bluuebunny/crossref_metadata_2025_split huggingface dataset:
Influence of Time and Temperature on Coagulation Analytes in Stored Plasma
Authors: Heil Wolfgang , Grunewald Regina , Amend Martin , Heins Michael 
Published: October 2004

Abstract
There is no comprehensive study on the stability of coagulation analytes in plasma. We therefore determined the influence of storage time and temperature on prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen, factors V and VIII, antithrombin III, protein C and S in plasma from 20 healthy subjects and 20 patients receiving heparin therapy. The stability in plasma, defined as the period during which there was a change of less than 10 % from the initial value, was 8 hours for activated partial thromboplastin time, 24 hours for prothrombin time, 48 hours for factor V and 7 days for thrombin time, fibrinogen, protein C and antithrombin III in healthy subjects at 6 °C. Factor VIII and protein S showed 19 and 12 % reduction in activity, respectively, after 8 hours.
In volunteers not treated with heparin therapy, activated partial thromboplastin time was stable for 8 hours; prothrombin time for 48 hours; and thrombin time, fibrinogen and antithrombin III for 7 days with sample storage at room temperature. Factor VIII showed a decrease of 18 % after 8 hours.
For patients receiving heparin therapy, the stability of the analytes in plasma stored at 6 °C was 8 hours for thrombin time, 24 hours for prothrombin time and activated partial thromboplastin time and 7 days for fibrinogen and antithrombin III. Factors V and VIII showed a decrease of 13 % and 20 % respectively after 8 hours.
When the plasma of these patients was stored at room temperature, factor V was stable for 8 hours, and prothrombin time for 24 hours, whereas fibrinogen and antithrombin III remained unchanged for 7 days. Activated partial thromboplastin time showed an increase of 13 %, thrombin time a fall of 16 %, and factor VIII a decrease of 18 % after 8 hours.
Biological Day-to-Day Variation and Daytime Changes of Testosterone, Follitropin, Lutropin and Oestradiol-17β in Healthy Men
Authors: Ahokoski Outi , Virtanen Arja , Huupponen Risto , Scheinin Harry , Salminen Eeva , Kairisto Veli , Irjala Kerttu 
Published: October 2004

Abstract
Information on biological day-to-day variation is needed for detecting within-subject changes over time. In this study the daytime changes and the biological day-to-day variation of serum testosterone, follitropin, lutropin and oestradiol-17β concentrations were investigated in 31 healthy males.
To analyse daytime changes, blood specimens were taken at 0800 h, 1200 h, 1600 h and 2000 h during one day (n=31) and two days (n=8). The day-to-day variation was analysed from blood specimens collected at 0800 h on days 1 and 2 (n=31) and additionally on days 3, 4, 6 and 9 (n=8). The evaluation of the day-to-day variation was based on calculations of the within-subject (CVA+I) and between-subject (CVG) coefficients of variation. When the within-subject day-to-day variances were not too heterogeneous, they were used for the calculation of 95 % reference change limits.
Serum testosterone and oestradiol-17βconcentrations showed a significant daytime variation; testosterone had higher serum concentrations at 0800 and 1200 h. A peak in the serum concentration of oestradiol-17β occurred at 1200 h with a decrease towards the evening. There were no clear daytime changes in the serum concentrations of follitropin or lutropin.
For different analytes the reference change limits were: serum testosterone ± 32.0 %, serum follitropin ± 24.1 % and serum oestradiol-17β ± 38.3 %. The reference change limit was not calculated for serum lutropin, as a high degree of heterogeneity and individuality was found.
The interpretation of the results of hormone measurements requires recognition of the biological daytime and day-to-day changes of hormones. The reference change limits determine what changes are significant when monitoring the patient.
Mutation Detection and Mutation Databases
Authors: Cotton Richard G. H. 
Published: October 2004

AbstractDetection of mutations in genes is vital throughout biology, however, this activity is time-consuming, expensive and requires a high degree of skill. This is unsatisfactory in a field which is increasing importance. Around 10–12 methods are commonly used with some predominating. All have their advantages and disadvantages and none is perfect. Sequencing is said to be the gold standard for detecting new mutations (and must be used to define it), but six or so methods have been described to make the search quicker to avoid sequencing the whole gene. These are called scanning methods. Other methods are used to detect known mutations and referred to as diagnostic methods. These methods will be briefly reviewed. Once mutations are described, they are usually published. The mutation lists are collected for convenience, analysis or research. In recent years, it has been realised that these lists are vital for research, patient care and commercial activities. These activities will be reviewed and the co-ordinating role of the HUGO Mutation Database Initiative will be outlined.
Oligonucleotide-Based Gene Therapy for Cardiovascular Disease
Authors: Morishita Ryuichi , Nakagami Hidenori , Taniyama Yoshiaki , Matsushita Hidetsugu , Yamamoto Kei , Tomita Naruya , Moriguchi Atsushi , Matsumoto Keiko , Higaki Jitsuo , Ogihara Toshio 
Published: October 2004

