A Complex Extracellular Sphingomyelinase of Pseudomonas aeruginosa Inhibits Angiogenesis by Selective Cytotoxicity to Endothelial Cells

The hemolytic phospholipase C (PlcHR) expressed by Pseudomonas aeruginosa is the original member of a Phosphoesterase Superfamily, which includes phosphorylcholine-specific phospholipases C (PC-PLC) produced by frank and opportunistic pathogens. PlcHR, but not all its family members, is also a potent sphingomyelinase (SMase). Data presented herein indicate that picomolar (pM) concentrations of PlcHR are selectively lethal to endothelial cells (EC). An RGD motif of PlcHR contributes to this selectivity. Peptides containing an RGD motif (i.e., GRGDS), but not control peptides (i.e., GDGRS), block the effects of PlcHR on calcium signaling and cytotoxicity to EC. Moreover, RGD variants of PlcHR (e.g., RGE, KGD) are significantly reduced in their binding and toxicity, but retain the enzymatic activity of the wild type PlcHR. PlcHR also inhibits several EC-dependent in vitro assays (i.e., EC migration, EC invasion, and EC tubule formation), which represent key processes involved in angiogenesis (i.e., formation of new blood vessels from existing vasculature). Finally, the impact of PlcHR in an in vivo model of angiogenesis in transgenic zebrafish, and ones treated with an antisense morpholino to knock down a key blood cell regulator, were evaluated because in vitro assays cannot fully represent the complex processes of angiogenesis. As little as 2 ng/embryo of PlcHR was lethal to ∼50% of EGFP-labeled EC at 6 h after injection of embryos at 48 hpf (hours post-fertilization). An active site mutant of PlcHR (Thr178Ala) exhibited 120-fold reduced inhibitory activity in the EC invasion assay, and 20 ng/embryo elicited no detectable inhibitory activity in the zebrafish model. Taken together, these observations are pertinent to the distinctive vasculitis and poor wound healing associated with P. aeruginosa sepsis and suggest that the potent antiangiogenic properties of PlcHR are worthy of further investigation for the treatment of diseases where angiogenesis contributes pathological conditions (e.g., vascularization of tumors, diabetic retinopathy).     

Identification of a major human serum DNA-binding protein as beta 1H of the alternative pathway of complement activation

The conformation of the molecule cyclo (Aha-$\text{Cys-Phe-D-Trp-Lys-Thr-Cys}$) was studied by empirical conformational energy calculations. The low-energy structure found contains a type II' bend centered at the D-Trp-Lys residues. The lowest energy conformer has the aromatic ring of DTrp positioned such that the γ-protons of the Lys side-chain are in the shielding region (i.e., perpendicular to the center of the aromatic ring). This is in agreement with the NMR results. A mechanism of action for the inhibition of GH release is presented which suggests a conformational change occurs in the D-Trp side-chain ring upon binding to the receptor. The resulting structure has the Phe-D-Trp ring-ring stacking suggested to be responsible for binding and agonist activity of model growth-hormone releasing peptides.     

The effect of phospholipases and proteases on the binding of γ-aminobutyric acid to junctional complexes of rat cerebellum

A preparation enriched in junctional complexes, as judged by marker enzymes and electron microscopy, was prepared from rat cerebellum. The junctional complexes were incubated with γ-amino[¹⁴C]butyric acid at 25°C for 10 min, using [³H]sucrose as a marker for entrapped space. Total binding was determined in the absence of, and non-specific binding in the presence of, an excess of unlabelled γ-aminobutyric acid. The difference between the two binding values, i.e. the specific binding, was saturable and reversible, and showed positive cooperativity with a Hill number of about 2. The specific binding was inhibited by N-methylbicuculline, picrotoxinine and imidazole-4-acetic acid, but not by curare, strychnine or l-2,4-diaminobutyric acid. The above compounds had little effect on the non-specific binding, but addition of ethylenediaminetetraacetic acid decreased non-specific binding by 80%. Trypsin, pronase, phospholipase A₂ (EC 3.1.1.4), lysolecithin and sodium dodecyl sulfate decreased binding. Phospholipase C (EC 3.1.4.3) increased the specific binding by 260%. Phospholipids competed with γ-aminobutyric acid for binding, with phosphatidylethanolamine being more potent than phosphatidylcholine. These results lend support for Watkins' hypothesis that phosphatidylethanolamine competes with γ-aminobutyric acid for binding to the receptor protein.     

