The papaya Kunitz-type trypsin inhibitor is a highly stable beta-sheet glycoprotein

The papaya Kunitz-type trypsin inhibitor, a 24-kDa glycoprotein, was purified to homogeneity. The purified inhibitor stoichiometrically inhibits bovine trypsin in a 1:1 molar ratio. Circular dichroism and infrared spectroscopy analyses demonstrated that the inhibitor contains extensive beta-sheet structures. The inhibitor was found to retain its full inhibitory activity over a broad pH range (1.5-11.0) and temperature (up to 80 degrees C), besides being stable at very high concentrations of strong chemical denaturants (e.g., 5.5 M guanidine hydrochloride). The inhibitor retained its compact structure over the pH range analyzed as shown by 8-anilino-1-naphtalenesulfonic acid binding characteristics, excluding the formation of some relaxed or molten state. Exposure to 2.5 mM dithiothreitol for 120 min caused a 33% loss of the inhibitory activity, while a loss of 75% was obtained in the presence of 20 mM of dithiothreitol during the same time period. A complete loss of the inhibitory activity was observed after incubation with 50 mM dithiothreitol for 5 min. Incubation of the inhibitor with general proteases belonging to different families revealed its extraordinary resistance to proteolysis in comparison with the soybean trypsin inhibitor, the archetypal member of the Kunitz-type inhibitors family. The inhibitor also exhibited a remarkable resistance to proteolytic degradation against pepsin for at least a 24-h incubation period. Instead, the soybean inhibitor was completely degraded after 2 h incubation with this aspartic protease. All these data demonstrated the high stability of the papaya trypsin inhibitor.     

Synthesis, characterisation and solution behaviour of fluoroalkyl phosphoryl complexes of tin tetrachloride

The synthesis, characterisation and solution behaviour of a series of octahedral complexes SnCl₄·2L (L = R₂NP(O)(OCH₂CF₃)₂; R = Me (1); Et (2) or L = P(O)(OCH₂R_f)₃; R_f = CF₃ (3); C₂F₅ (4)) are described. Complexes 1-4 were prepared from SnCl₄ and 2 equiv. of the ligand, L, in anhydrous CH₂Cl₂ solution. The adducts have been characterised by multinuclear (¹H, ³¹P and ¹¹⁹Sn) NMR, IR spectroscopy and elemental analysis. In dichloromethane solution, the NMR data showed the presence of a mixture of cis and trans isomers for 1 and 2 and only the cis isomer for 3 and 4. The difference could be interpreted in terms of the electronic effects of the substituents on the phosphorus atom of the ligand. In addition, the solution structure of the complexes studied by variable temperature ³¹P-{¹H} and ¹H NMR in the presence of excess ligand indicated that the ligand exchange on the cis isomer dominates the chemistry. The metal-ligand exchange barriers were estimated to be 13.38 and 11.39 kcal/mol for 1 and 3, respectively. The results are discussed and compared with those previously reported for the related hexamethylphosphoramide adduct, SnCl₄·2HMPA.     

Detrimental role of IL-33/ST2 pathway sustaining a chronic eosinophil-dependent Th2 inflammatory response,tissue damage and parasite burden during Toxocara canis infection in mice

Toxocariasis is a neglected disease that affects people around the world. Humans become infected by accidental ingestion of eggs containing Toxocara canis infective larvae, which upon reaching the intestine, hatch, penetrate the mucosa and migrate to various tissues such as liver, lungs and brain. Studies have indicated that Th2 response is the main immune defense mechanism against toxocariasis, however, there are still few studies related to this response, mainly the IL-33/ST2 pathway. Some studies have reported an increase in IL-33 during helminth infections, including T. canis. By binding to its ST2 receptor, IL-33 stimulating the Th2 polarized immune cell and cytokine responses. Thus, we aimed to investigate the role of the IL-33/ST2 pathway in the context of T. canis larval migration and the immunological and pathophysiological aspects of the infection in the liver, lungs and brain from Wild-Type (WT) BALB/c background and genetically deficient mice for the ST2 receptor (ST2^-/-). The most important findings revealed that the IL-33/ST2 pathway is involved in eosinophilia, hepatic and cerebral parasitic burden, and induces the formation of granulomas related to tissue damage and pulmonary dysfunction. However, ST2^-/- mice, the immune response was skewed to Th1/Th17 type than Th2, that enhanced the control of parasite burden related to IgG2a levels, tissue macrophages infiltration and reduced lung dysfunction. Collectively, our results demonstrate that the Th2 immune response triggered by IL-33/ST2 pathway mediates susceptibility to T. canis, related to parasitic burden, eosinophilia and granuloma formation in which consequently contributes to tissue inflammation and injury.     

Casein kinase 1δ activates human recombinant deoxycytidine kinase by Ser-74 phosphorylation, but is not involved in the in vivo regulation of its activity

Deoxycytidine kinase (dCK) is a key enzyme in the salvage of deoxynucleosides and in the activation of several anticancer and antiviral nucleoside analogues. We recently showed that dCK was activated in vivo by phosphorylation of Ser-74. However, the protein kinase responsible was not identified. Ser-74 is located downstream a Glu-rich region, presenting similarity with the consensus phosphorylation motif of casein kinase 1 (CKI), and particularly of CKI δ. We showed that recombinant CKI δ phosphorylated several residues of bacterially overexpressed dCK: Ser-74, but also Ser-11, Ser-15, and Thr-72. Phosphorylation of dCK by CKI δ correlated with increased activity reaching at least 4-fold. Site-directed mutagenesis demonstrated that only Ser-74 phosphorylation was involved in dCK activation by CKI δ, strengthening the key role of this residue in the control of dCK activity. However, neither CKI δ inhibitors nor CKI δ siRNA-mediated knock-down modified Ser-74 phosphorylation or dCK activity in cultured cells. Moreover, these approaches did not prevent dCK activation induced by treatments enhancing Ser-74 phosphorylation. Taken together, the data preclude a role of CKI δ in the regulation of dCK activity in vivo. Nevertheless, phosphorylation of dCK by CKI δ could be a useful tool for elucidating the influence of Ser-74 phosphorylation on the structure-activity relationships in the enzyme.     

