Effects of fat coated rumen bypass lysine and methionine on performance of dairy cows fed a diet deficient in lysine and methionine

Two experiments were conducted to evaluate the effect of fat coated rumen bypass lysine (RPLys) and methionine (RPMet) on the lactation performance of dairy cows. In Experiment 1, three lactating cows were supplied with RPLys and fat coated DL‐Met which was highly protected (H‐RPMet) as an indigestible marker, and total fecal emission was collected for 72 h following administration. Measuring the proportional difference in fecal excretion of lysine derived from RPLys relative to methionine derived from H‐RPMet, the intestinal availability of RPLys was estimated to be 66.2%. In Experiment 2, 20 multiparous Holstein cows producing approximately 40 kg/day of milk were assigned to two treatments; fed RPLys (16 g/day as lysine) and RPMet (6.5 g/day as methionine) or none (control) from 5 to 21 weeks postpartum. The consumption of dry matter, organic matter, crude protein, neutral detergent fiber and acid detergent fiber were significantly more in the control cows throughout the experimental period. Their milk protein yield, the contents of their milk protein and milk fat were higher by 0.03 kg (P = 0.03), 0.06% (P < 0.001) and 0.11% (P = 0.07), respectively, in the treatment group compared to the control. These results suggest that the RPLys and RPMet used in this study improved the lactation performance of dairy cows.     

An improved transformation system for Lombardy poplar (Populus nigra var. italica)

We report an improved transformation system for Lombardy poplar (Populus nigra var. italica). A new binary vector, pBF2, with 11 unique restriction enzyme sites and the normal neomycin phosphotransferase II (NPTII) gene was constructed for the transformation of Lombardy poplar. Genetically transformed adventitious shoots were directly regenerated after cocultivation of stem segments with Agrobacterium tumefaciens EHA105 that harbored a binary vector with genes for the NPTII and enhanced green fluorescent protein. Successful transformation was confirmed by fluorescence microscopy, immunoblotting and Southern blotting analyses, and resistance to kanamycin and geneticin. This transformation system requires less time than our previous method for the regeneration of transgenic shoots. When explants were incubated on a smedium containing dithiothreitol, the transformation frequency increased to approximately 20%.     

cDNA cloning and expression of a novel group IB phospholipase A2 in the intestine of the red sea bream,Pagrus (Chrysophrys) major

A cDNA encoding a novel phospholipase A₂ (PLA₂), which was named IN PLA₂, was cloned from the intestine of the red sea bream. The amino acid sequence of IN PLA₂ showed 49–75% homology with those of red sea bream group IB sPLA₂, hepatopancreas DE-1 and DE-2 PLA₂, and gill G-3 PLA₂. IN PLA₂ consists of a prepropeptide of 24 amino acid residues, followed by a mature protein. IN PLA₂ contains 14 cysteines, and includes Cys11, the calcium binding loop and the pancreatic loop that are commonly conserved in group IB sPLA₂ enzymes. In addition, IN PLA₂ is a cationic protein with a pI of 8.52. Therefore, IN PLA₂ was identified as a novel group IB sPLA₂ isoform in red sea bream. IN PLA₂ mRNA was found by northern blot analysis to be expressed mainly in the pyloric caeca and the intestine, and was detected in the goblet cells of the intestine by in situ hybridization. The expression level of IN PLA₂ mRNA was elevated by intravenous injection of lipopolysaccharide—the outer-membrane component of Gram-negative bacteria. These results suggest that IN PLA₂ is secreted from the goblet cells of the intestine in response to stimulus such as bacterial infection, and that it contributes to antimicrobial defense in addition to the digestion of dietary phospholipids in the gastrointestinal tract.     

