The bisnaphthalimides as new active lead compounds against Plasmodium falciparum

The bisquaternary bisnaphthalimides are a versatile class of compounds being active against the malaria parasite Plasmodium falciparum in the lower nanomolar range of concentration combined with no cytotoxicity. The series of compounds is designed as choline analogues and interfering agents of the phosphatidylcholine biosynthesis. The qualitative analysis of the structure–activity relationships (SAR) revealed the importance of a long methylene middle chain of at least 8 methylene groups between the two bisquaternary naphthalimides or a monoquaternary naphthalimide consisting of a long alkyl chain attached to the positively charged nitrogen atom. Since the SARs are different from reported biscationic antimalarial drugs the mode of action remains to be elucidated.     

Bisulfite compounds as metabolic inhibitors: nonspecific effects on membranes

Bisulfite compounds are shown to be nonspecific inhibitors ofphotosynthetic processes and of ion transport in green tissues.CO₂ fixation and light-dependent transient changes in externalpH are inhibited about 50% by 5x10^–4 M glyoxal-Na-bisulfite.Chloride uptake in the light and in the dark is inhibited tothe same extent at this concentration. At 5x10^–3 M theinhibitor reduces ATP levels in the light and in the dark, andeffects on glycolate oxidase and PEP carboxylase are observed.The extent of inhibition is dependent on time of treatment withglyoxal-Na-bisulfite and freshly prepared NaHSO₃ is equallyas effective as the addition compound. Possible explanations of the bisulfite effects and the relationshipsto SO₂ effects on photosynthesis are discussed. (Received September 1, 1971; )     

Mutational scanning of PCR products by subtractive oligonucleotide hybridization analysis

Here, we describe a new approach for mutational scanning of PCR products through hybridization analysis between complementary oligonucleotides. Sets of overlapping probe oligonucleotides complementary to wild-type (WT) sequence are hybridized to microbead-immobilized PCR products under solution-like conditions. Mismatch-hybridization situations between a mutant sample and probe oligonucleotides result in higher remaining concentrations in solution of involved probe oligonucleotides. Post-hybridization supernatants are subsequently analyzed for their probe oligonucleotide compositions using surface plasmon resonance-based biosensor technology. Relative remaining probe oligonucleotide concentrations are monitored in real-time through hybridization analysis between probe oligonucleotides and their corresponding sensor-chip immobilized complementary counterparts. This allows for the construction of composition diagrams revealing the existence and approximate location of a mutation within an investigated sample DNA sequence. Applied on PCR products derived from clinical samples of microdissected tumor biopsies, single mutations in exons 6 and 7 of the human p53 tumor-suppressor gene were successfully detected and approximately localized.     

Selection of protein epitopes for antibody production

Protein functional analysis in the post-genomic era is a huge task that has to be approached by different methods in parallel. The use of protein-specific antibodies in conjunction with tissue microarrays has proven to be one important technology. In this study, we present a strategy for the optimized design of protein subfragments for subsequent antibody production. The fragments are selected based on a principle of lowest sequence similarity to other human proteins, optimally to generate antibodies with high selectivity. Furthermore, the fragments should have properties optimized for efficient protein production in Escherichia coli. The strategy has been implemented in Bishop, which is a Java-based software enabling the high-throughput production of protein fragments. Bishop allows for the avoidance of certain restriction enzyme sites, transmembrane regions, and signal peptides. A Basic Local Alignment Search Tool (BLAST) scanning procedure permits the selection of fragments of a selected size with a minimal sequence similarity to other proteins. The software and the strategy were evaluated on a human test data set and verified to fulfill the requested criteria.     

Exercise hyperventilation, dyspnea sensation, and ergoreflex activation in lone atrial fibrillation

Lone atrial fibrillation may be associated with daily life disability and exercise limitation. The extracardiac pathophysiology of these effects is poorly explored. In 35 subjects with lone atrial fibrillation (mean age 67 +/- 7 yr), we investigated pulmonary function, symptom-limited cardiopulmonary exercise performance, muscle ergoreflex (handgrip exercise) contribution to ventilation, and brachial artery flow-mediated dilation (as a measure of endothelial function) before and after (average interval 20 +/- 5 days) restoring sinus rhythm with external cardioversion. Respiratory volumes and lung diffusing capacity at rest were within normal limits during both atrial fibrillation and after restoring sinus rhythm. Cardioversion was associated with the following changes: a decrease of the slope of exercise ventilation vs. CO2 production (from 35 +/- 5 to 29 +/- 3; P <0.01) and of dyspnea sensation (Borg score from 4 to 2) and an increase of peak oxygen uptake (Vo2; from 16 +/- 4 to 20 +/- 5 ml.min(-1).kg(-1); P <0.01), Vo2 at anaerobic threshold (from 11 +/- 2 to 13 +/- 2 ml.min(-1).kg(-1); P <0.05), and O2 pulse (from 8 +/- 3 to 11 +/- 3 ml/beat; P <0.01). After cardioversion, the observed improvement in ventilatory efficiency was accompanied by a significant peak end-tidal CO2 increase (from 33 +/- 2 to 37 +/- 2 mmHg; P <0.01) and no changes in dead space-to-tidal volume ratio (from 0.23 +/- 0.03 to 0.23 +/- 0.02; P=not significant). In addition, the ergoreflex contribution to ventilation was remarkably attenuated, and the brachial artery flow-mediated dilatation was significantly augmented (from 0.32 +/- 0.07 to 0.42 +/- 0.08 mm; P <0.01). Ten patients had atrial fibrillation relapse and, compared with values after restoration of regular sinus rhythm, invariably showed worsening of endothelial function, exercise ventilatory efficiency, and muscle ergoreflex contribution to ventilation. In subjects with lone atrial fibrillation, an impairment in ventilatory efficiency appears to be involved in the pathophysiology of exercise limitation, and to be primarily related with a demodulated peripheral control of ventilation.     

