bluuebunny/crossref_metadata_2025 huggingface dataset:
The binding sites of cytochalasin D. I. Evidence that they may be peripheral membrane proteins
Authors: Tannenbaum Janet , Tanenbaum Stuart W. , Godman Gabriel C.
Published: February 2005

Binding sites for tritiated cytochalasin D (3H‐CD) on the isolated plasma membrane from HEp‐2 cells were reversibly inactivated, but not dissociated from the membrane, by dialysis in 0.6 M KCl. Activity was restored by subsequent dialysis in 0.06 M KCl. Treatment with 0.2 mM ATP at low ionic strength also inactivated these sites, apparently irreversibly. Extraction of the membrane with 6% Triton X‐100 removed 75% of its protein, resulting in a two‐fold increase in specific binding activity for 3H‐CD. Both high and low affinity binding sites were retained by the detergent‐extracted membrane; at least 60% of the high affinity sites were resistant to this treatment. Evidence is presented for the attachment to the HEp‐2 plasma membrane of both actin and myosin. The results support the tentative conclusion that plasma membrane binding sites for 3H‐CD are peripheral proteins on the cytoplasmic face of the membrane. They are consistent with the hypothesis that myosin may be the location of the high affinity binding site and actomyosin may be the low affinity site. Comparison of these observations with those reported for the congeneric drug, cytochalasin B, suggests that CD binding sites differ from the high affinity site for cytochalasin B.
The relation of cycling of intracellular pH to mitosis in the acellular slime mould Physarum polycephalum
Authors: Gerson Donald F. , Burton Alan C.
Published: February 2005

The relation between intracellular pH and the mitotic cycle of Physarum polycephalum was studied by two independent techniques. Both techniques revealed a long term cycling of intracellular pH which has the same period as the mitotic cycle. Qualitative detection of the changes in intracellular pH was made by measuring the changes in fluorescence of 4‐methylesculetin which had been absorbed by the plasmodium. Quantitative measurements of intracellular pH were made throughout the mitotic cycle with antimony micro pH electrodes. The cycle of intracellular pH is sinusoidal in appearance. The maximum intracellular pH (pH 6.6) occurred at, or very near to, mitosis, and was approximately 0.6 pH units higher than the minimum pH, which occurred near the middle of the mitotic cycle.
Conditions controlling the proliferation of haemopoietic stem cells in vitro
Authors: Dexter T. M. , Allen T. D. , Lajtha L. G.
Published: February 2005

A liquid culture system is described whereby proliferation of haemopoietic stem cells (CFU‐S), production of granulocyte precursor cells (CFU‐C), and extensive granulopoiesis can be maintained in vitro for several months. Such cultures consist of adherent and non‐adherent populations of cells. The adherent population contains phagocytic mononuclear cells, “epithelial” cells, and “giant fat” cells. The latter appear to be particularly important for stem cell maintenance and furthermore there is a strong tendency for maturing granulocytes to selectively cluster in and around areas of “giant fat” cell aggregations. By “feeding” the cultures at weekly intervals, between 10 to 15 “population doublings” of functionally normal CFU‐S regularly occurs. Increased “population doublings” may be obtained by feeding twice weekly. The cultures show initially extensive granulopoiesis followed, in a majority of cases, by an accumulation of blast cells. Eventually both blast cells and granulocytes decline and the cultures contain predominantly phagocytic mononuclear cells.

Culturing at 33°C leads to the development of a more profuse growth of adherent cells and these cultures show better maintenance of stem cells and increased cell density.

When tested for colony stimulating activity (CSA) the cultures were uniformly negative. Addition of exogenous CSA caused a rapid decline in stem cells, reduced granulopoiesis and an accumulation of phagocytic mononuclear cells.
Cells regulate theri proliferation through alterations in transition probability
Authors: Shields Robert , Smith James A.
Published: February 2005

The proliferation of 3T3, 3T6 and SV3T3 cells was examined by time lapse cinephotography under a number of different growth conditions. It was found that the frequency distributions of intermitotic times of cells with widely different proliferation rates are qualitatively and quantitatively explained by the transition probability model of the cell cycle (Smith and Martin, 1973). The behaviour of quiescent cells was characterized by very low values of the transition probability. No “out of cycle” or G0 compartment of cells was detectable. From a consideration of these results and those in the literature it appears that the rate of cell proliferation is determined by the value of the “transition probability” (P), and that it is the biochemical manifestation of this parameter that regulates cell growth in vitro and in vivo.
Influence of flow velocity and cell concentration on dynamic adhesion of erythrocytes to glass surface
Authors: Kowalczynska Hanna M.
Published: February 2005

Comparative studies were carried out on dynamic adhesion of 51Cr‐labelled erythrocytes to the surface of glass beads in the presence of serum in the medium (50 μg of protein/ml) and in protein‐free medium. The influence of cell concentration (within the range 4 x 105 to 8 x 106/ml) and of cellular flow velocity (within the range 1.5–0.4 cm/min) on the value of adhesion was investigated.

It was found that when serum was present in the medium, the decisive influence on erythrocyte adhesion was exerted by the velocity with which the cells pass through the glass beads layer. Cell concentration under these conditions has only a very slight effect. When the medium does not contain serum, erythrocyte adhesion to the bead layer seems to depend on both cell concentration and flow velocity.