Abstract
Gene therapy is emerging as a potential strategy for the treatment of cardiovascular disease such as restenosis after angioplasty, vascular bypass graft occlusion, transplant coronary vasculopathy, homozygous familial hypercholesterolemia and cystic fibrosis, for which no known effective therapy exists. Gene therapy requires efficient in vivo gene transfer technology. During the past decade, many gene transfer methods including viral transfer techniques have been developed, and some are being applied clinically in human gene therapy studies. Molecular biology and pathophysiology of the cardiovascular system have started to emerge, and the time is ripe for the introduction of gene therapy to the management of cardiovascular disorders. In this review, we have focused on the future potential of oligonucleotide-based gene therapy for the treatment of cardiovascular disease.
Simple and Broadly Applicable Sample Preparation by Use of Magnetic Glass Particles
Authors: Bartl Knut , Wenzig Peter , Kleiber Joerg 
Published: October 2004

Abstract
A key issue in nucleic acid-based assays related to infectious disease testing and genetic testing is the sample preparation. This has to be convenient, efficient and suitable for automation.
We describe a new method based on the use of glass magnetic particles. The steps involved are the lysis of bacterial cells and viral particles by chaotropic salts, binding of released nucleic acids to the magnetic glass particles, the removal of inhibitors and the release of purified nucleic acids ready for PCR amplification. The method fulfills most of the requirements for sample preparation such as sensitivity, universality and suitability for automation.
Optimized Non-Radioactive Protein Truncation Test for Mutation Analysis of the Adenomatous Polyposis Coli (APC) Gene
Authors: Kirchgesser Michael , Albers Andreas , Vossen Rolf , den Dunnen Johan , van Ommen Gert-Jan , Gebert Johannes , Dupont Cecile , Herfarth Christian , von Knebel-Doeberitz Magnus , Schmitz-Agheguian Gudrun 
Published: October 2004

Abstract
Germline mutations in the adenomatous polyposis coli gene cause familial adenomatous polyposis, a colon cancer predisposition syndrome. More than 95% of the identified mutations result in the generation of stop codons or reading frame shifts and encode a truncated gene product, a mutation profile also found in other tumor predisposition genes such as the breast cancer or the hereditary non-polyposis coli. Therefore the protein truncation test is ideally suited for screening of mutations in these genes, starting from simple blood samples. Gene segments of interest are amplified from genomic DNA or mRNA, thereby incorporating a T7 promoter at the 5′-end. After in vitro transcription and translation of the PCR products, the resulting protein is analysed by gel electrophoresis. Truncated translation products indicate the presence of a stop mutation. We have developed a non-radioactive protein truncation test that uses a biotinylated Lys-t-RNA to label the translation products and allows a chemiluminescent detection instead of the standard radioactive method. This generic protein truncation test kit was then used to develop a parameter-specific protein truncation test for adenomatous polyposis coli. The adenomatous polyposis coli gene was divided in 5 overlapping segments, and primers were optimized to produce distinct bands with very low background in the protein truncation test. The assay was tested on 20 familial adenomatous polyposis patient samples, where 18 mutations were found, demonstrating the efficiency of this method.
Monitoring of Inhibitors of Enzymatic Amplification in Polymerase Chain Reaction and Evaluation of Efficacy of RNA Extraction for the Detection of Hepatitis C Virus Using the Internal Control
Authors: Miyachi Hayato , Masukawa Atsuko , Ohshima Toshio , Fusegawa Hisae , Hirose Toru , Impraim Chaka , Ando Yasuhiko 
Published: October 2004

