Development of a one-step immunochromatographic strip test for the detection of sennosides A and B

An immunochromatographic strip test was developed to detect sennoside A (1) and sennoside B (2) using anti-1 and anti-2 monoclonal antibodies. The qualitative assay was based on a competitive immunoassay in which the detector reagent consisted of colloidal gold particles coated with the respective sennoside antibodies. The capture reagents were 1- and 2-human serum albumin (HSA) conjugates immobilised on a nitrocellulose membrane on the test strip. The sample containing 1 and 2, together with detector reagent, passed over the zone where the capture reagents had been immobilised. The analytes in the sample competed for binding to the limited amount of antibodies in the detector reagent with the immobilised 1- and 2-HSA conjugates on the membrane and hence positive samples showed no colour in the capture spot zone. Detection limits for the strip test were 125 ng/mL for both sennosides. The assay system is useful as a rapid and simple screening method for the detection of 1 and 2 in plants, drugs and body fluids.     

A review on anti‐tuberculosis peptides: Impact of peptide structure on anti‐tuberculosis activity

Antibiotic resistance is a major public health problem globally. Particularly concerning amongst drug‐resistant human pathogens is Mycobacterium tuberculosis that causes the deadly infectious tuberculosis (TB) disease. Significant issues associated with current treatment options for drug‐resistant TB and the high rate of mortality from the disease makes the development of novel treatment options against this pathogen an urgent need. Antimicrobial peptides are part of innate immunity in all forms of life and could provide a potential solution against drug‐resistant TB. This review is a critical analysis of antimicrobial peptides that are reported to be active against the M tuberculosis complex exclusively. However, activity on non‐TB strains such as Mycobacterium avium and Mycobacterium intracellulare, whenever available, have been included at appropriate sections for these anti‐TB peptides. Natural and synthetic antimicrobial peptides of diverse sequences, along with their chemical structures, are presented, discussed, and correlated to their observed antimycobacterial activities. Critical analyses of the structure allied to the anti‐mycobacterial activity have allowed us to draw important conclusions and ideas for research and development on these promising molecules to realise their full potential. Even though the review is focussed on peptides, we have briefly summarised the structures and potency of the various small molecule drugs that are available and under development, for TB treatment.     

PAPP‐A: a new anti‐aging target?

This article focuses on the role of PAPP‐A in mammalian aging. It introduces PAPP‐A and a little of its history, briefly discusses the function of PAPP‐A in the insulin‐like growth factor (IGF) system and the regulators of PAPP‐A expression, and then reviews data concerning PAPP‐A in aging and age‐related diseases especially in regard to the PAPP‐A knockout (KO) mouse. The PAPP‐A KO mouse is a valuable new model to test hypotheses concerning the control of the tissue availability of IGF, independent from systemic levels, on healthspan as well as lifespan.     

Bovine lactoferricin is anti‐inflammatory and anti‐catabolic in human articular cartilage and synovium

Bovine lactoferricin (LfcinB) is a multi‐functional peptide derived from proteolytic cleavage of bovine lactoferrin. LfcinB was found to antagonize the biological effects mediated by angiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF‐2) in endothelial cells. However, the effect of LfcinB on human articular cartilage remained unknown. Here, our findings demonstrate that LfcinB restored the proteoglycan loss promoted by catabolic factors (interleukin‐1β) IL‐1β and FGF‐2 in vitro and ex vivo. Mechanistically, LfcinB attenuated the effects of IL‐1β and FGF‐2 on the expression of cartilage‐degrading enzymes (MMP‐1, MMP‐3, and MMP‐13), destructive cytokines (IL‐1β and IL‐6), and inflammatory mediators (iNOS and TLR2). LfcinB induced protective cytokine expression (IL‐4 and IL‐10), and downregulated aggrecanase basal expression. LfcinB specifically activated ERK MAPK and Akt signaling pathways, which may account for its anti‐inflammatory activity. We also revealed that LfcinB exerted similar protective effects on human synovial fibroblasts challenged by IL‐1β, with minimal cytotoxicity. Collectively, our results suggest that LfcinB possesses potent anti‐catabolic and anti‐inflammatory bioactivities in human articular tissues, and may be utilized for the prevention and/or treatment of OA in the future. J. Cell. Physiol. 228: 447–456, 2013. © 2012 Wiley Periodicals, Inc.     

