Effect of stressful conditions on the carotenogenic activity of a Colombian strain of Dunaliella salina

The objective was evaluate the carotenogenic activity of Dunaliella salina isolated from the artificial salt flats of municipality of Manaure (Department of La Guajira, Colombia). Two experimental testings were designed, in triplicate, to induce the reversibility of the cell tonality depending on the culture conditions. In the first test (A), to induce the reversibility from green to red tonality in D. salina cells, these were cultured in J/1 medium at a concentration of 4.0 M NaCl, 390 µmol m^?2 s^?1, 0.50 mM KNO₃. In the second test (B), to induce the reversibility from red to green cell tonality, the cultures were maintained in J/1 medium 1 M NaCl, 190 µmol m^?2 s^?1, 5.0 mM KNO₃ and pH 8.2. The population growth was evaluated by cell count and the pigment content was performed by spectrophotometric techniques. It was found that in both tests the culture conditions influenced the population growth and the pigments production of D. salina. There was a significant difference between the mean values of total carotenoids in the test A with 9.67 ± 0.19 μg/ml and second test with 1.54 ± 0.08 μg/ml at a significance level of p < 0.05. It was demonstrated that the culture conditions of test A induce the production of lipophilic antioxidants, among these carotenoids. The knowledge of the stressful conditions for the production of carotenoids from D. salina isolated from artificial saline of Manaure opens a field in implementation of this biotic resource for biotechnological purposes, production of new antibiotics, nutraceuticals and/or biofuels production.     

Influence of stress factors on growth and pigment production in three Dunaliella species cultivated outdoors in flat-plate photobioreactors

Abstract

Growth and production of carotenoid in three Dunaliella species (Dunaliella salina (Dunal) Teodoresco, Dunaliella bardawil Ben-Amotz & Avron and Dunaliella sp.) were investigated using flat-plate photobioreactors in outdoor conditions with two optical paths (3?cm and 5?cm). The experiment was conducted in duplicate and lasted four weeks during which light intensity, temperature, pH and optical density were checked daily. The pigment production (total carotenoid and chlorophyll a) was monitored every two days. To induce an additional stress besides temperature and light intensity, two different salt concentrations were used, i.e. 6% and 8% NaCl. The highest growth in all treatment groups was noticed for Dunaliella sp. followed by D. bardawil and D. salina. D. salina produced a higher content of carotenoid concentrations corresponding to 5?cm/8% and 5?cm/6% groups; 779.102?±?0.434?μg.mL^?1 and 694.326?±?0.098?μg.mL^?1 were registered at the end of the experiment. The same species had also greater content of β-carotene.     

Characterization of wheat root ribosomes isolated by Mg2+ precipitation

The Mg²⁺ precipitation method has been adapted for isolation of ribosomes from roots of wheat. The ribosomes prepared by this procedure show A₂₆₀/A₂₈₀ = 1.6 and A₂₆₀/A₂₃₅ = 1.3 and contain 44d% RNA and 56% ribosomal proteins. There are no detectable differences in the ribosomal protein complement and accessibility of the ribosomal proteins to phosphorylation between ribosomes isolated by this procedure and those prepared by classical ultracentrifugation methods. The ribosomes are active in a poly-U directed cell-free system for protein synthesis.     

Effect of culture medium on hydrogen production by sulfur-deprived marine green algae Platymonas subcordiformis

To study the effect of culture medium on hydrogen production by the marine green algae, Platymonas subcordiformis under sulfur deprivation, cell growth, hydrogen production, and starch and protein catabolism was investigated in the work. Algae cells cultured only in optimized medium required 6～8 days to reach the late logarithmic at the approximate density of (2.00 ± 0.18) × 10⁶ cells/mL, which in traditional medium needed 18～22 days to reach (1.85 ± 0.20) × 10⁶ cells/mL. Increased levels of Chlorophyll (10.74 ± 0.20 μg/mL), starch (149.50 ± 6.15 μg/mL), and protein (213.00 ± 7.36 μg/mL) were accumulated in optimized medium, which were 1.06, 1.47, and 1.87-fold of the algae cells cultured in traditional medium, respectively. The sealed culture of algae cells in sulfur-deprived optimized medium shifted to anaerobic conditions after 96 h of light illumination and produced 0.45 ± 0.12 mL H₂, but in traditional medium maintained aerobic condition and no hydrogen was produced. In addition, changes in starch and protein content during continuous light illumination indicated that more endogenous substrate was consumed in the sulfur-deprived optimized medium than that in the sulfur-deprived traditional medium.     

