In vitro accumulation and saturation of 3H-progestins in selected brain regions and in the adenohypophysis, uterus and pineal of the female rat

Tissue samples from adult ovariectomized female rats were bisected, weighed and incubated in media containing ³H-progesterone with or without additional unlabeled progesterone. In brain tissue incubated without unlabeled progesterone samples from the interpeduncular region of the mesencephalon accumulated more ³H-progestins than samples from occipital cortex, posterior and anterior hypothalamus and medial-dorsal hippocampus. Within this latter group of tissues uptake was approximately equal. Adenohypophysial uptake in media without unlabeled progesterone was similar to that of cortex, while uterine uptake was less and pineal uptake more than that of cortical samples. Unlabeled progesterone reduced ³H-progestin accumulation in the interpeduncular, hippocampal, pituitary and uterine samples.     

Isolation of Fucoxanthin from the Rhizoid of Laminaria japonica Aresch

Fucoxanthin was extracted from the intact rhizoid of Laminariajaponica Aresch with dimethyl sulfoxide （DMSO）, and then recovered from the DMSO extract by partitioning into ethyl acetate and subsequent evaporation. Some isolation conditions such as solvent volume and extraction time were screened. The quantity and quality of the extracted fucoxanthin were determined by spectral analysis （absorption spectra and fluorescence emission spectra）. The results indicated that： （1） the average total content of fucoxanthin was 122.1μg in 1 g of fresh L. japonica rhizoid; （2） in comparison with the widely used organic solvent, acetone, DMSO was much more effective for the extraction of fucoxanthin; （3） both DMSO volume and extraction time influenced extraction efficiency such as the recovery rate and purity of fucoxanthin （1 g of fresh L. japonica rhizoid treated with 4 mL DMSO for 60 min, yielded 〉 88% of the total fucoxanthin with purity 0.63）; （4） when （NH4）2SO4 concentration was in the range of 0.5-1.0 mol/L, the pigments rapidly and entirely moved from DMSO into the ethyl acetate phase; （5） the ethyl acetate and DMSO were recycled using a rotary evaporator.     

Comparative fermentability of enzymatic and acid hydrolysates of steam-pretreated aspenwood hemicellulose by Pichia stipitis CBS 5776

Summary Enzymatic hydrolysates of hemicellulose from steam-pretreated aspenwood were more fermentable than the acid hydrolysate after rotoevaporation or ethyl acetate extraction treatments to remove acetic acid and sugar- and lignin-degradation products prior to fermentation by Pichia stipitis CBS 5776. Total xylose and xylobiose utilization from 5.0% (w/v) ethyl acetate extracted enzymatic hydrolysate was observed with an ethanol yield of 0.47 g ethanol/g total available substrate and an ethanol production rate of 0.20 g·l^-1 per hour in 72 h batch fermentation.     

Acetic acid removal from corn stover hydrolysate using ethyl acetate and the impact on Saccharomyces cerevisiae bioethanol fermentation

Acetic acid is introduced into cellulose conversion processes as a consequence of composition of lignocellulose feedstocks, causing significant inhibition of adapted, genetically modified and wild‐type S. cerevisiae in bioethanol fermentation. While adaptation or modification of yeast may reduce inhibition, the most effective approach is to remove the acetic acid prior to fermentation. This work addresses liquid–liquid extraction of acetic acid from biomass hydrolysate through a pathway that mitigates acetic acid inhibition while avoiding the negative effects of the extractant, which itself may exhibit inhibition. Candidate solvents were selected using simulation results from Aspen Plus?, based on their ability to extract acetic acid which was confirmed by experimentation. All solvents showed varying degrees of toxicity toward yeast, but the relative volatility of ethyl acetate enabled its use as simple vacuum evaporation could reduce small concentrations of aqueous ethyl acetate to minimally inhibitory levels. The toxicity threshold of ethyl acetate, in the presence of acetic acid, was found to be 10 g L^?1. The fermentation was enhanced by extracting 90% of the acetic acid using ethyl acetate, followed by vacuum evaporation to remove 88% removal of residual ethyl acetate along with 10% of the broth. NRRL Y‐1546 yeast was used to demonstrate a 13% increase in concentration, 14% in ethanol specific production rate, and 11% ethanol yield. This study demonstrated that extraction of acetic acid with ethyl acetate followed by evaporative removal of ethyl acetate from the raffinate phase has potential to significantly enhance ethanol fermentation in a corn stover bioethanol facility. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:929–937, 2016     

