Characterization of an equine mannose-binding lectin and its roles in disease

The mannose-binding lectin (MBL), a pattern recognition serum protein, participates in the innate immune system of mammals as an opsonin. In humans, MBL plays a key role in first-line host defense against infection during the lag period prior to the development of a specific immune response. MBL also activates complement via the lectin pathway that requires a MBL-associated serine protease-2 (MASP-2). Homologues of human MBL (hMBL) have been identified in a variety of mammals, fish, and primitive animals such as ascidians. In this study, we report that equine MBL (eMBL) has properties that are similar to hMBL. In addition, we found low levels of MBL:MASP activity in sick horses compared to healthy horses. These results suggest that eMBL is involved in the immune response of the horse and that low MBL:MASP activity could be used to monitor immune function and clinical outcome.     

Biochemical characterization of a novel cycloisomaltooligosaccharide glucanotransferase from Paenibacillus sp. 598K

Cycloisomaltooligosaccharide glucanotransferase (CITase; EC 2.4.1.248), a member of the glycoside hydrolase family 66 (GH66), catalyzes the intramolecular transglucosylation of dextran to produce cycloisomaltooligosaccharides (CIs; cyclodextrans) of varying lengths. Eight CI-producing bacteria have been found; however, CITase from Bacillus circulans T-3040 (CITase-T3040) is the only CI-producing enzyme that has been characterized to date. In this study, we report the gene cloning, enzyme characterization, and analysis of essential Asp and Glu residues of a novel CITase from Paenibacillus sp. 598K (CITase-598K). The cit genes from T-3040 and 598K strains were expressed recombinantly, and the properties of Escherichia coli recombinant enzymes were compared. The two CITases exhibited high primary amino acid sequence identity (67%). The major product of CITase-598K was cycloisomaltoheptaose (CI-7), whereas that of CITase-T3040 was cycloisomaltooctaose (CI-8). Some of the properties of CITase-598K are more favorable for practical use compared with CITase-T3040, i.e., the thermal stability for CITase-598K (≤ 50 °C) was 10 °C higher than that for CITase-T3040 (≤ 40 °C); the k_cat/K_M value of CITase-598K was approximately two times higher (32.2 s^− 1 mM^− 1) than that of CITase-T3040 (17.8 s^− 1 mM^− 1). Isomaltotetraose was the smallest substrate for both CITases. When isomaltoheptaose or smaller substrates were used, a lag time was observed before the intramolecular transglucosylation reaction began. As substrate length increased, the lag time shortened. Catalytically important residues of CITase-598K were predicted to be Asp144, Asp269, and Glu341. These findings will serve as a basis for understanding the reaction mechanism and substrate recognition of GH66 enzymes.     

Neoculin, a taste-modifying sweet protein, accumulates in ripening fruits of cultivated Curculigo latifolia

Neoculin is a sweet protein with a taste-modifying activity of converting sourness to sweetness. It occurs in the fruits of Curculigo latifolia, a wild plant found in tropical Asia. We successfully cultivated the plant and evaluated the production of neoculin. The neoculin content of the fruit was high for 10 weeks after flowering, following which the yield decreased gradually. The optimal period for harvesting the fruits with sensory activity coincided with this 10-week peak period during which the amount of neoculin was 1-3mg in the whole fruit and 1.3mg/g of pulp. Immunohistochemical staining showed that neoculin occurred in the whole fruit, especially at the basal portion. Although it is known that neoculin comprises an acidic subunit (NAS) with an N-glycosylated moiety and a basic subunit (NBS), protein gel blot analysis revealed the presence of a non-glycosylated NAS species. This suggests the presence of multiple NAS-NBS heterodimers in our cultivar.     

Reexamination of the difference in susceptibility to adjuvant-induced arthritis among LEW/Crj, Slc/Wistar/ST and Slc/SD rats.

