Extract Low Molecular Weight Research Articles

Sea cucumber-derived extract can protect skin cells from oxidative DNA damage and mitochondrial degradation, and promote wound healing

Our skin serves as the primary barrier against external environmental insults, the latter of which can cause oxidative stress within cells, while various bioactive peptides sourced from natural resources hold promise in protecting cells against such oxidative stress. In this study, we investigate the efficacy of a low molecular weight extract from the sea cucumber Apostichopus japonicus, denoted as Sample-P, in facilitating cell migration and wound healing under oxidative stress conditions in skin cells. The naturally derived compound is a highly complex mix of peptides exhibiting antioxidative properties, as highlighted through liquid chromatography-mass spectrometry peptide screening and an in vitro antioxidant assay. Our results demonstrate that Sample-P is capable of promoting cell migration while preventing severe stress responses such as visible through mTOR expression. To further identify the molecular pathways underpinning the overall protective mechanism of Sample-P, we have utilised a proteomics approach. Our data reveal that Sample-P regulates protein expression associated with ribosomal pathways, glycolysis/gluconeogenesis and protein processing in the endoplasmic reticulum (ER), which help in preserving DNA integrity and safeguarding cellular organelles, such as mitochondria and the ER, under oxidative stress conditions in skin cells. In summary, in the presence of H2O2, Sample-P exhibits antioxidative properties at both molecular and cellular levels, rendering it a promising candidate for topical skin treatment to wound healing and to address age-related skin conditions.

Polydimethylsiloxane-coated macroporous silica adsorbent in thermal desorption gas chromatography

Polydimethylsiloxane (PDMS)-coated macroporous silica (MPSi) particles were developed for the extraction of volatile organic compounds (VOCs) from air samples in thermal desorption-gas chromatographic analysis. The gas sample was collected by a gas sampling pump at 100 mL/min. Different amounts of PDMS coating were prepared, and the fundamental extraction and desorption performances were investigated using even numbered n-alkanes of C8–C28. The extraction efficiency increased by increasing PDMS coating, and the desorption efficiency showed more than 99% at a desorption temperature of 310°C for all the investigated adsorbents. This remarkable desorption performance showed a higher peak area of the analytes than that of the Tenax TA-packed thermal desorption tube, especially for higher molecular weight compounds. Furthermore, an activated carbon particle of Carbopack X was introduced on the latter side of the PDMS-MPSi to extract low-molecular weight compounds. This double-bed-type extraction tube showed a successful determination of a wide range of VOCs. The double-bed-type extraction tube was then applied to the extraction of VOCs related to sick building syndrome (SBS). The results for the extraction and desorption performances of the SBS-related VOCs were well agreed with that of alkanes, and the applicability of the extraction tube to the determination of other types of conventional VOCs were confirmed.

RIAM: A Universal Accessible Protocol for the Isolation of High Purity DNA from Various Soils and Other Humic Substances.

A single universal open protocol RIAM (named after Research Institute for Agricultural Microbiology) for the isolation of high purity DNA from different types of soils and other substrates (high and low in humic, clay content, organic fertilizer, etc.) is proposed. The main features of the RIAM protocol are the absence of the sorption-desorption stage on silica columns, the use of high concentrations of phosphate in buffers, which prevents DNA sorption on minerals, and DNA precipitation using CTAB. The performance of RIAM was compared with a reference commercial kit and showed very good results in relation to the purity and quantity of DNA, as well as the absence of inhibitory activity on PCR. In all cases, the RIAM ensured the isolation of DNA in quantities much greater than the commercial kit without the effect of PCR inhibition up to 50 ng DNA per reaction in a volume of 15 µL. The latter circumstance along with the ability of the protocol to extract low molecular weight DNA fractions makes the method especially suitable for those cases where quantitative assessments, detection of minor components of soil microbiota, and completeness of isolation of all DNA fractions are required.

Dissolved organic metabolite extraction from high-salt media.

