Protein prosthesis: β‐peptides as reverse‐turn surrogates

The introduction of non‐natural modules could provide unprecedented control over folding/unfolding behavior, conformational stability, and biological function of proteins. Success requires the interrogation of candidate modules in natural contexts. Here, expressed protein ligation is used to replace a reverse turn in bovine pancreatic ribonuclease (RNase A) with a synthetic β‐dipeptide: β²‐homoalanine–β³‐homoalanine. This segment is known to adopt an unnatural reverse‐turn conformation that contains a 10‐membered ring hydrogen bond, but one with a donor–acceptor pattern opposite to that in the 10‐membered rings of natural reverse turns. The RNase A variant has intact enzymatic activity, but unfolds more quickly and has diminished conformational stability relative to native RNase A. These data indicate that hydrogen‐bonding pattern merits careful consideration in the selection of beneficial reverse‐turn surrogates.     

Risk factors of peripheral arterial disease: a case control study in Sri Lanka

Background

Peripheral artery disease (PAD) is an important global health problem and contributes to notable proportion of morbidity and mortality. This particular manifestation of systemic atherosclerosis is largely under diagnosed and undertreated. For sustainable preventive strategies in a country, it is mandatory to identify country-specific risk factors. We intended to assess the risk factors of PAD among adults aged 40–74 years.

Methods

This case control study was conducted in 2012–2013 in Sri Lanka. Seventy-nine cases and 158 controls in the age group of 40–74 years were selected for the study in order to have case to control ratio 1:2. The criterion for selecting cases and control was based on Ankle brachial pressure index (ABPI). Cases were selected from those who had ABPI 0.85 or less (ABPI ≤0.85) in either lower limb. Controls were selected from those ABPI score between 1.18 and 1.28 in both lower limbs. Only newly identified individuals with PAD were selected as cases. Controls were selected from the same geographical location and within the 5 year age group as cases.

Results

The history of diabetes mellitus more than 10 years (OR 5.8, 95% CI 2.2–14.2), history of dyslipidemia for more than 10 years (OR 4.9, 95% CI 2.1–16.2), history of hypertension for more than 10 years (OR 3.8, 95% CI 1.8–12.7) and smoking (OR 2.9, 95% CI 1.2–6.9), elevated HsCRP (OR 3.7, 95% CI 1.2–12.0) and hyperhomocysteinemia (OR 3.0, 95% CI 1.1–8.1) were revealed as country specific significant risk factor of PAD.

Conclusions

Diabetes mellitus, hypertension, dyslipidemia, smoking as well as elevated homocysteine and HsCRP found as risk factors of PAD. Longer the duration or higher level exposure to these risk factors has increased the risk of PAD. These findings emphasis the need for routine screening of PAD among patients with the identified risk factors.

     

The role of fish-poultry integration on fish growth performance,yields and economic benefits among smallholder farmers in sub-Saharan Africa,Tanzania

Aquaculture practices from sub-Saharan Africa are characterised by low production, owing to improper technology. Production can be increased through integrating fish farming with other existing on-farm activities, particularly livestock husbandry. We assessed the role of fish-poultry integration on all male Nile tilapia, Oreochromis niloticus growth performance, yields and economic benefits among smallholder farmers in sub-Saharan Africa, Tanzania. The study also compared phytoplankton species composition, abundance and biomass between the fish-poultry integration and non-integrated system. After 180 days of the experiment, all male O. niloticus cultured under fish-poultry integration exhibited significantly higher growth rates than those in the non-integrated system (p < 0.05). Gross fish yield (GFY), net fish yield (NFY) and net annual yields (NAY) obtained from fish-poultry integration were significantly higher than those from non-integrated system (p < 0.05). Partial enterprise budget analysis revealed that fish-poultry integration was more profitable than the non-integrated system. Moreover, fish-poultry integrated system produced significantly higher phytoplankton abundance and biomass than those from the non-integrated system. Results demonstrate that rural smallholder farmers can achieve higher growth rate, farm net yields and income by integrating all male O. niloticus with other on-farm activities than practising a stand-alone fish culture system.     

