Mitochondria inside acute myeloid leukemia cells hydrolyze ATP to resist chemotherapy.

Despite early optimism, therapeutics targeting oxidative phosphorylation (OxPhos) have faced clinical setbacks, stemming from their inability to distinguish healthy from cancerous mitochondria. Herein, we describe an actionable bioenergetic mechanism unique to cancerous mitochondria inside acute myeloid leukemia (AML) cells. Unlike healthy cells which couple respiration to the synthesis of ATP, AML mitochondria were discovered to support inner membrane polarization by consuming ATP. Because matrix ATP consumption allows cells to survive bioenergetic stress, we hypothesized that AML cells may resist cell death induced by OxPhos damaging chemotherapy by reversing the ATP synthase reaction. In support of this, targeted inhibition of BCL-2 with venetoclax abolished OxPhos flux without impacting mitochondrial membrane potential. In surviving AML cells, sustained polarization of the mitochondrial inner membrane was dependent on matrix ATP consumption. Mitochondrial ATP consumption was further enhanced in AML cells made refractory to venetoclax, consequential to downregulations in both the proton-pumping respiratory complexes, as well as the endogenous F1-ATPase inhibitor ATP5IF1. In treatment-naive AML, ATP5IF1 knockdown was sufficient to drive venetoclax resistance, while ATP5IF1 overexpression impaired F1-ATPase activity and heightened sensitivity to venetoclax. Collectively, our data identify matrix ATP consumption as a cancer-cell intrinsic bioenergetic vulnerability actionable in the context of mitochondrial damaging chemotherapy.

Neurovascular dysfunction associated with erectile dysfunction persists after long-term recovery from simulations of weightlessness and deep space irradiation.

There has been growing interest within the space industry for long-duration manned expeditions to the Moon and Mars. During deep space missions, astronauts are exposed to high levels of galactic cosmic radiation (GCR) and microgravity which are associated with increased risk of oxidative stress and endothelial dysfunction. Oxidative stress and endothelial dysfunction are causative factors in the pathogenesis of erectile dysfunction, although the effects of spaceflight on erectile function have been unexplored. Therefore, the purpose of this study was to investigate the effects of simulated spaceflight and long-term recovery on tissues critical for erectile function, the distal internal pudendal artery (dIPA), and the corpus cavernosum (CC). Eighty-six adult male Fisher-344 rats were randomized into six groups and exposed to 4-weeks of hindlimb unloading (HLU) or weight-bearing control, and sham (0Gy), 0.75 Gy, or 1.5 Gy of simulated GCR at the ground-based GCR simulator at the NASA Space Radiation Laboratory. Following a 12-13-month recovery, ex vivo physiological analysis of the dIPA and CC tissue segments revealed differential impacts of HLU and GCR on endothelium-dependent and -independent relaxation that was tissue type specific. GCR impaired non-adrenergic non-cholinergic (NANC) nerve-mediated relaxation in the dIPA and CC, while follow-up experiments of the CC showed restoration of NANC-mediated relaxation of GCR tissues following acute incubation with the antioxidants mito-TEMPO and TEMPOL, as well as inhibitors of xanthine oxidase and arginase. These findings indicate that simulated spaceflight exerts a long-term impairment of neurovascular erectile function, which exposes a new health risk to consider with deep space exploration.

Pan-tissue mitochondrial phenotyping reveals lower OXPHOS expression and function across cancer types.

Targeting mitochondrial oxidative phosphorylation (OXPHOS) to treat cancer has been hampered due to serious side-effects potentially arising from the inability to discriminate between non-cancerous and cancerous mitochondria. Herein, comprehensive mitochondrial phenotyping was leveraged to define both the composition and function of OXPHOS across various murine cancers and compared to both matched normal tissues and other organs. When compared to both matched normal tissues, as well as high OXPHOS reliant organs like heart, intrinsic expression of the OXPHOS complexes, as well as OXPHOS flux were discovered to be consistently lower across distinct cancer types. Assuming intrinsic OXPHOS expression/function predicts OXPHOS reliance in vivo, these data suggest that pharmacologic blockade of mitochondrial OXPHOS likely compromises bioenergetic homeostasis in healthy oxidative organs prior to impacting tumor mitochondrial flux in a clinically meaningful way. Although these data caution against the use of indiscriminate mitochondrial inhibitors for cancer treatment, considerable heterogeneity was observed across cancer types with respect to both mitochondrial proteome composition and substrate-specific flux, highlighting the possibility for targeting discrete mitochondrial proteins or pathways unique to a given cancer type.

