Toluidine blue binding capacity of heterochromatin and euchromatin of Triatoma injestans Klug

Summary Changes in the area of glutaraldehyde fixed 15 day p.c. mouse embryo limbs were recorded using a Quantimet 720 image analysing computer attached to a light microscope: during a period of treatment with an isotonic salt solution (mostly halides of the alkali or alkaline earth metals); a subsequent wash with distilled water; and dehydration through a 30, 50, 70, 80, 90, and 100% ethanol series. Pretreatment with NaCl, KCl, RbCl had no significant effect. Treatment with LiCl, LiNO₃, LiF (0.03 M), CsF and CsCl caused an increase (relative to Na, K or Rb treated samples) in the specimen volume during dehydration, which persisted in 100% ethanol. Li treated samples showed the largest post-critical-point-drying (CPD) volumes, followed by Cs treated tissue. Pretreatment with Be, Mg, Ca, Sr and Ba chlorides caused shrinkage and the 100% ethanol and post-CPD volumes of these samples were all lower than those treated with the monovalent cation containing salts.     

Disappearance of the Cartilage Interlacunar Network During Histological Dehydration

The interlacunar network connects adjacent chondrocytes in rapidly growing cartilage and with anionic dyes stains more intensely than the surrounding matrix. The network is seen in fresh-frozen sections and sections treated with a variety of fixatives, dehydration agents and embedding media. The network is not seen after using such common dehydrating fluids as ethanol and acetone. Polyanionic glycosaminoglycans (GAG) are abundant in the cartilage matrix and it has been suggested that the network is an artifact caused by a rearrangement of the matrix GAG during histological processing. Extraction of GAG from neonatal rat cartilage with guanidine hydrochloride removes stainable extracellular matrix but not the interlacunar network. However, after extraction of GAG and immersion of the cartilage in either 50% or 100% aqueous ethanol or acetone, the network was no longer visible. The network apparently is not an artifact formed by soluble matrix GAG, but a real structure which is extracted, collapsed or otherwise destroyed during dehydration in ethanol or acetone.     

Optimization of immunogold labeling TEM. An ELISA-based method for rapid and convenient simulation of processing conditions for quantitative detection of antigen.

We developed an ELISA-based method for rapid optimization of various tissue processing parameters in immunogold labeling for electron microscopy. The effects of aldehyde fixation, tannic acid, postfixation, dehydration, temperature, and antigen retrieval on antibody binding activity of Vitreoscilla hemoglobin (VHb) expressed in E. coli cells were assayed by ELISA and the results confirmed by quantitative immunogold labeling transmission electron microscopy (TEM). Our results demonstrated that low concentrations (0.2%) of glutaraldehyde fixation caused minimal loss in total binding compared to higher concentrations. Dehydration in up to 70% ethanol resulted in some distortion of cellular ultrastructure but better antibody binding activity compared to dehydration up to 100%. Postfixation or incorporation of tannic acid in the primary fixative caused almost total loss of activity, whereas antigen retrieval of osmium-postfixed material resulted in approximately 90-100% recovery. The sensitivity of detection of proteins by immunogold labeling electron microscopy depends on the retention of antibody binding activity during tissue processing steps, e.g., fixation and dehydration. Our study indicated that an ELISA-based screening method of various tissue processing procedures could help in rapid selection and optimization of a suitable protocol for immunogold localization and quantification of antigen by TEM.     

Dehydration reduces left ventricular filling at rest and during exercise independent of twist mechanics

