Demonstration of the Microvasculature of the Spinal Cord by Intravenous Injection of the Fluorescent Dye, Thioflavine S

In the past, thioflavine S has been used for visualizing blood vessels and patterns of blood flow (Schlegel 1949; Schlegel and Moses 1950; Oliver et al. 1951). Methods employed have involved an intravenous injection of the dye, immersion of hand-cut sections in glycerol and examination of sections under incident Wood's light. With improved techniques it is possible to obtain microtome-cut sections and to use a more intense light source for enhancing fluorescence and resulting visualization of small vessels. Occlusion of arterioles by undissolved dye particles is prevented by ultracentrifugation of the solution to be injected.     

Quinacrine Mustard—a Selective Fluorescent Stain for the Y Chromosome in Human Tissues for Routine Cytogenetic Screening

A simple sensitive and specific cytochemical method for detection of the Y chromosome in man (George 1970, Pearson et al. 1970) is based on staining by alkylating fluorochromes. The dye binds strongly to the heterochromatic regions of the Y chromosome, thus causing these regions to fluoresce very brightly under ultraviolet (uv) light. The Y chromosome could be identified easily at metaphase and also as a highly fluorescent body in interphase cells. Two bodies were seen by Pearson et at. in a subject having the XYY anomaly. In prophase cells, it appears as a fluorescing chromatin thread. Its presence has been demonstrable only in males; hence the usefulness of this technique for rapid cytogenetic screening is obvious. The constancy and reproducibility of the technique seems to rule out the possibility of unreliability caused by nonspecific binding of the dye. The present report gives techniques applied to tissues usually studied in cytogenetic screening     

A Staining Method for Light Microscopic Study of Nucleolar Structure in Plant Cells

Ever since the nucleolus was noted by Fontana (1781), a vast array of literature has accumulated on staining procedures for its study. However, most are for animal cells and there are few for plant cells. These techniques are reviewed by Busch and Smetana (1970). A method suggested by Das (1962) and later modified by Das and Alfert (1963) uses silver nitrate followed by photographic developers. Silver nitrate staining after hydroquinone-formalin fixation has been suggested for routine use (Gomez et al. 1969, Stockert et al. 1969). However, by these light microscopic techniques the entire nucleolus is darkly stained and intranucleolar structure is observed.     

Improved Stability of A Purified Glycol Methacrylate Preparation: Comments

The technical note from Bernier et al. (1984) presents additional observations on our procedure for purifying glycol methacrylate (GMA), a hydrophylic resin (Chappard et al. 1982). It is becoming increasingly popular and widely used as an embedding medium for light microscopic studies. GMA is prepared by esterification of methacrylic acid (MA), but about 1% of free unreacted MA remains in the monomer. MA can copolymerize with GMA and it also binds strongly to thiazin and other basic dyes (Tipett and O'Brien 1975) so as an undesirable impurity it must be removed.     

Use of Vital Dyes in Conjunction with the Semivitro Technic for the Detection of Pollen Tube Sperm Nuclei

The technique we describe here is a modification of that used by Hough et al. (1985), combined with “semivitro” pollen tube observations. With the semivitro technique, pollen tubes grow from the cut ends of pollinated styles (Brewbaker and Majumder 1961). Pollen of Nicotiana alata was presoaked for 15 min in simplified medium (Brewbaker and Kwack 1963) (10% sucrose, 300 ppm Ca(NO₃)₂, 100 ppm H₃BO₃ with the addition of 0.5 mg/ml of Hoechst 33258 stain from Serva Biochemicals, Heidelberg, Control H, purchased June 1983). (For germination of Nicotiana alata pollen in vitro, we use this same solution, except with 12% sucrose). After this prestaining, the pollen suspension was centrifuged for 5 min at 1200 × g, the pellet resuspended in control Brewbaker medium (i.e., no stain), recentrifuged and used to pollinate detached pistils. The pistils were then incubated at 25 C in a water-saturated atmosphere for 20 hr. At this time, the styles were cut just ahead of the front of the growing pollen tubes (Mulcahy and Mulcahy 1985) and the cut stylar ends each dipped in fresh control Brewbaker medium. Twelve to 24 hours later, tubes growing out of the cut styles were viewed by fluorescence microscopy (exciter filter, BG 12 + KV 418, beam splitter, 500 nm, and barrier filter OG 515). A distinct green fluorescence was seen in the generative and vegetative nuclei (Fig. 1).     

