In the past ten years there has been a growing interest in engineering Gram-positive bacteria for biotechnological applications, including vaccine delivery and production of recombinant proteins. Usually, bacteria are manipulated using plasmid expression vectors. The major limitation of this approach is due to the fact that recombinant plasmids are often lost from the bacterial culture upon removal of antibiotic selection. We have developed a genetic system based on suicide vectors on conjugative transposons allowing stable integration of recombinant DNA into the chromosome of transformable and non-transformable Gram-positive bacteria.Streptococcus gordonii to improve this genetic system making it suitable for expression of single-copy recombinant genes. To achieve this task, a promoterless gene encoding a chloramphenicol acetyltransferase (cat), was randomly integrated into the S. gordonii chromosome and transformants were selected for chloramphenicol resistance. Three out of eighteen chloramphenicol resistant transformants selected exhibited 100% stability of the phenotype and only one of them, GP215, carried the cat gene integrated as a single copy. A DNA fragment of 600 base pairs exhibiting promoter activity was isolated from GP215 and sequenced. The 5' end of its corresponding mRNA was determined by primer extention analysis and the putative -10 and a -35 regions were identified. To study the possibility of using this promoter (PP) for single copy heterologous gene expression, we created transcriptional fusions of PP with genes encoding surface recombinant proteins in a vector capable of integrating into the conjugative transposon Tn916. Surface recombinant proteins whose expression was controlled by the PP promoter were detected in Tn916-containing strains of S. gordonii and Bacillus subtilis after single copy chromosomal integration of the recombinant insertion vectors into the resident Tn916. The surface recombinant protein synthesized under the control of PP was also detected in Enterococcus faecalis after conjugal transfer of a recombinant Tn916 containing the transcriptional fusion.The aim of this work was to select a strong chromosomal promoter from S. gordonii chromosomal promoter. We demonstrated that this promoter can be used to direct expression of heterologous genes in different Gram-positive bacteria, when integrated in a single copy into the chromosome.We isolated and characterized a  An alternative approach is to integrate recombinant DNA molecules into the bacterial chromosome since this method allows increased in vivo stability of the genetic constructs. Therefore a lot of efforts have focused on the development of efficient expression systems based on chromosomal integration of expression cassettes [In the past ten years there has been a growing interest in engineering Gram-positive bacteria for biotechnological applications, including vaccine delivery. -4, and itectants  and micrtectants . A commotectants -9. Howevassettes ,11. Natu916 [916 [We have previously described a genetic system based on conjugative transposons allowing stable integration of recombinant DNA into the chromosome of transformable and non-transformable streptococci ,13. A se916  have bee916 [916 . The aimStreptococcus gordonii, we performed a random ligation of streptococcal DNA to a promoterless cat gene, conferring resistance to chloramphenicol (Cm). The ligation mixture was used to transform the naturally transformable S. gordonii «Challis» strain V288 and transformants were selected for Cm resistance. Chromosomal DNA flanking the promoterless cat gene provided the homology for the random integration of cat into the chromosome during transformation  transformants were isolated, presumably as a result of transcriptional fusions of streptococcal promoters to the promoterless cat gene. Eighteen CmR transformants were selected for further characterization. The strategy commonly used to select promoters is based on cloning random chromosomal DNA fragments in a promoter probe vector upstream of a promoterless reporter gene. However, integrating the promoterless reporter gene (cat) directly into the streptococcal chromosome, allowed us to select resident chromosomal promoters expressing cat after in vivo transcriptional fusion at a single locus on the chromosome. This is preferable when looking for promoters to express heterologous genes integrated into the chromosome in a single copy.To select resident promoters from the genome of ion Fig. . 71 Cm-rIn vivo analysis of promoter strength was determined in the eighteen selected transformants by measuring the minimum inhibitory concentration (MIC) of Cm. Fifteen transformants exhibited a MIC of 16 μg/ml, whereas the remaining three transformants exhibited a MIC of 8 μg/ml. The fifteen strains with higher MIC were tested for the stability of the chloramphenicol-resistance phenotype: after 50 generations of growth without selection, bacterial cultures were plated on non-selective medium and at least 200 colonies were picked and tested for Cm resistance. The stability of the resistance phenotype varied considerably among the different transformants (data not shown). Three strains (GP214, GP215 and GP216) showed a 100% stability and were chosen for further analysis.cat gene in GP214, GP215 and GP216 was analyzed by Southern blot. Only GP215 showed to have a single cat copy integrated into the chromosome, whereas in GP214 and GP216 integration occurred at multiple sites (data not shown). In order to clone the regions flanking cat integration site in GP215, the chromosome of this strain was cut with TaqI whose recognition sequence is absent inside the cat gene. The derivative fragments were ligated to pBLUESCRIPT, and the ligation mixture was used to transform Escherichia coli cells; transformants were then selected for CmR. All transformants analyzed for plasmid content showed to carry a plasmid of the same size. One of these transformants (GP334) was selected for further analysis and the transforming plasmid was named pVMB5. By restriction analysis we showed that pVMB5 contains a 2.2 kb TaqI insert where a 600 base pairs (bp) streptococcal DNA fragment was cloned upstream of the cat gene. This DNA fragment was stably maintained in E. coli, where it retained its promoter activity, conferring CmR to E. coli. This is of considerable interest since it is known that very often cloning streptococcal promoters in a high copy number plasmid results in the failure of that plasmid to be established [The structure of the integrated cat gene in pVMB5 was sequenced (GenBank accession number: U74080). Analysis of the sequence revealed the presence of an open reading frame (ORF1), preceded by a typical ribosome binding site (RBS)  of an uncharacterized protein conserved in bacteria and whose function is unknown. As indicated in figure dltD), a protein involved in D-alanine incorporation into lipoteichoic acid (LTA) [The streptococcal DNA upstream of the BS) Fig. . A BLASTid (LTA) , whereascat-specific mRNA was mapped by primer extension with a specific primer. Total RNA was isolated from E. coli GP334 (containing pVMB5) and S. gordonii GP215. In both strains, the position of the 5'-end of the mRNA was located at the same purine residue at position 423 of the 600 bp region of streptococcal DNA, 26 nucleotides upstream of the ORF1 translational start site  and E. coli (16 to 18 bp) promoters. Moreover, a TG nucleotide pair was found 1 bp upstream of the -10 region; such a structure is typical of Gram-positive bacteria promoters [To identify the sequences responsible for the observed promoter activity, the 5'-end of the ilis σ43  recognitli 16 to  bp promoStreptococcus pyogenes surface fibrillar M6 protein, as a partner for the construction of translational fusions to deliver foreign proteins on the surface of S. gordonii [emm6) in pSMB47, a suicide vector capable of integrating heterologous DNA into the conjugative transposon Tn916 via homologous recombinantion [916 insertion vector that could be used to express translational fusions between M6 and any heterologous proteins under the PP promoter control, a 900 bp AvrII-HindIII fragment internal to emm6 in pSMB139 was replaced by a 390 bp AvrII-HindIII fragment of pSMB55 containing a multiple cloning site [We have previously developed a genetic system based on the use of gordonii . To deteinantion . The recing site  Fig. 4)Streptocoemm6 gene was fused respectively with a DNA sequence encoding 339 aa of the chicken ovalbumin (OVA) (pSMB156), and a DNA sequence encoding 458 aa of the tetanus toxin fragment C (TTFC) (pSMB288) (see Methods). A schematic representation of the recombinant proteins is shown in Fig. pSMB148 was used to create two derivative vectors in which the S. gordonii, B. subtilis, and Enterococcus faecalis were three Gram-positive hosts used to analyze the capability of PP to direct transcription of the heterologous genes expressing M6, M6/TTFC and M6/OVA recombinant proteins.emm6, was introduced by natural transformation in S. gordonii GP201, a strain with a single copy of Tn916 integrated into the chromosome. One of the transformants (GP1241), in which the integrative suicide vector drove the integration of the PP-emm6 fusion into Tn916, was isolated and analyzed for M6 protein expression. Envelope fractions (containing surface associated proteins) prepared from equal amounts of cells grown to mid-log, early and late stationary phase, were analyzed by Western-blotting with an anti-M6 monoclonal antibody. Multiple bands could be detected in fractions of cells grown to mid-log and early stationary phase, whereas no band was detected in the fraction of cultures grown to late stationary phase  was analyzed for cell-surface expression of M6/OVA by flow-cytometric analysis using an anti-ovalbumin polyclonal antibody. The presence of recombinant M6/OVA on the surface of GP431 was clearly demonstrated by the increase of the fluorescence intensity in this strain, as compared to the parental control OG1SS  broth. For maintenance of plasmids, ampicillin (100 μg/ml), chloramphenicol (20 μg/ml) or erythromycin (100 μg/ml) was added to the growth medium. Streptococcal strains were cultured in Brain Heart Infusion medium (BHI, Difco) or Tryptic Soy Broth (TSB, Difco) in the presence of chloramphenicol (5 μg/ml), erythromicin (100 μg/ml) or streptomycin (500 μg/ml) whenever required. Transformation of naturally competent cells of S. gordonii V288 and GP201, scoring and genetic analysis of transformants was carried out as already described [B. subtilis strains were grown in LB broth with erythromicin (3 μg/ml) when it was required. Competent cells of B. subtilis GP800.2 were prepared and transformed according to described procedures [All strains and plasmids used in this work are listed in Table escribed ,22. B. socedures . Agar  was amplified by PCR from plasmid pVMB20 [GGATCCATCATATGGCTAAAAATAACACGAAT-3' (upstream primer, containing a BamHI site and introducing a NdeI site at the ATG translation initiation codon) and 5'-GCATGTCGACCATAATCATTAAATGTATCTCAT-3' . This 1648 bp PCR fragment was digested with BamHI and SalI and cloned in pVA891 [BamHIand SalI, resulting in plasmid pSMB89. A 451 bp region containing the streptococcal promoter PP was amplified from pVMB5 using the following primers: 5'-CGAGGATCCTTTAATCGATACTCATG-3' (upstream primer, containing a BamHI and a ClaI site) and 5'-CCGCATATGGTTCTCCTTTTTATTTGT-3' (downstream primers containing a NdeI site). After digestion with BamHI and NdeI, this PCR product was inserted between the BamHI and NdeI site of pSMB89 to obtain a transcriptional