Evaluation of peptones from chicken waste as a nitrogen source for micro-organisms.

In this study, chicken peptone was produced by hydrolysing inedible parts derived from chickens using endo-protease and exo-protease. The usefulness of chicken peptone as a nutrient source for bacteria was evaluated in comparison with other commercially produced peptones (animal, soy and casein-derived peptone). Escherichia coli and Bacillus subtilis were used as test strains to determine the effect of peptones from different sources on their growth ability. Both bacteria were successfully cultured in chicken peptone solution, which is similar to peptone solution containing commercial peptones apart from animal peptone. In chemical analysis, chicken peptone contained 12·0% nitrogen; this was similar to the nitrogen content from other commercial peptone sources, except for the 9·0% nitrogen found in soy peptones. The molecular weight of the peptone was determined by gel filtration chromatography, and those of all peptone, except animal-derived peptone, were found to be <5000 Da. In addition, when B. subtilis was cultured in a medium containing chicken peptone, it was shown that the protease activity was highest as compared with other commercial peptones. From these results, it is suggested that chicken peptone can be utilized for microbial culture, and this is an effective method to reuse chicken waste.

Effect of rice bran fermented with Saccharomyces cerevisiae and Lactobacillus plantarum on gut microbiome of mice fed high-sucrose diet.

To clarify the effect of rice bran (RB) and fermented RB (FRB) in a high-sucrose and low-dietary fibre diet on the gut microbiome, the in vitro bile acid-lowering capacity and caecal microbiota of ICR mice fed with 20% RB or FRB diets for two weeks were determined. The caecal microbiome was analysed by 16S rRNA gene amplicon sequencing. The in vitro bile acid-lowering capacity was high for FRB. In mouse experiments, triacylglycerol and total cholesterol were generally lower with FRB, although the faecal frequency was highest in mice fed with RB. The Shannon-Wiener and Simpson's indices for alpha-diversity in the microbiome of mice fed with RB and FRB, were higher than mice fed the control diet. At the phylum level in the caecal microbiome, Firmicutes and Bacteroidetes were high with FRB and RB, respectively. At the operational taxonomic unit level, some bacterial groups related to diabetes and gut toxicity, such as Lachnospiraceae and Enterorhabdus mucosicola, were high for RB but not for FRB diets. These results suggest that FRB, rather than RB, intake improve the intestinal environment and blood lipid condition.

Role of UCP2 polymorphisms on dietary intake of obese patients who underwent bariatric surgery.

Uncoupling protein 2 ( UCP2 ) plays an important role in body weight and energy metabolism and may be related to the control of food consumption. This study aimed to investigate the contribution of UCP2 gene variants on the dietary intake on a population after bariatric surgery. This study enrolled 150 obese patients (body mass index ≥ 35kg m(-2) ) who submitted to Roux-en-Y gastric bypass. Weight (kg), BMI (kg m(-2) ), energy (kcal d(-1) ) and macronutrients intake (g d(-1) ) of preoperative and 1-year postoperative period were collected from medical records. Ala55Val and -866G>A polymorphisms in the UCP2 gene were genotyped through allelic discrimination method in real-time polymerase chain reaction using the TaqMan pre-designed SNP Genotyping Assays kits. Hardy-Weinberg equilibrium, t-test and regression models were performed in statistical analysis (P<0.05).We found an allelic frequency of 0.44 for allele Val and 0.41 for allele A. In the postoperative period, patients with at least one rare allele for polymorphisms and with at least one rare allele for both polymorphisms together (haplotype) present a greater energy and carbohydrate intake, even after adjusting for gender, age and weight. Genetic variants in UCP2 gene were associated with the dietary consumption after Roux-En-Y gastric bypass.

In vitro cholesterol-lowering properties of Lactobacillus plantarum AN6 isolated from aji-narezushi.

