Enhanced Differentiation of Zaprionus Chromosomes by Prestaining Acid Hydrolysis

Two variations of orcein staining have been adapted to salivary gland chromosomes of Zaprionus. Method I: after dissection, glands are transferred to 1 N HCl at 60° C for 5 min, stained with 2.5% orcein in 60% acetic add for 15-20 min, and squashed in 60% acetic acid. Method II: after dissection, glands are transferred to 1 N HCl at 60° C for 5 min, transferred to a saturated solution of carmine in 45% acetic acid for 1 min, then to a mixture of 50 ml of 1% orcein in concentrated lactic acid and 50 ml of 30% acetic add for 5 min. They are squashed in the same mixture. The unproved differentiation of chromosomes from cytoplasm is attributed to the removal of cytoplasmic ribonucleic add by add hydrolysis.     

An Improved Pollen Grain Smear Method for Wheat

Anthers containing actively dividing pollen grains were treated 1 hour at 18-20° C. with 0.2% solution of colchicine, washed 1 hour in water, soaked in 0.002 M aqueous solution of 8-oxyquinoline at 10-14° C. for 1 hour, washed in water for 1 hour and then fixed in Carnoy's solution (alcohol, chloroform, acetic acid, 6:3:1) for 6 hours to overnight. They were washed successively in acetic-alcohol (1:1) 10-15 minutes, 70% alcohol 10-15 minutes and in water 30 minutes before hydrolysing them in bulk in 1 N HCl at 60° C. for 10-15 minutes. “Finally, they were stained in leuco-basic fuchsin for 15-30 minutes. Pollen grains were squeezed out of a stained anther in a small drop of egg albumen on a slide and the albumen smeared uniformly on the slide. The slide was dipped successively for a few seconds in glacial acetic acid and 45% acetic acid respectively. The smear was covered by a cover glass in a drop of aceto-carmine and pressed gently between folded filter papers. The cover glass was sealed with paraffin and stored overnight. To make the preparation permanent the paraffin was removed and the cover glass separated in a 1:1 mixture of acetic acid and n-butyl alcohol. The slide and the cover glass were then passed through n-butyl alcohol, 2 changes, and finally remounted in balsam.     

A Simple Histochemical Reaction for Aldehydes

A study has been made on the possibility of replacing leucofuchsin by colored basic fuchsin for the histochemical demonstration of aldehydes. Several tissues from mammals and various pertinent fixatives were used. Aldehydes were freed from carbohydrates by oxidation and from thymonucleic acid by hydrolysis.

It was found that the colored form and not necessarily the leucoform of basic fuchsin can be used histochemically in demonstrating aldehydes. The technic used is as follows: (1) Treat with 1.0-0.5% H₅IO₆ (or in 1% KIO₄ in M/1 H₂SO₄) for 5 to 10 min. and wash thoroughly. For thymonucleic acid hydrolize with N HCl 5 min. at room temperature, 10 min. at 60°C. and 5 min. at room temperature. (2) Stain for 2-3 min. with 0.05% basic fuchsin in 5% ethanol, 3% phenol. (3). Transfer immediately to 1 or 2 changes of 1% sodium bisulphite or potassium metabisulphite in 0.1-0.2 N H₂SO₄ for a total of 5 min. (4) Rinse with water and treat with M H₂SO₄ in 95% ethanol for 3-5 min. 6. Wash thoroughly in water and dehydrate, clear, and mount. For glycogen and mucin the following counterstaining solution is recommended: orange G, 0.25 g.; light green SFY, 0.10 g.; phosphotungstic acid 0.50 g.; 50% ethanol, 100 ml.; glacial acetic acid, 0.25 ml.     