Circular zymogens of human ribonuclease 1

The endogenous production of enzymes as zymogens provides a means to control catalytic activities. Here, we describe the heterologous production of ribonuclease 1 (RNase 1), which is the most prevalent secretory ribonuclease in humans, as a zymogen. In folded RNase 1, the N and C termini flank the enzymic active site. By using intein‐mediated cis‐splicing, we created circular proteins in which access to the active site of RNase 1 is obstructed by an amino‐acid sequence that is recognized by the HIV‐1 protease. Installing a sequence that does not perturb the RNase 1 fold led to only modest inactivation. In contrast, the ancillary truncation of residues from each terminus led to a substantial decrease in the catalytic activity of the zymogen with the maintenance of thermostability. For optimized zymogens, activation by HIV‐1 protease led to a > 10⁴‐fold increase in ribonucleolytic activity at a rate comparable to that for the cleavage of endogenous viral substrates. Molecular modeling indicated that these zymogens are inactivated by conformational distortion in addition to substrate occlusion. Because protease levels are elevated in many disease states and ribonucleolytic activity can be cytotoxic, RNase 1 zymogens have potential as generalizable prodrugs.     

Endowing human pancreatic ribonuclease with toxicity for cancer cells

Onconase is an amphibian protein that is now in Phase III clinical trials as a cancer chemotherapeutic. Human pancreatic ribonuclease (RNase 1) is homologous to Onconase but is not cytotoxic. Here, ERDD RNase 1, which is the L86E/N88R/G89D/R91D variant of RNase 1, is shown to have conformational stability and ribonucleolytic activity similar to that of the wild-type enzyme but > 10(3)-fold less affinity for the endogenous cytosolic ribonuclease inhibitor protein. Most significantly, ERDD RNase 1 is toxic to human leukemia cells. The addition of a non-native disulfide bond to ERDD RNase 1 not only increases the conformational stability of the enzyme but also increases its cytotoxicity such that its IC(50) value is only 8-fold greater than that of Onconase. Thus, only a few amino acid substitutions are necessary to make a human protein toxic to human cancer cells. This finding has significant implications for human cancer chemotherapy.     

Ribonuclease inhibitor as an intracellular sentry

Onconase® (ONC) is a homolog of RNase A that is in clinical trials as a cancer chemotherapeutic agent. The toxicity of ONC and RNase A variants relies on their ability to evade the cytosolic ribonuclease inhibitor protein (RI) and degrade cellular RNA. We find that these ribonucleases are more toxic for more rapidly growing cells. The enhanced cytotoxicity does not arise from variation in the endogenous level of RI, which is virtually constant. Overproduction of RI diminishes the potency of toxic RNase A variants, but has no effect on the cytotoxicity of ONC. Thus, RI constrains the cytotoxicity of RNase A. These data provide new insights for the development of an optimal ribonuclease-based cancer chemotherapy.     

An electronic effect on protein structure

The well-known preference of the peptide bond for the trans conformation has been attributed to steric effects. Here, we show that a proline residue with an N-formyl group (H(i-1)-C'(i-1)=O(i-1)), in which H(i-1) presents less steric hindrance than does O(i-1), likewise prefers a trans conformation. Thus, the preference of the peptide bond for the trans conformation cannot be explained by steric effects alone. Rather, an n --> pi* interaction between the oxygen of the peptide bond (O(i-1)), and the subsequent carbonyl carbon in the polypeptide chain (C'(i)) also contributes to this preference. The O(i-1) and C'(i) distance and O(i-1).C'(i)=O(i) angle are especially favorable for such an n --> pi* interaction in a polyproline II helix. We propose that this electronic effect provides substantial stabilization to this and other elements of protein structure.     

Efficient expression of exogenous genes in primary vascular cells using IRES-based retroviral vectors

Analysis of gene function in primary vascular cells has been particularly limited by low transfection efficiencies. Using internal ribosomal entry site (IRES)-based retroviral vectors, we demonstrate efficient infection (range of 45%-95%) of primary human endothelial and smooth muscle cells with genes varying in size from 1.3 to 4.5 kb. Because IRES vectors are designed to allow the expression of two genes from a single mRNA, we can show excellent correlation between the expression of a reporter gene and an inserted gene of interest. Reporter gene expression allows rapid (24-48 h) and unambiguous identification of transduced cells. Additionally, reporter gene expression can be used to isolate subpopulations of cells that express distinct levels of cistron 1 genes by flow cytometry, and sorted cells maintain relative levels of gene expression over multiple passages in culture. Two examples of the usefulness of these vectors to characterize gene function in primary vascular cells include (i) the inhibition of endothelial cell inflammatory responses in a polyclonal population by the expression of a dominant negative inhibitor of nuclear factor-kappaB and (ii) monitoring the in vitro evolution of smooth muscle cells provided with a selective growth advantage by transduction with telomerase. Potential applications of retroviral expression strategies in vascular biology are also discussed.