bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of Tumor‐Specific mRNA in Cell‐Free Bronchial Lavage Supernatant in Patients with Lung Cancer
Authors: ENGEL E , SCHMIDT B , CARSTENSEN T , WEICKMANN S , JANDRIG B , WITT C , FLEISCHHACKER M
Published: July 2004

Abstract: Bronchoscopy is a standard procedure in the workup of patients with suspicious pulmonary lesions. We wondered whether it is possible to isolate malignancy‐associated mRNA from cell‐free lavage supernatant. Extracellular mRNA from cell‐free lavage supernatant of 25 patients with lung cancer (23 with non‐small cell lung cancer, 2 with small cell lung cancer) was isolated, reverse‐transcribed, and amplified by reverse transcriptase polymerase chain reaction. The quantity and quality of the isolated RNA were checked after cDNA synthesis by amplification with β‐actin‐specific primers. Afterwards, a panel of eight genes known to be expressed in lung tumors was used for the detection of tumor‐associated mRNA expression in lavage supernatant and serum. mRNA coding for β‐actin could be isolated from lavage supernatant of all 25 patients. In addition, the expression of at least one tumor‐associated gene was detectable in all patients. These results show that intact mRNA can be isolated from cell‐free lavage supernatant and that its quantity and quality are sufficient for the detection of tumor‐associated gene expression alterations. This may open new possibilities for the diagnosis of lung cancer.
Real‐Time Quantitative PCR Measurement of Thyroglobulin mRNA in Peripheral Blood of Thyroid Cancer Patients and Healthy Subjects
Authors: LI DIANE , BUTT ASIF , CLARKE SUE , SWAMINATHAN R
Published: July 2004

Abstract: Follow‐up of recurrent differentiated thyroid carcinoma involves the measurement of serum thyroglobulin (Tg). However, Tg autoantibodies are present in a high proportion of thyroid carcinoma patients (up to 25%) and these can interfere with the Tg immunoassays. To overcome this obstacle, investigators have used real‐time quantitative reverse transcriptase polymerase chain reaction (RT‐PCR) to measure Tg mRNA in the blood of patients with differentiated thyroid cancer, with varying degrees of success. In the present study, we demonstrate the first reported use of the PAXgene Blood RNA collection tube and extraction kit method for the preparation of RT‐PCR‐quality RNA with subsequent deployment of the latter in the development of a specific, sensitive, and reproducible Taqman assay for the detection and quantification of thyroglobulin mRNA. Beta‐actin mRNA was also assayed and results are expressed as a ratio of Tg to β‐actin mRNA. The intra‐assay coefficient of variations (CVs) for Tg and β‐actin mRNA assay were 27.7% and 25.4%, respectively. Inter‐assay CVs were 20.8% and 28.8%, respectively, for the two assays. Tg mRNA was detected in all cancer subjects (n= 42) and healthy individuals (n= 20). Tg mRNA was significantly higher in cancer patients than in the healthy subjects (0.00169 ± 0.00013 vs. 0.00051 ± 0.00015; P<0.0001). Fourteen cancer patients had detectable levels of serum Tg, and Tg mRNA levels tended to be higher in these than in cancer subjects with undetectable serum Tg (0.00188 ± 0.00021 vs. 0.00157 ± 0.000178; P= 0.08). Circulatory Tg mRNA measurement may serve a useful role in the assessment of thyroid cancer.
Real‐Time Quantitative PCR Measurement of Circulatory Rhodopsin mRNA in Healthy Subjects and Patients with Diabetic Retinopathy
Authors: HAMAOUI KARIM , BUTT ASIF , POWRIE JAKE , SWAMINATHAN R
Published: July 2004

Abstract: Diabetic retinopathy is the commonest complication of diabetes and is the biggest single cause of registered blindness in the UK. No biochemical tests exist to determine the precise state and rate of change of the eyes in the diabetic patient. In the present study, using real‐time quantitative reverse transcriptase polymerase chain reaction (RT‐PCR), we measured mRNA encoding the retina‐specific pigment protein rhodopsin (RHO) in the peripheral blood of healthy individuals (n= 20) and diabetic patients (n= 46) with and without retinopathy. Beta‐actin mRNA was also assayed and results are expressed as a ratio of RHO to β‐actin mRNA. Peripheral blood was taken by venipucture directly into PAXgene Blood RNA collection tubes and RNA extracted by use of the PAXgene Blood RNA extraction kit, as per the manufacturer's (Qiagen) instructions. Diabetic patients were divided into three groups defined by the severity of retinopathy as assessed by fundoscopy: A, diabetic without retinopathy; B, background retinopathy; and C, preproliferative retinopathy. Medians of the ratios between groups were compared. RHO mRNA was successfully detected and quantified in peripheral blood in all healthy and diabetic groups, with levels shown to be significantly higher in diabetic patients than in healthy controls (2.54 × 10−5 vs. 1.29 × 10−5; P= 0.002). Significant differences in RHO mRNA levels were also seen between healthy control subjects and diabetic groups A (2.52 × 10−5; P= 0.022), B (1.98 × 10−5; P= 0.028), and C (5.08 × 10−5; P= 0.002). The results suggest that there is an increase in circulatory RHO mRNA with the severity of diabetic retinopathy.
Extracellular and Circulating Redox‐ and Metalloregulated eRNA and eRNP: Copper Ion‐Structured RNA Cytokines (Angiotropin Ribokines) and Bioaptamer Targets Imparting RNA Chaperone and Novel Biofunctions to S100‐EF‐Hand and Disease‐Associated Proteins
Authors: WISSLER JOSEF H.
Published: July 2004

