A population-based epidemiological study of neuromyelitis optica spectrum disorder in Hungary.

BACKGROUND AND PURPOSE: The goal of this study was to determine the prevalence and incidence of neuromyelitis optica spectrum disorder (NMOSD) in Hungary based on the 2015 International Panel of NMO Diagnosis (IPND) criteria. METHODS: A retrospective population-based cohort study was conducted of 6.4 million Hungarians (age ≥ 16 years) between 1 January 2006 and 31 December 2016. Possible NMOSD patients were selected via multistage re-evaluation from multiple sources. Crude and sex- and serostatus-specific prevalence (per 100 000 persons) and incidence rates (per 1 000 000 person-years) from 2006 to 2015 were estimated and age-adjusted rates were determined. RESULTS: Of 2262 study candidates, 154 NMOSD patients (age ≥ 16 years) with onset until 31 December 2016 were identified based on 2015 IPND criteria. The prevalence analysis on 1 January 2016 included 123 NMOSD living cases, resulting in a prevalence of 1.91 [95% confidence interval (CI) 1.52-2.28] per 100 000 persons. The 101 incident cases emerging from the observed 76 394 288 person-years provided an incidence rate of 1.32 (95% CI 1.08-1.61) per 1 000 000 person-years. Age-adjusted prevalence was 1.87 (95% CI 1.56-2.23) per 100 000 persons and incidence was 1.20 (95% CI 0.98-1.46) per 1 000 000 person-years. CONCLUSIONS: In this first report of a large population-based epidemiological study from an Eastern European Caucasian population using robust case validation, a greater prevalence and incidence of NMOSD was found compared to previous large studies in Caucasian populations.

[Formulation possibilities of self-emulsifying drug deliv- ery systems, microemulsions and nanoemulsions.] / Not available.

Objective The aim of this review is to introduce the self-emulsifying drug delivery systems which can be used to improved the bioavailability of poorly water soluble drug substances. Methods The review summarizes the most prominent results of the lipid based medicinal preparations, such as microemulsions and nanoemulsions developed in the last two decades. Results The analysis of the references details the lipid based formulation classification systems, the most common excipients, the quality attributes depending on the ingredients, as well as the differences in the characteristics of micro- and nanoemulsions. Conclusions The summary demonstrates the formulation possibilities of self-emulsifying drug delivery systems, which may increase the applicability and are promising to improve the therapeutic effectiveness.

Experiences with PMMA cement as a stand-alone intervertebral spacer: Percutaneous cement discoplasty in the case of vacuum phenomenon within lumbar intervertebral discs.

BACKGROUND: The treatment of symptomatic degenerative disc disease of the lumbar spine in elderly patients by standard surgical methods is often limited due to severe comorbidities (e.g., cardiopulmonary disease, hypertonia, diabetes). Minimally invasive procedures are more acceptable in this population, since they reduce surgical morbidity and the risk of complications. The percutaneous cement discoplasty (PCD) technique was introduced by the authors to treat dynamic (and angular) instability of the symptomatic lumbar segment by injecting bone cement (polymethylmethacrylate, PMMA) into the disc spaces showing vacuum phenomena via a posterolaterally positioned Jamshidi needle. The aim of this article is to describe the indication, method, and clinical results of PCD. METHOD: A total of 81 patients were treated with PCD in a tertiary care referral center over a 6-year period. The current study includes the first group of 47 consecutive patients to complete a pre- and postoperative questionnaire booklet regarding leg and back pain using the visual analog scale (VAS) and the Oswestry disability index (ODI) questionnaire. RESULTS: A total of 130 discs in these 47 patients were treated with PCD. The majority of patients reported a reduction in their lower back and leg pain (69% and 66%, respectively; p < 0.02) postoperatively. At 6-month follow-up, 61% of patients had a minimum 10-point reduction in their ODI scores (p < 0.01). CONCLUSION: Elderly patients with symptomatic dynamic foraminal stenosis and vacuum phenomenon in the intervertebral disc are suitable candidates for PCD, particularly if they represent high-risk patients for open surgery.

