Application of nox-restriction fragment length polymorphism for the differentiation of Brachyspira intestinal spirochetes isolated from pigs and poultry in Australia

Sixty-nine intestinal spirochetes isolated from pigs and poultry in eastern Australia were selected to evaluate the effectiveness of a species-specific PCR-based restriction fragment length polymorphism (RFLP) analysis of the Brachyspira nox gene. For comparative purposes, all isolates were subjected to species-specific PCRs for the pathogenic species Brachyspira hyodysenteriae and Brachyspira pilosicoli, and selected isolates were examined further by sequence analysis of the nox and 16S ribosomal RNA genes. Modifications to the original nox-RFLP method included direct inoculation of bacterial cells into the amplification mixture and purification of the PCR product, which further optimized the nox-RFLP for use in a veterinary diagnostic laboratory, producing sufficient product for both species identification and future comparisons. Although some novel profiles that prevented definitive identification were observed, the nox-RFLP method successfully classified 45 of 51 (88%) porcine and 15 of 18 (83%) avian isolates into 5 of the 6 recognized species of Brachyspira. This protocol represents a significant improvement over conventional methods currently used in veterinary diagnostic laboratories for rapid specific identification of Brachyspira spp. isolated from both pigs and poultry.

Visualizing levels of osteoblast differentiation by a two-color promoter-GFP strategy: Type I collagen-GFPcyan and osteocalcin-GFPtpz

A 3.9 kb DNA fragment of human osteocalcin promoter and 3.6 kb DNA fragment of the rat collagen type1a1 promoter linked with visually distinguishable GFP isomers, topaz and cyan, were used for multiplex analysis of osteoblast lineage progression. Three patterns of dual transgene, expression can be appreciated in primary bone cell cultures derived from the transgenic mice and by histology of their corresponding bones. Our data support the interpretation that strong pOBCol3.6GFPcyan alone is found in newly formed osteoblasts, while strong pOBCol3.6GFPcyan and hOC-GFPtpz are present in osteoblasts actively making a new matrix. Osteoblasts expressing strong hOC-GFPtpz and weak pOBCol3.6GF-Pcyan are also present and may or may not be producing mineralized matrix. This multiplex approach reveals the heterogeneity within the mature osteoblast population that cannot be appreciated by current histological methods. It should be useful to identify and isolate populations of cells within an osteoblast lineage as they progress through stages of differentiation.

Isoform specific changes in PPAR alpha and beta in colon and breast cancer with differentiation

To investigate the role of peroxisome proliferator-activated receptors (PPARs) chi and beta in the differentiation of colon cancer cells, we differentiated HT-29 cells using sodium butyrate (NaB) and culturing post-confluence and assessed differentiation using the marker intestinal alkaline phosphatase. While PPAR chi levels only changed with culturing post confluence, PPAR beta levels increased independent of the method of differentiation. To explore further the differences induced by NaB. we assessed changes in both PPAR isoforms in MCF-7 breast cancer cells cultured in the presence of NaB over 48 h. Again a very different expression pattern was observed with PPAR-1 increasing after 4 h and remaining elevated, while PPAR beta increased transiently. Our studies suggest that the expression of PPARs is dependent upon both the method of differentiation and on time. Moreover, these studies show that changes in levels are not required for the differentiation of colon cancer cell lines, whereas changes in PPAR beta are more closely associated with differentiation. (c) 2005 Elsevier Inc. All rights reserved.

Genetic differentiation in Scottish populations of the pine beauty moth, Panolis flammea (Lepidoptera : Noctuidae)

Pine beauty moth, Panolis flammea (Denis & Schiffermuller), is a recent but persistent pest of lodgepole pine plantations in Scotland, but exists naturally at low levels within remnants and plantations of Scots pine. To test whether separate host races occur in lodgepole and Scots pine stands and to examine colonization dynamics, allozyme, randomly amplified polymorphic DNA (RAPD) and mitochondrial variation were screened within a range of Scottish samples. RAPD analysis indicated limited long distance dispersal (F-ST=0.099), and significant isolation by distance (P < 0.05); but that colonization between more proximate populations was often variable, from extensive to limited exchange. When compared with material from Germany, Scottish samples were found to be more diverse and significantly differentiated for all markers. For mtDNA, two highly divergent groups of haplotypes were evident, one group contained both German and Scottish samples and the other was predominantly Scottish. No genetic differentiation was evident between P. flammea populations sampled from different hosts, and no diversity bottleneck was observed in the lodgepole group. Indeed, lodgepole stands appear to have been colonized on multiple occasions from Scots pine sources and neighbouring populations on different hosts are close to panmixia.

