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Game Stats
Game Stats
W: W. Rodriguez (3-3) L: K. Drabek (3-3)
S: M. Melancon (3)
|3rd Inning
Wallace singled to center, Pence scored, Lee to third.
|3rd Inning
Juan Rivera (4) solo shot to left off Rodriguez.
|5th Inning
Hunter Pence (6) 2 run shot to left off Drabek, Sanchez scored.
|6th Inning
J.P. Arencibia (8) solo shot to left center off Rodriguez.

subset: commoncrawl


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Switched at Birth
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Release:November 24, 1999
RunTime:1 hr 29 min
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Description: Watch Switched at Birth full movie online for free produced in 1999. Genres are Drama.
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Bankruptcy would test Sabres' arena lease
With all the uncertainty swirling around Adelphia Communications Corp., the Rigas family and their intertwined ownership of the Buffalo Sabres, those familiar with the team's lease with HSBC Arena are confident the National Hockey League team will remain in Buffalo.
Unless, of course, the Buffalo Sabres themselves file for some sort of bankruptcy protection because of the financial downfall of Adelphia and the Rigas family.
"Protections are in place, but limitations such as bankruptcy are still there," said Chris Hurley, a partner with Hurwitz & Fine, who represented the Erie County Industrial Development Agency during the lease talks.
The Sabres' lease with HSBC Arena, something that's covered in five Manhattan phone book-thick, red-covered volumes, holds the team to the downtown arena through 2022.
"Hopefully, none of this will have to be tested," said Arnold Gardner, a partner with Kavinoky & Cook, and one of the attorneys retained to negotiate the lease on Buffalo's behalf. "We worked hard to get what we think is a very good, non-relocation agreement."
The lease was written through a team of lawyers, both locally and in New York City, who represent everyone from the City of Buffalo, the primary lenders on the financing of the $125 million arena, to the NHL itself.
"It was very carefully negotiated and thought out by all the attorneys involved," said Michael Risman, Buffalo Corporation Counsel. "To the extent that they could, they protect all the public sector entities."
The lease is complex.
Non-relocation clauses were included to protect the public sector interests including Buffalo, Erie County, the state and the Erie County Industrial Development Agency.
There are equally strong non-relocation clauses that protect the interests of Marine Midland Bank (now HSBC Bank) and Key Bank of New York, who provided construction financing for the arena.
In one section of the document, it states:
"After the completion of the Crossroads Arena (the original name of the arena) and the commencement of the term of the Sabres lease, NFHLP (Niagara Frontier Hockey L.P.-the partnership who owned the Sabres in 1995) agrees that it will not apply to the NHL to relocate the Franchise to a playing site other than the Crossroads Arena and that it will not initiate or pursue proposals to relocate the Franchise prior to the expiration of the 27th Lease year....
"After the foregoing dates, NFHLP may explore and make inquiries, discuss relocation, apply to the NHL, if necessary and if necessary make arrangements to relocate the Franchise, but in no event shall NFHLP relocate prior to the expiration of the Term of this Agreement."
A similar clause is included in the public sector agreements.
Bernadette Mansur, NHL group vice president of communications, said the league believes the lease agreement is fairly airtight.
"It is a complex situation," Mansur said. "But no one has to worry about the future of the Sabres in Buffalo."
Where the lease gets tricky is that it gives the NHL the final say in the Sabres' ownership and location matters.
According to a section of the June 1, 1995 agreement negotiated between the NHL and local interests:
"...The NHL agrees that it will not enact any NHL Constitution and Agreements that specifically render the Public Sector Non-Relocation void and or unenforceable, provided that this sentence shall not limit in any way the NHL's rights to consider, act upon, or approve a voluntary application to relocate the club, subject to the Financing Participant(s)' right to seek injunctive relief as set forth in the preceding sentence."
As of June 5, a partnership headed by the Rigas family, Adelphia's founders, still owned the team. Any sale, should the Rigas' decide to sell the team, would be subject to NHL approval.
The team is maintaining a "business as usual" stance. It is renewing season ticket and luxury suite contracts. As of May 15, Sabres officials said their renewal rate is running at about 70 percent. The team also signed amateur prospects before their June 1 negotiating rights expired.
The Rigases remain under close scrutiny because of their off-the-books financial dealings involving Adelphia, the nation's sixth largest cable operator.
In the two-plus months since the Rigas' admitted to Adelphia having off-the-books debt totaling $3.1 billion, the company's stock has fallen from $22 a share to under one dollar and, ultimately, being delisted earlier this month.
According to various documents, the Rigases used as much as $150 million of Adelphia funds to purchase and operate the Sabres. The Rigas family partnership closed their deal to officially purchase the team two summers ago, although they had been investors in the team since 1994.
The Rigases have claimed that the team loses between $10 million and $15 million on an annual basis, although, some critics say those numbers aren't accurate.
Sources say if and when the Sabres come on the market, there are local investors who would be willing to acquire the team. Some of those investors have already begun behind-the-scenes maneuvering that would put them in a position to move quickly should the Sabres come up for sale.
The key, insiders agree, is to keep the team out of bankruptcy and allow for any sale, if the team comes up for sale, to take place outside the realm of a trustee or creditor's committee.
"The bottom line to all this, is that as long as the club stays out of bankruptcy, any form of relocation can be prevented," Hurley said.


text:
Town Square
Communities gear up for Independence Day celebrations
Original post made on Jun 30, 2011
Read the full story here Web Link posted Wednesday, June 29, 2011, 3:14 PM
Comments (1)
a resident of Danville
on Jun 30, 2011 at 1:26 pm
Safety Note:
Please, please leave Fido and Rover securely at home. The 4th of July is The Worst Day of the year for your family's Best Friends. The shotgun brigade at the Danville parade and the San Ramon fireworks are just not good places for Spot and Bowser.
Not a year goes by without seeing a panicked hound slipping its collar and taking off for parts unknown. The "lucky" ones make it to an uncertain, terrifying fate at a shelter -- others end up under the wheels. Every year. Some benighted shelters pre-kill for space, knowing they'll be deluged with post-4th strays.
Exercise your canine friends early, and leave them at home, preferably with some music on to dull the noise.
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text:
What does is the fact that the state Senate district he represented until 2010 is across the potomac river in Maryland.
river in eastern U.S., from Algonquian Patowmeck, originally the name of a native village in Virginia, perhaps literally "something brought."
A river that divides Washington, D.C., from Virginia.
Note: Sometimes used to refer loosely to Washington, D.C.: "Along the Potomac today, Congress met to vote on the budget."


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Familial isolated pituitary adenoma (FIPA) is an autosomal dominant condition with variable genetic background and incomplete penetrance. Germline mutations of the aryl hydrocarbon receptor interacting protein (AIP) gene have been reported in 15–40% of FIPA patients. Limited data are available on the functional consequences of the mutations or regarding the regulation of the AIP gene. We describe a large cohort of FIPA families and characterize missense and silent mutations using minigene constructs, luciferase and β-galactosidase assays, as well as in silico predictions. Patients with AIP mutations had a lower mean age at diagnosis (23.6±11.2 years) than AIP mutation-negative patients (40.4±14.5 years). A promoter mutation showed reduced in vitro activity corresponding to lower mRNA expression in patient samples. Stimulation of the protein kinase A-pathway positively regulates the AIP promoter. Silent mutations led to abnormal splicing resulting in truncated protein or reduced AIP expression. A two-hybrid assay of protein–protein interaction of all missense variants showed variable disruption of AIP-phosphodiesterase-4A5 binding. In summary, exonic, promoter, splice-site, and large deletion mutations in AIP are implicated in 31% of families in our FIPA cohort. Functional characterization of AIP changes is important to identify the functional impact of gene sequence variants. Hum Mutat 31:1–11, 2010. © 2010 Wiley-Liss, Inc.
The majority of pituitary adenomas are sporadic, but occasionally they may also occur in a familial setting [Melmed, 2006]. Familial isolated pituitary adenoma (FIPA; MIM 102200) is an increasingly recognized autosomal dominant disease with low or variable penetrance [Beckers and Daly, 2007; Chahal et al., 2010]. Heterozygous germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP; MIM 605555) have been identified in 15–40% of FIPA families, whereas in the majority of the families the disease-causing gene or genes are not known. AIP mutation-positive families most commonly present with somato-troph, or with both somatotroph and lactotroph adenomas, but rarely other types of pituitary tumors can be observed [Daly et al., 2007; Iwata et al., 2007; Leontiou et al., 2008; Toledo et al., 2007; Vierimaa et al., 2006]. Young onset, seemingly sporadic somatotroph adenoma patients can also harbor germline AIP mutations [Georgitsi et al., 2008a]. Patients have loss of heterozygosity (LOH) in the tumor tissue at the locus of the AIP gene at the 11q13 area [Gadelha et al., 2000; Soares et al., 2005; Vierimaa et al., 2006; Yamada et al., 1997]. However, LOH at 11q13 has been observed frequently in sporadic pituitary adenomas [Farrell and Clayton, 2000], and there are a number of FIPA families with 11q13 LOH, but no detectable AIP mutations [Leontiou et al., 2008; Soares et al., 2005]. Interestingly, the 11q13 locus also contains the Multiple Endocrine Neoplasia type 1 gene (MEN1; MIM 131100), 2.4 megabases upstream from the AIP gene, which can cause familial pituitary adenomas; however, the phenotype of MEN1 and FIPA is different.
