Cloning and characterization of a novel C-type lectin from Zhikong scallop Chlamys farreri

C-type lectin is a family of Ca2+ dependent carbohydrate-recognition proteins which play crucial roles in the innate immunity of invertebrates by mediating the recognition of host cells to pathogens and clearing microinvaders as a pattern recognition protein (PRP). The cDNA of Zhikong scallop Chlamys farreri C-type lectin (designated CFLec-1) was cloned by expressed sequence tag (EST) and RACE techniques. The full-length cDNA of CFLec-1 was 1785 bp, consisting of a 5'-terminal untranslated region (UTR) of 66 bp and an unusually long 3' UTR of 1040 bp with seven polyadenylation signal sequences AATAAA and a poly(A) tail. The CFLec-1 cDNA encoded a polypeptide of 221 amino acids with a putative signal peptide of 15 amino acid residues and a mature protein of 206 amino acids. Analysis of the protein domain features indicated a typical long-form carbohydrate-recognition domain (CRD) of 130 residues in the CFLec-1 deduced amino acid sequence. The expression pattern of CFLec-1 transcripts in healthy and bacterial challenged scallops was studied by semi-quantitative RT-PCR. mRNA transcripts of CFLec-1 could be mainly detected in the tissues of haemocytes, gill, gonad and mantle of unchallenged scallops, whereas the expression of CFLec-1 transcripts was increased in all the tested tissues after heat-killed Vibrio anguillarum challenge. The temporal expression of CFLec-1 mRNA in haemolymph challenged by Micrococcus luteus and V anguillarum was both up-regulated and reached the maximum level at 8 and 16 It post stimulation, respectively, and then dropped back to the original level. In order to investigate its immune functions, CFLec- I was recombined and expressed in Escherichia coli BL21(DE3)-pLysS as a fusion protein with thioredoxin. The recombinant CFLec-1 agglutinated bacteria E. coli JM109 in vitro, and the agglutination was Ca2+ dependent which could be inhibited by EDTA. But it did not agglutinate M. luteus, Candida lipolytica and animal erythrocytes including rabbit, rat, mouse, chicken, human group A, human group B, human group O. Meanwhile, the recombinant CFLec-1 could inhibit the growth of both E. coli JM 109 and M. luteus, but no inhibition activity against V anguillarum. These result indicated that CFLec-1 was a constitutive and inducible PRP which was involved in the reorganization and clearance of invaders in scallop. (c) 2006 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of a thioester-containing protein from Zhikong scallop Chlamys farreri

Thioester-containing proteins are a family of proteins characterized by the unique intrachain beta-cysteinyl-gamma-glutamyl thioester, which play important roles in innate immune responses. The cDNA of Zhikong scallop Chlamys farreri thioester-containing protein (designated as CfTEP) was cloned by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfTEP was of 4616 bp, consisting of a 5 '-terminal untranslated region (UTR) of 30 bp and a 3 ' UTR of 140 bp with a polyadenylation signal sequence AATAAA and a poly(A) tail. The CfTEP cDNA encoded a polypeptide of 1481 amino acids with the theoretical isoelectric point of 5.98 and the predicted molecular weight of 161.4 kDa. The deduced amino acid sequence of CfTEP contained the canonical thioester motif GCGEQ, nine potential N-glycosylation sites and a C-terminal distinctive cysteine signature. It also contained a presumed catalytic histidine and proteolytic cleavage sites that were similar to C3 molecules. The high similarity of CfTEP with the thioester-containing proteins in other organisms, such as the TEPs from insects, the complement component C3, C4, C5 and the protease inhibitor alpha(2)-macroglobulin indicated that CfTEP should be a member of TEP family. The phylogenetic analysis revealed that CfTEP was closely related to TEPs from mollusc, nematodes and insects, and they formed a separate branch apart from the branches of complements factors and alpha(2)-macroglobulins. The spatial expression of CfTEP transcripts in healthy and bacterial challenged scallops was examined by semi-quantitative RT-PCR. The CfTEP transcripts were mainly detected in the tissues of hepatopancreas and gonad, and remarkably up-regulated by Microbial challenge, which suggested that CfTEP was a constitutive and inducible acute-phase protein involved in immune defense. These results provided new insights into the role of CfTEP in scallop immune responses, as well as the evolutionary origin of this important, widespread and functionally diversified family of proteins. (c) 2007 Published by Elsevier Ltd.

