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R3HDM1 (R3H domain containing 1) is an uncharacterized RNA-binding protein that is highly expressed in the human cerebral cortex. We report the first case of a 12-year-old Japanese male with haploinsufficiency of R3HDM1. He presented with mild intellectual disability (ID) and developmental delay. He had a pericentric inversion of 46,XY,inv(2)(p16.1q21.3)dn with breakpoints in intron 19 of R3HDM1 (2q21.3) and the intergenic region (2p16.1). The R3HDM1 levels in his lymphoblastoid cells were reduced to approximately half that of the healthy controls. However, the expression of MIR128-1, in intron 18 of R3HDM1, was not affected via the pericentric inversion. Knockdown of R3HDM1 in mouse embryonic hippocampal neurons suppressed dendritic growth and branching. Notably, the Database of Genomic Variants reported the case of a healthy control with a 488-kb deletion that included both R3HDM1 and MIR128-1. miR-128 has been reported to inhibit dendritic growth and branching in mouse brain neurons, which directly opposes the novel functions of R3HDM1. These findings suggest that deleting both R3HDM1 and MIR128-1 alleviates the symptoms of the disease caused by loss-of-function mutations in R3HDM1 only. Thus, haploinsufficiency of R3HDM1 in the patient may be the cause of the mild ID due to the genetic imbalance between R3HDM1 and MIR128-1.

Inherited GPI (glycosylphosphatidylinositol) deficiencies (IGDs), a recently defined group of diseases, show a broad spectrum of symptoms. Hyperphosphatasia mental retardation syndrome, also known as Mabry syndrome, is a type of IGDs. There are at least 26 genes involved in the biosynthesis and transport of GPI-anchored proteins; however, IGDs constitute a rare group of diseases, and correlations between the spectrum of symptoms and affected genes or the type of mutations have not been shown. Here, we report four newly identified and five previously described Japanese families with PIGO (phosphatidylinositol glycan anchor biosynthesis class O) deficiency. We show how the clinical severity of IGDs correlates with flow cytometric analysis of blood, functional analysis using a PIGO-deficient cell line, and the degree of hyperphosphatasia. The flow cytometric analysis and hyperphosphatasia are useful for IGD diagnosis, but the expression level of GPI-anchored proteins and the degree of hyperphosphatasia do not correlate, although functional studies do, with clinical severity. Compared with PIGA (phosphatidylinositol glycan anchor biosynthesis class A) deficiency, PIGO deficiency shows characteristic features, such as Hirschsprung disease, brachytelephalangy, and hyperphosphatasia. This report shows the precise spectrum of symptoms according to the severity of mutations and compares symptoms between different types of IGD.

Cancer cells can migrate as collectives during invasion and/or metastasis; however, the precise molecular mechanisms of this form of migration are less clear compared with single cell migration following epithelial-mesenchymal transition. Elevated Na+/H+ exchanger1 (NHE1) expression has been suggested to have malignant roles in a number of cancer cell lines and in vivo tumor models. Furthermore, a metastatic human head and neck squamous cell carcinoma (HNSCC) cell line (SASL1m) that was isolated based on its increased metastatic potential also exhibited higher NHE1 expression than its parental line SAS. Time-lapse video recordings indicated that both cell lines migrate as collectives, although with different features, e.g., SASL1m was much more active and changed the direction of migration more frequently than SAS cells, whereas locomotive activities were comparable. SASL1m cells also exhibited higher invasive activity than SAS in Matrigel invasion assays. shRNA-mediated NHE1 knockdown in SASL1m led to reduced locomotive and invasive activities, suggesting a critical role for NHE1 in the collective migration of SASL1m cells. SASL1m cells also exhibited a higher metastatic rate than SAS cells in a mouse lymph node metastasis model, while NHE1 knockdown suppressed in vivo SASL1m metastasis. Finally, elevated NHE1 expression was observed in human HNSCC tissue, and Cariporide, a specific NHE1 inhibitor, reduced the invasive activity of SASL1m cells, implying NHE1 could be a target for anti-invasion/metastasis therapy.

