bluuebunny/crossref_metadata_2025 huggingface dataset:
Cerebral protection in traumatic brain injury
Authors: Tse Yat‐Hang , Leung Gilberto Ka‐Kit , Fan Yiu‐Wah
Published: October 2005

With advances in the understanding of the pathophysiology of traumatic brain injury, many novel cerebroprotective measures have been developed. Many of them have undergone preclinical trials and have shown promising results, but the results have not translated into clinical benefits. Evidence of these cerebroprotective measures including NMDA‐receptor antagonist, steroids, free radial scavengers, nimodipine, ziconotide, bradykinin receptor antagonist and dexanabinol has been reviewed. Problems encountered in clinical studies of traumatic brain injury are mainly related to the heterogenicity of traumatic brain injury and the design of clinical studies. Given all these difficulties, clear benefit of these measures cannot be shown and an optimum treatment strategy has yet been developed.
Polymorphisms in the interleukin‐4 and IL‐4 receptor genes and multiple sclerosis: a study in Spanish‐Basque, Northern Irish and Belgian populations
Authors: Suppiah V. , Goris A. , Alloza I. , Heggarty S. , Dubois B. , Carton H. , Antigüedad A. , Mendibe M. , McDonnell G. , Droogan A. , Hawkins S. , Graham C. , Vandenbroeck K.
Published: October 2005

Cytokine gene polymorphisms are known to influence susceptibility and disease course of many autoimmune diseases. Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system white matter characterized by inflammation, demyelination and axonal damage. We analysed both the well‐known intronic variable number of tandem repeat (VNTR) and +33 C/T single‐nucleotide polymorphisms (SNP) in the IL‐4 gene, as well as the functional Q551R SNP in the IL4‐R gene in a cohort of three distinct populations comprising sporadic cases and controls from the northern Spanish Basque Country and Northern Ireland, as well as family trios from Belgium. The IL‐4 +33 TT genotype was decreased in primary progressive (PP) versus relapsing‐remitting (RR) patients in the Northern Irish population (OR = 0.14; 95% CI = 0.018–1.09). Two‐marker haplotype distribution of the VNTR and +33 C/T SNP in PP patients differed from that seen in RR patients in Northern Ireland (P = 0.03). The R allele of the Q551R SNP was significantly under‐transmitted in the Belgian trio families (P = 0.003), although this effect was not seen in the Northern Irish and Basque data sets. We did not identify IL‐4–IL4‐R gene–gene interaction in determining susceptibility or clinical parameters of MS. Disease or genetic heterogeneity or both may be responsible for the observed lack of reproduction in different populations. Our data reinforce recent findings for a role of IL4‐R in susceptibility to MS.
A MULTIPLEX POLYMERASE CHAIN REACTION ASSAY FOR RAPID DETECTION OF ESCHERICHIA COLI O157:H7, LISTERIA MONOCYTOGENES, SALMONELLATYPHIMURIUM AND CAMPYLOBACTER JEJUNI IN ARTIFICIALLY CONTAMINATED FOOD SAMPLES
Authors: WANG H. , SLAVIK M.F.
Published: October 2005

A multiplex polymerase chain reaction (PCR) assay was evaluated for simultaneously detecting four major food‐borne pathogens – Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium and Campylobacter jejuni directly from 10 different artificially contaminated food samples. All food samples, including meat (ground beef, ground turkey and chicken wash solutions), ready‐to‐eat vegetables (fresh‐cut lettuce, broccoli and mushrooms), fruits (watermelon and grapes) and dairy products (cottage cheese and yogurt), were inoculated with the four pathogens at various concentrations. The inocula were allowed to set for 1 h at room temperature and then stomached with a 0.01 M phosphate‐buffered saline (pH 7.5) solution. A 50‐mL sample of each wash solution was centrifuged and the bacterial template DNA was extracted. Primers for the four pathogens were added to a single PCR super‐mix, the DNA was amplified at selected conditions (35 cycles, 52C for annealing temperature which fit all primers) and the products were visualized by ethidium bromide staining. Results indicate that all four pathogens in food samples tested can be detected in one reaction tube in a total test time of 6 h. The sensitivity of the multiplex PCR assay in the detection of all four bacteria in food samples is 104 cfu/PCR reaction. The multiplex PCR method is rapid and cost‐effective for food‐borne pathogen detection and can detect these four target pathogens in any combination by using only one assay.
QUANTIFYING METHIONINE WITH A GREEN FLUORESCENT ESCHERICHIA COLI METHIONINE AUXOTROPH
Authors: FROELICH C.A. , ZABALA DÍAZ I.B. , CHALOVA V.I. , KIM W.‐K. , RICKE S.C.
Published: October 2005

Methionine is one of the essential and primary limiting amino acids in animal nutrition. In this study, an Escherichia coli methionine auxotroph bacterial strain that exhibited a linear growth response to methionine concentrations was transformed with a plasmid containing genes encoding for a green fluorescent protein, in order to develop a fluorescent bioassay technique for methionine quantitation. Transformants were grown on arabinose‐supplemental media and kept at 27C. The strain was also tested in supplemented media with increasing methionine concentrations, and growth response (measured as optical density [OD]) was compared with fluorescence. The transformed E. coli methionine auxotroph exhibited low OD values, but the OD and fluorescent standard curves used for estimating methionine concentrations were linear with increasing increments of methionine concentration (OD, R2 = 0.97 and 0.89; fluorescent, R2 = 0.90 and 0.89) as were the corresponding fluorescent responses (OD versus fluorescent, R2 = 0.85 and 0.91). The transformed fluorescent strain also responded to enzymatic digests of selected animal feed proteins, yielding differences in available methionine among the different feed protein sources. Using the fluorescence‐based bacterial growth assay to quantify methionine in microtiter plates has potential for assessment of methionine levels in animal feed samples.
DEVELOPMENT OF A CULTURE‐INDEPENDENT POLYMERASE CHAIN REACTION‐BASED ASSAY FOR THE DETECTION OF LACTOBACILLI IN STRETCHED CHEESE
Authors: BARUZZI FEDERICO , MATARANTE ALESSANDRA , CAPUTO LEONARDO , MOREA MARIA
Published: October 2005

