A study of the interaction of Cr(III) complexes and their selective binding with B-DNA: a molecular modeling approach

Molecular modeling and energy minimisation calculations have been used to investigate the interaction of chromium(III) complexes in different ligand environments with various sequences of B-DNA. The complexes are [Cr(salen)(H(2)O)(2)](+); salen denotes 1, 2 bis-salicylideneaminoethane, [Cr(salprn)(H(2)O)(2)](+); salprn denotes 1, 3 bis- salicylideneaminopropane, [Cr(phen)(3)](3+); phen denotes 1, 10 phenanthroline and [Cr(en)(3)](3+); en denotes ethylenediamine. All the chromium(III) complexes are interacted with the minor groove and major groove of d(AT)(12), d(CGCGAATTCGCG)(2) and d(GC)(12) sequences of DNA. The binding energy and hydrogen bond parameters of DNA-Cr complex adduct in both the groove have been determined using molecular mechanics approach. The binding energy and formation of hydrogen bonds between chromium(III) complex and DNA has shown that all complexes of chromium(III) prefer minor groove interaction as the favourable binding mode.     

The processing of Holliday junctions by BLM and WRN helicases is regulated by p53

BLM, WRN, and p53 are involved in the homologous DNA recombination pathway. The DNA structure-specific helicases, BLM and WRN, unwind Holliday junctions (HJ), an activity that could suppress inappropriate homologous recombination during DNA replication. Here, we show that purified, recombinant p53 binds to BLM and WRN helicases and attenuates their ability to unwind synthetic HJ in vitro. The p53 248W mutant reduces abilities of both to bind HJ and inhibit helicase activities, whereas the p53 273H mutant loses these abilities. Moreover, full-length p53 and a C-terminal polypeptide (residues 373-383) inhibit the BLM and WRN helicase activities, but phosphorylation at Ser(376) or Ser(378) completely abolishes this inhibition. Following blockage of DNA replication, Ser(15) phospho-p53, BLM, and RAD51 colocalize in nuclear foci at sites likely to contain DNA replication intermediates in cells. Our results are consistent with a novel mechanism for p53-mediated regulation of DNA recombinational repair that involves p53 post-translational modifications and functional protein-protein interactions with BLM and WRN DNA helicases.     

Molecular characterization of L-CPT I deficiency in six patients: insights into function of the native enzyme.

Carnitine palmitoyltransferase I (CPT I) catalyzes the formation of acylcarnitine, the first step in the oxidation of long-chain fatty acids in mitochondria. The enzyme exists as liver (L-CPT I) and muscle (M-CPT I) isoforms that are encoded by separate genes. Genetic deficiency of L-CPT I, which has been reported in 16 patients from 13 families, is characterized by episodes of hypoketotic hypoglycemia beginning in early childhood and is usually associated with fasting or illness. To date, only two mutations associated with L-CPT I deficiency have been reported. In the present study we have identified and characterized the mutations underlying L-CPT I deficiency in six patients: five with classic symptoms of L-CPT I deficiency and one with symptoms that have not previously been associated with this disorder (muscle cramps and pain). Transfection of the mutant L-CPT I cDNAs in COS cells resulted in L-CPT I mRNA levels that were comparable to those expressed from the wild-type construct. Western blotting revealed lower levels of each of the mutant proteins, indicating that the low enzyme activity associated with these mutations was due, at least in part, to protein instability. The patient with atypical symptoms had approximately 20% of normal L-CPT I activity and was homozygous for a mutation (c.1436C-->T) that substituted leucine for proline at codon 479. Assays performed with his cultured skin fibroblasts indicated that this mutation confers partial resistance to the inhibitory effects of malonyl-CoA. The demonstration of L-CPT I deficiency in this patient suggests that the spectrum of clinical sequelae associated with loss or alteration of L-CPT I function may be broader than was previously recognized.     

The S7 ribosomal protein gene is truncated and overlaps a cytochrome c biogenesis gene in pea mitochondria

The pea mitochondrial genome contains a truncated rps7 gene lacking ca. 40 codons at its 5 terminus. This single-copy sequence is immediately downstream of and slightly overlapping an actively transcribed and edited reading frame of 744 bp (designated ccb248) homologous to the bacterial helC gene which encodes a subunit of the ABC-type heme transporter involved in cytochrome c biogenesis. This region of mitochondrial DNA appears recombinogenic, and the carboxy-termini of helC-type proteins are predicted to vary in sequence and length among plants. Sequences corresponding to the 5 coding region of rps7 were not detected elsewhere in the pea mitochondrial genome using wheat rps7 probes, and only a very short internal rps7 segment was observed in soybean mitochondrial DNA. The presence of rps7-homologous sequences in the nuclear genomes of pea and soybean is consistent with the recent transfer of a functional mitochondrial rps7 gene to the nucleus in certain plant lineages.     

Volatile aroma constituents of parsley leaves

The aroma volatiles of a desert parsley were analysed using routine procedures, and 45 constituents were positively identified, including 11 not previously reported as parsley leaf volatiles. One component, 2-(p-tolyl)propan-2-ol, is a new aroma volatile and, together with p-mentha- 1,3,8-triene, may be unique to parsley. Major constituents of the sample were 4-methoxy-6-(prop-2-enyl)benzo-1,3-dioxolan (myristicin) 4,7-dimethoxy-5-(prop-2-enyl)benzo-1,3 dioxolan (apiole), β-phellandrene, p-mentha-1,3,8-triene and 4-isopropenyl-1-methylbenzene. Aroma assessments during GC showed that apiole, in particular, had desirable parsley odour character.     

