Impact of external calcium and calcium sensors on ginsenoside Rb1 biosynthesis by Panax notoginseng cells.

The effects of external calcium concentrations on biosynthesis of ginsenoside Rb1 and several calcium signal sensors were quantitatively investigated in suspension cultures of Panax notoginseng cells. It was observed that the synthesis of intracellular ginsenoside Rb1 in 3-day incubation was dependent on the medium Ca2+ concentration (0-13 mM). At an optimal Ca2+ concentration of 8 mM, a maximal ginsenoside Rb1 content of 1.88 +/- 0.03 mg g(-1) dry weight was reached, which was about 60% and 25% higher than that at Ca2+ concentrations of 0 and 3 mM, respectively. Ca2+ feeding experiments confirmed the Ca2+ concentration-dependent Rb1 biosynthesis. In order to understand the mechanism of the signal transduction from external Ca2+ to ginsenoside biosynthesis, the intracellular content of calcium and calmodulin (CaM), activities of calcium/calmodulin-dependent NAD kinase (CCDNK) and calcium-dependent protein kinase (CDPK), and activity of a new biosynthetic enzyme of ginsenoside Rb1, i.e., UDPG:ginsenoside Rd glucosyltransferase (UGRdGT), in the cultured cells were all analyzed. The intracellular calcium content and CCDNK activity were increased with an increase of external Ca2+ concentration within 0-13 mM. In contrast, the CaM content and activities of CDPK and UGRdGT reached their highest levels at 8 mM of initial Ca2+ concentration, which was also optimal to the ginsenoside Rb1 synthesis. A similar Ca2+ concentration-dependency of the intracellular contents of calcium and CaM and activities of CCDNK, CDPK, and UGRdGT was confirmed in Ca2+ feeding experiments. Finally, a possible model on the effect of external calcium on ginsenoside Rb1 biosynthesis via the signal transduction pathway of CaM, CDPK, and UGRdGT is proposed. Regulation of external Ca2+ concentration is considered a useful strategy for manipulating ginsenoside Rb1 biosynthesis by P. notoginseng cells.

A new approach for the detection of multiple protein kinases using monoclonal antibodies directed to the highly conserved region of protein kinases.

To explore the protein kinase family enzymes expressed in cells, we attempted to generate antibodies that could detect a wide variety of protein kinases. For the production of such antibodies, synthetic peptides corresponding to amino acid sequences of a highly conserved subdomain (subdomain VIB) of the protein kinase family were used for immunization. Among the various peptide antigens, a peptide with 16 amino acids, CVVHRDLKPENLLLAS, effectively produced polyclonal antibodies with broad cross-reactivities to protein kinases. Two monoclonal antibodies, designated M8C and M1C, detected a variety of protein kinases such as calmodulin-dependent protein kinase II, calmodulin-dependent protein kinase IV, cAMP-dependent protein kinase, and mitogen-activated protein kinases, on Western blotting. The antibodies also immunoprecipitated various protein kinases in cell extracts. Furthermore, these antibodies could be used for detection of positive clones in the expression cloning of various protein kinases. Among 39 positive clones obtained from mouse brain cDNA library, 36 clones were identified as cDNA clones for various known and novel protein serine/threonine kinases, suggesting that the antibodies reacted highly specifically with various protein kinases. These results indicate that the present monoclonal antibodies directed to multiple protein kinases will be a powerful tool for the detection of a variety of known and novel protein kinases in cells.

Two epochs in the development of gamma-aminobutyric acidergic neurons in the ferret thalamus.

