Structural relatedness of lysis proteins from colicinogenic plasmids and icosahedral coliphages

The host-lysis-inducing functions of phi X174 protein E and MS2 protein L were recently shown to reside on the N-terminal and C-terminal halves of the two respective lysis proteins. In the present study it is shown that the small lysis proteins encoded in various colicinogenic plasmids share local sequence similarities and certain structural characteristics with the essential peptides of their coliphage-coded counterparts. Despite their dissimilar sizes and origins, it is suggested that the colicinogenic lysis proteins are functionally analogous and evolutionarily related to those of icosahedral single- stranded DNA and RNA phages.      

Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Effects of growth hormone releasing factor on pancreatic secretion in vivo and in vitro

Growth hormone releasing factor (GRF), a 44-residue peptide originally isolated from human pancreatic tumors, shows structural similarities to the members of the secretin-vasoactive intestinal peptide (VIP) peptides. This study was designed to determine the effects of human GRF (hGRF-(1-44] on pancreatic secretion in vivo in conscious dogs and in vitro in dispersed rat pancreatic acini. GRF given i.v. in graded doses in dogs caused a small but significant stimulation of pancreatic HCO3- and protein outputs and potentiated secretin- and cholecystokinin (CCK)-induced pancreatic HCO3- but not protein secretion. When given together with somatostatin, GRF failed to reverse the inhibitory action of this peptide on HCO3- and protein responses to secretin plus CCK in dogs. Studies in vitro dispersed rat pancreatic acini showed that GRF added to the incubation medium of these acini caused an increase in basal amylase release and shifted to the left the amylase dose-response curve to caerulein and urecholine but failed to affect the amylase response to VIP. This study indicates that GRF in vivo stimulates basal and augments secretin- or CCK-induced pancreatic HCO3- secretion and that this is probably due to direct stimulatory action of the peptide on pancreatic secretory cells.     

Comparison of helodermin, VIP and PHI in pancreatic secretion and blood flow in dogs

Helodermin, VIP and PHI, which share a high degree of homology with secretin, have been identified in the gut but their physiological role is unknown. In this study 3 series of tests were carried out to determine the actions of helodermin, VIP and PHI on pancreatic secretion in 6 conscious dogs and amylase release from the dispersed canine pancreatic acini and to correlate the alterations in pancreatic secretory and circulatory effects in 24 anesthetized dogs. Helodermin, VIP and PHI infused i.v. in graded doses (12.5-200 pmol/kg.h) resulted in a dose-dependent increase in pancreatic HCO3 secretion reaching, respectively, 100%, 7% and 2% of secretin maximum. When combined with constant dose infusion of CCK-8 (100 pmol/kg.h), helodermin but not VIP or PHI augmented dose-dependently the HCO3 secretion. When added in various concentrations (10(-10)-10(-5)M) to the incubation medium of dispersed pancreatic acini only helodermin but not VIP or PHI increased dose-dependently amylase release reaching about 50% of CCK-8 maximum. In anesthetized dogs, the pancreatic blood flow (PBF) measured by electromagnetic blood flowmetry showed an immediate and dose-dependent increase following the injections of various doses of helodermin, VIP, PHI and secretin, the peak blood flow preceding by about 1 min the peak secretory stimulation. This study shows that helodermin resembles secretin in its potent pancreatic HCO3 stimulation but differs from VIP or PHI which are poor secretagogues but potent vasodilators. We conclude that if tested peptides are released in the gut, helodermin, like secretin, may be involved in the hormonal stimulation of exocrine pancreas, whereas VIP and PHI may serve mainly as vasodilators in the pancreatic circulation.     

Comparison of organ uptake and disappearance half-time of human epidermal growth factor and insulin

Epidermal growth factor (EGF), which was originally identified in salivary glands and saliva, has been also found in the kidney and urine, suggesting that the kidney may be an alternate source of this peptide. Liver was considered as the major site of the degradation of EGF but the involvement of other organs has been little studied. Therefore, we carried out comparative studies on the organ uptake and the disappearance half-time of EGF and insulin (having similar molecular size) in the same model of anesthetized dog with arterial (from aorta) and venous (from mesenteric, portal, hepatic, renal, femoral and jugular veins) blood sampling from various organs. Basal plasma level of EGF (1.32 +/- 0.33 pmol/l) and insulin (62.1 +/- 13.8 pmol/l) in the aorta was not significantly different from that recorded at various sampling sites. During i.v. infusion of EGF at 41.6 and 166.6 pmol/kg/h, the respective arterial EGF concentrations averaged 103 +/- 21 and 240 +/- 49 pmol/kg/h and the percent reduction in plasma EGF after passage through the head, leg, intestines and liver was about 30-50% and that after passage through the kidney was about 95%. During insulin (6.9 pmol/kg/h) infusion, the arterial hormone level averaged 227 +/- 21 pmol/l and this level was significantly reduced (by 23-42%) after passage through the head, leg, intestine, liver and kidney but no significant difference was found between various venous sampling sites. EGF and insulin appearing in the urine during EGF or insulin infusion accounted for about 40 and 7% of the difference between the entering and leaving renal masses of the peptide. Mean disappearance half time on stopping of EGF and insulin infusion was, respectively, 2.32 +/- 0.58 and 6.88 +/- 1.25 min. We conclude that unlike insulin, which is removed to similar extent by various organs including the kidney and the liver, EGF is taken up mainly by kidney and EGF present in urine originates mainly from renal clearance of peptide.     

