Isozyme characterization of cattle (Bos taurus) and American buffalo (Bison bison) cell cultures.

Four Bovidae cell lines (BEK-1, MDBK, Bu and EBTr) were characterized by means of enzymatic biochemical markers. Out of 15 enzymatic systems, 3--adenosine deaminase (Ada), phosphoglucomutase (Pgm) and nucleoside phosphorylase (Np)--were found to be polymorphic and quite suitable for biochemical identification of each cell line. The Bu cell line has shown a Np phenotypic pattern which could be distinctive of the Bison bison species.     

Characterization of the phorbol 12, 13-dibutyrate (P(Bu)2) induced binding between human lymphocytes

The mechanisms, cell surface structures, and cell types involved in the phorbol 12,13-dibutyrate (P(Bu)₂)-induced binding between human lymphocytes were studied. Induction of cell aggregation by 20 min treatment with P(Bu)₂ required Ca²⁺, an intact membrane, functional microfilaments, and the possible participation of an esterase or, less likely, a protease. Trypsin-sensitive cell surface structures were needed and neuraminidase (NANase) treatment slightly increased the intercellular binding. Retinoic acid, an anti-tumor promoting agent, was inhibitory. Calmodulin-dependent processes, microtubules, phospholipid methylation, intracellular levels of cyclic adenosine monophosphate, and cellular secretion did not seem to be involved. Cell conjugation between 24 hr P(Bu)₂-treated and untreated cells required participation of trypsin-sensitive cell surface structures in each of the interacting cells and NANase treatment of one partner slightly increased the intercellular binding. Thymocytes, T cells, mature B and Epstein-Barr virus-transformed B cells aggregated while pre-B, early B, and intermediate B lymphocytes derived from representative malignancies did not. The lack of aggregation was not due to the absence of phorbol ester receptors. It is concluded that the P(Bu₂)-induced intercellular binding is mediated by cell surface proteins, depends on certain enzymatic activities and metabolic events and involves certain cell types.     

Reduction of Np(V) and precipitation of Np(IV) by an anaerobic microbial consortium

A combination of experimental, analytical, and modeling investigations shows that an anaerobic, sulfate-reducing consortium reduced Np(V) to Np(IV), with subsequent precipitation of a Np(IV) solid. Precipitation of Np(IV) during growth on pyruvate occurred before sulfate reduction began. H₂ stimulated precipitation of Np(IV) when added alone to growing cells, but it slowed precipitation when added along with pyruvate. Increasing concentrations of pyruvate also retarded precipitation. Accumulation of an intermediate pyruvate-fermentation product – probably succinate – played a key role in retarding Np(IV) precipitation by complexing the Np(IV). Hydrogen appears to have two roles in controlling Np precipitation: donating electrons for Np(V) reduction and modulating intermediate levels. That Np(V) is microbially reduced and subsequently precipitated under anaerobic conditions has likely beneficial implications for the containment of Np on lands contaminated by radionuclides, but complexation by fermentation intermediates can prevent immobilization by precipitation.     

Extraction of Np(IV), Pu(IV) and Am(III) by bidentate organophosphorus extractant

This paper summarizes the extraction of Np(IV), Pu(IV) and Am(III) with dihexyl-N,Ndiethyl carbamyl methylene phosphonate (DHDECMP)-diethyl benzene (DEB) in nitric acid.The distribution ratio of Np(IV), Pu(IV) and Am(III) was studied as a function of a number of parameters such as concentration of nitric acid, salting-out reagent in the aqueous phase, contact time and temperature. Stripping and separation of Np(IV), Pu(IV) and Am(III) from the pregnant organic phase were also studied. The suitable stripping and separation conditions were obtained. The enthalpy changes ΔH_Np, ΔH_Pu, ΔH_Am associated with their extraction process were estimated individually. The composition of extracted complex of Np(IV), Pu(IV) and Am(III) was determined.     

