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PubMed2006

Methylation status of a single CpG locus 3 bases upstream of TATA-box of receptor activator of nuclear factor-kappaB ligand (RANKL) gene promoter modulates cell- and tissue-specific RANKL expression and osteoclastogenesis.

Receptor activator of nuclear factor-kappaB ligand (RANKL) expression is tissue specific and limited to certain subsets of T-lymphocytes and stromal/osteoblastic cells. Even among osteoblasts, RANKL is expressed on about 20% of osteoblasts of the normal mouse. To clarify the mechanism of population-specific RANKL expression, we analyzed the effect of CpG methylation on its transcription, mRNA and protein expression as well as on osteoclastogenesis. Subpopulations of ST2 cells were used: P9, which expresses RANKL and supports osteoclastogenesis, and P16, which does not. By sodium bisulfite mapping, the rate of CpG methylation of the -65/+350 region, especially of CpG locus no. 1 three bases upstream of the TATA-box, was higher in P16 than in P9 ST2 cells. ChIP and gel shift assay showed that methylated CpG locus no. 1 was a target of MeCP2 binding that, in turn, blocked the binding of the TATA-box binding protein to the TATA-box. In vitro methylation by SssI of the promoter construct reduced its transcriptional activity at the steady state and its response to 1alpha,25(OH)2 vitamin D3. Conversely, treatment with DNA methylase inhibitor, 5-aza-2'-deoxycytidine, significantly restored RANKL expression and osteoclastogenesis in P16 cells. Except for primary cultured osteoblasts, CpG locus no. 1 was frequently methylated in various normal mouse tissues. We propose that the methylation status of the CpG locus three bases upstream of the TATA-box modulates the control of cell- and tissue-specific expression of RANKL gene and osteoclastogenesis. The heterogeneity of stromal/ osteoblastic cells in response to bone-resorbing stimuli may be attributed, in part, to the methylation status of the RANKL gene promoter.

Animals↗

PubMed1999

Basic helix-loop-helix proteins can act at the E-box within the serum response element of the c-fos promoter to influence hormone-induced promoter activation in Sertoli cells.

The Sertoli cell is a terminally differentiated testicular cell in the adult required to maintain the process of spermatogenesis. Previously basic helix-loop-helix (bHLH) factors and c-fos have been shown to influence Sertoli cell-differentiated functions. The induction of Sertoli cell differentiation appears to involve the serum response element (SRE) of the c-fos promoter to activate c-fos and intermediate bHLH factor(s) that regulate down-stream Sertoli cell-differentiated genes (e.g. transferrin expression). The SRE of the c-fos promoter is influenced through the serum response factor (SRF). Interestingly, an E-box nucleotide sequence is present within the SRE. bHLH proteins act through E-box elements, and the current study investigates the possibility that bHLH proteins may directly influence the SRE of the c-fos promoter. The activation of the c-fos promoter in Sertoli cells was found to be inhibited with the overexpression of the inhibitory HLH protein Id. Analysis of major response elements within the c-fos promoter demonstrated that the expression of Id specifically inhibited the activation of SRE in Sertoli cells and no other elements tested. Mutations in the E-box of the SRE also inhibited the activation of SRE, suggesting the direct role of bHLH proteins in regulating SRE activity in Sertoli cells. In contrast, the activation of SRE containing a mutated E-box was comparable to wild-type SRE in control stromal cells. Analysis of SRE oligonucleotide gel mobility shift assays with nuclear extracts from Sertoli cells demonstrated the presence of both the SRF and the ubiquitously expressed bHLH protein E12/E47. In contrast, no E12/E47 was detected in the SRE oligonucleotide gel shift using control stromal cell nuclear extracts. Observations suggest the binding of E12/E47 to SRE may be a cell-specific event. The SRF and bHLH proteins appear to bind to the SRE and activate the c-fos promoter in Sertoli cells. Observations provide evidence that a bHLH protein can interact with the SRE of the c-fos promoter to influence hormone-induced promoter activation. Cross-talk between these nuclear transcription factors appears to be instrumental in the control of Sertoli cell-differentiated functions.

