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It is known that moving volutin granules (“dancing bodies”), mechanism of which occurrence remains poorly understood, can be observed in yeast vacuoles. This study was performed to reveal the presence of a connection between moving volutin granules of Saccharomyces cerevisiae and polyphosphate metabolism in conditions of phosphoric starvation and hypercompensation. Methods. Cytological, biochemical, statistical methods were used in the study. Results. It was observed that the inactivation of the PPN1 gene, which encodes exopolyphosphatase Ppn1, resulted in a change in the number of cells with moving volutin granules (“dancing bodies” index) in the studied conditions. The index of “dancing bodies” was almost always lower in mutant CRN strain than in parent CRY strain. Using linear correlation analysis and factor analysis with the method of principal component, it was established that the “dancing bodies” index in both strains had significant correlation coefficients with exopolyphosphatase activity (EPPA) and the content of polyphosphate fractions (polyP). The difference was that this index in parent strain correlated better with the first three fractions of inorganic polyphosphates, while in mutant strain – with polyP4 and EPPA. Conclusions. Obtained data indicated the direct connection of motion of volutin granules with phosphoric metabolism in the studied conditions. It is assumed that the phenomenon of “dancing bodies” may be a consequence of the activity of vacuolar polyphosphatases.

The research is devoted to the development of color imaging information technology, which is relevant for evaluating the results of cytochemical research in both biology and medicine. The aim of this work was to study the validity of software algorithms and integrated information technology for the qualitative and quantitative analysis of metachromasia reaction of volutin granules of Saccharomyces cerevisiae UCM Y-517 yeast. Methods. The object of this study was Saccharomyces cerevisiae UCM Y-517 yeast from the Ukrainian collection of microorganisms. The yeast was cultivated for 24 hours at 28oC. The metachromatic reaction of volutin granules was detected microscopically by the preparation of yeast cells stained with methylene blue. Digital images of cells were transferred to a specialized web resource for computer processing. Results. The algorithm of computer analysis of images included 3 steps: (1) recognition of single volutin granules, (2) assessment of the magnitude of metachromasia for each granule, and (3) integral estimation of the magnitude of metachromasia for the whole image. Information technology was developed and tested to support the daily monitoring of the metachromasia phenomenon, including obtaining the digital images, their transfer to a remote server, and automatic processing. Information technology is integrated into the web portal of the large-scale biophysical experiment “Heliomed”. Conclusions. The proposed method of computer image processing gave a quantitative and qualitative assessment in the automatic mode of metachromatic staining of volutin granules and confirmed the previously proposed classification of this phenomenon on the basis of visual examination.

Summary The rate of polyphosphate accumulation in extraradical hyphae of an arbuscular mycorrhizal fungus was investigated by conventional histochemistry and a new enzymatic method using a bacterial enzyme, polyphosphate kinase. Marigold ( Tagetes patula cv. Bonanza Orange) was inoculated with Archaeospora leptoticha and grown under P-deficient conditions. Extraradical hyphae were harvested at 0, 1, 3 and 24 h after 1 m M P-application. PolyP levels were assessed by both metachromasy of Toluidine blue O and polyphosphate kinase which converted polyP to ATP followed by the ATP-luciferase assay. Percentage of hyphae with metachromatic granules was increased from 25 to 44% from 0 to 1 h, and a maximum of 50% was reach by 3 h. Polyphosphate content was doubled from 1 to 3 h after P-application (4.8‐10.0 µmol as Pi mg − 1 protein) at a rate of 46.4 ± 15.1 nmol min − 1 mg − 1 . The rate of polyphosphate accumulation in the hyphae was surprisingly rapid as those of polyphosphate-hyper accumulating microorganisms. The enzymatic method employed in the present study allows highly specific and sensitive assessment of polyphosphate in the mycorrhizal system.

Polyphosphate (PolyP), formerly referred to as volutin granules, is ubiquitous with a diverse range of functions. PolyP is present in substantial amounts in prokaryotes and has extensively been researched for various enzymes and pathways involved in its synthesis and degradation. Both the formation and degradation of PolyP are enzyme-mediated processes. Microorganisms utilize PolyP molecules not only as a source of energy and phosphate but also for various other diverse functions including motility, virulence, and gene expression. The present chapter focuses on the biogenesis of PolyP and its application in various fields. The application broadly varies from environmental bioremediation (as heavy metal and enhanced biological phosphate removal) to industrial and biotechnological values (as food additives, detergent additives, and enzyme technology). PolyPs also have applicability in the field of medicine for therapeutic drug design and drug delivery. This chapter later discusses numerous challenges toward the progress of PolyP formation and also the strategies to improve the yield. Metabolic strategies, classical accumulation methods, and lack of biotechnological advancements are considered to be a few of the constraints for PolyP production. To cope with these, many scientists have suggested different ideas such as acidic pH, osmotic stress, nutrient limitation, phosphate and amino acid starvation, presence of organic pollutants, and high-energy state in terms of adenosine triphosphate.

