The tissue-specific distribution of 3H-seocalcitol (EB 1089) and 3H-calcitriol in rats

Seocalcitol (EB 1089) is under development for the treatment of hepato-cellular carcinoma (HCC). The tissue distribution of 3H-seocalcitol was investigated in comparison to 3H-calcitriol in rats. Quantitative whole-body autoradiography was used to quantify the tissue distribution. The greatest difference in distribution between the two compounds was observed in the bloodstream. For most tissues the ratio seocalcitol/calcitriol varied between 0.2 and 3.1. The concentration of radioactivity in the liver was almost the same for the two compounds. For seocalcitol the concentration in the liver was 10 times higher than in serum. Assuming that the liver/serum concentration ratio is the same in rats and humans, the concentration of seocalcitol in the human liver is expected to be higher than the concentration resulting in more than 50% inhibition of cancer cell proliferation, and thus pharmacologically effective in HCC. It is questionable whether calcitriol would be present in the human liver in sufficient concentrations to be effective for the treatment of HCC, as the antiproliferative activity of calcitriol is generally more than 10-fold lower compared to that of seocalcitol and as calcitriol can only be administered at a dose that is ca. three-fold lower than the dose of seocalcitol.     

Learning of auditory equivalence classes for vowels by rats

Four male Long-Evans rats were trained to discriminate between synthetic vowel sounds using a GO/NOGO response choice task. The vowels were characterized by an increase in fundamental frequency correlated with an upward shift in formant frequencies. In an initial phase we trained the subjects to discriminate between two vowel categories using two exemplars from each category. In a subsequent phase the ability of the rats to generalize the discrimination between the two categories was tested. To test whether rats might exploit the fact that attributes of training stimuli covaried, we used non-standard stimuli with a reversed relation between fundamental frequency and formants. The overall results demonstrate that rats are able to generalize the discrimination to new instances of the same vowels. We present evidence that the performance of the subjects depended on the relation between fundamental and formant frequencies that they had previously been exposed to. Simple simulation results with artificial neural networks could reproduce most of the behavioral results and support the hypothesis that equivalence classes for vowels are associated with an experience-driven process based on general properties of peripheral auditory coding mixed with elementary learning mechanisms. These results suggest that rats use spectral and temporal cues similarly to humans despite differences in basic auditory capabilities.     

Homoeologous recombination within bread wheat to develop novel combinations of HMW-GS genes: transfer of the <Emphasis Type="Italic">Glu-A1</Emphasis> locus to chromosome 1D

In an attempt to improve the bread-making quality within hexaploid wheat by elaborating novel high-molecular weight glutenin subunits (HMW-GS) combinations useful in wheat-breeding programmes, a 1A chromosome fragment carrying the Glu-A1 locus encoding the subunit Ax2*, was translocated to the long arm of chromosome 1D. The partially isohomoeoallelic line, designated RR239, had a meiotic behaviour as regular as cv. Courtot. It was characterised using genomic in situ hybridization and microsatellite markers as well as biochemical and proteomic approaches. The translocated 1D chromosome had an interstitial 1AL segment representing in average 30% of the recombinant arm length that was confirmed by molecular analysis. The genetic length of the removed segment in chromosome 1DL was estimated to be at least 51 cM, and that of the interstitial 1AL translocation to be at least 33 cM. Proteome analysis performed on total endosperm proteins revealed variation in amounts, 8 spots and 1 spot being up- and downregulated, respectively. Quantitative variations in HMW-GS were observed for the Glu-A1 (Ax2*) and Glu-B1 (Bx7 + By8) loci in response to duplication of the Glu-A1 locus.     

Transgenic Fluorescent Plasmodium cynomolgi Liver Stages Enable Live Imaging and Purification of Malaria Hypnozoite-Forms

A major challenge for strategies to combat the human malaria parasite Plasmodium vivax is the presence of hypnozoites in the liver. These dormant forms can cause renewed clinical disease after reactivation through unknown mechanisms. The closely related non-human primate malaria P. cynomolgi is a frequently used model for studying hypnozoite-induced relapses. Here we report the generation of the first transgenic P. cynomolgi parasites that stably express fluorescent markers in liver stages by transfection with novel DNA-constructs containing a P. cynomolgi centromere. Analysis of fluorescent liver stages in culture identified, in addition to developing liver-schizonts, uninucleate persisting parasites that were atovaquone resistant but primaquine sensitive, features associated with hypnozoites. We demonstrate that these hypnozoite-forms could be isolated by fluorescence-activated cell sorting. The fluorescently-tagged parasites in combination with FACS-purification open new avenues for a wide range of studies for analysing hypnozoite biology and reactivation.     

A deubiquitylating complex required for neosynthesis of a yeast mitochondrial ATP synthase subunit

The ubiquitin system is known to be involved in maintaining the integrity of mitochondria, but little is known about the role of deubiquitylating (DUB) enzymes in such functions. Budding yeast cells deleted for UBP13 and its close homolog UBP9 displayed a high incidence of petite colonies and slow respiratory growth at 37°C. Both Ubp9 and Ubp13 interacted directly with Duf1 (DUB-associated factor 1), a WD40 motif-containing protein. Duf1 activates the DUB activity of recombinant Ubp9 and Ubp13 in vitro and deletion of DUF1 resulted in the same respiratory phenotype as the deletion of both UBP9 and UBP13. We show that the mitochondrial defects of these mutants resulted from a strong decrease at 37°C in the de novo biosynthesis of Atp9, a membrane-bound component of ATP synthase encoded by mitochondrial DNA. The defect appears at the level of ATP9 mRNA translation, while its maturation remained unchanged in the mutants. This study describes a new role of the ubiquitin system in mitochondrial biogenesis.     

