Resistance of Erythrocytes of Hibernating Mammals to Loss of Potassium during Hibernation and during Cold Storage

In two species of hibernators, hamsters and ground squirrels, erythrocytes were collected by heart puncture and the K content of the cells of hibernating individuals was compared with that of awake individuals. The K concentration of hamsters did not decline significantly during each bout of hibernation (maximum period of 5 days) but in long-term bouts in ground squirrels (i.e. more than 5 days) the K concentration of cells dropped significantly. When ground squirrels were allowed to rewarm the K content of cells rose toward normal values within a few hours. Erythrocytes of both hamsters and ground squirrels lose K more slowly than those of guinea pigs (nonhibernators) when stored in vitro for up to 10 days at 5°C. In ground squirrels the rate of loss of K during storage is the same as in vivo during hibernation, and stored cells taken from hibernating ground squirrels also lose K at the same rate. The rate of loss of K from guinea pig cells corresponded with that predicted from passive diffusion unopposed by transport. The actual rate of loss of K from ground squirrel cells was slower than such a predicted rate but corresponded with it when glucose was omitted from the storage medium or ouabain was added to it. Despite the slight loss of K that may occur in hibernation, therefore, the cells of hibernators are more cold adapted than those of a nonhibernating mammal, and this adaptation depends in part upon active transport.     

CYTOLOGICAL RESPONSES OF BROWN FAT TISSUE IN COLD-EXPOSED RATS

In young adult laboratory rats exposed to cold (6°C) the brown adipose tissue undergoes time-dependent increases in cellularity, vascular supply, and total mass. These changes are largely complete after 16 days in the cold and concurrent generally with the development of a thermoregulatory state not greatly dependent upon shivering. Histologically the brown fat changes from a tissue having both unilocular and multilocular fat cell types to one having almost exclusively the latter. During the first 6 to 12 hours in cold, the multilocular cells lose their lipid vacuoles and decrease in size, but these features are restored to normal by 24 hours. Cell proliferation, as estimated by the DNA synthetic index method (using tritiated thymidine autoradiography), appears in the reticuloendothelial cells of the brown fat at 1 day of cold exposure, becomes maximal at 4 days, and returns to the control level by 16 days. In animals injected with tritiated thymidine on the 3rd day of cold exposure and then maintained for 1 or more additional days in the cold, autoradiographs indicate that new brown fat (multilocular) cells arise by cytogenesis from reticuloendothelial progenitor cells and not by proliferation of existing brown fat cells. Throughout this and subsequent periods, cells of the epididymal white adipose tissue slowly decrease in size. Because a thermogenic role in cold acclimation has been established for the brown fat, the reported changes are regarded as adaptive responses to a cold environment.     

Cultured cells from renal cortex of hibernators and nonhibernators. Regulation of cell K+ at low temperature

Cells were grown as primary monolayer cultures from kidney cortex of guinea pigs (nonhibernators), hamsters and ground squirrels (both hibernating species). When plates of cells were placed at 5 °C, cells of guinea pigs lost 37% of their K⁺ in 2 h and those of the hibernator lost about 10%.Uptake of ⁴²K into the cells exhibited a simple, single exponential time course at both temperatures. Unidirectional efflux of K⁺ was equal to K⁺ influx in all cultures at 37 °C and, within limits of error, in hibernator cells at 5 °C. Efflux was 3- to 5-fold greater than influx in guinea pig cells at 5 °C.After 2 h in the cold the ouabain-sensitive K⁺ influx remaining (7–15% of that at 37 °C) was about the same in the cells of the 3 species. Cells from active hamsters and from hibernating ground squirrels, however, exhibited significantly greater pump activity after 45 min in the cold (19 and 14%, respectively). The stimulation of K⁺ influx by increasing [K⁺]_o did not show an increase in K_m⁺ at 5 °C in cells of guinea pigs and ground squirrels. Lowering [K⁺]_c and/or raising [Na⁺]_c by treatment in low- and high-K⁺ media caused only slight stimulation of K⁺ influx, except in cells of ground squirrels at 5 °C in which the stimulation was at least 11-times greater than at 37 °C or in cells of guinea pigs at either temperature.This altered kinetic response of K⁺ transport to cytoplasmic ion stimulation with cooling accounted for about one-third of the improved regulation of K⁺ at 5 °C in ground squirrel cells; the other two-thirds was attributable to a greater decrease in K⁺ leak with cooling. The inhibition of active transport by cold in all 3 species was much less severe than that previously seen in any (Na⁺ + K⁺)-ATPase of mammalian cells.     

