Employing directed evolution for the functional analysis of multi-specific proteins

Multi-specific proteins located at the heart of complex protein–protein interaction (PPI) networks play essential roles in the survival and fitness of the cell. In addition, multi-specific or promiscuous enzymes exhibit activity toward a wide range of substrates so as to increase cell evolvability and robustness. However, despite their high importance, investigating the in vivo function of these proteins is difficult, due to their complex nature. Typically, deletion of these proteins leads to the abolishment of large PPI networks, highlighting the difficulty in examining the contributions of specific interactions/activities to complex biological processes and cell phenotypes. Protein engineering approaches, including directed evolution and computational protein design, allow for the generation of multi-specific proteins in which certain activities remain intact while others are abolished. The generation and examination of these mutants both in vitro and in vivo can provide high-resolution analysis of biological processes and cell phenotypes and provide new insight into the evolution and molecular function of this important protein family.     

Current approaches in identification and isolation of cancer stem cells

Cancer stem cells (CSCs) are tumor cells with initiating ability, self-renewal potential, and intrinsic resistance to conventional therapeutics. Efficient isolation and characterization of CSCs pave the way for more comprehensive knowledge about tumorigenesis, heterogeneity, and chemoresistance. Also a better understanding of CSCs will lead to novel era of both basic and clinical cancer research, reclassification of human tumors, and development of innovative therapeutic strategies. Finding novel diagnostic and effective therapeutic strategies also enhance the success of treatment in cancer patients. There are various methods based on the characteristics of the CSCs to detect and isolate these cells, some of which have recently developed. This review summarized current techniques for effective isolation and characterization of CSCs with a focus on advantages and limitations of each method with clinical applications.     

Nerolidol,a sesquiterpene,attenuates oxidative stress and inflammation in acetic acid-induced colitis in rats

Targeting oxidative stress and inflammation by novel dietary compounds of natural origin convincingly appears to be one of the most important therapeutic strategies to keep inflammatory bowel diseases (IBD) such as ulcerative colitis disease in remission. It is imperative to investigate naturally occuring plant-derived dietary phytochemicals that are receiving attention for their therapeutic benefits to overcome the debilitating conditions of IBD. In the present study, the effect of nerolidol (NRD), a monocyclic sesquiterpene found in German Chamomile tea, was investigated in acetic acid-induced colitis model in Wistar rats. NRD was orally administered at a dose of 50 mg/kg/day either for 3 days before or 30 min after induction of IBD for 7 days, after intrarectal administration of acetic acid. The body weight, macroscopic, and microscopic analyses of the colon in different experimental groups were observed on days 0, 2, 4, and 7. Acetic acid caused significant reduction in body weight and induced macroscopic and microscopic ulcer along with a significant decline of antioxidants, concomitant to increased malondialdehyde (MDA), a marker of lipid peroxidation, and myeloperoxidase (MPO) activity, a marker of neutrophil activation. Treatment with NRD significantly improved IBD-induced reduction in body weight, improved histology, inhibited MDA formation, and restored antioxidants along with reduced MPO activity. Acetic acid also induced the release of pro-inflammatory cytokines and increased calprotectin, released by neutrophils under inflammatory conditions. NRD treatment significantly reduced calprotectin and pro-inflammatory cytokines. NRD treatment showed potential to improve disease activity and inhibit oxidative stress, lipid peroxidation, and inflammation along with histological preservation of the colon tissues.

     

Rhythmic behaviour and pattern-generating circuits in the locust: Key concepts and recent updates

There is growing recognition that rhythmic activity patterns are widespread in our brain and play an important role in all aspects of the functioning of our nervous system, from sensory integration to central processing and motor control. The study of the unique properties that enable central circuits to generate their rhythmic output in the absence of any patterned, sensory or descending, inputs, has been very rewarding in the relatively simple invertebrate preparations. The locust, specifically, is a remarkable example of an organism in which central pattern generator (CPG) networks have been suggested and studied in practically all aspects of their behaviour. Here we present an updated overview of the various rhythmic behaviours in the locust and aspects of their neural control. We focus on the fundamental concepts of multifunctional neuronal circuits, neural centre interactions and neuromodulation of CPG networks. We are certain that the very broad and solid knowledge base of locust rhythmic behaviour and pattern-generating circuits will continue to expand and further contribute to our understanding of the principles behind the functioning of the nervous system and, indeed, the brain.     

