The N-terminus of thrombospondin: the domain stands apart

Thrombospondin 1 (TSP1) was first recognized as a thrombin-sensitive protein associated with platelet membranes. It is secreted by numerous cell types and its expression is predominant in areas of active tissue remodeling. Thrombospondins 1 and 2 are large, trimeric, matricellular proteins, composed of multiple structural motifs which interact with a diverse array of receptors and molecules. Thrombospondin's capacity to bind multiple receptors renders it multifunctional. The functions of its isolated domains can be overlapping or contradictory. In this review, we focus on the N-terminus of the molecule, first recognized for its strong heparin binding properties and characterized by its susceptibility to proteolytic cleavage from the stalk region of thrombospondin. The N-terminus, called the heparin binding domain (HBD), interacts with a variety of macromolecules including heparan sulfate proteoglycans at the membrane and in the matrix, LDL receptor-related protein (LRP), sulfated glycolipids, calreticulin, and integrins. The HBD mediates endocytosis of thrombospondin. It functions both as a soluble and an insoluble modulator of cell adhesion and motility. In contrast to thrombospondin, the HBD has pro-angiogenic activity. We propose that the HBD of thrombospondins 1 and 2 are found primarily in the cellular microenvironment in conditions of cellular injury, stress and tissue remodeling and that the HBD conveys multiple signals involved in cellular adaptation to injury.     

Conformation and structure of polymeric contrast agents for medical imaging

The structure of Gd-DTPA-polylysine, Gd-DOTA-polylysine, Gd-SCN-Bz-DOTA-polylysine, and Gd-DTPA-poly(glu:lys) was investigated with circular dichroism, gel permeation chromatography, low angle light scattering, and proton longitudinal relaxivity. Molecular modeling calculations were performed and predicted helical secondary structure for charged Gd-chelator residues, i.e., Gd-DTPA, when the DTPA conjugation levels reached 90% and higher. This helical secondary structure was observed with circular dichroism. The conformational transition from coiled to extended linear was observed also by gel permeation chromatography and by proton relaxivity measurements. The helical secondary structure was not observed when the chelator was changed to DOTA. The residue charge interactions were eliminated in this case since the Gd-DOTA complex had no net charge. For this construct, the gel permeation and relaxivity measurements indicated a coiled conformation. An extended linear conformation was regained when the chelator complex was changed to Gd-SCN-Bz-DOTA, which had a net negative charge. The functional aspects of these structures were investigated by MR imaging of an animal tumor model. The linear extended polymer constructs gave 10-fold higher tumor signals then the coiled-collapsed constructs, indicating a much higher degree of trans-endothelial transport in the tumors.     

Mycoplasma haemocanis infection--a kennel disease?

Mycoplasma haemocanis (formerly Haemobartonella canis) is a red blood cell parasite that causes disease mainly in immunosuppressed and splenectomized dogs. Clinical outbreak of the disease resulted in failure of a large experimental project. We aimed to identify whether M. haemocanis has increased prevalence in kennel-raised dogs. In a prospective study, we compared the prevalence of M. haemocanis in whole blood (anti-coagulated by use of EDTA) collected from pet dogs (University of Illinois, Urbana Champaign, Ill.; n = 60) with that in blood from dogs raised in three distinct kennels in western Europe (WE; n = 23), eastern Europe (EE; n = 20), and North America (NA; n = 20). Screening included antibody testing and microscopy of blood smears. The presence of M. haemocanis was identified using a polymerase chain reaction (PCR) assay for specific DNA of the organism. None of the pet dogs (0%) was test positive for M. haemocanis DNA. Mycoplasma haemocanis was found in dogs tested at all of the kennels. Infection rate in the three kennels was 30, 35, and 87%, respectively (all P < 0.001 versus control, chi2-test). Latent infection with M. haemocanis was not a single observation in kennel-raised dogs. Prevalence may be higher than that in a pet dog population. The potential exists for these latent infections to adversely affect or confound research results.     

The differential tissue distribution of the citrus flavanone naringenin following gastric instillation

Citrus flavonoids have been investigated for their biological activity, with both anti-inflammatory and -carcinogenic effects being reported. However, little information is known on the bioavailability of these compounds in vivo. The objectives of this study were to determine the tissue distribution of naringenin after gastric gavage of [³H]-naringenin to rats. Unlabelled naringenin was also used to quantify the levels of naringenin and its major metabolites in tissues and eliminated in the urine and faeces. Significant radioactivity was detected in the plasma as well as all tissues examined 2 h post-gavage. After 18 h, higher levels of radioactivity were retained in plasma and tissues (55% of the administered radioactivity). Investigation of the nature of metabolites, using unlabelled naringenin, revealed that the glucuronides were the major components in plasma, tissues and urine, in addition to the colonic metabolite 3-(4-hydroxyphenyl) propionic acid, detected in the urine. The aglycone was the form extensively retained in tissues after 18 h post-gavage. Total identified metabolites detected after 18 h in most tissues were only 1-5% of the levels detected after 2 h. However, the brain, lungs and heart retained 27, 20 and 11%, respectively, relative to the total metabolites detected at 2 h. While radioactive detection suggests increased levels of breakdown products of naringenin after 18 h versus 2 h, the products identified using unlabelled naringenin are not consistent with this, suggesting that a predominant proportion of the naringenin breakdown products at 18 h are retained as smaller decomposition molecules which cannot yet be identified.     

