Hair Root Cells Research Articles

The Ultrastructure of Tissue Attached to Telogen Hair Roots

Most tissues encountered in forensic biology laboratories have been previously characterized with electron microscopy due to their medical importance. Anagen hair root cells, epithelial cells, erythrocytes, neutrophils, osteocytes, and spermatozoa have received considerable research attention at the ultrastructural level. There is no literature indicating that cells attached to removed telogen hair roots have been characterized with transmission electron microscopy. Nonetheless, telogen hairs are a frequent submission item to forensic laboratories for DNA typing. The amount of tissue attached to a telogen hair root usually determines whether that hair is suitable for nuclear DNA typing methods or mitochondrial DNA typing methods. This study used transmission and scanning electron microscopy to characterize the tissues found in three commonly occurring telogen hair root forms. The tissues were found to consist of keratinized remnant follicle, nonnucleated epithelial cells, nucleated epithelial cells, and trichilemmal keratin. These findings were consistent with the known principles of hair follicle regression. The recognition of the root structures that contain these specific tissue types may assist in the DNA typing of telogen hairs inasmuch as the quality of tissue present may be more important than the amounts of tissue present.

Endothelial progenitor cells display clonal restriction in multiple myeloma

BackgroundIn multiple myeloma (MM), increased neoangiogenesis contributes to tumor growth and disease progression. Increased levels of endothelial progenitor cells (EPCs) contribute to neoangiogenesis in MM, and, importantly, covary with disease activity and response to treatment. In order to understand the mechanisms responsible for increased EPC levels and neoangiogenic function in MM, we investigated whether these cells were clonal by determining X-chromosome inactivation (XCI) patterns in female patients by a human androgen receptor assay (HUMARA). In addition, EPCs and bone marrow cells were studied for the presence of clonotypic immunoglobulin heavy-chain (IGH) gene rearrangement, which indicates clonality in B cells; thus, its presence in EPCs would indicate a close genetic link between tumor cells in MM and endothelial cells that provide tumor neovascularization.MethodsA total of twenty-three consecutive patients who had not received chemotherapy were studied. Screening in 18 patients found that 11 displayed allelic AR in peripheral blood mononuclear cells, and these patients were further studied for XCI patterns in EPCs and hair root cells by HUMARA. In 2 patients whose EPCs were clonal by HUMARA, and in an additional 5 new patients, EPCs were studied for IGH gene rearrangement using PCR with family-specific primers for IGH variable genes (VH).ResultsIn 11 patients, analysis of EPCs by HUMARA revealed significant skewing (≥ 77% expression of a single allele) in 64% (n = 7). In 4 of these patients, XCI skewing was extreme (≥ 90% expression of a single allele). In contrast, XCI in hair root cells was random. Furthermore, PCR amplification with VH primers resulted in amplification of the same product in EPCs and bone marrow cells in 71% (n = 5) of 7 patients, while no IGH rearrangement was found in EPCs from healthy controls. In addition, in patients with XCI skewing in EPCs, advanced age was associated with poorer clinical status, unlike patients whose EPCs had random XCI.ConclusionOur results suggest that EPCs in at least a substantial subpopulation of MM patients are related to the neoplastic clone and that this is an important mechanism for upregulation of tumor neovascularization in MM.

Endothelial progenitor cell clonality and response to treatment in multiple myeloma

