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One of the prime objectives of the Rosetta Radio Science Investigations (RSI) experiment is the determination of the mass, the bulk density and the low degree and order gravity of the nucleus of comet P/Wirtanen, the target object of the international Rosetta mission. The RSI experiment will use the spacecraft's radio carrier frequencies at X-band (8.4 GHz) and S-band (2.3 GHz) in order to measure slight changes of the orbit velocity via the classical Doppler effect induced by the gravity attraction of the comet nucleus. Based on an estimate of the background Doppler noise, it is expected that a mass determination (assuming a representative radius of 700 m and a bulk density of 500 kg/m3) at an accuracy of 0.1% can be achieved if the spacecraft's orbit is iteratively reduced below 7 km altitude. The gravity field of degree and order two can be detected for reasonable tracking times below 5 km altitude. The major competing forces acting on the spacecraft are the radiation pressure and the gas mass flux from cometary activity. While the radiation pressure may be predicted, it is recommended to begin a gravity mapping campaign well before the onset of outgassing activity (>3.25 AU heliocentric distance). Radial acceleration by water outgassing is larger by orders of magnitude than the accelerations from the low degree and order gravity field and will mask the contributions from the gravity field.

The central role of the intracellular enzyme hormone-sensitive lipase (HSL) in regulating fatty acid metabolism makes it an interesting pharmacological target for the treatment of insulin resistant and dyslipidemic disorders where a decrease in delivery of fatty acids to the circulation is desirable, e.g., in individuals with type 2 diabetes, metabolic syndrome, or impaired glucose tolerance. On the basis of a lead structure from high throughput screening, we have identified a very potent type of carbamoyl-triazole inhibitors of HSL. As part of the lead optimization program, four new classes of carbamoyl-triazoles were synthesized and tested with respect to potency, efficacy and selectivity. Methyl-phenyl-carbamoyl-triazoles were identified as potent and efficacious HSL inhibitors. These compounds do not inhibit other hydrolases such as hepatic lipase, lipoprotein lipase, pancreatic lipase, and butyrylcholine esterase. However, the inhibitors 4b and 4g with IC50 values for HSL of 0.17 and 0.25 μM, respectively, were the only inhibitors selective against acetylcholine esterase. A reversible pseudosubstrate inhibition mechanism is proposed for this class of inhibitors.

Reversible phosphorylation and dephosphorylation of key proteins on tyrosine residues are important parts of intracellular signaling triggered by hormones and other agents. Recent knock-out studies in mice have identified PTP1B as a potential target for the treatment of diabetes and obesity. As a consequence, a number of academic and industrial groups are aggressively pursuing the development of selective PTP1B inhibitors. In addition, other protein−tyrosine phosphatases (PTPs) appear to be critically involved in major diseases such as cancer and autoimmunity. Given the diversity of PTPs and their potential as drug targets in different diseases, we have taken a broad approach to develop active site-directed selective inhibitors of specific members of this family of enzymes. Using a high throughput screening, we have previously identified 2-(oxalylamino)benzoic acid 3a as a relatively weak but classical competitive inhibitor of several PTPs.⁴ On the basis of our early studies, indicating that 3a might be used as a starting point for the synthesis of selective PTP inhibitors, we now present our efforts in expansion of this concept and provide here a number of new chemical scaffolds for the development of inhibitors of different members of the PTP family. Although the core structure of these inhibitors is charged, good oral bioavailability has been observed in rat for some compounds. Furthermore, we have observed enhancement of 2-deoxy-glucose accumulation in C2C12 cells with prodrug analogues.

Factor VII requires the cleavage of an internal peptide bond and the association with tissue factor (TF) to attain its fully active factor VIIa (FVIIa) conformation. The former event alone leaves FVIIa in a zymogen-like state of relatively low specific activity. We have designed a number of FVIIa mutants with the aim of mimicking the effect of TF, that is, creating molecules with increased intrinsic (TF-independent) enzymatic activity. Based on a possible structural difference between free and TF-bound FVIIa (Pike, A. C. W., Brzozowski, A. M., Roberts, S. M., Olsen, O. H., and Persson, E. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 8925--8930), we focused on the helical region encompassing residues 307-312 and residues in its spatial vicinity. For instance, FVIIa contains Phe-374 and Leu-305, whereas a Phe/Tyr residue in the position corresponding to 374 in homologous coagulation serine proteases is accompanied by Val in the position corresponding to 305. This conceivably results in a unique orientation of this helix in FVIIa. Substitution of Val for Leu-305 in FVIIa resulted in a 3--4-fold increase in the intrinsic amidolytic and proteolytic activity as compared with wild-type FVIIa, whereas the activity in complex with soluble TF remained the same. In accordance with this, L305V-FVIIa exhibited an increased rate of inhibition as compared with wild-type FVIIa, both by d-Phe-Phe-Arg-chloromethyl ketone and antithrombin III in the presence of heparin. The increased FVIIa activity upon replacement of Leu-305 by Val may be mediated by a movement of the 307--312 helix into an orientation resembling that found in factors IXa and Xa and thrombin. The corresponding shortening of the side chain of residue 374 (Phe --> Pro) had a smaller effect (about 1.5-fold increase) on the intrinsic activity of FVIIa. Attempts to increase FVIIa activity by introducing single or multiple mutations at positions 306, 309, and 312 to stabilize the 307-312 helix failed.

