Quantitative methods to characterize morphological properties of cell lines.

Descriptive terms are often used to characterize cells in culture, but the use of nonquantitative and poorly defined terms can lead to ambiguities when comparing data from different laboratories. Although recently there has been a good deal of interest in unambiguous identification of cell lines via their genetic markers, it is also critical to have definitive, quantitative metrics to describe cell phenotypic characteristics. Quantitative metrics of cell phenotype will aid the comparison of data from experiments performed at different times and in different laboratories where influences such as the age of the population and differences in culture conditions or protocols can potentially affect cellular metabolic state and gene expression in the absence of changes in the genetic profile.

Health status, infection and disease in California sea lions (Zalophus californianus) studied using a canine microarray platform and machine-learning approaches.

Conservation biologists face many challenges in assessing health, immune status and infectious diseases in protected species. These challenges include unpredictable sample populations, diverse genetic and environmental backgrounds of the animals, as well as the practical, legal and ethical issues involved in experimentation. The use of whole genome scale transcriptomics with animal samples obtained in a minimally invasive manner is an approach that shows promise for health assessment.

The transcriptomic response to viral infection of two strains of shrimp (Litopenaeus vannamei).

The extent to which data-intensive studies of the transcriptome can provide insight into biological responses is not well defined, especially in the case of species (such as shrimp) where much physiological and biochemical knowledge is missing. In this study we took a transcriptomic approach to gain insight into the response to viral infection of two strains of the Pacific whiteleg shrimp (Litopenaeus vannamei) that differ in their resistance to Taura Syndrome Virus (TSV). Changes in gene expression in the hepatopancreas following infection with TSV and Yellow Head Virus (YHV) were assessed using a cDNA microarray containing 2469 putative unigenes.

Non-specific activation of antiviral immunity and induction of RNA interference may engage the same pathway in the Pacific white leg shrimp Litopenaeus vannamei.

Many questions remain unanswered regarding RNAi-based mechanisms and dsRNA-induced antiviral immune responses in penaeid shrimp. In this study, we report the characterization in the white leg shrimp Litopenaeus vannamei of RNAi pathway associated proteins Lv-Ago 1 and Lv-Ago 2, two members of the Argonaute family of proteins, as well as Lv-sid 1, the first shrimp homologue of Sid-1, a membrane channel-forming protein implicated in the cellular import of dsRNA. To decipher their functional implication in RNAi-related phenomena, we monitored their relative expression following stimulation by specific and non-specific RNA duplexes of diverse length.

Modelling interactions of acid-base balance and respiratory status in the toxicity of metal mixtures in the American oyster Crassostrea virginica.

Heavy metals, such as copper, zinc and cadmium, represent some of the most common and serious pollutants in coastal estuaries. In the present study, we used a combination of linear and artificial neural network (ANN) modelling to detect and explore interactions among low-dose mixtures of these heavy metals and their impacts on fundamental physiological processes in tissues of the Eastern oyster, Crassostrea virginica. Animals were exposed to Cd (0.

Expression of IgM, IgD, and IgY in a reptile, Anolis carolinensis.

The reptiles are the last major group of jawed vertebrates in which the organization of the IGH locus and its encoded Ig H chain isotypes have not been well characterized. In this study, we show that the green anole lizard (Anolis carolinensis) expresses three Ig H chain isotypes (IgM, IgD, and IgY) but no IgA. The presence of the delta gene in the lizard demonstrates an evolutionary continuity of IgD from fishes to mammals.

A transcriptomic analysis of land-use impacts on the oyster, Crassostrea virginica, in the South Atlantic bight.

Increasing utilization and human population density in the coastal zone is widely believed to place increasing stresses on the resident biota, but confirmation of this belief is somewhat lacking. While we have solid evidence that highly disturbed estuarine systems have dramatic changes in the resident biota (black and white if you will), we lack tools that distinguish the shades of grey. In part, this lack of ability to distinguish shades of grey stems from the analytical tools that have been applied to studies of estuarine systems, and perhaps more important, is the insensitivity of the biological end points that we have used to assess these impacts.

Lack of evidence for Litopenaeus vannamei Toll receptor (lToll) involvement in activation of sequence-independent antiviral immunity in shrimp.

