Natural compound plumbagin based inhibition of hIAPP revealed by Markov state models based on MD data along with experimental validations.

Human islet amyloid polypeptide (amylin or hIAPP) is a 37 residue hormone co-secreted with insulin from ß cells of the pancreas. In patients suffering from type-2 diabetes, amylin self-assembles into amyloid fibrils, ultimately leading to the death of the pancreatic cells. However, a research gap exists in preventing and treating such amyloidosis. Plumbagin, a natural compound, has previously been demonstrated to have inhibitory potential against insulin amyloidosis. Our investigation unveils collapsible regions within hIAPP that, upon collapse, facilitates hydrophobic and pi-pi interactions, ultimately leading to aggregation. Intriguingly plumbagin exhibits the ability to bind these specific collapsible regions, thereby impeding the aforementioned interactions that would otherwise drive hIAPP aggregation. We have used atomistic molecular dynamics approach to determine secondary structural changes. MSM shows metastable states forming native like hIAPP structure in presence of PGN. Our in silico results concur with in vitro results. The ThT assay revealed a striking 50% decrease in fluorescence intensity at a 1:1 ratio of hIAPP to Plumbagin. This finding suggests a significant inhibition of amyloid fibril formation by plumbagin, as ThT fluorescence directly correlates with the presence of these fibrils. Further TEM images revealed disappearance of hIAPP fibrils in plumbagin pre-treated hIAPP samples. Also, we have shown that plumbagin disrupts the intermolecular hydrogen bonding in hIAPP fibrils leading to an increase in the average beta strand spacing, thereby causing disaggregation of pre-formed fibrils demonstrating overall disruption of the aggregation machinery of hIAPP. Our work is the first to report a detailed atomistic simulation of 22 µs for hIAPP. Overall, our studies put plumbagin as a potential candidate for both preventive and therapeutic candidate for hIAPP amyloidosis.

Investigating molecular interactions between human transferrin and resveratrol through a unified experimental and computational approach: Role of natural compounds in Alzheimer's disease therapeutics.

Disruptions to iron metabolism and iron homeostasis have emerged as significant contributors to the development and progression of Alzheimer's disease (AD). Human transferrin plays a key part in maintaining iron equilibrium throughout the body, highlighting its importance in AD. Many plant-derived compounds and dietary constituents show promise for preventing AD. Polyphenols that are abundant in fruits, vegetables, teas, coffee, and herbs possess neuroprotective attributes. Resveratrol is a natural polyphenol present in various plant sources like grapes, berries, peanuts, and red wine that has garnered research interest due to its wide range of biological activities. Notably, resveratrol exhibits neuroprotective effects that may help prevent or treat AD through multiple mechanisms. In the present study, we employed a combination of molecular docking and all-atom molecular dynamic simulations (MD) along with experimental approaches to unravel the intricate interactions between transferrin and resveratrol deciphering the binding mechanism. Through molecular docking analysis, it was determined that resveratrol occupies the iron binding pocket of transferrin. Furthermore, MD simulations provided a more profound insight into the stability and conformational dynamics of the complex suggesting that the binding of resveratrol introduced localized flexibility, while maintaining overall stability. The spectroscopic observations yielded clear evidence of substantial binding between resveratrol and transferrin, confirming the computational findings. The identified binding mechanism and conformational stability hold potential for advancing the development of innovative therapeutic approaches targeting AD through resveratrol, particularly concerning iron homeostasis. These insights serve as a platform for considering the natural compounds in the realm of AD therapeutics.

Severe acute respiratory syndrome coronavirus 2 infection reaches the human nervous system: How?

