Comparison of Guanidine Hydrochloride (GdnHCl) and Urea Denaturation on Inactivation and Unfolding of Human Placental Cystatin (HPC)

The activity and conformational change of human placental cystatin (HPC), a low molecular weight thiol proteinase inhibitor (12,500) has been investigated in presence of guanidine hydrochloride (GdnHCl) and urea. The denaturation of HPC was followed by activity measurements, fluorescence spectroscopy and Circular Dichroism (CD) studies. Increasing the denaturant concentration significantly enhanced the inactivation and unfolding of HPC. The enzyme was 50% inactivated at 1.5 M GdnHCl or 3 M urea. Up to 1.5 M GdnHCl concentration there was quenching of fluorescence intensity compared to native form however at 2 M concentration intensity increased and emission maxima had 5 nm red shift with complete unfolding in 4–6 M range. The mid point of transition was in the region of 1.5–2 M. In case of urea denaturation, the fluorescence intensity increased gradually with increase in the concentration of denaturant. The protein unfolded completely in 6–8 M concentration of urea with a mid-point of transition at 3 M. CD spectroscopy shows that the ellipticity of HPC has increased compared to that of native up to 1.5 M GdnHCl and then there is gradual decrease in ellipticity from 2 to 5 M concentration. At 6 M GdnHCl the protein had random coil conformation. For urea the ellipticity decreases with increase in concentration showing a sigmoidal shaped transition curve with little change up to 1 M urea. The protein greatly loses its structure at 6 M urea and at 8 M it is a random coil. The urea induced denaturation follows two-state rule in which Native→Denatured state transition occurs in a single step whereas in case of GdnHCl, intermediates or non-native states are observed at lower concentrations of denaturant. These intermediate states are possibly due to stabilizing properties of guanidine cation (Gdn⁺) at lower concentrations, whereas at higher concentrations it acts as a classical denaturant.     

Fungal metabolites of (E)-guggulsterone and their antibacterial and radical-scavenging activities

Fungal transformation of (E)-guggulsterone (= (17E)-pregna-4,17-diene-3,16-dione; 1) by Rhizopus stolonifer, Fusarium lini, Cunninghamella elegans, or Gibberella fujikuroi afforded ten hydroxylated metabolites (2-11; Scheme), which were fully characterized. Compounds 4-11 have not been described yet. Some of these novel hydroxylated metabolites, as well as acetylated derivatives thereof, exhibited significant antibacterial and radical-scavenging activities (Table 3).     

Microbial transformations of gelomulide G: a member of the rare class of diterpene lactones

Microbial transformation of gelomulide G (3beta,6beta-diacetoxy-8beta,14beta-epoxyabiet-13(15)-en-16,12-olide, 1) was carried out. Incubation of 1 with Aspergillus niger afforded two new metabolites, 3beta,6beta-diacetoxy-8beta,14beta-dihydroxyabiet-13(15)-en-16,12-olide (2) and 3beta,6beta-diacetoxy-14beta-hydroxyabieta-8(9),13(15)-dien-16,12-olide (3). While Cunninghamella elegans afforded the 14-epimer of 2, i.e., 3beta,6beta-diacetoxy-8beta,14alpha-dihydroxyabiet-13(15)-en-16,12-olide (4), along with 3beta-acetoxy-6beta-hydroxy-8beta,14beta-epoxyabiet-13(15)-en-16,12-olide (5). The structures of the transformed products 2-5 were deduced to be new on the basis of MS and NMR data.     

Stability‐indicating spectrofluorimetric method for the assay of riboflavin and photoproducts: Kinetic applications

A stability‐indicating spectrofluorimetric method has been developed for the simultaneous assay of riboflavin (RF) and photoproducts, formylmethylflavin (FMF), lumichrome (LC) and lumiflavin (LF) in aqueous solution. The method is based on the extraction of LC formed in acid solution and LC and LF formed in alkaline solution with chloroform at pH 2.0 and their assay by fluorescence measurements at 478 and 530 nm, respectively. The aqueous phase, on readjustment of the pH to 6.5, is used to extract FMF with chloroform and its assay is carried out at 530 nm. The aqueous phase is then used for the assay of RF at 530 nm. The proposed method gives more accurate results for the assay of RF compared to those of the United States Pharmacopeia (USP) spectrofluorimetric method which does not take into account the presence of RF photoproducts having similar fluorescence characteristics. The proposed method along with the USP method has been applied to the study of the kinetics of photolysis of RF, assay of stored commercial vitamin preparations and their radiated samples. The results show that the USP method does not distinguish between the fluorescence of RF and its photoproducts, and, therefore, gives erroneous results with about 11% excess in the quantity of the vitamin compared to that of the proposed method. This is due to the interference of the fluorescence of photoproducts in the assay of RF. The method has been validated for various analytical parameters according to the guideline of the International Council for Harmonization (ICH).     

