Antibody response to heat shock proteins and histopathology in mice infected with Trypanosoma cruzi and maintained at elevated temperature

Highly susceptible C3HeB/FeJ mice survive an otherwise lethal infection with a Brazil strain of Trypanosoma cruzi when held at an elevated environmental temperature of 36 C. The body temperature of these mice has been shown to increase 3-4 C to levels typical of a febrile response. In the present study, the synthesis of parasite heat shock proteins (hsp60, hsp70, and hsp90) was shown to be enhanced at a temperature of 39 C and the results of immunoprecipitation analysis indicated that parasite HSPs are highly immunogenic in T. cruzi-infected mice maintained at 36 C or room temperature (RT). Differences in the histopathology of cardiac and skeletal muscle in C3HeB/FeJ mice maintained at RT or 36 C at different times postinfection also were investigated in this study. The lower numbers of circulating parasites observed in mice maintained at 36 C were correlated with lower levels of tissue parasitism, inflammation, and tissue destruction. Finally, the transfer of infected mice from RT to an environment of 36 C at various times during infection was shown to increase the survival rate of infected mice and also resulted in a dramatic reduction in parasitemia levels. In light of the growing evidence for a beneficial effect of elevated temperature during experimental Chagas' disease, further studies seem warranted to determine if hyperthermia or fever therapy might also be beneficial in the treatment of humans infected with T. cruzi.     

RA-GEF, a novel Rap1A guanine nucleotide exchange factor containing a Ras/Rap1A-associating domain, is conserved between nematode and humans

A yeast two-hybrid screening for Ras-binding proteins in nematode Caenorhabditis elegans has identified a guanine nucleotide exchange factor (GEF) containing a Ras/Rap1A-associating (RA) domain, termed Ce-RA-GEF. Both Ce-RA-GEF and its human counterpart Hs-RA-GEF possessed a PSD-95/DlgA/ZO-1 (PDZ) domain and a Ras exchanger motif (REM) domain in addition to the RA and GEF domains. They also contained a region homologous to a cyclic nucleotide monophosphate-binding domain, which turned out to be incapable of binding cAMP or cGMP. Although the REM and GEF domains are conserved with other GEFs acting on Ras family small GTP-binding proteins, the RA and PDZ domains are unseen in any of them. Hs-RA-GEF exhibited not only a GTP-dependent binding activity to Rap1A at its RA domain but also an activity to stimulate GDP/GTP exchange of Rap1A both in vitro and in vivo at the segment containing its REM and GEF domains. However, it did not exhibit any binding or GEF activity toward Ras. On the other hand, Ce-RA-GEF associated with and stimulated GDP/GTP exchange of both Ras and Rap1A. These results indicate that Ce-RA-GEF and Hs-RA-GEF define a novel class of Rap1A GEF molecules, which are conserved through evolution.     

The putative brain photoperiodic photoreceptors in the vetch aphid,Megoura viciae

In an attempt to identify the brain photoreceptors that mediate the photoperiodic response of the vetch aphid, Megoura viciae, we utilised immunocytochemical techniques and employed 20 antibodies directed against invertebrate and vertebrate opsins and phototransduction proteins. A sub-set of these antibodies (to Drosophila rhodopsin 1: RH1-1; vertebrate cone opsins: COS-1; CERN-874; CERN-933; vertebrate rod opsin: CERN-901; vertebrate arrestin: AB-Arr; vertebrate transducin+arrestin+rhodopsin kinase+cGMP phosphodiesterase: CERN-911; and vertebrate cellular retinoid binding protein: CRALBP) consistently labelled an anterior ventral neuropile region of the protocerebrum. These anatomical findings, coupled with previous localised illumination and micro-lesion studies, provide strong evidence that this region of the aphid brain houses the photoperiodic photoreceptors. The present study also confirms that the medial (Group I) neurosecretory cells are not the photoperiodic photoreceptors.     

