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Background: The increasing use of the Internet and social network sites (SNS) has created a new domain of socio-emotional development for adolescents. The aim of this cross-sectional study was to explore cybervictimization across seven European countries, in relation to socio-demographic, Internet use and psychosocial variables., Methods: A cross-sectional school-based study was conducted in the participating countries: Germany, Greece, Iceland the Netherlands, Poland, Romania and Spain. Anonymous self-completed questionnaires included sociodemographic data, internet usage characteristics, school achievement, parental control, the Internet Addiction Test and Achenbach's Youth Self-Report., Results: The highest rate of cyber victimization was found in Romania (37.3%) and the lowest in Spain (13.3%). Multiple logistic regression analyses gave differing results between countries. In Romania, Poland and Germany cyberbullying victimization was associated with SNS use, whereas Internet use was associated with increased odds of cybervictimization only in Romania. Cybervictimization was associated with greater internalizing behavior problems in all countries analysed, and with externalizing problems in all except Romania., Conclusions: Cyberbullying victimization is an on-going problem, which is subject to country-specific socio-demographic factors and diverse patterns of current Internet use and its development. Preventive measures should emphasize the integration of Internet communication technology education in educational contexts, and focus on the consistent association between cybervictimization and internalizing and externalizing difficulties.

The nucleolar proteins which link cell proliferation to ribosome biogenesis are regarded to be potentially oncogenic. Here, in order to examine the involvement of an evolutionary conserved nucleolar protein SURF6/Rrp14 in proliferation and ribosome biogenesis in mammalian cells, we established stably transfected mouse NIH/3T3 fibroblasts capable of conditional overexpression of the protein. Cell proliferation was monitored in real-time, and various cell cycle parameters were quantified based on flow cytometry, Br-dU-labeling and conventional microscopy data. We show that overexpression of SURF6 accelerates cell proliferation and promotes transition through all cell cycle phases. The most prominent SURF6 pro-proliferative effects include a significant reduction of the population doubling time, from 19.8 ± 0.7 to 16.2 ± 0.5 hours (t-test, p < 0.001), and of the length of cell division cycle, from 17.6 ± 0.6 to 14.0 ± 0.4 hours (t-test, p < 0.001). The later was due to the shortening of all cell cycle phases but the length of G1 period was reduced most, from 5.7 ± 0.4 to 3.8 ± 0.3 hours, or by ∼30%, (t-test, p < 0.05). By Northern blots and qRT-PCR, we further showed that the acceleration of cell proliferation was concomitant with an accumulation of rRNA species along both ribosomal subunit maturation pathways. It is evident, therefore, that like the yeast homologue Rrp14, mammalian SURF6 is involved in various steps of rRNA processing during ribosome biogenesis. We concluded that SURF6 is a novel positive regulator of proliferation and G1/S transition in mammals, implicating that SURF6 is a potential oncogenic protein, which can be further studied as a putative target in anti-cancer therapy.

Adult neurons of the peripheral nervous system (PNS), in contrast to those of the central nervous system, have a remarkable capacity to repair themselves after injury, yet the mechanisms underlying this regenerative propensity of peripheral neurons are far from completely understood. Here we show that the transcription factor CCAAT enhancer binding protein delta (C/EBPδ) is necessary for the efficient axonal regeneration of dorsal root ganglia (DRG) neurons after sciatic nerve crush injury. Loss of C/EBPδ substantially impairs axonal growth in dissociated cultured DRG neurons. In addition, lack of C/EPBδ causes a major reduction in the regenerative response of DRG neurons to a conditioning lesion, which is a well known paradigm of injury that enhances axonal growth due to a transcription-dependent cell body response. C/EBPδ is required for the induction of selected regeneration-associated genes. For example, the expression of SPRR1A (small proline-rich repeat protein 1A) is greatly reduced in DRG neurons of C/EBPδ knockout mice during axonal regeneration compared to those in wild-type mice, while the expression of GAP-43 (growth associated protein-43) and galanin is not affected. Nevertheless, the expected prompt recovery of sciatic nerve function after injury is severely impaired in C/EBPδ knockout mice, having a delay time of approximately 1 month for reaching the full function of recovering wild-type mice, suggesting that a transcription mechanism mediated by C/EBPδ is required for efficient axonal regeneration. Taken together, our results identify C/EBPδ as a crucial component of the transcriptional regulatory machinery which underlies the intrinsic capacity of peripheral neurons for axonal regeneration., (Copyright © 2012 Elsevier Inc. All rights reserved.)

