Biology and conservation of elasmobranchs: an introduction to the collection.

Elasmobranchs, the taxonomic group comprising sharks, skates and rays, play important roles in society and marine ecology but several species in this subclass are under threat.  This collection aims to be an open access hub for articles concerning all areas of elasmobranch biology and conservation. The collection is indefinitely open to further submissions and so will continue to grow as additional articles are added.

Oxidized low density lipoprotein exposure alters the transcriptional response of macrophages to inflammatory stimulus.

Macrophage-derived foam cells in atherosclerotic lesions are generally thought to play a major role in the pathology of the disease. Because macrophages play a central role in the inflammatory response, and the atherosclerotic lesion has features associated with chronic inflammatory settings, we investigated foam cell inflammatory potential. THP-1-derived macrophages were treated with oxidized low density lipoprotein (OxLDL) for 3 days to lipid load the macrophages and establish a foam cell-like phenotype. The cells were then activated by treatment with lipopolysaccharide (LPS), and RNA was harvested at 0, 1, and 6 h after LPS addition. RNA from treated and control cells was hybridized to microarrays containing approximately 16,000 human cDNAs. Genes that exhibited a 4-fold or greater increase or decrease at either 1 or 6 h after LPS treatment were counted as LPS-responsive genes. Employing these criteria, 127 LPS-responsive genes were identified. Prior treatment of THP-1 macrophages with OxLDL affected the expression of 57 of these 127 genes. Among these 57 genes was a group of chemokine, cytokine, and signal transduction genes with pronounced expression changes. OxLDL pretreatment resulted in a significant perturbation of LPS-induced NF kappa B activation. Furthermore, some of the OxLDL effects appear to be mediated by the nuclear receptors retinoid X receptor and peroxisomal proliferator-activated receptor gamma because pretreatment of THP-1 macrophages with ligands for these receptors, followed by LPS treatment, recapitulates the OxLDL plus LPS results for several of the most significantly modulated genes.

Gene expression profiling of cardiovascular disease models.

Recent development of gene expression profiling technologies has enabled the large-scale analysis of gene expression changes during disease progression. Frequently, cardiovascular diseases involve complex interactions of multiple cell types over prolonged periods of time. A better understanding of the pathology of cardiovascular diseases and the potential identification of underlying genetic defects are currently being explored by using profiling methodologies in a number of animal and tissue-culture models.

Determination of temporal expression patterns for multiple genes in the rat carotid artery injury model.

Vascular injury induces extensive alteration to the extracellular matrix (ECM). These changes contribute to lesion formation and promote cell migration and proliferation. To elucidate ECM response to arterial injury, we used real-time polymerase chain reaction monitoring to quantitate the expression levels of 81 genes involved in the synthesis and breakdown of ECM as well as receptors and signaling proteins that communicate and respond to ECM molecules. The temporal regulation of gene expression in the carotid was measured at 1, 3, 5, 7, 9, 14, and 28 days postinjury. Among the 68 genes that showed detectable expression by our method, 47 (69%) were significantly induced or repressed over time, confirming the extensive ECM gene response in this model. More ECM-related genes (31) were regulated at day 1 than at any other time point, and the number of regulated genes decreased over time. However, 14 of the genes were still induced or repressed at day 28, indicating that return to preinjury expression patterns did not occur and no new steady state was achieved over 28 days. In spite of the large number of changes in gene expression, only a small number of expression patterns was observed, suggesting that ECM-related genes could potentially be coregulated.

Large scale gene expression analysis of cholesterol-loaded macrophages.

We conducted large scale gene expression analysis of the response of macrophages to exposure to oxidized low density lipoprotein (Ox-LDL). Much of the vessel wall lesion of atherosclerosis is composed of macrophages that have become engorged with cholesterol. These resulting "foam cells" contribute to the progression of vascular disease through several pathways. As a potential model of foam cell formation, we treated THP-1 cells with 12-O-tetradecanoylphorbol 13-acetate to differentiate them into a macrophage-like phenotype and subsequently treated them with oxidized low density lipoprotein for various time periods. RNA from Ox-LDL treated and time-matched control untreated cells was hybridized to microarrays containing 9808 human genes. 268 genes were found to be at least 2-fold regulated at one or more time points. These regulation patterns were classified into seven clusters of expression profiles. The data is discussed in terms of the overall pattern of gene expression, the thematic classification of the responding genes, and the clustering of functional groups in distinct expression patterns. The magnitude and the temporal patterns of gene expression identified known and novel molecular components of the cellular response that are implicated in the growth, survival, migratory, inflammatory, and matrix remodeling activity of vessel wall macrophages. In particular, the role of nuclear receptors in mediating the gene expression modulation by Ox-LDL is highlighted.

