Cell cycle-regulated modification of the ribosome by a variant multiubiquitin chain

Ubiquitin is ligated to L28, a component of the large ribosomal subunit, to form the most abundant ubiquitin-protein conjugate in S. cerevisiae. The human ortholog of L28 is also ubiquitinated, indicating that this modification is highly conserved in evolution. During S phase of the yeast cell cycle, L28 is strongly ubiquitinated, while reduced levels of L28 ubiquitination are observed in G1 cells. L28 ubiquitination is inhibited by a Lys63 to Arg substitution in ubiquitin, indicating that L28 is modified by a variant, Lys63-linked multiubiquitin chain. The K63R mutant of ubiquitin displays defects in ribosomal function in vivo and in vitro, including a dramatic sensitivity to translational inhibitors. L28, like other ribosomal proteins, is metabolically stable. Therefore, these data suggest a regulatory role for multiubiquitin chains that is reversible and does not function to target the acceptor protein for degradation.     

Retinoic acid resistance of the variant embryonal carcinoma cell line RAC65 is caused by expression of a truncated RARα

P19 embryonal carcinoma (EC) cells differentiate when treated with retinoic acid (RA). The P19 EC-derived mutant cell line RAC65 is resistant to the differentiation-inducing activity of RA. We show that these cells express a truncated retinoic acid receptor alpha(mRAR alpha-RAC65), probably due to the integration of a transposon-like element in the RAR alpha gene. This receptor lacks 71 C-terminal amino acids and terminates in the ligand-binding domain. In CAT assays in RAC65 cells, mRAR alpha-RAC65 fails to trans-activate the RAR beta promoter, which contains a RA-response element. In wild-type P19 EC cells mRAR alpha-RAC65 functions as a dominant-negative repressor of RA-induced RAR beta activation. Gel retardation assays demonstrate that mRAR alpha-RAC65 is still able to bind to the RA-response element of the RAR beta promoter, indicating that competition with functional RARs for the same binding site leads to the observed dominant-negative effect. In addition, in two RAC65 clones in which wild-type hRAR alpha was stably transfected RA-sensitivity was restored and in one RAR beta expression could be induced by RA. Taken together, these data show that the primary cause of RA-resistance of RAC65 cells is the expression of a defective RAR alpha, which prevents the trans-activation of RA-responsive genes and results in a loss of the ability to differentiate.     

Change in blood lactate decrease after submaximal exercise, after stay at moderate altitude]

The study of blood lactate concentration after a submaximum and steadfast exercise shows a lesser rise after three weeks at middle altitude. This can be related with an increase of maximum working capacity as previously demonstrated. The obtained modifications result in part from an increase in oxydative metabolism activity and perhaps from an enhancement of lactate oxydation.     

Cycle reset in a melanoma cell line caused by cooling

Abstract When cells in culture are released from G₀ into cycle by diluting into fresh medium there is a delay of many hours before they re-enter the cycle and start DNA synthesis. A mouse melanoma cell line designated HP2 has been used to investigate the effects of non-standard temperatures between the time of plating and DNA synthesis. When the cells were incubated in a 5% CO₂ box at 8^°C for periods during the G₀-G₁ transition there was an extra delay before the start of S, approximately equal to the time that the cells were held at 8^°C and independent of the time when the cold pulse was administered. When the cells were cooled to 25^°C the delay was longer than the time for which the cells had been kept at 25^°C, and this extra delay was also dependent on the point in G₀-G₁ when the cells were cooled, as though the cells could be reset to an earlier time by this treatment. It is suggested that a labile substance required for progression is destroyed faster than it is made at 25^°C but at 8^°C the rate of destruction is very low. Another phenomenon noted during these cooling experiments was that the peak height of the S phase profile, as measured by frequent pulse-thymidine incorporation experiments, was substantially higher for cells which had been cooled at a later stage in the G₀-G₁ transition, even though the overall times at 37^°C and at the colder temperature were identical. By varying the temperature of the cold pulse it was possible to separate the change in the peak height and the delay as separate entities.     