As a result of electron microscopic studies of morphogenesis in yeast Candida guilliermondii NP-4, the formation of new structures of volutin acidocalcisomes has been established within the cell cytoplasm. Under influence of X-irradiation, the changes in morphometric and electron-dense properties of yeast cells were identified: in yeast cytoplasm, the electron-dense volutin granules were increased up to 400 nm in size. After 24-h post-irradiation incubation of yeasts, the large volutin pellets are fragmented into smaller number particles in size up to 25-150 nm. The ATPase activity in yeast mitochondria was changed under X-irradiation. In latent phase of growth, ATPase activity was decreased 1·35-fold in comparison with non-irradiated yeasts. In logarithmic phase of growth, ATPase activity was three times higher than in latent phase, and in stationary phase of growth it has a value similar to the latent phase. Probably, the cells receive the necessary energy from alternative energy sources, such as volutin. Electron microscopy of volutin granule changes might serve as convenient method for evaluation of damages and repair processes in cells under influence of different environmental stress-factors.

Mast cell tumours (MCTs) are common in dogs and cats, but are only rarely reported in reptiles. This case documents the clinical and pathomorphological results from a Boa imperator with multiple cutaneous nodules, diagnosed as MCT based on histopathology, immunohistochemistry and electron microscopy approaches. Grossly, there were multifocal, poorly demarcated, mostly ulcerated nodules ~3 cm in diameter on the skin. Histologically, the dermis and the subcutaneous tissue were infiltrated by round cell populations with eosinophilic granules. Toluidine blue and Giemsa stain revealed metachromatic granules. Using immunohistochemistry, some cells exhibited cytoplasmic immunostaining positive for tryptase. Ultrastructurally, variable quantities of intracytoplasmic, spherical and electron-dense granules were also detected. The MCT literature on snakes is scarce, especially for the family Boidae, but MCTs should be considered a differential diagnosis for nodular skin lesions in reptiles.

The moving volutin (polyphosphate) granules known as "dancing bodies" can be observed in the vacuoles of the yeast cells. The aim of work was to study the effects of cultivation conditions and influences of physico-chemical factors on the motion of vacuolar volutin granules in Saccharomyces cerevisiae cells. The motion of granules is a non-Markovian process. It does not depend on the cell cycle phase, but depends on the growth stage. The maximal number of cells with "dancing bodies" was observed under cultivation of yeast at 25-28 °C and pH 5.4-5.8. Irradiation by non-ionizing electromagnetic radiation (EMR) of extremely high frequency (61.22 GHz, 100 μW, 30 min) had no effect on granule motion. After irradiation by non-ionizing EMR of very high frequency (40.68 MHz, 30 W, 30 min) the number of cells with "dancing bodies" decreased significantly and in 2 h restored almost to the control value. The possible nature of the moving volutin granules phenomenon due to metabolic processes is discussed.

Selenium (Se) respiration in bacteria was revealed for the first time at the end of 1980s. Although thermodynamically-favorable, energy-dense and documented in phylogenetically-diverse bacteria, this metabolic process appears to be accompanied by a number of challenges and numerous unanswered questions. Selenium oxyanions, SeO42− and SeO32−, are reduced to elemental Se (Se0) through anaerobic respiration, the end product being solid and displaying a considerable size (up to 500 nm) at the bacterial scale. Compared to other electron acceptors used in anaerobic respiration (e.g. N, S, Fe, Mn, and As), Se is one of the few elements whose end product is solid. Furthermore, unlike other known bacterial intracellular accumulations such as volutin (inorganic polyphosphate), S0, glycogen or magnetite, Se0 has not been shown to play a nutritional or ecological role for its host. In the context of anaerobic respiration of Se oxyanions, biogenic Se0 appears to be a by-product, a waste that needs proper handling, and this raises the question of the evolutionary implications of this process. Why would bacteria use a respiratory substrate that is useful, in the first place, and then highly detrimental? Interestingly, in certain artificial ecosystems (e.g. upflow bioreactors) Se0 might help bacterial cells to increase their density and buoyancy and thus avoid biomass wash-out, ensuring survival. This review article provides an in-depth analysis of selenium respiration (model selenium respiring bacteria, thermodynamics, respiratory enzymes, and genetic determinants), complemented by an extensive discussion about the evolutionary implications and the properties of biogenic Se0 using published and original/unpublished results.

In this work, the impact of space weather on the staining and the motility of volutin granules ("dancing bodies") in vacuoles of yeast Saccharoniyces cerevisiae with normal and changed phosphoric metabolism was studied. It was showed that the installed ear- lier relation between metachromatic reaction of volutin granules and galactic cosmic rays was confirmed by DAPI-staining of inorganic polyphosphates in cells S. cerevisiae UCM Y-517. The inverse correlation of efficiency of DAPI-staining of volutin granules with some solar activity indexes was observed. During the period of study, the rhythmicity of motile volutin granules in the studied cultures (S. cerevisiae UCM Y-517, S. cerevisiae CRY, S. cerevisiae CNX) was marked. The strains S. cerevisiae UCM Y-517 and CRY, both with unmodified phosphoric metabolism, showed the same rhythmicity of mobile volutin granules. However, this rhythmicity in the strain CNX, which cannot synthesize exopolyphosphatases PPX1 and PPN1 (CF 3.6.1.11), was changed. The volutin granule motion coincided with the rhythmicity of galactic cosmic rays within the period of 9 days. It can be suggested that "dancing bodies" are synchronized with rhythms of galactic cosmic rays. Observed differences between mutant and parental strains may indirectly indicate participation of polyphosphates in the reaction of cells on the changes of space weather pa- rameters. Thus, correlating variability of staining and motion of polyphosphate-containing volutin granules under action of cosmophysical factors may be an evidence of possible key role of phosphoric metabolism in sensitivity of yeast cells to space weather changes.

