Juan J. Bustamante

Hypoxia‐inducible factor‐dependent production of profibrotic mediators by hypoxic hepatocytes

Background/Aims: During the development of liver fibrosis, mediators are produced that stimulate cells in the liver to differentiate into myofibroblasts and to produce collagen. Recent studies demonstrated that the transcription factor, hypoxia‐inducible factor‐1α (HIF‐1α), is critical for upregulation of profibrotic mediators, such as platelet‐derived growth factor‐A (PDGF‐A), PDGF‐B and plasminogen activator inhibitor‐1 (PAI‐1) in the liver, during the development of fibrosis. What remains unknown is the cell type‐specific regulation of these genes by HIF‐1α in liver cell types. Accordingly, the hypothesis was tested that HIF‐1α is activated in hypoxic hepatocytes and regulates the production of profibrotic mediators by these cells.

Extraordinarily stable disulfide-linked homodimer of human growth hormone

Although a 22‐kDa human growth hormone (hGH) is the predicted protein product of the hGH‐N gene, a pleiotropic collection of uncharacterized molecular weight and charge isoforms is also produced. Using chromatography and preparative SDS‐PAGE under reducing conditions we isolated an unusually stable mercaptoethanol‐resistant (MER) 45‐kDa hGH. A 5‐h incubation at 100°C in the presence of 2‐mercaptoethanol was required to convert approximately 90% of MER‐45‐kDa hGH into a 22‐kDa hGH. Other reductants were not as effective in splitting MER‐45‐kDa hGH. After fracturing MER‐45‐kDa hGH, the 22‐kDa hGH fragments would spontaneously reassociate if the reductant was removed; however, alkylation of cysteine residues prevented their reassociation. Identical amino acid sequences for the first six N‐terminal residues were obtained for MER‐45‐kDa hGH and its 22‐kDa hGH cleavage product. Structural identity of MER‐45‐kDa hGH and 22‐kDa hGH was demonstrated by MALDI‐TOF mass spectrometry of tryptic digests. MER‐45‐kDa hGH did not break up upon incubation with EDTA and EGTA. The significance of this work to our understanding of the structure of hGH isoforms is that it demonstrates that MER‐45‐kDa hGH is not a single chain polypeptide but is instead a homodimer of 22‐kDa hGH monomers. The MER‐45‐kDa hGH dimer is held together by interchain disulfide bonds and not by divalent metal cation bridges. Additionally, MER‐45‐kDa hGHs interchain disulfide links are exceptionally resistant to reducing agents and thus confer extreme stability to the homodimer.

Gene profiling of maternal hepatic adaptations to pregnancy

Background: Maternal metabolic demands change dramatically during the course of gestation and must be co‐ordinated with the needs of the developing placenta and fetus. The liver is critically involved in metabolism and other important functions. However, maternal hepatic adjustments to pregnancy are poorly understood.

Pregnancy and lactation modulate maternal splenic growth and development of the erythroid lineage in the rat and mouse.

Maternal physiology changes dramatically during the course of gestation and lactation to meet the needs of the developing fetus and newborn. In the present study, we examined the influence of pregnancy and lactation on growth and erythroid gene expression patterns of the maternal spleen. Holtzman Sprague-Dawley rats and CD-1 mice were killed at various stages of gestation and post partum. We observed pregnancy dependent increases in spleen weight and spleen DNA content in both the rat and mouse. In the rat, spleen size was greatest at the end of pregnancy and regressed post partum. In contrast, mouse spleen size peaked by gestational Day 13 and regressed to its non-pregnant weight before parturition. Pregnancy dependent changes in the size of the spleen were primarily due to an increase in red pulp. Maternal spleen expression of erythroid-associated genes (erythroid Krüppel-like factor, erythroid 5-aminolevulinate synthase-2, beta-major globin) was influenced by pregnancy and lactation. A pregnancy dependent increase in erythroid progenitors was also observed. In summary, the demands of pregnancy and lactation cause marked adaptations in the maternal spleen. The maternal spleen increases in size and exhibits an expansion of the erythroid lineage.