Structure of a pilin monomer from Pseudomonas aeruginosa: implications for the assembly of pili

Type IV pilin monomers assemble to form fibers called pili that are required for a variety of bacterial functions. Pilin monomers oligomerize due to the interaction of part of their hydrophobic N-terminal alpha-helix. Engineering of a truncated pilin from Pseudomonas aeruginosa strain K122-4, where the first 28 residues are removed from the N terminus, yields a soluble, monomeric protein. This truncated pilin is shown to bind to its receptor and to decrease morbidity and mortality in mice upon administration 15 min before challenge with a heterologous strain of Pseudomonas. The structure of this truncated pilin reveals an alpha-helix at the N terminus that lies across a 4-stranded antiparallel beta-sheet. A model for a pilus is proposed that takes into account both electrostatic and hydrophobic interactions of pilin subunits as well as previously published x-ray fiber diffraction data. Our model indicates that DNA or RNA cannot pass through the center of the pilus, however, the possibility exists for small organic molecules to pass through indicating a potential mechanism for signal transduction.     

Reduced Position Effect in Mature Transgenic Plants Conferred by the Chicken Lysozyme Matrix-Associated Region

Matrix-associated regions may be useful for studying the role of chromatin architecture in transgene activity of transformed plants. The chicken lysozyme A element was shown to have specific affinity for tobacco nuclear matrices, and its influence on the variability of transgene expression in tobacco plants was studied. T-DNA constructs in which this element flanked either the [beta]-glucuronidase (GUS) reporter gene or both reporter and selection gene were introduced in tobacco. The variation in GUS gene activity was reduced significantly among mature first-generation transgenic plants carrying the A element. Average GUS activity became somewhat higher, but the maximum attainable level of gene expression was similar for all three constructs. Transient gene expression assays showed that the A element did not contain general enhancer functions; therefore, its presence seemed to prevent the lower levels of transgene expression. The strongest reduction in variability was found in plants transformed with the construct carrying the A elements at the borders of the T-DNA. In this population, expression levels became copy number dependent. The presence of two A elements in the T-DNA did not interfere with meiosis.     

Activation of LFA-1 through a Ca2(+)-dependent epitope stimulates lymphocyte adhesion

The leukocyte function-associated molecule-1 (LFA-1) plays a key role in cell adhesion processes between cells of the immune system. We investigated the mechanism that may regulate LFA-1-ligand interactions, which result in cell-cell adhesion. To this end we employed an intriguing anti-LFA-1 alpha mAb (NKI-L16), capable of inducing rather than inhibiting cell adhesion. Aggregation induced by NKI-L16 or Fab fragments thereof is not the result of signals transmitted through LFA-1. The antibody was found to recognize a unique Ca2(+)-dependent activation epitope of LFA-1, which is essentially absent on resting lymphocytes, but becomes induced upon in vitro culture. Expression of this epitope correlates well with the capacity of cells to rapidly aggregate upon stimulation by PMA or through the TCR/CD3 complex, indicating that expression of the NKI-L16 epitope is essential for LFA-1 to mediate adhesion. However, expression of the NKI-L16 epitope in itself is not sufficient for cell binding since cloned T lymphocytes express the NKI-L16 epitope constitutively at high levels, but do not aggregate spontaneously. Based on these observations we propose the existence of three distinct forms of LFA-1: (a) an inactive form, which does not, or only partially exposes the NKI-L16 epitope, found on resting cells; (b) an intermediate, NKI-L16+ form, expressed by mature or previously activated cells; and (c) an active (NKI-L16+) form of LFA-1, capable of high affinity ligand binding, obtained after specific triggering of a lymphocyte through the TCR/CD3 complex, by PMA, or by binding of NKI-L16 antibodies.     

