Gametic behavior in a marine green alga, Monostroma angicava: an effect of phototaxis on mating efficiency

The role of phototactic behavior of gametes was tested experimentally in the slightly anisogamous marine green alga Monostroma angicava Kjellman, and the effect of phototaxis on mating efficiency was discovered. Both male and female gametes showed positive phototaxis in response to a white light source. In contrast, they did not respond to a red light source. Their swimming velocity did not differ between these two illuminating light sources. It was, therefore, suggested that the search ability of the gamete itself might not vary between phototactic and non-phototactic conditions. The number of zygotes formed during the mating process may be expressed as the product of the number of encounters between male and female gametes and the fraction of encounters that result in sexual fusion. In this study, with high densities of male and female gametes mixed in test tubes, almost all minor (fewer in number) gametes fused sexually within 10 min. After dilution of the gamete suspensions by half, mating efficiency in test tubes illuminated by white light from above was higher than that in dark controls. This suggests that male and female gametes gathered at the water surface through their positive phototaxis, thus increasing the rate of encounters. Mating efficiency also decreased if the test tubes were illuminated from above by white light and also shaken. Since negative phototaxis is clearly shown in planozygotes, we suggest that positive phototaxis of male and female gametes in M. angicava is an adaptive trait for increasing the rate of gametic encounters rather than for the dispersal of zygotes as previously reported for zoospores of some marine algae. Received: 12 February 1999 / Revision accepted: 24 May 1999     

Plasmacytoid Dendritic Cells Sequester High Prion Titres at Early Stages of Prion Infection

In most transmissible spongiform encephalopathies prions accumulate in the lymphoreticular system (LRS) long before they are detectable in the central nervous system. While a considerable body of evidence showed that B lymphocytes and follicular dendritic cells play a major role in prion colonization of lymphoid organs, the contribution of various other cell types, including antigen-presenting cells, to the accumulation and the spread of prions in the LRS are not well understood. A comprehensive study to compare prion titers of candidate cell types has not been performed to date, mainly due to limitations in the scope of animal bioassays where prohibitively large numbers of mice would be required to obtain sufficiently accurate data. By taking advantage of quantitative in vitro prion determination and magnetic-activated cell sorting, we studied the kinetics of prion accumulation in various splenic cell types at early stages of prion infection. Robust estimates for infectious titers were obtained by statistical modelling using a generalized linear model. Whilst prions were detectable in B and T lymphocytes and in antigen-presenting cells like dendritic cells and macrophages, highest infectious titers were determined in two cell types that have previously not been associated with prion pathogenesis, plasmacytoid dendritic (pDC) and natural killer (NK) cells. At 30 days after infection, NK cells were more than twice, and pDCs about seven-fold, as infectious as lymphocytes respectively. This result was unexpected since, in accordance to previous reports prion protein, an obligate requirement for prion replication, was undetectable in pDCs. This underscores the importance of prion sequestration and dissemination by antigen-presenting cells which are among the first cells of the immune system to encounter pathogens. We furthermore report the first evidence for a release of prions from lymphocytes and DCs of scrapie-infected mice ex vivo, a process that is associated with a release of exosome-like membrane vesicles.     

Association between the ACCN1 gene and multiple sclerosis in Central East Sardinia

Multiple genome screens have been performed to identify regions in linkage or association with Multiple Sclerosis (MS, OMIM 126200), but little overlap has been found among them. This may be, in part, due to a low statistical power to detect small genetic effects and to genetic heterogeneity within and among the studied populations. Motivated by these considerations, we studied a very special population, namely that of Nuoro, Sardinia, Italy. This is an isolated, old, and genetically homogeneous population with high prevalence of MS. Our study sample includes both nuclear families and unrelated cases and controls. A multi-stage study design was adopted. In the first stage, microsatellites were typed in the 17q11.2 region, previously independently found to be in linkage with MS. One significant association was found at microsatellite D17S798. Next, a bioinformatic screening of the region surrounding this marker highlighted an interesting candidate MS susceptibility gene: the Amiloride-sensitive Cation Channel Neuronal 1 (ACCN1) gene. In the second stage of the study, we resequenced the exons and the 3' untranslated (UTR) region of ACCN1, and investigated the MS association of Single Nucleotide Polymorphisms (SNPs) identified in that region. For this purpose, we developed a method of analysis where complete, phase-solved, posterior-weighted haplotype assignments are imputed for each study individual from incomplete, multi-locus, genotyping data. The imputed assignments provide an input to a number of proposed procedures for testing association at a microsatellite level or of a sequence of SNPs. These include a Mantel-Haenszel type test based on expected frequencies of pseudocase/pseudocontrol haplotypes, as well as permutation based tests, including a combination of permutation and weighted logistic regression analysis. Application of these methods allowed us to find a significant association between MS and the SNP rs28936 located in the 3' UTR segment of ACCN1 with p = 0.0004 (p = 0.002, after adjusting for multiple testing). This result is in tune with several recent experimental findings which suggest that ACCN1 may play an important role in the pathogenesis of MS.     