The purpose of this study was to determine whether the reduction in stroke volume (SV), previously shown to occur with dehydration and increases in internal body temperatures during prolonged exercise, is caused by a reduction in left ventricular (LV) function, as indicated by LV volumes, strain, and twist ("LV mechanics"). Eight healthy men [age: 20 ± 2, maximal oxygen uptake (VO?max): 58 ± 7 ml·kg?1·min?1] completed two, 1-h bouts of cycling in the heat (35°C, 50% peak power) without fluid replacement, resulting in 2% and 3.5% dehydration, respectively. Conventional and two-dimensional speckle-tracking echocardiography was used to determine LV volumes, strain, and twist at rest and during one-legged knee-extensor exercise at baseline, both levels of dehydration, and following rehydration. Progressive dehydration caused a significant reduction in end-diastolic volume (EDV) and SV at rest and during one-legged knee-extensor exercise (rest: Δ-33 ± 14 and Δ-21 ± 14 ml, respectively; exercise: Δ-30 ± 10 and Δ-22 ± 9 ml, respectively, during 3.5% dehydration). In contrast to the marked decline in EDV and SV, systolic and diastolic LV mechanics were either maintained or even enhanced with dehydration at rest and during knee-extensor exercise. We conclude that dehydration-induced reductions in SV at rest and during exercise are the result of reduced LV filling, as reflected by the decline in EDV. The concomitant maintenance of LV mechanics suggests that the decrease in LV filling, and consequently ejection, is likely caused by the reduction in blood volume and/or diminished filling time rather than impaired LV function.     

Microwave processing for scanning electron microscopy

The normal processing of biological samples for Scanning Electron Microscopy, includes treatment with aldehyde (1 to 2 hours), postfixation with Osmium (1 hour), followed by dehydration in a ascending grade of ethanol (30 a 100%), 10 to 15 minutes in each step, and finally drying. This procedure takes at least 8 hours. In this work, samples of mosquitoes (Aedes), protozoa (Tritrichomonas muris), bacteria (Clostridium oceanicum), murine liver, and small intestine were processed in the same manner in a domestic microwave oven for two minutes at 20% of its maximum power. The complete procedure from the initial fixation to dehydration in 100% ethanol was reduced to one hour with good preservation of the ultrastructural details of the specimens.     

Selective dehydration of bio-ethanol to ethylene catalyzed by lanthanum-phosphorous-modified HZSM-5: influence of the fusel

Bio-ethanol dehydration to ethylene is an attractive alternative to oil-based ethylene. The influence of fusel, main byproducts in the fermentation process of bio-ethanol production, on the bio-ethanol dehydration should not be ignored. We studied the catalytic dehydration of bio-ethanol to ethylene over parent and modified HZSM-5 at 250°C, with weight hourly space velocity (WHSV) equal to 2.0/h. The influences of a series of fusel, such as isopropanol, isobutanol and isopentanol, on the ethanol dehydration over the catalysts were investigated. The 0.5%La-2%PHZSM-5 catalyst exhibited higher ethanol conversion (100%), ethylene selectivity (99%), and especially enhanced stability (more than 70 h) than the parent and other modified HZSM-5. We demonstrated that the introduction of lanthanum and phosphorous to HZSM-5 could weaken the negative influence of fusel on the formation of ethylene. The physicochemical properties of the catalysts were characterized by ammonia temperature-programmed desorption (NH(3)-TPD), nitrogen adsorption and thermogravimetry (TG)/differential thermogravimetry (DTG)/differential thermal analysis (DTA) (TG/DTG/DTA) techniques. The results indicated that the introduction of lanthanum and phosphorous to HZSM-5 could inhibit the formation of coking during the ethanol dehydration to ethylene in the presence of fusel. The development of an efficient catalyst is one of the key technologies for the industrialization of bio-ethylene.     

Purification of heparin,dermatan sulfate and chondroitin sulfate from mixtures by sequential precipitation with various organic solvents

Heparin, dermatan sulfate and chondroitin sulfate in mixtures were fractionated by sequential precipitation with methanol, ethanol and propanol. The recovered fractions from 0.1 to 2.0 volumes of various solvents were analyzed by agarose-gel electrophoresis and densitometric analysis. Heparins with different relative percentages of slow-moving and fast-moving components were precipitated from 0.5 to 0.7 volumes of methanol, and in this range of volumes, the amount of slow-moving component of heparin decreases and that of the fast-moving species increases. From 0.8 to 1.6 volumes of methanol, mixtures with different percentages of the fast-moving component, dermatan sulfate and chondroitin sulfate are precipitated. Heparin was precipitated from mixtures in the range of 0.1 to 0.4 volumes of ethanol, and from 0.5 to 0.8 volumes mixtures with different relative percentages of dermatan sulfate and chondroitin sulfate were precipitated. From 1.0 to 2.0 volumes of ethanol, high purity (about 100%) chondroitin sulfate can be precipitated. Propanol induces the precipitation of heparin from 0.3 to 0.4 volumes, whilst dermatan sulfate with a purity greater than 85% is precipitated at 0.5 and 0.6 volumes of propanol. 100% chondroitin sulfate is obtained with volumes greater than 0.8. Heparin and chondroitin sulfate from a bovine lung extract of glycosaminoglycans were purified by sequential precipitation with ethanol. The fraction precipitated with 0.4 volumes of ethanol shows greater than 90% heparin and that recovered from 0.9 to 2.0 volumes is composed of 100% chondroitin sulfate.     

