Alterations in the adhesion behavior of osteoblasts by titanium particle loading: inhibition of cell function and gene expression

Total joint replacement prostheses are required to withstand corrosive environments and sustain millions of loading and articulation cycles during their term of implantation. Wear debris generation has been implicated as one of the primary causes of periprosthetic osteolysis and subsequent implant loosening in total joint replacements. Particulate debris consisting of metals, polyethylene, ceramics, and bone cement have each been shown to provoke a biological response in joint tissues. The major cell types within the interfacial granulomatous fibrous tissues consist of fibroblasts, macrophages, lymphocytes, and foreign-body giant cells. Osteoblasts are one of the principal cell types in the bone tissue adjacent to prostheses, maintaining physiologic bone remodeling through the balanced coordination of bone formation and resorption in concert with osteoclasts. To date the phenomenon of osteoblast phagocytosis of titanium particles has been suggested, but has not been sufficiently studied or confirmed. This study seeks to clarify the influence of titanium particles on osteoblast adhesion, deformability, proliferation, and gene expression profile. These studies were accomplished by performing biorheological testing, Northern blot analysis and RNase protection assay. The uptake of metallic particles by the osteoblast resulted in a particle-filament complex formation, which induced a series of variations in cell function. Understanding these variations is critical to expanding our knowledge of implant loosening and elucidating the nature of prosthetic joint failure. This study suggests that the impact of titanium particles on osteoblast function and subsequent implant loosening may have been previously underestimated.     

Glutamate synthesis via photoreduction of NADP+ by photostable chlorophyllide coupled with polyethylene-glycol

Chlorophyllide a was coupled with alpha-(3-aminopropyl)-omega-methoxypoly(oxyethylene) (PEG-NH2) to form a PEG-chlorophyllide conjugate through an acid-amide bond. The conjugate catalyzed the reduction of methylviologen in the presence of 2-mercaptoethanol. It also catalyzed the photoreduction of NADP+ or NAD+ in the presence of ascorbate as an electron donor and ferredoxin-NADP+ reductase as the coupling enzyme. Utilizing the reducing power of NADPH generated by PEG-chlorophyllide conjugate under illumination, glutamate was synthesized from 2-oxoglutarate and NH4+ in the presence of glutamate dehydrogenase. PEG-chlorophyllide conjugate was quite stable toward light illumination compared with chlorophyll a. The increase in the molecular weight of PEG in the PEG-chlorophyllide conjugates was accompanied by the enhancement of photostability of the conjugate and also by the increased solubility in the aqueous solution.     

Improved oligosaccharide synthesis by protein engineering of beta-glucosidase CelB from hyperthermophilic Pyrococcus furiosus

Enzymatic transglycosylation of lactose into oligosaccharides was studied using wild-type beta-glucosidase (CelB) and active site mutants thereof (M424K, F426Y, M424K/F426Y) and wild-type beta-mannosidase (BmnA) of the hyperthermophilic Pyrococcus furiosus. The effects of the mutations on kinetics, enzyme activity, and substrate specificity were determined. The oligosaccharide synthesis was carried out in aqueous solution at 95 degrees C at different lactose concentrations and pH values. The results showed enhanced synthetic properties of the CelB mutant enzymes. An exchange of one phenylalanine to tyrosine (F426Y) increased the oligosaccharide yield (45%) compared with the wild-type CelB (40%). Incorporation of a positively charged group in the active site (M424K) increased the pH optimum of transglycosylation reaction of CelB. The double mutant, M424K/F426Y, showed much better transglycosylation properties at low (10-20%) lactose concentrations compared to the wild-type. At a lactose concentration of 10%, the oligosaccharide yield for the mutant was 40% compared to 18% for the wild-type. At optimal reaction conditions, a higher ratio of tetrasaccharides to trisaccharides was obtained with the double mutant (0.42, 10% lactose) compared to the wild-type (0.19, 70% lactose). At a lactose concentration as low as 10%, only trisaccharides were synthesized by CelB wild-type. The beta-mannosidase BmnA from P. furiosus showed both beta-glucosidase and beta-galactosidase activity and in the transglycosylation of lactose the maximal oligosaccharide yield of BmnA was 44%. The oligosaccharide yields obtained in this study are high compared to those reported with other transglycosylating beta-glycosidases in oligosaccharide synthesis from lactose.     