A PCR assay to specifically detect serovar 1a strains of Erysipelothrix rhusiopathiae and differentiate them from serovar 2 strains possessing an intact ERH_1440 gene

The Erysipelothrix rhusiopathiae ERH_1440 gene, which encodes CDP-glycerol:poly (glycerophosphate) glycerophosphotransferase, is conserved in serovar 1a strains. The gene is usually missing or truncated in other serovar strains and therefore has been used for PCR detection of serovar 1a strains. We have previously reported a rare case of an E. rhusiopathiae serovar 2 strain possessing an intact ERH_1440. In this study, we analyzed three additional serovar 2 strains with an intact ERH_1440 and developed a new PCR assay for the specific detection and differentiation of serovar 1a strains from these serovar 2 strains. PCR with primers designed based on serovar 1a-specific gene sequences upstream of ERH_1440 showed 100% specificity for four hundred thirty Erysipelothrix strains isolated from extensive origins.     

Bacterial degradation of antibiotic residues in marine fish farm sediments of Uranouchi Bay and phylogenetic analysis of antibiotic-degrading bacteria using 16S rDNA sequences

ABSTRACT:   Antibiotic residues in marine sediments of fish farms negatively influence microbial ecologic systems. The microbial degradation of antibiotic residues was experimentally examined in the marine sediments of Uranouchi Bay, to which one of five antibiotics was added. After incubation reducing physical factors, ampicillin, doxycycline, oxytetracycline, and thiamphenicol were significantly degraded, while josamycin maintained most of the initial amounts. The isolates resistant to ampicillin, josamycin, oxytetracycline, or thiamphenicol degraded each antibiotic in wide ranges of degrees, whereas the isolates degrading doxycycline were not obtained. Microbial degradation may contribute to the disappearance of ampicillin, doxycycline, oxytetracycline, and thiamphenicol in the fish farm. In contrast, the disappearance of josamycin would depend on physical factors, but the bacteria degrading josamycin at least exist in the marine sediments. Phylogenetic analysis using 16S rDNA sequences demonstrated that the antibiotic-resistant isolates formed several clusters in the Gram-positive bacterial group, the Flavobacterium–Cytophaga–Bacteroides group, and the proteobacteria subdivisions. The antibiotic-resistant bacterial population would be composed of various species including ubiquitous coastal bacterial groups. Several species of antibiotic resistant bacteria show antibiotic degradation activities, and appear to contribute to the disappearance of antibiotics in Uranouchi Bay.     

Effects of high-K+, Na+-deficient solution on contractility of the smooth muscles of the bovine trachea

A high-K+, Na+-deficient (I-154 K+) solution induced contraction followed by gradual relaxation of the smooth muscles of the bovine trachea, while hyperosmotic addition of 65 mM KCl induced a large sustained contraction. Exposure of the muscle to the I-154 K+ solution induced an increase in the ratio of cellular water content and a sustained increase in oxidized flavoprotein fluorescence or reduced pyridine nucleotide fluorescence. The I-154 K+ solution also induced a sustained increase in [Ca2+]i level. Decreases in developed tension and increases in cellular water content were both prevented by the addition of sucrose or NaCl but not pyruvate. Substitution of KI for KCl in the I-154 K+ solution produced a greater inhibition of contraction, while substitution with K-propionate produced no inhibition of contraction. Moreover, decreases in developed tension and increases in cellular water content were both prevented by addition of 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), but not 10 microM bumetanide or 1 mM acetazolamide. In conclusion, I-154 K+ solution induced-relaxation in the bovine trachea may be due to swelling of smooth muscle cells and the mechanism of swelling is probably involved in DIDS-sensitive anion movement.     

Effects of papaverine on carbachol- and high K+-induced contraction in the bovine abomasum

The effects of papaverine on carbachol (CCh) -and high K⁺- induced contraction in the bovine abomasum were investigated. Papaverine inhibited CCh (1 µM) -and KCl (65 mM) -induced contractions in a concentration-dependent manner. Forskolin or sodium nitroprusside inhibited CCh-induced contractions in a concentration-dependent manner in association with increases in the cAMP or cGMP contents, whereas papaverine increased cGMP contents only at 30 µM. Changes in the extracellular Ca²⁺ from 1.5 mM to 7.5 mM reduced verapamil-induced relaxation in high K⁺-depolarized muscles, but papaverine-induced relaxation did not change. Futhermore, papaverine (30 µM) and NaCN (300 µM) decreased the creatine phosphate contents. These results suggest that the relaxing effects of papaverine on the bovine abomasum are mainly due to the inhibition of aerobic energy metabolism.     