Identification of key residues for interaction of vasoactive intestinal peptide with human VPAC1 and VPAC2 receptors and development of a highly selective VPAC1 receptor agonist. Alanine scanning and molecular modeling of the peptide

The widespread neuropeptide vasoactive intestinal peptide (VIP) has two receptors VPAC(1) and VPAC(2). Solid-phase syntheses of VIP analogs in which each amino acid has been changed to alanine (Ala scan) or glycine was achieved and each analog was tested for: (i) three-dimensional structure by ab initio molecular modeling; (ii) ability to inhibit (125)I-VIP binding (K(i)) and to stimulate adenylyl cyclase activity (EC(50)) in membranes from cell clones stably expressing human recombinant VPAC(1) or VPAC(2) receptor. The data show that substituting residues at 14 positions out of 28 in VIP resulted in a >10-fold increase of K(i) or EC(50) at the VPAC(1) receptor. Modeling of the three-dimensional structure of native VIP (central alpha-helice from Val(5) to Asn(24) with random coiled N and C terminus) and analogs shows that substitutions of His(1), Val(5), Arg(14), Lys(15), Lys(21), Leu(23), and Ile(26) decreased biological activity without altering the predicted structure, supporting that those residues directly interact with VPAC(1) receptor. The interaction of the analogs with human VPAC(2) receptor is similar to that observed with VPAC(1) receptor, with three remarkable exceptions: substitution of Thr(11) and Asn(28) by alanine increased K(i) for binding to VPAC(2) receptor; substitution of Tyr(22) by alanine increased EC(50) for stimulating adenylyl cyclase activity through interaction with the VPAC(2) receptor. By combining 3 mutations at positions 11, 22, and 28, we developed the [Ala(11,22,28)]VIP analog which constitutes the first highly selective (>1,000-fold) human VPAC(1) receptor agonist derived from VIP ever described.     

Cytosol androgen receptor (AR) in human skin fibroblasts: characterization of the binding reaction and differentiation from androgen binding molecules of lower affinity

Androgen binding was studied in cytosol of human fibroblasts at 4 degrees C. When 5 alpha-dihydrotestosterone (DHT) was the ligand, a curvilinear Scatchard plot was seen, which was resolved into two components: I the androgen receptor (AR), Kd = 0.12-0.44 nM, and II a low affinity species, Kd = 6.3-28 nM. The same cytosol demonstrated only type I binding for 3H-methyltrienolone (MTr), Kd = 0.10-0.40 nM. The AR, i.e., 3H-MTr binding activity, eluted at 440,000 d by gel filtration chromatography in pre-labeling and post-labeling experiments. When the ligand was 3H-DHT, binding activity in the 10,000-45,000 d range was seen in addition to AR. Thus, saturable nonreceptor steroid binding was seen for DHT but not for MTr. The latter is the preferred ligand for the study of the AR in this system.     

Effects of Culture Fluids and Preinduction of Chitinase Production on Biocontrol of Bipolaris Leaf Spot by Stenotrophomonas maltophilia C3

Control of leaf spot, caused by Bipolaris sorokiniana, on tall fescue and perennial ryegrass by Stenotrophomonas maltophilia C3 was enhanced in growth chamber and field experiments by application of bacterial cells in culture fluids as compared to phosphate buffer. C3 population levels on leaves were up to 0.8 log units higher when applied with culture fluid than with phosphate buffer. Although fluids alone were inhibitory to conidial germination and leaf spot development, there was a synergistic effect when combined with C3 cells. Fluids from broth cultures with chitin as the carbon source were more inhibitory than those containing glucose, and the suppressiveness of a culture fluid was related to the age of the culture from which it was collected. Both of these effects were associated with the production of high levels of chitinase (EC 3.2.1.14), protease (EC 3.4.21-24), β-1,3-glucanase (EC 3.2.1.58), and lipase (EC 3.1.1.3) in the fluid. Culture fluids had a durable protective effect, inhibiting disease development even when applied 9 days before inoculation, and had a therapeutic effect if applied within 3 days after inoculation. When chitin was applied with C3 cells preinduced or noninduced for chitinase production (i.e., grown on chitin-containing or chitin-lacking media, respectively) biocontrol efficacy was significantly increased over either cell type without chitin, but the addition of chitin did not always increase colonization by C3. In general, preinduced cells were more effective than noninduced cells. The most effective field treatment was the combination of chitin with induced cells in culture fluid.     

