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PubMed1993

DNA typing with fluorescently tagged short tandem repeats: a sensitive and accurate approach to human identification.

Human identification through DNA analysis has faced tremendous changes in the past seven years. The advent of the polymerase chain reaction (PCR) technology coupled with the discovery of amplifiable minisatellites and microsatellites known as amplified fragment length polymorphisms and short tandem repeats (STRs), respectively, allow allelic profiles to be obtained with minute amounts of target DNA even in a degraded state. Very recently, a new dimension in DNA typing analysis was opened with the development of instruments for automated real-time analysis of fluorescent amplification products. In order to derive an automated approach to DNA typing, STR systems were evaluated for sensitivity and accuracy using the Gene Scanner and compared to other DNA typing methods currently in use. Eight different STR systems (encompassing tri-, tetra- and pentanucleotide repeats) were investigated, and conditions for their amplification with fluorescence-tagged primers, resolution on polyacrylamide gels and analysis on a fluorescent DNA fragment analyzer were optimized. Using these conditions, discrete allelic profiles were obtained following amplification of DNA extracted from various cell lines, liquid blood, dry bloodstains and hair samples. Amplification from serial dilutions of template DNA indicated that the minimal amount of target DNA required to detect a fluorescent signal on the Gene Scanner for any of the eight STR systems examined is approximately 100 picograms. The level of precision obtained for real-time allele size determination was observed to be +/- 0.2 to 0.5 base pair (intragel) and +/- 0.5 to 1.5 base pairs (intergel). Consequently, PCR-based DNA typing with fluorescent STR primers and automated analysis provides the enhanced level of precision, accuracy and sensitivity required for forensic casework analysis. Moreover, this approach offers significant advantages for the routine processing of large numbers of DNA samples, greatly facilitates and expedites the generation of allelic profile databases and enables investigators to perform the simultaneous survey of several different loci from single individuals and/or forensic samples.

Alleles↗
PubMed2002

DNA content and cell number determination in microdissected samples of breast carcinoma in situ.

Paraffin-embedded tissue (PET) is the specimen of choice for the histopathological diagnosis of cancer. PET has become a valuable resource for correlating cellular phenotype and genotype in microdissected lesions. A definitive improvement in this field has been the development of infra-red laser capture microdissection (LCM), which yields homogeneous populations of cells for DNA extraction and in vitro amplification by polymerase chain reaction (PCR). We report here a photographic and fluorescent technique for determining the number of nuclei and concentration of DNA obtained respectively by laser capture microdissection from paraffin-embedded breast cancer tissue. Breast biopsies containing carcinoma in situ were serially sectioned, mounted on plain glass slides and tumor cells were microdissected using a laser capture microscope. The DNA was extracted in digestion buffer and used directly as a template for PCR. In our protocols, each capture contained 21+/-5.4 nuclei with a DNA concentration of 115+/-5.3 pg. Also, a linear relationship was found between number of captures and DNA content (R2=0.9995). These results represent a novel contribution for a more precise correlation between phenotypic and genotypic diversity in cancer cells studied from microdissected paraffin-embedded tissue.

Breast Neoplasms↗
PubMed2005

Rationale for using Peltophorum africanum (Fabaceae) extracts in veterinary medicine.

