bluuebunny/crossref_metadata_2025 huggingface dataset:
A realistic approach to the sensitivity of PCR‐DGGE and its application as a sensitive tool for the detection of clonality in cutaneous T‐cell proliferations
Authors: Meyer J. C. , Hassam S. , Dummer R. , Muletta S. , Döbbeling U. , Dommann S. N. W , Burg G.
Published: April 2006

Abstract The practical value of the detection of clonality within the T‐cell receptor gamma locus by polymerase chain reaction for the diagnosis of cutaneous T‐cell lymphomas is well known. However, studies dealing with this subject so far, with special emphasis on the sensitivity of the technique in comparison to, for example, Southern blotting have used mixtures of DNA in various concentrations instead of using mixtures of the cells involved, which would reflect the in vivo situation in a more realistic scope. The purpose of this study was therefore to determine the sensitivity and the limitations of the PCR assay by dilution experiments, using mixtures of cells. Furthermore we studied its applicability to cutaneous T‐cell proliferative disorders. Two clonal T‐cell lines (MyLa and Jurkat) served as positive control. Dilutions of MyLa cells, cultured normal human keratinocytes and peripheral blood mononuclear cells from lymphoma negative volunteers were used to assess the sensitivity of the PCR‐DGGE assay. Skin samples from 4 patients with cutaneous T‐cell lymphoma, 1 lesional lymph node, 2 blood samples from a patient with Sézary syndrome and 4 lymphoma‐negative tissue samples were analysed. Two samples were uncertain for diagnosis of lymphoma. The PCR‐DGGE assay consisted of a 2‐round nested PCR with consensus primers within the TCR‐gamma locus followed by electrophoretic separation of the product along a denaturing urea/formamide gradient gel. PCR‐DGGE sensitivity was, to our knowledge, for the first time investigated for mixtures of lymphocytes (clonal and polyclonal) and keratinocytes. Clonal T‐cells were detected in a concentration between 1–0.1% in keratinocytes, whereas the sensitivity was generally lower upon dilution in peripheral blood mononuclear cells or in a mixture of keratinocytes and peripheral blood mononuclear cells. Nevertheless, T‐cell clonality was detected in 2 blood samples of a patient with Sézary syndrome, which were negative by Southern blot analysis. The crucial point of this work was the new approach to establish the sensitivity of the PCR‐DGGE, in a way which more closely mimics the condition of clinical specimens. Instead of mixing and amplifying DNA extracted from clonal T‐cell lines and polyclonal bone marrow cells, we amplified DNA from clonal and polyclonal cells which had been mixed in various ratios before DNA extraction. Polymerase chain reaction in conjunction with denaturing gradient gel electrophoresis is a sensitive and versatile molecular tool for the assessment of clonality of suspect cutaneous lesions. The determination of sensitivity using DNA extracted from premixed cells more closely corresponds to the actual test situation when testing skin samples.
Matrix metalloproteinases in skin
Authors: Kähäri Veli‐Matti , Saarialho‐Kere Ulpu
Published: April 2006

Abstract Matrix metalloproteinases (MMPs) are a family of zinc‐dependent endopeptidases collectively capable of degrading essentially all extracellular matrix components. These enzymes can be produced by several different types of cells in skin such as fibroblasts, keratinocytes. macro‐phages, endothelial cells, mast cells, and eosinophils and their activity can be specifically inhibited by TIMPs (tissue inhibitors of metalloproteinases), which bind to active MMPs with 1:1 stoichiometry. In general, MMPs are not constitutively expressed in skin but are induced temporarily in response to exogenous signals such as various cytokines. growth factors, cell‐matrix interactions and altered cell‐cell contacts. At present, more evidence is accumulating thai MMPs play an important role in proteolytic remodeling of extracellular matrix in various physiologic situations, including developmental tissue morphogenesis, tissue repair, and angiogenesis. On the other hand, MMPs play an important pathogenetic role in excessive breakdown of connective tissue components, e.g. in rheumatoid arthritis, oslteoarthritis, chronic ulcers, dermal photoageing, and periodontitis, as well as in tumor cell invasion and metastasis. In this review we discuss the role of MMPs and TIMPs in human skin based on new observations on the regulation of the expression of MMPs, on their substrate specificily, and MMP expression in physiologic and pathologic conditions of skin involving matrix remodeling. Furthermore, therapeutic modalities based on regulating MMP activity will be reviewed.
Correlation between hyperproliferation and suprabasal integrin expression in human epidermis reconstituted in culture
Authors: Rikimaru K. , Molès J‐P. , Watt F. M.
Published: April 2006

Abstract In normal epidermis integrin expression is largely confined to the basal layer. However, during wound healing and in psoriatic lesions suprabasal expression is observed. Although the potential importance of suprabasal integrin expression in the pathogenesis of psoriasis has been established, the cause of suprabasal expression is unknown. We now describe changes in integrin expression that occur with time when normal human keratinocytes are grown on two types of dermal equivalent, de‐epidermized dermis and collagen gels containing fibroblasts. We show that suprabasal integrin expression is correlated with suprabasal expression of the EGF receptor, but not with expression of keratin 10 or keratin 16. By quantitating the proportion of basal keratinocytes expressing the proliferation marker Ki‐67 we could show that suprabasal integrin expression is correlated with high proliferative activity within the basal layer. Taken together with our earlier work, these results suggest that suprabasal integrin expression is linked to hyperproliferation and not to abnormal terminal differentiation or to inflammation; they also establish dermal equivalent cultures as useful experimental models with which to manipulate keratinocyte integrin expression.
Modulation of MHC class II+ Langerhans cell numbers in corticosteroid treated epidermis by GM‐CSF in combination with TNF‐α
Authors: O'Sullivan Gabrielle M. , Halliday Gary M.
Published: April 2006

