Long-term iron depletion for CHC patients is a promising modality for lowering the risk of progression to HCC.

Several clinical studies have suggested that excess hepatic iron accumulation is a progressive factor in some liver diseases including chronic viral hepatitis and hemochromatosis. However, it is not known whether iron-induced hepatotoxicity may be directly involved in hepatitis, cirrhosis, and liver cancer. The Long-Evans Cinnamon (LEC) rat, which accumulates excess copper in the liver as in patients with Wilson's disease, is of a mutant strain displaying spontaneous hemolysis, hepatitis, and liver cancer. We found previously that LEC rats harbored an additional abnormality: accumulation of as much iron as copper in the liver. In the present study, we compared the occurrence of hepatitis and liver cancer in LEC rats fed an iron-deficient diet (ID) with those in rats fed a regular diet (RD). The RD group showed rapid increments of hepatic iron concentrations as the result of hemolysis, characteristics of fulminant hepatitis showing apoptosis, and a 53% mortality rate. However, no rats in the ID group died of fulminant hepatitis. Hepatic iron, especially "free" iron concentration and the extent of hepatic fibrosis in the ID group were far less than those of the RD group. At week 65, all rats in the RD group developed liver cancer, whereas none did in the ID group. These results suggest that the accumulation of iron, possibly by virtue of synergistic radical formation with copper, plays an essential role in the development of fulminant hepatitis, hepatic fibrosis, and subsequent hepatocarcinogenesis in LEC rats.

Calmodulin, a wide-spread eukaryotic Ca2+-binding protein, was phosphorylated at its tyrosine residues in Rous sarcoma virus (RSV)-transformed chicken and rat cells but not in normal chicken embryo fibroblasts. In contrast, serine and threonine phosphorylation of calmodulin was found to occur in both normal and virus-transformed cells. In an in vitro system containing purified src kinase from RSV-transformed cells, tyrosine phosphorylation of calmodulin by the src kinase was inhibited by Ca2+. Furthermore, the tyrosinephosphorylated calmodulin showed slower mobility than that of nonphosphorylated calmodulin in NaDodSO4/polyacrylamide gel electrophoresis when Ca2+ was present. These results suggest that the structure of calmodulin Ca2+ complex may be altered by tyrosine phosphorylation. It is thus inferred that Ca2+ may regulate the level of tyrosine phosphorylation of calmodulin in RSV-transformed cells, and phosphorylation in turn may attenuate the function of this protein in vivo.The product of the transforming src gene of Rous sarcoma virus (RSV) has tyrosine-specific protein kinase activity (1-4). Although it is not clear whether this kinase activity is responsible for the cellular transformation by RSV, phosphorylation of tyrosine residues in membrane-and/or cytoskeleton-associated proteins such as vinculin (5) and a 36-kDa protein (6, 7) has been found to occur upon transformation of RSV-infected cells. During the course of purification of the src kinase from RSV-transformed cells and subsequent analysis of proteins phosphorylated by the purified enzyme (8), it was found that calmodulin, a widespread eukaryotic Ca2l-binding protein (9), was phosphorylated at tyrosine residues by the purified src kinase. Since calmodulin is one of the cytoskeleton-associated proteins (10-12), we were interested in investigating further the tyrosine phosphorylation of calmodulin in normal and RSV-transformed cells. For these studies we took various precautions, because it is well known that the src kinase easily phosphorylates a large number of cellular proteins in vitro (13-18) and that the in vitro phosphorylation does not necessarily guarantee that the substrate used is indeed a physiological target of the src kinase in vivo. We believe that our experiments are valid, because (i) a rapid isolation procedure (19) and a specific identification method (20) for calmodulin have been established; (ii) calmodulin can easily be recovered after trichloroacetic acid treatment (21), which effectively suppresses phosphatase(s) in crude extracts; and (iii) calmodulin is a well-characterized protein (9), and the effect of its phosphorylation may be detected by using one of the sensitive analysis methods. We show below that calmodulin is phosphorylated at tyrosine residues in RSV-transformed chicken and rat cells and that calmodulin phosphorylation is inhibited by Ca2+ in an in vitro system with purified src kinase. A structural alteration of calmodulin upon tyrosine phosphorylation is also demonstrated.MATERIALS AND METHODS Mat...

Desmin and vimentin are intermediate filaments that play crucial roles in the maturation, maintenance and recovery of muscle fibers and mesenchymal cells. The expression of these proteins has not been investigated extensively in human fetuses. In the present study, we examined the immunohistochemical expression of intermediate filaments in skeletal muscles of the head, neck and thorax in 12 mid-term human fetuses at 9-18 weeks of gestation. We also used immunohistochemistry to localize the expression of the myosin heavy chain and silver impregnation to identify the fetal endomysium. Expression of desmin and vimentin was already detectable in intercostal muscle at 9 weeks, especially at sites of muscle attachment to the perichondrium. At this stage, myosin heavy chain was expressed throughout the muscle fibers and the endomysium had already developed. Beginning with punctate expression, the positive areas became diffusely distributed in the muscle fibers. At 15-18 weeks, intermediate filament proteins were extensively expressed in all of the muscles examined. Expression at the bone-muscle interface was continuous with expression along the intramuscular tendon fibres. These results suggest that the development of intermediate filaments begins in areas of mechanical stress due to early muscle contraction. Their initially punctate distribution, as observed here, probably corresponds to the earliest stage of fetal enthesis formation.

We demonstrated fetal peripheral lymphatic vessels (LVs) using D2-40 immunohistochemistry in a whole female fetus (18 weeks of gestation, CRL 155 mm) except for the head. There were abundant LVs in the thyroid gland, lung, stomach, small intestine, rectum and pancreas, whereas no LVs were seen in the parathyroid gland, spleen and adrenal cortex. In the liver, except for the gallbladder bed, LVs were still restricted to around hilar thick portal veins and around the hepatic vein terminals. Subcutaneous LVs were well developed throughout the body even in areas where no or few perforating LVs connected with the deep LVs. The diaphragm contained abundant, dilated LVs in the pleural half of its thickness. LVs were also seen not only along supplying arteries of muscles and cartilage but also along the epimysium and perichondrium. LVs ran in a space between the obliquus internus and transversus abdominis but not between the obliquus internus and obliquus externus. Some tight connective tissues such as the sacrotuberous ligament contained abundant LVs. The intervertebral foramen contained a lymphatic plexus. The present observations provide a better understanding of peripheral lymphatic development. The fetal lymphatic morphology seems not to represent a mini-version of the adult morphology.