Cytoadherence Properties of Plasmodium knowlesi-Infected Erythrocytes

Plasmodium knowlesi is responsible for zoonotic malaria infections that are potentially fatal. While the severe pathology of falciparum malaria is associated with cytoadherence phenomena by Plasmodium falciparum-infected erythrocytes (IRBC), information regarding cytoadherence properties of P. knowlesi-IRBC remained scarce. Here, we characterized the cytoadherence properties of RBC infected with the laboratory-adapted P. knowlesi A1-H.1 strain. We found that late-stage IRBC formed rosettes in a human serum-dependent manner, and rosettes hampered IRBC phagocytosis. IRBC did not adhere much to unexposed (unstimulated) human endothelial cell lines derived from the brain (hCMEC/D3), lungs (HPMEC), and kidneys (HRGEC). However, after being “primed” with P. knowlesi culture supernatant, the IRBC-endothelial cytoadherence rate increased in HPMEC and HRGEC, but not in hCMEC/D3 cells. Both endothelial cytoadherence and rosetting phenomena were abrogated by treatment of P. knowlesi-IRBC with trypsin. We also found that different receptors were involved in IRBC cytoadherence to different types of endothelial cells. Although some of the host receptors were shared by both P. falciparum- and P. knowlesi-IRBC, the availability of glycoconjugates on the receptors might influence the capacity of P. knowlesi-IRBC to cytoadhere to these receptors.

hC9ORF78 localizes to kinetochores and is required for proper chromosome segregation

C9ORF78 is a poorly characterized protein found in diverse eukaryotes. Previous work indicated overexpression of hC9ORF78 (aka HCA59) in malignant tissues indicating a possible involvement in growth regulatory pathways. Additional studies in fission yeast and humans uncover a potential function in regulating the spliceosome. In studies of GFP-tagged hC9ORF78 we observed a dramatic reduction in protein abundance in cells grown to confluence and/or deprived of serum growth factors. Serum stimulation induced synchronous re-expression of the protein in HeLa cells. This effect was also observed with the endogenous protein. Overexpressing either E2F1 or N-Myc resulted in elevated hC9ORF78 expression potentially explaining the serum-dependent upregulation of the protein. Immunofluorescence analysis indicates that hC9ORF78 localizes to nuclei in interphase but does not appear to concentrate in speckles as would be expected for a splicing protein. Surprisingly, a subpopulation of hC9ORF78 co-localizes with ACA, Mad1 and Hec1 in mitotic cells suggesting that this protein may associate with kinetochores or centromeres. Furthermore, knocking-down hC9ORF78 caused mis-alignment of chromosomes in mitosis. These studies uncover novel mitotic function and subcellular localization of cancer antigen hC9ORF78.SUMMARY STATEMENThC9ORF78 regulates chromosome segregation.

Extracellular Vesicles Support the Survival of Serum-dependent cells in vitro

Extracellular vesicles (EVs) are 30-150 nm in diameter and are released by cells into the extracellular environment. They facilitate intercellular communication and may be involved in cell survival. Although serum-dependent cells are not obviously affected by depletion of EVs from the serum, we previously reported that cells in serum that contain EVs were more resistant to the drugs, enoxacin and bis-enoxacin, compared to cells in serum without EVs. This change in resistance led us to examine whether EVs are sufficient to maintain serum dependent cells in vitro. Here, we tested the effect of EVs on the survival RAW 264.7 cells and on 4T1 murine breast cancer cells. EVs were isolated by differential centrifugation. EVs were counted over various time periods. Cells were grown in alpha or Dulbecco’s minimal essential media supplemented with FBS, EVs from various sources, or with no supplements. Cell growth was determined by cell counts. In media without supplementation, the RAW 264.7 cells and 4T1 cells died by three days. Media supplemented with EVs from FBS allowed the cells to survive in the absence of FBS for one week or more. Being able to perform experiments on cells supported by EVs will simplify the interpretation of experiments. Efforts are underway to determine their mechanism by which EVs support cell survival.

185 Serum-dependent and serum-independent effects of prolactin and progesterone during the completion of invitro maturation on metaphase II chromosomes in bovine oocytes

