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We have established the complete coding sequence of the human vimentin gene. It had 91% homology to the coding sequence of the Syrian hamster vimentin gene (Quax et al., Cell 35:215-223, 1983) and partial homology to several other sequences coding for intermediate filament proteins. The most striking difference between the Syrian hamster and human vimentin genes was in the 3' untranslated region, which was considerably longer in the Syrian hamster. Using RNA blots and a human vimentin cDNA clone from an Okayama-Berg library, we have established that expression of the vimentin gene was growth regulated. The steady-state levels of cytoplasmic vimentin mRNA in 3T3 cells were increased by serum and platelet-derived growth factor, but not by epidermal growth factor, insulin, or platelet-poor plasma. The increase in expression of the vimentin gene that occurred when G0-phase cells were stimulated to proliferate was detected in six different cell types from four different species. The expression of the vimentin gene was also increased when HL60 cells were induced to differentiate by phorbol esters; it decreased when differentiation was induced by retinoic acid.

We have investigated the expression of Mr 53,000 protein (p53) in total RNA isolated from human peripheral blood mononuclear cells stimulated by phytohemagglutinin, in serum-stimulated human diploid fibroblasts, and in normal and tumor cells of human epithelial colon tissue. We have found that the expression of p53 messenger RNA is growth regulated in human cells following kinetics similar to that previously shown in mouse 3T3 cells, and is increased in the large majority of colon adenocarcinomas in comparison to adjacent normal mucosa and adenoma. This increased expression of p53 is accompanied by a nearly proportional increase in the expression of histone H3. As the expression of histone H3 is restricted to the S phase of the cell cycle and therefore measures the growth fraction of a given population, we suggest that the increased expression of p53 observed in the large majority of colon tumors simply reflects the increased number of cycling cells frequently found in a neoplastic tissue. At variance with these findings a true overexpression of p53 was detected in one SV40-transformed human fibroblasts cell line.

The neuroendocrine bag cells in the abdominal ganglion of Aplysia generate a long-lasting synchronous afterdischarge upon brief stimulation of an afferent pathway. After this afterdischarge the cells become refractory to further synaptic stimulation. We find that synchrony, afterdischarge, and prolonged refractoriness are properties that can be expressed in the isolated asomatic neurites of the bag cells. We have distinguished two independent types of refractoriness. The first (type I) is seen as a failure of action potentials generated in the tips of bag cell neurites to invade cell somata. The second form of refractoriness (type II) controls the duration of afterdischarge such that stimuli after the first afterdischarge produce only very short afterdischarges or fail to elicit an afterdischarge. Type II refractoriness is sensitive to serotonin and certain of its analogues, and to dopamine and the methylxanthine phosphodiesterase inhibitors. Extracellularly applied serotonin suppresses an ongoing afterdischarge while dopamine and the phosphodiesterase inhibitors, when applied at the end of the first afterdischarge, generate a subsequent afterdischarge of long duration without further electrical stimulation. None of these compounds influenced the degree of type I refractoriness. We have shown that both serotonin and dopamine stimulate the formation of cyclic AMP in the bag cell clusters and in the pleurovisceral connectives and that the occurrence of an afterdischarge is associated with a specific increase in total cyclic AMP in bag cell bodies. Moreover, afterdischarges can be generated in unstimulated preparations by extracellular application of the cyclic AMP analogues, 8-benzylthio-cyclic AMP or 8-methylthio-cyclic AMP. Our data suggest that serotonin and/or dopamine may control bag cell activity and that activation of adenylate cyclase is linked to bag cell afterdischarge.

Figure 11 summarizes our present understanding of the relationships between the bag cells, the atrial gland, their respective peptides, the central nervous system, and reproductive behavior. There are some interesting aspects of the overall organization of the system. The three hormonal peptides (ELH and the two atrial gland peptides) have specific actions on the central nervous system not unlike what we are currently learning from mammalian systems (e.g., LHRH and TRH). ELH, in addition, has several specific peripheral targets, the details of which remain to be worked out. The fact that ELH and other hormones have multiple targets within the central nervous system as well as nonnervous peripheral targets raises the question of whether one or more different receptors exist for single hormone. We suggest that peptides larger than perhaps five residues may carry several "messages" or receptor binding sites encoded within the one molecule. Large peptides such as ELH could obviously have separate domains of charge distribution within the molecule, and these would have the advantage, over the classical small molecule transmitters, of activating a variety of very different targets. The atrial gland is a peripheral source of peptides with potent nervous system actions; this is reminiscent of peptides in mammals, e.g., substance P, gastrin, and somatostatin, all of which were initially isolated from the gut and which are now being found in and also have actions on the central nervous system. Such resemblances in the principles of organization between mammals and molluscs are constant reminders that neuropeptidergic systems are old tricks in the evolutionary bag and that what we learn from molluscs and other invertebrates about mechanisms and organization will likely apply to mammals.

