Ribulose bisphosphate carboxylase from methanol-grown Paracoccus denitrificans

Paracoccus denitrificans grows on methanol as the sole source of energy and carbon, which it assimilates aerobically via the reductive pentose phosphate cycle. This gram-negative bacterium grew rapidly on 50 mM methanol (generation time, 7 h, 30 degrees C) in excellent yield (3 g of wet-packed cells per liter of culture). Electron microscopic studies indicated that the late-log-phase cells were coccoid, having a thick envelope surrounding a layer of more diffuse electron-dense material and a relatively electron-transparent core. Ribulose bisphosphate carboxylase in the 15,000 X g supernatant of fresh cells had specific activities (micromoles of CO2 fixed per minute per milligram of protein) of 0.026, 0.049, 0.085, 0.128, and 0.034 during the lag, early, mild-, and late log, and late stationary phases, respectively. The enzyme was purified 40-fold by pelleting at 159,000 X g, salting out, sedimentation into a 0.2 to 0.8 M linear sucrose gradient, and elution from a diethylaminoethyl-Sephadex column. The enzyme was homogeneous by the criteria of electrophoresis on polyacrylamide gels polymerized from several acrylamide concentrations and sedimentation behavior. The molecular weight of the native enzyme, as measured by gel electrophoresis and gel filtration, averaged 525,000. Sodium dodecyl sulfate dissociated the enzyme into two types of subunits with molecular weights of 55,000 and 13,600. The S20,w of the enzyme was 14.0 Km values for ribulose bisphosphate and CO2 were 0.166 and 0.051 mM, respectively, and the enzyme was inhibited to the extent of 94% by 1 mM 6-phosphogluconate.     

C-n.m.r. spectral study of C reductively methylated glycopeptides derived from glycophorin A

¹³C-n.m.r. spectral data for ¹³C reductively methylated intact homozygous and heterozygous glycophorins A were compared with the ¹³C-n.m.r. spectral data for the ¹³C reductively methylated homozygous and heterozygous N-terminal glycopeptides derived from the trypsin digest of glycophorin A. The results indicate that pronounced aggregation of this glycoprotein in solution does not affect the structural differences that we have previously observed for glycophorins A^M and A^N at and/or near the N-terminal amino acid. Moreover, the data suggest that two structural states exist for glycophorin A^M.     

CCK-JMV-180, an analog of cholecystokinin, releases intracellular calcium from an inositol trisphosphate-independent pool in rat pancreatic acini.

In pancreatic acinar cells cholecystokinin and its analogs, caerulein and CCK-JMV-180, stimulate an increase in intracellular free [Ca2+] by releasing Ca2+ from non-mitochondrial intracellular pools. It is generally believed that the caerulein-induced release of Ca2+ is mediated by phospholipase C-catalyzed production of 1,4,5-inositol triphosphate (IP3). In this study we have investigated the source and mechanism of Ca2+ release induced by CCK-JMV-180 using streptolysin O-permeabilized pancreatic acinar cells. Caerulein-stimulated release of Ca2+ was completely blocked by either neomycin, an inhibitor of phospholipase C, or by heparin, an IP3 receptor antagonist. These observations are compatible with the conclusion that caerulein releases Ca2+ from an IP3-sensitive pool. In contrast to caerulein, however, CCK-JMV-180-stimulated release of Ca2+ was not blocked by either neomycin or by heparin, indicating that CCK-JMV-180 releases Ca2+ by mechanisms which do not involve the generation or action of IP3. CCK-JMV-180 stimulated the release of Ca2+ even after the IP3-sensitive pool had been completely emptied by prior exposure to a supramaximally stimulating concentration of IP3 (40 microM). Prestimulation of permeabilized acini with 20 mM caffeine did not abolish the CCK-JMV-180-induced Ca2+ release. These results indicate that CCK-JMV-180 stimulates release of Ca2+ from a hitherto uncharacterized intracellular storage pool which is insensitive to either IP3 or caffeine.     

Structural insights of resveratrol with its binding partners in the toll-like receptor 4 pathway

The benefits associated with resveratrol (Resv; 3,4′,5-trihydroxy-trans-stilbene) are known for a long time. The therapeutic properties of Resv are observed in diseases like cancer, neurological disorders, atherosclerosis, aging, inflammation, etc. Multiple studies suggest that the beneficial properties of Resv are due to its binding to targets in multiple pathways. The same has been reflected in inflammation, where Resv has been shown to inhibit nuclear factor κ light-chain enhancer of activated B cells in the toll-like receptor 4 (TLR4) pathway. There are multiple cellular targets which bind to Resv, however the mode and the key interactions involved remain elusive for many of them. In the current work, we have investigated the structural insights of Resv with three of its binding partners involved in the inflammatory TLR4 signaling pathway. Through a structure-based modelling and molecular dynamics study, we have unraveled the molecular and atomic interactions involved in the Resv-binary complexes of inhibitor of κB kinase, cyclooxygeanse-2, and tank-binding kinase I, all three of which are key players in TLR4 inflammatory signaling. This study is the latest addition to the investigations of the structural partners of Resv and its molecular interactions.     