Injection of non-specific dsRNA initiates a broad-spectrum innate antiviral immune response in the Pacific white shrimp, Litopenaeus vannamei, however, the receptor involved in recognition of this by-product of viral infections remains unknown. In vertebrates, dsRNA sensing is mediated by a class of Toll-like receptors (TLRs) and results in activation of the interferon system. Because a TLR (lToll) was recently characterized in L.

Sampling the skin transcriptome of the North Atlantic right whale.

As an initial step in defining the transcriptome of the North Atlantic right whale (Eubalaena glacialis) and developing functional genomic tools to study right whale health at the molecular physiological level, a cDNA library has been constructed from a skin biopsy. 2496 randomly selected clones (expressed sequence tags, ESTs) have been sequenced, and genes identified as important in the response to stress and immune challenges have been cloned by targeted RT-PCR from skin cDNA. The analysis of the EST collection (archived at www.

BOB.1 of the channel catfish, Ictalurus punctatus: not a transcriptional coactivator?

Expression of the immunoglobulin heavy chain (IGH) locus of the channel catfish (Ictalurus punctatus) is driven by the Emu3' enhancer, whose core region contains two octamer motifs and a muE5 site. Orthologues of the Oct1 and Oct2 transcription factors have been cloned in the channel catfish and shown to bind to the octamer motifs within the core enhancer. While catfish Oct2 is an activator of transcription, catfish Oct1 failed to drive transcription and may act as a negative regulator of IGH transcription.

Interaction between E-protein and Oct transcription factors in the function of the catfish IGH enhancer.

Transcriptional control of the immunoglobulin heavy chain (IGH) locus in the channel catfish, Ictalurus punctatus, is incompletely understood. It is, however, known that 2 variant octamer motifs and a microE5 motif in the core region of the enhancer (Emicro3') are important in driving transcription, and it has been suggested that interaction between transcription factors (Oct factors and E-proteins) bound to these sites contributes to enhancer function. In this study, the functional relationships between the microE5 motif, the proximal octamer motif, and the factors that bind them have been examined.

Anti-lipopolysaccharide factor in Litopenaeus vannamei (LvALF): a broad spectrum antimicrobial peptide essential for shrimp immunity against bacterial and fungal infection.

Antimicrobial peptides are an essential component of the innate immune system of most organisms. Expressed sequence tag analysis from various shrimp (Litopenaeus vannamei) tissues revealed transcripts corresponding to two distinct sequences (LvALF1 and LvALF2) with strong sequence similarity to anti-lipopolysaccharide factor (ALF), an antimicrobial peptide originally isolated from the horseshoe crab Limulus polyphemus. Full-length clones contained a 528bp transcript with a predicted open reading frame coding for 120 amino acids in LvALF1, and a 623bp transcript with a predicted open reading frame coding for 93 amino acids in LvALF2.

Function of E-protein dimers expressed in catfish lymphocytes.

E-proteins are essential class I bHLH transcription factors that play a role in lymphocyte development. In catfish lymphocytes the predominant E-proteins expressed are CFEB (a homologue of HEB) and E2A1, which both strongly drive transcription. In this study the role of homodimerization versus heterodimerization in the function of these catfish E-proteins was addressed through the use of expression constructs encoding forced dimers.

A cDNA microarray for Crassostrea virginica and C. gigas.

The eastern oyster, Crassostrea virginica, and the Pacific oyster, C. gigas, are species of global economic significance as well as important components of estuarine ecosystems and models for genetic and environmental studies. To enhance the molecular tools available for oyster research, an international group of collaborators has constructed a 27,496-feature cDNA microarray containing 4460 sequences derived from C.

Characterization of the immunoglobulin A heavy chain gene of the Atlantic bottlenose dolphin (Tursiops truncatus).

Immunoglobulin constant region heavy chain genes of the dolphin (Tursiops truncatus) have been described for IgM and IgG but not for IgA. Here, the heavy chain sequence of dolphin IgA has been cloned and sequenced as cDNA. RT-PCR amplification from blood peripheral lymphocytes was carried out using degenerate primers and a single sequence was detected.

Characterization of an Oct1 orthologue in the channel catfish, Ictalurus punctatus: a negative regulator of immunoglobulin gene transcription?

