Genomic potential of erythroid and leukocytic cells of Rana pipiens analyzed by nuclear transfer into diplotene and maturing oocytes

In order to determine whether differentiated somatic cells maintain genetic totipotency, nuclear transplantations from several differentiated somatic cell types into eggs and oocytes were performed previously in Rana pipiens and Xenopus laevis. The formation of postneurula embryos and tadpoles under the direction of the test nuclei demonstrated their genetic multipotency. In addition, Rana erythrocyte nuclei transplanted to oocytes directed more extensive tadpole development than those injected into eggs. We have extended our studies of the genomic potential of differentiated somatic nuclei from the peripheral blood of Rana pipiens. First, we show that the developmental potential of erythrocyte nuclei injected into oocytes at first meiotic metaphase was greater than those injected into diplotene oocytes. Second, we demonstrate that erythroblast and leukocyte nuclei transplanted to oocytes at first meiotic metaphase promoted more advanced tadpole development than those previously injected into Xenopus eggs. Third, erythrocyte nuclei were more successful in promoting advanced tadpole development compared with erythroblast and leukocyte nuclei. The results show that differentiated somatic nuclei transferred to the cytoplasm of oocytes at first meiotic metaphase display enhanced genomic and developmental potential over those transplanted to diplotene oocytes and eggs, at least for the three nuclear cell types tested from the peripheral blood.     

Comparative efficiencies of bispecific F(ab′γ)2 and chimeric mouse/human IgG antibodies in recruiting cellular effectors for cytotoxicity via Fcγ receptors

Summary The three forms of Fc receptor carried by monocytes (FcRI, II) and natural killer (NK) cells (FcRIII) are all capable of mediating cell lysis. Here we compare the use of F(ab)₂ bispecific antibodies, specifically targetting individual FcR, and chimeric IgG mouse/human antibodies which are capable of targetting all FcR, for their ability to mediate target cell destruction. The derivatives are prepared by linking hinge sulphydryl residues via tandem thioether bonds, using a bismaleimide crosslinker: Fab from an anti-FcR mAb linked to Fab from a common anti-target mAb (BsAb), or Fab from the common anti-target mouse antibody linked to human Fc (FabFc or bisFabFc). All the derivatives targetting chick red blood cells gave efficient lysis, although different effector cell donors yielded differences in both the lytic levels achieved and the comparative efficiencies of derivatives. In contrast, significant lysis of the guinea pig lymphoblastic leukaemia, L₂C, regularly resulted only via the anti-FcRIII BsAb and the chimeric derivatives. These results suggest that the chimeric, Fc-containing derivatives mediate tumour cell lysis principally through FcRIII on NK cells. This is in contrast to the situation with the chick red blood cells where the chimeric derivatives appear capable of lysing erythrocytes by utilizing either monocytes or NK cells, because significant (50%) lysis occurred with effector cell populations magnetically depleted through either FcRII or FcRIII. A major difference between these two types of antibody derivative was their ability to function in the presence of high concentrations of normal human Fc. The lysis mediated by BsAb reactive with FcRI or II was unaffected by the presence of human Fc at 2.5 mg/ml (a concentration comparable with that yielded by IgG in plasma) whereas the BsAb recognizing FcRIII and all the Fc-containing derivatives were completely inhibited.This work has been supported by Tenovus, the Cancer Research Campaign, the Leukaemia Research Fund, Italfarmaco, Milano, Italy and the Imperial Cancer Research Fund     

Behavioral,physiological, and morphological components of dominance and mate attraction in male green iguanas

This study investigated the morphological, physiological, and behavioral components of social dominance important for mate attraction in male green iguanas (Iguana iguana). A group of 9 male and 11 female adult green iguanas was studied in a large semi-natural enclosure during one reproductive season (October–January). Four of the nine males never initiated aggressive encounters; the other five were observed to display aggressively toward each other and were ranked in a linear dominance hierarchy. Head size was the most important factor influencing fighting success. Head size and display frequency were positively correlated with plasma testosterone levels. Dominance rank directly influenced ability to monopolize areas containing resources used by females. The quality of a male's home range, measured as his access to a large basking rock in the enclosure, was related to the proportion of potential mates found within his home range. One male greatly surpassed the others in his ability to defend a home range of high quality and attract potential mates. These data suggest that physiological and morphological factors, through their influence on social behavior, may ultimately affect male reproductive fitness. © 1992 Wiley-Liss, Inc.     

An interspecific linkage map of mouse chromosome 15 positioned with respect to the centromere

We have used an interspecific backcross between C57BL/6J and Mus spretus to derive a molecular genetic linkage map of chromosome 15 that includes 25 molecular markers and spans 93% of the estimated length of chromosome 15. Using a second interspecific backcross that was analyzed with a centromere-specific marker, we were also able to position our map with respect to the chromosome 15 centromere. This map provides molecular access to many discrete regions on chromosome 15, thus providing a framework for establishing relationships between cloned DNA markers and known mouse mutations and for identifying homologous genes in mice and humans that may be involved in disease.     

A molecular genetic linkage map of mouse chromosome 18 reveals extensive linkage conservation with human chromosomes 5 and 18

An interspecific backcross between C57BL/6J and Mus spretus was used to generate a molecular genetic linkage map of mouse chromosome 18 that includes 23 molecular markers and spans approximately 86% of the estimated length of the chromosome. The Apc, Camk2a, D18Fcr1, D18Fcr2, D18Leh1, D18Leh2, Dcc, Emb-rs3, Fgfa, Fim-2/Csfmr, Gnal, Grl-1, Grp, Hk-1rs1, Ii, Kns, Lmnb, Mbp, Mcc, Mtv-38, Palb, Pdgfrb, and Tpl-2 genes were mapped relative to each other in one interspecific backcross. A second interspecific backcross and a centromere-specific DNA satellite probe were used to determine the distance of the most proximal chromosome 18 marker to the centromere. The interspecific map extends the known regions of linkage homology between mouse chromosome 18 and human chromosomes 5 and 18 and identifies a new homology segment with human chromosome 10p. It also provides molecular access to many regions of mouse chromosome 18 for the first time.     

