Studies on the acid unfolded and molten globule states of catalytically active stem bromelain: a comparison with catalytically inactive form

We report the accumulation of an acid unfolded (UA) state and a molten globule (MG) state in the acid induced unfolding pathway of unmodified preparation of stem bromelain (SB) [EC 3.4.22.32], a cystein protease from Ananas cosmosus. The conformation of SB was examined over the pH 0.8-3 regions by circular dichroism, tryptophanyl fluorescence, 1-anilino-8-naphthalenesulfonate (ANS) binding, and tryptophanyl fluorescence quenching study. The pH 0.8-3.0 regions were selected to study the acid induced unfolding of SB because no autolysis of the enzyme was observed in these pH regions. The results show that SB at pH 2.0 is maximally unfolded and characterizes by significant loss of secondary structure ( approximately 80%) and almost complete loss of tertiary contacts. However, on further decreasing the pH to 0.8 a MG state was observed, with secondary structure content similar to that of native protein but no tertiary structure. We also made a comparative study of these acid induced states of SB with acid induced states of modified stem bromelain (mSB), reported by our group earlier [Eur. J. Biochem. (2002) 269, 47-52]. We have shown that modification of SB for inactivation significantly affects the N-UA transition but neither affects the UA-MG transition nor the stability of the MG state.     

Association of adiponectin gene functional polymorphisms (-11377C/G and +45T/G) with nonalcoholic fatty liver disease

Adiponectin levels are reduced in NAFLD patients and genetic variants of adiponectin have been frequently associated with type 2 diabetes and insulin resistance. To determine the genotypic frequencies of adiponectin functional polymorphisms (-11377C/G and +45T/G) and their subsequent effect on disease progression and plasma adiponectin levels in the patients with NAFLD. A total of 137 NAFLD patients and 250 matched controls were enrolled in the study. DNA sequencing and genotyping were performed to identify the genetic variants. The plasma adiponectin levels were assessed by ELISA. Homozygous mutant genotype of adiponectin SNPs, -11377C/G and +45T/G, were significantly more prevalent in NAFLD patients than controls (Bonferroni corrected p=0.014 and 0.018, respectively). Plasma adiponectin levels were significantly lower in the NAFLD patients as compared to controls. Moreover, presence of 'G' allele at position -11377C/G and +45T/G was found to be associated with necroinflammatory grade and reduced adiponectin levels, (p values 0.02 and 0.01) respectively. -11377G and +45G alleles are associated with severity of liver disease and hypoadiponectemia, in the patients with NAFLD, respectively.     

Salt-induced refolding in different domains of partially folded bovine serum albumin

In our earlier communication on urea denaturation of bovine serum albumin (BSA), we showed significant unfolding of domain III along with domain I prior to intermediate formation around 4.6-5.2 M urea based on the binding results of domain specific ligands:chloroform, bilirubin and diazepam for domains I, II and III, respectively. Here, we present our results on the salt-induced refolding of the two partially folded states of BSA obtained at 4.5 M urea and at pH 3.5, respectively. Both these states were characterized by significant unfolding of both domains I and III as indicated by decreased binding of chloroform and diazepam, respectively. Salt-induced stabilization of partially folded states of BSA was accompanied by nearly complete refolding of both domains I and III as the binding isotherms of chloroform and diazepam obtained in presence of approximately 1.0 M KCl were nearly identical to that obtained with native BSA at pH 7.4. From these observations, it can be concluded that the anion binding sites on serum albumin are not only confined to domain III (C-terminal region) but few sites are also present on domain I (or N-terminal region) of the protein.     

