Recognition of cellooligosaccharides by a family 28 carbohydrate-binding module

The crystal structures of a carbohydrate-binding module (CBM) family 28 domain of endoglucanase Cel5A from Clostridium josui have been determined in ligand-free and complex forms with cellobiose, cellotetraose, and cellopentaose as the first complex structures of this family. In the cleft of a β-sandwich fold, the ligands are recognized by stacking interactions and hydrogen bonds. Conformations of the bound cellooligosaccharides are similar to those in crystals and solution but clearly different from the cellulose structure. Interestingly, the glucan chain bound on CBM28 is in the opposite direction of that bound to CBM17, although these families share significant structural similarity.     

Metabolism of mono- and dichloro-dibenzo-p-dioxins by Phanerochaete chrysosporium cytochromes P450

The white-rot fungus Phanerochaete chrysosporium possesses biodegradative capabilities of polychlorinated dibenzo-p-dioxins (PCDDs). One hundred twenty yeast clones expressing individual P450s of P. chrysosporum (PcCYPs), generated in our previous efforts, were screened for transformation of dioxin, and 40 positive clones were obtained. Of these clones, six clones showed metabolism of 2-chloro-dibenzo-p-dioxin, and a microsomal PcCYP designated as PcCYP11a3 showed much higher activity than any other PcCYPs. The turnover numbers of hydroxylation activities of PcCYP11a3 toward 1-MCDD (58 min⁻¹) and 2-MCDD (13 min⁻¹) are more than 200 times higher than those of previously reported PcCYP65a2. In addition, PcCYP11a3 catalyzes hydroxylation of 2,3-dichloro-dibenzo-p-dioxin. To our best knowledge, PcCYP11a3 has the highest activity toward PCDDs among the known CYPs derived from microorganisms. Although PcCYP11a3 showed no detectable activity toward 2,7-dichloro-dibenzo-p-dioxin and 2,3,7-trichloro-dibenzo-p-dioxin, PcCYP11a3 is promising as a template whose activity would be enhanced by site-directed mutagenesis.     

Regulation of Arabidopsis defense responses against Spodoptera littoralis by CPK-mediated calcium signaling

Background

Genetic markers and linkage mapping are basic prerequisites for marker-assisted selection and map-based cloning. In the case of the key grassland species Lolium spp., numerous mapping populations have been developed and characterised for various traits. Although some genetic linkage maps of these populations have been aligned with each other using publicly available DNA markers, the number of common markers among genetic maps is still low, limiting the ability to compare candidate gene and QTL locations across germplasm.

Results

A set of 204 expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers has been assigned to map positions using eight different ryegrass mapping populations. Marker properties of a subset of 64 EST-SSRs were assessed in six to eight individuals of each mapping population and revealed 83% of the markers to be polymorphic in at least one population and an average number of alleles of 4.88. EST-SSR markers polymorphic in multiple populations served as anchor markers and allowed the construction of the first comprehensive consensus map for ryegrass. The integrated map was complemented with 97 SSRs from previously published linkage maps and finally contained 284 EST-derived and genomic SSR markers. The total map length was 742 centiMorgan (cM), ranging for individual chromosomes from 70 cM of linkage group (LG) 6 to 171 cM of LG 2.

Conclusions

The consensus linkage map for ryegrass based on eight mapping populations and constructed using a large set of publicly available Lolium EST-SSRs mapped for the first time together with previously mapped SSR markers will allow for consolidating existing mapping and QTL information in ryegrass. Map and markers presented here will prove to be an asset in the development for both molecular breeding of ryegrass as well as comparative genetics and genomics within grass species.     

Remarkable features of ovarian morphology and reproductive hormones in insulin-resistant Zucker fatty (fa/fa) rats

Background

Zucker fatty (fa/fa) rats are a well-understood model of obesity and hyperinsulinemia. It is now thought that obesity/hyperinsulinemia is an important cause of endocrinological abnormality, but to date there have been no reports on the changes in ovarian morphology or the ovarian androgen profile in rat models of obesity and insulin resistance.

Methods

In this study we investigated the effects of obesity and hyperinsulinemia on ovarian morphology and the hormone profile in insulin-resistant Zucker fatty rats (5, 8, 12 and 16 weeks of age, n = 6-7).

Results

Ovaries from 5-week-old fatty rats had significantly greater total and atretic follicle numbers, and higher atretic-to-total follicle ratios than those from lean rats. Ovaries from 12- and 16-week-old fatty rats showed interstitial cell hyperplasia and numerous cysts with features of advanced follicular atresia. In addition, serum testosterone and androstenedione levels significantly declined in fatty rats from age 8 to 16 weeks, so that fatty rats showed significantly lower levels of serum testosterone (12 and 16 weeks) and androstenedione (all weeks) than lean rats. This may reflect a reduction of androgen synthesis during follicular atresia. Serum adiponectin levels were high in immature fatty rats, and although the levels declined significantly as they matured, it remained significantly higher in fatty rats than in lean rats. On the other hand, levels of ovarian adiponectin and its receptors were significantly lower in mature fatty rats than in lean mature rats or immature fatty rats.

Conclusions

Our findings indicate that ovarian morphology and hormone profiles are significantly altered by the continuous insulin resistance in Zucker fatty rats. Simultaneously, abrupt reductions in serum and ovarian adiponectin also likely contribute to the infertility seen in fatty rats.     

