Concentration-dependent Stimulatory Effect Research Articles

Collagen/PCL Nanofibers Electrospun in Green Solvent by DOE Assisted Process. An Insight into Collagen Contribution.

Collagen, thanks to its biocompatibility, biodegradability and weak antigenicity, is widely used in dressings and scaffolds, also as electrospun fibers. Its mechanical stability can be improved by adding polycaprolactone (PCL), a synthetic and biodegradable aliphatic polyester. While previously collagen/PCL combinations were electrospun in solvents such as hexafluoroisopropanol (HFIP) or trifluoroethanol (TFE), more recently literature describes collagen/PCL nanofibers obtained in acidic aqueous solutions. A good morphology of the fibers represents in this case still a challenge, especially for high collagen/PCL ratios. In this work, thanks to preliminary rheological and physicochemical characterization of the solutions and to a Design of Experiments (DOE) approach on process parameters, regular and dimensionally uniform fibers were obtained with collagen/PCL ratios up to 1:2 and 1:1 w/w. Collagen ratio appeared relevant for mechanical strength of dry and hydrated fibers. WAXS and FTIR analysis showed that collagen denaturation is related both to the medium and to the electrospinning process. After one week in aqueous environment, collagen release was complete and a concentration dependent stimulatory effect on fibroblast growth was observed, suggesting the fiber suitability for wound healing. The positive effect of collagen on mechanical properties and on fibroblast biocompatibility was confirmed by a direct comparison of nanofiber performance after collagen substitution with gelatin.

Impact of Zinc Oxide Nanoparticles Amended Organic Manure on Arachis hypogaea Growth Response and Rhizosphere Bacterial Community

The effect of zinc oxide nanoparticle-organic manure amended ultisol and loam soils on plant growth response and rhizosphere bacterial community of peanut (Arachis hypogaea) was evaluated using standard methods under greenhouse conditions. Results indicate germination rates ranged between 30 and 100% in the amended soils compared to 50 and 70% in the controls. ZnO nanoparticles exerted concentration-dependent and varying effects on the plant root and shoot lengths, weights, nodules and pod formation in the two soil types. Heterotrophic bacterial counts ranged from 7.21 ± 0.51 to 7.38 ± 0.5 Log10CFUg-1 in the amended ultisol and 6.99 ± 0.55 Log10CFUg-1 in the control with a log reduction to 6.70 ± 0.39 Log10CFUg-1 in 500 mgkg⁻¹ ZnO spiked soil. Counts in the amended loam soil ranged between 6.59 ± 0.48 and 7.22 ± 0.41 Log10CFUg-1 relative to 6.80 ± 0.58 Log10CFUg-1 in the control. ZnO induced concentration-dependent effect on oxygen uptake rate relative to the controls. The organisms were members of the genera Lactobacillus, Pseudomonas, Bacillus, Rhizobium, Xanthobacter, Enterobacter, Citrobacter, Nitrosomonas and Agromyces. ZnO nanoparticle exerted concentration-dependent stimulatory and inhibitory effects on the plant growth response, oxygen uptake rate and induced temporal shifts in soil microbial abundance. It is challenging to generalize a consistent response of the plant or microorganisms because ZnO nanoparticles interacted with A. hypogaea and soil bacterial community in ways that differ in the ultisol and loam soil.

Oxidative damage to membrane lipids in the thyroid – no differences between sexes

It has long been observed that thyroid diseases are more prevalent in women than in men. However, there are limited experimental data demonstrating mechanisms by which sex differences in thyroid diseases may occur and exact molecular mechanisms involved are still far from clear. The aim of the study was to evaluate if there are sex differences concerning oxidative damage to membrane lipids in thyroid homogenates in response to Fenton reaction substrates, i.e., Fe2+ and/or H2O2, and, additionally, in response to potentially protective agent, i.e., melatonin. Homogenates of male or female thyroids collected from adult swine (Sus scrofa domesticus) at slaughter were incubated in the presence of H2O2 and/or Fe2+ without or with addition of melatonin. Malondialdehyde + 4-hydroxyalkenals concentration (LPO index) was measured spectrophotometrically. Neither H2O2 nor Fe2+, when used separately, did affect the level of lipid peroxidation in both male and female porcine thyroid homogenates. When H2O2 (0.5 mM) was used together with different concentrations of Fe2+, the level of lipid peroxidation increased significantly in both male and female porcine thyroid homogenates, with clear Fe2+ concentration-dependent stimulatory effect, but without differences between sexes. No sex-specific differences was found concerning oxidative damage to membrane lipids in porcine thyroid in response to Fenton reaction substrates and/or to melatonin. The lack of expected differences may be due to potentially lower sensitivity of membrane lipids comparing to other biological macromolecules to pro-/antioxidative agents in the thyroid. However, further studies should be performed to explain the discussed issue.

