bluuebunny/crossref_metadata_2025 huggingface dataset:
Preformed CD40 ligand exists in secretory lysosomes in effector and memory CD4+ T cells and is quickly expressed on the cell surface in an antigen-specific manner
Authors: Koguchi Yoshinobu , Thauland Timothy J. , Slifka Mark K. , Parker David C.
Published: June 2007

CD40 ligand (CD40L) is an essential effector cytokine for macrophage activation, dendritic cell licensing, and T-cell–dependent antibody responses. Although CD40L is known to be made de novo following antigen recognition, several reports have described surface mobilization of preformed, intracellular CD40L in certain CD4+ effector T cells. Here we show that rapid surface expression of preformed CD40L following antigen recognition is a general property of both effector and memory CD4+ T cells, including in vitro and in vivo activated T-cell–receptor transgenic T cells, memory phenotype CD4+ T cells from pathogen-free naive mice, and polyclonal virus–specific effector and memory T cells. Intracellular CD40L is stored in secretory lysosomes, and colocalizes more strongly with Fas ligand than with CTLA-4, two other molecules that are delivered to the cell surface following antigen recognition. Stimulated surface expression of preformed CD40L is found in memory CD4+ T cells from CD40-deficient mice, indicating that it does not depend on CD40-induced internalization for delivery to the secretory compartment. We suggest that delivery of preformed CD40L to antigen-presenting cells (APCs) could enable antigen-specific activation of APCs in transient interactions that are too brief to permit de novo synthesis of CD40L.
Presence of thiamine pyrophosphate in mammalian peroxisomes
Authors: Fraccascia Patrizia , Sniekers Mieke , Casteels Minne , Van Veldhoven Paul P
Published: June 2007

Background

Thiamine pyrophosphate (TPP) is a cofactor for 2-hydroxyacyl-CoA lyase 1 (HACL1), a peroxisomal enzyme essential for the α-oxidation of phytanic acid and 2-hydroxy straight chain fatty acids. So far, HACL1 is the only known peroxisomal TPP-dependent enzyme in mammals. Little is known about the transport of metabolites and cofactors across the peroxisomal membrane and no peroxisomal thiamine or TPP carrier has been identified in mammals yet. This study was undertaken to get a better insight into these issues and to shed light on the role of TPP in peroxisomal metabolism.

Results

Because of the crucial role of the cofactor TPP, we reanalyzed its subcellular localization in rat liver. In addition to the known mitochondrial and cytosolic pools, we demonstrated, for the first time, that peroxisomes contain TPP (177 ± 2 pmol/mg protein). Subsequently, we verified whether TPP could be synthesized from its precursor thiamine, in situ, by a peroxisomal thiamine pyrophosphokinase (TPK). However, TPK activity was exclusively recovered in the cytosol.

Conclusion

Our results clearly indicate that mammalian peroxisomes do contain TPP but that no pyrophosphorylation of thiamine occurs in these organelles, implying that thiamine must enter the peroxisome already pyrophosphorylated. Consequently, TPP entry may depend on a specific transport system or, in a bound form, on HACL1 translocation.
Definition of the zebrafish genome using flow cytometry and cytogenetic mapping
Authors: Freeman Jennifer L , Adeniyi Adeola , Banerjee Ruby , Dallaire Stephanie , Maguire Sean F , Chi Jianxiang , Ng Bee Ling , Zepeda Cinthya , Scott Carol E , Humphray Sean , Rogers Jane , Zhou Yi , Zon Leonard I , Carter Nigel P , Yang Fengtang , Lee Charles
Published: June 2007

Background

The zebrafish (Danio rerio) is an important vertebrate model organism system for biomedical research. The syntenic conservation between the zebrafish and human genome allows one to investigate the function of human genes using the zebrafish model. To facilitate analysis of the zebrafish genome, genetic maps have been constructed and sequence annotation of a reference zebrafish genome is ongoing. However, the duplicative nature of teleost genomes, including the zebrafish, complicates accurate assembly and annotation of a representative genome sequence. Cytogenetic approaches provide "anchors" that can be integrated with accumulating genomic data.

Results

Here, we cytogenetically define the zebrafish genome by first estimating the size of each linkage group (LG) chromosome using flow cytometry, followed by the cytogenetic mapping of 575 bacterial artificial chromosome (BAC) clones onto metaphase chromosomes. Of the 575 BAC clones, 544 clones localized to apparently unique chromosomal locations. 93.8% of these clones were assigned to a specific LG chromosome location using fluorescencein situhybridization (FISH) and compared to the LG chromosome assignment reported in the zebrafish genome databases. Thirty-one BAC clones localized to multiple chromosomal locations in several different hybridization patterns. From these data, a refined second generation probe panel for each LG chromosome was also constructed.

Conclusion

The chromosomal mapping of the 575 large-insert DNA clones allows for these clones to be integrated into existing zebrafish mapping data. An accurately annotated zebrafish reference genome serves as a valuable resource for investigating the molecular basis of human diseases using zebrafish mutant models.
Structural and functional definition of the specificity of a novel caspase-3 inhibitor, Ac-DNLD-CHO
Authors: Yoshimori Atsushi , Sakai Junichi , Sunaga Satoshi , Kobayashi Takanobu , Takahashi Satoshi , Okita Naoyuki , Takasawa Ryoko , Tanuma Sei-ichi
Published: June 2007

Background

The rational design of peptide-based specific inhibitors of the caspase family members using their X-ray crystallographies is an important strategy for chemical knockdown to define the critical role of each enzyme in apoptosis and inflammation. Recently, we designed a novel potent peptide inhibitor, Ac-DNLD-CHO, for caspase-3 using a new computational screening system named the Amino acid Positional Fitness (APF) method (BMC Pharmacol. 2004, 4:7). Here, we report the specificity of the DNLD sequence against caspase-3 over other major caspase family members that participate in apoptosis by computational docking and site-directed mutagenesis studies.

