bluuebunny/crossref_metadata_2025 huggingface dataset:
Membranes of animal cells. V. Biosynthesis of the surface membrane during the cell cycle
Authors: Gerner E. W. , Glick M. C. , Warren L.
Published: February 2005

KB cells grown in suspension culture were synchronized by using a double thymidine block. At various times throughout the life cycle aliquots of cells were pulsed with 14C‐L‐leucine, 14C‐D‐glucosamine and 14C‐choline for one hour periods. Surface membranes, cell particulates and soluble proteins were isolated and their 14C specific activities were determined. It was found that there was a marked increase in the rate of incorporation into surface membrane just after division. The pattern of incorporation was the same for all three isotopic precursors. The rate of incorporation of isotopic precursors into soluble proteins was constant throughout the cycle. Some increase in rate of incorporation of isotope into the particulate fraction was observed during division.
Thymidine as synchronizing agent. III. Persistence of cell cycle patterns of phosphatase activities and elevation of nuclease activity during inhibition of dna synthesis
Authors: Churchill Judy Rae , Studzinski George P.
Published: February 2005

Synchronous cultures of HeLa cells were obtained by selective detachment of cells in mitosis and fluctuations in enzyme activity were followed during the subsequent cell cycle. The enzymes measured were alkaline and acid phosphatases and a nuclease active on denatured DNA at alkaline pH (alkaline DNase). Each of these enzymes showed a different pattern of activity in the cell cycle, but a temporal relationship to the DNA synthetic phase was apparent in each case. Treatment of the cultures at the beginning of the cell cycle with 15 mM thymidine did not alter the subsequent pattern of fluctuations in activity of alkaline phosphatase or of acid phosphatase, although DNA synthesis was fully inhibited by this treatment. This indicates that the pattern of activity of some enzymes is not linked to DNA replication. On the other hand, the pattern of fluctuations in the activity of alkaline DNase was abolished by thymidine treatment, and elevation of the activity of this enzyme was observed. These results suggest complex and variable relationships between phases of the cell cycle and enzyme activity, and show that inhibition of DNA synthesis is not a suitable procedure for induction of culture synchrony if enzyme activities are to be studied.
Effects of phenethyl alcohol on transport reactions, nucleotide pools and macromolecular synthesis in novikoff rat hepatoma cells growing in suspension culture
Authors: Plagemann Peter G. W.
Published: February 2005

At cytostatic concentrations, phenethyl alcohol has immediate and reversible effects on multiple metabolic processes of Novikoff rat hepatoma cells growing in suspension culture. These include an inhibition of the transport of various low molecular weight substances into the cell, an inhibition of DNA and protein synthesis and the processing of ribosomal RNA, and a degradation of ribosomal RNA. All effects might be explained as resulting from an interaction of the chemical with cellular membranes. Phenethyl alcohol does not have an immediate effect on RNA synthesis per se. The immediate failure of phenethyl alcohol‐treated cells to incorporate uridine from the medium into RNA is due to an inhibition of the uridine transport reaction.
Synthesis by a clonal line of rat glial cells of a protein unique to the nervous system
Authors: Pfeiffer S. E. , Herschman H. R. , Lightbody J. , Sato G.
Published: February 2005

The synthesis of a protein unique to the nervous system, the “S100‐protein,” has been studied in a clonal line of rat glial cells. It has been shown that these cells do not begin to accumulate “S100‐protein” until the cultures enter a phase of density‐dependent inhibition of cell proliferation. Further experiments indicate that the regulation of “S100‐protein” accumulation resides at least in part in an interaction involving the cell surface.
Variation in the latency of acid phosphatase and ribonuclease throughout the growth cycle of Tetrahymena pyriformis
Authors: Lee Donald
Published: February 2005

The latency of both acid phosphatase and ribonuclease was studied throughout the growth cycle of the ciliate protozoan Tetrahymena pyriformis and was found to be remarkably low in cells in the logarithmic phase of growth. The latency increased progressively throughout the log phase until it reached a maximum just after the cells had entered the stationary phase.

The specific activity of acid phosphatase remained constant throughout the whole of the growth cycle while that of ribonuclease decreased as the cells began to leave log phase.

The possibility is discussed that all rapidly dividing cells have a high proportion of their acid hydrolases outside the lysosomes and that these free hydrolases may have a function in cell division.
Proteolytic enzymes in sea urchin eggs: Characterization, localization and activity before and after fertilization
Authors: Krischer Kenneth N. , Chambers Edward L.
Published: February 2005

The pH versus proteinase activity curve (casein or hemoglobin plus urea substrate) for homogenates of unfertilized Lytechinus eggs reveals two regions of maximum activity: one between pH 3.5 and 4.3, and another of far greater magnitude from pH 8.0 to 11.0. The two classes of proteinases can be separated on a sucrose density gradient. Both the acid and alkaline proteinases in homogenates prepared in isotonic monovalent salt solutions are remarkably stable at pH 7.4 and 0°C. Using synthetic peptide substrates, an enzyme with the specific esterase activity of chymotrypsin was demonstrated; this enzyme accounts for the major part of the proteinase activity at alkaline pH. In addition, an enzyme with specific esterase activity of trypsin was shown to be present, but of low activity. The proteinase activity at acid pH is largely due to an enzyme resembling cathepsin D. The data also suggest the presence of cathepsin B and cathepsin IV (or catheptic carboxypeptidase).

