An Improved Method for Embedding Brain Tissue in Albumin-Gelatin

A new modification of the Snodgrass-Dorsey (1963) albumin embedding method is described. Formalin fixed brains of various ages of rhesus monkey (Macaca mulatta) were sunk in 10% phosphate buffered formalin which contained 30% sucrose. and then embedded in a 3% gelatin, 30% egg albumin solution which had been centrifuged to ensure uniformity. The albumin-gelatin was hardened in formaldehyde fumes and blocks cut frozen at 10-40 μm. Sections thus prepared can be handled easily and mounted without damage to the tissue. Modifications of conventional cell and fiber stains produce high quality finished slides in which the stained brain tissue is surrounded by a colorless albumin-gelatin matrix.     

An Improved Field Method to Obtain DNA for Individual Identification From Wolf Scat

ABSTRACT Sampling of feces for genetic studies of wild populations can be problematic because of the low quality and quantity of template DNA obtained. We used cotton swabs in the field to isolate the mucous layer on the surface of fresh wolf (Canis lupus, C. lycaon, and their hybrids) scats followed by immediate preservation, and compared microsatellite genotyping of DNA from these fresh field swabs (FS) to that of previously frozen laboratory swabs (LS). In single polymerase chain reactions (PCRs) of 2 multiplexes, amplification at 8 loci was higher in the FS samples (FS = 50%, LS = 15%; P = 0.02) because proportion, quantity, and quality of large fragment wolf nuclear DNA from these samples was greater (2.5–25%, 6.25–62.5 ng/swab, 35% amplified at 1,000 base pairs [bp]) than from the LS samples (1.9%–10%, 4.7–25 ng/swab, 10% amplified at 1,000 bp). Paired blood and fresh field-swabbed samples had identical genotypes. In 84 multiplex PCRs we found no evidence of allelic dropout associated with low template quality or quantity. We conclude that field swabbing of fresh wolf scat facilitates field storage and reduces the need for multiple amplifications at single microsatellite loci, thereby reducing the genotyping costs for wildlife projects that use noninvasive samples.     

An Improved Method to Obtain High Molecular Weight DNA from Purified Micro- and Macronuclei of Tetrahymena thermophila

An improved method to obtain high molecular weight DNA from purified macro- and micronuclei of Tetrahymena thermophila is described. Micro- and macronuclear DNA obtained using previously described protocols was degraded and not suitable for the cloning of large (> 100 kb) DNA fragments. Based on the data reported here, we propose that DNA degradation is mainly due to nuclease activity; some micronuclear DNA degradation is due to mechanical shearing as a result of extended periods of blending. We have made modifications to reduce nuclease degradation by minimizing cell lysis, by the early addition of EDTA and by increasing the EDTA concentration (23 mM). To reduce mechanical shearing, cell and nuclear suspensions were blended for shorter periods. High molecular weight micro- and macronuclear DNA was obtained using the new protocol.     

Kinetics of K+-p-Nitrophenyl Phosphatase Stimulation by a Brain Soluble Fraction

We have already described the separation of two brain soluble fractions by Sephadex G-50, one of which stimulates (peak I) and the other inhibits (peak II) Na⁺, K⁺-ATPase and K⁺-p-nitrophenylphosphatase (K⁺-p-NPPase) activities. Here we examine the features of synaptosomal membrane p-NPPase activity in the presence and absence of brain peak I. It was observed that stimulation of Mg²⁺, K⁺-p-NPPase activity by peak I was concentration dependent, The ability of peak I to stimulate p-NPPase activity was lost by heat treatment followed by brief centrifugation. Pure serum albumin also stimulated enzyme activity. K⁺-p-NPPase stimulation by peak I proved dependent on K⁺ concentration but independent of Mg²⁺ and substrate p-nitrophenylphosphate concentrations. Since our determinations were performed in a non-phosphorylating condition reflecting the Na⁺, K⁺-ATPase Na⁺ site, it is suggested that peak I may stimulate the Na⁺-dependent enzyme phosphorylation known to take place from the internal cytoplasmic side.     

