Bioengineering of Nitrilases Towards Its Use as Green Catalyst: Applications and Perspectives

Nitrilases are commercial biocatalysts used for the synthesis of plastics, paints, fibers in the chemical industries, pharmaceutical drugs and herbicides for agricultural uses. Nitrilase hydrolyses the nitriles and dinitriles to their corresponding carboxylic acids and ammonia. They have a broad range of substrate specificities as well as enantio-, regio- and chemo-selective properties which make them useful for biotransformation of nitriles to important compounds because of which they are considered as ‘Green Catalysts’. Nitriles are widespread in nature and synthesized as a consequence of anthropogenic and biological activities. These are also present in certain plant species and are known to cause environmental pollution. Biotransformation using native organisms as catalysts tends to be insufficient since the enzyme of interest has very low amount in the total cellular protein, rate of reaction is slow along with the instability of enzymes. Therefore, to overcome these limitations, bioengineering offers an alternative approach to alter the properties of enzymes to enhance the applicability and stability. The present review highlights the aspects of producing the recombinant microorganisms and overexpressing the enzyme of interest for the enhanced stability at high temperatures, immobilization techniques, extremes of pH, organic solvents and hydrolysing dintriles to chiral compounds which may enhance the possibilities for creating specific enzymes for biotransformation.     

Treatment Default amongst Patients with Tuberculosis in Urban Morocco: Predicting and Explaining Default and Post-Default Sputum Smear and Drug Susceptibility Results

Setting

Public tuberculosis (TB) clinics in urban Morocco.

Objective

Explore risk factors for TB treatment default and develop a prediction tool. Assess consequences of default, specifically risk for transmission or development of drug resistance.

Design

Case-control study comparing patients who defaulted from TB treatment and patients who completed it using quantitative methods and open-ended questions. Results were interpreted in light of health professionals’ perspectives from a parallel study. A predictive model and simple tool to identify patients at high risk of default were developed. Sputum from cases with pulmonary TB was collected for smear and drug susceptibility testing.

Results

91 cases and 186 controls enrolled. Independent risk factors for default included current smoking, retreatment, work interference with adherence, daily directly observed therapy, side effects, quick symptom resolution, and not knowing one’s treatment duration. Age >50 years, never smoking, and having friends who knew one’s diagnosis were protective. A simple scoring tool incorporating these factors was 82.4% sensitive and 87.6% specific for predicting default in this population. Clinicians and patients described additional contributors to default and suggested locally-relevant intervention targets. Among 89 cases with pulmonary TB, 71% had sputum that was smear positive for TB. Drug resistance was rare.

Conclusion

The causes of default from TB treatment were explored through synthesis of qualitative and quantitative data from patients and health professionals. A scoring tool with high sensitivity and specificity to predict default was developed. Prospective evaluation of this tool coupled with targeted interventions based on our findings is warranted. Of note, the risk of TB transmission from patients who default treatment to others is likely to be high. The commonly-feared risk of drug resistance, though, may be low; a larger study is required to confirm these findings.     

The Small Molecule GMX1778 Is a Potent Inhibitor of NAD+ Biosynthesis: Strategy for Enhanced Therapy in Nicotinic Acid Phosphoribosyltransferase 1-Deficient Tumors