Stabilization and activity-enhancement of mandelate racemase from Pseudomonas putida ATCC 12336 by immobilization

Mandelate racemase [EC 5.1.2.2] from Pseudomonas putida ATCC 12336 was efficiently immobilized through ionic binding onto DEAE- and TEAE 23-cellulose. The activity of the immobilized enzyme was significantly enhanced as compared to the native protein, i.e., 2.7- and 2.5-fold, respectively. DEAE-cellulose-immobilized mandelate racemase could be efficiently used in repeated batch reactions for the racemization of (R)-mandelic acid under mild conditions.     

Structure-affinity relationships of 5'-aromatic ethers and 5'-aromatic sulfides as partial A1 adenosine agonists, potential supraventricular anti-arrhythmic agents

Atrial fibrillation (AF) is the most commonly encountered sustained clinical arrhythmia with an estimated 2.3 million cases in the US (2001). A(1) adenosine receptor agonists can slow the electrical impulse propagation through the atrioventricular (AV) node (i.e., negative dromotropic effect) resulting in prolongation of the stimulus-to-His bundle (S-H) interval to potentially reduce ventricular rate. Compounds that are full agonists of the A(1) adenosine receptor can cause high grade AV block. Therefore, it is envisioned that a compound that is a partial agonist of the A(1) adenosine receptor could avoid this deleterious effect. 5(') Phenyl sulfides (e.g., 17, EC(50)=1.26 microM) and phenyl ethers (e.g., 28, EC(50)=0.2 microM) are partial agonists with respect to their AV nodal effects in guinea pig isolated hearts. Additional affinity, GTPgammaS binding data suggesting partial activity of the A(1) adenosine receptor, and PK results for 5(') modified adenosine derivatives are shown.     

Effects of Ultraviolet Irradiation on the Cell Cycle

Cultured mouse Cloudman melanoma cells, EMT6 breast carcinoma cells, and 3T3 fibroblasts all accumulated in the G2/M phase of the cell cycle when exposed to UVB radiation. The effects of UVB were maximal at 20–30 mJ/cm² for all three cell lines, and could be observed by flow cytometry as early as 12 hr post irradiation. It has been known since the mid-1970s that MSH receptor binding activity is highest on Cloudman melanoma cells when they are in the G2/M phase of their cycle. Here we show that either UVB irradiation or synchronization of Cloudman cells with colchicine results in a stimulation of MSH binding within 24 hr following treatment, a time when both treatments have resulted in accumulation of cells in the G2/M phase of the cycle. Furthermore, the two treatments performed together on the melanoma cells stimulated MSH receptor activity to the same extent as either treatment performed separately, suggesting that each may be influencing MSH receptor activity solely through a G2/M accumulation of cells. Together, these results raise the possibility that an increase in the number of cells in the G2 phase of the cell cycle is a generalized cellular response to injury, such as UV irradiation. However, in the case of pigment cells this response includes a mechanism for increasing melanin formation, i.e., increased MSH receptor activity. Should this be the case, similar G2/M “injury responses” of other cell types might be expected, consistent with their differentiated phenotypes.     

