X-ray equatorial diffraction studies on the cross-sectional structure of Salmonella flagella

X-ray diffraction studies have been made on the cross-sectional structure of the normal Salmonella flagella. Two approaches have been made: one based upon small-angle equatorial scatterings (2θ 3°) and the other upon moderate-angle angle equatorial diffractions (3° 2θ 10°).Analysis of small-angle scattering data gives the radius of gyration of the flagella as 68 Å. Cylindrically averaged electron density of the cross-section of the flagella is obtained by means of the Fourier-Bessel transformation method. The average radius of the flagella is about 65 Å.In the investigation of the moderate-angle diffraction pattern, validity is examined of the model that a flagellum consits annularly arranged strands, of which each has a cylindrically symmetric structure. Features of the pattern observed in the range of 3° < 2θ < 10° can be interpreted fairly well by this model. Average radii of the flagella obtained for the 11 and 13 strands models are close to that obtained by the analysis of the small-angle scattering data.     

Mechanism of amyloid β-protein aggregation mediated by GM1 ganglioside clusters

It is widely accepted that the conversion of the soluble, nontoxic amyloid β-protein (Aβ) monomer to aggregated toxic Aβ rich in β-sheet structures is central to the development of Alzheimer's disease. However, the mechanism of the abnormal aggregation of Aβ in vivo is not well understood. We have proposed that ganglioside clusters in lipid rafts mediate the formation of amyloid fibrils by Aβ, the toxicity and physicochemical properties of which are different from those of amyloids formed in solution. In this paper, the mechanism by which Aβ-(1-40) fibrillizes in raftlike lipid bilayers composed of monosialoganglioside GM1, cholesterol, and sphingomyelin was investigated in detail on the basis of singular-value decomposition of circular dichroism data and analysis of fibrillization kinetics. At lower protein densities in the membrane (Aβ:GM1 ratio of less than ～0.013), only the helical species exists. At intermediate protein densities (Aβ:GM1 ratio between ～0.013 and ～0.044), the helical species and aggregated β-sheets (～15-mer) coexist. However, the β-structure is stable and does not form larger aggregates. At Aβ:GM1 ratios above ～0.044, the β-structure is converted to a second, seed-prone β-structure. The seed recruits monomers from the aqueous phase to form amyloid fibrils. These results will shed light on a molecular mechanism for the pathogenesis of the disease.     

Biliverdin IX delta and neobiliverdin IX delta, isolated from the ovaries of the marine snail, Turbo cornutus

The ovaries of the marine snail Turbo cornutus contain a number of pigments. So far, the presence of carotenoids and a chromoprotein with a bile pigment, called turboverdin (= 3(2)-hydroxy-mesobiliverdin IX alpha), as its prosthetic group are known. The present work describes the isolation and structure elucidation of two further bile pigments, biliverdin IX delta and neobiliverdin IX delta. This is the first report of naturally occurring bile pigments with IX delta structure.     

Production of anti-rat islet cell surface antibody in guinea pigs

Anti-rat islet serum was prepared in guinea pigs by multiple subcutaneous inoculations of rat islets homogenates emulsified in complete Freund's adjuvant (CFA). The anti-rat islet serum was cytotoxic against rat spleen cells in the presence of complement and the nonspecific antibodies were observed with homogenates of rat livers and spleens. After absorption, the serum lost the cytotoxicity against the rat spleen cells yet showed specific cytotoxicity against the rat islet cells. The binding capacity of anti-rat islet antibody was determined by the indirect immunofluorescence test using FITC conjugated rabbit anti-guinea pig IgG serum. As the guinea pig anti-rat islet serum contained anti-insulin antibody, the role of this antibody in this cytotoxic activity and surface immunofluorescence was studied. However, the anti-insulin antibody used as the control showed neither cytotoxicity nor surface immunofluorescence. After neutralizing the anti-insulin antibody in the antiserum with insulin, the serum remained cytotoxic to the rat islet cells and a surface immunofluorescence appeared. These data show that specific anti-rat islet cell surface antibody can be produced in guinea pigs by multiple inoculations of rat islets homogenates with CFA.     

A possible target protein for smg-25A/rab3A small GTP-binding protein.

The smg-25A/rab3A protein (smg p25A) is a small GTP-binding protein implicated in intracellular vesicle traffic, particularly in neurotransmitter release from the presynapse. In the present study, we attempted to identify a target protein in bovine brain crude membranes that might be interacted with the GTP-bound form of smg p25A. When the guanosine-5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of radioiodinated smg p25A and the crude membrane fraction of bovine brain were incubated with a cross-linker, disuccinimidyl suberate, and the sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography, one radioactive band with a M(r) of about 110,000 was detected. This radioactive band appeared to be composed of radioiodinated smg p25A and a molecule with a M(r) of about 86,000. This molecule, tentatively termed here smg p25A target, was extracted from the membranes by a detergent and highly purified by column chromatographies and sucrose density gradient ultracentrifugation. The purified smg p25A target was sensitive to heat boiling and tryptic digestion, indicating that smg p25A target is a protein molecule. The M(r) of the purified smg p25A target was estimated to be about 85,000-86,000 from SDS-PAGE and to be about 100,000 from the S value. The cross-linking of radioiodinated smg p25A with the purified smg p25A target was inhibited by the GTP gamma S-bound form of non-radioactive smg p25A with an IC50 of about 8 nM. The GDP-bound form of smg p25A was much less effective. Other small GTP-binding proteins, such as c-Ki-ras p21, rhoA p21, smg p21B, and rab11 p24 were ineffective. These results indicate that a protein with a M(r) of about 85,000-100,000 is a target for smg p25A.     

