Expression of the Hox gene complex in the indirect development of a sea urchin

Hox complex genes control spatial patterning mechanisms in the development of arthropod and vertebrate body plans. Hox genes are all expressed during embryogenesis in these groups, which are all directly developing organisms in that embryogenesis leads at once to formation of major elements of the respective adult body plans. In the maximally indirect development of a large variety of invertebrates, the process of embryogenesis leads only to a free-living, bilaterally organized feeding larva. Maximal indirect development is exemplified in sea urchins. The 5-fold radially symmetric adult body plan of the sea urchin is generated long after embryogenesis is complete, by a separate process occurring within imaginal tissues set aside in the larva. The single Hox gene complex of Strongylocentrotus purpuratus contains 10 genes, and expression of eight of these genes was measured by quantitative methods during both embryonic and larval developmental stages and also in adult tissues. Only two of these genes are used significantly during the entire process of embryogenesis per se, although all are copiously expressed during the stages when the adult body plan is forming in the imaginal rudiment. They are also all expressed in various combinations in adult tissues. Thus, development of a microscopic, free-living organism of bilaterian grade, the larva, does not appear to require expression of the Hox gene cluster as such, whereas development of the adult body plan does. These observations reflect on mechanisms by which bilaterian metazoans might have arisen in Precambrian evolution.

Differential Epitope Tagging of Actin in Transformed Drosophila Produces Distinct Effects on Myofibril Assembly and Function of the Indirect Flight Muscle

We have tested the impact of tags on the structure and function of indirect flight muscle (IFM)-specific Act88F actin by transforming mutant Drosophila melanogaster, which do not express endogenous actin in their IFMs, with tagged Act88F constructs. Epitope tagging is often the method of choice to monitor the fate of a protein when a specific antibody is not available. Studies addressing the functional significance of the closely related actin isoforms rely almost exclusively on tagged exogenous actin, because only few antibodies exist that can discriminate between isoforms. Thereby it is widely presumed that the tag does not significantly interfere with protein function. However, in most studies the tagged actin is expressed in a background of endogenous actin and, as a rule, represents only a minor fraction of the total actin. The Act88F gene encodes the only Drosophila actin isoform exclusively expressed in the highly ordered IFM. Null mutations in this gene do not affect viability, but phenotypic effects in transformants can be directly attributed to the transgene. Transgenic flies that express Act88F with either a 6x histidine tag or an 11-residue peptide derived from vesicular stomatitis virus G protein at the C terminus were flightless. Overall, the ultrastructure of the IFM resembled that of the Act88F null mutant, and only low amounts of C-terminally tagged actins were found. In contrast, expression of N-terminally tagged Act88F at amounts comparable with that of wild-type flies yielded fairly normal-looking myofibrils and partially reconstituted flight ability in the transformants. Our findings suggest that the N terminus of actin is less sensitive to modifications than the C terminus, because it can be tagged and still polymerize into functional thin filaments.

The Role of Glucosidase I (Cwh41p) in the Biosynthesis of Cell Wall β-1,6-Glucan Is Indirect

CWH41, a gene involved in the assembly of cell wall β-1,6-glucan, has recently been shown to be the structural gene for Saccharomyces cerevisiae glucosidase I that is responsible for initiating the trimming of terminal α-1,2-glucose residue in the N-glycan processing pathway. To distinguish between a direct or indirect role of Cwh41p in the biosynthesis of β-1,6-glucan, we constructed a double mutant, alg5Δ (lacking dolichol-P-glucose synthase) cwh41Δ, and found that it has the same phenotype as the alg5Δ single mutant. It contains wild-type levels of cell wall β-1,6-glucan, shows moderate underglycosylation of N-linked glycoproteins, and grows at concentrations of Calcofluor White (which interferes with cell wall assembly) that are lethal to cwh41Δ single mutant. The strong genetic interactions of CWH41 with KRE6 and KRE1, two other genes involved in the β-1,6-glucan biosynthetic pathway, disappear in the absence of dolichol-P-glucose synthase (alg5Δ). The triple mutant alg5Δcwh41Δkre6Δ is viable, whereas the double mutant cwh41Δkre6Δ in the same genetic background is not. The severe slow growth phenotype and 75% reduction in cell wall β-1,6-glucan, characteristic of the cwh41Δkre1Δ double mutant, are not observed in the triple mutant alg5Δcwh41Δkre1Δ. Kre6p, a putative Golgi glucan synthase, is unstable in cwh41Δ strains, and its overexpression renders these cells Calcofluor White resistant. These results demonstrate that the role of glucosidase I (Cwh41p) in the biosynthesis of cell wall β-1,6-glucan is indirect and that dolichol-P-glucose is not an intermediate in this pathway.

