Insulin activated endogenous protein kinase B alpha (also known as RAC/Akt kinase) activity 12-fold in L6 myotubes, while after transfection into 293 cells PKBalpha was activated 20- and 50-fold in response to insulin and IGF-1 respectively. In both cells, the activation of PKBalpha was accompanied by its phosphorylation at Thr308 and Ser473 and, like activation, phosphorylation of both of these residues was prevented by the phosphatidylinositol 3-kinase inhibitor wortmannin. Thr308 and/or Ser473 were mutated to Ala or Asp and activities of mutant PKBalpha molecules were analysed after transfection into 293 cells. The activity of wild-type and mutant PKBalpha was also measured in vitro after stoichiometric phosphorylation of Ser473 by MAPKAP kinase-2. These experiments demonstrated that activation of PKBalpha by insulin or insulin-like growth factor-1 (IGF-1) results from phosphorylation of both Thr308 and Ser473, that phosphorylation of both residues is critical to generate a high level of PKBalpha activity and that the phosphorylation of Thr308 in vivo is not dependent on phosphorylation of Ser473 or vice versa. We propose a model whereby PKBalpha becomes phosphorylated and activated in insulin/IGF-1-stimulated cells by an upstream kinase(s).

Type 1 diabetes (T1D) is a common autoimmune disorder that arises from the action of multiple genetic and environmental risk factors. We report the findings of a genome-wide association study of T1D, combined in a meta-analysis with two previously published studies. The total sample set included 7,514 cases and 9,045 reference samples. Forty-one distinct genomic locations provided evidence for association with T1D in the meta-analysis (P < 10(-6)). After excluding previously reported associations, we further tested 27 regions in an independent set of 4,267 cases, 4,463 controls and 2,319 affected sib-pair (ASP) families. Of these, 18 regions were replicated (P < 0.01; overall P < 5 × 10(-8)) and 4 additional regions provided nominal evidence of replication (P < 0.05). The many new candidate genes suggested by these results include IL10, IL19, IL20, GLIS3, CD69 and IL27.

Within- and cross-center test-retest studies were conducted to study the reliability of a new, semistructured, comprehensive, polydiagnostic psychiatric interview being used in a multisite genetic linkage study of alcoholism. Findings from both studies indicated that reliability for the Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA) was high for DSM-III-R substance dependence disorders, but less so for substance abuse disorders. Reliability of depression was good in both studies, but mixed for antisocial personality disorder (ASP). Findings are presented in terms of specific substance dependence and abuse diagnoses, as well as for depression and ASP. Criterion-specific reliabilities are examined by type of substance used. Although SSAGA was designed to provide for broad phenotyping of alcoholism, review of its new features suggests its suitability for a variety of family studies, not just those focusing on substance abuse.

Proteins that contain the Arg-Gly-Asp (RGD) attachment site, together with the integrins that serve as receptors for them, constitute a major recognition system for cell adhesion. The RGD sequence is the cell attachment site of a large number of adhesive extracellular matrix, blood, and cell surface proteins, and nearly half of the over 20 known integrins recognize this sequence in their adhesion protein ligands. Some other integrins bind to related sequences in their ligands. The integrin-binding activity of adhesion proteins can be reproduced by short synthetic peptides containing the RGD sequence. Such peptides promote cell adhesion when insolubilized onto a surface, and inhibit it when presented to cells in solution. Reagents that bind selectively to only one or a few of the RGD-directed integrins can be designed by cyclizing peptides with selected sequences around the RGD and by synthesizing RGD mimics. As the integrin-mediated cell attachment influences and regulates cell migration, growth, differentiation, and apoptosis, the RGD peptides and mimics can be used to probe integrin functions in various biological systems. Drug design based on the RGD structure may provide new treatments for diseases such as thrombosis, osteoporosis, and cancer.

Adenovirus contains a heterodimeric protein complex consisting of 186 kd fiber protein that mediates high affinity virus attachment to cells and a 400 kd pentavalent subunit (penton base) that contains five Arg-Gly-Asp sequences, implying a role for integrins in adenovirus infection. We demonstrate that the vitro-nectin-binding integrins alpha v beta 3 and alpha v beta 5 promote viral infection in a novel way since antibodies against these receptors or soluble penton base block virus internalization without affecting attachment. Moreover, adenovirus binds to cultured cells lacking alpha v integrins but fail to become internalized, thus restricting infection of these cells. Transfection of alpha v(-) cells with a cDNA encoding alpha v results in the expression of integrins alpha v beta 3 and alpha v beta 5 and allows virus internalization and infection. These data indicate that adenovirus attachment and uptake into cells are separate but cooperative events that result from the interaction of distinct viral coat proteins with a receptor for attachment and alpha v integrin receptors for internalization.