Collagen binding toEscherichia coli strain NG7C

Escherichia coli strain NG7C was shown to bind iodine-labeled human type IV collagen (Cn). The binding was rapid and saturable. The number of binding sites was estimated to be 1.5×10⁴ sites/cell and the dissociation constant 85 nM. The binding was inhibited by unlabeled type I, type IV, and type X Cn, gelatin and, at high doses by vitronectin and fibrinogen. Heat treatment of bacteria abolished the binding. A cell sonicate of strain NG7C inhibited the binding. Heat or protease treatment of the sonicate reduced its inhibitory activity by more than 50% Cell surface extracts of strain NG7C likewise inhibited Cn binding. Cells ofE. coli NG7C also bound to type IV Cn immobilized on microtiter plates. The Cn binding appears to be mediated by cell surface protein(s). Type IV Cn binding toE. coli NG7C differed from the earlier reported Cn binding mechanisms toE. coli, i.e., binding of soluble type II Cn, and from binding of immobilized type V Cn by enterobacteria.E. coli strains can thus produce different surface proteins which mediate binding to collagens. Expression of Cn binding byE. coli may enhance colonization of subepithelial tissues.     

Structure-activity relationships of steroid hormones on muscarinic receptor binding

A total of fifty steroidal compounds were tested for their inhibition on the binding of muscarinic receptor antagonist, [3H]quinuclidinyl benzilate ([3H](-)QNB), to the hypothalamic membranes prepared from male rats. Among the compounds tested, the active structures (with IC50 values less than or equal to 100 microM in parentheses) are: progesterone (40), 5 beta-pregnane-3,20-dione (40), deoxy-corticosterone (50), 5 beta-pregnane-17 alpha,21-diol-3,20-dione (30), 11-desoxy-17-hydroxycorticosterone (22), 17 alpha-hydroxyprogesterone (20), 5 beta-pregnan-17 alpha-ol-3,20-dione (24), 5 beta-androstane-3,17-dione (100), and 5 beta-dihydrotestosterone (100). By examining all the compounds tested, the following structure-activity relationship became apparent: (a) The ring A-reduced steroidal structures with a 5 beta-conformation were more potent than those with a 5 alpha-conformation; (b) 17 alpha-hydroxylation of the steroidal ring increased the steroid's inhibitory activity; (c) The C3 carbonyl group was essential for activity; (d) Reduction of the C3 carbonyl group or aromatization of the ring A abolished the steroid's inhibitory activity; (e) Oxidation of the C11 position of ring C resulted in a decrease or loss of inhibitory activity; and (f) Different modifications of the side chain of ring D by acetylation resulted in either an increase or a decrease in the inhibitory activity. The structure-activity relationship as revealed in this study might provide an insight for the synthesis of a steroidal molecule with a high affinity for the muscarinic receptor as well as for the search of a more potent and physiologically relevant steroidal metabolite possessing the ability to interact with the muscarinic receptor.     

Kynurenine hydroxylase mutants of the nematode Caenorhabditis elegans

Summary The relation of intestinal autofluorescence to tryptophan catabolism in the free-living nematode Caenorhabditis elegans has been investigated. L-Kynurenine hydroxylase (EC. 1.14.13.9) activity has been detected in normal (wild-type) individuals. Mutants in the gene flu-1 which are characterized by an altered autofluorescence of the intestine cells, i.e., more intense than wild type and bluish purple instead of light blue have also been examined. They show a markedly reduced activity of kynurenine hydroxylase. The finding supports the previously proposed model for altered fluorescence based on chromatographic identification of tryptophan catabolites present.     

Evidence for dual coupling of the murine luteinizing hormone receptor to adenylyl cyclase and phosphoinositide breakdown and Ca2+ mobilization. Studies with the cloned murine luteinizing hormone receptor expressed in L cells.

