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At least 235 records · Page 13Linked to original sources
PubMed1988

The rabbit autoperfusing heart-lung preparation.

The autoperfusing heart-lung preparation holds promise as a method for extended organ preservation. Further understanding of the hemodynamics, metabolism, and quality of tissue preservation of this preparation may increase the number of available organs for heart-lung transplantation. We describe the materials and operative technique for a small animal model of the autoperfusing heart-lung preparation. The method is easy to learn, the materials and animal subjects are inexpensive, and the preparation is fully monitored to provide consistent and reproducible data. The rabbit autoperfusing heart-lung preparation is an excellent vehicle for the investigator studying warm organ preservation.

Animals↗
PubMed1986

Reactivity and fate of benzene and formaldehyde in culture medium with and without fetal calf serum; relevance to in vitro mutagenicity testing.

Gas chromatographic-mass spectrometric analyses were performed to determine the reactivity and fate of benzene (BEN) and formaldehyde (FA) in culture medium. BEN (solubility in water: approximately 500 ppm) does not react with culture medium, either with or without fetal calf serum, but its volatility, even in closed vials, is so great that 90% of a 250-ppm solution is lost to the head space after 1 h at 24 degrees C. FA, as a 37% aqueous solution, is a complex mixture that changes composition after 15-min incubation at 38 degrees C. FA is extremely reactive in culture medium containing fetal calf serum, and is much less reactive with medium components in the absence of serum. There is a dramatic increase in the number of daughter products in FA-treated medium over time, such that those seen immediately after FA is added to medium have been replaced after 60-min incubation (38 degrees C in closed vials) by many other interaction products. Methods ensuring maximum solubilization and minimal volatilization of BEN during exposure are essential for obtaining reproducible data on the mutagenic potential of BEN. The volatilization of FA from stock formalin solutions, and, more importantly, the interaction product(s) formed by this highly reactive compound with medium components, especially those in serum, are probably the critical aspects of an effective testing protocol for FA.

Animals↗
PubMedDate not supplied

Cardiac S-100a0 protein: purification by a simple procedure and related immunocytochemical and immunochemical studies.

A simple procedure is described for the purification of the alpha alpha isoform of S-100 proteins (S-100a0) from porcine heart. Purification steps include the following: i) extraction of the tissue with a hypotonic medium containing EDTA; ii) ammonium sulfate fractionation (0-50%) of the extract; iii) Ca2+-dependent affinity chromatography of the supernatant obtained through the preceding step on phenyl-sepharose and elution of absorbed proteins through a two-chamber gradient of 1.0-0.0 mM CaCl2 and 0.0--1.0 mM EGTA, respectively; and iv) chromatography of the resultant S-100-containing fractions on Sephadex G-200. The yield is 20 mg S-100a0/kg porcine heart. The whole procedure takes five days and is highly reproducible. Data obtained from the phenyl-sepharose step suggest that the affinity of Ca2+ for S-100a0 increases by several orders of magnitude once the protein had interacted with that matrix. This observation is discussed in relation to the role of S-100 proteins in amplification of the Ca2+ signal. Immunocytochemical and immunoblotting analyses indicate that S-100a0 is exclusively found at the level of the sarcolemmal membranes, the membranes of the sarcoplasmic reticulum, the external mitochondrial membranes, and in the adjacent sarcoplasm. No evidence of S-100a0 being associated with the nuclei or with myofibrils has been obtained. Finally, the cardiac tissue does not contain the Triton X-100-extractable fraction of S-100 normally detected in the brain and in adipocytes. Our data suggest that S-100a0 behaves as a peripheral membrane protein in cardiac tissue.

Animals↗
PubMed1990

Measurement of atactic and paretic gait in neuropathies of rats based on analysis of walking tracks.

In 23 Lewis rats 2 forms of neuropathy both with atactic gait were studied by morphometric analysis of walking tracks. In one group of animals experimental allergic neuritis (EAN) was induced which leads to both sensory and motor dysfunction. Clinical symptoms are atactic gait and hindlimb paresis. In another group pyridoxine (vitamin B6) neuropathy was induced which is a purely sensory neuropathy clinically presenting with gait ataxia, too. Track analysis is a simple method that requires no visualisation process and produces significant and reproducible data. The findings were compared with the clinical scores and electrophysiological data. In EAN, toe spreading was impaired early, and at a later stage stride width, print length and outward rotation of the hind feet changed. Pyridoxine-induced neuropathy produced only an increase in stride width. Track analysis correlated well with clinical grading and electrophysiological recordings. We propose track analysis as a reliable and accurate indicator of neuropathy symptoms in rats.

