Liliana Gigova

Cancer Protective Action of Polysaccharide, Derived from Red Microalga Porphyridium Cruentum—A Biological Background

ABSTRACT The cell wall sulfated polysaccharide of the red microalga Porphyridium cruentum (Rhodophyta) (PcrPSH) exhibited strong antitumor activity against Graffi myeloid tumor in hamsters both in vitro and in vivo. When tested in vivo, depending on the concentration and the way of application, this polysaccharide decreased transplantability in all experimental groups till 20 days of observation and mortality rate. The tumor growth was retarded and the mean survival time was prolonged with 10 - 16 days. Applied in in vitro experiments the PcrPSH increased both—spreading and phagocytic ability of peritoneal macrophages from healthy and Graffi tumor bearing hamsters in a dose—dependent manner. Primary Graffi tumor cell culture, cultivated in the presence of PcrPSH showed significant decrease of cell viability, determined by MTT test, while in cells derived from bone marrow it was increased at the same conditions of cultivation and concentration of polysaccharide. Primary Graffi tumor cell culture, treated with PcrPSH showed the appearance of a characteristic DNA ladder on agarose gel electrophoresis, which is a biochemical hallmark of apoptosis. The manifested anticancer activity of PcrPSH could be associated with its immunostimulating action as well as with direct cytotoxic properties. Based on these results, we could suggest that the tested algal PcrPSH is a promising candidate as an antitumor agent. Further studies will be done to clarify the mechanisms of a biological action of PcrPSH.

RESPONSE OF TRACHYDISCUS MINUTUS (XANTHOPHYCEAE) TO TEMPERATURE AND LIGHT1

The effects of different temperatures and light intensities on growth, pigments, sugars, lipids, and proteins, as well as on some antioxidant and proteolytic enzymes of Trachydiscus minutus (Bourr.) H. Ettl, were investigated. The optimum growth temperature and light intensity were 25°C and 2 × 132 μmol photons · m−2 · s−1, respectively. Under these conditions, proteins were the main biomass components (33.45% dry weight [dwt]), with high levels of carbohydrates (29% dwt) and lipids (21.77% dwt). T. minutus tolerated temperatures between 20°C and 32°C, with only moderate changes in cell growth and biochemical composition. Extremely low (15°C) and high (40°C) temperatures decreased chl and RUBISCO contents and inhibited cell growth. The biochemical response of the alga to both unfavorable conditions was an increase in lipid content (up to 35.19% dwt) and a decrease in carbohydrates (down to 13.64% dwt) with much less of a change in total protein content (in the range of 30.51%–38.13% dwt). At the same time, the defense system of T. minutus was regulated differently in response to heat or cold treatments. Generally, at 40°C, the activities of superoxide dismutase (SOD), catalase (CAT), and proteases were drastically elevated, and three polypeptides were overexpressed, whereas the glutathione reductase (GR) and peroxidase (POD) activities were reduced. In contrast, at 15°C, all these enzymes except GR were suppressed. The effect of light was to enhance or decrease the temperature stress responses, depending on intensity. Our studies demonstrate the broad temperature adaptability of T. minutus as well as the potential for the production of valuable algal biomass.

Antitumor activity of C-phycocyanin from Arthronema africanum (Cyanophyceae)

ABSTRACT Pure C-phycocyanin (C-PC) was isolated from Arthronema africanum to evaluate its potential antitumor effects in vivo and in vitro. Experimental myeloid Graffi tumor in hamsters was used as a model. The cell proliferation assay showed that C-PC treatment, at concentration of 100 µg mL -1 for 24 h, significantly inhibited the growth of Graffi tumor cells (51.4% viability). Agarose gel electrophoresis of the genomic DNA of treated cells displayed time- and concentration-dependent fragmentation pattern, typical for apoptosis. Apoptotic process was related to the increase in cellular manganese and copper/zinc superoxide dismutases and glutathione reductase activities, coupled with a low catalase activity. In vivo C-PC administration (5.0 mg kg -1 body weight) suppressed the tumor transplantability and growth, while the mean survival time of the tumor-bearing hamsters was increased. The results revealed promising antitumor activities of A. africanum C-PC and suggested the potential of this natural biliprotein pigment for future pharmacological and medical applications. The study provided new data on the mechanism of the C-PC-induced apoptosis in which the imbalance of antioxidant enzymes that favoured hydrogen peroxide accumulation might play a leading role. Key words: Antitumor activity, Arthronema africanum , In vivo , In vitro , C-pycocyanin, Myeloid Graffi tumor