Proteins that permanently reside in the lumen of the endoplasmic reticulum (ER) must somehow be distinguished from newly synthesized secretory proteins, which pass through this compartment on their way out of the cell. Three luminal ER proteins whose sequence is known, grp78 ("BiP"), grp94, and protein disulphide isomerase, share the carboxy-terminal sequence Lys-Asp-Glu-Leu (KDEL). We show that deletion (or extension) of the carboxyl terminus of grp78 results in secretion of this protein when it is expressed in COS cells. Conversely, a derivative of chicken lysozyme containing the last six amino acids of grp78 fails to be secreted and instead accumulates in the ER. We propose that the KDEL sequence marks proteins that are to be retained in the ER and discuss possible retention mechanisms.

Accumulation of the amyloid-beta protein (Abeta) in the cerebral cortex is an early and invariant event in the pathogenesis of Alzheimer's disease. The final step in the generation of Abeta from the beta-amyloid precursor protein is an apparently intramembranous proteolysis by the elusive gamma-secretase(s). The most common cause of familial Alzheimer's disease is mutation of the genes encoding presenilins 1 and 2, which alters gamma-secretase activity to increase the production of the highly amyloidogenic Abeta42 isoform. Moreover, deletion of presenilin-1 in mice greatly reduces gamma-secretase activity, indicating that presenilin-1 mediates most of this proteolytic event. Here we report that mutation of either of two conserved transmembrane (TM) aspartate residues in presenilin-1, Asp 257 (in TM6) and Asp 385 (in TM7), substantially reduces Abeta production and increases the amounts of the carboxy-terminal fragments of beta-amyloid precursor protein that are the substrates of gamma-secretase. We observed these effects in three different cell lines as well as in cell-free microsomes. Either of the Asp ->> Ala mutations also prevented the normal endoproteolysis of presenilin-1 in the TM6 ->> TM7 cytoplasmic loop. In a functional presenilin-1 variant (carrying a deletion in exon 9) that is associated with familial Alzheimer's disease and which does not require this cleavage, the Asp 385 ->> Ala mutation still inhibited gamma-secretase activity. Our results indicate that the two transmembrane aspartate residues are critical for both presenilin-1 endoproteolysis and gamma-secretase activity, and suggest that presenilin 1 is either a unique diaspartyl cofactor for gamma-secretase or is itself gamma-secretase, an autoactivated intramembranous aspartyl protease.

Vaccination against human immunodeficiency virus type 1 (HIV-1) requires an immunogen which will elicit a protective immunity against viruses that show a high degree of genetic polymorphism. Therefore, the identification of neutralizing epitopes which are shared by many strains would be useful. In previous studies, we established a human monoclonal antibody (2F5) that neutralizes a variety of laboratory strains and clinical isolates of HIV-1. In the present report, we define the amino acid sequence Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) on the ectodomain of gp41 as the epitope recognized by this antibody. The sequence was found to be conserved in 72% of otherwise highly variable HIV-1 isolates. Escape mutants were not detected in cells infected with HIV-1 isolates MN and RF in the presence of antibody 2F5. Since sequence variability of neutralizing epitopes is considered to be a major obstacle to HIV-1 vaccine development, the conserved B-cell epitope described here is a promising candidate for inclusion in a vaccine against AIDS.

WD proteins are made up of highly conserved repeating units usually ending with Trp-Asp (WD). They are found in all eukaryotes but not in prokaryotes. They regulate cellular functions, such as cell division, cell-fate determination, gene transcription, transmembrane signalling, mRNA modification and vesicle fusion. Here we define the common features of the repeating units, and criteria for grouping such proteins into functional subfamilies.