Intrinsic adaptations in OXPHOS power output and reduced tumorigenicity characterize doxorubicin resistant ovarian cancer cells.

Although the development of chemoresistance is multifactorial, active chemotherapeutic efflux driven by upregulations in ATP binding cassette (ABC) transporters are commonplace. Chemotherapeutic efflux pumps, like ABCB1, couple drug efflux to ATP hydrolysis and thus potentially elevate cellular demand for ATP resynthesis. Elevations in both mitochondrial content and cellular respiration are common phenotypes accompanying many models of cancer cell chemoresistance, including those dependent on ABCB1. The present study set out to characterize potential mitochondrial remodeling commensurate with ABCB1-dependent chemoresistance, as well as investigate the impact of ABCB1 activity on mitochondrial respiratory kinetics. To do this, comprehensive bioenergetic phenotyping was performed across ABCB1-dependent chemoresistant cell models and compared to chemosensitive controls. In doxorubicin (DOX) resistant ovarian cancer cells, the combination of both increased mitochondrial content and enhanced respiratory complex I (CI) boosted intrinsic oxidative phosphorylation (OXPHOS) power output. With respect to ABCB1, acute ABCB1 inhibition partially normalized intact basal mitochondrial respiration between chemosensitive and chemoresistant cells, suggesting that active ABCB1 contributes to mitochondrial remodeling in favor of enhanced OXPHOS. Interestingly, while enhanced OXPHOS power output supported ABCB1 drug efflux when DOX was present, in the absence of chemotherapeutic stress, enhanced OXPHOS power output was associated with reduced tumorigenicity.

Bioenergetic Phenotyping of DEN-Induced Hepatocellular Carcinoma Reveals a Link Between Adenylate Kinase Isoform Expression and Reduced Complex I-Supported Respiration.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer worldwide. Increasing evidence suggests that mitochondria play a central role in malignant metabolic reprogramming in HCC, which may promote disease progression. To comprehensively evaluate the mitochondrial phenotype present in HCC, we applied a recently developed diagnostic workflow that combines high-resolution respirometry, fluorometry, and mitochondrial-targeted nLC-MS/MS proteomics to cell culture (AML12 and Hepa 1-6 cells) and diethylnitrosamine (DEN)-induced mouse models of HCC. Across both model systems, CI-linked respiration was significantly decreased in HCC compared to nontumor, though this did not alter ATP production rates. Interestingly, CI-linked respiration was found to be restored in DEN-induced tumor mitochondria through acute in vitro treatment with P1, P5-di(adenosine-5') pentaphosphate (Ap5A), a broad inhibitor of adenylate kinases. Mass spectrometry-based proteomics revealed that DEN-induced tumor mitochondria had increased expression of adenylate kinase isoform 4 (AK4), which may account for this response to Ap5A. Tumor mitochondria also displayed a reduced ability to retain calcium and generate membrane potential across a physiological span of ATP demand states compared to DEN-treated nontumor or saline-treated liver mitochondria. We validated these findings in flash-frozen human primary HCC samples, which similarly displayed a decrease in mitochondrial respiratory capacity that disproportionately affected CI. Our findings support the utility of mitochondrial phenotyping in identifying novel regulatory mechanisms governing cancer bioenergetics.

Scientific and standardization committee communications: classification and nomenclature of disintegrins isolated from snake venoms.

Viper venoms from the Elapidae, Hydrophiidae, Atractaspididae, Viperidae and Colubridae families contain at least 25 separate classes of biologically active compounds, including enzymes and non-enzymatic molecules. Nomenclature standardization of these by structure and function has been reported for prothrombin activators. No such agreement has been made for the nomenclature and classification of disintegrins. At the 51st Annual SSC Meeting in Sydney Australia (August 2005), we reported the variety of names given to the 78 disintegrins then known, and proposed a nomenclature standardization method for the naming of future members in this protein family. The summary of these recommendations will provide guidance as disintegrin discoveries move beyond high pressure liquid chromatography (HPLC) separation methods to mass spectrometry, proteomics and cloned cDNA.