These studies chart the development of gamma-aminobutyric acid (GABA)-ergic neurons in the three divisions of the thalamus (ventral thalamus, dorsal thalamus, and epithalamus). GABAergic neurons were identified by in situ hybridization to localize mRNA for 67-kDa glutamic acid decarboxylase (GAD(67)) and related to the morphological maturation of the thalamus in fetal and postnatal brains and to expression of transcription factors Gbx-2 and Tbr-1. Origins of GABAergic neurons were sought in in vitro slice preparations incubated in bromodeoxyuridine or injected with a carbocyanine dye. GABA neurons of ventral thalamus (reticular nucleus, ventral lateral geniculate nucleus, zona incerta, and nucleus of the fields of Forel) and of epithalamus appear at least 14 days before those intrinsic to dorsal thalamus. Ventral thalamus GABA cells are derived from a region connecting the ventricular zone of the third ventricle to the caudal ganglionic eminence. This region is delimited ventrally by the Tbr-1-expressing prethalamic eminence and dorsally by the Gbx-2-expressing part of the dorsal thalamus. GABA neurons of epithalamus are derived from the embryonic pretectum. Neurons continue to be added to the ventral thalamus, perireticular nucleus, entopeduncular nucleus, and substantia nigra from the ganglionic eminence as development proceeds. GAD(67)-expressing cells of dorsal thalamus become detectable only at birth and populate the thalamus from posterior to anterior over the first week of life. Although a very small number reaches the dorsal lateral geniculate nucleus from the caudal ganglionic eminence, there is no obvious new source of proliferating neurons at this stage. Intrinsic GABA cells of dorsal thalamus may, therefore, derive from an early generated population of cells that turns on a GABAergic phenotype only late in development.

Localization of CaMKIIalpha in rat primary sensory neurons: increase in inflammation.

This study investigates Ca(2+)/calmodulin kinase IIalpha (CaMKIIalpha) in primary sensory neurons. Immunohistochemical staining with a CaMKIIalpha antibody demonstrates 28% of dorsal root ganglion (DRG) cells are positively stained and have a diameter of 27 +/- 2.4 microm (mean +/- S.D.). Placement of tight ligatures around the sciatic nerve demonstrates a build up of immunoreaction product proximal to the ligatures indicating that CaMKIIalpha is transported into the peripheral processes of DRG cells. Immunostaining of lumbar dorsal roots at the electron microscopic level demonstrates reaction product in 15.4 +/- 2.1% of unmyelinated and 2.4 +/- 1.0% of myelinated axons, indicating that CaMKIIalpha is transported into the central processes of DRG cells. Electron microscopic analysis of normal digital nerves demonstrates CaMKIIalpha labeling in 3.3 +/- 0.3% of unmyelinated and 2.0 +/- 1.1% of myelinated cutaneous axons. These percentages increase significantly to 14.1 +/- 2.3% for unmyelinated and 5.1 +/- 1.4% for myelinated axons 48 h after complete Freund's adjuvant-induced inflammation of the hindpaw. The data indicate that CaMKIIalpha is present in small diameter primary sensory neurons, that it is transported into the peripheral and central processes of these cells and may play a role in processing noxious input, particularly in the inflamed state.

Low GSK-3beta immunoreactivity in postmortem frontal cortex of schizophrenic patients.

OBJECTIVE: Glycogen synthase kinase-3 (GSK-3) is a protein kinase that is highly abundant in the brain. It is involved in signal transduction cascades of multiple cellular processes, particularly neurodevelopment. In an attempt to explore possible involvement of GSK-3beta in psychiatric disorders, the authors examined its levels in postmortem brain tissue. METHOD: Western blot analysis was performed to measure GSK-3beta in the frontal cortex of 14 schizophrenic patients, 15 patients with bipolar disorder, 15 patients with unipolar depression, and 14 normal comparison subjects. RESULTS: GSK-3beta levels were 41% lower in the schizophrenic patients than in the comparison subjects. Other diagnostic groups did not differ from the comparison subjects. CONCLUSIONS: These results are consistent with the notion that schizophrenia involves neurodevelopmental pathology. It remains to be investigated whether the active fraction of GSK-3beta, or its activity, is also low in frontal cortex of schizophrenic patients and if this is also reflected in other brain regions.