Overexpression of ΔNp63 in a human nasopharyngeal carcinoma cell line downregulates CKIs and enhances cell proliferation

p63 belongs to a member of the tumor suppressor protein p53 family. Due to alternative promoter usage, two types of p63 proteins are produced. The ΔNp63 isoform lacks the N‐terminal transactivation domain and is thought to antagonize TAp63 and p53 in target gene regulation. ΔNp63 has been found to be overexpressed in numerous human squamous cell carcinomas, including nasopharyngeal carcinoma (NPC). However, the role of ΔNp63 overexpression in NPC pathogenesis has not been clear. In this study, we use a ΔNp63 overexpressing human NPC cell line (NPC‐076) to explore the possible roles of ΔNp63 in cell proliferation and cell‐cycle regulation. We found that the proliferation of NPC‐076 cell is greatly suppressed when the overexpressed ΔNp63 is silenced by specific ΔNp63 siRNA. Further studies show that ΔNp63 silencing results in the upregulation of CKIs, including p27^kip1 and p57^kip2 in both mRNA and protein levels. Cell‐cycle analysis shows that ΔNp63 silencing also results in an increased G1 phase cell and apoptotic cell population. Our findings indicate that ΔNp63 plays important roles in the regulation of NPC‐076 cell‐cycle progression, and may play a role in the maintenance of NPC‐076 tumor cell phenotype. J. Cell. Physiol. 219: 117–122, 2009. © 2008 Wiley‐Liss, Inc.     

Transfer of Np(V) nitrate from gastrointestinal segments of the adult rat

The transfer of soluble Np(V) nitrate was measured in gastrointestinal segments from adult rats by two procedures: instillation, in segments in which the physico-chemical form of Np might be modified by gastrointestinal factors; and perfusion, in segments in which the luminal state of Np remains constant. These assays allowed accurate measurement of the Np(V) transferred from the intestine to the whole body. The amount measured was proportional to segment length and to the duration of the experiments, which lasted for periods of 0.25 to 2 h. Under these experimental conditions, hourly transfer values were about 2 percent, both per millilitre of Np(V) solution instilled and per 10 cm of jejunum perfused. This flux is very much greater than that which may be deduced from studies in which Np was gavaged into intact rats. Intestinal transfer of Np was constant for Np concentrations ranging from 5 X 10(-12) M to 1 X 10-4) M. Raising the concentration of Np(V) to more than 1 X 10(-4) M reduced its intestinal transfer. Addition of Fe(II) also reduced it. The small intestine was the main site of Np(V) absorption, since the transfer from instilled jejunum was about 20 times that observed from the stomach, and no difference was noted between jejunal and duodenal transfer.     

Transmission of paternal chloroplasts in tobacco (Nicotiana tabacum)

Medgyesy et al. (1986, Mol. Gen. Genet. 204, 195–198) have described in Nicotiana plumbaginifolia and in an interspecific cross involving N. plumbaginifolia and N. tabacum a procedure for selecting cell lines derived from seedlings carrying paternal chloroplasts by taking advantage of a plastid-encoded mutation which confers resistance to streptomycin. We have extended their demonstration of occasional transmission of chloroplasts through pollen to the case of an intraspecific cross in N. tabacum. The line used as maternal parent, ITB19(sua), displayed a cytoplasmic male sterility due to the presence of a cytoplasm originating from N. suaveolens. The line used as paternal parent, SR1, was fertile and possessed mutant chloroplasts conferring resistance to streptomycin. From cell lines derived from 204 seedlings, three were regenerated into streptomycin-resistant buds. The plants derived from these three clones were male-sterile. Their progeny, after crossing with a wild type tobacco line, XHFD8, was resistant to streptomycin. Tests of resistance of the seedlings to tentoxin and restriction analyses of the chloroplast DNA indicated that two clones still had the maternal chloroplasts and were thus probably new streptomycin-resistant mutants, whereas the third one had acquired the chloroplasts of the paternal parent, but had retained the mitochondria of the maternal parent.Abbreviations cp-DNA chloroplast DNA - mt-DNA mitochondrial DNA - Np Nicotiana plumbaginifolia - Nt Nicotiana tabacum     