We describe considerations and strategies for developing a nuclear magnetic resonance (NMR) sample preparation method to extract low molecular weight metabolites from high-salt spent media in a model coculture system of phytoplankton and marine bacteria. Phytoplankton perform half the carbon fixation and oxygen generation on Earth. A substantial fraction of fixed carbon becomes part of a metabolite pool of small molecules known as dissolved organic matter (DOM), which are taken up by marine bacteria proximate to phytoplankton. There is an urgent need to elucidate these metabolic exchanges due to widespread anthropogenic transformations on the chemical, phenotypic, and species composition of seawater. These changes are increasing water temperature and the amount of CO2 absorbed by the ocean at energetic costs to marine microorganisms. Little is known about the metabolite-mediated, structured interactions occurring between phytoplankton and associated marine bacteria, in part because of challenges in studying high-salt solutions on various analytical platforms. NMR analysis is problematic due to the high-salt content of both natural seawater and culture media for marine microbes. High-salt concentration degrades the performance of the radio frequency coil, reduces the efficiency of some pulse sequences, limits signal-to-noise, and prolongs experimental time. The method described herein can reproducibly extract low molecular weight DOM from small-volume, high-salt cultures. It is a promising tool for elucidating metabolic flux between marine microorganisms and facilitates genetic screens of mutant microorganisms.

Therapeutic Effect of Spleen Low Molecular Weight Extracts on Leukopenia Caused by Epirubicin in Mice and Its Mechanism

To investigate the therapeutic effect of spleen low molecular weight extracts on epileptics hydrochloride-induced leukopenia in mice and explore its mechanism. The model of leukopenia in mice was established by the injection of epirubicin hydrochloride (10 mg/kg). After the injection of chemotherapeutic drugs, leukocytopenia mice were treated with different doses of spleen low molecular weight extract, Ganoderma oral solution and recombinant granulocyte colony stimulating factor (rhG-CSF). The general survival status indicators such as body weight, coat color and athletic ability of mice in each group were recorded; the tail vein blood of mice in each group was collected and the white blood cell count in them was calculated; bone marrow of mice was taken and bone marrow smears were observed. In the model group, the weight of the mice gradually decreased in the later period, their coat became dark and rough, and the ability to exercise decreased, while the mice in the treatment groups showed different degrees of improvement in their survival status except for the mice treated by rhG-CSF. There was no significant fluctuation in the white blood cell count of the blank control mice. After injection of epirubicin, the white blood cell count of peripheral blood in the model mice and treated mice were decreased. The white blood cell count was lower in the mice treated with high-dose low molecular weight extract and rhG-CSF than that in other experimental groups. Bone marrow smear showed that the proportion of bone marrow nucleated cells in the mice treated with the low molecular weight extract of the spleen was significantly higher than that of model mice (P<0.05). The low molecular weight spleen extracts can significantly improve the hematopoietic state of mouse bone marrow, promote the proliferation of inhibited bone marrow cells, and thus has the effect of treating leukopenia in mice.

Degradative solvent extraction of low-rank coals by the mixture of low molecular weight extract and solvent as recycled solvent

The authors have proposed a degradative solvent extraction method to dewater, upgrade and separate low-rank coals to upgraded coal, high molecular weight extract (Deposit), low molecular weight extract (Soluble), and a little amount of liquid and gas products. For the practical application of this extraction method, the solvent must be recycled. Furthermore, the separation of the Soluble and solvent, which was implemented by distillation, consumed much energy and added the cost of the whole system significantly. In order to solve these issues, the utilization of the Soluble and solvent as the recycled mixture solvent was proposed and investigated in this work. The results showed that the yields of the Deposit increased by more than two times with the mixture solvent recycle, and got almost stable after 5–7 extraction cycles. This made up for the loss of the part of the extraction product (Soluble) to a large extent. Furthermore, the ratios of O/C, N/C, and S/C of the Deposit decreased with the mixture solvent recycle, indicating that the Deposit was further upgraded. The aromaticity and reactivity of the Deposit decreased and increased with the mixture solvent recycle, respectively. The Cal/Car of the Deposits ranged from 0.35 to 0.84. The ash content of the Deposit increased slightly from 0.25% to 0.29%, and then got almost constant after 4 times of the mixture solvent recycle. Only 0.2–0.4% of the main inorganic elements were transformed from raw coal to the Deposit. The main inorganic elements in the Deposit were Si, Al and Fe. The relatively high contents of some inorganic elements in the Deposit were caused by their high contents and existing form in the raw coal. Thus, it was showed that using the Soluble and solvent as recycled mixture solvent is a feasible way for the practical application of the degradative solvent extraction of low-rank coals.

