The influence of skeletal muscle cell volume on carbohydrate metabolism in contracting skeletal muscle

This study investigated the regulation of carbohydrate metabolism through changes in skeletal muscle cell volume immediately post contraction and during recovery. Using an established in vitro isolated muscle strip model, soleus (SOL) and extensor digitorum longus (EDL) were dissected from male rats and incubated in an organ bath (perfused with 95% O2; 5% CO2, pH 7.4, temperature 25°C) containing medium- 199 altered to a target osmotic condition (iso-, hypo- or hyper-osmotic; 290, 1 80, 400 mmol/kg). Muscles were stimulated for 10 minutes (40 Hz SOL; 30 Hz EDL) and then either immediately flash frozen or allowed to recover for 20 minutes before subsequent metabolite and enzyme analysis. Results demonstrated a relative water decrease in HYPER vs. HYPOosmotic condition (n=8/group; p<0.05) regardless of muscle type. Specifically, the SOL HYPER condition had elevated metabolite concentrations after 10 minutes of stimulation in comparison to both HYPO and ISO (p<0.05), while EDL muscle did not show any significant difTerences between the HYPER or HYPO conditions. After 20 minutes of recovery, metabolic changes occurred in both SOL and EDL with the SOL HYPER condition showing greater relative changes in metabolite concentrations versus HYPO. The results of the current study have demonstrated that osmotic imbalance induces metabolic change within the skeletal muscle cell and muscle type may influence the mechanisms utilized for cell volume regulation.

Isolation and characterization of a chitinase-secreting mutant of mortierella pusilla with altered interaction with the mycoparasite piptocephalis virginiana

Mortierella pusilla is a susceptible host and supports good growth of the mycoparasite, Piptocephalis virginiana. Uninucleate spores of M. pusilla were sUbjected to N-methyl-N'-nitro-nitrosoguanidine (MNNG). To attain a high mutation frequency , a 1o-minute exposure to 10 mg/ml MNNG was used and lead to the survival of about 10 % of the spores. The exposed spores then were plated on chitin or milk plates. Approximately 30,000 colonies were examined after mutagenesis on the screening media. A strain, MUT23 , with abnormal slow growth morphology was found to delay parasitism by £. virginiana. The particular morphology was not due to auxotrophy, because this strain displayed normal hyphae when glucose was used as the sole carbon source. One interesting phenomenon was that MUT23 showed an extensive clearing zone around the colony on colloidal chitin agar after 20-25 d. On the same conditions, wild type strain did not show this phenotype. In addition, the MUT23 strain produced the same normal hypha as the wild type strain when it was grown on colloidal chitin agar. The MUT23 was also able to produce more spores on colloidal chitin agar than on malt-yeast extract and minimal media. The parasite germ tubes formed appressoria at the point of contact on the cell surface of wild type and MUT23 grown for 6 days cell surface but not on the cel surface of MUT23 grown for 2 days. Thus, interaction between MUT23 strain and the mycoparasite was dependent on MUT23 age. The effect of MUT23 filtrate on germination of the parasite was tested. Lysis of germinated spores of the parasite were observed in concentrated MUT23 filtered solution. MUT23 was compared to the wild type strain for their chitinase production in sUbmerged culture. The chitinase isozymes of both wild type and MUT23 were shown by immunoblotting. Eight distinct chitinase molecules were detected. MUT23 showed markedly higher chitinase activity than the wild type cultured in chitin-containing medium. Maximum chitinase activities of MUT23 were 13.5 fold higher at 20 day of the culture then that of wild type.