Comparative analysis of two slc11 (Nramp) loci in Takifugu rubripes

To study the evolution of the solute carrier family 11 (slc11; formerly Nramp) protein, we isolated and characterized two paralogs from the pufferfish Takifugu rubripes (Fugu). These teleost genes, designated Fugu slc11a-a and Fugu slc11a-b, comprise open reading frames of 1743 nucleotides (581 amino acids) and 1662 nt (554 aa), respectively. The proteins are 81% similar, and both exhibit signature features of the slc11 family of proteins including 12 transmembrane domains, a conserved transport motif and a glycosylated loop. Both Fugu paralogs are more Slc11a2-like based on sequence homology and phylogenetic studies. Analysis of gene environment placed both in the proximity of multiple loci syntenic to human chromosome 12q13, that is, within a SLC11A2 gene environment. However, Fugu slc11a-a also gave one match with chromosome 2q35, where human SLC11A1 resides. Functional diversification was suggested by differences in tissue distribution and subcellular localization. Fugu slc11a-a exhibits a restricted expression profile and a complex subcellular localization, including LAMP1 positive late endosomes/lysosomes in transiently transfected mouse macrophages. Fugu slc11a-b is expressed ubiquitously and localizes solely to late endosomes/lysosomes. This comparative analysis extends our understanding of the evolution and function of this important family of divalent cation transporters. [Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AJ496547/8/9 and AJ496550.]     

Presence of antibodies against Coxiella burnetii and risk of spontaneous abortion: a nested case-control study

Background and Aims

Q fever is a bacterial zoonosis caused by infection with Coxiella burnetii. It is well established that Q fever causes fetal loss in small ruminants. The suspicion has been raised that pregnant women may also experience adverse pregnancy outcome when the infection is acquired or reactivated during pregnancy. The purpose of this study was to assess the potential association between serologic markers of infection with C.burnetii and spontaneous abortion.

Methods

A nested case-control study within the Danish National Birth Cohort, a cohort of 100,418 pregnancies recruited from 1996–2002. Women were recruited in first trimester of pregnancy and followed prospectively. Median gestational age at enrolment was 8 weeks (25 and 75 percentiles: 7 weeks; 10 weeks). During pregnancy, a blood sample was collected at gestational week 6–12 and stored in a bio bank. For this study, a case sample of 218 pregnancies was drawn randomly among the pregnancies in the cohort which ended with a miscarriage before 22 gestational weeks, and a reference group of 482 pregnancies was selected in a random fashion among all pregnancies in the cohort. From these pregnancies, serum samples were screened for antibodies against C. burnetii in a commercial enzyme-linked immunosorbent assay (ELISA). Samples that proved IgG or IgM antibody positive were subsequently confirmatory tested by an immunofluorescence (IFA) test.

Results

Among cases, 11 (5%) were C. burnetii positive in ELISA of which one was confirmed in the IFA assay compared to 29 (6%) ELISA positive and 3 IFA confirmed in the random sample.

Conclusions

We found no evidence of a higher prevalence of C.burnetii antibodies in serum samples from women who later miscarried and the present study does not indicate a major association between Q fever infection and spontaneous abortion in humans. Very early first trimester abortions were, however, not included in the study.     

Allergy against chironomids (non biting midges) in adult atopic patients

Summary The aim of this investigation was to evaluate the prevalence of atopic sensitization to chironomids (CHI) in patients with asthma and/or rhinitis (A/R), and to study concomitant sensitization to CHI and other allergens. Skin prick tests were performed with 3 different CHI extracts as well as with common inhalant allergens in 600 consecutive patients, 495 of which had A/R. Allergen specific IgE antibodies in the sera against CHI, shell fish and cockroaches were analyzed with Magic Lite.59 (12%) of the patients with A/R had a positive skin test with CHI. Positive skin tests with house dust mites and a storage mite were more common in CHI allergic patients than in other atopic patients. Nasal or conjunctival provocation tests, performed on 23 of the patients with positive skin test with CHI, were clearly positive in 7 cases (30%), questionable in 8 (35%) and negative in 8 cases (35%).Magic Lite, performed on sera from 50 of the patients with positive skin test against CHI, was positive with CHI in 39 cases (78%), with crayfish in 33 (66%), shrimp 20 (40%), cockroach 21 (40%) and with crab in 3 cases (6%).It is concluded that sensitization against CHI is common in patients with A/R. The clinical relevance of the positive test results is, however, unknown. Concomitant sensitization with CHI, crustaceans and cockroach is common.     

Colitis induced by proteinase-activated receptor-2 agonists is mediated by a neurogenic mechanism

Proteinase-activated receptor-2 (PAR2) activation induces colonic inflammation by an unknown mechanism. We hypothesized that PAR2 agonists administered intracolonically in mice induce inflammation via a neurogenic mechanism. Pretreatment of mice with neurokinin-1 and calcitonin-gene-related peptide (CGRP) receptor antagonists or with capsaicin showed attenuated PAR2-agonist-induced colitis. Immunohistochemistry demonstrated a differential expression of a marker for the type-1 CGRP receptor during the time course of PAR2-agonist-induced colitis, further suggesting a role for CGRP. We conclude that PAR2-agonist-induced intestinal inflammation involves the release of neuropeptides, which by acting on their receptors cause inflammation. These results implicate PAR2 as an important mediator of intestinal neurogenic inflammation.