ACID-INDUCED FILAMENT ELONGATION IN IPOMOEA NIL (CONVOLVULACEAE)

It has been known for some time that morning glory filaments elongate in response to increases in concentration of gibberellins (Murakami, 1973) and decreases in ethylene production (Koning and Raab, in press), but many other aspects of their growth have remained unstudied. In the present work, the possible role of gibberellin-stimulated proton efflux in filament growth was examined. Although applied gibberellins stimulated extensive filament growth in vitro and the pH of the incubating medium became acidified during growth, gibberellin also induced growth in media buffered at alkaline pH values. Acidic buffers alone elicited only a very small amount of growth. Fusicoccin, a potent stimulator of proton efflux, initially stimulated the rate of filament growth but elicited only a small increment of growth. In fact, continued presence of fusicoccin poisoned sustained gibberellin-induced growth. Vanadate ions, believed to inhibit proton efflux, had little effect upon gibberellin-induced growth except at extremely high concentrations. Based upon these results, it appears that the acid-induced component of growth stimulation by gibberellin is relatively minor in Ipomoea filaments. These results are quite different from those reported for filament elongation in Gaillardia (Koning, 1983a).     

ROLE OF GROWTH SUBSTANCES IN THE FILAMENT GROWTH OF FUCHSIA HYBRIDA CV “BRILLIANT”

Filaments of Fuchsia hybrida cv “Brilliant” double in length within 24 hr after bud opening. Filament growth characterized by fresh wt increase and cell elongation was significantly inhibited in vitro by l-aminocyclopropane-l-carboxylic acid (ACC) but was not promoted by any growth regulator tested. Ions of Co²⁺ blocked the inhibitive effects of ACC in vitro suggesting that ethylene produced from ACC is the growth inhibiting substance. Ethylene levels surrounding the filaments within the closed bud decreased during development, and premature opening of the sepals which released the ethylene into the atmosphere resulted in rapid filament growth. The ACC levels were found to be much higher in the anthers than the filaments. This suggests that ethylene produced from floral organs other than filaments regulates filament elongation in Fuchsia. This is the first report of filament growth which cannot be promoted by application of growth regulators but which is inhibited by ethylene.     

Genetical genomics in humans and model organisms

Genetical genomics has been proposed to map loci controlling gene-expression differences (eQTLs) that might underlie functional trait variation. We briefly review the studies in model species and conclude that, although they successfully demonstrate the utility of genetical genomics, they are too limited to unlock the full potential of this approach and some results should be interpreted with caution. We subsequently elaborate on two recent studies that use this approach in humans. The many differences between these studies complicate meaningful comparisons between them. A joint analysis of the two experiments offers some scope for more powerful genetical genomics.     

Experimental evaluation of the power balance model of speed skating.

Prediction of speed skating performance with a power balance model requires assumptions about the kinetics of energy production, skating efficiency, and skating technique. The purpose of this study was to evaluate these parameters during competitive imitations for the purpose of improving model predictions. Elite speed skaters (n = 8) performed races and submaximal efficiency tests. External power output (P(o)) was calculated from movement analysis and aerodynamic models and ice friction measurements. Aerobic kinetics was calculated from breath-by-breath oxygen uptake (Vo(2)). Aerobic power (P(aer)) was calculated from measured skating efficiency. Anaerobic power (P(an)) kinetics was determined by subtracting P(aer) from P(o). We found gross skating efficiency to be 15.8% (1.8%). In the 1,500-m event, the kinetics of P(an) was characterized by a first-order system as P(an) = 88 + 556e(-0.0494t) (in W, where t is time). The rate constant for the increase in P(aer) was -0.153 s(-1), the time delay was 8.7 s, and the peak P(aer) was 234 W; P(aer) was equal to 234[1 - e(-0.153(t-8.7))] (in W). Skating position changed with preextension knee angle increasing and trunk angle decreasing throughout the event. We concluded the pattern of P(aer) to be quite similar to that reported during other competitive imitations, with the exception that the increase in P(aer) was more rapid. The pattern of P(an) does not appear to fit an "all-out" pattern, with near zero values during the last portion of the event, as assumed in our previous model (De Koning JJ, de Groot G, and van Ingen Schenau GJ. J Biomech 25: 573-580, 1992). Skating position changed in ways different from those assumed in our previous model. In addition to allowing improved predictions, the results demonstrate the importance of observations in unique subjects to the process of model construction.     

