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PubMed2004

Analysis of remnant reticulocyte mRNA reveals new genes and antisense transcripts expressed in the human erythroid lineage.

BACKGROUND AND OBJECTIVES: We studied the gene expression profile of human purified reticulocytes to provide a transcriptional basis for the study of erythroid biology, differentiation and hematologic disorders. DESIGN AND METHODS: We screened highly purified blood reticulocytes from ten healthy adult volunteers. We chose a modified protocol of serial analysis of gene expression (SAGE), the serial analysis of downsized extracts (SADE). RESULTS: Data analysis revealed that 64% of gene signatures (tags) matched with known genes; mainly hemoglobin. In addition to the abundant globin mRNA, SAGE analysis identified previously described genes and new transcripts. In reticulocytes, which are poor in mRNA, we also identified 9% of EST and 27% of tags that did not match with any known genes. Mining our data, 70% of the unknown tags and 39% of tags identifying EST were found to be specific to the reticulocyte. We demonstrated the presence of a mRNA that matched with the reverse sequence of the hemoglobin b (HBB) transcript. INTERPRETATION AND CONCLUSIONS: This is the first description of an antisense transcript of the human HBB gene suggesting regulation by way of sense-antisense pairing. The well-characterized genes found in the SAGE library were genes specific to the blood cell lineage, housekeeping genes and, interestingly, genes not previously described in the reticulocyte. Furthermore the study provides markers of the erythroid lineage regulated during the differentiation process as observed in in vitro experiments.

Adult↗
PubMed2004

[Gastrointestinal stromal tumors carrying PDGFRalpha mutations occur preferentially in the stomach and exhibit an epithelioid or mixed phenotype].

AIMS: Most gastrointestinal stromal tumors (GISTs) carry gain-of-function mutations of the KIT gene encoding the receptor tyrosine kinase KIT. However, in a subset of GISTs no activating mutations are detectable in KIT. Recently, PDGFRalpha mutations have been identified as alternative oncogenic mechanism. We studied a panel of 100 GISTs for mutations in hot spot regions of KIT (exons 9, 11, 13 and 17) and PDGFRalpha (exons 12 and 18) and compared the results with pathomorphological and immunohistochemical data. METHODS: DNA from formalin-fixed and paraffin-embedded tumor tissue was extracted after microdissection from serial sections. We performed single strand conformational polymorphism analysis and direct sequencing. RESULTS: We found PDGFRalpha mutations in 24 of 55 GISTs with wild-type sequence in exons 9, 11, 13 and 17 of KIT. All mutations were located in exon 18 of the PDGFRca gene which encodes the tyrosine kinase domain II. None of the 45 GISTs with detectable KIT mutation carried a mutation in the PDGFRalpha gene. Interestingly, all PDGFRalpha-mutated tumors were located in the stomach whereas GISTs with exon 9 and 13 KIT mutations occurred predominantly in the small bowel. Additionally, 21 of 24 GISTs carrying PDGFRalpha mutations displayed an epithelioid or mixed phenotype. In contrast, KIT-mutated GISTs exhibited almost always a spindled histology (38 of 45 cases). CONCLUSIONS: Our analysis provides evidence that GISTS represent distinctive entities with different genetic, biological and phenotypic features.

Diagnosis, Differential↗
PubMedDate not supplied

[Radiographic and histological study of a case of apexification in a human molar].

A case of apexification in a lower right second molar is described. Radiographs demonstrate apical closure with a different morphological pattern from that of the lower left second molar. Following extraction, after 15 months, serial histologic sections show calcified tissue obturating the apical foramen, well adapted to the initial dentin and cementum walls. Inside some small areas containing connective tissue with capillaries can be observed. The histologic and radiographic observations indicate that apical closure occurs as a result of differentiation of periodontal apical cells.

Adolescent↗
PubMed1983

[Alkaline phosphatase of the rat small intestine--purification of the enzyme and its physiological significance].