Inactivation of viable Ascaris eggs by reagents during enumeration.

Various reagents commonly used to enumerate viable helminth eggs from wastewater and sludge were evaluated for their potential to inactivate Ascaris eggs under typical laboratory conditions. Two methods were used to enumerate indigenous Ascaris eggs from sludge samples. All steps in the methods were the same except that in method I a phase extraction step with acid-alcohol (35% ethanol in 0.1 N H(2)SO(4)) and diethyl ether was used whereas in method II the extraction step was avoided by pouring the sample through a 38-microm-mesh stainless steel sieve that retained the eggs. The concentration of eggs and their viability were lower in the samples processed by method I than in the samples processed by method II by an average of 48 and 70%, respectively. A second set of experiments was performed using pure solutions of Ascaris suum eggs to elucidate the effect of the individual reagents and relevant combination of reagents on the eggs. The percentages of viable eggs in samples treated with acid-alcohol alone and in combination with diethyl ether or ethyl acetate were 52, 27, and 4%, respectively, whereas in the rest of the samples the viability was about 80%. Neither the acid nor the diethyl ether alone caused any decrease in egg viability. Thus, the observed inactivation was attributed primarily to the 35% ethanol content of the acid-alcohol solution. Inactivation of the eggs was prevented by limiting the direct exposure to the extraction reagents to 30 min and diluting the residual concentration of acid-alcohol in the sample by a factor of 100 before incubation. Also, the viability of the eggs was maintained if the acid-alcohol solution was replaced with an acetoacetic buffer. None of the reagents used for the flotation step of the sample cleaning procedure (ZnSO(4), MgSO(4), and NaCl) or during incubation (0.1 N H(2)SO(4) and 0.5% formalin) inactivated the Ascaris eggs under the conditions studied.     

Metabolic dysfunction and unabated respiration precede the loss of membrane integrity during dehydration of germinating radicles

This study shows that dehydration induces imbalanced metabolism before loss of membrane integrity in desiccation-sensitive germinated radicles. Using a photoacoustic detection system, responses of CO(2) emission and fermentation to drying were analyzed non-invasively in desiccation-tolerant and -intolerant radicles of cucumber (Cucumis sativa) and pea (Pisum sativum). Survival after drying and a membrane integrity assay showed that desiccation tolerance was present during early imbibition and lost in germinated radicles. However, tolerance could be re-induced in germinated cucumber radicles by incubation in polyethylene glycol before drying. Tolerant and polyethylene glycol (PEG)-induced tolerant radicles exhibited a much-reduced CO(2) production before dehydration compared with desiccation-sensitive radicles. This difference was maintained during dehydration. In desiccation-sensitive tissues, dehydration induced an increase in the emission of acetaldehyde and ethanol that peaked well before the loss of membrane integrity. Acetaldehyde emission from sensitive radicles was significantly reduced when dehydration occurred in 50% O(2) instead of air. Acetaldehyde/ethanol were not detected in dehydrating tolerant radicles of either species or in polyethylene glycol-induced tolerant cucumber radicles. Thus, a balance between down-regulation of metabolism during drying and O(2) availability appears to be associated with desiccation tolerance. Using Fourier transform infrared spectroscopy, acetaldehyde was found to disturb the phase behavior of phospholipid vesicles, suggesting that the products resulting from imbalanced metabolism in seeds may aggravate membrane damage induced by dehydration.     

