Ethacrynic acid analogues lacking the α,β-unsaturated carbonyl unit—Potential anti-metastatic drugs

A series of ethacrynic acid analogues, lacking the α,β-unsaturated carbonyl unit, was synthesized and subsequently evaluated for their ability to inhibit the migration of human breast cancer cells, MCF-7/AZ. Several of the analogues were already active in the low micromolar range, whereas ethacrynic acid itself shows no potential to inhibit the migration of these cancer cells. Preliminary studies show that the presence of one or more methoxy groups at the phenyl ring of ethacrynic acid is important in order for the ethacrynic acid analogues to demonstrate an inhibitory effect on the migration.     

Selective roles for tumor necrosis factor alpha-converting enzyme/ADAM17 in the shedding of the epidermal growth factor receptor ligand family: the juxtamembrane stalk determines cleavage efficiency

Epidermal growth factor (EGF) family ligands are derived by proteolytic cleavage of the ectodomains of integral membrane precursors. Previously, we established that tumor necrosis factor alpha-converting enzyme (TACE/ADAM17) is a physiologic transforming growth factor-alpha (TGF-alpha) sheddase, and we also demonstrated enhanced shedding of amphiregulin (AR) and heparin-binding (HB)-EGF upon restoration of TACE activity in TACE-deficient EC-2 fibroblasts. Here we extended these results by showing that purified soluble TACE cleaved single sites in the juxtamembrane stalks of mouse pro-HB-EGF and pro-AR ectodomains in vitro. For pro-HB-EGF, this site matched the C terminus of the purified human growth factor, and we speculate that the AR cleavage site is also physiologically relevant. In contrast, ADAM9 and -10, both implicated in HB-EGF shedding, failed to cleave the ectodomain or cleaved at a nonphysiologic site, respectively. Cotransfection of TACE in EC-2 cells enhanced phorbol myristate acetate-induced but not constitutive shedding of epiregulin and had no effect on betacellulin (BTC) processing. Additionally, soluble TACE did not cleave the juxtamembrane stalks of either pro-BTC or pro-epiregulin ectodomains in vitro. Substitution of the shorter pro-BTC juxtamembrane stalk or truncation of the pro-TGF-alpha stalk to match the pro-BTC length reduced TGF-alpha shedding from transfected cells to background levels, whereas substitution of the pro-BTC P2-P2' sequence reduced TGF-alpha shedding less dramatically. Conversely, substitution of the pro-TGF-alpha stalk or lengthening of the pro-BTC stalk, especially when combined with substitution of the pro-TGF-alpha P2-P2' sequence, markedly increased BTC shedding. These results indicate that efficient TACE cleavage is determined by a combination of stalk length and scissile bond sequence.     

Lean and Weight Stable: Behavioral Predictors and Psychological Correlates

Objective: To examine behavioral characteristics associated with being lean and weight stable during adulthood. Research Methods and Procedures: Participants included 165 white married couples who were part of a larger longitudinal study. Participants’ height and weight were measured on three occasions at 2‐year intervals. Men and women were classified as being lean and weight stable (the target group) if they had a BMI < 25 at baseline and maintained their weight within 5% of baseline at 2nd and 4th year follow‐up. Individuals not fulfilling these criteria were included in the comparison group. Group differences in background characteristics including childhood weight status, BMI at each occasion, dieting history, and mental and physical health history were examined. In addition, multiple measures of dietary intake and physical activity were obtained and used to predict the likelihood of being in the target group. Results: Men (N = 22) and women (N = 36) in the target group had lower mean BMI scores at each occasion, were less overweight during childhood, were less likely to have dieted in the past year, and rated themselves as being more healthy than men and women in the comparison group. No differences were identified in mental health. Relative to the comparison group, women in the target group reported higher levels of physical activity and higher levels of physical activity among their spouses, and men in the target group reported healthier dietary patterns. Discussion: Results from this study suggest that being lean and weight stable in adulthood is linked to childhood weight status in combination with dietary and activity patterns during adulthood.     

