Fugu rubripes, to identify nearly 1,400 highly conserved non-coding sequences. Given the evolutionary divergence between these species, it is likely that these sequences are found in, and furthermore are essential to, all vertebrates. Most, and possibly all, of these sequences are located in and around genes that act as developmental regulators. Some of these sequences are over 90% identical across more than 500 bases, being more highly conserved than coding sequence between these two species. Despite this, we cannot find any similar sequences in invertebrate genomes. In order to begin to functionally test this set of sequences, we have used a rapid in vivo assay system using zebrafish embryos that allows tissue-specific enhancer activity to be identified. Functional data is presented for highly conserved non-coding sequences associated with four unrelated developmental regulators (SOX21, PAX6, HLXB9, and SHH), in order to demonstrate the suitability of this screen to a wide range of genes and expression patterns. Of 25 sequence elements tested around these four genes, 23 show significant enhancer activity in one or more tissues. We have identified a set of non-coding sequences that are highly conserved throughout vertebrates. They are found in clusters across the human genome, principally around genes that are implicated in the regulation of development, including many transcription factors. These highly conserved non-coding sequences are likely to form part of the genomic circuitry that uniquely defines vertebrate development.In addition to protein coding sequence, the human genome contains a significant amount of regulatory DNA, the identification of which is proving somewhat recalcitrant to both in silico and functional methods. An approach that has been used with some success is comparative sequence analysis, whereby equivalent genomic regions from different organisms are compared in order to identify both similarities and differences. In general, similarities in sequence between highly divergent organisms imply functional constraint. We have used a whole-genome comparison between humans and the pufferfish,  Highly conserved non-coding sequences in vertebrate genomes are frequently located around genes involved in development and can direct tissue-specific gene expression in functional assays. The idea that animal development is controlled by cis-regulatory DNA elements (such as enhancers and silencers) is well established and has been elegantly described in invertebrates such as Drosophila and the sea urchin [cis-regulatory elements and their genomic locations. Unfortunately, initial in silico identification of such sequences is difficult, as current knowledge of their syntax or grammar is limited. By contrast, computational approaches for protein-coding exon prediction are well established, based on their characteristic sequence features, evolutionary conservation across distant species, and the availability of cDNAs and expressed sequence tags (ESTs), which greatly facilitate their annotation.Identification and characterisation of a urchin ,2,3,4. Tcis-regulatory elements. Comparing DNA sequences from different organisms provides a means of identifying common signatures that may have functional significance. Alignment algorithms optimise these comparisons so that slowly evolving regions can be anchored together and highlighted against a background of more rapidly evolving DNA that is free of any functional constraints.The completion of a number of vertebrate genome sequences ,6,7,8,9,One of the key decisions inherent in comparative genomics is the choice of organisms for which the comparison will be made. A number of successful pairwise and multiple-species sequence comparisons have already been carried out to identify novel enhancer elements in mammalian genomes ,18,19,20(Fugu rubripes) gene regions has readily identified highly conserved non-coding sequences that have been shown to have some function in vivo [Fugu last shared a common ancestor around 450 million years ago [Fugu genome has the added advantage of being highly compact, reducing intronic and intergenic distances almost 10-fold [Another highly successful approach to increasing the resolving power of comparative analyses is to use multi-species alignments combining both closely related and highly divergent organisms ,22,23,24 in vivo ,32,33,34ears ago , predati 10-fold ,36. WithFugu. A general strategy for testing whether non-coding regulatory sequences of this type are functionally relevant involves assaying their ability to up-regulate reporter gene expression in vivo. “Enhancer” assays using transgenic animals, in particular mouse, are both slow and laborious, but have provided some exciting data on the function of non-coding DNA around developmental genes (e.g., [(Danio rerio) embryos [As a first step towards identifying such networks in humans, we have used comparative genomics to identify and begin to characterise non-coding sequences that are highly conserved between human and s (e.g., ). An alt embryos ,38,39, wWe have adopted a medium-throughput strategy to test DNA sequences for enhancer activity in zebrafish embryos, whereby results may be cross-referenced and compared through a generalised scheme. We present functional data for 25 highly conserved non-coding sequences that are located around four unrelated developmental regulators, SOX21, PAX6, HLXB9, and SHH in order to demonstrate the suitability of this screen to a wide range of genes and expression patterns.Fugu genome assembly [Fugu genome. This is due to additional gene or genome duplications in the teleost lineage [To locate conserved non-coding sequences, we masked the majority of the coding and tRNA content of the assembly  and compassembly  with theassembly . From th lineage , where r lineage . To avoiFugu sequence. The zebrafish, chicken, mouse, and rat genomes are at different stages of completeness, and therefore missing sequence information may account for the missing CNEs (as well as the lower percent identity in zebrafish), although it may also reflect regulatory differences between the lineages.The set of CNEs comprise a total of 273 kb of sequence, with a maximum length of 736 bp (average = 199 bp) and identity ranging from 74% to 98% (average = 84.3%). This is considerably higher than the level of identity seen between coding regions in these two organisms. Unsurprisingly, virtually all of the CNEs are conserved in rodent and chicken genomes, as well as a majority in the zebrafish genome. Of the 1,373 CNEs, 1,365 are conserved in the mouse, 1,316 in rat, and 1,310 in chicken, aligning to the human sequence with average identities of 97% for mouse and rat and 96% for chicken; 1,093 are also found to be conserved in the zebrafish genome, aligning with an average identity of 87.6% to the −76  relate to transcriptional regulation and development (We then looked at the type of genes that are associated with CNEs in the human genome. For each CNE, we extracted the closest gene from Ensembl and submitted the resulting set of genes to GOstat  in orderelopment .trans-dev genes). Over 93% of the clusters (154/165) have a trans-dev gene located within 500 kb of one or more of its CNEs . Finally, there are three clusters that associate with two or more zinc finger genes.Whilst most of the clusters can be associated with one Trans-dev genes associated with CNE clusters tend to be located in regions of low gene density. We counted the number of genes situated within 500 kb upstream and downstream of a trans-dev gene, and compared this with the average for all human genes. Whereas the average for all human genes is 17, it is just six for the trans-dev genes. This is similar to the “gene desert” phenomenon described around the DACH gene [ACH gene . Interestrans-dev genes that we identified have previously been shown to have highly conserved cis-regulatory elements associated with them, including the Hox clusters [Fugu genomes and are located in a large gene desert on human Chromosome 22. Given that gene annotation and genomic sequencing of parts of the human genome are not yet fully complete, the discovery of CNEs here may well point to the existence in this region of an important transcriptional or developmental regulation gene with which they are associated. Indeed we find the largest number of CNEs (48) clustered around a relatively uncharacterised gene with zinc finger domains, ZNF503 on human Chromosome 10, the rat orthologue of which was recently characterised as a probable transcriptional regulator in brain development [A number of the clusters ,33, PAX6clusters , PAX9 [3clusters , SOX9 [2clusters , OTX2 [3clusters , SHH [30clusters , and DACclusters . Five CNelopment .All CNEs were compared with each other to look for local similarities. Forty-three elements show significant similarity to at least one other CNE, and in each case are situated close to genes with clear paralogous relationships, e.g., the HOX and IRX clusters. The remainder of the sequences appear to be unique in the human genome.trans-dev genes associated with CNE clusters, stringent whole-genome alignment located 408 CNEs, whereas MLAGAN identified over twice as many conserved regions (871) of at least 100 bp in length. The whole-genome analysis was more stringent in that we used a minimum exact word match of 20 bp, whereas MLAGAN uses short inexact words to create anchors from which a more sensitive (Needleman–Wunsch) alignment is carried out. It is important to note that similar alignments on genes that are not implicated in developmental regulation do not identify conserved non-coding sequence  alignment tool kit . This toe e.g., ,51)..trans-deFugu sequence, one CNE (SOX21_19) has 90% identity over 558 bp whilst another (SOX21_1) contains a 112-bp region of 100% identity  A. In mamidentity B, demonsCiona intestinalis, Drosophila melanogaster, and Caenorhabitis elegans, to see whether we could identify any of these highly conserved vertebrate sequences within the invertebrate lineage. Although many of the genes identified in our analysis have clear homologues within these genomes, we found no significant matches to any CNEs. More sensitive alignment using MLAGAN also failed to identify any conserved non-coding sequence similarity between vertebrates and non-vertebrates (including C. elegans, D. melanogaster and A. gambiae), whilst in each case the coding sequences were identified. This is surprising, given that the degree of identity between CNEs in vertebrates is higher than that of the coding regions for these genes. Thus, it is unlikely that the same set of sequences that appear to regulate important vertebrate trans-dev genes are found in invertebrates.Finally we searched invertebrate sequence databases, including the whole-genome sequences of We have assayed the ability of conserved non-coding sequences identified both from the whole-genome MegaBLAST analysis (CNEs) and from regional MLAGAN alignments (rCNEs) to up-regulate green fluorescent protein (GFP) reporter expression in zebrafish embryos see . We chosA total of 25 conserved non-coding regions were selected , and S1,Controls in which no element was injected , in which non-conserved, non-coding genomic DNA from the PAX6 or SOX21 regions was co-injected with the GFP reporter, or in which conserved, coding DNA from PAX6, SOX21, or SHH exons was co-injected with the GFP reporter produce essentially no up-regulation of GFP expression . When coIn order to build up a comprehensive picture of the GFP expression pattern induced by each of the elements, the expression profiles from multiple embryos positive for a given element were overlaid onto a schematic diagram, so providing a composite