Spontaneous cytotoxicity of human lymphoblast cell lines mediated by normal peripheral blood lymphocytes. I. Differential susceptibility of T-versus B-cell lines.

Human lymphoblast cell lines of B- and T-cell origin have been tested for their ability to serve as targets in a 4-hr ⁵¹Cr release microcytotoxicity assay using normal human peripheral blood lymphocytes as effector cells. Cell lines of T-cell origin were susceptible to lysis in this assay by effector lymphocytes from all normal donors tested. Cell lines of B-cell origin were repeatably lysed by normal lymphocytes from some, but not all donors. Spontaneous cytotoxicity of B-cell lines, when observed, was also quantitatively less than was obtained using T-cell lines as targets. One cell line (RPMI-7666), of B-cell origin, was not susceptible to spontaneous cytotoxicity by almost all of the normal lymphocyte effectors tested. Lymphocytes from patients with acute lymphoblastic leukemia in remission were less capable of effecting lysis in this assay.     

Macrophage activation in vivo and in vitro

Morphology, lysosomal enzyme activity and phagocytic ability were tested in peritoneal macrophage cultures after stimulation in vivo or in vitro with endotoxin, mineral oil or latex particles, and compared to the same parameters in normal peritoneal macrophages. Treatment with latex did not give changes in the parameters tested after in vivo or in vitro stimulation. In all other types of stimulation the cells displayed varying degrees of spreading and changes in granule content. Extensive ruffling of cell membrane was obvious in endotoxin-stimulated cells. The pattern of lysosomal enzyme activity was complex and depended on the means of stimulation. Acid phosphatase showed the greatest increase after both in vivo and in vitro stimulation, N-acetyl-glucosaminidase could not be increased in vitro. Internalization of opsonized red cells mediated by the Fc receptor increased after in vivo stimulation. No such change was observed after in vitro stimulation. Normal peritoneal macrophages do not internalize significantly via the C₃ receptor. In vivo stimulation triggered the capacity to internalize up to 45% of the attached red cells. A similar reaction was obtained in vitro when both endotoxin and FCS were added to the culture medium, but not when endotoxin or FCS were used alone. We conclude that the use of the term activation of macrophages should always be based on quantitative changes in well defined parameters. Changes in one parameter will not necessarily be accompanied by the whole range of biochemical and morphological perturbations. The capacity to ingest via the C₃ receptor may be the most useful parameter.     

Hageman factor substrates. Human plasma prekallikrein: mechanism of activation by Hageman factor and participation in hageman factor-dependent fibrinolysis.

Two molecular forms of prekallikrein can be isolated from pooled normal human plasma. Their approximate molecular weights by sodium dodecyl sulfate-gel electrophoresis are 88,000 and 85,000. The two bands observed are shown to represent prekallikrein by functional, immunochemical, and structural criteria. Both forms are cleaved by activated Hageman factor, they appear to share antigenic determinants, they are not interconvertible upon incubation with activated Hageman factor or kallikrein, and the ratio of kinin-generating, and plasminogen-activating activities of the preparations are independent of the relative proportion of each band. Activated Factor XII converts prekallikrein to kallikrein by limited proteolysis and two disulfide-linked chains designated kallikrein heavy chain (Mr = 52,000) and kallikrein light chains (Mr = 36,000 or 33,000) are formed. The active site is associated with the light chains as assessed by incorporation of [3H]diisopropyl fluorophosphate. No dissociable fragments were observed in the absence of reducing agents. However, kallikrein could digest prekallikrein to diminish its molecular weight by 10,000. In addition, two factors capable of activating plasminogen to plasmin have been isolated; one is identified as kallikrein. The second principle fractionates with Factor XI and is demonstrable in normal and prekallikrein-deficient plasma.     

Methanogenesis from acetate: a nonmethanogenic bacterium from an anaerobic acetate enrichment.