Abstract: Bioassays for cellular differentiation and tissue morphogenesis were used to design methods for isolation of bioactive redox‐ and metalloregulated nucleic acids and copper ion complexes with proteins from extracellular, circulating, wound, and supernatant fluids of cultured cells. In extracellular biospheres, diversities of nucleic acids were found to be secreted by cells upon activation. They may reflect nucleic acid biolibraries with molecular imprints of cellular history. After removal of protein components, eRNA prototypes exuded by activated cells were sequenced. They are small, endogenous, highly modified and edited, redox‐ and metalloregulated 5′‐end phosphorylated extracellular eRNA (∼2–200 bases) with cellular, enzymic, and bioaptamer functions. Fenton‐type OH* radical redox reactions may form modified nucleotides in RNA as wobbles eRNA per se, or as copper ion‐complex with protein (e.g., S100A12‐EF‐hand protein, angiotropin‐related protein, calgranulin‐C, hippocampal neurite differentiation factor) are shown to be bioactive in vivo and in vitro as cytokines (ribokines) and as nonmitogenic angiomorphogens for endothelial cell differentiation in the formation of organoid supracellular capillary structures. As bioaptamers, copper ion‐structured eRNA imparts novel biofunctions to proteins that they do not have on their own. The origin of extracellular RNA and intermediate precursors (up to 500 bases) was traced to intracellular parent nucleic acids. Intermediate precursors with and without partial homology were found. This suggests that bioaptamers are not directly retranslatable gene products. Metalloregulated eRNA bioaptamer function was investigated by domains (e.g. 5′…CUG…3′ hairpin loop) for folding, bioactivity, and binding of protein with copper, calcium, and alkali metal ion affinity. Vice versa, metalloregulated nucleic acid‐binding domains (K3H, R3H) in proteins were identified. Interaction of protein and eRNA docking potentials were visualized by 3D‐rapid prototyping of accurate molecular image models based on crystallographic or NMR data. For S100A12‐homologous proteins, receptor‐ and metalloregulated RNA chaperone‐shaped protein assemblies were investigated. They suggest insight into signaling cascades as to how eRNA transmits its cytokine (ribokine) bioinformation from the extracellular RNA biosphere into cells. Proteomics of the extracellular RNA biosphere demonstrate the presence of nucleic acid‐binding domain homologies in defense‐, aging‐, and disease‐associated neuronal and other proteins as targets for RNA orphans. By structural relationships found to transmissible processes, proteinaceous transfer (“infectivity”) and feedback of bioinformation beyond the central dogma of molecular biology are considered in terms of metalloregulated RNA bioaptamer function, nucleic acid‐binding domains, and protein conformation.
Improved Conditions for Isolation and Quantification of RNA in Urine Specimens
Authors: MENKE TIM B. , WARNECKE JENS M.
Published: July 2004

Abstract: There is clear evidence that the occurrence of specific mRNAs in plasma and serum is associated with cancer, while the usefulness of other body fluids for nucleic acid‐based cancer detection remains to be elucidated. Nevertheless, due to the principal advantages of urine (large sample quantities, easy to acquire), several attempts were made to use quantitative reverse transcriptase polymerase chain reaction (RT‐PCR)‐based detection of putative RNA tumor markers from urine as a tool for noninvasive tumor detection. Because most of the commercially available RNA isolation systems do not accommodate larger sample volumes, the majority of experiments were performed using urine pellets. During the centrifugation step, putative extracellular nucleic acids of low molecular weight as well as complexes containing nucleic acids with low density are lost. Furthermore, cells may be destroyed during this procedure, and the subsequently released nucleic acids will quickly be degraded by nucleases in the urine, which may give rise to inconsistent results. Therefore, we established an improved protocol for the isolation of RNA from urine and subsequent quantification steps. The isolation procedure was tested using a quantitative RT‐PCR specific for Ki‐67 RNA as well as a radioactive‐based reverse transcription approach.
Extracellular Ribonucleic Acids of Human Milk
Authors: SEMENOV DMITRY V. , KULIGINA ELENA V. , SHEVYRINA OLESYA N. , RICHTER VLADIMIR A. , VLASSOV VALENTIN V.
Published: July 2004

Abstract: Human milk has been shown to contain heterogeneous oligoribonucleotides varying in size from dimers to 100 mers. The sets of long oligonucleotides in milk samples from different donors and from different stages of lactation have some conservative elements. Sequences of some RNA oligonucleotides correspond to the 3′‐part of 5.8S human ribosomal RNA and to 3′‐parts of tRNAVal and tRNATyr. Primary structures of other oligo‐RNAs are homologous to fragments of human 18S and 28S rRNAs. Concentration of RNA in milk samples tends to decrease with lactation development. The change of RNA concentration and the low concentration of DNA in milk plasma suggest that the source of RNA in human milk may be active secretion of RNA by mammary gland cells.
Therapy Control in Oncology by Circulating Nucleosomes
Authors: HOLDENRIEDER S , STIEBER P
Published: July 2004

Abstract: The courses of circulating nucleosomes in the serum of patients with various solid tumors correlate with the clinical outcome after chemo‐ and radiotherapy. Already during the initial phase of treatment they showed considerable alterations consisting of a rapid increase followed by a decrease during the first therapeutic week. Among patients with advanced lung cancer undergoing chemotherapy, those patients who responded to therapy exhibited less pronounced increases and more complete decreases compared to those patients with insufficient response. In addition, response to therapy was correlated with stronger decreases of the precyclic baseline values from cycle 1 to 2 and from cycle 1 to 3. Thus, circulating nucleosomes are a valuable tool for the early prediction of therapeutic efficacy and can help to modulate therapy strategies early and on an individual basis.
Extracellular DNA in Breast Cancer: Cell‐Surface‐Bound, Tumor‐Derived Extracellular DNA in Blood of Patients with Breast Cancer and Nonmalignant Tumors
Authors: RYKOVA ELENA YU. , LAKTIONOV PAVEL P. , SKVORTSOVA TATYANA E. , STARIKOV ANDREY V. , KUZNETSOVA NINA P. , VLASSOV VALENTIN V.
Published: July 2004