overview for each element . This alOf the eight SOX21-associated elements tested in our functional assay, seven enhance GFP expression . Three oThree elements strongly enhance GFP expression in the sense organs: SOX21_4 and SOX21_19 direct GFP expression to the developing eye . PAX6 is associated with the loss-of-function disorder aniridia. Some aniridia cases show chromosomal rearrangements downstream of an intact PAX6 gene, indicating that cis-acting elements can influence PAX6 gene expression in the eye at a significant distance from the coding region [cis-acting elements in upstream, intronic, and downstream positions. For example, 5′ elements drive expression in the lens, pancreas, and parts of the neural tube [Significantly, PAX6 is a paired-box-containing transcription factor, expressed in and playing essential roles in the developing eye; it is also expressed in the forebrain, hindbrain, and spinal cord  and thWe assayed six elements associated with a genomic region containing the HLXB9 and KIAA0010 genes . Each ofhb9, is expressed in the notochord, hypochord, tail mesoderm, and tailbud [HLXB9 is a Mnx-class homeobox gene associated with autosomal dominant caudal defects . The zeb tailbud , paralleTwo of the four SHH-associated elements tested in this study  direct Gshh and its co-orthologue twhh are both expressed predominantly in midline structures, i.e., floorplate and notochord. Later expression domains include the branchial arches, pectoral fin buds, and the retina [shh/twhh expression overlap in the floorplate; however, most of the other domains of GFP expression . In zebe retina ,63. GFP Understanding the intricate and finely tuned process of gene regulation in vertebrate development remains a major challenge facing post-genomic research. In order to begin to understand how genomic information can coordinate regulatory processes, we have adopted an approach integrating comparative genomics and a medium-throughput functional assay. Nearly 1,400 non-coding DNA sequence elements were identified that exhibit extreme conservation throughout the vertebrate lineage. Despite a degree of overlap, less than half of the non-coding ultra-conserved regions (109 out of 256) identified from the mouse and human genomes  are presIn agreement with current GRN models, and given the function of many of the genes we have identified in our analysis, it is logical to speculate that CNEs consist of modules of binding sites for transcription factors. However, the model of CNEs as transcription factor binding sites, even for large numbers of transcription factors, does not fully explain their high sequence identity across vertebrates, given that transcription factor binding sites are generally rather short and exhibit a level of redundancy. Consequently, we have not ruled out the possibility that the CNEs may have a completely different mode of action or act in numerous different ways.Fugu) together with some degree of proportional compaction in the Fugu genome. This suggests that the CNEs might play a role in structuring the genomic architecture around trans-dev genes, which in turn may lead to an additional level of transcriptional control. Further evidence that genomic architecture may be important comes from the fact the trans-dev genes are generally located in regions of low gene density.The relative positions and order of CNEs within a cluster is completely conserved in all vertebrate genomes we have analysed .Having established a “map” of the major locations of CNEs in the genome, we were able to take a more sensitive alignment approach in a number of these regions in order to identify additional CNEs (rCNEs). The distinction between CNEs and rCNEs is purely a bioinformatics one, based on our search parameters, and we have no reason to believe that there is any functional distinction between the two sets of elements. We selected a number of elements (both CNEs and rCNEs) as candidates for functional analysis. Data from our functional assay of 25 elements from four different developmental genes demonstrate that a significant proportion can act as enhancers, inducing expression of a GFP reporter gene in a tissue-specific manner. The observed expression patterns differ among elements, but are reproducible for individual elements. Enhanced GFP expression domains frequently coincide with endogenous expression domains of the Fugu genomes. This is exacerbated by the fact that some CNEs may also be found within or around neighbouring genes. This phenomenon has been described for both the PAX6 [Fugu) than to SHH (1,000 kb in human and 60 kb in Fugu). Furthermore, a number of elements are located directly 5′ of the HLXB9 gene, whilst others are found located further upstream, in introns of the next gene, KIAA0010. Although we strongly suspect that all these elements are associated functionally with the HLXB9 gene (e.g., KIAA0010_1 directs expression prominently to the notochord, an expression domain of the zebrafish HLXB9 orthologue), we cannot rule out the possibility that they may associate with the SHH gene , which lies a few genes downstream. There are a number of cases where a CNE cluster is located close to more than one trans-dev gene, illustrating the value of correlating endogenous expression pattern with CNE enhancer activity. However, it should be noted that in order to build GRN maps for the elements, it is desirable but not essential to know which element associates functionally with which gene.Whilst it is straightforward to assign CNEs unequivocally to the SOX21 and PAX6 genes based on their location in the genome, the situation is more complex for elements in the vicinity of the SHH and HLXB9 genes, which are situated in close proximity to each other in the human, rodent, and the PAX6  and PAX9the PAX6  genes, athe PAX6 , where atrans-dev gene density.Our confidence in the correctness of our gene assignment for the elements tested in this study is borne out by the results of our functional analysis. For the elements that we have associated with PAX6 and SOX21, there is a good correlation between tissues that express the gene endogenously and tissues induced by the associated co-injected elements to express GFP, i.e., the major sites of endogenous gene expression are highly represented in our mosaically expressing embryos  to around 44% (SHH_6); this is probably due to many factors, e.g., variations in the embryonic stage at the time of injection and stochastic variations from embryo to embryo with regard to which cells the injected DNA is segregated into during cleavage. However, by combining expression data from a number of expressing embryos (an average of 30 embryos per positive element), we can gain insight into the overall pattern of reporter gene expression prescribed by each element.In addition to seeing GFP expression in “expected” domains (with respect to the associated gene), GFP expression was also often detected in tissues in which the associated gene is not normally expressed , subtle differences between CNE sequences may be tested functionally in this system. Zebrafish embryos are both readily produced and easily visualised, allowing convenient live screening throughout development. Their transparency makes the embryos ideally suited to GFP analysis and the problems associated with mosaicism in this screen are relatively easily overcome by injecting large numbers of embryos. Technical advances, such as the use of meganuclease injection, may facilitate this further.Fugu to the EMBL nucleotide database and are developing a publicly available relational database in order to store, curate, and analyse data from this study as well as data generated from ongoing identification and characterisation of rCNEs surrounding trans-dev genes.The combination of a comparative genomics approach together with functional screening of conserved elements produces a large and complex dataset. Efficient access, integration, and interrogation of this bioinformatics and functional data is crucial, and of increasing interest to the scientific community, to begin to characterise GRNs in vertebrates. To this end, we have submitted all CNE DNA sequences from We have identified an important set of highly conserved non-coding vertebrate sequences that associate with developmental regulators and have provided evidence that at least some of them demonstrate regulatory function. They are likely to be implicated in genetic disease, as has already been shown for the SHH gene . Their dFugu draft genome assembly . These predicted sequences were then masked in the Fugu sequence by supplying them as a “repeat library” to Repeatmasker35. The masked sequence was similarity searched against human genomic sequence from the Ensembl [Fugu sequences with alignments of 100 bp or over were selected to form the initial CNE sequence dataset.GENSCAN  . The GOstat program was used to find statistically over-represented GOs in this group of genes [The closest gene (using the transcription start site as defined in Ensembl) to the start of each CNE was determined from a list of all human genes supported by external evidence (“known” genes) downloaded using EnsMart, available from the Ensembl Web site  , D. melanogaster (v21.3.1), and Anopheles gambiae (v21.2.2) was carried out using MLAGAN as above.To look for the presence of CNEs in other available vertebrate genomes, CNEs were similarity searched against Ensembl mouse (v19.32.2), rat (v21.3.2), chicken (v22.1.1), and zebrafish (v21.3.2) genome sequences using BLASTn with default parameters. All invertebrate sequences in the EMBL database were searched in the same way using BLASTn with non-stringent parameters . More sensitive alignment of flanking orthologous sequence around the SOX21 gene (up to the coding sequence of the genes on either side) from Ensembl Zebrafish were raised and bred and embryos staged following standard protocols ,78; stagWe assayed for enhancer activity in embryos co-injected with candidate enhancer elements or control DNA and a minimal promoter–reporter construct in a method adapted from Muller and colleagues  as descrFugu genomic DNA (see For the preparation of DNA and micro-injection, CNEs, rCNEs, and negative controls were PCR-amplified from For screening of embryos and data collection, on the second day of development (approximately 26–33 hpf), injected embryos were anaesthetised in Tricaine  and analmscooper@uwashington.edu;data relating to tissue category was also recorded on a spreadsheet.GFP-expressing cells were classified according to the following tissue categories: forebrain, midbrain, hindbrain, spinal cord, eye, ear, notochord, muscle, blood (circulating)/blood islands, heart/pericardial region (Please note: Some cells classified in this category may be circulating blood cells), epidermis/EVL, or fins. Cells that did not fall into one of these major expression categories  were categorised as “other”. The location and tissue category of each GFP-expressing cell for each embryo was recorded schematically using Adobe Photoshop software , by manually drawing colour-coded schematised cells in appropriate positions onto an overlay of a camera lucida drawing of a 31-hpf embryo  using standard protocols  and the Figure S1Fugu DNA.Each sequence represents the PCR product used in the functional assay. Sequence in bold type represents the position of the conserved element or elements within the PCR product. All PCR products were generated from (61 KB DOC).Click here for additional data file.Table S1(40 KB XLS).Click here for additional data file.Table S2(67 KB DOC).Click here for additional data file.All 1,373 CNEs (CR846105 to CR847477) and 16 rCNEs (CR847478 to CR847493) have been submitted to the EMBL database.


