Geographical variation and successive adaptive radiations of yellow-floweredTeucrium (Labiatae) in the Mediterranean region

Study of the geographical variation of yellow-floweredTeucrium sect.Polium species using morphological, chemical, and cytological analyses shows that the group of North African taxa is both differentiated from the European group and homogeneous with it. It displays stable vegetative and floral characters that are common to all the taxa. The essential differences concern the indumentum structure and the habit of the plants. The diversity of environments has enhanced the expression of successive radiations of the group. The first doubtless originated in the Betic-Rif zone and in particular in the southeastern part of the Iberian Peninsula. These radiations probably first gave the groupT. aureum, now endemic in the Ibero-Provençal mountains. The species then became better differentiated in North Africa. Differentiation took place in the mountain ranges (Rif, Atlas, and Hoggar) and some Mediterranean islands (off the eastern coast of Tunisia) for the current species with narrow endemism and in desert regions (steppes and the Sahara) for those with wider endemism.     

Substrate specificity of acetoxy derivatives of coumarins and quinolones towards Calreticulin mediated transacetylation: investigations on antiplatelet function

Calreticulin transacetylase (CRTAase) is known to catalyze the transfer of acetyl group from polyphenolic acetates (PA) to certain receptor proteins (RP), thus modulating their activity. Herein, we studied for the first time the substrate specificity of CRTAase towards N-acetylamino derivatives of coumarins and quinolones. This study is endowed with antiplatelet action by virtue of causing CRTAase catalyzed activation of platelet Nitric Oxide Synthase (NOS) by way of acetylation leading to the inhibition of ADP/Arachidonic acid (AA)-dependent platelet aggregation. Among all the N-acetylamino/acetoxy coumarins and quinolones screened, 7-N-acetylamino-4-methylcoumarin (7-AAMC, 17) was found to be the superior substrate to platelet CRTAase and emerged as the most promising antiplatelet agent both in vitro and in vivo. Further it caused the inhibition of cyclooxygenase-1 (Cox-1) resulting in the down regulation of thromboxane A2 (TxA2), modulation of tissue factor and the inhibition of platelet aggregation. It was also found effective in the inhibition of LPS induced pro-thrombotic conditions.     

Nervilia pangteyana sp. nov., a terrestrial orchid from western Himalaya,India

Nervilia pangteyana J. S. Jalal, Kumar & G. S. Rawat (Orchidaceae), a new species from western Himalaya (Uttarakhand), India is described, illustrated and compared with its closest relative. In addition, a key is given to distinguish between species of Nervilia in the western Himalayas.     

Disease-related cardiac troponins alter thin filament Ca2+ association and dissociation rates

The contractile response of the heart can be altered by disease-related protein modifications to numerous contractile proteins. By utilizing an IAANS labeled fluorescent troponin C, [Formula: see text], we examined the effects of ten disease-related troponin modifications on the Ca(2+) binding properties of the troponin complex and the reconstituted thin filament. The selected modifications are associated with a broad range of cardiac diseases: three subtypes of familial cardiomyopathies (dilated, hypertrophic and restrictive) and ischemia-reperfusion injury. Consistent with previous studies, the majority of the protein modifications had no effect on the Ca(2+) binding properties of the isolated troponin complex. However, when incorporated into the thin filament, dilated cardiomyopathy mutations desensitized (up to 3.3-fold), while hypertrophic and restrictive cardiomyopathy mutations, and ischemia-induced truncation of troponin I, sensitized the thin filament to Ca(2+) (up to 6.3-fold). Kinetically, the dilated cardiomyopathy mutations increased the rate of Ca(2+) dissociation from the thin filament (up to 2.5-fold), while the hypertrophic and restrictive cardiomyopathy mutations, and the ischemia-induced truncation of troponin I decreased the rate (up to 2-fold). The protein modifications also increased (up to 5.4-fold) or decreased (up to 2.5-fold) the apparent rate of Ca(2+) association to the thin filament. Thus, the disease-related protein modifications alter Ca(2+) binding by influencing both the association and dissociation rates of thin filament Ca(2+) exchange. These alterations in Ca(2+) exchange kinetics influenced the response of the thin filament to artificial Ca(2+) transients generated in a stopped-flow apparatus. Troponin C may act as a hub, sensing physiological and pathological stimuli to modulate the Ca(2+)-binding properties of the thin filament and influence the contractile performance of the heart.     