A microarray-based high throughput molecular marker genotyping method: the tagged microarray marker (TAM) approach

A microarray-based method has been developed for scoring thousands of DNAs for a co-dominant molecular marker on a glass slide. The approach was developed to detect insertional polymorphism of transposons and works well with single nucleotide polymorphism (SNP) markers. Biotin- terminated allele-specific PCR products are spotted unpurified onto streptavidin-coated glass slides and visualised by hybridisation of fluorescent detector oligonucleotides to tags attached to the allele- specific PCR primers. Two tagged primer oligonucleotides are used per locus and each tag is detected by hybridisation to a concatameric DNA probe labelled with multiple fluorochromes.     

A growth regulatory loop that provides homeostasis to phytochrome a signaling

Phytochrome kinase substrate1 (PKS1) is a cytoplasmic protein that interacts physically with, and is phosphorylated by, the plant photoreceptor phytochrome. Here, we show that light transiently increases PKS1 mRNA levels and concentrates its expression to the elongation zone of the hypocotyl and root. This response is mediated by phytochrome A (phyA) acting in the very low fluence response (VLFR) mode. In the hypocotyl, PKS1 RNA and protein accumulation are maintained only under prolonged incubation in far-red light, the wavelength that most effectively activates phyA. Null mutants of PKS1 and its closest homolog, PKS2, show enhanced phyA-mediated VLFR. Notably, a pks1 pks2 double mutant has no phenotype, whereas overexpression of either PKS1 or PKS2 results in the same phenotype as the pks1 or pks2 single null mutant. We propose that PKS1 and PKS2 are involved in a growth regulatory loop that provides homeostasis to phyA signaling in the VLFR. In accordance with this idea, PKS1 effects are larger in the pks2 background (and vice versa). Moreover, the two proteins can interact with each other, and PKS2 negatively regulates PKS1 protein levels specifically under VLFR conditions.     

CD49d overexpression and T cell autoimmunity

D10.G4.1 (D10) cells, a murine conalbumin-reactive Th2 cell line, made to overexpress the beta(2) integrin LFA-1 by pharmacological manipulation or by transfection become autoreactive and are capable of inducing in vivo autoimmunity. However, whether this is specific to LFA-1 and whether overexpression of other T cell integrin molecules has the same effect are unknown. We examined the functional consequences of T cell CD49d (alpha(4) integrin) overexpression by transfecting murine CD49d cDNA into D10 cells. Similar to the LFA-1-transfected cells, the CD49d-overexpressing T cells are autoreactive and proliferate in response to APCs in an MHC class II-dependent manner in the absence of nominal Ag. Additionally, CD49d overexpression is associated with increased in vitro adhesion to endothelial cells and increased in vivo splenic homing. However, in contrast to LFA-1 overexpression, increased T cell CD49d expression is not associated with autoreactive cytotoxicity or the ability to induce in vivo autoimmunity. In addition to the novel observation that CD49d overexpression is sufficient to induce T cell autoreactivity, our results also support the hypothesis that the ability to induce in vivo autoimmunity is related to T cell cytotoxicity and not to T cell proliferation function in the D10 murine adoptive transfer model of autoimmunity.     

Either of the CD45RB and CD45RO isoforms are effective in restoring T cell,but not B cell,development and function in CD45-null mice

The protein tyrosine phosphatase CD45 is expressed as a series of isoforms whose tissue and differentiation stage specificity is broadly conserved in evolution. CD45 has been shown to be an important regulator of a variety of functions in many different hemopoietic lineages. We have chosen an in vivo genetic complementation strategy to investigate the differential functions between isoforms. In this study, we report the characterization of transgenic mice which express the isoforms CD45RO or CD45RB as their only CD45 molecules, at a variety of expression levels and in the majority of hemopoietic lineages. Both CD45RO and CD45RB isoforms reconstitute thymocyte development in a CD45-null mouse background when expressed above a threshold level. The resulting mature T cells populate the peripheral lymphoid organs where they are found at normal frequency. Both CD45RO and CD45RB isoforms also permit T cell function in the periphery, although the threshold for normal function here appears to be set higher than in the thymus. In contrast, neither isoform is capable of fully restoring peripheral B cell maturation, even at levels approaching those in heterozygous CD45(+/-) mice in which maturation is normal. In vitro activation of B cells by Ag-receptor stimulation is only minimally complemented by these CD45RO and CD45RB transgenes. Our results suggest that CD45 isoforms play unique roles which differ between the T and B lineages.