Measuring permeability with a whole cell-based biosensor as an alternate assay for angiogenesis: comparison with common in vitro assays

Angiogenesis plays cardinal role in normal developmental processes as well as in numerous pathologies. Multiple cytokines are released and act simultaneously to activate endothelial cells in vivo. The present study investigated the relative ability of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), tumor necrosis factor-alpha (TNF-alpha) in modulating cell monolayer permeability, migration, proliferation and tube formation individually and in combination. While the common methods for assaying angiogenesis were conducted for studying cell migration, proliferation and differentiation, endothelial cell monolayer permeability studies were carried out using a whole cell-based biosensor. The biosensor, consisting of a confluent monolayer of human umbilical vein endothelial cells (HUVECs) on a potassium ion-selective electrode, takes advantage of cell monolayer permeability dysfunction to detect the presence of small quantities of cytokines. When a confluent monolayer of cells was formed on the membrane surface, the response of the electrode toward the marker ion, potassium, was inhibited. The response obtained after exposing this sensor to different cytokines for 1 and 3h, can be attributed to the modulation of monolayer permeability by these cytokines. The present study demonstrated that at the concentrations experimented with, the relative change in permeability assay in the presence of cytokines compared to the control was much higher than that observed in other assays, thereby bolstering the potential of the biosensor to act as a quick screening tool for angiogenesis.     

Isoprenoid synthesis during the cell cycle. Studies of 3-hydroxy-3-methylglutaryl-coenzyme A synthase and reductase and isoprenoid labeling in cells synchronized by centrifugal elutriation

The activities of 3-hydroxy-3-methylglutaryl-coenzyme A synthase and reductase were assayed in exponentially growing LM fibroblasts and Friend murine erythroleukemia cells isolated at various stages of the cell cycle by centrifugal elutriation. The activities of these enzymes were similar in all phases of the cell cycle, regardless of whether the cells were cultured in the presence or absence of serum. These observations were confirmed in murine erythroleukemia cells synchronized by recultivation of pure populations of G1 cells. The incorporation of [14C]acetate or 3H2O into sterols decreased by 30-50% in later stages of the cell cycle, whereas the incorporation of [14C]acetate into ubiquinone increased as the cells progressed toward mitosis. Similar changes in the labeling of sterols compared to ubiquinone and dolichol were observed when [3H]mevalonate was used, suggesting that cell cycle-dependent alterations may occur in the flux of farnesyl pyrophosphate into the various branches of the isoprenoid pathway. Synchronized murine erythroleukemia cells incorporated [3H]mevalonate into protein-bound isoprenyl groups at all stages of the cell cycle, and there were no substantial changes in the electrophoretic profiles of these labeled polypeptides. The finding that the activities of the enzymes regulating mevalonate synthesis did not vary substantially during the cell cycle implies that changes in the endogenous mevalonate pool probably do not play a limiting role in regulating cell cycle traverse when cells are undergoing exponential growth. Although small cell cycle-dependent changes may occur in the relative activity of various post-mevalonate branches of the isoprenoid biosynthetic pathway, there is no evidence that synthesis of any major isoprenoid end product is confined exclusively to a specific phase of the cell cycle.     

Structure-activity relationship study of [1,2,3]thiadiazole necroptosis inhibitors

Necroptosis is a regulated caspase-independent cell death mechanism that results in morphological features resembling non-regulated necrosis. This form of cell death can be induced in an array of cell types in apoptotic deficient conditions with death receptor family ligands. A series of [1,2,3]thiadiazole benzylamides was found to be potent necroptosis inhibitors (called necrostatins). A structure–activity relationship study revealed that small cyclic alkyl groups (i.e. cyclopropyl) and 2,6-dihalobenzylamides at the 4- and 5-positions of the [1,2,3]thiadiazole, respectively, were optimal. In addition, when a small alkyl group (i.e. methyl) was present on the benzylic position all the necroptosis inhibitory activity resided with the (S)-enantiomer. Finally, replacement of the [1,2,3]thiadiazole with a variety of thiophene derivatives was tolerated, although some erosion of potency was observed.     