The purpose of this research was to develop a culture‐independent assay based on polymerase chain reaction (PCR) for the specific detection of lactobacilli present in very low amounts in natural microflora during the processing of traditional pasta filata cheese. The utility of casein micelle solubilization with Triton X‐100 was also evaluated before proceeding to DNA extraction and PCR detection. By using primers related to cell envelope proteinase and gassericin B genes, proteolytic lactobacilli were successfully detected in samples from which they had not previously been isolated by plate counting. The sensitivity of the method is estimated to be 1000 cfu/mL. The speed of the DNA extraction protocol and the sensitivity and reliability of the PCR assay even make it feasible to detect nondominant populations of Lactobacillus species in cheese.
New insights into central cardiovascular control during exercise in humans: a central command update
Authors: Williamson J. W. , Fadel P. J. , Mitchell J. H.
Published: October 2005

The autonomic adjustments to exercise are mediated by central signals from the higher brain (central command) and by a peripheral reflex arising from working skeletal muscle (exercise pressor reflex), with further modulation provided by the arterial baroreflex. Although it is clear that central command, the exercise pressor reflex and the arterial baroreflex are all requisite for eliciting appropriate cardiovascular adjustments to exercise, this review will be limited primarily to discussion of central command. Central modulation of the cardiovascular system via descending signals from higher brain centres has been well recognized for over a century, yet the specific regions of the human brain involved in this exercise‐related response have remained speculative. Brain mapping studies during exercise as well as non‐exercise conditions have provided information towards establishing the cerebral cortical structures in the human brain specifically involved in cardiovascular control. The purpose of this review is to provide an update of current concepts on central command in humans, with a particular emphasis on the regions of the brain identified to alter autonomic outflow and result in cardiovascular adjustments.
Heterogeneous chloride homeostasis and GABA responses in the median preoptic nucleus of the rat
Authors: Grob Magali , Mouginot Didier
Published: October 2005

The median preoptic nucleus (MnPO) is an integrative structure of the hypothalamus receiving periphery‐derived information pertinent to hydromineral and cardiovascular homeostasis. In this context, excitability of MnPO neurones is controlled by fast GABAergic, glutamatergic and angiotensinergic projection from the subfornical organ (SFO). Taking advantage of a brain slice preparation preserving synaptic connection between the SFO and the MnPO, and appropriate bicarbonate‐free artificial cerebrospinal fluid (CSF), we investigated a possible implication of an active outward Cl− transport in regulating efficacy of the GABAA receptor‐mediated inhibitory response at the SFO–MnPO synapse. When somata of the MnPO neurones was loaded with 18 mm chloride, stimulation of the SFO evoked outward inhibitory postsynaptic currents (IPSCs) in 81% of the MnPO neurones held at −60 mV. Accordingly, EIPSC was found 25 mV hyperpolarized from the theoretical value calculated from the Nernst equation, indicating that IPSC polarity and amplitude were driven by an active Cl− extrusion system in these neurones. EIPSC estimated with gramicidin‐based perforated‐patch recordings amounted −89.2 ± 4.3 mV. Furosemide (100 μm), a pharmacological compound known to block the activity of the neurone‐specific K+–Cl− cotransporter, KCC2, reversed IPSC polarity and shifted EIPSC towards its theoretical value. Presence of the KCC2 protein in the MnPO was further detected with immunohistochemistry, revealing a dense network of KCC2‐positive intermingled fibres. In the presence of a GABAB receptor antagonist, high‐frequency stimulation (5 Hz) of the SFO evoked a train of IPSCs or inhibitory postsynaptic potentials (IPSPs), whose amplitude was maintained throughout the sustained stimulation. Contrastingly, similar 5 Hz stimulation carried out in the presence of furosemide (50 μm) evoked IPSCs/IPSPs, whose amplitude collapsed during the high‐frequency stimulation. Similar reduction in inhibitory neurotransmission was also observed in MnPO neurones lacking the functional Cl− extrusion mechanism. We conclude that a majority of MnPO neurones were characterized by a functional Cl− transporter that ensured an efficient activity‐dependent Cl− transport rate, allowing sustained synaptic inhibition of these neurones. Pharmacological and anatomical data strongly suggested the involvement of KCC2, as an essential postsynaptic determinant of the inhibitory neurotransmission afferent to the MnPO, a key‐structure in the physiology of the hydromineral and cardiovascular homeostasis.
Apolipoprotein A‐IV is involved in detection of lipid in the rat intestine
Authors: Whited K. L. , Lu D. , Tso P. , Kent Lloyd K. C. , Raybould H. E.
Published: October 2005