A modified Neuhoff's colloidal Coomassie Blue G-250 stain is reported, dubbed "blue silver" on account of its considerably higher sensitivity, approaching the one of conventional silver staining. The main modifications, as compared to Neuhoff's protocol, were: a 20% increment in dye concentration (from 0.1% up to 0.12%) and a much higher level of phosphoric acid in the recipe (from 2% up to 10%). The "blue silver" exhibits a much faster dye uptake (80% during the first hour of coloration, vs. none with a commercial preparation from Sigma). Even at equilibrium (24 h staining), the "blue silver" exhibits a much higher sensitivity than all other recipes, approaching (but lower than) the one of the classical silver stain. Measurements of stain sensitivity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of bovine serum albumin (BSA) gave a detection limit (signal-to-noise ratio>> 3) of 1 ng in a single zone. The somewhat lower sensitivity of "blue silver" as compared to classical silvering protocols in the presence of aldehydes is amply compensated for by its full compatibility with mass spectrometry of eluted polypeptide chains, after a two-dimensional map analysis, thus confirming that no dye is covalently bound (or permanently modifies) to any residue in the proteinaceous material. It is believed that the higher level of phosphoric acid in the recipe, thus its lower final pH, helps in protonating the last dissociated residues of Asp and Glu in the polypeptide coils, thus greatly favoring ionic anchoring of dye molecules to the protein moiety. Such a binding, though, must be followed by considerable hydrophobic association with the aromatic and hydrophobic residues along the polypeptide backbone.

In this study, we have revised the rules and parameters for one of the most commonly used empirical pKa predictors, PROPKA, based on better physical description of the desolvation and dielectric response for the protein. We have introduced a new and consistent approach to interpolate the description between the previously distinct classifications into internal and surface residues, which otherwise is found to give rise to an erratic and discontinuous behavior. Since the goal of this study is to lay out the framework and validate the concept, it focuses on Asp and Glu residues where the protein pKa values and structures are assumed to be more reliable. The new and improved implementation is evaluated and discussed; it is found to agree better with experiment than the previous implementation (in parentheses): rmsd = 0.79 (0.91) for Asp and Glu, 0.75 (0.97) for Tyr, 0.65 (0.72) for Lys, and 1.00 (1.37) for His residues. The most significant advance, however, is in reducing the number of outliers and removing unreasonable sensitivity to small structural changes that arise from classifying residues as either internal or surface.

The structural basis for the divalent cation-dependent binding of heterodimeric alphabeta integrins to their ligands, which contain the prototypical Arg-Gly-Asp sequence, is unknown. Interaction with ligands triggers tertiary and quaternary structural rearrangements in integrins that are needed for cell signaling. Here we report the crystal structure of the extracellular segment of integrin alphaVbeta3 in complex with a cyclic peptide presenting the Arg-Gly-Asp sequence. The ligand binds at the major interface between the alphaV and beta3 subunits and makes extensive contacts with both. Both tertiary and quaternary changes are observed in the presence of ligand. The tertiary rearrangements take place in betaA, the ligand-binding domain of beta3; in the complex, betaA acquires two cations, one of which contacts the ligand Asp directly and the other stabilizes the ligand-binding surface. Ligand binding induces small changes in the orientation of alphaV relative to beta3.

This report describes the structural characterization of the recombinant envelope glycoprotein (rgp120) of human immunodeficiency virus type 1 produced by expression in Chinese hamster ovary cells. Enzymatic cleavage of rgp120 and reversed-phase high performance liquid chromatography were used to confirm the primary structure of the protein, to assign intrachain disulfide bonds, and to characterize potential sites for N-glycosylation. All of the tryptic peptides identified were consistent with the primary structure predicted from the cDNA sequence. Tryptic mapping studies combined with treatment of isolated peptides with Staphylococcus aureus V8 protease or with peptide:N-glycosidase F followed by endoproteinase Asp-N permitted the assignment of all nine intrachain disulfide bonds of rgp120. The 24 potential sites for N-glycosylation were characterized by determining the susceptibilities of the attached carbohydrate structures to peptide:N-glycosidase F and to endo-beta-N-acetylglucosaminidase H. Tryptic mapping of enzymatically deglycosylated rgp120 was used in conjunction with Edman degradation and fast atom bombardment-mass spectrometry of individually treated peptides to determine which of these sites are glycosylated and what types of structures are present. The results indicate that all 24 sites of gp120 are utilized, including 13 that contain complex-type oligosaccharides as the predominant structures, and 11 that contain primarily high mannose-type and/or hybrid-type oligosaccharide structures.