Protein trafficking in Plasmodium falciparum-infected red blood cells

Plasmodium falciparum inhabits a niche within the most highly terminally differentiated cell in the human body--the mature red blood cell. Life inside this normally quiescent cell offers the parasite protection from the host's immune system, but provides little in the way of cellular infrastructure. To survive and replicate in the red blood cell, the parasite exports proteins that interact with and dramatically modify the properties of the host red blood cell. As part of this process, the parasite appears to establish a system within the red blood cell cytosol that allows the correct trafficking of parasite proteins to their final cellular destinations. In this review, we examine recent developments in our understanding of the pathways and components involved in the delivery of important parasite-encoded proteins to their final destination in the host red blood cell. These complex processes are not only fundamental to the survival of malaria parasites in vivo, but are also major determinants of the unique pathogenicity of this parasite.     

MAHRP-1, a novel Plasmodium falciparum histidine-rich protein,binds ferriprotoporphyrin IX and localizes to the Maurer's clefts

Using a stage-specific cDNA library from Plasmodium falciparum we have identified a gene coding for a novel histidine-rich protein (MAHRP-1). The gene is exclusively transcribed during early erythrocyte stages and codes for a small transmembrane protein. The C-terminal region contains a polymorphic stretch of histidine-rich repeats. Fluorescence microscopy studies using polyclonal mouse sera revealed that MAHRP-1 is located at the Maurer's clefts, which represent parasite-induced structures within the cytosol of infected erythrocytes. Biochemical studies showed that recombinant MAHRP-1 binds the toxic hemoglobin degradation product, ferriprotoporphyrin (FP) with a submicromolar dissociation constant and a stoichiometry determined by the number of DHGH motifs. The bound FP has increased peroxidase-like activity and is 10-fold more susceptible to H2O2-induced degradation compared with unbound FP. These properties of MAHRP-1 suggest it may play a protective role against oxidative stress, and its location at the Maurer's clefts suggests a function in promoting the correct trafficking of exported proteins, such as P. falciparum erythrocyte membrane protein-1.     

Structures of lipopolysaccharides from Klebsiella pneumoniae. Eluicidation of the structure of the linkage region between core and polysaccharide O chain and identification of the residues at the non-reducing termini of the O chains

Deamination of LPSs from Klebsiella pneumoniae released O-chain polysaccharides together with a fragment of the core oligosaccharide. The structures of the products from serotypes O1, O2a, O2a,c, O3, O4, O5, and O12 were determined by NMR spectroscopy and chemical methods, identifying the linkage region between the O antigens and the core as well as novel residues at the non-reducing ends of the polysaccharides. All serotypes had an identical linkage between the O chain and core.     

Tumor necrosis factor-alpha converting enzyme (TACE) regulates epidermal growth factor receptor ligand availability

We previously implicated tumor necrosis factor-alpha converting enzyme (TACE/ADAM17) in the processing of the integral membrane precursor to soluble transforming growth factor-alpha (TGF-alpha), pro-TGF-alpha. Here we examined TGF-alpha processing in a physiologically relevant cell model, primary keratinocytes, showing that cells lacking TACE activity shed dramatically less TGF-alpha as compared with wild-type cultures and that TGF-alpha cleavage was partially restored by infection of TACE-deficient cells with TACE-encoding adenovirus. Moreover, cotransfection of TACE-deficient fibroblasts with pro-TGF-alpha and TACE cDNAs increased shedding of mature TGF-alpha with concomitant conversion of cell-associated pro-TGF-alpha to a processed form. Purified TACE accurately cleaved pro-TGF-alpha in vitro at the N-terminal site and also cleaved a soluble form of pro-TGF-alpha containing only the ectodomain at the C-terminal site. In vitro, TACE accurately cleaved peptides corresponding to cleavage sites of several epidermal growth factor (EGF) family members, and transfection of TACE into TACE-deficient cells increased the shedding of amphiregulin and heparin-binding EGF (HB-EGF) proteins. Consistent with the hypothesis that TACE regulates EGF receptor (EGFR) ligand availability in vivo, mice heterozygous for Tace and homozygous for an impaired EGFR allele (wa-2) were born with open eyes significantly more often than Tace(+/+)Egfr(wa-2)(/)(wa-2) counterparts. Collectively, these data support a broad role for TACE in the regulated shedding of EGFR ligands.     

Role of EP(2) and EP(3) PGE(2) receptors in control of murine renal hemodynamics

The kidney plays a central role in long-term regulation of arterial blood pressure and salt and water homeostasis. This is achieved in part by the local actions of paracrine and autacoid mediators such as the arachidonic acid-prostanoid system. The present study tested the role of specific PGE(2) E-prostanoid (EP) receptors in the regulation of renal hemodynamics and vascular reactivity to PGE(2). Specifically, we determined the extent to which the EP(2) and EP(3) receptor subtypes mediate the actions of PGE(2) on renal vascular tone. Renal blood flow (RBF) was measured by ultrasonic flowmetry, whereas vasoactive agents were injected directly into the renal artery of male mice. Studies were performed on two independent mouse lines lacking either EP(2) or EP(3) (-/-) receptors and the results were compared with wild-type controls (+/+). Our results do not support a unique role of the EP(2) receptor in regulating overall renal hemodynamics. Baseline renal hemodynamics in EP(2)-/- mice [RBF EP(2)-/-: 5.3 +/- 0.8 ml. min(-1). 100 g kidney wt(-1); renal vascular resistance (RVR) 19.7 +/- 3.6 mmHg. ml(-1). min. g kidney wt] did not differ statistically from control mice (RBF +/+: 4.0 +/- 0.5 ml. min(-1). 100 g kidney wt(-1); RVR +/+: 25.4 +/- 4.9 mmHg. ml(-1). min. 100 g kidney wt(-1)). This was also the case for the peak RBF increase after local PGE(2) (500 ng) injection into the renal artery (EP(2)-/-: 116 +/- 4 vs. +/+: 112 +/- 2% baseline RBF). In contrast, we found that the absence of EP(3) receptors in EP(3)-/- mice caused a significant increase (43%) in basal RBF (7.9 +/- 0.8 ml. min(-1). g kidney wt(-1), P < 0.05 vs. +/+) and a significant decrease (41%) in resting RVR (11.6 +/- 1.4 mmHg. ml(-1). min. g kidney wt(-1), P < 0.05 vs. +/+). Local administration of 500 ng of PGE(2) into the renal artery caused more pronounced renal vasodilation in EP(3)-/- mice (128 +/- 2% of basal RBF, P < 0.05 vs. +/+). We conclude that EP(3 )receptors mediate vasoconstriction in the kidney of male mice and its actions are tonically active in the basal state. Furthermore, EP(3) receptors are capable of buffering PGE(2)-mediated renal vasodilation.     

Antibody-Dependent Cellular Cytotoxicity Directed against Cells Expressing Human Immunodeficiency Virus Type 1 Envelope of Primary or Laboratory-Adapted Strains by Human and Chimpanzee Monoclonal Antibodies of Different Epitope Specificities