Preparation and in vitro evaluation of novel lipopeptide transfection agents for efficient gene delivery

Gene therapy by delivery of nonviral expression vectors is highly desirable, due to their safety, stability, and suitability for production as bulk pharmaceuticals. However, low transfection efficiency remains a limiting factor in application on nonviral gene delivery. Despite recent advances in the field, there are still major obstacles to overcome. In an attempt to construct more efficient nonviral gene delivery vectors, we have designed a series of novel lipopeptide transfection agents, consisting of an alkyl chain, one cysteine, 1 to 4 histidine and 1 to 3 lysine residues. The lipopeptides were designed to facilitate dimerization (by way of the cysteine residues), DNA binding at neutral pH (making use of charged lysine residues), and endosomal escape (by way of weakly basic histidine residues). DNA/lipopeptide complexes were evaluated for their biophysical properties and transfection efficiencies. The number and identity of amino acids incorporated in the lipopeptide construct affected their DNA/lipopeptide complex forming capacity. As the number of lysine residues in the lipopeptide increased, the DNA complexes formed became more stable, had higher zeta potential (particle surface charge), and produced smaller mean particle sizes (typically 110 nm at a charge ratio of 5.0 and 240 nm at a charge ratio of 1.0). The effect of inclusion of histidines in the lipopeptide moiety had the opposite effect on complex formation to lysine, but was necessary for high transfection efficiency. In vitro transfection studies in COS-7 cells revealed that the efficiency of gene delivery of the luciferase encoding plasmid, pCMV-Luc, mediated by all the lipopeptides, was much higher than poly(L-lysine) (PLL), which has no endosomal escape system, and in two cases was slightly higher than that of branched polyethylenimine (PEI). Lipopeptides with at least two lysine residues and at least one histidine residue produced spontaneous transfection complexes with plasmid DNA, indicating that endosomal escape was achieved by incorporation of histidine residues. These low molecular weight peptides can be readily synthesized and purified and offer new insights into the mechanism of action of transfection complexes.     

Study of the importance of Glu361 in the active site of cholesterol oxidase from<Emphasis Type="Italic">Rhodococcus</Emphasis> sp. PTCC 1633 by site-directed mutagenesis

Cholesterol oxidase is a monomeric flavoenzyme which catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. ThechoR gene was cloned in pET23a and used as the starting plasmid for Glu361Asn, Glu361Gln and Glu361Asp site-directed mutagenesis. The purified mutant proteins like the wild-type have a molecular mass of 55 kD. The specific activities of Glu361Gln and Glu361Asn mutants were 28 and 35 times less than the wild-type. Glu361Asp mutant showed nearly no catalytic activity and was not purified. These experiments clearly demonstrated the importance of Glu361 for the enzymatic reactions of cholesterol oxidaseRhodococcus sp.     

Severe suppression of Frzb/sFRP3 transcription in osteogenic sarcoma

Deciphering the molecular basis of cancer is critical for developing novel diagnostic and therapeutic strategies. To better understand the early molecular events involving osteogenic sarcoma (OGS), we have initiated a program to identify potential tumor suppressor genes. Expression profiling of total RNA from ten normal bone cell lines and eleven OGS-derived cell lines by microarray showed 135-fold lower expression of FRZB/sFRP3 mRNA in OGS cells compared to bone cells; this down-regulation of Frzb/sFRP3 mRNA expression was found to be serum-independent. Subsequently, fourteen OGS biopsy specimens showed nine-fold down-regulation of Frzb/sFRP3 mRNA expression compared to expression in eight normal bone specimens as determined by microarray. FRZB /sFRP3 protein level was also found to be at a very low level in 4/4 OGS cell lines examined. Quantitation by RT-PCR indicated approximately 70% and approximately 90% loss of Frzb/sFRP3 mRNA expression in OGS biopsy specimens and OGS-derived cell lines respectively, compared to expression in bone (p<0.0001). Hybridization experiments of a cDNA microarray containing paired normal and tumor specimens from nineteen different organs did not show any significant difference in the level of Frzb/sFRP3 mRNA expression between the normal and the corresponding tumor tissues. Exogenous expression of FRZB/sFRP3 mRNA in two OGS-derived cell lines lacking endogenous expression of the mRNA produced abundant mRNA from the exogenous gene, eliminating degradation as a possibility for very low level of FRZB/sFRP3 mRNA in OGS specimens. Results from PCR-based experiments suggest that the FRZB/sFRP3 gene is not deleted in OGS cell lines, however, karyotyping shows gross abnormalities involving chromosome 2 (location of the FRZB gene) in five of twelve OGS-derived cell lines. Together, these data suggest a tumor-suppressive potential for FRZB/sFRP3 in OGS.     

A new multi-wavelength model-based method for determination of enzyme kinetic parameters

Lineweaver-Burk plot analysis is the most widely used method to determine enzyme kinetic parameters. In the spectrophotometric determination of enzyme activity using the Lineweaver-Burk plot, it is necessary to find a wavelength at which only the substrate or the product has absorbance without any spectroscopic interference of the other reaction components. Moreover, in this method, different initial concentrations of the substrate should be used to obtain the initial velocities required for Lineweaver-Burk plot analysis. In the present work, a multi-wavelength model-based method has been developed and validated to determine Michaelis-Menten constants for some enzyme reactions. In this method, a selective wavelength region and several experiments with different initial concentrations of the substrate are not required. The absorbance data of the kinetic assays are fitted by non-linear regression coupled to the numeric integration of the related differential equation. To indicate the applicability of the proposed method, the Michaelis-Menten constants for the oxidation of phenanthridine, 6-deoxypenciclovir and xanthine by molybdenum hydroxylases were determined using only a single initial concentration of the substrate, regardless of any spectral overlap.