Background— Although we know much about the molecular makeup of the sinus node (SN) in small mammals, little is known about it in humans. The aims of the present study were to investigate the expression of ion channels in the human SN and to use the data to predict electrical activity. Methods and Results— Quantitative polymerase chain reaction, in situ hybridization, and immunofluorescence were used to analyze 6 human tissue samples. Messenger RNA (mRNA) for 120 ion channels (and some related proteins) was measured in the SN, a novel paranodal area, and the right atrium (RA). The results showed, for example, that in the SN compared with the RA, there was a lower expression of Na v 1.5, K v 4.3, K v 1.5, ERG, K ir 2.1, K ir 6.2, RyR2, SERCA2a, Cx40, and Cx43 mRNAs but a higher expression of Ca v 1.3, Ca v 3.1, HCN1, and HCN4 mRNAs. The expression pattern of many ion channels in the paranodal area was intermediate between that of the SN and RA; however, compared with the SN and RA, the paranodal area showed greater expression of K v 4.2, K ir 6.1, TASK1, SK2, and MiRP2. Expression of ion channel proteins was in agreement with expression of the corresponding mRNAs. The levels of mRNA in the SN, as a percentage of those in the RA, were used to estimate conductances of key ionic currents as a percentage of those in a mathematical model of human atrial action potential. The resulting SN model successfully produced pacemaking. Conclusions— Ion channels show a complex and heterogeneous pattern of expression in the SN, paranodal area, and RA in humans, and the expression pattern is appropriate to explain pacemaking.

A bacterial membrane protein, BacA, protects Sinorhizobium meliloti against the antimicrobial activity of host peptides, enabling the peptides to induce bacterial persistence rather than bacterial death.

Isoaspartate formation in extracellular matrix proteins, by aspartate isomerization or asparagine deamidation, is generally viewed as a degradation reaction occurring in vivo during tissue aging. For instance, non-enzymatic isoaspartate formation at RGD-integrin binding sites causes loss of cell adhesion sites, which in turn can be enzymatically "repaired" to RGD by protein-L-isoAsp-O-methyltransferase. We show here that isoaspartate formation is also a mechanism for extracellular matrix activation. In particular, we show that deamidation of Asn 263 at the Asn-Gly-Arg (NGR) site in fibronectin N-terminal region generates an ␣ v ␤ 3 -integrin binding site containing the L-isoDGR sequence, which is enzymatically "deactivated" to DGR by protein-L-isoAsp-O-methyltransferase. Furthermore, rapid NGRto-isoDGR sequence transition in fibronectin fragments generates ␣ v ␤ 3 antagonists (named "isonectins") that competitively bind RGD binding sites and inhibit endothelial cell adhesion, proliferation, and tumor growth. Time-dependent generation of isoDGR may represent a sort of molecular clock for activating latent integrin binding sites in proteins.Fibronectins are adhesive proteins that mediate a variety of cellular interactions with extracellular matrix and play important roles in hemostasis, thrombosis, inflammation, wound repair, angiogenesis, and embryogenesis (1, 2). About 20 isoforms of human fibronectin can be generated as a result of alternative splicing of the primary transcript (1, 3). Fibronectins are large glycoproteins (ϳ450 kDa) composed of two nearly identical disulfide-bonded subunits present in most body fluids and extracellular matrix of many tissues. Each subunit consists of three types of repeating homologous modules termed FN-I, FN-II, and FN-III repeats. Alternatively spliced modules, called EDA, EDB, and IIICS, can also be present (1, 3). Single modules or groups of modules may contain binding sites for different molecules, including sulfated glycosaminoglycans, DNA, gelatin, heparin, and fibrin (1, 3, 4). Furthermore, fibronectins contain binding sites for about half of the known cell surface integrin receptors (5, 6). In particular, the FN-III 10 repeat contains an RGD site that can bind, and ␣II b ␤ 3 integrins, while the FN-III 9 repeat contains the so-called "synergy site" PHSRN that cooperates with RGD in the binding of ␣ 5 ␤ 1 and ␣II b ␤ 3 (1, 7).Primary and tertiary structure analysis of human fibronectin showed that this protein contains two GNGRG loops, located in FN-I 5 and FN-I 7 modules, that are conserved in bovine, murine, rat, amphibian, and fish (8). Two additional NGR sites, less conserved, are also present in human FN-II 1 and FN-III 9 (see Fig. 1). Recent experimental work showed that peptides containing the NGR motif can inhibit ␣ 5 ␤ 1 -and ␣ v ␤ 1 -mediated cell adhesion to fibronectin (9).These notions prompted us to investigate the functional role of NGR in fibronectin. We observed that the NGR sequence of FN-I 5 (residues 263-265) promotes endothelial cell adhesion via an...

HIV-specific mucosal and cellular immunity was analyzed in heterosexual couples discordant for HIV status in serum and in HIV-unexposed controls. HIV-specific IgA but not IgG was present in urine and vaginal wash samples from HIV-exposed seronegative individuals (ESN), whereas both IgA and IgG were observed in their HIV-seropositive partners; antibodies were not detected in low-risk controls. Envelope protein (Env) peptide-stimulated interleukin-2 (IL-2) production by peripheral blood mononuclear cells (PBMCs) was detected in 9 out of 16 ESNs, 5 out of 16 HIV-infected patients and 1 out of 50 controls. Env peptide-stimulated PBMCs of ESNs produced more IL-2 and less IL-10 compared with those of HIV-infected individuals; no differences were observed in chemokine production or in CCR5 expression. These data demonstrate that a compartmentalized immune response to pathogens is possible in humans and raise the possibility of protective roles for cell-mediated immunity and mucosal IgA in HIV-seronegative individuals exposed to HIV.

Neuroblastoma, the most common solid tumor of infancy derived from the sympathetic nervous system, continues to present a formidable clinical challenge. Sterically stabilized immunoliposomes (SIL) have been shown to enhance the selective localization of entrapped drugs to solid tumors, with improvements in therapeutic indices. We showed that SIL loaded with doxorubicin (DXR) and targeted to the disialoganglioside receptor GD 2 [aGD 2 -SIL(DXR)] led to a selective inhibition of the metastatic growth of experimental models of human neuroblastoma. By coupling NGR peptides that target the angiogenic endothelial cell marker aminopeptidase N to the surface of DXR-loaded liposomes [NGR-SL(DXR)], we obtained tumor regression, pronounced destruction of the tumor vasculature, and prolonged survival of orthotopic neuroblastoma xenografts. Here, we showed good liposome stability, long circulation times, and enhanced timedependent tumor accumulation of both the carrier and the drug. Antivascular effects against animal models of lung and ovarian cancer were shown for formulations of NGR-SL(DXR). In the chick embryo chorioallantoic assay, NGR-SL(DXR) substantially reduced the angiogenic potential of various neuroblastoma xenografts, with synergistic inhibition observed for the combination of NGR-SL(DXR) with aGD 2 -SIL(DXR). A significant improvement in antitumor effects was seen in neuroblastoma-bearing animal models when treated with the combined formulations compared with control mice or mice treated with either tumor-or vascular-targeted liposomal formulations, administered separately. The combined treatment resulted in a dramatic inhibition of tumor endothelial cell density. Long-term survivors were obtained only in animals treated with the combined tumor-and vascular-targeted formulations, confirming the pivotal role of combination therapies in treating aggressive metastatic neuroblastoma.