Transmembrane-mediated changes in [Ca2+] are involved in the signaling pathway leading to macrophage cytocidal differentiation: implications of localized changes in intracellular [Ca2+] and of interferon priming on Ca2+ utilization.

Macrophage cytocidal activation requires the sequential impingement on the macrophage of a priming stimulus (interferon [IFN] alpha, beta, or gamma) and a triggering stimulus (such as polyinosinic acid:polycytidylic acid [poly [I:C]] or bacterial lipopolysaccharide). The mechanism of progression from the IFN-primed state to the cytocidal state is poorly understood. By quantifying the level of expression of a gene product (complement component factor B [Bf]) associated with cytocidal activation and through the use of phenotypically distinct populations of macrophages (unprimed and IFN-primed), we have investigated the functional necessity of changes in intracellular concentration of free calcium ions ([Ca2+]i) in signaling the transition from the primed to the cytocidal state. Elevating the [Ca2+]i by incubation of unprimed macrophages with the calcium ionophore, ionomycin, failed to induce the expression of Bf. By contrast, Bf was expressed at high levels when IFN-primed macrophages were exposed to ionomycin, suggesting that priming induced within the macrophages the capacity to respond to a nonspecific change in [Ca2+]i. Quantification of the [Ca2+]i in response to exposure to ionomycin revealed an initial transient elevation, followed by a secondary sustained component. No differences in these changes were observed between unprimed and IFN-primed macrophages. We therefore questioned if changes in [Ca2+]i were also implicated in the transition between the primed and the cytocidal state using the ligand, poly [I:C]. In contrast to ionomycin, incubation of IFN-primed macrophages with poly [I:C] did not sustain measurable increases in [Ca2+]i, yet fully stimulated the transition from the IFN primed to the cytocidal state. However, incubation of IFN-primed macrophages with poly [I:C] in the presence of 1) a Ca2+/ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffer calculated to clamp the extracellular concentration of free calcium ions to a value approximately equal to the resting [Ca2+]i; 2) the calcium channel blocker verapamil; or 3) the intracellular Ca2+ antagonists (W-7, W-13, and TMB-8) substantially inhibited the induction of Bf. Collectively, these data support the following conclusions. First, that changes in [Ca2+]i comprise an important element in the induction of progression from the IFN-primed to the cytocidal state. Second, the failure to detect global changes in [Ca2+]i in response to the ligand, poly [I:C], suggests that changes in [Ca2+]i or Ca2+ movement may occur in either a spatially restricted or in an asynchronous cyclical fashion and are not detected by population fluorescence measurements. Third, the source of the relevant Ca2+ is extracellular. Fourth, our findings suggest that priming influences macrophage functional responses at a locus that is distal to the changes in [Ca2+]i, thereby potentially allowing signaling processes to be utilized to initiate different cellular responses.     

Aggregation-dependent turnover of flagellar adhesion molecules in chlamydomonas gametes

Previous studies on flagellar adhesion in chlamydomonas (Snell, W. and S. Roseman. 1979. J. Biol. Chem. 254:10820-10829.) have shown that as gametes adhere to flagella isolated from gametes of the opposite mating type, the adhsiveness of the added flagella but not of the gametes is lost. The studies reported here show that the addition of protein synthesis inhibitors (cycloheximide [CH] or anisomycin) to the medium of such cell- flagella mixtures causes the cells to lose their adhesiveness. This loss, however, occurs only after the cells have interacted with 4-8 flagella/cell and does not occur if the cells are kept in CH (7 h) without aggregating. The availability of an impotent (imp) mating type plus (MT(+)) mutant (provided by U.W. Goodenough), which adheres but is unable to undergo the fusion that normally follows adhesion, made it possible to determine whether a similar loss of adhesiveness occurs in mixtures of matting type minus (mt(-)) and imp mt(+) gametes. In the absence of inhibitor, mt(-) and imp mt(+) gametes adhered to each other (without fusing) for several hours; however, in the presence of CH or anisomycin, the gametes began to de-adhere 35 min after mixing, and, by 90 min, 100 percent of the cells were single again. This effect was reversible, and the rapid turnover of cells were single again. This effect was reversible, and the rapid turnover of molecules involved in adhesion occurred only during adhesion inasmuch as gametes pretreated for 4 h with CH were able to aggregate in CH for the same length of time as nonpretreated cells aggregated in CH. By the addition of CH at various times after the mt(-) and imp mt(+) gametes were mixed, measurements were made of the “pool size” of the molecules involved in adhesion. The pool reached a minimum after 25 min of aggregation, rapidly increased for the next 25 min, and then leveled off at the premixing level. These results suggest that flagellar adhesion in chlamydomonas causes modification of surface molecules (receptors, ligands), which brings about their inactivation and stimulates their replacement.     