The AIP gene consists of six exons encoding a 330 amino acid protein with three typical tetratricopeptide repeat (TPR) domains and a final extended α-helix (α-7). AIP appears to function as a tumor suppressor gene: we have previously reported data showing that mutant AIP proteins lose the ability of wild-type (WT) AIP to decrease cell proliferation and are unable to bind protein partners [Leontiou et al., 2008]. Most of the described AIP mutations identified via standard sequencing techniques change the amino acid sequence due to nonsense, deletion, or insertion mutations, and result in a loss of the C-terminal end of the protein. Large gene deletions also severely disrupt the protein structure. Exon/intron junction mutations may affect splicing or RNA stability and promoter mutations affect RNA expression, but these have not been previously characterized.
We have systematically examined our large cohort of patients with FIPA specifically looking for large deletions, promoter, and splice-site mutations in addition to exonic mutations; we describe 11 new AIP mutation-positive families with six novel mutations. We present functional data on an AIP promoter mutation, a splice-mutation and a synonymous change, which leads to reduced AIP mRNA expression, as well as quantitative data on the effects of all the published missense mutations on the interaction of AIP with one of its binding proteins, phosphodiesterase-4A5 (PDE4A5). These data allow us to characterize the clinical features of FIPA and to provide the most accurate data for the functional impact of AIP variants, which has an important role in genetic counseling of patients and their families and will help inform the need for predictive testing and biochemical and imaging screening.
We studied 38 novel families with FIPA (Supp. Table S1) who were identified as having at least two family members with pituitary adenoma and no features of MEN1 or Carney complex (MIM 160980). All patients provided written informed consent, and institutional review board approval was obtained. For the clinical analysis we combined these novel families with our previously described cohort of 26 families (Supp. Table S2) [Leontiou et al., 2008]. The clinical features of some of these families have previously been described, as detailed in Supp. Table S1 and S2 [Gadelha et al., 1999, 2000; Georgitsi et al., 2007, 2008b; Leontiou et al., 2008; Matsuno et al., 1994; McCarthy et al., 1990; Pestell et al., 1989; Soares et al., 2005]. Direct sequencing of AIP (NM_003977.2) included the entire coding sequence, conserved splice sites (from the conserved A of the upstream branch site to + 10 downstream of each exon) and 1,200 base pairs of the promoter region. Nucleotide numbering throughout the manuscript reflects cDNA numbering with 11 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to the guidelines of the Human Genome Variation Society (http://www.hgvs.org/mutnomen). The initiation codon is codon 1. AIP sequencing data were compared with Caucasian (n 5 96) and Japanese (n =78) subjects from the general population, as previously described [Leontiou et al., 2008]. Multiplex ligation-dependent probe amplification (MLPA, P244-kit MRC-Holland, Amsterdam, The Netherlands) dosage analysis was carried out to look for partial or whole gene deletions in all the families that tested negative by direct sequencing for germline AIP mutations and for whom a suitable quality of DNA sample was available.
RNA from whole blood was extracted using PaxGene tube and extraction kit (Qiagen, Crawley, UK). RNA from rat pituitary cell line GH3 was extracted using the RNeasy Mini Kit (Qiagen). Both protocols include a DNase step. RT-PCR on 1μg RNA was performed as described previously [Leontiou et al., 2008]. Realtime PCR was performed with the TaqMan system using ready-made or custom-made (surrounding the c.807C > T mutation) AIP probe-primer kits (ABI, Warrington, UK). Reactions were performed in triplicate using β-actin or GAPDH as a housekeeping gene. Data were analyzed using the standard curve method. Rat somatomammotroph GH3 cell line was cultured and transfected as previously described [Leontiou et al., 2008].
The c.[−270_−269CG > AA;−220G > A] changes in the promoter area were identified in a Japanese somatotroph adenoma family (Family VI Supp. Table S2). These changes were not detected in Japanese (n =78) and Caucasian (n =96) individuals from the general population or in any of the studied family members (affected or unaffected AIP mutation carriers or their noncarrier family members or AIP mutation negative patients, n =150) of our familial cohort, or in any of our sporadic pituitary adenoma cases studied (n = 98), altogether 844 chromosomes. The transcription factor binding-site searching programs TESS (http://www.cbil.upenn.edu/cgi-bin/tess/tess) and Alibaba (http://www.gene-regulation.com/pub/programs/alibaba2/index.html) were used to identify possible binding site disruption caused by the c.[−270_−269CG > AA;−220G > A] sequence changes in the AIP promoter area (Fig. 1A and B). A 1.2-kb (upstream from the start codon) PCR fragment was amplified from gDNA (forward 5′TACAACCTCCATCTCCTGGG3′, reverse 5′GAGTCCGGAAGTTG-CCGAAA3′). The WT fragment was cloned into the SacI and SpeI sites of the promoterless firefly luciferase vector pGL3-basic (Promega, Southampton, UK). Three mutated constructs were prepared from the WT template using the QuikChange site-directed mutagenesis kit (Stratagene, LaJolla, CA): a dibasic −270_−269AA mutant, a single −220A mutant and a construct containing both −270_−269AA and −220A mutations (Fig. 1C). To study basal and stimulated activity, GH3 cells were transfected with the four different constructs and 48 hr later were treated with db-cAMP (2 mM), forskolin (10 μM), phorbol-12-myristate 13-acetate (PMA, 20 nM) and H89 (30 μM) for 5hr. A plasmid encoding renilla luciferase, pRL-CMV, was included to control for transfection efficiency and promoter activity was analyzed using the dual luciferase assay (Promega). Peripheral blood-derived cDNA samples were studied from the two affected patients and three ethnically matched controls.
The ALAMUT program (http://www.interactive-biosoftware.-com/alamut/doc/1.5/splicing.html) was used to predict whether the novel synonymous sequence variant c.249G > T, p. = (affecting the third nucleotide of codon 83 originally coding glycine) changes the splicing characteristics of the AIP gene (Family 28, Fig. 2A). The primers (forward 5′GCGGATATCATCGCAAGACT3′, reverse 5′CCTCATCTTCCACATGGAGA3′) were designed on exons 1 and 3 to detect aberrant splicing events.
The ALAMUT, the ESEfinder v.3.0 (http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi?process=home) and the RESCUE-ESE (http://genes.mit.edu/burgelab/rescue-ese/) programs were used to predict whether the synonymous c.807C > T, p. = (affecting the third member of codon 269 originally coding phenylalanine) sequence variant of exon 6 changes the splicing characteristics of the AIP gene (Fig. 3A) [Leontiou et al., 2008]. Conventional and real-time RT-PCRs were performed on patient blood-derived cDNA using primers spanning the exon 5–6 junction to compare AIP expression between patients carrying the mutation and control individuals. To further characterize the splicing events around this sequence abnormality we performed a minigene splicing study. To construct the minigene, gDNA from a patient harboring the c.807C > T mutation was PCR amplified with primers covering part of the exon 5, intron–exon region and part of exon 6 including the mutation (forward 5′AAGCTGGTGGTCGAGGAGTA3′, reverse 5′CAAAGTGCTGGAGCTGGAC3′). WT and mutant minigene constructs were prepared and fragments were cloned into the EcoRI site of pcDNA3.1+ (Invitrogen, Paisley, UK). GH3 cells were transiently transfected for 24–48 hr with WT and mutant minigenes separately or together in equal amounts. Transfection efficiency was followed by cotransfection with an expression plasmid coding for green fluorescent protein (GFP, pEGFP-N1) at a pcDNA3.1+with insert:pEGFP-N1ratio of 1:10. Expression of the minigene transcripts was detected using vector-specific primers.
Interactions between AIP and PDE4A5 were studied to clarify the role of missense sequence changes in protein-protein binding. PDE4A5 was cloned into the NotI-site of pLEXAN to generate a LexA DNA-binding-domain fusion [Bolger et al., 2003; Leontiou et al., 2008]. WT AIP and nine missense AIP sequence variants (c.47G > A, p.R16H [Buchbinder et al., 2008; Cazabat et al., 2007; Daly et al., 2007; Georgitsi et al., 2007; Yaneva et al., 2008]; c.145G > A, p.V49M [Iwata et al., 2007]; c.308A > G, p.K103R [Beckers et al., 2008], c.713G > A, p.C238Y [Leontiou et al., 2008], c.721A > G, p.K241E [Daly et al., 2007], c.769A > G, p.I257V [Montanana et al., 2009], c.811C > T, p.R271W [Daly et al., 2007; Igreja et al., 2010; Jennings et al., 2009], c.896C > T, p.A299V [Georgitsi et al., 2007], the hotspot mutation c.911G > A, p.R304Q, and a novel stop mutation (c.490C > T, p.Q164X) were cloned into the NotI-site of pGADN to generate GAL4 activation-domain fusions. Quantitative β-galactosidase assays were performed in the Saccharomyces cerevisiae strain L40 by the method of Guarente [1983] using O-nitrophenyl-β-D-galactopyranoside as a substrate. Each mutation was tested in at least two different yeast clones.
Location of AIP missense mutations were compared to the available consensus sequences of TPR proteins. A hypothetical model of AIP was constructed using the Phyre (Protein Homology/analogY Recognition Engine) program [Kelley and Sternberg, 2009], based on the crystal structure of FKBP51 (MIM 602623), the closest related protein with available crystal structure (c1kt0A). The alignment is showing 22% identical amino acids and 45% similar amino acids in the N-terminal region (amino acids 3–90 of AIP) and the TPR domain area (amino acids 165–328 of AIP) (Supp. Fig. S1). Alignment was performed with CLUSTAL 2.0.12 (http://www.clustal.org/) and BL2SEQ (http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi) sequence alignment tools.
