Prevalent founder mutation c.736T>A of LIPH in autosomal recessive woolly hair of Japanese leads to variable severity of hypotrichosis in adulthood.

BACKGROUND: Mutations in LIPH are a cause of autosomal recessive woolly hair (ARWH). Homozygous c.736T>A (p.Cys246Ser), and compound heterozygous c.736T>A and c.742C>A (p.His248Asn) have been reported in 5 and 7 Japanese children with ARWH respectively. The severity of hypotrichosis is known to be able to change in the clinical course, and the mutation patterns of LIPH do not always correlate with the severity of hypotrichosis in ARWH caused by other mutation sites of LIPH. However, all 12 Japanese children previously reported to have ARWH have shown similar severity of hypotrichosis. OBJECTIVE: In this study, we investigated the clinical features and molecular basis of ARWH in patients including three adults (three adults and two children) from five non-related Japanese families. METHODS: Five families of Japanese origin that presented with woolly hair were studied. The phenotype was confirmed by clinical examination. Direct automated DNA sequencing of the LIPH gene was performed to identify the mutations in our probands. RESULTS: All patients had had woolly hair since birth. Homozygous c.736T>A mutations were found in four patients, including three adult cases, and compound heterozygous c.736T>A and c.742C>A mutations were found in one child patient. The two adults and two children had only sparse scalp hair, although one adult woman had mild hypotrichosis with long hairs. CONCLUSION: Some patients with homozygous c.736T>A can have a mild hypotrichosis phenotype with long hairs in adulthood.

Herpes zoster of the nipple: rapid DNA-based diagnosis by the loop-mediated isothermal amplification method.

A 28-year-old Japanese man presented with grouped erosions and vesicles on an erythematous base affecting the right areola and the surrounding skin. A Tzanck smear from the vesicle revealed giant cells. An initial clinical diagnosis of mammary herpes simplex was considered but to explore the differential diagnosis, viral DNA was amplified by the loop-mediated isothermal amplification (LAMP) method. DNA replication was observed only in varicella zoster virus LAMP mixture, and this confirmed a diagnosis of herpes zoster. The patient was treated with 3000 mg of daily oral valacyclovir for seven days. After antiviral treatment, the lesion had healed and the pain had resolved completely.

Population pharmacokinetics of intravenous busulfan in patients undergoing hematopoietic stem cell transplantation.

A population pharmacokinetic analysis was performed in 30 patients who received an intravenous busulfan and cyclophosphamide regimen before hematopoietic stem cell transplantation. Each patient received 0.8 mg/kg as a 2 h infusion every 6 h for 16 doses. A total of 690 concentration measurements were analyzed using the nonlinear mixed effect model (NONMEM) program. A one-compartment model with an additive error model as an intraindividual variability including an interoccasion variability (IOV) in clearance (CL) was sufficient to describe the concentration-time profile of busulfan. Actual body weight (ABW) was found to be the determinant for CL and the volume of distribution (V) according to NONMEM analysis. In this limited study, the age (range 7-53 years old; median, 30 years old) had no significant effect on busulfan pharmacokinetics. For a patient weighting 60 kg, the typical CL and V were estimated to be 8.87 l/h and 33.8 l, respectively. The interindividual variability of CL and V were 13.6 and 6.3%, respectively. The IOV (6.6%) in CL was estimated to be less than the intraindividual variability. These results indicate high interpatient and intrapatient consistency of busulfan pharmacokinetics after intravenous administration, which may eliminate the requirement for pharmacokinetic monitoring.

Photoaffinity labeling of peroxisome proliferator binding proteins in rat hepatocytes; dehydroepiandrosterone sulfate- and bezafibrate-binding proteins.

To detect the cellular sites which directly interact with peroxisome proliferators (PPs) and mediate their inducing effect on peroxisomal enzymes in rat hepatocytes, two kinds of radiolabeled ligands, AD12 (7alpha-N-(4-azido-2-hydroxy-5-iodo[125I]benzyl)-aminomethyl-5-and rostene-3beta-ol-17-one-O-3-sulfate) and BZ5 (2-[p-[2-(4'-azido-3',5'-diiodo[125I]benzamido-2'-hydroxy)ethyl]phenoxy] -2-methylpropionic acid), were developed for photoaffinity labeling. These compounds were derivatives of dehydroepiandrosterone sulfate (DHEAS) and bezafibrate, respectively, with an azido group as the photoreactive functional group. Upon UV-irradiation following incubation with rat liver cytosol and nuclei, both the ligands effectively radiolabeled several proteins analyzed by SDS-polyacrylamide gel electrophoresis/radioluminography. When [125I]AD12 was used at a concentration of 0.2 microM, two cytosolic proteins with molecular masses of 55 and 28 kDa and a nuclear protein of 40 kDa were specifically labeled, as coincubation with a 1000-fold excess of DHEAS inhibited labeling. Photoaffinity labeling of the cytosolic 28-kDa protein was also affected by Wy-14,643, but not by unsulfated dehydroepiandrosterone or androsterone sulfate, consistent with our previous findings obtained in competitive binding studies of [3H]DHEAS-binding detected in rat liver cytosol (Yamada et al. (1994) Biochim. Biophys. Acta 1224, 139-146). On the other hand, [125I]BZ5 specifically labeled a cytosolic protein of 31 kDa, which was inhibited by coincubation with bezafibrate, clofibric acid and Wy-14,643, but not with DHEAS. Thus, [125I]AD12 and [125I]BZ5 labeled several proteins which recognized DHEAS and bezafibrate, respectively, in rat liver cytosol and nuclei, providing a useful means to investigate PP-binding proteins.

