The biomolecules of Euglena gracilis: Harnessing biology for natural solutions to future problems.

Over the past decade, the autotrophic and heterotrophic protist Euglena gracilis (E. gracilis) has gained popularity across the studies of environmental science, biosynthesis experiments, and nutritional substitutes. The unique physiology and versatile metabolism of E. gracilis have been a recent topic of interest to many researchers who continue to understand the complexity and possibilities of using E. gracilis biomolecule production. In this review, we present a comprehensive representation of recent literature outlining the various uses of biomolecules derived from E. gracilis across the fields of natural product biosynthesis, as a nutritional substitute, and as bioremediation tools. In addition, we highlight effective strategies for altering metabolite production using abiotic stressors and growth conditions. To better understand metabolite biosynthesis and its role in E. gracilis, integrated studies involving genomics, metabolomics, and proteomics should be considered. Together, we show how the ongoing advancements in E. gracilis related research continue to broaden applications in the biosynthetic sector and highlight future works that would strengthen our understanding of overall Euglena metabolism.

Effect of warming and flow rate conditions of blood warmers on red blood cell integrity.

BACKGROUND AND OBJECTIVES: Fluid warmers are routinely used to reduce the risk of hypothermia and cardiac complications associated with the infusion of cold blood products. However, warming blood products could generate haemolysis. This study was undertaken to compare the impact of temperature of blood warmers on the per cent haemolysis of packed red blood cells (RBCs) heated at different flow rates as well as non-flow conditions. MATERIALS AND METHODS: Infusion warmers used were calibrated at 41·5°C ± 0·5°C and 37·5°C ± 0·5°C. Cold RBC units stored at 4°C in AS-3 (n = 30), aged 30-39 days old, were divided into half units before being allocated under two different scenarios (i.e. infusion pump or syringe). RESULTS: Blood warmers were effective to warm cold RBCs to 37·5°C or 41·5°C when used in conjunction with an infusion pump at flow rate up to 600 ml/h. However, when the warmed blood was held in a syringe for various periods of time, such as may occur in neonatal transfusions, the final temperature was below the expected requirements with measurement as low as 33·1°C. Increasing the flow with an infusion pump increased haemolysis in RBCs from 0·2% to up to 2·1% at a flow rate of 600 ml/h regardless of the warming device used (P < 0·05). No relevant increase of haemolysis was observed using a syringe. CONCLUSIONS: The use of a blood warmer adjusted to 41·5°C is probably the best choice for reducing the risk of hypothermia for the patient without generating haemolysis. However, we should be cautious with the use of an infusion pump for RBC transfusion, particularly at high flow rates.

Blockade of angiotensin receptor subtypes in arcuate uterine artery of pregnant and postpartum rats.

During pregnancy, uterine circulation undergoes hypertrophy and hyperplasia. We investigated the effects of angiotensin (Ang) II receptor subtype (AT(1)/AT(2)) blockade on increased responses to the peptide during reversible remodeling of the uterine vasculature in pregnant and postpartum rats. Uterine arcuate arteries were set up in wire myographs for microvessel and submitted to a tension equivalent to 50 mm Hg transmural pressure. Cumulative concentration-response curves to Ang II were measured in the absence and presence of losartan on the same vascular segment. A similar protocol was repeated in the presence of PD 123,319, an AT(2) receptor blocker, again in the absence and presence of losartan. Responses to Ang II on the arcuate artery increased markedly during pregnancy and returned to the prepregnant level within 12 days postpartum. Losartan (10(-7) mol/L) produced a parallel right shift of the concentration-response curve to Ang II in all groups of tissues, but potency of the AT(1) receptor blocker was reduced at the end of pregnancy and in the early postpartum period. PD 123,319 (10(-7) mol/L) significantly increased maximum response to Ang II in arterial segments of the nonpregnant, term-pregnant, and 5 days postpartum rats. AT(1) receptor expression was decreased in arcuate arteries of term-pregnant rats. These results show that contractile responses to Ang II on the uterine arcuate artery of the rat are mediated by the AT(1) receptor and that blockade of AT(2) receptors potentiated responses to the peptide. They also indicate that, in uterine vessels, AT(2) receptor stimulation interferes with Ang II responses, but this effect is decreased in uterine arcuate arteries in the peripartum period.

