Study of the volt-ampere characteristics of ion channels by transmembrane current harmonics

A method is proposed for measuring the coefficient of non--linearity beta of current--voltage characteristics of the class i (U) approximately U (1 + beta U2) of ionic channels formed by grammicidine A (Gra) and amphotericine B by the 3rd harmonics of the membrane current. For Gra A beta depends on the concentration of electrolyte c increasing lg c from -17 B-2 at 0.03 M to 8 B-2 at 3.4 M KCl turning to 0 at c0 = 0.3 divided by 1 M. The membranes of egg lecithin and glycerylmonooleate (GMO) differ in c0 value. Substitution of K+ ion for Li+, of the membrane solvent (n-heptane for n-hexadecane) and freezing of the GMO membrane do not affect beta.     

Evidence for a redox-linked ionizable group associated with the [2Fe-2S] cluster of Thermus Rieske protein

The [2Fe-2S] clusters of Thermus Rieske protein, which were previously found to have nitrogen atoms coordinated directly to the iron (Cline, J.F., Hoffman, B.M., LaHaie, E., Ballou, D.P., and Fee, J.A. (1985) J. Biol. Chem. 260, 3251-3254), are now shown to have a tightly linked ionization that affects the spectral and redox properties of the cluster. The data are consistent with the reactions LH+, Fe3+ in equilibrium with L-Fe3+ +H+ and L-Fe3+ + H+ + e in equilibrium with LH+, Fe2+, where L is coordinated to Fe3+ but LH+ may not be, depending on its structure. The pKa of the protonic equilibrium is approximately 8 and the midpoint potential, Em7, is approximately 140 mV. Possible structures of L are suggested.     

Grand canonical Monte Carlo molecular and thermodynamic predictions of ion effects on binding of an oligocation (L8+) to the center of DNA oligomers.

Grand canonical Monte Carlo (GCMC) simulations are reported for aqueous solutions containing excess univalent salt (activities a +/- = 1.76-12.3 mM) and one of the following species: an octacationic rod-like ligand, L8+; a B-DNA oligomer with N phosphate charges (8 < or = N < or = 100); or a complex resulting from the binding of L8+ at the center of an N-mer (24 < or = N < or = 250). Simplified models of these multiply charged species are used in the GCMC simulations to predict the fundamental coulombic contributions to the following experimentally relevant properties: 1) the axial distance over which ligand binding affects local counterion concentrations at the surface of the N-mer; 2) the dependence on N of GCMC preferential interaction coefficients, gamma 32MC identical to delta C3/delta C2l a +/-, T, where C3 and C2 are, respectively, the molar concentrations of salt and the multiply charged species (ligand, N-mer or complex); and 3) the dependence on N of SaKobs identical to d in Kobs/d in a +/- = delta (magnitude of ZJ + 2 gamma 32J), where Kobs is the equilibrium concentration quotient for the binding of L8+ to the center of an N-mer and delta denotes the stoichiometric combination of terms, each of which pertains to a reactant or product J having magnitude of ZJ charges. The participation of electrolyte ions in the ligand binding interaction is quantified by the magnitude of SaKobs, which reflects the net (stoichiometrically weighted) difference in the extent of thermodynamic binding of salt ions to the products and reactants. Results obtained here from GCMC simulations yield a picture of the salient molecular consequences of binding a cationic ligand, as well as thermodynamic predictions whose applicability can be tested experimentally. Formation of the central complex is predicted to cause a dramatic reduction in the surface counterion (e.g., Na+) concentration over a region including but extending well beyond the location of the ligand binding site. For binding a cationic ligand, SaKobs is predicted to be negative, indicating net electrolyte ion release in the binding process. At small enough N, -SaKobs is predicted to decrease strongly toward zero with decreasing N. At intermediate N, -SaKobs appears to exceed its limiting value as N-->infinity.     

Ly1+ PRO-B lymphocyte clones. Phenotype, growth requirements and differentiation in vitro and in vivo.

