Search tips
Search criteria

Results 1-25 (3706)

Clipboard (0)

Select a Filter Below

more »
Year of Publication
more »
1.  PTEN Is a Negative Regulator of NK Cell Cytolytic Function 
Human NK cells are characterized by their ability to initiate an immediate and direct cytolytic response to virally infected or malignantly transformed cells. Within human peripheral blood, the more mature CD56dim NK cell efficiently kills malignant targets at rest, whereas the less mature CD56bright NK cells cannot. In this study, we show that resting CD56bright NK cells express significantly more phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein when compared with CD56dim NK cells. Consistent with this, forced overexpression of PTEN in NK cells resulted in decreased cytolytic activity, and loss of PTEN in CD56bright NK cells resulted in elevated cytolytic activity. Comparable studies in mice showed PTEN overexpression did not alter NK cell development or NK cell–activating and inhibitory receptor expression yet, as in humans, did decrease expression of downstream NK activation targets MAPK and AKT during early cytolysis of tumor target cells. Confocal microscopy revealed that PTEN overexpression disrupts the NK cell’s ability to organize immunological synapse components including decreases in actin accumulation, polarization of the microtubule organizing center, and the convergence of cytolytic granules. In summary, our data suggest that PTEN normally works to limit the NK cell’s PI3K/AKT and MAPK pathway activation and the consequent mobilization of cytolytic mediators toward the target cell and suggest that PTEN is among the active regulatory components prior to human NK cells transitioning from the noncytolytic CD56bright NK cell to the cytolytic CD56dim NK cells.
PMCID: PMC4319309  PMID: 25595786
2.  Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation 
British Journal of Cancer  2013;110(1):71-82.
Heat shock protein 90 (HSP90) has a key role in the maintenance of the cellular proteostasis. However, HSP90 is also involved in stabilisation of oncogenic client proteins and facilitates oncogene addiction and cancer cell survival. The development of HSP90 inhibitors for cancer treatment is an area of growing interest as such agents can affect multiple pathways that are linked to all hallmarks of cancer. This study aimed to test the hypothesis that targeting cysteine residues of HSP90 will lead to degradation of client proteins and inhibition of cancer cell proliferation.
Combining chemical synthesis, biological evaluation, and structure–activity relationship analysis, we identified a new class of HSP90 inhibitors. Click chemistry and protease-mass spectrometry established the sites of modification of the chaperone.
The mildly electrophilic sulphoxythiocarbamate alkyne (STCA) selectively targets cysteine residues of HSP90, forming stable thiocarbamate adducts. Without interfering with the ATP-binding ability of the chaperone, STCA destabilises the client proteins RAF1, HER2, CDK1, CHK1, and mutant p53, and decreases proliferation of breast cancer cells. Addition of a phenyl or a tert-butyl group in tandem with the benzyl substituent at nitrogen increased the potency. A new compound, S-4, was identified as the most robust HSP90 inhibitor within a series of 19 derivatives.
By virtue of their cysteine reactivity, sulphoxythiocarbamates target HSP90, causing destabilisation of its client oncoproteins and inhibiting cell proliferation.
PMCID: PMC3887302  PMID: 24322890
Hsp90; NRF2; cysteine; HSF1
3.  The Anti-HIV Microbicide Candidate RC-101 Inhibits Pathogenic Vaginal Bacteria Without Harming Endogenous Flora or Mucosa 
Vaginal microbicides represent a promising approach for preventing heterosexual HIV transmission. However, preclinical evaluation should be conducted to ensure that microbicides will be safe for human cells and healthy microflora of the female reproductive tract. One microbicide candidate, RC-101, has been effective and well-tolerated in preliminary cell culture and macaque models. However, the effect of RC-101 on primary vaginal tissues and resident vaginal microflora requires further evaluation.
Method of Study
We treated primary vaginal tissues and vaginal bacteria, both pathogenic and commensal, with RC-101 to investigate effects of this microbicide.
RC-101 was well-tolerated by host tissues, and also by commensal vaginal bacteria. Simultaneously, pathogenic vaginal bacteria, which are known to increase susceptibility to HIV acquisition, were inhibited by RC-101.
By establishing vaginal microflora, the specific antibacterial activity of RC-101 may provide a dual mechanism of HIV protection. These findings support advancement of RC-101 to clinical trials.
