Microbial communities in feed, bedding material, and bulk milk - experiences from a feeding trial.

There is an increasing interest in the microbiota of the dairy value chain, from field to fork. Studies to understand the effects of environmental, feed and management factors on the raw milk microbiota have been performed to elucidate the origin of the bacteria and find ways to control the presence or absence of specific bacteria. In this study, we explored the microbiota in feedstuff, bedding material and milk on a Swedish dairy farm to investigate the effects of feeding different silages on the bacterial compositions throughout the dairy value chain. Three ensiling treatments were evaluated: without additive, with acid treatment, and with inoculation of starter culture. The silage treatments were fed as partial mixed rations to 67 dairy cows for 3 weeks each, with one treatment fed twice to evaluate if a potential change in milk microbiota could be repeated. The highest average total bacteria counts were found in the used bedding material (9.6 log10 cfu/g), while milk showed the lowest (3.5 log10 cfu/g). Principal coordinate analysis of the weighted UniFrac distance matrix showed clear separation between 3 clusters of materials: 1) herbage, 2) silage and partial mixed ration, and 3) used bedding material and milk. Surprisingly, the expected effect of the ensiling treatments on silage microbiota was not clear. Transfer of major bacteria from the silages and resulting partial mixed rations to the used bedding material was observed, but rarely to milk. The milk microbiota showed most resemblance to that of the used bedding material. Lactobacillus was a major genus in both feed and milk, but investigations at amplicon sequence variant level showed that in most cases the sequences differed between materials. However, low total bacteria count in the milk in combination with a high diversity suggests that results may be biased due to environmental contamination of the milk samples. Considering that the study was performed on a research farm, strict hygienic measures during the feeding experiment may have contributed to the low transfer of bacteria from feed to milk.

Influence of leukotriene pathway polymorphisms on clinical responses to montelukast in Japanese patients with asthma.

WHAT IS KNOWN AND OBJECTIVE: Montelukast, a cysteinyl leukotriene receptor 1 antagonist, is safe and efficacious in patients with asthma. The mechanisms underlying the significant interpatient variability in response to montelukast are not clear but are believed to be, in part, because of genetic variability. METHODS: To examine the associations between polymorphisms in candidate genes in the leukotriene pathway and outcomes in patients with asthma on montelukast for 4-8 weeks, we evaluated the changes in peak expiratory flow (PEF), forced expiratory volume in 1 s (FEV(1·0) ) and patients' subjective symptom before and after montelukast treatment. DNA was collected from 252 Japanese participants. RESULTS AND DISCUSSION: Two single-nucleotide polymorphisms (SNPs) in the ALOX5 (rs2115819) and LTA4H (rs2660845) genes were successfully typed. There was no difference between members of the general population (n = 200) and patients (n = 52) in each genotype frequency. Significant associations were found between SNP genotypes in the LTA4H gene and changes in PEF and FEV(1·0) . The PEF and FEV(1·0) responses to montelukast in the A/A genotypes (n = 4) for the LTA4H SNP were significantly higher than those in the G allele carriers (A/G+G/G) (n = 17). WHAT IS NEW AND CONCLUSION: Despite the small sample size, our results suggest that genetic variation in leukotriene pathway candidate genes contributes to variability in clinical responses to montelukast in Japanese patients with asthma.

Histone acetylation determines the developmentally regulated accessibility for T cell receptor gamma gene recombination.