The murine receptor for luteinizing hormone (LHR) was cloned and expressed in L cells. This LHR (mature protein of 674 amino acids) is very similar to that of the rat (same length, 36 amino acid differences) but differs significantly more from that of man (673 amino acids, 109 differences). Expression of the murine LHR in L cells led to the appearance of binding sites for human chorionic gonadotropin (hCG) with a Kd of 150 pM and an LH- and hCG-stimulable adenylyl cyclase activity (EC50 = 50-100 pM hCG). Upon labeling pools of phosphoinositides with [3H]myo-inositol, L cells expressing the murine LHR responded to hCG with an increase in their rate of phosphoinositide hydrolysis (EC50 = 2,400 pM hCG). This was accompanied by an increase in intracellular Ca2+ [( Ca2+]i), as determined by the Fura2 method. This increase in [Ca2+]i in response to hCG was dependent on the LHR, for HCG did not affect [Ca2+]i in L cells not expressing the LHR. The effect was not due to the cAMP-forming activity of the LH receptor, for neither forskolin nor prostaglandin E1, which both increase cAMP levels in L cells, had a similar effect in either control or LHR-expressing cells and isoproterenol had no effect in L cells expressing a functionally active hamster beta-adrenergic receptor. The effect was also not due to overexpression of a Gs-coupled receptor, for L cells expressing 8-fold higher levels of the human V2 vasopressin receptor did not mimic the Ca(2+)-mobilizing response of the LH receptor. We conclude that the LH receptor has the capability of activating two intracellular signaling pathways: one leading to stimulation of adenylyl cyclase and resulting in increases in cAMP and a second leading to stimulation of phospholipase C and resulting in formation of inositol phosphates and elevations in [Ca2+]i. These data correlate positively with and provide a mechanistic explanation for previous reports on the ability of hCG to mobilize phosphoinositides and increasing [Ca2+]i in luteal and granulosa cells (e.g. Davis, J. S., West, L. A., and Farese, R. V. (1984) J. Biol. Chem. 259, 15028-15034).     

Laminin binding protein, 34/67 laminin receptor, carries stage-specific embryonic antigen-4 epitope defined by monoclonal antibody Raft.2

We previously produced monoclonal antibodies against the detergent-insoluble microdomain, i.e., the raft microdomain, of the human renal cancer cell line ACHN. Raft.2, one of these monoclonal antibodies, recognizes sialosyl globopentaosylceramide, which has the stage-specific embryonic antigen (SSEA)-4 epitope. Although the mouse embryonal carcinoma (EC) cell line F9 does not express SSEA-4, some F9 cells stained with Raft.2. Western analysis and matrix-assisted laser desorption ionization-time of flight mass spectrometry identified the Raft.2 binding molecule as laminin binding protein (LBP), i.e., 34/67 laminin receptor. Weak acid treatment or digestion with Clostridium perfringens sialidase reduced Raft.2 binding to LBP on nitrocellulose sheets and [(14)C]galactose was incorporated into LBP, indicating LBP to have a sialylated carbohydrate moiety. Subcellular localization analysis by sucrose density-gradient centrifugation and examination by confocal microscopy revealed LBP to be localized on the outer surface of the plasma membrane. An SSEA-4-positive human EC cell line, NCR-G3 cells, also expressed Raft.2-binding LBP.     

Coupling of a cloned rat dopamine-D2 receptor to inhibition of adenylyl cyclase and prolactin secretion.

We have previously described a cDNA which encodes a binding site with the pharmacology of the D2-dopamine receptor (Bunzow, J. R., VanTol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M., Machida, C., Neve, K. A., and Civelli, O. (1988) Nature 336, 783-787). We demonstrate here that this protein is a functional receptor, i.e. it couples to G-proteins to inhibit cAMP generation and hormone secretion. The cDNA was expressed in GH4C1 cells, a rat somatomammotrophic cell strain which lacks dopamine receptors. Stable transfectants were isolated and one clone, GH4ZR7, which had the highest levels of D2-dopamine receptor mRNA on Northern blot, was studied in detail. Binding of D2-dopamine antagonist [3H]spiperone to membranes isolated from GH4ZR7 cells was saturable, with KD = 96 pM, and Bmax = 2300 fmol/mg protein. Addition of GTP/NaCl increased the IC50 value for dopamine competition for [3H]spiperone binding by 2-fold, indicating that the D2-dopamine receptor interacts with one or more G-proteins. To assess the function of the dopamine-binding site, acute biological actions of dopamine were characterized in GH4ZR7 cells. Dopamine, at concentrations found in vivo, decreased resting intra- and extracellular cAMP levels (EC50 = 8 +/- 2 nM) by 50-70% and blocked completely vasoactive intestinal peptide (VIP) induced enhancement of cAMP levels (EC50 = 6 +/- 1 nM). Antagonism of dopamine-induced inhibition of VIP-enhanced cAMP levels by spiperone, (+)-butaclamol, (-)-sulpiride, and SCH23390 occurred at concentrations expected from KI values for these antagonists at the D2-receptor and was stereoselective. Dopamine (as well as several D2-selective agonists) inhibited forskolin-stimulated adenylate cyclase activity by 45 +/- 6%, with EC50 of 500-800 nM in GH4ZR7 membranes. Dopaminergic inhibition of cellular cAMP levels and of adenylyl cyclase activity in membrane preparations was abolished by pretreatment with pertussis toxin (50 ng/ml, 16 h). Dopamine (200 nM) abolished VIP- and thyrotropin-releasing hormone-induced acute prolactin release. These data show conclusively that the cDNA clone encodes a functional dopamine-D2 receptor which couples to G-proteins to inhibit adenylyl cyclase and both cAMP-dependent and cAMP-independent hormone secretion.     

