Band 15, Heft 3

The mechanism by which the mitochondrial large rRNA is involved in the restoration of the pole cell-forming ability in Drosophila embryos is still unknown. We identified a 15-ribonucleotide sequence which is conserved from the protobacterium Wolbachia to the higher eukaryotes in domain V of the mitochondrial large rRNA. This short sequence is sufficient to restore pole cell determination in UV-irradiated Drosophila embryos. Here, we provide evidence that the conserved 15-base sequence is sufficient to restore luciferase activity in vitro. Moreover, we show that the internal GAGA sequence is involved in protein binding and that mutations in this tetranucleotide affect the sequence’s ability to restore luciferase activity. The obtained results lead us to propose that mtlrRNA may be involved either in damaged protein reactivation or in protein biosynthesis during pole cell determination.

The PKB signaling pathway is essential for cell survival and the inhibition of apoptosis, but its functional mechanisms have not been fully explored. Previously, we reported that TPA effectively inhibited PKB activity and caused PKB degradation, which was correlated with the repression of PKB phosphorylation at Ser473. In this study, we focus on how PKB is regulated by TPA in gastric cancer cells. One of the TPA targets, PKCα, was found to mediate the inhibition of PKB phosphorylation and degredation caused by TPA. Furthermore, TPA induced the import of PKCα into the nucleus, where PKCα exerted an inhibitory effect on PKB expression and phosphorylation. As a result, cancer cell proliferation was arrested. Our study characterizes a novel function of PKCα in mediating the negative regulation of PKB by TPA, and suggests a potential application in the clinical treatment of gastric cancer.

Calpains and caspases are ubiquitous cysteine proteases that are associated with a variety of cellular pathways. Calpains are involved in processes such as long term potentiation, cell motility and apoptosis, and have been shown to cleave non-erythroid (brain) α- and β-spectrin and erythroid β-spectrin. The cleavage of erythroid α-spectrin by calpain has not been reported. Caspases play an important role in the initiation and execution of apoptosis, and have been shown to cleave non-erythroid but not erythroid spectrin. We have studied the effect of spectrin fragments on calpain and caspase activities. The erythroid and non-erythroid spectrin fragments used were from the N-terminal region of α-spectrin, and C-terminal region of β-spectrin, both consisting of regions involved in spectrin tetramer formation. We observed that the all spectrin fragments exhibited a concentration-dependent inhibitory effect on calpain, but not caspase activity. It is clear that additional studies are warranted to determine the physiological significance of calpain inhibition by spectrin fragments. Our findings suggest that calpain activity is modulated by the presence of spectrin partial domains at the tetramerization site. It is not clear whether the inhibitory effect is substrate specific or is a general effect. Further studies of this inhibitory effect may lead to the identification and development of new therapeutic agents specifically for calpains, but not for caspases. Proteins/peptides with a coiled coil helical conformation should be studied for potential inhibitory effects on calpain activity.

It was previously shown that the beta-spectrin ankyrin-binding domain binds lipid domains rich in PE in an ankyrin-dependent manner, and that its N-terminal sequence is crucial in interactions with phospholipids. In this study, the effect of the full-length ankyrin-binding domain of β-spectrin on natural erythrocyte and HeLa cell membranes was tested. It was found that, when encapsulated in resealed erythrocyte ghosts, the protein representing the full-length ankyrin-binding domain strongly affected the shape and barrier properties of the erythrocyte membrane, and induced partial spectrin release from the membrane, while truncated mutants had no effect. As found previously (Bok et al. Cell Biol. Int. 31 (2007) 1482–94), overexpression of the full-length GFP-tagged ankyrin-binding domain aggregated and induced aggregation of endogenous spectrin, but this was not the case with overexpression of proteins truncated at their N-terminus. Here, we show that the aggregation of spectrin was accompanied by the aggregation of integral membrane proteins that are known to be connected to spectrin via ankyrin, i.e. Na+K+ATP-ase, IP3 receptor protein and L1 CAM. By contrast, the morphology of the actin cytoskeleton remained unchanged and aggregation of cadherin E or N did not occur upon the overexpression of either full-length or truncated ankyrin-binding domain proteins. The obtained results indicate a substantial role of the lipid-binding part of the β-spectrin ankyrin-binding domain in the determination of the membrane and spectrin-based skeleton functional properties.

