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At least 289 records · Page 16Linked to original sources
PubMed2006

Aggregation of red blood cells in patients with Gaucher disease.

Gaucher disease is associated with increased red blood cell (RBC) aggregation, but the pathophysiological significance of this phenomenon and its correlation with disease manifestations are unclear. RBC aggregation was evaluated in 43 patients with Gaucher disease and 53 healthy controls. Dynamic RBC aggregation was examined in a narrow-gap flow chamber at varying shear stress. Compared with the controls, RBC aggregation in Gaucher disease was increased by 25%. Comparison of RBC aggregation in autologous plasma and in dextran (500 kDa) showed an increase both in plasma-dependent (extrinsic) and -independent (intrinsic) RBC aggregation. Subgroup analysis revealed that increased RBC aggregation was limited to patients with an intact spleen. RBC aggregation in patients did not correlate with plasma fibrinogen concentration, disease severity, enzyme replacement therapy or genotype. We conclude that RBC aggregation is increased in patients with Gaucher disease and an intact spleen, possibly reflecting the accumulation of glucocerebroside and other substances in the plasma and RBC membranes of these patients. Our results do not support a role for RBC aggregation in the pathogenesis of vascular complications of Gaucher disease.

Adult↗
PubMed1976

Metallocytochromes c: characterization of electronic absorption and emission spectra of Sn4+ and Zn2+ cytochromes c.

Tin (Sn4+) and zinc (Zn2+) derivatives of horse heart cytochrome c have been prepared and their optical spectra have been characterized. Zinc cytochrome c has visible absorption maxima at 549 and 585 nm and Soret absorption at 423 nm. Tin cytochrome c shows visible absorption maxima at 536 and 574 nm and Soret absorption at 410 nm. Unlike iron cytochrome c in which the emission spectrum of the porphyrin is almost completely quenched by the central metal, the zinc and tin derivatives of cytochrome c are both fluorescent and phosphorescent. The fluorescence maxima of zinc cytochrome c are at 590 and 640 nm and the fluorescence lifetime is 3.2 ns. The fluorescence maxima of Sn cytochrome are at 580 and 636 nm and the fluorescence lifetime is under 1 ns. The quantum yield of fluorescence is Zn greater than Sn while the quantum yield of phosphorescence is Sn greater than Zn. at 77 K the fluorescence and phosphorescence emission spectra of Sn and Zn cytochrome c show evidence of resolution into vibrational bands. The best resolved bands occur at frequency differences 750 cm-1 and 1540--1550 cm-1 from the O-O transition. These frequencies correspond with those obtained by resonance Raman spectroscopy for in-plane deformations of the porphyrin macrocycle.

Animals↗
PubMed1992

Expression of Erythrina corallodendron lectin in Escherichia coli.

The cDNA of the Erythrina corallodendron lectin (ECorL) has been expressed in Escherichia coli. For this purpose, an NcoI site was inserted into the cDNA coding for the lectin precursor [Arango, R., Rozenblatt, S. & Sharon, N. (1990) FEBS Lett. 264, 109-112] immediately before the codon GTG (103-105) which codes for the N-terminal valine of the mature lectin. This introduced an ATG codon for a methionine preceding the valine. The mutated cDNA was ligated into pUC-8, then subcloned into the expression vector pET-3d, which carries a strong promoter derived from gene 10 of the phage T7. The recombinant plasmid was introduced into the E. coli lysogenic strain BL21(DE3). Recombinant ECorL was expressed by growing the bacteria in the presence of isopropyl beta-D-thiogalactopyranoside. Most of the recombinant lectin was found in an insoluble aggregated form as inclusion bodies and only a small part was in the culture medium in a soluble active form. Functional recombinant lectin was recovered from the inclusion bodies by solubilization with 6 M urea in cyclohexylaminopropane sulfonate pH 10.5, renaturation by 10-fold dilution in the same buffer and further adjustment of the pH to 8.0. The recombinant lectin, obtained at a yield of 4-7 mg/l culture, had, by gel filtration, a slightly lower molecular mass (56 kDa) than the native lectin, and was devoid of covalently linked carbohydrate; it was, however, essentially indistinguishable from native ECorL by other criteria, including its dimeric structure, Western blot analysis with anti-ECorL polyclonal and monoclonal antibodies, and Ouchterlony double-diffusion analysis with polyclonal antibodies, as well as hemagglutinating activity and specificity for mono- or disaccharides.