Peltophorum africanum (Fabaceae) is a deciduous tree widespread in southern Africa. The plant has many ethnomedical and ethnoveterinary uses. Root and bark decoctions are used to treat diarrhoea, dysentery, sore throat, wounds, back and joint pains, HIV-AIDS, venereal diseases and infertility. Pastoralists and rural farmers use the root and bark extracts to treat diarrhoea, dysentery, infertility, and to promote well-being and resistance to diseases in cattle. To evaluate these ethnobotanical leads, dried leaves, stem bark and root bark were extracted with ethanol, acetone, dichloromethane and hexane. Polyphenols in the extract were determined by the Folin-Ciocalteu method with gallic acid as standard. Qualitative antioxidant activity was screened by spraying thin layer chromatograms (TLC) of the extracts with 0.2% 1,1-diphenyl-2-picryl hydrazyl (DPPH), and quantified with Trolox equivalent antioxidant capacity (TEAC) assay. Minimum inhibitory concentration (MIC) and total antibacterial activity (TAA) were determined by serial microplate dilution for Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis, with gentamicin as standard and tetrazolium violet as growth indicator. Acetone and ethanol extracted the largest quantity of material. Polyphenols concentration was 49.2% in acetone extract of the root and 3.8% in dichloromethane extract of the leaf. Antioxidant activity of at least 5 antioxidant compounds as measured by TEAC ranged from 1.34 (ethanol extract of the root) to 0.01 (hexane extract of the leaf). The total antibacterial activity (volume to which active compounds present in 1 g plant material can be diluted and still inhibit bacterial growth) was 1263 ml/g for ethanol extract of the root against S. aureus, and 800 ml/g for acetone extract of the root against P. aeruginosa. There was substantial activity against both Gram-positive and Gram-negative bacteria, with MIC values of 0.08 mg/ml for S. aureus and 0.16 mg/ml for P. aeruginosa. There is therefore a rationale for the traditional use of root and bark of P. africanum in treating bacterial infection related diseases.

Animals↗
PubMed1996

A major rearrangement of the VP6 gene of a strain of rotavirus provides replication advantage.

During coinfection of BSC-1 cells with bovine rotavirus B223 and human rotavirus 69M and subsequent serial passages at low multiplicity of infection (0.1 m.o.i.), a reassortant virus (BMR) with a rearranged VP6 gene became the predominant strain. At passage 24 virus extracted from 50 of 51 plaques (98%) contained the rearranged gene 6, which had been first observed in passage 19. The analyses of the clones obtained from passages before the appearance of the rearranged VP6 gene (passage 15) and after (passage 20) indicated that the B223 VP6 gene was the origin of the rearranged VP6 gene. To test whether the rearranged VP6 gene was responsible for the selection advantage observed, reassortant C11 was generated with BMR and WA rotavirus, containing the rearranged VP6 gene and the other 10 genes from WA. Coinfection of WA rotavirus and reassortant C11 and subsequent serial passages at low m.o.i. resulted in 100% of virus from clones extracted at passage 18 being identical to reassortant C11; demonstrating that the rearranged VP6 gene was once again selected over the normal VP6 gene. The selection advantage of the rearranged VP6 gene could not be explained by comparison of the growth curves of the viruses, as there was no significant difference between the growth cycles of rotavirus B223 and reassortant BMR, nor between rotavirus Wa and reassortant C11. However, the plaque and electropherotype analysis at passage 1 of Wa and C11 coinfection revealed that 85% of the progeny viruses contained the rearranged gene 6. These data show that the gene 6 rearrangement resulted in selection of the relevant reassortant, possibly by suppression of competitive strains, and may indicate a new mechanism for the evolution of rotavirus.

Animals↗
PubMed1996

Telomerase activity in human breast cancer and benign breast lesions: diagnostic applications in clinical specimens, including fine needle aspirates.

We analysed telomerase activity in normal, benign and malignant breast tissues and in fine needle aspirates by a PCR-based assay. The tissue samples we used in this assay consisted of 20 cryostat sections, 10 microns thick, from each breast biopsy. This method was used to obtain effective extraction from small samples and to confirm the histological identity of the specimen by microscopical examination of serial sections. Fifty-two of 71 breast carcinomas were positive for telomerase activity, and the intensity of this was strong in most cases, whereas all 6 samples of normal breast tissue and 17 of fibrocystic disease were negative and only 1 of 15 fibroadenomas was positive. Invasive ductal carcinomas were more frequently positive than invasive lobular carcinomas. There was no correlation of telomerase activity with tumour size or the occurrence of lymph node metastasis. Evaluation of our assay system showed that a signal of telomerase activity was detectable in extracts from single cryostat sections (< 1 mm2) of a cancer specimen and from as few as 4 cells of a human breast cancer cell line. On the basis of the above data, we applied this assay to fine needle aspirates of breast lesions. Ten of 15 aspirates which had been cytopathologically diagnosed as cancer were strongly positive, while 26 of 29 benign aspirates were totally negative and the remaining 3 showed only borderline activity. In 3 cases, the telomerase result could have helped establish a diagnosis when the cytological observations were inconclusive. Our results indicate that this sensitive assay could become a useful new modality for supplementing microscopic cytopathology in the detection of cancer cells in small tissue biopsies and fine needle aspirates.

