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PubMed1982

Genetic control of C6 polymorphism and C6 deficiency in rabbits.

The genetic control of the sixth component of complement (C6) in rabbits has been studied by quantitation of C6 functional and antigenic levels and identification of polymorphism by isoelectric focusing (IEF) in gels. Patterns of inheritance of C6 variants in families carrying a silent gene for C6 were examined, and it was found that 3 common plasma phenotypic variants, C6 A, C6 B, and C6 QO were under the genetic control of allelic genes, C6*A, C6*B, and C6*QO. In IEF patterns, C6 A could be identified by its isoelectric point that was slightly more acidic than that of C6 B. C6 QO was undetectable because it lacked functional and antigenic activity. The C6*A/C6*B genotype displayed a mixed IEF pattern with bands characteristic of both C6 A and C6 B. Functional and antigenic levels of C6 that were found in heterozygous C6*A/C6*QO and C6*B/C6*QO rabbits were approximately one-half of the C6 levels found in the corresponding homozygous animals. The phenotypic variation closely resembles that previously observed in humans and rhesus monkeys, as well as preliminary data in rabbits. The patterns of inheritance indicated that the two common C6 structural genes and the deficiency gene were allelic variants at the same genetic locus.

Animals↗
PubMed1993

Repair of N-methylpurines in the mitochondrial DNA of xeroderma pigmentosum complementation group D cells.

Previous work from our laboratory has shown that mitochondria are able to repair N-methylpurines formed by methylnitrosourea (MNU). However, it is unclear as to whether repair mechanisms that remove this type of lesion in nuclear DNA also remove these adducts in mitochondria. To address this question, we studied repair of MNU-induced N-methylpurines in the mitochondrial DNA from xeroderma pigmentosum complementation group D (XP-D) cells using quantitative Southern blot analysis and 32P-end-labeling techniques. These cells have been reported to be defective in the repair of this type of lesion in their nuclear genome. WI 38 cells were used as normal controls for these studies. Both XP-D fibroblasts and WI 38 cells were exposed to 0.5 mM MNU for 1 h. Following an 8 h repair period, 61% of N-methylpurines were repaired in the mitochondrial genome of XP-D cells and 39% of these lesions were repaired in WI 38 cells. After 24 h, XP-D cells had repaired 77% of the N-methylpurines in their mitochondrial genome, while WI 38 cells had 44% repair of this type of damage. During this same 24 h time period, 81.5% of the N7-methylguanines had been removed from the total cellular DNA of the WI 38 cells compared to only 38.3% repair of this lesion in the XP-D cells. Thus, XP-D cells, though deficient in the repair of N-methylpurines in their nuclear genome, are proficient in the repair of this type of damage in their mitochondria, suggesting that the mechanisms to repair N-methylpurines in the nuclear and mitochondrial genomes of these cells are different.

Blotting, Southern↗
PubMed1999

Evaluation of angiogenesis and perfusion of bone marrow lesions: role of semiquantitative and quantitative dynamic MRI.

Magnetic resonance imaging (MRI) is a noninvasive technique that complements computed tomography (CT), conventional X-ray, and bone marrow biopsies by sampling a large volume of musculoskeletal bone and providing information that aids the diagnosis, staging, and follow-up of various lesions. Although less sensitive to the mineral components of bones, the MRI appearance of physiologic bone marrow is mainly a reflection of the relative amounts of red marrow, yellow marrow, and trabecular bone. Therefore, use of T1-and T2-weighted MR sequences with or without fat suppression currently remains the most common approach to musculoskeletal bone lesion imaging. An additional imaging strategy to characterize various bone lesions is the application of contrast-enhanced dynamic MRI. This article examines semiquantitative and quantitative dynamic imaging, evaluation, and postprocessing techniques in various benign and malignant musculoskeletal lesions. Practical guidelines for performing a dynamic contrast-enhanced MR examination are proposed.

Bone Marrow Neoplasms↗
PubMed2005

Enzymatic detection of protein translocation.

Fundamental to eukaryotic cell signaling is the regulation of protein function by directed localization. Detection of these events has been largely qualitative owing to the limitations of existing technologies. Here we describe a method for quantitatively assessing protein translocation using proximity-induced enzyme complementation. The complementation assay for protein translocation (CAPT) is derived from beta-galactosidase and comprises one enzyme fragment, omega, which is localized to a particular subcellular region, and a small complementing peptide, alpha, which is fused to the protein of interest. The concentration of alpha in the immediate vicinity of omega correlates with the amount of enzyme activity obtained in a dose- and time-dependent manner, thus acting as a genetically encoded biosensor for local protein concentration. Using CAPT, inducible protein movement from the cytosol to the nucleus or plasma membrane was quantitatively monitored in multiwell format and in live mammalian cells by flow cytometry.

