Reutilisation of tritiated thymidine in studies of regenerating skeletal muscle

Summary Two different aspects of tritiated thymidine (³H-Tdr) reutilisation in skeletal muscle were examined. Injection of a high dose (7 Ci/g) of ³H-Tdr into mice prior to crush injury of skeletal muscle resulted in heavy labelling (grain counts) of myotube nuclei 9 d later. In contrast, myotube nuclei were essentially unlabelled when a low dose (1 Ci/g) of ³H-Tdr was injected at similar times with respect to injury. It was concluded that labelling seen after the high dose was due to reutilisation of ³H-Tdr. (Such ³H-Tdr reutilisation can account for the results of Sloper et al. (1970) which previously supported the concept of a circulating muscle precursor cell.) When replicating muscle precursors were labelled directly with ³H-Tdr 48 h after injury, the percentages of labelled myotube nuclei and the distribution of nuclear grain counts were similar with either high or low dose.We also investigated whether the light labelling seen in regenerated myotube nuclei after 9 d, when ³H-Tdr had been injected before the onset of myogenesis (as found by McGeachie and Grounds 1987), was due to ³H-Tdr reutilisation or, alternatively, to proliferation of local cells in the wound which subsequently gave rise to muscle precursors. Labelling of myotube nuclei was compared in mice injected with ³H-Tdr either 2 h before, or 2 h after injury. In another experiment, mice were injected 12 h after injury and lesions sampled 1, 12 or 36 h later, to see whether local cells were replicating 12 h after injury, and what labelled cells subsequently entered to wound. No difference was found in myotube labelling between mice injected before or after injury, and no cells replicating locally in the wound at 12 h after injury were observed. The results clearly show that the light labelling was due to ³H-Tdr reutilisation.     

High levels of manganese-containing superoxide dismutase and thermally induced DNA disruption in a dnaK7(Ts) mutant of Escherichia coli K12

Summary IndnaK7(Ts) mutant cells, scission of DNA strands occurred after temperature shift up. When cells at 30°C were labeled with [³H]-thymidine and then shifted to 46° or 49°C for 20 min, the profiles of sedimentation of thier cellular DNA in an alkaline sucrose gradient revealed a decrease in the size of DNA to a quarter of that at 30°C in the mutant, but not in wild-type cells. The level of manganese-containing superoxide dismutase (MnSOD) in the mutant was about twice that in wild-type cells, even at the permissive temperature, implying increased production of superoxide radical anion, which may cleave DNA strands directly or indirectly in the mutant. Moderate increase in the MnSOD level on temperature shift up was observed in both strains. These results indicated that some components of the DnaK protein participate in protection of cellular membrane functions from thermal damage resulting from elevated production of the superoxide anion radical.     

A new mutant class of soybean lacks urease in leaves but not in leaf-derived callus or in roots

Summary We reported earlier the recovery of two classes of soybean urease mutants in soybean (Glycine max L. Merr. cv. Williams). Class I mutants lack the embryo-specific urease while class II mutants lack the activities of both urease isozymes, the embryo-specific and the ubiquitous urease, the latter found in all tissues examined. We report here the recovery of a true-breeding mutant, aj3, which represents the third phenotypic class: normal levels of embryo-specific urease and little or no ubiquitous urease. Unlike class II mutant plants which lack urease in all tissue, aj3 lacks urease activity only in leaves (ca. 2% normal activity); its roots have near normal urease activity. Callus derived from leaves of aj3 has 14% to 40% the urease activity of Williams 82 callus. This partial reduction in urease activity in aj3 callus is sufficient to reduce growth with urea as sole nitrogen source and to confer resistance to 50 mM urea added to callus maintenance medium. Leaves of aj3 produce more than 40 times the urease antigen expected from their urease activity. The aj3 trait is due to a single recessive lesion which is not allelic with lesions at theEu2, Eu3 (class II) orEu1 (class I) loci. We designate the aj3 genotype aseu4/eu4.     

