Effects of curcumin on methyl methanesulfonate damage to mouse kidney

Methylmethane sulfonate (MMS) is an alkylating agent that may react with DNA and damage it. We investigated histological changes and apoptosis caused by MMS and the effects of curcumin on MMS treated mouse kidneys. Twenty-four mice were divided into four equal groups: controls injected with saline, a group injected with 40 mg/kg MMS, a group injected with 40 mg/kg MMS and given 100 mg/kg curcumin by gavage, and a group given 100 mg/kg curcumin by gavage. MMS caused congestion and vacuole formation, and elevated the apoptotic index significantly, but had no other effect on kidney tissue. Curcumin improved the congestion and vacuole formation caused by MMS and decreased the apoptotic index. Curcumin administered with MMS appears to decrease the deleterious effects of MMS on the kidney.     

Changes in purine specificity in tandem GAF chimeras from cyanobacterial cyaB1 adenylate cyclase and rat phosphodiesterase 2

The C-terminal catalytic domains of the 11 mammalian phosphodiesterase families (PDEs) are important drug targets. Five of the 11 PDE families contain less well-characterized N-terminal GAF domains. cGMP is the ligand for the GAF domains in PDEs 2, 5, 6 and 11, and cAMP is the ligand for PDE10. Structurally related tandem GAF domains signalling via cAMP are present in the cyanobacterial adenylate cyclases cyaB1 and cyaB2. Because current high-resolution crystal structures of the tandem GAF domains of PDE2 and cyaB2 do not reveal how cNMP specificity is encoded, we generated chimeras between the tandem GAF domains of cyaB1 and PDE2. Both bind the ligand in the GAF B subdomains. Segmental replacements in the highly divergent beta1-beta3 region of the GAF B subdomain of cyaB1 by the corresponding PDE2 regions switched signalling from cAMP to cGMP. Using 10 chimeric constructs, we demonstrated that, for this switch in purine specificity, only 11% of the sequence of the cyanobacterial GAF B needs to be replaced by PDE2 sequences. We were unable, however, to switch the purine specificity of the PDE2 tandem GAF domain from cGMP to cAMP in reverse constructs, i.e. by replacement of PDE2 segments with those from the cyaB1 GAF tandem domain. The data provide a novel view on the structure-function relationships underlying the purine specificity of cNMP-binding GAF domains and indicate that, as potential drug targets, they must be characterized structurally and biochemically one by one.     

Immunogold labeling of rosette terminal cellulose-synthesizing complexes in the vascular plant vigna angularis

The catalytic subunit of cellulose synthase is shown to be associated with the putative cellulose-synthesizing complex (rosette terminal complex [TC]) in vascular plants. The catalytic subunit domain of cotton cellulose synthase was cloned using a primer based on a rice expressed sequence tag (D41261) from which a specific primer was constructed to run a polymerase chain reaction that used a cDNA library from 24 days postanthesis cotton fibers as a template. The catalytic region of cotton cellulose synthase was expressed in Escherichia coli, and polyclonal antisera were produced. Colloidal gold coupled to goat anti-rabbit secondary antibodies provided a tag for visualization of the catalytic region of cellulose synthase during transmission electron microscopy. With a freeze-fracture replica labeling technique, the antibodies specifically localized to rosette TCs in the plasma membrane on the P-fracture face. Antibodies did not specifically label any structures on the E-fracture face. Significantly, a greater number of immune probes labeled the rosette TCs (i.e., gold particles were 20 nm or closer to the edge of the rosette TC) than did preimmune probes. These experiments confirm the long-held hypothesis that cellulose synthase is a component of the rosette TC in vascular plants, proving that the enzyme complex resides within the structure first described by freeze fracture in 1980. In addition, this study provides independent proof that the CelA gene is in fact one of the genes for cellulose synthase in vascular plants.     

Response surface methodology, an approach to predict the effects of a lactoperoxidase system, Nisin, alone or in combination, on Listeria monocytogenes in skim milk

Experimental designs using Response Surface Methodology (RSM) were used to determine effects and interactions of Nisin (0-200 i.u. ml-1), pH values (5.4-6.6), incubation time (0-36 h or 0-144 h) and the lactoperoxidase-thiocyanate-hydrogen peroxide system (LPS) on Listeria monocytogenes CIP 82110 in skim milk, at 25 degrees C. The LPS varied from level 0-2; LPS at level 1 consisted of lactoperoxidase (35 mg l-1), thiocyanate (25 mg l-1) and H2O2, which was supplied exogenously by glucose-oxidase (1 mg l-1) and glucose (0.2 g l-1); LPS activity was dependent on LPS level and incubation time. In the presence of LPS at level 1, a bacteriostatic phase was followed by growth, whereas at a higher level, a bactericidic phase was observed. Nisin response was time- and pH-dependent. Nisin was bactericidic at acidic pH values and for a short incubation time (12 h) only; then, a re-growth phase was observed. Nisin and LPS in combination gave an original response which lacked the transitory bactericidal effect of Nisin and had a continuously bactericidal affect, leading to 10 cfu ml-1 of L. monocytogenes at 144 h; the response was greatly affected by incubation time. Predicted values were in good agreement with experimental values. Response Surface Methodology is a useful experimental approach for rapid testing of the effects of inhibitors.     

