bluuebunny/crossref_metadata_2025 huggingface dataset:
Cord blood comprises antigen-experienced T cells specific for maternal minor histocompatibility antigen HA-1
Authors: Mommaas Bregje , Stegehuis-Kamp Janine A. , van Halteren Astrid G. , Kester Michel , Enczmann Jürgen , Wernet Peter , Kögler Gesine , Mutis Tuna , Brand Anneke , Goulmy Els
Published: October 2004

Umbilical cord blood transplantation is applied as treatment for mainly pediatric patients with hematologic malignancies. The clinical results show a relatively low incidence of graft-versus-host disease and leukemia relapse. Since maternal cells traffic into the fetus during pregnancy, we questioned whether cord blood has the potential to generate cytotoxic T cells specific for the hematopoietic minor histocompatibility (H) antigen HA-1 that would support the graft-versus-leukemia effect. Here, we demonstrate the feasibility of ex vivo generation of minor H antigen HA-1-specific T cells from cord blood cells. Moreover, we observed pre-existing HA-1-specific T cells in cord blood samples. Both the circulating and the ex vivo-generated HA-1-specific T cells show specific and hematopoietic restricted lysis of human leukocyte antigen-A2pos/HA-1pos (HLA-A2pos/HA-1pos) target cells, including leukemic cells. The cord blood-derived HA-1-specific cytotoxic T cells are from child origin. Thus, the so-called naive cord blood can comprise cytotoxic T cells directed at the maternal minor H antigen HA-1. The apparent immunization status of cord blood may well contribute to the in vivo graft-versus-leukemia activity after transplantation. Moreover, since the fetus cannot be primed against Y chromosome-encoded minor H antigens, cord blood is an attractive stem cell source for male patients. (Blood. 2005;105:1823-1827)
PCOGR: Phylogenetic COG ranking as an online tool to judge the specificity of COGs with respect to freely definable groups of organisms
Authors: Meereis Florian , Kaufmann Michael
Published: October 2004

Background

The rapidly increasing number of completely sequenced genomes led to the establishment of the COG-database which, based on sequence homologies, assigns similar proteins from different organisms to clusters of orthologous groups (COGs). There are several bioinformatic studies that made use of this database to determine (hyper)thermophile-specific proteins by searching for COGs containing (almost) exclusively proteins from (hyper)thermophilic genomes. However, public software to perform individually definable group-specific searches is not available.

Results

The tool described here exactly fills this gap. The software is accessible at http://www.uni-wh.de/pcogr and is linked to the COG-database. The user can freely define two groups of organisms by selecting for each of the (current) 66 organisms to belong either to groupA, to the reference groupB or to be ignored by the algorithm. Then, for all COGs a specificity index is calculated with respect to the specificity to groupA, i. e. high scoring COGs contain proteins from the most of groupA organisms while proteins from the most organisms assigned to groupB are absent. In addition to ranking all COGs according to the user defined specificity criteria, a graphical visualization shows the distribution of all COGs by displaying their abundance as a function of their specificity indexes.

Conclusions

This software allows detecting COGs specific to a predefined group of organisms. All COGs are ranked in the order of their specificity and a graphical visualization allows recognizing (i) the presence and abundance of such COGs and (ii) the phylogenetic relationship between groupA- and groupB-organisms. The software also allows detecting putative protein-protein interactions, novel enzymes involved in only partially known biochemical pathways, and alternate enzymes originated by convergent evolution.
Potentially inappropriate prescriptions for older patients in long-term care
Authors: Rancourt Carol , Moisan Jocelyne , Baillargeon Lucie , Verreault René , Laurin Danielle , Grégoire Jean-Pierre
Published: October 2004

Background

Inappropriate medication use is a major healthcare issue for the elderly population. This study explored the prevalence of potentially inappropriate prescriptions (PIPs) in long-term care in metropolitan Quebec.

Methods

A cross sectional chart review of 2,633 long-term care older patients of the Quebec City area was performed. An explicit criteria list for PIPs was developed based on the literature and validated by a modified Delphi method. Medication orders were reviewed to describe prescribing patterns and to determine the prevalence of PIPs. A multivariate analysis was performed to identify predictors of PIPs.

Results

Almost all residents (94.0%) were receiving one or more prescribed medication; on average patients had 4.8 prescribed medications. A majority (54.7%) of treated patients had a potentially inappropriate prescription (PIP). Most common PIPs were drug interactions (33.9% of treated patients), followed by potentially inappropriate duration (23.6%), potentially inappropriate medication (14.7%) and potentially inappropriate dosage (9.6%). PIPs were most frequent for medications of the central nervous system (10.8% of prescribed medication). The likelihood of PIP increased significantly as the number of drugs prescribed increased (odds ratio [OR]: 1.38, 95% confidence interval [CI]: 1.33 – 1.43) and with the length of stay (OR: 1.78, CI: 1.43 – 2.20). On the other hand, the risk of receiving a PIP decreased with age.

