Expression of the sucrose binding protein from soybean: renaturation and stability of the recombinant protein

The sucrose binding protein (SBP) belongs to the cupin family of proteins and is structurally related to vicilin-like storage proteins. In this investigation, a SBP isoform (GmSBP2/S64) was expressed in E. coli and large amounts of the protein accumulated in the insoluble fraction as inclusion bodies. The renatured protein was studied by circular dichroism (CD), intrinsic fluorescence, and binding of the hydrophobic probes ANS and Bis-ANS. The estimated content of secondary structure of the renatured protein was consistent with that obtained by theoretical modeling with a large predominance of beta-strand structure (42%) over the alpha-helix (9.9%). The fluorescence emission maximum of 303 nm for SBP2 indicated that the fluorescent tryptophan was completely buried within a highly hydrophobic environment. We also measured the equilibrium dissociation constant (K(d)) of sucrose binding by fluorescence titration using the refolded protein. The low sucrose binding affinity (K(d)=2.79+/-0.22 mM) of the renatured protein was similar to that of the native protein purified from soybean seeds. Collectively, these results indicate that the folded structure of the renatured protein was similar to the native SBP protein. As a member of the bicupin family of proteins, which includes highly stable seed storage proteins, SBP2 was fairly stable at high temperatures. Likewise, it remained folded to a similar extent in the presence or absence of 7.6M urea or 6.7 M GdmHCl. The high stability of the renatured protein may be a reminiscent property of SBP from its evolutionary relatedness to the seed storage proteins.     

Ordovician chitinozoans and acritarchs from southern and southeastern Turkey

Revision of the lithostratigraphy of Ordovician deposits in southern and southeastern Turkey led to a re-evaluation of the age assignments of formations identified in the subsurface and at outcrop. Previous datings were based on macrofauna (mainly trilobites and graptolites). The present paper focuses exclusively on organic-walled microfossils (chitinozoans and acritarchs), which provide numerous chronostratigraphical improvements, especially in successions barren or poor in macrofossils. Close to 200 samples were collected in the Taurus chain (i.e. from Kemer, Seydisehir, Ovacik, Kozan, to Sariz regions in southern Turkey) and in the Border Folds (Mardin and Hakkari regions), usually regarded as part of the Arabian Plate in palaeogeographical reconstructions. Many samples are productive and yield chitinozoans and/or acritarchs of extremely variable preservation, depending on their geographical and geological location. In the Taurus chain, the material is “coalified” and frequently fragmented whereas, in the Border Folds, maturation of the organic matter is much lower and preservation of the microfossils is good to excellent. Several Ordovician chitinozoan biozones (northern Gondwana zonation) as well as diagnostic acritarch assemblages are identified in southern and southeastern Turkey. These Ordovician formations are assigned here to the new global stages of the Ordovician chronostratigraphical scale. The Seydisehir (upper part), Sobova, and Kilgen Lake (lower part) formations are referred to the Darriwilian. The Kilgen Lake (upper part), Sort Tepe, and Bedinan formations are attributed to the Sandbian and to the Katian, and the Halevikdere Formation (glacio-marine part) is assigned to the Hirnantian. Reworking of Early Ordovician acritarchs is documented in pre-glacial and in glacial Late Ordovician deposits. They indicate that active erosive processes occurred during the Middle and Late Ordovician sedimentation. The organic-walled microfossils recorded in the Ordovician of south and southeastern Turkey belong to the northern Gondwana realm. Interestingly however, some Baltoscandian influences are noted in the Border Folds during Early Late Ordovician.     

Evidence for an interaction between the SH3 domain and the N-terminal extension of the essential light chain in class II myosins

The function of the src-homology 3 (SH3) domain in class II myosins, a distinct beta-barrel structure, remains unknown. Here, we provide evidence, using electron cryomicroscopy, in conjunction with light-scattering, fluorescence and kinetic analyses, that the SH3 domain facilitates the binding of the N-terminal extension of the essential light chain isoform (ELC-1) to actin. The 41 residue extension contains four conserved lysine residues followed by a repeating sequence of seven Pro/Ala residues. It is widely believed that the highly charged region interacts with actin, while the Pro/Ala-rich sequence forms a rigid tether that bridges the approximately 9 nm distance between the myosin lever arm and the thin filament. In order to localize the N terminus of ELC in the actomyosin complex, an engineered Cys was reacted with undecagold-maleimide, and the labeled ELC was exchanged into myosin subfragment-1 (S1). Electron cryomicroscopy of S1-bound actin filaments, together with computer-based docking of the skeletal S1 crystal structure into 3D reconstructions, showed a well-defined peak for the gold cluster near the SH3 domain. Given that SH3 domains are known to bind proline-rich ligands, we suggest that the N-terminal extension of ELC interacts with actin and modulates myosin kinetics by binding to the SH3 domain during the ATPase cycle.     

