Responses of the silkworm tyramine receptor to 2-phenylethylamines and 5-phenyloxazoles

Tyramine (TA), a biogenic amine, attenuates intracellular cAMP production by acting on its receptor in insects. Several non-biogenic amines were examined for their actions on native and heterologously expressed silkworm TA receptors. 5-(4-Hydroxyphenyl)oxazole, which showed an attenuating effect on cAMP production in silkworm-head membranes, did not attenuate forskolin-stimulated cAMP production in HEK-293 cells expressing the silkworm TA receptor, although the compound bound to the cloned receptor. 2-Phenylethylamines (2-PEAs), which showed positive and negative effects on cAMP production in silkworm-head membranes, inhibited [3H]TA binding to the cloned TA receptor. 2-Chloro-2-(4-chlorophenyl)ethylamine was the most potent inhibitor of [3H]TA binding among the 2-PEAs tested, with an IC50 of 30.4 nM. This compound acted as an antagonist and abolished TA-attenuation of forskolin-stimulated cAMP production in the cloned TA receptor. The discrepancy in the effects of the non-biogenic amines on the native and cloned TA receptors remains to be further examined. A newly synthesized 2-PEA, 2-chloro-2-(4-hydroxyphenyl)ethylamine, attenuated forskolin-stimulated cAMP production in the cloned TA receptor, indicating that the para-hydroxy group is important for the agonist action.     

Abrupt increase in UV sensitivity at late log-phase of growth in Paramecium tetraurelia

In this study, changes in UV sensitivity, a parameter of the clonal aging that has been studied in the daily reisolation culture, were examined in the logarithmically growing Paramecium culture. Cells in logarithmically growing cultures are thought to change the internal states under rapidly changing external conditions. In contrast, cells in daily reisolation cultures gradually change the internal states, the process being called clonal development and aging, under the external conditions that are kept almost constant. Cells were sampled at regular intervals, irradiated with UV, and examined for UV sensitivity assessed by the clonal survival. We found that log-phase cells showed low sensitivity to UV until they reached 2,000-3,000 cells/ml, and beyond that cell density, abruptly became highly UV sensitive. The extent of this increase in UV sensitivity was similar to that between two age groups, 130 fissions of clonal age apart. When cells from a culture of 2,000-3,000 cells/ml were resuspended in culture medium at various cell densities, they changed to UV sensitive only when the cultures reached over approximately 2,600 cells/ml. These results suggest that paramecia become UV sensitive in response to change in the nutrient level when cell density exceeds 2,000-3,000 cells/ml.     

Glutathionylation of two cysteine residues in paired domain regulates DNA binding activity of Pax-8

We reported that the first two cysteine residues out of three present in paired domain (PD), a DNA-binding domain, are responsible for redox regulation of Pax-8 DNA binding activity. We show that glutathionylation of these cysteines has a regulatory role in PD binding. Wild-type PD and its mutants with substitution of cysteine to serine were synthesized and named CCC, CSS, SCS, SSC, and SSS according to the positions of substituted cysteines. They were incubated in a buffer containing various ratios of GSH/GSSG and subjected to gel shift assay. Binding of CCC, CSS, and SCS was impaired with decreasing GSH/GSSG ratio, whereas that of SSC and SSS was not affected. Because [3H]glutathione was incorporated into CCC, CSS, and SCS, but not into SSC and SSS, the binding impairment was ascribed to glutathionylation of the redox-reactive cysteines. This oxidative inactivation of PD binding was reversed by a reductant dithiothreitol and by redox factor (Ref)-1 in vitro. To explore the glutathionylation in cells, Chinese hamster ovary cells overexpressing CSS and SCS were labeled with [35S]cysteine in the presence of cycloheximide. Immunoprecipitation with an antibody against PD revealed that treatment of the cells with an oxidant diamide induced the 35S incorporation into both mutants, suggesting the PD glutathionylation in cells. Since the two cysteine residues in PD are conserved in all Pax members, this novel posttranslational modification of PD would provide a new insight into molecular basis for modulation of Pax function.     

Improved catalytic and stereoselective glycosylation with glycosyl N-trichloroacetylcarbamate: application to various 1-hydroxy sugars

Efficient catalytic and stereoselective glycosylation was achieved by activating a glycosyl N-trichloroacetylcarbamate with a catalytic amount of Lewis acid in the presence of a glycosyl acceptor and 5 ? molecular sieves. Catalytic one-pot dehydrative glycosylation of a 1-hydroxy carbohydrate was achieved stereoselectively by reaction with trichloroacetyl isocyanate, followed by activation with a catalytic amount of activators.     

Crucial role of vinexin for keratinocyte migration in vitro and epidermal wound healing in vivo

In the process of tissue injury and repair, epithelial cells rapidly migrate and form epithelial sheets. Vinexin is a cytoplasmic molecule of the integrin-containing cell adhesion complex localized at focal contacts in vitro. Here, we investigated the roles of vinexin in keratinocyte migration in vitro and wound healing in vivo. Vinexin knockdown using siRNA delayed migration of both HaCaT human keratinocytes and A431 epidermoid carcinoma cells in scratch assay but did not affect cell proliferation. Induction of cell migration by scratching the confluent monolayer culture of these cells activated both EGFR and ERK, and their inhibitors AG1478 and U0126 substantially suppressed scratch-induced keratinocyte migration. Vinexin knockdown in these cells inhibited the scratch-induced activation of EGFR, but not that of ERK, suggesting that vinexin promotes cell migration via activation of EGFR. We further generated vinexin (−/−) mice and isolated their keratinocytes. They similarly showed slow migration in scratch assay. Furthermore, vinexin (−/−) mice exhibited a delay in cutaneous wound healing in both the back skin and tail without affecting the proliferation of keratinocytes. Together, these results strongly suggest a crucial role of vinexin in keratinocyte migration in vitro and cutaneous wound healing in vivo.     

