Effects of intravenous administration of neurokinin receptor subtype-selective agonists on gonadotropin-releasing hormone pulse generator activity and luteinizing hormone secretion in goats

Recent evidence suggests that neurokinin B (NKB), a member of the neurokinin (tachykinin) peptide family, plays a pivotal role in gonadotropin-releasing hormone (GnRH) pulse generation. Three types of neurokinin receptors (NKRs), NK1R, NK2R and NK3R, are found in the brain. Although NKB preferentially binds to NK3R, other NKRs are possibly also involved in NKB action. The present study examined the effects of intravenous administration of the NKR subtype-selective agonists GR73632 (NK1R), GR64349 (NK2R), and senktide (NK3R) on GnRH pulse generator activity and luteinizing hormone (LH) secretion. Multiple-unit activity (MUA) was monitored in ovariectomized goats (n = 5) implanted with recording electrodes. Characteristic increases in MUA (MUA volleys) were considered GnRH pulse generator activity. Although three NKR agonists dose-dependently induced an MUA volley and an accompanying increase in LH secretion, the efficacy in inducing the volley markedly differed. As little as 10 nmol of senktide induced an MUA volley in all goats, whereas a dose of 1000 nmol was only effective for the NK1R and NK2R agonists in two and four goats, respectively. When the treatment failed to evoke an MUA volley, no apparent change was observed in the MUA or LH secretion. Similar effects of the NK2R and NK3R agonists were observed in the presence of estradiol. The results demonstrated that NK3R plays a predominant role in GnRH pulse generation and suggested that the contributions of NK1R and NK2R to this mechanism may be few, if any, in goats.     

Geographical variation of skull size and shape in various populations in the black giant squirrel

We osteometrically examined the skulls of the black giant squirrel (Ratufa bicolor) from three mainland populations (M. Malayan Peninsula, V. South Vietnam, and B. Burma, India and North Thailand) and from two island populations (T. Tioman, and S. Sumatra Islands). The skull in the Malayan peninsula population was significantly smaller than that of the two other mainland populations. It is consistent with Bergmann's rule as shown in the gray-bellied squirrel. The two island populations did not show obvious differences in comparison with the Malayan population in many measurements. In the proportion analysis eliminating the size factor, the differences among populations were not easily confirmed and we concluded that the osteological characters peculiar to each population could not be shown in this species. The first and second principal component scores of M, S, and T populations were intermingled, whereas the V and B populations of V and B were not separated in the chart. We pointed out that the morphological differences were demonstrated between northern and southern groups of the Isthmus of Kra in the mainland populations, and that the two island populations did not show the island-isolation effect in comparison with the M population. The adaptational variation related to feeding and locomotion could not be confirmed among populations of the black giant squirrel as shown in the proportion analysis.     

Contribution of the grain size QTL GS3 to yield properties and physiological nitrogen-use efficiency in the large-grain rice cultivar ‘Akita 63’

The development of crop varieties with high nitrogen-use efficiency (NUE) is thought to be important in achieving sustainable cereal crop production. The high yield large-grain rice cultivar Oryza sativa L. ‘Akita 63’ (temperate japonica) has high physiological NUE (PNUE) for grain yield (GY). Our previous study revealed that a large-grain allele of GS3 is present in ‘Akita 63’. Here, we verified the influence of GS3 on the yield properties and PNUE for GY in ‘Akita 63’. The frequency distribution of brown rice length in F₂ crosses of ‘Iwate 75’ and ‘Akita 63’ showed a continuous distribution that could be explained by GS3. A near-isogenic line was developed to substitute the GS3 segment of ‘Koshihikari’, which harbours a normal-sized grain allele, in the genetic background of ‘Akita 63’ and the line was designated as Akita63NILGS3-Koshihikari. Compared with Akita63NILGS3-Koshihikari, ‘Akita 63’ exhibited a significantly increased grain length, single brown grain weight and GY, although no significant differences were observed in the nitrogen content and above-ground biomass per unit of cultivated area. These results indicate that the GS3 large-grain allele is a contributing factor to high PNUE for GY in ‘Akita 63’. These findings will facilitate the development of nitrogen-efficient rice varieties.     