Long chain triglyceride (>C12) in the intestinal lumen potently inhibits gastric emptying and acid secretion via the vagal afferent pathway. While the mechanism of inhibition involves the formation of chylomicrons, the essential role of the apolipoprotein apo A‐IV is unclear. Using apo A‐IV−/− mice, we tested the hypothesis that inhibition of gastric emptying and gastric acid secretion in response to dietary lipid is dependent upon apo A‐IV. As measured by nuclear scintigraphy in awake mice, gastric emptying of an ingested whole‐egg meal was significantly faster in apo A‐IV−/− knockout versus A‐IV+/+ controls (34 ± 1 versus 54 ± 3 min, P < 0.0001). In anaesthetized A‐IV+/+ mice, meal‐stimulated gastric acid secretion was 59% inhibited by intestinal lipid infusion; this was abolished in apo A‐IV−/− mice. Oral gavage of lipid in awake mice activated neurones throughout the nucleus of the solitary tract (NTS) in A‐IV+/+ mice, measured by immunohistochemical localization of Fos protein expression. However, in the mid region of the NTS (bregma −7.32 to −7.76 mm), Fos expression in response to intestinal lipid was significantly decreased by 50% in apo A‐IV−/− mice compared to A‐IV+/+ controls. We conclude that activation of the vagal afferent pathway and inhibition of gastric function in response to dietary lipid is partly dependent upon apo A‐IV.
Vascular endothelial growth factor increases the ultrafiltration coefficient in isolated intact Wistar rat glomeruli
Authors: Salmon Andrew H. J. , Neal Christopher R. , Bates David O. , Harper Steven J.
Published: October 2005

Vascular endothelial growth factor (VEGF) is expressed by the podocytes of renal glomeruli, and has profound influences on systemic microvascular permeability and haemodynamics. We describe an extensive refinement of a model that permits evaluation of the ultrafiltration coefficient (LpA) of isolated mammalian glomeruli, in the absence of circulating and haemodynamic influences, and tested the hypothesis that VEGF influences glomerular LpA via an effect on endothelial cells. Glomeruli were isolated by sieving Wistar rat renal cortical tissue, and individually loaded onto a suction micropipette. Flowing perifusate containing 1% bovine serum albumin (BSA) was rapidly switched to an oncopressive perifusate containing 8% BSA, eliciting transglomerular fluid efflux. The rate of the resultant reduction in glomerular volume was used to calculate glomerular LpA (1.07 ± 0.53 nl min−1 mmHg−1 (mean ±s.d.), n= 51), which compares favourably with those reported in the same rat strain using different techniques. A significant relationship between LpA and initial glomerular volume (Vi) (r= 0.72, n= 41, P < 0.0001) necessitated correction of LpA for Vi. The initial rate of change of glomerular volume, normalized for Vi, showed a strong positive correlation with applied oncotic gradient (Pearson r= 0.59, n= 28, P < 0.001), as predicted by Starling's law of filtration. A 60 min exposure of glomeruli to 1 nm VEGF increased glomerular LpA/Vi (1.19 ± 0.19 (n= 10) to 2.23 ± 0.33 (n= 9) min−1 mmHg−1 (mean ±s.e.m.); P < 0.02). Time‐ and concentration‐dependent relations between VEGF and LpA/Vi were observed. The VEGF‐induced elevation of LpA/Vi was blocked by the selective VEGF‐R2 inhibitor ZM323881. We suggest that glomerular VEGF contributes to the high physiological permeability of mammalian glomeruli to water through an action on endothelial cells.
Measuring Pump Efficiency: Uncertainty Considerations
Authors: Manring Noah D.
Published: October 2005

The objective of this paper is to analyze the uncertainty associated with pump efficiency measurements and to determine reasonable confidence intervals for these data. In the past, many industrial sales and some pieces of academic research have been based upon the experimental data of pump efficiencies; yet few have questioned the accuracy of the experimental data and no one has provided a confidence interval which reflects the range of uncertainty in the measurement. In this paper, a method for calculating this confidence interval is presented and it is shown that substantially large confidence intervals exist within the testing results of a pump. Furthermore, it is recommended that these confidence intervals be included with the efficiency data whenever it is reported.
An Order-of-Magnitude Estimate of Ocean Thermal Energy Conversion Resources
Authors: Nihous Gérard C.
Published: October 2005

Worldwide power resources that could be extracted from the steady-state operation of ocean thermal energy conversion (OTEC) plants are estimated using a simple model. This order-of-magnitude analysis indicates that about 3×109kW (3 TW) may be available, at most. This value is much smaller than estimates currently suggested in the technical literature. It reflects the scale of the perturbation caused by massive OTEC seawater flow rates on the thermal structure of the ocean. Not surprisingly, maximum OTEC power nearly corresponds to deep cold seawater flow rates of the order of the average abyssal upwelling representative of the global thermohaline circulation.
Optimum Design Parameters for Reciprocating Pumps Used in Natural Gas Wells
Authors: Rudolf Jeffrey J. , Heidrick Ted R. , Fleck Brian A. , Rajan V. S. V.
Published: October 2005

Experimental and theoretical investigation of a recently patented down-hole direct-acting reciprocating pump system is presented. The technology, (US Patent No. 5,860,795) consists of operating a gas well with gas and liquid phases being produced separately by using the gas phase to power a pump to bring the liquid phase to the surface. This would increase the duration of profitability of many gas wells in North America. Experiments and modeling were used to determine optimum design parameters to maintain flow at a minimum reservoir pressure; an optimum area ratio for the gas/liquid pistons is approximately 40. The effect of friction in the pumping system was predicted to have a small effect on this optimum design. The results of this investigation will now be used to design and construct a prototype for field testing.
Glycine-Rich Antifreeze Proteins from Snow Fleas
Authors: Graham Laurie A. , Davies Peter L.
Published: October 2005

We purified antifreeze proteins from winter-active snow fleas,
Hypogastrura harveyi
. These 6.5- and 15.7-kilodalton thermolabile proteins are glycine-rich (45% of the residues), and the short isoform is composed of the tripeptide repeat Gly-X-X. This makes them very different from other antifreeze proteins, including two from insects, suggesting independent adaptation to freezing environments.
Neurogenesis in the Hypothalamus of Adult Mice: Potential Role in Energy Balance
Authors: Kokoeva Maia V. , Yin Huali , Flier Jeffrey S.
Published: October 2005