GMX1777 is a prodrug of the small molecule GMX1778, currently in phase I clinical trials for the treatment of cancer. We describe findings indicating that GMX1778 is a potent and specific inhibitor of the NAD⁺ biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT). Cancer cells have a very high rate of NAD⁺ turnover, which makes NAD⁺ modulation an attractive target for anticancer therapy. Selective inhibition by GMX1778 of NAMPT blocks the production of NAD⁺ and results in tumor cell death. Furthermore, GMX1778 is phosphoribosylated by NAMPT, which increases its cellular retention. The cytotoxicity of GMX1778 can be bypassed with exogenous nicotinic acid (NA), which permits NAD⁺ repletion via NA phosphoribosyltransferase 1 (NAPRT1). The cytotoxicity of GMX1778 in cells with NAPRT1 deficiency, however, cannot be rescued by NA. Analyses of NAPRT1 mRNA and protein levels in cell lines and primary tumor tissue indicate that high frequencies of glioblastomas, neuroblastomas, and sarcomas are deficient in NAPRT1 and not susceptible to rescue with NA. As a result, the therapeutic index of GMX1777 can be widended in the treatment animals bearing NAPRT1-deficient tumors by coadministration with NA. This provides the rationale for a novel therapeutic approach for the use of GMX1777 in the treatment of human cancers.The cyanoguanidinopyridine GMX1778 (previously known as CHS828) is the active form of the prodrug GMX1777 and has potent antitumor activity in vitro and in vivo against cell lines derived from several different tumor origins (11). The antitumor activity of GMX1778 has been widely studied since its discovery (1, 11, 19-21, 24), but positive identification of the molecular target and the mechanism of action of GMX1778 has been elusive. Here, we demonstrate that GMX1778 exerts its antitumor activity via its potent and selective antagonism of NAD⁺ biosynthesis. GMX1777 is currently being assessed in phase I clinical trials for treatment of patients with refractory solid tumors.The pyridine nucleotide NAD⁺ plays a major role in the regulation of several essential cellular processes (7, 22, 25, 38). In addition to being a biochemical cofactor for enzymatic redox reactions involved in cellular metabolism, including ATP production, NAD⁺ is important in diverse cellular pathways responsible for calcium homeostasis (17), gene regulation (5), longevity (18), genomic integrity (33), and apoptosis (36). Cancer cells exhibit a significant dependence on NAD⁺ for support of the high levels of ATP production necessary for rapid cell proliferation. They also consume large amounts of this cofactor via reactions that utilize poly(ADP) ribosylation, including DNA repair pathways (10, 37, 39).In eukaryotes, the biosynthesis of NAD⁺ occurs via two biochemical pathways: the de novo pathway, in which NAD⁺ synthesis occurs through the metabolism of l-tryptophan via the kynurenine pathway, and the salvage pathway. The NAD⁺ salvage pathway can use either nicotinamide (niacinamide) (NM) or nicotinic acid (niacin) (NA) (via the Preiss-Handler pathway) as a substrate for NAD⁺ production. Saccharomyces cerevisiae species predominantly use NA as the substrate for NAD⁺ biosynthesis, through the deamidation of NM by the nicotinamidase PNC1 (25). However, mammalian cells do not express a nicotinamidase enzyme and use NM as the preferred substrate for the NAD⁺ salvage pathway. The mammalian NAD⁺ biosynthesis salvage pathway using NM is composed of NA phosphoribosyltransferase (NAMPT), which is the rate-limiting and penultimate enzyme that catalyzes the phosphoribosylation of NM to produce nicotinamide mononucleotide (NMN) (27, 29). NMN is subsequently converted to NAD⁺ by NMN adenyltransferases (NMNAT). The gene encoding NAMPT was originally identified as encoding a cytokine named pre-B-cell colony-enhancing factor (PBEF1) (30). NAMPT was also identified as a proposed circulating adipokine named visfatin (thought to be secreted by fat cells) and was suggested to function as an insulin mimetic; however, this role of NAMPT currently remains controversial (8). In mice, NAMPT has been shown to act as a systemic NAD⁺ biosynthetic enzyme that regulates insulin secretion from β cells (28). The molecular structure of NAMPT from human (15), rat (16) and mouse (35) tissue, containing either NMN or the inhibitor APO866, have been determined by X-ray crystallography. These structures revealed that NAMPT is a dimeric type II phosphoribosyltransferase.Here, we report that the anticancer compound GMX1778 is a specific inhibitor of NAMPT in vivo and in vitro and is itself a substrate for the enzyme. Phosphoribosylated GMX1778 inhibits NAMPT as potently as GMX1778 but is preferentially retained within cells. Finally, we have identified a novel anticancer strategy utilizing NA rescue of GMX1778 cytotoxicity to increase the therapeutic index of GMX1777 activity in tumors that are deficient in NA phosphoribosyltransferase 1 (NAPRT1).     