Variable/diversity/joining (V[D]J) recombination of the T cell receptor (TCR) and immunoglobulin (Ig) genes is regulated by chromatin accessibility of the target locus to the recombinase in a lineage- and stage-specific manner. Histone acetylation has recently been proposed as a molecular mechanism underlying the accessibility control. Here, we investigate the role for histone acetylation in the developmentally regulated rearrangements of the mouse TCR-gamma gene, wherein predominant rearrangement is switched from Vgamma3 to Vgamma2 gene during the fetal to adult thymocyte development. Our results indicate that histone acetylation correlates with accessibility, as histone acetylation at the fetal-type Vgamma3 gene in accord with germline transcription is relatively high in fetal thymocytes, but specifically becomes low in adult thymocytes within the entirely hyperacetylated locus. Furthermore, inhibition of histone deacetylation during the development of adult bone marrow-derived thymocytes by a specific histone deacetylase inhibitor, trichostatin A, leads to elevated histone acetylation, germline transcription, cleavage, and rearrangement of the Vgamma3 gene. These data demonstrate that histone acetylation functionally determines the chromatin accessibility for V(D)J recombination in vivo and that an epigenetic modification of chromatin plays a direct role in executing a developmental switch in cell fate determination.

Role of cell death ligand and receptor system on regulation of follicular atresia in pig ovaries.

Several hundred thousand primordial follicles are present in the mammalian ovary, however, only a limited number develop to the pre-ovulatory stage, and then finally ovulate. The others, more than 99%, will be eliminated through a degenerative process called 'atresia'. The endocrinological regulatory mechanisms involved in follicular development and atresia have been characterized to a large extent, but the precise temporal and molecular mechanisms involved in the regulation of these events have remained unknown. From many recent studies, it is suggested that the apoptosis in ovarian granulosa cells plays a crucial role in follicular atresia. Notably, death ligand-receptor interaction and subsequent intracellular signalling have been demonstrated to be the key mechanisms regulating granulosa cell apoptosis. In this review, we provide an overview of granulosa cell apoptosis regulated by death ligand-receptor signalling. The roles of death ligands and receptors [Fas ligand (FasL)-Fas, tumour necrosis factor (TNF)alpha-TNF receptor (TNFR), and TNFalpha-related apoptosis-inducing ligand (TRAIL)-TRAIL receptor (TRAILR)] and intracellular death-signal mediators [Fas-associated death domain protein (FADD), TNF receptor 1-associated death domain protein (TRADD), caspases, apoptotic protease-activating factor 1 (Apaf1), TNFR-associated factor 2 (TRAF2), and cellular FLICE-like inhibitory protein (cFLIP), etc.] in granulosa cells will be discussed.

Heritable translocations induced by dermal exposure of male mice to acrylamide.

Acrylamide (AA) is an important industrial chemical used mainly in the production of polymers. It can be absorbed through the skin. AA was shown to be a germ cell clastogen that entails a genetic risk for exposed workers. The genetic risk calculation was based on mouse heritable translocation test data obtained after acute intraperitoneal (ip) exposure (Adler et al., 1994). To obtain a correction factor between ip and dermal exposure, dominant lethal and heritable translocation tests were carried out with dermal exposure of male mice to AA. In the dominant lethal test, male (102/El x C3H/El)F1 mice were exposed by dermal application to the shaved backs of 50 mg/kg AA per day on five consecutive days or to five daily ip injections of 50 mg/kg AA. One day after the end of exposure, the males were mated to untreated females of the same hybrid stock for four days and females were changed every four days for a total of five matings. Dominant lethal effects were found during matings 1-3. For ip exposure, these values were 81.7, 85.7 and 45.4%, respectively; for dermal exposure the corresponding values were 22.1, 30.6 and 16.5%, respectively. In the heritable translocation assay, male C3H/El mice were treated with five dermal exposures of 50 mg/kg AA and mated 1.5-8.5 days after the end of exposure to untreated female 102/El mice. Pregnant females were allowed to come to term and all offspring were raised to maturity. Translocation carriers among the F1 progeny were selected by a sequential fertility testing and cytogenetic analysis including G-band karyotyping and M-FISH. A total of 475 offspring were screened and 41 translocation carriers were identified. The observed translocation frequency after dermal exposure was 8.6% as compared to 21.9% after similar ip exposure (Adler, 1990). The calculated ratio of ip vs. dermal exposure of 0.39 can be applied to obtain a more realistic calculation of genetic risk for dermally exposed workers.

