Characterization of white sapote (Casimiroa edulis Llave & Lex.) germplasm using floral morphology,RAPD and AFLP markers

Floral morphology, random amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP) were used to characterize and verify genetic diversity within a white sapote cultivar collection and to develop molecular markers for germplasm identification. On the basis of floral morphology, the cultivars were classified into three types: type I included 23 cultivars with large ovaries and small anthers; type II included 13 cultivars with small ovaries and large anthers; and type III included one cultivar, named ‘Maltby’, with a large ovary and large anthers. DNA was isolated from 39 cultivars of white sapote and subjected to RAPD and AFLP analysis using 24 and 7 primers, respectively. One hundred and sixty-eight RAPD and 286 AFLP bands were used to assess genetic characterization among white sapote. Sixty percent of the RAPD and 77% of the AFLP amplification products were polymorphic among accessions. RAPD or AFLP markers differentiated all white sapote cultivars effectively. Moreover, each flower type was characterized as specially associated with two RAPD bands. UPGMA dendrograms based on RAPD and AFLP data, showed the majority of the cultivars from flower type I and flower type II clustering together. Finally 101 RAPD markers and 220 AFLP markers were used to construct a neighbor-joining dendrogram. This showed that the 37 cultivars could be classified into six distinct clusters, between which the similarity coefficient was as low as 0.00–0.55, even though the cultivars were morphologically very similar. The remaining two cultivars namely ‘Smathers’ and ‘Maltby’ were found genetically very distant from the other cultivars in RAPD, AFLP or combined RAPD and AFLP based dendrograms. The results suggested that the level of genetic variation among white sapote cultivars is diverse and the morphological and molecular data may lead to representation of the cultivar relationships as well as flower type discrimination.     

The relationship between plasma leptin concentrations and the distribution of body fat in crossbred steers

The objective of the present study was to examine the relationship between plasma leptin levels and fat distribution in crossbred steers. Twenty-four crossbred steers (Japanese Black × Holstein) were used in this study. Six steers belonged to the growing phase (11 months of age), and 18 steers to the finishing phase (from 21 to 28 months of age). Plasma leptin levels correlated positively with body fat weight ( r  = 0.807, P  < 0.01), body fat percentage ( r  = 0.802, P  < 0.01), visceral fat weight ( r  = 0.807, P  < 0.01), visceral fat percentage ( r  = 0.780, P  < 0.01), subcutaneous fat weight ( r  = 0.745, P  < 0.01), subcutaneous fat percentage ( r  = 0.734, P  < 0.01), intermuscular fat weight ( r  = 0.793, P  < 0.01), intermuscular fat percentage (0.775, P  < 0.01), renal fat weight ( r  = 0.694, P  < 0.01), renal fat percentage ( r  = 0.642, P  < 0.01), subcutaneous fat thickness ( r  = 0.755, P  < 0.01), intermuscular fat thickness ( r  = 0.721, P  < 0.01), beef marbling score ( r  = 0.706, P  < 0.01) and the crude fat content of Musculus longissimus thoracis ( r  = 0.592, P  < 0.01). The location of fat deposits did not affect the correlation values. However, when steers were divided into growing ( n  = 6) and finishing ( n  = 18) groups, there was no significant correlation between plasma leptin levels and the carcass characteristics related to fat deposition (fat weight, fat percentage, carcass traits) in either group. These results suggest that plasma leptin levels in crossbred steers positively correlate with body fat content and carcass traits when these parameters vary widely.     

An intracytoplasmic injection of deionized bovine serum albumin immediately after somatic cell nuclear transfer enhances full-term development of cloned mouse embryos

In mouse somatic cell nuclear transfer (SCNT), polyvinylpyrrolidone (PVP) is typically included in the nuclear donor injection medium. However, the cytotoxicity of PVP, which is injected into the cytoplasm of oocytes, has recently become a cause of concern. In the present study, we determined whether bovine serum albumin deionized with an ion-exchange resin treatment (d-BSA) was applicable to the nuclear donor injection medium in SCNT as an alternative to PVP. The results obtained showed that d-BSA introduced into the cytoplasm of an enucleated oocyte together with a donor nucleus significantly enhanced the rate of in vitro development of cloned embryos to the blastocyst stage compared with that of a conventional nuclear injection with PVP in SCNT. We also defined the enhancing effects of d-BSA on the blastocyst formation rate when d-BSA was injected into the cytoplasm of oocytes reconstructed using the fusion method with a hemagglutinating virus of Japan envelope before oocyte activation. Furthermore, immunofluorescence experiments revealed that the injected d-BSA increased the acetylation levels of histone H3 lysine 9 and histone H4 lysine 12 in cloned pronuclear (PN) and 2-cell embryos. The injection of d-BSA before oocyte activation also increased the production of cloned mouse offspring. These results suggested that intracytoplasmic injection of d-BSA into SCNT oocytes before oocyte activation was beneficial for enhancing the in vitro and in vivo development of mouse cloned embryos through epigenetic modifications to nuclear reprogramming.     

