Expression and purification of polyhistidine-tagged rotavirus NSP4 proteins in insect cells

The rotavirus nonstructural NSP4 protein, a transmembrane endoplasmic reticulum-specific glycoprotein, has been described as the first viral enterotoxin. Purified NSP4 or a peptide corresponding to NSP4 residues 114-135 induces diarrhea in young mice. NSP4 has a membrane-destabilizing activity and causes an increase in intracellular calcium levels and chloride secretion by a calcium-dependent signalling pathway in eucaryotic cells. In this study, four recombinant baculoviruses were generated expressing the rotavirus NSP4 glycoprotein from the human strains Wa and Ito, the porcine strain OSU, and the simian strain SA11, which belong to two different NSP4 genotypes, A and B. The recombinant glycoproteins, expressed as polyhistidine-tagged molecules, were analyzed by Western blotting and immunoprecipitation. Newborn mice responded with diarrhea after inoculation with each of the recombinant NSP4 proteins.     

Expression of growth hormone-releasing hormone (GHRH) and splice variants of GHRH receptors in human experimental prostate cancers

The expression of mRNA for GHRH and splice variants (SVs) of GHRH receptors in LNCaP, MDA-PCa-2b and PC-3 human prostate cancers grown in nude mice was investigated by RT-PCR. The expression of mRNA for GHRH was detected in LNCaP and PC-3, but not in MDA-PCa-2b prostatic carcinoma. RT-PCR analyses of mRNA isolated from LNCaP, MDA-PCa-2b and PC-3 cancers, revealed the presence of 720 and 566 bp products, corresponding to SV(1) and SV(2) isoforms of GHRH receptors. In PC-3 tumor membranes a radiolabeled GHRH antagonist [125I]-JV-1-42 was bound to one class of high-affinity binding sites (K(d)=1.81+/-0.47 nM) and maximum binding capacity of 332.7+/-27.8 fmol/mg membrane protein. The in vivo uptake of [125I]-JV-1-42 was observed in all xenografts of human prostate cancer, the tracer accumulation being the highest in PC-3 tumors. These results indicate that GHRH and SVs of its receptors, different from those found in the pituitary, are present in experimental human prostate cancers and may form a local mitogenic loop. The antiproliferative effects of GHRH antagonists on growth of prostate cancer could be exerted in part by an interference with this local GHRH system.     

Iron deficiency causes changes in chlorophyll fluorescence due to the reduction in the dark of the Photosystem II acceptor side

Iron deficiency was found to affect the redox state of the Photosystem II acceptor side in dark-adapted, attached leaves of sugar beet (Beta vulgaris L.). Dark-adapted iron-deficient leaves exhibited relatively high F_o and F_pl levels in the Kautsky chlorophyll fluorescence induction curve when compared to the iron-sufficient controls. However, far-red illumination led to marked decreases in the apparent F_o and F_pl levels. Modulated fluorescence showed that far-red light decreased the fluorescence yield to the true F_o levels by increasing photochemical quenching, without inducing changes in the level of non-photochemical quenching. In dark-adapted, iron-deficient leaves, far-red illumination induced a faster fluorescence decay in the µs-ms time domain, indicating an improvement in the electron transport after the primary quinone acceptor in the reducing side of Photosystem II. All these data indicate that in iron-deficient leaves the plastoquinone pool was reduced in the dark. The extent of the plastoquinone reduction in sugar beet depended on the chlorophyll concentration of the leaf, on the time of preillumination and on the duration of dark adaptation. The dark reduction of plastoquinone was observed not only in sugar beet but also in other plant species affected by iron deficiency both in controlled conditions and in the field.     

