Myosin light-chain expression during avian muscle development

Monoclonal antibodies to adult chicken myosin light chains were generated and used to quantitate the types of myosin light-chain (MLC) isoforms expressed during development of the pectoralis major (PM), anterior latissimus dorsi (ALD), and medial adductor (MA) muscles of the chicken. These are muscles which, in the adult, are composed predominantly of fast, slow, and a mixture of fiber types, respectively. Three distinct phases of MLC expression characterized the development of the PM and MA muscles. The first identifiable pase occurred during the period of 5-7 d of incubation in ovo. Extracts of muscles from the pectoral region (which included the presumptive PM muscle) contained only fast MLC isoforms. This period of exclusive fast light-chain synthesis was followed by a phase (8- 12 d of incubation in ovo) in which coexpression of both fast and slow MLC isoforms was apparent in both PM and MA muscles. During the period, the composition of both fast and slow MLC isoforms in the PM and MA muscles was identical. Beginning at day 12 in ovo, the ALD was also subjected to immunochemical analyses. The proportion of fast and slow MLCs in this muscle at day 12 was similar to that present in the other muscles studied. The third development phase of MLC expression began at approximately 12 d of incubation in ovo and encompassed the transition in MLC composition to the isoform patterns incubation in ovo and encompassed the transition in MLC composition to the isoform patterns typical of adult muscle. During this period, the relative proportion of slow MLC rose in both the MA and ALD and fell in the PM. By day 16, the third fast light chain, LC(3f), was apparent in extracts of both the PM and MA. These results show that there is a developmental progression in the expression of MLC in the two avian muscles studied from day 5 in ovo; first, only fast MLCs are accumulated, then both fast and slow MLC isoforms are expressed. Only during the latter third of development in ovo is the final MLC isoform pattern characteristic of a particular muscle type expressed.     

Immunolocalization of plasma-membrane H+-ATPase and tonoplast-type pyrophosphatase in the plasma membrane of the sieve element-companion cell complex in the stem of Ricinus communis L

Plasma-membrane-located primary pumps were investigated in the sieve element (SE)-companion cell complex in the transport phloem of 2-week-old stems of Ricinus communis L. and, for comparison, in stems of Cucurbita pepo L. and in the secondary phloem of Agrobacterium tumefaciens-induced crown galls as a typical sink tissue. The plasma-membrane (PM) H⁺-ATPase and the tonoplast-type pyrophosphatase (PPase) were immunolocalized by epifluorescence and confocal laser scanning microscopy (CLSM) upon single or double labeling with specific monoclonal and polyclonal antibodies. Quantitative fluorescence evaluation by CLSM revealed both pumps in one membrane, the sieve-element PM. Different PM H⁺-ATPase antibody clones, raised against the PM H⁺-ATPase of Zea mays coleoptiles, induced in mouse and produced in mouse hybridoma cells, discriminated between different phloem cell types. Clones 30D5C4 and 44B8A1 labeled sieve elements and clone 46E5B11D5 labeled companion cells, indicating the existence of different phloem PM H⁺-ATPase isoforms. The results are discussed in terms of energization of SE transporters for retrieval of leaking sucrose, K⁺ and amino acids, as one of the unknown roles of ATP found in SEs. The function of the PPase could be related to phloem sucrose metabolism in support of ATP-requiring processes. Received: 3 July 2000 / Accepted: 12 October 2000     

A cross-sectional study of dehydroepiandrosterone sulfate in prepubertal children with myelomeningocele