Analogs of diadenosine tetraphosphate (Ap4A)

This review summarizes our knowledge of analogs and derivatives of diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A), the most extensively studied member of the dinucleoside 5',5"'-P1,Pn-polyphosphate (NpnN) family. After a short discussion of enzymes that may be responsible for the accumulation and degradation of Np4)N's in the cell, this review focuses on chemically and/or enzymatically produced analogs and their practical applications. Particular attention is paid to compounds that have aided the study of enzymes involved in the metabolism of Ap4A (Np4N'). Certain Ap4A analogs were alternative substrates of Ap4A-degrading enzymes and/or acted as enzyme inhibitors, some other helped to establish enzyme mechanisms, increased the sensitivity of certain enzyme assays or produced stable enzyme:ligand complexes for structural analysis.     

Synthesis, structural studies and solubility properties of zinc(II), nickel(II) and copper(II) complexes of bulky tris(triazolyl)borate ligands

Tris(triazolyl)borate (Ttz) ligands are sterically similar to tris(pyrazolyl)borate (Tp) but complexes of Ttz show improved solubility in water and alcohols due to their propensity for forming hydrogen bonds. Recently developed bulky tris(triazolyl)borate ligands can produce four and five coordinate transition metal complexes and serve as models for enzyme active sites in an aqueous environment. Herein we report the synthesis of such complexes, i.e. (Ttz^tBu,Me)ZnCl, (Ttz^tBu,Me)ZnBr, (Ttz^tBu,Me)NiCl, and (Ttz^tBu,Me)CuCl, which were analyzed by X-ray crystallographic and spectroscopic methods [Ttz^tBu,Me = tris(3-t-butyl-5-methyl-1,2,4-triazolyl)borate]. (Ttz^tBu,Me)ZnCl crystallizes as two different polymorphs with cubic and monoclinic symmetry. Both polymorphs of (Ttz^tBu,Me)ZnCl and (Ttz^tBu,Me)ZnBr have tetrahedral zinc atoms whereas the geometries at the metal in (Ttz^tBu,Me)NiCl and (Ttz^tBu,Me)CuCl are distorted tetrahedral. All complexes are methanol soluble and they also dissolve in methanol/water mixtures with up to 60% water.     

Partial expression of monoaminergic (serotoninergic) properties by the multipotent hypothalamic cell line F7. An example of learning at the cellular level

A serum-free medium supplemented with a glial conditioned medium, a brain extract from 8-to 10-day-old mice, hormones, and eye-derived growth factor has been devised which permitted the mouse primitive hypothalamic nerve cell line F7 to express some biochemical properties typical of monoaminergic neurons. Maximal expression was obtained when the culture conditions were applied for 2 days. Most (90–95%) cells then synthesized [³H]serotonin from [⁴H]5-hydroxytryptophan (but not from [³H]tryptophan). No synthesis was detected in the presence of carbidopa (20 μM), therefore suggesting the involvement of l-aromatic-amino-acid decarboxylase in this process. In addition, F7 cells cultured in such serum-free medium exhibited the capacity of accumulating exogenous serotonin by an ouabain-sensitive mechanism. These data further supported that active molecules in the cell environment can induce, in a primitive cell line, some of the enzymatic activities associated with monoaminergic neurons. Since other well-defined culture conditions can promote the differentiation of the same clone into oligodendrocytes (De Vitry et al., 1983), it can be concluded that the F7 cell has the properties of an embryonic stem cell of the CNS which, depending on external signals, may switch into different alternative developmental neural pathways. We postulate that the stabilization of neuron-like properties due to repetitive cell stimulation by active signals in the environment may represent an example of learning at the cellular level.     

