Glycation and phosphorylation of beta-lactoglobulin by dry-heating: effect on protein structure and some properties

Beta-lactoglobulin (beta-Lg) was glycated with maltopentaose and subsequently phosphorylated by dry-heating in the presence of pyrophosphate to investigate the structural and functional properties of phosphorylated beta-Lg. The circular dichroism spectra showed that the change of the secondary structure in the beta-Lg molecule by glycation and subsequent phosphorylation was small. The differential scanning calorimetry thermograms of beta-Lg showed that the denaturation temperature of the most stable domain was only slightly affected, whereas the retinol-binding activity of beta-Lg was somewhat reduced by glycation and subsequent phosphorylation. These results indicated that the conformational changes of the beta-Lg molecule by glycation and subsequent phosphorylation were mild. The anti-beta-Lg antibody response was somewhat reduced by glycation, but significant changes were not observed by phosphorylation. Although the stability of beta-Lg against heat-induced insolubility was improved by glycation alone, it was further enhanced by phosphorylation. The calcium phosphate solubilizing ability of beta-Lg was enhanced by phosphorylation following glycation.     

Improvement in isolation and identification of food-derived peptides in human plasma based on precolumn derivatization of peptides with phenyl isothiocyanate

For the isolation and detection of food-derived peptides in blood, an approach based on the derivatization of peptides with phenyl isothiocyanate (PITC) was developed. This approach allows hydrophilic peptides to be resolved and specifically detected by reversed-phase (RP) HPLC. For the rapid capturing and clarification of peptides in human plasma, solid-phase extraction by using a mini spin column (5 mmx5 mm) packed with a strong cation exchanger was used. The clarified peptide fraction was further fractionated by size-exclusion chromatography (SEC). The peptides in the SEC fractions were derivatized with PITC, and the derivatives were resolved by RP-HPLC by using an ammonium acetate buffer or a trifluoroacetic acid system. An automatic peptide sequencer based on Edman degradation with a modified program can directly analyze the resolved derivatives. Some synthetic peptides and food-derived peptides in human plasma were successfully isolated and identified by this approach.     

Storage of maitake mushroom (Grifola frondosa) culture medium after harvesting fruit bodies is an effective pretreatment for ethanol conversion

The storage of spent maitake culture medium (SMCM) under various conditions was investigated as a potential pretreatment of SMCM for increased ethanol conversion. When SMCM was stored at 25°C for 12?weeks, the glucose yield by enzymatic saccharification increased from 22 to 52%. Selective degradation of lignin and hemicellulose occurred in SMCM during storage. The optimal storage temperature of SMCM was at the active growing temperature of maitake mycelium, which is between 25 and 30°C. Storage of SMCM under anaerobic conditions did not increase the glucose yield compared to non-stored conditions. The glucose yield from the SMCM stored for 4?weeks at 25°C was increased by about 30% with either NaOH or vibrating ball milling pretreatment. After 12?weeks of storage, the glucose yield from SMCM without any other pretreatment was higher than that of non-stored SMCM with additional pretreatments. An ethanol yield of 42.1% was obtained from SMCM stored for 12?weeks by simultaneous saccharification and fermentation, which was comparable to yields after NaOH (41.3%) or vibrating ball milling (44.1%) pretreatments. Therefore, storage of SMCM is a very useful pretreatment for bioethanol production.     

CONTRAST AGENT Gd‐EOB‐DTPA (EOB·Primovist®) FOR LOW‐FIELD MAGNETIC RESONANCE IMAGING OF CANINE FOCAL LIVER LESIONS

Contrast‐enhanced magnetic resonance (MR) imaging with a new liver‐specific contrast agent gadolinium‐ethoxybenzyl‐diethylenetriamine penta‐acetic acid (Gd‐EOB‐DTPA; EOB·Primovist®) was studied in 14 normal beagles and 9 dogs with focal liver lesions. Gd‐EOB‐DTPA accumulates in normally functioning hepatocytes 20 min after injection. As with Gd‐DTPA, it is also possible to perform a dynamic multiphasic examination of the liver with Gd‐EOB‐DTPA, including an arterial phase and a portal venous phase. First, a reliable protocol was developed and the appropriate timings for the dynamic study and the parenchymal phase in normal dogs using Gd‐EOB‐DTPA were determined. Second, the patterns of these images were evaluated in patient dogs with hepatic masses. The optimal time of arterial imaging was from 15 s after injection, and the optimal time for portal venous imaging was from 40 s after injection. Meanwhile, the optimal time to observe changes during the hepatobiliary phase was from 20 min after injection. In patient dogs, 11 lesions were diagnosed as malignant tumors; all were hypointense to the surrounding normal liver parenchyma during the hepatobiliary phase. Even with a low‐field MR imaging unit, the sequences afforded images adequate to visualize the liver parenchyma and to detect tumors within an appropriate scan time. Contrast‐enhanced MR imaging with Gd‐EOB‐DTPA provides good demarcation on low‐field MR imaging for diagnosing canine focal liver lesions.     

