Circulating hyaluronan, chondroitin sulphate and dextran sulphate bind to a liver receptor that does not recognize heparin

Chondroitin sulphate, injected intravenously into rats and given prior to intravenous ¹²⁵I-labelled hyaluronan with a mean Mw of about 400 kDa, was shown to inhibit the rapid receptor-mediated uptake of hyaluronan by the liver. The labelled hyaluronan that remained in the circulation was shown, by size exclusion chromatography of serum and urine, to be rapidly degraded down to fragments of lower Mw and filtered out into the urine and tissues. When the uptake of ¹²⁵I-hyaluronan was inhibited by unlabelled hyaluronan, only very low degradation and urinary excretion were found. Liver uptake could also be inhibited by dextran sulphate but not by heparin. Unlabelled hyaluronan could inhibit the liver uptake of labelled chondroitin sulphate but not labelled heparin. Unlabelled chondroitin sulphate and dextran sulphate inhibited cell association of labelled hyaluronan to liver endothelial cells in culture more effectively than unlabelled hyaluronan. Our data show that the liver hyaluronan receptors also recognize and effectively bind chondroitin sulphate and dextran sulphate but not heparin and that a hyaluronan-specific saturable degradative mechanism exists in the circulation. Such a mechanism could explain why hyaluronan in the general circulation has a much lower Mw than the hyaluronan in lymph. The results also indicate that increased hyaluronan levels in serum, and increased urinary excretion of hyaluronan, may be secondary to increased outflow of chondroitin sulphate from the tissues during some pathological conditions. This revised version was published online in November 2006 with corrections to the Cover Date.     

PCR primed with minisatellite core sequences yields DNA fingerprinting probes in wheat

 Four minisatellite core sequences were used as primers in a polymerase chain reaction (PCR) technique, known as the directed amplification of minisatellite-region DNA (DAMD), to detect polymorphisms in three pairs of hexaploid/tetraploid wheat cultivars. In each pair, the tetraploid cultivar (genomic formula AABB) was extracted from its corresponding hexaploid (genomic formula AABBDD) parent. Reproducible profiles of the amplified products revealed characteristic bands that were present only in the hexaploid wheats but not in their extracted tetraploids. Some polymorphisms were observed among the hexaploid cultivars. Twenty-three DAMD-PCR amplified fragments were isolated and screened as molecular probes on the genomic DNA of wild wheat species, hexaploid wheat and triticale cultivars. Subsequently, 8 of the fragments were cloned and sequenced. The DAMD-PCR clones revealed various degrees of polymorphism among different wild and cultivated wheats. Two clones yielded individual-specific DNA fingerprinting patterns which could be used for species differentiation and cultivar identification. The results demonstrated the use of DAMD-PCR as a tool for the isolation of informative molecular probes for DNA fingerprinting in wheat cultivars and species. Received: 13 May 1996/Accepted: 11 October 1996     

Characterization of the Theileria parva sporozoite proteome

East Coast fever is a lymphoproliferative disease caused by the tick-borne protozoan parasite Theileria parva. The sporozoite stage of this parasite, harboured and released from the salivary glands of the tick Rhipicephalus appendiculatus during feeding, invades and establishes infection in bovine lymphocytes. Blocking this initial stage of invasion presents a promising vaccine strategy for control of East Coast fever and can in part be achieved by targeting the major sporozoite surface protein p67. To support research on the biology of T. parva and the identification of additional candidate vaccine antigens, we report on the sporozoite proteome as defined by LC–MS/MS analysis. In total, 4780 proteins were identified in an enriched preparation of sporozoites. Of these, 2007 were identified as T. parva proteins, representing close to 50% of the total predicted parasite proteome. The remaining 2773 proteins were derived from the tick vector. The identified sporozoite proteins include a set of known T. parva antigens targeted by antibodies and cytotoxic T cells from cattle that are immune to East Coast fever. We also identified proteins predicted to be orthologs of Plasmodium falciparum sporozoite surface molecules and invasion organelle proteins, and proteins that may contribute to the phenomenon of bovine lymphocyte transformation. Overall, these data establish a protein expression profile of T. parva sporozoites as an important starting point for further study of a parasitic species which has considerable agricultural impact.     

