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PubMed1992

Characterization and specifications of caramel colours: an overview.

An overview of the chemical characterization and specifications of the four classes of caramel colour, the historical development of the four classes, and the methods of manufacture is presented. The rationale for the approach used to characterize caramel colour and the derivation of the specifications are given. Results of the characterization programme and the derived specifications are presented in subsequent papers in this issue of Food and Chemical Toxicology.

Candy↗

PubMed1990

Sharing a common language about conditioning requires accurate characterizations of each others' positions: reply to Shanks.

Shanks' characterizations of the positions taken in papers that he comments on are inaccurate on a number of basic counts. For example, the papers were concerned with human autonomic Pavlovian conditioning, whereas Shanks refers to no autonomic evidence in his reply. Again, the two papers more specifically targeted by Shanks (Furedy, 1988b; Furedy & Riley, 1987) do not deny "that cognitive processes have any relevance for conditioning", but rather advocate that both cognitive and non-cognitive factors play roles that need to be empirically determined for different preparations and conditions. And the characterization of cognitive factors in those papers, contrary to Shanks, is not a teleological, intentional one, no matter how fashionable such teleological forms of cognitive psychology may be among many current philosophers and psychologists. We can proceed towards an empirical resolution of disagreements about the role of cognitive factors in human Pavlovian autonomic conditioning only if we both refer to the relevant autonomic conditioning evidence, and also characterize each others' positions with some accuracy.

Animals↗

PubMed1984

Distribution and characterization of cAMP-dependent protein kinase isoenzymes in bovine adrenal cells.

Subcellular localization and characterization of cAMP-kinase isoenzymes in fasciculata reticularis bovine adrenal cells has been investigated. Different subcellular fractions were purified on a Percoll gradient and characterized by marker enzymes. cAMP-kinase was located principally in cytosol and microsomes. In the low-speed particulate fractions cAMP-kinase was found associated mainly with plasma membrane but not with mitochondria. Characterization of isoenzyme patterns in subcellular fractions by conventional DEAE-cellulose chromatography and by anion-exchange HPLC gives essentially the same results. Isoenzyme I appears to be the main enzyme in cytosol whereas isoenzyme II predominates in solubilized microsome and plasma membrane enriched fraction. Photoaffinity labelling of chromatographic fractions demonstrated that HPLC separates both cAMP binding subunits. Photoaffinity labelling of the different subcellular fraction by 8-azido-[32P]cAMP confirmed the data obtained by anion-exchange chromatography. However, in microsomes this method revealed the presence of both isoenzymes and the preferential solubilization of isoenzyme II by Triton X-100. In summary, our results indicate a subcellular compartmentalization of cAMP-kinase in bovine adrenal cells with a preferential localization of isoenzyme I in cytosol and of isoenzyme II in membrane. However, the relation between the distribution and the role of each isoenzyme has so far not been documented.

Adrenal Glands↗

PubMed1991

Pain characterization in cancer patients and the analgetic response to epidural morphine.

In 48 patients with pain related to malignancy, a pain characterization was performed during oral opioid therapy. After an optimal epidural morphine regimen had been established, the alteration in pain relief was evaluated by means of a visual analogue scale. The CSF and plasma morphine concentrations at minimum steady state were then analysed in 28 patients and related to the degree of pain relief. The efficacy of the spinal treatment ranked in the following order: somatic greater than visceral greater than radiating = 0, but the difference was only significant between the somatic and radiating pain groups. There was a tendency for continuous pain to be better relieved than intermittent pain. No correlations were found between the CSF or plasma morphine concentrations and the degree of pain relief, suggesting that not all pain impulses are modulated in a dose-dependent manner by morphine at the spinal level. Pain characterization may be instrumental in providing an optimal spinal opioid analgesia in malignancy. Moreover, there is a need for better defined diagnostic criteria for clinical pain characterization.

Aged↗

PubMed1990

Autoradiographic characterization of putative excitatory amino acid transport sites.

