Cloning and expression of the major secreted cathepsin B-like protein from juvenile Fasciola hepatica and analysis of immunogenicity following liver fluke infection

The functions of the cathepsin B-like proteases in liver flukes are unknown and analysis has been hindered by a lack of protein for study, since the protein is produced in small amounts by juvenile flukes. To circumvent this, we isolated and characterized a cDNA encoding the major secreted cathepsin B from Fasciola hepatica. The predicted preproprotein is 339 amino acids in length, with the mature protease predicted to be 254 amino acids long, and shows significant similarity to parasite and mammalian cathepsin B. Only one of the two conserved histidine residues required for cathepsin B exopeptidase activity is predicted to be present. Recombinant preproprotein was produced in yeast, and it was shown that the recombinant proprotein can undergo a degree of self-processing in vitro to the mature form, which is active against gelatin and synthetic peptide substrates. The recombinant protein is antigenic in vaccinated rats, and antibodies to the protein are detected early after infection of rats and sheep with F. hepatica. The kinetics of the response to cathepsin B and cathepsin L after infection of sheep and rats confirm the temporal expression of these proteins during the life cycle of the parasite.     

Altered lymphocyte recognition repertoire during ageing. III. Changes in MHC restriction patterns in parental T lymphocytes and diminution in T suppressor function.

Irradiated C57BL/6 and (C57BL/6 X C3H)F1 mice have been reconstituted with bone marrow prepared from young (2 month) or aged (24 month) C57BL/6 donors. Indirect examination of the T cell receptor for H-2d alloantigens on H-2b splenocytes of these reconstituted mice, using the suppression of the H-2b anti-H-2d response induced by (H-2b X H-2d)F1 anti-(H-2b anti-H-2d) suppressor cells, suggests that the allo-receptor repertoire derived from bone marrow of aged mice is different from that of T cells derived from young bone-marrow precursors. These observations were supported by direct evidence, from rosette formation with murine erythrocytes, for changes in the T cell receptor of these different (radiation-chimaera) sources of H-2b-T cells. Along with these subtle changes in the allo-receptor repertoire of T cells derived from bone marrow of aged mice grown in irradiated F1 hosts, there is a decrease (compared with mice reconstituted with bone marrow from young donors) in the apparent frequency of T cells recognizing antigen in association with the new MHC-restricting elements in these parent F1 chimaeras. Analysis of those cell subsets reported to be involved in the regulation of MLC responses suggests that some of the differences observed between T cell differentiation from bone-marrow stem cells of young or aged donors may in part be explained by a diminution in the production from bone marrow of aged mice of those cells important for homeostasis within the immune system.     

Understanding the adaptation of Halobacterium species NRC-1 to its extreme environment through computational analysis of its genome sequence

The genome of the halophilic archaeon Halobacterium sp. NRC-1 and predicted proteome have been analyzed by computational methods and reveal characteristics relevant to life in an extreme environment distinguished by hypersalinity and high solar radiation: (1) The proteome is highly acidic, with a median pI of 4.9 and mostly lacking basic proteins. This characteristic correlates with high surface negative charge, determined through homology modeling, as the major adaptive mechanism of halophilic proteins to function in nearly saturating salinity. (2) Codon usage displays the expected GC bias in the wobble position and is consistent with a highly acidic proteome. (3) Distinct genomic domains of NRC-1 with bacterial character are apparent by whole proteome BLAST analysis, including two gene clusters coding for a bacterial-type aerobic respiratory chain. This result indicates that the capacity of halophiles for aerobic respiration may have been acquired through lateral gene transfer. (4) Two regions of the large chromosome were found with relatively lower GC composition and overrepresentation of IS elements, similar to the minichromosomes. These IS-element-rich regions of the genome may serve to exchange DNA between the three replicons and promote genome evolution. (5) GC-skew analysis showed evidence for the existence of two replication origins in the large chromosome. This finding and the occurrence of multiple chromosomes indicate a dynamic genome organization with eukaryotic character.     

Induction of B cell costimulatory function by recombinant murine CD40 ligand

T cell-dependent regulation of B cell growth and differentiation involves an interaction between CD40, a B cell surface molecule, and the CD40 ligand (CD40L) which is expressed on activated CD4⁺ T cells. In the current study, we show that recombinant membrane-bound murine CD40L induces B cells to express costimulatory function for the proliferation of CD4⁺ Tcells. CD40L- or lipopolysaccharide (LPS)-activated, but not control-cultured B cells were strong costimulators of anti-CD3 or alloantigen-dependent T cell responses. The molecular interactions responsible for the increased costimulatory functions were examined by analyzing the activated B cells for changes in the expression of two costimulatory molecules, B7 and heat-stable antigen (HSA), as well as by the use of antagonists of B7 and HSA (CTLA4.Fc and 20C9, respectively). The expression of both B7 and HSA was enhanced on B cells activated with LPS. As observed in previous studies, the costimulatory activity of the LPS-activated B cells was dependent on both B7 and HSA and was completely inhibited in the presence of a combination of CTLA4.Fc and 20C9. In contrast, activation of B cells with CD40L induced the expression of B7 but did not enhance the expression of HSA. In addition the costimulatory activity of the CD40L-activated B cells was partially, but not completely, inhibited by the combination of CTLA4.Fc and 20C9. These results demonstrate that CD40L regulates costimulatory function of B cells in part by inducing the expression of B7 and suggest that CD40L-activated B cells express an additional costimulatory activity that is not associated with LPS-activated B cells.     

