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.     

Identification of marker genes for intestinal immunomodulating effect of a fructooligosaccharide by DNA microarray analysis

Prebiotic fructooligosaccharides are noted for their intestinal immunodulating effects, and the identification of markers for the effects is a matter of great concern. This study aimed to identify marker genes for physiological effects of a particular fructooligosaccharide (FOS) on a host animal and also to define the target of its function in the small intestine. DNA microarray technology was used to screen candidate marker genes, and comprehensive changes in gene expressions in the ileum of mice fed with FOS were investigated. One of the major physiological effects of FOS was intestinal immunomodulation. Marker genes were then identified for major histocompatibility complex classes I and II, interferon, and phosphatidylinositol metabolites. Also, the ileum was segmented into Peyer's patch (PP) and the other ileal organ (DeltaPP), and these were analyzed by quantitative RT-PCR method, with the result that the site for recognizing the FOS function was the DeltaPP rather than the PP. This is the first paper showing the markers for the physiological effects of FOS in the small intestine at gene expression level. Applying these marker genes would make it possible to clarify the mechanisms of how the administration of dietary FOS and associated changes in the intestinal environment are recognized by host organisms as well as how its immunomodulating effects are expressed in the body.     

Cryptosporidium infection in juvenile pet rabbits

Cryptosporidium infection was confirmed by fecal examination for the first time in pet rabbits in a wholesale store located in Kanagawa Prefecture, Japan. Fecal samples were obtained postmortem from juvenile rabbits (n=66), which had died after developing diarrhea. Feces from healthy rabbits (n=30) were also collected and examined as controls. Two types of Cryptosporidium oocysts distinctive in size and shape were found (Type A and B). Types A and B oocysts were detected from 16.7% and 13.6% of the diarrheic, and 3.3% and 0% of the normal feces, respectively. Since Cryptosporidium oocysts were detected at a higher rate in the diarrheic rabbits than in the healthy rabbits, special caution should be taken when handling a pet rabbit presenting with diarrhea.     

Carrier rate of Factor XI deficiency in stunted Japanese black cattle

Blood examinations and genotyping of Factor XI (F11) were performed in growth retardation Japanese Black cattle and their dams. Genotyping of F11 revealed that the recessive homozygous and heterozygous genotype frequencies were 5.2% and 50.0% in the Claudin-16 (CL-16) deficiency group (n=58), 0% and 14.2% in the renal dysplasia group (n=7), 0% and 26.1% in the non-CL-16 deficiency nephritis group (n=23), 8.9% and 46.7% in the hypogenesis syndrome group (n=45), 6.2% and 25.0% in the neonatal weak calf syndrome group (n=32), 9.1% and 38.6% in the respective dams group (n=44), 0% and 23.1% in the normal cattle group (n=13), and 5.9% and 38.2% in total (n=222), respectively. These results showed that the carrier rate of F11 deficiency was high in Japanese Black cattle, and that the CL-16 deficiency, hypogenesis syndrome, neonatal weak calf syndrome, and dams groups had a large amount of recessive homozygous genotype than the other groups. No abnormal bleeding was observed clinically in the present study, and 4 of the recessive homozygous dams showed normal growth and parturition.     

Carcinogen-induced Thyroid Proliferative Lesions in Wistar Hannover GALAS Rats with Thyroid Dysplasia

Incidences and morphological features of thyroid proliferative lesions induced by carcinogens in Wistar Hannover GALAS rats (GALAS rats) showing normal growth with or without thyroid dysplasia were examined. All thyroid tissue samples were obtained from our recently conducted study using male GALAS rats treated with 5 carcinogens according to the medium-term multiorgan carcinogenicity bioassay protocol (called DMBDD treatment). In the DMBDD-treated rats, thyroid dysplasia was found in 9 out of 114 rats. Follicular cell adenomas were found in 5 out of 9 rats with thyroid dysplasia and in 7 out of 105 rats without thyroid dysplasia. The incidence of adenoma was significantly increased in rats with thyroid dysplasia (55.6%) compared with that in rats without thyroid dysplasia (6.7%). Adenomas in rats with thyroid dysplasia were observed as single or multiple nodules, well demarcated and composed of variously sized vacuolated cells or unvacuolated cells. These histopathological features and staining profiles of luminal colloid for PAS and thyroglobulin, together with PCNA-positive cells, were fundamentally similar to those of rats without thyroid dysplasia. On the other hand, the luminal colloid in adenomas of rats with thyroid dysplasia had a tendency to be poorly stained for T(4) compared with that of rats without thyroid dysplasia. From these findings, it appears that dysplastic thyroids of rats showing normal growth are more sensitive to carcinogens than normal thyroids. In addition, the morphological features of carcinogen-induced thyroid proliferative lesions in GALAS rats with thyroid dysplasia were fundamentally similar to those of rats without thyroid dysplasia, except for the vacuoles and T(4) staining profile.     

