Improved rapid and efficient method for Staphylococcus aureus DNA extraction from milk for identification of mastitis pathogens

A rapid and efficient DNA extraction method was developed for detecting mastitis pathogens in milk. The first critical step involved cell wall disruption by bead-beating, as physical disruption using beads was more effective for DNA extraction from Gram-positive bacteria, such as Staphylococcus aureus, than enzymatic disruption using proteinase K. The second critical step involves the use of acetic acid and ammonium sulfate in the purification process, as these reagents effectively and efficiently remove the lipids and proteins in milk. Using these methods, DNA suitable for loop-mediated isothermal amplification was obtained within 30 min. Also, the rapid and sensitive detection of S. aureus in milk was possible at levels as low as 200 cfu/ml.     

Effect of sevoflurane concentration on visual evoked potentials with pattern stimulation in dogs

The purpose of this study was to investigate the effects of sevoflurane concentration on canine visual evoked potentials with pattern stimulation (P-VEPs). Six clinically normal laboratory-beagle dogs were used. The minimum alveolar concentration (MAC) of sevoflurane was detected from all subjects by tail clamp method. The refractive power of the right eyes of all subjects was corrected to −2 diopters after skiascopy. For P-VEP recording, the recording and reference electrode were positioned at inion and nasion, respectively, and the earth electrode was positioned on the inner surface. To grasp the state of CNS suppression objectively, the bispectral index (BIS) value was used. The stimulus pattern size and distance for VEP recording were constant, 50.3 arc-min and 50 cm, respectively. P-VEPs and BIS values were recorded under sevoflurane in oxygen inhalational anesthesia at 0.5, 1.0, 1.5, 2.0, 2.5 and 2.75 sevoflurane MAC. For analysis of P-VEP, the P100 implicit time and N75-P100 amplitude were estimated. P-VEPs were detected at 0.5 to 1.5 MAC in all dogs, and disappeared at 2.0 MAC in four dogs and at 2.5 and 2.75 MAC in one dog each. The BIS value decreased with increasing sevoflurane MAC, and burst suppression began to appear from 1.5 MAC. There was no significant change in P100 implicit time and N75-P100 amplitude with any concentration of sevoflurane. At concentrations around 1.5 MAC, which are used routinely to immobilize dogs, sevoflurane showed no effect on P-VEP.     

Lethal limits of high temperature for two crayfishes, the native species Cambaroides japonicus and the alien species Pacifastacus leniusculus in Japan

ABSTRACT: Lethal limits of high temperature were studied to clarify the effects on the survival of the endangered Japanese crayfish species Cambaroides japonicus and the alien species Pacifastacus leniusculus . After the acclimation period for 2 weeks at 16°C, the temperature was raised at a rate of 1°C per week. As a result, the ultimate upper lethal temperatures of C.   japonicus and P.   leniusculus were 27.0 and 31.1°C, respectively, and the lethal temperature for P.   leniusculus was significantly higher than for C.   japonicus . The natural distributions of these two species are discussed in terms of the temperature tolerance.     

cDNA cloning of oyster matrix metalloproteinase and its possible involvement in hypoxic adaptation

ABSTRACT:   We have cloned a cDNA encoding the matrix metalloproteinase (MMP) from oyster Crassostrea gigas . The clone contains a 1797 bp open reading frame encoding a protein of 599 amino acids. Oyster MMP (Cg-MMP) has a transmembrane domain at its C-terminal and a furin/prohormone convertase cleavage site at the end of a propeptide domain, which are commonly observed in membrane-type MMPs (MT-MMPs). This suggests that Cg-MMP is an MT-MMP. The deduced amino acid sequence of oyster MMP shares approximately 30% identity with human MT4-MMP and MMPs from fruit fly and hydra. Cg-MMP mRNA was detected in the gill, mantle and adductor muscle, and more intense signals in the northern blot analysis were recognized in the gill and adductor muscle. Similar tissue distribution was observed for tissue inhibitor of metalloproteinase (Cg-TIMP) in oyster. In response to hypoxic stress, the abundance of Cg-MMP mRNA was elevated in the gill, while that of Cg-TIMP mRNA remained almost constant. These findings suggest that promotion of collagen metabolism may be implicated in the hypoxic adaptation in oyster.     