Plasma 2-hydroxycatecholestrogen responses to acute submaximal and maximal exercise in untrained women

De Crée, Carl, Peter Ball, Bärbel Seidlitz,Gerrit Van Kranenburg, Peter Geurten, and Hans A. Keizer. Plasma2-hydroxycatecholestrogen responses to acute submaximal and maximalexercise in untrained women. J. Appl.Physiol. 82(1): 364-370, 1997.Exercise-induced menstrual problems are accompanied by an increase in catecholestrogen (CE) formation. It has been hypothesized that hypoestrogenemia may besecondary to an increased turnover from estrogens to CE, which then maydisrupt luteinizing hormone release. In addition, the strong affinityof CE for the catecholamine-deactivating enzyme catechol-O-methyltransferase (COMT)has led to speculations about their possible role in safeguardingnorepinephrine from premature decomposition during exercise. Weinvestigated whether acute exercise on a cycle ergometer produces anychanges in CE homeostasis. Nine untrained eumenorrheic women (body fat,24.8 ± 3.1%) volunteered for this study. Baseline plasma CEaverages for total 2-hydroxyestrogens (2-OHE) were 218 ± 29 (SE)pg/ml during the follicular phase (FPh) and 420 ± 58 pg/ml duringthe luteal phase (LPh). 2-Methoxyestrogens (2-MeOE) measured 257 ± 17 pg/ml in the FPh and 339 ± 39 pg/ml in the LPh. Duringincremental exercise, total estrogens (E) increased, but 2-OHE and2-MeOE levels did not significantly change in either phase. The 2-OHE/Eratio (measure of CE turnover) decreased during exercise in bothmenstrual phases, whereas the 2-MeOE/2-OHE ratio (correlates with COMTactivity) did not significantly change. These findings suggest thatthere is insufficient evidence to conclude that brief incrementalexercise in untrained eumenorrheic females acutely produces increasedCE formation.

     

Can the QTL for late blight resistance on potato chromosome 5 be attributed to foliage maturity type?

We investigated the association between late blight resistance and foliage maturity type in potato by means of molecular markers. Two QTLs were detected for foliage resistance against Phytophthora infestans (on chromosomes 3 and 5) and one for foliage maturity type (on chromosome 5). The QTL for resistance to late blight and the QTL for foliage maturity type on chromosome 5 appeared to be mapped on indistinguishable positions. We were interested whether this genetic linkage was due to closely linked but different genes, or due to one (or more) gene(s) with pleiotropic effects. We therefore developed an approach to detect QTLs, in which resistance to late blight was adjusted for foliage maturity type. This analysis revealed the same two QTLs for resistance against P. infestans, but the effect of the locus on chromosome 5 was reduced to only half the original effect. This is a strong indication that the two indistinguishable QTLs for foliage maturity type and for late blight resistance on chromosome 5 may actually be one gene with a pleiotropic effect on both traits. However, there was still a significant effect on resistance against P. infestans on the locus on chromosome 5 after adjusting for foliage maturity type. Therefore we cannot rule out the presence of two closely linked QTLs on chromosome 5: one with a pleiotropic effect on both late blight resistance and foliage maturity type, and another with merely an effect on resistance. In addition, the two QTLs for resistance to late blight showed an important epistatic interaction, suggesting that QTLs for resistance affect each other's expression.     

Effects of a training program on resting plasma 2-hydroxycatecholestrogen levels in eumenorrheic women

