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We studied the structural and functional state of liver tissue during the regeneration process using multiphoton microscopy in combination with fluorescence lifetime imaging (FLIM) and second-harmonic generation microscopy, supplementing our findings with a mass spectrometry analysis. The regeneration process was induced by 30% or 70% partial hepatectomy (PH) of rats. The multiphoton microscopy and FLIM were used to evaluate the structure of the liver tissue and to assess the metabolic state of the hepatocytes, respectively. As a result, there was a statistically significant increase in the contribution of the bound forms of NAD(P)H during liver regeneration, indicating a metabolic shift towards oxidative phosphorylation and an increase in the intensity of biosynthesis processes in hepatocytes. Time-of-flight secondary ion mass spectrometry was used to study changes in the lipid and amino acid composition of liver tissue during regeneration. The approach used in the present work is effective for analyzing the dynamics of changes in the structural and functional state of liver tissue during the regeneration process. These results can be useful for the further development of criteria for the assessment of the regenerative potential of the liver and for predicting the dynamics of its recovery after the resection.

The emission spectra of krypton plasma in the range of 8 – 14 nm upon excitation of a pulsed gas jet by 1.06-mm Nd : YAG laser radiation with a pulse energy of 0.85 J, pulse duration of 5.2 ns, and repetition rate of 10 Hz are investigated. The krypton emission spectrum is a wide (8 – 14 nm) band, peaking at 10.3 nm, which is formed by a series of much narrower lines. The observed lines are identified, and the fraction of laser pulse energy converted into the (8 – 14)-nm emission band and emitted into half-space (2πsr) is determined. The maximum conversion efficiency is found to be 21 %. The expected throughputs of lithographic systems with sources based on Sn, Xe, and Kr ions for different wavelengths, corresponding to the emission peaks of ions of these materials, are compared.

In this study, we investigated in an androgenized rat model the involvement of autophagy and mitochondrial dynamics in granulosa cells in the pathogenesis of polycystic ovarian syndrome (PCOS) and its modulation by exogenous gonadotropin (eCG). We found 5α-dihydrotestosterone (DHT) treatment reduces ovarian length and weight with predominantly late antral and/or preovulatory stage follicles and no corpora lutea. DHT increased the population of large lysosomes (>50 micron) and macroautophagy, an event associated with granulosa cell apoptosis. Increased granulosa cell Dynamin Related Protein 1 (Drp1) content in the DHT group was accompanied by increased circular and constricted, but reduced rod-shaped, mitochondria. eCG eliminated all atypical follicles and increased the number of late antral and preovulatory follicles with less granulosa cell apoptosis. eCG-treated rats had a higher proportion of connected mitochondria, and in combination with DHT had a lower proportion of circular and constricted mitochondria than rats treated with DHT alone, suggesting that eCG induces mitochondrial fusion and attenuates fission in granulosa cells. In summary, we observed that DHT-induced up-regulation of Drp1 is associated with excessive mitochondrial fission, macroautophagy and apoptosis in granulosa cells at the antral stage of development in an androgenized rat model for PCOS, a response partially attenuated by exogenous gonadotropin.

An integrated microscopic study of the destruction of mouse Ehrlich ascites carcinoma (EAC) cells under starvation conditions has been carried out. It has been found that, in addition to apoptosis, necrosis, and apoptotic necrosis, already known for EAC, cell destruction can also occur through mitochondrial autolysis, which is proposed to be considered a new kind of mitoptosis. A mitoptosis in EAC is characterized by the appearance of many autolyzing mitochondria, the fusion of which leads to rupture of the cell membrane and the ejection of the nucleus from the cell. It is assumed that the polymorphism of EAC destruction patterns is explained by the different physiological state of the cells, which determines the "choice" of the cell death mechanism. This situation poses a challenge for researchers to develop complex inducers with the ability to stimulate all possible types of cancer cell death.

The changes in cell metabolism can affect the epigenome-modifying enzymes activity during iPSCs differentiation and thus control the functional potential of the final cell. Therefore, for therapeutic applications, the restoration of a fully functional mitochondrial network specific for the cell types derived from iPSCs will be required to support the energy and other mitochondrial factors. Recently, FLIM method allows to study the metabolic changes that accompanying cell differentiation noninvasively and without additional labels. In this study, we investigated the metabolic changes in iPSCs during neural differentiation using two-photon fluorescence microscopy and FLIM. Cellular metabolism was examined by monitoring the optical redox ratio (FAD/NAD(P)H), the fluorescence lifetime contributions of the free and bound forms of NADH and NADPH. Given that neural differentiation is also accompanied by synthetic processes and oxidative stress, this process was included in the scope of this work. We demonstrated an increased contribution of protein-bound NADH and NADPH in neuron associated with metabolic switch to oxidative phosphorylation and the biosynthetic processes or oxidative stress, respectively. We also found that the optical redox ratio FAD/NAD(P)H decreased during neural differentiation, and this was likely to be explained by the intensive lipid membrane synthesis or ROS generating and the enhanced NADPH production associated with them. The biochemical analysis was carried out to verify the metabolic status of iPSCs and their neural derivatives. Based on the data on glucose consumption, lactate and ATP amount we registered the trend to the metabolic pathways redistribution towards the oxidative phosphorylation in neuron.