Upgrading and multistage separation of rice straw by degradative solvent extraction

Upgrading and multi-stage separation (UMSS) of rice straw was conducted at different temperatures using 1-methylnaphthalene (1-MN) as solvent. Three main solid products were obtained: low molecular weight extract (soluble), high molecular weight extract (deposit) and extraction residue (residue). The elemental composition, chemical structure and physicochemical characteristic of each component were analyzed in detail. Alkali and alkaline earth metal (AAEM) contents of solid products and rice straw were also measured by ICP-MS. The results showed the yield of soluble increased with temperature, and the carbon-based yield of soluble reached 33.48% at 350°C. The carbon content and oxygen content of three solid products (soluble, deposit, and residue) increased and decreased with temperature, respectively. The carbon content of soluble and deposit reached up to 82.36% and 80.59% respectively. Meanwhile the oxygen contents of them were as low as 9.50% and 12.03% respectively. More than 86.99% oxygen of rice straw was removed as H2O and CO2. Soluble was almost free from ash, and the ash content of deposit was also less than 1.50%. The higher heating values (HHV) of three solid products were significantly higher than that of rice straw. The FT-IR results indicated that not only dehydration reaction and decarboxylation reaction occurred, but also including obvious aromatization reaction. The contents of Na, Mg and K of soluble and deposit were extremely low, and they decreased with temperature gradually. In conclusion, the degradative solvent extraction method realized dehydration, deoxygenation and multistage separation of biomass under mild condition, and obtained a variety of products of low ash and oxygen content as well as high carbon content and HHV.

Degradative solvent extraction of demineralized and ion-exchanged low-rank coals

Dehydration and upgrading are essential pretreatment methods for efficient utilization of low-rank coal. In previous works the authors employed degradative solvent extraction method to dehydrate and upgrade low-rank coals and fractionate them into several fractions. For further study of this method, two low-rank coals (MM and LY) were pretreated by acid washing for demineralization or acid washing and Na/Co ion-exchange. The pretreated and raw coals were then extracted by 1-methylnaphthalene (1-MN) at 350°C and fractionated into upgraded coal (UC), high molecular weight extract (deposit), low molecular weight extract (soluble), as well as a little H2O and gas products. The results show that both acid washing and ion-exchange enhance the yields and carbon contents of the two extracts. Ion-exchange obviously promotes the removal of oxygen-containing functional group during extraction. The yield of high molecular weight extract of demineralized MM increases from 3.5% to 9.5%, and the carbon content and oxygen content of low molecular weight extract of Na ion-exchanged LY are as high as 85.3% and less than 6.4%, respectively. Ion-exchange has a distinct influence on physical and chemical properties of the extracts. The influence of Na ion-exchange is especially remarkable. Thus, demineralization and ion-exchange have evident promotion for the degradative solvent extraction of low-rank coal.