Azure a-Schiff, Alcian Blue, HIO4-Schiff, Naphthol Yellow S—A Sequential Staining Method for Paraffin Sections

Paraffin sections from tissue fixed 4-12 hr in 10% formalin containing 0.5% cetyl pyridinium chloride, and washed 2 hr, were stained as follows: (1) Hydrolyze in 5 N HCl at room temperature for 8.5-9 min, or use standard Feulgen hydrolysis at 60 C. (2) Stain in azure A-Schiff, 0.5% in bisulfite bleach (1 N HCl, 5; 10% Na₂S₂O₅, 5; and distilled water 90—parts by volume) for 10 min. (3) Place in bisulfite bleach 2 changes, 2 min each; wash in water, 1-2 min. (4) Stain in Alcian blue (0.1% in 0.01 2V HCl, pH 2.0) for 10 min. (5) Place in 0.01 N HCl for 2-3 min; wash in water for 1-2 min. (6) Oxidize in 0.5% HIO₄ for 5 min; wash in water, 1-2 min. (7) Stain in Schiff's leucofuchsiu, 10 min. (8) Treat with bisulfite bleach as in step 3; wash in running water, 10 min. (9) Stain in naphthol yellow S (0.01% in 1% acetic acid) for 1-2 min. (10) Place in 1% acetic acid for 2 min, dehydrate in tertiary butanol, clear and cover. Result: DNA is deep blue; acidic mucins are light blue; neutral polysaccharides, red to magenta; and proteins, yellow. Proper timing of the hydrolysis for the Feulgen reaction is the most critical step. Overhydrolysis results in green nuclei (staining by naphthol yellow S) whereas purplish nuclei are the results of insufficient hydrolysis.     

Biosynthesis and properties of an extracellular metalloprotease from the Antarctic marine bacterium Sphingomonas paucimobilis

An extracellular protease from the marine bacterium Sphingomonas paucimobilis, strain 116, isolated from the stomach of Antarctic krill, Euphausia superba Dana, was purified and characterized. The excretion of protease was maximal at temperatures from 5 to 10°C, i.e. below the temperature optimum for the strain growth (15°C). The highly purified enzyme was a metalloprotease [sensivity to ethylenediaminetetraacetic acid (EDTA)] and showed maximal activity against proteins at 20–30°C and pH 6.5–7.0, and towards N-benzoyl-tyrosine ethyl ester (BzTyrOEt) at pH 8.0. At 0°C the enzyme retained as much as 47% of maximal activity in hydrolysis of urea denatured haemoglobin (Hb) (at pH 7.0), and at −5 and −10°C, 37 and 30%, respectively. The metalloprotease was stable up to 30°C for 15 min and up to 20°C for 60 min. These results indicate that the proteinase from S. paucimobilis 116 is a cold-adapted enzyme.     

Staining the Nucleus in Yeasts

Yeast cells kept young by repeated subculturing were centrifuged, washed twice in distilled water and smeared on slides coated with a little egg albumen. The cells were treated with 0.002 M 8-hydroxyquinoline for 1 hr, fixed first in OsO, vapour for 30 sec and then in chloroform for 30 sec. The slides were passed through descending grades of alcohol, washed in distilled water, then immersed in 0.17 M NaCl solution for 2 hr. at 57°C. They were again washed in distilled water and later hydrolysed in 1 N HCl at 60°C for 5-7 min. This was followed by washing in distilled water and in buffer. The slides were then kept for 3 hr in Giemsa stain comprising 96 ml of phosphate buffer of pH 7.0 and 4 ml of the stain. After dehydration, mounting was done in balsam. Nuclei were brightly stained and well differentiated; centrosomes were clear, and the process of nuclear division and movement to daughter cells could be studied. Pretreatment with 8-hydroxyquinoline increased the viscosity of the cytoplasm, while NaCl treatment and acid hydrolysis led to the complete removal of ribonucleic acid and basophilic material. A selective staining of chromatin was thus achieved. Structures resembling chromosomes could be seen when fixed and stained cells were squashed, soon after the removal of the slides from the stain, under a cover glass by applying uniform pressure with a rubber stopper. Fixation in osmic acid vapor and chloroform followed by acid hydrolysis and staining in leucobasic fuchsin also helps to obtain bright staining of the nucleus; however, the preparations are inferior to those obtained after 8-hydroxyquinoline, NaCl treatment and Giemsa staining.     