Identification and molecular analysis of a stress-inducible Hsp70 from Sciaenops ocellatus

Hsp70 proteins are a family of molecular chaperones that are involved in many aspects of protein homeostasis. In this study, an Hsp70 homologue (SoHsp70) was identified from red drum Sciaenops ocellatus and analyzed at molecular level. The open reading frame of SoHsp70 is 1920 bp and intronless, with a 5'-untranslated region (UTR) of 399 bp and a 3'-UTR of 241 bp. The deduced amino acid sequence of SoHsp70 shares 84-92% overall identities with the Hsp70s of a number of fish species. In silico analysis identified in SoHsp70 three conserved Hsp70 domains involved in nucleotide and substrate binding. The coding sequence of SoHsp70 was subcloned into Escherichia coli, from which recombinant SoHsp70 was purified and, upon ATPase assay, found to exhibit apparent ATPase activity. Expressional analysis showed that constitutive expression of SoHsp70 was detectable in heart, liver, spleen, kidney, brain, blood, and gill. Experimental challenges with poly(I:C) and bacterial pathogens of Gram-positive and Gram-negative nature induced SoHsp70 expression in kidney to different levels. Stress-responsive analysis of SoHsp70 expression in primary cultures of red drum hepatocytes showed that acute heat shock treatment elicited a rapid induction of SoHsp70 expression which appeared after 10 min and 30 min of treatment. Exposure of hepatocytes separately to iron, copper, mercury, and hydrogen peroxide significantly unregulated SoHsp70 expression in time-dependent manners. Vaccination of red drum with a Streptococcus iniae bacterin was also found to induce SoHsp70 expression. Furthermore, recombinant SoHsp70 enhanced the immunoprotective effect of a subunit vaccine. Taken together, these results suggest that SoHsp70 is a stress-inducible protein that is likely to play a role in immunity and in coping with environmental and biological stresses. (C) 2010 Elsevier Ltd. All rights reserved.

The cDNA cloning and mRNA expression of a potential selenium-binding protein gene in the scallop Chlamys farreri

Selenium binding proteins (SeBP) represent a family of proteins that are believed to be involved in controlling the oxidation/reduction in many physiological processes. The cDNA of Zhikong Scallop Chlamys farreri selenium binding protein (zSeBP) was cloned by expressed sequence tag (EST) and RACE techniques. The high similarity of zSeBP deduced amino acid sequence with the SeBP in other organisms, such as bird, fish, frog, mosquito, fruit fly, mammalian, and even nematode and microorganism indicated that zSeBP should be a member of SeBP family. The temporal expression of zSeBP in the hemocytes was measured by semi-quantitative RT-PCR after scallops were stimulated by either oxidative stress or microbial challenge. The expression of zSeBP was up-regulated progressively after stimulation, and then dropped gradually to the original level. Meanwhile, malondialdehyde (MDA) measured by the colorimetric method in the microbial challenged scallops increased immediately after scallops was challenged by microbes, and was significantly higher than that in the control scallops. Results indicated that the microbial infection could incense the disorder of oxidation/reduction and may result in high MDA production. The negative correlation between the expression level of zSeBP and the MDA content suggested that zSeBP could play an important role in mediating the anti-oxidation mechanisms and immune response in marine invertebrates. (c) 2005 Published by Elsevier Ltd.