When eggs are homogenized in isotonic NaCl plus KCl at pH 7.4, 0.02 M tris buffer at 0°C, all of the alkaline proteinase, and 85–90% of the acid proteinase activity is sedimented at 10,000 g. The presence of any proteinase activity in the supernatant phase represents an artifact of the preparative procedures used. The granules which possess the proteinase activity are contained entirely in the yolk fractions; and the acid proteinase is contained in a population of granules which sediment more readily than those which contain the alkaline proteinase. The acid proteinase resembles the lysosomal acid hydrolases in that it is readily released from the particulates; in contrast, the alkaline proteinase is bound relatively firmly.

In contradistinction to reports in the literature, no changes in proteinase activity nor intracellular distribution could be detected following fertilization.
Comparative aspects of poliovirus replication
Authors: Levintow Leon , Michael Bishop J.
Published: February 2005

To the extent that parallel experiments with picornaviruses and RNA bacteriophages can be compared, the results suggest that their mechanisms of RNA replication are basically similar, despite fundamental differences between the two groups of viruses with respect to the control of the expression of genetic information.

The significance of the extracted multi‐stranded replicative forms of viral RNA is discussed and it is concluded that, if not identical to the corresponding, intracellular forms, the extracted structures are meaningful derivatives of specific native forms.
Differential temperature sensitivity of normal and cancer cells in culture
Authors: Levine Elliot M. , Robbins Elliott B.
Published: February 2005

Serially‐propagated growing heteroploid and growing diploid cell cultures do not survive incubation at 42° for 24 hours, whereas contact‐inhibited diploid monolayers are still viable after at least nine days at this elevated temperature. Heat‐treated heteroploid HeLa cells and growing diploid cells exhibit a variety of morphologic abnormalities, but contact‐inhibited cells are only minimally affected. A similar differential temperature sensitivity exists in the synthesis of cellular macromolecular components such as DNA, RNA, and protein: incorporation of radioactive precursors is drastically reduced in growing diploid and heteroploid cells after 24 hours at 42°, but not in contact‐inhibited cells. Incorporation of labelled glucose, choline, or linolenic acid is actually enhanced in heat‐treated contact‐inhibited cells.
Modification of rabbit muscle aldolase in Vivo and in Vitro
Authors: Lai C. Y. , Horecker B. L.
Published: February 2005

Rabbit muscle aldolase in situ appears to undergo several modification reactions. One of these, specific deamidation of an asparagine residue near the COOH‐terminus, appears to account for the presence of two types of subunits in the enzyme isolated from the muscle of adult rabbits. Evidence for a second modification is the presence of approximately one equivalent of organic phosphorus in the crystalline enzyme preparations. The presence of this phosphate group may be related to the incomplete release of COOH‐terminal tyrosine residues from the enzyme protein with carboxypeptidase. Two reactions with substrate, both leading to the incorporation of organic phosphorus, have been demonstrated in vitro. A reaction with glyceraldehyde 3‐phosphate or erythrose 4‐phosphate leads to loss of catalytic activity and change in the susceptibility of COOH‐terminus to carboxypeptidase. The other reaction, with fructose 1,6‐diphosphate at low concentration, does not affect the activity of the enzyme, nor its susceptibility towards the action of carboxypeptidase. Either or both of these may be related to the changes which appear to occur during the life of the enzyme in vivo.
Biosynthesis of the α chains of collagen
Authors: Piez Karl A.
Published: February 2005

The α chains of collagen are synthesized like other proteins by the sequential addition of amino acids beginning at the amino‐terminal end and continuing for over 1000 amino acids. In addition to amino acid assembly, hydroxylation of certain prolyl and lysyl residues is required to complete the molecule. Approximately 4.8 minutes is necessary for the entire process. After extrusion from the cell, specific lysyl residues are oxidatively deaminated to the aldehyde allysine. An analogous conversion probably occurs with hydroxylysine. Cross‐linking results from the condensation of an aldehyde on one chain with an aldehyde or an ϵ‐amino group on another chain.
Control of macromolecular synthesis in proliferating and resting syrian hamster cells in monolayer culture. I. Ribosome function
Authors: Stanners C. P. , Becker H.
Published: February 2005

The rate of protein synthesis per cell in cultured hamster embryo fibroblasts in the stationary growth phase falls to about one third of the rate in the exponential growth phase. This reduction can be entirely accounted for by the following observations: (1) the average cell in stationary phase contains about one‐half the number of ribosomes per cell compared to the average cell in exponential phase; (2) only two thirds of the ribosomes are bound to polysomes in stationary phase, while nearly all of the ribosomes are polysome‐bound in exponential phase.