Analysis of the pre-S2 N- and O-linked glycans of the M surface protein from human hepatitis B virus

The surface antigen of hepatitis B virus comprises a nested set of small (S), middle (M), and large (L) proteins, all of which are partially glycosylated in their S domains. The pre-S2 domain, present only in M and L proteins, is further N-glycosylated at Asn-4 exclusively in the M protein. Since the pre-S2 N-glycan appears to play a crucial role in the secretion of viral particles, the M protein may be considered as a potential target for antiviral therapy. For characterization of the pre-S2 glycosylation, pre-S2 (glyco)peptides were released from native, patient-derived hepatitis B virus subviral particles by tryptic digestion, separated from remaining particles, purified by reversed-phase high performance liquid chromatography, and identified by amino acid and N-terminal sequence analysis as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Pre-S2 N-glycans were characterized by anion exchange chromatography, methylation analysis, and on target sequential exoglycosidase digestions in combination with MALDI-TOF-MS, demonstrating the presence of partially sialylated diantennary complex-type oligosaccharides. In addition, the pre-S2 domain of M protein, but not that of L protein, was found to be partially O-glycosylated by a Gal(beta1-3)GalNAcalpha-, Neu5Ac(alpha2-3)Gal(beta1-3)GalNAcalpha-, or GalNAcalpha-residue. The respective O-glycosylation site was assigned to Thr-37 by digestion with carboxypeptidases in combination with MALDI-TOF-MS and by quadrupole time-of-flight electrospray mass spectrometry. Analytical data further revealed that about 90% of M protein is N-terminally acetylated.     

Evolution of the species-rich Cape flora

The Cape Floristic Region ('fynbos biome') has very high levels of plant species diversity and endemism. Much of this diversity is concentrated in a relatively small number of clades centered in the region (Cape clades), and these form a vegetation called 'fynbos'. The general explanation for the origin of this diversity is that much of it evolved in the Pliocene and Late Miocene in response to progressive aridification. We present a phylogenetic analysis of an almost complete species sample of the largest clade of Restionaceae, the third largest Cape clade. This indicates that the radiation of the Restionaceae started between 20 and 42 Myr ago, and since then there were no, or at most gradual, changes in the speciation rate in this clade. For seven other clades, the estimated starting dates for their radiation ranges from 7 to 20 Myr ago. Combining the radiation patterns for these clades shows that ca. 15% of the modern species evolved during the Pleistocene, and almost 40% since the beginning of the Pliocene. We suggest that these clades might have radiated in response to the fynbos vegetation increasing its extent in the Cape as a result of climatic change.     

Podosomes: adhesion hot-spots of invasive cells

Podosomes are highly dynamic, actin-rich adhesion structures of monocyte-derived cells, certain transformed fibroblasts and carcinoma cells and have recently also been discovered in an increasing number of other cell types. Because they are found mainly in motile cells and control the activity of matrix metalloproteases, podosomes are thought to contribute to tissue invasion and matrix remodeling. Importantly, podosomes are physiologically relevant organelles because they can be found in ex vivo models of invasive cells. Regulators of podosome turnover include tyrosine kinases, RhoGTPases, actin regulators and the microtubule system. Podosomes might also serve as an attractive model to study how integration of various signaling pathways controls actin dynamics. Here, we summarize and discuss the known structural, regulatory and functional features of podosomes, our aim being to stimulate further research into these unique structures.     

Contrasting patterns of radiation in African and Australian Restionaceae

The floras of the Mediterranean-climate areas of southern Africa and southwestern Australia are remarkably species rich. Because the two areas are at similar latitudes and in similar positions on their respective continents, they have probably had similar Cenozoic climatic histories. Here we test the prediction that the evolution of the species richness in the two areas followed a similar temporal progression by comparing the rates of lineage accumulation for African and Australian Restionaceae. Restionaceae (Poales) are typical and often dominant elements in the fynbos vegetation of the Cape Floristic Region of southern Africa and the kwongan vegetation of the Southwestern Floristic Province of Western Australia. The phylogeny of the family was estimated from combined datasets for rbcL and trnL-F sequences and a large morphological dataset; these datasets are largely congruent. The monophyly of Restionaceae is supported and a basal division into an African clade (approximately 350 species) and an Australian clade (146 species) corroborated. There is also support for a futher subdivision of these two large sister-clades, but the terminal resolution within the African clade is very weak. Fossil pollen records provided a minimum age of the common ancestor of Australian and African Restionaceae as 64-71 million years ago, and this date was used to calibrate a molecular clock. A molecular clock was rejected by a likelihood ratio test; therefore, rate changes between the lineages were smoothed using nonparametric rate smoothing. The rate-corrected ages were used to construct a plot of lineages through time. During the Palaeogene the Australian lineage diversity increased consistent with the predictions of the constant birthrate model, while the African lineage diversity showed a dramatic increase in diversification rate in the Miocene. Incomplete sampling obscures the patterns in the Neogene, but extending the trends to the modern extant diversity suggests that this acceleration in the speciation rate continued in the African clade, whereas the Australian clade retained a constant diversification rate. The substantial morphological and anatomical similarity between the African and Australian Restionaceae appear to preclude morphological innovations as possible explanations for the intercontinental differences. Most likely these differences are due to the greater geographical extent and ecological variation in temperate Australia than temperate Africa, which might have provided refugia for basal Restionaceae lineages, whereas the more mountainous terrain of southern Africa might have provided the selective regimes for a more rapid, recent speciation.