The Effects of the Porosity of a Support and of Attachment on the Mode of Action of Immobilized Endopolygalacturonase

Endopolygalacturonase of Aspergillus sp. was immobilized by three different methods; viz. (a) via amino groups, (b) via carboxyl groups and (c) by means of epoxy groups to a nonporous microparticular silicon dioxide (Cabosil), functionalized by 3-(amino)-propyl groups and 3-(2',3'-epoxypropoxy)-propyl groups, respectively. The conjugates were compared in their mode of action with corresponding immobilized preparations based on microporous ceramics. The binding via amino groups and via carboxyl groups lead, by itself, to changes in the mode of action of the enzyme, consisting of a decrease in randomness of glycosidic linkage splitting. The changes were greater in microporous support conjugates due to additional size-exclusion effects. The action pattern of endopolygalacturonase bound by means of epoxy groups was modulated exclusively by the porosity of the support, whereas the binding alone did not play any role.     

The Effects of the Porosity of a Support and of Attachment on the Mode of Action of Immobilized Endopolygalacturonase

Endopolygalacturonase of Aspergillus sp. was immobilized by three different methods; viz. (a) via amino groups, (b) via carboxyl groups and (c) by means of epoxy groups to a nonporous microparticular silicon dioxide (Cabosil), functionalized by 3-(amino)-propyl groups and 3-(2′,3′-epoxypropoxy)-propyl groups, respectively. The conjugates were compared in their mode of action with corresponding immobilized preparations based on microporous ceramics. The binding via amino groups and via carboxyl groups lead, by itself, to changes in the mode of action of the enzyme, consisting of a decrease in randomness of glycosidic linkage splitting. The changes were greater in microporous support conjugates due to additional size-exclusion effects. The action pattern of endopolygalacturonase bound by means of epoxy groups was modulated exclusively by the porosity of the support, whereas the binding alone did not play any role.     

Atresia of the right atrial ostium of the coronary sinus

A case of asymptomatic congenital occlusion of the ostium of the coronary sinus is described. The myocardial venous drainage was maintained via a persistent left superior vena cava as well via ectatic, widened atrial veins of the dorsal wall of the left atrium. The study shows that complete ostial occlusion of the coronary sinus does not reduce cardiac venous drainage. The view of the literature allows a comparison with the comprehensive classification of coronary sinus anomalies.     

Conformational change of the chloroplast ATP synthase on the enzyme activation process detected by the trypsin sensitivity of the gamma subunit

Delta mu H(+) is known to stimulate the enzyme activity of chloroplast ATP synthase in addition to its important role as energy supply for ATP synthesis. In the present study, we focused on the relationship between the proton translocation via the membrane sector of ATP synthase, F(o), and the conformational change of the central stalk subunit gamma. The conformational change of CF(1) mainly at the gamma subunit was induced by the proton flow via F(o) in the absence of substrates. The effects of inhibitors on CF(o) or CF(1) for this conformational change were also examined. The observed conformational change was partially suppressed by ADP binding. From these results, we propose the Delta mu H(+)-dependent conformational change of CF(1) on the enzyme activation process, which is affected by both ADP binding to the catalytic sites and proton flow via F(o) portion.     

Glycoside and aglycone inotropism: Influence of temperature on the rate of onset

The onset of inotropism of a cardiac glycoside (strophanthin) and an aglycone (acetylstrophanthidin) was examined at three different temperatures (27, 32 and 37° C) in isolated rabbit left atria driven at a beat interval of 10 sec. The rate of onset of glycoside inotropism was highly dependent on temperature (the higher the temperature the faster the onset), while that of the aglycone was not. It is suggested that cardiac glycosides and aglycones may gain access to the “digitalis inotropic receptor” via different processes: glycosides via a highly temperature-dependent, and presumably active, process and aglycones via a temperature-insensitive, and presumably passive, process.     

The metabolism of fructose in the bivascularly perfused rat liver.