Aji-narezushi is a traditional lactic acid-fermented fish. In this study, we screened for lactose-utilizing, acidophilic, bile-resistant and cholesterol-lowering lactic acid bacteria (LAB) from aji-narezushi for use as starter strains for fermented foods, as well as for use as probiotics. Of the 301 LAB isolates, 277 fermented lactose, and among these, 171 grew in de Man, Rogosa and Sharpe broth adjusted to pH 3·5. Thirty-four of the isolates were grown in a broth containing 3% (w/v) bile. All of the isolates were lactobacilli. Seven isolates that demonstrated cholesterol-lowering activity in ethanolic solution were selected. All of the isolates were identified as Lactobacillus plantarum. Lactobacillus plantarum AN6 showed the highest cholesterol-lowering activity. AN6 was more resistant to acid, salt and bile than the type strain NBRC15891(T). One-half of the cholesterol-lowering effect remained after boiling AN6 for 10 min. The Fourier transform infrared (FT-IR) analysis indicated that the content of cell wall polysaccharides in AN6 is higher than ones in the type strain. These results indicate that Lact. plantarum AN6 can be used as a profitable starter organism and probiotic.

Cellular hydrophobicity of Listeria monocytogenes involves initial attachment and biofilm formation on the surface of polyvinyl chloride.

AIMS: To clarify the cellular properties of Listeria monocytogenes involved in adhesion to and biofilm formation on polyvinyl chloride, a widely used material in the food manufacturing process. METHODS AND RESULTS: A significant correlation between the ability of initial adherence to and biofilm formation on PVC was observed for 24 L. monocytogenes strains (Spearman rank-correlation coefficient, r(s) = 0.89). The swimming motility assay revealed no relationship between initial adherence and motility of L. monocytogenes. The microbial adhesion to solvent assay revealed an interaction of L. monocytogenes cells with nonpolar solvents, and a significant correlation was also observed between the degree of interaction with nonpolar solvents and initial adherence to PVC (r(s) = 0.87 and r(s) = 0.84, between initial adherence and affinities to decane and hexadecane, respectively). CONCLUSIONS: Results indicate that cellular hydrophobicity of L. monocytogenes is an important property involved in the initial adherence to and biofilm formation on PVC. SIGNIFICANCE AND IMPACT OF STUDY: This study clarified the factors involved in the adherence to and biofilm formation ability of L. monocytogenes strains with PVC.

[Peripheral intrapulmonary lipoma: report of a case].

A 62-year-old woman who had undergone a left mastectomy for a breast cancer consulted us for an abnormal chest shadow. Chest computed tomography showed a well-defined nodule of 1 cm diameter periphery in lower lobe of the right lung. The differential diagnosis included benign lung tumors, such as intrapulmonary lymph nodes, granuloma etc. However, because of her past history we needed to consider metastasis. To make diagnosis, a wedge resection of the pulmonary nodule was performed. The tumor was diagnosed as a lipoma. No malignant cells were seen. Although peripheral intrapulmonary lipoma is very rare, it should be kept in mind in the differential diagnosis of an intrapulmonary nodule.

Induction of the histidine decarboxylase genes of Photobacterium damselae subsp. damselae (formally P. histaminum) at low pH.

AIMS: To elucidate the detailed mechanism of histamine production by Photobacterium damselae subsp. damselae. METHODS AND RESULTS: Histidine decarboxylase and related genes of P. damselae subsp. damselae were cloned, and three open reading frames named as hdcT, hdcA and hisRS were identified. The hdcA gene encodes a polypeptide of 377 amino acids and is considered to be the pyridoxal-P dependent histidine decarboxylase. The hdcT gene is assumed to be a histidine/histamine antiporter, and the hisRS gene is considered to be a histidyl-tRNA synthetase. Recombinant Escherichia coli strains harbouring plasmids carrying the P. damselae hdc genes were shown to over-excrete histamine extracellularly. Northern blot analysis and quantitative RT-PCR revealed high levels of mono- and bi-cistronic transcripts of hdcA, hdcT and hisRS genes under conditions of low pH and histidine excess. CONCLUSIONS: The hdcA gene of P. damselae was constructed as an operon with putative histidine/histamine antiporter and histidyl-tRNA synthetase. Mono- and poly-cistronic transcripts and acid induction were detected. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of cloning the histidine decarboxylase gene cluster in gram-negative bacteria. Also, these genes were induced under acidic conditions and in the presence of excess histidine.