Animals↗
PubMed1984

Threonine uptake in Trypanosoma brucei.

Threonine uptake by the parasitic protozoan Trypanosoma brucei has been assessed using an oil-phase separation technique for measuring rapid amino acid fluxes. It was shown that the storage conditions for the organism were critical for the production of reproducible data. Using this method it has been shown that threonine uptake occurs rapidly, being linear for less than 30 s at 37 degrees C in contrast with previous reported results. Kinetic analysis of threonine uptake at 25 degrees C and at physiological plasma threonine levels (20-700 microM) gave a Km of 250 microM and a Vmax of 8 nmol mg-1 cell protein min-1. At these concentrations threonine uptake takes place against a concentration gradient.

Alanine↗
PubMed1991

A skin suction blister model in hairless rats: application to the study of anti-inflammatory and immunomodulatory drugs.

A suction blister model was developed in the hairless rat, in order to study the effects of various agents on the migration of polymorphonuclear leukocytes (PMN). A standardized abrasion, a suction blister, was formed by applying negative pressure to the skin and then separating the epidermis from the dermis. A migration chamber containing serum as the chemoattractant was placed over the wound. After 6 h of migration, the cells in the chamber were harvested, counted and identified. We evaluated PMN migration after treating the animals with active compounds: niflumic acid, and anti-inflammatory drug, and RU 41740, an immunomodulator. This in vivo model provided reproducible data and could be used to study further the functional properties of PMN. In addition, because this assay can also be used in man, a drug found to be effective in the animal system could then be tested for its activity in man.

Adjuvants, Immunologic↗
PubMed2004

Pharmacological characterization of human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3 in a fluorescence-based membrane potential assay.

We have expressed the human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3 stably in HEK293 cells and characterized the transporters pharmacologically in a conventional [(3) H]-d-aspartate uptake assay and in a fluorescence-based membrane potential assay, the FLIPR Membrane Potential (FMP) assay. The K(m) and K(i) values obtained for 12 standard EAAT ligands at EAAT1, EAAT2 and EAAT3 in the FMP assay correlated well with the K(i) values obtained in the [(3) H]-d-aspartate assay (r(2) values of 0.92, 0.92, and 0.95, respectively). Furthermore, the pharmacological characteristics of the cell lines in the FMP assay were in good agreement with previous findings in electrophysiology studies of the transporters. The FMP assay was capable of distinguishing between substrates and non-substrate inhibitors and to discriminate between "full" and "partial" substrates at the transporters. Taking advantage of the prolific nature of the FMP assay, interactions of the EAATs with substrates and inhibitors were studied in some detail. This is the first report of a high throughput screening assay for EAATs. We propose that the assay will be of great use in future studies of the transporters. Although conventional electrophysiology set-ups might be superior in terms of studying sophisticated kinetic aspects of the uptake process, the FMP assay enables the collection of considerable amounts of highly reproducible data with relatively little labor. Furthermore, considering that the number of EAAT ligands presently available is limited, and that almost all of these are characterized by low potency and a low degree of subtype selectivity, future screening of compound libraries at the EAAT-cell lines in the FMP assay could help identify structurally and pharmacologically novel ligands for the transporters.

Amino Acid Transport System X-AG↗
PubMed2004

Determination of toxic cyclic heptapeptides by liquid chromatography with detection using ultra-violet, protein phosphatase assay and tandem mass spectrometry.

