Role of water in protein kinase C catalysis and its binding to membranes

The role of hydration in the catalytic activity and membrane binding of rat brain protein kinase C (PKC) was investigated by modulating the activity of water with polyethylene glycols with molecular weights of 1000-20000 and dextran with a molecular weight of 20000. These polymers create an osmotic stress due to their exclusion from hydration shells and crevices on proteins, causing dehydration. Polymers larger than 1000 caused an activation of the PKC-catalyzed phosphorylation of histone, while PEG 1000 had no significant effect. The extent of activation by PEG and dextran 20000 was larger than that of PEG 6000 or 8000 when vesicles were composed of 1:1 POPS/POPC, suggesting the presence of at least two distinct regions of exclusion on PKC: one inaccessible to PEGs larger than 1000 and the other inaccessible only to PEGs of > 10000. The extent of activation was dependent on the composition of the vesicles used. If basal activity (without PEG) was low (e.g. with low PS content in membranes), then the extent of activation was similar for all polymers larger than 1000. Binding of PKC to membranes containing 50 mol % PS was unaffected by PEG 6000 but was inhibited by PEG 20000. At a low PS content of 10%, both PEG 6000 and 20000 inhibited binding. This suggests that PKC becomes hydrated upon binding to membranes. Under conditions in which all of the enzyme is membrane-bound, both Km and Vmax for the phosphorylation of histone increased linearly with osmotic stress induced by PEG 6000. Thus, PKC becomes hydrated with 2311 +/- 476 water molecules upon binding of histone and is dehydrated by 1349 +/- 882 water molecules in going to the transition state. Km and Vmax for phosphorylation of the MARCKS peptide also increase with osmotic stress induced by PEG 6000. When protamine sulfate was used as a substrate (cofactor-independent), Vmax for the reaction was unaffected, but Km decreased with osmotic pressure (with PEG 6000), suggesting that PKC becomes dehydrated upon binding protamine. Similar results were found with a peptide substrate derived from the pseudosubstrate site of PKC epsilon. Since dextran, a polymer unrelated in structure to PEG, could cause a similar activation of PKC, the effects seen are likely due to osmotic stress and not to specific binding of PEG to PKC. Also, results obtained with PE-linked PEG were opposite to those with free PEG. PE-linked PEGs of 2000 and 5000 caused an inhibition of PKC-catalyzed phosphorylation of histone when present in membranes. If a specific interaction occurred with PEG, this would be expected to occur even with PE-PEG. The effects observed with free PEG are also independent of ionic strength. Free PEG had no effect on the bilayer to hexagonal phase transition temperature of DEPE membranes, suggesting that the effects on PKC activity are not a consequence of changes in membrane properties at the osmotic pressures used.     

Light-induced tailoring of PEG-hydrogel properties

We have previously reported (Andreopoulos et al. J Am Chem Soc 118 (1996) 6235-6240) the synthesis of hydrogels via the photopolymerization of water-soluble PEG molecules. In this paper, PEG-hydrogel membranes were prepared by the irradiation (> 300 nm) of aqueous solutions of photosensitive 4-armed PEG (nominal molecular weight of 20000), in the absence of photo-initiators. The hydroxyl termini of the PEG's were functionalized with cinnamylidene acetate groups to form photosensitive PEG macromers (PEG-CA), which upon irradiation (>300 nm) formed crosslinks between adjacent cinnamylidene groups resulting in highly crosslinked networks (hydrogels) (Andreopoulos et al. J Am Chem Soc 118 (1996) 6235-6240). The hydrogel membranes were highly swellable with equilibrium volume fractions ranging from 0.02 to 0.05. Their swellability was a function of irradiation light (>300 nm) and degree of modification of the PEG molecules. The effect of light on the permeation fluxes of myoglobin (Mb), hemoglobin (Hb), and lactate dehydrogenase-L (LDH) through PEG membranes was also assessed and the diffusion coefficients of the proteins were determined accordingly. The PEG-CA membranes exhibited photoscissive behavior upon exposure to UV irradiation (254 nm). Therefore, UV light was used as a trigger to control the mesh size of the membranes, and thereby the permeation fluxes of Mb, Hb, and LDH. Equilibrium swelling experiments with membranes prepared under different irradiation conditions were performed, and the Flory-Huggins model was utilized to determine the mesh size and the average molecular weight between crosslinks of the synthesized hydrogels.     

