Correlation of CrystaL Growth with the Staining of Axons by the Golgi Procedure

The surface of the specimens subjected to a modified Golgi technique (formalin fixed material; specimens in the following solution for 8-10 days at 27 C: 3% K₂Cr₂O₇, 100 ml, with the addition of 2.5-10 ml of 10% formalin and 6-25 gm of sucrose; then in 0.75% AgNO₃ for at least 2 days at 27 C) is sometimes covered with a fur of filamentous crystals and sometimes with a powdery precipitate of laminar crystals. In a series of experiments in which about 500 blocks of tissue were treated with variations of the staining procedure, good axonal stain was positively correlated with the appearance of filamentous crystals. These filaments have a thickness of 1-4 μ and grow at a rate of 160-330 μ/hr, reaching a length of 2-7 mm.     

Controlled Formaldehyde-Catecholamine Condensation in Cryostat Sections to Show Adrenergic Nerves by Fluorescence

Three sets of sections of freshly removed tissue are cut at 18 μ in a cryostat and dried on slides for 1.5 hr over P₂O₅. Each set of sections is incubated with a differently hydrated paraformaldehyde (prepared by storing paraformaldehyde powder over 21%, 25% or 28% aqueous H₂SO₄ for 1 wk) at 80 C for 1 hr before being mounted in glycerol and viewed with a fluorescence microscope. At least one set of specimens shows optimal fluorescence. The entire procedure from removing the tissue to observing fluorescence microscopically is accomplished readily within 4-8 hr. Adrenergic axons in the medial muscle of the cat nictitating membrane, the myometrium of the cat uterus and the adventitia of arterial vessels in rat pancreas are demonstrated.     

Staining and Mounting Helminths

The following procedure is recommended: Fix ces-todes and trematodes (while held flat between glass slides) 0.5-2.0 hr. in the following mixture: formalin, 15; acetic acid (gl.), 5; glycerol, 10; 95% ethyl alcohol, 24; distilled H₂O, 46; all proportions by volume. After freeing them from the slides, wash thoroughly in running water and stain immediately thereafter. Stock staining solution: ferric ammonium alum (violet cryst.), 2 g.; distilled H₂O (cold) 100 ml.; after solution, add 2 ml. concentrated H₂SO₄, bring to a boil; add 1 g. coelestin blue B (Nat. Aniline), boil 3-5 min.; cool and add 10 ml. absolute methyl alcohol and 10 ml. glycerol. Dilute 1 vol. with 3 vol. distilled H20 for use. Stain 5-30 min., depending on size of specimens. Wash with 2 changes 0.5 hr. each of distilled H₂O, then 50% isopropyl alcohol 12-16 hr., 50% isopropyl alcohol 2 hr., followed by graded isopropyl alcohol for dehydration. Ether: ethyl alcohol (equal parts), 1 hr., is followed by embedding in celloidin in a sheet just thick enough to cover the specimens. Trim embedded specimens and dehydrate with isopropyl alcohol, 80%, 90% and absolute. Clear in beechwood creosote. Mount in balsam with cover glasses that overlap the edges of the celloidin 1-2 mm. While drying at 37°C, refill edges of mount with fresh balsam as needed. When dry, remove excess balsam and ring the edges with ordinary gloss enamel paint.     

Rapid Preparation of Uncoated Biological Specimens for Scanning Electron Microscopy

We have developed a relatively rapid glutarddehyde-tannic acid (GTA) and osmium tetroxide (OsO₄) fixation procedure which permits many types of uncoated biological specimens to he examined in the scanning electron microscope (SEM) at 40 kV without the occurrence of charging. Most specimens taken one day can be examined in the SEM the following afternoon. Types of specimens successfully treated were perfused adult and embryonic rat tissues, confluent human skin fibroblast tissue cultures, plant roots, flowers, seeds, some garden insects, and microcolonies of salivary streptococci. Cells in suspension and extracted human teeth did become electron conductive when treated with the GTA procedure. Most suspended cells must he centrifuged between each solution and the GTA procedure increases the preparation time for these cells. Extracted teeth are usually simply dried and coated. Therefore, the usual SEM preparation techniques are shorter and perhaps more useful for these types of specimens.     

