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PubMed2004

Quantitative assessment of osteoporosis from the tibia shaft by ultrasound techniques.

Bone mineral density (BMD) is used as a clinical estimate of the risk of fracture. Ultrasound provides an alternative or complement to X-ray based methods of bone densitometry for determining BMD. Among ultrasonic characteristics, the speed of sound (SOS) is a useful tool for assessment of osteoporosis because, as recently reported, it represents a combination of density and compressibility of bone tissue. Thus, it might provide better information on bone quality to estimate the fracture risk. In this paper, a dual-transducer ultrasound technique was employed to measure the mean ultrasound propagation speed of the cortical layer as well as the cancellous layer at the tibia shaft. Encouraging results from 18 outpatients showed a high correlation (r = 0.93) between measurements of BMD and those from dual energy X-ray absorptiometry (DEXA).

Absorptiometry, Photon↗
PubMed1983

Chemical analysis of organotypic cultures of mouse spinal cord in normal, demyelinative, and nondemyelinative conditions.

Several biochemical parameters were analyzed in cultured embryonic mouse spinal cord during various stages of normal myelinogenesis or demyelination. In cultures demyelinated by exposure to anti-whole CNS tissue serum plus complement, the activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37) was decreased 70%, whereas in cultures that did not show morphological changes with complement-inactivated anti-CNS serum or anti-myelin basic protein serum, the activity was 30% lower than in control cultures. The lipid composition of these cultures was quantitated by means of high-performance thin-layer chromatography densitometry technique. Cultures with normal nutrient medium alone or with the addition of 5% normal rabbit serum plus 10% guinea pig serum had 30% of the total lipid content of that present in newborn mouse spinal cord of the corresponding age. There were, however, relatively more lysophospholipids, cholesterol esters, triglycerides, and free fatty acids and less phosphatidylethanolamine and galactolipids in cultures as compared with normal spinal cord. Explants demyelinated by exposure to anti-CNS serum plus complement demonstrated principally a 70% decrease in the content of galactolipids with respect to normal cultures. When complement was inactivated, total lipids increased 42% (with increases of 40-70% in individual lipids). Inclusion of anti-myelin basic protein serum plus complement in the medium produced no significant changes in the lipid composition of the cultures.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗
PubMed1997

C5a-dependent up-regulation in vivo of lung vascular P-selectin.

Acute lung injury in rats following systemic activation of complement by i.v. infusion of cobra venom factor (CVF) is known to be P-selectin dependent. In the current studies, infusion of CVF caused the appearance in plasma of C5a (as revealed by ELISA analysis) together with neutrophil chemotactic activity, which was totally blocked by addition in vitro of anti-rat C5a. Using a detector 125I-labeled antibody to rat P-selectin, we have demonstrated quantitative up-regulation in vivo of lung vascular P-selectin in a time-dependent manner after infusion of CVF. This up-regulation was almost completely blocked by prior complement depletion or by the infusion of anti-rat C5a. Platelet depletion did not affect CVF-induced up-regulation of lung vascular P-selectin, indicating that platelets were not the source of P-selectin. These results demonstrate that complement and, specifically, C5a are necessary for up-regulation of lung vascular P-selectin after systemic activation of complement.

Animals↗
PubMed1977

[Heterogeneity of antinuclear antibodies (ANA), immunoglobulin- and complement levels in sera of patients with rheumatoid arthritis (RA) (author's transl)].