Effects of ethanol alone or after pretreatment with 20% ethanol on phospholipid metabolism in rat gastric mucosa

Changes in phospholipid metabolism in gastric mucosa caused by instillation of absolute ethanol (a cell-damaging agent) into the stomach of rats and the effects of pretreatment with 20% ethanol (a mild irritant) were investigated by using radioisotope-labeled fatty acids and glycerol. The labeled precursors were incorporated mainly into phosphatidylcholine and triacylglycerol, and also to lesser extents into phosphatidylethanolamine and phosphatidylinositol + phosphatidylserine. The instillation of absolute ethanol reduced the incorporation of fatty acids and glycerol into phospholipids within 15 min, indicating the inhibition by ethanol of de novo synthesis of phospholipids. Pretreatment with 20% ethanol caused the incorporation of fatty acids into phospholipids to be maintained after absolute ethanol instillation. These results suggest that the pretreatment with 20% ethanol may protect the cellular synthetic activity of phospholipids against damage by absolute ethanol. The incorporation of fatty acids into the free fatty acid fraction, monoacylglycerol and diacylglycerol was increased by absolute ethanol instillation, suggesting damage to the blood vessels of the gastric mucosa, and these changes were inhibited to some extent by the pretreatment with 20% ethanol.     

改良手工显微切割法获取特定细胞提取高质量RNA

探讨显微切割过程中有效保持RNA完整性的组织固定方法,建立一种简易的手工显微切割法.应用自制“T形板”辅助冰冻切片,100％无水乙醇一次性脱水固定,“排除切割法”获取目的细胞,用TRIzol提取RNA,琼脂糖凝胶电泳和RT-PCR分析RNA质量.“一步法”固定可长时间保存RNA的完整性;从食管癌标本5个特定阶段的细胞中提取的RNA,经电泳和RT-PCR分析均具有较高的质量.无水乙醇“一步法”固定,在显微切割的过程中可有效保持RNA的完整性;T形板和“排除切割法”简化了手工显微切割的操作,提取的RNA质、量均可满足后续分子水平研究的需要.     

Cytological and physiological changes in recalcitrant Chinese fan palm (<Emphasis Type="Italic">Livistona chinensis</Emphasis>) embryos during cryopreservation

Cytological and physiological changes during cryopreservation were investigated in Livistona chinensis embryos excised 42 weeks after flowering. Both dehydration and freezing caused numerous cellular ultrastructural alterations. Dehydration seriously impaired plasma membrane integrity, while freezing caused a further increase in electrolyte leakage. Damage to cellular ultrastructure and plasmalemma integrity had an inverse relationship with water content in unfrozen embryos and a positive relationship in frozen embryos. Changes in activity of antioxidant enzymes differed during cryopreservation. Dehydration and freezing had little effect on superoxide dismutase activity, although these treatments greatly reduced embryo viability. Activity of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) changed only slightly during dehydration, but dehydration markedly decreased activity of ascorbate peroxidase (APX) and catalase (CAT). Freezing further decreased APX and GR activity but increased CAT activity in dehydrated samples. A novel DHAR isozyme was induced during the freeze–thaw cycle. Membrane lipid peroxidation was detected in the control embryos, and was promoted by both dehydration and freezing. The malondialdehyde (MDA) content in post-thaw embryos increased by a maximum of 30%. Thus, changes in viability of embryos were closely related to damage to cellular ultrastructure and plasmalemma integrity, but were not directly related to antioxidant activity nor MDA accumulation.     