SURF-6 is an evolutionarily conserved nucleolar protein that is important for cell viability; however, its function in mammals still remains uncertain. The aim of this study is to generate monoclonal antibodies to human SURF-6 protein suitable for fundamental and biomedical research. The full-size human SURF-6 was expressed as a recombinant GST-fusion protein and used as an antigen to generate monoclonal antibodies, S79 and S148, specific for SURF-6. The monoclonal antibody produced by hybridoma clone S79 specifically recognizes endogenous SURF-6 by Western and immunofluorescence analyses in various cultured human cells, and by immunohistochemistry in paraffin-embedded sections of human breast cancer samples. Moreover, S79 immunoprecipitates protein complexes containing SURF-6 from HeLa cells extracts. The antibody S79 recognizes SURF-6 only in human cells; however, the antibody produced by hybridoma clone S148 can detect SURF-6 of human and mouse origin. Monoclonal antibodies to the nucleolar protein SURF-6 described in this work can be a useful tool for studies of ribosome biogenesis in normal and cancer cells.

The transcription factor CCAAT enhancer binding protein (C/EBP) is a key regulator of inflammation and immune responses, and recent studies suggest it is involved in inflammatory processes in the nervous system. We generated a transgenic reporter mouse model, carrying the luciferase (luc) gene under the transcriptional control of C/EBP, for visualising C/EBP activity in vivo. Real-time bioluminescence imaging reflecting C/EBP activity was performed in an acute inflammation model, after systemic administration of lipopolysaccharides (LPS), in C/EBP-luc mice. A striking activity of C/EBP was imaged predominantly in the brain of living C/EBP-luc mice in response to LPS, showing for the first time in vivo that C/EBP mediates the brain response to inflammation. Furthermore, dexamethasone, a potent anti-inflammatory agent, diminished the LPS-induced C/EBP activity demonstrating the physiological regulation of bioluminescence intensity in the brain of C/EBP-luc mice. Our results implicate that C/EBP reporter mice have the potential to be a valuable tool for studies on the mechanisms of brain inflammation in vivo and for the noninvasive preclinical evaluation of therapeutic agents targeting neuroinflammatory diseases., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Immunocytochemical staining with specific antibodies was used to study the expression of three nucleolar proteins (fibrillarin, B23/nucleofozmin, and SURF6), which were involved in pRNA maturation, in the lymphoid cells of healthy individuals and patients with lymphoproliferative diseases and to compare it with the expression of the known proliferation marker Ki-67 protein. The results indicated that fibrillarin was detectable at the comparable level in the lymphoid cells of the patients and in the peripheral blood lymphocytes of the healthy individuals. In one fourth of the patients, the proportion of cells containing B23/nucleofozmin was noticeably higher than that in the lymphocytes of donors; however, there was no great difference in patients with different types of the disease. The number of SURF6-positive cells was directly correlated with that of Ki-67-positive cells. The maximum level (47-67%) of SUR6-positive lymphoid cells was found in splenic lymphosarcomas and mantle cell lymphoma. The findings suggest that SURG6 protein may be of additional diagnostic and prognostic value.

Nuclear factor kappa B (NFkappaB) is a ubiquitous nuclear transcription factor that regulates the expression of a number of genes involved in cell survival, immune and inflammatory processes. It has been hypothesized that after nerve injury, the release of specific cytokines may provide a stimulus for activation of the transcription factor NF-kappaB in adult dorsal root ganglia (DRG) neurons exerting the protective effect on the sensory neurons. However, the complexity of this transcription factor has led to some misleading conclusions about NF-kappaB signalling in injured DRG neurons. The goal of the present study is to find out whether NF-kappaB is involved in the transcriptional regulation of genes in adult primary sensory neurons after peripheral nerve transection. In this series of experiments, we used a transgenic line of NF-kappaB reporter mice in which activation of NF-kappaB drives the expression of the lac-z gene. We show that the expression of beta-galactasidase (beta-gal) is not detected in injured DRG neurons and contralateral neurons. However, a strong beta-gal expression was detected in the muscle at the injury site. It may reflect the repressive influence of additional signalling cascades on NF-kappaB activity in sensory neurons.

