A Homozygous Frameshift Mutation in the HOXC13 Gene Underlies Pure Hair and Nail Ectodermal Dysplasia in a Syrian Family

Pure hair and nail ectodermal dysplasia (PHNED) is a rare genetic disorder characterized by hypotrichosis or complete alopecia, as well as nail dystrophy. Mutations in the type II hair keratin gene KRT85 and the HOXC13 gene on chromosome 12q have recently been identified in families with autosomal‐recessive PHNED. In the present study, we have analyzed a consanguineous Syrian family with an affected girl having complete alopecia and nail dystrophy since birth. The family clearly showed linkage to chromosome 12q13.13–12q14.3, which excluded the KRT85 gene. Sequencing of another candidate gene HOXC13 within the linkage interval identified a homozygous frameshift mutation (c.355delC; p.Leu119Trpfs*20). Expression studies in cultured cells revealed that the mutant HOXC13 protein mislocalized within the cytoplasm, and failed to upregulate the promoter activities of its target genes. Our results strongly suggest crucial roles of the HOXC13 gene in the development of hair and nails in humans.     

Antigenic variation of the Epstein–Barr virus nuclear antigen EBNA1 as revealed by monoclonal antibodies

The Epstein–Barr virus (EBV) nuclear antigen EBNA1 plays an essential role in the replication of EBV episomes in latently infected cells and is the only viral protein that is consistently expressed in all programs of latent EBV gene expression. In this study, four monoclonal antibodies (MoAbs) directed to a region (amino acid residues 442–530) of EBNA1 were generated. Competitive enzyme-linked immunosorbent assay (ELISA) experiments using biotinylated MoAbs showed that they recognized distinct epitopes. Reactivity of these MoAbs with various laboratory EBV strains and field EBV isolates was shown to be heterogeneous in that EBNA1 from certain strains (isolates) was recognized and that from others was not. All four MoAbs showed such heterogeneous reactivity, and moreover, each MoAb showed a distinct spectrum of reactivity with these EBV strains (isolates). These results demonstrate an extensive structural variation in this region of EBNA1 as predicted by previous sequencing studies. These MoAbs will be useful as probes to dissect this structural heterogeneity of EBNA1.     

Association of single nucleotide polymorphisms of the interleukin-10 promoter gene and susceptibility to primary biliary cirrhosis: immunogenetic differences in Italian and Japanese patients

Several lines of data suggest that genetic factors play an important role in the onset and/or progression of primary biliary cirrhosis (PBC). Since PBC is an autoimmune disease, it is reasoned to assume that genes encoding cytokines may confer susceptibility to disease. Amongst these factors, interleukin-10 (IL-10) has received significant attention. The promoter region of IL-10 gene has three single nucleotide polymorphisms (SNPs) at positions -1082, -819 and -592. To elucidate the association of the three SNPs of IL-10 promoter region with susceptibility of PBC in two different genetic populations, 159 unrelated patients with PBC (94 Italian and 65 Japanese) and 143 local controls (72 Italian and 71 Japanese) were enrolled. SNPs were determined using allele-specific PCR/RFLP. In Italian PBC patients, the frequency of homozygosity for G/G at position -1082 was significantly higher than that of local controls (p < 0.041, OR = 2.44, 95% C.I.; 1.02-5.86). The frequencies of haplotype GCC in PBC patients, possibly linked to higher IL-10 production, were also significant higher than local controls (p < 0.033). However, in Japanese population, there were no significant differences in the three SNPs and haplotypes between PBC patients and controls. Excessive production of IL-10 may play an important role in some populations in modulating the onset of PBC. Further, immunogenetic studies of PBC should take into account ethnic and geographic variations; this makes such studies in heterogeneous population, like the USA, more difficult.     

