Structural and functional homology between the 29 kD rat liver nucleoprotein and the high mobility group 1 protein

A 29 kD soluble rat liver nucleoprotein (p29) has increased binding affinity for the hormone responsive element (RE) of the rat haptoglobin (Hp) gene during the acute-phase reaction. In this work the possibility of its structural and functional homology to the high mobility group 1 (HMG1) nonhistone protein constituent of chromatin was examined. The results of two-dimensional gel electrophoresis, Southwestern and Western immunoblot analyses, showed that p29 and HMG1 are homologous protein species. On the basis of in vitro and in vivo phosphorylation/dephosphorylation experiments, we discuss the modulatory role of phosphate groups in view of the structure and function of p29.     

Caste formation in the polyembryonic wasp Copidosoma floridanum (Hymenoptera: Encyrtidae): in vivo and in vitro analysis

The polyembryonic wasp Copidosoma floridanum produces two morphologically distinct types of larvae in its host Trichoplusia ni. Reproductive larvae consume the host, pupate, and form adult wasps, whereas precocious larvae manipulate the sex ratios of the reproductive caste and defend the brood against interspecific competitors. The previous study indicated that morphogenesis of the reproductive caste was associated with a 9-day competency period, and that ecdysteroids of host origin were required for completion of embryogenesis. Here we investigated whether factors associated with the host environment mediate morphogenesis of precocious larvae and caste determination. Embryogenesis of precocious larvae was found to be synchronized with specific stages of the host first-fourth instars. However, development of precocious larvae did not depend on environmental factors specifically associated with these host stages. Elevation of the host juvenoid titer using the analogue methoprene induced T. ni to undergo a supernumerary sixth instar, but did not alter the proportion of wasp embryos that developed into precocious and reproductive larvae. In contrast, embryos competent to initiate morphogenesis developed into precocious larvae when transplanted into novel host stages such as pupae. Development of precocious larvae was arrested by ablation of the host's source of ecdysteroids, but could be rescued dose-dependently by injection of 20-hydroxyecdysone. In vitro rearing studies confirmed that completion of embryogenesis of the precocious caste required an exogenous pulse of 20-hydroxyecdysone. Combined with previous studies, our results indicate that embryos forming precocious and reproductive larvae acquire the competence to undergo morphogenesis at different times. However, we find no evidence to suggest that caste determination is mediated by environmental factors associated with a specific stage of the host.     

P2Y2 receptor promotes intestinal microtubule stabilization and mucosal re‐epithelization in experimental colitis

P2Y₂ receptor expression is increased in intestinal epithelial cells (IECs) during inflammatory bowel diseases (IBDs). In this context, P2Y₂ stimulates PGE₂ release by IECs, suggesting a role in wound healing. For this study, we have used the non‐cancerous IEC‐6 cell line. IEC‐6 cell migration was determined using Boyden chambers and the single‐edged razor blade model of wounding. The receptor was activated using ATP, UTP, or 2‐thioUTP. Pharmacological inhibitors, a blocking peptide, a neutralizing antibody and interfering RNAs were used to characterize the signaling events. Focal adhesions and microtubule (MT) dynamics were determined by immunofluorescence using anti‐vinculin and anti‐acetylated‐α‐tubulin antibodies, respectively. In vivo, the dextran sodium sulfate mouse model of colitis was used to characterize the effects of P2Y₂ agonist 2‐thioUTP on remission. We showed that P2Y₂ increased cell migration and wound closure by recruiting Go protein with the cooperation of integrin α_v. Following P2Y₂ activation, we demonstrated that GSK3β activity was inhibited in response to Akt activation. This leads to MT stabilization and increased number of focal adhesions. In vivo, P2Y₂ activation stimulates remission, as illustrated by a reduction in the disease activity index values and histological scores as compared to control mice. These findings highlight a novel function for this receptor in IECs. They also illustrate that P2Y receptors could be targeted for the development of innovative therapies for the treatment of IBDs. J. Cell. Physiol. 228: 99–109, 2013. © 2012 Wiley Periodicals, Inc.     

