Anatomical analysis of peach palm (Bactris gasipaes) leaves cultivated in vitro, ex vitro and in vivo

The present work characterized and compared the anatomical structures of the leaves of Bactris gasipaes (Arecaceae) plants grown under different cultivation conditions (in vitro, ex vitro and in vivo) with the goal of identifying the origins of the difficulties encountered in acclimatizing micro-plants. The Quant program was used to determine leaf tissue thicknesses and areas, and histochemical tests were performed on leaf sections and analyzed using light microscopy. Stomatal and trichome densities were determined using the epidermal impression method and by scanning electronic microscopy. Our results indicated that there were no discernible alterations of the anatomical characteristics of the leaves of micro-plants cultivated under differing conditions and that the thickening of the mesophyll and the vascular fibers indicated adaptive responses to ex vitro conditions. As such, the observed difficulties in acclimatizing peach palm micro-plants to ex vitro conditions cannot be attributed to plant anatomical characteristics acquired during in vitro cultivation.

In vitro and in vivo studies demonstrate non-mutagenicity of the herbicide metolachlor

The herbicide metolachlor was evaluated for genotoxic potential. Metolachlor did not induce micronuclei in mice, however at 40 mg/kg it significantly decreased the percentage of polychromatic erythrocytes, which is a cytotoxic effect. Metolachlor did not induce chromosomal aberrations in human lymphocytes in vitro, but 2.0 mug/ml culture medium resulted in cytotoxicity, decreasing the mitotic index significantly. The indirect exposure test was carried out by adding plasma from metolachlor-pretreated rats to the human lymphocyte cultures. There was no indication of clastogenicity by metolachlor metabolites. On the other hand, plasma of cyclophosphamide-pretreated rats had a significant clastogenic effect

Glycosphingolipid antigens from Leishmania (L.) amazonensis amastigotes: Binding of anti-glycosphingolipid monoclonal antibodies in vitro and in vivo

Specific glycosphingolipid antigens of Leishmania (L.) amazonensis amastigotes reactive with the monoclonal antibodies (MoAbs) ST-3, ST-4 and ST-5 were isolated, and their structure was partially elucidated by negative ion fast atom bombardment mass spectrometry. The glycan moieties of five antigens presented linear sequences of hexoses and N-acetylhexosamines ranging from four to six sugar residues, and the ceramide moieties were found to be composed by a sphingosine d18:1 and fatty acids 24:1 or 16:0. Affinities of the three monoclonal antibodies to amastigote glycosphingolipid antigens were also analyzed by ELISA. MoAb ST-3 reacted equally well with all glycosphingolipid antigens tested, whereas ST-4 and ST-5 presented higher affinities to glycosphingolipids with longer carbohydrate chains, with five or more sugar units (slow migrating bands on HPTLC). Macrophages isolated from footpad lesions of BALB/c mice infected with Leishmania (L.) amazonensis were incubated with MoAb ST-3 and, by indirect immunofluorescence, labeling was only detected on the parasite, whereas no fluorescence was observed on the surface of the infected macrophages, indicating that these glycosphingolipid antigens are not acquired from the host cell but synthesized by the amastigote. Intravenous administration of 125I-labeled ST-3 antibody to infected BALB/c mice showed that MoAb ST-3 accumulated significantly in the footpad lesions in comparison to blood and other tissues

