An estrogen responsive primary amphibian liver cell culture system

Amphibian hepatocytes have been prepared in both high yield and purity using a collagenase perfusion technique. The isolated cells attach efficiently in serum-free medium to collagen-coated culture dishes and subsequently form monolayers. These cultures can be maintained in an appropriate medium for over one week with minimal cell loss. The nuclear labelling index of cells exposed to [³H]thymidine indicates a very low level of cell growth. Twenty-four hour exposure to dexamethasone induces tyrosine aminotransferase activity throughout the culture period. Monolayers incorporate [³H]leucine linearly into acid-insoluble material with approx. 40% of all synthesis devoted to secreted protein. Polyacrylamide gel electrophoresis of proteins in the presence of sodium dodecyl sulfate shows the majority of proteins present in whole serum are synthesized and secreted by the cultured hepatocytes. The absolute rate of protein secretion on the first day of culture is approx. 73 μg/day/mg cell protein which subsequently declines and plateaus at 30% of this level by the 4th–5th day of culture. However, when hepatocytes are cultured in the continued presence of insulin, the drop in protein secretion is completely inhibited.Cultures of hepatocytes isolated from female frogs and subsequently exposed to 17-B estradiol in culture, synthesize and secrete the egg-yolk protein precursor vitellogenin. The protein initially appears as a minor component in the medium 1–2 days after hormone addition. Its rate of synthesis, relative to other secreted proteins, increases with time so that it ultimately constitutes the majority of protein being exported after 6 days of treatment. Parallel with vitellogenin induction is an increase in rate of total protein secretion reaching a 2-fold increase at maximal stimulation.The results show that viable, monolayer cultures of amphibian hepatocytes can be prepared which retain the ability to respond directly to added estrogen by synthesizing vitellogenin.     

Micromere Differentiation in the Sea Urchin Embryo: Two-Dimensional Gel Electrophoretic Analysis of Newly Synthesized Proteins

A method for large-scale culture of isolated blastomeres of sea urchin embryos in spinner flasks was developed. Micromeres and meso-, macromeres isolated from sea urchin embryos at the 16-cell stage were cultured by this method and the patterns of protein synthesis by their descendants were examined by two-dimensional gel electrophoresis of [³⁵S] methionine-labeled proteins. Six distinct proteins with molecular weights of 140–kDa, 105–kDa, 43–kDa, 32–kDa, and 28–kDa (two components) were specifically synthesized by differentiating micromeres. Quantitative analysis of the two-dimensional gel patterns demonstrated that all these proteins, except the 32–kDa protein, appeared at the time of ingression of primary mesenchyme cells (PMC's) in vivo , several hours earlier than the onset of spicule formation. The synthesis of 32–kDa protein was paralleled to active spicule formation and the uptake of Ca²⁺. Cell-free translation products directed by poly (A)⁺ RNAs isolated from descendant cells of micromeres and meso-, macromeres were compared by two-dimensional gel electrophoresis. Several spots specific to the micromere lineage were detected. However, none of them comigrated with the proteins synthesized specifically by the cultured micromeres. The results suggest that the expression of these proteins specific to differentiating micromeres may involve post-translational modification.     

Primary induction of vitellogenin synthesis in monolayer cultures of amphibian hepatocytes

Direct induction of vitellogenin production in cultured male amphibian hepatocytes by estradiol-17 beta has been accomplished. Liver cells were isolated from adult male bullfrogs by collagenase perfusion and maintained as monolayers in serum-free medium containing insulin and estradiol. Vitellogenin production was measured by direct immunoprecipitation from radioactively labeled secreted protein with a specific antiserum against vitellogenin. Significant quantities of vitellogenin were detected in the exported protein on the second day of hormone treatment. Vitellogenin production increased with duration of culture in the presence of estradiol until by the eighth day approximately 90% of secreted protein was vitellogenin. This response is largely comparable to that obtainable in vivo. Indirect immunofluorescence microscopy was used to identify cells synthesizing vitellogenin in response to estradiol. An increase in cytoplasmic fluorescence could be seen in cells throughout the cultures, with increasing time in the presence of estradiol. By the sixth day of treatment, the majority of cells showed significant fluorescence labeling. The results suggest that studies on the mechanisms underlying the primary activation of the vitellogenin gene may now be conducted under well defined conditions in a monolayer liver cell culture system.     