Ciliary neurotrophic factor (CNTF) induces weight loss in obese rodents and humans, and for reasons that are not understood, its effects persist after the cessation of treatment. Here we demonstrate that centrally administered CNTF induces cell proliferation in feeding centers of the murine hypothalamus. Many of the newborn cells express neuronal markers and show functional phenotypes relevant for energy-balance control, including a capacity for leptin-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3). Coadministration of the mitotic blocker cytosine-β-
d
-arabinofuranoside (Ara-C) eliminates the proliferation of neural cells and abrogates the long-term, but not the short-term, effect of CNTF on body weight. These findings link the sustained effect of CNTF on energy balance to hypothalamic neurogenesis and suggest that regulated hypothalamic neurogenesis in adult mice may play a previously unappreciated role in physiology and disease.
Recurrent Fusion of
TMPRSS2
and ETS Transcription Factor Genes in Prostate Cancer
Authors: Tomlins Scott A. , Rhodes Daniel R. , Perner Sven , Dhanasekaran Saravana M. , Mehra Rohit , Sun Xiao-Wei , Varambally Sooryanarayana , Cao Xuhong , Tchinda Joelle , Kuefer Rainer , Lee Charles , Montie James E. , Shah Rajal B. , Pienta Kenneth J. , Rubin Mark A. , Chinnaiyan Arul M.
Published: October 2005

Recurrent chromosomal rearrangements have not been well characterized in common carcinomas. We used a bioinformatics approach to discover candidate oncogenic chromosomal aberrations on the basis of outlier gene expression. Two ETS transcription factors,
ERG
and
ETV1
, were identified as outliers in prostate cancer. We identified recurrent gene fusions of the 5′ untranslated region of
TMPRSS2
to
ERG
or
ETV1
in prostate cancer tissues with outlier expression. By using fluorescence in situ hybridization, we demonstrated that 23 of 29 prostate cancer samples harbor rearrangements in
ERG
or
ETV1
. Cell line experiments suggest that the androgen-responsive promoter elements of
TMPRSS2
mediate the overexpression of ETS family members in prostate cancer. These results have implications in the development of carcinomas and the molecular diagnosis and treatment of prostate cancer.
Learning from the Aliens
Authors: Crawley Michael J.
Published: October 2005

Species Invasions
. Insights into Ecology, Evolution, and Biogeography.
Dov F. Sax, John J. Stachowicz, and Steven D. Gaines, Eds.
. Sinauer, Sunderland, MA, 2005. 509 pp. $74.95, £48.99. ISBN 0-87893-821-4. Paper, $49.95, £31.99. ISBN 0-87893-811-7.

The contributors use studies of invasive species to address basic research questions in the life sciences, with the hope that the resulting insights will also aid efforts to solve applied problems posed by non-native biota.
The Asian Tsunami: A Protective Role for Coastal Vegetation
Authors: Danielsen Finn , Sørensen Mikael K. , Olwig Mette F. , Selvam Vaithilingam , Parish Faizal , Burgess Neil D. , Hiraishi Tetsuya , Karunagaran Vagarappa M. , Rasmussen Michael S. , Hansen Lars B. , Quarto Alfredo , Suryadiputra Nyoman
Published: October 2005

The 26 December 2004 Indian Ocean tsunami had major effects on coastal communities and ecosystems. An assessment of coastlines after the tsunami indicates that coastal vegetation such as mangroves and beach forests helped to provide protection and reduce effects on adjacent communities. In recent years, mangroves and other coastal vegetation have been cleared or degraded along many coastlines, increasing their vulnerability to storm and tsunami damage. Establishing or strengthening greenbelts of mangroves and other coastal forests may play a key role in reducing the effect of future extreme events.
Relationship of Pleconaril Susceptibility and Clinical Outcomes in Treatment of Common Colds Caused by Rhinoviruses
Authors: Pevear Daniel C. , Hayden Frederick G. , Demenczuk Tina M. , Barone Linda R. , McKinlay Mark A. , Collett Marc S.
Published: October 2005

Pleconaril, a specific inhibitor of human picornaviruses, showed therapeutic efficacy against community-acquired colds caused by rhinoviruses in two placebo-controlled trials. Virological assessments were conducted during these trails, including virus culture and drug susceptibility testing. Nasal mucus samples collected from the enrolled patients were tested for the presence of picornavirus by reverse transcriptase PCR and culture. In total, 827 baseline nasal mucus samples were positive by virus culture (420 in the placebo group and 407 in the pleconaril group). Pleconaril treatment was associated with a more rapid loss of culturable virus. By study day 3, the number of samples positive by culture fell to 282 for the placebo-treated subjects and 202 for the pleconaril-treated subjects (P< 0.0001); and by day 6, the number of samples in the two groups positive by culture fell to 196 and 165, respectively (P= 0.07). The clinical benefit correlated strongly with the pleconaril susceptibility of the baseline virus isolate. Pleconaril-treated subjects infected with the more highly susceptible viruses (50% effective concentration ≤ 0.38 μg/ml) experienced a median 1.9- to 3.9-day reduction in symptom duration compared with that for the placebo-treated subjects. By contrast, subjects whose baseline virus isolate susceptibility was >0.38 μg/ml did not benefit from pleconaril treatment. These results indicate that the magnitude of symptomatic improvement in pleconaril-treated subjects with community-acquired colds is related to the drug susceptibility of the infecting virus, clearly linking the antiviral effects of the drug to clinical efficacy. Postbaseline virus isolates with reduced susceptibility or full resistance to pleconaril were recovered from 10.7% and 2.7% of drug-treated subjects, respectively. These patients shed low levels of virus and had no unusual clinical outcomes. Nevertheless, studies on the biologic properties and transmissibility of these variant viruses are warranted.
TAK-652 Inhibits CCR5-Mediated Human Immunodeficiency Virus Type 1 Infection In Vitro and Has Favorable Pharmacokinetics in Humans
Authors: Baba Masanori , Takashima Katsunori , Miyake Hiroshi , Kanzaki Naoyuki , Teshima Koichiro , Wang Xin , Shiraishi Mitsuru , Iizawa Yuji
Published: October 2005

