A combinatorial pattern discovery approach for the prediction of membrane dipping (re-entrant) loops

MOTIVATION: Membrane dipping loops are sections of membrane proteins that reside in the membrane but do not traverse from one side to the other, rather they enter and leave the same side of the membrane. We applied a combinatorial pattern discovery approach to sets of sequences containing at least one characterised structure described as possessing a membrane dipping loop. Discovered patterns were found to be composed of residues whose biochemical role is known to be essential for function of the protein, thus validating our approach. TMLOOP (http://membraneproteins.swan.ac.uk/TMLOOP) was implemented to predict membrane dipping loops in polytopic membrane proteins. TMLOOP applies discovered patterns as weighted predictive rules in a collective motif method (a variation of the single motif method), to avoid inherent limitations of single motif methods in detecting distantly related proteins. The collective motif method applies several, partially overlapping patterns, which pertain to the same sequence region, allowing proteins containing small variations to be detected. The approach achieved 92.4% accuracy in sensitivity and 100% reliability in specificity. TMLOOP was applied to the Swiss-Prot database, identifying 1392 confirmed membrane dipping loops, 75 plausible membrane dipping loops hitherto uncharacterised by topology prediction methods or experimental approaches and 128 false positives (8.0%).     

Impact of war on growth patterns in school children in Croatia

The objective of this paper is to present the growth patterns of school children in Osijek--the city which was exposed to severe attacks during the aggression on Croatia. The mean height and weight of Osijek schoolchildren aged 7 to 18 and the menarcheal age in girls in academic year 1995/96 were compared to the analogous data collected in 1980/81. The secular changes in height were heterogeneous. In older age groups from 12 in girls and 13 in boys, the mean height in 1995/96 increased markedly, whereas from 9 to 11 or 12, changes were undulating. In the youngest groups--at the age of 7 in both genders, and at 8 in boys, negative changes were observed. Markedly smaller height in this cohort was still pronounced in 1999/2000 when these children reached the age of 11. However, one year later (2000/01), at the age of 12, boys and girls caught up with their peers in the previous generations. These children during the war were approximately at the age of 2.5 to 4, a period when growth patterns are highly sensitive to adverse environmental influences. It might be possible that the emotional stress caused by a change of environment and separation from home, contributed to the deceleration of growth rate, i.e. the smaller height in a large part of childhood.     

Effects of morphine administration and its withdrawal on rat brain aminopeptidase activities

The endogenous opioid neuropeptide system seems to be involved in the neural processes which underlie drug addiction. Several studies have reported that the administration of morphine induces changes in the levels and/or activity of endogenous opioid peptides (enkephalin, dynorphin) and their precursors in specific brain regions of the adult CNS. The aim of this work was to study the effects of chronic morphine exposure and its withdrawal on certain aminopeptidases capable of degrading opioid peptides in brain areas including the amygdala, hypothalamus, hippocampus, striatum and brain cortices. In animals treated with morphine, aminopeptidase N presented higher enzyme activity levels in the striatum, the hypothalamus and the amygdala compared to control animals, although statistically significant differences were observed only in the case of the striatum. In addition, the activity of soluble puromycin-sensitive aminopeptidase (PSA) was found to be higher in the frontal cortex of these rats. In contrast, rats experiencing withdrawal symptoms presented decreased levels of aminopeptidase activity in certain brain areas. Thus, the activity of aminopeptidase N in the hippocampus and soluble puromycin-sensitive aminopeptidase in the frontal cortex were found to be lower in rats experiencing naloxone precipitated withdrawal symptoms, compared to the corresponding controls. Finally, the activity of the three studied aminopeptidases in vitro was unaltered by incubation with morphine, suggesting that the observed effects are not due to a direct action of this opioid upon the aminopeptidases. The results of the present report indicate that aminopeptidases may play an important role in the processes of tolerance and withdrawal associated with morphine administration.     

Occasional detection of Neospora caninum DNA in frozen extended semen from naturally infected bulls

Recently, the presence of Neospora caninum DNA in semen from naturally infected bulls was reported. In the present work, the presence and quantification of N. caninum by PCR techniques in frozen extended semen straws from naturally infected bulls was investigated. A total of 20 seropositive and five seronegative bulls raised for reproductive purposes in an AI centre were used. Ten extended semen straws from each bull obtained at different time-points during the previous 2 years were selected for Neospora testing. Eight of the seropositive bulls (40%) studied showed at least one positive straw to N. caninum DNA and 14 of their 180 semen straws examined (7.8%) were found to be positive. In all positive samples, N. caninum DNA was consistently detected in the cell fraction and not in the seminal plasma. However, the parasite number in each positive straw was under the detection level of real-time PCR. In parallel, 10 semen straws from each of the five seronegative bulls were also analyzed by the nested-PCR and no N. caninum DNA products were obtained. In order to check the consistent presence of N. caninum in a positive semen batch, three additional semen straws from the same batch of each positive straw from three seropositive bulls were analyzed but N. caninum DNA was only detected in one straw from one bull. In conclusion, we report the sporadic detection of N. caninum DNA in semen straws of naturally infected bulls but the low frequency of contaminated semen straws and the low parasite load observed indicate a minor chance of bovine neosporosis transmission by AI.     