Conclusion

Potentially inappropriate prescribing is a serious problem in the highly medicated long-term care population in metropolitan Quebec. Use of explicit criteria lists may help identify the most critical issues and prioritize interventions to improve quality of care and patient safety.
BIOKID: Randomized controlled trial comparing bicarbonate and lactate buffer in biocompatible peritoneal dialysis solutions in children [ISRCTN81137991]
Authors: Nau Barbara , Schmitt Claus P , Almeida Margarida , Arbeiter Klaus , Ardissino Gianluigi , Bonzel Klaus E , Edefonti Alberto , Fischbach Michel , Haluany Karin , Misselwitz Joachim , Kemper Markus J , Rönnholm Kai , Wygoda Simone , Schaefer Franz ,   European Pediatric Peritoneal Dialysis Study Group (EPPS)
Published: October 2004

Background

Peritoneal dialysis (PD) is the preferred dialysis modality in children. Its major drawback is the limited technique survival due to infections and progressive ultrafiltration failure. Conventional PD solutions exert marked acute and chronic toxicity to local tissues. Prolonged exposure is associated with severe histopathological alterations including vasculopathy, neoangiogenesis, submesothelial fibrosis and a gradual loss of the mesothelial cell layer. Recently, more biocompatible PD solutions containing reduced amounts of toxic glucose degradation products (GDPs) and buffered at neutral pH have been introduced into clinical practice. These solutions contain lactate, bicarbonate or a combination of both as buffer substance. Increasing evidence from clinical trials in adults and children suggests that the new PD fluids may allow for better long-term preservation of peritoneal morphology and function. However, the relative importance of the buffer in neutral-pH, low-GDP fluids is still unclear. In vitro, lactate is cytotoxic and vasoactive at the concentrations used in PD fluids. The BIOKID trial is designed to clarify the clinical significance of the buffer choice in biocompatible PD fluids.

Methods/design

The objective of the study is to test the hypothesis that bicarbonate based PD solutions may allow for a better preservation of peritoneal transport characteristics in children than solutions containing lactate buffer. Secondary objectives are to assess any impact of the buffer system on acid-base status, peritoneal tissue integrity and the incidence and severity of peritonitis.

After a run-in period of 2 months during which a targeted cohort of 60 patients is treated with a conventional, lactate buffered, acidic, GDP containing PD fluid, patients will be stratified according to residual renal function and type of phosphate binding medication and randomized to receive either the lactate-containing Balance solution or the bicarbonate-buffered Bicavera® solution for a period of 10 months. Patients will be monitored by monthly physical and laboratory examinations. Peritoneal equilibration tests, 24-h dialysate and urine collections will be performed 4 times. Peritoneal biopsies will be obtained on occasion of intraabdominal surgery. Changes in small solute transport rates, markers of peritoneal tissue turnover in the effluent, acid-base status and peritonitis rates and severity will be analyzed.
Single photon emission computed tomography (SPECT) of anxiety disorders before and after treatment with citalopram
Authors: Carey Paul D , Warwick James , Niehaus Dana JH , van der Linden Geoffrey , van Heerden Barend B , Harvey Brian H , Seedat Soraya , Stein Dan J
Published: October 2004

Background

Several studies have now examined the effects of selective serotonin reuptake inhibitor (SSRI) treatment on brain function in a variety of anxiety disorders including obsessive-compulsive disorder (OCD), posttraumatic stress disorder (PTSD), and social anxiety disorder (social phobia) (SAD). Regional changes in cerebral perfusion following SSRI treatment have been shown for all three disorders. The orbitofrontal cortex (OFC) (OCD), caudate (OCD), medial pre-frontal/cingulate (OCD, SAD, PTSD), temporal (OCD, SAD, PTSD) and, thalamic regions (OCD, SAD) are some of those implicated. Some data also suggests that higher perfusion pre-treatment in the anterior cingulate (PTSD), OFC, caudate (OCD) and antero-lateral temporal region (SAD) predicts subsequent treatment response. This paper further examines the notion of overlap in the neurocircuitry of treatment and indeed treatment response across anxiety disorders with SSRI treatment.

Methods

Single photon emission computed tomography (SPECT) using Tc-99 m HMPAO to assess brain perfusion was performed on subjects with OCD, PTSD, and SAD before and after 8 weeks (SAD) and 12 weeks (OCD and PTSD) treatment with the SSRI citalopram. Statistical parametric mapping (SPM) was used to compare scans (pre- vs post-medication, and responders vs non-responders) in the combined group of subjects.

Results

Citalopram treatment resulted in significant deactivation (p = 0.001) for the entire group in the superior (t = 4.78) and anterior (t = 4.04) cingulate, right thalamus (t = 4.66) and left hippocampus (t = 3.96). Deactivation (p = 0.001) within the left precentral (t = 4.26), right mid-frontal (t = 4.03), right inferior frontal (t = 3.99), left prefrontal (3.81) and right precuneus (t= 3.85) was more marked in treatment responders. No pattern of baseline activation distinguished responders from non-responders to subsequent pharmacotherapy.

Conclusions

Although each of the anxiety disorders may be mediated by different neurocircuits, there is some overlap in the functional neuro-anatomy of their response to SSRI treatment. The current data are consistent with previous work demonstrating the importance of limbic circuits in this spectrum of disorders. These play a crucial role in cognitive-affective processing, are innervated by serotonergic neurons, and changes in their activity during serotonergic pharmacotherapy seem crucial.
Pimecrolimus 1% cream for anogenital lichen sclerosus in childhood
Authors: Boms Stefanie , Gambichler Thilo , Freitag Marcus , Altmeyer Peter , Kreuter Alexander
Published: October 2004

Background

Lichen sclerosus is a chronic inflammatory disease with a predilection of the anogenital region. Because of the potential side effects of repeated local application of potent glucocorticosteroids, equally-effective, safer therapeutic options are required, especially in the treatment of children.