The effect of a potato fibre preparation on exocrine pancreatic secretions and on gastrointestinal hormone levels in plasma was studied in three 8 weeks old piglets that were surgically fitted with a jugular vein catheter for blood sampling, a pancreatic duct catheter and a T‐shaped duodenal cannula for collection of pancreatic juice. The animals were fed for 2 weeks a control diet (experimental period 1), thereafter for 2 weeks the control diet supplemented with 2%potato fibre (experimental period 2) and for another 2 weeks the control diet again (experimental period 3). Additionally, intraduodenal (i.d.) infusions of the experimental diet, the control diet and potato fibre as well as i.v. infusions of a solution containing cholecystokinin (CCK) and secretin were administered.Potato fibre in the diet evoked in tendency an increase in the volume of secretion of pancreatic juice and a significant increase both in the mean values of the total protein content and total activities of lipase, trypsin and a‐amylase when compared to the control diet. The i.d. infusion of the control diet, experimental diet and fibre infusate as well as the i.V. administration of the hormone infusate led to a spontaneous secretory response of the exocrine pancreas. Besides gastrointestinal hormones, such as CCK, other factors such as short chain fatty acids may be involved in the regulation of the exocrine pancreas.

Several protein-tyrosine phosphatases (PTPs) have been proposed to act as negative regulators of insulin signaling. Recent studies have shown increased insulin sensitivity and resistance to obesity in PTP1B knockout mice, thus pointing to this enzyme as a potential drug target in diabetes. Structure-based design, guided by PTP mutants and x-ray protein crystallography, was used to optimize a relatively weak, nonphosphorus, nonpeptide general PTP inhibitor (2-(oxalyl-amino)-benzoic acid) into a highly selective PTP1B inhibitor. This was achieved by addressing residue 48 as a selectivity determining residue. By introducing a basic nitrogen in the core structure of the inhibitor, a salt bridge was formed to Asp-48 in PTP1B. In contrast, the basic nitrogen causes repulsion in other PTPs containing an asparagine in the equivalent position resulting in a remarkable selectivity for PTP1B. Importantly, this was accomplished while retaining the molecular weight of the inhibitor below 300 g/mol.

The nucellus is a complex maternal grain tissue that embeds and feeds the developing cereal endosperm and embryo. Differential screening of a barley (Hordeum vulgare) cDNA library from 5-d-old ovaries resulted in the isolation of two cDNA clones encoding nucellus-specific homologs of the vacuolar-processing enzyme of castor bean (Ricinus communis). Based on the sequence of these barley clones, which are called nucellains, a homolog from developing corn (Zea mays) grains was also identified. In dicots the vacuolar-processing enzyme is believed to be involved in the processing of vacuolar storage proteins. RNA-blot and in situ-hybridization analyses detected nucellain transcripts in autolysing nucellus parenchyma cells, in the nucellar projection, and in the nucellar epidermis. No nucellain transcripts were detected in the highly vacuolate endosperm or in the other maternal tissues of developing grains such as the testa or the pericarp. Using an antibody raised against castor bean vacuolar-processing protease, a single polypeptide was recognized in protein extracts from barley grains. Immunogold-labeling experiments with this antibody localized the nucellain epitope not in the vacuoles, but in the cell walls of all nucellar cell types. We propose that nucellain plays a role in processing and/or turnover of cell wall proteins in developing cereal grains.

A transient expression system has been established to assay the regulation and impact of putative antifungal proteins at the single cell level in barley (Hordeum vulgareL.) epidermal leaf cells infected with powdery mildew (Erysiphe graminisf.sp.hordei).The system combines biolistic transformation with direct comparison of the fungal attack in transfected and wild type cells. Many epidermal cells showed detectable expression of marker genes including a novel improved version of green fluorescent protein (GFP). The marker was detectable for at least 10 days during which individual host cells supported an unaffected asexual life-cycle of the pathogen.

