bluuebunny/crossref_metadata_2025 huggingface dataset:
Centrifugal elutriation: Separation of spermatogenic cells on the basis of sedimentation velocity
Authors: Grabske Robert J. , Lake Suzanne , Gledhill Barton L. , Meistrich Marvin L.
Published: February 2005

Various types of cells from the testes of mice and hamsters were separated according to differences in sedimentation velocity by centrifugal elutriation, a counterflow centrifugation technique. Approximately 3 × 108 cells, prepared from six mouse testes or from one hamster testis, were separated into 11 fractions in less than two hours as compared to the 4–5 hours required for sedimentation at unit gravity (“Staput”). Fractions enriched in elongated spermatids and spermatozoa (100%), stages 1–8 spermatids (69%) and pachytene spermatocytes (58%) were obtained from mouse testis dispersions. Similarly enriched fractions were obtained from hamster cells. A single fraction enriched in stages 1–8 spermatids (mouse) was prepared in less than 30 minutes. As many as 2 × 109 cells were separated in a single procedure. Spermatogenic cells exhibited no evidence of structural damage with trypan blue and phase microscopy, and recovery was essentially 100%. Centrifugal elutriation had no effect on sperm motility or on the plating efficiency of CHO cells.
Passive cation movements in the ehrlich ascites tumor cell: The effects of 2,4,6‐trinitrobenzene sulfonic acid
Authors: Smith Thomas C. , Adams Ramona
Published: February 2005

We have investigated the effects of 2,4,6‐trinitrobenzene sulfonic acid (TNBS), an amino reactive reagent, on passive cation movements in Ehrlich ascites tumor cells. Incubation of tumor cells with TNBS (3 mM) results in a two phase association of TNBS with the cells. An initial, rapid phase, presumably at the level of the membrane, is independent of temperature, while the second phase increases linearly in time and is temperature dependent. Kinetic analyses of Na+ movements indicate that TNBS: (1) inhibits Na+ movement from a slowly exchanging cellular compartment, but is without effect on a more rapidly exchanging compartment; (2) does not alter net Na+ accumulation in transport‐inhibited cells; and (3) is without effect on non‐exchange Na+ efflux at 0°C. The actions of TNBS on K+ movements depend upon temperature and the continued presence of TNBS in the environment. At 22°C two minute exposure of the cells to TNBS leads to 77% inhibition of K+ efflux. With continued exposure to TNBS, the inhibition is only 42%. Reduction of the temperature to 0°C decreases K+ efflux in control cells by 82%. Two minute exposure to TNBS enhances K+ efflux by 50%, while continuous exposure increases it by 144%. These results suggest: (1) TNBS interacts with several classes of membrane sites which are involved with the regulation of passive cation movements; and (2) passive Na+ and K+ movements across the cell membrane proceed by different pathways.
Increasing content of poly A(+) mRNA of serum‐stimulated cells in the absence of ribosome synthesis
Authors: Johnson Lee F. , Penman Sheldon , Green Howard
Published: February 2005

When 3T6 cells undergo a serum‐induced transition from resting to growing state, the number of ribosomes and the amounts of mRNA and tRNA increase as the cells prepare for DNA synthesis. We have examined the effect of preventing ribosome synthesis during this transition. When resting cells are stimulated to grow in the presence of 5‐fluorouridine, mRNA accumulates normally during the first eight hours, though new ribosome formation is completely blocked by the drug. At later times, mRNA continues to accumulate, but at a reduced rate. The ratio of poly A(+) mRNA to rRNA increases from the value characteristic of resting 3T6 (1.8%) to that of growing 3T6 (2.7%) by five hours, and continues to increase to abnormally high values after this time.

Although labelling of tRNA is not affected after brief exposure of cells to fluorouridine, the drug prevents the later accumulation of tRNA that ordinarily occurs following serum stimulation of resting cells. This failure of accumulation is not the result of increased lability of fluorinated tRNA, but is probably due to failure of the transcription rate of pre‐tRNA to increase. It is possible that this effect might be due to a regulatory system coupling tRNA content to ribosome content.

In cultures stimulated with serum in the presence of fluorouridine the rate of protein synthesis increases with poly A(+) mRNA content during the first eight hours; it then fails to increase further, possibly because ribosomes become rate‐limiting.
The enhanced production of hyaluronic acid by cultured rat fibroblast cells treated with cyclic AMP and its dibutyryl derivative
Authors: Koyama Hideki , Tomida Mikio , Ono Tetsuo
Published: February 2005

Cells of a newly established rat fibroblast line (SEN) in culture synthesize mucopolysaccharides, which have been identified as hyaluronic acid, chondroitin‐4‐sulfate and heparan sulfate. Treatment of the cells with adenosine 3′:5′‐cyclic monophosphate resulted in a marked stimulation of production of hyaluronic acid, but not of the other mucopolysaccharides. Treated cells also showed increased activity of hyaluronic acid synthetase, a reduction in growth rate, and morphological alteration. In addition, 5‐bromodeoxyuridine was found to counteract greatly the cyclic AMP effect.
Stimulation of melanotic expression in a melanoma cell line by theophylline
Authors: Steinberg Mark L. , Whittaker J. R.
Published: February 2005

