Sleep‐related behaviors in zoo‐housed giraffes (Giraffa camelopardalis reticulata): Basic characteristics and effects of season and parturition

Despite increasing interest in the behavior of zoo animals, studies of nocturnal behavior of zoo animals are limited. In this study, we investigated the relationship between parturition, season, and the sleep‐related behaviors in captive reticulated giraffes to better understand the nocturnal life in giraffes. The subjects were two adult reticulated giraffes living in Kyoto City Zoo, Japan. Observations were made via an infrared camera that was mounted in the indoor enclosure between June 2007 and August 2009. We analyzed video clips that were recorded between 16:30 and 09:00 the next morning, over a total of 199 days. Sleep‐related behaviors were classified into two categories based on the posture of the giraffes; recumbent posture and paradoxical sleep. We also recorded the laterality of recumbent posture, which was coded based on the direction of the legs against the torso (right or left). Seasonal differences in sleep behaviors between summer and winter were observed in both individuals. They tended to start to lie down earlier in the winter than in the summer. Parturition also affected the behaviors as both individuals decreased the behaviors before and after the parturition of the female. Additionally, the female lay on her left side less frequently than her right when resuming a recumbent posture in the pre‐parturition period, while such laterality was not observed in the baseline and post‐parturition period. These results suggested that season and parturition are important factors for determining the sleep‐related behaviors in giraffes. Further studies are needed to understand how these changes in sleep affect other welfare parameters.     

Human umbilical cord-derived cells can often serve as feeder cells to maintain primate embryonic stem cells in a state capable of producing hematopoietic cells

Clinical application of human embryonic stem (ES) cells will require the establishment of methods for their culture, either in the presence or absence of human-derived feeder cells. We have tested the ability of non-immortalized cultured cells derived from human umbilical cord (HUC cells) to support ES cell culture. A primate ES cell line that had been established and maintained with mouse embryonic fibroblasts was cultured on HUC cells for >3 months (HUC-maintained ES cells). These cells retained their expression of alkaline phosphatase, SSEA-4, Oct-3/4, and to a lesser extent Nanog, but did not express Rex-1. Nevertheless, HUC-maintained ES cells could produce ectoderm-, mesoderm- and endoderm-derived cells in teratomata that they formed in immunodeficient mice. We show that HUC-maintained ES cells could give rise to hematopoietic cells, although this ability of HUC cells varied among HUC cell populations derived from different neonates. HUC cells are promising as human material with which to maintain ES cells in a state that retains their ability to produce mature cells, including hematopoietic cells.     

An Efficient reproductive method for Irs2-/- mice with C57BL/6JJcl genetic background

Efficient reproduction using natural mating and reproduction technology [in vitro fertilization (IVF) and embryo transfer (ET)] was investigated in IRS2 deficient mice with C57BL/6JJcl genetic background (Irs2(-/-) mice) as a typical type 2 diabetes model. From the results using various combinations of Irs2(-/-) and Irs2(-/+) mice, the combination of female Irs2(-/+) x male Irs2(-/-) was found to be more efficient than other combinations. In applications of reproduction technology using IVF and ET, the combination of female Irs2(-/+) x male Irs2(-/-) involves the possibility of Irs2(-/-) production by repeats using female Irs2(-/+) mice. However, reproductive continuity using this combination is difficult because of dependence on human technique and the cost of ET. Therefore, we concluded that Irs2(-/-) mice should be produced by embryo transfer using Irs2(-/-) mice from a colony consisting of female Irs2(-/+) x male Irs2(-/-).     