Cell kinetics of mouse urinary bladder epithelium. IV. Changes in cell proliferation, nuclear DNA content, and number of diploid, tetraploid and octoploid cells after a single dose of dibutylnitrosamine.

The initial effect of a single subcutaneous injection of 0.01 ml of dibutylnitrosamine on the mouse (hr/hr strain) urinary bladder epithelium was a block in DNA synthesis in the diploid cells followed by a regenerative reaction. This did not, however, lead to a subsequent wave of increased DNA synthesis among the tetraploid cells. Later, a new wave of DNA synthesis occurred among the diploid cells, and again there was no subsequent wave of tetraploid DNA synthesis. The total cell number was not affected. These disturbances resulted in periods of reduced numbers of octoploid cells. This effect was unlike that obtained in previously published experiments using cyclophosphamide, which led to considerable hyperplasia, especially of octoploid cells, and no disturbance of tetraploid DNA synthesis. Thus the action of a single dose of dibutylnitrosamine on the epithelial cells in the mouse urinary bladder is very different from that of cyclophosphamide in a single dose, but it is not possible to say whether this has anything to do with the carcinogenicity of the nitrosamine.     

Mouse targets undergo double-strand DNA fragmentation when exposed to syngeneic or xenogeneic LAK cells whereas human targets undergo single-strand breaks

A number of recent studies have shown that mouse target cells (TC) of hematopoietic origin, when exposed to cytotoxic lymphocytes, undergo double-stranded DNA fragmentation. The cause and relevance of the fragmentation remain controversial. In this study we generated a number of mouse (M-LAK) and human LAK (H-LAK) cells and exposed them to a variety of mouse and human TC. YAC and SP/2, 2 mouse TC underwent rapid and extensive fragmentation when lysed by either human or mouse LAK whereas K562 and Daudi, 2 human TC, under the same conditions did not. All 4 TC, however, were killed quite efficiently. Next we labeled TC with 125I-deoxyuridine, exposed them to LAK cells for up to 18 h and loaded the LAK:TC mixtures over an alkaline linear sucrose gradient. After lysing the cells with a lysis buffer containing Triton X-100 we showed that K562 that had been in contact with LAK cells for more than 1 h exhibited single-strand nicks. However, whereas double-strand fragmentation preceded chromium release (lytic activity), the appearance of single-strand nicks did not. Finally, protein synthesis was not required for either type of fragmentation. In summary, we have demonstrated that: (1) the ability to undergo DNA fragmentation is a property of the TC rather than the effector cells that mediated their death, and (2) K562 and Daudi, 2 human TC, undergo single-strand nicks when lysed by LAK cells whereas SP/2 and YAC, 2 mouse TC undergo double-strand fragmentation when exposed to the same syngeneic or xenogeneic effector cells.     

Enhancement and repression of somatic embryogenesis in cell cultures of carrot by cold pretreatment of stock plants

Roots of nine cultivars of carrot (Daucus carota L.) were exposed to one, two or three months cold treatment and cells isolated from cold-treated and control roots were assayed for the production of somatic embryos. Cells obtained from the one- or two-months cold treatments formed embryos earlier, produced embryos over a longer period of time and produced more embryos per callus than the controls. In contrast, cells obtained from roots exposed to three months cold displayed a reduction in all parameters of embryogenic competence and for some cultivars this treatment resulted in production of cells with no embryogenic competence. The relationship of cold treatment of stock plants to the induction of metastable and stable patterns of gene expression and the induction of somatic embryos are discussed.     

