Tryptophan depletion and depressive vulnerability.

BACKGROUND: Rapid and transient depletion of tryptophan (TRP) causes a brief depressive relapse in most patients successfully treated with and taking selective serotonin reuptake inhibitors, but little change in drug-free, symptomatic depressed patients. This study investigates the effects of TRP depletion in drug-free subjects in clinical remission from a prior major depressive episode (MDE). METHODS: Twelve subjects with a prior MDE, currently in clinical remission and drug-free for at least 3 months (patients), and 12 healthy subjects without personal or family history of Axis I disorder (controls), received TRP depletion. The study was conducted in a double-blind, controlled [full (102-g) and quarter-strength (25 g) 15-amino acid drinks], crossover fashion. Behavioral ratings and plasma TRP levels were obtained prior to, during, and after testing. RESULTS: All subjects experienced significant depletion of plasma TRP on both test-drinks, showing a significant dose-response relation. Healthy control subjects had minimal mood changes, but patients had a depressive response of greater magnitude. CONCLUSIONS: In the context of prior TRP depletion studies with antidepressant-treated, and drug-free symptomatic depressed patients, these results suggest that depression may be caused not by an abnormality of 5-HT function, but by dysfunction of other systems or brain regions modulated by 5-HT.

Variations on the staining method in quantitative indirect immunofluorescence assays for Bacillus spores, and the use of fluorescein--protein A.

Quantitative indirect immunofluorescence assays for B. anthracis and B. cereus spores fixed on multispot microscope slides have been performed using a microfluorometer to measure the fluorescence of individual bacteria. A study has been made of variations of the indirect assay sequence, in which the washing operation between application of 1st and 2nd antibody types was omitted. In one modification the addition of the indirect antibody was deferred, and in the second the direct and indirect reagents were added simultaneously to the spores at the start of the incubation period. This simultaneous addition method shows promise for wide application in diagnostic serology. Evidence is presented that fluorescein-protein A (F-PA) can be successfully substituted for fluorescein/sheep anti-rabbit antibody (F-SAR) in the indirect spore assay, and that it is far more active than the F-SAR on a weight basis. About twice as many F-PA molecules as F-SAR molecules are bound to the spore at saturation.

Comparison of immunoradiometric assays of Bacillus anthracis spores immobilised on multispot slides and on microtitre plates.

Indirect immunoradiometric assays (IRMA) for Bacillus anthracis spores are described in which the spores were heat fixed either on multispot microscope slides or on polyvinyl or polystyrene microtitre plates. Assays on plastic plates sometimes suffered from poor intra-experiment reproducibility. Signals were higher in assays in flat-bottomed microtitre wells than in assays in slides, but assay noise, due to non-specific absorption of antibody, was higher also, giving an overall disadvantage in signal-to-noise ratio. Elution of antibody with acid gave up to a doubling in signal-to-noise ratio. When small volumes of antibody reagents were used in assays in round-bottomed plastic wells, signal and noise characteristics were similar to those of the slide assay. While the assays on plastic wells tended to be less sensitive in terms of anti-spore antibody concentration or the number of spores detectable, the larger volume of antigen suspension that could be used in plastic wells gave them an advantage in terms of the minimum antigen concentration detectable.

Serum stimulation and repression of flow immunofluorescence staining of bacteria.

A flow cytometer was used to measure the fluorescence intensity of Bacillus anthracis spores, B. subtilis spores and Escherichia coli stained in suspension with specific rabbit fluorescein-conjugated antibody. The effect of normal sera and a number of other additives on the binding of conjugate to the surface of the homologous bacteria was assessed by measuring the median fluorescence intensity of the bacterial population in the reaction mixture. Non-ionic detergent depressed binding of one conjugate (anti-E. coli) by up to 22%. Bovine serum albumin, gelatin, foetal calf serum and normal rabbit serum did not affect the median fluorescence value for these 3 bacterial species by more than 14%. Normal serum from 5 goats reduced the specific staining of B. anthracis by up to two-thirds. Anti-B. anthracis antibodies were detected in goat serum by indirect immunofluorescence microscopy, and it is inferred that these goat antibodies were in competition with fluorescein conjugate for the bacterial antigens. Normal goat and sheep serum stimulated the specific staining of B. subtilis and E. coli measured by the cytometer; in the case of goat serum previous heating of the serum to 56 degrees C resulted in repression of staining of E. coli. Since anti-E. coli antibody was detected in this normal sera by indirect immunofluorescence assays, it is proposed that repression was caused by anti-bacterial antibodies and stimulation by a separate factor, heat-labile in the case of goat serum. The stimulatory factor was also apparently inactivated by increasing the NaCl concentration, suggesting that stimulation depends heavily on charge interactions. Preliminary evidence is presented that the stimulatory factor may be anti-antibody, possibly of the IgA or IgG class.