A Rapid Test for Soy Aeroallergens Exposure Assessment

BackgroundDetermining soy aeroallergens levels is extremely important in the assessment of health risks due to these airborne substances. Currently, soy aeroallergens exposure in the environment is monitored using enzyme immunoassays (EIA) which must be evaluated in a specialized laboratory by skilled personnel.ObjectiveTo describe the development and performance of a rapid immunochromatography assay for the detection of soy aeroallergens in environmental samples.MethodsA test strip using gold labeled anti-soy hull low molecular weight extract (SHLMWE) antibody for the rapid detection of soy aeroallergens in environmental samples was developed. One hundred nineteen airborne samples were analysed in parallel by the strip assay and the anti-SHLMWE sandwich EIA. The assay results were visually analysed by three independent observers who ranked samples as: -, + or ++. Strips were also scanned and analysed by densitometry.ResultsThe rapid test detected a range of concentrations from 6.25 to 25 ng/mL. Agreement in strip assay interpretations between evaluators was substantial (Kappa = 0.63; CI 0.544–0.715). Visual interpretation also gave a good concordance with EIA results, with sensitivity ranging from 77.3 to 100 and specificity from 65 to 83.5 depending on the observer. Furthermore, a strong correlation was observed between densitometry results of strip assay and EIA determinations.ConclusionsThe strip assay developed is rapid, simple, and sensitive and does not require expensive equipment or specific skills. It has considerable potential in the environmental monitoring field for screening soy aeroallergens levels in port cities where allergen measurements are not currently performed. Due to its simplicity, the test will improve the management of soy allergic patients by controlling environmental allergen exposure without the need for apparatus or skilled personnel.

The influence of porcine cathelicidins on neutrophils isolated from rabbits in the course of bone graft implantation

Antimicrobial peptides are important elements of host defence because of their direct antimicrobial activity and modulatory role in innate immune response. The purpose of the study was to determine whether porcine peptides PR-39, protegrins (PGs) and low molecular weight extract (LMWE) are able to influence the neutrophil response during bone graft implantation in rabbits. The study was conducted on 10 White New Zealand rabbits and neutrophil activity was assayed on the basis of elastase, myeloperoxidase, and alkaline phosphatase release as well as free radical generation. Our study showed that PR-39 and PGs inhibited enzyme release from neutrophils except for elastase, which is essential in the first phase of injury. Superoxide and nitric oxide generation under the influence of PR-39 and PGs were also decreased. Moreover, we found that unlike separated peptides PR-39 and PGs, LMWE acts proinflammatorily, intensifying the neutrophil secretory response and free radical generation. These results should be taken into account in treatment with natural antimicrobial peptides. The increased neutrophil responses in the first phase of inflammation during surgery may be useful in prevention of infection, but LMWE should not be used in conditions in which excessive neutrophil response is injurious.

Extraction and activity of regulatory peptides from sea urchin eggs

Experiments were performed to extract low molecular weight peptides from the eggs of the sea urchin Strongylocentrotus pallidus using sterilizing microfiltration and solid-phase extraction, along with analytical gel chromatography and spectroscopy. The peptides extracted had molecular weights of 360 – 1200 Dal and were divided into two fractions: acidic peptides contained a predominance of glutamic and aspartic acid residues, along with aromatic amino acid residues; alkaline peptides contained mainly lysine and arginine residues and lacked aromatic amino acids. Assessment of the biological activity of these extracts demonstrated high specificity in stimulating the development of explants in organotypic cultures of spleen, testicle, myocardium, and cerebral cortex tissues.

Selective extraction of low molecular weight proteins by mesoporous silica particles with modified internal and external surfaces

A new mesoporous silica material with modified external and internal surfaces (alkyl diol-vinyl-SiO(2)) was prepared and applied to selectively extract low molecular weight (LMW) proteins and peptides (less than 11 kDa) from biological samples. X-ray diffraction, N(2) adsorption, transmission electron microscopy, and Fourier transform infrared spectroscopy were used to characterize the alkyl diol-vinyl-SiO(2). This material was used to extract LMW proteins (peptides) from standard proteins, a mixture of peptides, and crude human plasma. We compared the amounts of different proteins adsorbed by alkyl diol-vinyl-SiO(2) and by vinyl-SiO(2) (vinyl group on the internal surface of the mesoporous silica material). The desorption recovery of alkyl diol-vinyl-SiO(2) by different eluents was also investigated by using a standard protein (insulin) as a model LMW protein. The material could efficiently extract LMW proteins and peptides from a mixture of standard proteins and crude human plasma with good extraction efficiency and desorption recovery. Furthermore, the alkyl diol-vinyl-SiO(2) had much better extraction selectivity for LMW proteins and peptides than a commercial C(18) solid phase extraction material.

