Amphotericin B delays both scrapie agent replication and PrP-res accumulation early in infection.

Amphotericin B delays the onset of clinical symptoms in hamsters infected with scrapie agent strain 263K. Here we show that accumulation of a scrapie-specific isoform of the prion protein (PrP-res) and agent replication were delayed early in amphotericin B-treated animals. By 8 weeks postinfection, only untreated animals exhibited clinical symptoms of scrapie infection whereas PrP-res levels and titers were similar in treated and untreated animals. This suggests that although PrP-res accumulation and agent replication are linked, they are not the sole factors required for the onset of clinical disease.     

A device for reducing the effects of wave action in shallow water

A device, consisting of two sets of perforated baffles, was designed to mediate the effects of mechanical wave action during tidal flooding. The construction, set-up and effectiveness of this device are described.     

Enhanced immune function does not depress reproductive output

Costs of reproduction might be mediated by a physiological (resource allocation) trade-off between immune function and reproductive effort, and several recent studies have shown that an experimental increase in reproductive effort is associated with decreased immune function. Here we test the complementary prediction of this hypothesis: that increased immune function (specific antibody production) depresses reproductive output. Female European starlings (Sturnus vulgaris) were injected with a non-pathogenic antigen (sheep red blood cells) following completion of laying of their first clutch, to stimulate an in vivo humoral immune response (primary antibody production). We induced laying of a second clutch by removing the first clutch, and assessed changes in reproductive performance in individual females pre- and post-treatment. Injection of sheep red blood cells produced a significant antibody response in 96% (n=29) of treated females, with titres comparable to previous studies (range 1 to 7). However, increased antibody production did not decrease primary or secondary female reproductive effort (re-laying interval, egg size, clutch size, chick growth or fledging success), compared with control, saline-injected birds (n=22). These data do not support a simple resource allocation model for the cost of reproduction, based on a reciprocal, negative relationship between resources allocated to immune function and reproduction.     

The mandibular organ of the lobster,Homarus americanus

The lobster mandibular organ is well vascularized and its polygonal cells are arranged loosely around blood vessels and blood sinuses. Numerous mitochondria and microbodies (peroxisomes) give the acidophilic cytoplasm a finely granular appearance, but there is no evidence of secretory granules. The abundant endoplasmic reticulum is almost entirely agranular and occurs in two morphologically distinct forms: tubular and cisternal. The tubular reticulum is randomly distributed and may represent the site of synthesis and transport of the mandibular organ product. The cisternal reticulum is frequently associated with microbodies. Both forms of endoplasmic reticulum proliferate during mid to late premolt. Mandibular organ ultrastructure closely resembles that of cells known to synthesize steroids or lipids, which suggests that this organ may have a similar function. There is no functional evidence of involvement in molt control in Homarus, but ultrastructural and other evidence suggests an analogy with insect corpus allatum.     

The early adaptive evolution of calmodulin

Interaction between gene duplication and natural selection in molecular evolution was investigated utilizing a phylogenetic tree constructed by the parsimony procedure from amino acid sequences of 50 calmodulin- family protein members. The 50 sequences, belonging to seven protein lineages related by gene duplication (calmodulin itself, troponin-C, alkali and regulatory light chains of myosin, parvalbumin, intestinal calcium-binding protein, and glial S-100 phenylalanine-rich protein), came from a wide range of eukaryotic taxa and yielded a denser tree (more branch points within each lineage) than in earlier studies. Evidence obtained from the reconstructed pattern of base substitutions and deletions in these ancestral loci suggests that, during the early history of the family, selection acted as a transforming force on expressed genes among the duplicates to encode molecular sites with new or modified functions. In later stages of descent, however, selection was a conserving force that preserved the structures of many coadapted functional sites. Each branch of the family was found to have a unique average tempo of evolutionary change, apparently regulated through functional constraints. Proteins whose functions dictate multiple interaction with several other macromolecules evolved more slowly than those which display fewer protein-protein and protein-ion interactions, e.g., calmodulin and next troponin-C evolved at the slowest average rates, whereas parvalbumin evolved at the fastest. The history of all lineages, however, appears to be characterized by rapid rates of evolutionary change in earlier periods, followed by slower rates in more recent periods. A particularly sharp contrast between such fast and slow rates is found in the evolution of calmodulin, whose rate of change in earlier eukaryotes was manyfold faster than the average rate over the past 1 billion years. In fact, the amino acid replacements in the nascent calmodulin lineage occurred at residue positions that in extant metazoans are largely invariable, lending further support to the Darwinian hypothesis that natural selection is both a creative and a conserving force in molecular evolution.      