Animals↗

PubMed1994

DNA sequence specificity of the v-erb A oncoprotein/thyroid hormone receptor: role of the P-box and its interaction with more N-terminal determinants of DNA recognition.

The viral erb A oncogene is a mutated allele of a normal cell gene for a thyroid hormone receptor. The DNA recognition properties of the v-erb A protein are altered from those of the thyroid hormone receptor, due in part to a point mutation in the P-box of the zinc-finger domain of the viral allele. We report here the effects of systematically varying this P-box codon; our results suggest that this P-box amino acid contributes to DNA specificity not by promoting recognition of the appropriate response elements, but rather by excluding binding of the erb A protein to inappropriate half-sites. In this manner, DNA recognition by the v-erb A protein appears to differ from that by the glucocorticoid receptor. A variety of P-box amino acids were compatible with recognition of the prototypic AGGTCA half-site; intriguingly, several of these mutant erb A proteins could also recognize a variety of alternative half-site sequences. Recognition of these alternative half-sites required a compatible amino acid sequence in the N terminus of the erb A protein. Our results begin to define a code by which the identity of the amino acids in the zinc-finger and N-terminal domains is reflected in the DNA recognition properties of the receptor.

Amino Acid Sequence↗

PubMed1995

Importance of the most proximal GC box for activity of the promoter of human thyroid hormone receptor beta 1.

We wish to localize the sequences required for transcriptional expression of the thyroid hormone receptor beta 1 (TR beta 1). Constitutive activity of the promoter of human thyroid hormone receptor beta 1 was assessed by transient transfection of deletion constructs attached to luciferase as reporter, into P19, GH3, HepG2, H19-7, and COS1 cells. A 40-base pair fragment of the beta 1 promoter including the TATA box induced minimal luciferase activity, which was considered basal activity. The activities of various lengths of the beta 1 promoter were estimated relative to the minimal promoter in five cell lines. The region between -130 and -40 was crucial for constitutive activity in all cell lines. Further deletion analysis in HepG2 cells showed that two regions mainly augmented the transcriptional activity of the minimal 40 base pair fragment. One region located at -115 to -93, which is highly GC-rich, included the most proximal of five putative GC boxes present in the whole 1325-base pair promoter. A second region contributing to expression of TR beta 1 in HepG2 cells is at -70 to -40. Mutation of the most proximal GC box strongly suppressed transactivity of the whole promoter in P19 and HepG2 cells. In contrast, mutations in the other GC boxes did not suppress transactivation in P19 cells and slightly suppress activation in HepG2 cells. In Schneider cells, which do not express Sp1, transactivity of the region distal to -40 is positively regulated by cotransfection with a vector expressing Sp1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Regulation of insulin-like growth factor I receptor gene expression by Sp1: physical and functional interactions of Sp1 at GC boxes and at a CT element.

The insulin-like growth factor I (IGF-I) receptor mediates signal transduction by the IGFs and plays a critical role in growth and development. The proximal promoter region of the rat IGF-I receptor gene contains multiple Sp1 consensus-binding sites (GC boxes). Various promoter fragments fused to a luciferase reporter gene were transiently cotransfected together with an Sp1 expression vector into Drosophila Schneider cells, which lack endogenous Sp1. A proximal promoter fragment containing 476 nucleotides of 5'-flanking region and 640 nucleotides of 5'-untranslated region was strongly activated by Sp1 (an average of 116-fold), and progressive 5'-deletions of the promoter that sequentially removed GC boxes reduced Sp1 activation to 15-fold over basal promoter activity. DNase I footprinting studies with purified Sp1 protein revealed four GC boxes in the 5'-flanking region of the promoter and one homopurine/homopyrimidine motif (CT element) in the 5'-untranslated region that bound Sp1. Mutation of the CT element reduced Sp1 activation by 70%. Taken together, these results demonstrate that Sp1 can regulate expression of the IGF-I receptor promoter by acting both on GC boxes in the 5'-flanking region of the promoter and on a CT element in the 5'-untranslated region.

Animals↗

PubMed2004

INCOMPOSITA: a MADS-box gene controlling prophyll development and floral meristem identity in Antirrhinum.