Polygalacturonase beta-subunit antisense gene expression in tomato plants leads to a progressive enhanced wound response and necrosis in leaves and abscission of developing flowers

Tomato (Lycopersicon esculentum var. Better Boy) plants were transformed with a tomato leaf wound-inducible polygalacturonase (PG) beta-subunit gene in the antisense orientation (PGbetaS-AS) under the control of the cauliflower mosaic virus 35S promoter. The leaves of the transgenic plants exhibited small localized lesions, which eventually enlarged and spread throughout the entire surfaces of the leaves, resulting in cell death. The same lesions were also observed in the peduncle of developing flowers, extending to the whole flower causing abscission, resulting in a sterile phenotype. Leaves of transgenic plants exhibited elevated levels of PG activity, hydrogen peroxide, and enhanced defense signaling in response to wounding and elicitor treatment. The defense signaling increased was accompanied by an increased resistance toward tobacco hornworm (Manduca sexta) larvae. The cumulative results suggest that in the absence of the beta-subunit protein in tomato leaves, an increase in PG activity occurred that led to an enhanced wound response, the formation of lesions leading to severe necrosis, and an abscission of developing flowers.     

Purification and characterization of S-adenosyl-L-methionine: caffeic acid 3-O-methyltransferase from suspension cultures of alfalfa (Medicago sativa L.)

Caffeic acid O-methyltransferase (COMT) is one of a group of proteins present in alfalfa cell cultures which can be photoaffinity labeled with S-adenosyl-L-[methyl-3H]methionine. The enzyme was purified to homogeneity from elicitor-treated suspension cultures and shown to exist as an active monomer of subunit Mr 41,000. COMT could be separated into two forms on the basis of their isoelectric points and relative affinities for S-adenosyl-methionine and S-adenosylhomocysteine. Both forms had equal affinities for caffeic acid, were highly specific for the 3-hydroxyl group of substituted cinnamic acids, and exhibited negligible activity toward flavonoid substrates. An antiserum raised against COMT from aspen immunoprecipitated alfalfa COMT activity. Peptide mapping studies indicated that the two forms of COMT and an isoflavone O-methyltransferase from alfalfa are closely related proteins. The extractable activity of COMT doubled over a 48-h period following exposure of alfalfa cell suspensions to a yeast elicitor preparation, and this was associated with a small change in the relative proportions of the two forms of the enzyme.     

Trypanosoma cruzi: subcellular distribution of glycolytic and some related enzymes of epimastigotes

The first six glycolytic enzymes in epimastigote Trypanosoma cruzi were shown to behave similarly during differential centrifugation, when maximum relative specific activity was found in the small granule fraction, and by isopycnic centrifugation, when the bulk of each activity coequilibrated on sucrose gradients with a modal density of 1.23 g/ml. All six showed substantial detergent latency in whole cell homogenates. Electron microscopic examination of fractions from a sucrose gradient with modal density 1.23 g/ml showed the presence of single membrane bound vesicles of diameter 0.2-0.8 micron. It was concluded that these six enzymes were contained in a microbodylike organelle, termed the "glycosome." Phosphoglucose isomerase (EC 5.3.1.9) also possessed substantial soluble activity. No microbody marker enzyme described in other sources could be detected. Peroxidase (EC 1.11.1.7) had an insignificant glycosomal component. Enzymes of amino acid and fatty acid metabolism were not detected in microbody fractions. Marker enzymes for the flagellar pocket and plasma membrane were suggested.     

Protease immobilization onto polyacrolein microspheres

Water-insoluble proteases were prepared by immobilizing papain and chymotrypsin onto the surface of polyacrolein microspheres with and without oligoglycines as spacer. The activity of immobilized proteases was found to be still high toward small ester substrates, but very low toward casein, a high-molecular-weight substrate. The relative activity of the immobilized proteases without spacer decreased gradually with the decreasing surface concentration of the immobilized proteases on the microspheres. On the contrary, the immobilized proteases with oligoglycine spacers gave an almost constant activity for the substrate hydrolysis within the surface concentration region studied and gave a much higher relative activity than those without any spacer. With the longer spacer, the immobilized enzymes showed a higher activity toward casein hydrolysis, whereas there was an optimum length for the spacer when hydrolysis was carried out toward the low-molecular-weight substrate. The thermal stability of the immobilized proteases was higher than that of the respective native proteases. The initial enzymatic activity of the immobilized proteases maintained almost unchanged without any elimination and inactivation of proteases, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.     

