Approaching the multifaceted nature of energy metabolism: inactivation of the cytosolic creatine kinases via homologous recombination in mouse embryonic stem cells

To study the physiological role of the creatine kinase/phosphocreatine (CK/PCr) system in cells and tissues with a high and fluctuating energy demand we have concentrated on the site-directed inactivation of the B- and M-CK genes encoding the cytosolic CK protein subunits. In our approach we used homologous recombination in mouse embryonic stem (ES) cells from strain 129/Sv. Using targeting constructs based on strain 129/Sv isogenic DNA we managed to ablate the essential exons of the B-CK and M-CK genes at reasonably high frequencies. ES clones with fully disrupted B-CK and two types of M-CK gene mutations, a null (M-CK^–) and leaky (M-CK¹) mutation, were used to generate chimaeric mutant mice via injection in strain C57BL/6 derived blastocysts. Chimaeras with the B-CK null mutation have no overt abnormalities but failed to transmit the mutation to their offspring. For the M-CK^– and M-CK¹ mutations successful transmission was achieved and heterozygous and homozygous mutant mice were bred. Animals deficient in MM-CK are phenotypically normal but lack muscular burst activity. Fluxes through the CK reaction in skeletal muscle are highly impaired and fast fibres show adaptation in cellular architecture and storage of glycogen. Mice homozygous for the leaky M-CK allele, which have 3-fold reduced MM-CK activity, show normal fast fibres but CK fluxes and burst activity are still not restored to wildtype levels.     

Metacercarial dispersion and intracellular parasitism in a strigeid trematode

Combes C. and Nassi H. 1977. Metacercarial dispersion and intracellular parasitism in a strigeid trematode. International Journal for Parasitology7: 501–503. The life cycle of Apatemon graciliformis Szidat, 1928 (Trematoda, Strigeidae), a parasite of Biorrphalaria glabrata in Guadeloupe, involves a novel mode of transmission, experimentally demonstrated, between the second intermediate host and the definitive host. The furcocercariae penetrate gravid females of the ovoviviparous fish, Poecilia reticulata, and develop into metacercariae in vitelline vesicles of the embryos where they encyst a short time before parturition. The young guppies are born infected with 1–3 metacercariae. It is considered that young infected fish are more prone to predation by the definitive host, thereby increasing the probability of the cycle being completed. Domestic ducks have been experimentally infected with these metacercariae. If cercariae penetrate non-gravid P. reticulata, they enter the oocytes; this represents a phase of intracellular parasitism.     

The influence of plant growth regulators on the growth of the embryonic axes of red- and far-red-treated lettuce seeds

The influence of several plant growth regulators on the growth of the embryonic axes from red- and far-red-(R- and FR-)treated lettuce (Lactuca sativa L., cv. Grand Rapids) seeds was examined; as shown previously, the water potential of the axes from R-treated seeds has been lowered by 3.5–5.6 bars compared to that in axes from FR-treated ones. Kinetin and abscisic acid (ABA), when included in the incubation medium, reduced the elongation of the axes whereas fusicoccin stimulated it; however, these effects were the same in axes of both R- and FR-treated seeds. In contrast, elongation of axes from FR-treated seeds was stimulated by gibberellic acid (GA₃, but elongation of axes from R-treated ones was not affected by this hormone. This latter result indicates that gibberellins may be involved in the phytochrome-mediated growth responses in lettuce axes.When the root caps of the embryos were removed prior to light treatment, R was still able to induce a water-potential decrease in the embryonic axes, indicating that at least a portion of the active Pfr resides in the axis and not the root cap.Abbreviations ABA abscisic acid - FR far red light - GA₃ gibberellic acid - PEG polyethylene glycol - Pfr far-red-absorbing form of phytochrome - R red light     

Developmental changes in the calcium currents in embryonic chick ventricular myocytes