fusion of PP with emm6. The resulting plasmid was named pSMB128. This plasmid was first cut with BamHI, treated with Klenow enzyme to generate blunt ends, and finally cut with SalI to obtain a 2.0 kbfragment containing PP-emm6 fusion. This fragment was gel-purified and ligated to the suicide integrative plasmid pSMB47 [HindIII, treated with Klenow enzyme to generate blunt ends, and finally cut with SalI. The resulting plasmid was named pSMB139  and 5'-TAAAGCTTTAGGGG AAACACATCTG-3' (downstream primer containing a HindIII site). After digestion with BglII and HindIII, this segment was introduced in pSMB148 previously digested with BglII and HindIII. The resulting plasmid, named pSMB156, contained a translational fusion of M6 with OVA. To create a translational fusion of M6 with the tetanus toxin fragment C (TTFC) a 1374 bp BglII-HindIII fragment encoding 458 aa of TTFC was isolated from pSMB158 [BglII and HindIII sites of pSMB148. The resulting plasmid was named pSMB288.A 1648 bp fragment containing the d pVMB20  using thn pVA891  previousm pSMB55  S. gordonii and B. subtilis cell envelope fractions  was performed as already described [Preparation of escribed ,30. The escribed  diluted E. faecalis was performed as already described [Flow-cytometric analysis of escribed ,32 usingescribed .The promoter containing fragment cloned in pVMB5 was sequenced by dideoxy chain termination method  as alreaS. gordonii and E. coli grown to late exponential phase (OD590 ≌ 0.5). Cells were harvested by centrifugation at 6000 × g at 4°C and lysed according to the following procedures. E. coli cells were first resuspended in hot (100°C) lysis buffer (50 mM Tris/HCl pH8, 1 mM EDTA, 1% SDS) and lysed by boiling the suspension for 5 minutes. S. gordonii cells were resuspended in lysozyme buffer (25 mM Tris/HCl (pH8), 10 mM EDTA, 50 mM glucose) and subjected to three cycles of freezing in liquid nitrogen and thawing at 52°C. After incubating with 0.2 mg/ml of lysozyme for 30 min at 37°C, one volume of hot (100°C) lysis buffer (100 mM Tris/HCl (pH8), 2 mM EDTA, 2% SDS) was added, and complete lysis was obtained by boiling cells for 5 minutes. After boiling, all lysates were cooled on ice for 5 min and total RNA was purified using the SV Total RNA Isolation System (Promega).Total RNA was isolated from a 50 ml cell culture of cat gene). Two pmol of the oligonucleotide, labeled with [γ-32P] ATP (3000 Ci/mmol, Amersham) using T4 polynucleotide kinase (New England Biolabs), were precipitated with 10 μg of RNA and the pellet was resuspended in 8 μl of Moloney Murine Leukemia Virus (M-MuLV) Reverse Transcriptase buffer (50 mM Tris/HCl (pH8.3), 8 mM MgCl2, 10 mM DTT). The mixture was heated at 65°C for 3 min, cooled rapidly at -80°C for 1 min and then transfered on ice until it was completely thawed. 1 μl of a 3.75 mM deoxynucleoside triphosphate solution and 10 U of M-MuLV Reverse Transcriptase (New England Biolabs) were added to the RNA-primer hybrid. The reaction mixture was incubated at 48°C for 30 min and terminated with 10 ml of stop solution (95% formamide, 20 mM EDTA pH8.0, 0.05% bromophenol blue, and 0.05% xylene cyanol FF). The reverse transcriptase reactions were analyzed by electrophoresis on a 6% polyacrylamide-7 M urea gel with sequencing reaction obtained with the same primer used as size standards.Primer extention analysis was performed with a synthetic oligonucleotide 5'-GTTCTTTACGATGCC-3' (position 47 to 61 relative to the initiation of the Conjugation experiments were performed on solid media as previously described .S. gordonii, writing of manuscript.RP, characterization of promoter, engineering of B. subtilis and E. faecalisTM, engineering of MRO, participation in experimental work, data evaluationRM, participation in experimental work, data evaluation, writing of manuscriptGP, design and coordination of the study, data evaluation, direct supervision of experimental work, writing of manuscriptAll authors read and approved the final manuscript.


Recently BRCA1 has been implicated in the regulation of gene expression from the X chromosome. In this study the influence of BRCA1 on expression of X chromosome genes was investigated. Complementary DNA microarrays were used to compare the expression levels of X chromosome genes in 18 BRCA1-associated ovarian cancers to those of the 13 "BRCA1-like" and 14 "BRCA2-like" sporadic tumors (as defined by previously reported expression profiling). Significance was determined using parametric statistics with P < 0.005 as a cutoff. Forty of 178 total X-chromosome transcripts were differentially expressed between the BRCA1-associated tumors and sporadic cancers with a BRCA2-like molecular profile. Thirty of these 40 genes showed higher mean expression in the BRCA1-associated samples including all 11 transcripts that mapped to Xp11. In contrast, four of 178 total X chromosome transcripts showed significant differential expression between BRCA1-associated and sporadic tumors with a BRCA1-like molecular profile. All four mapped to Xp11 and showed higher mean expression in BRCA1-associated tumors. Re-expression of BRCA1 in HCC1937 BRCA1-deficient breast cancer cell resulted in the repression of 21 transcripts. Eleven of the 21 (54.5%) transcripts mapped to Xp11. However, there was no significant overlap between these Xp11 genes and those found to be differentially expressed between BRCA1-associated and sporadic ovarian cancer samples. These results demonstrate that BRCA1 mediates the repression of several X chromosome genes, many of which map to the Xp11 locus. The mechanisms by which mutations in BRCA1 and BRCA2 tumor suppressor genes lead to carcinogenesis are incompletely understood. It remains to be established whether pathways involved in BRCA1 and BRCA2-associated tumorigenesis are also altered in sporadic cancers. Two recent reports demonstrated that BRCA1 and BRCA2-associated tumors have distinct expression profiles in both breast . These iThe goal of this study was to investigate further the influence of BRCA1 on the expression of transcripts mapped to the X chromosome. For this purpose the BRCA-associated and sporadic ovarian cancer gene expression data set was analyzed with respect to the expression of 178 transcripts mapped to the X chromosome. Additional in vivo and in vitro experiments employing an X chromosome enriched cDNA microarray were also performed to further evaluate the expression patterns of X chromosome genes in a more comprehensive manner. (under "Alternative Methods and Protocols").The first part of this investigation consisted of a de novo analysis of the large publicly available data set generated by previous microarray experiments with respect to the 178 X chromosome specific genes . Thus, tFor the second part of this investigation a recently developed cDNA microarray enriched in X chromosome transcripts was used. The developmental rationale and approach for this cDNA microarray are described in detail elsewhere . The X-e5 cells per 100 mm plate. The cells were infected at 250 plaque-forming units per cell with adenovirus encoding full-length human BRCA1 or green fluorescent protein (GFP) cDNAs (the latter used as an irrelevant infection control). Twenty-four hours later cells were harvested and RNA was purified using Trizol Reagent (Life Technologies, Inc.) according to manufacturer's instruction.Tissue culture techniques and adenoviral infection of HCC1937 cells was performed as described previously . BrieflyIn studies evaluating gene expression differences between ovarian tumor samples using the X-enriched cDNA microarrays, combined RNA from 10 human cell lines (Stratagene, La Jolla, CA) was used as the reference RNA. In studies involving the HCC1937 cell line, gene expression in BRCA1 virally infected cells was directly compared to that of GFP infected controls., consisting of a modified F test with P < 0.005 considered statistically significant. This stringent P value was selected in lieu of the Bonferroni correction for multiple comparisons, which was deemed excessively restrictive. For microarray experiments involving the HCC1937 cells infected with BRCA1 or GFP the geometric mean of BRCA1:GFP expression ratio from two separate microarray experiments was used. Genes exhibiting a mean expression ratio change of two-fold or greater were considered significant.The logarithmic expression ratios for the spots on each array were normalized by subtracting the median logarithmic ratio for the same array. Data were filtered to exclude spots with a size of less than 25 μm, an intensity of less than two times background, or less than 300 units in both red and green channels and to exclude any poor quality or missing spots. In addition, any features found to be missing in greater than 20% of the arrays were not included in the analysis. Statistical comparison between tumor groups was performed with the "BRB Array Tools" software 1 μg of total RNA was reverse transcribed in 50 μl reaction and 5 μl of cDNA was then used for PCR reaction according to Applied Biosystems technical manual. Separate reaction of the same samples with β-actin was performed for normalization purposes. The difference in threshold number of cycles between the ARAF1 and β-actin was then calculated and converted into real fold difference. All measurements were done in triplicates and the results were averaged. Probes for ARAF1 and β-actin were purchased from Applied Biosystems Inc.It was previously shown that when compared to sporadic cancers BRCA1-associated ovarian tumors were characterized by higher mean expression levels of genes mapped to Xp11 . This obThe significance of this differential pattern of gene expression between the BRCA1-associated and sporadic cancers is unclear at this time. However, higher expression from Xp11 may be related to the earlier age of presentation of epithelial ovarian cancers in BRCA1 mutation carriers compared to tumors in BRCA2 mutation carriers and patients with sporadic ovarian cancer . This obIn order to confirm and expand on the above noted differences in gene expression between BRCA1-associated and sporadic tumors, an X-chromosome enriched cDNA microarray was used . Due to We next sought to determine if differences in X chromosome gene expression between BRCA1-associated and sporadic tumors were directly mediated by BRCA1 as oppose to other, possibly confounding, features of these tumors. The HCC1937 breast cancer cells that are either homozygous or hemizygous for the BRCA15382insC mutation were used as a model. Gene expression in HCC1937 cells following virally mediated expression of wild-type BRCA1 was compared to gene expression following viral infection of GFP which was employed as an irrelevant infection control. Prior to using the X-chromosome enriched array, the validity of this approach was tested using a 7.5 K microarray whose features included BRCA1. This preliminary experiment demonstrated that viral infection did in fact result in a 3.4 fold higher BRCA1 expression compared to the GFP control (data not shown). BRCA1 expression was also demonstrated using Western blotting which confirmed BRCA1 protein expression following viral infection . Thus, the observed down regulation of genes does not appear to be a result of the X-enriched composition of the cDNA microarray used in these experiments nor can it be explained in terms of a global down regulation of all X chromosome transcripts. Rather, the observed repression appears to be a specific effect of BRCA1 expression on these 21 transcripts.Twenty-one X-chromosome transcripts demonstrated at least a two-fold mean expression change across two