In stationary phase, ribosomes which are polysome‐bound function with the same efficiency and produce proteins of approximately the same average length as in exponential phase. Experimental findings are presented which suggest that the generation of a higher proportion of free ribosomes in stationary phase in not due to a limitation in messenger RNA, but to a decreased attachment probability of ribosomes to messenger RNA.
Control of macromolecular synthesis in proliferating and resting syrian hamster cells in monolayer culture. II. Ribosome complement in resting and early G1 Cells
Authors: Becker H. , Stanners C. P. , Kudlow J. E.
Published: February 2005

Using a new, sensitive and quantitative technique for determining the ribosomal‐RNA content of a measured number of cells, the cellular ribosome complement was compared for cultured hamster embryo cells in the stationary growth phase and in the early G1 phase of the cell cycle. Cells from stationary phase cultures were found to contain less than 70% of the ribosome complement of the early G1 phase cells, though the volumes of the two cell types were similar. This would imply that the stationary phase cell is physiologically different from a cell merely arrested at some point in the cell cycle.
Characterization of the factor in L‐cell conditioned medium capable of stimulating colony formation by mouse marrow cells in culture
Authors: Austin P. E. , McCulloch E. A. , Till J. E.
Published: February 2005

Medium harvested from cultures of mouse L‐cells contains “conditioning factor activity” (CFA) that may be detected by its ability to stimulate colony formation by mouse marrow cells in culture. The active material has been characterized and appears to be a glycoprotein with properties similar to those reported for the colony‐stimulating activity in human urine. Characterization of trypsin‐digested material indicated that only part of the molecule is essential for biological activity.

The CFA has been partially purified from serum‐free L‐cell conditioned medium, and evidence has been obtained that radioactivity derived from labelled valine or glucosamine may be incorporated into the factor. L‐cell conditioned medium appears to be a convenient source of partially‐purified, highly active material, suitable for use in studies on its mechanism of action.
Cyclic AMP and calcium exchange in a cellular slime mold
Authors: Chi Yea‐Yuan , Francis David
Published: February 2005

Cyclic AMP is known to be an effective chemotactic agent for amebae of the cellular slime mold D. discoideum. A large amount of information from experiments on metazoa suggested that one cellular effect of cyclic AMP might be to alter the permeability of the cell membrane to Calcium or Sodium ions.

On the basis of this information experiments were designed to test the effect of cyclic AMP on outflow of labeled Calcium or Sodium ions from amebae of D. discoideum. It was found that addition of cyclic AMP at 10−4M resulted in a large increase of Ca45 outflow from cells at the pre‐aggregative or aggregative stage of development. No effect was found on Na22 outflow.

It is suggested that this effect on Calcium permeability of the membrane is related to the chemotactic influence of ATP by some action on the contractile mechanism for ameboid movement. The phenomenon may be distinct from the enzyme inductive activity of cyclic AMP known for bacteria, and perhaps occurring in the cellular slime molds as well.
Gene expression in synchronized lymphocytes: Studies on the control of synthesis of immunoglobulin polypeptides
Authors: Lerner R. A. , Hodge L. D.
Published: February 2005

A concept has been suggested that the role of immunogen is to stimulate resting cells to enter a phase of the cell cycle in which the synthesis of immunoglobulin is obligatory. This process conceivably involves the initial union of cells with immunogen followed by a subsequent transition from resting to proliferating cell. Several aspects of an in vitro cellular transition have been investigated using cultured WIL2 lymphocytes which are shown to enter the G1 phase of the cell cycle upon release from rest. This transition is associated with phenotypic changes in the cells manifested by differences in density of individual cells and the amount and profile of polyribosomes. An increase in the rate of synthesis of total protein and specific immunoglobulin polypeptides accompanies the G0 to G1 transition. Agents useful in bacterial and other mammalian cell systems to probe translational versus transcriptional control mechanisms are active in these lymphocytes. This cellular model appears to offer unique opportunities to approach regulatory problems in cell biology because large numbers of synchronized cells are obtainable in which specific messenger‐RNAs and their corresponding polypeptides can be isolated in relatively pure form.
Studies on the regulation of granulopoiesis. V. Effect of marrow cells on colony stimulating activity in serum
Authors: Morley Alec , Rickard Kevin A. , Quesenberry Peter , Garrity Marianne , Stohlman Frederick
Published: February 2005

Serum obtained from irradiated mice lost its colony stimulating activity when incubated with normal marrow cells. The loss of activity appeared to be due to absorption or inactivation of the active factor in the serum rather than to production of an inhibitor by the cells.
Energy transfer in a bioluminescent system
Authors: Morin James G. , Hastings J. W.
Published: February 2005

Many (but not all) of the bioluminescent systems in coelenter‐ates involve energy transfer from an excited product molecule of the calcium activated photoprotein to a second species, the green fluorescent protein, with emission at 508 nm from its excited state. Although all the luminescent coelen‐terates studied possess photoproteins, not all of them have the green fluorescent protein. This green fluorescent molecule is localized in the luminescent cells; they can thus be easily distinguished by fluorescence microscopy. The active components occur in subcellular particles; these have been isolated in an active form by homogenization in isotonic (to sea water) salt solutions.
Viral‐induced fusion of human cells. II. Heterokaryocytes between human cells from different donors, and between human cells and those from other species: Formation and properties
Authors: Velázquez Antonio
Published: February 2005

