A dramatic enhancing effect of InBr3 towards the oxidative Sonogashira cross-coupling reaction of 2-ethynylanilines.

The addition of InBr3 to the oxidative Sonogashira cross-coupling reaction of 2-ethynylaniline with (E)-trimethyl(3,3,3-trifluoroprop-1-enyl)silane led to a dramatic increase in the reactivity to afford the corresponding 1,3-enynes bearing a trifluoromethyl group on their terminal sp(2) carbon. The subsequent cyclization of these 1,3-enynes under palladium catalysis provides access to the corresponding indoles bearing a 3,3,3-trifluoroprop-1-enyl group at their 2-position.

Maternal IgG avidity, IgM and ultrasound abnormalities: combined method to detect congenital cytomegalovirus infection with sequelae.

OBJECTIVE: We used maternal immunoglobulin M (IgM), immunoglobulin G (IgG) avidity index (AI) and fetal ultrasonography (US) to effectively detect a congenital cytomegalovirus-infected fetus that would suffer neurological sequelae after birth. STUDY DESIGN: The detecting method was prospectively adapted to 1163 unselected pregnant women. IgM, IgG and IgG-AI were measured at the first prenatal examination (10.8±2.2 weeks of gestation). Advanced US was performed for the IgM-positive women at our center. The urine of 1163 neonates was examined via PCR. All infected neonates were followed for neurological development. RESULT: Most women (83.3%) were seropositive. Among them, 40 (4.1%) were IgM positive. Nine of forty (22.5%) had low AI, of which one showed abnormal US and suffered severe sequelae. The remaining eight had a normal US; however, one infant had hearing impairment. There were another three infected infants with normal development. Their mothers' serological results were: IgM positive with high AI (n=1); IgG positive; IgM negative with high AI (n=1); and both IgG and IgM negative (n=1). CONCLUSION: This method enabled us to detect infected fetuses having severe sequelae. However, the problem remains of detecting infected fetuses that only have a hearing impairment.

A new high-throughput analysis for drug metabolism profiling using liquid chromatography coupled with tandem mass spectrometry.

We developed 3 liquid chromatography (LC) methods coupled to tandem mass spectrometry for comprehensive high-throughput profiling of drug metabolism, employing different columns with gradient systems of aqueous 10 mmol/L ammonium acetate and acetonitrile. The methods were established using testosterone (TST), imipramine (IMP), acetaminophen (APAP) and salbutamol (SBM), which have markedly different values of partition coefficient, P. Method I, using an octadecylized silica (ODS) column (ACQUITY UPLC BEH C18) with a steep gradient, was suitable for analyzing TST and its metabolites, and IMP and its metabolites. These substrates could not be analyzed by Method II, using another ODS column (Atlantis dC18), or Method III, using an Atlantis HILIC silica column. APAP and its metabolites could be analyzed simultaneously by Method II, but not Method I or III. SBM and its metabolites could be detected simultaneously only by Method III. The developed analytical methods were further evaluated with a panel of 18 additional drugs. Based on the retention times and peak shapes, we propose general criteria, in terms of calculated LogP (cLogP) value, for method selection. Broadly speaking, drugs with high cLogP, those with intermediate cLogP and those with low cLogP were best analyzed by Methods I, II and III, respectively, although drugs with borderline values of cLogP could be evaluated by both the relevant methods. The injection cycle for each method was within 10 min, including up to 3 min conditioning time. These methods are expected be useful in high-throughput screening assays to examine biotransformations of candidate drugs.

Characterization of recombinant prolyl aminopeptidase from Aspergillus oryzae.

AIMS: Prolyl aminopeptidase (PAP) degrades only amino-terminal proline from peptides. The food-grade fungus Aspergillus oryzae produces this enzyme only in small amounts. In this paper, we present efficient production of recombinant PAP with an overexpression system of A. oryzae and characterization of its biochemical properties. METHODS AND RESULTS: The gene encoding PAP was overexpressed as a His-tag fusion protein under a taka-amylase gene (amyB) promoter with a limited expressing condition in A. oryzae. The PAP activity in the mycelia grown in rich medium containing glucose (repressing condition) was twice that in starch (inducing condition). The enzyme prepared as cell-free extract was partially purified through two-step column chromatography. The PAP was estimated to be a hexameric protein and exhibited salt tolerance against NaCl of up to 4 mol l(-1). CONCLUSIONS: Aspergillus oryzae PAP was produced under the repressing condition of amyB promoter in a PAP-overexpressing strain and purified 1800-folds. Overproduction of PAP under promoter-inducing conditions led to an increase in inactive PAP, possibly because of irregular folding. SIGNIFICANCE AND IMPACT OF THE STUDY: PAP with a high specific activity and salt tolerance may be used effectively in the manufacturing processes of fermented foods.