The Mpv17l protein has two isoforms, M-LPL and M-LPS, which both regulate the production of reactive oxygen species (ROS) and protect against mitochondrial oxidative stress and apoptosis. M-LPL is ubiquitously expressed, while M-LPS is expressed mostly in the kidney of aged animals. We identified a variety of transcripts of the Mpv17l gene that could encode novel isoforms by mapping expressed sequence tags on the mouse M-LP genomic locus. We analysed the expression and evolutionary conservation of a novel Mpv17l transcript (mpv17l-002) that is predicted to encode a new protein, termed M-LP2. The isoform M-LP2 has the full length of M-LPS plus six amino acids at the end of its amino terminus, which could be encoded by an alternative 5'-flanking sequence. We show that the mRNA of M-LP2 has a different pattern of expression than the mRNA of M-LPS, and sequences of both transcripts are conserved in the rodent genome; however, neither of these isoforms is detected in the human genome. These observations suggest that the functions of certain M-LP isoforms are tissue- and species-specific, implying that their potential involvement in ROS metabolism may be redundant or may be complemented by other members of the Mpv17 family.

Peripheral nerve transections cause much more neuronal death in embryonic and neonatal dorsal root ganglia (DRG) than in adult DRG. Here we used transgenic approaches to examine the hypothesis that NF-kappaB is an important intrinsic factor of adult DRG neurons for their in vivo capacity to survive after nerve injury. We generated transgenic mice expressing the NF-kappaB super-inhibitor (IkappaBalpha-SI), a multi-mutant form of IkappaBalpha, specifically in adult neurons. Adult DRG neurons in these transgenic animals are not abnormally susceptible to apoptosis after peripheral nerve injury, although there is a significant inhibition in the ability of NF-kappaB to translocate into their nucleus. We investigated the observed lack of NF-kappaB neuroprotective function at the level of NF-kappaB transcriptional activity using transgenic NF-kappaB/LacZ reporter mice. We show that the expression of the NF-kappaB reporter transgene is restricted in naïve and injured DRG neurons. However, NF-kappaB transcriptional activity in adult DRG neurons is evident upon exposure to Trichostatin A (TSA) which is a specific inhibitor of histone deacetylases. Taken together our results illustrate that the functions of NF-kappaB are limited in adult primary sensory neurons due to a transcriptional repression mechanism mediated by histone deacetylases, and that intrinsic neuroprotective factors other than NF-kappaB are responsible for the resistance of adult DRG neurons to apoptosis in response to nerve injury.

Transduction and activation of an inducible form of STAT3 (signal transducer and activator of transcription) sufficed to increase VIP (vasoactive intestinal protein) mRNA concentrations in neuroblastoma cells. Overexpression of SOCS3 (suppressor of cytokine signaling) inhibited and mutant SOCS3 (with an inactivating point mutation in amino acid 25) enhanced the induction of VIP mRNA by CNTF (ciliary neurotrophic factor). Because mutant SOCS3 did not augment the increase in STAT transcriptional activity following CNTF stimulation, the enhancement by mutant SOCS3 of the actions of CNTF cannot be attributed to changes in STAT3 signaling. Mutant SOCS3 increased AP-1 (activator protein) transcriptional activity and JNK (c-Jun N-terminal kinase) activity and SOCS3 bound to the scaffolding protein, JNK-interacting protein-1: these observations provide a plausible explanation for the enhancement by mutant SOCS3 of the actions of CNTF. We conclude that endogenous SOCS3 inhibits AP-1 activity through blocking of JNK phosphorylation.

