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Biomedical subjects

R Higuchi

Publications and source records attributed to R Higuchi.

At least 73 records · Page 4Linked to original sources
PubMed1992

Polymerase chain reaction (PCR) amplification and human leukocyte antigen (HLA)-DQ alpha oligonucleotide typing on biological evidence samples: casework experience.

The polymerase chain reaction (PCR) method of specific gene amplification was used in casework to synthesize millions of copies of the polymorphic second exon of the human leukocyte antigen (HLA)-DQ alpha (or DQA1) locus from a variety of evidence samples. The HLA-DQ alpha allelic variants in the amplified deoxyribonucleic acid (DNA) were determined in a rapid non-radioactive test by hybridization to sequence-specific oligonucleotide probes in both the dot-blot and reverse dot-blot formats. This genetic typing system has been subjected to blind proficiency testing; the performance of this test in the analysis of experimentally mixed samples was also evaluated. As of August 1990, over 250 cases have been tested and more than 2000 individual evidence (bloodstains, semen stains, individual hairs, bone fragments, and tissue sections) and reference samples have been analyzed. The first 198 of these cases are summarized in this paper; in 65% of the cases with conclusive results a suspect was included, and in 35%, all suspects were excluded. Individual cases as well as some of the general issues relating to forensic science analysis and this genetic typing system are discussed. The high rate of exclusion reported here combined with the ability of PCR to type old evidence samples suggests the relevance of this genetic test for postconviction review; two cases in which the convicted suspect was excluded are discussed.

Adolescent↗
PubMed1991

Nebulized vs. instilled exogenous surfactant in an adult lung injury model.

Three days after subcutaneous injection of N-nitroso-N-methylurethane (NNNMU) to induce lung injury, adult rabbits were mechanically ventilated and lung function was evaluated. Each animal then received either nebulized Survanta (Neb Surv), nebulized saline (Neb Saline), nebulized gas alone (Neb Gas), or tracheally instilled Survanta (Inst Surv). The ventilation efficiency index (VEI) value increased significantly compared with pretreatment values (P less than 0.01) over a 3-h treatment period for the Neb Surv animals, whereas VEI values for the other three groups decreased after treatment (P less than 0.05). Arterial PO2-to-fraction of inspired O2 ratios and dynamic compliance values significantly decreased after treatment for the Inst Surv group (P less than 0.05). Pressure-volume curves demonstrated a significantly greater volume at maximal pressure for the Neb Surv group compared with each of the other groups studied (P less than 0.01). The calculated quantity of surfactant recovered in lung tissue for the Neb Surv group was only 4.9 +/- 1.0 mg lipid/kg compared with 100 mg lipid/kg delivered to the Inst Surv group. Surfactant administered as an aerosol resulted in modest physiological improvements in this model of lung injury and was superior to the tracheal instillation technique.

Aerosols↗
PubMed1991

Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material.

Procedures utilizing Chelex 100 chelating resin have been developed for extracting DNA from forensic-type samples for use with the PCR. The procedures are simple, rapid, involve no organic solvents and do not require multiple tube transfers for most types of samples. The extraction of DNA from semen and very small bloodstains using Chelex 100 is as efficient or more efficient than using proteinase K and phenol-chloroform extraction. DNA extracted from bloodstains seems less prone to contain PCR inhibitors when prepared by this method. The Chelex method has been used with amplification and typing at the HLA DQ alpha locus to obtain the DQ alpha genotypes of many different types of samples, including whole blood, bloodstains, seminal stains, buccal swabs, hair and post-coital samples. The results of a concordance study are presented in which the DQ alpha genotypes of 84 samples prepared using Chelex or using conventional phenol-chloroform extraction are compared. The genotypes obtained using the two different extraction methods were identical for all samples tested.

Chelating Agents↗
PubMed1991

Report of the blind trial of the Cetus Amplitype HLA DQ alpha forensic deoxyribonucleic acid (DNA) amplification and typing kit.

