NITROGEN LIMITATION IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE) LEADS TO INCREASED SUSCEPTIBILITY TO DAMAGE BY ULTRAVIOLET‐B RADIATION BUT ALSO INCREASED REPAIR CAPACITY1

Chl fluorescence was used to measure the photosynthetic capacity of the green alga Dunaliella tertiolecta in order to investigate interactions between susceptibility to acute UV‐B radiation (UVBR, 280–320 nm) exposure and decreased nitrogen availability. Under UVBR exposure the decline in the fluorescence parameters F_v/F_m (the maximum effective quantum yield Φ_PSIIe‐max) and F_v′/F_m′ (the operational quantum yield of PSII, Φ_PSIIe) were enhanced with higher UVBR fluxes, with the data well described by the Kok model, inferring that a dynamic balance existed between damage and repair with the repair proportional to the pool size of inactivated targets. When UVBR exposure was coupled with nitrogen limitation, the inhibition of photosynthesis was intensified. Under the more severely N‐limited conditions, the damage rate increased. Unexpectedly, repair rates were also stimulated under N‐limited conditions, although this was insufficient to counteract the increase in damage, so the overall effect of N limitation was an enhancement of UVBR‐induced inhibition of photosynthesis.     

HLA haplotypes in dizygotic twin pairs: are dizygotic twins more similar than sibs?

It has been suggested that dizygotic twin pairs share two HLA haplotypes more often than ordinary siblings and thus might be genetically more alike. We tested this hypothesis in dizygotic twin pairs from the Danish Twin Registry. A total of 114 (60 female and 54 male) same-sexed healthy twin pairs aged 18-45 years participated. Dizygosity was established by means of DNA sequencing of nine polymorphic markers. HLA-A, B and Cw specificities were typed with serology, and if data were inconclusive, with DNA typing. If twin partners had the same HLA-types, they were assumed to share two haplotypes. If they had 1 HLA A, B and C antigen group in common they were assumed to share one haplotype and if they had no HLA types in common they were assumed to share zero haplotypes. Since HLA-types from parents were unavailable we could not test for identity-by-descent and thus had a risk of overestimating the number of twins sharing two haplotypes. A Chi-square test was used to compare observed numbers in each haplotype sharing group with the expected numbers. Twenty-nine (expected 28.5) twin pairs had two HLA-types in common, 52 (expected 57) had one HLA-type in common and 33 (expected 28.5) had zero HLA-types in common, p = 0.56. Our data show that DZ twins are not more similar than sibs from different pregnancies in general.     

Disturbance and reef topography maintain high local diversity in Ecklonia radiata kelp forests

Disturbance of competitive‐dominant plant and algae canopies often lead to increased diversity of the assemblage. Kelp forests, particularly those of temperate Western Australia, are habitats with high alpha diversity. This study investigated the roles of broad‐scale canopy loss and local scale reef topography on structuring the kelp‐dominated macroalgal forests in Western Australia. Eighteen 314 m² circular areas were cleared of their Ecklonia radiata canopy and eighteen controls were established across three locations. The patterns of macroalgal recolonisation in replicate clearances were observed over a 34 month period. Macroalgal species richness initially increased after canopy removal with a turf of filamentous and foliose macroalgae dominating cleared areas for up to seven months. A dense Sargassum canopy dominated cleared areas from 11 to 22 months. By 34 months, partial recovery of the kelp canopy into cleared areas had occurred. Some cleared areas did not follow this trajectory but remained dominated by turfing, foliose and filamentous algae. As kelp canopies developed, the initial high species diversity declined but still remained elevated relative to undisturbed controls, even after 34 months. More complex reef topography was associated with greater variability in the algal assemblage between replicate quadrats suggesting colonising algae had a greater choice of microhabitats available to them on topographically complex reefs. Shading by canopies of either Sargassum spp. and E. radiata are proposed to highly influence the abundance of algae through competitive exclusion that is relaxed by disturbance of the canopy. Disturbance of the canopy in E. radiata kelp forests created a mosaic of different patch types (turf, Sargassum‐dominated, kelp‐dominated). These patch types were both transient and stable over the 34 months of this study, and are a potential contemporary process that maintains high species diversity in temperate kelp‐dominated reefs.     

Development and validation of algorithms for measuring G-protein coupled receptor activation in cells using the LSC-based imaging cytometer platform.

BACKGROUND: A cell-based assay system (Transfluor) has been developed for measurement of G-protein coupled receptor (GPCR) activity by using cells transfected to express a fusion protein of arrestin plus green fluorescent protein (GFP) and the target GPCR. Upon agonist stimulation, the arrestin-GFP translocates to and binds the activated GPCR at the plasma membrane. The receptor/arrestin-GFP complexes then localize in clathrin-coated pits and/or intracellular vesicles. This redistribution of arrestin-GFP into condensed fluorescent spots is useful for visually monitoring the active status of GPCRs and its quantitation is possible with certain types of digital image analysis systems. METHODS: We designed two lines of image processing algorithms to carry out quantitative measurement of the arrestin-GFP movement on an inverted version of laser scanning cytometry (iCyte) as an imaging platform. We used a cell line expressing arrestin-GFP and the wild-type beta2-adrenergic receptor or a modified version of this receptor with enhanced affinity for arrestin. Each cell line was challenged with various concentrations of agonist. RESULTS: A dose-dependent signal was measured and half-maximal effective concentration values were obtained that agreed well with results determined by other methods previously reported. CONCLUSIONS: The results indicate that the combination of Transfluor, iCyte, and our algorithms is suitable for robust and pharmacologically relevant GPCR ligand exploration.     