Lysine decarboxylase of Vibrio parahaemolyticus: kinetics of transcription and role in acid resistance.

AIM: The aim of this study was to investigate the detailed mechanisms of acid resistance in Vibrio parahaemolyticus. METHODS AND RESULTS: All 11 strains of V. parahaemolyticus survived lethal acidic conditions following acid adaptation, and accumulation of cadaverine was detected. The addition of lysine improved survival, suggesting that lysine decarboxylase plays a role in the adaptive acid tolerance response. Two open reading frames (ORF) in V. parahaemolyticus, which are separated by a noncoding region, were found to be highly homologous to bacterial lysine decarboxylase (cadA) and lysine/cadaverine antiporter (cadB) genes. Transcriptional analyses of this operon revealed acid induction and enhanced induction by external lysine. The relative expression ratio of each transcript was found to follow the trend of cadA mRNA > cadB mRNA > cadBA bi-cistronic mRNA. A mutated strain, with a disrupted cadA gene, showed attenuated acid survival. CONCLUSIONS: We identified the lysine decarboxylase gene operon of V. parahaemolyticus. Expression of this operon was induced under acidic conditions. The cadA-mutated strain constructed in this study showed weaker tolerance to acidic conditions than the wild-type strain. SIGNIFICANCE AND IMPACT OF THE STUDY: Vibrio parahaemolyticus utilizes the lysine decarboxylation pathway for survival in acidic conditions.

[Surgically resected lung cancer in patients complicated with diffuse interstitial pulmonary shadow].

In 25 patients operated on for idiopathic interstitial pneumonia (IIP) associated with primary lung cancer, we clinically examined the predictive factors related to the acute exacerbation of IIP preoperatively and during operation. Most were male heavy smokers. Ages ranged 57 to 78 years. Standard surgery was performed in 11 patients, extended resection in 4 patients and limited resection in 10 patients. The incidence of postoperative acute exacerbation of IIP was 40% (10 patients). These patients were treated with steroid pulse therapy, 3 patients died due to acute exacerbation but 7 patients recovered. It seemed difficult to anticipate postoperative exacerbation of IIP based on preoperative patients evaluation and the degree of surgical invasiveness. Seven patients were alleviated with erythromycin before and after the operation and 4 patients were alleviated with high-dose steroid during or after surgery, with these patients not developing exacerbation of IIP. From these results, it was suggested that high-dose steroid administration during or after surgery and erythromycin before and after the operation were effective to avoid postoperative exacerbation of IIP.

[Solitary hemangioma of the rib; report of a case].

A case of solitary hemangioma which occurred in the rib. Tumor located in the right seventh rib, incidentally taken X-ray film demonstrated bone tumor, but she had no symptoms. Four years after, chest pain occurred and taken X-ray film, tumor size was inceeced. Computed tomography (CT) showed an expansile, well demarcated lesion, with thin corices and fine trabeclae. Chest wall, from the sixth rib to the eighth rib resection was performed and surgical margin was 4 cm. This case were diagnosed hemangioma of the rib. Hemangiomas of the bone are rare benign vascular tumors that account for less than 1% of all bony neoplasms. These lesions are most often occurring in the vertebral column or in the skull. The localization to the ribs is even more rare, with only sporadic case reports in the literature.

Marinospirillum insulare sp. nov., a novel halophilic helical bacterium isolated from kusaya gravy.

