Nonradioactive in situ nick translation combined with counterstaining: Characterization of C-band and silver positive regions in mouse testicular cells

The DNase I sensitivity of three different chromatin regions in mouse testicular cells was analysed by in situ nick translation with biotin-dUTP combined with various counterstaining techniques. The regions were: (i) the constitutive centromeric heterochromatin, (ii) an interstitial C-band positive insertion on chromosome 1, Is(HSR1;C5)1Lub, and (iii) the chromatin containing rDNA (designated nucleolar chromatin herein). Incorporated biotin was detected either by the horseradish peroxidase reaction with diaminobenzidine (DAB) or the alkaline phosphatase reaction with fast red. The latter resulted in a water insoluble red precipitate, which was easily removable by any organic solution thus allowing the application of various counterstaining protocols. DNase I sensitivity of the three chromatin regions was screened in different cell types of the mouse testis. The interstitial Is(HSR) region was highly DNase I sensitive when it was recognizable by strong mithramycin fluorescence. The centromeric heterochromatin was DNase I resistant when it was compacted into microscopically visible chromosomal structures (mitosis, pachytene, metaphase I and II). In interphase nuclei from Sertoli cells and spermatogonia it became highly DNase I sensitive. In round spermatids it displayed medium DNase I sensitivity. Nucleolar chromatin was not labelled by in situ nick translation when silver staining demonstrated strong protein production. Sperm cells were highly DNase I sensitive from stages 11 to 15, but resistant as mature spermatozoa.     

Actin organization and fibrin-clot retractile activity of cultured mouse fibroblasts

Summary It is known that human and animal fibroblasts are able to induce the retraction of a fibrin clot. In the present study the correlation between (i) fibrinclot retractile (FCR) activity, (ii) the number of actin stress-lines in mouse fibroblasts during growth in culture, and (iii) the sensitivity of actin stress-lines to a powerful actin-depolymerizing factor (ADF), present in plasma and serum of humans and laboratory animals was investigated. Fibroblasts at early passages (2–4) were tested for these parameters at various intervals after seeding (24, 96, and 168 h). The number of actin stress-lines was progressively higher, while the sensitivity to ADF action was progressively lower in cells cultured from 24 to 168 h; the FCR capacity was significantly decreased at 168 h. These data suggest that cells containing weakly polymerized and/or stabilized actin are more active than those containing highly polymerized and/or stabilized actin in triggering fibroblast contraction.     

Phylogenetic relationships among various helical bacteria

Helical bacteria from the generaSpirillum, Oceanospirillum, Aquaspirillum, andAzospirillum—as well asSerpens flexibilis—were characterized by oligonucleotide cataloging of 16S rRNA in order to establish their phylogenetic relationships to one another and to Gramnegative bacteria in general. The various genera of helical bacteria are not specifically related to one another (to the exclusion of nonhelical bacteria) and, where tested, the individual genera as presently constituted are not phylogenetically coherent (with the possible exception ofOceanospirillum, which may form a deep grouping).     

Improved high-performance liquid chromatography of the new antineoplastic agents bisantrene and mitoxantrone

Bisantrene and mitoxantrone are two new anthracene derivatives which have shown significant antitumor activity against a wide variety of animal tumors and in human phase I and II clinical trials. We have developed a rapid, simple and sensitive sample cleanup procedure and high-performance liquid chromatographic (HPLC) assay for both drugs. This method uses a commercially available mini-cartridge with C₁₈ reversed-phase packing to isolate the drugs from the biological matrix prior to HPLC. For both drugs the average recovery of the assay was 98 ± 6% with a coefficient of variation (C.V.) of less than 7%. Using this new method our assay sensitivity has improved to less than 10 ng/ml for bisantrene and 1 ng/ml for mitoxantrone, allowing us to document a prologned terminal phase plasma half-life for both bisantrene and mitoxantrone. Equilibrium dialysis studies showed that both drugs are highly protein bound. Mitoxantrone appears less stable in human plasma than bisantrene. Recoveries from plasma after a 24-h incubation at 25 and 37°C were 40 and 20% for mitoxantrone and 90 and 85% for bisantrene, respectively. Addition of ascorbic acid prior to incubation of mitoxantrone in human plasma at 37°C resulted in less than a 10% decrease in the latter's concentration over a 24-h period. To maintain sample integrity, all plasma samples should be fortified with ascorbic acid and kept frozen prior to analyses.     

The intracellular distribution of adenylate kinase in the leaves of spinach,wheat and barley

Of the total adenylate-kinase activity in 10-d-old barley and wheat leaves, 40–50% is localised in the chloroplasts, while in mature spinach leaves 50–70% of the enzyme is chloroplastic. The extra-chloroplastic adenylate-kinase activity is associated with the mitochondria, very little, if any, is freely soluble in the cytoplasm. The adenylate pool of the cytoplasm could have access to adenylate-kinase activity in the intermitochondrial space because of the free permeation of adenylates across the outer mitochondrial membrane. Thus the adenylate pool of the cytoplasm could be subject to adenylate-kinase equilibrium. The mitochondrial adenylate kinase appeared to the localised exclusively in the intermembrane space.     