Totally synthetic polymer with lectin-like function: induction of killer cells by the copolymer of 3-acrylamidophenylboronic acid with N,N-dimethylacrylamide

Lectin-like function was demonstrated in this study for a novel water-soluble polymer with phenylboronic acid residues (poly (AAPBA-DMAm)), which induced appreciable proliferation of murine spleen lymphocytes with an increased expression of interleukin-2 (IL-2) receptor on their surface. Consequently, boosted proliferation of lymphocytes with cytotoxic action to YAC-1 cells was achieved by concurrent addition of IL-2 with poly(AAPBA-DMAm) in the medium, indicating this boronate-containing polymer to be worked as an effective immuno-adjuvant for the induction of lymphokine-activated killer (LAK) cells. Flow-cytofluorimetry study revealed that poly(AAPBA-DMAm) competitively inhibited the cellular binding of N-acetylneuraminic acid-specific lectin (Limax Flavus Agglutinin) in a concentration-dependent manner, suggesting that phenylboronate moiety in the polymer may recognize N-acetylneur- aminic acid (sialic acid) residues existing on the plasma-membrane surface of lymphocytes to induce their proliferation.     

Chemostat modeling of Escherichia coli persistence in conventionalized mono-associated and streptomycin-treated mice

We have previously shown that Escherichia coli BJ4 has similar doubling time in mice that are mono-associated (having only the inoculated E. coli BJ4) or streptomycin-treated (having mainly gram-positive bacteria plus the inoculated E. coli BJ4). We also showed that when the mice were conventionalized (fed cecum homogenate from conventional mice or ones with a complete microbial flora), the introduction of complete flora in both cases increased the in vivo doubling time, while decreasing the colony counts in fecal samples. To determine whether the increase in doubling time could explain the decrease in colony counts, we analyzed our previous results by a chemostat model. The analysis shows that the increasing doubling time alone is sufficient to explain the decrease in colony counts in mono-associated mice, but not in the streptomycin-treated mice. The observed decreasing rate in colony counts in streptomycin-treated mice is slower than predicted. Furthermore, whereas the model predicted a decrease to extinction in both mice, the E. coli persist at a frequency 10-80 times higher in streptomycin-treated mice than in mono-associated mice. Thus, while a chemostat model is able to explain some of the population dynamics of intestinal bacteria in mice, additional factors not included in the model are stabilizing the system. Because we find that E. coli declines more slowly and to a higher stabilization frequency in streptomycin-treated mice, which have a more diverse flora before conventionalization, we take these results to suggest that the persistence of E. coli populations is promoted by species diversity. We propose that a mechanism for the persistence may be the presence of new E. coli niches created by keystone species in the more diverse flora.     

A comparison of the kinetics of low-density lipoprotein oxidation induced by copper or by gamma-rays: influence of radiation dose-rate and copper concentration

The oxidation of low-density lipoproteins is the first step in the complex process leading to atherosclerosis. The aim of our study was to compare the kinetics of low density lipoprotein oxidation induced by copper ions or by oxygen free radicals generated by 60Co gamma-rays. The effects of copper concentration and irradiation dose-rate on LDL peroxidation kinetics were also studied. The oxidation of LDL was followed by the measurement of conjugated diene, hydroperoxides, and thiobarbituric acid reactive substance formation as well as alpha-tocopherol disappearance. In the case of gamma irradiation, the lag-phase before the onset of lipid peroxidation was inversely correlated to the radiation dose-rate. The radiation chemical rates (nu) increased with increasing dose-rate. Copper-induced LDL peroxidation followed two kinetic patterns: a slow kinetic for copper concentrations between 5-20 microM, and a fast kinetic for a copper concentration of 40 microM. The concentration-dependent oxidation kinetics suggest the existence of a saturable copper binding site on apo-B. When compared with gamma-rays, copper ions act as drastic and powerful oxidants only at higher concentrations (> or = 40 microM).     

