bluuebunny/crossref_metadata_2025 huggingface dataset:
Sub-cellular localization and post-translational modifications of the Plasmodium yoelii enolase suggest moonlighting functions
Authors: Pal-Bhowmick Ipsita , Vora Hardeep K , Jarori Gotam K
Published: May 2007

Background

Enolase (2-Phospho-D-glycerate hydrolase; EC 4.2.1.11) is one of the glycolytic enzymes, whose levels are highly elevated in malaria parasite infected red blood cells. In several organisms, enolases have been shown to have diverse non glycolytic (moonlighting) biological functions. As functional diversity of a protein would require diverse sub-cellular localization, the possibility of involvement of Plasmodium enolase in moonlighting functions was examined by investigating its sub-cellular distribution in the murine malarial parasite, Plasmodium yoelii.

Methods

Cellular extracts of P. yoelii were fractionated in to soluble (cytosolic) and particulate (membranes, nuclear and cytoskeletal) fractions and were analysed by one and two-dimensional gel electrophoresis. These were probed by Western blotting using antibodies raised against recombinant Plasmodium falciparum enolase. Immunofluorescence assay was used for in situ localization. Fe+3 based metal affinity chromatography was used to isolate the phospho-proteome fraction from P. yoelii extracts.

Results

Apart from the expected presence of enolase in cytosol, this enzyme was also found to be associated with membranes, nuclei and cytoskeletal fractions. Nuclear presence was also confirmed by in situ immunofluorescence. Five different post translationally modified isoforms of enolase could be identified, of which at least three were due to the phosphorylation of the native form. in situ phosphorylation of enolase was also evident from the presence of enolase in purified phosphor-proteome of P. yoelli. Different sub-cellular fractions showed different isoform profiles.

Conclusion

Association of enolase with nuclei, cell membranes and cytoskeletal elements suggests non-glycolytic functions for this enzyme in P. yoelii. Sub-cellular fraction specific isoform profiles indicate the importance of post-translational modifications in diverse localization of enolase in P. yoelii. Further, it is suggested that post-translational modifications of enolase may govern the recruitment of enolase for non-glycolytic functions.
Concentrated oat β-glucan, a fermentable fiber, lowers serum cholesterol in hypercholesterolemic adults in a randomized controlled trial
Authors: Queenan Katie M , Stewart Maria L , Smith Kristen N , Thomas William , Fulcher R Gary , Slavin Joanne L
Published: May 2007

Background

Soluble fibers lower serum lipids, but are difficult to incorporate into products acceptable to consumers. We investigated the physiological effects of a concentrated oat β-glucan on cardiovascular disease (CVD) endpoints in human subjects. We also compared the fermentability of concentrated oat β-glucan with inulin and guar gum in a model intestinal fermentation system.

Methods

Seventy-five hypercholesterolemic men and women were randomly assigned to one of two treatments: 6 grams/day concentrated oat β-glucan or 6 grams/day dextrose (control). Fasting blood samples were collected at baseline, week 3, and week 6 and analyzed for total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, glucose, insulin, homocysteine and C-reactive protein (CRP). To estimate colonic fermentability, 0.5 g concentrated oat β-glucan was incubated in a batch model intestinal fermentation system, using human fecal inoculum to provide representative microflora. Fecal donors were not involved with the β-glucan feeding trial. Inulin and guar gum were also incubated in separate serum bottles for comparison.

Results

Oat β-glucan produced significant reduction from baseline in total cholesterol (-0.3 ± 0.1 mmol/L) and LDL cholesterol (-0.3 ± 0.1 mmol/L), and the reduction in LDL cholesterol were significantly greater than in the control group (p = 0.03). Concentrated oat β-glucan was a fermentable fiber and produced total SCFA and acetate concentrations similar to inulin and guar gum. Concentrated oat β-glucan produced the highest concentrations of butyrate at 4, 8, and 12 hours.

Conclusion

Six grams concentrated oat β-glucan per day for six weeks significantly reduced total and LDL cholesterol in subjects with elevated cholesterol, and the LDL cholesterol reduction was greater than the change in the control group. Based on a model intestinal fermentation, this oat β-glucan was fermentable, producing higher amounts of butyrate than other fibers. Thus, a practical dose of β-glucan can significantly lower serum lipids in a high-risk population and may improve colon health.
Calyculin A, an enhancer of myosin, speeds up anaphase chromosome movement
Authors: Fabian Lacramioara , Troscianczuk Joanna , Forer Arthur
Published: May 2007