In vitro generation of peroxynitrite by 2- and 4-hydroxyestrogens in the presence of nitric oxide.

Estrogen metabolism is altered in most, if not all, breast cancer tumors. These alterations primarily lead to the formation of the catechol estrogen metabolites, 2- and 4-hydroxyestrogens, which can generate superoxide anion radicals (O(2)(*)(-)) through the redox cycling of semiquinone/quinone derivatives. In breast cancer cells, the activity of nitric oxide synthase is also frequently elevated, resulting in an increased level of exposure to nitric oxide ((*)NO). Since (*)NO rapidly reacts with O(2)(*)(-) to produce the peroxynitrite anion (ONOO(-)), this study was undertaken to determine whether ONOO(-) can be generated when 2- and 4-hydroxyestrogens are incubated in vitro with (*)NO donor compounds. Using dihydrorhodamine 123 as a specific probe for ONOO(-) formation, a ratio of 100 microM dipropylenetriamine NONOate (DPTA/NO) to 10 microM 4-hydroxyestradiol (4-OHE(2)) gave an optimal ONOO(-) production of 11.9 +/- 1.9 microM (mean +/- SD). Quantification of ONOO(-) was not modified by mannitol, supporting the idea that the hydroxyl radical was not involved. This production of ONOO(-) required the presence of the catechol structure of estrogen metabolites since all methoxyestrogens that were tested were inactive. Hydroxyestrogen metabolites derived from estradiol showed the same efficiency in producing ONOO(-) as those originating from estrone. With DPTA/NO, the 4-hydroxyestrogens generated 30-40% more ONOO(-) than the 2-hydroxyestrogens. Optimal production of ONOO(-) was assessed with DPTA/NO and diethylenetriamine NONOate (initial (*)NO generation rates of 0.76 and 0.08 microM min(-1), respectively). With faster (*)NO-releasing compounds, such as diethylamine NONOate and spermine NONOate, lower levels of ONOO(-) were detected. These data suggest that once the optimal concentration of (*)NO was obtained, the reaction between (*)NO and 4-OHE(2) was saturated. The excess of (*)NO would probably react with aqueous oxygen to form nitrite (NO(2)(-)). Since the third-order reaction rate for the reaction between 2(*)NO and O(2) is 2 x 10(6) M(-2) s(-1), it can therefore be suggested that the reaction between (*)NO and 4-OHE(2) occurs at a faster rate.

[Comparison of four protocols for preoperative preparation in cattle]. / Comparaison de quatre protocoles de préparation préopératoire chez le bovin.

This study was designed to evaluate 4 preoperative skin preparations, that is, more specifically, to compare the efficacy of chlorhexidine gluconate (CG) and povidone-iodine (PI), as well as 2 hair removal techniques (clipper alone or clipper followed by razor) for preoperative skin preparation in cattle. The 4 protocols resulted in a significant decrease in the number of bacterial colony-forming units (cfu). Group 4 (clipping + shaving + CG) had a significantly lower number of preoperative cfu per gel plate compared with groups 1 (clipping + PI) and 3 (clipping + shaving + PI). Skin reaction frequency was significantly higher in groups 3 and 4 (47.8% for both protocols) than in groups 1 and 2 (clipping + PI or CG) (8.7% for both). Wound infection frequency was 4.3% (4/92) and no significant difference was observed between the 4 treatment groups. The 4 protocols tested were equivalent as to efficacy and satisfactorily decreased skin microflora. Clipping alone was shown to be preferable to clipping plus shaving as a method of hair removal in cattle, with fewer skin reactions and no more wound infections.

Thiols can either enhance or suppress DNA damage induction by catecholestrogens.