鼩鼹亚科(Talpidae:Uropsilinae)一新种

2006 年至2009 年,在四川西南地区开展小型兽类区系调查时,在普格县、美姑县、泸定县、九龙县采获了11 号鼩鼹类标本。其外形和头骨形态与其他鼩鼹类一致,但牙齿数量和齿式与已知鼩鼹类不同。这批标本上、下颌牙齿均为9 枚,齿式为i2/2,c1 /1,pm3 /3,m3 /3 =36,与少齿鼩鼹上颌9 枚、下颌8 枚(齿式:i2 /1,c1/1,pm3 /3,m3 /3 =34)不同,也不同于长吻鼩鼹与贡山鼩鼹(齿式:i2 /1,c1 /1,pm4/4,m3 /3 = 38)及峨眉鼩鼹(齿式:i2/2,c1 /1,pm4 /3,m3 /3 = 38)上颌10 枚、下颌9 枚,而不能归类于现有任何已知鼩鼹类物种。通过线粒体细胞色素b 基因构建的系统发育关系显示未归类标本形成一单系群,且与峨眉鼩鼹关系最近,构成姊妹关系。为此,我们认为采自于四川普格县、美姑县、泸定县、九龙县的鼩鼹类标本为鼩鼹亚科一新物种。根据新种上、下颌牙齿数量相等命名为等齿鼩鼹(Uropsilus aequodonenia)。     

Rapid plastic embedding is compatible with colorimetric detection following whole mount in situ hybridization in plant specimens

In performing in situ hybridizations, nonisotopic nucleic acid labeling coupled with colorimetric detection offers a safer, easier and more rapid alternative to using radioactively labeled nucleic acid probes and microscopic autoradiography. Whole mount in situ hybridization is also advantageous, because many samples can be processed identically and the reduced handling of specimens greatly reduces the risk of exposing tissues to RNase(s). The thickness of whole mount specimens, however, often prevents accurate determination of sites of expression within specific tissues. Although post-hybridization embedding and sectioning is a solution to this problem, the precipitate formed following the common colorimetric detection procedure is soluble in the organic solvents used for dehydration prior to embedding. We have developed a dehydration and embedding procedure that takes advantage of the compatibility of L.R. White^® resin containing 10% (v/v) polyethylene glycol 400, and heat polymerized. The addition of the plasticizer allows L.R. White^® embedded tissues to be sectioned at 10 μm providing excellent signal contrast.     

A modified technique for the measurement of sulfhydryl groups oxidized by reactive oxygen intermediates

This paper suggests a simple modification of the Ellman procedure when used to measure accurate changes in sulfhydryl (-SH) content induced by reactive oxygen intermediates (ROI). This modification became necessary when we found that the standard technique did not produce time invariant results in the presence of ROI-generating systems. Cysteine (cys; 20–100 μM) in 20 mM imidazole buffer (pH 7.0) containing 1.0 mM EDTA was reacted with excess (0.2 mM) 5,5′-dithiobis(2-nitrobenzoic acid), DTNB. The absorbance of the product (p-nitrothiophenol anion) was recorded at 412 nm (A₄₁₂). This A₄₁₂ was stable for 60 min and gave a linear relationship with cys concentrations used. ROI were generated either by 0.01 U xanthine oxidase (XO) + 0.01–1.0 mM hypoxanthine (HX), 0.01–1.0 mM H₂O₂, or H₂O₂ + 100 μM FeSO₄. In the presence of ROI, A₄₁₂ decreased with time and its rate of decrease was dependent upon the concentration of components of the ROI-generating system. This time-dependent decrease in A₄₁₂ was prevented completely by the addition of 100 U of catalase (CAT). Therefore, we modified the DTNB method as follows: -SH groups were reacted with ROI for 30 min; this was followed by the addition of 100 U of CAT to scavenge the excess unreacted ROI before the addition of DTNB to generate the product. Using this modification the ROI-induced decrease in A₄₁₂ was stable with time and was linearly related to the cys concentration. We further tested the modified procedure using metallothionein (MT) as a substrate for the ROI-induced changes in -SH content. MT, at concentrations of 2.5, 5.0, and 7.5 μM, was treated with XO + 100 μM HX. Using the modified procedure, an average decrease (as compared to the untreated control) of 15, 22, and 33 μM in -SH content was observed consistently at the respective MT concentrations. However, without the modification in the procedure, these average decrease were 20, 38, and 51 μM, respectively and continued to further increase with time. These discrepancies could give rise to errors ranging from 28 to 35% or higher in determination of the ROI-induced decrease in the -SH groups of MT. This data suggests that scavenging the unreacted H₂O₂ with C prior to the addition of DTNB to the assay mixture gives a stable and accurate estimate of the ROI-induced oxidative damage to -SH groups.     