L’entérotoxine STb d’Escherichia coli déloge la claudine-1 des jonctions serrées

Escherichia coli produit diverses entérotoxines thermolabiles et thermostables. STb est une toxine de faible poids moléculaire résistant à la chaleur chargée de la diarrhée chez les animaux de la ferme. Une étude antérieure a montré que les cellules ayant internalisé la toxine STb provoquent un dysfonctionnement de la barrière épithéliale par des changements dans les protéines des jonctions serrées (TJ). Ces modifications contribuent probablement à la diarrhée observée. Pour mieux comprendre le mécanisme de l'augmentation de la perméabilité intestinale, nous avons traité les cellules du côlon humain (T84) avec la toxine purifiée STb une fois que les cellules ont été récoltées et les protéines extraites. Après l'utilisation d'une solution contenant 1% de Nonidet P-40 (un détergent non dénaturant, non ionique), nous avons étudié la distribution de la claudine -1, une protéine majeure des TJs, responsable de l'imperméabilité de l'épithélium, entre la membrane (NP40-insoluble) et le cytoplasme (NP40-soluble). En utilisant l’immunoblot et la microscopie confocale, nous avons observé que le traitement des monocouches de cellules T84 avec STb induit la redistribution de la claudine-1. Après 24h, les cellules cultivées en milieu faible en Ca+ (5 uM) et traitées par STb, ont montré qu’environ 40 % de plus de la claudine-1 se sont délogées dans le cytoplasme par comparaison au contrôle. En passant d’un milieu faible à un milieu contenant des quantités physiologiques de Ca++ (1,8 mM) nous avons observé une augmentation du taux de claudine- 1 délogé, comme la délocalisation comparable et ce, après 6h. Un milieu supplémenté avec la même concentration de Mg++ ou Zn++ n'a pas affecté le taux de délogement comparé au milieu contenant une faible teneur en Ca++. En utilisant des anticorps anti-phosphosérine et anti-phosphothréonine, nous avons observé que la perte des claudines-1 de la membrane a été accompagnée par une déphosphorylation de cette protéine des TJs. Dans l'ensemble, nos résultats ont montré une importante redistribution de la claudine-1 dans les cellules traitées par la toxine STb. La perte de la claudine-1 phosphorylée de la membrane est susceptible d'être impliquée dans la perméabilité accrue observée. Les mécanismes par lesquels ces changements sont provoqués restent à élucider.

Carbohydrate preferences of Bifidobacterium species isolated from the human gut

The growth of nine species of Bifidobacterium on media containing glucose, xylose, xylooligosaccharides (XOS), xylan or fructooligosaccharides (FOS) as the sole carbon source were compared in pure culture. The bifidobacteria differed in fermentation profiles when tested on different carbohydrates. All species grew to their highest final optical density (OD) on a glucose containing medium, with the exception of B. catenulatum which demonstrated a preference for xylose over glucose, and XOS over FOS. B. bifidum grew to the highest OD on XOS compared to xylose suggesting a specific transport system for the oligosaccharide over the monomer. This is consistent with a lack of β-xylosidase activity present in the culture medium. Lactate, formate and acetate levels were determined and the ratios of these metabolites altered between and within species growing on different carbohydrates. In general, high lactate production correlated with low formate production and low lactate concentrations were obtained at higher levels of formate. Bifidobacteria may alter their metabolic pathways based upon the carbohydrates that are available for their use.

Role of glutaredoxin 2 and cytosolic thioredoxins in cysteinyl-based redox modification of the 20S proteasome

The yeast 20S proteasome is subject to sulfhydryl redox alterations, such as the oxidation of cysteine residues (Cys-SH) into cysteine sulfenic acid (Cys-SOH), followed by S-glutathionylation (Cys-S-SG). Proteasome S-glutathionylation promotes partial loss of chymotrypsin-like activity and post-acidic cleavage without alteration of the trypsin-like proteasomal activity. Here we show that the 20S proteasome purified from stationary-phase cells was natively S-glutathionylated. Moreover, recombinant glutaredoxin 2 removes glutathione from natively or in vitro S-glutathionylated 20S proteasome, allowing the recovery of chymotrypsin-like activity and post-acidic cleavage. Glutaredoxin 2 deglutathionylase activity was dependent on its entry into the core particle, as demonstrated by stimulating S-glutathionylated proteasome opening. Under these conditions, deglutathionylation of the 20S proteasome and glutaredoxin 2 degradation were increased when compared to non-stimulated samples. Glutaredoxin 2 fragmentation by the 20S proteasome was evaluated by SDS-PAGE and mass spectrometry, and S-glutathionylation was evaluated by either western blot analyses with anti-glutathione IgG or by spectrophotometry with the thiol reactant 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. It was also observed in vivo that glutaredoxin 2 was ubiquitinated in cellular extracts of yeast cells grown in glucose-containing medium. Other cytoplasmic oxido-reductases, namely thioredoxins 1 and 2, were also active in 20S proteasome deglutathionylation by a similar mechanism. These results indicate for the first time that 20S proteasome cysteinyl redox modification is a regulated mechanism coupled to enzymatic deglutathionylase activity.