17546 Background: In multiple myeloma (MM), increased levels of endothelial progenitor cells (EPCs) contribute to neoangiogenesis, covary with disease activity and response to treatment. To understand the mechanisms of enhanced neoangiogenic function by EPCs in MM, we investigated whether EPCs and tumor cells share clonal identity, and related this finding to clinical progression. EPC clonality was analyzed by X-chromosome inactivation (XCI) and the presence of clonotypic immunoglobulin heavy-chain (IGH) gene rearrangement. Methods: Hair root cells and confluent EPCs from 18 female patients were studied for XCI patterns by human androgen receptor assay (HUMARA). In 6 patients, EPCs were studied for the presence of IGH gene rearrangement using family-specific PCR primers for IGH variable genes (VH). Nine patients were treated with TDZ: thalidomide (100 mg/day), dexamethasone (10–40 mg for 4 days/3 weeks for 6 months, then reduced to 4 days/month), and zoledronate (4 mg/4 weeks). Results: Eleven of the 18 female patients displayed allelic XCI. Analysis of their EPCs for evidence of clonality revealed significant skewing (≥ 77% expression of a single allele) in 64% (n = 7). In 4 of these 7 patients, EPC XCI skewing was extreme (≥ 90% expression of a single allele) while in hair root cells XCI was random. Furthermore, sequencing of PCR products obtained with VH primers resulted in identification of the same product in EPCs and bone marrow cells in 67% (n = 4) of another 6 patients, while no IGH rearrangement was found in EPCs from healthy controls. 9 patients were subsequently treated with TDZ, 4 patients had clonal EPCs and Stage 2 MM, and 5 patients had non-clonal EPCs and Stage 3 MM. Despite having less advanced disease, patients whose EPCs were clonal had a significantly attenuated response to treatment with TDZ compared to patients whose EPCs were non-clonal (P = .05). Conclusions: Clonal identity between EPCs and neoplastic cells highlight the necessity to explore the thesis that MM cells and EPCs are derived from a progenitor cell capable of self-renewal and differentiation. The relationship between EPC clonality and impaired response to treatment underscores the pathogenic significance of EPCs in MM. No significant financial relationships to disclose.

Clonal Restriction in Endothelial Progenitor Cells in Patients with Multiple Myeloma.

In multiple myeloma (MM), neoplastic plasma cells and bone marrow microvascular cells have paracrine cross-regulatory interactions, and display abnormal features suggesting that these neoangiogenic cells are myeloma-related. Tumor neovascularization is closely linked to neoplastic growth. We have recently shown that circulating endothelial progenitor cells (EPCs) reflect disease severity as assessed by M protein and serum β2-microglobulin levels in MM. These observations linking circulating EPCs to disease activity in MM prompted us to study the genetic characteristics of EPCs using X-chromosome inactivation (XCI) analysis to determine their clonal restriction profile. In addition, we examined EPCs for the presence of genetic markers common to neoplastic MM cells by examining immunoglobulin heavy chain (IgH) rearrangement of germline DNA. Isolation and outgrowth of EPCs was done as previously described. Clonality within EPCs was evaluated by quantitation of XCI patterns in female patients by a human androgen receptor gene (AR) assay (HUMARA). DNA was prepared from EPCs, hair root, or PBMCs and digested by a methylase-sensitive restriction enzyme. The highly polymorphic X-linked human androgen receptor gene was amplified by PCR. In polyclonal populations, both alleles are seen as two distinct bands, while clonal populations yield a single band. Amplification of genomic DNA extracted from PBMCs showed that XCI status was informative in 7 of the 16 MM patients and in 7 of the 15 healthy controls. Next, EPCs and hair root cells from informative patients were studied for XCI patterns to determine clonality. DNA obtained from confluent EPCs outgrown from 7 of the 10 informative patients displayed extremely skewed XCI patterns (90% inactivation of one allele), while hair follicle cells in the same patients displayed a random pattern. These results suggest that EPCs share characteristics of clonal plasma cells in MM, and display clonal restriction that is lineage-specific. To further explore the genetic similarity between EPCs and MM cells, EPCs were assessed for clonotypic immunoglobulin heavy chain (IgH) VDJ sequences by PCR amplification of genomic DNA. In these experiments, DNA was extracted from bone marrow EPCs and used in a series of 7 PCR amplifications containing a specific VH forward primer and a consensus JH reverse primer. In EPCs from 2 patients, including one with skewed XCI, we found a single PCR product identical to the IgH VDJ rearrangement found in bone marrow containing 60% and 65 % plasma cells, respectively, suggesting that an identical IgH VDJ rearrangement was present in EPCs and plasma cells in the same patient. Controls for this experiment included HUVEC genomic DNA, which did not yield PCR products even when spiked with a positive control DNA (up to 50 %) extracted from the MM cell line U266, and PBMC genomic DNA from controls which yielded multiple bands produced from several of the VH primers. Our results show that EPCs in a population of MM patients display clonal restriction and genetic similarity to plasma cells. Thus, it is possible that neoplastic MM cells and EPCs are linked not only within a functional context by their paracrine cross-regulatory effects within bone marrow microenvironment, but also by their genetic ancestory. These conclusions await confirmation from ongoing molecular genetic comparisons of the two cell types.

