Extra- and Intracellular Mechanisms of Changes in Erythrocyte Aggregation

Reversible aggregation is a form of erythrocyte behavior. The presence of α- and β-adrenergic receptors on erythrocyte membranes suggests that aggregation can be influenced by agonists of these receptors. Human erythrocyte aggregation (EA) was studied in the presence of α- and β-agonists of adrenergic receptors (in the concentration range from 10⁻⁶ to 10⁻⁸ M). The α₂-agonist clonidine stimulated EA most strongly (by 163%, P < 0.01). The cell reaction decreased from the α-agonist clonidine to the β-agonist metaproterenol. The phosphodiesterase inhibitor papaverine and the penetrating cAMP analogue dibutyryl cAMP (dB-cAMP) increased the intracellular cAMP and decreased EA by 46–50% (P < 0.05). Clonidine-induced stimulation of EA sharply decreased upon erythrocyte incubation in the presence of clonidine + dB-cAMP and became even lower than in the control. Thus, α-agonists of adrenergic receptors markedly stimulated EA. The adenylate cyclase-cAMP system is likely involved as an intracellular signaling pathway.__________Translated from Fiziologiya Cheloveka, Vol. 31, No. 4, 2005, pp. 108–112.Original Russian Text Copyright © 2005 by A. V. Murav’ev, A. A. Murav’ev.     

一种同时提取水稻土壤中细胞外和细胞内形态DNA的方法

采用灭菌的水稻田土壤为基质,分别投加细菌基因组DNA和细菌活体,应用该方法提取细胞内和细胞外DNA。结果表明,DNA提取效率平均在75.4%-82.3%,DNA纯度OD260/280在1.75-1.85之间。用细菌16S rDNA通用引物PCR表明,DNA纯度能满足PCR要求,细胞内DNA与细胞外DNA互不污染。     