A simple method is described for effecting the formation of heterokaryocytes between different lines of human diploid fibroblasts, and between human diploid fibroblasts and cultured cells derived from other species. In the case of mixed monolayer cultures of human diploid fibroblasts exposed to UV‐inactivated Sendai virus, the proportion of nuclei in heterokaryocytes is between 25 and 35%. The heterokaryocytes engage in de novo protein synthesis. No evidence of hybrid enzymes was found in mixed cultures of human and mouse cells which had been exposed to Sendai virus and which therefore presumably contained mouse‐human heterokaryocytes. However, with the available data, it is not possible to distinguish between the absence of synthesis of hybrid enzymes and the synthesis of hybrid enzymes in amounts insufficient to permit their detection.
Enzyme kinetics in mammalian cells. III. Regulation of activities of galactokinase, galactose‐1‐phosphate uridyl transferase and uridine diphosphogalactose‐4‐epimerase in human erythrocytes
Authors: Hill Helene Z.
Published: February 2005

The sequential enzyme assay as previously described has been used to study various effects on the three enzymes in human red cells involved in the phosphorylation of galactose: galactokinase, galactose‐1‐phosphate uridyl transferase and uridine diphospho‐galactose‐4‐epimerase.

Enzyme activities in undiluted lysates appear to reflect the respective activities in whole cells.

Added extracellular Gal‐1‐P, G‐1‐P, UDPGal and UPDG do not affect enzyme activities in whole cells.

The kinase and transferase enzymes do not appear to be associated with the membrane fraction of the red cells.

Galactokinase activity is inhibited by G‐6‐P and Gal‐1‐P, but not by glucose, G‐1‐P, UDPG, UDPGal, UTP or NAD+. It is inhibited by ATP and ADP in high concentration.

Galactose‐1‐phosphate uridyl transferase activity is inhibited by G‐1‐P, G‐6‐P, UDPG, UDPGal, ATP, and ADP. It is not affected by UTP, NAD+, or galactose.

Uridine diphospho‐galactose‐4‐epimerase activity is inhibited by UDPG, ATP, ADP, UTP and NADH. It is stimulated by NAD+ and possibly by Gal‐1‐P. It is unaffected by G‐1‐P, G‐6‐P.

The rates of the three reactions decrease with decreasing temperature. The activities of transferase and epimerase are inactivated at the same rate, the kinase activity is inactivated more slowly.

Dilution experiments indicate the presence in lysates of a pool of UDPG (or, possibly UDPGal) which regulates the activities transferase and the epimerase enzymes.

Results of dilution experiments suggest that the radioactive product of the transferase enzyme is different from commercially available UDPGal‐u‐14C.

ATP, UTP and UDPG interact with some substance(s) in the red cell lysate to cause a time dependent inactivation of the epimerase. These interactions are the result of glucose metabolism.
Development of chick embryo liver during organ culture: Requirement for zinc‐insulin
Authors: Benzo Camillo A. , de la Haba Gabriel
Published: February 2005

Livers from five‐day chick embryos maintained as organ cultures on Eagle's minimum essential medium (MEM) develop an ultrastructure similar to more mature liver cells, except for glycogen deposits and the smooth endoplasmic reticulum (SER) normally associated with such deposits. The enzymes, glycogen synthetase and glycogen phosphorylase, failed to develop in these cells. The addition of zinc‐free insulin (insulin‐HCl) to MEM promoted the development of small amounts of SER in the cultured cells, as well as an increase in both glycogen synthetase and phosphorylase activities. The addition of zinc‐insulin also stimulated an increase in the activities of both enzymes, and promoted the development of greater amounts of SER and the deposition of glycogen, as well. In addition, both forms of insulin not only prevented the fall of total tissue protein which occurs during organ culture on MEM, but also stimulated net protein synthesis in the explanted liver (Benzo and de la Haba, '71).
Isolation of inclusion bodies from rabbit lung parenchyma
Authors: Hoffman Lee
Published: February 2005

The mitochondrial‐plus‐lysosomal fraction of rabbit lung parenchyma was studied by equilibrium density centrifugation in continuous sucrose density gradients (specific gravity 1.035 to 1.250). High concentrations of lysosomal marker enzymes were found both in a broad band at density 1.15–1.18, a density typical for lysosomes, and in a band at density 1.06–1.07. This light density band also had the highest specific activity of phospholipid, which thin layer and gas‐liquid chromatography showed to be primarily lecithin with a high content of palmitic acid residues. Electron microscopy of material from the light density band showed a homogeneous array of particles which bear a strong resemblance to the inclusion bodies of the type II alveolar epithelial cell as seen in electron micrographs of rabbit lung tissue sections. These data suggest that the light density band is an isolation of intact type II alveolar epithelial cell inclusion bodies, which previous studies have implicated as the storage site of the phospholipid moiety of pulmonary surfactant.
Correlative secretion of protein, lactose and K+ in milk of the goat
Authors: Silcock W. Ross , Patton Stuart
Published: February 2005

Rates of secretion of milk constituents (fat, protein, lactose, Na+ and K+) in the lactating goat were measured under normal circumstances and after injections of ouabain. In all experiments a close association was noted in the secretion rates for protein, lactose and K+. Under the influence of ouabain, the concentration of Na+ in the milk tended to rise and that of K+ to fall. The rate of milk fat secretion varied independently from the rates for the other constituents. It is reasonably assumed that the principal mechanism of milk protein secretion is by emptying of Golgi vesicles through the plasma membrane. The close correlation in rates for protein, lactose and K+ supports the contention that all three are assembled in Golgi vesicles and secreted by the same mechanism.
Mechanisms of acquired resistance to acute heat shock in cultured mammalian cells
Authors: Reeves O. Raymond
Published: February 2005

A clonal strain of mammalian cells with increased resistance to acute heat shock at 46° was compared with the heat‐sensitive parental line from which it was derived for possible differences that might account for this acquired resistance. Studies on synchronized populations of the two cell strains did not reveal any differential heat sensitivities in the various parts of the cell cycle (G1, S, G2) within either the sensitive or resistant populations. During thermal stress both cell types exhibited marked inhibition of ability to incorporate isotopically labeled precursors into macromolecular RNA, DNA, and protein.

