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At least 253 records · Page 14Linked to original sources
PubMed2003

Mutation identification in a 5-generation pedigree with autosomal dominant retinitis pigmentosa.

An extended 5-generation family has been investigated in which 32 of the 111 family members were diagnosed as having retinitis pigmentosa (RP). The proband was a 58-year old male in whom night-blindness was first observed in early childhood, with almost loss of vision by 52 years of age. The symptoms observed in other family members included night-blindness, impaired vision and visual field loss. Dementia, digital abnormalities, deaf-mutism and mental retardation were variously diagnosed in a number of individuals with RP. The affected and unaffected family members were tested for mutations in a range of candidate genes. The 8 exons of three candidate genes have been analyzed by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing techniques. A novel mutation was identified in the rhodopsin gene at codon 52 of exon 1 (TTC-TAC) that resulted in a substitution of Phe to Tyr.

DNA Mutational Analysis↗
PubMed2006

Effects of platelet-derived growth factor-BB on the metabolic function and morphologic features of equine tendon in explant culture.

OBJECTIVE: To evaluate the effects of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) on the metabolic function and morphologic features of equine superficial digital flexor tendon (SDFT) in explant culture. Animals-6 euthanized horses (2 to 5 years old). METHODS: Forelimb SDFT explants were cultured for 6 days as untreated control specimens or treated with rhPDGF-BB (1, 10, 50, or 100 ng/mL of medium). Treatment effects on explant gene expression were evaluated via real-time PCR analysis of collagen type I, collagen type III, PDGF-A, and PDGF-B mRNA. Explants were assayed for total collagen, glycosaminoglycan, and DNA content; histologic changes were assessed via H and E staining and immunohistochemical localization of collagen types I and III. RESULTS: No morphologic or proliferative changes were detected in tendon explant sections. After high-dose rhPDGF-BB treatment, gene expression of collagen types I and III was increased and decreased, respectively. Expression of PDGF-A and PDGF-B mRNA was significantly increased at 24 hours, but later decreased to have few or negative autoinductive effects. Although PDGF gene expression waned after 48 hours of culture, collagen type I gene expression was significantly increased at 48 hours and reached peak value on day 6. Glycosaminoglycan and DNA content of explants were unchanged with rhPDGF-BB treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that rhPDGF-BB use may be of benefit in the repair of equine tendon, particularly through induction of collagen type I mRNA. Positive autoinductive effects of PDGF-BB in equine SDFT explants were detected early following culture medium supplementation, but these diminished with time.

Animals↗
PubMed2002

Gene expression analysis using oligonucleotide arrays produced by maskless photolithography.

Microarrays containing 195,000 in situ synthesized oligonucleotide features have been created using a benchtop, maskless photolithographic instrument. This instrument, the Maskless Array Synthesizer (MAS), uses a digital light processor (DLP) developed by Texas Instruments. The DLP creates the patterns of UV light used in the light-directed synthesis of oligonucleotides. This digital mask eliminates the need for expensive and time-consuming chromium masks. In this report, we describe experiments in which we tested this maskless technology for DNA synthesis on glass surfaces. Parameters examined included deprotection rates, repetitive yields, and oligonucleotide length. Custom gene expression arrays were manufactured and hybridized to Drosophila melanogaster and mouse samples. Quantitative PCR was used to validate the gene expression data from the mouse arrays.

Animals↗
PubMed2006

Detection of HER2 amplification in breast carcinomas: comparison of Multiplex Ligation-dependent Probe Amplification (MLPA) and Fluorescence In Situ Hybridization (FISH) combined with automated spot counting.

In this study the detection of HER2 gene amplification was evaluated using Fluorescence In Situ Hybridization (FISH; PathVysion) in comparison with Multiplex Ligation-dependent Probe Amplification (MLPA), a PCR based technique. These two methods were evaluated on a series of 46 formalin fixed paraffin embedded breast carcinomas, previously tested for protein overexpression by HercepTest (grouped into Hercep 1+, 2+ and 3+). HER2 gene amplification (ratio > or =2.0) by FISH was found in 9/10, 10/30 and 0/6 in IHC 3+, 2+ and 1+/0 cases, respectively. Digitalized automated spot counting performed with recently developed CW4000 CytoFISH software was 100% concordant with manual FISH scoring. Using MLPA 18/46 samples showed a clear HER2 amplification. Comparing MLPA and IHC showed the same results as for FISH and IHC. All but one FISH positive cases (18/19) were confirmed by MLPA for the presence of the gene amplification. The overall concordance of detection of Her2 gene amplification by FISH and MLPA was 98% (45/46). Furthermore, both the level of amplification and equivocal results correlated well between both methods. In conclusion, MLPA is a reliable and reproducible technique and can be used as an either alternative or additional test to determine HER2 status in breast carcinomas.

