R120G alphaB-crystallin promotes the unfolding of reduced alpha-lactalbumin and is inherently unstable

alpha-Crystallin is the principal lens protein which, in addition to its structural role, also acts as a molecular chaperone, to prevent aggregation and precipitation of other lens proteins. One of its two subunits, alphaB-crystallin, is also expressed in many nonlenticular tissues, and a natural missense mutation, R120G, has been associated with cataract and desmin-related myopathy, a disorder of skeletal muscles [Vicart P, Caron A, Guicheney P, Li Z, Prevost MC, Faure A, Chateau D, Chapon F, Tome F, Dupret JM, Paulin D & Fardeau M (1998) Nat Genet20, 92-95]. In the present study, real-time 1H-NMR spectroscopy showed that the ability of R120G alphaB-crystallin to stabilize the partially folded, molten globule state of alpha-lactalbumin was significantly reduced in comparison with wild-type alphaB-crystallin. The mutant showed enhanced interaction with, and promoted unfolding of, reduced alpha-lactalbumin, but showed limited chaperone activity for other target proteins. Using NMR spectroscopy, gel electrophoresis, and MS, we observed that, unlike the wild-type protein, R120G alphaB-crystallin is intrinsically unstable in solution, with unfolding of the protein over time leading to aggregation and progressive truncation from the C-terminus. Light scattering, MS, and size-exclusion chromatography data indicated that R120G alphaB-crystallin exists as a larger oligomer than wild-type alphaB-crystallin, and its size increases with time. It is likely that removal of the positive charge from R120 of alphaB-crystallin causes partial unfolding, increased exposure of hydrophobic regions, and enhances its susceptibility to proteolysis, thus reducing its solubility and promoting its aggregation and complexation with other proteins. These characteristics may explain the involvement of R120G alphaB-crystallin with human disease states.     

Ticks associated with wild mammals in Ghana

The host ranges of a collection of 21 tick species found on wild mammals in the savanna, forests and coastal zone of Ghana suggested that most species were adapted to feeding mainly on host species within a single mammalian order, i.e. on artiodactyls (bovids/suids), carnivores, rodents or pholidotes (pangolins). Only a few species were dispersed evenly across a range of orders. Seven out of ten of the most common ticks on forest mammals were significantly associated with a particular host species or a group of closely related host species, which could be viewed as their major host or hosts, but they were also recorded much less frequently on a wide range of host species. Two other species were confined to their major hosts. Only one species appeared to be widely dispersed on forest mammals and to lack a particular major host. The majority of tick species therefore occurred on hosts with very distinctive biological, behavioural and ecological characteristics. The study provided no evidence to support the view that host specificity is an artefact of sampling. Finding that the tick species on Ghanaian wild mammals occurred on particular hosts, as well as in distinct habitats, indicated that tick-host associations are important for tick survival and confirmed the importance of climate and vegetation in tick distribution.     

Mechanistic differences in inhibition of ubiquinol cytochrome c reductase by the proximal Qo-site inhibitors famoxadone and methoxyacrylate stilbene

Famoxadone (FAM) is a newly commercialized antibiotic for use against plant pathogenic fungi. It inhibits mitochondria ubiquinol:cytochrome c oxidoreductase (EC 1.10.2.2, bc(1) complex) function by binding to the proximal niche of the quinol oxidation site on the enzyme. FAM has effects on the enzyme characteristic of both type Ia (E-beta-methoxyacrylates) and type Ic (stigmatellin) inhibitors. Steady-state and tight-binding inhibition kinetics; as well as direct binding measurements with famoxadone (FAM) and methoxyacrylate stilbene (MOAS), indicated that FAM is a non-competitive inhibitor of the enzyme while methoxyacrylate stilbene (MOAS) is better described as a mixed-competitive inhibitor with respect to substrate. Mixed-competitive and non-competitive inhibition kinetics predicts a ternary enzyme-substrate-inhibitor (ESI) intermediate in the reaction sequence. Current views of the Qo domain architecture propose substrate binding niches in both distal and proximal regions of the domain. Since both inhibitors bind within the proximal niche, the formation of an ESI complex implicates substrate binding within the distal niche near the iron-sulfur protein (ISP) and cytochrome c(1) (C1). In the presence of saturating FAM, addition of substrate led to a slow, nearly stoichiometric reduction of C1 that was enzyme dependent, and independent of O(2)(-) production. Similar experiments with saturating MOAS led to a slow, sub-stoichiometric reduction of C1 by substrate. A comparison of the stoichiometries of reduction, and the apparent second order rate constants (K(cat)/K(m)) indicated that saturating MOAS elicits two distinct enzyme-inhibitor (EI) intermediates. One form does not bind substrate, but the other does. In contrast, saturating FAM leads to a predominant EI form capable of binding substrate. We suggest that these differences can be correlated to the respective effects of each inhibitor on the position of the ISP, and the integrity of a distal substrate binding site. The results also indicate that binding of these inhibitory substrate analogues to the proximal niche of the Qo domain significantly increases the DeltaG(double dagger) for reduction of C1.     

