Symptomatic gastro-oesophageal reflux disease: double blind controlled study of intermittent treatment with omeprazole or ranitidine. The European Study Group

ObjectiveTo assess intermittent treatment over 12 months in patients with symptomatic gastro-oesophageal reflux disease.DesignRandomised, multicentre, double blind, controlled study. Patients with heartburn and normal endoscopy results or mild erosive changes received omeprazole 10 mg or 20 mg daily or ranitidine 150 mg twice daily for 2 weeks. Patients remaining symptomatic had omeprazole 10 mg or ranitidine dose doubled for another 2 weeks while omeprazole 20 mg was continued for 2 weeks. Patients who were symptomatic or mildly symptomatic were followed up for 12 months. Recurrences of moderate or severe heartburn during follow up were treated with the dose which was successful for initial symptom control.SettingHospitals and primary care practices between 1994 and 1996.Subjects677 patients with gastro-oesophageal reflux disease.Results704 patients were randomised, 677 were eligible for analyses; 318 reached the end of the study with intermittent treatment without recourse to maintenance antisecretory drugs. The median number of days off active treatment during follow up was 142 for the entire study (281 for the 526 patients who reached a treatment related end point). Thus, about half the patients did not require treatment for at least 6 months, and this was similar in all three treatment groups. According to outcome, 378 (72%) patients were in the best outcome ranks (no relapse or one (or more) relapse but in remission until 12 months); 630 (93%) had three or fewer relapses in the intermittent treatment phase. Omeprazole 20 mg provided faster relief of heartburn. The results were similar in patients with erosive and non-erosive disease.ConclusionsIntermittent treatment is effective in managing symptoms of heartburn in half of patients with uncomplicated gastro-oesophageal reflux disease. It is simple and applicable in general practice, where most patients are seen.

Key messages

• Symptomatic gastro-oesophageal disease can be managed successfully in half of patients with intermittent treatment with antisecretory drugs
• Omeprazole 20 mg once daily gives more rapid relief of symptoms than either omeprazole 10 mg once daily or ranitidine 150 mg twice daily. However, the choice of antisecretory drug has little effect on the overall outcome
• Relapses are relatively infrequent and can be managed with short courses of repeat treatment
• Starting intermittent treatment with omeprazole 20 mg once daily is more cost effective than a dose titration approach with omeprazole 10 mg once daily or ranitidine 150 mg twice daily
• An intermittent treatment strategy is simple and applicable in general practice, where most of these patients are seen

     

Effect of temperature on the saccharide composition obtained after alpha-amylolysis of starch

The hydrolysis of starch to low-molecular-weight products (normally characterised by their dextrose equivalent (DE), which is directly related to the number-average molecular mass) was studied at different temperatures. Amylopectin potato starch, lacking amylose, was selected because of its low tendency towards retrogradation at lower temperatures. Bacillus licheniformis alpha-amylase was added to 10% [w/w] gelatinised starch solutions. The hydrolysis experiments were done at 50, 70, and 90 degrees C. Samples were taken at defined DE values and these were analysed with respect to their saccharide composition. At the same DE the oligosaccharide composition depended on the hydrolysis temperature. This implies that at the same net number of bonds hydrolysed by the enzyme, the saccharide composition was different. The hydrolysis temperature also influenced the initial overall molecular-weight distribution. Higher temperatures led to a more homogenous molecular weight distribution. Similar effects were observed for alpha-amylases from other microbial sources such as Bacillus amyloliquefaciens and Bacillus stearothermophilus. Varying the pH (5.1, 6.2, and 7.6) at 70 degrees C did not significantly influence the saccharide composition obtained during B. licheniformis alpha-amylase hydrolysis. The underlying mechanisms for B. licheniformis alpha-amylase were studied using pure linear oligosaccharides, ranging from maltotriose to maltoheptaose as substrates. Activation energies for the hydrolysis of individual oligosaccharides were calculated from Arrhenius plots at 60, 70, 80, and 90 degrees C. Oligosaccharides with a degree of polymerisation exceeding that of the substrate could be detected. The contribution of these oligosaccharides increased as the degree of polymerisation of the substrate decreased and the temperature of hydrolysis increased. The product specificity decreased with increasing temperature of hydrolysis, which led to a more equal distribution between the possible products formed. Calculations with the subsite map as determined for the closely related alpha-amylase from B. amyloliquefaciens reconfirmed this finding of a decreased substrate specificity with increased temperature of hydrolysis. Copyright 1999 John Wiley & Sons, Inc.     