Development ofBradyrhizobium infections in supernodulating and non-nodulating mutants of soybean (Glycine max [L.] Merrill)

Summary The early events in the development of nodules induced byBradyrhizobium japonicum were studied in serial sections of a wild type (cv. Bragg), a supernodulating mutant (nts 382) and four non-nodulating mutants (nod49, nod139, nod772, andrj ₁) of soybean (Glycine max [L.] Merrill). Cultivar Bragg responded to inoculation in a similar manner to that described previously for cv. Williams; centres of sub-epidermal cell divisions were observed both with and without associated infection threads and most infection events were blocked before the formation of a nodule meristem. The non-nodulating mutants (nod49, nod772, andrj ₁) had, at most, a few centres of sub-epidermal cell divisions. In general, these were devoid of infection threads and did not develop beyond the very early stages of nodule ontogeny. Sub-epidermal cell divisions or infection threads were never observed on mutant nodl39. This mutant is not allelic to the other non-nodulating mutants and represents a defect in a separate complementation group or gene that is required for nodulation. The supernodulating mutant nts382, which is defective in autoregulation of nodulation, had a similar number of sub-epidermal cell divisions as the wild-type Bragg, but a much greater proportion of these developed to an advanced stage of nodule ontogeny. Mutant nts382, like Bragg, possessed other infection events that were arrested at an early stage of development. The results are discussed in the context of the progression of events in nodule formation and autoregulation of nodulation in soybean.Abbreviations nts nitrate tolerant symbiosis - RT root tip (i.e., position of the tap root tip at the time of inoculation) - SERH shortest emerging root hair (i.e., position of the shortest emerging root hair on the tap root at the time of inoculation) - SCD subepidermal cell divisions     

Appearance of lectin-binding sites during vascularization of the primordium of the central nervous system in 10 to 12-day-old mouse embryos

Summary In the present study lectin-binding sites were investigated for the lectins Ricinus communis agglutinin (RCA I), wheat germ agglutinin (WGA), soya bean agglutinin (SBA), concanavalin A (Con A), Lotus tetragonolobus(LTA) and Limulus polyphemus agglutinin (LPA) during the initial stages of vasculogenesis of the CNS-anlage in 10 to 12-day-old NMRI mouse embryos. Specific binding sites for the lectins RCA I (sugar specificity: -D-galactose, N-acetylgalactosamine), WGA (sugar specificity: N-acetylglucosamine, sialic acid), and SBA (sugar specificity: N-acetylgalactosamine, -D-galactose) were detected in the newly formed capillaries within the neuroepithelial cell layer. In contrast, binding sites for Con A, LTA and LPA could not be observed at the start of the vascularization of the CNS-anlage. From these results, the conclusion can be drawn that glycoconjugates containing D-galactose, N-acetylgalactosamine and N-acetyl-glucosamine moieties are involved in the early vasculogenesis of the embryonic CNS-anlage of the mouse.     

Vascular response in a non-uterine site to implantation-stage embryos following interspecies transfers between the rat,mouse, and guinea-pig

Summary Pre-implantation-stage embryos from rats, mice, and guinea-pigs were transferred to a non-uterine site — the anterior chamber of the eye — of female recipients. All 9 combinations of transfers were performed: 3 allogeneic (intraspecies) transfers as controls, and 6 xenogeneic (interspecies) transfers. Implantation, as judged by extravasation from blood vessels of the iris or ciliary body occurred with success rates of 90.4% per transfer in the control rat group, 76.9% in the control mouse group, and 81.8% in the control guinea-pig group. Significantly reduced implantation rates occurred in the rat to guinea-pig (0%), mouse to rat (46.9%), mouse to guinea-pig (6.7%), and guinea-pig to rat (0%) groups compared to controls. Reductions, although not significant, also occurred in the other 2 groups: rat to mouse (77.8%), and guinea-pig to mouse (44.4%). These results together with some ultrastructural and lightmicroscopical observations suggest a degree of species specificity involved in the vascular response to the implanting embryo. We propose that the peri-implantation embryo produces a signal(s) which is to some extent species specific and which in the normal allogeneic situation is responsible for the early vascular effects seen at implantation in most eutherian mammals.     

