Synthesis with good enantiomeric excess of both enantiomers of α-ketols and acetolactates by two thiamin diphosphate-dependent decarboxylases

In addition to the decarboxylation of 2-oxo acids, thiamin diphosphate (ThDP)-dependent decarboxylases/dehydrogenases can also carry out so-called carboligation reactions, where the central ThDP-bound enamine intermediate reacts with electrophilic substrates. For example, the enzyme yeast pyruvate decarboxylase (YPDC, from Saccharomyces cerevisiae) or the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1) can produce acetoin and acetolactate, resulting from the reaction of the central thiamin diphosphate-bound enamine with acetaldehyde and pyruvate, respectively. Earlier, we had shown that some active center variants indeed prefer such a carboligase pathway to the usual one [Sergienko, Jordan, Biochemistry 40 (2001) 7369–7381; Nemeria et al., J. Biol. Chem. 280 (2005) 21,473–21,482]. Herein is reported detailed analysis of the stereoselectivity for forming the carboligase products acetoin, acetolactate, and phenylacetylcarbinol by the E477Q and D28A YPDC, and the E636A and E636Q PDHc-E1 active-center variants. Both pyruvate and β-hydroxypyruvate were used as substrates and the enantiomeric excess was analyzed by a combination of NMR, circular dichroism and chiral-column gas chromatographic methods. Remarkably, the two enzymes produced a high enantiomeric excess of the opposite enantiomer of both acetoin-derived and acetolactate-derived products, strongly suggesting that the facial selectivity for the electrophile in the carboligation is different in the two enzymes. The different stereoselectivities exhibited by the two enzymes could be utilized in the chiral synthesis of important intermediates.     

A network of assembly factors is involved in remodeling rRNA elements during preribosome maturation

Eukaryotic ribosome biogenesis involves ∼200 assembly factors, but how these contribute to ribosome maturation is poorly understood. Here, we identify a network of factors on the nascent 60S subunit that actively remodels preribosome structure. At its hub is Rsa4, a direct substrate of the force-generating ATPase Rea1. We show that Rsa4 is connected to the central protuberance by binding to Rpl5 and to ribosomal RNA (rRNA) helix 89 of the nascent peptidyl transferase center (PTC) through Nsa2. Importantly, Nsa2 binds to helix 89 before relocation of helix 89 to the PTC. Structure-based mutations of these factors reveal the functional importance of their interactions for ribosome assembly. Thus, Rsa4 is held tightly in the preribosome and can serve as a “distribution box,” transmitting remodeling energy from Rea1 into the developing ribosome. We suggest that a relay-like factor network coupled to a mechano-enzyme is strategically positioned to relocate rRNA elements during ribosome maturation.     

Risk Factors for Buruli Ulcer in Ghana—A Case Control Study in the Suhum-Kraboa-Coaltar and Akuapem South Districts of the Eastern Region

Background

Buruli ulcer (BU) is a skin disease caused by Mycobacterium ulcerans. Its exact mode of transmission is not known. Previous studies have identified demographic, socio-economic, health and hygiene as well as environment related risk factors. We investigated whether the same factors pertain in Suhum-Kraboa-Coaltar (SKC) and Akuapem South (AS) Districts in Ghana which previously were not endemic for BU.

Methods

We conducted a case control study. A case of BU was defined as any person aged 2 years or more who resided in study area (SKC or AS District) diagnosed according to the WHO clinical case definition for BU and matched with age- (+/−5 years), gender-, and community controls. A structured questionnaire on host, demographic, environmental, and behavioural factors was administered to participants.

Results

A total of 113 cases and 113 community controls were interviewed. Multivariate conditional logistic regression analysis identified presence of wetland in the neighborhood (OR = 3.9, 95% CI = 1.9–8.2), insect bites in water/mud (OR = 5.7, 95% CI = 2.5–13.1), use of adhesive when injured (OR = 2.7, 95% CI = 1.1–6.8), and washing in the Densu river (OR = 2.3, 95% CI = 1.1–4.96) as risk factors associated with BU. Rubbing an injured area with alcohol (OR = 0.21, 95% CI = 0.008–0.57) and wearing long sleeves for farming (OR = 0.29, 95% CI = 0.14–0.62) showed protection against BU.

