Insulin-like growth factor-1 induces IRE1-XBP1-dependent endoplasmic reticulum biogenesis in bovine mammary epithelial cells.

Insulin-like growth factor-1 (IGF-1) plays a key role in proliferation and galactopoiesis in mammary epithelial cells (MEC), but its definitive functions on endoplasmic reticulum (ER) during protein synthesis remain unknown. The present study aimed to elucidate the effects of IGF-1 on ER biogenesis in MEC in vitro and examined the expression of ER biogenesis-associated genes in the mammary gland during early lactation. We treated mammary alveolar cells-large T antigen cells (immortalized bovine MEC line established via stable transfection with simian virus-40 large T-antigen) with IGF-1 and examined ER biogenesis using the fluorescence intensity of an ER tracker and quantitative real-time PCR. We found IGF-1 significantly increased ER tracker staining and upregulated mRNA levels of ER biogenesis-related genes, such as CHKA (choline kinase α), PCYT1A (choline-phosphate cytidylyltransferase A), and SURF4 (surfeit locus protein 4). We focused on unfolded protein response to explore molecular mechanisms by which IGF-1 induces ER biogenesis. We found IGF-1 significantly increased mRNA levels of the XBP1 splicing form (XBP1s). Based on western blot analysis, IGF-1 induced the expression of (inositol-requiring kinase 1 α) protein, upstream of XBP1s, and phosphorylated-IRE1α. The inhibition of IRE1 endoribonuclease activity with 4-methylumbelliferone 8-carbaldehyde (4µ8C) significantly suppressed the increase in ER tracker fluorescence and ER biogenesis-related gene expression induced by IGF-1. Also, IGF-1-induced XBP1s and ER biogenesis-associated gene expression was inhibited by rapamycin, an inhibitor of mTORC1 (mammalian target of rapamycin complex 1), indicating that IRE1-XBP1 activation by IGF-1 is mediated by mTORC1. Moreover, to clarify the expression of XBP1s and ER biogenesis-associated genes expression under normal physiological conditions, mammary gland tissue from biopsies of dairy cows during late gestation and lactation were analyzed. In vivo data highlighted the significant increases in the mRNA levels of XBP1s and ER biogenesis-related genes in mammary gland tissue immediately after calving through 6 wk of lactation. The mRNA levels of IGF1R (IGF-1 receptor) in mammary glands increased during 6 wk of lactation. Therefore, the present study indicated for the first time that IGF-1 induces ER biogenesis by activating the IRE1-XBP1 axis under the regulation of mTORC1 in bovine MEC line.

The unfolded protein response is involved in both differentiation and apoptosis of bovine mammary epithelial cells.

The unfolded protein response (UPR) describes a process involved in the homeostasis of endoplasmic reticulum (ER) and the differentiation of secretory cells. At present, the roles of UPR in the mammary gland tissue of dairy cattle are unknown. In the current study, we investigated the expression of UPR-related genes in Holstein cows during the developmental and lactating stages of the mammary gland tissue. To investigate the roles of UPR during the differentiation of mammary epithelial cells (MEC), we used MAC-T cells, a line of MEC. We collected samples of mammary gland tissue in dairy cows by biopsy during the late gestation and lactation periods and examined the expression of UPR-related genes by quantitative real-time PCR. Expression levels of the spliced X-box binding protein 1 (XBP1) and activating transcription factor 4 (ATF4) were found to be significantly higher in the mammary gland tissue 10 d before delivery compared with 40 d before delivery. An investigation before and after differentiation in MAC-T cells showed that the expression of ATF4 increased after differentiation of MEC, whereas that of the spliced XBP1 did not significantly change. Western blot analysis revealed that the differentiation-inducing stimulus induced phosphorylation of eukaryotic initiation factor 2α (eIF2α) but reduced that of protein kinase RNA-like endoplasmic reticulum kinase (PERK). Additionally, in ATF4-knockdown bovine MEC, differentiation was significantly suppressed; ATF4 knockdown also significantly suppressed the expression of glucocorticoid and insulin receptors. These results revealed that ER stress-independent ATF4 is involved in the cell differentiation mechanism, either directly or indirectly, via the control of the expression of lactogenic hormone receptors in bovine MEC. Immediately after parturition, gene expression levels of the spliced XBP1, ATF4, and C/EBP homologous protein (CHOP) markedly increased in mammary gland tissue, with a strong negative correlation between expression of CHOP and initial milk yield; CHOP is an apoptosis-related protein induced by ER stress. The above findings indicate that UPR is intrinsically associated with apoptosis of MEC, thus affecting the differentiation of these cells, as well as milk yield.