Actin and myosininhibitorsoften blocked anaphase movements in insect spermatocytes in previous experiments. Here we treat cells with anenhancerof myosin, Calyculin A, which inhibits myosin-light-chain phosphatase from dephosphorylating myosin; myosin thus is hyperactivated. Calyculin A causes anaphase crane-fly spermatocyte chromosomes to accelerate poleward; after they reach the poles they often move back toward the equator. When added during metaphase, chromosomes at anaphase move faster than normal. Calyculin A causes prometaphase chromosomes to move rapidly up and back along the spindle axis, and to rotate. Immunofluorescence staining with an antibody against phosphorylated myosin regulatory light chain (p-squash) indicated increased phosphorylation of cleavage furrow myosin compared to control cells, indicating that calyculin A indeed increased myosin phosphorylation. To test whether the Calyculin A effects are due to myosin phosphatase or to type 2 phosphatases, we treated cells with okadaic acid, which inhibits protein phosphatase 2A at concentrations similar to Calyculin A but requires much higher concentrations to inhibit myosin phosphatase. Okadaic acid had no effect on chromosome movement. Backward movements did not require myosin or actin since they were not affected by 2,3-butanedione monoxime or LatruculinB. Calyculin A affects the distribution and organization of spindle microtubules, spindle actin, cortical actin and putative spindle matrix proteins skeletor and titin, as visualized using immunofluorescence. We discuss how accelerated and backwards movements might arise.
Effect of BRAFV600E mutation on transcription and post-transcriptional regulation in a papillary thyroid carcinoma model
Authors: Cahill Susanne , Smyth Paul , Denning Karen , Flavin Richard , Li Jinghuan , Potratz Astrid , Guenther Simone M , Henfrey Richard , O'Leary John J , Sheils Orla
Published: May 2007

Background

microRNAs (miRNAs) are a group of non-coding single stranded RNAs measuring approximately 22 nucleotides in length that have been found to control cell growth, differentiation and apoptosis. They negatively regulate target genes and have recently been implicated in tumourigenesis. Furthermore, miRNA expression profiling correlates with various cancers, with these genes thought to act as both tumour suppressors and oncogenes. Recently, a point mutation in the BRAF gene leading to a V600E substitution has been identified as the most common genetic change in papillary thyroid carcinoma (PTC) occurring in 29–69% of cases. This mutation leads to aberrant MAPK activation that is implicated in tumourigenesis.

Aim

The aim of this study was to identify the effect that BRAF oncogene has on post-transcriptional regulation in PTC by using microRNA analysis.

Results

A unique miRNA expression signature differentiated between PTC cell lines with BRAF mutations and a normal thyroid cell line. 15 miRNAs were found to be upregulated and 23 miRNAs were downregulated. Several of these up/down regulated miRNAs may be involved in PTC pathogenesis. miRNA profiling will assist in the elucidation of disease pathogenesis and identification biomarkers and targets.
Calcium-activated potassium channels mediated blood-brain tumor barrier opening in a rat metastatic brain tumor model
Authors: Hu Jinwei , Yuan Xiangpeng , Ko MinHee K , Yin Dali , Sacapano Manuel R , Wang Xiao , Konda Bindu M , Espinoza Andres , Prosolovich Ksenia , Ong John M , Irvin Dwain , Black Keith L
Published: May 2007

Background

The blood-brain tumor barrier (BTB) impedes the delivery of therapeutic agents to brain tumors. While adequate delivery of drugs occurs in systemic tumors, the BTB limits delivery of anti-tumor agents into brain metastases.

Results

In this study, we examined the function and regulation of calcium-activated potassium (KCa) channels in a rat metastatic brain tumor model. We showed that intravenous infusion of NS1619, a KCa channel agonist, and bradykinin selectively enhanced BTB permeability in brain tumors, but not in normal brain. Iberiotoxin, a KCa channel antagonist, significantly attenuated NS1619-induced BTB permeability increase. We found KCa channels and bradykinin type 2 receptors (B2R) expressed in cultured human metastatic brain tumor cells (CRL-5904, non-small cell lung cancer, metastasized to brain), human brain microvessel endothelial cells (HBMEC) and human lung cancer brain metastasis tissues. Potentiometric assays demonstrated the activity of KCa channels in metastatic brain tumor cells and HBMEC. Furthermore, we detected higher expression of KCa channels in the metastatic brain tumor tissue and tumor capillary endothelia as compared to normal brain tissue. Co-culture of metastatic brain tumor cells and brain microvessel endothelial cells showed an upregulation of KCa channels, which may contribute to the overexpression of KCa channels in tumor microvessels and selectivity of BTB opening.

Conclusion

These findings suggest that KCa channels in metastatic brain tumors may serve as an effective target for biochemical modulation of BTB permeability to enhance selective delivery of chemotherapeutic drugs to metastatic brain tumors.
Bright/ARID3A contributes to chromatin accessibility of the immunoglobulin heavy chain enhancer
Authors: Lin Danjuan , Ippolito Gregory C , Zong Rui-Ting , Bryant James , Koslovsky Janet , Tucker Philip
Published: May 2007

Bright/ARID3A is a nuclear matrix-associated transcription factor that stimulates immunoglobulin heavy chain (IgH) expression and Cyclin E1/E2F-dependent cell cycle progression. Bright positively activates IgH transcriptional initiation by binding to ATC-rich P sites within nuclear matrix attachment regions (MARs) flanking the IgH intronic enhancer (Eμ). Over-expression of Bright in cultured B cells was shown to correlate with DNase hypersensitivity of Eμ. We report here further efforts to analyze Bright-mediated Eμ enhancer activation within the physiological constraints of chromatin. A system was established in which VH promoter-drivenin vitrotranscription on chromatin- reconstituted templates was responsive to Eμ. Bright assisted in blocking the general repression caused by nucleosome assembly but was incapable of stimulating transcription from prebound nucleosome arrays.In vitrotranscriptional derepression by Bright was enhanced on templates in which Eμ is flanked by MARs and was inhibited by competition with high affinity Bright binding (P2) sites. DNase hypersensitivity of chromatin-reconstituted Eμ was increased when prepackaged with B cell nuclear extract supplemented with Bright. These results identify Bright as a contributor to accessibility of the IgH enhancer.
HHV-8 encoded LANA-1 alters the higher organization of the cell nucleus
Authors: Stuber György , Mattsson Karin , Flaberg Emilie , Kati Emrah , Markasz Laszlo , Sheldon Julie A , Klein George , Schulz Thomas F , Szekely Laszlo
Published: May 2007