Biological activity of microalgae can be enhanced by manipulating the cultivation temperature and irradiance

The escalating levels of antibiotic resistance among pathogenic bacteria and the side effects of chemotherapeutic drugs in use forced the efforts of scientists to search for natural antimicrobial and anticancer substances with novel structures and unique mechanism of action. Focusing on bioproducts, recent trends in drug research have shown that microalgae (including the cyanobacteria) are promising organisms to furnish novel and safer biologically active compounds. Many microalgal metabolites have been found to possess potent antibacterial, antifungal, antiviral, anticancer and antiinflammatory activities, as well as antioxidant, enzyme inhibiting and immunostimulating properties. In this paper, the studies on the biological activity of microalgae associated with potential medical and pharmaceutical applications are briefly presented. Attention is focused on the impact of cultivation temperature, irradiance and growth stage on the biomass accumulation, activity and pathways of cell metabolism and the possibilities of using these variable factors to increase the diversity and quantity of biologically active substances synthesized by microalgae.

Dimerization of Rhizobium meliloti NifH protein in Saccharomyces cerevisiae cells requires simultaneous expression of NifM protein.

Compared to free living diazotrophs, the nitrogenase system of symbiotic microorganisms, like Rhizobium (Synorhizobium) meliloti, was poorly studied. The aim of our research was to investigate whether (by analogy with Klebsiella pneumoniae) the NifM product is required and sufficient to obtain active R. meliloti Fe-protein. We cloned nifH gene of R. meliloti and nifM gene of K. pneumoniae in suitable yeast vectors. When introduced into Saccharomyces cerevisiae cells, both genes were effectively expressed to proteins similar to the native products in its immunoreactivity and apparent molecular mass. The association of R. meliloti NifH protein into dimer structure required co-expression of NifM that also conferred stability of NifH polypeptide. However, the NifH protein synthesized in yeast did not show enzyme activity, suggesting that the NifM of K. pneumoniae is incapable of activating the NifH protein of R. meliloti.

Localization of nif genes on large plasmids in Rhizobium galegae

Plasmid DNA of six strains of Rhizobium galegae was blotted onto nitrocellulose and hybridized with the 4.8 kb PstI fragment of pRme4lb, a megaplasmid carrying the nifH and the nifD genes of Rhizobium meliloti. DNA sequences homologous to the nif genes were localized on the megaplasmid or on the large plasmid bands of the R. galegae strains tested. In three of the strains analysed the nif genes were located on the megaplasmids. In the other three strains investigated, which also possessed megaplasmids, the nif genes were located on the smaller plasmids.

Cytotoxic and Apoptogenic Potential of Red Microalgal Polysaccharides

ABSTRACT The efforts of scientists are aimed at finding anti-cancer agents of natural origin which have high biological activity, low toxicity and broad spectrum of therapeutic activity. This study was designed to determine the cytotoxic properties of polysaccharides derived from the red microalgae Dixoniella grisea and Porphyridium cruentum and to elucidate the mechanism of their action on two permanent human cell lines MCF-7 (breast adenocarcinoma) and HeLa (cervical carcinoma), as well as on primary culture from Graffi myeloid tumor in hamsters. Our investigations indicated that both algal polysaccharides showed high cytotoxic and apoptogenic activities on cancer cells and may be a promising alternative to synthetic substances.