A novel species that belongs to the genus Marinospirillum is described on the basis of phenotypic characteristics, phylogenetic analysis of 16S rRNA and gyrB gene sequences and DNA-DNA hybridization. Four strains of helical, halophilic, Gram-negative, heterotrophic bacteria were isolated from kusaya gravy, which is fermented brine that is used for the production of traditional dried fish in the Izu Islands of Japan. All of the new isolates were motile by means of bipolar tuft flagella, of small cell size, coccoid-body-forming and aerophilic; it was concluded that they belong to the same bacterial species, based on DNA-DNA hybridization values (>70% DNA relatedness). DNA G+C contents of the new strains were 42-43 mol% and they had isoprenoid quinone Q-8 as the major component. Phylogenetic analysis of 16S rRNA gene sequences indicated that the new isolates were members of the genus Marinospirillum; sequence similarity of the new isolates to Marinospirillum minutulum, Marinospirillum megaterium and Marinospirillum alkaliphilum was 98.5, 98.2 and 95.2%, respectively. Phylogenetic analysis based on the gyrB gene indicated that the new isolates had enough phylogenetic distance from M. minutulum and M. megaterium to be regarded as different species, with 84.7 and 78.7% sequence similarity, respectively. DNA-DNA hybridization showed that the new isolates had <36% DNA relatedness to M. minutulum and M. megaterium, supporting the phylogenetic conclusion. Thus, a novel species is proposed: Marinospirillum insulare sp. nov. (type strain, KT=LMG 21802T=NBRC 100033T).

Purification and properties of a histidine decarboxylase from Tetragenococcus muriaticus, a halophilic lactic acid bacterium.

AIMS: A histidine decarboxylase from Tetragenococcus muriaticus, a halophilic histamine-producing bacterium isolated from Japanese fermented squid liver sauce, was purified to homogeneity, for the first time. METHODS AND RESULTS: The enzyme was purified 16-fold from cell-free extract by ammonium sulphate precipitation, anion exchange chromatography and hydroxyapatite chromatography. The pure enzyme consisted of two polypeptide chains with molecular mass of 28.8 and 13.4 kDa. The N-terminal amino acid sequences of these polypeptides highly correlated with those of the alpha- and beta-chains of other Gram-positive bacterial histidine decarboxylases. The optimum and stable pH for the enzyme was 4.5-7.0 and 4.0-7.0, respectively. This enzyme did not decarboxylate lysine, arginine, tyrosine, tryptophan and ornithine. The enzyme activity decreased with the addition of NaCl. At pH 4.8, the Vmax and Km values were 16.8 micromol histamine min-1 mg-1 and 0.74 mmol l-1, respectively. CONCLUSIONS: The very similar physiological properties of this enzyme and almost identical N-terminal amino acid sequences to those from other Gram-positive bacteria indicated that this enzyme may be evolutionally highly conserved among Gram-positive bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on this enzyme could be useful for studying the mechanism of histamine accumulation in salted foods. In addition, the N-terminal amino acid sequence can be utilized to design oligonucleotide probes, which may prove valuable in the rapid monitoring of halophilic histamine producers in salted products.

Screening cardiac ultrasonographic examination in patients with suspected cardiac disease in the emergency department.

BACKGROUND/OBJECTIVE: Our purpose was to evaluate the utility of a brief screening cardiac ultrasonographic (SCU) examination. We prospectively compared the SCU with conventional clinical evaluation in 124 emergency department (ED) patients with suspected cardiac disease. Furthermore, we assessed the impact and quality of SCU examinations as obtained by briefly trained ED personnel (EP). METHODS: Patients underwent clinical evaluation by an ED physician and SCU examination by a sonographer or cardiologist. Patient disposition, hospital stay length, and the number of full echo examinations were compared with the presence of significant findings on SCU. In patients who received a full echocardiogram during hospitalization, results of the initial clinical examination were compared with results of the SCU examination in the diagnosis of significant findings. A similar analysis, but with quality assessment, was performed on only those SCU examinations acquired by 4 EP. RESULTS: Of the 124 patients enrolled in the main study, 40 of 124 (32%) had significant findings on SCU. Of patients with abnormal SCUs versus normal SCUs, 16 of 40 (40%) versus 18 of 84 (21%) had hospital stay lengths >2 days (P < or =.05). Using the 36 inpatient full echo studies obtained for standard indications during hospitalization as a gold standard, initial clinical examination identified only 7 of 30 (23%) significant findings and had 16 false-positive diagnoses, whereas SCU identified 22 of 30 (73%) with 8 false positives. Although similar study results occurred with interpretation of 68 SCUs obtained by EP, quality was achieved in only 55% ED personnel versus 97% of sonographer-obtained SCUs (P <.05). CONCLUSIONS: An SCU examination detects significant findings misdiagnosed on initial clinical evaluation in the ED and provides prognostic data regarding length of hospital stay.