Evaluation of a microfluorometer in immunofluorescence assays of individual spores of Bacillus anthracis and Bacillus cereus.

A microfluorometer was constructed by modifying a standard fluorescent microscope with a fibre optic eyepiece and a simple photometric system. It was evaluated in direct immunofluorescence assays of Bacillus anthracis and Bacillus cereus spores immobilised on multispot microscope slides. From measurements of stable fluorescent crystals comparable in size to the spores, it was inferred that the fluorescence intensity of a stained bacterium could be measured with good precision. Fluctuation of a exciting light from a mercury vapour lamp did not contribute significantly to the distribution of fluorescence measurements obtained when samples of 20 spores were assessed. Attempts to correlate spore size with fluorescence intensity suggest that spore fluorescence does not increase in a 1 : 1 ratio with surface area; it is therefore possible that the density of antigenic sites on the surface decreases with increasing spore size. It is concluded that differences in the observed fluorescence of individual spores truly reflect differences in fluorescent antibody binding, but the relative contribution of antigenic variability and of artefacts of the staining procedure remain unknown.

Limitations of flow cytometry for the specific detection of bacteria in mixed populations.

Flow immunofluorescence (FIF) techniques were established for the specific detection of the bacteria Escherichia coli, Legionella pneumophila and Bacillus anthracis spores after staining with fluorescein-conjugated antibacterial antibody. For each bacterial type, a comparison was made of gating on narrow forward angle (NFA) light scatter and on the red fluorescence (Red Flu) signal available from staining with the nucleic acid dye propidium iodide. No universal gating method was found, since Bacillus spores did not take up propidium iodide and only a part of the Legionella population gave detectable NFA scatter signals. The efficiency of detecting bacteria stained with antibody remained constant with differing concentrations of the specific bacterium, and the estimate of the count for specific bacteria expressed as a fraction of the total cytometer count fell sharply with bacterial concentration. This effect was apparently due to cytometer noise inherent in the high sensitivity of detection needed for particles as small as these bacteria. The noise did not originate in the photomultipliers and was evidently the result either of light scatter from sub-micron particles in the sheath fluid or scatter from optical components. Part of the noise could be removed by selective gating, but there remained a noise component overlapping with the NFA scatter and Red Flu signals from the heterologous bacteria, i.e., those not stained with specific antibody. In consequence, at the low bacterial concentrations used no meaningful cytometer count could be obtained for the excess of the unstained bacteria and the proportion of specific bacteria in the mixed population could not, therefore, be calculated.

Direct and indirect immunofluorescence analysis of bacterial populations by flow cytometry.

Bacillus anthracis spores and Escherichia coli were stained with fluorescein-conjugated antibody using direct and indirect methods, then analyzed by means of a commercial flow cytometer. To reduce the cytometer's fluorescence component resulting from unreacted conjugate, reaction mixtures were either diluted or were centrifuged through a sucrose solution using a moving zone technique. Evidence is produced that the fluorescence statistics for centrifuged samples closely represent the fluorescence distribution of stained single bacteria in the reaction mixture at the end of incubation; in particular, centrifugation did not cause aggregation of bacteria. Centrifugation is proposed as more effective than mere dilution for use with a wide range of bacterial concentrations, and the moving zone technique is to be preferred to conventional centrifugation in which bacteria tend to aggregate in the pellet. In indirect assays, it was shown that the washing step after reaction with antibacterial antibody may be omitted. The performance of direct and indirect staining methods was compared, including the use of either Staphylococcus aureus protein A or polyclonal sheep anti-rabbit antibody as the indirect reagent. When the bacterial concentration in reaction mixtures was increased the median fluorescence intensity fell, indicating that specific antibody had become limiting at low concentrations of the polyclonal antibody preparations. The implications of this for the design of flow cytometry assays of bacteria are discussed.

