Murine immune responses to a novel schistosome egg antigen, SmEP25

Pathology in schistosomiasis consists of granuloma formation around parasite eggs. There is considerable variation in the severity of disease in individuals with schistosomiasis, which may result from differential responses to egg antigens. The egg-induced immunopathology is mediated by CD4+ T helper cells sensitised to egg antigens. In this study, cellular responses to a 25-kDa fraction of egg proteins identified a novel T-cell antigen, SmEP25. The native SmEP25 elicited significant proliferative responses as well as gamma interferon (IFN-gamma), IL-2, IL-4, and IL-5 secretion in CD4+ cells from 8.5-week infected CBA and C57BL/6 mice. In C57BL/6 mice, proliferative responses to SmEP25 were relatively stronger than those directed against the major egg antigen Sm-p40, whereas in CBA mice the reverse was found. SmEP25 elicited stronger Th2 type response than Sm-p40 in both mouse strains. By comparison, recombinant SmEP25 elicited a smaller, Th1-polarised response, with significant IFN-gamma, low levels of IL-5 and essentially no IL-4. B-cell responses to SmEP25 coincided with the start of parasite egg production and SmEP25 protein was restricted to parasite eggs. The systematic identification of T-cell-sensitising egg components will lead to a better understanding of the processes involved in granuloma formation.     

HSP70 binds to the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA1a) and prevents thermal inactivation

This study examined whether HSP70 could bind to and protect against thermal inactivation of SERCA1a, the SERCA isoform expressed in adult fast-twitch skeletal muscle. Sarcoplasmic reticulum vesicles prepared from rat gastrocnemius muscle were incubated with purified HSP70 at both 37 and 41 degrees C for either 30, 60, or 120 min. Maximal SERCA1a activity (micromol/g protein/min) in the absence of HSP70 was reduced progressively with time, with greater reductions occurring at 41 degrees C compared with 37 degrees C. HSP70 protected against thermal inactivation of SERCA1a activity at 37 degrees C but not at 41 degrees C and only at 30 and 60 min but not at 120 min. HSP70 also protected against reductions in binding capacity for fluorescein isothiocyanate, a fluorescent probe that binds to Lys515 in the nucleotide binding domain of SERCA, at 30 and 60 min but not at 120 min, an effect that was independent of temperature. HEK-293 cells were co-transfected with cDNAs encoding rabbit SERCA1a and human HSP-EYFP and subjected to 40 degrees C for 1 h. Immunohistochemistry revealed nearly complete co-localization of SERCA1a with HSP70 under these conditions. Co-immunoprecipitation showed physical interaction between HSP70 and SERCA1a under all thermal conditions both in vitro and in HEK-293 cells. Modeling showed that the fluorescein isothiocyanate-binding site of intact SERCA1a in the E2 form lies in its close proximity to a potential interaction site between SERCA1a and HSP70. These results indicate that HSP70 can bind to SERCA1a and, depending on the severity of heat stress, protect SERCA1a function by stabilizing the nucleotide binding domain.     

Properties of the Dicyclohexylcarbodiimide-Binding Subunit of Cytochrome c Oxidase from Sweet Potato

Subunit IV of cytochrome c oxidase from sweet potato was boundwith dicyclohexylcarbodiimide and synthesized in isolated mitochondria.Thus, this subunit corresponds to subunit HI of the analogousmammalian and fungal enzymes. Subunit IV was a mixture of twopolypeptides (subunits IVa and IVb) which differed slightlyin structure. ¹Present address: Research Institute of Molecular Genetics,Shimane University, Matsue, 690 Japan. ²Present address: Institute of Low Temperature Science, HokkaidoUniversity, Sapporo, 060 Japan.     

Phospholipids differently modulate the activity of cytosolic protein-tyrosine kinase from porcine spleen

Effect of membrane phospholipids on the activity of cytosolic protein-tyrosine kinase from porcine spleen (CPTK-40) has been studied. Using poly(Glu Na, Tyr)4:1 as a substrate, phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine had stimulatory effects on that phosphorylation activity, however phosphatidic acid had inhibitory and phosphatidylinositol had no effects. Similar results were obtained using[Val5]angiotensin II as a substrate. On the other hand using basic protein (H2B histone and myelin basic protein) as substrates, phosphatidic acid stimulated the activity of CPTK-40, while phosphatidylinositol inhibited the activity. Phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine caused different effect on the activity of CPTK-40 depending on the substrate employed. However using acidic protein (tubulin and casein) as substrates, the activity of CPTK-40 was neither stimulated nor inhibited by any phospholipids. These results suggest that phospholipids may modulate the activity of CPTK-40.     