Statistical analysis was performed with StatsDirect software (Addison-Wesley-Longman, Cambridge, UK). Distribution of data was analyzed by the Shapiro-Wilk test. Comparisons were calculated with the Student t-test and the Kruskal-Wallis test followed by the Conover-Inman test, as appropriate. Data are shown as mean ± standard error, unless otherwise stated. Significance was taken at P < 0.05.
We report here 38 novel families with FIPA, including 11 families with an AIP mutation (Supp. Table S1). Six of these 11 families harbor a novel mutation including two complete gene deletions.
The novel mutations revealed via sequencing include a nonsense mutation (c.490C > T, p.Q164X), a single base-pair duplication (c.662dupC, p.E222X), a splice-site mutation (c.249G > T, p.G83AfsX15, see data below) and a complex deletion-insertion mutation (c.74_81delins7, p.L25PfsX130), all resulting in a frameshift and/or premature stop-codon. Three families have been found to have the previously well-described c.910C > T, p.R304X hotspot [Daly et al., 2007; Jaffrain-Rea et al., 2009; Leontiou et al., 2008; Vierimaa et al., 2006] mutation and one family the c.911G > A, p.R304Q hotspot [Daly et al., 2007; Leontiou et al., 2008] mutation. We also identified a previously described variant (c.896C > T, p.A299V) in one of our families that also harbors the p.R304X mutation.
We screened all affected patients in our cohort (Supp. Tables S1 and S2) for large deletions using MLPA, where no AIP mutations were identified by sequencing and suitable DNA was available. Three large deletions were identified among the 36 families (8.3%, Family 9, Family IV, and Family XXI; Supp. Tables S1 and S2), all three originally reported to have WT AIP sequences by conventional sequencing [Georgitsi et al., 2007; Leontiou et al., 2008]. Two of these resulted in the loss of the full length AIP gene and one was the previously described exon 2 deletion [Georgitsi et al., 2008b].
For clinical characteristics we analyzed the data from the currently reported 38 families together with the 26 families we have reported earlier [Leontiou et al., 2008]. In our cohort of 64 FIPA families with 160 affected subjects (Supp. Tables S1 and S2), the mean age (±SD) at diagnosis of pituitary adenoma was 33.7 ± 15.5 years (23.6 ± 11.2 years in the AIP mutant families and 40.4 ± 14.5 years in the AIP negative families; P < 0.00001). If we recalculate the age of onset only for the successfully performed MLPA negative families this was 38.4 ± 13.8 years (not significantly different from the full cohort of AIP negative families). Families with FIPA show variable penetrance. We are fully aware that penetrance calculations are subject to bias due to symptomatic patient referral and due to incomplete genealogical data as well as variable ages of the subjects studied, but these calculations can still provide some useful information. Penetrance was calculated in our families with taking into account affected and obligate carrier subjects as well as half of the subjects with 50% risk to inherit the mutation [Ogino et al., 2007]. We have a mean ± SD penetrance of 42 ± 21% for AIP-positive families with a minimum of 8% and a maximum of 83%. The penetrance in the AIP negative families was not significantly lower at 38 ± 16%, minimum 19% and maximum 67%. However, we observed a difference in the reported number of affected patients between AIP mutation-positive and -negative families, suggesting a difference in penetrance: the mean number of affected subjects in AIP mutation-positive families was 3.2 ± 1.8, whereas in AIP mutation-negative families it was 2.2 ± 0.4 (P < 0.001). Interestingly, 16 of our 20 families with an AIP mutation had at least one member with gigantism and/or disease onset < 18 years, whereas only 3 of our 44 AIP-negative FIPA families had a member with gigantism and/or disease onset < 18 years. In the 20 AIP-positive families 7 of the 63 affected subjects (11%) were diagnosed above the age of 35 years, three out of these (5%) were above 50 years. In the 44 AIP-negative families 56 of 96 patients (58%) were diagnosed above the age of 35 years, 24 of these (25%) above the age of 50 years. Five of 63 AIP mutation patients (8%) presented with pituitary apoplexy; three of them were in childhood. We studied whether the type of AIP mutation (presence or absence of C-terminal end of the protein) would influence penetrance. We have 16 families with mutations resulting in no protein or truncated proteins having 3.43 ± 1.9 patients per family and four families with full-length proteins having 2.5 ± 0.58 patients per family (no significant difference), suggesting the lack of genotype–phenotype correlation in these 20 AIP-positive families in terms of penetrance. Out of the 64 families, 59 had at least one member with a prolactinoma or acromegaly, whereas five families had pure nonfunctioning adenomas. Out of the 63 AIP mutation patients only 6 had "pure" prolactinoma, and all of these were macroadenomas. The AIP mutation-positive patients who clinically presented with a nonfunctioning adenoma all had positive GH and/or PRL staining in the adenoma tissue, suggesting that all subjects with AIP mutations have GH or PRL-synthesizing adenomas in our cohort.
Two mutations (c.[−270_−269CG>AA; −220G > A] in cis) had been identified in a Japanese FIPA family with two sisters suffering from gigantism (Family VII; Supp. Table S2, Fig. 1A and B) [Leontiou et al., 2008]. These changes are located 160 and 111 bp upstream of the 5′UTR. The transcription factor binding-site searching programs suggested that the −270_−269CG > AA promoter mutation causes disruption of several transcription factor binding sites. To investigate whether the observed sequence changes affected the transcriptional activity of the AIP promoter, we cloned a 1.2-kb upstream fragment of the AIP gene into the pGL3-basic luciferase reporter vector (WT construct). To identify which of the mutations were functional, promoter constructs were prepared with a dibasic change (−270_−269AA), a single base change (−220A), and a construct with both changes (−270_−269AA and −220A) (Fig. 1C). These were transiently transfected into GH3 cells and the activity of the promoter was measured using a luciferase assay.
Transfection of the WT promoter and the single −220A promoter construct resulted in an 18-fold increase of promoter activity compared to empty vector pGL3-basic (Fig. 1D). Compared to the WT promoter construct the dibasic −270_−269AA promoter construct and the double −270_−269AA and −220A promoter construct had significantly decreased promoter activities (P < 0.001). These results suggest that the dibasic –270_−269AA promoter mutation, but not the single −220A variant, reduces promoter activity, which is likely to have a functional impact in the patients carrying this dibasic mutation, and suggests that this area is important for the regulation of the AIP promoter.
Additional support for the reduced function of this AIP promoter was generated with real-time PCR study of the two affected subjects' leukocyte cDNA (heterozygote for the promoter mutation) compared to three healthy controls from the same ethnic background. This showed reduced AIP expression (AIP/GAPDH mRNA ratio, mean ± SEM, 0.86 ± 0.2 vs. 0.58 ± 0.1, P = 0.05), supporting the reduced activity of the AIP promoter in these cells.
The cyclic AMP pathway is important for somatotroph pathogenesis as its upregulation via somatic gsp mutations (MIM 139320) or via PRKAR1A (MIM 188830) mutations lead to somatotropinomas [Horvath and Stratakis, 2008]. cAMP stimulates AIP binding partner AhR [Oesch-Bartlomowicz et al., 2005]. In addition, the extracellular signal-regulated kinase (ERK) pathway has recently been shown to be deregulated in pituitary adenomas [Dworakowska et al., 2009]. No previous data are available about the regulation of the AIP promoter. We therefore treated GH3 cells transfected with the 1.2-kb AIP promoter constructs with db-cAMP (a cAMP analog which activates PKA), forskolin (an adenylate cyclase activator that activates PKA), and PMA (a protein kinase C activator leading to activation of the ERK/MAPK pathway) and measured luciferase activity. Treatment with db-cAMP and forskolin increased WT and −220A promoter activity compared to vehicle (Fig. 1E). The effect of forskolin on the WT promoter was inhibited by the PKA inhibitor H89 (Fig. 1F). However, db-cAMP and forskolin treatment did not have any effect on the dibasic −270_−269AA mutated promoter or the double −270_−269AA and −220A mutated promoter. Treatment with PMA did not alter AIP promoter activity in any of the four promoter constructs. These results implicate the involvement of the cAMP–PKA signaling pathway in regulating AIP expression, and suggest that the –270_–269 region is required for cAMP-induced AIP promoter activity.
A novel mutation (c.249G > T) was identified in a family (Family 28; Supp. Table S1) with two childhood-onset pituitary adenomas (a somatotrophinoma and a prolactinoma) and an adult-onset prolactinoma (Fig. 2A). The mutation at the end of exon 2 did not change the amino acid sequence, but in silico analysis using ALAMUT predicted that this mutation created a novel 5′ splice-site 32 bases upstream from the normal splice site resulting in a frameshift and a stop-codon after 35 novel amino acids (Fig. 2B). To determine whether this mutation indeed causes alternative splicing, we extracted RNA from blood of one of the patients. RT-PCR with primers upstream and downstream of the mutation was performed and compared to cDNA from a control individual. RT-PCR of cDNA from the patient heterozygous for the mutation generated the expected WT product plus an additional band that corresponded to the size of the predicted alternative product (425 bp; Fig. 2C). Sequencing of this smaller product confirmed the identity of the alternatively spliced transcript. These results confirmed the in silico prediction that the nucleotide change c.249G > T causes alternative splicing leading to a frameshift and a truncated AIP protein (p.G83AfsX15).