Efficient production and partial characterization of aspartyl aminopeptidase from Aspergillus oryzae.

AIMS: Aspartyl aminopeptidase (DAP) has a high degree of substrate specificity, degrading only amino-terminal acidic amino acids from peptides. Therefore, attention is focused here on the efficient production of this enzyme by a recombinant Aspergillus oryzae and characterization of its biochemical properties. METHODS AND RESULTS: The gene encoding DAP was overexpressed under a taka-amylase gene promoter, with His-tag linker in A. oryzae, during cultivation in a Co(2+)-containing medium. The enzyme was extracted from the mycelia and purified with immobilized nickel ion absorption chromatography using a buffer containing cobalt ion and imidazole. The active fraction was further purified with gel filtration chromatography. The resultant, electrophoretically pure enzyme displayed a molecular mass of 520 kDa. This enzyme displayed high reactivity towards peptide substrate rather than synthetic substrates. CONCLUSIONS: Recombinant A. oryzae DAP was purified to homogeneity with an increased specific activity, when cultivated in a Co(2+)-rich medium. Moreover, the use of suitable metal ions in microbial cultivation and purification processes may help in increasing the specific activity of other metalloproteases and their functional analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Recombinant DAP produced using a cobalt ion in culture media of A. oryzae and purification process allow high yield of the enzyme activity.

Development of ET, primary myelofibrosis and PV in mice expressing JAK2 V617F.

An acquired JAK2 V617F mutation is found in most patients with polycythemia vera (PV), and about half of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF). Mice transplanted with bone marrow cells in which JAK2 V617F was retrovirally expressed developed PV-like features, but not ET or PMF. To address the contribution of this mutation to the pathogenesis of these three MPDs, we generated two lines of JAK2 V617F transgenic mice. One line showed granulocytosis after 4 months of age. Among 43 mice, 8 (19%) showed polycythemia and 15 (35%) showed thrombocythemia. The second line showed extreme leukocytosis and thromobocytosis. They showed anemia that means Hb value from 9 to 10 g per 100 ml when 1 month old. Myeloid cells and megakaryocytes were predominant in the bone marrow of these animals, and splenomegaly was observed. The expression of JAK2 V617F mRNA in bone marrow cells was 0.45 and 1.35 that of endogenous wild-type JAK2 in the two lines, respectively. In vitro analysis of bone marrow cells from both lines showed constitutive activation of ERK1/2, STAT5 and AKT, and augmentation of their phosphorylations by cytokine stimulation. We conclude that in vivo expression of JAK2 V617F results in ET-, PMF- and PV-like disease.

Effects of ethyl-esterization, chain-lengths, unsaturation degrees, and hyperthermia on carcinostatic effect of omega-hydroxylated fatty acids.

AIM: To evaluate promotive effect of hyperthermia on the carcinostatic activity of synthesized omega-hydroxy fatty acids (omega HFAs) and their ethylesters agaist Ehrlich ascites tumor (EAT) cells. METHODS: EAT cells were cultured with either omegaHFAs or their ethylester derivatives in a water bath at either 37 degrees C or 42 degrees C for 30 min, followed by incubation in a CO2 incubator for 20 or 72 h. Mitochond-rial dehydrogenase-based WST-1 assay and trypan blue dye exclusion assay were then conducted after incubation. Morphological changes were observed by scanning electron microscopy (SEM). RESULTS: Omega-HFA having a saturated 16-carbon straight-chain (omega H16:0) was the most carcinostatic (at 37 degrees C - viability level: 60.0%; at 42 degrees C - 49.6% (WST-1)) among saturated and unsaturated omegaHFAs with 12, 15 or 16 carbon atoms, when administrated to EAT cells at 100 microM for 20 h. Carcinostatic activity was markedly enhanced by ethyl-esterization of saturated fatty acids, such as omegaH16:0 (at 37 degrees C - 42.3%; at 42 degrees C - 11.2%, ibid) and omegaH15:0 (at 37 degrees C - 74.6%; at 42 degrees C - 25.3%, ibid), and their unsaturated counterparts were extremely effective only in combination with hyperthermia. Prolongation of the incubation period to 72 h at the same concentration increased appreciably their carcinostatic effect (omega H16:0 ethylesther: 1.3%; omegaH15:0 ethylesther: 8.0%). These values were also supported by dye exclusion assay. The carcinostatic activity enhanced more markedly by hyperthermia (1.2%; 2.1%, ibid). SEM shows that omegaH16:0 ethylester-exposed EAT cells underwent extensive injury, such as deformation of cell structure or disappearance of microvilli. CONCLUSIONS: omega H16:0 ethylester possesses high carcinostatic activity in vitro in combination with hyperthermia and may be utilized as potent anticancer therapeutic agent.

