Hoxb8 is required for normal grooming behavior in mice.

Repertoires of grooming behaviors critical to survival are exhibited by most animal species, including humans. Genes that influence this complex behavior are unknown. We report that mice with disruptions of Hoxb8 show, with 100% penetrance, excessive grooming leading to hair removal and lesions. Additionally, these mice excessively groom normal cagemates. We have been unable to detect any skin or PNS abnormalities in Hoxb8 mutants. These observations suggest that the excessive, pathological grooming exhibited by these mice results from CNS abnormalities. Consistent with this interpretation, we demonstrate Hoxb8 expression in regions of the adult mouse CNS previously implicated in the control of grooming. The aberrant behavior observed in Hoxb8 mutants is not unlike that of humans suffering from the OC-spectrum disorder, trichotillomania. Interestingly, Hoxb8 is expressed in regions of the CNS known as the "OCD-circuit."     

Mutations in HOXD13 underlie syndactyly type V and a novel brachydactyly-syndactyly syndrome

HOXD13, the homeobox-containing gene located at the most 5' end of the HOXD cluster, plays a critical role in limb development. It has been shown that mutations in human HOXD13 can give rise to limb malformations, with variable expressivity and a wide spectrum of clinical manifestations. Polyalanine expansions in HOXD13 cause synpolydactyly, whereas amino acid substitutions in the homeodomain are associated with brachydactyly types D and E. We describe two large Han Chinese families with different limb malformations, one with syndactyly type V and the other with limb features overlapping brachydactyly types A4, D, and E and mild syndactyly of toes 2 and 3. Two-point linkage analysis showed LOD scores >3 (theta =0) for markers within and/or flanking the HOXD13 locus in both families. In the family with syndactyly type V, we identified a missense mutation in the HOXD13 homeodomain, c.950A-->G (p.Q317R), which leads to substitution of the highly conserved glutamine that is important for DNA-binding specificity and affinity. In the family with complex brachydactyly and syndactyly, we detected a deletion of 21 bp in the imperfect GCN (where N denotes A, C, G, or T) triplet-containing exon 1 of HOXD13, which results in a polyalanine contraction of seven residues. Moreover, we found that the mutant HOXD13 with the p.Q317R substitution was unable to transactivate the human EPHA7 promoter. Molecular modeling data supported these experimental results. The calculated interactions energies were in agreement with the measured changes of the activity. Our data established the link between HOXD13 and two additional limb phenotypes--syndactyly type V and brachydactyly type A4--and demonstrated that a polyalanine contraction in HOXD13, most likely, led to other digital anomalies but not to synpolydactyly. We suggest the term "HOXD13 limb morphopathies" for the spectrum of limb disorders caused by HOXD13 mutations.     

Purification of chondroitin precursor from Escherichia coli K4 fermentation broth using membrane processing

Recently the possibility of producing the capsular polysaccharide K4, a fructosylated chondroitin, in fed-batch experiments was assessed. In the present study, a novel downstream process to obtain chondroitin from Escherichia coli K4 fermentation broth was developed. The process is simple, scalable and economical. In particular, downstream procedures were optimized with a particular aim of purifying a product suitable for further chemical modifications, in an attempt to develop a biotechnological platform for chondroitin sulfate production. During process development, membrane devices (ultrafiltration/diafiltration) were exploited, selecting the right cassette cut-offs for different phases of purification. The operational conditions (cross-flow rate and transmembrane pressure) used for the process were determined on an ?KTA cross-flow instrument (GE Healthcare, USA), a lab-scale automatic tangential flow filtration system. In addition, parameters such as selectivity and throughput were calculated based on the analytical quantification of K4 and defructosylated K4, as well as the major contaminants. The complete downstream procedure yielded about 75% chondroitin with a purity higher than 90%.     

Macroinvertebrate community dynamics in a temperate European Atlantic river. Do they conform to general ecological theory?

Spatial and temporal dynamics of macroinvertebrate communities have usually been linked to several environmental and anthropic factors. The aim of this study is to elucidate how important are these factors in structuring macroinvertebrate communities from temperate regions. Regarding the macroinvertebrate number of taxa, the Habitat Template Model, the Dynamic Equilibrium Hypothesis and the Intermediate Disturbance Hypothesis will be tested in order to know how important the diversity of instream elements and the hydrological disturbance frequency are in defining the macroinvertebrate taxonomic richness. Thus, the structure and composition of macroinvertebrate communities were analysed in nine sites of the Pas River basin, a temperate Atlantic basin in northern Spain, during winter, spring, summer and autumn 2005, together with water physicochemical and environmental characteristics. Macroinvertebrate abundance increased downstream and during summer, probably favoured by lower hydraulic stress and water organic enrichment. As predicts the Habitat Template Model, the macroinvertebrate number of taxa was related to habitat heterogeneity. However, no clear relationship amongst macroinvertebrate richness and water quality was found. The macroinvertebrate taxonomic richness did not correspond exactly with the Dynamic Equilibrium Hypothesis and the Intermediate Disturbance Hypothesis because it was relatively high in the absence of hydrological disturbances (summer). Thus, disturbance events may play a secondary role in determining the seasonal dynamic of the number of taxa. However, hydrological disturbances can be considered the most important factors explaining the seasonal pattern of macroinvertebrate abundance. On the other hand, spatial patterns of macroinvertebrate community structure and composition were mainly determined by resource availability, hydraulic conditions, habitat heterogeneity and human alterations, whilst hydrological predictability and resource availability might play a major role in determining seasonal dynamics.     

