In vitro growth of murine T cells. IV. Use of T-cell growth factor to clone lymphoid cells

Murine bone marrow cells can suppress the in vitro primary antibody response of normal spleen cells without apparent cytotoxicity. The bone marrow cells suppress the response to both T-dependent (SRBC) and T-independent (DNP-Ficoll) antigens. When bone marrow cells are fractionated on a sucrose density gradient, the suppressive activity is found in the residue rather than the lymphocyte fraction. The suppressive activity is either unaffected or enhanced by treatment with anti-T- and anti-B-cell serums. Pretreatment of mice with phenylhydrazine which reduces the number of pre-B cells did not reduce the suppressive activity of their bone marrow cells. Suppressive activity is abolished by irradiation of the marrow cells in vitro with 1000 R prior to assay. The activity is present in the marrow of thymus deficient (nude) mice, infant mice, and mice which have been made polycythemic by transfusion. Furthermore, the suppressor cell can phagocytize iron carbonyl particles, is slightly adherent to plastic and Sephadex G-10, and can bind to EA monolayers. We conclude that the suppressor cell is not a mature lymphocyte or granulocyte nor a member of the erythrocytic series, but is likely to be an immature cell possibly of the myeloid series. We speculate on the physiologic role of this cell.     

Subunit composition of a high molecular weight oligomer: Limulus polyphemus hemocyanin

The hemocyanin of Limulus polyphemus is a 48-subunit aggregate. This 3.3 × 10⁶-dalton oligomer is composed of structurally and functionally heterogeneous subunits. Using polyacrylamide electrophoresis J. Markl, A. Markl, W. Schartau, and B. Linzen (J. Comp. Physiol. Ser. B130,283–292, 1979) observed 12 bands; while using immunoelectrophoresis, M. Hoylaerts, G. Preaux, R. Witters, and R. Lontie (Arch. Int. Physiol. Biochem.87, 417–418, 1979) and J. Lamy, J. Lamy, J. Weill, J. Bonaventura, C. Bonaventura, and M. Brenowitz. (Arch. Biochem. Biophys.196, 324–339, 1979) observed 8 subunits. To proceed with an analysis of subunit roles in assembly it is first necessary to determine the number of distinct subunits. Refinement of the chromatographic separation procedures has led to the isolation of 8 immunologically distinct subunits as well as additional charge isomers which cannot be distinguished immunologically. Alkaline electrophoresis revealed 15 bands and isoelectric focusing up to 17. On the basis of extensive control experiments, including composit acrylamide-agarose immunoelectrophoresis and checks for conformational isomers, aggregation, proteolysis, and other types of degradation, we conclude that the electrophoretic heterogeneity of immunologically identical subunits is not artifactual. We have extended the nomenclature used by Lamy et al. (1979) to include the electrophoretic heterogeneity by using primes (′) to denote electrophoretically distinguishable subunits which are immunologically identical. A number of patterns have become apparent by correlating the results obtained by the different techniques. For example, immunologically pure subunit II, which shows 3 bands on alkaline electrophoresis, is in fact a mixture of electrophoretically distinct subunits II, II′, II″. Except for subunits II, II′, and II″ immunoelectrophoretically identical subunits are typically homogeneous on sodium dodecyl sulfate-gels. However, slight differences in the apparent molecular weight are observed on high-resolution gels between immunologically unrelated subunits. The immunological identity and electrophoretic differences suggest that the charge isomers which are immunologically identical have similar antigenic surfaces. If a charge substitution is not in a critical location, we would expect the electrophoretically distinct but immunologically identical subunits to have identical assembly roles. Comparison of the results for Limulus hemocyanin with the hemocyanin of related species Eurypelma californicum and Androctanus australis, which have 7 and 8 immunologically distinct subunits, respectively, suggests that the calcium-mediated aggregation from 24 to 48 subunits of Limulus does not require more extensive subunit complexity.     

Electrophysiological evidence for the autoregulation of β-cell secretion by insulin

Electrophysiological studies of cultured rat pancreatic β-cells using intracellular microelectrodes show that exogenous insulin over the range of 0.1–10.0 μg/ml inhibits the electrical activity due to 27.8 mM glucose in a dose-related manner. This inhibitory effect is manifested by a mean increase of the membrane potential from about ?20 to ?30 mV and inhibition of the manner of cells impaled showing spike activity from 60 to less than 10%. The inhibitory influence of insulin is rapid occuring within 5 min for the highest level used. The results provide evidence for a negative feedback role of insulin in regulating its own release.     

