One hit wonder: A biomimetic transamination reaction has been developed that employs pyridoxal‐5‐phosphate to modify the N terminus of proteins and peptides under mild conditions. This technique ...introduces a uniquely reactive carbonyl group in a single location (see scheme), thus allowing further elaboration through oxime or hydrazone formation. This modification strategy is also compatible with proteins containing a free cysteine residue.
The diverse functional repertoire of proteins promises to yield new materials with unprecedented capabilities, so long as versatile chemical methods are available to introduce synthetic components at ...specific sites on biomolecule surfaces. As a demonstration of this potential, we have used site-selective strategies to attach antifreeze proteins found in Arctic fish and insects to polymer chains. This multivalent arrangement increases the thermal hysteresis activity of the proteins and leads to materials that can be cast into thin films. The polymer−protein conjugates retain the ability of the proteins to slow ice growth in subzero water and can inhibit ice formation after attachment to glass surfaces. These inexpensive materials may prove useful as coatings for device components that must function at low temperature without ice buildup. The polymer attachment also allows higher thermal hysteresis values to be achieved while using less protein, thus lowering the cost of these additives for biomedical applications.
Insulin resistance, a key feature of obesity, the metabolic syndrome and Type 2 diabetes mellitus, results in an array of metabolic and vascular phenomena which ultimately promote the development of ...atherosclerosis. Endothelial dysfunction is intricately related to insulin resistance through the parallel stimulatory effects of insulin on glucose disposal in metabolic tissues and NO production in the endothelium. Perturbations characteristic of insulin resistance, including dyslipidaemia, inflammation and oxidative stress, may jeopardize the structural or functional integrity of the endothelium. Recent evidence suggests that endothelial damage is mitigated by endogenous reparative processes which mediate endothelial regeneration. EPCs (endothelial progenitor cells) are circulating cells which have been identified as mediators of endothelial repair. Several of the abnormalities associated with insulin resistance, including reduced NO bioavailability, increased production of ROS (reactive oxygen species) and down-regulation of intracellular signalling pathways, have the potential to disrupt EPC function. Improvement in the number and function of EPCs may contribute to the protective actions of evidence-based therapies to reduce cardiometabolic risk. In the present article, we review the putative effects of insulin resistance on EPCs, discuss the underlying mechanisms and highlight potential therapeutic manoeuvres which could improve vascular repair in individuals with insulin resistance.
Low concentrations of insulin-like growth factor (IGF) binding protein-1 (IGFBP1) are associated with insulin resistance, diabetes, and cardiovascular disease. We investigated whether increasing ...IGFBP1 levels can prevent the development of these disorders. Metabolic and vascular phenotype were examined in response to human IGFBP1 overexpression in mice with diet-induced obesity, mice heterozygous for deletion of insulin receptors (IR(+/-)), and ApoE(-/-) mice. Direct effects of human (h)IGFBP1 on nitric oxide (NO) generation and cellular signaling were studied in isolated vessels and in human endothelial cells. IGFBP1 circulating levels were markedly suppressed in dietary-induced obese mice. Overexpression of hIGFBP1 in obese mice reduced blood pressure, improved insulin sensitivity, and increased insulin-stimulated NO generation. In nonobese IR(+/-) mice, overexpression of hIGFBP1 reduced blood pressure and improved insulin-stimulated NO generation. hIGFBP1 induced vasodilatation independently of IGF and increased endothelial NO synthase (eNOS) activity in arterial segments ex vivo, while in endothelial cells, hIGFBP1 increased eNOS Ser(1177) phosphorylation via phosphatidylinositol 3-kinase signaling. Finally, in ApoE(-/-) mice, overexpression of hIGFBP1 reduced atherosclerosis. These favorable effects of hIGFBP1 on insulin sensitivity, blood pressure, NO production, and atherosclerosis suggest that increasing IGFBP1 concentration may be a novel approach to prevent cardiovascular disease in the setting of insulin resistance and diabetes.
Creating a greener future: A strategy for creating hybrid materials of polymers and proteins relies on orthogonal reactions to activate the N and C termini of a protein concurrently. The protein is ...then used to cross‐link polymer chains and create a hydrogel (see picture). A model hybrid hydrogel containing enhanced green fluorescent protein is both biodegradable and responsive to changes in pH and temperature.