The latency-associated nuclear antigen (LANA-1) of Human Herpes Virus 8 (HHV-8), alternatively called Kaposi Sarcoma Herpes Virus (KSHV) is constitutively expressed in all HHV-8 infected cells. LANA-1 accumulates in well-defined foci that co-localize with the viral episomes. We have previously shown that these foci are tightly associated with the borders of heterochromatin [1]. We have also shown that exogenously expressed LANA-1 causes an extensive re-organization of Hoechst 33248 DNA staining patterns of the nuclei in non-HHV-8 infected cells [2]. Here we show that this effect includes the release of the bulk of DNA from heterochromatic areas, in both human and mouse cells, without affecting the overall levels of heterochromatin associated histone H3 lysine 9 tri-methylation (3MK9H3). The release of DNA from the heterochromatic chromocenters in LANA-1 transfected mouse cells co-incides with the dispersion of the chromocenter associated methylcytosin binding protein 2 (MECP2). The localization of 3MK9H3 to the remnants of the chromocenters remains unaltered. Moreover, exogeneously expressed LANA-1 leads to the relocation of the chromocenters to the nuclear periphery, indicating extensive changes in the positioning of the chromosomal domains in the LANA-1 harboring interphase nucleus. Using a series of deletion mutants we have shown that the chromatin rearranging effects of LANA-1 require the presence of a short (57 amino acid) region that is located immediately upstream of the internal acidic repeats. This sequence lies within the previously mapped binding site to histone methyltransferase SUV39H1. We suggest that the highly concentrated LANA-1, anchored to the host genome in the nuclear foci of latently infected cells and replicated through each cell generation, may function as "epigenetic modifier". The induction of histone modification in adjacent host genes may lead to altered gene expression, thereby contributing to the viral oncogenesis.
Genetic determinants of statin intolerance
Authors: Oh Jisun , Ban Matthew R , Miskie Brooke A , Pollex Rebecca L , Hegele Robert A
Published: May 2007

Background

Statin-related skeletal muscle disorders range from benign myalgias – such as non-specific muscle aches or joint pains without elevated serum creatinine kinase (CK) concentration – to true myositis with >10-fold elevation of serum CK, to rhabdomyolysis and myoglobinuria. The genetic basis of statin-related muscle disorders is largely unknown. Because mutations in the COQ2 gene are associated with severe inherited myopathy, we hypothesized that common, mild genetic variation in COQ2 would be associated with inter-individual variation in statin intolerance. We studied 133 subjects who developed myopathy on statin monotherapy and 158 matched controls who tolerated statins without incident or complaint.

Results

COQ2 genotypes, based on two single nucleotide polymorphisms (SNP1 and SNP2) and a 2-SNP haplotype, all showed significant associations with statin intolerance. Specifically, the odds ratios (with 95% confidence intervals) for increased risk of statin intolerance among homozygotes for the rare alleles were 2.42 (0.99 to 5.89), 2.33 (1.13 to 4.81) and 2.58 (1.26 to 5.28) for SNP1 and SNP2 genotypes, and the 2-SNP haplotype, respectively.

Conclusion

These preliminary pharmacogenetic results, if confirmed, are consistent with the idea that statin intolerance which is manifested primarily through muscle symptoms is associated with genomic variation in COQ2 and thus perhaps with the CoQ10 pathway.
Influence of venous emptying on the reactive hyperemic blood flow response
Authors: Bahadir Zeki , Tisdell Eric , Arce Esquivel Arturo A , Dobrosielski Devon A , Welsch Michael A
Published: May 2007

Background

Previous research indicates that venous emptying serves as a stimulus for vasodilation in the human forearm. This suggests the importance of recognizing the potential influence of venous volume on reactive hyperemic blood flow (RHBF) following occlusion. The purpose of this study was to examine the influence of venous emptying on forearm vascular function.

Methods

Forearm RHBF, venous capacitance and venous outflow were examined in 35 individuals (age = 22 ± 2 years), using mercury in-Silastic strain gauge plethysmography, at rest and following five minutes of upper arm occlusion using standard procedures (Control). In addition, the same measures were obtained following five minutes of upper arm occlusion preceded by two minutes of passive arm elevation (Pre-elevation).

Results

Average resting arterial inflow was 2.42 ± 1.11 ml·100 ml-1·min-1. RHBF and venous capacitance were significantly greater during Pre-elevation compared to Control (RHBF; Pre-elevation: 23.76 ± 5.95 ml·100 ml-1 ·min-1 vs. Control: 19.33 ± 4.50; p = 0.001), (venous capacitance; Pre-elevation: 2.74 ± 0.89 % vs. Control: 2.19 ± 0.97, p = 0.001). Venous outflow did not differ between the two conditions.