Without protective and/or therapeutic agents the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection known as coronavirus disease 2019 is quickly spreading worldwide. It has surprising transmissibility potential, since it could infect all ages, gender, and human sectors. It attacks respiratory, gastrointestinal, urinary, hepatic, and endovascular systems and can reach the peripheral nervous system (PNS) and central nervous system (CNS) through known and unknown mechanisms. The reports on the neurological manifestations and complications of the SARS-CoV-2 infection are increasing exponentially. Herein, we enumerate seven candidate routes, which the mature or immature SARS-CoV-2 components could use to reach the CNS and PNS, utilizing the within-body cross talk between organs. The majority of SARS-CoV-2-infected patients suffer from some neurological manifestations (e.g., confusion, anosmia, and ageusia). It seems that although the mature virus did not reach the CNS or PNS of the majority of patients, its unassembled components and/or the accompanying immune-mediated responses may be responsible for the observed neurological symptoms. The viral particles and/or its components have been specifically documented in endothelial cells of lung, kidney, skin, and CNS. This means that the blood-endothelial barrier may be considered as the main route for SARS-CoV-2 entry into the nervous system, with the barrier disruption being more logical than barrier permeability, as evidenced by postmortem analyses.

Evaluation of ThT augmentation and RLS inner filter effect caused by highly fluorescent coumarin derivative and establishing it as true inhibitor of amyloid fibrillation.

Screening of inhibitors that slow down or suppress amyloid fibrils formation relies on some simple but sensitive spectroscopy techniques. Thioflavin T (ThT) fluorescence assay is one of the most common, amyloid specific and sensitive method. However, if an inhibitor is itself fluorescent in the ThT fluorescence range, its screening becomes complicated and require complementary assays. One of such molecules, 6, 7-dihydroxycoumarin (6, 7-DHC, also known as aesculetin, esculetin, and cichorigenin) is fluorescent in the ThT emission range and absorbs in the ThT excitation range. Therefore, it can produce a subtractive effect attributed to primary inner filter effect and/or additive effect due to its self-fluorescence in ThT assay. Our study shows that 6, 7-DHC produces an additive effect in ThT fluorescence, which is minimized at high concentration of ThT and decrease in ThT fluorescence is solely due to its inhibitory effect against HSA fibrillation. These ThT fluorescence-based results are verified through other complementary assays, such as Rayleigh and dynamic light scattering and amyloid-specific Congo red binding assay. Furthermore, hydrophobicity reduction is studied through Nile red (NR) and kinetics through far-UV circular dichroism (far-UV CD) in place of the most commonly employed ThT assay owing to extremely high fluorescence of 6, 7-DHC during initial incubation period.

Inhibition and disruption of amyloid formation by the antibiotic levofloxacin: A new direction for antibiotics in an era of multi-drug resistance.

Neurodegenerative diseases are a group of debilitating maladies involving protein aggregation. To this day, all advances in neurodegenerative disease therapeutics have helped symptomatically but have not prevented the root cause of the disease, i.e., the aggregation of involved proteins. Antibiotics are becoming increasingly obsolete due to the rising multidrug resistance strains of bacteria. Thus, antibiotics, if put to different use as therapeutics against other diseases, could pave a new direction to the world of antibiotics. Hence, we studied the antibiotic levofloxacin for its potential anti-amyloidogenic behavior using human lysozyme, a protein involved in non-systemic amyloidosis, as a model system. At the sub-stoichiometric level, levofloxacin was able to inhibit amyloid formation in human lysozyme as observed by various spectroscopic and microscopic methods, with IC50 values as low as 8.8 ± 0.1 µM. Levofloxacin also displayed a retarding effect on seeding phenomena by elongating the lag-phase (from 0 to 88 h) at lower concentration, and arresting lysozyme fibrillation at the lag stage in sub-stoichiometric concentrations. Structural and computational analyses provided mechanistic insight showing that levofloxacin stabilizes the lysozyme in the native state by binding to the aggregation-prone residues, and thereby inhibiting amyloid fibrillation. Levofloxacin also showed the property of disrupting amyloid fibrils into a smaller polymeric form of proteins which were less cytotoxic as confirmed by hemolytic assay. Therefore, we throw new light on levofloxacin as an amyloid inhibitor and disruptor which could pave way to utilization of levofloxacin as a potential therapeutic against non-systemic amyloidosis and neurodegenerative diseases.