Genetic characterization of parental cell lines and biochemical analysis of somatic cell hybrids between mouse (RAG) cells and fibroblasts of Ateles paniscus chamek (primates,platyrrhini)

Mouse (RAG) cells, (deficient in hypoxanthine-phosphoribosyl-transferase), and Ateles paniscus chamek primary fibroblasts were used in fusion experiments to generate somatic cell hybrids. Both parental cell lines were genetically characterized by karyological and biochemical analyses with 27 isozyme systems. These procedures were useful for monitoring primate chromosome segregation in somatic cell hybrids, for detecting chromosome rearrangements of primate chromosomes, and for identifying individual primate chromosomes. These characterizations are necessary to distinguish between different hybrid cell lines and to generate a panel for gene mapping studies. This is achieved by selecting cell lines that segregate different sets of relatively few primate isozymes and chromosomes. Conversely, we eliminated hybrid cell lines either showing: (1) rearrangements between primate and mouse chromosomes, (2) extensive rearrangements of primate chromosomes, or (3) a large number of primate biochemical markers. © 1993 Wiley-Liss, Inc.     

The PHD domain of Np95 (mUHRF1) is involved in large-scale reorganization of pericentromeric heterochromatin

Heterochromatic chromosomal regions undergo large-scale reorganization and progressively aggregate, forming chromocenters. These are dynamic structures that rapidly adapt to various stimuli that influence gene expression patterns, cell cycle progression, and differentiation. Np95-ICBP90 (m- and h-UHRF1) is a histone-binding protein expressed only in proliferating cells. During pericentromeric heterochromatin (PH) replication, Np95 specifically relocalizes to chromocenters where it highly concentrates in the replication factories that correspond to less compacted DNA. Np95 recruits HDAC and DNMT1 to PH and depletion of Np95 impairs PH replication. Here we show that Np95 causes large-scale modifications of chromocenters independently from the H3:K9 and H4:K20 trimethylation pathways, from the expression levels of HP1, from DNA methylation and from the cell cycle. The PHD domain is essential to induce this effect. The PHD domain is also required in vitro to increase access of a restriction enzyme to DNA packaged into nucleosomal arrays. We propose that the PHD domain of Np95-ICBP90 contributes to the opening and/or stabilization of dense chromocenter structures to support the recruitment of modifying enzymes, like HDAC and DNMT1, required for the replication and formation of PH.     

Frozen storage (—20° and —80°C) of soybean Bradyrhizobium cell suspensions

Liquid cultures of Bradyrhizobium japonicum were added in a 1:1 ratio to 20% aqueous skim milk, or centrifuged and the cells resuspended in 10% skim milk. The suspensions were stored at —20° or —80°C for 7 months and cell survival assessed. At —20°C, there was a decrease in the viable count of about two logs in liquid culture whilst for cells resuspended in 10% skim milk the decrease was limited to one log. The temperature of —80°C was found to be in itself protective and the surviving rhizobial cells maintained their infectivity and effectiveness. Thus appropriate freezing conditions provide a suitable method to store soybean rhizobia cells prior to preparing the legume inoculant.     

Recombinant prosegment peptide acts as a folding catalyst and inhibitor of native pepsin

Porcine pepsin A, a gastric aspartic peptidase, is initially produced as the zymogen pepsinogen that contains an N-terminal, 44 residue prosegment (PS) domain. In the absence of the PS, native pepsin (Np) is irreversibly denatured and when placed under refolding conditions, folds to a thermodynamically stable denatured state. This denatured, refolded pepsin (Rp) state can be converted to Np by the exogenous addition of the PS, which catalyzes the folding of Rp to Np. In order to thoroughly study the mechanism by which the PS catalyzes pepsin folding, a soluble protein expression system was developed to produce recombinant PS peptide in a highly pure form. Using this system, the wild-type and three-mutant PS forms, in which single residue substitutions were made (V4A, R8A and K36A), were expressed and purified. These PS peptides were characterized for their ability to inhibit Np enzymatic activity and to catalyze the folding of Rp to Np. The V4A, R8A and K36A mutant PS peptides were found to have nanomolar inhibition constants, K_i, of 82.4, 58.3 and 95.6 nM, respectively, approximately a two-fold increase from that of the wild-type PS (36.2 nM). All three-mutant PS peptides were found to catalyze Np folding with a rate constant of 0.06 min^? 1, five-fold lower than that of the wild-type. The observation that the mutant PS peptides retained their inhibition and folding-catalyst functionality suggests a high level of resilience to mutations of the pepsin PS.     

