Cellular regulation of ADP-ribosylation of proteins. III. Selective augmentation of in vitro ADP-ribosylation of histone H3 in murine thymic cells after in vivo emetine treatment

Thymic cells were isolated at intervals of between 0 and 144 h from mice that received one intraperitoneal injection of emetine (33 mg/kg), and thymus weight, incorporation of [14C]leucine into proteins and [3H]thymidine into DNA in intact thymic cells, as well as initial rates of protein ADP-ribosylation in permeabilized cells [A. Sóoki-Tóth, F. Asghari, E. Kirsten, and E. Kun (1987) Exp. Cell Res. 170, 93] were simultaneously monitored. The effect of emetine as an inhibitor of protein synthesis [F. Antoni, N. G. Luat, I. Csuka, I. Oláh, A. Sóoki-Tóth, and G. Bánfalvi (1987) Int. J. Immunopharmacol. 9, 333] corresponds to the induction of sequential cellular events, such as cell exit and remigration, by other antimitotic agents [C. Penit and F. Vasseur (1988) J. Immunol. 140, 3315] and produces an activation of proliferation of cells reentering into this organ. Proliferation, as demonstrated by a large increase in DNA synthesis and entrance into S phase, was kinetically related to an apparent increase in poly(ADP-ribose) polymerase activity in thymic cells and a highly significant in vitro ADP-ribosylation of histone H3. Since no DNA fragmentation occurred in thymic cells, as tested by a fluorometric technique [C. Birnboim and J. J. Jevac (1981) Cancer Res. 41, 1889], it is probable that a selective activation of poly(ADP-ribose) polymerase may have been induced in cells that undergo differentiation and proliferation while repopulating the thymus.     

Conversion of disaccharides to 3-ketodisaccharides by nongrowing and immobilized cells ofAgrobacterium tumefaciens

High-performance liquid chromatography was used to estimate 3-ketolactose and 3-ketosucrose in cultures of agrobacteria. The activities of enzymes that convert the disaccharide substrate were evaluated during batch cultivation ofAgrobacterium tumefaciens on sucrose, maltose, and lactose. The highest activity of glucoside 3-dehydrogenase and a slight activity of disaccharide-hydrolyzing enzymes were found in cells grown on lactose. Nongrowing cells converted lactose to 3-ketolactose faster than immobilized cells did. Immobilization of cells into polysaccharide gels did not stabilize the activity of glucoside 3-dehydrogenase. Glutaraldehyde cross-linking of the cell content led to an inactivation of the respiratory chain but Fe³⁺ could be used as an electron acceptor. Cells treated with glutaraldehyde converted lactose faster than nongrowing ones but the activity of glucoside 3-dehydrogenase was not stable.     

The 76 kD cell-adhesion factor from crayfish haemocytes promotes encapsulation in vitro

Summary Semigranular cells from the crayfish, Pacifastacus leniusculus, were separated by Percoll gradient centrifugation and were used to study the encapsulation of foreign particles. The semigranular cells were found strongly to encapsulate glass beads coated with haemocyte lysate in which the prophenoloxidase-activating system had been activated with laminarin or with a low concentration of calcium ions. The granular cells only weakly encapsulated these particles. The encapsulationpromoting factor was purified from haemocyte lysates and found to be a 76 kD protein which was recognized by an antiserum to the previously described 76 kD cell-adhesion factor. After the last step in purification (Con A-Sepharose chromatography), the flowthrough consisted of several proteins, which had some, but less, encapsulation-promoting activity and contained a 30 kD band that was also recognized by the antiserum to the 76 kD cell-adhesion factor. If the haemocyte lysate prepared in low [Ca²⁺] was incubated with a -1,3-glucan prior to purification, no 76 kD protein could be isolated but only a 30 kD protein. The 30 kD protein thus seems to be a degradation product of the 76 kD cell-adhesion factor. We conclude that the 76 kD protein which is released from degranulating haemocytes, and to a lesser extent its 30 kD fragment, can promote encapsulation. Phenoloxidase did not have any encapsulation-promoting activity.     

A new facultatively nitrite oxidizing bacterium,Nitrobacter vulgaris sp. nov.

A total of 17 facultatively lithoautotrophic strains of Nitrobacter were investigated. They all were found to be related on the species level by DNA hybridizations. The G+C content of DNA ranged between 58.9 and 59.9 mol %. The isolates originated from divers environments. The cells were 0.5–0.8×1.2–2.0 m in size and motile by one polar to subpolar flagellum. Cell-division normally occurred by budding. Polar caps of intracytoplasmic membranes as well as carboxysomes were present. The cells tended to excrete extracellular polymers forming aggregates or biofilms. Heterotrophic growth was slower than mixotrophic but often faster than litoautotrophic growth. In the presence of nitrite and organic substances the organisms often showed diphasic growth. First nitrite and then the organic material was oxidized. In the absence of oxygen growth was possible by dissimilatory nitrate reduction. Nitrite, nitric and nitrous oxide as well as ammonia were formed. Depending on growth conditions the generation times varied from 12 to 140 h. The new Nitrobacter spec. may be one of the most abundant nitrite-oxidizing bacteria in soils, fresh waters and natural as well as artificial stones. For this organism the name Nitrobacter vulgaris is proposed.The type strain is filed with the culture collection of the Institut für Allgemeine Botanik, Universität Hamburg, FRG.     

