Chromosome Preparatons of Bone Marrow Cells without Prior In Vitro Culture or In Vivo Colchicine Administration

Bone marrow (about 0.5 ml) from au erythropoietic region is freed of blood clots by washing 1-3 min in 1 μg/ml colchicine solution (2-3 ml) and then soaking 1-2 hr at 20-30° C in a second change. For mammalian or avian marrows, the colchicine is made up in phosphate-buffered (pH 7) physiological NaCl solution; for amphibian, Ringer's A solution. Next the specimens are soaked about 20 min in a hypotonic solution as follows: for mammalian, 1% Na-citrate; for avian, a 1:4 dilution of the buffered NaCl solution by distilled water; and for amphibian, Ringer's A-distilled water, 1:1. Then they are heated in a mixture of 2% orcein in 45% acetic acid and 1 N HCl, 9:1. Immediately after heating, squash preparations are made with 2% acetic-orcein in the usual manner. An alternative method is to dissociate the marrow cells by agitating after colchicine treatment. Then, recovering the cells between changes by low-speed centrifugation, to carry out the hypotonic treatment and subsequent fixation in Carnoy's solution I (alcohol acetic, 3:1) before drying the cells onto slides from the fixative. After thorough drying the slides may be stained 10-20 min in acetic orcein, or by other suitable technics.     

CERTAIN EFFECTS OF SALTS ON THE PENETRATION OF BRILLIANT CRESYL BLUE INTO NITELLA

The effect of various substances on living cells may be advantageously studied by exposing them to such substances and observing their subsequent behavior in solutions of a basic dye, brilliant cresyl blue. The rate of penetration of the basic dye, brilliant cresyl blue, is decreased when cells are exposed to salts with monovalent cations before they are placed in the dye solution (made up with borate buffer mixture). This inhibiting effect is assumed to be due to the effect of the salts on the protoplasm. This effect is not readily reversible when cells are transferred to distilled water, but it is removed by salts with bivalent or trivalent cations. In some cases it disappears in dye made up with phosphate buffer mixture, or with borate buffer mixture at the pH value in which the borax predominates, and in the case of NaCl it disappears in dye containing NaCl. No inhibiting effect is seen when cells are exposed to NaCl solution containing MgCl₂ before they are placed in the dye solution. The rate of penetration of dye is not decreased when cells are previously exposed to salts with bivalent and trivalent cations. The rate is slightly increased when cells are placed in the dye solution containing a salt with monovalent cation and probably with bivalent or trivalent cations. In the case of the bivalent and trivalent salts the increase is so slight that it may be negligible.     

Covalent linkage between RNA and nascent DNA in the slime mold, Physarum polycephalum.

When alpha--32 P-labeled deoxyribonucleoside triphosphates are injected into plasmodia of the eukaryotic slime mold, Physarum polycephalum, they are incorporated initially into strands of DNA which are mostly less than 300 nucleotides long. Sixty minutes after injection incorporated deoxyribonucleoside triphosphates are found in much longer strands. If the short strands found two minutes after injection are denatured and centrifuged to equilibrium in a Cs(2)SO(4) density gradient, they migrate to a density slightly greater than that of single-stranded Physarum DNA. When these short strands are treated with alkali to hydrolyze RNA, a small fraction of the incorporated -32P is made acid-soluble and is identified as a mixture of the four ribonucleoside 2',3'-monophosphates. Such transfer of -32P to ribonucleotides occurs when any of the 4 alpha--32P-labeled deoxyribonucleoside triphosphates is used for injection, but the transfer is greatest with [alpha--32P]dGTP. We conclude that very short stretches of RNA are found linked through phosphodiester bonds to nascent DNA chains in Physarum polycephalum and that any of the 16 possible combinations of ribo- and deoxyribonucleotides can occur at the RNA-DNA junction.     

Digestion of Collagen and Reticulin in Paraffin Sections by Collagenase

Tissues are fixed in ethanol or in Carnoy's 6:3:1 mixture and embedded in paraffin after routine ethanol dehydration. Sections are taken to water and then covered with 0.2 ml of a 0.9% NaCl solution containing 1 mg/ml of collagenase, and incubated at 50° C for 45 min. After this, they were washed and then stained by the usual methods for connective tissue fibers. Control sections were made by substituting plain 0.9% NaCl solution for the collagenase solution. The collagenase used was from bacteria and obtained from Nutritional Biochemicals Corporation, Cleveland 28, Ohio.     