However, significant differences were observed between the sensitive and resistant cells in the amount of leakage of materials from the two cell types during heat stress and in their relative rates of recovery after stress. Sensitive cells pre‐labeled with 3H‐uridine released considerably more acid‐soluble (cold, 5% TCA) label‐containing materials during heat stress than did pre‐labeled resistant cells. This differential release of uridine‐containing materials was not paralleled by a generalized differential leakiness to other compounds. In addition, the resistant cells were found to regain the capacity to synthesize vital macromolecules sooner, and at initially faster rates, than the sensitive cells after stress.

These results suggest that permeability changes causing decreased leakage of uridine‐containing materials during heat stress combined with accelerated rates of recovery of synthesis of essential macromolecules after stress may be important cellular mechanisms in resistance to heat shock.
The carbohydrate content of control and virus‐transformed cells
Authors: Hartmann J. F. , Buck C. A. , Defendi V. , Glick M. C. , Warren L.
Published: February 2005

The levels of carbohydrates were measured in cells before and after transformation with DNA and RNA‐containing oncogenic virus. The most consistent finding was a markedly lower content of L‐fucose in all transformed cells relative to their normal counterpart. The cell pellet from a line of Chinese hamster cells transformed with polyoma or SV‐40 viruses showed a marked decrease in the level of carbohydrates. However, when the carbohydrate levels were calculated for the whole system (cells plus wash) the transformed cells except for L‐fucose had almost the same amount of carbohydrate as the controls. The levels of various carbohydrates in cells transformed by Rous sarcoma virus were the same or were elevated above the controls. Here consideration of the washes did not alter the results. Possible explanations for the results and causes of error in these comparative studies are discussed.
Ontogeny of the human lymphoid system: Study of the cytological maturation and the incorporation of tritiated thymidine and uridine in the foetal thymus and lymph node and in the infantile thymus
Authors: Papiernik Martine
Published: February 2005

The study of the cytological evolution of the foetal thymus enables us to distinguish an early foetal period (up to 20 weeks) from a late foetal period. The cytological maturation is achieved at 20 weeks of gestation. The thymus in the late foetal phase is comparable to that of the child.

The study of the lymphopoietic function shows a higher activity in the foetal thymus up to 20–22 weeks, lymphopoiesis is also slightly increased, in the thymus of the child up to the age of three years. The study of the contribution of the various cell types to the cellularity of the thymus, emphasizes the importance of the medium lymphocytes in the early foetal period. The small lymphocytes though to a lesser extent, are also capable of division in the foetal thymus and in the infantile thymus. Incorporation of uridine is also increased in the early foetal thymus.

Cytological composition and lymphopoiesis of the foetal thymus and lymph node, have been compared.
Sedimentation of single human red blood cells. Differences between normal and glutaraldehyde fixed cells
Authors: Jay Alfred W. L. , Canham Peter B.
Published: February 2005

The sedimentation behaviour of single human red blood cells fixed with glutaraldehyde at pH 7.4 and 6.4 was studied and compared to resiflts previously reported for normal fresh cells. The cells fixed at pH 7.4 were observed to have normal shapes while those fixed at pH 6.4 were more spherical and less disc‐like. Fixation of glutaraldehyde removed “membrane flicker,” resulting in increased stability as indicated by a decrease in the number of orientation changes per minute from 2.93 ± 0.16 (SEM) to 1.74 ± 0.10 (SEM). An orientation change was defined as a change of 45° in any direction. Fixation also increased the edge‐to‐flat time preference ratio from 2.5 to 4.7, and increased the sedimentation velocities in all three orientations, despite a measured 2.5% decrease in mean cell density.

Fixation of cells at pH 6.4 showed that the decrease in stability and preference for the on‐edge orientation was associated with an increase in the sphericity of the cell.
Hemoglobin synthesis in murine virus‐induced leukemic cells in vitro. III. Effects of 5‐bromo‐2′‐deoxyuridine, dimethylformamide and dimethylsulfoxide
Authors: Scher William , Preisler Harvey D. , Friend Charlotte
Published: February 2005

Erythroid differentiation of Friend leukemia cells is enhanced when the cells are grown for four days in the presence of dimethylsulfoxide (DMSO). Dimethylformamide (DMF) has a similar though less marked effect. 5‐Bromo‐2′‐deoxyuridine (BUdR) (10−5M) inhibits both DMF‐ and DMSO‐stimulated differentiation. For maximum inhibition, BUdR must be present during the first two days of growth, during which time DNA synthesis is maximal. The addition of BUdR after the third day has no effect.