The differentiation of endothelial cells from human iPSC has incontestable advantages in diseases research and therapeutic applications. However, the safe use of iPSC derivatives in regenerative medicine requires an enhanced understanding and control of factors that optimize in vitro reprogramming and differentiation protocols. Shifts in cellular metabolism associated with intracellular pH changes affect the enzymes that control epigenetic configuration, which impact chromatin reorganization and gene expression changes during reprogramming and differentiation. FLIM-based metabolic imaging of NADH and FAD is a powerful tool for measuring mitochondrial metabolic state and widely used diagnostic method for identification of neoplastic diseases, skin diseases, ocular pathologies and stem cells differentiation. Therefore, in this study, we used the potential of FLIM-based metabolic imaging and fluorescence microscopy of NADH and FAD to study the metabolic changes during iPSC differentiation in endothelial cells. The evaluation of the intracellular pH was carried out with the fluorescent pH-sensor SypHer-2 and fluorescence microscopy to obtain complete information about metabolic status of iPSC and their endothelial derivatives. Based on the FAD/NAD(P)H optical redox ratios increase and the contributions rise of the NAD(P)H fluorescence lifetime in iPSC during endothelial differentiation, we demonstrated an contribution increase of OXPHOS to cellular metabolism. Based on the shift toward more acidic intracellular pH in endothelial cell derived from iPSCs we verified their oxidative state.

Biomeshes based on decellularized bovine pericardium (DBP) are widely used in reconstructive surgery due to their wide availability and the attractive biomechanical properties. However, their efficacy in clinical applications is often affected by the uncontrolled immunogenicity and proteolytic degradation. To address this issue, we present here in vivo multiparametric imaging analysis of epoxy crosslinked DBPs to reveal their fate after implantation. We first analyzed the structure of the crosslinked DBP using scanning electron microscopy and evaluated proteolytic stability and cytotoxicity. Next, using combination of fluorescence and hypoxia imaging, X-ray computed microtomography and histology techniques we studied the fate of DBPs after subcutaneous implantation in animals. Our approach revealed high resistance to biodegradation, gradual remodeling of a surrounding tissue forming the connective tissue capsule and calcification of crosslinked DBPs. These changes were concomitant to the development of hypoxia in the samples within 3 weeks after implantation and subsequent induction of angiogenesis and vascularization. Collectively, presented approach provides new insights on the transplantation of the epoxy crosslinked biomeshes, the risks associated with its applications in soft-tissue reconstruction and can be transferred to studies of other types of implants.

Glutaredoxin 2 (GRX2), a mitochondrial glutathione-dependent oxidoreductase, is central to glutathione homeostasis and mitochondrial redox, which is crucial in highly metabolic tissues like the heart. Previous research showed that absence of Grx2, leads to impaired mitochondrial complex I function, hypertension and cardiac hypertrophy in mice but the impact on mitochondrial structure and function in intact cardiomyocytes and in humans has not been explored. We hypothesized that Grx2 controls cardiac mitochondrial dynamics and function in cellular and mouse models, and that low expression is associated with human cardiac dysfunction. Here we show that Grx2 absence impairs mitochondrial fusion, ultrastructure and energetics in primary cardiomyocytes and cardiac tissue. Moreover, provision of the glutathione precursor, N-acetylcysteine (NAC) to Grx2-/- mice did not restore glutathione redox or prevent impairments. Using genetic and histopathological data from the human Genotype-Tissue Expression consortium we demonstrate that low GRX2 is associated with fibrosis, hypertrophy, and infarct in the left ventricle. Altogether, GRX2 is important in the control of cardiac mitochondrial structure and function, and protects against human cardiac pathologies., Graphical abstract fx1

Current peroxisome proliferator-activated receptor (PPAR)-targeted drugs, such as the PPARγ-directed diabetes drug rosiglitazone, are associated with undesirable side effects due to robust agonist activity in non-target tissues. To find new PPAR ligands with fewer toxic effects, we generated transgenic zebrafish that can be screened in high throughput for new tissue-selective PPAR partial agonists. A structural analog of coenzyme Q10 (idebenone) that elicits spatially restricted partial agonist activity for both PPARα and PPARγ was identified. Coenzyme Q10 was also found to bind and activate both PPARs in a similar fashion, suggesting an endogenous role in relaying the states of mitochondria, peroxisomes and cellular redox to the two receptors. Testing idebenone in a mouse model of type 2 diabetes revealed the ability to reverse fatty liver development. These findings indicate new mechanisms of action for both PPARα and PPARγ, and new potential treatment options for nonalcoholic fatty liver disease (NAFLD) and steatosis. This article has an associated First Person interview with the first author of the paper., Summary: A zebrafish screen identifies a novel PPARα/γ ligand, idebenone, with potential for treatment of fatty liver diseases, as seen by testing it in a mouse model of type 2 diabetes.