De Crée, Carl, Peter Ball, BärbelSeidlitz, Gerrit Van Kranenburg, Peter Geurten, and Hans A. Keizer.Effects of a training program on resting plasma2-hydroxycatecholestrogen levels in eumenorrheic women.J. Appl. Physiol. 83(5):1551-1556, 1997.Catecholestrogens (CE) represent a majormetabolic pathway in estrogen metabolism. Previous information on CEand training is limited to two cross-sectional studies that did notinvolve standardized training. Our purpose, by means of a prospective design, was to evaluate the effects of a brief, exhaustive training program on resting plasma concentrations of 2-hydroxy CE. The experimental design spanned two menstrual cycles: a control cycle and atraining cycle. The subjects were nine previously untrained, eumenorrheic women [body fat: 24.8 ± 1.0 (SE) %]. Datawere collected during the follicular (FPh) and the luteal phases (LPh).Posttraining FPh and LPh tests were held the day after the last day ofa 5-day period of training on a cycle ergometer. Total2-hydroxyestrogens (2-OHE) averaged 200 ± 29 pg/ml during the FPhand 420 ± 54 pg/ml during the LPh(P < 0.05). Levels of total2-methoxyestrogens (2-MeOE) were 237 ± 32 pg/ml during the FPh and339 ± 26 pg/ml during the LPh (P < 0.05). After training, although the plasma levels of 2-OHEsignificantly decreased (21%;P < 0.05) during the LPh, the actualCE formation (as estimated from the 2-OHE-to-total estrogens ratio)increased (+29%; P < 0.05). CE activity, as expressed by the 2-MeOE-to-2-OHE ratio, showedsignificantly higher values in both phases (FPh, +14%; LPh, +13%;P < 0.05). At the same time, restinglevels of norepinephrine (NE) were increased by 42%(P < 0.05). CE strongly inhibitbiological decomposition of NE by catechol-O-methyltransferase (COMT).Results of the present study suggest that, in response to training, CEare increasingly competing with the enzyme COMT, thus preventingpremature NE deactivation.

     

The effect of diffusion on the binding of membrane-bound receptors to coated pits.

We have formulated a kinetic model for the primary steps that occur at the cell membrane during receptor-mediated endocytosis. This model includes the diffusion of receptor molecules, the binding of receptors to coated pits, the loss of coated pits by invagination, and random reinsertion of receptors and coated pits. Using the mechanistic statistical theory of nonequilibrium thermodynamics, we employ this mechanism to calculate the two-dimensional radial distribution of receptors around coated pits at steady state. From this we obtain an equation that describes the effect of receptor diffusion on the rate of binding to coated pits. Our equation does not assume that ligand binding is instantaneous and can be used to assess the effect of diffusion on the binding rate. Using experimental data for low density lipoprotein receptors on fibroblast cells, we conclude that the effect of diffusion on the binding of these receptors to coated pits is no more than 84% diffusion controlled. This corresponds to a dissociation rate constant for receptors on coated pits (k-) that is much less than the rate constant for invagination of the pits (lambda = 3.3 X 10(-3)/s) and a correlation length for the radial distribution function of six times the radius of a coated pit. Although the existing experimental data are compatible with any value of k-, we obtain a lower bound for the value of the binding constant (k+) of 2.3 X 10(-2)(micron)2/s. Comparison of the predicted radial distributions with experiment should provide a clear indication of the effect of diffusion on k+.     

Structure, dynamics and heparin binding of the C-terminal domain of insulin-like growth factor-binding protein-2 (IGFBP-2)

Insulin-like growth factor-binding protein-2 (IGFBP-2) is the largest member of a family of six proteins (IGFBP-1 to 6) that bind insulin-like growth factors I and II (IGF-I/II) with high affinity. In addition to regulating IGF actions, IGFBPs have IGF-independent functions. The C-terminal domains of IGFBPs contribute to high-affinity IGF binding, and confer binding specificity and have overlapping but variable interactions with many other molecules. Using nuclear magnetic resonance (NMR) spectroscopy, we have determined the solution structure of the C-terminal domain of IGFBP-2 (C-BP-2) and analysed its backbone dynamics based on 15N relaxation parameters. C-BP-2 has a thyroglobulin type 1 fold consisting of an alpha-helix, a three-stranded anti-parallel beta-sheet and three flexible loops. Compared to C-BP-6 and C-BP-1, structural differences that may affect IGF binding and underlie other functional differences were found. C-BP-2 has a longer disordered loop I, and an extended C-terminal tail, which is unstructured and very mobile. The length of the helix is identical with that of C-BP-6 but shorter than that of C-BP-1. Reduced spectral density mapping analysis showed that C-BP-2 possesses significant rapid motion in the loops and termini, and may undergo slower conformational or chemical exchange in the structured core and loop II. An RGD motif is located in a solvent-exposed turn. A pH-dependent heparin-binding site on C-BP-2 has been identified. Protonation of two histidine residues, His271 and His228, seems to be important for this binding, which occurs at slightly acidic pH (6.0) and is more significant at pH 5.5, but is largely suppressed at pH 7.4. Possible preferential binding of IGFBP-2 and its C- domain fragments to glycosaminoglycans in the acidic extracellular matrix (ECM) of tumours may be related to their roles in cancer.     

