Dopamine D4 Receptor Agonist Drastically Increases Delta Activity in the Thalamic Nucleus Reuniens: Potential Role in Communication between Prefrontal Cortex and Hippocampus.

Network oscillations are essential for all cognitive functions. Oscillatory deficits are well established in psychiatric diseases and are recapitulated in animal models. They are significantly and specifically affected by pharmacological interventions using psychoactive compounds. Dopamine D4 receptor (D4R) activation was shown to enhance gamma rhythm in freely moving rats and to specifically affect slow delta and theta oscillations in the urethane-anesthetized rat model. The goal of this study was to test the effect of D4R activation on slow network oscillations at delta and theta frequencies during wake states, potentially supporting enhanced functional connectivity during dopamine-induced attention and cognitive processing. Network activity was recorded in the prefrontal cortex (PFC), hippocampus (HC) and nucleus reuniens (RE) in control conditions and after injecting the D4R agonist A-412997 (3 and 5 mg/kg; systemic administration). We found that A-412997 elicited a lasting (~40 min) wake state and drastically enhanced narrow-band delta oscillations in the PFC and RE in a dose-dependent manner. It also preferentially enhanced delta synchrony over theta coupling within the PFC-RE-HC circuit, strongly strengthening PFC-RE coupling. Thus, our findings indicate that the D4R may contribute to cognitive processes, at least in part, through acting on wake delta oscillations and that the RE, providing an essential link between the PFC and HC, plays a prominent role in this mechanism.

[Anti-lung cancer effect of myeloid and plasmacytoid dendritic cell combined vaccines loaded with tumor cell lysates in vitro].

Objective: To investigate the feasibility of myeloid and plasmacytoid dendritic cell combined vaccines loaded with heat-treated Lewis lung cancer cell lysates for treatment of lung cancer in mice. Methods: Bone marrow cells were induced by the recombinant mouse fms-like tyrosine kinase receptor 3 ligand (rmFlt3-L) in vitro, myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC) were separated by magnetic beads. The mDC, pDC, and mDC∶pDC=1∶1 were stimulated with heat-treated Lewis lung cancer cell lysates, respectively. The effects of each group on stimulating of lymphocyte proliferation and inducing of T cell to kill tumor cells in vitro were compared. The alternations of the immunophenotypes of CD80, CD86, CD40 and major histocompatibility complex Ⅱ (MHC-Ⅱ) were detected by flow cytometry. The secretion of cytokines including interlukin-12 (IL-12), interlukin-6 (IL-6), and tumor necrosis factor α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). Results: The lymphocyte proliferation in mice stimulated with mDC+ pDC group loaded with heat-treated Lewis lung cancer cell lysates was 10.80±0.66, significantly higher than 8.63±0.65 of mDC group and 7.10±0.46 pDC group under the same culture conditions, respectively (P<0.05). When the ratio of effector cells: target cells (E∶T) was 10∶1, the killing rate of the mDC+ pDC group loaded with heat-treated tumor cell lysate was 31.68%±2.93%, significantly higher than 17.44%±0.97% of mDC group and 10.29%±1.33% of pDC group, respectively (P<0.05). When the ratio of E∶T was 20∶1, the killing rate of the mDC+ pDC group loaded with heat-treated tumor cell lysate was 54.77%±3.28%, significantly higher than 35.25%±1.51% of mDC group and 15.52%±0.73% of pDC group, respectively (P<0.05). When the ratio of E∶T was 40∶1, the killing rate of the mDC+ pDC group loaded with heat-treated tumor cell lysate was 73.01%±0.91%, significantly higher than 51.36%±0.58% of mDC group and 22.65%±1.28% of pDC group, respectively (P<0.05). With the rate of E∶T increased, the killing rate also increased. The mean fluorescence intensities of surface molecules including CD80, CD86, CD40 and MHC-Ⅱ of mDC: pDC=1 group pulsed with heat-treated Lewis lung cancer cell lysates were higher than those of mDC group and pDC group. The IL-6 cytokine concentrations of mDC+ pDC group, mDC group and pDC group loaded with heat-treated Lewis lung cancer cell lysates were (586.67±52.52) pg/ml, (323.33±67.14) pg/ml and (166.67±16.07) pg/ml, respectively. The concentrations of IL-12 in each group were (2 568.75±119.24) pg/ml, (2 156.25±120.55) pg/ml and (672.92±31.46) pg/ml, respectively. The concentrations of TNF-α in each group were (789.33±48.08) pg/ml, (584.89±116.49) pg/ml and (291.56±40.73) pg/ml, respectively. The concentrations of IL-6, IL-12 and TNF-α secreted by mDC+ pDC group were much higher than those of mDC group and pDC group under the same culture conditions (P<0.05). Conclusions: The mDCs and pDCs combined vaccines pulsed with heat-treated Lewis lung cancer cell lysates have synergistic effects on inducing of T lymphocyte proliferation and killing tumor cells in vitro. This synergistic anti-tumor effect is related with up-regulation of co-stimulatory molecules and increased secretion of cytokines.

