Noriko Shindo

Clinical and genetic spectrum of Birt–Hogg–Dubé syndrome patients in whom pneumothorax and/or multiple lung cysts are the presenting feature

Background Birt–Hogg–Dubé syndrome (BHDS) is an inherited autosomal genodermatosis characterised by fibrofolliculomas of the skin, renal tumours and multiple lung cysts. Genetic studies have disclosed that the clinical picture as well as responsible germline FLCN mutations are diverse. Objectives BHDS may be caused by a germline deletion which cannot be detected by a conventional genetic approach. Real-time quantitative polymerase chain reaction (qPCR) may be able to identify such a mutation and thus provide us with a more accurate clinical picture of BHDS. Methods This study analysed 36 patients with multiple lung cysts of undetermined causes. Denaturing high performance liquid chromatography (DHPLC) was applied for mutation screening. If no abnormality was detected by DHPLC, the amount of each FLCN exon in genome was quantified by qPCR. Results An FLCN germline mutation was found in 23 (63.9%) of the 36 patients by DHPLC and direct sequencing (13 unique small nucleotide alterations which included 11 novel mutations). A large genomic deletion was identified in two of the remaining 13 patients by qPCR (one patient with exon 14 deletion and one patient with a deletion encompassing exons 9 to 14). Mutations including genomic deletions were most frequently identified in the 3′-end of the FLCN gene including exons 12 and 13 (13/25=52.0%). The BHDS patients whose multiple cysts prompted the diagnosis in this study showed a very low incidence of skin and renal involvement. Conclusions BHDS is due to large deletions as well as small nucleotide alterations. Racial differences may occur between Japanese and patients of European decent in terms of FLCN mutations and clinical manifestations.

Stage-specific isoforms of Ascaris suum complex II: the fumarate reductase of the parasitic adult and the succinate dehydrogenase of free-living larvae share a common iron-sulfur subunit.

Complex II of adult Ascaris suum muscle exhibits high fumarate reductase (FRD) activity and plays a key role in anaerobic electron-transport during adaptation to their microaerobic habitat. In contrast, larval (L2) complex II shows a much lower FRD activity than the adult enzyme, and functions as succinate dehydrogenase (SDH) in aerobic respiration. We have reported the stage-specific isoforms of complex II in A. suum mitochondria, and showed that at least the flavoprotein subunit (Fp) and the small subunit of cytochrome b (cybS) of the larval complex II differ from those of adult. In the present study, complete cDNAs for the iron-sulfur subunit (Ip) of complex II, which with Fp forms the catalytic portion of complex II, have been cloned and sequenced from anaerobic adult A. suum, and the free-living nematode, Caenorhabditis elegans. The amino acid sequences of the Ip subunits of these two nematodes are similar, particularly around the three cysteine-rich regions that are thought to comprise the iron-sulfur clusters of the enzyme. The Ip from A. suum larvae was also characterized because Northern hybridization showed that the adult Ip is also expressed in L2. The Ip of larval complex II was recognized by the antibody against adult Ip, and was indistinguishable from the adult Ip by peptide mapping. The N-terminal 42 amino acid sequence of Ip in the larval complex II purified by DEAE-cellulofine column chromatography was identical to that of the mature form of the adult Ip. Furthermore, the amino acid composition of larval Ip determined by micro-analysis on a PVDF membrane is almost the same as that of adult Ip. These results, together with the fact, that homology probing by RT-PCR, using degenerated primers, failed to find a larval-specific Ip, suggest that the two different stage-specific forms of the A. suum complex II share a common Ip subunit, even though the adult enzyme functions as a FRD, while larval enzyme acts as an SDH.