Characterization of permeability and network structure of interfacially photopolymerized poly(ethylene glycol) diacrylate hydrogels

Hydrogel membranes formed by interfacially photopolymerizing poly(ethylene glycol) (PEG) diacrylate precursor solution were prepared from PEG diacrylate of molecular weights (MW) ranging from 2000 (2K) to 20000 (20K) with concentrations ranging from 10% to 30% w/w. The effects of PEG diacrylate MW and concentration in the membrane precursor solution upon the diffusivities of vitamin B12, myoglobin, ovalbumin, albumin, and IgG were determined. Regardless of the concentration of the PEG diacrylate in the precursor solution, hydrogels prepared with PEG 2K, 4K, and 8K diacrylate were impermeable to proteins with a size equal to or larger than myoglobin (22 kDa), while hydrogels prepared with PEG 20K diacrylate were impermeable to proteins with a size equal to or larger than ovalbumin (45 kDa). Similarities between hydrogels formed from PEG 2K, 4K, and 8K diacrylates were also seen in calculations of the molecular weight between crosslinks and the mesh size, with values in the range of 150-750 g/mol and 15-35 A, respectively, depending on PEG diacrylate concentration. In contrast, hydrogels formed from PEG 20K diacrylate had molecular weight between crosslinks ranging from 1150 to 2000 g/mol and mesh sizes ranging from 45-70 A, with larger values being observed in membranes polymerized from more dilute PEG diacrylate precursor.     

A novel source of wheat fiber and protein: effects on fecal bulk and serum lipids

BACKGROUND: Polyethylene glycol (PEG) 3350 is a non-absorbable, non-metabolised osmotic agent used in lavage solutions for gut cleansing. AIMS: To compare the efficacy of PEG and lactulose in chronic constipation. METHODS: A total of 115 patients with chronic constipation entered a multicentre, randomised, comparative trial. They initially received two sachets containing either PEG (13 g/sachet) or lactulose (10 g/sachet) and were given an option to change the dose to one or three sachets/day, depending on response. RESULTS: Ninety nine patients completed the trial. After four weeks, patients in the PEG group (n=50) had a higher number of stools and a lower median daily score for straining at stool than patients in the lactulose group (n=49). Overall improvement was greater in the PEG group. Clinical tolerance was similar in the two groups, but flatus was less frequently reported in the PEG group. The mean number of liquid stools was higher in the PEG group but the difference was significant only for the first two weeks. There were no serious adverse events and no significant change in laboratory tests in either group. At the end of the study, the number of sachets used by the patients was 1.6 (0.7)/day in the PEG group and 2.1 (0.7)/day in the lactulose group. Sixty one patients completed a further two months open study of one to three sachets PEG daily; there was no loss of efficacy and no serious toxicity. Conclusion: Low dose PEG 3350 was more effective than lactulose and better tolerated.     

Possible sources of right-to-left shunting in patients following a total cavopulmonary connection

Although bone wax is effective at achieving hemostasis, it is nonresorbable, causes a foreign body reaction, and inhibits osteogenesis. We report development of a polyethylene glycol/microfibrillar collagen composite (PEG/MFC) that has inherent hemostatic qualities, is biodegradable, and is compatible with bone repair. PEG/MFC composite (n = 42) was placed in 5 mm cranial defects in New Zealand white rabbits. Hemostasis and healing were compared to unfilled defects (n = 32) and defects filled with standard bone wax (n = 10). Both PEG/MFC and bone wax handled well and stopped bleeding. The polyethylene glycol component was resorbed by 8 h, and the microfibrillar collagen was resorbed over 2 months, eliciting only a minor inflammatory response during the first month. Defects filled with the PEG/MFC composite showed similar amounts of bony regeneration as did unfilled control defects. At 4 weeks, healing bone accounted for 43 +/- 13% in those treated with PEG/MFC and 47 +/- 19% defect area in untreated holes. In contrast, less than 1% of the area was bone in defects filled with bone wax (p < 0.05). PEG/MFC composite provided excellent bony hemostasis and did not inhibit bone growth.     