Squash Method for Meiosis in Ovules

The ordinary Feulgen or acetic-lacmoid squash tech-nic following fixation in freshly made Carnoy's fluid (alcohol, 6: chloroform, 3: glacial acetic acid, 1), provides an easy and reliable method of studying meiosis in ovules. After fixation for 1 day, the material was hardened in 95% ethyl alcohol for 1-2 days and taken to water by gradual hydration. For staining by the Feulgen method, the material was hydrolyzed 8-10 minutes in 1 N HO at 58-60°C., followed by staining in decolorized leuco basic fuchsin for 2 hours. The staining was intensified by transferring the material to water. After 15-20 minutes the water was replaced by 45% acetic acid. For staining by acetic-lacmoid, the ovules after fixation, hardening and hydration were transferred to standard acetic-lacmoid stain to which was added 1 drop of 1 N HCl to every 10 drops of stain. Gentle heat was applied till the stain started to give fumes. After allowing 20 minutes at room temperature the material was transferred to fresh acetic-lacmoid. Some 6-12 ovules were mounted either in a drop of 45% acetic acid or acetic-lacmoid, depending upon the Feulgen or acetic-lacmoid staining respectively. Gentle and repeated tapping over the cover glass by a blunt needle loosened the cells of integument and nucellus and finally left the megaspore mother cells undergoing meiosis, fully exposed to view. The process was carried out under constant observation using the low power of the microscope. The desired amount of flattening was brought about by light pressure over the cover glass and gentle heating. The preparations were made permanent by dehydrating in ethyl alcohol and mounting in Euparal.     

Chromosome Spreading Induced by Vegetable Oils

Seeds soaked in the oil extracted from castor beans (Ricinus communis) for 2 hr were germinated in petri dishes on moist filter papers. Root tips were fixed in acetic alcohol (1:3) at 10-14°C, for 24 hr, washed successively with 70% alcohol (15 min) and water (10 min), hydrolysed in 1 N HCl at 60°C for 15 min and stained in leucobasic fuchsin for 30 min. The stained tip was squashed under a cover glass in a drop of acetocarmine and sealed with paraffin wax. The slides were made permanent by separating the cover glass in a mixture of acetic acid and n-butyl alcohol (1:1), passing through 2 changes of n-butyl alcohol and mounting in balsam. Such a method leads to contraction and spreading of chromosomes, without affecting either the clarity of the constriction regions or the anaphase separation of chromosomes.     

Dihydroxamic acid complexes of iron (III): ligand pKa and coordinated water hydrolysis constants

A series of dihydroxamic acid ligands of the formula [RN(OH)C(O)]₂(CH₂)_n, (n = 2, 4, 6, 7, 8; R = CH₃, H) has been studied in 2.0 M aqueous sodium perchlorate at 25.0 °C. These ligands may be considered as synthetic analogs to the siderophore rhodotorulic acid. Acid dissociation constants (pK_a) have been determined for the ligands and for N-methylacetohydroxamic acid (NMHA). The pK_a1 and pK_a2 values are: n = 2, R = CH₃ (8.72, 9.37); N = 4, R = CH₃ (8.79, 9.37); N = 6, R = CH₃; N = 7, R = CH₃ (8.95, 9.47); N = 8, R = CH₃ (8.93, 9.45); N = 8, R = H (9.05, 9.58). Equilibrium constants for the hydrolysis of coordinated water (log K) have been estimated for the 1:1 feeric complexes of the ligands n = 2, 4, 8; R = CH₃. The N = 8 ligand forms a monomeric complex with Fe(III) while the n = 2 and 4 ligands form dimeric complexes. For hydrolysis of the n = 8 monomeric complex, log K₁ = −6.36 and log K₂ = −9.84. Analysis of the spectrophotometric data for the dimeric complexes indicates deprotonation of all four coordinated waters. The successive hydrolysis constants, log K_1–4, for the dimeric complexes are as follows: n = 2 (−6.37, −5.77, −10.73, −11.8); n = 4 (−5.54, −5.07, −11.57, −10.17). The log K₂ values for the dimers are unexpectedly high, higher in fact than log K₁, inconsistent with the formation of simple ternary hydroxo complexes. A scheme is proposed for the hydrolysis of the ferric dihydroxamate dimers, which includes the possible formation of μ-hydroxo and μ-oxo bridges.     