Physiological, biochemical, cyto-morphological properties of initial strain Candida albicans ATCC 10331, Saccharomyces sp. КНУ 1 and their variants, which were cultivated in medium Saburo with anionic surfactant (SAS) sodium dodecyl sulfate (SDS) have been compared. It was shown that the effect of SDS was accompanied by disappearance of possibility to assimilate a number of substrates, but did not affect antibiotics sensitivity. They changed its morphology after 1 passage, diminution quantity of volutin, appearance of fat inclusion. Morphology of cells partially restored after 4-th passage.

We describe a pulmonary mast cell tumor in a subsistence-harvested free-ranging Pacific walrus (Odobenus rosmarus divergens). Neoplastic cells effacing a focal area of pulmonary parenchyma were characterized by rare metachromatic granules and positive staining for C-kit. We also report co-occurrence of a peribronchial mass with a morphologic and immunohistochemical profile compatible with paraganglioma.

No previous records in the available literature about the cutaneous mast cell tumors in golden hamster. Two male golden hamsters with cutaneous lumps were brought to the Institute of Veterinary Pathology of Justus Leibig University of Giessen, Germany for diagnositic examination. Cutaneous lumps were taken and processed for histological, histochemical and immunohistochemical examination. Numerous oval and polygonal tumor cells with high mitotic count and basophilic metachromatic granules were observed; while the superjacent epidermis and adnexes were normal. Many unevenly proliferating cells showed positive proliferating cell nuclear antigen (PCNA) reaction and positive chymase activity. The reactions were negative to gene encoding KIT (c-Kit), Cyclooxygenase-2 (COX-2) and tryptase. These results indicated a well- differentiated grade I subcutaneous mast cell tumor. The results are discussed in relation to subcutaneous mast cell tumors in canines.

OBJECTIVE The aim of this study was to provide a description of a new haemogregarine, Haemogregarina sundarbanensis n. sp., from mud turtles collected from Sundarban regions, West Bengal, India. METHODS The turtles were collected from fish markets near Canning, South 24-Parganas District, West Bengal. Thin blood films were prepared using the blood drawn from the head and feet. The blood films were then air-dried, fixed in absolute methanol, and stained with Giemsa. RESULTS The erythrocytic stages could be differentiated into small and large forms. The young gamonts measured 7.896×3.469 μm, microgamonts measured 8.876×4.425 μm, and macrogamonts measured 8.919×4.272 μm. The cytoplasm of the macrogamonts was stained deep blue with Giemsa, and many metachromatic granules are found uniformly distributed. The oval or rounded nucleus was central in position and stained deep red with Giemsa stain, whereas in microgamonts, the nucleus was not at all compact but with a few dispersed chromatin granules. CONCLUSION The prevalence of infection was found to be 33.3% (5/15). The infected erythrocytes reveal shape alteration, marginal and atrophic nucleus, and were larger than non-parasitized erythrocytes. The cytoplasm of the infected erythrocytes stained darker than that of the uninfected erythrocytes.

Cyanobacteria synthesize a variety of chemically-different, high-value biopolymers such as glycogen (polyglucose), poly-β-hydroxybutyrate (PHB), cyanophycin (polyamide of arginine and aspartic acid) and volutin (polyphosphate) under excess conditions. Especially under unbalanced C to N ratios, glycogen and in some cyanobacterial genera also PHB are massively accumulated in the progression of the general nitrogen stress response. Several different technologies have been established for in situ and in vitro PHB analysis from different microbial sources. In this protocol, a rapid and reliable spectrophotometric method is described for PHB quantification in the cyanobacterium Synechocystis sp. PCC 6803 upon nitrogen deprivation as described in ( Damrow et al., 2016 ).

The budding yeast Saccharomyces cerevisiae serves as an effective model organism for many cellular pathways including phosphate transport, accumulation, and storage. In S. cerevisiae, phosphate is actively transported across the plasma membrane via several phosphate carriers and is then transported into the acidic vacuole (roughly equivalent to the mammalian lysosome with degradative functions but with additional storage functions, such as calcium) where it is synthesized into volutin, a storage form of polyphosphate, found in many organisms. We have been studying volutin granule formation in wild type cells to determine the physiological requirements for formation and in mutants to determine the pathway by which the volutin biosynthetic proteins are transported to the vacuole. Undertaking an analysis of volutin formation in yeast vacuoles by blocking vacuole function with pharmacological agents, such as ionomycin and CCCP, we see that vacuole pH as well as vacuolar calcium seems critical for volutin formation. Different blocks in vacuolar protein sorting have differential effects on volutin granule accumulation, with volutin granule formation seen in all mutant strains thus far tested, except for vps33, a mutant cell strain lacking all vacuolar structure. Our data are consistent with pleiotrophic effects of vacuolar physiological function blocks leading to a decrease in volutin formation.