Rapid Identification of Chromosomal Abnormalities in Human Neoplasia

Recent advances in banding techniques have led to the belief that certain chromosomal defects are consistently associated with specific types of human neoplasia. Based on the GTG technique, it has been suggested that the malignant cells of most neoplasias show chromosomal abnormalities (Yunis et al. 1983). From this recent publication of Yunis it appears that the majority of bands involved in carcinogenesis are G-negative, i.e., do not stain by the GTG technique, and it is therefore difficult to localize the breakpoints. In some of our recent publications we emphasized the importance of the RFA technique (Verma and Lubs 1975), which stains Giemsa-negative bands darkly, thus providing precise identification of chromosomal abnormalities (Verma and Dosik 1976). However, this technique cannot be applied until the slides have aged for at least 7 days. Therefore, we are reporting an alternative procedure using BrdU which provides “reverse” banding immediately when the slides are stained with acridness orange and examined with a fluorescence microscope.     

A New Method for Flat Embedding in Glycol Methacrylate

Glycol methacrylate (GMA) is a useful polymer for embedding tissue because of its stability, hydrophilic properties, and resistance to many solvents (Feder a d O'Brien 1968, Bennett et a/. 1976). Undue solvent extraction is also avoided as GMA contains water, making complete dehydration unnecessary (Cole and Sykes 1974). This property shows some evidence that GMA embedded sections may be useful in energy dispersive analysis by X-ray for some elements (DeNee et al. 1977). GMA also does not exclude water soluble dye molecules and has thus become a useful medium for histochemical studies (Bennett et al. 1976).     

Oxazine Dyes. 3. Simultaneous Nuclear and Cytoplasmic Staining from Single Solutions

Of 84 dyes tested, 26 were found to give a stable solution with celestine blue B dispersions which simultaneously stained nuclei and cytoplasm. The cytoplasmic dye is dissolved in celestine blue B dispersion prepared by the method of Cray et al. (1956). Croceine scarlet (C.I. 286), in the proportion of 0.38 gm to 114 ml of celestine blue B dispersion, gives results strikingly similar to hematoxylin-eosin when used for 2 min on a wide variety of tissues. No differentiation, other than that which occurs during dehydration, is necessary.     

Immunohistochemical evidence for neuronal and non-neuronal synthesis of GABA in the rat subcommissural organ

In the past few years, several studies have demonstrated in the rat subcommissural organ the presence of nerve endings and modified ependymocytes showing an uptake of [³H]GABA. The present work was performed to demonstrate in this cerebral zone the possibility of a GABA synthesis by the immunohistochemical localization of glutamate decarboxylase (GAD). GAD-positive reaction was detected with unlabelled antibody-enzyme peroxidase anti-peroxidase. Some nerve terminals containing either clear round vesicles, or sometimes clear round vesicles and some large granular vesicles, exhibited a positive staining. These terminals could belong to GABAergic inputs in the subcommissural organ. The few reactive terminals containing some granular vesicles could be related to the serotoninergic input as suggested previously (Gamrani et al., 1981). Several ependymocytes of this structure contained GAD-like positive reaction; these cells are also capable of taking up [³H]GABA (Gamrani et al., 1981) and present neuronal properties with regard to GABA. However, the presence in their cytoplasm of enolase, a specific glial marker, related them to glial elements. The presence of GABA in these ependymocytes suggests a modulating function of GABA on the secretory activity of the subcommissural organ.     