Phosphorylation of the kinase suppressor of ras by associated kinases

The kinase suppressor of Ras (KSR) is a loss-of-function allele that suppresses the rough eye phenotype of activated Ras in Drosophila and the multivulval phenotype of activated Ras in Caenorhabditis elegans. The physiological role of mammalian KSR is not known. We examined the mechanisms regulating the phosphorylation of this putative kinase in mammalian cells. Wild-type mouse KSR and a mutated KSR protein predicted to create a kinase-dead protein are phosphorylated identically in intact cells and in the immune complex. Phosphopeptide sequencing identified 10 in vivo phosphorylation sites in KSR, all of which reside in the 539 noncatalytic amino terminal amino acids. Expression of the amino terminal portion of KSR alone demonstrated that it was phosphorylated in the intact cell and in an immune complex in a manner indistinguishable from that of intact KSR. These data demonstrate that the kinase domain of KSR is irrelevant to its phosphorylation state and suggest that the phosphorylation of KSR and its association with a distinct set of kinases may affect intracellular signaling.     

Aromatase and COX-2 expression in human breast cancers

We have investigated aromatase and the inducible cyclooxygenase COX-2 expression using immunocytochemistry in tumors of a series of patients with advanced breast cancer treated with aromatase inhibitors. Aromatase was expressed in 58/102 breast cancers. This is similar to the percentage previously reported for aromatase activity. Interestingly, aromatase was expressed in a variety of cell types, including tumor, stromal, adipose, and endothelial cells. Since prostaglandin E₂ is known to regulate aromatase gene expression and is the product of COX-2, an enzyme frequently overexpressed in tumors, immunocytochemistry was performed on the tissue sections using a polyclonal antibody to COX-2. Aromatase was strongly correlated (P<0.001) with COX-2 expression. These results suggest that PGE₂ produced by COX-2 in the tumor may be important in stimulating estrogen synthesis in the tumor and surrounding tissue. No correlation was observed between aromatase or COX-2 expression and the response of the patients to aromatase inhibitor treatment. However, only 13 patients responded. Nine of these patients were aromatase positive. Although similar to responses in other studies, this low response rate to second line treatment suggests that tumors of most patients were no longer sensitive to the effects of estrogen. Recent clinical studies suggest that greater responses occur when aromatase inhibitors are used as first line treatment. In the intratumoral aromatase mouse model, expression of aromatase in tumors is highly correlated with increased tumor growth. First line treatment with letrozole was effective in all animals treated and was more effective than tamoxifen in suppressing tumor growth. Letrozole was also effective in tumors failing to respond to tamoxifen, consistent with clinical findings. In addition, the duration of response was significantly longer with the aromatase inhibitor than with tamoxifen, suggesting that aromatase inhibitors may offer better control of tumor growth than this antiestrogen.     

Bispecific and bifunctional single chain recombinant antibodies

Bispecific and bifunctional monoclonal antibodies as second generation monoclonals, produced by conventional chemical or somatic methods, have proved useful in the immunodiagnosis and immunotherapy of cancer and other diseases. Recombinant antibodies produced by genetic engineering techniques have also become available for use in preclinical and clinical studies. Furthermore, through genetic engineering, it is possible to remove or add on key protein domains in order to create designer antibody molecules with two or more desired functions. This review summarizes the strategies for development of single chain variable fragment (scFv) bifunctional and bispecific antibodies. The advantages and disadvantages as well as the problems of generating the various bispecific and bifunctional antibody constructs are reported and discussed. Since conventionally prepared bispecific and bifunctional monoclonal antibodies have already shown promise in clinical trials and results from preclinical studies of recombinant bispecific antibodies are encouraging, clinical trials in humans of recombinant bispecific and bifunctional antibodies, as a new generation of biologicals, are likely to be the thrust in the next decade and beyond.     