Resveratrol, a red wine polyphenol, attenuates ethanol-induced oxidative stress in rat liver

The involvement of oxidative stress in the pathogenesis of alcoholic diseases in the liver has been repeatedly confirmed. Resveratrol, a natural phytoalexin present in grape skin and red wine possesses a variety of biological activities including antioxidant. This study was conducted to evaluate whether resveratrol has a preventive effect on the main indicators of hepatic oxidative status as an expression of the cellular damage caused by free radicals, and on antioxidant defence mechanism during chronic ethanol treatment. Wistar rats were treated daily with 35% ethanol solution (3 g/kg/day i.p.) during 6 weeks and fed basal diet or basal diet containing 5 g/kg resveratrol. Control rats were treated with i.p. saline and fed basal diet. Experimentally, chronic ethanol administration leads to hepatotoxicity as monitored by the increase in the level of hepatic marker enzymes and the appearance of fatty change, necrosis, fibrosis and inflammation in liver sections. Ethanol also enhanced the formation of MDA in the liver indicating an increase in lipid peroxidation, a major end-point of oxidative damage, and caused drastic alterations in antioxidant defence systems. Particularly the activities of hepatic superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) were found reduced by ethanol treatment while glutathione reductase (GR) activity was unchanged. Dietary supplementation with resveratrol during ethanol treatment inhibited hepatic lipid peroxidation and ameliorated SOD, GPx and CAT activities in the liver. Conclusively, we can suggest that resveratrol could have a beneficial effect in inhibiting the oxidative damage induced by chronic ethanol administration, which was proved by the experiments that we conducted on rats.     

Tolerance and sensitization to endotoxin in Kupffer cells caused by acute ethanol involve interleukin-1 receptor-associated kinase

Ethanol changes sensitivity of Kupffer cells to endotoxin. Here, the hypothesis that interleukin-1 receptor-associated kinase (IRAK), a downstream signaling molecule of toll-like receptors, regulates the response to LPS in Kupffer cells after ethanol treatment was evaluated. C57BL/6 mice were given ethanol intragastrically, and LPS was injected 1 or 21 h later. One hour after ethanol treatment, serum transaminases after LPS were 60% of control, while ethanol increased these parameters about 3-fold 21 h after ethanol. Pretreatment with antibiotics blocked these effects of ethanol. In Kupffer cells from mice treated with ethanol 1 h earlier, LPS-induced TNFalpha production, and IRAK expression and activity and NFkappaB were decreased 50-60% of control. In contrast, in Kupffer cells from mice treated with ethanol 21 h earlier, LPS-induced TNFalpha production, expression and activity of IRAK were increased 1.5-fold over controls, while NFkappaB was elevated 3-fold. These data indicate that ethanol-induced tolerance and sensitization of Kupffer cells to endotoxin in mice involve IRAK.     

Enhancement of ethanol and biogas production from high‐crystalline cellulose by different modes of NMO pretreatment

Pretreatment of high‐crystalline cellulose with N‐methyl‐morpholine‐N‐oxide (NMO or NMMO) to improve bioethanol and biogas production was investigated. The pretreatments were performed at 90 and 120°C for 0.5–15 h in three different modes, including dissolution (85% NMO), ballooning (79% NMO), and swelling (73% NMO). The pretreated materials were then enzymatically hydrolyzed and fermented to ethanol or anaerobically digested to biogas (methane). The pretreatment at 85% NMO, 120°C and 2.5 h resulted in 100% yield in the subsequent enzymatic hydrolysis and around 150% improvement in the yield of ethanol compared to the untreated and water‐treated material. However, the best results of biogas production were obtained when the cellulose was treated with swelling and ballooning mode, which gave almost complete digestion in 15 days. Thus, the pretreatment resulted in 460 g ethanol or 415 L methane from each kg of cellulose. Analysis of the structure of treated and untreated celluloses showed that the dissolution mode can efficiently convert the crystalline cellulose I to cellulose II. However, it decreases the water swelling capacity of the cellulose. On the other hand, swelling and ballooning modes in NMO treatment were less efficient in both water swelling capacity and cellulose crystallinity. No cellulose loss, ambient pressure, relatively moderate conditions, and high efficiency make the NMO a good alternative for pretreatment of high‐crystalline cellulosic materials. Biotechnol. Bioeng. 2010; 105: 469–476. © 2009 Wiley Periodicals, Inc.     

