BMC Research Notes - Latest articles

Identification of a non-purple tartrate-resistant acid phosphatase: an evolutionary link to Ser/Thr protein phosphatases?

2008-09-04

Background: Tartrate-resistant acid phosphatases (TRAcPs), also known as purple acid phosphatases (PAPs), are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. The human enzyme is a major histochemical marker for the diagnosis of bone-related diseases. TRAcPs can occur as a small form possessing only the ~35 kDa catalytic domain, or a larger ~55 kDa form possessing both a catalytic domain and an additional N-terminal domain of unknown function. Due to its role in bone resorption the 35 kDa TRAcP has become a promising target for the development of anti-osteoporotic chemotherapeutics.FindingsA new human gene product encoding a metallohydrolase distantly related to the ~55 kDa plant TRAcP was identified and characterised. The gene product is found in a number of animal species, and is present in all tissues sampled by the RIKEN mouse transcriptome project. Construction of a homology model illustrated that six of the seven metal-coordinating ligands in the active site are identical to that observed in the TRAcP family. However, the tyrosine ligand associated with the charge transfer transition and purple color of TRAcPs is replaced by a histidine. Conclusions: The gene product identified here may represent an evolutionary link between TRAcPs and Ser/Thr protein phosphatases. Its biological function is currently unknown but is unlikely to be associated with bone metabolism.

Development of a nutrition and physical activity booklet to engage seniors

Linda Burke, Peter Howat, Andy H Lee, Jonine Jancey, Deb Kerr and Trevor Shilton — 2008-09-04

Background: This paper describes the development and process evaluation of an interactive booklet aimed at motivating older adults to improve their nutrition and physical activity. FindingsThe booklet was developed in consultation with seniors via focus groups, individual interviews and self administered questionnaires. The booklet was disseminated to a group of 114 seniors as the main component of a 12-week intervention program. Process evaluation was conducted during and at the end of the intervention period. A large proportion of participants (86%) were engaged in the program in that they had, as a minimum, read the booklet. The majority of the participants found the booklet provided them with useful and interesting advice in an easy-to-read and informative manner. Three quarters (76%) reported the materials to be motivating and increased their awareness of nutrition and physical activity, while 79% intended to continue with changes to their physical activity and diet after the program concluded.

Knowledge and attitudes towards HIV vaccines among Soweto adolescents

2008-08-29

Background: To explore adolescent HIV risk perception, HIV vaccine knowledge, willingness to participate in future HIV vaccine clinical trials, and the factors that influence willingness to participate among high school students in Soweto, South Africa, we recruited school-going youth through randomly selected local high schools. All pupils within the selected schools from whom parental consent and child assent could be obtained were eligible for participation. A self-administered, facilitated questionnaire was completed by all participants. FindingsPerception of adolescent HIV risk was high. Some misconceptions regarding vaccine research were common, particularly regarding placebo and potential eligibility criteria for prophylactic vaccine trials. Of 240 responses to the willingness item, 84 (35%) indicated they were "probably willing" and 126 (52.5%) that they were "definitely willing to participate". There were no significant differences in willingness by gender, age, school grade, or institution. Factors that were rated as "very important" in determining willingness included receiving current information about HIV research [n = 201 (88.9%)], doing something to honour people who have HIV or have died of AIDS [n = 168 (70.9%)], getting free counseling and testing [n = 167 (70.5)], that participants may receive some protection against HIV infection from the vaccine [n = 160 (70.2%)], and improving motivation to avoid risky behaviour [n = 134 (59%)]., Conclusions: Soweto school-going youth report high degrees of willingness to participate in HIV vaccine trials. This may be related to the high levels of adolescent HIV risk perception. Whether hypothetical willingness translates to participation will await data from adolescent HIV vaccine trials.

Drosophila phosphopantothenoylcysteine synthetase is required for tissue morphogenesis during oogenesis

Floris Bosveld, Anil Rana, Willy Lemstra, Harm H Kampinga and Ody C.M. Sibon — 2008-08-29

Background: Coenzyme A (CoA) is an essential metabolite, synthesized from vitamin B5 by the subsequent action of five enzymes: PANK, PPCS, PPCDC, PPAT and DPCK. Mutations in Drosophila dPPCS disrupt female fecundity and in this study we analyzed the female sterile phenotype of dPPCS mutants in detail. Results: We demonstrate that dPPCS is required for various processes that occur during oogenesis including chorion patterning. Our analysis demonstrates that a mutation in dPPCS disrupts the organization of the somatic and germ line cells, affects F-actin organization and results in abnormal PtdIns(4,5)P2 localization. Improper cell organization coincides with aberrant localization of the membrane molecules Gurken (Grk) and Notch, whose activities are required for specification of the follicle cells that pattern the eggshell. Mutations in dPPCS also induce alterations in scutellar patterning and cause wing vein abnormalities. Interestingly, mutations in dPANK and dPPAT-DPCK result in similar patterning defects. Conclusions: Together, our results, demonstrate that de novo CoA biosynthesis is required for proper tissue morphogenesis.