Histopathologic reaction of a calcium phosphate cement for alveolar ridge augmentation

The objective of the present study was to evaluate the feasibility of using a calcium phosphate cement (CPC) in the reconstruction of a defective alveolar ridge in conjunction with implant placement. The CPC consisted of an equimolar amount of tetracalcium phosphate and dicalcium phosphate anhydrous. At the beginning of the experiment, all mandibular premolar teeth of mature beagle dogs were extracted. After 1 month of healing, alveolar bone was reduced to make a space for a CPC block that was prefabricated from a CPC mixed with water at a powder/liquid ratio of 5 g/mL. After an additional month, 8-mm long hydroxyapatite-coated titanium implants were placed in such a way that the apical half was embedded into alveolar bone and the coronal half in the preformed CPC block. The dogs were sacrificed and biopsies were obtained at 1, 3, and 6 months after surgery. Sections that included implants were evaluated for integration of the CPC block to the alveolar bone and of the implant to the alveolar bone. Additional sections without the implants served as controls. The results obtained from this study show that the CPC ridge augmentation gradually is replaced by natural bone. Six months after surgery, histopathologic features of the augmentation area were quite similar to those of natural alveolar bone. The coronal half of the implants, previously surrounded by the CPC block, was firmly fixed by natural bone. Therefore, this method may be useful for increasing the height of the alveolar ridge.     

Cutaneous lesions in broiler chickens spontaneously affected with Marek's disease

A total of 91 8- to 9-week-old broiler chickens with Marek's disease (MD) skin tumours ("skin leukosis"), collected from 15 farms at the processing plants, were examined pathologically. Grossly, the skin lesions comprised various sizes and numbers of feather follicular nodules, which tended to fuse with each other as their size increased. Histologically, the lesions were classified into five types: type A was small lymphoid cell aggregates (LCA) consisting mostly of small lymphocytes with a few lymphoblasts and very rare mitotic figures; type B had large LCA consisting mainly of small lymphocytes with considerable numbers of lymphoblasts and very rare mitotic figures; type C was characterized by large coalesced LCA consisting almost equally of small lymphocytes and lymphoblasts with infrequent mitotic figures; type D exhibited very large coalesced LCA consisting mainly of lymphoblasts with some small lymphocytes and occasional mitotic figures; type E had very large coalesced LCA consisting almost completely of lymphoblasts with frequent mitotic figures. The histological MD cutaneous lesions were related to the size of gross skin nodules, the small feather follicular nodules consisted mainly of types B and C with type A, whereas large fused feather follicular nodules were composed mainly of types D and E. Nuclear inclusions were frequently found in the feather follicular epithelium in all skin nodules except for the largest fused ones. MD visceral lesions were more pronounced in birds having marked skin leukotic lesions. Feather pulp lesions (FPL) were more related to the visceral than the skin lesions; the constituent cells of FPL were compatible with those of the former lesions.     

Dual-probe assay for rapid detection of drug-resistant Mycobacterium tuberculosis by real-time PCR

Mutations in particular nucleotides of genes coding for drug targets or drug-converting enzymes lead to drug resistance in Mycobacterium tuberculosis. For rapid detection of drug-resistant M. tuberculosis in clinical specimens, a simple and applicable method is needed. Eight TaqMan minor groove binder (MGB) probes, which discriminate one-base mismatches, were designed (dual-probe assay with four reaction tubes). The target of six MGB probes was the rpoB gene, which is involved in rifampin resistance; five probes were designed to detect for mutation sites within an 81-bp hot spot of the rpoB gene, and one probe was designed as a tuberculosis (TB) control outside the rpoB gene hot-spot. We also designed probes to examine codon 315 of katG and codon 306 of embB for mutations associated with resistance to isoniazid and ethambutol, respectively. Our system was M. tuberculosis complex specific, because neither nontuberculous mycobacteria nor bacteria other than mycobacteria reacted with the system. Detection limits in direct and preamplified analyses were 250 and 10 fg of genomic DNA, respectively. The system could detect mutations of the rpoB, katG, and embB genes in DNAs extracted from 45 laboratory strains and from sputum samples of 27 patients with pulmonary TB. This system was much faster (3 h from DNA preparation) than conventional drug susceptibility testing (3 weeks). Results from the dual-MGB-probe assay were consistent with DNA sequencing. Because the dual-probe assay system is simple, rapid, and accurate, it can be applied to detect drug-resistant M. tuberculosis in clinical laboratories.     

