A role for initiation codon context in chloroplast translation

To study the role of initiation codon context in chloroplast protein synthesis, we mutated the three nucleotides immediately upstream of the initiation codon (the -1 triplet) of two chloroplast genes in the alga Chlamydomonas reinhardtii. In prokaryotes, the -1 triplet has been proposed to base pair with either the 530 loop of 16S rRNA or the extended anticodon of fMet-tRNA. We found that in vivo, none of the chloroplast mutations affected mRNA stability. However, certain mutations did cause a temperature-sensitive decrease in translation and a more dramatic decrease at room temperature when combined with an AUU initiation codon. These mutations disrupt the proposed extended base pairing interaction with the fMet-tRNA anticodon loop, suggesting that this interaction may be important in vivo. Mutations that would still permit base pairing with the 530 loop of the 16S rRNA also had a negative effect on translation, suggesting that this interaction does not occur in vivo. Extended base pairing surrounding the initiation codon may be part of a mechanism to compensate for the lack of a classic Shine-Dalgarno rRNA interaction in the translation of some chloroplast mRNAs.     

Effect of endothelial cell polarity on beta-amyloid-induced migration of monocytes across normal and AD endothelium

During normal aging and amyloid beta-peptide (Abeta) disorders such as Alzheimer's disease (AD), one finds increased deposition of Abeta and activated monocytes/microglial cells in the brain. Our previous studies show that Abeta interaction with a monolayer of normal human brain microvascular endothelial cells results in increased adherence and transmigration of monocytes. Relatively little is known of the role of Abeta accumulated in the AD brain in mediating trafficking of peripheral blood monocytes (PBM) across the blood-brain barrier (BBB) and concomitant accumulation of monocytes/microglia in the AD brain. In this study, we showed that interaction of Abeta(1--40) with apical surface of monolayer of brain endothelial cells (BEC), derived either from normal or AD individuals, resulted in increased transendothelial migration of monocytic cells (HL-60 and THP-1) and PBM. However, transmigration of monocytes across the BEC monolayer cultivated in a Transwell chamber was increased 2.5-fold when Abeta was added to the basolateral side of AD compared with normal individual BEC. The Abeta-induced transmigration of monocytes was inhibited in both normal and AD-BEC by antibodies to the putative Abeta receptor, receptor for advanced glycation end products (RAGE), and to the endothelial cell junction molecule, platelet-endothelial cell adhesion molecule-1 (PECAM-1). We conclude that interaction of Abeta with the basolateral surface of AD-BEC induces cellular signaling, promoting transmigration of monocytes from the apical to basolateral direction. We suggest that Abeta in the AD brain parenchyma or cerebrovasculature initiates cellular signaling that induces PBM to transmigrate across the BBB and accumulate in the brain.     

Effect of magnesium ions on the tertiary structure of the hepatitis C virus IRES and its affinity for the cyclic peptide antibiotic viomycin

A key ion-dependent folding unit within the hepatitis C IRES comprises the IIIef junction and pseudoknot. This region is also important in recruitment of the 40S ribosomal subunit. Here, circular dichroism is used to study the influence of metal ions on the structure and stability of this region. Comparison of the thermal stability of an IRES fragment encompassing subdomains IIIe/f and IV (named 3EF4) with that of a larger fragment also possessing subdomain IIId (3DEF4) indicates an additional stabilizing effect of Mg(2+) ions on the latter fragment. Magnesium and potassium ions stabilize both fragments through nonspecific counterion effects. The additional effect of magnesium on 3DEF4, observed in the absence or presence of 100 mM KCl, is attributed to a nonspecific but high-affinity site for metal ions created by a region of unusual high charge density. Subdomain IIId presumably participates in tertiary packing interactions that provide such a site. Viomycin binds to the full-length IRES and RNA fragments with K(d) values of 25-55 microM. Interestingly, viomycin binding to the two fragments is affected differently by Mg(2+); noncompetitive inhibition of binding to 3DEF4 is observed, whereas binding to 3EF4 is not impaired. Formation of a Mg(2+)-stabilized tertiary fold, involving subdomain IIId, may thereby hinder viomycin binding to 3DEF4 indirectly. Mutational and deletion studies locate viomycin binding within subdomains IIIe/f rather than within the pseudoknot. In pseudoknot mutants, Mg(2+) ions have different effects on viomycin binding and thermal stability, suggesting altered tertiary interactions involving subdomain IIId.     

