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PubMed1978

Inhibition of four human serine proteases by substituted benzamidines.

A series of substituted benzamidines has been examined for their inhibitory activity against the human serine proteases--trypsin, thrombin, plasmin, and C1s, a subunit of the first component of complement. The inhibition constants obtained for each enzyme were correlated with physical-chemical properties of the substituent group using the quantitative structure-activity relationship approach. This analysis indicated that plasmin and C1s are very similar in their interactions with substituted benzamidines. The binding of benzamidines in both enzymes was affected by electron donation from the substituent and its hydrophobicity. Thrombin-benzamidine interaction was affected only by the hydrophobicity of the substituent. Trypsin displayed a complex interaction with substituted benzamidines, and interaction was dependent on molar refractivity and molecular weight. Certain substituents deviated significantly from the interactions predicted by the analysis. These compounds, the (m- and p-amidinophenyl)pyruvic acids, when analyzed by computer modeling, suggested that direct interaction between the substituent and the enzyme surface is important in assessing the effect of substituent groups on inhibitory activity.

Amidines↗
PubMed2002

hairy: A quantitative trait locus for drosophila sensory bristle number.

Advances in medicine, agriculture, and an understanding of evolution depend on resolving the genetic architecture of quantitative traits, which is challenging since variation for complex traits is caused by multiple interacting quantitative trait loci (QTL) with small and conditional effects. Here, we show that the key developmental gene, hairy (h), is a QTL for Drosophila sternopleural bristle number, a model quantitative trait. Near-isoallelic lines (NIL) for the h gene region exhibited significant variation in bristle number and failed to complement a hairy mutation. Sequencing 10 h alleles from a single population revealed 330 polymorphic sites in approximately 10 kb. Genotypes for 25 of these and 14 additional sites in the flanking regions were determined for the 57 NIL and associated with variation in bristle number in four genetic backgrounds. A highly significant association was found for a complicated insertion/deletion polymorphism upstream of the transcription start site. This polymorphism, present in 17.5% of the h alleles, was associated with an increase of 0.5 bristle and accounted for 31% of the genetic variance in bristle number in the NIL.

Animals↗
PubMed1988

Site-directed mutagenesis of the cell-binding domain of human fibronectin: separable, synergistic sites mediate adhesive function.

Polypeptide sequences required for function of the cell-binding domain of human fibronectin were analyzed by site-directed mutagenesis. Site-specific deletion of the putative recognition sequence Arg-Gly-Asp-Ser or an Asp-to-Glu mutation decreased the adhesive activity of fibronectin fusion proteins expressed in E. coli by greater than or equal to 97%. A second functional site over 0.5 kb away was identified by deletion mutagenesis. These mutants also showed a greater than or equal to 96% loss of activity, indicating cooperativity between sites. The two classes of mutant protein displayed synergism of activity in a trans complementation assay. Effective actin microfilament bundle organization was also dependent on the combined function of both sites. Thus, fibroblast adhesion and intracellular response to the fibronectin cell-binding domain involve two synergistic sites, each of major quantitative importance.

Actins↗
PubMed2005

Complement activation after lumbosacral ventral root avulsion injury.

A lumbosacral ventral root avulsion (VRA) injury results in a pronounced loss of motoneurons, in part due to apoptosis. Caspase inhibitors may rescue motoneurons after a VRA in neonatal rats, but this treatment approach has been unsuccessful to protect motoneurons subjected to the same injury in adult rats. Other mechanisms may contribute to the retrograde motoneuron death encountered in adult animals. Here, we study whether the complement system, a part of the innate immune system, contributes to motoneuron death after a lumbosacral VRA. Adult Sprague-Dawley rats underwent a unilateral L5-S2 VRA injury. At 10 days postoperatively, quantitative immunohistochemical studies demonstrated that the lytic membrane attack complex (MAC) targeted approximately 38% of axotomized motoneurons. The MAC inhibitor Clusterin was concurrently expressed at significantly higher levels in astrocytes and de novo in 30% of the remaining motoneurons. Our data suggest that complement activation and necrosis contribute to motoneuron death after lumbosacral VRA injuries. We speculate that inhibition of MAC may constitute a potential neuroprotective strategy following cauda equina injuries.

