Human Hair Roots Research Articles

Evaluation of the Human Hair Root for DNA Typing Subsequent to Microscopic Comparison

Telogen human hairs are one of the most common useful evidence findings at crime scenes and/or on homicide victims. Occasionally, the microscopic characterization of the found telogen hair is the only physical evidence association to a victim or suspect. Recently efforts to characterize these hairs by mitochondrial DNA (mtDNA) methods have progressed. The nature of the telogen hair root morphology and ultrastructure has, however, been largely ignored. Examiners have recognized these hairs are unlikely to be typable by nuclear DNA (nuDNA) methods. Most forensic biologists have little knowledge of the complex cellular composition of anagen, catagen, and telogen hair roots or their morphogenesis. This paper reviews ex situ human hair root morphology as it relates to the likelihood of successful nuclear DNA typing. Dermatology texts of hair root morphology always demonstrate their microscopic appearance in the skin. This study investigates the use of fluorescence in situ hybridization (FISH) methods to sex type telogen head hairs, and it further investigates hair root morphology as it relates to the potential nuclear DNA content of evidence hairs. There is a need for the use of appropriate, consensus terminology for describing hair root morphology. There is also a need for standardized laboratory light microscopic methods in evaluating a hair root for DNA typing. FISH was found to be an unsuitable technique for sex determination of telogen hair clubs. It was determined that anagen/catagen hair roots without translucent sheath material are excellent candidates for nuDNA PCR-based typing and that hairs with telogen club root material only should not be submitted for nuDNA typing attempts.

Discovery of a second 15S-lipoxygenase in humans.

The lipoxygenase metabolism of arachidonic acid occurs in specific blood cell types and epithelial tissues and is activated in inflammation and tissue injury. In the course of studying lipoxygenase expression in human skin, we detected and characterized a previously unrecognized enzyme that at least partly accounts for the 15S-lipoxygenase metabolism of arachidonic acid in certain epithelial tissues. The cDNA was cloned from human hair roots, and expression of the mRNA was detected also in prostate, lung, and cornea; an additional 16 human tissues, including peripheral blood leukocytes, were negative for the mRNA. The cDNA encodes a protein of 676 amino acids with a calculated molecular mass of 76 kDa. The amino acid sequence has approximately 40% identity to the known human 5S-, 12S-, and 15S-lipoxygenases. When expressed in HEK 293 cells, the newly discovered enzyme converts arachidonic acid exclusively to 15S-hydroperoxyeicosatetraenoic acid, while linoleic acid is less well metabolized. These features contrast with the previously reported 15S-lipoxygenase, which oxygenates arachidonic acid mainly at C-15, but also partly at C-12, and for which linoleic acid is an excellent substrate. The different catalytic activities and tissue distribution suggest a distinct function for the new enzyme compared with the previously reported human 15S-lipoxygenase.

Polymorphism of a class 3 aldehyde dehydrogenase present in human saliva and in hair roots.

The types of aldehyde dehydrogenases (ALDH) present in human hair roots and in saliva were investigated. ALDH was detected by activity staining following separation of crude extracts by isoelectric focusing. Hair roots were found to express ALDH1, ALDH2, ALDH3, and ALDH4, whereas saliva expressed ALDH3. Two different patterns of ALDH3 were detected in hair roots collected from 42 donors, 40 expressed one pattern (variant I) and two another pattern (variant II) of activity staining. The variant I pattern of hair root ALDH3 changed with repetitive freezing and thawing of the sample, whereas the variant II pattern was stable. In contrast to hair root ALDH3, all patterns of ALDH3 activity in saliva were stable. The patterns of ALDH3 activity present in human hair roots that had been frozen and thawed twice matched those present in saliva collected from the same individual. Three polymorphisms of ALDH3 (variants I, II, and III) were detected in the 33 saliva samples analyzed. Variants I and II were inherited in each of three generations of a 10-member family.