[Experiences with PMMA cement as a stand-alone intervertebral spacer. Percutaneous cement discoplasty in the case of vacuum phenomenon within lumbar intervertebral discs]. / Erfahrungen mit PMMA-Zement als intervertebraler Stand-alone-Platzhalter. Perkutane Zement-Diskoplastie bei Vakuumphänomen der lumbalen Bandscheiben.

BACKGROUND: The treatment of symptomatic degenerative disc disease of the lumbar spine in elderly patients by standard surgical methods is often limited due to severe comorbidities (e.g., cardiopulmonary disease, hypertonia, diabetes). Minimally invasive procedures are more acceptable in this population, since they reduce surgical morbidity and the risk of complications. The percutaneous cement discoplasty (PCD) technique was introduced by the authors to treat dynamic (and angular) instability of the symptomatic lumbar segment by injecting bone cement (polymethylmethacrylate, PMMA) into the disc spaces showing vacuum phenomena via a posterolaterally positioned Jamshidi needle. The aim of this article is to describe the indication, method, and clinical results of PCD. METHOD: A total of 81 patients were treated with PCD in a tertiary care referral center over a 6-year period. The current study includes the first group of 47 consecutive patients to complete a pre- and postoperative questionnaire booklet regarding leg and back pain using the visual analog scale (VAS) and the Oswestry disability index (ODI) questionnaire. RESULTS: A total of 130 discs in these 47 patients were treated with PCD. The majority of patients reported a reduction in their lower back and leg pain (69 and 66 %, respectively; p < 0.02) postoperatively. At 6-month follow-up, 61 % of patients had a minimum 10-point reduction in their ODI scores (p < 0.01). CONCLUSION: Elderly patients with symptomatic dynamic foraminal stenosis and vacuum phenomenon in the intervertebral disc are suitable candidates for PCD, particularly if they represent high-risk patients for open surgery.

Antibody response against gastrointestinal antigens in demyelinating diseases of the central nervous system.

BACKGROUND: Antibodies against gastrointestinal antigens may indicate altered microbiota and immune responses in the gut. Recent experimental data suggest a connection between gastrointestinal immune responses and CNS autoimmunity. METHODS: Antibodies against gliadin, tissue transglutaminase (tTG), intrinsic factor (IF), parietal cells (PC) and Saccharomyces cerevisiae (ASCA) were screened in the sera of 45 patients with AQP4-seropositive neuromyelitis optica (NMO) and NMO spectrum diseases (NMO/NMO-SD), 17 patients with AQP4-seronegative NMO, 85 patients with clinically definite multiple sclerosis (MS), and 48 healthy controls (HC). RESULTS: Thirty-seven percentages of patients with AQP4-seropositive NMO/NMO-SD and 28% of patients with MS had at least one particular antibody in contrast to 8% of HC (P < 0.01, respectively). Antibodies were most common (46%) in AQP4-seropositive myelitis (P = 0.01 versus HS, P = 0.05 versus MS). Anti-gliadin and ASCA were more frequent in the AQP4-seropositive NMO-spectrum compared to controls (P = 0.01 and P < 0.05, respectively). CONCLUSION: Antibody responses against gastrointestinal antigens are common in MS and AQP4-seropositive NMO/NMO-SD, especially in longitudinally extensive myelitis.

Differential expression of PRLIPs, a pathogenesis-related gene family encoding class 3 lipase-like proteins in Arabidopsis.

In plants plenty of inducible defense-related proteins classified into 17 pathogenesis-related (PR) families have been described. Expression of homologous PR genes from the same family can be induced by the different defense hormones, like salicylic acid (SA), jasmonic acid (JA) or ethylene (ET), and are also regulated in a organ- or tissue-specific manner. A recently identified pathogenesis-related gene family, the PRLIP (pathogenesis-related lipase) has 9 members in Arabidopsis and their organization and expression pattern - as it is summarized in this study - is similar to the one of genes coding for other PR proteins. PRLIP3, PRLIP8 and PRLIP9 showed a relatively high expression in all tissues tested with a maximum in root (PRLIP3), stem (PRLIP8) or siliques (PRLIP9). The activity of PRLIP3 gene was further induced by SA and JA treatment. Other members (PRLIP1, PRLIP2, PRLIP4 and PRLIP6), however, were detected only in some of the tested organs. High levels of PRLIP1 mRNA occurred in all green tissues and in siliques, while in the latter PRLIP2 also displayed high expression. PRLIP6 and PRLIP4 exhibited root specific transcription while no mRNAs of PRLIP5 and PRLIP7 were detected in any plant tissues examined. In leaves SA treatment enhanced PRLIP1 and PRLIP2 expression, JA treatment induced PRLIP6 and ET treatment upregulated both PRLIP1 and PRLIP6. This organization and expression diversity of the PRLIP gene family is typical to plant PR genes suggesting the encoded proteins might serve essential functions in plant defense or priming.

A recommended treatment algorithm in relapsing multiple sclerosis: report of an international consensus meeting.

An International Working Group for Treatment Optimization in MS met to recommend evidence-based therapeutic options for the management of suboptimal responses or intolerable side-effects in patients treated with disease-modifying drugs (DMDs) for multiple sclerosis (MS). Several DMDs are now available for the treatment of MS that have been shown to alter the clinical course of the disease by decreasing disease activity and delaying the progression of disability. Nevertheless, many patients continue to experience disease activity whilst on treatment, and recommendations have been made on how the success of therapy in an individual patient can be assessed. However, even after having identified criteria for a suboptimal response to current treatments, clinicians require guidance on how to improve the outcomes. This report summarizes the conclusions from a workshop at which this issue was addressed. We suggest treatment pathways for optimizing therapy for those patients with suboptimal responses to DMDs, and therapeutic options for patients with unacceptable side-effects on their current therapy.

Potentiation of pathogen-specific defense mechanisms in Arabidopsis by beta -aminobutyric acid.

The nonprotein amino acids gamma-aminobutyric acid (GABA) and beta-aminobutyric acid (BABA) have known biological effects in animals and plants. Their mode of action has been the object of thorough research in animals but remains unclear in plants. Our objective was to study the mode of action of BABA in the protection of Arabidopis plants against virulent pathogens. BABA protected Arabidopsis against the oomycete pathogen Peronospora parasitica through activation of natural defense mechanisms of the plant such as callose deposition, the hypersensitive response, and the formation of trailing necroses. BABA was still fully protective against P. parasitica in transgenic plants or mutants impaired in the salicylic acid, jasmonic acid, and ethylene signaling pathways. Treatment with BABA did not induce the accumulation of mRNA of the systemic acquired resistance (SAR)-associated PR-1 and the ethylene- and jasmonic acid-dependent PDF1.2 genes. However, BABA potentiated the accumulation of PR-1 mRNA after attack by virulent pathogenic bacteria. As a result, BABA-treated Arabidopsis plants were less diseased compared with the untreated control. In the case of bacteria, BABA protected mutants insensitive to jasmonic acid and ethylene but was not active in plants impaired in the SAR transduction pathway. Thus, BABA protects Arabidopsis against different virulent pathogens by potentiating pathogen-specific plant resistance mechanisms. In addition, we provide evidence that BABA-mediated papilla formation after P. parasitica infection is independent of the SAR signaling pathway.

Binding of FITC-labelled lectins to the gastrointestinal epithelium of the rat.

Biotechnology uses lectin genes to transfect into crop plants for protection against insects and nematodes. On the other hand, the information is limited on lectin-binding properties of cells in the gastrointestinal tract. Therefore, binding of a panel of FITC-labelled plant lectins to gastrointestinal cells of the rat was studied. In the stomach, cytoplasmic staining of parietal cells by PHA appeared to be due to glycoproteins attached to the tubulovesicles. PNA also stained the parietal cells, but only in the isthmus and neck regions, reacting with desialylated glycoproteins. WGA bound to the mucous neck cells with higher affinity than to the surface and foveolar mucous cells. The mucous cells were also stained by SNA-I, UEA-I and, less intensively, by LCA. Chief cells did not show detectable reaction with any of the applied lectins. Binding of PHA to gastric cells showed differences when compared with the results of in vivostudies. Small intestinal brush border was stained with UEA-I and SNA-I, the latter lectin also strongly stained the surface of small intestinal crypts. Both lectins reacted with the mucus of goblet cells. In the large intestine UEA-I and SNA-I stained the goblet cells at the base and upper part of the crypts, respectively. Accordingly, we provided evidences for the unique lectin-binding phenotype of the various segments of the gastrointestinal tract.

Molecular characterization at the RNA and gene levels of U3 snoRNA from a unicellular green alga, Chlamydomonas reinhardtii.

A U3 snoRNA gene isolated from a Chlamydomonas reinhardtii (CRE:) genomic library contains putative pol III-specific transcription signals similar to those of RNA polymerase III-specific small nuclear (sn)RNA genes of higher plants. The 222 nt long CRE: U3 snoRNA was immunoprecipitated by anti-gamma-mpppN antisera, but not by anti-m(2,2,7)G antibodies, supporting the notion that it is a RNA polymerase III transcript. Tagged CRE: U3 snoRNA gene constructs were expressed in CRE: cells. Results of chemical and enzymatic structure probing of CRE: U3 snoRNA in solution and of DMS modification of CRE: U3 snoRNA under in vivo conditions revealed that the two-hairpin structure of the 5'-domain that is found in solution is no longer detected under in vivo conditions. The observed differences can be explained by the formation of several base pair interactions with the 18S and 5'-ETS parts of the pre-rRNA. A model that involves five intermolecular helices is proposed.

Interstrain variation in murine susceptibility to inhaled acid-coated particles.

Epidemiologic studies have demonstrated a positive correlation between concentration of acid aerosol and increased morbidity and mortality in many urban environments. To determine whether genetic background is an important risk factor for susceptibility to the toxic effects of inhaled particles, we studied the interstrain (genetic) and intrastrain (environmental) variance of lung responses to acid-coated particle (ACP) aerosol in nine strains of inbred mice. A flow-past nose-only inhalation system was used to expose mice to ACPs produced by the cogeneration of a carbon black aerosol-sulfur dioxide (SO(2)) mixture at high humidity. Three days after a single 4-h exposure to ACPs or filtered air, mice underwent bronchoalveolar lavage, and cell differentials and total protein were determined as indexes of inflammation and epithelial permeability, respectively. To determine the effect of ACPs on alveolar macrophage (AM) function, lavaged AMs were isolated from exposed animals and Fc receptor-mediated phagocytosis was evaluated. Compared with air-exposed animals, there was a slight but significant exposure effect of ACPs on the mean number of lavageable polymorphonuclear leukocytes in C3H/HeJ and C3H/HeOuJ mice. ACP exposure also caused a significant decrease in AM phagocytosis. Relative to respective air-exposed animals, Fc receptor-mediated phagocytosis was suppressed in eight of nine strains. The order of strain-specific effect of ACPs on phagocytosis was C57BL/6J > 129/J > SJL/J > BALB/cJ > C3H/HeOuJ > A/J > SWR/J > AKR/J. There was no effect of ACP exposure on AM phagocytosis in C3H/HeJ mice. The significant interstrain variation in AM response to particle challenge indicates that genetic background has an important role in susceptibility. The effects of ACPs on AM function, inflammation, and epithelial hyperpermeability were not correlated (i.e., no cosegregation). This model may have important implications concerning interindividual variation in particle-induced compromise of host defense.

Inhaled particle-bound sulfate: effects on pulmonary inflammatory responses and alveolar macrophage function.

Acid sulfate-coated solid particles are a significant environmental hazard produced primarily by the combustion of fossil fuels. We have previously described a system for the nascent generation of carbonaceous particles surface coated with approximately 140 microg/m(3) acid sulfate [cpSO(4)(2-); 10 mg/m(3) carbon black (CB) and 10 ppm sulfur dioxide (SO(2)) at 85% relative humidity (RH)]. The effects of inhaled cpSO(4)(2-) on pulmonary host defenses are assessed in the present work. Mice were acutely exposed (4 h) to either 10 mg/m(3) CB, 10 ppm SO(2), or their combination at 10% or 85% RH in a nose-only inhalation chamber. No evidence of an inflammatory response was found following any of the exposures as assessed by total cell counts and differential cell counts from bronchoalveolar lavage fluid. However, alveolar macrophage Fc receptor-mediated phagocytosis decreased only following exposure to 140 microg cpSO(4)(2-), significant suppression occurred after 24 h, maximal suppression occurred at 3 days postexposure, and recovery to preexposure levels required 7-14 days. Intrapulmonary bactericidal activity (IBA) was also suppressed only after exposure to 140 microg cpSO(4)(2-); suppression was maximal at 1 day postexposure and recovered by day 7. To assess the effects of lower cpSO(4)(2-) concentrations, mice were repeatedly exposed to 1 mg/m(3) CB and 1 ppm SO(2) at 85% RH ( approximately 20 microg/m(3) cpSO(4)(2-) for 4 h/day) for up to 6 days. A significant decrement in IBA was observed following 5 and 6 days of exposure. These studies indicated that acute or repeated exposure to cpSO(4)(2-) could alter pulmonary host defense mechanisms.

Endoscopic laser foraminoplasty on the lumbar spine--early experience.

Since April 1995 this endoscope assisted laser technique has been used for widening the lumbar exit route foramina. Patient selection included individuals with primarily unilateral sciatica as well as low back pain and referred buttock pain following various operative interventions. Endoscopic laser foraminoplasty (ELF) utilises an endoscope, a uniportal posterolateral approach and a side firing holmium laser probe. Under direct vision and within the protection of saline solution, epidural scarring, extruded and sequestrated disc protrusions and/or osteophytes are removed by holmium laser ablation. The patient is responsive and aware under neurolept analgesia and this ensures the protection and the integrity of the nerve root. A total of 219 such interventions has been performed up to January 1, 1997. The first 48 cases with a follow-up exceeding 12 months have been included in this preliminary review. Initial experience is encouraging. The ELF seems to provide another means of addressing sequelae of primary degenerative disorders as well as the common consequences of conventional spine surgery. ELF provides an alternative to minimal intervention fenestrectomy and open surgical undercutting for predominantly unisegmental and unilateral lateral recess stenosis and in selected cases it may prevent or delay the indication of spinal fusion.

Infectious in vivo and in vitro transcripts from a full-length cDNA clone of PVY-N605, a Swiss necrotic isolate of potato virus Y.

A full-length cDNA clone of the potato virus Y (PVY) genome was obtained after cloning difficulties in Escherichia coli were overcome. These difficulties were mainly due to the expression of the CI gene from upstream prokaryotic promoter-like elements within the PVY genome. To overcome this problem, PVY cDNA was maintained in two subclones which were ligated before infection. A plasmid in which these two fragments were contained could be propagated in some E. coli strains but was unstable and yielded only low levels of plasmid DNA. Replacement of the 35S promoter by the SP6 promoter slightly increased the stability of the plasmid and its RNA transcripts were infectious when capped with m7G(5')ppp(5')G. Using two inoculation methods (mechanical or biolistic) the cDNA and its RNA transcript proved infectious on three Nicotiana species and on Solanum tuberosum.

