Growth and spoilage metabolites production of a mesophilic Aeromonas salmonicida strain in Atlantic salmon (Salmo salar L.) during cold storage in modified atmosphere.

AIMS: The aim of the study was to quantify the growth kinetic parameters and spoilage-associated metabolites of an inoculated strain of Aeromonas salmonicida in pre-rigor filleted Atlantic salmon (Salmo salar L.) stored in vacuum (VP) or modified atmosphere (MAP 60/40% CO2 /N2 ) at 4 and 8°C. METHODS AND RESULTS: The maximum growth rate of A. salmonicida in VP salmon stored at 4°C was 0·56 ± 0·04 day-1 with no detectable lag-phase and the concentration of Aeromonas reached 8·33 log CFU per g after 10 days. The growth rates and maximum population density of Aeromonas in MAP salmon were lower but the applied atmosphere did not inhibit the growth. A selection of metabolites associated with fish spoilage were quantified using 1 H nuclear magnetic resonance (NMR) spectroscopy. The concentration of trimethylamine (TMA) was significantly affected by storage time and temperature, packaging atmosphere and inoculation with A. salmonicida (General Linear Model (GLM), P < 0·001 for all factors). CONCLUSION: The study presents preliminary results on A. salmonicida as a potential spoilage organism in vacuum-packaged salmon during cold storage. The combination of refrigeration and a packaging atmosphere consisting of 60/40 % CO2 /N2 did not completely inhibit the growth but prevented the formation of TMA. SIGNIFICANCE AND IMPACT OF THE STUDY: Little information is available on the spoilage potential of Aeromonas spp. in minimally processed salmon products under different packaging conditions. The study clearly demonstrates the importance of hurdle technology and provides data to further elucidate the significance of Aeromonas spp. as a spoilage organism.

Role of acid sphingomyelinase in the regulation of mast cell function.

BACKGROUND: Degranulation of mast cells is stimulated by store-operated Ca(2+) -entry (SOCE). In other cell types, Ca(2+) -entry is modified by ceramide. Exogenously added ceramide has been shown to trigger mast cell apoptosis. Effects of endogenously produced ceramide in mast cells remained, however, elusive. Ceramide may be produced from sphingomyelin by acid sphingomyelinase (Asm). OBJECTIVE: This study explored the impact of Asm on mast cell functions. METHODS: Mast cells were isolated from bone marrow (BMMCs) or peritoneal lavage of gene-targeted mice lacking Asm (asm(-/-)) and their wild-type littermates (asm(+/+)). BMMC maturation and apoptosis-associated annexin V binding were determined by flow cytometry. Asm activity was assessed enzymatically, cytosolic Ca(2+) activity ([Ca(2+)]i) utilizing Fura-2 fluorescence, current across the cell membrane by whole-cell patch clamp, degranulation from hexosaminidase-release and migration utilizing a transwell chamber. In vivo anaphylaxis was derived from decrease in body temperature. RESULTS: Peritoneal mast cell number, BMMC phenotype, spontaneous BMMC apoptosis as well as BMMC CD117, CD34 and FcεRI expression were similar in both genotypes. In asm(+/+) BMMCs, stimulation with antigen resulted in a fast ~2.5-fold increase in Asm activity. Release of Ca(2+) from internal stores and hence several Ca(2+) -dependent functions were strongly impaired in asm(-/-) BMMCs. Thus, antigen-induced increase in [Ca(2+)]i in IgE-sensitized cells, antigen- but not ionomycin-induced currents through Ca(2+) -activated K(+) -channels (KCa 3.1), IgE/antigen-triggered ß-hexosaminidase release, and antigen-induced migration were all lower in asm(-/-) BMMCs than in asm(+/+) BMMCs. Pharmacological inhibition of Asm by amitriptyline (500 nm, 3 h) in asm(+/+) BMMCs similarly decreased antigen-induced increase in [Ca(2+)]i , KCa 3.1 currents, ß-hexosaminidase release and migration. The decrease in body temperature upon the induction of systemic anaphylaxis was significantly less pronounced in asm(-/-) mice than in asm(+/+) mice, an observation pointing to in vivo significance of Asm. CONCLUSIONS AND CLINICAL RELEVANCE: Asm is a novel, powerful regulator of mast cell function and thus a potential target in the treatment of allergic reactions.

[Recombinant α-2 interferon in preventing the progression of melanoma].

