Basement membranes (BMs) are present in every tissue of the human body. All epithelium and endothelium is in direct association with BMs. BMs are a composite of several large glycoproteins and form an organized scaffold to provide structural support to the tissue and also offer functional input to modulate cellular function. While collagen I is the most abundant protein in the human body, type IV collagen is the most abundant protein in BMs. Matrigel is commonly used as surrogate for BMs in many experiments, but this is a tumor-derived BM-like material and does not contain all of the components that natural BMs possess. The structure of BMs and their functional role in tissues are unique and unlike any other class of proteins in the human body. Increasing evidence suggests that BMs are unique signal input devices that likely fine tune cellular function. Additionally, the resulting endothelial and epithelial heterogeneity in human body is a direct contribution of cell-matrix interaction facilitated by the diverse compositions of BMs.

Increased numbers of S100A4 + cells are associated with poor prognosis in patients who have cancer. Although the metastatic capabilities of S100A4 + cancer cells have been examined, the functional role of S100A4 + stromal cells in metastasis is largely unknown. To study the contribution of S100A4 + stromal cells in metastasis, we used transgenic mice that express viral thymidine kinase under control of the S100A4 promoter to specifically ablate S100A4 + stromal cells. Depletion of S100A4 + stromal cells significantly reduced metastatic colonization without affecting primary tumor growth. Multiple bone marrow transplantation studies demonstrated that these effects of S100A4 + stromal cells are attributable to local non-bone marrow-derived S100A4 + cells, which are likely fibroblasts in this setting. Reduction in metastasis due to the loss of S100A4 + fibroblasts correlated with a concomitant decrease in the expression of several ECM molecules and growth factors, particularly Tenascin-C and VEGF-A. The functional importance of stromal Tenascin-C and S100A4+ fibroblast-derived VEGF-A in metastasis was established by examining Tenascin-C null mice and transgenic mice expressing Cre recombinase under control of the S100A4 promoter crossed with mice carrying VEGF-A alleles flanked by loxP sites, which exhibited a significant decrease in metastatic colonization without effects on primary tumor growth. In particular, S100A4+ fibroblast-derived VEGF-A plays an important role in the establishment of an angiogenic microenvironment at the metastatic site to facilitate colonization, whereas stromal Tenascin-C may provide protection from apoptosis. Our study demonstrates a crucial role for local S100A4 + fibroblasts in providing the permissive "soil" for metastatic colonization, a challenging step in the metastatic cascade.stromal fibroblasts | metastasis-associated fibroblasts | tumor microenvironment | metastatic microenvironment A bout 90% of cancer deaths are attributable to systemic disease associated with metastasis (1). Among the steps involved in metastasis, the colonization step is considered the most challenging for an invading cancer cell (2). With metastatic disease as the leading cause of death among patients who have cancer (3), a greater need is emphasized for a better understanding of the metastatic process so as to identify efficacious cancer therapies. S100A4 (also known as CAPL, p9Ka, 42A, pEL98, mts1, metastasin, calvasculin, 18A2, or FSP1) is a member of the S100 calcium-binding family, which has a high prognostic significance for metastasis in patients with cancer (4). Several studies have demonstrated a correlation between increased numbers of S100A4 + cells and poor prognosis of patients for a variety of cancer types, including colorectal adenocarcinoma, non-small cell lung cancer, breast adenocarcinoma, gastric cancer, esophageal squamous carcinoma, bladder cancer, prostate adenocarcinoma, melanoma, and ovarian carcinoma. Although S100A4 + cells encompass a variety of cell types, including malignant c...