Quantification of mentolabial profile using lateral roentgen cephalography and profile reconstruction by mandibular osteotomy in healthy adults and patients with scar contracture following severe facial burns.

Here mentolabial form including lateral facial contour and bony contour in healthy adults was studied to refer to values of normal mentolabial form using lateral roentgen cephalography. In soft tissue of healthy adults, thickness of mentolabial soft tissue was greater for men than for women. Degree of mentolabial curvature was greater for men than for women. In severe facial burn patients, the micrognathia-like deformity was evaluated compared with the reference values. In soft tissue of severe facial burn patients, loss of mentolabial sulcus and eversion of the lower lip were characteristically analyzed, suspecting that the major cause was scar contracture. These statistical data contribute to reconstruction of micrognathia-like deformity after severe facial burn to lead to favorable mentolabial form. It is shown that osteotomized genio-advancement produced ideal mentolabial form in severe facial burn patients, who had not been satisfied with the form by conventional operative methods, free skin flap, skin flap or free skin graft. And that the analyzed data, especially in the parameter of thickness of mentolabial sulcus, was improved.

Keratinizing squamous epithelium associated with Syringocystadenoma papilliferum differentiates towards infrainfundibulum: case report with immunohistochemical study of cytokeratins.

We describe a case of Syringocystadenoma papilliferum (SCAP) with keratinizing squamous epithelium in a 26-year-old female presenting with a dark brown to black nodule on her forehead. After surgical excision, the specimen was examined immunohistochemically using antibodies against cytokeratin (CK) 1, 8, 10, 14, 17, 18 and 19. Within the keratinizing squamous epithelium, CK1, 10, 14 and 17 were present, whereas the other CKs were absent. Based on CK expression, keratinizing squamous epithelium in SCAP seems to differentiate towards the infrainfundibulum.

Usefulness of a new immuno-radiometric assay to detect hepatitis C core antigen in a community-based population.

A new immuno-radiometric assay (IRMA) to detect hepatitis C virus (HCV) core antigen (HCVcAg) has been developed. The aim of the present study was to investigate the sensitivity and specificity of this IRMA to measure HCV antigenemia, based on the detection of HCV RNA as the gold standard, and to assess the utility of the IRMA in a community-based population. Anti-HCV positive residents in a hyperendemic area of HCV infection in Japan were studied. Serum levels of HCVcAg were measured using IRMA, and the presence of HCV RNA was determined by a qualitative reverse transcription-polymerase chain reaction (RT-PCR) assay. The sensitivity and the specificity of the IRMA were 96.4 and 100%, respectively. The sensitivity of the IRMA was similar between serological HCV group I (HCV genotypes 1a and 1b) (97.6%) and group II (HCV genotypes 2a and 2b) (94.0%). There was a strong correlation between serum HCVcAg level and HCV-RNA measured by a quantitative RT-PCR (r = 0.832, P < 0.0001). There also was a very strong correlation of HCVcAg level between IRMA measurements performed on serum and those performed on plasma (r = 0.984, P < 0.0001). In conclusion, this new IRMA is useful for the detection of HCV core antigen in a community-based population.

Tissue reaction at the implantation of recombinant human bone morphogenetic protein-2 into the skeletal muscle.