Phosphorylation of eNOS initiates excessive NO production in early phases of portal hypertension

Akt, also known as protein kinase B, is a serine/threonine kinase. Akt becomes active when phosphorylated by the activation of receptor tyrosine kinases, G protein-coupled receptors, and mechanical forces such as shear stress. Studies in vitro have shown that Akt can directly phosphorylate endothelial nitric oxide (NO) synthase (eNOS) and activate the enzyme, leading to NO production. The aim of this study was to test the hypothesis that the phosphorylation of eNOS plays a role in the enhanced NO production observed in early portal hypertension. Male Sprague-Dawley rats were subjected to either sham or portal vein ligation (PVL), and mesenteric arterial beds were used for ex vivo perfusion studies. Mesenteric arterial beds from PVL rats had an approximately 60-70% decrease in response to methoxamine (an alpha(1)-agonist and vasoconstrictor) compared with the sham group (P < 0.01). When N(G)-monomethyl-L-arginine (a NOS inhibitor) was added to the perfusion, the difference in perfusion pressure between the two groups was abolished, suggesting that enhanced NO production in the PVL group blunted the response to the vasoconstrictor. The reduced responsiveness in PVL was not due to changes in eNOS expression but was due to an increase in enzyme-specific activity, suggesting posttranslational modification of eNOS. The phosphorylation of eNOS at Ser(1176) was significantly increased by twofold (P < 0.05) in the PVL group. Furthermore, PVL significantly increased Akt phosphorylation (an active form of Akt) by threefold (P < 0.05). When vessels were treated with wortmannin (10 nM) to block the phosphatidylinositol-3-OH-kinase/Akt pathway, NO-induced vasodilatation was significantly reduced. These results suggest that the phosphorylation of eNOS by Akt activates the enzyme and may be the first step leading to an initial increase in NO production in portal hypertension.     

Genetics of floral traits influencing reproductive isolation between Aquilegia formosa and Aquilegia pubescens

Abstract: Reproductive isolation between Aquilegia formosa and Aquilegia pubescens is influenced by differences in their flowers through their effects on pollinator visitation and pollen transfer. Here, we investigate the genetic basis of floral characters differentiating these species. We found that in addition to the effects of flower orientation and the length of nectar spurs previously described, other characters such as flower color or odor affect hawkmoth visitation. Repeatability of measurements in an F2 population ranged from 0.53 to 0.83 among five floral traits, indicating that using the means of multiple measures per plant will substantially increase the power of a quantitative trait locus (QTL) analysis. Integration of floral traits was indicated by significant correlations among traits in an F2 population. In a separate F2 population we found that QTL for different floral traits were often closely associated, indicating that linkage or pleiotropy cause at least some of this integration. In addition, we found QTL for all floral traits examined. Because Aquilegia species are largely interfertile and vary extensively in both floral morphology and ecology, they offer the opportunity for QTL studies of a wide range of characters affecting reproductive isolation.     

Identification,analysis, and utilization of conserved ortholog set markers for comparative genomics in higher plants

We have screened a large tomato EST database against the Arabidopsis genomic sequence and report here the identification of a set of 1025 genes (referred to as a conserved ortholog set, or COS markers) that are single or low copy in both genomes (as determined by computational screens and DNA gel blot hybridization) and that have remained relatively stable in sequence since the early radiation of dicotyledonous plants. These genes were annotated, and a large portion could be assigned to putative functional categories associated with basic metabolic processes, such as energy-generating processes and the biosynthesis and degradation of cellular building blocks. We further demonstrate, through computational screens (e.g., against a Medicago truncatula database) and direct hybridization on genomic DNA of diverse plant species, that these COS markers also are conserved in the genomes of other plant families. Finally, we show that this gene set can be used for comparative mapping studies between highly divergent genomes such as those of tomato and Arabidopsis. This set of COS markers, identified computationally and experimentally, may further studies on comparative genomes and phylogenetics and elucidate the nature of genes conserved throughout plant evolution.