Dosimetric evaluation and radioimmunotherapy of anti-tumour multivalent Fab́ fragments

We have been investigating the use of cross-linked divalent (DFM) and trivalent (TFM) versions of the anti-carcinoembryonic antigen (CEA) monoclonal antibody A5B7 as possible alternatives to the parent forms (IgG and F(ab́)2) which have been used previously in clinical radioimmunotherapy (RIT) studies in colorectal carcinoma. Comparative biodistribution studies of similar sized DFM and F(ab́)2 and TFM and IgG, radiolabelled with both 131I and 90Y have been described previously using the human colorectal tumour LS174T nude mouse xenograft model (Casey et al (1996) Br J Cancer 74: 1397–1405). In this study quantitative estimates of radiation distribution and RIT in the xenograft model provided more insight into selecting the most suitable combination for future RIT. Radiation doses were significantly higher in all tissues when antibodies were labelled with 90Y. Major contributing organs were the kidneys, liver and spleen. The extremely high absorbed dose to the kidneys on injection of 90Y-labelled DFM and F(ab́)2 as a result of accumulation of the radiometal would result in extremely high toxicity. These combinations are clearly unsuitable for RIT. Cumulative dose of 90Y-TFM to the kidney was 3 times lower than the divalent forms but still twice as high as for 90Y-IgG. TFM clears faster from the blood than IgG, producing higher tumour to blood ratios. Therefore when considering only the tumour to blood ratios of the total absorbed dose, the data suggests that TFM would be the most suitable candidate. However, when corrected for equitoxic blood levels, doses to normal tissues for TFM were approximately twice the level of IgG, producing a two-fold increase in the overall tumour to normal tissue ratio. In addition RIT revealed that for a similar level of toxicity and half the administered activity, 90Y-IgG produced a greater therapeutic response. This suggests that the most promising A5B7 antibody form with the radionuclide 90Y may be IgG. Dosimetry analysis revealed that the tumour to normal tissue ratios were greater for all 131I-labelled antibodies. This suggests that 131I may be a more suitable radionuclide for RIT, in terms of lower toxicity to normal tissues. The highest tumour to blood dose and tumour to normal tissue ratio at equitoxic blood levels was 131I-labelled DFM, suggesting that 131I-DFM may be best combination of antibody and radionuclide for A5B7. The dosimetry estimates were in agreement with RIT results in that twice the activity of 131I-DFM must be administered to produce a similar therapeutic effect as 131I-TFM. The toxicity in this therapy experiment was minimal and further experiments at higher doses are required to observe if there would be any advantage of a higher initial dose rate for 131I-DFM. © 1999 Cancer Research Campaig

A transcriptional reference map of defence hormone responses in potato

Phytohormones are involved in diverse aspects of plant life including the regulation of plant growth, development and reproduction, as well as governing biotic and abiotic stress responses. We have generated a comprehensive transcriptional reference map of the early potato responses to exogenous application of the defence hormones abscisic acid, brassinolides (applied as epibrassinolide), ethylene (applied as the ethylene precursor aminocyclopropanecarboxylic acid), salicylic acid and jasmonic acid (applied as methyl jasmonate). Of the 39000 predicted genes on the microarray, a total of 2677 and 2473 genes were significantly differentially expressed at 1 h and 6 h after hormone treatment, respectively. Specific marker genes newly identified for the early hormone responses in potato include: a homeodomain 20 transcription factor (DMG400000248) for abscisic acid; a SAUR gene (DMG400016561) induced in epibrassinolide treated plants; an osmotin gene (DMG400003057) specifically enhanced by aminocyclopropanecarboxylic acid; a gene weakly similar to AtWRKY40 (DMG402007388) that was induced by salicylic acid; and a jasmonate ZIM-domain protein 1 (DMG400002930) which was specifically activated by methyl jasmonate. An online database has been set up to query the expression patterns of potato genes represented on the microarray that can also incorporate future microarray or RNAseq-based expression studies

Expression and regulation of type 2A protein phosphatases and alpha4 signalling in cardiac health and hypertrophy