Aspartate modulates the circadian patterns of a few biochemical variables in Wistar rats

D-aspartate was used to demonstrate possible sources of excitatory input to the suprachiasmatic nucleus (SCN) in rats. Aspartate (50 mg/kg bodyweight) was orally administrated chronically for 60 days to Wistar rats and 24 h rhythmic patterns of glucose, cholesterol, total protein and aspartate transaminase (AST) were studied under light - dark (LD 12:12 h) cycle. Our results showed acrophase advances in glucose and delays in cholesterol and AST rhythms. Increased mesor and altered amplitude values were found in all rhythms; aspartate levels in the brain were found to be significantly increased in aspartate treated animals. We hypothesised that the altered biochemical rhythms in aspartate treated rats could be due to (1) modulation of neurotransmission in SCN, (2) behavioural rhythms and (3) feeding rhythms.     

Characterisation of three novel cationic lipids as liposomal complexes with DNA

Cationic lipids (CLs) are being increasingly exploited as transfection vectors for the delivery of DNA into eukaryotic cells. To obtain further insight to the complex formation and interactions between cationic liposomes and DNA, we characterised three novel cationic lipids, viz. bis[2-(11-phenoxyundecanoate)ethyl]-dimethylammonium bromide, N-hexadecyl-N-?10-[O-(4-acetoxy)-phenylundecanoate]ethyl?- dimethylammonium bromide, and bis[2-(11-butyloxyundecanoate)ethyl]dimethylammonium bromide. These lipids bear the same charged headgroup yet have different hydrophobic parts. Accordingly, we may anticipate their electrostatic interactions with DNA to be similar while differing in both thermal phase behaviour and physicochemical properties of their complexes with DNA. In keeping with the above all three lipids formed complexes with DNA as evidenced by light scattering, fluorescence spectroscopy and Langmuir film balance. Differential scanning calorimetry revealed very different phase behaviours for the binary mixtures of the three CLs with dimyristoylphosphatidylcholine and also provided evidence for DNA-induced lipid phase separation. These data were confirmed by compression isotherms and fluorescence microscopy of monolayers residing on an aqueous buffer, recorded both in the presence and absence of DNA. Importantly, binding to cationic liposomes appears to prevent thermal denaturation of DNA upon heating of the complexes. Likewise, renaturation of heat-treated DNA complexed with the cationic liposomes appears to be abolished as well.     

Identification of a Hsp70 recognition domain within the rubisco small subunit transit peptide

The interaction between SStp, the transit peptide of the precursor protein to the small subunit of Rubisco (prSSU) and two Hsp70 molecular chaperones, Escherichia coli DnaK and pea (Pisum sativum) CSS1, was investigated in detail. Two statistical analyses were developed and used to investigate and predict regions of SStp recognized by DnaK. Both algorithms suggested that DnaK would have high affinity for the N terminus of SStp, moderate affinity for the central region, and low affinity for the C terminus. Furthermore, both algorithms predicted this affinity pattern for >75% of the transit peptides analyzed in the chloroplast transit peptide (CHLPEP) database. In vitro association between SStp and these Hsp70s was confirmed by three independent assays: limited trypsin resistance, ATPase stimulation, and native gel shift. Finally, synthetic peptides scanning the length of SStp and C-terminal deletion mutants of SStp were used to experimentally map the region of greatest DnaK affinity to the N terminus. CSS1 displayed a similar affinity for the N terminus of SStp. The major stromal Hsp70s affinity for the N terminus of SStp and other transit peptides supports a molecular motor model in which the chaperone functions as an ATP-dependent translocase, committing chloroplast precursor proteins to unidirectional movement across the envelope.     

Role of exogenous inositol and phosphatidylinositol in glycosylphosphatidylinositol anchor synthesis of GP49 by Giardia lamblia

Although Giardia lamblia trophozoites are unable to carry out de novo phospholipid synthesis, they can assemble complex glycophospholipids from simple lipids and fatty acids acquired from the host. Previously, we have reported that G. lamblia synthesizes GP49, an invariant surface antigen with a glycosylphosphatidylinositol (GPI) anchor. It is therefore possible that myo-inositol (Ins), phosphatidylinositol (PI) and other GPI precursors are obtained from the dietary products of the human small intestine, where the trophozoites colonize. In this report, we have investigated the role of exogenous Ins and PI on GPI anchor synthesis by G. lamblia. The results demonstrate that [³H]Ins and PI internalized by trophozoites, metabolically transformed into GlcN(acyl)-PI and downstream GPI molecules. Further investigations suggest that G. lamblia expresses cytidine monophosphate (CMP)-dependent (Mg²⁺-stimulated) and independent (Mn²⁺-stimulated) inositol headgroup exchange enzymes, which are responsible for exchanging free Ins with cellular PI. We observed that 3-deoxy-3-fluoro-D-myo-inositol (3-F-Ins) and 1-deoxy-1-F-scyllo-Ins (1-F-scyllo-Ins), which are considered potent inhibitors of Mn²⁺-stimulated headgroup exchange enzyme, inhibited the incorporation of [³H]Ins into PI and GPI molecules significantly, suggesting that CMP-independent (Mn²⁺-stimulated) exchange enzyme may be important for these reactions. However, 3-F-Ins and 1-F-scyllo-Ins were not effective in blocking the incorporation of exogenously supplied [³H]PI into GPI glycolipids. Thus, it can be concluded that G. lamblia can use exogenously supplied [³H]PI and [³H]Ins to synthesize GPI glycolipids of GP49; while PI is directly incorporated into GPI molecules, free Ins is first converted into PI by headgroup exchange enzymes, and this newly formed PI participates in GPI anchor synthesis.