Newly raised anti‐VAChT and anti‐ChAT antibodies detect cholinergic cells in chicken embryos

The autonomic nervous system consists of sympathetic and parasympathetic nerves, which functionally antagonize each other to control physiology and homeostasis of organs. However, it is largely unexplored how the autonomic nervous system is established during development. In particular, early formation of parasympathetic network remains elusive because of its complex anatomical structure. To distinguish between parasympathetic (cholinergic) and sympathetic (adrenergic) ganglia, vesicular acetylcholine transporter (VAChT) and choline O‐acetyltransferase (ChAT), proteins associated with acetylcholine synthesis, are known to be useful markers. Whereas commercially available antibodies against these proteins are widely used for mammalian specimens including mice and rats, these antibodies do not work satisfactorily in chickens, although chicken is an excellent model for the study of autonomic nervous system. Here, we newly raised antibodies against chicken VAChT and ChAT proteins. One monoclonal and three polyclonal antibodies for VAChT, and one polyclonal antibody for ChAT were obtained, which were available for Western blotting analyses and immunohistochemistry. Using these verified antibodies, we detected cholinergic cells in Remak ganglia of autonomic nervous system, which form in the dorsal aspect of the digestive tract of chicken E13 embryos. The antibodies obtained in this study are useful for visualization of cholinergic neurons including parasympathetic ganglia.     

A novel human anti‐VCAM‐1 monoclonal antibody ameliorates airway inflammation and remodelling

Asthma is a chronic inflammatory disease induced by Type 2 helper T cells and eosinophils. Vascular cell adhesion molecule‐1 (VCAM‐1) has been implicated in recruiting eosinophils and lymphocytes to pathological sites in asthma as a regulatory receptor. Accordingly, monoclonal antibody (mAb) against VCAM‐1 may attenuate allergic inflammation and pathophysiological features of asthma. We attempted to evaluate whether a recently developed human anti‐VCAM‐1 mAb can inhibit the pathophysiological features of asthma in a murine asthma model induced by ovalbumin (OVA). Leucocyte adhesion inhibition assay was performed to evaluate the in vitro blocking activity of human anti‐VCAM‐1 mAb. OVA‐sensitized BALB/c mice were treated with human anti‐VCAM‐1 mAb or isotype control Ab before intranasal OVA challenge. We evaluated airway hyperresponsiveness (AHR) and bronchoalveolar lavage fluid analysis, measured inflammatory cytokines and examined histopathological features. The human anti‐VCAM‐1 mAb bound to human and mouse VCAM‐1 molecules and inhibited adhesion of human leucocytes in vitro. AHR and inflammatory cell counts in bronchoalveolar lavage fluid were reduced in mice treated with human anti‐VCAM‐1 mAb as compared with a control Ab. The levels of interleukin (IL)‐5 and IL‐13, as well as transforming growth factor‐β, in lung tissue were decreased in treated mice. Human anti‐VCAM‐1 mAb reduced goblet cell hyperplasia and peribronchial fibrosis. In vivo VCAM‐1 expression decreased in the treated group. In conclusion, human anti‐VCAM‐1 mAb attenuated allergic inflammation and the pathophysiological features of asthma in OVA‐induced murine asthma model. The results suggested that human anti‐VCAM‐1 mAb could potentially be used as an additional anti‐asthma therapeutic medicine.     