Inheritance of 15 microsatellites in the Pacific oyster Crassostrea gigas: segregation and null allele identification for linkage analysis

Microsatellites were screened in a backcross family of the Pacific oyster, Crassostrea gigas. Fifteen microsatellite loci were distinguishable and polymorphic with 6 types of allele-combinations. Null alleles were detected in 46.7% of loci, accounting for 11.7% of the total alleles. Four loci did not segregate in Mendelian Ratios. Three linkage groups were identified among 7 of the 15 segregating loci. Fluorescence-based automated capillary electrophoresis (ABI 310 Genetic Analyzer) that used to detect the microsatellite loci, has been proved a fast, precise, and reliable method in microsatellite genotyping.

Volumetric consequences of particle loss by grading entropy.

Chemical and biological processes, such as dissolution in gypsiferous sands and biodegradation in waste refuse, result in mass or particle loss, which in turn lead to changes in solid and void phase volumes and grading. Data on phase volume and grading changes have been obtained from oedometric dissolution tests on sand–salt mixtures. Phase volume changes are defined by a (dissolution-induced) void volume change parameter (Λ). Grading changes are interpreted using grading entropy coordinates, which allow a grading curve to be depicted as a single data point and changes in grading as a vector quantity rather than a family of distribution curves. By combining Λ contours with pre- to post-dissolution grading entropy coordinate paths, an innovative interpretation of the volumetric consequences of particle loss is obtained. Paths associated with small soluble particles, the loss of which triggers relatively little settlement but large increase in void ratio, track parallel to the Λ contours. Paths associated with the loss of larger particles, which can destabilise the sand skeleton, tend to track across the Λ contours.

Precise solutions of the one-dimensional Monge-Kantorovich problem

Plakhov, A.Y., (2004) 'Precise solutions of the one-dimensional Monge-Kantorovich problem', Sbornik: Mathematics 195(9) pp.1291-1307 RAE2008

RinA controls phage-mediated packaging and transfer of virulence genes in gram-positive bacteria

Phage-mediated transfer of microbial genetic elements plays a crucial role in bacterial life style and evolution. In this study, we identify the RinA family of phage-encoded proteins as activators required for transcription of the late operon in a large group of temperate staphylococcal phages. RinA binds to a tightly regulated promoter region, situated upstream of the terS gene, that controls expression of the morphogenetic and lysis modules of the phage, activating their transcription. As expected, rinA deletion eliminated formation of functional phage particles and significantly decreased the transfer of phage and pathogenicity island encoded virulence factors. A genetic analysis of the late promoter region showed that a fragment of 272 bp contains both the promoter and the region necessary for activation by RinA. In addition, we demonstrated that RinA is the only phage-encoded protein required for the activation of this promoter region. This region was shown to be divergent among different phages. Consequently, phages with divergent promoter regions carried allelic variants of the RinA protein, which specifically recognize its own promoter sequence. Finally, most Gram-postive bacteria carry bacteriophages encoding RinA homologue proteins. Characterization of several of these proteins demonstrated that control by RinA of the phage-mediated packaging and transfer of virulence factor is a conserved mechanism regulating horizontal gene transfer.

Performance Evaluation of Two-Shadow Speculative Concurrency Control

Speculative Concurrency Control (SCC) [Best92a] is a new concurrency control approach especially suited for real-time database applications. It relies on the use of redundancy to ensure that serializable schedules are discovered and adopted as early as possible, thus increasing the likelihood of the timely commitment of transactions with strict timing constraints. In [Best92b], SCC-nS, a generic algorithm that characterizes a family of SCC-based algorithms was described, and its correctness established by showing that it only admits serializable histories. In this paper, we evaluate the performance of the Two-Shadow SCC algorithm (SCC-2S), a member of the SCC-nS family, which is notable for its minimal use of redundancy. In particular, we show that SCC-2S (as a representative of SCC-based algorithms) provides significant performance gains over the widely used Optimistic Concurrency Control with Broadcast Commit (OCC-BC), under a variety of operating conditions and workloads.