Bone morphogenetic protein (BMP) is a unique cytokine that induces bony tissue in soft tissue. Tissue reactions at and around the implantation of recombinant human BMP-2 (rhBMP-2) into the soft tissue of rats and nonhuman primates were investigated. At the osteoinduced site of rats, massive trabeculae-lined osteoblasts and rich marrow were observed. At and around the nonosteoinduced sites of nonhuman primates, large clear nuclei were observed in reaction to rhBMP-2 implantation. The surrounding area was visually classified into zones 1, 2 and 3. Zone 3 was near the center of the implant. The area of nuclei, the major axis, the minor axis and the ratio of minor axis per major axis were image-analyzed in the histological views. In zones 1, 2 and 3, the nuclear areas were 18.0 (3.1) mean (SD); unit micron2, 33.4 (5.61) and 110.1 (23.7), respectively. The major axes of nuclear ellipses were 7.45 (0.22) (unit micron), 7.76 (0.26), and 13.9 (1.88), respectively. The minor axes were 3.07 (0.53), 5.59 (0.95) and 10.1 (1.35), respectively. The ratios of minor axis per major axis of nuclear ellipses were 0.4 (0.57), 0.72 (0.11) and 0.73 (0.11) in zones 1, 2 and 3, respectively. These results showed that in zones 2 and 3 cell and tissue reactions were marked against rhBMP-2 implantation.

Trichilemmoma: an immunohistochemical study of cytokeratins.

BACKGROUND: The histogenesis of trichilemmoma remains unclear. OBJECTIVES: To clarify the histogenesis of trichilemmoma by evaluating its cytokeratin (CK) expression. METHODS: In three cases of trichilemmoma, CK expression was studied immunohistochemically using seven antikeratin antibodies against CK1, 10, 14-17 and 19, respectively. RESULTS: CK1 and CK10 were present in keratinizing ductal epithelium. CK14 was present in the whole layer. CK15 was present in suprabasal layers in two cases. CK16 was present in the suprabasal layer, but was absent in keratinizing ductal epithelium. CK17 was present in suprabasal layers and the sebaceous duct-like structure. CK19 was totally absent. CONCLUSIONS: These results showed that trichilemmoma may differentiate mainly towards two directions: infundibular keratinization and proliferation of the outer root sheath with undifferentiated and pluripotent characteristics.

Preclinical study of recombinant human bone morphogenetic protein-2: application of hyperbaric oxygenation during bone formation under unfavourable condition.

The objective of this study was to evaluate the effect of hyperbaric oxygenation on bone formation by recombinant human bone morphogenetic protein-2 (rhBMP-2) under unfavourable conditions. The calf muscles of 10 rats with low-blood supply were prepared by ligating and cutting the right femoral artery, and 10 micro of rhBMP-2 was implanted in the calf muscle. Five rats each were randomly assigned to the hyperbaric oxygenation group and the control group (untreated). The rats in the hyperbaric oxygenation group were treated with hyperbaric oxygenation at 2.0 atmospheres absolute for 3 weeks. In the histologic evaluation, the number of osteoblasts in the hyperbaric oxygenation group was greater than that in control group. The area of the trabecular bone induced in the hyperbaric oxygenation group was significantly larger than that in the control group. The values of alkaline phosphatase activity and calcium contents in the hyperbaric oxygenation group were significantly higher than those in the control group. The results of the present study suggest that hyperbaric oxygenation increases the partial oxygen pressure in low blood supply tissue and accelerates the activity and rate of osteoinduction by rhBMP-2. Hyperbaric oxygenation therapy may increase the clinical application of rhBMP-2 to unfavourable condition.

Telomeric repeat sequence of Aspergillus oryzae consists of dodeca-nucleotides.

Four telomeres in the chromosomes of Aspergillus oryzae NFRI1599 were cloned and sequenced. The telomeric repeat sequence of A. oryzae consisted of dodeca-nucleotides: TTAGGGTCAACA. The length of the telomeric repeat tract was 114-136 bp, which corresponds to 9-11 repeats of the dodeca-nucleotide sequence. Compared to a chromosome internal control (18S rDNA), the telomeric sequences were found to be sensitive to BAL31 exonuclease digestion, thus proving that the identified telomeric repeat sequences were located at the most terminal tract of the chromosomes. The length of the telomeric repeat tract of A. oryzae is similar to that of Aspergillus nidulans, whose repeat unit is TTAGGG, indicating that the regulatory mechanism of telomere length might be conserved among Aspergillus species.

Osteoinduction by recombinant human bone morphogenetic protein-2 in muscles of non-human primates.