The metabolism of fructose was investigated in the bivascularly and hemoglobin-free perfused rat liver. Anterograde and retrograde perfusions were performed. In anterograde perfusion, fructose was infused at identical rates (19 mumols min-1 g-1) via the portal vein (all liver cells) or the hepatic artery (predominantly perivenous cells); in retrograde perfusion fructose was infused via the hepatic vein (all liver cells) or the hepatic artery (only periportal cells). The cellular water spaces accessible via the hepatic artery were measured by means of the multiple-indicator dilution technique. The following results were obtained. (i) Fructose was metabolized to glucose, lactate and pyruvate even when this substrate was infused via the hepatic artery in retrograde perfusion; oxygen consumption was also increased. (ii) When referred to the water spaces accessible to fructose via the hepatic artery in each perfusion mode, the rate of glycolysis was 0.99 +/- 0.14 mumols min-1 ml-1 in the retrograde mode; and, 2.05 +/- 0.19 mumols min-1 ml-1 in the anterograde mode (P = 0.002). (iii) The extra oxygen uptake due to fructose infusion via the hepatic artery was 1.09 +/- 0.16 mumols min-1 ml-1 in the retrograde mode; and, 0.51 +/- 0.08 mumols min-1 ml-1 in the anterograde mode (P = 0.005). (iv) Glucose production from fructose via the hepatic artery was 2.18 +/- 0.18 mumols min-1 ml-1 in the retrograde mode; and, 1.83 +/- 0.16 mumols min-1 ml-1 in the anterograde mode (P = 0.18). (v) Glucose production and extra oxygen uptake due to fructose infusion did not correlate by a single factor in all perfusion modes. It was concluded that: (a) rates of glycolysis are lower in the periportal area, confirming previous views; (b) extra oxygen uptake due to fructose infusion is higher in the periportal area; (c) a predominance of glucose production in the periportal area could not be demonstrated; and (d) extra oxygen uptake due to fructose infusion is not a precise indicator for glucose synthesis.     

Seasonal rhythms of the indolamine content in the epiphysis of rats

Pineal function was studied in Wistar pubertal male rats in summer and winter. It was demonstrated that the pineal weight is inversely related to the day length, increasing in winter and reducing in summer. In winter pineal serotonin is actively metabolized via N-acetylation and subsequent ortho-methylation to form N-acetyl serotonin and melatonin as well as via oxidative deamination followed by ortho-methylation to form 5-hydroxyindolacetic and 5-methoxyindolacetic acids. In summer serotonin metabolism in the pinealocytes changes to direct ortho-methylation of serotonin to form 5-methoxytryptamine. The decreased level of N-acetyltransferase limits the formation of melatonin in the pineal gland in summer.     

Neuronal background of positioning of the posterior tentacles in the snail Helix pomatia

The location of cerebral neurons innervating the three recently described flexor muscles involved in the orientation of the posterior tentacles was investigated by applying parallel retrograde Co- and Ni-lysine tracing via the olfactory and the peritentacular nerves. Their innervation patterns in the flexor muscles were studied by applying anterograde neurobiotin tracings via these nerves. The labeled neurons are clustered in eight groups in the cerebral ganglion. They send both common and distinct innervation pathways to the flexor and the tegumental muscles and to the tentacular retractor muscle. The common pathway reaches the muscles via the olfactory nerve, whereas the distinct pathways innervate via the internal and external peritentacular nerves. The three anchoring points of the three flexor muscles at the base of the tentacle outline the directions of three force vectors generated by the contraction of the muscles and enable the protracted tentacle to bend around a basal pivot. In the light of earlier physiological and the present anatomical findings, we suggest that the common innervation pathway to the muscles is required for tentacle withdrawal and the retractor mechanism, whereas the distinct pathways primarily serve the bending of the protracted posterior tentacles during foraging.     

Biosynthesis of anthraquinones in cell cultures of Cinchona 'Robusta' proceeds via the methylerythritol 4-phosphate pathway.

