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PubMed1993

Selective in situ hybridization histochemical analyses of alternatively spliced mRNAs encoding beta- and gamma-preprotachykinins in rat central nervous system.

The present study describes the development of an in situ hybridization histochemistry (ISHH) procedure which was employed to selectively monitor cellular distributions of the 2 major alternatively spliced beta- and gamma-species of mRNA encoding preprotachykinin (PPT) molecules found in rat CNS. For these purposes, 2 custom-designed oligodeoxynucleotide probes were synthesized corresponding to complementary sequences of beta- and gamma-PPT mRNAs. In particular, the gamma-selective probe was demonstrated to hybridize to the contiguous regions of RNA flanking the splice site formed by exclusion of exon 4. Initially, Northern blot analyses performed in conjunction with appropriate specificity controls demonstrated selective hybridization of the 32P-labeled beta- and gamma-selective probes to single bands of approximately 1.2-1.3 kilobases in size, consistent with previously established values for rat brain beta- and gamma-PPT mRNAs. In anatomical studies, results obtained from absorptions using competing nonradiolabeled oligonucleotides defined the specificity and selectivity of both probes for targeting their respective species of mRNA immobilized within sections of brain tissue. Extensive ISHH analyses using both beta- and gamma-selective probes demonstrated similar patterns of cellular labeling in all of the examined CNS areas. In addition, data obtained from analyses of adjacent thin sections of the dorsal root ganglia (DRG) indicated that beta- and gamma-PPT mRNAs were colocalized within individual DRG neurons, thereby suggesting generalized coexpression at the cellular level of both forms of mRNA. These data were complemented by semi-quantitative analyses which yielded cellular or intrinsic molar ratios of beta- to gamma-PPT mRNA of approximately 1:2-1:3, consistent with those values previously determined by nuclease protection analyses. In sum, a reasonable hypothesis evolving from the anatomical studies in combination with previous biochemical data supports the existence of a strong homeostatic mechanism involved in the maintenance of relatively constant intrinsic molar ratios of beta- to gamma-PPT mRNA by tachykinin-expressing neurons. The biological relevance of this putative fundamental relationship is discussed in the context of posttranslational processing of PPT molecules and of expression of mature tachykinins.

Animals↗
PubMed2004

The effect of venlafaxine on ongoing and experimentally induced pain in neuropathic pain patients: a double blind, placebo controlled study.

BACKGROUND AND AIM: The aim of this randomized double blind placebo controlled study was to investigate the effectiveness and the safety of venlafaxine XR 75 and 150 mg on ongoing pain and on quantitative sensory tests in 60 patients with neuropathic pain for 8 weeks. METHODS: Evaluation parameters consisted of ongoing pain intensity (VAS), patient satisfaction, side effects, global efficacy and tolerance. Quantitative sensory measurements taken from the affected area before and after the drug treatment included pin-prick hyperalgesia, allodynia, detection and pain thresholds to electrical and heat stimuli, temporal summation of repetitive electrical and heat stimuli. RESULTS: A total of 55 patients completed the study. VAS scores decreased significantly compared to the baseline measurements in all groups. There was no significant difference between the groups regarding pain intensity and escape medication. The areas of allodynia and pin-prick hyperalgesia decreased significantly in venlafaxine groups compared to the placebo. There was no significant difference between the groups regarding the detection thresholds (electrical and heat). The pain threshold and the summation threshold to electrical stimuli and the summation threshold to heat stimuli increased significantly following treatment in both venlafaxine groups. In addition, the degree of the temporal summation to electrical and heat stimuli decreased significantly following treatment in both venlafaxine groups compared to the placebo. CONCLUSION: The study showed significant effect of venlafaxine in the manifestations of hyperalgesia and temporal summation, but not on the ongoing pain intensity. Furthermore, the quantitative sensory tests provided complementing information to the clinical measures.

Adult↗
PubMed2004

Strain echocardiography tracks dobutamine-induced decrease in regional myocardial perfusion in nonocclusive coronary stenosis.