Isolation of total RNA from baker’s yeast

A simple method of production of total RNA from baker’s yeast was developed. Total RNA was isolated from yeast (Saccharomyces cerevisiae) biomass using lysis with sodium dodecyl sulfate at 100°C for 40–60 min and subsequent precipitation of the target product with 3 M NaCl. The preparation obtained was characterized in detail: yield of total RNA from 1 kg of pressed yeast, 9.25 g; optical density at 260 nm of 1 mg of RNA dissolved in 1 ml of water, 20.2 U; content of the acid-soluble fraction, 2.02%; and protein content, 1.8%. Total tRNA was isolated from total RNA by fractional precipitation with ethanol followed by gel filtration.     

Neutron scattering study of the 13 S fragment of 16 S RNA and its complex with ribosomal protein S4

A fragment with a molecular weight of 170,000 and a sedimentation coefficient of 13 S which is capable of specifically binding ribosomal protein S4 has been obtained by digestion of Escherichia coli 16 S RNA with ribonuclease A. The 13 S fragment of 16 S RNA and its complex with protein S4 have been studied by different physical methods; in the first place, by neutron scattering. It has been shown that this fragment is very compact in solution. The radii of gyration of this fragment (50 ± 3 Å) and of protein S4 within the complex (17 ± 3 Å) coincide, within the limits of experimental error, with the radii of gyration for the free RNA fragment (47 ± 2 Å) and the free ribosomal protein S4 in solution (18 ± 2 Å). Hence the conclusion is drawn that the compactness of the RNA fragment and the ribosomal protein does not change on complex formation. The compact 13 S fragment of 16 S RNA is shown to be contrast-matched in solvent containing 70% ²H₂O which corresponds to a value for the partial specific volume of RNA of 0.537 cm³/g.     

Structure of methemerythrin at 5 A resolution.

Rabbit globin messenger RNA was labelled in vitro with ¹²⁵I to specific activities in the range 20 to 200 × 10⁶ cts/min per μg. This ¹²⁵I-labelled mRNA bound to rabbit reticulocyte ribosomes with the kinetics and sensitivity to inhibitors expected from its participation in the normal process of the initiation of protein synthesis. Furthermore, when modified in 25% of its cytidine residues with unlabelled iodide, the mRNA coded for the same series of initiation peptides as did the unmodified mRNA. Using the techniques of RNA fingerprinting, the binding reaction was shown to select against contaminants and against “globin mRNA” molecules which lack a particular oligonucleotide implicated in the initiation process. When the ¹²⁵I-labelled mRNA was bound to ribosomes, both the initiating 40 S subunits and the 80 S ribosomes protected a fraction of the mRNA from digestion by pancreatic ribonuclease. Fingerprint analysis showed that highly specific regions of the mRNA were protected by the 40 S subunits and 80 S ribosomes and that these two protected regions were not identical.     

Changes in growth and composition of the marine microalgae <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> and <Emphasis Type="Italic">Nannochloropsis salina</Emphasis> in response to changing sodium bicarbonate concentrations