Several clones obtained from the bone marrow of a BALB/c mouse were found to contain the heavy and light chain Ig genes in the germline configuration, to express Ly1 and to carry the B cell lineage markers B-220, Lyb8 and BP-1; these clones are Pgp-1+, LFA-1+, J11d+, Mac-1+ and Thy1-, Lyt2-, L3T4-, GM1.2- and Ia-. Three clones analyzed in detail (Lyd9, LyH7 and Lyb9) have receptors for interleukin (IL) 2 and IL3 as assessed with the 7D4 and CC11 monoclonal antibodies respectively. They grow in rIL3 but not in rIL2 or rIL1; both rIL4 and rIL5 also promote their proliferation, albeit to a much lesser extent than rIL3. None of the interleukins tested alone or in various combinations promoted the clones to differentiate in vitro along the B cell pathway. Treatment with 5-Azacytidine (5-Aza) induced cell surface Ia expression but not rearrangement or expression of Ig genes. However, 5-Aza-treated Lyd9, LyH7 and Lyb9 cells co-cultured with X-ray irradiated accessory cells and LPS gave rise to Ly1+, IgM+ B lymphocytes (range 14-51%) including mu + kappa + (78-93%), and mu + lambda + (9-25%) B lymphocytes. In vivo, the Lyd9, LyH7 and Lyb9 clones gave rise to IgM+ B lymphocytes (8.5-17%) including mu + kappa +, and mu + lambda +, but not to Lyt2+ or L3T4+ T lymphocytes after 4-6 weeks of transfer into Scid mice. Our results indicate that Ly1+ IgM+ cells comprise a subpopulation of B lymphocytes that is derived from IL3-responsive Ly1+ PRO-B lymphocytes.     

Interaction of guanosine cyclic 3',5'-phosphate dependent protein kinase with lin-benzoadenine nucleotides

Using the activated cGMP-dependent protein kinase in the presence of the phosphorylatable peptide [[Ala34]histone H2B-(29-35)], we found that lin-benzoadenosine 5'-diphosphate (lin-benzo-ADP) was a competitive inhibitor of the enzyme with respect to ATP with a Ki (22 microM) similar to the Kd (20 microM) determined by fluorescence polarization titrations. The Kd for lin-benzo-ADP determined in the absence of the phosphorylatable peptide, however, was only 12 microM. ADP bound with lower affinity (Ki = 169 microM; Kd = 114 microM). With [Ala34]histone H2B-(29-35) as phosphoryl acceptor, the Km for lin-benzo-ATP was 29 microM, and that for ATP was 32 microM. The Vmax with lin-benzo-ATP, however, was only 0.06% of that with ATP as substrate [0.00623 +/- 0.00035 vs. 11.1 +/- 0.17 mumol (min.mg)-1]. Binding of lin-benzo-ADP to the kinase was dependent upon a divalent cation. Fluorescence polarization revealed that Mg2+, Mn2+, Co2+, Ni2+, Ca2+, Sr2+, and Ba2+ supported nucleotide binding to the enzyme; Ca2+, Sr2+, and Ba2+, however, did not support any measurable phosphotransferase activity. The rank order of metal ion effectiveness in mediating phosphotransferase activity was Mg2+ greater than Ni2+ greater than Co2+ greater than Mn2+. Although these results were similar to those observed with the cAMP-dependent protein kinase [Hartl, F. T., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 2347], major differences in the Vmax with lin-benzo-ATP as substrate and the effect of peptide substrates on nucleotide (both lin-benzo-ADP and ADP) binding were observed.     

Quantitative analysis of Fc gamma receptors on murine spleen cell populations by using dual parameter flow cytometry

The expression of Fc gamma R on subsets of mouse spleen cells was examined by dual parameter flow microfluorometry. B cells were detected by labeling them with antibodies against sIgM, sIgD, sIgG, or I-A; essentially all B cells expressed Fc gamma R. The number of Fc gamma R per cell on the sIgD+, sIgM+, and I-A+ cells averaged 2 X 10(4) receptors, and no correlation between the levels of expression of Fc gamma R and the B cell markers was evident. The sIgG+ B cells, however, expressed more Fc gamma R (8 X 10(4) receptors/cell) than sIgM+ and sIgD+ B cells. Fc gamma R on splenic macrophages were examined by double labeling spleen cells for Fc gamma R and Mac-1. The Mac-1+ cells (2 to 16% of the spleen cells) were 100% Fc gamma R+ and expressed threefold to fivefold higher numbers of Fc gamma R per cell than the sIgM+ or sIgD+ B cells. The Fc gamma R on T cells were studied on cells double labeled for Fc gamma R and Thy-1, Lyt-1, or Lyt-2. An average of 20% of the T cells expressed Fc gamma R and at least two subsets of Fc gamma R+ T cells were evident: Lyt-2- cells, most of which expressed intermediate (2 X 10(4) Fc gamma R/cell) levels of Fc gamma R, and Lyt-2+ cells, which expressed mainly high (8 X 10(4) Fc gamma R/cell) amounts of Fc gamma R. The levels of expression of Fc gamma R and sIgM increased dramatically in response to infection and were elevated in mice with genetic defects. We conclude that the level of Fc gamma R expression is a characteristic property of subsets of spleen cells from normal and infected mice.     