independent experiments following BRCA1 infection. It is notable that all 21 transcripts (representing 16 unigene clusters) showed decreased expression following BRCA1 transfection with a median repression of 2.4 fold. This uniform repression is particularly significant because median based normalization of logarithmic ratios was employed in microarray data analysis. Thus, the median log expression ratio for all 5,296 features on the array was adjusted to zero (corresponding to an expression ratio of 1.0). Following this median based normalization, the median log expression ratio of the transcripts mapped to the X chromosome  was also very close to zero (median logEleven of the 21 (52.4%) transcripts map to Xp11 and represent 8 unigene clusters . These ESTs are likely to represent as yet undiscovered members of this family of genes that are known for their characteristic pattern of expression, usually limited to the testes and tumors ,13. IntrAlthough not completely understood, the expression of cancer testis antigens is thought to be, at least partially regulated by DNA methylation . These dWhether any of these BRCA1-regulated X chromosome genes are involved in ovarian carcinogenesis and / or tumor progression remains to be determined. However, several lines of evidence support a possible connection between the X chromosome and ovarian neoplasia. First, the loss of a number of regions in the X-chromosome has been associated with ovarian agenesis or premature ovarian failure, which are commonly observed in Turner syndrome and related disorders ,20. ThusUntil recently, a mechanistic explanation for how BRCA1 may affect the expression of multiple genes on the X chromosome was lacking. Evidence for the existence of one such mechanism has been provided by Ganesan and colleagues who have demonstrated the co-localization and interaction between XIST and BRCA1 . This inOne of the unexplained features of germ-line BRCA1 mutations is the overwhelmingly disproportionate risk of cancer in female carriers. One hypothesis put forth to explain this observation is that estrogen is the inciting event by leading to deregulated proliferation and carcinogenesis in hormonally responsive tissues . An alte


We also determined whether subchronic treatment with pramipexole regulates dopamine transporter function, thereby reducing intracellular transport of the active metabolite of MPTP, 1-methyl-4-phenylpyridinium (MPP+).Our aim was to determine if pramipexole, a Dh). Mice were pretreated for 3 days and during the 2-day MPTP regimen with pramipexole (0.1 mg/kg/day) or saline. Stereological quantification of dopamine neuron number and optical density measurement of dopamine fiber loss were carried out at 1 week after treatment, using immunostaining for dopamine transporter (DAT) and tyrosine hydroxylase (TH). Additional wild-type (WT) and D3 receptor knockout (KO) mice were treated for 5 days with pramipexole (0.1 mg/kg/day) or vehicle. The kinetics of [3H]MPP+ and [3H]DA uptake (Vmax and Km) were determined 24 h later; and at 24 h and 14 days dopamine transporter density was measured by quantitative autoradiography.Ten 12-month old C57BL/6 mice were treated with MPTP  twice per day at 20 mg/kg s.c. (4 injections over 48 Vmax for [3H]DA and [3H]MPP+ uptake into striatal synaptosomes of WT mice. Pramipexole treatment lowered Vmax in WT but not D3 KO mice; however, D3 KO mice had lower Vmax for [3H]DA uptake. There was no change in DAT number in WT with pramipexole treatment or D3 KO mice at 24 h post-treatment, but there was a reduction in WT-pramipexole treated and not in D3 KO mice at 14 days post-treatment.Pramipexole treatment completely antagonized the neurotoxic effects of MPTP, as measured by substantia nigra and ventral tegmental area TH-immunoreactive cell counts. MPTP- induced loss of striatal innervation, as measured by DAT-immunoreactivity, was partially prevented by pramipexole, but not with regard to TH-IR. Pramipexole also reduced DAT- immunoreactivity in non-MPTP treated mice. Subchronic treatment with pramipexole lowered the + taken up into DA terminals via DAT. D3 receptor plays an important role in this regulation of transporter uptake and availability.These results suggest that protection occurs at clinically suitable doses of pramipexole. Protection could be due to a reduced amount of MPP An interesting development in the use of dopamine (DA) agonists for treatment of Parkinson's disease (PD) is that some of them have proven to be neuroprotective in animal models of PD. Antiparkinsonian agents that are direct DA agonists, such as apomorphine , bromocr2 receptor subtype [3 preferring agonist (+)-PD 128907 regulates extracellular DA levels via interactions with D3 autoreceptors. If D3 preferring agonists are potent autoreceptor agonists, then hypothetically long-term changes in expression of DAT or the functional properties of DAT might occur following subchronic treatment. Since intracellular accumulation of MPP+ following systemic injection of MPTP requires DAT [3 preferring agonists, this could result in lower intracellular accumulation of MPP+ and reduced neurotoxicity to MPTP.Dopaminergic neurons are tonically inhibited by dendritic and terminal autoreceptors, operating in interaction with DA transporters (DAT) and pharmacologically of the D subtype -19. Howe subtype  have repires DAT , then wh3 receptor preferring agonists, pramipexole and ropinirole, are the most potent of the DA agonists affording neuroprotection at 1 mg/kg for pramipexole against MPTP-induced neurodegeneration [3 receptor affinity such as apomorphine [The Dneration  and at 2neration . Doses 1morphine  are needmorphine , i.e. whmorphine ). Based 3-preferring agonists can protect against MPTP in vivo as well as against MPP+ in vitro [Male C57BL/6 mice of 8–10 months of age were pretreated with saline or pramipexole (0.1 mg/kg/day) followed by MPTP. At the end of the 7-day recovery period following the last injection of MPTP or vehicle were assessed for the degree of toxicity to the dopamine system by MPTP. MPTP produced a marked loss of tyrosine hydroxylase-immunoreactive (TH-IR) neurons in the substantia nigra (SN), but had less impact in the ventral tegmental area (VTA) Figs , Unbiasein vitro ,27.Visualization of DA fibers with dopamine transporter immunoreactivity, DAT-IR Fig , and TH-In contrast to DAT-IR, levels of TH-IR Figs  and 5B w3H]DA and [3H]MPP+ uptake through the DAT. Kinetics of [3H]DA uptake in mouse striata demonstrated a higher Vmax but a similar Km to that reported for rat fresh striatal postnuclear preparation [Km was similar due to inclusion of COMT inhibitor in the preparations [Vmax (P = 0.0008) and the Km (P = 0.016) for [3H]DA uptake was significantly decreased in pramipexole treated mice. The Vmax (P < 0.001) and the Km (P = 0.001) for [3H]MPP+ uptake were also significantly decreased in pramipexole treated mice.Since pramipexole treatment in non-MPTP treated mice altered levels of DAT-IR, it was important to identify if pramipexole treatment altered the kinetics of [paration . Uptake arations . C57BL/6Vmax and the Km for [3H]DA uptake by subchronic treatment with pramipexole was due to the D3 receptor, the values for Vmax and the Km for [3H]DA uptake was measured in WT (C57BL/6) and D3 KO littermate mice  and for Km values (F = 9.78, P = 0.0007). Synaptosomes prepared from D3 KO mice had significantly lower Vmax (P = 0.001, by 59%) and Km (P = 0.01, by 69 %), values for [3H]DA uptake than WT mice (Table Vmax (P = 0.001, by 65%) and Km (P = 0.05, by 50%) values for [3H]DA uptake compared with WT mice treated with saline. D3 KO mice treated with pramipexole had significantly lower Vmax (P = 0.001, by 57%) and Km (P = 0.051, by 77%) values for [3H]DA uptake than WT mice treated with saline, but D3 KO mice treated with pramipexole were not different from D3 KO mice treated with vehicle. WT mice treated with pramipexole were also not different from D3 KO mice treated with pramipexole.To determine if the reduction in  methods ), treateVmax and the Km for [3H]DA uptake by subchronic treatment with pramipexole at 24 h post-treatment was due to the D3 receptor regulation of the number of DAT sites, the density of DAT sites was measured in WT and D3 KO mice treated with vehicle or pramipexole for 5 days. ANOVA showed that there were no group differences -PD 128907 does interact with D3 autoreceptors to regulate extracellular DA levels. Those data are consistent with acute treatment with D2/D3 agonists leading to increased Vmax [The interesting observation that pramipexole down-regulated DAT-IR in saline treated mice suggests that DAT regulation plays a role in neuroprotective effects of pramipexole. Deceptors ,40, and eceptors -20,41,42eceptors -19. Zapased Vmax , but lesVmax, and Km, of [3H]DA and [3H]MPP+ uptake in mice at 24 h post-treatment. Interestingly, D3 receptor KO mice exhibited substantially lower Vmax and Km values of [3H]DA uptake than WT mice, and pramipexole did not further reduce Vmax and Km values in D3 receptor KO mice. However, the actual density of sites on the DA terminals was not reduced 24 h after pramipexole treatment in WT mice (or in D3 receptor KO mice), as determined by DAT autoradiography. The inability to modulate the number of DAT binding sites 12 h after termination of subchronic treatment with D2/D3 receptor agonists has previously been reported [Vmax and Km with pramipexole might be due to modification of the kinetics of DAT. However, pramipexole treatment reduced DAT-IR of DA fibers 7 days post-treatment, suggesting a reduction in DAT sites. Consistent with this, there was a reduction of [125I]RTI binding to DAT sites in pramipexole treated WT mice 14 days after pramipexole treatment, but not in D3 receptor KO mice. Thus, there might be both rapid and slower modifications in DAT function produced by D3/D2 agonist treatment, ultimately resulting in lower DAT number, and mediated by the D3 receptor.We observed that subchronic treatment with pramipexole reduced the reported , suggest2/D3 receptor agonists, and increased by the dopamine D2/D3 receptor antagonist, but not by D1 agonists and antagonists [2 receptor-deficient mice, which exhibit decreased striatal DA uptake [d-amphetamine increased internalization of surface DAT within 1 h. Treatment with DA in HEK-hDAT cells also reduced Vmax, due to a diminished presence of DAT at the surface of synaptosomes [3 receptor. The initial change in Vmax, and Km, could be related to a more rapid turnover of DAT, and the longer-term reduction in Bmax to greater internalization and/or reduced synthesis. Thus, D3 preferring agonists might be potent autoreceptor agonists, and long-term changes in expression of the DAT or the functional properties of DAT could occur following subchronic treatment. This, in turn, could lead to reduced MPP+ (and other neurotoxins) uptake into DA neurons, and reduced toxicity in animal models of PD. Ramirez and associates [3 antagonist A-437203. Furthermore, the neuroprotective effect of a low dose of pramipexole was attenuated in D3 transgenic knockout mice and protection by pramipexole was not further attenuated by treatment with a D3 antagonist. These in vivo data support an important role for the D3 receptor in the neuroprotective effects of DA agonists, and our data suggest that this, in part, is due to reduced MPP+ uptake into DA neurons.It is known that DAT half-life in the striatum is decreased by Dagonists . FurtherA uptake . The preA uptake  using hDptosomes . Subchrosociates  reported3 preferring agonists this could result in lower intracellular accumulation of MPP+ and reduced neurotoxicity to MPTP. The importance of knowing the targets of pramipexole, and other D3 preferring agonists, in neuroprotection in animal models of PD cannot be understated, given the possibility that this protection could be extended to humans [We have identified that subchronic treatment with a clinically relevant dose of pramipexole beginning before initiation of MPTP treatment affords neuroprotection against DA neuron loss and, to a lesser extent, DA fiber loss. This might involve down-regulation of DAT and reduced MPP+ uptake into DA fibers. Since intracellular accumulation of MPP+ following systemic injection of MPTP requires DAT , then ifo humans . Howevero humans ,48. Our o humans ,50.All animals were treated in accordance with a protocol approved by the Sun Health Research Institute Animal Care and Use Committee.We bred C57BL/6 mice from breeding pairs obtained from Jackson Laboratories. Eighteen male C57BL/6 mice 8–10 months of age were used for the experiments and were handled for 1 week prior to treatment. They had free access to food and water, and were maintained in a 12 h light/dark cycle prior to treatment. Mice were divided into 4 groups: the first received only vehicle  (SAL-SAL), the second received pramipexole plus vehicle (PPX-SAL), the third received vehicle plus MPTP (RBI, MA) (SAL-MPTP), and the fourth received pramipexole plus MPTP (PPX-MPT). For those receiving pramipexole , the drug was dissolved in 0.9% sterile saline and administered by i.p. injection (0.1 ml/10 mg body wt). A single daily dose of 0.1 mg/kg body weight of pramipexole was given for 5 days. Those not receiving pramipexole were given identical injections of saline., Following pramipexole or vehicle injections on days 4 and 5, mice were given injections of either MPTP  or vehicle  twice daily at 8 h intervals.After 7-day recovery period following the last injection of MPTP or vehicle, animals were euthanized by intracardiac perfusion with 4% paraformaldehyde in 0.15 M phosphate buffer (pH 7.2) following overdose with pentobarbital 120 mg/kg body weight i.p. Brains were removed, postfixed in the perfusion fixative for 24 h at 4°C and transferred to 30% sucrose solution for additional 24 h incubation at 4°C. The tissue was frozen on dry-ice and sectioned in cryostat at 20 μm thickness. Sections were placed in cryoprotectant solution for long-term storage at -80°C.th that of the midbrain processed for the visualization of TH-positive cell bodies using the avidin-biotin procedure [Every 10th section at the level of striatum was processed for visualization of DAT and tyrosine hydroxylase (TH), and every 5rocedure ,51. Immeth region of each section. Estimation of total neuron number was based upon actual cell counts, tissue thickness, total area of designated region, and the total number of sections analyzed per animal. Group means and variances were calculated.Unbiased stereological quantification of Nissl-stained and TH-IR neurons in the SNpc and VTA was used to estimate cell number in the midbrain. The general routine at low magnification involved use of a sampling grid for the SNpc and VTA. At high magnification the computer-based imaging system randomly selected a region of the grid and clearly definable neurons was counted within the 3-dimensional block. This was repeated for every 10Our routine quantitative measurement of the optical density of regions of the striatum stained for TH-IR and DAT-IR ,51 was e3 receptor knockout mice (25–30 g, 6 months), bred according to our previous methods [2 narcosis and the brains rapidly removed and snap-frozen in liquid nitrogen. Km and Vmax values for [3H]1-methyl-4-phenylpyridinium (MPP+) and [3H]DA uptake in synaptosomes derived from the striatum were deterimined by the method of Eshleman et al [Km and Vmax of [3H]MPP+ and DA uptake were made by t-test (0.05 level of significance).Fourteen C57/Bl6 mice (25–30 g, 6 months) were divided into 2 groups: one group received pramipexole (0.1 mg/kg) once a day for 5 days and the other group received the vehicle . An additional 10 WT and 10 Dan et al , with mi4 1.2 mM, pargyline 10 uM, glucose 0.2%, ascorbic acid 0.02%, pH7.4). To the 8 ml sample, butaclamol was added at a final concentration of 100 nM, 4 ml of sample was than removed and placed in a separate 15 ml centrifuge tube (VWR, Pennsylvania) and Mazindol (Sigma, Missouri) was added at a final concentration of 40 uM. 50μl of the samples were added to borosilicate tubes (Fisher, Texas) and placed in a 25°C water bath with the drugs for a 10 min preincubation. The assay was initiated by adding 50μl concentrations of unlabeled MPP+ or DA ranging from 0–300 nM with [3H]MPP+ or [3H]DA at a final concentration of 20 nM. The samples were incubated at 25°C for 10 min. Specific uptake was defined as the difference in uptake observed in the absence and presence of mazindol (40μM). Uptake was terminated after 5 min by filtration through Whatman GF/C filters presoaked in HEPES buffer. Scintillation fluid was added to each filtered spot and radioactivity remaining on the filters was determined using a Wallac β-scintillation spectrometer. Each experiment involved triplicate determinations, and 6 independent experiments for each drug competition curve were performed.Mouse striata were dissected and homogenized with a glass-Teflon homogenizer in ice-cold modified HEPES (1 ml). The sample was centrifuged at 1000 g, for 10 min at 4°C. The supernatant was collected and centrifuged at 14,000 g for 10 min at 4°C. The pellet was resuspended in 8 ml of HEPES buffer (HEPES 25 mM, NaCl 122 mM, CaCl 2.5 mM, MgSO3 receptor knockout mice (25–30 g, 6 months) were divided into 2 groups: one group received pramipexole (0.1 mg/kg) once a day for 5 days and the other group received the vehicle . Twenty-four h after the last injection of pramipexole or vehicle, the mice were overdosed as above and the brains rapidly removed and frozen on dry- ice. An additional group of 10 WT and 10 D3 receptor knockout mice (25–30 g, 6 months) were similarly treated but their brains were processed 14 days after the last treatment. Autoradiography of DAT sites were quantified following labeling with [125I]RTI-55 (3ß-(4-iodophenyl)tropan-2 ß-carboxylic acid methyl ester) (Dupont, New England Nuclear, Boston, MA) in the presence of 100 nM paroxetine (Smith Klein Beecham BRL 29060A) to block the serotonin transporter, according to published methodology [3H-Hyperfilm for 18 h for DAT. Autoradiographs were analyzed using a computer-based image analysis system (AIS, Imaging Research Inc., Ontario Canada) that converts transmitted optical density to the amount of radioligand bound in pmol per microgram of protein.10 WT and 10 D3 KO = D3 receptor knockoutDDA = dopamineDAT = dopamine transporterMPP+ = 1-methyl-4-phenylpyridiniumMPTP = 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinePD = Parkinson's diseaseSN = substantia nigraTH = tyrosine hydroxylaseVTA = ventral tegmental areaWT = wild typeThe author of this manuscript received a grant from Pharmacia Corporation to partially support the costs of the research. This company is no longer in existence.CW and HR carried out the MPTP treatment, performed the immunocytochemistry and participated in the statistical analysis. SB and DH carried out the uptake assays, performed the DAT autoradiography and participated in the statistical analysis. JNJ conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


Gastrocolic fistula is a rare presentation of both benign and malignant diseases of the gastrointestinal tract. Malignant gastrocolic fistula is most commonly associated with adenocarcinoma of the transverse colon in the Western World. Despite radical approaches to treatment, long-term survival is rarely documented.en-bloc surgery and adjuvant chemotherapy were performed. She is still alive ten years after treatment.We report a case of a 24-year-old woman who presented with the classic triad of symptoms associated with gastrocolic fistula. Radical en-bloc surgery with adjuvant chemotherapy may occasionally produce long-term survival.Gastrocolic fistula is an uncommon presentation of adenocarcinoma of the transverse colon. Radical  Gastrocolic fistula is a rare complication of both benign and malignant diseases of the gastrointestinal tract -6. In thThe authors report a 24-year-old woman who presented with a gastrocolic fistula secondary to an adenocarcinoma of the transverse colon and describe her treatment and long-term follow up.A 24-year-old woman presented to the surgical clinic with epigastric pain, feculent vomiting and post-prandial diarrhoea of three months duration; she had lost over one stone in weight. She was previously healthy and was not taking any regular medications. There was no history of peptic ulcer disease, inflammatory bowel disease, trauma or previous abdominal surgery. She had been investigated two years previously by a gastroenterologist for intermittent left-sided abdominal pain at which time the clinical examination and blood tests were normal. Irritable bowel syndrome had been diagnosed, although no colonic imaging was performed. Both her maternal grandfather and great-grandfather had suffered from colonic cancer.9/l; urea, electrolytes and liver function tests were all normal.An initial ultrasound scan of the abdomen revealed thickened bowel in the right upper quadrant with a dilated duodenum. A barium meal and follow through was then performed: this demonstrated a mucosal abnormality on the greater curvature of the stomach with a fistulous tract into the transverse colon Figure . Barium en-bloc resection was performed involving a subtotal gastrectomy, transverse colectomy and small bowel resection  and folinic acid (20 mg/m2) chemotherapy every four weeks for the following six months.In view of her symptoms, an exploratory laparotomy was undertaken. At surgery, a large mobile tumour of the distal transverse colon was identified; this was adherent to the greater curvature of the stomach, the mesentery and to several loops of jejunum. A radical n Figure . The patShe has been followed-up with two-yearly colonoscopy and five-yearly upper gastrointestinal endoscopy. She remains well with no signs of either local or distant recurrence more than ten years after initial diagnosis.Advanced neoplasms of the stomach and transverse colon are the commonest causes of a gastrocolic fistula: adenocarcinoma of the transverse colon is commoner in the Western World ,3,4, wheThe fistulous connection in a gastrocolic fistula usually arises between the greater curvature of the stomach and the distal half of the transverse colon because of their close anatomical proximity separated only by the gastrocolic omentum . Two theOur patient presented with the characteristic triad of symptoms associated with a gastrocolic fistula ,14: diarAlthough two stage approaches have been advocated historically for malignant gastrocolic fistula, in order to first correct nutritional deficiencies , most auGastrocolic fistula is an uncommon presentation of adenocarcinoma of the transverse colon. Radical en-bloc surgery with adjuvant chemotherapy may occasionally produce long-term survival.The author(s) declare that they have no competing interests.MJF collated the information, searched literature and wrote the manuscript.JKD assisted in literature search and writing of the manuscript.KM was responsible for long-term follow up of the patient and assisted in literature search.MCP managed the patient, helped in preparing the manuscript and edited the final version.All authors have read and approved the final version of the manuscript.