Heterogeneous human amniotic fluid contains various cell types. Herein, we report on the possibility of simultaneously isolating three subtypes of cells from one primary culture. Using a stainless steel instrument named a colony poculum, two of the three cell subtypes could be efficiently cultured, and these were further characterized. The results indicated that these two cell subtypes had different morphologies and were characterized by different cell marker expression profiles, including the differential expression of CD105, CD117 and EBAF. Furthermore, their gene expression array data revealed their different gene expression profiles. Although both cell types expressed several embryonic stem cell-specific markers, they were non-tumorigenic in vivo. This paper not only provides new insight into the heterogeneity of human amniotic fluid, it also presents a simple yet efficient cell isolation method. These results will contribute to the thorough investigation of the properties and potential future applications of human amniotic fluid-derived cells.

Retinoid resistance has limited the clinical application of retinoids as differentiation-inducing and apoptosis-inducing drugs. This study was designed to investigate whether celecoxib, a selective COX-2 inhibitor, has effects on retinoid sensitivity in human colon cancer cell lines, and to determine the possible mechanism of said effects. Cell viability was measured using the MTT assay. Apoptosis was detected via Annexin-V/PI staining and the flow cytometry assay. PGE2 production was measured with the ELISA assay. The expression of RARβ was assayed via western blotting. The results showed that celecoxib enhanced the inhibitory effect of ATRA in both COX-2 high-expressing HT-29 and COX-2 low-expressing SW480 cell lines. Further study showed the ATRA and celecoxib combination induced greater apoptosis, but that the addition of PGE2 did not affect the enhanced growth-inhibitory and apoptosis-inducing effects of the combination. Moreover, NS398 (another selective COX-2 inhibitor) did not affect the inhibitory effects of ATRA in the two cell lines. Western blotting showed that the expression of RARβ in HT-29 cell lines was increased by celecoxib, but not by NS398, and that the addition of PGE2 did not affect the celecoxib-induced expression of the retinoic acid receptor beta. In conclusion, celecoxib increased the expression of RARβ and the level of cellular ATRA sensitivity through COX-2-independent mechanisms. This finding may provide a potential strategy for combination therapy.

Hybrid cells derived from stem cells play an important role in organogenesis, tissue regeneration and cancer formation. However, the fate of hybrid cells and their range of function are poorly understood. Fusing stem cells and somatic cells induces somatic cell reprogramming, and the resulting hybrid cells are embryonic stem cell-like cells. Therefore, we hypothesize that fusion-induced hybrid cells may behave like ES cells in certain microenvironments. In this study, human hepatic cells were induced to apoptosis with H2O2, and then co-cultured with hybrid cells that had been derived from mouse ES cells and human hepatic cells using a transwell. After co-culturing, the degree of apoptosis was evaluated using Annexin-V/PI double-staining analysis, flow cytometry and Western-blot. We observed that H2O2-induced cell apoptosis was inhibited by co-culture. In addition, the activity of injury-related enzymes (GSH-Px, LDH and SOD) and the level of albumin release in the co-culture system trended toward the level of normal undamaged hepatic cells. The stably increased levels of secretion of ALB in the co-culture system also confirmed that co-culture with hybrid cells helped in recovery from injury. The fate of the hybrid cells was studied by analyzing their gene expression and protein expression profiles. The results of RT-PCR indicated that during co-culturing, like ES cells, hybrid cells differentiated into hepatic lineage cells. Hybrid cells transcripted genes from both parental cell genomes. Via immunocytochemical analysis, hepatic directional differentiation of the hybrid cells was also confirmed. After injecting the hybrid cells into the mouse liver, the GFP-labeled transplanted cells were distributed in the hepatic lobules and engrafted into the liver structure. This research expands the knowledge of fusion-related events and the possible function of hybrid cells. Moreover, it could indicate a new route of differentiation from pluripotent cells to tissue-specific cells via conditional co-culture.