Intravascular ultrasound assessment and endoluminal stenting of a stenosis in a 50-month-old coronary polytetrafluoroethylene graft.

We report the assessment and management of an 81-year-old patient with a rare stenosis in a 50-month-old polytetrafluoroethylene coronary graft. We concluded that atheromatous plaque was the cause for the stenosis based on the lesion location within the graft and its characteristics by intravascular ultrasound. Differential diagnoses are discussed under consideration of the literature. The lesion was successfully treated using endoluminal stenting.

[A focal usual interstitial pneumonia lesion: an important risk factor in diffuse alveolar damage--acute exacerbation of a focal usual interstitial pneumonia patient].

The aim of this study was to clarify whether the presence of a focal usual interstitial pneumonia lesion (F-UIP) is a risk factor for diffuse alveolar damage (DAD). Our subjects were 977 patients (681 males, 296 females, mean age, 64 years). The incidence of F-UIP increased significantly with age (between 50 and 60 years, 3% and 8%, p < 0.05; between 60 and 70 years, 8% and 14.1%, p < 0.05; and between 70 and 80 years, 14.1% and 27.9%, p < 0.01). The mean age of the non-UIP group was 64 years, and of the F-UIP group was 75 years, showing significance (p < 0.001). The incidence of DAD was higher in the F-UIP group (100 patients, 30%, p < 0.01) and the diffuse UIP group (60 patients, 30%, p < 0.01) than in the non-UIP group (817 patients, 5.3%). Although the causes of DAD were various, the presence of F-UIP was a important risk factor of DAD.

Comparison of TaqMan Salmonella amplification/detection kit with standard culture procedure for detection of Salmonella in meat samples.

We evaluated the TaqMan PCR Salmonella amplification/detection kit (PE Applied Biosystems) for rapid detection of Salmonella from a variety of meat samples. This system uses the 5' nuclease activity of Taq DNA polymerase, which digests an internal fluorogenic probe to monitor the amplification of the target gene. The detection sensitivity of the kit, using 2 kinds of DNA extraction protocols, was compared with that obtained with 4 protocols of official culture methods. A total of 98 meat samples (16 raw beef, 31 pork and 51 chicken) were tested. The results of the TaqMan PCR method and the combined results of the 4 cultural protocols showed excellent agreement. However, no single culture protocol showed optimal recovery of Salmonella comparable to the PCR method. These results suggest that the TaqMan PCR method is a reliable and rapid method useful for detecting Salmonella in meat products.

Protection against ischemia/reperfusion injury and myocardial dysfunction by antisense-oligodeoxynucleotide directed at angiotensin-converting enzyme mRNA.

Angiotensin-converting enzyme (ACE) activity in the myocardium and angiotensin-II (Ang-II) levels in plasma increase after myocardial ischemia, which lead to exacerbation of myocardial injury and cardiac dysfunction. We examined the protective role of novel antisense-oligodeoxynucleotide (AS-ODN) directed at ACE mRNA in myocardial ischemic injury. Sprague-Dawley rats were treated with ACE-AS-ODN (200 microg per rat, n = 8, i.v.) or inverted-ODN (IN-ODN, 200 microg per rat, n = 8, i.v.), given with 600 microg per rat of liposome DOTAP/DOPE. Hearts from AS-ODN- or IN-ODN-treated rats were excised, perfused in vitro, and subjected to 25 min of global ischemia followed by 30 min of reperfusion. Parallel groups of rats were given ACE inhibitor captopril (5 mg/kg, n = 8) or saline (n = 8) before excising the hearts. Ischemia/reperfusion resulted in myocardial dysfunction (increase in coronary perfusion pressure and LV end-diastolic pressure and a decrease in developed LV pressure) in the saline-treated rats. Myocardial dysfunction was associated with evidence of lipid peroxidation and enzyme leakage (MDA and LDH levels in the myocardium) and up-regulation of ACE protein expression. Administration of AS-ODN or captopril, but not IN-ODN, reduced Ang-II levels in the plasma, decreased ischemia/reperfusion-mediated cardiac functional deterioration and lipid peroxidation, and preserved LDH in the myocardium (all P < 0.05 versus the saline group). AS-ODN and captopril had equipotent effects on cardiac dynamics. ACE protein expression (western blot) was decreased in the hearts of the AS-ODN-treated group, but not in IN-ODN-treated rat hearts. In contrast, ACE protein expression was significantly increased in captopril-treated rat hearts. These observations suggest that AS-ODN directed at ACE mRNA can ameliorate myocardial dysfunction and injury after ischemia/reperfusion, and its use is associated with decreased expression of ACE protein in the ischemic myocardium.