Using small-angle x-ray scattering, we have observed the cGMPinduced elongation of an active, cGMP-dependent, monomeric deletion mutant of cGMP-dependent protein kinase (⌬ 1-52 PKG-I␤). On saturation with cGMP, the radius of gyration of ⌬ 1-52 PKG-I␤ increases from 29.4 ؎ 0.1 Å to 40.1 ؎ 0.7 Å, and the maximum linear dimension increases from 90 Å ؎ 10% to 130 Å ؎ 10%. The elongation is due to a change in the interaction between structured regulatory (R) and catalytic (C) domains. A model of cGMP binding to ⌬ 1-52 PKG-I␤ indicates that elongation of ⌬ 1-52 PKG-I␤ requires binding of cGMP to the low-affinity binding site of the R domain. A comparison with cAMP-dependent protein kinase suggests that both elongation and activation require cGMP binding to both sites; cGMP binding to the low-affinity site therefore seems to be a necessary, but not sufficient, condition for both elongation and activation of ⌬ 1-52 PKG-I␤. We also predict that there is little or no cooperativity in cGMP binding to the two sites of ⌬ 1-52 PKG-I␤ under the conditions used here. Results obtained by using the ⌬ 1-52 PKG-I␤ monomer indicate that a previously observed elongation of PKG-I␣ is consistent with a pure change in the interaction between the R domain and the C domain, without alteration of the dimerization interaction. This study has revealed important features of molecular mechanisms in the biochemical network describing PKG-I␤ activation by cGMP, yielding new insight into ligand activation of cyclic nucleotide-dependent protein kinases, a class of regulatory proteins that is key to many cellular processes.C yclic guanosine monophosphate (cGMP) is a second messenger signaling molecule that is central to the regulation of many physiological processes, including smooth muscle tone, visual transduction, platelet aggregation, bone growth, and electrolyte and fluid homeostasis (see reviews in refs. 1-3). Its known targets are ion channels, which can alter cellular cation or anion transport; phosphodiesterases, which degrade cyclic nucleotides; and the cGMP-dependent protein kinases (PKGs), which are believed to be responsible for most intracellular cGMP actions. The PKG [first discovered by Kuo and Greengard (4)] is a serine͞threonine protein kinase whose substrates include ion channels and pumps, receptors, and enzymes that control intracellular Ca 2ϩ concentrations. In its active form, PKG can transfer the ␥-phosphate from ATP to target proteins, preferentially targeting serines in substrates having consensus sequence RRXSX, with some exceptions (1, 5). Phosphorylation of target proteins by PKG effects smooth muscle relaxation by reduction of cellular Ca 2ϩ . The PKGs and cAMP-dependent protein kinases (PKA) are homologous enzymes, and the two enzyme families share many similarities in biochemical function and domain organization.PKG is a dimer comprised of two identical monomers (1-3). Each monomer of PKG contains a regulatory domain (R) and a catalytic domain (C) on a single polypeptide chain. The monomers dimerize via interactions ...

The fibulins are a family of secreted glycoproteins that are characterized by repeated epidermal-growth-factor-like domains and a unique C-terminus structure. Fibulins modulate cell morphology, growth, adhesion, and motility. Our initial basement membrane degradome screen using Cathepsin D, a tumor microenvironment-associated protease, contained fragments of fibulin-1 and full length fibulin-5. In this report, we evaluate the antiangiogenic activity of fibulin-1 and fibulin-5. Tumor studies demonstrate that both fibulin-1 and fibulin-5 suppress HT1080 tumor growth. CD31 labeling and TUNEL assay further reveal that fibulin-1 suppression of HT1080 tumor growth is associated with diminished angiogenesis and also enhanced apoptosis of endothelial cells and tumor cells. In contrast, fibulin-5 inhibits tumor angiogenesis with a minimal anti-apoptotic affect. Cathepsin D digestion of fibulin-1 produces a fragment with nearly the same molecular weight as fibulin-5, and this fragment (named Neostatin) inhibits endothelial cell proliferation. Additionally, degradation of basement membrane by cathepsin D liberates both fibulin-1 fragments and fibulin-5, which function to inhibit angiogenesis.

Goodpasture syndrome is an autoimmune vascular disease associated with kidney and lung failure, with pathogenic circulating autoantibodies targeted to a set of discontinuous epitope sequences within the noncollagenous domain-1 (NC1) of the ␣3 chain of type IV collagen (␣3(IV)NC1), the Goodpasture autoantigen. We demonstrate that basement membrane extracted NC1 domain preparations from Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio do not bind Goodpasture autoantibodies, while Xenopus laevis, chicken, mouse and human ␣3(IV)NC1 domains bind autoantibodies. The ␣3(IV) chain is not present in C elegans and Drosophila melanogaster, but is first detected in the Danio rerio. Interestingly, native Danio rerio ␣3(IV)NC1 does not bind Goodpasture autoantibodies. Next, we cloned, sequenced, and generated recombinant Danio rerio ␣3(IV)NC1 domain. In contrast to recombinant human ␣3(IV)NC1 domain, there was complete absence of autoantibody binding to recombinant Danio rerio ␣3(IV)NC1. Three-dimensional molecular modeling from existing x-ray coordinates of human NC1 domain suggest that evolutionary alteration of electrostatic charge and polarity due to the emergence of critical serine, aspartic acid, and lysine residues, accompanied by the loss of asparagine and glutamine, contributes to the emergence of the 2 major Goodpasture epitopes on the human ␣3(IV)NC1 domain, as it evolved from the Danio rerio over 450 million years. (Blood. 2006;107: 1908-1915