Glutamine enhances glucose-induced mesangial cell proliferation

The proliferation of mesangial cells (MC) in the presence of glutamine (0-20 mM) was determined in both low (5 mM) and high (25 mM) glucose-containing medium. Glutamine in a high glucose (HG) environment increased cell proliferation in a dose-dependent manner. Inhibition of glutamine:fructose 6-phosphate amidotransferase (GFAT) and of phosphodiesterase significantly reduced glutamine-induced proliferation. Supraphysiologic levels of glutamine increase MC proliferation in a HG milieu via GFAT and cAMP-dependent pathways, suggesting that glutamine could pose a risk for diabetic nephropathy.

Mechanism of Trypanosoma cruzi death induced by Cratylia mollis seed lectin

Incubation of T. cruzi epimastigotes with the lectin Cramoll 1,4 in Ca(2+) containing medium led to agglutination and inhibition of cell proliferation. The lectin (50 A mu g/ml) induced plasma membrane permeabilization followed by Ca(2+) influx and mitochondrial Ca(2+) accumulation, a result that resembles the classical effect of digitonin. Cramoll 1,4 stimulated (five-fold) mitochondrial reactive oxygen species (ROS) production, significantly decreased the electrical mitochondrial membrane potential (Delta I(m)) and impaired ADP phosphorylation. The rate of uncoupled respiration in epimastigotes was not affected by Cramoll 1,4 plus Ca(2+) treatment, but oligomycin-induced resting respiration was 65% higher in treated cells than in controls. Experiments using T. cruzi mitochondrial fractions showed that, in contrast to digitonin, the lectin significantly decreased Delta I(m) by a mechanism sensitive to EGTA. In agreement with the results showing plasma membrane permeabilization and impairment of oxidative phosphorylation by the lectin, fluorescence microscopy experiments using propidium iodide revealed that Cramoll 1,4 induced epimastigotes death by necrosis.

Effect of capacitation of stallion sperm with polyvinylalcohol or bovine serum albumin on penetration of bovine zona-free or partially zona-removed equine oocytes