An Improved Colorimetric Method to Quantify Sugar Content of Plant Tissue

A modification of the phenol-sulphuric acid method to determinethe bulk of soluble or insoluble sugars in plant tissue is proposed.The method is based on optimizing the phenol concentration toobtain equal colour yields for glucose, fructose and sucrose.The optimal phenol concentration is found to depend on the ethanolconcentration of the measuring solution. The method is quick,easy and its costs are low, permitting its use in routine analysis. Key words: Free sugars, colorimetry, routine analysis, plant tissue     

一种改进的牛体细胞核移植去核方法

为了提高传统的盲吸法牛体细胞核移植去核效率,将0.5mL离心管底部截掉,在截口处蒙上一层400目的细胞筛网,再与1 mL的离心管套在一起。实验1,将成熟的卵母细胞置于改造过的离心管内膜上,分别以1,000r/min、2,000r/min及3,000r/min离心10min,Hoechst 33342染色后,在荧光显微镜下计算第一极体与染色体的位置关系及去核效率;实验2,将卵母细胞以2,000r/min离心后去核做受体,颗粒细胞做供体进行核移植,检查重构胚胎的早期发育情况。结果表明:以2,000 r/min将卵母细胞离心10min后,有86.6%卵母细胞的极体与染色体之间夹角在20°以内,此时去核效率最高(87.4%);将卵母细胞离心后,对随后的重构胚发育无影响。因此,采用离心辅助去核的方法可以显著提高牛卵母细胞的去核效率。     

Specific Binding of the Syringolide Elicitors to a Soluble Protein Fraction from Soybean Leaves

Syringolides are glycolipid elicitors produced by Gram-negative bacteria expressing Pseudomonas syringae avirulence gene D. The syringolides mediate gene-for-gene complementarity, inducing the hypersensitive response only in soybean plants carrying the Rpg4 disease resistance gene. A site(s) for 125I-syringolide 1 was detected in the soluble protein fraction from soybean leaves, but no evidence for ligand-specific binding to the microsomal fraction was obtained. The Kd value for syringolide 1 binding with the soluble fraction was 8.7 nM, and binding was greatly reduced by prior protease treatment or heating. A native gel assay was also used to demonstrate ligand-specific binding of labeled syringolide 1 with a soluble protein(s). Competition studies with 125I-syringolide 1 and several structural derivatives demonstrated a direct correlation between binding affinity to the soluble fraction and elicitor activity. However, differential competition binding studies disclosed no differences in syringolide binding to soluble fractions from Rpg4/Rpg4 or rpg4/rpg4 soybean leaves. Thus, the observed binding site fulfills several criteria expected of an intracellular receptor for the syringolides, but it is most likely not encoded by the Rpg4 gene. Instead, the Rpg4 gene product may function subsequent to elicitor binding, possibly in intracellular signal transduction.     

An Improved Method for Embedding Hard Tissue in Poeymethyl Methacrylate

An improved routine method for embedding tissue, especially hard tissue, in poly-methyl methacrylate (pMMA) is described. The improvements were: the final dehydration step before MMA infiltration was performed with methanol in a Soxhlet apparatus; the stabilizer hydroquinone was not extracted from the monomer (MMA), and more important, the commonly used polymerization initiator, benzoyl peroxide (bpo), was replaced by the initiator, bis (4-tert-butylcyclohexyl)peroxydicarbonate (bbpd). Bbpd is preferred to bpo because it is not explosive, far less is needed and it has a suitable half life. Moreover, bbpd, as obtained from the manufacturer, needs no further purification, in contrast to bpo. Temperatures during bbpd initiated polymerization did not exceed 48 C. In bbpd initiated pMMA, bubbles were almost never generated.     