Abstract: A methylation‐specific polymerase chain reaction technique was used to investigate aberrant promoter methylation of RASSF1A and HIC‐1 genes in circulating extracellular DNA (exDNA) from the blood of breast cancer and fibroadenoma patients. Methylated DNA could be detected in the exDNA eluted from the surface of erythrocytes and leukocytes, even in the samples where no methylated DNA could be detected in plasma. The data obtained demonstrate that cell surface bound exDNA provides a valuable source of material for early noninvasive cancer diagnostics and monitoring.
Cell‐Surface‐Bound Nucleic Acids: Free and Cell‐Surface‐Bound Nucleic Acids in Blood of Healthy Donors and Breast Cancer Patients
Authors: LAKTIONOV PAVEL P. , TAMKOVICH SVETLANA N. , RYKOVA ELENA YU. , BRYZGUNOVA OLGA E. , STARIKOV ANDREY V. , KUZNETSOVA NINA P. , VLASSOV VALENTIN V.
Published: July 2004

Abstract: Concentrations of extracellular DNA and RNA in the blood of healthy donors and patients with malignant and nonmalignant breast tumors were investigated. Cell‐surface‐bound extracellular DNA and RNA were detached by PBS‐EDTA treatment or mild trypsin treatment of erythrocytes and leukocytes. In healthy donors, almost all extracellular nucleic acids (98%) are bound at the surface of blood cells. In the blood of cancer patients, extracellular nucleic acids were found in plasma and not at the cell surface. In patients with nonmalignant breast tumors, extracellular nucleic acids were found both at the surface of blood cells and in plasma. In healthy donors, the cell‐surface‐bound DNA is represented by 20‐kbp DNA fragments and smaller fragments that varied in amounts in different fractions.
Isolation of Nucleic Acid Binding Proteins: An Approach for Isolation of Cell Surface, Nucleic Acid Binding Proteins
Authors: CHELOBANOV BORIS P. , LAKTIONOV PAVEL P. , KHARKOVA MARIA V. , RYKOVA ELENA Y. , VLASSOV VALENTIN V.
Published: July 2004

Abstract: An approach for isolation of cell surface, nucleic acid binding proteins is described. This approach relies on affinity modification of the proteins of living cells with reactive oligonucleotides bearing a haptenic group. Covalently modified proteins were isolated by hapten‐specific affinity chromatography with subsequent SDS‐PAGE. Isolated 68‐kDa proteins responsible for the binding of oligonucleotides were MS/MS sequenced and identified as keratin K1, keratin K10, keratin K2e, and albumin.
Extracellular Nucleic Acids in Cultures of Long‐Term Cultivated Eukaryotic Cells
Authors: MOROZKIN EVGENIY S. , LAKTIONOV PAVEL P. , RYKOVA ELENA Y. , VLASSOV VALENTIN V.
Published: July 2004

Abstract: Investigation of the kinetics of nucleic acid release by HeLa (human cervical carcinoma cell line) and A431 (human squamous carcinoma cell line) cells is presented. The released DNA and RNA were shown to accumulate in culture medium and at the cell surface. A portion of cell surface bound RNA can be eluted with PBS/EDTA. Mild trypsin treatment is required for complete detachment of cell surface bound RNA and cell surface bound DNA. Electrophoretic analysis reveals characteristic patterns of cell‐associated and free RNA and DNA molecules.
Patterns of Circulating Hepatitis B Virus Serum Nucleic Acids during Lamivudine Therapy
Authors: HACKER HANS JÖRG , ZHANG WEI , TOKUS MEHMET , BOCK THOMAS , SCHRÖDER CLAUS H.
Published: July 2004

Abstract: Lamivudine treatment of individuals with chronic HBV infection leads to a rapid decline of hepatitis B virus (HBV) serum DNA. Because HBV replication quickly reaches pretreatment values following cessation of the drug, we addressed the question of whether changes during therapy in composition and amount of discernible circulating viral DNA and RNA might provide an explanation for this phenomenon. Nucleic acids were extracted from serial serum samples of two chronically infected patients. The first patient was treated with lamivudine for 14 weeks, whereas the second one, who displayed an HBV virus with a core gene mutation, received lamivudine for 10 weeks. Three sequence segments of the HBV genome synthesized successively during replication, namely, X, C, and X‐preC, were analyzed via competitive polymerase chain reaction (PCR) and reverse transcriptase (RT)/PCR. HBV transcripts were also analyzed for differential polyadenylation. At the start of treatment, identical DNA copy numbers (109/mL) were found for all three segments in the first patient. C segment DNA displayed the expected rapid decline. X‐preC, a target contiguous only on plus‐strand DNA, behaved similarly. In contrast, the X segment DNA copy numbers showed a less pronounced decrease, remaining at higher values (107/mL) than the C and X‐preC segments (both about 2 × 105/mL) at the end of therapy. X segment RNA displayed a persisting copy number of about 107/mL, whereas C and X‐preC RNA decreased to about 105 copies/mL. Polyadenylated HBV RNA, both full‐length and truncated, initially persisted at 105 but decreased to 104 to 103 copies/mL at the end of treatment. As expected, C segment DNA and RNA were not detected in the second patient, whereas X and X‐preC segments showed essentially the same pattern as the first patient, although at a slightly lower level. We conclude that: (1) actual numbers of HBV genome equivalents during lamivudine therapy can be assessed only via X segment DNA, because it is reverse transcribed first; (2) lamivudine induces coexistence of DNA and RNA for the C and X segments at similar levels, indicating drug‐arrested intermediates of reverse‐transcribed HBV DNA minus‐strand; and (3) packaged HBV RNA lacks a poly(A) tail, whereas polyadenylated RNA is likely not packaged.
A Serum Proteomics Approach to the Diagnosis of Ectopic Pregnancy
Authors: GERTON G L. , FAN X J , CHITTAMS J , SAMMEL M , HUMMEL A , STRAUSS J F , BARNHART K
Published: July 2004