Conclusion

This study identified the presence of wetland, insect bites in water, use of adhesive when injured, and washing in the river as risk factors for BU; and covering limbs during farming as well as use of alcohol after insect bites as protective factors against BU in Ghana. Until paths of transmission are unraveled, control strategies in BU endemic areas should focus on these known risk factors.     

Antitumor agents. 256. Conjugation of paclitaxel with other antitumor agents: evaluation of novel conjugates as cytotoxic agents

Sixteen different taxoid conjugates were prepared by linking various anticancer compounds, including camptothecin (CPT), epipodophyllotoxin (EP), colchicine (COL), and glycyrrhetinic acid (GA), at the 2'- or 7-position on paclitaxel (TXL, 1) through an ester, imine, amine, or amide bond. Newly synthesized conjugates were evaluated for cytotoxic activity against replication of several human tumor cell lines. Among them, TXL-CPT conjugates, 8-10, were more potent than TXL itself against the human prostate carcinoma cell line PC-3 (ED(50)=14.8, 3.1, 19.4nM compared with 55.5nM), and conjugate 10 was also 8-fold more active than TXL against the LN-CAP prostate cancer cell line. These compounds also possessed anti-angiogenesis ability as well as lower inhibitory effects against a normal cell line (MRC-5). Thus, conjugates 8-10 are possible antitumor drug candidates, particularly for prostate cancer.     

Toxic effects of mercury(II), cadmium(II) and lead(II) porphyrins on Trypanosoma brucei brucei growth

The effects of free mercury(II), cadmium(II) and lead(II) ions and their metalloporphyrin-derivatives on Trypanosoma brucei brucei growth in culture were studied. All experiments were conducted in the dark. IC(50) values on growth obtained in 24-h time-course experiments were 1.5 x 10(-7), 2.4 x 10(-6), 4.4 x 10(-6) and 2.6 x 10(-5) M for mercury(II) porphyrin, cadmium(II) porphyrin, lead(II) porphyrin and free base porphyrin, respectively. While the IC50 values for Hg2+, Cd2+ and Pb2+ were 3.6 x 10(-6), 1.5 x 10(-5) and 1.6 x 10(-5) M, respectively. These results clearly indicate that the toxicity of the metalloporphyrin complexes of mercury(II), cadmium(II) and lead(II) to T. b. brucei parasites was much higher compared to their free metal ions and free base porphyrin at low concentrations. It was also observed after 8 h incubation that the metalloporphyrins were effective in inhibiting the division of the parasites at concentrations >1.25 x 10(-7) M for mercury(II) porphyrin, concentrations >1.2 x 10(-6) M for cadmium(II) and lead(II) porphyrins and at concentrations >3.6 x 10(-6) M for Hg2+ ion. These observations were not detected in samples treated with the free metal ions and the free base porphyrin at the same concentrations. Interestingly, trypanosomes treated with metalloporphyrin complexes displayed different morphological features from those cells treated with free base porphyrin or metal ions. The chemotherapeutic potential of the metalloporphyrins of H2TMPyP for treatment of African trypanosomiasis is discussed.     

The intermediate chain of cytoplasmic dynein is partially disordered and gains structure upon binding to light-chain LC8

The N-terminal domain of dynein intermediate chain, IC(1-289), is highly disordered, but upon binding to dynein light-chain LC8, it undergoes a significant conformational change to a more ordered structure. Using circular dichroism and fluorescence spectroscopy, we demonstrate that the change in conformation is due to an increase in the helical structure and to enhanced compactness in the environment of tryptophan 161. An increase in helical structure and compactness is also observed with trimethylamine-N-oxide (TMAO), a naturally occurring osmolyte used here as a probe to identify regions with a propensity for induced folding. Global protection of IC(1-289) from protease digestion upon LC8 binding was localized to a segment that includes residues downstream of the LC8-binding site. Several smaller constructs of IC(1-289) containing the LC8-binding site and one of the predicted helix or coiled-coil segments were made. IC(1-143) shows no increase in helical structure upon binding, while IC(114-260) shows an increase in helical structure similar to what is observed with IC(1-289). Binding of IC(114-260) to LC8 was monitored by fluorescence and native gel electrophoresis and shows saturation of binding, a stoichiometry of 1:1, and moderate binding affinity. The induced folding of IC(1-289) upon LC8 binding suggests that LC8 could act through the intermediate chain to facilitate dynein assembly or regulate cargo-binding interactions.     