Amino Acid Sequence↗
PubMed1996

Mutational studies of the amino acid residues in the combining site of Erythrina corallodendron lectin.

High-resolution X-ray crystallography of the complex of the Gal/GalNAc-specific Erythrina corallodendron lectin with lactose identified the amino acid side chains that form contacts with the galactose moiety of the disaccharide. The contribution of these amino acids to the binding of different monosaccharides and oligosaccharides by the lectin was examined by site-directed mutagenesis. Replacement of Phe131, on which the galactose is stacked, by tyrosine, gave a mutant with the same hemagglutinating activity and carbohydrate specificity as the parent lectin, but replacement by alanine or valine resulted in loss of activity. Mutations of Ala88, Asp89, and Asn133 produced mutants that were also inactive whereas those of the other combining site residues, Tyr106, Ala218, and Gln219, were biologically active. None of the active mutants interacted with mannose or glucose. Thus, contrary to an earlier assumption. Ala218 is not responsible for the inability of E. corallodendron lectin to bind these sugars. Our findings also demonstrate that Gln219 is not involved in galactose binding in solution, even though this is implicated by the crystal data. Instead, our data suggest that Gln219 assists in the ligation of N-acetyllactosamine to the lectin, by interacting with the acetamide group of the disaccharide. Comparison with other legume lectins specific for mannose/glucose, galactose, N-acetylgalactosamine, L-fucose or N-acetylglucosamine, shows that only three of the combining site residues of E. corallodendron lectin occupy invariant positions both in their primary and tertiary structures. These residues are an aspartic acid and an asparagine corresponding to positions 89 and 133, respectively, in E. corallodendron lectin, and an aromatic residue, either phenylalanine (as Phe131 in this lectin), tyrosine or tryptophan. We therefore postulate that these three residues are essential for ligand binding by all such lectins, irrespective of their specificity.

Acetylgalactosamine↗
PubMed1997

Involvement of laser photo-CIDNP (chemically induced dynamic nuclear polarization)-reactive amino acid side chains in ligand binding by galactoside-specific lectins in solution.

For proteins in solution the validity of certain crystallographic parameters can be ascertained by a combination of molecular-dynamics (MD) simulations and NMR spectroscopy. Using the laser photo-CIDNP (chemically induced dynamic nuclear polarization) technique as a measure for surface accessibility of histidine, tyrosine and tryptophan, the spectra of bovine galectin-1 and Erythrina corallodendron lectin (EcorL) are readily reconcilable with the crystallographic data for these two proteins. The results emphasise the role of Trp68/Trp69 for carbohydrate binding in bovine galectin-1/chicken galectins and of Trp194 in murine galectin-3. This feature derived from the crystal structure of bovine galectin-1 is maintained in solution for the prototype human homologue, two avian galectins and the chimera-type murine galectin-3, as the spectra corroborate the CIDNP-inferable spatial parameters of the four calculated models for binding-site architecture. In EcorL, Tyr106/Tyr108 are constituents of the extended combining pocket, which can be shielded in solution by ligand presence. Discrepancies between results from modelling and CIDNP measurements concern primarily the lack of reactivity of histidine residues for human and avian prototype galectins and of Tyr82/Tyr229 of the plant lectin. Site-directed mutagenesis of EcorL is assumed to provide information on the role of a certain residue for functional aspects. When single-site mutants of EcorL ([Ala106]EcorL, [Ala108]EcorL, [Ala229]EcorL) were subjected to molecular-dynamics (MD) simulations, the apparent surface accessibilities even of spatially separated amino acid side chains could non-uniformly be affected. This conclusion is supported by the assessment of the spectra for the mutant proteins. On the basis of these CIDNP-results modelling of the binding-site architecture of the lectin indicates the occurrence of notable alterations in the orientation of Tyr106/Tyr108 phenyl rings. The implied potential effect of single-site mutations on conformational features of a protein will deserve attention for the interpretation of studies comparing wild-type and mutant proteins.

