Inactivation of dopamine β-monooxygenase by hydrogen peroxide and by ascorbate

The inactivation of the water-soluble form of bovine adrenal dopamine β-monooxygenase by H₂O₂ and by ascorbate was studied. Inactivation by H₂O₂ was slow for the copper-free apoenzyme, but addition of copper gave a rapid inactivation. The results presented indicate that the enzyme-bound copper during this inactivation catalyzes partial destruction of its own binding site. The reaction orders for the inactivation by H₂O₂ seem to be 1.0 with respect to the enzyme and in the range 0.6 to 0.8 with respect to H₂O₂. The rate of inactivation obtained in the presence of ascorbate increases with addition of copper and is faster than that obtained by similar concentrations of H₂O₂. The data could not, however, be used to decide whether the inactivation by ascorbate was catalyzed by the enzymebound copper. The inactivation reaction in the presence of ascorbate seems to be of first order with respect to ascorbate at ascorbate concentrations less than 40 μm and decreases toward zero as the ascorbate concentration is increased. Experiments with the Cu(I)-chelator, bathocuproine disulfonate, revealed that inactivation led to weaker binding of copper to the protein, and this effect was more pronounced with H₂O₂ than with ascorbate.     

Inhibition of proliferation of retrovirus-immortalized macrophages by LPS and IFN-γ: Possible autocrine down-regulation of cell growth by induction of IL1 and TNF

The GG₂EE macrophage tumor cell line was previously established by immortalization of C3H/HeJ mouse bone marrow cells with the J2 retrovirus which contains the v-myc and v-raf oncogenes. Studies on the control of GGZEE cell proliferationin vitro have recently been performed. We observed that the combination of 5–25 U/ml recombinant mouse interferon- (rmIFN-) plus 0.03 – 0.3 µg/ml lipopolysaccharide (LPS) markedly inhibited the proliferation of GG₂EE cells (by >95%)in vitro, while either agent alone inhibited only by <40% and 0–10%, respectively. Subsequent studies established that biologically active ILI-like (2–4 U/ml) and TNF-like (50–100 U/ml) activities were released into the supernatants of LPS-treated GG₂EE cells. The combination of IFN- + LPS induced more (6–8 U/ml) ILI release. These results suggested that the inhibition of proliferation of GG₂EE cells by IFN- + LPS could have been mediated in part by cytokines produced by the cells themselves. rhIL1 at a concentration of 10 U/ml inhibited GG₂EE proliferation by 25–30%, while rmIFN- (25 U/ml) + rhIL1 (10 U/ml) inhibited proliferation by 98%. Thus, 10 U/ml rhIL1 could completely replace LPS in the LPS + rmIFN- combination. Further, the combination of low doses of rhIL1 (0.1 to 1 U/ml) plus rmTNF (250 U/ml), which together inhibited proliferation by <20% synergized with doses of 5 to 25 U/ml rmIFN- to inhibit proliferation of GG₂EE cells by 98–99%. These results suggest that cytokines produced by the cells themselves can synergize with rmIFN- to inhibit the oncogene-driven proliferation of GG₂EE cells.     

Kinetics and mechanism of catalysis by proteolytic enzymes. The kinetics of hydrolysis of esters of γ-guanidino-l-α-toluene-p-sulphonamidobutyric acid by bovine trypsin and thrombin

1. Esters of gamma-guanidino-l-alpha-toluene-p-sulphonamidobutyric acid (alpha-N-toluene-p-sulphonyl-l-norarginine) have been synthesized and shown to be hydrolysed by bovine trypsin and thrombin. As substrates for these enzymes, they were better than esters of alpha-N-toluene-p-sulphonyl-l-homoarginine or of alpha-N-toluene-p-sulphonyl-l-ornithine but not as good as esters of alpha-N-toluene-p-sulphonyl-l-arginine. 2. With trypsin as catalyst, the methyl and propyl esters are hydrolysed at the same rate at high substrate concentrations and hence deacylation of the acyl-enzyme appears to be rate-determining. In the presence of thrombin, however, the methyl ester is hydrolysed much faster than the n-propyl ester. 3. The variation of k(0) with pH indicates that groups with pK((app.)) values of 7.05+/-0.02 and 6.53+/-0.02 must be dissociated in trypsin and thrombin respectively for hydrolysis to proceed. 4. Activation constants have been determined for the trypsin-catalysed hydrolysis of methyl gamma-guanidino-l-alpha-toluene-p-sulphonamidobutyrate and have been compared with the corresponding constants for the hydrolysis of homologous substrates. 5. Cholate increases k(0) and decreases K(m); the effects are more pronounced with thrombin than with trypsin.     