Breast Neoplasms↗
PubMed1990

Pediatric digital chest imaging.

The Philips Computed Radiography system performs well with pediatric portable chest radiographs, handling the throughout of a busy intensive care service 24 hours a day. Images are excellent and routinely provide a conventional (unenhanced) image and an edge-enhanced image. Radiation dose is decreased by the lowered frequency of repeat examinations and the ability of the plates to respond to a much lower dose and still provide an adequate image. The high quality and uniform density of serial PCR portable radiographs greatly enhances diagnostic content of the films. Decreased resolution has not been a problem clinically. Image manipulation and electronic transfer to remote viewing stations appear to be helpful and are currently being evaluated further. The PCR system provides a marked improvement in pediatric portable chest radiology.

Child↗
PubMed2005

Micro flow-through PCR in a PMMA chip fabricated by KrF excimer laser.

As the third PCR technology, micro flow-through PCR chip can amplify DNA specifically in an exponential fashion in vitro. Nowadays many academies in the world have successfully amplified DNA using their own-made flow-through PCR chip. In this paper, the ablation principle of PMMA at 248 nm excimer laser was studied, then a PMMA based flow-through PCR chip with 20 cycles was fabricated by excimer laser at 19 kv and 18 mm/min. The chip was bonded together with another cover chip at 105( composite function)C, 160 N and 20 minutes. In the end, it was integrated with electrical thermal thin films and Pt 100 temperature sensors. The temperature controllers was built standard PID digital temperature controller, the temperature control precision was +/- 0.2( composite function)C. The temperature grads between the three temperature zones were 16.5 and 22.2( composite function)C respectively, the gaps between the temperature zones could realize heat insulation.

Equipment Design↗
PubMed2000

Characterization of muscarinic receptors in human lens cells by pharmacologic and molecular techniques.

PURPOSE: Activation of muscarinic receptors has been implicated in an increased risk of cataract after anticholinesterase treatment for glaucoma. The purpose of the present study was to determine the acetylcholine muscarinic receptor subtype(s) present in native human lens epithelial cells (NHLECs) and a human lens cell line, HLE-B3, and to compare the distribution in other ocular cells. METHODS: Human lens cells were perfused with artificial aqueous humor (35 degrees C) after fura-2 incorporation, and calcium levels were measured using a fluorometric single-cell digital imaging system. Acetylcholine was the primary muscarinic agonist, and the receptor subtypes were elucidated by determining the relative effectiveness of pirenzepine and AF-DX 384 in blocking the agonist-induced response. The levels of expression of mRNA for the receptor subtypes M1 through M5 were determined by quantitative reverse transcription-polymerase chain reaction (QRT-PCR) using a sequence detection system (ABI Prism 7700; Perkin-Elmer, Foster City, CA). This was performed using total RNA extracted from native lens, retina, iris, and sclera and also cultured lens cells. RESULTS: Acetylcholine induced a similar concentration-dependent increase in peak-amplitude cytosolic calcium in the range 100 nM to 100 microM in both native and HLE-B3 cells. However, the kinetics of the response waveforms to 30-second pulses of acetylcholine were different in the two cell types. At higher concentrations (> 1 microM), a second phase appeared in the HLE-B3 cells that was absent in the NHLEC response. The 50% inhibitory concentration (IC50) values for blockade of a 1 microM acetylcholine response by pirenzepine and AF-DX 384 were 30 nM and 230 nM, respectively, for NHLECs, and 300 nM and 92 nM, respectively, for HLE-B3 cells. The QRT-PCR data showed that more than 90% of the total muscarinic receptor mRNA from NHLEC was of M1 origin. In the HLE-B3 cells, however, more than 95% of the mRNA was of M3 origin. mRNA for M3 was also in greatest abundance in other eye tissues, although there was a significant contribution from M1 in iris and sclera. CONCLUSIONS: Both NHLECs and HLE-B3 cells express muscarinic receptors that produce significant changes in cytosolic calcium in response to acetylcholine. Both pharmacologic and QRT-PCR evidence shows that whereas the M1 subtype predominates in NHLECs, M3 is the major contributor in HLE-B3 cells. In all other eye tissues, M3 appears to be the major contributor. These data should be taken into account when choosing particular models to investigate cataract mechanisms and also when designing muscarinic agonists to treat glaucoma.