ANA typing of RA patients according to the heavy- and light chains, complement fixing ability and immunofluorescent staining pattern was performed by the immunofluorescence technique and partially by qualitative immunoelectrophoresis. In the corresponding sera the immunoglobulins and complement components were quantiatively determined. Dependent on the ANA-immunoglobulin classes we found the following distribution: IgG-ANA; 40/93 (43%), IgM-ANA: 19/93 (20%), IgA-ANA: 2/93 (2%), and Ig-comb.-ANA:17/93 (18%). ANA only positive for polyspecific antimmunoglobulin serum: 15/93 (16%). After L-chain typing we found within a given ANA-Ig-class positivity for both L-chain subclasses (kappa and lambda): IgG-ANA: 19/40 (48%), IgM-ANA: 3/19 (16%), IgA-ANA:1/1 and Ig-comb.-ANA: 14/17 (82%). ANA cases within a given Ig-class only positive for the L-chain/type kappa:IgM-ANA:6/19 (32%), IgG-ANA: 9/40 (23%), Ig-comb.-ANA:2/12 (12%). ANA cases within a given Ig-class only positive for the L-chain type lambda: IgM-ANA: 7/19 (37%), IgG-ANA: 3/40 (8%), Ig-comb.-ANA:1/17 (6%) and IgA-ANA:1/1. In all cases of restricted ANA-positivity (only positive by use of polyspecific anti-immunoglobulin serum) no light chains were detected. In total we found approximatively 25% of complement fixing ANA, predominantly IgG-ANA (70%). Concerning the immunofluorescent nuclear staining we stated the following patterns: homogenous: 34/93 (36.6%), speckled: 29/93 (31.2%), mixed pattern : 22/93 (23.6%) peripheral: 5/93 (5.4%) and nucleolar: 3/93 (3.2%). The quantitative Ig-determination reveals a significant increase of IgG in ANA (single type) positive sera. IgG + IGM + IGA-ANA and IgG + IgM-ANA are found together with a significantly increased IgG- and IgM-serum levels, in the case of IgG + IgM-ANA we could demonstrate an additional relative decrease of the complement component C4. Sera with positive complement fixing ANA compared with non complement fixing ANA are characterized by an absolute increase of IgG and relative decrease of the complement component C4. No differences in serum-immunoglobulin- and -complement concentrations were registered dependent on ANA-immunofluorescent staining patterns.

Antibodies, Antinuclear↗
PubMed2006

An oxidoreductase is involved in cercosporin degradation by the bacterium Xanthomonas campestris pv. zinniae.

The polyketide toxin cercosporin plays a key role in pathogenesis by fungal species of the genus Cercospora. The bacterium Xanthomonas campestris pv. zinniae is able to rapidly degrade this toxin. Growth of X. campestris pv. zinniae strains in cercosporin-containing medium leads to the breakdown of cercosporin and to the formation of xanosporic acid, a nontoxic breakdown product. Five non-cercosporin-degrading mutants of a strain that rapidly degrades cercosporin (XCZ-3) were generated by ethyl methanesulfonate mutagenesis and were then transformed with a genomic library from the wild-type strain. All five mutants were complemented with the same genomic clone, which encoded a putative transcriptional regulator and an oxidoreductase. Simultaneous expression of these two genes was necessary to complement the mutant phenotype. Sequence analysis of the mutants showed that all five mutants had point mutations in the oxidoreductase gene and no mutations in the regulator. Quantitative reverse transcription-PCR (RT-PCR) showed that the expression of both of these genes in the wild-type strain is upregulated after exposure to cercosporin. Both the oxidoreductase and transcriptional regulator genes were transformed into three non-cercosporin-degrading bacteria to determine if they are sufficient for cercosporin degradation. Quantitative RT-PCR analysis confirmed that the oxidoreductase was expressed in all transconjugants. However, none of the transconjugants were able to degrade cercosporin, suggesting that additional factors are required for cercosporin degradation. Further study of cercosporin degradation in X. campestris pv. zinniae may allow for the engineering of Cercospora-resistant plants by using a suite of genes.

Ascomycota↗
PubMed1976

Studies on Epstein-Barr virus-related antigens. I. Indirect single radial immunodiffusion as a useful method for detection and assay of soluble antigen.

A useful method for the detection and assay of Epstein-Barr virus (EBV)-related soluble antigen has been developed by the application of the indirect single radial immunodiffusion technique which is frequently used for quantitative measurements of immunoglobulins and other soluble proteins. When the extracts of EBV-determined nuclear antigen (EBNA)-positive non-producer cells (Raji and NC-37) were applied to agar plates containing seropositive human serum, followed by overlay with anti-human IgG serum, ring-shaped precipitates with high specificity were clearly evident. The size of such precipitin rings was proportional to the amount of the antigen. This method is simple and applicable for a quantitative assay of a particular EBV-related soluble antigen and antibody and the sensitivity is equivalent to that seen with the complement fixation test.

Antibodies, Viral↗
PubMed2006

The modeling of global epidemics: stochastic dynamics and predictability.

