TEMPRANILLO is a regulator of juvenility in plants

Many plants are incapable of flowering in inductive daylengths during the early juvenile vegetative phase (JVP). Arabidopsis mutants with reduced expression of TEMPRANILLO (TEM), a repressor of FLOWERING LOCUS T (FT) had a shorter JVP than wild-type plants. Reciprocal changes in mRNA expression of TEM and FT were observed in both Arabidopsis and antirrhinum, which correlated with the length of the JVP. FT expression was induced just prior to the end of the JVP and levels of TEM1 mRNA declined rapidly at the time when FT mRNA levels were shown to increase. TEM orthologs were isolated from antirrhinum (AmTEM) and olive (OeTEM) and were expressed most highly during their juvenile phase. AmTEM functionally complemented AtTEM1 in the tem1 mutant and over-expression of AmTEM prolonged the JVP through repression of FT and CONSTANS (CO). We propose that TEM may have a general role in regulating JVP in herbaceous and woody species

Tissue factor in antiphospholipid antibody-induced pregnancy loss:a pro-inflammatory molecule

Fetal loss in patients with antiphospholipid antibodies (aPL) has been ascribed to thrombosis of placental vessels. However, we have shown that inflammation, specifically complement activation with generation of the anaphylotoxin C5a, is an essential mediator of fetal injury. We have analysed the role of tissue factor (TF) in a mouse model of aPL-induced pregnancy loss. TF is the major cellular activator of the coagulation cascade but also has cell signaling activity. Mice that received aPL-IgG showed strong TF staining throughout the decidua and on embryonic debris. This TF staining was not associated with either fibrin staining or thrombi in deciduas. The absence of fibrin deposition and thrombi suggests that TF-dependent activation of coagulation does not mediate aPL-induced pregnancy loss. We found that either blockade of TF with a monoclonal antibody in wild type mice or a genetic reduction of TF prevented aPL-induced inflammation and pregnancy loss indicated a pathogenic role for TF in aPL-induced pregnancy complications. In response to aPL-generated C5a, neutrophils express TF potentiating inflammation in the deciduas and leading to miscarriages. Importantly, we showed that TF in myeloid cells, but not fetal-derived cells (trophoblasts), was associated with fetal injury, suggesting that the site for pathologic TF expression is neutrophils. We found that TF expression in neutrophils contributes to respiratory burst and subsequent trophoblast injury and pregnancy loss induced by aPL. The identification of TF, acting as an important pro-inflammatory mediator in aPL-induced fetal injury, provides a new target for therapy to prevent pregnancy loss in the aPL syndrome

The secreted triose phosphate isomerase of Brugia malayi is required to sustain microfilaria production in vivo

Human lymphatic filariasis is a major tropical disease transmitted through mosquito vectors which take up microfilarial larvae from the blood of infected subjects. Microfilariae are produced by long-lived adult parasites, which also release a suite of excretory-secretory products that have recently been subject to in-depth proteomic analysis. Surprisingly, the most abundant secreted protein of adult Brugia malayi is triose phosphate isomerase (TPI), a glycolytic enzyme usually associated with the cytosol. We now show that while TPI is a prominent target of the antibody response to infection, there is little antibody-mediated inhibition of catalytic activity by polyclonal sera. We generated a panel of twenty-three anti-TPI monoclonal antibodies and found only two were able to block TPI enzymatic activity. Immunisation of jirds with B. malayi TPI, or mice with the homologous protein from the rodent filaria Litomosoides sigmodontis, failed to induce neutralising antibodies or protective immunity. In contrast, passive transfer of neutralising monoclonal antibody to mice prior to implantation with adult B. malayi resulted in 60–70% reductions in microfilarial levels in vivo and both oocyte and microfilarial production by individual adult females. The loss of fecundity was accompanied by reduced IFNγ expression by CD4+ T cells and a higher proportion of macrophages at the site of infection. Thus, enzymatically active TPI plays an important role in the transmission cycle of B. malayi filarial parasites and is identified as a potential target for immunological and pharmacological intervention against filarial infections

Testing the theory of immune selection in cancers that break the rules of transplantation

Modification of cancer cells likely to reduce their immunogenicity, including loss or down-regulation of MHC molecules, is now well documented and has become the main support for the concept of immune surveillance. The evidence that these modifications, in fact, result from selection by the immune system is less clear, since the possibility that they may result from reorganized metabolism associated with proliferation or from cell de-differentiation remains. Here, we (a) survey old and new transplantation experiments that test the possibility of selection and (b) survey how transmissible tumours of dogs and Tasmanian devils provide naturally evolved tests of immune surveillance

Homozygous CDA*3 is a major cause of life-threatening toxicities in gemcitabine-treated Japanese cancer patients

Among 242 Japanese pancreatic cancer patients, three patients (1.2%) encountered life-threatening toxicities, including myelosuppression, after gemcitabine-based chemotherapies. Two of them carried homozygous CDA*3 (CDA208G>A [Ala70Thr]), and showed extremely low plasma cytidine deaminase activity and gemcitabine clearance. Our results suggest that homozygous *3 is a major factor causing gemcitabine-mediated severe adverse reactions among the Japanese population

Successful Protein Extraction from Over-Fixed and Long-Term Stored Formalin-Fixed Tissues

One of the major breakthroughs in molecular pathology during the last decade was the successful extraction of full-length proteins from formalin-fixed and paraffin-embedded (FFPE) clinical tissues. However, only limited data are available for the protein extraction efficiency of over-fixed tissues and FFPE blocks that had been stored for more than 15 years in pathology archives. In this study we evaluated the protein extraction efficiency of FFPE tissues which had been formalin-fixed for up to 144 hours and tissue blocks that were stored for 20 years, comparing an established and a new commercial buffer system. Although there is a decrease in protein yield with increasing fixation time, the new buffer system allows a protein recovery of 66% from 144 hours fixed tissues compared to tissues that were fixed for 6 hours. Using the established extraction procedure, less than 50% protein recovery was seen. Similarly, the protein extraction efficiency decreases with longer storage times of the paraffin blocks. Comparing the two buffer systems, we found that 50% more proteins can be extracted from FFPE blocks that were stored for 20 years when the new buffer system is used. Taken together, our data show that the new buffer system is superior compared to the established one. Because tissue fixation times vary in the routine clinical setting and pathology archives contain billions of FFPE tissues blocks, our data are highly relevant for research, diagnosis, and treatment of disease

Homozygosity Mapping on Homozygosity Haplotype Analysis to Detect Recessive Disease-Causing Genes from a Small Number of Unrelated, Outbred Patients

Genes involved in disease that are not common are often difficult to identify; a method that pinpoints them from a small number of unrelated patients will be of great help. In order to establish such a method that detects recessive genes identical-by-descent, we modified homozygosity mapping (HM) so that it is constructed on the basis of homozygosity haplotype (HM on HH) analysis. An analysis using 6 unrelated patients with Siiyama-type α1-antitrypsin deficiency, a disease caused by a founder gene, the correct gene locus was pinpointed from data of any 2 patients (length: 1.2–21.8 centimorgans, median: 1.6 centimorgans). For a test population in which these 6 patients and 54 healthy subjects were scrambled, the approach accurately identified these 6 patients and pinpointed the locus to a 1.4-centimorgan fragment. Analyses using synthetic data revealed that the analysis works well for IBD fragment derived from a most recent common ancestor (MRCA) who existed less than 60 generations ago. The analysis is unsuitable for the genes with a frequency in general population more than 0.1. Thus, HM on HH analysis is a powerful technique, applicable to a small number of patients not known to be related, and will accelerate the identification of disease-causing genes for recessive conditions