Purification, molecular cloning, and genomic organization of human brain long-chain acyl-CoA hydrolase.

An acyl-CoA hydrolase, referred to as hBACH, was purified from human brain cytosol. The enzyme had a molecular mass of 100 kDa and 43-kDa subunits, and was highly active with long-chain acyl-CoAs, e.g. a maximal velocity of 295 micromol/min/mg and K(m) of 6.4 microM for palmitoyl-CoA. Acyl-CoAs with carbon chain lengths of C(8-18) were also good substrates. In human brain cytosol, 85% of palmitoyl-CoA hydrolase activity was titrated by an anti-BACH antibody, which accounted for over 75% of the enzyme activity found in the brain tissue. The cDNA isolated for hBACH, when expressed in Escherichia coli, directed the expression of palmitoyl-CoA hydrolase activity and a 44-kDa protein immunoreactive to the anti-BACH antibody, which in turn neutralized the hydrolase activity. The hBACH cDNA encoded a 338-amino acid sequence which was 95% identical to that of a rat homolog. The hBACH gene spanned about 130 kb and comprised 9 exons, and was mapped to 1p36.2 on the cytogenetic ideogram. These findings indicate that the long-chain acyl-CoA hydrolase present in the brain is well conserved between man and the rat, suggesting a conserved role for this enzyme in the mammalian brain, and enabling genetic studies on the functional analysis of acyl-CoA hydrolase.

Population pharmacokinetic modeling and model validation of a spicamycin derivative, KRN5500, in phase 1 study.

PURPOSE: KRN5500, a novel spicamycin derivative, shows the greatest activity against a human tumor xenograft model and the highest therapeutic index among spicamycin derivatives. KRN5500 is currently under clinical development in Japan and the United States. The objective of this study was to develop a population pharmacokinetic model that describes the KRN5500 plasma concentration versus time data. METHODS: Data were collected from 18 patients entered in a phase 1 study. These patients received KRN5500 3-21 mg/m2 as a 2-h infusion. A total of 219 concentration measurements were available. The data were analyzed using the nonlinear mixed effect model (NONMEM) program. In addition, the basic and final population pharmacokinetic models were evaluated using bootstrapping resampling. RESULTS: The basic model selected was a two-compartment model with a combination of additive and constant coefficient of variation error models. The basic model fitted well not only the original data, but also 100 bootstrap replicates generated from the original data set. With regard to the effect of covariates selected by generalized additive modeling analysis, gender (SEX) and performance status were found to be possible determinants of the volume of central compartment by NONMEM analysis. The final regression model for V1 was V1 = theta V1 (1--SEX x theta SEX), where V1 is the typical population value of the volume of central compartment, and SEX = 0 if the patient is male, otherwise SEX = 1. The final model was fitted to the 200 bootstrapped samples. The mean parameter estimates were within 15% of those obtained with the original data set. CONCLUSIONS: The KRN5500 plasma concentration versus time data obtained from the phase 1 study were well described by the population pharmacokinetic model. Further evaluation by bootstrapping showed that the population pharmacokinetic model was stable.

Expression of Lewis Y (Le(y)) antigen in human anagen hair follicles.

Lewis Y (Le(y)) antigen, a difucosylated tetrasaccharide found on type 2 blood group oligosaccharides of glycolipids and glycoproteins, is thought to be a phenotypic marker predictive of cell differentiation. The distribution of this antigen in human anagen hair follicles was examined by immunohistochemical staining using a monoclonal antibody (AH-6) to Le(y). In the bulbar and suprabulbar portion of anagen hair follicles, Le(y) antigen was detected in the three layers of the inner root sheath. Subsequently, the positive staining became translocated to the innermost layer of the outer root sheath in the middle part of the hair follicles. In the upper portion of the hair follicles, Le(y) antigen was found in the outer cells of the outer root sheath. These findings suggested that the expression of Le(y) antigen in the anagen hair follicles was correlated with the processes of keratinization or terminal differentiation.