Background: The enhancer (Emu3') of the immunoglobulin heavy chain locus (IGH) of the channel catfish (Ictalurus punctatus) has been well characterized. The functional core region consists of two variant Oct transcription factor binding octamer motifs and one E-protein binding muE5 site. An orthologue to the Oct2 transcription factor has previously been cloned in catfish and is a functionally active transcription factor.

Insights into the immune transcriptome of the shrimp Litopenaeus vannamei: tissue-specific expression profiles and transcriptomic responses to immune challenge.

Infectious disease constitutes a major obstacle to the sustainability of shrimp aquaculture worldwide and a significant threat to natural populations of shrimp and other crustacea. The study of the shrimp immune system, including the response to viral infection, has been hampered by a relative lack of molecular genetic information and of tools suitable for high-throughput assessment of gene expression. In this report, the generation of a cDNA microarray encompassing 2,469 putative unigenes expressed in gills, circulating hemocytes, and hepatopancreas of Litopenaeus vannamei is described.

Double-stranded RNA and antiviral immunity in marine shrimp: inducible host mechanisms and evidence for the evolution of viral counter-responses.

Double-stranded RNA (dsRNA) is a common virus-associated molecular pattern and a potent inducer of antiviral responses in many organisms. While it is clear that the specific RNA interference (RNAi) response, a phenomenon triggered by dsRNA, serves antiviral functions in invertebrates, innate (non-specific) antiviral immune reactions induced by dsRNA (e.g.

A dolphin peripheral blood leukocyte cDNA microarray for studies of immune function and stress reactions.

A microarray focused on stress response and immune function genes of the bottlenosed dolphin has been developed. Random expressed sequence tags (ESTs) were isolated and sequenced from two dolphin peripheral blood leukocyte (PBL) cDNA libraries biased towards T- and B-cell gene expression by stimulation with IL-2 and LPS, respectively. A total of 2784 clones were sequenced and contig analysis yielded 1343 unigenes (archived and annotated at ).

Enhancer and promoter activity in the JH to IGHM intron of the Pekin duck, Anas platyrhynchos.

A transcriptional enhancer, Emu, was defined in the IGH locus of the Pekin duck, Anas platyrhynchos. Regions of DNA from the JH to IGHM intron were cloned into reporter constructs containing the SV40 promoter and transiently transfected into chicken B and T lymphocytes. A strong transcriptional activity, of several hundred-fold greater than that of a reporter construct with the promoter alone, was localized to a 281bp region that contains 2 E-box motifs, CAGCTG.

Regulation of metallothionein genes in the American oyster (Crassostrea virginica): ontogeny and differential expression in response to different stressors.

Metallothioneins (MTs) are typically low molecular weight (6-7 kDa), metal-binding proteins with characteristic repeating cysteine motifs (Cys-X-Cys or Cys-Xn-Cys) and a prolate ellipsoid shape containing single alpha- and beta-domains. While functionally diverse, they play important roles in metals homeostasis, detoxification and the stress response. The present study, combined with previous observations (e.

Regulation of the immunoglobulin heavy chain locus expression at the phylogenetic level of a bony fish: transcription factor interaction with two variant octamer motifs.

Transcriptional control of the IGH locus in teleosts is not fully understood, but evidence from catfish and zebrafish indicates major roles for octamer-binding (Oct) and E-protein transcription factors. A pair of variant octamer motifs in the Emu3' enhancer of the catfish has been shown to be particularly important in driving expression, justifying detailed study of their function. These octamer motifs were examined to determine if they bound Oct2 POU domains in monomeric or dimeric (PORE and MORE) configurations.

Conservation and divergence of the Emicro3' enhancer in the IGH locus of teleosts.

The core region of the Emicro3' transcriptional enhancer that drives the expression of the teleost IGH locus has been characterized functionally in two species, the catfish (Ictalurus punctatus) and the zebrafish (Danio rerio). These studies have suggested important differences: whereas the catfish enhancer acts through an E-box and two octamer motifs, the zebrafish enhancer exerts its major effects through two E-box motifs alone. In this study, the function of the catfish enhancer was reexamined in a broader comparative context within the teleosts.