The secretory pattern and source of immunoreactive prolactin in pregnant African (Loxodonta africana) and Asian (Elephas maximus) elephants

The objective of the present study was to define the secretion of prolactin (PRL) in pregnant African and Asian elephants. Levels of immunoreactive (ir-) PRL in serum and placental homogenates were measured by a heterologous radioimmunoassay (RIA) based on an ovine and human RIA system, and the localization of ir-PRL in the placenta was detected by immunohistochemistry using anti-human PRL. Circulating ir-PRL clearly showed a biphasic pattern during pregnancy in African and Asian elephants. Serum levels of ir-PRL started to increase from the 4 - 6th month of gestation and reached the first peak level around the 11-14th month. A second peak of circulating ir-PRL levels was observed around the 18-20th month of gestation followed by an abrupt decline after parturition. In contrast, in a case of abortion of an African elephant, the second peak of ir-PRL was not observed, and the levels remained low for about four months until parturition. The weight of the fetus delivered at the 17th month of gestation was 23.5 kg, which was quite small compared with normal fetuses in previous reports. Ir-PRL was detected in placental homogenates, and immunolocalization was observed in trophoblasts in both the African and Asian elephants, indicating that the placenta is the source of ir-PRL during pregnancy in elephants. The present results clearly demonstrated that circulating ir-PRL shows a biphasic pattern during normal pregnancy and that the placenta appears to be an important source of circulating ir-PRL during pregnancy in both African and Asian elephants.     

Relationship between pH and Temperature in the Ruminal Fluid of Cows, Based on a Radio-Transmission pH-Measurement System

To assess the relationship between pH and temperature in the ruminal bottom fluid, circadian changes were monitored using cows fed a control diet (C diet) or a rumen acidosis-inducing diet (RAI diet) by using a wireless radio-transmission pH- measurement system. These two parameters were measured simultaneously at 10-min intervals on day 14 after commencement of feeding. Compared to the mean ruminal pH for 60 min immediately after the morning feeding (0 hr), significantly lower pH was noted 3-13 hr later (P<0.05) and 4-19 hr later (P<0.01) in cows fed the C and RAI diets, respectively, although the reduction in the latter was much higher than that in the former. In contrast, significantly higher ruminal temperature was found at 8 and 12-14 hr later (P<0.05) and 6, 8, and 10-19 hr later (P<0.01) in cows fed the C and RAI diets, respectively. A significant negative correlation was observed between the lowest ruminal pH and its corresponding ruminal temperature in cows fed the C and RAI diets (r=-0.722 and -0.650, P<0.01, respectively), suggesting active fermentation and volatile fatty acid production in the rumen. However, ruminal pH profiles may not be predictable by measuring only ruminal temperature because decreases in ruminal pH were preceded by increases in ruminal temperature, and circadian changes in pH and temperature were associated with ruminal fermentation.     

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.     

The evaluation of the curd forming ability of milk replacers

Inadequate milk curd formation in the abomasum of newborn calves causes malnutrition and diarrhea. In order to define the factors of inadequate curd formation, we compared the curd forming ability among 9 kinds of milk replacers, bulk milk (raw milk), and skim milk both in vitro and in vivo . When rennet was added, the raw milk and one milk replacer formed firm curds. The rest of the milk replacers and skim milk did not form any curd. When a solution of HCl was added, raw milk, three milk replacers and skim milk formed the curd at pH 4.5, but the other milk replacers did not. When HCl was added following the rennet, raw milk, one milk replacer and skim milk formed the curd. In vivo , raw milk, two milk replacers and skim milk showed good curd formation whereas the other milk replacers showed poor curd formation inside the abomasums of the calves. This study showed that most of the milk replacers sold in Japan could not form the curd with rennet.     

Pb/Ca in Thailand Coral Determined by LA-ICP-MS: Anthropogenic Pb Input of River Run-off into a Coral Reef from Urbanised Areas

In order to monitor lead pollution from urban areas to coral reefs in the Gulf of Thailand, linear and two-dimensional distributions of Pb in corals from Khang Khao Island, Thailand and Rukan-sho, Okinawa, were analysed with high resolution. Laser ablation inductively coupled plasma mass spectrometry was applied to measure Pb content in coral skeletons using synthetic Pb standards in a CaCO₃ matrix as calibration materials. Linear and two-dimensional ablation schemes were applied to determine the Pb content in corals collected from Khang Khao Island, Thailand and Rukan-sho, Okinawa. The coral skeleton was ablated by Nd:YAG laser (wavelength of 266 nm, beam diameter of 155 µm, scanning speed of 10 µm s^?1 and frequency of 10 Hz) along the growth axis, and ion counts for ²⁰⁸Pb were normalised to ⁴²Ca. Lead content in the corals was determined using a calibration curve obtained from the synthetic Pb standards (0–141 µg g^?1). The linear ablation track of the sample from Khang Khao showed over 30 peaks of Pb with an average value of 3.55 µg g^?1, while Pb content in the coral from Rukan-sho showed relatively small variation with an average value of 0.132 µg g^?1. Two-dimensional imaging of Pb in the coral skeletons was also carried out for an area of 7?×?20 mm on the sample from Khang Khao. The distribution patterns of Pb and Ba in the Thailand coral co-varied. These observations from the linear and imaging analyses suggest discontinuous inputs of anthropogenic Pb from rivers to the Gulf of Thailand.