Results

Ac-DNLD-CHO inhibits caspases-3, -7, -8, and -9 activities with Kiappvalues of 0.68, 55.7, >200, and >200 nM, respectively. In contrast, a well-known caspase-3 inhibitor, Ac-DEVD-CHO, inhibits all these caspases with similar Kiappvalues. The selective recognition of a DNLD sequence by caspase-3 was confirmed by substrate preference studies using fluorometric methylcoumarin-amide (MCA)-fused peptide substrates. The bases for its selectivity and potency were assessed on a notable interaction between the substrate Asn (N) and the caspase-3 residue Ser209 in the S3subsite and the tight interaction between the substrate Leu (L) and the caspase-3 hydrophobic S2subsite, respectively, in computational docking studies. Expectedly, the substitution of Ser209 with alanine resulted in loss of the cleavage activity on Ac-DNLD-MCA and had virtually no effect on cleaving Ac-DEVD-MCA. These findings suggest that N and L residues in Ac-DNLD-CHO are the determinants for the selective and potent inhibitory activity against caspase-3.

Conclusion

On the basis of our results, we conclude that Ac-DNLD-CHO is a reliable, potent and selective inhibitor of caspase-3. The specific inhibitory effect on caspase-3 suggests that this inhibitor could become an important tool for investigations of the biological function of caspase-3. Furthermore, Ac-DNLD-CHO may be an attractive lead compound to generate novel effective non-peptidic pharmaceuticals for caspase-mediated apoptosis diseases, such as neurodegenerative disorders and viral infection diseases.
Serology based disease status of Pakistani population infected with Hepatitis B virus
Authors: Alam Muhammad Masroor , Zaidi Soahil Zahoor , Malik Salman Akbar , Naeem Asif , Shaukat Shahzad , Sharif Salmaan , Angez Mehar , Khan Anis , Butt Javed Aslam
Published: June 2007

Background

The infection rate of hepatitis B virus is continuously increasing in Pakistan. Therefore, a comprehensive study of epidemiological data is the need of time.

Methods

A total of 1300 individuals were screened for HBV infection markers including HBsAg, anti-HBsAg, HBeAg and anti-HBcAg. The association of these disease indicators was compared with patients' epidemiological characteristics like age, socio-economic status and residential area to analyze and find out the possible correlation among these variables and the patients disease status.

Results

52 (4%) individuals were found positive for HBsAg with mean age 23.5 ± 3.7 years. 9.30%, 33.47% and 12% individuals had HBeAg, antibodies for HBsAg, and antibodies for HBcAg respectively. HBsAg seropositivity rate was significantly associated (p = 0.03) with the residing locality indicating high infection in rural areas. Antibodies titer against HBsAg decreased with the increasing age reflecting an inverse correlation.

Conclusion

Our results indicate high prevalence rate of Hepatitis B virus infection and nationwide vaccination campaigns along with public awareness and educational programs are needed to be practiced urgently.
Early prediction of median survival among a large AIDS surveillance cohort
Authors: Enanoria Wayne TA , Hubbard Alan E , van der Laan Mark J , Chen Mi , Ruiz Juan , Colford John M
Published: June 2007

Background

For individuals with AIDS, data exist relatively soon after diagnosis to allow estimation of "early" survival quantiles (e.g., the 0.10, 0.15, 0.20 and 0.30 quantiles, etc.). Many years of additional observation must elapse before median survival, a summary measure of survival, can be estimated accurately. In this study, a new approach to predict AIDS median survival is presented and its accuracy tested using AIDS surveillance data.

Methods

The data consisted of 96,373 individuals who were reported to the HIV/AIDS Reporting System of the California Department of Health Services Office of AIDS as of December 31, 1996. We defined cohorts based on quarter year of diagnosis (e.g., the "931" cohort consists of individuals diagnosed with AIDS in the first quarter of 1993). We used early quantiles (estimated using the Inverse Probability of Censoring Weighted estimator) of the survival distribution to estimate median survival by assuming a linear relationship between the earlier quantiles and median survival. From this model, median survival was predicted for cohorts for which a median could not be estimated empirically from the available data. This prediction was compared with the actual medians observed when using updated survival data reported at least five years later.

Results

Using the 0.15 quantile as the predictor and the data available as of December 31, 1996, we were able to predict the median survival of four cohorts (933, 934, 941, and 942) to be 34, 34, 31, and 29 months. Without this approach, there were insufficient data with which to make any estimate of median survival. The actual median survival of these four cohorts (using data as of December 31, 2001) was found to be 32, 40, 46, and 80 months, suggesting that the accuracy for this approach requires a minimum of three years to elapse from diagnosis to the time an accurate prediction can be made.