SURF-6 is a bona fide nucleolar protein comprising an evolutionary conserved family that extends from human to yeast. The expression of the mammalian SURF-6 has been recently found to be regulated during the cell cycle. In order to determine the importance of SURF-6 in mammalian cells, we applied the Tet-On system to regulate conditionally, in response to tetracycline, the expression of an antisense RNA (asRNA) that targets Surf-6 mRNA in mouse NIH/3T3 cells. Induced Surf-6 asRNA caused an effective depletion of SURF-6 protein resulted in cell death and in an apparent arrest in the G1 phase of the cell cycle. These results provide for the first time evidence that expression of SURF-6 is essential for mammalian cell viability, and suggest that SURF-6 might participate in the progression of cell cycle.

A conditioning lesion to peripheral axons of primary sensory neurons accelerates regeneration of their central axons in vivo or neurite outgrowth if the neurons are grown in vitro. Previous evidence has implicated neuropoietic cytokines and also cyclic AMP in regenerative conditioning. In experiments reported here, delivery through a lentivirus vector of ciliary neurotrophic factor to the appropriate dorsal root ganglion in rats was sufficient to mimic the conditioning effect of peripheral nerve injury on the regeneration of dorsal spinal nerve root axons. Regeneration in this experimental preparation was also stimulated by intraganglionic injection of dibutyryl cyclic AMP but the effects of ciliary neurotrophic factor and dibutyryl cyclic AMP were not additive. Dibutyryl cyclic AMP injection into the dorsal root ganglion induced mRNAs for two other neuropoietic cytokines, interleukin-6 and leukemia inhibitory factor and increased the accumulation of phosphorylated STAT3 in neuronal nuclei. The in vitro conditioning action of dibutyryl cyclic AMP was partially blocked by a pharmacological inhibitor of Janus kinase 2, a neuropoietic cytokine signaling molecule. We suggest that the beneficial actions of increased cyclic AMP activity on axonal regeneration of primary sensory neurons are mediated, at least in part, through the induction of neuropoietic cytokine synthesis within the dorsal root ganglion.

The localization of the specific protein Surf-6 from nucleoli of eukaryotic cells in mitosis and its sensitivity to the treatment of cells with RNase A and DNase I in situ were studied. It was shown that, in interphase nucleoli of 3T3 mouse cells, Surf-6 is probably associated with RNA and practically is not associated with DNA. In mitosis, Surf-6 appears in forming nucleoli after the known RNA-binding proteins fibrillarin and B23/nucleofozmin, which are involved in the early and late stages of the assembly of ribosomal particles, respectively. These observations and the regularities of migration of early and late proteins of ribosome assembly to nucleoli in the telophase of mitosis led us to the presumption that Surf-6 is involved in the terminal stages of the assembly of ribosomal particles in murine cells. An immunoblot analysis of the Surf-6 content in synchronized 3T3 cells showed for the first time that Surf-6 is present at all stages of the cell cycle but its content markedly decreases when cells enter the G0 period. Conversely, the activation of cells for proliferation is accompanied by an increase in the Surf-6 content. These observations allow one to regard Surf-6 as a marker of the cell proliferative state and suggest its implication in the regulation of the cell cycle. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.

Animal and clinical models of GHRH excess suggest that GHRH provides an important trophic drive to pituitary somatotrophs. We have adopted a novel approach to silence or ablate GHRH neurons, using a modified H37A variant of the influenza virus M2 protein ((H37A)M2). In mammalian cells, (H37A)M2 forms a high conductance monovalent cation channel that can be blocked by the antiviral drug rimantadine. Transgenic mice with (H37A)M2 expression targeted to GHRH neurons developed postweaning dwarfism with hypothalamic GHRH transcripts detectable by RT-PCR but not by in situ hybridization and immunocytochemistry, suggesting that expression of (H37A)M2 had silenced or ablated virtually all the GHRH cells. GHRH-M2 mice showed marked anterior pituitary hypoplasia with GH deficiency, although GH cells were still present. GHRH-M2 mice were also deficient in prolactin but not TSH. Acute iv injections of GHRH in GHRH-M2 mice elicited a significant GH response, whereas injections of GHRP-6 did not. Twice daily injections of GHRH (100 microg/d) for 7 d in GHRH-M2 mice doubled their pituitary GH but not PRL contents. Rimantadine treatment failed to restore growth or pituitary GH contents. Our results show the importance of GHRH neurons for GH and prolactin production and normal growth.