Fatty acid oxygenase activity of human hair roots

The extent to which fatty acid oxygenases are activated in the normal epidermis is not known. Characterization of the regio- and stereospecificity of the monohydroxylated derivatives of arachidonic and linoleic acid produced by human hair roots is needed to define the enzymatic origin of these compounds and to define a possible role for fatty acid oxygenases in growth, differentiation, and pathology of human hair. Hair roots epilated from normal human volunteers were incubated with radiolabeled arachidonic acid or linoleic acid and the monohydroxylated derivatives produced in vitro were characterized. Incubation of hair roots with 14C]arachidonic acid resulted in the production of 15(S)-[14C]hydroxyeicosatetraenoic acid and 12(S,R)-[14C]hydroxyeicosatetraenoic acid (mean S/R ratio, 2.5). 13(S)-[14C]hydroxyoctadecadienoic acid was the principal product of incubations with [14C]linoleic acid. No radiolabeled products were derived from incubations with heat-denatured hair roots. The fatty acid oxygenase activity of anagen hair roots was inhibited by nordihydroguaiaretic acid and was greatest in the hair root bulb. The strict S-stereospecificity and the regiospecificity of the n-6 oxygenase are strong evidence for the presence of a 15-lipoxygenase in human hair roots, similar to that identified in cultured human keratinocytes. The stereospecificity of the 12-HETE produced by human hair roots is not compatible with the sole action of 12-lipoxygenase.

Effect of minoxidil-sulfate on cultured human hair papilla cells (PCs) and root sheath fibroblasts(RSFs)

Effect of minoxidil-sulfate on cultured human hair papilla cells (PCs) and root sheath fibroblasts(RSFs)

In vitro effects of testosterone, dihydrotestosterone and estradiol on cell growth of human hair bulb papilla cells and hair root sheath fibroblasts.

The influence of testosterone, dihydrotestosterone (each 10 ng/ml up to 300 ng/ml) and estradiol (0.2 ng/ml up to 10 ng/ml) on the growth behaviour (cell count, [3H]thymidine uptake, cell doubling time) of subcultured human hair bulb papilla cells and hair root sheath fibroblasts was studied. Papilla cells and root sheath fibroblasts were isolated by microdissection from the same anagen hair follicles obtained from biopsies of androgen-sensitive scalp regions in 6 healthy male subjects. Dihydrotestosterone and testosterone concentrations above 30 ng/ml significantly reduced the growth of both cell types; lower doses had no effect. Estradiol had no distinct influence on the growth curves of either cell type up to 10 ng/ml, whereas higher concentrations significantly increased the growth of both cell types as shown by [3H]thymidine uptake. Papilla cells reacted more sensitively than root sheath fibroblast to dihydrotestosterone and testosterone, as shown by the growth curves, [3H]thymidine uptake, and cell doubling time.

In vitro effects of testosterone, dihydrotestosterone and estradiol on cell growth of human hair bulb papilla cells and hair root sheath fibroblasts.

The influence of testosterone, dihydrotestosterone (each 10 ng/ml up to 300 ng/ml) and estradiol (0.2 ng/ml up to 10 ng/ml) on the growth behaviour (cell count, [3H]thymidine uptake, cell doubling time) of subcultured human hair bulb papilla cells and hair root sheath fibroblasts was studied. Papilla cells and root sheath fibroblasts were isolated by microdissection from the same anagen hair follicles obtained from biopsies of androgen-sensitive scalp regions in 6 healthy male subjects. Dihydrotestosterone and testosterone concentrations above 30 ng/ml significantly reduced the growth of both cell types; lower doses had no effect. Estradiol had no distinct influence on the growth curves of either cell type up to 10 ng/ml, whereas higher concentrations significantly increased the growth of both cell types as shown by [3H]thymidine uptake. Papilla cells reacted more sensitively than root sheath fibroblast to dihydrotestosterone and testosterone, as shown by the growth curves, [3H]thymidine uptake, and cell doubling time.

DIA3 phenotyping in human tissues, dental pulps, and hair roots by isoelectric focusing.

The polymorphism of DIA3 was investigated in tissues of various human organs, dental pulps, and hair roots by isoelectric focusing. DIA3 types were demonstrated from tissues of brain, prostate, testis, ovary, and uterus, but not from tissues of spleen, pancreas, heart, liver, muscle, lung, skin, and kidney. Determination was possible from dental pulps stored at room temperature for up to 2 weeks and from fresh hair roots. The results show that the DIA3 typing by isoelectric focusing is useful for medicolegal individualization of brain, reproductive organs, teeth, and hairs.

The Morphology and Evidential Significance of Human Hair Roots

A discussion of the morphology of human hair roots is presented. In addition to descriptions of variants of the root appearance for hairs removed from follicles in the three classical growth phases, several other commonly occurring root configurations are described and illustrated with photomicrographs. The possible evidential significance of each in certain case situations is discussed.

