Isolation and characterization of a new Ca2+/calmodulin-dependent protein kinase from isoproterenol-stimulated proliferating rat parotid acinar cells.

A new Ca2+/calmodulin-dependent serine kinase was isolated from rat parotid gland acinar cells following chronic treatment with the beta-agonist isoproterenol. A single-step purification was performed on a calmodulin-agarose affinity column, following solubilization with Triton X-100. Among various substrates tested, bovine galactosyltransferase was the preferred substrate of the kinase, followed by glycogen synthetase greater than histone greater than phosphodiesterase greater than phenylalanine hydroxylase greater than phosphorylase b greater than bovine serum albumin. In comparison, a spleen preparation of Ca2+/calmodulin-dependent kinase did not show galactosyltransferase to be the preferred substrate. Thus, the enzyme would appear to be similar to the human galactosyltransferase-associated kinase. The kinase activity was saturable with 100 microM Ca2+ and 2 microM calmodulin. The molecular mass determined by nondenaturing and sodium dodecyl sulfate polyacrylamide gel electrophoreses was 75 kDa with a pI of 4.3. The Vmax was 3500 mumol/(min.mg protein) with a Km of 1.6 microM for the transferase substrate. Leukotriene C and prostaglandin E2 were found to be specific noncompetitive inhibitors of the rat galactosyltransferase-associated kinase.     

Role of protein phosphorylation and inositol phospholipid turnover in rat parotid gland proliferation

The involvement of protein phosphorylation in isoproterenol (ISO)-mediated proliferation in the rat parotid gland was investigated by labeling the cells with [³²P] orthophosphate. An increased (4–6 fold) incorporation of the radiolabel was noted in the total parotid gland homogenates of ISO-treated animals when compared to controls. Plasma membrane, nuclear membrane and cytoplasm were isolated, the proteins separated by SDS/PAGE and the phosphoproteins detected by autoradiography. Two phosphoproteins with apparent M_r of 45 and 170 kDa were identified in the cytoplasm while the 170 kDa phosphoprotein also appeared as part of plasma membrane. Transfer of these proteins to nitrocellulose followed by Western blot detection with an antiphosphotyrosine monoclonal antibody showed reactivity with the 170 kDa region of the plasma membrane and cytoplasm. Separate in vitro studies involving incubations of rat parotid slices with 0.2 mM ISO and [³H] myo-inositol for 1 min induced inositol phosphate hydrolysis resulting in a significant increase in inositol-bis and -tris phosphate production. Inositol phosphate production can be blocked by pre-incubation with a mixed -adrenergic receptor antagonist but not with physiological concentrations of - or ₁-specific adrenergic receptor antagonists, indicating the ISO effects are mediated through the ₂-adrenergic receptors. The inclusion of calmodulin antagonists along with ISO prevented the expression of cell-surface galactosyltransferase and retarded gland hypertrophy and hyperplasia. These results suggest that ISO treatment leads to the phosphorylation of target proteins which may be involved in signal transduction pathways leading to cell proliferation.Abbreviations InsP₁, InsP₂, InSP₃ inositol mono-, bis-, and tris-phosphates - UDP Uridine diphosphate - PMSF phenylmethylsulfonylfluoride - SDS sodium dodecyl sulfate - TFP Trifluoperazine - P-tyr phosphotyrosine - Gal Tase galactosyltransferase     

Method for lipase-catalyzed carbonate synthesis via one- and two-step alkoxycarbonylation reactions

Lipase-catalyzed alkoxycarbonylation methods offer potential advantages over the currently practiced industrial scale chemical synthesis of carbonates. We report a method for synthesis of organic carbonates via lipase-catalyzed alkoxycarbonylation between diphenyl carbonate and various alcohols in hexane. This method utilizes precursors that are readily available and does not involve extensive purification of the intermediate. In a two-step process, the two phenyl groups of diphenyl carbonate were substituted by two alcohol nucleophiles. The approach was demonstrated for two-step synthesis of 14 different disubstituted carbonate products. The rates of reaction for the two steps were much slower if the order of nucleophile addition was reversed. Under optimal conditions, complete conversion of diphenyl carbonate occurred within 8-15 h at 50 degrees C, which is a significant improvement from 50-90 h at 24 degrees C. A kinetic model for the alkoxycarbonylation reaction was derived based on the Michaelis-Menten equation, which simplified to first-order kinetics at low and equimolar concentration of substrates.     

Drebrin Regulates Neuroblast Migration in the Postnatal Mammalian Brain

After birth, stem cells in the subventricular zone (SVZ) generate neuroblasts that migrate along the rostral migratory stream (RMS) to become interneurons in the olfactory bulb (OB). This migration is crucial for the proper integration of newborn neurons in a pre-existing synaptic network and is believed to play a key role in infant human brain development. Many regulators of neuroblast migration have been identified; however, still very little is known about the intracellular molecular mechanisms controlling this process. Here, we have investigated the function of drebrin, an actin-binding protein highly expressed in the RMS of the postnatal mammalian brain. Neuroblast migration was monitored both in culture and in brain slices obtained from electroporated mice by time-lapse spinning disk confocal microscopy. Depletion of drebrin using distinct RNAi approaches in early postnatal mice affects neuroblast morphology and impairs neuroblast migration and orientation in vitro and in vivo. Overexpression of drebrin also impairs migration along the RMS and affects the distribution of neuroblasts at their final destination, the OB. Drebrin phosphorylation on Ser142 by Cyclin-dependent kinase 5 (Cdk5) has been recently shown to regulate F-actin-microtubule coupling in neuronal growth cones. We also investigated the functional significance of this phosphorylation in RMS neuroblasts using in vivo postnatal electroporation of phosphomimetic (S142D) or non-phosphorylatable (S142A) drebrin in the SVZ of mouse pups. Preventing or mimicking phosphorylation of S142 in vivo caused similar effects on neuroblast dynamics, leading to aberrant neuroblast branching. We conclude that drebrin is necessary for efficient migration of SVZ-derived neuroblasts and propose that regulated phosphorylation of drebrin on S142 maintains leading process stability for polarized migration along the RMS, thus ensuring proper neurogenesis.     