The three-dimensional structure of acetylcholinesterase from Torpedo californica electric organ has been determined by x-ray analysis to 2.8 angstrom resolution. The form crystallized is the glycolipid-anchored homodimer that was purified subsequent to solubilization with a bacterial phosphatidylinositol-specific phospholipase C. The enzyme monomer is an alpha/beta protein that contains 537 amino acids. It consists of a 12-stranded mixed beta sheet surrounded by 14 alpha helices and bears a striking resemblance to several hydrolase structures including dienelactone hydrolase, serine carboxypeptidase-II, three neutral lipases, and haloalkane dehalogenase. The active site is unusual because it contains Glu, not Asp, in the Ser-His-acid catalytic triad and because the relation of the triad to the rest of the protein approximates a mirror image of that seen in the serine proteases. Furthermore, the active site lies near the bottom of a deep and narrow gorge that reaches halfway into the protein. Modeling of acetylcholine binding to the enzyme suggests that the quaternary ammonium ion is bound not to a negatively charged "anionic" site, but rather to some of the 14 aromatic residues that line the gorge.

Histone deacetylases (HDACs) mediate changes in nucleosome conformation and are important in the regulation of gene expression. HDACs are involved in cell-cycle progression and differentiation, and their deregulation is associated with several cancers. HDAC inhibitors, such as trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), have anti-tumour effects, as they can inhibit cell growth, induce terminal differentiation and prevent the formation of tumours in mice models, and they are effective in the treatment of promyelocytic leukemia. Here we describe the structure of the histone deacetylase catalytic core, as revealed by the crystal structure of a homologue from the hyperthermophilic bacterium Aquifex aeolicus, that shares 35.2% identity with human HDAC1 over 375 residues, deacetylates histones in vitro and is inhibited by TSA and SAHA. The deacetylase, deacetylase-TSA and deacetylase-SAHA structures reveal an active site consisting of a tubular pocket, a zinc-binding site and two Asp-His charge-relay systems, and establish the mechanism of HDAC inhibition. The residues that make up the active site and contact the inhibitors are conserved across the HDAC family. These structures also suggest a mechanism for the deacetylation reaction and provide a framework for the further development of HDAC inhibitors as antitumour agents.

The crystal structure of a ternary complex of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) heterodimer (p66/p51), a 19-base/18-base double-stranded DNA template-primer, and a monoclonal antibody Fab fragment has been determined at 3.0 A resolution. The four individual subdomains of RT that make up the polymerase domains of p66 and p51 are named fingers, palm, thumb, and connection [Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A. & Steitz, T. A. (1992) Science 256, 1783-1790]. The overall folding of the subdomains is similar in p66 and p51 but the spatial arrangements of the subdomains are dramatically different. The template-primer has A-form and B-form regions separated by a significant bend (40-45 degrees). The most numerous nucleic acid interactions with protein occur primarily along the sugar-phosphate backbone of the DNA and involve amino acid residues of the palm, thumb, and fingers of p66. Highly conserved regions are located in the p66 palm near the polymerase active site. These structural elements, together with two alpha-helices of the thumb of p66, act as a clamp to position the template-primer relative to the polymerase active site. The 3'-hydroxyl of the primer terminus is close to the catalytically essential Asp-110, Asp-185, and Asp-186 residues at the active site and is in a position for nucleophilic attack on the alpha-phosphate of an incoming nucleoside triphosphate. The structure of the HIV-1 RT/DNA/Fab complex should aid our understanding of general mechanisms of nucleic acid polymerization. AIDS therapies may be enhanced by a fuller understanding of drug inhibition and resistance emerging from these studies.

cDNA clones encoding three classes of human actins have been isolated and characterized. The first two classes (gamma and beta, cytoplasmic actins) were obtained from a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA, and the third class (alpha, muscle actin) was obtained from a cDNA library constructed from adult human muscle mRNA. A new approach was developed to enrich for full-length cDNAs. The human fibroblast cDNA plasmid library was linearized with restriction enzymes that did not cut the inserts of interest; it was then size-fractionated on gels, and the chimeric molecules of optimal length were selected for retransformation of bacteria. When the resulting clones were screened for actin-coding sequences it was found that some full-length cDNAs were enriched as much as 50- to 100-fold relative to the original frequency of full-length clones in the total library. Two types of clones were distinguished. One of these clones encodes gamma actin and contains 100 base pairs of 5' untranslated region, the entire protein coding region, and the 3' untranslated region. The second class encodes beta actin, and the longest such clone contains 45 base pairs of 5' untranslated region plus the remainder of the mRNA extending to the polyadenylic acid tail. A third class, obtained from the human muscle cDNA library, encodes alpha actin and contains 100 base pairs of 5' untranslated region, the entire coding region, and the 3' untranslated region. Analysis of the DNA sequences of the 5' end of the clones demonstrated that although beta- and gamma-actin genes start with a methionine codon (MET-Asp-Asp-Asp and MET-Glu-Glu-Glu, respectively), the alpha-actin gene starts with a methionine codon followed by a cysteine codon (MET-CYS-Asp-Glu-Asp-Glu). Since no known actin proteins start with a cysteine, it is likely that post-translational removal of cysteine in addition to methionine accompanies alpha-actin synthesis but not beta- and gamma-actin synthesis. This observation has interesting implications both for actin function and actin gene regulation and evolution.