OBJECTIVES: This study was designed to determine whether strain echocardiography parameters reflect changes in regional myocardial perfusion during dobutamine stress. BACKGROUND: Strain echocardiography depicts regional myocardial mechanical activity. Ischemia has been shown to reduce systolic strain rate (sSR) and prolong the time to regional lengthening (T(RL)). In an experimental model, we tested whether sSR and T(RL) tracked dobutamine-induced changes in regional myocardial perfusion (regional myocardial blood flow [RMBF]), as measured by colored microspheres. METHODS: We used a closed-chest pig model of nonocclusive coronary stenosis (n = 14) created by inflating an angioplasty balloon in the proximal left anterior descending artery. Invasive hemodynamics, RMBF, and strain parameters were measured at baseline and peak dobutamine stimulation before and during the coronary stenosis. We compared segments with reduced RMBF versus those with preserved RMBF at peak dobutamine stimulation. RESULTS: Peak sSR correlated with RMBF (r = 0.70). In the absence of coronary stenosis, dobutamine stimulation caused a significant increase in RMBF and sSR and a decrease in T(RL). This response was blunted during coronary stenosis. Using the "best cutoff" method, the sensitivity and specificity for prediction of reduced RMBF (ischemia) was 81% and 91% for sSR and 65% and 91% for T(RL), respectively. These changes occurred in the absence of any change in global systolic and diastolic function (dP/dT(max), dP/dT(min), and tau). CONCLUSIONS: Novel strain parameters that depict regional myocardial mechanics are able to predict changes in RMBF during dobutamine stress. Quantitative strain parameters may complement current echocardiographic techniques for ischemia detection and potentially improve the accuracy and reproducibility of stress echocardiography.

Animals↗
PubMed2006

Gene-lifestyle interaction on risk of type 2 diabetes.

The descriptive epidemiology of type 2 diabetes suggests that gene-lifestyle interactions are critical to the development of the condition. However, unravelling the molecular detail of these interactions is a complex task. The existing literature is based on small intervention studies or cross-sectional observational quantitative trait studies. Our systematic review of the literature identified some evidence of interactions, most notably for a common variant in the PPAR-gamma gene which appears to interact with the nature of dietary fat intake. Other interactions have been reported for adrenoceptors, uncoupling proteins, fatty acid binding proteins, apolipoproteins and lipoprotein lipase. There are, to date, no reports based on the ideal study design which is a case-control study nested within a cohort. To limit the likelihood of false discovery, such studies would need to be large and the search for interaction should be restricted to a priori biologically driven hypotheses. Additional study designs that examine differential response to lifestyle change or test interaction in the context of quantitative trait studies would complement the nested case-control approach, but the emphasis here should be on precision of measurement of both phenotype and lifestyle behaviour.

Aged↗
PubMed1998

Correlation analysis of somatosensory evoked potential waveforms: clinical applications.

OBJECTIVE: To assess the validity of the correlation coefficient (CC) as a diagnostic tool in evaluating somatosensory evoked potential (SEP) waveforms. DESIGN: SEP waveforms were recorded over the spine and scalp in response to tibial nerve stimulation at the ankle for 30 patients. SETTING: A general community, ambulatory, private referral center. PATIENTS: Patients were all ambulatory and were selected randomly from among existing patient records at the medical center. MAIN OUTCOME MEASURE: The test for validity uses the CC to verify reproducibility. The test for side-to-side symmetry uses the CC to obtain a measure of similarity between left and right tibial scalp responses. For both tests, a table is presented with CCs for normal subjects. If the CC for each patient falls below the appropriate critical value from the table, then the CC is taken to be abnormal; p values are provided at specific levels in this table. RESULTS: A new data base for the means and critical values of CCs with corresponding p values is established for normal subjects. Most of the patient data reveal significant CC nonreproducibility and lack of symmetry in tibial scalp SEP waveforms. CONCLUSION: The CC provides objective and quantitative waveform information that complements latency and amplitude measurements in the clinical interpretation of tibial scalp SEP waveforms.

Algorithms↗
PubMed1984

Detection of pericardial heart disease by computed tomography.