The mutation c.807C > T, p. = at the beginning of exon 6 had been identified in a family with two somatotrophinomas (Family XVI; Supp. Table S2) [Leontiou et al., 2008]. In silico analysis (ESEfinder and RESCUE-ESE) suggested that the c.807C > T mutation might result in the loss of a binding site for splice enhancers SRp40 and SRp55 (splicing regulatory proteins that recognize exonic splicing enhancer sequences) in the final exon of AIP (Fig. 3A). We explored whether this mutation affected the splicing of the last AIP exon. cDNA obtained from affected or carrier individuals was amplified with different primer sets covering the various sections of the exon 5–3′UTR region and compared to the WT gene. PCR amplification with the different primer sets did not show any novel alternatively spliced transcripts, but instead, patients with the mutation showed decreased AIP mRNA expression compared to control individuals using both conventional (Fig. 3B) and real-time PCR (Fig. 3C). Thus, these data suggests that the c.807C > T mutation may have a functional impact by reducing AIP transcript levels.
To further investigate whether the c.807C > T mutation leads to decreased mRNA expression, we performed in vitro studies using a minigene construct. The minigene was constructed with an amplified DNA fragment from a patient carrying the mutation. AWT (807C) and a mutated (807T) minigene including the 3′ end of exon 5, the intron between exon 5 and 6, and the 5′-end of exon 6 were cloned into the pcDNA3.1(+) expression vector (Fig. 3D). The WT minigene and the mutated minigene were then transiently transfected into GH3 cells for 48 hr. To mimic the patient's heterozygous mutation, cells were cotransfected with equal amounts of the WT and mutated minigenes. Using conventional (Fig. 3E) and real-time PCR (Fig. 3F), we detected decreased transcriptional products of the minigene in the cells transfected with the mutated minigene (807T) and an intermediate amount with the cotransfection of WT and mutated minigenes compared to cells with the WT minigene construct. These data corroborate the in vivo data showing that the c.807C > T variant is associated with reduced levels of AIP mRNA, supporting the functional impact of the mutation.
In Family 10, two AIP changes were identified in two asymptomatic male carriers (70 and 65 years): the c.910C > T, p.R304X (a known pathogenic mutation) [Leontiou et al., 2008] and the c.896C > T, p.A299V (previously reported in a patient with sporadic acromegaly [Georgitsi et al., 2007], but in none of the general population subjects studied by us or others) variants. Other family members showed only the c.910C > T or the c.896C > T change. We cloned this region of the AIP gene from the gDNA of a subject carrying both mutations and sequenced several colonies. Some of the colonies carried the c.910C > T change while others the c.896C > T change. These data confirm that the two sequence changes reside on different chromosomes (in trans), and therefore the asymptomatic subjects are compound heterozygotes for these two AIP changes. Interestingly, one of the female carriers of the p.A299V variant, but not the p.R304X mutation, was diagnosed with a microprolactinoma at the age 30 years. Out of our seven "pure" prolactinoma patients found in our AIP mutation-positive families she is the only one with a microprolactinoma, and most of the others needed surgery and radiotherapy. We consider that her case might be a phenocopy, similar to the phenocopies reported in the Finnish Q14X mutation family [Vierimaa et al., 2006], however, as she carries the p.A299V AIP variant this is uncertain. We performed a functional assay to study the interaction of AIP and PDE > A5. The p.A299V variant of AIP did not show a profoundly reduced binding to PDE > A5 compared to WT controls (Fig. 4A). In summary, the clinical data on the compound heterozygote subjects support that the c.896C > T, p.A299V change might be a rare polymorphism and the in vitro data are compatible with this.
We decided to study the protein–protein interaction properties of all missense mutations reported in the literature. We have previously shown with a filter β-galactosidase assay that the R81X, R217X, and R304X nonsense mutations disrupt the binding between AIP and PDE > A5. Here we used a quantitative β-galactosidase assay to study all nine published missense mutations. Figure 4A shows that the K103R, C238Y, K241E, and R271W mutations completely disrupt the binding with values fivefold less than WT, similar to that of the novel Q164X nonsense mutation (P < 0.01 for each comparison). The V49M change showed no effect (mean ± SD, 97 ± 1% of WT), whereas the R16H, I257V, A299V, and R304Q variants showed a small drop in binding compared to WT (39 ± 8%, 35 ± 25%, 58 ± 13%, and 76 ± 17% of WT, respectively).
We used a combination of clinical, experimental, and in silico methods to analyze how AIP mutations would change the AIP protein structure and function. Out of the known 49 AIP variants 35 result in a truncated or no protein (12 nonsense mutations, 12 frameshifts, six splice site, four large deletions, and one promoter mutation) [Tahir et al., 2010]. Previous studies convincingly showed that loss of the C-terminal end of the protein results in loss of function [Leontiou et al., 2008; Petrulis and Perdew, 2002]. The in-frame insertion mutation of exon 6 has been shown to disrupt the antiproliferative effect of AIP [Leontiou et al., 2008]. No functional data are available for the four in-frame deletions. The nine missense changes result in full-length proteins (R16H, V49M, K103R, C238Y, K241E, I257V, R271W, A299V, and R304Q). The location of these missense mutations are shown on a model of AIP, which was based on the structure of FKBP51 (Fig. 4B).
R16H has been identified in control subjects as well as sporadic and familial patients, and no LOH was shown in a tumor sample [Buchbinder et al., 2008; Cazabat et al., 2007; Daly et al., 2007; Georgitsi et al., 2007; Yaneva et al., 2008]. A reduction in PDE > A5 binding was observed, but this was less than threefold difference from WT. It is important to note that the PDE > A5-AIP interaction is at the C-terminal end of the molecule [Bolger et al., 2003]. Although the R16 amino acid is conserved in vertebrates, the R16H change probably represents a rare polymorphism as previously suggested [Raitila et al., 2007].
The V49M change was found in a sporadic childhood-onset somatotroph adenoma case, but no LOH was found in the tumor sample [Iwata et al., 2007]. The PDE4A5 binding data was similar to WT, as expected, as PDE4A5 binds the C-terminal of the AIP molecule while this variant affects the N-terminal of the protein. This change is conserved in most vertebrates, but not in lower species. Although no further data are available, the clinical significance of this variant is uncertain.
The K103R variant was identified in a patient with a childhood-onset corticotroph adenoma [Beckers et al., 2008]. Although the K > R is conservative substitution, the K103 residue is a conserved amino acid in most species and the PDE binding assay suggests a profoundly reduced activity (8 ± 5% of WT), supporting the possibility that this mutation has a functional impact.
The C238Y mutation was found in a Mexican family with three acromegalic brothers [Leontiou et al., 2008]. It disrupts a consensus TPR motif amino acid. To understand the relevance of this amino acid change we need to examine the structure of the TPR domains. AIP has three TPR motifs (Fig. 4B and C). Each TPR motif is composed of a pair of antiparallel helices, termed helices A and B. The consecutive helices of TPR domains are packaged together so each helix shares two immediate helix neighbors [Das et al., 1998]. A typical TPR consensus sequence pattern of small and large hydrophobic residues has been defined [D'Andrea and Regan, 2003; Sikorski et al., 1990]. Consensus amino acids (TPR residue positions marked as numbered diamonds on Fig. 4C) are located at position 4, 7, 8, and 11 in helix A and position 20, 24, 27, and 32 in helix B [D'Andrea and Regan, 2003; Sikorski et al., 1990]. Small hydrophobic residues are commonly observed at positions 8, 20, and 27 within the TPR motif. Residues 8 and 20 are located at the position of closest contact between the A and B helices of a TPR, whereas residue 27 on helix B is located at the interface of three helices (A, B, and the A helix of the next TPR motif) within a three-helix bundle [Das et al., 1998]. The C238Y mutation is at position 8 of the A helix of the second TPR domain of AIP and its mutation could destabilize the A and B helix packaging of the second TPR domain (Fig. 4C) [D'Andrea and Regan, 2003; Hidalgo-de-Quintana et al., 2008]. Both the cell proliferation [Leontiou et al., 2008] and the PDE binding assays (Fig. 4A) show that this mutant loses its activity in these two different types of functional assays. Therefore, all the data strongly suggest that this mutation has a functional impact.
The K241E mutation was identified in a FIPA family [Daly et al., 2007]. Interestingly, the same amino acid can also be affected by a stop mutation [Beckers et al., 2008]. This large conserved residue is part of the TPR sequence consensus at position 11 (Fig. 4C), and is suggested to be required for the packing of adjacent TPR α-helices [D'Andrea and Regan, 2003; Hidalgo-de-Quintana et al., 2008]. Our experimental data on protein interaction show a reduced ability to interact with PDE4A5 supporting the possible functional impact of the variant.
The I257V change affects a conserved small residue in most vertebrates in the B helix of the second TPR domain at position 27, which is thought to be important in the packaging of the adjacent helices [Das et al., 1998], in this case, A and B helices of TPR2 and A helix of TPR3 (Fig. 4C). It was found in a sporadic patient with unusual cell type (TSHoma) [Montanana et al., 2009]. A reduction in PDE4A5 binding was observed, but this was less than threefold difference from WT. LOH data are not available; therefore, the possibility that it has a functional impact cannot be excluded.
The R271W is the second most common mutational hotspot in the AIP gene. This mutation has been identified in three independent families [Daly et al., 2007; Jennings et al., 2009] as well as in a sporadic giant [Igreja et al., 2010]. It disrupts a conserved amino acid in the third TPR; it corresponds to an arginine in several other TPR proteins at the equivalent location such as in protein phosphatase 5 (PP5A), AIP-L1, FKBP51, and FKBP52 proteins (Fig. 4C). Interestingly, this amino acid was selected for mutation to alanine experimentally even before human mutations were identified in this gene [Bolger et al., 2003; Laenger et al., 2009]. Both the human mutation R > W (Fig. 4A) and the experimental mutation of R > A [Bolger et al., 2003] completely disrupts the AIP–PDE binding. These experimental and clinical data strongly support the pathogenic role of this mutation.
