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Keratin-associated protein 8.1 (KAP8.1) is a hair protein whose structure, biochemical roles, and protein distribution patterns have not been well characterized. In this study, we generated a monoclonal antibody against human KAP8.1 to analyze the protein's roles and distribution in the human hair shaft. Using this antibody, we revealed that KAP8.1 was predominantly expressed in discrete regions of the keratinizing zone of the hair shaft cortex. The protein expression patterns paralleled the distribution of KAP8.1 mRNA and suggested that KAP8.1 plays a role associated with cells to control hair curvature. Cross-reactivity among species and epitope analysis indicated that the monoclonal antibody recognized a linear epitope shared among human, mouse, and sheep KAP8.1. The antibody failed to interact with sheep KAP8.1 in native conformation, suggesting that structural features of KAP8.1 vary among species.

We report a case of a 59-year-old woman who was diagnosed with gastric and small intestinal anisakiasis, which was successfully treated with endoscopic extraction and Gastrografin therapy. She was admitted to our hospital with epigastric pain and vomiting one day after eating raw fish. She exhibited tenderness in the epigastrium without obvious rebound tenderness or guarding. Computed tomography (CT) demonstrated segmental edema of the intestinal wall with proximal dilatation and a small number of ascites. Because enteric anisakiasis was suspected based on the patient’s history of recent raw fish consumption and abdominal CT, we performed gastroscopy and confirmed that nine Anisakis larvae were attached to the gastric mucosa. All of the Anisakis larvae were extracted via endoscopy, and the patient was diagnosed with gastric and enteric anisakiasis. Additionally, in the hospital, we performed ileography twice using Gastrografin, which led to shortened hospital stay. Based on the clinical results of this case, we suggest that Gastrografin therapy is a safe, convenient, and useful method to extract enteric Anisakis larvae.

All TGF-beta family members have a prodomain that is important for secretion. Lack of secretion of a TGF-beta family member GDF5 is known to underlie some skeletal abnormalities, such as brachydactyly type C that is characterized by a huge and unexplained phenotypic variability. To search for potential phenotypic modifiers regulating secretion of GDF5, we compared cells overexpressing wild type (Wt) GDF5 and GDF5 with a novel mutation in the prodomain identified in a large Pakistani family with Brachydactyly type C and mild Grebe type chondrodyslplasia (c527T>C; p.Leu176Pro). Initial in vitro expression studies revealed that the p.Leu176Pro mutant (Mut) GDF5 was not secreted outside the cells. We subsequently showed that GDF5 was capable of forming a complex with latent transforming growth factor binding proteins, LTBP1 and LTBP2. Furthermore, secretion of LTBP1 and LTBP2 was severely impaired in cells expressing the Mut-GDF5 compared to Wt-GDF5. Finally, we demonstrated that secretion of Wt-GDF5 was inhibited by the Mut-GDF5, but only when LTBP (LTBP1 or LTBP2) was co-expressed. Based on these findings, we suggest a novel model, where the dosage of secretory co-factors or stabilizing proteins like LTBP1 and LTBP2 in the microenvironment may affect the extent of GDF5 secretion and thereby function as modifiers in phenotypes caused by GDF5 mutations.

Summary Background Hypohidrotic ectodermal dysplasia (HED) is a rare condition characterized by hypotrichosis, hypohidrosis and hypodontia. A de novo heterozygous mutation in the tumour necrosis factor receptor-associated factor 6 gene (TRAF6) was recently identified in a patient with HED, while functional consequences resulting from the mutation remained unknown. Objectives To determine the mechanism by which the TRAF6 mutation results in HED. Methods We performed coimmunoprecipitation (co-IP) studies to determine whether the mutation would affect the interaction of TRAF6 with transforming growth factor β-activated kinase 1 (TAK1), TAK1-binding protein 2 (TAB 2) and ectodysplasin-A receptor-associated death domain protein (EDARADD). We then performed co-IP and glutathione S-transferase-pulldown assays to determine the TRAF6 binding sequences in EDARADD. In addition, we analysed the effect of the mutant TRAF6 protein on the affinity between wild-type TRAF6 and EDARADD, as well as on EDARADD-mediated nuclear factor (NF)-κB activation. Results The mutant TRAF6 protein was capable of forming a complex with TAK1 and TAB 2 in a similar way to wild-type TRAF6. However, the mutant TRAF6 protein completely lost the affinity to EDARADD, while the wild-type TRAF6 bound to the N-terminal domain of EDARADD. Furthermore, the mutant TRAF6 inhibited the interaction between the wild-type TRAF6 and EDARADD, and also potentially reduced the EDARADD-mediated NF-κB activity. Conclusions We conclude that the mutant TRAF6 protein shows a dominant negative effect against the wild-type TRAF6 protein, which is predicted to affect the EDARADD-mediated activation of NF-κB during the development of ectoderm-derived organs, and to lead to the HED phenotype.