Conclusion

The results of this study suggest that early and accurate prediction of median survival time after AIDS diagnosis may be possible using early quantiles of the survival distribution. The methodology did not seem to work well during a period of significant change in survival as observed with highly active antiretroviral treatment, but results suggest that it may work well in a time of more gradual improvement in survival.
Swedish social insurance officers' experiences of difficulties in assessing applications for disability pensions – an interview study
Authors: Ydreborg Berit , Ekberg Kerstin , Nilsson Kerstin
Published: June 2007

Background

In this study the focus is on social insurance officers judging applications for disability pensions. The number of applications for disability pension increased during the late 1990s, which has resulted in an increasing number of disability pensions in Sweden. A more restrictive attitude towards the clients has however evolved, as societal costs have increased and governmental guidelines now focus on reducing costs. As a consequence, the quantitative and qualitative demands on social insurance officers when handling applications for disability pensions may have increased. The aim of this study was therefore to describe the social insurance officers' experiences of assessing applications for disability pensions after the government's introduction of stricter regulations.

Methods

Qualitative methodology was employed and a total of ten social insurance officers representing different experiences and ages were chosen. Open-ended interviews were performed with the ten social insurance officers. Data was analysed with inductive content analysis.

Results

Three themes could be identified as problematic in the social insurance officers' descriptions of dealing with the applications in order to reach a decision on whether the issue qualified applicants for a disability pension or not: 1. Clients are heterogeneous. 2. Ineffective and time consuming waiting for medical certificates impede the decision process. 3. Perspectives on the issue of work capacity differed among different stakeholders. The backgrounds of the clients differ considerably, leading to variation in the quality and content of applications. Social insurance officers had to make rapid decisions within a limited time frame, based on limited information, mainly on the basis of medical certificates that were often insufficient to judge work capacity. The role as coordinating actor with other stakeholders in the welfare system was perceived as frustrating, since different stakeholders have different goals and demands. The social insurance officers experience lack of control over the decision process, as regulations and other stakeholders restrict their work.

Conclusion

A picture emerges of difficulties due to disharmonized systems, stakeholder-bound goals causing some clients to fall between two stools, or leading to unnecessary waiting times, which may limit the clients' ability to take an active part in a constructive process. Increased communication with physicians about how to elaborate the medical certificates might improve the quality of certificates and thereby reduce the clients waiting time.
Women's attitudes towards mechanisms of action of family planning methods: survey in primary health centres in Pamplona, Spain
Authors: de Irala Jokin , del Burgo Cristina Lopez , de Fez Carmen M Lopez , Arredondo Jorge , Mikolajczyk Rafael T , Stanford Joseph B
Published: June 2007

Background

Informed consent in family planning includes knowledge of mechanism of action. Some methods of family planning occasionally work after fertilization. Knowing about postfertilization effects may be important to some women before choosing a certain family planning method. The objective of this survey is to explore women's attitudes towards postfertilization effects of family planning methods, and beliefs and characteristics possibly associated with those attitudes.

Methods

Cross-sectional survey in a sample of 755 potentially fertile women, aged 18–49, from Primary Care Health Centres in Pamplona, Spain. Participants were given a 30-item, self-administered, anonymous questionnaire about family planning methods and medical and surgical abortion. Logistic regression was used to identify variables associated with women's attitudes towards postfertilization effects.

Results

The response rate was 80%. The majority of women were married, held an academic degree and had no children. Forty percent of women would not consider using a method that may work after fertilization but before implantation and 57% would not consider using one that may work after implantation. While 35.3% of the sample would stop using a method if they learned that it sometimes works after fertilization, this percentage increased to 56.3% when referring to a method that sometimes works after implantation. Women who believe that human life begins at fertilization and those who consider it is important to distinguish between natural and induced embryo loss were less likely to consider the use of a method with postfertilization effects.

Conclusion

Information about potential postfertilization effects of family planning methods may influence women's acceptance and choice of a particular family planning method. Additional studies in other populations are necessary to evaluate whether these beliefs are important to those populations.
The impact of different rehabilitation strategies after major events in the elderly: the case of stroke and hip fracture in the Tuscany region
Authors: Carinci Fabrizio , Roti Lorenzo , Francesconi Paolo , Gini Rosa , Tediosi Fabrizio , Di Iorio Tania , Bartolacci Simone , Buiatti Eva
Published: June 2007

Background

On a regional level, our aims were to describe rehabilitation patterns for elderly patients with stroke and hip fracture and to investigate mortality risk during the 6-month post acute period.

Methods

Data sources included administrative data relative to patients aged 65+ resident in Tuscany admitted in hospital for stroke or hip fracture between 2001 and 2003, traced up to 3 years before and 6 months following index admission. The study design involves computerized linkage of administrative data, and an exploratory analysis of the association between rehabilitation patterns and 6-month mortality, adjusting for clinical, demographic, and acute-related care characteristics using multivariate Cox regression.

Results

Rehabilitation patterns vary greatly across Tuscany with considerable cost implications. Six month mortality risk for stroke patients is significantly lower among residents of Local Health Authorities where patients are more frequently rehabilitated, specifically in extra-hospital settings.

Conclusion

Our study, targeting two crucial conditions for elderly patients, found a high variability of rehabilitation patterns across a region, albeit coherent between the two pathologies, associated with remarkable differences in average expenditure. Differences in hazard rates for 6-month mortality after stroke at population level were also found. These results need to be confirmed and further investigated through a more robust information framework.
Practice size, caseload, deprivation and quality of care of patients with coronary heart disease, hypertension and stroke in primary care: national cross-sectional study
Authors: Saxena Sonia , Car Josip , Eldred Darren , Soljak Michael , Majeed Azeem
Published: June 2007

Background

Reports of higher quality care by higher-volume secondary care providers have fuelled a shift of services from smaller provider units to larger hospitals and units. In the United Kingdom, most patients are managed in primary care. Hence if larger practices provide better quality of care; this would have important implications for the future organization of primary care services. We examined the association between quality of primary care for cardiovascular disease achieved by general practices in England and Scotland by general practice caseload, practice size and area based deprivation measures, using data from the New General Practitioner (GP) Contract.

