Studies on the different molecular forms of tyrosine aminotransferase from rat liver and hepatoma cells

In an attempt to clarify the significance of the separable forms of tyrosine aminotransferase, the enzyme from rat liver and from cultured hepatoma cells was studied by carboxymethyl-Sephadex chromatography. Our studies of the form conversion during the purification procedure of the enzyme, where all cellular components were quickly discarded, do not allow us to invoke a specific "converting factor", the existence of which in the particulate fraction has been suggested. Moreover the addition of serine protease inhibitors is not sufficient to prevent the classical conversion. More probably, several factors depending on the environmental conditions might influence different reactions which lead to a preferential conformation of the enzyme in vitro. The difference in the PO4- content of the various enzyme forms and the consecutive differences in negative charge may be the determining factor in the elution pattern of the three forms of the isolated soluble enzyme. This observation raises the possibility that phosphorylation might play a specific role in the regulation of tyrosine aminotransferase synthesis.     

Comparative cradle to grave environmental life cycle assessment of traditional and extensive vegetative roofs: an application for the Lebanese context

The International Journal of Life Cycle Assessment - Vegetative roofs (VRs) are fully planted roof spaces that offer aesthetic view, storm water management, sound insulation, energy savings, and...     

Selective and sensitive detection of pectin lyase activity using a colorimetric test: application to the screening of microorganisms possessing pectin lyase activity

Several methods have been described for the detection and quantification of polygalacturonase (PG) and pectin lyase (PL) activities. The most frequently used tests are the Nelson method using copper(II) and an arsenomolybdate reagent to detect PG activity, and the colorimetric method using thiobarbituric acid (TBA) to detect PL activity. We observed that none of these methods are suitable to differentiate between these two enzymatic activities. Therefore, we optimized the test conditions of the TBA method. As a result, the detection of the enzymatic beta-elimination (PL activity) became sensitive and selective. A basic pretreatment at 80 degrees C for 5 min of the solution which contains the pectin fragments of the PL activity furnished aldehydes which were condensed with TBA or its derivatives. After acidification of the medium, a pink fluorescent dye was detected spectrophotochemically (lambda = 550 nm). The interference of galacturonic acid or oligomers resulting from PG activity was completely eliminated. The most sensitive reagent was N-(pyridin-2-yl)-thiobarbituric acid. The application of this method with the new reagent was extended to the screening of microorganisms possessing the PL activity. The obtained results confirm that Aspergillus niger strain and a Saccharomyces cerevisiae SCPP strain possess this activity.     

Cultivation of explants of the marine sponge Crambe crambe in closed systems

Explants of the sponge Crambe crambe were cultured in natural seawater, with or without marine microalga (Phaeodactylum tricornutum) in discontinuous flow through systems and in continuous flow-through systems (DFTHS and CFTHS, respectively). Growth was measured as the increase in underwater weight. In the experiment carried out in the CFTHS, the explants average underwater weight increased by up to 1380% of the initial weight in 22-45 days. Growth in DFTHS was much slower producing a gain of up to an average value of 322% of the initial weight in 100-210 days. Growth kinetics varied considerably for different explants. Explants grew fastest in the first 10-days of subculture. The sponges grew better in CFTHS compared with the DFTHS. The high growth rates observed in CFTHS suggest that this technique is a promising method for culturing C. crambe in closed systems.     

A process for high yield and scaleable recovery of high purity eicosapentaenoic acid esters from microalgae and fish oil