Genetic modeling of susceptibility to nitrogen dioxide-induced lung injury in mice.

We investigated the mode of inheritance of susceptibility to nitrogen dioxide (NO2)-induced lung injury in inbred mice. Susceptible C57BL/6J (B6) and resistant C3H/HeJ (C3) mice, as well as F1, F2, and backcross (BX) populations derived from them, were exposed to 15 parts per million NO2 for 3 h. Six hours after exposure, animals were lavaged, and differential cell counts and cell viability (cytotoxicity) were measured. Statistically significant (P < 0.05) differences in numbers of lavageable macrophages, epithelial cells, and dead cells were found between inbred strains. Distributions of cellular responses in F1 progeny overlapped both progenitors, and mean responses were intermediate. In C3:BX progeny, ranges of responses to NO2 closely resembled C3 mice, and means were not significantly different between populations. Ranges of cellular responses to NO2 in B6:BX and intercross progeny overlapped both progenitors; mean responses of both populations were intermediate to progenitors. Segregation analyses tested goodness of fit of phenotyping data with various inheritance models, and the highest likelihood for each cell response to NO2 was for the hypothesis two-unlinked loci general. We conclude that there are likely two major unlinked genes that account for differential susceptibility to acute NO2 exposure. The chromosomal location of the genes is not known.

Chlamydomonas U2, U4 and U6 snRNAs. An evolutionary conserved putative third interaction between U4 and U6 snRNAs which has a counterpart in the U4atac-U6atac snRNA duplex.

The spliceosomal UsnRNAs U2, U4 and U6 from the green alga Chlamydomonas reinhardtii (Cre) were sequenced using a combination of RNA and cDNA sequencing methods and were compared to other sequenced UsnRNAs. The lengths of Cre U6 and Cre U2 RNAs are similar to those of their higher plant equivalents. Cre U4 RNA is shorter (139 nt) than its counterpart from higher plants (150-154 nt), and contains stem IV and loop D which are absent, with the exception of the Tetrahymena U4 RNA, from the U4 RNAs of other unicellular organisms studied to date. Base-pairing interactions between U6 and U4 RNAs and between U6 and U2 RNAs, identical to those described for mammalian and yeast systems, are structurally feasible in the Cre system. In addition, based on comparative analyses of the predicted U4/U6 RNA duplex from various species, an evolutionary conserved third putative U6-U4 interaction was found. Interestingly, it can also be formed with the recently discovered U6atac and U4atac RNAs. This is a strong support in favor of the possible biological significance of this third putative interaction. Based on comparative analysis, an extension of the earlier described U6-U2 interaction patterns is also proposed.

Rotational mobility of Ca2+-ATPase of sarcoplasmic reticulum in viscous media.

The rotational diffusion of Ca2(+)-ATPase [Ca2+,Mg2(+)-activated ATP phosphohydrolase E.C. 3.6.1.38] was studied in native sarcoplasmic reticulum membrane by saturation transfer ESR spectroscopy after covalent labelling of intramembranous sulfhydryl groups with nitroxyl derivative of maleimide (5-MSL) as a function of sucrose and glycerol in the suspending medium. The relative enzymatic activity of sarcoplasmic reticulum was followed by increasing the viscosity of the aqueous phase. The ATP hydrolysing activity of the enzyme decreased differently on adding sucrose and glycerol. In the case of sucrose the reciprocal of power dependence of viscosity was observed, whereas for glycerol an exponential decay law was obtained, indicating solvent-protein interaction. On increasing the viscosity of the aqueous phase by either sucrose or glycerol, no changes were observed in the intramembranous viscosity as measured using intercalated spin-labelled stearic acid (16-SASL). The effective rotational correlation time of the protein was measured, as a mobility parameter, using saturation transfer ESR spectroscopy and found to be increased linearly with the viscosity of the sucrose containing medium and for the extramembranous size a height of 6.8 nm was obtained, indicating that approx. 82% of the volume of Ca2(+)-ATPase protein is external to the sarcoplasmic reticulum. The addition of glycerol probably promoted protein-protein interaction, as indicated by the larger changes in rotational diffusion and non-linear viscosity dependence.

