An extracellular carboxylesterase from the basidiomycete Pleurotus sapidus hydrolyses xanthophyll esters

An extracellular enzyme capable of efficient hydrolysis of xanthophyll esters was purified from culture supernatants of the basidiomycete Pleurotus sapidus. Under native conditions, the enzyme exhibited a molecular mass of 430 kDa, and SDS-PAGE data suggested a composition of eight identical subunits. Biochemical characterisation of the purified protein showed an isoelectric point of 4.5, and ideal hydrolysis conditions were observed at pH 5.8 and 40 degrees C. Partial amino acid sequences were derived from N-terminal Edman degradation and from mass spectrometric ab initio sequencing of internal peptides. An 1861-bp cDNA containing an open reading frame of 1641 bp was cloned from a cDNA library that showed ca. 40% homology to Candida rugosa lipases. The P. sapidus carboxylesterase represents the first enzyme of the lipase/esterase family from a basidiomycetous fungus that has been characterised at the molecular level.     

Structural dissection of ATP turnover in the prototypical GHL ATPase TopoVI

GHL proteins are functionally diverse enzymes defined by the presence of a conserved ATPase domain that self-associates to trap substrate upon nucleotide binding. The structural states adopted by these enzymes during nucleotide hydrolysis and product release, and their consequences for enzyme catalysis, have remained unclear. Here, we have determined a complete structural map of the ATP turnover cycle for topoVI-B, the ATPase subunit of the archaeal GHL enzyme topoisomerase VI. With this ensemble of structures, we show that significant conformational changes in the subunit occur first upon ATP binding, and subsequently upon release of hydrolyzed P(i). Together, these data provide a structural framework for understanding the role of ATP hydrolysis in the type II topoisomerase reaction. Our results also suggest that the GHL ATPase module is a molecular switch in which ATP hydrolysis serves as a prerequisite but not a driving force for substrate-dependent structural transitions in the enzyme.     

Maternal control of integument cell elongation and zygotic control of endosperm growth are coordinated to determine seed size in Arabidopsis

We use Arabidopsis thaliana as a model to investigate coordination of cell proliferation and cell elongation in the three components that develop side by side in the seed. Two of these, the embryo and its nurturing annex, the endosperm, are placed under zygotic control and develop within the seed integument placed under maternal control. We show that integument cell proliferation and endosperm growth are largely independent from each other. By contrast, prevention of cell elongation in the integument by the mutation transparent testa glabra2 (ttg2) restricts endosperm and seed growth. Conversely, endosperm growth controlled by the HAIKU (IKU) genetic pathway modulates integument cell elongation. Combinations of TTG2 defective seed integument with reduction of endosperm size by iku mutations identify integument cell elongation and endosperm growth as the primary regulators of seed size. Our results strongly suggest that a cross talk between maternal and zygotic controls represents the primary regulator of the coordinated control of seed size in Arabidopsis.     

A new non-exercise-based Vo2max prediction equation for aerobically trained men

The purposes of the present study were to (a) modify previously published Vo(2)max equations using the constant error (CE = mean difference between actual and predicted Vo(2)max) values from Malek et al. (28); (b) cross-validate the modified equations to determine their accuracy for estimating Vo(2)max in aerobically trained men; (c) derive a new non- exercise-based equation for estimating Vo(2)max in aerobically trained men if the modified equations are not found to be accurate; and (d) cross-validate the new Vo(2)max equation using the predicted residual sum of squares (PRESS) statistic and an independent sample of aerobically trained men. One hundred and fifty-two aerobically trained men (Vo(2)max mean +/- SD = 4,154 +/- 629 ml.min(-1)) performed a maximal incremental test on a cycle ergometer to determine actual Vo(2)max. An aerobically trained man was defined as someone who had participated in continuous aerobic exercise 3 or more sessions per week for a minimum of 1 hour per session for at least the past 18 months. Nine previously published Vo(2)max equations were modified for use with aerobically trained men. The predicted Vo(2)max values from the 9 modified equations were compared to actual Vo(2)max by examining the CE, standard error of estimate (SEE), validity coefficient (r), and total error (TE). Cross-validation of the modified non-exercise-based equations on a random subsample of 50 subjects resulted in a %TE > or = 13% of the mean of actual Vo(2)max. Therefore, the following non-exercise-based Vo(2)max equation was derived from a random subsample of 112 subjects: Vo(2)max (ml.min(-1)) = 27.387(weight in kg) + 26.634(height in cm) - 27.572(age in years) + 26.161(h.wk(-1) of training) + 114.904(intensity of training using the Borg 6-20 scale) + 506.752(natural log of years of training) - 4,609.791 (R = 0.82, R(2) adjusted = 0.65, and SEE = 378 ml.min(-1)). Cross-validation of this equation on the remaining sample of 40 subjects resulted in a %TE of 10%. Therefore, the non-exercise-based equation derived in the present study is recommended for estimating Vo(2)max in aerobically trained men.     

