²H solid-state nuclear magnetic resonance investigation of whole Escherichia coli interacting with antimicrobial peptide MSI-78

A key aspect of the activity of antimicrobial peptides (AMPs) is their interaction with membranes. Efforts to elucidate their detailed mechanisms have focused on applying biophysical methods, including nuclear magnetic resonance (NMR), to AMPs in model lipid systems. However, these highly simplified systems fail to capture many of the features of the much more complex cell envelopes with which AMPs interact in vivo. To address this issue, we have designed a procedure to incorporate high levels of (2)H NMR labels specifically into the cell membrane of Escherichia coli and used this approach to study the interactions between the AMP MSI-78 and the membranes of intact bacteria. The (2)H NMR spectra of these membrane-deuterated bacteria can be reproduced in the absence and presence of MSI-78. Because the (2)H NMR data provide a quantitative measure of lipid disorder, they directly report on the lipid bilayer disruption central to the function of AMPs, in the context of intact bacteria. Addition of MSI-78 to the bacteria leads to decreases in the order of the lipid acyl chains. The molar peptide:lipid ratios required to observe the effects of MSI-78 on acyl chain order are approximately 30 times greater than the ratios needed to observe effects in model lipid systems and approximately 100 times less than the ratios required to observe inhibition of cell growth in biological assays. The observations thus suggest that MSI-78 disrupts the bilayer even at sublethal AMP levels and that a large fraction of the peptide does not actually reach the inner membrane.     

Post-translational modifications of the serotonin type 4 receptor heterologously expressed in mouse rod cells

G-protein-coupled serotonin receptor type 4 (5-HT(4)R) is a pharmacological target implicated in a variety of gastrointestinal and nervous system disorders. As for many other integral membrane proteins, structural and functional studies of this receptor could be facilitated by its heterologous overexpression in eukaryotic systems that can perform appropriate post-translational modifications (PTMs) on the protein. We previously reported the development of an expression system that employs rhodopsin's biosynthetic machinery in rod cells of the retina to express heterologous G-protein-coupled receptors (GPCRs) in a pharmacologically functional form. In this study, we analyzed the glycosylation, phosphorylation, and palmitoylation of 5-HT(4)R heterologously expressed in rod cells of transgenic mice. We found that the glycosylation pattern in 5-HT(4)R was more complex than in murine and bovine rhodopsin. Moreover, overexpression of this exogenous GPCR in rod cells also affected the glycosylation pattern of coexisting native rhodopsin. These results highlight not only the occurrence of heterogeneous PTMs on transgenic proteins but also the complications that non-native PTMs can cause in the structural and functional characterization of both endogenous and heterologous protein targets.     

Thrombospondin-1 interacts with Trypanosoma cruzi surface calreticulin to enhance cellular infection

Trypanosoma cruzi causes Chagas disease, which is a neglected tropical disease that produces severe pathology and mortality. The mechanisms by which the parasite invades cells are not well elucidated. We recently reported that T. cruzi up-regulates the expression of thrombospondin-1 (TSP-1) to enhance the process of cellular invasion. Here we characterize a novel TSP-1 interaction with T. cruzi that enhances cellular infection. We show that labeled TSP-1 interacts specifically with the surface of T. cruzi trypomastigotes. We used TSP-1 to pull down interacting parasite surface proteins that were identified by mass spectrometry. We also show that full length TSP-1 and the N-terminal domain of TSP-1 (NTSP) interact with T. cruzi surface calreticulin (TcCRT) and other surface proteins. Pre-exposure of recombinant NTSP or TSP-1 to T. cruzi significantly enhances cellular infection of wild type mouse embryo fibroblasts (MEF) compared to the C-terminal domain of TSP-1, E3T3C1. In addition, blocking TcCRT with antibodies significantly inhibits the enhancement of cellular infection mediated by the TcCRT-TSP-1 interaction. Taken together, our findings indicate that TSP-1 interacts with TcCRT on the surface of T. cruzi through the NTSP domain and that this interaction enhances cellular infection. Thus surface TcCRT is a virulent factor that enhances the pathogenesis of T. cruzi infection through TSP-1, which is up-regulated by the parasite.     