The A299V change was described in a sporadic acromegaly patient [Georgitsi et al., 2007] and in the current manuscript (see above). This variant, which is conserved in most vertebrates, is located at the beginning of the α-7 helix in the area that is relevant for PDE > A5 interaction but binding in vitro was less than threefold difference from WT. We identified this change in four subjects of a family where the R304X mutation was also detected. Two unaffected patients carried both changes; one unaffected patient carried only the A299V change while one young female carried only the A299V change and was diagnosed with a microprolactinoma at the age of 30 years. As AIP knockout mice are not viable [Lin et al., 2007], and the subjects with the compound heterozygote genotype with the certainly pathogenic R304X mutation are unaffected, this variant is unlikely to have a functional impact, although the patient with a possible phenocopy (small prolactinoma in a young female is not typical of AIP mutation patients) carrying this change makes the status of this variant uncertain.
The R304Q locus is part of a CpG island mutational hotspot. This amino acid change is relatively conservative changing a longer side chain positively charged amino acid to a slightly shorter, uncharged, hydrophilic residue in the α-7 helix. The amino acid is conserved in mammals, but not in lower species. Our protein interaction data show a less than threefold difference in PDE4A5 binding. The pathogenic role of R304Q, however, is overwhelmingly supported by the fact that it has been identified in several independent FIPA families as well as in sporadic patients [Cazabat et al., 2007; Georgitsi et al., 2007; Leontiou et al., 2008; Vargiolu et al., 2009] (and the current study).
In summary, our two-hybrid analysis of the missense mutations showed that they fell into two groups: mutations that were generally considered to have a functional impact had β-galactosidase activity values more than fivefold different from WT, whereas the mutants not generally considered to have a functional impact had β-galactosidase values less than threefold different from WT. Of note, none of the b-galactosidase values for the missense mutants fell between these two values, providing a clear distinction between functionally significant and functionally nonsignificant mutants in this PDE4A5-related assay.
We have analyzed clinical data and performed genetic and functional studies to investigate the possible functional impact of some of the reported AIP sequence changes. The main findings of this study are: (1) all our AIP-positive families have at least one member with GH/PRL-staining adenomas, have significantly higher number of subjects affected suggesting higher penetrance than in AIP-negative FIPA families, and have at least one childhood-onset case in the majority of the families. (2) Six novel AIP germline and five previously described mutations were identified using sequencing and MLPA, and our data support the suggestion that in addition to exon and exon–intron junction sequencing, study of the promoter area, and a technique suitable for the detection of large deletions, should be part of the DNA analysis of patients with FIPA. (3) Our data suggest a functional impact for the dibasic promoter mutation we have identified, as it causes decreased promoter activity compared to the WT promoter. In addition, we reveal that the cAMP–PKA signaling pathway positively regulates the activity of the AIP promoter. (4) The exonic synonymous mutations studied were shown to have a functional impact due to abnormal splicing or decreased AIP mRNA expression. (5) We combined clinical, in silico prediction and protein–protein binding assay data to characterize all the missense AIP changes identified in pituitary adenoma patients to help define those that may represent rare single nucleotide polymorphisms, and which may have a role in the disease process.
Previously described mutations affecting exon–intron junctions were hypothesized to cause abnormal AIP protein based on in silico predictions. Here we show actual data from patient cDNA samples regarding the consequences of the c.249G > T mutation, confirming the possible functional impact of this change. Studying another silent mutation (c.807C > T), we predicted the abnormal splicing of exon 6 due to the disrupted binding of the splicing regulatory proteins. As this abnormality affects the last exon, studying its role is not as straight forward as other splicing mutations at earlier exons. Although our search for alternative splice products using various sets of oligonucleotides did not reveal an alternative splice product, a minigene approach confirmed the lack of a normal exon 5–6 product and patient cDNA samples showed reduced AIP mRNA expression. We hypothesize that this mutation may either cause destabilization of the mRNA leading to reduced expression levels, or alternative splicing to a new isoform not detected by our RT-PCR assays.
AIP promoter regulation has not previously been studied. We suggest that the c. −270_−269 area is part of the basic promoter, as its disruption significantly reduces basal promoter activity in vitro. Based on prediction programs, the mutation at c.−270_−269 position would disrupt several transcription factor binding sites, including the zinc-finger protein SP1. SP1 can be activated by the cAMP–PKA pathway [Rohlff et al., 1997], which is important in proliferation and hormone release of somatotroph cells: gain-of-function mutations in the α-subunit of the G-protein (GNAS) and loss-of-function mutations of the regulatory subunit of PKA (PRKAR1A) lead to deregulation of the cAMP–PKA pathway and play a role in somatotroph tumorigenesis. Here we show that the activity of the AIP promoter can be upregulated by the stimulation of this pathway and the effect is inhibited by a PKA inhibitor. This first appears to be a paradox, as sporadic somatotroph adenomas show an overactive PKA pathway [Bertherat et al., 1995]. However, our data are compatible with increased AIP mRNA and protein expression described previously [Jaffrain-Rea et al., 2009; Leontiou et al., 2008] and corresponds to the fact that low AIP levels do not play a role in sporadic somatotroph tumorigenesis when AIP mutations are not present [Leontiou et al., 2008].
PDEs can also regulate the PKA pathway, and PDE4A5 and PDE2A are known partners of AIP [Bolger et al., 2003; de Oliveira et al., 2007]. The AIP–PDE4A5 interaction was shown to be lost in the presence of AIP mutations [Leontiou et al., 2008]. Here we further studied all the described missense AIP changes as well as our novel nonsense mutation with a quantitative assay. We found that a stop mutation and four missense mutations (K103R, C238Y, K241E, and R271W) completely disrupted the PDE4A5 binding. Three of these affected amino acids are known to be important for TPR structure [Das et al., 1998], and two have been experimentally shown to disrupt the function of the AIP protein [Bolger et al., 2003; Laenger et al., 2009; Leontiou et al., 2008]. Clinical data suggest, although do not prove, that the R16H (found in normals as well), V49M (lack of LOH in tumor sample), and the A229V (also identified in unaffected subjects with compound heterozygosity for the pathogenic R304X mutation) changes might not have a functional impact, and this is supported by the weak or no effect on PDE4A5 binding. The I257V change affects a conserved amino acid at a location known to be important for the α-helix packaging of the TPR domains, and despite the weak effect on PDE4A5 binding a functional impact is possible. The pathogenic role of the R304Q variant is beyond doubt. The lack of profound reduction in PDE4A5 binding suggests that this part of the molecule may not be important in this PDE4A5–AIP interaction. On the other hand, missense variants can often act by altering the stability or folding the protein, rather than affecting amino acids directly involved in a protein–protein interaction. Clearly, the lack of profoundly reduced binding to PDE4A5 does not rule out other types of dysfunction of these variant AIP molecules, and further studies are needed to clarify whether the reduced or lack of AIP–PDE4A5 interaction as measured in this in vitro assay is important for the pituitary pathogenic process.
We identified two asymptomatic carrier subjects with two changes in the AIP gene—p.R304X and p.A299V—and showed that these variants are located on opposite parental chromosomes. It has previously been demonstrated that the p.R304X hotspot mutation disrupts the function of the AIP molecule. The p.A299V change (first described in a sporadic patient with acromegaly) [Georgitsi et al., 2007] involves an amino acid substitution of alanine to valine. This residue is not part of the TPR-domain consensus. The PDE4A5 binding data did not support a profound loss of function due to this missense mutation. Also, if this change has functional impact, our subjects (65 and 70 years, both asymptomatic) would be compound-heterozygotes for AIP mutations, whereas AIP knockout mice are not viable (they die at embryonic age e10.5–e14.5 due to cardiac malformations) [Lin et al., 2007], thus suggesting this change has no functional impact. We, however, identified a p.A299V female carrier with a microprolactinoma. Combining all these data with the fact that her phenotype is not typical of AIP mutation positive patients, her case may represent a phenocopy. The A299V change is a variant with unknow significance, and further studies are needed to confirm that it has no functional impact.
More than two-thirds of FIPA families do not have a detectable mutation in the AIP gene, and show differences in age of onset and level of penetrance as well as heterogeneity of tumor types compared to AIP positive patients, suggesting that there is a phenotypic difference between FIPA families with AIP mutations and FIPA families where the disease is caused by another gene or genes. Currently, work is being carried out to identify these new disease loci.
In summary, 31% of our FIPA families harbor AIP mutations. They show a predilection for GH/PRL-synthesizing adenomas, younger age of onset, and higher penetrance of disease compared to AIP-negative FIPA families. The AIP promoter is positively regulated by the cAMP–PKA pathway, and functional characterization of AIP mutations reveal that our promoter mutation decreases promoter activity, whereas some synonymous changes result in abnormal splicing or reduced AIP expression. Searching for large deletions using MLPA and analysis of clinical data and functional characterization of rare AIP changes helps to clarify the pathogenic role of AIP in patients with familial and sporadic pituitary adenomas, and assists in the appropriate genetic counseling and clinical management of these patients and their families.