Harnessing Evolution to Elucidate the Consequences of Symbiosis

Many organisms harbor microbial associates that have profound impacts on host traits. The phenotypic effect of symbionts on their hosts may include changes in development, reproduction, longevity, and defense against natural enemies. Determining the consequences of associating with a microbial symbiont requires experimental comparison of hosts with and without symbionts. Then, determining the mechanism by which symbionts alter these phenotypes can involve genomic, genetic, and evolutionary approaches; however, many host-associated symbionts are not amenable to genetic approaches that require cultivation of the microbe outside the host. In the current issue of PLOS Biology, Chrostek and Teixeira highlight an elegant approach to studying functional mechanisms of symbiont-conferred traits. They used directed experimental evolution to select for strains of Wolbachia wMelPop (a bacterial symbiont of fruit flies) that differed in copy number of a region of the genome suspected to underlie virulence. Copy number evolved rapidly when under selection, and wMelPop strains with more copies of the region shortened the lives of their Drosophila hosts more than symbionts with fewer copies. Interestingly, the wMelPop strains with more copies also increase host resistance to viruses compared to symbionts with fewer copies. Their study highlights the power of exploiting alternative approaches when elucidating the functional impacts of symbiotic associations.Symbioses, long-term and physically close interactions between two or more species, are central to the ecology and evolution of many organisms. Though “Symbiosis” is more often used to define interactions that are presumed to be mutually beneficial to a host and its microbial partner, a broader definition including both parasitic and mutualistic interactions recognizes that the fitness effects of many symbioses are complex and often context dependent. Whether an association is beneficial can depend on ecological conditions, and mutation and other evolutionary processes can result in symbiont strains that differ in terms of costs and benefits to hosts (Fig. 1).Open in a separate windowFig 1The symbiosis spectrum.The costs and benefits of symbiosis for hosts are not bimodal but span a continuum. The benefit to cost ratio is mediated both by environmental conditions and by the strain of symbiont. For example, the bacteria Hamiltonella defensa increases aphid resistance to parasitoid wasps. When Hamiltonella loses an associated bacteriophage, protection is lost. Also, in aphids, Buchnera aphidicola is a bacterial symbiont that provisions its hosts with critical nutritional resources. However, alterations of the heat shock promoter in Buchnera lessen the fitness benefit of symbiosis for the hosts under elevated temperatures. Amplification of a region of the Wolbachia genome known as Octomom causes the bacteria to shorten the lifespan of its Drosophila fly hosts.Elucidating the effects of host-associated microbes includes, when possible, experiments designed to assay host phenotypes when they do and do not have a particular symbiont of interest (Fig. 2). In systems in which hosts acquire symbionts from the environment, hosts can be reared in sterile conditions to prevent acquisition [1]. If symbionts are passed internally from mother to offspring, antibiotic treatments can sometimes be utilized to obtain lineages of hosts without symbionts [2]. The impacts of symbiont presence on survival, development, reproduction, and defense can be quantified, with the caveat that these impacts may be quite different under alternative environmental conditions. While such experiments are sometimes more tractable in systems with simple microbial consortia, the same experimental processes can be utilized in systems with more complex microbial communities [3,4].Open in a separate windowFig 2Approaches to functionally characterize symbiont effects.The first step in functionally characterizing the phenotypic impacts of a symbiont on its host is to measure phenotypes of hosts with and without symbionts. Any effects need to be considered in the light of how they are modified by environmental conditions. Understanding the mechanisms underlying symbiont alteration of host phenotype can involve, and often combines, genomic, genetic, and evolutionary approaches. Solid arrows indicate the path leading to results highlighted in Chrostek and Teixeira’s investigation of Wolbachia virulence in this issue of PLoS Biology.Once a fitness effect of symbiosis is ascertained, determining the mechanistic basis of this effect can be challenging. A genomics approach sometimes provides informative insight into microbial function. Sequencing of many insect-associated symbionts, for example, has confirmed the presence of genes necessary for amino acid and vitamin synthesis [5–8]. These genomic revelations, in some cases, can be linked to phenotypic effects of symbiosis for the hosts. For example, aphids reared in the absence of their obligate symbiotic bacteria, Buchnera aphidicola, can survive when provisioned with supplemental amino acids but cannot survive without supplementation, suggesting that Buchnera’s provisioning of amino acids is critical for host survival [9,10]. The Buchnera genome contains many of the genes necessary for amino acid synthesis [5].Linking genotype to phenotype, however, can be complicated. Experiments are necessary to functionally test the insights garnered from genome sequencing. For example, just because a symbiont has genes necessary for synthesis of a particular nutrient does not mean that the nutrient is being provisioned to its host. Furthermore, in many systems we do not know what genetic mechanisms are most likely to influence a symbiont-conferred phenotype. For example, if hosts associated with a given microbe have lower fitness than those without the microbe, what mechanism mediates this phenotype? Is it producing a toxin? Is it using too many host resources? In these cases, a single genome provides even less insight.Comparative genomics can be another approach. This requires collection of hosts with alternative symbiont strains and then testing these strains in a common host background to demonstrate that they have different phenotypic effects. Symbiont genomes can then be sequenced and compared to identify differences. This approach was utilized to compare genomes of strains of the aphid bacterial symbiont Regiella insecticola that confer different levels of resistance to parasitoid