7-Ethoxyresorufin-O-deethylase activity in human hair roots: A potential marker for toxifying species of cytochrome P-450 isozymes

Assay systems for the evaluation of carcinogen interaction with human tissues are essential for assessing cancer risk. Human hair roots (HHR) are a readily obtainable epithelial tissue source that have been employed for investigating inherited enzyme activities. In this study HHR were found to possess cytochrome P-450-dependent 7-ethoxyresorufin-O-deethylase (ERD) activity which measures cytochrome P-450 isoenzymes that are highly specific (in the order of >95%) markers for the metabolic activation of many environmental carcinogenic substances such as the polycyclic aromatic hydrocarbons (PAHs). Topical application of PAHs (in liquor carbonis detergens) to the scalp of human volunteers was found to enhance the activity of this enzyme in freshly plucked hair roots. Oral and topical administration of ketoconazole to the same subjects resulted in an appreciable (up to 73%) inhibition of detectable enzyme activity. Our data suggest that measurement of ERD in HHR may be a useful marker for the study of toxifying species of cytochrome P-450 isozymes in human populations.

Effects of epidermal growth factor, fibroblast growth factor, minoxidil and hydrocortisone on growth kinetics in human hair bulb papilla cells and root sheath fibroblasts cultured in vitro.

Comparative studies on growth kinetics of cultivated human hair bulb papilla cells (PCs) and hair root sheath fibroblasts (RSFs) yielded evidence of some peculiarities of PCs in both proliferative behavior and morphological growth pattern. As the dermal papilla, essentially supporting the nutrition of matrix epithelium, can be considered a target tissue for agents influencing maintenance of hair growth, we studied the effects of epidermal growth factor (EGF), fibroblast growth factor (FGF), minoxidil (Mino), and hydrocortisone (HC) on the proliferation of PCs and RSFs, both gained from dissected hair follicles of scalp biopsy specimens of two male adults and separately cultured in vitro. EGF and FGF proved to increase proliferation of both PCs and RSFs most, yet at a different intensity for each cell group. HC slowed proliferation, and Mino failed to influence growth of PCs and RSFs.

α- l-fucosidase phenotyping in human tissues, dental pulps and hair roots

Phenotypes of α- l-fucosidase (Fu) were demonstrated from tissue samples of spleen, liver, lung and kidney stored for a few weeks at room temperature. Activity was fairly low in pancreas, heart, muscle, skin and brain, and typing was not reliable after 1 week storage. Fu types were detectable from dental pulp tissue of up to 1 week storage. Activity was present in hair root cells, but was extremely low. The results show the Fu typing may be applicable to individual identification of organ tissues and teeth.

Polymorphism of EsD by isoelectric focusing: phenotyping in human tissues, dental pulps, hair roots, and semen.

The polymorphism of EsD was investigated in tissues of various human organs, dental pulps, hair roots, and seminal stains by isoelectric focusing. The method yielded an excellent resolution of the isoenzyme components. The time limits of determination were: in organ tissues 3 weeks, in dental pulps 1 week, and in hair roots several days. The 7-1 type was less stable than the common types. Phenotyping was possible from fresh semen samples, but was unsuccessful from dried seminal stains after storage. The results show that the EsD typing by isoelectric focusing is of practical use for medicolegal individualization of organs, teeth, and hairs.

Comparative morphological and growth kinetics studies of human hair bulb papilla cells and root sheath fibroblasts in vitro

Dermal papillae isolated from anagen hair bulbs obtained from biopsy specimens from five subjects with normal hair pattern, and fibroblasts derived from the mesenchymal root sheaths (RSF) of the same hair follicles were separately grown in culture and the cell-cycle distribution pattern on different days was analyzed by applying DNA flow cytometry (FCM). Papilla cells (PC) exhibited distinctive morphological features by forming cell aggregates differing from RSF with respect to cell shape and growth pattern. They also proliferated remarkably more slowly than RSF. DNA-FCM analysis showed that both PC and RSF demonstrated synchronous fluctuations in the percentage of cells in G1/0, S and G2 + M phases during the period of subculture.

Simultaneous Typing of Erythrocyte Acid Phosphatase, Adenylate Kinase and Adenosine Deaminase in Human Hair Root Sheaths

Erythrocyte acid phosphatase (EAP), adenylate kinase (AK) and adenosine deaminase (ADA) were analyzed simultaneously in human hair root sheaths, collected, together with reference bloods, from sixty donors. In each case, hairs were typed first for these three genetic markers and then confirmed by typing the blood from the same donors. Hairs were checked under the stereoscope for root sheaths before subjecting them to electrophoresis. All the hairs with sheath material on them were typed accurately in EAP, AK and ADA.