The findings on computed tomography (CT) in 10 cases of pericardial disease, nine of which were surgically confirmed, are presented. The 10th patient (Case 10), whose CT diagnosis was 'pericarditis', was found to have a myxosarcoma at surgery. In some of these cases, echocardiographic and conventional radiographic investigation gave inconclusive information regarding the underlying pathology. Our results support the conclusion that CT complements echocardiography: in the quantitative and qualitative assessment of pericardial effusions, in the localisation of space-occupying lesions involving the pericardium and in diagnosis of constrictive pericarditis. As echocardiography is so very much operator-dependent, CT may well be a more reliable method of obtaining a result which indicates both the diagnosis and the extent of the disease.

Cysts↗
PubMed2001

An improved anti-C3/IgG ELISA for quantification of soluble immune complexes.

A semi-quantitative ELISA for complement-fixing, IgG-containing immune complexes (IC) is described. The assay is based on the insolubilization of IC by polyethyleneglycol, their capture by solid-phase anti-C3 antibodies, reaction with peroxidase-labeled anti-IgG antibodies and incubation with a chromogenic peroxidase substrate. It was markedly improved by the use of a single-step procedure which simultaneously washed and precipitated the insolubilized immune complexes. Intra-assay and inter-assay coefficients of variation were lower than 8.6 and 14.7%, respectively. As expected, higher levels of circulating immune complexes, in relation to healthy individuals, were found in patients with American visceral leishmaniasis (AVL), systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), with prevalences comparable to those described in the literature. The ELISA can be quickly assembled from reagents and plasticware widely available commercially, detects immune complexes fulfilling three different criteria and is more sensitive than a previously published method based on the same principles (detection limit for complement-sensitized aggregated IgG of 2 microg ml(-1) as compared with a detection limit above 16 microg ml(-1)).

Antigen-Antibody Complex↗
PubMed1987

Immunopathology of childhood idiopathic thrombocytopenic purpura.

Modern laboratory techniques have begun to elucidate the pathophysiology of chronic childhood ITP. Quantitative assays of PAIgG, complement, immune complexes, and platelet kinetic studies have all provided important information. Chronic ITP of childhood appears to be similar to adult ITP, with production of an antibody directed against platelets and megakaryocytes. Most of the antibody is produced in the spleen, but other parts of the RES can also produce antibody. Complement, immune complexes, and cell-mediated immunity may play a role in the pathogenesis. Sensitized platelets are cleared by the RES, particularly in the spleen. Platelet kinetic studies show that platelet turnover is usually rapid with compensatory increased thrombopoiesis, but there are some patients who have decreased thrombopoiesis. Acute ITP of childhood is a brief illness, characterized by abrupt onset of hemorrhagic symptoms and complete recovery. It often follows a viral illness, suggesting that immune complexes as well as antibodies are important in the pathogenesis. Both the spleen and liver may be important organs of immune clearance.

Acute Disease↗
PubMed2003

Caregiving near the end of life: unmet needs and potential solutions.

OBJECTIVE: A key aspect of the role of clinicians caring for patients in the setting of advanced illness focuses on attending to the needs of informal caregivers during the end-of-life period. The purpose of this study was twofold: (1) to complement and enrich existing quantitative findings regarding caregiver burden near the end of life, and (2) to identify potential solutions to caregivers' unmet needs in an effort to assist clinicians in the development of clinical interventions. METHODS: This qualitative study, using focus groups and content analysis of transcripts, was conducted in a comprehensive cancer center in Washington, DC. Seven focus groups were held: three with recently bereaved caregivers and four with active caregivers of patients with metastatic cancer and an expected survival of 6 to 12 months. RESULTS: Data were stratified into two broad categories: (1) general problems and (2) behaviors/activities that were helpful/would have been helpful in alleviating these problems. Within each of these two categories, five subcategories emerged: medical care (including provision of information, coordination of care, bedside manner, satisfaction with care), quality of life (including well-being, role adjustments), help from others (including practical assistance, social support), positives of caregiving, and unsolicited themes (including job flexibility, impact of the disease on the family, informational needs, relationship with patient). SIGNIFICANCE OF RESULTS: Results suggest caregivers may benefit from more information about patient prognosis and hospice, attention to quality-of-life issues, and enhanced, direct communication with clinicians. Although information of this nature is likely to be known to palliative care clinicians, the specific details and verbal insights provided by caregivers give an important voice to existing quantitative data and may provide more detailed information to assist palliative care clinicians seeking to develop interventions to meet caregiver needs during the period near the end of life.

