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Podocalyxin (Podxl) is broadly expressed on the luminal face of most blood vessels in adult vertebrates, yet its function on these cells is poorly defined. In the present study, we identified specific functions for Podxl in maintaining endothelial barrier function. Using electrical cell substrate impedance sensing and live imaging, we found that, in the absence of Podxl, human umbilical vein endothelial cells fail to form an efficient barrier when plated on several extracellular matrix substrates. In addition, these monolayers lack adherens junctions and focal adhesions and display a disorganized cortical actin cytoskeleton. Thus, Podxl has a key role in promoting the appropriate endothelial morphogenesis required to form functional barriers. This conclusion is further supported by analyses of mutant mice in which we conditionally deleted a floxed allele of Podxl in vascular endothelial cells (vECs) using Tie2Cre mice ( Podxl ΔTie2Cre ). Although we did not detect substantially altered permeability in naïve mice, systemic priming with lipopolysaccharide (LPS) selectively disrupted the blood-brain barrier (BBB) in Podxl ΔTie2Cre mice. To study the potential consequence of this BBB breach, we used a selective agonist (TFLLR-NH 2 ) of the protease-activated receptor-1 (PAR-1), a thrombin receptor expressed by vECs, neuronal cells, and glial cells. In response to systemic administration of TFLLR-NH 2 , LPS-primed Podxl ΔTie2Cre mice become completely immobilized for a 5-min period, coinciding with severely dampened neuroelectric activity. We conclude that Podxl expression by CNS tissue vECs is essential for BBB maintenance under inflammatory conditions., Competing Interests: The authors declare no conflict of interest.

The emergence of drug-resistant bacteria has compromised the use of many conventional antibiotics, leading to heightened interest in a variety of antimicrobial peptides. Although these peptides have attractive potential as antibiotics, their size, stability, tissue distribution, and toxicity have hampered attempts to harness these capabilities. To address such issues, we have developed small (molecular mass <1,000 Da) arylamide foldamers that mimic antimicrobial peptides. Hydrogen-bonded restraints in the arylamide template rigidify the conformation via hydrogen bond formation and increase activity toward Staphylococcus aureus and Escherichia coli. The designed foldamers are highly active against S. aureus in an animal model. These results demonstrate the application of foldamer templates as therapeutics.

Nine polypeptide peaks with antibiotic activity were resolved from human polymorphonuclear leukocyte azurophil granule membranes. All but 1 of the 12 constituent polypeptides were identified by N-terminal sequence analysis. Near quantitative recovery of protein and activity permitted an assessment of the contribution of each species to the overall respiratory-burst-independent antimicrobial capacity of the cell. Three uncharacterized polypeptides were discovered, including two broad-spectrum antibiotics. One of these, a defensin that we have designated human neutrophil antimicrobial peptide 4, was more potent than previously described defensins but represented less than 1% of the total protein. The other, named azurocidin, was abundant and comparable to bactericidal permeability-increasing factor in its contribution to the killing of Escherichia coli.

Human fibrosarcoma (HT-1080) cells, in contrast to normal fibroblasts, rapidly hydrolyze the glycoprotein, collagen, and elastin extracellular matrix (ECM) synthesized by cultured rat aortic smooth muscle cells. This degradation occurs at a rapid rate in the presence of serum, indicating that the cellular proteases responsible are relatively insensitive to serum proteinase inhibitors. Here it is shown that protease nexin I (PNI), a fibroblast-secreted inhibitor of urokinase, plasmin, and certain other serine proteinases, effectively inhibited the HT-1080 cell-mediated degradation of this ECM. PNI at 2.0 nM significantly inhibited matrix destruction for 1-2 days and at 0.2 microM caused a virtually complete inhibition that persisted for the entire 10-day period of observation. Inhibition of ECM destruction was accompanied by a transient arrest of HT-1080 cell proliferation that took place during the first 3 days after PNI addition. PNI did not inhibit the growth of normal fibroblasts and also did not inhibit the growth of HT-1080 cells that were seeded onto plastic dishes rather than onto ECM. Like many types of malignant cells, HT-1080 cells release large amounts of urokinase. Antibody against this plasminogen activator partially protected ECM from HT-1080 cell-mediated hydrolysis, indicating that it may have been a target of PNI. One potential physiological function of PNI could be to help maintain the integrity of connective tissue matrices, protection that malignant cells could overcome by secreting proteinases in excessive amounts.

We have examined the interaction of the estradiol receptor molecule with chromatin in MCF-7 cells, a human breast tumor cell line responsive to estradiol. Receptor was found associated with the various nucleosomal products produced by digestion with micrococcal nuclease. In order to determine whether these receptor binding sites were distributed in a random or nonrandom manner within the chromatin, we have fractionated MCF-7 cell chromatin into transcriptionally active and inactive fractions by limited micrococcal nuclease digestion followed by Mg(2+) precipitation. A comparison of the Mg(2+)-soluble and insoluble chromatin fractions showed that the Mg(2+)-soluble fraction: (i) was composed predominantly of mononucleosomes; (ii) was enriched in nonhistone proteins; (iii) apparently lacked histone H1; (iv) was enriched approximately 5-fold in transcribed sequences as measured by a cDNA probe to cytoplasmic poly(A)-RNA sequences; and (v) was depleted at least 5-fold of globin sequences, which is presumably a nontranscribed gene in these cells. When these cells were stimulated with beta-[(3)H]estradiol, the Mg(2+)-soluble fraction showed a significant enrichment in chromatin-bound estradiol receptor: the Mg(2+)-soluble mononucleosomes showed a 3- to 4-fold enrichment and the di- and trinucleosomes, a 7- to 19-fold enrichment, when compared to the corresponding subunits in the Mg(2+)-insoluble chromatin fraction. This cofractionation of chromatin enriched in transcribed sequences and bound estradiol receptor indicated that receptor binding to MCF-7 cell chromatin was not random but, rather, occurred preferentially in specific regions of the chromatin.

The urine alpha-fetoprotein (AFP) and serum albumin genes most probably arose in evolution as the consequence of a duplication of a common ancestral gene. They have both been previously mapped to chromosome 5 in the mouse. We now have evidence that these genes are closely linked. By using a unique copy DNA probe derived from previously cloned AFP 5' flanking DNA, a recombinant DNA phage has been isolated, from a bacteriophage DNA library, that contains sequences flanking the 5' end of the AFP gene and the 3' end of the albumin gene. Restriction endonuclease mapping and DNA sequence determination of the recombinant phage and comparison to total genomic DNA confirmed that the genes are in tandem, 13.5 kilobase pairs apart, with the albumin gene to the 5' side of the AFP gene. Thus, they are transcribed from the same strand of DNA.