Intertidal zonation of prosobranch gastropods: Analysis of densities of four co-existing species

The densities of populations of Nerita atramentosa Reeve, Austrocochlea constricta Lamarck, Bembiciuin nanum (Lamarck), and Cellana tramoserica (Sowerby) were recorded in 0.25 m² quadrats on landward and seaward halves of a sheltered and a moderately exposed rock platform from June 1972 to June 1973. The data have been subjected to variance and multiple regression analysis. Differences between densities of each species on the two shores and in the two areas of each shore are discussed with respect to the regressions on littoral height. Densities of Nerita on the sheltered shore, Austrocochlea and Bembicium on both shores, and Cellana, on the exposed shore, increased with height. The density of Cellana on the sheltered shore decreased as height increased. The density differences in the landward and seaward areas on the two shores were attributable to the effect of height for Nerita and Austrocochlea. Height was the major variable affecting the density of these two species on the sheltered shore. Bembicium and Cellana densities on both shores, and Austrocochlea density on the exposed shore, are not primarily determined by height.On the sheltered shore, Nerita and Cellana increased in density with increasing cover of water in rock pools. On the exposed shore, Bembicium and Cellana became sparser with increasing water cover. Bembicium on both shores increased in density with increasing cover of the alga Peyssonelia, as did Cellana on the exposed shore. Cellana and Austrocochlea tended to increase in density with Nerita on the sheltered shore. Cellana tended to be sparse where Bembicium was dense on the exposed shore. The biological effects of substrata and other species on the densities of each species remain unexplained.Significant time effects were found for variations in the densities of Nerita and Cellana on the sheltered shore and of Austrocochlea and Cellana on the exposed shore. The seasonal change in density of Nerita was parabolic with a minimum in summer, and is consistent with the known period of recruitment of this species. The lack of significant time effects on density of Austrocochlea on the sheltered shore is consistent with the known continuous recruitment of this species. The declining density of Austrocochlea on the moderately exposed shore was found, by size-frequency analysis, to be due to reduced recruitment compared with surrounding shores. The density of Cellana on both shores showed seasonal changes, with a maximum in summer, which is consistent with the known period of recruitment in Cellana. The lack of seasonal changes in density of Bembicium is briefly discussed.This type of multifactor analysis identifies biological interactions affecting variation in density of these species. It also corroborates the results of other ecological investigations and determines the relative importance of a variety of physical and biological factors in the distribution of density of intertidal gastropods.     

Genetic and biological characterisation of three cryptic Eimeria operational taxonomic units that infect chickens (Gallus gallus domesticus)

More than 68 billion chickens were produced globally in 2018, emphasising their major contribution to the production of protein for human consumption and the importance of their pathogens. Protozoan Eimeria spp. are the most economically significant parasites of chickens, incurring global costs of more than UK £10.4 billion per annum. Seven Eimeria spp. have long been recognised to infect chickens, with three additional cryptic operational taxonomic units (OTUs) first described more than 10 years ago. As the world’s farmers attempt to reduce reliance on routine use of antimicrobials in livestock production, replacing drugs that target a wide range of microbes with precise species- and sometimes strain-specific vaccines, the breakthrough of cryptic genetic types can pose serious problems. Consideration of biological characteristics including oocyst morphology, pathology caused during infection and pre-patent periods, combined with gene-coding sequences predicted from draft genome sequence assemblies, suggest that all three of these cryptic Eimeria OTUs possess sufficient genetic and biological diversity to be considered as new and distinct species. The ability of these OTUs to compromise chicken bodyweight gain and escape immunity induced by current commercially available anticoccidial vaccines indicates that they could pose a notable threat to chicken health, welfare, and productivity. We suggest the names Eimeria lata n. sp., Eimeria nagambie n. sp. and Eimeria zaria n. sp. for OTUs x, y and z, respectively, reflecting their appearance (x) or the origins of the first isolates of these novel species (y, z).     

A computational model of in vitro angiogenesis based on extracellular matrix fibre orientation

Recent interest in the process of vascularisation within the biomedical community has motivated numerous new research efforts focusing on the process of angiogenesis. Although the role of chemical factors during angiogenesis has been well documented, the role of mechanical factors, such as the interaction between angiogenic vessels and the extracellular matrix, remains poorly understood. In vitro methods for studying angiogenesis exist; however, measurements available using such techniques often suffer from limited spatial and temporal resolutions. For this reason, computational models have been extensively employed to investigate various aspects of angiogenesis. This paper outlines the formulation and validation of a simple and robust computational model developed to accurately simulate angiogenesis based on length, branching and orientation morphometrics collected from vascularised tissue constructs. Microvessels were represented as a series of connected line segments. The morphology of the vessels was determined by a linear combination of the collagen fibre orientation, the vessel density gradient and a random walk component. Excellent agreement was observed between computational and experimental morphometric data over time. Computational predictions of microvessel orientation within an anisotropic matrix correlated well with experimental data. The accuracy of this modelling approach makes it a valuable platform for investigating the role of mechanical interactions during angiogenesis.     

