BBA - General Subjects (v.1475, #2)

A genomic clone for 25-hydroxyvitamin D3-1α-hydroxylase (1α-hydroxylase) was isolated from a mouse embryonic stem cell P1 genomic library. It contains nine exons spanning 4.8 kb from the transcriptional start site. All the intron insertion sites are identical to that of the human 1α-hydroxylase and human vitamin D3 25-hydroxylase genes. A polyadenylation signal AUUAAA that differs from the consensus sequence at the second residue was identified 16 bp downstream of the 3′ end of the previously reported mouse cDNA. This element is the only common natural variant described. The 3′ end of the gene was determined using a RACE technique. Three poly(A) addition sites were observed 12, 15 and 22 bases from the AUUAAA element. Such distances are in agreement with what is required for maturation of mammalian pre-mRNAs. This molecular cloning makes possible the generation of transgenic mice in order to further investigate the role and importance of the 25-hydroxyvitamin D3-1α-hydroxylase (CYP1α).
Keywords: Vitamin D metabolism; Cytochrome P-450; Vitamin D hydroxylase; CYP1α; Gene structure;

Maple syrup urine disease is a metabolic disorder caused by mutations of the branched chain keto acid dehydrogenase complex, leading to accumulation of α-keto acids and their amino acid precursors in the brain. We now report that α-ketoisovaleric, α-keto-β-methyl-n-valeric and α-ketoisocaproic acids accumulated in the disease inhibit glutamate uptake into rat brain synaptic vesicles. The α-keto acids did not affect the electrochemical proton gradient across the membrane, suggesting that they interact directly with the vesicular glutamate carrier. Chloride anions have a biphasic effect on glutamate uptake. Low concentrations activate the uptake (0.2 to 8 mM), while higher concentrations are inhibitory. The α-keto acids inhibited glutamate uptake by a new mechanism, involving a change in the chloride dependence for the activation of glutamate uptake. The activation of glutamate uptake by low chloride concentrations was shifted toward higher concentrations of the anion in the presence of α-keto acids. Inhibition by α-keto acids was abolished at high chloride concentrations (20 to 80 mM), indicating that α-keto acids specifically change the stimulatory effect of low chloride concentrations. High glutamate concentrations also reduced the inhibition by α-keto acids, an effect observed both in the absence and in the presence of low chloride concentrations. The results suggest that in addition to their possible pathophysiological role in maple syrup urine disease, α-keto acids are valuable tools to study the mechanism of vesicular transport of glutamate.
Keywords: Synaptic vesicle; Glutamate uptake; Glutamate carrier; Maple syrup urine disease; Branched chain ketonuria;

A silent antifungal protein (AFP)-like gene lacking two introns in the mould Trichoderma viride by Jian-Jiang Hao; Jun-qiang Ye; Qiang Yang; Zhen-zhen Gong; Wang-Yi Liu; En-duo Wang (119-124).
In cells of the mould Trichoderma viride, the existence of an antifungal protein (AFP)-like gene consisting of 285 bp was confirmed by Southern analysis that genomic DNA of T. viride could hybridize with the cDNA of mature AFP of Aspergillus giganteus MDH 18894. Except for the absence of two introns, the nucleotide sequence of the AFP-like gene was identical to that of the AFP gene of A. giganteus in positions 336–479, 568–649, and 706–765. The AFP-like gene could not be transcribed into its mRNA in T. viride cells as examined by RT-PCR using total RNAs of T. viride as template. Furthermore, AFP could not be detected either directly from the culture medium of T. viride or by Western analysis. However, the AFP-like gene could be actively expressed like the cDNA of AFP in Escherichia coli cell. Recombinant AFP exhibited similar antifungal activity as native AFP.
Keywords: AFP-like gene; Intronless; Trichoderma viride;

