Purification of a cell growth inhibitor from bovine cerebral cortex cells

A glycopeptide, isolated from bovine cerebral cortex cells and added in only nanogram levels to cells in culture, has been shown to inhibit both cell protein synthesis and cell division. When purified by gel filtration and $\text{Ulex}$$\text{europaeus}$ lectin affinity chromatography, the radioiodinated preparation was subjected to high resolution isoelectric focusing and shown to contain three species of macromolecules. The glycopeptide focusing at pH 8.1 comprised over 75% of the radioiodinated material and possessed inhibitory activity against both cell protein synthesis and cell division. A second species that focused at pH 8.3 was also found to be inhibitory to cell metabolism and may have represented a variant of the major glycopeptide.     

A cell surface glycoprotein virus inhibitor that is not interferon

The possible relationship between a newly isolated glycoprotein virus inhibitor and interferon was assessed. Comparisons of the cell surface glycopeptide, obtained from mouse cerebral cortex, and interferon included antiviral activity, radioimmune assays, and the ability of antibodies raised against the brain cell surface glycoprotein (BCSG) and against mouse L cell interferon to precipitate the biological activity. BCSG was able to inhibit virus replication but only in a transient fashion. Although anti-BCSG precipitated a major portion of the radiolabelled inhibitor in a double antibody assay, anti-mouse interferon did not. Over 90% of the inhibitory activity was removed with anti-BCSG and $\text{Staphylococcus}$ protein A while anti-mouse interferon removed little, or none, of the activity under similar reaction conditions. Other properties of the BCSG that distinguish it from interferon are presented.     

Alcohol toxicity in cell culture

The purpose of this investigation was to study the mechanism of tissue toxicity induced by ethanol, which neither is metabolized nor interacts chemically with cell components. In concentrations that may be found in blood and tissues of humans, alcohol stimulated at low and impaired at higher concentrations the $\text{invitro}$ incorporation of DNA precursors into mitogen-stimulated mouse spleen cells in suspension. The degree of impairment varied directly with ethanol concentrations and duration of incubation. The impairment was demonstrable with several different mitogens and also when ethanol was added to the culture 2 hours after exposure to the mitogen. The impairment was irreversible when ethanol was removed in later stages of incubation. Because the cells did not metabolize ethanol under the conditions of this experiment, a direct physical effect of ethanol $\text{per se}$ on cell membranes is inferred. This conclusion is supported further by the finding that chlorpromazine $\text{in vitro}$ counteracted both stimulatory and inhibitory effects of ethanol.     

A dialyzable protein synthesis inhibitor released by mammalian cells in vitro

Macrophages, lymphocytes, and HeLa cells when incubated $\text{in vitro}$ at 37° in Krebs-Ringer phosphate buffer release a dialyzable, heat-stable, ninhydrin-reacting factor which inhibits protein synthesis by intact cells and isolated ribosomes. Release of the inhibitor appears to be dependent on metabolism. Partial purification of the inhibitor by Sephadex G-10 chromatography suggests it has a molecular weight of 400–600.     

Effect of amino acid and serum deprivation on the regulation of RNA synthesis in cultured Chinese hamster ovary cells

In a proline-requiring Chinese hamster ovary cell line, if both proline and serum are removed from the culture medium, net RNA synthesis is reduced to about 12 % of the unstarved control. This reduction in RNA synthesis is comparable to the stringent regulation of RNA in bacteria. A beta-globulin carbohydrate containing (3.5 % $\text{w}\text{w}$) protein factor was isolated and partially purified from fetal calf serum. The isolated serum factor is able to replace whole serum in stimulating cellular RNA synthesis and has an RNAase inhibitory effect in vitro. The effect of proline starvation and serum factor deprivation on RNA synthesis are independent and additive; each regulates about half of the total RNA synthesized. The regulation appears to affect the synthesis of all species of cytoplasmic RNA.     

Mechanism of action of cesalin, an antitumor protein.

A protein, cesalin, isolated from $\text{Caesalpinia}$$\text{gilliesii}$ is cytotoxic to KB cells in tissue culture. It has been shown to bind to the plasma membrane of this cell line and to inhibit Na⁺, K⁺-ATPase (ATP phosphohydrolase EC 3.6.1.3). Similar studies with HTC cells show no cytotoxicity or inhibition of plasma membrane Na⁺, K⁺-ATPase. The Na⁺, K⁺-ATPase of human erythrocytes and rat brain and kidney tissues are not inhibited. 5′-Nucleotidase and Mg⁺⁺-ATPase are not inhibited by cesalin in any cells tested.     