The course of malaria in mice: major histocompatibility complex (MHC) effects, but no general MHC heterozygote advantage in single-strain infections

A general MHC-heterozygote advantage in parasite-infected organisms is often assumed, although there is little experimental evidence for this. We tested the response of MHC-congenic mice (F2 segregants) to malaria and found the course of infection to be significantly influenced by MHC haplotype, parasite strain, and host gender. However, the MHC heterozygotes did worse than expected from the average response of the homozygotes.     

Crystal structure and solution NMR dynamics of a D (type II) peroxiredoxin glutaredoxin and thioredoxin dependent: a new insight into the peroxiredoxin oligomerism

Peroxiredoxins (Prxs) constitute a family of thiol peroxidases that reduce hydrogen peroxide, peroxinitrite, and hydroperoxides using a strictly conserved cysteine. Very abundant in all organisms, Prxs are produced as diverse isoforms characterized by different catalytic mechanisms and various thiol-containing reducing agents. The oligomeric state of Prxs and the link with their functionality is a subject of intensive research. We present here a combined X-ray and nuclear magnetic resonance (NMR) study of a plant Prx that belongs to the D-Prx (type II) subfamily. The Populus trichocarpa Prx is the first Prx shown to be regenerated in vitro by both the glutaredoxin and thioredoxin systems. The crystal structure and solution NMR provide evidence that the reduced protein is a specific noncovalent homodimer both in the crystal and in solution. The dimer interface is roughly perpendicular to the plane of the central beta sheet and differs from the interface of A- and B-Prx dimers, where proteins associate in the plane parallel to the beta sheet. The homodimer interface involves residues strongly conserved in the D (type II) Prxs, suggesting that all Prxs of this family can homodimerize. The study provides a new insight into the Prx oligomerism and the basis for protein-protein and enzyme-substrate interaction studies by NMR.     

Evidence from biosynthetically incorporated strontium and FTIR difference spectroscopy that the C-terminus of the D1 polypeptide of photosystem II does not ligate calcium

Recent FTIR studies have provided evidence that the C-terminal alpha-COO(-) group of the D1 polypeptide at D1-Ala344 is a unidentate ligand of a Mn ion in photosystem II [Chu, H.-A., Hiller, W., and Debus, R. J. (2004) Biochemistry 43, 3152-3166; Kimura, Y., Mizusawa, N., Yamanari, T., Ishii, A., and Ono, T.-A. (2005) J. Biol. Chem. 280, 2078-2083]. However, the FTIR data could not exclude Ca ligation. Furthermore, the recent approximately 3.5 A X-ray crystallographic structural model positions the alpha-COO(-) group of D1-Ala344 near a Ca ion [Ferreira, K. N., Iverson, T. M., Maghlaoui, K., Barber, J., and Iwata, S. (2004) Science 303, 1831-1838]. Therefore, to conclusively establish whether the alpha-COO(-) group of D1-Ala344 ligates Mn or Ca, the symmetric carboxylate stretching mode of the alpha-COO(-) group of D1-Ala344 was identified in the S(2)-minus-S(1) FTIR difference spectrum of PSII particles having Sr substituted for Ca. Cells of the cyanobacterium Synechocystis sp. PCC 6803 were propagated in media having Sr substituted for Ca and containing either l-[1-(13)C]alanine or unlabeled ((12)C) alanine. The S(2)-minus-S(1) FTIR difference spectra of the purified PSII particles show that substituting Sr for Ca alters several carboxylate stretching modes, including some that may correspond to one or more metal ligands, but importantly does not alter the symmetric carboxylate stretching mode of the alpha-COO(-) group of D1-Ala344. In unlabeled PSII particles, this mode appears at approximately 1356 cm(-)(1) in the S(1) state and at either approximately 1337 or approximately 1320 cm(-)(1) in the S(2) state, irrespective of whether the PSII particles contain Ca or Sr. These data are inconsistent with Ca ligation and show, therefore, that the C-terminal alpha-COO(-) group of the D1 polypeptide ligates a Mn ion. These data also show that substituting Ca with the larger Sr ion perturbs other unidentified carboxylate groups, at least one of which may ligate the Mn(4) cluster.     