Multivariate Genetic Analysis of Chronic Pelvic Pain and Associated Phenotypes

Chronic pelvic pain (CPP) is a common condition in women that is difficult to diagnose. Although heritability estimates have been published for some conditions potentially underlying pelvic pain, the heritability of CPP itself has never been investigated. Using data from 623 MZ and 377 DZ female twin pairs aged 29–50 from an Australian twin cohort, we found an increased CPP concordance among MZs compared to DZs, with tetrachoric correlations of 0.43 (95% CI: 0.26–0.58) and 0.11 (95% CI: –0.16–0.38), respectively. This corresponded to a heritability of 0.41 (95% CI: 0.25–0.56). Lack of correlations with environmental indicators suggested that violation of the equal environments assumption was not responsible for this effect. Multivariate Cholesky decomposition models incorporating CPP and significantly correlated phenotypes showed that the entire CPP heritability could be explained by genetic variance underlying endometriosis (38%), dysmenorrhoea (23%), fibroids (24%), and somatic distress (15%), the latter a possible indicator of increased nociception. CPP itself is unlikely to be a useful independent phenotype to conduct genetic aetiological studies; contributing conditions such as endometriosis and variation in nociception are likely to provide more useful phenotypes.     

Reversed diurnal variation in depression: associations with a differential antidepressant response, tryptophan: large neutral amino acid ratio and serotonin transporter polymorphisms

BACKGROUND: Although diurnal variation of mood is a widely recognized symptom of depression, the clinical, neurobiological and psychopharmacological significance of this symptom has not previously been reported. METHOD: A total of 195 depressed out-patients underwent a detailed clinical and neurobiological assessment, and were then randomized to treatment with either fluoxetine or nortriptyline. RESULTS: Of the 195 depressed patients, 62 had a pattern of reversed diurnal variation (i.e. worse in the evening). Those with reversed diurnal variation had a poorer response to a serotonergic anti-depressant, were less likely to have bipolar II disorder, had a higher tryptophan: large neutral amino acid ratio and had different allele frequencies of the polymorphisms in the promoter region of the serotonin transporter. CONCLUSIONS: These findings raise the possibility of serotonergic influence on diurnal variation, and that the symptom of reversed diurnal variation is of relevance to antidepressant prescribing.     

Regulation of the synthesis of Sindbis virus-specified RNA: role of the virion core protein

Cells infected with seven different RNA+ mutants of Sindbis virus were found to accumulate a virus-specified polypeptide of mol. wt. 144000 (p144) during incubation at the non-permissive temperature, while at the same time synthesis of the virus structural proteins was drastically reduced. Mapping of the tryptic peptides of p144 showed that it contained the amino acid sequences of all the virus structural proteins. At the non-permissive temperature cells infected with the same seven mutants (out of 28 examined) also showed increased synthesis of 26S RNA, the mRNA for the virus structural proteins, relative to 42S RNA, and the virus genome, compared with infections by wild-type virus. We propose that both these phenotypic effects are the results of a single mutational step and that the primary defect in the processing of the virus structural protein precursor induces the relatively increased rate of synthesis of structural protein mRNA. Temperature-shift experiments with mutant-infected cells showed that p144 itself is not the agent of this effect. The failure of exposure to zinc ions to alter the RNA ratio in wild-type virus-infected cells suggested that the virus envelope proteins are not involved either, since their synthesis is preferentially inhibited under these circumstances. It is possible that it is the failure to synthesize the proper quantity of core protein in the mutant-infected cells which causes the shift of RNA synthesis in favour of structural protein mRNA.     

Acute effect of phenobarbital on escape behavior

Rats were trained to run in a straight alley to escape shock. Subsequently, phenobarbital was administered and more trials given. Phenobarbital retards running at all doses tested in a curve that is positively accelerating as a function of dose on the first trial. The first trial is markedly slower than later trials with the greatest effect occurring at the higher dosages.     

p75(NTR) gene and suicide attempts in young adults with a history of childhood-onset mood disorder.

Recently, evidence has accumulated for the role of neurotrophic processes in mood disorders. Neurotrophins operate on receptors, one of which is the p75 neurotrophin receptor (p75(NTR)). We examined three p75(NTR) markers at the p75(NTR) gene, including a missense polymorphism that changes serine to leucine (S205L), for association with suicide attempt (SA) in 203 childhood-onset mood disorder (COMD) cases. There was no difference between COMD suicide attempters and COMD non-attempters with logistic regression models for any of the three markers. We also compared the three polymorphisms between 192 COMD cases and 192 matched healthy controls and found no significant differences between COMD and healthy controls. Our results do not support an association of the p75(NTR) S205L polymorphism with risk for COMD or SA in COMD.     

Differential ability of Th1 and Th2 T cells to express Fas ligand and to undergo activation-induced cell death

Stimulation of previously activated T cells through the antigenreceptor can result in the apoptotic death of the respondingcell, a process referred to as activation-induced cell death(AICD). This process appears to involve Fas (CD95) and tts ligand(Fas-L). The distribution of Fas and Fas-L on various T cellsubsets has not been extensively characterized. We have thereforeanalyzed cells committed to a T_h1- or T_h2-type differentiationpattern for the expression and function of Fas-L. Using botha sensitive bloassay and flow cytometry, we demonstrate thatcloned T_h1 cells express high levels of Fas-L, whereas clonedT_h cells express only low levels. The expression of Fas-L byT_h1 and T_h2 cells correlates with the relative abilities ofthese two cell types to undergo AICD. Whereas AICD is readilyobserved in cultures of cloned T_h1, but not T_h2 cells, T_h2 cellsare capable of undergoing apoptosls in the presence of T_h1 cellsexpressing Fas-L The ability of T cells to undergo AICD appearsto be unrelated to the presence of various cytokines. Thus,the Fas/Fas-L pathway appears to be critical for the inductionof AICD and this pathway is differentially regulated in cellscommitted to either T_h1 or T_h2 differentiation.