Synthetic Glycopeptide-Based Delivery Systems for Systemic Gene Targeting to Hepatocytes

Purpose. To design, synthesize, and test synthetic glycopeptide-baseddelivery systems for gene targeting to hepatocytes by systemicadministration.Methods. All peptides were synthesized by the solid phase methoddeveloped using Fmoc chemistry on a peptide synthesizer. The bindingof galactosylated peptides to HepG2 cells and accessibility of thegalactose residues on particle surface was demonstrated by acompetition assay using ¹²⁵I-labeleld asialoorosomucoid and RCA lectinagglutination assay, respectively. DNA plasmid encoding chloramphenicolacetyl transferase (CAT) gene was complexed with a tri-galactosylatedpeptide (GM245.3) or tri-galactosylated lipopeptide (GM246.3) in thepresence of an endosomolytic peptide (GM225.1) or endosomolyticlipopeptide (GM227.3) to obtain DNA particles of 100–150 nm insize. The plasmid/peptide complexes were added to HepG2 cell culturesor intravenously administered by tail vein injection into normal miceor rats. Plasmid uptake and expression was quantified by qPCR andELISA, respectively.Results. Multiple antennary glycopeptides that have the ability tocondense and deliver DNA plasmid to hepatocytes were synthesized andcomplexed with DNA plasmid to obtain colloidally stable DNA/peptidecomplexes. Addition of DNA/GM245.3/GM225.1 peptide complexes(1:3:1 (–/+/–)) to HepG2 cell cultures yielded CAT expression intransfected cells. The transfection efficiency was significantly reducedin the absence of galactose ligand or removal of endosomolytic peptide.Intravenous administration of DNA/GM245.3 peptide complexes (1:0.5(–/+)) into the tail vein of normal rats yielded DNA uptake in theliver. Substitution of GM245.3 by galactosylated lipopeptide GM246.3resulted in more stable DNA particles, and a 10-fold enhancement inliver plasmid uptake. CAT expression was detectable in liver followingintravenous administration of DNA/GM246.3 complexes. Addition ofendosomolytic lipopeptide GM227.3 into the complexes(DNA/GM246.3/GM227.3 (1:0.5:1 (–/+/–))) yielded a 5-fold increase inCAT expression. Liver expression was 8-fold and 40-fold higher thanlung and spleen, respectively, and localized in the hepatocytes only.The transfection efficiency in liver was enhanced by increasing DNAdose and injection volume. The plasmid uptake and expression in liverusing DNA/GM246.3/GM227.3 complexes was 100-200-fold higherthan DNA formulated in glucose. Tissue examination and serumbiochemistry did not show any adverse effect of the DNA/GM246.3/GM227.3 (1:0.5:1 (–/+/–)) complexes after intravenous delivery.Conclusions. Gene targeting to hepatocytes was achieved by systemicadministration of a well-tolerated synthetic glycopeptide-baseddelivery system. The transfection efficiency of this glycopeptide deliverysystem was dependent on peptide structure, endosomolytic activity,colloidal particle stability, and injection volume.     

Protein splicing in trans by purified N- and C-terminal fragments of the Mycobacterium tuberculosis RecA intein

Protein splicing involves the self-catalyzed excision of protein splicing elements, or inteins, from flanking polypeptide sequences, or exteins, leading to the formation of new proteins in which the exteins are linked directly by a peptide bond. To study the enzymology of this interesting process we have expressed and purified N- and C-terminal segments of the Mycobacterium tuberculosis RecA intein, each ≈100 amino acids long, fused to appropriate exteins. These fragments were reconstituted into a functional protein splicing element by renaturation from 6 M urea. When renaturation was carried out in the absence of thiols, the reconstituted splicing element accumulated as an inactive disulfide-linked complex of the two intein fragments, which could be induced to undergo protein splicing by reduction of the disulfide bond. This provided a useful tool for separately investigating the requirements for the reconstitution of the intein fragments to yield a functional protein splicing element and for the protein splicing process per se. For example, the pH dependence of these processes was quite different, with reconstitution being most efficient at pH 8.5 and splicing most rapid at pH 7.0. The availability of such an in vitro protein splicing system opens the way for the exploration of intein structure and the unusual enzymology of protein splicing. In addition, this trans-splicing system is a potential protein ligase that can link any two polypeptides fused to the N- and C-terminal intein segments.     