Regulation of RNA function by aminoacylation and editing?

The chemical modification of nucleic acids is a ubiquitous phenomenon. Aminoacylation of tRNAs by aminoacyl-tRNA synthetases (ARSs) is a reaction essentially devoted to protein synthesis but it is used also as an emergency mechanism to recycle stalled ribosomes, and it is required for genome replication in some RNA viruses. In several aminoacyl-tRNA synthetases a correction mechanism known as editing is present to prevent aminoacylation errors. Genome data reveal a growing number of open reading frames encoding ARS-like proteins. This strongly suggests the existence of a widespread and nonconventional machinery for aminoacylation and editing. Here we review the different biological functions of aminoacylation and editing; also we propose an evolutionary scenario for the origin of these two reactions, and hypothesize an extant role for RNA charging and editing outside the genetic code.     

Kinetics and mechanism of catalysis by proteolytic enzymes. A comparison of the kinetics of hydrolysis of synthetic substrates by bovine α- and β-trypsin

Several esters of the α-N-toluene-p-sulphonyl and α-N-benzoyl derivatives of S-(3-aminopropyl)-l-cysteine and the methyl ester of S-(4-aminobutyl)-N-toluene-p-sulphonyl-l-cysteine were synthesized. The kinetics of hydrolysis of these and esters of the α-N-toluene-p-sulphonyl and α-N-benzoyl derivatives of l-arginine, l-lysine, S-(2-aminoethyl)-l-cysteine and esters of γ-guanidino-l-α-toluene-p-sulphonamidobutyric acid and α-N-toluene-p-sulphonyl-l-homoarginine by α- and β-trypsin were compared. On the basis of values of the specificity constants (k_cat./K_m), the two enzymes display similar catalytic efficiency towards some substrates. In other cases α-trypsin is less efficient than β-trypsin. It is possible that α-trypsin possesses greater molecular flexibility than β-trypsin.     

Molecular recognition and enhancement of aqueous solubility and bioactivity of CD437 by β-cyclodextrin

CD437 (6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid) is a novel synthetic retinoic acid derivative that has been shown to selectively induce apoptosis in human lung cancer cells. This compound, however, is limited in its application due to its low solubility in aqueous solutions. One technique for increasing the solubility and bioavailability of a cytotoxic agent is the formation of inclusion complexes with cyclodextrins. Herein, we report the formation and characterization of a 2:1 complex between β-cyclodextrin (β-CD) and CD437. It is shown that CD437 is a tight binder of β-CD with an overall association constant of 2.6 ± 0.6 × 10⁷ M⁻². In addition, we demonstrate (a) that β-CD-derived complexation enhances the aqueous solubility of CD437, and (b) that a significant increase in the toxicity of CD437 against a human lung adenocarcinoma cell line can be achieved by co-treatment with β-CD.     

Characterization of the extracellular,water-insoluble α-D-glucans of oral streptococci by methylation analysis,and by enzymic synthesis and degradation

Methylation analysis of water-insoluble α-D-glucans synthesized from sucrose by culture filtrates from several strains of Streptococcus spp. has proved that all of the glucans were highly branched and that the chains contained (1→6)- and (1→3)-linked D-glucose residues not involved in branch points. Hydrolysis of the glucans with a specific endo-(1→3)-α-D-glucanase demonstrated that the majority of the (1→3)-linked glucose residues were arranged in sequences. D-Glucose was the major product of the hydrolysis, and a small proportion of nigerose was also released. The use of a specific endo-(1→6)-α-D-glucanase similarly indicated that the glucans also contained sequences of (1→6)-linked α-D-glucose residues, and that those chains were branched. Two D-glucosyltransferases (GTF-S and GTF-I), which reacted with sucrose to synthesize a soluble glucan and a water-insoluble glucan, respectively, were separated from culture filtrates of S. mutans OMZ176. The soluble glucan was characterized as a branched (1→6)-α-D-glucan, whereas the insoluble one was a relatively linear (1→3)-α-D-glucan. The hypothesis is advanced that the glucosyltransferases can transfer glucan sequences by means of acceptor reactions similar to those proposed by Robyt for dextransucrase, leading to the synthesis of a highly branched glucan containing both types of chain. The resulting structure is consistent with the evidence obtained from methylation analysis and enzymic degradations, and explains the synergy displayed when the two D-glucosyltransferases interact with sucrose. Variations in one basic structure can account for the characteristics of water-insoluble glucans from S. sanguis and S. salivarius, and for the strain-dependent diversity of S. mutans glucans.     

