Effects of preparation and fixation on three quantitative fluorescent cytochemical procedures

A series of experiments was undertaken in which cells dissociated from the abdominal lymph nodes of mice were lightly centrifuged into slides and fixed either wet or after drying in 70% ethanol, 1% glutaraldehyde, 1% formaldehyde, or neutral formalin. Three fluorescent cytochemical methods were evaluated: staining of DNA with mithramycin; fluorochroming of basic groups of proteins with brilliant sulfaflavine (BSF); and staining of sulfhydryl and disulfide groups with N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM). In the case of mithramycin, the best results were obtained after fixation in 70% ethanol without drying. Staining of dried preparations fixed in 1% glutaraldehyde also yielded reasonably consistent results, although the fluorescence was lower, and the variability higher, than in the group fixed without drying in 70% ethanol. The use of fixatives containing formaldehyde resulted in fluorescence values of only about one-third those of the other two groups, and the variability of the data was higher. In material stained with BSF, satisfactory results were obtained in preparations fixed without drying in neutral formalin containing mersalyl acid. Other fixatives could be used, but the resulting coefficients of variation were higher than those of formalin-fixed material. Sulfhydryl to disulfide ratios approaching those expected from biochemical evidence were obtained in DACM-stained material only after fixation without drying in neutral formalin containing mersalyl acid. Inverted sulfhydryl-disulfide ratios were observed in material fixed without drying in 70% ethanol; and in dried material fixed in 1% formaldehyde, neutral formalin, or 1% glutaraldehyde.     

Characteristics of alcohol/polyol dehydrogenases. The zinc-containing long-chain alcohol dehydrogenases

Sixteen characterized alcohol dehydrogenases and one sorbitol dehydrogenase have been aligned. The proteins represent two formally different enzyme activities (EC 1.1.1.1 and EC 1.1.1.14), three different types of molecule (dimeric alcohol dehydrogenase, tetrameric alcohol dehydrogenase, tetrameric sorbitol dehydrogenase), metalloproteins with different zinc contents (1 or 2 atoms per subunit), and polypeptide chains from different kingdoms and orders (mammals, higher plants, fungus, yeasts). Present comparisons utilizing all 17 forms reveal extensive variations in alcohol dehydrogenase, but with evolutionary changes that are of the same order in different branches and at different times. They emphasize the general importance of particular residues, suggesting related overall functional constraints in the molecules. The comparisons also define a few coincidences between intron positions in the genes and gap positions in the gene products. Only 22 residues are strictly conserved; half of these are Gly, and most of the remaining ones are Pro or acidic residues. No basic residue, no straight-chain hydrophobic residues, no aromatic residues, and essentially no branched-chain or polar neutral residues are invariable. Tentative consensus sequences were calculated, defining 13 additional residues likely to be typical of but not invariant among the alcohol dehydrogenases. These show a predominance of Val, charged residues, and Gly. Combined, the comparisons, which are particularly relevant to the data base for protein engineering, illustrate the requirements for functionally important binding interactions, and the extent of space restrictions in proteins with related overall conformations and functions.     

Microfluorometric estimates of proteins associated with murine hepatocyte and thymocyte nuclei, residual structures, and nuclear matrix derivatives

Isolated diploid hepatocyte and thymocyte nuclei and their derivatives ("residual structures" and nuclear matrices, as defined by Kaufmann et al. 1981) were evaluated by microfluorometry following reaction with the following fluorochromes: brilliant sulfaflavine (BSF) used at pH 2.8 for the demonstration of total protein; acridine orange (AO) used at pH 9.0 to reveal acidic groups of proteins; and 3-(4-maleimidylphenyl)-7-diethylamino-4-methylcoumarin (CPM) used under conditions required to demonstrate the sum of sulfhydryl (SH) and disulfide (SS) groups of proteins. The results suggested that the proteins reacting with AO and CPM differed from each other and from those revealed by fluorochroming with BSF. In every comparison, hepatocyte nuclei and their derivatives were more fluorescent than the respective populations of thymocyte nuclei and their derivatives. In material fluorochromed with BSF and AO, nuclear matrices were less fluorescent than residual structures, which, in turn, were less fluorescent than intact nuclei. In contrast, nuclear matrices fluorochromed with CPM were less fluorescent than intact nuclei but more fluorescent (paradoxically) than residual structures. The ratios of the total fluorescence values of hepatocyte and thymocyte nuclei fluorochromed with BSF changed significantly during extractions required to produce residual structures and nuclear matrices, while comparable ratios in material fluorochromed with AO or CPM did not change significantly. Comparisons of the ratios of the fluorescence values of intact nuclei and their derivatives in a variety of combinations yielded complex and variable results.(ABSTRACT TRUNCATED AT 250 WORDS)     

Yeasts in molecular biology. Spheroplast preparation withCanadida utilis,Schizosaccharomyces pombe andSaccharomyces cerevisiae

Efficient preparation of spheroplasts fromCandida utilis, Saccharomyces cerevisiae, andSchizosaccharomyces pombe, using a purified mixture of enzymes fromTrichoderma harzianum, is described. Limitations of other methods, and differences between yeasts are demonstrated.     