INCOMPOSITA (INCO) is a MADS-box transcription factor and member of the functionally diverse StMADS11 clade of the MADS-box family. The most conspicuous feature of inco mutant flowers are prophylls initiated prior to first whorl sepals at lateral positions of the flower primordium. The developing prophylls physically interfere with subsequent floral organ development that results in aberrant floral architecture. INCO, which is controlled by SQUAMOSA, prevents prophyll formation in the wild type, a role that is novel among MADS-box proteins, and we discuss evolutionary implications of this function. Overexpression of INCO or SVP, a structurally related Arabidopsis MADS-box gene involved in the negative control of Arabidopsis flowering time, conditions delayed flowering in transgenic plants, suggesting that SVP and INCO have functions in common. Enhanced flowering of squamosa mutants in the inco mutant background corroborates this potential role of INCO as a floral repressor in Antirrhinum. One further, hitherto hidden, role of INCO is the positive control of Antirrhinum floral meristem identity. This is revealed by genetic interactions between inco and mutants of FLORICAULA, a gene that controls the inflorescence to floral transition, together with SQUAMOSA. The complex regulatory and combinatorial relations between INCO, FLORICAULA and SQUAMOSA are summarised in a model that integrates observations from molecular studies as well as analyses of expression patterns and genetic interactions.

Alleles↗

PubMed1990

Pax8, a murine paired box gene expressed in the developing excretory system and thyroid gland.

Several mouse genes designated 'Pax genes' contain a highly conserved DNA sequence homologous to the paired box of Drosophila. Here we describe the isolation of Pax8, a novel paired box containing clone from an 8.5 day p.c. mouse embryo cDNA library. An open reading frame of 457 amino acids (aa) contains the 128 aa paired domain near the amino terminus. Another conserved region present in some other paired box genes, the octapeptide Tyr-Ser-Ile-Asn-Gly-Leu-Leu-Gly, is located 43 aa C-terminal to the paired domain. Using an interspecies backcross system, we have mapped the Pax8 gene within the proximal portion of mouse chromosome 2 in a close linkage to the surf locus. Several developmental mutations are located in this region. In situ hybridization was used to determine the pattern of Pax8 expression during mouse embryogenesis. Pax8 is expressed transiently between 11.5 and 12.5 days of gestation along the rostrocaudal axis extending from the myelencephalon throughout the length of the neural tube, predominantly in two parallel regions on either side of the basal plate. We also detected Pax8 expression in the developing thyroid gland beginning at 10.5 days of gestation, during the thyroid evagination. In the mesonephros and metanephros the expression of Pax8 was localized to the mesenchymal condensations, which are induced by the nephric duct and ureter, respectively. These condensations develop to functional units, the nephrons, of the kidney. These data are consistent with a role for Pax8 in the induction of kidney epithelium. The embryonic expression pattern of Pax8 is compared with that of Pax2, another recently described paired box gene expressed in the developing excretory system.

Amino Acid Sequence↗

PubMed2000

FOG-2, a novel F-box containing protein, associates with the GLD-1 RNA binding protein and directs male sex determination in the C. elegans hermaphrodite germline.

Male sex determination in the Caenorhabditis elegans hermaphrodite germline requires translational repression of tra-2 mRNA by the GLD-1 RNA binding protein. We cloned fog-2 by finding that its gene product physically interacts with GLD-1, forming a FOG-2/GLD-1/tra-2 3'untranslated region ternary complex. FOG-2 has an N-terminal F-box and a novel C-terminal domain called FTH. Canonical F-box proteins act as bridging components of the SCF ubiquitin ligase complex; the N-terminal F-box binds a Skp1 homolog, recruiting ubiquination machinery, while a C-terminal protein-protein interaction domain binds a specific substrate for degradation. However, since both fog-2 and gld-1 are necessary for spermatogenesis, FOG-2 cannot target GLD-1 for ubiquitin-mediated degradation. We propose that FOG-2 also acts as a bridge, bringing GLD-1 bound to tra-2 mRNA into a multiprotein translational repression complex, thus representing a novel function for an F-box protein. fog-2 is a member of a large, apparently rapidly evolving, C. elegans gene family that has expanded, in part, by local duplications; fog-2 related genes have not been found outside nematodes. fog-2 may have arisen during evolution of self-fertile hermaphroditism from an ancestral female/male species.

