Drowning is one of the most common causes accidental death worldwide, but its diagnosis remains a challenging task in forensic pathology. Several authors have suggested that diatom analysis be conducted via an enzymatic digestion method that uses proteinase K to provide objective evidence for drowning; we employed this method in our study because of its superior applicability as compared to the conventional disorganization methods. The purpose of this study was to examine the reclaiming ratio of diatoms from experimentally drowned animal organs, which could be influenced by diatom morphology. The authors injected 3 diatoms species (Cyclotella striata, Navicula incerta, and Pleurosigma angulatum) into a rat's airway and compared the detection rate to investigate the factors that influence the sensitivity of diatom analysis. The results are as follows: (1) Average reclaiming ratio in the lungs was 81.07 for Navicula incerta, 48.26 for Cyclotella striata, and 5.35 for Pleurosigma angulatum. (2) The detection rates from the closed organs in 15 experimental animals were highest in the kidney (73%, 11/15), followed by the heart (67%, 10/15), brain (60%, 9/15), and liver (53%, 8/15). (3) Two Cyclotella striata was detected in the kidney of postmortem control group which suggest the possibility of contamination during laboratory procedure. In conclusion, the authors propose that diatom size could be a significant influencing factor for diatom extraction from the organs of drowned bodies; therefore, the results of diatom analysis must be interpreted after considering the diatom population of the drowning medium at the scene and the possibility of contamination during the laboratory procedure.
Animal Structures ; Animals ; Brain ; Diatoms ; Digestion ; Drowning ; Endopeptidase K ; Forensic Pathology ; Heart ; Kidney ; Liver ; Lung

Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular bacterium that replicates in the cytosol of host cells. Although several protein antigens have been characterized and cloned, little information exists regarding the polysaccharide antigen of this bacterium. In this study, we characterized two monoclonal antibodies, NT19 and WT14, against the proteinase K-resistant antigen of O. tsutsugamushi. Western blot analysis showed that MAb NT19 and WT14 strongly recognized two antigenic bands with molecular masses of 20 kDa and 24 kDa, which were resistant to proteinase K digestion. We suggest that the proteinase-resistant antigen might be polysaccharide. One patient serum reacted with a 24 kDa band that was similar to a band observed by WT14, suggesting the possibility of the role of this proteinase-resistant antigen as an antigenic molecule in human infection.
Antibodies, Monoclonal ; Blotting, Western ; Clone Cells ; Cytosol ; Digestion ; Endopeptidase K ; Humans ; Orientia tsutsugamushi ; Scrub Typhus

BACKGROUND: The extraction methods of DNA from clinical samples are the major obstacle to use the PCR(Polymerase Chain Reaction) in routine labortary for early detection of M. tuberculosis. We tried to improve the extraction method of DNA from sputum for establishment of the PCR in routine labortary by reducing the possibility of cross contamination and performing it easily and safely. METHODS: We used the InstaGene(TM) DNA extraction kit(BioRad Co.) using Chelex 100 ion exchange resin for preparation of DNA. We compared InstaGene method in 100 cases of sputum from proteinase K method which is known as the most commonly used method for DNA purification(Experiment 1). And we compared InstaGene method in 98 cases of sputum from Microwave method developed by a company in Korea(Experiment 2). In experiment 1, 245bps of IS6110 were amplified and then 188bps were amplified by nested PCR. In experiment 2, 536bps in primary PCR and 276bps in nested PCR were amplified and analysed by agarose gel electrophoresis and EtBr staining. RESULTS: When we chose AFB smear, culture, or AFB smear and culture as a standard test, PCR had low specificity and positive predictive value in both experiments. The InstaGene method has higher value in sensitivity and negative predictive value significantly than proteinase K method. The InstaGene method and the Microwave methods were similar in sensitivity, specificity, positive predictive value and negative predictive value.. CONCLUSION: Even though both methods had lower possibility of cross contamination, shorter time requrirement, simplicity, and economic advantages than Proteinase K method, the InstaGene method was a little simpler than the Microwave method. Therefore, in terms of usfulness in clinical application, the Instagene method seems to be the most useful method in DNA extraction for detection of M. tuberculosis using PCR. The reliability of this method will be clarified by further studies with enough clinical samples.
DNA* ; Electrophoresis, Agar Gel ; Endopeptidase K ; Ion Exchange* ; Microwaves ; Polymerase Chain Reaction* ; Sensitivity and Specificity ; Sputum ; Tuberculosis*