Adult↗
PubMed1987

Shared active sites in oligomeric enzymes: model studies with defective mutants of aspartate transcarbamoylase produced by site-directed mutagenesis.

Many oligomeric enzymes are functional only in the assembled form, and it is often difficult to determine unambiguously why monomers are inactive. In some cases individual monomers cannot fold into stable correct ("native") conformations without contributions from interchain interactions. For other oligomers, catalysis requires the contributions of amino acid residues at the interface between adjacent polypeptide chains, and monomers are inactive because they cannot form complete active sites. A test for the presence of shared sites was devised that is based on the formation of active hybrid oligomers from appropriate inactive parental mutants produced by site-directed mutagenesis. This approach was applied in a study of the catalytic trimer of aspartate transcarbamoylase (aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli, using three mutants, in which Ser-52 was replaced by His, Lys-84 by Gln, or His-134 by Ala. Hybrid trimers formed from the virtually inactive Ser and Lys mutants were 10(5) more active than the parental proteins, and the specific activities of each hybrid were about 33% that of the wild-type trimer, as expected for the scheme based on shared sites. Hybrids from the His and Lys mutants had comparable specific activities. Moreover, one hybrid with approximately 33% activity had one high-affinity binding site for a bisubstrate analog as compared to about three for wild-type trimer. As a further test, hybrids were also formed from wild-type and double-mutant (Lys-84----Gln and His-134----Ala) trimers. The hybrid containing two chains with the double mutation and one wild-type chain had very little activity, and that composed of one double mutant and two wild-type chains had 32% the specific activity of wild-type trimers. This negative complementation experiment is in quantitative accord with the scheme based on shared sites at or near the interfaces between adjacent chains. The techniques used to demonstrate shared active sites in the catalytic subunits of aspartate transcarbamoylase can be applied generally to various types of oligomers (dimers, tetramers, etc.) to determine whether the participation of amino acid residues from adjoining chains is essential for forming active sites in oligomeric enzymes.

Alleles↗
PubMed2005

Physical and chemical characterization of beryllium particles from several workplaces in Québec, Canada--part B: time-of-flight secondary-ion mass spectroscopy.

The problems associated with detecting and characterizing beryllium (Be) particles in industrial samples from Québec were addressed in the companion article (Rouleau et al., 2005). The present study is a continuation of the work aimed at redefining the current occupational exposure level for beryllium. The goals were to determine the principal chemical forms and the principal physical characteristics of Be particles sampled in four Québec industries. Bulk particle chemistry was determined using inductively coupled plasma-mass spectroscopy (ICP-MS) and flame atomic absorption spectrophotometry (FAAS). Time-of-flight secondary-ion mass spectroscopy (TOF-SIMS) was used to characterize particle surface chemistry and physical particle size. The dust samples collected had Be concentrations varying from 58 to 146 microg/g. Results showed that numerous fine Be particles or aggregates were evenly dispersed throughout the samples. Thus, Be does not appear to be concentrated in large particles. However, it was not possible to confirm if these fine particles were combined to specific compounds, chemically or physically, or independent Be particles. Most of the particles containing Be were fine, with diameters less than 10 microm, which is important from an occupational health and safety standpoint. TOF-SIMS should be considered as an appropriate technique for qualitative characterization of Be particles, and a valuable complement to the recognized quantitative methods ICP-MS and FAAS.

Beryllium↗
PubMed1971

Immunochemical studies on blood groups. LI. A comparative study of the reaction of A 1 and A 2 blood group glycoproteins with human anti-A.