Abstract It is important to understand how dendritic cells (DC) are recruited, maintained and stimulated to migrate from tissues to lymph nodes. This is because DC are potent initiators of primary immune responses and candidates for vaccine development. Identification of factors which could lead to increased numbers of DC in tissues could affect immune responses by modulating their interaction with antigen which penetrates the tissue. To identify cytokines which could increase DC in tissues we tested the ability of GM‐CSF, TNF‐α and IL‐6 to partially prevent steroid depletion of Langerhans cells (LC) from the epidermis. Cytokines diluted in serum‐containing medium were compared with cytokines diluted in albumin‐containing, serum‐free medium in order to determine a minimum combination of cytokines required to increase LC and the effect of serum on the LC‐increasing activity of cytokines. In the presence of serum, GM‐CSF or TNF‐α could increase LC frequency compared to the control; but in the absence of serum neither of these cytokines were effective unless they were combined with each other. In the presence of serum the combination of GM‐CSF with TNF‐α was ineffective. The data support the hypotheses that GM‐CSF and TNF‐α are both important in regulating LC numbers in the epidermis in vivo. Serum may modulate how each of these cytokines, separately or in combination, affect LC frequency in the epidermis–GM‐CSF and TNF‐α separately probably interact with other factors present in serum to increase LC frequency, whereas in combination it is possible that these separate effects are cancelled in the presence of serum. TNF‐α and GM‐CSF together, in the absence of serum, form one combination of a minimum number of cytokines which can regulate LC frequency in the epidermis; and IL‐6 alone, or in combination with GM‐CSF, does not increase LC frequency.
Chronic UVA (365‐nm) irradiation induced scratching in hairless mice: dose‐time dependency and the effect of ketanserin
Authors: de Laat J. M. T. , Seité S. , Groenendijk M. , van Vloten W A. , de Gruijl F. R.
Published: April 2006

Abstract In a study on the dose–response relationship for longwave UVA (UVAI; 340–400 nm) carcinogenesis in hairless mice scratch marks appeared after months of daily exposure as an unwanted side effect. Tumor induction in the highest of the 4 tested dose groups (receiving a daily dose of 430 kJ/m2 of 365‐nm radiation) could not be determined because extensive scarification occurred prior to the development of any tumors. The induction of scratch marks could be scored and quantified in all 4 dose groups tested. The UVA I dose‐dependencies for the induction of tumors and scratch marks were compared. We found that the induction of scratch marks depended mainly on the cumulative UVA1 exposure, whereas tumor induction showed a lesser dose‐dependency. An attempt was made to prevent the apparent pruritogenic effect of UVA1 irradiation and to understand its mechanism. The influence of ketanserin, a serotonin/histamine antagonist, on the UVA1 induction of scratch marks was tested in groups of 8 mice daily irradiated with 430 kJ/m2. No difference was found between treated and untreated animals. Histological examination of skin biopsies from irradiated mice from the 430‐kJ/m2 dose group from the UVA1 careinogenic experiment, showed no changes in numbers of mast cells or other inflammatory features when compared to skin biopsies from unirradiated control mice. This indicated that UVA1‐induced scratching is not mediated through mast cell release of scrotonin and/or histamine. An adequate therapeutic treatment which can prevent UVA1‐induccd scratching would enable us to lest tumor induction with UVA1 over a larger close range, and may provide additional insight in how this radiation damages the skin. It remains conjectural whether there exists an analogous UVA‐induccd pruritus in human skin.
Reverse iontophoresis: monitoring prostaglandin E2 associated with cutaneous inflammation in vivo
Authors: Mize Nancy K. , Buttery Margaret , Daddona Peter , Morales Claudia , Cormier Michel
Published: April 2006

Abstract In response to topical application of irritants, increased concentrations of prostaglandin E2 (PGE2) are found in human skin exudate and in cultured dermal fibroblasts. In this study, PGE2 generated in response to transdermal delivery of irritant drug compounds was monitored in hairless guinea pig (HOP) by a non‐invasive method, reverse iontophoresis. Reverse iontophoresis is the movement of molecules from the skin under the influence of an applied electric field. Irritant drug compounds were applied with iontophoresis (electrotransport), and reverse iontophoresis of PGE2 from skin was monitored by radioimmunoassay (RIA) after extraction from the delivery system. Chlorpromazine was used as a model drug irritant. When chlorpromazine and saline were applied over a range of current densities from 0 to 200 μA/cm2, visual scores of erythema and edema yielded a correlation with measured skin efflux of PGE2 (r=0.86). Delivery of chlorpromazine resulted in greater efflux of PGE2 than delivery of non‐irritant saline controls under the same delivery conditions. Five drug compounds, chloroquine, promazine, chlorpromazine, tetracaine, metoclopramide, and saline were applied to hairless guinea pig skin. The 6 agents were similarly rank ordered by visual erythema/edema scores and by PGE2 efflux, indicating that the quantity of PGE2 effluxed reflects the intensity of skin irritation. In contrast, vasoconstriction or vasodilation produced by the local delivery of vasoactive agents did not correlate with PGE2 skin efflux, indicating that this measurement is specific for an inflammatory response. In summary, PGE2 generated in response to transdermally applied drug irritants can be monitored non‐invasively in vivo by reverse iontophoresis.
Identification of the glycine‐to‐arginine substitution G2043R in type VII collagen in a family with dominant dystrophic epidermolysis bullosa from Hungary
Authors: Cserhalmi‐Friedman P. B. , Karpati S. , Horvath A. , Christiano A. M.
Published: April 2006