Preliminary data were obtained concerning the release of 51Cr from the bead layer after erythrocyte adhesion.
Potassium transport and content during G1 and S phase following serum stimulation of 3T3 cells
Authors: Tupper Joseph T. , Zorgniotti Flavia , Mills Barry
Published: February 2005

The effect of serum stimulation on unidirectional and net K flux and their relationship to the initiation of DNA synthesis has been investigated in mouse 3T3 fibroblasts. Stimulation of quiescent 3T3 cells with 20% serum results in the initiation of S phase approximately ten hours after serum addition. During transition from G1 to S phase distinct changes in K transport and cellular K content occur. Total unidirectional K influx undergoes an immediate 2‐fold increase upon serum addition, an observation in qualitative agreement with previous results (Rozengurt and Heppel, 1975). This total increase in unidirectional K influx represents a proportional increase in the active, ouabain sensitive component and the K‐K exchange component. The initial increase in total flux is followed by a gradual decline over a 16‐hour period to levels approaching those of quiescent cells. Following the initial increase in unidirectional K influx is an approximately 75% increase in cell K on a per milligram protein basis or a 40% increase on a per volume basis. This increase peaks at four to five hours and then declines to initial levels at 10 to 14 hours. Populations of quiescent cells given 20% serum plus 0.5 mM ouabain simultaneously are totally blocked from entering S phase, as determined by the appearance of 3H‐thymidine labeled nuclei. However, if the ouabain is removed after six hours these cells then undergo the same changes in unidirectional K influx and content as serum stimulated cells with entrance into S phase retarded by five to six hours. If ouabain is added to serum stimulated cells at six hours, after the increase in K transport and K content have occurred, entrance into S phase is not entirely blocked. In cells stimulated with serum and 0.5 mM dBcAMP plus 1 mM theophylline simultaneously, entrance into S phase is greatly reduced as compared to serum stimulation only. However, the early and late changes in K flux and K content are not substantially altered. This indicates that the K transport events associated with G1 and early S phase are not directly regulated by changes in cAMP levels which follow serum stimulation.
The activity of the pneumococcal autolytic system and the fate of the bacterium during ingestion by rabbit polymorphonuclear leukocytes
Authors: Tomasz Alexander , Beckerdite Susan , McDonnell Maureen , Elsbach Peter
Published: February 2005

The extent to which autolytic microbial enzymes are involved in the fate of microorganisms ingested by phagocytes has not been determined. It is known, however, that activation of degradative enzymes occurs during certain microbicidal events.

We examined the possible role of the pneumococcal autolytic enzyme (an N‐acetylmuramyl‐L‐alanine amidase) in the loss of viability and degradation of pneumococci during phagocytosis by rabbit polymorphonuclear leukocytes. Three bacterial systems were compared: (a) wild type pneumococci with an active autolytic system; (b) wild type bacteria grown under conditions that block the endogenous autolytic activity and (c) a mutant strain defective in the major autolytic enzyme of this bacterium. No differences could be detected between the autolysis‐positive and negative bacteria in the rate of killing and in the fate of macromolecular cell constitutents during ingestion by rabbit peritoneal polymorphonuclear leukocytes.
3‐O‐methylglucose transport by rat thymocyte subpopulations
Authors: Reeves John P.
Published: February 2005

Rat thymocytes can be separated into two subpopulations by centrifugation for 20 minutes at 1,600 g in an 18/26/36% (w/v) discontinuous gradient of bovine serum albumin. Approximately 13% of the cells band at the 18/26% interface (light cells) while the remaining cells band at the 26/36% interface (heavy cells). In vitro and in vivo studies of 3H‐thymidine incorporation indicate that the light cells are 2‐ to 3‐fold enriched in the rapidly dividing lymphoblast subpopulation of thymocytes as compared to heavy cells. Light cells transport the non‐metabolizable glucose analogue 3‐O‐methylglucose (3‐MeGlc) approximately four times faster than heavy cells. The time course of 3‐MeGlc uptake is biphasic for light, heavy and unfractionated thymocytes. While the half‐times of the rapid (1 minute) and slow (20–45 minute) phases of uptake are similar for all three types of cells, the contributions of the rapid phase to total uptake are 50% for light cells, 20% for unfractionated thymocytes and 10% for heavy cells. The results show that 3‐MeGlc transport activity differs markedly within certain thymocyte subpopulations. The correlation between the contributions of the rapid phase of uptake and the proportion of lymphoblasts in the thymocyte fractions suggests that the lymphoblast and small lymphocyte subpopulations might be responsible for the rapid and slow phase of 3‐MeGlc uptake, respectively.
Hexose uptake and control of fibroblast proliferation
Authors: Naiditch William P. , Cunningham Dennis D.
Published: February 2005

The role of glucose uptake in control of cell growth was studied by experimentally varying the rate of glucose uptake and examining the subsequent effect on initiation and cessation of cell proliferation. The rate of glucose uptake was varied by adjusting the concentration of glucose in the culture medium. This permitted analysis of two changes in rate of glucose uptake which are closely related to the regulation of cell growth: (1) the rapid increase in glucose uptake that can be detected within several minutes after mitogenic stimulation of quiescent fibroblasts and (2) the decrease in glucose uptake which accompanies growth to a quiescent state.