Distribution and intracellular localization of a mouse homologue of Ca2+/calmodulin-dependent protein kinase Ibeta2 in the nervous system.

Ca2+/calmodulin-dependent protein kinases (CaMKs) are believed to play important roles in the development and function of the nervous system. We report here the identification and expression of mouse CaMKIbeta (mCaMKIbeta), in particular mCaMKIbeta2, an isoform of mCaMKIbeta. During embryogenesis, the mCaMKIbeta2 gene is expressed mainly in the nervous system, including brain, spinal cord, trigeminal ganglion, and retina. Within the CNS, the expression of mCaMKIbeta2 is detected in the mantle zone, but not in the ventricular zone, suggesting its possible involvement in the differentiation of neurons. In the adult brain, mCaMKIbeta2 transcripts are detected at high levels in the anterior olfactory nuclei, piriform cortex, septal nuclei, bed nuclei of the stria terminalis, hippocampal pyramidal cells, dentate granule cells, amygdala, hypothalamic nuclei, parabrachial nucleus, and nucleus of the solitary tract. The distinct gene expression pattern suggests that mCaMKIbeta2 may also be involved in different mature neuronal functions from other CaMKs. In addition, mCaMKI/beta2 proteins are localized to the cytoplasm and nuclei, but not to nucleoli, suggesting that mCaMKIbeta2 proteins might be involved in the cytoplasmic and nuclear signal transduction of the nervous system.

cDNA cloning and sequencing of Ca2+/calmodulin-dependent protein kinase IIalpha subunit and its mRNA expression in diisopropyl phosphorofluoridate (DFP)-treated hen central nervous system.

Diisopropyl phosphorofluoridate (DFP) produces delayed neurotoxicity, known as organophosphorus ester-induced delayed neurotoxicity (OPIDN), in hen, human, and other sensitive species. A single dose of DFP (1.7 mg/kg, se.) produces first mild ataxia followed by paralysis in 7-14 days in hens. DFP treatment also increases in vitro autophosphorylation of Ca2+ calmodulin-dependent protein kinase II (CaM kinase II) and the phosphorylation of several cytoskeletal proteins in the hen brain. To investigate whether increase in CaM kinase II activity is associated with increased expression of its mRNA, we cloned and sequenced CaM kinase II alpha subunit cDNA, and used it to study CaM kinase II expression in brain regions and spinal cord. Hen CaM kinase II alpha subunit differs in 7 amino acids from that of rat CaM kinase II. Its mRNA occurs predominantly as a 6.7 kb message, which is very close to that of human CaM kinase II alpha subunit. Northern blot analysis showed a transient increase in CaM kinase II alpha subunit mRNA in the cerebellum and spinal cord of DFP-treated chickens. The increase in CaM kinase II mRNA expression is consistent with the previously reported increase in its activity in brain and spinal cord, and its increased expression only in cerebellum and spinal cord, which are sensitive to the Wallerian-type degeneration characteristic of OPIDN, suggests the probable role of this enzyme in delayed neurotoxicity.

Characterization of calcium/calmodulin-dependent protein kinase II activity in the nervous system of the lobster, Panulirus interruptus.

Nervous system tissue from Panulirus interruptus has an enzyme activity that behaves like calcium/calmodulin-dependent protein kinase II (CaM KII) This activity phosphorylates known targets of CaM KII, such as synapsin I and autocamtide 3. It is inhibited by a CaM KII-specific autoinhibitory domain peptide. In addition, this lobster brain activity displays calcium-independent activity after autophosphorylation, another characteristic of CaM KII. A cDNA from the lobster nervous system was amplified using polymerase chain reaction. The fragment was cloned and found to be structurally similar to CaM KII. Serum from rabbits immunized with a fusion protein containing part of this sequence immunoprecipitated a CaM KII enzyme activity and a family of phosphoproteins of the appropriate size for CaM KII subunits. Lobster CaM KII activity is found in the brain and stomatogastric nervous system including the commissural ganglia, commissures, stomatogastric ganglion and stomatogastric nerve. Immunoblot analysis of these same regions also identifies bands at an apparent molecular weight characteristic of CaM KII.

p38-2, a novel mitogen-activated protein kinase with distinct properties.