Autoradiography↗
PubMed2004

Inhibition of Helicobacter pylori in vitro by various berry extracts, with enhanced susceptibility to clarithromycin.

The objective of this study was to evaluate the effects of various berry extracts, with and without clarithromycin on Helicobacter pylori. Resistance to clarithromycin by H. pylori has been reported, leading to interest in alternatives/adjuncts to therapy with clarithromycin. H. pylori American type culture collection (ATCC) strain 49503 was grown, cell suspensions were made in PBS and diluted 10-fold. One hundred microL of the suspension was then incubated for 18 h with extracts of raspberry, strawberry, cranberry, elderberry, blueberry, bilberry, and OptiBerry, a blend of the six berries, at 0.25-1% concentrations. Serially diluted cell suspensions were exposed for 1 h to clarithromycin at 15 microg/ml. Ten microl of bacterial samples from the 10(-7) dilution tube were plated and incubated for 18 h and the number of colonies were counted. Growth of H. pylori was confirmed by the CLO test. All berry extracts significantly (p < 0.05) inhibited H. pylori, compared with controls, and also increased susceptibility of H. pylori to clarithromycin, with OptiBerry demonstrating maximal effects.

Anti-Bacterial Agents↗
PubMed1994

Limitations of angiography for analyzing coronary atherosclerosis progression or regression.

PURPOSE: To analyze the utility and limitations of serial coronary angiography for determining atherosclerosis progression and regression. DATA SOURCES: A MEDLINE search of the English-language literature (1966 to January 1994) using the keywords atherosclerosis regression, atherosclerosis progression, lipid reduction therapy, and coronary angiography. STUDY SELECTION: Selected articles on the effects of cholesterol reduction and lifestyle modification on angiographic coronary artery disease, on the animal models of atherosclerosis progression and regression, and on the limitations of coronary angiography. DATA EXTRACTION: Independent extraction by two authors. RESULTS: Although several studies have reported that the rate of atherosclerosis progression, defined by serial coronary angiography, can be reduced and that luminal diameter can be improved somewhat by aggressive lipid modification, the reported changes are small (0.3 mm or 10% change) and have required a prolonged study duration (range, 1 to 10 years). More importantly, angiography simply does not measure atherosclerosis and cannot assess lesion composition. Angiography also underestimates the extent of atherosclerosis, especially in angiographically normal segments. In addition, difficulties with data acquisition, such as substantial variabilities in serial measurements of percent diameter stenosis and minimal luminal diameters, require large sample sizes to show statistically significant regression, even with computerized quantification. CONCLUSIONS: Given its current limitations, serial coronary angiography is not a satisfactory means of detecting atherosclerosis progression or regression.

Anticholesteremic Agents↗
PubMed2002

[Generation and analysis of a repeat DNA fragment of SeMNPV by serial undiluted passage in Se301 insect cells].

Two major clades, designated Groups I and II, of nucleopolyhedroviruses (NPVs) from insect hosts have been previously identified. In order to gain insight into DNA replication of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV, Groups II), the essential cis-acting DNA segments were studied. A strain, named Se-4, was plaque-purified from SeMNPV isolate US1. PCR, ELT-PCR and REN showed that Se-4 was genetically relatively homogeneous and retained the full-length of a hypervariable region which is usually prone to deletion from SeMNPV genome. To study the stability of this isolate in vitro, Se-4 was serially passaged in the Se301 cell line up to 10 times without dilution. Intracellular viral DNA extracted from every passage was analyzed by REN. A novel 3.5 kb PstI fragment was observed in passage 7 and the relative intensities of the bands increased with subsequent passages. In passage 10, the molar ratio of the fragment was much higher than those of any other viral DNA fragments. This fragment was thus expected to contain an important cis-acting element for SeMNPV DNA replication. The fragment was cloned and sequenced and it was found that it overlapped 3 525 bp with the published SeMNPV genome sequence (GenBank AF169823), from 81 014 nt to 84 538 nt. The region contained the SeMNPV non-hr origin (ori) of DNA replication and some ORFs including partial vlf-1, partial p26, Se84 as well asSe83, Se85, Se86, which are unique to SeMNPV. As compared to Autographa californica MNPV (Groups I), which also generated hypermolar DNA fragments containing the non-hr ori by serial undiluted passage in IPLB-SF-21 cell culture, our results provided in vitro evidence that the non-hr ori may also play an important role in the viral infection cycle of Groups II NPVs.