Case presentations

We report on the efficacy of twice-daily application of pimecrolimus 1% cream in four prepubertal girls (range of age: 4 to 9 years) who suffered from anogenital lichen sclerosus. After three to four-month treatment, all patients had almost complete clinical remission including relief from itch, pain and inflammation. Only minor improvement was observed for the white sclerotic lesions. No significant side effects have been observed.

Conclusions

Topical pimecrolimus appears to be an effective and safe treatment for children with anogenital lichen sclerosus. The clinical benefits observed in the four patient presented particularly include relief of pruritus, pain and inflammation. Vehicle-controlled studies on a larger number of patients are now warranted to substantiate our promising findings, and to investigate long-term efficacy and safety of topical pimecrolimus in anogenital lichen sclerosus.
Enhancing the African bioethics initiative
Authors: Ogundiran Temidayo O
Published: October 2004

Background

Medical ethics has existed since the time of Hippocrates. However, formal training in bioethics did not become established until a few decades ago. Bioethics has gained a strong foothold in health sciences in the developed world, especially in Europe and North America. The situation is quite different in many developing countries. In most African countries, bioethics – as established and practiced today in the west- is either non-existent or is rudimentary.

Discussion

Though bioethics has come of age in the developed and some developing countries, it is still largely "foreign" to most African countries. In some parts of Africa, some bioethics conferences have been held in the past decade to create research ethics awareness and ensure conformity to international guidelines for research with human participants. This idea has arisen in recognition of the genuine need to develop capacity for reviewing the ethics of research in Africa. It is also a condition required by external sponsors of collaborative research in Africa. The awareness and interest that these conferences have aroused need to be further strengthened and extended beyond research ethics to clinical practice. By and large, bioethics education in schools that train doctors and other health care providers is the hook that anchors both research ethics and clinical ethics.

This communication reviews the current situation of bioethics in Africa as it applies to research ethics workshops and proposes that in spite of the present efforts to integrate ethics into biomedical research in Africa, much still needs to be done to accomplish this. A more comprehensive approach to bioethics with an all-inclusive benefit is to incorporate formal ethics education into health training institutions in Africa.
Effects of recombinant adenovirus-mediated expression of IL-2 and IL-12 in human B lymphoma cells on co-cultured PBMC
Authors: Ebert Oliver , Wilbert Dorothee , Buttgereit Peter , Ziske Carsten , Flieger Dimitri , Schmidt-Wolf Ingo GH
Published: October 2004

Background

Modulation of the immune system by genetically modified lymphoma cell vaccines is of potential therapeutic value in the treatment of B cell lymphoma. However, the anti-tumor effect of any single immunogene transfer has so far been limited. Combination treatment of recombinant IL-2 and IL-12 has been reported to be synergistic for inducing anti-tumor responses in solid tumors but the potential of IL-2/IL-12 gene modified B cell lymphoma cells has not been explored yet.

Methods

Using three different human B cell lymphoma cell lines and primary samples from patients with B cell neoplasms, expression levels of the coxsackie B-adenovirus receptor (CAR) and alpha (v) integrins were analyzed by fluorescence-activated cell sorter (FACS). Adenoviral transduction efficiencies were determined by GFP expression analysis and IL-2 and IL-12 cytokine production was quantified by enzyme-linked immunosorbent (ELISA) assays. Proliferative activities of peripheral blood mononuclear cells (PBMC) stimulated with either cytokine derived from supernatants of transduced lymphoma cells were measured by cell proliferation (MTT) assays. An EuTDA cytotoxicity assay was used to compare cytotoxic activities of IL-2 and/or IL-12 stimulated PBMC against unmodified lymphoma cells.

Results

We found that B cell lymphoma cell lines could be transduced with much higher efficiency than primary tumor samples, which appeared to correlate with the expression of CAR. Adenoviral-expressed IL-2 and IL-12 similarly led to dose-dependent increases in proliferation rates of PBMC obtained from healthy donors. IL-2 and/or IL-12 transduced lymphoma cells were co-cultured with PBMC, which were assayed for their cytolytic activity against unmodified lymphoma cells. We found that IL-2 stimulated PBMC elicited a significant anti-tumor effect but not the combined effect of IL-2/IL-12 or IL-12 alone.

Conclusion

This study demonstrates that the generation of recombinant adenovirus modified lymphoma cell vaccines based on lymphoma cell lines expressing IL-2 and IL-12 cytokine genes is technically feasible, induces increases in proliferation rates and cytotoxic activity of co-cultured PBMC, and warrants further development for the treatment of lymphoma patients in the future.
The effect of chemotherapy combined with recombination mutant human tumor necrosis factor on advanced cancer
Authors: Liu Xing , Zhang Xiang-fu , Zheng Zhi-weng , Lu Huishan , Wu Xinyuan , Huang Changmin , Wang Chuan , Guang Guoxiang
Published: October 2004

Background

Past studies suggested that tumor necrosis factor (TNF) assisted anti-tumor treatment and intensified the sensitivity of chemotherapy. However its clinical application has been curbed because of its low purity, high dosage, and strong toxicity. This research, through perspective random clinical control experiment, observed the therapeutic effect of the treatment of late malignant tumor through the injection of recombinant mutant human tumor necrosis factor (rmhTNF) combined with general chemotherapy and its adverse reactions.

