Kinetics of protein degradation in diploid and trisomic human fibroblasts

The degradation rate of long-lived and short-lived proteins was determined in diploid fibroblasts and fibroblasts with trisomy 7 derived from human embryos. Two fractions of proteins were detected in the exponentially growing diploid fibroblasts with half-lives (T 1/2) 37 and 19 hours. The rate of protein degradation increases in diploid fibroblasts as they approach confluence and protein fractions with T 1/2 30, 18 and 12 hours appear. The rate of protein degradation in trisomic fibroblasts does not change for the long-lived and short-lived proteins and is the same in both exponential (T 1/2 31 and 14 hours) and stationary phase (T 1/2 33 and 17 hours). The relative amount of the short-lived proteins in trisomic fibroblasts in the stationary phase decreased as compared with the one in diploid fibroblasts. It is apparent that a mechanism of regulation of protein catabolism in trisomic fibroblasts is impaired.     

Variation in S phase in synchronous human cell lines.

Growth parameters of diploid and trisomic human fibroblasts were determined. The rate of growth of both classes of cells was examined in asynchronous cultures, and diploid and trisomic cells had similar growth rates. Synchronous cultures were developed using simple mitotic selection. The patterns and length of the DNA synthetic period (S phase) were found to be altered in trisomy 21 cells when compared to diploid human or to heteroploid HeLa cells. Early S-phase synthesis was absent or reduced and the overall length of the S phase was extended. However, the trisomic cells have apparently normal rates of DNA chain elongation and normal replicon sizes.     

Variation in S phase in synchronous human cell lines

Summary Growth parameters of diploid and trisomic human fibroblasts were determined. The rate of growth of both classes of cells was examined in asynchronous cultures, and diploid and trisomic cells had similar growth rates. Synchronous cultures were developed using simple mitotic selection. The patterns and length of the DNA synthetic period (S phase) were found to be altered in trisomy 21 cells when compared to diploid human or to heteroploid HeLa cells. Early S-phase synthesis was absent or reduced and the overall length of the S phase was extended. However, the trisomic cells have apparently normal rates of DNA chain elongation and normal replicon sizes. This work was supported by the United States Department of Energy and the National Institutes of Health.     

Lysosomal function in the degradation of defective collagen in cultured lung fibroblasts

Human fibroblasts when induced to make nonhelical , defective collagen have mechanisms for degrading up to 30% of their newly synthesized collagen intracellularly prior to secretion. To determine if at least a portion of the degradation of defective collagen occurs by lysosomes, extracts of cultured HFL-1 fibroblasts were examined for proteinases capable of degrading denatured type I [3H]procollagen. The majority of the proteolytic activity against denatured [3H]-procollagen had a pH optimum of 3.5-4; it was stimulated by dithiothreitol and inhibited 95% by leupeptin, 10% by pepstatin, and 98% by leupeptin and pepstatin together. Extracts of purified lysosomes from the fibroblasts were active in degrading denatured [3H]procollagen and were completely inhibited by leupeptin and pepstatin. To demonstrate directly that human lung fibroblasts can translocate a portion of their defective collagen to lysosomes, cultured cells were incubated with cis-4-hydroxyproline and labeled with [14C]proline to cause the cells to make nonhelical [14C]procollagen. About 3% of the total intracellular hydroxy[14C]proline was found in lysosomes. If, however, the cells were also treated with NH4Cl, an inhibitor of lysosomal function, 18% of the intracellular hydroxy[14C]proline was found in lysosomes. These results demonstrate that cultured human lung fibroblasts induced to make defective collagen are capable of shunting a portion of such collagen to their lysosomes for intracellular degradation.     

Uniparental disomy for chromosome 16 in humans.

The association between chromosomal mosaicism observed on chorionic villus sampling (CVS) and poor pregnancy outcome has been well documented. CVS mosaicism usually represents abnormal cell lines confined to the placenta and often involves chromosomal trisomy. Such confined placental mosaicism (CPM) may occur when there is complete dichotomy between a trisomic karyotype in the placenta and a normal diploid fetus or when both diploid and trisomic components are present within the placenta. Gestations involving pure or significant trisomy in placental lineages associated with a diploid fetal karyotype probably result from a trisomic zygote which has lost one copy of the trisomic chromosome in the embryonic progenitor cells during cleavage. Uniparental disomy would be expected to occur in one-third of such cases. Trisomy of chromosome 7, 9, 15, or 16 is most common among the gestations with these dichotomic CPMs. Nine pregnancies with trisomy 16 confined to the placenta were prenatally diagnosed. Pregnancy outcome, levels of trisomic cells in term placentas, and fetal uniparental disomy were studied. Intrauterine growth retardation (IUGR), low birthweight, or fetal death was observed in six of these pregnancies and correlated with high levels of trisomic cells in the term placentas. Four of the five cases of IUGR or fetal death showed fetal uniparental disomy for chromosome 16. One of the infants with maternal uniparental disomy 16 had a significant malformation (imperforate anus). All infants with normal intrauterine growth showed term placentas with low levels of trisomic cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibroblast procollagen production rates in vitro based on [3H]hydroxyproline production and procollagen hydroxyproline specific activity