It has been known that feeding of high-fat diets increases in intestinal and serum alkaline phosphatase (AP) activity in rats. This phenomenon is regarded as an indirect evidence for a participation of intestinal AP (I-AP) in regard to fat absorption. This study was performed to clarify the role of I-AP in the fat absorption. The results can be summarized as follows: I-AP was purified about 1,800-fold from rat intestinal mucosa by n-butanol extraction, acetone precipitation, and serial chromatographies with L-Phe-Sepharose, Sephadex G-200, and DEAE-Sephadex. The purity of the enzyme was about 90-95% as judged by SDS-PAGE. On SDS-PAGE under nonreducing conditions, the purified I-AP gave two bands corresponding to a Mw of 140,000 (major) and 120,000 (minor), respectively. Under reducing conditions, the enzyme dissociated into subunit with a Mw of 60,000. The isoelectric point of I-AP was 4.6, and the Km value for p-nitrophenylphosphate was estimated to be 0.7 mM. The enzyme activity was 50% inhibited by 4mM L-phenylalanine. A simple method for measuring I-AP isozyme specifically in rat serum was developed by the use of anti I-AP antibody-bound filter paper discs. This method has revealed that the I-AP activity normally accounts for about 80% of the total AP activity in rat serum. The I-AP activity in rat serum decreased to less than one-half the initial value after 24hr-fasting. After 48hr-fasting rats were fed with normal diets, the I-AP activity returned to normal level in about 12hr. Fasting rats were fed with 30 Cal of either corn oil or sucrose. In only the fat-fed rats, the serum I-AP activity increased gradually for 8hr. Rats were then allowed free access to the normal diets, the serum I-AP activity increased in both groups almost at the same rate. There was a great variability of serum and tissue I-AP activities in each rats. An inversed relationship was present between the tissue and serum I-AP activities in each rats. This phenomenon was more significant in jejunum than in duodenum. The meaning of these results will be discussed.

Alkaline Phosphatase↗
PubMed1980

Follow-up studies of porphyrin excretion in porphyria cutanea tarda treated with p-aminobenzoic acid.

The treatment of porphyria cutanea tarda (PCT) with p-aminobenzoic acid (PABA) was suggested because PABA is capable of reversing the porphyrinogenic action of 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC) in rats. Three patients with PCT were treated with 3 g of PABA daily during 6 and 12 months and the urinary and faecal porphyrin excretion were serially analyzed by solvent extraction techniques and by thin layer chromatography of their methyl esters. PABA treatment did not show any apparent effect on porphyrin excretion.

4-Aminobenzoic Acid↗
PubMed1995

Establishment of the sulfated 3,3'-diiodothyronine radioimmunoassay and its application in pregnant women.

Sulfation of iodothyronines is a major alternate pathway of thyroid hormone metabolism during fetal development. Sulfated 3,3'-diiodothyronine (T2S) is a low end metabolite of this pathway. Its clinical implications in the prenatal evaluation of fetal thyroid disorders are now being intensively investigated. A highly sensitive and reproducible radioimmunoassay (RIA) for T2S has been established in our laboratory. The detection threshold of the RIA approximated 5 pg T2S. The dose-response curve of T2S was essentially linear between 5-200 pg. An essentially parallel correlation of the dose-response curves for inhibition of binding of radiolabeled T2S and T2S antiserum was found between serial dilutions of serum extracts and the standards. The average intra- and inter-assay coefficients of variation were 7% and 15%, respectively. By applying the T2S RIA, we found that serum titers of T2S in pregnant women increased proportionately to the gestational age (first trimester vs. second trimester vs. third trimester: 30.1 +/- 1.4 vs. 41.6 +/- 1.9 vs. 98.0 +/- 3.9 ng/dL; p < 0.001 each). A high concentration of T2S was detected in cord and maternal serum at birth as compared to the results for non-pregnant women (165.1 +/- 10.3 vs. 113.7 +/- 5.6 vs. 7.5 +/- 0.9 ng/dL). The T2S levels decreased remarkably in maternal circulation 10 days after partuition. Four pregnant women who had two blood samplings four or more weeks apart during the third trimester showed invariable increases of serum T2S titers at later sampling times. Additionally, in the case of a pregnant woman who received two doses of T4 injections (200 micrograms/wk) intraamniotically for a previous Cretin birth, the maternal serum levels of T2S increased promptly from 47 ng/dL [corrected] to 96 ng/dL. Our findings imply that the established T2S RIA is clinically applicable, provide further evidence that the coincident increase of serum T2S titers in pregnant women may reflect ontogenesis of fetal thyroid hormone maturation, and provide a clue to the fetal thyroid status in the prenatal stage. However, more knowledge is still needed regarding the transfer and transformation of sulfated iodothyronine(s) from the fetal compartment to maternal circulation.

Analysis of Variance↗
PubMed1988

Quantitative determination of nuclear estrogen receptors by an enzyme immunoassay: applicability and caveats.

