bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Serological Diagnosis in Transitional Cell Bladder Cancer
Authors: VON KNOBLOCH ROLF , BRANDT HEIDRUN , HOFMANN RAINER
Published: July 2004

Abstract: There are no reliable serological tumor markers for transitional cell carcinoma (TCC) of the urinary bladder. Fluorescent microsatellite analysis (MSA) was used to detect serum‐DNA alterations in patients with bladder cancer. Prospectively, fresh tumor‐, peripheral blood‐, and serum‐specimens were collected from 58 consecutive patients treated for TCC of the bladder to obtain the corresponding DNA. DNA was extracted by the phenol‐chloroform method from tumors and blood lymphocytes. Serum DNA was isolated by a commercial kit. Fluorescent MSA was performed with a total of 17 polymorphic markers from chromosomal regions 5q, 8p, 9p, 9q, 13q, 14q, 17p, 17q, and 20q in the 58 cancer specimens as well as in specimens from 20 healthy controls. Detection of allelic imbalance and loss of heterozygosity in the tumor and serum specimens was carried out on an automated laser sequencer. Serum‐DNA alterations were identified in 79.3% (46/58). Four healthy controls displayed serum‐DNA artefacts rendering a specificity of 80%. The highest frequency of serum‐DNA alterations (38%) was detected for chromosomal region 8p. Chromosomes 5q, 9p, and 20q showed serum‐DNA alterations in 20% to 23%. Identification of tumor‐specific serum‐DNA alterations was stage independent (P >0.05), but was more frequent in high‐grade tumors (P= 0.08). To optimize specificity, simultaneous analysis of tumor DNA is advised to rule out artefacts resembling allelic imbalance in MSA of serum DNA. The method then has potential for application as a “serological marker” in the follow‐up of patients after radical surgical therapy for invasive TCC of the bladder.
Transrenal DNA as a Diagnostic Tool: Important Technical Notes
Authors: SU YING‐HSIU , WANG MENGJUN , BLOCK TIMOTHY M. , LANDT OLFERT , BOTEZATU IRINA , SERDYUK OL'GA , LICHTENSTEIN ANATOLY , MELKONYAN HOVSEP , TOMEI L DAVID , UMANSKY SAMUIL
Published: July 2004

Abstract: A small portion of DNA from apoptotic cells escapes complete degradation, appears in blood as oligonucleosomal‐size fragments, is excreted in the urine, and can be used for diagnostic purposes. More detailed study revealed that transrenal DNA (Tr‐DNA) belongs to a relatively low molecular‐weight (150–250 bp) fraction, thereby requiring more careful attention to methods employed for purification and analysis. For example, here it is demonstrated that the QIAamp blood kit purifies primarily high molecular‐weight DNA from serum, whereas the Guanidine/Promega Wizard Resin (GITC/WR) method purifies primarily low molecular‐weight DNA. As a result, sensitivity in detection of K‐RAS mutations in serum of patients with colorectal tumors is significantly higher with DNA isolated with the GITC/WR method than with the QIAamp kit. Amplicon size is also extremely important in analysis of Tr‐DNA, because the shorter the amplicon, the higher is the sensitivity of biomarker detection in Tr‐DNA. One hundred fifty‐seven and 87 bp amplicons were employed for detection of mutant K‐RAS in DNA isolated from 0.1 mL of urine obtained from 15 patients with pancreatic cancer. Mutant K‐RAS was found in Tr‐DNA of 3 and 10 patients with the long and short amplicons, respectively. The sensitivity and specificity of detection of mutant sequences are reduced in the presence of high excess of a respective wild‐type allele, but they can be significantly increased through application of enriched polymerase chain reaction (PCR), peptide nucleic acid (PNA) clamped PCR, and/or stencil‐aided mutation analysis (SAMA), based on selective pre‐PCR elimination of wild‐type sequences.
A Clinical Service in the UK to Predict Fetal Rh (Rhesus) D Blood Group Using Free Fetal DNA in Maternal Plasma
Authors: FINNING KIRSTIN , MARTIN PETER , DANIELS GEOFF
Published: July 2004

Abstract: Antenatal determination of fetal blood group is important in pregnancies with a significant risk of hemolytic anemia due to maternal alloimmunization. The International Blood Group Reference Laboratory (part of the National Blood Service) in Bristol, UK, provides a fetal blood group genotyping service to obstetricians caring for immunized pregnant women with heterozygous partners. Since 2001, fetal D typing has been offered using free fetal DNA in maternal plasma. Real‐time polymerase chain reaction (PCR) assays are performed to detect the RHD gene. To confirm the presence of fetal DNA when RHD is not detected, Y‐chromosome sequences are targeted. When a D‐negative female fetus is predicted, maternal buffy coat DNA is tested for eight insertion/deletion polymorphisms. Sequences that are absent from the maternal genome are then targeted in maternal plasma and are used to confirm the presence of free fetal DNA in the blood sample. Currently, 283 pregnancies have been tested, of which 50 are awaiting confirmatory results. Fetal D status was correctly predicted in 223 cases, and no result was obtainable in 7 cases. In three cases, serology on cord blood was discrepant with reported results, but all fetuses had received multiple intrauterine transfusions. The new test has significantly reduced the number of invasive procedures carried out in the UK for fetal D grouping. Antenatal anti‐D prophylaxis is currently being introduced in the UK to all D‐negative women; in the future, detection of fetal RHD sequences in maternal plasma may allow anti‐D to be restricted to pregnancies involving a D‐positive fetus.
Circulating Cell‐Free Fetal Nucleic Acid Analysis May Be a Novel Marker of Fetomaternal Hemorrhage after Elective First‐Trimester Termination of Pregnancy
Authors: WATAGANARA TUANGSIT , LESHANE ERIK S. , CHEN ANGELA Y. , SULLIVAN LISA M. , PETER INGA , BORGATTA LYNN , JOHNSON KIRBY L. , BIANCHI DIANA W.
Published: July 2004

