Effect of methenolone acetate on erythropoietin responsive cells in rat bone marrow

Bone marrow cells from methenolone acetate injected normal or hypertransfused polycythemic rats were cultured with erythropoietin. Heme synthesis rate in these cells was apparently increased as compared to control bone marrow cells similarly cultured. Plasma erythropoietin activity of methenolone treated rats was not detectable either by $\text{in vivo}$ nor by $\text{in vitro}$ assay methods. It was suggested that methenolone stimulates erythropoiesis by increasing the number and/or sensitivity of erythropoietin responsive cells.     

Inhibitor of heme synthesis in polycythemic rabbit serum

Sera from hypertransfused polycythemic rabbits were found to significantly inhibit ⁵⁹Fe incorporation into heme in erythroid cells in normal rabbit bone marrow cultures when compared with that of normal serum controls suggesting a higher concentration of this inhibitor in polycythemic serum. This serum inhibitor delayed the time of peak cumulative heme synthesis $\text{in}$$\text{vitro}$ and the delay in peak cumulative heme synthesis was increase with increasing concentrations of polycythemic serum. It is suggested from these studies that this serum inhibitor may be involved in a negative feedback system in the control of erythropoiesis and may act specifically on differentiated nucleated erythroid cells to delay their entry into the cell cycle, consequently inhibiting heme synthesis.     

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.     

Replacement of the B protein requirement of the E. coli quinolinate synthetase system by chemically-generated iminoaspartate

Two proteins (A and B) from $\text{Escherichia}\text{,}\text{coli}$ are required for $\text{in}\text{,}\text{vitro}$ synthesis of the NAD⁺ precursor, quinolinate, from L-aspartate and dihydroxyacetone phosphate. The requirement for B protein and L-aspartate in this system can be replaced by millimolar concentrations of oxaloacetate and ammonia if they are added together. This finding supports the concept that the B protein (L-aspartate oxidase) functions to form iminoaspartate which is condensed with dihydroxyacetone phosphate by the A protein to form quinolinate.     

Repetitive DNA of Gallus domesticus and its cytological locations

Cell death is a normal part of the morphogenesis of the chick limb. The posterior necrotic zone (PNZ) is located at the postaxial junction of the wing bud and body wall, and undergoes necrosis at stage 24 ($\text{4}\text{1}\text{2}$ days of incubation) in the chick embryo. The onset of necrosis in the PNZ is not preceded by any detectable cytologic changes in the cells which are to die. Necrosis of PNZ cells can be prevented by grafting prospectively necrotic cells to the dorsal surface of a wing bud prior to donor stage 22. However, if grafted at stage 22 or later, death will occur on schedule. At the same time, PNZ tissue grafted to the somites between stage 17 and 23 always die at stage 24. These results indicate that the commitment to death becomes irreversible at stage 22.An autoradiographic study was undertaken to examine the pattern of protein synthesis in prospectively necrotic PNZ cells. Tritiated amino acids were injected into chick embryos at various stages and their limb buds examined to determine the extent of protein synthesis. It was found that through stage 22 protein synthesis as indicated by amino acid incorporation was comparable in the cells of the PNZ and a control area which does not undergo cell death. However, a significant decrease in protein synthesis had occurred in cells of the PNZ by stage 23. The results reported show that the decrease in protein synthesis in prospectively necrotic cells of the PNZ occurs subsequent to the irreversible commitment to death. However, this decrease occurs prior to any cytologic evidence of the ensuing necrosis.     

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.     

Evaluation of commercial erythropoietin activity after preparative isoelectric focusing

The $\text{in}\text{vitro}$ study of erythropoiesis is often hampered by impurities associated with commerical preparations of erythropoietin. This report describes the activities associated with commercial erythropoietin, as well as a rapid and convenient method, preparative isoelectric focusing, to enhance the purity of low specific activity erythropoietin preparations.     

Extracellular matrix metabolism by chondrocytes III. Modulation of proteoglycan synthesis by extracellular levels of proteoglycan in cartilage cells in culture

Proteoglycan biosynthesis by cultured chondrocytes was shown to be depressed by extracellular concentrations of proteoglycan and partially degraded proteoglycan. This reduction in proteoglycan synthesis was reversible on removal of the added proteoglycan. $\text{Benzyl}\text{-β-}\text{D}\text{-}\text{xyloside}$, an exogenous acceptor of glycosaminoglycan synthesis, was used and it was shown that proteoglycan was inhibiting glycosaminoglycan synthesis. Proteoglycan had no effect on the overall protein synthesis by the cultured cells. It was concluded that the exogenous proteoglycan was inhibiting proteoglycan synthesis at the level of initiation or elongation of the glycosaminoglycan chains.     

Synthesis of quinolinate from D-aspartate in the mammalian liver-Escherichia coli quinolinate synthetase system.

Two proteins (A and B) from $\text{Escherichia coli}$ are required for the synthesis of the NAD precursor quinolinate from aspartate and dihydroxyacetone phosphate. Mammalian liver contains a FAD linked protein which replaces $\text{E. coli}$ B protein for quinolinate synthesis. D-aspartic acid but not L-aspartic acid is a substrate for quinolinic acid synthesis in a system composed of the B protein replacing activity of mammalian liver and $\text{E. coli}$ A protein. In contrast the $\text{E. coli}$ B protein-$\text{E. coli}$ A protein quinolinate synthetase system requires L-aspartic acid as substrate. The previous report that L-aspartate was a substrate in the liver-$\text{E. coli}$ system was due to contamination of commercially available [¹⁴C]L-aspartate with [¹⁴C]D-aspartate. These and other observations suggest that liver B protein is D-aspartate oxidase and $\text{E. coli}$ B protein is L-aspartate oxidase.     

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.     

The pharmacologic control of adrenal steroidogenesis

The control of cortisol secretion by ACTH and of aldosterone secretion by angiotensin is exerted upon separate cell populations in the adrenal cortex. Cells of the zona faciculata and the zona glomerulosa, while sharing common steroidogenic pathways, are affected differently by hormones and drugs. Fasciculata cells demonstrate increased cAMP formation and cortisol output primarily in response to ACTH. ACTH receptors, when occupied by hormone, transmit an activating signal to membrane-bound adenylate cyclase by a mechanism that may require the translocation of Ca²⁺. Although the precise way in which increased intracellular cAMP leads to increased steroidogenesis is unknown, protein phosphorylation and new protein synthesis are probably involved. Glomerulosa cells also respond to ACTH, but are uniquely responsive to physiological concentrations of angiotensin II and K⁺. The responsiveness of these cells to angiotensin may be governed by alterations in receptor number. Whether occupied angiotensin receptors activate steroidogenesis via cAMP is uncertain, but alterations in Ca²⁺ distribution within the cell may again be involved. Dopamine probably exerts a tonic inhibitory effect on glomerulosa cell function. Competitive inhibitory analogs for both ACTH and angiotensin II are available, but thus far all inhibitors have retained weak agonist properties. Because the regulatory processes for both cortisol and aldosterone are complex, a wide variety of drugs can affect rates of steroidogenesis $\text{in}$$\text{vivo}$.     

Enzyme secretion in E. coli K12 : studies on alkaline phosphatase synthesis using an unsaturated fatty-acid auxotroph.

The role of unsaturated fatty-acid starvation, and of the substitution of $\text{trans}$ for $\text{cis}$ fatty acids in the membrane phospholipid, on the secretion of alkaline phosphatase, has been investigated. A system in which alkaline phosphatase synthesis was initiated by a temperature shift has been used to obviate possible complications resulting from phosphate depletion. In contrast to earlier reports, we find (a) there is very little effect of unsaturated fatty-acid starvation on the synthesis of alkaline phosphatase; (b) the synthesis of both β-galactosidase and alkaline phosphatase synthesis was severely reduced below 27–30°C in cells grown on $\text{trans}\text{Δ}{}^9$ 16:1 fatty-acid, compared to cells grown on the $\text{cis}\text{Δ}{}^9$ 16:1 analogue. Thus no $\text{preferential}$ effect on alkaline phosphatase synthesis was observed.     

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.     

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.     

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.     

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.     

Studies on invitro DNA synthesis: II. Isolation of a protein which stimulates deoxynucleotide incorporation catalyzed by DNA polymerases of E.coli

Purified RNA polymerase, DNA polymerase III and unwinding protein of $\text{Escherichia}\text{coli}$ catalyze limited rifampicin sensitive fd or ØX 174 DNA-dependent DNA synthesis. A protein has been partially purified from $\text{E.}\text{coli}$ which stimulates rifampicin sensitive dXMP incorporation in this system 20 to 30 fold. This protein also stimulates DNA synthesis catalyzed by DNA polymerases I and II; the stimulation occurs in reactions primed with natural and synthetic DNAs as well as RNA-DNA hybrids. The protein is not a product of the known dna genes. In contrast to the above system of purified enzymes, rifampicin sensitive dXMP incorporation in crude extracts of $\text{E.}\text{coli}$ is specifically dependent on fd but not ØX 174 DNA. An additional factor has been isolated from extracts of $\text{E.}\text{coli}$ which restores specificity to the purified rifampicin sensitive system by preventing ØX 174 DNA from serving as a template.     

An inhibitor of protein synthesis prepared by protease treatment of mouse cerebral cortex cells

A substance was isolated from mouse brain cortical tissue that inhibits both cell division and protein synthesis by cells in culture. The inhibitor was released from cerebral cortex tissue by mild protease treatment. A single exposure of cells to as little as 1.25 μg of the isolated material was sufficient to inhibit BHK-21 cell protein synthesis by 20%. Higher concentrations and continual exposure resulted in 87% reduction in protein synthesis. The inhibition was shown to be independent of amino acid uptake and most effective against primary mouse embryo fibroblasts and neonatal mouse brain cell suspensions. Cells previously adapted to culture or transformed cells derived from the nervous system were less affected by, or refractory to, the inhibitor. The substance was shown to be nondialyzable, relatively resistant to thermal inactivation and the inhibitor activity was not removed by chloroform extraction. Two active fractions were identified by Bio-Gel P-100 chromatography and the protein synthetic inhibitor was removed by affinity chromatography with $\text{Ulex}$$\text{europus}$ agglutinin.     

Elevation of maternal glucocorticoid hormones alters neurotransmitter phenotypic expression in embryos

The tissue interaction between the notochord and the somites of the vertebrate embryo establishes the proper shape and constitution of the vertebral cartilage. Soon after somite formation, the somite differentiates into a cartilage-forming part, the sclerotome, and a muscle and skin-forming part, the dermamyotome. These components of the somite were dissected from $\text{3}\text{1}\text{2}\text{-}\text{day-old}$ chick embryos (stage $\text{18}\text{1}\text{2}\text{–19}$ and cultured in vitro in the presence or absence of notochord. It was found that the sclerotome cells respond to the notochord by an increased incidence of hyaline cartilage nodules, greater accumulation of sulfated glycosaminoglycans, synthesis of larger aggregates of proteoglycans, increased DNA accumulation, and accelerated DNA synthesis. The dermamyotome did not show these changes. These results indicate that the notochord enhances cartilage differentiation in the sclerotome. Under these conditions, the notochord did not elicit cartilage formation in the dermamyotome.