Increased synthesis of L-glycerol 3-phosphate dehydrogenase during in vitro differentiation of reaggregating cerebellar cells

Reaggregating cell cultures of neonatal mouse cerebellar cells express many of the differentiated properties of normal developing cerebellum, including the transition for the embryonic and adult isozymes of l-glycerol 3-phosphate dehydrogenase (EC 1.1.1.8). In order to determine the mechanism leading to increased levels of adult isozyme, aggregates in culture from 2 to 17 days were labeled with radioactive leucine and the relative rate of enzyme synthesis was measured after purification of the enzyme by affinity chromatography on Blue Sepharose 6B. During the course of in vitro differentiation, the relative rate of synthesis increased 100-fold, such that it represented 0.5% of the total protein synthesized in the cytoplasmic fraction of the cell. In vivo, $\text{BALB}\text{cBy}$ mice have twice the level of enzyme activity in the cerebellum as do $\text{C57BL}\text{6J}$ mice. Reaggregating cell cultures of cerebellar cells from these strains of mice also express a difference in the activity level, but only when the cerebellar cells are taken from mice 4 days of age or less. When the relative rates of synthesis of l-glycerol 3-phosphate dehydrogenase were measured in cultures expressing the strain-dependent difference in activity, these rates were found to be approximately twofold greater in cultures of $\text{BALB}\text{cBy}$ cells. In contrast, estimates of the relative rate of enzyme degradation by the double-isotope labeling technique indicate that neither specific enzyme degradation nor degradation of total protein is different in aggregates from the two strains of mice. The results suggest that the genetic mechanisms controlling the levels of l-glycerol 3-phosphate dehydrogenase in the cerebellum during development are intrinsic to the cells and, with the exception of serum factors, are independent of systemic influences.     

Isolation and characterization of minor glycosaminoglycans in the rabbit vitreous body

Protein synthesis in the microvascular system of the rabbit brain was inhibited following the elevation of body temperature by 2?2.5°C. $\text{In}$,$\text{vivo}$ labeling studies indicated that hyperthermia also induced the synthesis of a 74K protein in cerebral microvessels which is similar in molecular weight to one of the major heat shock proteins previously reported in tissue culture systems following elevation of ambient temperature. The present results suggest that a physiologically relevant increase in body temperature, similar to that attained during fever reactions, can induce the synthesis of a heat shock protein in the cerebral microvascular system of the intact mammal.     

Galactosyltransferase: confirmation of equilibrium-ordered mechanism.

A thermostable protein that strongly inhibits the soluble $\text{E. coli}$ D-alanine carboxypeptidase was isolated from a cell-free extract of $\text{E. coli B}$. The inhibitor was purified 140-fold by heat treatment, selective precipitation at pH 4.5, ion exchange chromatography on DEAE-cellulose and gel chromatography on Sephadex G-100. Inhibition of soluble D-alanine carboxypeptidase by this inhibitor is reversed by cations such as Mg⁺⁺ or Na⁺ and abolished by digestion of the inhibitor with proteolytic enzymes. The inhibitor does not affect either the particulate D-alanine carboxypeptidase of $\text{E. coli}$ or the growth of the bacteria.     

Inhibition of human erythrocyte Ca++-transport ATPase by phenothiazines and butyrophenones

Indirect immunofluorescent staining revealed that Prostatic Binding Protein, the major androgen-dependent protein in rat ventral prostate $\text{in}\text{vivo}$, is associated specifically with the epithelial cells in primary cell cultures derived from rat ventral prostate. The epithelial cells release Prostatic Binding Protein into the medium during primary culture. $\text{De}\text{novo}$ synthesis of Prostatic Binding Protein is demonstrable during early phases of cell culture. Prostatic Binding Protein is an excellent marker for the identification of functional prostate epithelial cells and for the study of regulation at the cellular level of the synthesis and secretion of a major androgen-dependent prostate protein.     

Requirement of protein synthesis in the initiation of meiosis and other nuclear changes in conjugation of Blepharisma.