A Direct Algorithm for the Type Interference in the Rank 2 Fragment of the Second--Order λ-Calculus

We study the problem of type inference for a family of polymorphic type disciplines containing the power of Core-ML. This family comprises all levels of the stratification of the second-order lambda-calculus by "rank" of types. We show that typability is an undecidable problem at every rank k ≥ 3 of this stratification. While it was already known that typability is decidable at rank ≤ 2, no direct and easy-to-implement algorithm was available. To design such an algorithm, we develop a new notion of reduction and show how to use it to reduce the problem of typability at rank 2 to the problem of acyclic semi-unification. A by-product of our analysis is the publication of a simple solution procedure for acyclic semi-unification.

Bounds on the Power of Constant-Depth Quantum Circuits

We show that if a language is recognized within certain error bounds by constant-depth quantum circuits over a finite family of gates, then it is computable in (classical) polynomial time. In particular, our results imply EQNC^0 ⊆ P, where EQNC^0 is the constant-depth analog of the class EQP. On the other hand, we adapt and extend ideas of Terhal and DiVincenzo [?] to show that, for any family

Ergodicity and Mixing Rate of One-Dimensional Cellular Automata

One-and two-dimensional cellular automata which are known to be fault-tolerant are very complex. On the other hand, only very simple cellular automata have actually been proven to lack fault-tolerance, i.e., to be mixing. The latter either have large noise probability ε or belong to the small family of two-state nearest-neighbor monotonic rules which includes local majority voting. For a certain simple automaton L called the soldiers rule, this problem has intrigued researchers for the last two decades since L is clearly more robust than local voting: in the absence of noise, L eliminates any finite island of perturbation from an initial configuration of all 0's or all 1's. The same holds for a 4-state monotonic variant of L, K, called two-line voting. We will prove that the probabilistic cellular automata Kε and Lε asymptotically lose all information about their initial state when subject to small, strongly biased noise. The mixing property trivially implies that the systems are ergodic. The finite-time information-retaining quality of a mixing system can be represented by its relaxation time Relax(⋅), which measures the time before the onset of significant information loss. This is known to grow as (1/ε)^c for noisy local voting. The impressive error-correction ability of L has prompted some researchers to conjecture that Relax(Lε) = 2^(c/ε). We prove the tight bound 2^(c1log^21/ε) ＜ Relax(Lε) ＜ 2^(c2log^21/ε) for a biased error model. The same holds for Kε. Moreover, the lower bound is independent of the bias assumption. The strong bias assumption makes it possible to apply sparsity/renormalization techniques, the main tools of our investigation, used earlier in the opposite context of proving fault-tolerance.

The Synthesis of Arbitrary Stable Dynamics in Non-linear Neural Networks II: Feedback and Universality

We wish to construct a realization theory of stable neural networks and use this theory to model the variety of stable dynamics apparent in natural data. Such a theory should have numerous applications to constructing specific artificial neural networks with desired dynamical behavior. The networks used in this theory should have well understood dynamics yet be as diverse as possible to capture natural diversity. In this article, I describe a parameterized family of higher order, gradient-like neural networks which have known arbitrary equilibria with unstable manifolds of known specified dimension. Moreover, any system with hyperbolic dynamics is conjugate to one of these systems in a neighborhood of the equilibrium points. Prior work on how to synthesize attractors using dynamical systems theory, optimization, or direct parametric. fits to known stable systems, is either non-constructive, lacks generality, or has unspecified attracting equilibria. More specifically, We construct a parameterized family of gradient-like neural networks with a simple feedback rule which will generate equilibrium points with a set of unstable manifolds of specified dimension. Strict Lyapunov functions and nested periodic orbits are obtained for these systems and used as a method of synthesis to generate a large family of systems with the same local dynamics. This work is applied to show how one can interpolate finite sets of data, on nested periodic orbits.

