Phosphotransbutyrylase Expression in Bacillus megaterium

The molecular analysis of a genomic region of B. megaterium revealed the presence of a gene coding for the enzyme phosphotransbutyrylase (Ptb). The enzyme activity was measured throughout the different phases of growth in B. megaterium, and its activity was found to be maximal in the late exponential growth phase. The branched amino acids isoleucine and valine activated Ptb expression. Ptb_Bm was capable of using butyryl-CoA and 2-methyl-propionyl CoA as substrates. Act_Bm, a sigma⁵⁴ regulator from B. megaterium whose gene is situated upstream from the ptb gene, activated its expression. Received: 14 September 2000 / Accepted: 13 October 2000     

trans activation of the Escherichia coliato structural genes by a regulatory protein from Bacillus megaterium : potential use in polyhydroxyalkanoate production

A Bacillus megaterium genomic fragment, which encoded an activator homologous to σ⁵⁴ regulators and which was capable of activating Escherichia coli ato genes in trans, was detected in a gene library of B.␣megaterium screened for β-ketothiolase activity. The fragment presented only one complete open reading frame (ORF1), which encoded a protein of 398 amino acids. The recombinant plasmid complemented mutations in the Escherichia coli atoC regulatory gene. The constitutive expression of the E. coli ato operon mediated by ORF1 could be useful for the synthesis of polyhydroxyalkanoates with different flexibility properties by recombinant E. coli strains. Received: 20 October 1997 / Received revision: 18 February 1998 / Accepted: 23 February 1998     

Binding of aflatoxins to the 20S proteasome: effects on enzyme functionality and implications for oxidative stress and apoptosis

Aflatoxins (AF) are contaminants of improperly stored foods; they are potent genotoxic and carcinogenic compounds, exerting their effects through damage to DNA. They can also induce mutations that increase oxidative damage. The goal of this study was to evaluate the possibility that a third mechanism could be involved in the carcinogenic action of aflatoxins, namely, direct binding to key enzymes involved in the regulatory pathways of the cell cycle, thereby modulating enzyme functionality. The 20S constitutive and immunoproteasome peptidase and proteolytic activities were assayed in the presence of aflatoxins B1, G1 and M1. All three toxins activated multiple peptidase activities of the proteasome. Aflatoxin (AF) M1 was the most potent activator of proteasome activity, while the constitutive 20S proteasome was specifically stimulated by AFG1. Furthermore, the effects of AFB1 on cultured hepatoma cells were investigated and the various proteasomal activities determined with cell lysates were differently affected. Taking into account the key role of the proteasome in cellular defense against oxidative stress, the carbonyl group content and the activities of antioxidant enzymes in cell lysates were analyzed. The proapoptotic effect of AFB1 was also investigated by measuring caspase-3 activity and cellular levels of p27 and IkappaBalpha.     

New oligo-/poly-meric forms for MX:dpex (1:1) complexes (M = Cu, Ag; X = (pseudo-)halide; dpex = Ph2E(CH2)xEPh2, E = (P), As; x = 1, 2)

Single-crystal X-ray structural characterizations of MX:dpam (1:1) (‘dpam’ = Ph₂AsCH₂AsPh₂) are reported for MX = AgCl, Br; CuI, CN/Cl (all isomorphous) and AgI, AgSCN, CuSCN arrays, all being of the novel form [(μ-X){M(μ-X)(As-dpam-As′)₂M′}]_∞, essentially the familiar M(E-dpem-E′)₂M′ binuclear array with both ‘bridging’ and (linking) ‘terminal’ (pseudo-)halides involved in the polymer. A different arrangement of bridging and linking entities is found with AgX:dpae (1:1)_2(∞|∞), X = Br, NCO, ‘dpae’ = Ph₂As(CH₂)₂AsPh₂, now comprising [M(μ-X)₂(As-dpae-As)M] kernels linked by As-dpae-As′, while in the thiocyanate analogue units are linked by the dpae ligands into a two-dimensional web. Synthetic procedures for all adducts have been reported. All compounds have been characterized both in solution (¹H, ¹³C, ³¹P NMR, ESI MS) and in the solid state (IR).     

20S proteasome mediated degradation of DHFR: implications in neurodegenerative disorders

The 20S proteasome is responsible for the degradation of protein substrates implicated in the onset and progression of neurodegenerative disorders, such as alpha-synuclein and tau protein. Here we show that the 20S proteasome isolated from bovine brain directly hydrolyzes, in vitro, the dihydrofolate reductase (DHFR), demonstrated to be involved in the pathogenesis of neurodegenerative diseases. Furthermore, the DHFR susceptibility to proteolysis is enhanced by oxidative conditions induced by peroxynitrite, mimicking the oxidative environment typical of these disorders. The results obtained suggest that the folate metabolism may be impaired by an increased degradation of DHFR, mediated by the 20S proteasome.     

