Iodoarene-catalyzed oxidative transformations using molecular oxygen.

Molecular oxygen serves as a useful oxidant for the glycol scission of 1,2-diols and the Hofmann rearrangement of primary amides using pentamethyliodobenzene as a catalyst. The use of isobutyraldehyde and Lewis basic nitriles under O2 enabled the iodine(i)/(iii) catalytic cycle, where in situ-generated peracid acts as a terminal oxidant.

BTK gene targeting by homologous recombination using a helper-dependent adenovirus/adeno-associated virus hybrid vector.

X-linked agammaglobulinemia (XLA) is one of the most common humoral immunodeficiencies, which is caused by mutations in Bruton's tyrosine kinase (BTK) gene. To examine the possibility of using gene therapy for XLA, we constructed a helper-dependent adenovirus/adeno-associated virus BTK targeting vector (HD-Ad.AAV BTK vector) composed of a genomic sequence containing BTK exons 6-19 and a green fluorescence protein-hygromycin cassette driven by a cytomegalovirus promoter. We first used NALM-6, a human male pre-B acute lymphoblastic leukemia cell line, as a recipient to measure the efficiency of gene targeting by homologous recombination. We identified 10 clones with the homologous recombination of the BTK gene among 107 hygromycin-resistant stable clones isolated from two independent experiments. We next used cord blood CD34⁺ cells as the recipient cells for the gene targeting. We isolated colonies grown in medium containing cytokines and hygromycin. We found that the targeting of the BTK gene occurred in four of the 755 hygromycin-resistant colonies. Importantly, the gene targeting was also observed in CD19⁺ lymphoid progenitor cells that were differentiated from the homologous recombinant CD34⁺ cells during growth in selection media. Our study shows the potential for the BTK gene therapy using the HD-Ad.AAV BTK vector via homologous recombination in hematopoietic stem cells.

Ghrelin enhancer, rikkunshito, improves postprandial gastric motor dysfunction in an experimental stress model.

BACKGROUND: Functional dyspepsia (FD) is one of the most common disorders of gastrointestinal (GI) diseases. However, no curable treatment is available for FD because the detailed mechanism of GI dysfunction in stressed conditions remains unclear. We aimed to clarify the association between endogenous acylated ghrelin signaling and gastric motor dysfunction and explore the possibility of a drug with ghrelin signal-enhancing action for FD treatment. METHODS: Solid gastric emptying (GE) and plasma acylated ghrelin levels were evaluated in an urocortin1 (UCN1) -induced stress model. To clarify the role of acylated ghrelin on GI dysfunction in the model, exogenous acylated ghrelin, an endogenous ghrelin enhancer, rikkunshito, or an α2 -adrenergic receptor (AR) antagonist was administered. Postprandial motor function was investigated using a strain gauge force transducer in a free-moving condition. KEY RESULTS: Exogenous acylated ghrelin supplementation restored UCN1-induced delayed GE. Alpha2 -AR antagonist and rikkunshito inhibited the reduction in plasma acylated ghrelin and GE in the stress model. The action of rikkunshito on delayed GE was blocked by co-administration of the ghrelin receptor antagonist. UCN1 decreased the amplitude of contraction in the antrum while increasing it in the duodenum. The motility index of the antrum but not the duodenum was significantly reduced by UCN1 treatment, which was improved by acylated ghrelin or rikkunshito. CONCLUSIONS & INFERENCES: The UCN1-induced gastric motility dysfunction was mediated by abnormal acylated ghrelin dynamics. Supplementation of exogenous acylated ghrelin or enhancement of endogenous acylated ghrelin secretion by rikkunshito may be effective in treating functional GI disorders.

Paediatric presentation and outcome of congenital protein C deficiency in Japan.

