Live-cell three-dimensional single-molecule tracking reveals modulation of enhancer dynamics by NuRD.

To understand how the nucleosome remodeling and deacetylase (NuRD) complex regulates enhancers and enhancer-promoter interactions, we have developed an approach to segment and extract key biophysical parameters from live-cell three-dimensional single-molecule trajectories. Unexpectedly, this has revealed that NuRD binds to chromatin for minutes, decompacts chromatin structure and increases enhancer dynamics. We also uncovered a rare fast-diffusing state of enhancers and found that NuRD restricts the time spent in this state. Hi-C and Cut&Run experiments revealed that NuRD modulates enhancer-promoter interactions in active chromatin, allowing them to contact each other over longer distances. Furthermore, NuRD leads to a marked redistribution of CTCF and, in particular, cohesin. We propose that NuRD promotes a decondensed chromatin environment, where enhancers and promoters can contact each other over longer distances, and where the resetting of enhancer-promoter interactions brought about by the fast decondensed chromatin motions is reduced, leading to more stable, long-lived enhancer-promoter relationships.

Antitumor activity of gamma-interferon in ascitic and solid tumor models of human ovarian cancer.

We have investigated the action of recombinant human gamma-interferon (rHuIFN-gamma) against human ovarian cancer xenografts growing as ascites or as bulky solid i.p. tumors in nude mice. Both forms of the disease responded to i.p. rHuIFN-gamma with significant increases in mouse survival time, and in 2 of 3 ascitic models the mice were cured of peritoneal disease. The activity of rHuIFN-gamma was dose and schedule dependent, and xenografts derived from 3 different patients showed a heterogeneity of response. Peak i.p. levels of rHuIFN-gamma in nude mice bearing multiple i.p. solid tumors were similar to those found in ovarian cancer patients receiving i.p. rHuIFN-gamma, but clearance was more rapid in the mice. Rat gamma-interferon had no antitumor activity at the same doses and schedules although it had some biological activity in the nude mice. Histological examination of treated tumors revealed increased necrosis and loss of cellular organization with large areas of hypocellular epithelial mucin. These changes were preceded by a fall in tumor tryptophan and a rise in tumor kynurenine. We conclude that rHuIFN-gamma has a direct dose related antitumor effect on ovarian cancer xenografts that is preceded by increased metabolism of tryptophan.

The Nucleosome Remodeling and Deacetylase Complex NuRD Is Built from Preformed Catalytically Active Sub-modules.

The nucleosome remodeling deacetylase (NuRD) complex is a highly conserved regulator of chromatin structure and transcription. Structural studies have shed light on this and other chromatin modifying machines, but much less is known about how they assemble and whether stable and functional sub-modules exist that retain enzymatic activity. Purification of the endogenous Drosophila NuRD complex shows that it consists of a stable core of subunits, while others, in particular the chromatin remodeler CHD4, associate transiently. To dissect the assembly and activity of NuRD, we systematically produced all possible combinations of different components using the MultiBac system, and determined their activity and biophysical properties. We carried out single-molecule imaging of CHD4 in live mouse embryonic stem cells, in the presence and absence of one of core components (MBD3), to show how the core deacetylase and chromatin-remodeling sub-modules associate in vivo. Our experiments suggest a pathway for the assembly of NuRD via preformed and active sub-modules. These retain enzymatic activity and are present in both the nucleus and the cytosol, an outcome with important implications for understanding NuRD function.

Recombinant human interleukin-1 beta primes human polymorphonuclear leukocytes for stimulus-induced myeloperoxidase release.

Recombinant human Interleukin-1 (rhIL-1) beta was found to enhance stimulus-induced granule exocytosis from human polymorphonuclear leukocytes (PMNs). PMNs were incubated with rhIL-1 beta and then stimulated with either heat-aggregated IgG (Hagg) or N-formyl-methionyl leucylphenylalanine (FMLP). The release of the azurophil enzyme myeloperoxidase (MPO) was measured. Low concentrations of stimuli (10 micrograms/ml Hagg, 2.5 X 10(-9) M FMLP) did not stimulate degranulation in the absence of rhIL-1 beta. However, such concentrations elicited marked degranulation from PMNs preincubated with rhIL-1 beta (0.2-100 ng/ml). The enhancement of degranulation was dependent on the concentration of rhIL-1 beta employed and on the period of incubation. In other experiments, the effect of rhIL-1 beta on the PMN oxidative response was determined. rhIL-1 beta did not directly stimulate the production of superoxide anions or enhance the oxidative response to Hagg or FMLP. It is suggested that in rheumatoid joints, IL-1 beta may potentiate PMN degranulation, but not their oxidative response.