Blood chimerism in monochorionic twins conceived by induced ovulation: Case report

A case of monochorionic twin boys delivered at 34 weeks of gestation following induced ovulation with clomiphene is described. One twin was typed as blood group AB and the other as B. Flow cytometry showed blood group chimerism. DNA polymorphism analysis of peripheral lymphocytes and hair root cells showed that the chimerism was confined to the blood cells and they were dizygotic.

A cluster of 21 keratin-associated protein genes within introns of another gene on human chromosome 21q22.3

Recently, we identified multiple unique sequences in the 21q22.3 region and predicted them to be a cluster of genes encoding hair-specific keratin-associated proteins (KAPs). Detailed computer-aided analysis of these clustered genes revealed that the cluster spans over 165 kb and consists of 21 KAP-related sequences including 16 putative genes and 5 pseudogenes. These were further divided into two subfamilies, KRTAP12 ( KRTAP12.1– 12.4 and KRTAP12.5P) and KRTAP18 ( KRTAP18.1– 18.12 and KRTAP18.13P– 18.16P). All 16 putative genes possess several intragenic repeat sequences and apparently belong to the high-sulfur KAP gene family (16–30% cysteine content) known for nonhuman mammalian species. Transcripts were detected by RT-PCR analysis for all 16 putative KAP genes and their expression was restricted to hair root cells (radix pili cells) and not found in 28 other tissues, including skin. All 16 KAP genes produced unspliced transcripts, indicating their nature to be that of active intronless genes. Interestingly, all these KAP-related genes are located within introns of the recently identified gene TSPEAR (approved gene symbol C21orf29), 214 kb in size. Surprisingly, the transcriptional direction of 8 of the 16 active genes is the same as that of C21orf29/TSPEAR. This finding suggests a novel transcription mechanism in which C21orf29/TSPEAR gene transcription passes over the multiple transcriptional termination sites of the KAP genes.

The Crystal Structure of Metal-free Human EF-hand Protein S100A3 at 1.7-Å Resolution

S100A3 is a unique member of the EF-hand superfamily of Ca(2+)-binding proteins. It binds Ca(2+) with poor affinity (K(d) = 4-35 mm) but Zn(2+) with exceptionally high affinity (K(d) = 4 nm). This high affinity for Zn(2+) is attributed to the unusual high Cys content of S100A3. The protein is highly expressed in fast proliferating hair root cells and astrocytoma pointing toward a function in cell cycle control. We determined the crystal structure of the protein at 1.7 A. The high resolution structure revealed a large distortion of the C-terminal canonical EF-hand, which most likely abolishes Ca(2+) binding. The crystal structure of S100A3 allows the prediction of one putative Zn(2+) binding site in the C terminus of each subunit of S100A3 involving Cys and His residues in the coordination of the metal ion. Zn(2+) binding induces a large conformational change in S100A3 perturbing the hydrophobic interface between two S100A3 subunits, as shown by size exclusion chromatography and CD spectroscopy.

Molecular sex diagnosis in castoridae

AbstractBeavers do not exhibit any external sexual trait. Therefore, a simple method was established to differentiate between males and females by verifying the male‐specific SRY gene. Genomic DNA was prepared from hair root cells. Male samples displayed an electrophoretic 157‐bp band derived from the SRY gene after polymerase chain reaction (PCR) amplification. This band is missing in female samples. A 375‐bp fragment of the mitochondrial cytochrome b gene served as internal control and was visible in all samples. The technique can easily be used as a standard monitoring method, and is noninvasive, quick, and reliable. Zoo Biol 21:305–308, 2002. © 2002 Wiley‐Liss, Inc.