Intramyocellular lipid content in type 2 diabetes patients compared with overweight sedentary men and highly trained endurance athletes

Recent evidence suggests that intramyocellular lipid (IMCL) accretion is associated with obesity and the development of insulin resistance and/or type 2 diabetes. However, trained endurance athletes are markedly insulin sensitive, despite an elevated mixed muscle lipid content. In an effort to explain this metabolic paradox, we compared muscle fiber type-specific IMCL storage between populations known to have elevated IMCL deposits. Immunofluorescence microscopy was performed on muscle biopsies obtained from eight highly trained endurance athletes, eight type 2 diabetes patients, and eight overweight, sedentary men after an overnight fast. Mixed muscle lipid content was substantially greater in the endurance athletes (4.0 +/- 0.4% area lipid stained) compared with the diabetes patients and the overweight men (2.3 +/- 0.4 and 2.2 +/- 0.5%, respectively). More than 40% of the greater mixed muscle lipid content was attributed to a higher proportion type I muscle fibers (62 +/- 8 vs. 38 +/- 3 and 33 +/- 7%, respectively), which contained 2.8 +/- 0.3-fold more lipid than the type II fibers. The remaining difference was explained by a significantly greater IMCL content in the type I muscle fibers of the trained athletes. Differences in IMCL content between groups or fiber types were accounted for by differences in lipid droplet density, not lipid droplet size. IMCL distribution showed an exponential increase in lipid content from the central region toward the sarcolemma, which was similar between groups and fiber types. In conclusion, IMCL contents can be substantially greater in trained endurance athletes compared with overweight and/or type 2 diabetes patients. Because structural characteristics and intramyocellular distribution of lipid aggregates seem to be similar between groups, we conclude that elevated IMCL deposits are unlikely to be directly responsible for inducing insulin resistance.     

DNA binding: a novel function of Pseudomonas aeruginosa type IV pili

The opportunistic pathogen Pseudomonas aeruginosa produces multifunctional, polar, filamentous appendages termed type IV pili. Type IV pili are involved in colonization during infection, twitching motility, biofilm formation, bacteriophage infection, and natural transformation. Electrostatic surface analysis of modeled pilus fibers generated from P. aeruginosa strain PAK, K122-4, and KB-7 pilin monomers suggested that a solvent-exposed band of positive charge may be a common feature of all type IV pili. Several functions of type IV pili, including natural transformation and biofilm formation, involve DNA. We investigated the ability of P. aeruginosa type IV pili to bind DNA. Purified PAK, K122-4, and KB-7 pili were observed to bind both bacterial plasmid and salmon sperm DNA in a concentration-dependent and saturable manner. PAK pili had the highest affinity for DNA, followed by K122-4 and KB-7 pili. DNA binding involved backbone interactions and preferential binding to pyrimidine residues even though there was no evidence of sequence-specific binding. Pilus-mediated DNA binding was a function of the intact pilus and thus required elements present in the quaternary structure. However, binding also involved the pilus tip as tip-specific, but not base-specific, antibodies inhibited DNA binding. The conservation of a Thr residue in all type IV pilin monomers examined to date, along with the electrostatic data, implies that DNA binding is a conserved function of type IV pili. Pilus-mediated DNA binding could be important for biofilm formation both in vivo during an infection and ex vivo on abiotic surfaces.