A brief history of interferon application in the treatment of cutaneous melanoma is represented.

[Potential of prevention of metastasizing with the aid of tumor-specific transfer-factor].

The report discusses our experimental data in support of biotherapy which uses chemotherapy and antitumor immune treatment with in vivo xenogenic transfer-factor polypeptides (TFP) isolated from lymphocytes sensitized to antigens of given tumor. After excision of primary tumor--lung carcinoma of Lewis--mice C57BL/6 were injected intraperitoneally with xenogenic TFP (200 pg/body, twice) and a cytostaic dose of cyclophosphamide. Such adjuvant chemotherapy was found to prevent metastases from spreading to the lung in 100%. The marked anti-metastatic effect of the treatment correlated with recovery of splenic cell mass and its cellular structure, higher levels of large granular lymphocytes in peripheral blood and enhanced functional activity of cytotoxic cells in vitro. Our results point to a possibility of raising efficacy of treating solid malignancies with adjuvant chemotherapy in combination with adoptive immune therapy.

[Transfer factor specific to mouse lung carcinoma of Lewis].

Factor of transfer was identified and sampled in the course of immunization of rats with cells or tissues of mouse lung carcinoma of Lewis and its antitumor action investigated on models of passive and spontaneous dissemination in mice C57BL/6. Samples obtained at the peak of immunological response (day 14) and immunological memory inception (day 60) were shown to be capable of marked antimetastatic effects: fall in metastatic frequency, number and size of metastases correlated with higher index of metastasis formation.

[Inhibition of tumor growth in rat liver induced by tumor-specific transfer factor].

A transfer factor (TF) specific to antigens of Geren's carcinoma in rat was developed using an original model of intraorganic growth. Intravenous injection (1pg/g body) inhibited primary tumor node growth in the liver by 78% and blocked dissemination to peritoneal viscera. Its effect was due to an immunospecific component promoting antitumor immunological response. The latter presented as generation of cytotoxic effector cells, vascular disorders in tumor parenchyma thus blocking tumor cell proliferation. Possible applications of tumor-specific TF for biotherapy of cancer patients are discussed.

[Antitumor immunity transfer by a factor isolated from lymphocytes of tumor bearer].

Low-molecular extracts (LMEs) of lymphocytes were obtained from spleen of noninbred rats using our experimental model of intraorganic growth of Guerin's carcinoma. They were intended to transfer immune reaction to tumor antigens in vitro. It was LMEs developed prior to tumor progression in spleen that showed immunospecific activity with respect to tumor cells. Also, they had marked antigen-independent immunopharmacological activity. Single intravenous injection of LMEs100 pg given on day 7 of tumor growth stimulated antitumor resistance in intact rats within a short time. It prevented tumor cells engrafting in 60% of tumors. A tumor-specific factor has been evolved capable of immune reaction transfer to tumor antigens in vitro thus preventing tumorigenesis in recipients in vivo.

[Morphological changes in immunogenesis organs in transplantation of Guerin carcinoma to the spleen]. / Morfologicheskie izmeneniia v organakh immunogeneza pri transplantatsii kartsinomy Gerena v selezenku.

The study was made in 30 male Wistar rats (b.m. 110+/-10 g) with transplanted Guerin carcinoma to their spleen. Morphological changes in the tumour, spleen, thymus and mesenteric lymph nodes on tumour growth day 7, 10, 14, 18, 22, 25 were studied. The elaborated experimental model of tumour growth in the spleen allowed to detect tumor-specific changes in organs of immunogenesis as well as the dynamics of their development. The findings show possibility of renewal of both cellular content in the tumour-activation of proliferation, and in tissue of the spleen-blast transformation with predominance of lymphoblasts. Morphologic changes in the thymus and regional lymph nodes are different this meaning that changes in the organs of immunogenesis are not of a systemic character, their reaction to the presence of tumour in organism is not always the same and has its own features.

[Immune response in non-inbred rats after implantation of Geren carcinoma into their spleens]. / Immunologicheskaia reaktivnost' u nelineinykh krys na modeli vnutriselëzenochnogo rosta kartsinomy Gerena.