Das Durchstanzproblem und die Querkrafttragfähigkeit wird für kurze Balken, Kreisplatten und Stützen erläutert und hieraus geeignete Bewehrungsformen abgeleitet. Der Einfluss von Aussparungen auf die Tragfähigkeit wird untersucht.

Über Spezialfälle bei der Schnittkraftermittlung von Platten.

The objective was to evaluate the influence of treatment with rhG‐CSF on allergic indexes. This was done by open trial of 5 days' treatment with rhG‐CSF (5 μg/kg/day s. c.). 10 patients (6 men), aged 28 to 54 years, with rhinoconjunctivitis due to grass pollen allergy, participated in the investigation. Main measures were blood count, basophil histamine release, skin test and conjunctival provocation test. Results: The treatment resulted in significant increases in numbers of neutrophils (590%), basophils (280%), eosinophils (250%) and lymphocytes (71%). Total blood histamine was increased, but basophil histamine releasability was decreased. Serum alkaline phosphatase increased 92% and serum lactate dehydrogenase increased 35% (both significant). There were no significant changes in the skin tests and the conjunctival provocation tests. Two months after the treatment all tests had returned to baseline levels. Five of the patients (50%) reported side effects, one withdrew. In conclusion treatment with rhG‐CSF increases the number of circulating blood cells other than neutrophils without causing changes in indexes of allergic reactivity.

Ten healthy men participated in a crossover study, and the experiments took place after 10 days of treatment (40 mg omeprazole every morning). Blood samples were drawn at fixed intervals during a complete migrating motor complex (MMC) cycle. The manometric pressure tube was removed after passage of the second duodenal phase III, and an omelet (1400 KJ) tagged with 99mTc was ingested, followed by 150 ml of water tagged with 111In-diethylenetriaminepentaacetic acid. Mean plasma gastrin (pmol/l) in phases I, II, and III in the omeprazole group was 18.8, 23.3, 19.9, respectively. The corresponding figures for the placebo group were 9.3, 9.6, 9.5, respectively. All mean values for the omeprazole group were significantly higher (p < 0.01). Mean plasma gastrin in the omeprazole group was significantly higher in phase II than in phase I (p < 0.05). Mean plasma secretin (pmol/l) in phases I, II, and III in the omeprazole group was 1.6, 1.4, 1.1, respectively. The corresponding figures for the placebo group were 2.0, 1.7, 2.2, respectively. Mean plasma secretin in the omeprazole group was significantly lower in phases I and III (p < 0.05). The mean incremental integrated postprandial gastrin response (pmol.30 min/l) was significantly higher in the omeprazole group (475.0 versus 97.5) (p < 0.05). The immediate postprandial mean value of secretin was significantly lower in the omeprazole group (p < 0.05). We conclude that 40 mg omeprazole elicits i) a phase-related increase in fasting plasma gastrin, ii) a decrease in secretin in phases I and III, iii) an augmented meal-stimulated gastrin response, and iv) a secretin response characterized by a significantly lower mean in the immediate postprandial period.

Background: Gastrin is an important stimulator of gastric lipase secretion in man. In advanced pancreatic insufficiency gastric lipases might compensate for the lack of pancreatic lipases, hut the role of gastrin in such compensation remains to be evaluated. The aim of this study was to examine the effect of gastrin on the gastric lipase secretion in patients with pancreatic insufficiency. Methods: Eight patients with pancreatic insufficiency secondary to alcohol abuse were studied, and six healthy subjects volunteered as controls for the study. All volunteers received identical doses of intravenous gastrin-17 (10, 30, and 60 pmol/kg/h). The gastric content was measured, using a nasogastric tube for aspiration, and the amount and activity of gastric lipase output were determined. Plasma concentrations of gastrin, secretin, and cholecystokinin were measured by radioimmunoassays. Results: The increased plasma levels of gastrin were accompanied by a dose-dependent increase in the amount and activity of gastric lipase in controls, but in the patients the response was almost abolished. Conclusions: Gastrin in postprandial concentrations does not influence the secretion of gastric lipase in patients with pancreatic insufficiency due to chronic pancreatitis.