A low expense process is developed for recovering esterified eicosapentaenoic acid (EPA) from microalgae and fish oil. Over 70% of the EPA content in the esterified crude extract of microalgae were recovered at purities exceeding 90%. The recovery scheme utilizes either wet or freeze-dried algal biomass. The process consists of only three main steps: 1) simultaneous extraction and transesterification of the algal biomass; 2) argentated silica gel column chromatography of the crude extract; and 3) removal of pigments by a second column chromatographic step. Argentated silica gel chromatography recovered about 70% of the EPA ester present in the crude fatty ester mixture of fish oil, but at a reduced purity ( approximately 83% pure) compared to the microalgal derived EPA. The optimal loading of the fatty ester mixture on the chromatographic support was about 3% (w/w) but loadings up to 4% did not affect the resolution significantly. The process was scaled up by a factor of nearly 320 by increasing the diameter of the chromatography columns. The elution velocity remained constant. Compared to the green alga Monodus subterraneus, the diatom Phaeodactylum tricornutum had important advantages as a potential commercial producer of EPA. For a microalgal EPA process to be competitive with fish oil derived EPA, P. tricornutum biomass (2.5% w/w EPA) needs to be obtained at less than $4/kg. If the EPA content in the alga are increased to 3.5%, the biomass may command a somewhat higher price. The quality of microalgal EPA compares favorably with that of the fish oil product. Compared to free fatty acid, EPA ester is more stable in storage. Shelf-life is extended by storing in hexane. The silver contamination in the final purified EPA was negligibly small (<210 ppb).     

Infectious Chikungunya Virus in the Saliva of Mice,Monkeys and Humans

Chikungunya virus (CHIKV) is a reemerging, ordinarily mosquito-transmitted, alphavirus that occasionally produces hemorrhagic manifestations, such as nose bleed and bleeding gums, in human patients. Interferon response factor 3 and 7 deficient (IRF3/7^-/-) mice, which are deficient for interferon α/β responses, reliably develop hemorrhagic manifestations after CHIKV infection. Here we show that infectious virus was present in the oral cavity of CHIKV infected IRF3/7^-/- mice, likely due to hemorrhagic lesions in the olfactory epithelium that allow egress of infected blood into the nasal, and subsequently, oral cavities. In addition, IRF3/7^-/- mice were more susceptible to infection with CHIKV via intranasal and oral routes, with IRF3/7^-/- mice also able to transmit virus mouse-to-mouse without an arthropod vector. Cynomolgus macaques often show bleeding gums after CHIKV infection, and analysis of saliva from several infected monkeys also revealed the presence of viral RNA and infectious virus. Furthermore, saliva samples collected from several acute CHIKV patients with hemorrhagic manifestations were found to contain viral RNA and infectious virus. Oral fluids can therefore be infectious during acute CHIKV infections, likely due to hemorrhagic manifestations in the oral/nasal cavities.     

Sustained growth of explants from Mediterranean sponge Crambe crambe cultured in vitro with enriched RPMI 1640

Marine sponges are potential sources of many unique metabolites, including cytotoxic and anticancer compounds. Natural sponge populations are insufficient or inaccessible for producing commercial quantities of metabolites of interest. It is commonly accepted that tissue (fragments, explants, and primmorphs) and in vitro cell cultivation show great potential. However, there is little knowledge of the nutritional requirements of marine sponges to carry out efficient and sustained in vitro culture and progress has been slow. In marine invertebrate fila many unsuccessful attempts have been made with in vitro cultures using typical commercial animal cell media based on sources of dissolved organic carbon (DOC) (e.g., DMEM, RPMI, M199, L-15, etc.). One of the reasons for this failure is the use of hardly identifiable growth promoters, based on terrestrial animal sera. An alternative is the use of extracts from marine animals, since they may contain nutrients necessary for growth. In this work we have cultivated in vitro explants of the encrusting marine sponge Crambe crambe. It is one of the most abundant sponges on the Mediterranean coastline and also possesses an array of potentially active metabolites (crambines and crambescidins). Initially a new approach was developed in order to show consumption of DOC by explants. Thus, different initial DOC concentrations (300, 400, 700 and 1200 mg DOC L(-1)) were assayed. Consumption was evident in all four assays and was more marked in the first 6 h. The DOC assimilation data were adjusted to an empirical model widely used for uptake kinetics of organic dissolved compounds in marine invertebrates. Second, a protocol was established to cultivate explants in vitro. Different medium formulations based on RPMI 1640 commercial medium enriched with amino acids and inorganic salts to emulate seawater salinity were assayed. The enrichment of this medium with an Octopus aqueous extract in the proportions of 10% and 20% (v/v) resulted in an evident sustained long-term growth of C. crambe explants. This growth enhancement produced high metabolic activity in the explants, as is confirmed by the high ammonium and lactate content in the medium a few days after its renewal and by the consumption of glucose. The lactate accumulation increased with the size and age of explants. Prior to these experiments, we successfully developed a robust new alternative method, based on digital image treatment, for accurate determination of the explant apparent volume as growth measure.     

