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Immunofluorescence (IFA) and immunoperoxidase (IPA) assays were developed for the titration of infectious hepatitis A virus. Both of these methods were found to be simple, rapid and quantitatively reproducible. The immunoperoxidase technique could be the method of choice for the assay of HAV in cell culture.
An assay is described for titration of human immunodeficiency virus type 1 (HIV-1) and for quantitative analysis of virus expression in vitro. The assay utilizes a liquid RNA-RNA hybridization method coupled with reversible target capture (RTC) on oligo(dT) derivatized magnetic particles. The assay provides a rapid, specific, and sensitive method for quantitation of HIV-1 RNA molecules present either in cells or in viral particles from cell-free culture media. Chronically infected monocytoid U1.1 cells were found to carry 52 pg HIV-1 RNA per 200,000 cells (160 HIV-1 RNA molecules per cell). In contrast, acutely infected CEM and H9 cells carried 3010 and 4370 pg HIV-1 RNA per 200,000 cells (9040 and 13,110 HIV-1 RNA molecules per cell, respectively). No hybridization was observed with uninfected cells or cells infected with HIV-2, HTLV-I, HTLV-II, or EBV. Use of liquid HIV-1 RNA hybridization in association with HIV-1 protein detection methods permits more complete characterization of HIV-1 expression in host cells than either method alone, and also provides a method for standardizing preparations of virus.
The feline immunodeficiency virus (FIV) readily produced syncytia in Crandell feline kidney (CrFK) cells adapted to a medium containing 0.5% fetal calf serum, a variety of growth factors and other supplements. This finding has been exploited to develop simple and sensitive virus titration and neutralization assays. High titre neutralizing antibodies were detected in cats infected naturally and experimentally with FIV, but not in uninfected animals.
A colorimetric end-point dilution assay was developed for the titration of rotavirus-containing samples that uses commercially available tetrazolium dyes as an indicator of virus infection. This assay offers several advantages over both plaque assays and traditional end-point dilution methods. The latter assays require manual counting of plaques or the scoring of wells for the presence of virus based on observed cytopathic effects. The colorimetric end-point dilution assay enables the scoring of wells based upon absorbance readings alone, thereby eliminating time-consuming and subjective manual screenings. This method also has the potential for automating the analysis of large numbers of samples. Virus titers of human-bovine rotavirus reassortants obtained using this method are comparable to those determined by plaque assay. The scoring of wells based on absorbance readings was also found to agree with manual scoring of cytopathic effects and with the production of viral antigen.
A sensitive and reproducible tissue culture biossay method was developed based on indirect immunofluorescence to titrate virus suspensions of the Junonia coenia densovirus (JcDNV) and to quantify transfections by its cloned genomic DNA. Four lepidopteran cell lines, the SPC-SL 52 from Spodoptera littoralis, the SPC-PL 40 and the SPC-PL 65 cells derived from Spodoptera litura ovaries and hemocytes, respectively, and the SC-LD 135 from Lymantria dispar were compared for their efficiency to support viral replication. The viral titres expressed as TCID50/ml averaged 10(5) for SPC-SL 52, SPC-PL 40 and SC-LD 135 cells, but were above 10(7) for SPC-PL 65 cells. Even with this most sensitive cell line, the rate of infected cells did not exceed 75% and decreased progressively by serial subcultures. Two transfection protocols were used to compare the sensitivity of the same four cell lines to a recombinant plasmid encompassing an infectious sequence of JcDNV genome. SPC-SL 52 cells were found to be the most sensitive, and the lipofection method resulted in about a 5-fold increase compared to the calcium phosphate precipitation protocol. The rescued virions proved to be infectious and the restriction profiles of their DNA were identical to that of wild type virions.
A titration method for determination of trypsin-like serine proteinase concentration has been developed by using ZArgONp and ZLysONp, two specific chromogenic amino-acid derivatives which show the characteristics of optimal active-site titrants. Active proteinase concentration has been estimated from the effect of titrant concentration on the amplitude, at time zero, of the time-course for the instantaneous release of p-nitrophenol, preceding the steady-state reaction (burst phase).