Construction of Neural Network Classification Expert Systems Using Switching Theory Algorithms

A new family of neural network architectures is presented. This family of architectures solves the problem of constructing and training minimal neural network classification expert systems by using switching theory. The primary insight that leads to the use of switching theory is that the problem of minimizing the number of rules and the number of IF statements (antecedents) per rule in a neural network expert system can be recast into the problem of minimizing the number of digital gates and the number of connections between digital gates in a Very Large Scale Integrated (VLSI) circuit. The rules that the neural network generates to perform a task are readily extractable from the network's weights and topology. Analysis and simulations on the Mushroom database illustrate the system's performance.

The role of the dopaminergic neurotrophin growth/differentiation factor-5 in the developing rat ventral midbrain

Growth differentiation factor-5 (GDF-5) is a member of the transforming growth factor-β superfamily, a family of proteins that play diverse roles in many aspects of cell growth, proliferation and differentiation. GDF-5 has also been shown to be a trophic factor for embryonic midbrain dopaminergic neurons in vitro (Krieglstein et al. 1995) and after transplantation to adult rats in vivo (Sullivan et al. 1998). GDF-5 has also been shown to have neuroprotective and neurorestorative effects on adult dopaminergic neurons in the substantia nigra in animal models of Parkinson’s disease (Sullivan et al. 1997, 1999; Hurley et al. 2004). This experimental evidence has lead to GDF-5 being proposed as a neurotrophic factor with potential for use in the treatment of Parkinson’s disease. However, it is not know if GDF-5 is expressed in the brain and whether it plays a role in dopaminergic neuron development. The experiments presented here aim to address these questions. To that end this thesis is divided into five separate studies each addressing a particular question associated with GDF-5 and its expression patterns and roles during the development of the rat midbrain. Expression of the GDF-5 in the developing rat ventral mesencephalon (VM) was found to begin at E12 and peak on E14, the day that dopaminergic neurons undergo terminal differentiation. In the adult rat, GDF-5 was found to be restricted to heart and brain, being expressed in many areas of the brain, including striatum and midbrain. This indicated a role for GDF-5 in the development and maintenance of dopaminergic neurons. The appropriate receptors for GDF-5 (BMPR-II and BMPR-Ib) were found to be expressed at high levels in the rat VM at E14 and BMPR-II expression was demonstrated on dopaminergic neurons in the E13 mouse VM. GDF-5 resulted in a three-fold increase in the numbers of dopaminergic neurons in cultures of E14 rat VM, without affecting the numbers of neurones or total cells. GDF-5 was found to increase the proportion of neurons that were dopaminergic. The numbers of Nurr1-positive cells were not affected by GDF-5 treatment, but GDF-5 did increase the numbers of Nurr1- positive cells that expressed tyrosine hydroxylase (TH). Taken together this data indicated that GDF-5 increases the conversion of Nurr1-positive, TH-negative cells to Nurr1-positive, TH-positive cells. In GDF-5 treated cultures, total neurite length, neurite arborisation and somal area of dopaminergic were all significantly increased compared to control cultures. Thus this study showed that GDF-5 increased the numbers and morphological differentiation of VM dopaminergic neurones in vitro. In order to examine if GDF-5 could induce a dopaminergic phenotype in neural progenitor cells, neurosphere cultures prepared from embryonic rat VM were established. The effect of the gestational age of the donor VM on the proportion of cell types generated from neurospheres from E12, E13 and E14 VM was examined. Dopaminergic neurons could only be generated from neurospheres which were prepared from E12 VM. Thus in subsequent studies the effect of GDF-5 on dopaminergic induction was examined in progentior cell cultures prepared from the E12 rat VM. In primary cultures of E12 rat VM, GDF-5 increased the numbers of TH-positive cells without affecting the proliferation or survival of these cells. In cultures of expanded neural progenitor cells from the E12 rat VM, GDF-5 increased the expression of Nurr1 and TH, an action that was dependent on signalling through the BMPR-Ib receptor. Taken together, these experiments provide evidence that GDF-5 is expressed in the developing rat VM, is involved in both the induction of a dopaminergic phenotype in cells of the VM and in the subsequent morphological development of these dopaminergic neurons