Quiescent cultures of mouse 3T3, human diploid foreskin and secondary chick embryo cells were switched to fresh serum‐containing medium with either the normal amount of glucose or a reduced level that lowered the rate of glucose uptake below the rate characteristic of quiescent control cells. The subsequent increases in cell number were equal in both media, demonstrating that the increase in glucose uptake, commonly observed after mitogenic stimulation, was not necessary for initiation of cell division. Measurements of intracellular D‐glucose pools after serum stimulation of quiescent cells revealed that the increase in glucose uptake was not accompanied by a detectable change in the intracellular concentration of glucose.

Nonconfluent growing cultures of mouse 3T3, human diploid foreskin and secondary chick embryo cells were switched to low glucose media, lowering the rate of glucose uptake below levels observed for quiescent cells. This did not affect rates of DNA synthesis or cell division over a several‐day period. Thus, the decrease in glucose uptake, which usually occurs at about the same time as the decrease in DNA synthesis as cells grow to quiescence, does not cause the decline in cell proliferation. Experiments indicated that there was no set temporal relationship between the decline in glucose uptake and DNA synthesis as cells grew to quiescence. The sequence was variable and probably depended on the cell type as well as culture conditions. Measurements of intracellular D‐glucose pools in secondary chick embryo cells demonstrated that the internal concentration of glucose in these cells did not significantly vary during growth to quiescence. Taken together, our results show that these fluctuations in the rate of glucose uptake do not lead to detectable changes in the intracellular concentration of glucose and that they do not control cell proliferation rates under usual culture conditions.
Methionine metabolism in BHK cells: The regulation of methionine adenosyltransferase
Authors: Caboche Michel
Published: February 2005

The regulation of methionine adenosyltransferase (MAT), an enzyme involved in SAM

Abbreviations: DEAE cellulose, diethylaminoethyl cellulose; Hydroxo‐B1,2, (5.6‐dimethylbenzimidazolyl) hydroxo‐cobamide; MAMP, methioninol AMP; MAT, ATP: L‐methionine S‐adenosyltransferase; MTS, methionly tRNA synthetase; SAM, S‐adenosyl L‐methionine; tRNAmet, L‐methionine acceptor transfer RNA.

biosynthesis was studied in baby hamster kidney cells. A 4‐fold increase of the specific activity of MAT gradually occurred when cells were transferred to a medium containing low amounts of methionine. In a methionine rich medium the MAT specific activity of previously induced cells slowly decreased (half inactivation time: 30 hours). Actinomycin D and cycloheximide blocked the induction of the enzyme. The analysis of the mechanism of repression of MAT biosynthesis by methionine strongly suggested that a post‐transcriptional process of regulation was involved. The induction of MAT was not affected by the growth phase of cells.

No evidence of a direct correlation between the intracellular amounts of aminoacylated tRNAmet and MAT repression was shown. A clone showing a reduced MTS activity was isolated by a (3H‐methyl) methionine suicide technique. The regulation of MAT remained unaffected in this clone. Cycloleucine, an inhibitor of in vivo SAM biosynthesis, induced the biosynthesis of MAT in a methionine rich medium. These results suggest that MAT is regulated by SAM, or a derivative of SAM, in hamster cells.

A Cycloleucine resistant clone was isolated. This clone showed increased intracellular SAM pool and MAT activity. No evidence for a structural modification of the enzyme was shown. A regulatory mutation might be involved in this clone.
Human fibroblast conditioned media contains growth‐promoting activities for low density cells
Authors: Millis Albert J. T. , Hoyle Marian , Field Brenda
Published: February 2005

Normal diploid human fibroblasts, cultured at high density (1–2 × 105 cells per cm2) release two growth promoting activities into the culture medium. The fibroblast proliferation activity‐conditioned medium facilitates the attachment of low density cells to the substrate. That activity resides in a non‐dialyzable material that is sensitive to proteolytic inactivation. A second activity is dialyzable and can be recovered in the dialysate. In the presence of serum it stimulates cell growth. After 168 hours of incubation conditioned medium cultures contain five times more cells than are present in comparable cultures without conditioned medium. A reproducible biological assay for each activity is described.
Polyamine biosynthesis and accumulation during the G1 to S phase transition
Authors: Fuller David J. M. , Gerner Eugene W. , Russell Diane Haddock
Published: February 2005

Ornithine decarboxylase, an important enzyme in growth regulation, is increased in CHO cells in G1 phase of the cell cycle and decreases as the cells progress into S phase. S‐adenosyl‐L‐methionine decarboxylase activity, which is dependent on either the presence of putrescine or spermidine for the synthesis of spermidine and spermine respectively, shows a maximal increase in late G1/early S phase which corresponds very closely with the cell cycle phase specific accumulation of spermidine and spermine during S phase. Total culture evaluation of spermidine and spermine, which included extracellular as well as intracellular concentrations, indicated that extracellular accumulations of these polyamines occurred only in G1 and that entry into S phase was concomitant with intracellular accumulation patterns. Hyperthermia (43°C for 1 hour) in mid‐G1 phase of the cell cycle resulted in rapid decreases in the activities of ornithine decarboxylase and S‐adenosyl‐L‐methionine decarboxylase. In these cells, DNA replication was also not detectable until nine hours after mitosis, a time at which there had been recovery of ornithine decarboxylase and S‐adenosyl‐L‐methionine decarboxylase activities. Previous data have further indicated a requirement for polyamine reaccumulation before control DNA replication rates are resumed. We therefore suggest that polyamine biosynthesis and intracellular accumulation are both temporal and quantitative prerequisites for transition through S phase.
The characterization of SV40‐transformed cell lines derived from mouse teratocarcinoma: Growth properties and differentiated characteristics
Authors: Topp W. , Hall J. D. , Rifkin D. , Levine A. J. , Pollack R.
Published: February 2005