wasps [11]; the protective and nonprotective Regiella genome differed in many respects. Comparing the genomes of Wolbachia strains with differential impacts on fly host fitness [12,13] revealed fewer differences, though none involved a gene with a function known to impact host fitness. Comparative genomics rarely uncovers a holy grail as the genomes of symbiont strains with alternative phenotypic effects rarely differ at a single locus of known function.Another approach, which is at the heart of studies of microbial pathogens, is to use genetic tools to manipulate symbionts at candidate loci (or randomly through mutagenesis) and compare the phenotypic effects of genetically-manipulated and unmanipulated symbionts. Indeed, this approach has provided insights into genes underlying traits of both pathogenic [14] and beneficial [15,16] microbes. There is one challenge. Many host-associated symbionts are not cultivable outside of their hosts, which precludes utilization of most traditional genetic techniques used to modify microbial genomes.An alternative approach to studying symbiont function leverages evolution. Occasionally, lineages that once conferred some phenotypic effect, when tested later, no longer do. If symbiont samples were saved along the way, researchers can then determine what in the genome changed. For example, pea aphids (Acyrthosiphon pisum) harboring the bacteria Hamiltonella defensa are more resistant to parasitoid wasps than those without the bacteria [17,18]. Toxin-encoding genes identified in the genome of a Hamiltonella-associated bacteriophage were hypothesized to be central to this defense [18,19]. However, confirmation of the bacteriophage’s role required comparing the insects’ resistance to wasps when they harbored the same Hamiltonella with and without the phage. No Hamiltonella isolates were found in nature without the phage, but bottleneck passaging of the insects and symbionts generation after generation in the laboratory led to the loss of phage in multiple host lineages. Experimental assays confirmed that in the absence of phage, there was no protection [20]. Similarly, laboratory passaging of aphids and symbionts serendipitously led to spread of a mutation in the genome of Buchnera aphidicola, the primary, amino acid-synthesizing symbiont of pea aphids. The mutation, a single nucleotide deletion in the promoter for ibpA, a gene encoding for a heat-shock protein, lowers aphid fitness under elevated temperature conditions [21]. The mutation is found at low levels in natural aphid populations, suggesting that laboratory conditions facilitate maintenance of the genotype.In the above cases, evolution was a fortunate coincidence. In this issue of PLoS Biology, Chrostek and Teixeira (2014) illustrate another alternative, directed experimental evolution. Previous work demonstrated that a strain of the symbiotic bacterium Wolbachia, wMelPop, is virulent to its Drosophila melanogaster hosts, considerably shortening lifespan while overproliferating inside the flies [22]. To investigate the mechanism of virulence, researchers compared the genomic content of an avirulent Wolbachia strain to that of the virulent wMelPop [12,13]. These comparisons revealed that the wMelPop genome contains a region with eight genes that is amplified multiple times; in avirulent strains there is only a single copy. This eight gene region was nicknamed “Octomom.” To functionally test whether Octomom mediates Wolbachia virulence, over successive generations, Chrostek and Teixeira selected for females with either high or low Octomom copy numbers to start the next generations. They found that copy number could evolve rapidly and was correlated with virulence. Flies harboring wMelPop with more copies of Octomom had shorter lifespans. This cost was reversed in the presence of natural enemies; flies harboring wMelPop with more copies of Octomom had higher resistance to viral pathogens. Thus, selection provided a functional link between genotype and phenotype in a symbiont recalcitrant to traditional microbial genetics approaches.In many respects, this is similar to the research on aphids and their symbionts, where protective phenotypes were lost through passaging of aphids and symbionts generation after generation, as part of standard laboratory maintenance. Chrostek and Teixeira simply used the tools of experimental evolution to select for altered symbionts in a controlled fashion. Comparison of the studies also highlights two potential approaches—select for a phenotype and determine the genotypic change, or select for a genotype of interest and determine the phenotypic effect.Why do we need to know the genetic mechanisms underlying symbiont-conferred traits? In terms of evolutionary dynamics, the maintenance of a symbiont’s effect in a population is predicated on the likelihood of it being maintained in the presence of mutation, drift, and selection. Symbiosis research often considers how ecological conditions influence symbiont-conferred traits but less often considers the instability of those influences due to evolutionary change. From the perspective of applied applications to human concerns, symbiont alteration of insect phenotypes are potential mechanisms to reduce vectoring of human and agricultural pathogens, either through directly reducing insect fitness or reducing the capacity of vectors to serve as pathogen reservoirs [23–28]. Short term field trials, for example, have demonstrated spread and persistence of Wolbachia in mosquito populations [29,30]. Because Wolbachia reduce persistence of viruses, including human pathogens, in insects [26,31–33], this is a promising pesticide-free and drug-free control strategy for insect-vectored diseases. Can we assume that Wolbachia and other symbionts will always confer the same phenotypes to their hosts? If the conferred phenotype is based on a region of the genome where mutation is likely (e.g., the homopolymeric track within the heat shock promoter of aphid Buchnera, the Octomom region in Drosophila wMelPop), then we have clear reason to suspect that the genotypic and phenotypic makeup of the symbiont population could change over time. We need to investigate how populations of bacterial symbionts evolve in host populations under natural ecological conditions, carefully screening for both changes in phenotype and changes in genotype over the course of such experimental observations. We then need to incorporate evolutionary changes when modeling symbiont maintenance and when considering the use of symbionts in applied applications.     