The fibrillar coat of Candida albicans is of interest as its significance in antigenicity, antiphagocytosis, and adherence to host tissues. The partial biochemical properties and ultrastructure of fibrillar coat induced by rabbit sera were examined. The induced fibrillar layer was destroyed by treatments of lyticase, proteinase K and dithiothreitol. The total protein concentration of fibrillar cell wall lysate was higher than that of non-fibrillar cell wall lysate, but the total sugar concentration was similar. On SDS-PAGE analysis, the protein profiles between in fibrillar cells and in non-fibrillar cells were shown to be different. In fibrillar cells, the major bands of cell wall lysate were 83, 66, 54, 47, 33, and 26 kDa in dithiothreitol-treated lysate. The proteins of 26 and 19 kDa were predominant in lyticase-treated lysate. Although the fibrillar thickness and protein amount of cell wall lysate were increased in according to the incubation time, the protein profiles did not changed. These results suggest that the proteins of 83, 66, 54, 47, 33, 26, and 19 kDa may be major constituents of fibrillar coat in C. albicans.
Candida albicans* ; Candida* ; Cell Wall ; Dithiothreitol ; Electrophoresis, Polyacrylamide Gel ; Endopeptidase K

The acid digestion method for extracting diatoms has been widely used to confirm death by drowning, but its reliability is still disputed because some diatoms can be destroyed during the extraction process due to treatment with strong acid and heat. There is a need to develop an efficient and reliable digestive method to overcome the limitation of the present analytical procedure. In this study, the reliability and efficacy of quantitative and qualitative diatom analysis from seawater by an enzymatic digestion method was evaluated. We confirmed the merit of the enzymatic method that used proteinase K instead of nitric acid in the conventional method. As a result, the enzymatic method showed a higher recovery ratio and better preservation of the diatom structure, which is essential for quantitative (diatom density) and qualitative (species) interpretation of diatom analysis. This result indicates that the enzymatic method can replace the conventional acid digestion method to confirm cases of death by drowning since it is more reliable and yields conclusive results.
Diatoms ; Digestion ; Drowning ; Endopeptidase K ; Hot Temperature ; Nitric Acid ; Plankton ; Seawater

Multilocus sequence typing (MLST) of Salmonella is useful method for replacing serotyping using antisera but is limited by difficulties associated with in polymerase chain reaction (PCR). We optimized the PCR reaction, especially annealing temperature and extension time (94degrees C for 2 min; 40 cycles at 94degrees C for 30 sec, 56.8degrees C for 1 min, 72degrees C for 2 min; and 72degrees C for 10 min). The degradation of PCR product by thermostable nucleases was inhibited by using template DNAs treated proteinase K or purified by a commercialized preparation kit. The resulting modified MLST was used as accurate and fast typing method.
DNA ; Endopeptidase K ; Immune Sera ; Multilocus Sequence Typing* ; Polymerase Chain Reaction ; Salmonella* ; Serotyping*

In order to study the physicochemical characteristics of cytosolic PrP (CytoPrP) and evaluate its possible influence on cell viability, a recombinant plasmid expressing human CytoPrP eukaryoticly was constructed and transfected into human neuroblastoma cell line SH-SY5Y transiently. Proteinase-resistant activities of CytoPrP were evaluated by a proteinase K (PK) digestion and cytotoxic effects of CytoPrP were tested by MTT assay and Trypan Blue cell-counting. The presence of CytoPrP in cytoplasm after transfection was controlled by the presence of protease inhibitor. Compared with wild-type PrP, CytoPrP possessed relatively stronger PK-resistant activities. Obvious cytotoxic effects were observed in the cells after inducement of CytoPrP in cytoplasm by protease inhibitor, showing a dose-dependent manner. The results provide useful scientific evidences for further studies of potential role of CytoPrP in pathological mechanism of prion disease.
Cell Line, Tumor ; Cell Survival ; Cytosol ; chemistry ; Endopeptidase K ; pharmacology ; Humans ; Prions ; genetics ; physiology ; Transfection

