Inulinase production in a batch bioreactor using agroindustrial residues as the substrate: experimental data and modeling

The production of inulinase employing agroindustrial residues as the substrate is a good alternative to reduce production costs and to minimize the environmental impact of disposing these residues in the environment. This study focused on the use of a phenomenological model and an artificial neural network (ANN) to simulate the inulinase production during the batch cultivation of the yeast Kluyveromyces marxianus NRRL Y-7571, employing a medium containing agroindustrial residues such as molasses, corn steep liquor and yeast extract. It was concluded that due to the complexity of the medium composition it was rather difficult to use a phenomenological model with sufficient accuracy. For this reason, an alternative and more cost-effective methodology based on ANN was adopted. The predictive capacity of the ANN was superior to that of the phenomenological model, indicating that the neural network approach could be used as an alternative in the predictive modeling of complex batch cultivations.     

Direct Rho-associated kinase inhibiton induces cofilin dephosphorylation and neurite outgrowth in PC-12 cells

Axons fail to regenerate in the adult central nervous system (CNS) following injury. Developing strategies to promote axonal regeneration is therapeutically attractive for various CNS pathologies such as traumatic brain injury, stroke and Alzheimer’s disease. Because the RhoA pathway is involved in neurite outgrowth, Rho-associated kinases (ROCKs), downstream effectors of GTP-bound Rho, are potentially important targets for axonal repair strategies in CNS injuries. We investigated the effects and downstream mechanisms of ROCK inhibition in promoting neurite outgrowth in a PC-12 cell model. Robust neurite outgrowth (NOG) was induced by ROCK inhibitors Y-27632 and H-1152 in a time-and dose-dependent manner. Dramatic cytoskeletal reorganization was noticed upon ROCK inhibition. NOG initiated within 5 to 30 minutes followed by neurite extension between 6 and 10 hours. Neurite processes were then sustained for over 24 hours. Rapid cofilin dephosphorylation was observed within 5 minutes of Y-27632 and H-1152 treatment. Re-phosphorylation was observed by 6 hours after Y-27632 treatment, while H-1152 treatment produced sustained cofilin dephosphorylation for over 24 hours. The results suggest that ROCK-mediated dephosphorylation of cofilin plays a role in the initiation of NOG in PC-12 cells.     

Effect of Y-24180 on Ca2+ movement and proliferation in renal tubular cells

In canine renal tubular cells, the effect of Y-24180, a presumed specific platelet activating factor (PAF) receptor antagonist, on intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by using fura-2 as a Ca(2+)-sensitive fluorescent probe. Y-24180 (0.1-10 microM) caused a rapid and sustained [Ca(2+)](i) rise in a concentration-dependent manner. The [Ca(2+)](i) rise was prevented by 30% by removal of extracellular Ca(2+), but was not changed by dihydropyridines, verapamil and diltiazem. Y-24180-induced Ca(2+) influx was confirmed by Mn(2+)-influx induced quench of fura-2 fluorescence. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the increasing effect of 5 microM Y-24180 on [Ca(2+)](i) was abolished; conversely, depletion of Ca(2+) stores with 5 microM Y-24180 abolished thapsigargin-induced [Ca(2+)](i) rise. U73122, an inhibitor of phoispholipase C, inhibited ATP-, but not Y-24180-induced [Ca(2+)](i) rise. Overnight treatment with Y-24180 did not alter cell proliferation rate. Collectively, these results suggest that Y-24180 acts as a potent, but not cytotoxic, Ca(2+) mobilizer in renal tubular cells, by stimulating both extracellular Ca(2+) influx and intracellular Ca(2+) release. Since alterations in Ca(2+) movement may interfere many cellular signaling processes unrelated to modulation of PAF receptors, caution must be applied in using this chemical as a selective PAF receptor antagonist.     