Extract of grapefruit-seed reduces acute pancreatitis induced by ischemia/reperfusion in rats: possible implication of tissue antioxidants.

Grapefruit seed extract (GSE) has been shown to exert antibacterial, antifungal and antioxidant activity possibly due to the presence of naringenin, the flavonoid with cytoprotective action on the gastric mucosa. No study so far has been undertaken to determine whether this GSE is also capable of preventing acute pancreatic damage induced by ischemia/reperfusion (I/R), which is known to result from reduction of anti-oxidative capability of pancreatic tissue, and whether its possible preventive effect involves an antioxidative action of this biocomponent. In this study carried out on rats with acute hemorrhagic pancreatitis induced by 30 min partial pancreatic ischemia followed by 6 h of reperfusion, the GSE or vehicle (vegetable glycerin) was applied intragastrically in gradually increasing amounts (50-500 microl) 30 min before I/R. Pretreatment with GSE decreased the extent of pancreatitis with maximal protective effect of GSE at the dose 250 microl. GSE reduced the pancreatitis-evoked increase in serum lipase and poly-C specific ribonuclease activity, and attenuated the marked fall in pancreatic blood flow and pancreatic DNA synthesis. GSE administered alone increased significantly pancreatic tissue content of lipid peroxidation products, malondialdehyde and 4-hydroxyalkens, and when administered before I/R, GSE reduced the pancreatitis-induced lipid peroxidation. We conclude that GSE exerts protective activity against I/R-induced pancreatitis probably due to the activation of antioxidative mechanisms in the pancreas and the improvement of pancreatic blood flow.     

Applications of DNA shuffling to pharmaceuticals and vaccines

DNA shuffling is a practical process for directed molecular evolution which uses recombination to dramatically accelerate the rate at which one can evolve genes. Single and multigene traits that require many mutations for improved phenotypes can be evolved rapidly. DNA shuffling technology has been significantly enhanced in the past year, extending its range of applications to small molecule pharmaceuticals, pharmaceutical proteins, gene therapy vehicles and transgenes, vaccines and evolved viruses for vaccines, and laboratory animal models.     

Comparison of gastric and intestinal antisecretory and protective effects of prostacyclin and its stable thia-amino-analogue (HOE 892) in consious rats

The effects of prostacyclin (PGI₂) and its stable thia-thimo-analogue (Hoe 892) on gastric and intestinal secretions and gastric mucosal lesions have been determined in conscious rats. Both PGI₂ and Hoe 892 given subcutaneously (s.c.) reduced dose-dependent gastric acid secretion, the ID₅₀ (dose producing 50% inhibition) being about 48.6 and 11.8 gmg/kg, respectively. In contrast, intragastric (i.g.) PGI₂ and Hoe 892 did not cause any change in gastric acid secretion at doses ranging from 1 to 100 gmg/kg. Both PGI₂ and Hoe 892 reduced significantly intestinal fluid secretion (antienteropooling activity). PGI₂ and Hoe 892 given i.g. or s.c. reduced dose-dependent gastric ulcer formation induced by acidified aspirin (ASA), Hoe 892 being somewhat less potent than PGI₂. Both PGI₂ and Hoe 892 were equally effective against mucosal necrosis induced by absolute ethanol and this effect was observed both after i.g. and s.c. administration of these agents. We conclude that stable thia-amino-PGI₂ analogue, Hoe 892, has similar gastric and intestinal antisecretory and protective activity as PGI₂ and may be useful in the prevention of gastric damage by various noxious agents.     

The effects of antagonists of receptors for gastrin, cholecystokinin and bombesin on growth of gastroduodenal mucosa and pancreas.

The effects of gastrin, cholecystokinin (CCK) and bombesin on the DNA synthesis, as a biochemical indicator of trophic action in the gastroduodenal mucosa and the pancreas have been examined in rats fasted for 48 h and in rats refed for 16 h with or without administration of specific receptor antagonists for bombesin, gastrin and CCK. Bombesin and gastrin administered three times daily for 48 h in fasted rats significantly increased the rate of DNA synthesis as measured by the incorporation of [3H] thymidine into DNA in each tissue tested. CCK significantly increased DNA synthesis in the duodenal mucosa and pancreatic tissue, but not in the gastric mucosa. The stimulation of DNA synthesis induced by bombesin in the gastroduodenal mucosa and pancreas was abolished by bombesin/GRP receptor antagonist, RC-3095. RC-3095 did not affect DNA synthesis stimulated by gastrin and CCK in these tissues. L-365,260, a receptor antagonist for gastrin suppressed the DNA synthesis induced by gastrin but not by CCK or bombesin in the gastrointestinal mucosa and pancreas. L-364,718 a specific antagonist for CCK receptors was effective only against CCK stimulated duodenal mucosa and pancreatic growth. Refeeding of 48 h fasting rats strongly enhanced the DNA synthesis in all tissues tested, and this effect was significantly reduced in the gastroduodenal mucosa by blocking only gastrin receptors (with L-365,260) and that in the duodenal mucosa and the pancreas by antagonizing of CCK receptors (with L-364,718). Antagonism of bombesin receptors (with RC-3095) did not significantly affect the stimulation of DNA synthesis induced by refeeding in all tissues tested. This study indicates that the stimulation of DNA synthesis can be achieved by exogenous gastrin, CCK and bombesin acting through separate receptor but that only gastrin and CCK play the major role in the postprandial stimulation of the growth of gastroduodenal mucosa and pancreatic tissue.