A novel alkaline alpha-galactosidase from melon fruit with a substrate preference for raffinose

The cucurbits translocate the galactosyl-sucrose oligosaccharides raffinose and stachyose, therefore, alpha-galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) is expected to function as the initial enzyme of photoassimilate catabolism. However, the previously described alkaline alpha-galactosidase is specific for the tetrasaccharide stachyose, leaving raffinose catabolism in these tissues as an enigma. In this paper we report the partial purification and characterization of three alpha-galactosidases, including a novel alkaline alpha-galactosidase (form I) from melon (Cucumis melo) fruit tissue. The form I enzyme showed preferred activity with raffinose and significant activity with stachyose. Other unique characteristics of this enzyme, such as weak product inhibition by galactose (in contrast to the other alpha-galactosidases, which show stronger product inhibition), also impart physiological significance. Using raffinose and stachyose as substrates in the assays, the activities of the three alpha-galactosidases (alkaline form I, alkaline form II, and the acid form) were measured at different stages of fruit development. The form I enzyme activity increased during the early stages of ovary development and fruit set, in contrast to the other alpha-galactosidase enzymes, both of which declined in activity during this period. In the mature, sucrose-accumulating mesocarp, the alkaline form I enzyme was the major alpha-galactosidase present. We also observed hydrolysis of raffinose at alkaline conditions in enzyme extracts from other cucurbit sink tissues, as well as from young Coleus blumei leaves. Our results suggest different physiological roles for the alpha-galactosidase forms in the developing cucurbit fruit, and show that the newly discovered enzyme plays a physiologically significant role in photoassimilate partitioning in cucurbit sink tissue.     

Structural basis of electron transfer modulation in the purple CuA center

The X-ray structure of an engineered purple CuA center in azurin from Pseudomonas aeruginosa has been determined and refined at 1.65 A resolution. Two independent purple CuA azurin molecules are in the asymmetric unit of a new P21 crystal, and they have nearly identical conformations (rmsd of 0.27 A for backbone atoms). The purple CuA azurin was produced by the loop-engineering strategy, and the resulting overall structure is unperturbed. The insertion of a slightly larger Cu-binding loop into azurin causes the two structural domains of azurin to move away from each other. The high-resolution structure reveals the detailed environment of the delocalized mixed-valence [Cu(1.5).Cu(1.5)] binuclear purple CuA center, which serves as a useful reference model for other native proteins, and provides a firm basis for understanding results from spectroscopic and functional studies of this class of copper center in biology. The two independent Cu-Cu distances of 2.42 and 2.35 A (with respective concomitant adjustments of ligand-Cu distances) are consistent with that (2.39 A) obtained from X-ray absorption spectroscopy with the same molecule, and are among the shortest Cu-Cu bonds observed to date in proteins or inorganic complexes. A comparison of the purple CuA azurin structure with those of other CuA centers reveals an important relationship between the angular position of the two His imidazole rings with respect to the Cu2S2(Cys) core plane and the distance between the Cu and the axial ligand. This relationship strongly suggests that the fine structural variation of different CuA centers can be correlated with the angular positions of the two histidine rings because, from these positions, one can predict the relative axial ligand interactions, which are responsible for modulating the Cu-Cu distance and the electron transfer properties of the CuA centers.     

Phosphatidylinositol-4,5-bisphosphate mediates the interaction of syndecan-4 with protein kinase C

