Increased production of serotonin by suspension and root cultures ofPeganum harmala transformed with a tryptophan decarboxylase cDNA clone fromCatharanthus roseus

Cell suspension and root cultures ofPeganum harmala were established expressing a tryptophan decarboxylase cDNA clone fromCatharanthus roseus under the control of the cauliflower mosaic virus (CaMV) 35S promoter and terminator sequences. The tryptophan decarboxylase activity of some of the transgenic lines was greatly enhanced (25–40 pkat/mg protein) as compared to control cultures (1–5 pkat per mg protein) and remained high during the growth cycle. While the levels of tryptamine, the product of the reaction catalysed by tryptophan decarboxylase, were unchanged in the transgenic lines, their serotonin contents were enhanced up to 10-fold, reaching levels of 1.5 to 2% dry mass. Thus, tryptamine produced by the engineered reaction was apparently immediately used for enhanced serotonin biosynthesis. The yields of serotonin in transgenic lines overexpressing tryptophan decarboxylase activity were further enhanced to 3–5% dry mass by feedingl-tryptophan, while no or only minor effects were seen when control cultures were fed. These data demonstrate that the production of a plant secondary metabolite can be enhanced greatly via genetic manipulation of the level of activity of the rate-limiting enzyme. The amounts of -carboline alkaloids, the other tryptamine-derived metabolites ofP. harmala, in contrast, were not affected by the overproduction of tryptamine. The information needed for successfully predicting manipulations that enhance production of a secondary metabolite is discussed.     

Synthesis and β-lactamase inhibitory activity of 6-fluoropenicillanic acids

The benzyl 6-fluoro-penicillanate sulfides 4a, 6a, 7a; and sulfones 6c, 7d were synthesized. The conversion to their free acids 4b, 4b, 6d, 7b, 7e and potassium salts 7c, 7f are described. These acids and salt 7c were evaluated as β- lactamase inhibitors using β-lactamase I from Bacillus cereus. The data indicate that substitution of the 6-hydrogen by a 6- fluorine atom on 6β-bromopenicillanic acid (1), leads to loss of β-lactamase inhibitory activity. In the case of the isomers 6β- and 6-fluoropenicillanic acids the 6β-enantiomer proved to be considerably more potent. Potassium salts of 6β- fluoropenicillanate sulfide and sulfone were unstable in solid state and in water solution. The fragmentation of the sulfone in two parts in water solution is consistent with the hydrolytic behavior of the penicillanic acid sulfone (2) with 0.5 N NaOH.     

Deconstructing the dimensions of alpha diversity in squamate reptiles (Reptilia: Squamata) across the Americas

Aim

Our aim is to document the dimensions of current squamate reptile biodiversity in the Americas by integrating taxonomic, phylogenetic and functional data, and assessing how this may vary across phylogenetic scales. We also explore the potential underlying mechanisms that may be responsible for the observed geographical diversity patterns.

Location

The Americas.

Time period

Present.

Major taxa

Squamate reptiles.

Methods

We used published data on the distribution, phylogeny, and body size of squamate reptiles to document the current dimensions of their alpha diversity in the Americas. We overlapped species ranges to estimate taxonomic diversity (TD) and calculated phylogenetic diversity (PD) using mean pairwise phylogenetic distance (MPD), speciation rate (DivRate) and Faith's phylogenetic index (PD). We estimated functional diversity (FD) as trait dispersion in the multivariate space using body size and leg development data. We implemented a deconstructive macroecological approach to understand how spatial mismatches between the three facets of diversity vary across phylogenetic scales, and the potential eco-evolutionary mechanisms driving these patterns across space.

Results

We found a strong latitudinal gradient of TD with a large accumulation in tropical regions. PD and FD patterns were largely similar likely due to the high phylogenetic signal in the traits used, and higher values tended to be concentrated in harsh and/or heterogeneous environments. We found differences between major clades within Squamata that display contrasting geographical patterns. Several regions across the continent shared the same spatial mismatches between dimensions across clades, suggesting that similar eco-evolutionary processes are shaping these regional reptile assemblages. However, we also found evidence that non-mutually exclusive processes can operate differently across clades.