Anthocyanin and proanthocyanidin contents, antioxidant activity, and in situ degradability of black and red rice grains.

OBJECTIVE: An experiment was conducted to assess the antioxidant contents and activities of colored rice grains and to evaluate their nutritive characteristics in terms of chemical composition and in situ ruminal degradation. METHODS: Ten cultivars of colored rice grains (Oryza sativa L.) collected from several areas of Japan were studied, and control rice without pigment, maize, barley, and wheat grains were used as control grains. Their chemical compositions, pigment, polyphenol contents, total antioxidant capacity (TAC), and degradation characteristics were determined. RESULTS: The starch contents of the colored rice grains were in the range of 73.5% to 79.6%, similar to that of the control rice grain. The black and red rice grains contained anthocyanin (maximum: 5,045.6 µg/g) and proanthocyanidin (maximum: 3,060.6 µg/g) at high concentrations as their principal pigments, respectively. There were significantly (p<0.05) positive relationships among the pigment contents, polyphenol content, and TAC values in the colored and control rice grains, indicating that the increase in pigment contents also contributed to the increased polyphenol content and TAC values in the colored rice grains. The dry matter and starch degradation characteristics, as represented by c (fractional degradation rate of slowly degradable fraction) and by the effective degradability, of the colored rice grains and the control rice grain were ranked as follows among commonly used grains: wheat>barley ≥rice>maize. The colored rice grains also included the most-digestible starch, since their potential degradable fraction and actual degradability at 48 h incubation were almost 100%. CONCLUSION: Colored rice grains have high potential to be used as antioxidant sources in addition to starch sources in ruminants.

Effect of harvesting strategy of second-cut orchardgrass silage on feed intake, digestion, and milk production in dairy cows.

We investigated the effects of regrowth interval and first-cut timing on the dietary characteristics of second-cut orchardgrass silage and feed intake and milk production in dairy cows fed second-cut orchardgrass silage. The second-cut grasses were harvested 7w after the first-cut at the early stage (E7w) or at the heading stage (H7w), or harvested 6w after the first-cut at the early stage (E6w) from orchardgrass sward, and then ensiled. We evaluated the effect of regrowth interval by comparing E7w and E6w, and the effect of first-cut timing by comparing E7w and H7w. Six multiparous Holstein cows were used in a replicated 3 × 3 Latin square design, with three dietary treatments: diets containing E7w, E6w, or H7w silage at 30% dietary dry matter. We observed that feeding E6w silage instead of E7w silage increased fiber digestibility, dry matter intake, and milk production; however, the first-cut timing (E7w vs. H7w) did not affect nutrient content and digestibility, feed intake, or lactation performance. These results show that harvesting at short regrowth intervals for second-cut orchardgrass can be an effective strategy for improving feed utilization and milk yield; however, the first-cut timing for second-cut orchardgrass has little impact.

Factors relating to doctors' desire to change hospitals in Japan.

PURPOSE: This study seeks to investigate doctors' desire to change the hospital where they work to sustain higher quality care. DESIGN/METHODOLOGY/APPROACH: Self-administered questionnaires were sent to doctors in Aichi Prefecture, Japan. Data were analyzed using univariate and logistic regression analysis and recursive partitioning. FINDINGS: Factors related to doctors' desire to change hospitals, according to logistic regression, were interaction between working hours and satisfaction with the hospital, evaluation, local government hospitals versus private ones, small vs large hospitals, ophthalmology versus internal medicine, desire to continue working as a hospital doctor and age. Additionally, working hours were also found to be related, based on recursive partitioning. RESEARCH LIMITATIONS/IMPLICATIONS: The response rate was low and sampling bias was observed--therefore results need careful interpretation. Also, because this was a cross-sectional study, causal relationships could not be identified. Desire to change hospitals, but not actual behavior, was measured. PRACTICAL IMPLICATIONS: Efforts to prevent doctors from changing hospitals should include considering job satisfaction and workload, doctor evaluation methods, support for career progression and organizational management. ORIGINALITY/VALUE: As the hospital doctor shortage in rural areas becomes more serious, exploring doctors' desire to leave their current hospital is meaningful for Japanese hospital managers and hospitals worldwide aiming to provide sustainable and higher quality care.