Methods

We analyzed data from 8,970 general practices with a total registered population of 55,522,778 patients in England and Scotland. We measured practice performance against 26 cardiovascular disease (coronary heart disease, left ventricular disease, and stroke) Quality and Outcomes Framework (QOF) indicators for patients on cardiovascular disease registers and linked this with data on practice characteristics and census data.

Results

Despite wide variations in practice list sizes and deprivation, the prevalence of was remarkably consistent, (coronary heart disease, left ventricular dysfunction, hypertension and cerebrovascular disease was 3.7%; 0.45%; 11.4% and 1.5% respectively). Achievement in quality of care for cardiovascular disease, as measured by QOF, was consistently high regardless of caseload or size with a few notable exceptions: practices with larger list sizes, higher cardiovascular disease caseloads and those in affluent areas had higher achievement of indicators requiring referral for further investigation. For example, small practices achieved lower scores 71.4% than large practices 88.6% (P < 0.0001) for referral for exercise testing and specialist assessment of patients with newly diagnosed angina.

Conclusion

The volume-outcome relationship found in hospital settings is not seen between practices in the UK in management of cardiovascular disorders in primary care. Further work is warranted to explain apparently poorer quality achievement in some aspects of cardiovascular management relating to initial diagnosis and management among practices in deprived areas, smaller practices and those with a smaller caseload.
In vitro activity of ferroquine (SSR 97193) against Plasmodium falciparum isolates from the Thai-Burmese border
Authors: Barends Marion , Jaidee Anchalee , Khaohirun Nopparat , Singhasivanon Pratap , Nosten François
Published: June 2007

Background

On the borders of Thailand, Plasmodium falciparum has become resistant to nearly all available drugs, and there is an urgent need to find new antimalarial drugs or drug combinations. Ferroquine (SSR97193) is a new 4-aminoquinoline antimalarial active against chloroquine resistant and sensitive P. falciparum strains in vivo and in vitro. This antimalarial organic iron complex (a ferrocenyl group has been associated with chloroquine) is meant to use the affinity of Plasmodium for iron to increase the probability for encountering the anti-malarial molecule.

The aim of the present study was to investigate the activity of ferroquine against P. falciparum isolates from an area with a known high multi-drug resistance rate.

Methods

Parasite isolates were obtained from patients with acute falciparum malaria attending the clinics of SMRU. In vitro cultures of these isolates were set-up in the SMRU-laboratory on pre-dosed drug plates, and grown in culture for 42 hours. Parasite growth was assessed by the double-site enzyme-linked pLDH immunodetection (DELI) assay.

Results

Sixty-five P. falciparum isolates were successfully grown in culture. The ferroquine mean IC50 (95% CI) was 9.3 nM (95% C.I.: 8.7 – 10.0). The mean IC50 value for the principal metabolite of ferroquin, SR97213A, was 37.0 nM (95% C.I.: 34.3 – 39.9), which is four times less active than ferroquine. The isolates in this study were highly multi-drug resistant but ferroquine was more active than chloroquine, quinine, mefloquine and piperaquine. Only artesunate was more active than ferroquine. Weak but significant correlations were found between ferroquine and its principal metabolite (r2 = 0.4288), chloroquine (r2 = 0.1107) and lumefantrine (r2 = 0.2364).

Conclusion

The results presented in this study demonstrate that the new ferroquine compound SSR97193 has high anti-malarial activity in vitro against multi-drug resistant P. falciparum.
A specific box switches the cell fate determining activity of XOTX2 and XOTX5b in the Xenopus retina
Authors: Onorati Marco , Cremisi Federico , Liu Yang , He Rong-Qiao , Barsacchi Giuseppina , Vignali Robert
Published: June 2007

Background

Otx genes, orthologues of the Drosophila orthodenticle gene (otd), play crucial roles in vertebrate brain development. In the Xenopus eye, Xotx2 and Xotx5b promote bipolar and photoreceptor cell fates, respectively. The molecular basis of their differential action is not completely understood, though the carboxyl termini of the two proteins seem to be crucial. To define the molecular domains that make the action of these proteins so different, and to determine whether their retinal abilities are shared by Drosophila OTD, we performed an in vivo molecular dissection of their activity by transfecting retinal progenitors with several wild-type, deletion and chimeric constructs of Xotx2, Xotx5b and otd.

Results

We identified a small 8–10 amino acid divergent region, directly downstream of the homeodomain, that is crucial for the respective activities of XOTX2 and XOTX5b. In lipofection experiments, the exchange of this 'specificity box' completely switches the retinal activity of XOTX5b into that of XOTX2 and vice versa. Moreover, the insertion of this box into Drosophila OTD, which has no effect on retinal cell fate, endows it with the specific activity of either XOTX protein. Significantly, in cell transfection experiments, the diverse ability of XOTX2 and XOTX5b to synergize with NRL, a cofactor essential for vertebrate rod development, to transactivate the rhodopsin promoter is also switched depending on the box. We also show by GST-pull down that XOTX2 and XOTX5b differentially interact with NRL, though this property is not strictly dependent on the box.

