Rho kinase regulates the intracellular micromechanical response of adherent cells to rho activation

Local sol-gel transitions of the cytoskeleton modulate cell shape changes, which are required for essential cellular functions, including motility and adhesion. In vitro studies using purified cytoskeletal proteins have suggested molecular mechanisms of regulation of cytoskeleton mechanics; however, the mechanical behavior of living cells and the signaling pathways by which it is regulated remains largely unknown. To address this issue, we used a nanoscale sensing method, intracellular microrheology, to examine the mechanical response of the cell to activation of the small GTPase Rho. We observe that the cytoplasmic stiffness and viscosity of serum-starved Swiss 3T3 cells transiently and locally enhances upon treatment with lysophosphatidic acid, and this mechanical behavior follows a trend similar to Rho activity. Furthermore, the time-dependent activation of Rho decreases the degree of microheterogeneity of the cytoplasm. Our results reveal fundamental differences between intracellular elasticity and cellular tension and suggest a critical role for Rho kinase in the regulation of intracellular mechanics.     

TRACTS: A program to map oligopurine.oligopyrimidine and other binary DNA tracts

A program to map the locations and frequencies of DNA tracts composed of only two bases ('Binary DNA') is described. The program, TRACTS (URL http://bioportal.weizmann.ac.il/tracts/tracts.html and/or http://bip.weizmann.ac.il/miwbin/servers/tracts) is of interest because long tracts composed of only two bases are highly over-represented in most genomes. In eukaryotes, oligopurine.oligopyrimidine tracts ('R.Y tracts') are found in the highest excess. In prokaryotes, W tracts predominate (A,T 'rich'). A pre-program, ANEX, parses database annotation files of GenBank and EMBL, to produce a convenient one-line list of every gene (exon, intron) in a genome. The main unit lists and analyzes tracts of the three possible binary pairs (R.Y, K.M and S;W). As an example, the results of R.Y tract mapping of mammalian gene p53 is described.     

Structure and function of an archaeal homolog of survival protein E (SurEalpha): an acid phosphatase with purine nucleotide specificity

The survival protein E (SurE) family was discovered by its correlation to stationary phase survival of Escherichia coli and various repair proteins involved in sustaining this and other stress-response phenotypes. In order to better understand this ancient and well-conserved protein family, we have determined the 2.0A resolution crystal structure of SurEalpha from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum (Pae). This first structure of an archaeal SurE reveals significant similarities to and differences from the only other known SurE structure, that from the eubacterium Thermatoga maritima (Tma). Both SurE monomers adopt similar folds; however, unlike the Tma SurE dimer, crystalline Pae SurEalpha is predominantly non-domain swapped. Comparative structural analyses of Tma and Pae SurE suggest conformationally variant regions, such as a hinge loop that may be involved in domain swapping. The putative SurE active site is highly conserved, and implies a model for SurE bound to a potential substrate, guanosine-5'-monophosphate (GMP). Pae SurEalpha has optimal acid phosphatase activity at temperatures above 90 degrees C, and is less specific than Tma SurE in terms of metal ion requirements. Substrate specificity also differs between Pae and Tma SurE, with a more specific recognition of purine nucleotides by the archaeal enzyme. Analyses of the sequences, phylogenetic distribution, and genomic organization of the SurE family reveal examples of genomes encoding multiple surE genes, and suggest that SurE homologs constitute a broad family of enzymes with phosphatase-like activities.     

In vivo gene therapy in young and adult RPE65-/- dogs produces long-term visual improvement

Defects in the RPE65 gene, which is selectively expressed in the retinal pigment epithelium (RPE), result in blindness and gradual photoreceptor cell degeneration. Experiments were conducted to assess the efficacy of gene replacement therapy in restoring retinal function in RPE65-/- dogs. Long-term effects of RPE65 gene therapy were assessed using visual behavioral testing and electroretinographic (ERG) recordings at 10-12 weeks and 6-9 months after surgery in five affected dogs. Subretinal injections of similar dosages of two constructs were performed in affected dogs at the ages of 4-30 months: rAAV.RPE65 into one eye and, in four of five dogs, rAAV.GFP contralaterally. Before surgery all RPE65-/- dogs were behaviorally blind with either no or very low-amplitude ERG responses to light stimuli. Marked improvements in visual behavior and ERG responses were observed as early as 4 weeks after surgery in affected animals. Except for light-adapted 50 Hz ERG flicker responses, all ERG parameters tested increased significantly in the eyes treated with the rAAV.RPE65 construct at the early follow-up. Gradual progressive improvements in ERG responses were observed in the RPE65-treated eyes over time. An unexpected finding was that on long-term follow-up, marked improvement of photopic ERG responses were also observed in the contralateral control eye in both young and older dogs. These results are promising for future clinical trials of human patients with retinal degenerative diseases, such as Leber congenital amaurosis, that result from RPE65 gene defects.     

