Molecular and biological constraints on ligand-binding affinity and specificity

Interactions between biological macromolecules have characteristic values of affinity and specificity that are set according to the biological function that is served by the interaction in the organism. Here we examine the molecular mechanisms that are used to achieve the required values of affinity and specificity in various biological systems. © 1997 John Wiley & Sons, Inc. 44: 181–198, 1997     

Effects of Nitrogen fertilisation and regrowth period on fatty acid concentrations in perennial ryegrass (Lolium perenne L.)

Information on lipids in forages is scarce, and effects of nitrogen (N) application level and regrowth period on the fatty acid (FA) concentration and composition of perennial ryegrass (Lolium perenne L.) were studied. N was applied at 0, 45 and 100 kg ha^?1, and swards were cut after various regrowth periods, resulting in six treatments designed as randomised blocks with three replicates. The stages of development ranged from vegetative to elongating swards, with herbage yield levels from 1.9 to 4.2 t dry matter (DM) ha^?1. Concentrations of individual FA were determined by gas chromatography, and canopy characteristics and herbage quality were assessed. The treatments resulted in canopies with contrasting DM yields and protein concentrations. Five FAs, representing 98% of total FAs, were studied in detail. On an average, the concentration of these major FAs in fresh grass was 15.1 g kg^?1 DM, and 69% of the major FAs consisted of C18:3. Regrowth period affected (P < 0.05) the total FA concentration, and significantly (P < 0.01) lower concentrations of C18:3 and C16:1 were found after a longer period of regrowth. N application resulted in higher (P < 0.001) concentrations of all FAs. The FA composition was not affected by N application, but a longer regrowth period significantly (P < 0.001) decreased the proportion of C18:3 and increased those of C18:2 and C16:0. A strong, positive overall linear relation was found between the concentrations of total FAs and C18:3 with the crude protein concentration in the herbage. These studies demonstrate opportunities to affect the FA concentration and composition of FA in herbage through management strategies, which could affect milk FA composition.     

Structure-activity relationships for the cardiotropic action of the Led-NPF-I peptide in the beetles Tenebrio molitor and Zophobas atratus.

We have examined the effects of the Led-NPF-I peptide (Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-amide) and a series of ten analogues on the heart contractile activity of Tenebrio molitor and Zophobas atratus, and the structure-activity relationships for cardioactive action of Led-NPF-I were established. A video microscopy technique and computer-based method of data acquisition and analysis were used to study the action of the peptides on continuously perfused heart preparations. Cardiac activity was progressively inhibited by Led-NPF-I when the peptide concentrations were increased from 10(-9) to 10(-5) M. Substitution of the L-proline residue at position 4 of the native peptide with hydroxyproline, valine or D-proline caused a loss of cardioinhibitory activity. Also, replacement of arginine residues at all three positions 2, 7 and 9 with another basic amino acid histidine, reduces cardioinhibitory action of Led-NPF-I. Some modifications of the C-terminal residues, as the Phe(4-NO2)-, Phe(4-NH2)- and Phe(4-NMe2)-analogues, resulted in agonistic peptides with biological activity similar to that of the native peptide. However, three other C-terminal analogues tested [Tyr10]-, [D-Phe10]-Led-NPF-I, and Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-OH were inactive in the heart bioassay, which suggests that this end of the amino acid chain may play an important role in bioactivity and interaction of the native peptide with its receptor on the myocardium.     

Intradiurnal periodicity of fungal spore concentrations (Alternaria, Botrytis, Cladosporium, Didymella, Ganoderma) in Cracow, Poland