We set out to explore regulatory domains of the cIRK1 promoter that enhance or inhibit expression of the gene in different cell types.Inward rectifier potassium channels (IRK) contribute to the normal function of skeletal and cardiac muscle cells. The chick inward rectifier KcIRK1. cIRK1 contains two exons with splice sites in the 5'-untranslated region, a structure similar to mouse and human orthologs. cIRK1 has multiple transcription initiation sites, a feature also seen in mouse. However, while the chicken and mouse promoter regions share many regulatory motifs, cIRK1 possesses a GC-richer promoter and a putative TATA box, which appears to positively regulate gene expression. We report here the identification of several candidate cell/tissue specific cIRK1 regulatory domains by comparing promoter activities in expressing (Qm7) and non-expressing (DF1) cells using in vitro transcription assays.We cloned and characterized the 5'-flanking region of cIRK1 expression are similar to those seen in mKir2.1, the cIRK1 promoter differs by the presence of a putative TATA box. In addition, several domains that regulate the gene's expression differentially in muscle (Qm7) and fibroblast cells (DF1) were identified. These results provide fundamental data to analyze cIRK1 transcriptional mechanisms. The control elements identified here may provide clues to the tissue-specific expression of this K+ channel.While multiple transcription initiation sites and the combinatorial function of several domains in activating  Using in vitro promoter assays with fragments of the cloned cIRK1 locus, we also identified several candidate control domains that may participate in regulating the channel's exquisite tissue-specific transcription.Kir2.1 expression is detected in excitable cells in brain, heart, and skeletal muscle in both mouse and chick -8. In adcIRK1 genomic clones from a chicken genomic DNA phage library. A series of overlapping clones were isolated by screening the library using full length cIRK1 cDNA as a specific probe. Approximately 6.5 kilobase pairs (kb) of the cIRK1 5'-flanking region were sequenced (Genbank AF375660), including exon 1 and a portion of the intron. cIRK1 contains two exons: exon 1 includes only upstream non-coding sequence (5'-untranslated region, 5'UTR) while exon 2 includes 5'UTR (216 bp), the full open reading frame (1,284 bp), and the 3'UTR (520 bp) . Sequence data also showed that the intron had consensus donor (GU) and acceptor (AG) sequences  Figure . The sinces Fig. . This ge mKir2.1 , which pframe 1,24 bp, andcIRK1 using primer extension and 5'RACE. When the specific reverse complementary sequence to the cIRK1 5'UTR at positions 61–41 was utilized as a primer for reverse transcription, a product of approximately 80 bases was generated from brain mRNA  5'-primer and the reverse primer R1. Subsequent nested PCR, using the supplied 5'-nested primer and a second reverse primer R2 designed at positions 64–41 of cIRK1 cDNA, produced multiple fragments. Those of approximately 110 bp were determined to be specific products of the primer set (N-R2) because they were not detected when the PCR was carried out using either the 5'-nested primer alone (N only) or the R2 primer alone , which occurred with similar frequencies. Therefore, the actual transcribed 5'-flanking regions were 16, 17, 18, or 36 nucleotides longer than the 5'-end of the clone originally isolated from a chick cochlea cDNA library [cIRK1 cDNA, was numbered +1. Our identification of multiple transcription initiation sites indicates that exon 1 of cIRK1 can be 119, 120, 121, or 139 bases in length.Before studying the motifs and elements of the promoter region, we sought to determine the transcription initiation sites of RNA Fig. . SpecifiRNA Fig. . To confcIRK1 5'-flanking region, based on the presumption that there might be significant divergence between chicken and mammal IRK1/Kir2.1 promoter regions that could account for their different gene expression patterns. The 5'-flanking region of cIRK1, shown in Fig. mKir2.1 promoter [Kir2.1 promoter shows high similarity to the mKir2.1 promoter (62.6% over1,017 bp), however, the hKir2.1 promoter also failed to show significant homology with the cIRK1 5'-flanking region. While both the cIRK1 5'-flanking region and the mKir2.1 promoter have high GC contents, the chick promoter is substantially more GC-rich (71.0% in chick and 62.8% in mouse from position -390 to -1) and contains 38 CpG dinucleotides as compared to 17 CpGs in mouse. The cIRK1 promoter region has four consensus Sp1 binding elements , absent in mKir2.1, at -56. Overall, while GC-rich domains and motifs are present in both the chick and mouse promoter regions, the extent and number of the motifs are different between the cIRK1 and mKir2.1 promoters. In addition, a TATA box-like sequence is present only in the chick promoter.We then analyzed the motifs contained in the promoter . The corp1 sites . Consensp1 sites  was founcIRK1 transcription. First, we tested whether the 5'-flanking region up to approximately 2 kb contained sufficient elements to activate cIRK1 transcription, and then we investigated the minimal promoter region by constructing a series of 5'-flanking region deletion fragments, including a portion of exon 1, inserted into plasmids upstream of the luciferase reporter  in this region increase promoter activity only in the myoblast cell line Qm7. Candidate elements within this region that might have cell-specific activity are an E box (CAGGTG) at -316 and an activating component of transcription activator NF-κB  at -296 [cIRK1 promoter construct 3B-531 did not enhance promoter activity (data not shown).While the results of our  at -296 ,23. Vari at -296  and MyoD at -296  are repo at -296 . HoweverDrosophila homeotic gene Antennapedia binding motif [+ conductance in inner hair cells [We also identified a region between -1115 and -727 with weak repressor activity that exhibited different properties in the two cell lines. Potential binding motifs present in this region are: a site for interferon regulatory factor-1 at -1030, an AP-4 site at -978, a thyroid hormone beta receptor binding element (TRE) at -962, another E box at -954, a binding site for the potent repressor CTF/NF-I at -859 , and a Dir cells .cIRK1 in DF1 cells which do not express the gene endogenously clearly suggests the existence of additional mechanisms to repress cIRK1 in DF1 cells. Since domains acting to strongly silence cIRK1 expression in DF1 cells were missing in the 5'-flanking region characterized in this study, additional distant control regions may lie further upstream, downstream, or in the intronic sequences. Although at this point clearly speculative, the existence of many CpG dinucleotides in cIRK1 suggests the possibility of gene repression through DNA methylation of CpG dinucleotides leading to heterochromatin condensation of the adjacent genomic locus [cIRK1 genomic locus through DNA methylation.Our observation of the transcriptional activation of ic locus . Tissue-cIRK1. These results provide fundamental data to further analyze cIRK1 transcriptional mechanisms. While the use of multiple transcription initiation sites and the combinatorial participation of multiple domains in activating cIRK1 expression are similar to those seen for mouse Kir2.1, the cIRK1 promoter is distinct in that it exhibits a higher GC-content than does the mKir2.1 promoter, and by the presence of a functional putative TATA box that is not observed in the mKir2.1 promoter. Transcriptional control domains identified here form the foundation of an in-depth analysis of tissue-specific expression of this K+ channel as well as the species-specific expression of cIRK1 in the chicken cochlea.We have identified multiple transcription initiation sites and several candidate regulatory elements in the chicken potassium channel gene IRK1 genomic clones were isolated by screening a chick genomic library in the lambda FIX II vector (Stratagene) using cIRK1 cDNA (GenBank U20216) as a screening probe. Briefly, the probe was random primer-labeled with 32P-α-dCTP and hybridized with phage plaques blotted onto HybondN nylon membranes (Amersham Biosciences) overnight at 65°C in 6x SSC, 250 μg/ml salmon sperm DNA, 5x Denhardt's solution, and 0.1% SDS. The membranes were washed in 1x SSC and 0.1% SDS, and exposed to x-ray film. Four positive clones were further analyzed following digestion with XbaI and/or EcoRI, and subcloning into pBlueScript-KS. The promoter region (AF375660) was sequenced from both ends. Comparisons between the putative human Kir2.1 promoter region (AC005242), the mouse Kir2.1 promoter region (AF072673), and the coding regions of mKir2.1 cDNA (AF021136) and hKir2.1 cDNA (U24055) were performed using the Wilbur-Lipman DNA alignment method. Transcription factor binding elements were predicted based on the TRANSFAC algorithm [.Chick 32P-end-labeled primer in 10 mM DTT, 50 μg/ml actinomycin D and 0.5 mM dNTP using Superscript II (Invitrogen) at 47°C for 60 min. The primer was designed at 61–41 of cIRK1 cDNA (U20216) [5'-TGT TAA GAT CCG CGG GGA CAC-3']. Reaction mixtures were fractionated on 7% polyacrylamide (PAA)/7 M Urea gels. The 3'-most transcription initiation site was numbered +1.Total RNA was extracted from tissues and cells using TRIZOL reagent (Invitrogen) and treated with DNaseI. Three micrograms of total RNA (brain, DF1, or yeast tRNA) were reverse transcribed with cIRK1 cDNA [5'-GCA GAG TTA GCT TAA CAA GTA ACC G-3'] at 42°C for 1 hr. The PCR was performed in a reaction mix containing 1x PCR buffer, 200 nM each of 5'-forward primer [5'-CGA CTG GAG CAC GAG GAC ACT GA-3'] and R1, 100 μM dNTPs, 5 μCi 32P-α-dCTP, 5% DMSO, and 1.25 U of AmpliTaq (Perkin-Elmer). Reaction conditions were; 94°C for 3 min, 5 cycles at 94°C for 1 min, 57°C for 5 min, 72°C for 2 min, and then 30 cycles at 94°C for 1 min, 57°C for 1 min, 72°C for 1 min, followed by 72°C for 5 min. Nested PCR was performed using the same conditions, with first round PCR products as template, and using the 5'-nested forward primer [5'-GGA CAC TGA CAT GGA CTG AAG GAG TA-3'] and R2 primer designed at 64-41 of cIRK1 cDNA [5'-GGG TGT TAA GAT CCG CGG GGA CAC-3']. Reaction products were eluted from the 7% non-denaturing PAA gel, re-amplified, then ligated into the pCR4-TOPO-TA cloning vector (Invitrogen) and sequenced.RNA ligase-mediated rapid amplification of 5' cDNA ends (5'RACE) was carried out using the GeneRacer kit (Invitrogen). In brief, 3 μg of total RNA were dephosphorylated, decapped and ligated with a 44-base RNA-oligo, according to manufacturer protocols. Next, the RNA was reverse transcribed using R1 primer designed at 146-122 of 32P-labeled cIRK1 cDNA fragment  overnight at 42°C, washed, and exposed to x-ray film. A quail glyceraldehyde-3-phosphate dehydrogenase (g3pdh) gene fragment  was used as an internal control. Message sizes were estimated by comparisons with RNA molecular weight markers (Invitrogen) run in the adjacent lane.Northern blotting was performed using poly A(+) RNA isolated using the FastTrak 2.0 kit (Invitrogen). Five micrograms of poly A(+) RNA were applied to each lane, fractionated on a 0.8% agarose gel, and transferred to a HybondN membrane. The membrane was hybridized with a random-primed cIRK1 5'-flanking region, AF375660), 3B-1229 (5228–6456 bp), 3B-531 (5926–6456 bp), and 3B-226 (6231–6456 bp) were generated by PCR, cloned into pBS-KS, and then subcloned into the multiple cloning site (between KpnI and XhoI) of pGL-3B (Promega). Constructs 3B-1089 (5368–6456 bp) and 3B-840 (5617–6456 bp) were obtained from a PCR fragment (1229 bp) with the 5'-region digested by SpeI and PvuII or XbaI, respectively. 