Multiplex PCR and Genescan analysis to detect immunoglobulin heavy chain gene rearrangement in feline B-cell neoplasms

Lymphoid neoplasms are usually diagnosed on the basis of cytological and histopathological findings. However, in some cases, discrimination of lymphoid neoplasms from reactive lymphoid proliferation is difficult. PCR amplification of complementarity-determining region 3 (CDR3) of the immunoglobulin heavy-chain variable region (IGHV) gene can be used to assess clonality of B-cell populations as a supportive diagnostic tool for B-cell neoplasms. Because of the sequence variation and possible somatic hypermutation of the IGHV gene, sensitivity of the PCR-based assay to detect clonal IGHV gene rearrangement largely depends on the sequences and numbers of primer sets. Prior to the development of an efficient assay, we cloned and sequenced 97 IGHV complementary DNAs (48 IGHV-1 and 49 IGHV-3 clones) from normal cat spleens. On the basis of these sequences, we designed 6 forward primers at the variable region and 5 reverse primers at the joining region. Using each of 6 forward primers and a mixture of 5 reverse primers, we amplified CDR3 of IGHV genes and analyzed the PCR products by conventional PAGE and Genescan analyses using fluorescence-labeled primers. Twenty-six feline B-cell neoplasms diagnosed by histopathological and immunohistochemical examinations were subjected to the newly developed analysis of IGHV gene rearrangement. Clonal IGHV gene rearrangement was detected in 22 of 26 (84%) samples by both PAGE and Genescan analyses. To reduce the number of PCR reactions, we constructed a multiplex PCR analysis system using a mixture of IGHV-1- and IGHV-3-specific primers as forward primers and a mixture of 5 joining region reverse primers. Results of the multiplex PCR were 100% concordant with those obtained by each of the singleplex PCRs. The multiplex PCR-based assay and Genescan analysis developed in the present study would be useful and practical tools to detect clonal IGHV gene rearrangement in feline B-cell neoplasms.     

Mycobacterium ulcerans infection in an Indian flap-shelled turtle (Lissemys punctata punctata)

We report an atypical mycobacterial infection in an Indian flap-shelled turtle, Lissemys punctata punctata, that died in an aquarium in Japan. At necropsy, the turtle showed multiple white nodules on the capsular surface and parenchyma of various organs such as the liver, spleen, intestine, and lung. Histologically, granulomatous inflammation surrounding a central zone of necrosis was observed. Sections stained by the Ziehl-Neelsen method revealed numerous acid-fast bacilli in the cytoplasm of macrophages and in the central area of necrosis. The organisms were identified as a mycobacterial species by PCR and nucleotide sequence analysis and revealed 98-100% homology to M. ulcerans. This is, to our knowledge, the first report of mycobacteriosis due to M. ulcerans in a turtle.     

Geographical distribution and seasonality of the prevalence of Leucocytozoon lovati in Japanese rock ptarmigans (Lagopus mutus japonicus) found in the alpine regions of Japan

In this study, we investigated the geographical distribution and seasonality of Leucocytozoon lovati infection in the Japanese rock ptarmigan (Lagopus mutus japonicus); this bird is one of the special natural monuments of Japan that inhabits the Japanese alpine regions. We examined blood samples from birds captured in the Kubiki, Hida, and Akaishi mountain ranges for three years from 2002 to 2005. Seventy-three blood samples from 42 males, 19 females, and 12 birds of unknown sex were used for this study. The rate of infection with L. lovati was 78.1% in the 73 birds examined. We demonstrated that the L. lovati infection was distributed across wide ranges of ptarmigan populations from the northern to the southern alpine zones. There was no sex bias in the prevalence ratio. The prevalence of L. lovati and the level of parasitization of the blood cells tended to increase from spring through summer; in contrast, a decrease was observed from summer through autumn. Although L. lovati infection was observed in a number of local populations inhabiting three mountainous regions, no infected birds were found in Mt. Johnen-dake and Mt. Maejohnen-dake. It is necessary to continue surveying the relationship between the population dynamics of the ptarmigan and the density of the arthropod vector from the perspective of in situ conservation of this endangered species.     

Effects of growth factors on development of fetal islet B-cells in vitro

To investigate the role of growth factors (epidermal growth factor [EGF], betacellulin, and activin A) in the development of islet B cells of rat fetal pancreatic explants in vitro, pancreases from rat fetuses at day 18 of gestation were cultured for 96 hr, with or without these growth factors. Culture medium was changed every 24 hr, and the level of insulin released in the culture medium was measured. After 72 hr of culture, pancreases were examined histologically. As a result, EGF promoted cell proliferation, but reduced B cell volume. Whereas, betacellulin and activin A inhibited cell division, but promoted increased B cell volume and insulin secretion, especially activin A, which stimulated insulin release in a time dependent manner. These results suggest that EGF, betacellulin, and activin A promote pancreatic cell proliferation, islet B-cell differentiation, and islet B-cell differentiation and functional maturation, respectively, and that EGF, betacellulin, and activin A, in this order, regulate islet B-cell neogenesis.     

