Displaceable binding of [3H]l-glutamic acid to non-receptor materials

[3H]L-glutamic acid binding to microfuge tubes and glass was investigated in four buffers. Background binding to these materials was negligible, but was increased by centrifugation or suction in Tris-HCl and Tris-citrate buffer. This binding was much less or eliminated when HEPES-KOH, or Tris-acetate buffer was used instead. [3H]L-glutamate binding to microfuge tubes was inhibited by L- but not D-isomers of glutamate and aspartate. DL-2-amino-7-phosphonoheptanoic acid also did not inhibit the binding. Other compounds which showed low to moderate inhibition were: N-methyl-D-aspartate, quisqualate, L-glutamic acid diethyl ester, N-methyl-L-aspartate, kainate, and 2-amino-4-phosphonobutyrate. Binding was inhibited by denatured rat brain membranes. A protein-dependent [3H]glutamate binding was obtained with a repeatedly frozen-thawed membrane preparation when binding was done in Tris-acetate buffer. It is recommended that Tris-acetate or HEPES-KOH buffer should be used in the glutamate binding assay. If Tris-HCl or Tris-citrate buffer is used, appropriate control experiment should be done to correct for binding to microfuge tubes or glass fiber filters.     

Carrier RNA enhancement of recovery of DNA from dilute solutions

A study of the use of carrier RNA to improve precipitation of DNA from dilute solutions was conducted to define the conditions which optimize DNA recovery. Replicate samples containing labeled pBR322 and increasing concentrations of commercially-available Torula yeast RNA were ethanol precipitated at -20 degrees C for 1 h in microfuge tubes obtained from various manufacturers. Nucleic acids were pelleted by centrifugation for either 5 or 30 min, dried and resuspended. Although recovery was not identical in each type of microfuge tube, in all cases the percent recovery increased when carrier was added. In most cases, extending centrifugation to 30 min did not significantly increase recovery. Recovery of unlabeled DNA's of heterogeneous molecular weight and conformation was also enhanced by the addition of carrier RNA. DNA's recovered by this method can be successfully digested with BamHI and ligated with T4 DNA ligase.     

Automated polymerase chain reaction in capillary tubes with hot air.

We describe a simple, compact, inexpensive thermal cycler that can be used for the polymerase chain reaction. Based on heat transfer with air to samples in sealed capillary tubes, the apparatus resembles a recirculating hair dryer. The temperature is regulated via thermocouple input to a programmable set-point process controller that provides proportional output to a solid state relay controlling a heating coil. For efficient cooling after the denaturation step, the controller activates a solenoid that opens a door to vent hot air and allows cool air to enter. Temperature-time profiles and amplification results approximate those obtained using water baths and microfuge tubes.     

On the disruption of biochemical and biological assays by chemicals leaching from disposable laboratory plasticware

Plastic consumables, used universally in bioscience laboratories, are presumed inert with respect to bioassay outcomes. However, it is clear that many pipette tips, microfuge tubes, and other plastic disposables leach bioactive compounds into assay solutions, profoundly affecting data and experimental interpretation. In this paper we discuss the nature and sources of leachates and review several examples of compromised bioassay data that speak to the probable widespread nature of this largely unrecognised source of error. Strategies for minimizing leachate interferences are discussed.     

A variable-tilt fermentation rack for screening organisms in microfuge tubes

Summary We designed a variable-tilt microfuge fermentation rack to screen liquid cultures of wild and mutant yeasts for ethanol production. The rack design allows for the evaluation of up to 40 cultures in the space normally required for three 125 mL Erlenmeyer flasks. Microfuge tubes containing 1.0 mL of inoculated medium were placed in the rack and incubated with shaking. This technique gave reproducible rates of ethanol formation in relation to sugar uptake.Visiting Scientist, D.F.R.L., Mysore-570011, INDIA     

A procedure for the small-scale isolation of plant RNA suitable for RNA blot analysis

A small-scale method for the isolation of total RNA from plant tissue is described. The method provides RNA of suitable quantity and quality from 0.2 g fresh tissue for the detection of mRNA species by RNA blot analysis. The entire procedure is adapted to 1.5-ml microfuge tubes and takes less than 5 h. This method is well suited for the isolation of RNA from large numbers of samples or from samples of limited quantity.     