A catalytic method for determination of trace amounts of iron in biological material

A rapid, simple, and reproducible method for determination of iron in biological material is suggested using the oxidation of p-phenetidine hydrochloride with hydrogen peroxide in a reaction catalyzed by Fe(III) and activated by 1,10-phenanthroline. The high sensitivity of the reaction allows a single determination to be carried out with as much as 1–5 mg fresh tissue.     

Analysis of the effects of cesium ions on potassium channel currents in biological membranes

Cesium ions block potassium channels in biological membranes in a voltage dependent manner. For example, external cesium blocks inward current with little or no effect on outward current. Consequently, it produces a characteristic N-shaped current-voltage relationship. We have modeled this result by single file diffusion of ions in a narrow channel spanning the membrane with a special blocking site in the channel for cesium ions. The model enables us to make detailed comparisons of the effects of cesium on potassium channels in different types of biological membranes.     

Myocardial hypertrophy, cardiac and urinary catecholamines during severe ethanol intoxication and withdrawal

Cardiomegaly was observed in rats severely intoxicated with ethanol for a 4 day period. It was apparent at 48 hours of treatment, a time at which cardiac protein was elevated and continued into withdrawal. During a 4 day abstinence period the degree of hypertrophy declined towards normal. Cardiac noradrenaline was reduced at the 48 hour time of intoxication, then increased gradually in the further experimental period. As the cardiac hypertrophy occurs at a time that urinary catecholamines are elevated and the adrenal medulla is intensely stimulated, it is proposed that increased levels of circulating catecholamines are largely responsible for the enlargement of the heart.     

Temperature-shift analysis of conidial development in Aspergillus nidulans

Temperature-shift experiments have been performed on spore-originated colonies of 11 thermosensitive aconidial mutants of Aspergillus nidulans in order to determine the latest time of shift to the restrictive temperature that prevents the initiation of conidiation. This time defines the beginning of the thermosensitive period (TSP) of the mutant. Eight of the mutants have TSPs that begin in the 7-hour period (32–39 hr) just prior to the first appearance of conidia-bearing structures, while 3 of the mutants have TSPs that begin later and very close to the time of onset of conidiation (45 hr). Thus no mutant of the set has a TSP that begins during the first 32 hr of vegetative growth of spore-originated colonies. For all mutants, an upshift performed after the beginning of the TSP allows initiation of conidiation at close to the normal time and at the normal rate, but results in an abrupt cessation of conidiation at some fixed time after upshift, characteristic of the mutant. The mutant whose TSP begins the earliest (aco-49) is exceptional in that, if conidiation is suppressed by growth of colonies in submerged culture, this mutant becomes thermoinsensitive during vegetative submerged growth; in contrast, the remaining 10 mutants become thermoinsensitive only after the suppressive condition has been relieved. We discuss the possibility that this exceptional mutant is defective in a function required for initiation of the process that ultimately results in the formation of conidia.     

Sequential elution of proteins in small volumes by preparative polyacrylamide gel electrophoresis

A simple flexible method for separation of proteins by polyacrylamide gel electrophoresis and sequential elution into dialysis bags has been devised. The system was applied to isolation of three glycoproteins from the peritoneal fluid of mice bearing Ehrlich ascites tumor.     

A near-infrared method for studying hydration changes in aqueous solution: Illustration with protease reactions and protein denaturation

Proteins undergoing protease reactions, heat denaturation, or interactions with sodium dodecyl sulfate (SDS) were used to demonstrate the effectiveness of a near-infrared method for the quantitative study of changes in hydration or water binding during such processes. The spectra of different proteins showed that the liberation of $\text{COO}{}^{\mathrm{?}}\text{and NH}{}_3{}^{\mathrm{+}}$ groups during a protease reaction is associated with a large increase in hydration and excluded volume. On the basis of experiments with model compounds, other spectral changes, including development of continuum absorbance between 1.55 and 1.85 μm and a band with a peak near 2.1 μm, were also attributed to the liberation of these groups. After heat denaturation or in the presence of SDS, the rate of proteolytic hydrolysis was markedly increased, consistent with the view that some preliminary denaturation is necessary for protease activity. The validity of the hydration changes calculated for protease reactions was supported by model studies with l-lysine, and with poly-l-lysine before and after hydrolysis. The near-infrared spectrum of the protein substrate with no added protease was largely unaffected by heat treatment alone, indicating that the hydration as such was not changed to a large extent by the structural modifications of denaturation. In contrast to the protease reaction, the interactions between SDS and the proteins resulted in a decrease in hydration. Results of this paper are compared with those obtained from other methods. Some unique advantages of the near-infrared method for the study of hydration changes during reactions in aqueous solution are described.