Physical properties of lipid-protein complexes formed by the interaction of dimyristoylphosphatidylcholine and human high-density apolipoprotein A-II

Apolipoprotein A-II (apoA-II) from human plasma high-density lipoproteins associates with dimyristoylphosphatidylcholine (DMPC) to give complexes whose structure is determined by the temperature at which the reaction is conducted. The temperature dependence is related to the gel leads to liquid crystalline transition temperature, Tc, of DMPC which occurs at 23.9 degrees C. At T less than Tc (20 degrees C), T = Tc, and T greater than Tc (30 degrees C), three different complexes can be isolated. At 20 degrees C, at 75:1 (molar ratio of lipid to protein) complex is formed. This complex has a molecular weight (Mt) of 343 000, a Stokes radius, Rs, of 65 A, and a partial specific volume (v) of 0.914 mL/g. At 24 degrees C, two different complexes may be formed. One is similar to the one formed at 20 degrees C and the other is a complex with a DMPC:apoA-II ratio of 241:1; the corresponding physical constants for the latter complex are Mr = 1580 000, Rs = 120 A, and v = 0.948 mL/g. This complex is asymmetric, having a frictional coefficient f/f0 = 1.20. AT 30 degrees C, a 45:1 complex was formed; for this complex, Mr = 229 000, Rs = 57 A, and v = 0.892 mL/g. Electron microscopy reveals that the negatively stained complexes are arranged in rouleaux having subunits with average dimensions of 175 x 60, 250 x 62, and 50 x 55 A for the 45:1, 75:1, and 240:1 complexes, respectively. The multiple lipid-protein species formed by apoA-II and DMPC suggest the possible existence of more than one macromolecular spices of lipid and apoA-II in the plasma.     

Neuraxial techniques for cancer pain: an opinion about unresolved therapeutic dilemmas

-Mast cells are present in the human arterial intima. To study whether mast-cell degranulation influences the rate of proliferation of smooth muscle cells, we cocultured sensitized (IgE-bearing) rat serosal mast cells and rat aortic smooth muscle cells (SMCs). When sensitized mast cells were stimulated to degranulate with antigen, the rate of proliferation of the cocultured SMCs decreased sharply. This inhibitory effect was found to be due mainly to the very high molecular weight (Mr) heparin proteoglycans (average Mr 750 000) released from the stimulated mast cells. When the heparin proteoglycans were purified from mast-cell granule remnants and added to the SMC culture, they were found to block the cell cycle at the G0-->S transition and the exit from the G2/M phase, their inhibitory effect resembling that of commercial heparin. However, in contrast to the reported dependence of the inhibitory effect of commercial heparin on the release of transforming growth factor-beta from serum, the inhibitory effect of the mast cell-derived heparin proteoglycans in the presence of serum was not transforming growth factor-beta dependent. Moreover, the effect of the mast cell-derived heparin proteoglycans was more efficient than that of commercial heparins of high (average Mr 15 000) and low (average Mr 5000) molecular weight. We also purified heparin glycosaminoglycans (average Mr 75 000) from the mast cell-derived heparin proteoglycans and found that they also inhibited SMC growth efficiently, although less strongly than their parent heparin proteoglycans. These results reveal, for the first time, that mast cells are able to regulate SMC growth. Thus, activated mast cells, by releasing heparin proteoglycans, possibly participate in the regulation of SMC growth in the human arterial intima, the site of atherogenesis.     