In vitro procollagen production rates can be determined by culturing cells in the presence of [3H]proline and measuring the subsequent formation of [3H]hydroxyproline. Values of actual procollagen production can be calculated if the total radioactivity and the specific activity of the newly synthesized procollagen is known. A simple microanalytical method for measuring procollagen specific activity in order to determine procollagen production by lung fibroblasts in vitro is reported. Confluent fibroblasts (IMR-90) were cultured in fresh medium containing [3H]proline, and [3H]hydroxyproline production and prolyl hydroxylation were measured. Hydroxyproline specific activity of nondialyzable procollagen in culture medium as well as extracellular and intracellular free proline specific activity were determined by an ultramicromethod in which the radiolabeled amino acids were reacted with [14C]dansyl chloride of known specific activity [Airhart et al. (1979) Anal. Biochem. 96, 45-55]. Procollagen production rates were readily determined by this method using 5 to 20 microCi [3H]proline and approximately 10(6) cells. It was found that 3H-procollagen production rate into culture medium was constant after a lag of 1.6 h, while procollagen production rate (0.23 pmol/microgram DNA . h) was constant from time zero to 9 h. The specific activities of extracellular and intracellular free proline were not constant during the labeling period, nor were they equal to procollagen specific activity. These data indicate that free proline pool specific activities are not a valid measure of procollagen specific activity. The experimental approach described obviates the need to define or characterize the proline precursor pool from which procollagen is synthesized, and may be readily applied to determine fibroblast procollagen production rates in vitro.     

Preliminary characterization of a Mg2+-ATPase in hamster sperm head membranes

The gene, IFRC, on human chromosome 21 has been considered, on the basis of indirect functional and antibody blocking evidence, to code for an interferon receptor. To obtain direct evidence for this conclusion, the specific binding of ¹²⁵I-HuIFN-αA to matched sets of fibroblasts diploid and aneuploid for chromosome 21 has been determined. Although the dissociation constant for IFN-α is the same for both trisomic and diploid cells, trisomic cells bind more IFN-α and monosomic cells less than do diploid cells. When the data for all determinations are combined, the monosomy 21:diploid:trisomy 21 binding ratios are 0.5:0.8:1.5, in good agreement with the values of 0.5:1.0:1.5 expected on the basis of strict gene dosage considerations for a product coded for by chromosome 21. We conclude, therefore, that the chromosome 21 gene product determined by IFRC and recognized by antibodies which block interferon action is truly a specific cell surface receptor for human interferon-α.     

Disturbances in collagen synthesis in trisomic cells from spontaneously aborted embryos

Summary Collagen synthesis in cells with trisomy 7 and 9 derived from human spontaneous abortuses was found to be lower (5.06% and 5.53% respectively) than in the control diploid cells (8.80%). The ratio of collagen types (I/III) in trisomic strains did not differ from the control data while the amount of the degraded procollagen in trisomic cells was increased.     

Decreased synthesis and increased intracellular degradation of newly synthesized collagen in freshly isolated chick tendon cells incubated with monensin

The synthesis and secretion of procollagen in embryonic chick tendon fibroblasts in suspension culture were inhibited with the carboxylic ionophore monensin. The synthesis of procollagen was inhibited by 50% in a 2-h exposure to 0.1 microM monensin and was inhibited by 70% in a 6-h exposure to 0.1 microM monensin. Secretion of procollagen was inhibited by greater than 90% in the 0.1 microM monensin-treated cultures and was totally inhibited by higher doses of the reagent. A cellular pool of collagenase-digestible peptides was demonstrated in the control cells, the level of which was elevated 3-4 times in the monensin-treated cultures. In order to determine whether the secretory and synthesis block caused by monensin inhibited intracellular degradation of newly synthesized collagen, the hydroxy[14C]proline in degraded collagen fragments present in control and monensin-treated cultures was determined and compared to the total hydroxy[14C]proline synthesized in each culture. The intracellular degradation of newly synthesized, pulse-labeled collagen was shown to proceed at rates comparable to those seen in the control cultures. The monensin-treated cells degraded pulse-labeled newly synthesized collagen nearly twice as long as the controls, resulting in an overall increase in the fraction of newly synthesized collagen that was degraded. These findings suggest that force generation in the activated cross-bridge cycle may occur as a result of an actin-attached cross-bridge transition between these two orientations.     

