N- and C-Terminal motifs in human alphaB crystallin play an important role in the recognition, selection, and solubilization of substrates

The functions of the interactive sequences in human alphaB crystallin that are involved in chaperone activity and complex assembly of small heat shock proteins need to be characterized to understand the mechanisms of action on unfolding and misfolding proteins. Protein pin arrays identified the hydrophobic N-terminal sequence (41STSLSPFYLRPPSFLRAP58) and the polar C-terminal sequence (155PERTIPITREE165) as interactive domains in human alphaB crystallin, which were then deleted to evaluate their importance in complex assembly and chaperone activity. Size exclusion chromatography determined that the complexes formed by the deletion mutants, Delta41-58 and Delta155-165, were larger and more polydisperse than the wild-type (wt) alphaB crystallin complex. In chaperone assays, the Delta41-58 mutant was as effective as wt alphaB crystallin in protecting partially unfolded betaL crystallin and alcohol dehydrogenase (ADH) and significantly less effective than wt alphaB crystallin in protecting unfolded citrate synthase (CS) from aggregation. Chaperone activity did not correlate with complex size but corresponded with the amount of substrate protein unfolding. The results confirmed the importance of N-terminal residues 41-58 in selective interactions with completely unfolded substrates. Poor solubility and limited or no chaperone activity for the three substrates characterized the Delta155-165 deletion mutant, which demonstrated the importance of C-terminal residues 155-165 in maintaining the solubility of unfolded substrates in a manner independent of the amount of substrate protein unfolding. The results presented in this report established that interactive domains in the N- and C-termini of human alphaB crystallin are important for the recognition, selection, and solubility of unfolding substrate proteins.     

Clinical and preclinical evidence of sex-related differences in anthracycline-induced cardiotoxicity

Anthracyclines are very effective chemotherapeutic agents that are widely used to treat pediatric and adult cancer patients. Unfortunately, the clinical utility of anthracyclines is limited by cardiotoxicity. There are several established risk factors for anthracycline-induced cardiotoxicity (AIC), including total cumulative dose, very young and very old age, concomitant use of other cardiotoxic agents, and concurrent mediastinal radiation. However, the role of sex as a risk factor for AIC is not well defined. In pediatric cancer patients, most studies support the notion that female sex is a significant risk factor for AIC. Conversely, there is anecdotal evidence that female sex protects against AIC in adult cancer patients. The lack of consistency in study designs and the different definitions of cardiotoxicity preclude reaching consensus regarding the role of sex as a risk factor for AIC in both pediatric and adult cancer patients. Therefore, more clinical research using reliable techniques such as cardiac magnetic resonance imaging is needed to determine if there truly are sex differences in AIC. In adult preclinical rodent studies, however, there is unequivocal evidence that female sex confers significant protection against AIC, with a possible protective effect of female sex hormones and/or a detrimental role of the male sex hormones. Although findings of these rodent studies may not perfectly mirror the clinical scenario in adult anthracycline-treated cancer patients, understanding the mechanisms of this significant sexual dimorphism may reveal important cardioprotective mechanisms that can be therapeutically targeted.     

HYDROPHOBIC INTERACTION CHROMATOGRAPHY ON OCTYL SEPHAROSE—AN APPROACH FOR ONE-STEP PLATFORM PURIFICATION OF CYCLODEXTRIN GLUCANOTRANSFERASES

Cyclodextrin glucanotransferase (CGTase) from Bacillus circulans ATCC 21783 was concentrated by ultrafiltration and subsequently purified by hydrophobic interaction chromatography on Octyl Sepharose 4 fast flow. The matrix was able to bind selectively to the enzyme at a very low ammonium sulfate concentration of 0.67 M and enzyme desorption was performed by decreasing gradient of the salt. The overall recovery was 80% with 689-fold purity. CGTases derived from four soil isolates and Toruzyme, the commercial preparation of CGTase, also bound to Octyl Sepharose under similar conditions at 0.67 M and eluted at 0.55–0.5 M of ammonium sulfate. Octyl Sepharose chromatography can thus be used as a platform approach for purification of CGTases from various bacterial sources. Long stretches of sequence predominated by hydrophobic amino acids are reportedly present in the starch binding domains of CGTases. Starch binding experiments indicated the binding of the enzymes to the octyl matrix through these domains.     

Breath volatile analysis from patients diagnosed with harmful drinking, cirrhosis and hepatic encephalopathy: a pilot study

Hepatic encephalopathy (HE) is a neuropsychiatric state potentially complicating cirrhosis following the accumulation of toxic compounds that cross the blood–brain barrier and affect brain function; the compounds may undergo alveolar gas exchange and be partially excreted by exhalation. Thus breath analysis as a non-invasive means of diagnosing HE, cirrhosis and harmful drinking was investigated in a pilot study. One litre samples of breath were collected from patients with alcohol-related cirrhosis (n = 34) with HE (n = 11) and without HE (n = 23), non-alcoholic cirrhosis without HE (n = 13), harmful drinkers without cirrhosis (n = 7), and healthy controls (n = 15) in a hospital setting. Breath compounds trapped on adsorbent tubes were released via thermal desorption and analysed by gas chromatography mass spectrometry for separation and detection. Multivariate discriminant analysis was used to identify volatile organic compounds to differentiate patients according to disease status and build models for disease classification. HE was correctly identified in 90.9 % of alcoholic cirrhotic patients and liver cirrhosis in 100 % of alcoholic patients. In patients without clinical HE, alcohol was correctly predicted as the cause of cirrhosis in 78.3 % of patients and non-alcoholic causes of cirrhosis were correctly determined in 69.2 %. Non-alcoholic cirrhosis, alcoholic cirrhosis, and harmful drinking could be discriminated from healthy controls with a sensitivity of 92.3, 97.1 and 100 %, respectively. Breath volatile analysis has the potential to aid the diagnosis of HE and a range of liver disorders.     