21.1.1, a novel activation marker of T and B cells.

A new rat mAb designated mAb 21.1.1 was raised against a T cell hybridoma of mouse origin, T2D4. This antibody, an IgG2b, immunoprecipitates from the membrane extracts of iodinated T2D4 cells a 56-kDa glycoprotein of apparent pI 4.6 which gives a 34-kDa polypeptide after treatment with endoglycosidase F. MAb 21.1.1 reacts with an antigen expressed on murine mitogen-activated thymocytes and T cells, and on B cells stimulated by anti-IgM antibodies. Cells isolated from the spleen, lymph nodes and bone marrow are negative, as are purified resting B cells or T cells. This antigen is strongly expressed on most day-16 fetal thymocytes whereas adult thymocytes are almost negative. mAb 21.1.1 may be useful for the study of activation and differentiation of T and B cells.

Adrenocorticotropin analogs and glucocorticoids in the hypophysectomized rat. II. Effects on cerebral cortex polyribosomes.

The capacity of free polyribosomes from rat cerebral cortex to incorporate labeled amino acids into protein (polyribosome activity) was compared in normal, hypophysectomized, and treated hypophysectomized rats. Polyribosome activity was measured in a cell-free system using a pH 5 supernatant from sham-operated rats. The polyribosome activity of hypophysectomized rats was 10-20% less than that of sham-operated rats. Subcutaneous treatment of hypophysectomized rats daily for 10 days with ACTH, corticotropic ACTH fragments [10-2- microgram of ACTH-(1-24), ACTH-(1-23), or Hoe 433 (ACTH-(1-17))], or glucocorticoids (1 mg corticosterone or 10 microgram dexamethasone) stimulated activity to a level 25-50% higher than that in sham-operated rats. Polyribosome aggregation, as measured by sucrose density gradient analysis, was also greater in treated hypophysectomized rats than in sham-operated rats. On the other hand, a daily sc dose of 100 microgram of the noncorticotropic fragment, ACTH-(1-10), did not stimulate brain polyribosome activity in hypophysectomized rats but merely restored it to the level observed in sham-operated rats. The present study suggests that stimulation of cerebral protein synthesis by ACTH and corticotropic ACTH analogs may be partly due to their ability to promote adrenal steroid secretion. Their stimulatory effect and that of glucocorticoids might explain their physiological roles during stress and learning.

Adrenocorticotropin analogs and glucocorticoids in the hypophysectomized rat. I. Effects on liver polyribosomes and rough endoplasmic reticulum.

Hypophysectomy decreased polyribosome aggregation and activity in rat liver. Polyribosome aggregation was measured by sucrose density gradient analysis and polyribosome activity was determined by the incorporation of labeled amino acids into protein in a cell-free system using pH 5 enzymes from sham-operated rats. Hypophysectomy also totally disorganized the rough endoplasmic reticulum of hepatocytes, as shown by electron micrographs. The sc administration of the ACTH fragments, Hoe 433 (corticotropic) and Org 2766 (noncorticotropic), and of glucocorticoids (dexamethasone or corticosterone) to hypophysectomized rats restored these parameters to the levels recorded in sham-operated rats in a dose-dependent fashion 5 h after injection; the effects were maintained until at least 24 h. The sc administration of the noncorticotropic but metabolically less stable fragment, ACTH-(1-10), had only a partial effect. The potency of a noncorticotropic ACTH fragment (Org 2766) in hypophysectomized rats suggests that ACTH may act on liver protein synthesis without the intervention of glucocorticoids or pituitary hormones.

Stability of 70S ribosomes in relation to misreading and antibacterial activity of aminoglycosides.

The anomeric aminoglycosides, RU 21886 and RU 23468, which both have a 2-deoxystreptamine residue, stabilize 70S ribosomes to similar extents at low magnesium ion concentrations. Only RU 21886, however, has marked antibacterial and bactericidal activity and gives rise to a high level of misreading in cell-free protein synthesizing systems. It would thus appear that the ability to stabilize the association of the two ribosomal subunits does not necessarily lead to errors in translation.

