Real-time monitoring of antibody secretion from hybridomas on a microchip by time-resolved luminescence anisotropy analysis

This article presents a real-time monitoring system for cellular analysis using micro total analysis systems technology. Time-resolved luminescence anisotropy analysis was adopted for real-time detection of small amounts of a target protein produced by a small number of cells. The system was tested by real-time monitoring of the antibody secretion by hybridomas. The cells were successfully cultivated in a micro-incubation chamber (240 nl) fabricated on a microchip. The quantification of the antibody was achieved using the Ru(II) complex-labeled Staphylococcus aureus protein A probe, which can bind specifically to the Fc region of the antibody. Using this system, we detected as little as 24 fmol of immunoglobulin G under physiological conditions without the bound/free separation protocol. We successfully achieved real-time and quantitative monitoring of small amounts of antibody production by approximately 200 hybridoma cells. This method could be applied to various cellular analyses using small numbers of cells.     

TrkB-T1 regulates the RhoA signaling and actin cytoskeleton in glioma cells

Recently, the truncated TrkB receptor, T1, has been reported to be involved in the control of cell morphology via the regulation of Rho proteins, through which T1 binds Rho guanine nucleotide dissociation inhibitor (Rho GDI) 1 and dissociates it in a brain-derived neurotrophic factor (BDNF)-dependent manner. However, it is unclear whether T1 signaling regulates the downstream of Rho signaling and the actin cytoskeleton. In this study, we investigated this question using C6 rat glioma cells, which express T1 endogenously. Rho GDI1 was dissociated from T1 in a BDNF-dependent manner, which also causes decreases in the activities of Rho-signaling molecules such as RhoA, Rho-associated kinase, p21-activated kinase, and extracellular-signal regulated kinase1/2. Moreover, BDNF treatment resulted in the disappearance of stress fibers in the cells treated with lysophosphatidic acid, an activator of RhoA, and in morphological changes in cells. Furthermore, a competitive assay with cyan fluorescent protein fusion proteins of T1-specific sequences reduced the effects of BDNF. These results suggest that T1 regulates the Rho-signaling pathways and the actin cytoskeleton.     

Mitochondrial DNA Diversity and Genetic Differentiation of the Honeybee (Apis cerana) in Thailand

Genetic diversity of the honeybee (Apis cerana) in Thailand collected from north, northeast, the central region, peninsular Thailand, and Samui Island (n = 181) was examined by PCR–RFLP of ATPase6–ATPase8. Interestingly, 78 individuals (43.09%) of the southern-latitude bees exhibited length heteroplasmy of the PCR product. The gel-eluted ATPase6–ATPase8 (825 bp) of each bee was restricted with TaqI, SspI, and VspI, respectively. Eight mitotypes were generated and revealed biogeographic differentiation between conspecific samples of A. cerana. AAA, ACA, AAD, BAA, ADA, and ABA were found only in the north-to-central samples (north, northeast, and central region); BBB and BBC were found in the southern-latitude bees; and BBC was restrictively found in the Samui sample. Large genetic distances were observed between each of the north-to-central samples and peninsular Thailand and Samui samples, but lower levels of genetic distance were found within each region. Geographic heterogeneity and phylogenetic analyses indicated that Thai A. cerana could be genetically differentiated into northern Thailand, peninsular Thailand, and Samui Island populations.     

CYP710A genes encoding sterol C22-desaturase in <Emphasis Type="Italic">Physcomitrella patens</Emphasis> as molecular evidence for the evolutionary conservation of a sterol biosynthetic pathway in plants

We have characterized cytochromes P450, CYP710A13, and CYP710A14, as the sterol C22-desaturase in the moss Physcomitrella patens. GC–MS analyses demonstrated that P. patens accumulated stigmasterol as the major sterol (56–60% of total sterol) and sitosterol to a lesser extent (8–12%); this sterol profile contrasts with those in higher plants accumulating stigmasterol as a minor component. Recombinant CYP710A13 and CYP710A14 proteins prepared using a baculovirus/insect cell system exhibited the C22-desaturase activity with β-sitosterol to produce stigmasterol, while campesterol and 24-epi-campesterol were not accepted as the substrates. The K _m values for β-sitosterol of CYP710A13 (1.0 ± 0.043 μM) and CYP710A14 (2.1 ± 0.17 μM) were at comparable levels of those reported with higher plant CYP710A proteins. In Arabidopsis T87 cells over-expressing CYP710A14, stigmasterol contents reached a level 20- to 72-fold higher than those in the basal level of T87 cells, confirming the C22-desaturase activity of this P450 enzyme. The occurrence of the end-products together with the enzymes involved in the last step of the pathway substantiated the presence of an entire sterol biosynthetic pathway in P. patens, providing evidence for the conservation of the sterol biosynthetic pathway through the evolutionary process of land plants. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mesenchymal to embryonic incomplete transition of human cells by chimeric OCT4/3 (POU5F1) with physiological co-activator EWS