Quantitative extraction of microgram amounts of lipid from cultured human cells

A simple and efficient procedure based on the solvent system, ethyl acetate:acetone, was developed to extract the principal cellular lipids quantitatively from monolayer cultures of human fibroblasts with populations of even less than one-third million cells. The method was suitable for both radioisotope incorporation studies and studies involving quantitative analysis of fatty acid composition by gas-liquid chromatography. In a modified form the extraction procedure also allowed a 99% recovery of radioactive cell lipid from serum-containing growth medium. Included are results and discussion on the validity of cell number data obtained from monolayer cell cultures which are detached by trypsin and counted by a hemocytometer and a Coulter counting apparatus.     

Proteomic profiling of the rat hypothalamus

Background

The hypothalamus plays a pivotal role in numerous mechanisms highly relevant to the maintenance of body homeostasis, such as the control of food intake and energy expenditure. Impairment of these mechanisms has been associated with the metabolic disturbances involved in the pathogenesis of obesity. Since rodent species constitute important models for metabolism studies and the rat hypothalamus is poorly characterized by proteomic strategies, we performed experiments aimed at constructing a two-dimensional gel electrophoresis (2-DE) profile of rat hypothalamus proteins.

Results

As a first step, we established the best conditions for tissue collection and protein extraction, quantification and separation. The extraction buffer composition selected for proteome characterization of rat hypothalamus was urea 7?M, thiourea 2?M, CHAPS 4%, Triton X-100 0.5%, followed by a precipitation step with chloroform/methanol. Two-dimensional (2-D) gels of hypothalamic extracts from four-month-old rats were analyzed; the protein spots were digested and identified by using tandem mass spectrometry and database query using the protein search engine MASCOT. Eighty-six hypothalamic proteins were identified, the majority of which were classified as participating in metabolic processes, consistent with the finding of a large number of proteins with catalytic activity. Genes encoding proteins identified in this study have been related to obesity development.

Conclusion

The present results indicate that the 2-DE technique will be useful for nutritional studies focusing on hypothalamic proteins. The data presented herein will serve as a reference database for studies testing the effects of dietary manipulations on hypothalamic proteome. We trust that these experiments will lead to important knowledge on protein targets of nutritional variables potentially able to affect the complex central nervous system control of energy homeostasis.     

Fluorometric determination of DNA in fixed tissue using ethidium bromide

The measurement of DNA in tissue samples fixed in ethanol/acetic acid is described. Small, fixed tissue samples are digested by warm alkaline treatment followed by neutralization with HCl, and DNA is determined by complex formation with the dye ethidium bromide (EB). When standard DNA from calf thymus was treated similarly, a hyperchromicity of 8–12% and a reduction in fluorescence intensity of the EB-DNA complex to 55% was observed. The NaOH concentration (0.5–2.0 mol/liter) or the temperature (50–60°C) used for the digestion of tissue, as well as subsequent ribonuclease or protease treatment had no effect on the observed tissue DNA concentrations.     

Behavioral and Morphological Studies of Fetal Neural Transplants into SCN-Lesioned Rats

We have studied the effects of fetal neuronal grafts on the temporal pattern of drinking behavior of suprachiasmatic nuclei (SCN)-lesioned adult rats. Additionally, in an independent set of animals, the immunohistochemical staining for vasopressin, vasoactive intestinal polypeptide, and neuropeptide Y and the retinal connections to the hypothalamus were studied. The behavioral experiments indicate that anterior hypothalamic transplants induced reorganization of the temporal pattern of drinking behavior when placed in the third ventricle of adult hosts bearing complete SCN lesions, but not when placed in a cavity in the occipital cortex. Such rhythmicity persists only when the animals were recorded under constant darkness but not under constant light, indicating that the restored rhythmicity was generated endogenously but that the oscillator was extremely sensitive to light. Fetal occipital cortex induced reorganization of the temporal pattern of previously arrhythmic hosts, but it disappeared when the animals were recorded under constant light or constant darkness. It is clear that this rhythmicity was exogenous. In contrast to the cortical transplants, the hypothalamic transplants showed a morphological organization similar to that found in the normal hypothalamus regardless of their placement in the host brain. From these observations it is concluded that development of neocortex is more affected by environmental factors than that of the hypothalamus. Both hypothalamic and cortical transplants induced sprouting of retinal fibers into the anterior hypothalamus and the grafted tissue. It is possible that such fibers could be the neuroanatomical substrate by which rhythmicity is induced by cortical tissue.     