Methodologies for the extraction and analysis of konjac glucomannan from corms of Amorphophallus konjac K. Koch

Here we present a comparison of commonly used methodologies for the extraction and quantification of konjac glucomannan (KGM). Compositional analysis showed that the purified konjac flour (PKF) produced using a modified extraction procedure contained 92% glucomannan, with a weight average molecular weight (M_w), polydispersity index (PDI) and degree of acetylation (DA) of 9.5 ± 0.6 × 10⁵ g mol⁻¹, 1.2 and 2.8 wt.%. These data, plus Fourier-transform infrared spectral (FTIR) and zero shear viscosity analyses of the extract (PKF) were all consistent with the literature. Comparison of three existing methodologies for the quantitative analysis of the KGM content of the PKF, namely 3,5-dinitrosalicylic acid (3,5-DNS), phenol-sulphuric acid and enzymatic colorimetric assays; indicated that the 3,5-DNS colorimetric assay was the most reproducible and accurate method, with a linear correlation coefficient of 0.997 for samples ranging from 0.5 to 12.5 mg/ml, and recoveries between 97% and 103% across three spiking levels (250, 500 and 750 μg/g) of starch. These data provide the basis of improved good laboratory practice (GLP) for the commercial extraction and analysis of this multifunctional natural polymer.     

Mir-206 Regulates Pulmonary Artery Smooth Muscle Cell Proliferation and Differentiation

Pulmonary Arterial Hypertension (PAH) is a progressive devastating disease characterized by excessive proliferation of the Pulmonary Arterial Smooth Muscle Cells (PASMCs). Studies suggest that PAH and cancers share an apoptosis-resistant state featuring excessive cell proliferation. MicroRNA-206 (miR-206) is known to regulate proliferation and is implicated in various types of cancers. However, the role of miR-206 in PAH has not been studied. In this study, it is hypothesized that miR-206 could play a role in the proliferation of PASMCs. In the present study, the expression patterns of miR-206 were investigated in normal and hypertensive mouse PASMCs. The effects of miR-206 in modulating cell proliferation, apoptosis and smooth muscle cell markers in human pulmonary artery smooth muscle cells (hPASMCs) were investigated in vitro. miR-206 expression in mouse PASMCs was correlated with an increase in right ventricular systolic pressure. Reduction of miR-206 levels in hPASMCs causes increased proliferation and reduced apoptosis and these effects were reversed by the overexpression of miR-206. miR-206 over expression also increased the levels of smooth muscle cell differentiation markers α-smooth muscle actin and calponin implicating its importance in the differentiation of SMCs. miR-206 overexpression down regulated Notch-3 expression, which is key a factor in PAH development. These results suggest that miR-206 is a potential regulator of proliferation, apoptosis and differentiation of PASMCs, and that it could be used as a novel treatment strategy in PAH.     

The Origin of the Vertebrate Eye

In his considerations of “organs of extreme perfection,” Charles Darwin described the evidence that would be necessary to support the evolutionary origin of the eye, namely, demonstration of the existence of “numerous gradations” from the most primitive eye to the most perfect one, where each such tiny change had provided a survival advantage (however slight) to the organism possessing the subtly altered form. In this paper, we discuss evidence indicating that the vertebrate eye did indeed evolve through numerous subtle changes. The great majority of the gradual transitions that did occur have not been preserved to the present time, either in the fossil record or in extant species; yet clear evidence of their occurrence remains. We discuss the remarkable “eye” of the hagfish, which has features intermediate between a simple light detector and an image-forming camera-like eye and which may represent a step in the evolution of our eye that can now be studied by modern methods. We also describe the important clues to the evolutionary origin of the vertebrate eye that can be found by studying the embryological development of our own eye, by examining the molecular genetic record preserved in our own genes and in the genes of other vertebrates, and through consideration of the imperfections (or evolutionary “scars”) in the construction of our eye. Taking these findings together, it is possible to discuss in some detail how the vertebrate eye evolved.     

Combined Psychophysiological Assessment of ADHD: A Pilot Study of Bayesian Probability Approach Illustrated by Appraisal of ADHD in Female College Students

Manifestations of ADHD are observed at both psychological and physiological levels and assessed via various psychometric, EEG, and imaging tests. However, no test is 100% accurate in its assessment of ADHD. This study introduces a stochastic assessment combining psychometric tests with previously reported (Consistency Index) and newly developed (Alpha Blockade Index) EEG-based physiological markers of ADHD. The assessment utilizes classical Bayesian inference to refine after each step the probability of ADHD of each individual. In a pilot study involving six college females with ADHD and six matched controls, the assessment achieved correct classification for all ADHD and non-ADHD participants. In comparison, the classification of ADHD versus non-ADHD participants was < 85% for any one of the tests separately. The procedure significantly improved the score separation between ADHD versus non-ADHD groups. The final average probabilities for ADHD were 76% for the ADHD group and 8% for the control group. These probabilities correlated (r = .87) with the Brown ADD scale and (r = .84) with the ADHD-Symptom Inventory used for the screening of the participants. We conclude that, although each separate test was not completely accurate, a combination of several tests classified correctly all ADHD and all non-ADHD participants. The application of the proposed assessment is not limited to the specific tests used in this study--the assessment represents a general paradigm capable of accommodating a variety of ADHD tests into a single diagnostic assessment.     

Development of isoform selective PI3-kinase inhibitors as pharmacological tools for elucidating the PI3K pathway

Using a parallel synthesis approach to target a non-conserved region of the PI3K catalytic domain a pan-PI3K inhibitor 1 was elaborated to provide alpha, delta and gamma isoform selective Class I PI3K inhibitors 21, 24, 26 and 27. The compounds had good cellular activity and were selective against protein kinases and other members of the PI3K superfamily including mTOR and DNA-PK.