Fourier transform infrared investigation of the Escherichia coli methionine aporepressor

This study represents the first physicochemical analysis of the recently cloned methionine repressor protein (Met aporepressor) from Escherichia coli. Infrared spectrometry was used to investigate the secondary structure and the hydrogen-deuterium exchange behavior of the E. coli Met aporepressor. The secondary structure of the native bacterial protein was derived by analysis of the amide I mode. The amide I band contour was found to consist of five major component bands (at 1625, 1639, 1653, 1665, and 1676 cm-1) which reflect the presence of various substructures. The relative areas of these component bands are consistent with a high alpha-helical content of the peptide chain secondary structure in solution (43%) and a small amount of beta-sheet structure (7%). The remaining substructure is assigned to turns (10%) and to unordered (or less ordered) structures (40%). The temperature dependence of the infrared spectra of native Met aporepressor in D2O medium over the temperature interval 20-80 degrees C indicates that there are two discrete thermal events: the first thermal event, centered at 42 degrees C, is associated with the hydrogen-deuterium exchange of the hard-to-exchange alpha-helical peptide bonds accompanied by a partial denaturation of the protein, while the second event, centered around 50 degrees C, represents the irreversible thermal denaturation of the protein.     

Distribution of heparinase covalently immobilized to agarose: Experimental and theoretical studies

An immobilized enzyme reactor has been developed to remove heparin, the anticoagulant that is required in all extracorporeal devices for patients undergoing open-heart surgery or kidney dialysis. The device uses the enzyme heparinase (EC 4.2.2.7), which is covalently linked to agarose with cyanogen bromide. A critical parameter in the development of a model for the degradation of heparin catalyzed by immobilized heparinase is the radial concentration profile of the enzyme within the agarose matrix. Experimental determinations of bound enzyme con centrations have been conducted previously for several enzyme systems using radioactive or fluorescent labels. For the development of the heparinase reactor it is necessary to use catalytically but not electrophoretically pure enzyme, and thus it is not possible to use the labeling techniques. To obtain information about the bound enzyme distribution, an experimental study of the intrinsic binding kinetics of heparinase to cyanogen bromide-activated agarose was conducted. The binding reaction was studied as a function of both the concentration of heparinase and the gel-reactive group. At conditions of functional group excess, the binding kinetics were pseudo first order in heparinase concentration with a rate constant equal to 0.12 C(c[triple chemical bond]n) (h(-1)), where C(c[triple chemical bond]n) is the gel-reactive group concentration. The reactive group concentration remained constant within the 2-4-h experiments. Competitive binding between heparinase and the protein contaminants was unimportant. A model was formulated for the immobilization procedure based on the diffusion of heparinase within the porous network and the binding kinetics as determined above. The model predicted the immobilization of heparinase to be kinetically controlled and the enzyme to distribute uniformly within the agarose matrix. These experimental techniques could be applied to predict the immobilized enzyme distribution for different enzyme systems that are not electrophoretically pure.     

Immobilized heparinase: In vitro reactor model

Heparinase immobilized to agarose has previously been shown to be useful in degrading heparin and thereby preventing thromboembolytic complications when this anticoagulant has been used in extracorporeal perfusions. The current study examined the kinetics of this immobilized enzyme. When heparinase is covalently bound to 8% agarose, the partition coefficient of heparin in the catalytic particle is 0.36 +/- 0.048 (N = 10). The immobilized enzyme has a K(m) of 0.15 +/- 0.03 mg/mL and an activation energy of 10.3 +/- 0.57 kcal/gmol (N = 5). These values are statistically indistinguishable from the values for the free enzyme. The immobilized enzyme showed a pH activity optimum between 7.0 and 7.4, compared to the optimum pH of 6.5 for the soluble enzyme. The activity optimum of immobilized heparinase with respect to salt concentration was between 0 and 0.1M. A reactor containing immobilized heparinase recirculating internally at 1300 mL/min behaved as a continuously stirred tank reactor (CSTR) when solutions at a flow rate of 120 mL/min were passed through the device. The residence time distribution was determined using blue dextran (molecular weight 2 x 10(6) daltons), which is sterically excluded from the agarose catalyst. A model of the heparinase reactor based on ideal CSTR behavior and the immobilized enzyme kinetic parameters was developed. It accurately predicted experimental conversions over a range of catalyst volumes, enzyme loadings, and substrate concentrations to within 7% in most cases and with a maximum deviation of 13%.     

Neuroanatomy of morphine-modulating peptides

Antisera against two mammalian peptides related to the molluscan cardioexcitatory peptide Phe-Met-Arg-Phe-NH2 were used to locate immunoreactive neurons in the rat brain, nerve fibres and terminals in the spinal cord, sympathetic ganglion cells and adrenal chromaffin cells. Immunoreactivity for the newly characterised octa- and octadecapeptide was detected in nerve cell bodies in the hypothalamic area, including parts of the dorsomedial, periventricular and paraventricular nuclei, and in the nucleus tractus solitarii. Nerve terminals in the superficial laminae of the spinal cord were also immunoreactive for these peptides, while the sensory ganglia were nonreactive. Some principal ganglion cells in the superior cervical ganglia exhibited bright immunofluorescence for the peptides, and a few adrenal medullary cells were immunoreactive. The presence of these peptides in the substantia gelatinosa of the spinal cord suggests that they may be involved in sensory neurotransmission, especially in the mechanisms mediating pain. In the hypothalamo-hypophysial system these peptides may be involved in the regulation of hormonal systems. They may also act as co-transmitters in the sympathetic nervous system.     