Influence of cytology development on frequency of pre-cancerous lesions and cervical cancer in east Croatia, 1978-2001

We analyzed the influence of cytology development to frequency of precancerosa (Cervical intraepithelial neoplasia grade 3 - CIN 3) and cervical cancer. The number of examined Pap smears increased significantly in the analyzed period. The calculated linear trend shows an average four-year increase of 17,283.35 smears. The number of detected CIN 3 increased accordingly. The calculated linear trend shows an average four-year increase by 45.03 and decrease of the rate of 0.07. A slight increase in cervical cancer was also noticed. The calculated linear trend shows an average four-year increase in cancer by 1.6 and decrease in the rate of 0.51. Analyses of cervical cancer by stages showed an increase in number of stage I and decrease of other stages. The calculated linear trend shows an average four-year increase of stage I by 4.94 or decrease in other stages of cervical cancer of 3.29 respectively. The rate of cervical cancer stage I decreased by 0.11 and of other stages by 0.39. Further analyses of the stage I showed that the linear trend of IA stage had an average four-year increase by 5.40 and decrease in stage IB of 0.48. The rate of the cervical cancer stage IA increased by 0.02, whereas the rate of IB decreased by 0.13. Cytology development in our country has resulted in detection of higher number of CIN 3 and the earliest stage (IA) of cervical cancer whereas the number of other stages (IB, II, III, IV) has decreased. However, a total number of cervical cancers haven't changed over the whole period. Thus, it is obvious that opportunistic program of detection, which has been using in Croatia, could not decrease frequency of cervical cancer. In order to achieve it, well-organized national program of detection is needed.     

Diagnostic value of cytology and colposcopy for squamous and glandular cervical intraepithelial lesions

The main objective of work was to determine a diagnostic value of cytology and colposcopy as a method of screening and differential diagnosis, as well as to determine the relative value of some colposcopic features of squamous and glandular cervical intraepithelial lesions. Cytological diagnosis and colposcopy findings is compared with histological ones for 187 patients with intraepithelial lesions (142 squamous and 45 glandular ones with or without squamous components) and determined the sensitivity and positive predictive value of cytology and colposcopy, including the types of colposcopic abnormalities associated with squamous/glandular intraepithelial lesions. The sensitivity of cytology as a screening method for SIL (squamous intraepithelial lesions) is 89% and for GIL (glandular intraepithelial lesions) 98%. Positive predictive value of differential cytology for SIL is 59% and for GIL 53%. Sensitivity of colposcopy for both lesions' type is 87%. Acetowhite epithelium occurs for more often with SIL, whereas atypical vessels and unequal, dilated gland openings with GIL (p < 0.05). Cytology and colposcopy as screening methods have a high sensitivity. Nevertheless, cytology is far more accurate in determining differential diagnosis of SIL than GIL and some colposcopy abnormalities suspicious of GIL should be further tested in praxis.     

Induction of a senescent-like phenotype does not confer the ability of bovine immortal cells to support the development of nuclear transfer embryos

Previously, we reported that cloned embryos derived from an immortalized bovine mammary epithelial cell line (MECL) failed to develop beyond 12- to 16-cell stage. To analyze whether induction of a senescent-like phenotype in MECL can improve their ability to support the development after transfer into enucleated oocytes, we treated MECL with DNA methylation inhibitor 5-aza-2-deoxycytidine (Aza-C), histone deacetylase inhibitors trichostatin A (TSA), sodium butyrate (NaBu), or 5-bromodeoxyuridine and used those cells for nuclear transfer. Primary bovine fetal fibroblasts (BFF) were used as control. All agents were capable to induce features of senescence including reduced cell proliferation, enlarged cell size with a considerable proportion of cells stained positive for acidic senescence-associated beta-galactosidase and G1/S cell cycle boundary arrest in MECL. Aza-C treatment induced genome demethylation. Acetylation of H3 and H4 was increased after TSA treatment in both MECL and BFF, whereas no obvious changes in global H3 or H4 acetylation were detected after NaBu treatment. Nuclear transfer experiments following diverse treatments demonstrated that the induced senescent-like phenotype of MECL did not confer their ability to support embryonic development, although 7.3% of reconstructed embryos derived from NaBu-treated cells developed to morula stage. Intriguingly, a much higher proportion of cloned embryos developed to blastocysts when using NaBu-treated BFF, compared with using untreated BFF (59% versus 26%). Our results suggest that the developmental failure of donor nuclei from bovine immortal cells could not be reversed by induction of senescent-like phenotype. The beneficial effect of NaBu on the developmental potential of cloned embryos reconstructed from BFF merits further studies.     

Vasa expression and germ-cell specification in the spider mite <Emphasis Type="Italic">Tetranychus urticae</Emphasis>

The specification of germ cells is an important process during the development of all animals. Expression of an evolutionarily conserved gene such as vasa can be used as a marker for germ cell fate. We have isolated a vasa-related gene from the two-spotted spider mite (Tetranychus urticae) and used it to examine the segregation of germ cells in this animal. In spider mites, vasa expression first appears in a group of cells that do not join the initial blastoderm surface. Instead, these cells remain in the interior of the blastoderm and then migrate to posterior regions of the embryo, where they form a cluster that appears in regions of the embryo consistent with the gonads. The expression pattern of this spider mite vasa homologue implies a novel process acts to specify germ cells in this species and that the specification of germ cells is an evolutionarily labile process.