bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Properties of the Plasmid Maintenance Proteins from theBorrelia burgdorferiLinear Plasmid lp17
Authors: Deneke Jan , Chaconas George
Published: March 2008

The Lyme disease spirocheteBorrelia burgdorfericarries more plasmids than any other bacterium, many of which are linear with covalently closed hairpin ends. These plasmids have also been referred to as mini-chromosomes and essential genetic elements and are integral components of its segmented genome. We have investigated two plasmid maintenance proteins, BBD14 (the replication initiator) and BBD21 (a presumptive ParA orthologue), encoded by the linear plasmid lp17; these proteins are representatives of paralogous families 62 and 32, respectively. We have purified recombinant 6-his-BBD21 and shown it possesses an ATPase activity. 6-his-BBD14 initially could not be overexpressed inEscherichia coliby itself. It was only effectively overproduced in recombinant form through coexpression with otherB. burgdorferiproteins and codon optimization. Although the mechanism for increased production through coexpression is not clear, this method holds promise for expression and purification of otherB. burgdorferiproteins, a number of which have remained recalcitrant to purification fromE. coli. Finally, we present evidence for the physical interaction of BBD14 and BBD21, a feature suggesting that BBD21 and the paralogous family 32 proteins are more likely involved in DNA replication than functioning as simple ParA orthologues as previously surmised based upon sequence homology. Such a role would not preclude a function in plasmid partitioning through interaction with the replication initiator.
Specific Partial Reduction of Geranylgeranyl Diphosphate by an Enzyme from the Thermoacidophilic Archaeon
Sulfolobus acidocaldarius
Yields a Reactive Prenyl Donor, Not a Dead-End Product
Authors: Sato Sho , Murakami Motomichi , Yoshimura Tohru , Hemmi Hisashi
Published: March 2008

Geranylgeranyl reductase from
Sulfolobus acidocaldarius
was shown to catalyze the reduction of geranylgeranyl groups in the precursors of archaeal membrane lipids, generally reducing all four double bonds. However, when geranylgeranyl diphosphate was subjected to the reductase reaction, only three of the four double bonds were reduced. Mass spectrometry and acid hydrolysis indicated that the allylic double bond was preserved in the partially reduced product derived from geranylgeranyl diphosphate. Thus, the reaction product was shown to be phytyl diphosphate, which is a substrate for archaeal prenyltransferases, unlike the completely reduced compound phytanyl diphosphate.
Autophosphorylation and Dephosphorylation by Soluble Forms of the Nitrate-Responsive Sensors NarX and NarQ from
Escherichia coli
K-12
Authors: Noriega Chris E. , Schmidt Radomir , Gray Michael J. , Chen Li-Ling , Stewart Valley
Published: March 2008

NarX-NarL and NarQ-NarP are paralogous two-component regulatory systems that control
Escherichia coli
gene expression in response to the respiratory oxidants nitrate and nitrite. Nitrate stimulates the autophosphorylation rates of the NarX and NarQ sensors, which then phosphorylate the response regulators NarL and NarP to activate and repress target operon transcription. Here, we investigated both the autophosphorylation and dephosphorylation of soluble sensors in which the maltose binding protein (MBP) has replaced the amino-terminal transmembrane sensory domain. The apparent affinities (

K
m

) for ADP were similar for both proteins, about 2 μM, whereas the affinity of MBP-NarQ for ATP was lower, about 23 μM. At a saturating concentration of ATP, the rate constant of MBP-NarX autophosphorylation (about 0.5 × 10
−4
s
−1
) was lower than that observed for MBP-NarQ (about 2.2 × 10
−4
s
−1
). At a saturating concentration of ADP, the rate constant of dephosphorylation was higher than that of autophosphorylation, about 0.03 s
−1
for MBP-NarX and about 0.01 s
−1
for MBP-NarQ. For other studied sensors, the published affinities for ADP range from about 16 μM (KinA) to about 40 μM (NtrB). This suggests that only a small proportion of NarX and NarQ remain phosphorylated in the absence of nitrate, resulting in efficient response regulator dephosphorylation by the remaining unphosphorylated sensors.
Genetic Analysis of the Methanol- and Methylamine-Specific Methyltransferase 2 Genes of
Methanosarcina acetivorans
C2A
Authors: Bose Arpita , Pritchett Matthew A. , Metcalf William W.
Published: March 2008

The entry of methanol into the methylotrophic pathway of methanogenesis is mediated by the concerted effort of two methyltransferases, namely, methyltransferase 1 (MT1) and methyltransferase 2 (MT2). The
mtaA1
,
mtaA2
, and
mtbA
genes of
Methanosarcina acetivorans
C2A encode putative methanol- or methylamine-specific MT2 enzymes. To address the in vivo roles of these genes in growth and methanogenesis from known substrates, we constructed and characterized mutants with deletions of each of these genes. The
mtaA1
gene is required for growth on methanol, whereas
mtaA2
was dispensable. However, the
mtaA2
mutant had a reduced rate of methane production from methanol. Surprisingly, deletion of
mtaA1
in combination with deletions of the genes encoding three methanol-specific MT1 isozymes led to lack of growth on acetate, suggesting that MT1 and MT2 enzymes might play an important role during growth on this substrate. The
mtbA
gene was required for dimethylamine and monomethylamine (MMA) utilization and was important, but not required, for trimethylamine utilization. Analysis of reporter gene fusions revealed that both
mtaA1
and
mtbA
were expressed on all methanogenic substrates tested. However,
mtaA1
expression was induced on methanol, while
mtbA
expression was down-regulated on MMA and acetate.
mtaA2
was expressed at very low levels on all substrates. The
mtaA1
transcript had a large 5′ untranslated region (UTR) (275 bp), while the 5′ UTR of the
mtbA
transcript was only 28 bp long.
Transcriptional Organization of the Region Encoding the Synthesis of the Flagellar Filament in
Pseudomonas fluorescens
Authors: Redondo-Nieto Miguel , Lloret Javier , Larenas Javiera , Barahona Emma , Navazo Ana , Martínez-Granero Francisco , Capdevila Silvia , Rivilla Rafael , Martín Marta
Published: March 2008