From the Cell to the Ecosystem: The Physiological Evolution of Symbiosis

Living organisms constantly interact with their habitats, selectively taking up compounds from their surroundings to meet their particular needs but also excreting metabolic products and thus modifying their environment. The small size, ubiquity, metabolic versatility, flexibility, and genetic plasticity (horizontal transfer) of microbes allow them to tolerate and quickly adapt to unfavorable and/or changing environmental conditions. The consumption of resources and the formation of metabolic products by spatially separated microbial populations constitute the driving forces that lead to chemical gradient formation. Communication and cooperation, both within and among bacterial species, have produced the properties that give these organisms a selective advantage. Observations of a wide range of natural habitats have established that bacteria do not function as individuals; rather, the vast majority of bacteria in natural and pathogenic ecosystems live in biofilms, defined as surface-associated, complex aggregates of bacterial communities that are attached to solid substrates and embedded in a polymer matrix of their own production. The spatial configurations of biofilms reach levels of complexity nearing those of multicellular eukaryotes. Microbial consortia have played important roles throughout the history of life on Earth, from the microbial mats (a type of biofilm) that were probably the first ecosystems in the early Archean, to the complex microbiota of the intestinal tract of different animals.     

The Evolution of Insect-Fungus Associations: From Contact to Stable Symbiosis

Insect-fungus interactions range from agonistic to mutualistic,and include several spectacular examples of complex symbioses.A potential benefit of mycophagy (the ingestion of fungal tissue)is the augmentation of digestive capacity by the ingestion offungal enzymes that remain active in the gut following ingestion.Cellulose digestion is mediated by ingested fungal enzymes inthe wood-boring larvae of cerambycid beetles and siricid woodwasps,in detritus-feeding stonefly nymphs, and in the workers of fungus-growingtermites. In this paper I discuss a plausible scenario for theevolution of stable symbiotic insect-fungus associations, inwhich the augmentation of digestive capacity through the ingestionof fungal enzymes is an important factor leading to the establishmentof interdependence between the interacting partners in a mutualism.Ingested fungal enzymes play a different role in the mutualisticassociation of the attine ants and their symbiotic fungi. Analyses of the associations of the siricid woodwasps, fungus-growingtermites, and fungus-growing ants with their symbiotic fungipermit the testing of Law's (1985) predictions concerning theconsequences of evolution in a mutualistic environment. As predicted,the rate of speciation has been slower in the protected partnerthan in the host partner, selection has favored asexual reproductionin the protected partner, and, at least in the attine ant-fungussymbiosis, the protected partner exhibits a low degree of specificitytoward different host species. Insect-fungus interactions provide rich material for the studyof both mechanistic and theoretical aspects of mutualism.     

Sulfur Species Investigation in Extra- and Intracellular Sulfur Globules of Acidithiobacillus ferrooxidans and Acidithiobacillus caldus

The sulfur chemical speciation in extracellular and intracellular sulfur globules of Acidithiobacillus ferrooxidans and Acidithiobacillus caldus were investigated with an integrated approach including scanning electron microscopy, transmission electron microscopy, energy dispersive spectroscopy and sulfur K-edge X-ray absorption near edge structure spectroscopy (XANES). The results indicated that both strains can accumulate extracellular sulfur globules when grown on thiosulfate, and the major sulfur chemical speciation of which were S₈ for A. ferrooxidans and mixture of ring sulfur and polythionate for A. caldus, respectively. In contrast, A. ferrooxidans can accumulate both linear sulfur and S₈ internally when grown with sulfur powder and thiosulfate, whereas A. caldus did not accumulate intracellular sulfur globules. In addition, the fitted results of sulfur K-edge XANES spectra indicated that the reduced glutathione (containing thiols groups) were involved in sulfur bio-oxidation of both strains and the tetrathionate were the intermediate products during thiosulfate metabolism by two strains.     

Molecular Evolution of Mollusc Shell Proteins: Insights from Proteomic Analysis of the Edible Mussel Mytilus

