolysable analogue of ATP. Diffraction data to 1.5 and 2.7 A, respectively, were collected at the European Synchrotron Radiation Facility (ESRF). Crystals grown in the presence of two ligands belong to space group P1, with unit-cell parameters a = 68.4, b = 71.4, c = 74.8 A, alpha = 73.4, beta = 80.5, gamma = 72.3 degrees. Crystals grown in the presence of UDP-MurNAc-L-Ala-D-Glu alone belong to space group P2(1), with unit-cell parameters a = 71.1, b = 129.4, c = 74.6 A, beta = 106.3 degrees.

Animals↗
PubMed2004

A functional MRI study of working memory task in euthymic bipolar disorder: evidence for task-specific dysfunction.

OBJECTIVES: Even when euthymic bipolar disorder patients can have persistent deficits in working memory, but the neural basis of this deficit remains unclear. We undertook an functional magnetic resonance imaging investigation of euthymic bipolar disorder patients performing two working memory paradigms; the two-back and Sternberg tasks, selected to examine the central executive and the phonological loop respectively. We hypothesized that neuronal dysfunction would be specific to the network underlying the executive rather than the phonological loop component of working memory. METHODS: Twelve right-handed euthymic bipolar I males receiving lithium carbonate monotherapy were matched with 12 controls. The two-back task comprised a single working memory load contrasted with baseline vigilance condition. The Sternberg paradigm used a parametric design incorporating variable working memory load with fixed delay between presentation of an array of items to be remembered and a target item. Functional activation data were acquired during performance of the tasks and were analysed to produce brain activation maps representing significant group differences in activation (ANOVA). Load-response curves were derived from the Sternberg task data set. RESULTS: There were no significant between-group differences (t-test) in performance of the two-back task, or in 2 x 5 group by memory load ANOVA for the performance data from Sternberg task. In the two-back task, compared with controls bipolar disorder patients showed reductions in bilateral frontal, temporal and parietal activation, and increased activations with the left precentral, right medial frontal and left supramarginal gyri. No between-group differences were observed in the Sternberg task at any working memory load. CONCLUSIONS: Our findings support the notion that, in euthymic bipolar disorder, failure to engage fronto-executive function underpins the core neuropsychological deficits.

Bipolar Disorder↗
PubMed2004

Phospho-N-acetyl-muramyl-pentapeptide translocase from Escherichia coli: catalytic role of conserved aspartic acid residues.

Phospho-N-acetyl-muramyl-pentapeptide translocase (translocase 1) catalyzes the first of a sequence of lipid-linked steps that ultimately assemble the peptidoglycan layer of the bacterial cell wall. This essential enzyme is the target of several natural product antibiotics and has recently been the focus of antimicrobial drug discovery programs. The catalytic mechanism of translocase 1 is believed to proceed via a covalent intermediate formed between phospho-N-acetyl-muramyl-pentapeptide and a nucleophilic amino acid residue. Amino acid sequence alignments of the translocase 1 family and members of the related transmembrane phosphosugar transferase superfamily revealed only three conserved residues that possess nucleophilic side chains: the aspartic acid residues D115, D116, and D267. Here we report the expression and partial purification of Escherichia coli translocase 1 as a C-terminal hexahistidine (C-His6) fusion protein. Three enzymes with the site-directed mutations D115N, D116N, and D267N were constructed, expressed, and purified as C-His6 fusions. Enzymatic analysis established that all three mutations eliminated translocase 1 activity, and this finding verified the essential role of these residues. By analogy with the structural environment of the double aspartate motif found in prenyl transferases, we propose a model whereby D115 and D116 chelate a magnesium ion that coordinates with the pyrophosphate bridge of the UDP-N-acetyl-muramyl-pentapeptide substrate and in which D267 therefore fulfills the role of the translocase 1 active-site nucleophile.

Amino Acid Sequence↗
PubMed2004

Hippocampal volume change in depression: late- and early-onset illness compared.

BACKGROUND: Evidence for structural hippocampal change in depression is limited despite reports of neuronal damage due to hypercortisolaemia and vascular pathology. AIMS: To compare hippocampal and white matter structural change in demographically matched controls and participants with early-onset and late-onset depression. METHOD: High-resolution volumetric magnetic resonance imaging (MRI) and rating of MRI hyperintensities. RESULTS: A total of 51 people with depression and 39 control participants were included. Participants with late-onset depression had bilateral hippocampal atrophy compared with those with early-onset depression and controls. Hippocampal volumes did not differ between control participants and those with early-onset depression. Age of depression onset correlated (negatively) with hippocampal volume but lifetime duration of depression did not. Hyperintensity ratings did not differ between groups. CONCLUSIONS: Results suggest that acquired biological factors are of greater importance in late-than in early-onset illness and that pathological processes other than exposure to hypercortisolaemia of depression underlie hippocampal atrophy in depression of late life.

Age Factors↗
PubMed2003

The pharmacological treatment of bipolar affective disorder: practice is improving but could still be better.

Pharmacological treatment of bipolar affective disorder was examined by case note review of 120 subjects under the care of four hospitals in the North-East of England and compared with that recommended in published consensus opinions and also with the findings of previous surveys. Although the only comparison available is with other geographical regions, it appears that lithium use has remained relatively constant in recent years (53% in this sample). The use of anticonvulsants (62%) was greater than in previous studies, as was use of mood stabilizers in combination (37% overall, 53% in rapid cycling subjects). Antidepressant use was found in 23% of subjects. Eleven percent of subjects taking antidepressants were not prescribed a mood stabilizer and 43% of antidepressants prescribed were tricyclics, possibly increasing the risk of switching to mania. Fifteen percent had no mood stabilizer and antipsychotic use was almost equally split between typical and atypical drugs. Practice in the population studied is nearer than treatment described in previous work to that suggested in the literature. This may result from either, or most probably a combination of, the local availability of a specialist mood disorder service and evolution of overall prescribing practice. However, it still falls short in a number of respects of such recommendations as exist and this requires further examination and monitoring.

Adult↗
PubMedDate not supplied

A volumetric study of MRI signal hyperintensities in late-life depression.

OBJECTIVE: An increase in white-matter lesions has been previously described in older subjects with depression. The authors investigated whether the regional location varied between depressed and normal subjects and determined the relationship of magnetic resonance (MR) signal hyperintensities to known clinical risk factors for vascular disease. METHODS: Authors used automated image-processing software to determine volumes of signal hyperintensities from MR brain scans of older people with depression (N=29; mean age: 76 years) and normal subjects of similar age (N=32). RESULTS: Overall, subjects with depression had a significantly greater frontal-lobe white-matter lesion volume than normal subjects (0.35% versus 0.22%). However, after excluding subjects with hypertension, diabetes, or ischemic heart disease (leaving 14 depressed and 15 normal subjects), we found even greater differences between groups, with a larger volume of MR signal hyperintensities in the frontal region of the depressed group, but no difference in the basal ganglia or parietal and occipital lobes. CONCLUSION: The results support the "vascular depression" hypothesis and suggest that those with depression but without traditional vascular risk factors may be much more susceptible to cerebrovascular disease than normal subjects. The mechanisms for this increased susceptibility remain to be determined.

Aged↗