Acetylcholine responses of identified neurons in Helix pomatia--III. Ionic composition of the depolarizing currents induced by acetylcholine

The ionic composition of the currents underlying the acetylcholine (ACh) depolarizations in the identified neurons B1 and B3 of the buccal ganglia of Helix pomatia was analysed. The equilibrium potential of the ACh responses was -2.8 +/- 0.6 mV (N = 49) and -4.0 +/- 0.7 mV (N = 79; mean +/- SEM) in the neurons B1 and B3, respectively. Replacement of NaCl in the bath solution by sucrose shifted the ACh equilibrium potential into the negative direction. A similar but less pronounced shift occurred when Ca2+ was substituted for Na+. Substitution of Cl- in the bath solution by propionate or an increase of the intracellular Cl- concentration did not affect the ACh equilibrium potential. Changes of K+ concentration in the bath between 1 and 50 mmol/l left the ACh equilibrium potential nearly unaffected when the Na+ concentration was at the control level. With a simultaneous reduction of extracellular Na+ an increase of K+ concentration shifted the ACh equilibrium potential towards more positive potentials. The findings are compatible with calculated K+ permeabilities if a K+ redistribution across the cell membrane is considered. In the neurons B1 and B3, channels operated by ACh are permeable for K+, Na+ and Ca2+, with the relative permeabilities 1.6:1.0:0.1.     

Determination of monoclonal antibody-induced alterations in Na+/K+-ATPase conformations using fluorescein-labeled enzyme

The fluorescein 5'-isothiocyanate (FITC)-labeled lamb kidney Na+/K+-ATPase has been used to investigate enzyme function and ligand-induced conformational changes. In these studies, we have determined the effects of two monoclonal antibodies, which inhibit Na+/K+-ATPase activity, on the conformational changes undergone by the FITC-labeled enzyme. Monitoring fluorescence intensity changes of FITC-labeled enzyme shows that antibody M10-P5-C11, which inhibits E1 approximately P intermediate formation (Ball, W.J. (1986) Biochemistry 25, 7155-7162), has little effect on the E1 in equilibrium E2 transitions induced by Na+, K+, Mg2+ Pi or Mg2+. ouabain. The M10-P5-C11 epitope, which appears to reside near the ATP-binding site, does not significantly participate in these ligand interactions. In contrast, we find that antibody 9-A5 (Schenk, D.B., Hubert, J.J. and Leffert, H.L. (1984) J. Biol. Chem. 259, 14941-14951) inhibits both the Na+/K+-ATPase and p-nitrophenylphosphatase activity. Its binding produces a 'Na+-like' enhancement in FITC fluorescence, reduces the ability of K+ to induce the E1 in equilibrium E2 transition and converts E2.K+ to an E1 conformation. Mg2+ binding to the enzyme alters both the conformation of this epitope region and its coupling of ligand interactions. In the presence of Mg2+, 9-A5 binding stabilizes an E1.Mg2+ conformation such that K+-, Pi- and ouabain-induced E1----E2 or E1----E2-Pi transitions are inhibited. Oubain and Pi added together overcome this stabilization. These studies indicate that the 9-A5 epitope participates in the E1 in equilibrium E2 conformational transitions, links Na+-K+ interactions and ouabain extracellular binding site effects to both the phosphorylation site and the FITC-binding region. Antibody-binding studies and direct demonstration of 9-A5 inhibition of enzyme phosphorylation by [32P]Pi confirm the results obtained from the fluorescence studies. Antibody 9-A5 has also proven useful in demonstrating the independence of Mg2+ ATP and Mg2+Pi regulation of ouabain binding. In addition, [3H]ouabain and antibody-binding studies demonstrate that FITC-labeling alters the enzyme's responses to Mg2+ as well as ATP regulation.     