Erythrocyte Acid Phosphatase in Human Hair Root Sheaths

Erythrocyte acid phosphatase (EAP) was observed in human hair root sheaths where hair sheaths and blood samples were collected from 150 donors. In each case, the hairs were typed first for EAP and then confirmed by typing the blood from the same donors. Of the 150 donors checked, EAP activity was found in 135 of the samples; the other 15 samples showed no activity because they contained no sheath cells. Of the 135 samples showing activity, 108 of those were typed accurately in EAP. The remaining 27 samples had EAP activity but with inconclusive findings. In this study, all six common phenotypes were encountered.

Two-dimensional electrophoresis of soluble and structure-bound proteins from cultured human fibroblasts and hair root cells: qualitative and quantitative variation.

Proteins from cultured human fibroblasts and native human hair root cells were investigated using the two-dimensional electrophoresis (2DE) technique. Cell material from 35 different healthy persons was examined. Proteins of different sources were separated: total proteins of fibroblasts (12 cell lines), soluble proteins of fibroblasts (12 cell lines), structure-bound proteins of fibroblasts (eight cell lines) and soluble proteins of hair root cells (12 subjects). The protein samples of different individuals were run in pairs through the electrophoresis procedure and the two patterns of each pair were compared. All changes in the electrophoretic mobility of polypeptide spots (qualitative variants) and all clearly visible differences in the staining intensity of the spots (quantitative variants) were scored. Less than 1% of the qualitative variants per pattern was found in total cell proteins and this percentage was not increased in soluble proteins. No qualitative variation was detected in structure-bound proteins. Quantitative variation occurred to a considerably higher degree in the 2DE patterns than qualitative changes. The incidence of quantitative variants was about three times higher in soluble proteins (11%) than in structure-bound proteins (3.5%); in the total cell proteins it lay in between (7%). Cultured cells (fibroblasts) and native cells (hair root cells) showed a similar degree of variation. A comparison of the data shown here with data obtained by an investigation on inbred strains of the mouse suggest that the major part of the quantitative variants observed in the 2DE patterns of proteins were genetically determined. The results presented here and the mouse data mentioned above lead us to the conclusion that the genetic variability of proteins may be characterized by quantitative changes rather than by qualitative changes, and that the genetic variability occurs to quite different degrees in different classes of proteins: structure-bound proteins less than soluble non-enzymatic proteins less than enzymes (certain groups).

Multiple Enzyme Systems Grouping of Human Hair Root Sheaths

A study of the reliability of the Esterase D (EsD), phosphoglucomutase (PGM), and Glyoxylase I (GLO I) enzyme systems in human hair root sheaths using the multiple systems method is described. Hair root sheaths from one hundred and fourteen individuals, including forty-four autopsy samples, were studied. The enzyme patterns of root sheaths were found to be as reliable as those of dried blood stains. Among three of the PGM 1 donors a slow or retarded “c” band was observed in the sheath PGM patterns. These were investigated in detail and the features which are thought to aid in the recognition of such PGM 1 patterns are summarized.

High-resolution protein mapping of human fibroblasts and hair root cells: a standardized reproducible procedure considering the effect of cell culture parameters.

The effect of different cell culture parameters on two-dimensional polypeptide maps of human fibroblasts is considered. Improved culture methods are introduced to get better resolution and reproducibility. Based on these investigations, highly standardized techniques for the protein mapping of this tissue and also of hair root cell lysates are presented. These methods seem to be quite suitable for analysis of cellular synthesized proteins and study of variability in normal subjects and in patients with genetic disorders.

Sex determination from plucked human hairs without epithelial root sheath III. Fluorescent Feulgen reaction using acriflavine

In order to determine sex from plucked human hair roots devoid of epithelial root sheath, the frequency of the X-chromatin was examined in hair cortex nuclei of plucked hairs stained by the fluorescent Feulgen reaction using acriflavine. There was a distinct difference between male and female hairs, the frequency ranging from 27% to 70% (average 45.8%) in female samples and from 0% to 8% (average 2.9%) in male samples. The difference in the frequency between male and female was detected in samples kept in a dried condition for 32 weeks.