Sigma binding sites in the brain; an emerging concept for multiple sites and their relevance for psychiatric disorders

An increasing amount of evidence suggests the existence of specific binding sites for psychotomimetic drugs from the opiate-benzomorphan and arylcyclohexylamine series. The sigma binding sites have preferential affinity for the dextrorotatory isomers of certain opiate benzomorphans, such as (+)SKF 10047, (+)cyclazocine and (+)pentazocine and also for some neuroleptics (e.g., haloperidol). The PCP receptor has preferential affinity for phencyclidine (PCP) analogs and other non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists. The physiological significance of the PCP receptor is associated with the blockade of the NMDA type of the glutamate receptor, implying a neuroprotective role of the PCP receptor. However, the significance of the sigma binding sites is less conspicuous. It is not only that drugs from distinct pharmacological classes display a certain degree of affinity for the "sigma/haloperidol" binding sites, but also that drugs which do not induce or block psychotomimetic activity, i.e., (+)3-(3-hydroxyphenyl)-N-(1-propyl) piperidine [(+)3-PPP] and 1,3-di-o-tolyl-guanidine (DTG), display relatively high affinity for the sigma binding sites. The diversity of the compounds which are proposed to interact with the sigma receptors and the variety of the responses elicited by these drugs suggest the existence of sigma receptor subtypes. The finding that the type A of monoamine oxidase (MAO) inhibitors, which are used in treatment of affective disorders, display high affinity for the sigma binding sites suggests their involvement in affective or schizoaffective disorders. Revealing the existence of sigma receptor subtypes may help to elucidate their association with various psychiatric disorders.     

The effect of phospholipases and proteases on the binding of gamma-aminobutyric acid to junctional complexes of rat cerebellum.

A preparation enriched in junctional complexes, as judged by marker enzymes and electron microscopy, was prepared from rat cerebellum. The junctional complexes were incubated with gamma-amino [14C]butyric acid at 25degreesC for 10 min, using [3H]sucrose as a marker for entrapped space, Total binding was determined in the absence of, and non-specific binding in the presence of, and excess of unlabelled gamma-aminobutyric acid. The difference bewteen the two binding values, i.e. the specific binding, was saturable and reversible, and showed positive cooperativity with a Hill number of about 2. The specific binding was inhibited by N-methylbicuculline, picrotoxinine and imidazole-4-acetic acid, but not by curare, strychnine or L-2,4-diaminobutyric acid. The above compounds had little effect on the non-specipic binding, but addition of ethylenediaminetetraacetic acid decreased non-specific binding by 80%. Trypsin, pronase, phospholipase A2 (EC 3.1.1.4), lysolecithin and sodium dodecyl sulfate decreased binding. Phospholipase C (EC 3.1.4.3) increased the specific binding by 260%. Phospholipids competed with gamma-aminobutyric acid for binding, with phosphatidylethanolamine being more potent than phosphatidylcholine. These results lend support for Watkins' hypothesis that phosphatidylethanolamine competes with gamma-aminobutyric acid for binding to the receptor protein.     