Experiments were conducted to study effects of macromolecules on stallion sperm capacitation and fertilization as determined by penetration of bovine zona-free and equine partially zona-removed oocytes. Stallion sperm were capacitated in TYH medium (modified Krebs-Ringer bicarbonate) supplemented with either 1 mg/mL of polyvinylalcohol (PVA) or 4 mg/ mL of BSA. Capacitation was induced with 8 bromoadenosine cyclic monophosphate (8BrcAMP; 0.5 mM) alone or in combination with 0.1 μM of ionomycin. Intraspecies gametes were co-incubated in TYH/PVA or TYH/ BSA for 18 to 20 h. For zona-free bovine oocytes, penetration rate (35%) with the combination of 8BrcAMP and ionomycin in PVA-containing medium was higher (P < 0.05) than any treatment in BSA-containing medium (5 to 6%). A similar study was conducted using equine oocytes with partially removed zonae. Sperm capacitated and used for in vitro fertilization (IVF) in PVA-containing medium had higher penetration rates (P < 0.01) than sperm in BSA-containing medium (54 vs. 11%). The effect of equine preovulatory follicular fluid on bovine oocyte penetration was assessed. Bovine oocytes were matured in tissue culture medium-199 with 0, 20, 50, or 100% equine preovulatory follicular fluid, and 1 IU/mL of equine chorionic gonadotropin. Stallion sperm were treated with 8BrcAMP + ionomycin in PVA- or BSA-containing media. The penetration rates of bovine zona-free oocytes by stallion sperm were again higher with PVA (47%) than BSA (18%; P < 0.01). Penetration rates of oocytes matured in 100% follicular fluid were higher (P < 0.05) than for oocytes matured with 0% follicular fluid. The effects of equine follicular fluid and PVA/BSA during sperm capacitation on standard bovine IVF were examined. Culture of bovine oocytes with equine follicular fluid did not affect oocyte maturation or penetration rates after IVF. Bovine sperm capacitated with heparin in PVA-containing medium yielded lower (P < 0.05) fertilization rates than those capacitated in BSA-containing medium when incubated with both zona-intact and zona-free bovine oocytes. In summary, PVA was superior to BSA for ionophore-induced capacitation of equine sperm for penetration of zona-free bovine oocytes or partially zona-removed equine oocytes, but not for standard bovine IVF with bovine sperm. Zona-free bovine oocytes may be useful for assaying in vitro capacitation and fertilization of stallion sperm. © 2003 American Society of Animal Science. All rights reserved.

Inibição da maturação nuclear pela butirolactona I durante o transporte de oócitos bovinos destinados à produção in vitro de embrões (PIV)

Pós-graduação em Medicina Veterinária - FCAV

Extracellular ATP triggers proteolysis and cytosolic Ca2+ rise in Plasmodium berghei and Plasmodium yoelii malaria parasites

Background: Plasmodium has a complex cell biology and it is essential to dissect the cell-signalling pathways underlying its survival within the host. Methods: Using the fluorescence resonance energy transfer (FRET) peptide substrate Abz-AIKFFARQ-EDDnp and Fluo4/AM, the effects of extracellular ATP on triggering proteolysis and Ca2+ signalling in Plasmodium berghei and Plasmodium yoelii malaria parasites were investigated. Results: The protease activity was blocked in the presence of the purinergic receptor blockers suramin (50 mu M) and PPADS (50 mu M) or the extracellular and intracellular calcium chelators EGTA (5 mM) and BAPTA/AM (25, 100, 200 and 500 mu M), respectively for P. yoelii and P. berghei. Addition of ATP (50, 70, 200 and 250 mu M) to isolated parasites previously loaded with Fluo4/AM in a Ca2+-containing medium led to an increase in cytosolic calcium. This rise was blocked by pre-incubating the parasites with either purinergic antagonists PPADS (50 mu M), TNP-ATP (50 mu M) or the purinergic blockers KN-62 (10 mu M) and Ip5I (10 mu M). Incubating P. berghei infected cells with KN-62 (200 mu M) resulted in a changed profile of merozoite surface protein 1 (MSP1) processing as revealed by western blot assays. Moreover incubating P. berghei for 17 h with KN-62 (10 mu M) led to an increase in rings forms (82% +/- 4, n = 11) and a decrease in trophozoite forms (18% +/- 4, n = 11). Conclusions: The data clearly show that purinergic signalling modulates P. berghei protease(s) activity and that MSP1 is one target in this pathway.