Behavioral and Morphological Studies of Fetal Neural Transplants into SCN-Lesioned Rats

We have studied the effects of fetal neuronal grafts on the temporal pattern of drinking behavior of suprachiasmatic nuclei (SCN)-lesioned adult rats. Additionally, in an independent set of animals, the immunohistochemical staining for vasopressin, vasoactive intestinal polypeptide, and neuropeptide Y and the retinal connections to the hypothalamus were studied. The behavioral experiments indicate that anterior hypothalamic transplants induced reorganization of the temporal pattern of drinking behavior when placed in the third ventricle of adult hosts bearing complete SCN lesions, but not when placed in a cavity in the occipital cortex. Such rhythmicity persists only when the animals were recorded under constant darkness but not under constant light, indicating that the restored rhythmicity was generated endogenously but that the oscillator was extremely sensitive to light. Fetal occipital cortex induced reorganization of the temporal pattern of previously arrhythmic hosts, but it disappeared when the animals were recorded under constant light or constant darkness. It is clear that this rhythmicity was exogenous. In contrast to the cortical transplants, the hypothalamic transplants showed a morphological organization similar to that found in the normal hypothalamus regardless of their placement in the host brain. From these observations it is concluded that development of neocortex is more affected by environmental factors than that of the hypothalamus. Both hypothalamic and cortical transplants induced sprouting of retinal fibers into the anterior hypothalamus and the grafted tissue. It is possible that such fibers could be the neuroanatomical substrate by which rhythmicity is induced by cortical tissue.     

Identification and quantitation of fatty acid ethyl esters in biological specimens

Fatty acid ethyl esters, recently described as enzymatic products of nonoxidative ethanol metabolism in the heart, may represent a mediator or marker of ethanol-induced organ pathology such as alcoholic cardiomyopathy. This study was designed to develop a method for the extraction, quantitation, and definitive identification of fatty acid ethyl esters formed both in biological specimens and during enzymatic incubations. First, several potential sources of error were identified and characterized. Tissue extraction with alcohols led to the time, temperature, and concentration-dependent nonenzymatic formation of fatty acid alcohol esters. Contamination of both substrates, [14C]ethanol and 14C-fatty acid, used to measure enzymatically mediated fatty acid ethyl ester synthesis, could be removed by purification. Accurate quantitation of fatty acid ethyl esters in tissue was achieved using acetone as an extraction solvent, after which isolated lipids were thin-layer chromatographed on silica gel developed with an apolar solvent system (petroleum ether:diethyl ether:acetic acid, 75:5:1). Gas chromatography and mass spectroscopy identified individual fatty acid ethyl esters. The reproducibility of this assay was high, as assessed by quintuplicate determinations of fatty acid ethyl esters formed in liver and heart homogenates, a method with standard deviations 4 to 11% of the mean.     

In vitro, release of cholecystokinin from hypothalamus and frontal cortex of Sprague-Dawley, Zucker lean (Fa/-) and obese (fa/fa) rats

Cholecystokinin (CCK) has been suggested as a putative satiety factor, whose site of action is in the hypothalamus. The genetically obese (fa/fa) Zucker rat has been proposed as a model of human obesity. Though hypothalamic tissue levels of CCK did not vary between the fa/fa rat and age-matched lean littermates (25.5 +/- 5.7 vs. 27.6 +/- 5.2 pmoles/g tissue) we sought to determine if the releasability of hypothalamic and cortical CCK was the same in lean and obese rats. The in vitro superfusion paradigm was used to study the release of CCK and substance P (sP) from hypothalamus, and CCK and vasoactive intestinal polypeptide (VIP) from frontal cortex. The potassium stimulated release of CCK from obese rat hypothalamic tissue was significantly higher than from lean rat hypothalamus (3.62 +/- 0.3 vs. 1.91 +/- 0.3 fmole equivalents CCK-8/mg tissue/10 min). Similarly, sP release was exaggerated in obese rats in a parallel fashion (5.56 +/- 0.44 vs. 2.761 +/- 0.46 fmoles/mg tissue/10 min). However, the potassium stimulated release of CCK and VIP from cortical tissue was the same in all three groups of rats. The obese Zucker rat thus, may have an anomalous release of CCK and sP from the hypothalamus, but not from the frontal cortex, an area not presumably associated with satiety.     