Acetylcholine↗
PubMed2006

Homozygous deletion of MKK4 in ovarian serous carcinoma.

Analysis of deleted chromosomal regions in tumors has historically led to the identification of tumor suppressor genes. In this study, we used digital karyotyping, a genome-wide, high-resolution technology, to search for chromosomal deletions in ovarian serous carcinoma, the most lethal gynecological malignancy in women. Five purified ovarian serous carcinomas were analyzed by digital karyotyping and small interstitial deletions at chromosome 17p were identified in two tumor samples. Aligning these two deletions identified an overlapping region that spanned 2.4 Mb which harbored a candidate tumor suppressor gene, mitogen-activated protein kinase kinase-4 (MKK4). Dual-color FISH analysis confirmed homozygous deletion of the MKK4 locus in both samples and RT-PCR demonstrated that both carcinomas lacked MKK4 transcript expression. Loss of heterozygosity of 17p occurred in 24 (86%) of 28 high-grade serous carcinomas including both cases with homozygous MKK4 deletion. Additionally, downregulation of MKK4 expression was found in 96 (75%) of 128 ovarian serous carcinomas as compared to benign ovarian tissues. These findings suggest that homozygous deletion or reduced expression of MKK4 may contribute to the development of ovarian serous carcinoma.

Female↗
PubMed2003

[Expression of transporter associated with antigen processing (TAP) in childhood acute leukemia and its clinical significance].

BACKGROUND & OBJECTIVE: Transporter associated with antigen processing(TAP) participates in immune surveillance, so it is probably relevant to carcinogenesis. Investigation of expression of TAP and its clinical significance in childhood acute leukemia will be helpful to clarify pathogenesis and to develop immunotherapy strategy. METHODS: RT-PCR analysis was used to detect the expression of TAP1 and TAP2 in leukemia cells from bone marrow in 34 inpatients with primary acute lymphoblastic leukemia (ALL), 15 inpatients with relapsed ALL, 20 inpatients with acute medullary leukemia (AML), and 20 surgical inpatients without systematic disorders as control. And then,the absorbance (A) values of expanded bands were measured by digital imaging analyzer and relative A values were worked out based on A value of GAPDH (positive inside control). RESULTS: The relative A values of TAP1(0.448+/-0.167 and 0.169+/-0.021,respectively) and TAP2(0.196+/-0.180 and 0.112+/-0.020, respectively) in primary ALL group and relapsed ALL group were lower than those in control group (P< 0.01). The A values of LMP2, LMP7, and PA28alpha (991.4+/-532.7, 686.3+/-663.8, and 2022.3+/-1622.3, respectively) in relapsed ALL group were lower than those in control group (P less than 0.01,0.01 and 0.05, respectively). The A value of LMP2 in relapsed group was lower than that in control (P< 0.01). The A values of LMP7 for the cases with no remission and relapse were both lower than that for the cases with constant complete remission in primary ALL group (P< 0.01). The relative A value of TAP2 in AML group was lower than that in control group (P< 0.05). The relative A value of TAP1 in relapsed ALL group was lower than that in primary ALL group (P< 0.05). In primary ALL group, the relative A value of TAP1 (0.215+/-0.159) for cases with relapse (6/34 cases) was lower than that (0.462+/-0.189) for those with constant complete remission (24/34 cases) (P< 0.05). CONCLUSION: There exists decreased expression of TAP in both childhood ALL and AML, which probably contributes to the escape of leukemia cells from immune surveillance. Decreased expression of TAP1 subunit is probably related to the relapse of ALL.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

Bioinformatics-based discovery of a novel factor with apparent specificity to colon cancer.