Severe heritable protein C (PC) deficiency is quite rare, although heterozygous PROC mutation is the second leading cause of genetic predisposition to thrombosis in Japanese adults. The aim of the study was to search the optimal management, the paediatric onset and outcomes of PC deficiency were characterized in Japan. The genetic study, postmarketing survey of activated PC(aPC) concentrate (Anact(®)C) and intensive review in Japan for 20 years enabled the analysis of the disease onset, genotype, treatment and prognosis. Symptomatic PC deficiency was determined in 27 Japanese children. All but two patients presented within 16 days after birth (three prenatal and six neonatal onsets). Postnatal-onset cases had normal growth at full-term delivery. Of the 27 patients, 19 suffered intracranial thrombosis or haemorrhage (ICTH) (three foetal hydrocephalies), 16 developed purpura fulminans (PF) and 10 had both at the first presentation. ICTH preceded PF in both affected cases. Low PC activities of 18 mothers and/or 12 fathers indicated 20 inherited PC deficiencies (2 homozygotes, 11 compound heterozygotes and 7 heterozygotes) and seven unidentified causes of PC deficiency. Nine of 11 patients studied had PROC mutations. Four unrelated patients (50%) carried PC nagoya (1362delG). No PC-deficient parents had experienced thromboembolism. Of the 18 patients with aPC therapy, two died and eight evaluable survivors had neurological sequelae. This first comprehensive study of paediatric PC deficiency suggested that perinatal ICTH was the major presentation, occurring earlier than neonatal PF. PC nagoya was prevalent in paediatric, but not adult, patients in Japan. Early maternal screening and optimal PC therapy are required for newborns at risk of PC deficiency.

Neonatal asphyxia and renal failure as the presentation of non-inherited protein C deficiency.

Inherited or acquired protein C (PC) deficiency leads to thromboembolic events. Plasma PC activity in infancy is physiologically lower than in adults. We describe a case of neonatal asphyxia and acute renal failure associated with isolated PC deficiency. A full-term male infant was born to a healthy mother by caesarean section because of fetal distress. The small-for-gestational age infant showed 2 and 7 of Apgar scores at 1 and 5 minutes, respectively. Hypercoagulability required repeated infusions of fresh frozen plasma. Coagulation study revealed PC activity, 6%, protein S activity, 61%, and high D-dimer levels, along with normal factor VII activity and absent vitamin K deficiency. Anticoagulant and activated PC therapy improved coagulopathy and nephropathy. Imaging analyses indicated no visceral infarctions. Renal function and PC activity have been slowly normalized until 6 months of age. He had no PROC mutation or PC-deficient parents. Selective PC deficiency may occur as an acquired cause of hypercoagulable crisis in the stressed newborn.

Long interspersed element-1 is differentially regulated by food-borne carcinogens via the aryl hydrocarbon receptor.

A single human cell contains more than 5.0 × 10(5) copies of long interspersed element-1 (L1), 80-100 of which are competent for retrotransposition (L1-RTP). Recent observations have revealed the presence of de novo L1 insertions in various tumors, but little is known about its mechanism. Here, we found that 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx), food-borne carcinogens that are present in broiled meats, induced L1-RTP. This induction was dependent on a cellular cascade comprising the aryl hydrocarbon receptor (AhR), a mitogen-activated protein kinase, and CCAAT/enhancer-binding protein ß. Notably, these compounds exhibited differential induction of L1-RTP. MeIQx-induced L1-RTP was dependent on AhR nuclear translocator 1 (ARNT1), a counterpart of AhR required for gene expression in response to environmental pollutants. By contrast, PhIP-induced L1-RTP did not require ARNT1 but was dependent on estrogen receptor α (ERα) and AhR repressor. In vivo studies using transgenic mice harboring the human L1 gene indicated that PhIP-induced L1-RTP was reproducibly detected in the mammary gland, which is a target organ of PhIP-induced carcinoma. Moreover, picomolar levels of each compound induced L1-RTP, which is comparable to the PhIP concentration detected in human breast milk. Data suggest that somatic cells possess machineries that induce L1-RTP in response to the carcinogenic compounds. Together with data showing that micromolar levels of heterocyclic amines (HCAs) were non-genotoxic, our observations indicate that L1-RTP by environmental compounds is a novel type of genomic instability, further suggesting that analysis of L1-RTP by HCAs is a novel approach to clarification of modes of carcinogenesis.

Congenital multiple pituitary hormone deficiency associated with hyperammonemia: a case report with a short review of the literature.

We herein report a case study of a female newborn with multiple pituitary hormone deficiencies who presented with generalized seizures, hypoglycemia and hyperammonemia at 18 h after birth. In addition, we review the association of hyperammonemia in neonates with multiple pituitary hormone deficiencies reported in the previous literature. This unrecognized association should be taken into account for the early diagnosis and treatment of these patients.

Genetic variation of vascular endothelial growth factor pathway does not correlate with the severity of retinopathy of prematurity.