Association between virus-specific T-cell responses and plasma viral load in human immunodeficiency virus type 1 subtype C infection.

Virus-specific T-cell immune responses are important in restraint of human immunodeficiency virus type 1 (HIV-1) replication and control of disease. Plasma viral load is a key determinant of disease progression and infectiousness in HIV infection. Although HIV-1 subtype C (HIV-1C) is the predominant virus in the AIDS epidemic worldwide, the relationship between HIV-1C-specific T-cell immune responses and plasma viral load has not been elucidated. In the present study we address (i) the association between the level of plasma viral load and virus-specific immune responses to different HIV-1C proteins and their subregions and (ii) the specifics of correlation between plasma viral load and T-cell responses within the major histocompatibility complex (MHC) class I HLA supertypes. Virus-specific immune responses in the natural course of HIV-1C infection were analyzed in the gamma interferon (IFN-gamma)-enzyme-linked immunospot assay by using synthetic overlapping peptides corresponding to the HIV-1C consensus sequence. For Gag p24, a correlation was seen between better T-cell responses and lower plasma viral load. For Nef, an opposite trend was observed where a higher T-cell response was more likely to be associated with a higher viral load. At the level of the HLA supertypes, a lower viral load was associated with higher T-cell responses to Gag p24 within the HLA A2, A24, B27, and B58 supertypes, in contrast to the absence of such a correlation within the HLA B44 supertype. The present study demonstrated differential correlations (or trends to correlation) in various HIV-1C proteins, suggesting (i) an important role of the HIV-1C Gag p24-specific immune responses in control of viremia and (ii) more rapid viral escape from immune responses to Nef with no restraint of plasma viral load. Correlations between the level of IFN-gamma-secreting T cells and viral load within the MHC class I HLA supertypes should be considered in HIV vaccine design and efficacy trials.

Magnitude and frequency of cytotoxic T-lymphocyte responses: identification of immunodominant regions of human immunodeficiency virus type 1 subtype C.

A systematic analysis of immune responses on a population level is critical for a human immunodeficiency virus type 1 (HIV-1) vaccine design. Our studies in Botswana on (i) molecular analysis of the HIV-1 subtype C (HIV-1C) epidemic, (ii) frequencies of major histocompatibility complex class I HLA types, and (iii) cytotoxic T-lymphocyte (CTL) responses in the course of natural infection allowed us to address HIV-1C-specific immune responses on a population level. We analyzed the magnitude and frequency of the gamma interferon ELISPOT-based CTL responses and translated them into normalized cumulative CTL responses. The introduction of population-based cumulative CTL responses reflected both (i) essentials of the predominant virus circulating locally in Botswana and (ii) specificities of the genetic background of the Botswana population, and it allowed the identification of immunodominant regions across the entire HIV-1C. The most robust and vigorous immune responses were found within the HIV-1C proteins Gag p24, Vpr, Tat, and Nef. In addition, moderately strong responses were scattered across Gag p24, Pol reverse transcriptase and integrase, Vif, Tat, Env gp120 and gp41, and Nef. Assuming that at least some of the immune responses are protective, these identified immunodominant regions could be utilized in designing an HIV vaccine candidate for the population of southern Africa. Targeting multiple immunodominant regions should improve the overall vaccine immunogenicity in the local population and minimize viral escape from immune recognition. Furthermore, the analysis of HIV-1C-specific immune responses on a population level represents a comprehensive systematic approach in HIV vaccine design and should be considered for other HIV-1 subtypes and/or different geographic areas.

Human immunodeficiency virus type 1 subtype C molecular phylogeny: consensus sequence for an AIDS vaccine design?

