Chitinolytic proteins secreted by Cellulosimicrobium sp. NTK2.

Cellulosimicrobium sp. NTK2 (NTK2 strain) was isolated as a chitinolytic bacterium from mature compost derived from chitinous waste. The growth of the NTK2 strain was enhanced by supplementation of the culture medium with 2% crystalline chitin. Approximately 70% of the supplemented crystalline chitin was degraded during cultivation. Whole genome analysis of the NTK2 strain identified eight chitinases and two chitin-binding proteins. The NTK2 strain secreted two bacterial extracellular solute-binding proteins, three family 18 glycosyl hydrolases and one lytic polysaccharide monooxygenase specifically in the presence of crystalline chitin. A chitinolytic enzyme with a molecular mass of 29 kDa on SDS-PAGE under native conditions was also secreted. This chitinolytic enzyme exhibited the largest band upon zymography but could not be identified. In an attempt to identify all the chitinases secreted by the NTK2 strain, we expressed recombinant versions of the proteins exhibiting chitinolytic activity in Escherichia coli. Our results suggest that the 29 kDa protein belonging to family 19 glycosyl hydrolase was expressed specifically in the presence of 2% crystalline chitin.

Multi-modal effects of BMP signaling on Nodal expression in the lateral plate mesoderm during left-right axis formation in the chick embryo.

During development of left-right asymmetry in the vertebrate embryo, Nodal plays a central role for determination of left-handedness. Bone morphogenetic protein (BMP) signaling has an important role for regulation of Nodal expression, although there is controversy over whether BMP signaling has a positive or negative effect on Nodal expression in the chick embryo. As BMP is a morphogen, we speculated that different concentrations might induce different responses in the cells of the lateral plate mesoderm (LPM). To test this hypothesis, we analyzed the effects of various concentrations of BMP4 and NOGGIN on Nodal expression in the LPM. We found that the effect on Nodal expression varied in a complex fashion with the concentration of BMP. In agreement with previous reports, we found that a high level of BMP signaling induced Nodal expression in the LPM, whereas a low level inhibited expression. However, a high intermediate level of BMP signaling was found to suppress Nodal expression in the left LPM, whereas a low intermediate level induced Nodal expression in the right LPM. Thus, the high and the low intermediate levels of BMP signaling up-regulated Nodal expression, but the high intermediate and low levels of BMP signaling down-regulated Nodal expression. Next, we sought to identify the mechanisms of this complex regulation of Nodal expression by BMP signaling. At the low intermediate level of BMP signaling, regulation depended on a NODAL positive-feedback loop suggesting the possibility of crosstalk between BMP and NODAL signaling. Overexpression of a constitutively active BMP receptor, a constitutively active ACTIVIN/NODAL receptor and SMAD4 indicated that SMAD1 and SMAD2 competed for binding to SMAD4 in the cells of the LPM. Nodal regulation by the high and low levels of BMP signaling was dependent on Cfc up-regulation or down-regulation, respectively. We propose a model for the variable effects of BMP signaling on Nodal expression in which different levels of BMP signaling regulate Nodal expression by a balance between BMP-pSMAD1/4 signaling and NODAL-pSMAD2/4 signaling.

Ontogeny of angiopoietin-like protein 1, 2, 3, 4, 5, and 7 genes during chick embryonic development.

Angiopoietin-like proteins (ANGPTLs) are secreted proteins possessing an amino-terminal coiled-coil domain and a carboxyl-terminal fibrinogen-like domain and are known as angiogenic factors. Several members of ANGPTLs also regulate lipid metabolism independently of angiogenic effects, but most of their functions during vertebrate development are not demonstrated. To ascertain their developmental functions, we examined the expression patterns of Angptl1, 2, 3, 4, 5, and 7 orthologues during chick development using whole-mount in situ hybridization. Angptl1 was first detected at embryonic day 3 (E3) in the somite. At E4, Angptl1 was expressed in somite-derivatives and limb mesenchyme. Angptl2 was first detected at E3 in the hindbrain. At E4, Angptl2 was expressed in neuroepithelium of forebrain and hindbrain and partly in the heart. Angptl3 was first detected at E3 and continued to be expressed in the liver and yolk sac at E4. Angptl4 was first detected at E3 in the somites and liver. At E4, Angptl4 was also observed in the heart. Angptl5 was not detected in these developmental stages. Angptl7 was first detected at E3 in the ectoderm overlying the lenses of the eyes. At E4, Angptl7 was specifically expressed in cornea. These data suggest that each member of the ANGPTL family could be related to angiogenesis during various organogeneses of the developing chick embryo.