NMR structure and functional characterization of a human cancer-related nucleoside triphosphatase

A screen of the human cancer genome anatomy project (CGAP) database was performed to search for new proteins involved in tumorigenesis. The resulting hits were further screened for recombinant expression, solubility and protein aggregation, which led to the identification of the previously unknown human cancer-related (HCR) protein encoded by the mRNA NM_032324 as a target for structure determination by NMR. The three-dimensional structure of the protein in its complex with ATPgammaS forms a three-layered alpha/beta sandwich, with a central nine-stranded beta-sheet surrounded by five alpha-helices. Sequence and three-dimensional structure comparisons with AAA+ ATPases revealed the presence of Walker A (GPPGVGKT) and Walker B (VCVIDEIG) motifs. Using 1D (31)P-NMR spectroscopy and a coupled enzymatic assay for the determination of inorganic phosphate, we showed that the purified recombinant protein is active as a non-specific nucleoside triphosphatase, with k(cat)=7.6x10(-3) s(-1). The structural basis for the enzymatic activity of HCR-NTPase was further characterized by site-directed mutagenesis of the Walker B motif, which further contributes to making the HCR-NTPase an attractive new target for further biochemical characterization in the context of its presumed role in human tumorigenesis.     

Formation of amyloids by Abeta-(1-42) on NGF-differentiated PC12 cells: roles of gangliosides and cholesterol

The conversion of soluble, non-toxic amyloid beta-protein (Abeta) to aggregated, toxic Abeta could be the key step in the development of Alzheimer's disease. Liposomal studies have proposed that Abeta-(1-40) preferentially recognizes a cholesterol-dependent cluster of gangliosides and a conformationally altered form of Abeta promotes the aggregation of the protein. Cell experiments using fluorescein-labeled Abeta-(1-40) supported this model. Here, the interaction of native Abeta-(1-42) with unfixed rat pheochromocytoma PC12 cells was visualized using the amyloid-specific dye Congo red. Abeta-(1-42) preferentially bound to ganglioside and cholesterol-rich domains of cell membranes and formed amyloids in a time-dependent manner. These observations corroborate the model involving ganglioside-mediated accumulation of Abeta. The NGF-induced differentiation of PC12 cells into neuron-like cells caused a marked increase in both gangliosides and cholesterol, and thereby greatly potentiated the accumulation and cytotoxicity of Abeta-(1-42). NGF-differentiated cells exposed to Abeta-(1-42) had degenerated neurites, in which ganglioside and cholesterol-rich domains were localized, preceding cell death. A reduction in the amount of cholesterol by the cholesterol synthesis inhibitor compactin almost nullified the formation of amyloids by Abeta-(1-42). Our system using NGF-differentiated PC12 cells and Congo red is useful for screening inhibitors of the formation of amyloids by and cytotoxicity of Abeta.     

S100B protein is released from rat neonatal neurons, astrocytes, and microglia by in vitro trauma and anti-S100 increases trauma-induced delayed neuronal injury and negates the protective effect of exogenous S100B on neurons

S100B protein is found in brain, has been used as a marker for brain injury and is neurotrophic. Using a well-characterized in vitro model of brain cell trauma, we have previously shown that strain injury causes S100B release from neonatal rat neuronal plus glial cultures and that exogenous S100B reduces delayed post-traumatic neuronal damage even when given at 6 or 24 h post-trauma. The purpose of the current studies was to measure post-traumatic S100B release by specific brain cell types and to examine the effect of an antibody to S100 on post-traumatic delayed (48 h) neuronal injury and the protective effect of exogenous S100B. Neonatal rat cortical cells grown on a deformable elastic membrane were subjected to a strain (stretch) injury produced by a 50 ms displacement of the membrane. S100B was measured with an ELISA kit. Trauma released S100B from pure cultures of astrocytes, microglia, and neurons. Anti-S100 reduced released S100B to below detectable levels, increased delayed neuronal injury in traumatized cells and negated the protective effect of exogenous S100B on injured cells. Heat denatured anti-S100 did not exacerbate injury. These studies provide further evidence for a protective role for S100B following neuronal trauma.     