Low N‐acetylating capacity in patients with Stevens‐Johnson syndrome and toxic epidermal necrolysis*

Low constitutive N-acetylating capacity has been implicated as a predisposing factor for the development of adverse reactions to certain drugs. This prompted us to investigate whether the N-acetylating capacity of patients with serious cutaneous adverse reactions, i.e., Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) differed from that of healthy control subjects. N-acetylating activity was measured in hair root cells by preparing a homogenate from freshly extracted hair roots and assessing acetyl-CoA-dependent N-acetylation by RP-HPLC using 2-aminofluorene as a substrate. Samples were obtained from hospitalized patients suffering from acute SJS and TEN or from healthy controls. All patients with SJS and TEN were found to have a low N-acetylating capacity (0.85 nmol/mg/min compared to 2.21 nmol/mg/min in controls, p < 0.05). Based on these findings, a low constitutive N-acetylating capacity may be one of the predisposing factors for the development of serious cutaneous adverse reactions to drugs that require N-acetylation in these patients.

Conidiogenesis in Oidiodendron periconioides and ultrastructure of ericoid mycorrhizas formed with Rhododendron brachycarpum

Oidiodendron periconioides was isolated from hair roots of seedlings of Rhododendron brachycarpum growing in pots containing a commercial peat moss in Japan. The unusual process of conidiogenesis in O. periconioides involved the formation of conidia from vesicle-like swellings that developed from vegetative hyphae or from the apex of a mononematous conidiophore. Scanning electron microscope examinations of mature conidia revealed that they were regularly dimpled, giving them the characteristic and previously reported radiate pattern of sculpturing visible with the light microscope. Oidiodendron periconioides formed ericoid mycorrhizas that were morphologically and ultrastructurally similar to previously studied ericoid systems involving Oidiodendron maius and Hymenoscyphus ericae, and a few other species of Rhododendron. Rough-walled hyphae grew appressed to the epidermal cells of young hair roots, penetrated the external walls via narrow penetration tubes, and invaginated the plasma membrane of the host cell during the process of forming a complex system of hyphal loops. Infection was restricted to the zone proximal to the active region of the meristem. Hyphal complexes were short-lived and degenerated as the epidermal cells matured and died. The physiological role of O. periconioides as a mycorrhizal endophyte is unknown. Key words: ericoid mycorrhiza, Oidiodendron periconioides, Rhododendron brachycarpum, conidiogenesis.

Sex chromatin in hair roots — 25 years later: fluorescence in situ hybridization of hair root cells for detection of numerical chromosome aberrations

The applicability of fluorescence in situ DNA hybridization to hair root preparations for the detection of numerical chromosome anomalies is demonstrated. This simple method yields a high number of scorable nuclei, allowing rapid identification of epithelial cell chromosome composition. In clinical genetics, this technique may become useful in cases where other types of cells are difficult to obtain.

Nuclear morphology and ploidy level in infected and uninfected hair root cells ofCalluna vulgaris andVaccinium myrtillus mycorrhizae

Morphometry, microfluorimetry and electron microscopy were used to investigate the size, ploidy level, and morphology of host nuclei in the ericoid mycorrhizal systemsCalluna vulgaris+Hymenoscyphus ericae andVaccinium myrtillus+H. ericae. Hypertrophy was observed in nuclei of mycorrhizal hair roots of both plants. This could not be explained by polyploidization but rather by a lower degree of chromatin condensation.

Louis-Bar syndrome: spontaneous and induced chromosomal aberrations in lymphocytes and micronuclei in lymphocytes, oral mucosa and hair root cells

Louis-Bar (L-B) syndrome, also called ataxia-telangiectasia, is cytogenetically characterized by an increased frequency of spontaneous and induced chromosomal aberrations (CA) in cultured lymphocytes and skin fibroblasts. However, it is not yet clear whether the chromosomal instability is also present in uncultured cells. The spontaneous and bleomycin-induced CA in peripheral lymphocytes of 8 L-B patients were evaluated. The micronucleus test was also performed, for the first time in lymphocytes by the cytokinesis-block method, and in uncultured cells of the oral cavity and hair root. The spontaneous frequency of CA and micronuclei in lymphocytes was about 3 times higher in L-B patients than in controls, these two cytogenetic parameters being highly correlated. Moreover, the induction by bleomycin of CA was higher in patients than in controls. The micronuclei in buccal and hair root cells of patients were normal. It remains to be determined whether the different responses obtained with cultured and uncultured cells are the result of the different L-B gene expression of chromosomal instability or whether they arise because of a particular cell sensitivity to culture conditions. The spontaneous and induced CA in lymphocytes of heterozygotes cultured in the presence of L-B serum were studied to evaluate a possible increased sensitivity of heterozygotes to a possible diffusible clastogenic factor present in the plasma of L-B patients. We could not demonstrate the presence of any factor that enhances CA in normal subjects or in heterozygote carriers.