Since BUdR is incorporated into DNA and thymidine prevents BUdR inhibition of DMSO‐stimulated differentiation, it is likely that BUdR acts by virtue of its incorporation into DNA. Although BUdR alone had little effect upon cell multiplication, in combination with DMSO, cell growth was inhibited up to 40%. Since the BUdR‐inhibition of the DMSO effect was approximately 70%, it is unlikely that its effect on differentiation is due to selective killing of those cells which are stimulated to differentiate.
Serum dependent growth of primary cultured differentiated fetal rat hepatocytes in arginine‐deficient medium
Authors: Leffert H. L. , Paul D.
Published: February 2005

Fetal rat hepatocytes in primary monolayer cultures multiply in arginine‐deficient medium. Both the “recovery efficiency” and the final cell density of the cultured cells are proportional to the concentration (0–15%, v/v) of dialyzed fetal bovine serum in the medium. Stationary‐phase cells divide again following addition of fresh serum to the culture. After two to three generations of growth, the chromosome number of these cells remains diploid [2N = 42].

Cross‐feeding (of a subpopulation of arginine‐requiring liver‐derived cells by parenchymal arginine‐synthesizing cells) and cellular degradation of various serum proteins do not account for sources of arginine required for cell multiplication in this culture system. Because these cultured hepatocytes utilize ornithine for arginine biosynthesis, and because ornithine enhances the rate and the amount of cell multiplication, it is more likely that the multiplying cells are parenchymal arginine‐synthesizing hepatocytes.

At least two classes of serum factors are required for the growth of cultured fetal rat hepatocytes: one stimulates cell multiplication; the other is required for cellular survival and/or attachment to the culture dish. These factors have been partially separated by fractionation with ammonium sulfate; they are non‐dialyzable, heat labile, and sensitive to changes in pH.
Studies on the nature of the subcellular localization of lactate dehydrogenase and glyceraldehyde‐3‐phosphate dehydrogenase in chicken skeletal muscle
Authors: Melnick Ronald L. , Hultin H. O.
Published: February 2005

The elucidation of the subcellular localization of enzymes by the classical technique of homogenization followed by differential centrifugation is limited in that it is difficult to determine the effect of the severe disruptive procedures on the normal relationship of the enzymes to their subcellular environment. Attempts have been made to study this problem under less severe limitations; one of the approaches used has been the use of pressure on whole muscle tissue to extract the cellular fluids. In this report we introduce the concept of “comparative extraction” for evaluation of results obtained by this procedure. By comparing the efflux of enzymes of similar solubility and similar size and shape, it is possible to determine the minimal amount of the less easily extractable enzyme which cannot be removed due to compartmentation or binding to cellular particulate structures.

Using this concept of “comparative extraction,” we show in this report that at least 35% of the lactate dehydrogenase of chicken breast muscle is restricted in its removal. The data do not definitely resolve the problem of whether the restriction is due to compartmentation of the enzyme within subcellular organelles or binding to subcellular structures.
Isolation and characterization of some of the proteins that shuttle between cytoplasm and nucleus in Amoeba proteus
Authors: Jelinek Warren , Goldstein Lester
Published: February 2005

The Rapidly Migrating Proteins (RMP) which shuttle nonrandomly between nucleus and cytoplasm and equilibrate in approximately equal amounts in each compartment, were isolated from Amoeba proteus by implanting 3H‐protein containing nuclei into unlabeled cells and some time later extracting the labeled material from the cytoplasms of such cells. The labeled material was subsequently fractionated by gel filtration in Sephadex G‐100 columns. The RMP are soluble in dilute salt solutions and appear as a heterogenous group of molecules, one component of which seems to be a single species of protein accounting for ca. one‐third of the RMP fraction. Because of its distinctness this component, called the LR fraction, received the major attention in this study. LR was found to comprise ca. 17% of the aqueous‐soluble proteins of the nucleus and ca. 3–4% of the total cell protein.

LR has a very low molecular weight as determined, e.g., by its elution from a Sephadex G‐100 column. Because of its low molecular weight, LR could be purified by taking advantage of the fact that LR is (1) soluble in a saturated Solution of ammonium sulfate and (2) insoluble in butanol, diethyl ether, and 10% trichloroacetic acid.

LR migrates toward the anode as a single band when subjected to electrophoresis on “standard disc” and SDS polyacrylamide gels. It does not enter a gel designed to separate basic proteins (at pH 4.0). When subjected to Sephadex G‐25 gel filtration LR migrates through the gel as a single band and elutes from the gel at a position in the middle of the linear separation range that indicates its molecular weight is ca. 2300. The only N‐terminal amino acid found in the LR fraction is proline.

Evidence is presented to show that LR is not the product of a non‐specific breakdown of protein produced during its isolation, but the possibility that it results from the cleavage of a single chemical bond of a larger polypeptide, has not been eliminated.