Role of GPR40 in fatty acid action on the beta cell line INS-1E

GPR40 is a G protein-coupled receptor expressed preferentially in beta cells, that has been implicated in mediating free fatty acid-stimulated insulin release. GPR40 RNAi impaired the ability of palmitic acid (PA) to increase both insulin secretion and intracellular calcium ([Ca2+]i). The PA-dependent [Ca2+]i increase was attenuated by inhibitors of Galphaq, PLC, and SERCA. Thus GPR40 activates the Galphaq pathway, leading to release of Ca2+ from the ER. Yet the GPR40-dependent [Ca2+]i rise was dependent on extracellular Ca2+ and elevated glucose, and was blocked by inhibition of L-type calcium channels (LTCC) or opening of the K(ATP) channel; this suggests that GPR40 promotes Ca2+ influx through up-regulation of LTCC pre-activated by glucose and membrane depolarization. Taken together, the data indicate that GPR40 mediates the increase in [Ca2+]i and insulin secretion through the Galphaq-PLC pathway, resulting in release of Ca2+ from the ER and leading to up-regulation of Ca2+ influx via LTCC.     

An updated list of the ticks of Ghana and an assessment of the distribution of the ticks of Ghanaian wild mammals in different vegetation zones

Twenty one species of ticks belonging to five genera of the family Ixodidae (Order Acari, sub-order Ixodida) - Amblyomma, Haemaphysalis, Hyalomma, Ixodes and Rhipicephalus (including the sub-genus Rhipicephalus (Boophilus)) - were collected from 1260 mammals, representing 29 species, 14 families and 6 orders, in four vegetation zones in Ghana during the period 1971-1978. Four other species were collected from humans in 1977. In all, eight species appeared to be new records for Ghana: Amblyomma tholloni Neumann; Dermacentor circumguttatus Neumann; Haemaphysalis houyi Nuttall & Warburton; Ixodes loveridgei Arthur; Ixodes oldi Nuttall; Ixodes vanidicus Schultze; Rhipicephalus complanatus Neumann; Rhipicephalus cuspidatus Neumann. The updated list of tick species in Ghana given here includes 41 species of ixodid ticks and four species of argasid ticks. Most species have been found in neighbouring regions of West Africa but 56 of the 121 different combinations of ixodid tick species and host species found in the collection described here have not apparently been reported before. The new combinations recorded here bring the total number of different combinations of ixodid tick species and mammalian host species now reported in Ghana to 151. The tick species found on wild mammals in Ghana mostly differed from those reported from domestic stock by other authors. The data showed that different tick species occurred in different vegetation zones and that most species displayed a pronounced preference for certain groups of related host species. Some tick species were found in the savanna feeding mainly on large bovids and/or suids; others were found in forests feeding mainly on small bovids, large rodents or small carnivores.     

Hyperactive spruce budworm antifreeze protein expression in transgenic Drosophila does not confer cold shock tolerance

Drosophila melanogaster, a freeze intolerant and cold shock sensitive insect, was transformed with the hyperactive insect antifreeze protein gene (AFP) from the spruce budworm, Choristoneura fumiferana. Transformation P-element constructs (pCasper) were made with CfAFP 337 isoform DNA using a strong constitutive promoter, Actin 5c. This is the first report of insect AFP used to transform another insect. Properly folded active insect AFP was only detected when signal sequences were used to target proteins to the endoplasmic reticulum for secretion into the hemolymph. The 18 residue Drosophila binding protein signal sequence (BiP) constructs resulted in transformed fly lines with significantly higher AFP expression in hemolymph than when the native C. fumiferana AFP signal sequence was used. The resultant transgene fly lines have the highest levels of thermal hysteresis, 0.8 degrees C, seen for any engineered Drosophila. Despite the high level of expression, even higher than some overwintering fish with natural levels of endogenous AFP, the transformants did not display any cold shock resistance compared to controls or low AFP expressing lines. These results indicate that insect AFP alone cannot protect Drosophila from cold shock and may not be useful for Drosophila cryopreservation.