Abstract Epidermolysis bullosa (EB) represents a group of genodermatoses characterized by fragility and easy blistering of the skin. In the dystrophic forms of EB (DEB), blisters occur below the basement membrane, at the level of the anchoring fibrils. In the dominantly inherited forms (DDEB), the predominant type of mutation detected thus far is the substitution of a glycine residue which occurs within the collagenous domain of the molecule characterized by the repeating Gly‐X‐Y amino acid sequence. In this study, we searched for mutations in DDEB in a family from Hungary, by PCR amplification of segments of COL7A1, followed by heteroduplex analysis. Examination of the PCR fragment corresponding to exon 73 revealed a heteroduplex in affected individuals from the family. Sequence analysis revealed a G‐to‐A transition at nucleotide 6127 in the triple‐helical domain of COL7A1, which converted a glycine residue at amino acid position 2043 to an arginine. This report represents the second incidence of this mutation. G2043R. described first in a family with DDEB from Italy.
The expression of the gap junctional protein Cx43 is restricted to proliferating and non differentiated normal and transformed keratinocytes
Authors: Gibson D. F. C. , Bikle D. D. , Harris J. , Goldberg G. S.
Published: April 2006

Abstract The passage of specific growth modulating signals through gap junctions may regulate the proliferation and differentiation of human keratinocytes. To investigate this, we correlated the proliferation of normal human keratinocytes and a transformed squamous cell carcinoma cell line, SCC4. with the expression of the gap junctional proteins Cx43. 31 and 31.1, known to be expressed by keratinocytes. Proliferation was con‐lined to preconfiuent and confluent cultures of normal keratinocytes, falling to undelectable levels once poslconfluency was achieved. Cx43. at both the message and protein levels, paralleled these changes, being elevated predominantly in prcconiluent and confluent cultures, and downregulated in postconfluency. Similar results were found for Cx31 and 31.1 at the message level. In contrast, the proliferation of SCC4 cells cultured in media supplemented with 5.0° FCS was maintained at n substantial level from preconfluency through 2 weeks postconfluency. Cx43, 31. and 31.1 RNA and Cx43 protein expression mirrored the levels of proliferation within SCC4 cultures. Cx26 and 32 were not found in normal keratinocytes or SCC4 cells at any stage of differentiation. These data, illustrating a tight correlation between proliferation and Cx43, 31 and 31.1 expression, suggest that these connexins may represent proliferation‐specific gap junctions within keratinocytes. and may therefore transmit signals that control keratinocyle division.
An Exploration Into the Internal Dynamics of a School‐Based Mental Health Collaboration
Authors: Kury Kenneth Wm. , Kury Gloria
Published: April 2006

Abstract: Schools are moving toward a human service approach that encompasses the physical and mental needs of its students on top of the traditional role of educating. School‐based collaboratives are one delivery model that satisfies this expanding role. Lacking in the research on this emerging setting, particularly in the realm of mental health, is information that gives insight into the interactions that occur between collaborative partners who may have opposing aims. This study explores 2 outcome measures, timeliness of implementation of the treatment plan and parental satisfaction with the treatment plan and the influence of the professional view of collaborative partners on these outcomes. Archival data were collected from patient charts on 101 Hispanic students, 73.3% of who were male and the sample mean age was 11.3 years. These data were analyzed using multiple regression techniques. Results revealed that time to implementation was impacted by the diagnosis alignment of participating partners, a proxy for professional view, as was parental satisfaction. Satisfaction was not influenced by timeliness, indicating alternative outcome desires. (J Sch Health. 2006;76(5):164‐168)
Health Behaviors of School‐Age Children: Evidence From One Large City
Authors: Cartland Jenifer , Ruch‐Ross Holly S.
Published: April 2006

Abstract: Relatively little is known about risk behaviors of elementary school children. A recent evaluation of a comprehensive school health education curriculum provided an opportunity to survey elementary school children about their perceived health status, knowledge, attitudes, health self‐efficacy, and health and risk behaviors. For the evaluation, a total of 4273 surveys were completed by 2 cohorts of school children, grades 2 and 4, in 24 schools in a large urban school district during the spring semesters of 2002; the cohorts were surveyed a second time when they were in third and fifth grade in spring 2003. Older children scored higher than younger ones on factors generally associated with improved health behaviors (such as health knowledge and refusal skills); yet, they scored lower than younger children on healthy behaviors, especially risk behaviors and those things presumed to be associated with later adolescent risk taking. The interrelationships among behavior, knowledge, age, and school‐level attributes are explored. As health knowledge, refusal skills, and other protective factors increase, health behaviors improve slightly, but the most important factor in explaining health behaviors is grade level. This article points to the need to study the development of risk behaviors throughout elementary school and the need for additional work on the measurement of risk behaviors during the elementary school years. (J Sch Health. 2006;76(5):175‐180)
Prevalence of Risk for Type 2 Diabetes in School Children
Authors: Urrutia‐Rojas Ximena , Menchaca John
Published: April 2006

Abstract: According to the Centers for Disease Control and Prevention, 1 in 3 children born in 2000 in the United States will become diabetic. The odds are higher for African American and Hispanic children as nearly 50% of them will develop diabetes. Random screening is not effective in identifying children at risk for type 2 diabetes mellitus (T2DM); therefore, there is a need to apply screening strategies that guide the development of appropriate primary prevention efforts. To assess the prevalence of risk factors for T2DM, 1066 fifth‐grade children were screened using American Diabetes Association guidelines. Overall, 22.6% were found at risk; African American and Hispanic children were almost 8 times more likely to be at risk when compared to Caucasians (odds ratio = 7.41 and 7.87). Children who reported watching TV/playing video games 2 or more hours/day were 73% more likely to be at risk. Children identified to be at risk were referred to their primary care provider and were invited to participate in a counseling session. The environmental risk factors for T2DM identified in this study are modifiable and should be targeted in preventive interventions at the school and community level to reduce overweight and consequently prevent T2DM in children, especially among minority children. (J Sch Health. 2006;76(5):189‐194)
Secretion of human glucocerebrosidase from stable transformed insect cells using native signal sequences
Authors: Sinclair Graham , Pfeifer Tom A , Grigliatti Thomas A , Choy Francis Y.M
Published: April 2006