In a previous study, a data mining tool called Digital Differential Display (DDD) from the Cancer Genome Anatomy Project (CGAP) was used to predict solid tumor- and organ-specific genes from the expressed sequence tag (EST) database. To validate the use of bioinformatics approaches in gene discovery, one of the ESTs, which was predicted to be colon tumor-specific, was chosen for further study. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis of matched sets of cDNAs from normal and colon tumor tissues indicated that the EST was specifically expressed in the majority of colon tumors. Expression was also detected in early adenomas. Among other normal tissues, EST expression was detected only in the small intestine. The colon tumor specificity of this EST was inferred from the lack of expression in carcinomas of the breast, lung, ovary, pancreas and prostate. To validate the computational prediction of specificity, a full-length cDNA encompassing the entire open reading frame was cloned and, in view of its apparent specificity to the colon tumors, this gene was termed Colon Carcinoma Related Gene (CCRG). CCRG encodes a novel cysteine-rich motif and a putative signal peptide sequence. Supernatant from COS cells transfected with the CCRG expression vector stimulated proliferation of colon cancer cells. Immunoreactive CCRG was also detected in the paraffin sections of colon tumor samples. CCRG belongs to a new class of growth factors and may be important in the diagnosis and treatment of colon cancers. Identification of CCRG using bioinformatics approaches validates gene discovery using computational approaches.

Amino Acid Sequence↗
PubMed2005

Molecular cloning and primary functional analysis of a novel human testis-specific gene.

In this study, a new data mining tool called Digital Differential Display (DDD) from the NCBI was used to predict testis-specific expressed genes from the expressed sequence tag (EST) database. DDD (digital differential display) was performed between nine testis libraries and seventy libraries derived from other tissues. We identified a new contig of ESTs (HS. 326528) which was from testis libraries. To validate the use of bioinformatic approaches in gene discovery, the ESTs (HS. 326528), which were predicted to be testis-specific, were chosen for further study. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis of matched sets of cDNAs from testis and other tissues indicated that the ESTs were specifically expressed in testis. This result was further validated by multi-tissue Northern blot. The full-length cDNA encompassing the entire open reading frame was cloned and, in view of its apparent specificity to testis, the gene was termed homo sapiens spermatogenesis-related gene 8---SRG8 (GenBank accession number: AY489187). The gene whose full cDNA length is 1 044 bp containing 3 exons and 2 introns is located in human chromosome 15q26.2, the cDNA encodes a novel protein of 105 amino acides with a theoretical molecular weight of 11.7 kD and isoelectric point of 10.09 which shares no significant homology with any known proteins in database. Real time PCR analysis of testis of different developmental periods revealed that SRG8 gene is significantly expressed in adult testis. The green fluorescent protein produced by pEGFP-C3/SRG8 was detected in the nucleus of HeLa cells after 24 h post-transfection. Cell cycle analysis showed that SRG8 can accelerate HeLa cells to traverse the S-phase and enter the G2-phase compared with the control without transfection of SRG8, which suggested that this gene plays an important role in the development of testis. The discovery of SRG8 showed that DDD combined with experiments is a feasible, time-saving strategy to identify new candidate genes for testis-specific development

Adult↗
PubMed2006

Bacterial diversity in a finished compost and vermicompost: differences revealed by cultivation-independent analyses of PCR-amplified 16S rRNA genes.

Bacterial communities are important catalysts in the production of composts. Here, it was analysed whether the diversity of bacteria in finished composts is stable and specific for the production process. Single-strand conformation polymorphism (SSCP) based on polymerase chain reaction amplified partial 16S rRNA genes was used to profile and analyse bacterial communities found in total DNA extracted from finished composts. Different batches of compost samples stored over a period of 12 years and a 1-year-old vermicompost were compared to each other. According to digital image analysis, clear differences could be detected between the profiles from compost and vermicompost. Differences between three different periods of compost storage and between replicate vermicompost windrows were only minor. A total of 41 different 16S rRNA genes were identified from the SSCP profiles by DNA sequencing, with the vast majority related to yet-uncultivated bacteria. Sequences retrieved from compost mainly belonged to the phyla Actinobacteria and Firmicutes. In contrast, vermicompost was dominated by bacteria related to uncultured Chloroflexi, Acidobacteria, Bacteroidetes and Gemmatimonadetes. The differences were underscored with specific gene probes and Southern blot hybridizations. The results confirmed that different substrates and composting processes selected for specific bacterial communities in the finished products. The specificity and consistency of the bacterial communities inhabiting the compost materials suggest that cultivation-independent bacterial community analysis is a potentially useful indicator to characterize the quality of finished composts in regard to production processes and effects of storage conditions.

Bacteria↗
PubMed2006

An inexpensive microslab gel DNA electrophoresis system with real-time fluorescence detection.