Improved disc SDS‐PAGE for extraction of low molecular weight proteins from serum

The low molecular weight proteins can provide a lot of valuable information of biomarkers. To study these proteins, the high abundance and high molecular weight proteins must be removed prior to analysis. In this work, a simple and inexpensive disc SDS-PAGE to extract low molecular weight proteins from human serum and cutoff proteins larger than 30 kDa was developed. Some experimental conditions were examined. The experimental results obtained by plate SDS-PAGE and MALDI-TOF MS showed that the molecular weight of extracted proteins was about in the range from 0.3 to 28 kDa. Some experiments, including precipitation of proteins in organic solvents, SPE and cytochrome C test, were carried out and the experimental results demonstrated successful recovery of proteins/peptides with molecular weight from several hundreds of dalton to about 30 kDa. The experimental results obtained by plate SDS-PAGE indicated the repeatability was satisfactory.

Cysteine sulphoxides, amino acids and alliinase activity of Allium nigrum

Allium nigrum L. belongs to the subgenus Melanocrommyum of the genus Allium. This subgenus is widely distributed in Southwest and Central Asia. However, A. nigrum is a typical plant of east Mediterranean islands. Many species of this subgenus were traditionally used. A. nigrum is reported to be active against helminthiasis. Typical for the genus Allium is a rather high content of cysteine sulphoxides. Most abundant is the cysteine sulphoxide methiin (1). Recently, an unusual cysteine sulphoxide containing a pyrrole ring system (2) could be reported [1]. Both compounds were also present in A. nigrum. Amino acid derivatives were analysed as corresponding o-phthaldialdehyde derivatives by means of HPLC-MS/MS. For methiin (1) and the pyrrole derivative (2), average concentrations of 0.04% and 0.01%, respectively, were found (concentrations related to the fresh weight of bulbs). If these cysteine sulphoxides would be incubated with the enzyme alliinase, a number of thiosulphinates can be expected. Enzymatic incubations were also performed for the low molecular weight extract of A. nigrum and resulting compounds were analyzed by HPLC-MS/MS. Interestingly, only the pyrrole derivative (3) could be detected. It is completely unclear in which manner methiin (1) reacts with the alliinase of A. nigrum. It can be supposed that this alliinase acts different from the well described alliinase of A. sativum (garlic).

Aqueous fish extract increases survival in the mouse model of cytostatic toxicity

BackgroundTreatment of cancer patients with anthracycline antibiotic doxorubicin (DOX) may be complicated by development of acute and chronic congestive heart failure (CHF), malignant arrhythmias and death. The aim of this study was to test whether an aqueous low molecular weight (LMW) extract from cod muscle decreases acute mortality in the mouse model of acute CHF caused by DOX.MethodsA LMW fraction (<500 Da) of the aqueous phase of cod light muscle (AOX) was used for treatment of male BALB/c mice (~25 g, n = 70). The animals were divided into four groups, DOX + AOX (n = 20), DOX + saline (NaCl) (n = 30), NaCl + AOX (n = 10) and NaCl only (n = 10). Echocardiography was performed in the separate subgroups (DOX treated n = 6 and controls n = 6) to verify the presence and the grade of acute CHF. The cod extract was delivered by subcutaneously implanted osmotic minipumps over the period of 2 weeks. High-dose injection of DOX was administered to randomly selected animals. The animals received single intraperitoneal injection of DOX (25 mg/kg) and were followed over two weeks for mortality.ResultsMortality rate was 68% lower (p < 0.05) in the mice treated with the extract. The analyses of cod extract have shown strong antioxidative effect in vitro.ConclusionThe aqueous LMW cod muscles extract decreases mortality in the mouse model of DOX induced acute CHF. This effect may be mediated by cardioprotection through antioxidative mechanisms.