Osteogenic potential of human adipose tissue-derived stromal cells as an alternative stem cell source

Adult bone marrow contains mesenchymal stem cells (bone marrow-derived mesenchymal stem cells; BMSCs) which contribute to the generation of mesenchymal tissue such as bone, cartilage, muscle and adipose. However, using bone marrow as a source of stem cells has the limitation of a low cell number. An alternate source of adult stem cells that could be obtained in large quantities, under local anesthesia, with minimal discomfort would be advantageous. Human adipose tissue obtained by liposuction was processed to obtain a fibroblast-like population of cells or adipose tissue-derived stromal cells (ATSCs). In this study, we compared the osteogenic differentiation of ATSCs with that of BMSCs. Both cell types were cultured in atelocollagen honeycomb-shaped scaffolds with a membrane seal (ACHMS scaffold) for three-dimensional culturing in a specific osteogenic induction medium. Optimal osteogenic differentiation in both cell types, as determined by alkaline phosphatase cytochemistry, secretion of osteocalcin, mineral (calcium phosphate) deposition and scanning electron microscopy, was obtained with the same three-dimensional culture. Furthermore, osteoblastic lining in vivo was examined using ATSC-seeded or BMSC-seeded scaffolds in nude mice. The present results show that ATSCs have a similar ability to differentiate into osteoblasts to that of BMSCs.     

Post-weaning mice fed exclusively milk have deficits in induction of long-term depression in the CA1 hippocampal region and spatial learning and memory

Previously, we have found that post-weaning mice fed exclusively milk display low-frequency exploratory behavior compared to mice fed a food pellet diet (Ishii et al., 2005a). Because cognitive functions play a key role in animal exploration, in the present study we examined the effect of an exclusively milk formula diet on spatial learning and memory in a water maze and also on induction of long-term potentiation (LTP) and long-term depression (LTD) at the Schaffer collateral-CA1 synapse in the hippocampus. Exclusively milk-fed mice exhibited slower learning and memory deficits in hidden water maze tests as compared with pellet-fed mice. Moreover, milk-fed mice showed a significant inhibition of LTD but a normal induction of LTP. Despite these functional deficits, adult neurogenesis in the dentate gyrus of the hippocampus, which has been proposed to have a causal relationship to spatial memory, was stimulated in milk-fed mice. These result suggest that an exclusively milk formula diet after weaning leads to a stimulation of hippocampal neurogenesis but causes deficits in the induction of LTD in the CA1 hippocampal region and impairment of spatial learning and memory.     

Quantitative assessment of central nervous system disorder induced by prenatal X-ray exposure using diffusion and manganese-enhanced MRI

Prenatal radiation exposure induces various central nervous system (CNS) disorders depending on the dose, affected region and gestation period. The goal of this study was to assess noninvasively a CNS development disorder induced by prenatal X-ray exposure using quantitative manganese-enhanced MRI (MEMRI) as well as apparent diffusion coefficient (ADC) and transverse relaxation time (T(2)) maps in comparison with immunohistological staining. The changes in ΔR(1) (increase in the longitudinal relaxation rate (R(1)) from before and after MnCl(2) administration.) induced by the Mn(2+) contrast agent were evaluated in the CNS of normal and prenatally irradiated rats. ADC and T(2) were also compared with the histological results obtained using hematoxylin and eosin (to estimate cell density), activated caspase-3 (apoptotic cells) and glial fibrillary acidic protein (proliferation of astrocytes/astroglia). We found the following: (i) the decreased Mn(2+) uptake (indicated by a smaller ΔR(1)) for radiation-exposed rats was predominantly correlated with a decrease in cell viability (apoptotic cytopathogenicity) and CNS cell density after prenatal radiation exposure; (ii) the longer T(2) and ADC were associated with a decrease in CNS cell density and apoptotic alteration after radiation exposure. In addition to the slight proliferation of astroglia (+58%), there was a substantial decrease in cell density (-78%) and an excessive increase in apoptotic cells (+613%) in our prenatal radiation exposure model. The results suggest that MEMRI in the prenatal X-ray exposure model predominantly reflected the decrease in cell density and viability rather than the proliferation of astroglia. In conclusion, quantitative MEMRI with ADC/T(2) mapping provides objective information for the in vivo assessment of cellular level alterations by prenatal radiation exposure, and has the potential to be used as a standard approach for the evaluation of the cellular damage of radiotherapy.