Effects of alkaloid precursor feeding on a Camptotheca acuminata cell line

To better understand the biosynthesis of Camptotheca acuminata alkaloids, the effect on camptothecin production of feeding with potential precursors of biosynthesis was studied (i.e., tryptamine and loganin combined, secologanin, and strictosidine). Two key enzymes in alkaloid biosynthesis 〚i.e., tryptophan decarboxylase (TDC; EC 4.1.1.28) and strictosidine synthase (STR; EC 4.3.3.2)〛 were also studied. The analyses were conducted using a C. acuminata CG1 cell line that does not produce alkaloids, which could be useful in better understanding the biosynthetic pathway and in identifying possible limiting factors. The activity of TDC was 5 pkat mg–1; the activity of STR was 1.1 pkat mg^–1. Feeding with strictosidine revealed that this precursor is easily biotransformed by two enzymes (i.e., a hydroxylase and a dehydrogenase) in hydroxystrictosidine and didehydrostrictosidine, but camptothecin was never detected. The indole pathway and the low level of STR activity could be limiting factors in the production of camptothecin in the cell line used.     

Identification of specific molecular structures of human immunodeficiency virus type 1 Tat relevant for its biological effects on vascular endothelial cells

Human immunodeficiency virus type 1 (HIV-1) Tat transactivates viral genes and is released by infected cells, acting as a soluble mediator. In endothelial cells (EC), it activates a proangiogenic program by activating vascular endothelial growth factor receptor type 2 (VEGFR-2) and integrins. A structure-activity relationship study was performed by functional analysis of Tat substitution and deletion variants to define the Tat determinants necessary for EC activation. Variants were made (i) in the basic and (ii) in the cysteine-rich domains and (iii) in the C-terminal region containing the RGD sequence required for integrin recognition. Our results led to the following conclusions. (i) Besides a high-affinity binding site corresponding to VEGFR-2, EC express low-affinity binding sites. (ii) The basic and the cysteine-rich variants bind only to the low-affinity binding sites and do not promote tyrosine phosphorylation of VEGFR-2. Furthermore, they have a reduced ability to activate EC in vitro, and they lack angiogenic activity. (iii) Mutants with mutations in the C-terminal region are partially defective for in vitro biological activities and in vivo angiogenesis, but they activate VEGFR-2 as Tat wild type. In conclusion, regions encoded by the first exon of tat are necessary and sufficient for activation of VEGFR-2. However, the C-terminal region, most probably through RGD-mediated integrin engagement, is indispensable for full activation of an in vitro and in vivo angiogenic program.     

Regulation of nitrate assimilation in the cyanobacterium Phormidium laminosum

The intracellular ratio of 2-oxoglutarate to glutamine has been analyzed under nutritional conditions leading to different activity levels of nitrate-assimilating enzymes in Phormidium laminosum (Agardh) Gom. This non-N₂-fixing cyanobacterium adapted to the available nitrogen source by modifying its nitrate reductase (NR; EC 1.7.7.2), nitrite reductase (NiR; EC 1.7.7.1) and glutamine synthetase (GS; EC 6.3.1.2) activities. The 2-oxoglutarate/glutamine ratio was similar in cells adapted to grow with nitrate or ammonium. However, metabolic conditions that increased this ratio [i.e., nitrogen starvation or l-methionine-d,l-sulfoximine (MSX) treatment] corresponded to high activity levels of NR, NiR, GS (except in MSX-treated cells) and glutamate synthase (GOGAT; EC 1.4.7.1). By contrast, metabolic conditions that diminished this ratio (i.e., addition of ammonium to nitrate-growing cells or addition of nitrate or ammonium to nitrogen-starved cells) resulted in low activity levels. The variation in the 2-oxoglutarate/glutamine ratio preceded the changes in enzyme activities. These results suggest that changes in the 2-oxoglutarate/glutamine ratio could be the signal that triggers the adaptation of P. laminosum cells to variations in the available nitrogen source, as occurs in enterobacteria.Abbreviations Chl chlorophyll - GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1) - GS glutamine synthetase (EC 6.3.1.2) - MSX l-methionine-d,l-sulfoximine - NiR nitrite reductase (EC 1.7.7.1) - NR nitrate reductase (EC 1.7.7.2) - TP total protein This work has been partially supported by grants from the Spanish Ministry of Education and Science (DGICYT PB88-0300 and PB92-0464) and the University of the Basque Country (042.310-EC203/94). M.I.T. was the recipient of a fellowship from the Basque Government.