An evolving dominance of human immunodeficiency virus type 1 subtype C (HIV-1C) in the AIDS epidemic has been associated with a high prevalence of HIV-1C infection in the southern African countries and with an expanding epidemic in India and China. Understanding the molecular phylogeny and genetic diversity of HIV-1C viruses may be important for the design and evaluation of an HIV vaccine for ultimate use in the developing world. In this study we analyzed the phylogenetic relationships (i) between 73 non-recombinant HIV-1C near-full-length genome sequences, including 51 isolates from Botswana; (ii) between HIV-1C consensus sequences that represent different geographic subsets; and (iii) between specific isolates and consensus sequences. Based on the phylogenetic analyses of 73 near-full-length genomes, 16 "lineages" (a term that is used hereafter for discussion purposes and does not imply taxonomic standing) were identified within HIV-1C. The lineages were supported by high bootstrap values in maximum-parsimony and neighbor-joining analyses and were confirmed by the maximum-likelihood method. The nucleotide diversity between the 73 HIV-1C isolates (mean value of 8.93%; range, 2.9 to 11.7%) was significantly higher than the diversity of the samples to the consensus sequence (mean value of 4.86%; range, 3.3 to 7.2%, P < 0.0001). The translated amino acid distances to the consensus sequence were significantly lower than distances between samples within all HIV-1C proteins. The consensus sequences of HIV-1C proteins accompanied by amino acid frequencies were presented (that of Gag is presented in this work; those of Pol, Vif, Vpr, Tat, Rev, Vpu, Env, and Nef are presented elsewhere [http://www.aids.harvard.edu/lab_research/concensus_sequence.htm]). Additionally, in the promoter region three NF-kappa B sites (GGGRNNYYCC) were identified within the consensus sequences of the entire set or any subset of HIV-1C isolates. This study suggests that the consensus sequence approach could overcome the high genetic diversity of HIV-1C and facilitate an AIDS vaccine design, particularly if the assumption that an HIV-1C antigen with a more extensive match to the circulating viruses is likely to be more efficacious is proven in efficacy trials.

Th2 cytokines and asthma. Interleukin-9 as a therapeutic target for asthma.

Asthma is a complex heritable inflammatory disorder of the airways in which the development of clinical disease depends on environmental exposure. It has been well established that T helper type 2 (TH2) lymphocytes and their cytokines have an important role in allergic asthma. Interleukin (IL)-9, a member of the TH2 cytokine family, has recently been implicated as an essential factor in determining mucosal immunity and susceptibility to atopic asthma. In this review we examine the critical experiments and observations that support this hypothesis. We also discuss these results in comparison with the experiments supporting the involvement of other T H2 cytokines such as IL-4, IL-5 and IL-13.

Characterization of a calcium-activated chloride channel as a shared target of Th2 cytokine pathways and its potential involvement in asthma.

Interleukin (IL)-9 is a T helper (Th) 2 cytokine recently implicated as an essential factor in determining susceptibility to asthma. Transgenic mice overexpressing IL-9 exhibit many features that are characteristic of human asthma. To better understand the mechanism by which IL-9 mediates the various biologic activities in asthma, we performed suppressive subtraction hybridization with whole lung from IL-9 transgenic and control mice. Here we report the identification of mCLCA3, a calcium-activated chloride channel that was specifically induced in the lung epithelium of IL-9 transgenic mice. Expression of mCLCA3 could also be induced by intratracheal administration of IL-9 or other Th2 cytokines (IL-4, IL-13), but not by interferon-gamma. Moreover, expression of mCLCA3 was induced in the lung of antigen-exposed mice, and this induction could be suppressed by neutralizing IL-9 antibody treatment, indicating IL-9 is both necessary and sufficient to induce mCLCA3 in this experimental model of asthma. Finally, we demonstrate that hCLCA1 is the human counterpart to mCLCA3 and is also induced in vitro in human primary lung cells by Th2 cytokine treatment. Together, these data strongly implicate the involvement of mCLCA3 (in mice) and hCLCA1 (in humans) in the pathogenesis of Th2 cytokine-mediated asthmatic disorders.

Identification of human immunodeficiency virus type 1 subtype C Gag-, Tat-, Rev-, and Nef-specific elispot-based cytotoxic T-lymphocyte responses for AIDS vaccine design.