The present work reveals the effect of kappa-, lambda- and kappa/beta-carrageenans on the immune modulation and supramolecular structure of lipopolysaccharide (LPS). The kappa/beta carrageenan was able to increase the synthesis of anti-inflammatory interleukin-10 (IL-10) in vitro, and at low concentrations, their activity in the mixture with LPS was higher than that of LPS alone. Kappa-carrageenan significantly inhibited LPS-induced upregulation of reactive oxygen species (ROS). The activation of cells by kappa-carrageenan occurs through TLR4 receptor specific for LPS. Carrageenans reduced (kappa-) or completely inhibited (lambda-) collagen-induced platelet aggregation and decreased their aggregation activity caused by cooperative effect of LPS and collagen. The transformation of ultrastructure of LPS by action of kappa- and kappa/beta-carrageenans is observed. Moreover, carrageenans changed both the sizes and ζ- potentials of the LPS.

Modern ideas on the biogenesis of plant peroxisomes, their involvement in the production and detoxification of reactive oxygen species, and the role in the processes they mediate are discussed. The data on the proliferation and degradation of these organelles in the cell during the oxidative stress and on their involvement in the generation of signaling molecules are considered.

Induced pluripotent stem cells (iPSC) are a promising tool for personalized cell therapy, in particular, in the field of dermatology. Metabolic plasticity of iPSC are not completely understood due to the fact that iPSC have a mixed mitochondrial phenotype, which still resembles that of somatic cells. In this study we investigated the metabolic changes in iPSC undergoing differentiation in two directions, dermal and epidermal, using two-photon fluorescence microscopy combined with FLIM. Directed differentiation of iPSC into dermal fibroblasts and keratinocyte progenitor cells was induced. Cellular metabolism was examined on the basis of the fluorescence of the metabolic cofactors NAD(P)H and FAD. The optical redox ratio (FAD/NAD(P)H) and the fluorescence lifetimes of NAD(P)H and FAD were traced using two-photon fluorescence microscopy combined with FLIM. Evaluation of the intracellular pH was carried out with the fluorescent pH sensor SypHer-2 and fluorescence microscopy. In this study, evaluation of the metabolic status of iPSC during dermal and epidermal differentiation was accomplished for the first time with the use of optical metabolic imaging. Based on the data on the FAD/NAD(P)H redox ratio and on the fluorescence lifetimes of protein-bound form of NAD(P)H and closed form of FAD, we registered a metabolic shift toward a more oxidative status in the process of iPSC differentiation into dermal fibroblasts and keratinocyte progenitor cells. Biosynthetic processes occurring in dermal fibroblasts associated with the synthesis of fibronectin and versican, that stimulate increased energy metabolism and lower the intracellular pH. No intracellular pH shift is observed in the culture of keratinocyte progenitor cells, which reflects the incomplete process of differentiation in this type of cells. Presented results provide the basis for further understanding the metabolic features of iPSC during differentiation process, which is essential for developing new treatment strategies in cell therapy and tissue engineering.

The activity of hydrolases (protease, RNase) in uninfected and tobacco mosaic virus-infected tobacco leaves of the Samsun variety, untreated and treated with disodium salt of 2-acetyl-4-hydroxycarbonyl-methylthio-5-chlorocyclopent-4-en-1,3-dione (DS), was determined. It was shown that treatment of leaves with this compound significantly increased the activity of hydrolases in them compared to untreated leaves. In infected leaves treated with DS one day before infection, along with an increased level of hydrolases, one revealed more viral particles exposed to destructive changes in infected, rather than untreated, leaves. It is assumed that the DS-caused activation of hydrolases promotes the destruction of viral particles and is therefore one of the cell defense mechanisms induced by this compound that prevents the intracellular accumulation of virus.

The study of destruction of mouse Ehrlich ascites carcinoma (EAC) cells demonstrated that the loss of cell viability occurred via apoptosis detected by AnnexinV staining of phosphatidylserine inverted from the inner monolayer in the outer layer of cyto� plasmic membrane and via necrosis identified by staining of nuclei of autolyzed cells with propidium iodide penetrating into the cells via membranes with disrupted barrier properties. However, in an experi� ment with cucumarioside A 2�2, we detected an increased number of cells destroyed by combined cel� lular destruction, including necrosis and apoptosis (8). Thus, the use of cucumarioside A2�2, probably involves stimulation of the socalled "apoptotic necrosis," which is one of the possible variants of cell death (9). For verification of this hypothesis, morpho� logical identification of all variants of EAC cell death induced by cucumarioside A2�2 is required.