Autocrine stimulation of interleukin 1 in human adherent synovial lining cells: down regulation by interferon gamma.

Interleukin 1 (IL-1) exerts biological properties on various immune and nonimmune cell types and tissues and thus may play an important role during chronic inflammatory processes. Here we have examined the IL-1 biosynthesis in adherent synovial lining cell (ASLC) cultures obtained from patients with rheumatoid arthritis (RA). We report that ASLCs in culture showed heterogeneous endogenous levels of IL-1 alpha and beta expression. Recombinant interleukin 1 (rIL-1) alpha or beta induced increases of IL-1 alpha and beta mRNA and proteins levels in ASLCs. Although IL-1 synthesis is enhanced by rIL-1 treatment, no soluble IL-1 alpha or beta could be detected by specific enzyme-linked immunosorbent assays. A pretreatment with recombinant IFN gamma (rIFN gamma) down-regulated the effect of rIL-1 on IL-1 synthesis in ASLCs. Actinomycin D suppressed the endogeneous and rIL-1-induced IL-1 mRNA expression Indomethacin, in the presence of rIL-1 alpha or beta, up-regulates the level of expression of IL-1 beta in ASLCs pretreated with rIFN gamma, but has the opposite effect in non-pretreated cells. The increase of IL-1 gene expression by rIL-1 in human ASLCs from RA patients may contribute as an amplification of the disease progress. These studies may also explain the beneficial effects of IFN gamma in experimental models of IL-1-induced bone and cartilage degradation and in patients with diseases involving IL-1.

Effects of low dose total body irradiation (LDTBI) and recombinant human interleukin-2 in mice.

10-16-Week-old female BALB/c mice received low dose total body irradiation (LDTBI) in one fraction immediately before the beginning of treatment with recombinant human interleukin-2 (rIL-2). LDTBI prevented in a dose-dependent manner the weight increase of the spleen, liver and lungs induced by fluid extravasation provoked by rIL-2 injections. It also limited the increase of the number of mononuclear cells in the spleen induced after in vivo treatment with rIL-2. Immunofluorescence analysis of spleen cells revealed that LDTBI decreased the relative sIgM+ cell number in spleen, while the relative numbers of Lyt-1+, Thy-1+ and L3T4+ cells were increased, indicating that a T and/or NK population, radioresistant to LDTBI, could still proliferate under rIL-2 stimulation in vivo. Such lymphocytes were capable of in vitro lysis of YAC cells in a 4-hour 51Cr release assay, as well as lymphokine-activated killer (LAK) cells obtained in mice treated with rIL-2 alone. Thus, LDTBI given prior to rIL-2, yet preserving the cytotoxic capacity of the LAK cells activated by rIL-2, could prevent the vascular leak syndrome toxicity induced by rIL-2 injection.

Interferon gamma is active on human lymphoblastoid Namalva cells without inducing an antiviral state.

We demonstrate the presence of high affinity receptors specific for interferon-gamma (IFN-gamma) in human lymphoblastoid Namalva cells. The presence of these receptors, whose binding affinity and cross-linking characteristics were not distinguishable from those of the corresponding receptors in sensitive cells, was not consistent with the lack of responsiveness of Namalva cells to IFN-gamma as regards growth inhibition, induction of 2'-5' oligoadenylate synthetase activity and inhibition of virus multiplication. Nevertheless, IFN-gamma enhanced the expression of two genes, HLA class II and c-myc. Although the mechanism of these IFN-gamma-mediated modifications is not understood, these results provide evidence that the IFN-gamma receptors present in Namalva cells are functional.

Developmental studies with the 14-3-2 antigen and the neuron specific enolase (NSE) associated activities-I.