POU5F1 (more commonly known as OCT4/3) is one of the stem cell markers, and affects direction of differentiation in embryonic stem cells. To investigate whether cells of mesenchymal origin acquire embryonic phenotypes, we generated human cells of mesodermal origin with overexpression of the chimeric OCT4/3 gene with physiological co-activator EWS (product of the EWSR1 gene), which is driven by the potent EWS promoter by translocation. The cells expressed embryonic stem cell genes such as NANOG, lost mesenchymal phenotypes, and exhibited embryonal stem cell-like alveolar structures when implanted into the subcutaneous tissue of immunodeficient mice. Hierarchical analysis by microchip analysis and cell surface analysis revealed that the cells are subcategorized into the group of human embryonic stem cells and embryonal carcinoma cells. These results imply that cells of mesenchymal origin can be traced back to cells of embryonic phenotype by the OCT4/3 gene in collaboration with the potent cis-regulatory element and the fused co-activator. The cells generated in this study with overexpression of chimeric OCT4/3 provide us with insight into cell plasticity involving OCT4/3 that is essential for embryonic cell maintenance, and the complexity required for changing cellular identity.     

Cold preservation of rat osteochondral tissues in two types of solid organ preservation solution, culture medium and saline

We compared Dulbecco’s modified Eagle’s medium (DMEM), saline, Euro-Collins (EC) solution and University of Wisconsin (UW) solution to determine which was best for cold preservation of rat osteochondral tissues (OCTs). After 7 days’ cold preservation, OCTs kept in UW solution had the highest relative viable cell number by the tetrazolium assay and the lowest activity of lactate dehydrogenase released from damaged cells. Histological evaluation revealed chondrocyte deformity, such as shrunken cytoplasm and pyknotic nuclei, particularly in the deeper layer of articular cartilage after preservation in saline and EC solution and predominantly in all layers if preserved in DMEM. In contrast, chondrocyte morphology in all layers of the articular cartilage preserved in UW solution was relatively unchanged and remained similar to fresh OCTs. It is therefore concluded that UW solution is the most suitable for cold preservation of rat OCTs as well as solid organs.     

The effect of cryopreservation or heating on the mechanical properties and histomorphology of rat bone-patellar tendon-bone

The effects of cryopreservation on tendon allograft have been reported, but remain unclear, particularly the potential effects on mechanical properties and histological changes by ice crystal formation. There are also few studies about effects of heating for sterilization of tendon. We evaluated the effect of cryopreservation or heating on the mechanical properties and histomorphology of rat bone-patellar tendon-bones (BTBs). BTBs were processed by cryopreservation at −80°C for 3 weeks, or heating at 80°C for 10 min. Tensile testing and histomorphological examination were performed. The cryopreservation of tendons showed less influences on their mechanical properties. When cryopreserved BTBs in frozen state were fixed by freeze-substitution method, many spaces were observed in interfibrillar substances. These results suggest that the collagen fibers of cryopreserved tendons were histomorphologically affected by ice crystals. The heating of tendons completely destroyed the collagen fibers of the tendons and is therefore thought to be inappropriate for the sterilization of BTBs.     