The global spread of emergent diseases is inevitably entangled with the structure of the population flows among different geographical regions. The airline transportation network in particular shrinks the geographical space by reducing travel time between the world's most populated areas and defines the main channels along which emergent diseases will spread. In this paper, we investigate the role of the large-scale properties of the airline transportation network in determining the global propagation pattern of emerging diseases. We put forward a stochastic computational framework for the modeling of the global spreading of infectious diseases that takes advantage of the complete International Air Transport Association 2002 database complemented with census population data. The model is analyzed by using for the first time an information theory approach that allows the quantitative characterization of the heterogeneity level and the predictability of the spreading pattern in presence of stochastic fluctuations. In particular we are able to assess the reliability of numerical forecast with respect to the intrinsic stochastic nature of the disease transmission and travel flows. The epidemic pattern predictability is quantitatively determined and traced back to the occurrence of epidemic pathways defining a backbone of dominant connections for the disease spreading. The presented results provide a general computational framework for the analysis of containment policies and risk forecast of global epidemic outbreaks.

Communicable Diseases↗
PubMed1993

Simulations of passive properties and action potential conduction in an idealized bullfrog atrial trabeculum.

This study investigates the properties of a distributed parameter model of an idealized trabeculum of cardiac muscle surrounded by a resistive-capacitive trabecular sheath. A mathematical approach is developed that permits the direct solution for the absolute potential in each medium [i.e., the intracellular (Vi), interstitial (Ve), and external (Vo) potentials), as opposed to obtaining solutions for the transmembrane potential V (where V identical to Vi-Ve). The mathematical description of the underlying individual cell is based upon quantitative whole-cell voltage-clamp measurements in bullfrog atrial myocytes. "Reduced" or "simplified" cell membrane models that lack the complete complement of transmembrane currents are compared with regard to their accuracy in representing the root, upstroke, and plateau regions of the propagated action potential in the complete model. The results show that a reduced cell membrane model must contain the sodium current INa, calcium current ICa, and background-rectifying K+ current IK1. A cell membrane model that contains a linear background K+ current IL instead of IK1 results in much poorer approximation to the upstroke, plateau, and conduction velocities of an action potential. The effects of varying the resistive-capacitive parameters of the trabecular sheath on both the passive properties (the time and space constants and the input resistance) and conduction parameters (time and space constants of the foot and conduction velocity of the action potential) of the trabeculum are also investigated. These simulations show that electrical activity within the trabeculum is much more sensitive to variations in the resistive component than in the capacitive component of the sheath. The trabecular sheath reduces the extracellular resistance seen by the cell by shunting current away from highly resistive interstitial medium into the volume conductor medium, which is of low resistance, and thereby increases conduction velocity. Finally, the addition of the cholinergic neurotransmitter acetylcholine to the extracellular medium reduces both the space constant of the trabeculum and the conduction velocity of propagated electrical activity.

Action Potentials↗
PubMed1988

Urinary protein analysis.

The differentiation and analysis of urinary proteins has substantially contributed to our knowledge of physiological and pathophysiological processes during glomerular filtration and tubular catabolism of plasma constituents. By use of high-resolution biochemical separation techniques, several urinary polypeptides could be identified as plasma proteins, tissue antigens, tubular enzymes and protein breakdown products. With regard to clinical application, the separation results of conventional gel chromatography and agarose electrophoresis were surpassed by fast protein liquid chromatography and polyacrylamide electrophoresis in one- and two-dimensional systems. In contrast to early one-dimensional polyacrylamide gel electrophoresis (PAGE) methods using homogeneous gels on a macro scale, modern gradient slab gels achieve better resolution over the entire relative molecular mass range of urinary proteins. For clinical demands, the use of micro-scale gradient gels, either laboratory-made or pre-cast, together with an improved Coomassie Brillant Blue staining, offers several advantages, including rapidity, sensitivity and economy. Isoelectric focusing and two-dimensional PAGE, combined with sensitive silver staining and immunoblotting methods, have proved to be valuable tools for the identification and characterization of urinary proteins in defined renal and extra-renal diseases. The quantitative determination of urinary indicator proteins such as albumin and alpha 1- and beta 2-microglobulin can be regarded as a reasonable complement to the pattern diagnosis, especially in the long-term course of renal diseases.

Animals↗
PubMed2005

Drug design using the example of the complement system inhibitors' development.

Undesired activation of the complement system, a part of the immune system, is a major pathogenic factor contributing to various diseases, such as ischemia-reperfusion injury, sepsis, asthma, allergic reactions, rheumatoid arthritis, Alzheimer's disease, myasthenia, multiple sclerosis and others. The history of the development of complement system inhibitors, preventing its destructive action on the body, represents the evolution of the main methods of drug design. This review illustrates the main approaches of drug design, ranging from screening and modification of natural products to structure-based ligand design, on the basis of complement inhibitors' creation. The current status of the field of complement inhibitors is also discussed.