Amyloid Oligomers, Protofibrils and Fibrils.

Amyloid diseases are of major concern all over the world due to a number of factors including: (i) aging population, (ii) increasing life span and (iii) lack of effective pharmacotherapy options. The past decade has seen intense research in discovering disease-modifying multi-targeting small molecules as therapeutic options. In recent years, targeting the amyloid cascade has emerged as an attractive strategy to discover novel neurotherapeutics. Formation of amyloid species, with different degrees of solubility and neurotoxicity is associated with the gradual decline in cognition leading to dementia/cell dysfunction. Here, in this chapter, we have described the recent scenario of amyloid diseases with a great deal of information about the structural features of oligomers, protofibrils and fibrils. Also, comprehensive details have been provided to differentiate the degree of toxicity associated with prefibrillar aggregates. Moreover, a review of the technologies that aid characterisation of oligomer, protofibrils and fibrils as well as various inhibition strategies to overcome protein fibrillation are also discussed.

Biophysical Insight into the Interaction of Human Lysozyme with Anticancer Drug Anastrozole: A Multitechnique Approach.

In the present study, we employ fluorescence spectroscopy, dynamic light scattering, and molecular docking methods. Binding of anticancer drug anastrozole with human lysozyme (HL) is studied. Binding of anastrozole to HL is moderate but spontaneous. There is anastrozole persuaded hydrodynamic change in HL, leading to molecular compaction. Binding of anastrozole to HL also decreased in vitro lytic activity of HL. Molecular docking results suggest the electrostatic interactions and van der Waals forces played key role in binding interaction of anastrozole near the catalytic site. Binding interaction of anastrozole to proteins other than major transport proteins in blood can significantly affect pharmacokinetics of this molecule. Hence, rationalizing drug dosage is important. This study also points to unrelated effects that small molecules bring in the body that are considerable and need thorough investigation.

Stabilizing proteins to prevent conformational changes required for amyloid fibril formation.

Amyloid fibrillation is associated with several human maladies, such as Alzheimer's, Parkinson's, Huntington's diseases, prions, amyotrophic lateral sclerosis, and type 2 diabetes diseases. Gaining insights into the mechanism of amyloid fibril formation and exploring novel approaches to fibrillation inhibition are crucial for preventing amyloid diseases. Here, we hypothesized that ligands capable of stabilizing the native state of query proteins might prevent protein unfolding, which, in turn, may reduce the propensity of proteins to form amyloid fibrils. We demonstrated the efficient inhibition of amyloid formation of the human serum albumin (HSA) (up to 85%) and human insulin (up to 80%) by a nonsteroidal anti-inflammatory drug, ibuprofen (IBFN). IBFN significantly increases the conformational stability of both HSA and insulin, as confirmed by differential scanning calorimetry (DSC). Moreover, increasing concentration of IBFN boosts its amyloid inhibitory propensity in a linear fashion by influencing the nucleation phase as assayed by thioflavin T fluorescence, transmission electron microscopy, and dynamic light scattering. Furthermore, circular dichroism analysis supported the DSC results, showing that IBFN binds to the native state of proteins and almost completely prevents their tendency to lose secondary and tertiary structures. Cell toxicity assay confirms that species formed in the presence of IBFN are less toxic to neuronal cells (SH-SY5Y). These results demonstrate the feasibility of using a small molecule to stabilize the native state of proteins, thereby preventing the amyloidogenic conformational changes, which appear to be the common link in several human amyloid diseases.

Explication of bovine serum albumin binding with naphthyl hydroxamic acids using a multispectroscopic and molecular docking approach along with its antioxidant activity.