We are very grateful for the patients and family members who provided samples. We thank Profs. David Barford and Carol Shoulders, as well as Drs. Chrysanthia Leontiou and Lou Metherell, for the helpful comments. HSC was supported by an MRC Training Fellowship. Projects in Prof Korbonits laboratory are supported by the Cancer Research Committee of St. Bartholomew's Hospital, by the Wellcome Trust and by the NIHR. Studies in Dr. Bolger's laboratory are supported by the Bolger Prostate Cancer Research Fund.
Akker, Scott, London, UK; Atkinson, Brew, Belfast, Northern Ireland; Aylwin, Simon, London, UK; Baldeweg, Stephanie, London, UK; Bevan, John, Aberdeen, UK; Cheetham, Tim, Newcastle, UK; Chew, Shern, London, UK; Choudry, Kiran, Texas, USA; Clayton, Richard, Stoke-On-Trent, UK; Damjanovic, Svetozar, Belgrade, Serbia; Darzy, Ken, Milton Keynes, UK; Dattani, Mehul, London, UK; Davis, Julian, Manchester, UK; Drake, Will, London, UK; Dzeranova, Larisa, Moscow, Russia; Edén, Britt, Engström, Sweden; Eguchi, Kuniki, Hiroshima, Japan; Fica, Simona, Bucharest, Romania; Flanagan, Daniel, Plymouth, UK; Frohman, Lawrence, Chicago, Illinois, USA; Gadelha, Monica, Rio de Janeiro, Brazil; Gallego, Patricia, Brisbaine, Australia; Gláz, Edit, Budapest, Hungary; Goldman, James, Boston, Massachustts, USA; Goldstone, Tony, London, UK; Howlett, Trevor, Leicester, UK; Inder, Warrick, Melbourne, Australia; Iwata, Takeo, Tokushima, Japan; Kaplan, Felicity, Stevenage, UK; Karavitaki, Niki, Oxford, UK; Laws, Ed, Boston, Massachusetts, USA; Lechan, Ron, Boston, Massachusetts, USA; Levy, Miles, Leicester, UK; Matsuno, Akira, Chiba, Japan; Miljic, Dragana, Belgrade, Serbia; Modenesi, Silvia, Belo Horizonte, Brazil; Molitch, Mark, Chicago, Illinois, USA; Musat, Madâlina, Bucharest, Romania; Orme, Steve, Leeds, UK; Patócs, Attila, Budapest, Hungary; Popovic, Vera, Belgrade, Serbia; Powell, Michael, London, UK; Quinton, Richard, Newcastle, UK; Randeva, Harpal, Warwick, UK; Ribeiro de Oliveira JR, Antônio, Belo Horizonte, Brazil; Schöfl, Christof, Erlangen, Germany; Soares, Beatriz, Belo Horizonte, Brazil; Spada, Anna, Milan, Italy; Strasburger, Christian, Berlin, Germany; Swords, Francesca, Norwich, UK; Tsagarakis, Stylianos, Athens, Greece; Vaks, Vladimir, Oxford, UK; Wass, John A.H., Oxford, UK; Widell, Hakan, Gotheborg, Sweden; Yarman, Sema, Istanbul, Turkey; Yoshimoto, Katsuhiko, Tokushima, Japan.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's Canada Institute for Scientific and Technical Information in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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White Cane Days raises money for eyesight preservation
- May 3, 2013 at 12:21PM
Members of the Poulsbo Noon Lions and Evening Pride Branch Lions clubs take to North Kitsap streets on Friday and Saturday to raise funds as part of the annual White Cane Days.
Sheriff's Log | April 25-29
- May 3, 2013 at 12:31PM
The Kitsap County Sheriff's Office responded to the following calls in North Kitsap April 25-29.
Donors share 200 joys with others | Neighbors Helping Neighbors
- May 3, 2013 at 12:00PM
It takes a certain kind of generous spirit to see within every act an opportunity to serve and help. Such was the case for a local couple who recently helped North Kitsap Fishline Food Bank in a most unusual way.
S'Klallam history presented Saturday
- May 3, 2013 at 12:30PM
The Poulsbo Historical Society hosts "A Historical Overview of The Port Gamble S'Klallam Tribe," Saturday, 7-8:30 p.m., in the Poulsbo City Hall Council Chambers.
Pace crowned Miss Viking Fest, Norton Little Miss
- May 3, 2013 at 12:26PM
Allison Pace was crowned Miss Viking Fest and Luci Norton was crowned Little Miss Viking Fest in the pageant April 27 at North Kitsap Auditorium.
Police Calls | Poulsbo PD | April 24-30
- May 3, 2013 at 12:32PM
The Poulsbo Police Department responded to the following calls April 24-30.
KHS fundraiser tees off May 10
- May 3, 2013 at 2:00PM
Kitsap Humane Society's upcoming golf tourney, "Fore the Animals," at Trophy Lake Golf & Casting in Port Orchard is Friday, May 10.
Lions, NKFFA host fishing derby
- May 3, 2013 at 2:00PM
The Poulsbo Noon Lions Club and North Kitsap Future Farmers of America will host Hooked on Fish Derby May 11.
Simpson competes in Wounded Warrior Games
- May 3, 2013 at 3:00PM
Four sailors based in the Bremerton area will compete on behalf of Team Navy at the fourth annual Wounded Warrior Games in Colorado Springs, Colo. May 11–16.
Local author Kristin Hannah at Sons of Norway
- May 4, 2013 at 9:00AM
Liberty Bay Books welcomes Kristin Hannah, who resides on Bainbridge Island, on her book tour for her latest novel, "Fly Away" May 7.
2013 Achievement Award for Gordon, Pearson, KHS
- May 4, 2013 at 10:00AM
Three schools in the North Kitsap School District were recognized Tuesday for earning the 2013 Washington Achievement Award.
There will be Vikings — and pancakes
- May 4, 2013 at 10:00AM
The Poulsbo Noon Lions Club will host its 42nd annual Viking Fest Lions Pancake Breakfast on May 18-19, in the North Kitsap High School cafeteria.
Michael Lord Nelson | Passages
- May 4, 2013 at 11:00AM
Michael Lord Nelson of Poulsbo passed away on April 25. He was 63.
Elmer Martens | Passages
- May 4, 2013 at 11:00AM
Elmer Arnold Martens, last surviving child of Petersburg, Alaska, pioneers Loui and Ragnhild Martens, died April 14 of natural causes with his family at his side.
Jan and Jerry Deardorff celebrate their 50th
- May 4, 2013 at 11:00AM
Jerry and Jan Deardorff of Poulsbo celebrated their 50th anniversary at the Edgewater Inn in Seattle, where they spent their wedding night on April 12, 1963.
Lions Park tennis courts look just like new
- May 4, 2013 at 2:00PM
Many thanks to the City of Poulsbo. The prompt response to citizens' request for maintenance at one of our city's beloved parks is truly appreciated.
Give SoundWorks a new home
- May 4, 2013 at 2:00PM
My family has been in Poulsbo since 1981. In all those years, the most delightful entertainment has been the unexpected mushrooming of "Bill Austin for mayor" signs last weekend.
Past time to end corruption 'tax'
- May 4, 2013 at 2:00PM
I just received my check from the mortgage fraud settlement agreement.
Coffee Oasis employee is a winner
- May 4, 2013 at 2:00PM
The North Kitsap Herald recently published a letter written by Ron Hirschi regarding the emotional devastation caused by a thief who stole his wife's purse in the few seconds she turned to help another person
Maintain funding for arts, music education
- May 4, 2013 at 2:00PM
Dear North Kitsap School District, Please include the community in your 2013-14 budget planning process.
2,000 is still greater than 33
- May 4, 2013 at 2:00PM
Last week, we saw two stark reactions to the rise of the alternative media.
A great example of sustainability
- May 4, 2013 at 2:00PM
On March 18, the Kitsap County Board of Commissioners held a community meeting to collect feedback from citizens on Olympic Property Group's preferred design for Port Gamble's master-planned development.
DNR, Kitsap Parks and Rec remove creosote debris
- May 5, 2013 at 9:00AM
The Washington State Department of Natural Resources and the Kitsap County Parks and Recreation Department are removing toxic creosote-treated debris from county park beaches through May 3.
Poulsbo a 'Tree City' for 16th consecutive year
- May 5, 2013 at 9:00AM
Poulsbo was named a "Tree City USA" for the 16th consecutive year, on Arbor Day.
Second location for Lucky Star Consignment
- May 5, 2013 at 12:00PM
Lucky Star Consignment of Kingston has opened a second store — at 3276 NW Plaza Road, No. 104, Silverdale
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Read the Jul 3
Green Edition
Browse the print edition page by page, including stories and ads.
Browse the archives.


text:
Jan Scruggs, the Vietnam veteran who founded the Vietnam Veterans Memorial Fund to raise money for the memorial, reads a letter left at the base of the much-visited wall on Nov. 5, 2002.
- Filed Under
GREENVILLE, Ind. -- Hours slipped by as they sipped coffee and talked about their brother. Virgil and Steve Deckard sometimes smiled, occasionally frowned and often paused to wipe their eyes as they spoke about Vietnam and the incident that took their sibling's ability to walk before eventually ending his life.
David "Larry" Deckard will be one of 11 veterans honored by having their names inscribed at the Vietnam Veterans Memorial in Washington on Memorial Day. Larry Deckard was driving a personnel carrier that was struck by a rocket March 24, 1969.
"He was the only one that survived, but he ...