Photoassimilatory and Photorespiratory Behaviour of Certain Drought Tolerant and Susceptible Tea Clones

Net photosynthetic rate (P _N) in the mother leaves was higher in the drought tolerant (DT) clones of tea (Camellia sinensis) while liberation of the fixed ¹⁴C in light from the mother leaves was higher in the drought susceptible (DS) clones. The DT clones translocated more photosynthates to the crop shoots (three leaves and a bud) from the mother leaf than the DS clones. Concentrations of RuBP carboxylase (RuBPC) or oxygenase (RuBPO) had no relationship with the drought tolerant nature of tea clones but their ratio correlated with the same. DT tea clones had higher catalase activity that could scavenge the hydrogen peroxide formed in the photorespiratory pathway and thereby reduced photorespiration rate (P _R). The ratio of RuBPC/RuBPO had a positive correlation with P _N and catalase activity. Negative correlation between RuBPC/RuBPO and P _R and between catalase activity and RuBPO activity was established.     

Correlates of parasite load in bumblebees in an Alpine habitat

Essentially, all animals face parasites, but little data are available on the rate of parasitism in wild animals, particularly in insects. Here, we report observations of more than 400 bumblebee workers collected at an Alpine site, including the parasites observed (Crithidia bombi, Nosema bombi, conopid parasitoid fly larvae and tracheal mites), as well as date of collection, bumblebee species and body variables (size, fat content, egg development and antibacterial activity). Among the 14 bumblebee species collected, C. bombi and tracheal mites reached a prevalence of approximately 10 and 6%, respectively, while conopids and N. bombi were almost absent. Correlations among the measured parameters suggest that larger workers are more likely to develop eggs and contain more tracheal mites. Across the season, we found a decrease in fat content but an increase in C. bombi and mite prevalence. Mites’ fitness was higher in fatter bees and lower in bees with more tracheal mites. Antibacterial activity was found in approximately 10% of the workers, suggesting at least sporadic infection with bacteria.     

Between-species correlations in the number of migrants at Ulmeth?chi (Switzerland)

We analysed the number of autumn migrants at a bird ringing station over 41 years in the Jura mountains of Switzerland. For 12 irruptive or potentially irruptive bird species, the correlations between their numbers per year were calculated and the species were clustered accordingly. We found high correlations in the number of migrants between the Coal Tit Periparus ater, Great Tit Parus major and Blue Tit Cyanistes caeruleus. Most correlations of passage number between species pairs changed dramatically over time. Only Blue Tit and Coal Tit showed continuously high correlation in this respect. The variation and changes over time in between-species correlations in the number of migrants needs more attention.     