Enzyme-Linked Immunosorbent Assay↗
PubMed1981

[Circulating immune complexes-like material and complement in rheumatoid arthritis and ankylosing spondylitis (author's transl)].

Circulating immune complexes (IC) in 52 rheumatoid arthritis (RA) seropositive, 25 RA seronegative and 32 ankylosing spondylitis (AS) patients were assayed by two methods: 1) precipitation with a final 2% polyethylene glycol (PEG) concentration with immunoglobulins (Igs) quantitation in the precipitate and 2) the ability of IC to consume complement, evaluating conversion products from human C3 by bidimensional rocket immunoelectrophoresis. CH50, C3, C4, C3PA and circulating C3 conversion products were quantitated. IC precipitates with 2% PEG contained three classes of Igs: IgG, IgM and IgA, the latter being the most rare. In the control group Igs level precipitated with 2% PEG were: 2% PEG-IgG 2.5 mg/100 ml +/- 2; 2% PEG-IgM 0.72 mg/100 ml +/- 1.09; 2% PEG-IgA 0.04 mg/100 ml +/- 0.18. With 2% PEG IC-like material was detected in 58.5% of RA seropositive patients but none generated C3 conversion products. With neither methods was IC-like material detected in the RA seronegative group and none with 2% PEG in AS, either. However, in the latter, 21.8% generated C3 conversion products and 18.7% circulating C3 conversión products. In the RA seropositive patients no correlation was observed between the levels of 2% PEG-IgG and 2% PEG-IgM and the titre of antinuclear antibodies (ANA) nor between 2% PEG-IgG and that of IgG in serum. However, there was a low correlation between 2% PEG-IgM and that of IgM in serum and between 2% PEG-IgG and 2% PEG-IgM with rheumatoid factor (RF). It should be stressed that the value of the PEG method in the detection of IC-like material must be reconsidered, since using a 2% final concentration and a specific evaluation of Igs and complement components in the precipitate, it may be a method easy to perform and useful for clinical purposes.

Adult↗
PubMed1977

Alteration of cell-surface antigenicity of the mouse plasmacytoma. II. Lack of correlation between synthesis of myeloma protein and alteration of surface antigen.

The correlation between the changes of immunoglobulin synthesis and the surface antigenicity was analyzed by cytotoxicity and quantitative antibody-absorption tests with the cells of immunoglobulin-producing and nonproducing mouse plasmacytoma. IgA-synthesizing BLAB/c plasmacytoma 58-8 and the non-IgA-synthesizing variant of the 58-8 (nonproducer) were killed with rabbit anti-58-8 plasmacytoma cell antiserum, C3H/He anti-BALB/c spleen cell antiserum, and (C57BL/6 X DBA/2)F1 anti-BALB/c plasmacytoma cell MOPC-31C antiserum plus complement, only when the cells were pretreated with pronase. Quantitative absorption tests revealed that the nonproducer, 58-8 had the same amount of plasmacytoma antigen of 58-8 and PC.1 antigen, and a greater amount of H-2d antigen, as did producer 58-8. The same analysis was carried out for the C3H mouse plasmacytoma X5563, which has an M-component of IgG2a. The nonproducer X5563 had a greater amount of H-2k antigen and a smaller amount of the plasmacytoma antigen of X5563 than did the producer X5563. No detectable PC.1 antigen was observed at surfaces of the producer and the nonproducer X5563 cells.

Animals↗
PubMed2003

Use of rapid rural appraisal and cross-sectional studies in the assessment of constraints in smallholder cattle production systems in the western Kenya highlands.

Assessment of livestock production constraints in the smallholder dairy systems in the western Kenya highlands was carried out using both qualitative and quantitative epidemiological methods. Rapid rural appraisals (qualitative) were conducted in rural and peri-urban areas. A cross-sectional survey (quantitative) was then conducted on a random sample of farms in the study area. Diseases, poor communication, lack of marketing of livestock produce, lack of artificial insemination services, and feed and water shortages during the dry season were identified as the major constraints to cattle production in both areas. Tick borne diseases (especially East Coast fever) were identified as the major constraint to cattle production. Qualitative methods were found to be more flexible and cheaper than the quantitative methods by a ratio of between 2.19-2.0. The two methods were found to complement each other. Qualitative studies could be applied in preliminary studies before initiating more specific follow up quantitative studies.

