Diverse functions of perlecan in central nervous system cells in vitro

Therapeutic treatment targeting one cell type is considered ineffective in remedying any injury to the central nervous system (CNS). Perlecan, a multi‐functional, heparan sulfate proteoglycan, shows diverse effects on distinct cell types, suggesting that it is one of the candidates that can augment the regenerative mechanisms in the injured CNS. Therefore, we examined the functions of perlecan in CNS cells in vitro by using perlecan purified from bovine kidney. Perlecan‐coated cell culture plates, unlike their type I/III collagen‐coated counterparts, did not inhibit the adhesion of neural stem/progenitor cells (NS/PCs) and neurons. The coated perlecan and the perlecan added to the culture medium suppressed astrocyte proliferation; however, perlecan added to the medium promoted NS/PC proliferation. Neurons were promoted to extend their neurites on the perlecan‐coated substrate, and perlecan added to the medium also showed a similar effect. NS/PC proliferation and neurite extension is a major regenerative reaction in CNS injury, whereas excess proliferation of astrocytes cause hypertrophy of glial scars, which repels neurons. Our in vitro study suggests that perlecan is an attractive candidate to promote regenerative mechanisms and to suppress reactions that hamper regenerative processes in cases of CNS injury.     

Disruption of endogenous perlecan function improves differentiation of rat articular chondrocytes in vitro

Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPG) are necessary for normal cartilage development and chondrocyte differentiation. However, recent studies demonstrated that HSPG accelerate dedifferentiation and catabolism in chondrocytes from degenerative cartilage. In this study, we investigated the inhibitory effect of HSPG on chondrocyte differentiation in vitro. Rat articular chondrocytes were cultured at low (0.3 × 10⁴ cells/cm²) and high (1.5 × 10⁵ cells/cm²) density in the presence or absence of heparitinase I, an HS degrading enzyme. Cells cultured at low density dedifferentiated and exhibited an elongated morphology, and treatment with heparitinase I precluded cell elongation. Conversely, populations of chondrocytes cultured at high density exhibited either a dedifferentiated or differentiated phenotype. Glycosaminoglycan accumulation increased in heparitinase I‐treated cells. To determine the function of perlecan, an important HSPG for cartilage development, in chondrocyte differentiation, rat chondrocyte cultures were exposed to an anti‐perlecan antiserum to inhibit perlecan function. Western blotting analysis indicated that preventing perlecan activity increased type II collagen synthesis. Our results suggest that HSPG are negative regulators of chondrocyte differentiation in vitro and that perlecan contributes to chondrocyte dedifferentiation in vitro.     

Reliability of estrous detection in Holstein heifers using a radiotelemetric pedometer located on the neck or legs under different rearing conditions

To evaluate the efficiency and accuracy of estrous detection using a new pedometry system that can measure the hourly activity of cattle, pedometers were attached to the neck and the hind legs of 15 Holstein heifers. Heifers were reared in pasture for grazing, an open paddock, or in a tie-stall barn (an additional pedometer was attached to a front leg of each of these heifers). The most recent 24 h-total number of steps was compared for each 1 h-interval with the mean value of the preceding days during the reference period (RP). The neck pedometer detected all 10 instances of estrous activity (100%) for the grazing heifers at 1.3 times the thresholds value for a 5-day RP but with only 32% accuracy. The hind leg pedometer, however, obtained 100% efficiency and 83% accuracy at 1.4 times the threshold value for a 7-day RP. The efficiencies and accuracies in detecting 12 instances of estrous activity under the paddock condition were 92 and 65% (neck, 1.3-fold, 7-day RP) and 92 and 100% (hind leg, 1.6- or 1.7-fold, 7-day RP), respectively. Under the tie stall condition, the neck pedometers detected 92% of 23 instances of estrous activity with 34% accuracy (1.2-fold, 3-day RP), and the efficiencies and accuracies of the leg pedometers were 78 and 78% (hind leg, 1.4-fold, 4- or 6-day RP) and 87 and 83% (front leg, 1.4-fold, 7-day RP), respectively. Prediction of ovulation time was more precisely with the leg pedometers than with those under the tie stall conditions. Our preliminary results indicate that this new pedometer system has practical value for estrous detection in heifers under different rearing conditions, which affect the criteria required for detection. Furthermore, they also indicate that a leg pedometer can reliably detect estrus and that a neck pedometer may only be capable of detecting estrus under paddock rearing conditions.     