Abstract: Analysis of cell‐free fetal DNA (fDNA) and RNA in maternal plasma could be useful in the diagnosis and management of complications of pregnancy. In this review, we discuss our studies to investigate the potential of fetal nucleic acid measurement in maternal plasma as a marker of fetomaternal hemorrhage (FMH) after elective first‐trimester termination of pregnancy (TOP). Using quantitative real‐time PCR amplification of the DYS1 sequence, elevation of plasma fDNA levels after TOP was observed, especially in the late first trimester. This corresponds with the functional development of the placental vascular structure and fetal hematopoiesis. This Y sequence‐based PCR amplification assay, however, limits the analysis to pregnant women carrying male fetuses. Therefore, we also developed a real‐time quantitative reverse‐transcriptase PCR assay of the γ‐globin transcript as a marker of fetal erythroid cells. Although plasma γ‐globin mRNA levels were decreased after TOP in many patients, an elevation was observed in some patients at greater than 9 weeks' gestation, which is consistent with the increase in plasma fDNA levels. Our data suggest that fetal hematopoietic cells contribute to the pool of fetal nucleic acids in the maternal circulation. Measurement of cell‐free fetal nucleic acid levels in maternal plasma may have clinical application as a novel marker of FMH after 9 weeks of gestation.
The Biology and Diagnostic Applications of Plasma RNA
Authors: LO Y M DENNIS , CHIU ROSSA W.K.
Published: July 2004

Abstract: The detection of cell‐free RNA in the plasma and serum of human subjects has opened up new investigational opportunities. Tumor‐derived RNA was detected in the plasma and serum of patients with various forms of cancer. Through the development of robust extraction and quantification protocols for circulating RNA, new information regarding its biology and characteristics has been obtained. Plasma RNA was shown to be particle associated and to be surprisingly stable. Recently, fetal RNA was also demonstrated in maternal plasma. The placenta was shown to be an important source of such circulating fetal RNA. This discovery has created new prenatal diagnostic and monitoring possibilities. It is expected that circulating RNA will find application in many other clinical scenarios, including emergency medicine.
The Use of Real‐Time Quantitative PCR to Detect Circulating Prostate‐Specific Membrane Antigen mRNA in Patients with Prostate Carcinoma
Authors: CHU DA‐CHANG , CHUANG CHENG‐KENG , LIOU YI‐FANG , TZOU RON‐DAR , LEE HSIAO‐CHUN , SUN CHIEN‐FENG
Published: July 2004

Abstract: Prostate‐specific antigen (PSA) has long been criticized for its lack of specificity in screening for the occurrence of prostate cancer. In this study, we tried to measure levels of another biomarker, prostate‐specific membrane antigen (PSM), in the peripheral circulation from subjects with either prostate cancer or benign prostatic hyperplasia (BPH). Total RNA was extracted from blood samples of 70 patients with prostate cancer and 19 with BPH. Reverse transcription was performed to convert mRNA to cDNA. The cDNA was analyzed with a novel real‐time quantitative polymerase chain reaction (PCR) protocol to measure PSM mRNA levels in the circulation. Melting curve analysis was adapted to assure that correct amplification data were obtained. Results showed that 41 of 70 prostate cancer patients had positive results, whereas 9 of 19 BPH cases were negative. Therefore, the sensitivity and specificity were determined to be 58.6% and 47.4%, respectively. For comparison, traditional nested PCR was performed to investigate whether the new method was superior. The sensitivity and specificity of nested PCR were determined to be 27.1% and 57.9%, respectively. The detection limits of these two methods were 0.0005 ng (for the real‐time quantitative PCR method) and 0.5 ng of PSM‐cDNA (for the nested PCR method). In conclusion, we have successfully developed a novel, noninvasive real‐time quantitative PCR method to detect the PSM mRNA levels in the peripheral circulation of prostate cancer subjects. This method may provide references for urologists diagnosing prostate cancer or monitoring the patient's condition after treatment.
Transcession of DNA from Bacteria to Human Cells in Culture: A Possible Role in Oncogenesis
Authors: ANKER PHILIPPE , ZAJAC VLADIMIR , LYAUTEY JACQUELINE , LEDERREY CHRISTINE , DUNAND CHRISTOPHE , LEFORT FRANÇOIS , MULCAHY HUGH , HEINEMANN JACK , STROUN MAURICE
Published: July 2004

Abstract: The human organism is continuously in close contact with microorganisms, especially bacteria. In the present work, by means of a real‐time polymerase chain reaction (PCR) technique, we looked for the presence of a distinct bacterial gene in human cells. To this end, we cultured a human cell line, HL60, in a supernatant in which bacteria (Bacillus subtilis) had been grown. A transient transcession of bacterial DNA into the human cells was observed.
Detection of Cell‐Free DNA in Bronchial Lavage Fluid Supernatants of Patients with Lung Cancer
Authors: CARSTENSEN TIM , SCHMIDT BERND , ENGEL EILEEN , JANDRIG BURKHARD , WITT CHRISTIAN , FLEISCHHACKER MICHAEL
Published: July 2004

