Benzodiazepine Binding to Cultured Human Pituitary Cells

Benzodiazepine receptors were investigated in a cell line of human pituitary cells (18-54,SF) grown in serum-free medium. Preparations of 18-54,SF whole cells and cell membranes were shown to possess saturable [3H]diazepam binding sites. Membrane sites were found to have a KD of 20 nM for diazepam while whole cells possessed a twofold higher value. The KD values determined from Rosenthal, Hill, and kinetic analyses were consistent for each preparation. Whole-cell binding of [3H]diazepam was observed to be more stable than binding to membranes at higher temperatures (37 degrees C) and when longer incubation times (60 min) were employed at 4 degrees C. The rank order potency of various benzodiazepines to inhibit [3H]diazepam binding to whole cells and membranes was Ro 5-4864, flunitrazepam, diazepam, and clonazepam. Representatives of other drug classes did not inhibit this benzodiazepine binding. When 18-54,SF cells were grown for 24 h with 100 nM diazepam and then extensively washed membranes prepared, the KD for diazepam increased to 38 nM whereas the Bmax was unchanged when compared with untreated controls. Overall, these findings indicate that pituitary cells possess a peripheral-type benzodiazepine receptor and that the whole cell receptor differs quantitatively when compared with the membrane receptor.     

Adhesion of Haemophilus paragallinarum to Cultured Chicken Cells

Adhesion of Haemophilus paragallinarum to chicken embryo fibroblasts (CEF) in vitro was investigated in correlation with its virulence. Under the scanning electron microscope, the organisms were seen adhering to CEF exposed to the bacteria. By transmission electron microscopy, the organisms appeared to attach to the plasma membrane of CEF by their fuzzy material. They were enclosed in membrane-limited vesicles and appeared morphologically intact. Specific fluorescence was seen in the cytoplasm of CEF in later stages of infection. There was a good correlation between pathogenicity of the organisms for chickens and their ability to adhere to CEF. This correlation was reinforced by the fact that the ability of a pathogenic strain to adhere to the mucosal surface of the chicken trachea was demonstrated by scanning electron microscopy.     

Somatic Embryogenesis in Cultured Carrot Cells

Somatic embryogenesis in cultured plant cells is an ideal system for investigating the whole process of differentiation and development from single cells to whole plants, and especially the molecular mechanism of expression of totipotency. This review reports recent progress the studies on somatic embryogenesis.     

Neurofilament Proteins in Cultured Chromaffin Cells

Antibodies were raised against the 200-kd, 145-kd, and 68-kd subunits of a rat neurofilament preparation. Immunoblots showed that each antibody was specific for its antigen and that it did not cross-react with any of the two other neurofilament polypeptides. Use of the three antibody preparations to stain bovine chromaffin cells in culture by the indirect immunofluorescence technique indicated that the three neurofilament polypeptides are present in chromaffin cells maintained in culture for 3 or 7 days. The three anti-neurofilament antibodies labelled the cells in a similar pattern: very thin filaments specifically localized around the nucleus were observed whereas neurites and growth cones, developed by cultured chromaffin cells, were generally not stained. Some fibroblasts were present in our cultures but they were never stained by any of the neurofilament antibodies. This indicated that the antibodies used do not react with vimentin, the major intermediate filament protein found in fibroblasts. The three neurofilament antibodies were also used to immunoprecipitate specifically three proteins of molecular weights 210 kd, 160 kd, 70 kd from solubilized extracts of cultured chromaffin cells that were radiolabelled with [35S]methionine. These proteins correspond in molecular weight to the neurofilament triplet found in bovine brain. Finally, the presence of neurofilaments in freshly isolated chromaffin cells was tested by immunoblotting using the 68-kd antibody. A 70-kd protein was specifically stained by this antibody, suggesting that neurofilaments are not only present in cultured chromaffin cells but also in the adrenal gland in vivo. It is concluded from these results that chromaffin cells contain completely assembled neurofilaments. This additional neuronal property again illustrates that chromaffin cells are closely related to neurons and therefore represent an attractive model system for the study of functional aspects of adrenergic neurons.     

Peroxisome Dynamics in Cultured Mammalian Cells

Despite the identification and characterization of various proteins that are essential for peroxisome biogenesis, the origin and the turnover of peroxisomes are still unresolved critical issues. In this study, we used the HaloTag technology as a new approach to examine peroxisome dynamics in cultured mammalian cells. This technology is based on the formation of a covalent bond between the HaloTag protein–a mutated bacterial dehalogenase which is fused to the protein of interest–and a synthetic haloalkane ligand that contains a fluorophore or affinity tag. By using cell-permeable ligands of distinct fluorescence, it is possible to image distinct pools of newly synthesized proteins, generated from a single genetic HaloTag-containing construct, at different wavelengths. Here, we show that peroxisomes display an age-related heterogeneity with respect to their capacity to incorporate newly synthesized proteins. We also demonstrate that these organelles do not exchange their protein content. In addition, we present evidence that the matrix protein content of pre-existing peroxisomes is not evenly distributed over new organelles. Finally, we show that peroxisomes in cultured mammalian cells, under basal growth conditions, have a half-life of approximately 2 days and are mainly degraded by an autophagy-related mechanism. The implications of these findings are discussed.     

