Characterization of follicular minoxidil sulfotransferase activity in a cohort of pattern hair loss patients from the Indian Subcontinent.

Several studies have established that sulfotransferase enzyme activity in the outer root sheath of plucked hair follicles predicts response to topical minoxidil in the treatment of pattern hair loss. However, the prevalence of this enzyme activity among Indian patients has not been studied. Additionally, no reports in the literature characterize sulfotransferase activity based on sex, age, duration of hair loss, grade of hair loss, and family history. In this study we utilized a sulfotransferase activity assay first reported by Goren et al. We characterize the follicular sulfotransferase activity of 120 pattern hair loss patients visiting a dermatology outpatient clinic in India. Overall, 40.8% of patients with pattern hair loss had low levels of sulfotransferase. Surprisingly, 49.3% of men had low levels of sulfotransferase compared to 26.6% of women. No correlation was found between sulfotransferase activity and age, duration of hair loss, grade of hair loss, or family history. A sub-analysis of patient reported outcomes (PRO) validated previous findings that sulfotransferase enzyme activity is a predictive marker for minoxidil response in pattern hair loss patients.

Alcohol Biomarkers in Clinical and Forensic Contexts.

BACKGROUND: Biomarkers of alcohol consumption are important not only in forensic contexts, e.g., in child custody proceedings or as documentation of alcohol abstinence after temporary confiscation of a driver's license. They are increasingly being used in clinical medicine as well for verification of abstinence or to rule out the harmful use of alcohol. METHODS: This review is based on pertinent publications that were retrieved by a selective literature search in PubMed concerning the direct and indirect alcohol markers discussed here, as well as on the authors' experience in laboratory analysis and clinical medicine. RESULTS: Alongside the direct demonstration of ethanol, the available markers of alcohol consumption include the classic indirect markers carbohydrate-deficient transferrin (CDT), gamma-glutamyltransferase (GGT), and mean corpuscular volume (MCV) as well as direct alcohol markers such as ethyl glucuronide (EtG) and ethyl sulfate (EtS) in serum and urine and EtG and fatty acid ethyl esters (FAEE) in hair. Phosphatidylethanol (PEth) is a promising parameter that com - plements the existing spectrum of tests with high specificity (48-89%) and sensi - tivity (88-100%). In routine clinical practice, the demonstration of positive alcohol markers often leads patients to admit previously denied alcohol use. This makes it possible to motivate the patient to undergo treatment for alcoholism. CONCLUSION: The available alcohol biomarkers vary in sensitivity and specificity with respect to the time period over which they indicate alcohol use and the minimum extent of alcohol use that they can detect. The appropriate marker or combination of markers should be chosen in each case according to the particular question that is to be answered by laboratory analysis.

iNOS inhibits hair regeneration in obese diabetic (ob/ob) mice.

Previous studies have shown that androgenic alopecia is associated with metabolic syndrome and diabetes. However, the detailed mechanism whereby diabetes causes alopecia still remains unclear. We focused on the inflammatory response that is caused by diabetes or obesity, given that inflammation is a risk factor for hair loss. Inducible nitric oxide synthase (iNOS) is known to be upregulated under conditions of acute or chronic inflammation. To clarify the potential role of iNOS in diabetes-related alopecia, we generated obese diabetic iNOS-deficient (ob/ob; iNOS-KO mice). We observed that ob/ob; iNOS-KO mice were potentiated for the transition from telogen (rest phase) to anagen (growth phase) in the hair cycle compared with iNOS-proficient ob/ob mice. To determine the effect of nitric oxide (NO) on the hair cycle, we administered an iNOS inhibitor intraperitoneally (compound 1400 W, 10 mg/kg) or topically (10% aminoguanidine) in ob/ob mice. We observed that iNOS inhibitors promoted anagen transition in ob/ob mice. Next, we administered an NO donor (S-nitrosoglutathione, GSNO), to test whether NO has the telogen elongation effects. The NO donor was sufficient to induce telogen elongation in wild-type mice. Together, our data indicate that iNOS-derived NO plays a role in telogen elongation under the inflammatory conditions associated with diabetes in mice.