[Analysis of new occupational diseases in Hengyang from 2006 to 2017].

Objective: To analyze the situation of new occupational diseases in Hengyang City from 2006 to 2017, and put forward prevention and control strategies. Methods: The data of the new occupational disease report of Hengyang city in the Occupational Disease and Occupational Health Information Monitoring System from January 1, 2006 to December 31, 2017 was collected, and the age, working years, and the region and industry of the new occupational disease patients were analyzed. Results: From 2006 to 2017, there were 7 categories, 30 kinds and 2 110 cases of new occupational diseases in Hengyang City, including 1 117 cases of pneumoconiosis, 951 cases of chronic occupational poisoning, 15 cases of acute occupational poisoning, 12 cases of occupational otolaryngological and stomatological diseases, 7 cases of occupational skin diseases, 6 cases of occupational diseases caused by physical factors, and 1 case of occupational eye diseases(cataracts), 1 case of occupational tumor (lung cancer and skin cancer caused by arsenic and its compounds). New occupational diseases were mainly concentrated in Changning and Leiyang County-level city (87.82%, 1 853/2110), among which occupational poisoning had the most incidence in Changning County-level city (97.83%, 945/966), and pneumoconiosis had the most incidence in Leiyang County-level city (67.05%, 749/1 117). New occupational diseases were mainly concentrated in the manufacturing and mining industries (95.59%, 2 017/2 110). Pneumoconiosis (63.74%, 712/1 117) and acute occupational poisoning (60.00%, 9/15) were mainly caused by small businesses. Chronic occupational poisoning (61.62%, 586/951) and occupational otolaryngological and stomatological diseases (75.00%, 9/12) were mainly caused by large enterprises. Conclusion: The new occupational diseases in Hengyang city are mostly pneumoconiosis and chronic occupational poisoning, and we should focus on strengthening the prevention and control of occupational diseases in the mining industry and manufacturing industry.

STAT3 regulates cytokine expression in peripheral blood mononuclear cells from asthma patients.

Asthma is a common long term inflammatory disease of the airways. This disease affected millions of people worldwide. Recently, it is demonstrated that signal transducer and activator of transcription 3 (STAT3) plays critical role in asthma occurrence. In the current study, we isolated peripheral blood mononuclear cells (PBMCs) from patients with mild and moderate asthma, and then determined the correlation between STAT3 and cytokine expression. We found that the concentration and mRNA level of cytokines was increased in PBMCs from asthma patients. The concentration and mRNA level of cytokines was altered by the regulation of STAT3 expression and the concentration and mRNA expression level of cytokines was positively correlated with STAT3 activation. Furthermore, phosphorylated STAT3 expression in PBMCs from asthma patients was increased compared with the control. Collectively, this study directly proved that STAT3 was correlated with cytokine expression in PBMCs from asthma patients, providing a potential linkage between STAT3 and pathogenesis of asthma.

Recruitment of trimeric proliferating cell nuclear antigen by G1-phase cyclin-dependent kinases following DNA damage with platinum-based antitumour agents.