OBJECTIVE: The aim of this study was to assess the genetic effects of the vascular endothelial growth factor (VEGF) pathway on retinopathy of prematurity (ROP). STUDY DESIGN: A prospective study from a tertiary center that enrolled 204 Japanese infants (<35 weeks of gestational age (GA)) having no anomalies. ROP developed in 127, but not in 77 infants. The relative severity was defined as non-severe, moderate and severe ROP for GA, based on the staging criteria. VEGF (g.-634G>C, g.+13553C>T) and VEGF-receptor (KDR g.+4422(AC)11 to 14, Flt-1 c.+6724(TG)13 to 23) gene polymorphisms and clinical variables were assessed by uni/multivariate analyses. RESULT: The frequency of polymorphisms did not differ between ROP and non-ROP patients. The TT genotype of g.+13553 showed a higher odds ratio for non-severe ROP than CC genotype (P=0.006). Multivariate analyses indicated that low birth weight, blood transfusion and respiratory distress syndrome, but not polymorphisms, were the risk factors of advanced ROP (≥ stage 3). CONCLUSION: A genotype of the VEGF pathway weakly affects the severity of ROP compared with other clinical factors.

Development of LC-MS method for detection of mutant uromodulin protein.

Mutations in the uromodulin gene cause the autosomal disorders familial juvenile hyperuricemic nephropathy (FJHN) and medullary cystic kidney disease type 2 (MCKD2). However, methods to detect the mutant form of the uromodulin protein have not been developed. In this study, we developed a liquid chromatography-mass spectrometry (LC-MS) method for detection of the mutated uromodulin peptide (C148W). Our method can distinguish the mutant peptide, GWHWE, from wildtype peptide, GWHC*E. Using MS/MS analysis with a selected reaction monitoring (SRM) mode, peptide-specific fragment ions (m/z 714 --> 381, 471, 567, and 679 for GWHWE and m/z 688 --> 381, 445, 541, and 653 for GWHC*E) were detected.

Development of improved SNAP25 endopeptidase immuno-assays for botulinum type A and E toxins.

Botulinum neurotoxins contain proteases that cleave specific intra-neural proteins essential for neurotransmitter release. Toxin types A, E and C1 intra-cellularly cleave SNAP25 resulting in a flaccid paralysis. As a consequence, various different endopeptidase assays have been developed to specifically detect the toxins enzymatic activity, however, many of these suffer from variability, low sensitivity or unwanted interference exerted by product specific excipients. The current studies utilised solid phase synthesized SNAP25(137-206) peptide substrate, and specific antibody to either the SNAP25(190-197) or (173-180) octapeptide epitopes that become exposed following cleavage by toxin types A or E respectively. Assay sensitivity was increased 50 fold by the use of an optimal 0.5% Tween 20 concentration in tandem to 0.1% albumin together with an improved, simplified assay design without a pre-activation / reduction step. Sensitivities capable of detecting 0.01 LD50/ml (40fg/ml or 0.3fM) of type A toxin was achieved with a linear dose response between 0.1 and 1 LD50/ml. This provides sufficient sensitivity and precision (inter assay GCV of < 2%) for monitoring activity within any current or newly marketed therapeutic products containing less units per vial and may also make it applicable for other applications. Both purified haemagglutinin free and complexed toxins could be detected equally. Unlike type A, type E activity could unexpectedly be detected in the complete absence of reducing conditions and the optimal assay had a limit of detection of 0.2LD50/ml (4.8pg/ml) with a linear dose response between 1 and 10LD50/ml. The principle of using a detecting antibody to a substrate sequence buried within the native substrates alpha-helix may be further expanded to other specific enzyme cleavage reactions in the future.

A novel "permanent" acid-type hair color made possible with dye-metal ion complex technology.

The advantages and disadvantages of oxidative permanent and acid-type semi-permanent hair colors are evident. The former provides a longlasting "permanent" color, while the latter imparts less damage to the hair. We developed a novel acid-type hair color technology that can allow an acid dye and a metal ion to form a complex inside the hair similar to the oxidative hair color. It is well known that acid dye diffuses into the hair and creates an ionic bond with the positively charged amino acid residues of hair protein. However, the dye can be extracted easily from the hair by daily shampooing due to the weakness of the bond. In order to strengthen this bond and to prevent the extraction of the dye by shampooing, an aluminum chloride ion was chosen as the metal ion component to form the dye-metal complex. A proper composition of penetration enhancers, benzyl alcohol and ethyl alcohol, was required to allow acid dyes to interact with the aluminum chloride ion after each component penetrates deeply into the hair to form a complex inside the hair. To provide color brightness and a color longevity effect to hair color, glycolic acid was also selected due to the observation that a weak acid with a small molecular weight would enhance those effects.