Abstract Cardiac physiology and hypertrophy are regulated by the phosphorylation status of many proteins, which is partly controlled by a poorly defined type 2A protein phosphatase-alpha4 intracellular signalling axis. Quantitative PCR analysis revealed that mRNA levels of the type 2A catalytic subunits were differentially expressed in H9c2 cardiomyocytes (PP2ACb[PP2ACa[PP4C[PP6C), NRVM (PP2ACb[PP2ACa = PP4C = PP6C), and adult rat ventricular myocytes (PP2ACa[ PP2ACb[PP6C[PP4C). Western analysis confirmed that all type 2A catalytic subunits were expressed in H9c2 cardiomyocytes; however, PP4C protein was absent in adult myocytes and only detectable following 26S proteasome inhibition. Short-term knockdown of alpha4 protein expression attenuated expression of all type 2A catalytic subunits. Pressure overload-induced left ventricular (LV) hypertrophy was associated with an increase in both PP2AC and alpha4 protein expression. Although PP6C expression was unchanged, expression of PP6C regulatory subunits (1) Sit4-associated protein 1 (SAP1) and (2) ankyrin repeat domain (ANKRD) 28 and 44 proteins was elevated, whereas SAP2 expression was reduced in hypertrophied LV tissue. Co-immunoprecipitation studies demonstrated that the interaction between alpha4 and PP2AC or PP6C subunits was either unchanged or reduced in hypertrophied LV tissue, respectively. Phosphorylation status of phospholemman (Ser63 and Ser68) was significantly increased by knockdown of PP2ACa, PP2ACb, or PP4C protein expression. DNA damage assessed by histone H2A.X phosphorylation (cH2A.X) in hypertrophied tissue remained unchanged. However, exposure of cardiomyocytes to H2O2 increased levels of cH2A.X which was unaffected by knockdown of PP6C expression, but was abolished by the short-term knockdown of alpha4 expression. This study illustrates the significance and altered activity of the type 2A protein phosphatase-alpha4 complex in healthy and hypertrophied myocardium

Movement of bymoviruses and functions of RNA2-encoded proteins of barley yellow mosaic virus

Observations and experimental data that were obtained from plants infected with bymoviruses such as barley yellow mosaic virus (BaYMV), barley mild mosaic virus (BaMMV), or wheat spindle streak mosaic virus (WSSMV) suggested that long-distance transport follows the phloem pathway. Inoculation of barley roots with BaMMV via viruliferous zoospores of Polymyxa graminis and mechanical inoculation of leaves revealed that viral transport from shoots to roots occurs in less than 5 d while movement from roots to shoots could only be detected after 5-6 weeks. A model for long-distance transport of bymoviruses was developed which shows that the bymovirus infection cycle passes through 4 main stages. During these stages an up- and downward movement may be important for sufficient replication and successful transmission of bymoviruses. The 28 kDa protein of BaYMV (RNA2) may have a function in either movement or replication as it was often found associated with virus particles. The other RNA2-encoded protein of BaYMV, a 70 kDa protein, could be involved in fungal transmission.Étude de la migration des bymovirus et fonctions possibles des protéines de transport codées par l'ARN2 du virus de la mosaïque jaune de l'orge. Les diverses observations réalisées et les données expérimentales obtenues à partir de plantes infectées par les bymoviruses, tel que le virus de la mosaïque jaune de l'orge (BaYMV), le virus de la mosaïque modérée de l'orge (BaMMV) ou le virus de la mosaïque striée en fuseaux du blé (WSSMV) suggèrent que leur transport à longue distance utilise les vaisseaux du phloème. L'inoculation des racines d'orge par le BaMMV, avec des zoospores de Polymyxa graminis contaminés par le virus, et l'inoculation mécanique de feuilles révèlent que le transport du virus des feuilles aux racines s'effectue en moins de 5 j, tandis que le mouvement inverse est détecté 5 à 6 sem après l'infection. Nous avons développé un modèle d'étude du transport à longue distance du bymovirus montrant que le cycle infectieux comprend 4 étapes principales. Le transport ascendant ou descendant des bymovirus pourrait jouer un rôle important pour leur réplication et leur transmission. La protéine de 28 kDa du BaYMV (ARN2) pourrait intervenir dans le transport ou la réplication du virus, car elle est souvent trouvée associée aux particules virales. L'ARN2 codant pour une seconde protéine de BaYMV de 70 kDa pourrait participer à la capacité vectrice du champignon virulifère

Studies of UK isolates of barley yellow mosaic virus

International audienc

Movement of Barley Mild Mosaic and Barley Yellow Mosaic-Viruses in Leaves and Roots of Barley

Leaves of barley plants were mechanically inoculated with barley mild mosaic virus (BaMMV) and roots were inoculated using viruliferous zoospores of the fungus vector Polymyxa graminis. At intervals after inoculation, leaves and roots were tested by different methods to detect virus coat protein (ELISA or Western blot) or nucleic acid (slot-blot or reverse transcriptase-PCR). Following inoculation with zoospores, virus could be detected in roots after 1 wk (Western blot or PCR) but not until 3-4 wk by ELISA. Virus moved to leaves in 5-6 wk but, except at temperatures of about 20 degrees C, plants had to be cut back close to soil level to stimulate virus movement. Following mechanical inoculation, virus could be detected in leaves of a susceptible cultivar within 5 days by ELISA and 3 days by the other methods. Western blots and PCR showed that virus was present in the roots by 5 days. BaMMV was not detected by any method in leaves or roots of a resistant cultivar, indicating that the virus did not multiply in it. When leaves were mechanically inoculated on a small area only, BaMMV capsid protein was detected below the inoculated site at 4 days and in young growing leaves and roots at 13 days after inoculation but never above the inoculation site or in older leaves. After stem extension began, new leaves of infected plants were free of symptoms. The results are compared to observations of plants infected with barley yellow mosaic virus (BaYMV). It is proposed that movement of BaMMV and BaYMV is strongly related to the phloem transport and to the source-sink pattern of winter barley plants