We have studied the cell cycle of resting T lymphocytes from long-term (LT) cultures following stimulation with phytohemagglutinin (PHA) and recombinant Interleukin 2 (IL-2). We examined the kinetics of entry into S phase by autoradiography, the accumulation of cellular RNA by microfluorometric techniques, and ultrastructural morphology by electron microscopy. In addition, we examined the expression at the mRNA level of six cell cycle-dependent growth-regulated genes (c-fos, c-myc, KC-1, JE-3, vimentin, and histone H3). We show that T lymphocytes of LT cultures respond differently to mitogenic stimulation than the T lymphocytes of freshly isolated peripheral blood mononuclear cell cultures. At the ultrastructural, biochemical, and molecular levels, resting T lymphocytes of LT cultures can be distinguished from physiological (G0) lymphocytes of peripheral blood.

We have studied the expression of seven cell cycle-dependent genes in phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells, in macrophage-depleted cultures and in macrophage-depleted cultures plus Interleukin-2 (IL-2). The expression of all seven genes is increased in PHA stimulated peripheral cells. Only two (2F1 and the IL-2 receptor) are increased in PHA-stimulated macrophage depleted cultures. Addition of IL-2 to these cultures increased the RNA levels of four genes (KC-1, c-myc, beta-actin and IL-2R), but has no effect on three others (4F1, 2F1, and JE-3). The results indicate that the expression of these cell cycle genes is regulated by different components of the mitogenic stimulus.

We constructed a stable cell line, 3T3A5, which carried a chimeric gene in which the simian virus 40 T-antigen-coding gene was under the control of the mouse ribosomal DNA promoter. These cells expressed T antigen when they were growing exponentially in 10% fetal calf serum, but they all became T negative when incubated for 5 days in low-concentration serum. The readdition of serum or platelet-poor plasma again induced the expression of T antigen, which was accompanied by an increase in steady-state levels of the corresponding RNA. Among the various growth factors tested for their ability to induce T-antigen expression in 3T3A5 cells, only insulinlike growth factor I (IGF-I) could induce T antigen at physiological concentrations. The effect of IGF-I or platelet-poor plasma was abolished by an antibody to IGF-I. Other growth factors, like insulin and epidermal growth factor, could induce the expression of T antigen in 3T3A5 cells, but only at concentrations far above the physiological range. Other growth factors were totally ineffective. These results indicate that exposure of cells to IGF-I can activate transcription from the ribosomal DNA promoter.

The bag cells in the abdominal ganglion of Aplysia californica control egg-laying behavior by releasing a polypeptide (ELH) during an afterdischarge of synchronous action potentials. We have used intracellular injection of Lucifer Yellow to study the morphology and interconnections of the bag cells. These neurosecretory cells are typically multipolar and their processes extend in all directions out from the bag cell clusters into the surrounding connective tissue, where they branch in a complex manner. In some of the dye injection experiments, dye transfer from the injected cell to neighboring cells was observed. Freeze fracture of the bag cell clusters and their surrounding connective tissue revealed numerous gap junctions on bag cell processes within the clusters as well as on more distal processes. We have also examined the morphology and coupling between bag cells in primary culture. As in the intact ganglion, bag cells in culture were found to be multipolar. All pairs of bag cells whose somata or processes had formed contacts in culture were electrically coupled. The strongest coupling was observed between pairs of cells whose somata appeared closely apposed. In these cases transfer of Lucifer Yellow between cells could also be observed. It is therefore likely that the synchrony of bag cell action potentials during a bag cell afterdischarge is a result of coupling between individual cells in the bag cell cluster.