Hierarchical recruitment by AMPA but not staurosporine of pro-apoptotic mitochondrial signaling in cultured cortical neurons: evidence for caspase-dependent/independent cross-talk

Excitotoxicity mediated via the ( S )-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of receptor for l -glutamate contributes to various neuropathologies involving acute brain injury and chronic degenerative disorders. In this study, AMPA-induced neuronal injury and staurosporine (STS)-mediated apoptosis were compared in primary neuronal cultures of murine cerebral cortex by analyzing indices up- and downstream of mitochondrial activation. AMPA-mediated apoptosis involved induction of Bax, loss of mitochondrial transmembrane potential (ΔΨ_m), early release of cytochrome c (cyt c ), and more delayed release of second mitochondrial activator of caspases (SMAC), Omi, and apoptosis-inducing factor (AIF) with early calpain and minor late activation of caspase 3. STS-induced apoptosis was characterized by a number of differences, a more rapid time course, non-involvement of ΔΨ_m, and relatively early recruitment of SMAC and caspase 3. The AMPA-induced rise in intracellular calcium appeared insufficient to evoke ΔΨ_m as release of cyt c preceded mitochondrial depolarization, which was followed by the cytosolic translocation of SMAC, Omi, and AIF. Bax translocation preceded cyt c release for both stimuli inferring its involvement in apoptotic induction. Inclusion of the broad spectrum caspase inhibitor zVAD-fmk reduced the AMPA-induced release of cyt c , SMAC, and AIF, while only affecting the redistribution of Omi and AIF in the STS-treated neurons. Only AIF release was affected by a calpain inhibitor (calpastatin) which exerted relatively minor effects on the progression of cellular injury. AMPA-mediated release of apoptogenic proteins was more hierarchical relative to STS with its calpain activation and caspase-dependent AIF redistribution arguing for a model with cross-talk between caspase-dependent/independent apoptosis.     

Effects of filamentous algal mats on sulfide invasion in eelgrass (Zostera marina)

The effect of filamentous algae invasion into Zostera marina meadows on water quality, sediment sulfur pools and sulfide invasion into plant tissues was studied experimentally. Sulfide invasion was assessed through analysis of sulfur isotopic composition (δ³⁴S) and total sulfur (TS) concentrations in plant tissues. The algal mats (5 and 10 cm thickness) depleted oxygen in the mats and increased the pools of sulfides in the sediments. Plants exposed to algal mats had δ³⁴S signals closer to the δ³⁴S of sediment sulfide, whereas plants with no mats present had δ³⁴S signals closer to the δ³⁴S of seawater sulfate, indicating a higher sulfide invasion in plants exposed to algal mats. The δ³⁴S varied between the plant tissues with the leaves having more positive δ³⁴S signals than roots and rhizomes, indicating that sulfide was invading into the roots and moved to the other tissues through the lacunae. TS concentrations were higher in plants exposed to algal mats suggesting that sulfur derived from sediment sulfide accumulated in the plants. F_sulfide showed that up to 50% of the sulfides in the plants were derived from sedimentary sulfides. The combined effect of water column anoxia in the lower parts of the meadow and high sulfide invasion into the plants lead to significantly reduced growth rates after 3 weeks and the below-ground tissues showed signs of degradation suggesting that algal mats invasion in to Zostera marina meadows can result in seagrass decline.     

Molecular phylogeny of parasitic Platyhelminthes based on sequences of partial 28S rDNA D1 and mitochondrial cytochrome c oxidase subunit I

The phylogenic relationships existing among 14 parasitic Platyhelminthes in the Republic of Korea were investigated via the use of the partial 28S ribosomal DNA (rDNA) D1 region and the partial mitochondrial cytochrome c oxidase subunit 1 (mCOI) DNA sequences. The nucleotide sequences were analyzed by length, G + C %, nucleotide differences and gaps in order to determine the analyzed phylogenic relationships. The phylogenic patterns of the 28S rDNA D1 and mCOI regions were closely related within the same class and order as analyzed by the PAUP 4.0 program, with the exception of a few species. These findings indicate that the 28S rDNA gene sequence is more highly conserved than are the mCOI gene sequences. The 28S rDNA gene may prove useful in studies of the systematics and population genetic structures of parasitic Platyhelminthes.