Biphasic and dosage-dependent regulation of osteoclastogenesis by β-catenin

Wnt/β-catenin signaling is a critical regulator of skeletal physiology. However, previous studies have mainly focused on its roles in osteoblasts, while its specific function in osteoclasts is unknown. This is a clinically important question because neutralizing antibodies against Wnt antagonists are promising new drugs for bone diseases. Here, we show that in osteoclastogenesis, β-catenin is induced during the macrophage colony-stimulating factor (M-CSF)-mediated quiescence-to-proliferation switch but suppressed during the RANKL-mediated proliferation-to-differentiation switch. Genetically, β-catenin deletion blocks osteoclast precursor proliferation, while β-catenin constitutive activation sustains proliferation but prevents osteoclast differentiation, both causing osteopetrosis. In contrast, β-catenin heterozygosity enhances osteoclast differentiation, causing osteoporosis. Biochemically, Wnt activation attenuates whereas Wnt inhibition stimulates osteoclastogenesis. Mechanistically, β-catenin activation increases GATA2/Evi1 expression but abolishes RANKL-induced c-Jun phosphorylation. Therefore, β-catenin exerts a pivotal biphasic and dosage-dependent regulation of osteoclastogenesis. Importantly, these findings suggest that Wnt activation is a more effective treatment for skeletal fragility than previously recognized that confers dual anabolic and anti-catabolic benefits.     

Overproduction and secretion of α-ketoglutaric acid by microorganisms

This mini-review presents a summary of research results of biotechnological production of alpha-ketoglutaric acid (KGA) by bacteria and yeasts. KGA is of particular industrial interest due to its broad application scope, e.g., as building block chemical for the chemical synthesis of heterocycles, dietary supplement, component of infusion solutions and wound healing compounds, or as main component of new elastomers with a wide range of interesting mechanical and chemical properties. Currently KGA is produced via different chemical pathways, which have a lot of disadvantages. As an alternative several bacteria and yeasts have already been studied for their ability to produce KGA as well as for conditions of overproduction and secretion of this intermediate of the tricarboxylic acid cycle. The aim of this mini-review was to summarize the known data and to discuss the potentials of biotechnological processes of KGA production.     

Substrate-selective and calcium-independent activation of CaMKII by α-actinin

Protein-protein interactions are thought to modulate the efficiency and specificity of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) signaling in specific subcellular compartments. Here we show that the F-actin-binding protein α-actinin targets CaMKIIα to F-actin in cells by binding to the CaMKII regulatory domain, mimicking CaM. The interaction with α-actinin is blocked by CaMKII autophosphorylation at Thr-306, but not by autophosphorylation at Thr-305, whereas autophosphorylation at either site blocks Ca(2+)/CaM binding. The binding of α-actinin to CaMKII is Ca(2+)-independent and activates the phosphorylation of a subset of substrates in vitro. In intact cells, α-actinin selectively stabilizes CaMKII association with GluN2B-containing glutamate receptors and enhances phosphorylation of Ser-1303 in GluN2B, but inhibits CaMKII phosphorylation of Ser-831 in glutamate receptor GluA1 subunits by competing for activation by Ca(2+)/CaM. These data show that Ca(2+)-independent binding of α-actinin to CaMKII differentially modulates the phosphorylation of physiological targets that play key roles in long-term synaptic plasticity.     