Animals↗
PubMed2001

Neuroprotective effects of Withania somnifera Dunn. in hippocampal sub-regions of female albino rat.

The neuroprotective effects of W. somnifera were studied on stressed adult female Swiss albino rats. Experimental rats were subjected to immobilization stress for 14 h and were treated with a root powder extract of W. somnifera available as Stresscom capsules (Dabur India Ltd). Control rats were maintained in completely, non stressed conditions. Thionin stained serial coronal sections (7 microm) of brain passing through the hippocampal region of stressed rats (E(1) group) demonstrated 85% degenerating cells (dark cells and pyknotic cells) in the CA(2) and CA(3) sub-areas. Treatment with W. somnifera root powder extract significantly reduced (80%) the number of degenerating cells in both the areas. The study thus demonstrates the antistress neuroprotective effects of W. somnifera.

Animals↗
PubMed2003

Solid-phase micro-extraction-gas chromatography-mass spectrometry and headspace-gas chromatography of tetrahydrocannabinol, amphetamine, methamphetamine, cocaine and ethanol in saliva samples.

In the present work, a method was developed aiming at the serial detection of tetrahydrocannabinol (THC), amphetamine, methamphetamine, cocaine and ethanol in saliva. Saliva samples were submitted to an initial headspace procedure for ethanol determination by gas chromatography/flame ionization detector (GC-FID). After this step, two consecutive solid-phase micro-extractions (SPME) were carried out: THC was extracted by submersing a polydimethylsiloxane fiber (100 micro m) in the vial for 20 min; amphetamine, methamphetamine and cocaine were subsequently extracted after alkalinization. Derivatization of the amphetamines was carried out directly in the solution by adding 2 micro l of butylchloroformate. Gas chromatography-mass spectrometry (GC-MS) was used to identify the analytes in selected ion monitoring (SIM) mode. Confidence parameters of validation of the method were: recovery, linearity, intra- and inter-assay precision as well as limits of detection and quantification of the analytes. The limits of quantification (LOQ) obtained were: ethanol (0.010 g/l); amphetamine (5.0 ng/ml); methamphetamine (0.5 ng/ml); cocaine (5 ng/ml) and THC (5 ng/ml). The method proved to be highly precise (coefficient of variation<8%) for all detected substances.

Amphetamine↗
PubMed2004

Improved specificity for Giardia lamblia cyst quantification in wastewater by development of a real-time PCR method.

The protozoan parasite Giardia lamblia is the most common cause of waterborne disease outbreaks associated with drinking water in the United States. The conventional method used for the enumeration of Giardia cysts in water is based on immunofluorescence with monoclonal antibodies. It is tedious and time-consuming and has the major drawback to be non-specific for the only species infecting humans, G. lamblia. We have developed a real-time polymerase chain reaction (PCR) method using fluorescent TaqMan technology, which improved the specificity of G. lamblia cyst quantification compared to the immunofluorescence assay (IFA). However, this PCR was not totally specific for G. lamblia species and amplified Giardia ardeae target as well. This method showed a sensitivity of 0.45 cysts per reaction and an efficiency of 95% in purified suspensions. We have then applied this quantification method to raw wastewater, a medium containing numerous debris, particles and PCR inhibitors. The adaptation to these environmental samples was realized by a screening of three cyst purification methods and six DNA extraction protocols. Real-time quantification was accomplished by the simultaneous amplification of unknown samples and a tenfold serial dilution of purified G. lamblia cysts. For all samples, the concentrations observed with TaqMan PCR method were compared to the IFA values. Giardia spp. cysts were detected in all non-spiked raw wastewater samples with IFA procedure and the concentrations of Giardia spp. cysts used for the comparison between the two methods ranged between 3.3x10(2)/l and 4.3x10(3)/l. The highest TaqMan PCR/IFA ratios were observed when Percoll/sucrose flotation was combined with DNA extraction protocol optimized for cyst wall lysis, impurities adsorption on a resin, and double step protein digestion and column purification. The concentrations observed with this TaqMan PCR method ranged from 2.5x10(2) to 2.4x10(3) G. lamblia cysts/l and only one sample resulted in a no amplification curve. Thus, we developed a TaqMan PCR method increasing the rapidity and specificity of G. lamblia cyst quantification. The combination of Percoll/sucrose flotation and DNA extraction optimized protocol before TaqMan assay has provided a good indication of the G. lamblia contamination level in raw sewage samples.