Damaging effect of 3H-uridine on tissue culture cells during the logarithmic and stationary phases of growth]

The Chinese hamster fibroblasts entered the stationary phase of growth after 5.5 days of cultivation. The induction of the culture proliferation occurred within the first 4 hrs of cultivation in the fresh nutrient medium. After addition of 3H-uridine in high concentration and with high specific activity (75 micron Cu/M and 23 Cu/mM), in the stationary phase a lesser number of cells with aberrations and a higher mitotic index were noted than in the logarithmic phase. 3H-uridine injured the cells in the stationary phase of growth but to a lesser extent than those in the logarithmic phase, i. e. during the intensive RNA synthesis in cells.     

Phosphofructokinase activity in fibroblasts aneuploid for chromosome 21

Summary Although the gene for the liver type (L) subunit of phosphofructokinase (PDK) is located on human chromosome 21 and PFKL subunits predominate in fibroblasts, an increase in PFK activity has not been reported in trisomy 21 fibroblasts. However, using well-matched pairs of trisomy 21 and diploid fibroblast strains, we observed an almost 1.5-fold increase in mean PFK activity of trisomic cells. In monosomy 21 fibroblasts we found an almost 0.5-fold decrease in mean PFK activity. Thus there appears to be a gene-dosage effect for the PFKL gene, as for other loci on chromosome 21. PFK activity in a cell strain deleted for the distal part of band 21q22.3 was not decreased, suggesting with other data that PFKL is located in the midportion of band 21q22.3.     

Fibroblast procollagen production rates in vitro based on [H]hydroxyproline production and procollagen hydroxyproline specific activity

In vitro procollagen production rates can be determined by culturing cells in the presence of [³H]proline and measuring the subsequent formation of [³H]hydroxyproline. Values of actual procollagen production can be calculated if the total radioactivity and the specific activity of the newly synthesized procollagen is known. A simple microanalytical method for measuring procollagen specific activity in order to determine procollagen production by lung fibroblasts in vitro is reported. Confluent fibroblasts (IMR-90) were cultured in fresh medium containing [³H]proline, and [³H]hydroxyproline production and prolyl hydroxylation were measured. Hydroxyproline specific activity of nondialyzable procollagen in culture medium as well as extracellular and intracellular free proline specific activity were determined by an ultramicromethod in which the radiolabeled amino acids were reacted with [¹⁴C]dansyl chloride of known specific activity [Airhart et al. (1979) Anal. Biochem. 96, 45–55]. Procollagen production rates were readily determined by this method using 5 to 20 μCi [³H]proline and approximately 10⁶ cells. It was found that ³H-procollagen production rate into culture medium was constant after a lag of 1.6 h, while procollagen production rate (0.23 pmol/μg DNA · h) was constant from time zero to 9 h. The specific activities of extracellular and intracellular free proline were not constant during the labeling period, nor were they equal to procollagen specific activity. These data indicate that free proline pool specific activities are not a valid measure of procollagen specific activity. The experimental approach described obviates the need to define or characterize the proline precursor pool from which procollagen is synthesized, and may be readily applied to determine fibroblast procollagen production rates in vitro.     

Synthesis of proline and hydroxyproline in human lung (WI-38) fibroblasts.

Human lung fibroblasts (WI-38) in late exponential phase of growth, in stationary phase after confluency was reached, and at high or low number of population doublings were used to investigate the synthesis of proline and hydroxyproline from glutamate or arginine. Glutamate was from two to five times as effective a precursor as arginine; glutamine did not seem to be involved in these metabolic pathways. Accumulation of protein-bound hydroxyproline in cell layers was observed only after confluency. Confluent cells synthesized more proline from glutamate than did cells in late exponential growth. Conversion of glutamate into intracellular free proline was conducted also to a greater extent in confluent cells at a high number of population doublings. Conversion of glutamate into proline or hydroxyproline in cell-layer protein was not affected significantly by the number of population doublings. Less total protein as well as less hydroxyproline accumulated with cells at a high number of population doublings.     

Dosage effects for superoxide dismutase-1 in nucleated cells aneuploid for chromosome 21.

Assays of the activity of chromosome 21 determined superoxide dismutase-1 (SOD-1) in lymphocytes and polymorphonuclear granulocytes have demonstrated 38% and 40% increases, respectively, in cells from individuals with trisomy 21. Similarly, SOD-1 activity in trisomic fibroblasts is increased by 81%, while cells monosomic for chromosome 21 have only 60% of normal activity. Taken together with the data on SOD-1 activities in trisomic erythrocytes and platelets, the present results firmly confirm the existence of a true dosage effect for this enzyme in cells aneuploid for chromosome 21. However, the results of assays of the activity of glutathione peroxidase in trisomic fibroblasts did not confirm the possibility previously reported of a chromosome 21 related dosage effect for this enzyme.     