Rapid and quantitative preparation of cytoplasmic RNA from small numbers of cells

An extremely simple procedure for preparing cytoplasmic RNA from small numbers of cells is described. Cells are lysed with the detergent NP-40 and efficient extraction of protein from the postnuclear cytoplasmic lysate is ensured by denaturation with sodium dodecyl sulfate and urea. This procedure is suitable for preparing RNA from many cell types. All procedures have been scaled down to be performed in 1.5-ml microfuge tubes and thus RNA may be prepared from small numbers of cells. The procedure is extremely rapid and RNA is ready for Northern gel analysis in less than 30 min. Because so few steps are involved, RNA recovery is quantitative.     

Ubiquinone binding protein used for determination of coenzyme Q

The conventional method of assaying for the ubiquinone (CoQ) content of biological samples is to partition CoQ into an organic phase and separate it from contaminants by high-performance liquid chromatography (HPLC). HPLC is an accurate method of quantifying CoQ content but is not ideal for routine clinical analyses. This paper describes the development of a rapid method for assaying the CoQ content of biological samples based on the binding of CoQ to a CoQ binding peptide. The 14-amino acid binding peptide was chemically synthesized, and conditions for immobilizing the peptide on microfuge tubes were established. CoQ could be selectively bound to the immobilized peptide, eluted, and determined spectrophotometrically. Limits of detection for the method were 0.25 to 5 nmol CoQ. To test biological samples, CoQ was isolated from cultures of Saccharomyces cerevisiae grown in oleic acid medium. The recovery of CoQ samples using the binding assay ranged from 99 to 102% of the values obtained with HPLC. The assay described here provides an inexpensive, rapid method for determining the CoQ content of large numbers of biological samples in a variety of laboratory settings.     

A microfuge tube technique for disrupting microquantities of fungal spores

A practical apparatus was assembled and through its use a technique was developed to disrupt microquantities of Ceratocystis ulmi spores. spores were placed in a microfuge tube with buffer and glass beads. The microfuge tube was sealed and placed inside a holding tube and then subjected to rapid vibrations over a Vortex mixer. By this technique 80–90% of the spores were disrupted and it was possible to extract a high-quality ribosomal RNA fraction from the supernatant fluid of the disrupted spore sample. The technique has also proved applicable to spores of fungi other than C. ulmi.     

Mechanism of sucrose uptake by isolated rat hepatocytes

The transport of molecules by nonspecific endocytosis has been described in many cell types, but it has not been characterized in hepatocytes. Because of its central role in the clearance of solutes from portal blood, endocytosis might represent a significant mode of cellular transport. We investigated the mechanism of sucrose uptake in an isolated hepatocyte system. Liver cells were isolated by perfusion and collagenization of rat liver, followed by differential centrifugation. Hepatocytes were then incubated with 14C-sucrose and harvested by spinning through oil in microfuge tubes. Radioactivity was standardized against DNA content. We found that sucrose uptake is concentration-dependent from 5 microM to 100 mM and follows first-order kinetics. Washout studies indicate that exocytosis is responsible for the dynamic equilibrium reached. Arrhenius analysis of temperature dependence yields a linear plot (Ea = 14.2 Kcal/mol). In addition, sucrose uptake is independent of cellular ATP levels. We conclude that sucrose is transported by fluid-phase micropinocytosis in isolated hepatocytes and that this transport mechanism may be important in the uptake of diverse molecules into liver cells.     