Animals↗
PubMed1998

Simultaneous determination of 5-fluorouracil and uracil by high-performance liquid chromatography using four serial columns.

A sensitive assay was developed for the quantitation of 5-fluorouracil (5-FU) and uracil using liquid-liquid extraction (LLE) and HPLC with UV detection. Analyses were performed with four microBondapak C18 columns connected in series using 20 mM acetic acid with 1% ACN as mobile phase. The calibration curves were linear across the range of 26-1000 ng ml(-1) (0.21-7.8 microM) for 5-FU and 1.0-14.0 microg ml(-1) (0.01-110 microM) for uracil. This assay has been implemented to determine the plasma concentrations for pharmacokinetic studies for 5-FU and uracil in conjunction with clinical trials.

Chromatography, High Pressure Liquid↗
PubMed1999

The involvement of phosphohydrolases in mineralization: studies on enzymatic activities extracted from red deer antler.

The process of growth of red deer antler "bone" is very similar to the endochondral growth of long bones. The growth zones of the antler are temporally organized from the tip, in a proximal direction, permitting sequential aspects of antler development to be followed in serial transverse sections from the tip to the base. The activities of alkaline phosphatase (ALKPase, phosphodiesterase I, nucleoside triphosphate pyrophosphohydrolase, and 5'-nucleotidase were measured in extracts of antler mineralizing tissue at different distances from the tip, and compared with mineralization, which peaked at ca. 60 mm. The activities of ALKPase and phosphodiesterase I were maximal at ca. 30 and 60 mm, respectively, suggesting that these enzymes are involved in different phases of mineralization. The patterns of distribution were similar to those determined from equivalent quantitative histochemical analyses, confirming the validity of both methods of assessment. The distribution of nucleoside triphosphate pyrophosphohydrolase also suggested that this enzyme is involved in mineralization, but the pattern of distribution was not as reproducible as for the other enzymes. 5'-Nucleotidase activity was distributed differently than the others and showed relatively constant levels at all distances. The results suggest that each of the four phosphohydrolases may have a different role in the process of mineralization. A tentative model suggesting functions for these phosphohydrolases and ATPases is presented and discussed.

Animals↗
PubMed1981

Fluorometric determination of tetracyclines in small blood and tissue samples.

A reported fluorometric method to determine tetracycline in biological material was modified to determine low levels of the antibiotics when using only small amounts of starting material. The method is applicable to tissue distribution studies in small experimental animals and also can be used for serial determinations in blood samples of these animals and for biopsy material. The tetracyclines are extracted in the presence of ethylenediaminetetraacetic acid, and the phosphate precipitation steps are omitted. This omission makes the procedures considerably less time consuming. The recoveries of the tetracyclines, oxytetracycline and doxycycline, are approximately 80-100%. The values obtained by this method were consistent with those from microbiological assays, and its reliability was shown further in a comparison with the fate of radioactive tetracycline.

Bacillus cereus↗
PubMed2006

Two-dimensional and serial column reversed-phase separation of phenolic antioxidants on octadecyl-, polyethyleneglycol-, and pentafluorophenylpropyl-silica columns.