OBJECTIVE: To assess biochemical characteristics of adrenarche in patients with myelomeningocele (MMC), we examined serum levels of dehydroepiandrosterone sulfate (DHEAS) in prepubertal MMC patients. PATIENTS AND METHODS: The study included a total of 54 prepubertal patients with MMC and shunted hydrocephalus: 13 patients (2 m, 11 f; aged 4.6-10.1 years, mean 8.1 +/- 0.4) with isolated pubarche (Tanner stage PH 2-4, B1 or testes volume < or =3 ml) and 41 prepubertal MMC patients without pubarche (17 m, 24 f; aged 2.0-11.9 years; mean 6.8 +/- 2.5). DHEAS levels were measured directly by chemiluminescence immunoassay (Nichols, USA). Auxological data (supine length, body mass index (BMI), arm span) and bone age (BA) were recorded. RESULTS: (mean +/- SD): Basal DHEAS levels correlated with chronological age (CA) (r = 0.32, p < 0.05), BA (r = 0.65, p < 0.01; n = 23), BMI (r = 0.54, p < 0.01) and pubic hair stage (PH1 vs. PH2-4, r = 0.49, p < 0.01). 10/11 patients aged 2-4 years had DHEAS levels in the normal range, whereas 18/40 (45.0%) of the 5- to 9-year-old patients showed elevated levels (>+2 SDS). Ten patients with isolated pubarche (10/13; 2 m, 8 f; CA 8.3 +/- 1.5 years) and 9 patients without pubarche (9/41; 6 m, 3 f; CA 6.9 +/- 2.1 years) had elevated DHEAS levels (+6.34 and +4.05 SDS, respectively). The values correlated with BA/CA ratio (p < 0.05, n = 23). There was a trend to higher BMI SDS levels in patients with elevated DHEAS levels. CONCLUSION: Our data show an early and increased activation of adrenal androgen secretion in MMC patients.     

Is the 6 kDa tobacco etch viral protein a bona fide ERES marker?

The claim that the 6 kDa viral protein (VP) of Tobacco Etch Virus is a marker for ER exit sites (ERES) has been investigated. When transiently expressed as a CFP tagged fusion construct in tobacco mesophyll protoplasts, this integral membrane protein co-localizes with both the COPII coat protein YFP-SEC24 and the Golgi marker Man1-RFP. However, when over-expressed the VP locates to larger spherical structures which co-localize with neither ER nor Golgi markers. Nevertheless, deletion of the COPII interactive N-terminal D(X)E motif causes it to be broadly distributed throughout the ER, supporting the notion that this protein could be an ERES marker. Curiously, whereas brefeldin A (BFA) caused a typical Golgi-stack response (redistribution into the ER) of the VP in leaf epidermal cells, in protoplasts it resulted in the formation of structures identical to those formed by over-expression. However, anomalous results were obtained with protoplasts: when co-expressed with the non-cycling cis-Golgi marker Man1-RFP, a BFA-induced redistribution of the VP-CFP signal into the ER was observed, but, in the presence of the cycling Golgi marker ERD2-YFP, this did not occur. High resolution images of side-on views of Golgi stacks in epidermal cells showed that the 6 kDa VP-CFP signal overlapped considerably more with YFP-SEC24 than with Man1-RFP, indicating that the VP is proportionately more associated with ERES. However, based on a consideration of the structure of its cytoplasmic tail, the scenario that the VP collects at ERES and is transported to the cis-Golgi before being recycled back to the ER, is supported.     

An Exo2 derivative affects ER and Golgi morphology and vacuolar sorting in a tissue-specific manner in arabidopsis

We screened a panel of compounds derived from Exo2 - a drug that perturbs post-Golgi compartments and trafficking in mammalian cells - for their effect on the secretory pathway in Arabidopsis root epidermal cells. While Exo2 and most related compounds had no significant effect, one Exo2 derivative, named LG8, induced severe morphological alterations in both the Golgi (at high concentrations) and the endoplasmic reticulum (ER). LG8 causes the ER to form foci of interconnecting tubules, which at the ultrastructural level appear similar to those previously reported in Arabidopsis roots after treatment with the herbicide oryzalin. In cotyledonary leaves, LG8 causes redistribution of a trans Golgi network (TGN) marker to the vacuole. LG8 affects the anterograde secretory pathway by inducing secretion of vacuolar cargo and preventing the brassinosteroid receptor BRI1 from reaching the plasma membrane. Uptake and arrival at the TGN of the endocytic marker FM4-64 is not affected. Unlike the ADP ribosylation factor-GTP exchange factor (ARF-GEF) inhibitor brefeldin A (BFA), LG8 affects these post-Golgi events without causing the formation of BFA bodies. Up to concentrations of 50 μm, the effects of LG8 are reversible.     