3B-399 (6058–6456 bp) was derived from 3B-531 by removing the 5'-region by SacI and SmaI digestion, and 3B-206 was generated by exonuclease III digestion of 3B-226 followed by recircularization. 3B-109 (6348–6456 bp) was generated by SpeI and NheI digestion of 3B-206. Construction of 3B-206 with a mutated putative TATA box (3B-206dT) was accomplished by replacing the putative TATA box (TATTAA) with a StuI site (AGGCCT) by amplifying the original construct using primer sets designed to be complementary to sequences just outside the elements of interest, yet including StuI sites at their 5'-ends.Primers were designed to amplify each promoter region of interest. Constructs 3B-2256  with 1.5 g/L sodium bicarbonate, 10% fetal calf serum, and 100 U/ml penicillin/streptomycin. The quail myoblast cell line Qm7 [2 at 37°C. The ability of Qm7 cells to differentiate into myotube-like morphology was confirmed periodically as previously described [2HPO4, 6 mM dextrose, 25 mM HEPES at pH = 7.15, and 120 mM of CaCl2), and were incubated with 2 × 104 cells in 24-well dishes for 8 hrs in 500 μl of DMEM. The transfection mixes were replaced with fresh growth media and incubation was continued for 48 hrs. In vitro promoter activities were determined using the Dual-Luciferase Reporter Assay System (Promega). Briefly, transfected cells in each well were lysed in 100 μl of 1x Passive Lysis Buffer for 15 min at room temperature. Lysates were centrifuged briefly, and 20 μl of the supernatant was mixed with 100 μl of Luciferase Assay Reagent II, followed by 100 μl of SG reagent. Luciferase activities reflecting cIRK1 promoter activities and control thymidine kinase promoter activities were measured for 10 seconds after premeasurement periods of 2 seconds. All experiments were performed in duplicate and repeated at least 3 times (n= 3–6). The data were analyzed using a t-test assuming equal variance between two samples. Results are shown as fold increases ± standard error when compared with the basal luciferase activities from cells transfected with mock vector pGL-3B.The chick fibroblast cell line DF1 was purchased from ATCC and cultured at 5% COline Qm7  was cultescribed . One miccIRK1. He also conducted 5'RACE, Northern blot, cell culture, construction of deletion mutant, in vitro transcription analysis, and drafted the manuscript. LCK mapped much of the genomic structure and determined some of the sequence. EL and NK participated in maintenance of the cell lines and in vitro transcription analysis. JCO participated in design and coordination of the study and finalized the manuscript.HM obtained the sequence of cIRK1 genomic DNA and did all the database searches to identify motifs and control regions in the 5'-flanking region of


It is generally recognized that there is a need for improved teaching of nutrition in medical schools and for increased education of the general population. A questionnaire, derived in part from a study of physician knowledge, was administered to first year medical students in order to assess their knowledge of various aspects of nutrition and metabolism, and as a teaching tool to transmit information about the subject. The performance of first year students was consistent with a generally educated population but there were surprising deficits in some fundamental areas of nutrition. Results of the questionnaire are informative about student knowledge, and immediate reinforcement from a questionnaire may provide a useful teaching tool. In addition, some of the subject matter can serve as a springboard for discussion of critical issues in nutrition such as obesity and markers for cardiovascular disease. A major barrier to improved teaching of nutrition is the lack of agreement on some of these critical issues and there are apparent inconsistencies in recommendations of government and health agencies. It seems reasonable that improved teaching should address the lack of knowledge of nutrition, rather than knowledge of official guidelines. Student awareness of factual information should be the primary goal. Like many medical schools, SUNY Downstate Medical Center has been trying to improve the teaching of nutrition in the curriculum. The study presented here had two goals. First, we wanted to assess the knowledge of the first year class on the subject and, second, we hoped to use the questionnaire method, with immediate feedback, as a mechanism for imparting information on the areas covered. Whereas some form of nutrition education is part of the curriculum of most medical schools , it is gPerhaps the most important problem in introducing nutrition into the medical curriculum is the lack of agreement on what should be taught. There is strong, even contentious debate about the most fundamental issues such as obesity, cardiovascular disease, and diabetes. Thus, concerns about inadequate physician knowledge frequently refer to ability to counsel patients according to standard guidelines -6. Here The quiz is presented twice: first, as given to medical students, and then, a second time, with the answers and the comments given to students immediately upon completion of the quiz. A follow-up email that was sent to students after results were tabulated has also been reproduced. Results and Discussion provides data on student performance and additional discussion.et al. [The following is a verbatim reproduction (with addition of references) of the questionnaire given to first year medical students at SUNY Downstate Medical Center. The first four questions were taken from Flynn, et al. . FollowiThis questionnaire is anonymous and does not affect your grade. Make up a User Name (in case there is a follow-up) that only you know and that is not common, like Sherlock-37)User Name _________________________________________________The questionnaire is designed to test your general knowledge, not anything you have learned in the course.1. A good source of monounsaturated fat is: _____ Butter_____ Canola Oil_____ Corn Oil_____ Flaxseed Oil_____ Olive Oil_____ Safflower Oil_____ Soybean Oil_____ don't know2. The diet component that is most likely to raise triglycerides is (select one)_____ Fat_____ Carbohydrate_____ Protein_____ don't know3. In general, what effect does a low-fat diet have on triglycerides?_____ Increase_____ Decrease_____ no change_____ don't know4. In general, what effect does a low-fat diet have on HDL-c (high density lipoprotein-cholesterol) ?_____ Increase_____ Decrease_____ No change_____ Don't know5. In the past thirty years, the per cent fat in the American Diet has:_____ Increased_____ Decreased_____ Stayed about the same.6. The most energy dense food  is:_____ Carbohydrate._____ Protein._____ Fat.7. High total blood cholesterol can be lowered significantly by:_____ Diet_____ Drugs such as statins_____ Diet or drugs are equally effective_____ Neitherincrease the risk of cardiovascular disease:8. The dietary change that is most likely to _____ unsaturated fat → saturated fat (that is, replace unsaturated fat with saturated fat)_____ unsaturated fat → carbohydrate_____ carbohydrate → unsaturated fat_____ carbohydrate → saturated fat_____ saturated fat → carbohydrate_____ saturated fat → unsaturated fat9. Glycemic Index measures the increase in blood sugar over 2 hours per gram of carbohydrate ingested, compared to glucose (=100). For each food indicate the approximate glycemic index as: H, high (70–100), M, Medium (40–70) or L, Low (< 40). You may enter a number if you think you know or can figure it out:_____ white bread_____ whole wheat bread_____ ice cream_____ carrots_____ sucrose (table sugar)_____ fructose_____ bran muffin_____ banana10. The substances in the following list that either are themselves or are considered to contain large amounts of complex carbohydrates._____ white bread_____ whole wheat bread_____ ice cream_____ fructose_____ sucrose (table sugar)_____ corn starch_____ fiber11. In the first list check the vitamins that are generally considered to have antioxidant activity. In the second list check the vitamins that are precursors for oxidative coenzymes. ANTIOXIDANTS_____ ascorbic acid (vitamin C)_____ niacin_____ riboflavin_____ thiamine6)_____ pyridoxal phosphate  that reported the results of a questionnaire designed to assess the level of knowledge of physicians. The authors were critical of the responses; the paper was entitled "Inadequate physician knowledge of the effects of diet on blood lipids and lipoproteins." In particular, "Physicians showed a poor understanding of the effects of changing the relative intake of carbohydrates and fats on triglycerides and HDL."The first four questions appeared in a recent paper (Flynn M, 106:3143–3421). The ATP III, compared to previous version, advocates lowering triglycerides as a secondary target to lowering LDL. The major conclusions:The rationale for the study were guidelines recommended by the Third Report of the National Cholesterol Education Program Adult Treatment Program  did not know that a low-fat diet, in general, would increase blood triglycerides.• Approximately three-quarters (70% of cardiologists vs. 77% of internists) did not know a low-fat diet would decrease HDL; almost half (45%) thought that a low-fat diet would not change HDL.• About one-half  did not know carbohydrate was the diet component most likely to raise triglycerides.1. A good source of monounsaturated fat is: _____ ButterX _ Canola Oil_ _____ Corn Oil_____ Flaxseed OilX _ Olive Oil_ _____ Safflower Oil_____ Soybean Oil_____ don't knowIt is generally considered that monounsaturated fats are protective of cardiovascular disease (as in the Mediterranean Diet). The effect of monounsaturated fat explains the anomaly in the so called Seven Countries study  which laSome people may be surprised when they actually look at the data Figure . High cois a plant in the Brassica family) and is the subject of popular controversy because of the possible production of trans-fatty acids during processing.You should also know that canola oil is named for CANadian OiL (there is no canola tree) and is a highly processed version of rapeseed oil (which 2. The diet component that is most likely to raise triglycerides is (select one)_____ FatX _ Carbohydrate_ _____ Protein_____ don't know3. In general, what effect does a low-fat diet have on triglycerides?X _ Increase_ _____ Decrease_____ no change_____ don't knowThe phenomenon of carbohydrate-induced hypertriglyceridemia is well established in the literature -15. It i4. In general, what effect does a low-fat diet have on HDL-c (high density lipoprotein-cholesterol)?