text: South Carolina Knocks off No. 17 Kentucky, Fans Storm Court in Celebration From Collegespun.com Mar 1, 2014 Share Tweet Next ARTICLE » Use your ← → (arrow) keys to browse more stories Lance King/Getty Images 66 Reads 0 Comments South Carolina is going to owe the SEC $25,000. Gamecocks fans and students probably don't care. Continue Reading on Collegespun.com »


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Katherine Williams would have never imagined the consequences of purchasing a ring on her birthday. Will an average college girl be able to handle the whispers leading to all the bad and evil Or are they misunderstood for guidance? Is it just about the whispers. If so then what does the Clown wants? Will she be able to choose between the love of her life or her life itself?
I have a few great stories and you can read them I havnt had comments and need inspiration
Stupid tablet, it does this to me all the time. Down meant down no meant or. Sorry
The story was great and I was wondering if you like stories that are finished NO a story with 6 chapters, what kind of stories down u like?
ayeI786 3 years ago
Awesome. I love it sooo far. Keep writing. Finally I made and account to read just your book. I loved it so far and am gonna read it further more.
shsa0009 3 years ago
I like it! I can totally relate to the lead. the lazy kath is just me. Her friend rose is cute too. even i have a cat. her name is moon. :)
plainjane20 3 years ago
That's a nice plot shag! your story has a good beginning but it is getting better with time... keep it up ;) And yea! this kath is soo much like you,almost in every way .omg i was picturing you in almost every scene....nina dobrev almost killed my constructive imagination........


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The Binding Chair: Or, a Visit from the Foot Emancipation Society (Paperback)
Email or call for price
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In poised and elegant prose, Kathryn Harrison weaves a stunning story of women, travel, and flight; of love, revenge, and fear; of the search for home and the need to escape it. Set in alluring Shanghai at the turn of the century, The Binding Chair intertwines the destinies of a Chinese woman determined to forget her past and a Western girl focused on the promises of the future.
About the Author
Kathryn Harrison is a graduate of Stanford University and the Iowa Writer's Workshop. She is the author of the novels The Binding Chair, Thicker Than Water, Exposure, and Poison. She has also written a memoir, The Kiss. Her personal essays have appeared in The New Yorker, Harper's Magazine, and other publications. She lives in New York with her husband, the novelist Colin Harrison, and their children.


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Are you Shirley Whittom?Claim your profile
Publications (4)16.32 Total impact
- SourceAvailable from: Yves Dauvilliers[Show abstract] [Hide abstract]
ABSTRACT: To test the hypothesis of autonomic nervous system dysfunction in patients with narcolepsy-cataplexy (NC) by assessing the physiologic activations associated with periodic limb movements during sleep (PLMS). Sleep and heart rate (HR) were recorded during 1 night of polysomnography. Data were collected at the Sleep Disorders Center, Sacre-Coeur Hospital, Montreal, Canada. Data from 14 patients with NC (6 men, 8 women, mean age: 52.5 ± 11.9 years) were compared with data from 14 healthy control subjects matched for age and sex. NA. Analyses included sleep stages, PLMS, microarousals, RR intervals converted into beats per minute on segments lasting 25 heartbeats (10 RR intervals before PLMS and 15 after), and cardiac-activation amplitudes. A Group-by-Heartbeat interaction was noted for PLMS without microarousals; the patients had a tachycardia of lower amplitude and a delayed and lower-amplitude bradycardia, compared with normal control subjects. Similar significant HR modifications were observed for PLMS with microarousals between patients with NC and control subjects. Patients with NC had a reduced magnitude of cardiac activation associated with PLMS with and without microarousals, as compared with control subjects. A negative correlation was noted between cardiac-activation amplitude and age in patients with NC, but no correlation with PLMS index was found in either patients with NC or control subjects. A significant reduction in the amplitude of PLMS-related HR responses in both tachycardia and bradycardia was found in patients with NC. These findings favor the physiologic relevance of the action of hypocretin on autonomic function that may be of clinical significance, i.e., increasing the risk of cardiovascular diseases.Sleep 01/2011; 34(2):219-23. · 5.06 Impact Factor
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ABSTRACT: A close temporal relationship was shown between the onset of melatonin secretion at night and the worsening of restless legs syndrome (RLS) symptoms, suggesting that melatonin may play a role in the genesis of this phenomenon. To test this hypothesis we studied the effects of the administration of exogenous melatonin and, conversely, the suppression of endogenous melatonin secretion by bright light exposure on the severity of RLS symptoms. Eight RLS subjects were studied in three conditions: at baseline, after administration of melatonin and during bright light exposure. The severity of RLS symptoms was assessed by the suggested immobilization test (SIT), which allows quantification of both sensory and motor manifestations (SIT-PLM) of RLS. Analyses showed a significant increase of SIT-PLM index when subjects received exogenous melatonin compared to both baseline and bright light conditions, but bright light exposure had no effect on leg movements compared to the baseline condition. Analyses also revealed a small but significant decrease in sensory symptoms with bright light exposure compared to baseline. Exogenous melatonin may have a detrimental effect on motor symptoms, and bright light exposure produced small but significant improvement of leg discomfort. The study shows the interest of using the SIT to measure outcome of intervention in RLS. Further studies will be needed to assess the therapeutic value of bright light in RLS.Sleep Medicine 03/2010; 11(4):351-5. DOI:10.1016/j.sleep.2009.12.008 · 3.10 Impact Factor
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ABSTRACT: To determine the distribution of age-at-onset in a large cohort of patients with restless legs syndrome (RLS) and to compare clinical and polysomnographic characteristics of patients with early and late age-at-onset of RLS. Two hundred and fifty patients with RLS were studied. Information on age-at-onset, etiology, familial history and symptoms severity of RLS was obtained. Age-at-onset density functions were determined from bootstrap methods and kernel density estimators. Age-at-onset showed a significant bimodal distribution with a large peak occurring at 20 years of age and a smaller peak in the mid-40s. Early- and late-onset RLS could be separated with a cut-off at 36 years of age. Distributions of age-at-onset differed as a function of presence/absence of a familial history and etiology of RLS. Age-at-onset clearly differentiated patients with a primary RLS (early onset) from those with secondary RLS. Finally, early-onset RLS was associated with increased RLS severity with higher indices of periodic leg movements in sleep (PLMS) associated with microarousals and periodic leg movements during wakefulness (PLMW). Early- and late-onset RLS could be distinguished depending on familial history and etiology of RLS. Our data suggest that different pathological processes are involved in these two groups, the early-onset group being highly genetically determined.Sleep Medicine 01/2008; 9(1):54-9. DOI:10.1016/j.sleep.2007.01.017 · 3.10 Impact Factor
Article: PLMS and PLMW in healthy subjects as a function of age: prevalence and interval distribution.[Show abstract] [Hide abstract]
ABSTRACT: Several studies have demonstrated a positive correlation between periodic leg movements during sleep (PLMS) and age in healthy subjects. However, little is known about periodic leg movements during wakefulness (PLMW) in this population. Although the definitions of PLMS and PLMW specify a typical intermovement interval of 20 to 40 seconds, scoring criteria allow an intermovement interval of 4 to 90 seconds. The aim of the present study was to look at the prevalence and interval distribution of PLMS and PLMW in relationship with age in a population of healthy subjects. Periodic leg movements were recorded during 1 night. Sleep laboratory, Hôpital du Sacré-Coeur de Montréal. Sixty-seven healthy subjects aged between 5 and 76 years (32 F, 35 M). N/A. The presence of PLMS was rare before the age of 40, but then the index increased dramatically. PLMW index was higher in younger subjects compared with middle-aged subjects. Interval histograms of PLMS did not revealed a clear peak in younger subjects. With advancing age, PLMS interval histograms show a peak around 15 to 35 seconds, which is not observed in younger subjects. On the other hand, despite high indexes, PLMW interval histograms do not show a clear peak for any age group. These results illustrate that interval evaluation is an important feature of the calculation of periodic movements to discriminate spontaneous motor activity from PLMS or PLMW.Sleep 10/2006; 29(9):1183-7. · 5.06 Impact Factor
Top Journals
- Sleep (1)
- Sleep Medicine (1)
- Sleep (1)
- Sleep Medicine (1)
Hôpital du Sacré-Coeur de Montréal
Montréal, Quebec, Canada
- Center for Advanced Research in Sleep Medicine
Université de Montréal
Montréal, Quebec, Canada
- Department of Psychology


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VpsAdmin Manages OpenVZ Instances
The developers of a Czech hosting company has released a Web interface for administering virtual servers under OpenVZ.
The vpsAdmin tool in version 1.0 is written in PHP 5.2 and uses a MySQL database. The Web interface shows statistics and allows central administration of countless OpenVZ instances on many physical computers. It allows installing, starting, and stopping virtual servers, migrating servers, and configuring their network interfaces.
The software, originally developed for the vpsFree.cz hosting service, is under GPLv3 licensing and currently only supports Debian Lenny officially. Further details and a tarball are available on the vpsAdmin homepage.
Issue 177/2015
Buy this issue as a PDF
RIP SSLv3
The Internet community officially banishes the notoriously unsafe Secure Sockets Layer protocol.
Mate 1.1 Arrives
Popular desktop environment continues the Gnome 2 legacy – with new support for the Gnome 3 toolkit.
US Government Requires HTTPS
The Obama White House has issued a memorandum telling all US government agencies they must use HTTPS for all websites and web communication.
Mozilla Signs Firefox Add-Ons
New program will dial up security for the Firefox browser.
Fedora 22 Arrives
Red Hat's community distro embraces the cloud.