A number of low molecular weight (LMW) fractions of carrageenans with different structural types were obtained by free radical depolymerization (H2O2), mild acid hydrolysis (HCl), and a specific enzyme. Three samples of carrageenans were depolymerized: kappa-carrageenan from Chondrus armatus, kappa-carrageenan from Kappaphycus alvarezii, and kappa/beta-carrageenan from Tichocarpus crinitus with initial molecular weights of 250, 390, and 400 kDa, respectively. The chemical depolymerization by two methods resulted to LMW derivatives of carrageenans with molecular weight from 1.2 to 3.5 kDa. Oligosaccharides of kappa- and kappa/beta-carrageenans with molecular weight of 2.2 and 4.3 kDa, respectively, were obtained after enzymatic depolymerization by recombinant kappa-carrageenase from Pseudoalteromonas carrageenovora. It was shown that the antiviral activity of high molecular weight carrageenans against tobacco mosaic virus was higher than that of their LWM derivatives independently on the depolymerization method. The method of depolymerization had some influence on the antiviral activity of carrageenan. LMW derivatives of kappa- and kappa/beta-carrageenans obtained by mild acid hydrolysis showed higher antiviral activity than the products of free radical depolymerization. The oligosaccharides prepared by enzymatic degradation possessed the lowest activity.

The effect of chitosan on the accumulation and state of tobacco mosaic virus (TMV) in mesophyll cells of Nicotiana tabacum L. var. Samsun leaves is studied in the early stage of the development of the infection (3 days after infection of leaves). In the cells of leaves treated with chitosan 24 h before infection, the virus accumulated to a lesser degree than in the control. With the use of chitosan, TMV-specific granular inclusions were often observed in infected cells, the presence of which is ascribed to the early stages of virus reproduction, whereas the control cells contained mainly tubular inclusions formed from granular inclusions at the late stages of the infectious processes. This shows that chitosan delays the development of the infection. In the phosphotungstic acid-treated juice preparation made from infected leaves, abnormal (swollen and thin), as well as normal, TMV particles were observed. The appearance of abnormal viral particles seems to result from the virus-induced activation of intracellular lytic processes. In chitosan-treated infected cells, the lytic activity was the highest and the number of abnormal viral particles increased compared to the control. It is suggested that the chitosan-mediated stimulation of lytic processes that cause the destruction of TMV particles may be one of the protective mechanisms that limit the accumulation of the virus in cells.

The activities of hydrolases (acid phosphatase, RNase, and proteases) in healthy and tobacco mosaic virus-infected leaves of Nicotiana tabacum L. var. Samsun, both untreated and treated with polysaccharides (PS) (1,3;1,6-β-D-glucan, fucoidan, and κ/β-carrageenan), were determined. The PS lead to substantial increase in the hydrolase level. The percentage of viral particles undergoing destructive change also increases in leaves treated with PS 24 h before infection. We suppose that the PS-mediated hydrolase activation promotes intracellular destruction of the viral particles and, thus, comprises one of the PS-induced protective mechanisms limiting intracellular viral accumulation.

Enzymatic (the action of lysozyme) and chemical (the action of hydrogen peroxide) hydrolysis of chitosans with various degree of acetylation (DA)—25, 17, and 1.5%—was performed. Purification and fractioning of the hydrolysis products were performed using dialysis, ultrafiltration, and gel-penetrating chromatography. Low-molecular (LM) derivatives of the polysaccharide with molecular masses from 17 to 2 kDa were obtained. The study of their antiviral activity against the tobacco mosaic virus (TMV) showed that these samples inhibited the formation of local necroses induced by the virus for 50–90%. The antiviral activity of the LM chitosans significantly increased with the lowering of their polymerization degree. Furthermore, the products of the enzymatic hydrolysis possessed lower activity than the chitosan samples obtained as a result of chemical hydrolysis. It was revealed that the exhibition of the antiviral activity weakly depended on the degree of acetylation of the samples.

The effect of κ/β-carrageenan from red alda Tichocarpus crinitus on the development of a potato virus X (PVX) infection in the leaves of Datura stramonium L. has been studied. The treatment of leaves with carrageenan stimulates a protein synthesis in the cells, causing an increase in the size of nucleoli and in the number of mitochondria and membranes of the rough endoplasmic reticulum. At the same time, such treatment slightly stimulates lytic processes, causing an increase in the number of smooth endoplasmic reticulum cisternae, dictyosomes, and cytoplasmic vacuoles and the formation of cytoplasmic electron-transparent zones. The carrageenan-induced stimulation of lytic processes results in the destruction of viral particles and can be considered as one of the defense mechanisms, preventing the intracellular accumulation of virus. The carrageenan-stimulated formation of PVX-specific laminar structures, able to bind viral particles and, therefore, prevent their intracellular translocation and reproduction, represents another carrageenan-induced mechanism of the antiviral defense in plant cells.