Methods

105 patients with advanced malignant tumor were randomly divided into trial group, 69 patients, and control group, 36 patients. Injection of rmhTNF 4 × 106u/m2 was given to the trial group, from the 1st to 7th days, the 11th to 17th days combined with chemotherapy course. The chemotherapy plan was as follows: CAP for patients with the NSCLC; FAM for patients with gastric cancer; FC for patients with colorectal cancer. One treatment cycle lasted for 21 days and two cycles were scheduled. The control group was given only the same chemotherapy as the trial group.

Results

In the trial group there was 1 CR case and 12 PR cases, and the response rate is 13/69 (18.84%); in the control group 1 PR case, the response rate 1/36 (2.78%). The response rate of the trial group was significantly higher than that of the control group (P = 0.022). The response rate for NSCLC in the trial group was 8/17 (47.06%), and 1/6 (16.67%) in the control group. The response rates for gastric cancer and colorectal cancer in the trial groups also were higher than those of the control groups. After the treatment the KPS is 89.00 ± 9.92 in the trial group, and 84.17 ± 8.84 in the control group, with a significant difference between the two groups (P = 0.028). The adverse reactions of rmhTNF injection included: pain in the injection area, chill, hardening and swelling and redness in the injection area, fever, ostealgia and myosalgia, and cold-like symptoms. All these adverse reactions were mild and bearable.

Conclusions

The administration of rmhTNF injection in combination with general chemotherapy is an effective and secure means in treating advanced malignant tumor.
The J-shape association of ethanol intake with total homocysteine concentrations: the ATTICA study
Authors: Pitsavos Christos , Panagiotakos Demosthenes B , Kontogianni Meropi D , Chrysohoou Christina , Chloptsios Yannis , Zampelas Antonis , Trichopoulou Antonia , Stefanadis Christodoulos
Published: October 2004

Background

Epidemiological studies suggest a non-monotonic effect of alcohol consumption on cardiovascular risk, while there is strong evidence concerning the involvement of homocysteine levels on thrombosis. The aim of this work was to evaluate the association between usual ethanol consumption and homocysteine levels, in cardiovascular disease free adults.

Methods

From May 2001 to December 2002 we randomly enrolled 1514 adult men and 1528 women, without any evidence of cardiovascular disease, stratified by age – gender (census 2001), from the greater area of Athens, Greece. Among the variables ascertained we measured the daily ethanol consumption and plasma homocysteine concentrations.

Results

Data analysis revealed a J-shape association between ethanol intake (none, <12 gr, 12 – 24 gr, 25 – 48 gr, >48 gr per day) and total homocysteine levels (mean ± standard deviation) among males (13 ± 3 vs. 11 ± 3 vs. 14 ± 4 vs. 18 ± 5 vs. 19 ± 3 μmol/L, respectively, p < 0.01) and females (10 ± 4 vs. 9 ± 3 vs. 11 ± 3 vs. 15 ± 4 vs. 17 ± 3 μmol/L, respectively, p < 0.01), after controlling for several potential confounders. The lowest homocysteine concentrations were observed with ethanol intake of < 12 gr/day (Bonferroni α* < 0.05). No differences were observed when we stratified our analysis by type of alcoholic beverage consumed.

Conclusion

We observed a J-shape relationship between homocysteine concentrations and the amount of ethanol usually consumed.
Expression of the largest CD97 and EMR2 isoforms on leukocytes facilitates a specific interaction with chondroitin sulfate on B cells
Authors: Kwakkenbos Mark J , Pouwels Walter , Matmati Mourad , Stacey Martin , Lin Hsi-Hsien , Gordon Siamon , Van Lier René A W , Hamann Jörg
Published: October 2004

The EGF-TM7 receptors CD97 and EMR2 are heptahelical molecules predominantly expressed on leukocytes. A characteristic of these receptors is their ability to interact with cellular ligands via the N-terminal epidermal growth factor (EGF)-like domains. The first two EGF domains of CD97 (but not EMR2) bind CD55 (decay-accelerating factor), while the fourth EGF domain of both CD97 and EMR2 interacts with the glycosaminoglycan chondroitin sulfate (CS). Using fluorescent beads coated with soluble recombinant CD97 and EMR2 protein, and isoform-specific monoclonal antibodies, we have determined the cellular and molecular characteristics of the interaction with CS. The fourth EGF domain of CD97 and EMR2 is expressed on activated lymphocytes and myeloid cells, whereas the ligand is specifically found on B cells within the peripheral blood. The interaction between CD97/EMR2 and CS may therefore play a role in the interaction of activated T cells, dendritic cells, and macrophages with B cells.
Dramatic Decrease of Innervation Density in Bone after Ovariectomy
Authors: Burt-Pichat B. , Lafage-Proust M. H. , Duboeuf F. , Laroche N. , Itzstein C. , Vico L. , Delmas P. D. , Chenu C.
Published: October 2004