The separation selectivity of octadecyl-silica (C18) and of bonded pentafluorophenylpropyl-silica (F5) and PEG-silica columns was compared for natural phenolic antioxidants. The separation selectivities for phenolic antioxidants on C18 and F5 columns are strongly correlated, but low selectivity correlation indicating strong differences in the retention mechanism was observed between the C18 and PEG columns. Hence, the combination of a C18 and a PEG column is useful for separation of phenolic antioxidants that are not fully separated on single columns. Two-dimensional comprehensive liquid chromatography using a short PEG-silica column in the first dimension and a conventional C18-silica in the second dimension has the advantage of on-column focusing of the fractions transferred onto the C18 column in the second dimension, as a weaker mobile phase is used in the first dimension than in the second dimension. However, a stop-flow set-up in the first dimension system is necessary after the transfer of each fraction to the second dimension. Peak capacity is considerably larger but the separation time is much longer than with serially coupled PEG and C18 columns, which were employed for separation of beer and hop extract samples in connection with coulometric detection.

Antioxidants↗
PubMed2007

Serial analysis of gene expression in sugarcane (Saccharum spp.) leaves revealed alternative C4 metabolism and putative antisense transcripts.

Sugarcane (Saccharum spp.) is a highly efficient biomass and sugar producing crop. Leaf reactions have been considered as potential rate-limiting step for sucrose accumulation in sugarcane stalks. To characterize the sugarcane leaf transcriptome, field-grown mature leaves from cultivar "SP80-3280" were analyzed using Serial Analysis of Gene Expression (SAGE). From 480 sequenced clones, 9,482 valid tags were extracted, with 5,227 unique sequences, from which 3,659 (70%) matched at least a sugarcane assembled sequence (SAS) with putative function; while 872 tags (16.7%) matched SAS with unknown function; 523 (10%) matched SAS without a putative annotation; and only 173 (3.3%) did not match any sugarcane ESTs. Based on gene ontology (GO), photosystem (PS) I reaction center was identified as the most frequent gene product location, followed by the remaining sites of PS I, PS II and thylakoid complexes. For metabolic processes, photosynthesis light harvesting complexes; carbon fixation; and chlorophyll biosynthesis were the most enriched GO-terms. Considering the alternative photosynthetic C(4) cycles, tag frequencies related to phosphoenolpyruvate carboxykinase (PEPCK) and aspartate aminotransferase compared to those for NADP(+)-malic enzyme (NADP-ME) and NADP-malate dehydrogenase, suggested that PEPCK-type decarboxylation appeared to predominate over NADP-ME in mature leaves, although both may occur, opposite to currently assumed in sugarcane. From the unique tag set, 894 tags (17.1%) were assigned as potentially derived from antisense transcripts, while 73 tags (1.4%) were assigned to more than one SAS, suggesting the occurrence of alternative processing. The occurrence of antisense was validated by quantitative reverse transcription amplification. Sugarcane leaf transcriptome provided new insights for functional studies associated with sucrose synthesis and accumulation.

Carbohydrate Metabolism↗
PubMed1984

Serial alterations in endothelial cell shape and pattern after intraocular surgery.

We serially examined the endothelial cellular pattern and thickness of the central cornea after intracapsular cataract extraction and penetrating keratoplasty. The endothelial cellular pattern was analyzed with a computer-assisted digitizer. We used the frequency of hexagonal cells as the index to describe the degree of hexagonality in the cellular pattern. In the intracapsular cataract extraction group (20 eyes), the frequency of hexagonal cells was 68% preoperatively. During the first four weeks this frequency decreased rapidly to a significantly lower level (52%). The corneal thickness was significantly increased within the first week (0.57 mm) and steadily decreased to preoperative values at four weeks (0.53 mm). During the period from four to 24 weeks, the frequency of hexagonal cells increased gradually to 64%. In the penetrating keratoplasty group (ten eyes), the frequency of hexagonal cells and the thickness at one month were 62% and 0.64 mm respectively. During the next six months the frequency of hexagonal cells decreased significantly to 53%, and the thickness decreased to 0.53 mm. There was a gradual increase in the frequency of hexagonal cells to 62% during the two years after penetrating keratoplasty.

Aged↗