Abstract: An ectopic pregnancy (EP) occurs when implantation of the embryo occurs outside of the uterus. If left untreated, the developing fetus will continue to grow, leading to life‐threatening consequences for the mother. A major difficulty with the diagnosis of ectopic pregnancy is that methods of detection are limited, and some, such as ultrasound, are not very reliable in the earliest days of gestation. Currently, no effective serum test exists to distinguish an ectopic pregnancy from a normal intrauterine pregnancy. The incidence of ectopic pregnancy is increasing and has doubled in the last 20 years. It is now the second most common cause of maternal death in the first trimester of pregnancy. To address this issue, we initiated a project to identify serum markers of ectopic pregnancy. The subjects for these studies presented at the Hospital of the University of Pennsylvania. We obtained over 140 serum samples from women with suspected ectopic pregnancy: women presenting with pain and/or bleeding in the first trimester of pregnancy. The approximate racial breakdown of the subjects is as follows: African American, 36%; Caucasian, 3%; Asian, 2%; Hispanic, 1%; unknown, 58%. Serum samples from 139 women (62 with ectopic pregnancy and 77 with a normal intrauterine pregnancy) were applied to WCX2 (weak ion exchange) protein chip surfaces and analyzed for serum markers using surface‐enhanced laser desorption/ionization time‐of‐flight mass spectrometry (SELDI‐TOF‐MS). Several proteins in the 7500–18,000 Da mass range were identified that may discriminate an ectopic pregnancy from an intrauterine pregnancy. The most promising markers were analyzed using classification and regression tree analysis (CART) with and without clinical variables (serum hCG value, length of amenorrhea). Two different algorithms were developed that classify the patients on the basis of sensitivity (number of EPs who screen positive/# of EPs) or specificity (# of healthy patients who screen negative/# of healthy). Our current approach is to refine these two “rule sets” to segregate patients into three groups: those who need immediate intervention for a probable ectopic pregnancy, those who appear to have a normal pregnancy, and those who need further monitoring for diagnosis.
Detection of Differentially Expressed Proteins in Early‐Stage Melanoma Patients Using SELDI‐TOF Mass Spectrometry
Authors: WILSON LORI L. , TRAN LINH , MORTON DONALD L. , HOON DAVE S.B.
Published: July 2004

Abstract: Tumor progression is a dynamic sequence of events that involves specific protein changes. We hypothesized that Surface Enhanced Laser Desorption/Ionization (SELDI) mass spectrometric analysis of sera from patients with AJCC stage I and II melanoma with negative loco‐regional lymph nodes could identify potential melanoma‐associated protein biomarkers of disease recurrence. Serum specimens were collected from 49 patients who developed recurrence (n= 25) or remained free of recurrence (n= 24) without evidence of disease following complete resection (AJCC stage I and II). Follow‐up was longer than 5 years. Serum proteins were denatured and applied onto two protein chip chemistry surfaces (weak cationic WCX2; metal‐binding, IMAC3‐Cu). SELDI ProteinChip™ mass spectrometry was then performed. SELDI data were analyzed, protein peak clustering and classification were performed, and a supervised classification algorithm was employed to classify the dataset. Multiple protein peaks ranging from 3.3 to 30 kDa were identified between patients with recurrence and those without recurrence, and the expression pattern differences of three proteins were used to generate the discriminating classification tree. The biomarkers were expressed with a high degree of reproducibility. In this early characterization study, melanoma recurrence was predicted with a sensitivity of 72% (18/25) and a specificity of 75% (18/24). This novel pilot study revealed three proteins that accurately identified patients who developed recurrence after curative resection of primary melanoma.
Phase III Study of Adjuvant Chemotherapy and Radiation Therapy Compared With Chemotherapy Alone in the Surgical Adjuvant Treatment of Colon Cancer: Results of Intergroup Protocol 0130
Authors: Martenson James A. , Willett Christopher G. , Sargent Daniel J. , Mailliard James A. , Donohue John H. , Gunderson Leonard L. , Thomas Charles R. , Fisher Barbara , Benson Al Bowen , Myerson Robert , Goldberg Richard M.
Published: July 2004

Purpose

Some patients with colon cancer have a high risk of local recurrence postoperatively. This trial was undertaken to determine whether radiation therapy added to an adjuvant chemotherapy regimen improves outcome in high-risk patients.

Patients and Methods

Patients with resected colon cancer with tumor adherence or invasion of surrounding structures, or with T3N1 or T3N2 tumors of the ascending or descending colon were randomly assigned to receive fluorouracil and levamisole therapy with or without radiation therapy. Patients who received chemotherapy and radiation therapy (chemoRT) received 45 to 50.4 Gy in 25 to 28 fractions beginning 28 days after starting chemotherapy. Patient enrollment was terminated because of slow accrual after 222 patients enrolled (original goal was 700 patients); 187 patients were assessable.