Genital herpes (GH) is a recurrent sexually transmitted infection (STI) that causes significant morbidity and is also the major source of herpes simplex virus (HSV) in cases of neonatal herpes. Vaccination is a current goal which has had limited success so far in preventing GH and microbicides offer an attractive alternative. Treatment of primary disease cannot prevent establishment of latent infections and thus, cannot prevent subsequent recurrent disease. Recently, many of the molecular events leading to entry of HSV into cells have been elucidated, resulting in the description of a number of herpesvirus entry mediators (HVEMs) that interact with HSV glycoprotein D (gD) on the surface of virions. Described here is a strategy for interrupting the spread of HSV based on interfering with these interactions. The hypothesis addressed in the current report was that single chain antibody variable fragments (scFv) that interrupt associations between gD and HVEMs would not only prevent infection in vitro but could also be used as microbicides to interfere with acquisition GH.Here we show that a scFv derived from a particular hybridoma, DL11, not only inhibits infection in vitro but also prevents development of GH in a guinea pig model when applied intravaginally in an inert vehicle. Comparison of different anti-gD single chain antibodies supported the hypothesis that the activity of DL11-scFv is based on its ability to disrupt the associations between gD and the two major receptors for HSV, nectin-1 and HveA. Further, the results predict that bacterial expression of active single chain antibodies can be optimized to manufacture inexpensively a useful microbicidal product active against HSV. GH is generally caused by HSV type 2 (HSV-2), though HSV type 1 (HSV-1) is increasingly recognized as a significant cause of primary infections . The sigth century, there were great strides in antiviral therapy for GH but unfortunately, treating primary disease does not prevent establishment of infection [In the latter half of the 20nfection  and thusnfection  owing tonfection . Hence cnfection . FurtherThus, the number of strategies for preventing sexual transmission of GH is limited. Recently, there has been considerable interest in topical microbicides as a potentially attractive alternative to vaccination for prevention of sexually transmitted infections, including GH . Women aAn evolving strategy that may be useful for preventing specific sexually transmitted viral infections is blocking of virus entry into cells or subsequent inhibition cell-to-cell spread. Many of the molecular events leading to entry of HSV into cells have now been unraveled, resulting in the description of two prominent cell-surface herpesvirus entry mediators  that interact with HSV glycoprotein D (gD) on the surface of virions -20. In aL and VH regions from each hybridoma were reverse transcribed using primers near the VH-CH and VL-CL junctions. For PCR cloning these primers were paired with a panel of degenerate primers derived from VH or VL signal sequences , which attaches hexa-His and V5 tags to expressed proteins for their isolation and identification. Bacteria were induced with IPTG, centrifuged and the supernatants tested for the presence of scFvs by western blotting using anti-His antibody figure . Bacteriin vitro by reacting with an epitope that disrupts the interface between gD and HVEMs was tested by comparing the activities of the various bacterially expressed anti-gD scFv in a Vero cell-based HSV-1 plaque reduction assay. A scFv directed against an epitope on carcinoembryonic antigen was included as an unrelated control. The results showed that pre-incubation of virus with DL11 and 1D3 scFvs inhibited plaque formation with decreasing efficiency. DL6 scFv showed minimal but reproducible activity (data not shown), whereas the other scFvs tested (DL2 and CEA) had no plaque reducing capability at all  were each instilled into the vaginas of guinea pigs . Approximately 20 seconds later the guinea pigs were challenged with 5 × 10t figure  showed t6 PFU of HSV-2, strain G and monitored daily as before. All except one animal were completely protected by DL11 scFv compared with DL2 scFv, all of which developed moderate to severe disease, scored as described in methods .Next a more ambitious test of microbicidal activity was attempted, using HSV-2 rather than HSV-1 and a longer interval (20 minutes) between scFv instillation and challenge  by the procedure described here allowed expeditious activity assessments to be made for the different constructs. Bacterial protein expression systems are widely used for the production of recombinant proteins and problems are often encountered with disulphide bond formation. Whilst there are no covalent bonds between heavy and light chain sequences in immunoglobulin hypervariable domains, intra-chain disulphide bonds can, to varying degrees among different antibodies, influence conformation of the antigen binding site . Thus, fEntry of HSV into cells is known to be mediated through interactions between gD and 3-O-sulfated heparan and one or more specific entry mediators, HveA, nectin-1 and nectin-2 . Overall2a monoclonal anti-gD antibody and its IgA switch variant. Here, these observations are extended in several respects. The epitope on gD recognized by the most effective scFv, that constructed from antibody DL11, was defined as one that interferes with binding of gD with two major mediators of herpesvirus entry into cells, namely HveA and nectin-1. In this respect, attention is drawn to the recent report of Linehan et al [The data presented here correlate with the prior finding that mice can be protected against HSV-2 by topical administration of antibody ,34 and aan et al  that necan et al  togetherIn summary, we believe that single chain antibodies against HSV merit further study and development as topical microbicides. The production of active molecules in bacteria makes their use a feasible and relatively inexpensive prospect.Single chain antibodies against HSV gD could be synthesized readily from several IgG secreting hybridomas using degenerate immunoglobulin heavy and light chain immunoglobulin primers that hybridized to regions flanking the complementary determining regions, which determine antigen specificity.Two mechanisms of interference with infection were evident when DL11 scFv was examined in detail. First, the number of plaques produced by virus could be inhibited by up to 90% when reacted with HSV prior to infection of Vero cells, indicating that scFv neutralized virus prior to establishment of productive infection. This result also suggested that nectin-1 and HveA, the binding of which are both blocked by DL11, are the main mediators of virus entry into Vero cells. 1D3, which interferes specifically with the interface of gD with HveA, was effective to a lesser extent. Second, in addition the striking ability of DL11 scFv to neutralize virus inoculums, this particular construct reduced plaque size significantly, from which it was concluded that cell-cell spread of HSV was also inhibited. This observation could have implications for therapeutic use of single chain antibodies in the future and may have enhanced the performance of DL11 scFv as a microbicide in the guinea pig model. This result was mediated by suboptimal scFv concentrations for virus neutralization, implying that lower concentrations of DL11 scFv may be required to interfere with intercellular spread of virus than to block entry.The finding that DL11 scFv was active for 20 minutes, the maximum time tested, when instilled into the vaginal vault was considered encouraging for future development of scFv as microbicides and the observation merits further consideration of the vehicle used. Slow release formulations may be appropriate depending on their cost. Overall, it appears that selected single chain antibodies are promising candidates for interfering with binding of gD to HVEMs and studies in a guinea pig model of GH suggest that they may comprise a plausible strategy for preventing transmission of GH.5 - 106 hybridoma cells were isolated using Trizol (Invitrogen, CA) and cDNAs were generated by reverse transcription with Taq polymerase . RT was primed with anti-sense oligonucleotides designed to anneal either to mouse kappa light chain or heavy chain constant region sequences, just downstream of the J-C junction (table L and VH) were amplified by priming 'sense' PCR reaction products with panels of oligonucleotides (OGNs) designed from Kabat database sequences to be complementary to kappa (light chain) and gamma (heavy chain) signal or framework sequences (table L and VH cDNAs were sequenced and new specific primers were designed each of which included 2/3 of the fifteen amino acid (Gly4Ser)3 flexible hinge region, allowing the variable regions to be amplified and spliced together reconstituting the antigen binding site on reconformation (figures 4Ser)2intervening segment, alternative glycine codons were used in each component of the hinge. Four of the scFvs were TA cloned into the bacterial expression vector pET101/D-TOPO (Invitrogen, Carlsbad, CA) which generates hexa-His tagged proteins after expression in vitro.Single chain antibodies were constructed from four anti-gD secreting hybridomas, DL11, DL6, DL2 and 1D3. An additional scFv, directed against carcinoembryonic antigen (CEA) served as an independent control. Messenger RNAs from 5 × 10Proteins were expressed in IPTG-induced E. Coli BL21 [DE3] (Invitrogen), released by sonication in PBS and inclusion bodies were separated by centrifugation. Proteins in inclusion bodies were solubulized with 6 M guanidine HCl and purified by metal chelation. A stepwise dialysis procedure with addition of GSSG  and L-arginine in the final two steps was used to assist in the formation of intra-chain disulphide bonds in order to optimize re-conformation and stability of the scFvs . ProteinMicrotiter plate wells were coated with soluble gD (6 µg/ml) and then blocked with 1% skimmed milk. After incubation with serial two-fold dilutions of scFv, binding was detected with anti-V5, the alternative tag on the scFv, because the recombinant gD used in the assay was, like the single chain antibodies, tagged with hexa-His. Binding ratios were calculated in relation to an irrelevant (CEA-specific) scFv.6 Vero cells for a further 1 hour the samples were plated in 6 cm dishes (Nunc) in a total volume of 5 mls of GM containing 2% carboxymethylcellulose (CMC). Plaques were enumerated after 3 days incubation at 37°C in a 5% CO2 atmosphere.HSV-1 (strain SC16) and HSV-2 (strain G) were grown and titrated in Vero cells as described ,37. Tite6 PFU HSV-1 (strain SC16) or HSV-2 (strain G). Over the ensuing two weeks lesions were scored on a scale of 0–4 . All experiments were done according to the guidelines laid down in The NIH Guide for Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee.The microbicidal properties of scFv were tested using a guinea pig model of GH. Female outbred Hartley guinea pigs weighing 350–400 grams were obtained from Charles River laboratories (Wilmington, MA). Prior to inoculation of each guinea pig with virus, the introitus was opened with a calcium alginate swab moistened in physiological saline and 1 ml of 1% CMC containing either DL2 scFv or DL11 scFv at a final concentration of 500 µg/ml, was instilled using a pipette with a plastic tip. CMC was used as a vehicle to facilitate retention of the scFv in the vaginal vault. At various times thereafter, animals were challenged with 10The author(s) declare that they have no competing interests.AS conceived and coordinated the work described and wrote the manuscript. JC was responsible for the experiments described and SKD provided technical support.