The immunochemical properties of purified A(1) and A(2) glycoproteins have been compared to ascertain whether their antigenic determinants differ. Quantitative precipitin and complement-fixation studies using several anti-A sera as well as purified gammaG anti-A antibodies clearly showed a specificity difference. This was also supported by absorption studies: A(2) substance specifically removed antibodies reacting with A(2) substance leaving anti-A(1) activity. A(1) substance was more effective than A(2) substance in dissolving an A(1) anti-A(1)-specific precipitate. Purified gammaM anti-A hemolyzed A(1) cells more readily than A(2) cells. Inhibition studies using mono- and difucosyl type 2 A-active oligosaccharides showed that type 2 difucosyl receptors are present in A(2) substance. The structural basis for the specificity difference between A(1) and A(2) would appear to be that A(2) substances lack type 1 A determinants; this would account for the observed higher H and Le(b) activity in A(2) substances.

ABO Blood-Group System↗
PubMed1991

Qualitative diatom analysis as a tool to diagnose drowning.

Since the 1950s, quantitative diatom analysis has been used successfully at the Department of Forensic Medicine at the University of Helsinki as a supportive method for diagnosing deaths by drowning. The reliability of the method was firmly established in 1986 by a study involving 107 probable cases of drowning. Since 1982, the quantitative analysis has been complemented with qualitative diatom analysis. This report presents potential applications of the latter method by describing its use in six cases of drowning.

Accidents↗
PubMed1976

A quantitative study on the activation of the alternative pathway of complement by mouldy hay dust and thermophilic actinomycetes.

Materials associated with the induction of farmer's lung were incubated with fresh normal human serum in the presence of magnesium ethylene glycol tetra-acetic acid (MgEGTA) or ethylene diamine tetra-acetic acid (EDTA) and results compared with material known to activate the alternative pathway of complement--zymosan. Results show that Micropolyspora faeni organisms are as active as zymosan in reducing complement (C) levels in the presence of MgEGTA, with a 50% reduction in CH50 at approximately 140 mug/ml. Thermoactinomyces vulgaris organisms produced a 50% CH50 reduction at approximately 1-25 mg/ml and two samples of respirable mouldy hay dust (MHD) at approximately 5-6 mg/ml whereas extracts of M. faeni and T. vulgaris reduced the CH50 titre by 17% and 39% respectively at 16 mg/ml in the presence of MgEGTA. Organisms and extracts did not reduce the CH50 titre in the presence of EDTA even at the maximum concentration quoted by more than 3%, thus it is considered that alternative pathway activation was responsible for C utilization in the presence of MgEGTA. Respirable MHD used less than 4% available C at 4 mg/ml in the presence of EDTA but at 8 mg/ml dust 11% and 28% available CH50 were used compared with 79% and 81% respectively in the presence of MgEGTA. The elution of immunoglobulin binding material from MHD may be responsible for apparent CH50 consumption in the presence of EDTA.

Allergens↗
PubMed1982

Protein components in saliva and plaque fluid from irradiated primates.

Irradiation of the major salivary glands of monkeys (Macaca mulatta) fed cariogenic diets leads to caries clinically indistinguishable from radiation caries in man. This study compares the organic composition of individual samples of plaque fluid and saliva from irradiated and control monkeys receiving the same cariogenic diet. Plaque and saliva were collected from fasting, tranquillised animals. Four irradiated animals were sampled repeatedly as were non-irradiated controls. Total protein, albumin, immunoglobulins A, G, and M, and the third component of complement (C'3) were quantitated in plaque fluid and whole saliva. Salivary amylase and peroxidase activities were also determined. Plaque fluid and saliva samples were also subjected to polyacrylamide gel electrophoresis. The total viable anaerobic count and numbers of Streptococcus mutans were determined in samples of plaque. The results suggest that the major effect of irradiation leading to increased numbers of S. mutans and caries susceptibility is in the amount, and not the composition, of the saliva produced by the residual gland tissue. The scanty flow of saliva may reduce the effectiveness of cleansing, buffering and lubrication mechanisms as well as resulting in a marked reduction in the total amount of specific and non-specific immune factors entering the mouth.