Superiority of hemoglobin to hemin for cultivation of Leishmania tropica promastigotes in serum-free media

Leishmania tropica promastigotes grew slowly but could be maintained for long periods in serum-free hemin-containing media formulated previously for other Leishmania species or in slightly simplified versions of these media. Replacement of hemin in the medium by hemoglobin resulted in a much longer log phase and a significant reduction in the doubling time. Cell counts in cultures started at 1 x 10(5) cells/ml increased 400-fold in less than 140 h in the hemoglobin-containing media. These media also proved suitable for growing L. donovani and L. enriettii promastigotes.     

THE INFLUENCE of A LOW ENVIRONMENTAL TEMPERATURE ON the STEM CELL POPULATION of the BONE MARROW and INTESTINAL MUCOSA

Protracted exposure of rats to a low environmental temperature (2° C) resulted in almost a two-fold increase in the number of colony forming units per femur. Following a dose of Vinblastine, return of CFUs in the bone marrow to the pretreatment level was more rapid in the cold exposed rats than in rats at a 25° C environment. the cell cycle time of the cells in the intestinal crypt was reduced for the cold exposed rats. These observations are thought to be the basis for the increased radioresistance and/or more rapid recovery from whole body irradiation previously reported.     

Studies on the Origin of the Traditional Chinese Drug Fuxiong and Its Relationships with Ligusticum chuanxiong and L. sinense

The vegetative characters of Ligusticum chuanxiong Hort. cv. Fuxiong are described in comparison with L. chuanxiong Hort. and L. sinense Oliv. The chromosome numbers and karyotypes of the three taxa were studied in root tip cells by Feulgen’s squash method. Their karyotypes are determined as follows: L. chuanxiong, K(2n)=22=16 m+ 4sm+2st (sat); L. chuanxiong cv. Fuxiong, K(2n)=33=24 m+6sm+3st (sat); L. sinense, K(2n)=22=12 m+6sm+2sm (sat)+2st (sat). The karyotypic similarities are found between L. chuanxiong and its cultivariety, butthe former is a diploid while the latter probably is a homologous triploid.     

NK recognition of target structures: is the transferrin receptor the NK target structure?

That the transferrin receptor acts as a target antigen for human NK cells has previously been suggested. In this study we used two models to examine the hypothesis that the transferrin receptor is recognized by NK cells. In the first model, we employed mouse cloned NK cells in conjunction with the species-specific monoclonal antibody R17 217, which binds to the murine transferrin receptor. We show that there is no correlation between the amount of transferrin receptor expressed on targets and the susceptibility of these targets to NK lysis or NK binding in cold target competition assays. In the second model, we used human NK cells and transferrin receptor-positive transformants as targets. These transformants were derived from mouse L cells transfected with human DNA and selected for the presence of human transferrin receptor. Results show that, in contrast to the mouse system, there is a correlation between the expression of the human transferrin receptor on targets and the ability of these targets to competitively inhibit the lysis of K562 by NK cells. However, because inhibition is not complete, other cell surface antigens probably play a role in human NK-target interactions.     