Regulation of protein synthesis by estradiol 17β, dexamethasone and insulin in primary cultured Xenopus hepatocytes

Changes in the protein synthesis of Xenopus hepatocytes caused by insulin, estradiol-17β (estradiol) and dexamethasone were studied by using a primary culture in serum-free medium. All of these hormones stimulated the synthesis of secretory and intracellular proteins. Dexamethasone induced or stimulated the synthesis of many proteins (though limited in number), whereas estradiol induced or stimulated relatively few proteins, including the yolk precursor protein vitellogenin. The majority of these proteins differed in molecular weight and/or isoelectric point. When hepatocytes were treated with both steroids, most of the proteins were synthesized at the rates expected from the single treatment of the respective steroids. Thus, each steroid selectively stimulated the synthesis of its specific proteins. However, exceptional proteins were observed, whose syntheses were stimulated only by double treatment. In contrast, insulin seemed to cause an overall increase in individual secretory protein synthesis.     

Regulation of protein synthesis by estradiol 17 beta, dexamethasone and insulin in primary cultured Xenopus hepatocytes

Changes in the protein synthesis of Xenopus hepatocytes caused by insulin, estradiol-17 beta (estradiol) and dexamethasone were studied by using a primary culture in serum-free medium. All of these hormones stimulated the synthesis of secretory and intracellular proteins. Dexamethasone induced or stimulated the synthesis of many proteins (though limited in number), whereas estradiol induced or stimulated relatively few proteins, including the yolk precursor protein vitellogenin. The majority of these proteins differed in molecular weight and/or isoelectric point. When hepatocytes were treated with both steroids, most of the proteins were synthesized at the rates expected from the single treatment of the respective steroids. Thus, each steroid selectively stimulated the synthesis of its specific proteins. However, exceptional proteins were observed, whose syntheses were stimulated only by double treatment. In contrast, insulin seemed to cause an overall increase in individual secretory protein synthesis.     

Growth Kinetics, Cell Shape, and the Cytoskeleton of Primary Astrocyte Cultures

Abstract: We examined correlations among growth kinetics, cell shape, and cytoskeletal protein content in rat astrocytes grown in primary culture. Cell suspensions from brains of newborn rats were seeded at densities from 0.2 to 3 × 10⁵/cm². At initial densities above 1 × 10⁵ the population increased to reach confluency by 10–12 days, after which cell number remained stable for many weeks. At low initial densities, 0.2–0.4 × 10⁵/cm², cells did not increase in number. Final density increased with increasing plating densities. High-density cells had small perikarya and several long cytoplasmic processes; low-density cells appeared flat and polygonal. All cultures were almost entirely astrocytic, as judged by immunofluorescent staining with antiserum against glial fibrillary acidic protein (GFAP). Cytoskeletal proteins were analyzed by gel electrophoresis after extraction from cells with nonionic detergent. Relative amounts of the proteins differed, in that low-density cells contained large amounts of cytoskeletal actin relative to the intermediate filament (IF) proteins vimentin and GFAP, whereas high-density cells contained relatively less actin and more IF proteins. Such differences in cytoskeletal proteins between the high- and low-density cultures were mirrored in the relative rates of synthesis of the cytoskeletal proteins. In the low-density cells amino acid incorporation into cytoskeletal-associated actin was more active than that into the IFs, whereas in the high-density cells higher rates of IF protein synthesis were observed.     