Removal of excitatory amino acids from the extracellular space is now postulated to occur through at least two distinct transport systems that are distinguished by their ionic dependency. Thus, both sodium-dependent and chloride-dependent systems have been described in the mammalian central nervous system. In this report we attempt to characterize these sites by autoradiography, using D-[3H]aspartate and L-[3H]glutamate as ligands. Previous studies have shown that sequestration of radioligand into membrane vesicles can be a potential artifact when examining transport sites. We have found that sequestration can be alleviated by incubation of tissue sections in xylenes prior to incubation with radioligand. Using in vitro autoradiography we have characterized the two binding sites with respect to their distribution, kinetics and pharmacology. Both appeared to have a single, saturable binding site with Kds in the low micromolar range. Sodium-dependent D-aspartate binding predominated, having a Bmax that was five times greater than chloride-dependent L-glutamate binding in whole brain. The levels of binding to the two sites varied between brain regions. Sodium-dependent D-aspartate binding was highest in the cerebellar molecular layer greater than dentate gyrus molecular layer greater than entorhinal cortex. Chloride-dependent L-glutamate binding was highest in the outer layers of cerebral cortex greater than dentate gyrus molecular layer greater than entorhinal cortex greater than striatum. Pharmacological characterization of these sites also showed major differences. Sodium-dependent D-aspartate binding was most potently inhibited by L-aspartate greater than threo-beta-hydroxyaspartate greater than L-cysteine sulfinic acid greater than L-cysteic acid. Chloride-dependent glutamate binding was most potently inhibited by L-glutamate greater than L-alpha-amino adipic acid greater than quisqualate greater than L-serine-o-sulfate. The differences in distribution, ligand binding properties and pharmacology of these sites suggest that a significant variable in excitatory amino acid circuitry may include heterogeneity in transporters associated with excitatory pathways.

Amino Acids↗

PubMed1990

Isolation and characterization of an expressed hypervariable gene coding for a breast-cancer-associated antigen.

A human gene and cDNA coding for a breast-cancer-associated antigen (H23Ag) were isolated and characterized. The gene contains two exons and one intron. Part of the second exon is a tandem repeat array (TRA) consisting of multiple 60-bp G + C-rich units. We report here the characterization of unique sequences that are found in the H23Ag gene and cDNA, in addition to the 60-bp repeats. Analysis of the cDNA sequences revealed a putative ATG start codon preceded by two overlapping initiation consensus sequences (CCACC). The open reading frame determines an amino acid (aa) sequence consisting of three regions. The first region contains an initiating methionine and a highly hydrophobic putative signal peptide. This is followed by a variable number of highly conserved 20-aa repeat units (TRA). The last region, C-terminal to TRA, contains four potential N-linked glycosylation sites. The genomic nucleotide sequences demonstrate a putative promoter region that includes a 'TATA' box. A putative estrogen regulatory element is located 5' to the promoter region. The characterization of the gene and cDNA coding for the H23Ag presented here, may help to elucidate its possible function in human breast cancer.

Amino Acid Sequence↗

PubMed1994

Construction of human chromosome-3-specific radiation hybrids and characterization by Alu-PCR.

Using a modification of the procedure developed by Cox et al. [Genomics 4 (1989) 397-407], we isolated and characterized 60 radiation hybrids (RH) prepared by fusing an X-ray-irradiated Chinese hamster-human chromosome 3 (Chr 3) cell line (Q314-2) with a UrdA Chinese hamster mutant cell line. The RH were screened for human DNA content by PCR amplification using primers directed to the human Alu repeat sequences. Over 80% (50/60) were scored as positive for the retention of human DNA. Of them, 18 were characterized with Chr-3-specific single-copy DNA probes of known map location. These experiments demonstrated that the RH analyzed contained distinct subregions of human Chr 3. The RH that we have produced constitute a bank of cellular clones containing small segments of Chr 3. In the accompanying paper [Atchison et al., Gene 151 (1994) 325-328], we present the construction of rare-restriction-site linking libraries and the sequence tagged site characterization of in situ localized clones.

Animals↗

PubMed1992

Principles of identifying and characterizing neurotoxicity.