The most severe human immunodeficiency virus type 1 (HIV-1) epidemic is occurring in southern Africa. It is caused by HIV-1 subtype C (HIV-1C). In this study we present the identification and analysis of cumulative cytotoxic T-lymphocyte (CTL) responses in the southern African country of Botswana. CTLs were shown to be an important component of the immune response to control HIV-1 infection. The definition of optimal and dominant epitopes across the HIV-1C genome that are targeted by CTL is critical for vaccine design. The characteristics of the predominant virus that causes the HIV-1 epidemic in a certain geographic area and also the genetic background of the population, through the distribution of common HLA class I alleles, might impact dominant CTL responses in the vaccinee and in the general population. The enzyme-linked immunospot (Elispot) gamma interferon assay has recently been shown to be a reliable tool to map optimal CTL epitopes, correlating well with other methods, such as intracellular staining, tetramer staining, and the classical chromium release assay. Using Elispot with overlapping synthetic peptides across Gag, Tat, Rev, and Nef, we analyzed HIV-1C-specific CTL responses of HIV-1-infected blood donors. Profiles of cumulative Elispot-based CTL responses combined with diversity and sequence consensus data provide an additional characterization of immunodominant regions across the HIV-1C genome. Results of the study suggest that the construction of a poly-epitope subtype-specific HIV-1 vaccine that includes multiple copies of immunodominant CTL epitopes across the viral genome, derived from predominant HIV-1 viruses, might be a logical approach to the design of a vaccine against AIDS.

A spermine-coupled cholesterol metabolite from the shark with potent appetite suppressant and antidiabetic properties.

OBJECTIVE: We describe the pharmacological properties of a novel spermine-cholesterol adduct, MSI 1436 (3beta-N-1(spermine)-7alpha, 24R-dihydroxy-5alpha-cholestane 24-sulfate), which causes reversible suppression of food and fluid intake in mammals resulting in profound weight loss, not associated with other signs or symptoms of illness, and which exhibits antidiabetic properties in genetically obese mice. METHODS: Wild-type rodents and strains with genetic obesity were studied. Effects on food and fluid intake, body weight and composition were examined along with pharmacological and toxicological parameters. RESULTS: MSI-1436 induces profound inhibition of food and fluid intake in rats and mice, resulting in significant weight loss. MSI-1436 is active when introduced directly into the third ventricle of the rat, suggesting the compound acts on central targets. Pair-feeding studies suggest that MSI-1436 causes weight loss by suppressing food intake. Fluid intake is also profoundly reduced but animals remain normally hydrated and defend both water and electrolyte balance from parenteral administration. MSI-1436 is active in ob/ob, db/db, agouti and MC4 receptor knockout mice. MSI-1436 has been administered to ob/ob mice over a 4 month period via a regimen that safely controls body weight, glucose homeostasis and serum cholesterol levels. Following MSI-1436 treatment, db/db mice preferentially mobilize adipose tissue and hyperglycemia is corrected. CONCLUSION: A naturally occurring spermine metabolite of cholesterol, isolated from the dogfish shark, Squalus acanthias, has been identified that induces profound reduction in food and fluid intake in rodents in a setting where thirst is preserved and fluid and electrolyte homeostasis appears to be functioning normally. MSI-1436 probably acts on a central target involving neural circuits that lie downstream from the leptin and the MC4 receptors. Although long-term administration can be accomplished safely in mice, the utility of this compound as a potential human therapeutic awaits an analysis of its pharmacological properties in man.

Squalamine treatment of human tumors in nu/nu mice enhances platinum-based chemotherapies.

