myo-Inositol phosphorothioates, phosphatase-resistant analogues of myo-inositol phosphates. Synthesis of DL-myo-inositol 1,4-bisphosphate and DL-myo-inositol 1,4-bisphosphorothioate.

Syntheses of a metabolite of the second messenger myo-inositol 1,4,5-trisphosphate, myo-inositol 1,4-bisphosphate, and an analogue, the 1,4-bisphosphorothioate, are reported, by using phosphite chemistry on (+/-)-1,2:4,5-di-isopropylidene-myo-inositol. The synthesis of (+/-)-1,2:4,5-di-isopropylidene 3,6-bis[di-(2-cyanoethyl)]phosphite provides an intermediate that can be oxidized to either the corresponding bisphosphate or bisphosphorothioate. myo-Inositol phosphorothioates are proposed as novel analogues of myo-inositol phosphates; their resistance to phosphatase-catalysed breakdown is reported.     

Water Balance in Cucumis Plants, Measured by Nuclear Magnetic Resonance, I

In this paper we demonstrate the study of plant water balanceby the non-invasive measurement of tissue water content andwater flow using proton nuclear magnetic resonance (NMR). Sapvelocity and flux were measured independently in the presenceof an excess of stationary tissue water. The instrumentationdescribed allows automated and unattended measurement of flow-and water content-variables in a well-defined region of theplant over periods of several days, with a time resolution betweensuccessive measurements of c. 5 s. Using this apparatus theeffect of changes in light intensity (day/night rhythm) andrelative humidity on stem tissue water content as well as onthe velocity and flux of xylem sap in the stem were investigatedin a cucumber plant. The results are in agreement with predictionsfrom a simple model for plant water balance, which is basedon water potential, flow rate and resistance to flow. As longas only transpiration is varied, flow rate and water content(or potential) are affected in opposite ways as demonstratedin this paper. In contrast, the model predicts that changesin uptake (resulting from changes in, for example, root resistance)will induce changes in water content and flow in the same direction.An experimental verification of this prediction is given ina subsequent paper, where, in addition, the NMR results arecompared to those obtained with a dendrometer. Key words: Water balance model, Cucumis sativus L., flow, water content, NMR, water balance measurement     

Behavioral and radioligand binding evidence for irreversible dopamine receptor blockade by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline

N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), an irreversible alpha adrenergic antagonist, also acts as a potent and longlasting in vivo antagonist of D-2 dopamine receptors. Rats given EEDQ 3-10 mg/kg i.p. exhibit catalepsy and greatly reduced apomorphine-induced stereotypy, behavioral effects associated with D-2 dopamine receptor blockade. These effects are apparent up to 4 days after drug administration, with scores returning to control level by day 7. In vitro receptor binding assays of striatal membrane preparations from these animals using the radioligand 3H-spiroperidol directly demonstrate that EEDQ is a potent D-2 dopamine receptor antagonist, revealing the apparent basis of the behavioral effects of EEDQ. This antagonism proceeds via a reduction in D-2 receptor Bmax, with no change in the observed KD for 3H-spiroperidol, and is resistant to extensive washing of the membrane preparation after in vivo EEDQ exposure. These observations suggest that EEDQ inhibition of D-2 receptors is irreversible. Administration of behaviorally active doses of EEDQ effect a reduction of 50-85% in D-2 receptor number. Recovery of this loss roughly parallels recovery of normal catalepsy and apomorphine stereotypy scores. These doses of EEDQ also reduce binding of 3H-flupentixol to D-1 and 3H-dopamine to D-3 type dopaminergic binding sites, putative dopamine receptors with no known behavioral correlates. Recovery of D-1 and D-3 binding also occurs with a similar timecourse. Because of the apparent covalent nature of its interaction with dopamine receptors and because of its activity after peripheral administration, EEDQ may prove useful in the study of the function and turnover of dopamine receptors.     