3' Untranslated Regions↗

PubMed1995

A supplemental measure of stay-times in the light box on step-through passive avoidance.

The avoidance behavior of rats in a typical step-through passive avoidance task was evaluated by using two types of time measurements that differed from traditional step-through latency (STL). The total stay-time in the light box (TL) was calculated as an index of the dark box avoidance throughout the retention trial, counting the time before and after the first step-through. The total stay-time in the far area of the light box (TF) was calculated as an index of the tendency to keep away from the dark box. TL, TF and the STL increased with electrical shock intensity at acquisition trials and gradually decreased through an extinction procedure. The discrepancy between our new measures and the STL was observed when the effects of a new drug, RS-8359, which was reported to ameliorate ischemic brain damage, were examined in ischemic animals. TL, TF and STL decreased in rats receiving brain ischemia, and we found that after treatment with RS-8359, TL and TF increased, but no effect was observed on STL. The discrepancy observed suggests that a short STL does not necessarily imply a loss of avoidance behaviour. Passive avoidance tests can be more revealing about a drug's effects when stay-time measures are used.

Animals↗

PubMed2003

The roles of endonucleolytic cleavage and exonucleolytic digestion in the 5'-end processing of S. cerevisiae box C/D snoRNAs.

Small nucleolar RNAs (snoRNAs) play important roles in ribosomal RNA metabolism. In Saccharomyces cerevisiae, box C/D snoRNAs are synthesized from excised introns, polycistronic precursors, or independent transcription units. Previous studies have shown that only a few independently transcribed box C/D snoRNAs are processed at their 5' end. Here we describe 12 additional independently transcribed box C/D snoRNAs that undergo 5'-end processing. 5' Extensions found in the precursors of these snoRNAs contain cleavage sites for Rnt1p, the S. cerevisiae homolog of RNase III, and unprocessed precursors accumulate in vivo in the absence of Rnt1p. Rnt1p cleavage products were identified in vivo when the 5' --> 3' exonucleases Xrn1p and Rat1p are inactivated (xrn1delta rat1-1) and in vitro using model RNA substrates and recombinant Rnt1p. Some of these snoRNAs show increased levels of unprocessed precursors when the rnt1Delta deletion is combined to the xrn1delta rat1-1 mutation, suggesting that these exonucleases participate in the 5' processing or the degradation of the snoRNA precursors. Unprocessed precursors are not significantly destabilized in the absence of the trimethylguanosine capping enzyme Tgs1p, suggesting that a 5' monomethyl cap is sufficient to ensure stabilization of these precursors. These results demonstrate that the majority of independently transcribed box C/D snoRNAs from the yeast genome undergo 5'-end processing and that the Rnt1p endonuclease and the Xrn1p and Rat1p 5' --> 3'exonucleases have partially redundant functions in the 5'-end processing of these snoRNAs.

Base Sequence↗

PubMed2005

The use of telemedicine in combination with a new stroke-code-box significantly increases t-PA use in rural communities.

BACKGROUND: The benefit of tissue plasminogen activator (t-PA) is strongly associated with the time to treatment. In Bavaria, Germany, only half of the population has the opportunity to be transferred to 1 of the 19 stroke units within the critical time window of less than 3 hours. The aim of this study was to investigate the benefit of a new stroke-code-box for t-PA thrombolysis combined with a telemedicine network system to increase the use of acute stroke thrombolysis. METHODS: Two specialized stroke centers in Germany established a 24-hour telemedicine network (Telemedicine Pilot Project of an Integrated Stroke Care [TEMPiS]) to advise 12 community hospitals in eastern Bavaria. These clinics are linked via telemedicine in a 24-hour/7-day service network that allows patients to be examined by experts via a videoconference system Additionally, a special stroke-code-box for acute t-PA thrombolysis was designed to reduce time in the application and documentation process. RESULTS: In the 12-month period before implementation of the TEMPiS network system, 10 patients had received systemic thrombolysis. In our 6-month study period (from July to December 2003) and after implementation of a stroke-code-box for t-PA thrombolysis within the telestroke network, 164 patients with acute stroke were presented with t-PA treatment indications. Of this patient population, 27.4% (45 of 164) received t-PA. CONCLUSIONS: Stroke care, including t-PA thrombolysis in non-urban areas, is feasible using a modern stroke unit concept within a telestroke network. With the expertise of specialized stroke centers accessed via telemedicine and the design of a stroke-code-box for t-PA thrombolysis, nearly one-third of patients presented with a possible indication for systemic thrombolysis can be treated with t-PA, thereby increasing the options for a successful stroke treatment.