The choice of methods for immunoglobulin IgG purification: yield and purity of antibody activity.

Six methods for the purification of immunoglobulin G (IgG) from serum were compared, using rabbit antiserum to Bacillus anthracis spores as a model. Antibody activity was monitored by a solid-phase immunoradiometric assay (IRMA). Salt precipitation/ion exchange chromatography and ethanol precipitation both resulted in IgG of high purity but there was considerable inactivation of antibody. Salt precipitation/affinity chromatography gave poor yields of antibody. PEG precipitation and gel filtration of Sephacryl S-300 gave moderate yields and purity of IgG, with little evidence of antibody inactivation. Salt precipitation was marginally more destructive than the last 2 methods, but is recommended for routine use on grounds of its simplicity. Should IgG prepared by salt precipitation prove inadequate for particular applications, gel filtration is recommended since it allows the balance of yield and purity to be altered at will.

Synthesis and pharmacology of some 2-aminotetralins. Dopamine receptor agonists.

A series of 2-amino-1,2,3,4-tetrahydronaphthalene compounds bearing substituents on the nitrogen and in the aromatic ring was synthesized from beta-tetralone intermediates. Compounds were screened in vivo for dopaminergic activity using tests in which apomorphine was especially active. It was found that apparent dopaminergic activity is inherent in 2-dialkylaminotetralins, the dipropylamine substitution being the most consistently productive amine group studies. Activity was greatly enhanced by proper substitution in the aromatic ring. The 5,6-dihydroxy group was the best potentiating group found. These data support the idea that the extended conformation for the phenylethylamine moiety of ampmorphine and dopamine is favorable for dopaminergic agonist activity. They also suggest that an unetherified catechol group may not be essential for such activity.

2,4-Diamino-5-benzylpyrimidines and analogues as antibacterial agents. 5. 3',5'-Dimethoxy-4'-substituted-benzyl analogues of trimethoprim.

Forty trimethoprim analogues in which the para substituent in the benzene ring was varied were prepared for antibacterial evaluation. All were very potent inhibitors of Escherichia coli dihydrofolate reductase. The similarity of their inhibitory activities strongly suggested that the side chains beyond the first two atoms were not in contact with the enzyme. However, among 38 ether derivatives which varied widely in their bulk and lipophilicity, very few approached trimethoprim in their broad-spectrum in vitro antibacterial activity. The 4'-methyl and 4'-ethyl analogues and the allyloxy and gamma-chloropropoxy ethers had activities fairly close to that of trimethoprim. The two ethers were chosen for further evaluation in vivo. Neither compound quite matched trimethoprim in efficacy in mice, and their half-lives, as well as that of the beta-methoxyethoxy analogue, were found to be shorter in dogs.

Reducing nursing students' anxiety level and increasing retention of materials.

The purpose of this descriptive study is to examine the effects active learning, collaboration and modified group testing have on reducing students' anxiety and increasing learning and retention of material. Subjects consist of 34 associate degree nursing students enrolled in the Advanced Adult Health nursing class at North Georgia College. Most of the students are married, have children and work part time. A self-reporting questionnaire suggests a reduction of the students' anxiety during the quarter. The attitudinal questionnaire reveals an atmosphere of collaboration among peers. Data evaluating learning and retention of material were analyzed using the parametric (T-test) and nonparametric (Wiley Rank Sum test) methods. Examination of the Null Hypotheses I and II suggests there were increased learning and retention of material as evidenced by higher grades on the comprehensive final examination than on the quizzes given after presentation of content. Principles of andragogy as defined by Knowles (1980) and cooperation with peers as described by Johnson, Johnson, Holabec, and Roy (1984), Johnson, Johnson, and Maruyama (1983), and Johnson and Johnson (1975) form the theoretical foundation.

