Aldimine to ketoamine isomerization (Amadori rearrangement) potential at the individual nonenzymic glycation sites of hemoglobin a: Preferential inhibition of glycation by nucleophiles at sites of low isomerization potential

The relative roles of the two structural aspects of nonenzymic glycation sites of hemoglobin A, namely the ease with which the amino groups could form the aldimine adducts and the propensity of the microenvironments of the respective aldimines to facilitate the Amadori rearrangement, in dictating the site selectivity of nonenzymic glycation with aldotriose has been investigated. The chemical reactivity of the amino groups of hemoglobin A forin vitro reductive glycation with aldotriose is distinct from that in the nonreductive mode. The reactivity of amino groups of hemoglobin A toward reductive glycation (i.e., propensity for aldimine formation) decreases in the order Val-1(), Val-1(), Lys-66(), Lys-61(), and Lys-16(). The overall reactivity of hemoglobin A toward nonreductive glycation decreased in the order Lys-16(), Val-1(), Lys-66(), Lys-82(), Lys-61(), and Val-1(). Since the aldimine is the common intermediate for both the reductive and nonreductive modification, the differential selectivity of protein for the two modes of glycation is clearly a reflection of the propensity of the microenvironments of nonenzymic glycation sites to facilitate the isomerization reaction (i.e., Amadori rearrangement). A semiquantitative estimate of this propensity of the microenvironment of the nonenzymic glycation sites has been obtained by comparing the nonreductive (nonenzymic) and reductive modification at individual glycation sites. The microenvironment of Lys-16() is very efficient in facilitating the rearrangement and the relative efficiency decreases in the order Lys-16(), Lys-82(), Lys-66(), Lys-61(), Val-1(), and Val-1(). The propensity of the microenvironment of Lys-16() to facilitate the Amadori rearrangement of the aldimine is about three orders of magnitude higher than that of Val-1() and is about 50 times higher than that of Val-1(). The extent of nonenzymic glycation at the individual sites is modulated by various factors, such as thepH, concentration of aldotriose, and the concentration of the protein. The nucleophiles—such as tris, glycine ethyl ester, and amino guanidine—inhibit the glycation by trapping the aldotriose. The nonenzymic glycation inhibitory power of nucleophile is directly related to its propensity to form aldimine. Thus, the extent of inhibition of nonenzymic glycation at a given site by a nucleophile directly reflects the relative role ofpK _a of the site in dictating the glycation at that site. The nonenzymic glycation of an amino group of a protein is an additive/synergestic consequence of the propensity of the site to form aldimine adducts on one hand, and the propensity of its microenvironment to facilitate the isomerization of the aldimines to ketoamines on the other. The isomerization potential of microenvironment plays the dominant role in dictating the site specificity of the nonenzymic glycation of proteins.     

Role of verocytotoxigenic Escherichia coli in cattle and buffalo calf diarrhoea

Abstract 273 Strains of Escherichia coli isolated from diarrhoeic and healthy control cattle and buffalo calves in Sri Lanka, were tested for Verocytotoxin (VT) and for heat-stable (ST) and heat-labile (LT) enterotoxins. VT and ST toxigenic E. coli were significantly associated with diarrhoea, accounting for 28% and 18% of diarrhoeic episodes, respectively. LT toxigenic E. coli were not significantly associated with diarrhoea.     

N-terminal and Central Segments of the Type 1 Ryanodine Receptor Mediate Its Interaction with FK506-binding Proteins

We used site-directed labeling of the type 1 ryanodine receptor (RyR1) and fluorescence resonance energy transfer (FRET) measurements to map RyR1 sequence elements forming the binding site of the 12-kDa binding protein for the immunosuppressant drug, FK506. This protein, FKBP12, promotes the RyR1 closed state, thereby inhibiting Ca²⁺ leakage in resting muscle. Although FKBP12 function is well established, its binding determinants within the RyR1 protein sequence remain unresolved. To identify these sequence determinants using FRET, we created five single-Cys FKBP variants labeled with Alexa Fluor 488 (denoted D-FKBP) and then targeted these D-FKBPs to full-length RyR1 constructs containing decahistidine (His₁₀) “tags” placed within N-terminal (amino acid residues 76–619) or central (residues 2157–2777) regions of RyR1. The FRET acceptor Cy3NTA bound specifically and saturably to these His tags, allowing distance analysis of FRET measured from each D-FKBP variant to Cy3NTA bound to each His tag. Results indicate that D-FKBP binds proximal to both N-terminal and central domains of RyR1, thus suggesting that the FKBP binding site is composed of determinants from both regions. These findings further imply that the RyR1 N-terminal and central domains are proximal to one another, a core premise of the domain-switch hypothesis of RyR function. We observed FRET from GFP fused at position 620 within the N-terminal domain to central domain His-tagged sites, thus further supporting this hypothesis. Taken together, these results support the conclusion that N-terminal and central domain elements are closely apposed near the FKBP binding site within the RyR1 three-dimensional structure.     

