Formation of heterophile antibodies by human tonsilar lymphocytes: II. In vitro stimulation with allogeneic cells

Short-term cultures of human tonsilar lymphocytes (HTL), 5 × 10⁶ cells/culture, in medium RPMI 1640 supplemented with human group AB serum were studied for the production of plaque-forming cells (PFC) against sheep (SRBC) and bovine (BRBC) red blood cells following in vitro stimulation by various allogeneic lymphoid cells. Of 55 HTL specimens examined, 48 produced a significant number (50–300/culture) of PFC against SRBC and/or BRBC following the in vitro stimulation. The optimal doses of the stimulator HTL and peripheral blood lymphocytes (PBL) were 10⁷ and 5 × 10⁶/culture, respectively. After the stimulation, PFC appeared in significant numbers on the third day, reached the peak number on the sixth day, and decreased sharply in number thereafter. Removal of E-rosetting cells from both stimulator and responder populations abolished the PFC formation. PFC formation against SRBC was inhibited by solubilized Forssman antigen, while PFC formation against BRBC was inhibited strongly by Hanganutziu-Deicher antigen, hardly by Paul-Bunnell antigen and not at all by Forssman antigen. Supernatants of mixed lymphocyte culture of PBL were shown to enhance PFC formation of HTL cultures stimulated by allogeneic lymphocytes. The results of this study indicated that in vivo primed B cells of the HTL were triggered in vitro by allogeneic stimulation for the heterophile antibody formation. Since these antibodies are apparently directed against Forssman and Hanganutziu-Deicher antigens, the “allo” nature of these antigens as well as their relationship to the previously described heterophile transplantation antigens have to be clarified.     

Male sexual behavior in deer mice (Peromyscus maniculatus) following castration and hormone replacement

Sexually experienced male deer mice (Peromyscus maniculatus bairdi) were castrated and tested for male sexual behavior. In the weeks following castration male sexual behavior decreased. Ejaculation disappeared first, followed by intromission and, finally, mounting. Castrated males failing to copulate were assigned to one of four treatment groups: 200 μg testosterone propionate (TP); 200 μg dihydrotestosterone propionate (DHTP); 2 μg estradiol benzoate (EB); or sesame oil (OIL). TP and DHTP were equally effective in restoring the complete male sexual behavior pattern. In contrast, EB was effective in stimulating mounting and minimally effective in stimulating intromissions (vaginal penetration), but did not stimulate ejaculatory responses. These data indicate that in deer mice testosterone may mediate male sexual behavior through reduction to dihydrotestosterone rather than through aromatization to estradiol.     

Neonatal thyroxine treatment enhances classical conditioning in the infant rat

Neonatal rats injected with either thyroxine (T₄) or vehicle (NaOH) on postnatal Days 1, 2, and 3 were given classical-conditioning pairings of an odor with footshock when 7, 9, or 11 days of age. In accord with the conventional acceleration of other indices of maturation following the T₄ treatment, 24-hr retention of the conditioned odor aversion was substantially enhanced among the 11 day-old rats given the earlier T₄ treatment. This effect was marginally significant among 9-day olds but not among 7-day olds.     

A fluorinated vitamin D3 analog with biopotency greater than 1 alpha, 25-dihydroxy vitamin D3

Vitamin D metabolites and analogs induce $\text{de novo}$ synthesis of a specific calcium-binding protein in embryonic chick duodenum maintained in organ culture. Using calcium-binding protein biosynthesis as a specific and sensitive biochemical indicator of intrinsic biopotency, 24,24-difluoro-1α,25-dihydroxy vitamin D₃ was found to be approximately four times more potent on a molar basis than the most active, naturally occurring metabolite, 1α,25-dihydroxy vitamin D₃.     

Purification and characterization of a highly acidic 2Fe-ferredoxin from Halobacterium of the dead sea

A 2Fe-ferredoxin from Halobacterium of the Dead Sea has been purified by chromatography on Sepharose and DEAE-cellulose, using decreasing concentration gradients of ammonium sulfate. Its amino acid composition reveals an extremely high excess of acidic amino acid residues: 44 glutamate and aspartate residues (of which 4 are in the amide form), compared to 6 lysines and arginines, as well as a high content of aromatic amino acids. The molecular weight of this ferredoxin was found to be 14,000 by amino acid composition, sedimentation equilibrium, and iron content. The millimolar coefficients at the maxima of the visible absorption spectrum are: 28.0 (277 nm), 12.2 (330 nm), 9.1 (420 nm), and 8.3 (465 nm). The optical properties—absorption and CD spectra in the visible region—of this ferredoxin are very similar to those of plant and algal ferredoxins, whereas its redox potential is much higher: ?345 ± 5 mV (at pH 7.3, 0.5 m NaCl). Although it is reduced by illuminated chloroplasts, it cannot mediate the photoreduction of NADP in their presence. Data reported elsewhere suggest that its physiological function might be to serve as an electron donor for nitrite reduction.     

In vitro immune blastogenesis during contact sensitivity in tumor-bearing mice: I. Description of progressive impairment and demonstration of splenic suppressor cells

T-cell responsiveness was measured by the DNA response of disassociated spleen and lymph node cells when exposed to antigen in vitro. Sensitized splenic lymphocytes from fibrosarcoma-bearing mice immunized with 2,4-dinitro-1,5-difluorobenzene (DN2FB) demonstrated a progressive decrease in T-cell responsiveness to the haptenprotein conjugate DNP-BSA. Hyporesponsiveness to the dinitrophenylated-protein conjugate appeared in the spleens but not lymph nodes of tumorous animals. Normal host lymph node cells (LNC) responded strongly 24 to 48 h after sensitization and subsequently declined with a corresponding increase in responsiveness in the spleen. Tumor-bearing hosts (TBH) had similar LNC kinetics during immunization, however, spleen cells were significantly suppressed when compared to normal BALB/c mice sensitization kinetics. Spleen cells from TBH were also capable of suppressing the in vitro response of normal primed lymphocytes to DNP-BSA when admixed. Results from these experiments suggest that in vitro measurement of contact sensitivity was affected by suppressor cells/products existing in the spleens but not lymph nodes of fibrosarcoma-bearing mice.     