Selective isolation and distribution of the genus Planomonospora in soils

For the screening of bioactive compounds and study of global distribution, a selective isolation method for Planomonospora strains by centrifugation from soil is examined. Planomonospora strains produced characteristic sporangia on the humic acid-trace salts gellan gum medium (pH 9.0) so that this genus was readily recognized on the isolation plate. High yields of motile spores were obtained by using a flooding solution containing 0.1% skim milk in 5 mM N-cyclohexyl-2-amino-ethanesulfonic acid (pH 9.0) followed by incubating the preparation at 32 degreesC for 90 min, centrifuging it at 1000 x g for 10 min, and further incubation at 32 degreesC for 60 min after centrifugation. By combining the techniques described above, we isolated 246 Planomonospora strains from 137 of the 1200 soil samples examined. Ninety-four percent of these strains were recovered from neutral to slightly alkaline soils (pH 7.0 to 9.0). Strains of P. venezuelensis group were obtained from 13 soil samples (1.1%), which were collected in Bolivia, Cyprus, Egypt, Greece, India, Japan, New Caledonia, and Turkey. Strains of this group appear widely distributed in the soil of tropical to temperate regions. To our knowledge, this is the first record that strains of this group have been isolated from a location other than Venezuela.     

Combinatorial evaluation of the chiral discrimination of permethylated carbohydrates using fast-atom bombardment mass spectrometry

The chiral discrimination abilities of several variously permethylated carbohydrates toward various amino acid 2-propyl esters were combinatorially evaluated from the relative peak intensity of the 1:1 diastereomeric complex ions with the deuterium-labeled L-amino acid 2-propyl ester protonated ion and with the unlabeled D-amino acid 2-propyl ester protonated ions in FAB mass spectrometry. The chiral discrimination abilities evaluated using FAB mass spectrometry approximately corresponded to the ratio of the association constants (K(R)/K(S)) toward each enantiomer in the solution. Therefore, this evaluation method is very useful for the screening of the chiral discrimination abilities of carbohydrates and their derivatives.     

Structural analysis of the exopolysaccharide from Burkholderia caribensis strain MWAP71

Burkholderia caribensis strain MWAP71 was isolated from rhizosphere soil microaggregates in Martinique. The extracellular polysaccharide produced by this strain was found to be composed of D-glucose (D-Glc), 6-deoxy-L-talose (L-6dTal), 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), and an O-acetyl group in a molar ratio of 2:1:1:1. The primary structure of the polysaccharide was shown by sugar analysis, electrospray mass spectrometry, partial acid hydrolysis and 1-D and 2-D NMR spectroscopy to consist of a tetrasaccharide repeating unit having the following structure: [structure in text].     

The vaso-contractile action of zooxanthellatoxin-B from a marine dinoflagellate is mediated via Ca2+ influx in the rabbit aorta

We previously showed that zooxanthellatoxin-B, isolated from dinoflagellate, caused a sustained contraction of the aorta in an external Ca2+-dependent manner. To clarify the role of Ca2+ in this action, we examined the effects of zooxanthellatoxin-B as well as a depolarizing stimulus (60 mM KCl), using the simultaneous recording for cytosolic Ca2+ level (fura-2) and developed tension in the rabbit aorta. KCl (60 mM) elicited a rapid cytosolic Ca2+ elevation followed by a pronounced contraction, and time required for half-maximum contraction was 2 min. Zooxanthellatoxin-B caused an increase in cytosolic Ca2+ followed by a gradual contraction, with a time for half-maximum contraction of 5-10 min in a concentration-dependent manner. We found a strong correlation between Ca2+ elevation and the contraction in zooxanthellatoxin-B action. In a Ca2+-free solution, zooxanthellatoxin-B caused neither the contraction nor the increase in cytosolic Ca2+. Furthermore, both pre- and post-treatment with verapamil, a voltage-operated Ca2+-channel blocker, partially suppressed both an increase in cytosolic Ca2+ and the contraction by zooxanthellatoxin-B. Zooxanthellatoxin-B-induced contraction was also inhibited by other voltage-operated Ca2+-channel blockers: nifedipine or diltiazem. These results suggest that zooxanthellatoxin-B-elicited contraction is caused by a Ca2+ influx into the smooth muscle cells, partially via voltage-operated Ca2+ channels.