Background: The mechanism behind the possible feedback regulation of pancreatic exocrine secretion is not well understood. Methods: Thirteen minipigs were prepared with fistulas to the pancreatic duct and the duodenum. Peripheral venous blood was obtained for determination of secretin and cholecystokinin (CCK) levels. Four different experiments were performed: 1) diversion and reinfusion of pancreatic juice; 2) intraduodenal infusion of NaHCO3 solution, with the same volume, bicarbonate concentration, and osmolality as the collected pancreatic secretion, and reinfusion of pancreatic juice; 3) reinfusion of pancreatic secretion for 1 h before and 2 h after a meal; and 4) diversion of pancreatic secretion and intraduodenal infusion of NaHCO3 solutions before and after a meal. Results: Reinfusing pancreatic juice significantly decreased pancreatic juice volume and bicarbonate output and slightly decreased the level of secretin in plasma. Alternating infusions of substitute NaHCO3 and pancreatic juice did not change pancreatic output of bicarbonate and protein, nor did it change the CCK and secretin levels in plasma. Replacing pancreatic juice with intraduodenal NaHCO3 infusions during a meal did not significantly modify the pancreatic secretion of bicarbonate and protein or the hormonal levels in blood. Conclusions: A negative feedback regulation of pancreatic exocrine secretion is present in starved minipigs. Duodenal acidity and plasma levels of secretin seem to be of importance, whereas duodenal enzymatic activity and the level of CCK in plasma probably are not. A postprandial negative feedback regulation through duodenal enzymatic activity and release of CCK into blood could not be shown.

We studied the effects of intraduodenal oleic acid on the release of somatostatin to plasma and the correlation between endogenous bile output and plasma somatostatin. In five normal persons infusion of 0, 5, 10, 20 and 40 mM oleic acid dose-dependently increased the levels of somatostatin during as well as after gallbladder emptying. The difference between somatostatin concentration during and after gallbladder emptying was not significant. The amylase secretion also was significantly correlated to the dose of fat, whereas the output of bile salts was the same for all fat doses used. Our observations indicate that intraduodenal oleic acid-and not bile salts-releases somatostatin from the gut.

Background: Gastric lipase is an important enzyme for dietary triglyceride digestion in normal subjects. Its regulation is unknown, as is the relation between the quantity and activity of the enzyme.Methods: In a dose-response study we investigated the effect of low doses of pentagastrin (less than 1000 ng/kg/h) on the output of gastric lipase measured by a kinetic assay and an enzyme-linked immunosorbent assay (ELISA).Results: In five healthy volunteers stepwise intravenous pentagastrin infusions of 0, 50, 100,500, and 1000 ng/kg/h resulted in a stepwise increase in the lipase output, as measured with ELISA. However, the lipolytic activity, measured with a kinetic assay, decreased as the pH of the gastric contents decreased.Conclusion: We conclude that secretion of the gastric lipase is stimulated by pentagastrin, but the simultaneous secretion of acid counteracts the lipolytic activity of the enzyme when food is not present.

1. Using the isolated mucosa from the colon of the adult hen, transport of galactose, leucine and lysine was studied through measurements of influx across the brush‐border membrane, unidirectional transmucosal fluxes, and of steady‐state mucosal uptake.

1. The isolated colonic mucosa from adult hens has been studied in vitro under short‐circuit conditions. Colonic mucosa was prepared from hens fed either a NaCl‐poor or a NaCl‐rich diet. 2. The unidirectional transmucosal fluxes of chloride, K and Na were measured. For the high NaCl colon the fluxes of lysine were also measured. The effects of galactose, glucose, leucine, lysine, amiloride (10(‐4) M) and acetazolamide (5 X 10(‐4) M) on the short‐circuit current (Isc) were examined. 3. The Isc of the high NaCl colon was stimulated by galactose, glucose, leucine and lysine in a manner which suggests that these substances were transported by the epithelium in co‐transport with Na. The Isc was in most cases insensitive to amiloride (mucosal side), but moderately stimulated by acetazolamide (serosal side). The Isc was closely matched by the sum of the net transmucosal fluxes of chloride, lysine, K and Na. The effects of galactose, leucine and lysine on the Isc were maintained only when glucose was present on the serosal side of the preparations. 4. The Isc of the low NaCl colon depended on the presence of glucose on the serosal (or mucosal) side. It was not stimulated by galactose or by lysine, but leucine did stimulate when added to the mucosal solution. Adding amiloride on the mucosal side invariably eliminated or, in most cases, led to an inversion of the Isc. Acetazolamide markedly stimulated the Isc; and, when added after amiloride, acetazolamide caused the Isc to become positive again. The data on Isc and net fluxes of chloride, lysine, K and Na are consistent with the presence of a process of bicarbonate absorption or H ion secretion. 5. There is a net secretion of K across both the high and the low Na colon, which is highest in the former although both Isc and net absorption of Na are highest in the latter. 6. The results from both types of colon suggest that the transport of chloride differs with the functional status of tissues.