Method to Obtain a Chlorophyll-free Preparation of Intact Mitochondria from Spinach Leaves

Mitochondria from green leaves of spinach have been prepared using a three-step procedure involving differential centrifugation, partition in an aqueous dextran polyethylene glycol two-phase system and Percoll gradient centrifugation. The mitochondrial fractions after the different steps of purification were compared. The final mitochondrial preparation was totally free from chloroplast material measured as chlorophyll content. The enrichment of mitochondria in relation to peroxisomes and microsomes was approximately 12 and 33 times, respectively, based on NAD:isocitrate dehydrogenase activity, glycolate oxidase activity, and NADPH:cytochrome c oxidoreductase activity. The apparent intactness of the inner and the outer mitochondrial membranes was higher than 90% as measured by latency of enzyme activities. The mitochondria showed high respiratory rates with respiratory control and the ADP/O ratios approached the theoretical limits.     

Identification of Nuclear Proteins that Are Developmentally Regulated in Embryonic Rat Brain

Abstract: To identify nuclear proteins that might play a role in the acquisition of neuronal phenotype, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to analyze nuclear proteins expressed over the course of embryonic rat brain development. Metabolically labeled rat brain nuclear proteins from embryonic day 14 (E14) were compared with proteins from embryonic day 20 (E20). Over this period, the rat brain develops from a collection of relatively homogeneous precursor cells into a complex structure containing many different classes of neurons. Computer-assisted analysis of 2D-PAGE fluorograms identified 11 proteins that show increases in their rate of synthesis between E14 and E20. Twenty proteins that consistently appear at E20 are not detectable on fluorograms of E14 nuclear proteins, even after long exposures, and thus may be considered to appear de novo. Fifty-eight proteins show consistent down-regulation between E14 and E20, and of these, 19 are not detectable on fluorograms of E20 nuclear proteins. The electrophoretic properties of many of these proteins suggest that they are previously unreported, developmentally regulated nuclear proteins. Some of the developmentally regulated, brain-enriched nuclear proteins identified here may play a role in regulating the expression of neural genes important for cellular differentiation in the mammalian CNS.     

Heterophilic NCAM-Mediated Cell Adhesion to Proteoglycans from Chick Embryonic Brain Membranes

The vertebrate neural cell adhesion molecule NCAM mediates adhesion by both homophilic and heterophilic mechanisms, with heparan sulfate proteoglycans (HSPGs) being likely heterophilic ligands. In this study, transfected chicken NCAM polypeptides expressed on mouse L cells mediated the adhesion of these cells to several different heparan sulfate proteoglycans in nonionic detergent extracts of Embryonic Day 10 chicken brain membranes. In addition, adhesion inhibition experiments suggested a hitherto-undetected role for chondroitin sulfate proteoglycans in the stimulation of NCAM-mediated adhesion to some, but not all, of the HSPG ligands. Our experiments support the view that NCAM is a multivalent adhesive molecule whose function is affected by interactions with extracellular matrix and cell surface molecules.     

An Improved Method of Making Tissue Carriers for TEM and SEM Fixation

Hazards in fixing small pieces of tissue for electron microscopy include damage, drying, or loss. Over the years, microstrainer tissue carriers have been developed to minimize these problems. Construction materials have included glass tubing, copper grids for electron microscopy, stainless steel screen, and bolting silk (Padawer 1951, Friend 1963, Bronskill 1970). Carriers made from plastic embedding molds (e.g., BEEM capsules) with either TEM grids attached to the conical tip (Buchanan 1965) or Nitex screen cloth held to one end by a retaining ring have proven to be inexpensive and popular, though the former has a very small filtration area and in the latter small tissues may be lost or crushed between the screen cloth and the bottom rim of the carrier. This note describes a carrier in which Nitex is permanently sealed to the bottom edee of a BEEM capsule cylinder.     

