Identification of dynein light chain 2 as an interaction partner of p21-activated kinase 1

p21-Activated kinase 1 (PAK1), a member of the evolutionarily conserved PAK family of serine/threonine kinases, is essential for a variety of cellular functions. Our previous studies showed that PAK1 participated in the apoptotic pathway mediated by p110C. To further investigate its functions, we used the yeast two-hybrid system to screen a human fetal brain cDNA library and identified dynein light chain 2 (DLC2)/myosin light chain (MLC) as an interacting partner of PAK1. The association of PAK1 with DLC2 was further confirmed by in vitro binding assay. With the stimulation of EGF, PAK1 interacted with HA-DLC2 in vivo and relocalized in cytoplasm near the perinuclear location in confocal microscope analysis. The deletion analysis showed that the interaction of DLC2 with PAK1 occurred within the residues 210-332 of PAK1. For that studies showed that DLC2 was a subunit of myosin complex, so it is possible that PAK1 binds to DLC2 and transports by myosin complex.     

Morphologies and conformation transition of lentinan in aqueous NaOH solution

Molecular morphologies and conformation transition of lentinan, a beta-(1-->3)-D-glucan from Lentinus edodes, were studied in aqueous NaOH solution by atomic force microscopy (AFM), viscometry, multiangle laser light scattering, and optical rotation measurements. The results revealed that lentinan exists as triple-helical chains and as single random-coil chains at NaOH concentration lower than 0.05M and higher than 0.08M, respectively. Moreover, the dramatic changes in weight-average molecular weight Mw, radius of gyration [s2](1/2), intrinsic viscosity [eta], as well as specific optical rotation at 589 nm [alpha]589 occurred in a narrow range of NaOH concentration between 0.05 and 0.08M NaOH, indicating that the helix-coil conformation transition of lentinan was carried out more easily than that of native schizophyllan and scleroglucan, and was irreversible. For the first time, we confirmed that the denatured lentinan molecule, which was dissolved in 0.15M NaOH to be disrupted into single coil chains, could be renatured as triple helical chain by dialyzing against abundant water in the regenerated cellulose tube at ambient temperature (15 degrees C). In view of the AFM image, lentinan in aqueous solution exhibited the linear, circular, and branched species of triple helix compared with native linear schizophyllan or scleroglucan.     

Rat kinesin light chain 3 associates with spermatid mitochondria

We recently discovered that in rat spermatids, kinesin light chain KLC3 can associate with outer dense fibers, major sperm tail components, and accumulates in the sperm midpiece. Here, we show that mitochondria isolated from rat-elongating spermatids have bound KLC3. Immunoelectron microscopy indicates that the association of KLC3 with mitochondria coincides with the stage in spermatogenesis when mitochondria move from the plasma membrane to the developing midpiece. KLC3 is able to bind in vitro to mitochondria from spermatids as well as somatic cells employing a conserved kinesin light chain motif, the tetratrico-peptide repeats. Expression of KLC3 in fibroblasts results in formation of large KLC3 clusters close to the nucleus, which also contain mitochondria: no other organelles were present in these clusters. Mitochondria are not present in KLC3 clusters after deletion of KLC3's tetratrico-peptide repeats. Our results indicate that the rat spermatid kinesin light chain KLC3 can associate with mitochondria.     

Antigen binding and stability properties of non-covalently linked anti-CD22 single-chain Fv dimers

By varying linker length and domain orientation three multivalent derivatives of a monovalent anti-CD22 single-chain fragment variable (scFv) antibody were generated. Shortening the linker of the V(H)-V(L) oriented scFv to 5 or 0 residues resulted in the formation of diabodies or a mixture of tetramers and trimers, respectively. Unexpectedly, a V(L)-0-V(H) scFv assembled to homogenous dimers, remained substantially more stable than the V(H)-5-V(L) diabody when incubated in human serum at 37 degrees C, and retained its dimeric state when concentrated up to 4 mg/ml. These properties suggest the V(L)-0-V(H) scFv could become an attractive vehicle for the selective delivery of multiple effector molecules to CD22(+) tumor cells.     