In vitro organogenesis using multipotent cells

The establishment of efficient methods for promoting stem cell differentiation into target cells is important not only in regenerative medicine, but also in drug discovery. In addition to embryonic stem (ES) cells and various somatic stem cells, such as mesenchymal stem cells derived from bone marrow, adipose tissue, and umbilical cord blood, a novel dedifferentiation technology that allows the generation of induced pluripotent stem (iPS) cells has been recently developed. Although an increasing number of stem cell populations are being described, there remains a lack of protocols for driving the differentiation of these cells. Regeneration of organs from stem cells in vitro requires precise blueprints for each differentiation step. To date, studies using various model organisms, such as zebrafish, Xenopus laevis , and gene-targeted mice, have uncovered several factors that are critical for the development of organs. We have been using X. laevis , the African clawed frog, which has developmental patterns similar to those seen in humans. Moreover, Xenopus embryos are excellent research tools for the development of differentiation protocols, since they are available in high numbers and are sufficiently large and robust for culturing after simple microsurgery. In addition, Xenopus eggs are fertilized externally, and all stages of the embryo are easily accessible, making it relatively easy to study the functions of individual gene products during organogenesis using microinjection into embryonic cells. In the present review, we provide examples of methods for in vitro organ formation that use undifferentiated Xenopus cells. We also describe the application of amphibian differentiation protocols to mammalian stem cells, so as to facilitate the development of efficient methodologies for in vitro differentiation.     

Connective tissue growth factor is a substrate of ADAM28

ADAM28, a member of the ADAM (a disintegrin and metalloproteinase) gene family, is over-expressed by carcinoma cells and the expression correlates with carcinoma cell proliferation and progression in human lung and breast carcinomas. However, information about substrates of ADAM28 is limited. We screened interacting molecules of ADAM28 in human lung cDNA library by yeast two-hybrid system and identified connective tissue growth factor (CTGF). Binding of CTGF to proADAM28 was demonstrated by yeast two-hybrid assay and protein binding assay. ADAM28 cleaved CTGF in dose- and time-dependent manners at the Ala¹⁸¹-Tyr¹⁸² and Asp¹⁹¹-Pro¹⁹² bonds in the hinge region of the molecule. ADAM28 selectively digested CTGF in the complex of CTGF and vascular endothelial growth factor₁₆₅ (VEGF₁₆₅), releasing biologically active VEGF₁₆₅ from the complex. RT-PCR and immunohistochemical analyses demonstrated that ADAM28, CTGF and VEGF are commonly co-expressed in the breast carcinoma tissues. These data provide the first evidence that CTGF is a novel substrate of ADAM28 and suggest that ADAM28 may promote VEGF₁₆₅-induced angiogenesis in the breast carcinomas by the CTGF digestion in the CTGF/VEGF₁₆₅ complex.     

Lineage-specific positive selection at the merozoite surface protein 1 (msp1) locus of Plasmodium vivax and related simian malaria parasites

Background

The 200 kDa merozoite surface protein 1 (MSP-1) of malaria parasites, a strong vaccine candidate, plays a key role during erythrocyte invasion and is a target of host protective immune response. Plasmodium vivax, the most widespread human malaria parasite, is closely related to parasites that infect Asian Old World monkeys, and has been considered to have become a parasite of man by host switch from a macaque malaria parasite. Several Asian monkey parasites have a range of natural hosts. The same parasite species shows different disease manifestations among host species. This suggests that host immune responses to P. vivax-related malaria parasites greatly differ among host species (albeit other factors). It is thus tempting to invoke that a major immune target parasite protein such as MSP-1 underwent unique evolution, depending on parasite species that exhibit difference in host range and host specificity.

Results

We performed comparative phylogenetic and population genetic analyses of the gene encoding MSP-1 (msp1) from P. vivax and nine P. vivax-related simian malaria parasites. The inferred phylogenetic tree of msp1 significantly differed from that of the mitochondrial genome, with a striking displacement of P. vivax from a position close to P. cynomolgi in the mitochondrial genome tree to an outlier of Asian monkey parasites. Importantly, positive selection was inferred for two ancestral branches, one leading to P. inui and P. hylobati and the other leading to P. vivax, P. fieldi and P. cynomolgi. This ancestral positive selection was estimated to have occurred three to six million years ago, coinciding with the period of radiation of Asian macaques. Comparisons of msp1 polymorphisms between P. vivax, P. inui and P. cynomolgi revealed that while some positively selected amino acid sites or regions are shared by these parasites, amino acid changes greatly differ, suggesting that diversifying selection is acting species-specifically on msp1.

Conclusions

The present results indicate that the msp1 locus of P. vivax and related parasite species has lineage-specific unique evolutionary history with positive selection. P. vivax and related simian malaria parasites offer an interesting system toward understanding host species-dependent adaptive evolution of immune-target surface antigen genes such as msp1.