The prognostic and therapeutic values of long noncoding RNA PANDAR in colorectal cancer

Long noncoding RNAs (lncRNAs) consist of 200 nucleotide sequences that play essential roles in different processes, including cell proliferation, and differentiation. There is evidence showing that the dysregulation of lncRNAs promoter of CDKN1A antisense DNA damage-activated RNA (PANDAR) leads to the development and progression in several cancers including colorectal cancer, via p53-dependent manner. This suggests that these lncRNAs may be of value as prognostic indices and a therapeutic target, as a high expression of lncRNAs PANDAR is associated with poor prognosis. Furthermore, modulating lncRNAs PANDAR has been reported to induce apoptosis and inhibit the tumor growth through modulation of cell cycle and epithelial-mesenchymal transition (EMT) pathway. The aim of the current review was to provide an overview of the prognostic and therapeutic values of lncRNAs PANDAR in colorectal cancer     

Selective fluorescent labeling at the α-amino group of bovine pancreatic trypsin inhibitor

Experimental investigation of protein structure and dynamics by spectroscopic methods using external probes requires attachment of a probe to a well-defined site and preparation of pure samples. Measurements of efficiency of nonradiative excitation energy transfer can yield very detailed information about the structure of proteins, provided that two different probes are selectively attached to well-defined sites. We have used specific protection of ε-amino groups using tert-butylazidoformate at high pH for covalent attachment of the fluorescent probe 2-naphthoxyacetic acid at the α-amino group of bovine pancreatic trypsin inhibitor (BPTI). The product is a chromatoraphically homogenous protein derivative that contains the probe at a dye to protein ratio of 1:1, specifically located at the N-terminus, and and that retains its full biological activity. The HPLC tryptic peptide map of BPTI has been analyzed, and all the peptide fragments have been identified. Analysis of tryptic fragments of the labled BPTI derivative showed that it was selectively labeled at the N-terminal amino acid. The probe absorbs in the 310–325-nm range, which is spectrally distinct from the absorption of the protein, and has a monoexponetial fluorescence decay. These and other charactristics make this probe a good energy donor in transfer-efficiency measurements.     

Polymyxin B is an inhibitor of insulin-induced hypoglycemia in the whole animal model. Studies on the mode of inhibitory action

The cyclic decapeptide, polymyxin B (PMXB), was found to inhibit hypoglycemia in mice receiving exogenous insulin (Amir, S., and Shechter, Y. (1985) Eur. J. Pharmacol. 110, 283-285). In this study, we have extended this observation to rats. Insulin-dependent hypoglycemia in rats is efficiently blocked at a 12:1 molar ratio of PMXB to insulin. This effect is highly specific, as it could not be mimicked by a variety of antibiotics or positively charged substances. Chemical modifications of PMXB have revealed that the ring structure, rather than the tail structure, is important for anti-insulin-like activity. Colistin A, which differs from PMXB by one conservative amino acid substitution in the ring structure, is devoid of this activity. Polymyxin B does not interact with insulin, nor does it alter the rate of insulin absorption and/or degradation, or the ability of insulin to bind to target tissues. This peptide inhibits hypoglycemia by blocking insulin-dependent activation of the hexose transport mechanism, as deduced by in vitro studies. The effect of insulin in stimulating hexose uptake (and subsequent glucose metabolism) in both isolated muscle tissue and adipocytes is blocked with little or no effect on the basal activities of these processes. Colistin A has no significant inhibiting effect. Other insulin-dependent activities, such as inhibition of lipolysis in adipocytes or synthesis of DNA in muscle cells, are not inhibited. It is concluded that PMXB inhibits, in a highly specific manner, the action of insulin in stimulating hexose transport and subsequent glucose metabolism, both in vitro and in the whole animal model.