Generalized pustular psoriasis (GPP) is a rare, potentially life threatening, and aggressive form of psoriasis, which is characterized by sudden onset with repeated episodic skin inflammation leading to pustule formation. Familial GPP is known to be caused by recessively inherited mutations in the IL36RN gene, which encodes interleukin 36 receptor antagonist (IL-36Ra). In this article, we performed mutation analysis of the IL36RN gene in 14 Japanese patients with GPP, and identified mutations in two of these patients analyzed. One patient was compound heterozygous for mutations c.115+6T>C and c.368C>G (p.Thr123Arg), whereas the other carried compound heterozygous mutations c.28C>T (p.Arg10*) and c.115+6T>C in the IL36RN gene. Expression studies using total RNA from the patients' skin revealed that the mutation c.115+6T>C resulted in skipping of exon 3, leading to a frameshift and a premature termination codon (p.Arg10Argfs*1). The protein structure analysis suggested that the missense mutation p.Thr123Arg caused misfolding and instability of IL-36Ra protein. In vitro studies in cultured cells showed impaired expression of the p.Thr123Arg mutant IL-36Ra protein, which failed to antagonize the IL-36 signaling pathway. Our data further underscore the critical role of IL36RN in pathogenesis of GPP.

Woolly hair (WH) is an abnormal variant of tightly curled hair, which is frequently associated with hypotrichosis. Non-syndromic forms of WH can show either autosomal-dominant WH (ADWH) or autosomal-recessive WH (ARWH) inheritance patterns. ARWH has recently been shown to be caused by mutations in either the lysophosphatidic acid receptor 6 (LPAR6) or lipase H (LIPH) gene. More recently, a mutation in the keratin K74 (KRT74) gene has been reported to underlie ADWH. Importantly, all of these genes are abundantly expressed in the inner root sheath (IRS) of human hair follicles. Besides these findings, the molecular mechanisms underlying hereditary WH have not been fully disclosed. In this study, we identified a Japanese family with ADWH and associated hypotrichosis. After exclusion of known causative genes, we discovered the heterozygous mutation c.422T>G (p.Phe141Cys) within the helix initiation motif of the IRS-specific keratin K71 (KRT71) gene in affected family members. We demonstrated that the mutant K71 protein led to disruption of keratin intermediate filament formation in cultured cells. To our knowledge, it is previously unreported that the KRT71 mutation is associated with a hereditary hair disorder in humans. Our findings further underscore the crucial role of the IRS-specific keratins in hair follicle development and hair growth in humans.

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Background: H syndrome is a rare autosomal recessive genetic disorder which involves the skin and other systemic organs and is caused by mutations in the SLC29A3 gene. Objectives: To disclose the molecular basis of H syndrome in two Syrian families, and to determine the localization of hENT3 in human skin. Methods: DNA from two Syrian families with H syndrome was analyzed through direct sequencing, and the expression of hENT3 in normal human skin was investigated by in situ hybridization and immunostaining. Results: We identified two novel mutations in the SLC29A3 gene: a homozygous splice site mutation IVS1+2T>G predicted to cause a splicing error, and a homozygous missense mutation c.1157G>A (p.R386Q) which substituted highly conserved amino acid residue in a transmembrane domain of hENT3. Furthermore, we demonstrate that hENT3 is expressed in histiocytes as well as in endothelium of blood and lymphatic vessels in normal human skin. Conclusions: Our results further enhance the mutation spectrum of the SLC29A3 gene for this rare genetic disorder, and also suggest potential pathomechanisms for the skin lesions resulting from SLC29A3 mutations.