Effects of feeding early-harvested orchardgrass-perennial ryegrass mixed silage instead of heading stage harvested timothy silage on digestion and milk production in dairy cows.

We evaluated the effects of replacement of heading stage harvested timothy silage with early-harvested orchardgrass-perennial ryegrass mixed (OP) silage while maintaining or reducing concentrate input on dry matter intake (DMI), milk production, nutrient digestibility, and N balance in dairy cows. Nine multiparous Holstein cows were used in a replicated 3 × 3 Latin square design with three dietary treatments: TYL, a diet containing timothy silage where forage-to-concentrate ratio (FC) was 50:50; OPL, a diet containing OP silage where FC ratio was 50:50; and OPH, a diet containing OP silage where FC ratio was 60:40. We observed that an equal replacement of timothy with OP silage increased DMI, milk yield, milk protein production, and nutrient digestibility but decreased milk fat content (TYL versus OPL). We observed that replacing timothy with OP silage while reducing concentrate input increased milk fat and protein yield, nutrient digestibility, and feed efficiency and reduced urinary N loss with no effect on DMI or milk fat content (TYL versus OPH). These results show that replacing timothy with OP silage can be a good approach to improve milk production, feed efficiency, and N utilization and reduce concentrate input. However, milk fat depression should be considered when an equal substitution is performed.

Effects of rice conservation methods on lactation, blood metabolites, and rumen fermentation in dairy cows.

We investigated the effect of rice grain conservation methods on feed intake, milk production, blood metabolites, and rumen fermentation in dairy cows. Raw rice grain was dried before crushing (DRY), ensiled after crushing (ENS-A), or ensiled before crushing (ENS-B). Twelve multiparous Holstein cows were used in a replicated 3 × 3 Latin square design with three dietary treatments comprising ad libitum access to one of three total mixed rations (TMRs; containing DRY, ENS-A, or ENS-B at 17% of dietary dry matter) plus a standard allowance of 2.0 kg/day of dairy concentrates. The dietary treatments did not affect the feed intake, milk yield, or milk composition. The selected blood constituents were not influenced by the rice conservation method. The ruminal lactic acid and total volatile fatty acid (VFA) concentrations and the VFA proportion in the cows were not influenced by the rice conservation method. These results demonstrate that the rice grain conservation method has little impact on lactation performance when cows are fed a TMR containing 17% treated rice grain (dry matter basis).

Effect of different methods for conserving rice grain on in situ ruminal degradation and in vivo nutrient digestion and rumen fermentation in steers.

We investigated the effects of different rice conservation techniques on in situ ruminal degradation and in vivo nutrient digestibility and rumen fermentation in steers. Raw rice grain was dried before crushing (DRY), ensiled after crushing (ENS-A), or ensiled before crushing (ENS-B). Six ruminally cannulated steers were used in a replicated 3 × 3 Latin square design with three dietary treatments: diets containing DRY, ENS-A, or ENS-B at 36% of the dietary dry matter. The in situ rapidly degradable fraction and effective ruminal degradability were higher for ensiled rice than for DRY, and higher for ENS-A than for ENS-B. The ruminal pH was lower and the lactic acid and total volatile acid concentrations were higher for the steers fed ensiled rice than those fed the DRY diet, but a treatment effect was not observed in the comparison between ENS-A and ENS-B. The whole-tract digestibility of crude protein and ether extract was improved when the rice grain was ensiled, but there were no differences in nutrient digestibility between ensiling methods. These results show that ensiling treatment can be a strategy to improve the nutrient value of rice grain, but the ensiling method has little impact on in vivo digestion.

Effect of ensiling process of total mixed ration on fermentation profile, nutrient loss and in situ ruminal degradation characteristics of diet.