Cloning,expression, and tissue distribution of bovine interleukin-21

Bovine interleukin-21 (IL-21) cDNA was cloned and sequenced from bovine peripheral blood lymphocytes (PBLs) stimulated with 10 microg/ml concanavalin A (ConA), 10 microg/ml phytohemagglutinin (PHA), and 50 ng/ml phorbol 12-myristate 13-acetate (PMA) for 48 h. The open reading frame of the bovine IL-21 cDNA is 459 bp in length and encodes 152 amino acids. The predicted amino acid sequence is 78.2 and 58.5% homologous to the human and murine IL-21 amino acid sequences, respectively. Recombinant bovine IL-21 was expressed by a baculovirus expression system. The bovine IL-21 was processed to the mature form in insect cells and secreted to the supernatant confirmed by N-terminal amino acid sequencing. The recombinant bovine mature IL-21 induced the proliferation of human IL-2-dependent cells, ILT-MAT. The mRNA expression for bovine IL-21 was observed in the spleen, but not in the brain, heart, lung, liver, and kidney. The bovine IL-21 identified in this study may provide new methods for the enhancement of innate immunity in cows.     

Macroscopic study of the functional significance of the forearm muscles in the giant panda

The extensor and flexor group muscles and their related muscles were functional-morphologically observed in the dead body of the giant panda to clarify the action of the forearm and the palm in the manipulation of the species. The Musculus flexor carpi ulnaris had two developed heads, however, we can conclude that the contraction of this muscle slightly changes the angle of the accessory carpal bone to the ulna. The data pointed out that the accessory carpal bone acts as a supporting post, when the giant panda seizes the object. The M. abductor digiti I longus possessed the well-developed origin in both ulna and radius. These findings suggest that this muscle may function as a supinator of the forearm. We also suggest that the well-developed M. pronator quadratus and M. pronator teres, and the proximal part of the M. abductor digiti I longus and the M. supinator may efficiently contribute to the pronator-spinator action of the forearm, when the giant panda brings the food to its mouth using the manipulation system equipped in the palm region.     

Large-scale production of porcine mature interleukin-18 (IL-18) in silkworms using a hybrid baculovirus expression system

In this report, a hybrid baculovirus expression system, which means a hybrid virus of the Autographa californica nuclear polyhedrosis virus and the Bombyx mori nuclear polyhedrosis virus, was used for the large-scale production of porcine mature interleukin-18 (IL-18) in silkworms. Two recombinant hybrid baculoviruses containing cDNA of the porcine precursor IL-18 and the porcine caspase-1 were constructed and were used to infect silkworm larvae. After the co-infection of the two viruses, porcine mature IL-18 was efficiently produced in the haemolymph. The concentration of IL-18 in the haemolymph was 80-100 microg/ml, as determined by porcine IL-18 specific ELISA. This yield was twenty-times more than that of the insect cell expression system described previously. The porcine mature IL-18 produced by the silkworms strongly induced interferon-gamma (IFN-gamma) production from porcine PBMC. An insect factory system for the large-scale production of useful cytokines for livestock animals will be available in the near future.     

An oligonucleotide probe for the detection of Erwinia herbicola and Erwinia ananas

An oligonucleotide probe targeting the rRNA of Erwinia herbicola and Erwinia ananas was designed to detect their cells using fluorescence in situ hybridization. The Cy3-labeled probe hybridized strongly with these species but very weakly with nontarget species such as Erwinia mallotivora, Erwinia nigrifluens, Erwinia cypripedii, Erwinia chrysanthemi, Erwinia carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Escherichia coli. This technique visualized E. herbicola cells after inoculation of kumquat fruits. The probe is promising as a tool for studying population dynamics of E. herbicola and E. ananas.     

High level expression and purification of bioactive bovine interleukin-18 using a baculovirus system

Bioactive recombinant bovine interleukin-18 (rboIL-18) was expressed using a baculovirus system. Normally, IL-18 is translated as a precursor form of a 24kDa polypeptide and processed by IL-1beta converting enzyme (ICE) to a mature bioactive form of 18kDa protein. Hence, to express active form IL-18, we constructed two recombinant baculoviruses containing boIL-18 and human ICE (hICE) genes, respectively, and superinfected these viruses into insect cells. Superinfection of both recombinant viruses into the cells resulted in the expression of a 24kDa precursor form and an 18kDa mature form detectable in the supernatant by immunoblotting using anti-porcine IL-18 antibody. Culture supernatant from the superinfected cells showed a synergistic effect with recombinant boIL-12 for production of interferon-gamma (IFN-gamma) in bovine peripheral mononuclear cells. By addition of histidine hexamer at the C-terminal of boIL-18, the mature IL-18 was purified. Bioactivity remained after purification.