Aerobiological monitoring enables the definition of seasonal fungal spore concentrations and also intradiurnal time when the highest concentrations of spores could cause or increase allergy symptoms. These data are useful to estimate symptoms of disease, duration of infection and how advanced the illness is in people suffering from fungal allergens. The aim of the study was to compare the concentrations of fungal spores (Alternaria, Botrytis, Cladosporium, Didymella, Ganoderma) during dry and rainy periods and to analyse their intradiurnal changes. Average daily spore concentrations in dry and rainy periods were compared, using z test, separately for each taxon, season and for a combined 3-year period. Intradiurnal periodicity of fungal spore concentrations was analysed on the basis of three complementary diagrams. These spore concentrations were presented using three curves for all, dry and rainy days in 1997–1999 (April–November). The spore percentage in particular hours was normalized in relation to the daily spore sum accepted as 100%. Two further diagrams enabled the more precise analysis of the highest concentrations in dry days. Daily Botrytis and Cladosporium spore concentrations did not show significant differences between dry and rainy periods. In the case of Didymella and Ganoderma spore concentrations, there were no significant differences between both weather types in the single years, although there was a significant difference when a 3-year period was considered. The differences between daily concentrations of Alternaria spores in dry and rainy periods occurred in 1997 and in a 3-year period. Intradiurnal periodicity of spore concentrations was different for ‘dry’ and ‘wet’ fungal spores. Dry spores are released from the spore-producing parts of the fungus under conditions of decreasing humidity and increasing airflow. Examples of dry spores are those from Alternaria, Cladosporium and Botrytis. Wet spores, such as those from many Ascomycetes (Didymella) and Basidiomycetes (Ganoderma), are released into the atmosphere by processes related to humidity conditions or rain. The highest concentrations of ‘dry’ spores were observed early in the afternoon, while highest values of ‘wet’ spore concentrations occurred in the predawn hours. Statistically non-significant differences between daily spore concentrations in dry and rainy periods of single seasons were found except for Alternaria. Statistically significant differences could occur when the studied period was longer than one season (Alternaria, Didymella, Ganoderma). The highest concentrations of Alternaria, Botrytis and Cladosporium spores were recorded at noon and early in the afternoon. Concentrations of Didymella and Ganoderma spores were highest in the predawn hours.     

The effect of long-term lactobacilli (lactic acid bacteria) enteral treatment on the central nervous system of growing rats

The aim of this study was to explore the relationship between consumption of large doses of lactic acid bacteria (LAB) and the behaviour and brain morphobiochemistry of normal growing rats. Four groups of rats were treated with LAB cultures twice daily for 6 months. The control group received 1 ml of saline per treatment, while two experimental groups received 1 ml of living bacteria (Lactobacillus plantarum and Lactobacillus fermentum, respectively) and the remaining group received a heat-treated (inactivated) L. fermentum culture. After 2 and 6 months of treatment, respectively, eight animals from each group were sacrificed, and specimens were taken for further analyses. The behaviour of the rats was evaluated five times in an open-field test at monthly intervals throughout the study. Lactobacilli treatment for 2 months induced changes in the motoric behaviour of the rats. The concentration of the astrocytesoluble and filament glial fibrillary acidic protein (GFAP) decreased in the posterior part of the hemispheres, including the thalamus, hippocampus and cortex of the rats treated with L. fermentum. A greater decrease in filament GFAP (up to 50%) was shown in the group receiving the live form of L. fermentum. In contrast, the GFAP in the live L. plantarum-treated group increased, showing elevated levels of the soluble and filament forms of GFAP in the posterior part of the hemispheres.A 60–66% decrease in the amount of the astrocyte-specific Ca-binding protein S-100b was shown in the posterior parts of the hemispheres and in the hindbrain of rats given LAB for 2 months.Prolonged feeding with LAB for 4 months up to full adulthood led to a further decrease in astrocyte reaction, reflected as an additional decrease in the amount of soluble GFAP and locomotor activity in all experimental groups. The changes in filament GFAP and S-100b appeared to disappear after prolonged feeding (total of 6 months) with LAB.In summary, LAB dietary treatment affected the ontogenetic development of the astrocytes, with the highest intensity observed in the early stages of rat development. It can be postulated that LAB treatment may play a preventive role in neurological diseases by decreasing astrocyte reaction and, consequently, lowering locomotor activity.     

Inhibitors of bacterial N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) and demonstration of in vitro antimicrobial activity

The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) is a critical bacterial enzyme for the construction of the bacterial cell wall. A screen biased toward compounds containing zinc-binding groups (ZBG’s) including thiols, carboxylic acids, boronic acids, phosphonates and hydroxamates has delivered a number of micromolar inhibitors of DapE from Haemophilus influenzae, including the low micromolar inhibitor l-captopril (IC₅₀ = 3.3 μM, K_i = 1.8 μM). In vitro antimicrobial activity was demonstrated for l-captopril against Escherichia coli.     

