Inverse modification of antibody responsiveness to RGG in lines of mice selected for high or low responses to somatic antigen of Salmonella

High (H/s) and low (L/s) antibody responder lines of mice selected according to their response to the somatic (s) antigen of Salmonella (Selection IV) have unexpected inverse capacity for antibody production to rabbit gamma globulin (RGG): H/s mice are low or even nonresponders to this antigen, whereas L/s mice are high responders. It was shown that the phenotypic variability within each line is due to environmental factors. RGG was a selection antigen in Selection V; the high (H/p) and low (L/p) responder mice are therefore considered as homozygous for the RGG genes. Responsiveness to RGG was investigated in F₁ and F₂ hybrids obtained by crossing the phenotypically similar RGG responder or nonresponder mice of Selections IV and V. The results support the hypothesis that the same genes control the response to RGG in L/s and H/p lines as well as in H/s and L/p lines. This means that the genes specific for RGG responsiveness were independent from those regulating responses to the s antigen. Unaffected by the selective breeding in Selection IV, they have been fixed by chance in an inverse way in H/s and L/s lines.     

Iron(III) binding in DNA solutions: complex formation and catalytic activity in the oxidation of hydrazine derivatives.

A strong interaction between iron(III) and calf thymus DNA at pH 7.4 was demonstrated in the present study by separation of the complex by column chromatography and by the slow kinetics of iron(III) removal from DNA by disodium-1,2-dihydroxybenzene-3,5-disulfonate (Tiron). An equilibrium constant of 2.1 x 10(14) was calculated by measurements of bound iron(III) by flame atomic absorption spectroscopy and assuming a one iron to two nucleotide stoichiometry. Graphic analysis of the interaction however, indicated that DNA has two binding sites for iron(III) characterized by a stoichiometry of one iron to 12 nucleotides and one iron to 2 nucleotides, and association constants of 4.8 x 10(12) and 2.3 x 10(11), respectively. The DNA-iron(III) complex isolated by column chromatography was shown to catalyze the oxidation of both 2-phenylethylhydrazine and methylhydrazine by spin-trapping experiments with alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN). By contrast, oxidation of 1,2-dimethylhydrazine was not catalyzed. Catalysis of 2-phenylethylhydrazine oxidation was confirmed by oxygen consumption studies. The results suggest that iron chelated to DNA may be significant in DNA damage induced by oxidizable chemicals.     

Pre-column derivatization of free bile acids for high-performance liquid chromatographic and gas chromatographic—mass spectrometric analysis

Pentachlorophenyl (PCP) esters of five free bile acids (FBA) were obtained by reacting the FBA and Kovacs' complex (KC) in a 1:8 molar ratio in acetone at 65°C, and were purified by column chromatography on silica gel. The esters were crystallized from benzene—hexane, derivatized as trimethylsilyl ethers for gas chromatography on a DB-1 capillary column and for gas chromatography—mass spectrometry with a DB-5 column, and mass spectrometry (MS) in the electron-impact (EI) positive-ion mode at 70 eV. The reaction is specific for FBA even in the presence of glycine and taurine conjugates of bile acids. The PCP esters were treated with benzylamine in chloroform or methanol to produce N-benzyl derivatives of FBA. The N-benzylamides were separated by high-performance liquid chromatography (HPLC) on a 4-μm Nova-Pak C₁₈ column, studied by thermospray—LC—MS, and in the direct insertion probe—EI positive-ion mode.     

Some biological properties of the human amniotic membrane interferon.

Human amniotic interferon was investigated to define the species specificity of its antiviral action and to compare its anti-cellular and NK cell stimulating activities with those of other human interferons. The antiviral effect was titrated in bovine (RV-IAL) and monkey (VERO) cells. Amniotic interferon exhibited, in bovine cells, 5% of the activity seen in monkey cells, while alpha interferon displayed 200%. No effect was detected with either beta or gamma interferon in bovine cells. Daudi cells were exposed to different concentrations of various interferons and the cell numbers were determined. The anticellular effect of the amniotic interferon reached its peak on the third day of incubation. Results suggested a higher activity for alpha and gamma interferons and a lower activity for beta when compared to amniotic interferon. Using total mononuclear cells as effector cells and K 562 as target cells in a 51Cr release assay, it was demonstrated that low concentrations of amniotic interferon consistently stimulated NK cell activity in cells derived from several donors, the results indicating a higher level of activity with this interferon than with alpha and beta interferons.     

One-step synthesis of Gly-Gly-PheNH2 from N-unprotected amino acid derivatives by papain in one-phase liquid media

Summary Papain was able to catalyze the one-step synthesis of Gly-Gly-PheNH₂, from N-unprotected amino acid derivatives. Maximum synthetic activity was obtained for a pH value of 6.5 and for a ]PheNH₂]/[Gly-GlyOEt] ratio of 6. The presence of an organic cosolvent, such as ethylene glycol, influenced the synthetic activity. Synthetic yield was higher than 65% for a 12.5 M cosolvent concentration.     

In vitro propagation of Amaryllis belladonna

Amaryllis belladonna L. plants were multiplied successfully by means of tissue culture techniques. Different plant parts were tested as explant material, but plantlets could only be generated from the twin-scales and immature scapes. These in vitro-formed plantlets were divided into four parts and used for further multiplication. The twin-scale explants had the highest multiplication rate when a medium with 22.2 M benzyladenine and 0.54 M naphthaleneacetic acid was used. The sucrose concentration played an important role in the initiation of new plantlets, and the best results were obtained when a sucrose concentration of 2–3% was used. Anatomical observations were made during the initiation of the new plantlets.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid - Benomyl (methyl [1-[(butylamino) carbonyl]-1H-benzimidazol-2-yl] carbamate) - Folpet (2-[(trichloromethyl)thio]-H-isoindole-1,3(2H)-dione phthalimide(I))     

Evaluation of different staining procedures for the quantification of fibroblasts cultured in 96-well plates.