Bax-type apoptotic proteins porate pure lipid bilayers through a mechanism sensitive to intrinsic monolayer curvature

During apoptosis, Bax-type proteins permeabilize the outer mitochondrial membrane to release intermembrane apoptogenic factors into the cytosol via a poorly understood mechanism. We have proposed that Bax and DeltaN76Bcl-x(L) (the Bax-like cleavage fragment of Bcl-x(L)) function by forming pores that are at least partially composed of lipids (lipidic pore formation). Since the membrane monolayer must bend during lipidic pore formation, we here explore the effect of intrinsic membrane monolayer curvature on pore formation. Nonlamellar lipids with positive intrinsic curvature such as lysophospholipids promoted membrane permeabilization, whereas nonlamellar lipids with negative intrinsic curvature such as diacylglycerol and phosphatidylethanolamine inhibited membrane permeabilization. The differential effects of nonlamellar lipids on membrane permeabilization were not correlated with lipid-induced changes in membrane binding or insertion of Bax or DeltaN76Bcl-x(L). Altogether, these results are consistent with a model whereby Bax-type proteins change the bending propensity of the membrane to form pores comprised at least in part of lipids in a structure of net positive monolayer curvature.     

Are laminin binding sites on tumor cell surface involved in the indomethacin-induced sensitivity to natural cytotoxic cells?

Prostaglandins are secreted by a variety of tumor cell lines. The prostaglandin synthesis inhibitor indomethacin (IND) inhibits 3LL tumor growth after both intramuscular or intrasplenic transplantation (45 and 72%, respectively). Moreover, when tumor cells were cultured with IND, the sensitivity of 3LL cells to natural cytotoxic (NC) effector cells was increased (30%) and a higher cytotoxicity was reached when both target and effector cells were treated. This effect was reversed partially or totally when the assay was performed in the presence of laminin or an octapeptide from the laminin B1 chain. In addition, we correlate the increased cytotoxicity mediated by IND with an enhanced ability of 3LL tumor cells to bind labeled laminin (55%). In summary, our results show that the blockage or modulation of cell surface laminin binding components could be directly correlated with the sensitivity of tumor target cells to be eliminated by way of natural cytotoxicity.     

Kinetics of tetrahydrobiopterin synthesis by rabbit brain dihydrofolate reductase.

Product identification and kinetic data are presented for the conversion of 7,8-dihydrobiopterin into tetrahydrobiopterin by purified rabbit brain dihydrofolate reductase.     

History, challenges and perspectives of cell microencapsulation

Cell microencapsulation continues to hold significant promise for biotechnology and medicine. The controlled, and continuous, delivery of therapeutic products to the host by immunoisolated cells is a potentially cost-effective method to treat a wide range of diseases. Although there are several issues that need to be addressed, including capsule manufacture, properties and performance, in the past few years, a stepwise analysis on the essential obstacles and limitations has brought the whole technology closer to a realistic proposal for clinical application. This paper summarizes the current situation in the cell encapsulation field and discusses the main events that have occurred along the way.     

Calpain I induces cleavage and release of apoptosis-inducing factor from isolated mitochondria

The translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus has been implicated in the mechanism of glutamate excitotoxicity in cortical neurons and has been observed in vivo following acute rodent brain injuries. However, the mechanism and time course of AIF redistribution to the nucleus is highly controversial. Because elevated intracellular calcium is one of the most ubiquitous features of neuronal cell death, this study tested the hypothesis that cleavage of AIF by the calcium-activated protease calpain mediates its release from mitochondria. Both precursor and mature forms of recombinant AIF were cleaved near the amino terminus by calpain I in vitro. Mitochondrial outer membrane permeabilization by truncated Bid induced cytochrome c release from isolated liver or brain mitochondria but only induced AIF release in the presence of active calpain. Enzymatic inhibition of calpain by calpeptin precluded AIF release, demonstrating that proteolytic activity was required for release. Calpeptin and the mitochondrial permeability transition pore antagonist cyclosporin A also inhibited calcium-induced AIF release from mouse liver mitochondria, implicating the involvement of an endogenous mitochondrial calpain in release of AIF during permeability transition. Cleavage of AIF directly decreased its association with pure lipid vesicles of mitochondrial inner membrane composition. Taken together, these results define a novel mechanism of AIF release involving calpain processing and identify a potential molecular checkpoint for cytoprotective interventions.     

Small-scale spatial variation in the interactions between Prunus mahaleb and fruit-eating birds

We studied fleshy fruit consumption by birds on individualPrunus mahaleb trees in four plots in northwestern Spain.We found large variation among trees in the relative contribution of differentbird species to fruit removal. This variation, which occurred on a scale ofmeters, was correlated with the distance from the tree to nests ofPhoenicurus ochruros, a major fruit consumer, and with thekind of microhabitat surrounding each tree. In the four plots studied, treeslocated farther away from P. ochruros nests receivedrelatively fewer visits by this species, and relatively more byTurdusmerula and Sylvia atricapilla. These three mainconsumer species differed in the kinds of microhabitats (rocks, loose stones,bare or grassy soil, and woody vegetation) where they flew after visitingP. mahaleb, and thus they probably created differentpatterns of seedfall. This study adds to the evidence that environmentalfactorsextrinsic to plants, and acting at different spatial scales, greatly influencethe identity of the fruit consumers visiting individual trees. This influenceundermines the chances that fruit traits become evolutionary modified so as toattract those consumers providing the best seed dispersalservicesto the plants.