Oils, carbohydrates, and fats generated by microalgae are being refined in an effort to produce biofuels. The research presented here examines two marine microalgae, Nannochloropsis salina (green alga) and Phaeodactylum tricornutum (diatom), when grown with 0 (no addition), 0.5, 1.0, 2.0, and 5.0 g L^?1 NaHCO₃ added to an f/2 medium during the growth phase (GP) and a nutrient induced (nitrate limitation) lipid formation phase (LP). We hypothesize that the addition of NaHCO₃ is a sustainable and practical strategy to increase cellular density and concentrations of lipids in microalgae as well as the rate of lipid accumulation. In N. salina, final cell densities were significantly (p?<?0.05) higher in the NaHCO₃-treated cells than the control while in P. tricornutum the cell densities were higher with >[NaHCO₃] during the GP. During the LP, cell densities were generally higher in the NaHCO₃-treated cells compared with controls. F _V/F _M (efficiency of photosystem II) patterns paralleled those for cell density with generally higher values with higher concentrations of NaHCO₃ and significantly different values between controls and 5.0 g L^?1 NaHCO₃ at the end of the GP (p?<?0.05). F _V/F _M was variable between treatments in P. tricornutum (0.3–0.65) but less so in N. salina for (0.5–0.7) regardless of [NaHCO₃]. The lipid index (measured with Nile red), used as a proxy for triacylglycerides (TAGs), was 10.2?±?6.5 and 4.4?±?2.9 (fluorescence units/OD cells ×1000) for N. salina and P. tricornutum, respectively, at the end of the GP. At the end of the LP, the lipid index was eight and four times higher than during the GP in the corresponding 5.0 g L^?1 NaHCO₃ treatments, revealing that N. salina was accumulating more lipid than P. tricornutum. Dry weights essentially doubled during LP compared with GP for N. salina; this was not the case for P. tricornutum. In general, the percentage of ash in dry weights was significantly higher in the LP relative to the corresponding GP treatments for P. tricornutum; this was not the case for N. salina. During the LP, there was also less soluble protein in N. salina compared to GP; differences were not significant in cells growing with 2.0 or 5.0 g L^?1 NaHCO₃. In P. tricornutum, faster growing cells had more soluble protein during the GP and LP; differences between treatments were significant. P. tricornutum generally accumulated significantly more crude protein than N. salina at higher [NaHCO₃]; there was three times more crude protein in the highest NaHCO₃ (5.0 g L^?1) treatment compared with the controls. C:N ratios (mol:mol) were similar across treatments during GP: 7.03?±?0.12 and 10.16?±?0.41 for N. salina and P. tricornutum, respectively. Further, C:N ratios increased with increasing [NaHCO₃] during LP. Species-specific fatty acid methyl ester (FAMEs) profiles were observed. While C16:0 was lower in P. tricornutum compared to N. salina, the diatom produced more C16:1 and C14 but not C18:3. Monounsaturated fatty acids (MUFA) significantly increased in N. salina in the LP compared to GP and in response to increasing [NaHCO₃] (t tests; p?<?0.05). Saturated fatty acids (SFA) responded similarly but to a lesser degree. There were more polyunsaturated fatty acids (PUFA) in N. salina than MUFAs or SFAs. In P. tricornutum, there were generally more SFAs, MUFAs and PUFAs in P. tricornutum during LP than GP in the corresponding NaHCO₃ treatments. These findings reveal the importance of considering NaHCO₃ as a supplemental carbon source in the culturing marine phytoplankton in large-scale production for biofuels.     

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in potentially heterogeneous specimens. Nucleic acids can be detected via several established approaches, including analytical methods that are spectrophotometric (e.g., A₂₆₀), fluorometric (e.g., binding of fluorescent dyes), or colorimetric (nucleoside-specific chromogenic chemical reactions).¹ Though it cannot readily distinguish RNA from DNA, the A₂₆₀/A₂₈₀ ratio is commonly employed, as it offers a simple and rapid² assessment of the relative content of nucleic acid, which absorbs predominantly near 260 nm and protein, which absorbs primarily near 280 nm. Ratios < 0.8 are taken as indicative of ''pure'' protein specimens, while pure nucleic acid (NA) is characterized by ratios > 1.5³.However, there are scenarios in which the protein/NA content cannot be as clearly or reliably inferred from simple uv-vis spectrophotometric measurements. For instance, (i) samples may contain one or more proteins which are relatively devoid of the aromatic amino acids responsible for absorption at ≈280 nm (Trp, Tyr, Phe), as is the case with some small RNA-binding proteins, and (ii) samples can exhibit intermediate A₂₆₀/A₂₈₀ ratios (~0.8 < ~1.5), where the protein/NA content is far less clear and may even reflect some high-affinity association between the protein and NA components. For such scenarios, we describe herein a suite of colorimetric assays to rapidly distinguish RNA, DNA, and reducing sugars in a potentially mixed sample of biomolecules. The methods rely on the differential sensitivity of pentoses and other carbohydrates to Benedict''s, Bial''s (orcinol), and Dische''s (diphenylamine) reagents; the streamlined protocols can be completed in a matter of minutes, without any additional steps of having to isolate the components. The assays can be performed in parallel to differentiate between RNA and DNA, as well as indicate the presence of free reducing sugars such as glucose, fructose, and ribose (Figure 1).     