Microcystins, toxic cyclic heptapeptides and nodularin-R, a toxic cyclic pentapeptide, were determined using liquid chromatography (LC) with detection using photo-diode array ultra-violet (PDA-UV) and protein phosphatase (PP) assay. Positive fractions were analysed for toxins using collision-induced dissociation (CID) and tandem MS/MS experiments which were carried out simultaneously using electrospray ion-trap instrumentation. Reversed-phase liquid chromatography (LC) using an acetonitrile/water gradient was used for the LC-MS(2) determination of six microcystins standards and nodularin. The molecular related ion species, [M+H](+)([M+2H](2+) in the case of MC-RR), were used as the precursor ions for MS(2) experiments. Optimum calibration and reproducibility data were obtained for MC-LR using LC-MS(2); 0.1-5.0 microg/ml, r2 = 0.992 (n = 3); % RSD < or =7.3 at 0.25 microg MC-LR/ml (n = 3). The detection limit (S/N = 3) was better than 0.1 ng. Water samples for microcystin analysis were first screened using protein phosphatase (PP) assays and positives were concentrated using C-18 solid-phase extraction. The developed method was applied to examine a lake in Ireland contaminated by Microcystis sp. and MC-LR and MC-LA were identified.

Chromatography, Liquid↗
PubMed2006

Myocardial strain and torsion quantified by cardiovascular magnetic resonance tissue tagging: studies in normal and impaired left ventricular function.

Accurate quantification and timing of regional myocardial function allows early identification of dysfunction, and therefore becomes increasingly important for clinical risk assessment, patient management, and evaluation of therapeutic efficacy. For this purpose, the application of tissue Doppler echocardiography has rapidly increased. However, echocardiography has some major inherent limitations. Cardiovascular magnetic resonance imaging with tissue tagging provides highly reproducible data on myocardial function, not only in longitudinal and radial directions, but also in the circumferential direction. Because of the development of faster imaging protocols, improved temporal resolution, less time-consuming postprocessing procedures, and the potential of quantifying myocardial deformation in 3 dimensions at any point in the heart, this technique may serve as an alternative for tissue Doppler echocardiography and is now ready for more widespread clinical use. This review discusses the clinical use of cardiovascular magnetic resonance tissue tagging for quantitative assessment of regional myocardial function, thereby underlining the specific features and emerging role of this technique.

Heart↗
PubMed2004

Airway glycoprotein secretion parallels production and predicts airway obstruction in pulmonary allergy.

BACKGROUND: Airway obstruction, perhaps the most relevant clinical feature of asthma, is typically assessed in allergic asthma models as airway hyperresponsiveness. Excess secretion of airway glycoproteins also contributes to airway obstruction in asthma but is not measured as part of most experimental models. OBJECTIVE: The purposes of this study were to develop a reliable, quantitative assay for detecting secreted airway glycoproteins and to assess the secretion of airway glycoproteins in comparison with other markers of airway obstruction resulting from allergic lung inflammation. METHODS: Two microtiter plate-based glycoprotein-detecting methods were developed, one using an antiglycoprotein antibody and the other using the glycoprotein-binding plant lectin, jacalin. Both methods were used to assess airway glycoprotein secretion in response to 2 defined agonists given intranasally, IL-13 and an allergen derived from Aspergillus fumigatus. Glycoprotein secretion was assessed concomitant with another measure of airway obstruction, airway hyperresponsiveness provoked by acetylcholine challenge, and a histologic method for quantitating glycoprotein production. RESULTS: Both assays were sufficient for quantitating airway glycoproteins over the full range of values encountered from murine bronchoalveolar lavage and yielded highly reproducible data. Secretion of airway glycoproteins increased commensurate with the detection of both airway hyperresponsiveness and airway glycoprotein production induced by IL-13 and allergen. CONCLUSION: Airway glycoprotein secretion is a consistent feature of the allergic lung phenotype and likely contributes to airway obstruction induced by allergen in both humans and rodents.

Airway Obstruction↗
PubMed2005

A new method to measure air-borne pyrogens based on human whole blood cytokine response.