Cisplatin and doxorubicin are widely used anticancer drugs that cause DNA damage, which activates the ATM-Chk2-p53 pathway in cancer cells. This activation leads to cell cycle block or apoptosis, depending on the nature of the DNA damage. In an attempt to enhance the effects of these agents, we inhibited ATM/ATR and Chk2, which are known upstream regulators of p53. The cancer cell lines A2780 and ARN8, bearing the wild-type p53 protein, were used to study changes in p53 activation and trans-activation. Our results suggest that the G1-checkpoint, normally activated by DNA damage, is functionally overcome by the action of kinase inhibitors that sensitize cells to apoptosis. Both inhibitors show these effects, albeit with variable intensity in different cell lines, which is promising for other studies and theoretically for use in clinical practice.

C-reactive protein (CRP) has two structurally distinct isoforms, the CRP pentamer and the CRP monomer. A role for the CRP monomer in atherosclerosis is emerging, but the underlying mechanisms are only beginning to be understood. Monocytes are an important contributor to atherosclerosis, and foam cell formation is the hallmark of atherogenesis. However, whether the CRP monomer can directly interact with the monocytes and modulate their responses remains unknown. Furthermore, although FcγRIII (CD16) has been identified as the receptor for the CRP monomer on neutrophils, its role in mediating the CRP monomer’s biological effects in other cell types has been questioned. In this study, we investigated the interaction of the CRP monomer with the monocytes using the U937 monocytic cell line. The CRP monomer specifically binds to U937 cells. This binding is unique in that it is independent of FcγRs and insensitive to protease digestion of the cell surface proteins. Further assays revealed that the CRP monomer directly incorporates into the plasma membrane. Interestingly, the presence of the CRP monomer efficiently retards oxidized low-density lipoprotein-induced foam cell formation of PMA-differentiated U937 macrophages and peripheral blood monocytic cell-derived macrophages. These findings provide additional evidence for the notion that the CRP monomer is an active CRP isoform that plays a role in atherogenesis via the direct modulation of the behavior of the monocytes.

Listeria monocytogenes enters non-phagocytic cells by binding its surface proteins inlA (internalin) and inlB to the host’s E-cadherin and Met, respectively. The two internalins play either separate or cooperative roles in the colonization of infected tissues. Here, we studied bacterial uptake into HeLa cells using an L. monocytogenes mutant strain (ΔinlA) carrying a deletion in the gene coding for inlA. The ΔinlA mutant strain showed the capability to invade HeLa cells. The monoclonal anti-β3- and anti-β1-integrin subunit antibodies prevented bacterial uptake into the cells, while the anti-β2- and anti-β4-integrin subunit antibodies failed to affect L. monocytogenes entry into HeLa cells. Three structurally distinct disintegrins (kistrin, echistatin and flavoridin) also inhibited bacterial uptake, showing different potencies correlated to their selective affinity for the β3- and β1-integrin subunits. In addition to inducing Met phosphorylation, infection of cells by the L. monocytogenes ΔinlA mutant strain promoted the tyrosine phosphorylation of the focal adhesion-associated proteins FAK and paxillin. Our findings provide the first evidence that β3- and β1-integrin receptors play a role in the inlB-dependent internalization of L. monocytogenes into host cells.

Skeletal muscle is a plastic tissue with known gender dimorphism, especially at the metabolic level. A proteomic comparison of male and female murine biceps brachii was undertaken, resolving an average of 600 protein spots of MW 15–150 kDa and pI 5–8. Twenty-six unique full-length proteins spanning 11 KOG groups demonstrated statistically significant (p<0.05) abundance differences between genders; the majority of these proteins have metabolic functions. Identified glycolytic enzymes demonstrated decreased abundance in females, while abundance differences in identified oxidative phosphorylation enzymes were specific to the proteins rather than to the functional group as a whole. Certain cytoskeletal and stress proteins showed specific expression differences, and all three phosphorylation states of creatine kinase showed significant decreased abundance in females. Expression differences were significant but many were subtle (≤ 2-fold), and known hormonally-regulated proteins were not identified. We conclude that while gender dimorphism is present in non-exercised murine skeletal muscle, the proteome comparison of male and female biceps brachii in exercise-naive mice indicates subtle differences rather than a large or obviously hormonal dimorphism.