One-step subcellular fractionation of rat liver tissue using a Nycodenz density gradient prepared by freezing-thawing and two-dimensional sodium dodecyl sulfate electrophoresis profiles of the main fraction of organelles

In the present study, we describe a new procedure using freezing‐thawing to density gradient solution of Nycodenz for one‐step separation of organelles from the rat liver and subsequent proteome analysis of subcellular fractions. To prepare two‐dimensional electrophoresis (2‐D PAGE) profiles of tissue organelles, we performed one‐step subcellular fractionation of rat liver homogenate using a density gradient of Nycodenz solution, which resulted in the separation of the cytosolic fraction from the postnuclear supernatant. The density gradient of Nycodenz was prepared from a 20% solution in a centrifuge tube by freezing‐thawing overnight at –20°C and at room temperature for a few hours without the initial centrifugation procedure. The shape of the gradient density curve was dependent on Nycodenz concentration and tube size. After fractionation, the protein profiles were examined using one‐dimensional sodium dodecyl sulfate (SDS)‐PAGE. The organelles were confirmed using Western blotting. Our results indicate that our procedure provides a simple method for the separation of organelle fractions from the rat liver tissue.

Transcriptome profile of Trypanosoma cruzi-infected cells: simultaneous up- and down-regulation of proliferation inhibitors and promoters

As Trypanosoma cruzi, the etiological agent of Chagas disease, multiplies in the cytoplasm of nucleated host cells, infection with this parasite is highly likely to affect host cells. We performed an exhaustive transcriptome analysis of T. cruzi-infected HeLa cells using an oligonucleotide microarray containing probes for greater than 47,000 human gene transcripts. In comparison with uninfected cells, those infected with T. cruzi showed greater than threefold up-regulation of 41 genes and greater than threefold down-regulation of 23 genes. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) of selected, differentially expressed genes confirmed the microarray data. Many of these up- and down-regulated genes were related to cellular proliferation, including seven up-regulated genes encoding proliferation inhibitors and three down-regulated genes encoding proliferation promoters, strongly suggesting that T. cruzi infection inhibits host cell proliferation, which may allow more time for T. cruzi to replicate and produce its intracellular nests. These findings provide new insight into the molecular mechanisms by which intracellular T. cruzi infection influences the host cell, leading to pathogenicity.

Optical resolution of phenylthiohydantoin-amino acids by capillary electrophoresis for protein sequencing

Abstract An advanced method is described for the complete separation of phenylthiohydantoin (PTH)- dl -amino acids for protein sequencing. Optical resolution of all standard PTH- dl -amino acids was successfully developed using some chiral selectors, although the resolution of only PTH- dl -His and Lys could not be confirmed, due to low reproducibility and the presence of impurities. In addition, mixed chiral selectors for making a single electrolyte with the ability to optically resolve all standard PTH- dl -amino acids were investigated. Using the only resulting electrolyte, sequence determination of [ d -Ala2]-methionine enkephalin, with dl differentiation, was performed.

Genomic causes of multiple cerebral cavernous malformations in a Japanese population

Cerebral cavernous malformation (CCM) is a hamartomatous vascular disease affecting the central nervous system. A fraction of CCM are thought to arise in association with genomic mutations in the cerebral cavernous malformation 1 (CCM1) (KRIT1), CCM2 (MGC4607), and CCM3 (PDCD10) genes. In the present study, 18 Japanese patients with multiple CCM (10 with familial type and eight with sporadic type), underwent genomic analysis for CCM1, CCM2 and CCM3 mutations with blood samples and surgical specimens. MRI showed CCM in the cerebral hemisphere in 17 patients, the cerebellum in 10, the brainstem in 10 and the spinal cord in eight. CCM2 mutations were the most prominent, followed by CCM1 and CCM3. CCM1, CCM2 and CCM3 mutations were not identified in seven patients. Among the 10 patients with familial CCM, CCM1, CCM2 and CCM3 mutations were found in two, three and one patient, respectively, whereas four patients lacked these mutations. Among the eight patients with sporadic CCM, these mutations were found in one, three, and one patients, respectively, whereas three patients lacked these mutations. Most of the patients had a stable course during the follow-up period. Genomic mutations other than CCM1, CCM2 and CCM3 may be frequent in patients with multiple CCM in the Japanese population.