Animals↗
PubMed2002

Single-neuron analysis using CE combined with MALDI MS and radionuclide detection.

Capillary electrophoresis (CE) has been combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and radionuclide detection to assay mass-limited biological samples. Nanovial sampling techniques enable injections into the CE capillary from 50 to 150-nL volume samples; after the separation, nanoliter fraction collection combines the CE effluent with a MALDI matrix and minimizes sample spreading, thus allowing both MALDI MS and radionuclide detection on the CE fractions. MALDI MS complements the elution time information of CE by providing accurate molecular mass data, and radionuclide detection provides zeptomole limits of detection with quantitative information. While MALDI MS detects all fully processed peptides at sufficient concentration, culturing the neuron in media containing 35S-Met provides selective radionuclide detection of newly synthesized methionine-containing peptides. The analysis and detection of the expected neuropeptides and hormones in a single 40-microm bag cell neuron from Aplysia californica with CE/MALDI MS/radionuclide detection demonstrates the ability of this hyphenated approach to work with chemically complex mass-limited samples.

Animals↗
PubMed2003

Two rate-limiting steps in the kinetic mechanism of the serine/threonine specific protein kinase ERK2: a case of fast phosphorylation followed by fast product release.

Extracellular regulated protein kinase 2 (ERK2) is a eukaryotic protein kinase whose activity is regulated by mitogenic stimuli. To gain insight into the catalytic properties of ERK2 and to complement structure-function studies, we undertook a pre-steady state kinetic analysis of the enzyme. To do this, ERK2 was quantitatively activated by MAPKK1 in vitro by monitoring the stoichiometry and site specificity of phosphorylation using a combination of protein mass spectrometry, tryptic peptide analysis, and (32)P radiolabeling. Using a quench-flow apparatus, MgATP(2-) was rapidly mixed (<1 ms) with both ERK2 and the protein substrate EtsDelta138 in the presence of a saturating total concentration (20 mM) of magnesium ion at 27 degrees C and pH 7.5. An exponential burst of product was observed over the first few milliseconds that followed mixing. This burst had an amplitude alpha of 0.44 and was followed by a slower linear phase. The pre-steady state burst is consistent with two partially rate-limiting enzymatic steps, which have the following rate constants: k(2) = 109 +/- 9 s(-1) and k(3) = 56 +/- 4 s(-1). These are attributed to rapid phosphorylation of EtsDelta138 and the process of product release, respectively. Single-turnover experiments provided an independent determination of k(2) (106 +/- 25 s(-1)). The observed catalytic constant (k(cat)(obs)) was found to be sensitive to the concentration of ERK2. The data fit a model in which ERK2 monomers form dimers and suggest that both the monomeric and dimeric forms of ERK2 are active with catalytic constants (k(cat)) of 25 and 37 s(-1), respectively. In addition, the model suggests that in the presence of saturating concentrations of both magnesium and substrates ERK2 subunits dissociate with a dissociation constant (K(d)) of 32 +/- 16 nM.

Amino Acid Sequence↗
PubMed2002

The application of 2D gel-based proteomics methods to the study of breast cancer.

The protein complement of breast cells consists of many thousands of proteins. Recent developments in 2D gel electrophoresis technology have made studies requiring the quantitative analysis of a differential proteome, such as comparison between normal and malignant cells or investigation of drug effects on cells, truly feasible. Computer software plays a central part in the comparisons between multiple gels required for such experiments. In addition, software tools allow patterns of coexpression of proteins to be studied, offering potential insights into protein regulation, interactions, and functions, especially when combined with complementary data on gene expression. In this paper, the technology and limitations of 2D gel-based proteomics are reviewed. Techniques for comparing sets of gels at a global level as well as identifying specific protein features that differentiate gels are discussed. Our own experience of studying the breast cell proteome is used to illustrate the difficulties and achievements of differential proteomics.

Breast Neoplasms↗
PubMed2005

Multimodal imaging and computer assisted diagnosis for functional tumour characterisation.

Radiological modalities, especially CT, mainly provide morphological and structural information with high spatial resolution covering large volumes. Novel developments, which are predominantly MR-based, also deliver 'functional' information, which can be used for individual characterisation of tumour biology. Both aspects and modalities, additionally complemented by ultrasound, have to be combined in the radiological workflow of cancer patients including volumetric visualisation, information extraction from multimodal imaging, quantitative surrogates, intelligent interpretation assistance and image-guided procedures. Based on volumetric visualisation and the generation of 3D+t maps, CAD tools have to address registration of different image series from different modalities, and extraction of quantitative surrogates. The latter will then serve tumour characterisation, therapeutic decision-making, image-guided procedures and efficacy evaluation.