_____ IncreaseX _ Decrease_ _____ No change_____ Don't know5. In the past thirty years the per cent fat in the American Diet has:_____ IncreasedX _ Decreased_ _____ Stayed about the same.trans fats) is being consumed although no effect of fat per se on obesity, independent of calories, has been found.This is the major point of attack on current guidelines since the obesity epidemic correlates with a decreased percentage of fat in the diet  and increased carbohydrate consumption ,24. Defe6. The most energy dense food  is:_____ Carbohydrate._____ Protein.X _ Fat._ The operational numbers in kcals/g are 4, 4, and 9 for carbohydrate, protein, and fat. This is the basis of traditional recommendations for low fat diets for obesity. The reduction in percentage of dietary fat and the obesity epidemic noted above, however, suggests that this is not a good universal principle. The role of macronutrient composition on satiety, taste, total consumption and effect on weight loss is largely unknown due to the multiple factors and individual differences, and some experimental support can be found for just about any idea.7. High total blood cholesterol can be lowered most significantly by:_____ DietX _ Drugs such as statins_ _____ Don't know_____ Diet or drugs are equally effective_____ NeitherDrugs such as statins (HMGCoA reductase inhibitors) are very effective at reducing cholesterol. Diet can also be effective but usually less so. Combination diet and drugs, however, may be most effective but there is, again, not universal agreement as to what that diet should be.increase the risk of cardiovascular disease:8. The dietary change that is most likely to _____ unsaturated fat → saturated fat (that is, replace unsaturated fat with saturated)X _ unsaturated fat → carbohydrate_ _____ carbohydrate → unsaturated fat_____ carbohydrate → saturated fat_____ saturated fat → carbohydrate_____ saturated fat → unsaturated fatIn addition to the effect on risk factors, epidemiologic evidence suggests that replacing fat with carbohydrate is deleterious. Replacing unsaturated fat with saturated fat will also increase the risk of cardiovascular disease ,9,14,25.9. Glycemic Index (GI) measures increase in blood sugar over 2 hrs per gram of carbohydrate, compared to glucose (=100). For each food indicate the approximate glycemic index as: H, high (60–100), M, Medium (40–60) or L, Low (< 40). You may enter a number if you think you know it:H (70) white breadM (52) whole wheat breadM (50) ice creamM (47) carrotsH (70) sucrose (table sugar)L (20) fructoseHM (60) bran muffinM (50) bananaThe GI is a very rough indicator of rise in blood sugar and is influenced by absorption and the concentration of glucose. Fructose has a low GI (20) indicating slow conversion to glucose in 2 hrs but far from being considered a "good" sugar, at high levels may be very deleterious. The concept of glycemic load (GL) which corrects for the amount of carbohydrate per serving may be a better parameter but runs into problems about serving size. So muffins and candy bars have GL = 15 and carrots only 3 but 80 g of carrots may not be a lot for some people -28.10. The substances in the following list that either are themselves or are considered to contain large amounts of complex carbohydrates.? _ white bread_ ? _ whole wheat bread_ NO ice cream_ NO fructose_ NO sucrose (Table Sugar)_ ? _ corn starch_ ? _ fiber_ If you had trouble with this, it is because nobody knows how the term should be used and, in fact, we recommend it not be used at all. The original chemical definition – to some extent still used in organic chemistry – is that a complex carbohydrate is a polysaccharide (not mono- (glucose, fructose) or di- (sucrose, lactose)). Any starch, e.g. corn starch or white bread (poly-glucose: amylose or amylopectin) fits the definition, and it used to be nutritional dogma that, at least in terms of raising blood glucose, complex carbohydrates (that is, polysaccharides) were better than simple sugars. When the dogma was finally tested this turned out not to be true and the concept of glycemic index arose. It is clear from question 9 that white bread is nutritionally similar to pure glucose. Probably because of the evocative nature of the word, the term "complex" is still used. Sometimes it means foods that have a low glycemic index due to poor absorption usually due to the presence of fiber, but it is never precise. When people say complex carbohydrate they usually mean the carbohydrate recommended in their diet and missing from somebody else's diet. Suggested nomenclature is "polysaccharides, starch, high fiber," although fiber itself is a heterogeneous category.11. In the first list check the vitamins that are generally considered to have antioxidant activity. In the second list check the vitamins that are precursors for oxidative coenzymes. ANTIOXIDANTSX _ ascorbic acid (vitamin C)_ _____ niacin_____ riboflavin_____ thiamine6)_____ pyridoxal phosphate (Vitamin B12_____ vitamin B_____ vitamin DX _ vitamin E_ REDOX PRECURSORS_____ ascorbic acidX _ niacin_ X _ riboflavin_ X _ thiamine_ 6)_____ pyridoxal phosphate . Megaloblastic anemia can also be caused by a B12. First, vitamin B12 is a cofactor in formation of the amino acid methionine from homocysteine and the folic acid derivative, methyl-tetrahydrofolic acid (methyl-THF): There are only two reactions in humans requiring vitamin BHomocysteine + methyl-THF → Methionine + THF This explains the relation between dietary folic acid and high homocysteine which is a marker for cardiovascular disease and potential birth defects.12, will prevent this reaction from occurring. The effect of B12 deficiency on folic acid is indirect: if methionine synthesis cannot be carried out, methyl-THF will build up ("methyl trap"). This is effectively a folic acid deficiency and anemia is the outcome. The second requirement for B12 involves organic acids and deficiencies can lead to neurologic damage. The anemia in a B12 deficiency may be successfully treated with folic acid, swamping out the methyl trap. Neurologic damage, however, may still occur unless the B12 deficiency is also treated. A dietary deficiency of B12 is rarely seen since little is needed and it is stored well. Deficiency is usually detected in the elderly due to decreased production of intrinsic factor, a protein required for absorption.A deficiency in folic acid or the cofactor, B12 deficiency could be masked. Since the major deficiency is not dietary but absorptive, the problem can't be solved by simply adding B12 as well.The considerations above bear on the recommendation to add folic acid to manufactured food. Critics point out that by preventing anemia, a BThe following analysis was sent as an Email to students after the scores on the quiz were tabulated.Nutrition Journal 2: 19) that reported the results of a questionnaire designed to assess the level of knowledge of physicians. Their conclusion was that "Physicians showed a poor understanding of the effects of changing the relative intake of carbohydrates and fats on triglycerides and HDL."As indicated in the answer sheet, the first four questions are taken from a recent paper  Interestingly the rationale for the study was determining "Physicians' ability to effectively counsel patients with elevated cholesterol to initiate a Therapeutic Lifestyle Changes Diet (TLC)"  which recommends lowering triglycerides as a secondary target to lowering LDL. The TLC Diet recommends total fat as 25–35 % and carbohydrate at 50–60 %. Paradoxically, Flynn, et al.'s assessment of physician knowledge focused on the deleterious effect of carbohydrate on triglycerides. Given this association, it is puzzling that ATP III would council people trying to lower triglycerides to undertake such a diet.In any case, your performance compared to their sample is shown in Figure Two questions of importance:Question 5:In the past thirty years, the per cent fat in the American diet has declined by about 10 %, close to the target level of 30% of total calories set back in the 70's. In order to explain why this has been associated with an obesity epidemic, many have blamed portion-size, the fast-food industry and consumers themselves. In any case, most people did not know that percent fat consumption had gone down.Answers were:Increased: 76 %Decreased 22 %Same 3 %Question 6. Again, it was surprising that so many people did not know the relative energy density. The answers:Carbohydrate 19 %Protein 7 %Fat 74 %). The discrimination coefficient varies between -1 and +1 and measures the extent to which performance on a particular question reflects performance on the quiz as a whole, that is, whether a question discriminates high performers from low performers. A typically high value of +0.4 means that the question was answered correctly by most students who did well on the exam and answered incorrectly by most who did not.Questionnaires were collected from 111 students, all but 6 of whom answered all the questions. Student answers were tabulated and discrimination coefficients were determined by point biserial correlations  using LXBecause the questions had different goals (knowledge assessment vs. motivation for accepting new information), and because some (e.g. questions 2–4) are interrelated, no measure of performance or statistics were carried out for the quiz as a whole.et al. used four questions that succinctly identified both practical and conceptual knowledge bearing on the ability to implement dietary recommendations from the Adult Training Program (ATP III) of the National Cholesterol Education Program (NCEP)[Results from the first eight questions and the data from Flynn, et al.  are showam (NCEP).First year students did not do as well as physicians at identifying sources of monounsaturated fats. On the other hand, the good discrimination coefficient indicates that knowledge of fat composition is a good indicator of overall knowledge .Although a substantial fraction of cardiologists polled by Flynn knew that carbohydrate raised triglycerides Figure , most intotal amount of fat has decreased, whereas for women there has been a slight increase consistent with the much larger increase in caloric intake among women [prima facie evidence of the effect of macronutrients. Despite the clear correlation between higher carbohydrate, lower fat and obesity, government and health agencies rarely question the appropriateness of the original guidelines and have continued to recommend still higher carbohydrate and still lower fat [Along the same lines, our questionnaire went beyond the area covered by Flynn to consider the changes in diet that have accompanied the epidemic of obesity. It has to be considered very surprising that only 22 % of an educated population knew that the per cent fat in the American diet has decreased Figure ; for menng women . The obsng women  to proviower fat ,30,31. SWe were surprised by some lapses in student knowledge revealed by the questionnaire. The most basic question – which macronutrient is the most energy dense – had only 74 % correct answers. To understand how surprising a response this is, it should be understood that the mean score on exams in this section of the medical course is typically 80 % and, on most exams, several questions are answered correctly by 98–100 % of the class. The National Board of Medical Examiners assumes knowledge of caloric value of macronutrients, and we had expected that it was common knowledge. The questionnaire result indicates that no fact in nutrition is too basic to be excluded from course material.and apple pie for high cholesterol.An overwhelming number of medical students believe that diet is as effective as drugs in lowering total cholesterol. This again attests to the pervasive message that diets control blood cholesterol, an idea continually reinforced by media advertisements, for example, for the cholesterol lowering effect of breakfast cereal. Whereas it is likely that diet is an important influence on CVD, there is, again, the problem of which diet and the question would probably have been better framed, as in questions 2–4, on specific lipid components. It is likely, for example, that many medical students would not know that dietary cholesterol is largely without effect on serum cholesterol. In any case, it is generally acknowledged that, on average, drugs such as statins have a greater impact on cholesterol than currently reported diet interventions. The general effectiveness of statins and the promotion by pharmaceutical companies has, most recently, led to a movement to reinforce the idea that genetics (which can't be controlled by diet) also plays a role. The competing financial interests have produced, in our view, bizarre and unpatriotic (?) television commercials blaming mother Few students in the first year medical class knew that replacing unsaturated fat with carbohydrate was the most damaging substitution in terms of an association with CVD risk. The data from Hu, et al., ,25 shownThese two questions Table  point ouWe recommend that the term complex carbohydrates not be used since, in practice, it has lost its original meaning of polysaccharide. It is interesting that, to some extent, student answers followed the original definition. Most students picked both white bread and whole wheat bred as complex although, with a slight preference for picking whole wheat over white bread as many health professionals and the lay public might.We credit the popular media with the generally good knowledge about the antioxidant vitamins shown in Table These questions were presented as a preview for an upcoming lecture on folate metabolism; therefore it was expected that students would not score very highly Table . We idenMany papers have been written on the need for, and the difficulty in implementing, improvements in teaching nutrition in medical schools ,3,33. SoWith respect to point 1) above, the first year medical school curriculum is undoubtedly very dense in content. Adding new material is difficult, especially if it is of the strictly factual type, e.g. macronutrient composition of particular foods. The "low pressure" quiz used here can, in theory, impart a certain amount of specific knowledge and generate student interest without interrupting the general flow of course work. The quiz provides a venue in which interested students can absorb the information, and become aware of the general area if they need to find the information later. Also, in our view, many subjects taught in basic science courses already have nutritional relevance, e.g., cofactors that come from vitamins, and these ideas should be better emphasized. We point out, when the NAD cofactors are introduced, that one would expect global effects of a deficiency disease because of the number of different enzymes that use these cofactors. Although vitamin deficiencies are rare in the absence of gross malnutrition, the emerging role of hypervitamin therapies  has greaWe see the question of what to teach as the most critical problem in introducing or expanding nutrition education in the medical school course. Individual faculty may be resistant to giving up their own interests, but this may depend on how well the case is made for changing to new topics.The original study by Flynn was designed to test physicians' knowledge and expand it to allow them to better implement ATP III recommendations on serum triglycerides. In combination with other questions that we have introduced, the general problem arises as to whether these recommendations or nutritional data should be taught. We feel that official recommendations, such as ATP III, have some inherent contradictions. Given that low fat diets tend to raise triglycerides, the associated recommendations to reduce dietary fat and to raise carbohydrate intake appear somewhat contradictory. The major focus of ATP III, however, is control of cholesterol but again, the literature is not clear-cut. Thus, whereas the association between cholesterol levels and CVD is generally accepted by all but a minority of critics, the effect of diet, especially reduced fat diets, on CVD, or even cholesterol, is far more controversial. The Chapter on "Diet and Coronary Heart Disease (CHD)" in Willett's Nutritional Epidemiology  is 40 paThe analysis above also bears on the role of low carbohydrate diets in educating students and physicians. We have previously indicated how such diets can be used to teach basic intermediary metabolism  and wherIn summary, what to teach remains very problematic. There are clear inconsistencies in the dietary recommendations of the ATP III and other professional agencies. This has to make one question whether students and physicians should be educated only in currently recommended practice, or whether we should instead emphasize understanding the underlying data. This is especially true, given the disclaimers in the American Heart statement  that "ThA questionnaire, derived in part from one previously published to assess physician knowledge, can be used to determine medical student awareness of nutritional facts. At the same time, such a quiz can be employed as a teaching device to reinforce earlier material, provide preview of new material, or expose students to factual information that is not easily incorporated into a formal course. One of the areas chosen, the effect of macronutrients on obesity and cardiovascular disease, can lead to discussion and focus on important current issues. The performance of first year medical students as well as the performance of the physicians in the previous study suggest that improvement is needed in imparting knowledge about some basic ideas in nutrition. We believe that the focus should be on these ideas rather than on official recommendations with which the ideas are sometimes in conflict.Finally, the questionnaire is intended as a practical method. The authors would be grateful for any information on the outcome of its use and/or any suggestions for improving the quiz itself.HDL: High Density LipoproteinLDL: Low Density LipoproteinTAG: TriacylglycerolTHF: Tetrahydrofolic acidTLC: Therapeutic Lifestyle Changes DietATP III: Third Report of the National Cholesterol Education Program Adult Treatment ProgramNCEP: National Cholesterol Education ProgramGI: glycemic indexGL: glycemic loadCVD: Cardiovascular Disease


Tiny protrusions on the surface of cells might be a more common mechanism for cell communication than previously expected During development, cells need to communicate with each other to establish properly organised and functional tissues. Cells communicate with each other in various ways, such as by secreting and receiving diffusible molecules (morphogens, hormones, and neurotransmitters) or by establishing intercellular connections (gap junctions and cell protrusions) to allow a direct exchange of instructive factors. A recent paper has shown that communication via tiny cell protrusions might be a more common mechanism than previously expected .Drosophila wing and eye imaginal discs (precursors of adult structures) that signals modulating the growth and patterning of one epithelial layer of cells are received through microtubule-based cell extensions arising from the apposing epithelium  on the protrusion could bind and activate receptors displayed on the surface of the receiving cell .The paper by Rustom and colleagues has provided a new outlook on the role of cell protrusions, by reporting a novel mechanism employed to transmit signals between cells connected by a protrusion. Surprisingly, they did not observe any of the mechanisms described above. Rather, transfer of molecules and organelles occurred directly from the cytoplasm of one cell to the other, passing through a protrusion that established membrane continuity between the connected cells.Using rat PC12 cells, Rustom and colleagues observed ultrafine protrusions  connecting sparse cells in culture . SimilarAiming to investigate how TNTs were linking cells, the authors performed scanning and transmission electron microscopy of TNTs. They observed a seamless transition between TNTs and the cells they were connected to, suggesting that indeed there was continuity between the membranes of the two connected cells. Rustom and colleagues then went on to test whether TNTs could be used to transmit signals between cells. The experimental approach used was to mark two populations of cells in a distinct way, either by introducing genes that encoded proteins tagged with green fluorescent proteins or by using dyes. The two different cell populations were then mixed, cocultured, and analysed for transfer of marked proteins or dye-stained organelles from one cell to another, between cells that were differently marked and connected by a TNT. Strikingly, soluble cytoplasmic molecules could not pass freely along the TNTs (with actin tagged with green fluorescent protein being the only exception), whereas membrane-bound proteins were transferred along TNTs and detected in the receiving cells, further supporting the likelihood of membrane continuity between connected cells. Rustom and colleagues also observed transport of vesicles, which seemed to be unidirectional. Finally, in transfer experiments performed at close to 0 °C, where endo-, exo-, and phagocytosis would be blocked, vesicle exchange still occurred, suggesting that these events are not required for vesicle transfer and further supporting the idea that membrane continuity exists between connected cells. By contrast, interfering with actin polymerization, using the drug latrunculin-B, led to protrusion removal and arrest in organelle transfer, indicating that actin is required both for protrusion biogenesis and organelle transport.Taken together, the experiments performed by Rustom and colleagues strongly suggest a role for cell protrusions in cell-to-cell communication. They also provide evidence, in culture, for a novel mechanism used by cell protrusions to transport molecules and organelles. It will be interesting to test whether TNTs also exist in living tissues and, if so, what molecules they transport. TNTs could be distinct from the protrusions known so far and could be responsible for establishing another type of connection between cells. They could connect all cells in a tissue, directly or indirectly, establishing a global interaction network potentially important in exchanging basic survival information as well as positional cues .Another interesting question is how connections such as TNTs are established. Rustom et al. have shown that, initially, many filopodial extensions arise from one cell and are directed toward a neighbour. As soon as one of them reaches the target, it is stabilised, while the others degenerate. It is possible that membrane fusion occurs between the tip of the protrusion and the planar plasma membrane of the target cell. However, membrane fusion can be more easily achieved if the tips of two cell protrusions fuse with each other, thus suggesting the participation, in the process of membrane fusion, of microvilli or other tiny protrusions belonging to the target cell. Fusion between two protrusions is reported to rely on the cylindrical shape and narrow diameter of cell protrusions and also on the localised concentration of adhesion/fusion molecules at the tips of the cell protrusions, such as microvilli, that display particular tip-specific membrane microdomains . The worIn my work as a graduate student, I am trying to address this question. We need to identify the types of cell protrusions that are present in tissues and the molecular complexes localizing on them as well as their functions. To then prove that cell protrusions are important in cell-to-cell communication in tissues, we would need to remove the protrusions and see how this affects tissue architecture and function. However, the necessary tools are still missing, given the lack of knowledge of the specific molecules important for the biogenesis of these protrusions. Thus far, the function of cell protrusions has been hypothesized mainly on the basis of their location in tissues and on crude attempts to remove them, for example by altering the actin cytoskeleton or even by removing the entire epithelium they belong to.The paper by Rustom et al. has shed some new light on these still mysterious cellular arms and has further boosted my interest in this emerging field of cell and developmental biology.