Self-association of des-(B26-B30)-insulin. The effect of Ca2+ and some other divalent cations

It has been confirmed by sedimentation equilibrium and sedimentation velocity experiments that des-(B26-B30)-insulin does not self-associate at neutral pH. Sedimentation equilibrium experiments at pH 7, 25 degrees C were conducted to investigate the effects of the structurally and physiologically important divalent cations Zn2+, Cd2+, Pb2+ and Ca2+ on the aggregation state of des-(B26-B30)-insulin (pig) in solution. It was found that all of these ions bring about association of this insulin analogue; Zn2+ and Cd2+ to a more marked degree than Pb2+ and Ca2+. The predominant species in solutions containing Zn2+ appear to be hexamers and hexameric aggregates, in those containing Cd2+, species up to and including tetramers, and in those containing Pb2+ and Ca2+, monomers and dimers of des-(B26-B30)-insulin appear to be the only species present. The possible significance of these findings, especially in relation to a role for Ca2+ in the action of insulin, is discussed.     

Osmoprotection of Escherichia coli by ectoine: uptake and accumulation characteristics.

Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is a cyclic amino acid, identified as a compatible solute in moderately halophilic bacteria. Exogenously provided ectoine was found to stimulate growth of Escherichia coli in media of inhibitory osmotic strength. The stimulation was independent of any specific solute, electrolyte or nonelectrolyte. It is accumulated in E. coli cells proportionally to the osmotic strength of the medium, and it is not metabolized. Its osmoprotective ability was as potent as that of glycine betaine. The ProP and ProU systems are both involved in ectoine uptake and accumulation in E. coli. ProP being the main system for ectoine transport. The intracellular ectoine pool is regulated by both influx and efflux systems.     

Interaction of metal ions with lupin seed conglutin gamma

Various metal ions were capable of aggregating and precipitating conglutin gamma, an oligomeric glycoprotein purified from Lupinus albus seeds, at neutral pH values. The most effective metal ions, at 60-fold molar excess to the protein, were Zn2+, Hg2+ and Cu2+; a lower influence on the physical status of conglutin gamma was observed with Cr3+, Fe3+, Co2+, Ni2+, Cd2+, Sn2+, and Pb2+, while Mg2+, Ca2+ and Mn2+ had no effect at all. The insolubilisation of the protein with Zn2+, which is fully reversible, strictly depended on both metal concentration and pH. with middle points of the sharp transitions at three-fold molar excess and pH 6.5, respectively. Conglutin gamma is also fully retained on a metal affinity chromatography column at which Zn2+ and Ni2+ were complexed. A drop of pH below 6.0 and the use of chelating agents, such as EDTA and imidazole, fully desorbed the protein. A slightly lower binding to immobilised Cu2+ and Co2+ and no binding with Mg2+, Cd2+ and Mn2+ were observed. The role of the numerous histidine residues of conglutin gamma in the binding of Zn2+ is discussed.     

Effects of pH and Ca2+ on monomer-dimer and monomer-tetramer equilibria of chromogranin A.

Chromogranin A is a high capacity, low affinity Ca2+ binding protein which undergoes Ca2+- and pH-dependent conformational changes, and has recently been suggested to play a Ca2+-buffering role in the secretory vesicle of adrenal medullary chromaffin cell, the major inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ store of chromaffin cell (Yoo, S.H., and Albanesi, J.P. (1990) J. Biol. Chem. 265, 13446-13448). In the present study, it is shown that chromogranin A exists in a monomer-dimer equilibrium at pH 7.5 and in a monomer-tetramer equilibrium at pH 5.5. The pH appears monomer-tetramer equilibrium at pH 5.5. The pH appears to be a necessary and sufficient factor determining the types of oligomers formed. Although Ca2+ did not change the type of oligomerization, it had a very significant effect on the values of the thermodynamic parameters characterizing the associations. The delta G0 values for a monomer-dimer equilibrium were -7 to -8 kcal/mol, while those for a monomer-tetramer equilibrium were -20 to -23 kcal/mol. At pH 5.5, the values of delta H0, delta S0, and delta C0p were large and negative in the absence of Ca2+ and large and positive in the presence of 35 mM Ca2+, implying markedly different reaction mechanisms. Extrapolation of the results to 37 degrees C and 1 mM chromogranin A suggests that chromogranin A is virtually 100% tetramer at pH 5.5 in the presence of 35 mM Ca2+ but is 96% dimer at pH 7.5 in the absence of Ca2+, the two conditions resembling those seen in vivo. These results suggest that chromogranin A is mostly dimer in the endoplasmic reticulum and cis-Golgi area and is essentially all tetramer in the vesicle.     