Evidence for No Proteolytic Processing during Transport of Isocitrate Lyase into Glyoxysomes in Castor Bean Endosperm

The amino-terminal sequence of isocitrate lyase purified fromcastor bean endosperm glyoxysomes was compared with that deducedfrom the nucleotide sequence of cDNA for the enzyme [Plant Mol.Biol. (1987) 8: 471]. The isolated active enzyme lacked sixamino acid residues in the amino terminus, although the enzymeimmunoselected from a tissue homogenate with trichloroaceticacid had the amino-terminal part. Thus the six amino acid residuesseem to be eliminated during enzyme purification and the enzymeis transported into glyoxysomes without proteolytic processing. (Received August 31, 1987; Accepted November 30, 1987)     

A Chloroplast-protein Possessing Photoreducible Disulfide Bond

The five strains of A. fumigatus examined showed marked variation in their ability to produce protease in submerged culture. Also marked differences in protease production were observed when individual strains were tested in different media. Yields of up to 3.7 EU_PH 10.0 per 1 were obtained in a liver-glucose medium with the one pathogenic strain included in the study.     

Adipose-derived stromal cells inhibit prostate cancer cell proliferation inducing apoptosis

Mesenchymal stem cells (MSCs) have generated a great deal of interest in the field of regenerative medicine. Adipose-derived stromal cells (AdSCs) are known to exhibit extensive proliferation potential and can undergo multilineage differentiation, sharing similar characteristics to bone marrow-derived MSCs. However, as the effect of AdSCs on tumor growth has not been studied sufficiently, we assessed the degree to which AdSCs affect the proliferation of prostate cancer (PCa) cell. Human AdSCs exerted an inhibitory effect on the proliferation of androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) human PCa cells, while normal human dermal fibroblasts (NHDFs) did not, and in fact promoted PCa cell proliferation to a degree. Moreover, AdSCs induced apoptosis of LNCaP cells and PC3 cells, activating the caspase3/7 signaling pathway. cDNA microarray analysis suggested that AdSC-induced apoptosis in both LNCaP and PC3 cells was related to the TGF-β signaling pathway. Consistent with our in vitro observations, local transplantation of AdSCs delayed the growth of tumors derived from both LNCaP- and PC3-xenografts in immunodeficient mice. This is the first preclinical study to have directly demonstrated that AdSC-induced PCa cell apoptosis may occur via the TGF-β signaling pathway, irrespective of androgen-responsiveness. Since autologous AdSCs can be easily isolated from adipose tissue without any ethical concerns, we suggest that therapy with these cells could be a novel approach for patients with PCa.     

Arctic mutant Aβ40 aggregates on α7 nicotinic acetylcholine receptors and inhibits their functions

Amyloid β protein (Aβ) plays a central role in the pathogenesis of Alzheimer's disease (AD). Point mutations within the Aβ sequence associated with familial AD (FAD) are clustered around the central hydrophobic core of Aβ. Several types of mutations within the Aβ sequence have been identified, and the ‘Arctic’ mutation (E22G) has a purely cognitive phenotype typical of AD. Previous studies have shown that the primary result of the ‘Arctic’ mutation is increased formation of Aβ protofibrils. However, the molecular mechanism underlying this effect remains unknown. Aβ42 binds to a neuronal nicotinic acetylcholine receptor subunit, neuronal acetylcholine receptor subunit alpha‐7 (CHRNA7), with high affinity and, thus, may be involved in the pathogenesis of AD. Therefore, to clarify the molecular mechanism of Arctic mutation‐mediated FAD, we focused on CHRNA7 as a target molecule of Arctic Aβ. We performed an in vitro binding assay using purified CHRNA7 and synthetic Arctic Aβ40, and demonstrated that Arctic Aβ40 specifically bound to CHRNA7. The aggregation of Arctic Aβ40 was enhanced with the addition of CHRNA7. Furthermore, the function of CHRNA7 was detected by measuring Ca²⁺ flux and phospho‐p44/42 MAPK (ERK1/2) activation. Our results indicated that Arctic Aβ40 aggregation was enhanced by the addition of CHRNA7, which destabilized the function of CHRNA7 via inhibition of Ca²⁺ responses and activation of ERK1/2. These findings indicate that Arctic Aβ mutation may be involved in the mechanism underlying FAD. This mechanism may involve binding and aggregation, leading to the inhibition of CHRNA7 functions.