Results

Overall 5-year survival was 62% for chemotherapy patients and 58% for chemoRT patients (P > .50); 5-year disease-free survival was 51% for both groups (P > .50). Toxicity (≥ grade 3) occurred in 42% of chemotherapy patients and 54% of chemoRT patients (P = .04). Leukopenia (≥ grade 3) occurred in 10% of chemotherapy patients and 22% of chemoRT patients (P = .02). No significant difference in nonhematologic toxicity (≥ grade 3) was observed between chemoRT and chemotherapy patients (35% v 44%; P = .26).

Conclusion

Patients who received chemotherapy or chemoRT had similar overall survival and disease-free survival. Toxicity was higher among chemoRT patients. These results must be interpreted with caution because of the high number of ineligible patients and the limited power of the study to detect potentially meaningful differences.
Phase I Study of an Immunomodulatory Thalidomide Analog, CC-4047, in Relapsed or Refractory Multiple Myeloma
Authors: Schey S.A. , Fields P. , Bartlett J.B. , Clarke I.A. , Ashan G. , Knight R.D. , Streetly M. , Dalgleish A.G.
Published: July 2004

Purpose

To assess the safety, efficacy, and immunomodulatory effects of CC-4047 (Actimid; Celgene, San Diego, CA) in patients with relapsed or refractory myeloma.

Patients and Methods

Twenty-four relapsed or refractory patients were treated with a dose-escalating regimen of oral CC-4047. Clinical responses and adverse effects were identified, and peripheral T-cell subsets, serum cytokines, and proangiogenic factors were evaluated.

Results

CC-4047 was tolerated with no serious nonhematologic adverse events. All patients were eligible for analysis. Toxicity criteria during the initial 4 weeks of study were used to define the maximum-tolerated dose (MTD). During this period, one patient withdrew with a deep vein thrombosis (DVT) probably caused by an undiagnosed primary melanoma with lymphadenopathy in the groin, one patient withdrew because of progressive disease (PD), and three patients discontinued with neutropenia. Nineteen of 24 patients continued on treatment beyond 4 weeks to PD or development of a serious adverse event. Three further patients developed a DVT at 4, 9, and 11 months. Treatment resulted in a greater than 25% reduction in paraprotein in 67% of patients, 13 patients (54%) experienced a greater than 50% reduction in paraprotein, and four (17%) of 24 patients entered complete remission. The MTD was 2 mg/d. All patients showed increased CD45RO expression on CD4+and CD8+cells, with a concomitant decrease in CD45RA+cells. CC-4047 treatment was associated with significantly increased serum interleukin (IL)-2 receptor and IL-12 levels, which is consistent with activation of T cells and monocytes and macrophages.

Conclusion

This study demonstrates the safety and efficacy of CC-4047. The MTD of CC-4047 orally was 2 mg/d. This is the first report demonstrating in vivo T-cell costimulation by this class of compound, supporting a potential role for CC-4047 as an immunostimulatory adjuvant treatment.
Inhibition of adipocyte differentiation by mechanical stretching through ERK-mediated downregulation of PPARγ2
Authors: Tanabe Yoshiyuki , Koga Masaru , Saito Maki , Matsunaga Yumi , Nakayama Koichi
Published: July 2004

This study investigated the effects of cyclic stretching on adipocyte differentiation of mouse preadipocyte 3T3-L1 cells. Confluent 3T3-L1 cells were treated with dexamethasone, 3-isobutyl-1-methylxanthine and insulin for 45 hours (induction period), followed by incubation with insulin for 9 additional days (maturation period). A transient burst of CCAAT/enhancer-binding protein (C/EBP) β and C/EBPδ at an early stage (∼3 hours) and a delayed induction (∼45 hours) of C/EBPα and PPARγ2 were sequentially provoked during the induction period. Application of cyclic stretching during the entire induction period or only during the final 15 hours of the induction period significantly retarded the induction of glycerol-3-phosphate dehydrogenase (GPDH) activity and the accumulation of intracellular triglycerides by the end of the maturation period. Cyclic stretching for the entire induction period, as well as that applied during the final 15 hours of the induction period, significantly reduced the expression of PPARγ2 mRNA, whereas reduction in the expression of C/EBPδ mRNA was only observed in response to stretching that had been applied during the entire induction period. The expression of C/EBPα and C/EBPβ mRNA did not change in response to stretching. Stretching induced the phosphorylation of extracellular-signal-regulated protein kinases 1 and 2 (ERK1/2), which are members of the mitogen-activated-protein kinase (MAPK) family, during the induction period. PD98,059, a MAPK/ERK kinase inhibitor, reversed the stretch-induced reduction of PPARγ2 at both mRNA and protein levels achieved during the induction period. PD98,059 also restored GPDH activity and lipid droplet accumulation. Furthermore, the differentiation inhibited by the stretching was also restored by synthetic PPARγ ligand. Collectively, these results suggest that the inhibition of adipocyte differentiation in response to stretching is mainly attributable to the reduced expression of PPARγ2, which is mediated by activation of the ERK/MAPK system.
KIN17 encodes an RNA-binding protein and is expressed during mouse spermatogenesis
Authors: Pinon-Lataillade Ghislaine , Masson Christel , Bernardino-Sgherri Jacqueline , Henriot Véronique , Mauffrey Philippe , Frobert Yveline , Araneda Silvia , Angulo Jaime F.
Published: July 2004

Genotoxic agents deform DNA structure thus eliciting a complex genetic response allowing recovery and cell survival. The Kin17 gene is up-regulated during this response. This gene encodes a conserved nuclear protein that shares a DNA-binding domain with the bacterial RecA protein. The KIN17 protein binds DNA and displays enhanced expression levels in proliferating cultured cells, suggesting a role in nuclear metabolism. We investigated this by studying the expression profile of KIN17 protein during mouse spermatogenesis. As expected, the expression level of Kin17 is higher in proliferating than in differentiated cells. KIN17 is selectively extracted from this tissue by detergents and a fraction was tightly associated with the nuclear matrix. Germinal cells ubiquitously express Kin17 and the protein is located mainly in the nucleus except in elongated spermatids where cytoplasmic staining is also observed. Sertoli and germ cells that are no longer mitotically active express KIN17, suggesting a general role in all testicular cell types. In adult testis a significant proportion of KIN17 co-purifies with polyadenylated RNA. KIN17 directly binds RNA, preferentially poly(G) and poly(U) homopolymers. These results together with the identification of KIN17 as a component of the human spliceosome indicate that this protein may participate in RNA processing.
CPD-photolyase adenovirus-mediated gene transfer in normal and DNA-repair-deficient human cells
Authors: Chiganças Vanessa , Sarasin Alain , Menck Carlos Frederico Martins
Published: July 2004

Cyclobutane pyrimidine dimers (CPDs) are the most frequent and deleterious lesions generated in the mammalian genome after UV-C irradiation. The persistence of these lesions in DNA can be toxic and mutagenic, and also represents a specific signal to apoptosis. To investigate the CPDs repair in situ and consequent UV-induced apoptosis in human cells, we generated a recombinant adenovirus vector containing the gene encoding a CPD-photolyase-EGFP fusion protein (Adphr-EGFP). Adphr-EGFP-infected cells are proficient in photorepair, which prevents apoptotic cell death in comparison with samples kept in the dark, indicating that the fusion protein is functional in CPD recognition and removal. By using local UV irradiation, foci of the photolyase fusion protein were observed in UV-damaged areas of the nuclei in colocalization with NER enzymes. Phr-EGFP migration to CPD sites and redistribution after photorepair was followed, and shown to present similar kinetics in normal or DNA-repair-deficient cells. To our knowledge, this is the first report of an investigation of CPDs repair in situ employing a CPD-photolyase-EGFP enzyme. The Adphr-EGFP vector can be an informative tool to investigate the repair and cellular consequences of UV-induced lesions in primary human cells.
Differential effects of a GTP-restricted mutant of Sar1p on segregation of cargo during export from the endoplasmic reticulum
Authors: Stephens David J. , Pepperkok Rainer
Published: July 2004

Export of cargo from the endoplasmic reticulum (ER) is the first membrane trafficking step in the secretory pathway. To date, all cargo proteins appear to use a common set of machinery for the initial stages of export, namely the COPII coat complex. Recent data from both yeast and mammalian systems have emerged suggesting that specific cargoes could be sorted from one another at the point of exit from the endoplasmic reticulum or immediately afterwards. Here, we have examined the mechanisms used for export of different types of cargo molecule from the endoplasmic reticulum. All cargoes examined utilise the COPII machinery, but specific differences are seen in the accumulation of cargo into ER-derived pre-budding complexes following expression of a GTP-restricted mutant of the Sar1p GTPase. Glycosylphosphatidylinositol (GPI)-anchored GFP is seen to be restricted to the ER under these conditions whereas other cargoes, including ts045-G and lumFP accumulate in pre-budding complexes. Following exit, GPI-FP, lumFP and ts045-G-FP all travel to the Golgi in the same vesicular tubular clusters (VTCs). These data show a differential requirement for efficient GTP hydrolysis by the Sar1p GTPase in export of cargo from the ER.
Junctions and vesicular trafficking duringDrosophilacellularization
Authors: Lecuit Thomas
Published: July 2004

The cellularization that converts the syncytial Drosophila embryo into thousands of distinct cells is a hybrid form of cleavage. It derives from cytokinesis and has acquired specific features required for epithelial biogenesis. Cellularization generates an epithelial layer in which adjacent cells are connected by apical adherens junctions. If this process goes awry, subsequent development is dramatically affected, in particular tissue remodelling during gastrulation. Cellularization is associated with the invagination of the plasma membrane between adjacent nuclei at the cell cortex, the formation of a basal-lateral surface and the assembly of apical adherens junctions. The regulated mobilization of intracellular pools of vesicles at defined sites of the plasma membrane underlies membrane growth and surface polarization. Genetic approaches have identified conserved core cellular pathways required for these processes, such as vesicular trafficking along the biosynthetic and endocytic routes, and vesicular insertion into the plasma membrane. The novel proteins Nullo and Slam, which are specifically induced during cellularization, represent developmental regulators of membrane growth during cellularization.
Measurements of Cavity Ringdown Spectroscopy of Acetone in the Ultraviolet and Near-Infrared Spectral Regions: Potential for Development of a Breath Analyzer
Authors: Wang Chuji , Scherrer Susan T. , Hossain Delwar
Published: July 2004

We report a study on the cavity ringdown spectroscopy of acetone in both the ultraviolet (UV) and the near-infrared (NIR) spectral regions to explore the potential for development of a breath analyzer for disease diagnostics. The ringdown spectrum of acetone in the UV (282.4–285.0 nm) region is recorded and the spectrum is in good agreement with those obtained by other spectral techniques reported in the literature. The absorption cross-section of the C–H stretching overtone of acetone in the NIR (1632.7–1672.2 nm) is reported for the first time and the maximum absorption cross-section located at 1666.7 nm is 1.2 × 10−21 cm2. A novel, compact, atmospheric cavity with a cavity length of 10 cm has been constructed and implemented to investigate the technical feasibility of the potential instrument size, optical configuration, and detection sensitivity. The detection limit of such a mini cavity employing ringdown mirrors of reflectivity of 99.85% at 266 nm, where acetone has the strongest absorption, is ∼1.5 ppmv based on the standard 3σ criteria. No real breath gas samples are used in the present study. Discussions on the detection sensitivity and background spectral interferences for the instrument development are presented. This study demonstrates the potential of developing a portable, sensitive breath analyzer for medical applications using the cavity ringdown spectral technique.
Thermodynamics of Complexation of Dimethyl Esters of Tere-, Iso-, and Phthalic Acids with α- and β-Cyclodextrins
Authors: Di Marino Antonio , Mendicuti Francisco
Published: July 2004