Characterization of endemic Shigella boydii strains isolated in Iran by serotyping, antimicrobial resistance, plasmid profile, ribotyping and pulsed-field gel electrophoresis

2008-08-29

Background: Shigellosis is one of the major causes of morbidity in children with diarrhea in Iran. The present study was undertaken to characterize apparently sporadic Shigella boydii strains isolated from pediatric patients in Iran.FindingsTen S. boydii strains isolated from pediatric cases of gastroenteritis and acute diarrhea in Tehran between December 2002 and November 2003 were submitted to serotyping, antimicrobial susceptibility testing, plasmid profile analysis, ribotyping and pulsed field gel electrophoresis (PFGE). Seven isolates were attributed to serotype 2, whereas the remaining three belonged to serotypes 14, 18, 19, respectively. Six drug resistance phenotypes (R1 to R6) were defined with R4 - streptomycin (STR), ampicillin (AMP), sulfamethoxazole-trimethoprim (SXT) - being the most prevalent. Plasmid analysis resulted in seven different plasmid profiles with one to five DNA bands. All strains, but one, shared the same ribotype, but PFGE differentiated them in four groups. Conclusion: Based upon ribotyping and PFGE results, endemic circulation of S. boydii in Tehran, Iran, could be attributed to a few clones. Resistance pattern and plasmid profile analysis proved to be very effective in discriminating apparently unrelated strains of S. boydii.

Characteristics of TIA and its management in a tertiary care hospital in Pakistan

2008-08-29

Background: Transient ischemic attack (TIA) is described as a brief episode of neurological dysfunction caused by focal brain ischemia, with clinical symptoms typically lasting less than an hour, and without evidence of acute infarction. Recent studies depict TIA as a particularly unstable condition. Risk of stroke is greater than 10% in the first 90 days after an index TIA. The presentation, prognosis and intervention for TIA have not been reported in South-Asians in a developing country. MethodA retrospective chart review was done for 158 patients who were admitted with the diagnosis of TIA, as defined by ICD 9 code 435, from January 2003 to December 2005 at the Aga Khan University Hospital, Karachi, Pakistan. The data was entered and analyzed in SPSS version 14.0.FindingsAmong 158 patients, 57.6% were male and 41.1% were female. The common presenting symptoms were motor symptoms (51.3%), speech impairment (43%), sensory impairment (34.8%) and loss of balance/vertigo (29.1%).The median delay in presenting to the hospital was 4 hours. Those with motor symptoms were found to present earlier. The study showed that only 60.8% of all the patients presenting with TIA received any immediate treatment out of which 44.7% received aspirin. Neuroimaging was used in 91.1% of the patients. Of all the TIA patients 9.1% converted to stroke with 50% doing so within the first 24 hours. Conclusion: The natural history of TIA from this developing nation is comparable to international descriptions. A large percentage of patients are still not receiving any immediate treatment as recommended in available guidelines, even in a tertiary care hospital.

Comparative phylogenomics and multi-gene cluster analyses of the Citrus Huanglongbing (HLB)-associated bacterium Candidatus Liberibacter

2008-08-28

Background: Huanglongbing (HLB, previously known as citrus greening), is associated with Candidatus Liberibacter species and is a serious threat to citrus production world-wide. The pathogen is a Gram negative, unculturable, phloem-limited bacterium with limited known genomic information. Expanding the genetic knowledge of this organism may provide better understanding of the pathogen and possibly develop effective strategies for control and management of HLB. Results: Here, we report cloning and characterization of an additional 14.7 Kb of new genomic sequences from three different genomic regions of the Candidatus Liberibacter asiaticus (Las). Sequence variation analyses among the available Ca. Liberibacter species sequences as well as the newly cloned 1.5 Kb of rpoB gene from different Ca. Liberibacter strains have identified INDELs and SNPs. Phylogenetic analysis of the deduced protein sequences from the cloned regions characterizes the HLB-associated Candidatus Liberibacter as a new clade in the sub-division of the alpha-proteobacteria. Conclusions: Comparative analyses of the cloned gene regions of Candidatus Liberibacter with members of the order Rhizobiales suggest overall gene structure and order conservation, albeit with minor variations including gene decay due to the identified pseudogenes. The newly cloned gene regions contribute to our understanding of the molecular aspects of genomic evolution of Ca. Liberibacter.

Quantification of circulating endothelial and progenitor cells: comparison of quantitative PCR and four-channel flow cytometry