Mechanism of formation of multilayered 2D crystals of the enzyme IIC-mannitol transporter

We have recently reported the crystallization by reconstitution into lipid bilayer structures of Enzyme IIC(mtl), the transmembrane C-domain of the mannitol transporter from E. coli. The projected structure was determined to a resolution of 0.5 nm [J. Mol. Biol. 287 5 (1999) 845]. However, further investigation proved that these crystals were multilamellar stacks instead of 2D crystals, and therefore were unsuitable for three-dimensional structural analysis by electron crystallography. Understanding the crystallogenesis of these crystals could reveal the mechanism of formation of multilayers. In the present study, cryo-electron microscopy (cryo-EM) and turbidimetry are used to study the successive steps of reconstitution of Enzyme IIC(mtl) into phospholipid-containing structures and its crystallization under different conditions. Our experimental approach enabled us to distinguish the separate steps of reconstitution and crystallization. The salt concentration especially influenced the nature of the vesicles, either half open unilamellar or aggregated multilamellar, formed during reconstitution of Enzyme IIC(mtl). The presence of DOPE and DOPC and the temperature influenced the type of lipid structures that were formed during the crystallization phase of Enzyme IIC(mtl). Cryo-EM showed that protein crystallization is closely associated with the formation of isotropic lipid (cubic) phases. We believe that DOPE is responsible for the formation of these lipid cubic phases, and that crystallization is driven by exclusion of protein from these phases and its concentration into the lamellar phases. This mechanism is inextricably associated with the formation of multilayers.     

Computer-assisted mass spectrometric analysis of naturally occurring and artificially introduced cross-links in proteins and protein complexes

A versatile software tool, VIRTUALMSLAB, is presented that can perform advanced complex virtual proteomic experiments with mass spectrometric analyses to assist in the characterization of proteins. The virtual experimental results allow rapid, flexible and convenient exploration of sample preparation strategies and are used to generate MS reference databases that can be matched with the real MS data obtained from the equivalent real experiments. Matches between virtual and acquired data reveal the identity and nature of reaction products that may lead to characterization of post-translational modification patterns, disulfide bond structures, and cross-linking in proteins or protein complexes. The most important unique feature of this program is the ability to perform multistage experiments in any user-defined order, thus allowing the researcher to vary experimental approaches that can be conducted in the laboratory. Several features of VIRTUALMSLAB are demonstrated by mapping both disulfide bonds and artificially introduced protein cross-links. It is shown that chemical cleavage at aspartate residues in the protease resistant RNase A, followed by tryptic digestion can be optimized so that the rigid protein breaks up into MALDI-MS detectable fragments, leaving the disulfide bonds intact. We also show the mapping of a number of chemically introduced cross-links in the NK1 domain of hepatocyte growth factor/scatter factor. The VIRTUALMSLAB program was used to explore the limitation and potential of mass spectrometry for cross-link studies of more complex biological assemblies, showing the value of high performance instruments such as a Fourier transform mass spectrometer. The program is freely available upon request.     

Highly efficient gluten degradation with a newly identified prolyl endoprotease: implications for celiac disease

Celiac disease is a T cell-driven intolerance to wheat gluten. The gluten-derived T cell epitopes are proline-rich and thereby highly resistant to proteolytic degradation within the gastrointestinal tract. Oral supplementation with prolyl oligopeptidases has therefore been proposed as a potential therapeutic approach. The enzymes studied, however, have limitations as they are irreversibly inactivated by pepsin and acidic pH, both present in the stomach. As a consequence, these enzymes will fail to degrade gluten before it reaches the small intestine, the site where gluten induces inflammatory T cell responses that lead to celiac disease. We have now determined the usefulness of a newly identified prolyl endoprotease from Aspergillus niger for this purpose. Gluten and its peptic/tryptic digest were treated with prolyl endoprotease, and the destruction of the T cell epitopes was tested using mass spectrometry, T cell proliferation assays, ELISA, reverse-phase HPLC, SDS-PAGE, and Western blotting. We observed that the A. niger prolyl endoprotease works optimally at 4-5 pH, remains stable at 2 pH, and is completely resistant to digestion with pepsin. Moreover, the A. niger-derived enzyme efficiently degraded all tested T cell stimulatory peptides as well as intact gluten molecules. On average, the endoprotease from A. niger degraded gluten peptides 60 times faster than a prolyl oligopeptidase. Together these results indicate that the enzyme from A. niger efficiently degrades gluten proteins. Future studies are required to determine if the prolyl endoprotease can be used as an oral supplement to reduce gluten intake in patients.     