Holocephalan (Chondrichthyes) dental plates with hypermineralized dentine as a substitute for missing teeth through developmental plasticity

All extant holocephalans (Chimaeroidei) have lost the ability to make individual teeth, as tooth germs are not part of the embryonic development of the dental plates or of their continuous growth. Instead, a hypermineralized dentine with a unique mineral, whitlockin, is specifically distributed within a dentine framework into structures that give the dental plates their distinctive, species-specific morphology. Control of the regulation of this distribution must be cellular, with a dental epithelium initiating the first outer dentine, and via contact with ectomesenchymal tissue as the only embryonic cell type that can make dentine. Chimaeroids have three pairs of dental plates within their mouth, two in the upper jaw and one in the lower. In the genera Chimaera, Hydrolagus and Harriotta, the morphology and distribution of this whitlockin within each dental plate differs both between different plates in the same species and between species. Whitlockin structures include ovoids, rods and tritoral pads, with substantial developmental changes between these. For example, rods appear before the ovoids and result from a change in the surrounding trabecular dentine. In Harriotta, ovoids form separately from the tritoral pads, but also contribute to tritor development, while in Chimaera and Hydrolagus, tritoral pads develop from rods that later are perforated to accommodate the vasculature. Nevertheless, the position of these structures, secreted by the specialized odontoblasts (whitloblasts), appears highly regulated in all three species. These distinct morphologies are established at the aboral margin of the dental plate, with proposed involvement of the outer dentine. We observe that this outer layer forms into serially added lingual ridges, occurring on the anterior plate only. We propose that positional, structural specificity must be contained within the ectomesenchymal populations, as stem cells below the dental epithelium, and a coincidental occurrence of each lingual, serial ridge with the whitlockin structures that contribute to the wear-resistant oral surface.     

Biannual Spawning and Temporal Reproductive Isolation in Acropora Corals

Coral spawning on the oceanic reef systems of north-western Australia was recently discovered during autumn and spring, but the degree to which species and particularly colonies participated in one or both of these spawnings was unknown. At the largest of the oceanic reef systems, the participation by colonies in the two discrete spawning events was investigated over three years in 13 species of Acropora corals (n = 1,855 colonies). Seven species spawned during both seasons; five only in autumn and one only in spring. The majority of tagged colonies (n = 218) spawned once a year in the same season, but five colonies from three species spawned during spring and autumn during a single year. Reproductive seasonality was not influenced by spatial variation in habitat conditions, or by Symbiodinium partners in the biannual spawner Acropora tenuis. Colonies of A. tenuis spawning during different seasons separated into two distinct yet cryptic groups, in a bayesian clustering analysis based on multiple microsatellite markers. These groups were associated with a major genetic divergence (G”_ST = 0.469), despite evidence of mixed ancestry in a small proportion of individuals. Our results confirm that temporal reproductive isolation is a common feature of Acropora populations at Scott Reef and indicate that spawning season is a genetically determined trait in at least A. tenuis. This reproductive isolation may be punctuated occasionally by interbreeding between genetic groups following favourable environmental conditions, when autumn spawners undergo a second annual gametogenic cycle and spawn during spring.     

PMR5, an acetylation protein at the intersection of pectin biosynthesis and defense against fungal pathogens

Powdery mildew (Golovinomyces cichoracearum), one of the most prolific obligate biotrophic fungal pathogens worldwide, infects its host by penetrating the plant cell wall without activating the plant's innate immune system. The Arabidopsis mutant powdery mildew resistant 5 (pmr5) carries a mutation in a putative pectin acetyltransferase gene that confers enhanced resistance to powdery mildew. Here, we show that heterologously expressed PMR5 protein transfers acetyl groups from [¹⁴C]‐acetyl‐CoA to oligogalacturonides. Through site‐directed mutagenesis, we show that three amino acids within a highly conserved esterase domain in putative PMR5 orthologs are necessary for PMR5 function. A suppressor screen of mutagenized pmr5 seed selecting for increased powdery mildew susceptibility identified two previously characterized genes affecting the acetylation of plant cell wall polysaccharides, RWA2 and TBR. The rwa2 and tbr mutants also suppress powdery mildew disease resistance in pmr6, a mutant defective in a putative pectate lyase gene. Cell wall analysis of pmr5 and pmr6, and their rwa2 and tbr suppressor mutants, demonstrates minor shifts in cellulose and pectin composition. In direct contrast to their increased powdery mildew resistance, both pmr5 and pmr6 plants are highly susceptibile to multiple strains of the generalist necrotroph Botrytis cinerea, and have decreased camalexin production upon infection with B. cinerea. These results illustrate that cell wall composition is intimately connected to fungal disease resistance and outline a potential route for engineering powdery mildew resistance into susceptible crop species.