Squalamine, an antiangiogenic aminosterol, is presently undergoing Phase II clinical trials in cancer patients. To broaden our understanding of the clinical potential for squalamine, this agent was evaluated in nu/nu mouse xenograft models using the chemoresistant MV-522 human non-small cell lung carcinoma and the SD human neuroblastoma lines. Squalamine was studied alone and in combination with either cisplatin or paclitaxel plus carboplatin. Squalamine alone produced a modest MV-522 tumor growth inhibition (TGI) and yielded a TGI with cisplatin that was better than cisplatin alone. Squalamine also significantly enhanced the activity of paclitaxel/carboplatin combination therapy in the MV-522 tumor model. Squalamine similarly improved the effectiveness of cisplatin in producing TGI when screened against the SD human neuroblastoma xenograft. Xenograft tumor shrinkage was seen for the MV-522 tumor in combination treatments including squalamine, whereas no tumor shrinkage was seen when squalamine was omitted from the treatment regimen. To gain a greater understanding of the mechanism by which squalamine inhibited tumor growth in the xenograft studies, in vitro experiments were carried out with vascular endothelial growth factor-stimulated human umbilical vein endothelial cells in culture exposed to squalamine. Squalamine treatment was found to retard two cellular events necessary for angiogenesis, inducing disorganization of F-actin stress fibers and causing a concomitant reduction of detectable cell the surface molecular endothelial cadherin (VE-cadherin). We propose that the augmentation by squalamine of cytotoxicity from platinum-based therapies is attributable to interference by squalamine with the ability of stimuli to promote endothelial cell movement and cell-cell communication necessary for growth of new blood vessels in xenografts after chemotherapeutic injury to the tumor.

New insights on disintegrin-receptor interactions: eristostatin and melanoma cells.

Viper venom disintegrins have been used frequently to study the cellular receptors which characterize various types of cells, including platelets, endothelial cells and cancer cells. While the majority of such analyses have pointed to involvement of integrin receptors alphavbeta3, alpha5beta1 or alphaIIbbeta3, this may not always be so. Eristostatin, from Eristocophis macmahoni, is a potent inhibitor of ADP-induced platelet aggregation as well as of human and murine melanoma metastases in mouse model systems. This disintegrin requires an RGDW motif, as well as an intact C-terminus, in order to interact with both platelets and four different types of melanoma cells. Eristostatin causes nonmetastatic SBc12 melanoma cells to show higher susceptibility to specific killing by NK-like TALL-104 cells. While it is known that eristostatin binds to alphaIIbbeta3 on platelets, the receptor with which eristostatin binds to the melanoma cells has not yet been identified.

Molecular cloning and biological characterization of full-length HIV-1 subtype C from Botswana.

Human immunodeficiency virus type 1 (HIV-1) subtype C is now responsible for more than half of all HIV-1 infections in the global epidemic and for the high levels of HIV-1 prevalence in southern Africa. To facilitate studies of the biological nature and the underlying molecular determinants of this virus, we constructed eight full-length proviral clones from two asymptomatic and three AIDS patients infected with HIV-1 subtype C from Botswana. Analysis of viral lysates showed that Gag, Pol, and Env structural proteins were present in the virions. In four clones, the analysis suggested inefficient envelope glycoprotein processing. Nucleotide sequence analysis of the eight clones did not reveal frameshifts, deletions, premature truncations, or translational stop codons in any structural, regulatory, or accessory genes. None of the subtype C clones were replication competent in donor peripheral blood mononuclear cells (PBMCs), macrophages, Jurkat(tat) cells, or U87. CD4.CCR5 cells. However, infection by two clones could be rescued by complementation with a functional subtype C envelope clone, resulting in a productive infection of PBMCs, macrophages, and U87. CD4.CCR5 cells.

HIV type 1 A/J recombinant with a pronounced pol gene mosaicism.

A nearly full-length genome sequence of a novel HIV-1 A/J recombinant with a complex structure of the pol gene has been analyzed. This virus was isolated in 1998 from a 35-year-old female from Botswana. The virus demonstrated a dual pattern for CXCR4/CCR5 coreceptor utilization. Using short-term enrichment of the donor's PBMCs, the 98BW21 isolate was long-range amplified, cloned, and sequenced. The sequence of the clone 98BW21.17 spanned 9103 bp from the PBS site to the U5 region of the 3' LTR. The phylogenetic relationship of the 98BW21.17 clone to HIV-1 sequences represented by M, N, and O groups and A-K subtypes of the M group was examined across the entire viral genome. The 98BW21.17 clone demonstrated a unique phylogenetic topology clustering within subtype A or subtype J reference sequences. However, the subtype origin of two regions within the pol gene (p51 RT and integrase) could not be identified. Recombination patterns of the 98BW21.17 clone were different from known AGJ/AGIJ-type viruses such as isolates BFP90 and 95ML84. This study demonstrated the existence and replication competence of a new dual-tropic X4/R5 recombinant form of HIV-1 on the subtype J backbone. The nucleotide sequence of the 98BW21.17 clone was submitted to GenBank under accession number AF192135.