提取方法和溶剂对薏苡仁油提取率的影响

为了提高薏苡仁油的提取效率,考察5种不同极性的溶剂（无水乙醇、丙酮、二氯甲烷、乙酸乙酯、环己烷）在3种不同的提取方法中对薏苡仁油提取率的影响,并分析其原因。结果表明,丙酮在加热回流提取法中提取率最高（8.17%）,无水乙醇在超声辅助提取和微波辅助提取法中得率均较高。3种提取方法中,超声辅助提取法的得油率最高（10.79%）,而微波法虽然提取效率高（0.0678g/min）,但得油率太低（6.78%）,不适于薏苡仁油的提取。     

Derivatization and gas chromatographic—mass spectrometric detection of anabolic steroid residues isolated from edible muscle tissues

A method was developed for the detection of anabolic steroid residues in edible muscle tissues. After enzymic digestion of the tissue and purification on disposable C₁₈ solid-phase extraction columns, the extract was injected onto a C₁₈ reversed-phase high-performance liquid chromatographic column. Three fractions or windows were collected, each containing specific analytes. After evaporation to dryness, the residues were subjected to a derivatization procedure which yielded suitable derivatives. After gas chromatographic—mass spectrometric analysis, both gas chromatographic retention data and mass spectral data were used for the detection and identification of nortestosterone, testosterone, estradiol, ethinylestradiol, trenbolone, methyltestosterone, chlormadinone acetate, medroxyprogesterone acetate and megestrol acetate.     

Spectrophotometric Methods for Monitoring the Microbial Transformation of Steroids: I. Determination of 9α-Fluorohydrocortisone and 9α-Fluoro-16α-Hydroxyhydrocortisone in Fermentation Broths

A method has been devised for the determination of 9alpha-fluorohydrocortisone and 9alpha-fluoro-16alpha-hydroxyhydrocortisone in fermentation broths. The method involves extraction of the two steroids from the broth with ethyl acetate, separation through the formation of the water-soluble borate complex of the vicinally hydroxylated steroid, and estimation of each steroid spectrophotometrically.     

Characterization of immunoreactive somatostatin released from rat nervous system in vitro.

There is considerable evidence that somatostatin is released from nerve terminals throughout the central nervous system in response to presynaptic stimulation, thus suggesting a neuromodulator role for the peptide. We here report the partial characterization of immunoreactive somatostatin released from rat nervous system in vitro (hypothalamus, spinal cord and hypothalamic, cortical, thalamic and striatal synaptosomes). Serial dilutions of released somatostatin immunoreactivity showed parallelism with dilutions of synthetic somatostatin standard. Somatostatin immunoreactivity released from all tissue areas coeluted with synthetic tetradecapeptide on Sephadex G-25 (fine grade) gel chromatography; more than 85% of this immunoreactivity bound to Sepharose-anti-somatostatin-serum immunoaffinity columns. In addition, immunoreactive material released from hypothalamus, spinal cord and hypothalamic and cortical synaptosomes inhibited somatotropin (growth hormone, 'STH', 'GH') release from perifused anterior pituitary in a dose-related manner, indicating biological similarity to synthetic somatostatin.     

Intracellular progesterone receptors are differentially regulated by sex steroid hormones in the hypothalamus and the cerebral cortex of the rabbit

