Cryopreservation and low-temperature storage of seeds of Phaius tankervilleae

In this study we established reliable methods for conservation of seeds of Phaius tankervilleae as an orchid genetic resource. The seeds, which were dehydrated to 5% water content and preserved at 4°C, showed no decrease in viability and germinability after three months. After storage for six months, however, the seeds showed a drastic decrease in germinability, even though survival rate was high. For long-term preservation of seeds of P. tankervilleae, cryopreservation is applied to the freshly harvested seeds. When the seeds were cryopreserved by the vitrification method for up to 12 months there was no apparent deterioration effect of storage time. These results indicate that cryopreservation by the vitrification method is useful for long-term conservation of P. tankervilleae seeds, which are difficult to preserve for more than three months under dry and low-temperature conditions.     

Yangtze finless porpoises along the main channel of Poyang Lake,China: Implications for conservation

Poyang Lake is the largest freshwater lake in China and flows into the Yangtze River. It is a traditional habitat for the endangered Yangtze finless porpoise, which has not been well investigated. To reveal the distribution of the porpoise in Poyang Lake, 12 passive acoustic surveys were conducted along 123 km of the main channel of the lake during different seasons (spring transition season, wet season, autumn transition season, and dry season) from 2008 to 2012. We counted the number of phonating porpoises encountered and calculated the detection rate (encountered individuals detected per kilometer). The median porpoise detection rates ranged from 0 to 0.65 individuals per kilometer during the different surveys. The highest median detection rate of 0.50 was detected in the autumn transition season. The seasonal shrinking of the lake during the dry season may cause a concentration of porpoises in the narrow channels and a high incidence of collisions with cargo ships and fishing boats. Conservation actions should be focused on the main channel of the lake during the dry and transition seasons. In addition, the expansion of the existing reserve to include areas with high porpoise detection rates is necessary.     

Role of numb in dendritic spine development with a Cdc42 GEF intersectin and EphB2

Numb has been implicated in cortical neurogenesis during nervous system development, as a result of its asymmetric partitioning and antagonizing Notch signaling. Recent studies have revealed that Numb functions in clathrin-dependent endocytosis by binding to the AP-2 complex. Numb is also expressed in postmitotic neurons and plays a role in axonal growth. However, the functions of Numb in later stages of neuronal development remain unknown. Here, we report that Numb specifically localizes to dendritic spines in cultured hippocampal neurons and is implicated in dendritic spine morphogenesis, partially through the direct interaction with intersectin, a Cdc42 guanine nucleotide exchange factor (GEF). Intersectin functions as a multidomain adaptor for proteins involved in endocytosis and cytoskeletal regulation. Numb enhanced the GEF activity of intersectin toward Cdc42 in vivo. Expression of Numb or intersectin caused the elongation of spine neck, whereas knockdown of Numb and Numb-like decreased the protrusion density and its length. Furthermore, Numb formed a complex with EphB2 receptor-type tyrosine kinase and NMDA-type glutamate receptors. Knockdown of Numb suppressed the ephrin-B1-induced spine development and maturation. These results highlight a role of Numb for dendritic spine development and synaptic functions with intersectin and EphB2.     

T2BP, a novel TRAF2 binding protein, can activate NF-kappaB and AP-1 without TNF stimulation.

TRAF2 is a key molecule involved in TNF signaling, which is crucial for the regulation of inflammatory processes. We have identified a novel TRAF2 binding protein, designated as T2BP (TRAF2 binding protein), by a mammalian two-hybrid screening approach. T2BP is a relatively small protein of 184 amino acids, which includes a forkhead-associated domain, the phosphopeptide binding motif. The interaction domain search showed that the TRAF domain in TRAF2 is required for the binding to T2BP whereas almost the entire protein in T2BP binds to TRAF2. The interaction was further confirmed by co-immunoprecipitation. Expression profiling for T2BP and TRAF2 revealed an ubiquitous expression in adult mouse tissues. Overexpression of T2BP in HEK293 cells activated NF-kappaB and AP-1 in a dose dependent manner as well as seen in the TNF-treated control cells. Our results suggest that T2BP is involved in the TNF-mediated signaling by its interaction with TRAF2.     

Light-Dark Difference in Arterial Pressure Variability During REM Sleep in the Rat

We have observed mean arterial pressure (MAP) variability during rapid eye movement (REM) sleep and brain temperature (Tb) in the rat during both light and dark periods over 24 h. MAP was measured using a telemetric device with a computer data capture and analysis system. As markers of MAP variability, the maximum and coefficient of variation (CV%) of MAP during REM sleep were determined. The following results were obtained: (a) there was a light-dark difference in MAP during non-REM (NREM) sleep and Tb during both NREM and REM sleep; (b) the increase of MAP in going from NREM to REM sleep in the light period was greater than that in the dark period, whereas the increase of Tb in the light period was not different from that in the dark period; (c) the maximum and CV% for MAP during REM sleep in the light period were greater than those in the dark period; (d) there was a negative correlation between the average Tb and MAP CV% during REM sleep. We suggest that phasic fluctuation of MAP during REM sleep may be influenced, in part, by a factor independent of sleep mechanisms.     

PURE ribosome display and its application in antibody technology

Ribosome display utilizes formation of the mRNA–ribosome–polypeptide ternary complex in a cell-free protein synthesis system to link genotype (mRNA) to phenotype (polypeptide). However, the presence of intrinsic components, such as nucleases in the cell-extract-based cell-free protein synthesis system, reduces the stability of the ternary complex, which would prevent attainment of reliable results. We have developed an efficient and highly controllable ribosome display system using the PURE (Protein synthesis Using Recombinant Elements) system. The mRNA–ribosome–polypeptide ternary complex is highly stable in the PURE system, and the selected mRNA can be easily recovered because activities of nucleases and other inhibitory factors are very low in the PURE system. We have applied the PURE ribosome display to antibody engineering approaches, such as epitope mapping and affinity maturation of antibodies, and obtained results showing that the PURE ribosome display is more efficient than the conventional method. We believe that the PURE ribosome display can contribute to the development of useful antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.