Diabetic nephropathy (DNP) is a common complication of type 1 and type 2 diabetes mellitus and the most common cause of kidney failure. While DNP manifests with albuminuria and diabetic glomerulopathy, its progression correlates best with tubular epithelial degeneration (TED) and interstitial fibrosis. However, mechanisms leading to TED in DNP remain poorly understood.We found that expression of scavenger receptor CD36 coincided with proximal tubular epithelial cell (PTEC) apoptosis and TED specifically in human DNP. High glucose stimulated cell surface expression of CD36 in PTECs. CD36 expression was necessary and sufficient to mediate PTEC apoptosis induced by glycated albumins (AGE-BSA and CML-BSA) and free fatty acid palmitate through sequential activation of src kinase, and proapoptotic p38 MAPK and caspase 3. In contrast, paucity of expression of CD36 in PTECs in diabetic mice with diabetic glomerulopathy was associated with normal tubular epithelium and the absence of tubular apoptosis. Mouse PTECs lacked CD36 and were resistant to AGE-BSA-induced apoptosis. Recombinant expression of CD36 in mouse PTECs conferred susceptibility to AGE-BSA-induced apoptosis.Our findings suggest a novel role for CD36 as an essential mediator of proximal tubular apoptosis in human DNP. Because CD36 expression was induced by glucose in PTECs, and because increased CD36 mediated AGE-BSA-, CML-BSA-, and palmitate-induced PTEC apoptosis, we propose a two-step metabolic hit model for TED, a hallmark of progression in DNP. The CD36 receptor may have a key role in the degeneration of the renal tubular epithelium, the first step in diabetic nephropathy Diabetic nephropathy (DNP) is a serious and common complication of type 1 and type 2 diabetes mellitus, leading to end-stage renal failure in up to 30% of individuals with diabetes. Early abnormalities of DNP affect glomeruli and include an increase in glomerular filtration rate, microalbuminuria, glomerular hypertrophy, and thickening of the glomerular basement membrane, followed by expansion of mesangial extracellular matrix and glomerulosclerosis .Combining detailed renal phenotype analysis with gene expression profiling of hyperglycemic mouse models of type 1 (streptozotocin [STZ]) and type 2 (db/db) diabetes, we recently reported that decreased mRNA levels of CD36 in kidneys were strongly correlated with albuminuria . CD36 isHere we report a novel functional role for CD36 scavenger receptor and AGE and FFA palmitate (PA) in tubular epithelial apoptosis associated with TED and progression of DNP. Specifically, we show that glucose stimulates CD36 cell surface expression in proximal tubular epithelial cells (PTECs), and increased CD36 renders PTECs susceptible to both AGE- and PA-induced PTEC apoptosis by mediating sequential activation of src kinase, proapoptotic p38 MAPK, and caspase 3. Based on these findings, we propose a new two-step metabolic hit model for TED in the progression of DNP.db/db, STZ-treated C57BL/6J, or STZ-treated 129SvJ mice and from age-matched control C57BLKS/J-leprdb/m, C57BL/6J, and 129SvJ mice as described [Kidneys were obtained from 28-wk-old C57BLKS/J-leprHuman proximal tubular cell line HK-2 and murine collecting duct cell line M1 were purchased from American Type Culture Collection (Manassas, Virginia, United States) and cultured according to the vendor's instructions. Mouse proximal tubular cell line MCT was provided by Fuad Ziyadeh (University of Pennsylvania, Philadelphia, Pennsylvania, United States). Transfections were performed with Fugene 6 (Roche Diagnostics, Indianapolis, Indiana, United States) according to manufacturer's protocol. CD36-containing plasmid was a kind gift of Nada Abumhrad (SUNY at Stony Brook, New York, United States). Cells were also co-transfected with EGFP (Clontech, Franklin Lakes, New Jersey, United States) to assess transfection efficiency. Cells were serum starved in 0.2% serum containing DMEM (1 g/l glucose) for at least 24 h prior to stimulation with AGE–bovine serum albumin (BSA), glucose, or FFA.Quantitative real-time PCR analysis of mouse and human CD36, HPRT1, and beta actin was performed as described previously . The folPrimary antibodies specific for the following proteins were used: monoclonal mouse anti-CD36 antibody, clone FA 6–152 (IgG) , clone SMO (IgM) , rabbit polyclonal anti-CD36 (Santa Cruz Biotechnology), rabbit polyclonal anti-aquaporin1, anti-aquaporin2, anti-Na/K/2Cl , rabbit polyclonal phospho38/MAPK and mouse monoclonal p38 , rabbit polyclonal p-src (Y418) , and mouse monoclonal anti-tubulin (Sigma, St. Louis, Missouri, United States). Immunostaining was performed on frozen sections with FITC- and Cy3-labeled secondary antibodies (Jackson Laboratories, USA), or on paraffin-embedded sections with immunoperoxidase, as described earlier [4) were analyzed by using a SCAN flow cytometer (BD, Franklin Lakes, New Jersey, United States), with appropriate gating. Flow cytometry data were analyzed using Cellquest (BD).Cells were incubated in 0.5 mM EDTA in PBS at 37 °C for 20 min, scraped, and then washed with 1% fetal bovine serum. Cells were then exposed to monoclonal anti-CD36 IgG FA6 (5 μg/ml), or control mouse IgG1 (5 μg/ml) (Sigma), for 45 min on ice in the presence of 10% fetal bovine serum then washed with PBS. This was followed by an incubation with phycoerythrin-conjugated goat anti-murine secondary antibody (Southern Biotechnology, Birmingham, Alabama, United States) 1:50 for 45 min on ice. Cells  was incubated at 37 °C for 2, 5, and 10 wk with D-glucose (500 mM) in a 0.4-M phosphate buffer containing EDTA, ampicillin, Fungazone, polymixin B, and protease inhibitors. Control preparations were treated identically except that glucose was omitted. Carboxymethyl-lysine (CML)–BSA was prepared as described earlier . BrieflyPalmitic acid (P5585), oleic acid, and FFA-free low-endotoxin BSA (A8806) were purchased from Sigma. Palmitic acid was dissolved at 12 mM in PBS containing 11% fatty-acid-free BSA, sonicated for 5 min, shaken overnight at 37 °C, and sonicated for 5 min again [In situ detection of DNA fragmentation was performed using the ApoTag TUNEL assay following the manufacturer's protocol (Intergen, Purchase, New York, United States) . ApoptotHuman kidney tissues  were obtained from archived kidney biopsy samples or from discarded nephrectomy specimens. All diabetic samples were from patients with biopsy-proven advanced DNP with serum creatinine ranging from 1.7 to 5.6 mg/dl (151 to 444 μM/l), heavy proteinuria (3+ by dipstick or 3–6 gr/d), and hypertension. All patients with FSGS were from patients with creatinine levels of 1.7 to 4.9 mg/dl (151 to 435 μM/l), heavy proteinuria (3+ by dipstick), and hypertension. The diagnosis of FSGS was made on Periodic acid–Schiff staining in the absence of immunodeposits on electron microscopy. The diagnosis of diabetic nephropathy was based on the presence of diabetes, proteinuria, and the characteristic light microscopy findings. Institutional Review Board approval was obtained for procurement of kidney specimens at the Thomas Jefferson University Hospital.t-tests). The Bonferroni correction was used for multiple comparisons. Significance for the quantification of the CD36 staining in human biopsy samples was calculated via the Wilcoxon Rank Sum Test.Data are reported as mean and standard error of the mean (SEM) for continuous variables. All cell culture experiments were performed at least three times and summarized. Standard software packages (SPSS and Excel for Windows) were used to provide descriptive statistical plots  for 20 wk A, or typdb/db mice (0.2 ± 0.1 TUNEL-positive cells per 100 tubular cells). Together, these findings indicate that CD36 expression in PTECs is associated with apoptotic events of proximal tubular cells and TED specifically in human DNP, but not in FSGS with matched functional and clinical abnormalities. These in vivo findings demonstrate a strong association of diabetes-induced CD36 expression and apoptosis in PTECs in human DNP, suggesting that CD36 may play a critical role in TED by mediating PTEC apoptosis in progressive human DNP.CD36 has been shown to mediate apoptosis signaling induced by TSP-1 in endothelial cells  and by oHigh ambient glucose has been shown to induce CD36 protein synthesis in macrophages . BecauseAGE albumin  and FFAsAmong the most abundant glucose-modified proteins detectable in the plasma of diabetic individuals are CML proteins , which aCD36 has been shown to transport fatty acids in adipocytes  and in mCD36 has previously been shown to trigger the activation of p59fyn, p38 MAPK, and caspase 3 (GeneID: 836) in response to thrombospondin in endothelial cells .In contrast with CD36-positive human HK-2 PTECs, we found that treatment of CD36-negative mouse MCT PTECs with AGE-BSA had no effect on rates of apoptotic nuclei (data not shown). To test whether CD36 was sufficient to mediate AGE-BSA-induced apoptosis, we transfected CD36-negative mouse MCT PTECs with a plasmid expressing human CD36 or empty control vector, followed by treatment with control BSA or AGE-BSA5. AGE-BSA5 treatment had no significant effect on rates of apoptotic nuclei in MCT PTECs transfected with control vector . In contAdvanced diabetic nephropathies in humans with type 1 or type 2 diabetes are uniformly characterized by TED, or tubular atrophy, and IF leading to renal failure ,30. AlthOur conclusions are supported by several key observations. First, we identify a new functional role for CD36 as an essential mediator of proximal tubular epithelial apoptosis, inducible by AGE-BSA, CMLmin-BSA, and FFA PA. Previous reports demonstrated a role for CD36 in mediating apoptosis induced by TSP-1 in endothelial cells and ox-LDL in macrophages ,14. In tCD36 and mouse Cd36 genes indicate a high degree of sequence and structural similarity in both coding and regulatory regions, suggesting that the mechanism or mechanisms that underlie our findings are likely determined by sequence-independent, epigenetically distinct response patterns to the diabetic milieu that differ between these species. It is also possible that dietary or metabolic factors account for the differences in CD36 expression, as mice were maintained on standard mouse chow characterized by significantly lower fat and cholesterol contents than typical western diets consumed by humans. However, dietary or other unknown environmental factors cannot explain the differential CD36 regulation by glucose in human and mouse PTECs. Thus, we are exploring whether biochemical or functional differences between mouse and human PTECs in glucose metabolism or glucose-induced signaling can be identified. However, current lack of understanding of the observed differential regulation between human and mouse does not diminish the translational research significance of our findings, with their clear therapeutic implications. Thus, the present study identifies a new CD36-dependent molecular signaling pathway that mediates tubular epithelial apoptosis, and may underlie TED and IF, hallmarks of disease progression, specifically in human diabetic nephropathy.Almost all TUNEL-positive apoptotic tubular epithelial cells showed increased expression of CD36, suggesting a strong correlation between upregulation of CD36 expression and increased apoptosis in PTECs specifically in human diabetic kidney in vivo. Importantly, biopsy samples from cases of FSGS that were matched for degree of proteinuria, renal function, and hypertension were characterized by TED, IF, and increased tubular epithelial apoptosis; however, proximal tubular CD36 expression was similar to that in normal human control kidney. Therefore, CD36 expression in PTECs is specifically associated with the diabetic condition and appears to be independent of degree of proteinuria and renal failure. Indeed, increased CD36 expression in PTECs in human DNP in vivo may be caused by hyperglycemia, as we show that high glucose concentration stimulates CD36 expression in vitro. It is intriguing that CD36 expression was not detected in PTECs in diabetic mice with longstanding hyperglycemia in vivo, although underlying mechanisms for the species-dependent differential regulation of CD36 in PTECs in vivo and in vitro between mouse and human remain unclear at this time. Comparisons of human Third, to our knowledge, our report provides the first controlled study demonstrating increased apoptosis specifically in PTECs in DNP with TED and IF. These findings are consistent with a recent uncontrolled case series of five patients with DNP , and witIn conclusion, we report a new functional role for CD36 scavenger receptor in tubular epithelial apoptosis associated with tubular degeneration and progression of DNP. Specifically, we show for the first time that both AGE and FFA PA induce PTEC apoptosis through CD36-mediated activation of src kinase, p38 MAPK, and caspase 3. Because high glucose stimulates CD36 expression in human PTECs and because CD36 expression is increased in apoptotic tubular epithelial cells in human DNP, we propose a two-step metabolic hit model relevant for TED, a hallmark of progression of human DNP.Figure S1Flow cytometric analysis of (A) human (HK-2) and (B) murine (M1) tubular epithelial cells incubated with control IgG (green curve) or with anti-CD36 antibody (FA6) (black curve) in medium containing 5 mM glucose (empty curve) or in medium containing 30 mM glucose (red curve) for 3 d.