Structure-function analysis of soluble forms of herpes simplex virus glycoprotein D.

Glycoprotein D (gD) of herpes simplex virus (HSV) is essential for virus entry. Truncated forms of gD lacking the transmembrane and cytoplasmic tail regions have been shown to bind to cells and block plaque formation. Using complementation analysis and a panel of gD mutants, we previously identified four regions of gD (regions I to IV) which are important for virus entry. Here, we used baculovirus vectors to overexpress truncated forms of wild-type gD from HSV type 1 (HSV-1) [gD-1(306t)] and HSV-2 [gD-2(306t)] and four mutants, gD-1(inverted delta 34t), gD-1(inverted delta 126t), gD-1(inverted delta 243t), and gD-1(delta 290-299t), each having a mutation in one of the four functional regions. We used an enzyme-linked immunosorbent assay and circular dichroism to analyze the structure of these proteins, and we used functional assays to study the role of gD in binding, penetration, and cell-to-cell spread. gD-1 and gD-2 are similar in antigenic structure and thermal stability but vary in secondary structure. Mutant proteins with insertions in region I or II were most altered in structure and stability, while mutants with insertions in region III or IV were less altered. gD-1(306t) and gD-2(306t) inhibited both plaque formation and cell-to-cell transmission of HSV-1. In spite of obvious structural differences, all of the mutant proteins bound to cells, confirming that binding is not the only function of gD. The region I mutant did not inhibit HSV plaque formation or cell-to-cell spread, suggesting that this region is necessary for the function of gD in these processes. Surprisingly, the other three mutant proteins functioned in all of the in vitro assays, indicating that the ability of gD to bind to cells and inhibit infection does not correlate with its ability to initiate infection as measured by the complementation assay. The region IV mutant, gD-1(delta 290-299t), had an unexpected enhanced inhibitory effect on HSV infection. Taken together, the results argue against a single functional domain in gD. It is likely that different gD structural elements are involved in successive steps of infection.     

Ecophysiological responses of the mangrove Avicennia marina to trace metal contamination

Growth responses of Avicennia marina seedlings to contamination by different concentrations of two essential (Cu, Zn) and two non-essential (Pb, Hg) trace metals were studied under glasshouse conditions. We tested the hypothesis that soil retention and root ultrafiltration would exclude most of the trace metals, and that those that are absorbed and translocated to the shoots would interfere with plant performance and be excreted via leaf salt glands. One-month-old seedlings were subjected to Cu, Zn, Pb and Hg at concentrations of 0, 40, 80, 120 and 160 μg g⁻¹ sediment for 12 months in a randomized complete block design (n = 6). Photosynthesis was measured at the end of 12 months of trace metal exposure with a portable gas exchange system and chlorophyll fluorescence with a pulse-modulated fluorometer. After morphometric measurements, plants were harvested and analyzed for Cu, Zn, Pb and Hg by atomic absorption spectroscopy. Total dry biomass decreased with increasing trace metal concentration for all metals. In the 160 μg g⁻¹ Cu, Zn, Hg and Pb treatments, total biomass was significantly lower than the control value by 43%, 37%, 42% and 40%, respectively. Decreases in plant height and number of leaves followed trends similar to those for total biomass and ranged from 37% to 60%, compared to the controls. Decreases in chlorophyll content in the trace metal treatments ranged from 50% to 58% compared to the control. Carbon dioxide exchange, quantum yield of photosystem II (PSII), electron transport rate (ETR) through PSII and photosynthetic efficiency of PSII (F_v/F_m) were highest in the control treatment and decreased with increasing trace metal concentrations. Decreases in CO₂ exchange in the 160 μg g⁻¹ treatments for all trace metals ranged from 50% to 60%. Concentrations of all trace metals in plant organs increased with increasing metal concentrations and were higher in roots than in shoots, with concentrations of Cu and Zn being considerably higher than those of Hg and Pb. Qualitative elemental analyses and X-ray mapping of crystalline deposits over the glands at the leaf surfaces indicated that Cu and Zn were excreted from the salt glands, while Hg and Pb were absent, at least being below the limits of detection. These results demonstrate that growth processes are sensitive to trace metals and therefore can be considered as a cost of metal tolerance, but salt glands of this mangrove species do contribute eliminating at least part of physiologically essential trace metals if taken up in excess.     