Translational machinery and protein folding: evidence of conformational variants of the estrogen receptor alpha

As an approach to understand how translation may affect protein folding, we analyzed structural and functional properties of the human estrogen receptor alpha synthesized by different eukaryotic translation systems. A minimum of three conformations of the receptor were detected using limited proteolysis and a sterol ligand-binding assay. The receptor in vitro translated in rabbit reticulocyte lysate was rapidly degraded by protease, produced major bands of about 34 kDa and showed a high affinity for estradiol. In a wheat germ translation system, the receptor was more slowly digested. Two soluble co-existing conformations were evident by different degradation patterns and estradiol binding. Our data show that differences in the translation machinery may result in alternative conformations of the receptor with distinct sterol binding properties. These studies suggest that components of the cellular translation machinery itself might influence the protein folding pathways and the relative abundance of different receptor conformers.     

Abnormal kinetochore-generated pulling forces from expressing a N-terminally modified Hec1

Background

Highly Expressed in Cancer protein 1 (Hec1) is a constituent of the Ndc80 complex, a kinetochore component that has been shown to have a fundamental role in stable kinetochore-microtubule attachment, chromosome alignment and spindle checkpoint activation at mitosis. HEC1 RNA is found up-regulated in several cancer cells, suggesting a role for HEC1 deregulation in cancer. In light of this, we have investigated the consequences of experimentally-driven Hec1 expression on mitosis and chromosome segregation in an inducible expression system from human cells.

Methodology/Principal Findings

Overexpression of Hec1 could never be obtained in HeLa clones inducibly expressing C-terminally tagged Hec1 or untagged Hec1, suggesting that Hec1 cellular levels are tightly controlled. On the contrary, a chimeric protein with an EGFP tag fused to the Hec1 N-terminus accumulated in cells and disrupted mitotic division. EGFP- Hec1 cells underwent altered chromosome segregation within multipolar spindles that originated from centriole splitting. We found that EGFP-Hec1 assembled a mutant Ndc80 complex that was unable to rescue the mitotic phenotypes of Hec1 depletion. Kinetochores harboring EGFP-Hec1 formed persisting lateral microtubule-kinetochore interactions that recruited the plus-end depolymerase MCAK and the microtubule stabilizing protein HURP on K-fibers. In these conditions the plus-end kinesin CENP-E was preferentially retained at kinetochores. RNAi-mediated CENP-E depletion further demonstrated that CENP-E function was required for multipolar spindle formation in EGFP-Hec1 expressing cells.

Conclusions/Significance

Our study suggests that modifications on Hec1 N-terminal tail can alter kinetochore-microtubule attachment stability and influence Ndc80 complex function independently from the intracellular levels of the protein. N-terminally modified Hec1 promotes spindle pole fragmentation by CENP-E-mediated plus-end directed kinetochore pulling forces that disrupt the fine balance of kinetochore- and centrosome-associated forces regulating spindle bipolarity. Overall, our findings support a model in which centrosome integrity is influenced by the pathways regulating kinetochore-microtubule attachment stability.     

The Beauty is a beast: Does leachate from the invasive terrestrial plant Impatiens glandulifera affect aquatic food webs?

Invasive alien species are a major threat to ecosystems. Invasive terrestrial plants can produce allelochemicals which suppress native terrestrial biodiversity. However, it is not known if leached allelochemicals from invasive plants growing in riparian zones, such as Impatiens glandulifera, also affect freshwater ecosystems. We used mesocosms and laboratory experiments to test the impact of I. glandulifera on a simplified freshwater food web. Our mesocosm experiments show that leachate from I. glandulifera significantly reduced population growth rate of the water flea Daphnia magna and the green alga Acutodesmus obliquus, both keystone species of lakes and ponds. Laboratory experiments using the main allelochemical released by I. glandulifera, 2‐methoxy‐1,4‐naphthoquinone, revealed negative fitness effects in D. magna and A. obliquus. Our findings show that allelochemicals from I. glandulifera not only reduce biodiversity in terrestrial habitats but also pose a threat to freshwater ecosystems, highlighting the necessity to incorporate cross‐ecosystem effects in the risk assessment of invasive species.     

Three conserved consensus sequences identify the NAD-binding site of ADP-ribosylating enzymes, expressed by eukaryotes, bacteria and T-even bacteriophages

It has been previously reported that the three-dimensional structures of the NAD-binding and catalytic site of bacterial toxins with ADP-ribosylating activity are superimposable, and that the key amino acids for the enzymatic activity are conserved. The model includes an NAD-binding and catalytic site formed by an α-helix bent over a β-strand, surrounded by two β-strands bearing a Glu and a His, or Arg, that are required for catalysis. We show here that the model can be extended to comprise all proteins with ADP-ribosylating activity known to date, including all eukaryotic mono- and poly-ADP-ribosyltransferases, the bacterial ADP-ribosylating enzymes which do not have toxic activity, and the analogous enzymes encoded by T-even bacteriophages. We show that, in addition to the common Glu and Arg/His amino acids previously identified, the conserved motifs can be extended as follows: (i) the Arg/His motif is usually arom-His/Arg (where 'arom' is an aromatic residue); (ii) in the sequences of the CT group the β-strand forming part of the 'scaffold' of the catalytic cavity has an arom-ph-Ser-Thr-Ser-ph consensus (where 'ph' represents a hydrophobic residue); and (iii) the motif centred in the key glutamic residue is Glu/Gln-X-Glu; while (iv) in the sequences of the DT group the NAD-binding motif is Tyr-X₁₀-Tyr. We believe that the model proposed not only accounts for all ADP-ribosylating proteins known to date, but it is likely to fit other enzymes (currently being analysed) which possess such an activity.