Diagnosis, Computer-Assisted↗
PubMed1993

Genetic and functional characterization of the gene cluster specifying expression of Pseudomonas aeruginosa pili.

The genetic organization of the gene cluster containing pilA, the structural gene for type IV pilin of Pseudomonas aeruginosa, as well as the accessory genes pilB, pilC, and pilD, has been studied. DNA sequences capable of initiating transcription when fused to a promoterless lacZ gene have been identified in the pilA-pilB and pilB-pilC intergenic regions. Unlike pilA, which requires rpoN (encoding the sigma 54 subunit of RNA polymerase) and products of two regulatory genes, pilS and pilR, expression of pilB, pilC, or pilD did not depend on any of these transcriptional regulators. Moreover, transcription of pilA from the tac promoter in an rpoN mutant background resulted in piliated bacteria, suggesting that the RpoN-based regulatory network is specific for pilA and does not control expression of any other genes necessary for formation of pili. Insertion of the omega fragment containing strong transcriptional terminators into pilB, pilC, and pilD failed to have a polar effect on expression of downstream genes, as determined by the ability of each cloned gene to complement, in trans, the corresponding insertionally inactivated chromosomal copy. Insertions into pilC, however, resulted in decreased synthesis of PilD as determined by quantitation of PilD enzymatic activity in processing prepilin in vitro and by immunoassay. This finding suggests that PilD may require PilC for its optimal stability or correct membrane localization.

Bacterial Outer Membrane Proteins↗
PubMed2002

Contributions of proteome profiling to the molecular analysis of cancer.

The proteome is the most functional compartment encoded for in the genome. Technologies for protein separation and quantitation, coupled with mass spectrometry for protein identification, have provided the means for proteome profiling of tumor cell lines and tissues that complement genomic and transcriptomic profiling. The application of established and novel proteomic technologies to the molecular analysis of cancer is reviewed.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1987

A comprehensive study on the mechanism of inhibition of serine proteases by benzamidines based on quantitative structure-activity relationship studies.

Based on quantitative structure-activity relationship studies, investigation is made on the mechanism of inhibition of serine proteases, i.e., thrombin, plasmin, trypsin, and complement, by benzamidines. It is found that inhibitions of all the four enzymes, thrombin, plasmin, trypsin and complement, involve hydrophobic interaction in general but they do not involve electronic interaction in all the cases. In the cases where the electronic interaction is involved the mode of interaction is not necessarily the same. The electronic interaction depends upon the kind and the source of the enzyme.

Amidines↗
PubMed1987

Anti-endothelial cell antibodies: detection and characterization using a cellular enzyme-linked immunosorbent assay.

Anti-endothelial cell antibodies (AECA) have been detected in autoimmune diseases such as systemic lupus erythematosus (SLE) and scleroderma (PSS) but their role in pathogenesis is unknown. Immunofluorescence, immunohistochemistry, complement-dependent antibody lysis, and radioimmunoassay have been used in the past to detect AECA. We have developed a rapid, sensitive, and quantitative cellular enzyme-linked immunosorbent assay (ELISA) to detect and characterize AECA. Sera were obtained from 28 normal volunteers, 28 patients with SLE, and 14 patients with PSS. We also performed studies in 47 patients with various monoclonal gammopathies. Endothelial cells (EC) were obtained from human umbilical veins by standard methods and subcultured on 96-well tissue culture plates without fixation. EC were then sequentially incubated with sera, peroxidase-conjugated goat anti-human Ig (IgG, IgM, or IgA), and substrate. Optical density readings were converted to arbitrary units by developing a standard curve. Heavy-chain specific antibodies were used to determine the class of AECA binding to EC. IgG was purified by using protein A columns and digested with pepsin to obtain F(ab')2 fragments. The mean units of AECA from normals were 19.3 for IgG and 12.5 for IgM. SLE sera showed significant levels of IgM AECA (37 units, P less than 0.001) but not IgG (29 units, P less than 0.1). PSS sera showed significant levels of both IgM AECA (38 units, P = 0.001) and IgG AECA (42.7 units, P less than 0.005). IgA AECA were not detected in normal, SLE, or PSS sera. Blocking Fc receptors with rabbit IgG did not affect the titer of IgG or IgM AECA.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Surface↗