Diagnostic approach to autoimmune blistering diseases using immunoblotting with chemiluminescence.

Autoantibodies in sera from patients with autoimmune blistering diseases were detected by immunoblotting with chemiluminescence. This method provided the high sensitivity in the detection of autoantibodies against human epidermal extract. It was useful for detecting low titers of autoantibodies, and identifying small amounts of autoantigens in antigen extracts.

Identification of programmed cell death in normal human skin tissues by using specific labelling of fragmented DNA.

Programmed cell death (PCD) in normal human skin tissues was studied by using in situ specific labelling of fragmented DNA. This labelling method clearly stained the nuclei of Henle's layer in the bulb of the anagen hair follicle in serial sections, and the nuclei of the inner root sheath cuticle cells and Huxley's layer cells showed positive staining in the upper part of the hair follicles. This staining pattern was consistent with the sequence of keratinization in the three layers. The nuclei of differentiated cells located at the centre of the sebaceous glands, and those of the granular keratinocyte layer, were also stained. These findings suggest that PCD might play a key role in the terminal differentiation of the epidermis and epidermal appendages.

Expression of transglutaminase I in human anagen hair follicles.

The expression of the transglutaminase I in human anagen hair follicles was studied by an immunohistochemical staining. In the bulbar and suprabulbar portions of anagen hair follicles, transglutaminase I was detected on the hair cuticle and the three layers of the inner root sheath. Subsequently, the positive staining became translocated to the inner site of the outer root sheath in the middle part of the hair follicle. In the upper portion of the hair follicle transglutaminase I was detected in the internal part of the outer root sheath and the surface epidermis. Therefore, it was suggested that the expression of transglutaminase I might be closely associated with the terminal keratinization in the anagen hair follicles.

Expression of heat shock protein 72 on peripheral blood mononuclear cells from patients with pustulosis palmaris et plantaris.

The expression of heat shock protein (HSP) 72 on peripheral blood mononuclear cells (PBMC) from patients with pustulosis palmaris et plantaris (PPP) was studied. PBMC isolated freshly from patients with PPP expressed HSP72, while those from psoriasis patients did not. PBMC from patients with PPP continued to express it in in vitro cultures at 37 degrees C. This expression was further augmented by in vitro heat stimulation at 43 degrees C. Immunofluorescence studies showed that approximately 20% of PBMC from patients with PPP were stained positively with anti-HSP72 antibody. HSP72 was expressed on both nonadherent and adherent cells of PBMC. These findings suggest that PBMC from patients with PPP may produce HSP72 spontaneously through their in vivo exposure to stressful agents.

Role of adhesion molecules in the development of pustular lesions in patients with pustulosis palmaris et plantaris.

The expression of adhesion molecules and the ligands on endothelial cells and infiltrating inflammatory cells in lesional skin specimens from patients with pustulosis palmaris et plantaris was studied. Intercellular adhesion molecule-1 and E-selectin were expressed on endothelial cells of microvessels in the papillary dermis. Intercellular adhesion molecule-1 was also expressed focally on keratinocytes in the epidermis of the lesional skin. On the other hand, lymphocyte function-associated antigen-1, Mac-1 and sialyl Lewis(x) were expressed on infiltrating inflammatory cells. Further, flow cytometric analysis demonstrated that circulating leukocytes in peripheral blood from patients with pustulosis palmaris et plantaris expressed the ligands of adhesion molecules. It is therefore suggested that the expression of adhesion molecules and the ligands on circulating leukocytes, endothelial cells, infiltrating inflammatory cells and keratinocytes might be closely related to the formation of pustular lesions in patients with pustulosis palmaris et plantaris.

Pitted keratolysis: clinical manifestations in 53 cases.

Pitted keratolysis (PK) has been reported to be more common among bare-footed people living in tropical regions. It is now known that the disease is not limited to the tropics but has a world-wide distribution. However, no study has previously been performed analysing the clinical manifestations of the disease in temperate countries. A survey of 53 patients revealed several distinctive clinical features. Hyperhidrosis is the most frequently observed symptom of this condition. Malodour and sliminess of the skin are also distinctive features, evident in 88.7% and 69.8% of the cases, respectively. The most common sites of onset of PK are the pressure-bearing areas, such as the ventral aspect of the toe, the ball of the foot and the heel. The next most common site is a friction area, the interface of the toes. Lesions are rarely seen on the non-pressure-bearing locations. Some of the primary lesions originate as a small defect along the plantar furrow, which gradually grows into the characteristics crateriform pit. Several clinical features are helpful in diagnosing PK.