mM In Absence Research Articles

Enzyme cascade for biocatalytic deracemization of D,L-phosphinothricin

Deracemization of D,L-phosphinothricin (D,L-PPT) is one of the most promising routes for preparation of optically pure L-PPT. In this work, an efficient multi-enzyme redox cascade was developed for deracemization ofPPT, which includes oxidative reaction and reductive reaction. The oxidative reaction catalyzing oxidative deamination of D-PPT to 2-oxo-4-[(hydroxy)(-methyl)phosphinyl]butyric acid (PPO) was performed by a D-amino acid oxidase and a catalase for removing H2O2. The reductive reaction catalyzing amination of PPO to L-PPT is achieved by a glufosinate dehydrogenase and a glucose dehydrogenase for cofactor regeneration. To avoid the inhibitory effect of glucose on the oxidative reaction, a “two stages in one-pot” strategy was developed to combine these two reactions in deracemization process. By using this strategy, the L-PPT was obtained with a high yield (89 %) and > 99 % enantiomeric excess at substrate loading of 300 mM in absence of addition of extra NADP+. These encouraging results demonstrated that the developed enzyme cascade deracemization process exhibits great potential and economical competitiveness for manufacture of L-PPT from D,L-PPT.

Simultaneous co-delivery of neuroprotective drugs from multi-loaded PLGA microspheres for the treatment of glaucoma

Glaucoma is a multifactorial neurodegenerative disorder and one of the leading causes of irreversible blindness globally and for which intraocular pressure is the only modifiable risk factor. Although neuroprotective therapies have been suggested to have therapeutic potential, drug delivery for the treatment of ocular disorders such as glaucoma remains an unmet clinical need, further complicated by poor patient compliance with topically applied treatments. In the present study we describe the development of multi-loaded PLGA-microspheres (MSs) incorporating three recognised neuroprotective agents (dexamethasone (DX), melatonin (MEL) and coenzyme Q10 (CoQ10)) in a single formulation (DMQ-MSs) to create a novel sustained-release intraocular drug delivery system (IODDS) for the treatment of glaucoma. MSs were spherical, with a mean particle size of 29.04 ± 1.89 μm rendering them suitable for intravitreal injection using conventional 25G–32G needles. >62% incorporation efficiency was achieved for the three drug cargo and MSs were able to co-deliver the encapsulated active compounds in a sustained manner over 30-days with low burst release. In vitro studies showed DMQ-MSs to be neuroprotective in a glutamate-induced cytotoxicity model (IC50 10.00 ± 0.94 mM versus 6.89 ± 0.82 mM in absence of DMQ-MSs) in R28 cell line. In vivo efficacy studies were performed using a well-established rodent model of chronic ocular hypertension (OHT), comparing single intravitreal injections of microspheres of DMQ-MSs to their equivalent individual single-drug loaded MSs mixture (MSsmix), empty MSs, no-treatment OHT only and naïve groups. Twenty one days after OHT induction, DMQ-MSs showed a significantly neuroprotective effect on RGCs compared to OHT only controls. No such protective effect was observed in empty MSs and single-drug MSs treated groups. This work suggests that multi-loaded PLGA MSs present a novel therapeutic approach in the management of retinal neurodegeneration conditions such as glaucoma.

P56 Use of the Tuberculin Skin Test and Tspot For Screening Prior to TNF Antagonist Therapy Identifies Additional Patients Eligible For Chemoprophylaxis Compared to Use of Risk Assessment Strategies Alone

Patientswith rheumatological disease who are treated with TNF antagonist therapy are at increased risk of reactivating latent tuberculosis infection (LTBI). It is believed that immunosuppressant therapy in this group may...

Intraductal Ultrasound in Evaluation of Biliary Strictures Without a Mass Lesion On CT Scan: Significance of Focal Wall Thickening and Extrinsic Compression At the Stricture Site

Background: Biliary intraductal ultrasound (IDUS) has been limited in its clinical utility to evaluate biliary strictures due to lack of easily recognized morphologic criteria to distinguish benign and malignant strictures. We evaluated the clinical significance of two easily assessed IDUS findings- wall thickness and extrinsic compression at stricture site. Patients and Methods: 48 patients who underwent ERCP/IDUS for evaluation of biliary strictures without an identifiable mass lesion on CT scan were retrospectively studied. Patients with strictures proximal to bifurcation of CHD or with periampullary tumors were excluded. IDUS pictures were reviewed specifically to measure wall thickness and look for extrinsic compression at stricture site. Final diagnosis was based on definitive cytology (biliary brush or EUS-FNA) diagnostic for malignancy or benign etiology and clinical follow up of at least 6 months. Results: The mean age of the patients was 64.7 ± 13.4 yrs (range 35-91 yrs). Thirty-three of 48 patients had jaundice at presentation and in 15 patients stricture was suspected on imaging. The mean length of biliary strictures was 15.4 ± 7.7 mm (range 5-40 mm). Twenty-six strictures were located in the distal CBD and 22 were located in the mid/proximal CBD or CHD (proximal strictures). Fifteen of 48 patients were finally diagnosed to be malignant strictures. 20 of 48 patients had extrinsic compression at stricture site. Of 28 patients without extrinsic compression , 7 had malignant strictures with wall thickness ranging from 9-17 mm and 21 patients with benign strictures had wall thickness ≤9 mm. Biliary wall thickness ≤7 mm in absence of extrinsic compression had a NPV of 100% for excluding malignancy in this cohort. Extrinsic compression in proximal strictures (n = 9) was due to malignancy in 4 patients and was benign due to vascular compression (n = 2), enlarged gallbladder with cholecystitis (n = 1), periductal abscess (n = 1) and scar tissue (n = 1) in 2 patients with history of liver transplantation. Extrinsic compression in distal strictures (n = 11) was due to chronic pancreatitis or pancreatic cancer (n = 4), which were indistinguishable by IDUS and were further evaluated by EUS-FNA. Conclusions: Evaluation of wall thickness and presence of extrinsic compression at the site of biliary strictures by IDUS can help in further management of these patients. Patients with biliary stricture due to extrinsic compression should be further evaluated by EUS-FNA except the ones due to vascular compression. In absence of extrinsic compression, wall thickness ≤7 mm is strongly predictive of a benign stricture.