The Role of the N-Terminus of the Myosin Essential Light Chain in Cardiac Muscle Contraction

To study the regulation of cardiac muscle contraction by the myosin essential light chain (ELC) and the physiological significance of its N-terminal extension, we generated transgenic (Tg) mice by partially replacing the endogenous mouse ventricular ELC with either the human ventricular ELC wild type (Tg-WT) or its 43-amino-acid N-terminal truncation mutant (Tg-Δ43) in the murine hearts. The mutant protein is similar in sequence to the short ELC variant present in skeletal muscle, and the ELC protein distribution in Tg-Δ43 ventricles resembles that of fast skeletal muscle. Cardiac muscle preparations from Tg-Δ43 mice demonstrate reduced force per cross-sectional area of muscle, which is likely caused by a reduced number of force-generating myosin cross-bridges and/or by decreased force per cross-bridge. As the mice grow older, the contractile force per cross-sectional area further decreases in Tg-Δ43 mice and the mutant hearts develop a phenotype of nonpathologic hypertrophy while still maintaining normal cardiac performance. The myocardium of older Tg-Δ43 mice also exhibits reduced myosin content. Our results suggest that the role of the N-terminal ELC extension is to maintain the integrity of myosin and to modulate force generation by decreasing myosin neck region compliance and promoting strong cross-bridge formation and/or by enhancing myosin attachment to actin.     

Lack of CFTR in Skeletal Muscle Predisposes to Muscle Wasting and Diaphragm Muscle Pump Failure in Cystic Fibrosis Mice

Cystic fibrosis (CF) patients often have reduced mass and strength of skeletal muscles, including the diaphragm, the primary muscle of respiration. Here we show that lack of the CF transmembrane conductance regulator (CFTR) plays an intrinsic role in skeletal muscle atrophy and dysfunction. In normal murine and human skeletal muscle, CFTR is expressed and co-localized with sarcoplasmic reticulum-associated proteins. CFTR–deficient myotubes exhibit augmented levels of intracellular calcium after KCl-induced depolarization, and exposure to an inflammatory milieu induces excessive NF-kB translocation and cytokine/chemokine gene upregulation. To determine the effects of an inflammatory environment in vivo, sustained pulmonary infection with Pseudomonas aeruginosa was produced, and under these conditions diaphragmatic force-generating capacity is selectively reduced in Cftr^−/− mice. This is associated with exaggerated pro-inflammatory cytokine expression as well as upregulation of the E3 ubiquitin ligases (MuRF1 and atrogin-1) involved in muscle atrophy. We conclude that an intrinsic alteration of function is linked to the absence of CFTR from skeletal muscle, leading to dysregulated calcium homeostasis, augmented inflammatory/atrophic gene expression signatures, and increased diaphragmatic weakness during pulmonary infection. These findings reveal a previously unrecognized role for CFTR in skeletal muscle function that may have major implications for the pathogenesis of cachexia and respiratory muscle pump failure in CF patients.     

A lectin affinity workflow targeting glycosite-specific, cancer-related carbohydrate structures in trypsin-digested human plasma

Glycans are cell-type-specific, posttranslational protein modifications that are modulated during developmental and disease processes. As such, glycoproteins are attractive biomarker candidates. Here, we describe a mass spectrometry-based workflow that incorporates lectin affinity chromatography to enrich for proteins that carry specific glycan structures. As increases in sialylation and fucosylation are prominent among cancer-associated modifications, we focused on Sambucus nigra agglutinin (SNA) and Aleuria aurantia lectin (AAL), lectins which bind sialic acid- and fucose-containing structures, respectively. Fucosylated and sialylated glycopeptides from human lactoferrin served as positive controls, and high-mannose structures from yeast invertase served as negative controls. The standards were spiked into Multiple Affinity Removal System (MARS) 14-depleted, trypsin-digested human plasma from healthy donors. Samples were loaded onto lectin columns, separated by HPLC into flow-through and bound fractions, and treated with peptide: N-glycosidase F to remove N-linked glycans. The deglycosylated peptide fractions were interrogated by ESI HPLC-MS/MS. We identified a total of 122 human plasma glycoproteins containing 247 unique glycosites. Importantly, several of the observed glycoproteins (e.g., cadherin 5 and neutrophil gelatinase-associated lipocalin) typically circulate in plasma at low nanogram per milliliter levels. Together, these results provide mass spectrometry-based evidence of the utility of incorporating lectin-separation platforms into cancer biomarker discovery pipelines.