Die allgemein bekannte und anschauliche Darstellung von schlanken Stäben, die durch Torsion beansprucht werden, auf Platten umzusetzen, die durch Drillmomente belastet sind, ist Ziel der Arbeit. Es wird gezeigt, daß prinzipiell für die Beanspruchung von Plattenecken durch Drillmomente kein Unterschied zwischen Torsionstheorie und Plattentheorie besteht.

Glad H, Svendsen P, Ainsworth MA, Olsen O, Rehfeld JF, Schaffalitzky de Muckadell OB. The effect of gastrin-releasing peptide on porcine pancreaticobiliary bicarbonate secretion is mediated by secretin. Scand J Gastroenterol 1994;29:195-202.The effect of gastrin-releasing peptide (GRP) (250, 500, 1000pmol/kg-h) on the pancreaticobiliary bicarbonate secretion, the pancreatic protein secretion, and the plasma concentrations of secretin and cholecystokinin (CCK) was studied in the anaesthetized pig. Infusion of GRP (1000 pmol/kg-h) increased the portal plasma concentrations of secretin from 0.9 to 13.6pmol/l and CCK from 1.2 to 38.4pmol/l, the pancreatic bicarbonate secretion from 0.01 to 5.6mmol/h, the hepatic bicarbonate secretion from 0.5 to 4.1mmol/h, and the pancreatic protein secretion from 3 to 680mg/h. Blocking of CCK-A receptors by MK-329 did not significantly change the effect of GRP, whereas prevention of secretin release by removal of the small intestine caused a 13-fold reduction in the GRP-induced pancreatic bicarbonate secretion and completely abolished the effect on hepatic bicarbonate secretion but did not change the effect on pancreatic protein secretion. We conclude that the effect of GRP on pancreaticobiliary bicarbonate secretion is not mediated through the release of CCK but more likely through the release of secretin and that the effect on pancreatic protein secretion is possibly a direct effect of GRP.

Ainsworth MA, Svendsen P, Glad H, Andersen NJ, Olsen O, Schaffalitzky de Muckadell OB. Scand J Gastroenterol 1994;29:889-896.Background: The purpose of the study was to examine the effect of stimulation and inhibition of duodenal mucosal bicarbonate secretion on pancreatic and hepatic bicarbonate secretion in response to acid. Methods: The effect of inhibition (indomethacin) or stimulation (misoprostol) of duodenal mucosal bicarbonate secretion on pancreatic and biliary bicarbonate secretion in response to intraduodenal infusion of HCl or intravenous infusion of secretin was studied in anaesthetized pigs. Results: The hepatic and pancreatic response to exogenous secretin was not significantly altered by stimulation/ inhibition of duodenal bicarbonate secretion. However, pancreatic and biliary bicarbonate secretion in response to duodenal acidification was significantly augmented by inhibition of duodenal mucosal bicarbonate secretion; conversely, it was reduced by stimulation of duodenal bicarbonate secretion. The increase in plasma secretin levels in response to duodenal acidification was reduced by stimulation and augmented by inhibition of duodenal mucosal bicarbonate secretion. Conclusions: Duodenal mucosal bicarbonate secretion can serve as a modulator of both pancreatic and biliary bicarbonate secretion in response to luminal acidification, possibly through regulation of the release of secretin.

In 20 normal persons we investigated the effects of duodenal osmolality on the release of secretin and cholecystokinin (CCK), pancreaticobiliary secretion, and fat absorption after intestinal infusion of emulsified oleic acid (pH 6.0). The release of CCK was found to be unaffected by the changes in osmolality, whereas the plasma levels of secretin were affected in parallel with volume and bicarbonate secretion. An inverse relation was found between fatty acid absorption and release of secretin and bicarbonate secretion but not between fatty acid absorption and release of CCK. It is suggested that the secretin and CCK cells respond differently to emulsified oleic acid.

Pancreatic and hepatic bicarbonate secretion and the disappearance rate of acid during duodenal acidification were measured simultaneously in anaesthetized pigs. Perfusion of the duodenum with HCI resulted in an increase in both hepatic and pancreatic bicarbonate secretion. During all acid loads hepatic bicarbonate secretion was significantly greater than pancreatic secretion. Furthermore, the disappearance rate of acid in the duodenum during diversion of both bile and pancreatic juice was significantly greater than the amount of acid which could be neutralized by the concomitant pancreatic bicarbonate secretion. Diversion of pancreatic juice from the duodenum did not affect the disappearance rate of acid at any acid load, whereas diversion of bile caused a significant decrease. Thus, in the anaesthetized pig the liver and the duodenal mucosa are of greater importance than the pancreas for the neutralization of acid in the duodenum. It is suggested that the importance of the pancreatic contribution to duodenal neutralization should be reevaluated in other species, including man.