DNA polymerase beta is not essential for the formation of palindromic (P) region of T cell receptor gene.

Formation of palindromic (P) region at the variable (V)-diversity (D)-joining (J) junction in DNA polymerase beta (pol-beta) deficient mice were investigated by sequencing of reverse transcriptase-polymerase chain reaction (RT-PCR) products of mRNAs encoding the beta chain of T cell receptor (TCR). Total 42 and 43 cDNA clones encoding V(beta8)-D(beta)-J(beta)-C(beta) from E18.5 embryonic thymocytes of pol-beta gene knocked-out and wild type control mouse, respectively, were sequenced. Among them five and six clones from pol-beta knocked-out and wild type, respectively, have P insertions of two nucleotides. This result unequivocally indicates that pol-beta, which is one of the repair-type DNA polymerases most abundantly expressed in thymus and spleen, is not essential for the formation of P region.

Dose response study for 1,3-butadiene-induced dominant lethal mutations and heritable translocations in germs cells of male mice.

Butadiene (BD) and its metabolites have extensively been studied in the EU sponsored research project "Multi-endpoint analysis of genetic damage induced by 1,3-butadiene and its major metabolites". Within this project a dominant lethal test and a heritable translocation test were performed with male mice to study the dose-response relationships for the respective endpoints. BD concentrations of 130 and 500 ppm were tested in the dominant lethal assay by exposing male mice on 6 h/day for five consecutive days resulting in doses of 3900 and 15,000 ppmh, respectively. Males were mated for four consecutive weeks at a ratio of 1:2 to untreated females. A positive dominant lethal effect was observed in the first mating week in the experiment with 15,000 ppmh but no dominant lethality was found with the lower dose of 3900 ppmh. The present dominant lethal data have to be viewed together with the data already published for a BD dose of 39,000 ppmh (1300 ppm at 6 h/day on 5 consecutive days) [1]. The main difference between results with the highest and the middle dose is that mating weeks one and two (sperm and late spermatids) showed an effect at 39,000 ppmh while only mating week one (sperm) showed an effect at 15,000 ppmh. In the heritable translocation assay, males mice were exposed with a BD dose of 15,000 ppmh and mated for one week to untreated females. Among 434 F1 offspring, we found 5 translocation carriers (1.15% vs. 0.05% in the historical control, p < 0.001). In the previous heritable translocation experiment with a BD dose of 39,000 ppmh of DB exposure, 2.7% of the offspring carried a reciprocal translocation [2]. These data can be used for quantification of genetic risk. The dose response for BD-induced heritable translocations in sperm and late spermatids of mice was linear (Y = 0.05 + 6.9 x 10(-5)X) and a doubling dose of 725 ppmh could be calculated.

Combined use of brushing cytology and endoscopic retrograde pancreatography for the early detection of pancreatic cancer.

Seventy-two cases of pancreatic cancer were examined by brushing cytology combined with endoscopic retrograde pancreatography. The results of this combined method were better than those reported for the exfoliative cytologic study of pancreatic fluid. The method detected a minute cancer of the main pancreatic duct that was not detected with any other method. The cells obtained by this technique had very well-preserved cytoplasm and nuclear chromatin, which facilitated making a correct diagnosis. Though this method can be applied only to the main pancreatic duct, it is effective for the diagnosis of ductal cell carcinoma, especially those located at the head of the pancreas, which is the most common site for pancreatic cancer. It is a safe procedure, with no complications seen in this series. The differentiation of carcinoma cells from the benign atypical cells of chronic pancreatitis is illustrated and emphasized.

[A clinical study on patients detected Pasteurella multocida from sputum].