Anthocyanin pigments and leaf flavonoids in the family araceae

Anthocyanins, variously identified in inflorescence, fruit, leaf or petiole of 59 representative species of the Araccae, are of a simple type. The most common pigment is cyanidin 3-rutinoside, while pelargonidin 3-rutinoside and cyanidin 3-glucoside are regularly present. Two rare pigments are: cyanidin 3-gentiobioside in Anchomanes and Rhektophyllum, both in the subfamily Lasioideae; and delphinidin 3-rutinoside in Schismatoglottis concinna. In a leaf survey of 144 species from 58 genera, flavone C-glycosides (in 82%) and proanthocyanidins (in 35–45%) were found as the major flavonoids. In the subfamily Calloideae, subtribe Symplocarpeae, flavonols replace glycoflavones as the major leaf components but otherwise flavonols are uncommon in the family (in 27% of the sample) and more usually co-occur with flavone C-glycosides. Two new flavonol glycosides were characterized from Lysichiton camtschatcense: kaempferol 3-(6-arabinosylgalactoside)and kaempferol 3-xylosylgalactoside. Simple flavones, luteolin and chrysoeriol (in 6%) were found only in the subtribes Arinae and Cryptocoryninae, subfamily Aroideae. Flavonoid sulphates were identified in only four taxa: glycoflavone sulphates in two Culcasia species and Philodendron ornatum and a mixture of flavone and flavonol sulphates in Scindapsus pictus. Caffeic ester sulphates were more common and their presence in Anthurium hookeri was confirmed. These results show that the Araceae are unusual amongst the monocots in their simple and relatively uniform flavonoid profile; no one subfamily is clearly distinguished, although at tribal level some significant taxonomic patterns are observed. The best defined groups are the subfamilies Lasioideae and Monsteroideae, and the tribes Symplocarpeae and Arophyteae, and the subtribe Arinae. The greatest chemical diversity occurs in Anthurium and Philodendron, but this may only reflect the fact that these are the two largest genera in the family. The origin and relationship of the Araccae to other monocot groups are discussed in the light of the flavonoid evidence.     

Asymmetric maturation of a dimeric transfer RNA precursor

Six of the eight transfer RNAs coded by bacteriophage T4 are synthesized via three dimeric precursor molecules. The sequences of two of these have been determined. Both of these precursors give rise to equimolar amounts of the cognate tRNA molecules in vivo. In contrast, even in wild-type infections, tRNA^Ile is present in ≤ 30% the amount of tRNA^Thr, with which it is processed from a common dimeric precursor.We have now determined the sequence of this dimer. In addition to the nucleotides present in tRNA^Thr and tRNA^Ile, it contains nine precursor-specific residues, located at the 5′ and 3′ termini and at the interstitial junction of the two tRNA sequences. While the three dimers share the majority of structural features in common, pre-tRNA^{Thr + Ile} is the only case in which an encoded tRNA 3′ -C-C-A terminus is present in the interstitial region.The processing of this dimer in various biosynthetic mutants has been analyzed in vivo and in vitro and shown to be anomalous in several respects. These results suggest that the apparent underproduction of tRNA^Ile can be explained by a novel processing pathway that generates a metabolically unstable tRNA^Ile product. Data from DNA sequence analysis of the T4 tRNA gene cluster (Fukada & Abelson, 1980) support the conclusion that the asymmetric maturation of this precursor is a consequence of the unique disposition of the -C-C-A sequence. These results argue that gene expression can be modulated at the level of RNA processing. The biological significance of this phenomenon is discussed in relation to evidence that tRNA^Ile has a unique physiological role.     

Late replication patterns in adult and embryonic mice carrying Searle's X-autosome translocation

Bromodeoxyuridine-dye technique analysis of X chromosome DNA synthesis in female adult and fetal mice carrying the balanced form of the T(X; 16) 16H translocation demonstrated that the structurally normal X chromosome was late replicating (and hence presumably inactive) in 93% of the adult cells and 99% of the 9-day embryo cells, with the X¹⁶ chromosome late replicating in the remaining cells. We conclude from these results that in T16H/+ females either there is preferential inactivation of the normal X chromosome or that, if inactivation is random, cell selection takes place before 9 days of development. Two 9-day female embryos with an unbalanced karyotype were also studied; both had two late-replicating chromosomes in most of their cells, one being the chromosome 16^X, the other a normal X chromosome. These results, together with the presence of a late-replicating X¹⁶ chromosome in T16H/+ adult and fetal mice, support the concept that more than one inactivation center is present on the X chromosome of the mouse because the X¹⁶ and the 16^x chromosomes can be late replicating.