Air-borne microorganisms, as well as their fragments and components, are increasingly recognized to be associated with pulmonary diseases, e.g. organic dust toxic syndrome, humidifier lung, building-related illness, "Monday sickness." We have previously described and validated a new method for the detection of pyrogenic (fever-inducing) microbial contaminations in injectable drugs, based on the inflammatory reaction of human blood to pyrogens. We have now adapted this test to evaluate the total inflammatory capacity of air samples. Air was drawn onto PTFE membrane filters, which were incubated with human whole blood from healthy volunteers inside the collection device. Cytokine release was measured by ELISA. The test detects endotoxins and non-endotoxins, such as fungal spores, Gram-positive bacteria and their lipoteichoic acid moiety and pyrogenic dust particles with high sensitivity, thus reflecting the total inflammatory capacity of a sample. When air from different surroundings such as working environments and animal housing was assayed, the method yielded reproducible data which correlated with other parameters of microbial burden tested. We further developed a standard material for quantification and showed that this assay can be performed with cryopreserved as well as fresh blood. The method offers a test to measure the integral inflammatory capacity of air-borne microbial contaminations relevant to humans. It could thus be employed to assess air quality in different living and work environments.

Air Microbiology↗
PubMed2005

Similar sequence-free amplification of human glyceraldehyde-3-phosphate dehydrogenase for real time RT-PCR applications.

One of the major applications of real time polymerase chain reaction (PCR) is relative quantification, where the expression of a target gene is determined as a ratio to a stably expressed reference gene, the so-called housekeeping gene. Glyceraldehyde-3-phosphate dehydrogenase (GAPD) is a glycolytic enzyme, which is active in all mammalian tissues and is frequently used as housekeeping gene in expression studies. The functional locus maps to human chromosome 12p13, but several GAPD-related sequences, including processed pseudogenes, GenBank homologous sequences and computationally predicted sequences are present along the human genome. Due to the high level of GAPD-related sequences it is very important to avoid genomic DNA amplification when GAPD is used as endogenous control in mRNA quantification. We have outlined a GAPD couple of primers that avoid any genomic DNA amplification for real time reverse transcription PCR applications by SYBR-Green Dye. These new designed primers are an useful and chip alternative to probe technologies, and can carry out specific and reproducible data in mRNA expression studies.

Base Sequence↗
PubMed2006

Early and transient alteration of adenosine A2A receptor signaling in a mouse model of Huntington disease.

Huntington Disease (HD) is characterized by choreic involuntary movements and striatal vulnerability. A2A receptors expressed on GABAergic striatal neurons have been suggested to play a pathogenetic role. Previous data demonstrated the presence of an aberrant alteration of A2A receptor-dependent adenylyl cyclase in an in vitro model of the disease (striatal cells expressing mutant huntingtin) and in peripheral circulating cells of HD patients. Here, we investigated whether this dysfunction is present in the R6/2 HD transgenic mouse model, by analyzing striatal A2A receptor-binding and adenylyl cyclase activity at different developmental stages in comparison with age-matched wild type animals. A transient increase in A2A receptor density (Bmax) and A2A receptor-dependent cAMP production at early presymptomatic ages (7-14 postnatal days) was found. Both alterations normalized to control values starting from postnatal day 21. In contrast, A2A receptor mRNA, as detected by real time PCR, dramatically decreased starting from PND21 until late symptomatic stages (12 weeks of age). The discrepancy between A2A receptor expression and density suggests compensatory mechanisms. These data, reproducing ex vivo the previous observations in vitro, support the hypothesis that an alteration of A2A receptor signaling is present in HD and might represent an interesting target for neuroprotective therapies.

Adenylyl Cyclases↗
PubMed2005

UV inactivation of adenovirus type 41 measured by cell culture mRNA RT-PCR.

Adenoviruses are among the most resistant waterborne pathogens to UV disinfection, yet of the 51 serologically distinct human adenoviruses, only a few have been evaluated for their sensitivities to UV irradiation. Human enteric adenoviruses (Ad40 and Ad41) are difficult to cultivate and reliably assay for infectivity, requiring weeks to obtain cytopathogenic effects (CPE). Inoculated cell cultures often deteriorate before the appearance of distinctive CPE making it difficult to obtain reliable and reproducible data regarding UV inactivation. Adenovirus is a double-stranded DNA virus and produces messenger RNA (mRNA) during replication in host cells. The presence of viral mRNA in host cells is definitive evidence of infection. We recently developed a rapid and reliable cell culture-mRNA RT-PCR assay to detect and quantify adenovirus infectivity. Viral mRNA recovered from cell cultures 5-7 days after infection was purified on oligo-dT latex, treated with DNase, and amplified by RT-PCR using the primers specific for a conserved region of the hexon late mRNA transcript. Treatment of approximately 10(4) Ad41 with different doses of 254 nm germicidal UV radiation resulted in a dose-dependent loss of infectivity. As UV doses were increased from 75 to 200 mJ/cm2, virus survival decreased and no virus infectivity (measured by detectable mRNA) was found at a dose of 225 mJ/cm2 or higher. Our results using the cell culture mRNA RT-PCR assay indicate that Ad41 is more resistant to UV radiation than in a previous study using a conventional cell culture infectivity assay. Results were more similar to those found for Ad 40 using CPE as a measure of infectivity in another previous study.