BACKGROUND: In cycling tumour cells, the binary cyclin-dependent kinase Cdk4/cyclin D or Cdk2/cyclin E complex is inhibited by p21 following DNA damage to induce G1 cell-cycle arrest. However, it is not known whether other proteins are also recruited within Cdk complexes, or their role, and this was investigated. METHODS: Ovarian A2780 tumour cells were exposed to the platinum-based antitumour agent 1R,2R-diaminocyclohexane(trans-diacetato)(dichloro)platinum(IV) (DAP), which preferentially induces G1 arrest in a p21-dependent manner. The Cdk complexes were analysed by gel filtration chromatography, immunoblot and mass spectrometry. RESULTS: The active forms of Cdk4 and Cdk2 complexes in control tumour cells have a molecular size of ~140 kDa, which increased to ~290 kDa when inhibited following G1 checkpoint activation by DAP. Proteomic analysis identified Cdk, cyclin, p21 and proliferating cell nuclear antigen (PCNA) in the inhibited complex, and biochemical studies provided unequivocal evidence that the increase in ~150 kDa of the inhibited complex is consistent with p21-dependent recruitment of PCNA as a trimer, likely bound to three molecules of p21. Although p21 alone was sufficient to inhibit the Cdk complex, PCNA was critical for stabilising p21. CONCLUSION: G1 Cdk complexes inhibited by p21 also recruit PCNA, which inhibits degradation and, thereby, prolongs activity of p21 within the complex.

Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation.

The centrosomes are thought to maintain genomic stability through the establishment of bipolar spindles during cell division, ensuring equal segregation of replicated chromosomes to two daughter cells. Deregulated duplication and distribution of centrosomes have been implicated in chromosome segregation abnormalities, leading to aneuploidy seen in many cancer cell types. Here, we report that STK15 (also known as BTAK and aurora2), encoding a centrosome-associated kinase, is amplified and overexpressed in multiple human tumour cell types, and is involved in the induction of centrosome duplication-distribution abnormalities and aneuploidy in mammalian cells. STK15 amplification has been previously detected in breast tumour cell lines and in colon tumours; here, we report its amplification in approximately 12% of primary breast tumours, as well as in breast, ovarian, colon, prostate, neuroblastoma and cervical cancer cell lines. Additionally, high expression of STK15 mRNA was detected in tumour cell lines without evidence of gene amplification. Ectopic expression of STK15 in mouse NIH 3T3 cells led to the appearance of abnormal centrosome number (amplification) and transformation in vitro. Finally, overexpression of STK15 in near diploid human breast epithelial cells revealed similar centrosome abnormality, as well as induction of aneuploidy. These findings suggest that STK15 is a critical kinase-encoding gene, whose overexpression leads to centrosome amplification, chromosomal instability and transformation in mammalian cells.

Safety and efficacy of using Judkins left 3.5 guiding catheters for transradial right coronary artery intervention.

OBJECTIVE: There is limited data about the use of a Judkins left (JL) 3.5 guiding catheter for routine transradial right coronary artery (RCA) percutaneous coronary intervention (PCI). This study investigated the safety and efficacy of JL3.5 for RCA PCI. PATIENTS AND METHODS: Patients with acute coronary syndrome (ACS) who underwent transradial RCA PCI between November 2019 and November 2020 at the Second Hospital of Shandong University were included. The study retrospectively compared JL 3.5 vs. other routine guiding catheters (GCs), including Judkins right (JR) 4.0 and Amplatz (left). Logistic multivariable analysis was used to analyze the factors associated with transradial RCA PCI success rate, in-hospital complications, and extra support. RESULTS: The study included 311 patients: 136 in the routine GC group and 175 in the JL 3.5 group. There were no significant differences between the two groups regarding in-hospital complications, extra support technics, or success. The multivariable analyses showed that coronary chronic total occlusion (CTO) was negatively associated with intervention success (OR = 0.06, 95% CI: 0.016-0.248, p < 0.001) but positively with extra support (OR = 8.74, 95% CI: 1.518-50.293, p = 0.015). Tortuosity was associated with extra support (OR = 16.50, 95% CI: 3.324-81.589, p = 0.001). In the JL 3.5 group, the left ventricular ejection fraction (OR = 1.11, 95% CI: 1.03-1.20, p = 0.006), CTO (OR = 0.07, 95% CI: 0.008-0.515, p = 0.009), and tortuosity (OR = 0.17, 95% CI: 0.03-0.95, p = 0.043) were independently associated with intervention success. CONCLUSIONS: JL 3.5 appears to be as safe and effective as the JR 4.0 and Amplatz (left) catheters for RCA PCI. When using the JL 3.5 catheter for RCA PCI, heart function, CTO, and tortuosity should be considered.

[A 5-year retrospective study of computer aided design and computer aided manufacturing ceramic endocrowns in endodontically treated posterior teeth].