The estrogen metabolites catecholestrogens (or hydroxyestrogens) are involved in carcinogenesis and the development of resistance to methotrexate. This induction of drug resistance correlates with the relative efficiency of catecholestrogens in the generation of reactive oxygen species (ROS) and the induction of DNA strand breaks. Although antioxidants can neutralize ROS, the generation of these reactive species by catecholestrogens can be enhanced by electron donors like NADH. Therefore, this study was undertaken to determine the ability of different thiol agents (GSH, NAC, DTT, DHLA) to either inhibit or enhance the level of DNA damage induced by the H(2)O(2) generating system 4-hydroxyestradiol/Cu(II). Our results show that GSH, DTT, and DHLA inhibited the induction of the 4-hydroxyestradiol/Cu(II)-mediated DNA damage, with GSH showing the best potential. In contrast, the GSH precursor NAC at low concentrations was able to enhance the level of oxidative damage, as observed with NADH. NAC can reduce Cu(II) to Cu(I) producing the radical NAC&z.rad;, which can generate the superoxide anion. However, the importance of this pathway appears to be relatively minor since the addition of NAC to the 4-hydroxyestradiol/Cu(II) system generates about 15 times more DNA strand breaks than NAC and Cu(II) alone. We suggest that NAC can perpetuate the redox cycle between the quinone and the semiquinone forms of the catecholestrogens, thereby enhancing the production of ROS. In conclusion, this study demonstrates the crucial importance of the choice of antioxidant as potential therapy against the negative biological effects of estrogens.

Insulin inhibits inducible nitric oxide synthase in skeletal muscle cells.

Recent studies have shown that cytokines and endotoxins impair insulin-stimulated glucose transport by activating the expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production in skeletal muscle cells. In this study, we investigated whether iNOS induction is modulated by insulin in L6 myocytes. Long term exposure of muscle cells to tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) greatly increased iNOS mRNA expression and NO production. Addition of insulin to the cytokine/LPS-treated muscle cells reduced (by approximately 40%) NO production. This inhibition was similar to that observed with the synthetic glucocorticoid dexamethasone, a known inhibitor of iNOS in several cell types. The combination of insulin and dexamethasone was more effective than either agent alone in reducing NO production. Dexamethasone greatly inhibited the effect of cytokines/LPS to induce cellular iNOS mRNA expression. In strong contrast, insulin failed to reduce iNOS mRNA expression under similar conditions. These results show that insulin is a novel inhibitor of iNOS-mediated NO production in skeletal muscle cells. Furthermore, our data indicate that unlike glucocorticoids, insulin does not inhibit NO production by suppression of iNOS gene transcription.

Induction by estrogens of methotrexate resistance in MCF-7 breast cancer cells.

Development of drug resistance is a major factor that limits the effectiveness of chemotherapy treatments. In this study, we determined whether estradiol or its metabolites 2-, 4- and 16alpha-hydroxyestrone could enhance the development of methotrexate resistance in the breast carcinoma cell line, MCF-7. Cells were incubated with the estrogens at a concentration of 10(-8) M for 12 cell doublings and enhancement of methotrexate resistance was measured with the Luria-Delbrück assay. The most efficient estrogens were the 4-hydroxyestrone and 16alpha-hydroxyestrone, which both stimulated methotrexate resistance by 88-fold as compared with the control without estrogen. 2-Hydroxyestrone had an enhancement factor of 33-fold, whereas estradiol showed a slight effect with an enhancement factor of 3.2-fold. To determine whether the estrogen receptor was involved in the development of resistance, expression of the pS2 gene, which contains an estrogen-responsive element, was measured. Both estradiol and 16alpha-hydroxyestrone stimulated expression of the pS2 gene. In contrast, 2- and 4-hydroxyestrone did not increase the level of pS2 mRNA. This suggests that tumors classified as estrogen receptor negative could also develop methotrexate resistance as the result of exposure to estrogens. The status of the tumor suppressor gene p53 was analyzed in methotrexate sensitive and resistant clones. In all the methotrexate resistant clones analyzed, the western blots indicated that the p53 protein was still present and transcriptionally competent, as measured by its capacity to stimulate transcription of the p21waf1/cip1 gene following UVB irradiation. However, the basal level of p53 was higher in resistant clones and addition of 2- or 4-hydroxyestrone increased p53 to levels equivalent to those observed following UVB irradiation. However, this induction of p53 accumulation by estrogens failed to stimulate the transcription of p21waf1/cip1, which indicates that a transcriptionally inactive form of p53 accumulated in methotrexate resistant cells.

Expression of nitric oxide synthase in skeletal muscle: a novel role for nitric oxide as a modulator of insulin action.