The first small-molecule CCR5 antagonist, TAK-779, could not be developed as an anti-human immunodeficiency virus type (anti-HIV-1) agent because of its poor oral bioavailability. TAK-652 is an orally bioavailable TAK-779 derivative with potent anti-HIV-1 activity. TAK-652 inhibited the binding of RANTES (regulated on activation, normal T-cell expressed and secreted), macrophage inflammatory protein 1α (MIP-1α), and MIP-1β to CCR5-expressing cells at nanomolar concentrations. TAK-652 could also suppress the binding of monocyte chemotactic protein 1 (MCP-1) to CCR2b-expressing cells. However, its inhibitory effect on ligand binding to other chemokine receptors was limited. TAK-652 was active against CCR5-using (R5) HIV-1 but totally inactive against CXCR4-using (X4) HIV-1. The compound was active against R5 HIV-1 clinical isolates containing reverse transcriptase and protease inhibitor-resistant mutations, with a mean 50% effective concentration (EC
50
) and EC
90
of 0.061 and 0.25 nM, respectively. In addition, recombinant R5 viruses carrying different subtype (A to G) envelope proteins were equally susceptible to TAK-652. A single oral administration of TAK-652 up to 100 mg was safe and well tolerated in humans. The compound displayed favorable pharmacokinetics, and its plasma concentration was 7.2 ng/ml (9.1 nM) even 24 h after the administration of 25 mg. Thus, TAK-652 is a promising candidate as a novel entry inhibitor of HIV-1.
Expression of the
mef
(E) Gene Encoding the Macrolide Efflux Pump Protein Increases in
Streptococcus pneumoniae
with Increasing Resistance to Macrolides
Authors: Wierzbowski Aleksandra K. , Boyd Dave , Mulvey Michael , Hoban Daryl J. , Zhanel George G.
Published: October 2005

Active macrolide efflux is a major mechanism of macrolide resistance in
Streptococcus pneumoniae
in many parts of the world, especially North America. In Canada, this active macrolide efflux in
S. pneumoniae
is predominantly due to acquisition of the
mef
(E) gene. In the present study, we assessed the
mef
(E) gene sequence as well as
mef
(E) expression in variety of low- and high-level macrolide-resistant, clindamycin-susceptible (M-phenotype)
S. pneumoniae
isolates (erythromycin MICs, 1 to 32 μg/ml; clindamycin MICs, ≤0.25 μg/ml). Southern blot hybridization with
mef
(E) probe and EcoRI digestion and relative real-time reverse transcription-PCR were performed to study the
mef
(E) gene copy number and expression. Induction of
mef
(E) expression was analyzed by Etest susceptibility testing pre- and postincubation with subinhibitory concentrations of erythromycin, clarithromycin, azithromycin, telithromycin, and clindamycin. The macrolide efflux gene,
mef
(E), was shown to be a single-copy gene in all 23 clinical
S. pneumoniae
isolates tested, and expression post-macrolide induction increased 4-, 6-, 20-, and 200-fold in isolates with increasing macrolide resistance (erythromycin MICs 2, 4, 8, and 32 μg/ml, respectively). Sequencing analysis of the macrolide efflux genetic assembly (mega) revealed that
mef
(E) had a 16-bp deletion 153 bp upstream of the putative start codon in all 23 isolates. A 119-bp intergenic region between
mef
(E) and
mel
was sequenced, and a 99-bp deletion was found in 11 of the 23 M-phenotype
S. pneumoniae
isolates compared to the published mega sequence. However, the
mef
(E) gene was fully conserved among both high- and low-level macrolide-resistant isolates. In conclusion, increased expression of
mef
(E) is associated with higher levels of macrolide resistance in macrolide-resistant
S. pneumoniae.
A Class of Pantothenic Acid Analogs Inhibits
Plasmodium falciparum
Pantothenate Kinase and Represses the Proliferation of Malaria Parasites
Authors: Spry Christina , Chai Christina L. L. , Kirk Kiaran , Saliba Kevin J.
Published: October 2005

The growth and proliferation of the human malaria parasite
Plasmodium falciparum
are dependent on the parasite's ability to obtain essential nutrients. One nutrient for which the parasite has an absolute requirement is the water-soluble vitamin pantothenic acid (vitamin B
5
). In this study, a series of pantothenic acid analogs which retain the 2,4-dihydroxy-3,3-dimethylbutyramide core of pantothenic acid but deviate in structure from one another and from pantothenic acid in the nature of the substituent attached to the amide nitrogen were synthesized using an efficient single-step synthetic route. Eight of 10 analogs tested inhibited the proliferation of intraerythrocytic
P. falciparum
parasites in vitro, doing so with 50% inhibitory concentrations between 15 and 200 μM. The compounds were generally selective, inhibiting the proliferation of a human cell line (the Jurkat cell line) only at concentrations severalfold higher than those required for inhibition of parasite growth. It was demonstrated that compounds in this series inhibited the phosphorylation of pantothenic acid by pantothenate kinase, the first step in the parasite's biosynthesis of the essential enzyme cofactor coenzyme A, doing so competitively, with

K
i

values in the nanomolar range.
Analysis of Binding Sites for the New Small-Molecule CCR5 Antagonist TAK-220 on Human CCR5
Authors: Nishikawa Masao , Takashima Katsunori , Nishi Toshiya , Furuta Rika A. , Kanzaki Naoyuki , Yamamoto Yoshio , Fujisawa Jun-ichi
Published: October 2005