Cycle reset in a melanoma cell line caused by cooling

When cells in culture are released from G0 into cycle by diluting into fresh medium there is a delay of many hours before they re-enter the cycle and start DNA synthesis. A mouse melanoma cell line designated HP2 has been used to investigate the effects of non-standard temperatures between the time of plating and DNA synthesis. When the cells were incubated in a 5% CO2 box at 8 degrees C for periods during the G0-G1 transition there was an extra delay before the start of S, approximately equal to the time that the cells were held at 8 degrees C and independent of the time when the cold pulse was administered. When the cells were cooled to 25 degrees C the delay was longer than the time for which the cells had been kept at 25 degrees C, and this extra delay was also dependent on the point in G0-G1 when the cells were cooled, as though the cells could be reset to an earlier time by this treatment. It is suggested that a labile substance required for progression is destroyed faster than it is made at 25 degrees C but at 8 degrees C the rate of destruction is very low. Another phenomenon noted during these cooling experiments was that the peak height of the S phase profile, as measured by frequent pulse-thymidine incorporation experiments, was substantially higher for cells which had been cooled at a later stage in the G0-G1 transition, even though the overall times at 37 degrees C and at the colder temperature were identical. By varying the temperature of the cold pulse it was possible to separate the change in the peak height and the delay as separate entities.     

Isolation and characterization of a variant myoblast cell line that is temperature sensitive for differentiation.

A new variant rat myogenic cell line, ts485, was isolated by subcloning the cell line ts3b2 (H. T. Nguyen, R. M. Medford, and B. Nadal-Ginard, Cell 34:281-293, 1983). Unlike the progenitor cell line, ts485 was thermosensitive for differentiation. Experiments with conditioned medium suggested that diffusible extracellular factors were not involved in dictating the differential phenotypes of ts485 cells cultured at the permissive and nonpermissive temperatures. Temperature shift experiments performed on cultures of ts485 cells indicated that the temperature-sensitive lesion was in a factor active during the growth phase and required to trigger a cascade of events leading to terminal differentiation.     

Cell toxicity caused by products of the p(L) operon of bacteriophage lambda.

Induction of a lambda prophage causes the death of the host cell even in the absence of phage replication and lytic functions due to expression of functions from the lambda p(L) operon. We genetically modified the lambda prophage to determine which lambda p(L) operon functions were involved in cell killing. Viability assays and flow cytometry were used to monitor cell death and filamentation. The kil gene was shown to cause cell death and filamentation as described previously. Another killing activity was mapped within the p(L) operon to the gam gene. Inspection of the DNA sequence showed that there are two possible translation start points for both kil and gam. In both cases, the shorter of the two possible products could cause cell killing. The shorter products were also sufficient for the known filamentation and recombination activities of the respective Kil and Gam functions. The expression level of the p(L) operon is down-regulated by Cro repressor. In the absence of Cro, higher p(L) expression levels allow either Kil or Gam to be lethal or growth inhibitory, whereas at lowered expression in Cro-repressed conditions, only Kil is lethal. The filamentation function of Kil and recombination activity of Gam are unaffected at Cro-repressed levels of expression.     

Cell surface glycosaminoglycans of a tumor cell line and its DNA transfected variant differing in their lung colonizing potential

A highly lung-colonizing cell line RMS/8² was obtained by DNA transfection from a low lung-colonizing line RMS/8, a clone of a rat rhabdomyosarcoma cell line. The cells were metabolically labeled with³H-glucosamine and³⁵S-sulfate. The newly synthesized pericellular glycosaminoglycans and the ability of the cells from the two lines to degrade extracellular matrix components were studied comparatively. The following conclusions were obtained: 1) Thein vitro proliferation rate is not a determinant in the modulation of the colonizing potential of these cells; 2) The strongly colonizing RMS/8² cells release more radioactivity from the radiolabeled extracellular matrix than their weakly colonizing counterparts; 3) The cells with a high colonizing potential incorporated less radioactivity into the cell surface glycosaminoglycans, and exhibited a lower heparan sulfate to chondroitin sulfate ratio than the weakly colonizing RMS/8 line.     

Treatment and prevention of experimental pneumonias, caused by L forms of bacteria]

On a model of experimental pneumonia in mice caused by the L-forms of bacteria against the background of diminished immunity a study was made of the therapeutic efficacy of penicillin, lincomycin, lysozyme and gamma-globulin. Lincomycin, particularly in combination with biologically-active preparations, proved to be expedience for the treatment and prophylaxis of pneumonia caused by the mentioned bacteria; a rapid arrest of pneumonic process occurred and more animals survived. In the greater percentage of cases the use of penicillin was accompanied by generalization of the process.     