Conclusion

Our data provide molecular evidence on how closely related homeodomain gene products can differentiate their functions to regulate distinct cell fates. A small 'specificity box' is both necessary and sufficient to confer on XOTX2 and XOTX5b their distinct activities in the developing frog retina and to convert the neutral orthologous OTD protein of Drosophila into a positive and specific XOTX-like retinal regulator. Relatively little is known of what gives developmental specificity to homeodomain regulators. We propose that this box is a major domain of XOTX proteins that provides them with the appropriate developmental specificity in retinal histogenesis.
Comparative analysis of transposed element insertion within human and mouse genomes reveals Alu's unique role in shaping the human transcriptome
Authors: Sela Noa , Mersch Britta , Gal-Mark Nurit , Lev-Maor Galit , Hotz-Wagenblatt Agnes , Ast Gil
Published: June 2007

Background

Transposed elements (TEs) have a substantial impact on mammalian evolution and are involved in numerous genetic diseases. We compared the impact of TEs on the human transcriptome and the mouse transcriptome.

Results

We compiled a dataset of all TEs in the human and mouse genomes, identifying 3,932,058 and 3,122,416 TEs, respectively. We than extracted TEs located within human and mouse genes and, surprisingly, we found that 60% of TEs in both human and mouse are located in intronic sequences, even though introns comprise only 24% of the human genome. All TE families in both human and mouse can exonize. TE families that are shared between human and mouse exhibit the same percentage of TE exonization in the two species, but the exonization level of Alu, a primate-specific retroelement, is significantly greater than that of other TEs within the human genome, leading to a higher level of TE exonization in human than in mouse (1,824 exons compared with 506 exons, respectively). We detected a primate-specific mechanism for intron gain, in which Alu insertion into an exon creates a new intron located in the 3' untranslated region (termed 'intronization'). Finally, the insertion of TEs into the first and last exons of a gene is more frequent in human than in mouse, leading to longer exons in human.

Conclusion

Our findings reveal many effects of TEs on these two transcriptomes. These effects are substantially greater in human than in mouse, which is due to the presence of Alu elements in human.
Toll-like receptors 1 and 6 are involved in TLR2-mediated macrophage activation by hepatitis C virus core and NS3 proteins
Authors: Chang Serena , Dolganiuc Angela , Szabo Gyongyi
Published: June 2007

Hepatitis C virus (HCV) is a leading cause of end-stage liver disease through sustained inflammation of the liver produced by the host’s immune system. The mechanism for HCV evasion or activation of the immune system is not clear. TLRs are cellular activators of the innate immune system. We recently reported that TLR2-mediated innate immune signaling pathways are activated by HCV core and NS3 proteins. TLR2 activation requires homo- or heterodimerization with TLR1 or TLR6. Here, we aimed to determine whether TLR2 coreceptors participated in cellular activation by HCV core or NS3 proteins. By designing small interfering RNAs targeted to TLR2, TLR1, and TLR6, we showed that knockdown of each of these receptors impairs pro- and anti-inflammatory cytokine activation by TLR-specific ligands as well as by HCV core and NS3 proteins in human embryonic kidney-TLR2 cells and in primary human acrophages. We found that HCV core and NS3 proteins induced TNF-α and IL-10 production in human monocyte-derived macrophages, which was impaired by TLR2, TLR1, and TLR6 knockdown. Contrary to human data, results from TLR2, TLR1, or TLR6 knockout mice indicated that the absence of TLR2 and its coreceptor TLR6, but not TLR1, prevented the HCV core and NS3 protein-induced peritoneal macrophage activation. In conclusion, TLR2 may use TLR1 and TLR6 coreceptors for HCV core- and NS3-mediated activation of macrophages and innate immunity in humans. These results imply that multiple pattern recognition receptors could participate in cellular activation by HCV proteins.
Endothelial selectins regulate skin wound healing in cooperation with L-selectin and ICAM-1
Authors: Yukami Toru , Hasegawa Minoru , Matsushita Yukiyo , Fujita Tomoyuki , Matsushita Takashi , Horikawa Mayuka , Komura Kazuhiro , Yanaba Koichi , Hamaguchi Yasuhito , Nagaoka Tetsuya , Ogawa Fumihide , Fujimoto Manabu , Steeber Douglas A , Tedder Thomas F , Takehara Kazuhiko , Sato Shinichi
Published: June 2007

Skin wound healing is mediated by inflammatory cell infiltration that is highly regulated by various adhesion molecules. Mice lacking intercellular adhesion molecule-1 (ICAM-1) delayed skin wound healing and mice lacking both L-selectin and ICAM-1 (L-selectin/ICAM-1−/−) show more delayed wound healing. Deficiency of both endothelial selectins (E-selectin or P-selectin) also delays wound healing. However, the relative contribution and interaction of selectins and ICAM-1 to the wound healing remain unknown. To clarify them, repair of excisional wounds was examined in L-selectin/ICAM-1−/− mice, wild-type mice with both E- and P-selectin blockade, and L-selectin/ICAM-1−/− mice with both E- and P-selectin blockade. Wild-type mice with both E- and P-selectin blockade showed delayed wound healing that was comparable with that in L-selectin/ICAM-1−/− mice. Combined E- and P-selectin blockade in L-selectin/ICAM-1−/− mice resulted in more significant delay. Mice lacking or blocked for adhesion molecules also showed suppressed keratinocyte migration, angiogenesis, granulation tissue formation, leukocyte infiltration, and cytokine expression, including transforming growth factor-β and interleukin-6. Application of basic fibroblast growth factor (bFGF) but not platelet-derived growth factor to the wounds significantly improved wound healing in L-selectin/ICAM-1−/− mice with both E- and P-selectin blockade. bFGF significantly increased the leukocyte infiltration and subsequent fibrogenic cytokine production, as well as keratinocyte migration, angiogenesis, and collagen synthesis despite the loss of four kinds of adhesion molecules. These results indicate that skin wound healing is regulated cooperatively by all selectins and ICAM-1 and may provide critical information for the therapy of skin wounds.
Gene expression profiling during differentiation of human monocytes to macrophages or dendritic cells
Authors: Lehtonen Anne , Ahlfors Helena , Veckman Ville , Miettinen Minja , Lahesmaa Riitta , Julkunen Ilkka
Published: June 2007