The lysosomal hydrolase, glucocerebrosidase (GBA), catalyses the penultimate step in the breakdown of membrane glycosphingolipids. An inherited deficiency of this enzyme activity leads to the onset of Gaucher disease, the most common lysosomal storage disorder. Affected individuals range from adults with hepatosplenomegaly, haematological complications, and bone pain (type 1 disease) to children and neonates with severe neuronopathy leading to neurological degradation and premature death (type 2 and type 3 disease). Enzyme replacement therapy has become the standard of treatment for type I Gaucher disease but remains an expensive option, in part because of the cost of recombinant enzyme production using mammalian cell culture. Using a nonlytic integrative plasmid expression system, we have successfully produced active human GBA in stable transformed Sf9 (Spodoptera frugiperda) cells. Both the 39 and 19 amino acid native GBA signal sequences were capable of endoplasmic reticulum targeting, which led to secretion of the recombinant protein, although approximately 30% more enzyme was produced using the longer signal sequence. The secreted product was purified to apparent electrophoretic homogeneity using hydrophobic interaction chromatography and found to be produced in a fully glycosylated and a hypoglycosylated form, both of which cross-reacted with a human GBA-specific monoclonal antibody. The pH optimum (at pH 5.5) for activity of the recombinant enzyme was as expected for human GBA using the artificial substrate 4-methyl-umbelliferyl-β-D-glycopyranoside. With initial nonoptimized expression levels estimated at 10–15 mg/L using small-scale batch cultures, stable transformed insect cells could provide a viable alternative system for the heterologous production of human GBA when grown under optimized perfusion culture conditions.Key words: Gaucher disease, glucocerebrosidase, protein expression, enzyme purification, Sf9 cells.
Cellular concentrations of glutamine synthetase in murine organs
Authors: van Straaten Henny W.M , He Youji , van Duist Marjan M , Labruyère Wil T , Vermeulen Jacqueline L.M , van Dijk Paul J , Ruijter Jan M , Lamers Wouter H , Hakvoort Theodorus B.M
Published: April 2006

Glutamine synthetase (GS) is the only enzyme that can synthesize glutamine, but it also functions to detoxify glutamate and ammonia. Organs with high cellular concentrations of GS appear to function primarily to remove glutamate or ammonia, whereas those with a low cellular concentration appear to primarily produce glutamine. To validate this apparent dichotomy and to clarify its regulation, we determined the GS concentrations in 18 organs of the mouse. There was a >100-fold difference in GS mRNA, protein, and enzyme-activity levels among organs, whereas there was only a 20-fold difference in the GS protein:mRNA ratio, suggesting extensive transcriptional and posttranscriptional regulation. In contrast, only small differences in the GS enzyme activity : protein ratio were found, indicating that posttrans lational regulation is of minor importance. The cellular concentration of GS was determined by relating the relative differences in cellular GS concentration, detected using image analysis of immunohistochemically stained tissue sections, to the biochemical data. There was a >1000-fold difference in cellular concentrations of GS between GS-positive cells in different organs, and cellular concentrations were up to 20× higher in subpopulations of cells within organs than in whole organs. GS activity was highest in pericentral hepatocytes (~485 µmol·g–1·min–1), followed in descending order by epithelial cells in the epididymal head, Leydig cells in the testicular interstitium, epithelial cells of the uterine tube, acid-producing parietal cells in the stomach, epithelial cells of the S3 segment of the proximal convoluted tubule of the kidney, astrocytes of the central nervous tissue, and adipose tissue. GS activity in muscle amounted to only 0.4 µmol·g–1·min–1. Our findings confirmed the postulated dichotomy between cellular concentration and GS function.Key words: mRNA, protein, enzyme activity, posttranscriptional regulation, quantitative immunohistochemistry.
Molten globule state of human placental cystatin (HPC) at low pH conditions and the effects of trifluoroethanol (TFE) and methanol
Authors: Rashid Fouzia , Sharma Sandeep , Baig M A , Bano Bilqees
Published: April 2006

Acid-induced conformational changes were studied in human placental cystatin (HPC) in terms of circular dichroism (CD) spectroscopy, the binding of hydrophobic dye 1-anilinonapthalene-8-sulphonic acid (ANS), and intrinsic fluorescence measurements. Our results show the formation of an acid-induced molten globule state at pH 2.0, with significant secondary and tertiary interactions that resemble the native state, exposed hydrophobic regions and the effects of trifluoroethanol (TFE) and methanol in conversion of the acid-denatured state of HPC to the alcohol-induced state, which is characterized by increased helical content, disrupted tertiary structure, and the absence of hydrophobic clusters. Alcohol-induced formation of α-helical structures at pH 2.0 is evident from the increase in the ellipticity values at 222 nm, with native-like secondary structural features at 40% TFE. The increase in helical content was observed up to 80% TFE concentration. The ability of TFE (40%) to refold acid-denatured HPC to native-state conformation is also supported by intrinsic and ANS fluorescence measurements.Key words: human placental cystatin, molten globule, acid-induced state, trifluoroethanol, methanol, CD spectroscopy, ANS fluorescence, pH, protein folding.
Differential abilities of mouse liver parenchymal and nonparenchymal cells in HDL and LDL (native and oxidized) association and cholesterol efflux
Authors: Lapointe Jany , Truong To Quyen , Falstrault Louise , Brissette Louise
Published: April 2006