Liposome-entrapped plasmid DNA: characterisation studies by Yvonne Perrie; Gregory Gregoriadis (125-132).
Plasmid DNA pRc/CMV HBS (5.6 kb) (100 μg) encoding the S (small) region of hepatitis B surface antigen was incorporated by the dehydration–rehydration method into liposomes composed of 16 μmol egg phosphatidylcholine (PC), 8 μmol dioleoylphosphatidylcholine (DOPE) and 1,2-diodeoyl-3-(trimethylammonium)propane (DOTAP) (cationic liposomes) or phosphatidylglycerol (anionic liposomes) in a variety of molar ratios. The method, entailing mixing of small unilamellar vesicles (SUV) with the DNA, followed by dehydration and rehydration, yielded incorporation values of 95–97 and 48–54% of the DNA used, respectively. Mixing of preformed cationic liposomes with 100 μg plasmid DNA also led to high complexation values of 73–97%. As expected, the association of DNA with preformed anionic liposomes was low (9%). Further work with cationic PC/DOPE/DOTAP liposomes attempted to establish differences in the nature of DNA association with the vesicles after complexation and the constructs generated by the process of dehydration/rehydration. Several lines of evidence obtained from studies on vesicle size and zeta-potential, fluorescent microscopy and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, interaction of DNA with preformed cationic SUV as above, or with cationic SUV made of DOPE and DOTAP (1:1 molar ratio; ESCORT Transfection Reagent), leads to the formation of large complexes with externally bound DNA. For instance, such DNA is accessible to and can be dissociated by competing anionic SDS molecules. However, dehydration of the DNA–SUV complexes and subsequent rehydration, generates submicron size liposomes incorporating most of the DNA in a fashion that prevents DNA displacement through anion competition. It is suggested that, in this case, DNA is entrapped within the aqueous compartments, in between bilayers, presumably bound to the cationic charges.
Keywords: Liposome; DNA vaccine; Plasmid DNA; Hepatitis B surface antigen; Microelectrophoresis; Gel electrophoresis;

Characterization of chloroquine-hematin μ-oxo dimer binding by isothermal titration calorimetry by Sudha Rani Vippagunta; Arnulf Dorn; Robert G. Ridley; Jonathan L. Vennerstrom (133-140).
Numerous studies indicate that a key feature of chloroquine’s (CQ) antimalarial activity is its interaction with hematin. We now characterize this CQ–hematin interaction in detail using isothermal titration calorimetry (ITC). Between pH 5.6 and 9.0, association constants (K a values) for enthalpy-driven CQ–hematin μ-oxo dimer binding fell in the narrow range of 2.3–4.4×105 M−1. It is therefore probable that CQ–hematin μ-oxo dimer binding affinity does not diminish at the pH range (4.8–5.4) of the parasite food vacuole. The binding affinity was unaffected by high salt concentrations, suggesting that ionic interactions do not contribute significantly to this complexation. With increasing ionic strength, the entropic penalty of CQ–hematin μ-oxo dimer binding decreased accompanied by increased hematin μ-oxo dimer aggregation. A stoichiometry (n) of 1:4 in the pH range 6.5–9.0 indicates that CQ binds to two hematin μ-oxo dimers. At pH 5.6, a stoichiometry of 1:8 suggests that CQ binds to an aggregate of four hematin μ-oxo dimers. This work adds further evidence supporting the hypothesis that CQ impedes hematin monomer incorporation into hemozoin by producing a forward shift in the hematin monomer-hematin μ-oxo dimer equilibrium, contributing to a destructive accumulation of soluble forms of hematin in the parasite and leading to its death by hematin poisoning.
Keywords: Chloroquine; Hematin; Hematin μ-oxo dimer; Association constant; Isothermal titration calorimetry;

Interaction of antibodies and antigens conjugated with synthetic polyanions: on the way of creating an artificial chaperone by Vladimir I. Muronetz; Sergey V. Kazakov; Maria B. Dainiak; Vladimir A. Izumrudov; Igor Yu. Galaev; Bo Mattiasson (141-150).
Recently we have initiated the use of synthetic polyelectrolytes to mimic the action of chaperones in living cells [Dainiak et al., Biochim. Biophys. Acta 1381 (1998) 279–285]. The next step in this direction is done by the synthesis of conjugates of poly(methacrylic acid) (PMAA) with antigen, denatured glyceraldehyde-3-phosphate dehydrogenase (dGAPDH), and with monoclonal antibodies specific for dGAPDH (but not for the native protein). The pH-dependent properties of the conjugates have been studied using turbidimetry and light scattering. The antibody–PMAA and dGAPDH–PMAA conjugates were shown to interact with free dGAPDH and antibodies respectively as well as with each other. Insoluble aggregates of dGAPDH with antibody–PMAA and of antibodies with dGAPDH–PMAA are formed in acidic media. The same situation occurs in the mixture of antibody–PMAA and dGAPDH–PMAA: precipitation takes place in acidic media, whereas soluble associates are formed in neutral solutions. The size of the soluble associates and the number of conjugates in the associate could be regulated by pH. The competition of free dGAPDH and dGAPDH–PMAA for binding with antibody–PMAA and the dynamic release of refolded GAPDH, with no affinity to antibody–PMAA, into solution could be used for simulating chaperone action.
Keywords: Artificial chaperone; Monoclonal antibody; Light scattering; Glyceraldehyde-3-phosphate dehydrogenase; Synthetic polyanion;

