Strategies for RNA folding and assembly

RNA is structurally very flexible, which provides the basis for its functional diversity. An RNA molecule can often adopt different conformations, which enables the regulation of its function through folding. Proteins help RNAs reach their functionally active conformation by increasing their structural stability or by chaperoning the folding process. Large, dynamic RNA-protein complexes, such as the ribosome or the spliceosome, require numerous proteins that coordinate conformational switches of the RNA components during assembly and during their respective activities.     

Impacts of bottom and suspended cultures of mussels <Emphasis Type="Italic">Mytilus</Emphasis> spp. on the surrounding sedimentary environment and macrobenthic biodiversity

The aim of this study was to quantify the effect of bottom and suspended mussel cultures, cultured in different physical environments, on the sedimentary environmental conditions and thereby the biodiversity structure of the associated macrofaunal community. We compared two bottom cultures (Limfjorden: microtidal, wind-driven; Oosterschelde: macrotidal) and one suspended culture (Ria de Vigo in an upwelling coastal region). The sedimentary environmental conditions (mud fraction, POC, PON, phosphorus content, chl a breakdown products) were significantly elevated underneath and surrounding bottom and suspended cultures compared to culture-free sediments that were nearby and hydrodynamically similar. The relative change in environmental conditions was more pronounced in the Oosterschelde compared to Limfjorden, most likely due to differences in hydrodynamic forcing and characteristics of the mussel bed. The effect of the suspended cultures in Ria de Vigo on the surrounding sediments was influenced by local topographic and hydrodynamic conditions. The impact of mussels on the benthic community due to biodeposition was clearly seen in the community structure. The species composition changed from species which are typically present in sandy environments to more small opportunistic species, which are typically present in organically enriched sediments. The impact of bottom cultures on the benthic community due to changes in the habitat under the presence of mussels was positive, especially in the Oosterschelde where an increase in the number of epibenthic species was seen. The influence of bottom cultures on the sedimentary environment and on the macrobenthic community seems to be very local. Within the mussel site in Limfjorden, differences were detected between sites where none or almost no mussels were present with sites where mussels were very abundant.     

Force-dependent detachment of kinesin-2 biases track switching at cytoskeletal filament intersections

Intracellular trafficking of organelles often involves cytoskeletal track switching. Organelles such as melanosomes are transported by multiple motors including kinesin-2, dynein, and myosin-V, which drive switching between microtubules and actin filaments during dispersion and aggregation. Here, we used optical trapping to determine the unitary and ensemble forces of kinesin-2, and to reconstitute cargo switching at cytoskeletal intersections in a minimal system with kinesin-2 and myosin-V motors bound to beads. Single kinesin-2 motors exerted forces up to ～5 pN, similar to kinesin-1. However, kinesin-2 motors were more likely to detach at submaximal forces, and the duration of force maintenance was short as compared to kinesin-1. In multimotor assays, force increased with kinesin-2 density but was not affected by the presence of myosin-V. In crossed filament assays, switching frequencies of motor-bound beads were dependent on the starting track. At equal average forces, beads tended to switch from microtubules onto overlying actin filaments consistent with the relatively faster detachment of kinesin-2 at near-maximal forces. Thus, in addition to relative force, switching probability at filament intersections is determined by the dynamics of motor-filament interaction, such as the quick detachment of kinesin-2 under load. This may enable fine-tuning of filament switching in the cell.     

Expressed murine and human CDR-H3 intervals of equal length exhibit distinct repertoires that differ in their amino acid composition and predicted range of structures

Immunoglobulin junctional diversity is concentrated in the third complementarity-determining region of the heavy chain (CDR-H3), which often plays a dominant role in antigen binding. The range of CDR-H3 lengths in mouse is shorter than in human, and thus the murine repertoire could be presumed to be a subset of the human one. To test this presumption, we analyzed 4751 human and 2170 murine unique, functional, published CDR-H3 intervals. Although tyrosine, glycine, and serine were found to predominate in both species, the human sequences contained fewer tyrosine residues, more proline residues, and more hydrophobic residues (p<0.001, respectively). While changes in amino acid utilization as a function of CDR-H3 length followed similar trends in both species, murine and human CDR-H3 intervals of identical length were found to differ from each other. These differences reflect both divergence of germline diversity and joining gene sequence and somatic selection. Together, these factors promote the production of a rather uniform repertoire in mice of tyrosine-enriched CDR-H3 loops with stabilized hydrogen bond-ladders versus a much more diverse repertoire in human that contains CDR-H3 loops sculpted by the presence of intra-chain disulfide bonds due to germline-encoded cysteine residues as well as the enhanced presence of somatically generated proline residues that preclude hydrogen bond ladder formation. Thus, despite the presumed need to recognize a similar range of antigen epitopes, the murine CDR-H3 repertoire is clearly distinct from its human counterpart in its amino acid composition and its predicted range of structures. These findings represent a benchmark to which CDR-H3 repertoires can be compared to better characterize and understand the shaping of the CDR-H3 repertoire over evolution and during immune responses. This information may also be useful for the design of species-specific CDR-H3 sequences in synthetic antibody libraries.     

