Structural studies of substrate and product complexes of 5-aminolaevulinic acid dehydratase from humans, Escherichia coli and the hyperthermophile Pyrobaculum calidifontis.

A number of X-ray analyses of an enzyme involved in a key early stage of tetrapyrrole biosynthesis are reported. Two structures of human 5-aminolaevulinate dehydratase (ALAD), native and recombinant, have been determined at 2.8 Šresolution, showing that the enzyme adopts an octameric quaternary structure in accord with previously published analyses of the enzyme from a range of other species. However, this is in contrast to the finding that a disease-related F12L mutant of the human enzyme uniquely forms hexamers [Breinig et al. (2003), Nature Struct. Biol. 10, 757-763]. Monomers of all ALADs adopt the TIM-barrel fold; the subunit conformation that assembles into the octamer includes the N-terminal tail of one monomer curled around the (α/ß)8 barrel of a neighbouring monomer. Both crystal forms of the human enzyme possess two monomers per asymmetric unit, termed A and B. In the native enzyme there are a number of distinct structural differences between the A and B monomers, with the latter exhibiting greater disorder in a number of loop regions and in the active site. In contrast, the second monomer of the recombinant enzyme appears to be better defined and the active site of both monomers clearly possesses a zinc ion which is bound by three conserved cysteine residues. In native human ALAD, the A monomer also has a ligand resembling the substrate ALA which is covalently bound by a Schiff base to one of the active-site lysines (Lys252) and is held in place by an ordered active-site loop. In contrast, these features of the active-site structure are disordered or absent in the B subunit of the native human enzyme. The octameric structure of the zinc-dependent ALAD from the hyperthermophile Pyrobaculum calidifontis is also reported at a somewhat lower resolution of 3.5 Å. Finally, the details are presented of a high-resolution structure of the Escherichia coli ALAD enzyme co-crystallized with a noncovalently bound moiety of the product, porphobilinogen (PBG). This structure reveals that the pyrrole side-chain amino group is datively bound to the active-site zinc ion and that the PBG carboxylates interact with the enzyme via hydrogen bonds and salt bridges with invariant residues. A number of hydrogen-bond interactions that were previously observed in the structure of yeast ALAD with a cyclic intermediate resembling the product PBG appear to be weaker in the new structure, suggesting that these interactions are only optimal in the transition state.

Extension of resolution and oligomerization-state studies of 2,4'-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP.

The enzyme 2,4'-dihydroxyacetophenone dioxygenase (DAD) catalyses the conversion of 2,4'-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid. This enzyme is a very unusual dioxygenase in that it cleaves a C-C bond in a substituent of the aromatic ring rather than within the ring itself. Whilst it has been shown that DAD is a tetramer in solution, the recently solved crystal structure of the Alcaligenes sp. 4HAP enzyme was in fact dimeric rather than tetrameric. Since the use of limited chymotrypsinolysis, which apparently results in removal of the first 20 or so N-terminal residues of DAD, was necessary for crystallization of the protein, it was investigated whether this was responsible for the change in its oligomerization state. Gel-filtration and analytical ultracentrifugation studies were conducted, which confirmed that chymotrypsinolysed DAD has an apparent molecular weight of around 40 kDa, corresponding to a dimer. In contrast, the native enzyme has a molecular weight in the 70-80 kDa region, as expected for the tetramer. The structural basis for tetramerization has been investigated by the use of several docking servers, and the results are remarkably consistent with the tetrameric structure of a homologous cupin protein from Ralstonia eutropha (PDB entry 3ebr).

Characterization of Clostridium Species from Food Commodities and Faecal Specimens in Lagos State, Nigeria.

BACKGROUND: Food-borne pathogens are a major public health challenge worldwide. These organisms' cause illnesses leading to time loss in the work place and reduced productivity.Clostridium species cause infections through the production of powerful toxins which are responsible for diarrhoea and cramping. Diarrhoeal diseases due to Clostridia are one of the commonest worldwide but have hardly been reported in Nigeria. OBJECTIVE: This study characterized Clostridium species from food commodities and human faeces in Lagos State MATERIALS AND METHODS: Four hundred and twenty samples comprising food (220) and faecal (200) specimens in Lagos state were included in this study. Isolates obtained were identified using API 20-A and confirmed by Polymerase Chain Reaction assay and 16S rRNA sequencing. The food samples included meat and meat products, ready to drink traditionally prepared milk products, fresh vegetables, canned foods and local honey. RESULTS: Seventy (16.7%) Clostridium species were identified, 50 from food and 20 from faeces. Majority of the isolates were obtained from vegetables (56%) and meat products (34%). Of the 70 Clostridial species, 38 (54.3%) were C. perfringens, 5 (7.1%) were C. difficile and 2 (2.9%) were C. botulinum. All 38 (100%) strains of C. perfringens possessed alpha (cpa) toxin gene. CONCLUSION: Clostridium species are present in our environment and contaminate food products posing potential risks to consumers. There is therefore a need for these traditionally made street vended foods to be monitored because they are potential sources of food borne pathogens.

