Thermal,Chemical and pH Induced Unfolding of Turmeric Root Lectin: Modes of Denaturation

Curcuma longa rhizome lectin, of non-seed origin having antifungal, antibacterial and α-glucosidase inhibitory activities, forms a homodimer with high thermal stability as well as acid tolerance. Size exclusion chromatography and dynamic light scattering show it to be a dimer at pH 7, but it converts to a monomer near pH 2. Circular dichroism spectra and fluorescence emission maxima are virtually indistinguishable from pH 7 to 2, indicating secondary and tertiary structures remain the same in dimer and monomer within experimental error. The tryptophan environment as probed by acrylamide quenching data yielded very similar data at pH 2 and pH 7, implying very similar folding for monomer and dimer. Differential scanning calorimetry shows a transition at 350.3 K for dimer and at 327.0 K for monomer. Thermal unfolding and chemical unfolding induced by guanidinium chloride for dimer are both reversible and can be described by two-state models. The temperatures and the denaturant concentrations at which one-half of the protein molecules are unfolded, are protein concentration-dependent for dimer but protein concentration-independent for monomer. The free energy of unfolding at 298 K was found to be 5.23 Kcal mol⁻¹ and 14.90 Kcal mol⁻¹ for the monomer and dimer respectively. The value of change in excess heat capacity upon protein denaturation (ΔC_p) is 3.42 Kcal mol⁻¹ K⁻¹ for dimer. The small ΔC_p for unfolding of CLA reflects a buried hydrophobic core in the folded dimeric protein. These unfolding experiments, temperature dependent circular dichroism and dynamic light scattering for the dimer at pH 7 indicate its higher stability than for the monomer at pH 2. This difference in stability of dimeric and monomeric forms highlights the contribution of inter-subunit interactions in the former.     

Lactam nonanic acid,a new substance from <Emphasis Type="Italic">Cleome viscosa</Emphasis> with allelopathic and antimicrobial properties

Cleome viscosa L. (Capparidaceae) is well known for its medicinal properties. Lactam nonanoic acid (LNA) [2-amino-9-(4-oxoazetidin-2-yl)-nonanoic acid; C₁₂H₂₂N₂O_3, mol. wt. 242] has been isolated and purified from the root exudates of Cleome viscosa. The aqueous solution of this pure compound has been tested on bacteria (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) and fungi (Aspergillus fumigatus, A. niger and A. tamarii). At a dosage of 500 ppm and above, P. aeruginosa and S. aureus were totally inhibited while E. coli remained unaffected. On the other hand, growth of A. niger and A. tamarii was stimulated while there was no effect on A. fumigatus. This pure compound showed concentration-dependent inhibitory activity on rice, gram and mustard seeds.     

Mutacins from Streptococcus mutans UA159 are active against multiple streptococcal species

Streptococcus mutans UA159, whose genome is completely sequenced, produces two nonlantibiotic mutacins, mutacin IV (encoded by nlmAB) and mutacin V (encoded by nlmC). In this study, we investigated the contribution of nlmA and nlmB to mutacin IV activity and demonstrated by performing genetic studies as well as by using semipurified molecules that, in contrast to a previous report, both of these genes are required for optimum mutacin IV activity. We also showed that mutacin IV is active against multiple Streptococcus species. In contrast, mutacin V displayed a narrower inhibitory range than mutacin IV. Our results suggest that mutacin IV and mutacin V may act synergistically to inhibit various organisms.     

Doubled haploid callus lines of Valencia sweet orange recovered from anther culture

Homozygous genotypes are valuable for genetic and genomic studies in higher plants. However, obtaining homozygous perennial plants using conventional breeding techniques is currently a challenge because of a long juvenile period, high heterozygosity and the substantial inbreeding depression. In vitro androgenesis has been used to develop haploid and doubled haploid plants. In this study, we report the regeneration of doubled haploid lines of Valencia sweet orange cv. Rohde Red (Citrus sinensis [L.] Osbeck) via anther culture. Anthers at the uninucleate stage were induced and two embryogenic calli were obtained that further regenerated to embryoids (2/400). Plantlets were obtained after transferring the embryoids to a shoot regeneration medium, but were short-lived. Ploidy analysis via both flow cytometry and chromosome counting verified that these two lines were diploids. Additionally, 43 simple sequence repeat (SSR) markers which showed to be heterozygous in the Valencia sweet orange donor line confirmed homozygosity and doubled haploids in the anther-derived lines. Furthermore, analysis of the doubled haploids via cleaved amplified polymorphic sequence (CAPS) markers and target region sequencing confirmed the allelic state of two genes (LCYE and LCYB) involved in the carotenoid biosynthesis of sweet oranges.     

