The involvement of cAMP signaling pathway in axis specification in Xenopus embryos

The cAMP signaling system has been postulated to be involved in embryogenesis of many animal species, however, little is known about its role in embryonic axis formation in vertebrates. In this study, the role of the cAMP signaling pathway in patterning the body plan of the Xenopus embryo was investigated by expressing and activating the exogenous human 5-hydroxytryptamine type 1a receptor (5-HT(1a)R) which inhibits adenylyl cyclase through inhibitory G-protein in embryos in a spatially- and temporally-controlled manner. In embryos, ventral, but not dorsal expression and stimulation of this receptor during blastula and gastrula stages induced secondary axes but were lacking anterior structures. At the molecular level, 5-HT(1a)R stimulation induced expression of the dorsal mesoderm marker genes, and downregulated expression of the ventral markers but had no effect on expression of the pan mesodermal marker gene in ventral marginal zone explants. In addition, ventral expression and stimulation of the receptor partially restored dorsal axis of UV-irradiated axis deficient embryo. Finally, the total mass of cAMP differs between dorsal and ventral regions of blastula and gastrula embryos and this is regulated in a temporally-specific manner. These results suggest that the cAMP signaling system may be involved in the transduction of ventral signals in patterning early embryos.     

Precise detection and tracing of Trichoderma hamatum 382 in compost-amended potting mixes by using molecular markers

Randomly amplified polymorphic DNA (RAPD) analysis and the PCR assay were used in combination with dilution plating on a semiselective medium to detect and enumerate propagules of Trichoderma hamatum 382, a biocontrol agent utilized in compost-amended mixes. Distinct and reproducible fingerprints were obtained upon amplification of purified genomic DNA of T. hamatum 382 with the random primers OPE-16, OPH-19, and OPH-20. Three amplified DNA fragments of 0.35 (OPE-16(0.35)), 0.6 (OPH-19(0.6)), and 0.65 (OPH-20(0.65)) kb were diagnostic for T. hamatum 382, clearly distinguishing it from 53 isolates of four other Trichoderma spp. tested. Some isolates of T. hamatum shared these low-molecular-weight fragments with T. hamatum 382. However, RAPD analysis of isolates of T. hamatum with all three random primers used in consecutive PCR tests distinguished T. hamatum 382 from other isolates of T. hamatum. These three RAPD amplicons were cloned and sequenced, and pairs of oligonucleotide primers for each cloned fragment were designed. Use of the primers in the PCR assay resulted in the amplification of DNA fragments of the same size as the cloned RAPD fragments from genomic DNA of T. hamatum 382. A combination of dilution plating on a semiselective medium for Trichoderma spp. and PCR, with the RAPD primers OPH-19, OPE-16, and OPH-20 or the three sequence-characterized primers, was used successfully to verify the presence of T. hamatum 382 propagules in nine different soil, compost, and potting mix samples. All 23 Trichoderma isolates recovered on semiselective medium from commercial potting mixes fortified with T. hamatum 382 were identified as T. hamatum 382, whereas 274 Trichoderma isolates recovered from the other nine samples were negative in the PCR assay. Thus, this highly specific combination of techniques allowed detection and enumeration of propagules of T. hamatum 382 in fortified compost-amended potting mixes. Sequence-characterized amplified region markers also facilitated the development of a very simple procedure to amplify DNA of T. hamatum 382 directly from fortified compost-amended potting mixes.     

Anti-HIV-1 activity of low molecular weight sulfated chitooligosaccharides

Chitooligosaccharides are nontoxic and water-soluble compounds obtained by enzymatic degradation of chitosan, which is derived from chitin by a deacetylation process. Chitooligosaccharides possess broad range of activities such as antitumour, antifungal, antibacterial activities. Sulfated chitooligosaccharides (SCOSs) with different molecular weights were synthesized by a random sulfation reaction. In the present study, anti-HIV-1 properties of SCOSs and the impact of molecular weight on their inhibitory activity were investigated. SCOS III (MW 3-5 kDa) was found to be the most effective compound to inhibit HIV-1 replication. At nontoxic concentrations, SCOS III exhibited remarkable inhibitory activities on HIV-1-induced syncytia formation (EC₅₀ 2.19 μg/ml), lytic effect (EC₅₀ 1.43 μg/ml), and p24 antigen production (EC₅₀ 4.33 μg/ml and 7.76 μg/ml for HIV-1_RF and HIV-1_Ba-L, respectively). In contrast, unsulfated chitooligosaccharides showed no activity against HIV-1. Furthermore, it was found that SCOS III blocked viral entry and virus-cell fusion probably via disrupting the binding of HIV-1 gp120 to CD4 cell surface receptor. These results suggest that sulfated chitooligosaccharides represent novel candidates for the development of anti-HIV-1 agent.     