Mouse teratocarcinoma cells derived from embryoid bodies of 129SVsl mice were cultured in vitro to permit their differentiation. These cells were then infected with simiam virus 40 (SV40) and 31 cloned cell lines (SVTER) were derived from these cultures. All 31 SVTER cell lines contained the SV40 tumor (T) antigen and grew as permanent lines in culture. Mock‐infected embryoid body cultures did not give rise to permanent cell lines. The morphology of each SVTER cell line was distinct and did not change during successive subclonings.

The growth properties and tumorigenic potential of all 31 SVTER cell lines were investigated. None of these lines produced tumors in 129SVsl mice. Each cell line was tested for its ability to (1) grow in medium containing 1% serum, (2) plate on a cell monolayer, and (3) form clones in methocel suspension. Only three of the SVTER cell lines were transformed with respect to all three of these criteria. Most of these cell lines were minimal transformation.

The SVTER cell lines were tested for creatine phosphokinase (CPK), an enzyme activity characteristic of mouse brain and muscle tissue, and the protease, plasminogen activator (PA) which is found in embryoid bodies and several differentiated cell types. Some of the SVTER cell lines contained high levels of CPK, while others had high levels of PA and a third group of cells contained neither enzyme activity. No SVTER cell line was found with high levels of both these enzyme activities. This result suggests that mutually exclusive sets of genes are expressed in these cells as might be expected from the distinct tissue distribution of the two enzyme activities studied. These SVTER cell lines may be useful in reconstructing developmental pathways of differentiating teratomas in vitro.
Decreased membrane potassium permeability and transport in human chronic leukemic and tonsillar lymphocytes
Authors: Segel George B. , Lichtman Marshall A.
Published: February 2005

Human blood T‐lymphocytes increase their potassium (K+) permeability and active K+ transport following lectin or antigen stimulation. We have studied the permeability and active transport of K+ by lymphocytes in chronic lymphocytic leukemia (CLL) to determine if their membrane K+ transport was similar to resting or lectin‐stimulated normal blood lymphocytes. K+ transport was assessed both by the rate of isotopic 42K+ uptake and by the rate of change in cell K+ concentration after inhibition of the K+ transport system with ouabain.

CLL lymphocytes had a marked decrease in membrane K+ permeability and active transport of K+ when compared to blood T lymphocytes. K+ transport in five subjects with CLL (10 mmol. 1 cell water−1. h−1) was half that in normal blood T‐lymphocytes (20 mmol. 1 cell water−1 h−1). Phytohemagglutinin (PHA) treatment of CLL lymphocytes did not increase significantly their active K+ transport, whereas K+ transport by normal T‐lymphocytes increased by 100%.

Since there were 73% T‐lymphocytes in normal blood and 14% in CLL blood, the difference in membrane K+ turnover could be related either to neoplasia or to the proposed B‐lymphocyte origin of CLL. We studied human tonsillar lymphocytes which contained a mean of 34% T‐cells. In five studies of tonsils, K+ transport was 14 mmol. 1 cell water−1. h−1 and treatment with PHA increased K+ transport only 30%. The intermediate values for basal K+ transport and K+ transport in response to PHA in tonsillar lymphocytes were consistent with the proportion of T‐lymphocytes present. These data suggest that B‐lymphocytes have reduced membrane permeability and active transport of K+. Thus the marked decrease in CLL lymphocyte membrane K+ permeability and transport may be a reflection of its presumed B‐cell origin, rather than a membrane alteration related to malignant transformation.
Effect of the growth state on protein turnover in two lines of cultured BHK cells
Authors: Tanaka Keiji , Ichihara Akira
Published: February 2005

Two BHK cell lines (21 and 21/13) showed different rates of protein synthesis and degradation during growth in monolayer culture. During cell growth the rate of protein synthesis decreased progressively in BHK 21 cells, whereas in BHK 21/13 cells it remained constant. Furthermore, use of cell free systems showed that polysomes of BHK 21 were disaggregated markedly in the stationary phase, whereas those of BHK 21/13 were not. The rates of translation for protein synthesis in the two cell lines were similar, and were not affected by the growth state. The translation rates were measured as the times of ribosome transit and of peptide chain completion. This suggests that the decreased rate of protein synthesis in BHK 21 in the stationary phase may be due to a decreased rate of chain initiation.

The curves for decay of proteins labeled with 3H‐leucine in 30 minutes were identical in the two cell lines in the growing state, showing a biphasic pattern due to rapidly degraded components (T1/2 = 16 h) and slowly degraded components (T1/2 = 65 h). However, in the stationary phase the half‐life of the rapidly degraded components of BHK 21/13 became 5 hours, while that of BHK 21 remained 16 hours. The half‐lives of slowly degraded proteins in the two cell lines were similar. It was also shown by the method of approach to equilibrium that the proteins of BHK 21/13 cells were degraded about twice as fast as those of BHK 21 cells in the stationary phase.