Recent studies have demonstrated that the cytoplasmic tail of syndecan-4, a widely expressed transmembrane proteoglycan, can activate protein kinase Calpha in vitro, in combination with phosphatidylinositol-4,5-bisphosphate (PI-4,5-P(2)). Syndecan-4 is involved in growth factor binding as well as in adhesion to extracellular matrix proteins, while PI-4,5-P(2) synthesis is modulated by growth factor and adhesion-generated signaling. The cooperative activation of PKCalpha by the proteoglycan and the phosphatidylinositol may constitute, therefore, an essential part of the cell's response to these extracellular signals. To characterize the activation mechanism of PKCalpha, we addressed here the nature of the interplay between syndecan-4, PI-4,5-P(2), and PKCalpha by measuring their mutual binding affinities and the specificity of their interactions. We found that the cytoplasmic tail of syndecan-4 is unlikely to bind directly to PKCalpha, and that this interaction critically depends on PI-4,5-P(2). The PI-4,5-P(2) specificity of the activation of PKCalpha is conferred by the cytoplasmic tail of syndecan-4, which has higher binding affinity for this phosphatidylinositol over phosphatidylinositol-3,4-bisphosphate and the -3,4,5-trisphospate. The activation is specific to PKCalpha and does not encompass the novel protein kinase C delta isoenzyme.     

Bacteria-Host Cell Interaction Mediated by Cellular Cholesterol/Glycolipid-Enriched Microdomains

Gram negative bacterial infection is a leading cause of fatality and is attributed, at least in part, to the bacteria's capacity to persist in the host in spite of appropriate antibiotic therapy. It has been suggested that bacteria evade antibiotics by hiding within host cells. We sought to investigate this important aspect of infections in mast cells, which are inflammatory cells found in close proximity to the host-environment interface and which have recently been reported to play a crucial role in the early innate immune response to bacteria. We examined mast cell interactions with FimH-expressing E. coli, one of the major opportunistic pathogens of humans. We determined that in serum free conditions, these bacteria were able to trigger mast cell uptake without loss of bacterial viability. CD48, a mannose containing GPI (glycosylphosphatidylinositol)-linked molecule was found to be the receptor of FimH-expressing E. coli in mouse mast cells. We found that the internalization via CD48 was blocked by filipin, a cholesterol binding drug known to disrupt cholesterol/glycolipid-enriched microdomains and the bacteria-encasing vacuoles were rich in cholesterol inside cells. Interestingly, we found that mast cells subsequently expelled majority of the intracellular bacteria in 24 hours. This expulsion process was blocked by lovastatin/cyclodextrin treatment, which is known to inhibit cellular trafficking of cholesterol/glycolipid-enriched microdomains. Thus, the bacterial entry into and expulsion from mast cells were critically dependent on cholesterol/glycolipid-enriched microdomains, which represents a novel mode of tussle between the pathogen and the mast cell occurring in opsonin deficient sites in the body or even at other sites in naive or immunocompromised hosts which have low systemic levels of E. coli specific antibody.     

Inhibition of Photosynthesis by Shift in the Balance of Excitation Energy Distribution Between Photosystems in Dithiothreitol Treated Soybean Leaves

Chlorophyll fluorescence kinetics was used to investigate the effect of 1,4-dithiothreitol (DTT) on the distribution of excitation energy between photosystem 1 (PS1) and photosystem 2 (PS2) in soybean leaves under high irradiance (HI). The maximum PS2 quantum yield (F_v/F_m) was hardly affected by the presence of DTT, however, photon-saturated photosynthesis was depressed distinctly. Photochemical efficiency of open PS2 reaction centres during irradiation (F_v/F_m) was enhanced by about 30–40 % by DTT treatment, whereas photochemical quenching (q_P) was depressed by about 40 % under HI. DTT treatment caused a 30 % decrease in allocation of excitation energy to PS1 under HI and a 20 % increase to PS2. An obvious shift in the balance of excitation energy distribution between photosystems was observed in DTT-treated leaves. Though high excitation pressure (1 - q_P) resulted from DTT treatment, non-photochemical quenching (q_N) was lower. DTT completely inhibited the formation of zeaxanthin and also distinctly depressed the state transition (q_T). The shift in the balance of excitation distribution between the two photosystems induced by DTT was mainly due to the enhancement of excitation energy capture by PS2 antenna and the inhibition of state transition. It might be the shift in the balance between the two photosystems that mainly induced the depression of photosynthesis. Thus, to keep high utilization efficiency of absorbed photon energy, it is necessary to maintain the balance of excitation distribution between PS2 and PS1.