Vanadate and copper induce overlapping oxidative stress responses in the vanadate-tolerant yeast Hansenula polymorpha by I. Mannazzu; E. Guerra; R. Ferretti; D. Pediconi; F. Fatichenti (151-156).
The mechanisms by which vanadate exerts a toxic effect on living organisms are not completely understood. This is principally due to the variety of intracellular targets of the metal and to the changes in the chemical form and oxidation states that vanadate can undergo, both in the external environment and intracellularly. In order to further elucidate the reasons for vanadate toxicity, and assuming that common detoxification mechanisms can be evoked by a general heavy metal response, we have compared some aspects of the cellular responses to vanadate and copper in the yeast Hansenula polymorpha. By means of 2D electrophoresis we show the existence of common determinants in the responses to vanadate- and copper-induced stresses. Moreover, we demonstrate that both metals induce significant increases in antioxidant enzyme levels, and that there are significant overlaps in the heavy metal and oxidative stress responses. Interestingly, vanadate induces an increase in catalase activity that is much higher than that seen with copper and, unlike copper, does not cause lipid peroxidation of cellular membranes. This suggests that H. polymorpha cells activate a further specific detoxification pathway against vanadate-induced oxidative insults.
Keywords: Vanadate toxicity; Heavy metal stress; Oxidative stress; Yeast; 2D electrophoresis; Hansenula polymorpha;

Interaction of DNA with [Cr(Schiff base)(H2O)2]ClO4 by Rajamanickam Vijayalakshmi; Mookandi Kanthimathi; Venkatesan Subramanian; Balachandran Unni Nair (157-162).
The binding of Schiff base complexes of chromium(III) of the type [Cr(salen)(H2O)2]+ and [Cr(salprn)(H2O)2]+, where salen denotes 1,2-bis(salicylideneamino)ethane and salprn denotes 1,3-bis(salicylideneamino)propane to calf thymus DNA has been investigated by absorption, emission, circular dichroism, melting temperature and viscosity measurements. These chromium(III) complexes showed absorption hyperchromicity accompanied by red shift in charge transfer band, fluorescence enhancement, increase in melting temperature, some structural changes in CD spectra and changes in specific viscosity when bound to calf thymus DNA. The binding constant K b has been determined from absorption measurements for both the complexes and found to be (2.5±0.4)×103 M−1 for [Cr(salen)(H2O)2]+ and (1.7±0.3)×104 M−1 for [Cr(salprn)(H2O)2]+. From the binding stoichiometry of DNA-[Cr(salprn)(H2O)2]+, the number of binding site size has been determined and found to be ten base pairs per bound complex molecule. The chromium(III) complexes also bring about single strand cleavage in plasmid DNA. The experimental results show that the chromium(III) complexes bind to DNA by non-intercalative mode. Major groove binding is the preferred mode of interaction for these Schiff base complexes of chromium(III).
Keywords: DNA; Chromium(III); Schiff base; Binding; Cleavage;

Interaction of Ni(II) and Cu(II) with a metal binding sequence of histone H4: AKRHRK, a model of the H4 tail by Maria Antonietta Zoroddu; Teresa Kowalik-Jankowska; Henryk Kozlowski; Henriette Molinari; Konstantin Salnikow; Limor Broday; Max Costa (163-168).
Chromatin proteins are believed to represent reactive sites for nickel binding. The unique structure of the N-terminal tail of histone H4 contains sites for post-translational modification close to a histidine residue capable of anchoring binding sites for metal ions. We have analyzed as a minimal model for the H4 tail, the blocked peptide CH3CO-AKRHRK-CONH2 for nickel and copper binding. Ultraviolet–visible, circular dichroism, electron paramagnetic resonance and nuclear magnetic resonance spectroscopic analysis showed that histidine acts as an anchoring metal binding site. A 1N complex is formed between pH=5–7 and 4–6 for Ni(II) and Cu(II), respectively, while at a higher pH a series of 4N complexes are formed. Above pH 8, the 2N high-spin octahedral resulted in a 4N low-spin planar Ni(II) complex. The stability constants of the Cu(II) (3N, 4N) and Ni(II) (4N) complexes with the peptide model of the H4 were distinctly higher than those for a similar blocked peptide with a histidine in the fourth position. Significant shifts in the αproton region in the 1H NMR spectrum of the 4N Ni-complex showed that the conformation of the peptide had been dramatically affected following Ni(II) complexation.
Keywords: Ni(II); Cu(II); Histone H4; AKRHRK peptide;