Modulation of adrenal cell functions by cadmium salts: 3. Sites affected by CdCl2 during stimulated steroid synthesis

In previous studies cadmium chloride (CdCl₂) nonlethally inhibited Y-1 mouse adrenal tumor cell 20-dihydroxyprogesterone (20DHP) secretion, affecting unstimulated and stimulated steroidogenic pathway sites differently. In addition, dibutyryl cAMP-stimulated 20DHP secretion was unaffected by CdCl₂, while the site of the unstimulated effect was indirectly shown to involve steps between endogenous cholesterol utilization and 20-hydroxycholesterol association with mitochondrial cytochrome P450 side-chain cleavage enzyme. In the present study we determined CdCl₂ effects on plasma membrane sites preceding pre-dbcAMP-stimulation of 20DHP secretion. Y-1 cells were incubated 0.5 h in medium with or without cadmium (using the concentration that inhibited adrenocorticotropin- (ACTH)-stimulated steroid secretion by 50%) together with exogenously added maximally stimulating concentrations of ACTH, cholera toxin, forskolin, or adenosine triphosphate Cholera toxin, forskolin and ATP bypass specific plasma membrane sites involved in the synthesis of intracellular cAMP and activate the steroid hormone biosynthetic pathway. Cadmium effects on ACTH-stimulated endogenous cAMP secretion were also examined. CdCl₂ significantly reduced Y-1 cell 20DHP secretion following exposure to ACTH, cholera toxin, forskolin, and ATP; it also significantly decreased endogenous cAMP secretion into culture medium. These data may be interpreted to suggest that CdCl₂ altered Y-1 cell regulation of adenyl cyclase activity, which reduced cAMP-activated cholesterol uptake by mitochondria as a consequence.Abbreviations ACTH adrenocorticotropin - ATP adenosine triphosphate - ANOVA analysis of variance - CdCl₂ or Cd²⁺ cadmium chloride - cAMP cyclic 3,5-adenosine monophosphate - CTX cholera toxin - dbcAMP dibutyryl cAMP,N,O-dibutyryl-3,5-adenosine monophosphate - EGTA ethylene glycol bis tetraacetic acid - FMEM serum-free Eagle's Minimum Essential Medium with all other supplements - FSK forskolin - Hepes N-2-hydroxyethylpiperazine-N-1,2-ethanesulfonic acid - IC_50' concentration inhibiting stimulated steroid secretion by 50% - IU international unit - MEM Eagle's Minimum Essential Medium - P450scc cytochrome P450 side-chain cleavage enzyme - PREG pregnenolone - PROG progesterone - SEM standard error of the mean - SMEM serum-containing Eagle's Minimum Essential Medium with supplements - 20DHP 20--hydroxy-4-pregnen-3-one     

A role for hippocampal Rho-ROCK pathway in long-term spatial memory

Morphological changes, including changes in size, shape, and number of synapses, in neurons have been observed in many species and are thought to be critical for long-term memory storage. Actin filaments are intimately involved in neuronal morphology and regulation of their dynamics can influence memory. Rho GTPase plays a prominent role in this process and has been implicated in both pre- and post-synaptic morphological changes. Therefore, we examined the effect of hippocampal manipulation of Rho and ROCK activity on performance in a spatial memory task. Post-training intrahippocampal infusion of an inhibitor of the downstream effector kinase p160ROCK impaired long-term memory. Furthermore, post-training activation of Rho using lysophosphatidic acid (LPA) enhanced long-term spatial memory. This memory enhancing effect of LPA was not mediated via the Erk cascade, as no change in Erk phosphorylation was observed as a result of its administration. Our results demonstrate a role for the Rho-ROCK pathway in hippocampus-dependent spatial memory.     