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Trichoderma reesei is a filamentous organism that secretes enzymes capable of degrading cellulose to cellobiose. The culture supernatant of T. reesei, however, lacks sufficient activity to convert cellobiose to glucose using β-glucosidase (BGL1). In this study, we identified a BGL (Cel3B) from T. reesei (TrCel3B) and compared it with the active β-glucosidases from Aspergillus aculeatus (AaBGL1). AaBGL1 showed higher stability and conversion of sugars to ethanol compared to TrCel3B, and therefore we chose to express this recombinant protein for use in fermentation processes. We expressed the recombinant protein in the yeast Saccharomyces cerevisiae, combined it with the superb T. reesei cellulase machinery and used the combination in a simultaneous saccharification and fermentation (SSF) process, with the hope that the recombinant would supplement the BGL activity. As the sugars were processed, the yeast immediately converted them to ethanol, thereby eliminating the problem posed by end product inhibition. Recombinant AaBGL1 activity was compared with Novozyme 188, a commercially available supplement for BGL activity. Our results show that the recombinant protein is as effective as the commercial supplement and can process sugars with equal efficiency. Expression of AaBGL1 in S. cerevisiae increased ethanol production effectively. Thus, heterologous expression of AaBGL1 in S. cerevisiae is a cost-effective and efficient process for the bioconversion of ethanol from lignocellulosic biomass.

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Background Clouston syndrome is a rare autosomal dominant condition characterized by hypotrichosis, nail dystrophy, and occasionally palmoplantar keratoderma. The disease is caused by mutations in GJB6 gene, which encodes a gap junction protein connexin 30 (Cx30). Objective To disclose the molecular basis of Clouston syndrome in a Lebanese-German family, and also to determine precise expression of Cx30 in normal skin of humans and mice, as well as transcriptional regulation for the GJB6 expression. Methods We searched for mutations in the GJB6 gene using DNA of the family members with Clouston syndrome. We performed immunostaining to localize the Cx30 expression in normal human skin and mouse embryos. In addition, we did a series of in vitro studies to investigate if the GJB6 could be a direct transcriptional target gene of p63. Results We identified a recurrent heterozygous mutation c.31G>C (p.Gly11Arg) in the GJB6 gene in the Lebanese-German family with Clouston syndrome. Immunostaining in normal human skin sections demonstrated predominant expression of Cx30 in hair follicles, nails, and palmoplantar epidermis, which partially overlapped with p63 expression. We also showed co-expression of Cx30 and p63 in developing mouse hair follicles and nail units. In cultured cells, the GJB6 expression was significantly upregulated by ΔNp63α isoform. Further in vitro analyses suggested that ΔNp63α was potentially involved in the GJB6 expression via binding to the sequences in intron 1 of the GJB6 gene. Conclusion Our data further underscore the crucial roles of Cx30 in morphogenesis and development of skin and its appendages.

Background: Hypohidrotic ectodermal dysplasia (HED) is a rare condition characterized by hypotrichosis, hypohidrosis and hypodontia. The disease shows X-linked recessive, autosomal-dominant or autosomal-recessive inheritance trait. X-linked form of HED is caused by mutations in the EDA gene, while autosomal forms are caused by mutations in either EDAR or EDARADD genes. Methods: We analyzed the DNA from a Japanese patient with HED through direct sequencing, and also performed functional studies for the mutation. Results: We identified a homozygous missense mutation c.1073G>A (p.R358Q) in the EDAR gene of the patient, which was a nonconservative amino acid substitution within the death domain of EDAR protein. We demonstrated that the p.R358Q mutant EDAR protein lost its affinity to EDARADD, leading to reduced activation of the downstream NF-ĸB. Conclusion: Our data further suggest the crucial role of the EDAR signaling in development of hair, teeth, and sweat gland in humans.

Hair keratin–associated proteins (KRTAPs) are one of the major structural components of the hair shaft. Approximately 100 KRTAP genes have been identified in humans to date, with each of the genes classified into a number of families based on their sequence homology and the nature of the repeat structures. The biophysical features of KRTAPs, however, have remained largely unknown. In this study, we investigated the characteristics of the human KRTAP2 family members at the DNA, RNA, and protein levels. We initially found that these genes had various size polymorphisms that were mainly due to differences in the length of the 3′-noncoding sequences. Reverse transcriptase–PCR experiments further detected the presence of KRTAP2 transcripts in plucked human hairs. Using indirect immunofluorescence with an anti-KRTAP2 antibody, we found that there was a predominant expression of the KRTAP2 proteins in the keratinizing zone of the human hair shaft cortex. In addition, we showed that the KRTAP2 proteins interacted with each other and preferentially bound to hair keratins, but not to epithelial keratins. Finally, we determined that the head domain of the hair keratins was essential for the affinity to KRTAP2 proteins. Our results further enhance the crucial roles of KRTAPs in hair shaft keratinization in humans.