Global Spectrum of Copy Number Variations Reveals Genome Organizational Plasticity and Proposes New Migration Routes

Global spectrum of CNVs is required to catalog variations to provide a high-resolution on the dynamics of genome-organization and human migration. In this study, we performed genome-wide genotyping using high-resolution arrays and identified 44,109 CNVs from 1,715 genomes across 12 populations. The study unraveled the force of independent evolutionary dynamics on genome-organizational plasticity across populations. We demonstrated the use of CNV tool to study human migration and identified a second major settlement establishing new migration routes in addition to existing ones.     

Mycobacterium tuberculosis MtrB Sensor Kinase Interactions with FtsI and Wag31 Proteins Reveal a Role for MtrB Distinct from That Regulating MtrA Activities

The septal association of Mycobacterium tuberculosis MtrB, the kinase partner of the MtrAB two-component signal transduction system, is necessary for the optimal expression of the MtrA regulon targets, including ripA, fbpB, and ftsI, which are involved in cell division and cell wall synthesis. Here, we show that MtrB, irrespective of its phosphorylation status, interacts with Wag31, whereas only phosphorylation-competent MtrB interacts with FtsI. We provide evidence that FtsI depletion compromises the MtrB septal assembly and MtrA regulon expression; likewise, the absence of MtrB compromises FtsI localization and, possibly, FtsI activity. We conclude from these results that FtsI and MtrB are codependent for their activities and that FtsI functions as a positive modulator of MtrB activation and MtrA regulon expression. In contrast to FtsI, Wag31 depletion does not affect MtrB septal assembly and MtrA regulon expression, whereas the loss of MtrB increased Wag31 localization and the levels of PknA/PknB (PknA/B) serine-threonine protein kinase-mediated Wag31 phosphorylation. Interestingly, we found that FtsI decreased levels of phosphorylated Wag31 (Wag31∼P) and that MtrB interacted with PknA/B. Overall, our results indicate that MtrB interactions with FtsI, Wag31, and PknA/B are required for its optimal localization, MtrA regulon expression, and phosphorylation of Wag31. Our results emphasize a new role for MtrB in cell division and cell wall synthesis distinct from that regulating the MtrA phosphorylation activities.     

Bioavailability and metabolism of fucoxanthin in rats: structural characterization of metabolites by LC-MS (APCI)

This study reports bioavailability and metabolism of fucoxanthin (FUCO) from brown algae Padina tetrastromatica in rats. Rats were divided into two groups (n = 25/group). Group one was fed basal diet (control) while the group two received retinol deficient diet (RD group) for 8 weeks. After confirmed RD in blood (0.53 μmol/l), rats were further sub-grouped (n = 5/sub group), intubated a dose of FUCO (0.83 μmol) and killed after 0, 2, 4, 6 and 8 h. The plasma levels (area under curve/8 h) of FUCO (fucoxanthinol (FUOH) + amarouciaxanthin (AAx)) was 2.93 (RD group) and 2.74 pmol/dl (control), respectively. No newly formed retinol was detected in RD rats intubated with FUCO. Besides FUOH (m/z 617 (M+H)⁺) and AAx (m/z 617 (M+H^?)⁺), other deacetylated, hydrolyzed and demethylated metabolites of bearing molecular mass at m/z 600.6 (FUOH–H₂O), m/z 597 (AAx–H₂O), m/z 579 (AAx–2H₂O+1), m/z 551 (AAx–2H₂O–2CH₃+2) and m/z 523 (AAx–2H₂O–4CH₃+4) were also detected in plasma and liver by LC-MS (APCI). Although biological functions of FUCO metabolites need thorough investigation, this is the first detailed report on FUCO metabolites in rats.     

Laccase production from oil palm industry solid waste: Statistical optimization of selected process parameters

Oil palm frond parenchyma tissue was used as a solid substrate for the production of laccase via solid‐state fermentation using the white rot fungus Pycnoporus sanguineus. With a rectangular aluminium tray as solid‐state fermentation bioreactor, process parameters such as bed height, moisture and supplemented nitrogen (as urea solution) levels were studied and optimized using a statistical design of experiment. The moisture level exerted a significant effect on the process. The interaction effect observed between bed height and supplemented nitrogen level suggested that uniform distribution of supplemented nitrogen into the substrate bed was important. The proposed regression model sufficiently predicted the process response over the experimental range tested. The optimum parameter combination for laccase production was a 3‐cm bed height, 72% w/w moisture and 0.21% w/v supplemented nitrogen. Laccase productivity remained constant when the tray size was increased from 1.4 to 3.4‐fold.