Raised lesions were present on the left nasal vestibule of a 20-month-old Japanese Brown heifer. The largest mass which caused partial nasal obstruction was removed surgically. Corynebacterium ulcerans was identified in the mass. 16S ribosomal RNA and RNA polymerase beta subunit genes were 100% and 98% identical to other C. ulcerans strains. Histologically, multiple foci of eosinophilic granuloma with Splendore-Hoeppli material were seen. Rod-shaped Gram-positive organisms were detected with metachromatic granules, producing diphtheria toxin with 5, 30 and 48 amino acid differences to another C. ulcerans strain, C. diphtheriae or C. pseudotuberculosis, respectively. The toxin is highly cytotoxic and may be responsible for the formation of abundant Splendore-Hoeppli material. The lesion was therefore judged to be an allergic reaction to bacterial antigens or diphtheria toxin.

Polyphosphate (polyP) is a biomolecule comprised of repeating units of inorganic phosphate residues linked by energy-rich phosphoanhydride bonds. PolyP is highly conserved throughout evolution and plays a vital role in the survival of prokaryotes and single-cell eukaryotes1 by functioning in processes such as motility, virulence and stress. Its role in mammalian systems was less well defined, however, identification of polyP in the dense granules of human platelets2 prompted analysis of the function of polyP in hemostasis. Stimulation of platelets by conventional agonists such as ADP, thrombin and collagen induces release of polyP, and promotes coagulation by acting at several points in the cascade3. These include 1) providing a surface for activation of the contact pathway protein factor XII, 2) augmenting conversion of factor V to factor Va which participates in the prothrombinase complex and 3) accelerating thrombin-mediated activation of factor XI4. The net outcome is a reduction in the lag time to thrombin formation thereby driving clot formation. The effect of polyP on thrombin generation has down-stream repercussions in terms of clot breakdown, as it augments the early activation of thrombin activatable fibrinolysis inhibitor (TAFI). There has been a degree of uncertainty and debate over whether polyP is capable of reaching adequate concentrations within the plasma milieu to elicit these profound effects on haemostasis. However, the observations that polyP can bind directly to fibrin5, allowing it to be incorporated into the forming clot, localise the biomolecule to its site of action. In addition, binding alters the structure of the fibrin clot altering its susceptibility to degradation by fibrinolytic enzymes6. These observations led to the hypothesis that polyP operates as a timed switch, being released from platelets upon activation thereby allowing rapid repair of an injury while simultaneously preventing clot breakdown until sufficient wound healing has occurred3. The ability of polyP to promote FXII-dependent coagulation has been reported in several studies but has been contested, as shorter soluble polymers, such as those found in platelets, are relatively inefficient in this process. Recent work from our group7 and others8 documents the retention of polyP on the surface of degranulated platelets. This pool of polyP is of longer chain length than the soluble secreted form and is complexed to divalent metal ions8 thereby augmenting its ability to stimulate factor XII activation. These studies highlight the potential that polyP may exert its biological cofactor activity when anchored to a surface such as platelets, fibrin and potentially other blood cells. Many questions remain as to how this polymer regulates its plethora of functions and whether the binding surface impacts on the target reaction and the half-life of the polymer. However, taken together these in vitro and in vivo studies have delineated a role for polyP in hemostasis and highlight the requirement for more efficient tools to quantify this interesting biomolecule in subsets of patients. References Kornberg A. Inorganic polyphosphate: a molecule of many functions. Prog Mol Subcell Biol. 1999;23:1-18. Ruiz FA, Lea CR, Oldfield E, Docampo R. Human platelet dense granules contain polyphosphate and are similar to acidocalcisomes of bacteria and unicellular eukaryotes. J Biol Chem. 2004;279(43):44250-44257. Smith SA, Mutch NJ, Baskar D, Rohloff P, Docampo R, Morrissey JH. Polyphosphate modulates blood coagulation and fibrinolysis. Proc Natl Acad Sci U S A. 2006;103(4):903-908. Choi SH, Smith SA, Morrissey JH. Polyphosphate is a cofactor for the activation of factor XI by thrombin. Blood. 2011;118(26):6963-6970. Whyte CS, Chernysh IN, Domingues MM, Connell S, Weisel JW, Ariens RA, Mutch NJ. Polyphosphate delays fibrin polymerisation and alters the mechanical properties of the fibrin network. Thromb Haemost. 2016;116(5):897-903. Mutch NJ, Engel R, Uitte de Willige S, Philippou H, Ariens RA. Polyphosphate modifies the fibrin network and down-regulates fibrinolysis by attenuating binding of tPA and plasminogen to fibrin. Blood. 2010;115(19):3980-3988. Mitchell JL, Lionikiene AS, Georgiev G, Klemmer A, Brain C, Kim PY, Mutch NJ. Polyphosphate colocalizes with factor XII on platelet-bound fibrin and augments its plasminogen activator activity. Blood. 2016;128(24):2834-2845. Verhoef JJ, Barendrecht AD, Nickel KF, Dijkxhoorn K, Kenne E, Labberton L, McCarty OJ, Schiffelers R, Heijnen HF, Hendrickx AP, Schellekens H, Fens MH, de Maat S, Renne T, Maas C. Polyphosphate nanoparticles on the platelet surface trigger contact system activation. Blood. 2017;129(12):1707-1717. Disclosures No relevant conflicts of interest to declare.