The effect of disodium salt of 2-acetyl-4-hydroxycarbonylmethylthio-5-chlorocyclopent-4-en-1,3-dione on development of the infection caused by tobacco mosaic virus in the leaves of two Nicotiana tabacum L. cultivars—an hypersensitive cultivar Xanthi-nc and systemically affected Samsun—was studied. The results suggest that this compound interferes with reproduction of the virus. This antiviral effect displayed the highest activity when applied in a mixture with the virus, presumably due to its action on both the plant and the virus.

In this study the negatively charged Proteus vulgaris O25 LPS was chosen for studying interaction with polycationic chitosan. The complex formation of LPS with chitosan was demonstrated using gradient centrifugation and laser interferometry method. The presented results have shown that laser interferometry method is sensitive enough for LPS–chitosan interaction studies. The changing in the ultra structure of LPS during binding with chitosan was observed by electronic microscope. The interaction of P. vulgaris O25 LPS with chitosan was shown to modulate significantly the biological activities of LPS. The toxicity of P. vulgaris O25 LPS decreased 10-fold after forming complexes with chitosan at injection to mice in the similar concentration of endotoxin. The complex LPS–chitosan was less effective than LPS alone in Limulus amabocyte lysate assay. Induction of TNF biosynthesis by LPS–chitosan complex was found to be 65% lower than that by parent LPS at concentration of 100 ng/ml.

The accumulation of potato virus X (PVX) in mesophyll cells of detached Datura stramonium L. leaves treated with fucoidan from brown algae Fucus evanescens C. Ag. have been evaluated by electron microscopy during the early infection period (three days after infection). It was found that cells of the leaves treated with fucoidan 24 h before infection accumulated virus less than untreated control. Ultrastructure-morphometric assay showed that fucoidan treatment causes an increase in the protein-synthesizing capability of cells (nucleolus dimention and amount of both mitochondria and rough endoplasmic reticulum membranes increased). At the same time, fucoidan treatment slightly activates the lytic compartment, which leads to the destruction of virus particles. Therefore, it may be considered that fucoidan induces a cellular defense mechanism that limits virus accumulation. Stimulation by the fucoidan of the formation of PVX-specific laminar structures able to bind virus particles may be another antiviral cell defense mechanism induced by the virus that prevents PVX reproduction and its intra- and intercellular transport.

We studied the influence of fucoidan from the brown alga Fucus evanescens on the development of infection induced by potato virus X (PVX) in Datura stramonium leaves. It is shown that 24 h after treatment of the leaves with fucoidan and following inoculation with PVX, the infectivity and amount of the virus in the leaves as well as the accumulation of viral particles in infected cells during early infection period were substantially less than those in the untreated control. Using ultrastructure-morphometric analysis, we established that the fucoidan treatment causes an increase in the protein-synthesizing capability of cells (nucleolus dimension and amount of both mitochondria and rough endoplasmic reticulum membranes increased). At the same time, the fucoidan treatment caused some stimulation of intracellular lytic processes that lead to destruction of virus particles and, therefore, may be considered as one of fucoidan-conditioned protective mechanisms limiting the virus accumulation in cells. Stimulation by fucoidan of the formation of PVX-specific laminated structures that may bind virus particles is another antiviral cell mechanism that limits reproduction and transportation of the virus.

The influence of κ/β-carrageenan from red marine alga Tichocarpus crinitus on the development of tobacco mosaic virus (TMV) infection in Xanthi-nc tobacco leaves was studied. It was shown that the number of necrotic lesions on the leaves inoculated with the mixture of TMV (2 μg/ml) and carrageenan (1 mg/ml) was reduced by 87%, compared to the leaves inoculated with the virus only. The suppression of virus infection was also observed when leaves were treated with carrageenan 24 h before or 24 h after leaf inoculation with TMV; however, in these cases, suppression was less evident than after inoculation with the virus-polysaccharide mixture. It is supposed that the antiviral activity of carrageenan applied together with TMV may be explained by its action not only on the plant but also on the virus itself. The inhibitory effect of carrageenan pretreatment can be explained by its favorable effect on tissue resistance to infection. The suppression of this resistance by actinomycin D indicates that carrageenan functions via its action on the cell genome.