In vivo selection of phage display libraries was used to isolate peptides that home specifically to tumor blood vessels. When coupled to the anticancer drug doxorubicin, two of these peptides-one containing an alphav integrin-binding Arg-Gly-Asp motif and the other an Asn-Gly-Arg motif-enhanced the efficacy of the drug against human breast cancer xenografts in nude mice and also reduced its toxicity. These results indicate that it may be possible to develop targeted chemotherapy strategies that are based on selective expression of receptors in tumor vasculature.

A comprehensive, unbiased inventory of synuclein forms present in Lewy bodies from patients with dementia with Lewy bodies was carried out using two-dimensional immunoblot analysis, novel sandwich enzyme-linked immunosorbent assays with modification-specific synuclein antibodies, and mass spectroscopy. The predominant modification of alpha-synuclein in Lewy bodies is a single phosphorylation at Ser-129. In addition, there is a set of characteristic modifications that are present to a lesser extent, including ubiquitination at Lys residues 12, 21, and 23 and specific truncations at Asp-115, Asp-119, Asn-122, Tyr-133, and Asp-135. No other modifications are detectable by tandem mass spectrometry mapping, except for a ubiquitous N-terminal acetylation. Small amounts of Ser-129 phosphorylated and Asp-119-truncated alpha-synuclein are present in the soluble fraction of both normal and disease brains, suggesting that these Lewy body-associated forms are produced during normal metabolism of alpha-synuclein. In contrast, ubiquitination is only detected in Lewy bodies and is primarily present on phosphorylated synuclein; it therefore likely occurs after phosphorylated synuclein has deposited into Lewy bodies. This invariant pattern of specific phosphorylation, truncation, and ubiquitination is also present in the detergent-insoluble fraction of brain from patients with familial Parkinson's disease (synuclein A53T mutation) as well as multiple system atrophy, suggesting a common pathogenic pathway for both genetic and sporadic Lewy body diseases. These observations are most consistent with a model in which preferential accumulation of normally produced Ser-129 phosphorylated alpha-synuclein is the key event responsible for the formation of Lewy bodies in various Lewy body diseases.

Nutrients and bioenergetics are prerequisites for proliferation and survival of mammalian cells. We present evidence that the cyclin-dependent kinase inhibitor p27(Kip1), is phosphorylated at Thr 198 downstream of the Peutz-Jeghers syndrome protein-AMP-activated protein kinase (LKB1-AMPK) energy-sensing pathway, thereby increasing p27 stability and directly linking sensing of nutrient concentration and bioenergetics to cell-cycle progression. Ectopic expression of wild-type and phosphomimetic Thr 198 to Asp 198 (T198D), but not unstable Thr 198 to Ala 198 (p27(T198A)) is sufficient to induce autophagy. Under stress conditions that activate the LKB1-AMPK pathway with subsequent induction of autophagy, p27 knockdown results in apoptosis. Thus LKB1-AMPK pathway-dependent phosphorylation of p27 at Thr 198 stabilizes p27 and permits cells to survive growth factor withdrawal and metabolic stress through autophagy. This may contribute to tumour-cell survival under conditions of growth factor deprivation, disrupted nutrient and energy metabolism, or during stress of chemotherapy.