Animals↗
PubMed2006

Determination of adiponectin in serum using a latex particle-enhanced turbidimetric immunoassay with an automated analyzer.

BACKGROUND: Adiponectin is an adipose-derived hormone that plays a role in regulating metabolic processes such as fat partitioning and lipid and glucose metabolism. Quantification of adiponectin is useful for obtaining information on metabolic syndrome, but there is no rapid method to measure adiponectin for clinical use. METHODS: We developed a rapid and sensitive latex particle-enhanced turbidimetric immunoassay (LTIA) using a latex bead-immobilized anti-adiponectin polyclonal antibody. The assay was performed on a Hitachi H7170 analyzer and evaluated for validity as a method to quantitate adiponectin, in parallel with the ELISA. RESULTS: Dilution tests using LTIA showed linearity from 0.25 to 30 microg/ml. Within-run CV and total CV were obtained in the range of 0.8-1.9% and 1.1-2.0%, respectively. No interference was observed in the testing of specimens containing potentially interfering substances such as bilirubin, ditaurobilirubin, hemoglobin triglyceride, rheumatoid factor, type IV collagen, fibronectin, and complement factor (C1q). A strong correlation between LTIA and ELISA was confirmed (n=30, r=0.990, y=0.95x+0.39). CONCLUSION: The LTIA assay is applicable to quantitating the serum concentration of adiponectin. This assay is more convenient and faster than ELISA and suitable for clinical routine analysis.

Adiponectin↗
PubMed1981

Development of an enzyme immunoassay to detect and quantitate tumor-associated antigens in the urine of sarcoma patients.

This study describes the development of an enzyme immunoassay (EIA) to quantitate antigen in the urine of sarcoma patients and compares its results with those of the authors' previously reported complement fixation assay. Three populations were studied for the presence of urinary antigen by EIA: (1) sarcoma patients who developed metastatic disease after resection of their primary tumor; (2) sarcoma patients who remain clinically disease-free two years after resection of their primary tumor; and (3) normal volunteers with no history of malignant disease. EAch group consisted of nine individuals. None of the urines from normal volunteers and none from sarcoma patients who remained free of disease for two years had elevated antigen titers detectable by EIA. However, all nine patients who developed pulmonary metastatic disease ahd significantly elevated levels of antigen in their urine prior to clinical evidence of disease recurrence. The day-to-day fluctuations in antigen titer detectable by EIA tended to parallel the levels detected by the complement fixation assay. However, EIA detected a significant elevation in antigen titer one to four months earlier than the complement fixation assay in four of the patients in whom disease recurred.

Antigens, Neoplasm↗
PubMed1984

Immune rejection mechanisms in murine leukemia. I. Timing of tumor cell rejection process relative to the development of humoral and cell-mediated cytotoxic immune responses.