Bifidogenic properties of cell-free extracts derived from probiotic strains of Bifidobacterium bifidum and Lactobacillus reuteri

Comprehensive study of the biological activity of structural components and metabolites of “beneficial” microorganisms opens the prospects of efficient and rational use of their biotechnological potential in the correction of microecological and related disorders. The study tested proliferative activity and biofilm formation by Bifidobacterium bifidum probiotic strain under the influence of cell-free extracts containing structural components and metabolites of the probiotic strains of B. bifidum and Lactobacillus reuteri. Cell-free extracts were obtained by disintegrating suspensions of probiotic cells by cyclic freezing-thawing, cultivating probiotic microorganisms in their own disintegrates and subsequent filtration of the obtained disintegrates and cultures. The proliferative activity and biofilm formation of the probiotic test culture were studied by spectrophotometric microtiter plate method with 10%vol, 30%vol and 50%vol content of cell-free extracts in the cultivation medium. All investigated extracts showed a significant concentration-dependent stimulatory effect on the proliferative activity of B. bifidum. According to the degree of stimulatory effect on the B. bifidum proliferation, cell-free extracts arranged in ascending order: MLG (filtrate of L. reuteri culture, grown in L. reuteri disintegrate supplemented with 0.8 M glycerol and 0.4 M glucose) < MB (filtrate of В. bifidum culture, grown in В. bifidum disintegrate) < B (filtrate of В. bifidum disintegrate) < ML (filtrate of L. reuteri culture, grown in L. reuteri disintegrate) < L (filtrate of L. reuteri disintegrate). With the same content in the culture medium, filtrates of disintegrates had a more pronounced stimulatory effect than filtrates of cultures grown in their own disintegrates. Cell-free extracts from L. reuteri (L and ML) exerted a more pronounced stimulatory effect than cell-free extracts from B. bifidum. Not all studied cell-free extracts stimulated the biofilm formation by B. bifidum. The effect of cell-free extracts on this process depended on their type and concentration. Extract L had a predominantly inhibitory effect on biofilm formation by B. bifidum. The most pronounced stimulatory effect on biofilm formation by B. bifidum came from extract MLG. ML, B and MB extracts stimulated this process approximately equally. The detection of significant bifidogenic effect of the studied cell-free extracts may contribute to their pharmaceutical applications. Cell-free extracts can be used as metabiotics or prebiotics for increasing the survival of the injected probiotic, facilitating its inoculation in the gastrointestinal tract when used together. The obtained data encourage further careful study of the biochemical composition of cell-free extracts and efforts to clarify the mechanism of their action.

Prokinetic activity of Prunus persica (L.) Batsch flowers extract and its possible mechanism of action in rats.

The peach tree, Prunus persica (L.) Batsch, is widely cultivated in China, and its flowers have been used for centuries in traditional Chinese medicine to treat gut motility disorders. But few studies have explored the pharmacological effect of Prunus persica (L.) Batsch flowers on gastrointestinal motility. In this study, the activities of different extracts from Prunus persica (L.) Batsch flowers on the smooth muscle contractions were evaluated using isolated colon model, and the ethyl acetate extract (EAE) showed the strongest effects in vitro. EAE (10−8–10−5 g/mL) caused a concentration-dependent stimulatory effect in rat colonic tissue. Additionally, ketotifen (100 µM), cimetidine (10 µM), and pyrilamine (1 µM) produced a significant inhibition of contractions caused by EAE. Furthermore, immunofluorescence and toluidine blue staining revealed increased numbers of mast cells in the EAE group, and EAE increased histamine release from the colonic tissues. These data indicate that EAE has significant prokinetic activity and acts by a mechanism that mainly involves mast cell degranulation. Our study provides a pharmacological basis for the use of an extract of Prunus persica (L.) Batsch flowers in the treatment of gut motility disorders.