Conditions influencing yield and analysis of 8-hydroxy-2'-deoxyguanosine in oxidatively damaged DNA

We have conducted studies to obtain practical knowledge regarding the stability, digestion, and analytical determination of the content of 8-hydroxy-2-deoxy-guanosine (8-OHdG) in oxidatively damaged DNA. Utilizing H2O2 plus uv light to form oxidatively damaged DNA, we found that storage of the DNA at -20 degrees C at alkaline pH caused a significant loss of 8-OHdG, whereas storage at -20 degrees C at neutral or acidic pH prevented loss of 8-OHdG. The 8-OHdG within DNA is stable at 100 degrees C for at least 15 min. Formation of 8-OHdG within DNA using uv light and H2O2 as a hydroxyl free radical-generating system yields the highest amounts when low levels of phosphate buffer are used; but the use of Tris or citrate buffers causes a lower yield of 8-OHdG because these buffers act as scavengers for the hydroxyl free radicals. Independent assessment of hydroxyl free radical flux by the use of salicylate trapping allows assessment of competitive radical reactions. Ethanol washing of plastic microfuge tubes prior to DNA enzymatic digestion improved the yield of 8-OHdG and reduced the variability between samples. Digestion of the oxidatively damaged DNA by the use of a method involving DNase I, endonuclease, phosphodiesterase, and alkaline phosphatase produced the highest yield of 8-OHdG.     

真菌菌丝体培养和提取DNA方法的改进

ANIMPROVEDPROTOCOLFORFUNGALDNAPREPARATIONHEYue-Qiu(KeyLaboratoryofPlantPathology,YunnanAgriculturalUniversity,Kunming650201)DNAfromfUngaJmyceliacanbeinexpensivelypreParedinandcrofugetUbescale.Thesmal1scaleoftheprocedaredescribedheremakesitPOssibleforonepeopletoconvenienUyhandleuPto6osamplesinlessthan5hoursandobtainlO~5OgofDNApersamPle,whichisequaltotheamountenoughtforseveralSouthernhybriclizahonsand5oO~25oOPCRbasedreachons.I.Medium:CaseinhydrolyteO.5g,sucrosel5g,…     

双斑切叶蜂的筑巢习性

【目的】明确双斑切叶蜂Megachile leachella在沙地上的筑巢环境及筑巢特点,旨在当地利用人工巢管诱引其筑巢。【方法】采用目测和拍照等方法对双斑切叶蜂的整个筑巢过程进行了连续观察; 采用挖掘、测量方法对双斑切叶蜂巢内结构进行了观测;在室内对蜂茧在土中和指形管中的羽化情况进行了初步观测。【结果】双斑切叶蜂一般在废弃的泥墙或者沙地上蚂蚁等废弃的巢穴中筑巢,有时候它也在沙地上挖掘新巢筑巢。双斑切叶蜂用叶片构建巢室,一巢多室,各巢室首尾相接排列在巢中,筑完巢后用叶片将巢口封住。双斑切叶蜂构建一个巢室需要切取11～14片叶子,为每巢室采集蜂粮6～9次,每巢室内产卵1粒;在内蒙古毛乌素沙地,该蜂一年2代,第1代在6月中下旬羽化,第2代大约在7月下旬至8月上旬羽化,主要寄生性天敌有尖腹蜂Coelioxy sp.、青蜂Chrysis sp.。双斑切叶蜂能在指形管中羽化。【结论】双斑切叶蜂不同个体之间的筑巢行为相似,雌蜂可以用人工巢管进行诱集和驯化。     

Different inhibitors of the gibberellin biosynthesis pathway elicit varied responses during in vitro culture of aspen (<Emphasis Type="Italic">Populus tremula</Emphasis> L.)

Several synthetic plant growth regulators (PGRs), including prohexadione-calcium (ProCa), paclobutrazol (PBZ), and chlormequat chloride (CCC), known for their ability to inhibit gibberellin (GA) biosynthesis, were investigated for their influence on Populus tremula L. (aspen) shoots grown in vitro. Changes in plant growth induced by these inhibitors were compared to the effects of exogenous gibberellins (GA₃ and GA_4/7). All PGRs were added to the nutrient medium at concentrations of either 1 or 5 μM. Stem segments with and without apical buds were excised from in vitro-grown shoot culture, and these explants were incubated either in test tubes or Petri dishes. In the presence of 5 μM ProCa, shoot growth and rooting were inhibited when grown in test tubes, while shoots grown in Petri dishes exhibited strongly enhanced shoot and root growth. PBZ suppressed shoot development both in test tubes and Petri dishes, although 1 μM PBZ promoted adventitious root formation when shoots were grown in test tubes. Five micromolars CCC suppressed shoot and root development in test tubes, but promoted shoot growth in Petri dishes.     