Relationship between NF-kappaB and trypsinogen activation in rat pancreas after supramaximal caerulein stimulation

Intra-acinar cell nuclear factor-kappaB (NF-kappaB) and trypsinogen activation are early events in secretagogue-induced acute pancreatitis. We have studied the relationship between NF-kappaB and trypsinogen activation in rat pancreas. CCK analogue caerulein induces early (within 15 min) parallel activation of both NF-kappaB and trypsinogen in pancreas in vivo as well as in pancreatic acini in vitro. However, NF-kappaB activation can be induced without trypsinogen activation by lipopolysaccharide in pancreas in vivo and by phorbol ester in pancreatic acini in vitro. Stimulation of acini with caerulein after 6 h of culture results in NF-kappaB but not trypsinogen activation. Protease inhibitors (AEBSF, TLCK, and E64d) inhibit both intracellular trypsin activity and NF-kappaB activation in caerulein stimulated acini. A chymotrypsin inhibitor (TPCK) inhibits NF-kappaB activation but not trypsin activity. The proteasome inhibitor MG-132 prevents caerulein-induced NF-kappaB activation but does not prevent trypsinogen activation. These findings indicate that although caerulein-induced NF-kappaB and trypsinogen activation are temporally closely related, they are independent events in pancreatic acinar cells. NF-kappaB activation per se is not required for the development of early acinar cell injury by supramaximal secretagogue stimulation.     

Measurement of fluid viscosity at microliter volumes using quartz impedance analysis

The purpose of this work was to measure viscosity of fluids at low microliter volumes by means of quartz crystal impedance analysis. To achieve this, a novel setup was designed that allowed for measurement of viscosity at volumes of 8 to 10 μL. The technique was based on the principle of electromechanical coupling of piezoelectric quartz crystals. The arrangement was simple with measurement times ranging from 2 to 3 minutes. The crystal setup assembly did not impose any unwanted initial stress on the unloaded quartz crystal. Quartz crystals of 5- and 10-MHz fundamental frequency were calibrated with glycerol-water mixtures of known density and viscosity prior to viscosity measurements. True frequency shifts, for the purpose of this work, were determined followed by viscosity measurement of aqueous solutions of sucrose, urea, PEG-400, glucose, and ethylene glycol at 25°C±0.5°C. The measured viscosities were found to be reproducible and consistent with the values reported in the literature. Minor inconsistencies in the measured resistance and frequency shifts did not affect the results significantly, and were found to be experimental in origin rather than due to electrode surface roughness. Besides, as expected for a viscoelastic fluid, PEG 8000 solutions, the calculated viscosities were found to be less than the reported values due to frequency dependence of storage and loss modulus components of complex viscosity. From the results, it can be concluded that the present setup can provide accurate assessment of viscosity of Newtonian fluids and also shows potential for analyzing non-Newtonian fluids at low microliter volumes.     

Diffusion and sedimentation interaction parameters for measuring the second virial coefficient and their utility as predictors of protein aggregation

The concentration-dependence of the diffusion and sedimentation coefficients (k_D and k_s, respectively) of a protein can be used to determine the second virial coefficient (B₂), a parameter valuable in predicting protein-protein interactions. Accurate measurement of B₂ under physiologically and pharmaceutically relevant conditions, however, requires independent measurement of k_D and k_s via orthogonal techniques. We demonstrate this by utilizing sedimentation velocity (SV) and dynamic light scattering (DLS) to analyze solutions of hen-egg white lysozyme (HEWL) and a monoclonal antibody (mAb1) in different salt solutions. The accuracy of the SV-DLS method was established by comparing measured and literature B₂ values for HEWL. In contrast to the assumptions necessary for determining k_D and k_s via SV alone, k_D and ks were of comparable magnitudes, and solution conditions were noted for both HEWL and mAb1 under which 1), k_D and k_s assumed opposite signs; and 2), k_D ≥ k_s. Further, we demonstrate the utility of k_D and k_s as qualitative predictors of protein aggregation through agitation and accelerated stability studies. Aggregation of mAb1 correlated well with B₂, k_D, and k_s, thus establishing the potential for k_D to serve as a high-throughput predictor of protein aggregation.     

Development of a new mouse model of acute pancreatitis induced by administration of L-arginine

The pathogenesis of acute pancreatitis is not fully understood. Experimental animal models that mimic human disease are essential to better understand the pathophysiology of the disease and to evaluate potential therapeutic agents. Given that the mouse genome is known completely and that a large number of strains with various genetic deletions are available, it is advantageous to have multiple reliable mouse models of acute pancreatitis. Presently, there is only one predominant model of acute pancreatitis in mice, in which hyperstimulatory doses of cholecystokinin or its analog caerulein are administered. Therefore, the aim of this study was to develop another mouse model of acute pancreatitis. In this study, C57BL/6 mice were injected intraperitoneally with L-arginine in two doses of 4 g/kg each, 1 h apart. Serum amylase, myeloperoxidase, and histopathology were examined at varying time points after injection to assess injury to the pancreas and lung. We found that injection of L-arginine was followed by significant increases in plasma amylase and pancreatic myeloperoxidase accompanied by marked histopathological changes. The injury to the pancreas was slow to develop and peaked at 72 h. Subsequent to peak injury, the damaged areas contained collagen fibers as assessed by increased Sirius red staining. In contrast, D-arginine or other amino acids did not cause injury to the pancreas. In addition, acute inflammation in the pancreas was associated with lung injury. Our results indicate that administration of L-arginine to mice results in severe acute pancreatitis. This model should help in elucidating the pathophysiology of pancreatitis.