Cell cycle control of Schwann cell proliferation: role of cyclin-dependent kinase-2.

Schwann cell proliferation is regulated by multiple growth factors and axonal signals. However, the molecules that control growth arrest of Schwann cells are not well defined. Here we describe regulation of the cyclin-dependent kinase-2 (CDK2) protein, an enzyme that is necessary for the transition from G1 to S phase. Levels of CDK2 protein were elevated in proliferating Schwann cells cultured in serum and forskolin. However, when cells were grown with either serum-free media or at high densities, CDK2 levels declined to low levels. The decrease in CDK2 levels was associated with growth arrest of Schwann cells. The modulation of CDK2 appears to be regulated at the transcriptional level, because CDK2 mRNA levels and its promoter activity both decline during cell cycle arrest. Furthermore, analysis of the CDK2 promoter suggests that Sp1 DNA binding sites are essential for maximal activation in Schwann cells. Together, these data suggest that CDK2 may represent a significant target of developmental signals that regulate Schwann cell proliferation and that this regulation is mediated, in part, through regulation of Sp1 transcriptional activity.

A new structural class of proteasome inhibitors that prevent NF-kappa B activation.

The multicatalytic proteinase or proteasome is a highly conserved cellular structure that is responsible for the ATP-dependent proteolysis of many proteins involved in important regulatory cellular processes. We have identified a novel class of inhibitors of the chymotrypsin-like proteolytic activity of the 20S proteasome that exhibit IC50 values ranging from 0.1 to 0.5 microgram/mL (0.1 to 1 microM). In cell proliferation assays, these compounds inhibit growth with an IC50 ranging from 5 to 10 micrograms/mL (10-20 microM). A representative member of this class of inhibitors was tested in other biological assays. CVT-634 (5-methoxy-1-indanone-3-acetyl-leu-D-leu-1-indanylamide) prevented lipopolysaccharide (LPS), tumor necrosis factor (TNF)-, and phorbol ester-induced activation of nuclear factor kappa B (NF-kappa B) in vitro by preventing signal-induced degradation of I kappa B-alpha. In these studies, the I kappa B-alpha that accumulated was hyperphosphorylated, indicating that CVT-634 did not inhibit I kappa B-alpha kinase, the enzyme responsible for signal-induced phosphorylation of I kappa B-alpha. In vivo studies indicated that CVT-634 prevented LPS-induced TNF synthesis in a murine macrophage cell line. In addition, in mice pretreated with CVT-634 at 25 and 50 mg/kg and subsequently treated with LPS, serum TNF levels were significantly lower (225 +/- 59 and 83 +/- 41 pg/mL, respectively) than in those mice that were treated only with LPS (865 +/- 282 pg/mL). These studies suggest that specific inhibition of the chymotrypsin-like activity of the proteasome is sufficient to prevent signal-induced NF-kappa B activation and that the proteasome is a novel target for the identification of agents that may be useful in the treatment of diseases whose etiology is dependent upon the activation of NF-kappa B.

CVT-313, a specific and potent inhibitor of CDK2 that prevents neointimal proliferation.

The activity of cyclin-dependent kinase 2 (CDK2) is essential for progression of cells from G1 to the S phase of the mammalian cell cycle. CVT-313 is a potent CDK2 inhibitor, which was identified from a purine analog library with an IC50 of 0.5 microM in vitro. Inhibition was competitive with respect to ATP (Ki = 95 nM), and selective CVT-313 had no effect on other, nonrelated ATP-dependent serine/threonine kinases. When added to CDK1 or CDK4, a 8.5- and 430-fold higher concentration of CVT-313 was required for half-maximal inhibition of the enzyme activity. In cells exposed to CVT-313, hyperphosphorylation of the retinoblastoma gene product was inhibited, and progression through the cell cycle was arrested at the G1/S boundary. The growth of mouse, rat, and human cells in culture was also inhibited by CVT-313 with the IC50 for growth arrest ranging from 1.25 to 20 microM. To evaluate the effects of CVT-313 in vivo, we tested this agent in a rat carotid artery model of restenosis. A brief intraluminal exposure of CVT-313 to a denuded rat carotid artery resulted in more than 80% inhibition of neointima formation. These observations suggest that CVT-313 is a promising candidate for evaluation in other disease models related to aberrant cell proliferation.