High-throughput screening of cell death inducible short peptides from TNF-related apoptosis-inducing ligand sequence

Therapeutic peptides and small molecules, rationally designed to trigger cell death have attracted strong attention. Cell death inducible peptides were screened from amino acid sequence of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Using Fmoc solid phase synthesis, cellulose membrane-bound octameric peptide library of TRAIL scan was prepared and cell viability assay was directly performed on peptide disk with Jurkat cells. Six peptide sequences that could induce cell death were found. Peptide sequence with RNSCWSKD (TRAIL(227-234)) that exist in the zinc-binding site revealed high cell death inducible activity. Apoptotic cell death was observed when cells were treated with soluble synthesized peptide.     

Rapid shifts in <Emphasis Type="Italic">Atta cephalotes</Emphasis> fungus-garden enzyme activity after a change in fungal substrate (Attini,Formicidae)

Fungus gardens of the basidiomycete Leucocoprinus gongylophorus sustain large colonies of leaf-cutting ants by degrading the plant material collected by the ants. Recent studies have shown that enzyme activity in these gardens is primarily targeted toward starch, proteins and the pectin matrix associated with cell walls, rather than toward structural cell wall components such as cellulose and hemicelluloses. Substrate constituents are also known to be sequentially degraded in different sections of the fungus garden. To test the plasticity in the extracellular expression of fungus-garden enzymes, we measured the changes in enzyme activity after a controlled shift in fungal substrate offered to six laboratory colonies of Atta cephalotes. An ant diet consisting exclusively of grains of parboiled rice rapidly increased the activity of endo-proteinases and some of the pectinases attacking the backbone structure of pectin molecules, relative to a pure diet of bramble leaves, and this happened predominantly in the most recently established top sections of fungus gardens. However, fungus-garden amylase activity did not significantly increase despite the substantial increase in starch availability from the rice diet, relative to the leaf diet controls. Enzyme activity in the older, bottom sections of fungus gardens decreased, indicating a faster processing of the rice substrate compared to the leaf diet. These results suggest that leaf-cutting ant fungus gardens can rapidly adjust enzyme activity to provide a better match with substrate availability and that excess starch that is not protected by cell walls may be digested by the ants rather than by the fungus-garden symbiont.     

Differential accumulation of xanthones in methyl-jasmonate- and yeast-extract-treated cell cultures of Centaurium erythraea and Centaurium littorale

Cell-suspension cultures of Centaurium erythraea and Centaurium littorale (Gentianaceae) respond to methyl jasmonate and yeast extract with a differential accumulation of xanthones. Methyl jasmonate induced the formation of 1-hydroxy-3,5,6,7-tetramethoxyxanthone, the amount of which increased in both cell cultures around 10 h after addition. A substantial increase in the activity of phenylalanine ammonia-lyase (PAL) was not observed. When challenged with yeast extract the cell cultures accumulated l,5-dihydroxy-3-methoxyxanthone. This appeared rapidly after addition of yeast extract in C. erythraea but its amount in C. littorale increased only after a lag phase of 25 h. While PAL activity in C. erythraea was strongly suppressed a fourfold increase in its activity was found in C. littorale. Both elicited xanthones accumulated intracellularly. A scheme for xanthone biosynthesis in the two cell cultures is proposed.     

Adaptogenic action of the complex of phenylpropanoids on Dioscorea deltoidea cell culture under abiotic stress

Biological activity of the extract from golden root (Rhodiola rosea L.) roots, containing the complex of phenylpropanoids (CPP), was studied on the cell culture of yam (Dioscorea deltoidea Wall) under normal conditions and abiotic stress. The high radical-binding capacity of CPP relative to anion- and hydroxyl-radicals was observed. Having a high level of antiradical protection, CPP at a high concentration(100 μM) exerted prooxidant effect, causing a decrease in D. deltoidea cell viability and a decrease in the activities of antioxidant enzymes: superoxide dismutase, guaiacol-dependent peroxidase, and catalase, with the exception of ascorbate peroxidase. At treatment with 100 μM CPP, oxidase (prooxidant) activity of peroxidase increased by three times. The low CPP concentration (2 μM) did not induce substantial changes in the activities of tested enzymes and also a substantial increase in the oxidase activity of peroxidase. Under conditions of oxidative stress induced by paraquat and high temperature, CPP manifested adaptogenic action, increasing cell viability; however, under hyperosmotic stress, it was not efficient. CPP was most efficient at a low concentration after cell pre-incubation with it for 5 days. In this case, the amount of primary and secondary POL products increased. Shortening pre-cultivation with CPP reduced its defensive effect.     