Abstract
Inhibitors of enzymatic amplification in serum may cause false-negative results for direct detection of hepatitis C virus (HCV) by polymerase chain reaction (PCR). This study was undertaken to demonstrate the importance of the internal control in a PCR assay for detection of HCV-RNA to monitor false-negatives due to inhibitors. HCV-RNA was extracted using RNA extraction kit (SepaGene RV-R, Sanko Junyaku) and a prototype instrument for automated specific capture of HCV-RNA with probes and magnetic bead/fluid separation (Roche Molecular Systems). The extracted HCV-RNA and internal control were detected by an automated PCR machine (Cobas Amplicor, Roche Diagnostic Systems). Addition of hemoglobin (up to 4.5 g/l) to the sera followed by RNA extraction with SepaGene RV-R had no inhibitory effect on the detection of either HCV-RNA or the internal control. In contrast, addition of heparin to the sera showed an inhibitory effect with a dose-dependent manner on the detection of both HCV-RNA and the internal control, with a greater effect at lower copy number of HCV. When HCV-RNA was extracted by the automated system, the inhibitory effect of heparin was successfully eliminated. In the assays of 65 serum samples positive for anti-HCV antibodies, positivity for the internal control indicated efficient amplification and validated 14 negative and two equivocal results for detection of HCV-RNA. Detection of the internal control was negatively correlated with viral copy number in sera suggesting competitive inhibition of high viral copy number on amplification of the internal control. Extraction, coamplification and detection of the internal control appears useful for estimating effects of inhibitors on amplification in each assay for the detection of HCV-RNA, and for evaluating efficacy of RNA extraction methods.
Automation of Polymerase Chain Reaction-Based Systems for Detection of Hepatitis C Virus RNA
Authors: Kessler Harald H. , Pierer Karen , Santner Brigitte I. , Vellimedu Srinivas K. , Stelzl Evelyn , Marth Egon 
Published: October 2004

Abstract
Polymerase chain reaction-based molecular assays are gaining increasing importance in the diagnosis and monitoring of infectious diseases. Over the past several years, the development and application of these techniques has initiated a revolution in the diagnosis and monitoring of hepatitis C infection. Presently, molecular assays are exclusively done in especially dedicated laboratories. Advances in automation will bring these technologies into routine diagnostic laboratories and will make them widely used.
Restriction Digest PCR (RD-PCR) for the Analysis of Gene Mutations. Application to Ki-ras
Authors: Huber Klaus Roland , Bittner Jürgen , Bauer Kurt , Trümper Lorenz , Sek Alexandra , Sebesta Christian , Rosen Harald , Tragl Karl-Heinz 
Published: October 2004

Abstract
The Kirsten-ras (onco)gene codes for a GTP-binding membrane protein that is involved in signal transduction. Activated ras triggers a cascade of protein-phosphorylations that ultimately lead to cell proliferation. Ras-mutations are the main cause for adenocarcinomas of the pancreas besides some mutations in the tumor suppressor gene p53 and the c-erbB-2 oncogene. The site of ras mutations in pancreatic cancer is restricted to codon 12 that normally encodes a glycine. For analysis of codon-12 mutations, DNA is extracted from cells in pancreatic fluid and amplified by PCR. Because most of these cells originate from normal tissue with only a few tumor cells in the fluid, “enrichment PCR” must be utilized: In a first round of the PCR, ras sequences from all cells are amplified. By utilizing an appropriate restriction enzyme, wild-type sequences can be digested and the remaining fragments containing mutated sequences be amplified again. An artificial restriction site must be introduced by the 5′ primer (…GGA C̱CT GGT…) for an enzyme (BstNI) (5′CC!WGG 3′) to differentiate between wild-type sequence (…GGA G̱CT GGT…) (during amplification, the G̱ is replaced by a C̱) and mutated sequences (̱…GGA GCT (GTT), (CGT), (CCT), etc.). The necessary manipulations pose a considerable risk for contamination for the second round of the PCR procedure. Therefore, we considered whether it would be feasible to perform the restriction digest simultaneously with the first PCR reaction, and avoiding the secound round altogether.
The results of our experiments demonstrate that one tumor cell in 1000 normal cells can be determined readily, paralleling the results with the original two step-assay. The restriction enzyme used to enrich mutated sequences is stable long enough to be included into the PCR procedure. By this, wild-type sequence amplicons are digested while they are formed and mutated sequences can be enriched selectively.
Direct Detection of Mycobacterium tuberculosis in Respiratory Specimens Using an Automated DNA Amplification Assay and a Single Tube Nested Polymerase Chain Reaction (PCR)
Authors: Yam Wing Cheong , Yuen Kwok Yung , Seto Wing Hong 
Published: October 2004