We reported ten cases, (four female and six male), whose sputum cultures positive for Pasteurella multocida from 1990 to 2000. In the past eleven years increasing numbers of cases have appeared in our hospital. The majority of the cases with P. multocida possessed some underlying pulmonary diseases (seven cases, 70%), inactive lung tuberculosis or bronchiectasis. There were compromised hosts such as high ages person, steroids dependent person and diabetes mellitus patients. P. multocida was almost susceptible to antibioticus (penicillin and cephalosporins), although some erythromycin resistant strains were identified. The cats' oral cavities in our two cases were cultured and P. multocida were isolated. In our survey the prevalence of this organism is as high as 85% in cats. Our data suggests that patients who are in the high infection risk category are easily infected to P. multocida.

[A survival case of severe Legionella longbeachae pneumonia].

A 56-year-old Japanese male was admitted to Toyohashi Municipal Hospital because of fever, cough, and dyspnea. Chest X-ray film showed bilateral alveolar infiltrates. He suffered from severe hypoxemia and was given a diagnosis of acute respiratory distress syndrome. He was also complicated with disseminated intravascular coagulation and pseudomembranous colitis. He fully recovered by intensive treatment with antibiotics, mechanical ventilation and endotoxin eliminating therapy. Legionella longbeachae was isolated from his respiratory specimens and was regarded as the etiologic agent of his pneumonia.

[An autopsy case of chronic granulomatous disease diagnosed by biopsy].

This report concerns a male patient aged 25 years, diagnosed at the age of 12 years as suffering from chronic granulomatous disease. This patient had p47-phox deficiency. He was admitted to this hospital because of fever and dyspnea accompanied by right spontaneous pneumothorax. He failed to respond to medical treatment. He died from respiratory failure four months after admission. Autopsy demonstrated pigmented lipid histiocytes characteristic of CGD. These characteristic pigmented cells were distributed in the spleen, liver, lymph nodes and in the small intestine. As for the nature of the pigment, lipofuschin-like compound were identified. Granulomatous component was seen in the mucosa of the stomach obtained by operation. The presence and characteristic distribution of such pigmented macrophages in tissue in young adults may suggest the diagnosis of CGD.

[A clinical study on ceftazidime in the treatment of intractable respiratory infections].

Ceftazidime (CAZ) was administered to 51 patients (37 males, 14 females) with respiratory infections including severe cases, accompanied by various underlying respiratory diseases. The clinical efficacy and side effects of CAZ were investigated. The mean age and body weight of these 51 cases were 62.6 years and 48.0 kg, respectively. CAZ was administered by intravenous drip infusion (daily dose of 2-4 g) for a mean of 14.7 days to a mean total dose of 56.7 g. Clinical efficacy rates were 64% (18 of 28 cases), 80% (16 of 20 cases) and 67% (2 of 3 cases) for airway and intermediary regional infections, pneumonia (including lung abscess) and pyothorax, respectively. In the bacteriological study, efficacy rates and bacterial eradication rates were 69% and 67%, 33% and 20%, 100% and 100%, and 100% and 100% for infections caused by Pseudomonas aeruginosa (13 cases), Staphylococcus aureus (6 cases), Streptococcus pneumoniae (6 cases) and Klebsiella pneumoniae (4 cases), respectively, and bacterial eradication was achieved in both of 2 cases of Peptostreptococcus anaerobius and 2 cases of Haemophilus influenzae, and 1 case each of Peptococcus sp., Fusobacterium necrophorum and Serratia marcescens. Side effects observed were eruption in 1 case (2%) and elevated GOT, GPT and Al-P values in 1 case (2%), but these cases tended to recover after CAZ treatment was discontinued.

[Effects of cepharanthin on leukopenia and thrombocytopenia induced by chemotherapy in lung cancer patients].