Microalgae respond differently to nitrogen availability during culturing

Variations in the exogenous nitrogen level are known to significantly affect the physiological status and metabolism of microalgae. However, responses of red, green and yellow-green algae to nitrogen (N) availability have not been compared yet. Porphyridium cruentum, Scenedesmus incrassatulus and Trachydiscus minutus were cultured in the absence of N in the medium and subsequent resupply of N to the starved cells. Culture growth and in-gel changes in isoenzyme pattern and activity of glutamate synthase, glutamate dehydrogenase, malate dehydrogenase, aspartate aminotransferase, superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase were studied. The results demonstrated that the algae responded to the fully N-depleted and N-replete culture conditions by species-specific metabolic enzyme changes, suggesting differential regulation of both enzyme activity and cellular metabolism. Substantial differences in the activities of the antioxidant enzymes between N-depleted and N-replete cells of each species as well as between the species were also found. In the present work, besides the more general responses, such as adjustment of growth and pigmentation, we report on the involvement of specific metabolic and antioxidant enzymes and their isoforms in the mechanisms operating during N starvation and recovery in P. cruentum, T. minutus and S. incrassatulus.

Growth, biochemical and enzymatic responses of thermal cyanobacterium Gloeocapsa sp. (Cyanophyceae) to temperature and irradiance

The present study describes a strain of Gloeocapsa sp. designated as Gacheva 2007/R‐06/1, originally isolated from a geothermal flow located in Rupite, Bulgaria. To evaluate whether this cyanobacterium is locally adapted to hot environment or has the ability to tolerate lower temperatures, its growth, biochemical composition, enzyme isoforms and activity of the main antioxidant enzymes and proteases were characterized under various temperatures and two irradiance levels. The strain was able to grow over the whole temperature range (15–40°C) under two different photon fluence densities – 132 μmol photons m−2 s−1 (unilateral, low light, LL) and 2 × 132 μmol photons m−2 s−1 (bilateral, high light, HL). The best growth occurred at either 34°C and LL or at 36°C and HL, but significant growth inhibition was noted at 15°C and 40°C. Low temperature treatment (15°C) resulted in higher levels of total protein and an increased activity of manganese superoxide dismutase (MnSOD) and glutathione reductase, as compared to optimum growth temperatures. After simultaneous exposure to 15°C and HL, increases in lipid content and activity of iron superoxide dismutase and catalase (CAT) were also observed. Cultivation of cells at 40°C enhanced MnSOD, CAT and peroxidase activities, regardless of irradiance level. Increased total protein content and protease activity at 40°C was only associated with the HL treatment. Overall, these results indicate that Gloeocapsa sp. strain Gacheva 2007/R‐06/1 used different strategies to enable cells to efficiently acclimate and withstand adverse low or high temperatures. This strain obviously tolerates a wide range of temperatures below its natural habitat temperature, and does not seem to be locally adapted to its original thermal regime. It behaved as a thermotolerant rather than a thermophilic cyanobacterium, which suggests its wider distribution in nature.

Free-living and symbiotic characteristics of plasmid-cured derivatives of Rhizobium galegae

Rhizobium galegae strain NBIMTC2250 contains two plasmids one of which (the larger one) is the symbiotic plasmid. To determine the participation of the smaller plasmid in cellular functions and in the symbiosis, we used a positive selection system for isolation of derivatives cured of this plasmid. The derivatives did not show differences from the wild type with respect to the following free-living cellular properties: antibiotic-resistance pattern, carbohydrates and organic acid utilization, growth rate and colony morphology in pure culture, lipopolysaccharide (LPS) profile, motility and sensitivity to extreme temperatures, which indicated that these functions were not plasmid encoded. Some of the cured derivatives differed from the wild type in the symbiotic characteristics, and the possible reason for the differences was discussed. In these cases reintroduction of the plasmid did not restore the derivatives to a wild type but the plasmid-free and plasmid-recontaining derivatives had similar symbiotic phenotype. Hybridization study showed that the smaller plasmid had been lost integrally in all cured derivatives. These results and the fact that almost half of the plasmid-cured derivatives have the symbiotic characteristics similar to those of the parent strain suggested that the smaller plasmid of strain Rhizobium galegae NBIMTC2250 did not participate in the symbiotically important functions of this strain.