Macrophages and dendritic cells (DC) are APC, which regulate innate and adaptive immune responses. Macrophages function locally mainly, maintaining inflammatory responses in tissues, whereas DC take up microbes, mature, and migrate to local lymph nodes to present microbial antigens to naïve T cells to elicit microbe-specific immune responses. Blood monocytes can be differentiated in vitro to macrophages or DC by GM-CSF or GM-CSF + IL-4, respectively. In the present study, we performed global gene expression analyses using Affymetrix HG-U133A Gene Chip oligonucleotide arrays during macrophage and DC differentiation. During the differentiation process, 340 and 350 genes were up-regulated, and 190 and 240 genes were down-regulated in macrophages and DC, respectively. There were also more that 200 genes, which were expressed differentially in fully differentiated macrophages and DC. Macrophage-specific genes include, e.g., CD14, CD163, C5R1, and FcγR1A, and several cell surface adhesion molecules, cytokine receptors, WNT5A and its receptor of the Frizzled family FZD2, fibronectin, and FcεR1A were identified as DC-specific. Our results reveal significant differences in gene expression profiles between macrophages and DC, and these differences can partially explain the functional differences between these two important cell types.
Expression, Structure, Function, and Evolution of Gonadotropin-Releasing Hormone (GnRH) Receptors GnRH-R1SHS and GnRH-R2PEY in the Teleost, Astatotilapia burtoni
Authors: Flanagan Colleen A. , Chen Chun-Chun , Coetsee Marla , Mamputha Sipho , Whitlock Kathleen E. , Bredenkamp Nicholas , Grosenick Logan , Fernald Russell D. , Illing Nicola
Published: June 2007

Multiple GnRH receptors are known to exist in nonmammalian species, but it is uncertain which receptor type regulates reproduction via the hypothalamic-pituitary-gonadal axis. The teleost fish, Astatotilapia burtoni, is useful for identifying the GnRH receptor responsible for reproduction, because only territorial males reproduce. We have cloned a second GnRH receptor in A. burtoni, GnRH-R1SHS (SHS is a peptide motif in extracellular loop 3), which is up-regulated in pituitaries of territorial males. We have shown that GnRH-R1SHS is expressed in many tissues and specifically colocalizes with LH in the pituitary. In A. burtoni brain, mRNA levels of both GnRH-R1SHS and a previously identified receptor, GnRH-R2PEY, are highly correlated with mRNA levels of all three GnRH ligands. Despite its likely role in reproduction, we found that GnRH-R1SHS has the highest affinity for GnRH2 in vitro and low responsivity to GnRH1. Our phylogenetic analysis shows that GnRH-R1SHS is less closely related to mammalian reproductive GnRH receptors than GnRH-R2PEY. We correlated vertebrate GnRH receptor amino acid sequences with receptor function and tissue distribution in many species and found that GnRH receptor sequences predict ligand responsiveness but not colocalization with pituitary gonadotropes. Based on sequence analysis, tissue localization, and physiological response we propose that the GnRH-R1SHS receptor controls reproduction in teleosts, including A. burtoni. We propose a GnRH receptor classification based on gene sequence that correlates with ligand selectivity but not with reproductive control. Our results suggest that different duplicated GnRH receptor genes have been selected to regulate reproduction in different vertebrate lineages.
Regulation of Hypothalamic Expression of KiSS-1 and GPR54 Genes by Metabolic Factors: Analyses Using Mouse Models and a Cell Line
Authors: Luque Raul M. , Kineman Rhonda D. , Tena-Sempere Manuel
Published: June 2007

It is well established that reproductive function is metabolically gated. However, the mechanisms whereby energy stores and metabolic cues influence fertility are yet to be completely deciphered. Recently, the hypothalamic KiSS-1/GPR54 system has emerged as a fundamental regulator of the gonadotropic axis, which conveys the modulatory actions of sex steroids to GnRH neurons. Evidence is also mounting that KiSS-1 neurons may also represent the link between systemic metabolic signals and central control of reproduction. To further explore this possibility, we examined the impact of changes in energy status and key metabolic regulators on the hypothalamic expression of KiSS-1 and GPR54 genes, using different mouse models and the hypothalamic cell line N6. Time-course analysis of the effects of short-term fasting revealed a rapid (12- and 24-h) decline in KiSS-1 and GPR54 mRNA levels, which preceded that of GnRH (48 h). In contrast, diet-induced obesity or obesity associated with leptin deficiency (ob/ob vs. wild-type mice) failed to induce overt changes in hypothalamic expression of KiSS-1 and GPR54 genes. However, leptin infusion of ob/ob mice evoked a significant increase in KiSS-1 and GPR54 mRNA levels compared with pair-fed controls. Moreover, leptin, but not insulin or IGF-I, stimulated KiSS-1 mRNA expression in the mouse hypothalamic cell line N6. In addition, neuropeptide Y (NPY) null mice showed decreased KiSS-1 mRNA levels at the hypothalamus, whereas exposure to NPY increased expression of KiSS-1 in hypothalamic N6 cells. In sum, our present data further characterize the functional relevance and putative key mediators (such as leptin and NPY) of the metabolic regulation of the hypothalamic KiSS-1 system in the mouse.
Daily Rhythm of Tryptophan Hydroxylase-2 Messenger Ribonucleic Acid within Raphe Neurons Is Induced by Corticoid Daily Surge and Modulated by Enhanced Locomotor Activity
Authors: Malek Zeina S. , Sage Dominique , Pévet Paul , Raison Sylvie
Published: June 2007