Albumins↗
PubMed2006

The mechanism of Salmonella entry determines the vacuolar environment and intracellular gene expression.

Macrophages are an important intracellular niche for Salmonella particularly for systemic infection. The interaction of Salmonella with these cells is mediated by two type III secretion systems (TTSS), encoded on Salmonella pathogenicity islands 1 and 2 (SPI1, SPI2), which mediate distinct phases of the pathogen-host cell interaction. The SPI1 TTSS mediates invasion whereas the SPI2 TTSS is required for intramacrophage survival. Importantly, however, Salmonella can enter macrophages by either SPI1-dependent invasion or host cell-mediated phagocytosis. Here, we investigated how the mechanism of internalization affects the intracellular environment and TTSS gene expression. Intracellular bacterial survival depended on the method of entry, because complement-opsonized and SPI1-induced Salmonella initiated replication within 8 h whereas immunoglobulin G (IgG)-opsonized and non-opsonized Salmonella were initially killed. Analysis of vacuolar pH showed that acidification of the Salmonella-containing vacuole occurred more rapidly for non-opsonized or SPI1-induced Salmonella compared with IgG-opsonized or complement-opsonized Salmonella. Finally, quantitative polymerase chain reaction was used to compare the transcriptional profiles of selected SPI1 and SPI2 regulon genes. We found that the magnitude of SPI2 gene induction depended on the mechanism of internalization. Unexpectedly, SPI1 genes, which are rapidly downregulated following SPI1-mediated invasion, were induced intracellularly following phagocytic uptake. These results reveal another level of complexity in pathogen-macrophage interactions.

Animals↗
PubMed2001

Dominance of virus over host factors in cross-species activation of human cytomegalovirus early gene expression.

Human cytomegalovirus (HCMV) exhibits a highly restricted host range. In this study, we sought to examine the relative significance of host and viral factors in activating early gene expression of the HCMV UL54 (DNA polymerase) promoter in murine cells. Appropriate activation of the UL54 promoter at early times is essential for viral DNA replication. To study how the HCMV UL54 promoter is activated in murine cells, a transgenesis system based on yeast artificial chromosomes (YACs) was established for HCMV. A 178-kb YAC, containing a subgenomic fragment of HCMV encompassing the majority of the unique long (UL) region, was constructed by homologous recombination in yeast. This HCMV YAC backbone is defective for viral growth and lacks the major immediate-early (IE) gene region, thus permitting the analysis of essential cis-acting sequences when complemented in trans. To quantitatively measure the level of gene expression, we generated HCMV YACs containing a luciferase reporter gene inserted downstream of either the UL54 promoter or, as a control for late gene expression, the UL86 promoter, which directs expression of the major capsid protein. To determine the early gene activation pathway, point mutations were introduced into the inverted repeat 1 (IR1) element of the UL54 promoter of the HCMV YAC. In the transgenesis experiments, HCMV YACs and derivatives generated in yeast were introduced into NIH 3T3 murine cells by polyethylene glycol-mediated fusion. We found that infection of YAC, but not plasmid, transgenic lines with HCMV was sufficient to fully recapitulate the UL54 expression program at early times of infection, indicating the importance of remote regulatory elements in influencing regulation of the UL54 promoter. Moreover, YACs containing a mutant IR1 in the UL54 promoter led to reduced ( approximately 30-fold) reporter gene expression levels, indicating that HCMV major IE gene activation of the UL54 promoter is fully permissive in murine cells. In comparison with HCMV, infection of YAC transgenic NIH 3T3 lines with murine cytomegalovirus (MCMV) resulted in lower (more than one order of magnitude) efficiency in activating UL54 early gene expression. MCMV is therefore not able to fully activate HCMV early gene expression, indicating the significance of virus over host determinants in the cross-species activation of key early gene promoters. Finally, these studies show that YAC transgenesis can be a useful tool in functional analysis of viral proteins and control of gene expression for large viral genomes.

3T3 Cells↗