Levels of germination proteins in dormant and superdormant spores of Bacillus subtilis

Bacillus subtilis spores that germinated poorly with saturating levels of nutrient germinants, termed superdormant spores, were separated from the great majority of dormant spore populations that germinated more rapidly. These purified superdormant spores (1.5 to 3% of spore populations) germinated extremely poorly with the germinants used to isolate them but better with germinants targeting germinant receptors not activated in superdormant spore isolation although not as well as the initial dormant spores. The level of β-galactosidase from a gerA-lacZ fusion in superdormant spores isolated by germination via the GerA germinant receptor was identical to that in the initial dormant spores. Levels of the germination proteins GerD and SpoVAD were also identical in dormant and superdormant spores. However, levels of subunits of a germinant receptor or germinant receptors activated in superdormant spore isolation were 6- to 10-fold lower than those in dormant spores, while levels of subunits of germinant receptors not activated in superdormant spore isolation were only ≤ 2-fold lower. These results indicate that (i) levels of β-galactosidase from lacZ fusions to operons encoding germinant receptors may not be an accurate reflection of actual germinant receptor levels in spores and (ii) a low level of a specific germinant receptor or germinant receptors is a major cause of spore superdormancy.     

Germination protein levels and rates of germination of spores of Bacillus subtilis with overexpressed or deleted genes encoding germination proteins

Deletion of Bacillus subtilis spores' GerA germinant receptor (GR) had no effect on spore germination via the GerB plus GerK GRs, and loss of GerB plus GerK did not affect germination via GerA. Loss of one or two GRs also did not affect levels of GRs that were not deleted. Overexpression of GRs 5- to 18-fold increased rates of germination via the overexpressed GR and slowed germination by other GRs up to 15-fold. However, overexpression of one or two GRs had no effect on levels of GRs that were not overexpressed. These results suggest that either interaction between different GRs reduces the activity of GRs in triggering spore germination or all GRs compete for interaction with a limiting amount of a downstream signaling molecule in the germination pathway. Overexpression or deletion of GRs also had no effect on spores' levels of the GerD protein needed for normal GR-dependent germination or of the SpoVAD protein likely involved in dipicolinic acid release early in germination. Loss of GerD also had no effect on levels of GRs or SpoVAD. Spores of a strain lacking the only B. subtilis prelipoprotein diacylglycerol transferase, GerF, also had no detectable GerD or the GerA's C subunit, both of which are most likely lipoproteins; GerA's A subunit was also absent. However, levels of GerB's C subunit, also almost certainly a lipoprotein, and GerK's A subunit were normal in gerF spores. These results with gerF spores were consistent with effects of loss of GerF on spore germination by different GRs.     

ETS-1 protein regulates vascular endothelial growth factor-induced matrix metalloproteinase-9 and matrix metalloproteinase-13 expression in human ovarian carcinoma cell line SKOV-3

Matrix metalloproteinase-mediated degradation of extracellular matrix is a crucial event for invasion and metastasis of malignant cells. The expressions of matrix metalloproteinases (MMPs) are regulated by different cytokines and growth factors. VEGF, a potent angiogenic cytokine, induces invasion of ovarian cancer cells through activation of MMPs. Here, we demonstrate that invasion and scattering in SKOV-3 cells were induced by VEGF through the activation of p38 MAPK and PI3K/AKT pathways. VEGF induced the expression of MMP-2, MMP-9, and MMP-13 and hence regulated the metastasis of SKOV-3 ovarian cancer cells, and the activities of these MMPs were reduced after inhibition of PI3K/AKT and p38 MAPK pathways. Interestingly, VEGF induced expression of ETS-1 factor, an important trans-regulator of different MMP genes. ETS-1 bound to both MMP-9 and MMP-13 promoters. Furthermore, VEGF acted through its receptor to perform the said functions. In addition, VEGF-induced MMP-9 and MMP-13 expression and in vitro cell invasion were significantly reduced after knockdown of ETS-1 gene. Again, VEGF-induced MMP-9 and MMP-13 promoter activities were down-regulated in ETS-1 siRNA-transfected cells. VEGF enriched ETS-1 in the nuclear fraction in a dose-dependent manner. VEGF-induced expression of ETS-1 and its nuclear localization were blocked by specific inhibitors of the PI3K and p38 MAPK pathways. Therefore, based on these observations, it is hypothesized that the activation of PI3K/AKT and p38 MAPK by VEGF results in ETS-1 gene expression, which activates MMP-9 and MMP-13, leading to the invasion and scattering of SKOV-3 cells. The study provides a mechanistic insight into the prometastatic functions of VEGF-induced expression of relevant MMPs.     