PURPOSE: P53 point mutation, especially G to T transversion at codon 249, has been found in many cases of the aflatoxin-related hepatocellular carcinoma(HCC). This study was aimed to exam the mutations of the third base at codon 249 of the p53 gene in Korean HCC. MATERIALS AND METHODS: Forty two patients with resectable HCC were enrolled. Genomic DNA was prepared by proteinase K digestion and phenol/chloroform extractions. A part of DNA including axon 7 of p53 was amplified by PCR. Restriction fragment polymorphism (RFLP) was analyzed after treatment of Hae III restriction enzyme. And the point mutation were confirmed using the direct sequencing method. RESULTS: P53 point mutation, G to T transversion at codon 249 was observed in only one tissue specimen(2.4%). CONCLUSION: P53 point mutation at codon 249 does not correlate with HCC in Korea.
Aflatoxins* ; Axons ; Carcinoma, Hepatocellular* ; Codon* ; Digestion ; DNA ; Endopeptidase K ; Genes, p53 ; Humans ; Korea ; Point Mutation* ; Polymerase Chain Reaction

BACKGROUND: To assess the clinical utility of new DNA extraction method, the authors attempted PCR using mycobacterial DNA extracted by Chelex 100 ion exchange resin method for 63 clinical samples in patients with pulmonary tuberculosis and compared with proteinase K method, simultaneously. METHODS: We used Chelex 100 ion exchange resin for preparation of DNA. Decontaminated sputums were mixed with resin and incubated at 56degrees C and 100degrees C without opening tube. After centrifugation, supernatants were used directly as template for PCR. 245 bps in primary PCR and 188 bps in nested PCR were amplified and analysed by agarose gel electrophoresis EtBr staining. RESULTS: Chelex 100 ion exchange resin method is more simple, rapid and reliable than proteinase K method, and during sample preparation, carry-over contamination loss of amplificated DNA, influence of organic solvents and cross-contamination are diminished. The results of PCR products are interpreted more distinctively in Chelex 100 ion exchange resin method than proteinase K method. CONCLUSIONS: In the basis of the results, it could be suggested that extraction of mycobacterial DNA by Chelex 100 ion exchange resin is more simple, rapid reliable method than that of conventional method for detection of mycobacterial DNA in patients with tuberculosis by polymerase chain reaction.
Centrifugation ; Diagnosis* ; DNA* ; Electrophoresis, Agar Gel ; Endopeptidase K ; Humans ; Ion Exchange ; Polymerase Chain Reaction* ; Solvents ; Sputum ; Tuberculosis* ; Tuberculosis, Pulmonary

BACKGROUND: A practical RNA in situ hybridization method using digoxigenin labeled RNA probes is described in order to evaluate the technical difficulties and problems in RNA in situ hybridization. METHODS: The paraffin sections, routinely processed in the Pathology Laboratory, were tested for the possibility of RNA in situ hybridization instead of the RNase free paraffin sections, fixed in 4% paraformaldehyde and prepared using RNase protection procedures. RESULTS: Most of the paraffin sections, fixed in 10% neutral formalin solution in fresh condition, showed relatively good reaction of RNA in situ hybridization, although the necrotic tissue and autopsy specimens showed poor reaction of RNA in situ hybridization. A refixation procedure using a 4% paraformaldehyde solution was evaluated for optimal expression of mRNA in the paraffin sections. CONCLUSION: The treatment of 4% paraformaldehyde before the treatment of proteinase K showed better in situ hybridization than did the treatment of 4% paraformaldehyde after the treatment of proteinase K. Also a new Polymerase Chain Reaction (PCR)-based method of RNA probe production showed consistently good results.
Autopsy ; Digoxigenin* ; Endopeptidase K ; Formaldehyde ; In Situ Hybridization* ; Paraffin ; Pathology ; Polymerase Chain Reaction ; Ribonucleases ; RNA Probes ; RNA* ; RNA, Messenger