Chemical Synthesis of 4-Trifluoromethylumbelliferyl-α- -N-acetylneuraminic Acid Glycoside and Its Use for the Fluorometric Detection of Poorly Expressed Natural and Recombinant Sialidases

When compared to bacterial or viral sialidases, eukaryotic sialidases are expressed at lower levels and frequently show poor specific activities. The identification and characterization of sialidases from eukaryotes have been slowed down due to the limited sensitivity of available sialidase substrates. Therefore, we chemically synthesized a fluorogenic compound, 4-trifluoromethylumbelliferyl-α- -N-acetylneuraminic acid (CF₃MU-Neu5Ac), and tested its use as a substrate for eight different sialidases, including enzymes from viral, bacterial, and eukaryotic sources. Kinetic analysis revealed CF₃MU-Neu5Ac to be a very sensitive sialidase substrate. Furthermore, this substance proves to be perfectly suitable for thein vivoexamination of sialidases and for the detection of recombinant sialidase by means of expression cloning.     

Respiratory transfer impedance between 8and 384Hz in guinea pigs before and after bronchial challenge

Sobh, Jamil F., Craig M. Lilly, Jeffrey M. Drazen, andAndrew C. Jackson. Respiratory transfer impedance between 8 and384 Hz in guinea pigs before and after bronchial challenge. J. Appl. Physiol. 82(1): 172-181, 1997.We report a forced oscillatory technique for noninvasivelymeasuring respiratory transfer impedance (Ztr) between 8 and 384 Hz inguinea pigs. This technique uses a device consisting of two chambers:one surrounding the animal's head that is used as a plethysmograph tomeasured flow through the airway opening and the other that surroundsthe animal's body and is used to apply pressure oscillations to thebody surface. Ztr was measured in spontaneously breathing awake guineapigs and while the animals were anesthetized in normal andmethacholine-challenged conditions. An eight-element model consistingof an airway compartment separated from a tissue compartment by a shuntgas compression compartment was fit to the data. Anesthesia increasedcentral and peripheral airway resistance and bronchial airway wallcompliance by 13, 31, and 44%, respectively, whereas it decreasedtissue compliance by 37%. Compared with the unanesthetized condition, the methacholine challenge (20 µg/kg) resulted in an increase incentral and peripheral airway resistance (69 and 319%, respectively) and a decrease in bronchial airway wall and tissue compliance (37 and79%, respectively). This technique is capable of measuring Ztr inanesthetized and awake guinea pigs. Analysis of these data with thiseight-element model provides reasonable estimates of airway and tissueparameters.

     

Relaxation effects in the gel electrophoresis of DNA in intermittent fields

The electrophoretic mobility of restriction fragments of lambda DNA in agarose gels declines if the field is intermittent rather than continuous, with a greater effect on the longer fragments. The changes are compatible with the assumption of two exponential relaxation processes for field-dependent configurational changes, one when the field is turned on and another when it terminates. The length dependence at the extrapolated limit of mobility for short pulses with long intervals corresponds closely to the simple inverse proportionality to length expected from theoretical considerations when the molecular configuration is not affected by the electric field. Simple intermittent fields would allow separation of longer molecules than can ordinarily be resolved. The relaxation times for both the change in conformation imposed by the field and the return to field-free conformation vary as approximately the second power of the length of the molecule, independent of the salt concentration or field strength and varying only slightly with gel density. These relations are not in good agreement with properties expected from reputation theory, and they suggest that a different mechanism must be invoked for the electrophoretic migration of long DNA molecules at ordinary values of field strength.     

Monitoring coastal wetland communities in north-eastern NSW using ASTER and Landsat satellite data

The coastal wetland communities of north-eastern New South Wales (NSW) Australia exist in a subtropical climate with high biodiversity and are affected by anthropogenic and natural stressors such as urbanization and climate change. The aim of the research is to map and monitor the coastal wetland communities in north eastern NSW using satellite data. Advanced Spaceborne Thermal Emission and Reflectance Radiometer, Landsat ETM+ and Landsat TM satellite imagery of November 2003, June 2001 and September 1989 respectively were used to identify and monitor the wetland communities. Supervised classification was performed using the maximum likelihood standard algorithm. Normalized Difference Vegetation Index was produced and the health of the wetland vegetation was evaluated. The wetland maps present significant changes in the coastal wetland communities in the months of September 1989, June 2001 and November 2003. This information could be used by coastal wetland managers in order to enhance the management of these ecosystems.     

Mannich reaction: an approach for the synthesis of water soluble mulundocandin analogues

Semisynthetic modifications at Hydroxy tyrosine (Htyr) unit of mulundocandin (1) were carried out to improve its aqueous solubility. A single step introduction of substituted aminomethyl groups at the ortho position(s) of phenolic hydroxyl of HTyr unit of mulundocandin has been achieved in 7-85% yield. The in vitro screening of Mannich products against Candida albicans and Aspergillus fumigatus, retained the in vivo activity of parent by oral and intraperitoneal route. Compound 20, showed significant improvement in activity over mulundocandin (1) and activity compares well with that of fluconazole.     