Theophylline, a phosphodiesterase inhibitor, was found to be a potent stimulator of melanogenesis in the RPMI 3460 hamster melanoma cell line. This stimulation was greater than that caused by either dibutyryl cyclic AMP (db‐cAMP) or another phosphodiesterase inhibitor, papaverine. Theophylline and db‐cAMP treatments also produced strikingly different morphologies in the monolayered cells. The theophylline effect on melanogenesis was diminished by db‐cAMP, whereas simultaneous treatment of cells with db‐cAMP and papaverine produced greater stimulation of melanotic activity than either agent acting alone. Theophylline, therefore, may have phenotypic effects that are at least partially independent of phosphodiesterase inhibition. Theophylline stimulated melanin biosynthesis, as measured by rates of 2‐ [2‐14C] thiouracil incorporation, and also caused an increase in the level of tyrosinase (EC 1.10.3.1) activity. This melanotic stimulation was prevented by the presence of cordycepin or cycloheximide. Theophylline inhibited DNA synthesis and mitosis in the melanoma cell cultures but stimulated protein synthesis. However, inhibition of proliferation and the first appearance of induced melanotic activity did not bear an immediate direct relationship to one another.
Differences in the incorporation of thymidine into DNA of normal and cystic fibrosis fibroblasts in vitro
Authors: Barranco S. C. , Bolton W. E. , Haenelt B. R. , Abell C. W.
Published: February 2005

Although similar fractions of cells were in the S phase of the cell cycle, normal human skin fibroblasts were shown to incorporate more than twice the 3HTdR into their DNA in vitro than did cells obtained from individuals with cystic fibrosis (CF). Obligate heterozygotes incorporated an intermediate amount of the DNA precursor. Studies were initiated to determine the basis of the differential incorporation of 3HTdR among the genotypes. An analog of thymidine, BUdR, produced varied effects on the growth kinetics of the three genotypes. The growth of cells in BUdR resulted in a 50% increase in the population doubling times of all three genotypes, and caused the cell morphology to change from a spindle shape to one in which the cells became broadened and flat, with numerous cytoplasmic projections extending for distances of several cell diameters. The activities of thymidine kinase and the participation of the exogenous and de novo pathways in the synthesis of TMP were found to be approximately the same in all three genotypes. The data suggest that an alteration in the transport of thymidine into the cells may account for the differences in TdR incorporation into DNA, and this may be associated with other changes in cystic fibrosis that are apparently membrane associated.
Turnover of nicotinamide adenine dinucleotide in cultures of human cells
Authors: Rechsteiner Martin , Hillyard David , Olivera Baldomero M.
Published: February 2005

The rate of turnover of nicotinamide adenine dinucleotide (NAD) in the human cell line, D98/AH2, has been estimated by measuring the rates of entry into and exit from NAD molecules of 14C‐adenine. In one set of experiments, cells were labeled by growth in medium containing 14C‐adenine for six hours and then shifted to medium without labeled adenine. The loss of 14C‐adenine from the adenine nucleotide and pyridine nucleotide pools was measured, and the data were analyzed using an analytical treatment which corrects for the relatively slow turnover of precursor pools. The loss of 14C‐adenine from the NAD pool and from the precursor ATP pool could be related to the absolute rate of NAD breakdown. Under the experimental conditions used, the rate of NAD turnover ranged from 83,000 to 126,000 molecules per second per cell. In a complementary experiment cells were grown in the presence of unlabeled adenine, then shifted into medium containing 14C‐adenine and the rate of entry of 14C‐adenine into adenine and pyridine nucleotides was measured. The data were treated using a similar analysis to relate the rate of entry of 14C‐adenine into NAD and the precursor ATP pools to the absolute turnover rate of NAD. This analysis gave a value for NAD turnover of 78,000 molecules per second per cell in excellent agreement with results from the pulse‐chase experiments. The results from both types of experiment indicate that within D98/AH2 cells the half‐life of an intact NAD molecule is 60 ± 18 minutes. Thus, in a human D98/AH2 cell growing with a generation time of 24 hours, NAD is turning over at twice the rate found in Escherichia coli with a generation time of half an hour.
Human epidermal growth factor and the proliferation of human fibroblasts
Authors: Carpenter Graham , Cohen Stanley
Published: February 2005

The effect of human epidermal growth factor (hEGF), a 5,400 molecular weight polypeptide isolated from human urine, on the growth of human foreskin fibroblasts (HF cells) was studied by measuring cell numbers and the incorporation of labeled thymidine. The addition of hEGF to HF cells growing in a medium containing 10% calf serum resulted in a 4‐fold increase in the final density. The presence of hEGF also promoted the growth of HF cells in media containing either 1% calf serum or 10% gamma globulin‐free serum. The addition of hEGF to quiescent confluent monolayers of HF cells, maintained in a medium with 1% calf serum for 48 hours, resulted in a 10‐ to 20‐fold increase in the amount of 3H‐thymidine incorporation after 20–24 hours. The stimulation of thymidine incorporation was maximal at an hEGF concentration of 2 ng/ml, was dependent on the presence of serum, and was enhanced by the addition of ascorbic acid. In confluent cultures of HF cells, subject to density dependent inhibition of growth, hEGF was able to stimulate DNA synthesis more effectively than fresh calf serum. Human EGF stimulated DNA synthesis in quiescent cultures, however, regardless of cell density. The addition of rabbit anti‐hEGF inhibited all effects of this growth factor on HF cells.
Carbohydrate preferences of mammalian cells
Authors: Burns Richard L. , Rosenberger Pamela G. , Klebe Robert J.
Published: February 2005

