Temperature effects on hatching and viability of Juvenile Gill Lice, Salmincola californiensis.

Salmonids of the genus Oncorhynchus, distributed throughout the Pacific Rim, can be infected by the gill lice species Salmincola californiensis (Dana, 1852), which makes them one of the most broadly distributed gill lice species. Despite their broad distribution and valuable obligate salmonid hosts, relatively little is known about S. californiensis. We evaluated effects of temperature on timing of S. californiensis hatching and survival of copepodids, and provide information on brood size and variability. Our results suggest that temperature was a primary driver of timing of S. californiensis hatching and post-hatching survival. Prior to this study, the free-swimming stage of S. californiensis was reported to survive approximately 2 days without a suitable host. We observed active copepodids 13 days after hatch with some individuals from most (>90%) viable egg sacs at all temperature treatments surviving ≥5 days. Our findings indicate that warmer temperatures could increase development rates of gill lice at certain life stages, potentially increasing fecundity. This information coupled with predictions that warmer water temperatures could intensify crowding of coldwater fishes, stress, and parasite transmission suggests that climate change could exacerbate negative effects of S. californiensis on ecologically and economically important salmonids.

Human adenovirus-vectored foot-and-mouth disease vaccines: establishment of a vaccine product profile through in vitro testing.

Next generation, foot-and-mouth disease (FMD) molecular vaccines based on replication deficient human adenovirus serotype 5 viral vectored delivery of FMD capsid genes (AdFMD) are being developed by the United States Dept. of Homeland Security and industry partners. The strategic goal of this program is to develop AdFMD licensed vaccines for the USA National Veterinary Stockpile for use, if needed, as emergency response tools during an FMD outbreak. This vaccine platform provides a unique opportunity to develop a set of in vitro analytical parameters to generate an AdFMD vaccine product profile to replace the current lot release test for traditional, inactivated FMD vaccines that requires FMDV challenge in livestock. The possibility of an indirect FMD vaccine potency test based on a serological alternative was initially investigated for a lead vaccine candidate, Adt.A24. Results show that serum virus neutralization (SVN) based serology testing for Adt.A24 vaccine lot release is not feasible, at least not in the context of vaccine potency assessment at one week post-vaccination. Thus, an in vitro infectious titer assay (tissue culture infectious dose 50, TCID50) which measures FMD infectious (protein expression) titer was established. Pre-validation results show acceptable assay variability and linearity and these data support further studies to validate the TCID50 assay as a potential potency release test. In addition, a quantitative physiochemical assay (HPLC) and three immunochemical assays (Fluorescent Focus-Forming Unit (FFU); tissue culture expression dose 50 (TCED50); Western blot) were developed for potential use as in vitro assays to monitor AdFMD vaccine lot-to-lot consistency and other potential applications. These results demonstrate the feasibility of using a traditional modified-live vaccine virus infectivity assay in combination with a set of physiochemical and immunochemical tests to build a vaccine product profile that will ensure the each AdFMD vaccine lot released is similar to a reference vaccine of proven clinical safety and efficacy.

Thematic roles assigned along the garden path linger.

In the literature dealing with the reanalysis of garden path sentences such as While the man hunted the deer ran into the woods, it is generally assumed either that people completely repair their initial incorrect syntactic representations to yield a final interpretation whose syntactic structure is fully consistent with the input string or that the parse fails. In a series of five experiments, we explored the possibility that partial reanalyses take place. Specifically, we examined the conditions under which part of the initial incorrect analysis persists at the same time that part of the correct final analysis is constructed. In Experiments 1a and 1b, we found that both the length of the ambiguous region and the plausibility of the ultimate interpretation affected the likelihood that such sentences would be fully reanalyzed. In Experiment 2, we compared garden path sentences with non-garden path sentences and compared performance on two different types of comprehension questions. In Experiments 3a and 3b, we constructed garden path sentences using a small class of syntactically unique verbs to provide converging evidence against the position that people employ some sort of "general reasoning" or pragmatic inference when faced with syntactically difficult garden paths. The results from these experiments indicate that reanalysis of such sentences is not always complete, so that comprehenders often derive an interpretation for the full sentence in which part of the initial misanalysis persists. We conclude that the goal of language processing is not always to create an idealized structure, but rather to create a representation that is "good enough" to satisfy the comprehender that an appropriate interpretation has been obtained.

Misinterpretations of garden-path sentences: implications for models of sentence processing and reanalysis.

Theories of sentence comprehension have addressed both initial parsing processes and mechanisms responsible for reanalysis. Three experiments are summarized that were designed to investigate the reanalysis and interpretation of relatively difficult garden-path sentences (e.g., While Anna dressed the baby spit up on the bed). After reading such sentences, participants correctly believed that the baby spit up on the bed; however, they often confidently, yet incorrectly, believed that Anna dressed the baby. These results demonstrate that garden-path reanalysis is not an all-or-nothing process and that thematic roles initially assigned for the subordinate clause verb are not consistently revised. The implications of the partial reanalysis phenomenon for Fodor and Inoue's (1998) model of reanalysis and sentence processing are discussed. In addition, we discuss the possibility that language processing often creates "good enough" structures rather than ideal structures.

