Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells

Mild sonication was used to obtain single cell suspensions of Paracoccidioides brasiliensis. These cells were intact by microscopic criteria. Direct cell counts in a given inoculum and colony formation on various media were used to determine plating efficiency. Sonicated and nonsonicated cell suspensions were used to study plating efficiency and to estimate viability by means of vital dyes. Methylene blue, Erythrosin B, and Janus green were unreliable when used with P. brasiliensis, but vital dyes were accurate when tested with Candida albicans.Acridine orange gave more meaningful results of viability. Estimates of viability, however, changed significantly as a result of relatively minor alterations in the composition of the suspending medium.In initial experiments, the plating efficiency of P. brasiliensis was dismally low. It descended abruptly with increasing dilution of inoculum. Efficiency was much improved if horse serum was added to brain heart infusion plates or if glucose glycine yeast extract (GGY) plates were incubated at room temperature and mycelial colonies were counted. With the technique we report, current plating efficiency of sonicated suspensions is of the order of 25 %. Our results and procedures have an important bearing upon those studies concerned with in vitro killing of P. brasiliensis in suspensions or with isolating this fungus from clinical or environmental specimens.     

Preparation of radioactive iodinated cholylhistamine for use in the radioimmunoassay of cholic acid

A major handicap in the development of simple and accurate radioimmunoassay procedures for bile acids has been the lack of a radioactive standard of high specific activity. To provide such a compound, we first synthesized cholylhistamine using the carbodiimide reaction. The hypothesized structure was confirmed by elemental analysis, thin-layer chromatography, infrared and mass spectral analysis. The cholylhistamine was then iodinated with 125I, using the choloramine-T method. The 125I-cholylhistamine was bound by antisera raised against a cholic acid-bovine serum albumin conjugate. This procedure should prove useful in preparing radioactive conjugates for all of the bile acids.     

Identification of mouse chromosomes required for murine leukemia virus replication.

The replication patterns of five ecotropic and two amphotropic strains of murine leukemia virus (MuLV) were studied by infecting 41 Chinese hamster x mounse hybrid primary clones segregating mouse (Mus musculus) chromosomes. Ecotropic and amphotropic strains replicated in mouse and some hybrid cells, but not in hamster cells, indicating that replication of exogenous virus requires dominantly expressed mouse cellular genes. The patterns of replication of the five ecotropic strains in hybrid clones were similar; the patterns of replication of the two amphotropic strains were also similar. When compared to each other, however, the replication patterns of ecotropic and amphotropic viruses were dissimilar, indicating that these two classes of MuLV require different mouse chromosomes for replication. Chromosome and isozyme analyses assigned a gene, Rec-1 (replication of ecotropic virus), to mouse chromosome 5 that is necessary and may be sufficient for ecotropic virus replication. Because of preferential retention of mouse chromosomes 15 and 17 in the hybrid clones, however, the possibility that these chromosomes carry genes that are necessary but not sufficient for ecotropic virus replication cannot be excluded. Similarly, the data indicate that mouse chromosome 8 (or possibly 19) carried a gene we have designated Ram-1 (replication of amphotropic virus) which is necessary and may be sufficient for amphotropic virus replication. Because chromosomes 8 and 19 tended to segregate together and two of the three clones excluding 19 have chromosome reaggrangements, we cannot exclude 19 as being independent of amphotropic virus replication. In addition, because of preferential retention, chromosomes 7, 12, 15, 16 and 17 cannot be excluded as being necessary but not sufficient. Hybrid cell genetic studies confirm the assignment of the Fv-1 locus to chromosome 4 previously made by sexual genetics. In addition, our results demonstrate that hybrid cells which have segregated mouse chromosome 4 but have retained 5 become permissive for replication of both N and B tropic strains of MuLV.     