Interactions of ANP and ANG II in tubular nephron acidification

(ANP, 1 µM) on the kinetics of bicarbonate reabsorption in the rat middle proximal tubule, we performed in vivo experiments using a stopped-flow microperfusion technique with the determination of lumen pH by Sb microelectrodes. These studies confirmed that ANG II added to the luminal or peritubular capillary perfusion fluid stimulates proximal bicarbonate reabsorption and showed that ANP alone does not affect this process, but impairs the stimulation caused by ANG II. We also studied the effects and the interaction of these hormones in cortical distal nephron acidification. Bicarbonate reabsorption was evaluated by the acidification kinetic technique in early (ED) and late (LD) distal tubules in rats during in vivo stopped-flow microperfusion experiments. The intratubular pH was measured with a double-barreled microelectrode with H+-sensitive resin. The results indicate that ANG II acted by stimulating Na+/H+ exchange in ED (81%) and LD (54%) segments via activation of AT1 receptors, as well as vacuolar H+-ATPase in LD segments (33%). ANP did not affect bicarbonate reabsorption in either segment and, as opposed to what was seen in the proximal tubule, did not impair the stimulation caused by ANG II. To investigate the mechanism of action of these hormones in more detail, we studied cell pH dependence on ANG II and ANP in MDCK cells using the fluorescent probe BCECF. We showed that the velocity of cell pH recovery was almost abolished in the absence of Na+, indicating that it is dependent on Na+/H+ exchange. ANP (1 µM) alone had no effect on this recovery but reversed both the acceleration of H+ extrusion at low ANG II levels (1 pM and 1 nM), and inhibition of H+ extrusion at higher ANG II levels (100 nM). To obtain more information on the mechanism of interaction of these hormones, we also studied their effects on the regulation of intracellular free calcium concentration, [Ca2+]i, monitored with the fluorescent probe Fura-2 in MDCK cells in suspension. The data indicate that the addition of increasing concentrations of ANG II (1 pM to 1 µM) to the cell suspension led to a progressive increase in [Ca2+]i to 2-3 times the basal level. In contrast, the addition of ANP (1 µM) to the cell suspension led to a very rapid 60% decrease in [Ca2+]i and reduced the increase elicited by ANG II, thus modulating the effect of ANG II on [Ca2+]i. These results may indicate a role of [Ca2+]i in the regulation of the H+ extrusion process mediated by Na+/H+ exchange and stimulated/impaired by ANG II. The data are compatible with stimulation of Na+/H+ exchange by increases of [Ca2+]i in the lower range, and inhibition at high [Ca2+]i levels

Eosinophil-active chemokines: assessment of in vivo activity

The selective recruitment of eosinophils in tissue is a striking feature of allergic diseases. Recently, a family of chemoattractant molecules, namely chemokines, has been described which potently activates eosinophil function in vitro. We have developed a murine model of eosinophil recruitment to compare the relative potency and efficacy of chemokines in vivo. Of the chemokines tested, only eotaxin and MIP-1a induced significant accumulation of eosinophils in vivo, but eotaxin was more effective than MIP-1a. Chemokines, especially eotaxin acting via the CCR-3 receptor, may have a fundamental role in determining selective eosinophil recruitment in vivo

Renal effects of uroguanylin and guanylin in vivo

Uroguanylin and guanylin are newly discovered endogenous heat-stable peptides that bind to and activate a membrane bound guanylyl cyclase signaling receptor (termed guanylyl cyclase C; GC-C). These peptides are not only found in blood but are secreted into the lumen of the intestine and effect a net secretion of electrolytes (Na+, K+, Cl-, HCO3-) and fluid into the intestine via a cyclic guanosine-3',5'-monophosphate (cGMP) mechanism. GC-C is also the receptor for Escherichia coli heat-stable enterotoxin (STa) and activation by STa results in a diarrheal illness. Employing mouse renal in vivo models, we have demonstrated that uroguanylin, guanylin, and STa elicit natriuretic, kaliuretic, and diuretic effects. These biological responses are time- and dose-dependent. Maximum natriuretic and kaliuretic effects are observed within 30-40 min following infusion with pharmacological doses of the peptides in a sealed-urethra mouse model. Our mouse renal clearance model confirms these results and shows significant natriuresis following a constant infusion of uroguanylin for 30 min, while the glomerular filtration rate, plasma creatinine, urine osmolality, heart rate, and blood pressure remain constant. These data suggest the peptides act through tubular transport mechanisms. Consistent with a tubular mechanism, messenger RNA-differential display PCR of kidney RNA extracted from vehicle- and uroguanylin-treated mice show the message for the Na+/K+ ATPase g-subunit is down-regulated. Interestingly, GC-C knockout mice (Gucy2c -/-) also exhibit significant uroguanylin-induced natriuresis and kaliuresis in vivo, suggesting the presence of an alternate receptor signaling mechanism in the kidney. Thus, uroguanylin and guanylin seem to serve as intestinal and renal natriuretic peptide-hormones influencing salt and water transport in the kidney through GC-C dependent and independent pathways. Furthermore, our recent clinical probe study has revealed a 70-fold increase in levels of urinary uroguanylin in patients with congestive heart failure. In conclusion, our studies support the concept that uroguanylin and guanylin are endogenous effector peptides involved in regulating body salt and water homeostasis.