The mucosa of the small intestine: development of the cellular lipid composition during enterocyte differentiation and postnatal maturation

Alterations in lipids linked to intestinal maturation and enterocyte differentiation were reviewed. The 3 main lipid components of cell membranes, ie cholesterol, phospholipids and glycolipids, were examined. Cell phospholipid content increases from the crypts to the mid-villus, which accounts for membrane development and organelle growth in differentiating cells. Changes in the proportion of phospholipid polar head groups occur in brush border membrane during postnatal maturation of the small intestine. The possibility that phospholipid fatty acid composition in differentiating cells might be altered by dietary lipids is discussed. Cholesterol biosynthesis mainly occurs in crypt and lower villus cells whereas its absorption from luminal content and esterification into lipoproteins occur in upper villus mature cells. Cholesterol cell content increases in mature cells in comparison to immature cells on the one hand, and in the distal by comparison with proximal parts of the intestine on the other. Increasing cholesterol content is generally correlated with decreasing membrane fluidity, which in turn could modulate functional properties of the mucosa. Glycosphingolipids are mainly found in the brush border membrane, which contains 20-30% glycolipids by weight of total lipids. These components tend to reinforce the membrane stability and significantly contribute to the surface properties of epithelial cells. The latter undergo noticeable changes during cell differentiation and postnatal maturation. Significant changes in both the glycosidic and lipophilic parts of glycosphingolipid molecules occur in differentiating cells and are of possible importance in the process of mucosal maturation. It is possible that the addition of a terminal sialic acid (sialyltransferase activity) instead of a terminal galactose (galactosyltransferase) to an endogenous acceptor (lactosylceramide) could constitute an important event in the differentiation process, and may account for the increasing content of hematosides along the intestinal villus of rat. Alterations in lipid counterpart mainly consist of hydroxylation of fatty acids in hematosides during postnatal maturation or in glucosylceramides during cell differentiation. Collectively these intestinal lipid changes may contribute in part to the development of mucosal barrier, selective permeability and functional properties of the mature intestinal mucosa.     

Changes in fatty acid composition during cell differentiation in the small intestine of suckling piglets.

Alterations of phospholipid fatty acid composition in the renewing intestine were studied in the infant piglet. Newborn piglets were fed from birth to 2 weeks of age a concentrated cow's milk which defined a standard supply of dietary fatty acids. Phospholipids were isolated from the whole mucosa, isolated intestinal cells and purified brush border membranes. Intestinal cells were isolated according to their position along the crypt-villus axis and cell phospholipids were extracted at each step of differentiation. Changes in fatty acid composition of cell phospholipids were related to those of lactase activity in the corresponding cell homogenates. In cell phospholipids, the relative content of linoleic and linoleic acids increased about 2-fold from crypt base to villus tip. Substantial contents of alkenylacyl glycerophospholipids (plasmalogens) were found in crypt cell phospholipids and in purified brush border membrane phosphatidylethanolamine (11 and 14% of alkenyl groups by weight of total fatty acids, respectively). The proportion of alkenylacyl glycerophospholipids decreased as cells ascended the villus column and became more differentiated. The results show that fatty acid compositional changes in differentiating cell phospholipids occurred in the immature intestine (before weaning) and suggest that these alterations might be related to the appearance of specific functions.     

Zooplankton Variability in 49 Shallow Tropical Reservoirs of Ivory Coast (West Africa)

Zooplankton samples were collected from 49 small reservoirs of northern Ivory Coast in April 1997. Thirty taxa were identified, including 20 rotifers, 3 copepods and 6 cladocerans. The number of taxa per lake ranged between 12 to 22 and decreased with the total abundance of zooplankton. Copepods dominated standing biomass. Coinertia analysis suggested the role of seston food abundance, oxygen depletion and turbidity for zooplankton abundance and community structure. Rotifers, and particularly Brachionus angularis, Polyarthra and Filinia, were more abundant than copepods in the most eutrophic, turbid and deoxygenated reservoirs. The role of oxygen as a determinant of community structure is probably linked to the specific tolerance of taxa, but turbidity role could not be evaluated with certainty in the absence of information on visual predators.     