Climate and infectious disease: Use of remote sensing for detection of Vibrio cholerae by indirect measurement

It has long been known that cholera outbreaks can be initiated when Vibrio cholerae, the bacterium that causes cholera, is present in drinking water in sufficient numbers to constitute an infective dose, if ingested by humans. Outbreaks associated with drinking or bathing in unpurified river or brackish water may directly or indirectly depend on such conditions as water temperature, nutrient concentration, and plankton production that may be favorable for growth and reproduction of the bacterium. Although these environmental parameters have routinely been measured by using water samples collected aboard research ships, the available data sets are sparse and infrequent. Furthermore, shipboard data acquisition is both expensive and time-consuming. Interpolation to regional scales can also be problematic. Although the bacterium, V. cholerae, cannot be sensed directly, remotely sensed data can be used to infer its presence. In the study reported here, satellite data were used to monitor the timing and spread of cholera. Public domain remote sensing data for the Bay of Bengal were compared directly with cholera case data collected in Bangladesh from 1992–1995. The remote sensing data included sea surface temperature and sea surface height. It was discovered that sea surface temperature shows an annual cycle similar to the cholera case data. Sea surface height may be an indicator of incursion of plankton-laden water inland, e.g., tidal rivers, because it was also found to be correlated with cholera outbreaks. The extensive studies accomplished during the past 25 years, confirming the hypothesis that V. cholerae is autochthonous to the aquatic environment and is a commensal of zooplankton, i.e., copepods, when combined with the findings of the satellite data analyses, provide strong evidence that cholera epidemics are climate-linked.

Indirect recognition of allopeptides promotes the development of cardiac allograft vasculopathy

Graft loss from chronic rejection has become the major obstacle to the long-term success of whole organ transplantation. In cardiac allografts, chronic rejection is manifested as a diffuse and accelerated form of arteriosclerosis, termed cardiac allograft vasculopathy. It has been suggested that T-cell recognition of processed alloantigens (allopeptides) presented by recipient antigen-presenting cells through the indirect pathway of allorecognition plays a critical role in the development and progression of chronic rejection. However, definitive preclinical evidence to support this hypothesis is lacking. To examine the role of indirect allorecognition in a clinically relevant large animal model of cardiac allograft vasculopathy, we immunized MHC inbred miniature swine with synthetic polymorphic peptides spanning the α1 domain of an allogeneic donor-derived swine leukocyte antigen class I gene. Pigs immunized with swine leukocyte antigen class I allopeptides showed in vitro proliferative responses and in vivo delayed-type hypersensitivity responses to the allogeneic peptides. Donor MHC class I disparate hearts transplanted into peptide-immunized cyclosporine-treated pigs not only rejected faster than unimmunized cyclosporine-treated controls (mean survival time = 5.5 +/−1.7 vs. 54.7 +/−3.8 days, P < 0.001), but they also developed obstructive fibroproliferative coronary artery lesions much earlier than unimmunized controls (<9 vs. >30 days). These results definitively link indirect allorecognition and cardiac allograft vasculopathy.

The contribution of trait-mediated indirect effects to the net effects of a predator

Many prey modify traits in response to predation risk and this modification of traits can influence the prey's resource acquisition rate. A predator thus can have a “nonlethal” impact on prey that can lead to indirect effects on other community members. Such indirect interactions are termed trait-mediated indirect interactions because they arise from a predator's influence on prey traits, rather than prey density. Because such nonlethal predator effects are immediate, can influence the entire prey population, and can occur over the entire prey lifetime, we argue that nonlethal predator effects are likely to contribute strongly to the net indirect effects of predators (i.e., nonlethal effects may be comparable in magnitude to those resulting from killing prey). This prediction was supported by an experiment in which the indirect effects of a larval dragonfly (Anax sp.) predator on large bullfrog tadpoles (Rana catesbeiana), through nonlethal effects on competing small bullfrog tadpoles, were large relative to indirect effects caused by density reduction of the small tadpoles (the lethal effect). Treatments in which lethal and nonlethal effects of Anax were manipulated independently indicated that this result was robust for a large range of different combinations of lethal and nonlethal effects. Because many, if not most, prey modify traits in response to predators, our results suggest that the magnitude of interaction coefficients between two species may often be dynamically related to changes in other community members, and that many indirect effects previously attributed to the lethal effects of predators may instead be due to shifts in traits of surviving prey.