A method is presented with which approx. 95% of nuclear estrogen receptors appear to be extracted from MCF-7 cells. Since both nuclear isolation and nuclear estrogen receptor extraction take place in a single test tube with only vortex mixing, loss of nuclear material is minimized. The amount of nuclear estrogen receptors in the nuclear extract was determined by direct [3H]estradiol labeling of monolayer cultures and with a commercially available estrogen receptor immunoassay (ER-EIA) kit. Since the ER-EIA kit was designed and calibrated for quantitative determination of cytosolic estrogen receptor isolated in low ionic strength buffer, the applicability of the ER-EIA to quantitative determination of estrogen receptor content in high ionic strength nuclear extraction buffer was tested. A linear relationship exists between the amount of nuclear estrogen receptor detected by the immunoassay, the amount of receptor present in serial dilutions of the nuclear extract and the amount of nuclear estrogen receptor detected in cells by [3H]estradiol labeling of monolayer cultures, the absolute amount of nuclear estrogen receptors determined by the immunoassay consistently exceeded the amount of receptor detected by [3H]estradiol labeling. The possibility that the enzyme immunoassay must be properly calibrated for the specific conditions of the nuclear estrogen receptor assay is discussed.

Blotting, Western↗
PubMed1996

Comparison of methods for extracting DNA from formalin-fixed paraffin sections for nonisotopic PCR.

DNA was extracted from unstained 5-microns sections of neutral buffered 10% formalin-fixed paraffin-embedded tissue by proteinase K digestion without detergents followed by boiling, proteinase K digestion with ionic detergents with and without phenol chloroform extraction and ethanol precipitation, sonication with proteinase K followed by boiling, or boiling alone. Serial 1:10 dilutions of the extracted DNA were subject to polymerase chain reaction (PCR) amplification of a 255-bp portion of the p53 gene. Digestion with proteinase K without ionic detergents followed by boiling (without phenol chloroform extraction) gave the best yield, enabling visualization of ethidium bromide-stained PCR product from a DNA dilution corresponding to 0.1 mm2 of tissue containing of the order of 10(3) nuclear profiles. Proteinase K digestion with detergents followed by phenol-chloroform extraction was no more effective than simple boiling. Although the success of PCR from preserved tissue will vary with the fixative and size of the amplified fragment, DNA extracted with this optimized method can be used for identification of viruses, loss of heterozygosity, and immunoglobulin gene rearrangements in paraffin-embedded tissue without radioisotopes.

Base Sequence↗
PubMed1998

Analysis of defective genomes of bombyx mori nucleopolyhedrovirus generated by serial undiluted passage in cell culture.

Viral DNA was extracted from cells infected with bombyx mori nucleopolyhedrovirus (BmNPV) D1 strain after 34 serial undiluted passages (P34). P34 DNA was subjected to restriction analysis and Southern blot hybridisation using standard D1 DNA and P34 DNA of BmNPV as probes. Based on hybridisation profiles, the BmNPV DNA regions retained in the P34 DNA were localised on HindIII and PstI restriction maps. Two regions of BmNPV DNA located at 0-12.8 and 40.2-65.0 map unit (m.u.) were highly conserved in P34 DNA. These regions contained two of three interspersed homologous sequences (ihss), but only one of five homologous regions (hrs). This suggests that ihss may have an essential role in BmNPV replication.

Animals↗
PubMed1986

Quantification and partial characterization of the hamster sperm proacrosin-acrosin system.

The proacrosin-acrosin proteinase system was measured and partially characterized in unpurified extracts of washed hamster epididymal sperm. Autoactivation experiments demonstrated that proacrosin accounted for greater than 98% of the acrosin activity in the sperm extracts from individual animals. Several bands of proteinase activity were observed on gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoretic (gelatin-SDS-PAGE) zymography. The major proteinase activities in the nonactivated extracts corresponded to relative molecular masses (Mr) of 51,000 to 56,000, while less distinct digestion occurred with relative molecular masses of 37,000 to 49,000. It was demonstrated that after a serial dilution of the sperm extract, the proteinase activity in as few as 6,000 sperm could readily be detected by the gelatin-SDS-PAGE methods. Time-course activation studies showed that the zymogen was completely converted to active proteinase in 45-60 min at pH 8.0 and 25 degrees C. This autoconversion process was markedly inhibited by calcium, sodium, and heparin. However, each of these compounds stimulated the proteolytic activity of acrosin. These studies demonstrate that the proacrosin-acrosin system can be investigated in extracts of nonpurified hamster epididymal sperm.

Acrosin↗
PubMed2004

Culicoides midge trap enhancement with animal odour baits in Scotland.