Metabolic characteristics of sea cucumber Apostichopus japonicus (Selenka) during aestivation

Metabolic characteristics of the sea cucumber Apostichopus japonicus (Selenka) during aestivation were studied in the laboratory. The effects of water temperature on oxygen consumption rate (OCR) and ammonia-N excretion rate (AER) in A. japonicus were determined by the Winkler and Hypobromite methods, respectively. Mature (large, 148.5 ± 15.4 g, medium 69.3 ± 6.9 g) and immature (small, 21.2 ± 4.7 g) individuals aestivated at water temperatures of 20 and 25 °C, respectively. The metabolic characteristics of mature individuals were different from immature individuals during this period. The OCR of mature sea cucumbers peaked at 20 °C, and then dropped significantly at higher temperatures, whereas the OCR of the immature animals continued to increase slightly, even beyond the aestivation temperature. The AER of mature individuals peaked at 20 °C, while that of the immature animals peaked at 25 °C. The relationships between dry weight (DW) and absolute oxygen consumption (R) and absolute ammonia-N excretion (N) could be described by the regression equation R or N = aW^b. With the exception of 15 °C, the O / N ratios (calculated in atomic equivalents) of large size sea cucumbers was close to 20 across the temperatures used in this study, indicating that their energy source was a combination of lipid and protein. On the other hand, apart from small individuals maintained at 10 °C, the O / N ratios of the medium and small sea cucumbers were close to 10, indicating that protein was their major energy source. The O / N ratios in all size groups remained unchanged after aestivation was initiated.     

Biotransformation of β-amino nitriles: the role of the N-protecting group

N-Tolylsulfonyl- and N-butyloxycarbonyl-protected β-amino nitriles were prepared to study the effect of the N-protecting group on the biotransformation of the β-amino nitriles to the corresponding β-amino amides and acids. The bioconversions were carried out by using whole cells of Rhodococcus sp. R312 and Rhodococcus erythropolis NCIMB 11540. The bioconversion products of five-membered carbocyclic nitriles were mainly the respective acids whereas the carbocyclic six-membered nitriles were accumulated at the stage of the amide. Benefits of the enzymatic compared with the chemical hydrolysis of β-amino nitriles are the mild reaction conditions for the transformation of the nitrile group in the presence of acid or base labile N-protecting groups. In the present work we concentrated on this chemoselectivity of the biotransformation rather than its potential enantioselectivity, which will be subject of future investigations. Thus, some new compounds were prepared: (±)-(2-cyano-cyclohexyl) carbamic acid tert-butyl ester (4a), (±)-(2-carbamoyl-cyclopentyl) carbamic acid tert-butyl ester (3b) and (±)-(2-carbamoyl-cyclohexyl) carbamic acid tert-butyl ester (4b).     

Modified chitosans carrying sulfonic acid groups

The sulfonic acid function was introduced into chitosan by reacting it with 5-formyl-2-furansulfonic acid, sodium salt, under the mild conditions of the Schiff reaction, thus avoiding polymer degradation and O-substitution. The reaction of chitosan (degree of deacetylation 0·58) with 5-formyl-2-furansulfonic acid, sodium salt produced a viscous solution that, upon hydrogenation, yielded N-sulfofurfuryl chitosan sodium salt. Infrared spectrometry, alkalimetry and elemental analysis provided evidence that the degree of substitution was 0·26. Circular dichroism measurements on solutions showed multiple Cotton bands in the pH interval 7·1–8·3, while at lower and higher pH values just one negative band was observed, thus providing indication of the polyampholyte nature of N-sulfofurfuryl chitosan. The ¹³C-NMR and FTIR spectra showed typical signals of furane carbons. Metal ion solutions at concentrations in the range 0·1–5·0 m , pH 6, promoted precipitation of metal ion complexes of N-sulfofurfuryl chitosan, with most effective removal from the solutions for Cu(II), Pb(II) and Ni(II). Sulfoethyl N-carboxymethyl chitosan was also synthesized from 2-chloroethanesulfonic acid in organic media: the sulfur content was similar (3·7%) in both polymers.     

