Cellulase activity of a haloalkaliphilic anaerobic bacterium, strain Z-7026

Summary The cellulolytic activity of an alkaliphilic obligate anaerobic bacterium, Z-7026, which was isolated from the microbial community of soda-lake sediments and belongs to the cluster III of Clostridia with low G+C content, was studied. The bacterium was capable of growing in media with cellulose or cellobiose as the sole energy sources. Its maximal growth rate on cellobiose (0.042–0.046 h^–1) was observed at an initial pH value of 8.5–9.0, whereas the maximal rate of cellulase synthesis, assayed by using a novel fluorimetric approach, was found to be 0.1 h^–1 at pH 8–8.5. Secreted proteins revealed high affinity for cellulose and were represented by two major forms of molecular masses of 75 and 84 kDa, whereas the general protein composition of the precipitated and cellulose-bound preparations was similar to cellulosome subunits of Clostridium thermocellum. The optimum pH of the partially purified enzyme preparation towards both amorphous and crystalline cellulose was in the range 6–9, with more than 70% and less than 50% of maximal activity being retained at pH 9.2 and 5.0, respectively.     

Overexpression of inducible nitric oxide synthase and cyclooxygenase-2 in rat zinc-deficient lung: Involvement of a NF-kappaB dependent pathway.

Reactive oxygen and nitrogen species have been implicated in the pathogenesis of pulmonary diseases. The goal of this study was to measure the response of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 enzymes (COX-2) in lung with moderate zinc deficiency. Adult male Wistar rats were divided into two groups receiving (1) a zinc-deficient diet (ZD) or (2) a zinc-adequate control diet. After 2 months of treatment, the zinc-deficient group showed a significant pulmonary edema. This was associated to a reduction of protein thiols and to a significant increase of metallothionein and glutathione disulfide levels. In addition, a higher serum and lung NO production in ZD group was positively related to the higher activity and expression of iNOS and COX-2 found in lungs. Western blot analysis revealed increased IkappaBalpha degradation, an indicator of NF-kappaB activation in ZD lungs. Anatomopathologic analysis of ZD lungs showed an increase of connective tissue fibers with an influx of polymorphonuclear cells. These cells and type II cells from the alveoli showed specific immunohistochemical signals for iNOS. The conclusion is that, during the development of zinc-deficiency, iNOS activity increases in lung and contributes to lung injury. Zinc deficiency implications must be taken into account to design therapies and public health interventions involving targeted zinc supplementation for high-risk subjects or certain diseases, such as asthma.     

Structural basis of DNA recognition by p53 tetramers

The tumor-suppressor protein p53 is among the most effective of the cell's natural defenses against cancer. In response to cellular stress, p53 binds as a tetramer to diverse DNA targets containing two decameric half-sites, thereby activating the expression of genes involved in cell-cycle arrest or apoptosis. Here we present high-resolution crystal structures of sequence-specific complexes between the core domain of human p53 and different DNA half-sites. In all structures, four p53 molecules self-assemble on two DNA half-sites to form a tetramer that is a dimer of dimers, stabilized by protein-protein and base-stacking interactions. The protein-DNA interface varies as a function of the specific base sequence in correlation with the measured binding affinities of the complexes. The new data establish a structural framework for understanding the mechanisms of specificity, affinity, and cooperativity of DNA binding by p53 and suggest a model for its regulation by regions outside the sequence-specific DNA binding domain.     

Pacemaker and network mechanisms of rhythm generation: cooperation and competition

The origin of rhythmic activity in brain circuits and CPG-like motor networks is still not fully understood. The main unsolved questions are (i) What are the respective roles of intrinsic bursting and network based dynamics in systems of coupled heterogeneous, intrinsically complex, even chaotic, neurons? (ii) What are the mechanisms underlying the coexistence of robustness and flexibility in the observed rhythmic spatio-temporal patterns? One common view is that particular bursting neurons provide the rhythmogenic component while the connections between different neurons are responsible for the regularisation and synchronisation of groups of neurons and for specific phase relationships in multi-phasic patterns. We have examined the spatio-temporal rhythmic patterns in computer-simulated motif networks of H-H neurons connected by slow inhibitory synapses with a non-symmetric pattern of coupling strengths. We demonstrate that the interplay between intrinsic and network dynamics features either cooperation or competition, depending on three basic control parameters identified in our model: the shape of intrinsic bursts, the strength of the coupling and its degree of asymmetry. The cooperation of intrinsic dynamics and network mechanisms is shown to correlate with bistability, i.e., the coexistence of two different attractors in the phase space of the system corresponding to different rhythmic spatio-temporal patterns. Conversely, if the network mechanism of rhythmogenesis dominates, monostability is observed with a typical pattern of winnerless competition between neurons. We analyse bifurcations between the two regimes and demonstrate how they provide robustness and flexibility to the network performance.     