A dynamic theory of coordination of discrete movement

The concepts of pattern dynamics and their adaptation through behavioral information, developed in the context of rhythmic movement coordination, are generalized to describe discrete movements of single components and the coordination of multiple components in discrete movement. In a first step we consider only one spatial component and study the temporal order inherent in discrete movement in terms of stable, reproducible space-time relationships. The coordination of discrete movement is captured in terms of relative timing. Using an exactly solvable nonlinear oscillator as a mathematical model, we show how the timing properties of discrete movement can be described by these pattern dynamics and discuss the relation of the pattern variables to observable end-effector movement. By coupling several such component dynamics in a fashion analogous to models of rhythmic movement coordination we capture the coordination of discrete movements of two components. We find the tendency to synchronize the component movements as the discrete analogon of in-phase locking and study its breakdown when the components become too different in their dynamic properties. The concept of temporal stability leads to the prediction that remote compensatory responses occur such as the restore synchronization when one component is perturbed. This prediction can be used to test the theory. We find that the discrete analogon to antiphase locking in rhythmic movement is a tendency to move sequentially, a finding that can also be subjected to empirical test.     

Forming sparse representations by local anti-Hebbian learning

How does the brain form a useful representation of its environment? It is shown here that a layer of simple Hebbian units connected by modifiable anti-Hebbian feed-back connections can learn to code a set of patterns in such a way that statistical dependency between the elements of the representation is reduced, while information is preserved. The resulting code is sparse, which is favourable if it is to be used as input to a subsequent supervised associative layer. The operation of the network is demonstrated on two simple problems.     

Fragile×expression and×inactivation

Summary The inactive fragile×chromosomes of a 47,fra(X),fra(X),Y male with a typical fragile×phenotype were successfully separated from the active homologues by means of somatic cell hybridization. It was shown by FUdR-induction and caffein-posttreatment that the separated inactive×chromosomes expressed their fragile sites and that the presence of an active mutated \sxchromosome was not a prerequisite for fragile X expression. The fragility seems to be an intrinsic property of the individual fragile site. This result is in favour of the classical concept that the fragile site at Xq27.3 has a primary pathogenetic function in this syndrome, although the fragility itself could represent a secondary phenomenon related to an unknown alteration of the DNA in this chromosome region. It is also concluded that inactivation of the fragile\sxchromosome in females is not responsible for either false negative fragile\sxfindings or the observation of fragile\sxnegative colonies isolated from fragile\sxpositive fibroblasts in heterozygotes.     

Susceptibility of autologous target cells to lysis by lymphokine-activated effectors from interferon-α-treated chronic myelogenous leukaemia patients

Summary Chronic myelogenous leukemia (CML) patients in chronic phase display compromised lymphokine-activated killer (LAK) cell induction, which is partly restored after therapy with interferon . However, the relative resistance of the leukemic cells from these patients to autologous or allogeneic LAK lysis is not affected by this treatment. In an attempt to render CML cells more susceptible to lysis or cytostasis, they were precultured in serum-free medium with or without recombinant growth factors. In eight patients studied, interleukin-3 (IL-3) significantly enhanced the spontaneous short-term (6-day) proliferation of CML cells, with retention of ability to form colonies in methylcellulose. Culture in either medium alone or IL-3 led to a significant enrichment of CD14⁺ and CD33⁺ cells but to a reduction in CD34⁺ cells. In contrast, culture of the same cells in IL-2 (to generate autologous LAK activity) resulted in a loss of CD14⁺ and CD33⁺ as well as CD34⁺ cells but in a significant increase in CD3⁺ and CD56⁺ cells. Despite similarities in their phenotypes, IL-3 cultured cells but not those cultured in medium alone acquired susceptibility to lysis by the IL-2-cultured autologous LAK cells. These results may have significance for the design of novel combination immunotherapy in CML.This work was supported in part by the Deutsche Forschungsgemeinschaft (SFB 120)     

Heterozygous expression of X-linked chondrodysplasia punctata

Summary Two females showing partial expression of X-linked chondrodysplasia punctata were identified in a family. Bone dysplasia was caused by an aberrant X chromosome that had an inverse duplication of the segment Xp21.2–Xp22.2 and a deletion of Xp22.3-Xpter. To characterise the aberrant X chromosome, dosage blots were performed on genomic DNA from a carrier using a number of X-linked probes. Anonymous sequences from Xp21.2–Xp22.2 to which probes D2, 99.61, C7, pERT87-15, and 754 bind were duplicated on the aberrant X chromosome. The proposita was heterozygous for all these markers. Dosage blots also showed that the loci for steroid sulfatase and the cell surface antigen 12E7 (MIC2) were deleted as expected from the cytogenetic results. Mouse human cell hybrids were constructed that retained the normal X in the active state. Analysis of these hybrid clones for the markers from Xp21.2–Xp22.2 revealed that all the alleles of the informative markers, present in a single dosage in the genomic DNA, were carried on the normal X chromosome of the proposita. The duplicated X chromosome therefore had two identical alleles, indicating that the aberration resulted from an intrachromosomal rearrangement.