High-resolution structure of a partially folded insulin aggregation intermediate

Insulin has long been served as a model for protein aggregation, both due to the importance of aggregation in the manufacture of insulin and because the structural biology of insulin has been extensively characterized. Despite intensive study, details about the initial triggers for aggregation have remained elusive at the molecular level. We show here that at acidic pH, the aggregation of insulin is likely initiated by a partially folded monomeric intermediate. High-resolution structures of the partially folded intermediate show that it is coarsely similar to the initial monomeric structure but differs in subtle details—the A chain helices on the receptor interface are more disordered and the B chain helix is displaced from the C-terminal A chain helix when compared to the stable monomer. The result of these movements is the creation of a hydrophobic cavity in the center of the protein that may serve as nucleation site for oligomer formation. Knowledge of this transition may aid in the engineering of insulin variants that retain the favorable pharamacokinetic properties of monomeric insulin but are more resistant to aggregation.     

Synthesis, crystal structure, magnetic property and oxidative DNA cleavage activity of an octanuclear copper(II) complex showing water-perchlorate helical network

A new octanuclear copper(II) complex has been synthesized and structurally characterized by X-ray crystallography: [Cu(8)(HL)(4)(OH)(4)(H(2)O)(2)(ClO(4))(2)].(ClO(4))(2).2H(2)O (1) (H(3)L=2,6-bis(hydroxyethyliminoethyl)-4-methyl phenol). The complex is formed by the linkage of two terminal bimetallic cationic units and a tetranuclear mu(3)-hydroxo bridged dicubane core by a very short intramolecular hydrogen bond (O-H...O, 1.48(3)A and the angle 175 degrees). The coordination sphere of the terminal copper atoms is square pyramidal, the apical positions being occupied by water and a perchlorate ion. Complex 1 self-assembles to form a new type of water-perchlorate helical network [(H(2)O)(2)(ClO(4))](infinity) involving oxygen atoms of coordinated perchlorate ion and the two lattice water molecules through hydrogen-bonding interaction. The variable temperature-dependent susceptibility measurement (2-300K) of 1 reveals a strong antiferromagnetic coupling, J(1)=-220cm(-1) and J(2)=-98cm(-1) (J(1) and J(2) representing the exchange constant within [Cu(2+)](4) and [Cu(2+)](2) units, respectively). The complex binds to double-stranded supercoiled plasmid DNA giving a K(app) value of 1.2x10(7)M(-1) and displays efficient oxidative cleavage of supercoiled DNA in the presence of H(2)O(2) following a hydroxyl radical pathway.     

Focused Screening and Treatment (FSAT): A PCR-Based Strategy to Detect Malaria Parasite Carriers and Contain Drug Resistant P. falciparum, Pailin, Cambodia

Recent studies have shown that Plasmodium falciparum malaria parasites in Pailin province, along the border between Thailand and Cambodia, have become resistant to artemisinin derivatives. To better define the epidemiology of P. falciparum populations and to assess the risk of the possible spread of these parasites outside Pailin, a new epidemiological tool named “Focused Screening and Treatment” (FSAT), based on active molecular detection of asymptomatic parasite carriers was introduced in 2010. Cross-sectional malariometric surveys using PCR were carried out in 20 out of 109 villages in Pailin province. Individuals detected as P. falciparum carriers were treated with atovaquone-proguanil combination plus a single dose of primaquine if the patient was non-G6PD deficient. Interviews were conducted to elicit history of cross-border travel that might contribute to the spread of artemisinin-resistant parasites. After directly observed treatment, patients were followed up and re-examined on day 7 and day 28. Among 6931 individuals screened, prevalence of P. falciparum carriers was less than 1%, of whom 96% were asymptomatic. Only 1.6% of the individuals had a travel history or plans to go outside Cambodia, with none of those tested being positive for P. falciparum. Retrospective analysis, using 2010 routine surveillance data, showed significant differences in the prevalence of asymptomatic carriers discovered by FSAT between villages classified as “high risk” and “low risk” based on malaria incidence data. All positive individuals treated and followed-up until day 28 were cured. No mutant-type allele related to atovaquone resistance was found. FSAT is a potentially useful tool to detect, treat and track clusters of asymptomatic carriers of P. falciparum along with providing valuable epidemiological information regarding cross-border movements of potential malaria parasite carriers and parasite gene flow.     

Lactobacillus rhamnosus GG secreting an antigen and Interleukin-2 translocates across the gastrointestinal tract and induces an antigen specific immune response

Lactobacillus rhamnosus strain GG (LGG) is a probiotic organism. In this present study, LGG that express the green fluorescence protein (LGG-GFP) and IL-2 and GFP as a fusion protein (LGG-IL-2-GFP) were used to examine bacterial uptake and the immune response induced by oral immunization. Using TEM to examine the intestinal tissue, the Lactobacilli were localized in M cells and in venules. After oral immunization, most of the bacteria were excreted in feces only a small fraction (0.15%) was retained in the intestine at 48 hr. However, more LGG-IL-2-GFP was found in the MLN and spleen than LGG-GFP. The loop ligation method was used to evaluate LGG uptake and both LGG-GFP and LGG-IL-2-GFP were found to translocate at the same rate. Analysis of LGG internalization in J774 macrophage cells indicated that IL-2 increased survival of LGG and this may explain the increased presence of these bacteria in the MLN for a longer period. After oral immunization, specific mucosal antibody production as well as GFP specific CTL activity was demonstrated. IL-2 co-expression with GFP further enhanced antibody production and CTL activity. In conclusion, Lactobacillus rhamnosus GG expressing an antigen could generate an effective immune response to the antigen and IL-2 improved the response generated probably by increasing LGG expressing antigen survival in immune cells.     

Unusual hepatic mitochondrial arginase in an Indian air-breathing teleost,Heteropneustes fossilis: Purification and characterization

A functional urea cycle with both cytosolic (ARG I) and mitochondrial (ARG II) arginase activity is present in the liver of an ureogenic air-breathing teleost, Heteropneustes fossilis. Antibodies against mammalian ARG II showed no cross-reactivity with the H. fossilis ARG II. ARG II was purified to homogeneity from H. fossilis liver. Purified ARG II showed a native molecular mass of 96 kDa. SDS–PAGE showed a major band at 48 kDa. The native enzyme, therefore, appears to be a homodimer. The pI value of the enzyme was 7.5. The purified enzyme showed maximum activity at pH 10.5 and 55 °C. The K_m of purified ARG II for l-arginine was 5.25 ± 1.12 mM. l-Ornithine and N^ω-hydroxy-l-arginine showed mixed inhibition with K_i values 2.16 ± 0.08 and 0.02 ± 0.004 mM respectively. Mn^+ 2 and Co^+ 2 were effective activators of arginase activity. Antibody raised against purified H. fossilis ARG II did not cross-react with fish ARG I, and mammalian ARG I and ARG II. Western blot with the antibodies against purified H. fossilis hepatic ARG II showed cross reactivity with a 96 kDa band on native PAGE and a 48 kDa band on SDS–PAGE. The molecular, immunological and kinetic properties suggest uniqueness of the hepatic mitochondrial ARG II in H. fossilis.