Maturation-induced cloaking of neutralization epitopes on HIV-1 particles

To become infectious, HIV-1 particles undergo a maturation process involving proteolytic cleavage of the Gag and Gag-Pol polyproteins. Immature particles contain a highly stable spherical Gag lattice and are impaired for fusion with target cells. The fusion impairment is relieved by truncation of the gp41 cytoplasmic tail (CT), indicating that an interaction between the immature viral core and gp41 within the particle represses HIV-1 fusion by an unknown mechanism. We hypothesized that the conformation of Env on the viral surface is regulated allosterically by interactions with the HIV-1 core during particle maturation. To test this, we quantified the binding of a panel of monoclonal antibodies to mature and immature HIV-1 particles by immunofluorescence imaging. Surprisingly, immature particles exhibited markedly enhanced binding of several gp41-specific antibodies, including two that recognize the membrane proximal external region (MPER) and neutralize diverse HIV-1 strains. Several of the differences in epitope exposure on mature and immature particles were abolished by truncation of the gp41 CT, thus linking the immature HIV-1 fusion defect with altered Env conformation. Our results suggest that perturbation of fusion-dependent Env conformational changes contributes to the impaired fusion of immature particles. Masking of neutralization-sensitive epitopes during particle maturation may contribute to HIV-1 immune evasion and has practical implications for vaccine strategies targeting the gp41 MPER.     

In Vivo Functions of CPSF6 for HIV-1 as Revealed by HIV-1 Capsid Evolution in HLA-B27-Positive Subjects

The host protein CPSF6 possesses a domain that can interact with the HIV-1 capsid (CA) protein. CPSF6 has been implicated in regulating HIV-1 nuclear entry. However, its functional significance for HIV-1 replication has yet to be firmly established. Here we provide evidence for two divergent functions of CPSF6 for HIV-1 replication in vivo. We demonstrate that endogenous CPSF6 exerts an inhibitory effect on naturally occurring HIV-1 variants in individuals carrying the HLA-B27 allele. Conversely, we find a strong selective pressure in these individuals to preserve CPSF6 binding, while escaping from the restrictive activity by CPSF6. This active maintenance of CPSF6 binding during HIV-1 CA evolution in vivo contrasts with the in vitro viral evolution, which can reduce CPSF6 binding to evade from CPSF6-mediated restriction. Thus, these observations argue for a beneficial role of CPSF6 for HIV-1 in vivo. CPSF6-mediated restriction renders HIV-1 less dependent or independent from TNPO3, RanBP2 and Nup153, host factors implicated in HIV-1 nuclear entry. However, viral evolution that maintains CPSF6 binding in HLA-B27+ subjects invariably restores the ability to utilize these host factors, which may be the major selective pressure for CPSF6 binding in vivo. Our study uncovers two opposing CA-dependent functions of CPSF6 in HIV-1 replication in vivo; however, the benefit for binding CPSF6 appears to outweigh the cost, providing support for a vital function of CPSF6 during HIV-1 replication in vivo.     

A Two-Step Path to Inclusion Formation of Huntingtin Peptides Revealed by Number and Brightness Analysis

Protein aggregation is a hallmark of several neurodegenerative diseases including Huntington's disease. We describe the use of the recently developed number and brightness method (N&B) that uses confocal images to monitor aggregation of Huntingtin exon 1 protein (Httex1p) directly in living cells. N&B measures the molecular brightness of protein aggregates in the entire cell noninvasively based on intensity fluctuations at each pixel in an image. N&B applied to mutant Httex1p in living cells showed a two-step pathway leading to inclusion formation that is polyQ length dependent and involves four phases. An initial phase of monomer accumulation is followed by formation of small oligomers (5-15 proteins); as protein concentration increases, an inclusion is seeded and forms in the cytoplasm; the growing inclusion recruits most of the Httex1p and depletes the cell leaving only a low concentration of monomers. The behavior of Httex1p in COS-7 and ST14A cells is compared.