Rad53 kinase activation-independent replication checkpoint function of the N-terminal forkhead-associated (FHA1) domain

Saccharomyces cerevisiae Rad53 has crucial functions in many aspects of the cellular response to DNA damage and replication blocks. To coordinate these diverse roles, Rad53 has two forkhead-associated (FHA) phosphothreonine-binding domains in addition to a kinase domain. Here, we show that the conserved N-terminal FHA1 domain is essential for the function of Rad53 to prevent the firing of late replication origins in response to replication blocks. However, the FHA1 domain is not required for Rad53 activation during S phase, and as a consequence of defective downstream signaling, Rad53 containing an inactive FHA1 domain is hyperphosphorylated in response to replication blocks. The FHA1 mutation dramatically hypersensitizes strains with defects in the cell cycle-wide checkpoint pathways (rad9Delta and rad17Delta) to DNA damage, but it is largely epistatic with defects in the replication checkpoint (mrc1Delta). Altogether, our data indicate that the FHA1 domain links activated Rad53 to downstream effectors in the replication checkpoint. The results reveal an important mechanistic difference to the homologous Schizosaccharomyces pombe FHA domain that is required for Mrc1-dependent activation of the corresponding Cds1 kinase. Surprisingly, despite the severely impaired replication checkpoint and also G(2)/M checkpoint functions, the FHA1 mutation by itself leads to only moderate viability defects in response to DNA damage, highlighting the importance of functionally redundant pathways.     

The cytoplasmic domain of Ig alpha is necessary and sufficient to support efficient early B cell development

The B cell receptor complex (BcR) is essential for normal B lymphocyte function, and surface BcR expression is a crucial checkpoint in B cell development. However, functional requirements for chains of the BcR during development remain controversial. We have used retroviral gene transfer to introduce components of the BcR into chicken B cell precursors during embryonic development. A chimeric heterodimer, in which the cytoplasmic domains of chicken Igalpha and Igbeta are expressed by fusion with the extracellular and transmembrane domains of murine CD8alpha and CD8beta, respectively, targeted the cytoplasmic domains of the BcR to the cell surface in the absence of extracellular BcR domains. Expression of this chimeric heterodimer supported all early stages of embryo B cell development: bursal colonization, clonal expansion, and induction of repertoire diversification by gene conversion. Expression of the cytoplasmic domain of Igalpha, in the absence of the cytoplasmic domain of Igbeta, was not only necessary, but sufficient to support B cell development as efficiently as the endogenous BcR. In contrast, expression of the cytoplasmic domain of Igbeta in the absence of the cytoplasmic domain of Igalpha failed to support B cell development. The ability of the cytoplasmic domain of Igalpha to support early B cell development required a functional Igalpha immunoreceptor tyrosine-based activation motif. These results support a model in which expression of surface IgM following productive V(D)J recombination in developing B cell precursors serves to chaperone the cytoplasmic domain of Igalpha to the B cell surface, thereby initiating subsequent stages of development.     

Fitness effects of parasite-mediated spatial heterogeneity within a swarm

The fitness consequences associated with the position an individualadopts within a dynamic group are not well understood. I investigatedmate acquisition by male chironomid midges using a simple swarmingmodel and empirically collected data on midge aerobatic ability.Previous work has suggested that the aerobatic ability of amale is an important predictor of his reproductive success,although there is contrary (and counterintuitive) evidence thatinfection with ectoparasitic mites increases a male's chanceof mating, despite having negative effects on flight speed.The model used here suggests that a male's location within theswarm, brought about passively through interindividual differencesin flight speed, may explain these contradictory results. Specifically,slower flying males (including those burdened with mites) adoptedpositions nearer the center of the swarm, whereas faster malestended to occupy the periphery. This in turn affected theiraccess to females because any mechanism that brought femalesnearer the swarm's center before capture (including high femaleflight speed and selective pairing by either males or females)significantly increased the relative reproductive success ofboth larger and parasitized males, with the benefits of parasitismpeaking at around 4 mites per host. There may be selective pressureon hosts and parasites to maintain this optimal mite densitybecause both are likely to benefit from the relationship: hostsenjoy an increased reproductive success, whereas only throughhost copulation can mites transfer to a female midge and returnto water (their next life-history stage) during host oviposition.     

Production and processing of a recombinant Fasciola hepatica cathepsin B-like enzyme (FhcatB1) reveals potential processing mechanisms in the parasite

The liver fluke, Fasciola hepatica, apparently uses a number of cysteine proteases during its life cycle, most likely for feeding, immune evasion and invasion of tissues. A cathepsin B-like enzyme (herein referred to as FhcatB1) appears to be a major enzyme secreted by the invasive, newly excysted juvenile flukes of this parasite. To examine the processing mechanisms for this enzyme, a recombinant form was expressed in Pichia pastoris and purified to yield a homogenous pool of the enzyme. The purified enzyme could be autoactivated at low pH via a bi-molecular mechanism, a process that was greatly accelerated by the presence of large, negatively charged molecules such as dextran sulfate. The enzyme could also apparently be processed to the correct size by an asparaginyl endopeptidase via cleavage in an unusual insertion N-terminal to the normal cleavage site used to yield the active form of the enzyme. Thus, there appear to be a number of ways in which this enzyme can be processed to its optimally active form prior to secretion by F. hepatica.     

Understanding women’s burdens: preliminary findings on psychosocial health among datoga and iraqw women of northern tanzania

This preliminary, community-based study examines major stressors identified by Iraqw and Datoga women of Mbulu District, Tanzania, and describes steps in creating a culturally specific questionnaire to assess mental health burdens. This area of Tanzania is remote, with limited access to goods and services, and is undergoing dramatic social and economic changes. Iraqw and Datoga reside in close proximity and often intermarry but have different cultural and subsistence responses to this rapid social change. Data were collected from May to October 2002, with 49 Datoga women and 64 Iraqw women interviewed. In-home interviews were conducted to have women (1) free-list their primary concerns and (2) answer questions from a translated (in Datoga and Iraqw) and modified standardized mental health questionnaire. Both groups of women identified hunger, the lack of animals, particularly cattle, and health/illnesses as the most common major stressors. Other frequently cited stressors included crop failure, general fears of violence, paying taxes, and no money for basic needs. Additional refinements are required for the mental health questionnaire, with strengths and limitations discussed. Such data, while preliminary, augment efforts to analyze the emotional burdens associated with dramatic social change.     

Identification of a catalytic exosite for complement component c4 on the serine protease domain of c1s

The classical pathway of complement is crucial to the immune system, but it also contributes to inflammatory diseases when dysregulated. Binding of the C1 complex to ligands activates the pathway by inducing autoactivation of associated C1r, after which C1r activates C1s. C1s cleaves complement component C4 and then C2 to cause full activation of the system. The interaction between C1s and C4 involves active site and exosite-mediated events, but the molecular details are unknown. In this study, we identified four positively charged amino acids on the serine protease domain that appear to form a catalytic exosite that is required for efficient cleavage of C4. These residues are coincidentally involved in coordinating a sulfate ion in the crystal structure of the protease. Together with other evidence, this pointed to the involvement of sulfate ions in the interaction with the C4 substrate, and we showed that the protease interacts with a peptide from C4 containing three sulfotyrosine residues. We present a molecular model for the interaction between C1s and C4 that provides support for the above data and poses questions for future research into this aspect of complement activation.