Studies were undertaken to investigate the relationship between cell-mediated and humoral immune responses in the rejection of L1210/MTX-Rev (LR) leukemia cells in CD2F1 mice. The anti-LR antibody (humoral) response was defined by both its cytotoxic antibody titer and isotype composition, assessed by a complement-dependent cytotoxicity assay and radioimmune assay, respectively. The cytotoxic thymus (T)-derived lymphocyte response in the spleen was quantitated by 125I release by 125I-IUdR-labelled target cells. Analysis of the sera of tumor-bearing mice indicated that the LR leukemia cells elicited a wide spectrum of anti-tumor antibody isotypes. In mice that mounted a rejection response, the IgM anti-LR response was transient, while in those that did not, the IgM response persisted. Antibody titers for all isotypes remained low until the onset of LR rejection. At that time, high titers of IgG anti-LR antibodies, predominantly of the IgG2a subclass, were detected in sera of tumor-free mice. Thus, the LR rejection response coincided best with the appearance of high titers of IgG2a anti-LR antibodies. A single i.p. injection of viable LR cells elicited a potent cell-mediated immune response; neither the appearance nor the magnitude of the cell-mediated immune response as measured in the spleen correlated well with the onset or the strength of the LR rejection response in the peritoneal cavity. The LR peritoneal cell population grew unabated in the presence of an intense spleen cell-mediated immune response, and the rejection process began at a time when little cellular immunity could be detected. These results suggest that in the rejection response IgM antibodies contribute little, if any, to the process, and the role of cytotoxic T lymphocytes is questionable (uncertain). The rejection of LR cells appears to be primarily mediated by IgG2a antibodies.

Animals↗
PubMed1975

Calcium pyrophosphate-induced pleurisy in rats: a new model of acute inflammation.

A model is described of acute inflammation in the pleural cavity of rats using calcium pyrophosphate as the irritant. This model would seem to simulate the pseudogout syndrome. It has been shown to be acute in onset, dominated by polymorphonuclear cells, complement independent. The advantage of the model is that volume of exudate, numbers and types of cells may be quantitated. Prostaglandins and cyclic AMP have been measured in the migrating cells. The significance of these findings has been discussed.

Animals↗
PubMed1991

Lysosomal cobalamin accumulation in fibroblasts from a patient with an inborn error of cobalamin metabolism (cblF complementation group): visualization by electron microscope radioautography.

Cobalamin (Cbl, vitamin B12) bound to transcobalamin II (TCII) enters cultured fibroblasts by receptor-mediated endocytosis. Following degradation of the TCII, Cbl is subsequently found in either the cytoplasm bound to methionine synthase or in the mitochondria bound to methylmalonyl CoA mutase. In fibroblasts from patients belonging to the cblF complementation group, Cbl is found free in the cell and is not transferred to the above two target enzymes. Quantitative EM radioautography was utilized to visualize intracellular Cbl in fibroblasts from cblF patients and from normal subjects. In cblF cells, 60% of all silver grains were assigned to lysosomes, with only 12.6% over cytoplasm and 1.2% over mitochondria. In contrast, in control cells, only 4.7% were assigned to lysosomes, with 47% to cytoplasm and 23.4% to mitochondria. Subcellular fractionation showed that in cblF cells, the majority of label was associated with clearly recognizable lysosomes. These studies conclusively demonstrate that secondary lysosomes accumulate Cbl in cblF disease.

Autoradiography↗
PubMed1979

Sensitive and convenient quantitation of antibody binding to cellular antigens using glutaraldehyde preserved cells.

As a preliminary step in the identification and isolation of antibodies to human cancers, we have developed a sensitive and convenient assay for antibody binding to cellular antigens. The basis for the method is antibody binding to glutaraldehyde-fixed cells (AbGfC) and quantitation with radioiodinated staphylococcal protein A (SpA). Glutaraldehyde fixation of intact cells, which does not appear to effect the ability to form antigen-antibody complexes, provides a convenient and standard supply of target cells which may be stored at 4 degrees C and used in the assay over a period of several months. The amount of antibody specifically bound to the cells is quantitated by the addition of 125I-labeled SpA. The sensitivity of the method was compared with two complement-dependent cytotoxicity methods (trypan blue exclusion and 51Cr release assays) and tested with two antisera to human lung cancer and one antiserum to a membrane antigen of a murine lymphoma. These comparisons indicated much greater sensitivity when compared with the trypan blue exclusion assay and equivalent sensitivity with greater dose response characteristics when compared with the 51Cr release assay.

Aldehydes↗
PubMed1998

Three-dimensional vascular imaging using Doppler ultrasound.