Evaluation of chromosomal aberrations in lymphocytes and micronuclei in lymphocytes, oral mucosa and hair root cells of patients under antiblastic therapy

In 7 patients undergoing antiblastic chemotheraphy for the first time, the structural chromosomal aberration (CA) test in peripheral lymphocytes was compared with the micronucleus (Mn) test in lymphocytes, in oral cavity cells and in hair root cells of the scalp. The last test is being proposed for the first time. The CA and Mn frequencies induced by chemotherapy were compared with the baseline (pretreatment) frequenies of the patients and with confidence limits calculated in 4 control groups studies for CA, Mn in lymphocytes, Mn in oral cavity cells and Mn in hair root cells, respectively. The studied chemotherapies induced a clear cytogentic effect in at least 2 of the tests studied with the exception of interferon-α 2b (patient 6) and interferon + low doses of cis-platinum (patient 2) which did not appear to cause evident chromosomal damage. The response to chemotheraphy is generaly characterized by an increase in Ca and Mn, reaching a peak value and then decreasing in the following weeks. The CA test proves to be the most sensitive despite the fact that CA were analyzed in an average of 100 cells per sample against the 500–3000 cells analyzed for Mn. The efficiency of Mn to detect CA in in the following order: Mn in lymphocytes > Mn in buccal cells > Mn in hair root cells. The last test appears to be very promising but, used following the current method, does not appear suitable to monitor acute exposure.

Ultrastructure of Mycorrhizas of Dracophyllum secundum R. Br. (Ericales:Epacridaceae)

Dracophyllum secundum R. Br. (Epacridaceae) often possessed ericoid mycorrhizas; fungal endophytes formed coils within cells of the epidermis of hair-roots. The plant plasma membrane extended around the hyphae. In some epidermal cells of hair-roots, both plant and fungal cells retained their structural integrity, both partners showing mitochondrial, vacuolar and lipid droplet profiles, and with much of the plant cytoplasm associated with the hyphal coils. In other epidermal cells of hair-roots, fungal coils were present but cytoplasmic features of both symbionts appeared to have broken down. Some epidermal cells showed no evidence of fungal infection. These three arrangements could occur in root-cells of the same age, and are interpreted as resulting from different stages in the development and degeneration of the infection by the mycorrhizal fungus. Two structural types of fungal endophyte here found in ericoid mycorrhizas in D. secundum: one with simple septa, Woronin bodies and two-layered walls (presumed to be an Ascomycete), and another with dolipore septa with imperforate parenthesomes (presumed to be a Basidiomycete). The possibilities that the mycorrhizas may be seasonal, and that mycorrhizal status varies from place to place, are discussed.

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.

Origin of human triosephosphate isomerase isozymes: further evidence for the single structural locus hypothesis with Japanese variants.

Four electrophoretic variants of human erythrocyte triosephosphate isomerase (TPI) have been studied to investigate the origin of the multiple forms of human TPI, in particular the constitutive TPI-B isozyme and the cell division-associated TPI-A isozyme. The variant phenotype expressed by the constitutive TPI-B isozyme in both erythrocytes and peripheral lymphocytes was also expressed by the cell division-associated isozymes in mitogen-stimulated lymphocytes and hair root cells. These results strongly support the hypothesis of Decker and Mohrenweiser (1981) that TPI-B and TPI-A originated from the same structural gene. We also found that the isozyme e is different from TPI-A with respect to both its electrophoretic mobility and heat stability. This finding is in contrast to the recent conclusion of Yuan et al. (1981) that both the isozyme e and TPI-A are deamidation products of TPI-B.

Early onset of menstruation in a 45,X girl

A girl with short stature developed breasts at age 10 and began menarche at 10.3 years. When examined by us at age 15 years, she had had regular menstrual cycles. Chromosome analyses using PHA- and EB virus-stimulated lymphocytes and cultured skin fibroblasts from both elbows all revealed an exclusively 45,X karyotype. Both buccal smears and hair root cells were negative for the X-chromatin.

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).

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.