Animal Feed↗
PubMed1989

What are the goals of magnetic resonance research?

Now that both magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) have reached their decadic majority, appropriate questions may be asked as to their accomplishments and prognostications for the future. This article emphasizes the approach of the metabolic biologists/physiologists to magnetic resonance biochemistry as indicated by the currently available multinuclear localized approaches. The viewpoint is emphasized that MRS is a critical care instrument where precipitious changes of oxidative metabolism lead to the well-known stroke, heart failure, liver failure, kidney failure, etc. Generally, the gradation between the classical metabolic steady state of life and the pathway leading to cell death is a narrow one and magnetic resonance in some cases is too finely tuned to delineate the gradations of stability and instability of cell metabolism. To this point, magnetic resonance can be supplemented by other modalities that sense tissue distress. An example of a most useful and predictive measure of hypoxic stress is optical spectrophotometry which uses time resolved ranging methods to measure optical path lengths to quantitate hemoglobin deoxygenation in tissues. With such a complement, the two methods emerge as one of general importance in diagnostic procedures.

Forecasting↗
PubMed1983

Extravascular immune complexes in experimental mycobacterial BCG granulomas.

Quantitative results of Mycobacterial BCG antigen, immunoglobulin and complement in rat skin lesions during the evolution of the granuloma reveal peak values of these factors at 49 days. The combination of antigen, Ig and complement, present extracellularly and in polymorphs at this time, indicates immune complex formation at an antigen-antibody ratio which may approximate to equivalence. This development coincides with mass lysis of host macrophages, and is followed by a sharp reduction in the antigen load. At 8 mth, surviving bacilli are coated with antibody and complement, and sequestrated in activated macrophages. It is possible that this antibody is non-specific and protective to the bacilli, leading to a second multiplication of organisms. But by 1 yr and 8 mth all the bacilli are dead, and immunoglobulins and complement are at very low levels. Although immune complex deposition and macrophage lysis was not associated with complete elimination of bacilli, it marked a turning point in the infection when the bacterial load was high and cell mediated immunity (CMI) was lacking. CMI may be important, especially at low antigen levels, but the crucial role here appears to have been played by complexed antibody. These experimental findings parallel those in human cutaneous leishmaniasis. They may explain some forms of necrosis in this condition and in tuberculosis.

Animals↗
PubMed1987

An enzyme-linked antiglobulin test for the detection of erythrocyte-bound antibodies in canine autoimmune haemolytic anaemia.

A method has been developed which allows identification and quantitation of red cell-bound immunoglobulins and complement in canine blood. The technique utilizes ELISA methodology and the assay identifies cases of autoimmune haemolytic anaemia which are negative by the Direct Coombs test. Further, the amount of antibody present on the red cells shows a close correlation to the haemoglobin level; suggesting that the degree of sensitization of the RBCs influences their rate of destruction. Favourable response to treatment correlates with a decrease in the levels of bound antibody and complement.

Anemia, Hemolytic, Autoimmune↗
PubMed1994

Strategies for overcoming linewidth limitations in quantitative petrophysical NMR measurements.

The simple pulse-acquire experiment has been used to evaluate the level of accuracy and precision achievable in NMR fluid saturation measurements for a range of rock core samples saturated with either brine or hydrocarbons. For a set of more than 70 cores measured at 0.66 T the mean error in the NMR measurement is only 0.35% porosity when the sample linewidths are less than 50 ppm. However, for a significant portion of cores, those with very broad NMR linewidths (> 50 ppm), difficulties associated with nonuniform excitation are encountered. The magnetic susceptibility difference between pore fluid and rock matrix translates into relatively broad NMR linewidths, and this feature of petrophysical samples is the major difficulty in performing quantitative NMR experiments. Numerical simulations are used to complement the experimental results in order to develop strategies for obtaining accurate NMR results with these difficult samples.

Hydrocarbons↗
PubMed2026

Structure-resolved virus-host interactomics by cross-linking mass spectrometry.