3D Cultures of Prostate Cancer Cells Cultured in a Novel High-Throughput Culture Platform Are More Resistant to Chemotherapeutics Compared to Cells Cultured in Monolayer

Despite monolayer cultures being widely used for cancer drug development and testing, 2D cultures tend to be hypersensitive to chemotherapy and are relatively poor predictors of whether a drug will provide clinical benefit. Whilst generally more complicated, three dimensional (3D) culture systems often better recapitulate true cancer architecture and provide a more accurate drug response. As a step towards making 3D cancer cultures more accessible, we have developed a microwell platform and surface modification protocol to enable high throughput manufacture of 3D cancer aggregates. Herein we use this novel system to characterize prostate cancer cell microaggregates, including growth kinetics and drug sensitivity. Our results indicate that prostate cancer cells are viable in this system, however some non-cancerous prostate cell lines are not. This system allows us to consistently control for the presence or absence of an apoptotic core in the 3D cancer microaggregates. Similar to tumor tissues, the 3D microaggregates display poor polarity. Critically the response of 3D microaggregates to the chemotherapeutic drug, docetaxel, is more consistent with in vivo results than the equivalent 2D controls. Cumulatively, our results demonstrate that these prostate cancer microaggregates better recapitulate the morphology of prostate tumors compared to 2D and can be used for high-throughput drug testing.     

Cultured Rat Astrocytes Give Rise to Neural Stem Cells

Previously, we reported the occurrence of neural stem cells (NSCs) around an area of damage after rat traumatic brain injury (TBI), but it was unclear if this was due to blastgenesis in astrocytes, or to NSCs migrating from the subventricular zone (SVZ). In this study, NSCs were isolated and cultured from cultured type 1 astrocytes taken from newborn rat cortex in which the subventricular zone and hippocampus had been discarded. All cultured type 1 astrocytes showed glial fibrillary acidic protein (GFAP) immunopositivity. Nestin immunopositive spheres were isolated from type 1 astrocytes and cultured in the presence of bFGF and EGF in the medium. Neurospheres differentiated into Tuj1-, GFAP- and A2B5-positive cells after 4 days of culture without bFGF and EGF. These results indicate that isolated neurospheres from brain cortex astrocytes can differentiate into neurons and glia and might contribute to neurogenesis and neuroplasticity.     

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births^1,2, 60-80% of all miscarriages^3,4, 10% of stillbirths^2,5, 13% of individuals with congenital heart disease⁶, 3-6% of infertility cases², and in many patients with developmental delay and birth defects⁷. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance^8,9.  Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents^10-13.Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy''s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)^14,15.     

Attempts to Stimulate Mitosis in Cultured Trophoblast Cells of Cattle Embryos

Trophoblast from Day-14 bovine embryos was cultured in medium containing mitogens to determine if the mitotic index could be altered. Trophoblast from each of 15 embryos was cultured in minimum essential medium (Eagles) with 20 % fetal calf serum (control) or in this medium supplemented with pokeweed mitogen (1 %, v/v), phytohemagglutinin (1 %, v/v), concanavalin A (1 %, v/v) or thymidine (2 mg/ml). No mitogenic effect was observed due to any of the treatments. However, mitotic indexes were significantly lower when pokeweed (P < 0.05) or thymidine (P < 0.01) was added to the medium. A highly significant (P < 0.001) variation in mitotic index between embryos was observed.     

Post-Translational Arginylation of Proteins in Cultured Cells

The aim of this study was to analyze the N-terminal post-translational incorporation of arginine into cytosolic proteins from cultured cells and the in vitro incorporation of arginine into soluble proteins of PC12 cells after serum deprivation. Arginine incorporation was measured in the presence of protein synthesis inhibitors. None of the inhibitors used affected significantly the arginylation reaction while the novo synthesis of protein was reduced by 98%. Under these conditions, we found that of the total [¹⁴C]arginine incorporated into the proteins, around 20% to 40% was incorporated into the N-terminal position of soluble proteins by a post-translational mechanism. These results suggest that this post-translational aminoacylation may be a widespread reaction in neuronal and non-neuronal cells. We also found that in PC12 cells, the in vitro post-translational arginylation was 60% higher in apoptotic cells with respect to control cells. These findings suggest that the post-translational arginylation of proteins may be involved in programmed cell death.     