Manipulation of inositol metabolism for improved plant survival under stress: a “network engineering approach”

Emergence of high-throughput sequencing tools and omics technologies paved the way for systems biology in last decade. While we have started to look at the biology of the plant in a more unified manner, the integration of such knowledge in agricultural biotechnology has become an arena of potential interest. The network of several central molecules operating in various life and developmental processes are now more adequately known, and fine tuning of such molecule pools, if connected to stress response, can result in enhanced stress tolerance of plants.This review interprets the potential of manipulation of myo-inositol and its derivatives in generation of transgenic crop plants. Being a molecule of central importance in plant life, inositol is connected to numerous life processes. The exploration of such pathways indicates that inositol itself and many of its derivatives can impart abiotic stress tolerance (against salinity, dehydration, chilling or oxidative stress) to plants when overexpressed. We propose that engineering inositol metabolic network is a potential approach towards stress-tolerant transgenic crop plant generation and thus its exploitation in agricultural biotechnology is the call of time.     

Ribozyme-mediated perlecan knockdown impairs chondrogenic differentiation of C3H10T1/2 fibroblasts

Perlecan (Pln) is an abundant heparan sulfate (HS) proteoglycan in the pericellular matrix of developing cartilage, and its absence dramatically disrupts endochondral bone formation. This study examined two previously unexamined aspects of the function of Pln in mesenchymal chondrogenesis in vitro. Using the well-established high-density micromass model of chondrogenic differentiation, we first examined the requirement for endogenous Pln synthesis and secretion through the use of Pln-targeted ribozymes in murine C3H10T1/2 embryonic fibroblasts. Second, we examined the ability of the unique N-terminal, HS-bearing Pln domain I (PlnDI) to synergize with exogenous bone morphogenetic protein-2 (BMP-2) to support later stage chondrogenic maturation of cellular condensations. The results provide clear evidence that the function of Pln in late stage chondrogenesis requires Pln biosynthesis and secretion, because 60%-70% reductions in Pln greatly diminish chondrogenic marker expression in micromass culture. Additionally, these data support the idea that while early chondrocyte differentiation can be supported by exogenous HS-decorated PlnDI, efficient late stage PlnDI-supported chondrogenesis requires both BMP-2 and Pln biosynthesis.     

Rac Inhibition Reverses the Phenotype of Fibrotic Fibroblasts

Background

Fibrosis, the excessive deposition of scar tissue by fibroblasts, is one of the largest groups of diseases for which there is no therapy. Fibroblasts from lesional areas of scleroderma patients possess elevated abilities to contract matrix and produce α−smooth muscle actin (α-SMA), type I collagen and CCN2 (connective tissue growth factor, CTGF). The basis for this phenomenon is poorly understood, and is a necessary prerequisite for developing novel, rational anti-fibrotic strategies.

Methods and Findings

Compared to healthy skin fibroblasts, dermal fibroblasts cultured from lesional areas of scleroderma (SSc) patients possess elevated Rac activity. NSC23766, a Rac inhibitor, suppressed the persistent fibrotic phenotype of lesional SSc fibroblasts. NSC23766 caused a decrease in migration on and contraction of matrix, and α−SMA, type I collagen and CCN2 mRNA and protein expression. SSc fibroblasts possessed elevated Akt phosphorylation, which was also blocked by NSC23766. Overexpression of rac1 in normal fibroblasts induced matrix contraction and α−SMA, type I collagen and CCN2 mRNA and protein expression. Rac1 activity was blocked by PI3kinase/Akt inhibition. Basal fibroblast activity was not affected by NSC23766.

Conclusion

Rac inhibition may be considered as a novel treatment for the fibrosis observed in SSc.     

Nuclear factor-kappa B localization and function within intrauterine tissues from term and preterm labor and cultured fetal membranes

Background  

The objective of this study was to quantify the nuclear localization and DNA binding activity of p65, the major transactivating nuclear factor-kappa B (NF-kappaB) subunit, in full-thickness fetal membranes (FM) and myometrium in the absence or presence of term or preterm labor.