The effect of phosphate deficiency on mitochondrial activity and adenylate levels in bean roots

Bean plants ( Phaseolus vulgaris ) were grown for 16–20 days with or without phosphate in Knop nutrient medium. It was found in previous experiments that for roots grown on a P_i-deficient medium respiration is mainly carried out by the cyanide-insensitive pathway. Mitochondria isolated from—P_i, roots had poor respiratory control and their respiration exhibited 62% inhibition by cyanide and was inhibited (30%) by salicylhydroxamic acid (SHAM). In contrast, mitochondria obtained with control (+P_i) roots had respiratory control and ADP/O ratios typical for succinate as the substrate; their respiration was inhibited to 95% by cyanide and insensitive to SHAM. The integrity of mitochondrial membranes was similar in both types of mitochondria. Cytochrome oxidase activity, however, was about 20% lower in -P_i mitochondria, but the cytochrome composition was the same in both types of mitochondria. The cytochrorae pathway was not operating at full capacity in mitochondria isolated from—P_i roots but the alternative oxidation pathway participated in a great part in mitochondrial respiration, similar to in vivo whole roots. The participation of the non-phosphorylating., alternative pathway decreased the respiratory control ratio in mitochondria and had an effect on the total adenine nucleotide pool and energy charge values which were lower (16 and 13% respectively) in -P_i roots. About 50% lower ADP and 20% lower ATP levels were observed whereas AMP levels were several times higher.     

Interactions of HIV-1 and HIV-2 envelope glycoproteins with sulphated polysaccharides and mannose-6-phosphate

Envelope glycoproteins of human immunodeficiency viruses (HIV-1and HIV-2) can interact with high-mannose glycans and with themannosyl or N-acetylglucosaminyl core of complex-type oligosaccharidicstructures. HIV-1 glycoproteins also specifically bind sulphatedpolysaccharides such as dextran sulphate (DS) and heparin. Here,we show that the latter property is shared by HIV-2 recombinantgp140 (rgpl40) precursor glycoprotein. Binding of rgpl40 andof corresponding rgp160 of HIV-1 to heparin- and DS-substituted(sulphated dextran beads; SDB) affinity matrices was inhibitedby the soluble specific ligand and also by fetuin, asialofetuinor the anionic simple carbohydrate derivative manncsse-6-phosphate(M6P). Interaction of HIV-1 rgpl20 subunit with the two affinitymatrices was also inhibited by M6P, but only rgpl20 bindingto heparin-agarose, and not that to SDB, was affected by fetuinand asialofetuin. These results suggest that HIV-1 and HIV-2envelope glycoproteins presumably display different sulphatedpolysaccharide and carbohydrate recognition sites. Some of thesemay be common or in close proximity: with respect to rgpl60,for example, the sites may be common on the gp41 moiety and/orin a region of gp120 which would be more accessible when expressedon rgpl60 than on processed gpl20, while they may be distincton the cleaved gpl20 subunit. Finally, because M6P is a markerof lysosomal enzymes, we verified that HIV-1 and HIV-2 envelopeglycoproteins could specifically bind in a M6P-inhibitable mannerto a representative lysosomal enzyme, bovine liver ß-glucuronidasecoupled to agarose, suggesting that they may possibly interferewith lysosomal enzyme sorting in HIV-infected cells. env glycoproteins HIV lectin mannose-6-phosphate sulphated polysaccharides     

The roles of calcium and manganese ions in the in vitro conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene by lentil root membranes

Ca²⁺ and Mn²⁺ activate the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) by root microsomes of Vicia lens as they do in other similar systems. The preparation of microsomes in the presence of Mn²⁺ greatly increases their ability to convert ACC into ethylene, without addition of Mn²⁺ in the reaction mixture. Ca²⁺ does not have this property. The effect could not be attributed to Mn²⁺ entrapping into membrane vesicles (sonication followed by repelleting had no effect) but, possibly, in part to Mn²⁺-mediated binding to microsomes of a soluble factor favouring the conversion of ACC to C₂H₄. Although no direct correlation could be established in vitro between ethylene-forming-enzyme (EFE) and peroxidase activities, some soluble peroxidases might be this soluble factor. Mn²⁺ favoured attachment to membranes of some peroxidase activity from the soluble fraction and from commercial HRP and lipoxygenase. This binding effect of Mn₂₊ cannot be readily distinguished from its role in the generation of a chain of free radicals and in redox mechanisms.     