Carbohydrate preferences of mammalian cells can be utilized to biochemically distinguish between different cell lines. Ninety‐three carbohydrates were examined of which (a) 15 supported cell proliferation and (b) 42 were toxic or growth inhibitory. The present investigation has employed an enzymatic system to eliminate trace glucose levels from reagents and glucose generated by serum enzymes.
Ouabain‐resistant human lymphoblastoid lines altered in the (Na+ + K+)‐dependent ATPase membrane transport system
Authors: Lever Julia E. , Seegmiller J. Edwin
Published: February 2005

Diploid human lymphoblastoid cells with altered response to ouabain inhibition of the (Na+ + K+)‐dependent ATPase transport system, manifest both in whole cells and in purified plasma membrane vesicles, were selected for their resistance to 0.1 m̈M ouabain. Ouabain‐resistant (OUAR) cells with normal growth at 50 times this dose were recovered at a frequency of 1 × 10−6. This frequency was increased 9‐fold after exposure to ethyl methane sulphonate but was decreased by the frameshift mutagen ICR‐191, under conditions where both increased the frequency of 8‐azaguanine‐resistant colonies. The ouabain resistance phenotype was stable after 200 population doublings in the absence of ouabain. OUAR clones showed 30–50% of the wild type amount of 3H‐ouabain bound per cell, with the same dissociation constant for ouabain, 0.1 m̈M at 0.5 mM K+, as observed in wild‐type cells. Both the initial rate of uptake of 86Rb+ in OUAR cells and the (Na+ + K+)‐dependent ATPase activity of OUAR plasma membranes showed decreased sensitivity to ouabain inhibition. However, growth and transport properties of OUAR cells in the absence of ouabain were unchanged compared with wild type cells.
The repair of the surface structure of animal cells
Authors: Buck Clayton A. , Warren Leonard
Published: February 2005

Experiments were performed to determine if animal cells in culture possess specific mechanisms to repair surface molecules damaged by enzymes. The surface membranes of a primary cell culture, chick fibroblasts, a permanent hamster cell line, BHK21/C13, and its virally transformed counterpart, C13/B4 were damaged by exposure to trypsin or to neuraminidase. Following digestion with trypsin, the incorporation of radioactive amino acids or sugars into purified surface membrane of cells was monitored. No differences were noted in rates of incorporation when control and trypsin‐damaged cells were compared. Neuraminidase damage to the surface of BHK21/C13 and C13/B4 cells was evidenced by altered gel filtration profiles of surface glycopeptides, i.e., delayed elution because of reduction in size. By labelling cells with 14C‐L‐fucose prior to neuraminidase treatment and following the incorporation of 3H‐L‐fucose into cell surface glycopeptides after neuraminidase digestion, we were able to monitor the synthesis and turnover of fucose‐containing glycopeptides in the same cells. Gel filtration profiles indicated that little or no desialylated glycoproteins were resialylated (repaired) by specific replacement of sialic acid. Comparing neuraminidase‐digested and control cells we observed no difference in rates of 3H‐L‐fucose incorporation or of 14C‐L‐fucose loss from these cells; nor did we find differences in the rate of incorporation of isotopic glucosamine into sialic acid. Neuraminidase treatment failed to alter the rate of cell growth or the pattern of isotopic incorporation into various cell surface components. These results support the suggestion that return of sialic acid (repair) was effected by turnover which serves as a non‐specific repair mechanism to replace damaged cell surface molecules (Warren and Glick '68; Warren, '69).
Purine uptake by azaguanine‐resistant chinese hamster cells
Authors: Parsons Marilyn , Morrow John , Stocco Douglas , Kitos Paul
Published: February 2005

In this study the resistance of a number of lines of Chinese hamster ovary cells to azaguanine is examined. Those which are drug resistant by virtue of a deficiency of hypoxanthine‐guanine phosphoribosyltransferase (HPRT) fail to take up any exogenous hypoxanthine or azaguanine. A second class of drug resistant cells which grow in the reverse selective HAT medium and have levels of HPRT in the range of the wild type parent line take up these purines at lower rates than the non‐resistant cells and incorporate smaller amounts of them into trichloracetic acid‐insoluble constituents. The results suggest that their basis for resistance resides in lowered incorporation of azaguanine into DNA and RNA, possibly due to a mofified HPRT molecule which accepts hypoxanthine, but not azaguanine as a substrate.
Control of normal differentiation of myeloid leukemic cells. XI. Induction of a specific requirement for cell viability and growth during the differentiation of myeloid leukemic cells
Authors: Fibach Eitan , Sachs Leo
Published: February 2005

Normal hematopoietic cells require the presence of a protein (MGI) in the appropriate conditioned medium (CM) for cell viability and growth and for differentiation to mature macrophages and granulocytes. Clones of myeloid leukemic cells have been established in culture (D+ clones) which require CM with this protein for differentiation, but not for cell viability and growth. It has been shown that these leukemic cells can be induced by CM to again require, like normal cells, the presence of CM for cell viability and growth. Induction of this requirement, which will be referred to as RVG, occurred before the D+ cells differentiated to mature granulocytes. Clones of myeloid leukemic cells (D− clones) that could not be induced to differentiate to mature cells, did not show the induction of RVG. The steroid hormones prednisolone and dexamethasone can induce some, but not all the changes associated with differentiation of D+ cells. Incubation with these steroids did not result in the induction of a requirement for these steroids for cell growth and viability. Studies with CM from different sources have shown, that all batches that induced RVG also induced differentiation of D+ cells and that both activities were inhibited after treating the CM with trypsin. It is suggested that the same protein (MGI) may be involved in both activities. Incubation of D+ cells with CM resulted in an increase in agglutinability by concanavalin A and this increase was maintained even in the absence of CM. This suggests, that the induction of RVG in D+ myeloid leukemic cells is associated with a change in the cell surface membrane.
Erythropoietin sensitivity as a differentiation marker in the hemopoietic system: Studies of three erythropoietic colony responses in culture
Authors: Gregory C. J.
Published: February 2005

A time course study of the sequential appearance of erythropoietin‐dependent colonies and bursts (derived from CFU‐E and BFU‐E, respectively) was performed on mouse hemopoietic cells cultured in methyl cellulose containing 2‐mercaptoethanol. A new type of small, short‐lived burst was found to be apparent by the third day in culture. By the sixth day most of these bursts had lysed. At the same time, differentiating erythroblasts began to be detectable in the large, late appearing bursts described previously.