Determination of macromolecular exchange and PO2 in the microcirculation: a simple system for in vivo fluorescence and phosphorescence videomicroscopy

We have developed a system with two epi-illumination sources, a DC-regulated lamp for transillumination and mechanical switches for rapid shift of illumination and detection of defined areas (250-750 µm²) by fluorescence and phosphorescence videomicroscopy. The system permits investigation of standard microvascular parameters, vascular permeability as well as intra- and extravascular PO2 by phosphorescence quenching of Pd-meso-tetra (4-carboxyphenyl) porphine (PORPH). A Pechan prism was used to position a defined region over the photomultiplier and TV camera. In order to validate the system for in vivo use, in vitro tests were performed with probes at concentrations that can be found in microvascular studies. Extensive in vitro evaluations were performed by filling glass capillaries with solutions of various concentrations of FITC-dextran (diluted in blood and in saline) mixed with different amounts of PORPH. Fluorescence intensity and phosphorescence decay were determined for each mixture. FITC-dextran solutions without PORPH and PORPH solutions without FITC-dextran were used as references. Phosphorescence decay curves were relatively unaffected by the presence of FITC-dextran at all concentrations tested (0.1 µg/ml to 5 mg/ml). Likewise, fluorescence determinations were performed in the presence of PORPH (0.05 to 0.5 mg/ml). The system was successfully used to study macromolecular extravasation and PO2 in the rat mesentery circulation under controlled conditions and during ischemia-reperfusion.

Role of Tamm-Horsfall protein in the binding and in vivo phagocytosis of type 1 fimbriated Escherichia coli by mouse peritoneal macrophages

Tamm-Horsfall glycoprotein (THP) contains manno-oligosaccharides that are recognized by type 1 fimbriae (F1) of Escherichia coli. In the present study, we examined the in vivo phagocytic activity of mouse peritoneal macrophages after treatment of bacteria with THP. At low THP concentrations (12.5 µg/ml and 50 µg/ml) no significant difference was observed in the phagocytosis of E. coli F1+. However, at high THP concentrations (500 µg/ml and 1250 µg/ml) we obtained a reduction of bacterial phagocytosis by mouse peritoneal macrophages.

In vivo and in vitro effect of imipramine and fluoxetine on Na+,K+-ATPase activity in synaptic plasma membranes from the cerebral cortex of rats

The effects of in vivo chronic treatment and in vitro addition of imipramine, a tricyclic antidepressant, or fluoxetine, a selective serotonin reuptake inhibitor, on the cortical membrane-bound Na+,K+-ATPase activity were studied. Adult Wistar rats received daily intraperitoneal injections of 10 mg/kg of imipramine or fluoxetine for 14 days. Twelve hours after the last injection rats were decapitated and synaptic plasma membranes (SPM) from cerebral cortex were prepared to determine Na+,K+-ATPase activity. There was a significant decrease (10%) in enzyme activity after imipramine but fluoxetine treatment caused a significant increase (27%) in Na+,K+-ATPase activity compared to control (P<0.05, ANOVA; N = 7 for each group). When assayed in vitro, the addition of both drugs to SPM of naive rats caused a dose-dependent decrease in enzyme activity, with the maximal inhibition (60-80%) occurring at 0.5 mM. We suggest that a) imipramine might decrease Na+,K+-ATPase activity by altering membrane fluidity, as previously proposed, and b) stimulation of this enzyme might contribute to the therapeutic efficacy of fluoxetine, since brain Na+,K+-ATPase activity is decreased in bipolar patients.

Comparison of the homeostasis model assessment and quantitative insulin sensitivity check index with data from forearm metabolic studies for the in vivo assessment of insulin sensitivity

The present study was designed to compare the homeostasis model assessment (HOMA) and quantitative insulin sensitivity check index (QUICKI) with data from forearm metabolic studies of healthy individuals and of subjects in various pathological states. Fifty-five healthy individuals and 112 patients in various pathological states, including type 2 diabetes mellitus, essential hypertension and others, were studied after an overnight fast and for 3 h after ingestion of 75 g of glucose, by HOMA, QUICKI and the forearm technique to estimate muscle uptake of glucose combined with indirect calorimetry (oxidative and non-oxidative glucose metabolism). The patients showed increased HOMA (1.88 ± 0.14 vs 1.13 ± 0.10 pmol/l x mmol/l) and insulin/glucose (I/G) index (1.058.9 ± 340.9 vs 518.6 ± 70.7 pmol/l x (mg/100 ml forearm)-1), and decreased QUICKI (0.36 ± 0.004 vs 0.39 ± 0.006 (µU/ml + mg/dl)-1) compared with the healthy individuals. Analysis of the data for the group as a whole (patients and healthy individuals) showed that the estimate of insulin resistance by HOMA was correlated with data obtained in the forearm metabolic studies (glucose uptake: r = -0.16, P = 0.04; non-oxidative glucose metabolism: r = -0.20. P = 0.01, and I/G index: r = 0.17, P = 0.03). The comparison of QUICKI with data of the forearm metabolic studies showed significant correlation between QUICKI and non-oxidative glucose metabolism (r = 0.17, P = 0.03) or I/G index (r = -0.37, P < 0.0001). The HOMA and QUICKI are good estimates of insulin sensitivity as data derived from forearm metabolic studies involving direct measurements of insulin action on muscle glucose metabolism.