A newly synthesized,potent tyrosinase inhibitor: 5-(6-Hydroxy-2-naphthyl)-1,2,3-benzenetriol

In searching for new agents with a depigmenting effect, we synthesized a derivative of resveratrol, 5-(6-hydroxy-2-naphthyl)-1,2,3-benzenetriol (5HNB) with a potent tyrosinase inhibitory activity. 5HNB inhibited mushroom tyrosinase with an IC₅₀ value of 2.95 μM, which is more potent than the well-known anti-tyrosinase activity of kojic acid (IC₅₀ = 38.24). The results of the enzymatic inhibition kinetics by Lineweaver–Burk analysis indicated 5HNB inhibits tyrosinase non-competitively when l-tyrosine was used as the substrate. Based on the strong inhibitory action of 5HNB, it is expected that 5HNB can suppress melanin production in which tyrosinase plays the essential role. Our expectation was confirmed by the experimentations with B16 melanoma cells in which 5HNB inhibited melanin production. We propose that 5HNB might have skin-whitening effects as well as therapeutic potential for treating skin pigmentation disorders.     

Novel 1H-pyrrolo[2,3-c]pyridines as acid pump antagonists (APAs)

A series of 1H-pyrrolo[2,3-c]pyridines as acid pump antagonists (APAs) was synthesized and the inhibitory activities against H⁺/K⁺ ATPase isolated from hog gastric mucosa were determined. After elaborating on substituents at N1, C5, and C7 position of 1H-pyrrolo[2,3-c]pyridine scaffold, we have observed that compounds 14f and 14g are potent APAs with H⁺/K⁺ ATPase IC₅₀ = 28 and 29 nM, respectively.     

Crystalline structure analysis of cellulose treated with sodium hydroxide and carbon dioxide by means of X-ray diffraction and FTIR spectroscopy

Crystalline structures of cellulose (named as Cell 1), NaOH-treated cellulose (Cell 2), and subsequent CO2-treated cellulose (Cell 2-C) were analyzed by wide-angle X-ray diffraction and FTIR spectroscopy. Transformation from cellulose I to cellulose II was observed by X-ray diffraction for Cell 2 treated with 15-20 wt% NaOH. Subsequent treatment with CO2 also transformed the Cell 2-C treated with 5-10 wt% NaOH. Many of the FTIR bands including 2901, 1431, 1282, 1236, 1202, 1165, 1032, and 897 cm(-1) were shifted to higher wave number (by 2-13 cm(-1)). However, the bands at 3352, 1373, and 983 cm(-1) were shifted to lower wave number (by 3-95 cm(-1)). In contrast to the bands at 1337, 1114, and 1058 cm(-1), the absorbances measured at 1263, 993, 897, and 668 cm(-1) were increased. The FTIR spectra of hydrogen-bonded OH stretching vibrations at around 3352 cm(-1) were resolved into three bands for cellulose I and four bands for cellulose II, assuming that all the vibration modes follow Gaussian distribution. The bands of 1 (3518 cm(-1)), 2 (3349 cm(-1)), and 3 (3195 cm(-1)) were related to the sum of valence vibration of an H-bonded OH group and an intramolecular hydrogen bond of 2-OH ...O-6, intramolecular hydrogen bond of 3-OH...O-5 and the intermolecular hydrogen bond of 6-O...HO-3', respectively. Compared with the bands of cellulose I, a new band of 4 (3115 cm(-1)) related to intermolecular hydrogen bond of 2-OH...O-2' and/or intermolecular hydrogen bond of 6-OH...O-2' in cellulose II appeared. The crystallinity index (CI) was obtained by X-ray diffraction [CI(XD)] and FTIR spectroscopy [CI(IR)]. Including absorbance ratios such as A1431,1419/A897,894 and A1263/A1202,1200, the CI(IR) was evaluated by the absorbance ratios using all the characteristic absorbances of cellulose. The CI(XD) was calculated by the method of Jayme and Knolle. In addition, X-ray diffraction curves, with and without amorphous halo correction, were resolved into portions of cellulose I and cellulose II lattice. From the ratio of the peak area, that is, peak area of cellulose I (or cellulose II)/total peak area, CI(XD) were divided into CI(XD-CI) for cellulose I and CI(XD-CII) for cellulose II. The correlation between CI(XD-CI) (or CI(XD-CII)) and CI(IR) was evaluated, and the bands at 2901 (2802), 1373 (1376), 897 (894), 1263, 668 cm(-1) were good for the internal standard (or denominator) of CI(IR), which increased the correlation coefficient. Both fraction of the absorbances showing peak shift were assigned as the alternate components of CI(IR). The crystallite size was decreased to constant value for Cell 2 treated at >or= 15 wt% NaOH. The crystallite size of Cell 2-C (cellulose II) was smaller than that of Cell 2 (cellulose I) treated at 5-10 wt% NaOH. But the crystallite size of Cell 2-C (cellulose II) was larger than that of Cell 2 (cellulose II) treated at 15-20 wt% NaOH.     