Changes in composition and structure of a tropical dry forest following intermittent cattle grazing

In northwestern Costa Rica, cattle are being used as a "management tool" to reduce the amount of combustible material, mainly dominated by Hyparrhenia rufa, an African grass. This project is being developed within Parque Nacional Palo Verde and Reserva Biológica Lomas Barbudal, both of which form part of the only remaining tropical dry forests in Mesoamerica. To determine the short-term effects of cattle grazing on the natural vegetation, we compared the floristic composition within Palo Verde in an area under intermittent cattle grazing with an area that has not been grazed. There were significantly fewer plant species in the area with intermittent cattle grazing compared to the area with no grazing. Floristic composition of these two habitats was different as reflected by both Fisher's alpha values and the Shannon index of diversity, both of which were significantly higher in the ungrazed site. The ungrazed area contained more plant species and was more similar to mature forest. The structure of the vegetation was significantly different between the intermittently grazed and ungrazed sites with more small stems (1-5 cm dbh) and fewer large stems (> 5 cm dbh) in the intermittently grazed habitat. These results indicate that cattle grazing has an impact on the dry forest by reducing the relative abundance and density of larger tree species and by changing the species composition and structure of the community. The current management plan implemented in Palo Verde and Lomas Barbudal is not appropriate because of the impact that cattle have on the structure of the natural vegetation and should not be considered a viable alternative in other protected areas of dry forest in the Neotropics. We suggest that alternative fire prevention measures be evaluated including hand-cutting H. rufa, the creation of more frequent and larger fire breaks, and the development of green breaks.     

Low frequency noise enhances cortisol among noise sensitive subjects during work performance

Salivary free cortisol concentration, rated stress and annoyance were determined in 32 subjects before, during and after carrying out a battery of performance tasks for 2 hours during exposure to ventilation noise, with dominant low frequencies (low frequency noise) or a flat frequency spectrum (reference noise). Both noises had a level of 40 dBA. All subjects were studied on two occasions and were exposed to both noises in strict rotation. Subjects were categorised as high- or low-sensitive to noise in general and low frequency noise in particular on the basis of questionnaires. Cortisol concentrations during the task were not significantly modulated by the noises or related to noise sensitivity alone. The normal circadian decline in cortisol concentration was however significantly attenuated in subjects high-sensitive to noise in general, when they were exposed to the low frequency noise. This noise was rated as more annoying and more disruptive to working capacity than the reference noise. The study showed physiological evidence of increased stress related to noise sensitivity and noise exposure during work. This is the first study to demonstrate an effect of moderate levels of noise on neuroendocrine activity. The impact of long-term exposure to moderate noise levels, and particularly low frequency noise, in the workplace deserves further investigation.     

The psychophysiology of nausea

Nausea is an unpleasant sensation usually referred to the stomach and sometimes followed by vomiting. Little is known about the subjective aspects of nausea because like pain and fatigue, it is a private sensation. We conceive of nausea as a complex control mechanism that signals us when not to eat. Our research in the areas of motion sickness and chemotherapy has led us to propose that we each have a dynamic threshold for nausea, which depends on the interaction of inherent factors and more changeable psychological factors, and that this threshold effects the individual's cognitive appraisal of both the nauseogenic stimulus and his/her bodily change in response to the nauseogenic stimulus. Inherent factors that are described are age, gender and race; psychological factors that are included are anxiety, expectation, anticipation and adaptation. The physiological responses that have been found to accompany nausea include an increase in sympathetic nervous system activity, a decrease in parasympathetic activity, an increase of abnormal dysrhythmic gastric activity, and an increase in plasma vasopressin. It is concluded that beneficial selective reduction of nausea will depend on a greater knowledge of the interaction of the psychological and physiological variables.     