Background Short-chain enoyl-CoA hydratase—ECHS1—catalyses many metabolic pathways, including mitochondrial short-chain fatty acid β-oxidation and branched-chain amino acid catabolic pathways; however, the metabolic products essential for the diagnosis of ECHS1 deficiency have not yet been determined. The objective of this report is to characterise ECHS1 and a mild form of its deficiency biochemically, and to determine the candidate metabolic product that can be efficiently used for neonatal diagnosis. Methods We conducted a detailed clinical, molecular genetics, biochemical and metabolic analysis of sibling patients with ECHS1 deficiency. Moreover, we purified human ECHS1, and determined the substrate specificity of ECHS1 for five substrates via different metabolic pathways. Results Human ECHS1 catalyses the hydration of five substrates via different metabolic pathways, with the highest specificity for crotonyl-CoA and the lowest specificity for tiglyl-CoA. The patients had relatively high (∼7%) residual ECHS1 enzyme activity for crotonyl-CoA and methacrylyl-CoA caused by the compound heterozygous mutations (c.176A>G, (p.N59S) and c.413C>T, (p.A138V)) with normal mitochondrial complex I–IV activities. Affected patients excrete large amounts of N-acetyl-S-(2-carboxypropyl)cysteine, a metabolite of methacrylyl-CoA. Conclusions Laboratory data and clinical features demonstrated that the patients have a mild form of ECHS1 deficiency harbouring defective valine catabolic and β-oxidation pathways. N-Acetyl-S-(2-carboxypropyl) cysteine level was markedly high in the urine of the patients, and therefore, N-acetyl-S-(2-carboxypropyl)cysteine was regarded as a candidate metabolite for the diagnosis of ECHS1 deficiency. This metabolite is not part of current routine metabolic screening protocols, and its inclusion, therefore, holds immense potential in accurate diagnosis.

Cancer cells can migrate as collectives during invasion and/or metastasis; however, the precise molecular mechanisms of this form of migration are less clear compared with single cell migration following epithelial-mesenchymal transition. Elevated Na

Mowat-Wilson syndrome (MWS) is a multiple congenital anomaly syndrome characterized by moderate or severe intellectual disability, a characteristic facial appearance, microcephaly, epilepsy, agenesis or hypoplasia of the corpus callosum, congenital heart defects, Hirschsprung disease, and urogenital/renal anomalies. It is caused by de novo heterozygous loss of function mutations including nonsense mutations, frameshift mutations, and deletions in ZEB2 at 2q22. ZEB2 encodes the zinc finger E-box binding homeobox 2 protein consisting of 1,214 amino acids. Herein, we report 13 nonsense and 27 frameshift mutations from 40 newly identified MWS patients in Japan. Although the clinical findings of all the Japanese MWS patients with nonsense and frameshift mutations were quite similar to the previous review reports of MWS caused by nonsense mutations, frameshift mutations and deletions of ZEB2, the frequencies of microcephaly, Hirschsprung disease, and urogenital/renal anomalies were small. Patients harbored mutations spanning the region between the amino acids 55 and 1,204 in wild-type ZEB2. There was no obvious genotype-phenotype correlation among the patients. A transfection study demonstrated that the cellular level of the longest form of the mutant ZEB2 protein harboring the p.D1204Rfs*29 mutation was remarkably low. The results showed that the 3'-end frameshift mutation of ZEB2 causes MWS due to ZEB2 instability.

Mutation of hypoxanthine guanine phosphoribosyltransferase (HPRT) gives rise to Lesch–Nyhan syndrome, which is characterized by hyperuricemia, severe motor disability, and self-injurious behavior, or HPRT-related gout with hyperuricemia. Four mutations were detected in two Lesch–Nyhan families and two families with partial deficiency since our last report. A new mutation of G to TT (c.456delGinsTT) resulting in a frameshift (p.Q152Hfs*3) in exon 3 has been identified in one Lesch–Nyhan family. In the other Lesch–Nyhan family, a new point mutation in intron 7 (c.532 + 5G > T) causing splicing error (exon 7 excluded, p.L163Cfs*4) was detected. In the two partial deficiency cases with hyperuricemia, two missense mutations of p.D20V (c.59A > T) and p.H60R (c.179A >G) were found. An increase of erythrocyte PRPP concentration was observed in the respective phenotypes and seems to be correlated with disease severity.