Main conclusions

The deconstructive approach implemented here is based on a solid macroecological framework. We can extend this to other taxonomic groups to establish whether there are particularities about how different eco-evolutionary mechanisms shape biodiversity facets in a spatially explicit context.     

Advanced mass spectrometry workflows for accurate quantification of trace-level host cell proteins in drug products: Benefits of FAIMS separation and gas-phase fractionation DIA

Therapeutic monoclonal antibodies (mAb) production relies on multiple purification steps before release as a drug product (DP). A few host cell proteins (HCPs) may co-purify with the mAb. Their monitoring is crucial due to the considerable risk they represent for mAb stability, integrity, and efficacy and their potential immunogenicity. Enzyme-linked immunosorbent assays (ELISA) commonly used for global HCP monitoring present limitations in terms of identification and quantification of individual HCPs. Therefore, liquid chromatography tandem mass spectrometry (LC-MS/MS) has emerged as a promising alternative. Challenging DP samples show an extreme dynamic range requiring high performing methods to detect and reliably quantify trace-level HCPs. Here, we investigated the benefits of adding high-field asymmetric ion mobility spectrometry (FAIMS) separation and gas phase fractionation (GPF) prior to data independent acquisition (DIA). FAIMS LC-MS/MS analysis allowed the identification of 221 HCPs among which 158 were reliably quantified for a global amount of 880 ng/mg of NIST mAb Reference Material. Our methods have also been successfully applied to two FDA/EMA approved DPs and allowed digging deeper into the HCP landscape with the identification and quantification of a few tens of HCPs with sensitivity down to the sub-ng/mg of mAb level.     

Alkaline phosphatase. A dominant enzyme of microvillus structure of cephalopod photoreceptors

The rhabdomeres of cephalopod photoreceptors, which are built up mainly of rhodopsin and phospholipid molecules, show a very high alkaline phosphatase activity. The enzyme has been partially characterized in purified rhodopsin vesicle fractions of the rhabdomeres by the following kinetic data: pH optimum 8.7; activation energy 9100 cal·m^?1; $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{= 2.5}$ μmol·min^?1·mg^?1; $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 1.5·10}{}^{\mathrm{?4}}\text{M}$; its activity depends on Mg²⁺. There is good evidence that the alkaline phosphatase is a membrane-bound enzyme with receptor sites presumably located on the inside of the membrane. This enzyme has not been purified but its high activity compared to that of other known alkalin phosphatases (see Table I) indicates that each mirovillus, the structural unit of the rhabdomere, contains 1–20 enzyme molecules. This finding supports the hypothesis that the alkaline phosphatase is involved in the biochemical amplification process of excitation, or adaptation.     

Enhancement of rat liver catalase activity dietary cholesterol

The effects of a fat-supplemented diet and clofibrate (ethylchlorophenoxyisobutirate) upon serum lipids and liver catalase activity were studied in male rats. A butter-supplemented diet produced a striking increase of serum triglycerides but did not affect the liver catalase activity. Cholesterol (1%, w/w), added to the butter supplemented diet markedly increased liver catalase activity. This diet produced a hypercholesterolemic state higher than that induced by a butter-supplemented diet only, although the hypertriglyceridemic effect was less pronounced. Clofibrate given a butter-supplemented diet produced a marked increase of liver catalase activity (about four-fold). When clofibrate is administered with the cholesterol-supplemented diet, the increment observed in the liver catalase activity was the same as that induced with the cholesterol supplemented diet alone. Clofibrate, in either lipid-rich diet, failed to induce a hypocholesterolemic response, although a clear hypotrigliceridemic effect was evident. This effect appears to be potentiated with clofibrate and the cholesterol supplemented diet. Thus the increment in liver catalase activity induced by dietary cholesterol and clofibrate seems to be related to a hypotriglyceridemic effect which gives support to a role of liver peroxisomes in lipid metabolism. The role that liver catalase would play, in this regard, remains unclear from these results.