Down-regulation of vinculin upon MK886-induced apoptosis in LN18 glioblastoma cells.

Glioblastomas are a type of malignant brain tumor and are among the most difficult cancers to treat. One strategy to treat aggressive cancers is the use of drugs that target multiple signaling pathways. MK886 is a drug known to inhibit both 5- lipoxygenase-activating-protein (FLAP) and peroxisome proliferator activated receptor-alpha (PPAR-alpha). The objectives of this study were to investigate the ability of MK886 to induce apoptotic cell death in LN18 glioblastoma cells and to characterize the cell death mechanisms. MK886 induced massive apoptotic LN18 cell death that was manifested by the release of nucleosomes, annexinV binding to phosphatidylserine in the absence of nuclear staining, and changes in the fluorescent intensity of Mito Tracker Deep Red 633 indicating changes in mitochondrial oxidative function and mass. The alteration of the mitochondrial function implied that MK886 induced apoptosis in LN18 cells via a mitochondrial pathway. The broad caspases inhibitor ZVAD-FMK inhibited MK886-induced nucleosome release, but not annexinV binding or MK886-altered mitochondrial function. Real time RT-PCR demonstrated that LN18 cells expressed significant levels of FLAP and PPAR- alpha mRNAs. A low level of arachidonate 5-lipoxygenase (ALOX-5) mRNA was detected, but little, if any, arachidonate 12- lipoxygenase (ALOX-12) mRNA was present. In addition, MK886-induced apoptosis in LN18 cells was accompanied by a decrease in the protein and mRNA levels of vinculin, but not other focal adhesion proteins. In summary, the data presented here indicate that disruption of the actin-vinculin-cell-cytoskeleton matrix of the LN18 glioblastoma is a component of the MK886 induced apoptosis. In addition, MK886 treated LN18 cells could provide one model in which to investigate drugs that target lipoxygenase and PPAR-alpha pathways in the chemotherapeutic treatment of glioblastomas.

Evolutionary-derived anatomical characteristics and universal attractiveness.

Hominid fossils illustrate how modern humans have evolved anatomically. Included in the fossils are traits no longer phenotypically prevalent in humans (primitive) and phenotypic traits that have become increasingly prevalent (derived). In this study, published paleontological information about the anatomical evolution of humans was used to create line drawings of human form. Survey data were accumulated by having 759 individuals evaluate more than 40 anatomical traits. Each anatomical trait was presented as a panel of three line drawings intended to express the trait in a primitive, intermediate, and derived form. For each panel of three drawings, subjects were instructed to select the drawing they considered most attractive and then select the drawing they considered least attractive. The survey data indicate that males and females of diverse ages, races, cultures, and from varied geographical regions show commonality in their judgements of beauty of human form. The individuals surveyed appeared to have a strong aversion to primitive traits, preferring proportions and characteristics that are intermediate or more derived. In many instances, the evaluators preferred drawings that were exaggeratedly derived. The data may have relevance to the ongoing debate of whether averageness or atypicality is the essence of human beauty. Also, there was high agreement in judging the attractiveness of shapes and proportions in line drawings that were not immediately recognized to be representations of human form. These data could indicate that our general aesthetic sense for art, architecture, and fashion may be based on a subliminal reference to derived anatomical shapes and proportions. Overall, the data support the hypothesis that derived traits that are universally shared by anatomically modern humans may be the standard for our innate sense of beauty of human form.

Why Barbie is perceived as beautiful.

The long-term acceptance and success of the Barbie doll suggests the physical characteristics of the doll are perceived as attractive. When viewed in the context of universal attractiveness, response to the doll raises the question why Barbie is perceived as attractive. Published paleontological data on hominid fossils indicate how the shapes and anatomical proportions of humans have evolved. Included in the fossils are phenotypic traits no longer prevalent in humans (primitive) and phenotypic traits that have become increasingly prevalent (derived). It is noted that the anatomical proportions of the Barbie doll are exaggerated and emphasize derived characteristics. It was proposed that in the perception of human form, derived traits are perceived as attractive while primitive traits are perceived as unattractive. Drawings and photographs were utilized to survey reactions to a comparison of primitive vs derived traits by 495 subjects, instructed to select the shape or proportion they considered more attractive. There was significant agreement among the subjects that derived anatomical traits were perceived as more attractive than primitive ones. The Barbie doll is illustrative of how human beauty has evolved and indicates elements of human form that appear beautiful. The doll emphasizes our derived evolutionary traits and, possibly, that is why the doll is perceived as attractive.