Vegetative characteristics of three low-lying Florida coastal rivers in relation to flow, light, salinity and nutrients

The Chassahowitzka, Homosassa and Crystal rivers along the central Gulf coast of Florida were studied from 1998 to 2000 to identify factors controlling the abundance and distribution of submersed aquatic vegetation (SAV). Each of these three low-lying coastal rivers are spring-fed and exhibit low to moderate absolute flow rates (flows in either direction because of tidal influences, 0.06–0.46ms^–1) with only 14 of the stations sampled for SAV having flow rates in excess of 0.25ms^–1. At those stations where flow rates exceeded 0.25ms–1, the substrate was generally comprised of exposed limestone outcroppings and did not provide a favorable habitat for either submersed macrophytes or macroalgae. The remaining sampling stations, where flow rates were less than 0.25ms–1, had suitable substrates (e.g. mud, mud/sand, and sand) for the colonization and subsequent growth of SAV. Light availability and salinity were determined to be major factors affecting the distribution and abundance of SAV. Sampling stations, where the percent of incident light at the surface reaching the substrate was less than 10, had little or no SAV biomass. Low SAV biomass was also linked to sites where annual average salinities exceeded 3.5. Nutrient loads and nutrient concentrations accounted for little variance in SAV biomass after accounting for flow and related substrate type, light and salinity. These latter factors control the distribution and abundance of SAV in these three Florida coastal rivers.     

Novel Phosphospecific Antibodies for Monitoring Phosphorylation of Proteins Encoded by the Myosin Light Chain Kinase Genetic Locus

Myosin light chain kinase (MLCK) and the kinase-related protein (KRP), also known as telokin, are the major independent protein products of the smooth muscle/non-muscle MLCK genetic locus. They share a common C-terminal part and major sites phosphorylated in vivo. Whereas MLCK is critically involved in myosin activation and contraction initiation in smooth muscle, KRP is thought to antagonize MLCK and to exert relaxation activity. Phosphorylation controls the MLCK and KRP activities. We generated two phosphorylation and site-specific antibodies to individually monitor levels of MLCK and KRP phosphorylation on critical sites. We quantified the level of KRP phosphorylation in smooth muscle before and after an increase in intracellular free Ca2+ and stimulation of adenylate cyclase, protein kinase C, and mitogen-activated protein kinases (MAP-kinases). Forskolin and phorbol-12,13-dibutyrate increased KRP phosphorylation at Ser13 from 25 to 100% but did not produce contraction in rat ileum. The level of Ser13 phosphorylation was not altered during Ca2+-dependent contraction evoked by KCl depolarization or carbachol, but subsequently increased to maximum during forskolin-induced relaxation. These data suggest that several intracellular signaling pathways control phosphorylation of KRP on Ser13 in smooth muscle and thus may contribute to relaxation. In contrast, phosphorylation level of Ser19 of KRP increased only slightly (from 30 to 40-45%) and only in response to MAP-kinase activation, arguing against its regulatory function in smooth muscle.     

Protein analysis with tetra-substituted sulfonated cobalt phthalocyanine by the technique of Rayleigh light scattering

This is the first report of cobalt-tetrasulfonatophthalocyanine (CoTSPc) as a probe of Rayleigh light scattering (RLS) to determine proteins at nanogram levels. A highly sensitive method has been developed for the determination of proteins by the light scattering technique on a common spectrofluorimeter, based on the fact that the weak RLS of CoTSPc can be greatly enhanced in the presence of proteins. Under optimum conditions, the linear ranges of the calibration curves were 0.10-34.3 microg x mL(-1) for both human serum albumin and bovine serum albumin, with detection limits of 15.5 and 13.9 ng x mL(-1), respectively. Moreover, there is almost no interference of any amino acids and metal ions. The method has been applied to the direct determination of total proteins in human serum samples, and the results were satisfactory with clinical data provided.     