Low‐dose radiation prevents type 1 diabetes‐induced cardiomyopathy via activation of AKT mediated anti‐apoptotic and anti‐oxidant effects

We investigated whether low‐dose radiation (LDR) can prevent late‐stage diabetic cardiomyopathy and whether this protection is because of the induction of anti‐apoptotic and anti‐oxidant pathways. Streptozotocin‐induced diabetic C57BL/6J mice were treated with/without whole‐body LDR (12.5, 25, or 50 mGy) every 2 days. Twelve weeks after onset of diabetes, cardiomyopathy was diagnosed characterized by significant cardiac dysfunction, hypertrophy and histopathological abnormalities associated with increased oxidative stress and apoptosis, which was prevented by LDR (25 or 50 mGy only). Low‐dose radiation‐induced cardiac protection also associated with P53 inactivation, enhanced Nrf2 function and improved Akt activation. Next, for the mechanistic study, mouse primary cardiomyocytes were treated with high glucose (33 mmol/l) for 24 hrs and during the last 15 hrs bovine serum albumin‐conjugated palmitate (62.5 μmol/l) was added into the medium to mimic diabetes, and cells were treated with LDR (25 mGy) every 6 hrs during the whole process of HG/Pal treatment. Data show that blocking Akt/MDM2/P53 or Akt/Nrf2 pathways with small interfering RNA of akt, mdm2 and nrf2 not only prevented LDR‐induced anti‐apoptotic and anti‐oxidant effects but also prevented LDR‐induced suppression on cardiomyocyte hypertrophy and fibrosis against HG/Pal. Low‐dose radiation prevented diabetic cardiomyopathy by improving cardiac function and hypertrophic remodelling attributed to Akt/MDM2/P53‐mediated anti‐apoptotic and Akt/Nrf2‐mediated anti‐oxidant pathways simultaneously.     

Losing anti‐predatory behaviour: A cost of translocation

Translocation of endangered species to habitats where exotic predators have been removed is now a common conservation practice around the world. Many of these translocated populations have thrived, and they are often used as sources for the harvesting of individuals for translocations to sites where exotic predators still exist, albeit at reduced densities. This study investigates how isolation from exotic predators affects the ability of individuals to recognize such predators using the North Island robin (Petroica longipes) as a model. The study was carried out in three robin populations in the North Island, New Zealand: a translocated population on Tiritiri Matangi Island, where exotic mammalian predators are absent; a population reintroduced from Tiritiri Matangi Island to Wenderholm Regional Park, a mainland site where these mammals are controlled to low densities; and a mainland population at Benneydale where exotic predatory mammals are common. The response intensity of robins to a model stoat was high at Benneydale and low at Tiritiri Matangi and Wenderholm. This result indicates that isolation from mammalian predators on Tiritiri Matangi has suppressed the ability of North Island robins to recognize these predators. It is possible that the low predatory mammal densities at Wenderholm have reduced robin contact with stoats, therefore reduced the opportunity for robins to learn to recognize stoats. Thus, translocation of individuals from populations without predators to places where key predators still exist could be unsuccessful if translocated individuals fail to perform appropriate anti‐predator behaviours.     

MXRA5 is a TGF‐β1‐regulated human protein with anti‐inflammatory and anti‐fibrotic properties