The neurosecretory bag cell neurons of the mollusk, Aplysia, control egg-laying behavior in the animal. In these cells, elevation of cAMP greatly enhances the height and width of action potentials. A similar enhancement of action potentials is seen during the bag cell afterdischarge, a 30 min period of repetitive activity that may be triggered by peptides from the reproductive tract or by brief extracellular stimulation. The enhancement of action potentials during an afterdischarge is well correlated with the observed elevations of cAMP. In the present study, we have examined the effects of forskolin (an activator of adenylate cyclase) and theophylline (a phosphodiesterase inhibitor) on the delayed outward currents that are likely to control repolarization of the action potential. Isolated bag cell neurons, maintained in primary culture, were studied with a whole-cell patch clamp technique. High intracellular concentrations of EGTA were used to block potassium current activated by calcium entry. Analysis of the remaining voltage-dependent delayed outward current revealed two major components, which could be separated on the basis of their different kinetic properties. Both currents (IK1 and IK2) were carried by potassium. IK1, which did not inactivate during 100 msec depolarizations, was reduced in amplitude by application of forskolin and theophylline. Ik2, a current defined by its faster kinetic properties, partially inactivated during 100 msec depolarizations. This inactivation was markedly speeded by application of forskolin and theophylline. It is suggested that such changes in outward current in response to cAMP could explain the enhancement of spike width seen during an afterdischarge in vivo.

A number of genes and cDNA sequences (including at least four oncogenes) are known to be expressed in a cell cycle-dependent manner, i.e. the levels of specific mRNAs vary with the phases of the cell cycle. In order to explore the significance of some of these sequences in the mitogenic response, we have investigated the expression of 8 cell cycle-dependent sequences (plus two control sequences, not expressed in a cell cycle-dependent manner) under a variety of conditions. These conditions included cells of different types, from different species, stimulated to proliferate by different mitogens. The genes (or sequences) studied included five cDNA clones whose sequences are preferentially expressed in early G1, i.e. two cDNA clones inducible by platelet-derived growth factor (JE-3 and KC-1), and three cDNA clones inducible by serum (2A9, 2F1, 4F1); and three oncogenes (c-myc, c-rasHa and p53) whose expression is known to be cycle-dependent. All of the tested genes, except 2A9, c-rasHa and the control genes, are expressed in a cell cycle-dependent manner in human peripheral blood mononuclear cells stimulated by phytohemagglutinin and in serum-stimulated mouse and Syrian hamster fibroblasts. The inducibility of these genes by different mitogens in cells of different types and from different species strongly suggests that these genes play a role in cell cycle progression. This conclusion is further supported by the known structural and functional similarities between cell-cycle dependent genes, oncogenes and genes coding for cell-cycle related molecules.

Protein kinase C is an enzyme whose activity is modulated by its lipid environment and which is fully activated by diacylglycerol in the presence of phosphatidyl serine and calcium ions. This kinase is highly enriched in the nervous systems of both vertebrates and invertebrates. The activity of protein kinase C can be stimulated in intact cells by certain synthetic diacylglycerols as well as by phorbol esters which substitute for endogenous diacylglycerol. The effects of such activators on the endogenous electrical properties of neurones, as well as on synaptic transmission, have recently been investigated in several vertebrate and invertebrate preparations of neurones. One example is that of the bag cell neurones of Aplysia which, in response to brief stimulation, generate a prolonged discharge during which the height of their action potentials is increased. Exposure of isolated bag cell neurones to activators of protein kinase C results in the enhancement of their action potentials through an increase in the amplitude of their voltage-dependent calcium current. This is caused by the unmasking of a previously inactive species of calcium channel in the plasma membrane.

A brief exposure of quiescent (Go) Swiss 3T3 mouse fibroblasts to inhibitors of protein synthesis can replace platelet-derived growth factor in the stimulation of cellular DNA synthesis. When 3T3 cells, after a 6 hr exposure to either cycloheximide or puromycin, are incubated with platelet-poor plasma, a significant percentage of cells enters DNA synthesis. Either inhibition of protein synthesis, or platelet poor plasma by themselves are totally ineffective. A possible mechanism by which inhibitors of protein synthesis may initiate cell cycle progression is through the activation of the c-myc gene.

The thymic atrophy developed in osteopetrotic grey-lethal (gl/gl) mouse during the last 10 days of life, and at 35 days of life gl/gl mice had only a few million thymic cells. Maximal survival of gl/gl mice was about 40 days. Earlier, at 20 days of age despite the presence of all signs of osteopetrosis their thymus had normal cellularity. This change in thymus cellularity was associated with relative increase in the frequency of cells forming thymic non-lymphoid (stromal) colonies in vitro. There were no qualitative differences in the presence of major cell types i.e. fibroblastoid, epithelioid and macrophages normally observed in these colonies. However, giant multinucleated cells resembling macrophages in size and appearance of nuclei were observed exclusively in cultures of the thymus from 20 day old control mice and not in cultures of gl/gl mice. On the other hand, in cultures of the thymuses of 35 day old gl/gl mice giant cells were observed that were nearly absent in cultures of the thymuses of control mice. These cells were either polynuclear or their nuclei were segmented and they resembled rather nuclei of epithelioid and fibroblastoid cells than nuclei of macrophages. The significance of all these findings is discussed.