Shell matrix proteins (SMPs) that are embedded within calcified layers of mollusc shells are believed to play an essential role in controlling the biomineral synthesis and in increasing its mechanical properties. Among the wide diversity of mollusc shell textures, nacro-prismatic shells represent a tremendous opportunity for the investigation of the SMP evolution. Indeed, nacro-prismatic texture appears early in Cambrian molluscs and is still present in the shell of some bivalves, gastropods, cephalopods and very likely also, of some monoplacophorans. One key question is to know whether these shells are constructed from similar matrix protein assemblages, i.e. whether they share a common origin. Most of the molecular data published so far are restricted to two genera, the bivalve Pinctada and the gastropod Haliotis. The shell protein content of these two genera are clearly different, suggesting independent origins or considerable genetic drift from a common ancestor. In order to describe putatively conserved mollusc shell proteins, here we have investigated the SMP set of a new bivalve model belonging to another genera, the edible mussel Mytilus, using an up-to-date proteomic approach based on the interrogation of more than 70,000 EST sequences, recently available from NCBI public databases. We describe nine novel SMPs, among which three are completely novel, four are homologues of Pinctada SMPs and two are very likely homologues of Haliotis SMPs. This latter result constitutes the first report of conserved SMPs between bivalves and gastropods. More generally, our data suggest that mollusc SMP set may follow a mosaic pattern within the different mollusc models (Mytilus, Pinctada, Haliotis). We discuss the function of such proteins in calcifying matrices, the molecular evolution of SMP genes and the origin of mollusc nacro-prismatic SMPs.     

A Macrophage Subversion Factor Is Shared by Intracellular and Extracellular Pathogens

Pathogenic bacteria have developed strategies to adapt to host environment and resist host immune response. Several intracellular bacterial pathogens, including Salmonella enterica and Mycobacterium tuberculosis, share the horizontally-acquired MgtC virulence factor that is important for multiplication inside macrophages. MgtC is also found in pathogenic Pseudomonas species. Here we investigate for the first time the role of MgtC in the virulence of an extracellular pathogen, Pseudomonas aeruginosa. A P. aeruginosa mgtC mutant is attenuated in the systemic infection model of zebrafish embryos, and strikingly, the attenuated phenotype is dependent on the presence of macrophages. In ex vivo experiments, the P. aeruginosa mgtC mutant is more sensitive to macrophage killing than the wild-type strain. However, wild-type and mutant strains behave similarly toward macrophage killing when macrophages are treated with an inhibitor of the vacuolar proton ATPase. Importantly, P. aeruginosa mgtC gene expression is strongly induced within macrophages and phagosome acidification contributes to an optimal expression of the gene. Thus, our results support the implication of a macrophage intracellular stage during P. aeruginosa acute infection and suggest that Pseudomonas MgtC requires phagosome acidification to play its intracellular role. Moreover, we demonstrate that P. aeruginosa MgtC is required for optimal growth in Mg²⁺ deprived medium, a property shared by MgtC factors from intracellular pathogens and, under Mg²⁺ limitation, P. aeruginosa MgtC prevents biofilm formation. We propose that MgtC shares a similar function in intracellular and extracellular pathogens, which contributes to macrophage resistance and fine-tune adaptation to host immune response in relation to the different bacterial lifestyles. In addition, the phenotypes observed with the mgtC mutant in infection models can be mimicked in wild-type P. aeruginosa strain by producing a MgtC antagonistic peptide, thus highlighting MgtC as a promising new target for anti-virulence strategies.     

VA菌根共生的起源和进化

ＶＡ菌根共生的起源和进化赵之伟（云南大学生物学系,昆明６５００９１）ＯｒｉｇｉｎａｎｄＥｖｏｌｕｔｉｏｎｏｆｔｈｅＶＡＭｙｃｏｒｒｈｉｚａｌＳｙｍｂｉｏｓｉｓ．ＺｈａｏＺｈｉｗｅｉ（ＢｉｏｌｏｇｙＤｅｐａｒｔｍｅｎｔｏｆＹｕｎｎａｎＵｎｉｖｅｒｓｉｔ...     

Glycosylation Directs Targeting and Activation of Cystatin F from Intracellular and Extracellular Sources

Cystatin F is a cysteine protease inhibitor that is selectively expressed in immune cells and unlike other cystatin family members is targeted to a significant extent to intracellular compartments. Initially made as an inactive glycosylated disulfide-linked dimer, cystatin F is converted to an active monomer by proteolytic cleavage following transport to the endosomal/lysosomal system. This active form of cystatin F targets cathepsin C/DPPI and probably other cathepsins in immune cells. We show that efficient targeting of cystatin F to the endocytic pathway is dependent not on its unique dimeric conformation but rather on its oligosaccharide chains. We demonstrate the unusual addition of N -linked sugars to an Asn-X-Cys motif in cystatin F and provide evidence that the mannose 6-phosphate sorting machinery is used to divert cystatin F from the secretory pathway and to mediate its uptake from extracellular pools. These studies identify a function for the oligosaccharides on cystatin F and raise the possibility that cystatin F might regulate proteases in trans by secretion in an inactive form by one cell and subsequent internalization and activation by another cell.     