In conjugation of Blepharismajaponicum, cell contact between complementary mating types induces meiosis and other nuclear changes. How long the cells must be in contact in order to be induced to undergo these nuclear changes (activated) can be ascertained by surgically separating the united cells at different times after the onset of cell union and then examining the occurrence of the nuclear changes. Applying this technique to cycloheximide-treated cells, we investigated the role of protein synthesis in the activation. Cycloheximide was used at the concentration which was found to inhibit most incorporation of amino acid into protein in this ciliate. Newly formed conjugant pairs were incubated with and without cycloheximide, washed free of the inhibitor and surgically separated. Although untreated controls were activated in 1.8 h after cell-cell contact, no activation was observed in cycloheximide-treated cells after 5 h of contact. Removal of cycloheximide from the paired cells resulted in an activation delayed by the interval of exposure time to the inhibitor. If the pairs were first incubated in normal medium and then exposed to cycloheximide, operated, activated cells appeared and increased very slowly (activation rate, about $\text{1}\text{10}$ of the control). Protein synthesis is therefore required for the initiation of meiosis and other nuclear changes. We propose that heterotypic cell union induces and maintains the synthesis of a protein, whose accumulation to a certain threshold is required for activation.     

Trypsin inhibitory activity of a polypeptide isolated from red kidney beans, that also enhances lymphocyte stimulation.

A polypeptide isolated from red kidney beans, $\text{Phaseolus}\text{vulgaris}$, which has previously been shown to stimulate RNA synthesis in cultures of mouse spleen lymphocytes and plasmolyzed $\text{E.}\text{coli}$, is here shown to be a potent inhibitor of trypsin and α-chymotrypsin. This polypeptide is compared with commercially available trypsin inhibitors with regard to their capacity to inhibit some proteolytic enzymes and to stimulate $\text{in}\text{vitro}$ cultures of lymphocytes. Similar to FV the lima been trypsin inhibitor was found to possess a stimulating effect on the RNA as well as the DNA synthesis in lymphocyte cultures.     

Protein synthesis in mitochondrial and microsomal fractions from rat brain and liver after acute or chronic ethanol administration

Incorporation of C¹⁴ Leucine was determined $\text{in vitro}$ or $\text{in vivo}$ in isolated mitochondria and microsomes of rat brain and liver after acute or chronic ethanol administration $\text{in vivo}$.The protein synthesis in mitochondrial and microsomal preparation was inhibited respectively by chloramphenicol and cycloeximide, specific inhibitors for the two systems tested. The experimental data demonstrate that the $\text{in vitro}$ protein synthesis in both systems, mitochondrial and microsomal, is strongly affected only after chronic treatment which produces significant activation at the mitochondrial and microsomal level in the liver and an inhibition on the same systems of the brain.The data for $\text{in vivo}$ protein synthesis instead show strong inhibition after acute administration, except for brain mitochondria, which are practically unaffected, while after chronic treatment no significant alterations are observed.     

Isolation and purification of the sexual agglutination substance of mating type a cells in Saccharomyces cerevisiae

The substance responsible for the sexual agglutinability was successfully solubilized by a newly established autoclaving method from the surface of mating type $\text{a}$ cells of $\text{Saccharomyces cerevisiae}$ and purified by DEAE cellulose chromatography, gel filtration, affinity chromatography and electrophoresis. The substance was found to consist of at least two different glycoprotein subunits. The molecular weight of the substance was estimated to be about 23,000 daltons by gel filtration. The substance was univalent in its biological activity and specifically masked the sexual agglutinability of the mating type α cells. The substance formed a complementary complex with the agglutination substance from α cells in vitro.     

Structural requirements of quassinoids for the inhibition of cell transformation

The effects of eleven quassinoids on Rous sarcoma virus induced cell transformation and on growth of normal cells were examined. At concentrations of 0.15-1 μg/ml they inhibited foci formation (76–99 %) without toxic effects on normal cells. The most active compounds also affected virus production by transformed cells. In intact normal and transformed cells, protein and DNA synthesis was equally affected after 3 hours of exposure to quassinoids of both cell types. RNA synthesis was not inhibited. This study has shown that the structural requirement of a C-15 ester in the quassinoids for antileukemic activity $\text{in vitro}$ and $\text{in vivo}$ is not essential for their antitransforming activity.     

DNA synthesis and the cell cycle in cultures of normal and mutant (mdg/mdg) embryonic mouse muscle cells.