It is known that the bio-astronomical effect of Chizhevsky-Velhover is based on the cytochemical metachromatic reaction of polyphosphatecontaining volutin granules of microorganisms. As a result of long-term monitoring (2001-2015 years) of this of cytochemical index in yeasts Saccharomyces cerevisiae, it was shown that metachromatic staining of the granules possessed rhythmicity and connection with space weather. Under laboratory conditions the change of metachromasy was achieved by applying different stress factors: anaerobiosis, low temperature, acidity. Simulation of metachromatic staining in vitro by using solutions of inorganic polyphosphate with other compounds showed that this reaction was possible for the low concentrations of these polymers. The effect of the low concentrations was more expressed with increasing chain length of polyphosphate. The inhibition of the metachromasy in solution with added protein and calcium chloride indicated that this of reaction involved the presence of the protonated sites in the polymers. It is assumed that metachromatic staining of volutin granules in vivo may depend on other conditions including conformational restructuring of a polymer or sol-gel phase transitions. Thus, the question whether the metachromatic reaction of intracellular polyphosphate granules to the space weather changes is driven by a response to associated stress or despite the apparent similarity of the effects they are of a different nature remains unclear.

Cutaneous mast cell tumours (MCTs) are the most common skin tumours in dogs. Due to the prevalence of canine MCTs and the variable biologic behavior of this disease, accurate prognostication and a thorough understanding of MCT biology are critical for the treatment of this disease. A cytologic diagnosis of mast cell tumor with evidence of prior hemorrhage was made, and the masses were surgically removed. Cytological evaluation of fine-needle aspirates from the cutaneous mass from the axillary comprised many well-differentiated, highly granulated mast cells with moderate numbers of eosinophils. Nuclei were varied in size and shape with high nuclear’to’cytoplasmic ratio, prominent nucleoli, marked atypical and mitotic figures. Microscopically, mass consisted of sheets of neoplastic round cells that formed nonencapsulated nodules in the dermis and infiltrated into the adjacent dermal collagen, and also there was diffuse subcutis invasion of round to pleomorphic tumor cells. Tumor cells had moderate to abundant cytoplasm, round to ovoid nuclei with scattered chromatin, and mitotic figures. In this tumor, cytoplasmic granules showed atypical metachromasia. In addition, eosinophils were scattered among the mast cells at the periphery of the nodules. The presence of eosinophils and the observation, at high magnification, of cells with cytoplasmic metachromatic granules. Invasion of the deep subcutaneous fat or cutaneous muscles were a common feature of grade III tumour. Finally, a diagnosis of grade III cutaneous mast cell tumor was made. The virtual slide(s) of this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4755249151157024 .

Titania/silica nanomaterials have many possible applications; however, they can be toxic to living organisms, particularly if the material accumulates in niche environments, e.g. areas colonised by actinomycetes. This study therefore investigated the effect of non-activated and UV light-activated titania/silica nanospheres on an environmental Streptomyces strain. The bacteria were incubated with the nanospheres and subsequently cultured on solid medium. The morphology and elemental composition were analysed using optical and electron microscopy (TEM, STEM) and energy-dispersive X-ray spectroscopy (EDX). The appearance of Streptomyces sp. in the experimental and control samples demonstrated that the nanospheres did not have bactericidal properties in the used dose. Furthermore, the observed strain not only survived in the presence of the nanomaterial but also appeared to play a role in its dissolution with an accumulation of the titanium in the intracellular globules of polyphosphate (volutin). Additionally, it was discovered that the UV light-activated titanium dioxide altered the ability of the bacteria to secrete humic acid. The reported phenomenon might be made possible through an accumulation of titanium in the volutin compounds. These findings suggest that streptomycetes could be employed to participate in the dissolution of nanomaterials which enter the natural environment.

norganic polyphosphates (polyP) are linear polymers of tens to hundreds orthophosphate residues linked by phosphoanhydride bonds. These fairly abundant biopolymers occur in all extant forms of life, from prokaryotes to mammals, and could have played a relevant role in prebiotic evolution. Since the first identification of polyP deposits as metachromatic or volutin granules in yeasts in the nineteenth century, an increasing number of varied physiological functions have been reported. Due to their "high energy" bonds analogous to those in ATP and their properties as polyanions, polyP serve as microbial phosphagens for a variety of biochemical reactions, as a buffer against alkalis, as a storage of Ca(2+) and as a metal-chelating agent. In addition, recent studies have revealed polyP importance in signaling and regulatory processes, cell viability and proliferation, pathogen virulence, as a structural component and chemical chaperone, and as modulator of microbial stress response. This review summarizes the current status of knowledge and future perspectives of polyP functions and their related enzymes in the microbial world.