In the present investigation, the protein-binding properties of naphthyl-based hydroxamic acids (HAs), N-1-naphthyllaurohydroxamic acid (1) and N-1-naphthyl-p-methylbenzohydroxamic acid (2) were studied using bovine serum albumin (BSA) and UV-visible spectroscopy, fluorescence spectroscopy, diffuse reflectance spectroscopy-Fourier transform infrared (DRS-FTIR), circular dichroism (CD), and cyclic voltammetry along with computational approaches, i.e. molecular docking. Alteration in the antioxidant activities of compound 1 and compound 2 during interaction with BSA was also studied. From the fluorescence studies, thermodynamic parameters such as Gibb's free energy (ΔG), entropy change (ΔS) and enthalpy change (ΔH) were calculated at five different temperatures (viz., 298, 303, 308, 313 or 318 K) for the HAs-BSA interaction. The results suggested that the binding process was enthalpy driven with dominating hydrogen bonds and van der Waals' interactions for both compounds. Warfarin (WF) and ibuprofen (IB) were used for competitive site-specific marker binding interaction and revealed that compound 1 and compound 2 were located in subdomain IIA (Sudlow's site I) on the BSA molecule. Conclusions based on above-applied techniques signify that various non-covalent forces were involved during the HAs-BSA interaction. Therefore the resulted HAs-BSA interaction manifested its effect in transportation, distribution and metabolism for the drug in the blood circulation system, therefore establishing HAs as a drug-like molecule.

Amino group of salicylic acid exhibits enhanced inhibitory potential against insulin amyloid fibrillation with protective aptitude toward amyloid induced cytotoxicity.

Protein misfolding and aggregation lead to amyloid generation that in turn may induce cell membrane disruption and leads to cell apoptosis. In an effort to prevent or treat amyloidogenesis, large number of studies has been paying attention on breakthrough of amyloid inhibitors. In the present work, we aim to access the effect of two drugs, that is, acetylsalicylic acid and 5-amino salicylic acid on insulin amyloids by using various biophysical, imaging, cell viability assay, and computational approaches. We established that both drugs reduce the turbidity, light scattering and fluorescence intensity of amyloid indicator dye thioflavin T. Premixing of drugs with insulin inhibited the nucleation phase and inhibitory potential was boosted by increasing the concentration of the drug. Moreover, addition of drugs at the studied concentrations attenuated the insulin fibril induced cytotoxicity in breast cancer cell line MDA-MB-231. Our results highlight the amino group of salicylic acid exhibited enhanced inhibitory effects on insulin fibrillation in comparison to acetyl group. It may be due to presence of amino group that helps it to prolong the nucleation phase with strong binding as well as disruption of aromatic and hydrophobic stacking that plays a key role in amyloid progression.

Capreomycin inhibits the initiation of amyloid fibrillation and suppresses amyloid induced cell toxicity.

Protein aggregation and amyloid fibrillation are responsible for several serious pathological conditions (like type II diabetes, Alzheimer's and Parkinson's diseases etc.) and protein drugs ineffectiveness. Therefore, a molecule that can inhibit the amyloid fibrillation and potentially clear amyloid fibrils is of great therapeutic value. In this manuscript, we investigated the antiamyloidogenic, fibril disaggregating, as well as cell protective effect of an anti-tuberculosis drug, Capreomycin (CN). Aggregation kinetics data, as monitored by ThT fluorescence, inferred that CN retards the insulin amyloid fibrillation by primarily targeting the fibril elongation step with little effect on lag time. Increasing the dose of CN boosted its inhibitory potency. Strikingly, CN arrested the growth of fibrils when added during the elongation phase, and disaggregated mature insulin fibrils. Our Circular Dichroism (CD) results showed that, although CN is not able to maintain the alpha helical structure of protein during fibrillation, reduces the formation of beta sheet rich structure. Furthermore, Dynamic Light Scattering (DLS) and Transmission Electronic Microscopy (TEM) analysis confirmed that CN treated samples exhibited different size distribution and morphology, respectively. In addition, molecular docking results revealed that CN interacts with insulin through hydrophobic interactions as well as hydrogen bonding, and the Hemolytic assay confirmed the non-hemolytic activity of CN on human RBCs. For future research, this study may assist in the rational designing of molecules against amyloid formation.

Exploration of ligand-induced protein conformational alteration, aggregate formation, and its inhibition: A biophysical insight.

The association of protein aggregates with plentiful human diseases has fascinated studies regarding the biophysical characterization of protein misfolding and ultimately their aggregate formation mechanism. Protein-ligand interaction, their mechanism, conformational changes by ligands, and protein aggregate formation have been studied upon exploiting experimental techniques and computational methodologies. Such studies for the exploration of ligand-induced conformational changes in protein, misfolding and aggregation, has confirmed drastic progresses in the study of aggregate formation pathways. This review comprises of an inclusive description of contemporary experimental techniques as well as theoretical improvements in the interpretation of the conformational properties of protein. We have also discussed various factors responsible for the microenvironment change around protein that sequentially causes amyloidoses. Biophysical techniques and cell-based assays to gain comprehensive understandings of protein-ligand interaction, protein folding, and aggregation pathways have also been described. The promising therapeutic methods used to inhibit the protein fibrillogenesis have also been discussed.

Elucidating the interaction of clofazimine with bovine liver catalase; a comprehensive spectroscopic and molecular docking approach.

Nowadays, understanding of interface between protein and drugs has become an active research area of interest. These types of interactions provide structural guidelines in drug design with greater clinical efficacy. Thus, structural changes in catalase induced by clofazimine were monitored by various biophysical techniques including UV-visible spectrometer, fluorescence spectroscopy, circular dichroism, and dynamic light scattering techniques. Increase in absorption spectra (UV-visible spectrum) confers the complex formation between drug and protein. Fluorescence quenching with a binding constants of 2.47 × 104  M-1 revealed that clofazimine binds with protein. Using fluorescence resonance energy transfer, the distance (r) between the protein (donor) and drug (acceptor) was found to be 2.89 nm. Negative Gibbs free energy change (ΔG°) revealed that binding process is spontaneous. In addition, an increase in α-helicity was observed by far-UV circular dichroism spectra by adding clofazimine to protein. Dynamic light scattering results indicate that topology of bovine liver catalase was slightly altered in the presence of clofazimine. Hydrophobic interactions are the main forces between clofazimine and catalase interaction as depicted by molecular docking studies. Apart from hydrophobic interactions, some hydrogen bonding was also observed during docking method. The results obtained from the present study may establish abundant in optimizing the properties of ligand-protein mixtures relevant for numerous formulations.

A biophysical and computational study unraveling the molecular interaction mechanism of a new Janus kinase inhibitor Tofacitinib with bovine serum albumin.

The interaction of a recently certified kinase inhibitor Tofacitinib (TFB) with bovine serum albumin (BSA) has been studied, by spectroscopic and molecular docking studies. Spectrofluorimetric measurements at 3 different temperatures (288, 298, and 310 K) showed that TFB quench the intrinsic fluorescence of BSA upon forming a nonfluorescent complex. The intrinsic fluorescence data showed that TFB binds to BSA with binding constant (Kb ) of approximately 104 M-1 , affirming a significant affinity of TFB with BSA. The decrease in Stern-Volmer quenching constant with increasing temperature exhibited the static mechanism of quenching. Negative value of ΔG (-6.94 ± 0.32 kcal·mol-1 ), ΔH (-7.87 ± 0.52 kcal·mol-1 ), and ΔS (-3.14 ± 0.42 cal·mol-1 ·K-1 ) at all 3 temperatures declared the reaction between BSA and TFB to be spontaneous and exothermic. Far-UV circular dichroism spectroscopy results demonstrated an increase in helical content of BSA in the presence of TFB. Moreover, dynamic light scattering measurements showed that TFB resulted into a decrease in the hydrodynamic radii (from 3.6 ± 0.053 to 2.9 ± 0.02 nm) of BSA. Molecular docking studies confirmed that TFB binds near site II on BSA, hydrogen bonding, and hydrophobic interaction were involved in the BSA-TFB complex formation. The present study characterizing the BSA-TFB interaction could be significant towards gaining an insight into the drug pharmacokinetics and pharmacodynamics and also in the direction of rational drug designing with better competence, against emerging immune-mediated diseases, ie, alopecia and rheumatoid arthritis.