Heteronuclear NMR identifies a nascent helix in intrinsically disordered dynein intermediate chain: implications for folding and dimerization

The intermediate chain of dynein forms a tight subcomplex with dimeric light chains LC8 and Tctex-1, and together they constitute the cargo attachment complex. There is considerable interest in identifying the role of these light chains in the assembly of the two copies of the intermediate chain. The N-terminal domain of the intermediate chain, IC1-289, contains the binding sites for the light chains, and is a highly disordered monomer but gains helical structure upon binding to light chains LC8 and Tctex-1. To provide insights into the structural and dynamic changes that occur in the intermediate chain upon light chains binding, we have used NMR spectroscopy to compare the properties of two distinct sub-domains of IC1-289: IC84-143 which is the light chains binding domain, and IC198-237, which contains a predicted coiled coil necessary for the increase in ordered structure upon light chain binding. Neither construct has stable secondary structure when probed by circular dichroism and amide chemical shift dispersion. Specific residues of IC84-143 involved in binding to the light chains were identified by their increase in resonance line broadening and the corresponding large intensity reduction in 1H-15N HSQC spectra. Interestingly, IC84-143 shows no sign of structure formation after binding to either LC8 or Tctex-1 or to both. IC198-237, on the other hand, contains a population of a nascent helix at low temperature as identified by heteronuclear NMR relaxation measurements, secondary chemical shifts, and sequential amide-amide connectivities. These data are consistent with a model for light chain binding coupled to intermediate chain dimerization through forming a coiled coil distant from the binding site.     

Multiple Recognition Motifs in Nucleoporin Nup159 Provide a Stable and Rigid Nup159-Dyn2 Assembly

Dyn2 is the yeast ortholog of the molecular hub LC8, which binds disordered proteins and promotes their self-association and higher order assembly. Dyn2 is proposed to dimerize and stabilize the Nup82-Nsp1-Nup159 complex of the nuclear pore assembly through its interaction with nucleoporin Nup159. Nup159 has six LC8 recognition motifs separated by short linkers. NMR experiments reported here show that the Dyn2 binding domain of Nup159 is intrinsically disordered and that binding of one equivalent of Dyn2 dimer aligns two Nup159 chains along the full Dyn2 binding domain to form a bivalent scaffold that promotes binding of other Dyn2 dimers. Isothermal titration calorimetry of Dyn2 binding to Nup constructs of increasing lengths determine that the third LC8 recognition motifs does not bind Dyn2. A new approach to identifying active LC8 recognition motifs based on NMR-detected β-sheet propensities is presented. Isothermal titration calorimetry experiments also show that, due to unfavorable entropy changes, a Nup-Dyn2 complex with three Dyn2 dimers is more stable than the wild-type complex with five Dyn2 dimers. The calorimetric results argue that, from a thermodynamics perspective, only three Dyn2 dimers are needed for optimal stability and suggest that the evolutionary adaptation of multiple tandem LC8 recognition motifs imparts to the complex other properties such as rigidity and a kink in the rod-like structure. These findings extend the repertoire of functions of intrinsically disordered protein to fine-tuning and versatile assembly of higher order macromolecular complexes.     

Extract of Ocimum canum lowers blood glucose and facilitates insulin release by isolated pancreatic β-islet cells

Aqueous extract of Ocimum canum Sim, (Lamiaceae) is used by some Ghanaians to manage diabetes mellitus. In vivo modulation of levels of fasting blood glucose by 0. canum extract was evaluated in type-II diabetes mellitus using the C57BL/KsJ db/db genetically diabetic animal model, and its effects on glucose-stimulated insulin release in vitro were monitored using isolated rat pancreatic beta-islet cells. The results showed that fasting blood glucose levels and body weight decreased significantly (p < 0.05) in diabetic and non-diabetic C57BL/KsJ mice, which were administered aqueous extract of 0. canum. In vitro, the 0. canum extract significantly enhanced insulin release from isolated rat pancreatic beta-islet cells. Insulin release was found to be dependent on glucose concentration and increased with increasing O. canum concentration in the incubation medium up to an optimum extract concentration of 0.03 mg/ml. Release of the hormone decreased beyond this concentration of extract in the medium. Addition to the medium of Desmodium adscendens, a plant preparation used to manage inflammatory disorders, did not increase but rather inhibited insulin secretion by the pancreatic beta-islet cells. These results could explain the use of 0. canum in Ghanaian folk medicine to manage diabetes mellitus.