Examples of the commercial trap Mosquito Magnet Pro (MMP emitting attractant 1-octen-3-ol in carbon dioxide 500 mL/min generated from propane fuel), were run 24 h/day on the Isle of Skye, Scotland, during June-August 2001 and evaluated for catching Culicoides biting midges (Diptera: Ceratopogonidae). From 30 days trapping, the catch averaged 2626 +/- 1358 Culicoides females/trap/day (mean +/- SE, range 558 +/- 139 to 6088 +/- 3597, for five sets of six consecutive nights), predominantly the pest Culicoides impunctatus Goetghebuer (68% overall), plus C. vexans (Staeger) > C. delta Edwards > C. pulicaris (L.) > C. lupicaris Downs & Kettle > C. albicans (Winnertz) > other Culicoides spp. Attempts were made to enhance the odour baiting system by adding hexane-extracts (2.1 mg/day) of hair samples from large host animals, resulting in the following effects on Culicoides collections: sheep - 53 %, red deer - 26 %, calf + 20%, pony + 40%, water buffalo + 262%, with greatest increases for C. impunctatus and C. pulicaris. Serial concentrations of these animal extracts (10(-1) - 10(-3) x 2.2 g/mL) were assayed on parous female C. impunctatus response in a Y-tube olfactometer (air-flow 150 mL/min), and by electroantennogram (EAG) on Culicoides nubeculosus Meigen laboratory-reared parous females. Positive behavioural responses to host odours were dose-dependent: the water buffalo extract being most active (threshold 0.22 g/mL), similar to deer, whereas other host extracts were > or = 10-fold less active. Correspondingly, the EAG threshold was lowest for water buffalo, 10-fold greater for deer, calf and pony, but not detected for sheep. If the active component(s) of these host extracts can be identified and synthesized, they might be employed to improve the capture of Culicoides midges for local control by removal trapping.

Animals↗
PubMed1992

Novel quantitative methods for the determination of biomaterial cytotoxicity.

Two novel methods for the determination of biomaterial cytotoxicity using cell culture are presented. The methods combine a standardized protocol for producing extracts from medical devices with either the established MTT assay or a new fluorimetric assay. The suitability of both methods for evaluating the toxicity of candidate materials was demonstrated by resolution of the differences in the toxic effects of serial dilutions of a PVC extract on BHK21 and HT1080 cells. The tests yield highly reproducible, quantitative results and can be applied to materials in the usual physical forms applicable to artificial organs.

Animals↗
PubMed1984

Proteolytic activity of rabbit incisor dentine.

The presence of proteolytic activity and plasminogen activator was demonstrated in extracts of the dentine from serial sections from all regions of the incisor. Maximal activity was at neutral pH, although some was also present at acidic pH. Preparations of dentine non-collagenous matrix (NCM) proteins were sensitive to the proteolytic activity; NCM prepared by EDTA extraction contained such activity.

Animals↗
PubMed2007

Variation in nuclear DNA concentrations during urination.

This study examined the cellular origin and concentration of nuclear DNA in human urine. Ten subjects provided two entire, first-morning voids: one as a single specimen and one as a consecutive series of samples. The serial samples were centrifuged, organically extracted, and quantified by slot-blot analysis. Total DNA concentrations ranged from 0.02 to 21.3 ng/mL for the males and 25.0 to 96.9 ng/mL for the females. The female samples were found to contain numerous vaginal epithelial cells. DNA was detected in all of the serial samples of nine subjects; however, the DNA concentrations varied considerably. With six subjects, the DNA concentration of the first serial sample was at least three times greater than that of the entire void. DNA was only detected in the first 21% of the void from one male subject. The results of this study have implications for the collection of urine samples.

Adult↗
PubMed1991

Selective growth and serial passage of mouse melanocytes from neonatal epidermis in a medium supplemented with bovine pituitary extract.

Suspensions of disaggregated epidermal cells from skins of newborn C57BL/10JHir mice were plated in a growth medium that consisted of Ham's F-10 plus bovine pituitary extract (BPE), insulin, and transferrin. Fetal bovine serum (FBS) was added to the culture medium at a concentration of 4% at the time of plating. On the second day of culture, a small number of melanocytes was randomly distributed among large sheets of keratinocytes. From the third day onward, FBS was excluded from the culture medium to prevent the proliferation of keratinocytes and fibroblasts. The melanocytes began to grow preferentially, and after 12 days pure and enriched populations of melanocytes could be harvested. In the absence of the proliferation of keratinocytes and fibroblasts, melanocytes could be serially passaged in the growth medium supplemented with a conditioned medium (CM) prepared from keratinocyte-enriched cultures, namely, those at the early stages of the primary culture. FBS was added at a concentration of 1% for the first day. These results suggest that both BPE and keratinocyte CM contain growth factors required for proliferation of melanocytes.

Animals↗
PubMed2001

Effects of triclosan on the cytotoxicity and fungal growth on a soft denture liner.