Amino Acid Sequence↗
PubMed1997

Synthesis of soybean agglutinin in bacterial and mammalian cells.

The cDNA of soybean agglutinin (SBA), a glycoprotein lectin, obtained from the mRNA of soybean seeds at mid-maturation, was cloned in a lambda gt 10 phage and subcloned in a pUC-8 plasmid. Probing with a fragment of the lectin gene [Vodkin, L. O., Rhodes, P. R. & Goldberg, R. B. (1983) Cell 34, 1023-1031] afforded a clone of 1012 nucleotides containing the complete coding region of 858 nucleotides for the precursor to soybean agglutinin. The deduced amino acid sequence contains the 253 residues of the mature lectin and an hydrophobic N-terminal signal peptide of 32 amino acids. Expression in Escherichia coli of the cDNA coding for the precursor to the lectin or for the mature lectin led to the accumulation of large quantities of inclusion bodies, from which mature SBA was isolated in small yield (up to 1 mg/l). It was identical with the native lectin in the hemagglutinating activity and carbohydrate specificity, N-terminal sequence and oligomeric structure, but, because it was not glycosylated, its subunit mass was lower by 2 kDa. Our findings show that pre-SBA is processed into the mature form in the bacteria, and that, contrary to what has been suggested [Nagai, K. & Yamaguchi, H. (1993) J. Biochem. (Tokyo) 113, 123-125], glycosylation is not essential for the folding of the lectin, nor for its subunit assembly into a biologically active tetramer. To obtain recombinant SBA in secreted form, the pre-SBA cDNA was subcloned in pTM1 vector and the construct inserted into vaccinia virus. When monkey BS-C-1 cells were infected by the virus, using a double expression protocol, recombinant lectin was secreted into the growth medium, from which it was isolated by immunoaffinity chromatography at a yield similar to that from the bacteria. Except for its lower hemagglutinating activity, the product was indistinguishable from native SBA in all properties tested. It was also susceptible to digestion by endo-beta-N-acetylglucosaminidase H or N-glycanase which caused a decrease of 2 kDa in its subunit mass and gave the same results on lectin blot analysis, indicating that it too is a glycoprotein with a single oligomannose unit.

Amidohydrolases↗
PubMed1997

Treatment planning 2000: an esthetically oriented revision of the original implant protocol.

Soft and hard tissue ridge deformities are prevalent in areas of tooth loss and trauma and can compromise esthetic outcomes. Only a full understanding of the severity of the dimensional defects, the reconstructive techniques available, and the functional and esthetic needs of the final implant restoration will allow the design of a treatment approach that will achieve the desired outcome. This article emphasizes the synergistic relation of the osseous, gingival, and restorative triad in treatment planning. In addition, a systematic approach to the enhancement of this triad, in the partially edentulous patient, is outlined as part of a revised protocol for achieving optimal esthetics in implant therapy.

Dental Implantation↗
PubMed1998

Fifty years of interdisciplinary site development: lessons and guidelines from periodontal prosthesis.

Just as "osseointegration" became synonymous with successful restoration of function in the fully edentulous patient during the 1980s, the term "implant site development" has become intricately associated in the 1990s with the techniques used to achieve esthetic results with implants in the partially edentulous patient. This article explores the roots of the concept of site development within the philosophy and principles of periodontal prosthesis. In addition, the myriad of techniques that are presently collectively referred to as site development are systematically classified into a sequential four-tiered approach that optimizes their efficient application as well as overall success.

Dental Prosthesis↗
PubMed1998

Esthetic proportions versus biologic width considerations: a clinical dilemma.