Dual-parameter scatter-flow immunofluorescence analysis of Bacillus spores.

Using a commercial flow cytometer (Cyto-fluorograf), narrow-forward-angle (NFA) light-scatter signals were detected for spore preparations of Bacillus anthracis Vollum, B. anthracis Sterne, B. cereus NCTC 8035, and B. subtilis var niger. In the flow immunofluorescence (FIF) analysis of spores stained with fluorescein-conjugated hyperimmune antibody to B. anthracis Vollum spores, fluorescence histograms could be acquired by selecting on NFA scatter. Fluorescence data selected on ninety degree scatter were rather noisier. Fluorescence analysis by dual parameter NFA scatter-FIF techniques was shown to have several advantages over the subtraction FIF method reported earlier. The implication from FIF analysis of spore suspensions and corresponding cell-free supernatants that the peak in the fluorescence histogram was caused by signals from fluorescing spores, was confirmed by use of the cell sorter and subsequent microscopy of the sorted samples. Although a proportion of spore aggregates was present in samples sorted from the right-hand tail of the fluorescence histogram, it was demonstrated that the majority of the observed distribution of fluorescence was not due to the formation of aggregates but was rather an expression of variation in the degree of staining of individual spores.

Comparison of direct and indirect immunoradiometric assays (IRMA) for Bacillus anthracis spores immobilised on multispot microscope slides.

A solid phase immunoradiometric assay (IRMA) is described in which Bacillus anthracis spores were heat fixed to the wells of glass multispot microscope slides. Assays for spores of B. anthracis Vollum and Sterne strains with 3H labels were evaluated in the direct and indirect versions. Neither single nor signal-to-noise characteristics of indirect assays were greatly improved by the use of immunopurified antibody (IPAB) or IgG anti-bacterial reagents rather than antiserum. However, the specificity of the direct and indirect assays for B. anthracis strains and B. cereus NCTC 8035 was altered by immunopurification of the anti-bacterial reagent. Although the signal-to-noise ratio was sometimes higher in indirect than in direct assays, signal values were usually no better. Evidence was produced that the overall ratio of the indirect : direct antibody molecules bound by preparations of B. anthracis spores rarely exceeded two but the antibody-molecular ratio for antigens on extracellular material in spore preparations was much higher than the ratio for antigens on the spores themselves.

Quantitative immunofluorescence studies of the serology of Bacillus anthracis spores.

A fluorescein-conjugated antibody against formalin-inactivated spores of Bacillus anthracis Vollum reacted only weakly with a variety of Bacillus species in microfluorometric immunofluorescence assays. A conjugated antibody against spores of B. anthracis Sterne showed little affinity for spores of several B. anthracis isolates including B. anthracis Vollum, indicating that more than one anthrax spore serotype exists.

Immunofluorescence analysis of bacillus spores and vegetative cells by flow cytometry.

A commercially available flow cytometer (Cytofluorograf) was used for the immunofluorescence (IF) analysis of spores of Bacillus anthracis, Bacillus cereus, and Bacillus subtilis, using fluorescein-labelled antispore conjugates. The cytometer was modified to allow analysis of known numbers of bacteria. In attempting to identify the region of the cytometer fluorescence histogram associated with the presence of stained spores, evidence was produced for signal components due to antibody bound to extracellular antigens. Under some reaction conditions these components were large enough partially or completely to obscure the fluorescence distribution imputed to the spores. The results support the hypothesis that the fluorescence histogram for a bacterial suspension can be modified by subtracting the histogram of the cell-free centrifugation supernatant to provide a fluorescence distribution more representative of the bacteria themselves. Spore and vegetative forms of B. anthracis could be differentiated in the flow IF assay by comparing the peak and area (integral) values of the photomultiplier output. The 90 degrees scatter histograms of the stained spores and their cell-free supernatants were so alike in shape that it was not possible to ascribe a unique peak to the spores themselves. Overall, these results confirm the considerable potential of flow cytometry for the rapid and quantitative IF assay of bacterial populations.