Gulf of fos (France): Main hydrological features (1976–1978)

Surrounded by a heavily industrialized area, subject to natural fresh water dilution (Etang de Berre, Rhône river), the Gulf of Fos is an unbalanced neritic zone. Its distrophic character is accentuated by numerous continental discharges; furthermore, the climatic instability which characterizes this region (irregular periods of winds, fluvial risings) reinforces the changing ecological conditions in the gulf waters. The present paper shows the major ecological conditions in that neritic area, and the main events occured during the 1976–1978 period, through and extensive sampling network. The principal regulating features are thus described by the mean of a mathematical data processing (Principal Components Analysis).     

Sedimentation modified by wind induced resuspension in a shallow tropical lagoon (Cote d'Ivoire)

In shallow environments, under certain conditions of fetch, wind velocity, bathymetry and bottom characteristics, resuspension can be generated by wind induced waves. In the tropical Ebrié lagoon, austral trade winds are dominant almost all year long, and their velocity shows a marked diel pattern with maximum speed between noon and midnight. Only austral trade winds with a speed >3 m s⁻¹ allow particle resuspension which is effective for depths<1.5 m. In these areas, significantly higher values of chlorophyll biomass and mineral seston are noted during the windy sequences. Granulometric and mineralogical analyses showed that only the surficial sediment (0–3 cm) was involved in resuspension. This process induces several effects: 1) an increase of the suspended matter concentration in the water and thus a light attenuation due to a higher turbidity, 2) a redistribution in the whole water column of nutrients from the pore water and 3) a removal of the finer fractions from the superficial sediment. On the contrary, for depths>1.5 m, particle sinking is permanent in depressions which are spontaneously transformed into anoxic systems. At the lagoon scale, sedimentation is significantly modified by wind induced resuspension. According to the bathymetry and the distance from a river, three sedimentary facies are recognized. Their grain size distributions are parabolic in areas where resuspension occurs, logarithmic in areas where no resuspension is possible and hyperbolic in the hollows and the main channels. Finally, a large part of the allochthonous inputs (from drainage and rivers) and autochthonous pelagic production is trapped into the Ebrié lagoon and less than 10% of the particles entering the lagoon are exported toward the Atlantic Ocean.     

Effects of a seasonal salinity change on periphyton biomass in a shallow tropical lagoon

In the Ebrié Lagoon (Cǒte d'Ivoire), growth of periphyton on bamboo stuck in the sediment is at the basis of the acadja, a low-cost system developed in order to enhance aquaculture of omnivorous fish like the tilapia Sarotherodon melanotheron. A survey based on hydrological, bacterial and algal data (including phytoplankton and periphyton) was conducted from November 1992 to June 1993 in an experimental structure. Climatic and hydrological seasonality was marked during the study. Succeeding to a rainy season, the oligohaline situation starting in November was characterized by low bacterial and chlorophyll biomass attached to the bamboo (respectively 1 and 18 mg m⁻²). The mesohaline situation observed from January featured a sharp increase in periphyton biomass, with a maximum in April (16 and 177 mg m⁻² for bacterial and algal biomass, respectively). The flood of the Agnéby river, induced in June by the local rains, originated again oligohaline conditions. The biomass decrease observed in May and June resulted from a shift towards low salinity (from 9.8 to 1.8 psu), a decrease in light availability (combined effects of a decreasing solar radiation during the rainy season and an increase of water turbidity due to the flood) and a lower phytoplankton biomass (therefore limiting the secondary epiphytism potentialities). In this shallow tropical environment characterized by high nutrient concentrations (due to local hydrology and organic nature of the substrate), the combination of seasonal variations of climatic (light availability), hydrological (salinity) and biological (abundance or lack of epiphytic algae) seems to control the periphyton biomass growing on bamboo. Therefore, marked seasonality in the production of resource available for the target fish would limit the interest of the acadja as an aquaculture system in brackish ecosystems.