Eubacterial isocitrate dehydrogenase with dual specificity for NAD and NADP from Rhodomicrobium vannielii

Abstract Cell-free extracts of the photosynthetic eubacterium Rhodomicrobium vannielii contained both NADP and NAD-linked isocitrate dehydrogenase activities. Apparent K _m values of 12 μM for NADP, 0.75 mM for NAD, 9.3 μM for isocitrate (NADP utilising) and 8.2 μM for isocitrate (NAD utilising) were determined in such extracts. Four lines of evidence indicated that one enzyme was responsible for the two activities; (i) non-additivity of reaction rates in the presence of both NADP and NAD (ii) the presence of one band which stained for activity with both cofactors on non-denaturing polyacrylamide gels (iii) identical heat inactivation kinetics for both activities (iv) co-elution of both activities after ion-exchange and hydrophobic interaction chromatography. This is the first report of a eubacterial isocitrate dehydrogenase with dual cofactor specificity.     

TASTE GROUP THRESHOLDS AND SENSORY EVALUATION OF SPANISH WINE VINEGARS

Wine vinegar is a product obtained from wine acidification which contains at least 5% by wt. of acetic acid, in general without any additives or colorings.
Aspects studied in this work include: the determination of the taste group thresholds (geometric mean of the individual best-estimate thresholds "BET") of two different acids (citric and acetic acids) in aqueous solution and spanish vinegars produced from table and sherry wines. The results obtained suggest that wine vinegar can be considered something more than just an acidulant agent.
In order to evaluate differences among wine vinegars, discriminant tests for twenty-five spanish vinegars (sherry, table and flavored vinegars) were applied. Six of the twelve attributes freely chosen by assessors allowed grouping of the spanish wine vinegars according to their sensory aspects.     

Antitumor response to recombinant murine interferon γ correlates with enhanced immune function of organ-associated,but not recirculating cytolytic T lymphocytes and macrophages

The mechanism of therapeutic activity for recombinant murine interferon-(rMu IFN) in the treatment of metastatic disease was investigated by comparing effector cell augmentation with therapeutic activity in mice bearing experimental lung metastases (B16-BL6 melanoma). Effector cell functions in spleen, peripheral blood, and lung (the tumor-bearing organ) were tested after 1 week and 3 weeks of rMu IFN administration (i.v. three times per week). Natural killer (NK), lymphokine-activated killer (LAK), cytolytic T lymphocyte (CTL) activities against specific and nonspecific targets, and macrophage tumoristatic activity were measured. rMu IFN demonstrated immunomodulatory activity in most assays of immune function. The optimal therapeutic protocol of rMu IFN (2.5×10⁶ U/kg, three times per week) prolonged survival and decreased the number of pulmonary metastatic foci. This therapeutic activity was correlated with specific CTL activity from pulmonary parenchymal mononuclear cells (PPMC), but not from spleen or blood. Macrophage tumoristatic activity in PPMC also correlated with therapeutic activity, but activity in alveolar macrophages did not. However, therapeutic activity did not correlate with NK or LAK activity at any site. These results demonstrate that the optimal therapeutic protocol is the same as the optimal immunomodulatory dose for pulmonary CTL and macrophage activities. Furthermore, while immunological monitoring may help to optimize treatment protocols, current monitoring procedures that use readily accessible sites, particularly peripheral blood, may not accurately predict the therapeutic efficacy of biological response modifiers in clinical trials.By acceptance of this article, the publisher or recipient acknowledges the right of the US. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the articleThis research was sponsored by the DHHS, under contract N01-23910 with Program Resources Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government     