Previous studies have shown that nitric oxide synthase (NOS), the enzyme that catalyzes the formation of nitric oxide (NO), is expressed in skeletal muscle. The aim of the present study was to test the hypothesis that NO can modulate glucose metabolism in slow- and fast-twitch skeletal muscles. Calcium-dependent NOS was detected in skeletal muscle, and the enzyme activity was greater in fast-type extensor digitorum longus (EDL) muscles than in slow-type soleus muscles. Both the neuronal-type (nNOS) and endothelial-type (eNOS) enzymes are expressed in resting skeletal muscles. However, nNOS protein was only detected in EDL muscles, whereas eNOS protein contents were comparable in soleus and EDL muscles. NOS expression in muscle cryosections (diaphorase histochemistry) was located in vascular endothelium and in muscle fibers, and the staining was greater in type IIb than in type I and IIa fibers. The macrophage-type inducible NOS (iNOS) was not detected in resting muscle, but endotoxin treatment induced its expression, concomitant with elevated NO production. iNOS induction was associated with impaired insulin-stimulated glucose uptake in isolated rat muscles. In vitro, NOS blockade with specific inhibitors did not affect basal or insulin-stimulated glucose transport in EDL or soleus muscles. In contrast, the NO donors GEA 5024 and sodium nitroprusside induced dose-dependent inhibition (up to 50%) of maximal insulin-stimulated glucose transport in both muscles with minor effects on basal uptake values. GEA 5024 also blunted insulin-stimulated glucose transport and amino acid uptake in cultured L6 muscle cells without affecting insulin binding to its receptor. On the other hand, the permeable cGMP analogue dibutyryl cGMP did not affect muscle glucose transport. These results strongly suggest that NO modulates insulin action in both slow- and fast-type skeletal muscles. This novel autocrine action of NO in muscle appears to be mediated by cGMP-independent pathways.

Cytokines modulate glucose transport in skeletal muscle by inducing the expression of inducible nitric oxide synthase.

The principal goal of the present study was to test the hypothesis that cytokines modulate glucose transport in skeletal muscle by increasing nitric oxide production. Cultured L6 skeletal muscle cells were incubated in the presence of tumour necrosis factor-alpha, interferon-gamma or lipopolysaccharide (LPS) alone or in combination for 24 h. Neither cytokines nor LPS alone induced NO production, as measured by nitrite concentrations in the medium. However, when used in combination, the two cytokines significantly stimulated NO production, and this effect was synergistically enhanced by the presence of LPS. Reverse transcriptase-PCR (RT-PCR) analysis revealed that NO release was associated with the induction of inducible (macrophage-type) NO synthase (iNOS). The increase in iNOS expression was confirmed at the protein level by Western-blot analysis and NADPH/diaphorase histochemical staining. Cytokines and LPS markedly increased basal glucose transport in L6 myocytes. Insulin also stimulated basal glucose transport, but significantly less in cells chronically exposed to cytokines/LPS. The sensitivity of L6 muscle cells to insulin-stimulated glucose transport was also significantly decreased by cytokines/LPS treatment. The NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME) inhibited nitrite production in cytokine/LPS-treated cells, and this prevented the increase in basal glucose transport and restored muscle cell responsiveness to insulin. Cytokines/LPS exposure significantly increased GLUT1 transporter protein levels but decreased GLUT4 expression in L6 cells. l-NAME treatment prevented the increase in GLUT1 protein content but failed to restore GLUT4 transporter levels. These results demonstrate that cytokines and LPS affect glucose transport and insulin action by inducing iNOS expression and NO production in skeletal muscle cells. The data further indicate that cytokines and LPS increase the expression of the GLUT1 transporter protein by an NO-dependent mechanism.

Successful training strategies to implement a workplace hazardous materials information system. An evaluation study at 80 plants.

According to recent regulations, Canadian companies must establish a Workplace Hazardous Materials Information System (WHMIS). Employee training is an essential facet of the system. The study looked at various training strategies and evaluated employees' comprehension about WHMIS and the quality of the WHMIS system at 80 manufacturing plants in the province of Québec in Canada. Comprehension was best when external experts trained all employees. It was lower when internal instructors were trained to teach WHMIS to other employees of the plant. Other components of WHMIS, such as labels and material safety data sheets, were of better quality in the plants having opted for training internal instructors. WHMIS was better in the plants that were initially good in occupational health and safety than in the marginal plants.

Efficacy of training procedures in implementing a legislated safety programme.

