Morphology and Viscoelasticity of Actin Networks Formed with the Mutually Interacting Crosslinkers: Palladin and Alpha-actinin

Actin filaments and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. Even though cells have multiple actin binding proteins (ABPs) that exist simultaneously to maintain the structural and mechanical integrity of the cellular cytoskeleton, how these proteins work together to determine the properties of actin networks is not clearly understood. The ABP, palladin, is essential for the maintenance of cell morphology and the regulation of cell movement. Palladin coexists with [Formula: see text]-actinin in stress fibers and focal adhesions and binds to both actin and [Formula: see text]-actinin. To obtain insight into how mutually interacting actin crosslinking proteins modulate the properties of actin networks, we characterized the micro-structure and mechanics of actin networks crosslinked with palladin and [Formula: see text]-actinin. We first showed that palladin crosslinks actin filaments into bundled networks which are viscoelastic in nature. Our studies also showed that composite networks of [Formula: see text]-actinin/palladin/actin behave very similar to pure palladin or pure [Formula: see text]-actinin networks. However, we found evidence that palladin and [Formula: see text]-actinin synergistically modify network viscoelasticity. To our knowledge, this is the first quantitative characterization of the physical properties of actin networks crosslinked with two mutually interacting crosslinkers.     

Selective alkylation of cycloheptaamylose

The verification of a number of assumptions made about cycloamylose structure, substrate binding and catalysis is dependent on the ability to 3-O-alkylate selectively these oligosaccharides. In the presence of barium oxide and barium hydroxide octahydrate, cycloheptaamylose reacts with 3-bromopropene in dimethylformamide and dimethyl sulfoxide to produce a high yield of tetradecakis-2,6-O-allylcycloheptaamylose, a compound which can serve as a highly versatile intermediate in the preparation of heptakis-3-O-alkylated cycloheptamyloses. Depending on the nature of the substituent to be introduced at the C-3 hydroxyl position, the intermediate allyl ether can be 3-O-alkylated and the allyl ether can be isomerized to the vinyl ether and cleaved; or, alternatively, the allyl ether can be first subjected to isomerization followed by alkylation, and then cleavage.     

Purification and Properties of Phospholipase D from Actinomadura sp. Strain No. 362

An extracellular phospholipase D from Actinomadura sp. Strain No. 362 was purified about 430-fold from the culture filtrate. The purified enzyme preparation was judged to be homogeneous on polyacrylamide gel electrophoresis. The molecular weight and isoelectric point of the enzyme were estimated to be about 50,000—60,000 and 6.4, respectively. The enzyme was most active at pH 5.5 and 50°C in the presence of Triton X-100, but showed the highest activity at pH 7.0 and 60 — 70°C in its absence. The enzyme was stable up to 30°C at pH 7.2 and also stable in the pH range of 4.0 to 8.0 on 2 hr incubation at 25°C. With regard to substrate specificity, this enzyme hydrolysed lecithin best among the phospholipids tested. It was activated by Fe^{3 +}, Al³⁺, Mn^{2 +}, Ca^{2 +}, diethyl ether, sodium deoxycholate and Triton X-100, but was inhibited by cetyl pyridinium chloride and dodecylsulfate.     

Studies on the Accumulation of Orotic Acid by Escherichia coli K12

The mechanism whereby Escherichia coli K12 accumulates orotic acid in culture fluid was studied. Pyrimidine compounds were incorporated effectively into cells of E. coli K12, stimulated the growth, and depressed the accumulation; while purine compounds were not so much consumed by the microorganism for its growth, and affected the accumulation to a lesser extent. On the other hand, E. coli B unable to accumulate orotic acid utilized less effectively pyrimidine compounds for its growth than strain K12.

It is supposed, therefore, that in the de novo pathway for pyrimidine synthesis in E. coli K12 the step from orotic acid to 5′-UMP is genetically depressed so that orotic acid is accumulated when pyrimidine compounds, that would cause a feedback inhibition of orotic acid synthesis upon incorporation, are not supplemented.     

Cleavage of the xylosyl serine linkage between a core peptide and a glycosaminoglycan chain by cellulases

We previously found that endo-beta-xylosidase from Patinopecten is an endo-type glycosidase that cleaves the xylosyl serine linkage between a glycosaminoglycan chain and its core protein (Takagaki, K., Kon, A., Kawasaki, H., Nakamura, T., Tamura, S., and Endo, M. (1990) J. Biol. Chem. 265, 854-860). Screening for endo-beta-xylosidase activity in several cellulases detected this activity in the enzymes from Aspergillus niger, Penicillium funiculosum, Trichoderma reesei, Trichoderma viride, and Irpex lacteus. The cellulase derived from A. niger was purified, and its molecular weight was determined to be 26,000 by SDS-PAGE. Examination of the specificity of the cellulase revealed that 1) the enzyme acts on the linkage region (xylosyl serine) between a core peptide and a glycosaminoglycan chain; 2) enzymatic activity is greater with shorter glycosaminoglycan chains; 3) the enzyme readily hydrolyzes the linkage in glycosaminoglycan peptides, but intact proteoglycan is cleaved only slowly; and 4) the activity is unaffected by the glycosaminoglycan component (chondroitin sulfate, dermatan sulfate, and heparan sulfate). Judging from these enzymatic characteristics, this cellulase is different from the endo-beta-xylosidase of Patinopecten. We believe that this cellulase will become a useful tool in the further development of glycotechnology, because, like the endo-beta-xylosidase of Patinopecten, it enables the release of intact glycosaminoglycans from glycosaminoglycan peptides.     

Characterization of a multidomain adaptor protein, p140Cap, as part of a pre-synaptic complex

p140Cap (Cas-associated protein) is an adaptor protein considered to play pivotal roles in cell adhesion, growth and Src tyrosine kinase-related signaling in non-neuronal cells. It is also reported to interact with a pre-synaptic membrane protein, synaptosome-associated protein of 25 kDa, and may participate in neuronal secretion. However, properties and precise functions of p140Cap in neuronal cells are almost unknown. Here we show, using biochemical analyses, that p140Cap is expressed in rat brain in a developmental stage-dependent manner, and is relatively abundant in the synaptic plasma membrane fraction in adults. Immunohistochemistry showed localization of p140Cap in the neuropil in rat brain and immunofluorescent analyses detected p140Cap at synapses of primary cultured rat hippocampal neurons. Electron microscopy further revealed localization at pre- and post-synapses. Screening of p140Cap-binding proteins identified a multidomain adaptor protein, vinexin, whose third Src-homology 3 domain interacts with the C-terminal Pro-rich motif of p140Cap. Immunocomplexes between the two proteins were confirmed in COS7 and rat brain. We also clarified that a pre-synaptic protein, synaptophysin, interacts with p140Cap. These results suggest that p140Cap is involved in neurotransmitter release, synapse formation/maintenance, and signaling.