When injected into non‐labeled amebae, purified radioactive LR concentrates in the nucleus — just as radioactive RMP concentrates in a recipient cell nucleus when an amino acid‐labeled nucleus is implanted into an unlabeled cell.
Conditionally lethal mutations in chinese hamster cells. I. Isolation of a temperature‐sensitive line and its investigation by cell cycle studies
Authors: Scheffler I. E. , Buttin G.
Published: February 2005

The isolation of a temperature sensitive cell line from the Chinese hamster line CCL39 of the American Type Culture Collection is described. At the nonpermissive temperature (39°C) the cells become attached to the surface of tissue culture dishes, but no microscopically observable colonies are formed upon prolonged incubation. Exposure to the high temperature for more than 24 hours leads to an almost complete loss in viability. A karyotypic analysis showed that this new line has lost one of the medium‐sized metacentric chromosomes, although no proof is available so far to show that this loss is not simply coincidental.

In nonsynchronized cultures transferred to 39°C DNA synthesis stops first, RNA synthesis shortly thereafter, while protein synthesis (turnover) continues for a longer time. After such a shift the cell number increases by less than 15% as measured with the Coulter counter.

Studies with synchronized cultures give the following results: (1) one round of DNA synthesis can occur at 39°C when the cells are released from serum starvation or a hydroxyurea block, or when mitotic cells are placed at 39°C; (2) the entry of cells into metaphase of mitosis at 39°C is almost normal when the preceding time interval at 39°C is only eight hours (release of cells from G1/S boundary), but considerably reduced when the cells spend an additional 12 to 15 hours at 39°C in G1 (release from serum starvation).

Infection by SV40 virus temporarily induces DNA synthesis after it has come to a stop at the nonpermissive temperature, but cells permanently transformed by SV40 still exhibit the temperature‐sensitive phenotype.
Investigations of the mechanism of decreased tumorigenicity of cells grown in BrdU
Authors: Rothschild Henry , Black Paul H.
Published: February 2005

Transplantable SV40‐transformed hamster cells cultivated in the presence of low concentrations of BrdU for prolonged periods of time and cells made deficient in the enzyme thymidine kinase (dTK) by continued exposure to BrdU became less tumorigenic. In both instances, when grown in BrdU the cells contained analog substituted DNA. The tumorigenicity of dTK+ cells exposed to low concentrations of BrdU, but not the dTK− cells, returned to control values when the cells were grown in medium devoid of BrdU. A tumorigenic mouse cell line made dTK deficient also had diminished oncogenicity. However, transformed hamster cells made deficient in another salvage pathway enzyme, hypoxanthineguanine phosphoribosyl‐transferase by growth in eight azaguanine, retained their tumorigenicity. Two of five revertant cell lines, in which thymidine kinase activity was restored, transplanted more readily to hamsters than the dTK− cells from which they were derived. It is concluded that there is a relative loss of tumorigenicity when BrdU is incorporated into the DNA of tumorigenic cell lines, or when there is a genetic modification of thymidine kinase activity.
Contact inhibition and gene expression in HTC/L cell hybrid lines
Authors: Levisohn Steven R. , Thompson E. Brad
Published: February 2005

Contact‐inhibited somatic cell hybrids were formed between two malignant cell lines lacking contact inhibition of growth. One cell line was HTC‐AR1, an azaguanine‐resistant subline from the rat hepatoma line HTC +; the other line was the BUDR‐resistant mouse L‐cell subline L‐B82. Hybrids were obtained from selective medium and characterized by chromosomal and enzymic analysis. The hybrids lacked the inducible rat enzyme tyrosine aminotransferase.
6,8‐Dihydroxypurine: A novel growth factor for mammalian cells in vitro, isolated from a commercial peptone
Authors: Yamane Isao , Murakami Osamu
Published: February 2005

A substance promoting the growth of mammalian cells in vitro has been isolated from Bacto‐peptone (Difco Labs., Detroit) and identified as 6,8‐dihydroxypurine. This compound, which is isomeric with xanthine(2,6‐dihydroxypurine), enhances the growth of one Chinese hamster and some other cell lines; xanthine itself is inactive. The biological action of the compound is discussed.
Automatic monitoring of suspension cultures
Authors: Roffe Lindsay M. , Mutton D. E. , Dawson K. B.
Published: February 2005

An apparatus is described for the continuous monitoring of the growth of suspension cultures of mammalian cells in normal conditions and following the administration of a drug.
Evidence for similar biochemical requirements for bioluminescence among the coelenterates
Authors: Cormier Milton J. , Hori Kazuo , Karkhanis Y. D. , Anderson James M. , Wampler John E. , Morin James G. , Hastings J. W.
Published: February 2005

We present evidence that the chemical requirements among all the bioluminescent coelenterates that have been examined are very similar or identical to those already described for Renilla by Cormier and associates. Components required for luminescence in Renilla were also found in a number of bioluminescent coelenterates examined such as Aequorea, Obelia, Cavernularia, Ptilosarcus, Stylatula, Acanthoptilum, Parazoanthus and Mnemiopsis. Depending on the organism these include one or more of the following: luciferyl sulfate, luciferase, and luciferin sulfokinase. These isolated components were found to be indistinguishable from those found in Renilla as evidenced by their reactivity in the Renilla bioluminescent system, by the spectral characteristics of the isolated luciferyl sulfates, by the molecular weights of the luciferases, and by the colors of the bioluminescence produced in vitro.
Stimulation of density‐inhibited cell cultures by insulin
Authors: Vaheri Antti , Ruoslahti Erkki , Hovi Tapani , Nordling Stig
Published: February 2005

Cell proliferation in density‐inhibited chick embryo cell cultures was induced by microgram quantities of insulin, neuraminidase, trypsin or papain. Other proteins tested, including albumin, fetuin, ribonuclease and hyaluronidase were inactive except in very high concentrations (> 100 μg/ml).