We have compared the rodent developmental pattern of the 14-3-2 antigen estimated by a microcomplement fixation technique with that of the cerebral enolases. Chromatographic separation of enolase isozymes on microcolumns demonstrates that the embryonic neuron specific enolase is firstly and mostly represented by the ?? isozyme. The most important increase in 14-3-2 antigen and ?? enolase occurs between post-natal days 7th and 15th. By post-natal day 30, adult levels have been reached. An interesting observation is-during embryonic development-the decrease in the specific activity of the cerebral enolase isozyme ??. This could be explained by the replacement-in neuroblasts-of ?? enolase by neuron specific enolase. A comparison between 14-3-2 antigen and neuron specific enolase (??) purified by completely different methods is presented. The 14-3-2 antigen exhibits an enolase specific activity comparable to that of purified enzyme and has the same electrophoretic mobility. Antibodies raised against either antigen have an identical specificity. Pre and post-natal developmental pattern in rodent brains are similar for both proteins. Thus neuron specific 14-3-2 antigen is identical to neuron specific enolase. Thus we have precisely described the ontogenic transition between the three cerebral enolase isozymes at the tissue level. This study is completed by the analysis of these transitions at the neuronal cell level, using homogenous cell lines (Part II of this paper).

Transition between isozymic forms of enolase during in vitro differentiation of neuroblastoma cells-II.

An analysis of enolase expression during differentiation of neuroblastoma clones in homogeneous culture is presented. The enolases expressed in these neuroblast-like cells are identical to those of mouse brain with respect to the examined properties. Our biochemical investigation has premitted us to demonstrate formally that neuroblastoma cells undergo a transition from the embryonic ?? form to the neuronal ?? form and contain both enolases as well as the ?? hybrid form during maturation. These results suggest that the same phenomenon must exist in vivo for neuroblasts. In neuroblastoma cells, an increase in both ?? and ?? neuron specific enolases is related to cell maturation and expression of the ?? form precedes that of the ?? form during differentiation. Modulation of neuronal enolase activities is similar in the various conditions of differentiation studied and appears not to be necessarily related with morphological differentiation, although concomitant with an arrest of cell division. The evolution of specific neuronal enolases in neuroblastoma cells parallels that observed in vivo, in brain from embryonic day 15 to post-natal day 7. Moreover, at least one treatment (dimethylsulfoxide) causes an important decrease in the high specific ?? activity of these cells as occurs in vivo. This enolase can therefore also be considered as a biochemical marker for neuroblastoma maturation. As observed with other markers and other cell types, neuroblastoma cells in culture express an immature biochemical differentiation of the enolase isozymes.

Detection and localization of 14-3-2 protein in primary cultures of embryonic rat brain.

The development of embryonic rat brain in cell cultures was studied by an immunocytochemical method based on the detection of 14-3-2 protein (neuron-specific enolase or NSE), a neuron-specific protein. This protein was already present in undifferentiated neurons (less than 5 days in culture), being dispersed throughout the cytoplasm, though seemingly concentrated in the vicinity of polyribosomal structures. It was not found in nuclei, in mitochondria or in the Golgi apparatus. During neuron differentiation, the location of 14-3-2 protein was related to neurite development insofar as it was detected along the axon and even in what could be taken to be the presynaptic region of numerous interneuron contacts. In contact areas, a thickening of the junction membrane was observed but the presence of 14-3-2 protein was always unilateral demonstrating the absence of a true synapse and reflecting the halt in neurite development observed after 15 days in culture. The presence of 14-3-2 protein in the cell cultures was confirmed by a microcomplement fixation assay. The protein detected in cell cultures had the same immunological properties as that found in the 17-day-old embryo, but was slightly different from that found in adult rat brain. This observation can be confronted with the lack of neuron maturation in the immunocytochemical studies.

Glucocorticoids modulate the response of ornithine decarboxylase to unilateral removal of the dorsal hippocampus.

The effect of unilateral removal of the dorsal hippocampus and of glucocorticoid administration was measured on the activity of ornithine decarboxylase (ODC) in the remaining contralateral hippocampus lobe. Unilateral hippocampectomy (Hx) resulted in a rapid rise of ODC activity in the contralateral lobe. The effect on ODC was maximal at 6 h after surgery and lasted two days. In the absence of the adrenals the effect of Hx on the enzyme was more potent and more prolonged. Elevated ODC activity was still detectable at 5 days after surgery, but not at 10 days. Chronic replacement with dexamethasone (DEX) offered in drinking water decreased the Hx-induced ODC response of ADX rats at 3 days after surgery to the level of enzyme activity observed in the S-ADX Hx subject. The effect of the steroid seemed related to the extent of occupation of the pool of glucocorticoid receptor sites in cytosol of rat hippocampus. In contrast, a single injection of a high dose of DEX to Hx-ADX animals at 3 days after surgery increased ODC activity in addition to the lesion-induced ODC in the contralateral lobe. It is concluded that after unilateral removal of the dorsal hippocampus ODC is a biochemical marker for cellular responses taking place in the contralateral lobe. Glucocorticoids modulate the lesion-induced ODC response.