Self-organization process in newborn skin organoid formation inspires strategy to restore hair regeneration of adult cells.

Organoids made from dissociated progenitor cells undergo tissue-like organization. This in vitro self-organization process is not identical to embryonic organ formation, but it achieves a similar phenotype in vivo. This implies genetic codes do not specify morphology directly; instead, complex tissue architectures may be achieved through several intermediate layers of cross talk between genetic information and biophysical processes. Here we use newborn and adult skin organoids for analyses. Dissociated cells from newborn mouse skin form hair primordia-bearing organoids that grow hairs robustly in vivo after transplantation to nude mice. Detailed time-lapse imaging of 3D cultures revealed unexpected morphological transitions between six distinct phases: dissociated cells, cell aggregates, polarized cysts, cyst coalescence, planar skin, and hair-bearing skin. Transcriptome profiling reveals the sequential expression of adhesion molecules, growth factors, Wnts, and matrix metalloproteinases (MMPs). Functional perturbations at different times discern their roles in regulating the switch from one phase to another. In contrast, adult cells form small aggregates, but then development stalls in vitro. Comparative transcriptome analyses suggest suppressing epidermal differentiation in adult cells is critical. These results inspire a strategy that can restore morphological transitions and rescue the hair-forming ability of adult organoids: (i) continuous PKC inhibition and (ii) timely supply of growth factors (IGF, VEGF), Wnts, and MMPs. This comprehensive study demonstrates that alternating molecular events and physical processes are in action during organoid morphogenesis and that the self-organizing processes can be restored via environmental reprogramming. This tissue-level phase transition could drive self-organization behavior in organoid morphogenies beyond the skin.

Alternative Sampling Strategies for Cytochrome P450 Phenotyping.

Interindividual variability in the expression and function of drug metabolizing cytochrome P (CYP) 450 enzymes, determined by a combination of genetic, non-genetic and environmental parameters, is a major source of variable drug response. Phenotyping by administration of a selective enzyme substrate, followed by the determination of a specific phenotyping metric, is an appropriate approach to assess the in vivo activity of CYP450 enzymes as it takes into account all influencing factors. A phenotyping protocol should be as simple and convenient as possible. Typically, phenotyping metrics are determined in traditional matrices, such as blood, plasma or urine. Several sampling strategies have been proposed as an alternative for these traditional sampling techniques. In this review, we provide a comprehensive overview of available methods using dried blood spots (DBS), hair, oral fluid, exhaled breath and sweat for in vivo CYP450 phenotyping. We discuss the relation between phenotyping metrics measured in these samples and those in conventional matrices, along with the advantages and limitations of the alternative sampling techniques. Reliable phenotyping procedures for several clinically relevant CYP450 enzymes, including CYP1A2, CYP2C19 and CYP2D6, are currently available for oral fluid, breath or DBS, while additional studies are needed for other CYP450 isoforms, such as CYP3A4. The role of hair analysis for this purpose remains to be established. Being non- or minimally invasive, these sampling strategies provide convenient and patient-friendly alternatives for classical phenotyping procedures, which may contribute to the implementation of CYP450 phenotyping in clinical practice.

Collagenase IV plays an important role in regulating hair cycle by inducing VEGF, IGF-1, and TGF-ß expression.