Histochemistry of Plasmalogen

Unfixed frozen sections of kidney, heart and brain from Wistar rats were used for the following tests: 1. relative reactivity of plasmalogen in different tissues to fuchsin-sulfurous acid (FSA); 2. effect of various mercury compounds in producing the plasmal reaction, and use of dithizone to verify the attachment of mercury to tissue plasmalogen; 3. effect of pH as shown by graded concentrations of HCl and by phosphate and acetate buffers; 4. effect of various cations; 5. solubility of plasmalogen in methanol, ethanol, isopropanol, n-butanol, t-butanol, n-pentanol and ethylene glycol; 6. effect of blocking reactions: acetal formation, bromination and iodination. The results showed that the amount of plasmalogen in brain is very much greater than that in heart muscle or in kidney and that the brain plasmalogen appears to be more reactive. All the highly ionized mercury compounds, HgCl₂, Hg(NO₃)₂, Hg(CH₃COO)₂, and HgNO₃ gave identical, immediate reactions. The moderately ionized K₂HgI₄ gave a less rapid reaction. Hg compounds which are unable to deliver any appreciable amount of mercuric ion into solution (nonionized or insoluble) did not react. Dithizone, which forms a deep red color in combination with Hg-containing compounds, gave a positive reaction at the same sites that gave the plasmal reaction. This indicates that the Hg combines with tissue plasmalogen. The hydrolysis of plasmalogen as a function of pH and time showed that 6 N HCl gave as rapid a reaction as HgCl₂. As the pH was increased the rate of plasmal formation decreased. At pH 4.0 no plasmal was evident in 2 hr. AuCl₃ and PdCl₂ gave reactions identical with that of HgCl₂. No other cation tested gave a plasmal reaction. Plasmalogen was rapidly dissolved by the short chain monohydric alcohols but not by ethylene glycol. Plasmalogen did not dissolve appreciably in 60% or lower grades of ethanol. Bromination or iodination abolished the plasmal reaction, but iodination required a longer time to do so. A 1.2 N HCl solution in ethylene glycol inhibited the HgCl₂-FSA reaction by the formation of an acetal which is relatively insensitive to HgCl₂, but it did not inhibit the FSA reaction when hydrolysis was by 6 N HCl instead of HgCl₂. The vinyl ether structure proposed by Rapport and associates (1957) for plasmalogen is supported by the specific reaction with HgCl₂, the hydrolysis with acid, the addition of iodine, of bromine, and the formation of an acetal.     

A Paradichlorobenzene, Saponin, Iron Mordant Sequence in the Staining of Meiotic Chromosomes of Ipomea

For the meiotic study of Ipomea spp., flower buds were stripped of the calyx and corolla and soaked in saturated aqueous paradichlorobenzene at about 28° C for 3 hr, transferred to acetic-alcohol (1:3) for 6 hr, then into 1% saponin solution and left overnight. They were mordanted in 1:3 acetic-alcohol saturated with ferric oxide for 24 hr and stained in a mixture of 1% aceto-carmine and 2% aceto-orcein with 1 N HCl in the proportion of 9:9:1. The preparations were mounted in 1% aceto-carmine for temporary use and made permanent by dehydration through the n-butanol schedule. The pollen mother cells had clear cytoplasm with deeply stained chromosomes.     

Acetylation of wheat straw hemicellulose B in a new non-aqueous swelling system

The acetylation of wheat straw hemicellulose B was carried out in a homogeneous N,N-dimethylformamide and lithium chloride system with acetic anhydride using 4-dimethylaminopyridine as a catalyst. The degree of substitution of hemicellulose B acetates ranged between 0.59 and 1.25 as a function of experimental conditions. Under an optimum condition (85°C, 60 h), approximately 75% of the free hydroxyl groups in native hemicellulose B were acetylated. The molecular weight measurements (31,890–34,090 g mol⁻¹) showed a controllable degradation of hemicellulose B chains during the reactions at temperature 60–85°C and duration of 2–60 h. It was found that the thermal stability of the products was increased by chemical modification.     