The relationship between cell fusion, DNA synthesis and the cell cycle in cultured embryonic normal and dysgenic ($\text{mdg}\text{mdg}$) mouse muscle cells has been determined by autoradiography. The experimental evidence shows that the homozygous mutant myotubes form by a process of cell fusion and that nuclei within the myotubes do not synthesize DNA or undergo mitotic or amitotic division. The duration of the total cell cycle and its component phases was statistically the same in 2-day normal and mutant ($\text{mdg}\text{mdg}$) myogenic cultures with the approximate values: T, 21.5 hr; G₁, 10.5 hr; S, 7.5 hr; and G₂, 2.5 hr. In both kinds of cultures, labeled nuclei appeared in myotubes 15–16 hr after mononucleated cells were exposed to [³H]thymidine, and the rate of incorporation of labeled nuclei into multinucleated muscle cells was comparable in control and dysgenic cultures. Thus, homozygous $\text{mdg}\text{mdg}$ muscle cells in culture are similar to control cells with respect to their mechanism of myotube formation and the coordinate regulation of DNA synthesis and the cell cycle during myogenesis.     

Preferential synthesis of yeast mitochondrial DNA in alpha factor-arrested cells

α factor is a diffusible substance produced by $\text{S. cerevisiae}$ cells of the $\text{α}$ mating type which inhibits cell division (1) and the initiation of nuclear DNA synthesis (2) in cells of the $\text{a}$ mating type. In this report, it is shown that mitochondrial DNA synthesis continues at a normal rate in $\text{a}$ cells for at least 6 hours in the presence of α factor, resulting in a 5-fold increase in the amount of mitochondrial DNA per cell. The continued synthesis of mitochondrial DNA in the absence of nuclear DNA synthesis allows specific labeling of yeast mitochondrial DNA.     

Isolation and partial characterization of “galactoprotein a” (LETS) and “galactoprotein b” from hamster embryo fibroblasts

The cell surface glycoproteins of hamster NIL cells, labeled with galactose oxidase and NaB³H4, were selectively solubilized by sequential extraction with Tris buffer containing 1) sucrose-ATP-EDTA, 2) zwitterionic detergent (Empigen BB), and 3) 8 M urea. The previously reported “galactoprotein b” $\text{(Gap b)}$ and “galactoprotein a” ($\text{Gap a}$ or LETS) were isolated by affinity chromatography on insoluble Ricinus communis lectin colums (RCA column) from extracts 2) and 3), respectively. The affinity-purified $\text{Gap a}$ contained an actin-like protein, whereas the other affinity-purified galactoproteins did not contain the actin-like protein. The isolated $\text{Gap b}$ was heterogeneous, and an additional glycoprotein, specific for NILpy cells was copurified on RCA-column with $\text{Gap b}$.     

A novel, inexpensive, and sensitive method for analysis of tyrosine hydroxylase activity in tissue samples.

Tyrosine hydroxylase activity in whole mouse brains was measured $\text{in v}\text{vitro}$. The L-dihydroxyphenylaline (L-DOPA) formed by the enzyme was quantitated by liquid chromatography with electrochemical detection (LCEC). An investigation of the incubation factors (added Fe⁺², DOPA decarboxylase inhibitor concentration, substrate concentration, amount of tissue, time of incubation) is reported. Under optimal conditions the activity was found to be 15.1 ± 0.6 (S.E.M.) nmol DOPA formed/hr./g. tissue.     

The sites of synthesis and the subsequent migration of newly synthesized protein in retina

Radioautographs of rabbit retinas fixed immediately after a 1 or 2 min exposure in vitro to ³H leucine revealed high rates of protein synthesis in receptor cell inner segments, perikarya of ganglion cells, and cells of the inner nuclear layer. If these brieflly labelled retinas were returned to unlabelled medium for periods of up to 6 hr, the radioautographs revealed a progressive dispersion of the labelled proteins from their sites of synthesis. This was largely completed by $\text{1}\text{1}\text{2}$ hr and appeared, in one instance at least, to involve processes other than simple diffusion. Superimposed on the dispersive phenomenon was a process of concentration of the newly formed proteins at two sites quite distant from their synthesis, that was apparent after $\text{1}\text{1}\text{2}$hr. One of these sites was the receptor cell outer segments, as has been previously described, the other was the outer plexiform layer.