These two types of burst, differing from each other and from CFU‐E derived colonies both in their ultimate size and morphology, as well as in their time course of appearance and lysis, were compared in other ways. It was found that early burst formation required about 100 times more erythropoietin than that needed to stimulate CFU‐E. On the other hand, early burst formation required less than one‐quarter of the amount of erythropoietin needed to obtain the large, late appearing bursts. Comparison of the distribution of early burst progenitors relative to pluripotent stem cells (CFU‐S) in individual spleen colonies gave a correlation coefficient that was also intermediate between that obtained comparing CFU‐S with CFU‐E and that obtained comparing CFU‐S with the progenitors of late bursts. These results suggest that decreasing proliferative capacity is associated with progressively increasing erythropoietin responsiveness as primitive erythropoietic progenitors move from a position close to pluripotent stem cells through several differentiation steps to reach a stage just prior to the onset of detectable hemoglobin synthesis.
Regulation of lipid synthesis from acetate in diploid fibroblast cultures —variation with passage level and stage of cell growth
Authors: Howard Barbara V. , Howard William J. , Kefalides Nicholas A.
Published: February 2005

Regulation of lipid synthesis from acetate in human diploid fibroblast cultures has been studied at various passage levels and at different stages of cell growth. When cultures were transferred to lipid free medium, a stimulation of [14C]acetate incorporation into lipid occurred within three to six hours after removal of exogenous lipid. In early passage cultures, this stimulation was observed whether cells were transferred to protein‐free medium or medium supplemented with delipidized serum protein. However, in late passage cultures the presence of delipidized serum protein was required for the stimulation of lipid synthesis. When logarithmically dividing and stationary phase cultures were compared, the cultures in log phase showed stimulation of acetate incorporation into lipid in the presence or absence of delipidized serum protein, whereas in the stationary cultures the delipidized serum protein was required. When cultures were partially synchronized by a thymidine block, stimulation of acetate incorporation into lipid in the blocked cells only occurred in the presence of delipidized serum protein; in released cells stimulation occurred in protein free medium. When inhibition of lipid synthesis from acetate was compared in young vs. old or dividing vs. stationary cultures, however, no differences were observed. The data indicate the response of diploid fibroblast cultures to change in exogenous lipid is dependent on passage level and state of growth.
Growth of cultured cells from patients with ataxia‐telangiectasia
Authors: Elmore Eugene , Swift Michael
Published: February 2005

Fibroblast cultures derived from skin biopsies of patients with ataxia‐telangiectasia had doubling times (mean of five lines: 29.9 ± 0.6 hours) significantly longer than normal controls (means of ten lines: 22.4 ± 0.5 hours).
A possible involvement of polyamines in the initiation of DNA synthesis by human WI‐38 and mouse BALB/3T3 cells
Authors: Boynton A. L. , Whitfield J. F. , Isaacs R. J.
Published: February 2005

Changing the medium, or adding fresh serum, induces a large proportion of the proliferatively quiescent cells in confluent monolayers of human WI‐38 and mouse BALB/3T3 cells to initiate a growth‐division cycle. Exposure at the time of the medium change or serum addition to MGBG (methyl glyoxal bis [guanylhydrazone]), an inhibitor of spermidine and spermine synthesis and function, reduces or stops the subsequent flow of cells into the DNA‐synthetic phase, without grossly affecting RNA synthesis. Mediation of MGBG action by an actual or functional shortage of spermidine or spermine (but not putrescine), and consequently an involvement of these polyamines in DNA synthesis, is strongly suggested by the reduction of the inhibitor's effectiveness by a brief (1‐hour), early prereplicative exposure of the treated cells to exogenous spermidine and spermine (but not putrescine).
Cell envelope proteins involved in the transport of maltose and sn‐glycerol‐3‐phosphate in Escherichia coli
Authors: Boos Winfried
Published: February 2005

Two types of proteins are discussed in their role of facilitating the transport of maltose and sn‐glycerol‐3‐phosphate in E. coli. The first protein is the receptor for phage δ, known to be an outer membrane protein. By facilitating the diffusion of maltose and the higher maltodextrins through the outer membrane the effect of the δ receptor is to decrease the Km of the transport system without influencing the Vmax of substrate flux.