(45 KB PPT).Click here for additional data file.http://www.ncbi.nlm.nih.gov/LocusLink/) accession numbers for the gene products discussed in this paper are caspase 3 (GeneID: 836), CD36 (GeneID: 948), MAPK (GeneID: 1432), p42/44 ERK MAPK (GeneID: 50689), p59fyn (GeneID: 2534), and Smad2 (GeneID: 4087).The LocusLink (The kidneys are often affected in people with diabetes. Around one in three people with type 1 (juvenile, or insulin-dependent) and one in ten people with type 2 (late onset, or non-insulin-dependent) diabetes will develop kidney disease . Diabetic nephropathy is one of the leading complications of diabetes and is the leading cause of kidney failure worldwide. Some risk factors make it more likely that certain people with diabetes will develop kidney disease—for example, kidney disease occurs more often in patients from South Asian or African backgrounds, in men, in patients with poor control of their blood sugar levels, and in those with high blood pressure or who smoke. However, the details of how, exactly, diabetes damages the kidneys are not clear.They studied samples taken from the kidneys of humans and mice with and without diabetes and looked at the effects of high glucose concentrations on the cells in the kidneys. They found that in one part of the human kidneys high glucose caused a change in the cell surface causing an increase in a protein called CD36. This change occurred in the samples from people with diabetes, but did not occur in the samples from mice with diabetes. The investigators also found that some substances that are often found in the blood of people with diabetes could join to CD36; in doing so, these substances triggered the death of these cells, which is one of the first steps that occurs in diabetic nephropathy.This particular protein (CD36) could have a central role in triggering diabetic nephropathy. Although there are no immediate clinical implications of this research for the treatment of people with kidney problems, this research helps in understanding how high glucose damages the kidney. In particular, it highlights how important it is to keep blood glucose levels as normal as possible.http://www.nlm.nih.gov/medlineplus/ency/article/000494.htmMedline Plus's article on diabetic nephropathy: http://www.diabetes.org.uk/infocentre/index.htmlDiabetes UK's online information centre: http://diabetes.niddk.nih.gov/National Diabetes Information Clearinghouse: http://www.amdcc.org/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Animal Models of Diabetic Complications Consortium (AMDCC):


The movements needed to read this synopsis—turning the page, tracking along the lines, sitting, breathing—all require myosin, a molecular motor in muscle that transforms chemical energy into small but deliberate motions. But beyond these macro-movements, myosin is also required for the micro-movements of individual cells and their organelles and for determining cellular architecture.There are many different myosins, but they all have the same general structure. At one end is a globular head, which is responsible for motor activity. This head binds ATP—the cell's power supply—and actin, an important component of the cytoskeleton of cells. Next comes a helical neck or lever region. Finally, there is a long helical tail, which has different and somewhat poorly understood functions in the different myosins.Myosin II, the classical form of myosin found in essentially all eukaryotic cells, is constructed from two heavy chains (which contain the three regions described above) and two pairs of light chains (which stabilize the neck region). The long helical tail of myosin II is formed by the two heavy chains wrapping around each other and is involved in getting myosin II to the right place in the cell, as well as in assembling it into filaments.Individual myosin II molecules can make tiny molecular motions. ATP cleavage induces a shape change in the globular head, which is transmitted to the lever region of the molecule. Angular rotation of this region moves the myosin along the actin filament. But to achieve the larger movements that are necessary to, for example, split cells apart during cell division, individual myosin II molecules group together to form highly regular bipolar structures called bipolar thick filaments (BTFs). In these, the globular myosin heads are positioned on either side of the filament, and the tail regions are clustered in the middle. This geometry enables myosin II molecules in thick filaments to pull from either side, generating contractile forces.Dictyostelium discoideum, an organism beloved by developmental and cellular biologists because of its simple development and ease of manipulation. In the present study, the researchers examined the physical properties of various fragments of the myosin tail to find out how the self-assembly and disassembly of the BTFs are regulated. They already knew that the addition of phosphate groups on three specific threonine amino acid residues in this region  is important for regulating BTF assembly; they knew this from studies showing that mutation of these residues to aspartic acid, which mimics phosphorylated threonine, inhibits BTF formation. Here, the researchers show that a specific tail fragment of the myosin heavy chain containing the three crucial threonine residues assembles into a structure with some, but not all, of the properties of BTFs. However, replacing these threonine residues with aspartic acid prevents any self-assembly of the fragment.James Spudich's team has been studying the assembly of these thick filaments in the slime mold Further experiments in which different tail regions were nibbled away and the assembly properties of the remaining fragments were determined suggest that the myosin tail contains a series of elements that correlate with the distribution of charged amino acids along the tail, some of which favor assembly and some of which favor disassembly. But it's not just the tail that is important. For myosin II to form fully fledged BTFs of a defined size, it seems that the addition of some kind of globular head—in these experiments one composed of green fluorescent protein so that it could be examined—is necessary. The overall result is a molecule that is finely poised to self-assemble into BTFs in response to one or two charge changes produced by phosphorylation. Consequently, the myosin contractile system can respond rapidly to environmental changes.Dictyostelium myosin II is somewhat different from vertebrate myosin II, the general principle by which myosin assembly and disassembly are regulated seems likely to hold for other myosins and so might throw light onto human disorders that involve myosin defects. But more fundamentally, similar principles may hold for spatial and temporal regulation of the many other macromolecular assemblies that are at the heart of cell and developmental biology.Although


One of the important challenges in microarray analysis is to take full advantage of previously accumulated data, both from one's own laboratory and from public repositories. Through a comparative analysis on a variety of datasets, a more comprehensive view of the underlying mechanism or structure can be obtained. However, as we discover in this work, continual changes in genomic sequence annotations and probe design criteria make it difficult to compare gene expression data even from different generations of the same microarray platform.We first describe the extent of discordance between the results derived from two generations of Affymetrix oligonucleotide arrays, as revealed in cluster analysis and in identification of differentially expressed genes. We then propose a method for increasing comparability. The dataset we use consists of a set of 14 human muscle biopsy samples from patients with inflammatory myopathies that were hybridized on both HG-U95Av2 and HG-U133A human arrays. We find that the use of the probe set matching table for comparative analysis provided by Affymetrix produces better results than matching by UniGene or LocusLink identifiers but still remains inadequate. Rescaling of expression values for each gene across samples and data filtering by expression values enhance comparability but only for few specific analyses. As a generic method for improving comparability, we select a subset of probes with overlapping sequence segments in the two array types and recalculate expression values based only on the selected probes. We show that this filtering of probes significantly improves the comparability while retaining a sufficient number of probe sets for further analysis.Compatibility between high-density oligonucleotide arrays is significantly affected by probe-level sequence information. With a careful filtering of the probes based on their sequence overlaps, data from different generations of microarrays can be combined more effectively. By providing a genome-wide view of gene expression, microarrays have become a common exploratory tool in many areas of biological and clinical studies -3. WhileLarge-scale gene expression data often contain a large amount of noise from various experimental factors. Fortunately, in most cases, the technical variability is relatively small compared to the biological one and its effect can be minimized by using a sufficient number of replicates -8. HowevSeveral issues have made this process difficult so far. First, different datasets have been processed using different procedures due to a lack of uniform standards, e.g., for background correction, normalization, and calculation of expression values. This makes it difficult to compare them directly. Raw data files are generally unavailable and, even if they are, reprocessing them requires substantial effort. Second, we have lacked datasets with enough controls and replicates, performed under a proper experimental design and with adequate annotations, in order to make proper comparisons. Third, possibly the most troublesome, the experiments have been performed on many different platforms, with significant differences among them. Even within a single platform, technological and algorithmic advances as well as the evolving annotations of the genomes have resulted in succeeding generations of arrays with substantial modification from one generation to the next. Until now, several studies have found varying degrees of disagreement between platforms, sometimes with large discrepancies that call into the question the reliability of certain conclusions reached in microarray studies -19. A coIn this work, we carry out a thorough examination of the comparability between the two generations of Affymetrix human GeneChip arrays, HG-U95Av2 and HG-U133A, both of which have been used extensively for studying human gene expression patterns. We then propose a method for enhancing their comparability. The analysis we carry out is made possible by a dataset consisting of the same tissue samples hybridized on both platforms. The procedure is illustrated in Figure The most common method of matching genes in cross-platform studies is to match the UniGene IDs among genes ,15-18. OAs a simple way to assess comparability, the Pearson correlation coefficient between each array pair from the same sample was calculated and the 14 correlation coefficients were averaged. The results are summarized in Table In a similar study , the autThere was a possibility that the lack of high correlation between the two versions was caused by a true inconsistency present in the data, perhaps due to RNA degradation between the times when the hybridizations on the two platforms were performed. To make sure that this was not the case, we investigated the quality of our data by examining the subset of probes which have the exactly same sequences between the array generations.When we examined about 5% of probes that have the same sequence between U95Av2 and U133A, the mean correlation coefficient of array pairs, calculated by PM intensity, was 0.967 ± 0.007.  