Effect of Sapindus trifoliatus on hyperalgesic in vivo migraine models

Phytotherapies have offered alternative sources of therapy for migraine and gained much importance in prophylactic treatment. Sapindus trifoliatus is a medium-sized deciduous tree growing wild in south India that belongs to the family Sapindaceae. The pericarp is reported for various medicinal properties. A thick aqueous solution of the pericarp is used for the treatment of hemicrania, hysteria or epilepsy in folklore medicine. We have investigated the antihyperalgesic effects of the lyophilized aqueous extract of S. trifoliatus in animal models predictive of experimental migraine models using morphine withdrawal-induced hyperalgesia on the hot-plate test and on 0.3% acetic acid-induced abdominal constrictions in adult male Swiss albino mice. The extract significantly (N = 10, P < 0.05) increased the licking latency in the hot-plate test when administered ip at 10 mg/kg (6.70 ± 0.39 s in saline control vs 18.76 ± 0.96 s in S. trifoliatus-treated animals) and significantly (N = 10, P < 0.001) reduced the abdominal constrictions when administered ip at 2 and 10 mg/kg (40.20 ± 1.36 in saline control vs 30.20 ± 1.33 and 23.00 ± 0.98 for 2 and 10 mg/kg, ip, respectively, in S. trifoliatus-treated animals). Furthermore, when administered ip at 20 and 100 mg/kg, the extract significantly (N = 10, P < 0.05) inhibited the apomorphine-induced climbing behavior in mice (climbing duration 15.75 ± 5.0 min for saline control vs 11.4 ± 1.28 and 3.9 ± 1.71 min for 20 and 100 mg/kg, respectively, in S. trifoliatus-treated animals). In receptor radioligand-binding studies, the extract exhibited affinity towards D2 receptors. The findings suggest that dopamine D2 antagonism could be the mechanism involved in the antihyperalgesic activity of the aqueous extract of S. trifoliatus.

Lycopene and ß-carotene protect in vivo iron-induced oxidative stress damage in rat prostate

It has been suggested that iron overload may be carcinogenic. In the present study, we evaluated the effect of plasma and prostate carotenoid concentration on oxidative DNA damage in 12-week-old Wistar rats treated with intraperitoneal (ip) ferric nitrilotriacetate (Fe-NTA) (10 mg Fe/kg). Plasma ß-carotene and lycopene concentrations were measured as a function of time after ip injection of carotenoids (10 mg kg-1 day-1 ß-carotene or lycopene) in rats. The highest total plasma concentration was reached 3 and 6 h after ip injection of lycopene or ß-carotene, respectively. After 5 days of carotenoid treatment, lycopene and ß-carotene were present in the 0.10-0.51 nmol/g wet tissue range in the prostate. Using a sensitive method to detected 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) by HPLC/EC, the level of 8-oxodGuo in rat prostate DNA was significantly higher (6.3 ± 0.6 residues/10(6) dGuo) 3 h after Fe-NTA injection compared with control rats (1.7 ± 0.3 residues/10(6) dGuo). Rats supplemented with lycopene or ß-carotene for 5 days prior to Fe-NTA treatment showed a reduction of about 70% in 8-oxodGuo levels to almost control levels. Compared with control rats, the prostate of Fe-NTA-treated animals showed a 78% increase in malondialdehyde accumulation. Lycopene or ß-carotene pre-treatment almost completely prevented lipid damage. Epidemiological studies have suggested a lower risk of prostate cancer in men reporting a higher consumption of tomato products. However, before associating this effect with tomato sauce constituents, more information is required. The results described here may contribute to the understanding of the protective effects of carotenoids against iron-induced oxidative stress.