Kidney transplantation in Canada, 1981-84.

The Canadian Renal Failure Register was established in 1980. Data have been collected annually for all Canadian patients in whom irreversible kidney failure developed and who required dialysis or transplantation. The authors present actuarial patient and graft survival rates for 1981-84. In 1984, patients with a functioning renal graft accounted for 43.9% of the patients with end-stage renal disease. The number of transplants performed increased from 482 in 1981 to 662 in 1984; however, 1,022 patients undergoing dialysis (25.2%) were on an active waiting list for a transplant at the end of 1984. Greater effort is needed to increase the transplantation rate.     

Effects of anhydrous benzoylation on staining with a variety of protein end-group methods

Summary The effects of anhydrous benzoylation were examined in connection with the following protein end-group methods: diazosulfanilic acid — Azure A, the Sakaguchi reaction and a fluorescent variant, the phenanthrequinone fluorescent method for arginine, the Millon reaction, the Morel-Sisley reaction, the mercury orange method, the alloxan and ninhydrinShiff methods, the Dansyl-chloride fluorescent method for lysine, the dimethylaminobenz-aldehydenitrite (DMAB) method for tryptophan, and the acid fluorochrome brilliant sulfoflavin used at pH 2.8 as a stain for basic groups. With all of these methods except the DMAB method for tryptophan, selective blockage of cytoplasmic proteins and staining of nuclei was obtained. With the DMAB reaction, the results were reversed: cytoplasmic staining exceeded nuclear staining after benzoylation.     

Some metabolites of 1-bromobutane in the rabbit and the rat

1. Rabbits and rats dosed with 1-bromobutane excrete in urine, in addition to butylmercapturic acid, (2-hydroxybutyl)mercapturic acid, (3-hydroxybutyl)mercapturic acid and 3-(butylthio)lactic acid. 2. Although both species excrete both the hydroxybutylmercapturic acids, only traces of the 2-isomer are excreted by the rabbit. The 3-isomer has been isolated from rabbit urine as the dicyclohexylammonium salt. 3. 3-(Butylthio)lactic acid is formed more readily in the rabbit; only traces are excreted by the rat. 4. Traces of the sulphoxide of butylmercapturic acid have been found in rat urine but not in rabbit urine. 5. In the rabbit about 14% and in the rat about 22% of the dose of 1-bromobutane is excreted in the form of the hydroxymercapturic acids. 6. Slices of rat liver incubated with S-butylcysteine or butylmercapturic acid form both (2-hydroxybutyl)mercapturic acid and (3-hydroxybutyl)mercapturic acid, but only the 3-hydroxy acid is formed by slices of rabbit liver. 7. S-Butylglutathione, S-butylcysteinylglycine and S-butylcysteine are excreted in bile by rats dosed with 1-bromobutane. 8. Rabbits and rats dosed with 1,2-epoxybutane excrete (2-hydroxybutyl)mercapturic acid to the extent of about 4% and 11% of the dose respectively. 9. The following have been synthesized: N-acetyl-S-(2-hydroxybutyl)-l-cysteine [(2-hydroxybutyl)mercapturic acid] and N-acetyl-S-(3-hydroxybutyl)-l-cysteine [(3-hydroxybutyl)mercapturic acid] isolated as dicyclohexylammonium salts, N-toluene-p-sulphonyl-S-(2-hydroxybutyl)-l-cysteine, S-butylglutathione and N-acetyl-S-butylcysteinyl-glycine ethyl ester.     

AF1, a sequenced bioactive neuropeptide isolated from the nematode Ascaris suum

An FMRFamide-like neuropeptide, named AF1, was isolated from head extracts of the nematode Ascaris suum using five steps of HPLC. AF1 is a heptapeptide with the amino acid sequence Lys-Asn-Glu-Phe-Ile-Arg-Phe-NH2. Synthetic AF1 (10(-9) to 10(-7) M) rapidly and reversibly abolished slow membrane potential oscillations of identified ventral and dorsal inhibitory motoneurons and selectively reduced their input resistances. Synaptic transmission was not blocked. In intact Ascaris, AF1 inhibited locomotory movements. This study indicates a potential physiological role for an endogenous neuropeptide in nematodes.