BACKGROUND: We have evaluated the efficacy of using three-dimensional reconstruction of amplitude Doppler imaging data to quantitatively assess carotid artery bifurcation stenoses. METHODS: Sixty-four consecutive frames of amplitude (power) Doppler images are stored to be reassembled into a three-dimensional image representing the patent lumen. These images can then be rotated by any angle necessary to clearly view the vascular anatomy and to make quantitative ultrasound caliper measurements of the stenotic lumen and normal vessel caliber. RESULTS: Three-dimensional Doppler images accurately classified 53 of 61 vessels (87%) into categories of stenosis compared with angiography. All stenoses with >60% diameter reduction were detected and classified as such, for a sensitivity of 100%. CONCLUSIONS: Three-dimensional vascular imaging based on amplitude (power) Doppler data provides an accurate noninvasive technique for quantitative diagnosis of carotid bifurcation atherosclerotic disease, with selectable viewing projections that eliminate vessel overlap and other artifacts, and complements the hemodynamic data already available with two-dimensional duplex ultrasound.

Aged↗
PubMed2002

Noise-to-harmonics ratio as an acoustic measure of voice disorders in boys.

This prospective study assessed the efficacy of computerized noise-to-harmonics ratio (NHR) to quantify perceptual and endoscopic findings of dysphonia and/or structural lesion of the vocal fold. Fifty Brazilian boys without vocal complaints were submitted to computerized, perceptual, and endoscopic examination. Thirty boys were dysphonic--3 were classified into the grade category, 5 into breathiness, 9 into roughness, and 15 into grade/breathiness. Vocal fold lesions were observed in 25 boys (17 nodules and 8 cysts). The Mann-Whitney U test revealed that NHR was significantly higher in boys with a structural lesion (p = 0.007) and in boys with dysphonia (p < 0.0001). However, according to a logistic regression model, only the occurrence of dysphonia was explained by NHR; the risk for having dysphonia increased approximately twice (odds ratio = 1.92, 95% confidence interval = 1.3-2.9) with each increase of 0.01 in NHR. Our results suggest that noise is a useful quantitative index to confirm a perceptual diagnosis of dysphonia and to evaluate quantitative changes in a dysphonic voice over time. However, we believe that computerized analysis should be used as a complement, rather than a substitute, for perceptual evaluation. Further studies with a larger sample are required to investigate the relationship between noise and lesions of the vocal folds.

Adolescent↗
PubMed2000

Electrodiagnosis of polyneuropathy.

Electrodiagnostic studies comprising electromyography (EMG) and nerve conduction studies (NCS) are well-established objective methods for the diagnosis, quantification and classification of polyneuropathies (PNP). This paper reviews examination techniques, their pathophysiological interpretation, examination strategies and diagnostic criteria for the diagnosis and classification of a PNP. The routine electrodiagnostic evaluation includes sensory NCSs performed with surface or needle electrodes, motor NCSs, F-wave studies and EMG by qualitative or quantitative techniques. Sensory NCSs and F-wave studies have a high sensitivity in PNPs and the different techniques complement each other. The distinction between a PNP with predominantly axonal loss and a PNP with predominantly demyelination is one of the major aims of the electrophysiological examination. There are, however, large variation in suggested criteria for predominantly demyelination. The degree of slowing in conduction taken to indicate demyelination varies between a decrease of 50 to 30% from mean of controls, distal latency prolongation criteria vary from 35% to 70% of mean of controls, F-wave latency prolongation criteria vary from 120% to 150% of upper limit of controls, and criteria for partial motor conduction block vary from 11 to 50% reduction of CMAP amplitude and/or area between proximal and distal stimulation. Needle EMG studies may be valuable in order to detect and quantify denervation activity, to assess chronicity by an evaluation of the extent of reinnervation, and to evaluate the topographical distribution of changes. It is concluded that electrodiagnostic studies are valuable in patients with suspected PNP and the results may have consequences for prognosis and therapy of individual patients. Large variation in examination techniques, strategies, interpretations and diagnostic criteria have been found among electromyographers and it is suggested that the value of electrodiagnostic studies may be further improved by international standardisation.