Adaptation strategies to climate change in marine systems

The world's oceans are highly impacted by climate change and other human pressures, with significant implications for marine ecosystems and the livelihoods that they support. Adaptation for both natural and human systems is increasingly important as a coping strategy due to the rate and scale of ongoing and potential future change. Here, we conduct a review of literature concerning specific case studies of adaptation in marine systems, and discuss associated characteristics and influencing factors, including drivers, strategy, timeline, costs, and limitations. We found ample evidence in the literature that shows that marine species are adapting to climate change through shifting distributions and timing of biological events, while evidence for adaptation through evolutionary processes is limited. For human systems, existing studies focus on frameworks and principles of adaptation planning, but examples of implemented adaptation actions and evaluation of outcomes are scarce. These findings highlight potentially useful strategies given specific social–ecological contexts, as well as key barriers and specific information gaps requiring further research and actions.     

Evolutionarily Related Dihydrofolate Reductases Perform Coequal Functions Yet Show Divergence in Their Trajectories

The enzyme dihydrofolate reductase (DHFR) catalyzes NADPH dependent reduction of dihydrofolate to tetrahydrofolate. It plays a crucial role in the DNA synthesis. The investigation of evolution of DHFR generates immense curiosity. It aids in predicting how the enzyme has adapted to the surroundings of various cell types. In spite of great similarity in the structure of E. coli DHFR and human DHFR, their primary sequences are divergent to a great extent, which is evident in variations in the kinetics mechanism of their catalysis. In presence of physiological levels of ligands, they possess distinct kinetics and different rate limiting steps. We have reviewed the process of their unfolding and refolding, their behaviour in denaturing conditions and in presence of various chaperones. Although there is structural similarity between these two homologous enzymes yet they have established distinct mechanisms to accomplish the coequal functions.     

Studies on the hormonal relationships of algae in pure culture

Summary The metabolism of L-tryptophan (methylene-C¹⁴) by Chlorogloea fritschii has been studied in sterile conditions. This organism can produce Indole-3-Acetic acid (IAA) from tryptophan. Tryptamine has been shown to be one of the intermediate compound in the conversion of tryptophan to IAA. The formation of Indole-3-pyruvic acid could not be demonstrated.     

Solvent based optimization for extraction and stability of thymoquinone from <Emphasis Type="Italic">Nigella sativa</Emphasis> Linn. and its quantification using RP-HPLC

The Nigella sativa pharmacological properties are mainly ascribed to its volatile oil, of which thymoquinone is an important bioactive component. Surprisingly, till date, no standard formulation or thymoquinone rich N. sativa extract is under clinical use probably due to its poor extraction and lesser stability in the already used solvents. In the present investigation solubility, extraction, percent composition and total antioxidant activity from the seeds of N. sativa was explored using five solvents. An HPLC method was standardized in an isocratic system (C-18 column, flow rate of 1.0 ml/min, mobile phase—water:methanol: 30:70, detection wavelength—254 nm, retention time—8.77 min) for quantification of thymoquinone. To further confirm the presence of thymoquinone in the respective extracts absorbance spectra analysis has been carried out and compared with pure thymoquinone. Additionally total antioxidant activity of Nigella sativa extracts has been evaluated using ascorbic acid as standard. Our results showed maximum percentage yield in aqueous extract while methanol having the least yield and the ethanol, benzene and hexane extracts exhibited moderate yields. A linear standard calibration curve of thymoquinone showed R² as 0.999 and % RSD as 7.166. The HPLC analysis revealed maximum percentage composition of thymoquinone in the benzene extract, whereas in the hexane and methanol extracts the content was less. Aqueous and ethanol extracts displayed insignificant thymoquinone content. Absorbance spectra analysis confirms the presence of thymoquinone peak in the benzene, hexane and methanol extracts while aqueous and ethanol extracts showed minimal absorbance. Maximum total antioxidant activity was observed in the aqueous extract while minimum was observed in the methanolic extract. Weak positive (+?0.3676) correlation was established between percent composition of thymoquinone and antioxidant activity among different extracts indicating that thymoquinone may not be the only factor for antioxidant activity, but other phytochemicals might also contribute. However, we for the first time demonstrated that the benzene extract of N. sativa has better solubility and percent composition of thymoquinone as compared to other solvents. It can be concluded that the solubility, differential composition of bioactive components among these extracts may have diverse effects on the total antiradical activity. Thus, our study provides insights on optimization and standardization of bioactive rich formulation of N. sativa.     