The aim of this study was to quantify the abilities of mouse liver parenchymal and nonparenchymal cells with respect to (i) cholesteryl ester (CE) selective uptake from low-density lipoproteins (LDL), oxidized LDL (OxLDL), and high-density lipoprotein (HDL); and (ii) their free cholesterol efflux to HDL. The preparations of cells were incubated with lipoproteins labelled either in protein with iodine-125 or in CE with3H-cholesterol oleate, and lipoprotein-protein and lipoprotein-CE associations were measured. The associations of LDL-protein and LDL-CE with nonparenchymal cells were 5- and 2-fold greater, respectively, than with parenchymal cells. However, in terms of CE-selective uptake (CE association minus protein association) both types of cell were equivalent. Similar results were obtained with OxLDL, but both types of cell showed higher abilities in OxLDL-CE than in LDL-CE selective uptake (on average by 3.4-fold). The association of HDL-protein with nonparenchymal cells was 3× that with parenchymal cells; however, nonparenchymal cells associated 45% less HDL-CE. Contrary to parenchymal cells, nonparenchymal cells did not show HDL-CE selective uptake activity. Thus parenchymal cells selectively take CE from the 3 types of lipoproteins, whereas nonparenchymal cells exert this function only on LDL and OxLDL. Efflux was 3.5-fold more important in nonparenchymal than in parenchymal cells.Key words: LDL, HDL, parenchymal, SR-BI, CD36, selective uptake, cholesterol.
ZNRD1 mediates resistance of gastric cancer cells to methotrexate by regulation of IMPDH2 and Bcl-2
Authors: Hong Liu , Qiao Taidong , Han Yu , Han Shuang , Zhang Xiaoyin , Lin Tao , Gao Juan , Zhao Pengtao , Chen Zhen , Fan Daiming
Published: April 2006

We previously showed that downregulation of a transcription-associated gene (ZNRD1) could reverse the resistant phenotype of gastric cancer cells through regulation of the transcription of multidrug resistance gene 1 (MDR1). In the present study, we determined both known and novel differentially expressed genes in VCR-induced multidrug resistant gastric cancer cell SGC7901/VCR transfected with ZNRD1 siRNA or empty vector control. Screening was performed using the Human Cancer Xpro(tm) HC-III plus arrays, containing 3072 cancer-related cDNAs. Ten genes, involved in cell cycle control, nucleic acid binding, and protein phosphorylation, among other functions, underwent more than 5-fold change. Of the downregulated genes we chose Inosine monophosphate dehydrogenase 2 (IMPDH2) for further validation by quantitative RT-PCR. In vitro and in vivo drug sensitivity analyses revealed that inhibition of ZNRD1 and IMPDH2 activity sensitized SGC7901/VCR cells to methotrexate. Additionally, inhibition of ZNRD1 could suppress adriamycin-induced apoptosis and significantly downregulate the expression of Bcl-2, but it did not alter the expression of the glutathione-S-transferase, or intracellular glutathione content. Taken together, the findings suggest that ZNRD1 could act as a modulator of methotrexate chemotherapy in gastric cancer cells through the regulation of IMPDH2 and Bcl-2.Key words: ZNRD1, IMPDH2, multidrug resistance, apoptosis, gastric cancer.
The structural basis for the serospecificity ofActinobacillus suisserogroup O:2
Authors: Rullo Anthony , Papp-Szabo Erzsebet , Michael Frank St , MacInnes Janet , Monteiro Mario A
Published: April 2006

Actinobacillus suis is an important bacterial pathogen of healthly pigs. An O-antigen (lipopolysaccharide; LPS) serotyping system is being developed to study the prevalence and distribution of representative isolates from both healthy and diseased pigs. In a previous study, we reported that A. suis serogroup O:1 strains express LPS with a (1→6)-β-D-glucan O-antigen chain polysaccharide that is similar in structure to a key cell-wall component in yeasts, such as Saccharomyces cerevisiae and Candida albicans. This study describes the O-antigen polysaccharide chemical structure of an O:2 serogroup strain, A. suis H91-0380, which possesses a tetrasaccharide repeating block with the structure: →3)-β-D-Galp-(1→4)-[α-D-Galp-(1→6)]-β-D-Glcp-(1→6)-β-D-GlcpNAc-(1→. Studies have shown that A. suis serogroup O:2 strains are associated with severely diseased animals; therefore, work on the synthesis of a glycoconjugate vaccine employing O:2 O-antigen polysaccharide to vaccinate pigs against A. suis serogroup O:2 strains is currently underway.Key words: Actinobacillus suis, lipopolysaccharide, serogroup O:2, vaccine.
Leptin biosynthetic pathway in white adipocytes
Authors: Cammisotto Philippe G , Bukowiecki Ludwik J , Deshaies Yves , Bendayan Moise
Published: April 2006