Optical resolution of phenylthiohydantoin-amino acids and identification of phenylthiohydantoin-d-amino acid residue of [d-Ala2]-methionine enkephalin by capillary electrophoresis

Abstract We propose a system of protein sequence analysis with dl differentiation using capillary electrophoresis (CE). This system consists of a protein sequencer and a CE. After fractionation of phenylthiohydantoin (PTH)-amino acids from the protein sequencer, optical resolution for each PTH-amino acid is performed by CE using some chiral selectors such as digitonin, o -trimethyl-β-cyclodextrin (TM-β-CD) and others. In addition, optical resolution of all standard PTH- dl -amino acids including PTH- dl -carboxymethyl-Cys (CM-Cys) and cysteic acid (CYA) except for PTH- dl -Lys was successfully developed. The resolution of PTH- dl -Lys could not be reconfirmed due to low reproducibility and the impurities. As a model peptide, [ d -Ala 2 ]-methionine enkephalin ( l -Tyr– d -Ala–Gly– l -Phe– l -Met), was used and the sequence with dl differentiation was completely determined.

A modification of determination for glucosamine and galactosamine in glycoprotein with the amino acid analyzer: Application to total acid hydrolyzate of rat renal glomerular basement membrane

Abstract A procedure was described for the rapid determination of glucosamine and galactosamine in total acid hydrolyzate of rat renal glomerular basement membrane (GBM) by the use of amino acid analyzer. Glucosamine and galactosamine, as well as other amino acids in glycoprotein hydrolyzate, were well identified and estimated simultaneously by using a short column of HITACHI I-PF-B spherical resin, eluted with a pH 6.09 buffer containing 8% methanol at 38°C. Total time consumed for elution of galactosamine was 60 min. This method is ideal for the separation of small amount of galactosamine from hydroxylysine-rich materials.

Identification of Chirality of Phenylthiohydantoin-D-Amino Acid Residue of [D-ala2]-Metthionine Enkephalin by Capillary Electrophoresis: Suppression and Control of Racemization Ratio in the Edman Sequencing Method

Abstract This paper describes the suppression and control of the racemization ratio (D or L/D+L) of phenylthiohydantoin (PTH) amino acids in the Edman sequencing method. Most of the racemization occurs in the cyclization/cleavage step. Although optimization of partial racemization using a mixture of TFA and boron trifluoride (BF3)-ethyl ether complex, which is effective in suppressing racemization in the cyclization/cleavage reaction. The partial racemization in PTH derivatization is often useful for DL differentiation, because a minor L- or D-peak produced by racemization can be used as an internal standard in CE. Using the partial racemization method with mixed acids as a cyclization/cleavage reagent, the sequence determination of [D-Ala2]-methionine enkephalin, with DL differentiation, was achieved on a sequencer.

Characterization of native and recombinant peptidyl prolyl cis-trans isomerases derived from Methanococcus thermolithotrophicus based on cDNA sequence.

It is important to establish whether a recombinant protein is an authentic copy of the predicted cDNA sequence. In this study, recombinant protein for native peptidyl prolyl cis‐trans isomerase (N‐PPIase) and double‐labeled (13C‐ and 15N‐) protein (DL‐PPIase) appeared on the sodium dodecyl sulfate (SDS) electropherograms as two bands for N‐PPIase and four bands for DL‐PPIase. Since the N‐terminal amino acid residues of all bands were the same, we characterized these bands using the peptide mapping method and amino acid composition analysis. Peptide mapping of the proteins seemed to be almost identical but they could not reflect the whole amino acid sequences of the protein. The bands on the polyvinylidene difluoride (PVDF) membrane, electroblotted after SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE), were hydrolyzed and their amino acid composition was analyzed using a highly sensitive 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate (AQC) amino acid analysis and compared with the cDNA sequences for proteins. The matching score (Σ(T%‐E%)2) for similarity of proteins was calculated by summation of the square difference between the theoretical (T%) and the experimental (E%) amino acid composition of the recombinant protein. The amino acid composition of all bands of both proteins showed more than 93% of the theoretical values. The major molecular weights of both proteins were 16 812 and 17 694 by electrospray ionization (ESI)‐mass spectrometry. However, the purified proteins also contained minor compounds with Mr of 3 721 for N‐PPIase and 5 285 for DL‐PPIase. These compounds were considered to be nonpeptidyl products that comigrated with the protein. Similarities of the amino acid composition of the four bands were more than 98%. Our results indicate that AQC amino acid analysis is the most suitable method for characterization of a recombinant protein.