A methanogenic acetate enrichment was initiated by inoculation of an acetate-mineral salts medium with domestic anaerobic digestor sludge and maintained by weekly transfer for 2 years. The enrichment culture contained a Methanosarcina and several obligately anaerobic nonmethanogenic bacteria. These latter organisms formed varying degrees of association with the Methanosarcina, ranging from the nutritionally fastidious gram-negative rod called the satellite bacterium to the nutritionally nonfastidious Eubacterium limosum. The satellite bacterium had growth requirements for amino acids, a peptide, a purine base, vitamin B12, and other B vitamins. Glucose, mannitol, starch, pyruvate, cysteine, lysine, leucine, isoleucine, arginine, and asparagine stimulated growth and hydrogen production. Acetate was neither incorporated nor metabolized by the satellite organism. Since acetate was the sole organic carbon source in the enrichment culture, organism(s) which metabolize acetate (such as the Methanosarcina) must produce substrates and growth factors for associated organisms which do not metabolize acetate.     

Microbial transformation of 14C-labeled 2,4,6-trinitrotoluene in an activated-sludge system.

The fate of 14C-labeled 2,4,6-trinitrotoluene (TNT) in an activated-sludge system was investigated. No [14C]TNT could be detected in the contents of an aerated reactor after 3 to 5 days of incubation. No significant 14CO2 was formed, and the radioactivity was about equally divided between the floc and the supernatant. The radioactive carbon present in the microflora was mainly associated with the lipid and protein components, but the characteristic constituents of these compounds (e.g., fatty acids and amino acids) were not radioactive. The major part of the 14C present in the lipid and protein fractions was found in precipitates that formed in both fractions. The solubility properties and infrared spectra of these precipitates suggested that they are macromolecular structures of the polyamide type formed by the reaction of TNT biotransformation products with lipids, fatty acids, and protein constituents of the microbial flora. This hypothesis is further supported by the correspondence of the infrared spectrum of the lipid precipitate with that of a model compound synthesized from TNT transformation products and lipid precursors. The resistance of these macromolecules to further biodegradation was paralleled by the reported resistance to microbial attack of polyamides containing similar linkages.     

The extent of mitochondrial F1-ATPase and adenine nucleotide carrier activity with ϵ-ATP

1. The use of 1,N⁶-ethenoadenosine 5′-triphosphate (?-ATP), a synthetic, fluorescent analog of ATP, by whole rat liver mitochondria and by submitochondrial particles produced via sonication has been studied.2. Direct [³H]adenine nucleotide uptake studies with isolated mitochondria, indicate the ?-[³H]ATP is not transported through the inner membrane by the adenine nucleotide carrier and is therefore not utilized by the 2,4-dinitrophenol-sensitive F₁-ATPase (EC 3.6.1.3) that functions in oxidative phosphorylation. However, ?-ATP is hydrolyzed by a Mg²⁺-dependent, 2,4-dinitrophenol-insensitive ATPase that is characteristic of damaged mitochondria.3. ?-ATP can be utilized quite well by the exposed F₁-ATPase of sonic submitochondrial particles. This ?-ATP hydrolysis activity is inhibited by oligomycin and stimulated by 2,4-dinitrophenol. The particle F₁-ATPase displays similar K_m values for both ATP and ?-ATP; however, the V with ATP is approximately six times greater than with ?-ATP.4. Since ?-ATP is a capable substrate for the submitochondrial particle F₁-ATPase, it is proposed that the fluorescent properties of this ATP analog might be employed to study the submitochondrial particle F₁-ATPase complex, and its response to various modifiers of oxidative phosphorylation.     

Soluble NADH-cytochrome b5 reductase from rabbit liver cytosol: partial purification and characterization.

A soluble form of NADH-cytochrome b5 reductase (NADH: ferricytochrome b5 oxidoreductase, EC 1.6.2.2) was found in the cytosolic fraction of rabbit liver. The partially purified enzyme was strictly specific for NADH. It catalyzed the reduction of several substrates such as the methemoglobin-ferrocyanide complex (Hegesh, E. and Avron, M. (1967) Biochim. Biophys. Acta 146, 91-101) (apparent Km: 8 micrometer), potassium ferricyanide (apparent Km: 10 micrometer) and ferricytochrome b5 (apparent Km: 15 micrometer). Upon acrylamide gel isoelectro-focusing followed by specific staining, the enzyme was resolved into four bands (isoelectric pH: 7.05, 6.70, 6.50 and 6.30). The optimum pH of activity with ferricytochrome b5 as a substrate was 6.5. The estimated molecular weight was 25 000--30 000. The enzyme was unsensitive to cyanide. It was strongly inhibited by p-hydroxymercuribenzoate. The cytosolic liver cytochrome b5 reductase was immunologically related to the soluble cytochrome b5 reductase from human and rabbit red-cells, and to the microsomal cytochrome b5 reductase from rabbit liver.     