The three-dimensional structure of the zona pellucida in growing and atretic ovarian follicles of the mouse

Summary The present study provides further details on the fine-structural three-dimensional architecture of the zona pellucida (ZP) in growing and atretic follicles of mice by use of ruthenium red in combination with the detergents Triton X100 and saponin. These detergents were used for extraction of the soluble fraction of the zonal proteins in an attempt to expose the structural zonal glycoproteins, which in turn can be viewed as minute three-dimensional networks upon transmission- and scanning electron-microscopic examination. By use of these methods, the ZP of growing follicles appeared to be formed by interconnected filaments which also bind to globular structures building up a three-dimensional lattice. In contrast, the ZP of stage I as well as other (II and III) stages of atretic follicles showed a structure characterized by the presence of closely packed granules connected with short filaments to form a close-mesh reticulum. This structural change of the ZP, which in the present study is also associated with the disappearance of gap junctions within the granulosa and cumulus cell population, might represent one of the early events involved in the onset of atresia. These changes, most probably depending on an altered secretory activity of both oocytes and follicle cells, might lead to a degradation of the ZP network structure and to its subsequent increased density (condensation). All these morphodynamic events eventually contribute to a sequestration of the oocyte in the early stage of atresia.     

Stage-specific localization of cytoskeletal actin mRNA in murine seminiferous tubules and intestinal epithelia as demonstrated by in-situ hybridization

Summary In-situ hybridization experiments have been performed using isoactin ( and )-specific riboprobes in various tissues of the rat and mouse. Distribution of the grains of actin mRNAs for both and types was similar throughout sections of the rat testis. Although both mRNAs were evenly distributed in the seminiferous tubule, extremely heavy labeling was observed in about 10% of the seminiferous tubules that could be identified as stage XII of spermatogenesis. At high magnification, grains of the mRNA were found in the cytoplasm of elongating spermatids and in the Sertoli cell cytoplasm at the adluminal side. Much higher density of the grains of mRNA was observed in the neck region of the spermatids at stage XII. Thus, the dense distribution of cytoskeletal actin mRNAs is stage-specific in the tubule during spermatogenesis in the rat. The high expression of both and actin mRNAs was also observed in the epithelial cells of the intestinal crypts.     

Topological orientation of mitochondrial GDPmannose: dolichyl-monophosphate mannosyltransferase in the outer membrane

Previous studies have shown the existence of an autonomous mitochondrial GDPmannose: dolichylmonophosphate mannosyltransferase, located in mitochondiral outer membrane of liver cells. As nothing is known about glycosylation sites in mitochondria, we have investigated the topological orientation of this enzyme in intact mitochondria, using controlled proteolysis with trypsin. Mitochondria were purified sequentially by mild ultrasonic treatment and sucrose density gradient. Purity and homogeneity of mitochondrial fraction were assessed by electron microscopy and specific marker enzymes measures. Our data provide evidence for a mitochondrial GDPmannose: dolichylmonophosphate mannosyltransferase facing the cytoplasmic side of the outer membrane. However, the exposure of this enzyme to the water phase has been shown to be dependent on the ionic strength of the environment.     

Thymic nurse cells: morphological study during their isolation from murine thymus

Summary Thymic nurse cells (TNC), which are multicellular complexes composed of epithelial cells and thymocytes, were obtained from C3H-mice thymuses. They were described by means of light and electron microscopy. The morphology of epithelial cells forming isolated TNC compared to that of small tissue fragments obtained by enzymatic digestion revealed that TNC could be derived from all parts of the thymus: cortex, corticomedullary junction and medulla, the cortex being their principal source. This variety of origin, the presence of several epithelial cells inside a single TNC, the presence of non-lymphoid cells, and the various locations of eleaved desmosomes confirmed that their aspect in vitro as round and sealed structures can be considered to be an artifact due to the isolation technique used. Indeed, during this procedure, they are formed by a process of wrapping of the epithelial cytoplasm around the tightly associated thymocytes. All three epithelial cell types: cortical reticular cells, medullary reticular cells, and medullary globular cells can form TNC.A portion of this work was presented at the first Thymus Workshop. Rolduc, Netherlands, April, 1988