Ascorbic acid inhibits human insulin aggregation and protects against amyloid induced cytotoxicity.

Protein aggregation into oligomers and fibrils are associated with many human pathophysiologies. Compounds that modulate protein aggregation and interact with preformed fibrils and convert them to less toxic species, expect to serve as promising drug candidates and aid to the drug development efforts against aggregation diseases. In present study, the kinetics of amyloid fibril formation by human insulin (HI) and the anti-amyloidogenic activity of ascorbic acid (AA) were investigated by employing various spectroscopic, imaging and computational approaches. We demonstrate that ascorbic acid significantly inhibits the fibrillation of HI in a dose-dependent manner. Interestingly ascorbic acid destabilise the preformed amyloid fibrils and protects human neuroblastoma cell line (SH- SY5Y) against amyloid induced cytotoxicity. The present data signifies the role of ascorbic acid that can serve as potential molecule in preventing human insulin aggregation and associated pathophysiologies.

A multitechnique approach to probe the interaction of a therapeutic tyrosine kinase inhibitor nintedanib and bovine serum albumin.

Drug and protein interaction provides a structural guideline in the rational drug designing and in the synthesis of new and improved drugs with greater efficacy. We have examined here the interaction tendency and mechanism of nintedanib (NTB), an anticancer drug (tyrosine kinase inhibitor) with bovine serum albumin (BSA), by spectroscopic techniques. The decline in Stern-Volmer quenching constants and binding constant with the temperature rise suggests that BSA forms a complex with NTB. Binding constant obtained by modified Stern-Volmer equation at 3 temperatures was realized to be of the order of ~104 M-1. Negative ΔG (~-5.93 kcal mol-1), ΔH (-3.74 kcal mol-1), and ΔS (-1.50 kcal mol-1) values exhibited a spontaneous and exothermic reaction between BSA and NTB. NTB molecule interacts with BSA by forming hydrogen bonds, as elucidated by fluorescence results. Moreover, a minor increment in the helical conformation of BSA upon its binding to NTB was observed by circular dichroism spectroscopy. The modification in protein's symmetry and a decline in hydrodynamic radii were observed in the presence of NTB (from ~3.6 to ~3 nm) as obtained by the dynamic light scattering measurement results.

Aggregation of intrinsically disordered fibrinogen as the influence of backbone conformation.

Fib having intrinsically disordered αC domains is involved in coagulation cascade and thrombosis. Fib molecules forms prefibrillar oligomers at 30%, and associate in 40 and 50% TFE to proceed α to ß transition, suggesting the formation of an intermolecular ß-structure. AFM images confirmed the nature of Fib aggregates at 40 and 50% TFE to be prefibrillar and fibrillar respectively. These aggregates possess high thioflavin T fluorescence with a shifted Congo red absorbance. Kinetics of Fib aggregation data at 50% TFE supports nucleation-dependent polymerization mechanism. At 60 and 70% TFE, no aggregation was observed. The inhibition of protein aggregation appears due to weakening of the hydrophobic interactions that were initially stabilizing the intermolecular ß-sheet structure in the protein aggregation. The loss of hydrophobic contacts seems to favor the formation of intramolecular hydrogen bonds over intermolecular hydrogen bonds leading to helix formation. To conclude, protein aggregation is accompanied by the formation of ß-sheet conformation, and induction of non-native helical segments in the protein inhibits aggregation. The discrepancy of the secondary structures on aggregation is proposed to stem from the disparity in the nature of the hydrogen bonds and packing of hydrophobic residues of the side chains in the ß-sheet and α-helix conformation.

Repositioning nordihydroguaiaretic acid as a potent inhibitor of systemic amyloidosis and associated cellular toxicity.