New immunoseparation-based homogeneous assay for HDL-cholesterol compared with three homogeneous and two heterogeneous methods for HDL-cholesterol

We evaluated four new commercial methods for HDL-cholesterol determination. The three completely homogeneous assays were an immunoseparation-based (IS) method from Wako, a polyethylene glycol-modified enzyme (PEG) method from Boehringer Mannheim, and a synthetic polymer-based (SP) method from Genzyme. The fourth method was a new heterogeneous method in which lipoproteins are removed using dextran sulfate-coated magnetic beads and Mg2+ (MB, Reference Diagnostics). We compared these methods with the conventional phosphotungstic acid/MgCl2 precipitation (PTA) procedure. The homogeneous assays had good intraassay imprecision with total CVs <2.3%, whereas the CVs of the MB assay were <5.9%. Adding HDL to serum to achieve HDL-cholesterol (HDL-C) concentrations up to 1000 mg/L revealed nearly complete recoveries in the IS, PEG, and MB assays, whereas the SP assay showed a lower recovery (approximately 70%). The SP HDL-C apparently increased at increasing LDL-cholesterol and VLDL-triglyceride concentrations, whereas the IS, PEG, and MB methods were not influenced by LDL-cholesterol up to 6000 mg/L (MB, 5000 mg/L) and VLDL-triglycerides up to 9000 mg/L. Free fatty acids above approximately 2 mmol/L produced falsely high HDL-C in the IS and SP assays, the error amounting to as much as 50% in some samples. An intermethod comparison in 291 fresh serum samples yielded correlation coefficients of at least r = 0.95 for all assays, when compared with the PTA procedure. The slopes and intercepts of the regression lines were 1.05 and 57 (IS), 1.12 and 9.9 (PEG), 1.00 and 39 (SP), and 1.0 and 38 mg/L (MB), respectively. The new assays are precise and simplify the determination of HDL-C, but in part they lack specificity or are susceptible to interferences, resulting in discrepancies when compared with the established PTA procedure.     

Association of air pollution caused by sulphur dioxide and total number of particles in suspension and daily mortality in the city of Madrid (1986-1992)

The structure of brain extracellular space resembles foam. Diffusing molecules execute random movements that cause their collision with membranes and affect their concentration distribution. By measuring this distribution, the volume fraction (alpha) and the tortuosity (lambda) can be estimated. The volume fraction indicates the relative amount of extracellular space and tortuosity is a measure of hindrance of cellular obstructions. Diffusion measurements with molecules <500 Mr show that alpha approximately 0.2 and lambda approximately 1.6, although some brain regions are anisotropic. Molecules > or =3000 Mr show more hindrance, but molecules of 70000 Mr can move through the extracellular space. During stimulation, and in pathophysiological states, alpha and lambda change, for example in severe ischemia alpha = 0.04 and lambda = 2.2. These data support the feasibility of extrasynaptic or volume transmission in the extracellular space.     

Interfacial thickness of liposomes containing poly(ethylene glycol)-cholesterol from electrophoresis

The electrophoretic mobilities of liposomes incorporating a polyethylene glycol (PEG) headgroup coupled to cholesterol for PEG of average chain index 3.0, 13.2, and 22.3 have been determined as a function of PEG-cholesterol mole fraction between 5% and 40% and ionic strength between 2 and 200 mM. The liposome compositions were 40 mole % cholesterol plus PEG-cholesterol, 10 mole % 1,2-dipalmitoyl-sn-glyerco-3-phosphoglycerol, and 50 mole % egg phosphatidylcholine. The mobilities were fit to a model in which the PEG forms a surface layer of polymer subject to viscous drag arising from electroosmotic flow within this layer. The model provides estimates of the average layer thickness that are comparable to those determined from contemporary models of surface-attached polymer.     