The structural organization of parasites has been the subject of investigation by many groups and has lead to the identification of structures and metabolic pathways that may represent targets for anti-parasitic drugs. A specific group of organelles named acidocalcisomes has been identified in a number of organisms, including the apicomplexan parasites such as Toxoplasma and Plasmodium, where they have been shown to be involved in cation homeostasis, polyphosphate metabolism, and osmoregulation. Their structural counterparts in the apicomplexan parasite Eimeria have not been fully characterized. In this work, the ultrastructural and chemical properties of acidocalcisomes in Eimeria were characterized. Electron microscopy analysis of Eimeria parasites showed the dense organelles called volutin granules similar to acidocalcisomes. Immunolocalization of the vacuolar proton pyrophosphatase, considered as a marker for acidocalcisomes, showed labeling in vesicles of size and distribution similar to the dense organelles seen by electron microscopy. Spectrophotometric measurements of the kinetics of proton uptake showed a vacuolar proton pyrophosphatase activity. X-ray mapping revealed significant amounts of Na, Mg, P, K, Ca, and Zn in their matrix. The results suggest that volutin granules of Eimeria parasites are acidic, dense organelles, and possess structural and chemical properties analogous to those of other acidocalcisomes, suggesting a similar functional role in these parasites.

The isolation and identification of hydrogen production bacteria(HPB)with high yield and high evolution rate is an important foundation of fermented hydrogen production process by anaerobic digesting high strength organic wastewater. An improved Hungater rolling tubes technique and a plate method of culture bottle (PMCB) were employed to enumerate and isolate the anaerobes. The HPB-RL medium was designed specially for isolating and culturing anaerobic hydrogen bacteria. Culture temperature was at 37°C and 5.5 of pH. About 90 strains of bacteria were isolated, in which, a strain of the hydrogen producer with high yield and high evolution rate was isolated and named Rennanqi12. The isolates were straight rods, Grams positive, strictly anaerobic, no spore-forming bacteria, whole bacteria flagella, having capsule, 3~4 metachromatic granules. The strain of Rennanqi12 could produce hydrogen using molasses wastewater as the carbon resource. The isolate Rennanqi12 has the closest relationship with the strain C. cellulose for the 94% homology of 16S rDNA. The strain of Rennanqi12 might be a new species that belongs to a new genus. The amount of hydrogen-producing (YH2)is 1880.5ml/L medium and the maximum hydrogen-producing rate(QH2)is 28.2 mmol/g•(dry•cell)•h when R12 digested substrates were glucose.

Polyphosphate (polyP) has been abundant on Earth long before life began. In living organisms, polyphosphate can be stored in electron-dense granules that have long been referred to as volutin granules. After having being ignored for a long time, this polymer has proven to have numerous unsuspected functions in living organisms and is receiving more attention. The ubiquitous presence of polyP in nature and its structural and physico-chemical characteristics appear to be the drivers for the evolutionary selection of this versatile molecule as a component of cellular processes. Most of our knowledge about the functions of polyP comes from the microbial world. PolyP can influence the transcription and translation of specific genes, act as a metabolic regulator, provide an alternative source of energy, and modulate several stress responses. In plant systems, although the presence of polyP has long been known, its roles in cellular processes have begun to emerge only recently. The purpose of this mini-review is to provide a brief overview of the proposed biological roles of polyphosphates in plants and their roles in microbial/plant interactions.

Canine mast cell tumours (MCTs) may be graded microscopically for prognostic purposes. Grade I (well-differentiated) and grade II (intermediate differentiation) tumours have an abundance of metachromatic granules within the cytoplasm; however, grade III (poorly differentiated) MCTs may be difficult to diagnose as they frequently have fewer discernable granules. Herein we report that a cross-reactive anti-human CD1a monoclonal antibody (clone O10) may be used in immunohistochemistry to identify canine MCTs of all grades. The antibody was applied to tissue sections from 48 canine MCTs of different histological grades. Serial sections from each tumour were stained with toluidine blue and safranin O to compare diagnostic sensitivity. All MCTs were labelled positively by the CD1a antibody, but histochemical staining was often equivocal and identification of mast cells was extremely difficult in some cases. This antibody did not label neoplastic cells in cases of canine histiocytoma, plasmacytoma or amelanotic melanoma; therefore, the reagent may be a valuable marker for the diagnosis of canine MCTs, especially those tumours of histological grade III.

The caecilians (Amphibia, Gymnophiona) constitute one of the least known groups of terrestrial vertebrates because most species live underground in quite inaccessible environments. Siphonops annulatus is an exclusively fossorial species and is the most extensively distributed caecilian in South America. Little is known of this order concerning circulating granulocytes, including their morphological and cytochemical structure and ultrastructure. This paper is part of a project covering the study of granulocytes in representative species of the order Amphibia. Blood extensions were carried out and submitted to Leishman, Toluidine Blue, Periodic acid Schiff, Sirius Red and hydrogen o-toluidine peroxide methods. Part of the samples was prepared for conventional transmission electron microscopy. Among granular leukocytes, mature and immature neutrophils and eosinophils were identified, plus basophils. The most frequent granulocyte encountered in S. annulatus peripheral blood is the neutrophil. This is a cell with a hyper-segmented nucleus and with a very clear cytoplasm when compared to the eosinophil, which presents large cytoplasmic acidophilic granules. On the other hand, the basophils present basophilic and metachromatic granules. Glycogen was detected in the cytoplasm of the neutrophils and eosinophils, while basic protein rich in amino acids was observed in the eosinophil’s granules. Myeloperoxidase activity was detected in the cytoplasm of the neutrophils and eosinophils. Neutrophils were ultrastructurally detected with three types of small granules: eosinophils with large and small spherical granules and basophils with large spherical granules with lamellate structures.

