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Separation of high-molecular-weight polymers differing just by one monomeric unit remains a challenging task. Here, we describe a protocol using metal-organic frameworks (MOFs) for the efficient separation and purification of mixtures of polymers that differ only by their terminal groups. In this process, polymer chains are inserted by threading one of their extremities through a series of MOF nanowindows. Selected termini can be adjusted by tuning the MOF structure, and the insertion methodology. Accordingly, MOFs with permanently opened pores allow for the complete separation of poly(ethylene glycol) (PEG) based on steric hindrance of the terminal groups. Excellent separation is achieved, even for high molecular weights (20 kDa). Furthermore, the dynamic character of a flexible MOF is used to separate PEG mixtures with very similar terminal moieties, such as OH, OMe, and OEt, as the slight difference of polarity in these groups significantly changes the pore opening kinetics.

The design of monomer sequences in polymers has been a challenging research subject, especially in making vinyl copolymers by free-radical polymerization. Here, we report a strategy to obtain sequence-regulated vinyl copolymers, utilizing the periodic structure of a porous coordination polymer (PCP) as a template. Mixing of Cu 2+ ion and styrene-3,5-dicarboxylic acid (S) produces a PCP, [Cu(styrene-3,5-dicarboxylate)] n , with the styryl groups periodically immobilized along the one-dimensional channels. After the introduction of acrylonitrile (A) into the host PCP, radical copolymerization between A and the immobilized S is performed inside the channel, followed by decomposing the PCP to isolate the resulting copolymer. The predominant repetitive SAAA sequence in the copolymer is confirmed by monomer composition, NMR spectroscopy and theoretical calculations. Copolymerization using methyl vinyl ketone also provides the same type of sequence-regulated copolymer, showing that this methodology has a versatility to control the copolymer sequence via transcription of PCP periodicity at the molecular level.

Methylmalonic acidemia (MMA) is caused by a deficiency in the activity of L: -methylmalonyl-CoA mutase (MCM), a vitamin B12 (or cobalamin, Cbl)-dependent enzyme. Apoenzyme-deficient MMA (mut MMA) results from mutations in the nuclear gene MUT. Most of the MUT mutations are thought to be private or restricted to only a few pedigrees. Our group elucidated the spectrum of mutations of Japanese mut MMA patients by performing mutation and haplotype analyses in 29 patients with mut MMA. A sequence analysis identified mutations in 95% (55/58) of the disease alleles. Five mutations were relatively frequent (p.E117X, c.385 + 5G > A, p.R369H, p.L494X, and p.R727X) and four were novel (p.M1V, c.753_753 + 5delGGTATA, c.1560G > C, and c.2098_2099delAT). Haplotype analysis suggested that all of the frequent mutations, with the exception of p.R369H, were spread by the founder effect. Among 46 Japanese patients investigated in the present and previous studies, 76% (70/92) of the mutations were located in exons 2, 6, 8, and 13. This finding - that a limited number of mutations account for most of the mutations in Japanese mut MMA patients - is in contrast with results of a previous study in Caucasian patients.

Carbamoylphosphate synthetase I deficiency (CPS1D) is a urea-cycle disorder characterized by episodes of life-threatening hyperammonemia. Correct diagnosis is crucial for patient management, but is difficult to make from clinical presentation and conventional laboratory tests alone. Enzymatic or genetic diagnoses have also been hampered by difficult access to the appropriate organ and the large size of the gene (38 exons). In this study, in order to address this diagnostic dilemma, we performed the largest mutational and clinical analyses of this disorder to date in Japan. Mutations in CPS1 were identified in 16 of 18 patients with a clinical diagnosis of CPS1D. In total, 25 different mutations were identified, of which 19 were novel. Interestingly, in contrast to previous reports suggesting an extremely diverse mutational spectrum, 31.8% of the mutations identified in Japanese were common to more than one family. We also identified two common polymorphisms that might be useful for simple linkage analysis in prenatal diagnosis. The accumulated clinical data will also help to reveal the clinical presentation of this rare disorder in Japan.

Carnitine palmitoyltransferase I (CPT I) is one of the carnitine cycle enzymes that plays a role in the transportation of long-fatty acids into the mitochondria for beta-oxidation. Hepatic carnitine palmitoyltransferase I (CPT IA) is one of the isozymes of CPT I, and its deficiency results in an autosomal recessive mitochondrial fatty acid oxidation disorder. To date, 19 patients with CPT IA deficiency and 9 CPT IA mutations have been reported. Recently, six novel mutations in the CPT IA gene were reported in Japanese patients with CPT I deficiencies who were clinically diagnosed as having a Reye-like syndrome. One of these mutations was a missense mutation, 1079A>G (E360G). The other was a splicing mutation, 2027-2028+2delAAGT, which caused aberrant splicing transcripts, whereas 1876-2028del, 2027-2028insGTCTCTTCC ACTTCTTCC, and 2026-2028del were three aberrant transcripts that kept reading in-frame. In this report, an expression assay using SV40 transformed fibroblasts was performed to investigate the consequences of these two mutations on enzyme activity and protein levels. Molecular analysis in this study revealed that the two mutations 1079A>G and 2028+2delAAGT were the disease-causing mutations.