Characterization of the human cyclin-dependent kinase 2 gene. Promoter analysis and gene structure.

Cyclin-dependent kinase 2 is a serine/threonine protein kinase essential for progression of the mammalian cell cycle from G1 to S phase. CDK2 mRNA has been shown to be induced by serum in several cultured cell types. Therefore, we set out to identify elements that regulate the transcription of the human CDK2 gene and to characterize its structure. This paper describes the cloning of approximately 2.4-kilobase pair genomic DNA fragment from the upstream region of the human CDK2 gene. This fragment contains five transcription initiation sites within a 72-nucleotide stretch. A 200-base pair sub-fragment that confers 70% of maximal basal promoter activity was shown to contain two synergistically acting Sp1 sites. However, a much larger DNA fragment containing approximately 1.7 kilobase pairs of upstream sequence is required for induction of promoter activity following serum stimulation. The intron exon boundaries of seven exons in this gene were also identified, and this information will be useful for analyzing genomic abnormalities associated with CDK2.

Functional analysis of the human cyclin D2 and cyclin D3 promoters.

The D-type cyclins promote progression through the G1 phase of the cell cycle and may provide a link between growth factors and the cell cycle machinery. We determined the nucleotide sequence of the 5'-flanking region of the human cyclin D2 and cyclin D3 genes and identified the transcription start sites. Analysis of the upstream sequences required for transcription of the cyclin D2 and cyclin D3 genes in continuously dividing cells revealed marked differences in their regulatory elements. In the cyclin D2 gene positive elements were localized between positions -306 and -114 relative to the ATG codon at +1. Additional positive elements were localized between -444 and -345, whereas sequences that reduced transcription were identified between nucleotides -1624 and -892. In the cyclin D3 gene all of the positive elements required for maximal transcription were localized between nucleotides -366 and -167, and no negative elements were found. The activities of a reporter gene linked to the upstream regulatory sequences of the cyclin D2 gene but not the cyclin D3 gene were induced when starved cells were serum stimulated. This suggests that although the abundance of both the cyclin D2 and cyclin D3 mRNAs is increased by serum stimulation, only the cyclin D2 gene is up-regulated at the transcriptional level. Sequences between nucleotides -306 and -1624 of the cyclin D2 gene were necessary for serum inducibility.

Role of the imp operon of the Streptomyces coelicolor genetic element SLP1: two imp-encoded proteins interact to autoregulate imp expression and control plasmid maintenance.

The Streptomyces coelicolor genetic element SLP1 can exist either integrated into the host chromosome or as an autonomously replicating plasmid. The integrated form of SLP1 includes a locus (imp, for inhibition of plasmid maintenance) that can act both in cis and in trans to prevent propagation of SLP1 as an extrachromosomal replicon (S. R. Grant, S. C. Lee, K. Kendall, and S. N. Cohen, Mol. Gen. Genet. 217:324-331, 1989). We report here that a 1.8-kb Eco47III DNA fragment previously shown to encode the Imp+ phenotype contains two genes (impA and impC) that must be expressed in cis to each other and whose products interact functionally and probably physically to interfere with SLP1 plasmid maintenance and repress expression of the imp operon. Partial repression of the imp promoter (P(imp)), which is located immediately 5' of impA, by the 29.7-kDa ImpA protein is enhanced by the impC gene product. Gel shift analysis indicates that ImpA binds to a 16-bp sequence located within the DNA segment containing P(imp) and that ImpC interferes with this binding. Our data suggest that binding of ImpA to the P(imp) region mediates DNA looping in this region.

Reconstruction of a Streptomyces linear replicon from separately cloned DNA fragments: existence of a cryptic origin of circular replication within the linear plasmid.