Some peculiarities of the lipid composition of the outer membrane of the marine Gram-negative bacterium Chryseobacterium indoltheticum CIP 103168T have been studied. It is found that C. indoltheticum cells do not contain phosphatidylglycerol. Lipopolysaccharide could not be extracted by conventional methods; therefore, lipid A was isolated by hydrolysis of whole bacterial cells with 10% acetic acid with subsequent treatment with a chloroform: methanol mixture. The isolated lipid A has an unusual monosaccharide structure and constitutes 1-phosphate-D-glucosamine acylated with (R)-3-hydroxy-15-methylhexadecanoic and (R)-3-hydroxy-13-methyltetradecanoic acids at C2 and C3 atoms, respectively. The C. indoltheticum cells are shown to have a capsule; its polysaccharide apparently fulfils the LPS function.

This study examines the effects of selenium in concentrations of 0.01, 0.5, 1, 5, and 10 mg/liter on the growth and ultrastructure of the microalga Dunaliella salina. Selenium in concentrations of 0.01 and 0.5 mg/liter stimulated cell population growth, while the number of ultrastructural alterations was the same as in the control cells. At a selenium concentrations of 1 mg/liter, cell population growth slightly decreased by the end of the experiment, and there was some increase in the number of cells with damaged organoids and in the number of completely destroyed cells. As well, the excretory function of cell vacuoles was suppressed, and the autophagic activity of these vacuoles was activated to destroy the cytoplasm and nucleus. Concentrations of 5 and 10 mg/liter were toxic to D. salina, suppressing cell population growth and promoting extensive destructive changes. The threshold concentration of selenium for D. salina was 1 mg/liter, which is 1000 times greater than the maximum permissible concentration (MPC). The fact that the microalga was able to survive for several days in this concentration is indicative of its high resistance to selenium.

The effect of fucoidan (1.3; 1.4-α-L-fucan), a sulfated polysaccharide from the brown alga Fucus evanescens on the formation of specific granular and tubular inclusions induced by tobacco mosaic virus (TMV) and consisted presumably of the virus-coded protein components of the viral replicase was investigated in the TMV-infected leaves of tobacco (Nicotiana tabacum L.). In four days after inoculation of the leaves with a TMV preparation (1 mg/ml), the signature of infection in a presence of fucoidan (1 mg/ml) was a preferential formation of intracellular granular inclusions, which were related to early stages of the virus reproduction. When infected leaves were not treated with fucoidan, their cells contained mainly tubular inclusions, which were presumably formed from the granular ones on the last stages of the infection process. These observations demonstrated that fucoidan delayed the development of the TMV-induced infection.

The influence of culture conditions and plasmids on immunoglobulin (Ig)-binding activity of two isogenic strains of Yersinia pseudotuberculosis (plasmid-free strain 48(-)82(-) and strain 48(+)82(+) bearing plasmids pYV48 and pVM82) was studied. The highest activity was observed in the bacteria grown on glucose-containing liquid medium in the stationary growth phase. The Ig-binding activity of the bacteria cultured on the liquid medium at pH 6.0 was about 1.5-fold higher than that of the bacteria grown at pH 7.2. Expression of the Ig-binding proteins (IBPs) was most influenced by temperature of cultivation. The IBP biosynthesis was activated in the bacteria grown at 4 degrees C and markedly decreased in those grown at 37 degrees C. The Ig-binding activity of lysates from the bacteria was caused by proteins with molecular weights of 7-20 kD. The activities of the plasmid-free and plasmid-bearing Y. pseudotuberculosis strains (48(-)82(-) and 48(+)82(+), respectively) were analyzed, and the plasmids were shown to have no effect on the IBP expression and biosynthesis, which seemed to be determined by chromosomal genes.

The effect of fucoidan from the brown alga Fucus evanescens on the spread of infection induced by tobacco mosaic virus (TMV) was investigated in the leaves of tobacco (Nicotiana tabacum L.) of two cultivars (Ksanti-nk and Samsun). In the leaves of cv. Ksanti-nk inoculated with a mixture of TMV preparation (2 μg/ml) and fucoidan (1 mg/ml), the number of local necrotic lesions induced by the virus decreased by more than 90% as compared with the leaves inoculated with the virus alone. In tobacco leaves of cv. Samsun, virulence and the concentration of the virus 3 days after inoculation with the same mixture of TMV and fucoidan were by 62 and 66%, respectively, lower than in the leaves inoculated with TMV alone. As the infection spread, the inhibitory effect of fucoidan decreased. When the leaves were treated with fucoidan before and after the inoculation with TMV, its antiviral activity was less pronounced than when a mixture of the virus and the polysaccharide was used as inoculum. Electron microscopic investigation of TMV mixed with fucoidan often showed agglutinated virions. The highest virulence of the mixture (TMV preparation, 12 μg/ml, plus fucoidan, 1 mg/ml) was observed upon its twofold dilution, and after that it decreased. It was concluded that, when the leaves were inoculated with the mixture of TMV and fucoidan, the latter affected not only the plant but the virus as well. Treatment of tobacco leaves, cv. Ksanti-nk, with actinomycin D (10 μg/ml) 24 h before the inoculation with TMV almost completely suppressed the effect of fucoidan, indicating that fucoidan acted at a gene level.