There is currently considerable interest in the neurotoxic effects of environmental pollutants. Some of this interest is due to epidemiological, clinical and laboratory studies showing that the nervous system is a target for many toxic substances. The interest is also due to a realization of how little is actually known about the neurotoxicity of most environmental pollutants. Laboratory research in neurotoxicology can be viewed as having two distinct approaches that focus on either the identification or the characterization of neurotoxic substances. Research on the identification of neurotoxicity deals mainly with the screening of chemicals for neurotoxicity. There has been a long-standing tradition of screening chemicals for neurotoxicity. There have, however, been several recent developments that are likely to improve our ability to identify neurotoxic substances, including more detailed assessments of a variety of behavioral and neurological functions. Research on the characterization of neurotoxicity deals mainly with efforts to discover the mechanism(s) of action of neurotoxic substances. This type of research has in some cases significantly advanced our knowledge of neurotoxic effects (e.g., acrylamide, n-hexane). Characterization-based research addresses many of the extrapolation issues of concern in toxicology (e.g., acute to chronic, high-dose to low-dose), and specifically attempts to interrelate the cellular, molecular and functional (neurophysiological, neurobehavioral) effects of toxic substances. These two research approaches represent critical elements of a tiered testing approach that could ultimately lead to more efficient testing protocols and a more comprehensive understanding of pollutant-induced neurotoxic risk in human populations.

Animals↗

PubMed1995

Mass spectrometric approaches to molecular characterization of protein-nucleic acid interactions.

The recent development of 'soft' ionization-desorption methods has lead to a breakthrough for the mass spectrometric analysis of biomacromolecules such as proteins and nucleic acids. In particular, the feasibility of electrospray-ionization mass spectrometry (ESI-MS) for the direct characterization of non-covalent supramolecular complexes is opening new analytical perspectives. Examples hitherto analyzed by ESI-MS include enzyme-substrate and -inhibitor complexes, homo- and heterodimers/trimers of leucine zipper polypeptides, and several other DNA- and RNA-binding proteins. Furthermore, the characterization of double-stranded and higher-order oligo- and polynucleotide complexes by negative-ion ESI has been demonstrated. Ions specific of non-covalent protein and oligonucleotide complexes can be selectively dissociated by changing the solution conditions and by increasing the desolvation potential. These results form the basis for the molecular characterization of protein-nucleotide interactions, thus complementing protein-chemical approaches, and other methods of structure determination.

Amino Acid Sequence↗

PubMed1981

Spectro-temporal characterization of auditory neurons: redundant or necessary.

For neurons in the auditory midbrain of the grass frog the use of a combined spectro-temporal characterization has been evaluated against the separate characterizations of frequency-sensitivity and temporal response properties. By factoring the joint density function of stimulus intensity, I (f, t), preceding a spike, into two marginal density functions I1(f) and I2(t) one may under the assumption of statistical independence reconstruct the joint density by multiplication: I1(f).I2(t). The reconstructed I(f, t) is compared to the original I(f, t) for 83 neurons: in 23% thereof the I(f, t) appeared to be vastly different from I(f, t). These units appeared to be located dominantly in the ventral parts of the auditory midbrain and had a latency exceeding 30 ms. On the basis of the action-potential wave forms the absence of non-separable I(f, t) in the incoming nerve fiber population is concluded. A spectro-temporal characterization of auditory neurons seems mandatory for investigations in and central from the auditory midbrain.

Action Potentials↗

PubMed1995

Characterization of biodegradable poly(D,L-lactide-co-glycolide) polymers and microspheres.

Characterization of biodegradable polymers used for controlled drug delivery is essential to ensure reproducibility of in vitro and in vivo performance. Selected characterization techniques established for poly(D,L-lactide-co-glycolide) (PLGA) copolymers included DSC to analyse thermal behavior, 13C-NMR to determine exact comonomer ratios and comonomer sequencing, cloud point titration to establish solubility, SEC to monitor molecular weight averages and polydispersity, SEM to observe surface morphology, BET gas adsorption to analyse surface area, tapped bulk density measurements to suggest internal pore structure and porosity and finally in vitro degradation to analyse degradation times and profiles. Comonomer ratios of 50:50 PLGAs were found to be closer to stated values for Boehringer Ingelheim polymers than for polymers from two other suppliers. Implementing such a characterization program for biodegradable polymers ensures the production of reproducible and reliable controlled drug delivery systems.

Adsorption↗

PubMed1995

Characterization of return cycle responses predictive of successful pacing-mediated termination of ventricular tachycardia.

