Mutations in B3GALT6, which encodes a glycosaminoglycan linker region enzyme, cause a spectrum of skeletal and connective tissue disorders.

Proteoglycans (PGs) are a major component of the extracellular matrix in many tissues and function as structural and regulatory molecules. PGs are composed of core proteins and glycosaminoglycan (GAG) side chains. The biosynthesis of GAGs starts with the linker region that consists of four sugar residues and is followed by repeating disaccharide units. By exome sequencing, we found that B3GALT6 encoding an enzyme involved in the biosynthesis of the GAG linker region is responsible for a severe skeletal dysplasia, spondyloepimetaphyseal dysplasia with joint laxity type 1 (SEMD-JL1). B3GALT6 loss-of-function mutations were found in individuals with SEMD-JL1 from seven families. In a subsequent candidate gene study based on the phenotypic similarity, we found that B3GALT6 is also responsible for a connective tissue disease, Ehlers-Danlos syndrome (progeroid form). Recessive loss-of-function mutations in B3GALT6 result in a spectrum of disorders affecting a broad range of skeletal and connective tissues characterized by lax skin, muscle hypotonia, joint dislocation, and spinal deformity. The pleiotropic phenotypes of the disorders indicate that B3GALT6 plays a critical role in a wide range of biological processes in various tissues, including skin, bone, cartilage, tendon, and ligament.

An activated medium with high durability and low nonspecific adsorption: application to protein A chromatography.

Activated media allow the user to easily synthesize a variety of affinity media. We have developed a novel activated medium based on porous silica modified with phosphorylcholine (PC) and N-hydroxysuccinimide (NHS) groups for the purpose of high-throughput purification and reducing nonspecific protein adsorption. The PC groups function as suppressors of nonspecific protein adsorption, whereas the NHS groups are able to covalently bind to the primary amino groups of ligands. Because protein A affinity medium is the most frequently used affinity medium, we prepared protein A media in which a recombinant protein A was bound to the NHS groups of the activated media and evaluated its utility. After optimizing various factors in the synthetic process, the resultant protein A medium showed improved durability at a high flow rate over 300 purification cycles and reduced nonspecific protein adsorption compared with commercially available protein A media.

High-sensitivity simultaneous analysis of ubiquinol-10 and ubiquinone-10 in human plasma.

A method to determine ubiquinol-10 and ubiquinone-10 in human serum was developed by using high-performance liquid chromatography consisting of a semi-microcolumn switching system and an electrochemical detector (ECD), which requires minimized sample pre-treatments. A linear dynamic range was obtained from 1.0 to 5000 ng/mL, and recovery values of 89-105% were observed in a low-concentration region of 10-50 ng/mL. In a long operation test, a good precision was maintained during 5100 runs without any maintenance on ECD or columns. In addition, retention behaviors of other ubiquinone homologues were examined.

ADAM17 regulates TNF-α expression upon lipopolysaccharide stimulation in oral keratinocytes.

A disintegrin and metalloprotease 17 (ADAM17) is a tumor necrosis factor (TNF)-converting enzyme and was first identified as the enzyme that cleaves the prodomain of TNF-α, a proinflammatory cytokine that plays a central role in immune regulation and a variety of inflammatory responses in destructive periodontal disease. The aim of the present study was to verify the presence of ADAM17 in the gingival epithelium and elucidate its involvement in the release of TNF-αin oral keratinocytes. Immunohistochemical analyses of ADAM17 were performed in gingival tissues obtained from patients and in human oral keratinocytes (HOKs). Additionally, levels of TNF-α and ADAM17 in HOKs exposed to lipopolysaccharide (LPS) were assessed using enzyme-linked immunosorbent assays. Moreover, the effects of ADAM17 inhibitor, matrix metalloproteinase (MMP) inhibitor, and ADAM17 siRNA on TNF-α concentration were assessed. Strong immunoreactivity for ADAM17 was observed in the epithelium of the inflamed gingival tissues and in HOKs. Furthermore, treatment with either ADAM17 inhibitor or ADAM17 siRNA inhibited the generation of TNF-α induced by LPS in HOKs. The present study demonstrates that ADAM17 is strongly expressed in the epithelium of gingival tissues and suggests that ADAM17 may be a key enzyme that regulates the generation of TNF-α in oral keratinocytes.

Leukoencephalopathy around a tumor cyst following intracystic methotrexate injection.

A 4-year-old female with choroid plexus carcinoma developed progressive disturbance of consciousness 2 years after postoperative treatment with radiotherapy, chemotherapy, and focal methotrexate injection into a residual tumor cyst. Magnetic resonance imaging revealed white matter lesions localized around the expanding large cyst. A malignant recurrence of choroid plexus carcinoma with a propensity of cerebrospinal fluid overproduction was suspected. However, daily drainage of cerebrospinal fluid from the cyst and treatment with glycerol and dexamethasone achieved improvement. Diffuse hypoperfusion over the lesions on single-photon emission computed tomography denied the possibility of residual tumor aggravation and together with subsequent atrophic changes confirmed that the lesions reflect localized leukoencephalopathy, possibly associated with methotrexate forced into the parenchyma as a result of the expanding intracystic high pressure.

Myelin transcription factor 1 (MyT1) immunoreactivity in infants with periventricular leukomalacia.