Cellular immunity to Coccidioides immitis: In vitro lymphocyte response to spherules,arthrospores, and endospores

In vitro stimulation of incorporation of tritiated thymidine by human peripheral lymphocytes in response to two soluble antigens and three different intact but nonviable fungal forms of Coccidioides immitis was studied. Lymphocytes were obtained from three groups of subjects: healthy skin test positive, healthy skin test negative, and disseminated disease. Dose-response relationships to the intact forms (endospores, arthrospores, and spherules) were determined. Responses of lymphocytes from healthy skin test-positive subjects and subjects with disseminated disease were similar. Ranking of antigens by “potency” gave the following results: endospores = spherulin > mycelial filtrate > arthrospores = spherules. Endospores were the most potent of the intact forms in 10 of 11 subjects. The clear superiority of endospores over spherules is not due to differences in the total particle surface area available for presentation to the leukocytes. All antigens tested except spherules could discriminate between skin test-positive and skin test-negative subjects in this in vitro system. A T-cell-enriched, B-cell- and mono-cyte-depleted cell population demonstrated an active response to spherulin and to endospores. The variance of these finding with animal studies demonstrating spherules to be immunogenically superior when compared to endospores is discussed. This may have importance in future studies in humans of vaccines to C. immitis.     

The treatment of the hepatorenal syndrome with intra-renal administration of prostaglandin E1

Three patients with the hepatorenal syndrome were treated with prostaglandin E₁ administered through a selective renal arterial catheter. Prostaglandin E₁ was given in progressively increasing doses (2 to 100 ng/kg/min) over a 60-minute period. Control plasma prostaglandin E levels were elevated in all three patients, 0.98, 0.91, and 0.83 ng/ml, respectively. At the end of the infusion, plasma prostaglandin E levels had risen to 10.4, 2.63, and 10.3 ng/ml in the three patients respectively. Plasma renin activity increased during the course of the infusion in two of the patients. The plasma aldosterone concentration did not change during the prostaglandin E₁ infusion. Intrarenal prostaglandin E₁ failed to increase urine volume or urinary sodium concentration in three patients with the hepatorenal syndrome.     

Modification of the C-terminal octapeptide of cholecystokinin with a high-specific-activity iodinated imidoester: preparation, characterization, and binding to isolated pancreatic acinar cells

Proteoglycans were separated by high-performance liquid chromatography (HPLC), using two coupled Aquapore columns containing glycerylpropylsilane groups covalently linked to large-pore (50–100 nm) silica spheres. This two-column HPLC system was effective in separating cartilage proteoglycan aggregates and monomers, without altering their biochemical integrity. This system was also effective in resolving small amounts of isotopically labeled proteoglycans synthesized by cultured mammalian cells. The small sample size, short analysis time, and high reproducibility represent improvements in the study of proteoglycans over conventional soft-gel chromatography.     

Effects of water conductivity on electrocommunication in the weak-electric fish Brienomyrus niger (Mormyriformes)

A characteristic electric organ discharge display in social encounters between mormyrid fish is a temporary discharge cessation. Using this response, we have investigated the useful range of electrocommunication under different water conductivity conditions in the mormyrid Brienomyrus niger. An individual fish was confined to a porous ceramic shelter tube and moved from a starting distance of 380 cm toward a similarly confined conspecific until discharge, cessation occurred. The moved fish was subsequently returned to its original, position. Water conductivity affects the peak-to-peak source voltage of the electric organ and the sensitivity of the fish's electroreceptors. Within a range of 10 to 36 000 μS/cm, the peak-to-peak amplitude of the electric organ discharge declined as a power function. At 120 μS/cm, the amplitude was 50%, and at 300μS/cm, 30% of the 10 μS/cm value. The interfish distance at which discharge cessation occurred and the associated electric field gradients were dependent on water conductivity and upon the spatial orientation of the two fish (end-to-end or parallel orientations of their shelter tubes). The respective ranges were from 135 cm and 0.02 mV/cm at 52 μS/cm (parallel orientation) to 22 cm and 0.36 mV/cm at 678 μS/cm (end-to-end orientation). When the data for both tube orientations were combined, the relationship between water conductivity (x) and the distance at which discharge cessation occurred (y) could be expressed by a power function, y=K·x^a (with K=10^2.97 and a=?0.56). When an electrically ‘silent’ fish was moved away from its conspecific, a discharge resumption in the form of a high-frequency rebound occasionally effected changes in the other fish's discharge activity at distances up to 157 cm (with an associated electric, field gradient of 0.01 mV/cm under the lowest conductivity condition).     