The mammalian SURF-6 protein is localized in the nucleolus, yet its function remains elusive in the recently characterized nucleolar proteome. We discovered by searching the Protein families database that a unique evolutionary conserved SURF-6 domain is present in the carboxy-terminal of a novel family of eukaryotic proteins extending from human to yeast. By using the enhanced green fluorescent protein as a fusion protein marker in mammalian cells, we show that proteins from distantly related taxonomic groups containing the SURF-6 domain are localized in the nucleolus. Deletion sequence analysis shows that multiple regions of the SURF-6 protein are capable of nucleolar targeting independently of the evolutionary conserved domain. We identified that the Saccharomyces cerevisiae member of the SURF-6 family, named rrp14 or ykl082c, has been categorized in yeast databases to interact with proteins involved in ribosomal biogenesis and cell polarity. These results classify SURF-6 as a new family of nucleolar proteins in the eukaryotic kingdom and point out that SURF-6 has a distinct domain within the known nucleolar proteome that may mediate complex protein-protein interactions for analogous processes between yeast and mammalian cells.

The type 1A GH secretagogue (GHS) receptor (GHSR) has been proposed to mediate the effects of ghrelin on GH release, food intake, and body composition. We have overexpressed GHSR in GH-producing GC cells and GHRH neurons in an attempt to enhance signaling via this pathway selectively, in the GH axis. Constitutive overexpression of human GHSR in rat GC cell lines resulted in increased basal phosphoinositol turnover and rendered them responsive to GHS ligands. We then generated transgenic mice overexpressing human GHSR in GHRH neurons using a 38-kb rat GHRH cosmid promoter. GHRH-GHSR transgenic mice showed increased hypothalamic GHRH expression, pituitary GH contents, and postweaning growth rates. Body weights of the transgenic mice became similar in adulthood, whereas adipose mass was reduced, particularly so in female GHRH-GHSR mice. Organ and muscle weights of transgenic mice were increased despite chronic exposure to a high fat diet. These results suggest that constitutive overexpression of GHSR in GHRH neurons up-regulates basal activity in the GHRH-GH axis. However, GHRH-GHSR mice showed no evidence of increased sensitivity to acute or chronic treatment with exogenous GHS ligands. Food intake and adipose tissue responses to chronic high fat feeding and treatment with GHS ligands were unaffected, as were locomotor and anxiety behaviors, although GHRH-GHSR mice remained significantly leaner than wild-type littermates. Thus, constitutive overexpression of GHSR can up-regulate basal signaling activity in the GHRH/GH axis and reduce adiposity without affecting other GHSR-mediated signals.

Analysis of small dorsal root ganglion (DRG) neurons revealed novel functions for vanilloid receptor 1 (VR1) in the regulation of cytosolic Ca(2+). The VR1 agonist capsaicin induced Ca(2+) mobilization from intracellular stores in the absence of extracellular Ca(2+), and this release was inhibited by the VR1 antagonist capsazepine but was unaffected by the phospholipase C inhibitor xestospongins, indicating that Ca(2+) mobilization was dependent on capsaicin receptor binding and was not due to intracellular inositol-1,4,5-trisphosphate generation. Confocal microscopy revealed extensive expression of VR1 on endoplasmic reticulum, consistent with VR1 operating as a Ca(2+) release receptor. The main part of the capsaicin-releasable Ca(2+) store was insensitive to thapsigargin, a selective endoplasmic reticulum Ca(2+)-ATPase inhibitor, suggesting that VR1 might be predominantly localized to a thapsigargin-insensitive endoplasmic reticulum Ca(2+) store. In addition, VR1 was observed to behave as a store-operated Ca(2+) influx channel. In DRG neurons, capsazepine attenuated Ca(2+) influx following thapsigargin-induced Ca(2+) store depletion and inhibited thapsigargin-induced inward currents. Conversely, transfected HEK-293 cells expressing VR1 showed enhanced Ca(2+) influx and inward currents following Ca(2+) store depletion. Combined data support topographical and functional diversity for VR1 in the regulation of cytosolic Ca(2+) with the plasma membrane-associated form behaving as a store-operated Ca(2+) influx channel and endoplasmic reticulum-associated VR1 possibly functioning as a Ca(2+) release receptor in sensory neurons.