A novel method for the measurement of elemental selenium produced by bacterial reduction of selenite

The measurement of elemental selenium (Se⁰) is needed to assess the rate and magnitude of bacteria reduction of selenite or selenate. We have developed a spectrophotometric method for the measurement Se⁰ that is rapid and can be employed to measure the quantity of Se⁰ produced by bacterial cultures. This method employs the use of 1 M Na₂S to convert the insoluble elemental selenium to a red-brown solution and with this method there is a direct correlation between concentration of elemental selenium and the absorption at 500 nm. To demonstrate the utility of this assay, we have followed the reduction of selenite to Se⁰ by Moraxella bovis, and by bacterial consortia in soil and water samples.     

Distinct early molecular responses to mutations causing vLINCL and JNCL presage ATP synthase subunit C accumulation in cerebellar cells

Variant late-infantile neuronal ceroid lipofuscinosis (vLINCL), caused by CLN6 mutation, and juvenile neuronal ceroid lipofuscinosis (JNCL), caused by CLN3 mutation, share clinical and pathological features, including lysosomal accumulation of mitochondrial ATP synthase subunit c, but the unrelated CLN6 and CLN3 genes may initiate disease via similar or distinct cellular processes. To gain insight into the NCL pathways, we established murine wild-type and CbCln6 ^nclf/nclf cerebellar cells and compared them to wild-type and CbCln3 ^{Δex7/8/Δex7/8} cerebellar cells. CbCln6 ^nclf/nclf cells and CbCln3 ^{Δex7/8/Δex7/8} cells both displayed abnormally elongated mitochondria and reduced cellular ATP levels and, as cells aged to confluence, exhibited accumulation of subunit c protein in Lamp 1-positive organelles. However, at sub-confluence, endoplasmic reticulum PDI immunostain was decreased only in CbCln6 ^nclf/nclf cells, while fluid-phase endocytosis and LysoTracker® labeled vesicles were decreased in both CbCln6 ^nclf/nclf and CbCln3 ^{Δex7/8/Δex7/8} cells, though only the latter cells exhibited abnormal vesicle subcellular distribution. Furthermore, unbiased gene expression analyses revealed only partial overlap in the cerebellar cell genes and pathways that were altered by the Cln3 ^Δex7/8 and Cln6 ^nclf mutations. Thus, these data support the hypothesis that CLN6 and CLN3 mutations trigger distinct processes that converge on a shared pathway, which is responsible for proper subunit c protein turnover and neuronal cell survival.     

Development and validation of a highly sensitive LC-MS/MS method for organic cation transporter (OCT) substrate tetraethylammonium (TEA) in rabbits

Tetraethylammonium is widely used as a probe in organic cation transporters studies. A simple, highly sensitive, and specific method using direct protein precipitation was developed using Hydrophilic Interaction Liquid Chromatography coupled with positive electrospray ionization tandem mass spectrometry for the determination of tetraethylammonium (TEA) in rabbit plasma. Isocratic separation was achieved using a ZIC-HILIC column with acetonitrile and 5mM ammonium acetate in the ratio of 8:2 containing 0.1% formic acid. Acquisition was performed in multiple reaction monitoring mode with the transitions: m/z 130→100 and 130→86 for TEA and m/z 276.1→142.2 for internal standard (homatropine). This method was validated to determine selectivity, linearity, sensitivity, precision, accuracy, recovery and stability. A good linearity was found within a range of 1.53-784.6 ng/mL. The above method has been demonstrated for its capability to estimate the plasma levels of TEA after its topical instillation in rabbit eyes. This method provides an accurate, precise and sensitive tool for determining TEA levels for transporter studies.