Advantages of Epoxy Resin as a Mounting Medium for Light Microscopy

The need for very durable mounting is especially felt in the teaching of parasitology and mycology; otherwise, the availability of microscope slides may depend on the use of fresh specimens. Resinous mounts and those in aqueous media sealed with fingernail lacquer, paraffin or asphalt do not preserve specimens satisfactorily. Polyvinylpyrrolidone (pvp), a water-soluble mounting medium described by Burstone (1962), cannot be applied directly for mounting of insects and certain other parasites which have water-repelling integuments; moreover, pvp bleaches eosin. Grimley et al. (1965) prepared large epoxy sections of tissues from which areas for electron microscopy could be selected. This procedure however is designed for electron microscopic techniques whereas the present paper describes a direct epoxy mounting method to produce permanent mounts for light microscopy.     

Preparation of Fossil Bone Specimens for Scanning Electron Microscopy

A method is described for preparing fossil bone specimens for scanning electron microscopy. To obtain bone surfaces suitable for study, material was embedded in Epon 812 and selected faces exposed by grinding were subjected to controlled etching with a 4:1 mixture of 5% HNO₃ and 1% OsO₄, Surfaces thus prepared were further processed by the so-called clearing replicas technique. As a result of this procedure the bone surfaces revealed a network of anastomosing vascular canals the inner surface of whose walls could be examined in the scanning electron microscope. By etching extremely thin ground sections of bone stuck to plastic tape the contents of vascular canals as well as osteocytes can be isolated. This method ensures the good preservation of spatial relations between bone elements essential for studies of fossil bones, which an sometimes very brittle.     

Combined Aldehyde-Fuchsin and Periodic Acid-Schiff Staining of the Pituitary

Aldehyde-fuchsin and the periodic acid-Schiff procedure can be applied in sequence to the same tissue section and combined with a stain for acidophils, such as orange G, for routine analysis of the pituitary. This is made possible by fixation in a mixture containing 5% chrome-alum, 5% HgCl₂ and 5% formalin; control of the pH and the activity of the aldehyde-fuchsin; and increased sensitivity of the Schiff reagent, effected by reducing its SO₂ content. Although designed especially for the pituitary, the procedure is also applicable to other histological problems.     

Fluorescence in Paraffin Sections of Dye-Labelled Protein Administered in vivo: Effect of HgCl2 Fixation

Lung and liver slices, 2-3 mm thick, from guinea pigs injected intravenously with fluorescent dye-protein conjugate are fixed for 15-30 min in saturated aqueous HgCl₂, dehydrated in ethanol, cleared in xylene and embedded in paraffin at 60 C. Mercurial deposits are removed with I₂KI from 5 μ sections taken to water, and the iodine then removed with 5% Na₂S₂O₃. Sections are mounted from xylene into permanent nonfluorescent mounting medium. This procedure gives optimal fluorescence which is not decreased by the technic of removing mercurial precipitates. Longer fixation, fixation in phosphate-buffered formalin, or in an HgCl₂-formalin mixture gives inferior results.     

A Glutaraldehyde-Dichromate-Silver Sequence for Differentiating Norepinephrine Cells from Epinephrine Cells in Neonatal and Adult Rats—Paraffin Sections

A glutaraldehyde-K₂Cr₂O₇ procedure intensified by silver staining enabled norepinephrine and epinephrine cells to be distinguished readily in paraffin sections of the adrenal glands of rats 8 days after birth. The technique involved fixation in 0.1 M cacodylate-buffered 5% glutaraldehyde (6-24 hr), treatment with 3.5% K₂Cr₂O₇ (6-12 hr) and routine preparation of paraffin sections. The sections were deparaffinised, brought to water and immersed in Fontana's solution (24 hr), prepared by adding concentrated NH₄OH drop by drop to 5% AgNO₃ until the precipitate formed just redissolved; more 5% AgNO₃ was then added until a permanent cloudiness just developed. After a rinse in distilled water, the sections were treated with 0.5% gold chloride (5 min) and Na₂S₂O₃ (5 min), then mounted in Depex. This sequence resulted in an intense black cytoplasmic colouration in norpinephrine-containing cells of both the adult and 8-day-old animals whereas epinephrine-containing cells remained colourless. The glutaraldehyde-K₂CrO₇ procedure, without intensification, gave very clear results in the adult: a yellow cytoplasmic colour in the norepinephrine cells with epinephrine cells colourless. A glutaraldehyde-OsO₄ sequence gave a less well defined separation of these cell types in the adult and failed to distinguish the cell types in the neonate.     