Autosomal dominant GH deficiency type II (IGHDII) is often associated with mutations in the human GH gene (GH1) that give rise to products lacking exon-3 ((Deltaexon3)hGH). In the heterozygous state, these act as dominant negative mutations that prevent the release of human pituitary GH (hGH). To determine the mechanisms of these dominant negative effects, we used a combination of transgenic and morphological approaches in both in vitro and in vivo models. Rat GC cell lines were generated expressing either wild-type GH1 (WT-hGH-GC) or a genomic GH1 sequence containing a G->A transition at the donor splice site of IVS3 ((Deltaexon3)hGH-GC). WT-hGH-GC cells grew normally and produced equivalent amounts of human and rGH packaged in dense-cored secretory vesicles (SVs). In contrast, (Deltaexon3)hGH-GC cells showed few SVs but accumulated secretory product in amorphous cytoplasmic aggregates. They produced much less rGH and grew more slowly than WT-hGH-GC cells. When cotransfected with an enhanced green fluorescent protein construct (GH-eGFP), which copackages with GH in SVs, WT-hGH-GC cells showed normal electron microscopy morphology and SV movements, tracked with total internal reflectance fluorescence microscopy. In contrast, coexpression of (Deltaexon3)hGH with GH-eGFP abolished the vesicular targeting of GH-eGFP, which instead accumulated in static aggregates. Transgenic mice expressing (Deltaexon3)hGH in somatotrophs showed an IGHD-II phenotype with mild to severe pituitary hypoplasia and dwarfism, evident at weaning in the most severely affected lines. Hypothalamic GHRH expression was up-regulated and somatostatin expression reduced in (Deltaexon3)hGH transgenic mice, consistent with their profound GHD. Few SVs were detectable in the residual pituitary somatotrophs in (Deltaexon3)hGH transgenic mice, and these cells showed grossly abnormal morphology. A low copy number transgenic line showed a mild effect relatively specific for GH, whereas two severely affected lines with higher transgene copy numbers showed early onset, widespread pituitary damage, macrophage invasion, and multiple hormone deficiencies. These new in vitro and in vivo models shed new light on the cellular mechanisms involved in IGHDII, as well as its phenotypic consequences in vivo.

Our previous study demonstrated that transduction of murine embryonic stem (ES) cells with a human erythropoietin (Epo) receptor (R) cDNA resulted in enhanced erythropoiesis in developing embryonic bodies (EBs). To address possible mechanisms of gene regulation, we compared gene expression between hEpoR cDNA-transduced ES (ES-hEpoR) cells and parental ES cells during in vitro differentiation induced by withdrawal of leukemia inhibitory factor (LIF) and cultured in the absence of Epo using differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR). A total of 48 differentially expressed cDNA fragments were found; 12 were sequenced and five were confirmed by Northern blot analysis to be up- or down-regulated in ES-hEpoR cells during differentiation compared to parental ES cells. In a GenBank search of the five putatively regulated cDNA fragments, two fragments shared high sequence homology to two known genes: the Surf-6 gene and the gene for calcyclin binding protein. Northern blot analysis demonstrated that 2.5-kb and 0.3-kb transcripts of the Surf-6 gene were expressed in undifferentiated ES-hEpoR and parental ES cells at a low level, but this expression was enhanced from day 2 to 14 of differentiation after withdrawal of LIF and culture in the presence of Epo. Furthermore, the enhanced expression of these two transcripts was also noticed in EML-C1 cells, a murine multipotential hematopoietic cell line that has erythroid differentiation potential in response to Epo. In summary, our results demonstrate that Surf-6 gene expression is regulated during differentiation of hematopoietic stem/progenitor cells in response to Epo, suggesting a possible role for Surf-6 gene in erythropoiesis.