The second protein is a periplasmic protein that is induced by growth on glycerol and is essential for transport of sn‐glycerol‐3‐phosphate in whole cells but not in membrane vesicles. This protein has solely been identified by the use of a two‐dimensional polyacrylamide gel electrophoresis of periplasmic proteins in wild‐type and mutants defective in sn‐glycerol‐3‐phosphate transport.
Regulation of sugar transport in chick embryo fibroblasts and in fibroblasts transformed by a temperature‐sensitive mutant of the rous sarcoma virus
Authors: Kletzien Rolf F. , Perdue James F.
Published: February 2005

The mode of induction of sugar transport by serum‐stimulation of growth and hexose‐starvation in chick embryo fibroblasts (CEF) has been studied using metabolic inhibitors. We have concluded from these studies that the sugar transport increases induced by serum‐stimulation are regulated by post‐transcriptional and post‐translational mechanisms while sugar transport increases induced by hexose‐starvation are regulated by a transcriptional mechanism. CEF infected with a temperature‐sensitive mutant of the Rous sarcoma virus, Ts68, and incubated at the nonpermissive temperature for transformation, 41°, retain the capacity to regulate sugar transport in a manner similar to uninfected CEF. However, Ts68‐infected CEF maintained at the permissive temperature for transformation, 37°, have lost the ability to regulate sugar transport at the post‐transcriptional and post‐translational levels.
Loss of the post‐translational control of nutrient transport in in vitro and in vivo virus‐transformed chicken cells
Authors: Perdue James F.
Published: February 2005

The removal of serum from the medium of uninfected fibroblasts decreased the rate of uptake of uridine, 2‐deoxyglucose, α‐aminoisobutyrate and thymidine. Its subsequent addition rapidly and reversibly stimulated the uptake of all the nutrients but thymidine and this response was not inhibited by treatment of the cells with cycloheximide. The cycloheximide insensitive, rapid increase in the rate of transport has been designated post‐translational control. The nutrient transport systems in chick embryo fibroblasts transformed in vitro with avian sarcoma viruses and virus‐induced cultured chicken tumor cells do not respond to serum removal or addition. Two possible levels for the control of nutrient transport, i.e., mitogen receptor occupancy and mitogen‐induced activation of the transport system, are presented to explain these observations.
Saturable and nonsaturable process of sugar uptake: Effect of oncogenic transformation in transport and uptake of nutrients
Authors: Hatanaka Masakazu
Published: February 2005

Uptake of sugars into cells by a saturable process increased enormously during and after transformation, and uptake by a nonsaturable process increased significantly but less remarkably compared to controls. The drastic change of uptake rates, observed at around 5 × 10−3 M sugar during and after transformation, emphasizes the significant observation that transition of the sugar uptake system from a saturable to a nonsaturable process occurs near the physiological concentration of D‐glucose normally seen in animal blood. At concentrations higher than 5 × 10−3 M, where a saturable process is barely involved, nonsaturable uptakes of D‐glucose, D‐mannose, D‐galactose, 2‐deoxy‐D‐glucose and 3‐O‐methyl‐D‐glucose proceed tens to hundreds fold faster than the rate of simple diffusion of L‐glucose.

These findings suggest that nonsaturable uptake of the sugars known to be substrates for the saturable transport carrier system may not be a physical process or simple diffusion, as observed for L‐glucose uptake. Rather, the nonsaturable uptake might be part of the total physiological process which, along with the saturable process, is controlled by a membrane‐coordination mechanism.

A plausible mechanism is discussed in which negative cooperativity of nutrient uptake, such as that found in bacteria, is involved.
Low‐ and high‐affinity concanavalin A binding to thymocyte plasma membrane vesicles
Authors: Wallach Donald F. H. , Schmidt‐Ullrich Rupert
Published: February 2005

Binding of concanavalin A to isolated thymocyte membrane vesicles occurs through (a) numerous (∼6 × 106/cell equivalent) low‐affinity sites (Ka = 1.3 × 105 M−1) and (b) fewer (∼0.4 × 106/cell equipment) specific receptors (Ka = 6.8 × 106 M−1) defined as 55,000 D glycoprotein and its multimers. Specific binding is positively‐cooperative, with a Hill coefficient of∼1.8. Low concentrations of glutaraldehyde selectively crosslink the 55,000 D glycoprotein with replacement of positively‐cooperative sites by high‐affinity sites. It is proposed that concanavalin A‐binding induces multimerization of the 55,000 D glycoprotein.
Regulation of plasma membrane protein phosphorylation in two mammalian cell types
Authors: Avruch Joseph , Fairbanks Grant , Crapo Lawrence M.
Published: February 2005

The appreciation of protein phosphorylation as a ubiquitous mechanism for the post‐translational control of protein function has drawn our attention to the phosphorylation of plasma membrane proteins. We have studied this phenomenon in the human erythrocyte and rat adipocyte, and have observed several features, common to the two systems, which may be of general significance. In examining protein phosphorylation in intact cells incubated with 32Pi, it is evident that the 32P‐polypeptides of the plasma membrane are among the most highly labelled species in the cell, despite their minor contribution to overall protein content. The addition of epinephrine (to adipocytes) or cAMP

The following abbreviations are used: cAMP, adenosine 3′:5′‐cyclic monophosphate; DBcAMP, N6, O2′‐dibutyryl adenosine 3′:5′‐cyclic monophosphate; HEPES, N‐2‐hydroxyethylpiperazine‐N1‐2‐ethanesulfonic acid; SDS, sodium dodecyl sulfate.

(to erythrocytes) increases the phosphorylation of certain peptides, whereas others are unaffected. The protein kinases mediating these phosphorylations are present in the plasma membrane as isolated, and can be divided into two groups–‐cAMP dependent and cAMP independent. These two classes of kinase differ markedly in their substrate specificity toward endogenous and exogenous polypeptide substrates. Two classes of protein kinases with similar properties can be detected in the cytoplasm. The relationship between the membrane‐bound and cytoplasmic enzymes is uncertain.