Insulin Regulates Islet α-Cell Function by Reducing KATP Channel Sensitivity to Adenosine 5′-Triphosphate Inhibition

Glucose regulates pancreatic islet alpha-cell glucagon secretion directly by its metabolism to generate ATP in alpha-cells, and indirectly via stimulation of paracrine release of beta-cell secretory products, particularly insulin. How the cellular substrates of these pathways converge in the alpha-cell is not well known. We recently reported the use of the MIP-GFP (mouse insulin promoter-green fluorescent protein) mouse to reliably identify islet alpha- (non-green cells) and beta-cells (green cells), and characterized their ATP-sensitive K(+) (K(ATP)) channel properties, showing that alpha-cell K(ATP) channels exhibited a 5-fold higher sensitivity to ATP inhibition than beta-cell K(ATP) channels. Here, we show that insulin exerted paracrine regulation of alpha-cells by markedly reducing the sensitivity of alpha-cell K(ATP) channels to ATP (IC(50) = 0.18 and 0.50 mM in absence and presence of insulin, respectively). Insulin also desensitized beta-cell K(ATP) channels to ATP inhibition (IC(50) = 0.84 and 1.23 mM in absence and presence of insulin, respectively). Insulin effects on both islet cell K(ATP) channels were blocked by wortmannin, indicating that insulin acted on the insulin receptor-phosphatidylinositol 3-kinase signaling pathway. Insulin did not affect alpha-cell A-type K(+) currents. Glutamate, known to also inhibit alpha-cell glucagon secretion, did not activate alpha-cell K(ATP) channel opening. We conclude that a major mechanism by which insulin exerts paracrine control on alpha-cells is by modulating its K(ATP) channel sensitivity to ATP block. This may be an underlying basis for the proposed sequential glucose-insulin regulation of alpha-cell glucagon secretion, which becomes distorted in diabetes, leading to dysregulated glucagon secretion.

Dependence of critical micelle concentration of a zwitterionic detergent on ionic strength: implications in receptor solubilization

The zwitterionic detergent, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), is mild, non-denaturing, and extensively used for solubilizing membrane proteins and receptors. We report here that the critical micelle concentration (CMC) of CHAPS is dependent on the concentration of NaCl in the solution. Thus, the CMC of CHAPS decreases from 6.41 mM in absence of any salt to 4.10 mM in presence of 1.5 M NaCl. The logarithm of the CMC values appear to have a linear relationship with the salt concentration. Such changes in CMC with ionic strength have important implications in solubilization of membrane-bound neuronal receptors. This is shown by optimal solubilization of the serotonin receptor type 1A (5-HT 1A) from bovine brain hippocampal crude (native) membrane by CHAPS at premicellar concentration under high salt conditions.

Influence of insulin on phosphate uptake by brush border membranes from human placenta

Regulation of phosphate transport by insulin was investigated in brush border membranes from human placenta at term. At 22°C, a 45 min incubation of the total tissue with 10 −6 M insulin significantly decreased both the initial rate and the peak of sodium-dependent phosphate uptake by the corresponding brush border membranes. In contrast, Na + transport was not influenced by the hormone. Increasing the insulin concentration from 0 to 10 −5 M resulted in a dose-dependent inhibition of phosphate uptake with half-maximal effect at 1.1 × 10 −9 M. The hormone decreased PO 4 transport by decreasing the affinity of the carrier for the substrate ( K m = 0.180 ± 0.010 mM and 0.215 ± 0.015 mM in absence and presence of 10 −6 M insulin respectively, P < 0.05). The inhibitory effect of insulin required the presence of Mn 2+ whereas neither Mn 2+ nor insulin alone had any influence on PO 4 uptake. It is therefore assumed that receptor phosphorylation, which needs the presence of Mn 2+, is an intermediate step of insulin action on PO 4 uptake by the subsequently isolated brush border membranes. In contrast, insulin had no effect on PO 4 uptake when the membranes were directly incubated with the hormone prior to the transport measurement, suggesting that an intracellular messenger is needed for the inhibitory effect. This messenger is not cAMP since insulin at 10 −6 M concentration has no effect on cAMP content of the total placental tissue. Since insulin binding occurs predominantly if not exclusively on the brush border membrane facing the maternal blood compartment, it is assumed that only the maternal insulin influences the phosphate transport through the placenta.