STATEMENT OF PROBLEM: Contamination of removable prostheses with microorganisms, particularly Candida albicans, is a common clinical problem. Microban, a broad-spectrum antimicrobial containing triclosan, recently has been proposed to inhibit microbial growth. PURPOSE: This study aimed to determine whether the addition of Microban to PermaSoft denture liner prevents the growth of C albicans and affects the cytotoxicity of the PermaSoft material. MATERIAL AND METHODS: Experimental specimen disks (5 x 1 mm each) with and without incorporated Microban were fabricated aseptically (n = 6) against polyester film to produce a smooth surface. To assess the cytotoxic effect of Microban, the MTT assay was used. To determine the effect of Microban on the growth of C albicans, disks were placed in Transwell dishes, covered with Sabouraud's broth containing an ATCC strain of C albicans, and incubated at 37 degrees C for 24 hours. Wells containing fluorocarbon resin disks or broth alone served as controls. The disks were rinsed to remove unattached C albicans and then sonicated in sterile water to remove surface organisms. Serial dilutions of the water extracts were plated on Sabouraud's agar and returned to the incubator for 24 hours. Colonies were counted with a Brunswick Colony Counter. Growth of C albicans in the internal aspects of the specimens was determined in a manner as previously described, with the exception that the specimens were sonicated to remove surface organisms, minced, and sonicated once more before making serial dilutions. The results were compared with ANOVA and Tukey intervals (alpha=.05). RESULTS: The number of colonies formed ranged from 17 to 31 x 10(5) (mean = 23 +/- 4 x 10(5)) and 14 to 69 x 10(5) (mean = 32 +/- 20 x 10(5)) for the PermaSoft with and without Microban groups, respectively. There was no statistically significant difference between PermaSoft with and without Microban. CONCLUSION: The addition of Microban did not significantly alter the cytotoxicity of the PermaSoft denture lining material or reduce the adherence of viable C albicans to the surface of PermaSoft material after 24 hours.

Analysis of Variance↗
PubMed1981

Immunochemical characterization and distribution of calcitonin in the lizard.

Immunological and chromatographic methods were used to investigate the distribution of calcitonin (CT) in various tissues of Lacerta muralis, a common wall lizard. Salmon CT-like immunoreactivity was found in high concentration in extracts of ultimobranchial gland (UBG) and in significant amounts in lung and brain extracts, but not in other tissues (oesophagus, stomach, duodenum, liver, skin, muscle and thyroid). Serial dilution of UBG, lung and brain extracts gave parallel displacement curves to that of synthetic salmon CT (sCT), but no reactivity was found in the human CT assay. On Sephadex G-50, UBG, lung and brain extracts contained an sCT-like immunoreactive peak which co-eluted with synthetic sCT. However, on high performance liquid chromatography (HPLC) the sCT-like peak in UB, lung and brain extracts eluted 2 ml later than synthetic sCT. This paper describes the first immunochemical characterization of CT in the UBG of the lizard, demonstrates the distribution of extra-ultimobranchial CT and throws some light on the evolution of the calcitonins.

Animals↗
PubMed2003

Detection of nonmelanoma skin cancer micrometastases in lymph nodes by using reverse transcriptase-polymerase chain reaction for keratin 19 mRNA.

BACKGROUND: A new sensitive method for the detection of skin cancer micrometastases in lymph nodes is based on amplification of keratin 19 (K19) mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). OBJECTIVES: To compare results of RT-PCR with those of histological examination in terms of the detection rate of skin cancer micrometastases. METHODS: Twenty-six lymph nodes obtained from 13 patients with squamous cell carcinoma (SCC), eccrine porocarcinoma and Paget's disease were investigated by histological examination (haematoxylin and eosin sections) and RT-PCR. RT-PCR was performed on extracted RNA by using K19 primer pairs. RT-PCR products were visualized by ethidium bromide staining and confirmed by non-radioactive hybridization with K19-specific probes. RESULTS: All of 10 histologically positive lymph nodes yielded the expected 460-bp band. Of the 16 histologically negative lymph nodes, one (6%) was found by RT-PCR to express K19 mRNA, indicating the presence of micrometastases which could not be detected by histological examination. A serial dilution study using RNA extracted from SCC cells mixed with RNA extracted from normal lymph node cells showed a detection sensitivity of K19 RT-PCR of 10-5 micro g cancer cell RNA in 1 micro g lymph node RNA. Nested RT-PCR showed a detection sensitivity of one tumour cell in 106 lymphocytes. CONCLUSIONS: These results demonstrate the usefulness of K19 RT-PCR for the detection of skin cancer micrometastases in lymph nodes.

Biomarkers, Tumor↗