Viruses depend on host protein networks to replicate, assemble progeny, and spread between cells and organisms. Defining these virus-host protein interactions is challenging because they are highly dependent on infection stage, cell type, species, and because mechanistic interpretation requires information about structural interfaces and conformational states. Cross-linking mass spectrometry (XL-MS) addresses these challenges by adding a spatial and structural dimension to virus-host interactomics in native systems. In this review, we discuss how XL-MS has advanced from targeted analysis of viral protein complexes to structure-resolved mapping of virion architecture and infected-cell virus-host interactomes. We highlight how XL-MS complements AP-MS, cryo-EM/cryo-ET, quantitative proteomics, genetic perturbation, and structure prediction to connect physical proximity with molecular mechanisms. Finally, we discuss current limitations in sensitivity, chemical coverage, temporal resolution, and model interpretation, and outline how future quantitative and integrative XL-MS workflows may enable systems-level structural virology.

Mass Spectrometry↗
PubMed2003

Rat retinal ganglion cells upregulate the pro-apoptotic BH3-only protein Bim after optic nerve transection.

Increased expression of Bim, a pro-apoptotic member of the Bcl-2 family, has been shown to be critical for neuronal apoptosis. To study the involvement of Bim in injury-induced cell death in retina, Bim expression was studied in normal rat retina and in retina after optic nerve transection using quantitative RT-PCR and immunohistochemistry. As a complement to this, the apoptotic regulators Bax, Bcl-2, caspase-3 and phosphorylated c-jun were studied. The relative levels of Bim mRNA in retina were significantly higher 4 days after optic nerve transection and below normal levels at 14 days after transection. A parallel increase in the number of Bim-immunoreactive cells in the retinal ganglion cell layer could be seen. Bim-immunoreactivity localized to retrogradely True Blue-labeled retinal ganglion cells. The relative mRNA levels for both Bax and Bcl-2 were higher at 4 days after transection when compared to normal. Immunoreactivity for Bax, Bcl-2 as well as for caspase-3 and phosphorylated c-jun, indicative of cell death, localized to True Blue-identified retinal ganglion cells 4 days after injury. Bcl-2 immunoreactivity was also seen on other cells, most likely Müller glia cells. In addition, optic nerve transection caused an increase in Bim, Bax, and Bcl-2 mRNA levels in optic nerve and superior colliculus. Our results suggest that Bim is involved in injury-induced retinal ganglion cell death and indicate that the increase in Bim and Bax expression promote cell death of axotomized retinal ganglion cells whereas the elevation in Bcl-2 in retina may contribute to the control of the extent of apoptosis after the optic nerve transection.

Animals↗
PubMed2003

The upper body segmental movements during walking by young females.

OBJECTIVES: To characterise the physiological pattern of trunk and shoulders movements during walking and provide a reference for further studies on spine deformities. DESIGN: Implementation of a model for measuring spine and shoulder girdle movements during gait. Data collection on a population of eighteen, young, healthy, female subjects. BACKGROUND: The analysis of gait modifications in subjects with idiopathic scoliosis could offer an insight to better understand the functional relationship with the pathology. METHODS: Retroreflective markers were positioned on the main spine processes and acromions to be detected by a TV-based motion analysis system. A model of kinematic computation was implemented and integrated in a previously developed protocol for multifactorial gait analysis. Movements in the main reference planes and in relation to pelvis were analysed. RESULTS: The trunk was on average bent forward by 3.4 degrees with respect to standing; of the two physiological curves in the sagittal plane only lordosis changed during walking; in the frontal plane, a dynamic spine deformation appeared, that was maximum at heel strike-early stance; the trunk was bent controlaterally of the foot on the ground, while the shoulders remained stable; in the horizontal plane, the shoulders rotated contralaterally to the pelvis. CONCLUSIONS: In our population all the segmental movements analysed were smaller than 5 degrees during gait, except the angle of proximal curvature in the frontal plane, shoulder rotation, and angle between shoulders and pelvis; all the measured angles were far below their possible ranges of motion. RELEVANCE: Quantitative data on upper body kinematics as a complement to gait analysis can help understanding movement disorders and compensation strategies in several pathologies.

Adolescent↗
PubMed2000

Biodistribution of liposomes and C3 fragments associated with liposomes: evaluation of their relationship.

