Entamoeba histolytica: gene linkage groups and relevant features of its karyotype

We identified some gene linkage groups in Entamoeba histolytica using a 4-M urea improved transversal alternating field electrophoresis (TAFE) method. Complex rosette-structured DNA molecules were found trapped along the gel lanes, explaining the fuzziness of the patterns. Using several episomal probes, including 16 S, 5.8 S, and 25 S ribosomal (r)Dna genes, an autonomous replication sequence (ARS), and EhVR1, we identified a complete ribosomal episome linkage group (CELG) at the 1.2-Mb position. Three other incomplete groups were found: IELG-1, formed by EhVR1,16 S, 5.8 S, and 25 S genes; IELG-2 formed by EhVR1, 16 S and 25 S; and IELG-3 formed only by 5.8 S. Ehadh3, Ehpfo, and Ehredox genes migrated at the 1.8-Mb position, forming the non-ribosomal linkage group, NRLG-1.8, while the Ehenl-1 gene migrated at 1.6 Mb forming the NRLG-1.6 group. Ehhk was located at 1.2, 0.8, and 0.17 Mb in three different groups: NRLG-1.2, IELG-3-0.8, and NRLG-0.17. Putative lineal chromosomes were also identified using an heterologous telomeric probe. By in situ hybridization experiments, the rDNA and Ehhk genes were located in both nucleus and cytoplasm, while the Ehpfo and Ehredox genes were found mainly in the nucleus. We propose a model hypothezising that the 16 S and 25?S genes are in a linear molecule, duplicated in two inverted repeats, which may be looped out of the linear DNA to form an episome probably lacking or not the 5.8 S sequence, which could be added later by recombination.     

Growth Hormone Treatment of Hypophysectomized Rats Increases Catecholamine-induced Lipolysis and the Number of β-Adrenergic Receptors in Adipocytes: No Differences in the Effects of Growth Hormone on Different Fat Depots

Growth hormone (GH) has a lipolytic effect in adipose tissue but this effect may differ in adipose tissue from various fat depots. This latter possibility was investigated in the present study, in which the effects of GH in vivo on catecholamine-induced lipolysis and the number of β-adrenergic receptors in isolated adipocytes from different fat depots of hypophysectomized rats were investigated. Female and male Sprague-Dawley rats were hypophysectomized or sham-operated at 45 days of age. One week after the operation, hormonal replacement therapy with L-thyroxine and hydrocortisone acetate was given. In addition, groups of rats were treated with GH (1.33 mg/kg per day, given as two daily subcutaneous injections). After 1 week of hormonal treatment, adipocytes were isolated from the parametrial, epididymal and inguinal fat pads, and glycerol release after catecholamine-stimulation and ¹²⁵I-cyanopindolol binding were measured. Hypophysectomy resulted in a marked decrease in the lipolytic response to catecholamines. GH treatment significantly increased catecholamine-induced lipolysis with similar effects in adipocytes from parametrial or epididymal and inguinal fat depots in both female and male rats. There were no differences between norepinephrine compared with isoproterenol-induced responses. ¹²⁵I-cyanopindolol binding was reduced after hypophysectomy and normalized by GH treatment, without differences between parametrial and inguinal adipose tissue regions. We conclude that the lipolytic effects of GH in the rat may partly be mediated by a stimulatory effect on β-adrenergic receptors in adipocytes. In addition, GH exerted similar effect on catecholamine-induced lipolysis and β-adrenergic receptors in adipocytes from parametrial, epididymal and inguinal fat depots.     

Extended repertoire of permissible peptide ligands for HLA-B*2702.

Recognition of self peptides bound to the class I major histocompatibility complex molecule HLA-B27 is thought to trigger proliferation of autoreactive T cells and result in autoimmune arthritic diseases. Previous work from other laboratories established that a predominant feature of endogenous peptides eluted from purified B27 is an arginine at position 2. We studied the binding of peptides containing both natural and unnatural amino acids by the subtype HLA-B*2702, with the goal of gaining insight into peptide binding by this B27 subtype that is associated with susceptibility to arthritic disease. A soluble from of B*2702 was depleted of endogenous peptides. We tested the binding of peptides substituted with cysteine, homocysteine, or an alpha-amino-epsilon-mercapto hexanoic acid side chain (Amh) instead of the naturally occurring arginine at position 2, to determine whether the peptide sulfhydryl residue could be covalently linked to cysteine 67 in the B*2702 binding cleft. Although none of the altered peptide sequences bound covalently to B*2702, the affinities of the homocysteine- and Amh-substituted peptides were close to that of the native peptide sequence. Substitutions at position 2 with other side chains, such as glutamine and methionine, also resulted in peptides that bound with only slightly reduced affinity. These results demonstrate that peptide side chains other than arginine at position 2 can be accomodated within the B*2702 peptide binding site with only minor reductions in affinity. This extended repertoire of permissible B27-binding peptides should be taken into account for a consideration of disease-associated peptide sequences.     

