DNA profiling of complex bacterial populations: toxic cyanobacterial blooms

Cyanobacteria are prokaryotic photosynthetic living organisms that inhabit our planet for over three billion years. With a worldwide distribution, they can be found in all types of environments: fresh, brackish and saltwater as well as terrestrial. Though beneficial in the development of life on earth, they also constitute a serious risk to our ecosystems since they can biologically produce harmful secondary metabolites named cyanotoxins. When studying cyanobacteria and their cyanotoxins, several methodologies have been applied with an increasing relevance to molecular methods. Therefore, the aim of this review is to describe alternative molecular methods that can be used as alternative methods for the identification of cyanobacteria. More traditional chemotaxonomic methods are discussed briefly as are the standard and somewhat dated techniques for assessing genome content for taxonomic classification schemes. The use of DNA amplification technology has been applied to the systematics and phylogeny of many bacterial groups, and the optimisation of methods for rapid identification and classification of cyanobacteria are presented. Together with novel methods developed for these photosynthetic microorganisms, the generated DNA profiles have been utilised to study cyanobacterial bloom population diversity and prediction of strain toxigenicity. Finally, the genotypes found were applied to a variety of phylogenetic analyses; trees were reconstructed and compared to the current morphological system of classification. The ecology and diversity of the cyanobacteria is discussed with respect to the derived molecular phylogenies and systematics.     

Bacteria,Archaea, and Crenarchaeota in the Epilimnion and Hypolimnion of a Deep Holo-Oligomictic Lake

In a deep, subalpine holo-oligomictic lake, the relative abundance of Archaea and Crenarchaeota, but not that of Bacteria, increases significantly with depth and varies seasonally. Cell-specific prokaryotic productivity is homogeneous along the water column. The concept of active Archaea observed in the deep ocean can therefore be extended to a deep oxic lake.The abundance, activity, and community composition of epilimnetic and hypolimnetic prokaryotes have been less thoroughly investigated in deep lakes than in oceans. Strong evidence that the depth gradient plays a role in modulating the balance between the domains of Bacteria and Archaea has been found in various marine systems (8, 12, 13, 20). It has been shown that the percentage of Bacteria in the deep marine hypolimnion decreases (up to 5,000 m) while, conversely, the percentage of Archaea increases. The percentage of Crenarchaeota is also higher in the mesopelagic zone than in surface waters (10).Although Archaea have been found in a variety of freshwater habitats (3), little has thus far been published on differentiating between Bacteria, Archaea, and Crenarchaeota in the hypolimnion of deep lakes. An exception is a study of the high-altitude ultraoligotrophic Crater Lake (21, 22), where group I marine Crenarchaeota were observed in deep-water populations (22). This study and another study of various lakes from three continents (9) are based on summer sampling, making it impossible to ascertain the effects of temporal variability on the vertical distribution of Archaea and Crenarchaeota, as has been done for marine systems and shallow lakes (for examples, see references 8 and 11).Our primary objective was to follow variations in the relative abundance of Bacteria, Archaea, and Crenarchaeota found in the hypolimnetic waters of a deep holo-oligomictic lake with a permanent oxic hypolimnion and compare them with those in the epilimnetic assemblages. We used the catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) technique and compared the data thus obtained with prokaryotic productivity.     