This is similar to the conclusion in  that theTo determine the extent to which the analysis results from the two versions of the arrays agree, we employ the two most frequent tools for exploratory analysis: cluster analysis and identification of differentially expressed genes. For evaluating the compatibility in terms of cluster analysis, we combined the datasets from U95Av2 and U133A by Best Match. Then, the 28 samples were clustered by agglomerative hierarchical clustering method with the Pearson correlation coefficient as the distance measure. Figure t-test with unequal variances (the Wilcoxon test gives very similar results). If the two generations of arrays were comparable, the lists of differentially expressed genes should contain many overlapping genes. To increase the possibility of overlaps, we filtered out non-expressed genes by deleting those in which more than 75% of the samples received Absent calls in both U95Av2 and U133A arrays. When we examine the list of genes identified in common in the two cases, however, its length is disappointingly small. When we look at the list of length 100 or smaller, the percentage of overlap does not exceed 25%. The plot of the percentage of genes common in both lists as a function of the list size is virtually identical to the dashed line in Figure 7b . This low overlap indicates that the two array types give highly inconsistent results and brings into question the reliability of the highly ranked genes in either platform. We do note, however, that this result must be interpreted in terms of the sample size and other characteristics of the specific dataset. A low percentage is often partially due to the presence of a large number of genes that are differentially expressed to a similar extent in a particular dataset, in which case a ranking of the genes would be expected to be somewhat unstable.Next, we identified differentially expressed genes between the DMs and other myopathies from each dataset (5 vs 9 samples), using the two-sample To understand the reason for the discordance observed in Figure A simple solution to this problem is to scale expression values for each gene across samples, for instance, making the mean to be 0 and the standard deviation to be 1. The effect of this gene scaling on the gene pair from Figure While gene scaling was effective in cluster analysis, it is limited to evening the influence of different genes in a global analysis by focusing on their patterns. It does not enhance the comparability, for instance, in terms of identifying differentially expressed genes in most algorithms. For that case, some simple filtering schemes could enhance reproducibility instead. One way is to consider only the genes that exhibit strong correlations between the two versions. To see the impact of this on the selection of differentially expressed genes, we calculated the overlap for the 1,000 genes whose profiles on the two array versions were highly correlated. The result is plotted in Figure We now describe a more general method for improving comparability by filtering at the probe level, instead of at the probe set level. We have already observed that the probes with exactly the same sequences on the two generations give highly reproducible values  and 9 had other inflammatory myopathies including necrotizing myopathy, inclusion body myositis, granulomatous myositis, and polymyositis. Because the molecular profile of DM is sufficiently different from those of the rest, we can think of the DMs as one group and the rest as the other group in a two-group comparison . Total RAlthough they belong to the same oligonucleotide array platform, the changes from the older version (U95Av2) to the newer one (U133A) were substantial: 1) Main source of probe selection region is different ; 2) The number of probe pairs was reduced from 16 to 11 for a single gene; and 3) Probe selection method was improved . The annFor considering variations in the probe sets for the same transcript between different array versions, Affymetrix provides the probe set matching tables for comparative analysis. These matching tables were constructed based on the sequence information of probe sets as follows . First,  as a stand-alone program) because it appears to be more accurate and faster than others for matching short sequences with high sequence identity (more than 90%). BLAT has been used previously for annotating the probe sets of HG-U95Av2 in GeneAnnot system from Weizmann Institute of Science [[For improving compatibility between U95Av2 and U133A, those probes whose sequence overlapped with any of the probes for the same gene on the other platform were selected. The extent of overlap necessary is described in the Results section. First, all the perfect match (PM) probes were aligned to the coding regions of the genome. Of commonly used short sequence alignment tools such as SIM4 , SPIDEY  Science . The aliScience [). We ranThe length of the overlap between probe sequences (1 bp ~ 25 bp) was used as a criterion for choosing probes for comparative analysis. The expression values were recomputed each time using only the selected probes by masking out the other probes from the raw (.cel) files. The values were calculated by the Statistical Expression Analysis Algorithm using Microarray Suite version 5.0 (MAS 5.0) (Affymetrix, Santa Clara, CA) without linear scaling to target intensity. MAS 5.0 is a robust estimator of expression index based on one-step biweight estimation algorithm, considering both perfect match (PM) and mismatch (MM) probes. This algorithm alleviates the problem of unstable expression values to some extent when a fraction of the probes is eliminated in our analysis.KBH carried out probe set matching, performed BLAT searches as well as statistical analysis, and drafted the manuscript. SWK carried out the raw data processing, performed statistical analysis, and provided input on drafts of the manuscript. SAG participated in the design of the study as well as providing the microarray data set for this study. PJP conceived the original idea of this study, participated in its design and coordination, and wrote sections of the manuscript. All authors read and approved the final manuscript.Supplementary material for the paper "Combining gene expression data from different generations of oligonucleotide arrays" Supplementary figures for the paperClick here for file


The incidence of fractures in rural populations is lower than in urban populations, although the reason for this difference is unclear. This cross-sectional study was designed to examine the difference in bone mineral density (BMD), a primary predictor of fracture risk, between urban and rural Thai populations.Femoral neck and lumbar spine BMD was measured by dual-energy X-ray absorptiometry (GE Lunar, Madison, WI) in 411 urban and 436 rural subjects (340 men and 507 women), aged between 20 and 84 years. Body mass index (BMI) was calculated from weight and height.P < 0.001), but the difference was not observed at the lumbar spine. After stratifying by sex, age group, and BMI category, the urban-rural difference in femoral neck BMD became more pronounced in men and women aged <50 years and with BMI ≥ 25 kg/m2.After adjusting for age and body weight in an analysis of covariance model, femoral neck BMD in rural men and women was significantly higher than those in urban men and women (These data suggest that femoral neck BMD in rural men and women was higher than their counterparts in urban areas. This difference could potentially explain part of the urban-rural difference in fracture incidence. Osteoporosis and its ultimate consequence of low traumatic fracture pose a major public health problem, because it incurs significant costs and increased risk of mortality -3. OsteoMeasurement of bone mineral density (BMD) is considered the primary predictor of fracture risk . TherefoThe pace of urbanization in developing countries is more pronounced than in developed countries. Therefore, developing countries are ideal settings for studying the urban-rural difference in BMD. The aim of this study was to examine the difference in BMD between an urban population and a rural population in Thailand.The present study was designed as a cross-sectional, population-based investigation. The setting was Bangkok city and Khon Kaen province in Thailand. Bangkok is the capital city with a population of 5.7 million and Khon Kaen is a rural province, located 445 km northeast of Bangkok with a population of 1.8 million and is largely an agricultural community. Further details of this study have been described elsewhere The study included 872 Thai men and women, aged between 20 and 84 years, of whom 422 subjects were from Bangkok and 450 subjects from Khon Kaen. In Khon Kaen, subjects were recruited from 2 villages in the Muang district. There were 14 hamlets in the two villages. In each hamlet, a full list of subjects was obtained, from which 40 subjects were randomly selected by the village's administrator. The selected subjects were then sent a letter of invitation to participating in the study. The response rate was 80.3%. In Bangkok, subjects were recruited via a media campaign, and the sampling technique was similar to the scheme used in Khon Kaen, where subjects were randomly selected from 5 districts within the city of Bangkok.All Khon Kaen subjects were farmers, while Bangkok subjects were office workers, factory workers or house workers. Twenty-one subjects were excluded from analysis because of bone disorders, chronic diseases, history of taking medications that are deemed to affect calcium and bone metabolism, such as the use of steroids or thyroid hormone; and 4 women were excluded on the basis of pregnancy, lactation, delivery or abortion within the previous 3 months, previous history of oophorectomy and premature menopause. The study was conducted in accordance with the Helsinski Declaration in 1975 and as revised in 1983 and was approved by the Ethics Committee of Faculty of Medicine Ramathibodi Hospital Mahidol University (Bangkok) and Khon Kaen University (Khon Kaen), and written informed consent was obtained from all subjects.2, was measured by dual-energy X-ray absorptiometry with a Lunar DXP-IQ densitometer (GE Lunar Radiation Corp, Madison, WI, USA). The two study sites (Bangkok and Khon Kaen) used the same model of the DXA machine and the same protocol of measurements. The radiation dose with this method is < 0.1 μGy. The coefficient of variation of BMD for normal subjects is 0.96 and 0.98 at proximal femur and lumbar spine, respectively.BMD at the femoral neck and lumbar spine (L2-4) in g/cmBody weight (including light indoor clothing) was measured using an electronic balance (accuracy 0.1 kg) and standing height (without shoes) with a standiometer (nearest 0.1 cm). Body mass index (BMI) was calculated as ratio of weight (in kg) over height (in meter squared).Descriptive statistics were computed for each residential region and sex separately. In order to test for difference between urban and rural regions, an analysis of covariance (ANCOVA) model was performed. In this model, BMD was treated as outcome variables; age and weight (or BMI) were treated as covariates; and residence  was the factor. Interactions between age and BMI or age and residence variable were also considered in the model. Estimates of the model parameters were based on the least square method via the SPSS version 9.0 (SPSS, Inc, Chicago).P < 0.001), whereas urban women had a greater height (P < 0.001) but equivalent weight (P = 0.72) compared with rural women (Table After excluding 25 subjects, data from 847 subjects (340 men and 507 women) were analysed. There was no significant difference between urban and rural subjects with respect to age or sex distribution. The mean age was 49 and 50 years old in men and women, respectively. However, urban men had higher weight and greater height than rural men P < 0.00, whereasr = 0.13, P = 0.017 for femoral neck, and r = 0.37, P < 0.001 for lumbar spine) and in women . On the other hand, advancing age was associated with a significant reduced BMD in men  and women .In the entire sample, higher weight was associated with higher BMD in men  BMD in urban subjects .However, the strength of relationship between age and BMD in urban subjects was less pronounced than in rural subjects, such that rural women had a higher cross-sectional "rate of bone loss" than urban women, particularly at the femoral neck. For example, in women, each 5-year increase in age was estimated to associate with a 2.1% and 1.2% decrease in femoral neck BMD for rural and urban group, respectively; in men, the respective rate of decrease was 1.3% and 0.8%. As a result, among those aged 502 significantly higher (P < 0.001) than in urban men and women, respectively; but the difference was lower for the lumbar spine BMD (0.12 g/cm2 in men, P = 0.017 and 0.05 g/cm2 in women, P = 0.293). The statistical significance of the age-by-residence interaction term in the ANCOVA model suggested that the urban-rural difference in BMD decreased with advancing age . This trend was consistent for men and women. However, for lumbar spine BMD, no significant urban-rural difference was observed in most subgroups, with the exception of women aged ≥ 50 years and BMI < 25 kg/m2 in whom BMD was lower in the rural group compared to the urban group  and higher BMI . This finding was partially consistent with a previous study [The present study's data and design can not elucidate any underlying factors that are responsible for the difference but some propositions could be put forward. The urban-rural difference in femoral neck BMD could be due to the difference in the peak of bone mass levels. In this study, both rural men and women aged between 20 and 30 years had significantly higher BMD than urban counterparts. For example, young rural men and women had significantly higher than urban subjects  declare that they have no competing interests.Chatlert Pongchaiyakul had an active role in the conduct of this study, obtained and analysed data, and drafted the manuscript. Tuan V Nguyen was involved in the conceptual discussion of this study, and had an active role in data analysis, drafting of the manuscript. Vongsvat Kosulwat, Nipa Rojroongwasinkul, and Somsri Charoenkiatkul had an active role in the study design, and was involved in the conceptual discussion. Rajata Rajatanavin conducted and established this study. All authors contributed to the last version of the manuscript.The pre-publication history for this paper can be accessed here: