Millifluidic magnetophoresis-based chip for age-specific fractionation: evaluating the impact of age on metabolomics and gene expression in yeast.

A novel millifluidic process introduces age-based fractionation of S. pastorianus var. carlsbergensis yeast culture through magnetophoresis. Saccharomyces yeast is a model organism for aging research used in various industries. Traditional age-based cell separation methods were labor-intensive, but techniques like magnetic labeling have eased the process by being non-invasive and scalable. Our approach introduces an age-specific fractionation using a 3D-printed millfluidic chip in a two-step process, ensuring efficient cell deflection in the magnetic field and counteracting magnetic induced convection. Among various channel designs, the pinch-shaped channel proved most effective for age differentiation based on magnetically labeled bud scar numbers. Metabolomic analyses revealed changes in certain amino acids and increased NAD+ levels, suggesting metabolic shifts in aging cells. Gene expression studies further underlined these age-related metabolic changes. This innovative platform offers a high-throughput, non-invasive method for age-specific yeast cell fractionation, with potential applications in industries ranging from food and beverages to pharmaceuticals.

Induced expression of the alcohol acetyltransferase gene ATF1 in industrial yeast Saccharomyces pastorianus TUM 34/70.

Targeted induced gene expression for industrial fermentation processes in food and beverage production could fulfill future demands. To avoid metabolic burden and disturbances owing to the fermentation procedure, induced gene expression is necessary for combating stress, such as that caused by temperature shifts that occur during the transition from fermentation to maturation in the brewing process. The aim of this study was to target gene expression in industrial yeast using stress-responsive promoters and homologues of the selection marker SMR1. Self-cloning strains of the industrial brewing yeast Saccharomyces pastorianus TUM 34/70 were constructed to overexpress the alcohol acetyltransferase (ATF1) gene under the control of inducible promoters P SSA3, P HSP104 and P UBI4. Transcription analysis shows the highest induction after 72 h of shock situation for P HSP104 with 1.3-fold and P UBI4 with 2.2-fold. Further, at the end of shock situation the concentrations of ethyl acetate were 1.2- and 1.3-fold higher than the wild type for P HSP104 and P UBI4, respectively. In addition, the influence of the final temperature and temporal sequence of temperature shock to 4°C had a major impact on expression patterns. Therefore, these data show that temperature-induced gene expression of self-cloning industrial yeast could be an option for optimization of the beverage fermentation.

A novel CGRP-neutralizing Spiegelmer attenuates neurogenic plasma protein extravasation.

BACKGROUND AND PURPOSE: Calcitonin gene-related peptide (CGRP) plays an important role in the pathology of migraine, and recent clinical trials suggest the inhibition of CGRP-mediated processes as a new therapeutic option in migraine. In this study, we describe the generation of NOX-L41, a CGRP-neutralizing mirror-image (L-)aptamer (Spiegelmer) and investigate its in vitro and in vivo function. EXPERIMENTAL APPROACH: A CGRP-binding Spiegelmer was identified by in vitro selection. Binding studies were performed using surface plasmon resonance (SPR), and the inhibitory activity was determined in cell-based assays. The pharmacokinetic profile comparing i.v. and s.c. dosing was analysed in rats. Intravital two-photon microscopy was employed to follow extravasation from meningeal vessels. Finally, in vivo efficacy was tested in a model of electrically evoked meningeal plasma protein extravasation (PPE) in rats. KEY RESULTS: We identified NOX-L41, a novel CGRP-neutralizing Spiegelmer. SPR studies showed that NOX-L41 binds to human and rat/mouse CGRP with sub-nanomolar affinities and is highly selective against related peptides such as amylin. In vitro, NOX-L41 effectively inhibited CGRP-induced cAMP formation in SK-N-MC cells. In rats, NOX-L41 had a plasma half-life of 8 h. Pharmacodynamic studies showed that NOX-L41 extravasates from blood vessels in the dura mater and inhibits neurogenic meningeal PPE for at least 18 h after single dosing. CONCLUSIONS AND IMPLICATIONS: This is the first description of the CGRP-neutralizing Spiegelmer NOX-L41. Preclinical studies confirmed a role for CGRP in neurogenic PPE and provided proof-of-concept for the potential use of this new drug candidate for the treatment or prevention of migraine.