The interferon regulatory factors (IRF) consist of a growing family of related transcription proteins first identified as regulators of the alpha beta interferon (IFN-alpha/beta) gene promoters, as well as the interferon-stimulated response element (ISRE) of some IFN-stimulated genes. IRF-3 was originally identified as a member of the IRF family based on homology with other IRF family members and on binding to the ISRE of the ISG15 promoter. IRF-3 is expressed constitutively in a variety of tissues, and the relative levels of IRF-3 mRNA do not change in virus-infected or IFN-treated cells. In the present study, we demonstrate that following Sendai virus infection, IRF-3 is posttranslationally modified by protein phosphorylation at multiple serine and threonine residues, which are located in the carboxy terminus of IRF-3. A combination of IRF-3 deletion and point mutations localized the inducible phosphorylation sites to the region -ISNSHPLSLTSDQ- between amino acids 395 and 407; point mutation of residues Ser-396 and Ser-398 eliminated virus-induced phosphorylation of IRF-3 protein, although residues Ser-402, Thr-404, and Ser-405 were also targets. Phosphorylation results in the cytoplasm-to-nucleus translocation of IRF-3, DNA binding, and increased transcriptional activation. Substitution of the Ser-Thr sites with the phosphomimetic Asp generated a constitutively active form of IRF-3 that functioned as a very strong activator of promoters containing PRDI-PRDIII or ISRE regulatory elements. Phosphorylation also appears to represent a signal for virus-mediated degradation, since the virus-induced turnover of IRF-3 was prevented by mutation of the IRF-3 Ser-Thr cluster or by proteasome inhibitors. Interestingly, virus infection resulted in the association of IRF-3 with the CREB binding protein (CBP) coactivator, as detected by coimmunoprecipitation with anti-CBP antibody, an interaction mediated by the C-terminal domains of both proteins. Mutation of residues Ser-396 and Ser-398 in IRF-3 abrogated its binding to CBP. These results are discussed in terms of a model in which virus-inducible, C-terminal phosphorylation of IRF-3 alters protein conformation to permit nuclear translocation, association with transcriptional partners, and primary activation of IFN- and IFN-responsive genes.

Retroviral proteins are synthesized as polyprotein precursors that undergo proteolytic cleavages to yield the mature viral proteins. The role of the human immunodeficiency virus (HIV) protease in the viral replication cycle was examined by use of a site-directed mutation in the protease gene. The HIV protease gene product was expressed in Escherichia coli and observed to cleave HIV gag p55 to gag p24 and gag p17 in vitro. Substitution of aspartic acid residue 25 (Asp-25) of this protein with an asparagine residue did not affect the expression of the protein, but it eliminated detectable in vitro proteolytic activity against HIV gag p55. A mutant HIV provirus was constructed that contained the Asn-25 mutation within the protease gene. SW480 human colon carcinoma cells transfected with the Asn-25 mutant proviral DNA produced virions that contained gag p55 but not gag p24, whereas virions from cells transfected with the wild-type DNA contained both gag p55 and gag p24. The mutant virions were not able to infect MT-4 lymphoid cells. In contrast, these cells were highly sensitive to infection by the wild-type virions. These results demonstrate that the HIV protease is an essential viral enzyme and, consequently, an attractive target for anti-HIV drugs.

Pulmonary emphysema, a significant global health problem, is characterized by a loss of alveolar structures. Because VEGF is a trophic factor required for the survival of endothelial cells and is abundantly expressed in the lung, we hypothesized that chronic blockade of VEGF receptors could induce alveolar cell apoptosis and emphysema. Chronic treatment of rats with the VEGF receptor blocker SU5416 led to enlargement of the air spaces, indicative of emphysema. The VEGF receptor inhibitor SU5416 induced alveolar septal cell apoptosis but did not inhibit lung cell proliferation. Viewed by angiography, SU5416-treated rat lungs showed a pruning of the pulmonary arterial tree, although we observed no lung infiltration by inflammatory cells or fibrosis. SU5416 treatment led to a decrease in lung expression of VEGF receptor 2 (VEGFR-2), phosphorylated VEGFR-2, and Akt-1 in the complex with VEGFR-2. Treatment with the caspase inhibitor Z-Asp-CH(2)-DCB prevented SU5416-induced septal cell apoptosis and emphysema development. These findings suggest that VEGF receptor signaling is required for maintenance of the alveolar structures and, further, that alveolar septal cell apoptosis contributes to the pathogenesis of emphysema.

The mechanisms underlying neoplastic epithelial cell killing by ionizing radiation are largely unknown. We discovered a novel response to radiation manifested by autophagy and the development of acidic vesicular organelles (AVO). Acidification of AVO was mediated by the vacuolar H+-ATPase. Staining with the lysosomotropic agent acridine orange enabled us to quantify AVO accumulation and to demonstrate their time- and dose-dependent appearance. The appearance of AVO occurred in the presence of the pan-caspase inhibitor z-Val-Ala-Asp(Ome)-fluoromethyl ketone, but was inhibited by 3-methyladenine, an inhibitor of autophagy. The accretion of AVO in surviving progenies of irradiated cells, and the increased incidence of clonogenic death after inhibition of vacuolar H+-ATPase suggest that formation of acidic organelles represents a novel defense mechanism against radiation damage.