Probing the structure and mobility of Pseudomonas aeruginosa azurin by circular dichroism and dynamic fluorescence anisotropy.

The UV dynamic fluorescence and CD of several Pseudomonas aeruginosa azurins bearing single amino acid mutation have been studied. Two classes of mutants were examined. In the first class, two hydrophobic residues in the core of the protein, Ile 7 and Phe 110, nearest to the azurin single tryptophan Trp 48, were substituted by a serine (mutants 17S and F110S). In the second class, two residues in the outer sphere of the copper ligand field were changed, obtaining the following mutants: M44K, H35F, H35L, and H35Q. All these proteins showed two fluorescence lifetimes in the copper-containing form, but only one in the copper-free form. The lifetime of the latter derivatives was different from either those of the metal-bound samples, definitely ruling out the presence of apo-like species in the holo protein. Copper-free 17S and F110S showed a more complex fluorescence decay profile requiring a distribution of lifetimes rather than a single lifetime. Holo F110S was also better fitted, in the limit of confidence, with two distributions rather than a pair of lifetimes. Time-resolved anisotropy of these two mutants as well as of wild-type (wt) protein showed two components (rotational times for wt < or = 200 ps and 7 ns, respectively). These components were not affected significantly by copper removal in the case of wt protein. Instead, the short rotational component of the mutants dropped dramatically to values near zero, indicating a much greater mobility of the tryptophanyl residue in the mutant apo azurins. These data were supported by CD measurements showing a small effect of the copper presence in the region below 250 nm, i.e., in the secondary structure, but almost a collapse of the aromatic asymmetry at 270-295 nm related to a relaxation of the structural constraint around the tryptophan. Altogether these data show that copper does not play a structural role in wt azurin, whereas it is crucial in the stabilization of 17S and F110S mutants. Furthermore, although the metal site geometry is rigidly kept in wt apo-azurin, it regains the native form only in the presence of the metal in the "core" mutants. This finding is important for the theory of entatic states in metalloproteins (Williams RJP, 1995, Eur J Biochem 234:363-381).     

Conformational stability of apoflavodoxin.

Flavodoxins are alpha/beta proteins that mediate electron transfer reactions. The conformational stability of apoflavodoxin from Anaboena PCC 7119 has been studied by calorimetry and urea denaturation as a function of pH and ionic strength. At pH > 12, the protein is unfolded. Between pH 11 and pH 6, the apoprotein is folded properly as judged from near-ultraviolet (UV) circular dichroism (CD) and high-field 1H NMR spectra. In this pH interval, apoflavodoxin is a monomer and its unfolding by urea or temperature follows a simple two-state mechanism. The specific heat capacity of unfolding for this native conformation is unusually low. Near its isoelectric point (3.9), the protein is highly insoluble. At lower pH values (pH 3.5-2.0), apoflavodoxin adopts a conformation with the properties of a molten globule. Although apoflavodoxin at pH 2 unfolds cooperatively with urea in a reversible fashion and the fluorescence and far-UV CD unfolding curves coincide, the transition midpoint depends on the concentration of protein, ruling out a simple two-state process at acidic pH. Apoflavodoxin constitutes a promising system for the analysis of the stability and folding of alpha/beta proteins and for the study of the interaction between apoflavoproteins and their corresponding redox cofactors.     

Through-bond correlation of adenine H2 and H8 protons in unlabeled DNA fragments by HMBC spectroscopy

Summary A new application of the HMBC experiment is presented that provides a useful means to discriminate between H2 and H8 proton resonances, to assign the base proton resonances to the various residue types and, most importantly, to correlate the H2 and H8 protons for adenine or inosine residues in natural abundance ¹³C fragments. The utility of this experiment is demonstrated for an unlabeled DNA 20-mer. Thanks to the obtained results, preliminary conclusions could be drawn regarding the molecular conformations of the non-canonical G/I-A base pairs in the hairpin formed by this fragment.     

A Basic Peroxidase Isoenzyme, Marker of Resistance Against Plasmopara viticola in Grapevines, is Induced by an Elicitor from Trichoderma viride in Susceptible Grapevines

The constitutive expression of basic peroxidase isoenzymes in the Plasmopara viticola -resistant ( Vitis vinifera × Vitis rupestris) × Vitis riparia crossing and in the P. viticola -susceptible V. vinifera parent species was studied. The results illustrate that both leaves and stems of the ( V. vinifera × V. rupestris) × V. riparia crossing showed the differential expression of a basic peroxidase isoenzyme B₃ (pl = 8.9), this being almost completely absent from the P. viticola -susceptible V. vinifera parent species. To test whether the basic peroxidase isoenzyme B₃ may be considered as a molecular marker of disease resistance in Vitis spp., suspension cell cultures derived from the P. viticola -susceptible V. vinifera parent species were treated with an elicitor (cellulase Onoztika R-10) from the soil fungus Trichoderma viride , a specific and well-known elicitor of disease resistance reactions in grapevines. The results showed that treatment with the elicitor induces, simultaneously with the activation of the disease resistance mechanism, the appearance of B₃ in the cell cultures. These results suggest that the basic peroxidase isoenzyme B₃ may be considered as a marker of disease resistance in Vitis species since it is present in the P. viticola -resistant ( V. vinifera × V. rupestris) × V. riparia hybrid and is induced by the elicitor Onozuka R-10 in cell cultures of the P. viticola -susceptible Vitis vinifera parent species. This conclusion is supported by the presence of this isoenzyme in other resistant and its absence in other susceptible Vitis spp.     

