PMP22-related disease: A novel splice site acceptor variant and intrafamilial phenotype variability.

PMP22 is the most frequent mutated gene in Charcot-Marie-Tooth disease (CMT) type 1A. Another phenotype, hereditary neuropathy with pressure palsies (HNPP), could be caused by PMP22 mutations. PMP22 encodes a peripheral myelin protein with molecular weight 22-kDa. Various pathomechanisms have been postulated in PMP22-related disease, including dysfunction due to missense mutations, and alteration of a gene dose due to duplication/deletion mutations. We identified a novel PMP22 splice site acceptor variant, c.179-1G>A, in a patient with adult-onset chronic generalized polyneuropathy and two asymptomatic family members. Pathophysiological features of the members mainly overlapped with those reported in HNPP, but broad intrafamilial clinical variations were observed. PMP22 transcripts lacking of exon 4 were produced by the variant, presumably leading to in-frame deletion of 47 amino acids. The variant was also shown to exert effect on dosage of PMP22 mRNA. The complex molecular pathology would lead to the unique clinical and pathophysiological conditions.

Efficient refolding and immobilization of PMMA-tag-fused single-chain Fv antibodies for sensitive immunological detection on a PMMA plate.

In this study, we investigated the efficient refolding and site-specific immobilization of single-chain variable fragments (scFvs) genetically fused with a poly(methylmethacrylate)-binding peptide (PMMA-tag). According to the results of an aggregation test of a scFv-PM in the presence of 0.5 M urea, aggregation was hardly detectable at a weak-alkaline pH (8.5) with lower concentrations of NaCl. Consequently, more than 93% recovery of the anti-RNase scFv-PM model was attained, when it was refolded by dialysis against 50 mM TAPS (pH8.5). These results suggested that the apparent isoelectric point (pI) of a target scFv was decreased to a great extent by the genetic fusion of a PMMA-tag containing 5 acidic amino acids, and, thus, the solubility of the scFv-PM in its semi-denatured form was considerably improved. We also designed alternative peptide-tags composed of plural aspartic acid residues (D5, D10 and D15-tags) to decrease the apparent pI value of the fusion protein. As a consequence, scFv-D5, scFv-D10 and scFv-D15 were also efficiently refolded with yields of more than 95%. It is noteworthy that even scFv-PS-D15, which had both a positively charged polystyrene-binding peptide (PS-tag) and a negatively charged D15-tag, was serially connected at the C-terminal region of scFvs, and also refolded with a yield of 96.1%. These results clearly indicate that controlling the apparent pI value of scFvs by the fusion of oligo-peptides composed of acidic amino acids at the C-terminus resulted in a high degree of recovery via dialysis refolding. According to the results of a sandwich ELISA using scFv-PMs, scFv-D15 and scFv-PS-D15 as ligands, high antigen-binding signals were detected from both the PMMA and phi-PS plates immobilized with scFv-PMs. Furthermore, the high antigen-binding activity of scFv-PMs was maintained in an adsorption state when it was immobilized on the surface of not only PMMA, but also hydrophilic PS (phi-PS) and polycarbonate (PC). These results strongly suggested that a PMMA-tag introduced at the C-terminus of scFvs preferably recognizes ester and/or carboxyl groups exposed on the surface of plastics. The scFv-PM developed in the present study has advantages such as being a ligand antibody, compared with whole Ab and the conventional PS-tag-fused scFvs (scFv-PS), and, thus, it is considerably useful in a sandwich ELISA as well as in various immuno-detection and immuno-separation systems.

[Clinical features of tetanus: a review with case reports].

Tetanus is a CNS disorder characterized by muscle spasms that is caused by the exotoxin of an anaerobic bacterium, Clostridium tetani. This disease is killing tens of thousands of neonates in developing countries. Although the incidence and mortality of tetanus has dramatically dropped in developed countries due to effective vaccination, appropriate wound management, and recent advances in intensive care, treatments remain difficult. From among developed countries, Japan has had a relatively high incidence of tetanus, and prevention is the problem especially in the elderly. We analyzed the data from 12 patients admitted to our hospital during 1997-2010. Their age ranged from 50-82 years; median 72 years with male to female ratio 5: 7, and all patients lacked a reliable history of toxoid immunization. Five patients out of 12 had history of cancer and one each had diabetes mellitus, pulmonary emphysema and Sjogren syndrome. In some cases, the patients had been initially misdiagnosed with ENT disorders, dental problems, or psychosis. Therefore to date, observation of clinical symptoms such as difficulty in mouth opening is considered the most crucial for diagnosis. The shorter was a period from onset to generalized convulsion (onset time), the longer was a duration of hospital stay. A notable complication was intramuscular hemorrhage in the lumbar muscles, which occurred in 2 patients. No patients died due to ICU managements, in which the most useful were propofol for spasm control and sedation, and magnesium sulfate for autonomic overactivity. Retrospectively, the Tetanus Severity Score (TSS) for mortality proposed by Thwaites et al (2006) is considered a useful tool also for predicting the clinical outcome at discharge. Although tetanus has been traditionally classified into the generalized, local, and cephalic types, a simpler severity-based classification into "severe", "moderate", and "mild" types may be more practical with regard to disease management.