UNICELLULAR CYANOBIONTS IN OPEN OCEAN DINOFLAGELLATES,RADIOLARIANS, AND TINTINNIDS: ULTRASTRUCTURAL CHARACTERIZATION AND IMMUNO‐LOCALIZATION OF PHYCOERYTHRIN AND NITROGENASE1

Cyanobacterial symbionts (cyanobionts) have been identified forming associations with various open ocean eukaryotic host genera, including two dinophysoid genera, Histioneis sp. and Ornithocercus sp., two radiolarians, Spongastaurus and Dictyocoryne truncatum, sp., and a tintinnid, Codonella sp. The TEM analysis revealed that single individual hosts were closely associated with one to two different cyanobacterial morphotypes (cyanobionts) and two hosts had in addition to cyanobionts, one to two bacterial cell types. Eleven significantly (P<0.01) different cell types were identified as cyanobionts, with cell diameters ranging 0.5±0.38–3.7±0.66 μm. Using immunogold‐labeling techniques coupled to the TEM, four of the five cell types contained phycoerythrin (PE) at high levels (>71±28 gold particles·μm^?2). Immunolabeling‐TEM using nitrogenase antisera demonstrated a significant (P<0.01) nitrogenase content in cell type four cyanobionts of Histioneis sp. host 1 (39±34 gold particles·μm^?2). The cyanobionts of the radiolarians were of a cell diameter (0.5–0.8 μm) and showed ultrastructural characters (peripheral thylakoids) reminiscent of Prochlorococcus sp. Also, an open ocean tintinnid, Codonella sp., was shown to contain cyanobacteria as symbionts for the first time. In all cyanobionts, glycogen storage was obvious, no cellular degradation was visible, cells were observed in the process of cellular division, and antisera localization was apparent. These observations suggest that the relationship between host eukaryote and cyanobacteria is an active one, and likely symbiotic.     

Novel reversible methionine aminopeptidase-2 (MetAP-2) inhibitors based on purine and related bicyclic templates

The natural product fumagillin 1 and derivatives like TNP-470 2 or beloranib 3 bind to methionine aminopeptidase 2 (MetAP-2) irreversibly. This enzyme is critical for protein maturation and plays a key role in angiogenesis. In this paper we describe the synthesis, MetAP-2 binding affinity and structural analysis of reversible MetAP-2 inhibitors. Optimization of enzymatic activity of screening hit 10 (IC₅₀: 1 μM) led to the most potent compound 27 (IC₅₀: 0.038 μM), with a concomitant improvement in LLE from 2.1 to 4.2. Structural analysis of these MetAP-2 inhibitors revealed an unprecedented conformation of the His339 side-chain imidazole ring being co-planar sandwiched between the imidazole of His331 and the aryl-ether moiety, which is bound to the purine scaffold. Systematic alteration and reduction of H-bonding capability of this metal binding moiety induced an unexpected 180° flip for the triazolo[1,5-a]pyrimdine bicyclic template.     

Importance of a hypervariable active-site residue in human Mu class glutathione transferases catalyzing the bioactivation of chemotherapeutic thiopurine prodrugs

Glutathione transferases (GSTs) catalyze the bioactivation of the thiopurine prodrugs azathioprine, cis-6-(2-acetylvinylthio)purine (cAVTP) and trans-6-(2-acetylvinylthio)guanine (tAVTG), thereby releasing the antimetabolites 6-mercaptopurine and 6-thioguanine. In the GST Mu class, GST M1-1 has the highest catalytic efficiency, whereas GST M2-2 and other enzymes are less active. In the evolution of Mu class GSTs, residue 210 appears hypervariable and has particular functional significance. We demonstrate that the catalytic activity of GST M1-1 with cAVTP or tAVTG is successively diminished when wild-type Ser-210 is mutated into Ala followed by Thr. Conversely, mutating wild-type Thr-210 in GST M2-2 into Ala and Ser enhanced the corresponding activities. Comparisons were also made with GST M2-2 distinguished by Gly or Pro in position 210, as well as wild-type GSTs M4-4 and M5-5. The results suggest that the hydroxyl group of Ser in position 210 stabilizes the transition state of the GST-catalyzed reaction. The low activity of GSTs containing Thr in position 210 is probably due to steric hindrance caused by the beta-methyl group of the side chain. The ratios of the different catalytic efficiencies were translated into differences in the Gibbs free energies of transition state stabilization. The effects of the mutations were qualitatively parallel for the alternative substrates, but vary significantly in magnitude. From the evolutionary perspective the data show that a point mutation can alternatively enhance or attenuate the activity with a particular substrate and illustrate the functional plasticity of GSTs.     