FRET or no FRET: a quantitative comparison

Fluorescence resonance energy transfer (FRET) is a technique used to measure the interaction between two molecules labeled with two different fluorophores (the donor and the acceptor) by the transfer of energy from the excited donor to the acceptor. In biological applications, this technique has become popular to qualitatively map protein-protein interactions, and in biophysical projects it is used as a quantitative measure for distances between a single donor and acceptor molecule. Numerous approaches can be found in the literature to quantify and map FRET, but the measures they provide are often difficult to interpret. We propose here a quantitative comparison of these methods by using a surface FRET system with controlled amounts of donor and acceptor fluorophores and controlled distances between them. We support the system with a Monte Carlo simulation of FRET, which provides reference values for the FRET efficiency under various experimental conditions. We validate a representative set of FRET efficiencies and indices calculated from the different methods with different experimental settings. Finally, we test their sensitivity and draw conclusions for the preparation of FRET experiments in more complex and less-controlled systems.     

DISCO--a general computer program for the computation of acid dissociation constants of polyprotic molecules in water and biological fluids,from nuclear magnetic resonance data: application to polyamines

A new computer program, DISCO, running under Windows, has been developed under the project CSA98P22 falling within the Competitive Support Activities initiative launched within the EU 4th Framework Programme. DISCO allows the calculation of the stepwise acid dissociation constants of polyprotic molecules in water and in complex media (i.e., biofluids, etc.) from nuclear magnetic resonance (NMR) data (chemical shifts) by means of two derivative-free methods: Pit-mapping and Simplex. DISCO performances were tested using simulated-unaffected by experimental error-data sets, for systems having up to seven equilibrium constants and experimental NMR data of spermine, 6-monofluorospermine, and 6,6-difluorospermine, dissolved in D(2)O and in physiological solution (D(2)O/NaCl). Results demonstrated that (i) DISCO enables the determination of pK(A) values with high precision even when small-sized raw data sets are employed, when chemical shifts are measured with low precision (the usual condition in biofluids due to the impossibility to obtain narrow line shape), and when the guess solution, necessary as an initial step of the mathematical iterative process, is fixed within a large interval of variation; (ii) DISCO always converges to the root; (iii) DISCO permits the calculation of pK(A) values which lie within the observed pH range, independent of the narrowness of the pH range.     

Molecular cloning and characterization of the gene encoding rat submandibular gland apomucin,Mucsmg

Mucin glycoproteins are a major constituent of salivary secretions and play a primary role in the protection of the oral cavity. Rat submandibular glands (RSMG) synthesize and secrete a low molecular weight (114 kDa) mucin glycoprotein. We have isolated, partially sequenced, and characterized the gene which encodes the RSMG apomucin. The gene is encoded by three exons of 106 nt, 69 nt, and 991 nt, separated by introns of 921 nt and 12.5 kb. CAAT and TATA elements are present, at –68 and –26, respectively, in the 5 flanking sequence of the RSMG apomucin gene. The tandem repeat domain present in exon III consists of ten tandem repeats of 39 nt encoding the consensus sequence PTTDSTTPAPTTK. Sequence comparison and organization of the nucleic acid sequence encoding the tandem repeats of two alleles for this gene suggests that the apomucin gene has undergone recombinational events during its evolution. No significant sequence similarity was found with other mucin genes, or with other known salivary gland-specific genes. The gene was localized to rat chromosome 14 using somatic cell hybrids that segregate rat chromosomes. Since this, to our knowledge, represents the first RSMG mucin gene cloned, we have designated this geneMucsmg.Abbreviations RSMG rat submandibular gland - RSM rat salivary mucin - GRP glutamine-glutamic-acid rich protein - nt nucleotide - kb kilobase Sequences reported herein have been assigned GenBank accession numbers U33441 and U33442.     

Urodynamic parameters and plasma LH/FSH in spayed Beagle bitches before and 8 weeks after GnRH depot analogue treatment

The pathophysiology of urinary incontinence due to spaying remains unknown. Incontinent bitches can be treated successfully with depot preparations of GnRH-analogues and there are differences in plasma gonadotropin levels between continent and incontinent spayed bitches. It is therefore assumed that the supraordinated hormones, GnRH, FSH, and/or LH, have an effect on the urodynamic parameters. In this study, the potential influence of these hormones on the lower urinary tract was investigated by measuring urethral pressure profiles and cystometry. Simultaneously, plasma concentrations in 10 spayed Beagle bitches were determined 5 weeks prior to and 8 weeks after treatment with the GnRH analogue leuprolide. Within 1 week of GnRH analogue administration, plasma FSH and LH levels decreased from 72.5 and 7.7 to 7.75 and 0.72ng/mL, respectively. These plasma gonadotropin levels correspond with those of intact bitches during anoestrus. Urethral pressure profiles indicated that the treatment had no significant effect on maximum urethral closure pressure, functional and total length of the urethra, or area of the closure pressure curve. The data obtained by cystometry regarding mean bladder threshold volume showed a significant increase from 109 to 172mL. The improvement in bladder function after the application of GnRH-application is presumably a direct effect of the GnRH as a relationship between the plasma gonadotropin levels and the urodynamic parameters could not demonstrated.