In vivo growth-inhibition of Sarcoma 180 by piplartine and piperine, two alkaloid amides from Piper

Piplartine {5,6-dihydro-1-[1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]-2(1H)pyridinone} and piperine {1-5-(1,3)-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]piperidine} are alkaloid amides isolated from Piper. Both have been reported to show cytotoxic activity towards several tumor cell lines. In the present study, the in vivo antitumor activity of these compounds was evaluated in 60 female Swiss mice (N = 10 per group) transplanted with Sarcoma 180. Histopathological and morphological analyses of the tumor and the organs, including liver, spleen, and kidney, were performed in order to evaluate the toxicological aspects of the treatment with these amides. Administration of piplartine or piperine (50 or 100 mg kg-1 day-1 intraperitoneally for 7 days starting 1 day after inoculation) inhibited solid tumor development in mice transplanted with Sarcoma 180 cells. The inhibition rates were 28.7 and 52.3% for piplartine and 55.1 and 56.8% for piperine, after 7 days of treatment, at the lower and higher doses, respectively. The antitumor activity of piplartine was related to inhibition of the tumor proliferation rate, as observed by reduction of Ki67 staining, a nuclear antigen associated with G1, S, G2, and M cell cycle phases, in tumors from treated animals. However, piperine did not inhibit cell proliferation as observed in Ki67 immunohistochemical analysis. Histopathological analysis of liver and kidney showed that both organs were reversibly affected by piplartine and piperine treatment, but in a different way. Piperine was more toxic to the liver, leading to ballooning degeneration of hepatocytes, accompanied by microvesicular steatosis in some areas, than piplartine which, in turn, was more toxic to the kidney, leading to discrete hydropic changes of the proximal tubular and glomerular epithelium and tubular hemorrhage in treated animals.

Nucleotide-sugar transporters: structure, function and roles in vivo

The glycosylation of glycoconjugates and the biosynthesis of polysaccharides depend on nucleotide-sugars which are the substrates for glycosyltransferases. A large proportion of these enzymes are located within the lumen of the Golgi apparatus as well as the endoplasmic reticulum, while many of the nucleotide-sugars are synthesized in the cytosol. Thus, nucleotide-sugars are translocated from the cytosol to the lumen of the Golgi apparatus and endoplasmic reticulum by multiple spanning domain proteins known as nucleotide-sugar transporters (NSTs). These proteins were first identified biochemically and some of them were cloned by complementation of mutants. Genome and expressed sequence tag sequencing allowed the identification of a number of sequences that may encode for NSTs in different organisms. The functional characterization of some of these genes has shown that some of them can be highly specific in their substrate specificity while others can utilize up to three different nucleotide-sugars containing the same nucleotide. Mutations in genes encoding for NSTs can lead to changes in development in Drosophila melanogaster or Caenorhabditis elegans, as well as alterations in the infectivity of Leishmania donovani. In humans, the mutation of a GDP-fucose transporter is responsible for an impaired immune response as well as retarded growth. These results suggest that, even though there appear to be a fair number of genes encoding for NSTs, they are not functionally redundant and seem to play specific roles in glycosylation.

In vivo antithrombotic properties of a heparin from the oocyte test cells of the sea squirt Styela plicata(Chordata-Tunicata)

In the ascidian Styela plicata, the oocytes are surrounded by two types of accessory cells named follicle cells and test cells. A heparin-like substance with an anticoagulant activity equivalent to 10% of mammalian heparin and about 5% as potent as the mammalian counterpart for the inhibition of thrombin by antithrombin was isolated from the oocyte test cells. In the present study, we compared the antithrombotic and hemorrhagic effects of sea squirt oocyte test cell heparin with those of porcine heparin in rat models of venous thrombosis and blood loss. Intravenous administration of the oocyte test cell heparin to Wistar rats (both sexes, weighing ~300 g, N = 4 in each group) at a dose of 5.0 mg/kg body weight, which produced a 1.8-fold increase in plasma activated partial thromboplastin time, inhibited thrombosis by 45 ± 13.5% (mean ± SD) without any bleeding effect. The same dose of porcine heparin inhibited thrombosis by 100 ± 1.4%, but produced a blood loss three times greater than that of the saline-treated control. However, 10-fold reduction of the dose of porcine heparin to 0.5 mg/kg body weight, which produced a 5-fold increase in plasma-activated partial thromboplastin time, inhibited thrombosis by 70 ± 13% without any bleeding effect. The antithrombotic properties of a new heparin isolated from test cells of the sea squirt S. plicata, reported here for the first time, indicate that, although sea squirt oocyte test cell heparin was a poor anticoagulant compared to porcine heparin, it had a significant antithrombotic effect without causing bleeding.