Biophysical studies of DNA modified with conformationally constrained nucleotides: comparison of 2'-exo (north) and 3'-exo (south) 'locked' templates

The biophysical properties of oligodeoxyribonucleotides (ODNs) selectively modified with conformationally ‘locked’ bicyclo[3.1.0]hexane pseudosugars (Maier,M.A., Choi,Y., Gaus,H., Barchi,J.J. Jr, Marquez,V.E., Manoharan,M. (2004) Synthesis and characterization of oligonucleotides containing conformationally constrained bicyclo[3.1.0]hexane pseudosugar analogs Nucleic Acids Res., 32, 3642–3650) have been studied by various techniques. Six separate synthetic ODNs based on the Dickerson Drew dodecamer sequence (CGCGAAT*T*CGCG) were examined where each one (or both) of the thymidines (T*) were substituted with a bicyclic pseudosugar locked in either a North (2′-exo) or South (3′-exo) ring pucker. Circular dichroism spectroscopy, differential scanning calorimetry and ¹H NMR spectroscopy were used to examine the duplex stability and conformational properties of the ODNs. Replacement of one or both thymidines with North-locked sugars (RNA-like) into the dodecamer did not greatly affect duplex formation or melt temperatures but distinct differences in thermodynamic parameters were observed. In contrast, incorporation of South-locked sugar derivatives that were predicted to stabilize this standard B-DNA, had the unexpected effect of causing a conformational equilibrium between different duplex forms at specific strand and salt concentrations. Our data and those of others suggest that although DNA can tolerate modifications with RNA-like (North) nucleotides, a more complicated spectrum of changes emerges with modifications restricted to South (DNA-like) puckers.     

Crystallographic, thermodynamic, and molecular modeling studies of the mode of binding of oligosaccharides to the potent antiviral protein griffithsin

The mode of binding of oligosaccharides to griffithsin, an antiviral lectin from the red alga Griffithsia sp., was investigated by a combination of X-ray crystallography, isothermal titration calorimetry, and molecular modeling. The structures of complexes of griffithsin with 1-->6alpha-mannobiose and with maltose were solved and refined at the resolution of 2.0 and 1.5 A, respectively. The thermodynamic parameters of binding of 1-->6alpha-mannobiose, maltose, and mannose to griffithsin were determined. Binding profiles of 1-->6alpha-mannobiose and mannose were similar with Kd values of 83.3 microM and 102 microM, respectively. The binding of maltose to griffithsin was significantly weaker, with a fourfold lower affinity (Kd = 394 microM). In all cases the binding at 30 degrees C was entropically rather than enthalpically driven. On the basis of the experimental crystal structures, as well as on previously determined structures of complexes with monosaccharides, it was possible to create a model of a tridentate complex of griffithsin with Man9GlcNAc2, a high mannose oligosaccharide commonly found on the surface of viral glycoproteins. All shorter oligomannoses could be modeled only as bidentate or monodentate complexes with griffithsin. The ability to mediate tight multivalent and multisite interactions with high-mannose oligosaccharides helps to explain the potent antiviral activity of griffithsin.     

Mycobacterial STAND adenylyl cyclases: The HTH domain binds DNA to form biocrystallized nucleoids

Mycobacteria harbor a unique class of adenylyl cyclases with a complex domain organization consisting of an N-terminal putative adenylyl cyclase domain fused to a nucleotide-binding adaptor shared by apoptotic protease-activating factor-1, plant resistance proteins, and CED-4 (NB-ARC) domain, a tetratricopeptide repeat (TPR) domain, and a C-terminal helix-turn-helix (HTH) domain. The products of the rv0891c-rv0890c genes represent a split gene pair, where Rv0891c has sequence similarity to adenylyl cyclases, and Rv0890c harbors the NB-ARC-TPR-HTH domains. Rv0891c had very low adenylyl cyclase activity so it could represent a pseudoenzyme. By analyzing the genomic locus, we could express and purify Rv0890c and find that the NB-ARC domain binds ATP and ADP, but does not hydrolyze these nucleotides. Using systematic evolution of ligands by exponential enrichment (SELEX), we identified DNA sequences that bound to the HTH domain of Rv0890c. Uniquely, the HTH domain could also bind RNA. Atomic force microscopy revealed that binding of Rv0890c to DNA was sequence independent, and binding of adenine nucleotides to the protein induced the formation of higher order structures that may represent biocrystalline nucleoids. This represents the first characterization of this group of proteins and their unusual biochemical properties warrant further studies into their physiological roles in future.     

Recent advances in cellular effects of fluoride: an update on its signalling pathway and targeted therapeutic approaches

Molecular Biology Reports - Fluoride is a natural element essential in minute quantities in human’s to maintain dental and skeletal health. However, the disease fluorosis manifests itself due...