Approaches to interval mapping of QTL in a multigeneration pedigree: the example of porcine chromosome 4

Quantitative trait loci (QTLs) have been mapped in many studies of F2 populations derived from crosses between diverse lines. One approach to confirming these effects and improving the mapping resolution is genetic chromosome dissection through a backcrossing programme. Analysis by interval mapping of the data generated is likely to provide additional power and resolution compared with treating data marker by marker. However, interval mapping approaches for such a programme are not well developed, especially where the founder lines were outbred. We explore alternative approaches to analysis using, as an example, data from chromosome 4 in an intercross between wild boar and Large White pigs where QTLs have been previously identified. A least squares interval mapping procedure was used to study growth rate and carcass traits in a subsequent second backcross generation (BC2). This procedure requires the probability of inheriting a wild boar allele for each BC2 animal for locations throughout the chromosome. Two methods for obtaining these probabilities were compared: stochastic or deterministic. The two methods gave similar probabilities for inheriting wild boar alleles and, hence, gave very similar results from the QTL analysis. The deterministic approach has the advantage of being much faster to run but requires specialized software. A QTL for fatness and for growth were confirmed and, in addition, a QTL for piglet growth from weaning at 5 weeks up to 7 weeks of age and another for carcass length were detected.     

A hierarchy of gene expression accompanying induction of the primitive streak by Vg1 in the chick embryo

In the chick embryo, two secreted factors have recently be shown to cooperate in inducing the first axial structure, the primitive streak: cWnt8C (normally expressed around the circumference of the embryo, in the marginal zone) and the TGF beta superfamily member cVg1 (expressed in the posterior part of the marginal zone) (Development 128 (2001) 2915). Misexpression of Vg1 in the anterior marginal zone induces an ectopic primitive streak and recapitulates the morphological changes associated with normal primitive streak formation. Here, we analyse the time-course of appearance and disappearance of expression of 12 genes (cVg1, Lef1, Nodal, FGF8, cWnt8C, cBra, cNot1, goosecoid, HNF3 beta, Chordin, Otx2 and Sox3, whose normal expression is also polarized at early stages of development) in response to cVg1 misexpression in the anterior marginal zone. We show that a hierarchy of gene expression accompanies induction of the ectopic axis, reminiscent of the order in which the same genes begin to be expressed in the normal embryo.     

A Rab8-specific GDP/GTP exchange factor is involved in actin remodeling and polarized membrane transport

The mechanisms mediating polarized delivery of vesicles to cell surface domains are poorly understood in animal cells. We have previously shown that expression of Rab8 promotes the formation of new cell surface domains through reorganization of actin and microtubules. To unravel the function of Rab8, we used the yeast two-hybrid system to search for potential Rab8-specific activators. We identified a coil-coiled protein (Rabin8), homologous to the rat Rabin3 that stimulated nucleotide exchange on Rab8 but not on Rab3A and Rab5. Furthermore, we show that rat Rabin3 has exchange activity on Rab8 but not on Rab3A, supporting the view that rat Rabin3 is the rat equivalent of human Rabin8. Rabin8 localized to the cortical actin and expression of Rabin8 resulted in remodeling of actin and the formation of polarized cell surface domains. Activation of PKC by phorbol esters enhanced translocation of both Rabin8 and Rab8-specific vesicles to the outer edge of lamellipodial structures. Moreover, coexpression of Rabin8 with dominant negative Rab8 (T22N) redistributes Rabin8 from cortical actin to Rab8-specific vesicles and promotes their polarized transport to cell protrusions. The C-terminal region of Rabin8 plays an essential role in this transport. We propose that Rabin8 is a Rab8-specific activator that is connected to processes that mediate polarized membrane traffic to dynamic cell surface structures.