Histone Acetylation-independent Effect of Histone Deacetylase Inhibitors on Akt through the Reshuffling of Protein Phosphatase 1 Complexes

Despite advances in understanding the role of histone deacetylases (HDACs) in tumorigenesis, the mechanism by which HDAC inhibitors mediate antineoplastic effects remains elusive. Modifications of the histone code alone are not sufficient to account for the antitumor effect of HDAC inhibitors. The present study demonstrates a novel histone acetylation-independent mechanism by which HDAC inhibitors cause Akt dephosphorylation in U87MG glioblastoma and PC-3 prostate cancer cells by disrupting HDAC-protein phosphatase 1 (PP1) complexes. Of four HDAC inhibitors examined, trichostatin A (TSA) and HDAC42 exhibit the highest activity in down-regulating phospho-Akt, followed by suberoylanilide hydroxamic acid, whereas MS-275 shows only a marginal effect at 5 microm. This differential potency parallels the respective activities in inducing tubulin acetylation, a non-histone substrate for HDAC6. Evidence indicates that this Akt dephosphorylation is not mediated through deactivation of upstream kinases or activation of downstream phosphatases. However, the effect of TSA on phospho-Akt can be rescued by PP1 inhibition but not that of protein phosphatase 2A. Immunochemical analyses reveal that TSA blocks specific interactions of PP1 with HDACs 1 and 6, resulting in increased PP1-Akt association. Moreover, we used isozyme-specific small interfering RNAs to confirm the role of HDACs 1 and 6 as key mediators in facilitating Akt dephosphorylation. The selective action of HDAC inhibitors on HDAC-PP1 complexes represents the first example of modulating specific PP1 interactions by small molecule agents. From a clinical perspective, identification of this PP1-facilitated dephosphorylation mechanism underscores the potential use of HDAC inhibitors in lowering the apoptosis threshold for other therapeutic agents through Akt down-regulation.

A rapid assay to conduct in-vitro polyomavirus DNA replication

Traditionally, the HIRT extraction method using phenol and chloroform, is employed to selectively extract low-molecular weight DNA to conduct polyomavirus DNA replication assays. Moreover, DNA replication results obtained with this approach are typically inconsistent between triplicate specimens. To hasten the procedure of polyomavirus DNA replication assay and to enhance reproducibility, we compared the DNA quality and yield using Qiagen Spin Column technology and the HIRT extraction method. CV-1 cells were transfected with SV40 DNA, cells were harvested at days 2, 4 and 6, and DNA was extracted using the Qiagen Spin Column and the HIRT extraction methods. Southern hybridization was performed using a 32P labeled linear full-length SV40 DNA. Viral DNA replication was quantitated using the BioRad phosphorimager and results obtained with the two procedures were compared. Additionally, B-cells and Rat2 cells were spiked with 500 ng of JC polyomavirus linear DNA and processed as described above, except 32P labeled full-length linear JCV DNA was employed in the Southern hybridization step. Southern blot analysis revealed consistent SV40 and JCV DNA recovery using the Qiagen Spin Column technology, and SV40 DNA replication was consistent between triplicate specimens at all time points. In addition, SV40 DNA recovery was greater using the Qiagen Spin Column technology as judged by Southern blot analysis. The traditional HIRT extraction method employed to conduct polyomavirus DNA replication assays takes approximately 24 hr, whereas the Qiagen Spin Column technology reduces the time required to obtain good quality DNA to less than one hr. These modifications to the assay will improve the determination of polyomavirus DNA replication activity, while reducing exposure of the investigator to toxic organic compounds.

Semiochemical strategies for sea louse control: host location cues.

The development of behavioural bioassays and electrophysiological recording techniques has enabled the role of semiochemicals to be investigated for the first time in Lepeophtheirus salmonis (Krøyer). Adult male sea lice L salmonis were activated by, and attracted to, salmon-conditioned water (SCW) and SCW extract. Non-host fish odours, turbot-conditioned water (TCW) and turbot-conditioned water extract (TCW extract), elicited activation but no attraction in the lice. Solid-phase extraction techniques were developed to extract low molecular weight components of fish odours, and biological activity was shown to be confined to these. Electrophysiological activity from aesthetascs in the first antennae was found and neural responses to odours were recorded from the antennal nerves. Preliminary experiments on individual chemicals (isophorone, 1-octen-3-ol) linked to salmon revealed behavioural activation and electrophysiological responses in adult male L salmonis. Isophorone was shown to be significantly attractive to sea lice when placed in a slow-release system in a perfused tank of seawater in a choice situation. Proof of concept has been established for successfully extracting odour cues from seawater, analysing their biological activity and applying these to slow-release technologies for field trapping of lice. Future work involving linked GC-MS techniques using behaviour, electrophysiological responses, and organolepsis will establish further host location cues specific to Atlantic salmon.