Induction of ornithine decarboxylase in macrophages by bacterial lipopolysaccharides (LPS) and mycobacterial cell wall material

Lipopolysaccharide from $\text{E. Coli}$ (LPS) and BCG cell walls (BCGcw) are recognized immunoadjuvants that directly stimulate some macrophage functions. The macrophage cell line J774.1 and peritoneal exudate cells (PEC) from mice can be stimulated by LPS or other adjuvants $\text{in vitro}$ to synthesize and release protein factor(s) that activate thymus-derived lymphocytes. We have utilized J774.1 cells and PEC to demonstrate that an increase in ornithine decarboxylase (ODC) activity is a marker of early biochemical changes in adjuvant-stimulated macrophages. BCGcw and LPS increased ODC within 2 hours in J774.1 cells as well as murine peritoneal exudate macrophages. Maximal increases in ODC were detected 4 hours after the addition of adjuvants to J774.1 cells. The marked increases (12–23 fold) in ODC observed with BCGcw (20 μg/ml) did not appear to involve an effect on cell proliferation which was suppressed by this adjuvant. Cycloheximide inhibited the induction of ODC by LPS and BCGcw in the macrophage cell line. Evidence that the induction of ODC may be promoted by an increase in cyclic AMP was provided by experiments demonstrating that prostaglandin E₁ (PGE₁) and 8-bromo-adenosine-3′:5′-monophosphate (8Br-cyclic AMP) can mimic the effects of LPS and BCGcw in J774.1 cells. These observations indicate that one of the early biochemical changes in macrophages promoted by adjuvants is an induction of ODC.     

Crystal structures and coordination-site exchange reactions of palladium(II) and platinum(II) complexes containing tris[2-(tert-butylthio)ethyl]amine

Palladium(II) and platinum(II) complexes, [PdX(NS₃ ¹Bu)]BPh₄ (X=Cl, Br, I; NS₃ ¹Bu=tris[2-(tert-butylthio)ethyl]amine) and [PtCl(NS₃ ¹Bu)]BPh₄, were prepared, and their structures were determined by X-ray analyses. The geometry around the palladium and platinum atoms is square planar. The NS₃ ¹Bu ligand functions as a tridentate ligand and one sulfur atom is not coordinated to the metal. The ¹H NMR spectrum of [PdCl(NS₃ ¹Bu)]BPh₄ in acetone-d₆ exhibited a dynamic behavior. At 20°C the spectrum showed a singlet signal at 1.60 ppm that can be assigned to tert-butyl protons, whereas at −70°C three singlet signals were observed at 1.36, 1.61, and 1.70 ppm with an intensity ratio of 1: 0.25: 2. The signals at 1.36 and 1.70 ppm are assigned to the tert-butyl protons in the square-planar structure, and these signals are consistent with the X-ray structure. The signal at 1.61 ppm can be assigned to the tert-butyl protons in a trigonal-bipyramidal structure where the three tert-butyl groups are magnetically equivalent. Thus, we concluded that the coordination-site exchange occurred via a trigonal-bipyramidal intermediate. The square-planar and trigonal-bipyramidal species of [PdCl(NS₃ ¹Bu)]BPh₄ are in equilibrium in acetone-d₆. The equilibrium was shifted toward the square-planar species on decreasing the temperature. The ¹H NMR spectra for [PdX(NS₃ ¹Bu)]BPh₄ (X=Cl, Br, and I) were similar to one another at the same temperature, suggesting that the site-exchange process is insensitive to the kind of coexisting halogen ligand. The site exchange reaction of [PtCl(NS₃ ¹Bu)]BPh₄ seems to occur more slowly than that of the palladium(II) analogue.