Modulation of urinary kallikrein and plasma renin activities does not affect established hypertension in the fawn-hooded rat.

Fawn-hooded (FH) rats develop low-renin hypertension which is preceded by a decrease in urinary kallikrein. We examined urinary excretion of active and inactive kallikrein in hypertensive FH male rats and matched animals of the ancestral, normotensive Wistar strain. To determine the effects of modulation of salt intake on the kallikrein profile, rats were given standard rat chow (0.39% NaCl), a low-salt diet (0.02% NaCl), or a high-salt diet (standard chow plus water with 1% NaCl). Control FH rats excreted less active kallikrein (p less than 0.02), had similar amounts of inactive kallikrein, and had a higher inactive/active kallikrein ratio (p less than 0.02) than control Wistar rats. Low salt intake increased active kallikrein 136% (p less than 0.002) and 54% (p less than 0.035) in FH and Wistar rats, respectively, but did not change the level of inactive kallikrein or the inactive/active kallikrein ratio. High salt intake had no effect on kallikrein excretion in either strain. Low salt intake did not change blood pressure in either strain in spite of significant changes in plasma renin activity, angiotensin II and active kallikrein excretion. The low urinary active kallikrein and the high inactive/active kallikrein ratio in FH rats do not appear to play a role in the established hypertension in the FH rat, since modulation of these parameters did not cause a significant change in the elevated blood pressure.

Characterisation of IgE-mediated histamine release from equine basophils in vitro.

In vitro IgE-mediated histamine release by equine blood basophils was characterised as the basis for a screening test for immediate hypersensitivity responses in horses. The responses are initiated by inducing agents that are capable of crosslinking or bridging the membrane-bound IgE molecules. The release process is complete within 40 mins. In vitro histamine release is dose-dependent, with a submaximal response at less or greater than the optimal dose of inducing agent. Exogenous calcium is required but not magnesium; the optimal release calcium concentration is 1.0 to 1.5 mM. If an IgE-mediated inducing agent is added in the absence of exogenous calcium, the basophils become desensitised. The pH and temperature optima for release are physiological (pH 7.4, 37 degrees C). Histamine release is potentiated by deuterium oxide.

Contrasting effects of early and late orchiectomy on hypertension and renal disease in fawn-hooded rats.

Fawn-hooded (FH) rats, primarily males, develop spontaneous low-renin hypertension associated with reduced urinary excretion of kallikrein as early as 2 months of age, followed by progressive glomerular sclerosis and proteinuria as early as 3 months of age. In the present study we determined the effects of early (5-7 weeks) or late (5 months) orchiectomy on the blood pressure and nephropathy of FH rats, compared to sham-operated (control) FH males. Early orchiectomy reduced significantly the progression of glomerular sclerosis and of proteinuria and ameliorated the hypertension but had no significant effect on excretion of urinary kallikrein. Late orchiectomy, in contrast, had no significant effect on the progression of glomerular sclerosis or proteinuria but did significantly reduce the blood pressure and marginally increase the excretion of urine kallikrein. These results suggest that (a) male sex hormones may play a role in the pathogenesis of hypertension and nephropathy in the FH rats and (b) renal disease in this strain progresses in spite of improvement in blood pressure.

Increased catecholamine output in the hypertensive fawn-hooded rat.