Stigmatic and stylar incompatibility in Prunus avium L.: Morphological and ultrastructural aspects

Abstract

Stigmatic and stylar structures of-sweet cherry (Prunus avium L.) «Malizia» were examined with light and electron microscopes (S.E.M. and T.E.M.).

It was demonstrated that the transmitting tissue, situated in the central portion of the style, extends below the stigmatic papillae and secretes an electrondense material accumulating in the intercellular spaces. Pollen tubes which germinated on the stigma, reached the ovules passing through this substance which facilitates their passage while trophic relations are established.

Some ultrastructural aspects of the pollen tubes observed inside the style were examined and discussed in relation to the phenomenon of self-incompatibility.     

Effect of Broccoli (Brassica oleracea) Tissue, Incorporated at Different Depths in a Soil Column, on Meloidogyne incognita

Brassicas have been used frequently for biofumigation, a pest-management strategy based on the release of biocidal volatiles during decomposition of soil-incorporated tissue. However, the role of such volatiles in control of plant-parasitic nematodes is unclear. The goal of this study was to determine the direct localized and indirect volatile effects of amending soil with broccoli tissue on root-knot nematode populations. Meloidogyne incognita-infested soil in 50-cm-long tubes was amended with broccoli tissue, which was mixed throughout the tube or concentrated in a 10-cm layer. After three weeks at 28°C, M. incognita populations in the amended tubes were 57 to 80% smaller than in non-amended tubes. Mixing broccoli throughout the tubes reduced M. incognita more than concentrating broccoli in a 10-cm layer. Amending a 10-cm layer reduced M. incognita in the non-amended layers of those tubes by 31 to 71%, probably due to a nematicidal effect of released volatiles. However, the localized direct effect was much stronger than the indirect effect of volatiles. The strong direct effect may have resulted from the release of non-volatile nematicidal compounds. Therefore, when using biofumigation with broccoli to control M. incognita, the tissue should be thoroughly and evenly mixed through the soil layer(s) where the target nematodes occur. Effects on saprophytic nematodes were the reverse. Amended soil layers had much greater numbers of saprophytic nematodes than non-amended layers, and there was no indirect effect of amendments on saprophytic nematodes in adjacent non-amended layers.     

Rapid purification of DNA from agarose gels by centrifugation through a disposable plastic column

A simple and rapid method for purifying DNA from agarose gels is described. Agarose slices containing DNA are placed in a disposable plastic column and the DNA is separated from the agarose by centrifugation in a microfuge. Recoveries averaging 25% are obtained for DNA of 14 kb or less. The recovered DNA can be labeled to high specific activity, cleaved with restriction endonucleases, and ligated efficiently using standard cloning vectors.     

A microtiter well assay for quantitative measurement of estrogen receptor binding to estrogen-responsive elements

Reproducible, rapid measurement of estrogen receptor (ER) binding to DNA was accomplished in microtiter wells treated so that ER-DNA complexes or DNA bound in preference to free ER. Mixtures of 35S-labeled DNA and [3H]estrogen-charged ER ([3H]ER), incubated to equilibrium in microfuge tubes, were transferred to microtiter wells previously treated with histone followed by gelatin. After binding of the DNA or ER-DNA complex to the treated wells, free ER was removed by washing. Radioactivity retained in each well was measured by placing individual wells from snap-apart microtiter plates directly in scintillation fluid. Binding of DNA was saturable, and ER-DNA complex binding was complete within 2 h at 4 C. The use of 35S-labeled DNA and [3H]ER allowed stoichiometric determination of ER bound to DNA. The amount of ER specifically bound to a consensus estrogen-responsive element (ERE) containing the inverted repeat GGTCAgagTGACC was determined by comparing ER bound to plasmid containing or lacking the ERE. At saturating concentrations of ER, plasmids bearing one, two, and four EREs in tandem bound approximately one, two, and four dimeric ER molecules, respectively. Scatchard analysis of saturation binding data revealed a Kd of 0.15 nM for specific ER binding to a single ERE site. Thus, the assay detects ER retaining both DNA-binding and estrogen-binding functions. ER complexed with DNA in the well was also detected using a monoclonal antibody specific for the receptor. Simple modifications of this method would allow study of other DNA-protein interactions.     

