Differential Effects of Lysophosphatidic Acid and Phosphatidylinositol 4,5-Bisphosphate on Actin Dynamics by Direct Association with the Actin-binding Protein Villin

We have previously reported that the epithelial cell-specific actin-binding protein villin directly associates with phosphatidylinositol 4,5-bisphosphate (PIP₂) through three binding sites that overlap with actin-binding sites in villin. As a result, association of villin with PIP₂ in hibits actin depolymerization and enhances actin cross-linking by villin. In this study, we demonstrate that these three PIP₂-binding sites also bind the more hydrophilic phospholipid, lysophosphatidic acid (LPA) but with a higher affinity than PIP₂ (dissociation constant (K_d) of 22 μm versus 39.5 μm for PIP₂). More interestingly, unlike PIP₂, the association of villin with LPA inhibits all actin regulatory functions of villin. In addition, unlike PIP₂, LPA dramatically stimulates the tyrosine phosphorylation of villin by c-Src kinase. These studies suggest that in cells, selective interaction of villin with either PIP₂ or LPA could have dramatically different outcomes on actin reorganization as well as phospholipid-regulated cell signaling. These studies provide a novel regulatory mechanism for phospholipid-induced changes in the microfilament structure and cell function and suggest that LPA could be an intracellular regulator of the actin cytoskeleton.     

Environmental forcing as a main determinant of bloom dynamics of the Chrysochromulina algae

In this paper, we demonstrate that the seasonal dynamics in the abiotic factors, without including seasonal changes in the biological relationships, can appropriately account for the seasonal dynamics of Chrysochromulina spp. This is through the analysis of data on the population dynamics of Chrysochromulina spp. off southern Norway that is evaluated in relation to environmental factors and season by the analyses of 12 year monthly time-series. Chrysochromulina spp. abundance, nutrient concentrations, hydrographical properties, as well as current and wind data were analysed on a monthly scale by means of autoregressive moving average models, principal component analyses (PCA), and linear and nonlinear regression models. Seasonal development of the Chrysochromulina assemblage was well predicted from regression models forced with two PCA components representing seasonal variation in nutrient and chlorophyll a levels and ratios, inflow of North Seawater to the Skagerrak and northeasterly wind along the Norwegian coast. Assuming these to be general results, we might hypothesis that marine algal communities are governed by seasonally varying abiotic factors to a large extent.     

Expression and localization of locally produced growth factors regulating lymphangiogenesis during different stages of the estrous cycle in corpus luteum of buffalo (Bubalus bubalis)

Recent experiments using expression, immunolocalization, and cell culture approaches have provided leading insights into regulation of luteal angiogenesis by different growth factor systems and its role in the function of corpus luteum (CL) in buffalo. On the contrary, lymphangiogenesis and its regulation in the CL are still poorly understood. The aim of this study was to evaluate the expression and localization of lymphangiogenic factors (vascular endothelial growth factor [VEGF]-C and VEGFD), their receptor (VEGFR3), and lymphatic endothelial marker (LYVE1) in bubaline CL during different stages of the estrous cycle and to investigate functional role of VEGFC and VEGFD in luteal lymphangeogenesis. The mRNA and protein expression of VEGFC, VEGFD, and VEGFR3 was significantly greater in mid and late luteal phases, which correlated well with the expression of LYVE1. The lymphangiogenic factors were localized in luteal cells, exclusively in the cytoplasm. Immunoreactivity of VEGFC was greater during midluteal phase and that of VEGFD was greater during the mid and late luteal phases. Luteal cells were cultured in vitro and treated for different time duration (24, 48, and 72 hours) with VEGFC and VEGFD each at 50, 100, and 150 ng/mL concentration and VEGFC with VEGFD at 100 ng/mL concentration. The temporal increase in LYVE1 mRNA expression was significant (P < 0.05) in VEGFC and VEGFC with VEGFD treatment and no significant change was seen in VEGFD treatment. Thus, it seems likely that VEGFD itself has little role in lymphangiogenesis but along with VEGFC it might have a synergistic effect on VEGFR3 receptors for inducing lymphangiogenesis. In summary, the present study provided evidence that VEGFC and VEGFD, and their receptor VEGFR3, are expressed in bubaline CL and are localized exclusively in the cell cytoplasm, suggesting that these factors have a functional role in lymphangiogenesis of CL in buffalo.     

Design and in vitro evaluation of new rpoB-DGGE primers for ruminants

Two new primer sets based on the rpoB gene were designed and evaluated with bovine and ovine rumen samples. The newly developed rpoB-DGGE primer set was used along with the 16S rRNA gene-V3, and another (old) rpoB-DGGE-based primer set from a previous study to in vitro compare the bovine and ovine rumen ecosystems. The results indicate a significant (P<0.001) difference in the microbial population between the two ruminants irrespective of the primers used in the analysis. Qualitative comparison of the data provides evidence for the presence of similar phyla profiles between the 16S rRNA gene and the newly developed rpoB primers. A comparison between the two rpoB-based primer sets (old and new) showed that the old rpoB-based primers failed to amplify phylum Bacteroidetes (a common phylum in the rumen) in both bovine and ovine rumen samples. The old and new rpoB-DGGE-based primers amplified a large number of clones belonging to phylum Proteobacteria, providing a useful insight into the microbial structure of the rumen. ChaoI, ACE, Simpson, and Shannon-Weaver index analysis estimated the bovine rumen to be more diverse than the ovine rumen for all three primer sets. These results provide a new insight into the community structure among ruminants using the newly developed primers in this study.     