A Simple Method for Estimation of Neutral Detergent-Soluble Fibre

A method was developed to estimate the neutral detergent-soluble fibre (NDSF) content of feeds. Citrus pulp, sugar beet pulp, soybean hulls, separated leaves and stems from mature and immature alfalfa, red clover, reed canarygrass and timothy were analysed. Half-gram samples were stirred at room temperature for 4 h with 100 ml of 90:10 (v/v) ethanol/water to extract low molecular weight substances, crude protein (CP) and ash to produce ethanol-insoluble residues (EIR). Samples were refluxed for 1 h with neutral detergent solution and heat stable α-amylase to extract low molecular weight substances, NDSF, starch, CP and ash to produce neutral detergent residues (NDF). The EIR and NDF were analysed for organic matter (OM; EIROM; NDFOM) and CP (EIRCP; NDFCP), and EIR was analysed for starch. Values were expressed as proportions of the original sample dry matter. The NDSF was calculated as the difference in OM mass between EIR and NDF after correction for CP and starch by the equation: EIROM-NDFOM-EIRCP+NDFCP-EIR starch. Extractions with 90:10 (v/v) ethanol/water gave a higher yield of EIR and NDSF than did 80: 20 (v/v) ethanol/water. The method gave good precision and is convenient for assessing NDSF content of feeds. © 1997 SCI.

Neuronal cholecystokinin release: morphine and naloxone effects on rat periaqueductal grey

The periaqueductal grey area (PAG) of rat brain is strongly indicated as a major site of opiateand stimulation-induced analgesia. The PAG is thought to integrate perception and response to nociceptive stimuli; afferent projections include major imputs from limbic and sensory areas; efferent projections include those to the spinal cord and nucleus raphe magnus (Beitz, 1982). Apart from its content of opioid peptides, the PAG also contains cholecystokinin octapeptide (CCK), which may be involved in processing of nociceptive stimuli since it reverses morphine-, /I-endorphinand footshock-induced analgesia (Faris et al., 1983). Furthermore, at higher doses CCK is an antinociceptive agent in its own right (Jurna & Zetler, 1981; Sheehan & de Belleroche, 1984). A further indication of a possible interaction between CCK and opiate analgesia systems at the level of the PAG is the finding that the firing of CCK neurons in the EdingerWestphal nucleus of the PAG is reduced by noxious stimuli or intravenous morphine (Innis & Aghajanian, 1985). The following experiments were designed to ascertain whether there is a direct action of opiates on CCKcontaining neurons in the PAG as measured by CCK release. Male CFY rats were killed, their brains removed and the PAG dissected out and chopped (tissue slices 0.3mm thick). The slices were incubated at 37°C in Krebs-bicarbonate medium, pH 7.4, containing 0.5 mMbacitracin and gassed with 95% 0 , / 5 % CO,. After one 10 min preincubation the slices were sequentially transferred to drug-containing medium for a further IOmin, to evaluate basal release and then, for another 5 min, to a third chamber containing the drug plus high K + ion concentration (34 mM) to evoke neurotransmitter release. After incubation, tissue slices were placed in 90% CH30H/IO% H,O, O'C, and sonicated to extract low molecular weight forms of CCK. The methanolic extracts were dried under nitrogen and rehydrated before assay. The supernatants from the incubation vials were frozen until used for assay. Tissue and released levels of CCK were measured by radioimmunoassay with antisera Ab2717 (a generous gift from Professor J. Rehfeld) against monoiodinated human gastrin (2-17) as a tracer. Tissue slices were exposed to morphine hydrochloride ( 10-'0-10-' M) or naloxone hydrochloride ( l o ' 1 0 6 ~ ) . Two Caz+ ion concentrations were used for the experiments with morphine, 0.6 mM or 2.5 mM, to evaluate whether any morphine action was Ca2+ dependent.