Enantioselective metabolism of primaquine by human CYP2D6

Given the threat of resistance of human malaria parasites, including to artemisinin derivatives, new agents are needed. Chloroquine (CQ) has been the most widely used anti-malarial, and new analogs (CQAns) presenting alkynes and side chain variations with high antiplasmodial activity were evaluated. Six diaminealkyne and diaminedialkyne CQAns were evaluated against CQ-resistant (CQ-R) (W2) and CQ-sensitive (CQ-S) (3D7) Plasmodium falciparum parasites in culture. Drug cytotoxicity to a human hepatoma cell line (HepG2) evaluated, allowed to calculate the drug selectivity index (SI), a ratio of drug toxicity to activity in vitro. The CQAns were re-evaluated against CQ-resistant and -sensitive P. berghei parasites in mice using the suppressive test. Docking studies with the CQAns and the human (Hss LDH) or plasmodial lactate dehydrogenase (Pf LDH) enzymes, and, a β-haematin formation assay were performed using a lipid as a catalyst to promote crystallization in vitro. All tested CQAns were highly active against CQ-R P. falciparum parasites, exhibiting half-maximal inhibitory concentration (IC50) values below 1 μΜ. CQAn33 and CQAn37 had the highest SIs. Docking studies revealed the best conformation of CQAn33 inside the binding pocket of Pf LDH; specificity between the residues involved in H-bonds of the Pf LDH with CQAn37. CQAn33 and CQAn37 were also shown to be weak inhibitors of Pf LDH. CQAn33 and CQAn37 inhibited β-haematin formation with either a similar or a 2-fold higher IC50 value, respectively, compared with CQ. CQAn37 was active in mice with P. berghei, reducing parasitaemia by 100%. CQAn33, -39 and -45 also inhibited CQ-resistant P. berghei parasites in mice, whereas high doses of CQ were inactive. The presence of an alkyne group and the size of the side chain affected anti-P. falciparum activity in vitro. Docking studies suggested a mechanism of action other than Pf LDH inhibition. The β-haematin assay suggested the presence of an additional mechanism of action of CQAn33 and CQAn37. Tests with CQAn34, CQAn37, CQAn39 and CQAn45 confirmed previous results against P. berghei malaria in mice, and CQAn33, 39 and 45 were active against CQ-resistant parasites, but CQAn28 and CQAn34 were not. The result likely reflects structure-activity relationships related to the resistant phenotype.     

Agrobacterium-mediated transformation and plant regeneration of Brassica oleracea var. capitata

An efficient protocol for Agrobacterium tumefaciens-mediated transformation of four commercial cultivars of Brassica oleracea var. capitata is described. A strain of A. tumefaciens LBA4404 with the neomycin phosphotransferase gene (nptII) and a CaMV 35S-peroxidase gene cassette were used for co-cultivation. Preliminary selection of regenerated transgenic plants was performed on kanamycin-containing medium. The frequency of transgenic plants was calculated on the basis of GUS (β-glucuronidase) activity detected by the histochemical X-gluc test. Tissue-specific GUS expression driven by the peroxidase gene promoter in transgenic plants was analysed by GUS staining. The transformation rates of the commercial cultivars of B. oleracea was higher than in previous reports. Southern blot analysis revealed that integration of marker genes occurred in single and multiple loci in the genome. All transgenic plants grew normally after a brief vernalization period and showed stable inheritance of the marker gene. The present study demonstrates that morphologically normal, fertile transgenic plants of B. oleracea can be obtained. Received: 24 August 1999 / Revision received: 23 November 1999 / Accepted: 3 December     

A synthetic nonapeptide corresponding to the NH2-terminal sequence of C3d-K causes leukocytosis in rabbits

Numerous biologically active fragments have been described that are derived from the C3 molecule. Recently, a polypeptide (Mr 41,000) generated from the alpha chain of human iC3b by limited proteolysis with plasma kallikrein was shown to exhibit several biological functions. This C3-derived cleavage product, C3d-K, suppresses mitogen- and antigen-induced proliferation of human T-lymphocytes and induces leukocytosis in rabbits. We have identified and synthesized a portion of C3d-K that is associated with the leukocytosis phenomenon. A nonapeptide corresponding to the amino-terminal nine residues of C3d-K was synthesized using conventional Merrifield solid-phase peptide chemistry; the structure of this peptide is Thr-Leu-Asp-Pro-Glu-Arg-Leu-Gly-Arg (TLDPERLGR). At a final concentration of 4 X 10(-6) M, both the nonapeptide and the des-Arg octapeptide (TLDPERLG) were capable of inducing leukocytosis in rabbits. Additionally, both peptides enhance vascular permeability when injected in guinea pig skin. These activities are similar to those previously attributed to a C3 fragment identified as C3e by Ghebrehiwet and Müller-Eberhard (Ghebrehiwet, B., and Müller-Eberhard, H.J. (1979) J. Immunol. 123, 616-621). We conclude that the nonapeptide TLDPERLGR represents the active center of the C3-derived leukocytosis factors C3e and C3d-K. This active synthetic analogue of C3d-K should prove valuable in elucidating the mechanism of action for complement-dependent leukocyte mobilization in vivo.