Corynebacterium glutamicum forms inorganic polyphosphate (poly P) that may occur as soluble (cytosolic) poly P and/or as volutin granules. A suitable method for monitoring soluble and granular poly P in C. glutamicum was developed and applied to C. glutamicum cells cultivated under different growth conditions. Under phosphate-limiting conditions, C. glutamicum did not accumulate poly P, but it rebuilt its poly P storages when phosphate became available. The poly P content of C. glutamicum growing on glucose minimal medium with sufficient phosphate varied considerably during growth. While the poly P content was minimal in the midexponential growth phase, two maxima were observed in the early exponential growth phase and at entry into the stationary growth phase. Cells in the early exponential growth phase primarily contained granular poly P, while cells entering the stationary growth phase contained soluble, cytosolic poly P. These results and those obtained for C. glutamicum cells cultivated under hypo- or hyperosmotic conditions or during glutamate production revealed that the poly P content of C. glutamicum and the partitioning between cytosolic and granular forms of poly P are dynamics and depend on the growth conditions.

Received 14 January 2005; returned 15 May 2005; revised 24 May 2005; accepted 7 October 2005Objectives: Investigation of the mode of action of the naphthoimidazole N1, obtained from the reaction ofb-lapachonewithbenzaldehyde,whichamong45semi-syntheticderivativesofnaphthoquinonesisolatedfrom Tabebuia sp. was one of the most active compounds against Trypanosoma cruzi trypomastigotes.Methods: Quantification of the effect of N1 against the proliferative forms of T. cruzi, and investigation ofpotential targets in the parasite using electron microscopy and flow cytometry techniques.Results:N1presentedthefollowingorderofactivity:amastigotes>trypomastigotes>epimastigotes.Theeffectonintracellularformswas 25timeshigherthanonmacrophagesandheartmusclecells.N1-treatedparasites presented an abnormal chromatin condensation and mitochondrial damage. In epimastigotes,alterations of reservosomes were observed, and in trypomastigotes, a decrease in the electron density ofacidocalcisomeswasobserved.Inepimastigotes,thenaphthoimidazoleinhibitedtheactivityofsuccinatecytochrome c reductase. Labelling with rhodamine 123 or Acridine Orange was decreased in both formstreated with N1.Conclusions:Theresultssuggestthatepimastigotes,reservosomes,mitochondrion,andnucleuscontainN1 targets. In trypomastigotes, in which reservosomes are absent, the organelles affected by thecompound were also the mitochondrion and nucleus, as well as acidocalcisomes, in which the decreasein electron density could be due to the use of polyphosphate as an alternative energy supply.Keywords: T. cruzi, chemotherapy, naphthoquinones, parasites

Mucopolysaccharidosis (MPS) is a group of lysosomal storage disorders in which there is deficiency of specific enzymes. Depending upon the enzyme which is deficient and the nature of the material that accumulates at various tissues, the MPS is divided into 8 types (MPS I to MPS VIII). In MPS VI, deficiency of aryl B sulfatase leads to the accumulation of dermatan sulfate. Mucopolysaccharidosis VI, also called as Maroteaux-Lamy syndrome, in its severe form presents with bony lesions, corneal clouding, hepatosplenomegaly, cardiovascular abnormalities, and central nervous system deterioration. This form of MPS features the most striking abnormal granulation in the circulating white blood cells. Mucopolysaccharidosis VI has an estimated global incidence of 1 in 340 000. The number of cases showing abnormal granules in the cytoplasm of leucocytes is still rarer. We report a case of MPS VI with abnormal granules in the circulating blood leukocytes.

Inorganic polyphosphate (polyP) has been identified and measured in human platelets. Millimolar levels (in terms of Pi residues) of short chain polyP were found. The presence of polyP of approximately 70-75 phosphate units was identified by 31P NMR and by urea-polyacrylamide gel electrophoresis of platelet extracts. An analysis of human platelet dense granules, purified using metrizamide gradient centrifugation, indicated that polyP was preferentially located in these organelles. This was confirmed by visualization of polyP in the dense granules using 4',6-diamidino-2-phenylindole and by its release together with pyrophosphate and serotonin upon thrombin stimulation of intact platelets. Dense granules were also shown to contain large amounts of calcium and potassium and both bafilomycin A1-sensitive ATPase and pyrophosphatase activities. In agreement with these results, when human platelets were loaded with the fluorescent calcium indicator Fura-2 acetoxymethyl ester to measure their intracellular Ca2+ concentration ([Ca2+]i), they were shown to possess a significant amount of Ca2+ stored in an acidic compartment. This was indicated by the following: 1) the increase in [Ca2+]i induced by nigericin, monensin, or the weak base, NH4Cl, in the nominal absence of extracellular Ca2 and 2) the effect of ionomycin, which could not take Ca2+ out of acidic organelles and was more effective after alkalinization of this compartment by the previous addition of nigericin, monensin, or NH4Cl. All of these characteristics of the platelet dense granules, together with their known acidity and high density (both by weight and by electron microscopy), are similar to those of acidocalcisomes (volutin granules, polyP bodies) of bacteria and unicellular eukaryotes. The results suggest that acidocalcisomes have been conserved during evolution from bacteria to humans.