The effect of infusion of secretin alone or in combination with cholecystokinin (CCK) on pancreatic, hepatic, and duodenal mucosal bicarbonate secretion was studied in anaesthetized pigs. After laparotomy, catheters were inserted into the common bile duct, the pancreatic duct, and both ends of the duodenum. Pancreatic, hepatic, and duodenal mucosal secretions were collected during intraportal infusion of increasing doses of secretin, either alone or in combination with CCK. During infusion of secretin in doses that caused physiologic increases in plasma secretin concentrations the liver produced significantly more bicarbonate than the pancreas. A physiologic dose of CCK augmented the effect of secretin on both hepatic and pancreatic bicarbonate secretion, but the hepatic production of bicarbonate was still larger than the pancreatic production. Neither secretin alone nor secretin combined with CCK caused any changes in duodenal mucosal bicarbonate secretion. These results suggest that the liver plays an important role in the neutralization of acid in the duodenum.

Coagulation factor VIIa belongs to a family of homologous enzymes, including factors IXa and Xa and activated protein C, composed of two epidermal growth factor-like domains located between an N-terminal domain rich in gamma-carboxyglutamic acid residues and a C-terminal serine protease domain. The first epidermal growth factor-like domain in factor VIIa contains a Ca2+ binding site, the function of which is largely unknown. Site-directed mutagenesis of two Ca2+-liganding Asp residues in this domain abolished Ca2+ binding and resulted in a 2-3-fold decrease in amidolytic activity at optimal Ca2+ concentrations. The lower amidolytic activity persisted in complex with soluble tissue factor, apparently due to a lower kcat of the mutant factor VIIa. Mutant and wild-type factor VIIa bound to lipidated tissue factor were equally efficient activators of factor X. The dissociation constants, derived from amidolytic activity and surface plasmon resonance measurements, were 2-5 nM and 50-60 nM for the interactions between wild-type and mutant factor VIIa, respectively, and soluble tissue factor. Binding to lipidated tissue factor was characterized by dissociation constants of 7.5 pM for factor VIIa and 160 pM for the factor VIIa mutant. Hence, a functional Ca2+ binding site in the first epidermal growth factor-like domain added 7-8 kJ/mol to the total binding energy of the interaction with both lipidated and soluble tissue factor.

The pathogenicity of the bacteriumErwinia carotovorasubsp.atroseptica, which causes potato soft rot, is triggered by short oligogalacturonates released by enzymic degradation of plant cell wall pectin. In the first stage unsaturated digalacturonate (uDG), produced by the action of pectate lyases, is degraded by oligogalacturonide lyase (OGL) to keto-deoxyuronate (DKI). The OGL encoding gene fromE. carotovoraand the corresponding recombinant enzyme were characterized. Measuring the changes in plant cell viability and tissue maceration during soft rot pathogenesis in tissue slices of sprouting potato tubers, it was observed that exposure to uDG and DKI, produced by recombinant OGL, killed up to 30% of the plant cells over a period of 16h. This protected the tissue against maceration byE. carotovorasubsp.atroseptica. Endogenous OGL activity was detected in extracts from sprouting tubers where it may be involved in the conversion of uDG into cell toxic compounds. The results indicate that an additional function of small, diffusable digalacturonates is to induce plant cell death during the rotting process, thus contributing to defence reactions againstE. carotovorasubsp.atroseptica.

To study the effect of emulsified oleic acid on pancreatic secretion and concentrations of secretin and cholecystokinin (CCK) in plasma, eight normal subjects received three sets of duodenal perfusates containing peptone pH 6.0 or pH 2.7, with and without 20 mM oleic acid. Pancreatic secretion was measured by an indicator dilution technique. At pH 6.0, peptone and oleic acid was about as effective as peptone pH 2.7 in stimulating secretin release. However, oleic acid in addition produced a three-fold increase in plasma CCK and was five times as effective as pH 2.7 in stimulating duodenal flow. Also, at pH 2.7, oleic acid augmented pancreatic secretion and concentrations of CCK and secretin in plasma. Duodenal output of amylase and bile salts was independent of the pH of infusate. Low pH alone was a very weak stimulant of CCK release and did not stimulate output of amylase and bile. Emulsified oleic acid is a potent releaser of secretin and CCK and augments the acid-induced pancreatic secretion.