Glucoxylan-mediated green synthesis of gold and silver nanoparticles and their phyto-toxicity study

A green synthesis of gold and silver nanoparticles having exceptional high stability is reported. The synthesis involves the use of glucoxylans isolated from seeds of Mimosa pudica and excludes the use of conventional reducing and capping agents. The average particle sizes were 40 and 6nm for gold and silver, respectively. The size of gold particles obtained in this work is suitable for drug delivery as they are non-cytotoxic. In phyto-toxicity tests the gold and silver nanoparticles did not show any significant effect on germination of radish seeds, whereas in radish seedling root growth assay the two particles behaved differently. The silver nanoparticles exhibited a concentration-dependent stimulatory effect on root length, whereas the gold nanoparticles had no significant effect in this test. The likely mechanism of these effects is discussed.

Species and tissue-specificity of prokinetic, laxative and spasmodic effects of Fumaria parviflora

BackgroundFumaria parviflora Linn. (Fumariaceae), is a small branched annual herb found in many parts of the world including Saudi Arabia and Pakistan. This study was designed to provide pharmacological basis for the medicinal use of Fumaria parviflora in gut motility disorders.MethodsThe in-vivo prokinetic and laxative assays were conducted in mice. Isolated intestinal preparations (ileum and jejunum) from different animal species (mouse, guinea-pig and rabbit) were separately suspended in tissue baths containing Tyrode's solution bubbled with carbogen and maintained at 37°C. The spasmogenic responses were recorded using isotonic transducers coupled with PowerLab data acquisition system.ResultsThe aqueous-methanol extract of Fumaria parviflora (Fp.Cr), which tested positive for the presence of alkaloids, saponins, tannins and anthraquinones showed partially atropine-sensitive prokinetic and laxative activities in the in-vivo in mice at 30 and 100 mg/kg. In the in-vitro studies, Fp.Cr (0.01-1 mg/ml) caused a concentration-dependent atropine-sensitive stimulatory effect both in mouse tissues (jejunum and ileum), and rabbit jejunum but had no effect in rabbit ileum. In guinea-pig tissues (ileum and jejunum), the crude extract showed a concentration-dependent stimulatory effect with higher efficacy in ileum and the effect was partially blocked by atropine, indicating the involvement of more than one types of gut-stimulant components (atropine-sensitive and insensitive). This could be a plausible reason for the greater efficacy of Fp.Cr in gut preparations of guinea-pig than in rabbit or mouse.ConclusionsThis study shows the prokinetic, laxative and spasmodic effects of the plant extract partially mediated through cholinergic pathways with species and tissue-selectivity, and provides a sound rationale for the medicinal use of Fumaria parviflora in gut motility disorders such as, indigestion and constipation. This study also suggests using different species to know better picture of pharmacological profile of the test material.

The slower the better: how sperm capacitation and acrosome reaction is modified in the presence of estrogens

In order for mammalian sperm to obtain a fertilizing ability, they must undergo a complex of molecular changes, called capacitation. During capacitation, steroidal compounds can exert a fast nongenomic response in sperm through their interaction with plasma membrane receptors, and activate crucial signaling pathways leading to time-dependent protein tyrosine phosphorylation (TyrP). Estrogen receptor beta was detected in epididymal mouse sperm; therefore, the effect of 17B-estradiol, estrone, estriol, and 17A-ethynylestradiol on mouse sperm capacitation in vitro was investigated. The effect was evaluated by positive TyrP in sperm heads and in the whole sperm lysates. Simultaneously, the state of the acrosome after the calcium ionophore-induced acrosome reaction was assessed. Generally, estrogens displayed a time and concentration-dependent stimulatory effect on sperm TyrP during capacitation. In contrast, the number of sperm that underwent the acrosome reaction was lower in the experimental groups. It has been demonstrated that both natural and synthetic estrogens can modify the physiological progress of mouse sperm capacitation. The potential risk in the procapacitation effect of estrogens can also be seen in the decreased ability of sperm to undergo the acrosome reaction. In conclusion, the capacitating ability of sperm can be significantly lowered by increasing the level of estrogens in the environment.