Burnout among doctors in residency training in a tertiary hospital.

The mental health of doctors is an issue of growing concern all over the world as it frequently interplays with their professional trainings and responsibilities. This study was done to determine the pattern and correlates of burnout among 204 doctors undergoing residency training. Eligible participants were interviewed using designed questionnaire, General Health Questionnaire (GHQ-12) and Maslach Burnout Inventory (MBI). The mean age of participants was 33.44±4.50. Ninety-three (45.6%) respondents reported burnout in the dimension of emotional exhaustion (EE), 118 (57.8%) in the dimension of depersonalization (D), and 126 (61.8%) in the dimension of reduced personal accomplishment (RPA). Factors that were significantly associated with all the dimensions of burnout were perceived heavy workload and presence of emotional distress (based on GHQ score of ≥3). The perception of call duty as being not stressful was negatively predictive of burnout in the emotional exhaustion subscale (odds ratio [OR]=0.52; 95%confidence interval [CI]=0.29-0.97; p=0.03), while emotional distress was a positive predictor (OR=6.97; 95%CI=3.28-14.81; p<0.001]. Absence of doctor-to-doctor conflict negatively predicted burnout in the depersonalization subscale (OR=0.36; 95%CI=0.17-0.76); p<0.01), while older age (OR=0.66; 95%CI=0.47-0.95; p=0.03) and adequate support from the management (OR=0.45; 95%CI=0.22-0.90; p=0.02) constituted negative predictors of burnout in the reduced personal accomplishment subscale. Burnout is highly prevalent among resident doctors. Evolvement of comprehensive mental health services, training supports, conflict de-escalation/resolution mechanisms, and periodic assessment are indicated to mitigate work related distress with burn out among resident doctors, while improving their productivity.

Structural evidence for the partially oxidized dipyrromethene and dipyrromethanone forms of the cofactor of porphobilinogen deaminase: structures of the Bacillus megaterium enzyme at near-atomic resolution.

The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor, which is covalently linked by a thioether bridge to an invariant cysteine residue (Cys241 in the Bacillus megaterium enzyme). The cofactor is extended during the reaction by the sequential addition of the four substrate molecules, which are released as a linear tetrapyrrole product. Expression in Escherichia coli of a His-tagged form of B. megaterium PBGD has permitted the X-ray analysis of the enzyme from this species at high resolution, showing that the cofactor becomes progressively oxidized to the dipyrromethene and dipyrromethanone forms. In previously solved PBGD structures, the oxidized cofactor is in the dipyromethenone form, in which both pyrrole rings are approximately coplanar. In contrast, the oxidized cofactor in the B. megaterium enzyme appears to be in the dipyrromethanone form, in which the C atom at the bridging α-position of the outer pyrrole ring is very clearly in a tetrahedral configuration. It is suggested that the pink colour of the freshly purified protein is owing to the presence of the dipyrromethene form of the cofactor which, in the structure reported here, adopts the same conformation as the fully reduced dipyrromethane form.

Dengue virus serotype-2 impairs proliferation of healthy donors' T lymphocytes.

OBJECTIVES: T lymphocytes are not infected by dengue virus (DENV), nevertheless it is possible that exposure to DENV may affect their function. T lymphocytes from DENV-infected individuals are impaired in their proliferative capacity, although this effect has been attributed to altered function of antigen-presenting cells rather than to an intrinsic defect on T lymphocytes. Here we analyzed whether T lymphocytes from healthy donors became impaired in their proliferative capacity following in vitro exposure to DENV serotype-2 (DENV-2), as well as the possible mechanisms for this. METHODS: Isolated CD4+ and CD8+ T lymphocytes from healthy donors were in vitro exposed to DENV-2, before polyclonal activation, cell proliferation, IL-2 synthesis. IL-2Rα expression, nuclear translocation of NF-AT and NF-κB, and intracellular calcium flux were assessed. RESULTS: In vitro exposure of both CD4+ and CD8+ T lymphocytes from healthy donors to DENV-2 impairs cell proliferation, IL-2 synthesis, and IL-2Rα (CD25) cell membrane expression. Signalling wise, exposure to DENV-2 impairs the nuclear translocation of NF-AT, downstream of intracellular calcium mobilization, as well as that of NF-κB. CONCLUSION: In the course of a dengue infection, direct exposure of T lymphocytes to DENV could affect cell-mediated immune responses.