OBJECTIVES: The purpose of this study was to characterize response patterns during overdrive pacing that predict successful termination of ventricular tachycardia. BACKGROUND: Overdrive pacing during ventricular tachycardia typically results in entrainment at slow pacing rates and in termination or acceleration at faster rates. The factors that determine the critical paced cycle length that results in tachycardia termination have not been extensively studied. METHODS: Ventricular tachycardias in 14 patients with coronary artery disease were studied with overdrive pacing at several cycle lengths. Return cycles were measured after each additional paced beat at each paced cycle length. The return cycle responses during pacing trials that resulted in tachycardia termination and those that resulted in entrainment were compared. RESULTS: Three return cycle responses were identified: flat, plateau and increasing. Twenty trials of overdrive pacing resulted in tachycardia termination; all were characterized by an increase in the return cycle with the delivery of each successive beat in the pacing drive until the tachycardia terminated (increasing response). Thirty-four pacing trials resulted in entrainment and not termination; these were characterized either by a constant return cycle (flat response) or an initial increase in return cycle followed by a longer, constant return cycle (plateau response) with the delivery of additional paced beats. The longest paced cycle length that resulted in tachycardia termination correlated with the relative refractory period of the circuit, defined as the tachycardia cycle length minus the fully excitable gap (r2 = 0.764, p = 0.0001). Tachycardia termination was not observed unless the paced cycle length was shorter than the relative refractory period of the circuit. CONCLUSIONS: The critical paced cycle length that causes termination of ventricular tachycardia depends on the relative refractory period of the circuit because this factor determines whether the nth + 1 beat of the pacing drive will encounter partially recovered tissue. These data provide insights into the mechanism of pacing-mediated tachycardia termination and entrainment and are applicable to the development of improved antitachycardia pacing algorithms.

Aged↗

PubMed1995

Increased echodensity of myocardial wall in the diabetic heart: an ultrasound tissue characterization study.

OBJECTIVES: We sought to characterize myocardial echodensity in asymptomatic patients with insulin-dependent diabetes and normal conventional two-dimensional echocardiographic findings to determine whether ultrasound tissue characterization can detect ultrastructural changes in myocardium, such as an increase in collagen content. BACKGROUND: Fibrosis alters the acoustic properties of the heart in animals and humans, and these changes are detectable by cardiac tissue characterization with ultrasound. Early changes detected in the diabetic heart include increased interstitial collagen deposition. METHODS: Using two-dimensional echocardiography, we evaluated 26 asymptomatic patients with insulin-dependent diabetes with normal regional and global rest function, and 17 age- and gender-matched control subjects. By selection, all diabetic patients were normotensive and had negative maximal exercise stress test results to avoid the confounding effects of hypertension and coronary artery disease. Using an echocardiographic instrument implemented at the Institute of Clinical Physiology, we performed an on-line radiofrequency analysis to obtain quantitative operator-independent measurements of the integrated back-scatter signal of the ventricular septum and posterior wall. The integrated values of the radiofrequency signal from the myocardial wall were normalized for those from the pericardial interface and were expressed as percentages (integrated backscatter index). RESULTS: Diabetic patients showed a significant increase in myocardial echodensity both in the septum ([mean +/- SD] 36.6 +/- 8.1 vs. 23.6 +/- 4.4, p < 0.0001) and posterior wall (21.2 +/- 5.3 vs. 18.4 +/- 3.7, p < 0.001). By individual patient analysis, 17 patients exceeded the 95% confidence limits for normal myocardial echocardiographic reflectivity found in normal subjects, and only 3 had a relatively abnormal transmitral Doppler filling pattern (E/A ratio), mainly consisting of an abnormally increased late peak flow velocity (65% vs. 11%, p < 0.001). The increased myocardial intensity was similar in patients with (n = 16) and without (n = 10) noncardiac complications, such as retinopathy or nephropathy (37.5 +/- 7.9% vs. 35.0 +/- 8.3%, p = 0.35). CONCLUSIONS: Abnormally increased myocardial echodensity, possibly related to collagen deposition, can be detected in asymptomatic diabetic patients with normal rest function. Theoretically, this finding might be considered a very early preclinical alteration potentially related to subsequent development of diabetic cardiomyopathy.