Inherited GPI (glycosylphosphatidylinositol) deficiencies (IGDs), a recently defined group of diseases, show a broad spectrum of symptoms. Hyperphosphatasia mental retardation syndrome, also known as Mabry syndrome, is a type of IGDs. There are at least 26 genes involved in the biosynthesis and transport of GPI-anchored proteins; however, IGDs constitute a rare group of diseases, and correlations between the spectrum of symptoms and affected genes or the type of mutations have not been shown. Here, we report four newly identified and five previously described Japanese families with PIGO (phosphatidylinositol glycan anchor biosynthesis class O) deficiency. We show how the clinical severity of IGDs correlates with flow cytometric analysis of blood, functional analysis using a PIGO-deficient cell line, and the degree of hyperphosphatasia. The flow cytometric analysis and hyperphosphatasia are useful for IGD diagnosis, but the expression level of GPI-anchored proteins and the degree of hyperphosphatasia do not correlate, although functional studies do, with clinical severity. Compared with PIGA (phosphatidylinositol glycan anchor biosynthesis class A) deficiency, PIGO deficiency shows characteristic features, such as Hirschsprung disease, brachytelephalangy, and hyperphosphatasia. This report shows the precise spectrum of symptoms according to the severity of mutations and compares symptoms between different types of IGD.

Lesch-Nyhan disease (LND) is a rare X-linked recessive disorder caused by deficiency of the purine salvage enzyme hypoxanthine–guanine phosphoribosyltransferase (HPRT), encoded by the HPRT1. To date, nearly all types of mutations have been reported in the whole gene; however, duplication mutations are rare. We here report the case of a 9-month-old boy with LND. He showed developmental delay, athetosis, and dystonic posture from early infancy, but no self-injurious behaviors. Hyperuricemia was detected, and his HPRT enzyme activity in erythrocytes was completely deficient. A novel duplication mutation (c.372dupT, c.372_374 TTT > c.372_375 TTTT) was identified in exon 4 of the HPRT1, which causes aberrant splicing. This is the third case of a duplication mutation in the HPRT1 that causes splicing error.

Squamous cell carcinoma antigens 1 and 2 (SCCA1 and 2, SERPIN B3 and B4), members of the ovalbumin serpin (ov-serpin)/clade B serpin family, were originally discovered as tumor-specific antigens and are used as tumor markers for various kinds of squamous cell carcinomas. Recently, our understanding of the underlying mechanisms of how SCCA1/2 enhance tumor growth has greatly increased. Moreover, it has been shown that SCCA1/2 are involved in the pathogenesis of several inflammatory diseases: asthma, psoriasis, and atopic dermatitis (AD). IL-22 and IL-17, signature cytokines of type 17 inflammation, as well as IL-4 and IL-13, signature cytokines of type 2 inflammation, both of which are positively correlated with the pathogenesis of psoriasis and allergic diseases, respectively, can induce expression of SCCA1/2 in airway epithelial cells and/or keratinocytes, leading to high expression of SCCA1/2 in these diseases. Based on these findings, several trials have been performed to examine the potential of applying SCCA1/2 to biomarkers for these diseases. The findings show that SCCA2 is useful to aid diagnosis, estimate clinical severity and disease type, and assess responses to treatment in psoriasis and AD. These results suggest that SCCA2 has emerged as a novel biomarker for skin inflammatory diseases.

Upper-GI variceal bleeding is an important complication of portal hypertension. Endoscopic injection sclerotherapy (EIS) and endoscopic variceal ligation (EVL) are widely accepted as primary therapies for esophageal and gastric varices. Balloonoccluded retrograde transvenous obliteration (B-RTO) has been introduced recently as a new interventional modality to prevent fatal bleeding from gastric fundic varices. Compared with esophageal and gastric varices, duodenal varices are less common in portal hypertension. And, although bleeding is often severe and fatal, a definitive treatment for bleeding duodenal varices has not been established. A case of bleeding duodenal varices successfully controlled by EVL and B-RTO is presented, and the usefulness of these procedures in the management of this condition is discussed.