Comparison of the Lipids of Intracellular and Extracellular Rabies Viruses

The lipid composition of both intracellular and extracellular forms of the ERA strain of rabies virus grown in BHK/21 cells was determined. The lipids from purified preparations of both intracellular and extracellular virus yielded 57 and 58% neutral lipid, respectively. The phospholipids of the intracellular and extracellular virus constituted 43 and 42%, respectively. Triglyceride and cholesterol appear to be the major neutral lipids, whereas sphingomyelin, phosphatidylethanolamine, and phosphatidylcholine comprise the major bulk of phospholipid in both virus types. The molar ratio of cholesterol to phospholipid was 0.87 (intracellular) and 0.92 (extracellular). On the basis of the data presented, it is reasonable to assume that the lipids of both intracellular and extracellular rabies virus are similar.     

Extracellular Calcium Sensing by Glial Cells: Low Extracellular Calcium Induces Intracellular Calcium Release and Intercellular Signaling

Abstract: Glial cells in primary mixed cultures or purified astrocyte cultures from mouse cortex respond to reduced extracellular calcium concentration ([Ca²⁺]_e) with increases in intracellular calcium concentration ([Ca²⁺]_i) that include single-cell Ca²⁺ oscillations and propagated intercellular Ca²⁺ waves. The rate and pattern of propagation of low [Ca²⁺]_e-induced intercellular Ca²⁺ waves are altered by rapid perfusion of the extracellular medium, suggesting the involvement of an extracellular messenger in Ca²⁺ wave propagation. The low [Ca²⁺]_e-induced Ca²⁺ response is abolished by thapsigargin and by the phospholipase antagonist U73122. The low [Ca²⁺]_e-induced response is also blocked by replacement of extracellular Ca²⁺ with Ba²⁺, Zn²⁺, or Ni²⁺, and by 100 µM La³⁺. Glial cells in lowered [Ca²⁺]_e(0.1–0.5 mM) show an increased [Ca²⁺]_i response to bath application of ATP, whereas glial cells in increased [Ca²⁺]_e (10–15 mM) show a decreased [Ca²⁺]_i response to ATP. These results show that glial cells possess a mechanism for coupling between [Ca²⁺]_e and the release of Ca²⁺ from intracellular stores. This mechanism may be involved in glial responses to the extracellular environment and may be important in pathological conditions associated with low extracellular Ca²⁺ such as seizures or ischemia.     

Extracellular Matrix Stiffness and Architecture Govern Intracellular Rheology in Cancer

Little is known about the complex interplay between the extracellular mechanical environment and the mechanical properties that characterize the dynamic intracellular environment. To elucidate this relationship in cancer, we probe the intracellular environment using particle-tracking microrheology. In three-dimensional (3D) matrices, intracellular effective creep compliance of prostate cancer cells is shown to increase with increasing extracellular matrix (ECM) stiffness, whereas modulating ECM stiffness does not significantly affect the intracellular mechanical state when cells are attached to two-dimensional (2D) matrices. Switching from 2D to 3D matrices induces an order-of-magnitude shift in intracellular effective creep compliance and apparent elastic modulus. However, for a given matrix stiffness, partial blocking of β1 integrins mitigates the shift in intracellular mechanical state that is invoked by switching from a 2D to 3D matrix architecture. This finding suggests that the increased cell-matrix engagement inherent to a 3D matrix architecture may contribute to differences observed in viscoelastic properties between cells attached to 2D matrices and cells embedded within 3D matrices. In total, our observations show that ECM stiffness and architecture can strongly influence the intracellular mechanical state of cancer cells.     