Impaired niacin sensitivity in acute first-episode but not in multi-episode schizophrenia

Niacin (vitamin B3) flushing--a marker of altered prostaglandin signaling--is indirectly linked to the phospholipid-prostaglandin metabolism. Diminished skin flushing was repeatedly found in schizophrenia, but has not been systematically investigated at different stages of disorder as yet. We compared niacin sensitivity of 32 first-episode and 32 multi-episode patients (mainly on stable medication) with age and gender matched healthy controls. Methylnicotinate was applied in three concentrations onto the inner forearm skin. Flush response was assessed in 3 min intervals over 15 min using optical reflection spectroscopy. Whereas first-episode patients showed significantly diminished flush response as compared to controls, comparable differences were not found between multi-episode patients and controls. Comparison of niacin sensitivity at different stages of schizophrenia support the notion of altered prostaglandin signaling primarily at the onset of disorder. Longitudinal studies have to rule out possible long-term effects of neuroleptic medication.     

A critical role for cyclin E in cell fate determination in the central nervous system of Drosophila melanogaster

We have examined the process by which cell diversity is generated in neuroblast (NB) lineages in the central nervous system of Drosophila melanogaster. Thoracic NB6-4 (NB6-4t) generates both neurons and glial cells, whereas NB6-4a generates only glial cells in abdominal segments. This is attributed to an asymmetric first division of NB6-4t, localizing prospero (pros) and glial cell missing (gcm) only to the glial precursor cell, and a symmetric division of NB6-4a, where both daughter cells express pros and gcm. Here we show that the NB6-4t lineage represents the ground state, which does not require the input of any homeotic gene, whereas the NB6-4a lineage is specified by the homeotic genes abd-A and Abd-B. They specify the NB6-4a lineage by down-regulating levels of the G1 cyclin, DmCycE (CycE). CycE, which is asymmetrically expressed after the first division of NB6-4t, functions upstream of pros and gcm to specify the neuronal sublineage. Loss of CycE function causes homeotic transformation of NB6-4t to NB6-4a, whereas ectopic CycE induces reverse transformations. However, other components of the cell cycle seem to have a minor role in this process, suggesting a critical role for CycE in regulating cell fate in segment-specific neural lineages.     

Fetal loss in homozygous mutant Norrie disease mice: a new role of Norrin in reproduction

Mutations in the Norrie disease pseudoglioma gene (NDP) are known to cause X-linked recessive Norrie disease. In addition, NDP mutations have been found in other vasoproliferative retinopathies such as familial exudative vitreoretinopathy, retinopathy of prematurity, and Coats disease, suggesting a role for Norrin in vascular development. Here we report that female mice homozygous for the Norrie disease pseudoglioma homolog (Ndph) knockout allele exhibit almost complete infertility, while heterozygous females and hemizygous males are fertile. Histological examinations and RNA in situ hybridization analyses revealed defects in vascular development and decidualization in pregnant Ndph-/- females from embryonic day 7 (E7) onwards, resulting in embryonic loss. Using RT-PCR analyses we also demonstrate, for the first time, the expression of Ndph in mouse uteri and deciduae as well as the expression of NDP in human placenta. Taken together, these data provide strong evidence for Norrin playing an important role in female reproductive tissues.     

Saccharomyces cerevisiae Npc2p is a functionally conserved homologue of the human Niemann-Pick disease type C 2 protein, hNPC2

Niemann-Pick Disease Type C (NP-C) is a fatal neurodegenerative disease, which is biochemically distinguished by the lysosomal accumulation of exogenously derived cholesterol. Mutation of either the hNPC1 or hNPC2 gene is causative for NP-C. We report the identification of the yeast homologue of human NPC2, Saccharomyces cerevisiae Npc2p. We demonstrate that scNpc2p is evolutionarily related to the mammalian NPC2 family of proteins. We also show, through colocalization, subcellular fractionation, and secretion analyses, that yeast Npc2p is treated similarly to human NPC2 when expressed in mammalian cells. Importantly, we show that yeast Npc2p can efficiently revert the unesterified cholesterol and GM1 accumulation seen in hNPC2-/- patient fibroblasts demonstrating that it is a functional homologue of human NPC2. The present study reveals that the fundamental process of NPC2-mediated lipid transport has been maintained throughout evolution.     

Respiration and motor activity of cercariae of three trematode species from the intertidal mollusc Littorina littorea L (Gastropoda) from the White sea

The age dynamics in oxygen consumption of the cercariae Himasthla elongata, Cryptocotyle lingua and Cercaria parvicaudata (Renicola sp.) was studied using modified Winkler's method. It was detected that under stable temperature and water salinity conditions the rate of oxygen uptake depends directly on cercariae size. The highest intensity of energetic metabolism was recorded in the first few hours of cercariae life when their movement activity was maximal. The following reduction of oxygen consumption passed unequally in three cercariae species studied. Large, long-lived H. elongata cercariae after relatively short period of active swimming turn to crawling on the bottom. The rate of oxygen uptake in such cercariae was two times less than in free-swimming ones and remained approximately invariable up to the cercariae death. The smaller sized, short-lived Renicola sp. cercariae swim actively in the water and during this time the level of their energetic metabolism remains more or less stable. It decreased drastically after cercariae sinking to the bottom, after that they perished very soon. Also relatively small C. lingua cercariae alternate the active and passive phases of swimming. Thanks to that they consume the energetic resources economically and prolong their longevity. During free-swimming period the rate of oxygen uptake of C. lingua cercariae remains more or less stable. As in the case of Renicola sp. cercariae, it decreased drastically after the cercariae sinking to the bottom. Apparently such cercariae lose their ability to infect the second intermediate host (fish).