DEVELOPMENT OF PROTOPLASTS OF ULVA FASCIATA (CHLOROPHYTA) FOR ALGAL SEED STOCK

The aim of this study was to isolate and cultivate protoplasts of the green alga Ulva fasciata Delile and subsequently induce them to form a microthallus suspension for algal seed stock. The protoplasts were covered with secreted mucilage following 6 h of culture when viewed with SEM. The mucilage fused to form thick layers during day 1 of culture. Microfibrillar cell walls were deposited into the thick layers of mucilage on the 5th day of culture. An average of about 10% of the freshly isolated protoplasts began to divide at 6–14 days. These protoplasts subsequently developed varied morphologies, depending on the time of collection during the year. Protoplasts isolated from U. fasciata collected in March to June developed frond thalli or microthalli when they were cultured in low or high densities (cells/area), respectively. The microthallus suspension was cultured for more than two years at 10–40 μ mol·m^{− 2} ·s^{− 1} . Frond thalli formed when the suspension was cultivated at 100–160 μ mol·m^{− 2} ·s^{− 1} . Therefore, microthallus suspension can serve as a seed stock of U. fasciata .     

Phenotype modulation in primary cultures of arterial smooth-muscle cells

Abstract. The effects of prostaglandin E₁ (PGE₁) on the phenotypic state of enzymatically isolated arterial smooth-muscle cells in primary culture were studied by transmission electron microscopy, thymidine autoradiography, and cell counting. Early in culture (day 0–2), PGE₁, stimulated conversion of the cells from contractile (less euchromatic nucleus and cytoplasm dominated by myofilament bundles) to synthetic state (more euchromatic nucleus and cytoplasm dominated by cisternae of rough endoplasmic reticulum and a large Golgi complex). The rate of entrance of the cells into DNA synthesis and mitosis was also increased at this time. Later on (day 3–6), when the majority of the cells had entered synthetic state, PGE₁ inhibited DNA synthesis and cellular proliferation. These observations indicate that the effect of prostaglandins on arterial smooth muscle is dual in nature and dependent on the state of differentiation of the cells.     

Sucrose inhibits vascular smooth muscle cell proliferation/migration--protein synthesis is maintained

Abstract.  We have previously shown that the onset of smooth muscle cell proliferation in tissue cultures is triggered independently of serum. The aim of the present study was to investigate if this process was affected by osmotic stress. Vascular explants from 8-month-old male rats were cultured under serum-free conditions using collagen I as migration substrate. Sucrose was added to the culture medium in concentrations varying from 1 to 3% (30–90 mOsM). Cell migration from aortic explants onto the culture dishes was totally inhibited at a sucrose concentration of 90 mOsM. A significant dose-dependent decline in proliferation was shown for cells in explants pulse labelled with ³H-thymidine. In contrast, pulse labelling with ³⁵S-methionine revealed that protein synthesis was maintained in the presence of sucrose. The results indicate that osmotic pressure affects smooth muscle cell protein synthesis, proliferation and migration.     

Lactate Dependent Protein Synthesis in Round Spermatids from Rat Testes

Lactate (20 mM) markedly increased protein labeling of round spermatids (steps 1–8) from rat testes. The stimulatory effect of lactate on protein labeling was also observed to some degree in spermatocytes and late spermatids (steps 13–16), but not in Leydig cells and 7 day-old testis cell suspznsions. In the lactate-treated spermatids, 51 % of the labeled proteins was found in the water-soluble fraction (the 105,000 × g 1 hr supernatant), whereas only 21%, in the control cells. The labeled proteins did not break down for at least 90 minutes in the presence of lactate. Actinomycin D (20 μg/ml) had no effect on [³H]leucine incorporation into the water-soluble proteins of spermatids, while labeling of the water-insoluble proteins (the 105,000 × g 1 hr pellet) was decreased by 23%.
These findings suggest that the round spermatids might be the most susceptible for the lactate-induced stimulation of protein synthesis among various types of testicular cells, and that lactate increases the synthesis of water-soluble proteins which may be regulated in the translational level of protein synthesis.     