Robustaquinone B was found as a major anthraquinone in cell cultures of Cinchona 'Robusta' after treatment with a fungal elicitor. Anthraquinones in Cinchona are considered to be of the Rubia type, i.e. rings A and B are derived from chorismate and alpha-ketoglutarate, whereas ring C is formed from isopentenyl diphosphate (IPP). To determine the origin of IPP, either formed via the mevalonic acid pathway or the 2-C-methyl-D-erythritol 4-phosphate pathway, the incorporation of [1-13C]glucose into robustaquinone B was studied. The 13C labeling of robustaquinone B was analyzed by one- and two-dimensional NMR spectroscopy and the labeling pattern was compared with the hypothetical labeling patterns obtained via the different biosynthetic pathways. The results clearly show that the IPP, constituting the ring C of robustaquinone B, is biosynthesized via the 2-C-methyl-D-erythritol 4-phosphate pathway. Moreover, the data also confirm that rings A and B of robustaquinone B are formed from chorismate and alpha-ketoglutarate via o-succinylbenzoate.     

Regulation of alternative pathway activity in plant mitochondria: nonlinear relationship between electron flux and the redox poise of the quinone pool

The dependence of respiratory flux via the alternative pathway on the redox poise of the ubiquinone (Q) pool was investigated in soybean cotyledon mitochondria. A marked nonlinear relationship was observed between Q-pool reduction level and O2 uptake via the alternative oxidase. Significant engagement of the alternative pathway was not apparent until Q-pool reduction level reached 35-40% but increased disproportionately on further reduction. Similar results were obtained with electron donation from either Complex 1 or Complex 2. Close agreement was obtained over a range of experimental conditions between the estimated contribution of the alternative pathway to total respiratory flux, as measured with salicylhydroxamic acid, and that predicted from the redox poise of the Q-pool. These results are discussed in terms of existing models of the regulation of respiratory flux via the alternative pathway.     

Structural organization of the compensation process in the lymph nodes in venous stasis

Transmission and scanning electron microscopy was used to study the structural organization of the lymph nodes during venous congestion. At the early period (1 h of venous congestion) one could see an increased number of the fenestrated cells in the venous part of the capillary whereas at the later period (after 6 h to 3 days) a rise of the microvesicular transport via endotheliocytes of the arterial part of the capillary and via fenestras of the venous part. Within 7 to 14 days one could see activation of the microvesicular transport via the indicated parts of the capillary. At all the stages of venous congestion the fluid contained by the parenchyma of the lymph node penetrates via fenestras and intercellular fissures to the marginal sinus, while only via fenestra to the cerebral sinuses.     

Maximum airflow through the nose in humans

Inspiratory and expiratory flow via the nose and via the mouth during maximum-effort vital capacity (VC) maneuvers have been compared in 10 healthy subjects. Under baseline conditions maximum flow via the nose was lower than that via the mouth in the upper 50-60% of the VC on expiration and throughout the VC on inspiration. The mean ratio of maximum inspiratory to maximum expiratory flow at mid-VC was 1.38 during mouth breathing and 0.62 during nasal breathing. Inspiratory flow limitation with no increase in flow through the nose as driving pressure was increased above a critical value (usually between 12 and 30 cmH2O) was found in all six subjects studied. Stenting the alae nasi in seven subjects increased peak flow via the nose from a mean of 3.49 to 4.32 l/s on inspiration and from 4.83 to 5.61 l/s on expiration. Topical application of an alpha-adrenergic agonist in seven subjects increased mean peak nasal flow on inspiration from 3.25 to 3.89 l/s and on expiration from 5.03 to 7.09 l/s. Further increases in peak flow occurred with subsequent alan stenting. With the combination of stenting and topical mucosal vasoconstriction, nasal peak flow on expiration reached 81% and, on inspiration, 79% of corresponding peak flows via the mouth. The results demonstrate that narrowing of the alar vestibule and the state of the mucosal vasculature both influence maximum flow through the nose; under optimal conditions, nasal flow capacity is close to that via the mouth.     

External Pathway of NADH Oxidation in the Liver of the River Lamprey Lampetra fluviatilis

A possibility of exogenous NADH oxidation via the external pathway has been shown on homogenates and isolated liver cells of the lamprey Lampetra fluviatilis in the presence of rotenone and antimycin A. The homogenates were incubated in isotonic and hypotonic sucrose media, while cells, in isotonic salt medium. At incubating the tissue preparations in isotonic media, digitonin was used to enhance membrane permeability to NADH and cytochrome c. In homogenates, the maximal rate of NADH oxidation via the external pathway in the presence of cytochrome c and digitonin was 5.3 nmol O₂/min/10 mg wet weight. This value in the cells amounted to 12.6, while without addition of exogenous NADH and cytochrome c, to 11.0 nmol O₂/min/10 million cells. Cyanide inhibited completely the NADH oxidation via the external pathway both in homogenates and in cells. The intact lamprey hepatocytes, unlike homogenates, are suggested to contain sufficient concentrations of cytochrome c and extramitochondrial NADH to provide maximal NADH oxidation rate in mitochondria through external pathway. This allows thinking that potential possibilities of NADH oxidation via the external pathway in Cyclostomata and mammals are qualitatively and quantitatively close.     