Effect of rIFN-gamma and IL-2 treatments in mouse and nude rat infections with Toxoplasma gondii.

Mice and nude rats lethally infected with T. gondii and treated with recombinant rat interferon-gamma (rIFN-gamma) or recombinant human interleukin-2 (rIL-2) were protected against death, when compared with untreated infected controls. In mice rIFN-gamma and rIL-2 played an important role in "prophylactic treatment", but not in "curative therapy". The survival rate was 42% in mice treated with 3 doses of 20,000 U of rIFN-gamma at days -2, -1, 0 before challenge and up to 66% in mice treated with 3 doses of 10,000 U of rIFN-gamma at days -2, 0, +2 before and after infection. Whereas the survival rate was 33% in mice that received 3 doses of 500 U rIL-2 at days -2, -1, 0 before infection, or -2, 0, +2 before and after infection respectively, up to 50% of the mice treated with 3 doses of 1,000 U rIL-2 at days -2, -1, 0 survived. In nude rats rIFN-gamma had a slight effect in "prophylactic treatment", whereas rIL-2 was active only in "curative treatment". The survival rate was 25% both in nude rats treated with doses of 400,000 U of rIFN-gamma at days -3, 0 before challenge, or with doses of 5,000 U of rIL-2 at days +2, +6, +9 after infection. These results lead us to hypothesise that the mechanism by which the lymphokine treatment exerts a protective effect on Toxoplasma infected mice is different from that on nu/nu rats. We conclude that these cytokines may play a notable role in modulating the host's immune defence against T. gondii infection.

Melting of cross-linked DNA: I. Model and theoretical methods.

Covalent and strong coordination binding to DNA of a large number of antitumour drugs and other compounds leads to interstrand cross-link formation. To investigate cross-link influence on double helix stability, two methods are developed for the calculation of melting curves. The first method is based on Poland's approach. It requires computer time proportional to u.N, where u is the average distance (in base pairs) between neighboring cross-links and N is the number of base pairs in the DNA chain. The method is more suitable when u is not large, and small loops formed by interstrand cross-links in melted regions strongly affect DNA melting. The computer time for the second method, based on the Fixman-Freire approach, does not depend on the number of cross-links and is proportional to I.N (I is the number of exponential functions used for a decomposition of the loop entropy factor). It is more appropriate when N and u are large, and therefore particular values of the entropy factors of small loops do not influence DNA melting behavior.

Long-range interactions between ligands bound to a DNA molecule give rise to adsorption with the character of phase transition of the first kind.

Influence of long-range interactions between ligands bound to DNA molecule on the character of their adsorption is studied using computer modeling. For this investigation, two calculation procedures are developed. They are based upon the method of the free energy minimum and on the partition function method. The both procedures demonstrate that in the case of a strong enough attraction between all the bound ligands their binding to DNA has the character of phase transition of the first kind. There is a break in the binding curve c(c0) where c - relative concentration of bound ligands, c0 - molar concentration of free ligands. The break occurs because there is an interval of central degrees of binding (approximately 50% of the maximum c value) that are prohibited for individual DNA molecules. Such a transition might be caused by some types of DNA condensation. Attraction between the neighboring ligands only, adjacent or/and separated by double helix regions, does not cause this effect.

Further evidence for the presence of specific binding sites for prolactin in the rabbit brain. Preferential distribution in the hypothalamus and substantia nigra.

In the present research we have extended our work on the presence of binding sites for prolactin in the rabbit brain focusing our attention on the brain areas with high dopamine cell bodies density. Among these areas the hypothalamus showed the highest specific binding of labeled ovine prolactin (oPRL). Clearly detectible specific binding was observed also in substantia nigra, whereas in other brain regions the specific binding was very small, except for the striatum where a low but not negligible binding was found in female rabbits. The binding of 125I-oPRL showed a hormonal specificity and Scatchard analysis of the binding showed no clear difference in dissociation constant (Kd) between hypothalamus, nigra and striatum.