The relationship between gastric acid secretion and gastric H+,K+-ATPase activity

The H+,K+-ATPase has been postulated to be the enzyme responsible for H+ secretion by the parietal cell. Omeprazole has been shown to be an inhibitor of acid secretion in vivo, but also in in vitro test models for acid secretion, including partly purified H+,K+-ATPase, the inhibitory action of omeprazole has been demonstrated (Wallmark, B., Jaresten, B. M., Larsson, H., Ryberg, B., Br?ndstr?m, A., and Fellenius, E. (1983) Am. J. Physiol. 245, G64-G71). It was thus possible to use this compound to demonstrate a correlation between H+,K+-ATPase activity in rat oxyntic mucosa and in vivo H+ secretion. Two results were found. (a) Increasing oral doses of omeprazole progressively inhibited acid secretion, H+,K+-ATPase activity, and phosphoenzyme formation of a microsomal fraction isolated from the inhibited rat mucosa. Furthermore, a Mg2+-stimulated ATPase activity, associated with the H+,K+-ATPase membrane fraction, was not affected by the omeprazole treatment. (b) Recovery of H+,K+-ATPase activity following complete omeprazole inhibition was correlated with the appearance of acid secretion. The results indicate a strict relationship between the activity of the gastric H+,K+-ATPase in the microsomal fraction and gastric acid secretion.     

Genetic Study of Cationic ATPase Activities and Audiogenic Seizure Susceptibility in Recombinant Inbred and Congenic Strains of Mice

Total, Mg2+, and Na+, K+ ATPase activities were studied in fresh brain homogenates of the audiogenic seizure (AGS)-resistant C57BL/6J (B6) and AGS-susceptible DBA/2J (D2) inbred strains and in 13 B6 X D2 (BXD) recombinant inbred (RI) strains. These activities were also studied in the D2.B6-Iasb congenic mice, that are similar genetically to D2 mice, except for the Iasb gene which inhibits the spread of AGS activity. The total and Mg2+ ATPase activities of the brainstem were significantly lower in the D2 than in the B6 mice at 21 days of age. No differences were found between these strains for Na+,K+ ATPase activity. The total, Mg2+, and Na+,K+ ATPase activities in the B6 brainstem did not change noticeably from 21 to 80 days of age. In the D2 brainstem, however, the Mg2+ activity increased with age, and the Na+,K+ ATPase activity decreased from 30 to 80 days of age. No genetic associations could be found between AGS susceptibility and total or Mg2+ ATPase activities in the D2.B6-Iasb mice or among the 13 BXD RI strains. Hence, differences in genetic background, rather than differences in AGS susceptibility, can account for the lower ATPase activities in 21-day-old D2 mice. Further, the Mg2+ and Na+,K+ ATPase activities appear to be regulated by more than one gene. This study emphasizes the utility of RI and congenic strains for testing the biochemical basis of AGS susceptibility in mice.     

Analysis of human bone marrow with monoclonal antibodies

In order to study the antigenic phenotype of different hemopoietic cells, we used a series of monoclonal antibodies to investigate normal bone marrow in a standard immunofluorescence assay. The antibodies detected the following antigens: HLA-ABC, beta 2-microglobulin (beta 2m), HLA-DR (Ia), a lymphocyte subset and specific antigen (T and B) HuLy-m2, m3, T lymphocyte antigen (HuLy-m1), lymphocyte T200 antigen (HuLy-m4), a viral-associated antigen (HuLy-m5), and platelet-specific glycoproteins IIb-IIIa (HuPl-m1). The following results were obtained: (a) normoblasts were weakly HLA-ABC+, beta 2m+ and Ia-; all other lymphocyte and platelet antigens were not detected. (b) Myeloid cells at all stages of differentiation (promyelocytes, myelocytes, metamyelocytes, and neutrophils) were HLA-ABC+; beta 2m+; HuLy-m1-, m2-, m3+/- (20%), m4+, m5+/- (20%); HuPl-m1-; in addition, promyelocytes and myelocytes were Ia+ but neutrophils and metamyelocytes were Ia-. (c) Lymphocytes were HLA-ABC+, beta 2m+, Ia+/- (20-30%), HuLy-m1+/- (40-50%), m2+/- (60-70%), m3+, m4+, m5+; Pl-m1-. (d) Platelets and megakaryocytes were HLA-ABC+; beta 2m+; Ia-; HuLy-m1+-, m2-, m3-, m4-, m5-, HuPl-m1+, and the putative "megakaryocyte precursors" were HuPl-m1+, Ia-, HuLy-m1-. The different cell types in bone marrow could readily be distinguished, particularly cells of the myeloid series (Ia and HuLy-m4, m5), lymphocytes (Ia and HuLy-m1, m2, m3), and platelets and their precursor cells (HuPl-m1). This simple method of defining cellular phenotypes in bone marrow has demonstrated the practicality of using monoclonal antibodies to identify marrow cells and should be of diagnostic value.     