The final stages of IVM are of great importance for the developmental competence of mammalian oocytes. The goal of the present work was to study effects of prolactin (PRL) and progesterone (PG) during the completion of IVM in serum-containing and serum-free media on destructive and apoptotic changes of MII chromosomes in bovine oocytes. Cumulus-oocyte complexes (COCs) were matured for 16h in TCM-199 containing 10% fetal calf serum (FCS), 10μgmL−1 porcine FSH, and 10μgmL−1 ovine LH at 38.5°C and 5% CO2. Thereafter, the COCs were transferred to and cultured for either 8 or 26h in the following systems: TCM-199 containing 10% FCS (Control-1) and TCM-199 containing 3mgmL−1 bovine serum albumin (BSA; Control-2). In both systems, the medium of experimental groups was supplemented with 50ngmL−1 bovine PRL or 50ngmL−1 PG. All treatments were repeated 4 to 5 times using 80 to 108 oocytes per group. The state of the oocyte nuclear material was evaluated by Tarkowski's method. Apoptosis in MII oocytes was detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. Arcsine-transformed data were analysed by ANOVA. At the end of culture, the rate of MII oocytes was similar in all the groups (83.4-93.9%). Following 8h of IVM, the frequency of MII chromosome abnormalities (decondensation, adherence, clumping) was reduced (P<0.01) in the PRL-1 and PG-1 groups compared with Control-1 (Table 1). This effect was still observed in the PG-1 group (P<0.001) but disappeared in the PRL-1 group after 26h of prolonged oocyte culture. In contrast, a decrease in the rate of MII oocytes with abnormal chromosomes occurred only in the PG-2 group by the end of 8h of culture (P<0.01), whereas the decrease was revealed in both the PG-2 and PRL-2 groups after 26h of culture (P<0.001). Furthermore, the rate of MII oocytes with apoptotic signs following 8h was lower in the PRL-1 and PG-1 groups than in Control-1 (P<0.05). During the 26-hculture, this rate increased in all groups, being higher in Control-1 than in the PG-1 group (P<0.05). The rate of apoptotic oocytes was 2.8 times (8h) and 1.8 times (26h) lower in Control-2 than in Control-1 (P<0.01). Neither PRL nor PG affected oocyte apoptosis in system 2. Thus, during the completion of IVM, PRL can exert a short-term and serum-dependent inhibitory effect on destructive changes of MII chromosomes in bovine oocytes, whereas the similar effect of PG is long-term and serum-independent. Anti-apoptotic effects of both PRL and P4 on the oocytes are determined by serum. Table 1.Effects of prolactin (PRL) and progesterone (PG) on MII chromosomes in bovine oocytes Group % of MII oocytes With abnormal chromosomes With apoptotic signs 8 h 26 h 8 h 26 h Control-1 36.5±3.1a 68.0±2.2a 19.4±1.1a 65.5±2.9a PRL-1 23.6±3.2bc 68.3±2.6a 9.8±3.3b 51.2±7.8abc PG-1 23.4±2.4bc 49.4±0.6b 8.0±3.1b 53.6±0.7b Control-2 31.7±2.7ac 62.7±1.0a 6.9±1.3b 37.3±3.3cd PRL-2 32.6±2.5ac 45.9±0.9b 7.4±2.9b 28.1±2.1d PG-2 20.0±2.5b 47.2±1.6b 6.8±0.9b 29.9±3.9cd a-dValues within a column differ (at least P<0.05). This study was supported by RFBR (No. 17-29-08035).

Serum-dependent and -independent regulation of PARP2

PARP2 belongs to a family of proteins involved in cell differentiation, DNA damage repair, cellular energy expenditure, and chromatin modeling. In addition to these overlapping functions with PARP1, PARP2 participates in spermatogenesis, T-cell maturation, extra-embryonic endoderm formation, adipogenesis, lipid metabolism, and cholesterol homeostasis. Knowledge of the functions of PARP2 is far from complete, and the mechanism(s) by which the gene and protein are regulated are unknown. In this study, we found that two different mechanisms are used in vitro to regulate PARP2 levels. In the presence of serum, PARP2 is degraded through the ubiquitin–proteasome pathway; however, when serum is removed or dialyzed with a 3.5 kDa molecular cut membrane, PARP2 rapidly becomes sodium dodecyl sulphate- and urea-insoluble. Despite the presence of a putative serum response element in the PARP2 gene, transcription is not affected by serum deprivation, and PARP2 levels are restored when serum is replaced. The loss of PARP2 affects cell differentiation and gene expression linked to cholesterol and lipid metabolism. These observations highlight the critical roles that PARP2 plays under different physiological conditions, and reveal that PARP2 is tightly regulated by distinct pathways.

Serum-dependent and independent regulation of PARP2

PARP2 belongs to a family of proteins involved in cell differentiation, DNA damage repair, cellular energy expenditure, chromatin modeling and cell differentiation. In addition to these overlapping functions with PARP1, PARP2 participates in spermatogenesis, T-cell maturation, extraembryonic endoderm formation and adipogenesis. The function(s) of PARP2 is far from complete, and the mechanism(s) by which the gene and protein are regulated are unknown. In this study, we found that two different mechanisms are used in vitro to regulate PARP2 levels. In the presence of serum, PARP2 is degraded through the ubiquitin-proteasome pathway, however, when serum is removed, PARP2 is rapidly sequestered into an SDS- and urea-insoluble fraction. This sequestration is relieved by serum in a dose-dependent manner, and again PARP2 is detected by immunoblotting. Furthermore, and despite the presence of a putative serum response element in the PARP2 gene, transcription is not affected by serum deprivation. These observations that PARP2 is tightly regulated by distinct pathways highlights the critical roles PARP2 plays under different physiological conditions.