The potential roles of the plasma membrane cAMP dependent protein kinases are evident from the diverse effects of cAMP on surface properties; however, the prevalence of plasma membrane proteins phosphorylated via cAMP independent pathways is striking. Thus, elucidation of the regulatory properties of the plasma membrane cAMP independent protein kinases may give new insight into the control of a variety of surface phenomena not mediated by cAMP.
The mechanism of interaction between high‐affinity probes and the uridine transport system of mammalian cells
Authors: Eilam Yael , Cabantchik Ioav
Published: February 2005

The carrier of uridine transport in hamster cells in culture is highly susceptible to the inhibitory effect of probes like S‐benzylated derivatives of mercaptopurine nucleosides. The interaction between the probes and the carrier is competitive and reversible and it takes place at a site different from the substrate binding site. The Ki for the most potent deravative p‐nitrobenzyl‐6‐mercaptoinosine is 0.15 n Molar at 20°C. The effect of the probes is interpreted in terms of a conformational change induced on the carrier upon binding of the probe. The carrier assumes distinct conformations depending on whether it is probe‐free (form A) or probe bound (form B).

Kinetic as well as chemical evidence supports the predictions of the allosteric carrier model. A single component of kinetics is observed either in the absence of inhibitor (Km form A) or at high concentrations of inhibitor (Km form B). A two component kinetics is observed at intermediate concentrations of inhibitor (some carriers in form B and others in form A). The two forms have distinct Km values for uridine: form A 50 μMolar and form B 250 μMolar. The two forms have also different susceptibilities to the action of organomercurials: form A is insensitive whereas form B is highly inhibited by the chemical modifier of SH groups.

The existence of putative allosteric sites in carriers is discussed in terms of modifier sites capable of modulating transport activities as a result of specific membrane‐ligand interactions.
Carchesium stalk fibrillar matrix as a highly filled polymer network
Authors: Hawkes Richard
Published: February 2005

Glycerolated stalks of the sessile peritrich ciliate Carchesium sp. were treated with 10−6 g ion/1 Ca2+ to disrupt the contractile spasmoneme. The resulting preparation consisted primarily of the fibrillar matrix, a dense extracellular meshwork of microfibrils. Some mechanical properties of this preparation have been investigated. The matrix tensile force‐extension ratio relation for an initial stretch was characteristic of a soft, swollen polymer network, elastic modulus in young stalks 1.7 × 105 Nm−2, in mature stalks 4.0 × 105 Nm−2. The higher elastic modulus in mature stalks implies an increase in the interchain cross‐link frequency. In young stalks, elastic modulus was found to be independent of the ambient Ca2+ concentration in the threshold range for spasmonemal contraction. Stalk relaxation was pronouncedly irreversible, showing stress softening and permanent hysteresis on repeated loading. Hysteresis was time independent and stiffness was not recovered after four hours at zero strain. Hysteresis was enhanced by repeated loading to the same tensile force. Stress‐strain hysteresis at a low extension ratio is characteristic of highly filled polymer networks in which polymer chains are interconnected via rigid filler particles as well as directly cross‐linked.
Cytotoxicity of ascorbate and other reducing agents towards cultured fibroblasts as a result of hydrogen peroxide formation
Authors: Peterkofsky Beverly , Prather Willie
Published: February 2005

Several types of cultured fibroblasts, including chick embryo, human and mouse, were killed by the addition of sodium ascorbate at final concentrations of 0.05–0.25 mM to cultures at the time of inoculation or to attached cells. Ascorbate did not affect the attachment of cells to the substratum. The effect on chick embryo fibroblasts was visible by fours hours and by six hours almost all cells had swelled and were becoming detached. By 24 hours detached cells had either lysed or become crenated in appearance. Other end‐diol reducing agents and also glutathione and cysteine were effective while gulonolactone, a non‐reducing analogue of ascorbate, was ineffective. Preincubation of medium containing ascorbate but no cells, conditions which result in degradation of the vitamin, led to loss of toxicity, indicating that a degradation product was not the lethal agent and that a component of the medium was not converted to a lethal substance. The lethal effect of both ascorbate and glutathione was prevented by the addition of catalase to the medium, suggesting that H2O2 formed by intracellular reactions and then excreted into the medium was the cytotoxic agent. This conclusion was supported by the findings that 0.05 mM H2O2 added to chick embryo fibroblasts was lethal and that the effect of this compound on cellular morphology was almost identical to that of ascorbate.
The in vivo effect of dimethyl sulphoxide (DMSO) on protein synthesis and the polyribosome profile in paramecium
Authors: Reisner A. H. , Bucholtz Carolyn
Published: February 2005

When Paramecium tetraurelia in log phase growth is treated with 4% dimethyl sulphoxide (DMSO) for five minutes the amount of polyribosomes is reduced 3‐ to 4‐fold while there is a corresponding increase in 80s ribosomal material. Reducing the concentration of DMSO to 1% allows immediate reversal of the condition.

Paramecium polyribosomes subjected to 4% DMSO either in whole cell homogenates or during purification through sucrose density gradients appear unaffected while cycloheximide at concentrations up to 100 μg/ml did not prevent DMSO from exerting its effect in vivo.

Analyses of 14C amino acid incorporation experiments indicated a strict correspondence between the effect of DMSO on polyribosomes and overall protein synthesis. The reduction of acid precipitable radioactivity in the polyribosomal region after DMSO treatment was associated with a corresponding increase in radioactivity in the 80s region. There was no comparable increase in the acid precipitable radioactivity in the soluble fraction.