BACKGROUND: It has been reported that collagenases (matrix metalloproteinase 2 [MMP-2] and matrix metalloproteinase 9 [MMP-9]) are associated with hair cycle, whereas the mechanism of the association is largely unknown. METHODS: The mice were randomly allocated into four groups: saline, and 5, 10, and 15 nM SB-3CT. Immunohistochemical analysis was employed to examine MMP-2 and MMP-9 protein. Real-time polymerase chain reaction and enzyme-linked immunosorbent assay were performed to determine mRNA and protein levels of VEGF, IGF-1, TGF-ß, and GAPDH. Growing hair follicles from anagen phase III-IV were scored based on hematoxylin and eosin staining. Hair regrowth was also evaluated. RESULTS: Results showed that mRNA expressions of enzymes changed with a peak at late anagen and a trough at telogen after depilation. Immunostaining showed that the highest expression of MMP-2 was more than that of MMP-9, and the highest expression of enzymes changed during anagen. The localizations of MMP-2 changed from dermal papilla, keratinocyte strand, out of root sheath, and basal plate at early anagen, to hair bulb, inner root sheath, and outer root sheath at late anagen. The localization of MMP-9 changed from partial keratinocyte to dermal papilla at early anagen and to outer root sheath at late anagen. VEGF, IGF-1, and TGF-ß have been shown to regulate hair growth. We found mRNA and protein expressions of VEGF and IGF-1 fluctuated with a peak at anagen and a decrease at catagen to telogen. In contrast, mRNA and protein expressions of TGF-ß changed with highest and lowest levels at anagen and telogen, respectively. With selective inhibitor of collagenase IV, SB-3CT, mice showed significant suppressed hair growth and decreased expression of VEGF, IGF-1, and TGF-ß. The MMPs agonist also significantly increased expression of VEGF, IGF-1, and TGF-ß. Meanwhile, SB-3CT treatment significantly suppressed hair growth. CONCLUSION: All these data suggest that the type IV collagenases, MMP-2 and MMP-9, play important roles in hair cycle, and this could be mediated by induced expression of VEGF, IGF-1, and TGF-ß.

Diverse selective regimes shape genetic diversity at ADAR genes and at their coding targets.

A-to-I RNA editing operated by ADAR enzymes is extremely common in mammals. Several editing events in coding regions have pivotal physiological roles and affect protein sequence (recoding events) or function. We analyzed the evolutionary history of the 3 ADAR family genes and of their coding targets. Evolutionary analysis indicated that ADAR evolved adaptively in primates, with the strongest selection in the unique N-terminal domain of the interferon-inducible isoform. Positively selected residues in the human lineage were also detected in the ADAR deaminase domain and in the RNA binding domains of ADARB1 and ADARB2. During the recent history of human populations distinct variants in the 3 genes increased in frequency as a result of local selective pressures. Most selected variants are located within regulatory regions and some are in linkage disequilibrium with eQTLs in monocytes. Finally, analysis of conservation scores of coding editing sites indicated that editing events are counter-selected within regions that are poorly tolerant to change. Nevertheless, a minority of recoding events occurs at highly conserved positions and possibly represents the functional fraction. These events are enriched in pathways related to HIV-1 infection and to epidermis/hair development. Thus, both ADAR genes and their targets evolved under variable selective regimes, including purifying and positive selection. Pressures related to immune response likely represented major drivers of evolution for ADAR genes. As for their coding targets, we suggest that most editing events are slightly deleterious, although a minority may be beneficial and contribute to antiviral response and skin homeostasis.

Prostaglandin D2 inhibits hair growth and is elevated in bald scalp of men with androgenetic alopecia.

Testosterone is necessary for the development of male pattern baldness, known as androgenetic alopecia (AGA); yet, the mechanisms for decreased hair growth in this disorder are unclear. We show that prostaglandin D(2) synthase (PTGDS) is elevated at the mRNA and protein levels in bald scalp compared to haired scalp of men with AGA. The product of PTGDS enzyme activity, prostaglandin D(2) (PGD(2)), is similarly elevated in bald scalp. During normal follicle cycling in mice, Ptgds and PGD(2) levels increase immediately preceding the regression phase, suggesting an inhibitory effect on hair growth. We show that PGD(2) inhibits hair growth in explanted human hair follicles and when applied topically to mice. Hair growth inhibition requires the PGD(2) receptor G protein (heterotrimeric guanine nucleotide)-coupled receptor 44 (GPR44), but not the PGD(2) receptor 1 (PTGDR). Furthermore, we find that a transgenic mouse, K14-Ptgs2, which targets prostaglandin-endoperoxide synthase 2 expression to the skin, demonstrates elevated levels of PGD(2) in the skin and develops alopecia, follicular miniaturization, and sebaceous gland hyperplasia, which are all hallmarks of human AGA. These results define PGD(2) as an inhibitor of hair growth in AGA and suggest the PGD(2)-GPR44 pathway as a potential target for treatment.

LIPH expression in skin and hair follicles of normal coat and Rex rabbits.

Natural mutations in the LIPH gene were shown to be responsible for hair growth defects in humans and for the rex short hair phenotype in rabbits. In this species, we identified a single nucleotide deletion in LIPH (1362delA) introducing a stop codon in the C-terminal region of the protein. We investigated the expression of LIPH between normal coat and rex rabbits during critical fetal stages of hair follicle genesis, in adults and during hair follicle cycles. Transcripts were three times less expressed in both fetal and adult stages of the rex rabbits than in normal rabbits. In addition, the hair growth cycle phases affected the regulation of the transcription level in the normal and mutant phenotypes differently. LIPH mRNA and protein levels were higher in the outer root sheath (ORS) than in the inner root sheath (IRS), with a very weak signal in the IRS of rex rabbits. In vitro transfection shows that the mutant protein has a reduced lipase activity compared to the wild type form. Our results contribute to the characterization of the LIPH mode of action and confirm the crucial role of LIPH in hair production.

Normal fur development and sebum production depends on fatty acid 2-hydroxylase expression in sebaceous glands.

2-Hydroxylated fatty acid (HFA)-containing sphingolipids are abundant in mammalian skin and are believed to play a role in the formation of the epidermal barrier. Fatty acid 2-hydroxylase (FA2H), required for the synthesis of 2-hydroxylated sphingolipids in various organs, is highly expressed in skin, and previous in vitro studies demonstrated its role in the synthesis of HFA sphingolipids in human keratinocytes. Unexpectedly, however, mice deficient in FA2H did not show significant changes in their epidermal HFA sphingolipids. Expression of FA2H in murine skin was restricted to the sebaceous glands, where it was required for synthesis of 2-hydroxylated glucosylceramide and a fraction of type II wax diesters. Absence of FA2H resulted in hyperproliferation of sebocytes and enlarged sebaceous glands during hair follicle morphogenesis and anagen (active growth phase) in adult mice. This was accompanied by a significant up-regulation of the epidermal growth factor receptor ligand epigen in sebocytes. Loss of FA2H significantly altered the composition and physicochemical properties of sebum, which often blocked the hair canal, apparently causing a delay in the hair fiber exit. Furthermore, mice lacking FA2H displayed a cycling alopecia with hair loss in telogen. These results underline the importance of the sebaceous glands and suggest a role of specific sebaceous gland or sebum lipids, synthesized by FA2H, in the hair follicle homeostasis.

A deletion in exon 9 of the LIPH gene is responsible for the rex hair coat phenotype in rabbits (Oryctolagus cuniculus).

The fur of common rabbits is constituted of 3 types of hair differing in length and diameter while that of rex animals is essentially made up of amazingly soft down-hair. Rex short hair coat phenotypes in rabbits were shown to be controlled by three distinct loci. We focused on the "r1" mutation which segregates at a simple autosomal-recessive locus in our rabbit strains. A positional candidate gene approach was used to identify the rex gene and the corresponding mutation. The gene was primo-localized within a 40 cM region on rabbit chromosome 14 by genome scanning families of 187 rabbits in an experimental mating scheme. Then, fine mapping refined the region to 0.5 cM (Z = 78) by genotyping an additional 359 offspring for 94 microsatellites present or newly generated within the first defined interval. Comparative mapping pointed out a candidate gene in this 700 kb region, namely LIPH (Lipase Member H). In humans, several mutations in this major gene cause alopecia, hair loss phenotypes. The rabbit gene structure was established and a deletion of a single nucleotide was found in LIPH exon 9 of rex rabbits (1362delA). This mutation results in a frameshift and introduces a premature stop codon potentially shortening the protein by 19 amino acids. The association between this deletion and the rex phenotype was complete, as determined by its presence in our rabbit families and among a panel of 60 rex and its absence in all 60 non-rex rabbits. This strongly suggests that this deletion, in a homozygous state, is responsible for the rex phenotype in rabbits.