Autonomic Nervous System↗
PubMed2005

Simple reporter gene-based assays for hairpin poly(amide) conjugate permeability and DNA-binding activity in living cells.

Hairpin poly(amide)s (HPs) are sequence specific DNA-binding compounds that have engendered considerable interest as potential pharmacological agents to manipulate the expression of specific genes. However, recent reports have indicated that the ability of HP conjugates to pass through cell membranes is sensitive to the cell type employed and the nature of the conjugate. Furthermore, while binding of HPs to DNA sequences in vitro is relatively well understood, packing of DNA into chromatin in living cells makes predicting the efficiency with which a given poly(amide) will bind its cognate site less certain. Previous methods to evaluate HP permeability and binding in vivo, while effective, are somewhat tedious and qualitative. We report here two related reporter gene-based assays that provide a more convenient and quantitative measure of poly(amide) permeability and DNA binding activity in living cells. We anticipate that these methods will complement existing tools and facilitate the development of HP conjugates with the desired biological activity.

Base Sequence↗
PubMed2004

Reduced beta-amyloid production and increased inflammatory responses in presenilin conditional knock-out mice.

Mutations in presenilins (PS) 1 and 2 are the major cause of familial Alzheimer's disease. Conditional double knock-out mice lacking both presenilins in the postnatal forebrain (PS cDKO mice) exhibit memory and synaptic plasticity impairments followed by progressive neurodegeneration in the cerebral cortex. Here we further investigate the molecular events that may underlie the observed phenotypes and identify additional neuropathological markers in the PS cDKO brain. Enzyme-linked immunosorbent assay analysis showed reduced levels of the toxic beta-amyloid (Abeta) peptides in the cerebral cortex of PS cDKO mice. Interestingly, the reduction in Abeta40 and Abeta42 peptides is similar in PS1 conditional knock-out and PS cDKO mice. We further examined the gene expression profile by oligonucleotide microarrays in the PS cDKO cerebral cortex and found that a high number of genes are differentially expressed, most notably a group of up-regulated inflammatory genes. Quantitative real-time reverse transcription PCR and Western analyses confirmed the elevated levels of glial fibrillary acidic protein, complement component C1q, and cathepsin S, up-regulation of which has been associated with inflammatory responses in various neurodegenerative processes. Immunohistochemical analysis revealed that the increase in complement component C1q is confined to the hippocampal formation, whereas glial fibrillary acidic protein and cathepsin S are up-regulated throughout the entire neocortex and hippocampus. In addition, strong microglial activation occurs in the hippocampus and the deeper cortical layers of PS cDKO mice. These results indicate that the memory impairment and neurodegeneration in PS cDKO mice are not caused by Abeta accumulation and that loss of PS function leads to differential up-regulation of inflammatory markers in the cerebral cortex.

Amyloid beta-Peptides↗
PubMed2003

The interaction between human PEX3 and PEX19 characterized by fluorescence resonance energy transfer (FRET) analysis.