Abstract
The performance of an automated DNA amplification assay (Roche Cobas® Amplicor™ Mycobacterium Tuberculosis Test (aPCR) was compared with an in-house single tube nested polymerase chain reaction (nPCR) for the direct detection of Mycobacterium tuberculosis in respiratory specimens. Among 385 specimens, 56 were culture positive for mycobacteria (44 positive for Mycobacterium tuberculosis and 12 positive for nontuberculosis mycobacteria). The diagnostic sensitivities of aPCR and nPCR were 86 % and 91 % whereas a 100 % diagnostic specificity of both assays was attained. By aPCR, inhibitors were detected in 6% of the clinical samples. For nPCR, the usage of a new thermostable DNA polymerase facilitated pre-PCR decontamination using uracil-N-glycosylase and “hot start” in single step procedure. The results of the study indicated that DNA amplification assays, either manual or automated, were rapid and specific tools for diagnosing pulmonary tuberculosis.
Angiotensin Converting Enzyme Gene Polymorphism in Coronary Artery Disease: The Indian Scenario
Authors: Joseph Ansamma , Nair Kappiareth Gopal , Ashavaid Tester Framroze 
Published: October 2004

Abstract
Angiotensin converting enzyme is a key component of the renin angiotensin system that plays an important role in cardiovascular regulation. It seems to modulate cardiovascular growth by virtue of its role in the conversion of angiotensin I to angiotensin II and degradation of kinins. A deletion polymorphism localized in intron 16 of the human angiotensin converting enzyme gene, corresponding to a 287 bp long Alu repetitive sequence, was found to be associated with increased risk of myocardial infarction in various subgroups, including European, French and Japanese coronary patients. This angiotensin converting enzyme gene I/D polymorphism was examined by the polymerase chain reaction in a cross-sectional study of 201 healthy Indian subjects and 150 patients (angiographically proven cases of coronary artery disease) whose serum angiotensin converting enzyme levels were concomitantly measured. The D/D, I/D and I/I genotypes were found in 20.66 %, 46.66 % and 32.66 % of the Indian coronary heart disease patients and in 23.38 %, 49.75% and 26.86 % of controls respectively. One of the reasons for not finding an association between the D allele and coronary artery disease in this study could be the ethnic heterogeneity and disease status heterogeneity among the patients and controls. However the phenotypic variance of serum angiotensin converting enzyme levels is strongly influenced by this polymorphism. In the Indian population, the angiotensin converting enzyme gene I/D polymorphism is not associated with risk for coronary artery disease although it is associated with plasma angiotensin converting enzyme activity. Hence the angiotensin converting enzyme gene I/D polymorphism does not seem to be a useful marker for coronary artery disease in the Indian population.
Mutation C677T of Methylenetetrahydrofolate Reductase Gene Is Not Associated with Coronary Artery Disease, but Possibly with Albuminuria, in Type 2 Diabetic Patients
Authors: Wirta Valtteri , Huang Xiao-Hong , Wirta Ole , Rantalaiho Vappu , Pasternack Amos , Jokela Hannu , Koivula Timo , Lehtimäki Terho 
Published: October 2004

Abstract
The missense mutation in the 677th nucleotide (C677T) of methylenetetrahydrofolate reductase gene causes substitution of valine (V) for alanine (A) resulting in three genotypes VV, VA and AA. The VV genotype causes hyperhomocysteinemia and may be a risk factor for coronary artery disease. We determined genotypes by polymerase chain reaction and subsequent restriction fragment length analysis and compared them in 84 patients with type 2 diabetes and in 115 non-diabetic subjects with and without coronary disease. Fractional urinary excretion rate of albumin was assessed by nephelometry. The VV, VA, and AA frequencies in the diabetic and in the control groups were 0.095, 0.357, 0.548 and 0.061, 0.417, 0.522, respectively (p = NS, diabetic vs. controls, χ2 test). Genotype frequencies did not differ in either diabetic or control subjects between those with or those without coronary disease (χ2 test). The fractional urinary excretion rate of albumin (mean ±SD) in diabetic patients with the VV genotype i.e. 1.59 ± 0.71 was lower (Kruskall-Wallis test p = 0.002) than in the other genotypes i.e. VA 5.98 ± 9.75 and AA 3.75 ± 4.77, respectively (post-hoc Mann-Whitney test VV vs. VA p = 0.005 and VV vs. AA p = 0.054, respectively). We found that in patients with type 2 diabetes the methylenetetrahydrofolate reductase VV genotype was associated with a low urinary albumin excretion but not with coronary artery disease or diabetes per se.
Analysis of Clonality in T-Lymphoproliferative Diseases by Multiplex PCR
Authors: Zadro Renata , Sučić Mirna , Aurer Igor , Metelko-Kovačević Jasminka , Labar Boris , Stavljenić Rukavina Ana 
Published: October 2004