Adenoviruses, Human↗
PubMed1988

Body contouring. Suction-assisted lipolysis and fat transplantation techniques.

Suction lipolysis is a technique of blunt aspiration of fat that allows contouring and shaping of fatty distributions in a variety of areas of the body. Sufficient experience with the technique exists so that it may be considered an acceptable and safe procedure in the armamentarium of the plastic and reconstructive surgeon. It is a basically low-risk surgical procedure that usually is performed on an outpatient basis, and its results are permanent. Autologous fat transplantation or microlipoinjection is a recent outgrowth of liposuction surgery. It involves gentle aspiration of free fat cells from one area of the body and reinjection of these cells into other areas to accomplish either cosmetic or reconstructive soft-tissue augmentation. It is still investigational in nature, and accurate, reproducible data demonstrating its prolonged efficacy are not yet available.

Adipose Tissue↗
PubMed1990

Blood cell labeling with 99mTc: progress and perspectives.

Blood cell labeling with 99mTc has progressed through various developmental phases. In the case of red cell labeling, the science seems to have matured sufficiently, although minor refinements in the procedures will no doubt continue to be made. During the last 3 to 5 years, there has been a resurgence of interest in labeling leukocytes and platelets with 99mTc. As a result of these efforts, the techniques for these cell types appear to be developing slowly, having finally come out of their infancy. Progress in these directions over the last 3 1/2 years is summarized and discussed in this article. Emerging techniques that offer the promise of combining simplicity and convenience with reliable and reproducible data are highlighted. Mechanisms involved in the various labeling approaches, if studied and understood, are included. Recent efforts on cell labeling with 99mTc using the monoclonal antibody approach are summarized. Although results in this area are quite preliminary, the monoclonal antibody approach holds the greatest promise for labeling leukocytes and platelets in vivo, and thus overcoming the biggest drawback of current techniques, ie, cell separation and handling before labeling. This is a US government work. There are no restrictions on its use.

Animals↗
PubMed1999

In vivo iron mobilisation evaluation of hydroxypyridinones in 59Fe-ferritin-loaded rat model.

Although there are a number of well-characterised animal models available for testing and comparing the efficacy of iron chelators, most are expensive to operate and are not capable of providing rapid and reproducible results. The method described herein is based on the labelling of rat liver with 59Fe using rat 59Fe-ferritin. This method produces highly reproducible data of the type necessary for dose-response investigations, comparison of the efficacies of different administration routes, and structure activity studies.

Animals↗
PubMed1994

Temperature dependence of the inhibitory effects of orthovanadate on shortening velocity in fast skeletal muscle.

We have investigated the effects of the orthophosphate (P(i)) analog orthovanadate (Vi) on maximum shortening velocity (Vmax) in activated, chemically skinned, vertebrate skeletal muscle fibers. Using new "temperature-jump" protocols, reproducible data can be obtained from activated fibers at high temperatures, and we have examined the effect of increased [Vi] on Vmax for temperatures in the range 5-30 degrees C. We find that for temperatures < or = 20 degrees C, increasing [Vi] inhibits Vmax; for temperatures > or = 25 degrees C, increasing [Vi] does not inhibit Vmax. Attached cross-bridges bound to Vi are thought to be an analog of the weakly bound actin-myosin.ADP-P(i) state. The data suggest that the weakly bound Vi state can inhibit velocity at low temperature, but not at high temperature, with the transition occurring over a narrow temperature range of < 5 degrees C. This suggests a highly cooperative interaction. The data also define a Q10 for Vmax of 2.1 for chemically skinned rabbit psoas fibers over the temperature range of 5-30 degrees C.

Actins↗