C4GEM, a genome-scale metabolic model to study C4 plant metabolism

Leaves of C(4) grasses (such as maize [Zea mays], sugarcane [Saccharum officinarum], and sorghum [Sorghum bicolor]) form a classical Kranz leaf anatomy. Unlike C(3) plants, where photosynthetic CO(2) fixation proceeds in the mesophyll (M), the fixation process in C(4) plants is distributed between two cell types, the M cell and the bundle sheath (BS) cell. Here, we develop a C(4) genome-scale model (C4GEM) for the investigation of flux distribution in M and BS cells during C(4) photosynthesis. C4GEM, to our knowledge, is the first large-scale metabolic model that encapsulates metabolic interactions between two different cell types. C4GEM is based on the Arabidopsis (Arabidopsis thaliana) model (AraGEM) but has been extended by adding reactions and transporters responsible to represent three different C(4) subtypes (NADP-ME [for malic enzyme], NAD-ME, and phosphoenolpyruvate carboxykinase). C4GEM has been validated for its ability to synthesize 47 biomass components and consists of 1,588 unique reactions, 1,755 metabolites, 83 interorganelle transporters, and 29 external transporters (including transport through plasmodesmata). Reactions in the common C(4) model have been associated with well-annotated C(4) species (NADP-ME subtypes): 3,557 genes in sorghum, 11,623 genes in maize, and 3,881 genes in sugarcane. The number of essential reactions not assigned to genes is 131, 135, and 156 in sorghum, maize, and sugarcane, respectively. Flux balance analysis was used to assess the metabolic activity in M and BS cells during C(4) photosynthesis. Our simulations were consistent with chloroplast proteomic studies, and C4GEM predicted the classical C(4) photosynthesis pathway and its major effect in organelle function in M and BS. The model also highlights differences in metabolic activities around photosystem I and photosystem II for three different C(4) subtypes. Effects of CO(2) leakage were also explored. C4GEM is a viable framework for in silico analysis of cell cooperation between M and BS cells during photosynthesis and can be used to explore C(4) plant metabolism.     

Overlooked post-translational modifications of proteins in Plasmodium falciparum: N- and O-glycosylation -- a review

Human malignant malaria is caused by Plasmodium falciparum and accounts for almost 900,000 deaths per year, the majority of which are children and pregnant women in developing countries. There has been significant effort to understand the biology of P. falciparum and its interactions with the host. However, these studies are hindered because several aspects of parasite biology remain controversial, such as N- and O-glycosylation. This review describes work that has been done to elucidate protein glycosylation in P. falciparum and it focuses on describing biochemical evidence for N- and O-glycosylation. Although there has been significant work in this field, these aspects of parasite biochemistry need to be explored further.     

The inheritance of chemical phenotype in Cannabis sativa L

Four crosses were made between inbred Cannabis sativa plants with pure cannabidiol (CBD) and pure Delta-9-tetrahydrocannabinol (THC) chemotypes. All the plants belonging to the F(1)'s were analyzed by gas chromatography for cannabinoid composition and constantly found to have a mixed CBD-THC chemotype. Ten individual F(1) plants were self-fertilized, and 10 inbred F(2) offspring were collected and analyzed. In all cases, a segregation of the three chemotypes (pure CBD, mixed CBD-THC, and pure THC) fitting a 1:2:1 proportion was observed. The CBD/THC ratio was found to be significantly progeny specific and transmitted from each F(1) to the F(2)'s derived from it. A model involving one locus, B, with two alleles, B(D) and B(T), is proposed, with the two alleles being codominant. The mixed chemotypes are interpreted as due to the genotype B(D)/B(T) at the B locus, while the pure-chemotype plants are due to homozygosity at the B locus (either B(D)/B(D) or B(T)/B(T)). It is suggested that such codominance is due to the codification by the two alleles for different isoforms of the same synthase, having different specificity for the conversion of the common precursor cannabigerol into CBD or THC, respectively. The F(2) segregating groups were used in a bulk segregant analysis of the pooled DNAs for screening RAPD primers; three chemotype-associated markers are described, one of which has been transformed in a sequence-characterized amplified region (SCAR) marker and shows tight linkage to the chemotype and codominance.     

Inhibitors of glycosyl-phosphatidylinositol anchor biosynthesis

Glycosyl-phosphatidylinositol (GPI) is a complex glycolipid structure that acts as a membrane anchor for many cell-surface proteins of eukaryotes. GPI-anchored proteins are particularly abundant in protozoa such as Trypanosoma brucei, Leishmania major, Plasmodium falciparum and Toxoplasma gondii, and represent the major carbohydrate modification of many cell-surface parasite proteins. Although the GPI core glycan is conserved in all organisms, many differences in additional modifications to GPI structures and biosynthetic pathways have been reported. Therefore, the characteristics of GPI biosynthesis are currently being explored for the development of parasite-specific inhibitors. In vitro and in vivo studies using sugars and substrate analogues as well as natural compounds have shown that it is possible to interfere with GPI biosynthesis at different steps in a species-specific manner. Here we review the recent and promising progress in the field of GPI inhibition.     

