Triauxic growth of an oleaginous red yeast <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> on waste ‘extract’ for enhanced and concomitant lipid and β-carotene production

Background

Vegetable ‘mandi’ (road-side vegetable market) waste was converted to a suitable fermentation medium for cultivation of oleaginous yeast Rhodosporidium toruloides by steaming under pressure. This cultivation medium derived from waste was found to be a comparatively better source of nutrients than standard culture media because it provided more than one type of usable carbon source(s) to yeast.

Results

HPLC results showed that the extract contained glucose, xylose and glycerol along with other carbon sources, allowing triauxic growth pattern with preferably usage of glucose, xylose and glycerol resulting in enhanced growth, lipid and carotenoid production. Presence of saturated and unsaturated fatty acid methyl esters (FAMEs) (C_14-20) in the lipid profile showed that the lipid may be transesterified for biodiesel production.

Conclusion

Upscaling these experiments to fermenter scale for the production of lipids and biodiesel and other industrially useful products would lead to waste management along with the production of value added commodities. The technique is thus environment friendly and gives good return upon investment.

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Recent trends and advancements in microbial tannase-catalyzed biotransformation of tannins: a review

The outburst of green biotechnology has facilitated a substantial upsurge in the usage of enzymes in a plethora of industrial bioconversion processes. The tremendous biocatalytic potential of industrial enzymes provides an upper edge over chemical technologies in terms of safety, reusability, and better process control. Tannase is one such enzyme loaded with huge potential for bioconversion of hydrolysable tannins to gallic acid. Tannins invariably occur in pteridophytes, gymnosperms, and angiosperms and predominately cumulate in plant parts like fruits, bark, roots, and leaves. Furthermore, toxic tannery effluents from various tanneries are loaded with significant levels of tannins in the form of tannic acid. Tannase can be principally employed for debasing the tannins that predominately occur in the toxic tannery effluents thus providing a relatively much cheaper measure for their biodegradation. Over the years, microbial tannase-catalyzed tannin degradation has gained momentum. The plentious availability of tannin-containing agro- and industrial waste paves a way for efficient utilization of microbial tannase for tannin degradation eventually resulting into gallic acid production. Gallic acid has received a great deal of attention as a molecule of enormous therapeutic and indusrial potential. The current worldwide demand of gallic acid is 8000 t per annum. As a matter of fact, bioconversion of tannins into gallic acid through fermentation has not been exploited completely. This necessitates further studies for development of more efficient, economical, productive processes and improved strains for gallic acid production so as to meet its current demand.     

(±)-2-(N-tert-Butylamino)-3'-[(125)I]-iodo-4'-azidopropiophenone: a dopamine transporter and nicotinic acetylcholine receptor photoaffinity ligand based on bupropion (Wellbutrin, Zyban)

Towards addressing the knowledge gap of how bupropion interacts with the dopamine transporter (DAT) and nicotinic acetylcholine receptors (nAChRs), a ligand was synthesized in which the chlorine of bupropion was isosterically replaced with an iodine and a photoreactive azide was added to the 4'-position of the aromatic ring. Analog (±)-3 (SADU-3-72) demonstrated modest DAT and α4β2 nAChR affinity. A radioiodinated version was shown to bind covalently to hDAT expressed in cultured cells and affinity-purified, lipid-reincorporated human α4β2 neuronal nAChRs. Co-incubation of (±)-[(125)I]-3 with non-radioactive (±)-bupropion or (-)-cocaine blocked labeling of these proteins. Compound (±)-[(125)I]-3 represents the first successful example of a DAT and nAChR photoaffinity ligand based on the bupropion scaffold. Such ligands are expected to assist in mapping bupropion-binding pockets within plasma membrane monoamine transporters and ligand-gated nAChR ion channels.     