This experiment aimed to determine the changes in chemical composition, fermentation profile, in situ disappearance characteristics, and nutrient losses of ensiled total mixed ration (TMR) containing steam-flaked corn or brown rice (BR) during storage. TMRs for dairy cows, containing either steam-flaked corn or BR at 31.9% with 15.2% rye silage, 40.5% alfalfa silage, 5.0% beet pulp and 7.0% soybean meal, were prepared (dry matter (DM) basis). Each TMR was placed in a plastic drum silo, stored at 23°C in an air-conditioned room and sampled 0, 7, 14, 30, 90 and 210 days after preparation. In both grain sources, the fermentation products increased, while DM and starch storage losses increased and starch content greatly decreased during storage. The rapidly degradable fraction and effective ruminal degradability of DM, crude protein and starch increased during storage. These changes of dietary characteristics were large during 30 days of storage, but small after 90 days of storage. Replacing corn with BR led to increased fermentation products, starch loss and effective ruminal degradability of the ensiled TMR. These results indicate that the ensiling process of TMR changes the dietary characteristics and replacing corn with BR in TMR had a large impact on these dietary characteristics.

Rapid identification of Ochroconis gallopava by a loop-mediated isothermal amplification (LAMP) method.

Ochroconis gallopava is a species of dematiaceous fungi recognized as a causative agent of zoonotic and emerging fungal infections. It affects the central nervous system and respiratory tracts of humans, birds and cats. We designed O. gallopava species-specific primer sets to aid in its identification by a loop-mediated isothermal amplification (LAMP) method based on the D1/D2 domain of the LSU rDNA sequence. The LAMP method successfully detected the gene from both fungal DNA and experimentally infected brains and spleens of mice and will be helpful in the diagnosis of O. gallopava infection.

Reexamination of Coccidioides spp. reserved in the Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, based on a multiple gene analysis.

The Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University is the only organization in Japan to possess a series isolates of Coccidioides spp., which are the most virulent pathogenic fungi and which are treated as biosafety level 3 microorganisms. Recently, the genus Coccidioides has been classified into two species, C. immitis and C. posadasii, based on their endemic areas and genotyping; the former species is endemic to the state of California, and the latter is endemic to other parts of North and South America. We reevaluated 19 isolates of Coccidioides immitis stored in our center using a multiple gene analysis. Five isolates were identified as C. immitis and 14 as C. posadasii. Their sequence information in GenBank will help to identify the two genospecies of Coccidioides spp.

Lactation and digestion in dairy cows fed ensiled total mixed ration containing steam-flaked or ground rice grain.

The objective of this experiment was to investigate the effect of feeding ensiled total mixed ration (TMR) containing steam-flaked (SF) or ground brown rice (Oryza satira L.; BR) on feed intake, lactation performance, digestion, ruminal fermentation and nitrogen (N) utilization in dairy cows. Eight multiparous Holstein cows were used in a crossover design with two dietary treatments: diets containing either SF or fine ground (FG) BR at 24% of dietary dry matter. Dietary treatment did not affect dry matter intake or milk yield and composition. The whole-tract digestibility of organic matter and fiber decreased, and the digestibility of starch increased with the replacement of SF with FG in ensiled TMR, but these differences were small between diets. Crude protein digestibility was not different between diets. The processing method of BR did not affect ruminal pH, total volatile acid concentration, or volatile fatty acid proportion in cows. The N intake, milk N secretion, fecal and urinary N excretion and N retention were not influenced by dietary treatment. These results show that feeding ensiled TMR containing FG instead of SF reduces fiber digestibility but has little impact on lactation and N utilization when diets contained 24% on a dry matter basis.

Effect of time at pasture and herbage intake on profile of volatile organic compounds of dairy cow milk.

Volatile organic compounds (VOCs) in milk were investigated as quantitative markers of herbage intake (HI) at pasture. Eight Holstein cows were fed indoors with concentrate and conserved forages (grass silage, corn silage and hay) (NG), then were divided into three treatments according to the duration of access to pasture: 4 h (G4), 8 h (G8), and 20 h (G20) per day. The HIs were 4.3, 8.6, and 13.0 kg dry matter/day for the G4, G8 and G20 treatments, respectively. Milk from cows was sampled and analyzed VOCs by the steam distillation-extraction method and gas chromatography-mass spectrometry (GC-MS). From the intensity of the GC peak area, the levels of 1-phytene (3,7,11,15-tetramethyl-1-hexadecene) and 2-phytene (3,7,11,15-tetramethyl-2-hexadecene) were lowest in NG treatment and markedly increased with grazing time at pasture. With simple regression analysis on the HI to each diterpenoid, a strong correlation was found between the intensity of 1-phytene in the milk and the HI (r = 0.807, P < 0.001). 1-phytene content in milk could be useful as a quantitative marker of the HI of grazing cows.