Expression of genes related to corticotropin production and glucocorticoid feedback in corticotroph adenomas of dogs with Cushing's disease

Cushing's disease caused by pituitary corticotroph adenoma is a common endocrine disease in dogs. A characteristic biochemical feature of corticotroph adenomas is their relative resistance to negative feedback by glucocorticoids. In this study, we examined gene expression related to adrenocorticotropic hormone (ACTH) production and secretion, and the negative feedback by glucocorticoids in canine corticotroph adenoma. We used resected corticotroph adenomas from 10 dogs with Cushing's disease. In order to investigate the alteration of gene expression between corticotroph adenoma and normal corticotrophic cells, ACTH-positive cells in the anterior lobe were microdissected using a laser-capture microdissection system, and mRNA levels of proopiomelanocortin (POMC), corticotropin releasing hormone receptor 1 (CRHR1), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and 11 beta hydroxysteroid dehydrogenase (11HSD) type 1 and type 2 were determined using real-time RT-PCR. POMC, CRHR1, and 11HSD2 mRNA levels in corticotroph adenoma were greater than those in normal corticotrophic cells (POMC, 5.5-fold; CRHR1, 4.9-fold; 11HSD2, 4.2-fold, P<0.01, respectively). MR and 11HSD1 mRNA levels in corticotroph adenoma were lower than those in normal corticotrophic cells (MR, 2.2-fold; 11HSD1, 2.9-fold, P<0.01, respectively). GR mRNA levels did not differ between corticotroph adenoma and normal corticotrophic cells. Our results may help to understand the increased ACTH production and the resistance to negative feedback suppression by glucocorticoids in canine corticotroph adenomas. These changes in gene expression may have a role in the growth of canine corticotroph adenoma, and help elucidate the pathophysiology of dogs with Cushing's disease.     

Plasma hormone and metabolite concentrations involved in the somatotropic axis of Japanese Black heifers in association with growth hormone gene polymorphism

Bovine growth hormone (bGH) gene polymorphism of leucine (Leu)-threonine (Thr) (allele A), valine (Val)-Thr (allele B), and Val-methionine (Met) (allele C) at codons 127 and 172 was shown to relate with carcass trait variations in Japanese Black cattle. In this study, 10-mo-old Japanese Black heifers with growth hormone (GH) genotypes AA, AB, BB, AC, BC, and CC (N = 141) were compared for basal GH, insulin-like growth factor-1 (IGF-1), insulin, ghrelin, glucose, and nonesterified fatty acid (NEFA) concentrations. Growth hormone release was also measured as response to growth hormone–releasing hormone (GHRH) (0.4 μg/kg body weight [BW]) using 18 heifers with GH genotypes AA, BB, and CC (n = 6 for each group). The genotype AA heifers showed the greatest BW among genotypes (P < 0.05). Genotype AC, BC, and CC heifers showed greater GH concentrations than genotype AA, AB, or BB heifers, in which genotype CC heifers had the highest concentrations (P < 0.05). However, IGF-1 concentrations did not significantly differ. The genotype AA and BB heifers had a greater GH release at 60 min following GHRH injection than did the genotype CC heifers. The area under the curve (AUC; P < 0.07) and incremental area (IA; P < 0.08) of GH responses to the GHRH challenge tended to be the highest in the genotype AA heifers and the lowest in the genotype CC heifers. In conclusion, GH gene polymorphism altered GH, which may have contributed to differences in BW and carcass traits among genotypes.     

Scallop shell extract promotes recovery from UV-B-induced damage in rat skin epidermal layer

ABSTRACT:   It has been previously reported that scallop shell extract can protect keratinocyte cells against UV-B-induced damage in vitro by acting as an antioxidant and a growth promoter. To establish the growth promoting activity of scallop shell extract, the effect was investigated in vivo. Exposure of rat dorsal skin to UV-B caused the formation of erythema and eschar. Either the scallop shell extract or vehicle (water) was applied once a day for 5 days to the injured dorsal area and the area of the erythema and eschar was estimated using National Institute of Health (NIH) image software. Wound healing of the erythema and eschar was clearly promoted after application of scallop shell extract when compared to application of the vehicle control. Histological studies indicated that scallop shell extract promoted the recovery of the epidermal layer. These results suggest that scallop shell extract activates rat keratinocyte cells and promotes the turnover of skin stratum corneum. This conclusion was also supported by measurement of the turnover rate of the stratum corneum. Therefore, scallop shell extract may be effective in protecting skin against UV-B.     

Dairy cattle behavior classifications based on decision tree learning using 3‐axis neck‐mounted accelerometers

Demand has been increasing recently for an automated monitoring system of animal behavior as a tool for the management of livestock animals. This study investigated the association between the behavior of dairy cattle and the acceleration data collected using three‐axis neck‐mounted accelerometers, as well as the feasibility of improving the precision of behavior classifications through machine learning. In total 38 Holstein dairy cows were used, and kept in four different farms. A logger was mounted to each collar to obtain acceleration data for calculating the activity level and variations. At the same time the behavior of the cattle was observed visually. Characteristic acceleration waves were recorded for eating, rumination, and lying, respectively; and the activity level and variations were significantly different among these behaviors (p < 0.01). Decision tree learning was performed on the data set from Farm A and validated its precision; which proved to be 99.2% in cross‐validation, and 100% in test data sets from Farms B to D. This study showed that highly precise classifications for eating, rumination, and lying is possible by using decision tree learning to calculate the activity level and variations of cattle based on the data obtained by three‐axis accelerometers mounted to a collar.