Xq28 duplication syndrome including MECP2 is a neurodevelopmental disorder characterized by axial hypotonia at infancy, severe intellectual disability, developmental delay, mild characteristic facial appearance, epilepsy, regression, and recurrent infections in males. We identified a Japanese family of Xq28 duplications, in which the patients presented with cerebellar ataxia, severe constipation, and small feet, in addition to the common clinical features. The 488-kb duplication spanned from L1CAM to EMD and contained 17 genes, two pseudo genes, and three microRNA-coding genes. FISH and nucleotide sequence analyses demonstrated that the duplication was tandem and in a forward orientation, and the duplication breakpoints were located in AluSc at the EMD side, with a 32-bp deletion, and LTR50 at the L1CAM side, with "tc" and "gc" microhomologies at the duplication breakpoints, respectively. The duplicated segment was completely segregated from the grandmother to the patients. These results suggest that the duplication was generated by fork-stalling and template-switching at the AluSc and LTR50 sites. This is the first report to determine the size and nucleotide sequences of the duplicated segments at Xq28 of three generations of a family and provides the genotype-phenotype correlation of the patients harboring the specific duplicated segment.

Mutations of two enzyme genes, HPRT1 encoding hypoxanthine guanine phosphoribosyltransferase (HPRT) and PRPS1 encoding a catalytic subunit (PRS-I) of phosphoribosylpyrophosphate synthetase, cause X-linked inborn errors of purine metabolism. Analyzing these two genes, we have identified three HPRT1 mutations in Lesch–Nyhan families following our last report. One of them, a new mutation involving the deletion of 4224 bp from intron 4 to intron 5 and the insertion of an unknown 28 bp, has been identified. This mutation resulted in an enzyme polypeptide with six amino acids deleted due to abnormal mRNA skipping exon 5. The other HPRT1 mutations, a single base deletion (548delT, 183fs189X), and a point mutation causing a splicing error (532+1G>A, 163fs165X) were detected first in Japanese patients but have been reported in European families. On the other hand, in the analysis of PRPS1, no mutation was identified in any patient.

PLEKHA5 (pleckstrin homology domain-containing protein family A, member 5) belongs to the PLEKHA family (PLEKHA1–6); however, the properties of this protein remain poorly characterized. We have identified and characterized two forms of PLEKHA5 mRNA. The long form of PLEKHA5 (L-PLEKHA5) contains 32 exons, encodes 1282 amino acids, and is specifically expressed in the brain; the short form of PLEKHA5 (S-PLEKHA5) is generated by alternative splicing of L-PLEKHA5, contains 26 exons, encodes 1116 amino acids, and is ubiquitously expressed. Both forms of the protein contain putative Trp–Trp (WW) and pleckstrin homology (PH) domains and are located mainly in the cytosol. Developmental and age-dependent expression studies in the mouse brain have shown that Plekha5 is the most abundantly expressed protein at E13.5 with S-Plekha5 dominancy. L-Plekha5 levels increased gradually with the decrease in total Plekha5 levels; moreover, L-Plekha5 became the dominant protein at E17.5, maintaining its dominance throughout adulthood. Protein-lipid overlay assays have indicated that the PH domain of PLEKHA5 specifically interacts with PI3P, PI4P, PI5P, and PI(3,5)P2. These results suggest that the S- to L-conversion of PLEKHA5 (Plekha5) may play an important role in brain development through association with specific phosphoinositides.

CHD6 is an ATP-dependent chromatin-remodeling enzyme, which has been implicated as a crucial component for maintaining and regulating chromatin structure. CHD6 belongs to the largest subfamily, subfamily III (CHD6-9), of the chromodomain helicase DNA (CHD-binding protein) family of enzymes (CHD1-9). Here we report on a female patient with a balanced translocation t(4;20)(q33;q12) presenting with severe mental retardation and brachydactyly of the toes. We identified the translocation breakpoint in intron 27 of CHD6 at 20q12, while the 4q33 breakpoint was intergenic. Northern blot analysis demonstrated the CHD6 mRNA in the patient's lymphoblastoid cells was decreased to ∼50% of the control cells. To investigate the cellular mechanism of diseases resulting from decreased CHD subfamily III proteins, we knocked down CHD6 or CHD7 by RNA interference in HeLa cells and analyzed chromosome alignment. The both CHD6- and CHD7-knockdown cells showed increased frequency of misaligned chromosomes on metaphase plates. Moreover, an elevated frequency of aneuploidy, the major cause of miscarriages and mental retardation, was observed in patients with CHD6 and CHD7 haploinsufficiency. These results suggest that CHD6 and CHD7 play important roles in chromatin assembly during mitosis and that mitotic delay and/or impaired cell proliferation may be associated with pathogenesis of the diseases caused by CHD6 or CHD7 mutations.