Arcobacter, an emerging opportunistic food borne pathogen--A review.

BACKGROUND: Arcobacters, emerging aetiologic agents of food-borne diarrhoeal illness in humans and animals are more frequently isolated in meat, especially poultry meat, pork and beef. Though human infection may exist, it has not been documented in Nigeria. AIM: This review presents an update of scientific information in Nigeria on arcobacters as an emerging food-borne pathogen of public health significance in Nigeria. METHODS: A comprehensive reviews of literatures was adopted to give an update on scientific findings on the disease in Nigeria. RESULTS AND CONCLUSIONS: The review revealed scientific evidences attributing the cause of human and animal illness to Arcobacter spp. It also highlights efforts towards the development of animal models where in virulence and pathogenicity of primarily A. butzleri and A. cryaerophilus isolated from human diarrhoeal stool samples were tested. This in turn elucidated the public health significance of this emerging food-borne pathogen. The review canvases for more investigation as to the role of arcobacters in food contamination and unrecognised food-borne disease in Nigeria.

Are we discussing SUDEP?--A retrospective case note analysis.

PURPOSE: Sudden unexplained death in epilepsy (SUDEP) is uncommon. Discussing the risk of SUDEP can be difficult, particularly in those where the risk is considered low, and previous studies have suggested that clinical practice varies widely. The Scottish Intercollegiate Guidelines Network (SIGN) suggest information on SUDEP is "essential" and National Institute of Clinical Excellence (NICE) recommend that "tailored information on the person's relative risk of SUDEP should be part of the counselling process…". The study aimed to evaluate if discussion of SUDEP risk is being documented in clinical records and to determine if there is an association between documented discussion and risk factors for SUDEP. METHODS: A retrospective case note review was undertaken in those with an established diagnosis of epilepsy attending clinic between 1st January 2009 and 30th June 2009. RESULTS: Overall, a documented SUDEP discussion was noted in 14/345 (4%) cases. Patients were statistically more likely to have a documented SUDEP discussion if they had ongoing generalised tonic-clonic seizures, with a trend also towards informing those non-compliant with medication. CONCLUSION: Patients were more likely to be informed of SUDEP if they had potentially modifiable risk factors identified. There was, however, no documented evidence to suggest that SUDEP is being discussed in the majority of cases.

An analysis of subdomain orientation, conformational change and disorder in relation to crystal packing of aspartic proteinases.

The analysis reported here describes detailed structural studies of endothiapepsin (the aspartic proteinase from Endothia parasitica), with and without bound inhibitors, and human pepsin 3b. Comparison of multiple crystal structures of members of the aspartic proteinase family has revealed small but significant differences in domain orientation in different crystal forms. In this paper, it is shown that these differences in domain orientation do not necessarily correlate with the presence or absence of bound inhibitors, but appear to stem at least partly from crystal contacts mediated by sulfate ions. However, since the same inherent flexibility of the structure is observed for other enzymes in this family such as human pepsin, the native structure of which is also reported here, the observed domain movements may well have implications for the mechanism of catalysis.

Silencing of the polyamine catabolic key enzyme SSAT prevents CDK inhibitor-induced apoptosis in Caco-2 colon cancer cells.

Roscovitine and purvalanol are purine derivative cyclin-dependent kinase (CDK) inhibitors that induce apoptosis in various types of cancer cells. However, their impact on the apoptotic cell death mechanism requires further elucidation. Natural polyamines putrescine, spermidine and spermine play essential roles in the regulation of cell growth and proliferation. Increased levels of polyamines in cells are considered to be involved in cancer progression. Intracellular polyamine levels are under the control of several catabolic enzymes, such as spermidine/spermine-N-acetyl transferase (SSAT), acetylpolyamine oxidase (APAO) and spermine oxidase (SMO), which could be altered by several therapeutic drugs. However, the possible role of polyamines in drug-induced apoptosis has yet to be clarified. In the present study, our aim was to determine the modulation of the polyamine catabolic pathway related to CDK inhibitor-induced apoptosis in Caco-2 cells. We found that roscovitine and purvalanol (each 20 µM) induced apoptosis by activating caspase-9 and -3, and inhibiting the mitochondrial membrane potential in Caco-2 cells. CDK inhibitors decreased the intracellular putrescine and spermine levels without affecting spermidine levels. Although both roscovitine and purvalanol induced SSAT expression, they did not exert a significant effect on the APAO expression profile. SSAT transient silencing prevented roscovitine-induced apoptosis compared to parental cells. Thus, we concluded that roscovitine and purvalanol significantly induce apoptosis in Caco-2 cells by modulating the polyamine catabolism, and that SSAT could be an important target in evaluating the potential role of polyamines in apoptotic cell death.