We report here the reconstruction of a functional linear replicon, the 12-kilobase Streptomyces clavuligerus plasmid pSCL, from separate DNA fragments cloned in Escherichia coli on the pUC19 plasmid. Protein-free DNA molecules containing the full-length pSCL sequence, an internally inserted thiostrepton-resistance gene, and adventitious nucleotides external to the pSCL termini were introduced into Streptomyces lividans, where the synthesis and functional attachment of replication proteins occurred and pSCL was established as an extrachromosomal linear replicon. Transformation of S. lividans with uncut supercoilded pUC19/pSCL DNA from E. coli or with a circularized 8-kilobase internal fragment of pSCL yielded circular replicons, indicating the existence of a cryptic origin of circular replication within the linear plasmid. Insertion mutations at sites that prevented the replication of pSCL linear plasmids also interfered with its replication in the circular mode.

High level expression in Escherichia coli of isopenicillin N synthase genes from Flavobacterium and Streptomyces, and recovery of active enzyme from inclusion bodies.

A T7 promoter-based vector was used to express the isopenicillin N synthase (IPNS) genes of Flavobacterium sp. 12,154 and Streptomyces jumonjinensis in Escherichia coli. Most of the IPNS synthesized at 37 degrees C, and representing some 22% and 51% of the total cell protein respectively, occurred in an insoluble, enzymatically inactive form. Active IPNS was recovered in a rapid and simple two-step procedure in which the insoluble material was first denatured in 5 M urea and then refolded by passing the solubilized IPNS through a G-25 Sephadex sizing column. Further chromatography on DEAE-Sepharose resulted in highly active IPNS preparations. This procedure was found to be well suited for scaling up to produce large amounts of IPNS.

Evolution of isopenicillin N synthase genes may have involved horizontal gene transfer.

The isopenicillin N synthase genes from three fungal species, three Gram-positive species, and one Gram-negative bacterial species share an unusually high sequence similarity. A phylogenetic analysis was carried out to determine which type of evolutionary scenario best accounts for this similarity. The most plausible scenario is one in which a horizontal gene-transfer event, from the prokaryotes to the eukaryotes, occurred at a time close to the divergence between the Gram-positive and the Gram-negative bacteria.

Microbial isopenicillin N synthase genes: structure, function, diversity and evolution.

Clinically and economically, penicillins and cephalosporins are the most important class of the beta-lactam antibiotics. They are produced by a wide variety of microorganisms including numerous species of Streptomyces, some unicellular bacteria and several filamentous fungi. A key step common to their biosynthetic pathways is the conversion of a linear, cysteine-containing tripeptide to a bicyclic beta-lactam antibiotic by isopenicillin N synthase. Recent successes in the cloning and expression of isopenicillin N synthase genes now permit production of a plentiful supply of this enzyme, which may be used for structural and mechanistic studies, or for biotechnological applications in the creation of novel beta-lactam compounds from peptide analogues. New ideas concerning the evolution and prevalence of the penicillin and cephalosporin biosynthetic genes have emerged from studies of isopenicillin N synthase genes.

Cloning and comparative sequence analysis of the gene coding for isopenicillin N synthase in Streptomyces.

The genes coding for isopenicillin N synthase (IPNS) in Streptomyces jumonjinensis and S. lipmanii were isolated from recombinant phage lambda libraries using the S. clavuligerus IPNS gene as a heterologous probe. The S. jumonjinensis IPNS gene has an open reading frame coding for 329 amino acids, identical in size to that of the previously cloned S. clavuligerus IPNS gene. A partial nucleotide sequence was also determined for the S. lipmanii IPNS gene. Comparison of the predicted amino acid sequences of all three streptomycete IPNS proteins shows that they exhibit more than 70% similarity, close to that found in comparisons among fungal IPNS proteins and significantly greater than that found, approximately 60%, between Streptomyces and fungal IPNS proteins. We conclude that procaryotic and eucaryotic IPNS genes are subgroups of a single family of microbial IPNS genes. Hybridization probes prepared from IPNS genes of the above streptomycete species were used to detect analogous genes in eight other strains that included both penicillin and cephalosporin producers and non-producers. Each producer strain responded with all three probes implying the presence of an IPNS gene. Surprisingly, several non-producer strains also responded with one or two of the probes. Our results suggest that IPNS-related genes may be more prevalent in Streptomyces than previously believed.