Rational design of Striga hermonthica-specific seed germination inhibitors

The obligate hemiparasitic weed Striga hermonthica grows on cereal roots and presents a severe threat to global food security by causing enormous yield losses, particularly in sub-Saharan Africa. The rapidly increasing Striga seed bank in infested soils provides a major obstacle in controlling this weed. Striga seeds require host-derived strigolactones (SLs) for germination, and corresponding antagonists could be used as germination inhibitors. Recently, we demonstrated that the common detergent Triton X-100 is a specific inhibitor of Striga seed germination by binding noncovalently to its receptor, S. hermonthica HYPO-SENSITIVE TO LIGHT 7 (ShHTL7), without blocking the rice (Oryza sativa) SL receptor DWARF14 (OsD14). Moreover, triazole ureas, the potent covalently binding antagonists of rice SL perception with much higher activity toward OsD14, showed inhibition of Striga but were less specific. Considering that Triton X-100 is not suitable for field application and by combining structural elements of Triton and triazole urea, we developed two hybrid compounds, KK023-N1 and KK023-N2, as potential Striga-specific germination inhibitors. Both compounds blocked the hydrolysis activity of ShHTL7 but did not affect that of OsD14. Binding of KK023-N1 diminished ShHTL7 interaction with S. hermonthica MORE AXILLARY BRANCHING 2, a major component in SL signal transduction, and increased ShHTL7 thermal specificity. Docking studies indicate that KK023-N1 binding is not covalent but is caused by hydrophobic interactions. Finally, in vitro and greenhouse tests revealed specific inhibition of Striga seed germination, which led to a 38% reduction in Striga infestation in pot experiments. These findings reveal that KK023-N1 is a potential candidate for combating Striga and a promising basis for rational design and development of further Striga-specific herbicides.

Designed strigolactone antagonists inhibit Striga seed germination.     

On the mechanism of the okadaic acid-induced inhibition of phosphatidylcholine biosynthesis in isolated rat hepatocytes.

The mechanism of inhibition of phosphatidylcholine biosynthesis by okadaic acid was investigated in suspension cultures of isolated rat hepatocytes. Cells were pulsed with [methyl-3H]choline and chased in the absence or presence of 1 microM okadaic acid for up to 120 min. Phosphatidylcholine biosynthesis was inhibited after 15 min of chase. To see if okadaic acid altered the degree of phosphorylation of cytidylyltransferase (CT), hepatocytes were incubated with 32P(i) and chased in the absence or presence of okadaic acid. Okadaic acid caused a rapid (within 15 min) increase in the phosphorylation state of the cytosolic enzyme. Two-dimensional peptide map analysis revealed an increase in the phosphorylation of several peptides in okadaic acid-treated hepatocytes compared with controls. After 15 min of incubation of hepatocytes with okadaic acid, membrane CT activity was decreased and a corresponding increase in cytosolic CT activity was observed. In hepatocytes incubated with okadaic acid and oleate a correlation between membrane CT activity, diacylglycerol level, and phosphatidylcholine biosynthesis was observed. These data suggest that the concentration of diacylglycerol is responsible for the increase in membrane CT activity and subsequently phosphatidylcholine biosynthesis in oleate-treated cells. We postulate that the okadaic acid-induced decrease in phosphatidylcholine biosynthesis is due to an increase in the phosphorylation state of CT which promotes a translocation of CT activity from the membranes to the cytosol.     

A PLETHORA/PIN-FORMED/auxin network mediates prehaustorium formation in the parasitic plant Striga hermonthica

The parasitic plant Striga (Striga hermonthica) invades the host root through the formation of a haustorium and has detrimental impacts on cereal crops. The haustorium results from the prehaustorium, which is derived directly from the differentiation of the Striga radicle. The molecular mechanisms leading to radicle differentiation shortly after germination remain unclear. In this study, we determined the developmental programs that regulate terminal prehaustorium formation in S. hermonthica at cellular resolution. We showed that shortly after germination, cells in the root meristem undergo multiplanar divisions. During growth, the meristematic activity declines and associates with reduced expression of the stem cell regulator PLETHORA1 and the cell cycle genes CYCLINB1 and HISTONE H4. We also observed a basal localization of the PIN-FORMED (PIN) proteins and a decrease in auxin levels in the meristem. Using the structural layout of the root meristem and the polarity of outer-membrane PIN proteins, we constructed a mathematical model of auxin transport that explains the auxin distribution patterns observed during S. hermonthica root growth. Our results reveal a fundamental molecular and cellular framework governing the switch of S. hermonthica roots to form the invasive prehaustoria.

The parasitic plant Striga hermonthica forms its invasive organ, the prehaustorium, by inducing differentiation of the radicle by arresting cell division.     

Biased signaling: potential agonist and antagonist of PAR2

Protease activated receptor 2 (PAR2) has emerged as one of the promising therapeutic targets to inhibit rapidly metastasizing breast cancer cells. However, its elusive molecular mechanism of activation and signaling has made it a difficult target for drug development. In this study, in silico methods were used to unfold PAR2 molecular mechanism of signaling based on the concept of GPCR receptor plasticity. Although, there are no conclusive evidences of the presence of specific endogenous ligands for PAR2, the efficacy of synthetic agonist and antagonist in PAR2 signaling has opened up the possibilities of ligand-mediated signaling. Furthermore, it has been proved that ligands specific for one GPCR can induce signaling in GPCRs belonging to other subfamilies. Therefore, the aim of this study was to identify potential agonists and antagonists from the GPCR ligand library (GLL), which may induce biased signaling in PAR2 using the concept of existence of multiple ligand-stabilized receptor conformations. The results of our in silico study suggest that PAR2 may show biased signaling mainly with agonists of serotonin type 1, β-adrenergic type 1,3 and antagonists of substance K (NK1), serotonin type 2, dopamine type 4, and thromboxane receptors. Further, this study also throws light on the putative ligand-specific conformations of PAR2. Thus, the results of this study provide structural insights to putative conformations of PAR2 and also gives initial clues to medicinal chemists for rational drug design targeting this challenging receptor.