Proteomic map of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> CL Brener: the reference strain of the genome project

In this work two-dimensional gel electrophoresis combined with mass spectrometry was carried out in order to start the construction of a map of soluble proteins from epimastigote form of Trypanosoma cruzi CL Brener. This strain is a hybrid organism derived from two genotypes, T. cruzi I and T. cruzi II and was chosen for genome sequencing. The two-dimensional gel electrophoresis showed that most of proteins focused at 4–7 pH range. The identification demonstrated that several proteins were in multiple isoforms, such as tubulin and heat shock proteins. Potential targets for development of chemotherapeutic agents like arginine kinase, an enzyme absent from mammalian tissues that is involved in the energy supply of the parasite, were also detected.     

CCHCR1 Is Up-Regulated in Skin Cancer and Associated with EGFR Expression

Despite chronic inflammation, psoriatic lesions hardly ever progress to skin cancer. Aberrant function of the CCHCR1 gene (Coiled-Coil α-Helical Rod protein 1, HCR) within the PSORS1 locus may contribute to the onset of psoriasis. As CCHCR1 is expressed in certain cancers and regulates keratinocyte (KC) proliferation in a transgenic mouse model, we studied its relation to proliferation in cutaneous squamous cell cancer (SCC) cell lines by expression arrays and quantitative RT-PCR and in skin tumors by immunohistochemistry. CCHCR1 protein was detected in the pushing border of SCC and lining basal cell carcinoma islands. Different from psoriasis, Ki67 had a similar expression pattern as CCHCR1. The most intense CCHCR1 staining occurred in areas positive for epidermal growth factor receptor (EGFR). Expression of CCHCR1 mRNA was upregulated 30–80% in SCC lines when compared to normal KCs and correlated positively with Ki67 expression. The most aggressive and invasive tumor cell lines (RT3, FaDu) expressed CCHCR1 mRNA less than non-tumorigenic HaCaT cells. Moreover, the tumor promoters okadaic acid and menadione downregulated CCHCR1 mRNA. We conclude that both in psoriasis and the early stages of KC transformation, CCHCR1 may function as a negative regulator of proliferation, but beyond a certain point in oncogenesis cannot control this phenomenon any longer.     

Investigating the binding of β‐1,4‐galactan to Bacillus licheniformis β‐1,4‐galactanase by crystallography and computational modeling

Microbial β‐1,4‐galactanases are glycoside hydrolases belonging to family 53, which degrade galactan and arabinogalactan side chains in the hairy regions of pectin, a major plant cell wall component. They belong to the larger clan GH‐A of glycoside hydrolases, which cover many different poly‐ and oligosaccharidase specificities. Crystallographic complexes of Bacillus licheniformis β‐1,4‐galactanase and its inactive nucleophile mutant have been obtained with methyl‐β(1→4)‐galactotetraoside, providing, for the first time, information on substrate binding to the aglycone side of the β‐1,4‐galactanase substrate binding groove. Using the experimentally determined subsites as a starting point, a β(1→4)‐galactononaose was built into the structure and subjected to molecular dynamics simulations giving further insight into the residues involved in the binding of the polysaccharide from subsite ?4 to +5. In particular, this analysis newly identified a conserved β‐turn, which contributes to subsites ?2 to +3. This β‐turn is unique to family 53 β‐1,4‐galactanases among all clan GH‐A families that have been structurally characterized and thus might be a structural signature for endo‐β‐1,4‐galactanase specificity. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Palmitate and inflammatory state additively induce the expression of PTP1B in muscle cells

The factors responsible for up-regulation of PTP1B, a negative regulator of insulin signaling, in insulin resistance state are not well understood. We performed a series of experiments in C2C12 muscle cells to determine the role of palmitate and an inflammatory state in regulation of PTP1B. Palmitate (0.75 mM) induced PTP1B mRNA and protein level only at 16 h. The combination of palmitate and macrophages, accompanied by a great increase of TNF-α and IL-6 in the culture media, additively caused a higher level of PTP1B protein levels in the muscle. Higher concentrations of palmitate reduced insulin stimulated glucose uptake in myotubes. A specific inhibitor of PTP1B partly increased insulin stimulated glucose uptake in palmitate treated cells. In conclusion, our results showing the additive influence of palmitate and the inflammatory state in the expression of PTP1B imply the involvement of these factors in the overexpression of PTP1B in insulin resistance state. We further provided the evidence suggesting the mediatory role for PTP1B in palmitate induced insulin resistance in myotubes.     

Mycophagy and its influence on habitat use and ranging patterns in Callimico goeldii

Mycophagy has been documented in a number of species of marmosets and lion tamarins (Callitrichinae) but its effect on ranging behavior is not known. We present the results of 10 years of research on five groups of Goeldi's monkey (Callimico goeldii) at a field site in northwestern Bolivia. We studied the diet and ranging behavior of two of the groups. On average, groups contained 4.5 individuals (range 2.0–9.0), but they gradually decreased in size until only the breeding female remained in the home range. The annual diet was composed of fungi (31.1–34.9%), fruits (34.0–40.6%), prey (17.4–30.1%), and exudates (1.0–10.9%). They had large home ranges (114–150 ha) and over time individuals tended to shift their core areas of use. They used secondary and bamboo forest and forest with dense understories more than expected based on availability. We suggest that the large home ranges and shifting core areas used by C. goeldii are components of a foraging strategy to track patchy, low density, and ephemeral fungal fruiting bodies. Our results, along with data published on other callitrichines, indicate that groups of Leontopithecus, Callithrix, and Callimico that eat fungi have larger home ranges than those that do not. Mycophagy is one of the several factors that evidently affect home range size in callitrichines. Fungi are clearly an important food source for a number of populations, but additional studies are needed to determine why some eat fungi frequently while others do not. Am J Phys Anthropol, 2010. © 2010 Wiley‐Liss, Inc.     