Objective: To evaluate the clinical outcomes of computer aided design and computer aided manufacturing (CAD/CAM) ceramic endocrowns in endodontically treated posterior teeth after five years by a retrospective study. Methods: Patients who received CAD/CAM ceramic endocrowns after endodontically treatment in Department of Endodontics, School of Stomatology, The Fourth Military Medical University between January 2016 and June 2017 were invited for this clinical study. Clinical performance was evaluated in the aspect of color match, anatomic form,marginal adaptation, restoration integrity and secondary caries. Survival rate of the restorations was calculated by the use of Kaplan-Meier method. Log-rank test was applied as well for the sake of analyzing the effect of tooth position, sex and materials to the survival rate of the restorations. Results: Seventy-four patients, 25 men and 49 women with age of (38.8±10.2) years, participated in this study for a total of 101 CAD/CAM ceramic endocrowns after observation period of (62.8±12.0) months. There were 8 failed cases among 101 restorations, 5 were loss of retention, 2 were ceramic fracture and 1 was secondary caries respectively. In particular, 93% (89/96) restorations got score A on anatomic form and 95% (91/96) restorations got score A on marginal adaptation, while 38% (36/96) restorations showed the good color match compared with the abutment teeth. The estimated cumulative survival rate of CAD/CAM ceramic endocrowns in endodontically treated posterior teeth after 5 years was 93.0% (95%CI: 87.9%-98.1%). The single-factor Log-rank analysis demonstrated that there was no statistically significant difference in the survival rate of CAD/CAM ceramic endocrowns among men and women, premolars and molars, position in the dental arch, or different materials (χ²<0.01, P=0.957; χ²=0.64, P=0.422; χ²=0.69, P=0.407; χ²=0.88, P=0.349). Conclusions: Based on this clinical study, the clinical performance of CAD/CAM ceramic endocrowns in endodontically treated posterior teeth after five years is reliable, which could be a general option to restore nonvital teeth.

Scanning chimeragenesis: the approach used to change the substrate selectivity of fatty acid monooxygenase CYP102A1 to that of terpene omega-hydroxylase CYP4C7.

CYP102A1 is a highly active, water-soluble, bacterial monooxygenase enzyme that contains both substrate-binding heme and diflavin reductase subunits, both in a single polypeptide. Recently we developed a procedure which uses the known structure of the substrate-bound heme domain of CYP102A1 and its sequence homology with a cytochrome P450 of unknown structure, both of which react with a common substrate but produce different products, to create recombinant enzymes which have substrate selectivity different from that of CYP102A1, and produce the product of the enzyme of unknown structure. Insect CYP4C7, a terpene hydroxylase from the cockroach, was chosen as the cytochrome P450 of unknown structure, and farnesol was chosen as the substrate. CYP102A1 oxidizes farnesol to three products (2,3-epoxyfarnesol, 10,11-epoxyfarnesol, and 9-hydroxyfarnesol), whereas CYP4C7 produces 12-hydroxyfarnesol as the major product. In earlier work it was found that the chimera C(78-82,F87L) showed a change in substrate selectivity from fatty acids to farnesol, and was approximately sixfold more active than wild-type CYP102A1 (Chen et al. in J Biol Inorg Chem 13:813-824, 2008), but neither it nor any other earlier chimera produced 12-hydroxyfarnesol. In this work we added amino acid residues 327-332, to create six new full-length, functional chimeric proteins. Four of these, the most active of which was C(78-82,F87L,328-330), produce 12-hydroxyfarnesol as the major product, with approximately twofold increase in turnover number as compared with wild-type CYP102A1 toward farnesol. Methylfarnesoate was metabolized to 12-hydroxymethylfarnesoate (70%) and 10,11-epoxymethylfarnesoate (juvenile hormone III) (30%). The latter is metabolized to 65% 12-hydroxy-10,11-epoxymethylfarnesoate and 35% 15-hydroxy-10,11-epoxymethylfarnesoate. Substitution of residues 328-330, APA, by VPL was crucial to accomplishing this change in product.

At least two kinases phosphorylate the MPM-2 epitope during Xenopus oocyte maturation.