Involvement of Rho/Rho-kinase signalling in the contractile activity and acetylcholine release in the mouse gastric fundus

In this study effects of Rho kinase inhibitors have been examined on the mouse gastric fundal smooth muscle reactivity and neurotransmitter (acetylcholine) release. Two Rho-kinase inhibitors, Y-27632 and fasudil (HA-1077), conspicuously suppressed the contractile responses to carbachol (CCh) and KCl as well as electrical field stimulation (EFS, 40 V, 0.5 ms, and 20 s). pEC(50) value for CCh and EC(50) value for KCl were 6.68+/-0.15 M and 10.4+/-2.8 mM, respectively. EFS induced reproducible contraction (38.3+/-4.75 mN/g tissue) which was almost abolished and potentiated in the presence of atropine (10(-6)M) and eserine (10(-6)M), respectively. The Rho-kinase inhibitors relaxed the fundic strips preconstricted by submaximal concentration of CCh or KCl in a concentration dependent manner. With CCh-elicited contraction, the pEC(50) values of Y-27632 and fasudil were 5.45+/-0.14 and 5.11+/-0.14 M, respectively (p>0.05). However, the pEC(50) values for Y-27632 and fasudil on KCl-induced tone were 6.09+/-0.1 and 5.35+/-0.06 M (p<0.001), respectively. Moreover, [3H]acetylcholine ([3H]ACh) release upon EFS from the gastric fundus was measured and it was found that Y-27632 (10(-4)M) significantly impaired the release. At 3 Hz the radioactivity ratio obtained after and before EFS (S(2)/S(1) ratio) was 0.88+/-0.03 in control but 0.63+/-0.08 in the presence of 10(-4)M Y-27632 (p<0.05). These results suggest that Rho kinase inhibitors can not only relax the gastric fundus but also modulate CCh, cholinergic nerve stimulation, and KCl-induced contraction. Furthermore, Rho/Rho kinase signalling may play a role in the neurotransmitter (ACh) release in the mouse gastric fundus.     

Calmodulin-Binding Proteins in Human Y-79 Retinoblastoma and HTB-14 Glioma Cell Lines

The Y-79 human retinoblastoma cell line has been used as a model system for studying differentiation of primitive neuroectodermal cells into either glial-like (glial fibrillary acidic protein positive) or neuron-like (neuron-specific enolase-positive) cells. To determine whether Y-79 retinoblastoma cells express neuronotypic calmodulin-binding proteins, Y-79 cells were either treated with butyrate or dibutyryl cyclic AMP (dbcAMP) in serum-containing medium or were maintained in serum-free media. Using a biotinylated calmodulin blot overlay technique, we found that Y-79 cells treated with dbcAMP or butyrate expressed low levels of membrane-bound calmodulin-binding proteins of 150, 147, 127, and 126 kilodaltons (kDa); butyrate-treated cells also expressed a calmodulin-binding peptide of 135 kDa. Since butyrate treatment of Y-79 cells induces the expression and the secretion of interphotoreceptor retinoid-binding protein (IRBP, 140 kDa), we tested the hypothesis that the calmodulin-binding protein of 135 kDa induced by butyrate treatment was IRBP. Purified bovine IRBP did not bind calmodulin; further, the 135-kDa calmodulin binding protein was not immunoreactive with antisera directed against IRBP. Since dbcAMP and butyrate induce some glial-like characteristics in Y-79 cells, we compared the calmodulin-binding protein pattern in these cells with that seen in human HTB-14 glioma cells. The HTB-14 line did not express calmodulin-binding proteins, even after treatments with agents that induce morphologic change in these cells. Thus, we conclude that Y-79 cells express membrane-bound calmodulin-binding proteins, but in a pattern different from that seen with adult, differentiated neurons or from human HTB-14 glioma cells.     

Modulation of adrenal cell functions by cadmium salts. 4. Ca2+-dependent sites affected by CdCl2 during basal and ACTH-stimulated steroid synthesis