The process of peroxisome biogenesis involves several PEX genes that encode the machinery required to assemble the organelle. Among the corresponding peroxins the interaction between PEX3 and PEX19 is essential for early peroxisome biogenesis. However, the intracellular site of this protein interaction is still unclear. To address this question by fluorescence resonance energy transfer (FRET) analysis, we engineered the enhanced yellow fluorescent protein (EYFP) to the C-terminus of PEX3 and the enhanced cyan fluorescent protein (ECFP) to the N-terminus of PEX19. Functionality of the fusion proteins was shown by transfection of human PEX3- and PEX19-deficient fibroblasts from Zellweger patients with tagged versions of PEX3 and PEX19. This led to reformation of import-competent peroxisomes in both cell lines previously lacking detectable peroxisomal membrane structures. The interaction of PEX3-EYFP with ECFP-PEX19 in a PEX3-deficient cell line during peroxisome biogenesis was visualized by FRET imaging. Although PEX19 was predominantly localized to the cytoplasma, the peroxisome was identified to be the main intracellular site of the PEX3-PEX19 interaction. Results were confirmed and quantified by donor fluorescence photobleaching experiments. PEX3 deletion proteins lacking the N-terminal peroxisomal targeting sequence (PEX3 34-373-EYFP) or the PEX19-binding domain located in the C-terminal half of the protein (PEX3 1-140-EYFP) did not show the characteristic peroxisomal localization of PEX3, but were mislocalized to the cytoplasm (PEX3 34-373-EYFP) or to the mitochondria (PEX3 1-140-EYFP) and did not interact with ECFP-PEX19. We suggest that FRET is a suitable tool to gain quantitative spatial information about the interaction of peroxins during the process of peroxisome biogenesis in single cells. These findings complement and extend data from conventional in vitro protein interaction assays and support the hypothesis of PEX3 being an anchor for PEX19 at the peroxisomal membrane.

Bacterial Proteins↗
PubMed1995

teosinte branched1 and the origin of maize: evidence for epistasis and the evolution of dominance.

Two quantitative trait loci (QTL) controlling differences in plant and inflorescence architecture between maize and its progenitor (teosinte) were analyzed. Complementation tests indicate that one of these, which is on chromosome arm 1L, is the locus for the maize mutant teosinte branched1 (tb1). This QTL has effects on inflorescence sex and the number and length of internodes in the lateral branches and inflorescences. This QTL has strong phenotypic effects in teosinte background but reduced effects in maize background. The second QTL, which is on chromosome arm 3L, affects the same traits as the QTL on 1L. We identify two candidate loci for this QTL. The effects of this QTL on several traits are reduced in both maize and teosinte background as compared to a maize-teosinte F2 population. Genetic background appears to affect gene action for both QTL. Analysis of a population in which both QTL were segregating revealed that they interact epistatically. Together, these two QTL substantially transform both plant and inflorescence architecture. We propose that tb1 is involved in the plant's response to local environment to produce either long or short branches and that maize evolution involved a change at this locus to produce short branches under all environments.

Biological Evolution↗
PubMed1989

Neutrophil-dependent, oxygen-radical mediated lung injury associated with acute pancreatitis.

Cerulein-induced acute pancreatitis in rats is associated with a reversible lung injury that is characterized by alveolar capillary endothelial-cell injury, increased microvascular permeability, interstitial edema formation, and intraalveolar hemorrhage and fibrin deposition. The role of mediators in this injury was analyzed using gravimetric data, microvascular permeability indices, electron microscopy, and a quantitative morphometric analysis. Neutrophil depletion induced by a specific antibody was highly protective against lung injury. Interruption of the complement pathway (using low dose Naja naja cobra venom factor) also protected against lung injury. Catalase and superoxide dismutase were also protective. The iron chelator deferoxamine and the hydroxyl radical scavenger, dimethylsulfoxide, were not protective against acute lung injury. These data suggest that complement, neutrophils, and neutrophil-derived (H2O2-dependent) oxygen products mediate lung injury that occurs secondary to cerulein-induced pancreatitis. In contrast to other models of neutrophil-dependent, oxygen-radical-mediated lung injury, this lung injury does not appear to be an iron-dependent and hydroxyl-radical mediated injury. We postulate that the process of acute pancreatitis leads to complement activation followed by neutrophil recruitment, sequestration, and adherence to alveolar capillary endothelial cells. Ultimately lung injury appears to result from local endothelial-cell injury secondary to neutrophil-generated oxygen products that may be myeloperoxidase dependent.

Acute Disease↗