Tryptophan hydroxylase (TPH, the rate-limiting enzyme of serotonin synthesis) protein and mRNA levels display a circadian expression in the rat dorsal and median raphe. These patterns suggest a rhythmic synthesis of serotonin under the control of the master clock of suprachiasmatic nuclei. In the present study, we examined the involvement of endocrine and behavioral output signals of the master clock upon the Tph2 mRNA levels by quantitative in situ hybridization. In the absence of adrenals, a complete suppression of Tph2 mRNA rhythm was observed in dorsal and median raphe over 24 h. The restoration of corticosterone daily variations in adrenalectomized rats induced a Tph2 mRNA rhythmic pattern de novo, indicating that Tph2 mRNA rhythm is dependent upon daily fluctuations of glucocorticoids. Enhanced voluntary locomotor activity during 6 wk increased the level of Tph2 mRNA in both raphe nuclei of control rats without concomitant increase of corticosterone plasma levels. Moreover, this long-term enhanced locomotor activity was able to restore significant variation of Tph2 mRNA in adrenalectomized rats. In conclusion, both endocrine and behavioral cues can modulate Tph2 expression in dorsal and median raphe. The corticosterone surge acts as a rhythmic cue that induces the rhythmic expression of Tph2 in the raphe neurons. On the other hand, long-term exercise modulates the expression levels of this gene. Thus, the serotonin neurons are a target for both endocrine and behavioral circadian cues, and the serotoninergic input to the suprachiasmatic nuclei might feedback and influence the functioning of the clock itself.
Plasma Osteopontin Levels and Expression in Adipose Tissue Are Increased in Obesity
Authors: Gómez-Ambrosi Javier , Catalán Victoria , Ramírez Beatriz , Rodríguez Amaia , Colina Inmaculada , Silva Camilo , Rotellar Fernando , Mugueta Carmen , Gil María J. , Cienfuegos Javier A. , Salvador Javier , Frühbeck Gema
Published: June 2007

Context: Obesity acts as a cardiovascular risk factor by mechanisms that are not fully understood. Osteopontin (OPN) is a proinflammatory mediator involved in tissue remodeling that plays a role in atherosclerosis and diabetes.

Objective: The aim of the present study was to compare the circulating concentrations of OPN and its mRNA expression in omental adipose tissue of lean, overweight, and obese individuals and to analyze the effect of weight loss.

Subjects and Methods: Plasma concentrations of OPN were measured in 77 volunteers. OPN mRNA expression in omental adipose tissue obtained from 12 women was quantified by real-time PCR. In addition, the concentrations of OPN in 12 obese men were measured before and after weight loss following a dietetic program.

Setting: The study was conducted at a University Hospital.

Results: Obese and overweight patients exhibited significantly increased circulating OPN concentrations as compared with lean subjects (obese 72.6 ± 28.5, overweight 68.2 ± 20.8, lean 42.7 ± 27.9 ng/ml; P < 0.001). A significant positive correlation was found between OPN levels and body fat (r = 0.45; P < 0.0001). Obese individuals showed significantly increased (P < 0.05) mRNA expression of OPN in omental adipose tissue as compared with lean volunteers, which was further increased in obese diabetic patients. Diet-induced weight loss significantly decreased OPN concentrations from 64.7 ± 22.1 to 36.6 ± 20.1 ng/ml (P = 0.006).

Conclusions: These findings represent the first observation that plasma OPN and mRNA expression of OPN in omental adipose tissue are increased in overweight/obese patients with the latter being further elevated in obesity-associated diabetes. Moreover, weight loss reduces OPN concentrations, which may contribute to the beneficial effects accompanying weight reduction. Measurement of OPN might be useful for evaluating the outcomes of various clinical interventions for obesity-related cardiovascular diseases.
Age-Related Changes in Serum Testosterone and Sex Hormone Binding Globulin in Australian Men: Longitudinal Analyses of Two Geographically Separate Regional Cohorts
Authors: Liu Peter Y. , Beilin Jonathan , Meier Christian , Nguyen Tuan V. , Center Jacqueline R. , Leedman Peter J. , Seibel Markus J. , Eisman John A. , Handelsman David J.
Published: June 2007

Background: Cross-sectional studies from different populations show a variable decline in blood testosterone concentrations as men age. Few population representative cohorts have been followed up over time.

Objective: The objective of the study was to quantify longitudinally the change in serum testosterone and SHBG concentrations with age in two well-defined, representative but geographically widely separated regional Australian cohorts.