利用内部信息的农用自动引导行走车的研究(第3报)——农业环境内位置检测系统

采用内部传感器（压电式陀螺仪角速度计和雷达式速度计）来检测行走车的角速度和速度，通过对检测量的积分可分别得到方向角和移动距离，并可由位置计算模型获得行走车的方位。考虑到农业环境内车辆的纵摇和横摇的影响，使用３个角速度计构成相互垂直的直角坐标系，依此对角速度进行修正。该位置检测系统获得了较好的检测精度。     

Measurement of the maximum amount of water-extractable phosphorus in animal manure compost by continuous and sequential water extraction

Measuring the phosphorus (P) solubility in animal manure compost (AMC) is important to estimate both the risk of P loss from agricultural land and the P availability for crops in agriculture that use AMC as fertilizer. Water-extractable phosphorus (WEP) has most often been used to estimate P solubility in AMC. A single water extraction with a high ratio of compost to water is usually used to determine WEP, but in many cases, this may not evaluate the maximum WEP. In this study, we extracted the P included in AMC by using a continuous extraction method with a large amount of water, and tested this approach for 16 AMCs: four cattle manure composts, five swine manure composts, five layer (chicken) manure composts, and two broiler (chicken) litter composts. The P dissolution patterns were fitted to kinetic models, and the maximum WEP (WEP_max) was determined by the coefficient of the non-linear regression equation. The WEP_max values corresponded to the sum of H₂O- and sodium bicarbonate (NaHCO₃)-extractable P measured using the modified Hedley sequential extraction method. The result also suggests that the maximum amount of WEP from AMC can be estimated rapidly using a sequential extraction (the Hedley method) that has been widely used to characterize P in manure or AMC. The maximum water-extractable magnesium (Mg) (WEMg_max) was significantly positively correlated with WEP_max (r = 0.854, P < 0.01). This suggests that WEP_max in the AMCs is affected by water-soluble magnesium compounds.     

A weeding-duration model for Abies sachalinensis plantations in Hokkaido, northern Japan

I developed a weeding-duration model for Sakhalin fir (Abies sachalinensis (Fr. Schmidt) Masters) plantations that employs a generalized linear model. The number of years following planting that weeding is necessary is the response variable, and elevation, slope steepness, maximum snow depth, annual precipitation, geology, soil, site index, slope aspect, and vegetation type are explanatory variables. Among the explanatory variables, geology, soil, slope aspect, and vegetation type are categorical data. A Poisson distribution is assumed for the response variable, with a log-link function. Elevation, slope steepness, maximum snow depth, annual precipitation, site index, and vegetation type had a significant effect on weeding duration. Among the eight models with the smallest Akaike information criterion (AIC), I chose the model with no multicollinearity among the explanatory variables. The weeding-duration model includes site index, maximum snow depth, slope steepness (angle) and vegetation type as explanatory variables; elevation and annual precipitation were not included in the selected model because of multicol- linearity with maximum snow depth. This model is useful for cost-benefit analyses of afforestation or reforestation with Abies sachalinensis.     

Dynamic responses of nailed plywood-timber joints under a band-limited white-noise wave

In this study, dynamic tests of nailed plywood-timber joints are conducted under a band-limited white-noise wave using a uniaxial hydraulic shaking table. The principal results are as follows: dynamic responses of nailed plywood-timber joints under a band-limited white-noise wave are reasonably related to the static load-slip relationships and the dynamic responses under harmonic waves of them. When nailed plywood-timber joints are shaken under a composite wave of wide frequency range, they resonate transitionally with one of the frequency components one after another decreasing their equivalent resonant frequencies along their static load-slip curves. Nailed plywood-timber joints are in danger of ultimate failures even though the input maximum accelerations do not exceed their damage limits, if they are shaken for long times with or without intermittences. In this case, nailed plywood-timber joints are prone to fail in low-cyclic bending fatigue failure of nails. Nailed plywood-timber joints, on the other hand, are prone to fail in typical static failure modes when they are shaken under strong input maximum accelerations equivalent to their ultimate limits.     

Vitrification procedure decreases inositol 1,4,5‐trisphophate receptor expression,resulting in low fertility of pig oocytes

Although cryopreservation of mammalian oocytes is an important technology, it is well known that unfertilized oocytes, especially in pigs, are highly sensitive to low temperature and that cryopreserved oocytes show low fertility and developmental ability. The aim of the present study was to clarify why porcine in vitro matured (IVM) oocytes at the metaphase II (MII) stage showed low fertility and developmental ability after vitrification. In vitro matured cumulus oocyte complexes (COCs) were vitrified with Cryotop and then evaluated for fertility through in vitro fertilization (IVF). Although sperm‐penetrated oocytes were observed to some extent (30–40%), the rate of pronuclear formation was low (9%) and none of them progressed to the two‐cell stage. The results suggest that activation ability of cryopreserved oocytes was decreased by vitrification. We examined the localization and expression level of the type 1 inositol 1,4,5 trisphosphate receptor (IP₃R1), the channel responsible for Ca²⁺ release during IVF in porcine oocytes. Localization of IP₃R1 close to the plasma membrane and total expression level of IP₃R1 protein were both decreased by vitrification. In conclusion, our present study indicates that vitrified‐warmed porcine COCs showed a high survival rate but low fertility after IVF. This low fertility seems to be due to the decrease in IP₃R1 by the vitrification procedure.