Faculty Bibliography

Ceruloplasmin, a blue copper oxidase circulating in serum of all vertebrates, is a glycoprotein synthesized mainly in hepatocytes and secreted into plasma with six tightly bound atoms of copper per molecule. Many aspects of the mechanisms by which synthesis and secretion of this protein are regulated by copper are still not known. In HepG2 hepatocarcinoma cells this fine regulation is not maintained; we have then utilized Met-murine-hepatocytes (MMH), isolated from the liver of transgenic mice expressing a truncated form of c-Met (hepatocyte growth factor receptor), that are immortalized but not transformed. Copper deficiency was induced by treatment of cells with bathocuproine disulphonate. Experiments of metabolic labeling with 35S-methionine-cysteine and of Western blotting followed by immunostaining, demonstrated that maturation and secretion of ceruloplasmin but not its synthesis are affected by copper availability. In this paper we have shown that in copper deficiency ceruloplasmin accumulates in a pre-Golgi compartment, in which the protein is still in a Endo H sensitive form, and where presumably copper binding to the apo-protein takes place. Moreover, we found that treatment of copper-deficient cells with the proteasomal inhibitor lactacycstin leads to immature ceruloplasmin accumulation in the cell. We have optimized conditions to induce in vitro copper deficiency and found that MMH-D3 cells represent a suitable model to study in detail the molecular mechanism of copper-regulated ceruloplasmin synthesis, secretion and degradation.

A 4-year-old girl presented with a 3-year history of demarcated, salmon-pink, hyperkeratotic plaques, which were symmetrically distributed on the elbows, knees, ankles, and dorsal aspects of the hands and feet. A diffuse, orange-pink palmoplantar keratoderma was also evident. Clinical and histologic findings were consistent with a diagnosis of pityriasis rubra pilaris (PRP), type IV (circumscribed juvenile). Type IV PRP develops in prepubertal children, is typically localized to the distal aspects of the extremities, and has an unpredictable course. Although ultraviolet (UV) radiation can potentially exacerbate PRP, our patient has improved with broad-band UVB phototherapy

A 13-year-old boy presented with a lifelong history of tightly-adherent, brown, polygonal scales that covered the extensor surfaces of the extremities, lateral aspects of the trunk, and neck. The clinical presentation and the history of a similar skin condition in the patient's male maternal relatives helped establish the diagnosis of X-linked recessive ichthyosis (XLI). Systemic manifestations of the steroid sulfatase (STS) deficiency underlying XLI include cryptorchidism, asymptomatic corneal opacities, and maternal failure to progress during labor. Most cases of XLI are caused by deletions of the STS gene, and contiguous gene syndromes may occur when the deletions extend to neighboring genes on the distal short arm of the X chromosome

A 3-year-old girl presented with a 6-month history of multiple, light-pink, flat-topped papules over the dorsal aspects of the metacarpophalangeal and interphalangeal joints of the hands and feet. Nailfold telangiectases, ragged cuticles, and a heliotrope color of the upper eyelids were also evident, but there was no clinical evidence of muscle weakness and levels of muscle enzymes were normal. A biopsy specimen from one of the papules showed a vacuolar interface dermatitis consistent with a diagnosis of dermatomyositis. This report draws attention to juvenile amyopathic dermatomyositis, which is an uncommon subtype of dermatomyositis with an excellent prognosis

Gene products provided by the mother to the embryo determine the body axes in most animals. A recent study in zebrafish proposes that the TGFss signal Squint is one such factor.

The biochemistry of early stages of hematopoietic differentiation is difficult to study because only relatively small numbers of precursor cells are available. The murine EML cell line is a multipotential cell line that can be used to model some of these steps. We found that the lineage- EML precursor cells can be separated into two populations based on cell surface markers including CD34. Both populations contain similar levels of stem cell factor (SCF) receptor (c-Kit) but only the CD34+ population shows a growth response when treated with SCF. Conversely, the CD34- population will grow in the presence of the cytokine IL-3. The human beta-globin locus control region hypersensitive site 2 plays different roles on beta-globin transcription in the CD34+ and CD34- populations. The two populations are present in about equal amounts in culture, and the CD34+ population rapidly regenerates the mixed population when grown in the presence of SCF. We suggest that this system may mimic a normal developmental transition in hematopoiesis.

Nodes of Ranvier are regularly placed, nonmyelinated axon segments along myelinated nerves. Here we show that nodal membranes isolated from the central nervous system (CNS) of mammals restricted neurite outgrowth of cultured neurons. Proteomic analysis of these membranes revealed several inhibitors of neurite outgrowth, including the oligodendrocyte myelin glycoprotein (OMgp). In rat spinal cord, OMgp was not localized to compact myelin, as previously thought, but to oligodendroglia-like cells, whose processes converge to form a ring that completely encircles the nodes. In OMgp-null mice, CNS nodes were abnormally wide and collateral sprouting was observed. Nodal ensheathment in the CNS may stabilize the node and prevent axonal sprouting.

Overcoming the androgen independence of prostate tumors is considered the most critical therapeutic end point for improving survival in patients with metastatic prostate cancer. Normal epithelial and endothelial cells can undergo apoptosis when detached from the extracellular matrix (ECM), via the anoikis phenomenon. In contrast, tumor cells upon detachment from the ECM are capable of evading anoikis and metastasizing to different distant organs. Is the biological repertoire of the epithelial and endothelial cells sufficient to account for the events associated with the process of anoikis during prostate cancer metastasis? Although there is no clear answer to this question, what has become increasingly evident from the existing evidence is that molecules that induce anoikis in tumor epithelial and endothelial cells provide exciting new leads into effective therapeutic targeting as well as markers of prostate cancer progression and prediction of therapeutic resistance. This review analyzes recent findings on anoikis regulators and discusses the relevance of this unique apoptosis mode in the development of metastatic prostate cancer and identification of molecular signatures for treatment of advanced disease.

Until recently, studies in this field of signal transduction have involved the 'what' and 'when' of signaling. Who talks to whom and for how long? With the advent of genetically encoded fluorescent proteins, it has become possible to monitor signaling events in living cells in real time. This has added the dimension of 'where' to the study of cellular signaling. This lecture, which is a part of 'Cell Signaling Systems: A Course for Graduate Students,' provides a survey of how green fluorescent protein (GFP)-tagged probes for signaling events have been used to elucidate new pathways, to describe the kinetics of signaling events at the single-cell level, and to reveal upon which subcellular compartments these events take place. Some of the findings confirm previous ones using biochemical techniques, and others have been surprising. Examples include those utilizing protein localization, relocalization, fluorescence recovery after photobleaching (FRAP), and fluorescence resonance energy transfer (FRET). The design of FRET probes is described. The detection of small guanosine triphosphatase (GTPase) signaling in living cells is used as an example to explore the creative and diverse ways investigators have developed to look at this system

During tRNA translocation on the ribosome, an arc-like connection (ALC) is formed between the G' domain of elongation factor G (EF-G) and the L7/L12-stalk base of the large ribosomal subunit in the GDP state. To delineate the boundary of EF-G within the ALC, we tagged an amino acid residue near the tip of the G' domain of EF-G with undecagold, which was then visualized with three-dimensional cryo-electron microscopy (cryo-EM). Two distinct positions for the undecagold, observed in the GTP-state and GDP-state cryo-EM maps of the ribosome bound EF-G, allowed us to determine the movement of the labeled amino acid. Molecular analyses of the cryo-EM maps show: (1) that three structural components, the N-terminal domain of ribosomal protein L11, the C-terminal domain of ribosomal protein L7/L12, and the G' domain of EF-G, participate in formation of the ALC; and (2) that both EF-G and the ribosomal protein L7/L12 undergo large conformational changes to form the ALC