Glycine betaine aldehyde dehydrogenase from Bacillus subtilis: characterization of an enzyme required for the synthesis of the osmoprotectant glycine betaine

Production of the compatible solute glycine betaine from its precursors choline or glycine betaine aldehyde confers a considerable level of tolerance against high osmolarity stress to the soil bacterium Bacillus subtilis. The glycine betaine aldehyde dehydrogenase GbsA is an integral part of the osmoregulatory glycine betaine synthesis pathway. We strongly overproduced this enzyme in an Escherichia coli strain that expressed a plasmid-encoded gbsA gene under T7φ10 control. The recombinant GbsA protein was purified 23-fold to apparent homogeneity by fractionated ammonium sulfate precipitation, ion-exchange chromatography on Q-Sepharose, and subsequent hydrophobic interaction chromatography on phenyl-Sepharose. Molecular sieving through Superose 12 and sedimentation centrifugation through a glycerol gradient suggested that the native enzyme is a homodimer with 53.7-kDa subunits. The enzyme was specific for glycine betaine aldehyde and could use both NAD⁺ and NADP⁺ as cofactors, but NAD⁺ was strongly preferred. A kinetic analysis of the GbsA-mediated oxidation of glycine betaine aldehyde to glycine betaine revealed K _m values of 125 μM and 143 μM for its substrates glycine betaine aldehyde and NAD⁺, respectively. Low concentrations of salts stimulated the GbsA activity, and the enzyme was highly tolerant of high ionic conditions. Even in the presence of 2.4 M KCl, 88% of the initial enzymatic activity was maintained. B. subtilis synthesizes high levels of proline when grown at high osmolarity, and the presence of this amino acid strongly stimulated the GbsA activity in vitro. The enzyme was stimulated by moderate concentrations of glycine betaine, and its activity was highly tolerant against molar concentrations of this osmolyte. The high salt tolerance and its resistance to its own reaction product are essential features of the GbsA enzyme and ensure that B. subtilis can produce high levels of the compatible solute glycine betaine under conditions of high osmolarity stress. Received: 2 May 1997 / Accepted: 2 July 1997     

Polymorphism of human C2 detected by immunoblotting

Summary To allotype human complement component C2, thin layer agarose gel isoelectric focusing of human serum and/or EDTA-plasma was performed followed by direct immunofixation or by immunoblotting with a specific antihuman-C2 antibody. Using reference samples for C2 BC phenotypes and local samples from an HLA, C4, and Bf genotyped family, a differentiation of the C2^*B and C2^*C variants segregating with the respective HLA haplotype was achieved. The C2 BC phenotype is characterized by a double banding pattern similar to that observed in the haemolytic overlay assay usually used for the detection of C2 polymorphism.An homozygous C2^*Q0 reference sample determined by functional assays was shown to be biochemically deficient, as demonstrated by immunofixation and immunoblotting. The visual interpretation of C2 phenotypes was definitely easier after immunofixation and immunoblotting than after an haemolytic overlay assay. In addition, the method for C2 allotyping described here has several advantages, in particular it saves time and tolerates repeated thawing and freezing of the samples.     

A symplasmic flow of sucrose contributes to phloem loading in Ricinus cotyledons

External sucrose, supplied by the endosperm in vivo, is the physiological source of sucrose for Ricinus communis L. seedlings. It is taken up by the cotyledons and exported via the sieve tubes to the growing hypocotyl and root. Two parallel pathways of external sucrose to the sieve tubes, directly via the apoplasm and indirectly after transit through the mesophyll, have already been established (G. Orlich and E. Komor, 1992). In this study, we analysed whether a symplasmic flow of sucrose contributes to phloem loading. Uptake of external sucrose into the mesophyll and into the sieve tubes, and export of total sucrose were measured with intact and exuding seedlings in the presence of p-chloromercuribenzenesulfonic acid (PCMBS). Sucrose uptake into the mesophyll and into the sieve tubes was inhibited by 80–90%. Consequently, export of total sucrose slowed down. However, after the addition of PCMBS, sucrose was transiently exported in such a high amount that could not be accounted for by the residual uptake activity nor by the amount of sucrose confined to the sieve element-companion cell complex (seccc). From the results, we conclude that most of the sucrose exported transiently had moved to the sieve tubes from a symplasmic domain larger than the seccc, comprising at least all the cells of the bundle including the bundle sheath. We suggest that the symplasmic flow of sucrose observed is a mass flow driven by a turgor pressure. As a structural prerequisite for a symplasmic flow, plasmodesmata interconnect all the cells from the bundle sheath to the sieve tubes and also occur between the bundle sheath and the mesophyll. The phloem loading pathway of Ricinus cotyledons can thus be classified as a combination of three different routes. Received: 17 October 1997 / Accepted: 9 March 1998