Since there is no difference in the protein contents of the two cell lines at any stage of cell growth, it seems that during cell culture protein turnove of BHK 21 cells is controlled mainly by the rate of protein degradation.
Preparation of colony stimulating activity from large batches of human urine and production of antisera against it
Authors: Laukel H. , Gassel W.‐D. , Dosch H.‐M. , Schmidt W. , Havemann K.
Published: February 2005

In vitro induction of myelopoetic colonies from mouse bone marrow has been used for measurement of leucopoetic colony stimulating activity (CSA) isolated from large batches of human urine. After high flow dialysis in artificial kidneys and immediate adsorption to DEAE‐Cellulose, followed by purification on Con A‐Sepharose, treatment with insoluble Papain and gelfiltration on Sephadex G 100, enrichment of CSA was about 6,000‐fold. An important step of the enrichment procedure was the separation from a CSA‐inhibiting protein, probably combining with CSA.

Specific activity was further increased by preparative polyacrylamide gel electrophoresis to 5.3 × 106 units per mg protein. The total enrichment exceeded 25,000‐fold.

The final purification product consisted of a group of closely related proteins with high specific activity.

Antisera raised with one of the electrophoretic fractions suppressed bioactivity in each of the different purification steps including the final CSA fractions differing in electrophoretic mobility. The antisera furthermore inhibited CSA in human lung and monocyte conditioned media but had only very little effect on partially purified CSA from stimulated human lymphocytes as well as CSA derived from mouse lung conditioned medium.
Antibody‐mediated suppression of bone marrow colony formation in vitro
Authors: Meyer‐Hamme Sabine , Bluestein Harry G.
Published: February 2005

Antisera to mouse brain reacts with hematopoietic stem cells in the mouse bone marrow. We have examined the effect of anti‐mouse brain serum (AMBS) on the development of in vitro colonies from mouse bone marrow cells. The addition of 5% AMBS to the cultures markedly decreased the numbers of colonies formed to an average of 10% of the number obtained with normal rabbit serum. AMBS suppressed formation induced by colony stimulating factors (CSF) derived from three different sources; serum from endotoxin treated mice, mouse L‐cell conditioned media, and human peripheral blood mononuclear cell conditioned media. The suppressive activity was quantitatively recovered in the IgG fraction of AMBS. Divalent F (ab′)

AHBS, rabbit anti‐human brain serum; AMBS, rabbit anti‐mouse brain serum; BM, bone marrow; CFU‐C, colony forming unit in vitro; CFU‐S, spleen colony forming unit; CSF, colony stimulating factor; FCS, fetal calf serum; MEM, minimal essential medium; NRS, normal rabbit serum; PBS, 0.01 M sodium phosphate, 0.15 M sodium chloride, pH 7.4.

fragments were as effective as the intact IgG in decreasing colony formation. Fab fragments were not suppressive. These results suggest that colony formation is induced via a dynamic interaction between CSF and the progenitor cell membrane, and that antibody directed at cell membrane antigen(s) interferes with the generation of the induction signal.
A hypotetraploid human T lymphoid cell line established by cell fusion
Authors: Ishii Kazuhiro , Yodoi Junji , Hanaoka Masao , Furuyama Jun‐Ichi
Published: February 2005

A human T lymphoid cell line was established by cell hybridization technique from peripheral blood leucocytes of a patient with Sezary syndrome. The cells beared the surface antigens of human T lymphocyte specificity as demonstrated by immune cytolysis tests, but did not form E rosettes with sheep red blood cells. Isozyme patterns of enzymes in this line such as lactate dehydrogenase, glucose 6‐phosphate dehydrogenase and esterase were of human type. The line had 79 chromosomes in modal number. This case supports the proposal that the production of tetraploids is favourable for establishment of cell lines.
Control of normal differentiation of myeloid leukemic cells. XII. Inducibility for some stages of differentiation by dimethylsulfoxide and its disassociation from inducibility by MGI
Authors: Maeda Sakan , Sachs Leo
Published: February 2005

There are clones of myeloid leukemic cells that can be induced to differentiate by the normal differentiation‐inducing protein MGI to form Fc and C3 rosettes, mature macrophages and granulocytes. One of these clones (MGI+DMSO+) was also inducible by dimethylsulfoxide (DMSO) for C3 but not Fc rosettes, and for mature macrophages but not for mature granulocytes. Other clones (MGI+DMSO−) were inducible by MGI but not DMSO and a third type of clone (MGI−DMSO−) was not inducible by either compound. Clones that differed in their inducibility by DMSO showed a similar inhibition of cell multiplication by DMSO. The results indicate, that some stages of differentiation can be induced by DMSO in an appropriate clone of myeloid leukemic cells and that there are different cellular sites for induction by DMSO and MGI.
Effects of an acridine half‐mustard (ICR 191) on growth and ploidy of frog cells in culture
Authors: Viceps‐Madore Dace
Published: February 2005