Membrane-associated protein synthesis of mammalian cells. II. Isopycnic separation of membrane-bound polyribosomes

The membrane-bound ribosomes of HeLa cells were analyzed in CsCl buoyant-density gradients. There exist two classes: a heavy class with a density of 1.55 g/ cm³ and a light class heterogeneously distributed with a mean density of 1.49 g/cm³. The lower density of the light class is due to an increased ratio of protein to RNA. This additional protein is not removed with increased ionic strength. Both classes of ribosomes incorporate amino acids at a rate comparable to non-membrane-bound ribosomes. The heavy and light ribosomes appear to correspond to the EDTA-sensitive and the EDTA-resistant ribosomes, respectively.     

Purification and partial characterization of the antiviral protein from Phytolacca americana which inhibits eukaryotic protein synthesis.

The antiviral protein from the pokeweed plant (Phytolacca americana) which inhibits eukaryotic protein synthesis has been purified to homogeneity and its molecular weight has been determined by two physical methods. The protein consists of a single polypeptide chain of an approximate molecular weight of 27,000. The inhibitory effect of this protein on the synthesis of polyphenylalanine in a cell-free system from Artemia salina suggests that this protein acts in an enzymatic manner on eukaryotic ribosomes. It is also demonstrated that polyphenylalanine synthesis on A. salina ribosomes is more sensitive to inhibition by this protein than on rabbit reticulocyte ribosomes.     

Follicle-stimulating hormone stimulated differentiation and progesterone production of bovine granulosa cells in culture

Progesterone production of granulosa cells cultured in vitro is stimulated and cell differentiation increased, by follicle-stimulating hormone (FSH). This study examined whether the increased progesterone production observed when bovine granulosa cells are cultured occurs because (1) progesterone production by undifferentiated and/or differentiated cells is increased or (2) the differentiation of granulosa cells is stimulated. Viable bovine granulosa cells (2−3×10⁵) from follicles 5–8 mm in diameter were cultured in the presence of 0, 1, 10 and 100 μu FSH (1 μu ≡ 1 μg NIH-FSH-S1) for 6 days at 37°C in a humidified atmosphere of 5% CO₂ in air in 1 ml of a 1:1 mixture of Dulbecco's modified Eagle medium: Ham's F10 medium supplemented with 365 μg ml⁻¹ l-glutamine, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin. Progesterone production, total DNA and protein, and cell diameter were determined sequentially over the culture period. The increases in progesterone production (ng μg⁻¹ DNA per 24 h), cytoplasmic:nuclear ratio (μg protein μg⁻¹ DNA) and cell diameter (μm) over 6 days culture indicated that granulosa cells underwent differentiation in the presence of FSH. Progesterone production of undifferentiated granulosa cells (diameter 14 μm or less) was stimulated by FSH (P < 0.01) in a dose dependent manner (1.0±0.2, 2.9±0.3, 3.7±0.3 and 4.9±0.4 ng μg⁻¹ DNA per 24 h for 0, 1, 10 and 100 μu ml⁻¹ FSH respectively) but remained constant within dose (P > 0.05) during a 6 day culture period. FSH stimulated (P < 0.05) the rate of granulosa cell differentiation (10±3%, 53±13%, 74±21% and 82±10% differentiating cells per well for 0 μu, 1 μu, 10 μu and 100 μu ml⁻¹ FSH respectively) but did not stimulate (P > 0.05) progesterone production by differentiating granulosa cells (8.7±0.5 ng μg⁻¹ DNA per 24 h). In conclusion, the increase in progesterone production of FSH-stimulated granulosa cells cultured in vitro appears to be mainly due to an increase in the number of differentiating cells with a constant rather than an increasing progesterone production per cell.     