Effect of metal ions on the activity of the catalytic domain of calcineurin

Calcineurin (CN) is a heterodimer, composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). There are four functional domains present in CNA, which are catalytic domain (CNa), CNB-binding domain (BBH), CaM-binding domain (CBH) and autoinhibitory domain (AI). It has been shown previously that the in vitro activity of calcineurin is relied primarily on the binding of metal ions. Mn2+ and Ni2+ are the most crucial cation-activators for this enzyme. In order to determine which domain(s) in CN is functionally regulated by metal ions, the rat CNA alpha subunit and its catalytic domain (CNa) were cloned and expressed in E. coli. The effects of Mn2+, Ni2+ and Mg2+ on the catalytic activity of these purified proteins were examined. Our results demonstrate that all the metal ions tested in this study activated either CNA or CNa. However, the activation degree of CNa by the metal ions was much higher than that of CNA. In term of different metal ions, the activating extents to CNA and CNa were different. To CNA, the activating order from high to low was Mg2+ > > Ni2+ > Mn2+, but Mn2+ > Ni2+ > > Mg2+ to CNa. No effect of CaM/Ca2+ and CNB/Ca2+ on the activity of CNa was observed in our experiments. Moreover, a weak interaction (or untight coordination binding) between metal ions and the enzyme molecule was also identified. These results suggest that the activation of these enzymes by the exogenous metal ions might be via both regulating fragment of CNA (including BBH, CBH and AI) and catalytic domain (CNa), and mainly via regulating fragment to CNA and mainly via catalytic domain to CNa. The activating extents of metal ions via catalytic domain were higher than that via regulating fragment. The results obtained in this study should be very useful for understanding the molecular mechanism underlying the interaction between calcineurin and metal ions, especially Mn2+, Ni2+ and Mg2+.     

Metabolic flux analysis of the mixotrophic metabolisms in the green sulfur bacterium Chlorobaculum tepidum

The photosynthetic green sulfur bacterium Chlorobaculum tepidum assimilates CO(2) and organic carbon sources (acetate or pyruvate) during mixotrophic growth conditions through a unique carbon and energy metabolism. Using a (13)C-labeling approach, this study examined biosynthetic pathways and flux distributions in the central metabolism of C. tepidum. The isotopomer patterns of proteinogenic amino acids revealed an alternate pathway for isoleucine synthesis (via citramalate synthase, CimA, CT0612). A (13)C-assisted flux analysis indicated that carbons in biomass were mostly derived from CO(2) fixation via three key routes: the reductive tricarboxylic acid (RTCA) cycle, the pyruvate synthesis pathway via pyruvate:ferredoxin oxidoreductase, and the CO(2)-anaplerotic pathway via phosphoenolpyruvate carboxylase. During mixotrophic growth with acetate or pyruvate as carbon sources, acetyl-CoA was mainly produced from acetate (via acetyl-CoA synthetase) or citrate (via ATP citrate lyase). Pyruvate:ferredoxin oxidoreductase converted acetyl-CoA and CO(2) to pyruvate, and this growth-rate control reaction is driven by reduced ferredoxin generated during phototrophic growth. Most reactions in the RTCA cycle were reversible. The relative fluxes through the RTCA cycle were 80～100 units for mixotrophic cultures grown on acetate and 200～230 units for cultures grown on pyruvate. Under the same light conditions, the flux results suggested a trade-off between energy-demanding CO(2) fixation and biomass growth rate; C. tepidum fixed more CO(2) and had a higher biomass yield (Y(X/S), mole carbon in biomass/mole substrate) in pyruvate culture (Y(X/S) = 9.2) than in acetate culture (Y(X/S) = 6.4), but the biomass growth rate was slower in pyruvate culture than in acetate culture.     