Effects of preformed proline and proline amino acid precursors (including glutamine) on collagen synthesis in human fibroblast cultures

A technique of derivatizing proline and 4-hydroxyproline with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole was used to measure the radioactivities, concentrations and specific activities of proline and hydroxyproline. The technique was used to study the conditions of procollagen synthesis in cultured human foreskin fibroblasts. Procollagen synthesis appeared to be independent of the proline concentration in the medium, in the presence of glutamine, when monitored by the assay of non-dialyzable hydroxyproline, but not when monitored by [14C]proline incorporation. In the absence of unlabelled proline added to labelled proline in the medium, the specific activity of the secreted procollagen did not reach a plateau over a 24-h period. When the medium was supplemented with glutamine, glutamic acid, or aspartic acid, both the radioactivity and concentration of intracellular free proline decreased. Pyrrolidone-2-carboxylic acid and ornithine both induced a slight increase in concentration of the intracellular free proline. Glutamine competed with [14C]proline for incorporation into prolyl-tRNA and procollagen, independently of free intracellular proline, and it stimulated the biosynthesis of procollagen (expressed as non-dialyzable hydroxyproline) by a factor of 2.3.     

Trisomy 9 mosaicism in a girl with multiple malformations

A one-year-old girl with a mosaicism for an extra chromosome 9 is reported. Clinical findings included severe growth and mental retardation, frequent respiratory infections, peculiar face, skeletal and craniofacial abnormalities, seizures, spasticity, cardiopulmonary, gastrointestinal and genitourinary alterations. These findings were compared to those of the 10 other previously reported cases of trisomy 9 mosaicism. This helps to define the most constant phenotypical characteristics and most frequent major malformations which occur in trisomy 9 mosaicism. It is noteworthy that the reported percentage of trisomic cells was different in lymphocytes and in fibroblasts in each case.     

Trisomics from triploid-diploid crosses in self-incompatible Lycopersicum peruvianum

Summary The transmission rate of trisomy was determined for two primary trisomic types, triplo-1 and triplo-3, of the self-incompatible species Lycopersicum peruvianum. Chromosome counts in somatic metaphases of root-tip squashes from 112 progeny plants showed that 8 individuals (7.2 %) were trisomic and 104 (92.8%) were diploid. The average frequency of transmission approximated 2.6% in triplo-1 and 8.6% in triplo-3. Data are presented on the karyotype and the morphological features of the 8 trisomics detected in the progenies of triplo-1 and triplo-3 and the various factors affecting the transmission rate of trisomy are discussed.The transmission rate of trisomy was also determined for the trisomic plant 269 which displayed a complete deletion of the satellited part of chromosome 2 and was characterized by ovate fruits. Out of 18 progeny plants analysed, 8 (44.4%) were trisomic and 10 (55.6%) were diploid. Cytological and morphological analyses of the 8 trisomic individuals revealed that only two of them (11.1 %) resembled the parental trisomic. A number of diploid and trisomic progenies exhibited a partial or a complete deletion of the satellited segment of chromosome 2.This work has been supported by a contract between the European Communities and the CNEN. This publication is contribution n ° 484 from The Division Applicazioni delle Radiazioni del CNEN and contribution n ° 1482 from the Biology Radioprotection Medical Research programme of the Directorate General XII of the European communities     

Characteristics of DNA replication in the long-term culture of human cells at the stationary phase

Peculiarities of DNA replication in cultured human diploid fibroblasts in logarithmic and stationary phases were studied using DNA autoradiography. The rate of DNA replication fall from 30-36 mu/hour at active proliferative phase to 18-20 mu/hour at late stationary phase. This phenomenon is characteristic of stationary cultures after stimulation to proliferate by changing medium as well as by culturing without stimulation. Possible mechanisms of DNA replication rate alteration in senescent human cells are discussed.     

Growth phase regulation of the main folate transporter of Leishmania infantum and its role in methotrexate resistance

The protozoan parasite Leishmania relies on the uptake of folate and pterin from the environment to meet its nutritional requirements. We show here that a novel gene (folate transporter 1 (FT1)) deleted in a Leishmania infantum methotrexate-resistant mutant corresponds to the main folate transporter (K(m), 410 nM). FT1 was established as the main folate transporter by both gene transfection and by targeted gene deletion. Modulation of the expression of FT1 by these manipulations altered the susceptibility of Leishmania cells to methotrexate. Folate transport was stage-regulated with higher activity in the logarithmic phase and less in the stationary phase. FT1 fused to green fluorescent protein led to the observation that FT1 was located in the plasma membrane in the logarithmic phase but was retargeted to an intracellular organelle followed by a degradation of the protein in stationary phase. Leishmania has several folate transporters with different characteristics, and the growth stage-related activity of at least one transporter is regulated post-translationally.