The molecular basis of cystinuria: the role of the rBAT gene

Summary The cDNAs of mammalian amino acid transporters already identified could be grouped into four families. One of these protein families is composed of the protein rBAT and the heavy chain of the cell surface antigen 4F2 (4F2hc). The cRNAs of rBAT and 4F2hc induce amino acid transport activity via systems b^0,+ -like and y⁺L -like inXenopus oocytes respectively. Surprisingly, neither rBAT nor 4F2hc is very hydrophobic, and they seem to be unable to form a pore in the plasma membrane. This prompted the hypothesis that rBAT and 4F2hc are subunits or modulators of the corresponding amino acid transporters. The association of rBAT with a light subunit of ~40kDa has been suggested, and such an association has been demonstrated for 4F2hc.The b^0,+-like system expressed in oocytes by rBAT cRNA transports L-cystine, L-dibasic and L-neutral amino acids with high-affinity. This transport system shows exchange of amino acids through the plasma membrane ofXenopus oocytes, suggesting a tertiary active transport mechanism. The rBAT gene is mainly expressed in the outer stripe of the outer medulla of the kidney and in the mucosa of the small intestine. The protein localizes to the microvilli of the proximal straight tubules (S3 segment) of the nephron and the mucosa of the small intestine. All this suggested the participation of rBAT in a high-affinity reabsorption system of cystine and dibasic amino acids in kidney and intestine, and indicated rBAT (named SLC3A1 in Gene Data Bank) as a good candidate gene for cystinuria. This is an inherited aminoaciduria due to defective renal and intestinal reabsorption of cystine and dibasic amino acids. The poor solubility of cystine causes the formation of renal cystine calculi. Mutational analysis of the rBAT gene of patients with cystinuria is revealing a growing number (~20) of cystinuria-specific mutations, including missense, nonsense, deletions and insertions. Mutations M467T (substitution of methionine 467 residue for threonine) and R270X (stop codon at arginine residue 270) represent approximately half of the cystinuric chromosomes where mutations have been found. Mutation M467T reduces transport activity of rBAT in oocytes. All this demonstrates that mutations in the rBAT gene cause cystinuria.Three types of cystinuria (types, I, II and III) have been described on the basis of the genetic, biochemical and clinical manifestations of the disease. Type I cystinuria has a complete recessive inheritance; type I heterozygotes are totally silent. In contrast, type II and III heterozygotes show, respectively, high or moderate hyperaminoaciduria of cystine and dibasic amino acids. Type III homozygotes show moderate, if any, alteration of intestinal absorption of cystine and dibasic amino acids; type II homozygotes clearly show defective intestinal absorption of these amino acids. To date, all the rBAT cystinuria-specific mutations we have found are associated with type I cystinuria (~70% of the chromosomes studied) but not to types II or III. This strongly suggests genetic heterogeneity for cystinuria. Genetic linkage analysis with markers of the genomic region of rBAT in chromosome 2 (G band 2p16.3) and intragenic markers of rBAT have demonstrated genetic heterogeneity for cystinuria; the rBAT gene is linked to type I cystinuria, but not to type III. Biochemical, genetic and clinical studies are needed to identify the additional cystinuria genes; a low-affinity cystine reabsortion system and the putative light subunit of rBAT are additional candidate genes for cystinuria.     

Redox-Bohr effect in the tetrahaem cytochrome c3 from Desulfovibrio vulgaris: a model for energy transduction mechanisms

 Using potentiometric titrations, two protons were found to participate in the redox-Bohr effect observed for cytochrome c ₃ from Desulfovibrio vulgaris (Hildenborough). Within the framework of the thermodynamic model previously presented, this finding supports the occurrence of a concerted proton-assisted 2e^– step, ideally suited for the coupling role of cytochrome c ₃ to hydrogenase. Furthermore, at physiological pH, it is shown that when sulfate-reducing bacteria use H₂ as energy source, cytochrome c ₃ can be used as a charge separation device, achieving energy transduction by energising protons which can be left in the acidic periplasmic side and transferring deenergised electrons to sulfate respiration. This mechanism for energy transduction, using a full thermodynamic data set, is compared to that put forward to explain the proton-pumping function of cytochrome c oxidase.