Attenuation of hypertension and heart hypertrophy by adeno-associated virus delivering angiotensinogen antisense.

Angiotensinogen (AGT), one of the major components in the renin-angiotensin system, has been linked to hypertension in humans and animals. We have previously systemically administered antisense oligonucleotides and plasmid vectors with DNA that targeted AGT and attenuated hypertension in spontaneously hypertensive rats. The aim of the present study was to prolong the effect of antisense treatment by the use of a recombinant adeno-associated viral (rAAV) vector targeted to AGT. Using a model of lifelong hypertension in which 5-day-old spontaneously hypertensive rats are treated, a single intracardiac injection of rAAV-AGT-antisense (rAAV-AGT-AS) delayed the onset of hypertension for 91 days and significantly attenuated hypertension in adulthood for up to 6 months. Systolic blood pressure was always lower, by up to 23 mm Hg in the AS-treated group. The vector was stable and expressed a reporter gene in liver, kidney, and heart. The rAAV-AGT-AS treatment significantly decreased left ventricular hypertrophy (P=0.01) and also lowered levels of AGT in the liver (2.78+/-0.61 microgram/g tissue versus 5.23+/-0.41 microgram/g tissue for the sense-treated group, P<0.01). Measurement of liver transaminases showed no evidence for liver toxicity. We conclude that rAAV-AGT-AS offers a safe, stable approach for gene therapy of hypertension.

Rapid, quantitative PCR monitoring of growth of Clostridium botulinum type E in modified-atmosphere-packaged fish.

A rapid, quantitative PCR assay (TaqMan assay) which quantifies Clostridium botulinum type E by amplifying a 280-bp sequence from the botulinum neurotoxin type E (BoNT/E) gene is described. With this method, which uses the hydrolysis of an internal fluoregenic probe and monitors in real time the increase in the intensity of fluorescence during PCR by using the ABI Prism 7700 sequence detection system, it was possible to perform accurate and reproducible quantification of the C. botulinum type E toxin gene. The sensitivity and specificity of the assay were verified by using 6 strains of C. botulinum type E and 18 genera of 42 non-C. botulinum type E strains, including strains of C. botulinum types A, B, C, D, F, and G. In both pure cultures and modified-atmosphere-packaged fish samples (jack mackerel), the increase in amounts of C. botulinum DNA could be monitored (the quantifiable range was 10(2) to 10(8) CFU/ml or g) much earlier than toxin could be detected by mouse assay. The method was applied to a variety of seafood samples with a DNA extraction protocol using guanidine isothiocyanate. Overall, an efficient recovery of C. botulinum cells was obtained from all of the samples tested. These results suggested that quantification of BoNT/E DNA by the rapid, quantitative PCR method was a good method for the sensitive assessment of botulinal risk in the seafood samples tested.

Histamine formation by Tetragenococcus muriaticus, a halophilic lactic acid bacterium isolated from fish sauce.

We examined histamine formation in cultures of Tetragenococcus muriaticus, a halophilic lactic acid bacterium isolated from fish sauce. T. muriaticus formed histamine in low acidity (pH 5.8), O2 limiting conditions with optimal NaCl and glucose concentrations of 5-7% (w/v) and above 1%, respectively. Histamine formation could not be prevented even at 20% (w/v) NaCl, indicating that NaCl could not prevent histamine formation by this bacterium. A conspicuous amount of histamine accumulated only during the late stationary phase regardless of the growth conditions. Studies of cell suspension experiments confirmed the results obtained from cultured cells.