Aged↗

PubMed1994

Developmental regulation of DEAD box proteins and cloning of putative RNA helicase genes from Dictyostelium discoideum.

RNA secondary structure is essential for RNA function in pre-mRNA splicing, mRNA translation, ribosome assembly and RNA stability. The involvement of DEAD/H RNA helicases in the regulation of these processes has been demonstrated in some cases. To investigate the repertoire of DEAD box proteins expressed in Dictyostelium discoideum, we used PCR techniques to clone two cDNAs coding for DEAD box proteins with high similarity to known yeast proteins: Dictyostelium Hel2A is about 45% identical to Saccharomyces cerevisiae DBP2 and S. pombe dbp2, the yeast homologues of human p68. Dictyostelium Hel2B is about 43% identical to the S. cerevisiae splicing factor PRP28, but has a different domain at the N-terminus, which is unique for Dictyostelium discoideum. Using a polyclonal antibody directed against a DEAD box peptide we show differential expression of three DEAD box proteins during the developmental cycle of Dictyostelium.

Amino Acid Sequence↗

PubMed1990

The gene for the beta subunit of porcine LH: clusters of GC boxes and CACCC elements.

The gene for the beta subunit of porcine LH (LH-beta) was cloned from a genomic library constructed in EMBL3. The nucleotide sequence was determined for the entire gene transcriptional unit of porcine LH-beta in addition to 1277 and 372 bp of the 5'- and 3'-flanking regions respectively. Southern blot analysis of the porcine genomic DNA indicated that the LH-beta gene is present as a single copy. The transcriptional unit of porcine LH-beta spanned 1107 bp and contained three exons interrupted by two introns of 326 and 289 bp. The short untranslated sequence in the first exon and the location of the exon/intron junctions at amino acid residues -16/-15 and +41/+42 were highly conserved in the rat, human and bovine LH-beta genes. In the 5'-flanking region, one TATA box and two CCAAT boxes were present. The steroid-responsive element was not found up to 1277 bases upstream of the transcription start site. The potential AP-2 factor-responsive elements appeared nine times within the sequence that was determined, and four of them were located in the 5'-flanking region. Two distal AP-2 elements were arranged in an inverted repeat forming a 16 bp palindromic sequence. This feature suggested that hypothalamic gonadotrophin-releasing hormone stimulates expression of the LH-beta gene, predominantly by a signal-transduction system with the protein kinase C cascade and a mediator, the AP-2 factor. A further characteristic feature of the porcine LH-beta gene was the presence of clusters of GC boxes and CACCC elements in the 5'-flanking region and the downstream sequence. Co-existence of these regulatory elements with other elements, such as the AP-2 element or CCAAT box, was also found. The porcine LH-beta gene shows a structure distinct from the porcine FSH-beta and common alpha genes, which are counterparts of the LH-beta gene, reflecting differential control of their synthesis during gametogenesis.

Amino Acid Sequence↗

PubMed1997

Effects of ambient light and view box luminance on the detection of calcifications in mammography.