Dinuclear silver(I) complexes of 24-membered bibracchial tetraimine Schiff base macrocycles derived from 2,5-diformylthiophene

The cyclocondensation of 2,5-diformylthiophene and the amines N,N-bis-(2-aminoethyl)-2-phenylethylamine, N,N-bis-(2aminoethyl)-t-butyl-amine and N,N-bis-(2-aminoethyl)-t-butyl-amine in the presence of silver(I) salts yields homodinuclear bibracchial tetraimine Schiff base macrocyclic complexes. The structures of two such complexes are also reported. The complex Ag₂L⁴(NO₃)(PF₆) (2) crystallises in the triclinic space group , No. 2) and has unit-cell dimensions a = 12.834(6), B = 13.183(6), C = 14.588(7) Å, = 64.86(4), β = 79.77(4), γ = 69.44(3)° with Z = 2; there is a monodentate and singly bridging nitrate anion present and the Ag---Ag separation is 4.161 Å. The complex Ag₂L⁴(CH₃CN)₂(BF₄)₂·CH₃CN (9) crystallises in the triclinic space group , No. 2) and has unit-cell dimensions a = 9.297(4), B = 12.985(3), C = 21.770(5) Å, = 91.570(10), β = 92.33(3), γ = 97.92(3) ° with Z = 2; there is a strongly bonded acetonitrile molecule coordinated to each silver atom and the Ag---Ag separation is 4.920 Å.     

Einfluß der Fixierung und der Säurekonzentration auf die Feulgen-Hydrolyse bei 28° C

Zusammenfassung Es wurde eine für Routinezwecke geeignete Feulgenreaktion für die cytophotometrische DNS-Bestimmung ausgearbeitet, die gegen geringe Schwankungen der Temperatur, der Säurekonzentration und der Zeitdauer der HCl-Hydrolyse weniger empfindlich ist als die 60°-Standardhydrolyse. Dazu wurden die Feulgen-Hydrolysekurven von alkohol-, formalin- und methanol-formalin-eisessigfixierten Hühnererythrocyten nach Behandlung mit 5 N, 4 N und 2 N HCl bei 28° C, bzw. mit 1 N HCl bei 60° C geprüft und miteinander verglichen. Als besonders brauchbar erwies sich die Fixierung mit 70%igem Isopropylalkohol (20 min Hydrolyse in 5 N HCl oder 45 min in 4 N HCl) und die MethanolFormalin-Eisessig-Fixierung (105 min Hydrolyse in 4 N HCl). Reine Formalinfixierung erwies sich als ungeeignet, da eine starke Kernschrumpfung mit Extinktionen größer als 0,75 beobachtet wurden.An alkoholfixierten Leberzellausstrichen wurde ein gleichartiger Verlauf der Hydrolysekurven von di-, tetra- und oktoploiden Leberzellkernen festgestellt. Die relativen Farbstoffmengen (AE-Werte) dieser Kerne verhielten sich bei allen geprüften Hydrolysezeiten wie 124.

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Influence of fixation and add concentration on the Feulgen hydrolysis at 28° C

Summary A routine Feulgen procedure for quantitative cytophotometric absorption measurements of DNA at the integrating microdensitometer should be established, which is less alterable by minor deviations in temperature, acid concentration and in duration of hydrolysis than is the 60° C standard treatment. Feulgen hydrolysis curves of alcohol-, formalin- and methanol-formalin-glacialacidicacid-fixed fowl erythrocytes have been examined after hydrolysis in 5 N, 4 N and 2 N HCl at 28° C, as well as in 1 N HCl at 60° C. Fixation in 70% isopropylalcohol and hydrolysis in 5 N HCl for 20 minutes or in 4 N HCl for 45 minutes proved to be particularly useful. Fixation in a mixture of 85% methanol, 10% formalin and 5% glacialacidicacid gave good results, too, but hydrolysis time had to be chosen considerably longer for maximum staining (105 minutes in 4 N HCl). Formalin fixation proved not to be suitable because of a considerable shrinkage of the nuclei resulting in extinctions well above 0.75.Identical hydrolysis curves have been obtained for di-, tetra- and octoploid liver cell nuclei from alcohol-fixed liver smears. The relative dye contents (AE-values) of these nuclei were in the ratio 124 at all hydrolysis times examined.