The overall results of the study suggest that DMSO acts on polyribosomes indirectly through some unknown primary reaction with cell constituents, and that the mode of action is such as to cause the release of ribosomes from messenger RNA (mRNA) rather than to prevent initiation of the ribosome‐mRNA complex. Our data suggest that the effect may be selective.

Finally, it is of interest that high concentrations of DMSO (above 8%) appear to have the opposite effect of lower concentrations of DMSO, i.e., they appear to “freeze” the ribosomes to mRNA.
Enzyme inactivation by a cellular neutral protease: Enzyme specificity, effects of ligands on inactivation, and implications for the regulation of enzyme degradation
Authors: Levy Michael R. , McConkey Charles L.
Published: February 2005

A protease from Tetrahymena pyriformis inactivated eight of nine commercially available enzymes tested, including lactate dehydrogenase, isocitrate dehydrogenase (TPN‐specific), glucose‐6 phosphate dehydrogenase, D‐amino acid oxidase, fumarase, pyruvate kinase, hexokinase, and citrate synthase. Urate oxidase was not inactivated. Inactivation occurred at neutral pH, was prevented by inhibitors of the protease, and followed first order kinetics. In those cases tested, inactivation was enhanced by mercaptoethanol. Most of the enzyme‐inactivating activity was due to a protease of molecular weight 25,000 that eluted from DEAE‐Sephadex at 0.3 M KCl. A second protease of this molecular weight, which was not retained by the gel, inactivated only isocitrate dehydrogenase and D‐amino acid oxidase. These two proteases could also be distinguished by temperature and inhibitor sensitivity. Two other protease peaks obtained by DEAE‐Sephadex chromatography had little or no enzyme inactivating activity, while another attacked only D‐amino acid oxidase.

At least six of the enzymes could be protected from proteolytic inactivation by various ligands. Isocitrate dehydrogenase was protected by isocitrate, TPN, or TPNH, glucose‐6‐dehydrogenase by glucose‐6‐P or TPN, pyruvate kinase by phosphoenolypyruvate or ADP, hexokinase by glucose, and fumarase by a mixture of fumarate and malate. Lactate dehydrogenase was not protected by either of its substrates or coenzymes. Citrate synthase was probably protected by oxalacetate.

Our data suggest that the protease or proteases discussed here may participate in the inactivation or degradation of at least some enzymes in Tetrahymena. Since the inactivation occurs at neutral pH, this process could be regulated by variations in the cellular levels of substrates, coenzymes, or allosteric regulators resulting from changes in growth conditions or growth state. Such a mechanism would permit the selective retention of enzymes of metabolically active pathways.
Quantitation of mitochondrial DNA, RNA, and protein in starved and starved‐refed rat liver
Authors: Stocco D. M. , Cascarano J. , Wilson M. A.
Published: February 2005

The purpose of this study was to investigate the problem of mitochondrial biogenesis in rat liver. The approach consisted of isolating mitochondria from control, 6 day starved and 6 day starved‐5 day refed rats and comparing their DNA, RNA and protein content. This was performed by isolating the mitochondria by reorienting rate zonal centrifugation in sucrose gradients. It was found that six days of starvation resulted in a loss of 30% of the body weight, 55% of the liver weight, 40% of the mitochondrial protein, 60% of the mitochondrial RNA, but only 20% of the mitrochondrial DNA. It was also shown that refeeding of the rats for five days resulted in a restoration to normal or near normal levels in all the parameters measured. Further experiments employing the incorporation of 3H‐TTP into isolated mitochondria indicated that the maintenance of mitochondrial DNA was not the result of continuous DNA synthesis.
Accumulation of polyploid cells and G2‐phase cells during ascites tumor growth
Authors: Andersson Gunnar
Published: February 2005

Ehrlich ascites tumor cells from the plateau phase of growth were transplanted into new hosts, pulse‐labeled with tritiated thymidine and blocked with repeated injections of vinblastine. When unlabeled cells were analyzed for their cellular DNA content utilizing a cytophotometric technique it was found that in relation to the total number of cells (labeled plus unlabeled), 13% had a 2C DNA content, 36% a 4C DNA content and 5% an 8C DNA content at 0.5 hours after transplantation. By 24 hours the distributions changed dramatically: the initially unlabeled 2C cells were now 4C, the 36% of the cells that were initially 4C partitioned into 24% that were still 4C and 12% that progressed to 8C, and the initial 8C cells remained 8C. These studies indicate that the accumulation of 4C cells during the plateau phase of growth is due to a combination of G2 diploid and G1 tetraploid cells.
Peritoneal exudate cells. IV. Characterization of colony forming cells
Authors: Lin Hsiu‐San , Freeman Pamela G.
Published: February 2005

The adherence, phagocytic activity and buoyant density of mouse peritoneal exudate colony forming units (CFU‐PE) were investigated. There was a significant enrichment in the proportion of CFU‐PE in the adherent cells population, defined as cells adhering to a plastic surface within 30 minutes of incubation. The phagocytic activity of CFU‐PE was studied by incubating exudate cells with iron particles for 45 minutes. The cells were then separated into phagocytic and non‐phagocytic cell fractions by passing the incubation mixture through a magnetic field. A significant enrichment of CFU‐PE was seen in the phagocytic cell fraction. When exudate cells were fractionated in a Ficoll discontinuous density gradient, more than 88% of CFU‐PE were recovered at the 16/18% and 18/20% interfaces. It is concluded that CFU‐PE are adherent cells, have strong phagocytic activity and have a buoyant density between 1.0562 and 1.0703. When bone marrow cells were studied by these techniques, the committed stem cells for both granulocytes and macrophages (CFU‐C) were enriched in both non‐adherent cell and non‐phagocytic cells populations. In the Ficoll density gradient, CFU‐C banded at a heavier density region than CFU‐PE.
The effect of colony stimulating factor on the synthesis of ribonucleic acid by mouse bone marrow cells in vitro
Authors: Burgess Antony W. , Metcalf Donald
Published: February 2005