Effect of Fas+ and Fas− Target Cells on the Ability of NK Cells to Repeatedly Fragment DNA and Trigger Lysis via the Fas Lytic Pathway

We and others have recently shown that human NK cells express the Fas ligand (FasL) constitutively and that they can trigger the lysis of Fas positive (Fas+) target cells (TC) by apoptosis. We have also previously demonstrated that NK cells exposed to sensitive TC temporarily lose their ability to lyse sensitive TC via the granule-mediated pathway and that this loss is recovered when inactivated NK cells (NKi) are incubated in medium supplemented with IL-2, IL-12 or IL-15. In this study, we investigated the fate of the Fas-lytic pathway in NK cells exposed to either Fas+ or Fas– TC. To this end, we exposed NK cells to Jurkat (Fas–) or Jurkat (Fas+) TC for up to 6 h, separated NK cells from the TC and assessed the residual lytic activity against K562, a traditional human NK cell target, Jurkat Fas+ and Jurkat Fas– TC. Fas lytic activity was determined in calcium free medium, in the presence or absence of two distinct Fas-blocking monoclonal antibodies and a Fas.Fc fusion protein. In parallel experiments, the extent of DNA fragmentation in the three TCs was also assayed by the JAM test. Our results indicate that: (i) NK cells exposed to susceptible Fas+ TC temporarily lose most of their lytic potential due to the granule-mediated pathway, while only partially losing the Fas-lytic pathway. They also partially lose their ability to fragment DNA. (ii) NK cells exposed to Fas+ TC completely recover the Fas lytic pathway and the ability to fragment DNA via the Fas/Fas ligand when incubated in medium supplemented with IL-2 for 18 h.     

The effects of embryo stage and cell number on the composition of mouse aggregation chimaeras

Studies with intact preimplantation mouse embryos and some types of chimaeric aggregates have shown that the most advanced cells are preferentially allocated to the inner cell mass (ICM) rather than the trophectoderm. Thus, differences between 4-cell and 8-cell stage embryos could contribute to the tendency for tetraploid cells to colonise the trophectoderm more readily than the ICM in 4-cell tetraploid<-->8 cell diploid chimaeras. The aim of the present study was to test whether 4-cell stage embryos in 4-cell diploid<-->8-cell diploid aggregates contributed equally to all lineages present in the E12.5 conceptus. These chimaeras were compared with those produced from standard aggregates of two whole 8-cell embryos and aggregates of half an 8-cell embryo with a whole 8-cell embryo. As expected, the overall contribution of 4-cell embryos was lower than that of 8-cell embryos and similar to that of half 8-cell stage embryos. In the 4-cell<-->8-cell chimaeras the 4-cell stage embryos did not contribute more to the trophectoderm than the ICM derivatives. Thus, differences between 4-cell and 8-cell embryos cannot explain the restricted tissue distribution of tetraploid cells previously reported for 4-cell tetraploid<-->8-cell diploid chimaeras. It is suggested that cells from the more advanced embryo are more likely to contribute to the ICM but, for technical reasons, are prevented from doing so in simple aggregates of equal numbers of whole 4-cell and whole 8-cell stage embryos.     

Male meiosis in polyploid silkworms,Bombyx mori L. (Lepidoptera : Bombycidae)

Tetraploid and hexaploid silkworms, Bombyx mori L. (Lepidoptera Bombycidae) were induced by applying a cold shock to diploid and triploid eggs at the first cleavage stage. Male meiosis in the primary spermatocytes of these silkworms having a different ploidy was observed. In the polyploid cells, chromosome bridges, 2 for the tetraploid and 3 for the hexaploid, occurred between the newly formed daughter nuclei at telophase. Observation in the living spermatocytes showed that tetraploid cells needed a longer time than the diploid ones to complete the first meiotic division. The delay in the cell division may be responsible for the high sterility of the tetraploid males.     