Improved Visualization of Cell Nuclei in Unstained Cultured Cells

Nuclear morphology is useful in tissue culture studies in determining the presence and grade of transformed cells as well as in determining the heterogeneity of the cell population (Grogan el al. 1981, Hustin 1976, Siracky et al. 1978, Siracky 1979). The ratio of long and short nuclear axes provides a useful numerical expression of nuclear shape (Hustin 1976). Clear visualization of nuclei is critical for making the necessary measurements.     

Notes on Technic: Simple, Reliable Detection of Latex Microspheres in High Quality Tissue Sections

Latex microspheres used in biological research have been visualized by light microscopy in mounts of cell suspensions, disrupted cells, or cleared tissues (Mishima et al 1987, Koonce et al 1986, LeFevre et al 1978); in unembedded coverslip monolayers (Koerten et al 1980); in fixed (Cornwall and Phillipson 1988) or unfixed (Wells et al 1988) frozen sections; in paraffin sections cleared and deparaffinized with n-butyl alcohol (Callebaut and Meeussen 1989); and in tissues embedded in resins suitable for transmission electron microscopy, such as Spurr's (Hampton et al 1987), Epon (Herzog and Miller 1979), or Ladd Low Viscosity Epon (LeFevre et al 1985). Paraffin embedding, and some plastic embedments, are impractical for demonstration of latex beads because the beads are dissolved by such organic solvents as xylene, dioxane, or chloroform (Van Furth and Diesselhoff-Den Dulk 1980), propylene oxide (Lentzen et al 1984), amyl acetate (Okada et al 1981), or toluene, the solvent in commonly used mounting media such as Fisher Permount (personal observation). The space remaining after dissolution of a bead is not maintained with paraffin embedding as it is with resin embedding. Even after plastic embedding, the resolving power of light microscopes may be inadequate to distinguish such spaces from other spaces found in and between cells. Latex beads are stable in methanol (Van Furth and Diesselhoff-Den Dulk 1980), ethanol. and n-butyl alcohol (Callebaut and Meeussen 1989).     

微生物新型防御系统的系统性发现与展望

微生物防御系统是生物技术创新与发展的重要工具库,细菌限制性内切酶的发现催生了现代分子克隆技术,CRISPR系统的开发利用则使基因组编辑技术取得革命性突破。基于上述原因,微生物新型防御系统的发现与研究已引起各国科学家的重视,一些新的防御系统如pAgos和DISARM等相继被发现和研究。为进一步挖掘微生物中可能蕴藏着的其他未知防御系统,最近以色列科学家Sorek等报道了从海量的微生物基因组序列中系统性发现新型防御系统的研究策略,并且通过合成生物学思路鉴定了10种新型系统的抗病毒或抗质粒功能。本文将首先介绍Sorek团队系统性发现新型防御系统的研究工作,进而总结目前已知的主要微生物新型防御系统的可能机制,并对该领域的发展态势与挑战进行分析和展望。     

Malnutrition increases insoluble-to-soluble tubulin ratio and in vitro incorporation of 32ATP in rat cerebral cortex.

Wistar rats were fed a normal protein (25% casein) or an isoenergetic low protein (8% casein) diet from the day of birth to weaning on day 21. Litters were killed at weaning and cerebral cortex was removed. Tubulin was prepared by centrifugation at 100,000 g, 4°C, as described by Shelansky et al. [Proc. Natn. Acad. Sci. U.S.A. 70, 765–768 (1973)]. Cold-insoluble tubulin was recovered in the pellet (Pl) fraction and cold-soluble tubulin in the supernatant (Sl) fraction. Alpha and beta tubulin were quantified by electrophoretic and immunological methods in both fractions. Our results indicated that malnutrition enhanced the ratio of cold-insoluble-tubulin-to-cold-soluble-tubulin. Furthermore malnutrition induced an increased in vitro incorporation of ³²P into both soluble and insoluble tubulins. Although tubulin phosphorylation has been related to tubulin stability properties, we cannot unequivocally ascribe the increased insoluble/soluble tubulin ratio with malnutrition to increased in vitro incorporation of ³²P.     