The effects of an acridine half‐mustard, ICR 191, on the growth rate and ploidy of four haploid and two diploid lines of Rana pipiens cells in culture were studied. Growth curves indicate that the haploid and diploid cell lines were equally resistant to a 4‐hour exposure of this drug (0.1 μM to 10 μM). ICR 191 treatment induced the haploid cell cultures to become diploid. The proportion of diploid cells increased progressively with respect to time after the 4‐hour exposure period. The greater the concentration of ICR 191 applied, the more rapid the rate of conversion. Autoradiographic determinations of percent labelled nuclei indicate that DNA synthesis was not inhibited in haploid or in diploid cells. Therefore, the increased proportion of diploid cells did not originate from the small percentage of diploid cells in the initial population. Instead, the haploid cells were converted to diploid cells. Time lapse cinematography indicated that the conversion mechanism was other than cell fusion. Conversion to higher ploidy did not occur when diploid cell cultures were exposed to ICR 191.
Effects of cortisol, 17β‐estradiol and thyroliberin on prolactin and growth hormone production, cell grwoth and cell cycle distribution in cultured rat pituitary tumour cells
Authors: Clausen O. P. F. , Gautvik K. M. , Haug E.
Published: February 2005

Prolactin and growth hormone production were measured in a rat pituitary tumour cell strain (GH3) after treatment with cortisol (5 × 10−6 M), thyroliberin (2.5 × 10−6 M) and 17β‐estradiol (10−6 M). The changes in hormone production were related to alterations in cell growth rate and cell cycle distribution. Cortisol inhibited prolactin production, stimulated growth hormone production and reduced the cellular growth rate measured two days after start of treatment (maximum about 40% inhibition). Flow‐micro fluorometric analysis of DNA distributions showed that cortisol treatment reduced the relative number of cells in S phase (maximum effect about 50%) with a compensatory increase of the proportion of cells in G1 phase. The lack of inhibition of prolactin production after three days of cortisol treatment may partly be related to the increased number of cells in G1 phase. Thyroliberin and 17β‐estradiol did not significantly affect cell growth after six days of treatment, although the fraction of cells in S phase was reduced by approximately 40% with a corresponding increase of cells in G1 phase. For thyroliberin and 17β‐estradiol, the stimulatory effect on prolactin production and the inhibitory effect on growth hormone production witin a period of treatment of six days cannot be explained by a shift in cell cycle distributions. None of the three hormones influenced the growth fraction which was equal to unity.

In conclusion, thyroliberin and 17β‐etradiol are able to change prolactin and growth hormone production without altering the cell cycle distribution. However, the effects of cortisol on prolactin and growth hormone production may partly be due to an alteration in cell cycle traverse resulting in an increased number of cells in the G1 phase.
Nature of cells forming erythroid colonies in agar after stimulation by spleen conditioned medium
Authors: Johnson G. R. , Metcalf D.
Published: February 2005

Erythroid colony formation in agar cultures of CBA cells was stimulated by the addition of pokeweed mitogen‐stimulated C57BL spleen conditioned medium. Both 48‐hour colonies (“48‐hour benzidine‐positive aggregates”) and day 7 large burst or unicentric erythroid colonies (“erythroid colonies”) developed, together with many neutrophil and/or macrophage colonies.

In CBA mice, the cells forming erythroid colonies occurred with maximum frequency (650/105 cells) in 10‐ to 11‐day‐old yolk sac and fetal liver but were present also in fetal blood, spleen and bone marrow. The frequency of these cells fell sharply with increasing age and only occasional cells (2/105 cells) were present in adult marrow. A marked strain variation was noted, CBA mice having the highest levels of erythroid colony‐forming cells.

The erythroid colony‐forming cells in 12‐day CBA fetal liver were radiosensitive (Do 110–125 rads), mainly in cycle and were non‐adherent, light density, cells sedimenting with a peak velocity of 6–9 mm/hr. These properties are similar to those of other hemopoietic progenitor cells in fetal tissues. The relationship of these apparently erythropoietin‐independent erythroid colony‐forming cells to those forming similar colonies after stimulation by erythropoietin remains to be determined.
The role of adenosine 3′, 5′‐cyclic monophosphate in the density‐dependent regulation of growth and tyrosinase activity of b‐16 melanoma cells
Authors: Wade David R. , Burkart Mary E.
Published: February 2005

Incubation of cultured B‐16 melanoma cells with 1‐methyl‐3‐isobutyl xanthine (MIX) produced a sustained rise in intracellular adenosine 3′,5′‐cyclic monophosphate (cAMP) which preceded an increase in the specific activity of tyrosinase (EC 1.10.3.1). Cultures of two clones of melanoma cells, one having a mean population doubling time twice that of the other, showed density‐dependent inhibition of growth. The tyrosinase activity of each line increased progressively during logarithmic growth, reaching maximal values shortly after the cultures achieved confluence. Intracellular cAMP levels fell during logarithmic growth, being minimal in confluent cultures. The stimulatory effects of MIX and confluence on tyrosinase activity were additive. Cells plated at high density had a lower tyrosinase activity than cells allowed to achieve a similar density by successive division from sparsely planted cultures although the intracellular cAMP levels of such cultures were not different. We support the observations of other investigators that agents which increase intracellular cAMP concentrations can both inhibit cell division and stimulate tyrosinase activity. There are, however, mechanisms for increasing tyrosinase activity and inhibiting cell division which are expressed as B‐16 melanoma cells approach confluence and which are not mediated by an increase in intracellular cAMP concentrations.
Epithelioid and fibroblastic rat kidney cell clones: Epidermal growth factor (EGF) receptors and the effect of mouse sarcoma virus transformation
Authors: De Larco Joseph E. , Todaro George J.
Published: February 2005