G protein-coupled receptor CCR5 is the main coreceptor for macrophage-tropic human immunodeficiency virus type 1 (HIV-1), and various small-molecule CCR5 antagonists are being developed to treat HIV-1 infection. It has been reported that such CCR5 antagonists, including TAK-779, bind to a putative binding pocket formed by transmembrane domains (TMs) 1, 2, 3 and 7 of CCR5, indicating the importance of the conformational changes of the TMs during virus entry. In this report, using a single-round infection assay with human CCR5 and its substitution mutants, we demonstrated that a new CCR5 antagonist, TAK-220, shares the putative interacting amino acid residues Asn252 and Leu255 in TM6 with TAK-779 but also requires the distinct residues Gly163 and Ile198 in TMs 4 and 5, respectively, for its inhibitory effect. We suggested that, together with molecular models of the interactions between the drugs and CCR5, the inhibitory activity of TAK-220 could involve direct interactions with amino acid residues in TMs 4, 5, and 6 in addition to those in the previously postulated binding pocket. The possible interaction of drugs with additional regions of the CCR5 molecule would help to develop a new small-molecule CCR5 antagonist.
Activities of Flucytosine, Fluconazole, Amphotericin B, and Micafungin in a Murine Model of Disseminated Infection by
Candida glabrata
Authors: Mariné Marçal , Serena Carolina , Fernández-Torres Belkys , Pastor F. Javier , Guarro Josep
Published: October 2005

We compared the efficacies of amphotericin B, fluconazole, flucytosine, and micafungin in a systemic murine infection by three isolates of
Candida glabrata.
Amphotericin B showed the best results, although none of the drugs dramatically reduced mortality or tissue burden in liver or spleen.
AmpC and OprD Are Not Involved in the Mechanism of Imipenem Hypersusceptibility among
Pseudomonas aeruginosa
Isolates Overexpressing the
mexCD
-
oprJ
Efflux Pump
Authors: Wolter Daniel J. , Hanson Nancy D. , Lister Philip D.
Published: October 2005

Pseudomonas aeruginosa
strains that overexpress
mexCD
-
oprJ
become hypersusceptible to imipenem. Disruption of AmpC induction has been suggested to cause this phenotype. However, data from this study demonstrate that hypersusceptibility to imipenem can develop without changes in
ampC
expression or AmpC activity. Furthermore, hypersusceptibility is not caused by changes in expression of the outer membrane porin, OprD.
Differential Antibiotic Susceptibilities of Starved
Mycobacterium tuberculosis
Isolates
Authors: Xie Zhifang , Siddiqi Noman , Rubin Eric J.
Published: October 2005

We compared the efficacies of 17 agents against rapidly growing and starved
Mycobacterium tuberculosis
H37Rv. Few compounds have significant activity at attainable concentrations. However, two phenothiazine compounds at similar concentrations were bactericidal for starved and growing cells. These drugs appear to target a process important in both replicating and nonreplicating bacteria.
Impact of Mass Distribution of Azithromycin on the Antibiotic Susceptibilities of Ocular
Chlamydia trachomatis
Authors: Solomon Anthony W. , Mohammed Zeena , Massae Patrick A. , Shao John F. , Foster Allen , Mabey David C. W. , Peeling Rosanna W.
Published: October 2005

In a community of Tanzania where trachoma is endemic, we cultured conjunctival swabs from all residents who had active trachoma and were PCR positive for ocular
Chlamydia trachomatis
, both before (43 isolates) and 2 months after (9 isolates) mass antibiotic treatment. No clinically or programmatically significant increase in azithromycin or tetracycline resistance was observed.
Gene Silencing and Overexpression of Porcine Peptidoglycan Recognition Protein Long Isoforms: Involvement in β-Defensin-1 Expression
Authors: Sang Yongming , Ramanathan Balaji , Ross Christopher R. , Blecha Frank
Published: October 2005

Peptidoglycan recognition proteins (PGRPs) are a group of newly identified proteins with emerging functions in mammalian innate immunity. Here we report the identification and characterization of two long isoforms of porcine PGRP. Their complete cDNA sequences encode predicted peptides of 252 and 598 residues and are named pPGRP-L1 and pPGRP-L2, respectively. These porcine isoforms share identical PGRP domains at their C terminus, which are highly conserved with human and mouse orthologs. pPGRP-L1 is expressed constitutively in several tissues, including bone marrow, intestine, liver, spleen, kidney, and skin. pPGRP-L2 is highly expressed in the duodenum and liver, and expression in intestinal tissues is increased bySalmonellainfection. In intestinal cells, expression of both pPGRP-L1 and pPGRP-L2 is increased by bacterial infection. Recombinant pPGRP-L1 and pPGRP-L2 haveN-acetylmuramoyl-L-alanine amidase activity. Loss-of-function and gain-of-function experiments indicate that these two pPGRPs are involved in expression of the antimicrobial peptide β-defensin-1. Silencing of pPGRP-L2 in intestinal cells challenged withListeria monocytogenesresults in downregulation of β-defensin-1. Conversely, overexpression of pPGRP-L1 or pPGRP-L2 dramatically upregulates expression of β-defensin-1. Collectively, these findings suggest that porcine PGRPs are involved in antimicrobial peptide expression.
Identification of
gmhA
, a
Yersinia pestis
Gene Required for Flea Blockage, by Using a
Caenorhabditis elegans
Biofilm System
Authors: Darby Creg , Ananth Sandya L. , Tan Li , Hinnebusch B. Joseph
Published: October 2005