Signalling via rat dopamine D2-receptors expressed in mouse fibroblasts is not influenced by compounds binding to the sigma sites of these cells

The mouse fibroblast cell line LZR-1 is a well-established test system used to characterize the intrinsic activity of dopamine D₂-receptor ligands (Neve et al., Mol. Pharmac., 1989). This cell line is transfected with a eucaryotic expression vector containing the gene of the rat dopamine D_2B-receptor (short isoform of the D₂-receptor) (Bunzow et al., Nature, 1988). In addition to the expression of high levels of the rat dopamine D₂-receptor, these mouse cells also express sigma binding sites. Binding affinities of BMY 14,802, DTG, haloperidol, ( + )-pentazocine, ( + )-3-PPP, and ( + )-SKF 10,047 for the sigma binding sites as well as for the D₂-dopamine receptor in membranes of LZR-1 cells were determined. Using intact LZR-1 cells, it was found that the influence of sigma ligands on signalling via the dopamine D₂-receptor can be explained by their affinity for the latter receptor. Specific sigma ligands did not influence dopaminergic signal transduction in LZR-1 cells. It is therefore concluded that the LZR-1 cell is a suitable test model for determination of the intrinsic activity of dopamine D₂-receptor ligands even if these compounds have affinity for sigma binding sites.     

Endpoint error in smoothing and differentiating raw kinematic data: An evaluation of four popular methods

‘Endpoint error’ describes the erratic behavior at the beginning and end of the computed acceleration data which is commonly observed after smoothing and differentiating raw displacement data. To evaluate endpoint error produced by four popular smoothing and differentiating techniques, Lanshammar's (1982, J. Biomechanics 15, 99–105) modification of the Pezzack et al. (1977, J. Biomechanics, 10, 377–382) raw angular displacement data set was truncated at three different locations corresponding to the major peaks in the criterion acceleration curve. Also, for each data subset, three padding conditions were applied. Each data subset was smoothed and differentiated using the Butterworth digital filter, cubic spline, quintic spline, and Fourier series to obtain acceleration values. RMS residual errors were calculated between the computed and criterion accelerations in the endpoint regions. Although no method completely eliminated endpoint error, the results demonstrated clear superiority of the quintic spline over the other three methods in producing accurate acceleration values close to the endpoints of the modified Pezzack et al. (1977) data set. In fact, the quintic spline performed best with non-padded data (cumulative error=48.0 rad s⁻²). Conversely, when applied to non-padded data, the Butterworth digital filter produced wildly deviating values beginning more than the 10 points from the terminal data point (cumulative error=226.6 rad s⁻²). Each of the four methods performed better when applied to data subsets padded by linear extrapolation (average cumulative error=68.8 rad s⁻²) than when applied to analogous subsets padded by reflection (average cumulative error=86.1 rad s⁻²).     

Reactions of Nitric Oxide with Nitronyl Nitroxides and Oxygen: Prediction of Nitrite and Nitrate Formation by Kinetic Simulation

Nitric oxide reacts with nitronyl nitroxides (NNO) to form imino nitroxides (INO) and this transformation can be monitored using electron spin resonance spectroscopy. Recently, Akaike et al., reported that NNO such as 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO) and its derivatives (e.g., carboxy-PTIO) react with nitric oxide (·NO) in a 1:1 stoichiometry forming 2-phenyl-4,4,5,5-tetra-methylimidazoline-1-oxyl (PTI) or the respective product (e.g., carboxy-PTI) together with nitrite and nitrate (Akaike et al., Biochemistry 32, 827-832, 1993). In this paper, we reevaluate their results and show that the stoichiometry of the reaction between PTIO and ·NO is 0.63 ± 0.06:1.0. The reason for this discrepancy is due to an erroneous assumption by Akaike et al., that the stoichiometry for the reaction between ·NO and O₂ is 2:1 in aqueous solution. If the data reported by Akaike et al., were recalculated using a 4:1 stoichiometry established for the aqueous oxidation of ·NO, the reaction between ·NO and PTIO would give a stoichiometry of 0.5:1.0 in closer agreement with our data. We propose a mechanism for the reaction between PTIO and ·NO in aqueous solution. This mechanism predicts that the stoichiometry between carboxy-PTIO and ·NO is dependent on the rate of generation of ·NO and is 1:1 only at low rates of ·NO generation (i.e., 10^-13 M/s). However the stoichiometry approaches 0.5:1.0 at higher rates of ·NO production or when it is added as a bolus. The ratio between nitrite and nitrate also varies as a function of the rate of generation of ·NO. The model agrees with previous experimental observations that the aqueous oxidation of ·NO in air saturated solutions will exclusively form nitrite and predicts that ·NO will only generate substantial amounts of nitrate if it is released at a rate less than 10^-17 M/s. This may have important consequences in cellular systems where the concentration of ·NO is typically measured from nitrite production.     