Metabolic profiles and lipoprotein lipid concentrations in non-obese and obese patients with polycystic ovarian disease

Clinical parameters, androgen status and lipoprotein lipid profiles were assessed in 10 non-obese and 10 obese patients with polycystic ovarian disease (PCOD) and reference subjects matched for age, height and weight. Both obese and non-obese women with PCOD had significantly higher androgen levels when compared to the reference groups. When comparison of lipoprotein lipid profiles were made between groups, non-obese women with PCOD had significantly higher total cholesterol, triglycerides and LDL-cholesterol levels than non-obese reference subjects. Obese PCOD women manifested significantly higher total cholesterol, LDL-cholesterol, cholesterol/HDL, and LDL/HDL values than did obese reference subjects. Correlations between serum androgens and lipoprotein lipid concentrations in PCOD and normal women were unhelpful. Both non-obese and obese patients with PCOD had significantly higher systolic and diastolic blood pressures (BPs) than the reference groups. Thus, both non-obese and obese women with PCOD manifest hyperandrogenaemia which may result in a male pattern of lipoprotein lipid concentrations.     

Photic signaling by cryptochrome in the Drosophila circadian system

Oscillations of the period (per) and timeless (tim) gene products are an integral part of the feedback loop that underlies circadian behavioral rhythms in Drosophila melanogaster. Resetting this loop in response to light requires the putative circadian photoreceptor cryptochrome (CRY). We dissected the early events in photic resetting by determining the mechanisms underlying the CRY response to light and by investigating the relationship between CRY and the light-induced ubiquitination of the TIM protein. In response to light, CRY is degraded by the proteasome through a mechanism that requires electron transport. Various CRY mutant proteins are not degraded, and this suggests that an intramolecular conversion is required for this light response. Light-induced TIM ubiquitination precedes CRY degradation and is increased when electron transport is blocked. Thus, inhibition of electron transport may "lock" CRY in an active state by preventing signaling required either to degrade CRY or to convert it to an inactive form. High levels of CRY block TIM ubiquitination, suggesting a mechanism by which light-driven changes in CRY could control TIM ubiquitination.     

Comparative metabolism as a key driver of wildlife species sensitivity to human and veterinary pharmaceuticals

Human and veterinary drug development addresses absorption, distribution, metabolism, elimination and toxicology (ADMET) of the Active Pharmaceutical Ingredient (API) in the target species. Metabolism is an important factor in controlling circulating plasma and target tissue API concentrations and in generating metabolites which are more easily eliminated in bile, faeces and urine. The essential purpose of xenobiotic metabolism is to convert lipid-soluble, non-polar and non-excretable chemicals into water soluble, polar molecules that are readily excreted. Xenobiotic metabolism is classified into Phase I enzymatic reactions (which add or expose reactive functional groups on xenobiotic molecules), Phase II reactions (resulting in xenobiotic conjugation with large water-soluble, polar molecules) and Phase III cellular efflux transport processes. The human–fish plasma model provides a useful approach to understanding the pharmacokinetics of APIs (e.g. diclofenac, ibuprofen and propranolol) in freshwater fish, where gill and liver metabolism of APIs have been shown to be of importance. By contrast, wildlife species with low metabolic competency may exhibit zero-order metabolic (pharmacokinetic) profiles and thus high API toxicity, as in the case of diclofenac and the dramatic decline of vulture populations across the Indian subcontinent. A similar threat looms for African Cape Griffon vultures exposed to ketoprofen and meloxicam, recent studies indicating toxicity relates to zero-order metabolism (suggesting P450 Phase I enzyme system or Phase II glucuronidation deficiencies). While all aspects of ADMET are important in toxicity evaluations, these observations demonstrate the importance of methods for predicting API comparative metabolism as a central part of environmental risk assessment.     

Relationship between morphological and physiological responses to waterlogging and salinity in Sporobolus virginicus (L.) Kunth

The effects of waterlogging and salinity on morphological and physiological responses in the marsh grass Sporobolus virginicus (L.) Kunth were investigated in a 4×2 factorial experiment. Plants were subjected to four salinity levels (0, 100, 200 and 400 mol m^–3 NaCl) and two soil inundation conditions (drained and flooded) for 42 days. Flooding at 0 mol m^–3 NaCl caused initiation of adventitious surface roots, increased internal acration and plant height, induced alcohol dehydrogenase activity (ADH), and decreased belowground biomass and the number of culms per plant. Salinity increase from 0 to 400 mol m^–3 NaCl under drained conditions increased leaf and root proline concentrations and decreased photosynthesis, aboveground biomass, number of culms per plant and number of internodes per culm. Concurrent waterlogging and salinity induced ADH activity and adventitious surface roots but decreased plant height and aboveground biomass. Internal air space increased with waterlogging from 0 to 100 mol m^–3 NaCl but further increases in salinity to 400 mol m^–3 reduced air space. Combined waterlogging and salinity stresses, however, had no effect on photosynthesis or on the concentrations of proline in leaves or roots. These results are discussed in relation to the widespread colonization by S. virginicus of a wide range of coastal environments varying in soil salinity and in the frequency and intensity of waterlogging.     