Conclusion

Venous emptying prior to upper arm occlusion results in a significant greater RHBF response and venous capacitance. Recognition of the influence of venous volume on RHBF is particularly important in studies focusing on arterial inflow, and also provides further evidence for the interplay between the venous and arterial system.
Takotsubo cardiomyopathy in a Caucasian Italian woman: Case report
Authors: Lisi Matteo , Zacà Valerio , Maffei Silvia , Casucci Francesca , Maggi Marianna , Lunghetti Stefano , Aitiani Paolo , Carrera Arcangelo , Castellani Debora , Favilli Roberto , Pierli Carlo , Mondillo Sergio
Published: May 2007

Background

Takotsubo cardiomyopathy is an acute cardiac syndrome characterized by transient LV regional wall motion abnormalities (with peculiar apical ballooning appearance), chest pain or dyspnea, ST-segment elevation and minor elevations of cardiac enzyme levels

Case presentation

A 68-year-old woman was admitted to the Emergency Department because of sudden onset chest pain occurred while transferring her daughter, who had earlier suffered a major seizure, to the hospital. The EKG showed sinus tachycardia with ST-segment elevation in leads V2–V3 and ST-segment depression in leads V5–V6, she was, thus, referred for emergency coronary angiography. A pre-procedural transthoracic echocardiogram revealed regional systolic dysfunction of the LV walls with hypokinesis of the mid-apical segments and hyperkinesis of the basal segments. Coronary angiography showed patent epicardial coronary arteries; LV angiography demonstrated the characteristic morphology of apical ballooning with hyperkinesis of the basal segments and hypokinesis of the mid-apical segments. The post-procedural course was uneventful; on day 5 after admission the echocardiogram revealed full recovery of apical and mid-ventricular regional wall-motion abnormalities.

Conclusion

Takotsubo cardiomyopathy is a relatively rare, unique entity that has only recently been widely appreciated. Acute stress has been indicated as a common trigger for the transient LV apical ballooning syndrome, especially in postmenopausal women. The present report is a typical example of stress-induced takotsubo cardiomyopathy in a Caucasian Italian postmenopausal woman.
Coronary artery-pulmonary artery fistula: case report
Authors: Quatrini Ilaria , Zacà Valerio , Mondillo Sergio
Published: May 2007

Background

Coronary artery fistulas are rare congenital or acquired coronary artery anomalies that can originate from any of the three major coronary arteries and drain in all the cardiac chambers and great vessels.

Case presentation

An 11-year-old boy was referred for evaluation of an exertional dyspnoea. He reported recent history of few episodes of shortness of breath associated with moderate entity physical activity. At physical examination a mild continuous murmur could be heard mainly at the level of the second intercostal space of the left parasternal area. A transthoracic echocardiogram showed a continuous flow at color Doppler analysis in the high parasternal short axis view, originating from a small entry site on the wall of the main pulmonary artery. A selective left coronary angiography revealed a fistula connecting the proximal portion of the left anterior descending coronary artery with the main pulmonary artery.

Conclusion

A combination like the one described in the present case is unusual since fistulas originate from the left coronary artery in about 35% of cases and drainage into the pulmonary artery occurs in only 17%.
Perfluorochemical (PFC) liquid enhances recombinant adenovirus vector-mediated viral interleukin-10 (AdvIL-10) expression in rodent lung
Authors: Li John T , Bonneau Laura L , Zimmerman Jerry J , Weiss Daniel J
Published: May 2007

Adenovirus and cationic liposome mediated transfer of Interleukin-10 (IL-10), a potent anti-inflammatory cytokine, has been shown to decrease pro-inflammatory cytokine levels and overall lung inflammation in models of lung transplantation and injury. Limitations to current approaches of IL-10 gene therapy include poor vector delivery methods and pro-inflammatory properties of human IL-10 under certain conditions. We hypothesize that using perfluorochemical (PFC) liquid to deliver the highly homologous viral IL-10 (vIL-10), which is predominantly anti-inflammatory with minimal pro-inflammatory activities, can potentially be a more effective strategy to combat inflammatory lung diseases. In this study, we compare the use of PFC liquid versus aerosolized method to deliver adenovirus encoding the vIL-10 gene (AdvIL-10) in C57Bl6 mice. Detectable vIL-10 levels were measured from bronchoalveolar lavage fluid and lung homogenates at one, four, ten and thirty days after AdvIL-10. Furthermore, we determined if use of PFC liquid could allow for the use of a lower dose of AdvIL-10 by comparing the levels of detectable vIL-10 at different doses of AdvIL-10 delivered +/- PFC liquid. Results showed that PFC liquid enhanced detectable vIL-10 by up to ten fold and that PFC liquid allowed the use of ten-fold less vector. PFC liquid increased detectable vIL-10 in lung homogenates at all time points; however, the increase in detectable vIL-10 in BAL fluid peaked at four days and was no longer evident by thirty days after intratracheal instillation. In summary, this is the first report utilizing PFC liquid to enhance the delivery of a potentially therapeutic molecule, vIL-10. We believe this strategy can be used to perform future studies on the use of the predominantly anti-inflammatory vIL-10 to treat inflammatory lung diseases.
Outcomes of resection for rectal cancer in India: The impact of the double stapling technique
Authors: Shrikhande Shailesh V , Saoji Rajesh R , Barreto Savio G , Kakade Anagha C , Waterford Stephen D , Ahire Sanjay B , Goliwale Fahim M , Shukla Parul J
Published: May 2007

Background

The introduction of circular staplers into colorectal surgery has revolutionized anastomotic techniques stretching the limits of sphincter preservation. Data on the double-stapling technique (DST) has been widely published in the West where the incidence of colorectal cancer is high. However studies using this technique and their results, in the Indian scenario, as well as the rest of Asia, have been few and far between.

