Studies of proteins in heme and iron metabolism

One aim of the study was to investigate the regulation of the murine erythroid isoform of aminolevulinate synthase (eALAS) protein involved in heme synthesis. Another aim was to characterize the structure of the rat hemochromatosis gene (HFE) gene and its expression in conjunction with other genes involved in the regulation of iron homeostasis during inflammation and iron overload in the rat liver. Most of the iron in the body is consumed via heme synthesis starting with eALAS. We found a new protease activity in bone marrow cells, which proteolytically cleaves an 8-kDa segment of the eALAS protein from its N-terminus. More differentiated erythroid cells isolated from peripheral blood exhibit very little of this protein truncation. We also demonstrated that the newly discovered short form of eALAS is located in the mitochondrial membrane of bone marrow erythroid cells. Most common protease inhibitors did not affect this proteolytic activity. The specific function of eALAS short protein is not clear and needs additional studies. To further investigate eALAS regulation we analyzed the effect of heme on eALAS synthesis in natural erythroid cells. Incubation with heme (5-100 ìM) clearly inhibits eALAS synthesis in bone marrow cells. This inhibitory effect of heme could also be observed in peripheral blood cells at higher concentrations. However, low heme concentrations (1-10 ìM) increased eALAS synthesis. Addition of heme also increased the levels of the cytosolic preform of eALAS in blood cells. This suggests that eALAS synthesis is controlled via a composite of regulatory mechanisms including the iron regulatory proteins (IRPs). Regulation of eALAS in bone marrow cells also seems more tightly controlled in order to prevent oxidative cell damage. While eALAS plays a major role in iron consumption via the heme synthesis pathway there are several other proteins and genes responsible for the regulation of iron homeostasis in the body. HFE has been shown to be involved in iron metabolism but little is known about its regulation. Cloning of the rat HFE gene using library screening and PCR showed that the rat HFE gene contains six exons and five introns. Determination of HFE mRNA levels by Northern blot (tissues) and real-time PCR (isolated liver cells) showed that the HFE gene is expressed in multiple tissues in the rat, including bone marrow, with the highest expression in the liver. In contrast to earlier beliefs our experiments on isolated rat liver cells demonstrated the highest expression of the HFE gene in rat hepatocytes. High levels were also found in Kupffer cells. To better understand the process of hemochromatosis, we investigated the expression of HFE and other genes involved in iron metabolism (TfR1, TfR2, DMT1, IREG1, HAMP), in rat liver during inflammation and iron overload. Expression of HAMP in whole liver was higher in iron-loaded than in normal animals. mRNA expression levels of HAMP in cultured hepatocytes from normal rats were also higher than in freshly isolated hepatocytes. Levels were however clearly lower in cultured hepatocytes from iron-loaded rats, arguing that its expression depends on surrounding cells or factors not present in tissue culture. TfR1 had a different regulation than TfR2 during inflammation and iron overload in hepatocytes. The expression of the other studied genes in animals with both normal and iron diets followed the idea that inflammation and iron loading causes iron to be bound intracellularly

A Population-based Investigation of the Autoantibody Profile in Mothers of Children with Atrioventricular Block

The objective of the study was to investigate the antigen specificity and occurrence of individual autoantibodies in mothers of children diagnosed with atrioventricular (AV) block in a nation-wide setting. Patients with AV block detected before 15 years of age were identified using national quality registries as well as a network of pediatric and adult cardiologists and rheumatologists at the six university hospitals in Sweden. Patients with gross heart malformations, surgically or infectiously induced blocks were excluded. Blood samples were obtained from the mothers and maternal autoantibody profile, including the occurrence of antibodies against Ro52, Ro60, La, SmB, SmD, RNP-70k, RNP-A, RNP-C, CENP-C, Scl-70, Jo-1, ribosomal RNP and histones was investigated in 193 mothers of children with AV block by immunoblotting and ELISA. Autoantibody reactivity was detected in 48% (93/193) of the mothers of children with AV block. In autoantibody-positive mothers, the vast majority, 95% (88/93), had antibodies against Ro52, while 63% (59/93) had autoantibodies to Ro60 and 58% (54/93) had autoantibodies to La. In addition, 13% (12/93) of the autoantibody-positive mothers had antibodies to other investigated antigens besides Ro52, Ro60 and La, and of these anti-histone antibodies were most commonly represented, detected in 8% (7/93) of the mothers. In conclusion, this Swedish population-based study confirms that maternal autoantibodies may associate with heart block in the child. Further, our data demonstrate a dominant role of Ro52 antibodies in association with AV block.Funding Agencies|KIRCNET (Karolinska Institutet Circulation and Respiratory Research Network)||Magn Bergvalls Foundation||Jerringfoundation||Stiftelsen Samariten||Karolinska Institute||Royal Swedish Academy of Sciences||Swedish Research Council||Goran Gustafsson Foundation||Torsten and Ragnar Soderberg Foundation||King Gustaf the V:th 80-year foundation||Swedish Foundation for Strategic Research||Heart-Lung Foundation||Swedish Rheumatism Association|

Development of heart block in children of SSA/SSB-autoantibody-positive women is associated with maternal age and displays a season-of-birth pattern

Objective Congenital heart block may develop in the fetuses of Ro/SSA-positive and La/SSB-positive mothers. Recurrence rates of only 10-20% despite persisting maternal antibodies indicate that additional factors are critical for the establishment of heart block. The authors investigated the influence of other maternal and fetal factors on heart block development in a Swedish population-based cohort. less thanbrgreater than less thanbrgreater thanMethods The influence of fetal gender, maternal age, parity and time of birth on heart block development was analysed in 145 families, including Ro/La-positive (n=190) and Ro/La-negative (n=165) pregnancies. less thanbrgreater than less thanbrgreater thanResults There was a recurrence rate of 12.1% in Ro/La-positive women, and no recurrence in Ro/La-negative women. Fetal gender and parity did not influence the development of heart block in either group. Maternal age in Ro/La-positive pregnancies with a child affected by heart block was, however, significantly higher than in pregnancies resulting in babies without heart block (pandlt;0.05). Seasonal timing of pregnancy influenced the outcome. Gestational susceptibility weeks 18-24 occurring during January-March correlated with a higher proportion of children with heart block and lower vitamin D levels during the same period in a representative sample of Swedish women and a corresponding higher proportion of children with heart block born in the summer (pandlt;0.02). Maternal age or seasonal timing of pregnancy did not affect the outcome in Ro/La-negative pregnancies. less thanbrgreater than less thanbrgreater thanConclusion This study identifies maternal age and seasonal timing of pregnancy as novel risk factors for heart block development in children of Ro/La-positive women. These observations may be useful for counselling when pregnancy is considered.Funding Agencies|KIRCNET (Karolinska Institutet Circulation and Respiratory Research Network)||Magn. Bergvalls Foundation||Jerring Foundation||Stiftelsen Samariten||Karolinska Institute||Royal Swedish Academy of Sciences||Swedish Research Council||Goran Gustafsson Foundation||Torsten and Ragnar Soderberg Foundation||King Gustaf Vth 80-Year Foundation||Swedish Foundation for Strategic Research||Heart-Lung Foundation||Swedish Rheumatism Association|

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Development of heart block in children of SSA/SSB-autoantibody-positive women is associated with maternal age and displays a season-of-birth patter