Serological diagnosis of enzootic pneumonia of swine by a double-sandwich enzyme-linked immunosorbent assay using a monoclonal antibody and recombinant antigen (P46) of Mycoplasma hyopneumoniae

To facilitate the control of enzootic pneumonia (EP) of swine caused by Mycoplasma hyopneumoniae, the complement fixation (CF) test has been used for the detection of M. hyopneumoniae antibodies. However, the CF test is a cumbersome and time-consuming technique and cross-reactivity are major drawbacks associated with this method. To circumvent these drawbacks, we have developed a double-sandwich enzyme-linked immunosorbent assay (ELISA), consisting of purified monoclonal antibody (Mab) against the 46 kDa surface antigen (P46) of M. hyopneumoniae and recombinant P46 protein expressed in Escherichia coli, for the detection of antibodies to M. hyopneumoniae in serum samples from pigs experimentally inoculated with M. hyopneumoniae and from naturally infected pigs, and compared the practical usefulness of ELISA using the CF test. In experimentally inoculated pigs, the CF and ELISA antibodies were detected at almost the same time, and a good correlation was demonstrated between the CF test and the ELISA. In a survey conducted on field samples, the seropositivity by ELISA in pigs of age 2-6 months was increased. At the time of slaughter, approximately 80% of the animals were seropositive for ELISA. However, a gradual decrease in the prevalence of ELISA positive samples was observed in sows with increasing parity. No correlation was seen between the results obtained with the two methods in the clinical samples. The CF test appears to have limited value for the diagnosis of EP in conventional herds because nonspecific reactions were frequently observed. Therefore, this ELISA is a useful alternative to the CF test currently used for the diagnosis of EP.     

Detection of New Ethylene-Producing Bacteria, Pseudomonas syringae pvs. cannabina and sesami, by PCR Amplification of Genes for the Ethylene-Forming Enzyme

ABSTRACT Strains of Pseudomonas syringae (78 strains and 43 pathovars) and other strains (79) of plant and insect origin were examined for the presence of the ethylene-forming enzyme gene (efe) by polymerase chain reaction (PCR) assay. The sequence of the efe gene of P. syringae pv. phaseolicola PK2 was used to design two primer sets for amplification of the gene. In addition to P.syringae pv. phaseolicola (the "kudzu strain") and P.syringae pv. glycinea, which were efficient ethylene producers, several strains of P.syringae pvs. sesami and cannabina generated PCR products of the predicted size. A DNA probe of the efe gene, isolated from strain PK2, hybridized to these PCR products, indicating homology to the P.syringae pv. phaseolicola efe gene. PCR restriction fragment length polymorphism analyses suggested that these four pathovars harbor a similar efe gene. Furthermore, the probe hybridized to an indigenous plasmid of P.syringae pv. cannabina, suggesting that the efe gene could be located on a plasmid in this pathovar, but did not hybridize to plas-mids of P.syringae pv. sesami strains. P.syringae pvs. sesami and cannabina strains produced ethylene in King's medium B at levels similar to those of P.syringae pvs. phaseolicola and glycinea. Thus, two new ethylene-producing bacteria were detected by the PCR assay.     

Hong Kong consumer preferences for Japanese beef: Label knowledge and reference point effects

The excellent flavor of Wagyu is becoming increasingly popular all over the world. However, the popularity of Wagyu has encouraged competition for authentic Japanese Wagyu, resulting in the appearance of inauthentic Wagyu beef. To ward off this export competition, Japanese Wagyu producers need to improve and differentiate their value‐added beef. As hardly any past studies focus on the consumption of Japanese Wagyu in Hong Kong, this paper uses a choice experiment to examine the valuation of beef by Hong Kong consumers in terms of country of origin. Data from 250 Hong Kong consumers obtained through a web questionnaire were used to analyze the beef preferences. In addition to the beef's country of origin, its marbling level, the Japanese Wagyu label and reference point effects were considered. The results indicate that Hong Kong consumers place a significant premium on Japanese Wagyu over Australian or American Wagyu. That premium is greater among consumers who have seen the Japanese Wagyu label. Reference price effects were also statistically confirmed. To promote Japanese Wagyu beef consumption, therefore, it is important to make the consumer aware of the advantages of Japanese Wagyu.     