Current therapy for chronic kidney disease (CKD) is unsatisfactory because of an insufficient understanding of its pathogenesis. Matrix remodelling‐associated protein 5 (MXRA5, adlican) is a human protein of unknown function with high kidney tissue expression, not present in rodents. Given the increased expression of MXRA5 in injured tissues, including the kidneys, we have suggested that MXRA5 may modulate kidney injury. MXRA5 immunoreactivity was observed in tubular cells in human renal biopsies and in urine from CKD patients. We then explored factors regulating MXRA5 expression and MXRA5 function in cultured human proximal tubular epithelial cells and explored MXRA5 expression in kidney cancer cells and kidney tissue. The fibrogenic cytokine transforming growth factor‐β1 (TGFβ1) up‐regulated MXRA5 mRNA and protein expression. TGFβ1‐induced MXRA5 up‐regulation was prevented by either interference with TGFβ1 activation of the TGFβ receptor 1 (TGFBR1, ALK5) or by the vitamin D receptor agonist paricalcitol. By contrast, the pro‐inflammatory cytokine TWEAK did not modulate MXRA5 expression. MXRA5 siRNA‐induced down‐regulation of constitutive MXRA5 expression resulted in higher TWEAK‐induced expression of chemokines. In addition, MXRA5 down‐regulation resulted in a magnified expression of genes encoding extracellular matrix proteins in response to TGFβ1. Furthermore, in clear cell renal cancer, von Hippel–Lindau (VHL) regulated MXRA5 expression. In conclusion, MXRA5 is a TGFβ1‐ and VHL‐regulated protein and, for the first time, we identify MXRA5 functions as an anti‐inflammatory and anti‐fibrotic molecule. This information may yield clues to design novel therapeutic strategies in diseases characterized by inflammation and fibrosis.     

A structural model of anti‐anti‐σ inhibition by a two‐component receiver domain: the PhyR stress response regulator

PhyR is a hybrid stress regulator conserved in α‐proteobacteria that contains an N‐terminal σ‐like (SL) domain and a C‐terminal receiver domain. Phosphorylation of the receiver domain is known to promote binding of the SL domain to an anti‐σ factor. PhyR thus functions as an anti‐anti‐σ factor in its phosphorylated state. We present genetic evidence that Caulobacter crescentus PhyR is a phosphorylation‐dependent stress regulator that functions in the same pathway as σ^T and its anti‐σ factor, NepR. Additionally, we report the X‐ray crystal structure of PhyR at 1.25 Å resolution, which provides insight into the mechanism of anti‐anti‐σ regulation. Direct intramolecular contact between the PhyR receiver and SL domains spans regions σ₂ and σ₄, likely serving to stabilize the SL domain in a closed conformation. The molecular surface of the receiver domain contacting the SL domain is the structural equivalent of α4‐β5‐α5, which is known to undergo dynamic conformational change upon phosphorylation in a diverse range of receiver proteins. We propose a structural model of PhyR regulation in which receiver phosphorylation destabilizes the intramolecular interaction between SL and receiver domains, thereby permitting regions σ₂ and σ₄ in the SL domain to open about a flexible connector loop and bind anti‐σ factor.     

Lactoferricin mediates anti‐inflammatory and anti‐catabolic effects via inhibition of IL‐1 and LPS activity in the intervertebral disc

The catabolic cytokine interleukin‐1 (IL‐1) and endotoxin lipopolysaccharide (LPS) are well‐known inflammatory mediators involved in degenerative disc disease, and inhibitors of IL‐1 and LPS may potentially be used to slow or prevent disc degeneration in vivo. Here, we elucidate the striking anti‐catabolic and anti‐inflammatory effects of bovine lactoferricin (LfcinB) in the intervertebral disc (IVD) via antagonism of both IL‐1 and LPS‐mediated catabolic activity using in vitro and ex vivo analyses. Specifically, we demonstrate the biological counteraction of LfcinB against IL‐1 and LPS‐mediated proteoglycan (PG) depletion, matrix‐degrading enzyme production, and enzyme activity in long‐term (alginate beads) and short‐term (monolayer) culture models using bovine and human nucleus pulposus (NP) cells. LfcinB significantly attenuates the IL‐1 and LPS‐mediated suppression of PG production and synthesis, and thus restores PG accumulation and pericellular matrix formation. Simultaneously, LfcinB antagonizes catabolic factor mediated induction of multiple cartilage‐degrading enzymes, including MMP‐1, MMP‐3, MMP‐13, ADAMTS‐4, and ADAMTS‐5, in bovine NP cells at both mRNA and protein levels. LfcinB also suppresses the catabolic factor‐induced stimulation of oxidative and inflammatory factors such as iNOS, IL‐6, and toll‐like receptor‐2 (TLR‐2) and TLR‐4. Finally, the ability of LfcinB to antagonize IL‐1 and LPS‐mediated suppression of PG is upheld in an en bloc intradiscal microinjection model followed by ex vivo organ culture using both mouse and rabbit IVD tissue, suggesting a potential therapeutic benefit of LfcinB on degenerative disc disease in the future. J. Cell. Physiol. 228: 1884–1896, 2013. © 2013 Wiley Periodicals, Inc.     