次碳酸铋分光光度法测定酸铜镀液中聚乙二醇

针对传统次硝酸铋法测定酸铜镀液中聚乙二醇(PEG)含量时,铜离子和酸度干扰大、方法检出限高和常规检测试剂极易水解,从而导致实验结果非线性和重复性差等问题,试验通过加入氢氧化钠等碱性试剂对电镀液进行前处理,消除了铜离子和酸度的干扰,利用BiI4-络合物与质子化聚乙二醇离子结合,形成在515 nm波长处有特征吸收的PEG-...     

Quantitative analysis of polyethylene glycol (PEG) in PEG-modified proteins/cytokines by aqueous two-phase systems

Covalent attachment of poly(ethylene glycol) (PEG) to proteins produces conjugates with altered/improved physicochemical and biological properties which depend upon the number of PEG chains linked. Quantification of the attached PEG is however not a trivial issue. The partition coefficient, K, of the PEG-protein conjugate in PEG/dextran two-phase systems provides a quantitative measure for the degree of modification. A linear relationship between log K and the number of PEG chains was observed in fractionated PEG-modified-granulocyte-macrophage colony stimulating factor conjugates having 1 to 3 substitutions. Furthermore, in mixtures of PEG-bovine-serum-albumin conjugates with increasing degrees of modification, a linear relationship was found between log K and n, the average substitution. The increment in log K per PEG chain added is protein specific and this suggests that the interactions between the PEG-protein conjugate and the polymers in the phase system are more complex than just a simple affinity of the PEG for the PEG-rich top phase. Increasing the polymer concentration in the phase system produces larger increments in log K per PEG molecule attached and the proportionality between log K and number of PEG molecules is only compromised for conjugates with high degree of substitution when partitioned in biphasic systems of high concentration of polymers.     

Inhibitors of acrosin and granulocyte proteinases from human genital tract secretions

Human seminal plasma contains two acid-stable proteinase inhibitors, HUSI-II (Mr approximately 6500) and HUSI-I, (Mr approximately 11 000) with different inhibition specificities. The inhibitory activity of HUSI-II is strongly limited to trypsin and acrosin; both enzyme-inhibitor complexes are very stable (e.g. bovine trypsin-HUSI-II complex: Ki = 1 x 10(-10)M; human acrosin-HUSI-II complex: Ki = 2.7 x 10(-10)M). The inhibitor from human seminal plasma HUSI-II may therefore be seen as the natural antagonist of the sperm protease acrosin. In addition to pancreatic trypsin and alpha-chymotrypsin, HUSI-I forms strong complexes with neutral proteases of the lysosome-like granules from human granulocytes, for example, the elastase (Ki = 2.5 x 10(-9)M) and cathepsin G, the chymotrypsin like protease (Ki = 7 x 10(-8)M).     

Crystallization and preliminary X-ray analysis of a truncated family A alkaline endoglucanase isolated from Bacillus sp. KSM-635

The catalytic domain of an alkaline endo-1,4-beta-glucanase (family A) isolated from Bacillus sp. KSM-635 (Mr = 40.2 kDa) was crystallized using the hanging drop vapor diffusion method. Two different crystal forms were obtained. Form 2 crystals (trigonal space group R3 with cell dimensions of a = b = 111.9 and c = 207.1 angstroms in a hexagonal lattice) were found to be more stable than form 1 ones upon X-ray irradiation. A full data set for form 2 crystals has been collected up to 3.3 angstroms resolution.     

高分子保护固相法制备纳米氧化镁

以六水氯化镁和草酸钠为原料,用聚乙二醇作保护剂,通过室温固相化学反应制备了纳米氧化镁的前驱物,真空干燥后,在500℃焙烧前驱物3 h,得到产物纳米氧化镁。采用热分析仪、红外光谱仪、X-射线粉末衍射仪和透射电镜等研究了纳米氧化镁的形成过程和结构;并考察了焙烧温度、焙烧时间和高分子用量对粒径大小的影响。结果表明:高分子保护固相法制备的纳米氧化镁为球形立方晶系结构,纯度高,粒径小,分布范围窄,分散性好,无硬团聚,平均粒径约7.8 nm;高分子保护固相法制备纳米氧化镁的适宜工艺条件为:焙烧温度500℃,焙烧时间3 h,高分子用量3 mL。     