An infant with cartilage-hair hypoplasia due to a novel homozygous mutation in the promoter region of the RMRP gene associated with chondrodysplasia and severe immunodeficiency.

Cartilage-hair hypoplasia (CHH) is a rare autosomal-recessive disorder characterized by short-limbed dwarfism, sparse hair, and immune deficiency. It is caused by mutations in the RMRP gene, which encodes the RNA component of the mitochondrial RNA-processing ribonuclease (RNase MRP). Several mutations have been identified in its promoter region or transcribed sequence. However, homozygous mutations in the promoter region have been only reported in a patient with primary immunodeficiency without other features of CHH. We report on a Thai girl who first presented with chronic diarrhea, recurrent pneumonia, and severe failure to thrive, without apparently disproportionate dwarfism. The diagnosis of CHH was made after the severe wasting was corrected, and disproportionate growth became noticeable. The patient had the typical features of CHH, including sparse hair and metaphyseal abnormalities. The immunologic profiles were consistent with combined immune deficiency. Mutation analysis identified a novel homozygous mutation, g.-19_-25 dupACTACTC, in the promoter region of the RMRP gene. Identification of the mutation enabled us to provide a prenatal diagnosis in the subsequent pregnancy. This patient is the first CHH case with the characteristic features due to the homozygous mutation in the promoter region of the RMRP gene. The finding of severe immunodeficiency supports that promoter mutations markedly disrupt mRNA cleavage function, which causes cell-cycle impairment.

Acidic catalase in human skin in vivo: a new marker of permanent damage.

Malignant melanoma incidence is increasing rapidly in Western countries. Its prevention requires a deep knowledge of the biological basis of the neoplasm leading to the identification of new biological risk markers. In in-vitro and ex-vivo models we demonstrated that catalase was modified not only in its activity but also in its charge properties after ultraviolet A irradiation through pheomelanin. Here we focus on the electrophoretic behaviour of catalase in the human skin in vivo, in association with cutaneous phototype. Zymographic analysis of the enzyme on skin biopsies from Caucasian population (phototype I-IV), collected from the trunk in autumn-winter, to exclude possible influences of an acute photoexposure, evidenced a protein doublet, representing the coexistence of two active isoforms of catalase with different charge properties. In the skin from low-phototype subjects, the percent contribution of the more acidic component of the doublet was prevalent, inversely correlated with total melanin concentration in hair, and associated with a high number of melanocytic nevi. In summary, this study shows for the first time the existence of an acidic catalase in association with clinically defined risk characteristics in low phototype skin in vivo, contributing to the knowledge of a new biochemical marker of cutaneous photosusceptibility.

In vitro analysis of LIPH mutations causing hypotrichosis simplex: evidence confirming the role of lipase H and lysophosphatidic acid in hair growth.

Hypotrichosis simplex (HS) is a group of isolated alopecias that can be inherited as an autosomal-dominant or an autosomal-recessive trait. Hair loss usually begins in early childhood, and is diffuse and progressive. Mutations in LIPH, which encodes lipase member H, have recently been shown to cause an autosomal-recessive form of HS. Here we describe an Austrian HS patient who was found to be carrying compound heterozygous mutations in the LIPH gene: a 7-bp frameshift duplication (c.403_409dup; p.Gln137HisfsX1) and a recently reported 30-amino acid in-frame duplication (c.280_369dup; p.Gly94_Lys123dup). To examine the impact of LIPH mutations on lipid metabolism, we established an in vitro assay to measure the action of this phospholipase in a cell-based system. Both the 7-bp duplication frameshift mutation and all known in-frame mutations were observed to reduce the in vitro activity of the lipase in response to the addition of phosphatidic acid, the substrate of lipase H. The reduced production of lysophosphatidic acid (LPA) led to a reduced response of cells expressing the human G-protein-coupled receptor p2y5 (p2y5) receptor. Our study increases the spectrum of known LIPH mutations and provides biochemical evidence for the important role of lipase H and its product LPA in human hair growth.