Mitogen-activated protein (MAP) kinases are involved in many cellular processes. Here we describe the cloning and characterization of a new MAP kinase, p38-2. p38-2 belongs to the p38 subfamily of MAP kinases and shares with it the TGY phosphorylation motif. The complete p38-2 cDNA was isolated by polymerase chain reaction. It encodes a 364-amino acid protein with 73% identity to p38. Two shorter isoforms missing the phosphorylation motif were identified. Analysis of various tissues demonstrated that p38-2 is differently expressed from p38. Highest expression levels were found in heart and skeletal muscle. Like p38, p38-2 is activated by stress-inducing signals and proinflammatory cytokines. The preferred upstream kinase is MEK6. Although p38-2 and p38 phosphorylate the same substrates, the site specificity of phosphorylation can differ as shown by two-dimensional phosphopeptide analysis of Sap-1a. Additionally, kinetic studies showed that p38-2 appears to be about 180 times more active than p38 on certain substrates such as ATF2. Both kinases are inhibited by a class of pyridinyl imidazoles. p38-2 phosphorylation of ATF2 and Sap-1a but not Elk1 results in increased transcriptional activity of these factors. A sequential kinetic mechanism of p38-2 is suggested by steady state kinetic analysis. In conclusion, p38-2 may be an important component of the stress response required for the homeostasis of a cell.

A MAP kinase homologue from the human malaria parasite, Plasmodium falciparum.

Pfmap-1, a gene encoding a novel protein kinase, has been identified in the human malaria parasite Plasmodium falciparum, using the polymerase chain reaction with degenerate oligodeoxyribonucleotides designed to hybridise to conserved regions of cdc2-related kinases. Computer comparison with other protein kinases strongly suggests that the protein encoded by this gene is closely related to mitogen-activated protein (MAP) kinases, which play important roles in eukaryotic adaptative response and signal transduction. In addition to the conserved MAP kinase catalytic domain, Pfmap-1 contains a highly charged C-terminal extension that includes two sets of repeated amino acid motifs. Pfmap-1 is located on chromosome 14 of P.falciparum, and its mRNA has a size of 3.7 kb.

Localization of alpha type II calcium calmodulin-dependent protein kinase at glutamatergic but not gamma-aminobutyric acid (GABAergic) synapses in thalamus and cerebral cortex.

The alpha subunit of type II calcium/calmodulin-dependent protein kinase (CAM II kinase-alpha) plays an important role in longterm synaptic plasticity. We applied preembedding immunocytochemistry (for CAM II kinase-alpha) and postembedding immunogold labeling [for glutamate or gamma-aminobutyric acid (GABA)] to explore the subcellular relationships between transmitter-defined axon terminals and the kinase at excitatory and inhibitory synapses in thalamus and cerebral cortex. Many (but not all) axon terminals ending in asymmetric synapses contained presynaptic CAM II kinase-alpha immunoreactivity; GABAergic terminals ending in symmetric synapses did not. Postsynaptically, CAM II kinase-alpha immunoreactivity was associated with postsynaptic densities of many (but not all) glutamatergic axon terminals ending on excitatory neurons. CAM II kinase-alpha immunoreactivity was absent at postsynaptic densities of all GABAergic synapses. The findings show that CAM II kinase-alpha is selectively expressed in subpopulations of excitatory neurons and, to our knowledge, demonstrate for the first time that it is only associated with glutamatergic terminals pre- and postsynaptically. CAM II kinase-alpha is unlikely to play a role in plasticity at GABAergic synapses.

Alpha calcium/calmodulin-dependent protein kinase II immunoreactivity in corticospinal neurons: combination of axonal transport method and immunofluorescence.