Differentiation of Sea Urchin Micromeres: Correlation between Specific Protein Synthesis and Spicule Formation

Sea urchin micromeres were isolated from the 16-cell stage embryos and cultured until they differentiated into spicule-forming cells. Electrophoretic analysis of proteins labeled with [³⁵S]-methionine showed that the differentiation accompanied the synthesis of five cell-specific proteins. These proteins appeared prior to spicule formation and were synthesized continuously or maintained stably while the cultured micromeres formed spicules. In contrast, these proteins were hardly detectable during development of the meso- and macromeres. Correlation between synthesis of the specific proteins and spicule formation was further examined in culture conditions which inhibit spicule formation. In Zn²⁺ -containing or serum-free medium, the micromere descendants failed to form spicules and exhibited markedly reduced synthesis of one of the specific proteins (32 K daltons). After removal of Zn²⁺, or addition of serum, however, spicules were formed with delay but concomitantly with an increase in the synthesis of this protein. This clear correlation suggests the participation of the 32 K protein in the process of spicule formation.     

Acceleration of Retinol-Induced Epidermal Mucous Metaplasia by Stimulating the Dermal Adenylate Cyclase-cAMP System in Chick Embryonic Skin: Appearance of cAMP-Dependent Phosphorylated Proteins in Dermis of Retinol-Pretreated Skin after 2 h-Treatment with cAMP

Epidermal mucous metaplasia of cultured 13-day-old chick embryonic tarsometatarsal skin can be induced by culture in medium containing excess retinol (20 μM) for only 8–24 h and then in a chemically defined medium with Bt₂cAMP (0.2–2 mM) and without retinoids or serum for 2 days. In this work, stimulation of the adenylate cyclase-cAMP system in retinol-pretreated skin by forskolin, pertussis toxin, cholera toxin or AIF₄^– was found to accelerate the synthesis of epidermal sulfated glycoprotein (mucin). In skin induced toward mucous metaplasia by retinol, treatment with forskolin for 1 day increased the cAMP content 10-fold in the dermis but only 2-fold in the epidermis over the control levels. The cAMP level of Bt₂cAMP (0.2 mM)-treated skin was 18 times higher in the dermis but rather lower in the epidermis than untreated skin. These results suggest the importance of an adenylate cyclase-cAMP system in the dermis of skin in stimulating mucous metaplasia induced by retinoids. In fact, cAMP-dependent protein phosphorylation was seen only in the dermis of retinol-pretreated skin after 2 h-treatment with cAMP. As no transfer of cAMP from the dermis to the epidermis of forskolin-treated skin was detected, there may be no gap junctional communication between the epidermis and the dermis, while the basement membrane becomes discontinuous during mucous metaplasia.     

Protein Synthesis in a Cell-free System from Rat Brain Sensitive to ACTH-like Peptides

Abstract: Protein synthesis was measured using a cell-free system obtained from subcortical rat brain tissue. The concentrations of Mg²⁺ and K⁺ and the amount of tissue, during both the preparation and the final assay, were critical to the incorporation of amino acids as expressed per milligram protein. Even under optimal conditions mainly elongation of growing peptide chains was measured. Behaviorally active fragments of ACTH modulated the activity of the system in a biphasic manner; i.e., at a low concentration (10⁻⁸ M) of ACTH a stimulation of between 10 and 70% was found; a high concentration (10⁻⁴ M) was inhibitory (50 to 70%). Structure-activity studies revealed that the stimulatory effect was confined to the N-terminus of the peptide (1–24), whereas the C-terminal sequence was responsible for the inhibition. The stimulation by ACTH_1–24 was dependent on Ca²⁺ and Mg²⁺. Cyclic AMP (10⁻⁵ M) stimulated the amino acid incorporation too. When a similar cell-free extract was prepared from brain tissue of hypophysectomized rats, the lower in vivo protein synthesis in these animals was preserved in the present cell-free system. The data are discussed in terms of a possible direct intracellular effect of ACTH on brain protein synthesis.     