Detection of gp43 of Paracoccidioides brasiliensis by the loop-mediated isothermal amplification (LAMP) method.

Paracoccidioidomycosis is a deep mycosis caused by the thermo-dependent dimorphic fungus Paracoccidioides brasiliensis and is prevalent in Latin American countries. We detected the species specific gp43 gene of P. brasiliensis by loop-mediated isothermal amplification (LAMP) in 22 clinical and seven armadillo-derived isolates. The amplified DNA appeared as a ladder with a specific banding pattern. The advantage of the LAMP method is speed; only 3 h were necessary for identification of the organism and diagnosis of the disease. We were also able to obtain positive results from DNA extracted from a paraffin-embedded tissue sample of paracoccidioidomycosis, suggesting that this method may achieve clinical application in the near future.

Diagnosis of histoplasmosis by detection of the internal transcribed spacer region of fungal rRNA gene from a paraffin-embedded skin sample from a dog in Japan.

The lesions of histoplasmosis in dogs in Japan differ from those in dogs in North America. Affected dogs in Japan have had multiple granulomatous or ulcerated foci in skin or gingiva and have not had pulmonary or gastrointestinal lesions. The present report introduces a polymerase chain reaction (PCR) diagnosis of canine histoplasmosis and the characteristic of disease in Japan. The surgically removed skin ulcerate samples from a 5-years-old female Shiba-inu native to Japan without traveling out of the country were evaluated. Tissue samples had many yeast-like organisms in the macrophages. DNA was extracted from paraffin-embedded tissue samples. A nested PCR technique was applied. The detected sequence of the internal transcribed spacer of ribosomal RNA gene had 99.7% in homology with Ajellomyces capsulatus (the teleomorph of Histoplasma capsulatum). Clinical manifestations, historical background of equine epizootic lymphangitis in Japan, and a human autochthonous case of histoplasmosis farciminosi indicated that this dog might have been infected with H. capsulatum var. farciminosum as a heteroecism.

[Canine histoplasmosis in Japan].

Histoplasmosis is a fungal infection caused by Histoplasma capsulatum and is distributed a worldwide. Although the disease has been treated as an imported mycosis, some autochthonous human, 1 equine and 4 canine cases suggested that the disease is endemic. Histoplasmosis is classified depending on the variety of causative agent. Histoplasmosis farciminosi known as pseudofarcy, is manifested only in Perissodactyla where it invades lymph nodes and lymph ducts, and is recognized by isolation from horses. Historically, Japan was one of the endemic areas of pseudofarcy before World War II, and more than 20,000 cases were recorded in horses used by the military. Interestingly, Japanese canine histoplasmosis uniformly showed skin ulcers and granulomatous lesions on the skin without pulmonary or gastrointestinal involvement, both of which were very similar to pseudofarcy. It was diagnosed as histoplasmosis by the detection of internal transcribed spacer legions of rRNA gene of H. capsulatum from paraffin embedded tissue samples. Furthermore, the fungal isolate from the human case with no history of going abroad or immigrating was identified as H. capsulatum var. farciminosum by a gene sequence. These facts indicated that pseudofarcy is not only an infectious disease in horses, but also a zoonotic fungal infection. Japanese autochthonous canine histoplasmosis might be a heteroecism of pseudofarcy because of its likeness to the human case, the similarity of clinical manifestations and the historical background at this stage.

Cytotoxicity of Aspergillus fumigatus culture filtrate against macrophages.