PMCID: PMC3541468  PMID: 23167830
Bacterial Vaginosis; Lactobacilli; RC-101; Theta-Defensin; Vaginal Microbicide
4.  A Compensatory Mutation Provides Resistance to Disparate HIV Fusion Inhibitor Peptides and Enhances Membrane Fusion 
PLoS ONE  2013;8(2):e55478.
Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide, are peptides designed to competitively inhibit the viral fusion protein gp41. With the emergence of drug resistance, there is an increased need for effective and unique alternatives within this class of antivirals. One such alternative is a class of cyclic, cationic, antimicrobial peptides known as θ-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the θ-defensin analog RC-101 is being developed as a microbicide due to its specific antiviral activity, lack of toxicity to cells and tissues, and safety in animals. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development. In previous studies, we identified a mutant, R5-tropic virus that had evolved partial resistance to RC-101 during in vitro selection. Here, we report that a secondary mutation in gp41 was found to restore replicative fitness, membrane fusion, and the rate of viral entry, which were compromised by an initial mutation providing partial RC-101 resistance. Interestingly, we show that RC-101 is effective against two enfuvirtide-resistant mutants, demonstrating the clinical importance of RC-101 as a unique fusion inhibitor. These findings both expand our understanding of HIV drug-resistance to diverse peptide fusion inhibitors and emphasize the significance of compensatory gp41 mutations.
PMCID: PMC3564752  PMID: 23393582
5.  Identification and Characterization of Bacterial Vaginosis-Associated Pathogens Using a Comprehensive Cervical-Vaginal Epithelial Coculture Assay 
PLoS ONE  2012;7(11):e50106.
Bacterial vaginosis (BV) is the most commonly treated female reproductive tract affliction, characterized by the displacement of healthy lactobacilli by an overgrowth of pathogenic bacteria. BV can contribute to pathogenic inflammation, preterm birth, and susceptibility to sexually transmitted infections. As the bacteria responsible for BV pathogenicity and their interactions with host immunity are not understood, we sought to evaluate the effects of BV-associated bacteria on reproductive epithelia. Here we have characterized the interaction between BV-associated bacteria and the female reproductive tract by measuring cytokine and defensin induction in three types of FRT epithelial cells following bacterial inoculation. Four BV-associated bacteria were evaluated alongside six lactobacilli for a comparative assessment. While responses differed between epithelial cell types, our model showed good agreement with clinical BV trends. We observed a distinct cytokine and human β-defensin 2 response to BV-associated bacteria, especially Atopobium vaginae, compared to most lactobacilli. One lactobacillus species, Lactobacillus vaginalis, induced an immune response similar to that elicited by BV-associated bacteria, stimulating significantly higher levels of cytokines and human β-defensin 2 than other lactobacilli. These data provide an important prioritization of BV-associated bacteria and support further characterization of reproductive bacteria and their interactions with host epithelia. Additionally, they demonstrate the distinct immune response potentials of epithelial cells from different locations along the female reproductive tract.
PMCID: PMC3499514  PMID: 23166828
6.  [F-18]FDDNP microPET imaging correlates with brain Aβ burden in a transgenic rat model of Alzheimer disease: Effects of aging, in vivo blockade, and anti-Aβ antibody treatment 
Neurobiology of disease  2011;43(3):565-575.
In vivo detection of Alzheimer's disease (AD) neuropathology in living patients using positron emission tomography (PET) in conjunction with high affinity molecular imaging probes for β-amyloid (Aβ) and tau has the potential to assist with early diagnosis, evaluation of disease progression, and assessment of therapeutic interventions. Animal models of AD are valuable for exploring the in vivo binding of these probes, particularly their selectivity for specific neuropathologies, but prior PET experiments in transgenic mice have yielded conflicting results. In this work, we utilized microPET imaging in a transgenic rat model of brain Aβ deposition to assess [F-18]FDDNP binding profiles in relation to age-associated accumulation of neuropathology. Cross-sectional and longitudinal imaging demonstrated that [F-18]FDDNP binding in the hippocampus and frontal cortex progressively increases from 9 to 18 months of age and parallels age-associated Aβ accumulation. Specificity of in vivo [F-18]FDDNP binding was assessed by naproxen pretreatment, which reversibly blocked [F-18]FDDNP binding to Aβ aggregrates. Both [F-18]FDDNP microPET imaging and neuropathological analyses revealed decreased Aβ burden after intracranial anti-Aβ antibody administration. The combination of this non-invasive imaging method and robust animal model of brain Aβ accumulation allows for future longitudinal in vivo assessments of potential therapeutics for AD that target Aβ production, aggregation, and/or clearance. These results corroborate previous analyses of [F-18]FDDNP PET imaging in clinical populations.
PMCID: PMC3144750  PMID: 21605674
[F-18]FDDNP; positron emission tomography; amyloid; transgenic rat; naproxen; immunotherapy
7.  Characterization of the Retrocyclin Analogue RC-101 as a Preventative of Staphylococcus aureus Nasal Colonization▿ 
Antimicrobial Agents and Chemotherapy  2011;55(11):5338-5346.
Nasal colonization of Staphylococcus aureus is a risk factor for pathogenic autoinfection, particularly in postoperative patients and the immunocompromised. As such, standardized preoperative nasal decolonization of S. aureus has become a major consideration for the prevention of nosocomial infection. However, only a few treatment options for nasal decolonization are currently available, with resistance to these approaches already a concern. Here we have identified the macrocyclic θ-defensin analogue RC-101 as a promising anti-S. aureus agent for nasal decolonization. RC-101 exhibits bactericidal effects against S. aureus with the use of in vitro epithelium-free systems, while also preventing the pathogen's proliferation and attachment in an ex vivo human nasal epithelial cell adhesion model and an organotypic model of human airway epithelia. Peptide concentrations as low as 2.5 μM elicited significant reductions in S. aureus growth in epithelium-free systems, with 10 μM concentrations being completely bactericidal for all strains tested, including USA300. In ex vivo nasal colonization models, RC-101 significantly reduced adherence, survival, and proliferation of S. aureus on human nasal epithelia. Reductions in S. aureus viability were evident in these assays, with as little as 1 μg of peptide per tissue, while 10 μg of RC-101 completely prevented adhesion of all strains tested. Furthermore, RC-101 did not exhibit cellular toxicity to human nasal epithelia at concentrations up to 200 μM, nor did it induce a proinflammatory response in these cells. Collectively, the findings of this study identify RC-101 as a potential preventative of S. aureus nasal colonization.
PMCID: PMC3195007  PMID: 21825301
8.  Exoproteome of Staphylococcus aureus reveals putative determinants of nasal carriage 
Journal of proteome research  2011;10(4):2064-2078.
Due to the increasing prevalence of nosocomial and community-acquired antibiotic resistant Staphylococcus aureus (SA), understanding the determinants of SA nasal carriage has become a major imperative. Previous research has revealed many host and bacterial factors that contribute to SA nasal carriage. To assess bacterial factors that facilitate nasal carriage, we compared the exoproteome of a nasal carrier strain of SA to a genetically similar non-carrier strain. Additionally, the carrier strain biofilm exoproteome was also compared against its planktonic counterpart. Using high throughput proteomics, it was observed that the carrier strain of SA secretes a greater number of proteins that may promote successful colonization of the human nose, including cell attachment and immunoevasive proteins, than the non-carrier strain. Similarly, SA carrier strain biofilm exoproteome contains a greater number of immunoevasive proteins than its planktonic counterpart. Analysis of the most abundant immunoevasive proteins revealed that Staphylococcal protein A was present at significantly higher levels in carrier than in non-carrier strains of SA, suggesting an association with nasal carriage. While further analyses of specific differences between carrier and non-carrier strains of SA are required, many of the differentially expressed proteins identified can be considered to be putative determinants of nasal carriage.
PMCID: PMC3070068  PMID: 21338050
Innate immunity; bacteria/bacterial immunity; comparative proteomics; nasal colonization; exoproteome
9.  HIV-Neutralizing Activity of Cationic Polypeptides in Cervicovaginal Secretions of Women in HIV-Serodiscordant Relationships 
PLoS ONE  2012;7(2):e31996.
HIV exposed seronegative (HESN) women represent the population most in need of a prophylactic antiviral strategy. Mucosal cationic polypeptides can potentially be regulated for this purpose and we here aimed to determine their endogenous expression and HIV neutralizing activity in genital secretions of women at risk of HIV infection.
Methodology/Principal Findings
Cervicovaginal secretions (CVS) of Kenyan women in HIV-serodiscordant relationships (HESN, n = 164; HIV seropositive, n = 60) and low-risk controls (n = 72) were assessed for the cationic polypeptides HNP1–3, LL-37 and SLPI by ELISA and for HIV neutralizing activity by a PBMC-based assay using an HIV primary isolate. Median levels of HNP1–3 and LL-37 in CVS were similar across study groups. Neither HSV-2 serostatus, nor presence of bacterial vaginosis, correlated with levels of HNP1–3 or LL-37 in the HESN women. However, an association with their partner's viral load was observed. High viral load (>10,000 HIV RNA copies/ml plasma) correlated with higher levels of HNP1–3 and LL-37 (p = 0.04 and 0.03, respectively). SLPI was most abundant in the low-risk group and did not correlate with male partner's viral load in the HESN women. HIV neutralizing activity was found in CVS of all study groups. In experimental studies, selective depletion of cationic polypeptides from CVS rendered the remaining CVS fraction non-neutralizing, whereas the cationic polypeptide fraction retained the activity. Furthermore, recombinant HNP1–3 and LL-37 could induce neutralizing activity when added to CVS lacking intrinsic activity.
These findings show that CVS from HESN, low-risk, and HIV seropositive women contain HIV neutralizing activity. Although several innate immune proteins, including HNP1–3 and LL-37, contribute to this activity these molecules can also have inflammatory properties. This balance is influenced by hormonal and environmental factors and in the present HIV serodiscordant couple cohort study we show that a partner's viral load is associated with levels of such molecules.
PMCID: PMC3289637  PMID: 22389677
10.  Nanoscopy – Pushing the Limits of Light Microscopy 
Nanoscopy has been a rapidly expanding field over the last 5 years. Different modalities have been developed based on optical, computational and combined optical/computational techniques that can be used to circumvent the diffraction barrier of visible light. Stimulated emission depletion (STED) microscopy is a purely optical technique that was originally developed by Stephane Hell and has been recently commercialized by Leica Microsystems. This technique uses two superimposed laser beams. One beam excites a point of fluorescence while the second beam – a donut shape – depletes the fluorescence emission around the spot through stimulated emission. The result is a point source of excitation which can be scanned across a fluorescence sample giving resolutions on the order of 20–40 nm. Single molecule localization microscopy is a purely computation technique that comes under many flavors (PALM, F-PALM, STORM, dSTORM). Each technique relies on the repetitive excitation and localization of a small subset of fluorescent molecules within a sample. Single molecules are imaged as Gaussian spots and with sufficient signal-to-noise can each be fit with a precision approaching 20 nm. Finally, structured illumination uses a combination of optics and image processing. Grid patterns are super-imposed on fluorescent samples with 15–25 different angles and z-axis positions. The result is a translation of high frequency sample information into low frequency Moire patterns. The 15–25 images are post processed based on the imposed patterns to de-convolve out fine sample details with at least twice the resolution limit of a conventional light microscope. This overview talk will introduce these various nanoscopy techniques.
PMCID: PMC3630589
11.  Formulation Development of Retrocyclin 1 Analog RC-101 as an Anti-HIV Vaginal Microbicide Product▿ 
RC-101 is a synthetic microbicide analog of retrocyclin, which has shown in vitro activity against X4 and R5 HIV-1. In an effort to develop a safe and effective RC-101 vaginal microbicide product, we assessed safety in ex vivo macaque and human models and efficacy using in vitro and ex vivo models. A polyvinyl-alcohol vaginal film containing RC-101 (100 μg/film) was developed. Formulation assessment was conducted by evaluating disintegration, drug content, mechanical properties, and stability. Efficacy was evaluated by in vitro peripheral blood mononuclear cells (PBMC) assay and ex vivo human ectocervical tissue explant model. Ex vivo safety studies were conducted by exposing RC-101 to an excised monkey reproductive tract and excised human ectocervical tissue. RC-101 100 μg films were shown to be safe to human and monkey tissue and effective against HIV-1 in vitro and ex vivo in human ectocervical tissue. The 90% inhibitory concentration (IC90) for RC-101 films at 2,000 μg (IC90 = 57.5 μM) using an ex vivo model was 10-fold higher than the IC90 observed using an in vitro model (IC90 = 5.0 μM). RC-101 films were stable for 1 month at 25°C, with in vitro bioactivity maintained for up to 6 months. RC-101 was developed in a quick-dissolve film formulation that was shown to be safe in an ex vivo model and effective in in vitro and ex vivo models. RC-101 film formulations were shown to maintain bioactivity for a period of 6 months. Findings from the present study contribute to the development of a safe and effective topical microbicide product.
PMCID: PMC3088226  PMID: 21321138
12.  Site-specific introduction of an acetyl-lysine mimic into peptides and proteins by cysteine alkylation 
Protein acetylation on Lys residues is recognized as a significant post-translational modification in cells, but it is often difficult to discern the direct structural and functional effects of individual acetylation events. Here we describe a new tool, methylthiocarbonyl-aziridine, to install acetyl-Lys mimics site-specifically into peptides and proteins by alkylation of Cys residues. We demonstrate that the resultant thiocarbamate modification can be recognized by the Brdt bromodomain and site-specific anti-acetyl-Lys antibodies, is resistant to histone deacetylase cleavage, and can confer activation of the histone acetyltransferase Rtt109 by simulating autoacetylation. We also use this approach to obtain functional evidence that acetylation of CK2 protein kinase on Lys102 can stimulate its catalytic activity.
PMCID: PMC2912447  PMID: 20608637
13.  HIV-1 Enhancing Effect of Prostatic Acid Phosphatase Peptides Is Reduced in Human Seminal Plasma 
PLoS ONE  2011;6(1):e16285.
We recently reported that HIV-1 infection can be inhibited by innate antimicrobial components of human seminal plasma (SP). Conversely, naturally occurring peptidic fragments from the SP-derived prostatic acid phosphatase (PAP) have been reported to form amyloid fibrils called “SEVI” and enhance HIV-1 infection in vitro. In order to understand the biological consequence of this proviral effect, we extended these studies in the presence of human SP. PAP-derived peptides were agitated to form SEVI and incubated in the presence or absence of SP. While PAP-derived peptides and SEVI alone were proviral, the presence of 1% SP ablated their proviral activity in several different anti-HIV-1 assays. The anti-HIV-1 activity of SP was concentration dependent and was reduced following filtration. Supraphysiological concentrations of PAP peptides and SEVI incubated with diluted SP were degraded within hours, with SP exhibiting proteolytic activity at dilutions as high as 1∶200. Sub-physiological concentrations of two prominent proteases of SP, prostate-specific antigen (PSA) and matriptase, could degrade physiological and supraphysiological concentrations of PAP peptides and SEVI. While human SP is a complex biological fluid, containing both antiviral and proviral factors, our results suggest that PAP peptides and SEVI may be subject to naturally occurring proteolytic components capable of reducing their proviral activity.
PMCID: PMC3024420  PMID: 21283773
14.  Grifonin-1: A Small HIV-1 Entry Inhibitor Derived from the Algal Lectin, Griffithsin 
PLoS ONE  2010;5(12):e14360.
Griffithsin, a 121-residue protein isolated from a red algal Griffithsia sp., binds high mannose N-linked glycans of virus surface glycoproteins with extremely high affinity, a property that allows it to prevent the entry of primary isolates and laboratory strains of T- and M-tropic HIV-1. We used the sequence of a portion of griffithsin's sequence as a design template to create smaller peptides with antiviral and carbohydrate-binding properties.
The new peptides derived from a trio of homologous β-sheet repeats that comprise the motifs responsible for its biological activity. Our most active antiviral peptide, grifonin-1 (GRFN-1), had an EC50 of 190.8±11.0 nM in in vitro TZM-bl assays and an EC50 of 546.6±66.1 nM in p24gag antigen release assays. GRFN-1 showed considerable structural plasticity, assuming different conformations in solvents that differed in polarity and hydrophobicity. Higher concentrations of GRFN-1 formed oligomers, based on intermolecular β-sheet interactions. Like its parent protein, GRFN-1 bound viral glycoproteins gp41 and gp120 via the N-linked glycans on their surface.
Its substantial antiviral activity and low toxicity in vitro suggest that GRFN-1 and/or its derivatives may have therapeutic potential as topical and/or systemic agents directed against HIV-1.
PMCID: PMC3002932  PMID: 21179548
15.  The Formulated Microbicide RC-101 Was Safe and Antivirally Active Following Intravaginal Application in Pigtailed Macaques 
PLoS ONE  2010;5(11):e15111.
RC-101 is a congener of the antiretroviral peptide retrocyclin, which we and others have reported is active against clinical HIV-1 isolates from all major clades, does not hemagglutinate, and is non-toxic and non-inflammatory in cervicovaginal cell culture. Herein, film-formulated RC-101 was assessed for its antiviral activity in vitro, safety in vivo, retention in the cervix and vagina, and ability to remain active against HIV-1 and SHIV after intravaginal application in macaques.
Methodology/Principal Findings
RC-101 was formulated as a quick-dissolving film (2000 µg/film), retained complete activity in vitro as compared to unformulated peptide, and was applied intravaginally in six pigtailed macaques daily for four days. At one and four days following the final application, the presence of RC-101 was assessed in peripheral blood, cervicovaginal lavage, cytobrushed cervicovaginal cells, and biopsied cervical and vaginal tissues by quantitative western blots. One day following the last film application, cervical biopsies from RC-101-exposed and placebo-controlled macaques were collected and were subjected to challenge with RT-SHIV in an ex vivo organ culture model. RC-101 peptide was detected primarily in the cytobrush and biopsied cervical and vaginal tissues, with little to no peptide detected in lavage samples, suggesting that the peptide was associated with the cervicovaginal epithelia. RC-101 remained in the tissues and cytobrush samples up to four days post-application, yet was not detected in any sera or plasma samples. RC-101, extracted from cytobrushes obtained one day post-application, remained active against HIV-1 BaL. Importantly, cervical biopsies from RC-101-treated animals reduced RT-SHIV replication in ex vivo organ culture as compared to placebo-treated animals.
Formulated RC-101 was stable in vivo and was retained in the mucosa. The presence of antivirally active RC-101 after five days in vivo suggests that RC-101 would be an important molecule to develop further as a topical microbicide to prevent HIV-1 transmission.
PMCID: PMC2993972  PMID: 21124745
16.  In Vitro Enzymatic Characterization of Near Full Length EGFR in Activated and Inhibited States 
Biochemistry  2009;48(28):6624-6632.
The epidermal growth factor receptor (EGFR) is a single-pass transmembrane protein with an extracellular ligand-binding region and a cytoplasmic tyrosine kinase. Ligand binding activates the tyrosine kinase, which in turn initiates signaling cascades that influence cell proliferation and differentiation. EGFR activity is essential for normal development of many multicellular organisms, and inappropriate activation of EGFR is associated with multiple human cancers. Several drugs targeting EGFR activity are approved cancer therapies, and new EGFR-targeted therapies are being actively pursued. Much of what is known about EGFR structure and function is derived from studies of soluble receptor fragments. We report here development of an approach to producing an active, membrane-spanning form of EGFR of suitable purity, homogeneity, and quantity for structural and functional studies. We show that EGFR is capable of direct autophosphorylation of tyrosine 845, which is located on its kinase activation loop, and that the kinase activity of EGFR is ~500-fold higher in the presence of EGF vs. the inhibitory anti-EGFR antibody Cetuximab. The potencies of the small molecule EGFR kinase inhibitors erlotinib and lapatinib for various forms of EGFR were measured, and the therapeutic and mechanistic implications of these results considered.
PMCID: PMC2747374  PMID: 19518076
17.  Reawakening Retrocyclins: Ancestral Human Defensins Active Against HIV-1 
PLoS Biology  2009;7(4):e1000095.
Human alpha and beta defensins contribute substantially to innate immune defenses against microbial and viral infections. Certain nonhuman primates also produce theta-defensins—18 residue cyclic peptides that act as HIV-1 entry inhibitors. Multiple human theta-defensin genes exist, but they harbor a premature termination codon that blocks translation. Consequently, the theta-defensins (retrocyclins) encoded within the human genome are not expressed as peptides. In vivo production of theta-defensins in rhesus macaques involves the post-translational ligation of two nonapeptides, each derived from a 12-residue “demidefensin” precursor. Neither the mechanism of this unique process nor its existence in human cells is known. To ascertain if human cells retained the ability to process demidefensins, we transfected human promyelocytic cells with plasmids containing repaired retrocyclin-like genes. The expected peptides were isolated, their sequences were verified by mass spectrometric analyses, and their anti-HIV-1 activity was confirmed in vitro. Our study reveals for the first time, to our knowledge, that human cells have the ability to make cyclic theta-defensins. Given this evidence that human cells could make theta-defensins, we attempted to restore endogenous expression of retrocyclin peptides. Since human theta-defensin genes are transcribed, we used aminoglycosides to read-through the premature termination codon found in the mRNA transcripts. This treatment induced the production of intact, bioactive retrocyclin-1 peptide by human epithelial cells and cervicovaginal tissues. The ability to reawaken retrocyclin genes from their 7 million years of slumber using aminoglycosides could provide a novel way to secure enhanced resistance to HIV-1 infection.
Author Summary
Defensins are a large family of small antimicrobial peptides that contribute to host defense against a broad spectrum of pathogens. In primates, defensins are divided into three subfamilies—alpha, beta, and theta—on the basis of their disulfide bonding pattern. Theta-defensins were the most recently identified defensin subfamily, isolated initially from white blood cells and bone marrow of rhesus monkeys. They are the only known cyclic peptides in mammals and act primarily by preventing viruses such as HIV-1 from entering cells. Whereas theta-defensin genes are intact in Old World monkeys, in humans they have a premature stop codon that prevents their expression; they thus exist as pseudogenes. In this work, we reveal that, upon correction of the premature termination codon in theta-defensin pseudogenes, human myeloid cells produce cyclic, antiviral peptides (which we have termed “retrocyclins”), indicating that the cells retain the intact machinery to make cyclic peptides. Furthermore, we exploited the ability of aminoglycoside antibiotics to read-through the premature termination codon within retrocyclin transcripts to produce functional peptides that are active against HIV-1. Given that the endogenous production of retrocyclins could also be restored in human cervicovaginal tissues, we propose that aminoglycoside-based topical microbicides might be useful in preventing sexual transmission of HIV-1.
Repairing an ancestral human pseudogene by mutagenesis, or by the application of aminoglycosides to suppress the termination codon, can restore the production of retrocyclins, which are peptides of the defensin family that are remarkable inhibitors of HIV-1 entry into cells.
PMCID: PMC2672613  PMID: 19402752
18.  Field production and functional evaluation of chloroplast-derived interferon-α2b 
Plant biotechnology journal  2007;5(4):511-525.
Type I interferons (IFNs) inhibit viral replication and cell growth and enhance the immune response, and therefore have many clinical applications. IFN-α2b ranks third in world market use for a biopharmaceutical, behind only insulin and erythropoietin. The average annual cost of IFN-α2b for the treatment of hepatitis C infection is $26 000, and is therefore unavailable to the majority of patients in developing countries. Therefore, we expressed IFN-α2b in tobacco chloroplasts, and transgenic lines were grown in the field after obtaining United States Department of Agriculture Animal and Plant Health Inspection Service (USDA-APHIS) approval. Stable, site-specific integration of transgenes into chloroplast genomes and homoplasmy through several generations were confirmed. IFN-α2b levels reached up to 20% of total soluble protein, or 3 mg per gram of leaf (fresh weight). Transgenic IFN-α2b had similar in vitro biological activity to commercially produced PEG-Intron™ when tested for its ability to protect cells against cytopathic viral replication in the vesicular stomatitis virus cytopathic effect (VSV CPE) assay and to inhibit early-stage human immunodeficiency virus (HIV) infection. The antitumour and immunomodulating properties of IFN-α2b were also seen in vivo . Chloroplast-derived IFN-α2b increased the expression of major histocompatibility complex class I (MHC I) on splenocytes and the total number of natural killer (NK) cells. Finally, IFN-α2b purified from chloroplast transgenic lines (cpIFN-α2b) protected mice from a highly metastatic tumour line. This demonstration of high levels of expression of IFN-α2b, transgene containment and biological activity akin to that of commercial preparations of IFN-α2b facilitated the first field production of a plant-derived human blood protein, a critical step towards human clinical trials and commercialization.
PMCID: PMC2596645  PMID: 17490449
antitumour; antiviral; gene containment; molecular ‘pharming’; plant-made cytokine
19.  Total energy expenditure in small for gestational age infants. 
AIM: To measure total energy expenditure and body composition in small for gestational age (SGA) infants in order to investigate proposed hypermetabolism in such babies. METHODS: A cross sectional study of 52 SGA infants measured at 5 weeks of age was made, using existing data from appropriate for gestational age (AGA) infants as controls. The double labelled water technique was used to assess both total energy expenditure and body composition. RESULTS: Multiple regression analysis showed that expressing energy expenditure per kg fat free mass adjusts for body composition in infants of this age. The relation between total energy expenditure and fat free mass differed between the two groups. CONCLUSION: These data indicate that for a given fat free mass the total energy expenditure of SGA infants is greater than that of AGA infants. Such data should be taken into account when energy requirements for SGA infants are being considered.
PMCID: PMC2528347  PMID: 8777687
20.  Total energy expenditure in small for gestational age infants. 
OBJECTIVE: To measure total energy expenditure and body composition in small for gestational age (SGA) infants, to investigate hypermetabolism. METHODS: A cross-sectional study was performed in 52 small for gestational age (SGA) measured at 5 weeks of age, using existing data from appropriate for gestational age (AGA) infants as controls. The doubly-labelled water technique was used to assess both total energy expenditure and body composition in both cohorts of infants. RESULTS: Multiple regression analysis revealed that expressing energy expenditure per kg fat free mass adjusts for body composition in infants of this age. Regression analysis also showed that the relation between total energy expenditure and fat free mass differed between the two groups. CONCLUSION: These data indicate that for a given fat free mass, the total energy expenditure of SGA infants is greater than that of AGA infants. Such data should be taken into account when energy requirements for SGA infants are being considered.
PMCID: PMC1061150  PMID: 8795356
21.  Down's syndrome. 
PMCID: PMC1294981  PMID: 7853297
22.  Bone age, social deprivation, and single parent families. 
Archives of Disease in Childhood  1992;67(10):1281-1285.
It is well known that deprivation affects bone growth. The study was set up to investigate what aspects of deprivation are of greatest importance. Bone ages of 1593 child trauma patients aged 0-19 years from Middlesbrough General Hospital, Cleveland, were related to local authority ward indices of socioeconomic status (51 wards). After adjustment for chronological age and sex, the mean bone ages in each ward were highly significantly negatively associated with five ward indices of deprivation: the rate of single parent families, low care ownership, unemployment, rented housing, and overcrowding. There was a mean four month deficit in bone age among children living in wards with the highest single parent family rates. The inverse association between deprivation and bone age is unlikely to be causal throughout childhood, as older and younger children were affected to the same extent. However the bone age deficit could be caused by deprivation retarding skeletal maturation during a critical period in early life.
PMCID: PMC1793949  PMID: 1444529
23.  Avermectins, New Family of Potent Anthelmintic Agents: Isolation and Chromatographic Properties 
The avermectins, a family of new anthelmintic agents, were isolated from the mycelia of Streptomyces avermitilis. Four closely related major components and four homologous minor components were separated from the complex. Solvent extraction, solvent partition, and adsorption methods were used to isolate and purify the complex; novel partition chromatography systems using Sephadex LH-20 were used to separate the components. A reverse-phase high-pressure liquid chromatography assay for the quantitative determination of all components was used extensively to monitor the purification methods.
PMCID: PMC352667  PMID: 464562
The Journal of General Physiology  1936;19(4):609-623.
The alternating current resistance and capacity of suspensions of unfertilized eggs of Asterias forbesi have been measured at frequencies from one thousand to sixteen million cycles per second. The plasma membrane of the egg has a static capacity of 1.10µf/cm.2 which is practically independent of frequency. The suspensions show a capacity dependent on frequency at low frequencies which may be attributable to surface conductance. The specific resistance of the cytoplasm is between 136 and 225 ohm cm. (4 to 7 times sea water), indicating a relatively high concentration of non-electrolytes. At frequencies above one million cycles there is definite evidence of another element of which the nucleus is presumably a part.
PMCID: PMC2141462  PMID: 19872951
The Journal of General Physiology  1936;19(4):625-632.
The alternating current resistance and capacity of suspensions of unfertilized and fertilized eggs of Arbacia punctulata have been measured at frequencies from 103 to 1.64 x 107 cycles per second. The unfertilized egg has a static plasma membrane capacity of 0.73 µf./cm.2 which is practically independent of frequency. The fertilized egg has a static membrane capacity of 3.1 µf./cm.2 at low frequencies which decreases to a value of 0.55 µf./cm.2 at high frequencies. The decrease follows closely the relaxation dispersion of the dielectric constant if the dissipation of such a system is ignored. It is considered more probable that the effect is due to a fertilization membrane of 3.1 µf./cm.2 capacity lifted 1.5 µ. from the plasma membrane, the interspace having the conductivity of sea water. The suspensions show a frequency-dependent capacity at low frequencies which may be attributable to surface conductance. The equivalent low frequency internal specific resistance of both the unfertilized and fertilized egg is about 186 ohm cm. or about 6 times that of sea water, while the high frequency data extrapolate to a value of about 4 times sea water. There is evidence at the highest frequencies that the current is penetrating the nucleus and other materials in the cytoplasm. If this effect were entirely due to the nucleus it would lead to a very approximate value of 0.1 µf./cm.2 for the capacity of the nuclear membrane. The measurements do not indicate any change in this effect on fertilization.
PMCID: PMC2141460  PMID: 19872952

Results 1-25 (3706)