Stigma and style anatomy and ultrastructure in Italian Oxalis pes-caprae L. and their possible connection with self-incompatibility

The South-African Oxalis pes-caprae with trimorphic flowers is naturalised in many Mediterranean countries. In Italy only short-styled (S) populations are known for certain and the plant is believed to reproduce only asexually, due to self- and intramorph-incompatibility. This study aims to clarify anatomical, biochemical and ultrastructural features of the stigma and style of an Italian S population, also to define their possible role in incompatibility. Additional observations were also carried out on other Italian population and on short-, long- (L) and mid-styled (M) flowers from plants of South African origin. Morphological and biochemical features of flowers collected in different phenological stages during the whole flowering season were observed both under LM and TEM. In S flowers, three different zones could be distinguished in each stigma-style complex: zone I (stigmatic), zone II (substigmatic), zone III (stylar). The main differences concern the transmitting tissue: in zones I and III this is composed of loosely arranged cells with thick walls, with an abundant soft matrix which at anthesis is rich in mucopolysaccharides and lacks pectins. In zone II, it is more compact, with a less abundant wall matrix, at anthesis containing both mucopolysaccharides and pectins. In S flowers, subjected to illegitimate pollination, many pollen tubes penetrate the stigmatic papillae but apparently are arrested in zone II; only few—and mostly at the end of flowering period—succeed reaching zone III, where they encounter no further resistance to growth. Differently, after legitimate pollinations, pollen tubes succeed in crossing transmitting tissue of zone II, where cell walls of cells lying close to pollen tubes show a considerably reduced pectin content. In L and M flowers of South African origin, no peculiar transmitting tissue could be noticed in the substigmatic zone. In such flowers, pollen tubes seemingly grow easily from stigma to style both after legitimate and illegitimate pollinations. Results suggest that in S flowers the rigid transmitting tissue in zone II acts as a mechanical barrier for illegitimate pollen tubes, as the spaces between cells are narrower than the tube diameter and pectins maintain the rigidity of the cell walls, preventing cells from separating from one another. This obstacle can be overcome by legitimate pollen tubes, which make their way between cells, possibly releasing or activating specific pectinases. However, the blocking of illegitimate tubes is not absolute: a few of such tubes grow beyond zone II and reach the ovules, so that occasional fertilisation and embryo formation can be observed. In M and L flowers, different self-incompatibility mechanisms can be hypothesised.     

Factors influencing the in vitro growth of Mycobacterium leprae: effect of oxygen.

In our efforts to evaluate factors responsible for in vitro growth of Mycobacterium leprae in DH medium and also to improve the system, effects of oxygen tension on in vitro growth of M. leprae were determined. This was achieved by varying the ratio between DH medium and free air space above the medium in the culture tubes. Growth-competent M. phlei (ATCC 11758) could tolerate all the oxygen it can get in the medium. On the other hand, M. leprae seemed to be of microaerophilic nature. In vivo-grown M. leprae cells were more sensitive than their counterparts that were adapted to in vitro environment. In vivo-grown cells grew better when 70% of the space in culture tube was occupied by DH medium. These in vitro-adapted cells gave optimum growth in subcultures when the air spaces in the culture tubes were 40-50%. The role of oxygen tensions in the development of lesions in leprosy patients and armadillos has been discussed.