Inhalation of acid coated carbon black particles impairs alveolar macrophage phagocytosis.

A flow-past nose-only inhalation system was used for the co-exposure of mice to carbon black aerosols (CBA) and sulfur dioxide (SO2) at varying relative humidities (RH). The conversion of SO2 to sulfate (SO4(-2)) on the CBA, at a fixed aerosol concentration, was dependent on RH and SO2 concentration. The effect of the aerosol-gas mixture on alveolar macrophage (AM) phagocytosis was assessed three days following exposure for 4 h. Exposure to 10 mg/m3 CBA alone at low RH (10%) and high RH (85%), to 10 ppm SO2 alone at both RH, and to the mixture at low RH had no effect on AM phagocytosis. In contrast, AM phagocytosis was significantly suppressed following co-exposure at 85% RH, the only circumstance in which significant chemisorption of the gas by the aerosol and oxidation to SO4(-2) occurred. The results suggest that fine carbon particles can be an effective vector for the delivery of toxic amounts of SO4(-2) to the periphery of the lung.

Influence of various domains of protein kinase C epsilon on its PMA-induced translocation from the Golgi to the plasma membrane.

Subcellular redistribution (translocation) was initiated by treatment of NIH 3T3 cells overexpressing different epitope-tagged fragments of PKC epsilon with PMA, and was analyzed by immunocytochemistry. The PMA-induced translocation of holo PKC epsilon, as well as fragments epsilon 2 (zinc finger domain + pseudosubstrate domain) and epsilon 7 (zinc finger domain + hinge region) from the Golgi to the plasma membrane was rapid (<10 min), while translocation of fragment epsilon 3 (zinc finger domain) was much slower (30-60 min). These results, combined with results of studies carried out at 20 degrees C to inhibit exocytotic vesicle traffic, indicated that PMA-induced translocation from the Golgi to the plasma membrane may proceed by two distinct mechanisms: a rapid, vesicle independent process noted with holo PKC epsilon (which requires the presence of the pseudosubstrate and/or hinge regions), and a slow, vesicle-dependent pathway observed with the zinc finger fragment.

Protein kinase C epsilon subcellular localization domains and proteolytic degradation sites. A model for protein kinase C conformational changes.

Protein kinase C (PCK) epsilon has been found to have unique properties among the PCK isozymes in terms of its membrane association, oncogenic potential, and substrate specificity. Recently we have demonstrated that PKC epsilon localizes to the Golgi network via its zinc finger domain and that both the holoenzyme and its zinc finger region modulate Golgi function. To further characterize the relationship between the domain organization and the subcellular localization of PKC epsilon, a series of NIH 3T3 cell lines were created, each overexpressing a different truncated version of PKC epsilon. The overexpressed proteins each were designed to contain an epsilon-epitope tag peptide at the COOH terminus to allow ready detection with an antibody specific for the tag. The subcellular localization of the recombinant proteins was analyzed by in vivo phorbol ester binding, immunocytochemistry, and cell fractionation followed by immunoblotting. Results revealed several regions of PKC epsilon that contain putative subcellular localization signals. The presence either of the hinge region or of a 33-amino-acid region including the pseudosubstrate sequence in the recombinant proteins resulted in association with the plasma membrane and cytoskeletal components. The catalytic domain was found predominantly in the cytosolic fraction. The accessibility and thus the dominance of these localization signals is likely to be affected by the overall conformation of the recombinant proteins. Regions with putative proteolytic degradation sites also were identified. The susceptibility of the overexpressed proteins to proteolytic degradation was dependent on the protein conformation. Based on these observations, a model depicting the interaction and hierarchy of the suspected localization signals and proteolytic degradation sites is presented.