Membrane type-1 matrix metalloproteinases and tissue inhibitor of metalloproteinases-2 RNA levels mimic each other during Xenopus laevis metamorphosis

Matrix metalloproteinases (MMPs) and their endogenous inhibitors TIMPs (tissue inhibitors of MMPs), are two protein families that work together to remodel the extracellular matrix (ECM). TIMPs serve not only to inhibit MMP activity, but also aid in the activation of MMPs that are secreted as inactive zymogens. Xenopus laevis metamorphosis is an ideal model for studying MMP and TIMP expression levels because all tissues are remodeled under the control of one molecule, thyroid hormone. Here, using RT-PCR analysis, we examine the metamorphic RNA levels of two membrane-type MMPs (MT1-MMP, MT3-MMP), two TIMPs (TIMP-2, TIMP-3) and a potent gelatinase (Gel-A) that can be activated by the combinatory activity of a MT-MMP and a TIMP. In the metamorphic tail and intestine the RNA levels of TIMP-2 and MT1-MMP mirror each other, and closely resemble that of Gel-A as all three are elevated during periods of cell death and proliferation. Conversely, MT3-MMP and TIMP-3 do not have similar RNA level patterns nor do they mimic the RNA levels of the other genes examined. Intriguingly, TIMP-3, which has been shown to have anti-apoptotic activity, is found at low levels in tissues during periods of apoptosis.     

Dynamic energy budget models with size-dependent hazard rates

We formulate and analyze two dynamic energy budget models, a net assimilation model with constant allocation strategy and a net production model with a 2-stage allocation strategy, with the objective of determining strategies that maximize the expected lifetime reproductive energy. The per capita death rate depends on the organisms size, as for example when the main cause of death is predation. In the analysis of the net production model, the size at maturity is calculated along with the probability of reaching that size. We show that a small probability of survival to maturity is incompatible with the simple assumption of an exponential survival probability. We demonstrate that when the hazard rate is significantly greater for small individuals than large ones, it is possible for the optimum strategy to be for an individual to grow to a large size in spite of an arbitrarily small probability of survival to maturity. Numerical simulations indicate how the optimal allocation strategies depend on the parameter values.     

Lipid rafts in cytokine signaling

Lipid rafts are established as critical structures for a variety of cellular processes, including immune cell activation. Beyond their importance for initial immune cell activation at the immunological synapse, lipid rafts are now also being recognized as important sites for cytokine and growth factor signal transduction, both in immune cells as part of secondary regulatory processes, and in non-immune cells. This review summarizes current knowledge regarding the roles of rafts in cytokine signaling and emphasizes the need for measures to better standardize the study of rafts.     

The in vitro and in vivo effects of anti-galactose antibodies on endothelial cell activation and xenograft rejection

We have previously produced a series of antigalactose (anti-Gal) hybridomas and characterized their heavy chain gene usage. Here we have quantified the affinity of these Abs for the alpha-Gal epitope and characterized their in vitro effects on endothelial cell activation and apoptosis. We report that anti-Gal mAbs derived from Gal(-/-) mice show a range of affinity for the alpha-Gal epitope, and that affinity was generally increased as the V(H) gene usage transitioned from germline sequences to sequences exhibiting somatic maturation. Despite an 85-fold range in affinity, all the anti-Gal mAbs examined induced alpha-Gal-specific endothelial cell activation, and after prolonged exposure induced endothelial cell apoptosis in a complement-independent manner. Only murine anti-Gal mAbs of the IgM or IgG3 subclass, but not IgG1, were effective at initiating complement-dependent cell lysis. Using a novel rat to mouse xenograft model, we examined the in vivo ability of these mAbs to induce xenograft rejection and characterized the rejection using histology and immunohistochemistry. Infusion of complement-fixing IgG3 mAbs resulted in either hyperacute rejection or acute vascular rejection of the xenograft. Surprisingly, infusion of an equal amount of a high affinity anti-Gal IgG1 mAb, that fixed complement poorly also induced a rapid xenograft rejection, which we have labeled very acute rejection. These studies emphasize the importance of in vivo assays, in addition to in vitro assays, in understanding the role of anti-Gal IgG-mediated tissue injury and xenograft rejection.     

Association of the calpain/calpastatin network with subcellular organelles

The calcium-activated cysteine protease calpain is intimately involved in modulating cell adhesion and migration. The two ubiquitous isoforms of this protease, calpain I and II, are considered to be cytosolic proteins that can translocate to both focal complexes/adhesions or the plasma membrane. Using confocal microscopy and isopycnic density centrifugation, the results demonstrate that calpain I and II, the 30kDa regulatory subunit, and calpastatin associate with the endoplasmic reticulum and Golgi apparatus. Confocal microscopy reveals that calpain II colocalizes with the subcellular proteins calnexin and Rab6 in cells bound to laminin. To further verify this association, cell lysates prepared from laminin stimulated and unstimulated cells were subjected to isopycnic density centrifugation. The results reveal an increased association of calpain I, II, calpastatin, and the 30kDa regulatory subunit with the endoplasmic reticulum and Golgi apparatus as evidenced by their position in the gradient relative to calnexin, Rab6, caveolin, and beta1 integrin after laminin stimulation. This correlates with the accumulation of inducible calpain activity at the endoplasmic reticulum-Golgi apparatus interface. Further experiments established that calpain II colocalizes with phosphatidylinositol 4,5-bisphosphate. Finally, calpain II associates with membrane lipid rafts. These results provide new insights into how the calpain/calpastatin network is spatially and temporally regulated in cells binding to the extracellular matrix.     

Cholesterol modification of sonic hedgehog is required for long-range signaling activity and effective modulation of signaling by Ptc1

Sonic hedgehog (Shh) signaling from the posterior zone of polarizing activity (ZPA) is the primary determinant of anterior-posterior polarity in the vertebrate limb field. An active signal is produced by an autoprocessing reaction that covalently links cholesterol to the N-terminal signaling moiety (N-Shh(p)), tethering N-Shh(p) to the cell membrane. We have addressed the role played by this lipophilic modification in Shh-mediated patterning of mouse digits. Both the distribution and activity of N-Shh(p) indicate that N-Shh(p) acts directly over a few hundred microns. In contrast, N-Shh, a form that lacks cholesterol, retains similar biological activity to N-Shh(p), but signaling is posteriorly restricted. Thus, cholesterol modification is essential for the normal range of signaling. It also appears to be necessary for appropriate modulation of signaling by the Shh receptor, Ptc1.     

The Evolution of the Ribonucleotide Reductases: Much Ado About Oxygen

Ribonucleotide reduction is the only known biological means for de novo production of deoxyribonucleotides, the building blocks of DNA. These are produced from ribonucleotides, the building blocks of RNA, and the direction of this reaction has been taken to support the idea that, in evolution, RNA preceded DNA as genetic material. However, an understanding of the evolutionary relationships among the three modern-day classes of ribonucleotide reductase and how the first reductase arose early in evolution is still far off. We propose that the diversification of this class of enzymes is inherently tied to microbial colonization of aerobic and anaerobic niches. The work is of broader interest, as it also sheds light on the process of adaptation to oxygenic environments consequent to the evolution of atmospheric oxygen.