In this paper, we describe the construction of a simple yet powerful gel electrophoresis apparatus that can be used to perform size-selective separations of DNA fragments in virtually any laboratory. This system employs a microslab gel format with a novel gel casting technique that eliminates the need for delicate combs to define sample loading wells. The compact size of the microslab gel format allows rapid separations to be performed at low voltages using submicroliter sample volumes. Real time fluorescence detection of the migrating DNA fragments is accomplished using an inexpensive digital microscope that directly connects to any PC with a USB interface. The microscope is readily adaptable for this application by replacing its white light source with a blue light-emitting diode (LED) and adding an appropriate emission filter. Both polyacrylamide and agarose gels can be used as separation matrices. Separation performance was characterized using standard dsDNA ladders, and correct sizing of a 191 bp PCR product was achieved in 15 min. The low cost and simplicity of this system makes it ideally suited for use in a variety of laboratory and educational settings.

DNA↗
PubMed2005

[Protective effects of amygdalin on hyperoxia-exposed type II alveolar epithelial cells isolated from premature rat lungs in vitro].

OBJECTIVE: To analyze the effect of hyperoxia on the proliferation and surfactant associated protein messenger RNA levels of type II alveolar epithelial cells (AECIIs) of premature rat, and to investigate the effect of amygdalin on the change resulted from hyperoxia in AECIIs isolated from premature rat lung in vitro. METHODS: The lung tissue of 20-day fetal rat was digested by trypsin and collagenase. AECIIs and lung fibroblasts (LFs) were isolated and purified at different centrifugal force and different adherence, then cultured. The nature of the cultures was identified by cytokeratin staining, vimentin staining and transmission electron micrography. For establishing hyperoxia-exposed cell model, purified AECIIs were cultured for 24 hours after culture flasks were filled with 95% oxygen-5% CO2 at 3 L/min for 10 min, and then sealed. Oxygen concentrations were tested in CYS-1 digital oxygen monitor after 24 hours of exposure. A sample was discarded if its oxygen concentration was < 90%. Cell proliferating vitality was examined by MTT assay after treatment with amygdalin at various concentrations. DNA content, protein expression of proliferating cell nuclear antigen (PCNA) and mRNA levels of SPs of AECIIs were analyzed with flow cytometric assay, Western blot and reverse transcription polymerase chain reaction (RT-PCR) respectively after 24 hours of air or hyperoxia exposure in the presence or absence of 200 micromol/L amygdalin. RESULTS: Excellent yields of highly purified, culturable AECIIs could be obtained from 20-day fetal lungs. The expression of cytokeratin in AECIIs was positive and that of vimentin negative by immunocytochemistry. Those, however, in LFs were just opposite. Lamellar bodies in purified AECIIs were revealed by transmission electron micrography. The established hyperoxia-exposed cell model assured the oxygen concentrations of culture flasks more than 90%. Amygdalin at the concentration range from 50 micromol/L to 200 micromol/L stimulated the proliferation of AECIIs in a dose-dependent manner; however, at the concentration of 400 micromol/L inhibited the proliferation of AECII. Flow cytometric analysis showed that the apoptosis rate and G0/G1 phase percentage increased significantly (P < 0.01), S phase and G2/M phase percentage decreased significantly (P < 0.01), in hyperoxia group compared with that of air group. The apoptosis rate of air plus 200 micromol/L amygdalin group, compared with air group, was not significantly different (P > 0.05); however, G0/G1 phase percentage decreased markedly, S phase percentage increased significantly, G2/M phase percentage did not significantly change (P > 0.05). The apoptosis rate of hyperoxia plus 200 micromol/L amygdalin group was not significantly different (P > 0.05) from that of hyperoxia group, S phase and G2/M phase percentage increased significantly (P < 0.01), G0/G1 phase percentage decreased significantly (P < 0.01). Western blot analysis showed that the protein expression levels of PCNA in all group was significantly different, in turn, hyperoxia group < hyperoxia plus 200 micromol/L amygdalin < air group < air puls 200 micromol/L amygdalin (P < 0.01). SPs mRNA levels were significantly decreased in hyperoxia group, as compared with air group (P < 0.01). After amygdalin was added, SPs mRNA levels were elevated in air plus amygdalin group and hyperoxia plus amygdalin group, as compared with hyperoxia group (P < 0.01, P < 0.05, respectively), but compared with air group, SP mRNA levels were not significantly elevated (P > 0.05). CONCLUSION: AECIIs of premature rats were isolated, purified and cultured successfully. Hyperoxia-exposed cell model was established in AECIIs of premature rat in this experiment. Amygdalin promotes the proliferation of premature rat AECII exposed to air or hyperoxia, the concentration of amygdalin with the best effect was 200 micromol/L. Hyperoxia inhibited the proliferation and decreased SPs mRNAs levels in AECIIs in vitro, which may contribute to hyperoxia-induced lung injury in premature rats. Amygdalin could inhibit the changes of SPs mRNAs levels and cell proliferation of AECIIs resulted from hyperoxia and may play partial protective role in hyperoxia-induced premature lung injury.