The use of complementary and alternative products including Biological Based Therapy (BBT) has increased among patients with various medical illnesses and conditions. The studies assessing the prevalence of BBT use among patients with cardiovascular diseases are limited. Therefore, an evaluation of BBT in this patient population would be beneficial. This was a survey designed to determine the effects of demographics on the use of Biological Based Therapy (BBT) in patients with cardiovascular diseases. The objective of this study was to determine the effect of the education level on the use of BBT in cardiovascular patients. This survey also assessed the perceptions of users regarding the safety/efficacy of BBT, types of BBT used and potential BBT-drug interactions.The survey instrument was designed to assess the findings. Patients were interviewed from February 2001 to December 2002. 198 inpatients with cardiovascular diseases (94 BBT users and 104 non-users) in a university hospital were included in the study.Users had a significantly higher level of education than non-users (college graduate: 28 [30%] versus 12 [12%], p = 0.003). Top 10 BBT products used were vitamin E [41(43.6%)], vitamin C [30(31.9%)], multivitamins [24(25.5%)], calcium [19(20.2%)], vitamin B complex [17(18.1%)], fish oil [12(12.8%)], coenzyme Q10 [11(11.7%)], glucosamine [10(10.6%)], magnesium [8(8.5%)] and vitamin D [6(6.4%)]. Sixty percent of users' physicians knew of the BBT use. Compared to non-users, users believed BBT to be safer (p < 0.001) and more effective (p < 0.001) than prescription drugs. Forty-two potential drug-BBT interactions were identified.Incidence of use of BBT in cardiovascular patients is high (47.5%), as is the risk of potential drug interaction. Health care providers need to monitor BBT use in patients with cardiovascular diseases. The use of complementary and alternative medicine (CAM), defined by the United States National Center for CAM as a group of diverse medical and health care systems, practices, and products that are not presently considered to be a part of conventional medicine, has grown tremendously in the United States -4. A recSimilar to other chronic medical conditions, patients suffering from a variety of cardiovascular diseases including coronary heart disease, congestive heart failure, stroke, arrhythmia and congenital cardiovascular defects, may also be looking to CAM to prevent or treat their illnesses. This is particularly likely since a number of BBT products including vitamin E, vitamin C, beta-carotene, fish oils, and coenzyme Q10 have been evaluated for prevention and/or treatment of cardiovascular diseases -25.Despite a wide array of available BBT for cardiovascular conditions, studies evaluating the prevalence of usage of these agents are limited -25. As CThe primary objective of this study was to determine the effect of the education level on the use of BBT in cardiovascular patients. The study also investigated the attitudes and beliefs towards BBT by patients with cardiovascular diseases. In addition, patient perceptions regarding the safety and efficacy of BBT, common BBT used and a list of potential BBT-drug interactions were reviewed.This is a cross-sectional, descriptive study utilizing structured interviews to assess the level of education, usage, beliefs and attitudes towards BBT among inpatients with cardiovascular diseases. The study was conducted in the Cardiac Care Center at the Mount Sinai Hospital, New York, USA from February 2001 to December 2002. Participants provided informed consent and were interviewed by one of the investigators. To maintain consistency of the interview and to prevent interviewer bias, a scripted letter was drafted for the investigators to invite the patients to participate in the study and to explain the process of the study. After a patient was enrolled, an investigator read the survey questions verbatim to the patient and tried not to elaborate whenever possible. Patients were included in the study if they had at least one of the following diagnoses: cardiovascular disease(s) including congestive heart failure, coronary heart disease, thromboembolic diseases, valvular heart disease, arrhythmia, vascular aneurysm, peripheral vascular disease, pulmonary hypertension, congenital heart disease and post heart transplant. Additional inclusion criteria included being 18 years of age or older, English speaking, no documented cognitive deficits precluding the patient from understanding the interviewer, and willingness to provide an informed consent. Prior to patient contact, the attending physicians of the eligible patients were contacted and informed about the study. If the attending physicians chose not to have their patients participate in the study, the patients were not included. Upon agreement of the physicians, subjects were invited to participate and were asked to sign an informed consent at their convenience prior to being interviewed. This study was approved by the Institutional Review Boards of Mount Sinai Hospital and Long Island University.® Database [Utilizing a structured instrument see , eligiblDatabase . The pat® for Windows, Rel. 10.01 1999).For this study to have an 80 percent power to detect a 20 percent clinically significant difference in determining factors of BBT use such as education level between the users and non-users of BBT, and establishing a p value of < 0.05 as the level of statistical significance, approximately 200 patients (100 patients in each group) needed to be enrolled. For demographic parameters, continuous variables were compared between the two groups using Students' t-test and categorical variables were compared using chi-square. Attitudes and beliefs regarding the safety and efficacy of BBT were compared between the two groups using a chi-square test for categorical data and Students' t test for Likert-type scale questions. The BBT products used by cardiovascular patients were recorded and the likelihood of potential drug interactions between BBT and other medications the patients were taking were described. Statistical analyses were conducted using Statistical Product and Service Solutions program  reported BBT use at some point in their lifetime and 84 (42%) reported using such products during the immediate 12 months prior to the survey. Of the 94 patients who used BBT, 32% reported using the products all the time.The demographic and socioeconomic characteristics of the total sample are presented in Table Overall, education level significantly influenced the use of BBT, p = 0.0006. Among users, more patients had college degrees (28 [30%]) as compared with nonusers, (12 [12%], p = 0.003). In contrast, 42 (40%) nonusers finished high school versus 23 (24%) of users, p = 0.023. There were no significant differences between users and nonusers in other demographic variables.The distribution of cardiovascular and noncardiovascular diseases reported by the sample is presented in Table The mean number of traditional prescription and nonprescription medications taken by the users of BBT (7 [range 1–15]) and nonusers (7 [range 0–18]) was not significantly different, p = 0.445. The most common cardiovascular medications prescribed for both users and nonusers were diuretics (71%, 73%), followed by aspirin (56%, 43%) and beta-blockers (49%, 50%). The cardiovascular and noncardiovascular medications taken by both groups were not different.The BBT products utilized by cardiovascular patients are presented in Table The patients reported that their physicians were aware of their BBT use in 60% of the instances and pharmacists were aware in 32% of the cases. Only 33% of users reported that they were asked about BBT use during a history/physical examination by a health care professional. The patients were not surveyed about their pharmacists' assessments of BBT use.A greater percentage of users (74.5%) of BBT reported that they believed these products to be safe substances as compared with nonusers (26.2%), p < 0.001. Likewise, significantly more users believed that BBT was effective (70.2%) compared to nonusers (30.1%), p < 0.001. More nonusers (72%) than users (45%) did not know whether BBT products work better, as well as, or worse than traditional medications, p < 0.001. More users than nonusers believed that BBT works as well as traditional medications (30.9% versus 9.7%, p < 0.001). Concerning adverse effects, more users (44.7%) of BBT than nonusers (17.5%) believed that BBT causes fewer side effects than traditional medications, p < 0.001. At the same time, 32.2% of users and 62.1% of nonusers did not know whether BBT causes more or fewer side effects than traditional prescription medications, p < 0.001.Examination of patients' prescription and nonprescription medication profiles and BBT utilized, revealed 42 potential drug-BBT interactions. The onset of the interaction and the degree of severity were classified according to that used by the MicroMedex HealthCare Series Integraded Index  and the Despite a wide array of available BBT for cardiovascular conditions, studies evaluating the prevalence of their usage are limited. A review of the literature at the time of study initiation (January 2001) and more recently (June 2004) identified five studies in this area, and only a few included a broad spectrum of cardiovascular disease patients -25.Wood et al. conducted a telephone survey in 107 patients randomly selected from the "Improving Cardiovascular Outcomes in Nova Scotia" database . A majorIn our study, the lifetime prevalence of BBT use in the sample of 198 cardiovascular patients was 47.5%, which is very similar to the results reported by Eisenberg and colleagues in the general population (42%) . CompariSimilar to Ackman's study , the mosSimilar to other studies ,3,6,21, Among the demographic variables collected in this analysis, no parameter other than the level of education had a significant impact on whether patients used BBT. The present investigation revealed that the education level among users (63% received some college or college and graduate school education) of BBT was higher than that of the nonusers (32%), p < 0.001. This finding has also been reported in other studies [The majority of BBT users (as compared to nonusers) in the current investigation believed that BBT products are safe, effective and cause fewer side effects as compared to prescription medications. These results are consistent with the findings by other investigators ,21, suppSeveral limitations of the study need to be addressed. Those that are intrinsically related to survey data collection in general include potential bias of responders and nonresponders. Since participation in this study was voluntary, patients who chose to participate in the study may be more motivated and knowledgeable about the subject of BBT as compared to those who refused to participate. Therefore, the results of this study may not be applicable to the entire cardiovascular patient population. Also, a cross-sectional nature of this study precludes drawing any definitive conclusions regarding cause and effect relationships. For example, one cannot definitively conclude that more education will significantly effect one's decision to take BBT because other factors such as exposure to these types of products may have influenced more educated consumers to take these products.As with other surveys, recall bias cannot be eliminated as data collection relied on patients' self-report, rather than objectively documenting BBT use. Additionally, with surveys there is always a possibility of misunderstanding the questions and responses, miscommunication between the interviewer and the patients, and inaccurate recording of the information. The majority of the survey collected factual information (i.e., demographics and BBT usage history). However, the questions that evaluated patients' perception on the safety and efficacy of conventional medicine and BBT were not validated.This study was conducted in a large urban inner city hospital; the results may not be extrapolated to other cities or clinical settings. Another limitation may be the exclusion of non-English speaking patients, which may be particularly important since there are certain ethnic groups who are documented to use more BBT than are other ethnic groups . Finally, terminology varies greatly in the published literature. Some authors use the term CAM in all instances, others differentiate CAM from BBT, etc. As a result, it is very difficult to compare published reports with certainty.In recent years, the interest of using BBT in disease management has increased dramatically in the medical and layman communities. The amount of valid scientific research in this area of therapy continues to increase. Yet, there are still many unknowns concerning BBT, especially in the area of adverse effects and drug interactions. The finding of a high prevalence of BBT (47.5%) use among cardiovascular patients and the lack of communication between patients and their physicians/pharmacists should be addressed by the health care community. Higher education level, as shown in the present study and other previous investigations ,3,6,22, The author(s) declare that they have no competing interests.LC participated in literature review, protocol development, collected and analyzed data, and submitted the manuscript for publication. DB collected the data. JWMC participated in study design, data analysis and preparation of manuscript. VR participated in design development and data analysis. HLK proposed the project and reviewed the manuscript. GCC, JM and BM participated in protocol implementation and manuscript review. All authors read and approved the manuscript.The pre-publication history for this paper can be accessed here:Appendix A – Biological based therapy survey, It's a survey tool utilized in the study to collect patient data.Click here for file