The aim of this study was to determine through morphological and biochemical means the biosynthetic and secretory pathway followed by leptin in adipocytes. Immunocytochemistry revealed the presence of leptin in the rough endoplasmic reticulum, the Golgi apparatus, and in numerous small vesicles along the plasma membrane of white adipo cytes. In vitro, isolated adipocytes under nonstimulated conditions (basal) continuously secreted leptin while their intra cellular content remained unchanged. When adipocytes were stimulated with insulin, leptin cellular content and secretion increased in parallel and were significantly different from basal secretion only after 45 min. L-leucine and L-glutamate also strongly stimulated leptin synthesis and secretion. These stimulating effects were abolished by cycloheximide and brefeldin A. The transcriptional inhibitor actinomycin D did not have any effects in either basal or stimulated conditions. Leptin mRNA levels were not affected by any stimulating or inhibiting agents. Finally, norepinephrine, isoproterenol, CL316243, and palmitate inhibited the effects of insulin, L-leucine, and L-glutamate on leptin synthesis. We thus conclude that (i) adipocytes continuously synthesize and secrete leptin along a rough endoplasmic reticulum–Golgi secretory vesicles pathway, (ii) an increase in leptin secretion requires increased de novo synthesis, and (iii) short-term leptin secretion does not involve changes in mRNA levels.Key words: leptin, vesicles, constitutive secretion, de novo synthesis, transcription.
Selenium Accumulation in Prostate Tissue During a Randomized, Controlled Short-term Trial of l-Selenomethionine: a Southwest Oncology Group Study
Authors: Sabichi Anita L. , Lee J. Jack , Taylor Robert J. , Thompson Ian M. , Miles Brian J. , Tangen Catherine M. , Minasian Lori M. , Pisters Louis L. , Caton John R. , Basler Joseph W. , Lerner Seth P. , Menter David G. , Marshall James R. , Crawford E. David , Lippman Scott M.
Published: April 2006

Purpose: Epidemiologic and clinical data suggest that selenium could prevent prostate cancer, but it has not been shown that supplemental selenium leads to an increased concentration of selenium in prostate tissue compared with adjacent tissue.

Experimental Design: We conducted a randomized, controlled, short-term trial of l-selenomethionine (SeMet) versus observation in men with organ-confined prostate cancer. The primary endpoint was the measurement of selenium concentration in prostate tissue and seminal vesicle (SV). We assessed baseline selenium levels in serum and in toenail specimens (reflecting long-term intake) and post-intervention selenium levels in serum, and in prostate and SV tissues using hydride generation atomic fluorescence spectroscopy.

Results: Sixty-six eligible patients were randomly assigned to the SeMet (n = 34) or observation (n = 32) arm; both arms had similar baseline patient characteristics. Baseline serum selenium was similar in the two groups (P = 0.64). Baseline toenail selenium levels were slightly higher in the SeMet group than in the control group (P = 0.07). After the intervention, the mean serum selenium level increased 15% in the SeMet arm and was higher than in the observation arm (P = 0.001). The selenium concentration in prostate tissue was 22% higher in the SeMet arm (n = 26) than in the observation arm (n = 25; 1.80 versus 1.47 ppm; P = 0.003, Wilcoxon rank sum test) and remained significantly higher after adjusting for chronic selenium intake (P = 0.021, ANCOVA). SV selenium concentration was similar in both groups (P = 0.384) and was lower than in prostate tissue.

Conclusions: The present study is the first to show that selenium taken as oral supplementation accumulates preferentially in the human prostate gland as opposed to the SV. These findings support the hypothesis that oral selenium supplementation may contribute to the cancer preventive effects of selenium.
Polymorphisms in Genes of Nucleotide and Base Excision Repair: Risk and Prognosis of Colorectal Cancer
Authors: Moreno Victor , Gemignani Federica , Landi Stefano , Gioia-Patricola Lydie , Chabrier Amélie , Blanco Ignacio , González Sara , Guino Elisabet , Capellà Gabriel , Canzian Federico ,   Bellvitge Colorectal Cancer Study Group
Published: April 2006

Objectives: We have undertaken a comprehensive study of common polymorphisms in genes of DNA repair, exploring both the risk of developing colorectal cancer and the prognosis of patients.

Methods: Subjects from a case-control study (377 cases and 329 controls) designed to assess gene-environment interactions were genotyped by use of an oligonucleotide microarray and the arrayed primer extension technique. Twenty-eight single nucleotide polymorphisms in 15 DNA repair genes were included. The candidate genes belong to different DNA repair pathways: base excision repair (OGG1, LIG3, APEX, POLB, XRCC1, PCNA, and MUTYH), nucleotide excision repair (ERCC1, ERCC2, ERCC4, and ERCC5), double-strand breaks repair (XRCC2, XRCC3, and XRCC9), and reversion repair (MGMT) genes.

Results: Polymorphism OGG1 S326C was associated with an increased risk of colorectal cancer [odds ratio (OR), 2.3; 95% confidence interval (95% CI), 1.1-5.0], the risk being higher in younger individuals. A haplotype of ERCC1 was associated with increased risk (OR, 2.3; 95% CI, 1.0-5.3). POLB P242R was also associated with decreased risk (OR, 0.23; 95% CI, 0.05-0.99), although the number of variant allele carriers was low. In the univariate analysis, adjusted for age, sex, and Dukes' stage, three polymorphisms were significantly associated with better prognosis: XRCC1 R399Q [hazard ratio (HR), 0.38; 95% CI, 0.17-0.85], XRCC3 T141M (HR, 0.66; 95% CI, 0.45-0.97), and MGMT L84F (HR, 0.14; 95% CI, 0.02-0.99). ERCC1 19007T>C was associated with worse prognosis (HR, 1.51; 95% CI, 1.01-2.27). In a multivariate analysis, only XRCC1 R399Q and ERCC1 19007T>C remained significant. These associations were stronger among patients receiving adjuvant chemotherapy.