Karyotype of North American shortnose sturgeon Acipenser brevirostrum with the highest chromosome number in the Acipenseriformes

The shortnose sturgeon Acipenser brevirostrum was revealed to have a larger number of chromosomes than previously reported for other sturgeon species. Its chromosome number ranged from 362 to 372 (of ten specimens examined), showing intraindividual variation. The karyotype of metaphase with the highest chromosome number (372) consisted of 89 pairs of macrochromosomes and 97 pairs of microchromosomes (fundamental number; NF=550). Although the microchromosomes were relatively shorter than the macrochromosomes, most of them had discernible arms and centromeres. Silver-stained nucleolar organizer regions (Ag-NORs) were localized on the telomeric regions of 5 pairs of chromosomes (Ag-NORs=10): 4 were made up of small meta/submetacentrics and 1 of acrocentrics. Polyploidy of A. brevirostrum should be hexaploid based on the karyotype, numerous chromosomes, Ag-NORs, and previously reported large genome size (ca. 13pg DNA/cell).Supplementary material to this paper is available in electronic format at http://dx.doi.org/10.1007/s10228-004-0257-z     

NF-kappaB activation is required for the development of cardiac hypertrophy in vivo

In the present study, we examined whether NF-kappaB activation is required for cardiac hypertrophy in vivo. Cardiac hypertrophy in rats was induced by aortic banding for 1, 3, and 5 days and 1-6 wk, and age-matched sham-operated rats served as controls. In a separate group of rats, an IkappaB-alpha dominant negative mutant (IkappaB-alphaM), a superrepressor of NF-kappaB activation, or pyrrolidinedithiocarbamate (PDTC), an antioxidant that can inhibit NF-kappaB activation, was administered to aortic-banded rats for 3 wk. The heart weight-to-body weight ratio was significantly increased at 5 days after aortic banding, peaked at 4 wk, and remained elevated at 6 wk compared with age-matched sham controls. Atrial natriuretic peptide and brain natriuretic peptide mRNA expressions were significantly increased after 1 wk of aortic banding, reached a maximum between 2 and 3 wk, and remained increased at 6 wk compared with age-matched sham controls. NF-kappaB activity was significantly increased at 1 day, reached a peak at 3 wk, and remained elevated at 6 wk, and IKK-beta activity was significantly increased at 1 day, peaked at 5 days, and then decreased but remained elevated at 6 wk after aortic banding compared with age-matched sham controls. Inhibiting NF-kappaB activation in vivo by cardiac transfection of IkappaB-alphaM or by PDTC treatment significantly attenuated the development of cardiac hypertrophy in vivo with a concomitant decrease in NF-kappaB activity. Our results suggest that NF-kappaB activation is required for the development of cardiac hypertrophy in vivo and that NF-kappaB could be an important target for inhibiting the development of cardiac hypertrophy in vivo.     

Comparative analysis of the secretory proteome of human adipose stromal vascular fraction cells during adipogenesis

Adipogenesis is a complex process that is accompanied by a number of molecular events. In this study, a proteomic approach was adopted to identify secretory factors associated with adipogenesis. A label‐free shotgun proteomic strategy was implemented to analyze proteins secreted by human adipose stromal vascular fraction cells and differentiated adipocytes. A total of 474 proteins were finally identified and classified according to quantitative changes and statistical significances. Briefly, 177 proteins were significantly upregulated during adipogenesis (Class I), whereas 60 proteins were significantly downregulated (Class II). Changes in the expressions of several proteins were confirmed by quantitative RT‐PCR and immunoblotting. One obvious finding based on proteomic data was that the amounts of several extracellular modulators of Wnt and transforming growth factor‐β (TGF‐β) signaling changed during adipogenesis. The expressions of secreted frizzled‐related proteins, dickkopf‐related proteins, and latent TGF‐β‐binding proteins were found to be altered during adipogenesis, which suggests that they participate in the fine regulation of Wnt and TGF‐β signaling. This study provides useful tools and important clues regarding the roles of secretory factors during adipogenic differentiation, and provides information related to obesity and obesity‐related metabolic diseases.