The biodistribution of liposomes with two different kind phospholipids (hydrogenated egg phosphatidylcholine and egg phosphatidylcholine) plus cholesterol (CHOL) were investigated after intravenous administration to rats. Elimination of liposomes from blood circulation was affected by the lipid composition. It appeared that the inclusion of CHOL in liposomes accelerates the rate of liposome uptake by liver, resulting in rapid elimination of liposomes. The amount of C3 fragments bound to liposomes was quantitatively determined to assess the contribution of the complement system to liposome accumulation into organs and liposome destabilization in vivo and in vitro. The amount of bound C3 fragments was directly proportional to CHOL content, and the amount was also proportional to the CLh, CLs as well as CLrel. This relationship suggests that the complement system is responsible for the elimination of liposomes from blood circulation, presumably as a consequence of opsonization by C3 fragments and assembly of membrane attack complex (MAC) onto liposomes. In addition, substitution of cholesteryl methyl ether into the liposome formulation for CHOL significantly diminished not only the binding of C3 fragments but also the CLh, CLs and CLrel, resulting in increased mean resident time (MRT) of the liposomes. This result suggests that the hydroxyl-group on CHOL is a binding site for C3 fragments on the liposomes and that CHOL in a liposome formulation promotes the accumulation of liposomes into the liver and spleen, probably due to their uptake by phagocytic cells, and impairs the stability of the liposomes in blood circulation, via a mechanism involving the complement system.

Animals↗
PubMed2005

An integrative genomics approach to infer causal associations between gene expression and disease.

A key goal of biomedical research is to elucidate the complex network of gene interactions underlying complex traits such as common human diseases. Here we detail a multistep procedure for identifying potential key drivers of complex traits that integrates DNA-variation and gene-expression data with other complex trait data in segregating mouse populations. Ordering gene expression traits relative to one another and relative to other complex traits is achieved by systematically testing whether variations in DNA that lead to variations in relative transcript abundances statistically support an independent, causative or reactive function relative to the complex traits under consideration. We show that this approach can predict transcriptional responses to single gene-perturbation experiments using gene-expression data in the context of a segregating mouse population. We also demonstrate the utility of this approach by identifying and experimentally validating the involvement of three new genes in susceptibility to obesity.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗
PubMed1997

Ultrasonographic texture characterization of salivary and neck masses using two-dimensional gray-scale clustering.

OBJECTIVES: To evaluate the clinical usefulness of texture analysis in ultrasonography (US) of head and neck masses. METHODS: Sixty-one cases with salivary or neck masses were examined by US with a linear 7.5MHz array. Images were digitized and quantitatively evaluated by two-dimensional gray-scale clustering using two texture indices, M and SD, as measures of coarseness and homogeneity respectively. RESULTS: Coarseness of Warthin's tumors was significantly higher than that of the other salivary tumors. Malignant lymphomas had finer texture values than other lymph node lesions. CONCLUSIONS: Quantitative texture analysis appears to be a useful complement in diagnostic US.

Diagnosis, Differential↗
PubMed2004

Dual targeting of yeast catalase A to peroxisomes and mitochondria.

Yeast catalase A (Cta1p) contains two peroxisomal targeting signals (SSNSKF) localized at its C-terminus and within the N-terminal third of the protein, which both can target foreign proteins to peroxisomes. In the present study we demonstrated that Cta1p can also enter mitochondria, although the enzyme lacks a classical mitochondrial import sequence. Cta1p co-targeting was studied in a catalase A null mutant after growth on different carbon sources, and expression of a Cta1p-GFP (green fluorescent protein)-fusion protein or a Cta1p derivative containing either a c-Myc epitope (Cta1p(myc)) or a SKF-extended tag (Cta1p(myc-SKF)). Peroxisomal and mitochondrial co-import of catalase A were tested qualitatively by fluorescence microscopy and functional complementation of a Delta cta1 null mutation, and quantitatively by subcellular fractionation followed by Western blot analysis and enzyme activity assays. Efficient Cta1p import into peroxisomes was observed when cells were cultivated under peroxisome-inducing conditions (i.e. growth on oleate), whereas significant co-import of Cta1p-GFP into mitochondria occurred when cells were grown under respiratory conditions that favour oxygen stress and ROS (reactive oxygen species) accumulation within this organelle. In particular, when cells were grown on the non-fermentable carbon source raffinose, respiration is maximally enhanced, and catalase A was efficiently targeted to the mitochondrial matrix where it presumably functions as scavenger of H2O2 and mitochondrial-derived ROS.

Catalase↗