Evidence for two chemosensory pathways in Rhodobacter sphaeroides

In contrast to enteric bacteria, chemotaxis in Rhodobacter sphaeroides requires transport and partial metabolism of chemoattractants. Although a chemotaxis operon has been identified containing homologues of the enteric cheA, cheW, cheR genes and two homologues of the cheY gene, deletion of the entire chemotaxis operon had only minor effects on chemotactic behaviour under the conditions tested. Responses to sugars were enhanced in tethered cells but in all other chemotaxis assays behaviour of the operon deletion mutant was wild type. The mutant also showed wild-type responses to weak organic acids such as acetate and propionate, the dominant chemoattractants for this organism, under all conditions. This is in direct contrast to the enterics in which CheA, CheW and CheY are absolutely essential for taxis to PTS sugars, oxygen and MCP-dependent chemoeffectors. The operon deletion mutant was subjected to Tn5 transposon mutagenesis and new mutants selected using a chemotaxis and phototaxis screen. One mutant, JPA203, was non-chemotactic on swarm plates and showed inverted responses when tethered or subjected to changes in light intensity. Characterization of the Tn5 insertion in JPA203 identified a second chemotaxis operon in R. sphaeroides that contains homologues of cheY, cheA and cheR, the first homologue of cheB and two homologues of cheW. The new genes were labelled orf10, cheY_III, cheA_II, cheW_II, cheW_III, cheR_II, cheB and tlpC. When introduced into a wild-type background, deletion of cheA_II produced a chemotaxis minus phenotype in R. sphaeroides, suggesting that cheA_II forms part of a dominant chemotactic pathway, whereas the earlier identified operon plays only a minor role under laboratory conditions. The data presented here support the existence of two chemosensory pathways in R. sphaeroides, a feature that so far is unique in bacterial chemotaxis.     

Arginine deprivation in KB cells: I. Effect on cell cycle progress

When exponentially growing KB cells were deprived of arginine, cell multiplication ceased after 12 h but viability was maintained throughout the experimental period (42-48 h). Although tritiated thymidine ([(3)H]TdR) incorporation into acid-insoluble material declined to 5 percent of the initial rate, the fraction of cells engaged in DNA synthesis, determined by autoradiography, remained constant throughout the starvation period and approximately equal to the synthesizing fraction in exponentially growing controls (40 percent). Continous [(3)H]TdR-labeling indicated that 80 percent of the arginine-starved cells incorporated (3)H at some time during a 48-h deprivation period. Thus, some cells ceased DNA synthesis, whereas some initially nonsynthesizing cells initiated DNA synthesis during starvation. Flow microfluorometric profiles of distribution of cellular DNA contents at the end of the starvation period indicated that essentially no cells had a 4c or G2 complement. If arginine was restored after 30 h of starvation, cultures resumed active, largely asynchronous division after a 16-h lag. Autoradiographs of metaphase figures from cultures continuously labeled with [(3)H]TdR after restoration indicated that all cells in the culture underwent DNA synthesis before dividing. It was concluded that the majority of cells in arginine-starved cultures are arrested in neither a normal G1 nor G2. It is proposed that for an exponential culture, i.e. from most positions in the cell cycle, inhibition of cell growth after arginine with withdrawal centers on the ability of cells to complete replication of their DNA.     

VH restriction among human cold agglutinins. The VH4-21 gene segment is required to encode anti-I and anti-i specificities.

We previously reported that human autoantibodies with cold agglutinin activity contained a single human VH gene segment (VH4-21) which was also responsible for the cross-idiotypic specificity characteristic of the cold agglutinin response. To confirm and extend this observation we have analyzed at the nucleotide level the H and L chains of six new cold agglutinin molecules derived from different patients. We found that regardless of whether the antibody recognizes the i or the I red cell Ag, restriction at the VH gene segment level is absolute. We also found that even in the absence of somatic mutation the VH4-21 gene segment can encode both anti-i and anti-I specificities. Finally, although the VH4-21 gene segment is essential for cold agglutinin activity, the other genetic elements that contribute to the V region of the antibody molecules can be extremely diverse. The structural information provided in this report sharply restricts the requirement for encoding pathogenic cold agglutinin activity to one of the components of the H chain V region, specifically the VH gene segment. The implications of this apparently absolute requirement for a single VH gene segment, unprecedented in the human autoimmune response, are discussed.