Prokinetic and laxative activities of Lepidium sativum seed extract with species and tissue selective gut stimulatory actions

To provide ethnopharmacological basis for the medicinal use of Lepidium sativum seeds in indigestion and constipation. The in vivo studies were conducted in mice, while isolated tissues of mouse, guinea-pig and rabbit were suspended in tissue bath to measure isotonic contractions. The aqueous-methanolic extract of Lepidium sativum seeds (Ls.Cr) at 30 and 100mg/kg showed atropine-sensitive prokinetic and laxative activities in mice, which were partially sensitive to atropine. In isolated gut preparations of mouse and guinea-pig, Ls.Cr (0.1-1mg/mL) caused a concentration-dependent stimulatory effects both in jejunum and ileum, which was blocked in the presence of atropine. In rabbit jejunum, the stimulant effect of Ls.Cr remained unchanged in the presence of atropine, pyrilamine or SB203186, while in rabbit ileum, the stimulatory effect was partially blocked by atropine. The Ls.Cr was more efficacious in gut preparations of rabbit than in guinea-pig or mouse. The phytochemical analysis of the plant extract detected alkaloids, saponins and anthraquinones as plant constituents. This study showed the prokinetic and laxative effects of Lepidium sativum in mice, which were partially mediated through a cholinergic pathway. The in vitro spasmodic effect of the plant extract mediated through a similar mechanism with species and tissue-selectivity, provides a rationale for the medicinal use of the seeds of Lepidium sativum in indigestion and constipation, and suggests studying the plant extracts on more than one species to get the wider picture.

Effect of salts on luminescence of natural and recombinant luminescent bacterial biosensors

Effect of cations K+, Na+, Mg2+, and Ca2+ and anions SO4(2-), HCO3(-), and CO3(2-) on the luminescence intensity of the marine luminescent bacterium Photobacterium phorphoreum (Microbiosensor B-17 677f) and the recombinant strain Escherichia coli with cloned lux operon of P. leiognathi (Ekolyum-9). It is found that small concentrations of chlorides and sulfates of the cations studied had a concentration-dependent stimulatory effect on bacterial bioluminescence; as the concentration of agents increased, activation was succeeded by quenching. The strength of the inhibitory effect, which is characterized by EC50, decreased in the series Ca2+ > Na+ > Mg2+ > K+. Carbonates and hydrocarbonates had a pronounced inhibitory effect on the bioluminescence intensity, determined by an increase in pH. We showed that some types of highly mineralized water with a high hydrocarbonate content have a marked inhibitory effect on the luminescence intensity of microbial luminescent biosensors, mimicking the effect of chemical pollutants.

Stanniocalcin-1 secretion and receptor regulation in kidney cells

Kidney collecting duct principal cells are the main source of stanniocalcin-1 (STC-1) production and secretion. From there, the hormone targets thick ascending limb and distal convoluted tubule cells, as well as collecting duct cells. More specifically, STC-1 targets their mitochondria to exert putative antiapoptotic effects. Two distal tubule cell lines serve as models of STC-1 production and/or mechanism of action. Madin-Darby canine kidney-1 (MDCK-1) cells mimic collecting duct cells in their synthesis of STC-1 ligand and receptor, whereas inner medullary collecting duct-3 (IMCD-3) cells respond to additions of STC-1 by increasing their respiration rate. In the present study, MDCK cell STC-1 secretion was examined under normal and hypertonic conditions, vectorally, and in response to hormones and signal transduction pathway activators/inhibitors. STC-1 receptor regulation was monitored in both cell lines in response to changing ligand concentration. The results showed that NaCl-induced hypertonicity had concentration-dependent stimulatory effects on STC-1 secretion, as did the PKC activator TPA. Calcium and ionomycin were inhibitory, whereas calcium receptor agonists had no effect. Angiotensin II, aldosterone, atrial natriuretic factor, antidiuretic hormone, and forskolin also had no effects. Moreover, STC-1 secretion exhibited no vectoral preference. STC-1 receptors were insensitive to homologous downregulation in both cell lines. In contrast, they were upregulated when STC-1 secretion was inhibited by calcium. The findings suggest that hypertonicity-induced STC-1 secretion is regulated through PKC activation and that high intracellular calcium levels are a potent inhibitor of release. More intriguingly, the results suggest that the receptor may not accompany STC-1 in its passage to the mitochondria.