Effect of substitution inert metal complexes on calcineurin.

As a substitute for M(H2O)2+6, Co(NH3)3+6 was found to activate calcineurin with para-nitrophenyl phosphate as substrate. Kinetics for calcineurin catalyzed hydrolysis of para-nitrophenyl phosphate at pH 7.0 with Mn2+, Mg2+, Co2+, and Co(NH3)3+6 were compared. Although kcat and Km were different with the metals, values of kcat/Km were nearly identical for Mn2+ and Mg2+, but lower for Co2+ and Co(NH3)3+6. The concentration of each metal providing half-maximal activation, designated Kact, was evaluated as 15.9 mM for Co(NH3)3+6, compared to Kact = 0.17 mM for Mn2+ and Co2+ and 6.3 mM for Mg2+, respectively. Comparing kcat/Kcat showed that Co(NH3)3+6 was a 170-fold poorer activator of calcineurin than was Mn2+, but only 1.5-fold poorer than Mg2+. Activation by Co(NH3)3+6 indicated that activation of calcineurin by exogenous metal ions can proceed via an outer coordination sphere reaction mechanism with no requirement for the direct coordination of substrate by metal. Because Co(NH3)3+6 was found to support calcineurin activity, the related compound [Co-(ethylenediamine)3]3+ (or Co(en)3+3) was tested as a possible activator. Co(en)3+3 did not support calcineurin activity but did inhibit calcineurin. Co(en)3+3 showed competitive inhibition kinetics with either Mn2+ or pNPP as the varied ligand and the other at a fixed, subsaturating concentration. Inorganic phosphate was used as a known competitive inhibitor to pNPP (B. L. Martin and D. J. Graves, J. Biol. Chem. 261, 14545-14550, 1986) and showed uncompetitive inhibition with Mn2+ as the varied ligand. These patterns are consistent with the mechanism of ligand binding to calcineurin being ordered with metal preceding substrate. Prior formation of a metal-substrate complex was not required for association with calcineurin.     

Natural killer cells in the thymus. Studies in mice with severe combined immune deficiency

The relationship between NK cell and T cell progenitors was investigated by using mice with severe combined immune deficiency (scid). Scid mice are devoid of mature T and B cells because they cannot rearrange their Ig and TCR genes. However, they have normal splenic NK cells. Thymus of scid mice, although markedly hypocellular, contains cells that lyse YAC-1, an NK-sensitive tumor cell. By flow cytometry, two populations of cells were identified in the scid thymus. Eighty percent of the cells were Thy-1+, IL-2R(7D4)+, J11d+, CD3-, CD4-, CD8- whereas the remaining were IL-2R-, J11d-, CD3-, CD4-, and CD8-. By cell sorting, all NK activity was found in the latter population, which is phenotypically similar to splenic NK cells. To determine if the thymus contains a bipotential NK/T progenitor cell, J11d+, IL-2R+ cells were cultured and analyzed for the generation of NK cells in vitro. These cells were used because they resemble 15-day fetal and adult CD4- CD8- thymocytes that are capable of giving rise to mature T cells. Cultured J11d+ thymocytes acquired non-MHC-restricted cytotoxicity, but in contrast to mature NK cells, the resulting cells contained mRNA for the gamma, delta, and epsilon-chains of CD3. This suggests that J11d+ cells are early T cells that can acquire the ability to kill in a non-MHC-restricted manner, but which do not give rise to NK cells in vitro. The differentiative potential of scid thymocytes was also tested in vivo. Unlike bone marrow cells, scid thymocytes containing 80% J11d+ cells failed to give rise to NK cells when transferred into irradiated recipients. Together these results suggest that mature NK cells reside in the thymus of scid mice but are not derived from a common NK/T progenitor.