Decision-making critical amino acids: role in designing peptide vaccines for eliciting Th1 and Th2 immune response

CD4 T cells play a cardinal role in orchestrating immune system. Differentiation of CD4 T cells to Th1 and Th2 effector subsets depends on multiple factors such as relative intensity of interactions between T cell receptor with peptide-major histocompatibility complex, cytokine milieu, antigen dose, and costimulatory molecules. Literature supports the critical role of peptide’s binding affinity to Human Leukocyte Antigens (HLAs) and in the differentiation of naïve CD4 T cells to Th1 and Th2 subsets. However, there exists no definite report addressing very precisely the correlation between physicochemical properties (hydrophobicity, hydrophilicity), pattern, position of amino acids in peptide and their role in skewing immune response towards Th1 and Th2 cells. This may play a significant role in designing peptide vaccines. Hence in the present study, we have evaluated the relationship between amino acid pattern and their influence in differentiation of Th1 and Th2 cells. We have used a data set of 320 peptides, whose role has been already established experimentally in the generation of either Th1 or Th2 immune response. Further, characterization was done based on binding affinity, promiscuity, amino acid pattern and binding conformation of peptides. We have observed that distinct amino acids in peptides elicit either Th1 or Th2 immunity. Consequently, this study signifies that alteration in the sequence and type of selected amino acids in the HLA class II binding peptides can modulate the differentiation of Th1 and Th2 cells. Therefore, this study may have an important implication in providing a platform for designing peptide-based vaccine candidates that can trigger desired Th1 or Th2 response.     

Mutational analysis of the (p)ppGpp synthetase activity of the Rel enzyme of Mycobacterium tuberculosis

Rel_Mtb, a GTP pyrophosphokinase encoded by the Mycobacterium tuberculosis (Mtb) genome, catalyzes synthesis of (p)ppGpp from ATP and GDP(GTP) and its hydrolysis to GDP(GTP) and pyrophosphate to mediate stringent response, which helps bacteria to survive during nutrient limitation. Like other members of Rel_Spo homologs, Rel_Mtb has four distinct domains: HD, Rel_Spo (RSD), TGS and ACT. The N-terminal HD and RSD are responsible for (p)ppGpp hydrolysis and synthesis, respectively. In this study, we have dissected the rel _Mtb gene function and determined the minimal region essential for (p)ppGpp synthetic activity. The Rel_Mtb and its truncated derivatives were expressed from an arabinose inducible promoter (P _BAD ), and in vivo functional analyses were done in a (p)ppGpp null Escherichia coli strain. Our results indicate that only 243 amino acids (188–430 residues) containing fragment are sufficient for Rel_Mtb (p)ppGpp synthetic activity. The results were further confirmed by in vitro assays using purified proteins. We further characterized the RSD of Rel_Mtb by substituting several conserved amino acids with structurally related residues and identified six such residues, which appeared to be critical for maintaining its catalytic activity. Furthermore, we have also extended our analysis to an RSD encoding gene rv1366 of Mtb, and experimental results indicated that the encoded protein Rv1366 is unable to synthesize (p)ppGpp.     

Aromatic-aromatic interactions: analysis of π-π interactions in interleukins and TNF proteins

Aromatic-aromatic hydrogen bonds are important in many areas of chemistry, biology and materials science. In this study we haveanalyzed the roles played by the π-π interactions in interleukins (ILs) and tumor necrosis factor (TNF) proteins. Majority of π-πinteracting residues are conserved in ILs and TNF proteins. The accessible surface area calculations in these proteins reveal thatthese interactions might be important in stabilizing the inner core regions of these proteins. In addition to π-π interactions, thearomatic residues also form π-networks in ILs and TNF proteins. The results obtained in the present study indicate that π-πinteractions and π-π networks play important roles in the structural stability of ILs and TNF proteins.     

C-reactive Protein Exists in an NaCl Concentration-dependent Pentamer-Decamer Equilibrium in Physiological Buffer

C-reactive protein (CRP) is an acute phase protein of the pentraxin family that binds ligands in a Ca²⁺-dependent manner, and activates complement. Knowledge of its oligomeric state in solution and at surfaces is essential for functional studies. Analytical ultracentrifugation showed that CRP in 2 mm Ca²⁺ exhibits a rapid pentamer-decamer equilibrium. The proportion of decamer decreased with an increase in NaCl concentration. The sedimentation coefficients s_20,w⁰ of pentameric and decameric CRP were 6.4 S and in excess of 7.6 S, respectively. In the absence of Ca²⁺, CRP partially dissociates into its protomers and the NaCl concentration dependence of the pentamer-decamer equilibrium is much reduced. By x-ray scattering, the radius of gyration R_G values ranged from 3.7 nm for the pentamer to above 4.0 nm for the decamer. An averaged K_D value of 21 μm in solution (140 mm NaCl, 2 mm Ca²⁺) was determined by x-ray scattering and modeling based on crystal structures for the pentamer and decamer. Surface plasmon resonance showed that CRP self-associates on a surface with immobilized CRP with a similar K_D value of 23 μm (140 mm NaCl, 2 mm Ca²⁺), whereas CRP aggregates in low salt. It is concluded that CRP is reproducibly observed in a pentamer-decamer equilibrium in physiologically relevant concentrations both in solution and on surfaces. Both 2 mm Ca²⁺ and 140 mm NaCl are essential for the integrity of CRP in functional studies and understanding the role of CRP in the acute phase response.