The mitogen-activated protein kinases (MAPKs) have been shown to participate in a wide array of cellular functions. A role for some MAPKs  has been documented in response to certain physiological stimuli, such as ischemia, visceral pain and electroconvulsive shock. We recently demonstrated that restraint stress activates the Erk MAPK pathway, but not c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK) or p38MAPK, in several rat brain regions. In the present study, we investigated the effects of a different stressor, acute forced swim stress, on the phosphorylation (P) state of these MAPKs in the hippocampus, neocortex, prefrontal cortex, amygdala and striatum. In addition, effects on the phosphorylation state of the upstream activators of the MAPKs, their respective MAPK kinases , were determined. Finally, because the Erk pathway can activate c-AMP response element (CRE) binding (CREB) protein, and swim stress has recently been reported to enhance CREB phosphorylation, changes in P-CREB were also examined.A single 15 min session of forced swimming increased P-Erk2 levels 2–3-fold in the neocortex, prefrontal cortex and striatum, but not in the hippocampus or amygdala. P-JNK levels (P-JNK1 and/or P-JNK2/3) were increased in all brain regions about 2–5-fold, whereas P-p38MAPK levels remained essentially unchanged. Surprisingly, levels of the phosphorylated MAPKKs, P-MEK1/2 and P-MKK4 (activators of the Erk and JNK pathways, respectively) were increased in all five brain regions, and much more dramatically . Consistent with the lack of forced swim on phosphorylation of p38MAPK, there appeared to be no change in levels of its activator, P-MKK3/6. P-CREB was increased in all but cortical  areas.Swim stress specifically and markedly enhanced the phosphorylation of the MAPKKs P-MEK1/2 and P-MKK4 in all brain regions tested without apparent alteration in the phosphorylation of P-MKK3/6. Curiously, phosphorylation of their cognate substrates (Erk and JNK) was increased to a much more modest extent, and in some brain regions was not altered. Similarly, there was a region-specific discrepancy between Erk and CREB phosphorylation. Possible explanations for these findings and comparison with the effects of restraint stress will be discussed. Thus, activation of the Erk pathway via both a cAMP/PKA-dependent mechanism leading to activation of CREB, and a non-cyclase-linked pathway, leading to activation of CREB and/or the serum response element (SRE) binding protein Elk-1, have been shown to occur in the hippocampus during acquisition of learning, memory consolidation and long-term potentiation (LTP) [Increasing evidence implicates stress as an important factor in the vulnerability to depressive and other mental illnesses -3. Conseon (LTP) -8. It ison (LTP) .The term MAPK is used to denote a family of signal transduction mediators, extensively distributed throughout the central nervous system ,11, thatc-fos gene and/or Fos protein expression in many brain regions) [c-fos and zif286. P-CREB and P-Elk-1, by binding to these elements, enhance the transcription of the IEGs and regulate gene expression [c-fos expression, and on the other, that certain stressful stimuli (see above) activated Erk, it was reasonable to predict that acute physiological stressors would activate the Erk (and perhaps other) MAPK pathway(s). The brain regions most likely to show such an effect would be those implicated in the response to stress, including the hippocampus [Subjecting rats or mice to acute stressors such as restraint or forced swim results in activation of regions) -24. CRE pression ,26. Recepression . Thus, gpocampus , prefronpocampus -32 and opocampus ,33, and pocampus ,34-36. Ipocampus . The prepocampus .An acute 15 min session of forced swimming produced obvious large increases in the phosphorylation of the MAPKK MEK1/2 in the hippocampus, neocortex, prefrontal cortex, amygdala and striatum  in the hippocampus  may account for the difference in results. Note, moreover, that the regional pattern of changes in P-CREB , except for the increase in P-MEK1/2 in neocortex, which peaked at 5 min Fig. . The levThe results presented establish unambiguously that forced swim stress elicits a rapid and profound but relatively transient increase in the phosphorylation (and consequently the presumed activation) of the MEK-Erk and MKK4-JNK signaling pathways in, at the least, the five brain regions examined. These effects displayed specificity in that the MKK3/6-p38MAPK pathway was essentially unchanged.  and the corresponding MAPKs (P-Erk and P-JNK). It is obvious from Figs. Nevertheless, differences in phosphorylation stoichiometry cannot explain why, in some brain regions  increases in P-Erk levels in hippocampus and prefrontal and cingulate cortex . As for c-fos gene expression) [via NMDA glutamate receptor-stimulated calcium influx [The nature of the extracellular signaling molecule(s) that are recruited upon subjecting animals to stress are unknown. The pathways leading from stimulation of a particular receptor on the cell surface to activation of MAPK signaling cascades are diverse and complex ,14,47-50ression) -24, and m influx -56. Strem influx , which mm influx . Thus, im influx ). NMDA rm influx -60. Intem influx . In prelAcute swim stress initiated a rapid increase in phosphorylation of the MAPKKs MEK1/2 and MKK4 in hippocampus, neocortex, prefrontal cortex, amygdala and striatum. Concomitantly, their corresponding substrates Erk and JNK were also phosphorylated, but not always in register with the changes in the MAPKKs. Moreover, the magnitude of the increase in phosphorylation of MEK1/2 and MKK4 was much greater than for their cognate substrates. While it is clear that stressors such as forced swim and restraint activate these signaling pathways, much work will be required to define the mechanism(s) responsible for these effects, and to relate potential alterations in the activity of these molecules to stress-related influences on the development of anxiety and depressive disorders.Guide for the Care and Use of Laboratory Animals, and were approved by the NYU School of Medicine Institutional Animal Care and Use Committee.Male Wistar rats  were used throughout. Rats were housed 2/cage and maintained on a 12 h light/dark cycle with food and water ad libitum; they were acclimated to handling and transportation for 2–3 weeks before being used in experiments. All experimental procedures were carried out in accordance with the NIH ® protease inhibitor cocktail was from Roche Molecular Biochemicals, Indianapolis, IN and colored molecular weight markers were purchased from Bio-Rad, Hercules, CA. Biotinylated protein ladder marker and anti-biotin were from Cell Signaling Technology (CST), Beverly, MA. West Pico Super Signal enhanced chemiluminescence (ECL) reagent was obtained from Pierce Biotechnology, Rockford, IL, and Re-Blot Plus stripping buffer was from Chemicon International, Temecula, CA. All primary antibodies were rabbit polyclonal except as noted. Because suppliers often offer more than a single antibody for the same antigen, catalog numbers are specified in parentheses. P-Erk , pan Erk (sc-93) and pan MKK4/SEK1 (sc-964) were all from Santa Cruz Biotechnologies, Santa Cruz, CA. The following antibodies were obtained from CST: P-MEK1/2 (9121), pan MEK1/2 (9122), P-MKK4/SEK1 (9151), P-JNK , pan JNK (9252), P-MKK3/6 (9231), pan CREB (9192) and pan p38MAPK (9212). P-CREB (06-519) was from Upstate Biotechnology, Inc. , while P-p38 was either from CST (9212) or Chemicon (AB3828). Control cell extracts for P-MKK3/6 were obtained from CST: +/- UV-treated NIH/3T3 cell lysates (9233) and +/- anisomycin-treated C6 glioma cell lysates (9213).Precast 10% polyacrylamide gels were obtained from Cambrex BioScience, Rockland, ME. CompleteGroups of rats (n = 4–6) were subjected to forced swim stress in a round glass tank (24 cm W × 44 cm H) filled to a depth of 30 cm with water (25 ± 1°C). In most experiments rats were forced to swim for 15 min and sacrificed by decapitation immediately after narcotization with carbon dioxide for 20 sec. In some experiments the duration of swim stress (2–15 min) or the post-swim interval (0–60 min) was varied as described in the legends to Figures. Control rats were not treated and were sacrificed in parallel as above.Brains were rapidly removed, briefly chilled in saline and placed in a stainless steel brain matrix . Sections of 1–2 mm in thickness were cut, frozen quickly on dry ice, and brain areas of interest  were micropunched using the atlas of Paxinos and Watson  as a gui3VO4, 1 mM NaF, 0.5 μM okadaic acid and Complete® protease inhibitor cocktail). After centrifugation at 30,000 g for 20 min, lysates were mixed with 5X sodium dodecyl sulfate (SDS) sample buffer, boiled for 5 min and stored at -80°C. Protein content of lysates was determined using the bicinchoninic acid assay kit (Pierce).Tissues were mechanically disrupted, in glass homogenizers using a Teflon pestle, in 10–20 volumes of buffer (50 mM Tris-HCl, pH 7.4 containing 300 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM EDTA, 1 mM NaProteins (10–40 μg/lane) were separated by SDS-PAGE on precast polyacrylamide gels. Gels were also loaded with colored molecular weight markers to assess electrophoretic transfer, and biotinylated protein ladder marker to estimate molecular weights of bands of interest. Following electrophoretic transfer to nitrocellulose, blots were incubated in blocking buffer ) for 1 hr at room temperature (RT), washed 3 × 10 min in TBST and incubated with primary phospho-specific antibodies overnight at 4°C . Blots were washed 3× in TBST, incubated with the appropriate secondary antibody plus anti-biotin for 1 hr at RT, washed again, treated with ECL reagent and exposed to film. Blots were then stripped by incubation for 15 min at RT with Re-Blot Plus, re-blocked, washed and incubated for 1 hr at RT with the corresponding pan antibody which recognizes total antigen protein (phosphorylated and nonphosphorylated). This was followed by incubation for 1 hr at RT with the appropriate secondary antibody plus anti-biotin. Antigens were again visualized by treatment with ECL reagent and exposure to film.A computerized image analysis system (MCID-M4, Imaging Research, St. Catherines, Ontario, Canada) was utilized to analyze immunoblots. Image analysis was carried out as described previously . For eact test (SigmaStat, ver. 2.03, SPPS, Inc., Chicago, IL).Control and swim-stressed groups were compared using Student's C-PS conducted the majority of the immunoblotting studies. YT performed some of the experiments and quantitated many of the films using image analysis. CS assisted in the design of some of the experiments, carried out the MKK3/6 experiments, and assisted in the writing of the manuscript. EM designed the study, supervised and facilitated the experiments, analyzed the data and wrote the manuscript.