Abstract: Recently, it was shown that it is possible to isolate free circulating DNA from plasma/serum of patients with benign and malignant diseases. In addition, several groups were able to detect tumor‐associated alterations in these nucleic acids. We wondered whether any nucleic acids are detectable in cell‐free bronchial lavage supernatants, which until now have been discarded after cell harvest. Additionally, we wanted to find out if it is possible to detect tumor‐associated alterations in these DNA molecules. DNA was isolated from cell‐free lavage supernatants from 30 lung cancer patients, and the DNA was examined for microsatellite alterations. Intact DNA could be isolated from all cell‐free bronchial lavage supernatants. Microsatellite alterations were found in lavage supernatants of 12 of 30 patients and in lavage cells of 6 of 30 patients. Altogether, alterations were found in 14 of 30 patients. Thus, we could demonstrate for the first time that it is possible to isolate intact DNA from cell‐free bronchial lavage supernatants. Their quantity and quality are sufficient for further amplification via polymerase chain reaction. Altogether, tumor‐associated changes were detected in the DNA of 47% of the patients that were analyzed.
Cell‐Free Plasma DNA as a Prognostic Marker in Intensive Treatment Unit Patients
Authors: WIJERATNE S , BUTT A , BURNS S , SHERWOOD K , BOYD O , SWAMINATHAN R
Published: July 2004

Abstract: Recent evidence suggests that cell‐free plasma DNA has potential use as a prognostic marker in many clinical settings. The aim of the present study was to evaluate the prognostic role of cell‐free plasma DNA in the prediction of clinical outcome in intensive treatment unit (ITU) patients. Cell‐free plasma DNA was measured by real‐time polymerase chain reaction assay for the β‐globin gene and SOFA score, APACHE II score, CRP concentrations, and clinical outcome (duration of stay, ventilation time, and mortality) were noted in 94 patients on admission to the ITU. The median plasma DNA concentration in ITU patients was 5493 GE/mL and this was significantly (P <0.001) higher than the DNA concentration in healthy subjects (1970 GE/mL). DNA concentration demonstrated a significant correlation with serum C‐reactive protein (CRP) (r= 0.363) concentration and S epsis‐related O rgan F ailure A ssessment (SOFA) (r= 0.360) score (P <0.001 for both by Pearson correlation) but not with A cute Physiology And Chronic Health Evaluation (APACHE II) score. Patients on ventilation had significantly higher DNA concentrations compared to nonventilated patients (7362 GE/mL versus 4479 GE/mL; P= 0.004). The median DNA concentration in nonsurvivors was 9148 GE/mL, and this was 2.3‐fold greater than that in survivors (3921 GE/ml, P <0.001). ROC analysis of the data indicated a sensitivity of 85% and a specificity of 80% when DNA concentration of 6109 GE/mL was taken as a predictor of death. The data suggest that cell‐free plasma DNA concentration is potentially useful as a prognostic marker in ITU patients.
High Sensitivity Scanning of Colorectal Tumors and Matched Plasma DNA for Mutations in APC, TP53, K‐RAS, and BRAF Genes with a Novel DHPLC Fluorescence Detection Platform
Authors: LILLEBERG S L , DUROCHER J , SANDERS C , WALTERS K , CULVER K
Published: July 2004

Abstract: Tumor‐associated DNA has been detected in plasma of colorectal cancer (CRC) patients using various techniques but with limited gene or mutation coverage. We report a highly sensitive scanning methodology for mutational assessment of the APC and TP53 genes, which typically pose an analytical challenge because of their significant genotypic heterogeneity as well as specific mutational scoring assays for K‐RAS and BRAF. Plasma DNA isolated from 20 CRC patients were scanned for mutations in these targets without knowledge of the molecular or pathological analyses of the matched primary tumors. We chose mutation scanning technology and these molecular targets to provide a comprehensive screen for somatic mutations known to be associated with sporadic CRC. Mutations were identified with a novel denaturing high‐performance liquid chromatography (DHPLC) platform that uses post‐separation fluorescence technology to enable the detection of variants that represent <0.1% of the total analyzed DNA. Mutant allele specific amplification (MASA) followed by detection with the same platform was used to identify low‐level target mutations (mutation scoring) in K‐RAS codons 12, 13, and 61, and BRAF codon 599. Using this combined scanning and scoring approach, we were able to identify at least one mutational event in 20/20 (100%) CRC patients. The thoroughness of a mutation scanning and scoring panel may have important implications for CRC screening and disease monitoring during and following therapy.
A New Technology for Mutation Detection
Authors: SHACKELFORD W , DENG S , MURAYAMA K , WANG J
Published: July 2004

Abstract: A sensitive, accurate, and simple method, called shifted termination assay (STA), was developed for detection of genetic mutations. The STA technology can be used to detect genetic mutations in polymerase chain reaction (PCR)‐amplified samples of tissue, and plasma and serum that include circulating DNA containing point mutations, insertions, deletions, translocations, or single nucleotide polymorphisms (SNPs). STA is a multiple‐base and multiple‐cycle primer extension based detection method that can identify mutant DNA in samples containing as little as 1% mutant DNA in a mixture with 99% wild‐type DNA. On the basis of the STA method, a mutation detection kit, called Mutector, was developed. Mutector is a 96‐well microplate format test kit with colorimetric or luminescent detection options. An initial study shows that Mutector is more accurate and sensitive than sequencing and PCR restriction enzyme analysis methods in detecting DNA mutations from clinical samples.
Clinical Role of Circulating Epstein‐Barr Virus DNA as a Tumor Marker in Lymphoepithelioma‐Like Carcinoma of the Lung
Authors: NGAN ROGER K.C. , YIP TIMOTHY T.C. , CHENG W W , CHAN JOHN K.C. , CHO WILLIAM C.S. , MA VICTOR W.S. , WAN K K , AU Joseph S.K. , LAW C K
Published: July 2004

Abstract: Nineteen Chinese patients with lymphoepithelioma‐like carcinoma (LELC) of the lung were tested for Epstein‐Barr virus (EBV) DNA in their serum samples by a quantitative polymerase chain reaction (PCR) technique. There was prospective serial monitoring of the serum in seven patients with advanced inoperable or relapsing disease. Five other patients at first diagnosis and two patients at relapse had only a single serum sample available. Serum samples were also taken from three other patients who had prior curative surgery and two patients with prolonged disease remission. Measurable levels of EBV DNA were detected in 11 of 12 patients with a pre‐therapy serum sample and a clinically evident tumor. A low level of EBV DNA was also detectable in one of the two other patients whose first serum samples were obtained after some chemotherapy. There was no detectable EBV DNA in the five patients without evidence of tumor. The longitudinal serum EBV DNA profile of seven patients showed consistent correlation with response to therapy and clinical outcome. Patients with a pre‐therapy serum EBV DNA >10,000 copies/mL had significantly inferior overall survival. This study suggests that circulating serum EBV DNA can be used as a tumor marker in the clinical management of patients with LELC of the lung.
Activation of HHV‐6 in Lymphoproliferative Disorders: A Polymerase Chain Reaction‐Based Study
Authors: TAILOR P B , SAIKIA T K , ADVANI S H , MUKHOPADHYAYA R
Published: July 2004

Abstract: HHV‐6 is a latent herpes virus persisting throughout the adult life of the infected host in an integrated form and is often activated in immunocompromised situations. Detection of HHV‐6 DNA in the plasma of an individual indicates the presence of active viral replication in the host. Because lymphomas are known to be associated frequently with host immunosupression, we studied activation of HHV‐6 in 98 patients diagnosed with Hodgkin's disease (HD) or non‐Hodgkin's lymphoma (NHL). HHV‐6 activation was documented in 34% of cases of non‐Hodgkin's lymphoma and 39% of those of Hodgkin's disease; however, no correlation of activation status with pathological types of Hodgkin's disease and between copy numbers in peripheral blood mononuclear cell DNA and the corresponding plasma DNA was noticeable.
Cancer Proteomics: Serum Diagnostics for Tumor Marker Discovery
Authors: RAI ALEX J. , CHAN DANIEL W.
Published: July 2004

Abstract: Cancer proteomics is an exciting field that is witnessing many new developments in recent years. It is hoped that these advances will result in decreased cancer death rates, which have not declined dramatically in the last several decades. Some of the problems with current tumor markers include the lack of sensitivity and specificity, factors that prevent their use in population‐based screening of disease. Thus, there is an urgent need to identify novel biomarkers that can faithfully detect the disease state. As we are now in the post‐genome era, many opportunities have been created. Genomic sequence data are available for human, as well as several other species. We are now poised to mine these data and to determine the functions of the encoded proteins constituting the human genome. Proteomics affords this opportunity by providing enhanced procedures and tools for discovery and also a framework for understanding these components in terms of pathogenesis. New technologies and improvements in existing methodologies will allow for the rapid growth in the identification and characterization of peptides and proteins that are unique to various clinical states. This technology can be successfully applied to clinical specimens for the identification of new tumor markers.
Clinical Proteomics and Biomarker Discovery
Authors: JOHANN DONALD J. , MCGUIGAN MICHAEL D. , PATEL AMIT R. , TOMOV STANIMIRE , ROSS SALLY , CONRADS THOMAS P. , VEENSTRA TIMOTHY D. , FISHMAN DAVID A. , WHITELEY GORDON R. , PETRICOIN EMANUEL F. , LIOTTA LANCE A.
Published: July 2004

Abstract:Early detection of disease generally provides much‐improved outcomes by a definitive medical procedure or through lifestyle modification along with specific medical management strategies. For serum biomarkers, which are central to the diagnosis of many diseases, to become truly useful sentinels of pathogenesis, their sensitivity and specificity in both early detection and recurrence monitoring must be improved. Currently, the detection and monitoring of disease markers is based on solitary proteins, and this approach is not always reliable. New classes of biomarkers derived from mass spectroscopy analysis of the low molecular weight proteome have shown improved abilities in the early detection of disease and hence in patient risk stratification and outcome. The development of a modular platform technology with sufficient flexibility and design abstractions allowing for concurrent experimentation, test, and refinement will help speed the progress of mass spectroscopy‐derived proteomic pattern‐based diagnostics from the scientific laboratory to the medical clinic. For acceptance by scientists, physicians, and regulatory personnel, new bioinformatic tools are essential system components for data management, analysis, and intuitive display of these new and complex data. Clinically engineered mass spectroscopy systems are essential for the further development and validation of multiplexed biomarkers that have shown tremendous promise for the early detection of disease.
Upregulation of the Tissue Inhibitor of Metalloproteinase-1 Protein Is Associated With Progression of Human Non–Small-Cell Lung Cancer
Authors: Aljada Ibrahim S. , Ramnath Nithya , Donohue Kathleen , Harvey Shashi , Brooks John J. , Wiseman Sam M. , Khoury Thaer , Loewen Gregory , Slocum Harry K. , Anderson Timothy M. , Bepler Gerold , Tan Dongfeng
Published: July 2004

Purpose

Tissue inhibitors of metalloproteinases (TIMPs) are naturally occurring inhibitors of matrix metalloproteinases (MMPs). It has been shown that TIMP-1 may be a multifunctional protein. Little is known about the role of TIMP-1 in progression and metastasis of human lung cancer (tumor inhibiting or tumor promoting), although studies using a variety of techniques have analyzed the expression of TIMP-1 mRNA and/or protein in human cancers.

Patients and Methods

We examined the expression of TIMP-1 protein by immunohistochemistry in patients (n = 160) with primary respectable (stage I to IIIA) non–small-cell lung cancer (NSCLC).

Results

Twenty-seven percent of the tumors (43 of 160) demonstrated elevated expression of this protein. We demonstrate that overexpression of TIMP-1 protein is associated with an adverse outcome. In addition, disease stage, patient's age, and performance status were all significantly related to survival. In multivariate analyses, patients with high TIMP-1 expression had a 90% increased risk of death when compared with those with low expression (relative risk, 1.92; 95% CI, 1.19 to 3.09; P = .008). TIMP-1 expression did not correlate with expression of MMP-2 and MMP-9.

Conclusion

These results suggest that TIMP-1, independent of its inhibiting activity of MMPs, may have other function(s) critical for NSCLCs. The significance of our results is two-fold. The adverse outcome in patients with overexpression of TIMP-1 indicates its potential prognostic value in NSCLC. Thus, TIMP-1 overexpression may serve to help identify patients with particularly aggressive disease for adjuvant treatments. In addition, the TIMP-1 molecule may represent a novel therapeutic target for treatment of some NSCLCs.
Calcium binding protein 1 of the protozoan parasiteEntamoeba histolyticainteracts with actin and is involved in cytoskeleton dynamics
Authors: Sahoo Nivedita , Labruyère Elisabeth , Bhattacharya Sudha , Sen P. , Guillén Nancy , Bhattacharya Alok
Published: July 2004

Blocking expression of EhCaBP1, a calmodulin-like, four EF-hand protein from the protozoan parasite Entamoeba histolytica, resulted in inhibition of cellular proliferation. In this paper we report that EhCaBP1 is involved in dynamic changes of the actin cytoskeleton. Both endocytosis and phagocytosis were severely impaired in cells where EhCaBP1 expression was blocked by inducible expression of the antisense RNA. In wild-type cells both actin and EhCaBP1 were found to co-localize in phagocytic cups and in pseudopods. However, in antisense-blocked cells the phagocytic cup formation is affected. Analysis of the staining patterns in the presence and absence of actin dynamics inhibitors, jasplakinolide and cytochalasin D suggested that EhCaBP1 and polymerized F-actin co-localize on membrane protrusions. Direct interaction between soluble EhCaBP1 and F-actin was further demonstrated by a co-sedimentation assay. A variant of EhCaBP1 did not bind F-actin showing the specificity of the interaction between EhCaBP1 and actin. There is no significant change in the kinetics of in vitro polymerization of actin in presence of EhCaBP1, indicating that EhCaBP1 does not affect filament treadmilling. In addition, using atomic force microscopy; it was found that filaments of F-actin, polymerized in presence of EhCaBP1, were thinner. These results indicate that EhCaBP1 may be involved in dynamic membrane restructuring at the time of cell pseudopod formation, phagocytosis and endocytosis in a process mediated by direct binding of EhCaBP1 to actin, affecting the bundling of actin filaments.
Synergistic interaction between the Arp2/3 complex and cofilin drives stimulated lamellipod extension
Authors: DesMarais Vera , Macaluso Frank , Condeelis John , Bailly Maryse
Published: July 2004

Both the Arp2/3 complex and cofilin are believed to be important for the generation of protrusive force at the leading edge; however, their relative contributions have not been explored in vivo. Our results with living cells show that cofilin enters the leading edge immediately before the start of lamellipod extension, slightly earlier than Arp2/3, which begins to be recruited slightly later as the lamellipod is extended. Blocking either the Arp2/3 complex or cofilin function in cells results in failure to extend broad lamellipods and inhibits free barbed ends, suggesting that neither factor on its own can support actin polymerization-mediated protrusion in response to growth factor stimulation. High-resolution analysis of the actin network at the leading edge supports the idea that both the severing activity of cofilin and the specific branching activity of the Arp2/3 complex are essential for lamellipod protrusion. These results are the first to document the relative contributions of cofilin and Arp2/3 complex in vivo and indicate that cofilin begins to initiate the generation of free barbed ends that act in synergy with the Arp2/3 complex to create a large burst in nucleation activity.
N4WBP5A (Ndfip2), a Nedd4-interacting protein, localizes to multivesicular bodies and the Golgi, and has a potential role in protein trafficking
Authors: Shearwin-Whyatt Linda M. , Brown Darren L. , Wylie Fiona G. , Stow Jennifer L. , Kumar Sharad
Published: July 2004

N4WBP5A (Ndfip2) belongs to an evolutionarily conserved group of Nedd4-interacting proteins with two homologues in mammalian species. We have previously shown that N4WBP5A expression in Xenopus oocytes results in increased cell-surface expression of the epithelial sodium channel. N4WBPs are characterized by one or two amino terminal PPxY motifs and three transmembrane domains. Here we show that both PPxY motifs of N4WBP5A mediate interaction with WW domains of Nedd4 and that N4WBP5A can physically interact with the WW domains of several Nedd4-family proteins. N4WBP5A is ubiquitinated and ubiquitination does not significantly affect the turnover of N4WBP5A protein. Ubiquitination of N4WBP5A is enhanced by Nedd4 and Nedd4-2 expression. N4WBP5A localizes to the Golgi, vesicles associated with the Golgi complex and to multivesicular bodies. We show that the ectopic expression of N4WBP5A inhibits receptor-mediated endocytosis of labelled epidermal growth factor. N4WBP5A overexpression inhibits accumulation of EGF in large endocytic/lysosomal vesicles suggestive of a role for N4WBP5A in protein trafficking. We propose that N4WBP5A acts as an adaptor to recruit Nedd4 family ubiquitin-protein ligases to the protein trafficking machinery.
DrosophilaKlp67A is required for proper chromosome congression and segregation during meiosis I
Authors: Savoian Matthew S. , Gatt Melanie K. , Riparbelli Maria G. , Callaini Giuliano , Glover David M.
Published: July 2004