Chemical clearing and dehydration of GFP expressing mouse brains

Generally, chemical tissue clearing is performed by a solution consisting of two parts benzyl benzoate and one part benzyl alcohol. However, prolonged exposure to this mixture markedly reduces the fluorescence of GFP expressing specimens, so that one has to compromise between clearing quality and fluorescence preservation. This can be a severe drawback when working with specimens exhibiting low GFP expression rates. Thus, we screened for a substitute and found that dibenzyl ether (phenylmethoxymethylbenzene, CAS 103-50-4) can be applied as a more GFP-friendly clearing medium. Clearing with dibenzyl ether provides improved tissue transparency and strikingly improved fluorescence intensity in GFP expressing mouse brains and other samples as mouse spinal cords, or embryos. Chemical clearing, staining, and embedding of biological samples mostly requires careful foregoing tissue dehydration. The commonly applied tissue dehydration medium is ethanol, which also can markedly impair GFP fluorescence. Screening for a substitute also for ethanol we found that tetrahydrofuran (CAS 109-99-9) is a more GFP-friendly dehydration medium than ethanol, providing better tissue transparency obtained by successive clearing. Combined, tetrahydrofuran and dibenzyl ether allow dehydration and chemical clearing of even delicate samples for UM, confocal microscopy, and other microscopy techniques.     

Effect of protein synthesis inhibitors on the activity of mitochondrial enzymes of Saccharomyces cerevisiae during desiccation

The work deals with the effect of carbon sources, presence of protein synthesis inhibitors (cycloheximide and chloramphenicol) and dehydration regime on the enzyme activity of the dried yeast Saccharomyces cerevisiae. The yeast grown on molasses and dried by aeration demonstrated a notable increase of the NADH-dehydrogenase and succinate dehydrogenase activities as compared with the analogously treated yeast grown on ethanol. The latter showed a notable rise only in the activity of NADH-dehydrogenase during slow drying. Addition of protein synthesis inhibitors into the cultivation medium caused no decrease of activities of the above mentioned enzymes of the dried yeasts in any variant under study.     

Changes in lymphocyte and macrophage subsets due to morphine and ethanol treatment during a retrovirus infection causing murine AIDS

Infection by LP-BM5 murine leukemia virus (MuLV) suppressed significantly the percentage of peripheral blood cells showing surface markers for macrophages, lymphocytes and activated lymphoid cells. Chronic administration of a 7% (36% calories) ethanol diet or injection of 1.9 mg/mouse/day of morphine for a 7 day period were followed by 3 week periods of abstinence and then 1 week periods of consumption of 5% ethanol diets or morphine injection to female C57BL/6 mice resulted in changes in the numbers of macrophages and lymphocyte subsets. The number of lymphocytes of various subsets were not significantly changed by the ethanol exposure except those showing activation markers which were reduced. The percentage of peripheral blood cells showing markers for macrophage functions and their activation were significantly reduced after "binge" use of ethanol. Ethanol retarded suppression of cells by retroviral infection. However by 25 weeks of infection there was a 8.6% survival in the ethanol fed mice infected with retrovirus which was much less than virally infected controls (45.0%). Morphine treatment also increased the percentage of cells with markers for macrophages and activated macrophages in virally infected mice, while suppressing them in uninfected mice. The second and third morphine injection series suppressed lymphocyte T-helper and T-suppressor cells, but not total T cells. However, suppression by morphine was significantly less during retroviral disease than suppression caused by the virus only. At 25 weeks of infection 44.8% of morphine treated, infected mice survived. Morphine treatment also caused deaths such that the survival in morphine treated, retrovirally infected was higher than would have been expected if the death rate in virally infected, and morphine injected animals were combined during combined treatment. Thus these drugs of abuse can modulate peripheral blood lymphoid subsets, suppression caused by retroviral infection, and survival.     

Localization of plant lipids for light microscopy using p-phenylenediamine in tissues of Arachis hypogaea L.

p-Phenylenediamine (pPD) can be used en bloc to preserve and differentiate cell lipids in aldehyde-fixed peanut plant tissues treated with osmium tetroxide during dehydration in 70% ethanol. Semithin plastic sections for light microscopy need no further staining and can be mounted in Histoclad after drying on a slide. Brown staining above background differentiates lipid-containing structures. Nonspecific staining can be distinguished in control preparations extracted en bloc with lipid solvents.