Carboxylmethylation of Calmodulin in Cultured Pituitary Cells

We have used fast protein liquid chromatography (FPLC) and reverse-phase HPLC to rapidly resolve carboxylmethylated proteins in cultured pituitary GH3 cells. This procedure preserves labile carboxylmethyl esters, which are lost under the usual procedures employed for protein fractionation. GH3 cells were incubated with [methyl-3H]-methionine in culture and incorporation of label into the soluble fraction, total cell protein, and protein carboxylmethyl esters was determined; protein carboxylmethyl ester formation was shown to be resistant to cycloheximide. Fractionation of protein carboxylmethyl esters from GH3 cells by gel permeation FPLC, anion-exchange FPLC, and reverse-phase HPLC in the presence of calcium and in the presence of EGTA identified two proteins that are major substrates for protein carboxylmethyltransferase and indicated that one of these proteins is calmodulin. Similar results were obtained when a cytosolic fraction from GH3 cells was incubated with S-adenosyl-L-[methyl-3H]methionine. These results indicate that rapid chromatography at low temperature and low pH is useful for the analysis of eucaryotic carboxylmethylated proteins and that contrary to reports obtained in other systems, calmodulin is carboxylmethylated in intact pituitary cells.     

K-Casein Suppresses Melanogenesis in Cultured Pigment Cells

The effects of bovine milk proteins on melanogenesis in B16 cells were examined. Both whey protein isolate and casein exhibited depigmenting properties. Among the major protein components of milk—including β-lactoglobulin, α-lactalbumin, α-, β-, and k-casein—only K-casein exhibited the depigmenting effect. However, the carboxyl terminal peptide of K-casein, glycomacropeptide, did not show this activity. Also, K-casein promoted the proliferation of the cells and inhibited the activity of tyrosinase in the cells. These results indicate that K-casein acts as a melanogenesis-suppressing modulator.     

The Dynamics of Microtubules in Cultured Cells

The behavior of microtubules in cultured cells in a cooled matrix after the microinjection of fluorescent tubulin was studied using a frame recording with a digital camcorder. In the cell lamella, the positive ends of individual microtubules extend and shorten at random. The histograms of rate distribution have an almost normal distribution with a mode close to 0. The maximum rate of lengthening and shortening reaches 30 and 50 m/min, respectively. The positive ends of microtubules in PtK cells were in an equilibrium state, while in murine embryonic fibroblasts and Vero cells, they were usually displaced to the cell edge. Free microtubules were present in the cells of all three cultures. In the epithelial cells, they were numerous and relatively stable, while in the fibroblasts, they occurred rarely and were depolymerized at the proximal end. Free microtubules in PtK cells appeared mostly due to spontaneous assembly in the cytoplasm (not in the relationship with the preexisting microtubules) and, more rarely, due to breakage of long microtubules. Separation of microtubules from the centrosome is a very rare event. Unlike positive ends that were characterized by dynamic instability, negative ends were stable and were sometimes depolymerized. When long microtubules were broken, new negative ends were formed that were, as a rule, stable, while in the lamella of fibroblasts (in murine embryonic fibroblasts and Vero cells), new negative ends were immediately depolymerized: free microtubules existed in these cells no more than 1–2 min. A diffusion model has been proposed where the behavior of microtubule ends is considered as unidimensional diffusion. The coefficient of diffusion of positive ends in the epithelial cells is several times less than in the fibroblasts, thus suggesting a higher rate of tubulin metabolism in the fibroblasts as compared to the epithelium. The results obtained indicate that for the exchange of long microtubules, the dynamic instability is not sufficient. In the fibroblasts, their exchange takes place mostly at the expense of depolymerization of the liberating negative ends, which agrees with the previously proposed conveyer hypothesis of microtubule assembly on the centrosome.     

Troponin in Cultured Chicken Breast Muscle Cells

The onset of troponin accumulation and the localization of troponin in cultured chick embryo skeletal muscle cells were studied by means of indrect immunofluorescent microscopy. At 31 hr after plating, troponin components were detected in 54–62% of total mononucleated myogenic cells and in all myotubes as longitudial fibrous structures. ³H-thymidine incorporation stduy coupled with the immunofluorescent microscopy showed that mononucleated myogenic cells at the mitotic stage did not contain troponin. As myotube maturation proceeds, the troponin-containing fibers were organized into cross-striated structures. At the myotube stage, muscle cells were labeled with ³⁵S-methionine and proteins synthesized were analyzed by two-dimensional gel electrophoresis. It was found that myotubes in culture synthesized both fast and slow types of troponin-I and-C. Our results suggest that fast and slow types of troponin components are synthesized in cultured skeletal muscle cells before or at the early phase of myofibrillogenesis.     