2008-08-28

Background: Circulating endothelial cells (CEC) and endothelial precursor cells (CEP) have been suggested as surrogate markers for angiogenesis and response to antiangiogenic therapy in cancer. However, CEC/CEP represent a tiny and heterogeneous cell population, rendering their enumeration in peripheral blood samples difficult. CEC/CEP quantification by flow cytometry is currently the most commonly applied method but technically demanding and poorly standardized. Thus, we investigated whether quantitative PCR-based detection might overcome these limitations. Methods: To test the sensitivity of both assays autologous endothelial colony forming cell clones (ECFC) and cord blood derived CD45- CD34+ progenitor cells were spiked into peripheral blood mononuclear cells (PBMNC) of healthy volunteers. Expression of CD45, CD31, CD34, KDR and CD133 was analyzed by 4-color flow cytometry. Furthermore, absolute gene expression of progenitor and endothelial cell markers was quantified in spiked samples, PBMNC from healthy controls (n=30), newly-diagnosed locally advanced rectal cancer patients (n=20) and patients with metastatic non-small cell lung cancer (NSCLC, n=25). Results: Applying flow cytometry, spiked ECFC and progenitor cells were detectable only at frequencies 0.01%, whereas using qPCR a detection limit of 0.001% was achievable. Moreover, qPCR revealed significantly increased gene transcripts of CD34 (p=0.028) and KDR (p=0.002) in patients with metastatic NSCLC but not in patients with locally advanced rectal cancer. Conclusions: QPCR may overcome the limitations of flow cytometry for the detection of CEC/CEP. However, further studies using qPCR-based assays as well as more specific CEC/CEP markers are needed to validate and improve the detection of these rare cell types.

Differential susceptibility of PCR reactions to inhibitors: an important and unrecognised phenomenon

2008-08-28

Background: PCR inhibition by nucleic acid extracts is a well known yet poorly described phenomenon. Inhibition assessment generally depends on the assumption that inhibitors affect all PCR reactions to the same extent; i.e. that the reaction of interest and the control reaction are equally susceptible to inhibition. To test this assumption we performed inhibition assessment on DNA extracts from human urine samples, fresh urine and EDTA using different PCR reactions. Results: When co-purified inhibitors were assessed using two different PCR reactions, one reaction appeared to be inhibited whilst the other was not. Further experiments, adding various concentrations of urine to inhibit six different PCR reactions revealed that susceptibility to inhibition was highly variable between reactions. Similar results were obtained using EDTA as the PCR inhibitor. We could find no obvious explanation as to why one reaction should be more susceptible to inhibition than another, although a possible association with amplicon GC content was noted. Conclusions: These findings have serious implications for all PCR-based gene expression studies, including the relatively new PCR array method, and for both qualitative and quantitative PCR-based molecular diagnostic assays, suggesting that careful consideration should be given to inhibition compatibility when conducting PCR analyses. We have demonstrated unequivocally that it is not possible to assume that different PCR reactions are equally susceptible to inhibition by substances co-purified in nucleic acid extracts.

Barriers to healthy eating by National Health Service (NHS) hospital doctors in the hospital setting: results of a cross-sectional survey

James Winston, Carol Johnson and Sue Wilson — 2008-08-28

Background: With high levels of obesity and related illness, improving the health of the nation is a major public health concern. This study aimed to identify factors that prevent healthy eating among doctors, and that are associated with satisfaction with catering services. Findings Methods: Cross-sectional survey of 328 NHS doctors working in two NHS Trusts with on-site hospital canteen. Questionnaire to establish perceived barriers to healthy eating, weekly use and satisfaction with the hospital canteen, lifestyle and dietary habits, gender, age, height, weight, job details, and affect. Results: 70% of doctors reported using their hospital canteen each week, with 2 visits per week on average. Canteen opening times, lack of selection and lack of breaks were the most commonly perceived barriers to healthy eating. Availability of healthy options caused the most dissatisfaction. Only 12% felt the NHS was supportive of healthy eating. 74% did not feel their canteen advocated healthy eating. Canteen use is associated with younger age (r= -0.254, p<0.0001) and health score (r=0.123, p=0.049). Conclusions: Interventions to encourage regular meal breaks, eating breakfast and drinking more water each day need developing. Improved canteen accessibility and availability of healthy options at evenings and weekends may be beneficial.

The transcriptional cofactor MIER1-beta negatively regulates histone acetyltransferase activity of the CREB-binding protein

Tina M Blackmore, Corinne F Mercer, Gary D Paterno and Laura L Gillespie — 2008-08-22

Background: Mier1 encodes a novel transcriptional regulator and was originally isolated as a fibroblast growth factor early response gene. Two major protein isoforms have been identified, MIER1 alpha and beta, which differ in their C-terminal sequence. Previously, we demonstrated that both isoforms recruit histone deacetylase 1 (HDAC1) to repress transcription. To further explore the role of MIER1 in chromatin remodeling, we investigated the functional interaction of MIER1 with the histone acetyltransferase (HAT), Creb-binding protein (CBP).FindingsUsing GST pull-down assays, we demonstrate that MIER1 interacts with CBP and that this interaction involves the N-terminal half (amino acids 1-283) of MIER1, which includes the acidic activation and ELM2 domains and the C-terminal half (amino acids 1094-2441) of CBP, which includes the bromo-, HAT, C/H3 and glutamine-rich domains. Functional analysis, using HEK293 cells, shows that the CBP bound to MIER1 in vivo has no detectable HAT activity. Histone 4 peptide binding assays demonstrate that this inhibition of HAT activity is not the result of interference with histone binding. Conclusions: Our data indicate that an additional mechanism by which MIER1 could repress transcription involves the inhibition of histone acetyltransferase activity.