Recent studies have demonstrated that bone is highly innervated and contains neuromediators that have functional receptors on bone cells. However, no data exist concerning the quantitative changes of innervation during bone loss associated with estrogen withdrawal. To study the involvement of nerve fibers in the regulation of bone remodeling, we have evaluated the modifications of innervation in a classical in vivo model of osteopenia in rats, ovariectomy (OVX). Skeletal innervation was studied by immunocytochemistry using antibodies directed against specific neuronal markers, neurofilament 200 and synaptophysin, and the neuromediator glutamate. Sciatic neurectomy, another model of bone loss due to limb denervation and paralysis, was used to validate our quantitative image analysis technique of immunostaining for nerve markers. Female Wistar rats at 12 wk of age were sham-operated (SHAM) or ovariectomized (OVX). Bone mineral density measurement and bone histomorphometry analysis of tibiae 14 d after surgery demonstrated a significant bone loss in OVX compared with SHAM. We observed an important reduction of nerve profile density in tibiae of OVX animals compared with SHAM animals, whereas innervation density in skin and muscles was similar for OVX and control rats. Quantitative image analysis of immunostainings demonstrated a significant decrease of the percentage of immunolabeling per total bone volume of neurofilament 200, synaptophysin, and glutamate in both the primary and secondary spongiosa of OVX rats compared with SHAM. These data indicate for the first time that OVX-induced bone loss in rat tibiae is associated with a reduction in nerve profile density, suggesting a functional link between the nervous system and the bone loss after ovariectomy.
Structural Determinants for G Protein Activation and Selectivity in the Second Intracellular Loop of the Thyrotropin Receptor
Authors: Neumann Susanne , Krause Gerd , Claus Maren , Paschke Ralf
Published: October 2004

The TSH receptor (TSHR) activates mainly two signal transduction pathways, cAMP production and phosphoinositide turnover, mediated by Gs and Gq coupling, respectively. Several activating deletion and point mutations within intracellular loop 3 (ICL3) and the adjacent portion of transmembrane domain 6 (TM6) support a direct G protein activation by this receptor domain. The ICL3, however, is predicted by modeling to interact with other receptor domains, primarily ICL2, to form a pocket for G protein binding and to allow optimum interaction. Systematic mutagenesis was used to identify important sites within ICL2 and potential interactions between ICL2 and ICL3 of the TSHR required for G protein coupling. Deletions of four or five residues and their corresponding multiple alanine substitutions were introduced into ICL2. Residues I523-D530, comprising mainly the N-terminal half of ICL2, appeared to be critical for Gs- and Gq-mediated signaling. A single alanine substitution screening within ICL2 revealed hydrophobic residue M527 in particular and, to lesser extents, F525, R528, L529, and D530 as residues that selectively abolished or strongly impaired Gq activation. Molecular modeling suggests that F525 interacts with ICL3. To test this hypothesis, ICL2/ICL3 double mutants introducing strong complementary properties were constructed and tested for functional rescue of Gq-mediated signaling. Our results indicate that ICL2 interacts with ICL3 in close vicinity to F525 and T607, suggesting a conformational cooperation between ICL2 and ICL3 during Gq activation by TSHR.
Estrogen Acidifies Vaginal pH by Up-Regulation of Proton Secretion via the Apical Membrane of Vaginal-Ectocervical Epithelial Cells
Authors: Gorodeski George I. , Hopfer Ulrich , Liu Chung Chiun , Margles Ellen
Published: October 2004

The objective of this study was to assess estrogen-dependent cellular mechanisms that could contribute to the acid pH of the vaginal lumen. Cultures of normal human cervical-vaginal epithelial (hECE) cells and endocervical cells were grown on filters, and acidification of the extracellular solutions on the luminal (L-pHo) and contraluminal (CL-pHo) sides was measured. The hECE cells and endocervical cells decreased CL-pHo from 7.40 to 7.25 within 20–30 min of incubation in basic salt solution. Endocervical cells also produced a similar decrease in L-pHo. In contrast, hECE cells acidified L-pHo down to pH 7.05 when grown as monoculture and down to pH 6.05 when grown in coculture with human cervical fibroblasts. This enhanced acid secretion into the luminal compartment was estrogen dependent because removal of endogenous steroid hormones attenuated the effect, whereas treatment with 17β-estradiol restored it. The 17β-estradiol effect was dose dependent (EC50 0.5 nm) and could be mimicked by diethylstilbestrol and in part by estrone and tamoxifen. Preincubation with ICI-182780, but not with progesterone, blocked the estrogen effect. Preincubation of cells with the V-ATPase blocker bafilomycin A1, when administered to the luminal solution, attenuated the baseline and estrogen-dependent acid secretion into the luminal solution. Treatment with EGTA, to abrogate the tight junctional resistance, blocked the decrease in L-pHo and stimulated a decrease in CL-pHo, indicating that the tight junctions are necessary for maintaining luminal acidification. We conclude that vaginal-ectocervical cells acidify the luminal canal by a mechanism of active proton secretion, driven in part by V-H+-ATPase located in the apical plasma membrane and that the baseline active net proton secretion occurs constitutively throughout life and that this acidification is up-regulated by estrogen.
c-Src Regulates Clathrin Adapter Protein 2 Interaction with β-Arrestin and the Angiotensin II Type 1 Receptor during Clathrin- Mediated Internalization
Authors: Fessart Delphine , Simaan May , Laporte Stéphane A.
Published: October 2004