Receptor-Mediated Recognition and Uptake of Iron from Human Transferrin by Staphylococcus aureus and Staphylococcus epidermidis

Staphylococcus aureus and Staphylococcus epidermidis both recognize and bind the human iron-transporting glycoprotein, transferrin, via a 42-kDa cell surface protein receptor. In an iron-deficient medium, staphylococcal growth can be promoted by the addition of human diferric transferrin but not human apotransferrin. To determine whether the staphylococcal transferrin receptor is involved in the removal of iron from transferrin, we employed 6 M urea–polyacrylamide gel electrophoresis, which separates human transferrin into four forms (diferric, monoferric N-lobe, and monoferric C-lobe transferrin and apotransferrin). S. aureus and S. epidermidis but not Staphylococcus saprophyticus (which lacks the transferrin receptor) converted diferric human transferrin into its apotransferrin form within 30 min. During conversion, iron was removed sequentially from the N lobe and then from the C lobe. Metabolic poisons such as sodium azide and nigericin inhibited the release of iron from human transferrin, indicating that it is an energy-requiring process. To demonstrate that this process is receptor rather than siderophore mediated, we incubated (i) washed staphylococcal cells and (ii) the staphylococcal siderophore, staphyloferrin A, with porcine transferrin, a transferrin species which does not bind to the staphylococcal receptor. While staphyloferrin A removed iron from both human and porcine transferrins, neither S. aureus nor S. epidermidis cells could promote the release of iron from porcine transferrin. In competition binding assays, both native and recombinant N-lobe fragments of human transferrin as well as a naturally occurring human transferrin variant with a mutation in the C-lobe blocked binding of ¹²⁵I-labelled transferrin. Furthermore, the staphylococci removed iron efficiently from the iron-loaded N-lobe fragment of human transferrin. These data demonstrate that the staphylococci efficiently remove iron from transferrin via a receptor-mediated process and provide evidence to suggest that there is a primary receptor recognition site on the N-lobe of human transferrin.     

What survival benefits do premenopausal patients with early breast cancer need to make endocrine therapy worthwhile?

BACKGROUND: Adjuvant endocrine therapies such as tamoxifen, goserelin, and oophorectomy improve survival for premenopausal women diagnosed with early-stage breast cancer. However, these treatments often result in menopausal symptoms, sexual dysfunction, permanent infertility, or the need to delay pregnancy. We aimed to quantify the survival gains that premenopausal patients with early-stage breast cancer require to justify the side-effects and inconvenience of adjuvant endocrine treatments. METHODS: Participants consisted of 102 women who had been diagnosed with early-stage (stage I-II) breast cancer 6-60 months previously, who were aged 40 years or younger at diagnosis, and who had been treated for a minimum of 3 months with endocrine therapy (67 with tamoxifen alone, seven with goserelin alone, and 28 with tamoxifen and goserelin or oophorectomy). 76 patients also received chemotherapy, and 75 received radiotherapy. Participants attended a face-to-face patient-preference interview, in which they were presented with four hypothetical clinical scenarios that were used to quantify the gains in survival rate and life expectancy that women judged necessary to make their endocrine therapy worthwhile. They also completed a questionnaire on standard psychological measures. FINDINGS: About half of participants thought that adjuvant endocrine therapy was worthwhile for an absolute gain in survival of 2% from a baseline of either 65% or 85%, and for a gain in life expectancy of 3 months from a baseline of 5 years and of 6 months for a baseline of 15 years. Women who had had more severe endocrine side-effects required larger gains to make endocrine therapy worthwhile (univariate p=0.02, multivariate p=0.04). INTERPRETATION: Modest gains in survival are sufficient to make adjuvant endocrine treatment worthwhile for premenopausal women with early-stage breast cancer. Knowing and incorporating what women think should enhance shared decision-making.     