Transglutaminase-3 enzyme: a putative actor in human hair shaft scaffolding?

The family of transglutaminases (TGase) is known to be involved in terminal differentiation processes in the epidermis. These enzymes contribute also to the physical resistance and the preservation of the hair follicle structure. Our particular interest in hair fiber keratinization led us to focus on the TGase 3, exclusively expressed in the hair shaft. To date its function is still to be elucidated, thus we have developed a multidisciplinary approach in order to define the localization, activity, and substrates of TGase 3. The hair fiber is characterized by the expression of specific proteins essentially consisting of keratin intermediate filaments and keratin-associated proteins (KAPs), which are essential for the formation of a rigid hair shaft through their extensive disulfide cross-links. Gel electrophoresis combined with mass spectrometry experiments revealed an unexpected protein migration pattern, suggesting the existence of covalent interactions other than disulfide bonds. Western blot and amino-acid analysis revealed the presence of gamma-glutamyl-epsilon-lysine isopeptide linkages that could constitute this second covalent network. Our hypothesis is that TGase 3-driven specific isopeptide bonds between intermediate filaments and KAPs participate to the progressive scaffolding of the hair shaft.

Dissecting the non-canonical functions of telomerase.

It is now well established that the canonical function of telomerase protects the telomere repeats from erosion and the consequent induction of replicative senescence or apoptosis. In the absence of key cell cycle checkpoint proteins, the canonical function of telomerase also prevents chromosome fusions and immortalizes human cells. The canonical function of telomerase requires both the telomerase reverse transcriptase enzyme (TERT) which adds telomere (TTAGGG) repeats to the chromosome ends and the telomerase RNA component (TERC), which provides the template for TERT. However, there is growing evidence that telomerase has other (non-canonical) functions. These functions can be divided further into those that require telomerase activity but not telomere lengthening (non-canonical I or NC I) and those that require neither telomerase activity nor telomere lengthening (non-canonical II or NC II). NC I functions are associated with the induction of neoplasia in both epidermis and mammary gland, the correct response to DNA damage, and insensitivity to transforming growth factor beta. In contrast, NC II functions are not sufficient for the induction of neoplasia and are associated with the activation of the WNT and MYC signaling pathways in keratinocytes and a more general resistance to the induction of apoptosis by a variety of stimuli. The overexpression of either TERT or TERC appears to be capable of providing NC I functions but NC II functions require neither TERC nor the integrity of the TERT catalytic site. The molecular mechanisms underpinning both NC I and NC II are largely obscure but transcriptional profile changes have been reported by some groups. In this article, we will discuss the proposed mechanisms of NC I and NC II and their relevance to normal and neoplastic cell functions.

Autosomal ichthyosis with hypotrichosis syndrome displays low matriptase proteolytic activity and is phenocopied in ST14 hypomorphic mice.