β-Arrestins are multifunctional adapters involved in the internalization and signaling of G protein-coupled receptors (GPCRs). They target receptors to clathrin-coated pits (CCPs) through binding with clathrin and clathrin adapter 2 (AP-2) complex. They also act as transducers of signaling by recruiting c-Src kinase to certain GPCRs. Here we sought to determine whether c-Src regulates the recruitment of AP-2 to β-arrestin and the angiotensin II (Ang II) type 1 receptor (AT1R) during internalization. We show that the agonist stimulation of native AT1R in vascular smooth muscle cells (VSMCs) induces the formation of an endogenous complex containing c-Src, β-arrestins and AP-2. In vitro studies using coimmunoprecipitation experiments and a yeast three-hybrid assay reveal that c-Src stabilizes the agonist-independent association between β-arrestin2 and the β-subunit of AP-2 independently of the kinase activity of c-Src. However, although c-Src expression promoted the rapid dissociation of AP-2 from both β-arrestin and AT1R after receptor stimulation, a kinase-inactive mutant of c-Src failed to induce the dissociation of AP-2 from the agonist-occupied receptor. Thus, the consequence of c-Src in regulating the dissociation of AP-2 from the receptor was also examined on the internalization of AT1R by depleting c-Src in human embryonic kidney (HEK) 293 cells using a small interfering RNA strategy. Experiments in c-Src depleted cells reveal that AT1R remained mostly colocalized with AP-2 at the plasma membrane after Ang II stimulation, consistent with the observed delay in receptor internalization. Moreover, coimmunoprecipitation experiments in c-Src depleted HEK 293 cells and VSMCs showed an increased association of AP-2 to the agonist-occupied AT1R and β-arrestin, respectively. Together, our results support a role for c-Src in regulating the dissociation of AP-2 from agonist-occupied AT1R and β-arrestin during the clathrin-mediated internalization of receptors and suggest a novel function for c-Src kinase in the internalization of AT1R.
Peripheral-Type Benzodiazepine Receptor-Mediated Action of Steroidogenic Acute Regulatory Protein on Cholesterol Entry into Leydig Cell Mitochondria
Authors: Hauet Thierry , Yao Zhi-Xing , Bose Himangshu S. , Wall Christopher T. , Han Zeqiu , Li Wenping , Hales Dale B. , Miller Walter L. , Culty Martine , Papadopoulos Vassilios
Published: October 2004

Hormone-induced steroid biosynthesis begins with the transfer of cholesterol from intracellular stores into mitochondria. Steroidogenic acute regulatory protein (StAR) and peripheral-type benzodiazepine receptor (PBR) have been implicated in this rate-determining step of steroidogenesis. MA-10 mouse Leydig tumor cells were treated with and without oligodeoxynucleotides (ODNs) antisense to PBR and StAR followed by treatment with saturating concentrations of human choriogonadotropin. Treatment with ODNs antisense but not missense for both proteins inhibited the respective protein expression and the ability of the cells to synthesize steroids in response to human choriogonadotropin. Treatment of the cells with either ODNs antisense to PBR or a transducible peptide antagonist to PBR resulted in inhibition of the accumulation of the mature mitochondrial 30-kDa StAR protein, suggesting that the presence of PBR is required for StAR import into mitochondria. Addition of in vitro transcribed/translated 37-kDa StAR or a fusion protein of Tom20 (translocase of outer membrane) and StAR (Tom/StAR) to mitochondria isolated from control cells increased pregnenolone formation. Mitochondria isolated from cells treated with ODNs antisense, but not missense, to PBR failed to form pregnenolone and respond to either StAR or Tom/StAR proteins. Reincorporation of in vitro transcribed/translated PBR, but not PBR missing the cholesterol-binding domain, into MA-10 mitochondria rescued the ability of the mitochondria to form steroids and the ability of the mitochondria to respond to StAR and Tom/StAR proteins. These data suggest that both StAR and PBR proteins are indispensable elements of the steroidogenic machinery and function in a coordinated manner to transfer cholesterol into mitochondria.
Hunting for Disease Genes in Multi-Functional Diseases
Authors: Pang Chi-Pui , Baum Larry , Lam Dennis S. C.
Published: October 2004

Disease genes may be identified through functional,
positional, and candidate gene approaches. Although
extensive and often labor-intensive studies such as
family linkage analysis, functional investigation of
gene products and genome database searches are
usually involved, thousands of human disease genes,
especially for monogenic diseases with Mendelian
transmission, have been identified. However, in diseases
caused by more than one gene, or by a combination
of genetic and environmental factors, identification
of the genes is even more difficult. Common
examples include atherosclerosis, cancer, Alzheimer's
disease, asthma, diabetes, glaucoma, and age-related
macular degeneration. There have been conflicting reports
on the roles of associated genes. Even with population-based case-control studies and new statistical
methods such as the sib-ship disequilibrium test and
the discordant alleles test, there is no agreement
on whether α2-macroglobulin (A2M) is a gene for Alzheimer's
disease. Another example is the inconsistent
association between age-related macular degeneration
and ATP-binding cassette transporter (ABCR).
Ethnic variation causes further complications. In our
investigation of LDL-receptor variants in familial hypercholesterolemia,
and the trabecular meshwork inducible
glucocorticoid response protein, or myocillin
(TIGR-MYOC) mutation pattern in primary open angle
glaucoma, we did find dissimilar results in Chinese
compared to Caucasians. New information from the
Human Genome Project and advancements in technologies
will aid the search for and confirm identification
of disease genes despite such challenges.
Gene Amplification as Means for Determining Therapeutic Strategies in Human Cancers
Authors: Tsongalis Gregory J. , Cartun Richard W. , Ricci Andrew
Published: October 2004