Summary Using the patch-clamp technique, we recorded whole-cell calcium current from isolated cardiac myocytes dissociated from the apical ventricles of 7-day and 14-day chick embryos. In 70% of 14-day cells after 24 hr in culture, two component currents could be separated from totalI _Ca activated from a holding potential (V _h) of –80 mV. L-type current (I _L) was activated by depolarizing steps fromV _h –30 or –40 mV. The difference current (I _T) was obtained by subtractingI _L, fromI _Ca.I _T could also be distinguished pharmacologically fromI _L in these cells.I _T was selectively blocked by 40–160 m Ni²⁺, whereasI _L was suppressed by 1 m D600 or 2 m nifedipine. The Ni²⁺-resistant and D600-resistant currents had activation thresholds and peak voltages that were near those ofI _T andI _L defined by voltage threshold, and resembled those in adult mammalian heart. In 7-day cells,I _T andI _L could be distinguished by voltage threshold in 45% (S cells), while an additional 45% of 7-day cells were nonseparable (NS) by activation voltage threshold. Nonetheless, in mostNS cells,I _Ca was partly blocked by Ni²⁺ and by D600 given separately, and the effects were additive when these agents were given together. Differences among the cells in the ability to separateI _T andI _L by voltage threshold resulted largely from differences in the position of the steady-state inactivation and activation curves along the voltage axis. In all cells at both ages in which the steady-state inactivation relation was determined with a double-pulse protocol, the half-inactivation potential (V _1/2) of the Ni²⁺-resistant currentI _L averaged –18 mV. In contrast,V _1/2 of the Ni²⁺-sensitiveI _T was –60 mV in 14-day cells, –52 mV in 7-dayS cells, and –43 mV in 7-day NS cells. The half-activation potential was near –2 mV forI _L at both ages, but that ofI _T was –38 mV in 14-day and –29 mV in 7-day cells. Maximal current density was highly variable from cell to cell, but showed no systematic differences between 7-day and 14-day cells. These results indicate that the main developmental change that occurs in the components ofI _Ca is a negative shift with, embryonic age in the activation and inactivation relationships ofI _T along the voltage axis.     

Numbers and size of sperm storage tubules and the duration of sperm storage in birds: a comparative study

Estimates of the numbers of sperm storage tubules (SSTs) in the utero-vaginal junction of 11 bird species are presented. Numbers of SSTs varied by a factor of 40 between species, and ranged from 500 to 20000. Body mass accounted for over 50% of the variation in SST mumbers. SST length was positively correlated with the length of spermatozoa across species. The duration of sperm storage was not correlated with the number of SSTs or the volume of sperm storage tissue. However, the number of 'active' SSTs appears to vary between species and it was not possible to make allowance for this. Sperm storage duration was weakly, positively correlated wth clutch size, but showed a significant positive relationship with the number of days over which laying occurred. The number of SSTs was also positively correlated with the number of sperm per ejaculate. The best predictor of sperm storage duration was a multiple regression equation using the spread of laying and the length of sperm storage tubules. The duration of sperm storage in birds which remain together during the pre-laying period is such that a single insemination immediately before the start of laying could fertilize the entire clutch.     

Developmental patterns of ornithine decarboxylase activity in organogenesis phase rat embryos in culture and in utero

Summary Ornithine decarboxylase activity was measured during organogenesis in rat embryos grown in utero and whole rat conceptuses maintained in an in vitro culture system. Ornithine decarboxylase levels in vivo showed a distinct peak at embryonic age 10.5 d. Despite identical morphology, protein content, crown rump length and numbers of somites cultured embryos displayed a different developmental pattern and possessed less than half the ODC activity of that in vivo. The data suggest that the normal embryonic programming of ODC activity is significantly altered by the culture environment and that further biochemical comparisons of embryos growing in utero and in vitro may be required to evaluate properly the applicability of this technique to detailed studies of teratogenesis and developmental biology. This work was supported by NIH-5-507-RR5359-17 and a 1980 Research Starter Grant from the Pharmaceutical Manufacturers Association Foundation.     