Quality control of diphtheria tetanus acellular pertussis combined (DTaP) vaccines in Japan.

Diphtheria tetanus acellular pertussis combined (DTaP) vaccines have been successfully used in Japan by controlling their potencies and toxicities with animal models. In accordance with the recent practical introduction of DTaP vaccines of various formulations, a question has been raised in other nations as to the efficacy of a quality control system based on animal tests and standard preparations. The World Health Organization issued its guidelines on the production and quality control of acellular pertussis vaccines in 1998 along with the concept of quality control by ensuring that production lots were consistent with clinical trial lots, rather than by comparing them with standard preparations in traditional laboratory tests. However, because it is not feasible to evaluate the combined use of vaccines from different manufacturers in a clinical study, the alternative trend of quality control may give rise to a difficulty in rationalizing the practical immunizations to use vaccines of different brands in a mixed consequence. A standardized national regulation system to ensure the equivalence of approved products would be essential for such an immunization practice. The success of the Japanese DTaP vaccination suggests the possibility of an effective quality control of DTaP vaccines by means of standardized test systems.

Psoas abscess and cellulitis of the right gluteal region resulting from carcinoma of the cecum.

Although retroperitoneal or psoas abscess is an unusual clinical problem, the insidious and occult characteristics of this abscess sometimes cause diagnostic delays, resulting in considerably high morbidity and mortality. In particular, psoas abscess caused by perforated colon carcinoma is uncommon. We report a case of psoas abscess caused by a carcinoma of the cecum. A 72-year-old Japanese woman was admitted to our hospital, with pain in the right groin and buttock. The pain had appeared 6 months before admission, and the symptoms had then been relieved by oral antibiotics. On March 25, 1999, inflammatory signs in the right buttock indicated localized cellulitis, and incision and drainage was performed at a local hospital. The patient was referred to our hospital on the same day. On admission to our hospital, computed tomography (CT) scan revealed a thick right-sided colonic wall and enlargement of the right ileopsoas muscle. Barium enema and colonofiberscopy revealed an ulcerated tumor occupying the entire circumference of the cecum. A retroperitoneal abscess and fistula had been formed by the retroperitoneal perforation of cecum carcinoma: surgical resection was performed after remission of the local inflammatory signs. Operative findings indicated that the cancerous lesion and its surrounding tissues were firmly attached to the right iliopsoas and major psoas muscle, and en-bloc resection, including adjacent muscular tissue, was performed. The fact that carcinoma of the colon could be a cause of psoas abscess and cellulitis in the gluteal region should be considered when an unexplained psoas abscess is diagnosed.

A quantitative in vitro assay for detecting biological activity of endotoxin using rabbit peripheral blood.

The pyrogen test or the endotoxin test has been playing a crucial role in detecting endotoxin in parenteral drugs. The current test methods, however, have disadvantages such as requiring a relatively high number of animals or an inadequacy in direct evaluation of in vivo activity. We made an attempt to establish a new in vitro assay method that can overcome the shortcomings of the current assay methods. We standardized the system of tumor necrosis factor-alpha (TNF-alpha) induction from the peripheral blood of rabbits for assaying endotoxin activity. This in vitro assay showed a linear dose-response regression between 0.1 and 5.0 endotoxin units per milliliter of endotoxin and a definite homogeneity of variance by logarithmically transforming the endotoxin and TNF-alpha concentrations in the reaction mixtures at 5 h of incubation at 37 degrees C. The assay showed a definite correlation with the pyrogen test but not with the endotoxin test when endotoxins from various bacteria were tested.

Limitations of successive transradial approach in the same arm: the Japanese experience.

The transradial approach (TRA) has been used for diagnostic and interventional cardiology. It has not previously been determined how many times the same radial artery can be cannulated without complications. A total of 812 patients (502 men and 310 women) underwent angiography or angioplasty via the TRA between 1997 and 1999 at our institution with a total of 1,438 procedures. Sheaths were 5 (55%) or 6 Fr (45%). Dropout rates of 3.5% and 7.9% were found at the second TRA attempt in the men and the women, respectively. Of the 62 TRA failures, 56 (90%) were due to narrowing or occlusion of the radial artery after the previous TRA procedure. A third TRA procedure was possible in 90% of the men and 80% of the women. A fifth TRA procedure was possible in 70% of the men and 50% of the women. The dropout rates for TRA increased as successive punctures were performed. This was primarily due to vessel narrowing and occlusion occurring as a function of multiple punctures.

