Identification of two human WAVE/SCAR homologues as general actin regulatory molecules which associate with the Arp2/3 complex.

WAVE/SCAR protein was identified as a protein which has similarity to WASP and N-WASP, especially in its C terminal. Recently, WAVE/SCAR protein has been shown to cooperate with the Arp2/3 complex, a nucleation core for actin polymerization in vitro. However, in spite of its general function, WAVE/SCAR expression is mainly restricted to the brain, suggesting the existence of related molecule(s). We here identified two human WAVE/SCAR homologues, which cover other organs. We named the original WAVE1 and newly identified ones WAVE2 and WAVE3. WAVE2 had a very wide distribution with strong expression in peripheral blood leukocytes and mapped on chromosome Xp11.21, next to the WASP locus. WAVE3 and WAVE1 had similar distributions. WAVE3 was strongly expressed in brain and mapped on chromosome 13q12. WAVE1 was mapped on chromosome 6q21-22. Ectopically expressed WAVE2 and WAVE3 induced actin filament clusters in a similar manner with WAVE1. These actin cluster formations were suppressed by deletion of their C-terminal VPH (verproline homology)/WH2 (WASP homology 2) domain. Further, WAVE2 and WAVE3 associate with the Arp2/3 complex as does WAVE1. Our identification of WAVE homologues suggests that WAVE family proteins have general function for regulating the actin cytoskeleton in many tissues.     

Fluorescence energy transfer between Cys-10 residues in F-actin filaments

Fluorescence energy transfer was measured between Cys-10 residues in an F-actin filament using 5-[2-((iodoacetyl)amino)-ethyl]aminonaphthalene-1-sulphonic acid (1,5-IAEDANS) as a fluorescence energy donor and 4-dimethylaminophenylazophenyl-4'-maleimide (DABMI) as the acceptor. Both labels were covalently attached to Cys-10 residues in an F-actin filament. Taking the helical structure of the F-actin filament into consideration, the radial coordinate of Cys-10 was calculated to be 23 A. This corresponds to a distance between adjacent sites along the long pitch helix of 56.1 A and along the genetic helix of 53.3 A.     

Removing the two C-terminal residues of actin affects the filament structure.

We define conditions under which the two C-terminal residues of actin, Cys-374 and Phe-375, can be selectively removed by proteolysis with trypsin. This modification had little effect on the secondary structure of actin detected by Fourier-transform infrared spectroscopy. However, removing these residues caused small but significant decreases in the critical concentration of actin, in its ability to activate myosin ATPase, and in its interaction with tropomyosin and troponin. Removing residues 374-375 caused dramatic changes in the actin filament as seen by electron microscopy. The filaments had a much greater and more irregular curvature and were intertwined into disordered multifilament bundles. Removing 374-375 also significantly lowered the flow viscosity of filamentous-actin solutions. These data suggest an increase in the flexibility and fragility of the filament, supporting the idea that the C-terminus forms one of the major intermonomer contacts in the filament.     

The relationship between physical property and function of highly activated mutants of thermolysin

Thermolysin mutants having a variety of amino acid at the 119th position are designed by considering electrostatic field effect upon the active area. The most activated mutant has five times higher hydrolytic activity than the wild type. Negative correlation between the activity and the thermal stability is observed. A combined effect of the flexibility of the substrate binding site and the negative electrostatic field around the site is suggested as a key to enhance the activity.     

Chromosome sorting of primates: Isolation and identification of Y chromosome in green monkeys

Single laser flow cytometry was applied to the karyotype analysis of green monkeys. Clear sex difference in flow karyotype was recognized in this monkey, because Y chromosome could be identified as a single peak in the histogram of male specimens. We could isolate Y chromosome of this species by the use of a cell sorter, and demonstrate by polymerase chain reaction that the sorted-out chromosomes contained the Y chromosome specific nucleotide sequence (SRY). This chromosome sorting technique provides a powerful strategy for constructing the DNA library specific to Y chromosome in this species.     

Pyruvate kinase isozymes: Ancient diversity retained in modern plant cells

Pyruvate kinase is a key regulatory enzyme of glycolysis. Phylogenetic analyses of the sequences coding for pyruvate kinase from plants and other organisms revealed unexpected diversity of this glycolytic enzyme in the progenitor of the present-day eukaryotes. Plants contain an ancient lineage of cytosolic pyruvate kinase, which may have diverged from the animal pyruvate kinase genes prior to the plant-animal divergence. The plant cytosolic pyruvate kinase genes are no more closely related to the animal and fungal pyruvate kinase genes than to the prokaryotic pyruvate kinase genes. The results suggest that the plant pyruvate kinase genes and the animal-fungal pyruvate kinase genes have descended from divergent isozymes which existed in the progenitor of the present-day eukaryotes and prokaryotes.     

Co-induction of DNA relaxation and synthesis of DnaK and GroEL proteins inEscherichia coli by expression of LetD (CcdB) protein, an inhibitor of DNA gyrase encoded by the F factor

We examined the influence of overexpression of LetD (CcdB) protein, an inhibitor of DNA gyrase encoded by the F factor ofEscherichia coli, on DNA supercoiling and induction of heat shock proteins. Cells were transformed with a plasmid carrying the structural gene for LetD protein under control of thetac promoter, and LetD protein was induced by adding isopropyl-d-thiogalactopyranoside (IPTG) to the culture medium. Analysis by agarose gel electrophoresis in the presence of chloroquine revealed relaxation of plasmid DNA in cells depending on the concentration of IPTG employed for induction. Protein pulse-labeling experiments with [³⁵S]methionine and cysteine revealed that synthesis of DnaK and GroEL proteins was also induced by IPTG, and concentrations necessary for DNA relaxation and induction of the heat shock proteins were much the same. Expression of mutant LetD protein lacking two amino acid residues at the C-terminus induced neither DNA relaxation nor the synthesis of DnaK and GroEL proteins. Induction of wild-type LetD protein but not mutant LetD protein markedly enhanced synthesis of ³². We interpret these results to mean that DNA relaxation in cells caused by the expression of LetD protein induces heat shock proteins via increased synthesis of ³².     

Effect of macroscopic deformation on lung microstructure

Butler, James P., Hiroshi Miki, Stephanie Squarcia, Rick A. Rogers, and John L. Lehr. Effect of macroscopic deformation onlung microstructure. J. Appl. Physiol.81(4): 1792-1799, 1996.Using an anisotropic theory of diffuselight scattering in lungs, we measured the fractional changes ingeometric mean linear intercepts in orthogonal directions when freshlyexcised rabbit lungs were subjected to isovolume uniaxial strains.Results from the optical technique were compared with morphometricestimates of fractional changes in mean linear intercepts from the samestrained and unstrained (control) lobes, with the conclusion thatdiffuse light scattering is adequate to estimate changes in mean freepaths in different directions. We compared optical estimates offractional changes in mean linear intercepts with the macroscopicstrain field measured by displacements of pleural markers; thisrelationship did not significantly differ from the line of identity. Weconclude that the microscopic strain field is closely matched to themacroscopic strain field during uniaxial distortion. This suggests thatsurface reorientation may not play a large role in the origin of thelow shear modulus of the lung, but this cannot be definitively stated without comparison of these experimental results to specific model predictions of the changes in mean linear intercepts in shear deformation.