This study examines the effect of training procedures, as measured by the results of a test, on the level of assimilation of a new safety programme in 80 Quebec companies. The Workplace Hazardous Materials Information System (WHMIS) was introduced in the province in 1989. The bipartite safety association representing the manufacturers of transportation equipment and machinery offered two types of WHMIS training services to their members: (a) 4 h employee training courses, and (b) two-day trainer training courses. Companies were free to choose from these and other services to comply with the WHMIS training requirements. A test was issued to a stratified sample of 862 employees approximately one year after the training. On average, the scores were best in plants having employees trained directly by experts from the safety association (type a). The training of internal trainers for companies produced slightly inferior learning results (type b).

Binocular Interactions in the Lateral Suprasylvian Visual Area of Strabismic Cats Following Section of the Corpus Callosum.

Visually responsive neurons have been recorded in the lateral suprasylvian area (LSA) of cats raised with either a convergent or a divergent strabismus. In contrast to areas 17 and 18, where many studies have documented a profound loss of binocularly activated neurons following early strabismus, in the LSA the majority of cells could still be binocularly driven. Acute or chronic section of the splenium of the corpus callosum reduced but did not abolish binocularity in the LSA. We propose that the widespread callosal connections, the large size of the receptive fields and the peculiar internal circuitry of the LSA all concur in permitting the maintenance of binocular coding in spite of early misalignment of the eyes.

Cardiac strain among women workers in an industrial laundry.

Occupational heat exposure standards define permissible thermal conditions on the basis of metabolic heat production. However, even when energy expenditure is low, upright posture and repetitive upper limb motion may influence thermoregulatory behaviour through increased cardiovascular stress. The present study was undertaken to examine the relationship between work activity, thermal exposure and cardiac strain among women laundry workers engaged in sedentary, repetitive work activity. Ambient temperatures, work activity and heart rate were recorded during complete work shifts for 11 women over three days in summer and three days in winter. Workstation temperatures were significantly higher in summer. Analysis of continuous recordings of heart rate with respect to work activity showed (i) parallel increases in pulse rate and dominant arm movement frequency; (ii) different patterns of heart rate fluctuations for the two work cycles, with peaks during particular sub-tasks and when arms were in elevated positions. Recommended limits for cardiac strain indices were surpassed in both seasons, although in summer they were exceeded significantly more frequently. The part of cardiac effort attributable to thermoregulatory adjustments was also higher in summer while the fraction reflecting metabolic needs did not change with the season. These findings demonstrate high levels of cardiac strain in this work situation and raise the question of redefining heat exposure standards to include the prevention of excessive cardiac strain resulting from cumulative effects of heat load and ergonomic stressors.

Lack of binocular activation of cells in area 19 of the Siamese cat.

Single cells were recorded in area 19 of 8 Siamese cats. Receptive fields (RFs) were typical for this area in terms of size, directional specificity and type. However, 69 out of the 70 units found were monocularly driven through the contralateral eye. Moreover, the amount of excursion of RFs into the ipsilateral visual field was more limited than that generally demonstrated for areas 17 and 18, extending to a maximum of 5 degrees with very few cells having RFs situated completely within the ipsilateral hemifield.

Properties of triiodothyronine-binding proteins in liver cytosol of rat.

The electrophoretic mobility and the sedimentation coefficient were determined in partially purified preparations of both rat liver cytosol and serum triiodothyronine (T3)-binding proteins. Crude cytosol and serum, each labeled with [125I]T3, were filtered through a Sephadex G-100 column. The cytosol yielded a single T3-binding peak, whereas three binding components were recognized in the serum. Protamine sulfate precipitated the cytosol T3-binding protein, but had no effect on the serum T3-binders. The cytosol protein and the three binding proteins from serum were analyzed by polyacrylamide gel electrophoresis and sucrose density gradient centrifugation. The cytosol binder migrated as a single peak on gel electrophoresis with an Rf of 0.53, whereas the serum proteins had RfS between 0.27-0.33. The sedimentation coefficient of the cytosol protein was 6.3 S, whereas it was 4.1 S for the major binding protein of the serum. These data indicate that: (i) preliminary purification by gel chromatography is a useful step for better characterization of the T3-binding proteins of the cytosol and serum; (ii) the cytosol binder is an acidic protein with completely different properties from those of the serum T3-binding proteins.