Regulation of lysine ɛ-aminotransferase by carbon source and lack of control by phosphate in Streptomyces clavuligerus

Cephalosporin production by Streptomyces clavuligerus is known to be negatively regulated by carbon sources, e.g., glycerol and starch, and by phosphate at high concentrations. Formation of lysine ɛ-aminotransferase (LAT) activity, the first enzyme of the biosynthetic pathway, was affected by a high concentration of carbon source. Whereas 3% starch more than doubled LAT activity production as compared to 1% starch, 3% glycerol repressed LAT activity formation by 20%–30%. LAT activity production was not affected by 100 mM K₂HPO₄. Our results thus show that the negative effects of 2% glycerol and 3% starch and 100 mM phosphate on cephalosporin production are not due to an effect on production of LAT activity. However, repression of LAT activity by 3% glycerol would be expected to play a negative role in antibiotic production. Received: 13 June 1997 / Received revision: 20 August 1997 / Accepted: 25 August 1997     

Regulation of TGF-β signal transduction by mono- and deubiquitylation of Smads

Polyubiquitylation leading to proteasomal degradation is a well-established mechanism for regulating TGF-β signal transduction components such as receptors and Smads. Recently, an equally important role was suggested for monoubiquitylation of both Smad4 and receptor-associated Smads that regulates their function without protein degradation. Monoubiquitylation of Smads was discovered following the identification of deubiquitylases required for TGF-β signaling, suggesting that continuous cycles of Smad mono- and deubiquitylation are required for proper TGF-β signal transduction. Here we summarize and discuss recent work on Smad mono- and deubiquitylation.     

Inhibition of calf thymus DNA polymerase α and of normal and cancer cell growth by butylanilinouracil and butylphenylguanine

6-(p-n-Butylanilino)uracil and N²-(p-butylphenyl)guanine inhibited the activity of DNA polymerase α from calf thymus but had no effect on other eukaryotic polymerases (DNA polymerases β and γ) or Escherichia coli DNA polymerase I. Inhibition was competitive with deoxyguanosine 5′-triphosphate and did not occur in the reaction of DNA polymerase α with a template that did not contain cytosine residues. The results support a mechanism which involves hydrogen bonding of inhibitors with cytosines in the DNA template and binding with an inhibitor specific site on the enzyme. A screen of inhibitor effects on normal and cancer cell growth in culture showed that cells were not uniformly sensitive to these compounds, a mouse lymphoma line being least sensitive and a human lung cancer line being most sensitive. It is suggested that these inhibitors may be useful to probe possible structural differences among DNA polymerases α.     

Evolution of primate α and θ defensins revealed by analysis of genomes

Defensins are endogenous peptides with cysteine-rich antimicrobial ability that contribute to host defence against bacterial, fungal and viral infections. There are three subfamilies of defensins in primates: α, β and θ-defensins. α-defensins are most present in neutrophils and Paneth cells; β-defensins are involved in protecting the skin and the mucous membranes of the respiratory, genitourinary and gastrointestinal tracts; and θ-defensins are physically distinguished as the only known fully-cyclic peptides of animal origin, which are first isolated from rhesus macaques. All three kinds of defensins have six conserved cysteines, three intramolecular disulfide bonds, a net positive charge, and β-sheet regions. α and θ-defensins are closely related, comparative amino acid sequences showed that the difference between them is that θ-defensins have an additional stop codon limits the initial defensin domain peptides to 12 residues. Humans, chimpanzees and gorillas do not produce θ-defensin peptides due to a premature stop codon present in the signal sequence of all θ-defensin pseudogenes. By using comprehensive computational searches, here we report the discovery of complete repertoires of the α and θ-defensin gene family in ten primate species. Consistent with previous studies, our phylogenetic analyses showed all primate θ-defensins evident formed one distinct clusters evolved from α-defensins. β-defensins are ancestors of both α and θ-defensins. Human has two copies of DEFA1 and DEFT1P, and two extra DEFA3 and DEFA10P genes compared with gorilla. As different primates inhabit in quite different ecological niches, the production of species-specific α and θ-defensins and these highly evolved θ-defensins in old world monkeys would presumably allow them to better respond to the specific microbial challenges that they face.     