Pseudohypophosphatasia: aberrant localization and substrate specificity of alkaline phosphatase in cultured skin fibroblasts.

We explored the biochemical basis for the disorder pseudohypophosphatasia (PsHYPT) in one patient by examining the substrate specificity and localization of alkaline phosphatase (ALP) in cultured dermal fibroblasts. Despite substantial ALP activity, in cell homogenates, toward the artificial substrate 4-methyl-umbelliferyl phosphate (4-MUP), there was a marked deficiency in ALP activity toward the natural substrates pyridoxal 5'-phosphate (PLP) and phosphoethanolamine (PEA), indicating altered substrate specificity. Furthermore, although 4-MUP phosphatase (4-MUP-P'tase) activity was predominantly localized as an ecto-enzyme, the small amount of PLP phosphatase (PLP-P'tase) activity was intracellular. This differential localization was apparent in intact cells, since (1) brief acidification of the medium at 4 degrees C inactivated a majority of the 4-MUP-P'tase activity but only 15% of the PLP-P'tase activity (in contrast to greater than 85% inactivation of both in homogenates), (2) greater than 70% of the 4-MUP-P'tase activity but only 30% of the PLP-P'tase activity was released by phosphatidylinositol-specific phospholipase C (PI-PLC) digestion, and (3) degradation of extracellular PLP was less than 35% of that of disrupted cells. Both 4-MUP- and PLP-P'tase activities were predominantly in a lipid-anchored form that could be converted to a soluble, lipid-free form by PI-PLC digestion. Our findings suggest that the clinical and biochemical presentation of this PSHPT patient results from the production of two aberrant ALP species. One form of ALP has appropriate ectoorientation but is preferentially deficient in activity toward natural substrates; the other ALP species has appropriate substrate specificity but is sequestered from substrates by its intracellular location.     

Increased activity of plasminogen activators during involution of the rat ventral prostate.

Plasminogen activator was measured in the ventral prostates of non-castrated, castrated, and androgen-treated rats to determine whether changes in this activity correlated with the process of glandular involution. While the activity was very low in cytosolic extracts from the prostates of non-castrated rats, 2 days following castration the plasminogen activator activity increased in a near-linear fashion such that by day 7 it was 10-fold higher in terms of specific activity (per mg of protein) and cellular concentration (per mg of DNA). During this interval there was a rapid decrease in the cell population of the prostates. Treatment of the 7-day castrated rats with the potent androgen, dihydrotestosterone, both reduced the plasminogen activator activity and restored the cell number in a dose-related manner. Gel electrophoretic analysis revealed two major bands of plasminogen activator activity in the cytosolic extracts from 4- and 7-day castrated rats, plus additional minor bands in samples from 10- and 14-day castrated rats. Approx. 10% of the cellular concentration of plasminogen activator activity was recovered in association with an 18000g pellet fraction from the prostates; this fraction showed less heterogeneity of the plasminogen activator forms as observed by gel electrophoresis. Inhibitor studies indicated that the 18000g pellet fraction from the prostates of non-castrated rats possessed some plasminogen activator inhibitor activity, but the relative concentration of the inhibitor activity was small. We conclude that the involution of the prostate is probably associated with increased synthesis of plasminogen activators through a de-repression process which may involve loss of androgen receptors.     