Suggested Counterstains for Davenport Reduced Silver Preparations of Peripheral Nerves

—Peripheral nerves which have been fixed in a mixture of formaldehyde and acetic acid and stained according to the method of Davenport can be successfully counterstained for demonstration of myelin sheaths and stroma. After mounted sections have been silvered, reduced and toned, the coating of nitrocellulose is removed by passing thru two changes of acetone. Following brief washes in 100,95,85 and 75% alcohols they are stained in an acidified aqueous solution of azo carmine for 30 to 60 minutes. Excess azo carmine is extracted with anilin alcohol followed by acetic alcohol after which the sections are mordanted for 15 to 60 minutes in a 5% aqueous solution of phosphotungstic acid. Without washing they are transferred to a stain mixture of either anilin blue and orange G (acidified) or light green and orange G (acidified) where they remain from 1 to 5 hours. After destaining in 95% alcohol and dehydration in absolute alcohol the sections are mounted in dammar. Result: axons stain black; sheath and fibroblast nuclei, red; myelin sheaths, orange; and connective tissue, blue or green. When the counterstains are applied to ganglia, cytological details of individual cells are demonstrated.     

Propidium iodide and S1 nuclease: tools for studying DNA reassociation kinetics

A method for the determination of the amount of double-stranded DNA in a reassociation mixture is described. Reassociated DNA resistant to S1 nuclease digestion is measured fluorometrically using propidium iodide. A direct comparison is made between this method and an established method in which radiolabeled Escherichia coli DNA resistant to S1 digestion is measured by scintillation counting after separation of nucleotides by Sephadex G-100 chromatography. Reassociation curves determined for calf thymus and E. coli DNA are presented.     

Carbowax for Embedding and Serial Sectioning of Botanical Material

Plant material infiltrated with gradually increasing concentrations of Carbowax 400, followed by Carbowax 1540 and finally a 19:1 embedding mixture of Carbowax 1540 and 4000 showed minimum shrinkage. Quantitative measurements of shrinkage in tissue of potato tubers gave the following amounts: fixation and washing, about 4%; transfer from water directly to 70% Carbowax 400, 5176; from water through a graded series (5, 10, 15, 20, 30, 40, 50 and 60% Carbowax) to 70%, only 2.5% shrinkage; with an additional 1.5% occurring in transition to the embedding mixture. Dry ribbons are placed on adhesive-coated (gelatin, 5 gm; water, 120 ml; glycerol, 40 ml; phenol, 2 gm) slides in a humidity chamber. In 10-15 min enough moisture is absorbed by the ribbon to float the sections out gently and bring them in contact with the adhesive. Slides are then dried 5-10 min at room temperature. To remove minor wrinkles, the sections are subsequently flooded with water, then redried 12-24 hr; after which, they are ready for staining.     

Carbowax for Embedding and Serial Sectioning of Botanical Material

Plant material infiltrated with gradually increasing concentrations of Carbowax 400, followed by Carbowax 1540 and finally a 19:1 embedding mixture of Carbowax 1540 and 4000 showed minimum shrinkage. Quantitative measurements of shrinkage in tissue of potato tubers gave the following amounts: fixation and washing, about 4%; transfer from water directly to 70% Carbowax 400, 5176; from water through a graded series (5, 10, 15, 20, 30, 40, 50 and 60% Carbowax) to 70%, only 2.5% shrinkage; with an additional 1.5% occurring in transition to the embedding mixture. Dry ribbons are placed on adhesive-coated (gelatin, 5 gm; water, 120 ml; glycerol, 40 ml; phenol, 2 gm) slides in a humidity chamber. In 10-15 min enough moisture is absorbed by the ribbon to float the sections out gently and bring them in contact with the adhesive. Slides are then dried 5-10 min at room temperature. To remove minor wrinkles, the sections are subsequently flooded with water, then redried 12-24 hr; after which, they are ready for staining.     