Amygdalin↗
PubMed1996

Regulation of retinal hemodynamics in diabetic rats by increased expression and action of endothelin-1.

PURPOSE: To investigate the role of endogenous endothelin-1 (ET-1) expression and its interaction with the ETA receptor in the physiologic regulation of vascular tone as well as in the development of abnormal retinal hemodynamics in diabetes. METHODS: Retinal blood flow, using digitized video fluorescein angiography recordings, was quantitated after intravitreous injections of ET-1; BQ-123, an ETA receptor antagonist; and phosporamindon, an endothelin converting enzyme inhibitor in the eyes of diabetic and nondiabetic rats. A total of 154 rats were used for these experiments. Message levels of preproendothelin-1 (preproET-1) were measured from the retina of diabetic and nondiabetic rats using competitive polymerase chain reaction (PCR) techniques. RESULTS: Retinal blood flow was reduced (33%, P < 0.001) in diabetic rats compared to nondiabetic rats. BQ-123, an ETA receptor antagonist, but not saralasin, an angiotensin receptor antagonist, increased retinal blood flow in a dose-dependent manner in diabetic (EC50 of 8 x 10(-7) M) and in nondiabetic rats (EC50 of 8 x 10(-8) M). Besides being resistant to BQ-123, the maximal response in diabetic animals occurred 20 minutes later than in nondiabetic animals. Decreasing ET-1 levels by inhibiting endothelin-converting enzyme with phosphoramidon normalized retinal blood flow in diabetic rats. In nondiabetic rats, the intravitreous injection of exogenous ET-1 (10(-8) M) resulted in retinal blood flow decreases comparable to those measured in diabetic animals, and the subsequent injection of 10(-4) M BQ-123 produced retinal blood flow changes comparable to those measured in BQ-123 injected diabetic rats. Comparison of preproET-1 messenger RNA expression in the retina, brain and lung of control and diabetic rats using quantitative PCR and Northern blot analysis showed 2.0- and 1.7-fold increases in the retina and the brain, respectively, without changes in the lung. CONCLUSIONS: These data suggest that ET-1 is involved in the regulation of retinal blood flow in normal physiologic outcome, and an increase in the endogenous expression of ET-1 contributes to the reduction of retinal blood flow reported in the early stages of diabetes mellitus.

Animals↗
PubMed1997

Effects of toluidine blue and destaining reagents used in sexual assault examinations on the ability to obtain DNA profiles from postcoital vaginal swabs.

Toluidine blue is an important tool to detect and document genital and perianal injuries following sexual assault. Application of toluidine blue dye and its subsequent removal from unstained areas by means of a destaining reagent, such as diluted acetic acid or a lubricant has been shown to increase the detection rate of posterior fourchette lacerations from 16% to 40% in adult rape victims. Currently, limited information on toluidine blue positive findings in sexually active control groups imposes some limitation on the interpretation of these injuries. Because injuries could otherwise be attributed to improper handling of an examination speculum or the improper insertion of the examining finger, the toluidine blue test should be performed prior to any digital or speculum examination and thus prior to the collection of forensic evidence. For forensic DNA identity testing, it becomes pertinent to determine whether toluidine blue and the destaining reagents used in a sexual assault examination have an adverse effect on the recovery of high molecular weight DNA from postcoital vaginal swabs and thereby have an impact on restriction fragment length polymorphism (RFLP) analysis or PCR-based tests. It is known that some of the lubricants used can have a destructive effect on sperm motility. In order to investigate the potential effects, postcoital vaginal swabs were taken 6 h after sexual intercourse and exposed directly to 1% toluidine blue in aqueous solution, 1-10% acetic acid, and various surgical and vaginal lubricants. Subsequently, the DNA was isolated and DNA identity typing (RFLP and PCR-based) was performed. The results demonstrate, that these reagents have no negative effect on the ability to obtain DNA profiles, either RFLP or PCR-based, from shallow and deep vaginal swabs. The quantity and quality of extractable high molecular weight DNA obtained was comparable with that from uncontaminated postcoital vaginal swabs. RFLP patterns and PCR-based typing results on the D1S80, HUMTH01, TPOX, and CSF1PO loci were consistent with the uncontaminated control swabs and the corresponding whole blood samples of the donors. Therefore, evidentiary material inadvertently contaminated with these reagents can be successfully typed.