Postembryonic segregation of the germ line in sea urchins in relation to indirect development.

The four small micromeres of the sea urchin embryo contribute only to the coelomic sacs, which produce major components of the adult body plan during postembryonic development. To test the proposition that the small micromeres are the definitive primordial germ cell lineage of the sea urchin, we deleted their 4th cleavage parents, and raised the deleted embryos through larval life and metamorphosis to sexual maturity. Almost all of the experimental animals produced functional gametes, excluding the possibility that the germ cell lineage arises exclusively and obligatorily from descendants of the small micromeres; rather, the germ cell lineage arises during the postembryonic development of the rudiment. A survey of the literature indicates that there is no known case of an embryonic primordial germ cell lineage in a bilaterian species that displays maximal indirect development.

Recognition of diverse sequences by class I zinc fingers: asymmetries and indirect effects on specificity in the interaction between CF2II and A+T-rich elements.

The Drosophila CF2II protein, which contains zinc fingers of the Cys2His2 type and recognizes an A+T-rich sequence, behaves in cell culture as an activator of a reporter chloramphenicol acetyltransferase gene. This activity depends on C-terminal but not N-terminal zinc fingers, as does in vitro DNA binding. By site-specific mutagenesis and binding site selection, we define the critical amino acid-base interactions. Mutations of single amino acid residues at the leading edge of the recognition helix are rarely neutral: many result in a slight change in affinity for the ideal DNA target site; some cause major loss of affinity; and others change specificity for as many as two bases in the target site. Compared to zinc fingers that recognize G+C-rich DNA, CF2II fingers appear to bind to A+T-rich DNA in a generally similar manner, but with additional flexibility and amino acid-base interactions. The results illustrate how zinc fingers may be evolving to recognize an unusually diverse set of DNA sequences.

Indirect coupling of phosphate release to de novo tension generation during muscle contraction.

A key question in muscle contraction is how tension generation is coupled to the chemistry of the actomyosin ATPase. Biochemical and mechanochemical experiments link tension generation to a change in structure associated with phosphate release. Length-jump and temperature-jump experiments, on the other hand, implicate phase 2slow, a significantly faster, markedly strain-sensitive kinetic process in tension generation. We use a laser temperature jump to probe the kinetics and mechanism of tension generation in skinned rabbit psoas fibers--an appropriate method since both phosphate release and phase 2slow are readily perturbed by temperature. Kinetics characteristic of the structural change associated with phosphate release are observed only when phosphate is added to fibers. When present, it causes a reduction in fiber tension; otherwise, no force is generated when it is perturbed. We therefore exclude this step from tension generation. The kinetics of de novo tension generation by the temperature-jump equivalent of phase 2slow appear unaffected by phosphate binding. We therefore propose that phosphate release is indirectly coupled to de novo tension generation via a steady-state flux through an irreversible step. We conclude that tension generation occurs in the absence of chemical change as the result of an entropy-driven transition between strongly bound crossbridges in the actomyosin-ADP state. The mechanism resembles the operation of a clock, with phosphate release providing the energy to tension the spring, and the irreversible step functions as the escapement mechanism, which is followed in turn by tension generation as the movement of the hands.