Redox Control of 20S Proteasome Gating

The proteasome is the primary contributor in intracellular proteolysis. Oxidized or unstructured proteins can be degraded via a ubiquitin-and ATP-independent process by the free 20S proteasome (20SPT). The mechanism by which these proteins enter the catalytic chamber is not understood thus far, although the 20SPT gating conformation is considered to be an important barrier to allowing proteins free entrance. We have previously shown that S-glutathiolation of the 20SPT is a post-translational modification affecting the proteasomal activities. Aims: The goal of this work was to investigate the mechanism that regulates 20SPT activity, which includes the identification of the Cys residues prone to S-glutathiolation. Results: Modulation of 20SPT activity by proteasome gating is at least partially due to the S-glutathiolation of specific Cys residues. The gate was open when the 20SPT was S-glutathiolated, whereas following treatment with high concentrations of dithiothreitol, the gate was closed. S-glutathiolated 20SPT was more effective at degrading both oxidized and partially unfolded proteins than its reduced form. Only 2 out of 28 Cys were observed to be S-glutathiolated in the proteasomal alpha 5 subunit of yeast cells grown to the stationary phase in glucose-containing medium. Innovation: We demonstrate a redox post-translational regulatory mechanism controlling 20SPT activity. Conclusion: S-glutathiolation is a post-translational modification that triggers gate opening and thereby activates the proteolytic activities of free 20SPT. This process appears to be an important regulatory mechanism to intensify the removal of oxidized or unstructured proteins in stressful situations by a process independent of ubiquitination and ATP consumption. Antioxid. Redox Signal. 16, 1183-1194.

Entwicklung neuer Biomarker zur Detektion von Umweltbelastung

Es wurde bis heute meist mit relativ aufwendigen Methoden nach dem Vorkommen von Strangbrüchen in der DNA gesucht. Die hier verwendete Fast-Micromethod steht jetzt neben mehreren bereits etablierten Methoden zur qualitativen und quantitativen Erfassung von DNA-Strangbrüchen zur Verfügung. Die Methode wurde zur schnellen und empfindlichen Messung von DNA-Schäden und deren Reparatur unter Verwendung von PicoGreen, einem spezifisch an DNA-Einzelstränge bindenden Fluoreszenzfarbstoff, durchgeführt. Die Methode wurde in der vorliegenden Arbeit für verschiedene Zellarten wie HeLa-Zellen und Schwamm-Zellen mit Schwermetallionen verwendet. Sie wurde ebenfalls bei verschiedenen Strangbruch-induzierenden Stoffen wie NQO und physikalischen Faktoren wie UV-Bestrahlung und Gammastrahlung erfolgreich eingesetzt. Die Strangbrüche wurden bei HeLa-Zellen mit 10 µM NQO nach 90 Minuten Inkubation und ebenso bei Gammastrahlung mit 16 Gy gemessen. An Schwammzellen wurden Effekte von Schwermetallionen und UV, auch einschließlich nachgeschalteter Reparaturaktivität, gemessen. Dabei war die Reparatur jedoch nicht ganz vollständig. Durch die Fast-Micromethod wurde DNA-Schädigung durch UV-Bestrahlung und Gentoxizität von Schwermetall-Ionen an HeLa-Zellen nachgewiesen. Die Schwammzellen wurden mit verschiedenen Schwermetall-Ionen in verschiedenen Konzentrationen getestet und mit UV bei verschiedenen Dosen bestrahlt. Teilweise wurde nach dem Einfluss die DNA-Reparatur erfasst. Schwammzellen wurden in den drei häufig verwendeten Medien CMFSW, CMFSW+EDTA und Seewasser aufgenommen. Im Seewasser waren die Zellen nach 60 Minuten Inkubationszeit aggregiert (Primmorphe). Im CMFSW+EDTA Medium lagen dagegen nur Einzellen vor, da die Zellen im EDTA-Medium dissoziiert wurden und auch nicht mehr proliferierten.

