Biophysical studies of a transmembrane peptide derived from the T cell antigen receptor

Core peptide (CP) is a unique peptide derived from thetransmembrane sequence of T cell antigen receptor (TCR)-alpha chain and is capable of inhibiting the immuneresponse both in vitro and in animal models of Tcell mediated inflammation. The structure of CP, withsequence GLRILLLKV, is similar to the amphipathic regionof many peptides. Unlike antimicrobial peptides,however, which damage cell membranes, electron microscopyand propidium iodide exclusion assays on cell membranessuggest that CP does not create pores and may act byinterfering with signal transduction at the membranelevel. To investigate this effect further we report theresults of ³¹P and ²H solid-state NMRspectroscopy of CP on model membranes. As predicted,even at high concentrations of CP, the structure of modelmembranes was not significantly perturbed. Only at thevery high peptide-to-lipid molar ratio of 1:10significant effects on the model membranes were observed. We conclude that CP does not destroy the integrity of thelipid bilayer.     

The Application of Acetolysis for Releasing Leaf Cuticular Membranes of Pandanus in Taxonomic Studies

Leaf cuticular membranes can be released cleanly and in one piece after a 10 min treatment of leaf in a 100 C acetolysis mixture: acetic anhydride, 9; and concentrated H₂SO₄,1 v/v. This method has been used till now only for studying pollen and spore morphology but was found quite appropriate for a taxonomic study of the leaf surface of the genus Pandanus.     

The CSF-1 receptor fashions the intestinal stem cell niche

Gastrointestinal (GI) homeostasis requires the action of multiple pathways. There is some controversy regarding whether small intestine (SI) Paneth cells (PCs) play a central role in orchestrating crypt architecture and their relationship with Lgr5 + ve stem cells. Nevertheless, we previously showed that germline CSF-1 receptor (Csf1r) knock out (KO) or Csf1 mutation is associated with an absence of mature PC, reduced crypt proliferation and lowered stem cell gene, Lgr5 expression. Here we show the additional loss of CD24, Bmi1 and Olfm4 expression in the KO crypts and a high resolution 3D localization of CSF-1R mainly to PC. The induction of GI-specific Csf1r deletion in young adult mice also led to PC loss over a period of weeks, in accord with the anticipated long life span of PC, changed distribution of proliferating cells and this was with a commensurate loss of Lgr5 and other stem cell marker gene expression. By culturing SI organoids, we further show that the Csf1r^?/? defect in PC production is intrinsic to epithelial cells as well as definitively affecting stem cell activity. These results show that CSF-1R directly supports PC maturation and that in turn PCs fashion the intestinal stem cell niche.     

M?ssbauer characterization of the tetraheme cytochrome c3 from Desulfovibrio baculatus (DSM 1743). Spectral deconvolution of the heme components.

M?ssbauer spectroscopy was used to study the tetraheme cytochrome c3 from Desulfovibrio baculatus (DSM 1743). Samples with different degrees of reduction were prepared using a redoxtitration technique. In the reduced cytochrome c3, all four hemes are reduced and exhibit diamagnetic M?ssbauer spectra typical for low-spin ferrous hemes (S = 0). In the oxidized protein, the hemes are low-spin ferric (S = 1/2) and exhibit overlapping magnetic M?ssbauer spectra. A method of differential spectroscopy was applied to deconvolute the four overlapping heme spectra and a crystal-field model was used for data analysis. Characteristic M?ssbauer spectral components for each heme group are obtained. Hyperfine and crystal-field parameters for all four hemes are determined from these deconvoluted spectra.     

Can dispersal buffer against salinity-driven zooplankton community change in Great Plains' lakes?

1. The North American Great Plains contains thousands of lakes that vary in salinity from freshwater to hypersaline. Paleolimnological studies show that salinity levels in these lakes are tightly linked with climate, and current projections point to a more arid future in the region due to natural and anthropogenic climate change, potentially influencing lake salinity.
2. Many zooplankton species are sensitive to changes in salinity, and their position near the base of the aquatic food web makes it important to understand how they might respond to increasing salinity levels. Zooplankton communities in lakes with rising salinity levels may exhibit changes in structure, including a shift toward more salinity-tolerant species and a reduction in abundance, species richness, and diversity. However, it is possible that dispersal of zooplankton among lakes could mitigate such community changes when migrant populations replace sensitive zooplankton with those that are locally adapted to higher salinities.
3. To test if dispersal could reduce salinity-induced changes in zooplankton communities, we ran a field enclosure experiment at a freshwater lake in southern Saskatchewan where we manipulated salinity levels and zooplankton dispersal. We evaluated how salinity and dispersal influenced species identities and relative abundances (community structure) using multivariate statistics and comparing taxonomic and functional compositions among the different treatments (richness, diversity, and evenness).
4. We found that increasing salinity levels in our enclosures above that in our study lake resulted in lower zooplankton abundances and species richness levels, primarily due to the loss of cladoceran species. However, patterns in our multivariate analyses suggested that cladocerans were maintained in enclosures with salinity levels of 2.5 and 5.0 g/L when those enclosures received immigration from nearby lakes.
5. In contrast, our univariate analyses failed to find evidence that immigration affected community structure (richness, diversity, evenness). The lack of significant statistical differences could suggest that dispersal does not have an effect, or it may have been a problem with statistical power, as a power analysis suggested that fairly large effect sizes would have been required to achieve statistical significance.
6. Based on our results, we were unable to reach a definitive conclusion on the role that dispersal might play in buffering zooplankton communities against salinity-driven changes. However, our study provides two important insights for planning future work. First, our power analyses indicated that more replication may be needed given the variability among our experimental enclosures. Second, the patterns in our multivariate analyses suggested that cladocerans could be maintained in lakes undergoing salinity increases if they receive immigration from surrounding lakes with higher salinities. Future work examining how inter- and intraspecific salinity tolerance varies across lakes with a gradient of salinities would be helpful for understanding the role that dispersal might play in buffering against salinity-driven losses of cladoceran zooplankton.

     

Prediction and confirmation of a switch-like region within the N-terminal domain of hSIRT1

Many proteins display conformational changes resulting from allosteric regulation. Often only a few residues are crucial in conveying these structural and functional allosteric changes. These regions that undergo a significant change in structure upon receiving an input signal, such as molecular recognition, are defined as switch-like regions. Identifying these key residues within switch-like regions can help elucidate the mechanism of allosteric regulation and provide guidance for synthetic regulation. In this study, we combine a novel computational workflow with biochemical methods to identify a switch-like region in the N-terminal domain of human SIRT1 (hSIRT1), a lysine deacetylase that plays important roles in regulating cellular pathways. Based on primary sequence, computational methods predicted a region between residues 186–193 in hSIRT1 to exhibit switch-like behavior. Mutations were then introduced in this region and the resulting mutants were tested for allosteric reactions to resveratrol, a known hSIRT1 allosteric regulator. After fine-tuning the mutations based on comparison of known secondary structures, we were able to pinpoint M193 as the residue essential for allosteric regulation, likely by communicating the allosteric signal. Mutation of this residue maintained enzyme activity but abolished allosteric regulation by resveratrol. Our findings suggest a method to predict switch-like regions in allosterically regulated enzymes based on the primary sequence. If further validated, this could be an efficient way to identify key residues in enzymes for therapeutic drug targeting and other applications.