In previous studies, nonlethal CdCl₂ concentrations apparently inhibited basal Y-1 mouse adrenal tumor cell endogenous mitochondrial cholesterol conversion to pregnenolone. In addition, CdCl₂ inhibited all agents stimulating both plasma membrane-dependent cAMP synthesis and 20-hydroxy-4-pregnen-3-one (20DHP) secretion. Bypassing the plasma membrane using dibutyryl-cAMP (dbcAMP) stimulated cytoplasmic cholesterol metabolism and 20DHP secretion in the presence of CdCl₂. Since CdCl₂ competed at metabolic steps requiring Ca²⁺ in other tissues, experiments were designed to examine Cd²⁺ competition with Ca²⁺ during steroidogenesis. Sets of cells incubated with either medium or adrenocorticotropin (ACTH) with or without CdCl₂ were also treated with 0, 1.0, 5.0 or 10.0 mmol/L CaCl₂ in the presence or absence of EGTA, a relatively specific Ca²⁺, but not Cd²⁺, chelating agent. Another experimental cell set incubated with either medium or ACTH, with or without CdCl₂, was treated with or without 1 mmol/L A23187, an ionophore specifically facilitating extracellular Ca²⁺ transfer across plasma membranes. Besides determining Ca²⁺ involvement in steroidogenesis using steroid secretion as an endpoint, we directly measured Ca²⁺ concentrations using intracellular fura-2 fluorescence. Following loading with 2 mol/L fura-2, cells remained untreated or medium was infused with CdCl₂, ACTH, ACTH/CdCl₂ or ACTH followed after 50 s by CdCl₂. Using Ca²⁺-supplemented media, we observed that Cd²⁺ inhibition of ACTH-stimulated 20DHP secretion was completely reversed. Standard Ca²⁺-containing medium supplemented with Ca²⁺ also enhanced maximally stimulated 20DHP secretion by ACTH. 20DHP secretion by ACTH-treated and ACTH/Cd²⁺-treated cells was only reduced by EGTA, when Ca²⁺ was not supplemented. The ionophore A23187 increased basal and ACTH-stimulated 20DHP secretion by Cd²⁺-treated cells, suggesting that extracellular Ca²⁺ resources may compete against Cd²⁺ effects on plasma membrane cAMP synthesis and on basal cholesterol metabolism by mitochondria. No time-dependent change in Ca²⁺ concentrations occurred within untreated cell suspensions. ACTH stimulation caused a 25 s burst in Ca²⁺ concentrations before returning to basal, steady-state levels. Cd²⁺ also stimulated intracellular fura-2 fluorescence. Untreated cell suspensions infused with Cd²⁺ exhibited a continuous rise in intracellular fluorescence. ACTH/CdCl₂-treated cells exhibited a hyperbolic rise in intracellular fluorescence over the 300 s study period. Cells treated with Cd²⁺ 50 s after ACTH treatment initially exhibited the 25 s fluorescence burst followed by a Cd²⁺-induced hyperbolic rise in intracellular Cd²⁺. These fluorescence measurements suggested that cytoplasmic Ca²⁺ changes do not appear to be necessary for basal 20DHP synthesis and secretion; only a 25 s burst in intracellular Ca²⁺ is necessary to a slightly higher plateau level for stimulated 20DHP synthesis and secretion. Cd²⁺ freely enters the cell under basal conditions and Cd²⁺ entry is accelerated by ACTH stimulation. Data were consistent with Ca²⁺ being required for optimal stimulated steroid production and Cd²⁺ probably competing with Ca²⁺ during basal mitochondrial cholesterol metabolism and plasma membrane ACTH-stimulated cAMP generation.     

Alternative Cultures for Human Pluripotent Stem Cell Production,Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.     

Retinoic Acid Increases Hydroxyindole-O-Methyltransferase Activity and mRNA in Human Y-79 Retinoblastoma Cells

Abstract: Hydroxyindole- O -methyltransferase (HIOMT) plays an important role as the final enzyme in the synthesis of melatonin. Here we present the first evidence that retinoic acid (RA) stereoisomers are potent regulators of HIOMT in the human retinoblastoma-derived Y-79 cell line. Treatment with all- trans -, 13- cis -, and 9- cis -RA induced a gradual 10-fold increase in HIOMT activity and mRNA, without changing the levels of mRNA encoding glyceraldehyde-3-phosphate dehydrogenase, actin, S-antigen, and interphotoreceptor retinoid-binding protein. These findings point to the possibility that RA may play a physiological role in the regulation of human HIOMT.