Extrachromosomal Circular DNA from Nuclear Fraction of Higher Plants

To determine the cellular location of extrachromosomal circularDNA in higher plants, wheat (Triticum aestivum) and tobacco(Nicotiana tabacum) nuclei were isolated and purified, and theircircular DNAs were examined by electron microscopy. Covalentlyclosed circular (ccc) DNAs were found in nuclear fractions fromboth species. They showed a heterogeneous size distributionranging from 0.1 µm to more than 5 µm in contourlength, with a mean of 1.7 µm (5.3 kbp) for T. aestivumand 1.5 µm (4.7 kbp) for N. tabacum, respectively. Thisdistribution is significantly different from that for smallcircular DNAs in mitochondria. Small polydisperse circular (spc)DNA/protein complexes were also observed by a rapid microscalemethod of mica-press-adsorption for electron microscopy. Complexesof spcDNA/protein showed a similar size distribution to cccDNA.The average number of spcDNA/protein complexes per nucleus wasestimated at more than hundred. The origin and biological functionsof the nuclear circular DNAs are discussed. (Received February 13, 1985; Accepted August 8, 1985)     

An Improved Method for In Situ Freezing of Cat Brain for Metabolic Studies

This study introduces a new method for rapid freezing of the cat brain. The method employed a Styrofoam box which was fitted around the head of the animal. Liquid nitrogen was poured into the box until the head was submerged. Temperature changes in three brain sites (ventral hypothalamus, the fourth ventricle, and the corpus callosum) and levels of labile carbohydrate metabolites (glycogen, glucose, ATP, P-creatine, and lactate) in five brain regions (cortex, thalamus, midbrain, cerebellum, and pons) frozen by the box method were compared with those frozen by a conventional cup method in which liquid nitrogen was poured into a hollow Styrofoam cup placed on top of the skull. The box method shortened the time of arrival of the freezing front and improved the freezing rate. The time required to bring the tissue to -20 degrees C was shortened, from 20 min at the ventral hypothalamus and 10-12 min at the fourth ventricle with the cup method, to less than 5 min at both sites with the box technique. Continued perfusion of brainstem prior to freezing was demonstrated. Levels of metabolites frozen by either method were similar. Lactate levels in any of the five brain regions studied by either method were not elevated, indicating no ischemic change.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate Specificity of [3H] Muscimol Binding to a Particulate Fraction of a Neuron-enriched Culture of Embryonic Rat Brain

Abstract: The effects of some GABA analogues and some drugs on the binding of [³H]muscimol (3.08 nM) to thoroughly washed subcellular particles prepared from a neuron-enriched culture of embryonic rat brain were examined using Na+-free Tris-citrate medium and a centrifugation method. Competition for [³H]muscimol binding sites by excess(10^?5 M) unlabelled GABA provided estimates of “specific” binding. In accord with in vivo neuropharmacological studies on GABA receptors and with in vitro studies on cerebral membrane preparations, [³H]muscimol binding was potently inhibited by muscimol itself (IC₅₀, 2.5 nM), GABA (1C₅₀, 43 nM), isoguvacine (IC₅₀, 61 nM), and 3-aminopropanesulphonic acid (IC₅₀, 160 nM), and less potently inhibited by the GABA antagonist bicuculline methobromide (IC₅₀, 800 nM). δ- Aminovaleric acid (IC₅₀, 2.6 μM), the glycinelp-alanine antagonist strychnine (IC₅₀, 6.6 μM), and the predominantly glial GABA uptake inhibitors β-alanine (IC₅₀, 23 μM) and p-proline (IC₅₀, 66 μM) also inhibited [³H]muscimol binding. Other inhibitors of Na+-dependent GABA uptake, (±)-nipecotic acid, L- 2,4-diaminobutyric acid, and guvacine, as well as picrotoxinin, were relatively inactive as inhibitors of [³H]muscimol binding (IC₅₀≥ 1 mM). In addition to revealing that GABA receptors are present on neuronal membranes before the formation of most synapses, this binding of [³H]muscimol that occurs to neuronal, but not to glial, membranes might be useful as a “neuronal marker” and for the further characterization and isolation of GABA receptors.     