Zebrafish KLF4 is essential for anterior mesendoderm/pre-polster differentiation and hatching

Gene knockout studies of Kruppel-like factors (KLFs) in mice have shown essential roles in organogenesis. A screen for KLF family members in zebrafish identified many KLFs. One of these, zebrafish KLF4 (zKLF4) is the homologue of neptune, a Xenopus laevis KLF. zKLF4 is expressed from approximately 80% epiboly a patch of dorsal/anterior mesendodermal cells called the pre-polster and, subsequently, in the polster and hatching gland. Here we investigate the function of zKLF4 using morpholino-based antisense oligonucleotides. Knockdown of zKLF4 resulted in complete absence of hatching gland formation and subsequent hatching in zebrafish. In addition, there was early knockdown of expression of the pre-polster/anterior mesendoderm markers CatL, cap1, and BMP4. These results indicate zKLF4 is expressed within the pre-polster, an early mesendodermal site, and that it plays a critical role in the differentiation of these cells into hatching gland cells.

The colony-stimulating factor 1 receptor is expressed on dendritic cells during differentiation and regulates their expansion

The lineage of dendritic cells (DC), and in particular their relationship to monocytes and macrophages, remains obscure. Furthermore, the requirement for the macrophage growth factor CSF-1 during DC homeostasis is unclear. Using a transgenic mouse in which the promoter for the CSF-1R (c-fms) directs the expression of enhanced GFP in cells of the myeloid lineage, we determined that although the c-fms promoter is inactive in DC precursors, it is up-regulated in all DC subsets during differentiation. Furthermore, plasmacytoid DC and all CD11c(high) DC subsets are reduced by 50-70% in CSF-1-deficient osteopetrotic mice, confirming that CSF-1 signaling is required for the optimal differentiation of DC in vivo. These data provide additional evidence that the majority of tissue DC is of myeloid origin during steady state and supports a close relationship between DC and macrophage biology in vivo.

Loyalty without Conformity: Tailoring Self-perception as a Means of Balancing Belonging and Differentiation

We argue that members of individualist cultures balance their desire to belong with their desire to be different by maintaining a self-image as being loyal but relatively immune to group influence. Consistent with this, in Study 1 there was a strong tendency for people to rate themselves as being more independent (i.e., less conformist) than other people in their college. College students also rated themselves as being highly loyal to the group, however no self - other discrepancies were found on this dimension. This is despite the fact that traits of loyalty were rated more positively than were traits of independence. Study 2 provided evidence that culture influences the pattern of self - other discrepancies. Whereas people from individualist countries self-enhance on independence dimensions, people from collectivist countries self-enhance on loyalty dimensions. Again, these effects could not be explained as being a function of how positive these traits were seen to be, suggesting a cultural explanation rather than a straight forward superiority bias explanation for the observed discrepancies in self - other ratings. Results are discussed in relation to the SCENT model.

Differentiation between protective reflexes: Cardiac defense and startle

Rise time and duration are two parametric characteristics of the eliciting stimulus frequently used to differentiate among psychophysiological reflexes. The present research varied the duration (study 1) and rise time (study 2) of an intense acoustic stimulus to dissociate cardiac defense and cardiac startle using the eyeblink response as the external criterion of startle. In each study, 100 participants were presented with five white noise stimuli of 105 dB under one of five duration (50, 100, 250, 500, and 1000 ms) or rise time (0, 24, 48, 96, and 240 ms) conditions. Cardiac defense was affected by stimulus duration, present only in the 500- and 1000-ms conditions, but not by stimulus rise time, present in all rise time conditions. Rise time affected blink startle, but did not selectively alter the short latency accelerative component of the heart rate response, thus questioning whether it reflects startle.