Ammoniated Silver Carbonate for the Demonstration of Lysosomes

Hortega's ammoniated silver carbonate method was used to demonstrate lysosomes in the central nervous system and kidney of adult rats. Formol-CaCl₂, (10%:1%) fixed, frozen sections were impregnated for 10 min in Hortega's solution: 30 ml of 10% AgNO₂ and 90 ml of 5% Na₂CO₃, with concentrated NH₄OH added until the precipitate dissolved, then distilled water to make 400 ml. This procedure revealed silver-positive cytoplasmic structures whose form, shape and distribution were similar to that seen by staining adjacent sections for acid phosphatase. A short fixation of 18-24 hr appears to be essential. A useful, nonenzymatic method for the demonstration of lysosomes is thereby available.     

Stomatal frequency of Betula pubescens and Pinus sylvestris shows no proportional relationship with atmospheric CO2 concentration

We investigated the relationship between stomatal frequency and a range of atmospheric CO₂ concentrations ([CO₂]_atm) in Betula pubescens and Pinus sylvestris , two important boreal trees in Scandinavia. If strong relationships exist, they can be used to reconstruct past [CO₂]_atm from stomatal frequency of fossil Betula and Pinus leaves. Responses of epidermal characters (stomatal density (SD), epidermal cell density (ED), stomatal index (SI)) to different CO₂ concentrations were investigated utilising (1) the lower partial pressure of CO₂ at increasing altitudes for B. pubescens , and in herbarium specimens of B. pubescens and P. sylvestris collected during the post-industrial rise of [CO₂]_atm from c. 280 ppmv to c. 360 ppmv in 1997 and (2) concentrations (560 ppmv) and temperatures (3° summer) above present day in the CLIMEX greenhouse experiment. All the results show no clear relationship between SD or SI and [CO₂] _atm for either B. pubescens or P. sylvestris. Most likely there are stronger genetically and environmentally induced factors that affect the development of the leaves. Problems with collecting representative samples from herbarium specimens are discussed. Since the effects of changes in [CO₂]_atm cannot be statistically modelled, B. pubescens and P. sylvestris are not suitable for reconstructing past atmospheric CO₂ concentrations from fossil leaves using stomatal density or stomatal index     

Avian Microchromosomes; As Shown by Prefixation Treatment with Colchicine, Squashing, and Haematoxylin Staining

Small pieces of bird tissue from developing feathers, embryos, embryonic gonads and adult testes are treated before fixation in a 0.25% solution of colchicine diluted with an equal volume of hypotonic Pannett and Compton solution containing KCI, 0.336 gm; CaCl₂, 0.160 gm; Na₂HP0₄. 12H₂O, 0.172 gm; and NaH₂PO₄. 4H₂O, 0.0172 gm per 1000 ml of double distilled water, for 20-30 min at 37° C. This material is subsequently centrifuged at 1000 rev/min for 1-3 min, fixed with acetic alcohol (1:3) and stained with either Gomori's haematoxylin or by Feulgen's procedure. Preparations are frozen in the freezing chamber of a refrigerator for the separation of the cover slips, then mounted in euparal after dehydration in alcohols. With this technique, unclumped and clear preparations of both macrochromosomes and microchromosomes are obtained regularly.     

A New Synthesis Of Coenzymically Active Water-Soluble Macromolecular Nad And Nadp Derivatives

Based on an unexpected transformation of N (1)-(2-aminoethyl)-NAD(P) to N⁶-(2-aminoethy1)-NAD(P) under mild aqueous conditions (pH 6.0-6.5, 50°C) synthesis of uniform macromolecular derivatives of N⁶-alkylated NAD and N⁶-alkylated NADP was possible, with, in most cases, acceptable overall yields (6-37%). The usual steps of (a) the chemical reduction with Na₂S₂O₄,(b) the Dimroth rearrangement under harsh alkaline conditions and (c) the enzymatic or chemical oxidation were omitted. This represents a significant simplification of the procedure. A common procedure for the synthesis of macromolecular N⁶-(2-aminoethyl)-NAD(P) derivatives was pursued, coupling N⁶-(2-aminoethyl)-NAD(P) to several water-soluble copolymers containing maleic acid anhydride. PEG (M_r = 20000)-N⁶-(2-aminoethl)-NAD, polyvinylpyrrolidone (M_r,= 160000)-N⁶-(2-aminoethylNAD and dextran (M_r= 70000)-N⁶-(2-aminoethyl)-NAD were synthesized by covalently binding N⁶-(2-aminoethyl)-NAD to the corresponding carboxylated polymers by the carbodiimide method. PEG (M_r= 4000 and 20000-N⁶-(2-aminoethyl)-NADP was efficiently synthesized by covalent attachment of N⁶-(2-aminoethyl)-NADP to N-hydroxy-succinimide activated carboxylate PEG (M_r= 4000 and 20000), avoiding the carbodiimide method, which would lead simultaneously to 2'3'-cyclic NADP derivatives. Except for the macromolecular cofactor derivatives based on copolymers containing maleic acid anhydride, the total enzymatic reducibility of the macromolecular N-(2-aminoethyl)-NAD(P) derivatives was satisfactory (90-95%).     