The effect of granulocyte‐macrophage colony stimulating factor (GM‐CSF) on the synthesis of RNA in liquid cultures of mouse bone marrow, spleen, thymus, peritoneal, peripheral blood leukocytes and lymph node cells was investigated. GM‐CSF appeared to stimulate RNA‐synthesis in syngeneic bone marrow cells within ten minutes of adding it to the culture. In the presence of GM‐CSF bone marrow cultures maintained their initial rate of RNA synthesis for approximately ten hours. GM‐CSF had no apparent effect on the uptake of3H‐uridine into bone marrow cells. This stimulation was still observed in the presence of puromycin and cycloheximide, but was abrogated by actinomycin D. The magnitude of the stimulation was not affected by the density of cells between 1 and 20 × 106cells/ml but was slightly smaller at 0.1 and 40 × 106cells/ml. Increasing concentration of GM‐CSF (up to 2 × 105units per ml) led to increased stimulation of RNA synthesis in bone marrow cells, but a significant stimulation could be detected at concentrations as low as 800 units/ml.

GM‐CSF did not significantly stimulate RNA synthesis in spleen, thymus, mesenteric or subcutaneous lymph node cells. However a small stimulation was observed in peripheral blood leukocytes and peritoneal cells.

Autoradiographic studies showed that GM‐CSF stimulated RNA synthesis in blast cells, myelocytes, metamyelocytes and polymorphs. Nucleated erythroid cells showed no increased labeling with GM‐CSF. Labeling in lymphoid‐like cells was highly variable but the level of labeling did not appear to be influenced by GM‐CSF.
Studies on a fractionated murine fibrosarcoma: A reproducible method for the cautious and a caution for the unwary.
Authors: Sheridan J. W. , Finlay‐Jones J. J.
Published: February 2005

A technique is described for the dissociation and fractionation by isopycnic centrifugation (4,000 × g, 60 minutes, 4°C) in linear bovine albumin density gradients (12 ml, pH 5.2 real osmolality 333 mmol/Kg water, 1.030–1.075 g/cm3) of cells (⩽ 3 × 107/gradient) released by a strictly standardised combination of mechanical and enzymatic means from a transplantable methylcholanthrene induced BALB/c fibrosarcoma. Optimal conditions for reproducible localisation of cell bands with maintenance of both satisfactory resolution and satisfactory viable cell recovery (> 80%) were established by means of a series of simultaneous double fractionation experiments. When rebanding was performed under these conditions the density of the median of the cell count of the refractioned cells shifted less than 0.0005 g/cm3. Experiments also showed that close adherence to certain aspects of the tissue dissociation and fractionation protocol was necessary to avoid reduced cell yields and viabilities, density‐dependent selective cell losses, reductions in resolution and shifts in the location of cell bands. Other aspects of the protocol were tolerant to variation without introducing artefacts.
The effects of neuraminidase on concanavalin a agglutination of erythrocytes: Evidence for adsorption of neuraminidase to erythrocyte membrane
Authors: Lamont J. Thomas , Isselbacher Kurt J.
Published: February 2005

Neuraminidase‐treated human erythrocytes, but not untreated erythrocytes, were agglutinated by concanavalin A. The degree of concanavalin A agglutinability was not directly related to sialic acid removal by neuraminidase. While maximal sialic acid release was obtained with 5 units neuraminidase/2 × 109 erythrocytes, maximal concanavalin A agglutination was only obtained after exposure to 20 units neuraminidase. Binding of 3H‐concanavalin A by erythrocytes was 10‐fold higher with rabbit compared to human red cells.Neuraminidase treatment of human erythrocytes caused a relative increase in 3H‐concanavalin binding, but the absolute amount was still 10‐fold less than that bound to rabbit erythrocytes. Specific adherence of neuraminidase to Con A‐Agarose could not be demonstrated. There was no evidence for contamination of the neuraminidase preparation with proteases using a sensitive assay. These studies suggest that neuraminidase adsorbs to erythrocyte membranes and leads to concanavalin A agglutination of human erythrocytes by a mechanism other than removal of sialic acid.
Interrelationships between water and cellular metabolism in Artemia cysts. VI. RNA and protein synthesis
Authors: Clegg James S.
Published: February 2005

Using 14CO2 as a labelled precursor the relationship between the initiation of protein and RNA synthesis, and water concentration, has been examined in cysts (encysted embryos) of the brine shrimp, Artemia salina. Although incorporation of radioactivity into amino acids and nucleotides occurred in cysts at hydrations as low as 0.3 g H2O/g dried cysts, incorporation into proteins and RNA was not measurable until the cysts had achieved a hydration in the range of 0.6–0.6 g/g. In no case was radioactivity detected in DNA of unemerged cysts. Fully hydrated cysts (about 1.3 g/g) that were actively synthesizing proteins and RNA, stopped doing so when dehydrated to levels below the same hydration range: thus, the hydration dependence does not involve appreciable hysteresis. The hydration range required to initiate synthesis of these macromolecules is essentially the same as that previously shown to initiate embryonic development.