Drosophila Klp67A belongs to the Kip3 subfamily of Kinesin-type microtubule catastrophe factors. In primary spermatocytes, loss of klp67A leads to defects in karyokinesis and cytokinesis. We show that these cells formed disorganised, bipolar spindles that contained increased numbers of microtubules. The kinetochore fibres were wavy and bent, whereas astral microtubules appeared abnormally robust and formed cortical bundles. Time-lapse studies revealed that during biorientation, the chromosomes in klp67A mutant cells continued to reorient for about twice as long as those in control cells. Metaphase plates were poorly defined in the mutants and often formed at non-equatorial positions. Consistent with the above abnormalities in chromosome congression, we found that in wild-type cells Klp67A associated with prometaphase/metaphase kinetochores before redistributing to the central spindle at anaphase onset. Although the timing of this redistribution of kinetochores argues against a role in anaphase chromosome segregation, dyads in the mutants disjoined but exhibited greatly diminished poleward velocities. They travelled on average at approximately 34% of the velocity of their wild-type counterparts and often decondensed at non-polar locations. Hypomorphic mutations of klp67A may lead to segregation defects.
Tyrosine phosphorylation activates surface chaperones facilitating sperm-zona recognition
Authors: Asquith Kelly L. , Baleato Rosa M. , McLaughlin Eileen A. , Nixon Brett , Aitken R. John
Published: July 2004

Mammalian spermatozoa undergo a series of molecular and biochemical changes collectively termed capacitation prior to acquiring the ability to fertilise the oocyte. Although phosphorylation of sperm proteins on tyrosine residues has been recognised as an important component of this process, the precise relationship between the phosphorylation status of mammalian spermatozoa and their capacity for fertilisation has remained unclear. In this study we demonstrate a causal relationship between tyrosine phosphorylation in spermatozoa and sperm-zona interaction. The phosphotyrosine expression associated with sperm capacitation localised to internal flagellar structures in permeabilised cells but could also be detected on the exterior surface of the sperm head in live cells. Importantly, almost all spermatozoa bound to the zona pellucida demonstrated this pattern of phosphoprotein localisation, compared to fewer than 15% of the free-swimming population. These data suggest that tyrosine phosphorylation plays a significant role in remodelling the sperm surface, so that these cells are able to recognise the zona pellucida. Phosphoproteome analysis yielded the first evidence of molecular chaperones, endoplasmin (erp99) and heat shock protein 60 (hsp60), as targets for phosphorylation on the surface of mouse spermatozoa, whereas immunofluorescence localised these proteins to the precise region of the sperm head that participates in zona recognition. Based on these results, we propose a novel mechanism for mammalian gamete interaction whereby the activation of sperm-surface chaperones by tyrosine phosphorylation during capacitation may trigger conformational changes facilitating the formation of a functional zona pellucida receptor complex on the surface of mammalian spermatozoa.
The receptor for activated C-kinase-I (RACK-I) anchors activated PKC-β on melanosomes
Authors: Park Hee-Young , Wu Heng , Killoran Christina E. , Gilchrest Barbara A.
Published: July 2004

Protein kinase C (PKC), a family of at least eleven isoforms, mediates numerous cell functions. In human melanocytes, α, β, δ, ϵ and ζ isoforms of PKC are expressed, but uniquely PKC-β activates tyrosinase, the key and the rate-limiting enzyme in melanogenesis, by phosphorylating specific serine residues on its cytoplasmic domain. To investigate the mechanism by which only PKC-β phosphorylates tyrosinase, we examined the expression of receptor for activated C-kinase-I (RACK-I), a receptor specific for activated PKC-β, on the surface of melanosomes, the specialized organelle in which melanogenesis occurs. Immunoblot analysis of purified melanosomes revealed that RACK-I is readily detectable. Immunoprecipitation of RACK-I from purified melanosomes, followed by immunoblot analysis using antibody against PKC-β, revealed abundant PKC-β, whereas PKC-α was not detected when immunoblot analysis was performed using antibody against PKC-α. Activation of PKC in melanocytes increased the level of PKC-β co-immunoprecipitated with RACK-I, while the level of melanosome-associated RACK-I decreased when melanocytes were treated chronically with the 12-0-tetradecanoyl-phorbol 13-Acetate (TPA), a condition known to deplete PKC and reduce tyrosinase activity. Immunoprecipitation with RACK-I antibody co-precipitated fewer PKC-β in the presence of UV-activated 1, 1′-decamethylenebis-4-aminoquinaldinium di-iodide (DECA), known to disrupt the interaction between activated PKC-β and RACK-I. Treatment of intact melanocytes with DECA also decreased tyrosinase activity. Moreover, suppression of RACK-I expression by transfecting melanocytes with siRNA against RACK-I reduced the basal tyrosinase activity and blocked TPA-induced increases in tyrosinase activity. Taken together, these results demonstrate that RACK-I anchors activated PKC-β on the melanosome membrane, allowing PKC-β to phosphorylate tyrosinase.
Cytosolic, nuclear and nucleolar localization signals determine subcellular distribution and activity of the NF-κB inducing kinase NIK
Authors: Birbach Andreas , Bailey Shannon T. , Ghosh Sankar , Schmid Johannes A.
Published: July 2004