Menkes disease is an X-linked recessive disorder of the copper membrane transport system caused by mutations in the ATP7A gene. While various mutations in the ATP7A gene have been reported, a genotype-phenotype correlation has not been clearly defined. A novel mutation in the ATP7A gene in a Japanese patient with classical Menkes disease was identified via analysis of reverse-transcriptase polymerase chain reaction products and genomic DNA of the ATP7A gene. The nonsense mutation, L718X, was found to result in premature termination and immature ATP7A protein, unlikely to have normal functioning. Therefore, this nonsense mutation of the ATP7A gene is proposed to play a causative role in presenting the classical Menkes phenotype. Furthermore, four novel polymorphisms, C1535T (L464L), C2151T (T669I), G2253A (R703H), and C3677T (H1178Y) were also identified.

Carnitine/acylcarnitine translocase (CACT) transports acylcarnitines into mitochondria in exchange for free carnitine, and is therefore an essential component within the fatty acid beta-oxidation pathway. CACT deficiency is an autosomal recessive disease caused by a mutation of the CACT gene. We have identified two novel mutations of the CACT gene in a patient with CACT deficiency. The first, a deletion mutation (146 del T), leads to premature termination and results in a very immature CACT protein. The second, a splicing mutation (261-10T > G), results in either skipping of exons 3 and 4, or of exon 3 alone, and leads to truncation of the protein. Each of these mutations is hypothesized to destroy the function of the CACT protein. We propose that each of these mutations of the CACT gene play a causative role in the disease.

Menkes disease is an X-linked recessive disorder of the copper membrane transport system caused by mutations to the Menkes (MNK) gene. We identified three novel mutations of the MNK gene in three unrelated Japanese patients with classical Menkes disease by analyzing reverse-transcriptase polymerase chain reaction products and genomic DNA of the MNK gene. Firstly, an insertional mutation was found, 1173 ins A, which led to a premature termination and resulted in a very immature Menkes protein. Secondly, we found a point mutation, T2763G, resulting in a leucine-to-arginine conversion, which we predicted would cause a change in the secondary structure of the Menkes protein. Finally, we identified a splicing mutation, 2317 + 5G > C, which resulted in the skipping of both exons 8 and 9 or exon 9 only, and led to a truncation of the protein. Each of these mutations is hypothesized to destroy copper-ATPase-mediated copper transport. We propose that each of these mutations in the MNK gene plays a causative role in the disease.

An isoleucine/valine polymorphism was observed at codon 1464 of the ATP7A gene, which is thought to encode a copper transporting adenosine triphosphatase (ATPase). The frequency of Val1464 was estimated to be 5.7% in the Japanese population. This polymorphism may be useful in genetic studies of Menkes disease.

Two novel mutations in the gene encoding T-protein, a component of the glycine cleavage system, were identified in a Japanese family with nonketotic hyperglycinemia. The proband had two affected sibs, and enzymatic analysis of the liver sample from the proband revealed the T-protein deficiency. The first mutation, 183delC, was found in exon 1. One of six cytidine residues (base position 183-188) was deleted. The deletion was located in a coding region of the mitochondrial leader peptide and was deduced to create a truncated peptide with 94 amino acids. The second mutation was a base substitution from G to C at position 955 in exon 7. The G955C substitution caused an amino acid change from aspartate to histidine at position 276 (D276H). Aspartic acid at position 276 is evolutionarily conserved among human, bovine, chicken, and pea genes, and replaced by glutamic acid in Escherichia coli, suggesting that the presence of an acidic amino acid at 276 may be crucial for the enzymatic function. No base change other than the 183delC and the G955C was observed in the sequencing analysis. Familial analysis revealed that the 183delC and the D276H mutations were inherited from the father and the mother, respectively. This is the first report of T-protein gene mutation in Oriental patients with nonketotic hyperglycinemia.

Ornithine transcarbamylase (OTC) is located in the mitochondrial matrix of the liver and small intestine and catalyzes the second step of the urea cycle. OTC deficiency (OTCD) is an X-linked inborn error of metabolism and causes hyperammonemia. We reported in 1992 the A152V and G195R mutations in patients with OTCD. These mutant OTC cDNAs were prepared by site-directed mutagenesis using the polymerase chain reaction (PCR). The wild-type and mutant cDNAs were transiently expressed in COS-7 cells. The wild-type cDNA gave an OTC activity of 1180 +/- 47 nmol/min per mg protein. The OTC activities of the A152V and the G195R mutants were 3.7% and 2.5% of that of wild-type, respectively. Immunoblot analysis showed that the quantities of OTC proteins in the A152V and G195R mutants were 29% and 12% of that of wild-type, respectively. In pulse-labeling and pulse-chase experiments, the precursor form of OTC was synthesized and processed to the mature form. The A152V mutant OTC was processed to the mature form as rapidly as the wild-type precursor. However, the processed, mature form of the mutant OTC was rapidly degraded, presumably in the mitochondrial matrix. These results indicate that OTCD with the A152V mutation is due both to rapid degradation of the processed, mature form, and to a lower specific activity of the remaining protein.