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Supplementary Table and Figures - PDF file 237K, Supplementary Table 1. Clinical characteristics of cancer patients. Supplementary Fig. 1. Isolation of CD45+/CD31- cells, CD45+/CD31bright cells, and CD45-/CD31+ cells from PBMCs using FACSAria flow cytometer. Supplementary Fig. 2. A Venn diagram showing the expression of various antigens in the endothelial cells, platelets, and monocytes

Purpose: Circulating endothelial cells (CEC) have been widely used as a prognostic biomarker and regarded as a promising strategy for monitoring the response to treatment in several cancers. However, the presence and biologic roles of CECs have remained controversial for decades because technical standards for the identification and quantification of CECs have not been established. Here, we hypothesized that CECs detected by flow cytometry might be monocytes rather than endothelial cells.Experimental Design: The frequency of representative CEC subsets (i.e., CD45−/CD31+, CD45−/CD31+/CD146+, CD45−/CD31+/CD105+) was analyzed in the peripheral blood of patients with gynecologic cancer (n = 56) and healthy volunteers (n = 44). CD45−/CD31+ cells, which are components of CECs, were isolated and the expression of various markers (CD146, CD105, vWF, and CD144 for endothelial cells; CD68 and CD14 for monocytes) was examined by immunocytochemistry.Results: CD45−/CD31+/CD105+ cells were significantly increased in the peripheral blood of patients with cancer, whereas evaluation of CD45−/CD31+/CD146+ cells was not possible both in patients with cancer and healthy controls due to the limited resolution of the flow cytometry. Immunocytochemistry analyses showed that these CD45−/CD31+/CD105+ cells did not express vWF and CD146 but rather CD144. Furthermore, CD45−/CD31+/CD105+ cells uniformly expressed the monocyte-specific markers CD14 and CD68. These results suggest that CD45−/CD31+/CD105+ cells carry the characteristics of monocytes rather than endothelial cells.Conclusions: Our data indicate that CD45−/CD31+/CD105+ circulating cells, which are significantly increased in the peripheral blood of patients with gynecologic cancer, are monocytes rather than endothelial cells. Further investigation is required to determine the biologic significance of their presence and function in relation with angiogenesis. Clin Cancer Res; 19(19); 5340–50. ©2013 AACR.

p57kip2, a KIP family cyclin-dependent kinase (Cdk) inhibitor, blocks the cell cycle by acting on multiple cyclin-Cdk complexes. To investigate the role of p57kip2 in human fertility, the expression of p57kip2 was investigated in testes from normal and obstructive azoospermic male patients who were positive for p57kip2 mRNA. In the seminiferous tubule, strong immunoreactivity of p57kip2 was found in nuclei of early spermatocytes, but not in the spermatogonia. The p57kip2 immunoreactivity in spermatocytes was markedly heterogeneous. Preleptotene spermatocytes showed strong p57kip2 immunoreactivity, but no visible signal was found in late pachytene spermatocytes. Nuclei of the elongating spermatids was also positive for p57kip2 immunoreactivity. Taken together, this suggests that p57kip2 may play a role in the regulation of meiotic progression of early spermatocytes and cell cycle arrest and differentiation of spermatids. p57kip2 immunoreactivity was found in the perinuclear region of the peritubular cells, but not in the Sertoli cells. In Leydig cells, moderate immunoreactivity of p57kip2 was largely found in the cytoplasm, suggesting the noble function of p57kip2 in the differentiation of adult Leydig cells.

The effects of progesterone on the acrosome reaction, as well as the effects of RU486 on the progesterone-induced acrosome reaction in capacitated boar spermatozoa, were investigated. Progesterone, a major steroid that is secreted by the cumulus cells of oocyte, clearly induced the acrosome reaction in a dose-dependent manner in capacitated boar spermatozoa, even though it failed to show similar effects in non-capacitated spermatozoa. RU486, a potent antiprogestin, significantly reduced the effects of progesterone on the progesterone-induced acrosome reaction; however, when treated alone, it showed no inhibitory effects on the acrosome reaction. The inhibitory effects of RU486 were also shown to be dose dependent. These results imply that in addition to the wellknown inducer of the acrosome reaction, zona pellucida, progesterone can also induce the acrosome reaction through its specific receptors on spermatozoa after the spermatozoa undergo capacitation.

The purified 32 kDa boar sperm protease, known as the 32 kDa sperminogen, was identified by reverse transcription polymerase chain reaction (RT-PCR) and Northern blot analysis following its partial peptide sequencing. The 32 kDa boar sperm protease was purified from the acid extracts of boar spermatozoa and subjected to CNBr-digestion. The most prominently digested 30 kDa product was purified by HPLC and its peptide sequence was analyzed. NCBI Blast search of the analyzed 21 amino acid sequence revealed that the sequence matched 91% with that of proacrosin. DNA primer was deduced from the analyzed peptide sequence and the 32 kDa protease was further identified by RT-PCR. Upon RT-PCR, 1 Kbp DNA fragment was amplified, which is the expected length if the product was amplified from the proacrosin mRNA, implying that there is no separate mRNA for the 32 kDa sperminogen. To confirm these results, Northern blot analysis was performed. Four DNA probes generated from the exons of proacrosin genomic DNA sequence all detected a single species of mRNA, suggesting that there is no separate mRNA for the 32 kDa sperminogen which might be produced either from the potential separate 32 kDa sperminogen gene or by differential splicing from proacrosin mRNA. These results strongly suggest that the 32 kDa protease is part of the proacrosin/acrosin system, and that the 32 kDa sperminogen might be formed from post-translational processing of proacrosin.

The current state of knowledge concerning the expression of the nuclear genes that code the light-harvesting chlorophyll a/b-binding polypeptides of photosystem II is presented. This review covers the structure of these genes, the complex multistep pathway involved in their expression, and the environmental and other factors which regulate their expression. Some of the effects of these factors are mediated, at least in part, at the level of transcription, but other effects can be explained only by the existence of multiple posttranscriptional regulatory steps.

Summary Dark-grown Euglena gracilis strain Z were exposed to white light which induces chloroplast development including a massive formation of thylakoid membranes. Thylakoid membranes were isolated from greening cells at various times from 12 to 72 h following light-exposure. The temporal appearance in the membranes of the three main chlorophyll-protein complexes (CP1, CPa, LHCP) and the N,N 1 ?dicyclohexylcarbodiimide (DCCD)-binding CF 0 -IH subunit of the coupling-factor ATPase was assessed. When the cells were greened on a medium containing the readily-metabolized carbon source, ethanol, LHCP was detected as early as 12 h, and both CP1 and CPa were detected in low quantity up to 36 h and then increased. CP1, CPa and LHCP were detected as green complexes on polyacrylamide gels earalier during greening when cells were greened on resting medium compared to cells greened in the presence of ethanol. Photosystem I activity was detected at 12 h, photosystem II activity at 18 to 24 h, and water-splitting and whole electron transport chain activities after 24 h. Correlations are made between the temporal appearances of these activities and of the chlorophyll-protein complexes. The DCCD- binding CF 0 -IH was detected in the membrane at 12 h and increased in amount thereafter.