Responses of the saltmarsh rush Juncus kraussii to salinity and waterlogging

Juncus kraussii Hochst., an important saltmarsh macrophyte, is intensively harvested for many commercially orientated products and current populations are under threat of overexploitation. In saline, intertidal mud banks, this species occurs on higher ground, suggesting that it is adapted to lower salinities and less frequent inundation. The objectives of this study were to determine biomass accumulation, as well as morphological and physiological adaptations of J. kraussii to salinity and waterlogging stresses. Plants collected from the field were subjected to 0.2, 10, 30, 50 and 70% seawater under drained or flooded conditions for three months. Measurements were made of biomass accumulation, CO₂ exchange, chlorophyll fluorescence, ion and water relations. Furthermore, seed germination responses to a range of salinities were investigated. Total dry biomass accumulation, as well as the number and height of culms, decreased with increase in salinity under both flooded and drained conditions. Generally, CO₂ exchange, stomatal conductance, Photosystem II (PSII) quantum yield and electron transport rate (ETR) through PSII declined with increase in salinity in both the flooded and drained treatments. Predawn and midday ψ in culms decreased with increase in salinity, being lower under drained than flooded conditions. Inorganic solute concentrations in culms increased with increase in salinity, with Na⁺ and Cl⁻ being the predominant ions. Na⁺/K⁺ ratios in culms increased significantly with increase in salinity. Proline concentrations in roots and culms, which increased with salinity, were considerably higher under drained than flooded conditions. Germination was best at salinities less than 20% seawater and decreased significantly with further increase in salinity to 110% seawater. Transfer of ungerminated salt-treated seeds to distilled water stimulated germination. This study has demonstrated that J. kraussii is a highly salt and flood tolerant species, being able to grow and survive in salinities up to 70% seawater, under both drained and flooded conditions. Maximal growth occurred at low salinities (<10% seawater) under flooded condition.     

Ultrastructural responses of the desiccation tolerant plants Xerophyta viscosa and X. retinervis to dehydration and rehydration

This paper compares the changes in water content, chlorophyll a fluorescence and leaf ultrastructure during dehydration and rehydration in two desiccation tolerant plants Xerophyta viscosa and X. retinervis. Both species showed decreasing quantum efficiency of photosystem 2 (F_v/F_m) with decreasing water content. Extreme water loss observed after 25 d of dehydration resulted in considerable damage of leaf tissue ultrastructure. After rehydration, both species need several days to reconstitute their photosynthetic machinery.     

Phenotypes and Karyotypes of Human Malignant Mesothelioma Cell Lines

Background

Malignant mesothelioma is an aggressive tumour of serosal surfaces most commonly pleura. Characterised cell lines represent a valuable tool to study the biology of mesothelioma. The aim of this study was to develop and biologically characterise six malignant mesothelioma cell lines to evaluate their potential as models of human malignant mesothelioma.

Methods

Five lines were initiated from pleural biopsies, and one from pleural effusion of patients with histologically proven malignant mesothelioma. Mesothelial origin was assessed by standard morphology, Transmission Electron Microscopy (TEM) and immunocytochemistry. Growth characteristics were assayed using population doubling times. Spectral karyotyping was performed to assess chromosomal abnormalities. Authentication of donor specific derivation was undertaken by DNA fingerprinting using a panel of SNPs.

Results

Most of cell lines exhibited spindle cell shape, with some retaining stellate shapes. At passage 2 to 6 all lines stained positively for calretinin and cytokeratin 19, and demonstrated capacity for anchorage-independent growth. At passage 4 to 16, doubling times ranged from 30–72 hours, and on spectral karyotyping all lines exhibited numerical chromosomal abnormalities ranging from 41 to 113. Monosomy of chromosomes 8, 14, 22 or 17 was observed in three lines. One line displayed four different karyotypes at passage 8, but only one karyotype at passage 42, and another displayed polyploidy at passage 40 which was not present at early passages. At passages 5–17, TEM showed characteristic features of mesothelioma ultrastructure in all lines including microvilli and tight intercellular junctions.

Conclusion

These six cell lines exhibit varying cell morphology, a range of doubling times, and show diverse passage-dependent structural chromosomal changes observed in malignant tumours. However they retain characteristic immunocytochemical protein expression profiles of mesothelioma during maintenance in artificial culture systems. These characteristics support their potential as in vitro model systems for studying cellular, molecular and genetic aspects of mesothelioma.