Heparin, other sulphated glycosaminoglycans and histamine were extracted from various dissected organs of Anomalocardia brasiliana, a mollusc from the South Atlantic, and quantified. A good correlation between heparin and histamine content was found in the labial palp, intestine, ctenidium, mantle and foot tissues. The tissue location of metachromatic cells, putatively containing heparin, was identified histologically with Alcian Blue, Toluidine Blue, Masson trichrome, Haematoxylin–Eosin and PAS. Except for the foot, cells containing metachromatic granules were found in the epithelium surfaces of all the organs analysed. An in situ identification of heparin using nitrous acid and heparinase degradation has established unequivocally the presence of this compound in the metachromatic cells. The location of 'mast-like' cells at the epithelium surface of mollusc tissues exposed to the environment are very similar to the distribution of mammalian and other vertebrate mast cells and gives support to the suggestion for a role of mast cells in defense mechanisms.

This study investigates the number and the distribution of mast cells in the normal human uterus. Reliability of results was ensured by prompt tissue fixation and the use of biotin-labelled lectins in conjunction with the avidin-biotin peroxidase complex (ABC) method. This design revealed that mast cells are, indeed, normal constituents of the human uterus. They occur in large numbers in the myometrium, but are only scanty in the endometrium where they tend to be confined to the stratum basalis. The mean mast cell counts per high power field (MC/HPF), after staining with Canavalia ensiformis agglutinin (Con A), were 17.9MC/HPF in the inner half of the myometrium, and 8.3MC/HPF in the outer half of the myometrium; 2.7MC/HPF in the basalis, and 0.3MC/HPF in the functionalis (P

Ectomycorrhizas produced between Pisolithus tinctorius and Eucalyptus pilularis under axenic conditions were rapidly frozen, freeze-substituted in tetrahydrofuran and embedded anhydrously, and dry-sectioned for X-ray microanalysis. The vacuoles of the sheath and Hartig net hyphae were rich in phosphorus and potassium. They also contained sulfur and variable amounts of chlorine. In anhydrously processed freeze-substituted mycorrhizas, dispersed electron-opaque material filled the fungal vacuoles. X-ray maps indicated that P was distributed evenly throughout the entire vacuole profile and was not concentrated in spherical bodies or subregions of the vacuole. There were no electron-opaque granules surrounded by electron-lucent areas, such as are commonly seen in chemically fixed material. The fungal vacuoles were also rich in K, which similarly gave a signal from the entire vacuolar profile. Such P-rich vacuoles occurred in both the mycorrhizal sheath and Hartig net hyphae. Stained sections of ether-acrolein freeze-substituted mycorrhizas also showed only dispersed material in the fungal vacuoles as, in most cases, did acetone-osmium freeze-substituted material. Precipitation of metachromatic granules by ethanol suggested that large amounts of polyphosphate are stored in these regions under the conditions of our experiments, as well as in the tips of actively growing hyphae of the same fungus. The higher plant vacuoles of ectomycorrhizas gave a much lower signal for K, and P was barely detectable. Much more K was located in the vacuoles of the root exodermal cells than in epidermal cells. The analysis of element distribution between the vacuole and cytoplasm in root cells agrees well with that found for other plant species using other techniques. We conclude that polyphosphate is indeed present in the vacuoles of the fungal cells of these ectomycorrhizas, but that in vivo it is in a dispersed form, not in granules.

Spontaneous mastocytomas studied in 18 axolotls (Ambystoma mexicanum) and six tiger salamanders (Ambystoma tigrinum) were gray-white, uni- to multilobular cutaneous protrusions from 2 mm to 2 cm in diameter. Tumors were moderately cellular unencapsulated masses that usually infiltrated the dermis and hypodermis with the destruction of intervening tissues. Some tumors were invading superficial bundles of the underlying skeletal muscle. Tumors consisted of mitotically active cells derived from a single lineage but showing a range of differentiation. Immature cells had nearly smooth to lightly cleft or folded basophilic nuclei bordered by a band of cytoplasm with few cytoplasmic processes and containing a few small uniform eccentric granules. Mature cells had basophilic nuclei with deep clefts or folds and abundant eosinophilic cytoplasm with multiple long intertwining cytoplasmic extensions packed with metachromatic granules. The axolotls were old individuals from an inbred laboratory colony. The tiger salamanders were wild animals from a single polluted pond. They could have been old and inbred. Both groups were neotenic. These are the first mastocytomas discovered in cold-blooded animals.