Myelin transcription factor 1 (MyT1) is a zinc-dependent, DNA-binding protein, and is known to be expressed in early progenitors of oligodendrocytes. We examined the immunoreactivity of MyT1 in developing human brains and brains with periventricular leukomalacia (PVL) to understand the relationship between the expression of MyT1 and myelination in PVL brains. MyT1-positive glial cells were first detected at 19 gestational weeks (GWs) and then gradually increased until 26-29 GWs in the control group. Then they decreased and became very rare at 1 year of age. The expression of MyT1 immunoreactivity shifted from the nucleus to the cytoplasm of the glial cells in the developmental time course. In the chronic stage of PVL, MyT1-positive cells were significantly increased around necrotic foci and some of the regions were coincident with increasing MBP and PLP immunoreactivity. These results may reflect myelin repair on dysmyelination around PVL areas. Therefore, MyT1 may play an important role in the myelin repair in PVL regions.

Characteristic developmental expression of amyloid beta40, 42 and 43 in patients with Down syndrome.

We immunohistochemically studied the expression of beta-amyloid precursor protein (APP), Abeta40, Abeta42, and Abeta43 in the frontal lobes of 20 Down syndrome (DS) patients and 13 controls. The immunoreactivity for each antibody was different in the degree of intensity and the chronological pattern of expression. APP and Abeta43 immunoreactivity was increased in neurons initially, and then Abeta43 and 42 immunoreactivity appeared in diffuse plaques from 32 years of age. APP and Abeta43 were characteristically observed in axons around senile plaques. Finally, Abeta40 immunoreactivity was detected in the cores of senile plaques. This time course of immunoreactive expression may be related to the pathogenetic process of Alzheimer-type dementia in DS, and the axonal damage in senile plaques may lead to the formation of neurofibrillary tangles (NFT) or neuronal death through axonal flow disturbance and accumulation of Abeta43 in cortical neurons.

[A case of Menkes disease with urinary bladder hemorrhage].

We report a case of Menkes disease with urinary bladder hemorrhage. Diverticulums were found at 1 year and 3 months of age. He had repetitive urinary tract infections at 2 years old and died of urinary hemorrhage at 2 years and 9 months. Large hematoma and diverticulum were found at necropsy. In patients with Menkes disease, attention should paid to urinary complications.

Phosphorylcholine (PC)-bonded protein A affinity chromatographic medium for high-throughput purification with reduced non-specific protein adsorption.

A protein A affinity chromatographic medium based on porous silica modified with phosphorylcholine (PC) groups and amino groups (PNSP) was synthesized. The PC groups functioned as suppressors of non-specific protein adsorption. Recombinant protein A was bound to the amino groups on PNSP with a glutaraldehyde used as a spacer (PNSP-PA). The PC groups and amino groups were immobilized on porous-silica particles using two silane coupling reagents, PC-bound silane, and 3-aminopropyltrimethoxysilane. After optimizing various factors in the synthetic process, the resultant protein A medium showed improvements in non-specific protein adsorption, dynamic binding capacity, and chemical stability under basic conditions compared with conventional protein A affinity media.

A protein recycling system for nuclear magnetic resonance-based screening of drug candidates.

A sample-treating system for nuclear magnetic resonance (NMR)-based interaction screening between drug candidates (small molecules) and a protein of interest was developed by applying high-performance liquid chromatography (HPLC) technology. The system prepares a test solution by mixing a (15)N-labeled protein solution and a solution of each candidate compound, loads it to a flow cell-type NMR probe, and recycles the protein after the data acquisition. The system was designed to behave differently according to the information obtained in NMR measurements. In a test operation with a 100-compound library, the system could single out known interacting substances properly. Recovery values of the protein and one representative compound were 75 and 71%, respectively, and the recovered protein was found intact as intended.

A hippocampal lesion detected by high-field 3 tesla magnetic resonance imaging in a patient with temporal lobe epilepsy.

Nearly 80% of patients with temporal lobe epilepsy have some types of lesion identified by conventional 1.5 tesla (T) magnetic resonance imaging (MRI). We performed high-field 3 T MRI in a 5-year-old patient with recurrent complex partial seizures who was diagnosed as having right temporal lobe epilepsy based on the results of single photon emission computed tomography and ictal video-electroencephalogram monitoring, because 1.5 T MRI failed to detect any abnormalities in the suspected region. High-field 3 T MRI revealed a small high-intensity lesion on fast spin-echo short inversion time inversion-recovery images of the hippocampus, possibly responsible for the seizures. This is the first report detecting a hippocampal lesion by 3 T MRI, which could not be found by conventional 1.5 T MRI.

Choroid plexus carcinoma presented with spinal dysfunction caused by a drop metastasis: a case report.

A 2-year-old girl demonstrating gait disturbance and dysuria was evaluated and showed two large remote tumors at the left lateral ventricle and lower spinal canal. Pathological analysis demonstrated both of the tumors to be choroid plexus carcinoma (CPC) with high MIB-1 labeling index. The enhanced mitotic propensity would have contributed to an early stage of drop metastasis from the primary site to the sacral sac and following accelerated formation of a longitudinal tumor, which had grown in the subarachnoid space conforming to the spinal canal and finally caused the presenting symptoms of spinal dysfunction. This report shows that CPC can develop exophytically in the subarachnoid space as well as in the ventricle simultaneously before appearance of clinical symptoms and confirms the importance of extensive neuroimaging in its evaluation.