Enhanced Stability and Thickness‐Independent Oxygen Evolution Electrocatalysis of Heterostructured Anodes with Buried Epitaxial Bilayers

Achieving high oxygen evolution reaction (OER) activity while maintaining performance stability is a key challenge for designing perovskite structure oxide OER catalysts, which are often unstable in alkaline environments transforming into an amorphous phase. While the chemical and structural transformation occurring during electrolysis at the electrolyte–catalyst interface is now regarded as a crucial factor influencing OER activity, here, using La_0.7Sr_0.3CoO_3?δ (LSCO) as an active OER catalyst, the critical influence of buried layers on the oxidation current stability in nanoscopically thin, chemically and structurally evolving, catalyst layers is revealed. The use of epitaxial thin films is demonstrated to engineer both depletion layer widths and chemical stability of the catalyst support structure resulting in heterostructured anodes that maintain facile transport kinetics across the electrolyte–anode interface for atomically thin (2–3 unit cells) LSCO catalyst layers and greatly enhanced oxidation current stability as the perovskite structure OER catalysts chemically and structurally transform. This work opens up an approach to design robust and active heterostructured anodes with dynamically evolving ultrathin OER electrocatalyst layers for future green fuel technologies such as conformal coatings of high‐density 3D anode topologies for water splitting.     

Interleukin-15 effectively potentiates the in vitro tumor-specific activity and proliferation of peripheral blood γδT cells isolated from glioblastoma patients

γδT cells play a regulatory role in both primary and metastatic tumor growth in humans. The mechanisms responsible for the activation and proliferation of circulating γδT cells should be fully understood prior to their adoptive transfer to cancer patients. We have examined in vitro functional effects of interleukin-15 (IL-15) on highly purified γδT cells isolated from glioblastoma patients. γδT cells constitutively express the heterotrimeric IL-2 receptor (IL-2R) αβγ, but the levels of IL-2Rβ or γ expression were not increased by incubation with saturating amounts of IL-15. IL-15 was shown to induce a maximal γδT cell proliferation, although at much higher concentrations (at least 2000 U/ml) than IL-2 (100 U/ml). Submaximal concentrations of IL-15 plus low concentrations of IL-2 produced an additive proliferative response. In contrast to the IL-2-induced response, this activity was completely or partially abrogated by anti-IL-2Rβ, or anti-IL-2Rγ antibodies, but not by anti-IL-2Rα antibodies. Incubation of γδT cells in the presence of IL-15 resulted not only in the appearance of NK and LAK activity, but also in specific autologous tumor cell killing activity, an additive effect being seen with IL-15 and IL-2. This IL-15-induced tumor-specific activity could be significantly blocked by anti-IL-2Rγ and anti-IL-2R-β mAb, but not by anti-IL-2Rα mAb. Thus, in contrast to IL-2, IL-15 activates tumor-specific γδT cells through the components of IL-2Rβ and IL-2Rγ, but not IL-2Rα. These enhanced in vitro tumor-specific and proliferative responses of γδT cells seen with IL-15 suggest a rational adjuvant imunotherapeutic use of γδT cells in cancer patients. Received: 23 January 1998 / Accepted: 20 May 1998     

Protein kinase C delta regulates the phosphorylation of heat shock protein 27 in human hepatocellular carcinoma

We have recently reported that attenuated phosphorylation of heat shock protein (HSP) 27 correlates with tumor progression in patients with hepatocellular carcinoma (HCC). In the present study, we investigated what kind of kinase regulates phosphorylation of HSP27 in human HCC-derived HuH7 cells. 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleoyl-2-acetylglycerol, direct activators of protein kinase C (PKC), markedly strengthened the phosphorylation of HSP27. Bisindorylmaleimide I, an inhibitor of PKC, suppressed the TPA-induced levels of HSP27 phosphorylation in addition to its basal levels. Knock down of PKCdelta suppressed HSP27 phosphorylation, as well as p38 mitogen-activated protein kinase (MAPK) phosphorylation. SB203580, an inhibitor of p38 MAPK, suppressed the TPA-induced HSP27 phosphorylation. Our results strongly suggest that activation of PKCdelta regulates the phosphorylation of HSP27 via p38 MAPK in human HCC.     

Diversity in the degree of sulfation and chain length of the glycosaminoglycan moiety of urinary trypsin inhibitor isomers

Five isomers with different electric charge were fractionated from human urinary trypsin inhibitor (UTI) by anion exchange HPLC. Intact low-sulfated chondroitin 4-sulfate chains from the isomers were analyzed by HPLC and mass spectrometry. Unsaturated disaccharide composition analysis of the chondroitin sulfate chain revealed that the five isomers differ in the numbers of 4-sulfated disaccharide units. Intriguingly, we detected the presence of multiple novel isomers with different numbers of non-sulfated disaccharide units even in the same charge isomer fraction. Our results demonstrate that UTI can vary in terms of both the degree of sulfation and the length of the low-sulfated chondroitin 4-sulfate chain.