The physicochemical properties and morphology of κ, κ/β, κ/ι, and λ-carrageenans from red algae of the families Gigartinaceae and Tichocarpaceae were analyzed and compared. The highest molecular weights (527 kDa) and negative electrokinetic potentials (–36.4 mV) were obtained for x-carrageenan, which had the conformation of a disordered coil whereas fine threads of κ and κ/β-carrageenans formed long fibrils of width up to 16 nm that were more heterogeneous in the latter instance whereas κ/ι-carrageenan formed a fibrous network structure.

It was found that in the Graafian oocytes of laboratory mice Mus musculus the population of electron-dense bodies contains two patterns of structures. One of these, designated as cortical granules, originated from the Golgi complex and was surrounded by a membrane. The other was discovered as cristae-containing mitochondrial derivatives lacked an outer membrane. It was found that the mitochondrial derivatives underwent progressive condensation and transformed into electron-dense bodies similar to germinal bodies of metazoan animals. Based on examination of Graafian follicle oocytes from 5 female individuals, about 15% of electron-dense bodies were cortical granules. However, about 85% of electron-dense bodies were condensing mitochondrial derivatives transforming into electron-dense bodies.

Certain physicochemical properties of glycoglycerolipids from marine alga Laminaria japonica (monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol) and their ability to be incorporated into immunostimulating complexes (ISCOMs) used for delivery of microbial and tumor antigens in vesicular form were comparatively described. These glycolipids proved to considerably differ by fatty acid composition, degree of unsaturation, and phase transition temperatures. Production of modified ISCOMs through incorporation of these glycolipids into the vesicle instead of the glycolipid component was demonstrated. Preliminary data demonstrated no significant increase in immune response to Yersinia pseudotuberculosis porin in the modified (with monogalactosyldiacylglycerol) and classical (with phosphatidylcholine) ISCOMs as compared to pure porin.

Some physicochemical properties of glycoglycerolipids (monogalactosyldiacylglycerol, digalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol) from the sea algae Laminaria japonica, as well as their ability to become incorporate into immunostimulating complexes (ISCOMs), used as a delivery system of microbial and tumor antigens in vesicular form, were studied. These glycolipids were found to differ essentially in fatty acid composition, unsaturation index and thermotropic behavior. The possibility of ISCOM modification by embedding the glycolipids studied instead of a phospholipid component in vesicles was shown. A preliminary research of the immunogenicity of the pore-forming protein from Yersinia pseudotuberculosis in modified (by monogalactosyldiacylglycerol) and typical (egg phosphatidylcholine) ISCOMs did not reveal a significant enhancement of immune response in comparison with that of isolated protein.

The effect of chitosan on the development of infection caused by Tobacco mosaic virus (TMV) in leaves of Nicotiana tabacum L. cv. Samsun has been studied. It was shown that the infectivity and viral coat protein content in leaves inoculated with a mixture of TMV (2 μg/mL) and chitosan (1 mg/mL) were lower in the early period of infection (3 days after inoculation), by 63% and 66% respectively, than in leaves inoculated with TMV only. Treatment of leaves with chitosan 24 h before inoculation with TMV also caused the antiviral effects, but these were less apparent than when the virus and polysaccharide were applied simultaneously. The inhibitory effects of the agent decreased as the infection progressed. Inoculation of leaves with TMV together with chitosan considerably enhanced the activity of hydrolases (proteases, RNases) in the leaves, in comparison with leaves inoculated with TMV alone. Electron microscope assays of phosphotungstic acid (PTA)-stained suspensions from infected tobacco leaves showed that, in addition to the normal TMV particles (18 nm in diameter, 300 nm long), these suspensions contained abnormal (swollen, "thin" and "short") virions. The highest number of abnormal virions was found in suspensions from leaves inoculated with a mixture of TMV and chitosan. Immuno-electron microscopy showed that "thin" virus particles, in contrast to the particles of normal diameter, lost the ability to bind to specific antiserum. It seems that the chitosan-induced activation of hydrolases stimulates the intracellular degradation of TMV particles and hence hydrolase activation may be considered to be one of the polysaccharide-mediated cellular defense mechanisms that limit virus accumulation in cells.