Hydrogels derived from decellularized lungs are promising materials for tissue engineering in the development of clinical therapies and for modeling the lung extracellular matrix (ECM)
. ...Characterizing and controlling the resulting physical, biochemical, mechanical, and biologic properties of decellularized ECM (dECM) after enzymatic solubilization and gelation are thus of key interest. As the role of enzymatic pepsin digestion in effecting these properties has been understudied, we investigated the digestion time-dependency on key parameters of the resulting ECM hydrogel. Using resolubilized, homogenized decellularized pig lung dECM as a model system, significant time-dependent changes in protein concentration, turbidity, and gelation potential were found to occur between the 4 and 24 h digestion time points, and plateauing with longer digestion times. These results correlated with qualitative scanning electron microscopy images and quantitative analysis of hydrogel interconnectivity and average fiber diameter. Interestingly, the time-dependent changes in the storage modulus tracked with the hydrogel interconnectivity results, while the Young's modulus values were more closely related to average fiber size at each time point. The structural and biochemical alterations correlated with significant changes in metabolic activity of several representative lung cells seeded onto the hydrogels with progressive decreases in cell viability and alterations in morphology observed in cells cultured on hydrogels produced with dECM digested for >12 and up to 72 h of digestion. These studies demonstrate that 12 h pepsin digest of pig lung dECM provides an optimal balance between desirable physical ECM hydrogel properties and effects on lung cell behaviors.
The diverse functional repertoire of proteins promises to yield new materials with unprecedented capabilities, so long as versatile chemical methods are available to integrate biomolecules with ...synthetic components. As a demonstration of this potential, we have used site-selective strategies to cross-link polymer chains using the N- and C-termini of a metallothionein derived from a pea plant. This arrangement directly relates the swelling volume of the polymer to the folded state of the protein. The material retains the protein’s ability to remove heavy metal ions from contaminated water samples, and can be regenerated through the subsequent addition of inexpensive chelators. The change in hydrogel volume that occurs as metal ions are bound allows the detection of contaminants through simple visual inspection. The utility of this bulk property change is demonstrated in the construction of a low-cost device that can report heavy metal contamination with no external power requirements. Most importantly, the generality of the protein modification chemistry allows the immediate generation of new hybrid materials from a wide range of protein sequences.
Chemical reactions that facilitate the attachment of synthetic groups to proteins are useful tools for the field of chemical biology and enable the incorporation of proteins into new materials. We ...have previously reported a pyridoxal 5′-phosphate (PLP)-mediated reaction that site-specifically oxidizes the N-terminal amine of a protein to afford a ketone. This unique functional group can then be used to attach a reagent of choice through oxime formation. Since its initial report, we have found that the N-terminal sequence of the protein can significantly influence the overall success of this strategy. To obtain short sequences that lead to optimal conversion levels, an efficient method for the evaluation of all possible N-terminal amino acid combinations was needed. This was achieved by developing a generalizable combinatorial peptide library screening platform suitable for the identification of sequences that display high levels of reactivity toward a desired bioconjugation reaction. In the context of N-terminal transamination, a highly reactive alanine−lysine motif emerged, which was confirmed to promote the modification of peptide substrates with PLP. This sequence was also tested on two protein substrates, leading to substantial increases in reactivity relative to their wild-type termini. This readily encodable tripeptide thus appears to provide a significant improvement in the reliability with which the PLP-mediated bioconjugation reaction can be used. This study also provides an important first example of how synthetic peptide libraries can accelerate the discovery and optimization of protein bioconjugation strategies.
Therapies for patients with chronic heart failure caused by left ventricular systolic dysfunction have advanced substantially over recent decades. The cumulative effect of these therapies on ...mortality, mode of death, symptoms, and clinical characteristics has yet to be defined.
This study was a comparison of 2 prospective cohort studies of outpatients with chronic heart failure caused by left ventricular systolic dysfunction performed between 1993 and 1995 (historic cohort: n=281) and 2006 and 2009 (contemporary cohort: n=357). In the historic cohort, 83% were prescribed angiotensin-converting enzyme inhibitors and 8.5% were prescribed β-adrenoceptor antagonists, compared with 89% and 80%, respectively, in the contemporary cohort. Mortality rates over the first year of follow-up declined from 12.5% to 7.8% between eras (P=0.04), and sudden death contributed less to contemporary mortality (33.6% versus 12.7%; P<0.001). New York Heart Association class declined between eras (P<0.001). QTc dispersion across the chest leads declined from 85 ms (SD, 2) to 34 ms (SD, 1) and left ventricular end-diastolic dimensions declined from 65 mm (SD, 0.6) to 59 mm (SD, 0.5) (both P<0.001).
Survival has significantly improved in patients with chronic heart failure caused by left ventricular systolic dysfunction over the past 15 years; furthermore, sudden death makes a much smaller contribution to mortality, and noncardiac mortality is a correspondingly greater contribution. This has been accompanied by an improvement in symptoms and some markers of adverse electric and structural left ventricular remodeling.
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