Substance P regulates the expression of matrix metalloproteinases and tissue inhibitors of metalloproteinase in cultured human gingival fibroblasts

Substance P may play a role in the pathogenesis of periodontal disease; however, its mechanisms of modulation are not clear. This study evaluated the effect of two concentrations of Substance P on the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in cultured human gingival fibroblasts. Fibroblasts were stimulated for 48 h with 10(-4) or 10(-9) m Substance P; untreated fibroblasts served as controls. The expression of MMP-1, -2, -3, -7 and -11 and of TIMP-1 and -2 was evaluated using real-time polymerase chain reaction and western blotting. There was a significant, concentration-dependent stimulatory effect of Substance P on MMP-1, -2, -3 and -7 and TIMP-2 gene expression (p < 0.05), and a probable effect on MMP-11 (p = 0.06). At the higher concentration (10(-4) m Substance P), MMP-1, -2, -3, -7 and -11 and TIMP-2 showed the greatest up-regulation; at the lower concentration (10(-9) m Substance P), MMP-1, -3 and -7 and TIMP-2 exhibited diminished up-regulation, with MMP-2 and -11 showing down-regulation (p < 0.05). Expression of TIMP-1 was not affected by Substance P (p > 0.05). Western blotting confirmed that Substance P up-regulated MMP-1, -2, -3 and -11 and TIMP-2. MMP-1, -3 and -11 and TIMP-2 showed greater up-regulation at the higher Substance P concentration and diminished up-regulation at the lower concentration. MMP-2 was up-regulated to a similar degree at both Substance P concentrations. In gingival fibroblast cells, Substance P at the higher concentration (10(-4) m) induced greater up-regulation of MMP-1, -3 and -11 and TIMP-2 expression, but at the lower concentration (10(-9) m) induced diminished up-regulation, which may represent a mechanism for modulating periodontal breakdown.

Insulin Releasing Properties of the Temporin Family of Antimicrobial Peptides

Temporin-1Vb, -1Oe, -1DRb, and -1TGb (10(-8) -10(-6)M) produced significant (p<0.05) and concentration-dependent stimulatory effects on insulin secretion from clonal rat BRIN-BD11 cells without increased release of lactate dehydrogenase. Temporin-1Va and temporin-1Vc (10(-8) - 10(-6)M) also stimulated insulin-release but were cytotoxic at 10(-6)M. Temporin-1DRa was without effect. The temporins at 10(-7) M had no effect on intracellular calcium concentrations suggesting that they stimulate insulin release via a K(ATP) channel- independent pathway.

The respiratory effects of stanniocalcin-1 (STC-1) on intact mitochondria and cells: STC-1 uncouples oxidative phosphorylation and its actions are modulated by nucleotide triphosphates

Stanniocalcin-1 (STC-1) is one of only a handful of hormones that are targeted to mitochondria. High affinity receptors for STC-1 are present on cytoplasmic membranes and both the outer and inner mitochondrial membranes of nephron cells and hepatocytes. In both cell types, STC-1 is also present within the mitochondrial matrix and receptors presumably enable its sequestration. Furthermore, studies in bovine heart sub-mitochondrial particles have shown that STC-1 has concentration-dependent stimulatory effects on electron transport chain activity. The aim of the present study was to determine if the same effects could be demonstrated in intact, respiring mitochondria. At the same time, we also sought to demonstrate the functionality, if any, of an ATP binding cassette that has only recently been identified within the N-terminus of STC-1 by Prosite analysis. Intact, respiring mitochondria were isolated from rat muscle and liver and exposed to increasing concentrations of recombinant human STC-1 (STC-1). Following a 1 h exposure to 500 nM STC-1, mitochondria from both organs displayed significant increases in respiration rate as compared to controls. Moreover, STC-1 uncoupled oxidative phosphorylation as ADP:O ratios were significantly reduced in mitochondria from both tissues. The resulting uncoupling was correlated with enhanced mitochondrial 45Ca uptake in the presence of hormone. Respiratory studies were also conducted on a mouse inner medullary collecting cell line, where STC-1 had time and concentration-dependent stimulatory effects within the physiological range. In the presence of nucleotide triphosphates such as ATP and GTP (5 mM) the respiratory effects of STC-1 were attenuated or abolished. Receptor binding studies revealed that this was due to a four-fold decrease in binding affinity (KD) between ligand and receptor. The results suggest that STC-1 stimulates mitochondrial electron transport chain activity and calcium transport, and that these effects are negatively modulated by nucleotide triphosphates.