O português de herança em território fronteiriço: a LH em Olivença como arma para preservação de um grupo minoritário

Esta pesquisa tem como objetivo investigar a língua portuguesa nativa e de herança sobreviventes em território fronteiriço de língua oficial espanhola. Como locus de pesquisa selecionamos Olivença, uma cidade da Espanha em que a língua portuguesa se faz em situações específicas de uso. A relevância desta pesquisa traduz-se no fato de que espaços fronteiriços mantêm sobrepostas em espaços geográficos contíguos algumas realidades que se sobrepõem: a realidade da oficialidade linguística e a realidade do sentimento de pertença sociolinguística. Nesta tese, partimos da identificação, em trabalho de campo, da presença da língua portuguesa no território atualmente espanhol (mas historicamente português) e na constatação de que há uma flutuação de identificação-identidade linguística. No espaço geográfico em que fizemos incursão científica, duas cidades sobrepõem-se historicamente e duas geografias políticas, por outro lado, avizinham-se e roçam-se continuamente. Ao tomar contato com essa cidade, hipotetizamos que o sentimento de pertença linguística estaria presente entre os falantes mais velhos, que manteriam a herança de traços lusitanos em sua comunicação, mesmo ao falar o espanhol. A justificativa é que, logo de chegada, já avistáramos recintos comerciais com nomes portugueses e, contrariamente, não ouvíamos o som lusitano nas ruas. Sabemos que o domínio espanhol numa cidade outrora portuguesa tenderia a apagar vestígios portugueses. No entanto, em grupos íntimos pressupúnhamos o português como língua corrente. Durante o trabalho de campo, identificamos fortes valores culturais sendo empunhados como armas de resistência entre descendentes de portugueses, fazendo correr numa velocidade acentuada a reorganização dos valores lusitanos em redutos da cidade espanhola. Essa força e essa velocidade pareciam ser as molas propulsoras de uma mudança linguística muito sorrateira, que impactava o sentimento de unidade de um segmento social da comunidade sociolinguística. Isso nos inspirou a dar um passo investigativo seguinte em direção aos mais jovens, que tinham o espanhol como língua materna, mas tinham o português como língua de herança. À pergunta central sobre a força do português como língua de herança buscamos respostas por meio de duas outras questões mais indiretas feitas aos sujeitos entrevistados: será que os mais jovens percebiam-se como portugueses? será que os elementos culturais lusitanos presentes nas ruas eram reconhecidos como vinculados à língua de herança? Foi assim que passamos a recolher pistas sobre os traços de resiliência do português como língua incrustada na região espanhola de Olivença.

Direct versus indirect channels: Differentiated loss aversion in a high‐involvement, non‐frequently purchased hedonic product

Purpose – This article aims to investigate whether intermediaries reduce loss aversion in the context of a high-involvement non-frequently purchased hedonic product (tourism packages). Design/methodology/approach – The study incorporates the reference-dependent model into a multinomial logit model with random parameters, which controls for heterogeneity and allows representation of different correlation patterns between non-independent alternatives. Findings – Differentiated loss aversion is found: consumers buying high-involvement non-frequently purchased hedonic products are less loss averse when using an intermediary than when dealing with each provider separately and booking their services independently. This result can be taken as identifying consumer-based added value provided by the intermediaries. Practical implications – Knowing the effect of an increase in their prices is crucial for tourism collective brands (e.g. “sun and sea”, “inland”, “green destinations”, “World Heritage destinations”). This is especially applicable nowadays on account of the fact that many destinations have lowered prices to attract tourists (although, in the future, they will have to put prices back up to their normal levels). The negative effect of raising prices can be absorbed more easily via indirect channels when compared to individual providers, as the influence of loss aversion is lower for the former than the latter. The key implication is that intermediaries can – and should – add value in competition with direct e-tailing. Originality/value – Research on loss aversion in retailing has been prolific, exclusively focused on low-involvement and frequently purchased products without distinguishing the direct or indirect character of the distribution channel. However, less is known about other types of products such as high-involvement non-frequently purchased hedonic products. This article focuses on the latter and analyzes different patterns of loss aversion in direct and indirect channels.

Causal Maps and Indirect Influences Analysis in the Diagnosis of Second-Home Tourism Impacts

This paper proposes a method for diagnosing the impacts of second-home tourism and illustrates it for a Mediterranean Spanish destination. This method proposes the application of network analysis software to the analysis of causal maps in order to create a causal network model based on stakeholder-identified impacts. The main innovation is the analysis of indirect relations in causal maps for the identification of the most influential nodes in the model. The results show that the most influential nodes are of a political nature, which contradicts previous diagnoses identifying technical planning as the ultimate cause of problems.