Ubiquitylation of ε-COP by PIRH2 and regulation of the secretion of PSA

Ubiquitylation appears to be involved in the membrane trafficking system including endocytosis, exocytosis, and ER-to-Golgi transport. We found that PIRH2, which was identified as an interacting protein for androgen receptor or p53, interacts with and ubiquitylates the ε-subunit of coatmer complex, ε-COP. PIRH2 promotes the ubiquitylation of ε-COP in vitro and in vivo and consequently promotes the degradation of ε-COP. The interaction between PIRH2 and ε-COP is affected by the presence of androgen, and PIRH2 in the presence of androgen promotes ubiquitylation of ε-COP in vivo. Furthermore, overexpression of the wild type of PIRH2 in prostate cancer cells causes downregulation of the secretion of prostate-specific antigen (PSA), a secretory protein in prostate epithelial cells and one of diagnostic markers for prostate cancer. Our results indicate that PIRH2 functions as a regulator for COP I complex.     

Preparation of octyl β-d-xylobioside and xyloside by xylanase-catalyzed direct transglycosylation reaction of xylan and octanol

1-Octyl -d-xylobioside and xyloside were prepared by direct transglycosylation reaction of xylan and 1-octanol using purified xylanase from Aureobasidium pullulans. 2-Ethylhexyl -d- xylobioside and xyloside were also prepared in the same way. The maximum yields of 2-ethylhexyl -d-xylobioside and 2-ethylhexyl -d-xyloside were 110 and 54 mg/g xylan, respectively. The proposed mechanism for production of octyl xylobioside and xyloside involved the reaction of xylan and octanol by xylanase to produce octyl xylobioside and xylotrioside, the latter of which was simultaneously hydrolyzed by xylanase into octyl xyloside and xylobiose.     

Activation of PI3Kα by physiological effectors and by oncogenic mutations: structural and dynamic effects

PI3Kα, a heterodimeric lipid kinase, catalyzes the conversion of phosphoinositide-4,5-bisphosphate (PIP₂) to phosphoinositide-3,4,5-trisphosphate (PIP₃), a lipid that recruits to the plasma membrane proteins that regulate signaling cascades that control key cellular processes such as cell proliferation, carbohydrate metabolism, cell motility, and apoptosis. PI3Kα is composed of two subunits, p110α and p85, that are activated by binding to phosphorylated receptor tyrosine kinases (RTKs) or their substrates. The gene coding for p110α, PIK3CA, has been found to be mutated in a large number of tumors; these mutations result in increased PI3Kα kinase activity. The structure of the complex of p110α with a fragment of p85 containing the nSH2 and the iSH2 domains has provided valuable information about the mechanisms underlying the physiological activation of PI3Kα and its pathological activation by oncogenic mutations. This review discusses information derived from x-ray diffraction and theoretical calculations regarding the structural and dynamic effects of mutations in four highly mutated regions of PI3K p110α, as well as the proposed mechanisms by which these mutations increase kinase activity. During the physiological activation of PI3Kα, the phosphorylated tyrosine of RTKs binds to the nSH2 domain of p85, dislodging an inhibitory interaction between the p85 nSH2 and a loop of the helical domain of p110α. Several of the oncogenic mutations in p110α activate the enzyme by weakening this autoinhibitory interaction. These effects involve structural changes as well as changes in the dynamics of the enzyme. One of the most common p110α mutations, H1047R, activates PI3Kα by a different mechanism: it increases the interaction of the enzyme with the membrane, maximizing the access of the PI3Kα to its substrate PIP₂, a membrane lipid.     

Combinatorial morphogenesis of dendritic spines and filopodia by SPAR and α-actinin2

Rap small GTPases regulate excitatory synaptic strength and morphological plasticity of dendritic spines. Changes in spine structure are mediated by the F-actin cytoskeleton, but the link between Rap activity and actin dynamics is unclear. Here, we report a novel interaction between SPAR, a postsynaptic inhibitor of Rap, and α-actinin, a family of actin-cross-linking proteins. SPAR and α-actinin engage in bidirectional structural plasticity of dendritic spines: SPAR promotes spine head enlargement, whereas increased α-actinin2 expression favors dendritic spine elongation and thinning. Surprisingly, SPAR and α-actinin2 can function in an additive rather than antagonistic fashion at the same dendritic spine, generating combination spine/filopodia hybrids. These data identify a molecular pathway bridging the actin cytoskeleton and Rap at synapses, and suggest that formation of spines and filopodia are not necessarily opposing forms of structural plasticity.