Functional Analysis of Mouse and Monkey Multidrug Resistance-Associated Protein 2 (Mrp2)

We investigated the intrinsic transport activity of mouse and monkey Mrp2 and compared it with that of rat and dog Mrp2 reported previously. Mrp2 cDNAs were isolated from BALB/c and Macaca fascicularis liver, respectively, and vesicle transport studies were performed using recombinant Mrp2s expressed in insect Sf9 cells. ATP-dependent transport of [3H]leukotriene C4 (LTC4), [3H]17beta-estradiol 17-(beta-D-glucuronide) (E217betaG), [3H]bromosulfophthalein (BSP), and [3H]cholecystokinin octapeptide (CCK-8) were readily detected for all Mrp2s. A species difference in the intrinsic transport activity was apparent for LTC4 (monkey > mouse, dog > rat) and BSP (rat, dog, monkey > mouse). In addition to the difference in the transport activity, complex kinetic profiles were also evident in CCK-8, where a cooperative transport site was observed. Moreover, the transport of [3H]E217betaG by mouse and monkey Mrp2 was quite different from that of rat and dog Mrp2 in that 1) there was practically only nonsaturable uptake for [3H]E217betaG and 2) 4-methylumbelliferon glucuronide (Mrp2 modulator) showed a concentration-dependent stimulatory effect on the transport of [3H]E217betaG in mouse and monkey Mrp2, whereas rat and dog transport activity was inhibited by the modulator. In conclusion, although the substrate specificity is similar, the intrinsic transport activity differs from one species to another. This is due not only to the difference in the Km and Vmax values, but also the qualitatively different mode of substrate and modulator recognition exhibited by different species.

Phytohormone signalling pathways interact with sugars during seed germination and seedling development

Exogenous glucose delays seed germination in Arabidopsis thaliana not only in wild type (WT), but also in a number of mutants in hormone signalling pathways. This study demonstrates that the ABA Insensitive 3 (ABI3) gene in the ABA signalling pathway and the RGA-like 2 (RGL2) and SPINDLY (SPY) genes in the GA signalling pathways all play important roles in the glucose-induced delay of seed germination. Transcription of the ABI3 and RGL2 genes is up-regulated by glucose. This study also supports the idea that different sugars such as the hexose stereoisomers, glucose, and mannose, delay or inhibit seed germination via different branches of the hormone signalling pathways. Analysis of post-germination seedling development of wild-type plants indicates that exogenous glucose supplied after germination may have a concentration-dependent stimulatory effect on root and shoot growth. Comparison of WT and spy seedling growth on different glucose concentrations suggests that the stimulatory effect of glucose is partially exerted via the GA or cytokinin signalling pathways. The effects of glucose on plant growth and development may be stimulatory or inhibitory depending on the developmental stage. The inhibitory effect on seed germination seems to be accomplished via the activation of the ABA signalling pathway, through ABI3, and inactivation of the GA signalling pathway through RGL2 and SPY. On the other hand, the stimulatory effect of glucose on seedling growth may involve the GA and/or cytokinin signalling pathways.

Putrescine as a modulator of the level of RNA polymerase σS subunit in Escherichia coli cells under acid stress

Metabolites accumulated in the culture medium of Escherichia coli cells induce expression of the rpoS gene encoding the alternative sigmaS subunit of RNA polymerase, which controls adaptation of E. coli to acid stress during growth in glucose-mineral medium. The effect of acetate and succinate as end products of E. coli metabolism has been investigated on the levels of transcription, translation, and sigmaS protein stability. These end products mainly influenced the stability of the RNA polymerase sigmaS subunit. Under conditions of acid stress caused by acetate addition, the content of polyamines in the cells and medium decreased, whereas artificial rpoS gene switch-off by antisense RNA was accompanied by increase in polyamine level. Addition of polyamine to E. coli cells treated with acetate and especially with succinate caused a significant concentration-dependent stimulatory effect on rpoS expression. Thus, induction of the rpoS regulon depends on the combined action of the investigated metabolites determining adequate control of gene expression under conditions of acid stress. A scheme for metabolic pathways describing the role of putrescine in the maintenance of intracellular pH and polyamine pool homeostasis during E. coli adaptation to acid stress is proposed.

Leptin-mediated inhibition of the insulin-stimulated increase in fatty acid uptake in differentiated 3T3-L1 adipocytes

The effects of insulin and leptin on fatty acid uptake in differentiated (adipocytes) and undifferentiated 3T3-L1 cells were investigated. It was demonstrated that in undifferentiated 3T3-L1 cells, insulin and leptin have no effect on fatty acid uptake. In differentiated 3T3-L1 adipocytes, insulin had a concentration-dependent stimulatory effect on fatty acid uptake, whereas leptin on its own had no effect. Leptin, when coincubated with 10 nmol/L insulin, resulted in a concentration-dependent inhibition of the insulin-stimulated fatty acid uptake in differentiated 3T3-L1 cells. These results indicate that leptin has a direct inhibitory effect on the stimulation of fatty acid uptake by insulin in differentiated murine adipocytes.