Cellular retinoic acid binding protein I (CRABP-I) and cellular retinoic acid binding protein II (CRABP-II) are small, cytoplasmic proteins which bind all-trans-retinoic acid with high affinity. Both of these proteins belong to a family of intracellular proteins which bind amphiphilic lipids, including fatty acids, bile salts, and retinoids. Because CRABP-I and -II exhibit different tissue distributions and differential transcriptional regulation, they are proposed to serve different functions. The binding properties of mouse CRABP-I and -II purified from Escherichia coli were examined to further understand their role in intracellular retinoic acid processing. Fluorescence titrations were performed using nanomolar protein concentrations, near the obtained dissociation constants, and analyzed by direct mathematical fitting to raw data, in order to extend the range and accuracy of binding constant determination. The apparent dissociation constants, K'd, of mouse CRABP-I and CRABP-II binding all-trans-retinoic acid were determined to be 0.4 +/- 0.3 nM and 2 +/- 1 nM respectively, stronger binding than previously reported. The K'd of mCRABP-I and mCRABP-II complexing with acitretin, a pharmacologically active synthetic retinoid used in the treatment of psoriasis, was 3 +/- 1 nM and 15 +/- 11 nM. Both CRABPs bound 9-cis-retinoic acid with a K'd of roughly 200 nM, and neither exhibited significant binding of 13-cis-retinoic acid.
Substance P (SP) was injected intrathecally (10 or 100 micrograms) into cats previously implanted with nerve-stimulating electrodes and the effect on shock titration thresholds was evaluated. Elevated shock thresholds were observed in 5 of 8 cats following the 100 micrograms dose of SP. In addition, one cat exhibited a decreased threshold and two cats showed a triphasic (increase, decrease, increase) response. Overt behavioral effects of intrathecal SP were mild, but suggested that injection of the drug was aversive.
By means of the macrophage electrophoretic mobility technique a striking digestive system cancer-associated human lymphocyte response to CEA has been found during a large-scale study including tests in 499 individuals. The question to be answered by this study was whether this response is really CEA-specific. Titration experiments with 3 different CEA preparations in lymphocytes from 5 colorectal cancer patients showed that the threshold dose of CEA necessary to induce lymphocyte responses amounts to 50-100 ng CEA per ml and 10(6) lymphocytes, regardless of the CEA origin and its state of purity. The CEA specificity of the responses was proved by neutralization experiments with 3 CEA-specific monoclonal antibodies. When allowed to react with CEA before lymphocyte incubation, the MABs prevented CEA from inducing lymphocyte responses. Appropriate murine control myeloma protein did not influence these responses. The reactivity of these lymphocyte samples to a teratocarcinoma extract could not be prevented by treating this material with CEA-specific MABs before incubation. Preliminary attempts to enrich the lymphokine(s) released after CEA stimulation resulted in recovery of the activity within 2 arbitrarily cut Sephadex G-100 fractions comprising the mol. wt range of 3000-47,000.
An analysis is given of the theoretical approach to the quantitative description of proton dissociation curves for weak polyacids. The basic model of the counterion condensation theory has been used, with the modification reported in the preceding study (S. Paoletti, A. Cesàro, C. Arce Samper and J.C. Benegas, Biophys. Chem. 34 (1989) 301). In this paper we demonstrate the effect of relaxing the hypothesis of a rigid conformation on the polyelectrolytic properties of weak polyacids. As an application of the present approach, a description is given of the titration curves for two weak polyacids, poly(DL-glutamic acid) (PDLGA) and poly(L-aspartic acid) (PLAA).
The cooperative conformational transition of poly(L-glutamic acid) induced by pH is monitored by the titration curves from literature. The polyelectrolytic approach described in the preceding article (A. Cesàro, S. Paoletti and J.C. Benegas, Biophys. Chem. 39 (1991) 1) is used to fit the experimental curves under various conditions of ionic strength and temperature, with the sole assumption that each polymeric state is characterized by a proper conformational flexibility. The helix-coil transition of the system becomes molecularly defined by the balance between the non-ionic conformational energy and the repulsive electrostatic energy of the two forms. Implications of the results of the theoretical model on the energetics of the cooperative order-disorder transition are discussed.
The present work outlines various kinetic parameters of the interaction between bGH (bovine growth hormone) and a receptor located on the membranes of rabbit liver. A dissociation procedure, which strips the hormone off its receptor has been worked out, by varying MgCl2 concentrations and times of contact; exposure to 4 M MgCl2 for 15--30 min was found optimal for dissociation, without denaturing the receptor, as shown by the possibility of rebinding the hormone to the desaturated membranes. This method has been applied to titrate growth hormone receptors in rabbit liver, during pregnancy and lactation. The first half of pregnancy is characterized by a gradual increase of receptor levels and low saturation by endogenous hormone; conversely at parturition, there occurs a striking increase in receptors, which, however, may be demonstrated only after desaturation. During the lactational period, the receptors reamin in part saturated. The results suggest that growth hormone levels increase at parturition and during lactation in the rabbit and hence may play an important role during lactation. The factors which modulate receptor levels at the same period remain unknown.