Aim

To evaluate the feasibility of the DST in Indian patients with low rectal cancers and assess its impact on anastomotic leak rates, covering colostomy rates, level of resection and morbidity in patients undergoing low anterior resection (LAR).

Methods

A comparative analysis was performed between retrospectively acquired data on 78 patients (mean age 53.2 ± 13.5 years) undergoing LAR with the single-stapling technique (SST) (between January 1999 and December 2001) and prospective data acquired on 138 LARs (mean age 50.3 ± 13.9 years) performed using the DST (between January 2003 – December 2005).

Results

A total of 77 out of 78 patients in the SST group had Astler Coller B and C disease while the number was 132/138 in the DST group. The mean distance of the tumor from anal verge was 7.6 cm (2.5–15 cm) and 8.0 cm (4–15 cm) in the DST and SST groups, respectively. In the DST group, there were 5 (3.6%) anastomotic failures and 62 (45%) covering stomas compared to 7 (8.9%) anastomotic failures and 51 (65.4%) covering stomas in the SST group. The anastomotic leak rate, though objectively lower in the DST group, did not attain statistical significance (p = 0.12). Covering stoma rates were significantly lower in DST group (p = 0.006). There was 1 death in the DST group due to cardiac causes (unrelated to the anastomosis) and no mortality in the SST group. The LAR and abdominoperineal resection (APR) rates were 40% and 60%, respectively, during 1999–2001. In 2005, these rates were 55% and 45%, respectively.

Conclusion

This study, perhaps the first from India, demonstrates the feasibility of the DST in a country where the incidence of colorectal cancer is increasing. Since the age at presentation is at least a decade younger than the Western world, consideration of sphincter preservation assumes greater significance. The observed improvement of surgical outcomes with DST needs further studies to significantly prove these findings in a population where the tumors at presentation are predominantly Astler Coller Stage B and C.
Endoscopically assisted resection of a scapular osteochondroma causing snapping scapula syndrome
Authors: Fukunaga Satoru , Futani Hiroyuki , Yoshiya Shinichi
Published: May 2007

Background

Osteochondroma is the most common benign bone tumor in the scapula. This condition might lead to snapping scapula syndrome, which is characterized by painful, audible, and/or palpable abnormal scapulothoracic motion. In the present case, this syndrome was successfully treated by use of endoscopically assisted resection of the osteochondroma.

Case presentation

A 41-year-old man had a tolerable pain in his scapular region over a 10 years' period. The pain developed gradually with shoulder motion, in particular with golf swing since he was aiming a professional golf player career. On physical examination, "clunking" was noted once from 90 degrees of abduction to 180 degrees of shoulder motion. A trans-scapular roentgenogram and computed tomography images revealed an osteochondroma located at the anterior and inferior aspect of the scapula. Removal of the tumor was performed by the use of endoscopically assisted resection. One portal was made at the lateral border of the scapula to introduce a 2.7-mm-diameter, 30 degrees Hopkins telescope. The tumor was resected in a piece-by-piece manner by the use of graspers through the same portal. Immediately after the operation pain relief was obtained, and the "clunking" disappeared. CT images showed complete tumor resection. The patient could start playing golf one week after the surgery.

Conclusion

Endoscopically assisted resection of osteochondroma of the scapula provides a feasible technique to treat snapping scapula syndrome and obtain early functional recovery with a short hospital stay and cosmetic advantage.
Semaphorin-4D (Sema-4D), the Plexin-B1 ligand, is involved in mouse ovary follicular development
Authors: Regev Avital , Goldman Shlomit , Shalev Eliezer
Published: May 2007

Background

Human Plexin-B1 is expressed in two truncated forms. The long form encodes a trans-membranal protein, while the short form, which is bound to the cell surface and partially secreted, possibly serves as a decoy receptor. Plexin receptors are trans-membrane proteins. The sema domain, found in the extracellular region, is common to all plexins, semaphorins, and the scatter factor receptors and is crucial for the biological activity and plexin receptor specificity. Semaphorin-4D/Plexin-B1 binding provides attractive and repulsive cues for the navigation of axonal growth cones, and new studies suggest that this system also plays a role in the regulation of the biological functions of endothelial cells, specifically in the control of angiogenesis. In a previous study, we have demonstrated the expression and possible role of Plexin-B1 in the mouse ovary. The present study was designed to test the hypothesis that Plexin-B1 effects are mediated by Semaphorin-4D.

Methods

In vivo expression and localization of mouse ovarian Sema-4D were tested by immunohisto-chemistry. The role of Sema-4D in follicular development was examined by in vitro growth of preantral follicles in the presence or absence of Semaphorin-4D, with or without neutralizing antibodies against Plexin-B1. Follicular growth and steroid hormone secretion rates were tested.

Results

Semaphorin-4D is expressed in the mouse ovary in vivo mostly in the granulosa cells and and its expression is modulated by PMSG and hCG. In the presence of Semaphorin-4D, in-vitro constant growth was observed as indicated by follicular diameter during the culture period and elevated steroid hormone secretion rates compared with control. These effects were abolished after addition of neutralizing antibodies against Plexin-B1.

