Structure, function and evolution of three-finger toxins: Mini proteins with multiple targets

Snake venoms are complex mixtures of pharmacologically active peptides and proteins. These protein toxins belong to a small number of superfamilies of proteins. Three-finger toxins belong to a superfamily of non-enzymatic proteins found in all families of snakes. They have a common structure of three β-stranded loops extending from a central core containing all four conserved disulphide bonds. Despite the common scaffold, they bind to different receptors/acceptors and exhibit a wide variety of biological effects. Thus, the structure-function relationships of this group of toxins are complicated and challenging. Studies have shown that the functional sites in these ‘sibling’ toxins are located on various segments of the molecular surface. Targeting to a wide variety of receptors and ion channels and hence distinct functions in this group of mini proteins is achieved through a combination of accelerated rate of exchange of segments as well as point mutations in exons. In this review, we describe the structural and functional diversity, structure-function relationships and evolution of this group of snake venom toxins.     

CDR substitutions of a humanized monoclonal antibody (CC49): contributions of individual CDRs to antigen binding and immunogenicity

One of the major obstacles in the successful clinical application of monoclonal antibodies has been the development of host immune responses to murine Ig constant and variable regions. While the CDR grafting of MAbs may alleviate many of these problems, the potential remains that one or more murine CDRs on the human Ig backbone of a "humanized" MAb may still be immunogenic. Studies were undertaken employing a MAb of potential clinical utility, CC49, to define those CDRs that are essential for antigen binding and those that may be immunogenic in humans. We previously developed a humanized CC49 (HuCC49) by grafting the MAb CC49 hypervariable regions onto frameworks of human MAbs. To identify those CDRs essential for binding, a panel of variant HuCC49 MAbs was generated here by systematically replacing each of the murine CDRs with their human counterparts. The relative affinity constant of each variant was determined. Serum from a patient who received murine CC49 was used to determine the potential immunogenicity of each CDR in humans. The serum was shown to react with the anti-CC49 variable region. Results showed that patients' anti-idiotypic responses are directed mainly against LCDR3 and moderately against LCDR1 and HCDR2. These studies demonstrate for the first time that variants containing individual CDR substitutions of a humanized MAb can be constructed, and each CDR can be defined for the two most important properties for potential clinical utility: antigen binding and immunogenicity.     

In vitro affinity maturation of a specificity-determining region-grafted humanized anticarcinoma antibody: isolation and characterization of minimally immunogenic high-affinity variants.

PURPOSE: HuCC49V10 (V10), a humanized anticarcinoma monoclonal antibody (Ab) CC49, was generated by grafting only the specificity-determining regions (SDRs) of CC49 onto the variable light and variable heavy frameworks of the human Abs LEN and 21/28'CL, respectively. SDRs are those residues of the complementarity-determining regions that are most critical for antigen (Ag) binding. Compared with HuCC49, which was developed by conventional complementarity-determining region grafting, V10 has lower reactivity to the sera from patients who were previously given murine CC49 in clinical trials, although its Ag-binding affinity is 2-3-fold lower than that of HuCC49. To generate variants of V10 with higher Ag-binding affinity and lower sera reactivity, in vitro affinity maturation of V10 was carried out using phage display technique. EXPERIMENTAL DESIGN: A limited library of Fabs was generated by replacing some of the SDRs with all possible residues located at the corresponding positions in human Abs. The library was enriched, by several rounds of panning, in Fabs that have high affinity for the TAG-72 Ag. The clones encoding the best binders were expressed in insect cells as whole Abs that were purified and characterized. RESULTS: Competition radioimmunoassay and surface plasmon resonance measurements showed that two of the isolates, V14 and V15, have higher binding affinity than that of V10. In addition, the surface plasmon resonance analysis showed that the variants V14 and V15, compared with the parental V10, have lower reactivity to the anti-V region Abs using sera from patients who received murine CC49. CONCLUSIONS: The two isolates, V14 and V15, which show higher Ag-binding reactivity and lower sera reactivity than the parental V10 Ab, are potentially more useful clinical reagents. These results demonstrate that phage display can be used to isolate variants of an Ab that are potentially less immunogenic in patients than the parental Ab from which they are derived.     

Novel analogues of degarelix incorporating hydroxy-, methoxy-, and pegylated-urea moieties at positions 3, 5, 6 and the N-terminus. Part III

Novel degarelix (Fe200486) analogues were screened for antagonism of GnRH-induced response (IC(50)) in a reporter gene assay. Inhibition of luteinizing hormone release over time was measured in the castrated male rat. N(omega)-Hydroxy- and N(omega)-methoxy-carbamoylation of Dab and Dap at position 3 (3-6), and N(omega)-hydroxy-,N(omega)-methoxy-carbamoylation and pegylation of 4Aph at positions 5 and 6 (7-10, 15-17, 22-25) were carried out. Modulation of hydrophobicity was achieved using different acylating groups at the N-terminus (11-14, 18-21, 26-28). Analogues 8, 15-17, 22, and 23 were equipotent to acyline (IC(50) = 0.69 nM) and degarelix (IC(50) = 0.58 nM) in vitro. Analogues 7, 17, and 23 were shorter acting than acyline, when 9, 11, 13, 15, 16, and 22 were longer acting. Only 9 and 14 were inactive at releasing histamine. No analogue exhibited a duration of action comparable to that of degarelix. Analogues with shorter and longer retention times on HPLC (a measure of hydrophilicity) than degarelix were identified.     

Outcomes of hospital-acquired acute kidney injury in elderly patients: a single-centre study

International Urology and Nephrology - HAAKI is a common clinical problem in hospitalized patients. Its incidence is high in older patients and carries worse prognosis. The presence of multiple...     

Hypocholesterolemic activity of indigenous probiotic isolate Saccharomyces cerevisiae ARDMC1 in a rat model

The aim of this study was to investigate probiotic attributes of Saccharomyces cerevisiae ARDMC1 isolated from traditional rice beer starter cake and its hypocholesterolemic effects on Wistar rats fed a high-cholesterol diet. The indigenous isolate ARDMC1 showed potential probiotic characteristics such as tolerance to simulated gastrointestinal stress conditions, autoaggregation properties, and adhesion to intestinal epithelium Caco-2 cell line. In addition, ARDMC1 isolate exhibited in vitro cholesterol assimilation properties in media supplemented with cholesterol. Furthermore, administration of probiotic isolate to rats fed a hypercholesterolemic diet resulted in significant reduction of serum total cholesterol, low-density lipoprotein cholesterol, and triglyceride at the end of 42 days. The present study envisages ARDMC1 as a promising starter culture for the preparation of functional foods with properties to combat cardiovascular diseases.