Sonochemical synthesis of a new nano-plate lead(II) coordination polymer constructed of maleic acid

A new nano-sized lead(II) coordination polymer of maleic acid (H₂Mal), [Pb(μ₇-Mal)]_n (1), has been synthesized by sonochemical method and characterized by scanning electron microscopy, X-ray powder diffraction, elemental analyses and IR spectroscopy. The compound 1 was structurally characterized by single-crystal X-ray diffraction. Thermal stability of nano and bulk samples of compound 1 were studied and compared with each other. After calcination of nano-sized compound 1 at 600 °C, pure phase micro-sized lead(II) oxide has been produced.     

Complex Role of Collybistin and Gephyrin in GABAA Receptor Clustering

Gephyrin and collybistin are key components of GABA_A receptor (GABA_AR) clustering. Nonetheless, resolving the molecular interactions between the plethora of GABA_AR subunits and these clustering proteins is a significant challenge. We report a direct interaction of GABA_AR α2 and α3 subunit intracellular M3–M4 domain (but not α1, α4, α5, α6, β1–3, or γ1–3) with gephyrin. Curiously, GABA_AR α2, but not α3, binds to both gephyrin and collybistin using overlapping sites. The reciprocal binding sites on gephyrin for collybistin and GABA_AR α2 also overlap at the start of the gephyrin E domain. This suggests that although GABA_AR α3 interacts with gephyrin, GABA_AR α2, collybistin, and gephyrin form a trimeric complex. In support of this proposal, tri-hybrid interactions between GABA_AR α2 and collybistin or GABA_AR α2 and gephyrin are strengthened in the presence of gephyrin or collybistin, respectively. Collybistin and gephyrin also compete for binding to GABA_AR α2 in co-immunoprecipitation experiments and co-localize in transfected cells in both intracellular and submembrane aggregates. Interestingly, GABA_AR α2 is capable of “activating ” collybistin isoforms harboring the regulatory SH3 domain, enabling targeting of gephyrin to the submembrane aggregates. The GABA_AR α2-collybistin interaction was disrupted by a pathogenic mutation in the collybistin SH3 domain (p.G55A) that causes X-linked intellectual disability and seizures by disrupting GABA_AR and gephyrin clustering. Because immunohistochemistry in retina revealed a preferential co-localization of collybistin with α2 subunit containing GABA_ARs, but not GlyRs or other GABA_AR subtypes, we propose that the collybistin-gephyrin complex has an intimate role in the clustering of GABA_ARs containing the α2 subunit.     

The co-chaperone BAG3 interacts with the cytosolic chaperonin CCT: New hints for actin folding

It has been recently hypothesized that BAG3 protein, a co-chaperone of Hsp70/Hsc70, is involved in the regulation of several cell processes, such as apoptosis, autophagy and cell motility. Following the identification of Hsc70/Hsp70, further BAG3 molecular partners such as PLC-γ and HspB8 were likewise identified, thus contributing to the characterization of the mechanisms and the biological roles carried out by this versatile protein. By using a His-tagged BAG3 protein as bait, we fished out and identified the cytosolic chaperonin CCT, a new unreported BAG3 partner. The interaction between BAG3 and CCT was confirmed and characterized by co-immunoprecipitation experiments and surface plasmon resonance techniques. Furthermore, our analyses showed a slower CCT association and a faster dissociation with a truncated form of BAG3 containing the BAG domain, thus indicating that other protein regions are essential for a high-affinity interaction. ATP or ADP does not seem to significantly influence the chaperonin binding to BAG3 protein. On the other hand, our experiments showed that BAG3 silencing by small interfering RNA slowed down cell migration and influence the availability of correctly folded monomeric actin, analyzed by DNAse I binding assays and latrunculin A depolymerization studies. To our knowledge, this is the first report showing a biologically relevant interaction between the chaperonin CCT and BAG3 protein, thus suggesting interesting involvement in the folding processes regulated by CCT.     

Roles of trehalose and magnesium sulfate on structural and functional stability of firefly luciferase

Firefly luciferase is widely used in many analytical techniques. However, the enzyme is unstable, so that its relative inactivation results in low sensitivity of those techniques. In this study, we have investigated the effects of MgSO₄ and trehalose on the structural stability and function of luciferase from Photinus pyralis using circular dichroism (CD), conventional and stopped-flow fluorescence spectroscopy and bioluminescence assay. The secondary structural content, compactness and its melting temperature are also studied, which showed that the stability of luciferase increased in the presence of additives. Measurements of refolding rate constants under conditions that favor folding, show that MgSO₄ accelerates the folding of enzyme, on the contrary, refolding rate constant decreases in the presence of trehalose which can be attributed to its high viscosity. Finally, combined with remaining activity assay we concluded that magnesium sulfate and trehalose can be used for short- and long-term stabilization, respectively.