Dental variation among four prehispanic Mexican populations

In this paper, the dental morphology of prehispanic Mesoamerican populations is described, compared, and examined within the context of New World dental variation. Twenty-eight morphological dental traits were studied and compared in four samples of prehispanic Mexican populations. After eliminating intra- and interobserver error, the dental morphological characteristics observed show evidence of heterogeneity among the populations. In particular, the oldest population, Tlatilco (1300–800 BC ), was significantly different from the other three groups, Cuicuilco (800–100 BC ), Monte Albán (500 BC –700 AD ) and Cholula (550–750 AD ). When the four samples were compared to other Mongoloid populations, either univariately or multivariately, it was observed that the Mexican groups did not follow a strict Sinodont (characteristic of Northeast Asia)/Sundadont (characteristic of Southeast Asia) classification (Turner [1979] Am. J. Phys. Anthropol. 51:619–636). From the traits examined, 27% presented frequencies consistent with Sinodont variation, while 73% of the traits showed similar incidence to Southeast Asian groups. Multivariately, the Mexican populations were found to fit an overall Sundadont classification. These results indicate that there is more dental morphological variation among American Indian populations than previously shown. © 1996 Wiley-Liss, Inc.     

Universal foliage-stem scaling across environments and species in dicot trees: plasticity, biomechanics and Corner's Rules

Trees range from small-leaved, intricately branched species with slender stems to large-leaved, coarsely branched ones with thick stems. We suggest a mechanism for this pattern, known as Corner's Rules, based on universal scaling. We show similar crown area–stem diameter scaling between trunks and branches, environments, and species spanning a wide range of leaf size and stem biomechanics. If crown and stem maintain metabolically driven proportionality, but similar amounts of photosynthates are produced per unit crown area, then the greater leaf spacing in large-leaved species requires lower density stem tissue and, meeting mechanical needs, thicker stems. Congruent with this scenario, we show a negative relationship between leaf size and stem Young's modulus. Corner's Rules emerge from these mutual adjustments, which suggest that adaptive studies cannot consider any of these features independently. The constancy of scaling despite environmental challenges identifies this trait constellation as a crucial axis of plant diversification.     

Nest architecture, activity pattern, worker density and the dynamics of disease transmission in social insects

The role of disease in the organization of insect colonies has become an important focus of research in evolutionary pathobiology, in which the relationship of sociality and disease transmission can be comparatively and experimentally analysed. In this paper we use an individual-based model of disease transmission to assess how an epidemic is influenced by worker density and activity level, the probability of disease transmission, and the structural organization of the nest. First, we observed in our model a nonlinear interaction between worker density and the probability of disease transmission: high levels of both factors interact to enhance the likelihood of an epidemic. Additionally, when we incorporated in our model the empirical observation that only a fraction of the worker population in social insect colonies is active at any given point in time, results suggested that relatively low levels of worker movement can have a significant impact on the spread of disease, slowing its transmission through the colony. Finally, we found that nests having even a simple spatial separation of chambers could delay the spread of infection and diminish the severity of an outbreak. The effect of nest structure in delaying infection spread became more pronounced as nest architecture became increasingly unidimensional, as in the case of simple gallery nests. Therefore, nest architecture and worker activity patterns might indeed exert considerable influence on the dynamics of epidemics in social insects and should be incorporated into models of disease transmission.     

Ecto- and exo-protein kinases in Schistosoma mansoni: regulation of surface phosphorylation by acetylcholine and identification of the alpha subunit of CKII as a major secreted protein kinase

The Schistosoma mansoni parasite life cycle involves complex developmental processes that enable it to cause severe hepatic damage. Protein phosphorylation has previously been implicated in the transformation of cercariae to schistosomula of S. mansoni. Here, we studied the possible involvement of surface (ecto) and shed (exo) protein kinases (PKs) in this developmental process. We found that ecto-PKs are indeed located on the surface of the schistosomula and can phosphorylate up to 5 distinct proteins at this location. Surface phosphorylation was sensitive to acetylcholine, which increased phosphorylation of 3 proteins and reduced phosphorylation of the other 2. The ecto-PKs can be shed from the surface into the incubation medium during parasite differentiation. The main exo-PK is CKII, as concluded from the substrate specificity of the PK, its inhibition by heparin, activation by spermin, and recognition by antibody directed to the anti--alpha-subunit of CKII in the incubation medium of the schistosomula. In spite of its similarity to the ecto-PKs, the activity of the exo-PK is not affected by addition of acetylcholine. These results indicate that ecto- and exo-PKs could be involved in the parasite's development or host-parasite interactions.     