Enhanced target-specific accumulation of radiolabeled antibodies by conjugating arginine-rich peptides as anchoring molecules

We have devised and estimated a new strategy to prolong the residence time of radiolabeled antibodies in tumor in which an octaarginine peptide (R?) was used as an anchoring molecule to fix antibodies against CD20 (NuB2; IgG2a) on tumor cells. Conjugation of R? with antibodies was performed by maleimide-thiol chemistry using thiol groups generated by reducing the disulfide bonds of the antibody. The R?-conjugated NuB2 was then reacted with succinimidyl meta-[12?I]iodobenzoate to prepare [12?I]SIB-NuB2(I) (0.92 R?/NuB2) and [12?I]SIB-NuB2(III) (3.38 R?/NuB2). Both SIB-NuB2(I) and SIB-NuB2(III) exhibited size-exclusion HPLC elution profiles and immunoreactivity to CD20-positive cells similar to those of NuB2. NuB2(I) also possessed isoelectric focusing (IEF) profile similar to NuB2. However, NuB2(III) registered a broad IEF band toward higher pI. When incubated with CD20-positive cells, [12?I]SIB-NuB2(I) and [12?I]SIB-NuB2(III) exhibited 1.4 and 4.0 times higher cell-associated radioactivity than [12?I]SIB-NuB2. After the cells were washed and reincubated in a fresh medium for 3 h, [12?I]SIB-NuB2(I) and [12?I]SIB-NuB2(III) exhibited significantly higher cell-associated radioactivity than [12?I]SIB-NuB2. In biodistribution studies in normal mice, while both [12?I]SIB-NuB2(I) and [12?I]SIB-NuB2 exhibited similar biodistribution profiles, [12?I]SIB-NuB2(III) showed faster clearance from the blood and higher hepatic radioactivity levels than [12?I]SIB-NuB2. In SCID mice bearing CD20-positive xenografts, [131I]SIB-NuB2(I) exhibited significantly higher radioactivity in xenografts than those of [12?I]SIB-NuB2 with no significant increase being observed in other tissues. The findings indicate that appropriate R? modification of antibodies satisfies both specific targeting ability of antibody and strong cell-association property of R?, which was reflected in the increased radioactivity levels in tumor. These findings supported the applicability of this approach to enhance target-specific accumulation of radiolabeled antibodies.     

Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories.     

Structure of the cadherin-related neuronal receptor/protocadherin-alpha first extracellular cadherin domain reveals diversity across cadherin families

The recent explosion in genome sequencing has revealed the great diversity of the cadherin superfamily. Within the superfamily, protocadherins, which are expressed mainly in the nervous system, constitute the largest subgroup. Nevertheless, the structures of only the classical cadherins are known. Thus, to broaden our understanding of the adhesion repertoire of the cadherin superfamily, we determined the structure of the N-terminal first extracellular cadherin domain of the cadherin-related neuronal receptor/protocadherin-alpha4. The hydrophobic pocket essential for homophilic adhesiveness in the classical cadherins was not found, and the functional significance of this structural domain was supported by exchanging the first extracellular cadherin domains of protocadherin and classical cadherin. Moreover, potentially crucial variations were observed mainly in the loop regions. These included the protocadherin-specific disulfide-bonded Cys-X(5)-Cys motif, which showed Ca(2+)-induced chemical shifts, and the RGD motif, which has been suggested to be involved in heterophilic cell adhesion via the active form of beta1 integrin. Our findings reveal that the adhesion repertoire of the cadherin superfamily is far more divergent than would be predicted by studying the classical cadherins alone.     

Expression of isoforms of NADPH oxidase components in rat pancreatic islets

Increased oxidative stress plays a role in the pathogenesis of beta-cell dysfunction and death. We studied isoforms of NADPH oxidase components in islets of Langerhans isolated from rat pancreas and tumoral rat beta-cell line RINm5F cells by RT-PCR and sequencing of its products. RT-PCR revealed that isolated islets constitutively expressed mRNA of NADPH oxidase components, Nox1, Nox2, Nox4 and p22(phox) as membrane-associated components and p47(phox), Noxo1 (homologue of p47(phox)), Noxa1 (homologue of p67(phox)), and p40(phox) as cytosolic components. RINm5F cells showed a similar pattern of expression but Nox2 mRNA was not detected. Expression of Nox1, Nox4, Noxo1 and Noxa1 was confirmed by sequencing the PCR products. Immunohistochemistry revealed the expression of NADPH oxidase component in beta-cells of rat pancreatic islets. Glucose-stimulated insulin secretion from isolated islets was suppressed by diphenyleneiodonium, a flavocytochrome inhibitor, but not by apocynin, an inhibitor of p47(phox) translocation to membranes. Our results suggest that the functional significance of NADPH oxidase in insulin secretion may merit further investigation.     

Bi-phasic activation of eNOS in response to uni-axial cyclic stretch is mediated by differential mechanisms in BAECs

We investigated the signaling mechanism of stretch-induced NO (Nitric oxide) production in bovine arterial endothelial cells (BAECs). BAECs cultured on an elastic silicone chamber coated with fibronectin were subjected to uni-axial cyclic stretch (1 Hz, 20% in length) and the amount of produced NO was measured by a cGMP assay. NO production increased in a bi-phasic manner and peaked at 5 min and 20 min after stretch onset. Correspondingly, the activities of endothelial nitric oxide synthase (eNOS) and Akt/PKB (measured by phosphorylation at serine 1,177 and serine 473, respectively), showed two peaks over time. Application of Gd(3+), a potent SA channel blocker, and depletion of external Ca(2+) exclusively inhibited the first peaks of eNOS and Akt activity, but exerted little effect on the second peak. On the other hand, the PI3K inhibitors, Wortmannin, LY294002, almost completely inhibited the second peak but not the first. These results suggest that up-regulation of eNOS in response to cyclic stretch was mediated by two distinct pathways, [Ca(2+)](i) increases via the SA channel in an early phase (partially Akt/PKB), and PI3K-Akt/PKB pathways in a late phase.