A combination of either retrograde or anterograde fluorescent tracer and immunofluorescence histochemistry using the monoclonal antibody specific for the alpha isoform of calcium/calmodulin-dependent protein kinase II (CaM kinase II alpha) was employed to test whether CaM kinase II alpha is expressed in somata of corticospinal neurons and their axons over their whole course. After the injection of carbocyanine dye DiI into the hindlimb area of the primary motor cortex of the rat, corticospinal axons and their terminal arbors were anterogradely labeled: DiI-labeled corticospinal fibers proceeded caudally in the ipsilateral internal capsule, cerebral peduncle and medullary pyramid, crossed at the pyramidal decussation and descended in the ventralmost area of the contralateral dorsal funiculus of the spinal cord. These DiI-labeled corticospinal axons expressed strong CaM kinase II alpha immunoreactivity along their course. However, their terminal arbors within the gray matter of the lumbar cord were very weakly immunostained. With the injection of Fast Blue into the lumbar enlargement of the rat, somata of corticospinal neurons in layer V of the motor cortex were retrogradely labeled. The subsequent immunofluorescent histochemistry revealed that more than 80% of Fast Blue-labeled corticospinal neurons were immunostained with CaM kinase II alpha antibody. The present immunohistochemical study demonstrated that CaM kinase II alpha is strongly expressed in both somata and axons of a majority of corticospinal neurons, although we could not detect this enzyme in the corticospinal terminals in the spinal target areas.

Evidence of redox-linked signaling for producing a giant signal complex.

Previously we showed that a thiol-reactive heavy metal, HgCl2, crosslinked multiple cell surface receptors through a ligand-independent pathway, which produced massive aggregates of phosphotyrosine (PTYR)-containing proteins beneath plasma membrane [Nakashima et al. (1994): J Immunol 152: 1064-1071]. In this study we characterized these unique aggregates at the molecular level. The lysates in Brij 96 of thymocytes treated with HgCl2 were separated into the supernatant and pellet fractions by simple centrifugation. Selected PTYR-containing proteins and p56lck appeared in the pellet fraction as quickly as 5 s after exposure to HgCl2, and were further increased in amount by 5 min. Although the mechanism of triggering these events was redox-linked, the majority of proteins in the Brij 96-insoluble aggregates were dissociated in SDS-PAGE under nonreducing condition. This suggested that PTYR-containing proteins and p56lck themselves do not form dimer or polymer directly by thiol-mediated bond. The pellet fraction was further found to include some other signal delivery elements, such as GTPase activating protein, phosphatidylinositol 3 kinase, and mitogen-activated protein kinase. Finally, all of these signal elements and selected PTYR-containing proteins were collected in the same fraction by the sucrose density gradient centrifugation. These results suggest a unique redox-linked pathway of formation of a giant signal complex.

Fast and slow axonal transport-different methodological approaches give complementary information: contributions of the stop-flow/crush approach.

This 'minireview' describes experiments in short term crush operated rat nerves, to study endogenous substances in anterograde and retrograde fast axonal transport. Immunofluorescence was used to recognize transported antigens, and cytofluorimetric scanning was employed to quantitate different antigens which had accumulated proximal and distal to the crushes. Vesicle membrane components p38 (synaptophysin) and SV2 accumulated on both sides of a crush. This was expected from a number of studies from different laboratories. Surface associated molecules, however, like synapsins and rab3a, have been studied by other groups with biochemical methods, and suggested to be transported with slow transport. The crush method, however, revealed that a considerable fraction of these two substances are transported with the fast transport system, and, thus, associated with fast transported organelles in the living neuron. Evidently, more than one technique is required to give a more complete picture of intraneuronal transport related events.

Terminal differentiation and senescence in the human melanocyte: repression of tyrosine-phosphorylation of the extracellular signal-regulated kinase 2 selectively defines the two phenotypes.