Formalin fixation strongly influences biomechanical properties of the spine

As fresh human cadaveric spine specimens for in vitro testing are hard to obtain and carry a potential risk of infection, the possibility of using embalmed spine specimens has been considered. The cross-linking effect of formalin fixation, however, raises uncertainties regarding the biomechanical likeness of preserved specimens. They have been reported to be stiffer, but no quantitative data exist.

The purpose of this study was to determine the biomechanical differences between fresh and formalin-fixed spine specimens, using L1–2 motion segments from six 16-week-old calf spines. The range of motion and neutral zone were determined in flexion-/extension, left/right axial rotation, and right/left lateral bending.

The range of motion decreased in the formalin fixed specimens by as much as 80%, and the neutral zone by as much as 96%. The results of this study therefore imply that, for biomechanical testing, formalin-fixed specimens are not representative of the in vivo conditions.     

High-performance liquid chromatographic method for the determination of insulin synthesis in biological systems

This paper reports a two-step high-performance liquid chromatographic procedure which permits the study of the incorporation of [³H]leucine into insulin in biological systems. The first step of the procedure was size exclusion chromatography, performed on a GPC-100 column, which was eluted with 0.1 M KH₂PO₄—methanol (9:1, v/v). By this step the bulk of both protein and radioactivity was separated from tritiated insulin. The second step, which employs reversed-phase chromatography on an octadecylsilyl column, permits the separation of insulin from other contaminants by means of a linear gradient of acetonitrile. This simple and reproducible method was employed to test insulin synthesis in cultured human retinoblastoma Y79 cells.     

The Pmr1 protein, the major yeast Ca2+-ATPase in the Golgi, regulates intracellular levels of the cadmium ion

Cadmium is a nonessential, highly toxic heavy metal that shows ionic properties similar to calcium. These ionic similarities imply that the cadmium ion, Cd²⁺, is a calcium ion, Ca²⁺, receptor-agonist, affecting the same biochemical pathways involved in Ca²⁺ homeostasis. In the yeast Saccharomyces cerevisiae , the PMC1 and PMR1 genes encode vacuolar and Golgi Ca²⁺-ATPases, respectively. The PMR1 protein product Pmr1p is involved in both Ca²⁺ and Mn²⁺ homeostasis. This study investigated the importance of Pmc1p and Pmr1p for Cd²⁺ cellular detoxification. Using the standard techniques of yeast molecular research and a multielemental procedure named particle-induced X-ray emission, Pmr1p was identified as a protein that directly participates in the detoxification of Cd²⁺, possibly through the secretory pathway. The results allow us to posit a model of Cd²⁺ detoxification where Pmr1p has a central role in cell survival in a Cd²⁺-rich environment.     

The chemical microenvironment of the symbiotic planktonic foraminifer Orbulina universa

Microsensor measurements of CO₂, O₂, pH and Ca²⁺ in the vicinity of the symbiont-bearing planktonic foraminifer Orbulina universa showed major light-modulated changes in the chemical microenvironment due to symbiont photosynthesis, respiration of the holobiont, and precipitation of the calcite shell. Under saturating light conditions, microprofiles measured towards the shell surface showed an O₂ increase of up to 220% air saturation, a decrease in CO₂ concentration to 4.9 μM, and a pH increase to 8.8 due to symbiont photosynthesis. The Ca²⁺ concentration decreased to ∼9.6 mM in two specimens, while it increased to 10.2-10.8 mM in three other specimens kept in light. In darkness, the respiration of the community decreased the O₂ concentration to 82% of air saturation, CO₂ increased up to 15 μM, the pH decreased to 8.0, and the Ca²⁺ concentration increased up to 10.4 mM. These data, and derived calculations of the distribution of HCO₃^- and CO₃^2- near the shell, showed that the carbonate system in the vicinity of O. universa was significantly different from conditions in the surrounding seawater, both in light and darkness, due to the metabolism of the foraminifer and its associated algae. Experimental light-dark cycles indicated a sufficient CO₂ supply sustaining high carbon fixation rates of the symbiotic algae via conversion of HCO₃^- or via CO₂ release from calcification and host respiration. Our findings on irradiance-dependent CO₂ and pH changes in the vicinity of symbiont-bearing planktonic foraminifera give direct experimental evidence for the predictions of isotope fractionation models used in palaeoclimatology stating that metabolic processes affect the isotopic carbon signal (δ¹³C) in foraminifera.