Effects of starvation on RNA : DNA ratio, glycogen content, and C : N ratio in columellar muscle of the Japanese turban shell Turbo (Batillus) cornutus (Gastropoda)

ABSTRACT: The validity of using the RNA : DNA ratio, glycogen content, and C : N ratio in the columellar muscle as indicators of the nutritional condition in the Japanese turban shell Turbo cornutus was examined. These biochemical indices were compared between fed and starved animals. The fed animals were fed brown algae to satiation for 108 days as a control, whereas the starved animals were not fed for the first 75 days and then fed for next 33 days. All three indices declined during the starvation period, and the values for the starved group were significantly lower than those for the fed group during days 20–75. At day 108, after the re-feeding period, the indices for the starved group were found to have increased. Among the indices, only the RNA : DNA ratio for the starved males responded rapidly to starved conditions and became significantly lower even at day 4, and only the RNA : DNA ratio for the starved males and females recovered to those levels of the fed animals at day 108. The results indicated that the RNA : DNA ratio is the most rapid indicator of nutritional stress among the three indices. However, it is recommended that glycogen content and C : N ratio be used in addition to the RNA : DNA ratio for monitoring the health of T. cornutus as the RNA : DNA ratio showed large variations.     

Population structure of pigs determined by single nucleotide polymorphisms observed in assembled expressed sequence tags

We have collected more than 190 000 porcine expressed sequence tags (ESTs) from full‐length complementary DNA (cDNA) libraries and identified more than 2800 single nucleotide polymorphisms (SNPs). In this study, we tentatively chose 222 SNPs observed in assembled ESTs to study pigs of different breeds; 104 were selected by comparing the cDNA sequences of a Meishan pig and samples of three‐way cross pigs (Landrace, Large White, and Duroc: LWD), and 118 were selected from LWD samples. To evaluate the genetic variation between the chosen SNPs from pig breeds, we determined the genotypes for 192 pig samples (11 pig groups) from our DNA reference panel with matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry. Of the 222 reference SNPs, 186 were successfully genotyped. A neighbor‐joining tree showed that the pig groups were classified into two large clusters, namely, Euro‐American and East Asian pig populations. F‐statistics and the analysis of molecular variance of Euro‐American pig groups revealed that approximately 25% of the genetic variations occurred because of intergroup differences. As the F_IS values were less than the F_ST values_, the clustering, based on the Bayesian inference, implied that there was strong genetic differentiation among pig groups and less divergence within the groups in our samples.     

Development and age‐related change of cerebrovascular noradrenergic (NA) innervation in the Japanese quail

Development of cerebrovascular noradrenergic (NA) innervation was investigated in the Japanese quail, using histofluorescence technique and quantitative analysis. Cerebral perivascular NA nerves arose from the cerebral carotid (CCA), internal ethmoidal (IEA) and vertebral (VA) arteries before hatching, with the first appearance on the CCA and IEA at embryonic day (E) 8 and of the VA at E14. Nerves arising from the IEA were greater in number and spread more rapidly than those from the CCA and VA. On E16, fluorescent fiber bundles lying on the anterior circulation subdivided rapidly into thin fibers. Consequently, the number of NA nerves over the arterial system increased greatly at hatching, particularly along the distal portion of the anterior ramus. At the same developmental stage, all the major arteries of the anterior and posterior circulation were almost entirely covered by NA nerves. The abrupt reorganization of cerebrovascular NA innervation in newborn quails may be related to some specific trophic and vasomotor roles for structural and functional improvement of the cerebral circulation that is required for its brain differentiation at this critical period of development. The supply of NA nerves to the anterior and posterior circulations sequentially increased during development from posthatching day (P) 1–15. Plexuses of NA nerves in each of the major cerebral arteries at P22 and P42–50 were similar in meshwork construction to each other, and to those seen at P15. Likewise, there was no clear statistical difference in the nerve density of the corresponding arteries among the three posthatching ages, except for the caudal basilar artery.     

运用RAPD法识别杨树品种技术的研究

在正确提取ＤＮＡ后,探讨了在成本最低、效果最好的基础上,得出了ＤＮＡ最佳增殖反应液的组成（１０Ｘｂｕｆｆｅｒ１．５０、ｄｎｔｐｓ ０．３０、ＭｇＣｌ２ ０．６０、灭菌水 １０．５７、Ｐｒｉｍｅｒ１．００、Ｔａｇ０．０３、ＤＮＡ１．００）,其次用 ６０个引物对杨柳科属和杨属的 １３个品种进行了人工分析,发现多态标记 ３２个,用其中的任意 １３个标记就能区别 １３品种。