Integrated response of the upper and lower respiratory tract of asthmatic subjects to frigid air.

To evaluate the influence of cold air hyperpnea on integrated upper and lower airway behavior, 22 asthmatic volunteers hyperventilated through their mouths (OHV) and noses (NHV) while pulmonary and nasal function were determined individually and in combination. In the isolated studies, OHV at a minute ventilation of 65 +/- 3 l/min lowered the 1-s forced expiratory volume (FEV(1)) 24 +/- 2% (P < 0. 001) and NHV (40 l/min) induced a 31 +/- 9% (P < 0.001) increase in nasal resistance (NR). In the combined studies, oral hyperpnea reduced the FEV(1) (DeltaFEV(1) 26 +/- 2%, P < 0.001) and evoked a significant rise in NR (DeltaNR 26 +/- 9%, P = 0.01). In contrast, NHV only affected the upper airway. NR rose 33 +/- 9% (P = 0.01), but airway caliber did not change (DeltaFEV(1) 2%, P = 0.27). The results of this investigation demonstrate that increasing the transfer of heat and water in the lower respiratory tract alters bronchial and nasal function in a linked fashion. Forcing the nose to augment its heat-exchanging activity, however, reduces nasal caliber but has no effect on the intrathoracic airways.

EC3, a heterodimeric disintegrin from Echis carinatus, inhibits human and murine alpha4 integrin and attenuates lymphocyte infiltration of Langerhans islets in pancreas and salivary glands in nonobese diabetic mice.

The venom of Echis carinatus suchoreki contains a monomeric disintegrin echistatin (Mr 5,500 Da) that strongly inhibits alphaIIbbeta3, alphavbeta3, and alpha5beta1 integrins and a heterodimeric disintegrin called EC3 (M(r) 14,762 Da). At nanomolar concentration, EC3 inhibits adhesion of human cell lines expressing alpha4beta1 and alpha4beta7 to immobilized VCAM-1; it has a lower inhibitory effect on alpha5beta1-mediated cell adhesion. In this study, we demonstrated that EC3, in contrast to echistatin, inhibited binding of monoclonal anti-alpha4 and anti-alpha5 antibodies to cells expressing alpha4beta7. In a dose-dependent manner and to the same extent, EC3 inhibited adhesion of Jurkat cells and murine splenic lymphocytes to immobilized VCAM-1, whereas echistatin was not active. EC3 injected intraperitoneally into nonobese diabetic (NOD mice) suppressed development of insulitis and sialoadenitis, whereas echistatin had no significant effect. We propose that the effect of EC3 is mediated, at least, in part, by blocking alpha4beta1 and alpha4beta7 on murine lymphocytes.

Structural requirements of echistatin for the recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins.

There are key differences between the amino acid residues of the RGD loops and the C termini of echistatin, a potent antagonist of alpha(IIb)beta(3), alpha(v)beta(3) and alpha(5)beta(1), and eristostatin, a similar disintegrin selectively inhibiting alpha(IIb)beta(3). In order to identify echistatin motifs required for selective recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins, we expressed recombinant echistatin, eristostatin, and 15 hybrid molecules. We tested them for their ability to inhibit adhesion of different cell lines to fibronectin and von Willebrand factor and to express ligand-induced binding site epitope. The results showed that Asp(27) and Met(28) support recognition of both alpha(v)beta(3) and alpha(5)beta(1). Replacement of Met(28) with Asn completely abolished echistatin's ability to recognize each of the integrins, while replacement of Met(28) with Leu selectively decreased echistatin's ability to recognize alpha(5)beta(1) only. Eristostatin in which C-terminal WNG sequence was substituted with HKGPAT exhibited new activity with alpha(5)beta(1), which was 10-20-fold higher than that of wild type eristostatin. A hypothesis is proposed that the C terminus of echistatin interacts with separate sites on beta(1) and beta(3) integrin molecules.