Biosynthesis and uptake of thiamine (vitamin B1) in bloodstream form Trypanosoma brucei brucei and interference of the vitamin with melarsen oxide activity

Bloodstream forms of Trypanosoma brucei brucei were cultivated in the presence and absence of thiamine (vitamin B1) and pyridoxine (vitamin B6). The vitamins do not change growth behaviour, indicating that Trypanosoma brucei is prototrophic for the two vitamins even though in silico no bona-fide thiamine-biosynthetic genes could be identified in the T. brucei genome. Intracellularly, thiamine is mainly present in its diphosphate form. We were unable to detect significant uptake of [3H]thiamine and structural thiamine analogues such as pyrithiamine, oxithiamine and amprolium were not toxic for the bloodstream forms of T. brucei, indicating that the organism does not have an efficient uptake system for thiamine and its analogues. We have previously shown that, in the fission yeast Saccharomyces pombe, the toxicity of melarsen oxide, the pharmacologically active derivative of the frontline sleeping sickness drug melarsoprol, is abolished by thiamine and the drug is taken up by a thiamine-regulated membrane protein which is responsible for the utilization of thiamine. We show here that thiamine also has weak effects on melarsen oxide-induced growth inhibition and lysis in T. brucei. These effects were consistent with a low affinity of thiamine for the P2 adenosine transporter that is responsible for uptake of melaminophenyl arsenicals in African trypanosomes.     

Trypanosoma brucei: a survey of pyrimidine transport activities

Purine uptake has been studied in many protozoan parasites in the last few years, and several of the purine transporters have been cloned. In contrast, very little is known about the salvage of preformed pyrimidines by protozoa, and no pyrimidine transporters have been cloned, yet chemotherapy based on pyrimidine nucleobases and nucleosides has been as effective as purine antimetabolites in the treatment of infectious and neoplastic disease. Here, we surveyed the presence of pyrimidine transporters in Trypanosoma brucei brucei. We could not detect any mediated uptake of thymine, thymidine or cytidine, but identified a very high-affinity transporter for cytosine, designated C1, with a K(m) value of 0.048+/-0.009 microM. We also confirmed the presence of the previously reported U1 uracil transporter and found it capable of mediating uridine uptake as well, with a K(m) of 33+/-5 microM. A higher-affinity U2 uridine transporter (K(m)=4.1+/-2.1 microM) was also identified, but efficiency of the C1 and U2-mediated transport was low. Pyrimidine antimetabolites were tested as potential trypanocidal agents and only 5-fluorouracil was found to be effective. This drug was efficiently taken up by bloodstream forms of T. b. brucei.     

Selective cytotoxic T-lymphocyte targeting of tumor immune escape variants

Defects in major histocompatibility complex (MHC) class I-restricted antigen presentation are frequently observed in human cancers and result in escape of tumors from cytotoxic T lymphocyte (CTL) immune surveillance in mice. Here, we show the existence of a unique category of CTLs that can prevent this escape. The CTLs target an alternative repertoire of peptide epitopes that emerge in MHC class I at the surface of cells with impaired function of transporter associated with antigen processing (TAP), tapasin or the proteasome. These peptides, although derived from self antigens such as the commonly expressed Lass5 protein (also known as Trh4), are not presented by normal cells. This explains why they act as immunogenic neoantigens. The newly discovered epitopes can be exploited for immune intervention against processing-deficient tumors through adoptive T-cell transfer or peptide vaccination.