Host Cell-Virus Cross Talk: Phosphorylation of a Hepatitis B Virus Envelope Protein Mediates Intracellular Signaling

Phosphorylation of cytosolic pre-S domains of the duck hepatitis B virus (DHBV) large envelope protein (L) was identified as a regulatory modification involved in intracellular signaling. By using biochemical and mass spectrometric analyses of phosphopeptides obtained from metabolically radiolabeled L protein, a single phosphorylation site was identified at serine 118 as part of a PX(S/T)P motif, which is strongly preferred by ERK-type mitogen-activated protein kinases (MAP kinases). ERK2 specifically phosphorylated L at serine 118 in vitro, and L phosphorylation was inhibited by a coexpressed MAP kinase-specific phosphatase. Furthermore, L phosphorylation and ERK activation were shown to be induced in parallel by various stimuli. Functional analysis with transfected cells showed that DHBV L possesses the ability to activate gene expression in trans and, by using mutations eliminating (S→A) or mimicking (S→D) serine phosphorylation, that this function correlates with L phosphorylation. These mutations had, however, no major effects on virus production in cell culture and in vivo, indicating that L phosphorylation and transactivation are not essential for hepadnavirus replication and morphogenesis. Together, these data suggest a role of the L protein in intracellular host-virus cross talk by varying the levels of pre-S phosphorylation in response to the state of the cell.     

Herpes Simplex Virus Type 1 Glycoprotein gC Mediates Immune Evasion In Vivo

Many microorganisms encode proteins that interact with molecules involved in host immunity; however, few of these molecules have been proven to promote immune evasion in vivo. Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) binds complement component C3 and inhibits complement-mediated virus neutralization and lysis of infected cells in vitro. To investigate the importance of the interaction between gC and C3 in vivo, we studied the virulence of a gC-null strain in complement-intact and C3-deficient animals. Using a vaginal infection model in complement-intact guinea pigs, we showed that gC-null virus grows to lower titers and produces less severe vaginitis than wild-type or gC rescued virus, indicating a role for gC in virulence. To determine the importance of complement, studies were performed with C3-deficient guinea pigs; the results demonstrated significant increases in vaginal titers of gC-null virus, while wild-type and gC rescued viruses showed nonsignificant changes in titers. Similar findings were observed for mice where gC null virus produced significantly less disease than gC rescued virus at the skin inoculation site. Proof that C3 is important was provided by studies of C3 knockout mice, where disease scores of gC-null virus were significantly higher than in complement-intact mice. The results indicate that gC-null virus is approximately 100-fold (2 log₁₀) less virulent that wild-type virus in animals and that gC-C3 interactions are involved in pathogenesis.     

Synthesis and Assembly of Simian Virus 40 I. Differential Synthesis of Intact Virions and Empty Shells

Intact virions and empty shells of simian virus 40 may be rapidly separated from each other and from cell contaminants by a procedure employing a CsCl cushion. This approach permits quantitation of their respective syntheses in infected cells labeled with radioactive amino acids. As much as 5 to 10% of the total acid-precipitable radioactive lysine in infected cell extracts was incorporated into viral particles in a two-hour pulse late in infection. Evidence for multiple origins of empty shells is presented. Some of the empty shells result from breakdown of intact virions. However, empty shells can also form independently of intact virions. First, labeling for periods of 15 min to 2 hr late in the course of infection results in preferential incorporation of (3)H-lysine into empty shells. Secondly, treatment with the deoxyribonucleic acid inhibitor cytosine-beta-d-arabinofuranoside late in infection results in a 50% inhibition in the rate of formation of intact virions with minimal reduction in the rate of appearance of empty shells.     

A competitive labelling method for determining the ionization constants and reactivity of individual histidine residues in proteins. The histidines of α-chymotrypsin

A competitive labelling method (Kaplan et al., 1971), using tritiated 1-fluoro-2,4-dinitrobenzene as the labelling reagent, is described for determining the ionization constants and reactivities of individual histidine residues in proteins. When this method was applied to the two histidines of alpha-chymotrypsin, histidine-57 was found to have pK(a) 6.8 and a reactivity ten times that of alpha-N-acetyl-l-histidine. Histidine-40 had pK(a) 6.7 and a reactivity approximately six times that of alpha-N-acetyl-l-histidine. Between pH7.5 and 8 the reactivities of both histidines decrease simultaneously to approximately that of alpha-N-acetyl-l-histidine. The high reactivities of the histidines are attributed to hydrogen bonding, which increases the nucleophilicity of the imidazole ring. The sharp decrease in reactivity between pH7.5 and 8 is attributed to a conformational change that disrupts the hydrogen bonding by these residues. The reactivity data support the proposal of a charge-relay mechanism involving histidine-57 (Blow et al., 1969), which makes serine-195 more nucleophilic but indicates that this system is fully operative only in the enzyme-substate complex.