Inherited mutation of hypoxanthine guanine phosphoribosyltransferase (HPRT) gives rise to Lesch-Nyhan syndrome or HPRT-related gout. On the other hand, PRPS1 mutations cause PRPP synthetase superactivity associated with hyperuricemia and gout, sometimes including neurodevelopmental abnormalities. We have identified two mutations in two Lesch-Nyhan families after our last report. One of them, a new single nucleotide substitution (130GT) resulting in a missense mutation D44Y was detected in exon 2 of HPRT1. RT-PCR amplification showed not only a cDNA fragment with normal size, but also a small amount of shorter fragment skipping exons 2 and 3. The other missense mutation F74L (222CA) was detected in a Japanese patient but has been reported previously in European families. In four hyperuricemic patients with mild neurological abnormality, no mutations responsible for partial HPRT deficiency were identified in HPRT1. In these four patients, we also performed molecular analysis of PRPS1, but no mutations in PRPP synthetase were found.

A novel 13C nuclear magnetic resonance (NMR) method is described for the detection of subtle structural differences between mouse immunoglobulins carrying different allotypes. Fc fragments of mouse IgG1 antibodies carrying allotypes a and j have been selectively labeled with [1-13C]methionine. 13C-NMR spectra have shown that the microenvironment around Met-398 is significantly different for the two kinds of allotypes. Peptide mapping and amino acid sequence analyses have revealed that Val-406 of IgG1 carrying allotype a is substituted for Ile in the case of allotype j. X-ray crystallographic data indicate that Met-398 is in close spatial proximity to Val (Ile)-406. We therefore conclude that the 13C-NMR method can provide us with a novel spectroscopic probe for the structural characterization of allotypic markers.

Inherited mutation of hypoxanthine guanine phosphoribosyltransferase, (HPRT) gives rise to Lesch-Nyhan syndrome or HPRT-related gout. We have identified a number of HPRT mutations in patients manifesting different clinical phenotypes, by analyzing all nine exons of the HPRT gene (HPRT1) from genomic DNA and reverse transcribed mRNA using the PCR technique coupled with direct sequencing. Recently, we detected two novel mutations: a single nucleotide substitution (430C > T) resulting in a nonsense mutation Q144X, and a deletion of HPRT1 exon 1 expressing no mRNA of HPRT. Furthermore, we summarized the spectrum of 56 Japanese HPRT mutations.

Inherited mutations of a purine salvage enzyme, hypoxanthine guanine phosphoribosyltransferase (HPRT, EC 2.4.2.8), give rise to Lesch-Nyhan syndrome or HPRT-related gout. We have identified a number of HPRT mutations in Asian patients manifesting different clinical phenotypes, by analyzing all nine exons of the HPRT gene (HPRT1) from genomic DNA and reverse-transcribed mRNA using the PCR technique coupled with direct sequencing. In this study, we update the spectrum of mutations with nine novel mutations. Two missense mutations (T124P and D185G) were detected in patients with HRH (HPRT-related hyperuricemia). In a patient having a severe partial deficiency of HPRT with neurological dysfunction (HRND: HPRT-related neurological dysfunction), a single nucleotide substitution (27+5G > A) causing a splicing error was found in intron 1. The mutation resulted in a remarkably decreased level of normal mRNA, and production of an abnormal mRNA with a 49-bp insert at the 5'-end of intron 1, which caused the frame-shift of an amino acid codon (10fs27X). In six typical Lesch-Nyhan families, we found two 3-bp deletions responsible for single amino acid deletions (V8del and Y28del), two 1-bp deletions (440delA and 635delG) generating a frame-shift, an insertion of two amino acids (159insKV), and a 4,131-bp deletion from introns 4 to 6 resulting in two types of abnormal mRNA. Including these nine mutations, 42 HPRT1 mutations have been identified among 47 Asian families with deficiency of HPRT.

We have identified six novel mutations in Japanese families of HPRT deficiency manifesting different clinical phenotypes. A missense mutation (Y195C) was identified in a patient with hyperuricemia and gout (Case 1). In Cases 2 and 3 with mild neurological symptoms, missense mutations L147P and K159E were detected, respectively. A point mutation 532+2T>C causing splicing error was found in a severe form of partial HPRT deficiency (Case 4). Two mutations causing splicing error, 609+1delGT (Case 5) and 610-−1G>A (Case 6), resulted in the classic Lesch-Nyhan syndrome.