Conclusions: Although the overall effect of DNA repair genes in colorectal cancer etiology seems limited, their influence in the response to chemotherapy and prognosis may be more relevant. This knowledge may help to clarify the utility of specific adjuvant treatments according to the individual genetic background.
Quantitative Analysis of Human Telomerase Reverse Transcriptase in Pancreatic Cancer
Authors: Ohuchida Kenoki , Mizumoto Kazuhiro , Yamada Daisuke , Yamaguchi Hiroshi , Konomi Hiroyuki , Nagai Eishi , Yamaguchi Koji , Tsuneyoshi Masazumi , Tanaka Masao
Published: April 2006

Although telomerase activity is a promising diagnostic marker, clinical introduction of this marker for cancer diagnosis is still problematic due to the lack of means of evaluating sample quality. Human telomerase reverse transcriptase (hTERT), one of the subunits of telomerase, is also a promising diagnostic marker. In the present study, we did large-scale analysis of 88 pancreatic juice samples to determine the feasibility of quantitative analysis of hTERT mRNA for diagnosis of pancreatic cancer. We found significant differences in hTERT expression among carcinoma-derived, intraductal papillary mucinous neoplasm (IPMN)–derived, and chronic pancreatitis–derived juice samples. Results showed that quantitative analyses of hTERT mRNAs are more useful in discriminating carcinoma from IPMN than from chronic pancreatitis. When the specificity was set at 100%, the sensitivity for differentiation between carcinoma and IPMN was 43.5%, whereas the sensitivity of cytologic examination was 22.0%. There were significant differences in hTERT expression among carcinoma cells, IPMN cells, and normal ductal cells isolated from pancreatic tissues by microdissection. Lymphocytes and hyperplastic epithelial cells isolated from tissues with the histologic appearance of pancreatitis showed various expression levels of hTERT. Our results suggest that quantitative analysis of hTERT mRNA in pancreatic juice is advantageous over cytologic analysis for differentiation between carcinoma and IPMN but probably not for differentiation between carcinoma and chronic pancreatitis.
Bead-Based ELISA for Validation of Ovarian Cancer Early Detection Markers
Authors: Scholler Nathalie , Crawford Meghan , Sato Alicia , Drescher Charles W. , O'Briant Kathy C. , Kiviat Nancy , Anderson Garnet L. , Urban Nicole
Published: April 2006

Purpose: Efforts to validate ovarian cancer early detection biomarkers with immunoassays are challenged by the limited specimen volumes available. We sought to develop a specimen-efficient assay to measure CA125 in serum, assess its reproducibility, validity, and performance, and test its potential for multiplexing and combining with human epididymis protein 4 (HE4), a promising novel ovarian cancer marker.

Experimental Design: Four pairs of commercially available anti-CA125 antibodies and one pair of anti-HE4 antibodies were evaluated for accuracy in measuring known concentrations of antigen on a bead-based platform. The two best pairs were further assessed for reproducibility, validity, and the ability to discriminate between blinded serum samples obtained from ovarian cancer cases (n = 66) and women without ovarian cancer (n = 125).

Results: Suitability for use in a bead-based assay varied across CA125 antibody pairs. Two CA125 bead-based assays were highly reproducible (overall correlations between replicates ≥ 0.95; coefficients of variation < 0.2) and strongly correlated with the research standard CA125II RIA (correlations ≥ 0.9). Their ability to distinguish ovarian cancer cases from non-cases based on receiver operating characteristic analyses (area under the curve, AUC, of 0.85 and 0.84) was close to that of the CA125II RIA (AUC, 0.87). The HE4 bead-based assay showed lower reproducibility but yielded an AUC of 0.89 in receiver operating characteristics analysis. Multiplexing was not possible but a composite marker including CA125 and HE4 achieved an AUC of 0.91.

Conclusion: Optimization procedures yielded two bead-based assays for CA125 that perform comparably to the standard CA125II RIA, which could be combined with an HE4 bead-based assay to improve diagnostic performance, and requires only 15 μL of sample each.
Quantitative Effect of Gender, Age, Liver Function, and Body Size on the Population Pharmacokinetics of Paclitaxel in Patients with Solid Tumors
Authors: Joerger Markus , Huitema Alwin D.R. , van den Bongard Desiree H.J.G. , Schellens Jan H.M. , Beijnen Jos H.
Published: April 2006

Background: The aim of this study was to quantitatively assess the effect of anthropometric and biochemical variables and third-space effusions on paclitaxel pharmacokinetics in solid tumor patients.

Materials and Methods: Plasma concentration-time data of paclitaxel were collected in patients with non–small cell lung cancer (n = 84), ovarian cancer (n = 40), and various solid tumors (n = 44), totaling 168 patients. Paclitaxel was given as a 3-hour infusion (n = 163) at doses ranging from 100 to 250 mg/m2, or as a 24-hour infusion (n = 5) at a dose of 135 or 175 mg/m2. Data were analyzed using nonlinear mixed-effect modeling.

Results: A three-compartment model with saturable elimination and distribution was used to describe concentration-time data. Male gender and body surface area were positively correlated with maximal elimination capacity of paclitaxel (VMEL); patient age and total bilirubin were negatively correlated with VMEL (P < 0.005 for all correlations). Typically, male patients had a 20% higher VMEL; a 0.2 m2 increase of body surface area led to a 9% increase of VMEL; a 10-year increase of patient age led to a 5% decrease of VMEL; and a 10-μmol increase of total bilirubin led to a 14% decrease of VMEL. Third-space effusions were not correlated with paclitaxel pharmacokinetics.