It has been shown previously that the transcription factor NF-κB and its inhibitor IκBα shuttle constitutively between cytosol and nucleus. Moreover, we have recently demonstrated nucleocytoplasmic shuttling of the NF-κB-inducing kinase NIK, a component of the NF-κB pathway, which is essential for lymph node development and B-cell function. Here we show that nuclear NIK also occurs in nucleoli and that this localization is mediated by a stretch of basic amino acids in the N-terminal part of the protein (R143-K-K-R-K-K-K149). This motif is necessary and sufficient for nucleolar localization of NIK, as judged by nuclear localization of mutant versions of the full-length protein and the fact that coupling of these seven amino acids to GFP also leads to accumulation in nucleoli. Using fluorescence loss in photobleaching (FLIP) and fluorescence recovery after photobleaching (FRAP) approaches, we demonstrate a dynamic distribution between nucleoli and nucleoplasm and a high mobility of NIK in both compartments. Together with the nuclear export signal in the C-terminal portion of NIK that we have also characterized in detail, the nuclear/nucleolar targeting signals of NIK mediate dynamic circulation of the protein between the cytoplasmic, nucleoplasmic and nucleolar compartments. We demonstrate that nuclear NIK is capable of activating NF-κB and that this effect is diminished by nucleolar localization. Thus, subcellular distribution of NIK to different compartments might be a means of regulating the function of this kinase.
Platelet adhesion signalling and the regulation of thrombus formation
Authors: Gibbins Jonathan M.
Published: July 2004

Platelets perform a central role in haemostasis and thrombosis. They adhere to subendothelial collagens exposed at sites of blood vessel injury via the glycoprotein (GP) Ib-V-IX receptor complex, GPVI and integrin α2β1. These receptors perform distinct functions in the regulation of cell signalling involving non-receptor tyrosine kinases (e.g. Src, Fyn, Lyn, Syk and Btk), adaptor proteins, phospholipase C and lipid kinases such as phosphoinositide 3-kinase. They are also coupled to an increase in cytosolic calcium levels and protein kinase C activation, leading to the secretion of paracrine/autocrine platelet factors and an increase in integrin receptor affinities. Through the binding of plasma fibrinogen and von Willebrand Factor to integrin αIIbβ3, a platelet thrombus is formed. Although increasing evidence indicates that each of the adhesion receptors GPIb-V-IX and GPVI and integrins α2β1 and αIIbβ3 contribute to the signalling that regulates this process, the individual roles of each are only beginning to be dissected. By contrast, adhesion receptor signalling through platelet endothelial cell adhesion molecule 1 (PECAM-1) is implicated in the inhibition of platelet function and thrombus formation in the healthy circulation. Recent studies indicate that understanding of platelet adhesion signalling mechanisms might enable the development of new strategies to treat and prevent thrombosis.
Preparation and Properties of Tetra- and Octa-substituted Phthalocyanines with Cationic Trimethylaniliniumyl Groups
Authors: Sugimori Tamotsu , Nojima Jun , Ozawa Tomohiro , Handa Makoto , Kasuga Kuninobu
Published: July 2004

Substituted magnesium(II) phthalocyanines modified with dimethylaminophenyl and trimethylaniliniumyl groups were prepared and characterized. The tetra- and octa-substituted phthalocyanines with the cationic trimethylaniliniumyl groups were soluble in water, and the octa-substituted one existed as a photoactive monomer in water.
Determination of Nitrogen in Sand Using Laser-Induced Breakdown Spectroscopy
Authors: Harris Ronny D. , Cremers David A. , Ebinger Michael H. , Bluhm Brian K.
Published: July 2004

The use of laser-induced breakdown spectroscopy (LIBS) to detect a variety of elements in soils has been demonstrated and instruments have been developed to facilitate these measurements. The ability to determine nitrogen in soil is also important for applications ranging from precision farming to space exploration. For terrestrial use, the ideal situation is for measurements to be conducted in the ambient air, thereby simplifying equipment requirements and speeding the analysis. The high concentration of nitrogen in air, however, is a complicating factor for soil nitrogen measurements. Here we present the results of a study of LIBS detection of nitrogen in sand at atmospheric and reduced pressures to evaluate the method for future applications. Results presented include a survey of the nitrogen spectrum to determine strong N emission lines and determination of measurement precision and a detection limit for N in sand (0.8% by weight). Our findings are significantly different from those of a similar study recently published regarding the detection of nitrogen in soil.
On-Line Fermentation Monitoring by Mid-Infrared Spectroscopy
Authors: Mazarevica Gunta , Diewok Josef , Baena Josefa R. , Rosenberg Erwin , Lendl Bernhard
Published: July 2004

A new method for on-line monitoring of fermentations using mid-infrared (MIR) spectroscopy has been developed. The method has been used to predict the concentrations of glucose and ethanol during a baker's yeast fermentations. A completely automated flow system was employed as an interface between the bioprocess under study and the Fourier transform infrared (FT-IR) spectrometer, which was equipped with a flow cell housing a diamond attenuated total reflection (ATR) element. By using the automated flow system, experimental problems related to adherence of CO2 bubbles to the ATR surface, as well as formation of biofilms on the ATR surface, could be efficiently eliminated. Gas bubbles were removed during sampling, and by using rinsing steps any biofilm could be removed from the ATR surface. In this way, constant measuring conditions could be guaranteed throughout prolonged fermentation times (∼8 h). As a reference method, high-performance liquid chromatography (HPLC) with refractive index detection was used. The recorded data from different fermentations were modeled by partial least-squares (PLS) regression comparing two different strategies for the calibration. On the one hand, calibration sets were constructed from spectra recorded from either synthetic standards or from samples drawn during fermentation. On the other hand, spectra from fermentation samples and synthetic standards were combined to form a calibration set. Differences in the kinetics of the studied fermentation processes used for calibration and prediction, as well as the precision of the HPLC reference method, were identified as the main chemometric sources of error. The optimal PLS regression method was obtained using the mixed calibration set of samples from fermentations and synthetic standards. The root mean square errors of prediction in this case were 0.267 and 0.336 g/L for glucose and ethanol concentration, respectively.
Study of the Interactions between Sucrose and Metal Ions (Mg2+ and K+) and Their Simultaneous Quantification in Ternary Mixture by Mid-Infrared and 13C Nuclear Magnetic Resonance Spectroscopies
Authors: Rondeau Philippe , Jhurry Dhanjay , Sers Sandrine , Cadet Frédéric
Published: July 2004