β2-Adrenergic receptor polymorphisms: pharmacogenetic response to bronchodilator among African American asthmatics

Beta2-adrenergic receptor (beta2AR) gene polymorphisms have been reported to be associated with various asthma-related traits in different racial/ethnic populations. However, it is unknown whether beta2AR genetic variants are associated with asthma in African Americans. In this study, we have examined whether there is association between beta2AR genetic variants and asthma in African Americans. We have recruited 264 African American asthmatic subjects and 176 matched healthy controls participating in the Study of African Americans, Asthma, Genes and Environments (SAGE). We genotyped seven known and recently identified beta2AR SNP variants, then tested genotype and haplotype association of asthma-related traits with the beta2AR SNPs in our African American cohort with adjustment of confounding effect due to admixture background and environmental risk factors. We found a significant association of the SNP -47 (Arg-19Cys) polymorphism with DeltaFEF(25-75), a measure of bronchodilator drug responsiveness, in African American asthmatics after correction for multiple testing (P = 0.001). We did not observe association of the SNP +46 (Arg16Gly) variant with asthma disease diagnosis and asthma-related phenotypes. In contrast to previous results between the Arg16Gly variant and traits related to bronchodilator responsiveness, our results indicate that the Arg-19Cys polymorphism in beta upstream peptide may play an important role in bronchodilator drug responsiveness in African American subjects. Our findings highlight the importance of investigating genetic risk factors for asthma in different populations.     

Application of mass spectrometry to the discovery of biomarkers for detection of prostate cancer

There has been an impressive emergence of mass spectrometry based technologies applied toward the study of proteins. Equally notable is the rapid adaptation of these technologies to biomedical approaches in the realm of clinical proteomics. Concerted efforts toward the elucidation of the proteomes of organ sites or specific disease state are proliferating and from these efforts come the promise of better diagnostics/prognostics and therapeutic intervention. Prostate cancer has been a focus of many such studies with the promise of improved care to patients via biomarkers derived from these proteomic approaches. The newer technologies provide higher analytical capabilities, employ automated liquid handling systems, fractionation techniques and bioinformatics tools for greater sensitivity and resolving power, more robust and higher throughput sample processing, and greater confidence in analytical results. In this prospects, we summarize the proteomic technologies applied to date in prostate cancer, along with their respective advantages and disadvantages. The development of newer proteomic strategies for use in future applications is also discussed.     