The effect of sodium bicarbonate supplementation on growth and biochemical composition of marine microalgae cultures

The addition of bicarbonate (NaHCO₃; 0, 1, or 2 g L^?1) to microalgal cultures has been evaluated for two species (Tetraselmis suecica and Nannochloropsis salina) in respect of growth and biochemical composition. In batch cultures, addition of bicarbonate (1 g L^?1) resulted in significantly (P?<?0.05) higher final mean cell abundances for both species. No differences in specific growth rates (SGRs) were recorded for T. suecica between treatments; however, increasing bicarbonate addition decreased SGR values in N. salina cultures. Bicarbonate addition (1 g L^?1) significantly improved nitrate utilisation from the external media and photosynthetic efficiency (F _v /F _m ) in both species. For both T. suecica and N. salina, bicarbonate addition significantly increased the cellular concentrations of total pigments (3,432–3,587 and 19–37 fg cell^?1, respectively) compared to cultures with no additional bicarbonate (1,727 and 11 fg cell^?1, respectively). Moreover, final concentrations of total cellular fatty acids in T. suecica and N. salina cultures supplemented with 2 g L^?1 bicarbonate (7.6?±?1.2 and 1.8?±?0.1 pg cell^?1, respectively) were significantly higher than those cells supplemented with 0 or 1 g L^?1 bicarbonate (3.2–3.5 and 0.9–1.0 pg cell^?1, respectively). In nitrate-deplete cultures, bicarbonate addition caused species-specific differences in the rate of cellular lipid production, rates of change in fatty acid composition and final lipid levels. In summary, the addition of sodium bicarbonate is a viable strategy to increase cellular abundance and concentrations of pigments and lipids in some microalgae as well as the rate of lipid accumulation in nitrate-deplete cultures.     

Accumulation of mitochondrial DNA during oogenesis in Xenopus laevis

The mitochondrial DNA (mtDNA) content of Xenopus laevis oocytes at various stages of oogenesis has been determined by molecular hybridization with ³H-labeled complementary RNA (cRNA). The previtellogenic oocyte less than 250 μm in diameter (stage 1) contains 0.95 ± 0.47 ng of mtDNA. Accumulation of mtDNA proceeds until stage 4 (500–750 μm diameter oocyte), by which time a steady-state level of 4.28 ± 0.40 ng/oocyte is attained. Using the hybridization assay, the stage 6 (full-grown) Xenopus oocyte contains 4.51 ± 0.69 ng of mtDNA, compared to the previously reported value of 3.8 ng determined by direct measurement on the unfertilized egg. There appears to be a reasonable correlation, therefore, between the termination of mtDNA accumulation and the dispersal of the juxtanuclear, mitochondrial aggregate (Balbiani body) at the onset of vitellogenesis in Xenopus. It is concluded that the enormous complement of oocyte mitochondria is accumulated well before the end of oocyte growth and is maintained at a constant level during the remainder of oogenesis, through maturation, fertilization, and on into early development.     

Determination of the nuclear DNA content of Saccharomyces cerevisiae and implications for the organization of DNA in yeast chromosomes.

The DNA content of the nucleus of the yeast Saccharomyces cerevisiae has been determined by both renaturation kinetics and DNA per cell measurements. Renaturation kinetics experiments were performed by following the decrease of optical hyperchromicity at 260 nm and by hydroxyapatite chromatography. DNA per cell measurements were made by the diaminobenzoic acid method and by the ethidium bromide method of Klotz &; Zimm (1972b). The conclusion from the above experiments is that the S. cerevisiae nucleus contains 9 × 10⁹ ± 2 × 10⁹ daltons of DNA. Previously we (Lauer &; Klotz, 1975) had measured the molecular weight of the largest piece of DNA in the yeast nucleus to be 2 × 10⁹ ± 0.2 × 10⁹. Here we extend this work by using a more highly protein-denaturing buffer system and conclude that the largest piece of DNA in the S. cerevisiae nucleus contains 1.5 × 10⁹ to 2.2 × 10⁹ daltons of DNA in both haploid and diploid cell lysates. From genetics, the largest yeast chromosome should contain 13% of the genome, or 0.9 × 10⁹ to 1.5 × 10⁹ daltons of DNA (using our DNA per cell range). Thus, the large DNA we measure contains from one to two times the amount of the DNA predicted from genetics to be in the largest chromosome. In light of these new data, viscoelastic measurements on yeast DNA are now consistent with the idea that each chromosome contains one piece of DNA.     