Pseudomonas fluorescens
F113 is motile by means of type b flagella. Analysis of the region encoding the synthesis of the flagellar filament has shown a transcriptional organization different from that of type a flagella. Additionally to the promoters driving
fliC
,
fliD
, and
fleQ
expression, we have found promoters upstream of the
flaG
gene and the
fliST
operon. These promoters were functional in vivo. Both promoters have been mapped and appear to be dependent on the vegetative sigma factor and independent of FleQ, the master regulator of flagellum synthesis.
Genome Sequence of the Streptomycin-Producing MicroorganismStreptomyces griseusIFO 13350
Authors: Ohnishi Yasuo , Ishikawa Jun , Hara Hirofumi , Suzuki Hirokazu , Ikenoya Miwa , Ikeda Haruo , Yamashita Atsushi , Hattori Masahira , Horinouchi Sueharu
Published: March 2008

We determined the complete genome sequence ofStreptomyces griseusIFO 13350, a soil bacterium producing an antituberculosis agent, streptomycin, which is the first aminoglycoside antibiotic, discovered more than 60 years ago. The linear chromosome consists of 8,545,929 base pairs (bp), with an average G+C content of 72.2%, predicting 7,138 open reading frames, six rRNA operons (16S-23S-5S), and 66 tRNA genes. It contains extremely long terminal inverted repeats (TIRs) of 132,910 bp each. The telomere's nucleotide sequence and secondary structure, consisting of several palindromes with a loop sequence of 5′-GGA-3′, are different from those of typical telomeres conserved among otherStreptomycesspecies. In accordance with the difference, the chromosome has pseudogenes for a conserved terminal protein (Tpg) and a telomere-associated protein (Tap), and a novel pair of Tpg and Tap proteins is instead encoded by the TIRs. Comparisons with the genomes of two related species,Streptomyces coelicolorA3(2) andStreptomyces avermitilis, clarified not only the characteristics of theS. griseusgenome but also the existence of 24Streptomyces-specific proteins. TheS. griseusgenome contains 34 gene clusters or genes for the biosynthesis of known or unknown secondary metabolites. Transcriptome analysis using a DNA microarray showed that at least four of these clusters, in addition to the streptomycin biosynthesis gene cluster, were activated directly or indirectly by AdpA, which is a central transcriptional activator for secondary metabolism and morphogenesis in the A-factor (a γ-butyrolactone signaling molecule) regulatory cascade inS. griseus.
Sodium Chloride Affects
Helicobacter pylori
Growth and Gene Expression
Authors: Gancz Hanan , Jones Kathleen R. , Merrell D. Scott
Published: March 2008

Epidemiological evidence links high-salt diets and
Helicobacter pylori
infection with increased risk of developing gastric maladies. The mechanism by which elevated sodium chloride content causes these manifestations is unclear. Here we characterize the response of
H. pylori
to temporal changes in sodium chloride concentration and show that growth, cell morphology, survival, and virulence factor expression are all altered by increased salt concentration.
Functional Characterization of a Vitamin B
12
-Dependent Methylmalonyl Pathway in
Mycobacterium tuberculosis
: Implications for Propionate Metabolism during Growth on Fatty Acids
Authors: Savvi Suzana , Warner Digby F. , Kana Bavesh D. , McKinney John D. , Mizrahi Valerie , Dawes Stephanie S.
Published: March 2008

Mycobacterium tuberculosis
is predicted to subsist on alternative carbon sources during persistence within the human host. Catabolism of odd- and branched-chain fatty acids, branched-chain amino acids, and cholesterol generates propionyl-coenzyme A (CoA) as a terminal, three-carbon (C
3
) product. Propionate constitutes a key precursor in lipid biosynthesis but is toxic if accumulated, potentially implicating its metabolism in
M. tuberculosis
pathogenesis. In addition to the well-characterized methylcitrate cycle, the
M. tuberculosis
genome contains a complete methylmalonyl pathway, including a
mutAB
-encoded methylmalonyl-CoA mutase (MCM) that requires a vitamin B
12
-derived cofactor for activity. Here, we demonstrate the ability of
M. tuberculosis
to utilize propionate as the sole carbon source in the absence of a functional methylcitrate cycle, provided that vitamin B
12
is supplied exogenously. We show that this ability is dependent on
mutAB
and, furthermore, that an active methylmalonyl pathway allows the bypass of the glyoxylate cycle during growth on propionate in vitro. Importantly, although the glyoxylate and methylcitrate cycles supported robust growth of
M. tuberculosis
on the C
17
fatty acid heptadecanoate, growth on valerate (C
5
) was significantly enhanced through vitamin B
12
supplementation. Moreover, both wild-type and methylcitrate cycle mutant strains grew on B
12
-supplemented valerate in the presence of 3-nitropropionate, an inhibitor of the glyoxylate cycle enzyme isocitrate lyase, indicating an anaplerotic role for the methylmalonyl pathway. The demonstrated functionality of MCM reinforces the potential relevance of vitamin B
12
to mycobacterial pathogenesis and suggests that vitamin B
12
availability in vivo might resolve the paradoxical dispensability of the methylcitrate cycle for the growth and persistence of
M. tuberculosis
in mice.
The Carriage Population of
Staphylococcus aureus
from Mali Is Composed of a Combination of Pandemic Clones and the Divergent Panton-Valentine Leukocidin-Positive Genotype ST152
Authors: Ruimy Raymond , Maiga Aminata , Armand-Lefevre Laurence , Maiga Ibrahim , Diallo Amadou , Koumaré Abdel Karim , Ouattara Kalilou , Soumaré Sambou , Gaillard Kevin , Lucet Jean-Christophe , Andremont Antoine , Feil Edward J.
Published: March 2008