Purification and characterization of alpha-glucosidase I from Japanese honeybee (Apis cerana japonica) and molecular cloning of its cDNA

alpha-Glucosidase (JHGase I) was purified from a Japanese subspecies of eastern honeybee (Apis cerana japonica) as an electrophoretically homogeneous protein. Enzyme activity of the crude extract was mainly separated into two fractions (component I and II) by salting-out chromatography. JHGase I was isolated from component I by further purification procedure using CM-Toyopearl 650M and Sephacryl S-100. JHGase I was a monomeric glycoprotein (containing 15% carbohydrate), of which the molecular weight was 82,000. Enzyme displayed the highest activity at pH 5.0, and was stable up to 40 degrees C and in a pH-range of 4.5-10.5. JHGase I showed unusual kinetic features: the negative cooperative behavior on the intrinsic reaction on cleavage of sucrose, maltose, and p-nitrophenyl alpha-glucoside, and the positive cooperative behavior on turanose. We isolated cDNA (1,930 bp) of JHGase I, of which the deduced amino-acid sequence (577 residues) confirmed that JHGase I was a member of alpha-amylase family enzymes. Western honeybees (Apis mellifera) had three alpha-glucosidase isoenzymes (WHGase I, II, and III), in which JHGase I was considered to correspond to WHGase I.     

A protein associated with toll-like receptor 4 (PRAT4A) regulates cell surface expression of TLR4

TLRs recognize microbial products. Their subcellular distribution is optimized for microbial recognition. Little is known, however, about mechanisms regulating the subcellular distribution of TLRs. LPS is recognized by the receptor complex consisting of TLR4 and MD-2. Although MD-2, a coreceptor for TLR4, enhances cell surface expression of TLR4, an additional mechanism regulating TLR4 distribution has been suggested. We show here that PRAT4A, a novel protein associated with TLR4, regulates cell surface expression of TLR4. PRAT4A is associated with the immature form of TLR4 but not with MD-2 or TLR2. PRAT4A knockdown abolished LPS responsiveness in a cell line expressing TLR4/MD-2, probably due to the lack of cell surface TLR4. PRAT4A knockdown down-regulated cell surface TLR4/MD-2 on dendritic cells. These results demonstrate a novel mechanism regulating TLR4/MD-2 expression on the cell surface.     

Decrease in Epstein-Barr virus-positive AIDS-related lymphoma in the era of highly active antiretroviral therapy

Recent introduction of highly active antiretroviral therapy (HAART) is reported to have reduced the incidence of lymphoma among HIV-infected individuals. A clinicopathological study was performed on 86 AIDS-related lymphoma patients who were treated in Tokyo area from 1987 to 2005. The incidence of lymphoma detected by autopsy was 27% (53 cases/198 autopsies). Diffuse large B cell lymphoma was the most predominant histological subtype throughout the period (78%). Burkitt's lymphoma (BL) increased from 2% in the pre-HAART era (before end-1997) to 13% in the HAART era, whereas incidence of BL did not vary between HAART users and non-users. Epstein-Barr virus (EBV)-positive lymphoma decreased from 88% in the pre-HAART era to 58% in the HAART era, but did not differ significantly between HAART users (73%) and non-users (74%). Nodal involvement of lymphoma increased from 14% in the pre-HAART era to 50% in the HAART era; however, central nervous system involvement decreased from 62 to 38%. Kaposi's sarcoma-associated herpesvirus infection was rare (4%) among all cases. These data suggest that HAART might play a partial role in these changes, and the alteration in immunological backgrounds, such as EBV prevalence, is suggested as another leading cause of these changes in Japanese AIDS-related lymphoma.     

IAA-Ala Resistant3, an Evolutionarily Conserved Target of miR167, Mediates Arabidopsis Root Architecture Changes during High Osmotic Stress

The functions of microRNAs and their target mRNAs in Arabidopsis thaliana development have been widely documented; however, roles of stress-responsive microRNAs and their targets are not as well understood. Using small RNA deep sequencing and ATH1 microarrays to profile mRNAs, we identified IAA-Ala Resistant3 (IAR3) as a new target of miR167a. As expected, IAR3 mRNA was cleaved at the miR167a complementary site and under high osmotic stress miR167a levels decreased, whereas IAR3 mRNA levels increased. IAR3 hydrolyzes an inactive form of auxin (indole-3-acetic acid [IAA]-alanine) and releases bioactive auxin (IAA), a central phytohormone for root development. In contrast with the wild type, iar3 mutants accumulated reduced IAA levels and did not display high osmotic stress–induced root architecture changes. Transgenic plants expressing a cleavage-resistant form of IAR3 mRNA accumulated high levels of IAR3 mRNAs and showed increased lateral root development compared with transgenic plants expressing wild-type IAR3. Expression of an inducible noncoding RNA to sequester miR167a by target mimicry led to an increase in IAR3 mRNA levels, further confirming the inverse relationship between the two partners. Sequence comparison revealed the miR167 target site on IAR3 mRNA is conserved in evolutionarily distant plant species. Finally, we showed that IAR3 is required for drought tolerance.