OBJECTIVE: Viewing conditions can affect diagnostic performance differently depending on background optical densities. We quantified detection accuracy when viewing calcifications in glandular tissue under recommended viewing conditions versus accuracy with lower view box luminance and higher ambient lighting. MATERIALS AND METHODS: A phantom with adipose, 50% adipose and 50% glandular, and glandular-simulating material was imaged, and images were interpreted by five medical imaging physicists using two lighting conditions: the recommended one, high view box luminance (4365 nits) with low ambient light (25 lx), and a suboptimal one, low view box luminance (1763 nits) with moderate ambient lighting (290 lx). Then, a dense (Breast Imaging Reporting and Data System breast composition pattern type 4) unfixed cadaveric breast with numerous native calcifications was imaged 28 times. Nineteen of the films had added clusters of simulated calcifications. Three radiology fellows, each with 11 months of training in mammography, identified the added calcification clusters in the images under the two lighting conditions. Changes in phantom analysis and accuracy of the clinical diagnosis were compared for each lighting condition. RESULTS: On mammograms of the phantom, both speck and fibril identification were degraded by an average of 1.4 objects for the adipose-simulating section (with its darker optical density). For medium optical densities, found in the section with the simulation of 50% glandular and 50% adipose tissue, suboptimal lighting conditions had little or no effect on speck and fibril identification. For sections of the phantom that simulated glandular tissue, an average of 0.6 specks or fibers were not seen when lighting was suboptimal. With the dense cadaveric breast, the fraction of added calcification clusters detected by the three observers improved by an average of 17% when low luminance viewers and high ambient light were replaced with recommended viewing conditions; individual scores of the observers improved significantly: p values ranged from .02 to .05. CONCLUSION: Luminance of the view box and ambient lighting significantly affect detection of calcifications in dense breasts when images are interpreted by radiologists with about 1 year of training in mammography. Detection of calcifications in phantoms is primarily degraded for adipose tissue with its darker optical density. However, when lighting conditions are suboptimal, some observers also have trouble detecting calcifications in glandular tissue with its low optical density.

Adipose Tissue↗

PubMed1992

Predicting percentage of retail yield from carcass measurements, the yield grading equation, and closely trimmed, boxed beef weights.

Over the past 3 yr, 100 carcasses (64 steers, 24 bulls, and 12 heifers) were fabricated into closely trimmed (6 mm maximum fat cover), boxed beef and further evaluated for percentage of retail yield at the Iowa State University Meat Laboratory. Hot carcass weight ranged from 235 to 399 kg with a least squares mean (LSM) and standard error across all sex classes of 318 +/- 3 kg. Additionally, fat cover ranged from .30 to 1.78 cm with an average of .91 +/- .05 cm. The LSM for longissimus muscle area (LMA) across all sex classes was 81.6 +/- 1.0 cm2. Bulls had significantly less subcutaneous fat (P less than .01) and greater LMA (P less than .01) than did either steers or heifers. Retail yield from the boxed chuck, expressed as a percentage of cold carcass weight, was 19.2 for bulls and 14.8 for steers. This difference was due primarily to a reduction of intermuscular fat. Similarly, bulls had a greater yield (P less than .01) of the boxed round than did steers. When cattle of differing frame sizes were compared, only percentage of retail yield of the boxed round was significant (P less than .01): large-framed cattle yielded 14.3 +/- .2%, compared with 12.8 +/- .2% for the small-framed cattle. When all possible regression analyses were run, sex class differences accounted for 25.7% of the variation in retail yield. The current USDA retail yield equation accounted for only 37.2% of the variation. Percentage of closely trimmed, boneless round had an R2-value of .57.(ABSTRACT TRUNCATED AT 250 WORDS)

Abattoirs↗

PubMed2000

Microbiological testing of raw, boxed beef in the context of hazard analysis critical control point at a high-line-speed abattoir.

The efficacy of cold storage of raw, bagged, boxed beef was assessed microbiologically at a high-line-speed abattoir (270 carcasses per h). At the time of this study, plant management was in the process of creating a hazard analysis critical control point plan for all processes. Aerobic bacteria, coliforms, and type 1 Escherichia coli were enumerated (5 by 5-cm excision samples, hydrophobic grid membrane filter technology) before and after cold storage of this final product produced at six fabrication tables. In addition, the temperature-function integration technique (TFIT) was used to calculate the potential number of generations of E. coli during the first 24 or 48 h of storage of the boxed beef. Based on the temperature histories (total of 60 boxes, resulting from 12 product cuts, five boxes from each of two fabrication tables on each of 6 sampling days, and six types of fabrication tables), TFIT did not predict any growth of E. coli (with or without lag) for the test period. This was verified by E. coli mean log10 values of 0.65 to 0.42 cm2 (P > 0.05) determined by culture before and after the cooling process, respectively. Counts of aerobic bacteria and coliforms were significantly reduced (P < 0.001 and P < 0.05, respectively) during the initial period of the cooling process. There were significant microbiological differences (P < 0.05) between table-cut units.