Conclusions: This extended retrospective population analysis showed patient gender to significantly and independently affect paclitaxel distribution and elimination. Body surface area, total bilirubin, and patient age were confirmed to affect paclitaxel elimination. This pharmacokinetic model allowed quantification of the covariate effects on the elimination of paclitaxel and may be used for covariate-adapted paclitaxel dosing.
Diffuse Large B-Cell Lymphoma with Overexpression of Cyclin E Substantiates Poor Standard Treatment Response and Inferior Outcome
Authors: Tzankov Alexandar , Gschwendtner Andreas , Augustin Florian , Fiegl Michael , Obermann Ellen C. , Dirnhofer Stephan , Went Philip
Published: April 2006

Purpose: Gold standard to predict survival and stratify patients for risk-adapted therapy in diffuse large B-cell lymphoma (DLBCL) is the international prognostic index, although it does not consider the molecular heterogeneity of DLBCL. Deregulation of cyclin E (CCNE) is a strong predictor of poor prognosis in some neoplastic diseases. In tumor cells, it induces chromosomal instability with an increased rate of aneuploidy/polyploidy.

Experimental Design: We analyzed in this retrospective study the prognostic value of immunohistochemical CCNE expression on a validated tissue microarray containing 101 de novo DLBCLs and, in 9 cases, the CCNE-induced chromosomal instability as assessed by cytometry.

Results: Forty-six of 98 evaluable DLBCLs expressed CCNE in a mean proportion of 20 ± 29% of tumor cells; 38 cases expressed CCNE in ≥20% of tumor cells. CCNE-positive samples were aneuploid compared with near tetraploidy in CCNE-negative cases. Multivariate analysis showed CCNE expression in ≥20% of tumor cells to be an international prognostic index–independent, Adriamycin-based treatment-independent, and BCL2-independent prognostic factor for poor disease-specific survival. CCNE expression in ≥80% of tumor cells was associated with dismal short-term prognosis. CCNE expression in ≥50% of tumor cells emerged as an independent predictive factor for standard CHOP treatment resistance.

Conclusions: CCNE expression assessment is easy on paraffin-embedded tissue. The high prognostic value of CCNE expression in DLBCL may be the basis for future prospective trials. In addition, a high CCNE expression hints at the presence of a possible target for individualized cancer therapy.
Physiologic Levels of 2-Methoxyestradiol Interfere with Nongenomic Signaling of 17β-Estradiol in Human Breast Cancer Cells
Authors: Vijayanathan Veena , Venkiteswaran Sripriya , Nair Sandhya K. , Verma Arti , Thomas T.J. , Zhu Bao Ting , Thomas Thresia
Published: April 2006

Purpose: The purpose of this investigation is to determine the effects of physiologic levels (10-50 nmol/L) of 2-methoxyestradiol (2ME) on the growth of estrogen receptor (ER)–positive breast cancer cells and provide insights into its mechanism(s) of action.

Experimental Design: Using the ERα-positive breast cancer cells, we studied the effects of 2ME on cell proliferation and cell signaling. Our hypothesis is that 17β-estradiol (E2) and 2ME can affect shared cell signaling pathways, leading to different outcomes in cell proliferation, depending on the absence/presence of E2.

Results: E2 stimulated the growth of MCF-7 and T-47 D cells and induced Akt phosphorylation, a nongenomic signaling pathway. In the absence of E2, 10 to 50 nmol/L of 2ME enhanced cell growth and Akt phosphorylation. However, in the presence of E2, 2ME inhibited E2-induced cell growth and prevented E2-induced Akt phosphorylation. Confocal microscopic studies showed that 2ME inhibited subcellular distribution of ERα in response to E2 in MCF-7 and T-47D cells. 2ME also down-regulated E2-induced increases in cyclic AMP and ornithine decarboxylase activity. In addition, treatment of MCF-7 cells with 2ME in the presence of E2 resulted in a decrease in ERα level by 72 hours. Accelerated down-regulation of ERα may contribute to growth inhibition in the presence of E2/2ME combinations. In contrast, a concentration of up to 2.5 μmol/L 2ME had no effect on the growth of ER-negative SK-BR-3 cells, either in the presence or absence of E2.

Conclusions: Our results provide evidence for the nongenomic action of 2ME in ER-positive cells. In the presence of E2, 2ME suppressed E2-induced cell growth, Akt signaling, and generation of cyclic AMP, whereas it acted as an estrogen in the absence of E2. The intriguing growth-stimulatory and growth-inhibitory effects of 2ME on breast cancer cells suggests the need for its selective use in patients.
Coengagement of CD16 and CD94 Receptors Mediates Secretion of Chemokines and Induces Apoptotic Death of Naive Natural Killer Cells
Authors: Jewett Anahid , Cacalano Nicholas A. , Head Christian , Teruel Antonia
Published: April 2006

Down-modulation of CD16 (FcγRIII) receptors and loss of natural killer (NK) cell function have been observed in oral cancer patients. However, neither the mechanisms nor the significance of the decrease in CD16 receptors have been fully understood. The cytotoxic activity and survival of NK cells are negatively regulated by antibodies directed against CD16 surface receptor. The addition of anti-CD94 antibody in combination with either F(ab′)2 fragment or intact anti-CD16 antibody to NK cells resulted in significant inhibition of NK cell cytotoxic function and induction of apoptosis in resting human peripheral blood NK cells. Addition of interleukin-2 to anti-CD16 and/or anti-CD94 antibody-treated NK cells significantly inhibited apoptosis and increased the function of NK cells. There was a significant increase in tumor necrosis factor-α (TNF-α) but not IFN-γ secretion in NK cells treated either with anti-CD16 antibody alone or in combination with anti-CD94 antibodies. Consequently, the addition of anti-TNF-α antibody partially inhibited apoptosis of NK cells mediated by the combination of anti-CD94 and anti-CD16 antibodies. Increase in apoptotic death of NK cells also correlated with an increase in type 2 inflammatory cytokines and in the induction of chemokines. Thus, we conclude that binding of antibodies to CD16 and CD94 NK cell receptors induces death of the NK cells and signals for the release of chemokines.