Hereditary lymphedema type I (Milroy disease) is a rare autosomal dominant disease resulting from mutations of FLT4 encoding the vascular endothelial growth factor receptor-3. Patients develop edema of the legs and feet, resulting in chronic swelling of the lower extremities from the neonatal period. Here we report a Japanese family with 10 affected members of five generations of hereditary lymphedema type I. We identified a previously reported missense mutation of G857R in one allele of FLT4 from three affected individuals of three generations, the mother of whom presented only hemi-lymphedema of the left foot. Thus, the clinical features of hereditary lymphedema type I caused by a FLT4 mutation are heterogeneous and it would be appropriate to consider FLT4 mutations even in a patient with hemi-lymphedema of the foot.

A 45 kDa protein, which is recognized by IgE antibodies in sera of food-allergic patients, was purified and characterized as an allergenic protein from the tomato. The IgE-binding protein purified from tomato extract was found to be a glycoprotein with a molecular weight of approximately 45,000, an isoelectric point of 4.2, and no free N-terminal amino group. Furthermore, it was shown that the purified protein had peroxidase activity. From the amino acid sequence of a peptide fragment prepared by lysylendopeptidase digestion, the allergenic protein was identified to be the tomato suberization-associated anionic peroxidase 1 known as one of the pathogenesis-related proteins widely distributed in plants. These properties suggested the protein isolated from tomato to be a new allergenic protein in plant foodstuffs.

OBJECTIVE—We investigated the effect of glimepiride, a third-generation sulfonylurea hypoglycemic agent, on insulin resistance in elderly patients with type 2 diabetes, in connection with plasma adiponectin and 8-epi-prostagrandin F2α (8-epi-PGF2α), an oxidative stress marker. RESEARCH DESIGN AND METHODS—A total of 17 elderly patients with type 2 diabetes received 12 weeks of treatment with glimepiride. Homeostasis assessment model of insulin resistance (HOMA-IR), homeostasis assessment model of β-cell function, HbA1c, C-peptide in 24-h pooled urine (urine CPR), and plasma concentrations of 8-epi-PGF2α, tumor necrosis factor-α (TNF-α), plasminogen activator inhibitor type 1, and adiponectin were measured at various times. The metabolic clearance rate of glucose (MCR-g) was also assessed by a hyperinsulinemic-euglycemic clamp. RESULTS—After 8 weeks of glimepiride treatment, significant reductions were observed in HbA1c (from 8.4 ± 1.9 to 6.9 ± 1.0%), HOMA-IR (from 2.54 ± 2.25 to 1.69 ± 0.95%), and plasma TNF-α concentrations (from 4.0 ± 2.0 to 2.6 ± 2.5 pg/ml). MCR-g was significantly increased from 3.92 ± 1.09 to 5.73 ± 1.47 mg · kg−1 · min−1. Plasma adiponectin increased from 6.61 ± 3.06 to 10.2 ± 7.14 μg/ml. In control subjects, who maintained conventional treatment, no significant changes were observed in any of these markers. CONCLUSIONS—Glimepiride remarkably improved insulin resistance, suggested by a significant reduction in HOMA-IR, an increase in MCR-g, and a reduction in HbA1c without changing extrapancreatic β-cell function and urine CPR. Increased plasma adiponectin and decreased plasma TNF-α may underlie the improvement of insulin resistance with glimepiride.

Helicobacter pylori infection is recognized to be a pathogen of various gastroduodenal disease. Eradication therapy of H. pylori reduces the recurrence of gastro-duodenal ulcer, improves histological gastritis, and is suggested to act a certain role in protection against gastric carcinogenesis. Although, several studies show uncomfortable results arise after H. pylori infection was cured. These studies suggest that gastro-esophageal reflux disease (GERD) and gastro-duodenal erosion may increase after successful eradication of H. pylori. Recently, adenocarcinoma of the gastric cardia and esophagus increase in incidence. Reflux esophagitis and Barrett's esophagus are recognized as precancerous lesion of esophageal adenocarcinoma. It is uncertain the association of newly occurrence of GERD after H. pylori eradication and increase of esophageal adenocarcinoma. GERD may cause adenocarcinoma development, though long term observations is necessary after H. pylori eradication.