We studied the effects of Cepharanthin (CEP) on bone marrow suppression induced by chemotherapy in 18 primary lung cancer patients (14 NSCLC, 4 SCLC). NSCLC patients received IP (IFM+CDDP) therapy and SCLC patients received ION (IFM+VCR+ACNU) therapy. For the control, we chose the first course and we administered CEP (1 mg/kg) during the second course. The rate of leukopenia and neutropenia was significantly lower during the CEP course than during the control (p less than 0.01). The recovery rate (at 3 weeks) of leukopenia and neutropenia was significantly higher during the CEP course than during the control (p less than 0.05). But, obvious effects of CEP for lymphopenia and thrombocytopenia were not obtained. Side effects by CEP were not observed in this study. These data suggest that the large dose of CEP contributes to the prevention of leukopenia, especially neutropenia, in patients who receive a sufficient amount of anticancer drugs.

Effect of diet on milk allantoin and its relationship with urinary allantoin in dairy cows.

The present study examined the effects of diet on the concentration and excretion of allantoin in milk and the relationship between allantoin in milk and urine of dairy cows. Results are reported from three experiments. In Experiment 1, four diets with two different protein percentages and two different rumen degradabilities were fed to 12 cows in a continuous trial. In Experiment 2, four diets with different protein balance values in the rumen were fed to four cows in a 4 x 4 Latin square design. In Experiment 3, four diets with different contents of concentrate and fat were fed to four cows in two incomplete 2 x 2 Latin squares. The excretion of allantoin N in milk increased as dry matter intake increased (Experiment 1) and as the concentrate in the diet increased (Experiment 3). In Experiments 1 and 3, the excretion of allantoin in milk was correlated with its concentration in milk and with its excretion in urine. In the three experiments, allantoin excretion in milk was closely correlated with milk yield. The amount of allantoin secreted in milk represented a small proportion (0.63 to 1.34%) of the total excretion in urine and milk. The proportion of allantoin secreted in milk was negatively correlated with the urinary excretion of allantoin in Experiments 2 and 3 and positively correlated with the excretion of allantoin in milk in Experiment 1. In Experiments 1 and 2, the proportion of allantoin excreted in milk was not constant but increased as milk yield increased.

[A case of bronchocentric granulomatosis presenting as a tumorous shadow on chest X-ray film].

A 57-year-old woman was admitted to our hospital with cough, sputum and abnormal chest X-ray. In summer, 1989, she developed cough which gradually worsened in autumn. In November, the chest X-ray revealed a tumorous shadow in the left suprahilar region. On admission, there were no symptoms of bronchial asthma. Chest X-ray revealed a subpleural tumorous shadow in the left upper lung field. X-ray findings suggested that the tumorous shadow in the suprahilar region moved to the left peripheral upper lung field. Left B1+2 orifice obstruction with necrotic tissue was seen on fiberoptic bronchoscopy. Transbronchial biopsies failed to yield specific diagnostic findings, except for bronchitis with exudate containing eosinophils. In February, 1990, she developed hemosputum and left chest pain. Chest X-ray showed consolidation in the left apical lung field. Left upper lobectomy was performed. Histological examination disclosed many granulomas with central necrosis around the bronchi, and aspergillus hyphae were seen. These findings are compatible with bronchocentric granulomatosis without asthma.

[Malignant fibrous histiocytoma of the lung].

A 68-year-old man had a malignant fibrous histiocytoma of the lung that was found at autopsy. The patient was admitted to our hospital because of exertional dyspnea. A chest X-ray film and chest CT scan showed atelectasis in the left upper lobe. Examination with a fiberoptic bronchoscope revealed a necrotic mass in the left mainstem bronchus. Microscopic examination of a biopsy specimen disclosed several atypical giant cells but was not diagnostic. He underwent chemotherapy with CBDCA, IFM and VP-16, because a malignant tumor of the lung was strongly suspected. After three cycles of chemotherapy, the tumor had shrunk, as demonstrated on repeated bronchoscopy. The patient's condition improved temporarily, but he began to complain of dyspnea, and died of respiratory insufficiency despite radiotherapy. Postmortem examination was done. The tumor in the lung was mainly composed of spindle-shaped fibroblast-like cells and several pleomorphic giant cells with multiple nuclei, with storiform and fascicular patterns. Immunohistochemically, these tumor cells were shown to contain vimentin, alpha-1-antitrypsin, and LN-5. These findings were compatible with malignant fibrous histiocytoma of the lung.