SummaryThe germ plasm-related structures (GPRS) and the transformation that occurs to them during the spermatogenesis of the sea urchin Anthocidaris crassispina were studied by electron microscopy and morphometry. The GPRS were observed in spermatogonia and spermatocytes, but not in spermatids and sperm, which suggests an important role for these structures during the onset of meiosis. It was proposed that the germinal granules are fragmented into the compact electron-dense nuage, and fragments of the latter penetrate into the periphery of the compact electron-lucent nuage. The process of nuage integration is completed with the formation of the combined nuage, which aggregates some mitochondria into clusters. Once formed, the mitochondrial clusters undergo dissemination and assume the appearance of the dispersed nuage with mitochondrial derivatives, which in turn develops into the scattered nuage. The scattered nuage, which presumably presents the composite mixture saturated with mitochondrial matrix, terminates the GPRS transformation.

The fragmentation of the biologically active 1,3;1,6-beta-D-glucan Antivir by endo-1,3-beta-D-glucanase LIV from crystalline styles of the marine mollusk Spisula sachalinensis was carried out. It was found that low molecular mass oligomers possessing a stabilizing effect on membranes and anti-viral activity against tobacco mosaic virus appeared in the process of enzymatic hydrolysis of Antivir. Biological activity of 1,3;1,6-beta-D-glucooligo- and polysaccharides was found to be associated with molecular mass (polymerization degree (n) not less than 14) and with presence of intralinked beta-1,6-connected monosaccharide residues. Probably, decrease in molecular mass is compensated by increase in number of intralinked beta-1,6-connected monosaccharide residues.

Glucan preparations were obtained by transformation of laminaran from the alga Laminaria cichorioides with endo-β-1,3-glucanase from marine mollusks. These preparations, like those of other sources, have an inhibitory effect on tobacco mosaic virus (TMV) infection of detached leaves of local and systemic host tobacco plants.

Phagocytic resorption during spermatogenesis was studied in the sea urchin Anthocidaris crassispina. Nutritive phagocytes in gonad absorbed both waste sperm cells and residual bodies discarded from maturing spermatids, and these materials were subsequently compartmented in heterophagosomes. Based on 180 heterophagosomes examined by transmission electron microscopy, over 99% of heterophagosomes contained either residual bodies or sperm cells only. Simultaneous resorption of sperm cells and residual bodies in a heterophagosome was uncommon, with only ∼0.56% occurrence, suggesting that heterophagosomes have a selective resorption ability in nutritive phagocytes.

Actinomycin D (AMD), 50, 10, and 1 μg/ml, stimulated potato virus X (PVX) multiplication in detached Datura stramonium L. leaves floated on water. Increased virus synthesis was observed upon treatment of the leaves with AMD at 2, 6, and 24 hr before, or at the time of, inoculation as well as 24 hr later. Treatment of leaves with AMD prior to inoculation proved most effective. The stimulating effect depended on both AMD concentration and time; it increased with concentration changes of from 1 to 50 μg/ml, as well as with lengthier treatment in AMD solutions up to 24 hr. Increased PVX multiplication in AMD-treated leaves was observed by bioassay of virus on Gomphrena globosa L. leaves, testing both the homogenates and a partially purified viral preparation obtained from inoculated D. stramonium leaves. The stimulated PVX multiplication in AMD-treated D. stramonium leaves was observed in the presence of suppressed cellular metabolism; this was testified to by data obtained with the aid of electronic paramagnetic resonance (EPR) measurements. Possibly, increased PVX multiplication in AMD-treated D. stramonium leaves occurs as a result of the DNA function being suppressed by the antibiotic causing an interruption in cellular syntheses, thus leading to the primary use of the cell energy resources and metabolic precursors in viral synthesis.

It is shown that κ/s-carrageenan obtained from red marine alga Tichocarpus crinitus possesses antiviral activity against Tobacco mosaic virus (TMV). Two days after inoculation of Nicotiana tabacum L. cv. ‘Samsun’ detached leaves with a mixture of TMV (1 μ/ml) and carrageenan (1 mg/ml), the infectivity and virus content in the leaves as well as the intracellular accumulation of virus particles were significantly inhibited in comparison with leaves inoculated with TMV only. The carrageenan applied 24 h prior to inoculation with TMV also exhibited the antiviral effects but to a lower degree than when the virus and polysaccharide were inoculated together. As the infection developed, the inhibitory effects of carrageenan decreased. Treatment of the leaves with carrageenan affected the formation of TMV-specific intracellular inclusions (granular and tubule) known to consist of the virus-encoded protein components of the viral replicase. A characteristic of the infection development in the presence of carrageenan, 4 days after infection of the leaves with TMV, was the predominant formation in cells of granular inclusions famous to appear at early stages of TMV multiplication. At the same time, cells of the leaves inoculated with TMV alone contained for the most part tubule inclusions considered to arise from granular ones on the advanced infection stages. Taken together, our observations indicate that the κ/s-carrageenan inhibits TMV infection in detached tobacco leaves at early stages.