The rate of gene transcription in endosperm nuclei up to the formation of the first cell layers was investigated by pulse-labelling young fertilized barley (Hordeum vulgare L.) ovules with [3H]uridine. Quantitative autoradiographic studies of silver grains accumulating over the nuclei of wild-type endosperm demonstrated that the rate of transcription increased sixfold in the period from 3 to 7 d after pollination (DAP). Based on this observation, and the fact that cell-wall formation is initiated at 6 DAP, it is concluded that at least a proportion of the transcripts encode proteins involved in cell-wall formation. A similar study was also undertaken with the two barley sex mutants B7 and B15, in which developmental arrest at the syncytical stage leads to a complete lack of endosperm cell walls. This study showed that [3H]uridine is incorporated into the nuclei of the mutant syncytia, although at a rate different from that in the wild-type. Thus, the lack of cell-wall formation is not caused by a total block of gene transcription in these mutants, but rather by the lack of a gene product essential for cell-wall formation in the endosperm.

Erwinia carotovorabacteria cause blackleg and soft-rot of potatoes by enzymic maceration of host tissue. Pectate lyases (PLs), the mainE. carotovoraenzymes, depolymerize pectic substances of plant cell walls. The degradation products, unsaturated oligogalacturonates, have been proposed to induce plant defence reactions. The expression of the isoenzyme PL3in plantaand its effect on the soft-rot resistance of transgenic potatoes was investigated. The DNA sequence encoding the mature enzyme was fused to the promoter of the potato patatin B33 gene and to the 35S promoter of the cauliflower mosaic virus. The expression cassettes were inserted into the binary vector pBin19. The plasmids pB33-PL3 and p35S-PL3 were used forAgrobacterium-mediated transformation of potato, cv. Dèsirèe. Enzyme production in plant lines transformed with plasmid pB33-PL3 was confined to tuber tissue, yielding a maximum of 340 mU ml−1extract. Plants harbouring the 35S-PL3 chimeric gene exhibited constitutive expression of PL in various tissues, with activities up to 280 mU ml−1in leaf extracts and 50 mU ml−1in tuber tissue. After wounding the tuber tissue, the liberated PL3 caused a decrease of cell viability. Compared with non-transformed plants, the transgenic lines which synthesized PL3 were more resistant to tissue maceration byE. carotovoraor its enzymes. Wounding induced the transcription of the plant defence related gene encoding phenylalanine ammonia-lyase to a high level in tubers of a PL3-expressing transgenic line. The possible implications of PLs and their enzymatic products for protection against invading microbes are discussed.

The molecular basis for the absence of anthocyanins and proanthocyanidins in four independent sodium azide-induced ant18 mutants of barley was examined by sequencing the gene encoding dihydroflavonol 4-reductase in these mutants. Sodium azide generated 21 base substitutions, which corresponds to 0.17% of the 12,704 nucleotides sequenced. Of the substitutions, 86% were nucleotide transitions, and 14% were transversions. A.T-->G.C base pair transitions were about 3 times more frequent than G.C-->A.T transitions. No deletions or mutation hot spots were found. The absence of dihydroflavonol 4-reductase activity in ant18-159, ant18-162, and ant18-164 plants is caused by missense mutations in the respective genes. By using microprojectile bombardment, a plasmid harboring the wild-type Ant18 gene was introduced into ant18-161 mutant cells and resulted in the development of anthocyanin pigmentation, which demonstrates that the mutation is corrected by expression of the introduced gene. On the other hand, a plasmid derivative with the two ant18-161-specific base transitions at the 5' splice site of intron 3 prevented complementation. It is concluded that the absence of detectable mRNA for dihydroflavonol 4-reductase in ant18-161 cells is due to the mutations in the pre-mRNA splice donor site.

In order to study the effect of welding fumes on lung function, 74 high-exposed welders and 31 age-matched electricians were examined in 1982. None had ever smoked tobacco or been exposed to known or potentially noxious agents to the lungs. A significant difference was found between the welders and the control group in vital capacity, total lung capacity, forced expiratory volume in one second, peak expiratory flow rate, maximal expiratory flow rate at 75% of vital capacity, diffusion capacity and slope of the alveolar plateau. The lungs of the welders were physiologically 10–15 years older than those of the control group. Thirty percent of the welders had a well defined respiratory disease. Their lung function impairment was predominantly obstructive (16 persons), but restrictive patterns were also seen (6 persons).