Food Intake and Circadian Rhythms of Activity of Red-Winged Blackbirds (Agelaius phoeniceus). A Time-Course Study on the Effects of Alpha-Chloralose and Secobarbital

Anesthetic drugs like alpha-chloralose and secobarbital are used to capture wild birds. The doses are usually chosen on the basis of low mortality rates and high capture success. However, little information is available on the time necessary for the birds to fully recover from the drugs' effects after they have regained consciousness. In this work, we used circadian rhythms of activities to study the long-term effects of a mixture of alpha-chloralose and secobarbital on adult male Red-winged Blackbirds. The birds were housed individually in sound-proof boxes for 30 days, during which they received alpha-chloralose and secobarbital mixed with corn, either on day 3 or on day 15. On the day of treatment, the birds experienced a significant decrease in activity level, including feeding activities, which lasted more than 22 days for the birds treated on day 3, compared to about 8 days for the birds treated on day 15. Moreover, typical circadian distribution of activity was lost and many birds became aperiodic. Two weeks were needed for the birds treated on day 3 to recover normal rhythms, compared to less than 9 days for those treated on day 15. These results indicate that circadian parameters can be used to detect after-effects of intoxication long after the birds have regained consciousness.     

Complementary Behaviors of Maternal and Offspring <Emphasis Type="Italic">Spodoptera littoralis</Emphasis>: Oviposition Site Selection and Larval Movement Together Maximize Performance

The selection of oviposition and feeding sites within cotton plants by Spodoptera littoralis was investigated in the field in 2 years, 2007 and 2008. The female moths exhibited significant oviposition preference for young leaves (YL), particularly the 3rd and 4th leaves from top. The larvae originating from egg batches deposited on YL fed mostly in situ for about 5 days, after which they gradually moved their feeding site toward fully expanded or mature leaves on the same individual plant or on neighboring plants. Larvae hatching from batches deposited on fully expanded leaves (FE) fed in situ only for about 2 days, after which they moved toward younger leaves, where they fed for about 3 more days. After the fifth day, however, larvae of the two groups dispersed mainly downward and outward from their hatching site until the end of a 12-day observation. Larvae hatching from eggs deposited on mature or pre-senescent leaves (MP) moved mainly horizontally to other plants after a slight upward shift. The YL and FE larvae grew significantly faster than MP larvae, both in the field and in a laboratory experiment. In the laboratory experiment, the larval period was shorter and the pupal weight was higher when the animals were offered young leaves or young and fully expanded leaves, than when the animals were offered mature and pre-senescent leaves during the first 5 days after hatching. Possible causes and advantages of the exhibited oviposition preference, as well as the apparent ability of larvae to correct for small egg misplacements made by the females, are discussed.     

Cell therapy prospects in Duchenne muscular dystrophy

Skeletal muscle is made of multinucleated postmitotic fibers which are the agents of contraction. These fibers arise from mononuclear precursor cells which fuse after having migrated from the somites to the site of myogenesis. The cascade of events which result in muscle differentiation is well known nowadays (Sabourin & Rudnicki, 2000). Some precursor cells present in adults muscle are termed satellite cells (Mauro, 1961), because of their location between the plasma membrane and the extracellular matrix of muscle fibers. Since they have a role in muscle growth and regeneration, these cells were the first candidates for treating muscle dystrophies. Despite a large corpus of positive experimental data about transplantation of these cells, no hard clinical result has been obtained to date, However these investigations have led to fruitful thinking about heterogeneity of muscle cell precursors and the possible ways to use them for therapy.     

Molecular interactions between micellar polysialogangliosides and affinity-purified tetanotoxins in aqueous solution

Two-affinity purified tetanotoxin forms, TeToA and TeToB, with different affinities for gangliosides were characterized by analytical ultracentrifuge, circular dichroism (CD), and amino acid composition. Both toxin forms share a common sedimentation coefficient of about 6-7 S and similar alpha-helicity values, but they vary in amino acid composition. Incubation of TeToB with micellar polysialogangliosides results in formation of high (21-24 S) and medium (13-15 S) size toxin-micellar ganglioside aggregates as revealed by analytical ultracentrifuge technique. At TeToB/[N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl- [N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosylceramide (GT1b) molar ratios of greater than 26, high molecular weight aggregates (Mr greater than or equal to 700,000) which contain between 3 and 5 toxin monomers are formed, whereas at molar ratios less than 15, about 1-2 monomers are present. TeToA does not form aggregates in the presence of gangliosides. A marked increase in the alpha-helix from about 20 to 39% is apparent in the CD spectrum of TeToB after interaction with ganglioside mixture (G1b). Cerebrosides, sulfatides, sphingomyelin, and phosphatidylserine also increase the alpha-helix, presumably because of an overall effect of lipids on the protein. TeToA and fragment B but not C also undergo similar changes in the presence of G1b, suggesting that the effect of ganglioside is not specific. The polarity of the CD spectra of a number of gangliosides is shifted from a negative to a positive value after interaction with tetanotoxin. The data are consistent with the interpretation of a discrete hydrophobic domain on the toxin heavy chain which interacts with micellar gangliosides to form macromolecular complexes.     