The insulin chick embryo model was selected for detailed analysis of the initiation of proliferation. Insulin insolubilized by conjugation with Sepharose particles was also active, but only in so far as it was released in soluble form from the particles. This was measured by a radioimmunoassay. Under the conditions giving maximal cell proliferation less than 0.002‐0.2% of insulin was taken up by the cells. This suggests that an interaction of insulin with the cell surface only is sufficient to stimulate the cells. Insulin released the density‐inhibited cells from G1 phase to produce an almost synchronous wave of proliferation. The following sequence of events was characteristic of the cells after stimulation by insulin: an early increase in sugar uptake and decrease in leucine uptake, increase in cell volume, stimulation of RNA and protein synthesis, increase in thymidine uptake, DNA synthesis, mitosis and cell division.
Ionic basis of volume regulation in mammalian cells following osmotic shock
Authors: Shank Brenda Buckhold , Rosenberg Howard M. , Horowitz Carolyn
Published: February 2005

Previous studies with mammalian cultured cells have shown that volume regulation in hypotonic medium requires active Na transport. In the present study, determinations of intracellular Na and K content were made in cultured mouse lymphoblasts during the process of swelling and subsequent shrinking (volume regulation) in hypotonic medium. Na and K content were measured in cells in which the shrinking phase was inhibited by the cardiac glycoside, ouabain. In osmotically‐shocked cells, an initial permeability increase to K, and not Na, was observed, which allowed K to diffuse out rapidly, down its gradient. Na, meanwhile, rapidly flowed inward with water entry during the swelling process, and was later lost with the same kinetics as the cell shrinkage. This loss of Na was prevented in the presence of ouabain. The results imply that volume regulation is achieved by pumping Na gained during swelling out of the cells, while any K taken up by the pump is rapidly lost through a more permeable membrane. The loss of osmotically active Na, presumably with accompanying anions, allows water to passively diffuse down its osmotic gradient, reducing cell volume subsequent to the initial passive swelling, during which K was rapidly lost.
Effects of colchicine and 2‐Br‐α‐Ergocryptine‐methane‐sulfonate (CB 154) on the release of prolactin and growth hormone by functional pituitary tumor cells in culture
Authors: Gautvik Kaare M. , Hoyt Richard F. , Tashjian Armen H.
Published: February 2005

The effects of colchicine and 2‐Br‐α‐ergocryptine‐methane‐sulfonate (CB 154) on the release of prolactin and growth hormone have been studied in a clonal strain of rat pituitary tumor cells (GH3) in monolayer culture. These cultures produce both prolactin and growth hormone and release both proteins spontaneously into the medium without storing them in large amounts. Immunological methods were used to measure both intracellular and extracellular concentrations of the hormones. Colchicine (5 × 10−6 M for 3 hours) caused a 2‐ to 3‐fold increase in intracellular concentrations of prolactin and growth hormone but, under basal conditions, had little or no measurable effect on the amounts of hormone accumulated in the medium during the course of the standard three hour treatment period. This latter finding evidently is due to a lag in the onset of drug action. Colchicine had little or no effect on accumulation of extracellular prolactin during the first two hours of treatment whereas such accumulation was depressed by over 60% during the third hour of treatment. Previous studies have shown that treatment of GH3 cells with thyrotropin releasing hormone (TRH) and hydrocortisone (HC) increases both intra and extracellular levels of prolactin and growth hormone, respectively. In cultures treated with TRH (5 × 10−8 M), colchicine (5 × 10−6 M for 3 hours) increased intracellular prolactin by about 70% and decreased extracellular hormone by 10%. In cultures treated with HC (3 × 1O−6 M), colchicine increased intracellular growth hormone by more than 100% and decreased medium concentrations of the hormone by 15%. Colchicine did not significantly alter total hormone (intracellular + extracellular) accumulation, cellular uptake of 3H‐amino acids, or total cell protein synthesis. The synthetic ergot alkaloid, CB 154, (3.3 × 10−6 M for 3 hours) caused an 80% increase in intracellular, and a nearly 50% decrease in extracellular, prolactin without affecting the accumulation of growth hormone, the uptake of 3H‐labeled amino acids, or overall protein synthesis in the cultures. Elevation of medium potassium concentration from a basal value of 5.3 mM to 3–5 × 10−2 M (by addition of KCl) decreased intracellular levels of prolactin by 85% and growth hormone by 55%. These effects of high potassium were blocked by colchicine and by CB 154. We conclude that colchicine, after a lag period of two hours, acts to inhibit the release of prolactin and growth hormone from GH3 cells. By the end of three hours of treatment, this inhibition is over 60% complete in the case of prolactin. The qualitatively different effects of colchicine and CB 154 on prolactin and growth hormone release suggest that these two secretory blocking agents probably act on GH3 cells by different mechanisms.