Carboxypeptidase N from pig serum

Carboxypeptidase N has been purified 865-fold from pig serum. The enzyme has a molecular weight of approximately 315 000. In the presence of dodecylsulfate and mercaptoethanol, it dissociates into three subunits of Mr = 90 000, 50 000, 30 000, respectively. The native enzyme and the subunit of Mr = 90 000 contain carbohydrate; no carbohydrate is found in the subunits of Mr = 50 000 and 30 000. Trypsin transforms carboxypeptidase N into a form having a smaller molecular weight and enhanced activity.     

Utility of a rectal suppository containing the antiepileptic drug zonisamide

A suppository of zonisamide (ZNS) was investigated from the viewpoint of pharmaceutical evaluation, pharmacokinetics and pharmacological effect. Two types of ZNS suppositories were prepared. One used Witepsol (H-15:S-55 = 3:1) as a lipophilic base and the other polyethylene glycol (PEG, 4000:1500 = 4:1) as a hydrophilic base. The in vitro release rate of ZNS from the PEG suppository was significantly rapid compared with that of ZNS from Witepsol. Male Wistar rats were administered ZNS (20 mg/kg) using an intravenous, oral or rectal (PEG or Witepsol) route. The absorption of ZNS from the PEG suppository was more rapid than that of ZNS from the Witepsol suppository or from the oral preparation. The peak plasma concentration (Cmax) after a rectal administration of ZNS with Witepsol or PEG suppository was significantly higher than that after the oral administration of ZNS. However, the bioavailability of the three preparations was approximately 100%. Male ICR mice were administered ZNS (80 mg/kg) using the oral or rectal (PEG or Witepsol) route. A positive correlation was observed between the electroshock seizure (ES) threshold and ZNS concentration in plasma or brain. Further, there was no significant difference in the ES threshold or the ZNS concentration in plasma or brain among the three preparations. These results indicate that a ZNS suppository is a very useful preparation from the viewpoint of both pharmacokinetics and pharmacological action.     

以铀矿渣为载体联合PEG2000的细菌固定化培养

以铀矿渣为载体,采用逐次降低接种率循环培养的方法,开展了氧化亚铁硫杆菌的固定化培养试验,研究了聚乙二醇2000（PEG2000）对游离细菌和铀矿渣载体柱中固定化细菌生长的影响。试验结果表明：90 mg/L PEG2000对游离细菌的生长促进作用最大。铀矿渣和K3环材料均可作为载体进行细菌固定化培养。固定化完成时,其亚铁离子的平均氧化速率分别稳定在0.6和0.5 g/(L?h)附近。90 mg/L PEG2000对循环培养和连续培养阶段的固定化细菌生长均具有促进作用。循环培养阶段,可使Fe2+氧化为Fe3+的时间缩短约1/3。连续培养阶段,PEG2000能够促进固定化细菌对亚铁离子的氧化速率,在0.5 L/h进液流量时,未加PEG2000的亚铁离子氧化速率为7.04 g/(L?h),而加PEG2000的,达到了8.18 g/(L?h),亚铁离子氧化效率提高了16.2%。     

Cloning, expression, and crystallization of a hyperthermophilic protein that is homologous to the eukaryotic translation initiation factor, eIF5A

A gene coding for a protein homologous to a translation initiation factor of eukaryotes, eIF5A, was cloned from Methanococcus jannaschii, a hyperthermophile with an optimum growth temperature of 85 degrees C. The protein was overexpressed, purified and crystallized. The crystals were obtained by vapor diffusion method with 8% PEG 4000 as precipitant and belong to space group P4(1)22 with unit cell dimensions a = b = 45.52 A and c = 155.59 A. These crystals diffract to at least 2.2 A resolution.