Differential Expression of MARCKS and Other Calmodulin-Binding Protein Kinase C Substrates in Cultured Neuroblastoma and Glioma Cells

Abstract: Expression of the protein kinase C substrate MARCKS and other heat-stable myristoylated proteins have been studied in four cultured neural cell lines. Amounts of MARCKS protein, measured by [³H]myristate labeling and western blotting, were severalfold higher in rat C6 glioma and human HTB-11 (SK-N-SH) neuroblastoma cells than in HTB-10 (SK-N-MC) or mouse N1E-115 neuroblastoma cells. Higher levels of MARCKS mRNA were also detected in the former cell lines by S1 nuclease protection assay. At least two additional ³H-myristoylated proteins of 50 and 40–45 kDa were observed in cell extracts heated to >80°C or treated with perchloric acid. The 50-kDa protein, which bound to calmodulin in the presence of Ca²⁺, was more prominent in cells (N1E-115 and HTB-10) with less MARCKS, whereas neuromodulin (GAP-43) was detected in N1E-115 and HTB-11 cells only. Heating resulted in a fourfold increase in the detection of MARCKS by western blotting; this was not paralleled by a similar increase in [³H]myristate-labeled MARCKS and may be due to a conformational change affecting the C-terminal epitope or enhanced retention of the protein on nitrocellulose. Addition of β-12- O -tetradecanoylphorbol 13-acetate resulted in three- to fourfold increased phosphorylation of MARCKS in HTB-11 cells, with little increase noted in HTB-10 cells. These results indicate that MARCKS, neuromodulin, and other calmodulin-binding protein kinase C substrates exhibit distinct levels of expression in cultured neurotumor cell lines. Of these proteins, only MARCKS appears to be correlated with phorbol ester stimulation of phosphatidylcholine turnover in these cells.     

Quantitation of vitellogenin messenger RNA in the liver of male xenopus toads during primary and secondary stimulation by estrogen

From livers of estrogen-stimulated female Xenopus toads, large quantities of estrogen-induced, poly(A)-containing RNA could be isolated, showing the same characteristics as vitellogenin mRNA obtained from hormone-treated males.Using cDNA hybridization, vitellogenin mRNA was monitored in the cytoplasmic poly(A)-containing RNA of the liver of male toads during 13 days of primary and the initial phase of secondary stimulation with estrogen.During primary stimulation, low amounts of vitellogenin mRNA, not exceeding 0.18% of the cytoplasmic poly(A)-containing RNA, were first detected after 12 hr of hormone treatment, and vitellogenin mRNA was found to increase on the average to 34% of the cytoplasmic poly(A)-containing RNA on the seventh day of hormone treatment. After 3 days of primary stimulation, accumulation of vitellogenin mRNA leveled off, showing no significant increase in the cytoplasm up to 13 days of hormone treatment. As judged from incorporation of ³²PO₄ into blood plasma proteins of males during primary stimulation, vitellogenin was first detected after 1 day, and its synthesis was found to increase dramatically until the thirteenth day of hormone treatment. This implies that there is a coincidence between appearance and extent of synthesis of vitellogenin and the abundance of vitellogenin mRNA in the cytoplasm, but there is evidence that during later phase of primary stimulation (day 3–13), the increase in synthesis of vitellogenin cannot be attributed anymore to a significant accumulation of vitellogenin mRNA.In male Xenopus, estrogen-induced synthesis of vitellogenin is no more detectable 41 days after hormone injection, and the concentration of vitellogenin mRNA was found to be <0.03% of the cytoplasmic poly(A)-containing RNA. Secondary stimulation by estrogen of these animals results in an at least 30 fold faster accumulation of vitellogenin mRNA in the cytoplasm within the initial 12 hr of hormone treatment. This may explain the faster appearance of vitellogenin in the blood plasma.     

Regulation of the Ca2+ Sensitivity of Exocytosis by Rab3a

Abstract: Ca²⁺ ions trigger the release of hormones and neurotransmitters and contribute to making the secretory vesicles competent for fusion. Here, we present evidence for the involvement of the GTP-binding protein Rab3a in the sensitivity of the exocytotic process to internal [Ca²⁺]. The secretory activity of bovine adrenal chromaffin cells was elicited by Ca²⁺ dialysis through a patch-clamp pipette and assayed by monitoring changes in cell membrane capacitance. Microinjection of antisense oligonucleotides directed to rab3a mRNA increased the secretory activity observed at low (0.2–4 µ M ) [Ca²⁺], but did not change the maximal activity observed at 10 µ M free [Ca²⁺]. Moreover, after a train of depolarizing stimuli, the secretory activity of antisense-injected cells dialyzed with 10 µ M [Ca²⁺] was increased significantly compared with that of control cells. This result suggests that the activity of either Rab3a or its partners might change upon stimulation. We conclude that Rab3a, together with its partners, participates in the Ca²⁺ dependence of exocytosis and that its activity is modulated further in a stimulus-dependent manner. These findings should provide some clues to elucidate the role of Rab3a in synaptic plasticity.     