Extracellular and Intracellular Correlates of Organ Initiation in the Embryonic Chick Thyroid

Apical and basal bundles of microfilaments have been suggestedas intracellular structures which might cause cellular contractionsleading to bending movements during organogenesis. Microfilamentbundles may have a different role in some organ primordia. Theembryonic chick thyroid develops as an evagination of the pharyngealfloor at the level of the anterior attachment of the heart.The developing thyroid is confined to a pocket surrounded bythe developing aortic arches, ventral aorta, and truncus arteriosus.Initiation of thyroid evagination is accompanied by the formationof grooves as two concentric rings in the basal surface of theplacode near its margins. The cells surrounding the groovescontain longitudinally oriented bundles of microfilaments andmicrotubules. Other microfilament bundles are present at thecell apices and bases. The primordium almost doubles in volumeduring the period of evagination, but the area of pharyngealfloor that it occupies undergoes virtually no increase. A modelis presented which describes the role of microfilament bundlesduring thyroid evagination as stabilizing cellular dimensions.The bending movements are thought to result from cell elongation,increase in cell numbers, and confinement of the thyroid toa limited space by the developing truncus arteriosus and itsbranches.     

Cytological damage to the red alga Griffithsia pacifica from ultraviolet radiation

Continuous exposure for 7–10 days to 60% of ambient levels (sea level at mid-day in December) of UV-A and UV-B radiation caused cytological damage to regenerating fragments of Griffithsia pacifica under laboratory conditions. There was high mortality of individual cells and entire fragments in UV treated filaments. Rhizoid initiation was slower and rhizoids grew more slowly following UV treatment. After 7 days, UV radiated thalli showed chloroplast and nuclear degeneration. In addition, filaments tended to disarticulate so that single or groups of apparently healthy cells were common in the medium. These data suggest that the subtidal habitat of G. pacifica is based in part on lack of tolerance to UV radiation, and that UV protection mechanisms are not inducible or insufficient to prevent the accumulation of damage in this species.     

Extracellular and Intracellular Polyphenol Oxidases Cause Opposite Effects on Sensitivity of Streptomyces to Phenolics: A Case of Double-Edged Sword

Many but not all species of Streptomyces species harbour a bicistronic melC operon, in which melC2 encodes an extracellular tyrosinase (a polyphenol oxidase) and melC1 encodes a helper protein. On the other hand, a melC-homologous operon (melD) is present in all sequenced Streptomyces chromosomes and could be isolated by PCR from six other species tested. Bioinformatic analysis showed that melC and melD have divergently evolved toward different functions. MelD2, unlike tyrosinase (MelC2), is not secreted, and has a narrower substrate spectrum. Deletion of melD caused an increased sensitivity to several phenolics that are substrates of MelD2. Intracellularly, MelD2 presumably oxidizes the phenolics, thus bypassing spontaneous copper-dependent oxidation that generates DNA-damaging reactive oxygen species. Surprisingly, melC⁺ strains were more sensitive rather than less sensitive to phenolics than melC ⁻ strains. This appeared to be due to conversion of the phenolics by MelC2 to more hydrophobic and membrane-permeable quinones. We propose that the conserved melD operon is involved in defense against phenolics produced by plants, and the sporadically present melC operon probably plays an aggressive role in converting the phenolics to the more permeable quinones, thus fending off less tolerant competing microbes (lacking melD) in the phenolic-rich rhizosphere.     

Mixotrophic in vitro cultivations: the way to go astray in plant physiology

Rate of photosynthesis and related plant carbohydrate status are crucial factors affecting plant vigor. Sugars providing carbon and energy sources serve also as important signaling molecules governing plant growth and development through a complex regulatory network. These facts are often neglected when mixotrophic cultivation of plants in vitro is used, where artificial exogenous sugar supply hinders studies of metabolism as well as sugar‐driven developmental processes. We compared the growth, selected gas‐exchange parameters and sugar metabolism characteristics in four model plants, potato (Solanum tuberosum ‘Lada’), tobacco (Nicotiana tabacum ‘Samsun’), rapeseed (Brassica napus ‘Asgard’) and strawberry (Fragaria vesca), under both photomixotrophic (PM) and photoautotrophic (PA) conditions. To ensure PA conditions, we used our improved sun caps that serve as gas and light permeable covers for cultivation vessels. We found bigger biomass accumulation, larger leaf areas, higher stomatal conductance and higher instantaneous water use efficiency and lower root sugar contents in PA plants compared to PM ones. However, for other characteristics (root biomass, root/shoot ratio, pigment contents, leaf sugar and starch levels and transpiration rates), a strong species‐dependent reactions to the exogenous sugar supply was noted, which does not allow to create a general view on the overall impact of PM nutrition under in vitro conditions.