Staphylococcus aureus
is an important human pathogen, but it appears more commonly in asymptomatic colonization of the nasopharynx than in cases of invasive disease. Evidence concerning the global population structure of
S. aureus
is limited by the overrepresentation in the multilocus sequence testing database of disease isolates recovered from Western Europe, the Americas, Australia, and Japan. We address this by presenting data from the
S. aureus
carriage population in Mali, the first detailed characterization of asymptomatic carriage from an African population. These data confirm the pandemic spread of many of the common
S. aureus
clones in the carriage population. We also note the high frequency (∼24%) of a single divergent genotype, sequence type 152 (ST152), which has not previously been recovered from nasal carriage isolates but corresponds to a sporadic Panton-Valentine leukocidin (PVL)-positive, community-acquired methicillin-resistant
S. aureus
clone noted mostly in Central Europe. We show that 100% of the ST152 isolates recovered from nasal carriage samples in Mali are PVL positive and discuss implications relating to the emergence and spread of this virulent genotype.
CabC, an EF-Hand Calcium-Binding Protein, Is Involved in Ca2+-Mediated Regulation of Spore Germination and Aerial Hypha Formation inStreptomyces coelicolor
Authors: Wang Sheng-Lan , Fan Ke-Qiang , Yang Xu , Lin Zeng-Xi , Xu Xin-Ping , Yang Ke-Qian
Published: March 2008

Ca2+was reported to regulate spore germination and aerial hypha formation in streptomycetes; the underlying mechanism of this regulation is not known.cabC, a gene encoding an EF-hand calcium-binding protein, was disrupted or overexpressed inStreptomyces coelicolorM145. On R5− agar, the disruption ofcabCresulted in denser aerial hyphae with more short branches, swollen hyphal tips, and early-germinating spores on the spore chain, whilecabCoverexpression significantly delayed development. Manipulation of the Ca2+concentration in R5− agar could reverse the phenotypes ofcabCdisruption or overexpression mutants and mimic mutant phenotypes with M145, suggesting that the mutant phenotypes were due to changes in the intracellular Ca2+concentration. CabC expression was strongly activated in aerial hyphae, as determined by Western blotting against CabC and confocal laser scanning microscopy detection of CabC::enhanced green fluorescent protein (EGFP). CabC::EGFP fusion proteins were evenly distributed in substrate mycelia, aerial mycelia, and spores. Taken together, these results demonstrate that CabC is involved in Ca2+-mediated regulation of spore germination and aerial hypha formation inS. coelicolor. CabC most likely acts as a Ca2+buffer and exerts its regulatory effects by controlling the intracellular Ca2+concentration.
A new predictive indicator for development of pressure ulcers in bedridden patients based on common laboratory tests results
Authors: Hatanaka N , Yamamoto Y , Ichihara K , Mastuo S , Nakamura Y , Watanabe M , Iwatani Y
Published: March 2008

Background:

Various scales have been devised to predict development of pressure ulcers on the basis of clinical and laboratory data, such as the Braden Scale (Braden score), which is used to monitor activity and skin conditions of bedridden patients. However, none of these scales facilitates clinically reliable prediction.

Aims:

To develop a clinical laboratory data-based predictive equation for the development of pressure ulcers.

Methods:

Subjects were 149 hospitalised patients with respiratory disorders who were monitored for the development of pressure ulcers over a 3-month period. The proportional hazards model (Cox regression) was used to analyse the results of 12 basic laboratory tests on the day of hospitalisation in comparison with Braden score.

Results:

Pressure ulcers developed in 38 patients within the study period. A Cox regression model consisting solely of Braden scale items showed that none of these items contributed to significantly predicting pressure ulcers. Rather, a combination of haemoglobin (Hb), C-reactive protein (CRP), albumin (Alb), age, and gender produced the best model for prediction. Using the set of explanatory variables, we created a new indicator based on a multiple logistic regression equation. The new indicator showed high sensitivity (0.73) and specificity (0.70), and its diagnostic power was higher than that of Alb, Hb, CRP, or the Braden score alone.

Conclusions:

The new indicator may become a more useful clinical tool for predicting presser ulcers than Braden score. The new indicator warrants verification studies to facilitate its clinical implementation in the future.
The role of nurses in euthanasia and physician-assisted suicide in The Netherlands
Authors: van Bruchem-van de Scheur G G , Arend A J G van der , Abu-Saad H Huijer , Spreeuwenberg C , van Wijmen F C B , ter Meulen R H J
Published: March 2008

Background:

Issues concerning legislation and regulation with respect to the role of nurses in euthanasia and physician-assisted suicide gave the Minister for Health reason to commission a study of the role of nurses in medical end-of-life decisions in hospitals, home care and nursing homes.

Aim:

This paper reports the findings of a study of the role of nurses in euthanasia and physician-assisted suicide, conducted as part of a study of the role of nurses in medical end-of-life decisions. The findings for hospitals, home care and nursing homes are described and compared.