Partial characterization of a specific high affinity binding macromolecule for 24R,25 dihydroxyvitamin D3 in differentiating skeletal mesenchyme

Cytosol preparations and cells from 6-day old cultured differentiating chick limb-bud mesenchyme, which consist of a high proportion of chondrocytes, were shown to specifically bind 24R,25 dihydroxycholecalciferol. Nuclei from identical cultures also showed specific binding for 24R,25 dihydroxycholecalciferol. On the contrary, similar preparations of limb-bud mesenchyme cells (6-day old cultures) pretreated on day one by 5-bromodesoxyuridine which induced a fibroblast phenotypic expression, failed to show any specific binding for either 24R,25 or 1α,25 dihydroxycholecalciferol. Pronase treatment of the cytosol indicated that the receptor was protein-like in nature. The chromatographic properties of the protein-receptor on diethylaminoethyl cellulose and Sephadex G-100 columns were similar to those of the protein receptor found for 1α,25 dihydroxycholecalciferol. This report is the first demonstration that a cytosol protein receptor for 24R,25 dihydroxycholecalciferol exists in developing skeletal tissue. 24,25 dihydroxyvitamin D₃ but not any of the other metabolites was shown to induce DNA synthesis after 24 h by almost two-fold and protein synthesis after 5 h by 240%. These results suggest an important physiological role for 24R,25 dihydroxyvitamin D₃ in the development of skeletal tissue.     

Neonatally induced tolerance to soluble protein antigens. I. Analysis of B-cell and T helper cell function in mice tolerant to bovine serum albumin or fowl gamma-globulin

High dose tolerance to either bovine serum albumin (BSA) or fowl γ-globulin (FGG) was induced in CBA mice by neonatal injection. Tolerance to BSA lasted about 9 weeks, and that to FGG, about 18 weeks. Splenic B-cell function was analyzed using quantitative in vivo assays and in vitro limiting dilution analysis. Tolerogen-specific IgM- and non-IgM-producing B cells are depleted at least threefold in the spleens of tolerant mice. Tolerogen-specific T-helper-cell function was examined by immunization with haptenated antigens. Analysis of the recovery from tolerance indicates that the return to normal function in the tolerogen-specific B-cell and T helper fractions coincides with the return to normal responsiveness by the whole animal.     

Azodipyrroles of unconjugated and conjugated bilirubin using diazotized ethyl anthranilate in dimethyl sulfoxide

We have developed a diazotization technique in which both conjugated and unconjugated bilirubin react completely. The method represents a crucial modification of the ethyl anthranilate diazo reaction originally described by K. P. M. Heirwegh, J. Fevery, J. A. T. P. Meuwissen, and J. de Groote (1974, Methods Biochem. Anal.22, 205–250). In the presence of dimethyl sulfoxide (2 ml/ml of sample and diazo reagent), conjugated and unconjugated bilirubin in human serum and human, rat, and mouse bile reacted rapidly and completely. The azopigments were stable for at least 4 h. Addition of human serum to unconjugated bilirubin, bilirubin monoglucuronide, and human bile did not influence azopigment formation. Because the reaction solution was optically clear, total azopigments could be measured by spectrophotometry or separated and quantitated by high-performance liquid chromatography without prior extraction into nonpolar solvents. Alternatively, the pigments could also be extracted into 2-pentanone for analysis by thin-layer or high-performance liquid chromatography. This method allows the quantitation of total bilirubin and analysis of individual ethyl anthranilate azopigments after a single diazotization step.