Effects of Acidification on Salmonid Spawning Behavior

We studied the effects of acidification on female sexual behavior in brown trout (Salmo trutta) and compared the results with those in hime (land-locked sockeye) salmon (Oncorhynchus nerka) (Kitamura and Ikuta, 2000). The results were similar to those of sockeye salmon. Spawning brown trout were extremely sensitive to the acidity of ambient water, and nest-digging behavior was severely inhibited (p<0.05) by very slight acidification (pH below 6.4). However, there were some differences between the two species. Female trout and salmon showed almost no digging below pH 5.0 and 6.0 (Kitamura and Ikuta, 2000), respectively. When the ambient water was returned to nearly neutral (pH6.6) conditions, digging in hime salmon reappeared in 4 of the 6 fish tested (Kitamura and Ikuta, 2000), whereas digging in brown trout reappeared in all 6 fish tested. The above-mentioned differences in behavioral response between the two species appear to reflect the species difference in terms of vulnerability to acidification (Ikuta et al., 1992). Avoidance of slightly acidic water in selection of spawning site or cessation of spawning behavior in weakly acidic environments may be the most potent cause of the reduction of salmonid populations in the early stages of acidification.     

Determination and Speciation of Aluminum in Environmental Samples by Cation Exchange High-Performance Liquid Chromatography with High Resolution ICP-MS Detection

A cation-exchange high-performance liquid chromatography with high resolution inductively coupled plasma mass spectrometric detection (CE-HPLC/ICP-MS) was developed for the determination and the speciation of aluminum in environmental samples. Three types of aluminum species (AlL_x ^<+2, AlL_x ²⁺, Al³⁺) were separated from one another, and were determined with the present system. The comparison of the present system with an established CE-HPLC with fluorimetric detection using 5-sulfo-8-quinolinol (CE-HPLC/FL) was described. The present system showed better sensitivity for aluminum than CE-HPLC/FL. Moreover, the analytical results for soil extract and lake water samples obtained with both methods were in good agreement with each other.     

Effects of hypoxia and glucose-removal condition on muscle contraction of the smooth muscles of porcine urinary bladder

To elucidate the dependence of aerobic energy metabolism and utilization of glucose incontraction of urinary bladder smooth muscle, we investigated the changes in the reducedpyridine nucleotide (PNred) fluorescence, representing glycolysis activity, and determinedthe phosphocreatine (PCr) and ATP contents of the porcine urinary bladder duringcontractions induced by high K⁺ or carbachol (CCh) and with and without hypoxia(achieved by bubbling N₂ instead of O₂) or in a glucose-freecondition. Hyperosmotic addition of 65 mM KCl (H-65K⁺) and 1µM CCh induced a phasic contraction followed by a tonic contraction. Aglucose-free physiological salt solution (PSS) did not change the subsequent contractileresponses to H-65K⁺ and CCh. However, hypoxia significantly attenuatedH-65K⁺- and CCh-induced contraction. H-65K⁺ and CCh induced asustained increase in PNred fluorescence, representing glycolysis activity. Hypoxiaenhanced H-65K⁺- and CCh-induced increases in PNred fluorescence, whereasglucose-free PSS decreased these increases, significantly. In the presence ofH-65K⁺, hypoxia decreased the PCr and ATP contents; however, the glucose-freePSS did not change the PCr contents. In conclusion, we demonstrated that highK⁺- and CCh-induced contractions depend on aerobic metabolism and that anendogenous substrate may be utilized to maintain muscle contraction in a glucose-free PSSin the porcine urinary bladder.