Ink Perfusion for Displaying Capillaries in the Chicken

A chicken was anesthetized with Na-phenobarbital, 130 mg/kg, and the external ischiatic artery was exposed. Heparin, 90 units/kg, was injected into the brachial vein, after which a cannula was inserted into the exposed ischiatic artery. Heat was applied to the head to produce vasodilation. Perfusion through the cannula was made at about 150 mm Hg pressure and consisted of 30 ml of a biological type of Pelikan ink to which was added NaNO₂ to make a 0.02% solution. After the bird was killed by an overdose of anesthetic, the skin around the perimeter of the femoral feather tract was cauterized, the tract removed, attached to a stainless steel wire net, fixed for 1 hr in 10% formic acid, then dehydrated in 4 changes of acetone, 1 hr each, followed by a 5th change for 12 hr. The dehydrated specimens were placed in methyl benzoate, the 1st change for 1 hr and the 2nd until clear (3-15 hr). This procedure exhibits small vessels and capillary networks within the integument, but if only the vessels larger than capillaries are to be studied, they can be shown by slowly injecting 15-30 ml of the perfusion mixture into a peripheral vein. The still beating heart will distribute the ink to other regions of the body, thus allowing the vessels to be traced in excised tissue.     

A simple procedure for large-scale preparation of pure plasmid DNA free from chromosomal DNA from bacteria

A very simple, inexpensive procedure for preparing pure plasmid DNA from bacteria is described. In this method, lysozyme-induced spheroplasts are made in presence of 833 micrograms/ml of ethidium bromide which are then lysed by a mixture of Brij 58 and sodium deoxycholate, and the lysate is centrifuged at 48,000 g for 25 min whereby about 99.9% of total chromosomal DNA is pelleted. From the supernatant containing plasmid DNA, the proteins are removed by phenol extraction and the major part of RNA by CaCl2 precipitation, and finally the small amount of residual RNA is removed by RNase treatment. The average yield of pBR322 DNA from 1 liter of amplified culture by this procedure is 2 to 2.5 mg and the preparation is highly pure, containing only about 0.005% of total yield as chromosomal DNA contaminant. Moreover, the substrate activity and the transforming ability of the plasmid DNA prepared by this method remain unaffected.     

AN ELECTRON MICROSCOPE STUDY OF TOBACCO MOSAIC VIRUS LESIONS IN NICOTIANA GLUTINOSA L

Ultra-thin sections of Nicotiana glutinosa L. leaves inoculated with a concentrated solution of tobacco mosaic virus were made at short intervals from 0 to 78 hours after inoculation. Eight hours after inoculation, the size of starch grains increased. This was followed by rupture of cytoplasmic and chloroplast membranes. At about 24 hours there was a great increase in number of mitochondria, which persisted until about 60 hours, when some became electron opaque while others appeared to disintegrate. Finally, the cell contents were compressed into one area of the cell, where they became electron opaque. This was accompanied by collapse of the rest of the cell and tearing away of the cell walls from adjacent cells. The nucleus remained stable and intact for as long as observations could be made. No identifiable virus particles were seen.     

Effet de l'application d'un mélange lambda-cyhalothrin (pesticide chimique) et de spores de Metarhizium anisopliae (flavoviride) var. acridum Driver and Milner (biopesticide) appliqué sur les larves de sauteriaux au Mali

A mixture of lambda-cyhalothrin (lambda-cyhalothrin: chemical insecticide) and Metarhizium anisopliae ( flavoviride ) var. acridum Driver and Milner, an entomopathogenic fungus (bioinsecticide) was used for grasshopper control in Mali. An oil-based formulation of Metarhizium anisopliae ( flavoviride ) var. acridum Driver and Milner has been developed by LUBILOSA a collaborative project for locust and grasshoppers control. It takes 6 to 10 days for the biopesticide to kill the hosts, which is not a problem for larvae in fallows because they will die before reaching the farmers' fields. However, if crops are infested by adults, the farmers can not wait for 6 to 10 days. An experiment was conducted in Mali using a mixture of a biopesticide and chemical pesticide. The mixture of lambda-cyhalothrin (chemical insecticide) and Metarhizium anisopliae ( flavoviride ) var. acridum (biopesticide: oil-based entomopathogenic fungus spore suspension) was applied to nymphs of Sahelian grasshoppers, using ultra low volume (ULV) sprayers. Both the mixture and lambda-cyhalothrin alone gave quick mortality, with slightly higher mortality for the mixture. Mortality due to the Metarhizium treatments began 2 days after application and subsequently reached similar levels of mortality to the lambda-cyhalothrin mixture treatments. The efficacy of the mixture was greater than Metarhizium alone. The efficacy of lambda-cyhalothrin reached 80% on the day following application, but declined after 10 days, due probably to immigration of untreated grasshoppers.     