Method:

A questionnaire was sent to 1509 nurses, employed in 73 hospitals, 55 home care organisations and 63 nursing homes. 1179 responses (78.1%) were suitable for analysis. The questionnaire was pilot-tested among 106 nurses, with a response rate of 85%.

Results:

In 37.0% of cases, the nurse was the first person with whom patients discussed their request for euthanasia or physician-assisted suicide. Consultation between physicians and nurses during the decision-making process took place quite often in hospitals (78.8%) and nursing homes (81.3%) and less frequently in home care situations (41.2%). In some cases (12.2%), nurses administered the euthanatics.

Conclusions:

The results show substantial differences between the intramural sector (hospitals and nursing homes) and the extramural sector (home care), which are probably linked to the organisational structure of the institutions. Consultation between physicians and nurses during the decision-making process needs improvement, particularly in home care. Some nurses had administered euthanatics, although this task is by law exclusively reserved to physicians.
Ethical issues arising from the requirement to sign a consent form in palliative care
Authors: Plu I , Purssell-François I , Moutel G , Ellien F , Hervé C
Published: March 2008

French healthcare networks aim to help healthcare workers to take care of patients by improving cooperation, coordination and the continuity of care. When applied to palliative care in the home, they facilitate overall care including medical, social and psychological aspects. French legislation in 2002 required that an information document explaining the functioning of the network should be given to patients when they enter a healthcare network. The law requires that this document be signed. Ethical issues arise from this legislation with regard to the validity of the signature of dying patients. Signature of the consent form by a guardian or trustee, a designated person—the Person of Trust—transforms the doctor–patient relationship into a triangular doctor–patient–third-party relationship.
Ethical decision-making about older adults and moral intensity: an international study of physicians
Authors: Malloy D C , Williams J , Hadjistavropoulos T , Krishnan B , Jeyaraj M , McCarthy E F , Murakami M , Paholpak S , Mafukidze J , Hillis B
Published: March 2008

Through discourse with international groups of physicians, we conducted a cross-cultural analysis of the types of ethical dilemmas physicians face. Qualitative analysis was used to categorise the dilemmas into seven themes, which we compared among the physicians by country of practice. These themes were a-theoretically-driven and grounded heavily within the text. We then subjected the dilemmas to an analysis of moral intensity, which represents an important (albeit novel within healthcare research) theoretical perspective of ethical decision making. These constructs (ie, culture and moral intensity) represent salient determinants of ethical behaviour and our cross-cultural sample afforded us the opportunity to consider both the pragmatic aspects of culture, as they are perceived by physicians, as well as the theory-driven concept of moral intensity. By examining both culture and moral intensity, we hope to better elucidate the complexities of ethical decision-making determinants among physicians in their daily practice. Doing so may potentially have practical implications for ethics training of medical students and foreign physicians.
Ethics in a scientific approach: the importance of the biostatistician in research ethics committees
Authors: Atici E , Erdemir A D
Published: March 2008

In medical practice and research it is necessary to consider the rights of the researcher or physician and of the subject or patient, to conform to scientific standards and to examine the appropriateness with respect to laws and moral values. Research ethics committees have an important role to play in ensuring the ethical standards and scientific merit of research on human subjects. Research of no scientific value is also against ethical principles. To obtain valid and reliable results from biomedical research, it is a scientific and ethical obligation to make use of the science of statistics. Therefore, for research to be evaluated using biostatistics intensively from ethical and scientific points of view, a biostatistics expert is necessary on research ethics committees. Developments in Turkey are used as examples.
Should ethics consultants help clinicians face scarcity in their practice?: Figure 1
Authors: Hurst S A , Reiter-Theil S , Slowther A-M , Pegoraro R , Forde R , Danis M
Published: March 2008

In an international survey of rationing we have found that European physicians encounter scarcity-related ethical difficulties, and are dissatified with the resolution of many of these cases. Here we further examine survey results to explore whether ethics support services would be potentially useful in addressing scarcity related ethical dilemmas. Results indicate that while the type of help offered by ethics support services was considered helpful by physicians, they rarely referred difficulties regarding scarcity to ethics consultation. We propose that ethics consultants could assist physicians by making the process less difficult, and by contributing to decisions being more ethically justifiable. Expertise in bringing considerations of justice to bear on real cases could also be useful in recognising an unjust limit, as opposed to a merely frustrating limit. Though these situations are unlikely to be among the most frequently referred to ethics support services, ethics consultants should be prepared to address them.
Thinking ethically about genetic inheritance: liberal rights, communitarianism and the right to privacy for parents of donor insemination children
Authors: Burr J , Reynolds P
Published: March 2008

The issue of genetic inheritance, and particularly the contradictory rights of donors, recipients and donor offspring as to the disclosure of donor identities, is ethically complicated. Donors, donor offspring and parents of donor offspring may appeal to individual rights for confidentiality or disclosure within legal systems based on liberal rights discourse. This paper explores the ethical issues of non-disclosure of genetic inheritance by contrasting two principle models used to articulate the problem—liberal and communitarian ethical models. It argues that whilst the latter provides a more constructive avenue to providing an ethics for donation than the competing and contradictory positions represented in a liberal rights approach, it raises issues of ethical judgement and authority that remain problematic. This ethical discussion is supported by a field study, funded by the Wellcome Trust, exploring the perceptions and experiences of recipients of donor sperm and their partners towards donor anonymity. The field study provides the empirical basis of an argument for making ethical judgements on the grounds of the community good rather than individual rights, that nevertheless recognises that both are inherently problematic.
Malawians permit research bronchoscopy due to perceived need for healthcare
Authors: Mtunthama N , Malamba R , French N , Molyneux M E , Zijlstra E E , Gordon S B
Published: March 2008

Objectives:

Bronchoalveolar lavage obtained at bronchoscopy is useful for research on pulmonary defence mechanisms. Bronchoscopy involves some discomfort and risk to subjects. We audited the process of consent, experienced adverse effects and reasons for participation among research bronchoscopy volunteers.