The total 24 hour urinary outputs of the catecholamines norepinephrine (NE), epinephrine (E), dopamine (DA) and the DA metabolite homovanillic acid (HVA) were measured in hypertensive fawn-hooded rats and compared to the ancestral strain of normotensive Wistar rats. The hypertensive fawn-hooded rats demonstrated significantly higher urinary outputs of the catecholamines NE and DA, and of the DA metabolite HVA. Following treatment with the antihypertensive, debrisoquin sulfate, the blood pressure of the fawn-hooded rats decreased until it approached the levels observed in normotensive Wistar rats. By inhibiting sympathetic nervous activity and monoamine oxidase, the debrisoquin treatment significantly decreased the output of DA, NE and HVA but not E. The data suggest the fawn-hooded rat is a model of neurogenic hypertension which is characterized by an increased sympathetic output.

Preparation and evaluation of a soluble derivative of the antiallergic compound trans-2, 3b, 4, 5, 7, 8b, 9, 10-octahydronaphtho [1, 2-c:5, 6-c'] dipyrazole (LC-6).

In the present report we describe the preparation of LC-6.2HCl, a soluble derivative of the synthetic bispyrazole LC-6. Because the latter was practically insoluble in aqueous and organic media, experiments which indicated that it had antiallergic activity were confined to in vivo studies following its oral administration. The availability of soluble LC-6.2HCl made it possible to administer the drug i.v. or i.p. Through these routes it exhibited greater antiallergic activity than by the oral route, as judged by lower ID50's and by the achievement of 100% PCA inhibition. The latter result had not previously been attained orally with the base. Furthermore, when injected i.v. or i.p. LC-6.2HCl showed prolonged inhibitory activity, a valuable attribute of the parent compound. The in vivo activity of the soluble salt, which we describe, further establishes the potential therapeutic value of the drug.

Differences in the occurrence of hypertension among (NZB X NZW)F1, MRL-lpr, and BXSB mice with lupus nephritis.

Lupus-prone (NZB X NZW)F1 (B X W) mice and MRL-lpr and BXSB mice were examined for the prevalence of hypertension and levels of plasma renin activity (PRA). Hypertension (greater than 145 mmHg) was observed only in female and male B X W mice with severe nephritis; in female MRL-lpr and male BXSB mice severe nephritis developed without blood pressure elevation (80-135 mmHg). The B X W parental strains, NZB and NZW, and the MRL-lpr congenic partners, MRL- +, did not become hypertensive as they aged. Other strains of mice, aged 3-32 months (A/HeN, BALB/cJ, BALB/cByJ, B10.S/Sg, B10.D2/ oSn , CBA/J, C3H/HeJ, SJL/J and [SJL X NZW]F1), also had normal blood pressure (98-122 mmHg). All mice with lupus nephritis had low PRA, even those with hypertension; furthermore, the MRL-lpr strain had low or undetectable PRA (2 +/- 1 ng/ml/hr), even when kidneys were normal. NZB, NZW, and MRL- + mice had normal PRA (10-16 ng/ml/hr). Thus, B X W mice frequently developed low renin hypertension during the last phase of their renal disease; whereas MRL-lpr and BXSB mice died from renal disease without observable increases in blood pressure.

Evidence for lipoxygenase activity in induction of histamine release from rat peritoneal mast cells by chelated iron.

The ferric iron-desferrioxamine B chelate effectively induced histamine release from rat peritoneal mast cells. The release was maximum at exogenous ferric iron concentrations of 10-100 microM, and the chelate was non-toxic, as determined by trypan blue uptake. In many aspects the chelate-induced histamine release paralleled IgE-mediated release. The kinetics, temperature, and Ca2+ dependence resembled antigen-induced release. Phosphatidylserine potentiated the release in Wistar rats but not in fawn-hooded rats, a strain which does not respond to phosphatidylserine potentiation. The chelate-induced histamine release was blocked by the metabolic inhibitors dinitrophenol, potassium cyanide, 2-deoxyglucose, and antimycin A. Lipoxygenase inhibitors also effectively blocked release, indicating an involvement of fatty acid metabolism via the lipoxygenase pathway. Free radical scavengers and antioxidants antagonistic to lipid peroxidation also inhibited the chelate-induced histamine release. Overall the data raise the possibility that endogenous cellular iron may be involved in the generation of free radicals and lipid peroxidation and that these may be early events in IgE-mediated release of histamine.