Photosensitizing activity of hematoporphyrin on Staphylococcus aureus cells by Giulio Bertoloni; Federico M Lauro; Giuliana Cortella; Michèle Merchat (169-174).
The photosensitizing action of hematoporphyrin (Hp) on two Staphylococcus aureus strains was investigated to determine if the photoprocess induces in vivo damage in DNA in addition to that occurring at the level of the cytoplasmic membrane. The results obtained demonstrate that the photokilling is dependent on the Hp dose even though the two strains, having a similar Hp-binding capacity, show different levels of photosensitivity. The electrophoretic analysis of cytoplasmic membrane proteins and DNA (chromosomal and plasmidial) suggests that the membrane represents the primary target of the photoprocess, while the DNA, that is damaged both in vivo and in vitro only at relatively long irradiation time, might be a secondary target. Moreover, the photoprocess results in mutagenesis for Salmonella typhimurium tester strains. This information is particularly important in view of the potential use of photodynamic therapy for the treatment of microbial infections.
Keywords: Hematoporphyrin; Photosensitization; Electrophoresis; Protein; DNA; Staphylococcus aureus;

Inhibition of Fas-mediated apoptosis by Trypanosoma cruzi infection by Junko Nakajima-Shimada; Chunbin Zou; Masatoshi Takagi; Masato Umeda; Takeshi Nara; Takashi Aoki (175-183).
Trypanosoma cruzi-infected and normal control mammalian cells were subjected to analysis of Fas-mediated apoptosis stimulated by an agonistic anti-Fas monoclonal antibody. The infected cells showed markedly hampered apoptotic changes in nuclear morphology, phosphatidylethanolamine translocation from the inside to the outside of the plasma membrane, and DNA fragmentation into multiples of 180 bp, relative to normal control cells. Upstream of these morphological and biochemical consequences, the caspase-3 activity was elevated by the Fas stimulation in a significantly greater proportion of intact control cells, but at a highly reduced rate of infected cells. The rapid elevation of caspase-8 activity in control, apoptotic cells was completely inhibited in infected cells. In an examination of the specificity of other stimulants, X-ray radiation or chemicals such as hydrogen peroxide, colchicine or etoposide did not cause significant differences in apoptotic rates between control and infected cells; tumor necrosis factor-α, however, induced a high rate of apoptosis in control cells, with an extremely lowered rate in infected cells. This study demonstrates, for the first time, that T. cruzi infection inhibits one of the earliest steps of death receptor-mediated apoptosis, an effect that most probably involves the inhibition of caspase-8. Differential apoptotic responses in cells infected with T. cruzi and other intracellular parasites are discussed.
Keywords: Fas-mediated apoptosis; Inhibition of apoptosis; Caspase-8; Intracellular parasite; Trypanosoma cruzi;

Evidence for the regulation of β-N-acetylhexosaminidase expression during pregnancy in the rat by Brunella Tancini; Carla Emiliani; Simona Mencarelli; Cristina Cavalieri; John L. Stirling; Aldo Orlacchio (184-190).
It is believed that the lysosomal glycohydrolase β-N-acetylhexosaminidase plays a part in several important processes of reproduction and it has been postulated that this enzyme is subject to hormonal regulation. During pregnancy, activity levels of the enzyme are strongly increased in both human and rat serum. However, little is known about the expression of this enzyme in the female reproductive apparatus and there is no evidence linking the production of hexosaminidase α- and β-subunits to pregnancy. To clarify these aspects better, we examined the enzyme activity, isoenzyme subunit composition and distribution, as well as steady state levels of α- and β-subunit mRNAs in the female reproductive organs and in other selected tissues of pregnant and non-pregnant rats. Among the female rat tissues tested, the ovary and kidney had the highest specific activity. Pregnancy modulated the hexosaminidase activity differently in the tissues examined. In pregnant rats, the activity decreased in the ovary but increased significantly in the uterus, liver and to a lesser extent in other tissues. The decreased hexosaminidase activity in the ovary from pregnant rats appeared to be accompanied by a disproportionately large decrease in β-subunit mRNA abundance, whereas in the uterus and liver, an increased abundance of this transcript was detectable. The abundance of α-subunit mRNA was comparable in pregnant and control rat tissues. Hexosaminidase histochemical staining of tissue sections clearly demonstrates that the greatly increased activity of hexosaminidase in the uterus during pregnancy is largely due to the enzyme in the endometrium, and not to the uterus as a whole. The overall results provide evidence that, during pregnancy, a mechanism(s) of regulation of β-N-acetylhexosaminidase expression is in operation, and that the enzyme is differentially regulated in rat tissues.
Keywords: β-N-Acetylhexosaminidase; Regulation; Female reproductive apparatus; Pregnancy;