The use of the fluorescent probe alpha-parinaric acid to determine the physical state of the intracytoplasmic membranes of the photosynthetic bacterium, Rhodopseudomonas sphaeroides.

alpha-Parinaric acid has been used to determine the degree of ordering of the hydrocarbon region of purified intracytoplasmic membranes of Rhodopseudomonas sphaeroides. The usefulness of alpha-parinaric acid as a probe of membrane fluidity was established by comparison of its fluorescent properties in phosphatidylcholine vesicles with those of the more commonly used fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene. Both fluorescent probes were shown to monitor similar environments in the phosphatidylcholine vesicles when the phospholipids were maintained at temperatures above their phase transition temperature. The rotational mobility of alpha-parinaric acid in the intracytoplasmic membranes was determined from 0 to 50 degrees C, a region where no phase transitions were detectable. The rotational mobility of alpha-parinaric acid dissolved in vesicles formed from total extracted intracytoplasmic membrane phospholipids, was 2--3-fold greater than that measured in the intact intracytoplasmic membranes; demonstrating that the presence of protein greatly reduces the mobility of the phospholipid acyl chains of the intracytoplasmic membranes. Due to the high protein content of these membranes, the perturbing effect of protein on acyl chain mobility may extend to virtually all the intracytoplasmic membrane phospholipid.     

Effect of heat and 2-mercaptoethanol on intracytoplasmic membrane polypeptides of Rhodopseudomonas sphaeroides.

Solubilization at 75 degrees C of Rhodopseudomonas sphaeroides chromatophores in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (beta-ME) resulted in the selective absence of reaction center B and C polypeptides from SDS-polyacrylamide gel electrophoresis profiles. A newly identified, chromatophore-specific polypeptide, with a mass of 35.2 kdaltons, was also missing under these conditions of chromatophore solubilization. Solubilization at 27 degrees C in the presence of SDS and beta-ME also resulted in the disappearance of these three polypeptides, but at much slower rates. Disappearance of either endogenous or exogenously supplied reaction center polypeptides B and C during SDS solubilization of whole chromatophores at either 27 or 75 degrees C was shown to be entirely dependent upon the presence of beta-ME. After chromatophore solubilization in the presence of beta-ME and subsequent SDS-polyacrylamide gel electrophoresis, exogenously added reaction centers B and C could be localized in a complex of no less than 100 to 200 kdaltons. However, the precise size of the complex was influenced by the stoichiometry of the reacting components. The disappearance of the 35.2-kdalton polypeptide was neither dependent upon the presence of beta-ME nor dependent upon the presence of any additional chromatophore polypeptides. The 35.2-kdalton polypeptide underwent a heat-induced oligomerization to yield several high-molecular-weight species.     

Synthesis of photopigments and electron transport components in synchronous phototrophic cultures of Rhodopseudomonas sphaeroides.

The kinetics of synthesis and incorporation of the photosynthetic pigments and several of the major oxidative and photosynthetic electron transport components of Rhodopseudomonas sphaeroides have been studied during synchronous and asynchronous phototrophic growth. The photosynthetic pigments and cytochromes c and b, measured spectroscopically, exhibited continuous patterns of synthesis and incorporation into the membrane particulate fraction in both synchronous and asynchronous cultures. Succinic dehydrogenase and NADH-oxidase activities, present at low levelnous growth. In a previous paper, Leuking, D.R., Fraley, R.T., and Kaplan, S. ((1978) J. Biol. Chem. 253, 451-457) have shown that total cellular phospholipid is also accumulated discontinuously during synchronous growth. A continuously incorporated membrane component is thus subject to a wide variation in the membrane protein/lipid ratio. The significance of this ratio in regulating the activity of membrane proteins is discussed and the distinction between protein incorporation and function is drawn with particular reference to the photosynthetic pigments and cytochrome components and the oxidative activities measured. It is suggested that a dependence of membrane protein activity on the membrane protein to lipid ratio in vivo is of possible significance in the control of membrane synthesis and cell division.