水翁花蕾和水翁叶精油的化学成分研究

水翁的花营和鲜叶经水蒸汽蒸馏得到一种淡黄色的精油,前者出油率为0.18％。后者为0.08％。我们应用毛细管气相色谱,气相色谱/质谱联用,红外光谱和紫外光谱等方法,对两种精油进行化学分成分析,分别鉴定出35个和27个已知化学成分。两者相同的化学成分有:β-罗勒烯(Z)、β-罗勒烯(E)、α-蒎烯、β-蒎烯、月桂烯、小茴香烯、香叶醇、顺式-丁香烯、橙花叔醇等23个化学成分,分别占花营精油全油的90％和叶精油95％以上。     

Localization and Pattern of Graviresponse across the Pulvinus of Barley Hordeum vulgare

Pulvini of excised stem segments from barley (Hordeum vulgare cv `Larker') were pretreated with 1 millimolar coumarin before gravistimulation to reduce longitudinal cell expansion and exaggerate radial cell enlargement. The cellular localization and pattern of graviresponse across individual pulvini were then evaluated by cutting the organ in cross-section, photographing the cross-section, and then measuring pulvinus thickness and the radial width of cortical and epidermal cells in enlargements of the photomicrographs. With respect to orientation during gravistimulation, we designated the uppermost point of the cross-section 0° and the lowermost point 180°. A gravity-induced increase in pulvinus thickness was observable within 40° of the vertical in coumarin-treated pulvini. In upper halves of coumarin-treated gravistimulated pulvini, cells in the inner cortex and inner epidermis had increased radial widths, relative to untreated gravistimulated pulvini. In lower halves of coumarin-treated pulvini, cells in the central and outer cortex and in the outer epidermis showed the greatest increase in radial width. Cells comprising the vascular bundles also increased in radial width, with this pattern following that of the central cortex. These results indicate (a) that all cell types are capable of showing a graviresponse, (b) that the graviresponse occurs in both the top and the bottom of the responding organ, and (c) that the magnitude of the response increases approximately linearly from the uppermost point to the lowermost. These results are also consistent with models of gravitropism that link the pattern and magnitude of the graviresponse to graviperception via statolith sedimentation.     

Thrombolysis using liposomal-encapsulated streptokinase: an in vitro study

The clot-lysing ability of streptokinase (SK) was examined using membrane-bound thrombi. Encapsulation of SK in large unilamellar phospholipid vesicles (liposomes) resulted in entrapping approximately 30% of its original activity. Measurements of streptokinase activity for liposomal-encapsulated streptokinase (LESK) indicated little loss of activity or leakage in Tris-buffered saline over a 24-hr period at temperatures of 4 and 23 degrees C. However, incubation of free SK and LESK in platelet-poor plasma (PPP) at 37 degrees C resulted in a decrease of SK activity. The retention of SK activity in LESK was considerably higher than that of unentrapped SK. Clot-dissolving time (CDT) was measured by monitoring the pressure drop during slow filtration in plasma through membrane-bound thrombi. The results indicated that both LESK and free SK were able to activate the fibrinolytic system. Without prior incubation in PPP at 37 degrees C, the CDT of a SK and PPP mixture (SK/PPP) was 10.7 +/- 1.9 min (n = 12), while that of a LESK and PPP mixture (LESK/PPP) was 12.4 +/- 1.7 min (n = 12). The CDT-detected clot-lysing abilities of both SK and LESK were diminished by incubation in PPP, but to different extents. After 15- and 30-min incubations, the CDT of SK/PPP increased significantly to 15.5 +/- 1.5 and 24.1 +/- 2.4 min (n = 5, P less than 0.05), respectively. In contrast, the CDT of LESK/PPP increased to 13.3 +/- 0.8 min (n = 5) after 15 min of incubation and to 16.0 +/- 1.1 min (n = 5, P less than 0.05) after a 30-min incubation. These results suggest that entrapment of SK in liposomes preserves the thrombolytic potential of the plasminogen activator by limiting its exposure to the components of the plasma.     

Influence of sucrose concentration and phosphate limitation on citric acid production by immobilized cells of Aspergillus niger

Summary Immobilized cells of Aspergillus niger needed a lower initial sucrose concentration than free cells in order to obtain maximal yields of citric acid production. High sucrose concentrations led to reduced yields and increased polyol formation (glycerol, erythritol, arabitol). Continuous fermentation with media containing low sugar concentrations prevented the formation of polyols. The change from nitrogen-limited to phosphate-limited precultivation of immobilized spores significantly increased the productivity of the mycelium. The ratio of citric acid to residual sugar in the effluent distinctly lay in the direction of citric acid. Inside the alginate beads mainly large bulbous cells were observed.     

IgG from antiserum against endogenous mammalian FMRF-NH2-related peptides augments morphine- and stress-induced analgesia in mice

Two mammalian FMRF-NH2-like peptides have been isolated from bovine brain; an octapeptide with the structure Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (F-8-F-NH2) and an octadecapeptide, Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe- NH2 (A-18-F-NH2). In the present study determinations were made of the effects of intracerebroventricular administration of IgG prepared from antisera raised against these peptides on nociception and morphine- and immobilization-induced opioid analgesia in mice. Both F-8-F-NH2-IgG and A-18-F-NH2-IgG antisera increased nociception (thermal response latency) and significantly augmented morphine- and immobilization-induced analgesia in a naloxone reversible manner, with F-8-F-NH2-IgG antisera having a greater effect than A-18-F-NH2-IgG antisera. These results provide further evidence that mammalian FMRF-NH2-like peptides function as endogenous opiate antagonists and have a role in the mediation of antinociception.