     

Effect of nifedipine on core cooling in rats during tail cold water immersion

Male rats (450 g, n=11/group) were heated at an ambient temperature of 42°C until a rectal temperature of 42.8°C was attained. Rats, then received either saline (30°C)+tail ice water immersion (F+I) or saline (30°C)+tail ice water immersion+Nifedipine, a peripheral vasodilator, (F+I+N) to determine cooling rate effectiveness and survivability. The time to reach a rectal temperature of 42.8°C averaged 172 min in both groups resulting in similar heating rates (0.029°C/min). The cooling rates in group F+I and F+I+N were not significantly different from each other. We conclude that since Nifedipine did not improve cooling rates when combined with fluid+tail ice water immersion, its use as a cooling adjunct does not seem warranted.     

Induction of lacI mutations in Big Blue Rat-2 cells treated with 1-(2-hydroxyethyl)-1-nitrosourea: a model system for the analysis of mutagenic potential of the hydroxyethyl adducts produced by 1,3-bis (2-chloroethyl)-1-nitrosourea.

We have investigated the genotoxic effects of 1-(2-hydroxyethyl)-1-nitrosourea (HENU). We have chosen this agent because of its demonstrated ability to produce N7-(2-hydroxyethyl) guanine (N7-HOEtG) and O⁶-(2-hydroxyethyl) 2′-deoxyguanosine (O⁶-HOEtdG); two of the DNA alkylation products produced by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). For these studies, we have used the Big Blue Rat-2 cell line that contains a lambda/lacI shuttle vector. Treatment of these cells with HENU produced a dose dependent increase in the levels of N7-HOEtG and O⁶-HOEtdG as quantified by HPLC with electrochemical detection. Treatment of Big Blue Rat-2 cells with either 0, 1 or 5 mM HENU resulted in mutation frequencies of 7.2±2.2×10⁻⁵, 45.2±2.9×10⁻⁵ and 120.3±24.4×10⁻⁵, respectively. Comparison of the mutation frequencies demonstrates that 1 and 5 mM HENU treatments have increased the mutation frequency by 6- and 16-fold, respectively. This increase in mutation frequency was statistically significant (P<0.001). Sequence analysis of HENU-induced mutations have revealed primarily G:C→A:T transitions (52%) and a significant number of A:T→T:A transversions (16%). We propose that the observed G:C→A:T transitions are produced by the DNA alkylation product O⁶-HOEtdG. These results suggest that the formation of O⁶-HOEtdG by BCNU treatment contributes to its observed mutagenic properties.     

Staining Plant and Animal Chromosomes by the Feulgen-Acetocarmine Sequence

A technic, some fundamentals of which were first worked out on brome grass, has been considerably extended and adapted to the somatic chromosomes of salmon. Fresh salmon eggs were quickly pierced in 45% acetic acid and fixed therein for 4 minutes. The eggs were then placed in N HCl at 60°C. for 8 minutes and thereafter transferred to Feulgen stain for 30 to 45 minutes. Subsequently, each stained embryo was dissected out and divided in two, each half being placed on a slide in a drop of acetocarmine stain. The pieces were well macerated and, after covering with a cover slip, maceration was completed by tapping. Heavy pressure was gradually applied to the cover slip in order to flatten the chromosome complements. A square screw-type laboratory hose clamp was then used to maintain this pressure while a liquid gelatin seal was applied around the edges. The slide, with the clamp on, was placed in the refrigerator overnight. Before the slide was scanned, the clamp was removed permanently. After each scanning period the slide was returned to the refrigerator. Photomicrographs of well-spread chromosomes in one optical plane were enlarged and tracings made from them. These tracings together with the photomicrographs were used for chromosome analysis.