Combining harmonious proportions of final restorations with periodontal health and stability surrounding these restorations is a basic fundamental in esthetic dentistry. However, when one can adversely affect the other, a clinical dilemma arises. This article describes a case with unfavorable proportions that may be worsened due to an existing unfavorable preparation design. A simple approach for diagnosis and treatment of such a situation is presented.

Adult↗
PubMedDate not supplied

Retardation of organic contaminants in natural fractures in chalk.

Transport of a conservative compound and two sorbing compounds through fractured chalk was studied using flow-through columns consisting of chalk cores with a single subvertical fracture. Two types of chalk matrix were compared, an oxidized white chalk with low organic carbon content (0.2%), and a gray chalk with a higher organic carbon content (1.3%). Initial rapid breakthrough followed by a delayed approach to a relative concentration of unity for the conservative compound (2,6-difluorobenzoic acid [DFBA]) was clear evidence for diffusion into the porous chalk matrix. Matrix diffusion of DFBA was apparently much greater in the gray chalk columns than in the white chalk columns. Breakthrough curves (BTCs) of the sorbing compounds (2,4,6-tribromophenol [TBP] and ametryn [AME]) were retarded in all cases as compared to the conservative compound. Sorption retardation was far greater in the gray chalk as compared with the white chalk, in good agreement with results from batch sorption experiments. BTCs for the conservative compound were relatively nonhysteretic for both white and gray chalk columns. In contrast, BTCs for the sorbing compounds were hysteretic in all cases, demonstrating that sorption was not at equilibrium before desorption began. These experiments suggest that on a field scale, transport of contaminants through fractures in chalk and other fractured porous media will be attenuated by diffusion and sorption into the matrix.

Adsorption↗
PubMedDate not supplied

Characterization of the hydraulic properties of fractures in chalk.

The fracture systems intersecting Eocene chalk formations in the Negev desert, Israel, and their hydraulic properties were characterized using a variety of geologic and hydrologic techniques. These included identification of the prevailing directions of fracture systems in outcrops, in cores retrieved from inclined coreholes, in coreholes using video logs, and in trenches. The orientation and inclination of these fracture systems were determined, and evidence of ground water flow on the fracture surfaces was described and ranked. Their hydraulic conductivity was determined through slug and pumping tests performed at discrete intervals. Temperature, electrical conductivity, caliper, gamma and heat-pulse logs were conducted in the same coreholes. The results from the logs, tests, and core descriptions were compared to identify reliable and cost-effective tools for investigating the hydraulic characteristics of fracture systems. We concluded that in the study area: (1) fracture mapping in outcrops and coreholes (including downhole video and caliper logs) must be supplemented by hydraulic testing of the mapped fracture sets in the coreholes; (2) inclined coreholes provide information regarding the orientation of the hydraulically active fracture systems that cannot be obtained from vertical boreholes; (3) hydraulic testing of unpacked holes provides a reasonable estimate of the maximum hydraulic conductivity; and (4) the hydraulic conductivity distribution with depth is log normal and all significant ground water flow takes place within the upper 25 m.

Environmental Monitoring↗
PubMedDate not supplied

Correlating fracture trends and hydraulic head using semivariogram cloud analysis.

Fracture trends (defined as kilometer-scale linear features interpolated between field observations of fractures along their strikes) often have a dominant orientation. Finding a correlation between this orientation and hydraulic data could shed light on their hydraulic influence. A significant correlation between head residuals from first-order regional drift and the orientation of 2- to 4-km-long fracture trends was found in a study site in the Negev, Israel, using the semivariogram cloud analysis. Correlation of head residuals rather than the head itself implies that the orientation of the fracture trends controls the anisotropy and heterogeneity at this scale, mainly because the fracture trends define the orientation of blocks, which differ in their hydraulic properties. Preferential transmissive pathways are probably shorter than the full extent of the fracture trends, causing a relatively high head difference along the trends on the 2- to 4-km scale. Fracture trend density and additional data from short-range hydraulic tests helped characterize two blocks separated by a fault zone. The identification of hydraulic features on a kilometer scale is necessary for better modeling of regional ground water flow and transport. Hydraulic tests at this scale are not feasible, thereby rendering combined analyses of head and structural data, such as the one presented here, essential.