The AmpliType HLA DQ alpha forensic DNA amplification and typing kit is designed for the qualitative analysis of the human leukocyte antigen (HLA) DQ alpha alleles present in deoxyribonucleic acid (DNA) extracted from forensic samples. The AmpliType kit is the first forensic DNA typing product based on the GeneAmp polymerase chain reaction (PCR) process. The kit was evaluated by five forensic science laboratories (test sites) to assess their ability to perform DNA typing using PCR on sample types typically encountered by forensic laboratories. None of the DNA-containing samples was mistyped. Of the 180 DNA-containing samples analyzed, results were reported for 178 (98.9%). Of the 178 samples with results, all were correctly typed. Two sites did not report a result for one sample each. Four of the five laboratories experienced no significant levels of contamination in the DNA-containing samples. At the one site with the highest number of DNA-containing samples with contamination, the typing results were not compromised. This site was able to correct the contamination problem through simple procedural changes and stricter attention to sterile technique. Blank controls were important to monitor contamination. In conclusion, the trial demonstrated that forensic science laboratories are capable of setting up a PCR-based DNA typing laboratory and successfully using the AmpliType HLA DQ alpha forensic DNA amplification and typing kit to analyze forensic samples.

Alleles↗
PubMed1990

Identification of a mislabeled fixed specimen by DNA analysis.

Formalin-fixed, paraffin embedded surgical specimens can be identified by genotypic analysis. DNA was extracted from single sections of fixed tissue and amplified by the polymerase chain reaction for the human leukocyte antigen (HLA) DQ alpha and low density lipoprotein receptor genes. These two loci are polymorphic and fixed specimens can be distinguished based on differences between these alleles. The examination can be performed in 2 days and the HLA DQ alpha test utilized a nonisotopic color detection method. In this case report, a mislabeled specimen was identified as a section from a mastectomy because they had identical genotypes. Molecular genetic analysis of the DNA present in fixed specimens can yield information not otherwise evident from the histology.

Breast↗
PubMed1989

Avoiding false positives with PCR.

The exquisite sensitivity of the polymerase chain reaction means DNA contamination can ruin an entire experiment. Tidiness and adherence to a strict set of protocols can avoid disaster.

False Positive Reactions↗
PubMed1989

Negative ion fast atom bombardment mass spectrometry for native gangliosides using a neutral matrix.

A hexamethylphosphoric triamide proved to be a useful solvent for negative ion fast atom bombardment mass spectrometry (FABMS) of underivatized gangliosides, using a conventional glycerol matrix. Analysis of the gangliosides using a hexamethylphosphoric triamide was more convenient and more efficient not only for molecular weight determination but also for elucidating the structure of the carbohydrate sequence. We also noted the significance of the high polarity of the solvent as well as the electron pair donicity of the matrix system for negative ion FABMS of underivatized gangliosides.

Animals↗
PubMed1989

Hyperthermic effects in vitro on the peripheral lymphocytes isolated from three cases of malignant lymphoma/leukemia.

We have carried out in vitro studies of hyperthermic effects on the viability, blastogenesis, and ultrastructural changes of atypical lymphocytes isolated from the peripheral blood of three patients with malignant lymphoma/leukemia. One was lymphocytic lymphoma/leukemia (T-cell origin), and the others were adult T-cell lymphoma/leukemia (both acute and smoldering type). In the cases of adult T-cell lymphoma/leukemia, the peripheral lymphocytes were found to be more sensitive to hyperthermia than those of non-ATL patients in terms of trypan-blue exclusion ability and blastogenesis ability with concanavalin A challenge. When heated under sublethal conditions, lymphocytes of both ATL and non-ATL patients exhibited the following characteristics: production of multilayered nuclear membranes, increase in vesicles (lysosomes), swelling of mitochondria, and cytoplasmic vacuolization.