The recently identified novel protein SURF-6 is shown to be a component of the nucleolar matrix. Immunofluorescence analysis demonstrated that SURF-6 was localized in residual nucleoli of in situ nuclear matrix preparations of mouse fibroblast cells (NIH 3T3), which were depleted of soluble and chromatin related proteins. Immunoblot analysis of biochemical nucleolar subfractions confirmed that SURF-6 was present in the nucleolar matrix fraction, and was absent from the fractions of soluble proteins released by DNase or RNase. The capacity of SURF-6 to bind nucleic acids was investigated in vitro. Both endogenous SURF-6 from nuclear extracts and recombinant SURF-6 exhibited a strong binding capacity for nucleic acids. It was shown that SURF-6 bound to both DNA and RNA, however, it showed stronger binding to RNA. The presence and nuclear distribution of SURF-6 during the cell cycle was explored by immunofluorescence analysis. It was shown that SURF-6 was always found in the nucleolus regardless of the phase of the cell cycle suggesting that it is a structural protein constitutively present in nucleolar substructures. The colocalization of SURF-6 with the major nucleolar proteins B23 and fibrillarin, which are known to be involved in the processing of ribosomal RNA (rRNA), was examined both in interphase and mitosis by double immunolabeling of cells. SURF-6 was found to be largely coincident with both proteins in interphase and it was distributed in the same cellular locations, namely the perichromosomal layer, the cytoplasm and prenucleolar bodies, in mitosis. However, colocalization of SURF-6 with fibrillarin and B23 was only partial in interphase, and the dynamics of its localization was not completely the same as those of either fibrillarin or B23 during mitosis. Taken together, these results indicate that SURF-6 is a novel nucleolar matrix component and imply that SURF-6 might support nucleolar matrix structure and function(s) via its association with nucleic acids. We propose that SURF-6 may be involved in processing of rRNA, based on its cytological characteristics, but at stages in ribosomal biogenesis which are different from those for fibrillarin and B23.

The Surfeit locus contains the tightest cluster of mammalian genes so far described. The five Surfeit genes (Surf-1 to -5) that have been previously isolated and characterized do not share any DNA or amino acid sequence homology. These Surfeit genes appear to be housekeeping genes, with the Surf-3 gene encoding the 1.7a ribosomal protein and the Surf-4 gene encoding an integral membrane protein most likely associated with the endoplasmic reticulum. In this work, we have isolated the Surf-6 gene, a sixth member of the Surfeit locus. The Surf-6 gene contains four exons spanning a genomic region of 14 kb and specifies a mRNA of 2,571 bases. Surf-6 has features common to housekeeping genes because its transcript is present in every tissue tested, its 5' end is associated with a CpG-rich island, and its promoter does not contain a canonical TATA box. The Surf-6 long open reading frame encodes a novel highly basic polypeptide of 355 amino acids (28% Arg and Lys). By immunofluorescence and immunoblot analyses, the Surf-6 protein has been found to be located in the nucleolus and by immunocytochemical microscopy to be localized predominantly in the nucleolar granular component, a structure that is involved in ribosome maturation. These results indicate that the novel Surf-6 gene is involved in a nucleolar function.

Previous studies have demonstrated that the expression of the alpha-amylase gene is repressed by dietary glucose in Drosophila melanogaster. Here, we show that the alpha-amylase gene of a distantly related species, D. virilis, is also subject to glucose repression. Moreover, the cloned amylase gene of D. virilis is shown to be glucose repressible when it is transiently expressed in D. melanogaster larvae. This cross-species, functional conservation is mediated by a 330-bp promoter region of the D. virilis amylase gene. These results indicate that the promoter elements required for glucose repression are conserved between distantly related Drosophila species. A sequence comparison between the amylase genes of D. virilis and D. melanogaster shows that the promoter sequences diverge to a much greater degree than the coding sequences. The amylase promoters of the two species do, however, share small clusters of sequence similarity, suggesting that these conserved cis-acting elements are sufficient to control the glucose-regulated expression of the amylase gene in the genus Drosophila.