Comparison of published methods for the quantification of adherent cell numbers by the measurement of absorbance of bound stain indicates a wide variation in their sensitivity. This study aimed at comparing the sensitivities of five different staining procedures (Coomassie brilliant blue G in perchloric acid, Coomassie brilliant blue G in phosphoric acid, methylene blue, crystal violet, and toluidine blue) applied to three separate types of cultured fibroblasts (3T3 cells, Vero cells, and human gingival fibroblasts) at concentrations from 0.125 x 10(4) to 10 x 10(4) per well in 96-well microplates. Absorbance values of Coomassie blue-stained cells were measured in situ. Those of the remaining cells were measured after solubilization of the dye with 1% sodium dodecyl sulfate. All absorbance values were measured using an Elisa reader at 620 or 570 nm for crystal violet. The relationship between cell number and absorbance over the entire cell concentration range was best fitted with quadratic regression analysis, in contrast with the linear relationship described elsewhere. The order of sensitivity of the staining procedures was the same for each cell type: Coomassie blue in perchloric acid less than Coomassie blue in phosphoric acid less than methylene blue less than crystal violet less than toluidine blue. With the latter two stains absorbance values began to plateau at approximately 8 x 10(4) cells per well. However, staining with Coomassie blue in perchloric acid and methylene blue resulted in an almost linear relationship between cell number and absorbance over the entire concentration range tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Basal Gastric Acid Secretion on the Pharmacodynamics of Ranitidine

Twelve patients with inactive ulcer disease were administered placebo and ranitidine via bolus and continuous intravenous infusions, at doses ranging from 50 every 8 h, to 12.5 mg/h for 24 h. Gastric acid was collected for 20 min each h for 24 h, and ranitidine serum concentrations were measured ± every 2 h, during each of the six study periods. Cosinor analysis of gastric acid secretion during placebo treatment revealed a significant circadian rhythm in all subjects. Mesor acid output ranged from 1.7 to 11.6 mmol/h (mean 5.6 ± 2.8 mmol/h) and the amplitude ranged from 0.7 to 6.5 mmol/h (mean 2.8 ±1.6 mmol/h). Peak acid output (acrophase) occurred at 10 p.m. ± 3 h. A pharmacodynamic model, relating ranitidine serum concentration to hourly acid secretion, was derived, which incorporated the circadian change in basal acid output. Data for this fractional response model included basal acid secretion-as determined by time of day, measured acid secretion, and associated serum ranitidine concentration. The 50% inhibitory concentration (IC₅₀) for ranitidine ranged from 10-75 ng/ml, with a mean of 44 ng/ml. The variation in IC₅₀ and in basal acid secretion combined to produce a wide variation in the pharmacodynamic response to ranitidine. The model-predicted serum concentrations, required to maintain acid secretion at 0.1 mmol/h, ranged from 250 to 1550 ng/ml, at the time of peak evening acid secretion. Despite a constant degree of acid inhibition by ranitidine during the day, higher serum concentrations are required during times of peak acid output to maintain adequate suppression of hydrogen ion secretion.     

The mechanism of action of Cu2+ on the frog skin.

The mechanism of action of Cu2+ when applied to the external side of the frog skin preparation was investigated. Cu2+ acts most probably on the external barrier of this preparation, since it increases the transport pool of Na+ proportionally to the increase in the short circuit current (Isc). Cu2+ does not open new routes for the Na+ entry since the stimulated Isc is still completely abolished by amiloride. The Isc dependence of Na+ concentration in the external medium is modified by copper, since the Km value increases in addition to changes in V. It is suggested that copper acts at the external barrier Na channels in a way similar to that proposed by Zeiske and Lindemann ((1974) Biochim. Biophys. Acta 352, 323--326) for benzoylimidazole-2 guanidine and benzoylthiazole-2 guanidine and by Dick and Lindemann ((1975) Pflügers Arch. ges. Physiol. 355, R72) for para-chloromercuribenzenosulfonate and para-chloromercuribenzoate.     

The effect of copper on frog skin. The role of sulphydryl groups

1. 1. Cu²⁺ at a concentration of 10⁻⁴ M, when applied to the external side of the frog skin produces an increase in the short-circuit current (I_sc).

2. 2. This effect was studied in skins of Rana temporaria adapted to cold (5°C) and room temperature (20°C), skins of Rana pipiens adapted to cold, and the results compared with those obtained previously with Rana ribibunda.

3. 3. The observed effect is less dependent upon the adaptation to cold than upon the functional state of the skin: skins with low short circuit currents have a bigger response to Cu²⁺ than skins with high I_sc.

4. 4. A species difference cannot be ruled out since skins of Rana ribibunda exhibiting high I_sc give good responses to Cu²⁺.

5. 5. 5,5′-dithiobis(2-nitrobenzoic acid), a sulphydryl-oxidizing reagent, produces an effect similar to that of Cu²⁺, and dithiothreitol an SH-reducing agent, reverses the effect of this ion.

6. 6. Cu²⁺ also induces an increase in the unidirectional K⁺ fluxes and unmasks a net outward potassium flux.

7. 7. The outward K⁺ flux induced by Cu²⁺ is sensitive to ouabain.

8. 8. It is concluded that Cu²⁺ increases the permeability of the external barrier of the frog skin to Na⁺ and K⁺, probably by reacting with SH groups.