Yersinia pestis
, the cause of bubonic plague, blocks feeding by its vector, the flea. Recent evidence indicates that blockage is mediated by an in vivo biofilm.
Y. pestis
and the closely related
Yersinia pseudotuberculosis
also make biofilms on the cuticle of the nematode
Caenorhabditis elegans
, which block this laboratory animal's feeding. Random screening of
Y. pseudotuberculosis
transposon insertion mutants with a
C. elegans
biofilm assay identified
gmhA
as a gene required for normal biofilms.
gmhA
encodes phosphoheptose isomerase, an enzyme required for synthesis of heptose, a conserved component of lipopolysaccharide and lipooligosaccharide. A
Y. pestis gmhA
mutant was constructed and was severely defective for
C. elegans
biofilm formation and for flea blockage but only moderately defective in an in vitro biofilm assay. These results validate use of the
C. elegans
biofilm system to identify genes and pathways involved in
Y. pestis
flea blockage.
Studies of Fibronectin-Binding Proteins of
Streptococcus equi
Authors: Lannergård Jonas , Flock Margareta , Johansson Staffan , Flock Jan-Ingmar , Guss Bengt
Published: October 2005

Streptococcus equi
subsp.
equi
is the causative agent of strangles, a disease of the upper respiratory tract in horses. The initiation of
S. equi
subsp.
equi
infection is likely to involve cell surface-anchored molecules mediating bacterial adhesion to the epithelium of the host. The present study describes the cloning and characterization of FNEB, a fibronectin-binding protein with cell wall-anchoring motifs. FNEB can thus be predicted as cell surface located, contrary to the two previously characterized fibronectin-binding proteins in
S. equi
subsp.
equi
, FNE and SFS. Assays of antibody titers in horses and in experimentally infected mice indicate that the protein is immunogenic and expressed in vivo during
S. equi
subsp.
equi
infection. Using Western ligand blotting, it was shown that FNEB binds to the N-terminal 29-kDa fragment of fibronectin, while SFS and FNE both bind to the adjacent 40-kDa fragment.
S. equi
subsp.
equi
is known to bind fibronectin to a much lower degree than the closely related
S. equi
subsp.
zooepidemicus
, but the binding is primarily directed to the 29-kDa fragment. Inhibition studies using
S. equi
subsp.
equi
cells indicate that FNEB mediates cellular binding to fibronectin in this species.
LruA and LruB, Novel Lipoproteins of PathogenicLeptospira interrogansAssociated with Equine Recurrent Uveitis
Authors: Verma Ashutosh , Artiushin Sergey , Matsunaga James , Haake David A. , Timoney John F.
Published: October 2005

Recurrent uveitis as a sequela toLeptospirainfection is the most common infectious cause of blindness and impaired vision of horses worldwide. Leptospiral proteins expressed during prolonged survival in the eyes of horses with lesions of chronic uveitis were identified by screening a phage library ofLeptospira interrogansDNA fragments with eye fluids from uveitic horses. Inserts of reactive phages encoded several known leptospiral proteins and two novel putative lipoproteins, LruA and LruB. LruA was intrinsically labeled during incubation ofL. interrogansin medium containing [14C]palmitic acid, confirming that it is a lipoprotein.lruAandlruBwere detected by Southern blotting in infectiousLeptospira interrogansbut not in nonpathogenicLeptospira biflexa. Fractionation data from culturedLeptospiraindicate that LruA and LruB are localized in the inner membrane. Uveitic eye fluids contained significantly higher levels of immunoglobulin A (IgA) and IgG specific for each protein than did companion sera, indicating strong local antibody responses. Moreover, LruA- and LruB-specific antisera reacted with equine ocular components, suggesting an immunopathogenic role in leptospiral uveitis.
Selection of Protective Epitopes for
Brucella melitensis
by DNA Vaccination
Authors: Yang Xinghong , Hudson Mary , Walters Nancy , Bargatze Robert F. , Pascual David W.
Published: October 2005

The
Brucella melitensis
16M genome was examined for proteins in excess of 100 amino acids and for immunogenicity-associated genes. One subset of 32 annotated genes or open reading frames was identified, and each of these were cloned into the eukaryotic vector pcDNA3.1. Purified recombinant plasmids were used to intramuscularly (i.m.) immunize BALB/c mice. After challenge with
B. melitensis
16M strain, two protective antigens were found: the periplasmic protein, bp26, and the chaperone protein, trigger factor (TF). Protective efficacy was confirmed with DNA vaccines for these two
B. melitensis
proteins and, when combined, protection against wild-type challenge was significantly enhanced. Both proteins were found to be immunogenic since elevated serum immunoglobulin G (IgG) antibodies without a specific IgG subclass bias were induced subsequent to i.m. DNA immunization. Antigen-restimulation assays revealed that bp26 and TF stimulated gamma interferon and only bp26 induced interleukin-4 (IL-4), IL-5, and IL-6 cytokines as measured by cytokine enzyme-linked immunospot assay. These collective results suggest that both bp26 and TF are excellent candidates for use in future vaccination studies against brucellosis.
Endotoxin Priming Improves Clearance ofPseudomonas aeruginosain Wild-Type and Interleukin-10 Knockout Mice
Authors: Varma Tushar K. , Durham Megan , Murphey Erle D. , Cui Weihua , Huang Zhiyu , Lin Cheng Y. , Toliver-Kinsky Tracy , Sherwood Edward R.
Published: October 2005