Effect of food restriction on the insulin signalling pathway in rat skeletal muscle and adipose tissue

We tried to elucidate the effects of a brief and severe model of food restriction on insulin sensitivity in female rats, focusing on key proteins involved in the insulin signalling pathway in skeletal muscle and adipose tissue after 5, 10 and 15 days of food restriction. Using euglycemic clamp, we detected that food-restricted rats are significantly less sensitive to insulin action than control rats. However, the time of restriction promotes a progressive increase on insulin sensitivity. The analysis of the insulin signalling pathway showed a tissue-specific regulation of several proteins involved in insulin signalling. In skeletal muscle, insulin receptor substrate 1 and Glut4 are up-regulated at the end of the food restriction period, just the opposite of what we found in adipose tissue. In conclusion, a 50% reduction of food intake modulates insulin sensitivity through a tissue-specific regulation of the insulin signalling pathway in the main target tissues for this hormone.     

Selection for virulent dengue viruses occurs in humans and mosquitoes

Dengue is the most common mosquito-borne viral disease in humans. The spread of both mosquito vectors and viruses has led to the resurgence of epidemic dengue fever (a self-limited flu-like syndrome) and the emergence of dengue hemorrhagic fever (severe dengue with bleeding abnormalities) in urban centers of the tropics. There are no animal or laboratory models of dengue disease; indirect evidence suggests that dengue viruses differ in virulence, including their pathogenicities for humans and epidemic potential. We developed two assay systems (using human dendritic cells and Aedes aegypti mosquitoes) for measuring differences in virus replication that correlate with the potential to cause hemorrhagic dengue and increased virus transmission. Infection and growth experiments showed that dengue serotype 2 viruses causing dengue hemorrhagic fever epidemics (Southeast Asian genotype) can outcompete viruses that cause dengue fever only (American genotype). This fact implies that Southeast Asian genotype viruses will continue to displace other viruses, causing more hemorrhagic dengue epidemics.     

Iron deficiency interrupts energy transfer from a disconnected part of the antenna to the rest of Photosystem II

Iron deficiency changed markedly the shape of the leaf chlorophyll fluorescence induction kinetics during a dark-light transition, the so-called Kautsky effect. Changes in chlorophyll fluorescence lifetime and yield were observed, increasing largely the minimal and the intermediate chlorophyll fluorescence levels, with a marked dip between the intermediate and the maximum levels and loss of the secondary peak after the maximum. During the slow changes, the lifetime-yield relationship was found to be linear and curvilinear (towards positive lifetime values) in control and Fe-deficient leaves, respectively. These results suggested that part of the Photosystem II antenna in Fe-deficient leaves emits fluorescence with a long lifetime. In dark-adapted Fe-deficient leaves, measurements in the picosecond-nanosecond time domain confirmed the presence of a 3.3-ns component, contributing to 15% of the total fluorescence. Computer simulations revealed that upon illumination such contribution is also present and remains constant, indicating that energy transfer is partially interrupted in Fe-deficient leaves. Photosystem II-enriched membrane fractions containing different pigment-protein complexes were isolated from control and Fe-deficient leaves and characterized spectrophotometrically. The photosynthetic pigment composition of the fractions was also determined. Data revealed the presence of a novel pigment-protein complex induced by Fe deficiency and an enrichment of internal relative to peripheral antenna complexes. The data suggest a partial disconnection between internal Photosystem II antenna complexes and the reaction center, which could lead to an underestimation of the Photosystem II efficiency in dark-adapted, low chlorophyll Fe-deficient leaves, using chlorophyll fluorescence. This revised version was published online in August 2006 with corrections to the Cover Date.