A study of Sertoli-spermatid tubulobulbar complexes in selected mammals

Tubulobulbar complexes (TBCs) were found in nine mammalian species (opossum, vole, guinea-pig, mouse, hamster, rabbit, dog, monkey and human) primarily originating from the plasma membrane overlying the acrosome of late spermatids. Fewer complexes (4–10) were noted in these species than has been previously reported for the rat (up to 24). TBCs were not seen emanating from round spermatids or those elongated spermatids located within the deep recesses of the Sertoli cell, but they appeared as the spermatids came to reside much closer to the tubular lumen in preparation for release. TBCs developed in areas deficient or lacking in Sertoli filaments and endoplasmic reticulum (ectoplasmic specialization). In general their structural configuration was similar to that shown in the rat, although minor differences were noted. Fine fibrils were observed connecting the distal portion of the spermatid tube with the Sertoli plasma membrane forming a bristle-coated pit. The length of TBCs from most species studied was 1–2 μm, whereas those of the opossum extended 6–8 μm into an apical Sertoli process. TBCs were degraded within the Sertoli cell by its lysosomes prior to sperm release, and for most species there was evidence indicating that formation of more than one generation of TBCs occurred. As sperm release approached, TBCs formed preferentially from the leading edge of spermatids with spatulate heads. The Sertoli cell gradually withdrew from around the spermatid head until only the tip of the head was embedded within the Sertoli cell. This region of contact frequently demonstrated TBCs. The proposed functions of TBCs are reviewed and discussed in light of these findings from other species.     

5-Azacytidine induces changes in electrophysiological properties of human mesenchymal stem cells

Previously, mouse bone marrow-derived stem cells （MSC） treated with the unspecific DNA methyltransferase inhibitor 5-azacytidine were reported to differentiate into cardiomyocytes. The aim of the present study was to investigate the efficiency of a similar differentiation strategy in human mononuclear cells obtained from healthy bone marrow donors. After 1-3 passages, cultures were exposed for 24 h to 5-azacytidine （3 μM） followed by 6 weeks of further culture. Drug treatment did not induce expression of myogenic marker MyoD or cardiac markers Nkx2.5 and GATA-4 and did not yield beating cells during follow-up. In patch clamp experiments, approximately 10-15% of treated and untreated cells exhibited L-type Ca^2＋ currents. Almost all cells showed outwardly rectifying K^＋ currents of rapid or slow activation kinetics. Mean current amplitude at ＋60 mV doubled after 6 weeks of treatment compared with time-matched controls. Membrane capacitance of treated cells was significantly larger than in controls 2 weeks after treatment and remained high after 6 weeks, Expression levels of mRNAs for the K^＋ channels Kv 1,1, Kv 1,5, Kv2,1, Kv4,3 and KCNMA 1 and for the Ca^2＋ channel Cav 1.2 were not affected by 5-azacytidine. Treatment with potassium channel blockers tetraethylammonium and clofilium at concentrations shown previously to inhibit rapid or slowly activating K^＋ currents of hMSC inhibited proliferation of these cells. Our results suggest that despite the absence of differentiation ofhMSC into cardiomyocytes, treatme.nt with 5-azacytidine caused profound changes in current density.     

Viral-induced fusion of human cells. II. Heterokaryocytes between human cells from different donors, and between human cells and those from other species: formation and properties

A simple method is described for effecting the formation of heterokaryocytes between different lines of human diploid fibroblasts, and between human diploid fibroblasts and cultured cells derived from other species. In the case of mixed monolayer cultures of human diploid fibroblasts exposed to UV-inactivated Sendai virus, the proportion of nuclei in heterokaryocytes is between 25 and 35%. The heterokaryocytes engage in de novo protein synthesis. No evidence of hybrid enzymes was found in mixed cultures of human and mouse cells which had been exposed to Sendai virus and which therefore presumably contained mouse-human heterokaryocytes. However, with the available data, it is not possible to distinguish between the absence of synthesis of hybrid enzymes and the synthesis of hybrid enzymes in amounts insufficient to permit their detection.     

The nature of thymidine kinase in the human-mouse hybrid cell

In order to characterize the thymidine kinase in human-mouse somatic cell hybrids derived from mouse parental cells lacking thymidine kinase, we have examined the electrophoretic migration on starch gel and the heat sensitivity of this enzyme in human, mouse, and hybrid cells. The enzyme of hybrid cells migrates similarly to that of human fetal liver and human diploid fibroblasts and faster than that of either L or A₉ mouse cells. It is less sensitive to heat than that of the mouse cells. Therefore, the human group E chromosome provides human thymidine kinase for the hybrid cell. The electrophoresis of thymidine kinase makes possible the search for variants.Aided by U.S.P.H.S. Grant No. HD 00486.     