Solubility, adsorption, and the thermodynamics of the cutoff effect

For many years, the expression "cutoff effect of anesthesia," has been used to denote the failure of the higher alcohols or paraffins to produce anesthesia. As such, it is used to assess the plausibility of specific models, proposed for anesthesia. However, the uses were shown, in many respects, to be problematic. This article augments the notion of the cutoff to fit for all cases in which only some of the molecules in a homologous series are anesthetics. We find that the location of the cutoff points is affected by three free energy quantities: that of the adsorption of the agent to the anesthetic "site" (f(sl,site)), that of the perturbation of the site (f(ll,site)), and that of the evaporation of the agent from its pure condensed phase (Deltamu degrees (evaporation)). This outcome indicates that the cutoff cannot be attributed to a single parameter. In addition, the analyses that attribute the cutoff to the failure of compounds to obey the much-used Meyer-Overton correlation will have to be amended. This article shows that cutoff results can be used to elucidate the structure of a site.     

A virulence attenuated amoebapore-less mutant of Entamoeba histolytica and its interaction with host cells

Entamoeba histolytica, the protozoan parasite which causes amoebiasis, is an exclusively human pathogen so developing a vaccine could effectively impact the spread of the disease. Recently we developed a genetically modified avirulent strain, termed G3, from the virulent E. histolytica strain HM-1:IMSS. The new strain lacks the important virulence factor, the amoebapore-A. The objective of our current study was to investigate the avirulence of the attenuated strain as well as to examine the antigenic and immunogenic responses of these trophozoites as potential candidates for a live vaccine. Functional assays were conducted to characterise the virulent behaviour of the G3 strain. This behaviour was compared to the virulent strain HM-1:IMSS and the non-virulent strain Rahman. Western blots were conducted to confirm the lack of amoebapore-A in the E. histolytica G3 strain and to demonstrate that it had no influence on the presence of other virulence factors. Results of these two sets of tests proved the G3 strain to be phenotypically similar to the avirulent Rahman strain while antigenically identical to the virulent HM-1:IMSS, apart from the lack of the amoebapore-A protein. Intraperitoneal immunisation of hamsters with G3 trophozoites compared to sham immunised hamsters resulted in IgG anti-HM-1:IMSS antibodies. The level of humoral response was variable and further testing has to take place before introducing this new strain as a vaccine.     

Phylogenetic position of Sorogena stoianovitchae and relationships within the class Colpodea (Ciliophora) based on SSU rDNA sequences

The ciliate Sorogena stoianovitchae, which can form a multicellular fruiting body, has been classified based upon its ultrastructure and morphology: the oral and somatic infraciliature of S. stoianovitchae most closely resemble those of members of the order Cyrtolophosidida in the class Colpodea. We characterized the small subunit ribosomal DNA (SSU rDNA) gene sequence from S. stoianovitchae and compared this sequence with those from representatives of all ciliate classes. These analyses placed S. stoianovitchae as either sister to members of the class Nassophorea or Colpodea. In an in-group analysis, including all SSU rDNA sequences from members of the classes Nassophorea and Colpodea and representatives of appropriate outgroups, S. stoianovitchae was always sister to Platyophrya vorax (class Colpodea, order Cyrtolophosidida). However, our analyses failed to support the monophyly of the class Colpodea. Instead, our data suggest that there are essentially three unresolved clades: (1) the class Nassophorea; (2) Bresslaua vorax, Colpoda inflata, Pseudoplatyophrya nana, and Bursaria truncatella (class Colpodea); and (3) P. vorax and S. stoianovitchae (class Colpodea).     

A novel device for the simple and efficient refinement of liposuctioned tissue

In short, our device allows a surgeon who is harvesting adipose tissue for autologous fat transplantation to immediately, easily, efficiently, and sterilely isolate adipose tissue from the unwanted waste components that are associated with primary liposuction effluent. It does so by "trapping" the fat tissue contained within raw liposuction effluent. Once the tissue fraction has been separated, the device design then allows for direct implantation or subsequent washing/rinsing of the tissue with saline/buffer of choice in preparation for tissue reimplantation.     

Prosthetic crowns and other clinical risk indicators of caries among old-old Swedish adults: findings from the KEOHS Project. Kungsholmen Elders Oral Health Study

Objectives: The Kungsholmen Elders Oral Health Study (KEOHS) evaluated the oral health status of generally healthy, community‐dwelling persons over the age of 80 living in Kungsholmen, Sweden. This paper explored possible clinical risk indicators of coronal and root caries among the KEOHS subjects. Design: In this cross‐sectional study, dentate KEOHS subjects received a caries assessment using defined visual, tactile criteria. Setting: Examinations were carried out in two local clinics by standardized examiners. Subjects: One hundred twenty‐nine dentate persons were examined. Main Outcome Measures: The examination identified decayed and filled surfaces, prosthetic crowns, and missing teeth. Results: More root than coronal surfaces had untreated decay, and secondary root caries contributed the greatest number of decayed surfaces. Ninety percent of the examined dentate subjects had at least one prosthetic crown. Root surfaces exposed to crown margins were more likely to have caries than root surfaces not so exposed, particularly among women. The presence of untreated coronal caries (yes/no) was positively associated with having untreated root caries and an intermediate number (14–20) of teeth, but inversely associated with having 4+ prosthetic crowns. Active root caries (yes/no) was positively associated with having untreated coronal caries, 14–20 teeth, and 4+ prosthetic crowns. Nearly 20% of identified root lesions were present at or below the gingival margin, and most (88%) were secondary caries associated with crown margins (65%) or other restorations (23%). Conclusions: Our findings suggest that some dental characteristics, including the presence of prosthetic crowns, are risk indicators for the presence of untreated coronal and root caries.