NF‐κB/NKILA signaling modulates the anti‐cancerous effects of EZH2 inhibition

A wealth of evidence supports the broad therapeutic potential of NF‐κB and EZH2 inhibitors as adjuvants for breast cancer treatment. We contribute to this knowledge by elucidating, for the first time, unique regulatory crosstalk between EZH2, NF‐κB and the NF‐κB interacting long non‐coding RNA (NKILA). We define a novel signaling loop encompassing canonical and non‐canonical actions of EZH2 on the regulation of NF‐κB/NKILA homeostasis, with relevance to breast cancer treatment. We applied a respective silencing approach in non‐transformed breast epithelial cells, triple negative MDA‐MB‐231 cells and hormone responsive MCF‐7 cells, and measured changes in EZH2/NF‐κB/NKILA levels to confirm their interdependence. We demonstrate cell line‐specific fluctuations in these factors that functionally contribute to epithelial‐to‐mesenchymal transition (EMT) remodelling and cell fate response. EZH2 inhibition attenuates MDA‐MB‐231 cell motility and CDK4‐mediated MCF‐7 cell cycle regulation, while inducing global H3K27 methylation and an EMT phenotype in non‐transformed cells. Notably, these events are mediated by a cell‐context dependent gain or loss of NKILA and NF‐κB. Depletion of NF‐κB in non‐transformed cells enhances their sensitivity to growth factor signaling and suggests a role for the host microenvironment milieu in regulating EZH2/NF‐κB/NKILA homeostasis. Taken together, this knowledge critically informs the delivery and assessment of EZH2 inhibitors in breast cancer.     

High efficacy and minimal peptide required for the anti‐angiogenic and anti‐hepatocarcinoma activities of plasminogen K5

Kringle 5(K5) is the fifth kringle domain of human plasminogen and its anti‐angiogenic activity is more potent than angiostatin that includes the first four kringle fragment of plasminogen. Our recent study demonstrated that K5 suppressed hepatocarcinoma growth by anti‐angiogenesis. To find high efficacy and minimal peptide sequence required for the anti‐angiogenic and anti‐tumour activities of K5, two deletion mutants of K5 were generated. The amino acid residues outside kringle domain of intact K5 (Pro452‐Ala542) were deleted to form K5mut1(Cys462‐Cys541). The residue Cys462 was deleted again to form K5mut2(Met463‐Cys541). K5mut1 specifically inhibited proliferation, migration and induced apoptosis of endothelial cells, with an apparent two‐fold enhanced activity than K5. Intraperitoneal injection of K5mut1 resulted in more potent tumour growth inhibition and microvessel density reduction than K5 both in HepA‐grafted and Bel7402‐xenografted hepatocarcinoma mouse models. These results suggested that K5mut1 has more potent anti‐angiogenic activity than intact K5. K5mut2, which lacks only the amino terminal cysteine of K5mut1, completely lost the activity, suggesting that the kringle domain is essential for the activity of K5. The activity was enhanced to K5mut1 level when five acidic amino acids of K5 in NH₂ terminal outside kringle domain were replaced by five serine residues (K5mut3). The shielding effect of acidic amino acids may explain why K5mut1 has higher activity. K5, K5mut1 and K5mut3 held characteristic β‐sheet spectrum while K5mut2 adopted random coil structure. These results suggest that K5mut1 with high efficacy is the minimal active peptide sequence of K5 and may have therapeutic potential in liver cancer.