Adult↗
PubMed2001

Feline cutaneous fibropapillomas: clinicopathologic findings and association with papillomavirus infection.

Twenty-three feline cutaneous fibropapillomas with histologic features similar to equine sarcoids were diagnosed. They were characterized by dermal fibroblastic proliferation with overlying, often ulcerated hyperplastic epidermis. Electron microscopic findings supported the fibroblastic nature of the neoplastic cells. The 23 tumors came from 20 cats and were submitted from veterinary clinics in Wisconsin and Minnesota. These tumors occurred most commonly in young cats and were found primarily on the head, neck, and digits. Fifteen of the 17 cats for which breed was reported were domestic shorthair cats. In 11/20 cases, there was confirmed exposure to cattle. Local recurrence of the tumor following surgical excision was reported in 7 of the 18 cats for which follow-up information was available. Metastasis was not documented in any of the cases. Two of the 19 tumors tested by polymerase chain reaction (PCR) had no amplifiable DNA. The remaining 17 were positive for papillomavirus by PCR. No papillomavirus DNA was detected in three other feline skin tumors (cutaneous mast cell tumor, malignant lymphoma, and fibrosarcoma) that served as controls. This is the first report of detection of papillomavirus in feline tumors that have clinicopathologic features similar to equine sarcoids.

Animals↗
PubMed2006

[A DNA duplication at chromosome 10q24.3 is associated with split-hand split-foot malformation in a Chinese family].

OBJECTIVE: To identify the disease-causing genetic alteration of split-hand/split-foot malformation (SHFM) in a Chinese family. METHODS: Three of the 5 affected individuals from a four-generation Chinese SHFM family were examined physically and radiologically. Peripheral blood samples were collected from Digital photographs of the malformed hands and feet were taken. Peripheral blood samples were collected from 2 affected individuals, and lymphocytes were isolated to undergo high resolution G-banding. Genomic DNA was extracted from the whole blood samples of 4 available family members, including the 3 affected individuals. All 16 exons and their flanking intronic sequences of the TP63 gene were amplified using polymerase chain reaction (PCR) and sequenced directly. Microsatellite markers from the five SHFM loci were analyzed in the available family members by PCR, polyacrylamide gel electrophoresis and silver staining. For semi-quantitative determination of the allele copy number, the polymorphic PCR-amplified fragments representing genetic markers from the SHFM3 locus at chromosome 10q24.3 were sequenced in the affected individuals using normal individuals with identical genotypes as controls. RESULTS: All 3 existing affected individuals showed absence of 3 radial fingers, 2 affected individuals had a deep central cleft and central ray deficiency in the feet, and 1 affected individual had a fibular monodactyli, all limb malformations being bilateral and consistent with the phenotype of typical SHFM. G-banding showed normal karyotypes in the 3 affected individuals and no visible cytogenetic abnormality was found. Moreover, no mutation was identified in the TP63 gene. While no haplotype sharing was observed in the markers from loci SHFM1, SHFM4 and SHFM5, potential haplotype sharing was detected in the markers from two loci, SHFM2 and SHFM3, indicating possible causative mutation at SHFM2 or SHFM3. Furthermore, obviously biased silver density toward the allele fragments shared by the 3 affected individuals was observed in the markers from the SHFM3 locus. Comparative sequencing showed roughly one-fold increase of fluorescent signal of the shared fragments in the affected individuals. These results suggested a large-scale DNA duplication within the SHFM3 locus. CONCLUSION: A large-scale DNA duplication within the SHFM3 locus at chromosome 10q24.3 has been identified as the pathogenic genetic change in Chinese patients with SHFM.

Base Sequence↗