Melanocytes are pigmented cells distributed in humans in several organs like the epidermis, the leptomeninges, the eye, and the inner ear. Epidermal melanocytes, whether derived from adult or neonatal skin, proliferate well in a medium supplemented with phorbol esters and other mitogens before they undergo senescence. Potent cAMP inducers like cholera toxin are also growth promoters for neonatal melanocytes but only transient growth stimulators for cells derived from adults. We used this cellular system to delineate biochemical pathways involved in proliferation and in terminal differentiation. Here we show that after a period of 4-8 wk of sustained proliferation in the presence of cholera toxin, the adult melanocytes became round, flat, and enlarged. These changes were associated with terminal growth and preceded by a five- to sixfold increase in cAMP levels and an 8- to 10-fold increase in melanin content. The simultaneous addition of phorbol esters and cholera toxin did not prevent cells from reaching terminal differentiation. Identified targets for phorbol esters are protein kinase C (PKC) and the mitogen-activated kinases (MAPKs), also called extracellular signal-regulated kinases (ERKs). PKC was found to be similarly regulated in proliferating and in terminally differentiated melanocytes. Proliferating melanocytes in early or late passage showed identical activation of the kinase ERK2. This kinase was rapidly phosphorylated upon phorbol 12-myristate 13-acetate (PMA) addition and specifically accumulated in the nucleus of the cells, whereas in unstimulated cells it had a perinuclear distribution. In contrast, senescent and terminally differentiated cells were unable to phosphorylate tyrosine residues of the ERK2 gene product in spite of presenting normal amounts of ERK2 protein. In addition, ERK2 did not show the nuclear accumulation observed in proliferating melanocytes after PMA activation and remained localized in the perinuclear area. These results demonstrate that senescent and terminally differentiated melanocytes share a common block in a critical pathway thought to integrate multiple intracellular signals transmitted by various second messengers and specifically prevent the continuation of the signal transduction cascade initiated by PMA activation of PKC.

Alpha calcium/calmodulin-dependent protein kinase II selectively expressed in a subpopulation of excitatory neurons in monkey sensory-motor cortex: comparison with GAD-67 expression.

In situ hybridization histochemistry and immunocytochemistry, including double immunofluorescence, were used to study the populations of neurons expressing the alpha subunit of type II calcium/calmodulin-dependent protein kinase (CAM II kinase-alpha) or glutamic acid decarboxylase (GAD) in the somatic sensory and motor areas of the macaque monkey cerebral cortex. Sections were subjected to in situ hybridization using radioactive, complementary RNA probes specific for monkey CAM II kinase-alpha or 67 kDa GAD mRNAs. Others were stained immunocytochemically for CAM II kinase-alpha and/or GABA. CAM II kinase-alpha and GAD-67 are expressed in different populations of cells, with no colocalization. CAM II kinase-alpha is expressed in pyramidal cells of layers II-VI, especially layers II and III, as well as in certain small nonpyramidal cells of layer IV in areas 3a, 3b, 1, and 2 and of middle regions of area 4. Both cell types produce excitatory amino acid transmitters. Therefore, as in subcortical regions, CAM II kinase-alpha will be found on the presynaptic side of excitatory synapses but on the postsynaptic side only when these synapses occur on excitatory neurons in the sensory-motor cortex. Quantitative examination showed that CAM II kinase-alpha immunoreactive cells form, on average, approximately 50% of the total neuronal population in each area, while GABA immunoreactive or GAD cRNA hybridized cells form approximately 25-30%. Thus, CAM II kinase-alpha expressing cells cannot account for the total population of non-GABAergic cortical cells, and a certain proportion of the pyramidal cells probably do not express it. In other cortical areas, gene expression for the two molecules is regulated by afferent activity. Therefore, the present results form a necessary basis for studies aimed at determining the role of activity-dependent changes in the balance of excitation and inhibition as a mechanism underlying plasticity of representational maps in the primate sensory-motor cortex.