Forecasting↗
PubMed1989

Inhibitory activity of cranberry juice on adherence of type 1 and type P fimbriated Escherichia coli to eucaryotic cells.

Inhibition of bacterial adherence to bladder cells has been assumed to account for the beneficial action ascribed to cranberry juice and cranberry juice cocktail in the prevention of urinary tract infections (A. E. Sobota, J. Urol. 131:1013-1016, 1984). We have examined the effect of the cocktail and juice on the adherence of Escherichia coli expressing surface lectins of defined sugar specificity to yeasts, tissue culture cells, erythrocytes, and mouse peritoneal macrophages. Cranberry juice cocktail inhibited the adherence of urinary isolates expressing type 1 fimbriae (mannose specific) and P fimbriae [specific for alpha-D-Gal(1----4)-beta-D-Gal] but had no effect on a diarrheal isolate expressing a CFA/I adhesin. The cocktail also inhibited yeast agglutination by purified type 1 fimbriae. The inhibitory activity for type 1 fimbriated E. coli was dialyzable and could be ascribed to the fructose present in the cocktail; this sugar was about 1/10 as active as methyl alpha-D-mannoside in inhibiting the adherence of type 1 fimbriated bacteria. The inhibitory activity for the P fimbriated bacteria was nondialyzable and was detected only after preincubation of the bacteria with the cocktail. Cranberry juice, orange juice, and pineapple juice also inhibited adherence of type 1 fimbriated E. coli, most likely because of their fructose content. However, the two latter juices did not inhibit the P fimbriated bacteria. We conclude that cranberry juice contains at least two inhibitors of lectin-mediated adherence of uropathogens to eucaryotic cells. Further studies are required to establish whether these inhibitors play a role in vivo.

Agglutination↗
PubMed2001

Search for correlates of protective immunity conferred by anthrax vaccine.

Vaccination by anthrax protective antigen (PA)-based vaccines requires multiple immunization, underlying the need to develop more efficacious vaccines or alternative vaccination regimens. In spite of the vast use of PA-based vaccines, the definition of a marker for protective immunity is still lacking. Here we describe studies designed to help define such markers. To this end we have immunized guinea pigs by different methods and monitored the immune response and the corresponding extent of protection against a lethal challenge with anthrax spores. Active immunization was performed by a single injection using one of two methods: (i) vaccination with decreasing amounts of PA and (ii) vaccination with constant amounts of PA that had been thermally inactivated for increasing periods. In both studies a direct correlation between survival and neutralizing-antibody titer was found (r(2) = 0.92 and 0.95, respectively). Most significantly, in the two protocols a similar neutralizing-antibody titer range provided 50% protection. Furthermore, in a complementary study involving passive transfer of PA hyperimmune sera to naive animals, a similar correlation between neutralizing-antibody titers and protection was found. In all three immunization studies, neutralization titers of at least 300 were sufficient to confer protection against a dose of 40 50% lethal doses (LD(50)) of virulent anthrax spores of the Vollum strain. Such consistency in the correlation of protective immunity with anti-PA antibody titers was not observed for antibody titers determined by an enzyme-linked immunosorbent assay. Taken together, these results clearly demonstrate that neutralizing antibodies to PA constitute a major component of the protective immunity against anthrax and suggest that this parameter could be used as a surrogate marker for protection.

Animals↗
PubMed1986

Incorporation and modification of exogenous phosphatidylcholines by mycoplasmas.