The aim of this study was to examine the role of sex steroid hormones in the regulation of intracellular progesterone receptors (PR) in the rabbit central nervous system. We determined PR concentration in cytosol preparations from the hypothalamus, the frontal, tempo-parietal and occipital cortex, by using the specific binding of the synthetic progestin [³H]ORG 2058. PR concentration was higher in the hypothalamus of intact adult females than in that of adult males and prepubertal females, whereas no significant differences were observed in the cerebral cortex of these animals. PR concentration was similar in the three cortical regions analyzed, indicating a homogeneous distribution of PR in the cerebral cortex. The administration of estradiol to ovariectomized animals increased PR concentration in the hypothalamus but not in the cortex. The administration of progesterone to ovariectomized rabbits did not modify PR concentration in any region, however when progesterone was administered after estradiol, it induced a significant diminution in hypothalamic PR concentration without effects in the cortex. These findings suggest that in the rabbit, PR are estrogen regulated in the hypothalamus but not in the cerebral cortex. In the latter, PR are not regulated by progesterone, whereas in the former the estrogen-induced PR are down-regulated by progesterone. Interestingly, hypothalamic PR constitutively expressed in ovariectomized animals are progesterone-insensitive.     

Responses of pyramidal tract and corticorubral neurons to stimulation of lateral hypothalamic structures in cats

Responses of pyramidal tract neurons and corticorubral and unidentified neurons in the pericruciate area of the cortex to electrical stimulation in the posterior, tuberal, and anterior zones of the lateral hypothalamus and also to electrodermal stimulation of all four limbs were studied in cats anesthetized with chloralose. The proportion of pyramidal tract, corticorubral, and unidentified neurons which responded to hypothalamic stimulation was 73.3, 55.7, and 79.1% respectively. Data on the possibility of monosynaptic activation of some pyramidal tract neurons and unidentified cells were obtained. The presence of less stable and longer-latency responses of corticorubral neurons indicated the absence of a monosynaptic pathway for realization of ascending hypothalamic influences on neurons of the cortical extrapyramidal system. Some cortical neurons responded to stimulation of more than one zone of the hypothalamus. Pyramidal tract neurons and corticorubral neurons with axon collaterals running into the region of the hypothalamus were discovered for the first time. It was shown that most neurons of the pericruciate area of the cortex to which the ascending influence of the hypothalamus is directed have a large bilateral receptive field and respond to electrodermal stimulation of several limbs.L. A. Orbeli Institute of Physiology, Academy of Sciences of the Armenian SSR, Erevan. Translated from Neirofiziologiya, Vol. 14, No. 3, pp. 298–306, May–June, 1982.     

Analysis of urinary 3-methoxy-4-hydroxyphenylglycol by high-performance liquid chromatography and electrochemical detection

A high-performance liquid chromatographic method with electrochemical detection for the quantitation of total 3-methoxy-4-hydroxyphenylglycol (MHPG) in human urine is described. Existing methods for deconjugation and extraction have been optimized. The present method is simpler than existing methods with a high precision. Urinary MHPG is deconjugated enzymatically and subsequently extracted with ethyl acetate. The organic layer is extracted with acetic acid and a sample of the aqueous layer is injected into a reversed-phase column. In one run 90 samples can be processed. The critical parameters of deconjugation, extraction and chromatography are described. Data for reproducibility and selectivity are presented.     

Aneuploidy-inducing chemicals in yeast evaluated by the micronucleus test

Chinese hamsters were exposed to acetone, methyl ethyl ketone, ethyl acetate and 2-methoxy ethyl acetate, known to be strong inducers of aneuploidy in the yeast Saccharomyces cerevisiae. All solvents yielded negative results in the micronucleus test, whereas the vinca alkaloid vindesine--used as a positive control substance--proved to act as a spindle poison in mammals in vivo.     

Induction of chlorosis, ROs generation and cell death by a toxin isolated from Pyricularia oryzae

The ethyl acetate extract of the conidia germination fluid from an Avena isolate (Br58) of Pyricularia oryzae had chlorosis-inducing activity on oat leaf segments. The same activity was also present in the acetone extract of an oatmeal agar culture of Br58. Fungal cultures were used for a large-scale preparation. A series of acetone and ethyl acetate extraction monitored by chromatography was used to isolate an active fraction. The active principle was purified by HPLC. We show by NMR and LC/MS that the toxin was an oxidized C18 unsaturated fatty acid named Mag-toxin. Mag-toxin induced chlorosis on oat leaf segments incubated in the light but not in the dark. Reactive oxygen species (ROS) and cell death were induced by Mag-toxin in oat cells. The sub-cellular localization of ROS generation induced by the toxin treatment was correlated with the location of mitochondria. Interestingly, the induction of ROS generation and cell death by Mag-toxin was light-independent.