Crystallographic analysis of reversible metal binding observed in a mutant (Asp153-->Gly) of Escherichia coli alkaline phosphatase.

Here we present the refined crystal structures of three different conformational states of the Asp153-->Gly mutant (D153G) of alkaline phosphatase (AP), a metalloenzyme from Escherichia coli. The apo state is induced in the crystal over a 3 month period by metal depletion of the holoenzyme crystals. Subsequently, the metals are reintroduced in the crystalline state in a time-dependent reversible manner without physically damaging the crystals. Two structural intermediates of the holo form based on data from a 2 week (intermediate I) and a 2 month soak (intermediate II) of the apo crystals with Mg2+ and Zn2+ have been identified. The three-dimensional crystal structures of the apo (R = 18.1%), intermediate I (R = 19.5%), and intermediate II (R = 19.9%) of the D153G enzyme have been refined and the corresponding structures analyzed and compared. Large conformational changes that extend from the mutant active site to surface loops, located 20 A away, are observed in the apo structure with respect to the holo structure. The structure of intermediate I shows the recovery of the entire enzyme to an almost native-like conformation, with the exception of residues Asp 51 and Asp 369 in the active site and the surface loop (406-410) which remains partially disordered. In the three-dimensional structure of intermediate II, both Asp 51 and Asp 369 are essentially in a native-like conformation, but the main chain of residues 406-408 within the loop is still not fully ordered. The D153G mutant protein exhibits weak, reversible, time dependent metal binding in solution and in the crystalline state.(ABSTRACT TRUNCATED AT 250 WORDS)

A molecular sensor system based on genetically engineered alkaline phosphatase.

Binding and signaling proteins based on Escherichia coli alkaline phosphatase (AP; EC 3.1.3.1) were designed for the detection of antibodies. Hybrid proteins were constructed by using wild-type AP and point mutants of AP [Asp-101 --> Ser (D101S) and Asp-153 --> Gly (D153G)]. The binding function of the hybrid proteins is provided by a peptide epitope inserted between amino acids 407 and 408 in AP. Binding of anti-epitope antibodies to the hybrid proteins modulates the enzyme activity of the hybrids; upon antibody binding, enzyme activity can increase to as much as 300% of the level of activity in the absence of antibody or can decrease as much as 40%, depending on the presence or absence of the point mutations in AP. The fact that modulation is altered from inhibition to activation by single amino acid changes in the active site of AP suggests that the mechanism for modulation is due to structural alterations upon antibody binding. Modulation is a general phenomenon. The properties of the system are demonstrated by using two epitopes, one from the V3 loop of human immunodeficiency virus type 1 gp120 protein and one from hepatitis C virus core protein, and corresponding monoclonal antibodies. The trend of modulation is consistent for all hybrids; those in wild-type AP are inhibited by antibody, while those in the AP mutants are activated by antibody. This demonstrates that modulation of enzyme activity of the AP-epitope hybrid proteins is not specific to either a particular epitope sequence or a particular antibody-epitope combination.

Modulation of enzyme activity by antibody binding to an alkaline phosphatase-epitope hybrid protein.

An epitope from the HIV-1 gp120 protein V3 loop has been inserted onto the surface of bacterial alkaline phosphatase at different positions in the vicinity of the enzyme active site, creating hybrid proteins that can bind to an anti-gp120 monoclonal antibody. One of the hybrid proteins, API1, has a 13 amino acid V3 loop sequence inserted between residues 407 and 408 of alkaline phosphatase. The enzymatic activity of this protein is modulated upon antibody binding. API1 maintains the full activity of the wild type alkaline phosphatase but in the presence of the anti-gp120 antibody, the enzyme activity is inhibited by 40-50%. Thus, the hybrid enzyme can be used to detect the presence of antibody in solution. The concept of signalling proteins may have a wide application. Two models for the mechanism of modulation, steric hindrance and allosteric regulation, are discussed.

Protection against feline infectious peritonitis by intranasal inoculation of a temperature-sensitive FIPV vaccine.

Cats vaccinated intranasally (i.n.) with a temperature sensitive feline infectious peritonitis virus (ts-FIPV) vaccine were protected against an FIP-inducing challenge. Seventeen of 20 vaccinated cats (85%) survived a rigorous virulent FIPV challenge that caused FIP in 12 of 12 non-vaccinated cats (100%), 10 (83%) of which died. Intranasal vaccination stimulated serum IgG and serum and salivary IgA antibody responses (measured by ELISA), FIPV-neutralizing antibody (VN), and a cell-mediated immune (CMI) response as measured by lymphocyte proliferation. The serum antibody response to vaccination was not associated with protection. In fact, the IgG, IgA and VN titres were much higher in control cats than in vaccinated cats following challenge suggesting an immune-mediated pathogenesis. In contrast, stimulation of a mucosal IgA response to vaccination was related to protection. The in vitro proliferation of peripheral blood lymphocytes in response to virulent FIPV was observed in vaccinated cats, in vaccinated and challenged cats but not in non-vaccinated challenged cats.