The organotypic culture of human skin keratinocytes and fibroblasts to achieve form and function

We describe an organotypic model of human skin comprised of a stratified layer of human epidermal keratinocytes and dermal fibroblasts within a contracted collagen lattice. Feasible and reproducible production of the skin construct has required the use of traditional as well as specialized culture techniques. The configuration of the construct has been engineered to maintain polarity and permit extended culture at the air-liquid interface. Morphological, biochemical and kinetic parameters were assessed and functional assays were performed to determine the degree of similarity to human skin. Light and ultrastructural morphology of the epidermis closely resembled human skin. The immunocytochemical localization of a number of differentiation markers and extracellular matrix proteins was also similar to human skin. Kinetic data showed a transition of the epidermal layer to a morein vivo-like growth rate during the development of the construct at the air-liquid interface. The barrier properties of the construct also increased with time reaching a permeability to water of less than 2%·h after approximately 2 weeks at the air-liquid interface which is still on average 30-fold more water-permeable than normal human skin. The construct is currently used forin vitro research and testing and is also being tested in clinical applications.     

Reduction of predation risk under the cover of darkness: Avoidance responses of mayfly larvae to a benthic fish

Summary Mayfly larvae of Paraleptophlebia heteronea (McDunnough) had two antipredator responses to a nocturnal fish predator (Rhinichthys cataractae (Valenciennes)): flight into the drift and retreat into interstitial crevices. Drift rates of Paraleptophlebia abruptly increased by 30 fold when fish were actively foraging in the laboratory streams but, even before fish were removed, drift began returning to control levels because larvae settled to the substrate and moved to areas of low risk beneath stones. This drifting response was used as an immediate escape behavior which likely decreases risk of capture from predators which forage actively at night. Surprisingly, drift most often occurred before contact between predator and prey, and we suggest that in darkness this mayfly may use hydrodynamic pressure waves for predator detection, rather than chemical cues, since fish forage in an upstream direction. Although drifting may represent a cost to mayfly larvae in terms of relocation to a new foraging area with unknown food resources, the immediate mortality risk probably out-weighs the importance of staying within a profitable food patch because larvae can survive starvation for at least 2 d. In addition to drifting, mayflies retreated from upper, exposed substrate surfaces to concealed interstitial crevices immediately after a predator encounter, or subsequent to resettlement on the substrate after predator-induced drift. A latency period was associated with this response and mayflies remained in these concealed locations for at least 3 h after dace foraging ceased. Because this mayfly feeds at night and food levels are significantly lower in field refugia under stones, relative to exposed stone surfaces, predator avoidance activity may limit foraging time and, ultimately, reduce the food intake of this stream mayfly.     

High plating efficiency and plant regeneration frequency in low density protoplast cultures derived from an embryogenic Brassica napus cell suspension

Protoplasts were isolated from an embryogenic cell suspension culture derived from microspores of Brassica napus cv. Jet Neuf. Protoplast yield varied with the cell suspension growth medium. Optimization of protoplast plating density, manipulation of culture medium, carbon source and medium matrix, and inclusion of Ficoll resulted in protoplast plating efficiencies close to 30%. Placement of the protoplasts close to the gas interface contributed greatly to the elevated plating efficiency. Low density cultures could be induced to regenerate calli at optimum plating efficiencies if grown in the presence of nurse culture. This is of great advantage for manipulation of individual protoplasts or for microinjection. Plants were regenerated directly from the cell suspension or from the protoplast cultures.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid     

The spo0A gene is implicated in the maintenance of non-complementing diploids in Bacillus subtilis

Bacillus subtilis can exist in a diploid state in which two genetically distinct chromosomes co-exist in the same cell and yet only one of them is expressed, thereby determining the phenotype. Such cells are called non-complementing diploids (Ncds). In this study, two types of experiments are reported which indicate that a previously known pleiotropic gene, spo0A, plays a role in the maintaining the diploid state, as follows. (i) When protoplasts of two Spo0A mutant strains were fused, the resulting products continued to segregate cells of both parental phenotypes for many more divisions than had been reported previously. (ii) When a stable Ncd (an Ncd in which the unexpressed markers are not spontaneously activated at a detectable level) harbouring a chloramphenicol acetyltransferase gene on the silent chromosome was transformed with spo0A null alleles the transformants often expressed chloramphenicol acetyltransferase activity. Together these results indicate that the spo0A gene is involved in maintenance of the diploid state in both unstable and stable Ncds.     

Identification and characterization of a pregnane steroid recognition site that is functionally coupled to an expressed GABAA receptor

Conclusion Based on the pharmacological and biochemical evidence to date, especially that derived from the recombinantly expressed receptor studies, the suggestion that a novel GBRC-linked steroid recognition site exists becomes a cogent argument. The high affinity of the steroid site for certain naturally occurring metabolites of progesterone and glucocorticoids favors a physiologic role for these steroids in the regulation of brain excitability. Clearly, investigations of such a regulatory role is warranted. If present, it provides an important example of endocrine control of a major inhibitory neurotransmitter in the CNS. Moreover, as we gain a greater understanding of the molecular organization of the GBRC, the putative steroid site provides a novel target for the rational design of therapeutic agents for the treatment of anxiety, epilepsy, and insomnia.Special issue dedicated to Dr. Eugene Roberts.