Pharmacogenomic analysis aspires to identify individuals
with specific genetic characteristics in order to
predict a positive response or reduce a negative response
to a therapeutic modality. While the search
continues for the many single nucleotide polymorphisms
which will be used in such genetic analyses,
other genetic alterations in specific cell types have
proven useful in determining the potential for response
to therapy. One such genetic alteration is amplification
of entire gene sequences which results in
overexpression of a gene product or protein. Amplification
of the HER2 (neu, erbB-2) oncogene is found in
up to 35% of human breast cancers and is associated
with a poor prognosis. In addition, this genetic alteration
may predict response to various therapeutic
modalities. Assays are available to detect the HER2
protein receptor or copies of the HER2 gene sequence
to determine eligibility for Herceptin treatment or adriamycin
treatment in node positive patients, respectively.
This model represents a somatic event used in
the functional determination of a therapeutic strategy.
Drug-Metabolizing Enzymes, Polymorphisms and Interindividual Response to Environmental Toxicants
Authors: Nebert Daniel W.
Published: October 2004

Individual risk of toxicity or cancer reflects the amount
of exposure to environmental agents, combined with
one's underlying genetic predisposition. More than six
dozen human ecogenetic polymorphisms have been
described; whereas some of these have been demonstrated
to be associated with altered risks of toxicity or
cancer, others presently remain equivocal and require
further study. Thus, genetic differences in the regulation,
expression and activity of “environmental susceptibility
genes” can be decisive in defining susceptibility
to toxicity or cancer.

“Drug-metabolizing enzymes” (DMEs) are regarded
as one class of environmental susceptibility genes.
DME genes have actually existed on this planet for
more than 2.5 billion years, and might more appropriately
be named “effector-metabolizing enzymes.” Receptors
controlling DME levels have been called “DME
receptors.” DMEs have functioned in many critical life
processes in prokaryotes and, more recently, in countless
basic functions in plants and animals—events that
evolved long before the existence of pharmaceutical
companies and apothecaries. DME genes exist in every
eukaryotic cell and probably in all prokaryotes. Virtually
all environmental agents act as either agonists or
antagonists—in competing with endogenous ligands
that bind to DME receptors and/or competing as substrates
for the DMEs. Over the past decade it has become
clear that each of us has our own “individual fingerprint”
of unique alleles coding for DMEs. The
underlying genetic predisposition of each patient will
reflect combinations of poor- and extensive-metabolizer
phenotypes; if these enzymes cooperate in the
same metabolic pathway for any given drug or environmental
agent, such ecogenetic variability might be
synergistic and lead to as much as 30- or >40-fold differences
in activation or degradation. The end result
can be large interindividual differences in risk of environmentally
caused toxicity or cancer.
How to Manage Individualized Drug Therapy: Application of Pharmacogenetic Knowledge of Drug Metabolism and Transport
Authors: Meisel Christian , Roots Ivar , Cascorbi Ingolf , Brinkmann Ulrich , Brockmöller Jürgen
Published: October 2004

Significant fractions of health budgets must be spent
for treatment of drug side effects and for inefficient
drug therapy. Hereditary variants in drug metabolizing
enzymes, drug transporters, and drug targets are important
determinants of drug response and toxicity
and may therefore aid in selection and dosage of
drugs. Today there is extensive knowledge of genetic
polymorphisms of cytochrome P450 (CYP) enzymes
2A6, 2C9, 2C19, and 2D6; of phase-2 enzymes such as
thiopurine S-methyltransferase; and more recently of
drug transporters such as the MDR-1 gene-product P-glycoprotein,
affecting a significant share of currently
used drugs. However, application of pharmacogenetic
knowledge to clinical routine is limited in current practice.
To promote the application of pharmacogenetic
knowledge in clinical routine, research on genotype-based
dose adjustments is still necessary—as is the
promotion of faster and cheaper genotype analyses.
Furthermore, the benefits of CYP genotype-directed
drug therapy should be evaluated in properly designed
prospective studies. Once such steps have been successfully
taken, drug therapy could well become more
prevention-directed and patient-tailored than it is possible
today, replacing the current “one drug in one
dose for one disease” strategy by a more individualized
approach.
P-Glycoprotein and Bioavailability-Implication of Polymorphism
Authors: Liu Yan , Hu Ming
Published: October 2004

P-Glycoprotein (P-gp) may have a significant impact
on systemic and tissue/cellular bioavailability of drugs
because it functions as an “anti-absorption” mechanism
that effluxes drug molecules out of the lipid bilayer
and cytoplasm. The ability to reduce bioavailability
at the tissue/cellular level was first discovered
during the investigation of the causes of multidrug resistance
(MDR) in cancer chemotherapy. Initially, it
was thought that MDR is only caused by P-gp. Recently,
many other transporters such as multidrug resistance-related protein (MRP) have also been identified.
The ability of P-gp to impact systemic drug
bioavailability was only recently recognized. Dr. Alfred
Schinkel's group was first to show a significant improvement
in the systemic bioavailability of several
drugs in the MDR1 knockout mice. The same group
also discovered that the blood-brain barrier (BBB) has
a very high expression level of P-gp, and that this protein
is necessary to restrict the entrance of various
drug molecules into the central nervous system (CNS).

Polymorphism in the normal human cells has not
been reported, but it has been discovered in human
cancer cells. Functional implication of P-gp polymorphism
in changing the tissue bioavailability has been
studied in rodents. These studies strongly support the
role of P-gp in restricting tissue bioavailability of anticancer
drugs. These studies also support the effectiveness
of P-gp in limiting CNS toxicity of the cytotoxic
drugs.