Pneumococcal Carriage in Children under Five Years in Uganda-Will Present Pneumococcal Conjugate Vaccines Be Appropriate?

BackgroundPneumonia is the major cause of death in children globally, with more than 900,000 deaths annually in children under five years of age. Streptococcus pneumoniae causes most deaths, most often in the form of community acquired pneumonia. Pneumococcal conjugate vaccines (PCVs) are currently being implemented in many low-income countries. PCVs decrease vaccine-type pneumococcal carriage, a prerequisite for invasive pneumococcal disease, and thereby affects pneumococcal disease and transmission. In Uganda, PCV was launched in 2014, but baseline data is lacking for pneumococcal serotypes in carriage.ObjectivesTo study pneumococcal nasopharyngeal carriage and serotype distribution in children under 5 years of age prior to PCV introduction in UgandaMethodsThree cross-sectional pneumococcal carriage surveys were conducted in 2008, 2009 and 2011, comprising respectively 150, 587 and 1024 randomly selected children aged less than five years from the Iganga/Mayuge Health and Demographic Surveillance Site. The caretakers were interviewed about illness history of the child and 1723 nasopharyngeal specimens were collected. From these, 927 isolates of S. pneumoniae were serotyped.ResultsOverall, the carriage rate of S. pneumoniae was 56% (957/1723). Pneumococcal carriage was associated with illness on the day of the interview (OR = 1.50, p = 0.04). The most common pneumococcal serotypes were in descending order 19F (16%), 23F (9%), 6A (8%), 29 (7%) and 6B (7%). One percent of the strains were non-typeable. The potential serotype coverage rate for PCV10 was 42% and 54% for PCV13.ConclusionAbout half of circulating pneumococcal serotypes in carriage in the Ugandan under-five population studied was covered by available PCVs.     

Norepinephrine Metabolism in Humans Studied by Deuterium Labelling: Turnover of 4–Hydroxy-3–methoxyphenylglycol

Abstract: D, L(±)-4-Hydroxy-3-methoxyphenylglycol (HMPG) labelled with three deuterium atoms was used to study turnover of plasma free HMPG following an intravenous injection. Ten healthy men were given a pulse dose of either 4.3 μmol or 2.2 μmol of labelled HMPG ([²H₃]HMPG piperazine salt). Plasma and urine levels of both endogenous and labelled HMPG were subsequently followed by gas chromatography-mass spectrometry with selected ion detection. Kinetic calculations based upon a single-compartment model were consistent with a monoexponential elimination of plasma free HMPG. The half-life of HMPG was 0.46 and 0.78 h (mean values in the two dose groups). The HMPG production rate was 2.01 and 2.35 μmol/hour, and the urinary excretion rate of HMPG (free and conjugated) was 0.48 and 0.47 μmol/h. The endogenous plasma level of free HMPG was 25 and 33 nmol/L. The results show that HMPG turns over rapidly and that HMPG is further metabolized extensively. About one-fourth of the HMPG produced is excreted in urine as free and conjugated HMPG.     

Thyroxine as a Mediator of Metamorphosis of Atlantic Halibut, Hippoglossus Hippoglossus