Home health care nurses as a new channel for smoking cessation treatment: outcomes from project CARES (Community-nurse Assisted Research and Education on Smoking)

BACKGROUND: Clinical guidelines for smoking cessation may not be sufficient for helping some subgroups of smokers quit. Incorporating smoking cessation into home-based medical care can proactively reach high-risk smokers who may not have access to (or spontaneously seek) smoking cessation. METHOD: Home health care nurses (N = 98) were randomly assigned to deliver either Motivational Enhancement (ME; Motivational Interviewing + Carbon Monoxide Feedback) or Standard Care (AHCPR Guidelines for smoking cessation) to their patients. Seventy percent of patients were eligible and willing to participate (N = 273; 54% female, mean age = 57 years, 83% Caucasian, 41% < high school education). The study was conducted in Providence, RI, USA from 1998 to 2003. RESULTS: Biochemically verified continuous abstinence rates at the 12-month follow-up were 4.2% (SC) and 8.7% (ME) for intent to treat analyses, and 5.2% (SC) and 11.8% (ME) using all available cases (P > 0.05). ME reported more quit attempts and significantly greater reductions in the number of cigarettes smoked per day at all follow-ups through 12 months of post-treatment (all P values < 0.05). CONCLUSIONS: Use of an existing public health channel such as home health care to reach smokers who vary in their motivation to quit could have the potential for large public health impact.     

Mothers reframing physical activity: family oriented politicism, transgression and contested expertise in Australia

Mothers of young children are a population sub-group with one of the lowest levels of physical activity. This paper presents the findings from a qualitative study with 40 Australian mothers of children under school age. The research aimed to understand the tensions, dilemmas and trade-offs which women experience around physical activity within the contexts of their everyday lives as mothers of young children. The analysis shows that, in contrast to health promotion messages which frame physical activity as a positive and healthy behaviour, mothers of young children perceive activity as being both enhancing and threatening to their health and social relationships. Restrictive stereotypes of the 'good' mother make it difficult for many women to prioritise their own physical activity needs over their childrearing and domestic responsibilities. Nevertheless, women's involvement in physical activity is often underpinned by the maternal 'ethic of care' as something which can help them cope better with the challenges of being a mother and contribute to the wellbeing of the family. This article takes as its departure point the notion that the maternal 'ethic of care' creates previously unrecognised opportunities in relation to physical activity. For many mothers, physical activity can also be a way of challenging hegemonic discourses and extending what it means to be a good mother in contemporary society. Although largely overlooked by contemporary health promotion, it is women's family-oriented politicism and resistance to dominant meanings about motherhood, health and the 'ideal' body which create alternative possibilities for their participation and enjoyment of physical activity during early motherhood.     

Prehospital prediction of intensive care unit stay and mortality in blunt trauma patients

BACKGROUND: The success of a trauma system relies on transfer of patients from the field to the most appropriate hospital for definitive care. However, no consensus has been reached regarding the best criteria or triage tool for identifying patients injured seriously enough to warrant transfer to a trauma center. METHODS: Predictors of mortality and intensive care unit stay were identified and prediction models developed in a design data set. The performance of these models was evaluated in a test data set and compared with current trauma triage guidelines, derived from the American College of Surgeons model. RESULTS: The newly developed prediction models performed comparably with the current trauma triage guidelines. CONCLUSION: Although the performance of newly developed triage models was promising, their performance did not exceed that of the current trauma triage guidelines. In particular, the anatomic injury criteria appeared to be the key component of the current trauma triage guidelines.