On calcium-buffer dynamics within the excess buffer regime

In intracellular calcium signaling, calcium buffers has been recognized for their role in reshaping and localizing the calcium concentration profile in the vicinity of the channel, as well as reducing the effective diffusion of free calcium. In the presence of an excess of endogenous or exogenous buffers, linearization of the reaction-diffusion system describing the calcium-buffer dynamics has been instrumental in understanding the extent of the microdomain formation and in quantifying the apparent diffusion of the free calcium. In these linearized models, the conclusions are usually drawn from the steady-state solutions upon the opening of the channel. In this work, using the joint Laplace-Fourier method, we give an explicit integral transient solution, as well as, the long-time asymptotic behavior of the linearized calcium-buffer dynamics. The results confirm and emphasize the long stated intuitions on the diffusive character of the calcium-buffer dynamics. Numerical validations of our analytical results will be discussed.     

Variation in Volatile Leaf Oils of 13 Eucalyptus Species Harvested from Souinet Arboreta (Tunisia)

Hydrodistillation of the dried leaves of 13 species of the genus Eucalyptus L' Hér ., viz., E. bicostata Maiden, Blakely & Simmonds , E. cinerea F. Muell . ex Benth ., E. exerta F. Muell ., E. gigantea Hook . f ., E. gunnii Hook . f ., E. macarthurii Deane & Maiden ., E. macrorrhyncha F. Muell ., E. maidenii F. Muell ., E. odorata Behr ., E. pauciflora Sieber ex Sprengel , E. sideroxylon A. Cunn . ex Woolls , E. tereticornis Sm ., and E. viminalis Labill ., harvested from Souinet arboreta (region of Ain Draaham, north of Tunisia) in June 2006, afforded essential oils in yields varying from 0.5±0.2 to 3.9±0.4%, dependent on the species. E. cinerea and E. exerta provided the highest and the lowest percentage of essential oil amongst all the species examined, respectively. Analysis by GC (RI) and GC/MS allowed the identification of 142 components, representing 81.5 to 98.9% of the total oil. The contents of the different samples varied according to the species. The main components were 1,8‐cineole ( 1 ), followed by cryptone, spathulenol ( 4 ), p‐cymene ( 2 ), viridiflorol ( 6 ), globulol ( 7 ), β‐eudesmol, α‐terpineol ( 5 ), limonene ( 8 ), D ‐piperitone, α‐pinene ( 3 ), cuminal, and γ‐eudesmol. The principal component and the hierarchical cluster analyses separated the 13 Eucalyptus leaf essential oils into three groups, each constituting a chemotype.     

Cedrus libani (A. Rich) distribution in Lebanon: Past,present and future

Long-term vegetation studies are needed to better predict the impact of future climate change on vegetation structure and distribution. According to the IPCC scenario, the Mediterranean region is expected to undergo significant climatic variability over the course of this century. Cedrus libani (A. Rich), in particular, is currently distributed in limited areas in the Eastern Mediterranean region, which are expected to be affected by such climate change. In order to predict the impact of future global warming, we have used fossil pollen data and model simulations. Palaeobotanical data show that C. libani has been affected by both climate change and human activities. Populations of C. libani survived in refugial zones when climatic conditions were less favourable and its range extended during periods of more suitable climate conditions. Simulations of its future geographical distribution for the year 2100 using a dynamic vegetation model show that only three areas from Mount Lebanon may allow its survival. These results extrapolated for cedar forests for the entire Eastern Mediterranean region show that forests in Syria are also threatened by future global warming. In southern Turkey, cedar forests seem to be less threatened. These results are expected to help in the long-term conservation of cedar forests in the Near East.     

Combination of quantification and observation methods for study of "Side Population" cells in their "in vitro" microenvironment.

BACKGROUND: Qualitative and quantitative analyses of the rare phenotypic variants in in vitro culture systems is necessary for the understanding of cell differentiation in cell culture of primary cells or cell lines. Slide-based cytometry combines image acquisition and data treatment, and associates the power of flow cytometry (FCM) and the resolution of the microscopic studies making it suitable for the analysis of cells with rare phenotype. In this paper we develop a method that applies these principles to a particularly hot problem in cell biology, the study of stem cell like cells in cultures of primary cells, cancer cells, and various cell lines. METHODS: The adherent cells were labeled by the fluorescent dye Hoechst 33342. The images of cell populations were collected by a two-photon microscope and processed by a software developed by us. The software allows the automated segmentation of the nuclei in a very dense cell environment, the measurement of the fluorescence intensity of each nucleus and the recording of their position in the plate. The cells with a given fluorescence intensity can then be located easily on the recorded image of the culture plate for further analysis. RESULTS: The potential of our method is illustrated by the identification and localization of SP cells in the cultures of the C2C12 cell line. Although these cells represent only about 1% of the total population as calculated by flow cytometry, they can be identified in the culture plate with high precision by microscopy. CONCLUSION: Cells with the rare stem-cell like phenotype can be efficiently identified in the undisturbed cultures. Since the fluorescence intensity of rare events and the position of thousands of surrounding cells are recorded at the same time, the method associates the advantage of the FCM analysis and the microscopic observation.