Biomedical subjects
Publications and source records attributed to L D Lee.
Blood samples from approximately 200 Scottish Caucasian individuals were typed at conventional loci (PGM, Gc and EAP) and also with a four locus STR multiplex. Tests of the data are described which demonstrate that the assumptions of between locus independence are robust for use in forensic casework.
Data for four STR loci have been collected from 400 samples taken from complainers and suspects encountered in casework at the Strathclyde Police Forensic Science Laboratory (SPFSL). This paper describes statistical testing which demonstrates that its use will provide operationally robust procedures. Comparisons made with data collected from other British samples confirmed no practical differences between the different frequency distributions. This work provides further confirmation of the reliability of the so-called "product rule' in estimating the frequency of multilocus genotypes in British forensic casework.
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The Tris-urea-mercaptoethanol (pH 9) soluble fibrous proteins of hair and stratum corneum have been compared in a number of animals. The 2 tissues show different urea (pH 8.3) and SDS polyacrylamide gel electrophoretic patterns and vary in amino acid composition. Cyanogen bromide cleavage was done on human stratum corneum and hair fibrous proteins and found to give different fragments by electrophoresis. Antibodies to stratum corneum protein did not react with hair protein and vice versa. Although the 2 types of fibrous proteins have the same helical structure as shown by x-ray diffraction analysis, their differences in composition may be important for specific interactions with other macromolecules.
The alpha-keratins, the principal components of the tonafilaments, were extracted, characterized and compared in bovine hoof and snout epidermis. The alpha-fibrous proteins of these tissues are similar with respect to their molecular weights, amino acid composition and percentage of helical structure. However, distinct differences in the polypeptides comprising these proteins were observed. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these proteins consistently showed that the polypeptide chain in snout, designated as band B (mol.wt. 67,000), was completely absent from hoof preparations. This was confirmed with several alternative preparative procedures. The peptides produced by digestion of the intact keratins from hoof and snout with CNBr were distinctly different. Finally, digestion of keratins from hoof and snout with trypsin yielded products that differed in size and resistance to further digestion. Thus, in addition to the interspecies polypeptide heterogeneity documented in the literature, this report establishes the intraspecies heterogeneity of keratins and suggests that these differences are due to either the expression of different gene products or differences in post-translational modifications in these two tissues.
Thymidine phosphorylase (thymidine : orthophosphate deoxyribosyltransferase, EC 2.4.2.4) has been purified 1500-fold from extracts of human amniochorion. The purified enzyme catalyzes the phosphorolysis of deoxythymidine and to a lesser extent deoxyuridine but not deoxycytidine nor uridine. Discontinuous gel electrophoresis of the freshly purified enzyme shows a band containing 95% of the stainable protein. Gradient gel electrophoresis resolves the preparations into an active fraction with an apparent molecular weight of about 120 000 and a heavier less active or inactive fraction of about 180 000. Storage of the enzyme results in a decrease of the 120 000 dalton component, a loss in activity, and an apparent increase in the high molecular weight component. Sodium dodecyl sulfate gel electrophoresis shows only a single subunit of about 58 000 daltons which does not change on storage. These data are consistent with an active enzyme dimeric in structure which is capable of being converted to a less active form larger in molecular weight and possibly trimeric or tetrameric in structure.
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Modifications of the Laurel rocket technique for assaying antigen antibody reactions are described. These procedures allow insoluble proteins to be dissolved in a variety of denaturing solvents (e.g., SDS and urea) and subjected to electroimmunoassay without loss of sensitivity or specificity. Methods are also presented for obtaining rockets using gel slices from polyacrylamide gel electrophoresis either in the presence of urea or SDS. Results obtained using keratins, the insoluble proteins of epidermis, hair and nail are summarized.
The polyacrylamide SDS electrophoretic pattern of protein extracted from the stratum corneum obtained by scraping the surface of involved skin of patients with psoriasis was different from that of uninvolved skin and normal controls. The pattern from superficial scales was similar to that of whole stratum corneum in the case of involved psoriatic epidermis but different in uninvolved and normal epidermis. These data indicate that the changes which are observed in the structural proteins during normal keratinization are not seen in involved psoriatic epidermis. In addition, the relative proportion of keratin polypeptides was different in involved psoriatic epidermis compared to normal skin. That these changes are not specific for psoriasis was shown by finding similar electrophoretic patterns with stratum corneum proteins from patients with other keratinizing disorders.
The fibrous proteins of the malpighian layer of human epidermis (prekeratin) have been isolated with citrate buffer, pH 2.65, and shown to consist of 7 polypeptide chains varying in molecular weight from 45,000 daltons to 67,000. Some variation in the number and amount of the components was observed in prekeratin prepared from the epidermis of different individuals. The fibrous proteins of the stratum corneum were isolated with Tris buffer, pH 9.0, containing 6 M urea and 0.1 M mercapto-ethanol and were found to have a pattern similar to prekeratin but not identical to it. However, fibrous protein isolated from the superficial layers of the stratum showed a considerably different pattern indicating that there was post-translational modification of the protein in the late stages of keratinization. These data show that human keratin has the same heterogeneity which was observed previously in cow epidermis. This was further confirmed by studying the polypeptide chain content of prekeratin from a large number of lesions showing benign epidermal hyperplasia, where considerable variation in composition was observed.
Modifications of the classical technique for double diffusion in agar plates are presented. These procedures allow insoluble proteins to be dissolved in a variety of denaturing solvents and antibody production specificity to be monitored by the appearance of precipitin lines in agarose gels as in the conventional Ouchterlony method. Results obtained using the insoluble proteins of bovine epidermis are summarized.
A previously reported electrophoretic variant in hair keratin was looked for in a number of different racial groups. Only Caucasian samples showed the variant with the exception of one American black and one American Indian where Caucasian admixture is likely.
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The proteins of cow snout stratum corneum can be extracted in part with Tris buffer, pH 9.0, containing 6 M urea. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis shows that about half the extracted material migrates as prekeratin when the extract is treated with both SDS and mercaptoethanol; only trace amounts of the proteins migrating as prekeratin are seen when SDS alone is used. The urea extract gives precipitin lines to an antibody against prekeratin. After exhaustive extraction with the Tris-urea buffer an additional amount of protein is solubilized by extraction with buffer containing mercaptoethanol. This extract shows an electrophoretic pattern similar but not identical to prekeratin. These results suggest that the stratum corneum contains fibrous proteins with different degrees of cross-linking. An analogous system has also been observed in human stratum corneum.
This report describes the preparation and detection of antibodies to chemically unmodified prekeratin and stratum corneum proteins of cow and human epidermis. The antibodies are specific for epidermal fibrous proteins and do not cross-react with those of hair and nail which have the same molecular configuration but but distinctive physical and chemical properties. The antibodies did cross-react with epidermal fibrous proteins from a number of other vertebrate sources indicating an immunologic relationship among epidermal proteins whose polypeptide compositions and amino acid contents are somewhat dissimilar. Both antibodies give intense immunofluorescence localized to the malpighian layers but not to the stratum corneum. The antigenic sites in the native configuration of the stratum corneum layer may be buried, since denatured stratum corneum proteins react readily with the antibody. These antibodies have permitted the first detection of a form of keratin whose solubility properties are quite different from either prekeratin or stratum corneum proteins.
A patient with a congenital claw-like lesion of the finger was studied to determine the nature of the tissue. A variety of biochemical techniques and histological examinations indicated that the lesion was an example of rudimentary polydactyly covered with stratum corneum rather than an accessory nail.