Fibroblastic and epithelioid clones have been isolated from the normal rat kidney line, NRK. These clones were studied for their ability to bind epidermal growth factor (EGF), susceptibility to transformation by mouse sarcoma virus (MSV), and alteration in EGF binding upon sarcoma virus transformation. The epithelioid clones bound much more EGF than the fibroblastic clones; Scatchard plots on two of these clones, one epithelioid and one fibroblastic, showed that the higher EGF binding (1.3 × 105 molecules per cell for the epithelioid clone and 1.3 × 104 molecules per cell for the fibroblastic clone) was due to a greater number of receptors on the epithelioid cells rather than to a difference in the apparent affinity constant. When the clones were transformed by Moloney murine sarcoma virus the EGF binding decreased, the effect being greater with the fibroblastic clones. In 20 out of 20 independently isolated sarcoma virus transformed fibroblastic clones, the level of EGF binding was either greatly reduced or completely eliminated. In contrast to EGF, another growth factor, multiplication stimulating activity (MSA), bound to a greater extent to the fibroblastic clones than the epithelioid clones, and its binding was not decreased by sarcoma virus transformation. The results show that loss of EGF binding ability correlates with expression of the murine sarcoma virus transformation.
Serum‐stimulated changes in calcium transport and distribution in mouse 3T3 cells and their modification by dibutyryl cyclic AMP
Authors: Tupper Joseph T. , Rosso Mario Del , Hazelton Bonnie , Zorgniotti Flavia
Published: February 2005

Serum stimulation of quiescent 3T3 cells returns the cells to a proliferative state. Changes in Ca content, transport and distribution during the transition through G1 and S phase have been investigated following serum stimulation of these cells. 45Ca exchange data indicate at least two kinetically defined cellular compartments for Ca; a rapidly exchanging component presumably representing surface Ca which is removable by EGTA and a slowly exchanging component presumably representing cytoplasmically located Ca. Previous studies (Tupper and Zorgniotti, 1977) indicate that the approach to quiescence in the 3T3 cells is characterized by a large increase in the surface Ca component. The present data demonstrate that this component is rapidly lost following serum stimulation. Furthermore, the serum induces an 8‐fold increase in Ca influx into the cytoplasmic compartment and a reduction in the unidirectional efflux rate coefficient for Ca. The increased Ca uptake peaks at approximately six hours (mid G1) and is accompanied by a parallel increase in cellular Ca. Prior to entrance of the cells into S phase (10—12 hours), Ca uptake declines. This is followed by a slower decline in cytoplasmic Ca levels. Simultaneous addition of fresh serum plus 0.5 mM dibutyryl cAMP inhibits the entrance of the cells into S phase. Under these conditions the loss of surface Ca is not blocked. However, the presence of 0.5 mM dibutyryl cAMP inhibits the increase in Ca uptake and, in turn, diminishes the increase in cellular Ca following serum stimulation. In contrast, a low level of dibutyryl cAMP (0.1 mM) enhances progression through G1 phase but also reduces both Ca uptake and Ca content of the cells. The data suggest that the serum induced changes in Ca content and transport are linked to intracellular cyclic nucleotide levels and progression through G1 phase and that extracellular cAMP elevating agents may enhance or inhibit these interactions in a concentration dependent manner.
The effect of tobacco smoke on the metabolism and function of rat alveolar macrophages
Authors: Drath David B. , Harper Annabel , Gharibian Jane , Karnovsky Manfred L. , Huber Gary L.
Published: February 2005

Alveolar macrophages harvested by bronchopulmonary lavage from rats exposed to tobacco smoke for 30 days (“smokers”) showed alterations in oxidative metabolism, lactate production and phagocytosis of inert starch particles when compared with control macrophages. Phagocytosis of viable Staphylococcus aureus was unaffected by tobacco smoke. Glucose oxidation measured by conversion of glucose‐1‐14C to 14CO2 was moderately affected while oxidation of glucose‐6‐14C to 14CO2 was not. Smokers routinely yielded fewer cells than controls, though these cells contained approximately 17% more protein than did controls. Opsonization of particles was not necessary for macrophages from either smoker or control animals to manifest a respiratory burst and increased superoxide and hydrogen peroxide release during phagocytosis. The glycolytic inhibitors, sodium fluoride and iodoacetamide, while effectively blocking glycolysis, did not inhibit phagocytosis by macrophages from either group. The results reported clearly distinguish alveolar macrophages from other phagocytic cells (peritoneal macrophages and polymorphonuclear leukocytes) and suggest a state of non‐specific activation caused by exposure to tobacco smoke.
Retention of transformant specific type III collagen in dibutyryl cAMP treated Kirsten sarcoma virus transformed BALB 3T3 cells and in a flat revertant
Authors: Hata Ryu‐Ichiro , Peterkofsky Beverly
Published: February 2005

