Transport of soluble proteins through the Golgi occurs by diffusion via continuities across cisternae.

The mechanism of transport through the Golgi complex is not completely understood, insofar as no single transport mechanism appears to account for all of the observations. Here, we compare the transport of soluble secretory proteins (albumin and α1-antitrypsin) with that of supramolecular cargoes (e.g., procollagen) that are proposed to traverse the Golgi by compartment progression-maturation. We show that these soluble proteins traverse the Golgi much faster than procollagen while moving through the same stack. Moreover, we present kinetic and morphological observations that indicate that albumin transport occurs by diffusion via intercisternal continuities. These data provide evidence for a transport mechanism that applies to a major class of secretory proteins and indicate the co-existence of multiple intra-Golgi trafficking modes.

Physiological implications of NTBI uptake by T lymphocytes.

In iron overload disorders a significant fraction of the total iron circulates in the plasma as low molecular weight complexes not bound to transferrin, known as non-transferrin-bound iron (NTBI). By catalyzing the formation of free radicals, NTBI accumulation results in oxidative stress and cellular damage, being a major cause of organ toxicity. NTBI is rapidly and preferentially cleared from circulation by the liver and the myocardium, the main disease targets in iron overload conditions. We have recently demonstrated that human peripheral blood T lymphocytes take up NTBI in vitro, with a pattern that resembles that of hepatocytes. Since T lymphocytes constitute a numerically important component of the circulating cell pool, these findings support a putative role for this cell type in the systemic protection against iron toxicity. Here we tested the hypothesis that the circulating peripheral blood T lymphocyte pool constitutes an important storage compartment for NTBI and is thus a modifier of NTBI deposition in target organs. First we show that NTBI uptake by human T lymphocytes increases the expression of the iron-storage protein ferritin and of the iron exporter ferroportin via an IRE-dependent mechanism. NTBI retention by T lymphocytes is shown to be critically controlled by the hepcidin-mediated modulation of ferroportin both in vitro and in vivo. Finally, the protective effect of T lymphocytes was tested by analyzing the patterns of iron accumulation in the T lymphocyte-deficient mouse model Foxn1(nu) before and after reconstitution with T lymphocytes by adoptive transfer. The results confirmed a significant increase of liver and pancreas iron accumulation in T lymphocyte-deficient mice. NTBI accumulation in the liver and spleen was prevented by reconstitution with syngeneic T lymphocytes. Altogether, our results demonstrate that T lymphocytes are important components of a circulating "NTBI storage compartment" and show its physiological relevance as a modifier of tissue iron overload.

Non-transferrin-bound iron (NTBI) uptake by T lymphocytes: evidence for the selective acquisition of oligomeric ferric citrate species.

Iron is an essential nutrient in several biological processes such as oxygen transport, DNA replication and erythropoiesis. Plasma iron normally circulates bound to transferrin. In iron overload disorders, however, iron concentrations exceed transferrin binding capacity and iron appears complexed with low molecular weight molecules, known as non-transferrin-bound iron (NTBI). NTBI is responsible for the toxicity associated with iron-overload pathologies but the mechanisms leading to NTBI uptake are not fully understood. Here we show for the first time that T lymphocytes are able to take up and accumulate NTBI in a manner that resembles that of hepatocytes. Moreover, we show that both hepatocytes and T lymphocytes take up the oligomeric Fe3Cit3 preferentially to other iron-citrate species, suggesting the existence of a selective NTBI carrier. These results provide a tool for the identification of the still elusive ferric-citrate cellular carrier and may also open a new pathway towards the design of more efficient iron chelators for the treatment of iron overload disorders.

Pattern shape control for heat treatment purification of electron-beam-induced deposition of gold from the Me2Au(acac) precursor.

Gold structures can be created in a scanning electron microscope (SEM) from the Me(2)Au(acac) precursor by direct writing with the electron beam. The as-deposited purity is usually poor, and a common purification approach is a post-annealing step that indeed is effective but also induces a volume reduction because of carbon loss and an undesirable reconfiguration of the gold structure, resulting in the loss of the original shape. We studied the shape change as a result of such purification, and to minimize this effect, the application of a tantalum and chromium buffer layer was investigated. These buffer materials are well-known for their good adhesion properties. We confirm by dedicated SEM, atomic force microscopy (AFM), and transmission electron microscopy (TEM) analysis that, for the creation of a uniform Au structure, tantalum is a better buffer layer material than chromium. Post-annealing of the Au electron-beam-induced deposition (EBID) patterns for 1 h at 600 °C in air resulted in a dramatic purity increase (from 8-12 atomic % Au to above 92 atomic % Au). The uncovered part of the tantalum layer can be easily etched away, resulting in a well-defined, high-purity, gold structure.