Functional proteometrics for cell migration.

BACKGROUND: Advances in living cellular fluorescence biosensors and computerized microscopy enable a vision of fully automated high-resolution measurements of the detailed intracellular molecular dynamics directly linked to cellular behaviors. Given the heterogeneity of cell populations, a statistically relevant study of molecular-cellular dynamics is a key motivation for improved automation. METHODS: We explored automating computerized, microscope-based data extraction and analyses that monitor cell locomotion, rates of mitoses, and spatiotemporal activities of intracellular proteins via ratiometric fluorescent biosensors in mouse fibroblasts. Novel image processing methods included K-means clustering segmentation preprocessing followed by modified discrete, normalized cross-correlational alignment of two-color images; ratiometric processing for fluorescence resonance energy transfer (FRET) measurements; and intracellular spatial distribution measurements of RhoA GTPase activity. RESULTS: The interdivision time was 19.4 h (mean) +/- 6.0 h (SD) (n = 7) for the GFP-histone cells in the two-by-two field that was scanned for 72 h. After registration and ratioing of the cells with the RhoA biosensor, increases in both cell protrusion and retraction were coincident with to increases in RhoA activity. CONCLUSIONS: These advances lay the foundation for extracting and correlating measurements characterizing the functional relationships of spatial localization and protein activation with features of cell migration such as velocity, polarization, protrusion, retraction, and mitosis.     

Silencing survivin gene expression promotes apoptosis of human breast cancer cells through a caspase-independent pathway

Survivin is recognized as an attractive target in cancer therapy because of its selective overexpression in the majority of tumors. Upregulated expression of this protein correlates with increased tumor grade, recurrence risk and decreased cancer patients survival. In this study, we assessed the efficacy of two survivin-specific small interfering RNA (siRNA) constructs to inhibit T47D human breast cancer cell growth. After siRNA transfection, T47D cells showed a significant reduction in proliferation and survival exhibiting clear signs of apoptosis. pSil_1 that targeted exon 1 exhibited a stronger inhibitory effect on cell growth, and increased cell apoptosis compared to pSil_30 that targeted exon 4. Cell apoptosis was found to be mediated by translocation of the mitochondrial apoptosis inducing factor (AIF), while no changes were observed in caspase-3 activation and Bid cleavage. Thus, silencing survivin expression using siRNA strategies represents a suitable therapeutic approach to selectively modulate the survival and growth of human breast cancer cells.     

Catalytic properties and classification of cellobiose dehydrogenases from ascomycetes

Putative cellobiose dehydrogenase (CDH) genes are frequently discovered in various fungi by genome sequencing projects. The expression of CDH, an extracellular flavocytochrome, is well studied in white rot basidiomycetes and is attributed to extracellular lignocellulose degradation. CDH has also been reported for plant-pathogenic or saprotrophic ascomycetes, but the molecular and catalytic properties of these enzymes are currently less investigated. This study links various ascomycetous cdh genes with the molecular and catalytic characteristics of the mature proteins and suggests a differentiation of ascomycete class II CDHs into two subclasses, namely, class IIA and class IIB, in addition to the recently introduced class III of hypothetical ascomycete CDHs. This new classification is based on sequence and biochemical data obtained from sequenced fungal genomes and a screening of 40 ascomycetes. Thirteen strains showed CDH activity when they were grown on cellulose-based media, and Chaetomium atrobrunneum, Corynascus thermophilus, Dichomera saubinetii, Hypoxylon haematostroma, Neurospora crassa, and Stachybotrys bisbyi were selected for detailed studies. In these strains, one or two cdh-encoding genes were found that stem either from class IIA and contain a C-terminal carbohydrate-binding module or from class IIB without such a module. In several strains, both genes were found. Regarding substrate specificity, class IIB CDHs show a less pronounced substrate specificity for cellobiose than class IIA enzymes. A pH-dependent pattern of the intramolecular electron transfer was also observed, and the CDHs were classified into three groups featuring acidic, intermediate, or alkaline pH optima. The pH optimum, however, does not correlate with the CDH subclasses and is most likely a species-dependent adaptation to different habitats.