Cell death caused by hyper-expression of a secretory exoglucanase in Escherichia coli

Induced expression of a gene fusion between the ompA leader sequence and the Cellulomonas fimi cex gene encoding a secretory exoglucanase, Exg, engineered in the Tac-cassette excretion vector was lethal to Escherichia coli. An exponentially growing culture harboring the recombinant construct suffered slow growth and 99.9% of its cells died within 60-100 min after induction. This abnormality was found to have a close correlation with the rapid increase in the relative amount of the OmpA/Exg fusion precursor (Pre-Exg) compared to its processed product (Mat-Exg). Analysis of subcellular fractions revealed the presence of Pre-Exg in the inner membrane of cultures expressing high levels but not low levels of Pre-Exg. As only Pre-Exg but not Mat-Exg was detectable in the cytoplasm, and Exg was shown by cross-linking experiments to be physically associated with the Sec proteins, it was concluded that secretion and processing of Pre-Exg took place in the SecYEG translocation machinery. The results were in line with the previous speculation that accumulation of unprocessed precursor proteins in the cytoplasmic membrane was detrimental, and supported the idea that cell death was caused by some unusual tie-up of Pre-Exg with the SecYEG translocation machinery, thus imposing an inhibitory effect on the secretion of endogenous secretory proteins. A new model, designated "Saturated Translocation," was proposed to explain the interchangeable lethal and non-lethal properties of Pre-Exg, and to address the possible scenarios that might occur in the course of cell death triggered by secretion of Pre-Exg.     

Extensive chromosome aberrations caused by [3H]thymidine incorporation in a diploid monkey cell line DBS-FRhL-2.

Extensive chromosome aberrations were induced in a diploid monkey cell line designated as DBS-FRhL-2 after exposure to [3H]thymidine ([3H]Tdr) for either 30 or 60 min at a dose of 1 muCi per ml of medium. Cultures exposed to [3H]Tdr for a longer period had significantly larger numbers of aberrations than those exposed for a shorter period. The most common type of aberrations were chromatid breaks. The majority of aberrations were observed in cells which were in contact with [3H]Tdr during S phase, especially the middle S. Cells from cultures of early and late passages exposed to [3H]Tdr were affected to a similar extent when chromosomes were examined. No clear relationship between sites of breakage and intensity of labeling could be established.     

Regulation of the peripheral body temperature by foods: a temperature decrease induced by the Japanese persimmon (kaki,Diospyros kaki)

We investigated whether the ingestion of the Japanese persimmon (kaki, Diospyros kaki) could lower the human peripheral body temperature. It was found that the temperatures recorded at the foot and wrist were depressed after kaki consumption compared to after the same amount of water consumption. The effects of ingesting freeze-dried kaki and eating a cookie (as its nutritional counterpart) containing the same amount of carbohydrate, protein, fat, and water were compared. A similar temperature-reducing effect of kaki was observed. The recovery of finger temperature after soaking the finger in ice-cooled water was also studied. The temperature recovery was delayed after kaki consumption. It was thus quantitatively demonstrated that ingesting kaki indeed had the effect of lowering (or repressing the rise) of the peripheral human body temperature, as has been traditionally believed in China for many hundreds of years.     

Mechanism of the decrease in hexose transport by mouse mammary epithelial cells caused by fasting.

The basal carrier-mediated uptake of 0.5 mM-3-O-methylglucose by mammary epithelial cells from lactating mice was calculated to be 227 +/- 9 pmol/min per microgram of DNA (mean +/- S.E.M., n = 11). Fasting the mice for 16 h overnight resulted in a decrease in this rate to 65 +/- 4 pmol/min per microgram of DNA (n = 10). Refeeding the fasted mouse for 3 h before isolation of the cells restored the transport activity to 230 +/- 12 pmol/min per microgram of DNA (n = 12). The Vmax. for equilibrium exchange entry of 3-O-methylglucose by intact cells was decreased from 6.6 +/- 0.4 to 0.9 +/- 0.2 nmol/min per microgram of DNA (mean +/- S.E.M., n = 3) by fasting. The number of D-glucose-inhibitable cytochalasin-B-binding sites in a plasma-membrane-enriched fraction of the cells was also decreased from 5.7 +/- 1.5 to 1.7 +/- 0.1 pmol/mg of membrane protein (mean +/- S.E.M., n = 3). Again, refeeding the fasted mouse for 3 h reversed both these effects. These results are consistent with a decrease in the number of functional glucose carriers in the plasma membrane of the mammary epithelial cells. Since the restoration of transporter activity after refeeding does not appear to require the synthesis of new protein, the effect of fasting probably involves not a loss of transporters, but a change in their orientation within the plasma membrane or a redistribution within the cell.