Biosynthetic exchange of bromide for chloride and strontium for calcium in the photosystem II oxygen-evolving enzymes

The active site for water oxidation in photosystem II goes through five sequential oxidation states (S(0) to S(4)) before O(2) is evolved. It consists of a Mn(4)Ca cluster close to a redox-active tyrosine residue (Tyr(Z)). Cl(-) is also required for enzyme activity. To study the role of Ca(2+) and Cl(-) in PSII, these ions were biosynthetically substituted by Sr(2+) and Br(-), respectively, in the thermophilic cyanobacterium Thermosynechococcus elongatus. Irrespective of the combination of the non-native ions used (Ca/Br, Sr/Cl, Sr/Br), the enzyme could be isolated in a state that was fully intact but kinetically limited. The electron transfer steps affected by the exchanges were identified and then investigated by using time-resolved UV-visible absorption spectroscopy, time-resolved O(2) polarography, and thermoluminescence spectroscopy. The effect of the Ca(2+)/Sr(2+) and Cl(-)/Br(-) exchanges was additive, and the magnitude of the effect varied in the following order: Ca/Cl < Ca/Br < Sr/Cl < Sr/Br. In all cases, the rate of O(2) release was similar to that of the S(3)Tyr(Z)(.) to S(0)Tyr(Z) transition, with the slowest kinetics (i.e. the Sr/Br enzyme) being approximately 6-7 slower than in the native Ca/Cl enzyme. This slowdown in the kinetics was reflected in a decrease in the free energy level of the S(3) state as manifest by thermoluminescence. These observations indicate that Cl(-) is involved in the water oxidation mechanism. The possibility that Cl(-) is close to the active site is discussed in terms of recent structural models.     

Comparison of real-time and nested PCR assays for detection of herpes simplex virus DNA

We performed a real-time PCR assay to detect herpes simplex virus (HSV) DNA, and compared it prospectively with a nested PCR assay in 164 clinical samples (109 cerebrospinal fluid and 55 sera) from patients suspected of having neonatal HSV infection or HSV encephalitis. In 25 of 164 samples, HSV DNA was detected by the nested PCR assay. All samples positive for HSV DNA in the nested PCR assay were also positive in the real-time PCR assay, and all but two samples negative for HSV DNA in the nested assay were negative in the real-time assay. The real-time PCR assay thus had a sensitivity of 100% and a specificity of 99%, when compared with the nested assay. Sequential assays in a case of disseminated HSV showed that a decrease in HSV DNA paralleled clinical improvement. Quantification of HSV DNA by real-time PCR was useful for diagnosing and monitoring patients with HSV encephalitis and neonatal HSV infection.     

Patency of the preterm fetal ductus arteriosus is regulated by endothelial nitric oxide synthase and is independent of vasa vasorum in the mouse

Patency of the fetal ductus arteriosus (DA) is maintained in an environment of low relative oxygen tension and a preponderance of vasodilating forces. In addition to prostaglandins, nitric oxide (NO), a potent vasodilator in the pulmonary and systemic vasculatures, has been implicated in regulation of the fetal DA. To further define the contribution of NO to DA patency, the expression and function of NO synthase (NOS) isoforms were examined in the mouse DA on days 17-19 of pregnancy and after birth. Our results show that endothelial NOS (eNOS) is the predominant isoform expressed in the mouse DA and is localized in the DA endothelium by in situ hybridization. Despite rapid constriction of the DA after birth, eNOS expression levels were unchanged throughout the fetal and postnatal period. Pharmacological inhibition of prostaglandin vs. NO synthesis in vivo showed that the preterm fetal DA on day 16 is more sensitive to NOS inhibition than the mature fetal DA on day 19, whereas prostaglandin inhibition results in marked DA constriction on day 19 but minimal effects on the day 16 DA. Combined prostaglandin and NO inhibition caused additional DA constriction on day 16. The contribution of vasa vasorum to DA regulation was also examined. Immunoreactive platelet endothelial cell adhesion molecule and lacZ tagged FLK1 localized to DA endothelial cells but revealed the absence of vasa vasorum within the DA wall. Similarly, there was no evidence of vasa vasorum by vascular casting. These studies indicate that eNOS is the primary source of NO in the mouse DA and that vasomotor tone of the preterm fetal mouse DA is regulated by eNOS-derived NO and is potentiated by prostaglandins. In contrast to other species, mechanisms for DA patency and closure appear to be independent of any contribution of the vasa vasorum.     