Subjects and Setting: The Busselton cohort comprises individuals aged 18–90 yr residing in Western Australia assessed prospectively since 1981. Sera were assayed from 910 men, from whom further samples were available 14 yr later in 480. The Dubbo cohort involves individuals aged 61–90 yr living in Eastern Australia. Baseline sera were collected from 610 men and additional sera on a second (n = 370) and third (n = 200) occasion from 1989 to 2004. Men from both cohorts are community dwelling and of predominately European origin.

Results: Longitudinal analyses show the following: 1) total testosterone declines comparably (P > 0.9) by 1.3% (Busselton) and 0.9% (Dubbo) per annum with the same rates of decline when analyses were restricted to men older than 60 yr of age; 2) annual changes in SHBG were also very similar in age-restricted analyses (2.3% vs. 2.5%, P = 0.48); and 3) the annual increase in SHBG was steeper in middle-aged and older men (P < 10−3vs. young men). These longitudinal changes were all up to 4-fold greater in magnitude, compared with cross-sectional analyses of baseline data.

Conclusion: In two separate regional Australian populations, blood testosterone fell and SHBG increased comparably with age. Age-related changes in blood testosterone and SHBG previously described in urban-dwelling men are the same in men who reside in smaller regional cities of another continent.
Insulin-Like Growth Factor Binding Protein-5 Interacts with the Vitamin D Receptor and Modulates the Vitamin D Response in Osteoblasts
Authors: Schedlich Lynette J. , Muthukaruppan Anita , O’Han Michelle K. , Baxter Robert C.
Published: June 2007

The 1,25 dihydroxyvitamin D3 [1,25(OH)2D3]-induced differentiation of osteoblasts comprises the sequential induction of cell cycle arrest at G0/G1 and the expression of bone matrix proteins. Reports differ on the effects of IGF binding protein (IGFBP)-5 on bone cell growth and osteoblastic function. IGFBP-5 can be growth stimulatory or inhibitory and can enhance or impair osteoblast function. In previous studies, we have shown that IGFBP-5 localizes to the nucleus and interacts with the retinoid receptors. We now show that IGFBP-5 interacts with nuclear vitamin D receptor (VDR) and blocks retinoid X receptor (RXR):VDR heterodimerization. VDR and IGFBP-5 were shown to colocalize to the nuclei of MG-63 and U2-OS cells and coimmunoprecipitate in nuclear extracts from these cells. Induction of osteocalcin promoter activity and alkaline phosphatase activity by 1,25(OH)2D3 were significantly enhanced when IGFBP-5 was down-regulated in U2-OS cells. Moreover, we found IGFBP-5 increased basal alkaline phosphatase activity and collagen α1 type 1 expression, and that 1,25(OH)2D3 was unable to further induce the expression of these bone differentiation markers in MG-63 cells. Expression of IGFBP-5 inhibited MG-63 cell growth and caused cell cycle arrest at G0/G1 and G2/M. Furthermore, IGFBP-5 reduced the effects of 1,25(OH)2D3 in blocking cell cycle progression at G0/G1 and decreased the expression of cyclin D1. These results demonstrate that IGFBP-5 can interact with VDR to prevent RXR:VDR heterodimerization and suggest that IGFBP-5 may attenuate the 1,25(OH)2D3-induced expression of bone differentiation markers while having a modest effect on the 1,25(OH)2D3-mediated inhibition of cell cycle progression in bone cells.
Glucocorticoid-Induced Degradation of Glycogen Synthase Kinase-3 Protein Is Triggered by Serum- and Glucocorticoid-Induced Protein Kinase and Akt Signaling and Controls β-Catenin Dynamics and Tight Junction Formation in Mammary Epithelial Tumor Cells
Authors: Failor Kim L. , Desyatnikov Yelena , Finger Lindsay A. , Firestone Gary L.
Published: June 2007

Glucocorticoid hormones stimulate adherens junction and tight junction formation in Con8 mammary epithelial tumor cells and induce the production of a stable nonphosphorylated β-catenin protein localized exclusively to the cell periphery. Glycogen synthase kinase-3 (GSK3) phosphorylation of β-catenin is known to trigger the degradation of this adherens junction protein, suggesting that steroid-activated cascades may be targeting this protein kinase. We now demonstrate that treatment with the synthetic glucocorticoid dexamethasone induces the ubiquitin-26S proteasome-mediated degradation of GSK3 protein with no change in GSK3 transcript levels. In transfected cells, deletion of the N-terminal nine amino acids or mutation of the serine-9 phosphorylation site on GSK3-β prevented its glucocorticoid-induced degradation. Expression of stabilized GSK3 mutant proteins ablated the glucocorticoid-induced tight junction sealing and resulted in production of a nonphosphorylated β-catenin that localizes to both the nucleus and the cell periphery in steroid-treated cells. Serine-9 on GSK3 can be phosphorylated by Sgk (serum- and glucocorticoid-induced protein kinase) and by Akt. Expression of dominant-negative forms of either Sgk- or Akt-inhibited glucocorticoid induced GSK3 ubiquitination and degradation and disrupted the dexamethasone-induced effects on β-catenin dynamics. Furthermore, the steroid-induced tight junction sealing is attenuated in cells expressing dominant-negative forms of either Sgk or Akt, although the effect of blunting Sgk signaling was significantly greater. Taken together, we have uncovered a new cellular cascade in which Sgk and Akt trigger the glucocorticoid-regulated phosphorylation, ubiquitination, and degradation of GSK3, which alters β-catenin dynamics, leading to the formation of adherens junctions and tight junction sealing.