Relative levels of the two mammalian Rad23 homologs determine composition and stability of the xeroderma pigmentosum group C protein complex

Mammalian cells express two Rad23 homologs, HR23A and HR23B, which have been implicated in regulation of proteolysis via the ubiquitin/proteasome pathway. Recently, the proteins have been shown to stabilize xeroderma pigmentosum group C (XPC) protein that is involved in DNA damage recognition for nucleotide excision repair (NER). Because the vast majority of XPC forms a complex with HR23B rather than HR23A, we investigated possible differences between the two Rad23 homologs in terms of their effects on the XPC protein. In wild-type mouse embryonic fibroblasts (MEFs), endogenous XPC was found to be relatively stable, while its steady-state level and stability appeared significantly reduced by targeted disruption of the mHR23B gene, but not by that of mHR23A. Loss of both mHR23 genes caused a strong further reduction of the XPC protein level. Quantification of the two mHR23 proteins revealed that in normal cells mHR23B is actually approximately 10 times more abundant than mHR23A. In addition, overexpression of mHR23A in the mHR23A/B double knock out cells restored not only the steady-state level and stability of the XPC protein, but also cellular NER activity to near wild-type levels. These results indicate that the two Rad23 homologs are largely functionally equivalent in NER, and that the difference in expression levels explains for a major part the difference in complex formation with as well as stabilization effects on XPC.     

An EF-hands protein, centrin-1, is an EGTA-sensitive SUMO-interacting protein in mouse testis

A multifunctional calcium‐binding protein, centrin‐1, is specifically expressed in male germ cells, certain neurons and ciliated cells. We identified centrin‐1 as a protein interacting with SUMO‐2/3 using yeast two‐hybrid screening of a mouse testicular cDNA library. In bead halo assays, the interaction between centrin‐1 and SUMO‐2/3 was reduced in the presence of EGTA and facilitated by the addition of CaCl_2. immunostaining of seminiferous tubules in 35‐day‐old mouse testes revealed that cells in the layer containing spermatogonia showed colocalization of SUMO‐2/3 with centrin‐1 in cytoplasmic spots. Identification of centrin‐1 as the EGTA‐sensitive SUMO‐2/3‐interacting protein indicates the possible role of calcium in modulating the centrin‐1–SUMO‐2/3 interaction and suggests the importance of this interaction in mouse testis. Copyright © 2010 John Wiley & Sons, Ltd.     

Limited Survival Period of Cold Acclimatized Trees in Frozen Ambient Temperatures

Cold-acclimatized twigs of poplar exhibited metabolic dysfunctionwithin a year at –10°C. The symptoms were depressionof glucose-6-phosphate and reduced glutathione levels, followedby a decrease in a number of enzyme activities, particularlyin the xylem, suggesting that the injury to the cambial zoneof the twigs came from the xylem side. (Received April 14, 1986; Accepted June 25, 1986)     

Cytochemical Evidence for the Occurrence in Plants of a Novel Microbody that Contains Peroxidase

A cytochemical study with 3,3'-diaminobenzidine revealed thepresence of peroxidase in the microbodies which were recentlyfound in the xylem parenchyma cells of wintering poplar (Sagisakaand Asada 1981). The peroxidative reaction was not inhibitedby 3-amino-l,2,4-triazole, but was completely inhibited by KCN.There was no detectable staining in the absence of added H₂O₂. (Received March 6, 1986; Accepted August 27, 1986)     

Amino Acid Pools in Perennial Plants at the Wintering Stage and at the Beginning of Growth

Free amino acids in plants at the stages of wintering and thebeginning of growth were analyzed under natural conditions.From the major amino acid contents, the pools were separatedinto the following types: 1) a group which accumulated argininealone (11 out of 31 samples); 2) a group which accumulated prolinealone (4 out of 31) and 3) a group which accumulated arginineand proline (15 out of 31). Comparison of the compositions atstages of wintering and the beginning of budding indicated thatthe increase in the proline level is related to the onset ofgrowth. Synthesis of arginine and roline and translocation ofproline would seem to indicate that these substances play therole of filling-in materials in any particular tissue. For plants which require chilling temperatures for growth, theaccumulation of arginine and proline was considered to be anormal event, since temporal changes in the major amino acidsfrom arginine (and proline) through glutamate to asparagineplus aspartate were generally observed. Uncoupling between aminoacid syntheses and the commencement of development was alsoobserved in plants which were housed in a greenhouse duringwinter. A possible function of proline as a natural cryoprotectantis discussed. ¹ Contribution No. 2270 from the Institute of Low TemperatureScience, Hokkaido University, Sapporo, Japan. (Received August 2, 1982; Accepted January 5, 1983)     

Dormancy Breaking is Followed by Mitochondria Proliferation in Poplar and Apple Trees in Milieu of Midwinter

In the early stage soon after the dormancy breaking, proliferationof mitochondria commenced in twigs of nongrowing poplar andapple trees under sub-zero temperatures. Since the profile ofproliferation could be observed every year, the proliferationmay be associated annually with the cytological events in theperennials in midwinter. (Received July 1, 1988; Accepted November 4, 1988)     

Changes in Parenchyma Cells of Poplar Xylem during Transition from Growing to Wintering Stages

Ultrastructural changes in xylem parenchyma cells during transitionfrom growing to wintering stages were investigated. Changesin the fine structures of parenchyma cells in differentiatedxylem started in mid-August. At this time, dictyosomes werefrequently found and vesicles were abundant in the cells inwhich active formation of cellular structures took place. Thecytosol was filled with polysomes. In mid-September, the cytoplasmof the parenchyma cells gradually began to fill up with microbodies,and the endoplasmic reticulum cisternae had already startedto decrease. Dictyosomes and vesicles were still abundant atthis stage and microbody-associated vesicles could be seen,indicating participation of the vesicles in the formation ofmicrobodies at this stage. A direct structural connection withrespect to the biogenesis of the microbodies remained unclear.In the early stage, the microbodies showed a dense center surroundedby a less dense outer surface, and in the latter stage in November,the outer surface exhibited an increased accumulation of osmiophilicmaterials. There were few further changes in the overall patternof the fine structures until mid-winter. (Received July 2, 1987; Accepted November 30, 1987)     

Increase in Enzyme Activities Related to Ascorbate Metabolism during Cold Acclimation in Poplar Twigs

Activities of ascorbate free radical reductase, ascorbate peroxidase,dehydroascorbate reductase, ascorbate oxidase and catalase inthe twigs of poplar, Populus gelrica, were measured throughouta year. Activity levels of the first three enzymes were highduring the wintering period of the life cycle, and the changesin the three enzyme activities occurred simultaneously. In fall,when the growth and enlargement of the tissues began to cease,the activities began to increase. In contrast to the activitiesof the above three enzymes, catalase activity began to increaseas the growth and enlargement proceeded, and the activity droppedby early November. Ascorbate levels in the twigs were measured,and the living bark and the xylem tissue were found to containsimilar levels of ascorbate (8 to 20 µmol and 2 to 14µmol per gram dry weight, respectively). From these results,it was suggested that in the growing period, deleterious H₂O₂produced in such organelles as the endoplasmic reticulum isdecomposed by catalase, and that in winter, oxidation and reductionreactions of ascorbate function not only to detoxify the peroxideproduced in the tissues, but also serve as a respiratory processto regenerate NADPH in the non-photosynthetic tissues of perennials. ¹Contribution No. 2624 from the Institute of Low TemperatureScience, Hokkaido University, Sapporo 060, Japan. (Received January 25, 1984; Accepted June 1, 1984)