Specificity of antierythrocyte autoantibodies secreted by a NZB-derived hybridoma and NZB peritoneal cells

The specificity of monoclonal IgM antierythrocyte autoantibody produced by a NZB-derived hybridoma and the specificity of autoantibodies produced by uninduced NZB peritoneal cells in culture were determined. Supernatant fluids from cultures of hybridoma and peritoneal cells reacted in direct hemagglutination assays with bromelin-treated mouse erythrocytes, and, to a lesser extent, with sheep red blood cells; no agglutination was observed with intact mouse red blood cells or human O⁺ erythrocytes. These results suggest the presence of previously characterized anti-HB, but not anti-X or cold reactive autoantibodies, with a cross-reaction between antigenic constituents on sheep and bromelin-treated mouse erythrocytes. Specificity was affirmed by neutralization of agglutination or of direct hemolysis of bromelin-treated mouse erythrocytes with partially purified SEA-HB, the soluble plasma analog of the erythrocyte-bound HB autoantigen. Plaque formation in direct plaque-forming cell assays by both hybridoma and peritoneal cells was specifically inhibited by SEA-HB. These results demonstrate that NZB-derived hybridoma as well as NZB peritoneal cells secrete anti-HB autoantibody, an autoantibody that spontaneously appears in the serum of NZB as well as other strains of mice.     

In vitro expression of factor-mediated cytotoxic activity generated during the immune response to Chlamydia in the mouse

Supernatant fluid (SF) prepared by mitogen incubation of spleen cells from A/J mice previously immunized against lethal challenge by the 6BC strain of Chlamydia psittaci was cytotoxic for mouse fibroblasts (L cells) infected with 6BC, as detected by the [3H]thymidine release assay and the trypan blue exclusion test. In contrast, SF prepared from spleen cells taken from unimmunized animals (controls) was not cytotoxic when added to infected L cells. No cytotoxicity was observed when SF was added to uninfected L cells. Maximal levels of cytotoxicity were observed only from cells infected with 6BC for at least 26 hr and exposed to SF for greater than 20 hr. Furthermore, the degree of cytotoxicity was dependent on both the dose of Chlamydia administered and the concentration of SF in the medium. We conclude that the capacity to secrete a spleen cell cytotoxic factor is an aspect of the immune response against the obligate intracellular prokaryotic pathogen Chlamydia. Our results indicate that SF-mediated cytotoxicity is induced subsequent to immunization with Chlamydia, and is significantly more pronounced against infected as opposed to uninfected L cells.     

BALB and kirsten murine sarcoma viruses alter growth and differentiation of EGF-dependent BALB/c mouse epidermal keratinocyte lines

Clonal BALB/c mouse epidermal keratinocyte (BALB/MK) cell lines were established in tissue culture. Despite their aneuploid nature, the lines were nontumorigenic, and retained in vitro properties similar to those of primary diploid keratinocytes. These included the constitutive expression of keratin and terminal differentiation in response to a calcium concentration greater than 1.0 mM in the medium. The cells also demonstrated an absolute requirement for nanomolar concentrations of epidermal growth factor (EGF) for their proliferation. BALB or Kirsten murine sarcoma viruses are acute transforming retroviruses, which have been shown to transform fibroblastic and hematopoietic cells. Infection of BALB/MK or its clonal sublines with either virus leads to the rapid acquisition of EGF-independent growth. The cells concomitantly lose their sensitivity to calcium-induced terminal differentiation. Thus these retroviruses can rapidly confer upon epithelial keratinocytes in culture growth properties that resemble those of malignant epidermoid carcinoma cells.