Molten globule-like folding intermediate of asialofetuin at acidic pH

In our earlier communication on acid-induced unfolding of bovine serum fetuin (BSF), we showed the existence of a molten globule (MG)-like state of BSF at pH 1.8. The MG state was characterized by higher content of secondary structure than native and almost complete loss of tertiary structure and more solvent exposed hydrophobic surface [Biochim. Biophys. Acta 1649 (2003) 164]. In this work we have shown the presence of an MG-like partially folded intermediate of asialofetuin at around pH 1.8, which is much different from the MG state observed in BSF in secondary structure contents. The results show that asialofetuin at pH 1.8 retains approximately 45% secondary structure, as evident from far-UV CD spectra. The near-UV CD spectra showed almost complete loss of tertiary structure. The intrinsic fluorescence and acrylamide quenching of the lone tryptophan residue showed that in acid-induced state, it is buried in the interior in a nonpolar environment. The temperature dependence of far-UV CD signal of asialofetuin at pH 1.8 exhibits a weak cooperative thermal transition. A significant increase in ANS fluorescence showed extensive solvent exposure of nonpolar cluster. Size exclusion chromatography (SEC) indicates a slight increase in the hydrodynamic size of acid-induced protein. These results suggest that asialofetuin at pH 1.8 represents the MG-like folding intermediate. Moreover, our results showed that glycosylation might play a role in stabilization of secondary structure during acid and/or thermal denaturation.     

Alkali-induced conformational transition in different domains of bovine serum albumin

Alkaline pH induced conformational changes in different domains of bovine serum albumin were studied by using domain specific ligands: chloroform, bilirubin and diazepam for domains I, II and III respectively. The effect of alkaline pH on the secondary structure of BSA was monitored by far-UV CD in the range 250 nm to 200 nm. The pH profiles of BSA in the alkaline region showed a two-step change, one corresponding to N<-->B transition (pH 7.5 to 9.0) and the other to B --> U (pH 11.0 to 13.5). Binding of chloroform decreased continuously on increasing pH, whereas binding of diazepam, remained unchanged up to pH 9 and decreased thereafter. In contrast, binding of bilirubin gradually increased up to pH 11.0 and decreased thereafter reaching a value similar to one obtained with native BSA at pH 11.5. Above pH 11.5, bilirubin binding decreased and was abolished completely at pH 12.5. In the pH region 7.5 to 11.0, a continuous decrease in chloroform binding (pH 7.5 to 9.5) and a late decrease in diazepam binding (pH 9.5 to 11.0) suggested major loss of native conformation of domain I followed by domain III during alkaline induced unfolding of BSA. However, a significant increase in bilirubin binding showed a favorable conformational rearrangement in domain II in this pH region (pH7.5 to 11.0). Further, a nearly complete abolishment of bilirubin binding to BSA and significant loss of secondary structure around pH 12.5 indicated that domain II was more resistant to alkaline pH and unfolds only at extreme alkalinity. Taken together, these data suggest that unfolding of three domains of BSA follow the following order of susceptibility towards alkaline denaturation of BSA domain I>domain III>domain II.     

Urea induced unfolding of F isomer of human serum albumin: a case study using multiple probes

The human serum albumin is known to undergo N <==> F (neutral to fast moving) isomerization between pH 7 and 3.5. The N < ==> F isomerization involves unfolding and separation of domain III from rest of the molecule. The urea denaturation of N isomer of HSA shows two step three state transition with accumulation of an intermediate state around 4.8-5.2 M urea concentration. While urea induced unfolding transition of F isomer of HSA does not show the intermediate state observed during unfolding of N isomer. Therefore, it provides direct evidence that the formation of intermediate in the unfolding transition of HSA involves unfolding of domain III. Although urea induced unfolding of F isomer of HSA appears to be an one step process, but no coincidence between the equilibrium transitions monitored by tryptophanyl fluorescence, tyrosyl fluorescence, far-UV CD and near-UV CD spectroscopic techniques provides decisive evidence that unfolding of F isomer of HSA is not a two state process. An intermediate state that retained significant amount of secondary structure but no tertiary structure has been identified (around 4.4 M urea) in the unfolding pathway of F isomer. The emission of Trp-214 (located in domain II) and its mode of quenching by acrylamide and binding of chloroform indicate that unfolding of F isomer start from domain II (from 0.4 M urea). But at higher urea concentration (above 1.6 M) both the domain unfold simultaneously and the protein acquire random coil structure around 8.0 M urea. Further much higher KSV of NATA (17.2) than completely denatured F isomer (5.45) of HSA (8.0 M urea) suggests the existence of residual tertiary contacts within local regions in random coil conformation (probably around lone Trp-214).     