Thermodynamics of inclusion of α- and β-cyclodextrins (CDs) with three diester precursors of polyesters, dimethyl terephthalate (DMT), dimethyl isophthalate (DMI), and dimethyl phthalate (DMP) were studied with different fluorescence spectroscopy techniques. The stoichiometry, association constants, and other thermodynamics parameters were investigated from the fluorescence intensity decrease that takes place upon CD addition and temperature changes. Apart from DMP, which did not form a complex with αCD, all other systems showed 1:1 stoichiometry complexes with different stabilities. The complexes formed with the DMT guest are the most stable, whereas the stability constant for DMP: βCD is the lowest. Binding constants are also larger for complexes formed with βCD than for those formed with αCD. Fluorescence polarization, quenching, and lifetime measurements provide information about the structure of the complexes formed. This geometry explains the values of enthalpy and entropy changes during complexation. Van der Waals interactions seem to be involved in the formation of these complexes.
In-Line Reaction Monitoring of a Methyl Methacrylate and N,N-Dimethylacrylamide Copolymerization Reaction Using Near-Infrared Spectroscopy
Authors: Beyers C. P. , Boelens H. F. M. , Klumperman L. , Westerhuis J. A.
Published: July 2004

Fast and accurate monitoring of monomer concentration during copolymerization reactions is of much interest. It is known that near-infrared spectroscopy (NIRS) can be used to monitor polymerization reactions. Here, a free radical solution copolymerization reaction between methyl methacrylate and N,N-dimethylacrylamide is considered. NIR spectra were measured in-line with a transflectance probe. The spectra of both involved monomers are very similar, making monitoring with NIRS challenging. It is shown that the NIRS calibration can be set up with only a few (5) off-line measured mixtures. Several validation methods for such a NIRS calibration model are discussed and tested. NIRS is used to follow conversion of the two monomers in a copolymerization reaction on-line.
Self‐Consistent Hybrid Method: Theory and Applications to Ultrafast Electron Transfer Reactions in Condensed Phases
Authors: Wang Haobin , Thoss Michael
Published: July 2004

The self‐consistent hybrid method is an efficient approach for simulating quantum dynamics in complex systems. In this method, the overall system is partitioned into a core and a reservoir. The former is treated via a numerically exact quantum mechanical method and the latter is treated via a more approximate approach. An iterative convergence procedure is then introduced to systematically improve the accuracy of the result for the overall quantum dynamics. In this article, we review the methodological development and practical implementations of the self‐consistent hybrid method, as well as its applications to the study of ultrafast electron transfer reactions in condensed phases.
Quantum Rate Constant Calculations for Polyatomic Reactions
Authors: Huarte‐Larrañaga Fermín , Manthe Uwe
Published: July 2004

In this paper we review an efficient method for the accurate quantum mechanical (QM) calculation of thermal rate constant. The approach employs flux correlation functions to directly calculate the thermal rate constant, without having to perform a full reactive scattering calculation. All quantum effects such as tunneling or zero point energy are correctly included. This method provides therefore a rigorous quantum description of the reactive process. The combination of the flux correlation function formalism with the MultiConfigurational Time Dependent Hartree (MCTDH) wave packet scheme has given a tool capable of accurately simulating the dynamics of polyatomic reactions. In this article, the theoretical outlines of the methodology are given together with some explanatory examples and applications that illustrate its potentiality in the field of polyatomic reaction dynamics.
Pneumatic conveying of granular solids in horizontal and inclined pipes
Authors: Zhu Kewu , Wong Chun Kit , Madhusudana Rao S. , Wang Chi‐Hwa
Published: July 2004

Computational fluid dynamics simulations are used to investigate the pneumatic conveying of granular solids through an inclined pipe at different inclinations. Model predictions agreed reasonably well with the measurements reported by Tsuji and Morikawa in 1982 for mean gas and solids velocities. The results of influence of model parameters, inclination angle, and feeding conditions on the flow patterns were also reported. Particle–wall collisions were found to have a very significant effect on the solids distribution over the cross section of the conveying tube for large particles. Upon introducing finite‐amplitude sinusoidal fluctuations in the gas velocity at the inlet of the pipe, solids density waves were observed to move along the conveying line with significant axial dispersions. © 2004 American Institute of Chemical Engineers AIChE J, 50: 1729–1745, 2004
Measurement of granular temperature and stresses in risers
Authors: Tartan Mehmet , Gidaspow Dimitri
Published: July 2004