Myoepithelial calls: Pathology, cell separation and markers of myoepithelial differentiation

Until recently the myoepithelial cell has been studied relatively little in terms of its role in breast cancer. A number of malignancies showing myoepithelial differentiation have been reported in the literature, although they are still thought to be relatively rare and only limited studies are published. As a result of recent expression profiling experiments, one type of tumor with myoepithelial features, the so-called 'basal' breast cancer, has received a renewed interest, although it has been known to pathologists for more than two decades. These tumors, which express markers of both luminal and myoepithelial cells, are now being studied using antibodies against some new molecules that have emerged from studies of sorted normal luminal and myoepithelial cells. These immunohistochemical data, combined with genomic studies, may lead to better identification and management of patients with 'basal' tumors.

Genetics and genomics of osteoclast differentiation: Integrating cell signaling pathways and gene networks

The regulation of osteoclast differentiation in the bone microenvironment is critical for normal bone remodeling, as well as for various human bone diseases. Over the last decade, our knowledge of how osteoclast differentiation occurs has progressed rapidly. We highlight some of the major advances in understanding how cell signaling and transcription are integrated to direct the differentiation of this cell type. These studies used genetic, molecular, and biochemical approaches. Additionally, we summarize data obtained from studies of osteoclast differentiation that used the functional genomic approach of global gene profiling applied to osteoclast differentiation. This genomic data confirms results from studies using the classical experimental approaches and also may suggest new modes by which osteoclast differentiation and function can be modulated. Two conclusions that emerge are that osteoclast differentiation depends on a combination of fairly ubiquitously expressed transcription factors rather than unique osteoclast factors, and that the overlay of cell signaling pathways on this set of transcription factors provides a powerful mechanism to fine tune the differentiation program in response to the local bone microenvironment.

Reduced genetic diversity and significant genetic differentiation after translocation: Comparison of the remnant and translocated populations of bridled nailtail wallabies (Onychogalea fraenata)

Loss of genetic diversity and increased population differentiation from source populations are common problems associated with translocation programmes established from captive-bred stock or a small number of founders. The bridled nailtail wallaby is one of the most endangered macropods in Australia, having been reduced to a single remnant population in the last 100 years. A translocated population of bridled nailtail wallabies was established using animals sourced directly from the remnant population (wild-released) as well as the progeny of animals collected for a captive breeding programme (captive-bred). The aims of this study were to compare genetic diversity among released animals and their wild-born progeny to genetic diversity observed in the remnant population, and to monitor changes in genetic diversity over time as more animals were released into the population. Heterozygosity did not differ between the translocated and remnant population; however, allelic diversity was significantly reduced across all released animals and their wild-born progeny. Animals bred in captivity and their wild-born progeny were also significantly differentiated from the source population after just four generations. Wild-released animals, however, were representative of the source population and several alleles were unique to this group. Both heterozygosity and allelic diversity among translocated animals decreased over time with the additional release of captive-bred animals, as no new genetic stock was added to the population. Captive breeding programmes can provide large numbers of animals for release, but this study highlights the importance of sourcing animals directly from remnant populations in order to maintain genetic diversity and minimise genetic drift.

The matricellular protein SPARC is internalized in Sertoli, Leydig, and germ cells during testis differentiation

The gene encoding the matricellular protein secreted protein, acidic and rich in cysteine (SPARC) was identified in a screen for genes expressed sex-specifically during mouse gonad development, as being strongly upregulated in the male gonad from very early in testis development. We present here a detailed analysis of SPARC gene and protein expression during testis development, from 11.5 to 15.5 days post coitum (dpc). Section in situ hybridization analysis revealed that SPARC mRNA is expressed by the Sertoli cells in the testis cords and the fetal Leydig cells, found within the interstitial space between the testis cords. Immunodetection with anti-SPARC antibody showed that the protein was located inside the testis cords, within the cytoplasm of Sertoli and germ cells. In the interstitium, SPARC was present intracellularly within the Leydig cells. The internalization of SPARC in Sertoli, Leydig, and germ cells suggests that it plays an intracellular regulatory role in these cell types during fetal testis development.