Design:

100 consecutive volunteer research subjects attending for bronchoscopy, repeat bronchoscopy or routine recruitment clinic were interviewed. Information was gathered about volunteer motivation, perception of the consent process and adverse effects of bronchoscopy. Suggestions for improvement were requested. Responses were themed by a second investigator prior to data analysis.

Results:

81 bronchoscopy-experienced subjects (total of 263 procedures) and 19 new volunteers were interviewed. 19 subjects (21%) reported adverse symptoms during or after bronchoscopy, but no symptoms were of sufficient severity that they would not repeat the procedure. The frequency of symptoms was not related to gender, the quality of the lavage or the HIV status of the subject. 76 subjects (94%) reported that the information given pre-procedure was useful and adequate but 43 (56%) had further questions mostly relating to their own results. The reasons given for research participation were access to health assessment (75 subjects), access to treatment when ill (61 subjects), desire to participate in research (15 subjects) and remuneration (6 subjects). 7 subjects complained that the remuneration was inadequate.

Conclusions:

The main incentive to participation in research bronchoscopy was access to healthcare. Informed consent and procedure technique were adequate but subjects would value more feedback about individual and project results.
The junior doctor as ethically unique
Authors: McDougall R
Published: March 2008

This paper argues that the professional situation of junior doctors is unique in ethically important ways and thus that ethics work focusing on junior doctors specifically is necessary. Unlike the medical student or the more senior doctor, the doctor in his or her early postgraduate years is simultaneously a responsible health professional, a subjugate learner and a human resource. These multiple roles generate the set of ethical issues faced by junior doctors, a set that has some overlaps with that faced by medical students and with that faced by more experienced doctors but is far from completely continuous with either. Further, the multiple roles that junior doctors play affect their options for negotiating the ethical challenges that they face. Their position determines not only the content of the set of ethical issues that they encounter, but also the kinds of actions they can take in the face of these challenges. Thus considering junior doctors only in combination with medical students or more senior doctors fails on two fronts. Firstly, only a very incomplete set of the ethical issues faced by junior doctors will be addressed, and, secondly, the constraints associated with the specific professional situation of junior doctors will not be adequately considered, limiting the practical applicability for these agents of any such analyses.
Exposure to nicotine and a tobacco-specific carcinogen increase with duration of use of smokeless tobacco
Authors: Hecht S S , Carmella S G , Edmonds A , Murphy S E , Stepanov I , Luo X , Hatsukami D K
Published: March 2008

Background:

Smokeless tobacco is an efficient delivery vehicle for nicotine and can contain significant amounts of carcinogens. However, few studies have examined factors that might moderate levels of nicotine or carcinogen exposure.

Aims:

To determine the effect of duration of smokeless tobacco use on the uptake of nicotine and a tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

Methods:

Questionnaires on use of smokeless tobacco were administered, and urine samples from 212 smokeless tobacco users were analysed for biomarkers of uptake of nicotine and NNK. The biomarkers were cotinine and total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). Male smokeless tobacco users were recruited for studies designed to investigate methods of reducing smokeless tobacco use. The questionnaire and biomarker data were obtained at baseline, prior to reduction.

Results:

Levels of cotinine (p<0.001) and total NNAL (p<0.001) were significantly correlated with duration (in years) of use of smokeless tobacco products. Median cotinine and total NNAL were 2.4 and 2.1 times higher, respectively, in the ⩾21 years of use than in the 0–5 years of use category.

Conclusions:

Smokeless tobacco users adjust their intensity of use with experience in order to increase their nicotine dose, resulting in a corresponding increase in exposure to NNK, a powerful carcinogen. These results indicate the importance of educating smokeless tobacco users about the effects of prolonged use of these products.
Adsorption of vapor-phase VOCs (benzene and toluene) on modified clays and its relation with surface properties
Authors: López-Cortés C , Osorio-Revilla G , Gallardo-Velázquez T , Arellano-Cárdenas S
Published: March 2008

A mexican bentonite was used to prepare three modified clays: an organoclay (OC–CPC) by intercalating cetylpyridinium chloride (CPC), an aluminum-pillared clay (Al-PILC), and an inorganic–organic clay (IOC–CPC) prepared from Al-PILC intercalating CPC. The infrared and thermogravimetric analyses enabled us to differentiate between the structures obtained. Interlayer distance was assessed through X-ray diffraction; the OC–CPC was the clay that showed the highest value (12.4 Å). The natural clay’s specific area was 43.5 m2/g, increasing to 157.2 m2/g for Al-PILC. The OC–CPC and IOC–CPC specific areas were 13.27 m2/g and 14.5 m2/g, respectively. Al-PILC was a microporous material; whereas natural clays, OC–CPC and IOC–CPC were non-porous materials. Toluene and benzene adsorption on OC–CPC was higher than in IOC–CPC and Al-PILC. Natural clay showed no adsorption capacity for these compounds. Comparison of the GC retention times for non polar and low-polarity compounds (octyne and benzene) in columns packed with OC–CPC and a commercial non polar column (squalene) showed that the OC–CPC possessed a higher organophilic (non polar) nature than squalene. This explains the higher benzene and toluene adsorption capacity of the OC–CPC compared with the other modified clays. The toluene and benzene adsorption isotherm for OC–CPC was well-described by the GAB (Guggenheim–Anderson–De Boer) model for the whole range of relative pressure used in this work (0–0.9) describing multilayer adsorption and capillary condensation.Key words: organoclay, inorgano–organo clay, vapor phase VOC’s adsorption, clay surface properties.
Zinc oxide-tetrabutylammonium bromide tandem as a highly efficient, green, and reusable catalyst for the Michael addition of pyrimidine and purine nucleobases to α,β-unsaturated esters under solvent-free conditions
Authors: Zare Abdolkarim , Hasaninejad Alireza , Beyzavi Mohammad Hassan , Parhami Abolfath , Zare Ahmad Reza Moosavi , Khalafi-Nezhad Ali , Sharghi Hashem
Published: March 2008

An efficient procedure for the synthesis of carboacyclic nucleosides via microwave-assisted Michael addition of pyrimidine and purine nucleobases to α,β-unsaturated esters in the presence of catalytic amounts of zinc oxide (ZnO) and tetrabutylammonium bromide (TBAB) is described. The reactions proceed rapidly in good to excellent yields.Key words: ZnO–TBAB, green chemistry, Michael addition, nucleobase, solvent-free, microwave.
Intramolecular cyclization reaction — Palladium(0)-catalyzed cyclization is more effective than tin hydride mediated reaction
Authors: Majumdar Krishna C , Debnath Pradip , Taher Abu , Pal Amarta K
Published: March 2008

Intramolecular CH arylation of pyrone, pyridone, uracil, imidazole, and benzimidazole derivatives are carried out in the presence of a Pd catalyst to form potentially bioactive fused heterocyclic compounds, whereas the n-Bu3SnH-mediated aryl radical cyclization of the precursors 3 afforded mainly halogen-reduced uncyclized products.Key words: Pd(0) catalyst, CH arylation, intramolecular cyclization, regioselectivity.
Characterization of the structural and functional changes in the myocardium following focal ischemia-reperfusion injury
Authors: Ojha Navdeep , Roy Sashwati , Radtke Jared , Simonetti Orlando , Gnyawali Surya , Zweier Jay L. , Kuppusamy Periannan , Sen Chandan K.
Published: March 2008

High-resolution (11.7 T) cardiac magnetic resonance imaging (MRI) and histological approaches have been employed in tandem to characterize the secondary damage suffered by the murine myocardium following the initial insult caused by ischemia-reperfusion (I/R). I/R-induced changes in the myocardium were examined in five separate groups at the following time points after I/R: 1 h, day 1, day 3, day 7, and day 14. The infarct volume increased from 1 h to day 1 post-I/R. Over time, the loss of myocardial function was observed to be associated with increased infarct volume and worsened regional wall motion. In the infarct region, I/R caused a decrease in end-systolic thickness coupled with small changes in end-diastolic thickness, leading to massive wall thickening abnormalities. In addition, compromised wall thickening was also observed in left ventricular regions adjacent to the infarct region. A tight correlation ( r2 = 0.85) between measured MRI and triphenyltetrazolium chloride (TTC) infarct volumes was noted. Our observation that until day 3 post-I/R the infarct size as measured by TTC staining and MRI was much larger than that of the myocyte-silent regions in trichrome- or hematoxylin-eosin-stained sections is consistent with the literature and leads to the conclusion that at such an early phase, the infarct site contains structurally intact myocytes that are functionally compromised. Over time, such affected myocytes were noted to structurally disappear, resulting in consistent infarct sizes obtained from MRI and TTC as well as trichrome and hematoxylin-eosin analyses on day 7 following I/R. Myocardial remodeling following I/R includes secondary myocyte death followed by the loss of cardiac function over time.
Voltage-dependent K+ channels regulate the duration of reactive hyperemia in the canine coronary circulation
Authors: Dick Gregory M. , Bratz Ian N. , Borbouse Léna , Payne Gregory A. , Dincer Ü. Deniz , Knudson Jarrod D. , Rogers Paul A. , Tune Johnathan D.
Published: March 2008

We previously demonstrated a role for voltage-dependent K+ (KV) channels in coronary vasodilation elicited by myocardial metabolism and exogenous H2O2, as responses were attenuated by the KV channel blocker 4-aminopyridine (4-AP). Here we tested the hypothesis that KV channels participate in coronary reactive hyperemia and examined the role of KV channels in responses to nitric oxide (NO) and adenosine, two putative mediators. Reactive hyperemia (30-s occlusion) was measured in open-chest dogs before and during 4-AP treatment [intracoronary (ic), plasma concentration 0.3 mM]. 4-AP reduced baseline flow 34 ± 5% and inhibited hyperemic volume 32 ± 5%. Administration of 8-phenyltheophylline (8-PT; 0.3 mM ic or 5 mg/kg iv) or NG-nitro-l-arginine methyl ester (l-NAME; 1 mg/min ic) inhibited early and late portions of hyperemic flow, supporting roles for adenosine and NO. 4-AP further inhibited hyperemia in the presence of 8-PT or l-NAME. Adenosine-induced blood flow responses were attenuated by 4-AP (52 ± 6% block at 9 μg/min). Dilation of arterioles to adenosine was attenuated by 0.3 mM 4-AP and 1 μM correolide, a selective KV1 antagonist (76 ± 7% and 47 ± 2% block, respectively, at 1 μM). Dilation in response to sodium nitroprusside, an NO donor, was attenuated by 4-AP in vivo (41 ± 6% block at 10 μg/min) and by correolide in vitro (29 ± 4% block at 1 μM). KV current in smooth muscle cells was inhibited by 4-AP (IC50 1.1 ± 0.1 mM) and virtually eliminated by correolide. Expression of mRNA for KV1 family members was detected in coronary arteries. Our data indicate that KV channels play an important role in regulating resting coronary blood flow, determining duration of reactive hyperemia, and mediating adenosine- and NO-induced vasodilation.
Mechanisms and potential therapeutic targets for folic acid in cardiovascular disease
Authors: Moens An L. , Vrints Christiaan J. , Claeys Marc J. , Timmermans Jean-Pierre , Champion Hunter C. , Kass David A.
Published: March 2008