The dissociation constant of verapamil estimated from its effect on Ca concentration-tension curves in guinea-pig tracheal muscle.

Concentration-tension curves for calcium ions (Ca2+) were studied in indomethacin-treated guinea-pig tracheal muscle in the presence of different concentrations of carbachol in media containing 5.9 mM K+ or 40 mM K+. The effect of verapamil was investigated taking into account the steepness (the Hill coefficient) of the Ca2+ curve. When carbachol (1 microM) was added to 40 mM K+ solution, the Ca2+ concentration to produce half maximum tension (EC50) was reduced from 0.2 mM to 0.08 mM and the Hill coefficient was increased from 1.4 to 2.0, respectively. In the presence of carbachol (1 microM), the Ca2+ concentration-tension curve was not much influenced by increasing the K+ concentration from 5.9 to 40 mM K+. Verapamil (0.5 microM) shifted the Ca2+ concentration-tension curve to the right in a parallel manner under all experimental conditions, the shift being greater with curves having a smaller Hill coefficient. The dissociation constant of verapamil was not altered by carbachol when estimated from the shift of the curve if the Hill coefficient is taken into consideration. It is concluded that the relatively low susceptibility of carbachol-induced contractions to verapamil in the presence of 40 mM K+, compared with these produced by K+ alone, is not due to a decreased verapamil affinity but to improved Ca(2+)-response coupling.

[Two cases of human chorionic gonadotropin-producing large cell carcinoma of the lung accompanied with gynecomastia].

Two cases of human chorionic gonadotropin (HCG)-producing lung carcinoma are reported. Both were male, aged 66 and 65, respectively histological examination of percutaneous lung biopsy revealed large cell carcinoma. The patients both developed bilateral gynecomastia during their clinical course. Endocrine function tests demonstrated high levels of HCG, HCG-beta, luteinizing hormone, estrone, estradiol, progesterone in the blood. They were given only anticancer chemotherapy with no effect and died. Autopsy revealed extensive metastases of lung cancer and the presence of HCG in tumor cells was demonstrated by immunohistochemical technique in both cases.

Portal net appearance of amino acids in growing pigs fed a barley-based diet with inclusion of three different forage meals.

The net absorption of amino acids (AA) in young pigs fed a barley-based control diet (C) and diets where barley was replaced by 200 g/kg fresh weight of dried lucerne (Medicago sativa; L20), white clover (Trifolium repens; W20) or perennial ryegrass (Lolium perenne; PR20) meal was studied. Castrated male pigs were fitted with permanent catheters in the hepatic portal vein and mesenteric artery, and the hepatic portal net absorption of AA was estimated from the porto-arterial plasma concentration differences and the hepatic portal-vein blood flow. In general, the essential AA (EAA) concentrations in the hepatic portal vein reached peak levels 90 min after feeding and thereafter exhibited a transient decline. Maximum porto-arterial differences were reached between 1 and 3 h postprandially for most of the AA. The cumulative net absorption of non-essential AA (NEAA) and EAA did not differ significantly between the barley-based diet and diets W20 and PR20. Due to a lower intake of AA on diet L20, the cumulative net absorption of NEAA and EAA was significantly (P < 0.05) lower than diet C. With the exceptions of the EAA arginine, cystine and valine, and the NEAA glutamic acid + glutamine and glycine, there were no significant differences in the absorption coefficients for the EAA and NEAA between the diets. In addition, the pattern of the total EAA in the mixture absorbed postprandially did not differ significantly between the diets. The present study gives support to the contention that the replacement of barley AA with forage meal AA in a barley-based diet for growing pigs should be expected to result in minor differences in the net portal flux of AA.