Changes in walking parameters of milking cows after hoof trimming

We examined the walking parameters of milking cows in relation to hoof trimming. The hooves of eight Holstein cows were trimmed. Two days before and after the hoof trimming, walking behavior, limb angles, back posture and vertical movement of the back while walking were measured. Walking rate (1.27 vs 0.87 m/s; P = 0.003), step length (1.46 vs 1.33 m; P = 0.009) and stepping rate (0.78 vs 0.65 steps/s; P = 0.046) were found to increase significantly after hoof trimming. Limb angles at the start and end of the support phase changed significantly after hoof trimming, thus indicating an improvement in the ‘on tiptoe’ appearance. The parameters relating to backline indicated that the marked arched shape of the back diminished after hoof trimming. Before hoof trimming, the positions of the withers varied widely and irregularly while walking; however, it changed after hoof trimming as the sinusoid curve and the range of vertical movement of withers decreased significantly. Overall, it has been quantitatively indicated that walking characteristics are improved after hoof trimming. The parameters used in the present study are expected to be useful for monitoring the walking characteristics of cows.     

Dynorphin-Kappa Opioid Receptor Signaling Partly Mediates Estrogen Negative Feedback Effect on LH Pulses in Female Rats

Accumulating evidence suggests that the arcuate nucleus (ARC) kisspeptin/neurokinin B (NKB)/dynorphin (KNDy) neurons play a role in estrogen negative feedback action on pulsatile gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release. The present study aimed to determine if dynorphin (Dyn) is involved in estrogen negative feedback on pulsatile GnRH/LH release. The effect of the injection of nor-binaltorphimine (nor-BNI), a kappa-opioid receptor (KOR) antagonist, into the third cerebroventricle (3V) on LH pulses was determined in ovariectomized (OVX) adult female rats with/without replacement of negative feedback levels of estradiol (low E₂). The mean LH concentrations and baseline levels of LH secretion in nor-BNI-injected, low E₂-treated rats were significantly higher compared with vehicle-treated controls. On the other hand, the nor-BNI treatment failed to affect any LH pulse parameters in OVX rats without low E₂ treatment. These results suggest that Dyn is involved in the estrogen negative feedback regulation of pulsatile GnRH/LH release. The low E₂ treatment had no significant effect on the numbers of ARC Pdyn (Dyn gene)-,Kiss1- and Tac2 (NKB gene)-expressing cells. The treatment also did not affect mRNA levels of Pdyn and Oprk1 (KOR gene) in the ARC-median eminence region, but significantly increased the ARC kisspeptin immunoreactivity. These findings suggest that the negative feedback level of estrogen suppresses kisspeptin release from the ARC KNDy neurons through an unknown mechanism without affecting the Dyn and KOR expressions in the ARC. Taken together, the present result suggests that Dyn-KOR signaling is a part of estrogen negative feedback action on GnRH/LH pulses by reducing the kisspeptin release in female rats.     

Relationships between the age and blood test results or body sizes in Noma horses

The objective of this study is to analyze the relationships between the age and blood test results or body sizes in Noma horses by using the results of periodical health examination. Out of 45 hematological or physical items examined, statistically significant, but loose correlations were observed in 14 items. Red blood cell count, activities of aspartate aminotransferase, alkaline phosphatase, and creatinine kinase, concentrations of calcium and inorganic phosphorus decreased with aging. Conversely, mean corpuscular volume, mean corpuscular hemoglobin, lipase activity, γ-globulin and chloride concentrations, body height, chest circumference and cannon bone circumference increased with aging. The changes in a few items seemed unique to Noma horse. However, most age-related changes found in this study might be considered as a common trend in horse breeds rather than distinctive characteristic in Noma horse.     

Influence of osmotic pressure on somatic embryo maturation in Pinus densiflora

The effect of an osmoticum, polyethylene glycol (PEG), on somatic embryo production was examined using embryogenic cells of Pinus densiflora. In the basal medium containing 30 μM abscisic acid and 6% maltose, the quality of the embryos formed was poor even though somatic embryos were produced. The addition of PEG with molecular weight of 4000 or 8000 significantly enhanced the development of both the quality and quantity of somatic embryos. Furthermore, higher levels of a constant osmotic pressure with PEG 8000 in a range from about 300 to 450 mmol/kg could remarkably enhance the morphogenesis of somatic embryos and their number of embryos produced. A higher stable osmotic pressure with an appropriate molecular weight of PEG is a key factor for the production of good quality somatic embryos in P. densiflora.     

Harpinpsta from Pseudomonas syringae pv. tabaci Is Defective and Deficient in Its Expression and HR-inducing Activity

To elucidate the role of harpins produced by Pseudomonas syringae, the corresponding hrpZ gene was isolated from P. s. pv. tabaci. The sequence information revealed that this gene carries a serious mutation with 326 bp lacking in the central region and potentially encodes only 140 N-terminal amino acids because of a frame shift. The investigation of biological properties using recombinant harpin indicated harpin_psta was incapable of inducing HR in both host and nonhost plants. Based on an immunoblot analysis to detect harpin from P. s. pathovars in hrp-inducing medium, the truncated harpin_psta was neither expressed nor secreted into the culture medium. These results suggest that harpin is not the sole determinant of the host-parasite specificity in P. s. pv. tabaci. Received 10 August 2000/ Accepted in revised form 21 December 2000