Adult↗

PubMed1992

The identification and characterization of KRAB-domain-containing zinc finger proteins.

The zinc finger motif is a highly conserved tandemly repeated sequence of 28-30 amino acids that was first identified in transcription factor TFIIIA from Xenopus laevis. Subsequently, similar motifs were found and characterized in many genes from mammalian genomes and the genomes of lower eukaryotes such as Drosophila and yeast, thereby defining a large superfamily of genes. Non-finger-coding modules conserved among members of subfamilies of zinc finger genes have been described in the murine genome (finger-associated boxes, or FAX domain) and the human genome (Krüppel-associated boxes, or KRAB domain). Here we report the identification and partial characterization of more members of the human KRAB-containing subfamily of genes. Based on Southern blot hybridization experiments, they also are zinc-finger-coding genes. All members share a highly homologous 42-amino-acid-long A element of the described KRAB domain. The conservation extends to the murine developmentally expressed zinc finger gene, mKr2. The homologous sequences, however, are part of the 5'-untranslated region. In all cases for which there is adequate information, the KRAB domain is found at the NH2-terminus of the respective protein. In one zinc-finger-encoding cDNA clone that we characterized further in this work, BRc1744 (ZNF45), the KRAB domain most probably constitutes the entire second exon of the gene. Based on the data, it is tempting to speculate that the FAX- and KRAB-containing zinc finger genes define subfamilies of genes with overlapping functions that participate in the regulation of common or similar developmental programs.

Amino Acid Sequence↗

PubMed1996

Slice cultures of the imprinting-relevant forebrain area medio-rostral neostriatum/hyperstriatum ventrale of the domestic chick: immunocytochemical characterization of neurons containing Ca(2+)-binding proteins.

The forebrain area medio-rostral neostriatum/hyperstriatum ventrale, a presumed analogue to the mammalian prefrontal cortex, displays a variety of synaptic changes during auditory filial imprinting. In order to study the underlying basic mechanisms of this synaptic plasticity we developed slice cultures of the medio-rostral neostriatum/hyperstriatum ventrale from newly hatched chicks. As a prerequisite for these investigations and in order to test the suitability of this system for future studies, we performed a thorough characterization of the in vitro tissue, of its cellular components and some of their biochemical features in comparison with in situ tissue. Since in situ the medio-rostral neostriatum/hyperstriatum ventrale has been previously shown to contain three distinct neuron populations characterized by the activity-regulated Ca(2+)-binding proteins parvalbumin, calbindin D28K and calretinin, we used these proteins as neuronal markers to study the survival and preservation of the morphological features of medio-rostral neostriatum/hyperstriatum ventrale neurons in vitro. In agreement with in vivo studies the three Ca(2+)-binding proteins are confined to neuronal cells and they are not colocalized, i.e. they appear to characterize three different neuron populations. The immunoreactive neurons in medio-rostral neostriatum/hyperstriatum ventrale cultures to a certain extent appear to form synaptic contacts with each other, shown by the double immuncytochemical experiments. One difference between cells in vivo and in vitro is their soma size, which is much larger in vitro than in vivo. This and our previous study on neuronal morphology demonstrates that morphologically and biochemically intact neurons can be maintained in medio-rostral neostriatum/hyperstriatum ventrale slice cultures, which may thus provide a suitable in vitro system for further studies of neuronal and synaptic plasticity in vitro.

Animals↗

PubMed1994

Characterization of biomembranes by spectral ellipsometry, surface plasmon resonance and interferometry with regard to biosensor application.

Phospholipid bilayers with transport proteins and antigen/antibody interfaces are considered to be suitable biosensor systems. The quality of such membranes or interfaces depends on the properties of the layers. Optical methods have proved to be an appropriate tool for characterizing those layers in situ and in a non-destructive manner. Two systems with potential for biosensor applications are characterized by some of these methods: phospholipid bilayer membranes spread from vesicle solution and protein-antigens both adsorbed on planar solid support. The results of spectral ellipsometry, surface plasmon resonance (SPR) and spectral interferometry are compared with respect to quality of characterization, expenditure of sample preparation and measurement, and time resolution. The phospholipid membranes adsorbed show a relatively low refractive index and a relatively high thickness. Bruggeman effective medium approximation is used to calculate the effective layer thickness. This result is compared to SPR measurements. A correlation between thickness and vesicle concentration may be detected. Further, the test protocol of an immunoassay is examined by spectral interferometry and SPR. Thicknesses determined are compared to results obtained by applying spectral ellipsometry. The data measured by ellipsometry are in agreement with the molecular dimensions of the immunoglobulins. Differences between details can be explained by physical considerations.