In human, there are three fundamental tissue-specific isozymes of AMP deaminase (EC 3.5.4.6),1,2 and three different genes for the isozymes have been identified:3–6 AMPD1 encoding isozyme M found in skeletal muscle, AMPD2 encoding L in liver, and AMPD3 encoding erythrocyte AMP deaminase (El). The erythrocyte AMP deaminase deficiency that we initially identified,7,8 is clinically asymptomatic. Erythrocyte ATP contents in the complete deficiency are about 150% of normal,8 which can be explained by impairment of the degradation of AMP by way of AMP deaminase. The inheritance of the deficiency was autosomal recessive and the heterozygote frequency was estimated at about 1/30 in Japan, Korea and Taiwan.7,8 The deficiency was also reported in Europe,9 and the frequency in northern Poland was almost the same as that in east Asia. The nonsense mutation on AMPD1 responsible for human myoadenylate deaminase (muscle type AMP deaminase) deficiency was found in 12% of Caucasians and 19% of African-Americans, whereas none of the 106 Japanese subjects surveyed has the mutant allele.10 Human erythrocyte AMP deaminase deficiency in Japanese is associated with 75% of the same mutation (R573C) and 25% of other heterogeneous mutations on AMPD3.11,12 It is of interest to know whether the same mutation occurs in Poles, since their frequency of the enzyme deficiency was not significantly different from Japanese.

For the purpose of engineering the antibody combining site, mapping residues that are involved in antigen binding provide us with valuable information. By use of13C NMR spectroscopy with selectively13C-labeled Fv fragments, we have established a general strategy to identify the residues that are perturbed upon binding of small antigen (hapten) molecules [(1990) Biochemistry 30, 6604–6610]. In the present paper, we demonstrate that this strategy can be extended to molecular structural analyses of the complexes of an Fab fragment and a larger antigen molecule such asPseudomonas aeruginosa exotoxin A with a molecular mass of 67 kDa.

Hirschsprung disease (HSCR) is sometimes associated with a set of characteristics including mental retardation, microcephaly, and distinct facial features1,2,3, but the gene mutated in this condition has not yet been identified. Here we report that mutations in SIP1, encoding Smad interacting protein-1, cause disease in a series of cases. SIP1 is located in the deleted segment at 2q22 from a patient with a de novo t(2;13)(q22;q22) translocation. SIP1 seems to have crucial roles in normal embryonic neural and neural crest development.

Mouse monoclonal antibodies (MAbs) against Pseudomonas aeruginosa exotoxin A (Ex-A) were established, and 4 of 20 MAbs were extensively studied for analysis of the structure-function relationship of Ex-A. IN vivo experiments demonstrated that MAb Ex-3C7 protected mice either injected with Ex-A or infected with Ex-A-producing P. aeruginosa from death caused by Ex-A at the highest rate, followed by MAbs Ex-4F2 and Ex-8H5, in that order. MAb Ex-2A10 failed to rescue the mice. MAb Ex-3C7 (immunoglobulin G1 [IgG1]) inhibited incorporation of Ex-A into target cells and strongly neutralized cytotoxicity in cell culture but did not inhibit an enzymatic activity of Ex-A, ADP-ribosyltransferase, at all. The MAb also bound Ex-A, even at a low pH of 4, and recognized amino acid residues 241 to 297 (domain Ia/II), suggesting that MAb Ex-3C7 can interfere with the conformational change and/or processing of Ex-A by keeping a complex of Ex-A and antibody stable at low pH in the phagolysosome. MAb Ex-4F2 (IgG1), which recognizes residues 550 to 590 (domain III), strongly inhibited Ex-A incorporation and neutralized cytotoxicity in cell culture but only weakly inhibited ADP-ribosyltransferase. MAb Ex-8H5 (IgG1), which recognizes residues 591 to 613 (domain III), also inhibited cytotoxicity in cell culture, but weakly. In contrast to the above three MAbs, MAb Ex-2A10 (IgG2b) greatly inhibited ADP-ribosyltransferase but showed no inhibition of Ex-A incorporation and no neutralizing activity against cell toxicity. A line of evidence indicates that (i) domain Ia/II plays an important role in the pathogenesis of Ex-A and (ii) MAbs that inhibit an intracellular postbinding process, such as conformational change, processing, and translocation of Ex-A in target cells, can display potent inhibitory activity against cytotoxicity in vivo, as well as in cell culture, and would be a good candidate for therapy of pseudomonal infections.