Summary The endosperm of cereal grains develops as a multinucleate mass of wall-less cytoplasm (syncytium) that lines the periphery of the central cell before becoming cellular. The pattern of initial wall formation is precisely oriented and is followed by a round of precisely oriented formative cell division that gives rise to initials for the two tissues of endosperm. The initial anticlinal walls form at boundaries of nuclear-cytoplasmic domains (NCDs) defined by radial microtubules emanating from nuclei in the syncytium. Polarized growth of the NCDs in axes perpendicular to the embryo sac wall and centripetal elongation of the anticlinal walls results in a single layer of open ended alveoli overtopped by the remaining syncytial cytoplasm. This arboreal stage, so named because the elongate nucleate columns of cytoplasm resemble an orchard of trees, predicts the division polarity of the imminent formative division. Mitosis occurs as a wave which, like polarization, moves in both directions from ventral to dorsal. Spindles are oriented parallel to the long axis of the alveoli and cell plates give rise to periclinal walls. The outer daughter nuclei (aleurone initials) are thus completely enclosed by walls and the inner nuclei (starchy endosperm initials) are in alveoli adjacent to the central vacuole.

The aim was to estimate the quantitative impact of working conditions on cardiovascular diseases in Denmark.

To estimate quantitatively (the aetiological fraction) the impact of poor social network on premature death from cardiovascular disease in middle aged, white men.

This paper reviews recent data from our laboratories on the early phases of endosperm development in barley. First, we provide a description of endosperm morphogenesis based on immunohistochemical studies using the confocal laser scanning microscope. In the second part, the barley Ltp2 gene, which is exclusively expressed in the aleurone layer of developing grains, is characterized. In the last part, we describe the barley gene encoding the small subunits of the AGPase in the starchy endosperm and leaf tissue.

Renal reabsorption of bicarbonate was examined in Merino ewes. In all experiments the bicarbonate reabsorption was directly related to that of sodium. When the rate of bicarbonate reabsorption was expressed as the ratio of bicarbonate over sodium reabsorption, no tubular maximum for bicarbonate could be demonstrated. Arterial pH or $$P_{CO_2 } $$ did not affect the reabsorption of bicarbonate except through alterations in the plasma bicarbonate concentration. The ratio of bicarbonate over sodium reabsorption was directly proportional to the plasma concentration of bicarbonate over a wide range. This evidence suggests that the ionic reabsorption of bicarbonate constitutes a major part of total bicarbonate reabsorption. It does not, however, exclude a contribution from an active hydrogen ion secretory process, as the kinetic properteis of such a process are as yet undetermined.

MK-329 (formerly L-364,718) is a new nonpeptide antagonist for the peripheral (type-A) Cholecystokinin (CCK) receptor, which has proved effective in blocking the actions of both exogenous and endogenous CCK in several species. To evaluate the effect of MK-329 on CCK-stimulated pancreaticobiliary output in man, six normal subjects received 10 mg MK-329 or placebo orally in a randomized, crossover fashion, before a background intravenous infusion of secretin (5 pmol/kg/h) and two doses of CCK-8 (approximately 15 and 40 pmol/kg/h, each for 1 h). Gastric and duodenal juice were aspirated separately via two double-lumen tubes, with 51Cr-ethylenediaminetetraacetic acid as a duodenal marker. After placebo treatment the background infusion of secretin produced maximum plasma concentrations of secretin similar to postprandial values, averaging about 5pM. After placebo treatment the low dose CCK-8 infusion (15 pmol/kg/h) increased circulating CCK concentrations from basal levels of 1.8 ± 0.2 pM to levels similar to those observed postprandially, averaging 9.2 ± 1.3pM, and the high dose of CCK-8 (40 pmol/kg/h) induced supraphysiologic levels of CCK, averaging 23.4 ± 3.2 pM. Plasma concentrations of secretin and CCK were not significantly different during MK-329 treatment. As expected, infusion of CCK-8 at both doses stimulated pancreatic exocrine secretion and gallbladder contraction in placebo controls, as indicated by increases in the output of trypsin, amylase, bicarbonate, and bilirubin. Whereas MK-329 did not significantly reduce basal pancreatic secretion, the integrated incremental output of trypsin, amylase, and bicarbonate in response to stimulation with the low (physiologic) CCK dose was inhibited by 74% (p < 0.01), 89% (NS), and 75% (p < 0.05), respectively. Basal bilirubin output was virtually abolished after treatment with MK-329, and the response to the low dose of CCK was reduced by 98% (p < 0.01), indicating almost complete inhibition of gallbladder contraction at physiologic circulating concentrations of CCK. It is concluded that MK-329 is an orally active antagonist of CCK-stimulated pancreaticobiliary output in man and could thus be utilized to explore the physiologic regulation of the exocrine pancreas and gallbladder by CCK.