Causes of shear sensitivity of the toxic dinoflagellate Protoceratium reticulatum

Dinoflagellates have proven extremely difficult to culture because they are inhibited by low‐level shear forces. Specific growth rate of the toxic dinoflagellate Protoceratium reticulatum was greatly decreased compared with static control culture by intermittent exposure to a turbulent hydrodynamic environment with a bulk average shear rate that was as low as 0.3 s^?1. Hydrodynamic forces appeared to induce the production of reactive oxygen species (ROS) within the cells and this caused peroxidation of cellular lipids and ultimately cell damage. Exposure to damaging levels of shear rate correlated with the elevated level of lipoperoxides in the cells, but ROS levels measured directly by flow cytometry did not correlate with shear induced cell damage. This was apparently because the measured level of ROS could not distinguish between the ROS that are normally generated by photosynthesis and the additional ROS produced as a consequence of hydrodynamic shear forces. Continuously subjecting the cells to a bulk average shear rate value of about 0.3 s^?1 for 24‐h caused an elevation in the levels of chlorophyll a, peridinin and dinoxanthin, as the cells apparently attempted to counter the damaging effects of shear fields by producing pigments that are potential antioxidants. In static culture, limitation of carbon dioxide produced a small but measureable increase in ROS. The addition of ascorbic acid (0.1 mM) to the culture medium resulted in a significant protective effect on lipid peroxidation, allowing cells to grow under damaging levels of shear rates. This confirmed the use of antioxidant additives as an efficient strategy to counter the damaging effects of turbulence in photobioreactors where shear sensitive dinoflagellates are cultivated. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Producing drugs from marine sponges

Marine sponges are potential sources of many unique metabolites, including cytotoxic and anticancer compounds. Natural sponge populations are insufficient or inaccessible for producing commercial quantities of metabolites of interest. This review focuses on methods of producing sponge biomass to overcome supply limitations. Production techniques discussed include aquaculture in the sea, the controlled environments of aquariums, and culture of sponge cells and primmorphs. Cultivation in the sea and aquariums are currently the only practicable and relatively inexpensive methods of producing significant quantities of sponge biomass. In the future, metabolite production from cultured sponge cells and primmorphs may become feasible. Obtaining a consistent biomass yield in aquariums requires attention to many factors that are discussed in this work.     

Structural organization of pLP1, a cryptic plasmid from Lactobacillus plantarum CCM 1904

To construct shuttle vectors based on an endogenous replicon, we isolated a small cryptic plasmid (pLP1) from Lactobacillus plantarum CCM 1904. The nucleotide sequence (2093 bp, 38.25 GC mol%) revealed one major open reading frame encoding for a 317 amino acid protein (Rep). Comparisons with proteins encoded by other Gram-positive bacteria plasmids strongly suggest that the protein encoded by pLP1 has a replicative role. The presence of a consensus sequence including a tyrosine residue known to be the replication protein binding site to the DNA (in phage φX174) strengthens this hypothesis. The DNA sequence contains also a sequence similar to the pC194 origin nick sequence, which initiates the plasmid replication at the plus origin, characteristic of plasmids which replicate following a rolling circle mechanism via single-stranded DNA intermediates. A set of 13 direct repeats of 17 bp could be involved in the expression of the incompatibility or in the copy number control as in the other plasmids. A promoter sequence located at the rep 5′ region has been identified and is functional in Bacillus subtilis.