The orally active antiallergic compound, LC-6 (trans-2,3b,4,5,7,8b,9,10-octahydronaphtho[1,2-c:5,6-C] dipyrazole) inhibits the arachidonate lipoxygenase enzyme.

Trans-2,3,4,5,7,8b,9,10-octahydronaphtho[1,2-C:5,6-C]dipyrazole (LC-6), which is active in vivo when administered orally, inhibits histamine release from peritoneal rat mast cells and human basophils in vitro. LC-6.2Cl blocks both IgE-mediated and non-IgE-mediated histamine release. Histamine release induced by antigen, dextran, compound 48/80, or the Ca2+ ionophore A23187 was effectively inhibited. The in vitro data indicate that LC6.2HCl has the capacity to inhibit the mediator release phase of anaphylaxis. The drug has the ability to inhibit arachidonate lipoxygenase activity. The IC50 for IgE-mediated histamine release inhibition is in good agreement with the IC50 for inhibition of arachidonate lipoxygenase activity. The data indicate that LC-6 acts in vivo by blocking the release of mediators; its activity may be explained by its ability to stop the synthesis of lipoxygenase-derived arachidonic acid products.

Effect of sulfhydryl-reactive ATPase inhibitors upon mast cell and basophil activation.

Ethacrynic acid is an inhibitor of Ca2+-Mg2+-activated ATPases which also inhibits histamine release. By testing analogs of ethacrynic acid, the molecular structural requirements for ATPase inhibition and for inhibition of histamine release were compared. The results indicated that effective inhibition involves several structural features of the molecule. Analogs void of chlorine atoms were ineffective as inhibitors of histamine release and ATPase activity. Inhibition of both ATPase and histamine release requires a sulfhydryl-reactive olefinic bond, but sulfhydryl reactivity alone is not sufficient, as certain analogs which have a high capacity to react with sulfhydryl moieties were not active. Replacement of carboxy by highly ionized moieties like sulfo, rendered the molecules ineffective as an inhibitor of histamine release. Compounds which did not inhibit ATPase activity did not inhibit histamine release. The data indicate that mast cells and basophils require an intact ATPase system for histamine release, and raises the question of whether both ecto and endo ATPases are essential.

Spontaneous hypertension in fawn-hooded rats.

By 2 to 3 months of age, many fawn-hooded rats, particularly males, had indirect systolic blood pressures of greater than 145 mm Hg; by 4 months of age, most males had persistent elevations in blood pressure (greater than 160 mm Hg). Female fawn-hooded rats developed hypertension at 4-5 months of age. Concurrently, fawn-hooded rats develop a severe form of focal glomerular sclerosis. The causes of the hypertension and focal glomerular sclerosis are yet not known, nor has a relation been found between these abnormalities.

Plasma renin activity decrease precedes spontaneous focal glomerular sclerosis in aging rats.

An association between the activity of the renin-angiotensin system and the development of spontaneous focal glomerular sclerosis (FGS) in rats has been observed: the onset is preceded by a decrease in plasma renin activity (PRA). This observation was facilitated by the use of Fawn-hooded rats, which develop spontaneous FGS at an early age. Male Fawn-hooded rats develop severe FGS as early as 3 months of age. Male Wistar rats do not develop similar lesions until after 1 year of age. Correspondingly the PRA drops much sooner in Fawn-hooded than in Wistar rats. The low PRA appears to be due to low plasma renin rather than a limitation of the renin substrate, angiotensinogen, which appears to be present in the Fawn-hooded plasma in nonlimiting quantities. In the FH male rats renin content of the kidney drops only after severe glomerular pathology is evident, implying that the low PRA may be due to a decrease in renin secretion by the chromaffin cells of the juxtaglomerular apparatus.