Folic acid (FA) is a member of the B-vitamin family with cardiovascular roles in homocysteine regulation and endothelial nitric oxide synthase (eNOS) activity. Its interaction with eNOS is thought to be due to the enhancement of tetrahydrobiopterin bioavailability, helping maintain eNOS in its coupled state to favor the generation of nitric oxide rather than oxygen free radicals. FA also plays a role in the prevention of several cardiac and noncardiac malformations, has potent direct antioxidant and antithrombotic effects, and can interfere with the production of the endothelial-derived hyperpolarizing factor. These multiple mechanisms of action have led to studies regarding the therapeutic potential of FA in cardiovascular disease. To date, studies have demonstrated that FA ameliorates endothelial dysfunction and nitrate tolerance and can improve pathological features of atherosclerosis. These effects appear to be homocysteine independent but rather related to their role in eNOS function. Given the growing evidence that nitric oxide synthase uncoupling plays a major role in many cardiovascular disorders, the potential of exogenous FA as an inexpensive and safe oral therapy is intriguing and is stimulating ongoing investigations.
Cellular kinetics and modeling of bronchioalveolar stem cell response during lung regeneration
Authors: Nolen-Walston R. D. , Kim C. F. , Mazan M. R. , Ingenito E. P. , Gruntman A. M. , Tsai L. , Boston R. , Woolfenden A. E. , Jacks T. , Hoffman A. M.
Published: March 2008

Organ regeneration in mammals is hypothesized to require a functional pool of stem or progenitor cells, but the role of these cells in lung regeneration is unknown. Whereas postnatal regeneration of alveolar tissue has been attributed to type II alveolar epithelial cells (AECII), we reasoned that bronchioalveolar stem cells (BASCs) have the potential to contribute substantially to this process. To test this hypothesis, unilateral pneumonectomy (PNX) was performed on adult female C57/BL6 mice to stimulate compensatory lung regrowth. The density of BASCs and AECII, and morphometric and physiological measurements, were recorded on days 1, 3, 7, 14, 28, and 45 after surgery. Vital capacity was restored by day 7 after PNX. BASC numbers increased by day 3, peaked to 220% of controls ( P < 0.05) by day 14, and then returned to baseline after active lung regrowth was complete, whereas AECII cell densities increased to 124% of baseline (N/S). Proliferation studies revealed significant BrdU uptake in BASCs and AECII within the first 7 days after PNX. Quantitative analysis using a systems biology model was used to evaluate the potential contribution of BASCs and AECII. The model demonstrated that BASC proliferation and differentiation contributes between 0 and 25% of compensatory alveolar epithelial (type I and II cell) regrowth, demonstrating that regeneration requires a substantial contribution from AECII. The observed cell kinetic profiles can be reconciled using a dual-compartment (BASC and AECII) proliferation model assuming a linear hierarchy of BASCs, AECII, and AECI cells to achieve lung regrowth.
Human type II pneumocyte chemotactic responses to CXCR3 activation are mediated by splice variant A
Authors: Ji Rong , Lee Clement M. , Gonzales Linda W. , Yang Yi , Aksoy Mark O. , Wang Ping , Brailoiu Eugen , Dun Nae , Hurford Matthew T. , Kelsen Steven G.
Published: March 2008

Chemokine receptors control several fundamental cellular processes in both hematopoietic and structural cells, including directed cell movement, i.e., chemotaxis, cell differentiation, and proliferation. We have previously demonstrated that CXCR3, the chemokine receptor expressed by Th1/Tc1 inflammatory cells present in the lung, is also expressed by human airway epithelial cells. In airway epithelial cells, activation of CXCR3 induces airway epithelial cell movement and proliferation, processes that underlie lung repair. The present study examined the expression and function of CXCR3 in human alveolar type II pneumocytes, whose destruction causes emphysema. CXCR3 was present in human fetal and adult type II pneumocytes as assessed by immunocytochemistry, immunohistochemistry, and Western blotting. CXCR3-A and -B splice variant mRNA was present constitutively in cultured type II cells, but levels of CXCR3-B greatly exceeded CXCR3-A mRNA. In cultured type II cells, I-TAC, IP-10, and Mig induced chemotaxis. Overexpression of CXCR3-A in the A549 pneumocyte cell line produced robust chemotactic responses to I-TAC and IP-10. In contrast, I-TAC did not induce chemotactic responses in CXCR3-B and mock-transfected cells. Finally, I-TAC increased cytosolic Ca2+and activated the extracellular signal-regulated kinase, p38, and phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B kinases only in CXCR3-A-transfected cells. These data indicate that the CXCR3 receptor is expressed by human type II pneumocytes, and the CXCR3-A splice variant mediates chemotactic responses possibly through Ca2+activation of both mitogen-activated protein kinase and PI 3-kinase signaling pathways. Expression of CXCR3 in alveolar epithelial cells may be important in pneumocyte repair from injury.
Cigarette smoke-mediated inflammatory and oxidative responses are strain-dependent in mice
Authors: Yao Hongwei , Edirisinghe Indika , Rajendrasozhan Saravanan , Yang Se-Ran , Caito Samuel , Adenuga David , Rahman Irfan
Published: March 2008