The uptake and modification of exogenous phosphatidylcholine (PC) by several Mycoplasma and Spiroplasma species was investigated. While in most Mycoplasma species and in all Spiroplasma species tested the PC appears to be incorporated unchanged from the growth medium, the PC of M. gallisepticum, M. pulmonis, and M. pneumoniae was disaturated PC, apparently formed by modification of 1-saturated-2-unsaturated PC from the growth medium. The modification of the exogenous PC by M. gallisepticum was inhibited by chloramphenicol under conditions that did not affect de novo synthesis of phosphatidylglycerol. A low activity of an endogenous phospholipase A was detected in native M. gallisepticum membranes. The activity was markedly stimulated by treating the membranes with low concentrations of the nonionic detergents. The PC modification was affected by the fatty acid composition of the exogenous PC species. Diunsaturated, 1-saturated-2-unsaturated, and 1-unsaturated-2-saturated PCs were modified to various extents, whereas the disaturated dipalmitoyl PC (DPPC) was not. Both modified and unmodified PCs were incorporated by the cells, but the unmodified DPPC was incorporated at a lower rate and to a lesser extent. The possibility that the incorporation of DPPC into M. gallisepticum cells is associated with the formation of intracytoplasmic membranes is discussed.

Chloramphenicol↗
PubMed1992

Formation of the LuxR protein in the Vibrio fischeri lux system is controlled by HtpR through the GroESL proteins.

The transcription of the luminescence (lux) system of Vibrio fischeri is regulated by the LuxR protein and an autoinducer. We previously showed that apart from these regulatory elements, the transcription of the lux system is negatively controlled by the LexA protein and positively controlled by the HtpR protein (sigma 32). This study was conducted in order to elucidate the mode of action of the HtpR protein. Using luxR-lacZ fused genes, we showed that the HtpR protein is essential for the maximum expression of beta-galactosidase activity in Escherichia coli lac mutant cells. Using this construct, we also demonstrated that luxR is preferentially expressed toward the end of the logarithmic phase of growth. Starvation and addition of ethanol significantly advanced the appearance of beta-galactosidase activity in htpR+ cells. The luminescence system of E. coli htpR+ cells harboring the pChv1 plasmid with a deletion in the luxI gene is induced in the presence of low and constant concentrations (150 pg/ml) of the inducer only at a late stage of the logarithmic phase of growth. When the cellular LuxR content is reduced, following 23 generations of exponential growth in Luria broth, a mid-log-phase culture does not respond to the inducer (150 pg/ml). On the basis of the above observations we suggest that the HtpR protein controls the formation of V. fischeri LuxR protein. Preliminary findings indicate that the HtpR protein acts through the chaperonins GroESL. E. coli htpR/pChv1 cells retained their full level of in vivo and in vitro luciferase activities in the presence of multiple copies of groESL genes. The possibility that GroESL proteins stabilize the native form of LuxR protein is discussed.

Bacterial Proteins↗
PubMed2000

The transmembrane mutation G380R in fibroblast growth factor receptor 3 uncouples ligand-mediated receptor activation from down-regulation.

A point mutation, Gly380Arg, in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3) leads to achondroplasia, the most common form of genetic dwarfism in humans. This substitution was suggested to enhance mutant receptor dimerization, leading to constitutive, ligand-independent activation. We found that dimerization and activation of the G380R mutant receptor are predominantly ligand dependent. However, using both transient and stable transfections, we found significant overexpression only of the mutant receptor protein. Metabolic pulse-chase experiments, cell surface labeling, and kinetics of uptake of radiolabeled ligand demonstrated a selective delay in the down-regulation of the mutant receptor. Moreover, this receptor was now resistant to ligand-mediated internalization, even at saturating ligand concentrations. Finally, transgenic mice expressing the human G380R mutant receptor under the mouse receptor transcriptional control demonstrated a markedly expanded area of FGFR3 immunoreactivity within their epiphyseal growth plates, compatible with an in vivo defect in receptor down-regulation. We propose that the achondroplasia mutation G380R uncouples ligand-mediated receptor activation from down-regulation at a site where the levels and kinetics of FGFR3 signals are crucial for chondrocyte maturation and bone formation.

Amino Acid Substitution↗