MPM-2 antigens, a discrete set of phosphoproteins that contain similar phosphoepitopes (the MPM-2 epitope), are associated with various mitotically important structures. The central mitotic regulator cdc2 kinase has been proposed to induce M-phase by phosphorylating many proteins which might include the MPM-2 antigens. To clarify the relationship of cdc2 kinase and the MPM-2 antigens, we developed an in vitro assay that enabled us to specifically detect the kinases that phosphorylate the MPM-2 epitope (ME kinases) in crude cell extracts. Two different ME kinase activities were identified in unfertilized Xenopus eggs, neither of which was cdc2 kinase, but both appeared to be activated by the introduction of cdc2 kinase into oocytes or oocyte extract. The two ME kinases differed in molecular size, substrate specificity, peptide components, and MPM-2 reactivity. The larger one, ME kinase-H, phosphorylated several MPM-2 antigens, while the smaller one, ME kinase-L, phosphorylated mainly one. We purified ME kinase-L to near homogeneity by sequential chromatography and showed that it has the characteristics of the 42-kD microtubule-associated protein (MAP) kinase. Our results support the previous finding that MAP kinase is activated during Xenopus oocyte maturation and suggest that MAP kinase may contribute to oocyte maturation induction by phosphorylating one subtype of MPM-2 epitope.

Sequence-specific and phosphorylation-dependent proline isomerization: a potential mitotic regulatory mechanism.

Pin1 is an essential and conserved mitotic peptidyl-prolyl isomerase (PPIase) that is distinct from members of two other families of conventional PPIases, cyclophilins and FKBPs (FK-506 binding proteins). In response to their phosphorylation during mitosis, Pin1 binds and regulates members of a highly conserved set of proteins that overlaps with antigens recognized by the mitosis-specific monoclonal antibody MPM-2. Pin1 is here shown to be a phosphorylation-dependent PPIase that specifically recognizes the phosphoserine-proline or phosphothreonine-proline bonds present in mitotic phosphoproteins. Both Pin1 and MPM-2 selected similar phosphorylated serine-proline-containing peptides, providing the basis for the specific interaction between Pin1 and MPM-2 antigens. Pin1 preferentially isomerized proline residues preceded by phosphorylated serine or threonine with up to 1300-fold selectivity compared with unphosphorylated peptides. Pin1 may thus regulate mitotic progression by catalyzing sequence-specific and phosphorylation-dependent proline isomerization.

A "human knockout" model to investigate the influence of the α-actinin-3 protein on exercise-induced mitochondrial adaptations.

Research in α-actinin-3 knockout mice suggests a novel role for α-actinin-3 as a mediator of cell signalling. We took advantage of naturally-occurring human "knockouts" (lacking α-actinin-3 protein) to investigate the consequences of α-actinin-3 deficiency on exercise-induced changes in mitochondrial-related genes and proteins, as well as endurance training adaptations. At baseline, we observed a compensatory increase of α-actinin-2 protein in ACTN3 XX (α-actinin-3 deficient; n = 18) vs ACTN3 RR (expressing α-actinin-3; n = 19) participants but no differences between genotypes for markers of aerobic fitness or mitochondrial content and function. There was a main effect of genotype, without an interaction, for RCAN1-4 protein content (a marker of calcineurin activity). However, there was no effect of genotype on exercise-induced expression of genes associated with mitochondrial biogenesis, nor post-training physiological changes. In contrast to results in mice, loss of α-actinin-3 is not associated with higher baseline endurance-related phenotypes, or greater adaptations to endurance exercise training in humans.

Unraveling the discrepancies in size dependence of hardness and thermal stability in crystalline/amorphous nanostructured multilayers: Cu/Cu-Ti vs. Cu/HfO2.

Crystalline/amorphous interfaces (CAIs) confer outstanding mechanical properties on crystalline/amorphous nanostructured multilayers (C/ANMs), which are widely used in micro/nanodevices, because their unique interfacial structure possesses high strain compatibility. In this study, Cu/X (X = Cu-Ti, HfO2) C/ANMs with equal layer thicknesses (h) were comparatively investigated in terms of size-dependent hardness (H) and thermal stability to uncover the fundamental difference(s) between Cu/Cu-Ti and Cu/HfO2. It was found that both as-deposited Cu/Cu-Ti and Cu/HfO2 C/ANMs exhibited a maximum hardness at a critical thickness of h ∼30 nm, which was caused by a transition from confined dislocation gliding to dislocation transmission across the interface. Specifically, the Cu/Cu-Ti C/ANMs exhibited annealing hardening, whereas the Cu/HfO2 C/ANMs exhibited annealing softening associated with a minimum softening at h ∼ 30 nm, which was closely correlated with their thermal stability. In comparison with monolithic amorphous X thin films, the glassy X nanolayers in the present Cu/X C/ANMs exhibited reduced thermal stability and a trend that smaller sizes led to higher stability. The underlying mechanism of the size-dependent crystallization behavior of X nanolayers is discussed in terms of the constraining effects of the interface. These findings provide deep insights into the design of Cu/metallic-glass and Cu/ceramic-glass C/ANMs with optimal performance.