Ancient diversity and geographical sub-structuring in African buffalo Theileria parva populations revealed through metagenetic analysis of antigen-encoding loci

An infection and treatment protocol involving infection with a mixture of three parasite isolates and simultaneous treatment with oxytetracycline is currently used to vaccinate cattle against Theileria parva. While vaccination results in high levels of protection in some regions, little or no protection is observed in areas where animals are challenged predominantly by parasites of buffalo origin. A previous study involving sequencing of two antigen-encoding genes from a series of parasite isolates indicated that this is associated with greater antigenic diversity in buffalo-derived T. parva. The current study set out to extend these analyses by applying high-throughput sequencing to ex vivo samples from naturally infected buffalo to determine the extent of diversity in a set of antigen-encoding genes. Samples from two populations of buffalo, one in Kenya and the other in South Africa, were examined to investigate the effect of geographical distance on the nature of sequence diversity. The results revealed a number of significant findings. First, there was a variable degree of nucleotide sequence diversity in all gene segments examined, with the percentage of polymorphic nucleotides ranging from 10% to 69%. Second, large numbers of allelic variants of each gene were found in individual animals, indicating multiple infection events. Third, despite the observed diversity in nucleotide sequences, several of the gene products had highly conserved amino acid sequences, and thus represent potential candidates for vaccine development. Fourth, although compelling evidence for population differentiation between the Kenyan and South African T. parva parasites was identified, analysis of molecular variance for each gene revealed that the majority of the underlying nucleotide sequence polymorphism was common to both areas, indicating that much of this aspect of genetic variation in the parasite population arose prior to geographic separation.     

Rational design of a monomeric and photostable far‐red fluorescent protein for fluorescence imaging in vivo

Fluorescent proteins (FPs) are powerful tools for cell and molecular biology. Here based on structural analysis, a blue‐shifted mutant of a recently engineered monomeric infrared fluorescent protein (mIFP) has been rationally designed. This variant, named iBlueberry, bears a single mutation that shifts both excitation and emission spectra by approximately 40 nm. Furthermore, iBlueberry is four times more photostable than mIFP, rendering it more advantageous for imaging protein dynamics. By tagging iBlueberry to centrin, it has been demonstrated that the fusion protein labels the centrosome in the developing zebrafish embryo. Together with GFP‐labeled nucleus and tdTomato‐labeled plasma membrane, time‐lapse imaging to visualize the dynamics of centrosomes in radial glia neural progenitors in the intact zebrafish brain has been demonstrated. It is further shown that iBlueberry can be used together with mIFP in two‐color protein labeling in living cells and in two‐color tumor labeling in mice.     

On robustness and model flexibility in survival analysis: transformed hazard models and average effects

Yin and Ibrahim (2005a, Biometrics 61, 208-216) use a Box-Cox transformed hazard model to acknowledge uncertainty about how a linear predictor acts upon the hazard function of a failure-time response. Particularly, additive and proportional hazards models arise for particular values of the transformation parameter. As is often the case, however, this added model flexibility is obtained at the cost of lessened parameter interpretability. Particularly, the interpretation of the coefficients in the linear predictor is intertwined with the value of the transformation parameter. Moreover, some data sets contain very little information about this parameter. To shed light on the situation, we consider average effects based on averaging (over the joint distribution of the explanatory variables and the failure-time response) the partial derivatives of the hazard, or the log-hazard, with respect to the explanatory variables. First, we consider fitting models which do assume a particular form of covariate effects, for example, proportional hazards or additive hazards. In some such circumstances, average effects are seen to be inferential targets which are robust to the effect form being misspecified. Second, we consider average effects as targets of inference when using the transformed hazard model. We show that in addition to being more interpretable inferential targets, average effects can sometimes be estimated more efficiently than the corresponding regression coefficients.