Code.org and College Board Team Reach Out for Talented High School Coders
New partnership will bring more and better CS training to US schools
A Peek Inside TeslaCrypt Ransomware
Criminals offer online help over Tor network
Mumblehard Still Spams
Sophisticated malware is still present on Joomla and WordPress sites around the world.
Launchpad Gets Git
Future versions of Ubuntu's code service will support the popular Git version control system used with Linux and other open source projects.
AMD Posts Code for Linux GPU Driver
New release marks the arrival of AMD's unified driver strategy.


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"There were multiple color options for customization, it featured a unique design and the invitations were delivered quickly."
"Love this invitation! I was looking for a princess themed product for my daughter's third birthday party. Beautiful thick paper. I liked that this one had a good bit of room for my text. I bought the extra scalloped edges and love how it turned out. I have received several complements. Quick shipping."
"This invite is even better in person! I usually use photo invitations, but this time I put two photos on the back. It's perfect! Love the soft pink color."
"As always, prompt service, beautiful cards and a happy customer. This one is particular is super cute and am so happy with the quality. Thanks Tiny Prints!"
"This card is perfect for pre teen or really any age "girly girl" at heart. The colors are so vibrant. I chose a pearl top finish that gives a hint of shimmer. We also ordered the silver envelopes and matching address labels. They are awesome about letting you customize all these little details so it reflects your taste and is exactly what you want! My daughter loves these and was so exited to send them to her friends."


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1957 Chevy tail fins... No automotive detail more thoroughly captures 1950s style and the spirit of Americana. The sight of these tail... More
1954 Chevrolet Bel Air 2 Door Sedan Chevrolet introduced the Bel Air line in 1950 setting an automotive design standard that would last for decades. Although... More
This frame off restoration was done about 8 years ago and it has been driven about 4,200 miles since. The restoration was done under the... More
Just arrived, beautiful four door hardtop with upgraded drivetrain and vintage air conditioning. Great car for shows or drive anywhere... Excellent dual stage repaint in White over... More
Complete Frame off RestorationOnly 300 Miles Since RestorationPower Top/Steering/Brakes/Dual ExhaustFender Skirts/Spare Tire/Jack and ToolsOwned by a longtime customer, the 55 Chevy has been part... More
WORLDWIDE VINTAGE AUTOS CALL OUR SALES TEAM AT 720 457 9145 MONDAY-FRIDAY 10AM-6PM SATURDAY 10AM-4PM AS ONE... More
V-8 ENGINE, WIDE WHITE-WALL TIRES... More
1956 Chevrolet Bel Air hardtop, 396, 4 speed, straight body, no rust, extensive recent professional upgrades inc.new wiring, steering column, power steering, power disc... More
1956 Chevy named ~ The Ghost ~ car originally from West Virginia, owned since 1999 , Jasper 350 cu. in, Edelbrock manifold, and 750... More
This 1955 Chevrolet Bel Air Convertible has a 265 C.I. V8 (Date Code Correct), Cast Iron Power Glide Transmission, P55 Rear End w/ 3.55:1... More
Cars like this 1957 Chevrolet Bel Air convertible remain popular year after year, and it's easy to understand why. They're handsome, comfortable, reliable cruisers... More
FRAME-OFF AND PERFECT This fully restored (frame off) Chevrolet Bel Air convertible is ready to go!! It is painted in the beautiful Tropical Turquoise and... More
For sale at Gateway Classic Cars is a 1955 Chevrolet Bel Air 2 door hardtop. This is 55 is painted in a classic... More
1956 Chevrolet Bel-Air 4 door sedan in orginal condition. Features a V8 with power pack, automatic transmission, power steering, power brakes, windshield washer, gas... More
June 20th we will be auctioning off a great selection of classic cars! It will be located at 3056 E Baseline Rd. in Evansville,... More


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Ranging from 16-24 credits, all our certificates may be completed in less than a year, 100% online.
Note: Federal financial aid is only available for associate and bachelor's degree programs. Standalone certificate programs are ineligible for financial aid.
Champlain offers the following online professional certificates:
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text:
Hot Dog Thursday
July 2, 2015
WhenFirst Thursday of Each Month
April through October
11:30 a.m. - 1:30 p.m.
Next Hot Dog Thursday:
Thursday July 2, 2015.
WherePacific Coast Air Museum
Santa Rosa (directions)
Cost$5 includes Admission,
large Hot Dog, chips,
& soda or water
Chili available at selected events for $1.00 extra.
Ice cream available for $1.00 extra.
Big Dog$8 includes two Hot Dogs!
Members get $1 offYou can't find a better or more affordable lunch anywhere, and what a setting!
Bring friends, families and guests too!
The Pacific Coast Air Museum is grateful to Sanderson Ford and JDH Wealth Management for sponsoring the July 2015 Hot Dog Thursday.
We would like to thank all prior monthly Hot Dog Thursday sponsors throughout 2015:
Silviera GMC
Sonoma Jet Center
La Tortilla Factory
Sonoma County Office of Education
Task Mortgage and Investments
North Coast Air


text:
- 800.878.7323
More copies of this ISBN
Other titles in the Bible series:
Adobe Indesign Cs3 Bible (Bible)by Galen Gruman
Synopses & Reviews
Publisher Comments:
Publish anything in print or online—here's the whole story
Whether you're designing a complete book, creating an eye-catching ad, or producing your own newsletter, InDesign can make the job easier and more professional. More than a software how-to, this essential reference guides you through the complete process of publishing a document from start to finish. If you're a veteran of previous versions, here's the fast track to every upgrade; if you're a recent InDesign convert, everything you need to get going is right here.
Apply colors and gradients with quick and easy methods
Choose from a variety of effects to get exactly the right look
Manipulate text and images with expert attention to detail
Companion Web site
Visit www.indesigncentral.com for additional tools, updates, resources, tips, and more.
While QuarkXPress has been the most-widely used page-layout program in the world, Adobe InDesign is rapidly gaining ground with better typography and transparency features, speedier performance, and more overall control. Learn InDesign now from respected InDesign and QuarkXPress expert Galen Gruman. He packs this book with real-world insights from publishing pros who use InDesign in critical projects, shows you the program's innovative architecture and functionality, and tells you everything you need to know to get up to speed. Whether an experienced designer, a novice, or somewhere in between, you'll find all the real-world tips and techniques you're looking for in this comprehensive reference.
About the Author
Galen Gruman is principal of the content and consulting firm The Zango Group and is a veteran desktop-publishing guru. He has written two dozen books on InDesign, QuarkXPress, and PageMaker, including QuarkXPress to InDesign: Face to Face. A former editor of Macworld, Gruman regularly contributes tips and publishing techniques to Macworld, Layers Magazine, and other publications.
Table of Contents
About the Author.
Quick Start: Building a Document from Start to Finish.
Part I: Welcome to InDesign.
Chapter 1: What InDesign Can Do for You.
Chapter 2: Inside the InDesign Interface.
Chapter 3: Getting InDesign Ready to Go.
Part II: Document Fundamentals.
Chapter 4: Creating, Opening, and Saving Documents.
Chapter 5: Working with Pages.
Chapter 6: Working with Layers.
Chapter 7: Creating Layout Standards.
Chapter 8: Defining Colors, Tints, and Gradients.
Part III: Object Fundamentals.
Chapter 9: Adding Frames and Lines.
Chapter 10: Manipulating Objects.
Chapter 11: Transforming Objects.
Chapter 12: Applying Effects to Objects.
Chapter 13: Orchestrating Objects.
Part IV: Text Fundamentals.
Chapter 14: Importing Text Files.
Chapter 15: Flowing Text through a Document.
Chapter 16: Editing and Formatting Text.
Chapter 17: Specifying Character Attributes.
Chapter 18: Specifying Paragraph Attributes.
Chapter 19: Creating Special Text Formatting.
Chapter 20: Setting Up Styles.
Chapter 21: Using Special Characters.
Part V: Business Document Fundamentals.
Chapter 22: Setting Up Tabs and Tables.
Chapter 23: Automating Text.
Chapter 24: Working with Footnotes, Indexes, and TOCs.
Chapter 25: Creating Multi-Document Projects.
Part VI: Graphics Fundamentals.
Chapter 26: Importing Graphics.
Chapter 27: Fitting Graphics and Frames Together.
Chapter 28: Drawing Free-Form Shapes and Curved Paths.
Part VII: Output Fundamentals.
Chapter 29: Preparing for Color Prepress.
Chapter 30: Preparing for Printing.
Chapter 31: Printing Techniques.
Chapter 32: Creating Prepress Files.
Part VIII: Electronic Publishing Techniques.
Chapter 33: Creating Electronic Documents.
Chapter 34: Working with XML.
Part IX: Workgroup Publishing Techniques.
Chapter 35: Working with Others.
Chapter 36: Workgroup Editing with InCopy.
Part X: Extending InDesign.
Chapter 37: Using Plug-Ins.
Chapter 38: Using Scripts.
Part XI: Appendixes.
Appendix A: Installing or Upgrading InDesign.
Appendix B: What's New in InDesign CS3.
Appendix C: Switching from QuarkXPress.
Appendix D: Switching from PageMaker.
Appendix E: More Resources.
Appendix F: Shortcuts Cheat Sheet.
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text:
hi there,
i want to ask is there a skin creator/editor or something to create X-Port compatible skins like for zsnexbox?
i mean something to move the graphics by drag&drop instead blind editing the ini you know?
thanks in advance!
X-port (zsnexbox) Skin Creator?
Started by nexxxus86 , Aug 21 2008 07:38 AM
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