Heterotopic osteoinduction in a muscle of a medium-sized, non-human primate (Japanese macaque monkey; Macaca fuscata) was investigated with recombinant human bone morphogenetic protein-2 (rhBMP-2) mixed with atelopeptide type I collagen as the carrier. Nine monkeys were divided into three groups of three: groups I (1.25 mg rhBMP-2), II (250 micrograms rhBMP-2) and III (50 micrograms rhBMP-2). Four weeks after implanting into the calf muscle pouch, the implant was examined radiographically and histologically. In one specimen of three in group I, marked radio-opaque shadow, massive chondrogenesis and partial osteogenesis were observed. In the other two specimens, only microscopic calcification signs were recognized. In groups II and III, no findings of heterotopic osteoinduction were radiographically observed; however, nuclei from muscle bundles reacted to rhBMP-2 and were large and round, as in muscle bundles near the site of osteogenesis in group I. A positive control study using rats was carried out in parallel. This was a dose-finding study, with the monkeys in group III acting as a sub-effective dose (placebo) control, and rats acting as an active control, or verum, to show that the techniques are sufficiently sensitive. Bone morphogenetic protein appears to osteoinduce less bony material in soft tissue in primates than in rats.

Immunohistochemical study of cytokeratins in hidradenitis suppurativa (acne inversa).

In 14 cases of hidradenitis suppurativa, cytokeratin (CK) expression was studied immunohistochemically, using six antikeratin antibodies against CK1, CK10, CK14, CK16, CK17 and CK19, respectively. The draining sinus tract epithelium of hidradenitis suppurativa lesions was divided into three components: infundibular-like keratinized epithelium (type A), non-infundibular keratinized epithelium (type B), and non-keratinized epithelium (type C). In type A samples, CK17 (which is found in normal infundibulum) was not detected, suggesting fragility of this epithelial type. Keratin expression in types B and C epithelia was similar to that observed in the outer root sheath in normal hair follicles. Our results suggest that the draining sinus epithelium may possess characteristics of fragility, undifferentiation and hyperproliferation, as shown with CK expression.

Cytokeratin expression in pilonidal sinus.

BACKGROUND: Pilonidal sinus (PS) is considered to belong in the category of follicular occlusion diseases (acne triad). OBJECTIVES: The aim of our study was to elucidate the pathogenesis of PS by evaluating its cytokeratin (CK) expression. METHODS: CK expression in nine cases of PS was studied immunohistochemically using six antikeratin antibodies. RESULTS: Infundibular-like epithelium contained CK1, 10 and 14 similar to normal infundibulum, but it did not contain CK17. In non-infundibular-like epithelium, CK14, 16 and 17 were detected similar to that in normal outer root sheath. CK expression in PS was similar to that in hidradenitis suppurativa, suggesting that sinus epithelium may be fragile, hyperproliferative and undifferentiated. CONCLUSIONS: PS can be classified in the same entity as follicular occlusion diseases based on CK expression.

A non-toxic heat shock protein 70 inducer, geranylgeranylacetone, suppresses apoptosis of cultured rat hepatocytes caused by hydrogen peroxide and ethanol.

BACKGROUND/AIMS: A stress-inducible heat shock protein 70 (HSP70) is one of the best-known endogenous factors protecting cell injury under various pathological conditions. The aim of this study was to examine anti-apoptotic actions of a non-toxic HSP70 inducer, geranylgeranylacetone (GGA), on hepatocytes exposed to hydrogen peroxide (H2O2) or ethanol. METHODS: Primary cultures of rat hepatocytes were treated with different concentrations of GGA and exposed to 0.5 mM H202 or 100 mM ethanol. The heat shock response was assessed by measuring the activation of heat shock factor 1 (HSF1), HSP70 mRNA expression, and accumulations of HSP70, HSP90, and HSP27. Apoptosis was evaluated by DNA fragmentation. RESULTS: Pretreatment with 1 microM GGA for 2 h enhanced nuclear translocation and phosphorylation of HSF1, HSF1-DNA binding, HSP70 mRNA expression, and its accumulation, when the cells were exposed to H202 or ethanol. In association with this accelerated response, GGA suppressed the insult-induced activation of c-Jun N-terminal kinases, caspase 9, and caspase 3-like proteases, leading to significant inhibition of apoptosis. CONCLUSIONS: GGA exerted anti-apoptotic actions, at least in part, by priming hepatocytes for enhanced HSP70 induction. Our results suggest that GGA may have a potential benefit for the treatment of alcoholic and ischemia-reperfusion liver injuries.