A competitive nucleotide binding inhibitor: in vitro characterization of Rab7 GTPase inhibition

Mapping the functionality of GTPases through small molecule inhibitors represents an underexplored area in large part due to the lack of suitable compounds. Here we report on the small chemical molecule 2-(benzoylcarbamothioylamino)-5,5-dimethyl-4,7-dihydrothieno[2,3-c]pyran-3-carboxylic acid (PubChem CID 1067700) as an inhibitor of nucleotide binding by Ras-related GTPases. The mechanism of action of this pan-GTPase inhibitor was characterized in the context of the Rab7 GTPase as there are no known inhibitors of Rab GTPases. Bead-based flow cytometry established that CID 1067700 has significant inhibitory potency on Rab7 nucleotide binding with nanomolar inhibitor (K(i)) values and an inhibitory response of ≥97% for BODIPY-GTP and BODIPY-GDP binding. Other tested GTPases exhibited significantly lower responses. The compound behaves as a competitive inhibitor of Rab7 nucleotide binding based on both equilibrium binding and dissociation assays. Molecular docking analyses are compatible with CID 1067700 fitting into the nucleotide binding pocket of the GTP-conformer of Rab7. On the GDP-conformer, the molecule has greater solvent exposure and significantly less protein interaction relative to GDP, offering a molecular rationale for the experimental results. Structural features pertinent to CID 1067700 inhibitory activity have been identified through initial structure-activity analyses and identified a molecular scaffold that may serve in the generation of more selective probes for Rab7 and other GTPases. Taken together, our study has identified the first competitive GTPase inhibitor and demonstrated the potential utility of the compound for dissecting the enzymology of the Rab7 GTPase, as well as serving as a model for other small molecular weight GTPase inhibitors.     

Biodegradation of 2,4,6-trinitrotoluene (TNT): An enzymatic perspective

Enzymatic degradation of TNT by aerobic bacteria is mediated by oxygen insensitive (Type 1) or by oxygen sensitive nitroreductases (Type II nitroreductases). Transformation by Type I nitroreductases proceeds through two successive electron reductions either by hydride addition to the aromatic ring or by direct nitro group reduction following a ping pong kinetic mechanism. TNT is reduced to the level of hydroxylaminodinitrotoluenes and aminodinitrotoluenes by pure enzyme preparations without achieving mineralization. Interestingly, database gene and amino acid sequence comparisons of nitroreductases reveal a close relationship among all enzymes involved in TNT transformation. They are all flavoproteins which use NADPH/NADH as electron donor and reduce a wide range of electrophilic xenobiotics. TNT degradation by fungi is initiated by mycelia bound nitroreductases which reduce TNT to hydroxylaminodinitrotoluenes and aminodinitrotoluenes. Further degradation of these products and mineralization is achieved through the activity of oxidative enzymes especially lignin degrading enzymes (lignin and manganese peroxidases).     

A comparison of skin temperature and EMG training for primary dysmenorrhea

Eleven female volunteers completed a 6-month treatment program consisting of a 2-month baseline phase, 2 months of biofeedback training ( number of sessions=12.9), and 2 months of follow-up data collection. Subjects were assigned to one of two treatment groups: skin temperature training or EMG training of the frontalis muscle. Self-report data were gathered by means of the Symptom Severity Scale. Results, which were analyzed according to a 2×3 (treatment×phase) split-plot factorial design, indicate a highly significant overall treatment effect (F=19.32,p<.001). There was no significant difference between treatments (F=.47) and no significant interaction effect (F=1.74).     

Characterization of Cadmium Binding, Uptake, and Translocation in Intact Seedlings of Bread and Durum Wheat Cultivars

High Cd content in durum wheat (Triticum turgidum L. var durum) grain grown in the United States and Canada presents potential health and economic problems for consumers and growers. In an effort to understand the biological processes that result in excess Cd accumulation, root Cd uptake and xylem translocation to shoots in seedlings of bread wheat (Triticum aestivum L.) and durum wheat cultivars were studied. Whole-plant Cd accumulation was somewhat greater in the bread wheat cultivar, but this was probably because of increased apoplastic Cd binding. Concentration-dependent ¹⁰⁹Cd²⁺-influx kinetics in both cultivars were characterized by smooth, nonsaturating curves that could be dissected into linear and saturable components. The saturable component likely represented carrier-mediated Cd influx across root-cell plasma membranes (Michaelis constant, 20–40 nm; maximum initial velocity, 26–29 nmol g⁻¹ fresh weight h⁻¹), whereas linear Cd uptake represented cell wall binding of ¹⁰⁹Cd. Cd translocation to shoots was greater in the bread wheat cultivar than in the durum cultivar because a larger proportion of root-absorbed Cd moved to shoots. Our results indicate that excess Cd accumulation in durum wheat grain is not correlated with seedling-root influx rates or root-to-shoot translocation, but may be related to phloem-mediated Cd transport to the grain.