The present investigations were performed to assess the differences among rat colonies commonly used for neurophysiological research regarding the development of complete Freund's adjuvant (CFA)-induced arthritis. Inflammatory signs including edema in the paw fluctuated remarkably among individual Wistar (Slc/Wistar/ST) and Sprague-Dawley (Slc/SD) rats, while the inflammatory signs of Lewis (LEW/Crj) rats appeared earlier and was severer and more consistent than Slc/Wistar/ST and Slc/SD rats. Edema in the hind paw developed in 100% of LEW/Crj rats with the lowest dose of CFA (0.6 mg/rat) used as compared with 64% of Slc/Wistar/ST (CFA 1 mg/rat) and 38% of Slc/SD rats (CFA 1.2 mg/rat). Retardation of weight gain was observed in Slc/Wistar/ST and Slc/SD rats in contrast to a severe weight decrease in inflamed LEW/Crj rats after the development of arthritis.     

Fe-Si biominerals in the Vilyuchinskie hot springs, Kamchatka Peninsula, Russia.

The micromorphological structure of microbial mats (biomats) from the hot springs of the Vilyuchinskaya hydrothermal system, Kamchatka Peninsula, Russia, were investigated. The Vilyuchinskie hot springs had a discharge temperature of 55-56 degrees C and Na-Ca-HCO3-type waters rich in silicic and boric acids. Water and biomats had high concentrations of Fe, Mn, Sr, and As. Enumeration of total bacterial abundance (TBA) demonstrated a low density of bacterial populations. However, the fractions of metabolically active bacteria and respiring iron-oxidizing bacteria in the hot-spring water were high, comprising 68 and 21% of TBA, respectively. Scanning electron microscopy equipped with an energy dispersive X-ray spectrometer (SEM-EDX) showed that unicellular rod-shaped bacteria about 5-microm long predominated in the brown biomats. The mineral capsules of these bacteria contained large amounts of Fe and Si. Extracellular and intracellular particles were observed by transmission electron microscopy. Fe-oxidizing bacteria were isolated from the biomats on agar plates with selective medium. Therefore, it can be concluded that microorganisms inhabiting the biomats of the Vilyuchinskie hot springs are essential for the deposition of Fe-minerals at neutral pH.     

Production of enokipodins A, B, C, and D: a new group of antimicrobial metabolites from mycelial culture of Flammulina velutipes

Antimicrobial compounds enokipodins A, B, C, and D were originally isolated from the culture filtrates of Flammulina velutipes mycelial culture. Analysis of antibacterial activity by the paper disk method and quantification of enokipodins A–D by high performance liquid chromatography (HPLC) showed that F. velutipes mycelia produced enokipodins A–D in their late growing phase. Great genetic variability in production of these compounds was observed among ten strains of F. velutipes in analyses of antimicrobial activity by the hole-plate diffusion method and quantification by HPLC. Enokipodins A–D demonstrated antimicrobial activity mainly against the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus. Evaluation of minimum inhibitory doses (MIDs) showed that MIDs of enokipodins A and C for B. subtilis were as low as that of the penicillin G antibiotic.     

Amino acid sequence variations of signaling lymphocyte activation molecule and mortality caused by morbillivirus infection in cetaceans

Morbillivirus infection is a severe threat to marine mammals. Mass die‐offs caused by this infection have repeatedly occurred in bottlenose dolphins (Turiops truncatus) and striped dolphins (Stenella coeruleoalba), both of which belong to the family Delphinidae, but not in other cetaceans. However, it is unknown whether sensitivity to the virus varies among cetacean species. The signaling lymphocyte activation molecule (SLAM) is a receptor on host cells that allows morbillivirus invasion and propagation. Its immunoguloblin variable domain‐like (V) region provides an interface for the virus hemagglutinin (H) protein. In this study, variations in the amino acid residues of the V region of 26 cetacean species, covering almost all cetacean genera, were examined. Three‐dimensional (3D) models of them were generated in a homology model using the crystal structure of the marmoset SLAM and measles virus H protein complex as a template. The 3D models showed 32 amino acid residues on the interface that possibly bind the morbillivirus. Among the cetacean species studied, variations were found at six of the residues. Bottlenose and striped dolphins have substitutions at five positions (E68G, I74V, R90H, V126I, and Q130H) compared with those of baleen whales. Three residues (at positions 68, 90 and 130) were found to alternate electric charges, possibly causing changes in affinity for the virus. This study shows a new approach based on receptor structure for assessing potential vulnerability to viral infection. This method may be useful for assessing the risk of morbillivirus infection in wildlife.