Conclusion

In the ovarian follicle, the effect of Plexin-B1 is mediated by sema-4D.
Investigating the spatial risk distribution of West Nile virus disease in birds and humans in southern Ontario from 2002 to 2005
Authors: Beroll Heidi , Berke Olaf , Wilson Jeffrey , Barker Ian K
Published: May 2007

Background

The West Nile virus (WNv) became a veterinary public health concern in southern Ontario in 2001 and has continued to threaten public health. Wild bird mortality has been shown to be an indicator for tracking the geographic distribution of the WNv. The purpose of this study was to investigate the latent risk distribution of WNv disease among dead birds and humans in southern Ontario and to compare the spatial risk patterns for the period 2002–2005. The relationship between the mortality fraction in birds and incidence rate in humans was also investigated.

Methods

Choropleth maps were created to investigate the spatial variation in bird and human WNv risk for the public health units of southern Ontario. The data were smoothed by empirical Bayesian estimation before being mapped. Isopleth risk maps for both the bird and human data were created to identify high risk areas and to investigate the potential relationship between the WNv mortality fraction in birds and incidence rates in humans. This was carried out by the geostatistical prediction method of kriging. A Poisson regression analysis was used to model regional human WNv case counts as a function of the spatial coordinates in the east and north direction and the regional bird mortality fractions. The presence of disease clustering and the location of disease clusters were investigated by the spatial scan test.

Results

The isopleth risk maps exhibited high risk areas that were relatively constant from year to year. There was an overlap in the bird and human high risk areas, which occurred in the central-west and south-west areas of southern Ontario. The annual WNv cause-specific mortality fractions in birds for 2002 to 2005 were 31.9, 22.0, 19.2 and 25.2 positive birds per 100 birds tested, respectively. The annual human WNv incidence rates for 2002 to 2005 were 2.21, 0.76, 0.13 and 2.10 human cases per 100,000 population, respectively. The relative risk of human WNv disease was 0.72 times lower for a public health unit that was 100 km north of another public health unit. The relative risk of human WNv disease increased by the factor 1.44 with every 10 positive birds per 100 tested. The scan statistic detected disease cluster in the bird and human data. The human clusters were not significant, when the analysis was conditioned on the bird data.

Conclusion

The study indicates a significant relationship between the spatial pattern of WNv risk in humans and birds.
CMV pp65 and IE-1 T cell epitopes recognized by healthy subjects
Authors: Slezak Stefanie L , Bettinotti Maria , Selleri Silvia , Adams Sharon , Marincola Francesco M , Stroncek David F
Published: May 2007

Background

Adoptive immune and vaccine therapies have been used to prevent cytomegalovirus (CMV) disease in recipients of hematopoietic progenitor cell transplants, but the nature of T cell responses to CMV have not been completely characterized.

Methods

Peptide pools and individual peptides derived from the immune-dominant CMV proteins pp65 and IE-1 and antigen-specific, cytokine flow cytometry were used to characterize the prevalence and frequency of CD4+ and CD8+ memory T cells in 20 healthy CMV-seropositive subjects.

Results

CD8+ T cell responses to pp65 were detected in 35% of subjects and to IE-1 in 40% of subjects. CD4+ T cell responses to pp65 were detected in 50% of subjects, but none were detected to IE-1. Several new IE-1 HLA class I epitopes were identified, including 4 restricted to HLA-C antigens. One region of IE-1 spanning amino acids 300 to 327 was rich in class I epitopes. The HLA class I restrictions of IE-1 peptides were more promiscuous than those of pp65 peptides.

Conclusion

Since naturally occurring CD4+ and CD8+ T cell responses to pp65 were detectable in many subjects, but only CD8+ T cell responses to IE-1 were detected, pp65 may be better than IE-1 for use in vaccine and adoptive immune therapies.
Generation of clinical grade dendritic cells with capacity to produce biologically active IL-12p70
Authors: Zobywalski Anke , Javorovic Miran , Frankenberger Bernhard , Pohla Heike , Kremmer Elisabeth , Bigalke Iris , Schendel Dolores J
Published: May 2007

Background

For optimal T cell activation it is desirable that dendritic cells (DCs) display peptides within MHC molecules as signal 1, costimulatory molecules as signal 2 and, in addition, produce IL-12p70 as signal 3. IL-12p70 polarizes T cell responses towards CD4+ T helper 1 cells, which then support the development of CD8+ cytotoxic T lymphocytes. We therefore developed new maturation cocktails allowing DCs to produce biologically active IL-12p70 for large-scale cancer vaccine development.

Methods

After elutriation of leukapheresis products in a closed bag system, enriched monocytes were cultured with GM-CSF and IL-4 for six days to generate immature DCs that were then matured with cocktails, containing cytokines, interferon-gamma, prostaglandin E2, and a ligand for Toll-like receptor 8, with or without poly (I:C).

Results

Mature DCs expressed appropriate maturation markers and the lymph node homing chemokine receptor, CCR7. They retained full maturity after culture for two days without maturation cocktails and following cryopreservation. TLR ligand stimulation induced DCs capable of secreting IL-12p70 in primary cultures and after one day of coculture with CD40L-expressing fibroblasts, mimicking an encounter with T cells. DCs matured with our new cocktails containing TLR8 ligand, with or without poly (I:C), induced alloresponses and stimulated virus-specific T cells after peptide-pulsing. DCs matured in cocktails containing TLR8 ligand without poly (I:C) could also be loaded with RNA as a source of antigen, whereas DCs matured in cocktails containing poly (I:C) were unable to express proteins following RNA transfer by electroporation.