ADRENOMEDULLIN IS OVEREXPRESSED IN PANCREATIC CANCER AND STIMULATES CELL GROWTH AND INVASION.

Background: Pancreatic cancer is a major oncological challenge, due to its aggressive growth and metastasis. The molecular mechanisms underlying it’s deadly behavior are not well understood. We have shown that adrenomedullin (ADM) is highly expressed in pancreatic cancer (Cancer Res. 63:2649, 2003). In this study, we investigated the role of ADM on pancreatic cancer cell growth, migration, and invasion. Methods: RT-PCR and immunohistochemistry (IHC) were used to assess ADM expression. An ELISA kit was used to detect ADM secretion. Human ADM was stably over-expressed in Panc-1 cells and silenced in MPanc96 cells using shRNA. Growth was assessed by MTS assays, migration by Cell Migration assays, and invasion by Matrigel Invasion assays. Results: ADM was expressed 4.2 fold higher in pancreatic cancer compared to normal pancreas and pancreatitis, with elevated expression in 8/10 pancreatic cancer cell lines. RT-PCR confirmed the microarray data and IHC revealed expression of ADM in neoplastic cells, with 90% (43/48) of pancreatic adenocarcinoma samples on a tissue array over-expressing ADM. ADM secretion in pancreatic cancer cell lines correlated well with the microarray data. ADM had a concentration dependent stimulatory effect on Panc1 cell growth, migration and invasion. ADM (200 nM) stimulated Panc1 cell growth by 242±25%, migration by 370±0.7% and invasion by 272±6.4% of control (p<0.05) at 48h, while ADM blocker (1uM) blocked growth by 75±10%, migration by 55±2.1% and invasion by 63±7.7%. Panc-1 cells over-expressing ADM stimulated cell growth 163±4.5%, migration 150±5.6% and invasion 150±3.8% compared to empty vector, while ADM blocker inhibited migration by 96±2.5% and invasion by 85±1.5% (p<0.05). ADM silenced MPanc96 cells had a decrease in cell growth (50±3.8%), migration (65±5.1%) and invasion (58±10%) vs. control (p<0.05). Conclusions: ADM is highly expressed in pancreatic cancer cells and may act as an autocrine factor to stimulate pancreatic cancer cell growth, migration and invasion.

Effects of chlorpromazine on excitation-contraction coupling events in fast-twitch skeletal muscle fibres of the rat.

1. Single mechanically skinned fibres from the rat extensor digitorum longus muscle, which allow access to intracellular compartments, were used to examine the effects of 0.5-100 microM chlorpromazine hydrochloride (CPZ) on the major steps of the excitation-contraction (E-C) coupling to elucidate the involvement of skeletal muscle in the neuroleptic malignant syndrome (NMS). 2. At 1 microM, CPZ caused a 20-30% increase in the force response induced by t-system depolarisation and a marked increase in the rate of caffeine-induced SR Ca(2+) release. At [CPZ]> or =2.5 microM, there was an initial increase followed by a marked decrease of the t-system depolarisation-induced force responses, while the potentiating effect on the caffeine-induced SR Ca(2+) release remained. These effects were reversible. 3. CPZ had no effect on the maximum Ca(2+)-activated force, but caused reversible, concentration-dependent increases in the Ca(2+) sensitivity of the contractile apparatus at [CPZ] > or =10 microM, with a 50% predicted shift of 0.11 pCa (-log [Ca(2+)]) units at 82.3 microM CPZ. 4. CPZ did not alter the rate of SR-Ca(2+) loading at 1 and 10 microM, but reversibly reduced it by approximately 40% at 100 microM by reducing the SR Ca(2+) pump. Nevertheless, the SR Ca(2+) content was greater when fibres became unresponsive to t-system-induced depolarisation in the presence than in the absence of 100 microM CPZ. 5. The results show that CPZ has concentration-dependent stimulatory and inhibitory effects on various steps of the E-C coupling, which can explain the involvement of skeletal muscle in NMS and reconcile previous divergent data on CPZ effects on muscle.