Aspergillus fumigatus causes serious, life-threatening human infection, and is one of the most important pathogenic fungi. Little is known, however, about its mechanism of infection or its virulence factors. To learn about its virulence factors, the effect of the culture filtrate of A. fumigatus on macrophages was studied. When cocultured with A. fumigatus in 96-well microplates, murine peritoneal macrophages showed significant morphological changes indicating serious cellular damage, even when the macrophages were not in direct contact with the fungus. Then culture filtrates of Aspergillus spp., A. fumigatus, A. flavus, A. terreus and A. niger, were prepared by culturing the fungus in 96-well or 24-well microplates for 24 h, and the effect of the culture filtrates was determined by culturing macrophages with or without culture filtrate. When cultured with the culture filtrate of A. fumigatus at a concentration of 1% or higher, macrophages demonstrated significant morphological changes, leading to their death. Treatment with heat greatly lowered the activity of the culture filtrate. In contrast, culture filtrates of A. terreus and A. flavus showed no detectable effect on macrophages, whereas A. niger did display a similar, but much weaker effect. Our study strongly suggests that A. fumigatus releases a toxic product (s) in the medium very rapidly, and this may be critically involved as the virulence factor in human infection, at least in part, by causing serious injury to macrophages.

[Culture filtrate of Aspergillus fumigatus and its cytotoxic activity against leukocytes].

Aspergillosis has become the biggest cause of death among mycoses in Japan. The main causative agent is Aspergillus fumigatus and its virulence factors have been variously investigated, yet much remains to be clarified. We recently found that the culture filtrate of A. fumigatus has potent anti-leukocyte activity. It seriously damages macrophages and hampers the function of polymorphonuclear neutrophils. The filtrate is also active in vivo: when injected intraperitoneally, the culture filtrate lowered the survival of mice infected with A. fumigatus. This activity becomes overt within 24 hours of culture of the fungus. Although the mechanism of pathogenicity of A. fumigatus is believed to be somewhat complex, we suggest the activities shown in the culture filtrate may play a crucial role in the development of the infection.

[Fungicidal activity of liranaftate against Trichophyton rubrum].

The fungicidal activities of thiocarbamate antifungal agent liranaftate were studied by determining the MIC and the MCC against Trichophyton rubrum with the Milliflex -100 Test System and by determining the time-kill curve in comparison to that of six reference agents. Liranaftate and lanoconazole both showed excellent fungistatic activity against the conidia of T. rubrum. For each of these agents, the MIC after 14 days of contact was 0.009 g/ml. The liranaftate-induced decrease in the MCC occurred from 9 days onwards; MCC at 14 days was 0.039 g/ml. The MCC for tolciclate was also reduced from 9 days onwards, but that of amorolfine, lanoconazole, neticonazole, clotrimazole and bifonazole was not lowered up to 14 days. Similar results were obtained when the studies were performed with germinated conidia. The time-kill curves showed that both liranaftate and tolciclate, at concentrations ranging from 2 to 32 times the MICs, achieved a decrease in viable counts to below the detection limit within 7 to 9 days. In experiments with low levels of inoculum, only amorolfine produced a decrease to below the detection level, and that occurred at 14 days; no reduction in viable counts was observed up to 14 days with the four azole agents. Our data suggest that antifungal agents of the thiocarbamate class possess the most potent fungicidal activity against dermatophytes; these are followed in order by morpholine and azole antifungals.

The role of chlamydospores of Paracoccidioides brasiliensis.

The role of chlamydospores in the conversion process from a mycelial-to-yeast form using the slide culture method was studied. Three clinical isolates and two other isolates from armadillo, belonging to the fungal species Paracoccidioides brasiliensis, were cultured on Sabouraud dextrose agar (SDA), potato dextrose agar (PDA) and brain heart infusion dextrose agar (BHIDA). Initially, the mycelial forms of each isolate were grown at 25 degrees C for 7, 14, 30 or 60 days on slide cultures and then the temperature was shifted to 35 degrees C. Interestingly, the slide cultures of all the isolates at 25 degrees C formed chlamydospores on either SDA or BHIDA, whereas, on PDA medium, aleurioconidia were formed. If the slide cultures on BHIDA were incubated at 35 degrees C for 7 to 14 days, multiple budding forms could be observed. This phenomenon was not evident in the slide cultures of SDA or PDA. The results of this morphological study indicate that in P. brasiliensis, chlamydospores may play an important role in the conversion process from a mycelial-to-yeast form.