Conclusion

Our new maturation cocktails allowed easy DC harvesting, stable maturation and substantial recoveries of mature DCs after cryopreservation. Our procedure for generating DCs is easily adaptable for GMP-compliance and yields IL-12p70-secreting DCs suitable for development of cancer vaccines using peptides or RNA as sources of immunizing antigens.
Homocysteine and lipid profile in children with Juvenile Idiopathic Arthritis
Authors: Gonçalves Marcela , D'Almeida Vânia , Guerra-Shinohara Elvira M , Galdieri Luciano C , Len Claudio A , Hilário Maria Odete E
Published: May 2007

Background

An increased concentration of plasma homocysteine (Hcy) has toxic effects on vascular endothelium. This seems to be a risk factor of cardiovascular disease, premature stroke and venous thrombosis. The risk is higher in coincidence with other factors like chronic diseases and familiar hypercholesterolemia. The aim of our study was to evaluate plasma Hcy concentration in patients with juvenile idiopathic arthritis (JIA) and its correlation with methotrexate (MTX) therapy, serum folate and B12 vitamin, and hyperlipidemia.

Methods

Fifty-one patients (37 females; mean age 11.3 years, range 2.3–17 years) with JIA and 52 healthy controls (42 females; mean age 12.5 years; range 3–18 years) were included in the study. Thirty-two patients were using weekly MTX (mean doses: 0.1–1 mg/kg). For statistical analysis both JIA and control groups were distributed in three subgroups according to age (3 – 7, 8 – 12 and 13 – 18 years). The laboratory investigation included measurement of erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), plasma Hcy, serum folate, vitamin B12, triglycerides, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL). For data analysis, we considered raised Hcy values ≥ 12.56 μmol/L, which corresponds to the 90th percentile observed in the control group.

Results

The mean plasma Hcy concentration was 9.3 ± 3.16 μmol/L in JIA patients and 8.9 ± 2.42 μmol/L in healthy controls (p = 0.615). Higher concentration of Hcy was observed in the subgroup of 13 – 18 years (patients and controls, p < 0.001). We did not find correlation between MTX use and plasma Hcy concentration. With regard to vitamin B12 concentration, we detected normal values in both patients and controls while serum folate concentration was higher in patients (p < 0.001). With regard to the lipidogram, lower concentration of HDL was found in patients (p = 0.007) and higher levels of VLDL (p = 0.014) and triglycerides (p = 0.001) were observed in controls. We did not observe correlation among plasma Hcy concentration, clinical findings, ESR and CRP.

Conclusion

JIA patients do not present significant increased concentration of Hcy despite the use of MTX, probably due to the folate supplementation. The mild abnormalities in the lipidogram may reflect a current concern with diet and health.
Reactive arthritis of the temporomandibular joints and cervical spine in a child
Authors: Arabshahi Bita , Baskin Kevin M , Cron Randy Q
Published: May 2007

Background

Temporomandibular joint (TMJ) arthritis is frequently seen in children with chronic arthritis. It has rarely been described in a non-infectious acute setting. We report a case of reactive arthritis isolated to the TMJs and cervical spine.

Case presentation

A 6-year-old Native American boy hospitalized for treatment of lymphadenitis and aseptic meningitis had an incidental brain magnetic resonance imaging (MRI) finding of effusions in the TMJs, as well as the atlanto-occipital and C1–C2 articulations. Repeat TMJ and cervical spine MRI four weeks later showed resolution of effusions. Reactive TMJ arthritis has been previously reported in adults but not in children.

Conclusion

This report represents the first pediatric case of reactive arthritis isolated to the cervical spine and TMJs. Arthritis of the TMJ should be considered in the differential diagnosis of children with reactive arthritides.
Pygo1 and Pygo2 roles in Wnt signaling in mammalian kidney development
Authors: Schwab Kristopher R , Patterson Larry T , Hartman Heather A , Song Ni , Lang Richard A , Lin Xinhua , Potter S Steven
Published: May 2007

Background

Thepygopusgene ofDrosophilaencodes an essential component of the Armadillo (β-catenin) transcription factor complex of canonical Wnt signaling. To better understand the functions ofPygopus-mediated canonical Wnt signaling in kidney development, targeted mutations were made in the two mammalian orthologs,Pygo1andPygo2.

Results

Each mutation deleted >80% of the coding sequence, including the critical PHD domain, and almost certainly resulted in null function.Pygo2homozygous mutants, with rare exception, died shortly after birth, with a phenotype including lens agenesis, growth retardation, altered kidney development, and in some cases exencephaly and cleft palate.Pygo1homozygous mutants, however, were viable and fertile, with no detectable developmental defects. DoublePygo1/Pygo2homozygous mutants showed no apparent synergy in phenotype severity. The BAT-gal transgene reporter of canonical Wnt signaling showed reduced levels of expression inPygo1-/-/Pygo2-/-mutants, with tissue-specific variation in degree of diminution. ThePygo1andPygo2genes both showed widespread expression in the developing kidney, with raised levels in the stromal cell compartment. Confocal analysis of the double mutant kidneys showed disturbance of both the ureteric bud and metanephric mesenchyme-derived compartments. Branching morphogenesis of the ureteric bud was altered, with expanded tips and reduced tip density, probably contributing to the smaller size of the mutant kidney. In addition, there was an expansion of the zone of condensed mesenchyme capping the ureteric bud. Nephron formation, however, proceeded normally. Microarray analysis showed changed expression of several genes, includingCxcl13,Slc5a2,Klk5,Ren2andTimeless, which represent candidate Wnt targets in kidney development.