Actin acetylation in Drosophila tissue culture cells

In a permanent cell line derived from Drosophila embryos, cytoplasmic actin is produced as an unstable precursor, which is subsequently converted to a stable form. This conversion results in a reduction in isoelectric point, with no apparent change in molecular weight. The conversion involves an enzymatic acetylation, and results in an insensitivity to aminopeptidase digestion, suggesting N-terminal blockage. Both the acetylated and unacetylated actins can participate in the assembly of F-actin, but with different efficiencies.This work was supported by a grant from the NIH (GM 22866).     

The cultivation of the exoerythrocytic stages ofPlasmodium berghei from sporozoites

Summary Cultures of embryonic rat brain and liver, and embryonic turkey brain were inoculated with sporozoites ofPlasmodium berghei. Sporozoites succeeded in establishing exoerythrocytic infections in approximately 10% of the cultures. The exoerythrocytic parasites developed to a late schizont stage with some showing early segmentation although free merozoites were not observed. The morphology and rate of development of the exoerythrocytic parasites in culture appear similar to that seen in vivo. This work was supported by ONR Contract No. N00014-76-C-1132 and Naval Medical Research and Development Command, Research Work Unit No. M0095PN.002.5058. the opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The experiments reported herein were conducted according to the principles set forth in theGuide for the Care and Use of Laboratory Animals, Institute of Laboratory Resources, National Research Council, DHEW, Pub. No. (NIH) 74-23.     

Strain distribution and linkage relationship of a mouse embryonic hemoglobin variant

Search for structural variants of three globin chains (x, y, z), synthesized only during mouse embryonic hematopoiesis, was carried out by electrophoretic analysis of blood from 12-day embryos, all with C57BL/6 mothers, and fathers from 115 inbred stocks selected for their diverse genetic origins. Structure of the -chains of adult hemoglobins differed among the tested strains, with 57 carrying the Hbb ^sallele, 56 the Hbb ^dallele, and two the Hbb ^pallele. The search revealed no x- or z-chain variants but confirmed and extended knowledge of a previously described y-chain variant. Blood of all embryos sired by males from the 57 Hbb ^sstrains contained only y¹-chains, while blood of all embryos sired by Hbb ^dor Hbb ^pmales contained y2-chains as well as the y¹-chains inherited from their C57 BL/6 mother. The locus controlling structure of the y-chain of mouse embryonic hemoglobins is thus extremely closely linked to the locus controlling structure of adult hemoglobin -chain, with maximum possible recombination frequency less than 0.019.This work was supported in part by Grants CA-01074 from the National Cancer Institute, USPHS, and GM 18684 from the National Institute of General Medical Sciences, in part by Grant ACS-VC58 from The American Cancer Society, in part by grants to the Jackson Laboratory from the Bushrod H. Campbell and Adah F. Hall Charity Fund and the Robert Sterling Clark Foundation, and in part by the Jackson Laboratory Endowment Fund. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Biotechnological challenges of bioartificial kidney engineering

With the world-wide increase of patients with renal failure, the development of functional renal replacement therapies have gained significant interest and novel technologies are rapidly evolving. Currently used renal replacement therapies insufficiently remove accumulating waste products, resulting in the uremic syndrome. A more preferred treatment option is kidney transplantation, but the shortage of donor organs and the increasing number of patients waiting for a transplant warrant the development of novel technologies. The bioartificial kidney (BAK) is such promising biotechnological approach to replace essential renal functions together with the active secretion of waste products. The development of the BAK requires a multidisciplinary approach and evolves at the intersection of regenerative medicine and renal replacement therapy. Here we provide a concise review embracing a compact historical overview of bioartificial kidney development and highlighting the current state-of-the-art, including implementation of living-membranes and the relevance of extracellular matrices. We focus further on the choice of relevant renal epithelial cell lines versus the use of stem cells and co-cultures that need to be implemented in a suitable device. Moreover, the future of the BAK in regenerative nephrology is discussed.