Abattoirs↗

PubMed2006

A novel mycoplasma detected in association with upper respiratory disease syndrome in free-ranging eastern box turtles (Terrapene carolina carolina) in Virginia.

Clinical signs of upper respiratory tract disease-like syndrome (URTD-LS) were observed in free-ranging eastern box turtles (Terrapene carolina carolina) from Virginia, USA (May 2001-August 2003), some of which also had aural abscesses. After a Mycoplasma sp. was detected by polymerase chain reaction (PCR), a study was undertaken to better define the range of clinical signs of disease and to distinguish mycoplasma-associated URTD-LS from other suspected causes of URTD-LS and aural abscessation in box turtles. Nasal and/or ocular swabs (from turtles possessing URTD-LS) or nasal washes (from asymptomatic turtles) were collected from turtles May 2001-August 2003; samples were assayed for Mycoplasma spp., chelonian herpesvirus, and iridoviruses by PCR testing. A partial DNA sequence (933 bases) of the small ribosomal subunit (16S rRNA) of the box turtle Mycoplasma sp. was analyzed to determine its phylogenetic relatedness to other Mycoplasma spp. of veterinary interest. Mycoplasma sp. was detected in seven (six with clinical signs of URTD-LS; one asymptomatic) of 23 fortuitously collected animals from six of 11 Virginia counties. Clinical signs in Mycoplasma sp.-infected animals included unilateral to bilateral serous to mucopurulent nasal discharge, epiphora, ocular edema, and conjunctival injection. Five Mycoplasma sp.-positive animals possessed aural abscesses; two did not. Analysis of the mycoplasma 16S rRNA gene sequence from one asymptomatic and three symptomatic animals representing four counties revealed a consensus Mycoplasma sp. sequence closely related to, but distinct from, M. agassizii. None of the samples collected contained viral DNA of chelonian herpesviruses or invertebrate and vertebrate (including FV3) iridoviruses. In conclusion, a new Mycoplasma sp. was associated with URTD-LS in native box turtles from Virginia that was not codetected with other suspected causes of chelonian upper respiratory disease; there was no proof of a direct relationship between aural abscessation and the Mycoplasma sp.

Animals↗

PubMed1999

Transcriptional up-regulation of matrix metalloproteinase-9 expression during spontaneous epithelial to neuroblast phenotype conversion by SK-N-SH neuroblastoma cells, involved in enhanced invasivity, depends upon GT-box and nuclear factor kappaB elements.

Spontaneous epithelial (S) to neuroblast (N) conversion enhanced the capacity of SK-N-SH neuroblastoma (NB) cells to invade reconstituted basement membrane in vitro. This involved a switch to matrix metalloproteinase (MMP) activity, in particular MMP-9, and was associated with the induction of MMP-9 expression. N-type-specific MMP-9 expression was herbimycin A inhibitable tyrosine kinase (possibly c-src) dependent and was regulated transcriptionally through GT-box (-52), and nuclear factor kappaB (NFkappaB; -600) elements within the MMP-9 gene. GT-box function was associated with elevated levels of specific nuclear GT-box binding complexes in N-type cells. NFkappaB function was associated with specific p50- and p65-containing nuclear NFkappaB binding complex(es). No function could be attributed to the proximal AP-1 (-79) element, and minimal function was attributed to the SP-1 (-560), ets (-540), or distal AP-1 (-533) elements. This was despite elevated levels of specific junD/fra-1 containing proximal AP-1 element binding complex(es) in N-type cells. Our data highlight a pivotal role for the GT-box, in concert with the NFkappaB element, in the transcriptional up-regulation of MMP-9 expression during spontaneous S to N phenotype conversion by SK-N-SH cells involved in enhanced basement membrane invasivity.

Benzoquinones↗