A promising strain of endophytic <Emphasis Type="Italic">Streptomyces</Emphasis> sp. for biological control of cucumber anthracnose

In a survey for effective biocontrol agents of Colletotrichum orbiculare, causal agent of cucumber anthracnose, 43 (MBCu series) and 135 (MBPu series) endophytic actinomycete strains were recovered from surface-sterilized organs of cucumber (Cucumis sativus) and pumpkin (Cucurbita moschata) plants, respectively. The strains were cultured with C. orbiculare on IMA-2 agar medium to determine their in vitro antagonistic ability. Eleven strains that strongly inhibited hyphal growth of the pathogen were selected as potent antagonists. Detached cotyledons of cucumber were soaked in a spore suspension of an antagonistic strain 1 day before challenge-inoculation with the pathogen, and six of these antagonists (MBCu-32, 36, 42, 45, and 56, and MBPu-75) significantly reduced the number and size of the lesions on the cotyledons compared to the untreated control. In the same way, these six strains inhibited lesion development on attached leaves of 3-week-old cucumber seedlings. Strain MBCu-56, the most suppressive of the strains, was selected for further tests. Its suppressiveness increased as concentrations increased; pretreatment of leaves with the strain at 10⁷, 10⁸ and 10⁹ cfu/ml suppressed disease by 72, 79 and 93%, respectively. These results strongly indicated that MBCu-56 has strong potential for controlling cucumber anthracnose. Based on the taxonomic characteristics and 16S rDNA sequence, MBCu-56 was identified as Streptomyces sp. With scanning electron microscopy, the substrate mycelia of the strain were seen to colonize the surface of leaves above the cuticle. Some hyphae also penetrated and grew underneath the cuticle.     

Production of triploid eastern little tuna,Euthynnus affinis (Cantor, 1849)

Herein, we developed a triploidization method using spawned eggs collected immediately after spawning of eastern little tuna (ELT), Euthynnus affinis. ELT broodstock induced the spawning by hormonal treatment in the tank, with the resulting spawned eggs being used for the triploidy induction. Under optimal conditions, the mean ± SEM triploidization and hatching rates were 97.2 ± 2.8% and 84.5 ± 10.3%, respectively. Although triploid ELT showed growth performance equivalent to that of diploids, the triploids died at a higher rate than the diploids during 2–4 weeks post‐hatching when triploids and diploids were reared in the same tank. Therefore, we propose that it would be necessary in a practical operation to use triploid‐only ELT seedlings to avoid selective cannibalism by the diploids. The ELT triploids exhibited an all‐female phenotype. Because previous studies have reported that female triploids show a greater probability of sterility than male triploids, this characteristic could be a major advantage. Since this triploidization method, using spawned eggs, can be performed without handling the broodstock, it is possible to avoid the physical damage caused by the process of artificial insemination, making it possible to repeatedly produce triploid populations without valuable broodstock loss. Thus, we have developed an efficient method to produce ELT triploids, although further study is essential to evaluate sterility of the triploid ELT.     

Application of surrogate broodstock technology in aquaculture

Fisheries Science - Surrogate broodstock technology facilitates the production of donor-derived gametes in surrogates, and comprises transplanting germ cells of a donor into recipients of a...     

Development of a simple method for sperm cryopreservation of Scombridae fishes in outdoor environments

We developed a simple dry shipper method for cryopreserving the sperm of Scombridae fish in outdoor environments. First, we undertook a preliminary study to optimize the sperm cryopreservation conditions using bullet tuna, Auxis rochei (Risso, 1810) sperm. We found that the optimum cryomedium contained 90% foetal bovine serum (FBS) or 300 mM trehalose as an external cryoprotectant and 10% dimethyl sulfoxide (DMSO) as an internal cryoprotectant. Under these optimized conditions, the post‐thaw sperm had a duration of motility of 500 s and a motility rate of >70%. We then performed practical trials of the optimized protocol in various outdoor environments (e.g., fishing boats and ports) using the sperm of five Scombridae species: chub mackerel, Scomber japonicus (Houttuyn, 1782); blue mackerel, S. australasicus (Cuvier, 1832); skipjack tuna, Katsuwonus pelamis (Linnaeus, 1758); longtail tuna Thunnus tonggol, (Bleeker, 1851) and Pacific bluefin tuna, T. orientalis (Temminck & Schlegel, 1844). The post‐thaw sperm of all five of these species had a duration of motility of 650 s and a motility rate of >70%, indicating that this simple method can be used to obtain high‐quality cryopreserved sperm of various Scombridae species in outdoor environments.