A static laser light scattering assay for surfactant-induced tau fibrillization

Static light scattering is an important solution-based method for assaying spontaneous protein aggregation reactions. But the reliability of the measurements when conducted in the presence of fibrillization inducers has been questioned. Here the utility of static laser light scattering for quantitative assay of anionic micelle-induced protein fibrillization was characterized using tau protein, the major component of neurofibrillary lesions of Alzheimer's disease. Both inducer micellization and tau fibrillization made significant contributions to light scattering intensity. The intensity arising solely from micellization was quantified using proteins that promoted inducer micellization but could not fibrillize, such as mixed histones and assembly-incompetent mutant htau40(I277P/I308P). When corrected for micellization, reaction progress curves for wild-type tau fibrillization were sigmoidal and correlated well with measurements of total filament length made by transmission electron microscopy. The utility of the improved laser light scattering assay was demonstrated by quantifying the effect of inducer concentration on tau assembly kinetics using a three-parameter Gompertz growth function. Results showed that alkyl sulfate detergent accelerated tau nucleation as reflected by shorter lag times and modulated pre-nuclear equilibria to yield more filament mass at reaction equilibrium.     

Actin filaments play an essential role for transport of nascent HIV-1 proteins in host cells

To investigate the role of actin filaments (F-actin) for human immunodeficiency virus type 1 (HIV-1) production in host cells, the effect of mycalolide B that is a novel actin-depolymerizing marine toxin was examined. Mycalolide B blocked the production of HIV-1 from primary infected T-lymphoblastoid and clonically infected monocytoid cells in a concentration-dependent manner. In the presence of 10 microM of mycalolide B, F-actins were disorganized and mostly disappeared in the host cells, and viral envelope- and capsid-proteins did not reach the plasma membrane, but were distributed in the cytoplasm forming aggregates. In electron micrographs, no HIV-1 virions were detected on the cell surface, but many lysosome-like vesicles containing electron dense granules were observed in the cytoplasm, implying that mycalolide B did not disturb the synthesis of viral proteins, but rather inhibited their transport processes of HIV-1 in the host cells.     

Human chromosome 21q22.2-qter carries a gene(s) responsible for downregulation of mlc2a and PEBP in Down syndrome model mice

Congenital heart disease (CHD) is a major clinical manifestation of Down syndrome (DS). We recently showed that chimeric mice containing a human chromosome 21 (Chr 21) exhibited phenotypic traits of DS, including CHD. Our previous study showed that myosin light chain-2a (mlc2a) expression was reduced in the hearts of chimeric mice and DS patients. We found that phosphatidylethanolamine binding protein (PEBP) was also downregulated in Chr 21 chimeras in this study. As mlc2a is involved in heart morphogenesis, and PEBP controls the proliferation and differentiation of different cell types, these genes are candidates for involvement in DS-CHD. The DS-CHD candidate region has been suggested to span between PFKL and D21S3, which is the STS marker near the ETS2 loci. To identify gene(s) or a gene cluster on Chr 21 responsible for the downregulation of mlc2a and PEBP, we fragmented Chr 21 at the EST2 loci, by telomere-directed chromosome truncation in homologous recombination-proficient chicken DT40 cells. The modified Chr 21 was transferred to mouse ES cells by microcell-mediated chromosome transfer (MMCT), via CHO cells. We used ES cell lines retaining the Chr 21 truncated at the ETS2 locus (Chr 21E) to produce chimeric mice and compared overall protein expression patterns in hearts of the chimeras containing the intact and the fragmented Chr 21 by two-dimensional electrophoresis. While mouse mlc2a and PEBP expression was downregulated in the chimeras containing the intact Chr 21, the expression was not affected in the Chr 21E chimeras. Therefore, we suggest that Chr 21 gene(s) distal from the ETS2 locus reduce mouse mlc2a and PEBP expression in DS model mice and DS. Thus, this chromosome engineering technology is a useful tool for identification or mapping of genes that contribute to the DS phenotypes.