Protein kinase C and calcium/calmodulin-dependent protein kinase II phosphorylate three-repeat and four-repeat tau isoforms at different rates

All six isoforms of the microtubule-associated protein tau are present in hyperphosphorylated states in the brains of patients with Alzheimer's disease (AD). It is presently unclear how such hyperphosphorylation of tau is controlled. In a previous study (Singh et al. Arch Biochem Biophys 328: 43-50, 1996) we have shown that three-repeat taus containing two N-terminal inserts were phosphorylated to higher levels and at different sites compared to those either lacking or containing only one such insert. We have extended these observations in this study by comparing the phosphorylation of tau isoforms containing three-repeats (t3, t3L) and four-repeats (t4, t4L). In the absence of N-terminal inserts in tau structure (t3, t4) both CaM kinase II and C-kinase phosphorylated four-repeat tau (t4) to a higher extent than three-repeat tau (t3). When two N-terminal inserts are present in tau structure (t3L, t4L), then three-repeat tau (t3L) is phosphorylated to a higher extent than four-repeat tau (t4L) by these kinases. CK-1 and GSK-3 phosphorylated each of the above pairs of three-repeat and four-repeat taus to the same extents. However, after an initial prephosphorylation of the taus by CaM kinase II, GSK-3 differentially phosphorylated three-repeat and four-repeat taus. Under these conditions thr 231, ser 235, ser 396, and ser 404 were phosphorylated to greater extents in four-repeat tau (t4) compared to three-repeat tau (t3) in the absence of N-terminal inserts. In the presence of such inserts these sites were phosphorylated to greater extents in three-repeat (t3L) compared to four-repeat (t4L) tau. Our results indicate that the extents to which tau isoforms are phosphorylated in normal and AD brain depends on (a) the number of repeats (3 or 4), (b) the number of N-terminal inserts (0, 1, or 2), and (c) the initial phosphorylation state of tau.     

Post-thaw plasma membrane integrity of bull spermatozoa separated with a Sephadex ion-exchange column

Previous experiments have established that filtration of bovine semen through a Sephadex ion-exchange column improves its quality before and after freezing. The present study was conducted to determine the post-thaw membrane integrity of bull spermatozoa separated with a Sephadex ion-exchange column and to determine the kind of protection to spermatozoa is provided by glycerol during freezing and thawing. Semen from Holstein bulls diluted in TEST-yolk extender (with and without glycerol) was filtered through a Sephadex ion-exchange column and frozen in liquid nitrogen (-196 degrees C). After thawing, there were more normal acrosomes in filtered spermatozoa than nonfiltered (P < 0.01). Post-thaw plasma membrane integrity and swelling ability in a hypoosmotic solution revealed that the filtered spermatozoa had a stronger (P < 0.005) plasma membranes than the nonfiltered. Filtered spermatozoa demonstrated higher zona-free hamster oocyte penetration than the nonfiltered (30.5 vs 11.5%; P < 0.0005). Spermatozoa extended in TEST-yolk without glycerol had the lowest (P < 0.001) normal acrosomes, intact plasma membranes and swelling ability. Plasma membrane over the post-acrosomal region of the head and post-midpiece region of the tail was more sensitive to damages caused by freezing and thawing than acrosomal and midpiece regions of spermatozoa. Glycerol in the extender provided significant (P < 0.05) protection to the sensitive regions of filtered and nonfiltered spermatozoa during freezing and thawing. Filtered plus glycerolated spermatozoa had the highest (P < 0.01) normal acrosomes, intact plasma membranes and swelling ability. In conclusion, the pre-freezing filtration of bovine semen harvested the spermatozoa possessing stronger plasma membranes which enabled them to endure freezing and thawing stresses. The addition of glycerol to the extender protected the post-acrosomal region of the head and post-midpiece region of the tail of spermatozoa from freezing and thawing shocks.