Adult↗
PubMed1988

A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions.

Specific, end-labeled DNA fragments can be simply and rapidly prepared using the polymerase chain reaction (PCR). Such fragments are suitable for use in DNase I protection footprint assays, chemical sequencing reactions, and for the production and analysis of paused RNA polymerase transcription complexes. Moreover, a general means of introducing a specific mutation at any position along the length of such PCR-generated fragments is described. These procedures, which can circumvent the need for large-scale phage or plasmid growths, preparative gel-electrophoresis and the screening of molecular clones, can facilitate the rapid study of sequence-specific interactions of proteins and DNA. A rapid means of removing excess oligonucleotide primers from completed PCRs is also described.

Base Sequence↗
PubMed1988

DNA typing from single hairs.

The characterization of genetic variation at the DNA level has generated significant advances in gene and disease mapping, and in the forensic identification of individuals. The most common method of DNA analysis, that of restriction fragment length polymorphism (RFLP), requires microgram amounts of relatively undegraded DNA for multi-locus typing, and hundreds of nanograms for single-locus comparisons. Such DNA frequently cannot be obtained from forensic samples such as single hairs and blood stains, or from anthropological, genetic or zoological samples collected in the field. To detect polymorphic DNA sequences from single human hairs, we have used the polymerase chain reaction (PCR), in which specific short regions of a gene can be greatly amplified in vitro from as little as a single molecule of DNA. We have detected genetically variable mitochondrial and nuclear DNA sequences from the root region of shed, as well as freshly-plucked, single hairs; mitochondrial DNA (mtDNA) sequences have been detected in a sample from a single hair shaft. We have used three different means of DNA typing on these samples: the determination of amplified DNA fragment length differences, hybridization with allele-specific oligonucleotide probes, and direct DNA sequencing.

Alleles↗
PubMed1988

Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.

A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.

Cloning, Molecular↗
PubMed1987

A new concept of basal cell epitheliomas based on the three-dimensional growth pattern of the superficial multicentric type.

Little is known about the growth characteristics of basal cell epitheliomas (BCEs), largely because of the difficulty in studying these tumors, both in situ and in vitro. In this study, the authors report that a scanning electron microscopic examination of 2 cases of superficial BCEs reveals that the tumor proliferates centrifugally as a monolayer beneath the epidermis as well as over the surfaces of hair follicles, while eroding and replacing the original basal architecture. Behind the leading edge of this monolayer, the cells split away from the epidermis to form a round border resembling a carpet being rolled up. This border then develops into a mass lesion, which represents the tumor cell foci seen in routine paraffin sections. These findings have led the authors to propose a fundamentally different explanation for the pathogenesis of these tumors: BCEs are conditioned neoplasms that require contact with the epidermal or hair follicular epithelium in order to grow. This hypothesis could explain the behavior of BCEs as well as the clinical and pathologic varieties observed in clinical practice.

Aged↗
PubMed1981

Human ribosomal RNA gene spacer sequences are found interspersed elsewhere in the genome.

A cloned EcoRI fragment containing human 18 S rRNA gene sequences was used to screen a gene library to obtain a set of 8 overlapping cloned DNA segments extending into the non-transcribed spacer region of the human ribosomal RNA gene cluster. 19.4 kb of the approx. 43-kb rDNA repeat was obtained in cloned form and mapped with restriction endonucleases. None of the clones obtained extended into 28 S rRNA sequences. A 7-kb region of non-transcribed spacer DNA shared in common between five independent clones was subjected to comparative restriction digests. It was estimated that sequences among the five different spacer isolated varied by not more than 1.0%, if all the observed differences are assumed due to point mutation. HaeII-restriction fragments from within this same 7-kb region contain sequences carried not only within the tandem repeats of the gene cluster but interspersed elsewhere in the genome. Some of these sequences correspond to the Alu family of highly repeated interspersed sequences.

Base Sequence↗