Mutational analysis of receptor binding mediated by the Dr family of Escherichia coli adhesins

The fimbrial and afimbrial adhesins of the Dr family mediate the adherence of uropathogenic and diarrhoea-associated Escherichia coli to decay-accelerating factor (DAF) present on erythrocytes and other cell types. The Dr haemagglutinin binds type IV collagen and, unlike other members of the Dr family, mediates an adherence inhibited in the presence of chloramphenicol. We examined the ability of other members of the Dr family—AFAI, AFAIII, and F1845—to bind to type IV collagen, and demonstrated that the collagen-binding phenotype was unique to the Dr haemagglutinin. We employed site-directed mutagenesis to demonstrate the requirement of a negatively charged amino-acid at position 54 of the Dr haemagglutinin subunit for chloramphenicol sensitivity of binding. Mutations at position 32, 40, 54, 90, and 113 differently affected type IV collagen binding and chloramphenicol sensitivity of binding, while retaining DAF-binding capability. These results suggest the existence of a conformational receptor-binding domain in the major structural subunit of Dr family adhesins and demonstrate that chloramphenicol sensitivity of binding and adherence to type IV collagen were independent and separable phenotypes. Finally, we showed that the two conserved cysteine residues of Dr family structural subunits form a disulphide bond and that mutations of these residues abolish haemagglutination and binding to type IV collagen.     

Serine Transhydroxymethylase: A Simplified Radioactive Assay; Purification and Stabilization of Enzyme Activity Employing Affi-Gel Blue

An improved radioactive assay has been developed for serine transhydroxymethylase. This assay involves the direct measurement of the [¹⁴C]HCH0 which is generated when [3-¹⁴C]-serine is employed as the substrate. The new assay eliminates 14 the need for a solvent extraction of a [C]HCHO-dimedon adduct which is the basis of the assay devised by Taylor and Weissbach.

The enzyme has been purified employing Affi-Gel Blue. The purified enzyme retains full activity when bound to this affinity chromatography matrix and can be stored in this state at 4° indefinitely.     

Residence times of neutrally-buoyant matter such as larvae,sewage or nutrients on coral reefs

Coral reef flushing times at an individual reef scale are specified and a general formula to determine these times is developed. The formula is confirmed by comparison with residence times predicted by numerical small-scale reef models, including those from a 4 month unsteady current simulation of John Brewer Reef on Australia's Great Barrier Reef. The method proves to be a satisfactory alternative to the numerical modelling. When neutrally-buoyant material around a reef is removed by the currents, the concentrations decay exponentially. The decay rate depends primarily on free stream current and reef dimensions. Secondary factors are the tidal excursion, shelf depth, lagoon size and residual current in the lee of the reef. These factors, when combined into a decay coefficient, specify the rate of loss of neutrally-buoyant material (e.g. some larvae, pollutants and sewage) from a coral reef and its surrounds. The analytical formula can be used to predict the flushing rates or the percentage of material still remaining on a reef after a selected time interval. We demonstrate that material can remain on or near typical reefs in common weather conditions for several weeks.     

The Effect of Space Flight on Monoclonal Antibody Synthesis in a Hybridoma Mouse Cell Line

The hybridoma cell line, 3G10G5, producing a monoclonal antibody to the major capsid protein VP1 from the avian polyomavirus budgerigar fledgling disease virus, was produced from a Balb/C mouse. This cell line was used to test the effects of microgravity on cellular processes, specifically protein synthesis. A time course study utilizing incorporation of [³⁵S]methionine into newly synthesized monoclonal antibody was performed on STS-77. After 5.5 days, it was observed that cell counts for the samples exposed to microgravity were lower than those of ground-based samples. However, radiolabel incorporation of the synthesized monoclonal antibody was similar in both orbiter and ground control samples. Overall, microgravity does not seem to have an effect on this cell line's ability to synthesize IgG protein.     

Effects of resource quality on the population dynamics of the Indian meal moth Plodia interpunctella and its granulovirus

Although the Plodia interpunctella-granulovirus system is one of the most studied models for insect-pathogen interactions, there are relatively few precise data on the dynamics of the virus in coexisting populations of these two organisms. Previous work has suggested that resource quality, in terms of the diet supplied to P. interpunctella, has a strong effect on the population dynamics of host and pathogen. Here we investigate the impact of resource-dependent host patterns of abundance on pathogen dynamics and prevalence. In the laboratory, three populations of P. interpunctella feeding on a good quality food and infected with a granulovirus were compared with three populations also infected with a granulovirus but feeding on poor quality food. Populations feeding on good quality food produced larger adult moths, and had greater numbers of adult moths, healthy larvae, and virus-infected larvae. A higher proportion of larvae in these good quality populations were infected with virus, and adult moths exhibited cyclic fluctuations in abundance, unlike those on poor quality food. This cyclic behaviour was shown to be associated with cycles in the age structure of the larval population. Previous theoretical work suggests that these cycles may result from asymmetric competition between young and old larvae. Cyclic fluctuations in the proportion of infected larvae, that occurred on good, but not on poor quality food, were also shown to be related to cycles in the age structure of the larval population.     

A <Emphasis Type="Italic">var2</Emphasis> leaf variegation suppressor locus, <Emphasis Type="Italic">SUPPRESSOR OF VARIEGATION3</Emphasis>, encodes a putative chloroplast translation elongation factor that is important for chloroplast development in the cold

Background  

The Arabidopsis var2 mutant displays a unique green and white/yellow leaf variegation phenotype and lacks VAR2, a chloroplast FtsH metalloprotease. We are characterizing second-site var2 genetic suppressors as means to better understand VAR2 function and to study the regulation of chloroplast biogenesis.     

Hippocampal Lesions Impair Rapid Learning of a Continuous Spatial Alternation Task

The hippocampus is essential for the formation of memories for events, but the specific features of hippocampal neural activity that support memory formation are not yet understood. The ideal experiment to explore this issue would be to monitor changes in hippocampal neural coding throughout the entire learning process, as subjects acquire and use new episodic memories to guide behavior. Unfortunately, it is not clear whether established hippocampally-dependent learning paradigms are suitable for this kind of experiment. The goal of this study was to determine whether learning of the W-track continuous alternation task depends on the hippocampal formation. We tested six rats with NMDA lesions of the hippocampal formation and four sham-operated controls. Compared to controls, rats with hippocampal lesions made a significantly higher proportion of errors and took significantly longer to reach learning criterion. The effect of hippocampal lesion was not due to a deficit in locomotion or motivation, because rats with hippocampal lesions ran well on a linear track for food reward. Rats with hippocampal lesions also exhibited a pattern of perseverative errors during early task experience suggestive of an inability to suppress behaviors learned during pretraining on a linear track. Our findings establish the W-track continuous alternation task as a hippocampally-dependent learning paradigm which may be useful for identifying changes in the neural representation of spatial sequences and reward contingencies as rats learn and apply new task rules.