Hematopoietic stem and progenitor cell mobilization in mice and humans by a first-in-class mirror-image oligonucleotide inhibitor of CXCL12.

NOX-A12 is a PEGylated mirror-image oligonucleotide (a so-called Spiegelmer) that binds to CXCL12 (stromal cell-derived factor-1, SDF-1) with high affinity thereby inhibiting CXCL12 signaling on both its receptors, CXCR4 and CXCR7. In animals, NOX-A12 mobilized white blood cells (WBCs) and hematopoietic stem and progenitor cells (HSCs) into peripheral blood (PB). In healthy volunteers, single doses of NOX-A12 had a benign safety profile and also dose-dependently mobilized WBCs and HSCs into PB. HSC peak mobilization reached a plateau at five times the baseline level at an i.v. dose of 5.4 mg/kg. In accordance with the plasma half-life of 38 h, the duration of the WBC and HSC mobilization was long lasting and increased dose-dependently to more than 4 days at the highest dose (10.8 mg/kg). In conclusion, NOX-A12 may be appropriate for therapeutic use in and beyond mobilization of HSCs, e.g., in long-lasting mobilization and chemosensitization of hematological cancer cells.

Human Toll-like receptor 2 mediates induction of the antimicrobial peptide human beta-defensin 2 in response to bacterial lipoprotein.

Recognition of pathogens by Drosophila Toll or human Toll-like receptors results in translocation of Dorsal or its human homologue NF-kappaB, respectively; in Drosophila, this is followed by the production of antimicrobial peptides serving as antimicrobial effector system of the innate immune response. We investigated whether human Toll-like receptors also mediate induction of the synthesis of antimicrobial peptides. We found that HEK293 cells transfected with Toll-like receptor 2, but not wild-type cells responded to stimulation with bacterial lipoprotein by production of human beta-defensin 2. Furthermore, the human lung epithelial cell line A549 was found to constitutively express Toll-like receptor 2 and to produce beta-defensin 2 in response to bacterial lipoprotein. This response was abrogated by blocking the signaling pathway activated through Toll-like receptors by transfecting the A549 cells with a dominant-negative form of IRAK-2. Thus, exposure of human cells to bacterial lipoprotein elicits production of the antimicrobial peptide beta-defensin 2 through Toll-like receptor 2.

The inflammatory action of CD40 ligand (CD154) expressed on activated human platelets is temporally limited by coexpressed CD40.

Recently, we have demonstrated that human platelets carry preformed CD40 ligand (CD154) molecules, which rapidly appear on the platelet surface following stimulation by thrombin. Once on the surface, platelet CD154 induces an inflammatory reaction of CD40-bearing endothelial cells. This study shows that strong platelet agonists other than thrombin also lead to the expression of CD154 on the platelet surface. At the same time, several lines of evidence are presented that together indicate that thrombotic events in the vasculature are generally accompanied by activation of the inflammatory potential of platelet CD154. This study also reports the constitutive expression of CD40, the receptor for CD154, on platelets. The binding of CD154 to coexpressed CD40 in the platelet aggregate leads within minutes to hours to the cleavage of membrane-bound surface CD154 and the release of an 18-kd soluble form of the molecule. Soluble CD154 (sCD154), in contrast to transmembrane CD154, can no longer induce an inflammatory reaction of endothelial cells. These findings indicate that the interaction of platelet CD154 with CD40 on neighboring cells is temporally limited to prevent an uncontrolled inflammation at the site of thrombus formation. Thus, similar to the very tight regulation of the CD154-CD40 interaction in the immune system, an effective mechanism controls the inflammatory potential of platelet CD154 in the vascular system. (Blood. 2001;98:1047-1054)

Near-infrared spectroscopy--not useful to monitor cerebral oxygenation after severe brain injury.

Since its development more than twenty years ago, non-invasive near-infrared-spectroscopy (NIRS) has been widely used to monitor cerebral oxygenation. Despite of its growing number of users, the diagnostic value of near-infrared spectroscopy still remains unclear, especially in case of acute brain injury and long-term neuromonitoring, necessary during intensive care therapy. To evaluate quality and sensitivity of NIRS measurements compared to invasive ICP-, CPP- and regional brain tissue--pO2 (p(ti)O2) monitoring, 31 patients, suffering from severe brain injury due to subarachnoid hemorrhage or severe head injury, were studied. NIRS measurements were only possible in 80% (using the INVOS oximeter) and in 46% (using the CRITIKON monitor), while good data quality was obtained in 100% from ICP, CPP and p(ti)O2. Major reasons for the failure of NIRS measurements were: (1) a wet chamber between sensor and skin, (2) galea hematoma or (3) subdural air after craniotomy. Different tests were performed to compare the sensitivity of regular oxygen saturation (NIRS) with the sensitivity of invasively determined p(ti)O2. Only induced hyperoxia (FiO2 = 1.0) revealed a significant correlation between both parameters (r = 0.67, p < 0.01). Lower or no correlation was found after changing paCO2 and administration of mannitol. The high failure rate and the limited sensitivity does not make the clinical use of near-infrared spectroscopy suitable as a part of neuromonitoring after acute brain injury at the present time.

Protein kinase calpha targeting is regulated by temporal and spatial changes in intracellular free calcium concentration [Ca(2+)](i).

Protein kinase C (PKC) isoforms exert specific intracellular functions, but the different isoforms display little substrate specificity in vitro. Selective PKC isoform targeting may be a mechanism to achieve specificity. We used a green fluorescent fusion protein (GFP) to test the hypothesis that local changes in [Ca(2+)](i) regulate translocation of PKCalpha and that different modes of Ca(2+) and Ca(2+) release play a role in PKCalpha targeting. We constructed deletion mutants of PKCalpha to analyze the Ca(2+)-sensitive domains and their role in targeting. Confocal microscopy was used and [Ca(2+)](i) was measured by fluo-3. The fusion protein PKCalpha-GFP was expressed in vascular smooth muscle cells and showed a cytosolic distribution similar to the wild-type PKCalpha protein. The Ca(2+) ionophore ionomycin induced a speckled cytosolic PKCalpha-GFP distribution, followed by membrane translocation, while depolarization by KCl induced primarily membrane translocation. Selective voltage-operated Ca(2+) channel opening led to a localized accumulation of PKCalpha-GFP near the plasma membrane. Opening Ca(2+) stores with InsP(3), thapsigargin, or ryanodine induced a specific PKCalpha-GFP targeting to distinct intracellular areas. The G-protein-coupled receptor agonist thrombin induced a rapid translocation of the fusion protein to focal domains. The tyrosine kinase receptor agonist PDGF induced Ca(2+) influx and led to a linear PKCalpha-GFP membrane association. PKCalpha-GFP deletion mutants demonstrated that the C2 domain, but not the catalytic subunit, is necessary for Ca(2+)-induced PKCalpha targeting. Targeting was also abolished when the ATP binding site was deleted. We conclude that PKCalpha can rapidly be translocated to distinct intracellular or membrane domains by local increases in [Ca(2+)](i). The targeting mechanism is dependent on the C2 and ATP binding site of the enzyme. Localized [Ca(2+)](i) changes determine the spatial and temporal targeting of PKCalpha.

Analysis of the subcellular distribution of protein kinase Calpha using PKC-GFP fusion proteins.

One important factor for the determination of the specific functions of protein kinase C (PKC) isoforms is their specific subcellular localization. In NIH 3T3 fibroblasts phorbol esters induce translocation of PKCalpha to the plasma membrane and the nucleus. In order to investigate PKCalpha's subcellular distribution and especially its nuclear accumulation in more detail we used fusion proteins consisting of PKCalpha and the green fluorescent protein (GFP). Purified GFP-PKCalpha from baculovirus-infected insect cells undergoes nuclear accumulation without any further stimuli in digitonin-permeabilized cells. Interestingly, permeabilization appears to be a trigger for PKCalpha's nuclear translocation, since the fusion protein also translocates to the nucleus in transiently transfected cells following permeabilization. This suggests that PKCalpha has a high nuclear binding capacity even in the case of large protein amounts. In contrast to endogenous PKCalpha, overexpressed GFP-PKCalpha as well as overexpressed PKCalpha itself translocates mainly to the plasma membrane and only to a smaller extent to the nucleus following stimulation with phorbol ester. Use of fusion proteins of GFP and different mutants of PKCalpha enabled determination of motifs involved PKCalpha's subcellular distribution: A25E and K368R point mutations of PKCalpha showed enhanced affinity for the plasma membrane, whereas sequences within the regulatory domain probably confer PKCalpha's nuclear accumulation.

Genetic and molecular complexity of the position effect variegation modifier mod(mdg4) in Drosophila.

mod(mdg4), also known as E(var)3-93D, is involved in a variety of processes, such as gene silencing in position effect variegation (PEV), the control of gypsy insulator sequences, regulation of homeotic gene expression, and programmed cell death. We have isolated a large number of mod(mdg4) cDNAs, representing 21 different isoforms generated by alternative splicing. The deduced proteins are characterized by a common N terminus of 402 amino acids, including the BTB/POZ-domain. Most of the variable C termini contain a new consensus sequence, including four positioned hydrophobic amino acids and a Cys(2)His(2) motif. Using specific antibodies for two protein isoforms, we demonstrate different distributions of the corresponding proteins on polytene chromosomes. Mutations in the genomic region encoding exons 1-4 show enhancement of PEV and homeotic transformation and affect viability and fertility. Homeotic and PEV phenotypes are enhanced by mutations in other trx-group genes. A transgene containing the common 5' region of mod(mdg4) that is present in all splice variants known so far partially rescues the recessive lethality of mod(mdg4) mutant alleles. Our data provide evidence that the molecular and genetic complexity of mod(mdg4) is caused by a large set of individual protein isoforms with specific functions in regulating the chromatin structure of different sets of genes throughout development.

Molecular cloning and characterization of murine ICOS and identification of B7h as ICOS ligand.

Human ICOS (huICOS) is a T cell-specific molecule structurally related to CD28 and CTLA-4 with potent co-stimulatory activities on T cell proliferation, cytokine induction and T cell help for B cells. We have now cloned and characterized murine ICOS (muICOS). muICOS mRNA of 1.5 kb and 3.3 kb encodes a protein with a deduced molecular mass of 20.3 kDa, which is 71.7 % identical to huICOS. On the cell surface, muICOS is expressed as a disulfide-linked, glycosylated homodimer of 47-57 kDa, with subunits of approximately 26 kDa. With a panel of monoclonal antibodies we have determined the expression of muICOS in vitro and in vivo. Following activation of splenic T cells via CD3, muICOS became detectable at 12 h and reached a maximum of expression at around 48 h, thus exhibiting expression kinetics similar to huICOS. In vivo, muICOS was found to be substantially expressed in the thymic medulla and in the germinal centers and T cell zones of lymph nodes and Peyer's patches. Non-lymphoid tissue was ICOS negative. The muICOS gene was mapped to a region of chromosome 1 also harboring the CD28 and CTLA-4 genes. Using recombinant chimeric muICOS-Ig we determined that B7h, a recently cloned B7-like molecule, is a ligand for muICOS.

The role of protein kinase C in the regulation of cell growth and in signalling to the cell nucleus.

The protein kinase C (PKC) family of serine/threonine kinases consists of at least 11 mammalian isoforms, which show slight differences in their molecular structure and enzymatic properties. PKC isoforms are involved in a wide variety of intracellular signalling events and play an important role in tumour promotion and cell growth control in general. Studies of expression levels in cancer cells and studies using overexpression of single isoforms or expression of dominant negative isoforms reveal that, depending on the cellular background, PKC isoforms can either promote or inhibit cell growth. To understand the role of PKC isoforms in growth control, it is essential to understand how PKC functions in the intracellular signalling cascades towards the cell nucleus. Recent work has shown that PKC isoforms can act either in the cytoplasm, and cause nuclear effects indirectly by triggering signalling pathways directed towards the cell nucleus, or, after translocation and activation, can themselves act in the cell nucleus.

Induction, binding specificity and function of human ICOS.

Recently, we have identified the inducible co-stimulator (ICOS), an activation-dependent, T cell-specific cell surface molecule related to CD28 and CTLA-4. Detailed analysis of human ICOS presented here shows that it is a 55-60-kDa homodimer with differently N-glycosylated subunits of 27 and 29 kDa. ICOS requires both phorbol 12-myristate 13-acetate and ionomycin for full induction, and is sensitive to Cyclosporin A. ICOS is up-regulated early on all T cells, including the CD28- subset, and continues to be expressed into later phases of T cell activation. On stimulation of T cells by antigen-presenting cells, the CD28/B7, but not the CD40 ligand/CD40 pathway is critically involved in the induction of ICOS. ICOS does not bind to B7-1 or B7-2, and CD28 does not bind to ICOS ligand; thus the CD28 and ICOS pathways do not cross-interact on the cell surface. In vivo, ICOS is expressed in the medulla of the fetal and newborn thymus, in the T cell zones of tonsils and lymph nodes, and in the apical light zones of germinal centers (predominant expression). Functionally, ICOS co-induces a variety of cytokines including IL-4, IL-5, IL-6, IFN-gamma, TNF-alpha, GM-CSF, but not IL-2, and superinduces IL-10. Furthermore, ICOS co-stimulation prevents the apoptosis of pre-activated T cells. The human ICOS gene maps to chromosome 2q33 - 34.

Structure, distribution, and biological activity of novel members of the allatostatin family in the crayfish Orconectes limosus.

In the central and peripheral nervous system of the crayfish, Orconectes limosus, neuropeptides immunoreactive to an antiserum against allatostatin I (= Dipstatin 7) of the cockroach Diploptera punctata have been detected by immunocytochemistry and a sensitive enzyme immunoassay. Abundant immunoreactivity occurs throughout the central nervous system in distinct interneurons and neurosecretory cells. The latter have terminals in well-known neurohemal organs, such as the sinus gland, the pericardial organs, and the perineural sheath of the ventral nerve cord. Nervous tissue extracts were separated by reverse-phase high-performance liquid chromatography and fractions were monitored in the enzyme immunoassay. Three of several immunopositive fractions have been purified and identified by mass spectroscopy and microsequencing as AGPYAFGL-NH2, SAGPYAFGL-NH2, and PRVYGFGL-NH2. The first peptide is identical to carcinustatin 8 previously identified in the crab Carcinus maenas. The others are novel and are designated orcostatin I and orcostatin II, respectively. All three peptides exert dramatic inhibitory effects on contractions of the crayfish hindgut. Carcinustatin 8 also inhibits induced contractions of the cockroach hindgut. Furthermore, this peptide reduces the cycle frequency of the pyloric rhythms generated by the stomatogastric nervous system of two decapod species in vitro. These crayfish allatostatin-like peptides are the first native crustacean peptides with demonstrated inhibitory actions on hindgut muscles and the pyloric rhythm of the stomatogastric ganglion.

Age-dependent differences in glutamate-induced phosphorylation systems in rat hippocampal slices.

Glutamate receptor induced changes in the activity of different phosphorylation systems were measured in hippocampal slices from 12- and 56-day-old rats, by determining the endogenous phosphorylation of 2.5% perchloric acid (PCA) soluble proteins. We identified among these proteins an 85, 80 kDa and the tau protein as specific substrates for protein kinase A (PKA), MARCKS, and neurogranin as specific substrates for protein kinase C (PKC), and prostaglandin-D-synthase as substrate for casein kinase II (CKII). In addition, a 35 kDa protein was phosphorylated by calcium/calmodulin dependent kinase II and protein kinase C and a 21 kDa protein was a substrate for all investigated kinases. The basal endogenous phosphorylation of 2.5% PCA soluble proteins changed during development qualitatively and quantitatively. Thus, the phosphorylation degree of nearly all proteins declines during maturation. Activation of mGluR induced an increased phosphorylation of PKA, PKC, and CKII substrates in hippocampal slices from 12-day-old rats, but in slices of 56-day-old rats only PKA and to a lower extent PKC substrates were affected. In contrast, stimulation of NMDA receptors led to an enhancement of CKII and PKA dependent phosphorylation only in slices of young animals, whereas the endogenous phosphorylation of some proteins in adult slices was actually decreased. These data showing developmental changes in the coupling of metabotropic and ionotropic glutamate receptors to different phosphorylation systems are discussed in the light of altered physiological properties of the mature hippocampus.

Isoform-specific translocation of protein kinase C following glutamate administration in primary hippocampal neurons.

High concentrations of glutamate, the major excitatory neurotransmitter in the mammalian brain, lead to intracellular calcium overload resulting in excitotoxic damage and death of neurons. Since protein kinase C (PKC) is involved in neuronal degeneration resulting from cerebral ischemia and from glutamate excitotoxicity, we investigated the effect of glutamate on changes in the cellular distribution of various PKC isoforms in cultured hippocampal neurons in comparison with the effects elicited by the PKC activator phorbol ester. Out of the expressed PKC isoforms alpha, gamma, epsilon, zeta and lambda only the conventional isoforms PKC alpha and gamma responded to glutamate. Using subcellular fractionation and Western blotting with isoform-specific antibodies and immunocytochemical localization with confocal laser scanning microscopy, we observed that phorbol ester and glutamate have different effects on PKC isoform redistribution: Whereas phorbol ester resulted in translocation of PKC alpha and PKC gamma toward a membrane fraction, the glutamate-mediated rise in intracellular calcium concentration induced a translocation mainly toward a detergent-insoluble, cytoskeletal fraction. Immunocytochemical analysis revealed an isoform-specific translocation following glutamate treatment: PKC gamma was translocated mainly to cytoplasmic, organelle-like structures, whereas PKC alpha redistributed to the plasma membrane and into the cell nucleus. The latter result is of special interest, as it indicates that nuclear PKC may play a role in processes of excitotoxic cell damage.

Nuclear import of protein kinase C occurs by a mechanism distinct from the mechanism used by proteins with a classical nuclear localization signal.

Protein kinase C does not have any known nuclear localization signal but, nevertheless, is redistributed from the cytoplasm to the nucleus upon various stimuli. In NIH 3T3 fibroblasts stimulation with phorbol ester leads to a translocation of protein kinase C alpha to the plasma membrane and into the cell nucleus. We compared the mechanism of protein kinase C alpha's transport into the nucleus with the transport mechanism of a protein with a classical nuclear localization signal at several steps. To this end, we co-microinjected fluorescently labeled bovine serum albumin to which a nuclear localization signal peptide was coupled, together with substances interfering with conventional nuclear protein import. Thereafter, the distribution of both the nuclear localization signal-bearing reporter protein and protein kinase C alpha was analyzed in the same cells. We can show that, in contrast to the nuclear localization signal-dependent transport, the phorbol ester-induced transport of protein kinase C alpha is not affected by microinjection of antibodies against the nuclear import factor p97/importin/karyopherin beta or microinjection of non-hydrolyzable GTP-analogs. This suggests that nuclear import of protein kinase C alpha is independent of p97/importin/karyopherin beta and independent of GTP. At the nuclear pore there are differences between the mechanisms too, since nuclear transport of protein kinase C alpha cannot be inhibited by wheat germ agglutinin or an antibody against nuclear pore complex proteins. Together these findings demonstrate that the nuclear import of protein kinase C alpha occurs by a mechanism distinct from the one used by classical nuclear localization signal-bearing proteins at several stages.

Intracellular targeting and protein kinase C in vascular smooth muscle cells: specific effects of different membrane-bound receptors.

Protein kinase C is an important second messenger system, which is translocated from the cytosol to the cell membrane upon cell stimulation. We used confocal microscopy to study the spatial distribution of protein kinase C isoforms after stimulation of cultured vascular smooth muscle cells with different agonists. First, we analysed the effects of angiotensin II and platelet-derived growth factor (PDGF). Confocal microscopy showed a rapid assembly of PKC alpha along cytosolic fibres followed by a translocation towards the nucleus with angiotensin II. PDGF engendered a similar, but much slower response; however, a cytoskeletal distribution was not observed. We then investigated the effects of thrombin and bFGF on nuclear translocation. bFGF induced a rapid translocation of the isoform towards the perinuclear region and into the nucleus. bFGF had a similar effect on PKC epsilon. In contrast, thrombin had a smaller effect on nuclear translocation of PKC alpha and did not influence PKC epsilon, but instead induced a rapid nuclear translocation of PKC zeta. Thus, tyrosine kinase receptor activation via bFGF induces a rapid association of PKC alpha and epsilon within nuclear structures. Our results show that agonists cause, not only a translocation of protein kinase C isoforms into the cell membrane but also into the cell nucleus. Lastly, we analyzed the nuclear immunoreactivity of the PKC isoforms, alpha, delta, epsilon and zeta in vascular smooth muscle cells during the cell cycle. Resting cells were stimulated with foetal calf serum (FCS, 10%), which translocated PKC alpha and epsilon to the perinuclear region and into the nucleus, while PKC delta and zeta showed no increase in nuclear immunoreactivity. After 4 h of FCS, the nuclear immunoreactivity for PKC alpha and epsilon was reduced to or below control values. At 8 h, increased nuclear expression of isoforms alpha, epsilon and zeta was observed, while isoform delta was not affected. Our results demonstrate a complex spatial and temporal regulation of PKC isoforms in response to vasoactive hormones and growth factors. We suggest that protein kinase C may be important for nuclear signaling and demonstrate that nuclear translocation of PKC isoforms is differentially regulated during the cell cycle.

Signal transduction and protein kinases: the long way from the plasma membrane into the nucleus.

All living cells must be able to receive information from the extracellular space and to react to it by processing and converting it into intracellular effects. If the properties of cells are to change in the long term, some signals must reach the nucleus in order to bring about changes in gene transcription. Three of the pathways, beginning with an extracellular signal and ending with the nucleus, serve to illustrate some principles of signal transduction such as signal conversion, signal cascade, cross-talk, and on/off switch. One element common to most of the pathways is the activation of protein kinases. One example of these kinases, the protein kinase C, is discussed as a vehicle of signal transport toward the nucleus and as a means of cross-talk between different signaling pathways.

Large-conductance cation channels in the envelope of nuclei from rat cerebral cortex.

Eucaryotic nuclei are surrounded by a double-membrane system enclosing a central cisterna which is continuous with the endoplasmic reticulum and serves as a calcium store for intracellular signaling. The envelope regulates protein and nucleic acid traffic between the nucleus and the cytoplasm via nuclear pores. These protein tunnels cross through both nuclear membranes and are permeable for large molecules. Surprisingly, patch clamp recordings from isolated nuclei of different cell species have revealed a high resistance of the envelope, enabling tight seals and the resolution of single ion channel activity. Here we present for the first time single-channel recordings from nuclei prepared from neuronal tissue. Nuclei isolated from rat cerebral cortex displayed spontaneous long-lasting large conductances in the nucleus-attached mode as well as in excised patches. The open times are in the range of seconds and channel activity increases with depolarization. The single-channel conductance in symmetrical K+ is 166 pS. The channels are selective for cations with PK/PNa = 2. They are neither permeable to, nor gated by Ca2+. Thus, neuronal tissue nuclei contain a large conductance ion channel selective for monovalent cations which may contribute to ionic homeostasis in the complex compartments surrounding these organelles.