Synthesis of a cytosine nucleoside of 2-amino-2-deoxy-beta-D-xylofuranose.

1-(2-Amino-2-deoxy-beta-D-xylofuranosyl)cytosine (13) was synthesized by three routes: (a) coupling of 2-deoxy-3,5-di-O-p-nitrobenzoyl-2-(trifluoroacetamido)-D-xylofuranosyl chloride (5) with 2,4-dimethoxypyrimidine and subsequent treatment with methanolic ammonia, (b) coupling of 5 with 4-N-acetyl-2-O,4-N-bis(trimethylsilyl)cytosine followed by treatment with methanolic ammonia, and (c) thiation of 1-[3,5-di-O-acetyl-2-deoxy-2-(trifluoroacetamido)-beta-D-xylofuranosyl]uracil (6) by treatment with phosphorus pentasulfide in pyridine followed by amination of the resulting 4-thionucleoside 12 with metanolic ammonia. The best yield was obtained via route (a).     

The enthalpy of oxidation of flavin mononucleotide: Temperature dependence of in vitro bacterial luciferase bioluminescence

The enthalpy of the bioluminescent reaction

$\text{FMNH}{}_2\text{+ RCHO + O}{}_2\text{→}\text{luciferase}\text{FMN + RCOO + H}{}_3\text{O}{}^{\mathrm{+}}\text{+ hv}$

has been studied by direct calorimetric methods. Bacterial luciferase, isolated from Beneckea harveyi (formerly strain MAV) has been used to catalyze the oxidation of reduced flavin mononucleotide (FMNH₂) and a long chain aliphatic aldehyde (dodecanal, RCHO) by molecular oxygen to give the indicated products and blue-green light. The enthalpy measured for this process was found to be ΔH_L = ?338.9 k.J (mol FMN)^?1 (?81.0 kcal) at 25.00 °C and ?402.9 kJ (mol FMN)^?1 (?96.3 kcal) at 7.00 °C. Calculations based on redox electrode potentials indicate a corresponding value of the free energy change, ΔG_L = ?464.8 kJ (mol FMN)^?1 (?111.1 kcal), at 25 °C. Measurements were performed in 0.15 m phosphate buffer, pH 7.0 and the values were arrived at by correcting the observed heats for the heat associated with the autoxidation process: FMNH₂ + O₂ ? FMN + H₂O₂; ΔH_D = ?158.5 kJ (mol FMN)^?1 (?37.8). These data and a detailed thermodynamic analysis have demonstrated the need for two parameters, referred to as the intrinsic free energy, ΔG₁, and intrinsic enthalpy, ΔH₁, which are functionally defined by the relations ΔG_I = ΔG_L ? uhvΔH_I = ΔH_L ? uhv, where u is the quantum yield of the reaction expressed in einsteins mole^?1.These parameters reflect the thermochemistry of the bioluminescent reaction corrected for emitted photons. Thus, they are useful for comparing the thermochemistry of a chemiluminescent process. Their values for the bacterial luciferase system at 25 °C and pH 7.0 are ?391.6 and ?266.9 kJ (mol FMN)^?1 (?93.6 and ?63.8 kcal), respectively, assuming a value of 0.3 for the quantum yield. The calorimetric data also suggest the existence of a long-lived species which persists after photon emission.     

Effects of net charge on the hydrogen-deuterium exchange parameters of lysozyme.

Possible effects of changes in net charge on protein hydrogen exchange rates were investigated by desalting hen egg-white lysozyme, which allowed its net charge to increase with decreasing pH in the acid region. Chloride ion-binding ratios, expressed as ratios of free to total Cl^?, were measured with a chloride-specific electrode at pH 5 on a 2.4% solution of a five-time-desalted product. This ratio was used to show a 97% reduction of the 11% Cl^? present in a commercial lysozyme preparation upon three passes of the enzyme through a column of ion-retardation resin. Net charges on the purified product were assigned from a combination of electrophoretic mobility and proton titration data gathered under minimal ionic strength conditions. The net charge on the desalted product increased by 1.64 units between pH 5.0 and 3.0. Hydrogendeuterium exchange studies on the purified lysozyme in D₂O were obtained using the near-infrared region of a Cary 14R spectrophotometer. The rate-pD profile for k₂, the rate constant for the intermediate class of exchanging hydrogens, showed a decrease in the apparent pD of minimum exchange rate of 0.3 units, when compared to that obtained earlier in 0.2 m added NaCl. However, the rate of exchange at pD minimum and the number of hydrogens in the class remained largely unaffected. A similar shift was observed for the rate-pD profile of the class 1 hydrogens. Thus, the effect of an increase in net positive charge is to shift the rate-pD profile to a lower pD. Moreover, the effect extended to the interior peptide hydrogens of this globular protein. Consequently, the exchange rates of all the observable hydrogens are altered by the net charge changes, and the effect appeared uniform. The shift can be accounted for quantitatively by applying electrostatic interaction terms to the acid and base catalytic constants characterizing the exchange process. The calculated electrostatic interaction factors in minimal salt and 0.2 m added NaCl were found to be 29 and 18% lower, respectively, than those obtained theoretically. Therefore, under conditions where changes in net charge may occur for a globular protein, the effect on hydrogen exchange rates can be estimated fairly well theoretically, especially at moderate ionic strengths.