We previously reported that Kirsten sarcoma virus transformed BALB 3T3 (Ki‐3T3) cell cultures contained mainly type I collagen and about 30% of another type designated by us as Y and which appears to be type III collagen, [α1 (III)]3. Clones of BALB 3T3 which exhibited contact‐inhibition were found to contain mainly type I collagen [α1(I)]2α2, and about 25% of another type (X) which was composed of three α1 chains differing from those of type III (Hata, R. and B. Peterkofsky, 1977 Proc. Nat. Acad. Sci. (U.S.A.), 74: 2933—2937). Since dibutyryl 3′:5′ cyclic adenosine monophosphate (dbcAMP) increases collagen synthesis and alters other transformation specific properties of Ki‐3T3 cells, we determined whether treatment of Ki‐3T3 cells with this compound restored the normal collagen phenotype. We also analyzed the collagen of a revertant of Ki‐3T3 which exhibits properties similar to those of the dbcAMP treated transformant. Procollagen labeled with radioactive proline was isolated from the medium or cells of cultures and was converted to collagen with pepsin; the collagen was analyzed by carboxymethyl cellulose (CMC) chromatography or gel electrophoresis under denaturing conditions. Ki‐3T3 cells treated with 0.5 mM dbcAMP continued to accumulate type III collagen but there was an increase in the number of α1 chains eluting from CMC columns in the same position as α1 (I) suggesting increased accumulation of type X collagen. Although the revertant was similar to dbcAMP treated cells in that it exhibited a flattened morphology and a high relative rate of collagen synthesis, the collagen profile was similar to that of the transformant, consisting mainly of types I and III. These results indicate that accumulation of type III collagen is unaffected by dbcAMP but suggest that cAMP may be involved in the regulation of type X collagen. The failure of dbcAMP or reversion to affect the occurrence of type III collagen supports the mechanism of cell selection as a means of explaining the specific occurrence of type III collagen in sarcoma virus transformed 3T3 cells.
The effect of protein degradation on cellular growth characteristics
Authors: Baxter G. C. , Stanners C. P.
Published: February 2005

The role of protein degradation in cellular proliferation was investigated by measurements of the rates of degradation of labile and stable proteins for a number of cell types under various growth conditions. The rate of protein degradation was found to be a relatively invariant parameter in that it did not change after strong inhibition of protein synthesis with cycloheximide or histidinol, it was the same in both exponential and stationary phase, and it did not correlate with the presence or absence of malignant tranformation.

Using three different cell types with widely differing division rates, the rate of cell division and DNA synthesis (in %/hr) was found to be precisely equal to the rate of protein accumulation (in %/hr), i.e., to the rate of protein synthesis minus the rate of protein degradation. Division rates between the different cell types appeared to be determined chiefly by the rate of protein synthesis though, especially at low division rates, the rate of protein degradation could represent a large component of the protein accumulation rate.
Localization of pyruvate kinase isozymes in bovine kidney and comparison of these patterns with those of lactate dehydrogenases and aldolases
Authors: Cardenas Janet M. , Richards Thomas C. , Gabourel Linda
Published: February 2005

Electrophoretic and immunofluorescence analysis were used to study the distribution of pyruvate kinase isozymes in the bovine kidney. Electrophoretic analysis demonstrated the presence of large amounts of K4 plus small amounts of K‐M hybrids in cortical, medullary, and papillary sections cut from the kidney. Nearly all of the K‐L hybrids seen in whole kidney extracts were found in cortical sections. Immunofluorescence of frozen sections revealed the presence of type L subunits in the tubules but the complete absence of this subunit type in glomeruli. Glomeruli do contain large quantities of pyruvate kinase isozymes, probably K4 and K‐M hybrids, that cross‐react with antibodies produced against type M pyruvate kinase.

Type L‐containing forms of pyruvate kinase and aldolase type B both appear to be found in cell types thought to be capable of catalyzing gluconeogenesis, while type K pyruvate kinase and type A aldolase are found in predominantly glycolytic cell types of the kidney. Lactate dehydrogenase isozymic patterns appear to be less closely correlated with glycolytic versus gluconeogenic functions of the kidney but may be determined more directly by other metabolic functions.
Intracellular plasminogen activator activity in growing and quiescent cells
Authors: Loskutoff David J. , Paul Dieter
Published: February 2005

Intracellular plasminogen activator (PA) was examined in 3T3 and transformed 3T3 cells under various growth conditions to determine whether expression of this activity changes with the growth state. During exponential growth, SV40 and benzpyrene (BP) transformed 3T3 cells exhibited 3‐ to 5‐fold more intracellular PA activity than untransformed 3T3 cells. This relationship changed as the cells exhausted serum factors and arrested in G1. The specific activity of intracellular PA in cells that have retained a serum‐sensitive restriction point in G1 (G0) (3T3 and BP 3T3) increased 200‐ and 20‐fold, respectively, at this time, while the level in cells that have lost most growth control mechanisms (SV3T3) remained constant. At confluency, 3T3 cells had considerably more PA than either of their transformed counterparts. Sparse cultures of 3T3 and BP3T3 cells arrest at G1 following serum depravation, and also accumulate high intracellular PA activity. The addition of serum or purified epidermal growth factor to these cultures initiated cell proliferation and resulted in a rapid, actinomycin D‐sensitive loss of this activity. Less than 50% of the original activity remained 30 minutes after growth stimulation. This loss of intracellular PA activity did not appear to result from the presence of serum or cellular inhibitors. Intracellular PA activity remained low following growth stimulation. It increased again as the cells traversed through G1. These findings indicate that intracellular PA activity fluctuates with the growth state of cells, and may be related to the cell cycle. Culture conditions which place cells, whether normal or transformed, in G1 arrest lead to increased intracellular PA, while factors that initiate growth again result in a rapid loss of this activity. This behavior is lacking in cells not subject to density‐dependent inhibition of growth. Like many other correlates of transformation, comparison of intracellular PA in normal and transformed cells must be defined in terms of the growth state of the cells in question.