We have investigated the expression of six growth-regulated genes (c-myc, c-myb, p53, 4F1, 2F1, and ornithine decarboxylase) and the S-phase-specific histone H3 gene in acute myeloid and lymphoid leukemic cells. We have purposely chosen three growth-regulated protooncogenes that share similar biological features and three gene sequences that have in common the cell cycle dependence of their expression in cells of different tissue and in different species. The level of expression was determined by measuring the amounts of specific RNA by Northern blot analysis. Levels of expression of the six growth-regulated genes were compared to the level of expression of the S-phase-specific H3 gene and among themselves. This method distinguishes the increased expression of a growth-regulated gene due to a true altered activation from over-expression which simply reflects an increase in the fraction of cycling cells. We have found that six of 14 patients with acute leukemias have markedly high ratios of c-myc/H3, c-myc/p53, and c-myc/c-myb expression. Two patients with altered c-myc expression have also a high ratio p53/H3. Within the group of cell cycle-dependent genes the ratios of expression seem in the overall much more regular with the clear exception of a patient with acute myelogenous leukemia in which the ratios 4F1/H3 and 2F1/H3 are significantly increased. A possible interpretation of these findings is that the fraction of noncycling leukemic cells that often constitute the majority of the entire leukemic population is in some cases in a true resting state, whereas in other cases heterogeneous degrees of growth arrest might occur. The altered expression of c-myc seems the feature most commonly associated with this putative growth arrest of leukemic cells suggesting that this gene may contribute to the impairment of proliferative control that is associated with the leukemic phenotype.

This article summarizes common features of activation of different types of cells along different physiological lines such as proliferation, differentiation, and execution of function of terminally differentiated cells. The common basis of many of these phenomena includes (i) first messengers (growth factors, cytokines, neurotransmitters, etc.) acting on membrane receptors, (ii) second messengers (cAMP, IP3, DAG, Ca2+) spreading an activating signal inside the cell, and (iii) elevated expression of some genes (c-fos, c-myc, ornithine decarboxylase). The role of the genetic correlate in cell activation is emphasized, and it is concluded that the aforementioned genes (their protein products) should be called third messengers, whose function is mediation of long-term phenotypic changes.

Egg laying in Aplysia involves a well-characterized series of behaviors that can last for several hours. The behaviors are controlled by two bilateral clusters of peptidergic neurons in the abdominal ganglion. Following brief stimulation, these neurons, which have been termed the bag cell neurons, undergo a sequence of changes in their excitability lasting many hours. The bag cell neurons have served as a model system for studying the molecular mechanisms involved in the synthesis, processing, and release of neuroactive peptides and in the regulation of prolonged changes in neuronal excitability.

An unusually high level of expression of the c-myc protooncogene was observed in peripheral blood lymphocytes of a patient with prolymphocytic leukemia (atypical chronic lymphocytic leukemia). The overexpression of c-myc could not be attributed to a high level of proliferating activity of the leukemic cells in the blood. Treatment with cytosine-arabinoside at high doses abolished this altered expression of c-myc and resulted in a twofold increase in the expression of a gene sequence encoding the invariant gamma-chain of class II histocompatibility antigens, preferentially expressed in resting B lymphocytes. These observations suggest that the leukemic cells may have been arrested in the cell cycle outside the G0 phase. Our findings demonstrate that growth-regulated genes can be useful molecular markers of diseases with altered mechanisms of cellular proliferation.

The concentration of taurine and the activities of cysteinesulphinate decarboxylase and glutamate decarboxylase have been measured in rat brain. During development, taurine exhibited a decrease in concentration unrelated to the activity of cysteinesulphinate decarboxylase which increased during the same period. The distribution of taurine in subcellular fractions of adult and 7-day-old rat brain was typical of most amino acids, whereas half of the cysteinesulphinate decarboxylase activity was found in the nerve-ending cytoplasm. In anatomical distribution, taurine displayed great regional heterogeneity but both cysteinesulphinate decarboxylase and glutamate decarboxylase were more evenly distributed. Hypertaurinaemia was shown to have no effect on the entry of glycine into the brain or on its utilization in protein synthesis.