Biosensing Techniques↗

PubMed1989

6,4,4'-trimethylangelicin photoadduct formation in DNA: production and characterization of a specific monoclonal antibody.

The photochemical reactions of 6,4,4'-trimethylangelicin (TMA) with calf thymus DNA and an octanucleotide containing a single thymine have been characterized. HPLC analyses of enzymatically hydrolyzed TMA-DNA showed that isomeric forms of 4',5'-furan-side monoadducts were the major products. To develop monoclonal antibodies Balb c mice were immunized with the TMA-DNA complexed with methylated bovine serum albumin. The resultant antibodies were characterized by enzyme-linked immunosorbent assays (ELISA). The most sensitive antibody (7E3) has high specificity for TMA-DNA, very low cross-reactivity with DNA modified with either 4',5'-dimethylangelicin or 4'-methylangelicin and no cross-reactivity with non-modified DNA or with DNA modified with either 4'-aminomethyl-4,5',8-trimethylpsoralen or 8-methoxypsoralen. To characterize further this antibody, oligonucleotides containing specific TMA photoadducts were isolated from the photoreaction mixture by polyacrylamide gel electrophoresis and used as competitive inhibitors in the ELISA. Autoradiography of the gel showed an intense band corresponding to the 4',5'-monoadduct and two weaker unidentified bands. Antibody 7E3 reacted only with the 4',5'-monoadduct band as would be expected since this photoadduct was the principal photoadduct in the original antigen.

Antibodies, Monoclonal↗

PubMed1992

Characterization of recombinant antistasin secreted by Saccharomyces cerevisiae.

Secretion from recombinant yeast was used as a potential source of large quantities of the leech protein antistasin (ATS), a potent and highly specific inhibitor of the serine protease coagulation factor Xa. Mature recombinant ATS (r-ATS) is obtained after intracellular cleavage by the yscF protease of the mating factor alpha-1 pre-proleader from the fusion protein at the Lys-Arg sequence junction. Production levels are relatively low (ca. 1 mg/liter). Purification of the secreted product from a complex growth medium involved cell removal by microfiltration and diafiltration, cation-exchange capture and concentration on S-Sepharose Fast Flow, C-4 reverse-phase high-performance liquid chromatography (RP-HPLC), and HPLC cation-exchange chromatography step, and RP-HPLC concentration and desalting. The process was scaled up from the 16- to the 250-liter level with a corresponding increase in amount of r-ATS. From the 250-liter fermentation two major forms, r-ATS-I and r-ATS-II, distributed approximately 60:40, and a minor form, r-ATS-minor (ca. 1% of the purified r-ATS), were characterized. Limited N-terminal sequence analysis by Edman degradation indicated that r-ATS-I has the predicted mature N-terminus starting with Gln, that r-ATS-II is N-terminally blocked with pyroglutamate, and that r-ATS-minor is an incompletely processed form. RP-HPLC, hydrophilic-interaction HPLC, cation-exchange HPLC analysis, and electrophoresis results are consistent with the differences observed by sequencing. Preliminary in vitro characterization by intrinsic Ki determination for factor Xa inhibition indicated that the yeast r-ATS forms are indistinguishable from each other as well as from r-ATS expressed by the insect baculovirus host-vector system. Nevertheless, r-ATS-I and r-ATS-II appear less potent than insect-derived r-ATS in the activated partial thromboplastin time clotting assay. Further characterization indicated that C-terminal cleavage at Pro-116 had occurred in r-ATS-I and r-ATS-II as well as oxidation of methionine residues to methionine sulfoxide. The possible role of the C-terminus in inhibition of the prothrombinase complex is discussed.

Amino Acid Sequence↗