Abstract
Distinction between benign and malignant T-cell lymphoproliferative diseases can be difficult using morphological criteria. Using multiplex polymerase chain reaction system we have tested a series of patients with various lymphoproliferative disorders to detect clonal T-lymphocyte populations.
Results show that clonal amplification products were obtained from all 10 patients with T-cell lymphoproliferative disorders while the amplification of DNA samples from B-cell neoplasms and normal individuals revealed polyclonal amplification products.
By splitting the multiplex primer mix, the patient specific T-cell receptor γ rearrangement was determined: five out of ten patients showed the exclusive presence of a single T-cell receptor γ gene rearrangement. Three patients exhibited two rearranged T-cell receptor γ genes, while in two patients positive reactions were obtained with three pairs of primers for variable and joining segments.
Molecular analysis of rearranged T-cell receptor genes by multiplex polymerase chain reaction represents a useful and rapid tool for confirming diagnosis, to determine the extent of disease and to monitor the response to therapy.
The Impact of Molecular Diagnosis on Familial Colorectal Cancer
Authors: Bennett Graeme , Rudzki Zbigniew , Cole Steve , Young Graeme , Suthers Graeme 
Published: October 2004

Abstract
We have shown the impact of molecular diagnostics related to mutation detection in an extensive family with a strong history of colorectal cancer. The nature and presentation of the cancers suggested that hereditary nonpolyposis colorectal cancer was the most likely cause. The strategies employed have enabled the detection and characterisation of the causative mutation in the proband and predictive testing in the remaining relatives where requested.
Using the chemical cleavage of mismatches technique and direct sequencing, the MSH2 and MLH1 genes of the proband were investigated. A single base substitution, C→T at nucleotide 350, codon 117, of the MLH1 gene was identified. Across the family pedigree at specific points, 22 other relatives have been tested for the mutation by direct DNA sequencing from genomic DNA. Of the total of 23 patients tested to date, 11 have the mutation.
In conjunction with appropriate genetic counselling, this service has clarified the genetic status of many individuals within this family. Predictive information provided prior to the development of symptoms enables individuals to make informed choices regarding disease management and the future, removing the anxiety associated with the unknown.
CGG Repeat Interruptions in the FMR1 Gene in Patients with Infantile Autism
Authors: Poon Priscilla M. K. , Chen Qian L. , Lai Kelly Y. C. , Wong Chung-Kwong , Pang Chi-Pui 
Published: October 2004

Abstract
We determined the CGG repeat length and AGG interruptions in the FMR1 gene in normal Chinese subjects and patients with infantile autism and mild mental retardation. Genomic DNA was investigated by PCR and Southern hybridisation for CGG repeat number and PCR with Mnl I restriction analysis for AGG interruption. Both the normal subjects and the patients with autism have 53 CGG repeats in FMR1, and the majority have two interspersed AGG. Our normal Chinese subjects have a similar number of interspersed AGG as other populations. When compared with the normal subjects, the autism patients have less AGG interruptions and a different pattern of AGG distribution. There was a significant difference in the CGG configurations between normal subjects and patients with autism. The latter had less interspersed AGG, as in fragile X patients, but they did not have fragile X. A study on mentally retarded patients with no infantile autism should also be carried out to ascertain whether mental retardation alone may have contributed to such AGG pattern.
Genetic Basis of Congenital Hypothyroidism: Abnormalities in the TSHβ Gene, the PIT1 Gene, and the NIS Gene
Authors: Tatsumi Ke-ita , Miyai Kiyoshi , Amino Nobuyuki 
Published: October 2004

Abstract
We have elucidated the molecular pathology of three types of congenital hypothyroidism. Thyrotropin (TSH) is the major regulator of thyroid function. In cases of isolated congenital TSH deficiency, we found that they are caused by a missense mutation in the conserved CAGYC region of the TSHβ gene. Pit-1/GHF-1 is a pituitary specific POU-domain DNA binding factor, which transactivates the growth hormone (GH), prolactin (PRL), TSHβ genes, and the PIT1 gene itself. In cases of combined deficiency of GH, PRL, and TSH, we found that they are caused by abnormalities in the PIT1 gene, either recessively or dominantly. Sodium dependent iodide symporter (NIS) actively transports iodide into the thyroid cells to produce thyroid hormones. In cases of iodide transport defect, we elucidated that a missense mutation in the transmembrane region of the NIS gene caused them.