The characteristics of antibody-dependent cellular cytotoxicity (ADCC) directed by a panel of human and chimpanzee antienvelope (anti-Env) monoclonal antibodies (MAbs) of different epitope specificities were studied; this was accomplished by using target cells expressing human immunodeficiency virus type 1 (HIV-1) Envs of either primary or laboratory-adapted strains. Human MAbs of similar apparent affinities (1 × 10⁹ to 2 × 10⁹ liters/mol) against either a “cluster II”-overlapping epitope of gp41 or against the CD4 binding site, V3 loop, or C5 domain of gp120 directed substantial and comparable levels of specific lysis against targets infected with laboratory-adapted strains of HIV-1. As expected, those MAbs specific for relatively conserved regions of Env generally exhibited ADCC activity against a broader range of HIV-1 strains than those directed against variable epitopes. Significant ADCC activities of selected MAbs against primary isolate Env-expressing cells were demonstrated. In addition, a new ADCC epitope in the V2 domain of gp120 was defined. CD56⁺ cells were demonstrated to be the effector cells in these studies by fluorescence-activated cell sorting followed by ADCC assays. Notably, all anti-Env MAbs tested in this study, including MAbs directed against each of the known neutralization epitope clusters in gp120, directed significant levels of ADCC against targets expressing Env of one or more HIV-1 strains. These results imply that many, if not most, HIV-1-neutralizing human Abs of high affinity (≥3 × 10⁸ liters/mol in these studies) and of the immunoglobulin G1 (IgG1) subclass (i.e., the predominate IgG subclass) are capable of directing ADCC. Since neutralizing Abs have been associated with long-term survival following HIV-1 infection, this suggests that ADCC activity may be beneficial in vivo.The in vivo role(s) of antibodies (Abs) that can direct antibody-dependent cellular cytotoxicity (ADCC) against human immunodeficiency virus type 1 (HIV-1) Env-expressing cells in vitro remains unclear. In ADCC, anti-Env Abs direct effector cells to kill target cells bearing HIV-1 envelope on their surfaces; this is accomplished via specific binding of the Abs’ antigen-binding sites to Envs and their Fc regions to Fc receptors on the effector cells. Broadly strain reactive, ADCC-directing Abs arise early in the immune response to HIV-1 infection in vivo (14) and may be partially responsible for the initial clearance of viremia.Earlier in the HIV-1 epidemic, concerns were raised that shed soluble gp120 in HIV-1-infected individuals might bind to CD4⁺ cells, including uninfected ones, and could target these cells for “innocent bystander” killing by ADCC (6). However, effector cells armed with serum Abs able to direct ADCC in vitro against either innocent bystanders or HIV-1-infected cells were found at highest frequency in asymptomatic, seropositive individuals; patients with AIDS-related complex and AIDS showed progressively diminished reactivities (20). Furthermore, in a recent study (1), the ability of monoclonal Abs (MAbs) against three distinct gp120 epitopes to direct ADCC against uninfected CD4⁺ cells to which rgp120_SF2 had been adsorbed (i.e., innocent bystanders) was demonstrated to be less efficient by at least an order of magnitude than their ability to direct ADCC against HIV-1-infected cells.The existing data from in vivo studies (reviewed in reference 1) supports the efficacy, rather than the pathogenicity, of ADCC-directing Abs against HIV-1. Consistent with this data is our recent characterization of two MAbs, 42F and 43F, isolated from a long-term survivor of HIV-1 infection (1); these MAbs directed significant levels of ADCC and defined a new, conserved ADCC epitope in the C5 domain of HIV-1 gp120. Preliminary evidence indicated that concentrations of 42F- and 43F-like Abs in the serum of the donor were in the range required to direct high levels of ADCC, and these MAbs were shown to bind both oligomeric primary-isolate and laboratory-adapted Env efficiently (1).Because of the potential importance of ADCC-directing Abs against HIV-1, in this study we have evaluated ADCC directed against cells expressing HIV-1 Envs of primary or laboratory-adapted strains by a panel of human and chimpanzee anti-Env MAbs of different epitope specificities. Significant ADCC activities of selected MAbs against primary-isolate Env-expressing cells were demonstrated, and a new ADCC epitope in the V2 domain of gp120 was defined. Finally, a MAb’s ability to direct ADCC against a specific target cell type was shown to be dependent on additional factors beyond its ability to efficiently bind antigen on the target cell and its possession of an Fc region of the appropriate isotype to engage FcγR on effector cells.