Characterization of an attenuated temperature sensitive feline infectious peritonitis vaccine virus.

Intranasal administration of a ts-FIPV vaccine protected cats against two rigorous challenges of immunity. Investigations showed that ts-FIP viral RNA synthesis was normal at 39 degrees C and structural proteins were synthesized, but not expressed at the cell surface. Lack of surface expression combined with decreased virus titer indicate that, although structural viral proteins were initially synthesized, they were not packaged into intact virions at the nonpermissive temperature. The ts-FIP vaccine virus was shown to replicate exclusively in the upper respiratory tract, where lower temperatures allow maturation of the virus. Viral proteins expressed on cells in the upper respiratory tract probably stimulate the development of local IgA and CMI responses and a systemic CMI response which in turn may stop the dissemination of virulent FIPV if it crosses the mucosal barrier. Investigations are ongoing to study the protective mechanism of ts-FIPV induced immunity.

Characterization of a temperature sensitive feline infectious peritonitis coronavirus.

The characteristics of a temperature sensitive feline infectious peritonitis virus (TS-FIPV) were examined. TS-FIPV, unlike its parent strain, DF2 wild type FIPV (WT-FIPV), propagated at 31 degrees C (permissive temperature) but not at 39 degrees C (nonpermissive temperature). This temperature preference of TS-FIPV was also demonstrated in cats by the ability of the virus to replicate only at the lower temperature in the upper respiratory tract and not at systemic sites where higher temperatures (38-39 degrees C) prevail. Viral structural proteins and RNA were synthesized at 39 degrees C but some undefined maturational defect prevented the formation of infectious TS-FIPV at its nonpermissive temperature. TS-FIPV was more thermolabile than WT-FIPV which indicated alterations in the structural proteins of TS-FIPV, and a difference in the envelope protein of the two viruses was revealed by Western blot analysis. Plaque assay characterization showed that TS-FIPV produced small plaques in comparison to the large plaques of WT-FIPV. These unique characteristics possessed by TS-FIPV may account for its nonvirulent nature and ability to stimulate protective immune responses in cats.

Transport and processing of staphylococcal enterotoxin A.

A larger-molecular-weight precursor of enterotoxin A was found in membranes of Staphylococcus aureus and was shown to be the kinetic precursor to the extracellular form of the toxin. Subcellular fractionation revealed that mature enterotoxin A was transiently associated with the cell wall before being released to the extracellular environment.

Structural and biochemical differentiation of the guinea-pig colon during foetal development.

We have studied some aspects of the morphological and biochemical differentiation of the foetal guinea-pig colonic epithelium. At day 40 the epithelium was organised in ridges and appeared pseudo-stratified. Folding of the epithelium, followed by villus formation, occurred between days 45 and 55, and by day 50 mucus-secreting goblet cells appeared at the bases of the colonic villi. By day 55 most epithelial cells, including goblet cells, possessed numerous microvilli which, by day 65, had become organised into well developed brush-borders. Between day 55 and term (day 65-68) mucosal depth increased markedly and the colon attained its final glandular morphology. Biochemical studies showed the specific activities of the microvillar hydrolases to be much lower in the washed colon than in either foetal meconium or small intestine at all times during development. Furthermore, a membrane fraction highly enriched in microvillus hydrolase activities was prepared from foetal colonic meconium using techniques originally devised to isolate the foetal small intestinal microvillus membrane. This meconial subfraction was almost identical in polypeptide composition to the highly-purified foetal small intestinal microvillus membrane. Identification of the colonic microvillus membrane was hampered by the absence of reliable membrane markers. Nevertheless, a fraction 14-fold enriched in aminopeptidase activity was prepared from day 40 foetal colon and its polypeptide composition compared by SDS-PAGE to that of the small intestinal microvillus membrane at the same age.

Transport and processing of staphylococcal alpha-toxin.

Two larger precursors to staphylococcal alpha-toxin were identified and partially characterized. Both precursor proteins were present on the cell membrane at very low levels and appeared to be rapidly processed to the mature form. Dinitrophenol inhibited processing such that the two precursors accumulated in the membranes, whereas little extracellular (mature) alpha-toxin is formed. The peptide maps of the 35S-labeled peptides from extracellular alpha-toxin and the two precursors were almost identical. The larger precursor protein contained four additional peptides and the smaller precursor protein contained three additional peptides not found in the extracellular toxin.