Formation of specific immune response of Geren's carcinoma (GC) versus its biological properties was studied in non-inbred rat spleen. GC growth was accompanied by mounting immune response to tumor-associated antigens (TAA) which was evaluated by macrophage adherence inhibition test. Increase in cell-mediated response to such antigens passed through stages determined by the peculiarities of GC growth in spleen tissues. Immune response to TAA was reported in half the animals as the cells were taking. Enhanced proliferative pool of tumor, aneuploidy and DNA index offset by the low level of apoptosis were characteristic of tumor progression stage. The combination of those factors served as a backdrop for immunologic tolerance development in all the animals. Immune response resurfaced as apoptosis intensified during generalized tumor growth when cell proliferation in the parenchyme declined.

Sodium channel activity in leukemia cells is directly controlled by actin polymerization.

The actin cytoskeleton has been shown to be involved in the regulation of sodium-selective channels in non-excitable cells. However, the molecular mechanisms underlying the changes in channel function remain to be defined. In the present work, inside-out patch experiments were employed to elucidate the role of submembranous actin dynamics in the control of sodium channels in human myeloid leukemia K562 cells. We found that the application of cytochalasin D to the cytoplasmic surface of membrane fragments resulted in activation of non-voltage-gated sodium channels of 12 picosiemens conductance. Similar effects could be evoked by addition of the actin-severing protein gelsolin to the bath cytosol-like solution containing 1 microm [Ca(2+)](i). The sodium channel activity induced by disassembly of submembranous microfilaments with cytochalasin D or gelsolin could be abolished by intact actin added to the bath cytosol-like solution in the presence of 1 mm MgCl(2) to induce actin polymerization. In the absence of MgCl(2), addition of intact actin did not abolish the channel activity. Moreover, the sodium currents were unaffected by heat-inactivated actin or by actin whose polymerizability was strongly reduced by cleavage with specific Escherichia coli A2 protease ECP32. Thus, the inhibitory effect of actin on channel activity was observed only under conditions promoting rapid polymerization. Taken together, our data show that sodium channels are directly controlled by dynamic assembly and disassembly of submembranous F-actin.

Influence of coprecipitation and interactions with anionic surfactants on the activity of plant phospholipase D.

The effects of two surfactants (sodium dodecyl sulfate and sodium dodecyl dioxyethylene sulfate) of similar structure but differing water solubility (of their calcium salts) on the enzymatic activity of cabbage phospholipase D have been studied. The solubility difference is insignificant because the two surfactants activate phospholipase D similarly. To elucidate the mechanism of their influence on the enzyme, the phase behavior in the reaction media and the interactions of the surfactants with the enzyme were investigated by potentiometry and by light scattering and UV spectroscopy. Calcium dodecyl dioxyethylene sulfate (which is more soluble in water than calcium dodecyl sulfate) precipitates in the presence of phosphatidylcholine, the substrate of the enzymatic reaction. In the reaction media phospholipase D was involved into a precipitate consisting of calcium salts of the surfactants and phosphatidylcholine that might be interpreted as its immobilization. In addition, the surfactants were adsorbed on the enzyme, unfolding the globular enzyme molecule due to electrostatic repulsion between adsorbed surfactant anions. The observed increase in the functional activity of phospholipase D is accounted for by transfer to an optimal tertiary structure for the enzyme molecule in the course of consecutive conformational transitions induced by the surfactants.

Effects of surfactants on the activity of phospholipase D.

We studied the dependence of the activity of cabbage phospholipase A on the substrate (phosphatidylcholine) the aggregated state of which is regulated by addition of either anionic (sodium dodecyl sulfate, cholate or oleate) or cationic (cetyl-trimethylammonium bromide) surfactants. Activation of the enzyme induced by anionic surfactants was shown to correlate with the size of their polar groups. The phospholipase hydrolase activity correlated with the transformation of multilayer liposomes into micelles. The relationship between the processes was of a complex character. The dependence of the amount of enzymically released choline on the calcium concentration passed through a sharp maximum in the presence of the anionic detergents and monotonically increased in the presence of the cationic detergent. In the former case, the sharp increase in the enzyme activity was suggested to be caused by precipitation of phospholipase D with the anionic detergent calcium salt, which can be considered as a specific type of immobilization.

[The clinico-immunological characteristics of myasthenia patients]. / Kliniko-immunologicheskaia kharakteristika bol'nykh miasteniei.

Data are submitted of examination of 480 patients with myasthenia. The host protective mechanisms were studied, with the immunodeficiency (both cellular and humoral) having been ascertained. The above event were found out to be associated with severity of myasthenia, being more apparent in gravely ill patients.