Non-fucosylated therapeutic antibodies: the next generation of therapeutic antibodies

Therapeutic antibody IgG1 has two N-linked oligosaccharide chains bound to the Fc region. The oligosaccharides are of the complex biantennary type, composed of a trimannosyl core structure with the presence or absence of core fucose, bisecting N-acetylglucosamine (GlcNAc), galactose, and terminal sialic acid, which gives rise to structural heterogeneity. Both human serum IgG and therapeutic antibodies are well known to be heavily fucosylated. Recently, antibody-dependent cellular cytotoxicity (ADCC), a lytic attack on antibody-targeted cells, has been found to be one of the critical effector functions responsible for the clinical efficacy of therapeutic antibodies such as anti-CD20 IgG1 rituximab (Rituxan^®) and anti-Her2/neu IgG1 trastuzumab (Herceptin^®). ADCC is triggered upon the binding of lymphocyte receptors (FcγRs) to the antibody Fc region. The activity is dependent on the amount of fucose attached to the innermost GlcNAc of N-linked Fc oligosaccharide via an α-1,6-linkage, and is dramatically enhanced by a reduction in fucose. Non-fucosylated therapeutic antibodies show more potent efficacy than their fucosylated counterparts both in vitro and in vivo, and are not likely to be immunogenic because their carbohydrate structures are a normal component of natural human serum IgG. Thus, the application of non-fucosylated antibodies is expected to be a powerful and elegant approach to the design of the next generation therapeutic antibodies with improved efficacy. In this review, we discuss the importance of the oligosaccharides attached to the Fc region of therapeutic antibodies, especially regarding the inhibitory effect of fucosylated therapeutic antibodies on the efficacy of non-fucosylated counterparts in one medical agent. The impact of completely non-fucosylated therapeutic antibodies on therapeutic fields will be also discussed.     

Participation of hup gene product in ori2-dependent replication of fertility plasmid F

The fertility plasmid F'gal was not stably maintained in a hupA-hupB double mutant of Escherichia coli. Moreover, mini-F plasmids pFZY1, pFTC1 and pFTC2 were unable to transform the double mutant, though these plasmids efficiently transformed cells harboring a hupA or hupB single mutation. The composite plasmid pFHS1, which consists of the f5 DNA fragment of F plasmid and the whole DNA of a pSC101 derivative that carries a temperature-sensitive mutation for DNA replication, was not stably maintained in the hup double mutant at 42°C. These findings strongly suggest that HU protein is required for ori2-dependent replication of the F plasmid.     

Establishment of hepatic stem-like cell lines from normal adult porcine liver in a poly-D-lysine-coated dish with nair-1 medium

The existence, origin, and bipotency of the hepatic stem cell (HeSC) have been investigated. However, the isolation and culture of HeSCs from adult liver tissue is not yet well established, and the mechanism by which HeSCs differentiate into mature cells remains unclear. On the other hand, the development of HeSC-isolating and -culturing methods and the in vitro clonal analysis of their mechanism of differentiation are required to enable clinical applications of regenerative medicine in the liver. For the purpose of providing HeSCs for these studies, we attempted to establish an HeSC line from a normal adult porcine liver using a unique culture system, a poly-D-lysine-coated culture dish with NAIR-1 medium (the PDL-NAIR-1 culture system). Moreover, we examined the differentiating capacity of HeSCs in vitro. We demonstrated that it was possible in the culture system that immature epithelial cells capable of proliferating grew selectively into aggregates and that two hepatic stem-like cell lines, PHeSC-A1 and PHeSC-A2, were established. The results from our data suggest that these hepatic stem-like cell lines were capable of self-renewing and differentiating into hepatocytes or biliary epithelial cells and show that the PDL-NAIR-1 culture system offers the immense advantage of isolating and culturing HeSCs from a normal adult liver. Furthermore, because of the ability to use a clonal analysis in vitro, these cell lines are useful for the investigation of various mechanisms in which HeSCs seem to participate and their application in the study of regenerative medicine in the liver.