A variety of mouse models have been used to study the pathogenesis of pulmonary emphysema/chronic obstructive pulmonary disease. The effect of cigarette smoke (CS) is believed to be strain dependent, because certain mouse strains are more susceptible or resistant to development of emphysema. However, the molecular basis of susceptibility of mouse strains to effects of CS is not known. We investigated the effect of CS on lungs of most of the commonly used mouse strains to study the molecular mechanism of susceptibility to effects of CS. C57BL/6J, A/J, AKR/J, CD-1, and 129SvJ mice were exposed to CS for 3 consecutive days, and various parameters of inflammatory and oxidative responses were assessed in lungs of these mice. We found that the C57BL/6J strain was highly susceptible, the A/J, AKR/J, and CD-1 strains were moderately susceptible, and the 129SvJ strain was resistant to lung inflammatory and oxidant responses to CS exposure. The mouse strain that was more susceptible to effects of CS showed augmented lung inflammatory cell influx, activation of NF-κB and p38 MAPK, and increased levels of matrix metalloproteinase-9 and NF-κB-dependent proinflammatory cytokines compared with resistant mouse strains. Similarly, decreased levels of glutathione were associated with increased levels of lipid peroxidation products in susceptible mouse strains compared with resistant strains. Hence, we identified the susceptible and resistant mouse strains on the basis of the pattern of inflammatory and oxidant responses. Identification of sensitive and resistant mouse strains could be useful for studying the molecular mechanisms of effects of CS on inflammation and pharmacological interventional studies in CS-exposure mouse models.
The Signatures of Autozygosity among Patients with Colorectal Cancer
Authors: Bacolod Manny D. , Schemmann Gunter S. , Wang Shuang , Shattock Richard , Giardina Sarah F. , Zeng Zhaoshi , Shia Jinru , Stengel Robert F. , Gerry Norman , Hoh Josephine , Kirchhoff Tomas , Gold Bert , Christman Michael F. , Offit Kenneth , Gerald William L. , Notterman Daniel A. , Ott Jurg , Paty Philip B. , Barany Francis
Published: March 2008

Previous studies have shown that among populations with a high rate of consanguinity, there is a significant increase in the prevalence of cancer. Single nucleotide polymorphism (SNP) array data (Affymetrix, 50K XbaI) analysis revealed long regions of homozygosity in genomic DNAs taken from tumor and matched normal tissues of colorectal cancer (CRC) patients. The presence of these regions in the genome may indicate levels of consanguinity in the individual's family lineage. We refer to these autozygous regions as identity-by-descent (IBD) segments. In this study, we compared IBD segments in 74 mostly Caucasian CRC patients (mean age of 66 years) to two control data sets: (a) 146 Caucasian individuals (mean age of 80 years) who participated in an age-related macular degeneration (AMD) study and (b) 118 cancer-free Caucasian individuals from the Framingham Heart Study (mean age of 67 years). Our results show that the percentage of CRC patients with IBD segments (≥4 Mb length and 50 SNPs probed) in the genome is at least twice as high as the AMD or Framingham control groups. Also, the average length of these IBD regions in the CRC patients is more than twice the length of the two control data sets. Compared with control groups, IBD segments are found to be more common among individuals of Jewish background. We believe that these IBD segments within CRC patients are likely to harbor important CRC-related genes with low-penetrance SNPs and/or mutations, and, indeed, two recently identified CRC predisposition SNPs in the 8q24 region were confirmed to be homozygous in one particular patient carrying an IBD segment covering the region. [Cancer Res 2008;68(8):2610–21]
Silencing Bcl-2 in models of mantle cell lymphoma is associated with decreases in cyclin D1, nuclear factor-κB, p53, bax, and p27 levels
Authors: Tucker Catherine A. , Kapanen Anita I. , Chikh Ghania , Hoffman Brad G. , Kyle Alastair H. , Wilson Ian M. , Masin Dana , Gascoyne Randy D. , Bally Marcel , Klasa Richard J.
Published: March 2008

Molecular mechanisms responsible for lymphoma resistance to apoptosis often involve the bcl-2 pathway. In this study, we investigated the cell signaling pathways activated in bcl-2-overexpressing human mantle cell lymphoma cell lines (JVM-2 and Z-138) that have been treated with oblimersen, a molecular gene silencing strategy that effectively suppresses bcl-2 in vitro and in vivo. Z-138 cells expressed higher levels of bcl-2 and were more sensitive to the effects of bcl-2 silencing, mediated by oblimersen or bcl-2 small interfering RNA, in vitro. Tumors derived following injection of Z-138 cells were sensitive to oblimersen as judged by decreases in tumor growth rate and decreases in cell proliferation (as measured by Ki-67). Immunohistochemistry and Western blot analysis of oblimersen-treated Z-138 tumors revealed a dose-dependent decrease in bcl-2 levels and an associated increase in the proapoptotic proteins caspase-3 and caspase-9. Silencing bcl-2 in Z-138 xenografts revealed an associated dose-dependent suppression of bax, a decrease in nuclear factor-κB and phospho-nuclear factor-κB, and transient loss of p53 levels. Coimmunoprecipitation studies suggest that the latter observation is mediated by an association between bcl-2 and phospho-mdm2. Bcl-2 silencing also led to p27 down-regulation and coimmunoprecipitation studies point to a role for bcl-2 in regulation of p27 localization/degradation. Bcl-2 silencing was also correlated with loss of cyclin D1a protein levels but not cyclin D1b levels. Coimmunoprecipitation studies indicate that bcl-2 may mediate its effects on cyclin D1a via interaction with p38 mitogen-activated protein kinase as well as a previously unreported interaction between bcl-2 and cyclin D1a. [Mol Cancer Ther 2008;7(4):749–58]