Low 25-OH serum vitamin D (VitD) is pervasive in older adults and linked to functional decline and progression of frailty. We have previously shown that chronic VitD insufficiency in “middle-aged” mice results in impaired anaerobic exercise capacity, decreased lean mass, and increased adiposity. Here, we examine if VitD insufficiency results in similar deficits and greater frailty progression in old-aged (24 to 28 months of age) mice. Similar to what we report in younger mice, older mice exhibit a rapid and sustained response in serum 25-OH VitD levels to differential supplementation, including insufficient (125 IU/kg chow), sufficient (1000 IU/kg chow), and hypersufficient (8000 IU/kg chow) groups. During the 4-month time course, mice were assessed for body composition (DEXA), physical performance, and frailty using a Fried physical phenotype-based assessment tool. The 125 IU mice exhibited worse grip strength (p = 0.002) and inverted grip hang time (p = 0.003) at endpoint and the 8000 IU mice transiently displayed greater rotarod performance after 3 months (p = 0.012), yet other aspects including treadmill performance and gait speed were unaffected. However, 125 and 1000 IU mice exhibited greater frailty compared to baseline (p = 0.001 and p = 0.038, respectively), whereas 8000 IU mice did not (p = 0.341). These data indicate targeting higher serum 25-OH vitamin D levels may attenuate frailty progression during aging.

BACKGROUND To minimize the risk of pneumonia many anesthesiologists delay anesthesia-requiring procedures when patients exhibit signs of viral upper respiratory tract infection. Post-influenza secondary bacterial pneumonias (SBP) are a major cause of morbidity and mortality. Increased host susceptibility to SBP post-influenza has been attributed to physical damage to the pulmonary epithelium, but flu-induced effects on the immune system are being shown to also play an important role. We demonstrate that halothane mitigates risk of SBP post-flu through modulation of the effects of type I interferon (IFN). METHODS Mice (n=6–15) were exposed to halothane or ketamine and treated with influenza and S. pneumoniae. Bronchoalveolar lavage (BAL) and lung homogenate was procured for measurement of inflammatory cells, cytokines, chemokines, albumin, MPO, and bacterial load. RESULTS Halothane exposure resulted in decreased bacterial burden (7.9 ± 3.9 × 105 vs 3.4 ± 1.6 × 108 colony forming units, p<0.01), clinical score (0.6 ± 0.2 vs 2.3 ± 0.2, p<0.0001) and lung injury (as measured by BAL albumin, 1.5 ± 0.7 vs 6.8 ± 1.6 mg/ml, p<0.01) in CD-1 mice infected with flu for 7 days and challenged with S. pneumoniae on day 6 post-flu. IFN receptor A1 knockout mice similarly infected with flu and S. pneumoniae, but not exposed to halothane, demonstrated a reduction of lung bacterial burden equivalent to that achieved in halothane-exposed wild-type mice. CONCLUSIONS These findings indicate that the use of halogenated volatile anesthetics modulate the type I IFN response to influenza and enhance post-infection anti-bacterial immunity.

Three cases of IgA kappa cold agglutinins (CAs) were studied. One had anti-Pr1 specificity, one had anti-Pra, and one had anti-Sa. The CAs recognize O-glycans of glycophorins. The findings supplement previous data on anti-Pr1 specificities of four IgA kappa CAs. Because all IgA kappa CAs described recognize O-glycans of glycophorins, a close association between the CA IgA isotype and specificities for O-glycans becomes apparent. It is unlikely, however, that the striking association reflects interrelations between IgA CA structure and specificity, because anti-Sa specificity and all anti-Pr subspecificities were originally defined with IgM CAs.