The thermodynamics associated with the Mn2- -ATP, Mn-ADP- and Mn-AMP complex formation reactions determined from K potentiometric measurements at I = 0.2 are reported for the temperature range 1--45 degrees C. The K values increase with the length of the phosphate chain and with temperature. The limits and the best conditions for use of the "pH stat" titration method are discussed. Comparison with the results obtained by potentiometric and calorimetric methods in the case of Mg-nucleotide complexes is made.
Cigarette smokers were presented with puffs from either high (2.5 mg), medium (1.5 mg) or low (.5 mg) nicotine cigarettes in order to determine their ability to discriminate nicotine delivery in tobacco smoke. Puffs were presented in random order during each of two conditions and tar content was controlled by using research cigarettes and a smoke mixing device that varied only nicotine. The first condition allowed olfactory stimuli to be used in discrimination, while the second condition blocked olfaction by occluding subjects' nostrils. In both conditions, subjects discriminated between the nicotine content of different puffs, with higher nicotine puffs rated as significantly stronger (by roughly 50%). Subjective desirability ratings did not vary with nicotine delivery. The implications of the magnitude of change in subjects' ratings for theories of nicotine titration are discussed.
A method of utilizing vitamin C in amounts just short of the doses which produce diarrhea is described (TITRATING TO BOWEL TOLERANCE). The amount of oral ascorbic acid tolerated by a patient without producing diarrhea increase somewhat proportionately to the stress or toxicity of his disease. Bowel tolerance doses of ascorbic acid ameliorate the acute symptoms of many diseases. Lesser doses often have little effect on acute symptoms but assist the body in handling the stress of disease and may reduce the morbidity of the disease. However, if doses of ascorbate are not provided to satisfy this potential draw on the nutrient, first local tissues involved in the disease, then the blood, and then the body in general becomes deplete of ascorbate (ANASCORBEMIA and ACUTE INDUCED SCURVY). The patient is thereby put at risk for complications of metabolic processes known to be dependent upon ascorbate.
The wheal is used for assessment of the Provocation-Neutralisation Response in Clinical Titration. It is the third, slowest, and most indirect measure of the Antigen-Antibody Reaction as demonstrated in the Triple Response. The flare is produced by an axone reflex. It appears rapidly as a direct neurological response to antigen challenge. It is associated with local changes in Direct Current Potential. These are transmitted distally through the d.c. Perineural Analogue System. Neutralisation values appear in a harmonic series with increasing dilutions of antigen. Passive Allergenic Neutralisation does not require active antigen administration into the patient's tissues. Passive Neutralisation can be performed in the absence of allergenic material. It is concluded that the wheal need not be associated with the Provocation-Neutralisation technique, and that a neuropsychological basis for the Provocation-Neutralisation response should be explored.
The OmpR protein of Escherichia coli is a positive regulator specific for the ompF and ompC genes. The function of OmpR is modulated through phosphotransfer signaling mediated by the kinase, EnvZ. We previously demonstrated that OmpR contains two functional domains, which are physically separable; one is responsible for the interaction with EnvZ, whereas the other participates in interactions with cognate promoter DNAs. In this study, these domains of OmpR were overproduced in wild-type cells harboring the endogenous intact ompR gene on their chromosome. It was found that when the N-terminal domain of OmpR, which contains the phosphorylation site, was overproduced, expression of the ompF and ompC genes was markedly inhibited, irrespective of the osmolarity of the growth medium. Based on our current model for the molecular mechanism underlying signal transduction through Envz-OmpR phosphotransfer (T. Mizuno and S. Mizushima, Mol. Microbiol. 4, (1990), 1077-1082), we provide evidence that this phenomenon is best interpreted by the concept of 'signal titration' in the phosphotransfer signaling pathway.
An end point dilution microtitration assay is described that can be used for the titration of both cytopathic and non-cytopathic isolates of bovine virus diarrhoea-mucosal disease virus. Indirect immunofluorescence is used to detect infected MDBK cells in the wells of Terasaki plates. The virus titre is derived from the number of uninfected wells, using the Poisson distribution. The assay is simple, fast and economical. Titres of cytopathic virus determined by the microtitration assay and standard plaque assay are equivalent.