Human autosomal recessive ichthyosis with hypotrichosis (ARIH) is an inherited disorder recently linked to homozygosity for a point mutation in the ST14 gene that causes a G827R mutation in the matriptase serine protease domain (G216 in chymotrypsin numbering). Here we show that human G827R matriptase has strongly reduced proteolytic activity toward small molecule substrates, as well as toward its candidate epidermal target, prostasin. To further investigate the possible contribution of low matriptase activity to ARIH, we generated an ST14 hypomorphic mouse strain that displays a 100-fold reduction in epidermal matriptase mRNA levels. Interestingly, unlike ST14 null mice, ST14 hypomorphic mice were viable and fertile but displayed a spectrum of abnormalities that strikingly resembled ARIH. Thus, ST14 hypomorphic mice developed hyperproliferative and retention ichthyosis with impaired desquamation, hypotrichosis with brittle, thin, uneven, and sparse hair, and tooth defects. Biochemical analysis of ST14 hypomorphic epidermis revealed reduced prostasin proteolytic activation and profilaggrin proteolytic processing, compatible with a primary role of matriptase in this process. This work strongly indicates that reduced activity of a matriptase-prostasin proteolytic cascade is the etiological origin of human ARIH and provides an important mouse model for the exploration of matriptase function in ARIH, as well as multiple other physiological and pathological processes.

Functional barrel cortex in 'hairless', nude mice.

Although nude mice are not truly hairless, they demonstrate abnormal hair structure and growth patterns, which are related to their genetic state. Whereas wild-type mice are born with visible vibrissae, nude mice are distinguishable at birth by the lack of visible vibrissae, which do not appear until approximately postnatal day 6. Additionally, adult nude mice have abnormal whisker cycling patterns in which structurally normal whisker follicles produce fragile whiskers which break or fallout leaving follicles whiskerless for several days before a fine replacement whisker appears and develops. The current study shows that despite these abnormal periods of whisker deprivation, the barrel cortex of nude mice develops a normal structural appearance viewed with cytochrome oxidase staining. Additionally, intrinsic optical imaging studies of barrel cortex responses to single whisker stimulation do not appear altered from normal despite periodic loss of adjacent whiskers.

Molecular beacon genotyping for globoid cell leukodystrophy from hair roots in the twitcher mouse and rhesus macaque.

Rapid and accurate genotype determination is ideal for the maintenance of breeding colonies of laboratory animal models of genetic disease. The rhesus macaque and murine (twitcher) models of globoid cell leukodystrophy have a dinucleotide deletion or single nucleotide substitution, respectively, which abolish ceramide beta-galactosidase activity and are authentic models of Krabbe disease. We report a molecular beacon PCR assay for each species which allows unambiguous determination of the genotype in under 4h. The assay works reliably with DNA extracted from hair roots using Chelex-100 in a 20 min, 100 degrees C incubation. We demonstrate that genotyping from hair roots is a preferred alternative to collecting blood or tissue for DNA extraction because it reduces animal distress, uses an inexpensive reagent, and is simpler and faster. Following amplification on a standard thermocycler with a 96-well plate format, these molecular beacon assays can be read on a standard laboratory fluorescent plate reader, eliminating the need to use a real-time thermocycler or to open the plate for subsequent restriction enzyme digestion and gel electrophoresis. The multiplexed ratio of fluorescence from wild-type- and mutant-specific beacons reporting at 560 nm and 535 nm wavelengths is distinct for each genotype.

Hair cycle-dependent changes of alkaline phosphatase activity in the mesenchyme and epithelium in mouse vibrissal follicles.

Alkaline phosphatase (ALP) activity was detected in the restricted mesenchymal and epithelial regions in mouse vibrissal follicles. Its localization and strength dramatically changed during the hair cycle. Activity in the dermal papilla (DP) was moderate in very early anagen, reached a maximal level in early anagen, decreased at the proximal region of DP after mid anagen, and was kept at a low level during catagen. The bulbar dermal sheath showed intense ALP activity only in early anagen. Although most bulbar epithelium did not show ALP activity, germinative epidermal cells that were adjacent to the ALP-negative DP cells became ALP-positive in mid anagen and rearranged in a single layer so as to encapsulate the DP in mid catagen. During catagen, the outermost layer of bulbar epithelium became ALP-positive, which could be follicular epithelial precursors migrating from the bulge. Before the initiation of hair formation, ALP activity in the bulbar epithelium rapidly decreased and that in DP increased. These dynamic changes of ALP expression might be related to DP's functions in hair induction and also to reconstruction of the bulbar structure during the hair cycle.