Summary Human monoclonal antibody HI-1A4 (IgG3, λ) neutralized a toxicity caused by pseudomonal exotoxin A (Ex-A) in cell culture and in vivo, and was effective in experimental Pseudomonas aeruginosa infections in mice. HI-1A4 inhibited an Ex-A catalyzed ADPribosylation of elongation factor 2 but did not inhibit an incorporation of toxin into a target cell at all. One molecule of HI-1A4 neutralized at least 2 molecules of Ex-A. HI-1A4 retained its binding activity at pH 4.0. The epitope region for HI-1A4 was demonstrated to be a carboxyl terminal end of amino acid residues 591–613 of Ex-A. HI-1A4 might bind to Ex-A carboxyl terminal region outside a target cell, be incorporated into cells as a complex with Ex-A, and inhibit the intracellular function in which the carboxyl terminal part of Ex-A was involved, resulting in the interruption of intoxication of Ex-A.

We have cloned immunoglobulin (Ig) genes from an Epstein-Barr (EB) virus-transformed human B cell line, FK-001, which produced a monoclonal human IgM that specifically binds Pseudomonas aeruginosa exotoxin A (Ex-A). Cloned Ig genomic DNA was introduced into Ig non-producing mouse myeloma cells, P3-X63-Ag-8-6.5.3. (653) and Sp2/0-Ag14 (Sp2/0), by protoplast fusion or electroporation using a series of pSV2 vectors. Transfectant-derived recombinant antibodies (r-Abs) and antibodies produced by the original cell FK-001 did not differ in binding specificity or affinity to Ex-A. Recombinant antibodies and those of FK-001 were pentameric although joining (J) genes of human origin were not introduced into the mouse myeloma cells. Heterogeneity in the molecular weights of r-Abs expressed in different mouse myeloma cells was observed, and shown to be due to differential N-glycosylation of the μ-chain polypeptides. Thus, human Ig μ and κ genomic DNA with its own promoters and enhancers can function efficiently in mouse myeloma cells in vivo resulting in transfectants that synthesize active human IgM.

Double-stranded DNA complementary to the partially purified mRNA prepared from 3-methylcholanthrene (MC)-treated rat liver was constructed and cloned in Escherichia coli. Twenty clones were verified to carry a complementary DNA (cDNA) insert coding for MC-inducible cytochrome P-450 by positive hybridization translation assay and immunochemical assay with anti-cytochrome P-450 antibody. The identified cDNA clones were divided into at least two groups on the basis of comparison of restriction maps of the cDNA inserts. A clone pAU157 whose cDNA insert was approximately 2.7 kb in length contained nearly full-length mRNA information for cytochrome P-450MC or P-450c, which is the major form of MC-inducible cytochrome P-450. Other cDNA clones pTZ286-pTZ330 contained the 1.2 kb sequence complementary to cytochrome P-450d mRNA. RNA blot analysis revealed that pAU157 and pTZ286-pTZ330 cDNA clones were derived from 22S and 18S mRNAs, respectively, both of which were induced in rat liver by MC treatment. Sequence analysis revealed that there were closely homologous sequence regions in pAU157 and pTZ286-pTZ330 cDNA inserts and most of the homologous sequences were localized in two limited coding regions of the two cytochrome P-450 species. pAU157 encoded the total amino acid sequence of cytochrome P-450MC or P-450c and pTZ286-pTZ330 coded for the C-terminal 368 amino acid residues of cytochrome P-450d. Two highly homologous regions were found in the amino acid sequences of these cytochrome P-450 species.

The acid catalyzed ring opening of trans-stilbene oxide was investigated in DMSO, DMF, THF, and dioxane and the products, meso and dl-hydrobenzoins and diphenylacetaldehyde, were determined. In the solvents of high polarity, inversion of configuration was observed in most products, and the opposite was true in the solvents of low polarity. There was no direct relation between rearrangement products and polarity of solvents.