[3H]Epibatidine Photolabels Non-equivalent Amino Acids in the Agonist Binding Site of Torpedo and ��4��2 Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo α₂βγδ and expressed human α4β2 nAChRs with [³H]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of αTyr¹⁹⁸ in agonist binding site Segment C of the principal (+) face in both α subunits and of γLeu¹⁰⁹ and γTyr¹¹⁷ in Segment E of the complementary (−) face, with no labeling detected in the δ subunit. For affinity-purified α4β2 nAChRs, [³H]epibatidine photolabeled α4Tyr¹⁹⁵ (equivalent to Torpedo αTyr¹⁹⁰) in Segment C as well as β2Val¹¹¹ and β2Ser¹¹³ in Segment E (equivalent to Torpedo γLeu¹⁰⁹ and γTyr¹¹¹, respectively). Consideration of the location of the photolabeled amino acids in homology models of the nAChRs based upon the acetylcholine-binding protein structure and the results of ligand docking simulations suggests that epibatidine binds in a single preferred orientation within the α-γ transmitter binding site, whereas it binds in two distinct orientations in the α4β2 nAChR.Nicotinic acetylcholine receptors (nAChRs)³ are prototypical members of the Cys loop superfamily of neurotransmitter-gated ion channels that mediate the actions of the neurotransmitter acetylcholine (1). nAChRs from vertebrate skeletal muscle and the electric organs of Torpedo rays are heteropentamers of homologous subunits with a stoichiometry of 2α:β:γ(ϵ):δ that are arranged pseudosymmetrically around central cation-selective ion channels (1, 2). There are 12 mammalian neuronal nAChR subunit genes: nine neuronal α subunits (α2–α10) and three neuronal β subunits (β2–β4). The α4β2 nAChR is the most abundant and widely distributed nAChR subtype expressed in the brain and is a major target for potential therapeutic agents for neurological diseases and conditions, including nicotine dependence and Alzheimer and Parkinson diseases (3, 4). Although the ratio of α4 to β2 subunit in vivo is uncertain, expressed receptors containing either three α4 or three β2 subunits have distinct pharmacological properties (5, 6).The agonist binding sites (ABS) of nAChRs are located within the amino-terminal extracellular domain at the interface of adjacent subunits (α-γ and α-δ in the Torpedo nAChR), and different nAChR subunit combinations form ABS with distinct physical and pharmacological properties (3, 7). Affinity labeling studies with Torpedo nAChR and site-directed mutational analyses of muscle and neuronal nAChRs identified key amino acids delineating the ABS from three noncontiguous stretches of the α subunit (Segments A-C, the principal component (+ face)) and three noncontiguous regions of the non-α subunit (Segments D–F, the complementary component (− face)) (8, 9). The three-dimensional structure of the ABS in the absence and presence of nAChR agonists or competitive antagonists has been determined for snail acetylcholine-binding proteins (AChBPs) that are soluble homopentamers homologous to the extracellular (amino-terminal) domain of a nAChR (10–12). In the AChBP, four aromatic amino acids from Segments A–C that are conserved within α subunits, along with a conserved Trp in Segment D, form a core aromatic “pocket” with a dimension optimal for accommodation of a trimethylammonium group. The other amino acids in the non-α subunits closest to the aromatic pocket, which are generally not conserved among γ, δ, or neuronal β subunits, are on three antiparallel β strands. The AChBP structure was used to refine the structure of the Torpedo nAChR in the absence of agonist to 4 Å resolution (13). In this structure, there is a reorientation of Segments A–C, resulting in the absence of a well defined core aromatic binding pocket.Analysis of agonist interactions with mutant nAChRs containing fluorine-substituted core aromatic residues provides evidence that cation-π interactions, particularly with αTrp¹⁴⁹ in Segment B, are important determinants of agonist binding affinity (14) and for the higher affinity binding of nicotine to α4β2 nAChRs compared with α₂βγδ nAChRs (15). Mutational analyses and molecular docking calculations have also provided evidence that two molecules of very similar structure may actually bind to a single receptor in very different orientations, as seen for two high affinity antagonists, d-tubocurarine and its quaternary ammonium analog metocurine, binding to the AChBP and to the muscle nAChR (16, 17).Photoaffinity labeling provides an alternative means to identify amino acids contributing to a drug binding site (18, 19) and has been used to determine the orientation of drugs bound in the ABS of Torpedo nAChR (20). Epibatidine binds with very high affinity (∼10 pm) to heteromeric neuronal nAChRs (e.g. α4β2) and with nanomolar affinity to α7 and muscle-type/Torpedo nAChRs (3). Utilizing a photoreactive analogue of epibatidine (azidoepibatidine; Fig. 1) and mass spectrometry, Tomizawa et al. (21) identified photolabeled amino acids in the Aplysia AChBP (Tyr¹⁹⁵ in Segment C and Met¹¹⁶ in Segment E), establishing an orientation for bound azidoepibatidine consistent with the orientation of epibatidine in an AChBP crystal structure (12).Open in a separate windowFIGURE 1.Structure of [³H]epibatidine (top) and azidoepibatidine (bottom).In this report, we use [³H]epibatidine as a photoaffinity reagent to identify the amino acids photolabeled in an expressed α4β2 nAChR and in the Torpedo α₂βγδ nAChR. Comparisons of the labeled amino acids seen in the Torpedo nAChR α-γ binding site and in the α4β2 nAChR, in conjunction with the results of docking calculations for epibatidine binding to homology models of the α₂βγδ and α4β2 nAChRs, suggests that epibatidine binds in a single orientation in the α-γ site but in two orientations in the α4β2 ABS.