In this paper, a ternary aqueous mixture of sucrose and two metal ions (Mg2+ and K+) has been examined by mid-infrared spectroscopy coupled with principal component analysis (PCA) and the partial least-squares regression method (PLS). PCA was first used for the description of Fourier transform mid-infrared (mid-FTIR) spectral data of the complex samples. The resulting factorial map, set up with the two most influential component axes, features distinct concentration distribution specific to each component. Prediction equations that linked sucrose, magnesium, and potassium concentrations to the spectral data were established by the partial least-squares regression method. A quite good correlation was obtained between the first 5 axes and the concentration variables, with coefficient values ranging from 0.984 to 0.997. It was thus possible to predict specifically both metal ion concentrations in the ternary mixture with relatively good accuracy. The ternary mixtures of sucrose, Mg2+, and K+ were also subjected to 13C NMR (nuclear magnetic resonance) analysis. From the relative displacements of chemical shifts of the carbon atoms of sucrose, it was possible to determine the influence of each metal ion present in the mixture.
Controlled Dissolution for Elemental Analysis of Sample Layers by Inductively Coupled Plasma—Mass Spectrometry: A Feasibility Study
Authors: Li Fumin , Houk R. S.
Published: July 2004

Aqueous acid mixtures at room temperature are used to partially dissolve steel samples. The dissolved elements are washed off the surface, diluted, and then determined by inductively coupled plasma–mass spectrometry (ICP-MS) using a magnetic sector mass analyzer. The amount of material removed is measured from the amount of Fe dissolved and increases linearly with HNO3 concentration in the etch acid. Analyte concentrations in the solid are determined from the signal ratio of analyte ion/Fe+. The shape of a plot of mass of element removed vs. nitric acid concentration yields information about the efficiency of the removal process and the likely chemical form of the element in the sample. For elements like Mn, Al, and W in steel, these plots have the same linear shape as that for the major element (Fe), and the measured concentrations agree well with the certified values. For problem elements like Nb and Ta, the plots have two linear regions with different slopes, and measured concentrations are lower than the certified values. Laser ablation ICP-MS and scanning electron microscopy (SEM) measurements show these elements to be associated together in the solid in refractory grains that are not dissolved to the same extent as the Fe matrix. For steel, the amount of Fe dissolved corresponds to an average depth of at least 4 μm, or 20 000 atomic layers.
Fuzzy Logic for Identifying Pigments Studied by Raman Spectroscopy
Authors: Ramos Pablo Manuel , Ferré Joan , Ruisánchez Itziar , Andrikopoulos Konstantinos S.
Published: July 2004

Fuzzy logic and linguistic variables are used for the automatic interpretation of Raman spectra obtained from pigments found in cultural heritage art objects. Featured bands are extracted from a Raman spectrum of a reference pigment and the methodology for constructing the library is illustrated. An unknown spectrum is then interpreted automatically and a process for identifying the corresponding pigment is described. A reference library consisting of 32 pigments was built and the effectiveness of the algorithm was tested by the Raman spectroscopic analysis of 10 pigments that are known to have been extensively used in Byzantine hagiography. Binary mixtures of these pigments were also tested. The algorithm's level of identification was good even though extra peaks, noise, and background signals were encountered in the spectra.
Apertureless Tip-Enhanced Raman Microscopy with Confocal Epi-Illumination/Collection Optics
Authors: Bulgarevich Dmitry S. , Futamata Masayuki
Published: July 2004

It is demonstrated that confocal epi-illumination/collection optics can be effectively used to generate surface-enhanced Raman scattering at the near-field region of a gold-coated tip for an atomic force microscope operated in semi-contact tapping mode. When the tip, with a 50-nm apex radius, was illuminated by a highly focused laser beam at 532 nm and approached the isolated diamond particle, with a size of ∼1 μm, the Raman signal was enhanced by ∼103. This result is in good agreement with numerical simulations performed by the finite difference time-domain method. Since our apertureless microscope is based on readily available conventional components, there is wide room for improvements and modifications by common users in various applications of micro-Raman analysis.
Phosphorescence Properties of p-Aminobenzoic Acid Immobilized on a Nylon Membrane
Authors: Escandar Graciela M.
Published: July 2004

The properties of nylon as a new material for obtaining room-temperature phosphorescence from p-aminobenzoic acid (PABA) have been investigated. Although PABA shows native phosphorescence when adsorbed on a nylon membrane, a significant improvement in both sensitivity and limit of detection is achieved in the presence of α-cyclodextrin and heavy atoms. An additional enhancement of the phosphorescence signal is attained when the measurements are carried out under a nitrogen atmosphere. The analytical figures of merit obtained under the best experimental conditions are: linear calibration range from 0.6 to 6 ng/spot (the lowest value corresponds to the quantitation limit); correlation coefficient, 0.998 for 18 data points; relative standard deviation, 2.2% ( n = 5) at a level of 2.4 ng/spot; and limit of detection, 0.2 ng/spot (calculated according to Clayton's definition).