Normal ranges of some blood chemistry parameters in adult farmed Atlantic salmon, Salmo salar

Blood samples from healthy adult Atlantic salmon fed an optimal diet in net sea pens were collected at intervals from October to May. Haematological determinations and biochemical serum analyses were carried out on 20 fish in each of seven samples. The ranges of haemato-logical values for sample means were: haematocrit 44–49%, haemoglobin 8.9–10.4 g 100ml⁻¹, red blood cell count 0.85–1.10 × 10¹² l⁻¹, MCV 441–553 × 10⁻¹⁵ 1, MCH 94–106 × 10⁻⁶ g, MCHC 19.4–21.7 g 100ml⁻¹ and leucocrit 0.43–0.96%. The ranges of enzyme activities in serum, for sample means, were: alkaline phosphatase 647–988Ul⁻¹, aspartate aminotrans-ferase 202–351 Ul⁻¹ and alanine aminotransferase 4–8 Ul⁻¹. The ranges of the other parameters analyzed in serum were: total protein 41.6–56.6 gl⁻¹, albumin 18.3–24.3 gl⁻¹, albumin/total protein ratio 39.3–44.0%, creatinine 26–46 μmol, triglycerides 2.53–4.98 μmol and cholesterol 9.3–12.8 μmol. These values are considered to be the normal ranges in healthy fish. Variations due to seasonal changes, and the clinical significance of the selected parameters, are discussed. Data showing the reproducibility of the biochemical analyses in serum are presented.     

Indispensability of Iron for the Growth of Cultured Chick Cells

In order to clarify the role of iron in the growth promoting effect of transferrin (Tf), the effects of the following substances were examined in cultured chick skeletal myogenic cells: transition metal ions (Fe²⁺, Fe³⁺, Cr³⁺, Cu²⁺, Mn²⁺, Co²⁺, Cd²⁺, Zn²⁺ and Ni²⁺), Tf complexes with these metals and metal-free apoTf.
The cells did not grow well when incubated in a culture medium composed of Eagle's minimum essential medium and horse serum. But they grew well in the presence of Fe²⁺ or Fe³⁺ (10–100 μM) or iron-bound Tf (10–500 nM) in the medium. None of the transition metal ions other than iron was effective. Neither apoTf nor Tf complexes with these metals showed the growth promoting effect. The generality of the requirement of iron for cell growth was ascertained in the primary culture of other types of chick embryonic cells: fibroblasts, cardiac myocytes, retinal pigment cells and spinal nerve cells.
The results show that iron is one of the indispensable substances for cell growth and suggest that Tf protein plays a role in facilitating the transport of iron into the cells.     

Dose-related effects of 17βoestradiol (E2) on liver weight, plasma E2, protein, calcium and thyroid hormone levels, and measurement of the binding of thyroid hormones to vitellogenin in rainbow trout, Salmo gairdneri Richardson

Administration of 17β-oestradiol (E₂) to rainbow trout, in the form of hydrogenated coconut oil implants produced a stable, long-term elevation in plasma E₂ levels. The elevation was doserelated (over the range 1–10mg kg-¹ body weight) both 4 and 8 weeks after implantation. Dose-related increases were also observed with respect to liver weight-body weight ratios and plasma protein levels. Plasma T₃ and total calcium levels were depressed and elevated, respectively, by E₂ treatment but the responses were not linearly related to the dose of E₂ administered; there was no significant effect of E₂ on plasma T₄ levels.
E₂ induced a shift in the binding of T₃ to plasma proteins, with T₃ binding to smaller molecular weight proteins; neither T₄ nor T₃ bound to vitellogenin which was present at high levels in the plasma of E₂-treated fish.