Development of an Optimized Method to Obtain a Limonene-Rich Concentrate from the Discarded Lemon Peels

In this study, lemon peels were used as volatile component source. Automatic solvent extraction has been used for the recovery of limonene rich citrus volatile extract for the first time. The process parameters (amount of raw material, immersion time and washing time) were analyzed to optimize the process by means of Box-Behnken design via response surface methodology. The optimum conditions were achieved by ~10 g fresh lemon peel, and ~15 min immersion time and ~13 min washing time. The difference between the actual (89.37 mg/g limonene) and predicted (90.85 mg/g limonene) results was satisfactory (<2 %). α-Terpinene, β-pinene, citral, ɣ-terpinene and linalool were determined as other major volatiles in the peel extract. FT-IR and ¹H- and ¹³C-NMR spectroscopies were applied to verify the identified volatile compounds.     

一种获得转基因芥菜的简便方法

随着转基因技术的不断革新,更简便高效的转基因方法逐渐发展起来.通过对花序浸染法的探索和改进,采取用移液枪点滴重悬液的方式转化芥菜(雪里蕻,Brassica juncea var.crispifolia)花蕾,最终获得了转基因芥菜.同时通过对影响转化效率的蔗糖浓度、表面活性剂浓度、菌液OD600值等进行研究,发现用5%的蔗糖(W/V)+0.03%的Silwet-77(V/V)重悬农杆菌,使重悬液的菌液浓度OD600为0.8时,可以获得较高的转化效率,转化率为2.3%.该方法避免了真空操作及浸染时间的影响,使得操作更加简便,且转化周期更短,为芥菜的遗传转化提供了一种更为简便的转化方法.     

Effectiveness of Spray Congealing to Obtain Physically Stabilized Amorphous Dispersions of a Poorly Soluble Thermosensitive API

An amorphous phase produced by micronization up to the molecular or colloidal level of a poorly soluble drug having low lipophilicity can distinctly enhance its solubility characteristics. However, though dispersing the molten mass of a poorly water-soluble drug within polymeric matrix has been found to be most effective in formation of molecular dispersions, the drug molecules which melt at high temperature also accompanied by decomposition, such as acetazolamide, are difficult to formulate as molecular dispersions. Hence, a method is proposed to obtain molecular dispersions of acetazolamide with poloxamer-237 by spray congealing under optimal heat treatment. Uniform molecular and/or colloidal dispersions of the drug were achieved with instantaneous solvent evaporation by mixing a drug solution with molten mass of the plasticizer matrix. Immobilization of dispersed drug molecules was effected subsequently through rapid solidification by spray congealing. Initial characterization of 1:1, 1:1.5, and 1:2 ratios of solid dispersions and devitrification study of an optimized (1:2) ratio ensured efficacy of the proposed method in formation of physically stabilized amorphous systems without thermal degradation and hence resulted in more than ninefold rise in solubility and more than 90% dissolution within initial 10 min. With 1:2 ratio, molecular dispersions could be achieved by initial solvent evaporation stage, which when subjected to spray congealing produced physically stable amorphous systems, without signs of thermal degradation. This study also proposes an opportunity for selection of those polymers with which the drug is immiscible in their fluid state, yet obtaining molecular dispersions.KEY WORDS: low molecular lipophilicity, melt with decomposition, rapid solidification, solid solutions     

The Oxidation of Iso-citrate by a Particulate Fraction Obtained from Swede Storage Tissue

A particulate fraction has been prepared from swede storagetissue which possesses both an NAD and an NADP linked iso-citricdehydrogenase. Such preparations have been used to study thepathway of oxidation of iso-citrate and the reduced coenzyme—cytochromec. reductase activity. The activity of the enzyme systems associatedwith the particles varies with pH, at pH 7.1 the NAD linkediso-citric dehydrogenase exhibits optimal activity whilst theoptimum pH for the NADP linked dehydrogenase is pH 8.3. At eitherpH the major pathway of oxidation of iso-citrate appears tobe through the NAD linked iso-citric dehydrogenase, NADH₂-cytochromec. reductase system. The preparations are apparently unableto transfer electrons from reduced coenzyme, produced by dehydrogenaseactivity, to cytochrome c. at the same rate as they are ableto transfer electrons from reduced coenzyme supplied as a substrate.The results are discussed in relation to mitochondrial structureand a comparison is made with the results of work carried outon the iso-citric dehydrogenases of animal tissues.