Proteins binding to DNA and their Relation to Growth in Cultured Mammalian Cells

Analysis of the proteins of mouse fibroblasts which can bind to DNA suggests that one of them may control DNA synthesis.     

Resistance Mechanism of Cultured Plant Cells to Tobacco Mosaic Virus

In somaclonal tissues obtained from systemically TMV-infected tobacco plants, a relation between changes of TMV amounts and the callus growth was examined. The culture medium was suitable for maintaining a constant concentration of TMV as well as active callus growth. By using the shake-culture method, somaclonal tissues were separated into two classes on the basis of callus sizes. In large callus tissues, TMV amounts were constant during subculturing but the tissues did not either grow or release the newly divided cells after the last subculture. On the other hand, smaller callus tissues grew markedly and the TMV amounts were conspicuously lowered. After shake-subculture of smaller tissues, they were successfully regenerated to plantlets. None of the plantlets expressed any mosaic symptoms, while plantlets from the original somaclones showed severe mosaic symptoms of TMV in leaflets. Thus, the present report describes the successful production of virus-free plantlets from infected somaclonal callus cultures.     

Proteins Associated with Adaptation of Cultured Tobacco Cells to NaCl

Cultured tobacco cells (Nicotiana tabacum L. cv Wisconsin 38) adapted to grow in medium containing high levels of NaCl or polyethylene glycol (PEG) produce several new or enhanced polypeptide bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The intensities of some of the polypeptide bands (molecular weights of 58, 37, 35.5, 34, 26, 21, 19.5, and 18 kilodaltons) increase with increasing levels of NaCl adaptation, while the intensities of other polypeptide bands (54, 52, 17.5, and 16.5 kilodaltons) are reduced. Enhanced levels of 43- and 26-kilodalton polypeptides are present in both NaCl and PEG-induced water stress adapted cells but are not detectable in unadapted cells. In addition, PEG adapted cells have enhanced levels of 29-, 17.5-, 16.5-, and 11-kilodalton polypeptides and reduced levels of 58-, 54-, 52-, 37-, 35.5-, 34-, 21-, 19.5-, and 18-kilodalton polypeptide bands.

Synthesis of 26-kilodalton polypeptide(s) occurs at two different periods during culture growth of NaCl adapted cells. Unadapted cells also incorporate ³⁵S into a 26-kilodalton polypeptide during the later stage of culture growth beginning at midlog phase. The 26-kilodalton polypeptides from adapted and unadapted cells have similar partial proteolysis peptide maps and are immunologically cross-reactive. During adaptation to NaCl, unadapted cells synthesize and accumulate a major 26-kilodalton polypeptide, and the beginning of synthesis corresponds to the period of osmotic adjustment and culture growth. From our results, we suggest an involvement of the 26-kilodalton polypeptide in the adaptation of cultured tobacco cells to NaCl and water stress.

     

Digitoxin Biosynthesis in Isolated Mesophyll Cells and Cultured Cells of Digitalis

In the laminae of Digitalis, most of the digitoxin present isfound in the mesophyll. A new method for determining the amountof digitoxin biosynthesis using a digitoxin antibody was devisedto estimate this activity in isolated mesophyll cells and culturedcells. Isolated mesophyll cells showed significant activity,which suggests that the site of biosynthesis and the accumulationof cardenolides in a lamina of Digitalis is mainly in the mesophyllcells. Of five liquid cultures of D. purpurea; green shoot-formingcultures, white shoot-forming cultures, root-forming cultures,undifferentiated green cells and undifferentiated white cells,the green shoot-forming cultures had the highest activity. Thewhite shoot-forming cultures had about one-third the activityof the green shoot-forming cultures, and the other three cultureshad very low activity. No stimulatory effect of light was foundduring the 48-h incubation. (Received January 19, 1984; Accepted June 8, 1984)     

Mineral Composition of Cultured Ginseng Cells

The contents of macroelements and microelements in ginseng roots and callus cultures was determined by atom absorption spectroscopy. Ginseng cells and tissues were shown to accumulate considerable amounts of microelements. The content of six of the eleven mineral components studied (K, Ca, Na, Mo, Mn, and Cr) in callus cultures was higher than that in roots of agricultural ginseng plants. We revealed good correlations between the contents of microelements (K, Ca, and Mg), as well as between the concentrations of macroelements (Mo, Li, Cu, and Cr), in ginseng cultures. The ability to accumulate elements varied between ginseng species, which was probably related to their genetic features. Our findings indicate that cultured ginseng cells hold much promise as a source of microelements.