The response of morphological, histological and endocrinological development to exogenous 1-thyroxine (T₄) and to water depth during metamorphosis in Atlantic halibut, Hippoglossus hippoglossus, was investigated. Exogenous T₄ was given in daily doses of 0.1, 0.05 ppm or a control treatment to halibut larvae at 550 daydegrees (posthatch, premetamorphic) for 14 days. Water depths of 40 cm, 10 cm or 1.5 cm were used to rear halibut larvae from 590 daydegrees for 21 days. Halibut larvae given exogenous T₄ at 0.1 ppm had accelerated eye migration relative to MH in fish given 0.05 ppm and in control fish. Pigmentation was correlated with dosage after 14 days. The volume of thyroid tissue was expressed in a dose-dependent manner and exhibited a size-dependency within each treatment. However, the follicles were atypical with reduced colloid, increased lumen and low epithelial cells even in the control group. The results indicate that T₄ is a mediator in halibut metamorphosis. In the water depth experiment, only cortisol levels of larvae reared in 1.5 cm water were significantly affected after 21 days, but this was not correlated with metamorphic rate. Hormone profiles, morphological changes and size suggest the existence of a window of opportunity for metamorphosis in halibut extending from about 16 mm and tapering off about 21 mm SL. The pooled hormone profiles indicate the commencement of a hormonal cascade similar to that of other flatfishes during metamorphosis. The results indicate that growth, neural and skeletal transformation, and pigmentation are biochemically separate processes in the metamorphosis of Atlantic halibut.     

Antibacterial peptides in stimulated human granulocytes: characterization of ubiquitinated histone H1A.

Antibacterial peptides were isolated from human peripheral granulocytes of a healthy donor who had been treated with granulocyte-colony stimulating factor (G-CSF) and cortisol. Peptides were solubilized in acidified chloroform/methanol, and partitioned in chloroform/methanol/water. Water- soluble polypeptides were separated by cation-exchange and reversed-phase chromatography. Several previously characterized antibacterial polypeptides were identified; defensins 1-3, defensin 4, lysozyme, eosinophil cationic protein, and calgranulin A. In addition, several histone fragments were isolated and exhibited activity against the Gram- positive bacterium Bacillus megaterium strain Bm11. These fragments included two C-terminal fragments of histone H1A, three C-terminal fragments of histone H1D, one fragment of histone H1B, and two fragments of histone H4. The molecular masses of both histone H1A fragments, as determined by electrospray (ES) MS, were 270 Da higher than those calculated from their amino acid sequences. The two histone H1A fragments corresponded to Lys152-Lys222 (7527 +/- 1 Da) and Lys167-Lys222 (6023 +/- 1 Da). Tandem MS (MS/MS) of the 7.5 kDa and 6.0 kDa fragments indicated that the post-translational modification is on Lys222, the epsilon-amino group of which was conjugated with the alpha-carboxyl group of the tripeptide Arg-Gly-Gly. This finding was substantiated by digestion of the 7.5-kDa polypeptide with trypsin and analysis of the resulting peptides by ES MS and MS/MS. The tripeptide Arg-Gly-Gly corresponded uniquely to the three C-terminal residues of ubiquitin, demonstrating the presence of ubiquitinated histone H1A.     

Auxin-mediated induction of GAL promoters by conditional degradation of Mig1p improves sesquiterpene production in Saccharomyces cerevisiae with engineered acetyl-CoA synthesis

The yeast Saccharomyces cerevisiae uses the pyruvate dehydrogenase-bypass for acetyl-CoA biosynthesis. This relatively inefficient pathway limits production potential for acetyl-CoA-derived biochemical due to carbon loss and the cost of two high-energy phosphate bonds per molecule of acetyl-CoA. Here, we attempted to improve acetyl-CoA production efficiency by introducing heterologous acetylating aldehyde dehydrogenase and phosphoketolase pathways for acetyl-CoA synthesis to enhance production of the sesquiterpene trans-nerolidol. In addition, we introduced auxin-mediated degradation of the glucose-dependent repressor Mig1p to allow induced expression of GAL promoters on glucose so that production potential on glucose could be examined. The novel genes that we used to reconstruct the heterologous acetyl-CoA pathways did not sufficiently complement the loss of endogenous acetyl-CoA pathways, indicating that superior heterologous enzymes are necessary to establish fully functional synthetic acetyl-CoA pathways and properly explore their potential for nerolidol synthesis. Notwithstanding this, nerolidol production was improved twofold to a titre of ˜ 900 mg l⁻¹ in flask cultivation using a combination of heterologous acetyl-CoA pathways and Mig1p degradation. Conditional Mig1p depletion is presented as a valuable strategy to improve the productivities in the strains engineered with GAL promoters-controlled pathways when growing on glucose.