The contribution of endophytic bacteria to Albizia lebbeck-mediated phytoremediation of tannery effluent contaminated soil

Toxicity of chromium often impairs the remediation capacity of plants used in phytoremediation of polluted soils. In this study, we have identified Albizia lebbeck as a prospective chromium hyperaccumulator and examined cultivable diversity of endophytes present in chromium-treated and control saplings. High numbers (22–100%) of endophytic bacteria, isolated from root, stem, and leaf tissues, could tolerate elevated (1–3 mM) concentrations of K₂CrO₇. 16S rRNA gene sequence-based phylogenetic analysis showed that the 118 isolates obtained comprised of 17 operational taxonomic units affiliated with the proteobacterial genera Rhizobium (18%), Marinomonas (1%), Pseudomonas (16%), and Xanthomonas (7%) but also with members of Firmicutes genera, such as Bacillus (35%) and Salinococcus (3%). The novel isolates belonging to Salinococcus and Bacillus could tolerate high K₂CrO₇ concentrations (3 mM) and also showed elevated activity of chromate reductase. In addition, majority (%) of the endophytic isolates also showed production of indole-3-acetic acid. Taken together, our results indicate that the innate endophytic bacterial community assists plants in reducing heavy metal toxicity.     

2,4,6-Triphenylaniline nanoemulsion formulation,optimization, and its application in type 2 diabetes mellitus

The secondary metabolite 2,4,6-triphenylaniline (TPA) was isolated from an endophytic fungi Alternaria longipes strain VITN14G of mangrove plant Avicennia officinalis, that exhibited satisfactory in vitro antidiabetic activity for type 2 diabetes mellitus (T2DM). The TPA was encapsulated using nanoemulsion (NE) to overcome the problem of stability and permeability to increase its therapeutic applications. Response surface methodology (RSM) was used for the optimization of the variables given, such as hydrodynamic diameter, surface charge, and polydispersity index (PDI). TPA was encapsulated using an optimized ratio of olive oil and tween 80 (2:1) significantly affected the response variables including particle size (124.8 nm), ζ potential (−46.0 mV), and PDI (0.396), and the encapsulation efficiency was found to be 95.93%. The TPA-loaded NE after MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) analysis showed nontoxic effects on L929 normal cell lines (areolar and adipose subcutaneous connective tissue of Mus musculus) with a viable percentage of 92%. In vitro release study revealed the slow and sustained release of the TPA over 48 hrs from NE under the Fickian diffusion mechanism and followed the Higuchi model for release kinetics. Further, the percentage of α-glucosidase and α-amylase inhibition rate of TPA-loaded NE was found to be 78.5 and 43.42%, respectively. The present study, therefore, can aid in the development of a novel drug delivery system as a therapeutic approach to T2DM.     

A non-canonical transferable signal mediates nuclear import of simian immunodeficiency virus Vpx protein

Protein transport into the nucleus is generally considered to involve specific nuclear localization signals (NLS) though it is becoming increasingly evident that efficient and well controlled import of proteins which lack a canonical NLS also occurs in cells. Vpx, a 112 amino acid protein from human immunodeficiency virus type 2 (HIV-2) and the closely related simian immunodeficiency virus (SIV) is one such protein, which does not have an identifiable canonical NLS and is yet efficiently imported to the nuclear compartment. Here we report that Vpx protein is imported to the nucleus independently of virus-encoded cofactors. When fusions of truncated versions of Vpx with full-length beta-galactosidase (beta-Gal) were tested, the region from Vpx 61 to 80 was found to be sufficient to mediate the import of the heterologous cytoplasmic protein to the nucleus. Inactivation of Vpx NLS precluded nuclear import of Vpx and reduced virus replication in non-dividing macrophage cultures, even when functional integrase and Gag matrix proteins implicated in viral nuclear import were present. Importantly, we identified and characterized a novel type of 20 amino acid transferable nuclear import signal in Vpx that is distinct from other import signals described. In addition, we show that the minimal nuclear targeting domain identified here overlaps with helical domain III (amino acid (aa) 64-82) and the structural integrity of this helical motif is critical for the nuclear import of Vpx. Taken together, these data suggest that Vpx is imported to the nucleus via a novel import pathway that is dependent on its 20 amino acid unique nuclear targeting signal, and that the nuclear import property of Vpx is critical for the optimal virus replication in non-dividing cells such as macrophages.