Enzyme destruction by a protease contaminant in bacitracin

Bacitracin, as purchased from biochemical supply companies, is a mixture of more than 30 different substances. The major antibiotic isoforms A and B account for about 60% of the mixture. A newly identified impurity in some, but not all, of the bacitracin lots is a powerful subtilisin-type protease capable of cleaving many proteins including protein disulfide isomerase (PDI), myosin, and a variety of artificial substrates Thus, it is important for investigators who use bacitracin as a protease or other enzyme inhibitor to determine if the bacitracin they are using is contaminated with a protease enzyme. If it is present, they may have to reinterpret their results and retest with an enzyme-free bacitracin reagent.     

Food Intake and Circadian Rhythms of Activity of Red-Winged Blackbirds (Agelaius phoeniceus). A Time-Course Study on the Effects of Alpha-Chloralose and Secobarbital

Anesthetic drugs like alpha-chloralose and secobarbital are used to capture wild birds. The doses are usually chosen on the basis of low mortality rates and high capture success. However, little information is available on the time necessary for the birds to fully recover from the drugs' effects after they have regained consciousness. In this work, we used circadian rhythms of activities to study the long-term effects of a mixture of alpha-chloralose and secobarbital on adult male Red-winged Blackbirds. The birds were housed individually in sound-proof boxes for 30 days, during which they received alpha-chloralose and secobarbital mixed with corn, either on day 3 or on day 15. On the day of treatment, the birds experienced a significant decrease in activity level, including feeding activities, which lasted more than 22 days for the birds treated on day 3, compared to about 8 days for the birds treated on day 15. Moreover, typical circadian distribution of activity was lost and many birds became aperiodic. Two weeks were needed for the birds treated on day 3 to recover normal rhythms, compared to less than 9 days for those treated on day 15. These results indicate that circadian parameters can be used to detect after-effects of intoxication long after the birds have regained consciousness.     

Extramembraneous particles and structural variations of tubular myelin figures in rat lung surfactant

Tubular myelin figures of pulmonary surfactant were examined by electron microscopy after fixation in glutaraldehyde and postfixation in an osmium tetroxide-ferrocyanide mixture. Bilayered membranes were seen as parallel arrays or as lattices with spacings varying from about 36 to 50 nm. This method also produced good visualization of drumstick-like particles, 5 nm in diameter and about 15 nm in length. The particles were regularly spaced at intervals of 16 nm in rows along the rectangular angles of myelin membranes. Depending on the size of the tubules the particles contacted each other in the center of the tubules at low diameters (tubular diameter less than 40 nm) and formed a continuous filamentous central core, or they were separated from one another (tubular diameter greater than 40 nm). In the latter case the central core had a hollow appearance. Based on further findings employing tannic acid, lipid extraction with 2,2-dimethoxypropane, and a ruthenium red-osmium tetroxide technique for the demonstration of polyanionic proteins it is suggested that these particles are protein in nature and that they are involved in the formation and maintenance of the structure of tubular myelin. A new concept of the ultrastructure of tubular myelin figures is proposed.     

Fetal cells in mother rats contribute to the remodeling of liver and kidney after injury

Fetal microchimerism indicates a mixture of cells of maternal and fetal origin seen in maternal tissues during and after pregnancy. Controversy exists about whether persistent fetal microchimerism is related with some autoimmune disorders occurring during and after pregnancy. In the current experiment, an animal model in which EGFP positive cells were taken as fetal-origin cells was designed to detect the fetal microchimerism in various maternal organs. Ethanol drinking and gentamicin injection were adopted to induce liver and kidney injury simultaneously. EGFP positive cells were engrafted not only in the maternal circulation and bone marrow, but also in the liver and kidney as hepatocytes and tubular cells, respectively. These results indicate that fetal cells are engrafted to maternal hematopoietic system without apparent injury and they also contribute to the repairing process of maternal liver and kidney.