The functional implication of P-gp on systemic
bioavailability is much less well defined in humans, although
it appears to be quite obvious in MDR1 knockout
mice. Pharmacokinetic models clearly suggest that
a change in the absorption rate will have a significant
impact on systemic blood level of a drug. However,
whether functionally significant polymorphisms of P-gp
exist in humans has not been determined. If they do
exist, they will surely impact on both systemic bioavailability
and drug interaction potentials of many drugs.

In the drug development process, several models
may be used to select a lead compound that may or
may not interact with P-gp, depending on whether the
interaction is desirable. Several inhibitors of P-gp are
currently on the clinical trial stage. Natural inhibitors
of P-gp have also been discovered. There is no doubt
that these new developments will have significant impact
on the bioavailability of a variety of anticancer
and CNS drugs in the next decades.
Cancer Therapy and Polymorphisms of Cytochromes P450
Authors: MacLeod Stewart L. , Nowell Susan , Massengill Joyce , Jazieh Abdul , McClure Gail , Plaxco Jason , Kadlubar Fred F. , Lang Nicholas P.
Published: October 2004

Cytochrome P450 (CYP) enzymes are important in the
metabolism of some endogenous compounds, environmental
and dietary xenobiotics and many drugs.
Many of these enzymes have genetic polymorphisms
that produce significant changes in metabolic activity,
however the function of other polymorphisms is unknown.
Genetic polymorphisms have important influences
on variability in human pharmacokinetics, including
intra-individual differences in drug toxicity,
drug interactions and response to chemotherapy.
Other factors that influence drug metabolism include
differences in enzyme expression due to differences in
age, gender, smoking status, exposure to dietary or
environmental xenobiotics or co-administration of
other drugs. In addition, some xenobiotics and drugs
can directly inhibit or induce the activity of CYPs. All of
these factors can produce differences in metabolic capacities
among individuals which can produce toxicity
in some patients and sub-effective dosing in others.
Maximum clinical benefit will require a more complete
understanding of the influence of these polymorphisms
on allele function and their interaction with inducers
and inhibitors of enzyme expression or activity.
This effort will permit the pharmacogenetic screening
of patients before the administration of drugs and result
in the identification of individuals who are prone
to adverse reactions or poor response, resulting in
more effective individualized therapy.
Hypervariable Region 1 of Hepatitis C Virus Genome and Response to Interferon Therapy
Authors: Grahovac Blaženka , Bingulac-Popović Jasna , Vucelić Boris , Hrstić Irena , Ostojić Rajko , Dražić Vesna , Balija Melita , Grgičević Damir
Published: October 2004

The relationship between the complexity of the hypervariable
region 1 (HVR1) quasispecies of hepatitis C
virus (HCV) and responsiveness to interferon-α (IFN)
therapy was studied in patients with chronic hepatitis
C. Twelve HCV-RNA-positive patients were treated
daily with high dose IFN and ribavirin for 4 weeks, and
then with IFN 3 MIU (Million International Units) TIW
(three times per week) and ribavirin for 6 months. The
HVR1 quasispecies complexity was analyzed by
nested polymerase chain reaction-mediated single-strand
conformation polymorphism (SSCP). The baseline
HCV-RNA levels in the study group ranged from
106 to 107 copies/ml. All patients exhibited HCV genotype
1 b. Initial SSCP analysis revealed four (33.3%) patients
with a low complexity pattern (SSCP bands ≤4)
and eight (66.6%) patients with high complexity pattern
(SSCP bands >4). After 4 weeks of IFN therapy,
one patient became HCV negative, and among those
remaining positive, the HCV-RNA levels decreased by 2
to 3 logs and the number of SSCP decreased by 2 to 3
bands per sample. After 6 months of IFN therapy, five
(41.7%) patients became HCV-RNA-negative. Seven
(58.3%) patients did not respond to IFN therapy with
sustained viral load from 103 to 105 copies/ml, and high
complexity SSCP patterns. Our data support the HVR
quasispecies complexity to be an independent predictive
factor for IFN responsiveness in patients infected
with HCV.
Influence of Glutathione S-Transferase M1 and T1 Genotypes on Larynx Cancer Risk among Korean Smokers
Authors: Hong Young-Joon , Lee Jin-Kyung , Lee Guk-Haeng , Hong Seok-Il
Published: October 2004

Glutathione S-transferase (GST) isoenzymes are involved
in the detoxification of major carcinogens present
in tobacco smoke. It is thus conceivable that deficiency
in GST activity due to homozygous deletions of
the GSTM1 and GSTT1 genes (the null genotypes) may
modulate susceptibility to smoking-induced cancers.
The influence of the GSTM1 and GSTT1 null genotypes
on larynx cancer risk among the Korean population
were evaluated using peripheral blood DNA from 82
larynx cancer patients and 63 healthy controls, all of
whom were male current smokers. Increased larynx
cancer risk was related to the GSTM1 null genotype
(odds ratio (OR)=3.53, 95% confidence interval
(CI)=1.27−9.83). The OR associated with the GSTT1 null
genotype was also increased, but did not reach statistical
significance (OR=1.83, 95% CI=0.70−4.79). Individuals
lacking both the GSTM1 and GSTT1 genes were at
a significantly higher risk for larynx cancer than individuals
with both genes present (OR=4.04, 95%
CI=1.33−12.30). These data confirm that the GSTM1
null genotype is an important risk modifier for larynx
cancer among Korean smokers and combined GSTM1
and GSTT1 null genotypes could be a useful predictor
of genetic susceptibility to larynx cancer.