On the origin of plastids

The buoyant density in CsCl of ribosomes from chloroplasts of the green algaChlorella pyrenoidosa and two species of higher plants,Pisum sativum andChenopodium album, has been studied. From the relative protein content it was calculated that 70S ribosomes from chloroplasts are much smaller than 80S cytoplasmic ribosomes (3.0–3.1×10⁶ and 4.0×10⁶ daltons) and slightly larger than 70S ribosomes from abcteriaE. coli 2.5×10⁶ daltons). Chloroplast ribosomes from pea seedlings were analyzed by two-dimensional polyacrylamide gel electrophoresis. They appear to contain 71 proteins. This indicates that chloroplast ribosomes contain a larger number of proteins than do the ribosomes fromE. coli and other species of Enterobacteriaceae. Further study will permit a probable evaluation of the validity of Mereschkowsky's hypothesis that the photosynthetic plastids of eukaryotic plant cells are the evolutionary descendants of endosymbiotic blue-green algae.     

Preparation and characterization of monoclonal antibodies to human adiponectin for its quantitative measurement in serum

Mouse Monoclonal antibodies against human adiponectin were produced by the routine method and the specificity of antibodies was verified. These monoclonal antibodies (MoAbs) interacted with the monomeric and trimeric forms of recombinant adiponectin according to the results of a Western blot analysis. Human blood serum was fractionated by gel filtration, and the protein of these fractions was stained using labeled MoAbs. It was established that a single high-molecular-weight form (HMW) of endogenous adiponectin was detected by this method. The use of competitive enzymelinked immunoassay on the basis of the obtained MoAbs allowed us to show that the sera of healthy male donors contains lower adiponectin concentrations than that of female donors (8.42 ± 1.59 μg/ml vs. 11.01 ± 2.58 μg/ml, p = 0.01). We also detected statistically significant lower adiponectin levels in the serum of patients with coronary artery disease for both men (6.01 ± 2.73 μg/ml vs. 8.42 ± 1.59 μg/ml, p = 0.015) and women (5.79 ± 2.98 μg/ml vs. 11.01 ± 2.58 μg/ml, p = 0.0003). Therefore, the developed methods for the analysis of the HMW form of adiponectin can be helpful in the diagnostics of the possible implications and assessment of unfavorable prognoses in patients with cardiovascular disorders.     

Lipophorin from Rhodnius prolixus: Purification and partial characterization

The lipophorin of adult females of Rhodnius prolixus was radioactively labelled with ³²P exclusively in the phospholipid moiety and purified on a KBr ultracentrifugation gradient. The density of purified [³²P]phospholipid labelled lipophorin on the fifth day after a blood meal was 1.1211 ± 0.0017 g/ml. By weight it contained 51.7% protein, 0.7% sugar and 47.6% lipid. The protein moiety was composed of three apoproteins of 226 ± 11, 86 ± 2 and 16 ± 1 kDa. Mannose and N-acetylglucosamine were the only sugars detected. Among the lipids, 66.3% were neutral lipids and 33.8% were phospholipids. Analysis by thin-layer chromatography showed that in the total phospholipids fraction ³²P was distributed as follows: phosphatidylethanolamine (54.4%), phosphatidylcholine (44.7%), cardiolipin (2.1%), phosphatidylserine (0.7%), phosphatidylinositol (0.4%), sphingomyelin (0.3%) and phosphatidic acid (0.2%). The total phosphate content was 0.53 ± 0.03 nmol/μg of protein.