Glycosylphosphatidyl-inositols in murine malaria: Plasmodium yoelii yoelii

Glycosylphosphatidyl-inositols (GPIs) are vital major glycoconjugates in intraerythrocytic stages of Plasmodium. Here, we report on the biosynthesis and the characterization of GPIs synthesized by the murine malarial parasite P. yoelii yoelii YM. Parasitized erythrocytes were labeled in vivo and in vitro with either radioactive nucleotide sugar precursors, ethanolamine or glucosamine. The pathway leading to the formation of GPI precursors was found to resemble that described for P. falciparum; however, in P. yoelii, the formation of an additional hydrophilic precursor containing an acid-labile modification was detected. The data suggest that this modification is linked to the fourth mannose attached to the trimannosyl backbone in an alpha1-2 linkage. The modification was susceptible to hydrofluoric acid (HF), but not to nitrous acid (HNO(2)). Data obtained from size-exclusion chromatography on Bio-Gel P4, and Mono Q analysis of the fragments generated by HNO(2) deamination suggest that the modification is due to the presence of an additional ethanolamine linked to the fourth mannose via a phosphodiester bond.     

Autosomal recessive hypercholesterolemia protein interacts with and regulates the cell surface level of Alzheimer's amyloid beta precursor protein

The familial Alzheimer's disease gene product amyloid beta protein precursor (A beta PP) is sequentially processed by beta- and gamma-secretases to generate the A beta peptide. Although much is known about the biochemical pathway leading to A beta formation, because extracellular aggregates of A beta peptides are considered the cause of Alzheimer's disease, the biological role of A beta PP processing is only recently being investigated. Cleavage of A beta PP by gamma-secretase releases, together with A beta, a COOH-terminal A beta PP intracellular domain, termed AID. Hoping to gain clues about proteins that regulates A beta PP processing and function, we used the yeast two-hybrid system to identify proteins that interact with the AID region of A beta PP. One of the interactors isolated is the autosomal recessive hypercholesterolemia (ARH) adapter protein. This molecular interaction is confirmed in vitro and in vivo by fluorescence resonance energy transfer and in cell lysates. Moreover, we show that reduction of ARH expression by RNA interference results in increased levels of cell membrane A beta PP. These data assert a physiological role for ARH in A beta PP internalization, transport, and/or processing.     

Trimetazidine improves left ventricular function in diabetic patients with coronary artery disease: a double-blind placebo-controlled study

Background

Patients with diabetic cardiomyopathy have an impaired myocardial glucose handling and distal distribution of coronary atherosclerosis. Trimetazidine, an anti-ischemic metabolic agent, improves myocardial glucose utilization though inhibition of fatty acid oxidation. Aim of the present study was to evaluate whether the metabolic effect of trimetazidine on left ventricular function in patients with diabetic cardiomyopathy.

Methods

32 patients (24 males and 8 females, mean (SE) age = 67 ± 6 years) with type 2 diabetes and ischemic cardiomyopathy were randomized to receive either trimetazidine (20 mg, t.d.s.) or placebo (t.d.s.) for six months in a randomized parallel study. Patients performed an echocardiogram at baseline and after 6 months.

Results

Demographic data were comparable between the two groups. After six month baseline left ventricular end-diastolic diameters increased from 62.4 ± 1.7 to 63 ± 2.1 mm in the placebo group, while decreased from 63.2 ± 2.1 to 58 ± 1.6 mm (p < 0.01 compared to baseline) in the trimetazidine group. Compared to baseline, left ventricular ejection fraction increased by 5.4 ± 0.5% (p < 0.05) in the trimetazidine group while remained unchanged in the placebo group -2.4 ± 1.1% (NS), p < 0.01 between groups. A significant improvement in wall motion score index and in the E/A wave ratio was detected in patients treated with trimetazidine, but not in those receiving placebo.

Conclusion

in diabetic patients with ischemic heart disease trimetazidine added to standard medical therapy has beneficial effect on left ventricular volumes and on left ventricular ejection fraction compared to placebo. This effect may be related to the effect of trimetazidine upon cardiac glucose utilization.