ASTAXANTHIN PRODUCTION FROM HAEMATOCOCCUS PLUVIALIS UNDER VARIOUS LIGHT INTENSITIES AND CARBON DIOXIDE CONCENTRATIONS

The microalga Haematococcus pluvialis was cultivated in MES-volvox medium at various light intensities and CO2 concentrations. It was found that CO2 concentrations of 10 and 15%, in combination with high irradiance at initial pH =6.7, accelerate astaxanthin accumulation in H. pluvialis cells but obstruct cell growth. The purpose of this research study was to devise a one-stage process consisting of the simultaneous cultivation of H. pluvialis and astaxanthin production using high light intensity and high CO2 concentration. This could be achieved at 200 µE/m2s and 15% CO2 in growth medium at initial pH = 4.3. Compared to the traditional two-stage H. pluvialis cultivation system, this one-step process can save up to 8-9 days of astaxanthin production time. The astaxanthin content in H. pluvialis cells induced with high light intensity only or with a combination of high light intensity and high CO2 concentration had comparable astaxanthin content; 94 and 97 mg/g dry biomass, respectively. However, it was extremely low in nitrate-free medium at high irradiance alone or combined with high CO2 concentration, with an average value of 4 mg/g dry biomass. Cell density was 40% less in cultures under discontinuous illumination compared to continuous illumination. This process could serve as a microalgal CO2 mitigation system after further understanding of the CO2 fixation ability of H. pluvialis has been gained.

The role of cell death and myofibrillar damage in contractile dysfunction of long-term cultured adult cardiomyocytes exposed to doxorubicin

In failing hearts cardiomyocytes undergo alterations in cytoskeleton structure, contractility and viability. It is not known presently, how stress-induced changes of myofibrils correlate with markers for cell death and contractile function in cardiomyocytes. Therefore, we have studied the progression of contractile dysfunction, myofibrillar damage and cell death in cultured adult cardiomyocytes exposed to the cancer therapy doxorubicin. We demonstrate, that long-term cultured adult cardiomyocytes, a well-established model for the study of myofibrillar structure and effects of growth factors, can also be used to assess contractility and calcium handling. Adult rat ventricular myocytes (ARVM) were isolated and cultured for a total of 14 days in serum containing medium. The organization of calcium-handling proteins and myofibrillar structure in freshly isolated and in long-term cultured adult cardiomyocytes was studied by immunofluorescence and electron microscopy. Excitation contraction-coupling was analyzed by fura 2 and video edge detection in electrically paced cardiomyocytes forming a monolayer, and cell death and viability was measured by TUNEL assay, LDH release, MTT assay, and Western blot for LC3. Adult cardiomyocytes treated with Doxo showed apoptosis and necrosis only at supraclinical concentrations. Treated cells displayed merely alterations in cytoskeleton organization and integrity concomitant with contractile dysfunction and up-regulation of autophagosome formation, but no change in total sarcomeric protein content. We propose, that myofibrillar damage contributes to contractile dysfunction prior to cell death in adult cardiomyocytes exposed to clinically relevant concentrations of anthracyclines.

Mice mutant for glucokinase regulatory protein exhibit decreased liver glucokinase: A sequestration mechanism in metabolic regulation

The importance of glucokinase (GK; EC 2.7.1.12) in glucose homeostasis has been demonstrated by the association of GK mutations with diabetes mellitus in humans and by alterations in glucose metabolism in transgenic and gene knockout mice. Liver GK activity in humans and rodents is allosterically inhibited by GK regulatory protein (GKRP). To further understand the role of GKRP in GK regulation, the mouse GKRP gene was inactivated. With the knockout of the GKRP gene, there was a parallel loss of GK protein and activity in mutant mouse liver. The loss was primarily because of posttranscriptional regulation of GK, indicating a positive regulatory role for GKRP in maintaining GK levels and activity. As in rat hepatocytes, both GK and GKRP were localized in the nuclei of mouse hepatocytes cultured in low-glucose-containing medium. In the presence of fructose or high concentrations of glucose, conditions known to relieve GK inhibition by GKRP in vitro, only GK was translocated into the cytoplasm. In the GKRP-mutant hepatocytes, GK was not found in the nucleus under any tested conditions. We propose that GKRP functions as an anchor to sequester and inhibit GK in the hepatocyte nucleus, where it is protected from degradation. This ensures that glucose phosphorylation is minimal when the liver is in the fasting, glucose-producing phase. This also enables the hepatocytes to rapidly mobilize GK into the cytoplasm to phosphorylate and store or metabolize glucose after the ingestion of dietary glucose. In GKRP-mutant mice, the disruption of this regulation and the subsequent decrease in GK activity leads to altered glucose metabolism and impaired glycemic control.