Increased leukocyte activity as a predictor for flow-limiting coronary lesions in patients with angina pectoris.

Myocardial ischemia initiates an acute inflammatory response, in which polymorphonuclear leukocytes (PMNs) are major participants. We investigated whether increased PMN activity in the peripheral blood is a marker for high-grade coronary artery stenosis in patients with angina pectoris. PMN activity was examined in 45 patients by measuring the luminol-dependent chemiluminescence (CL) response of the peripheral blood upon addition of phorbol myristate acetate (PMA). The CL of patients with angiographic filling delay was 1.6-fold higher than in patients without delay (480+/-54 vs. 302+/-33 counts/PMN, P<0.01). The CL of patients with a filling delay of the left anterior descending artery (LAD) was 1.7-fold higher than in patients without an LAD filling delay (537+/-79 vs. 317+/-27 counts/PMN, P<0.01). With the CL cut-off at 400 counts/PMN, the sensitivity and specificity for detecting an angiographic filling delay of the LAD were 69 and 75%, respectively. These data indicate that PMN activity in the peripheral blood is increased with the presence of a flow-limiting coronary lesion showing an angiographic filling delay. Measuring PMN activity may be a useful approach to assess indications for and timing of angiography and/or adjunctive invasive therapies in patients with angina pectoris.

Thermal and photochemical solvolysis of (E)- and (Z)-2-phenyl-1-propenyl(phenyl)iodonium tetrafluoroborate: benzenium and primary vinylic cation intermediates.

The thermal and photochemical solvolysis of the two stereoisomeric 2-phenyl-1-propenyl(phenyl)iodonium tetrafluoroborates has been investigated in alcoholic solvents of varying nucleophilicity. The product profiles and rates of product formation in the thermal reaction are all compatible with a mechanism involving cleavage of the vinylic C-I bond assisted by the group in the trans position (methyl or phenyl), always leading to rearranged products. Depending on the nucleophilicity of the solvent, the primarily formed cations may or may not further rearrange to more stable isomers. The less reactive Z compound also yields some unrearranged vinyl ether product in the more nucleophilic solvents via an in-plane S(N)2 mechanism. The mechanism of the photolysis involves direct, unassisted cleavage of the vinylic, and aromatic, C-I bond in an S(N)1 mechanism. This produces a primary vinyl cation, which is partially trapped prior to rearrangement in methanol. The unrearranged vinyl ethers are mainly formed with retention of configuration via a lambda3-iodonium/solvent complex in an S(N)i mechanism. Thermal and photochemical solvolyses of iodonium salts are complementary techniques for the generation of different cation intermediates from the same substrate.

High susceptibility of Scid mice to colon carcinogenesis induced by azoxymethane indicates a possible caretaker role for DNA-dependent protein kinase.

Severe combined immunodeficiency (Scid) mice have defects in V(D)J recombination and DNA double-strand breaks repair caused by an inherited genetic defect in the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Scid mice are highly susceptible to development of T-cell lymphomas, and because of the nature of its association with DNA repair and recombination, DNA-PKcs is considered to belong to the caretaker class of tumor suppressor genes. In the present study, the susceptibility of Scid mice to colon carcinogenesis due to administration of azoxymethane (AOM) was investigated. Significantly higher susceptibility in terms of induction of both aberrant crypt foci (ACFs), putative pre-cancerous lesions of the colon and colon cancers was observed as compared with the isogenic strain, C.B-17 mice. The incidences of colon tumors, either adenomas or adenocarcinomas, in Scid and C.B-17 mice after administration of AOM (10 mg/kg body weight/week) for 6 weeks were 87% (26 of 30) and 50% (15 of 30), respectively, by experimental week 22 (P < 0.01). The multiplicity of colon tumors in Scid mice was also significantly higher than in C.B-17 mice, being 2.2 +/- 1.5 and 0.9 +/- 1.2, respectively (P < 0.001). The present study clearly demonstrated high susceptibility of Scid mice to colon carcinogenesis, which might be attributable to disruption of the caretaker role of DNA-PK in colonic epithelial cells.