Clearance of surfactant phosphatidylcholine via the upper airways in rabbits

A possible route of clearance of surfactant phosphatidylcholine from the lungs is via the airways. To quantify surfactant loss via this pathway, latex bags were surgically placed into the abdomens of adult rabbits such that secretions cleared via the esophagus could be collected. The rabbits then were given treatment or trace doses of radiolabeled phosphatidylcholine-surfactant by tracheal injection and/or intravascular radiolabeled precursors of phosphatidylcholine. Labeled saturated phosphatidylcholine was measured in all fluids that were collected from the bags at 2-h intervals for 24 h and in alveolar washes and lung tissues at 24 h. No more than 7% of either treatment or trace doses of intratracheal surfactant-saturated phosphatidylcholine was lost via clearance up the airways over 24 h. Clearances of endogenously synthesized and secreted saturated phosphatidylcholine were estimated to be no more than 3% of the flux of labeled saturated phosphatidylcholine through the alveolar pool. These experiments demonstrate that surfactant phosphatidylcholine clearance via movement up the airways is not a major pathway leading to surfactant catabolism.     

Dual mechanisms for shedding of the cellular prion protein

The cellular prion protein (PrP(C)) is essential for the pathogenesis and transmission of prion diseases. Whereas the majority of PrP(C) is bound to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor, a secreted form of the protein has been identified. Here we show that PrP(C) can be shed into the medium of human neuroblastoma SH-SY5Y cells by both protease- and phospholipase-mediated mechanisms. The constitutive shedding of PrP(C) was inhibited by a range of hydroxamate-based zinc metalloprotease inhibitors in a manner identical to the alpha-secretase-mediated shedding of the amyloid precursor protein, indicating a proteolytic shedding mechanism. Like amyloid precursor protein, this zinc metalloprotease-mediated shedding of PrP(C) could be stimulated by phorbol myristate acetate and by copper ions. The lipid raft-disrupting agents filipin and methyl-beta-cyclodextrin promoted the shedding of PrP(C) via a distinct mechanism that was not inhibited by hydroxamate-based inhibitors. Filipin-mediated shedding of PrP(C) is likely to occur via phospholipase cleavage of the GPI anchor, since a transmembrane polypeptide-anchored PrP construct was not shed in response to filipin treatment. Collectively, our data indicate that shedding of PrP(C) can occur via both secretase-like proteolytic cleavage of the protein and phospholipase cleavage of the GPI anchor moiety.     

The involvement of angiotensins in the control of prostatic epithelial cell proliferation in the rat.

The effects of captopril (the inhibitor of the angiotensin-converting enzyme) and of angiotensins II and IV (3-8 fragment of angiotensin II) on cell proliferation of the prostatic epithelium was investigated in the rat. The incorporation of bromodeoxyuridine into cell nuclei was used as an index of cell proliferation. It was found that the treatment with captopril resulted in the suppression of prostatic epithelial cell proliferation. The antiproliferative effect of captopril was reversed (at least partially) by a simultaneous treatment with either angiotensin II or angiotensin IV. The effects of angiotensins were not blocked by the administration of losartan--AT1 angiotensin receptor blocker. These findings suggest the involvement of angiotensins in the control of prostatic growth, acting via the receptors different from the AT1-subtype (presumably via AT4 receptors).     

Genotoxic and cytotoxic effects in the bone marrow of rats exposed to a low dose of paraquat via the dermal route

The genotoxic effect of the herbicide paraquat was studied in rat bone-marrow by means of the micronucleus assay. Paraquat at dose levels of 6, 15 and 30 mg/kg body weight was given to rats in a single application via the dermal route. Marrow was collected at 24, 48 and 72 h after the application. The micronucleus assay was done as recommended by standard procedures. Paraquat gave rise to an increase in the number of micronuclei in a dose-dependent manner. The number of micronucleated polychromatic erythrocytes showed a maximum at 48 h and the toxicity was further prolonged, as there was no complete recovery at 72 h. These findings suggest a genotoxic effect of paraquat even after exposure via dermal application.