Although the cure of amyloid related neurodegenerative diseases, non-neuropathic amyloidogenic diseases and non-neuropathic systemic amyloidosis are appealing energetic research attempts, beneficial medication is still to be discovered. There is a need to explore intensely stable therapeutic compounds, potent enough to restrict, disrupt or wipe out such toxic aggregates. We had performed a comprehensive biophysical, computational and cell based assay, that shows Nordihydroguaiaretic acid (NA) not only significantly inhibits heat induced hen egg white lysozyme (HEWL) fibrillation but also disaggregates preformed HEWL fibrils and reduces the cytoxicity of amyloid fibrils as well as disaggregated fibrillar species. The inhibitory potency of NA was determined by an IC50 of 26.3 µM. NA was also found to effectively inhibit human lysozyme (HL) fibrillation. NA interferes in the amyloid fibrillogenesis process by interacting hydrophobically with the amino acid residues found in highly prone amyloid fibril forming region of HEWL as explicated by molecular docking results. The results recommend NA as a probable neuroprotective and promising inhibitor for the therapeutic advancement prospective against amyloid related diseases.

Structural elucidation and molecular characterization of Marinobacter sp. α-amylase.

Halophiles have been perceived as potential source of novel enzymes in recent years. The interest emanates from their ability to catalyze efficiently under high salt and organic solvents. Marinobacter sp. EMB8 α-amylase was found to be active and stable in salt and organic solvents. A study was carried out using circular dichroism (CD), fluorescence spectroscopy, and bioinformatics analysis of similar protein sequence to ascertain molecular basis of salt and solvent adaptability of α-amylase. Structural changes recorded in the presence of varying amounts of NaCl exhibited an increase in negative ellipticity as a function of salt, confirming that salt stabilizes the protein and increases the secondary structure, making it catalytically functional. The data of intrinsic and extrinsic fluorescence (using 1-anilinonaphthalene 8-sulfonate [ANS] as probe) further confirmed the role of salt. The α-amylase was active in the presence of nonpolar solvents, namely, hexane and decane, but inactivated by ethanol. The decrease in the activity was correlated with the loss of tertiary structure in the presence of ethanol. Guanidine hydrochloride and pH denaturation indicated the molten globule state at pH 4.0. Partial N-terminal amino acid sequence of the purified α-amylase revealed the relatedness to Pseudoalteromonas sp. α-amylase. "FVHLFEW" was found as the N-terminal signature sequence. Bioinformatics analysis was done using M. algicola α-amylase protein having the same N-terminal signature sequence. The three-dimensional structure of Marinobacter α-amylase was deduced using the I-TASSER server, which reflected the enrichment of acidic amino acids on the surface, imparting the stability in the presence of salt. Our study clearly indicate that salt is necessary for maintaining the secondary and tertiary structure of halophilic protein, which is a necessary prerequisite for catalysis.

The pharmacological features of bilirubin: the question of the century.

This review looks at the toxicity and metabolism of bilirubin in terms of its pharmacological potential. Its role has gained importance as more research has revealed the functional significance and interrelationship between the gasotransmitters nitric oxide and carbon monoxide. The biological actions of bilirubin have mostly been characterized in the high micromolar range where toxic effects occur. However, it could also prove to be an important cytoprotector for brain tissue, which is inherently less equipped for antioxidant defense. Plasma bilirubin levels negatively correlate to a number of disease states. Higher levels of bilirubin that are still within the normal range provide a protective effect to the body. The effects on various disorders could be tested using controlled pharmacological upregulation of the molecule with animal models. At nanomolar concentrations, considerable benefits have been obtained when the molecule was delivered pharmacologically under in vitro or in vivo test conditions, particularly in neurodegenerative disorders and after tissue or organ transplantation. The induction of heme oxygenase-1 (HMOX-1) via the activation of nuclear factor erythroid 2-related factor or the use of bile pigments in the harvesting of diseased tissue are novel applications, and like every new therapy, should be used with caution. HMOX-1 is tissue specific, and in exceptional states, such as schizophrenia and specific types of renal disorder, the same therapy may have disastrous effects.