Conclusion

The mammalianPygopusgenes are required for normal branching morphogenesis of the ureteric bud during kidney development. Nevertheless, the relatively mild phenotype observed in the kidney, as well as other organ systems, indicates a striking evolutionary divergence ofPygopusfunction between mammals andDrosophila. In mammals, thePygo1/Pygo2genes are not absolutely required for canonical Wnt signaling in most developing systems, but rather function as quantitative transducers, or modulators, of Wnt signal intensity.
In vitroprediction of stop-codon suppression by intravenous gentamicin in patients with cystic fibrosis: a pilot study
Authors: Sermet-Gaudelus Isabelle , Renouil Michel , Fajac Anne , Bidou Laure , Parbaille Bastien , Pierrot Sébastien , Davy Nolwen , Bismuth Elise , Reinert Philippe , Lenoir Gérard , Lesure Jean François , Rousset Jean Pierre , Edelman Aleksander
Published: May 2007

Background

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which acts as a chloride channel activated by cyclic AMP (cAMP). The most frequent mutation found in 70% of CF patients is F508del, while premature stop mutations are found in about 10% of patients. In vitro aminoglycoside antibiotics (e.g. gentamicin) suppress nonsense mutations located in CFTR permitting translation to continue to the natural termination codon. Pharmacologic suppression of stop mutations within the CFTR may be of benefit to a significant number of patients. Our pilot study was conducted to determine whether intravenous gentamicin suppresses stop codons in CF patients and whether it has clinical benefits.

Methods

A dual gene reporter system was used to determine the gentamicin-induced readthrough level of the most frequent stop mutations within the CFTR in the French population. We investigated readthrough efficiency in response to 10 mg/kg once-daily intravenous gentamicin perfusions in patients with and without stop mutations. Respiratory function, sweat chloride concentration, nasal potential difference (NPD) and CFTR expression in nasal epithelial cells were measured at baseline and after 15 days of treatment.

Results

After in vitro gentamicin incubation, the readthrough efficiency for the Y122X mutation was at least five times higher than that for G542X, R1162X, and W1282X. In six of the nine patients with the Y122X mutation, CFTR immunodetection showed protein at the membrane of the nasal epithelial cells and the CFTR-dependent Cl- secretion in NPD measurements increased significantly. Respiratory status also improved in these patients, irrespective of the gentamicin sensitivity of the bacteria present in the sputum. Mean sweat chloride concentration decreased significantly and normalised in two patients. Clinical status, NPD and sweat Cl- values did not change in the Y122X patients with no protein expression, in patients with the other stop mutations investigated in vitro and those without stop mutations.

Conclusion

Suppression of stop mutations in the CFTR gene with parenteral gentamicin can be predicted in vitro and is associated with clinical benefit and significant modification of the CFTR-mediated Cl- transport in nasal and sweat gland epithelium.
Mutation in the loop C-terminal to the cyclophilin A binding site of HIV-1 capsid protein disrupts proper virus assembly and infectivity
Authors: Abdurahman Samir , Höglund Stefan , Höglund Anders , Vahlne Anders
Published: May 2007

We have studied the effects associated with two single amino acid substitution mutations in HIV-1 capsid (CA), the E98A and E187G. Both amino acids are well conserved among all major HIV-1 subtypes. HIV-1 infectivity is critically dependent on proper CA cone formation and mutations in CA are lethal when they inhibit CA assembly by destabilizing the intra and/or inter molecular CA contacts, which ultimately abrogate viral replication. Glu98, which is located on a surface of a flexible cyclophilin A binding loop is not involved in any intra-molecular contacts with other CA residues. In contrast, Glu187 has extensive intra-molecular contacts with eight other CA residues. Additionally, Glu187 has been shown to form a salt-bridge with Arg18 of another N-terminal CA monomer in a N-C dimer. However, despite proper virus release, glycoprotein incorporation and Gag processing, electron microscopy analysis revealed that, in contrast to the E187G mutant, only the E98A particles had aberrant core morphology that resulted in loss of infectivity.
Centrosome and retroviruses: The dangerous liaisons
Authors: Afonso Philippe V , Zamborlini Alessia , Saïb Ali , Mahieux Renaud
Published: May 2007

Centrosomes are the major microtubule organizing structures in vertebrate cells. They localize in close proximity to the nucleus for the duration of interphase and play major roles in numerous cell functions. Consequently, any deficiency in centrosome function or number may lead to genetic instability. Several viruses including retroviruses such as, Foamy Virus, HIV-1, JSRV, M-PMV and HTLV-1 have been shown to hamper centrosome functions for their own profit, but the outcomes are very different. Foamy viruses, HIV-1, JSRV, M-PMV and HTLV-1 use the cellular machinery to traffic towards the centrosome during early and/or late stages of the infection. In addition HIV-1 Vpr protein alters the cell-cycle regulation by hijacking centrosome functions. Enthrallingly, HTLV-1 Tax expression also targets the functions of the centrosome, and this event is correlated with centrosome amplification, aneuploidy and transformation.