The morphology and histochemistry of adult rats neurocytes after BCNU administration

Studies were performed on adult rats of Wistar strain given four 7-days-spaced intraperitoneal doses of BCNU with single dose resembling to used in clinical practice. The animals were sacrificed at the seventh day after the last dose of the drug. Morphological alterations were evaluated in H + E or cresyl violet stained sections. In frozen microtome sections histoenzymatic reactions were performed to detect enzymatic activity of some phosphatases and esterases. Karyo- and cytophotometric measurements of pyramidal cell nuclei in frontal and parietal cortex and of motor neurons in trigeminal nerve nucleus were performed in sections subjected to Feulgen reaction, using automatic microscopic image analyzer "Morphoquant" (VEB Carl Zeiss, Jena). The performed studies showed that administration of multiple therapeutic doses of BCNU lead to primary injury of vascular wall as oedema and proliferation of endothelium and small perivascular haemorrhages. The cytostatic drug induced a decrease in NsE and AlkP enzymatic activities, increased activity of AChE, ChE, AcP and ATPase and topographically variable changes in intensity of TPPase enzymatic reaction. Several karyo- and cytophotometric alterations were observed also in neurocyte cell nuclei which became elongated and acquired a more round shape. This was associated with a decrease in relative DNA content, loosening of nuclear chromatin structure and with shifting chromatin lumps toward periphery of cell nuclei.     

Developmental expression of G proteins that differentially modulate adenylyl cyclase activity in mouse brain.

Changes in the relative abundance of the G protein alpha subunits were observed during early mouse development Gs alpha was almost exclusively present as a large form (Gs-1) in prenatal brain. Postnatally with a substantial increase in Gpp[NH]p stimulated adenylyl cyclase activity, the small form (Gs.s) increased in amount while Gs-1 decreased. These results suggest that the Gs-s may be the more effective cyclase activator and that changes in alternative splicing are developmentally regulated. Gi1 and Go appeared before birth whereas Gi2 developed postnatally. Opiate stimulation of GTPase and inhibition of adenylyl cyclase were fully expressed prenatally.     

High affinity 17alpha-substituted estradiol derivatives: synthesis and evaluation of estrogen receptor agonist activity

We synthesized four derivatives of 17beta-estradiol (E2) with an azide substitution on a 17alpha-side chain of varying length, namely 17alpha-(azidopropargyl)-3,17beta-estradiol (5), its 17beta-azido derivative (diazide 7), 17alpha-(5-azido-pent-1-ynyl)-3,17beta-estradiol (6) and 17alpha-(azidopentyn-2-yl)-3,17beta-estradiol (10). While most of the derivatives had low (7) or marginal (6 and 10) relative binding affinity (RBA) for both types of estrogen receptor (ERalpha and ERbeta), the RBAalpha and RBAbeta of 5 were practically identical to those of E2. The estrogenic activity of the derivatives was assessed using estrogen-responsive breast (MCF-7) and endometrial cancer (Ishikawa) cells. While 5 was a potent and effective inducer of alkaline phosphatase in Ishikawa cells and 7 was less potent but as effective as 5, 6 was marginally active and 10 was totally inactive in this respect. In the presence of 0.1 nM E2, however, 6 exhibited some ER antagonist activity at the highest concentration tested (1 microM). Similar results were obtained as regards the potency and efficacy of stimulation of MCF-7 cell proliferation and induction of luciferase gene expression in MCF-7:D5L cells, a clone stably transfected with an estrogen-responsive form of the gene. These data suggest that, while 5, 6, 7 and 10 interact with either type of ER in isolation, only 5 and 7 exhibit substantial ER agonist activity in the different estrogen-target cells examined, which could provide for photoaffinity labelling of the receptor in the cell as well as in isolation.     

Biphasic kinetics of a methanotrophic community is a combination of growth and increased activity per cell

Because methane-oxidizing bacteria (MOB) are the only biological sink for the greenhouse gas methane, knowledge of the functioning of these bacteria in various ecosystems is needed to understand the dynamics observed in global methane emission. The activity of MOB is commonly assessed by methane oxidation assays. The resulting methane depletion curves often follow a biphasic pattern of initial and induced methane oxidation activity, often interpreted as representing the in situ active and total MOB community, respectively. The application of quantitative-PCR on soil incubations, which were stopped before, at and after the transition point in the methane-depletion curve, demonstrated that both pmoA -mRNA was produced as well as substantial cell growth took place already in the initial phase. In addition, type Ia and II MOB displayed markedly different behaviour, which can be interpreted as ecologically different strategies. For the correct interpretation of methane oxidation assays, the use of small time windows is recommended to calculate methane oxidation activities to avoid substantial cell growth.