Synthesis, spectroscopy (IR, multinuclear NMR, ESI-MS), diffraction, density functional study and in vitro antiproliferative activity of pyrazole-beta-diketone dihalotin(IV) compounds on 5 melanoma cell lines

Novel 4-acylpyrazolon-5-ato-dihalotin(IV) complexes, [Q2SnX2], (X = F, Cl, Br or I); HQ = HQ(CHPh2) (1,2-dihydro-3-methyl-1-phenyl-4-(2,2-diphenylacetyl)pyrazol-5-one), HQ(Bn) (1,2-dihydro-3-methyl-1-phenyl-4-(2-phenylacetyl)pyrazol-5-one) or HQ(CF3,py) (4-(2,2,2-trifluoroacetyl)-1,2-dihydro-3-methyl-1-(pyridin-2-yl)pyrazol-5-one) have been synthesized and characterized by spectroscopic (IR, 1H, 13C, 19F and 119Sn NMR, electrospray ionisation mass spectrometry (ESI-MS)), analytical and structural methods (X-ray and density functional theory). 119Sn chemical shifts depend on the nature of the halides bonded to tin. Isomer conversion, detected in solution by NMR spectroscopy, is related to the acyl moiety bulkiness while the cis(Cl)-cis(acyl)-trans(pyrazolonato) scheme is found in the solid state. The in vitro antiproliferative tests of three derivatives on three human melanoma cell lines (JR8, SK-MEL-5, MEL501) and two melanoma cell clones (2/21 and 2/60) show dose-dependent decrease of cell proliferation in all cell lines. The activity correlates with the nature of the substituent on position 1 of pyrazole, decreasing in the order pyridyl>Ph>methyl. The activity for (Q(CF3,py))2SnCl2 on the SK-MEL-5 cell line is IC50 = 50 microM.     

Sequence and Copy Number Analyses of HEXB Gene in Patients Affected by Sandhoff Disease: Functional Characterization of 9 Novel Sequence Variants

Sandhoff disease (SD) is a lysosomal disorder caused by mutations in the HEXB gene. To date, 43 mutations of HEXB have been described, including 3 large deletions. Here, we have characterized 14 unrelated SD patients and developed a Multiplex Ligation-dependent Probe Amplification (MLPA) assay to investigate the presence of large HEXB deletions. Overall, we identified 16 alleles, 9 of which were novel, including 4 sequence variation leading to aminoacid changes [c.626C>T (p.T209I), c.634C>A (p.H212N), c.926G>T (p.C309F), c.1451G>A (p.G484E)] 3 intronic mutations (c.1082+5G>A, c.1242+1G>A, c.1169+5G>A), 1 nonsense mutation c.146C>A (p.S49X) and 1 small in-frame deletion c.1260_1265delAGTTGA (p.V421_E422del). Using the new MLPA assay, 2 previously described deletions were identified. In vitro expression studies showed that proteins bearing aminoacid changes p.T209I and p.G484E presented a very low or absent activity, while proteins bearing the p.H212N and p.C309F changes retained a significant residual activity. The detrimental effect of the 3 novel intronic mutations on the HEXB mRNA processing was demonstrated using a minigene assay. Unprecedentedly, minigene studies revealed the presence of a novel alternative spliced HEXB mRNA variant also present in normal cells. In conclusion, we provided new insights into the molecular basis of SD and validated an MLPA assay for detecting large HEXB deletions.     

Synthesis, spectroscopic and structural characterization of Cu(II) derivatives of tris(pyrazol-1-yl)methanes

The reaction between CuX₂ (X=ClO₄, NO₃, Cl, Br and CH₃COO) and excess of tris(pyrazol-1-yl)methane ligands L (L=CH(pz)₃, CH(4-Mepz)₃, CH(3,5-Me₂pz)₃, CH(3,4,5-Me₃pz)₃ or CH(3-Mepz)₂(5-Mepz)) yields [CuX₂(L)], [{CuX₂}₃(L₂)₂] or [Cu(L₂)]X₂-type complexes. The ligand to metal ratio is dependent on the number and disposition of the Me substituents on the azole-type ligand and mainly on the nature of the counter-ion X. All complexes have been characterized in the solid state as well as in solution (IR and UV spectra, and conductivity determinations). The solid-state structures of [Cu{(3,5-Me₂pz)₃CH}₂](NO₃)₂, [Cu{(3,5-Me₂pz)₃CH}₂](ClO₄)₂·0.5H₂O, [Cu{(3,4,5-Me₃pz)₃CH}₂](NO₃)₂·H₂O, [Cu{(4-Mepz)₃CH}₂]Br₂·3H₂O have been determined by single crystal X-ray studies.