Cross-linked stem bromelain: A more stabilized active preparation

Cross-linking of the protease stem bromelain (bromelain) with 0.25 and 1.25% glutaraldehyde (GTA) results in the formation of a large molecular mass, multimeric and soluble aggregate having comparable activity to the unmodified bromelain. Both 0.25 and 1.25% GTA cross-linked (CL) bromelain preparations were more stable against urea, guanidine hydrochloride (GdnHCl) and temperature-induced inactivation, and exhibited slightly better storage stability compared to the unmodified protease. Such a high molecular weight, soluble, active and stable preparation may be useful in industry, i.e. in the textile industry for improving the properties of a fabric without loss of fabric strength and shape.     

Heavy drinking relates to positive valence ratings of alcohol cues

A positive family history of alcohol use disorders (FH) is a robust predictor of personal alcohol abuse and dependence. Exposure to problem-drinking models is one mechanism through which family history influences alcohol-related cognitions and drinking patterns. Similarly, exposure to alcohol advertisements is associated with alcohol involvement and the relationship between affective response to alcohol cues and drinking behavior has not been well established. In addition, the collective contribution that FH, exposure to different types of problem-drinking models (e.g. parents, peers) and personal alcohol use have on appraisal of alcohol-related stimuli has not been evaluated with a large sample. We investigated the independent effects of FH, exposure to problem-drinking models and personal alcohol use on valence ratings of alcohol pictures in a college sample. College students (n = 227) completed measures of personal drinking and substance use, exposure to problem-drinking models, FH and ratings on affective valence of 60 alcohol pictures. Greater exposure to non-familial problem-drinkers predicted greater drinking among college students (beta = 0.17, P < 0.01). However, personal drinking was the only predictor of valence ratings of alcohol pictures (beta = -0.53, P < 0.001). Personal drinking level predicted valence ratings of alcohol cues over and above FH, exposure to problem-drinking models and demographic characteristics. This suggests that positive affective responses to alcohol pictures are more a function of personal experience (i.e. repeated heavy alcohol use) than vicarious learning.     

Searching for conditions to form stable protein oligomers with amyloid-like characteristics: The unexplored basic pH

Conversion of peptides and proteins from their native states into amyloid fibrillar aggregates is the hallmark of a number of pathological conditions, including Alzheimer's disease and amyloidosis. Evidence is accumulating that soluble oligomers, as opposed to mature fibrils, mediate cellular dysfunction, ultimately leading to disease onset. In this study, we have explored the ability of alkaline pH solutions, which have remained relatively unexplored so far, to form a partially folded state of the N-terminal domain of the Escherichia coli protein HypF (HypF-N), which subsequently assembles to form stable soluble oligomers. Results showed that HypF-N unfolds at high pH via a two-state process. Characterization of the resulting alkaline-unfolded state by near- and far-UV circular dichroism, intrinsic and ANS-derived fluorescence and DLS indicated characteristics of a monomeric, premolten globule state. Interestingly, alkaline-unfolded HypF-N aggregates, at high concentration in the presence of low concentrations of TFE, into stable oligomers. These are able to bind amyloid-specific dyes, such as Congo red, ThT, and ANS, contain extensive β-sheet structure, as detected with far-UV circular dichroism, and have a height of 2.0–3.9 nm when analysed using atomic force microscopy. This study, which complements our previous one in which morphologically, structurally, and tinctorially similar oligomers were formed at low and nearly neutral pH values by the same protein, offers opportunities to explore the fine differences existing in the mechanism of formation of these species under different conditions, in their precise molecular structure and in their ability to cause cellular dysfunction.     

Exposure of Candida to p-anisaldehyde inhibits its growth and ergosterol biosynthesis

p-Anisaldehyde (4-methoxybenzaldehyde), an extract from Pimpinella anisum seeds, is a very common digestive herb of north India. Antifungal activity of p-anisaldehyde was investigated on 10 fluconazole-resistant and 5 fluconazole-sensitive Candida strains. Minimum inhibitory concentrations (MIC(90)) ranged from 250 μg/ml to 600 μg/ml for both sensitive and resistant strains. Ergosterol content was drastically reduced by p-anisaldehyde-62% in sensitive and 66% in resistant strains-but did not corelate well with MIC(90) values. It appears that p-anisaldehyde exerts its antifungal effect by decreasing NADPH routed through up-regulation of putative aryl-alcohol dehydrogenases. Cellular toxicity of p-anisaldehyde against H9c2 rat cardiac myoblasts was less than 20% at the highest MIC value. These findings encourage further development of p-anisaldehyde.