Detailed experimental velocity, particle concentration, and stresses for flow of particles in a vertical pipe, riser are needed for verification of various CFD models for multiphase flow. This is the principle‐unsolved task of the Fluid Dynamics Multiphase Flow Consortium, organized by Dow Chemical Company. This study provides such information for flow of 530 μm glass beads in the fully developed flow region of a 7 m symmetric riser with a splash plate. Instantaneous particle‐velocity distributions were obtained using a particle velocity imaging technique, and a probe inserted into the riser, while the particle concentrations were measured with a gamma‐ray densitometer. Time‐averaged particle‐velocity distributions can be well represented by a parabolic‐ velocity distribution, with the mean velocity obtained from flux divided by the measured bulk density. The flow is very anisotropic. The radial granular‐temperature profiles agree with an analytical expression similar to the thermal‐temperature distribution in Poiseuille flow with viscous heat generation. A numerical solution for the standard isotropic model developed shows that the approximations made in the analytical solutions are reasonable. In the core, the normal Reynolds stresses are much smaller than the velocity averaged particle stresses, whereas near the wall the time averaged normal Reynolds stresses are large. © 2004 American Institute of Chemical Engineers AIChE J, 50: 1760–1775, 2004
Aerated vibrofluidization of silica nanoparticles
Authors: Nam Caroline H. , Pfeffer Robert , Dave Rajesh N. , Sundaresan Sankaran
Published: July 2004

Vigorous homogeneous fluidization of 12‐nm silica particles was easily achieved by coupling aeration with vibration. Vibration (with frequency in the range of 30 to 200 Hz, and vibrational acceleration in the range of 0 to 5 g) was found to be necessary to achieve smooth fluidization. The minimum fluidization velocity, defined as the lowest gas velocity at which the pressure drop across the bed reaches a plateau, was approximately 0.3–0.4 cm/s, and essentially independent of the vibrational acceleration. However, the bed expanded almost immediately after the air was turned on, reaching bed expansions of three times the initial bed height or higher. Thus the bed appeared to exhibit a fluidlike behavior at velocities much lower than the minimum fluidization velocity. Fluidization of nanoparticles was achieved as a result of the formation of stable, relatively large, and very porous agglomerates. Practically no bubbles or elutriation of particles was observed. A fractal analysis combined with a modified Richardson–Zaki approach is proposed for prediction of agglomerate size and voidage. © 2004 American Institute of Chemical Engineers AIChE J, 50: 1776–1785, 2004
Analytical treatment of fragmentation‐diffusion population balance
Authors: Kostoglou M. , Karabelas Anastasios J.
Published: July 2004

The quest for advanced models to realistically simulate various processes involving populations of “particles” (aerosols, dispersions of all kinds, minerals, cultures of microorganisms) necessitates the extension of the classical population balance formulation, which is of zero dimension (lumped) in external and one‐dimensional (1‐D) (single particle‐characterization variable) in internal coordinate space, to more dimensions in both spaces. In order to contribute in this direction, this work is focused on a spatially distributed fragmentation equation, the spatial nonuniformity being governed by a diffusion mechanism and by deposition of particles on solid boundaries. The combined effect of diffusion and fragmentation on the particle‐size distribution is of interest here. The linear form of the fragmentation‐diffusion equation allows a convenient analytical manipulation, based on decomposition of solutions in spatial and temporal modes. Generalized solutions are derived for the above equation for arbitrary spatial domains, fragmentation functions, deposition rates and particle sources. The above approach can be combined with the well‐known approaches for the numerical solution of the lumped fragmentation equation to derive solutions for the fragmentation‐diffusion equation under arbitrary conditions. This study, as a first step, is restricted to obtaining explicit results for some rather simple cases, for example, power law fragmentation and deposition rates, fragmentation in equal sizes and simple shapes of the domain under consideration. © 2004 American Institute of Chemical Engineers AIChE J, 50: 1746–1759, 2004
On the reliability of recent tests of the Out of Africa hypothesis for modern human origins
Authors: Bräuer Günter , Collard Mark , Stringer Chris
Published: July 2004

In this paper we critique two recent studies that have been claimed to disprove the Out of Africa hypothesis for modern human origins (Hawks et al., 2000; Wolpoff et al., 2001). We show that the test prediction employed by Hawks et al. (2000) and Wolpff et al. (2001) is not relevant to many versions of the Out of Africa hypothesis, and that the key specimens they used are problematic in terms of morphological representativeness. We also show that there are significant problems with the character state datasets employed in the studies. Lastly, we highlight evidence that the main method used in the studies (pairwise difference analysis) is not reliable when applied to the type of data employed by Hawks et al. (2000) and Wolpoff et al. (2001). In view of the foregoing, we contend that Hawks et al.'s (2000) and Wolpoff et al.'s (2001) claim to have disproved the Out of Africa hypothesis cannot be sustained. © 2004 Wiley‐Liss, Inc.
Distribution of calcitonin gene‐related peptide at the neuromuscular junction of mdx mice
Authors: Marques Maria Julia , Minatel Elaine , Guimarães Alessandra Oliveira , Neto Humberto Santo
Published: July 2004

In normal skeletal muscle, the protein dystrophin is associated with plasma membrane glycoproteins and may be involved in the stabilization of the sarcolemma. Mutant mdx mice are markedly deficient in dystrophin and show muscle fiber necrosis followed by regeneration. Changes in the distribution of acetylcholine receptors (AChRs) have been reported at the neuromuscular junction of mdx mice possibly as a result of alterations in the release or response to neural trophic factors. One such factor is calcitonin gene‐related peptide (CGRP), which has been implicated in AChR synthesis and function. In this study, we used rhodamine‐α‐bungarotoxin and anti‐CGRP IgG FITC to study AChR and CGRP distribution at the neuromuscular junction of mdx mice. Using laser scanning fluorescence confocal microscopy, it was possible to see that CGRP‐like immunoreactivity had a presynaptic distribution, covering the AChRs. Thirty‐four percent of dystrophic junctions were found to be labeled with CGRP compared to 80% of control endplates. Since CGRP‐positive and ‐negative fibers showed similar changes in AChR distribution, it is suggested that CGRP is probably not directly involved in the altered pattern of AChR seen in dystrophin‐deficient muscle fibers of mdx mice. © 2004 Wiley‐Liss, Inc.