Endotoxin (lipopolysaccharide [LPS]) tolerance is an altered state of immunity caused by prior exposure to LPS, in which production of many cytokines, including gamma interferon (IFN-γ) and interleukin-12 (IL-12), are reduced but secretion of the anti-inflammatory cytokine IL-10 is increased in response to a subsequent LPS challenge. This pattern of cytokine production is also characteristic of postinflammatory immunosuppression. Therefore, we hypothesized that LPS-primed mice would exhibit an impaired ability to respond to systemic infection with the opportunistic pathogenPseudomonas aeruginosa. We further hypothesized that depletion of IL-10 would reverse the endotoxin-tolerant state. To test this hypothesis, systemic clearance ofPseudomonas aeruginosawas measured for LPS-primed wild-type and IL-10-deficient mice. LPS-primed wild-type mice exhibited significant suppression of LPS-induced IFN-γ and IL-12 but increased IL-10 production in blood and spleen compared to levels exhibited by saline-primed wild-type mice. The suppressed production of IFN-γ and IL-12 caused by LPS priming was ablated in the spleens, but not blood, of IL-10 knockout mice. LPS-primed wild-type mice clearedPseudomonas aeruginosafrom lungs and blood more effectively than saline-primed mice. LPS-primed IL-10-deficient mice were particularly efficient in clearingPseudomonas aeruginosaafter systemic challenge. These studies show that induction of LPS tolerance enhanced systemic clearance ofPseudomonas aeruginosaand that this effect was augmented by neutralization of IL-10.
Borrelia burgdorferiRegulates Expression of Complement Regulator-Acquiring Surface Protein 1 during the Mammal-Tick Infection Cycle
Authors: von Lackum Kate , Miller Jennifer C. , Bykowski Tomasz , Riley Sean P. , Woodman Michael E. , Brade Volker , Kraiczy Peter , Stevenson Brian , Wallich Reinhard
Published: October 2005

During the natural mammal-tick infection cycle, the Lyme disease spirocheteBorrelia burgdorfericomes into contact with components of the alternative complement pathway.B. burgdorferi, like many other human pathogens, has evolved the immune evasion strategy of binding two host-derived fluid-phase regulators of complement, factor H and factor H-like protein 1 (FHL-1). The borrelial complement regulator-acquiring surface protein 1 (CRASP-1) is a surface-exposed lipoprotein that binds both factor H and FHL-1. Analysis of CRASP-1 expression during the mammal-tick infectious cycle indicated thatB. burgdorferiexpresses this protein during mammalian infection, supporting the hypothesized role for CRASP-1 in immune evasion. However, CRASP-1 synthesis was repressed in bacteria during colonization of vector ticks. Analysis of cultured bacteria indicated that CRASP-1 is differentially expressed in response to changes in pH. Comparisons of CRASP-1 expression patterns with those of other infection-associatedB. burgdorferiproteins, including the OspC, OspA, and Erp proteins, indicated that each protein is regulated through a unique mechanism.
Murine Macrophages Kill the Vegetative Form of
Bacillus anthracis
Authors: Kang Tae Jin , Fenton Matthew J. , Weiner Matthew A. , Hibbs Stephen , Basu Subhendu , Baillie Les , Cross Alan S.
Published: October 2005

Anti-protective antigen antibody was reported to enhance macrophage killing of ingested
Bacillus anthracis
spores, but it was unclear whether the antibody-mediated macrophage killing mechanism was directed against the spore itself or the vegetative form emerging from the ingested and germinating spore. To address this question, we compared the killing of germination-proficient (gp) and germination-deficient (Δ
gerH
) Sterne 34F2 strain spores by murine peritoneal macrophages. While macrophages similarly ingested both spores, only gp Sterne was killed at 5 h (0.37 log kill). Pretreatment of macrophages with gamma interferon (IFN-γ) or opsonization with immunoglobulin G (IgG) isolated from a subject immunized with an anthrax vaccine enhanced the killing of Sterne to 0.49 and 0.73 log, respectively, but the combination of IFN-γ and IgG was no better than either treatment alone. Under no condition was there killing of Δ
gerH
spores. To examine the ability of the exosporium to protect spores from macrophages, we compared the macrophage-mediated killing of nonsonicated (exosporium
+
) and sonicated (exosporium
−
) Sterne 34F2 spores. More sonicated spores than nonsonicated spores were killed at 5 h (0.98 versus 0.37 log kill, respectively). Pretreatment with IFN-γ increased the sonicated spore killing to 1.39 log. However, the opsonization with IgG was no better than no treatment or pretreatment with IFN-γ. We conclude that macrophages appear unable to kill the spore form of
B. anthracis
and that the exosporium may play a role in the protection of spores from macrophages.
A Strong Antigen-Specific T-Cell Response Is Associated with Age and Genetically Dependent Resistance to Avian Enteric Salmonellosis
Authors: Beal Richard K. , Powers Claire , Wigley Paul , Barrow Paul A. , Kaiser Pete , Smith Adrian L.
Published: October 2005

Chicken genetics and age affect resistance to enteric infection withSalmonella entericaserovar Typhimurium and were used to identify the immune responses that may contribute to rapid clearance. When birds were infected at 40 days of age, line 61chickens cleared the infection more effectively than line N chickens, whereas when birds were infected at 10 days of age, both chicken lines were highly susceptible to infection. Antibody levels, T-cell responsiveness, and cytokine mRNA levels were all elevated during infection. A negative correlation between resistance and antigen-specific antibody production was observed in older chickens. However, this finding was not replicated for age-related resistance; we found that older chickens exhibited a stronger and more rapid antibody response than younger chickens. The levels of interleukin-1β (IL-1β) and gamma interferon (IFN-γ) mRNA were similar in the spleens and cecal tonsils of both line 61and line N chickens, except for higher levels of IL-1β in the spleens of line 61chickens at 6 days postinfection. Differences in the levels of IFN-γ and IL-1β 1β mRNA between the lines were more apparent in younger chickens, but while the increases were greater than those observed in the older chickens, the clearance of entericS. entericaserovar Typhimurium was much slower. The level of antigen-specific proliferation of splenocytes was associated with increased resistance in both experimental systems, and the strongest responses were observed in older and genetically resistant chickens. The data presented here implicate T-cell responses in the clearance ofS. entericaserovar Typhimurium from the intestine of infected chickens.