Embedding Free-Floating Cells in Stained Agar for Rapid Scanning and Subsequent Ultramicrotomy

Methods we have previously used for epoxy embedding of suspended tissue culture cells, white blood cells and oral mucosal scrapings did not provide a simple means for selecting the desired specimens and facilitating their subsequent handling through embedding in an epoxy resin. We have therefore modified the agar embedding technique previously recommended for the processing of microorganisms by Kellenberger et al. (1958).     

Combined Staining by Thiolacetic Acid and Bielschowsky Methods

Tongues of mice were fixed in 10% Baker's Ca-formalin and sectioned at 50 μ with a freezing microtome. The thiolacetic acid method (Wachstein et al., J. Histochem. Cytochem., 9: 325-39) was used for motor endplates and after this cholinesterase staining, the Bielschowsky method was applied for nerve fibers. Thus, both motor endplates and nerve fibers were fully demonstrated.     

A molecular model for the interaction between vorozole and other non-steroidal inhibitors and human cytochrome P450 19 (P450 Aromatase)

In a previous study (Vanden Bossche et al., Breast Cancer Res. Treat. 30 (1994) 43) the interaction between (+)-S-vorozole and the I-helix of cytochrome P450 19 (P450 aromatase) has been reported. In the present study we extended the “I-helix model” by incorporating the C-terminus of P450 aromatase. The crystal structures of P450 101 (P450 cam), 102 (P450 BM-3) and 108 (P450 terp) reveal that the C-terminus is structurally conserved and forms part of their respective substrate binding pocket. Furthermore, the present study is extended to the interaction between P450 aromatase and its natural substrate androstenedione and the non-steroidal inhibitors (−)-R-vorozole, (−)-S-fadrozole, R-liarozole and (−)-R-aminoglutethimide. It is found that (+)-S-vorozole, (−)-S-fadrozole and R-liarozole bind in a comparable way to P450 aromatase and interact with both the I-helix (Glu³⁰² and Asp³⁰⁹) and C-terminus (Ser⁴⁷⁸ and His⁴⁸⁰). The weak activity of (−)-R-aminoglutethimide might be attributed to a lack of interaction wit the C-terminus.     

Insect nervous system [H]saxitoxin binding sites: characterization and target size

Saxitoxin (STX) has proved useful in the isolation and characterization of vertebrate and invertebrate voltage-operated sodium channels. Membrane extracts from the nervous system of the cockroach Periplaneta americana contain a saturable component of specific [³H]STX binding. Scatchard analysis yields a K_D of 0.84 nM, similar to that (3.0 nM) determined in electrophysiological studies on axons in the same tissue (Sattelle et al., 1979). The maximum number of binding sites, B_max (8.25 pmol/mg protein), was higher than previously observed. The specific binding component was blocked by STX and tetrodotoxin (TTX), but not by scorpion (Leiurus quinquestriatus) venom, aconitine, veratridine, sea anemone toxin and deltamethrin, which act at different sites on the channel molecule. Unlabelled STX samples prepared from different sources (Mytilus, Saxidomus and Gonyaulax) were all effective as inhibitors of [³H]STX binding. Radiation inactivation was employed to determine the molecular target size of the [³H]STX binding molecule in membranes prepared from the cockroach nervous system. By this means M_r = 171,400 ± 25,000 was estimated for the insect sodium channel.