Effect of inhibitors of arachidonic acid metabolism upon IgE and non-IgE-mediated histamine release.

Inhibitors of arachidonic acid metabolism were tested for their ability to block histamine release from human basophils. Eighteen inhibitors of lipoxygenases, cyclo-oxygenases, prostaglandin isomerases and thromboxane synthetases were tested. Agents inhibitory to the activity of lipoxygenases were effective blockers of IgE and non-IgE-mediated histamine release; agents antagonistic to cyclo-oxygenases, isomerases and thromboxane synthetases were not. These findings indicate that a functioning lipoxygenase pathway is essential for basophil activation and secretion and that the cyclo-oxygenase, isomerase and thromboxane synthetase pathways are not. Compounds antagonistic to phospholipase A also block histamine release, as does the intracellular Ca2+ antagonist TMB-8. The data are consistent with the idea that mast-cell and basophil activation and secretion involve phospholipase A generation of arachidonic acid, which is metabolized via the lipoxygenase pathway.

Histamine release from fawn-hooded rat mast cells is not potentiated by phosphatidylserine.

Histamine release from the peritoneal mast cells of the Fawn-hooded rat strain is not potentiated by phosphatidylserine (PS) or lysophosphatidylserine (Lyso-PS). When compared with Wistar rats the Fawn-hooded rats produce normal levels of IgE. Except for not being potentiated, the mast cells of Fawn-hooded rats release histamine similarly to Wistar rats when challenged with a variety of IgE and non-IgE mediated inducing agents. Mast cells of Fawn-hooded rats, however, do not respond significantly to dextran challenge which is not improved by PS of Lyso-PS. In the absence of PS the Ca2+ dependence of release from the Fawn-hooded mast cells is similar to the Wistar strain and both are similarly altered in their Ca2+ dependence by the presence of PS; with PS the mast cells of both rat strains release maximum percentages of histamine at levels of Ca2+ which are inhibitory in the absence of PS. The kinetics of release is similar in the two strains. However, when the mast cells are challenged with inducing agent in the absence of Ca2+ the rate of desensitization is slowed by PS in the Wistar strain but not the Fawn-hooded strain.

Creatine phosphokinase in rat mast cells.

The soluble cytoplasmic fraction of an homogenate from peritoneal rat mast cells, demonstrated a considerable amount of catalytic activity which promotes the transfer of phosphate from creatine phosphate to ADP. The plasma membrane, mitochondrial and microsomal fractions show negligible amounts of the catalyst. Enzyme activity is maximal at 37 degrees showing little activity below 17 degrees or above 45 degrees. The enzyme is strongly Mg2+-dependent, whereas it is only slightly activated by Ca2+. pH values between 7 and 8 are optimal and the enzyme is irreversibly inactivated below pH 4. The overall behaviour of the catalyst indicates it to be a creatine phosphokinase (CPK), an enzyme considered important to muscle and nerve tissues. The CPK is probably not encapsulated within the mast cells' perigranular membranes and is retained in the soluble cytoplasm during exocytosis. The possible role of CPK, as to whether it is assisting in maintaining proper levels of intracellular ATP during exocytosis, and/or whether it is associated with components of the mast cells' contractile apparatus, is discussed.

Ethacrynic acid inhibitable Ca2+ and Mg2+-activated membrane adenosine triphosphatase in rat mast cells.

A crude plasma membrane fraction from the homogenate of purified rat mast cells demonstrates a high degree of Ca2+-dependent and Mg2+-dependent adenosine triphosphatase (ATPase) activity. The microsomal and mitochondrial fractions show negligible amounts of the Ca2+ and Mg2+-activated ATPases. The broad ATPase inhibitor, ethacrynic acid, effectively blocks the mast cell ATPase activity while ouabain demonstrates little inhibitory effect. Correspondingly, ethacrynic acid inhibits histamine release from antigen-challenged mast cells while ouabain does not. Both ATPase inhibition and histamine release inhibition by ethacrynic acid require the presence of the olefinic bond in the ethacrynic acid molecule.