From topography to hydrology—The modifiable area unit problem impacts freshwater species distribution models

Species distribution models (SDMs) are statistical tools to identify potentially suitable habitats for species. For SDMs in river ecosystems, species occurrences and predictor data are often aggregated across subcatchments that serve as modeling units. The level of aggregation (i.e., model resolution) influences the statistical relationships between species occurrences and environmental predictors—a phenomenon known as the modifiable area unit problem (MAUP), making model outputs directly contingent on the model resolution. Here, we test how model performance, predictor importance, and the spatial congruence of species predictions depend on the model resolution (i.e., average subcatchment size) of SDMs. We modeled the potential habitat suitability of 50 native fish species in the upper Danube catchment at 10 different model resolutions. Model resolutions were derived using a 90‐m digital‐elevation model by using the GRASS‐GIS module r.watershed. Here, we decreased the average subcatchment size gradually from 632 to 2 km². We then ran ensemble SDMs based on five algorithms using topographical, climatic, hydrological, and land‐use predictors for each species and resolution. Model evaluation scores were consistently high, as sensitivity and True Skill Statistic values ranged from 86.1–93.2 and 0.61–0.73, respectively. The most contributing predictor changed from topography at coarse, to hydrology at fine resolutions. Climate predictors played an intermediate role for all resolutions, while land use was of little importance. Regarding the predicted habitat suitability, we identified a spatial filtering from coarse to intermediate resolutions. The predicted habitat suitability within a coarse resolution was not ported to all smaller, nested subcatchments, but only to a fraction that held the suitable environmental conditions. Across finer resolutions, the mapped predictions were spatially congruent without such filter effect. We show that freshwater SDM predictions can have consistently high evaluation scores while mapped predictions differ significantly and are highly contingent on the underlying subcatchment size. We encourage building freshwater SDMs across multiple catchment sizes, to assess model variability and uncertainties in model outcomes emerging from the MAUP.     

<Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> chloroplasts express an orally immunogenic protein targeting the p210 epitope implicated in atherosclerosis immunotherapies

Key message

An algae-based vaccine model against atherosclerosis was developed with positive findings in terms of antigen yield and immunogenicity in mouse.

Abstract

Several immunotherapies against atherosclerosis have been evaluated at the preclinical level thus far, with some of them currently under evaluation in clinical trials. In particular, the p210 epitope from ApoB100 is known to elicit atheroprotective responses. Considering that Chlamydomonas reinhardtii is an attractive host for the production and delivery of subunit vaccines, in this study a chimeric protein consisting of the B subunit of the cholera toxin and the p210 epitope from ApoB100 (CTB:p210) has been expressed in C. reinhardtii chloroplast as an attempt to establish an oral vaccine candidate against atherosclerosis. The Chlamydomonas-made CTB:p210 protein was successfully expressed at levels of up to 60 µg per g of fresh weight biomass. The antigenic activity of the CTB and the p210 moiety was preserved in the CTB:p210 chimera. Moreover the algae-made CTB:p210 showed an immunogenic activity, when orally administered to BALB/c mice, as evidenced the presence of anti-p210 serum antibodies in mice treated with the algae-derived CTB:p210. The antibody response lasts for at least 80 days after the last boost. This experimental model is proposed as a convenient tool in the development of low cost atherosclerosis vaccines of easy compliance and friendly delivery. Further studies will determine the therapeutic potential of this algae-made vaccine in atherosclerosis animal models.