Systematic identification of mitotic phosphoproteins.

BACKGROUND: Cyclin-dependent kinases (CDKs) are thought to initiate and coordinate cell division processes by sequentially phosphorylating key targets; in most cases these substrates remain unidentified. RESULTS: Using a screen that scores for phosphorylation of proteins, which were translated from pools of cDNA plasmids in vitro, by either phosphoepitope antibody recognition or electrophoretic mobility shifts, we have identified 20 mitotically phosphorylated proteins from Xenopus embryos, 15 of which have sequence similarity to other proteins. Of these proteins, five have previously been shown to be phosphorylated during mitosis (epithelial-microtubule associated protein-115, Oct91, Elongation factor 1gamma, BRG1 and Ribosomal protein L18A), five are related to proteins postulated to have roles in mitosis (epithelial-microtubule associated protein-115, Schizosaccharomyces pombe Cdc5, innercentrosome protein, BRG1 and the RNA helicase WM6), and nine are related to transcription factors (BRG1, negative co-factor 2alpha, Oct91, S. pombe Cdc5, HoxD1, Sox3, Vent2, and two isoforms of Xbr1b). Of 16 substrates tested, 14 can be directly phosphorylated in vitro by the mitotic CDK, cyclin B-Cdc2, although three of these may be physiological substrates of other kinases activated during mitosis. CONCLUSIONS: Examination of this broad set of mitotic phosphoproteins has allowed us to draw three conclusions about how the activation of CDKs regulates cell-cycle events. First, Cdc2 itself appears to directly phosphorylate most of the mitotic phosphoproteins. Second, during mitosis most of the substrates are phosphorylated more than once and a number may be targets of multiple kinases, suggesting combinatorial regulation. Third, the large fraction of mitotic phosphoproteins that are presumptive transcription factors, two of which have been previously shown to dissociate from DNA during mitosis, suggests that an important function of mitotic phosphorylation is to strip the chromatin of proteins associated with gene expression.

Finely tuned regulation of cytoplasmic retention of Xenopus nuclear factor 7 by phosphorylation of individual threonine residues.

Xenopus nuclear factor 7 (xnf7) is a maternal gene product that functi ons in dorsal/ventral patterning of the embryo. The xnf7 protein is stored in the oocyte nucleus germinal vesicle in a hypophosphorylated state. At oocyte maturation, xnf7 is hyperphosphorylated and released into the cytoplasm, where it is anchored until the midblastula stage, where it is dephosphorylated and enters the nucleus. We demonstrated that cytoplasmic anchoring of xnf7 was regulated by changes in the phosphorylation status of four threonines within two sites, site 1 (Thr-103) and site 2 (Thr-209, Thr-212, and Thr-218), which function in an additive manner. A mutant form of xnf7 (xnf7thr-glu) in which the threonines at sites 1 and 2 were mutated to glutamic acids to mimic a permanent state of phosphorylation was retained in the cytoplasm in oocytes and embryos through the gastrula stage. The cytoplasmic form of xnf7 was detected in a large 670-kDa protein complex probably consisting of xnf7 and several other unknown protein components. Anchoring of xnf7 was not dependent on association with either microtubule or microfilament components of the cytoskeleton, since treatment with cytochalasin B and nocodazole did not affect cytoplasmic retention. Both wild-type xnf7 and xnf7thr-glu form dimers in the yeast two-hybrid system; however, homodimerization was not required for cytoplasmic retention. We suggest that the cytoplasmic retention of xnf7 depends on the phosphorylation state of the protein whereas the cytoplasmic anchoring machinery appears to be constitutively present in oocytes and throughout development until the gastrula stage.

cdc25 is one of the MPM-2 antigens involved in the activation of maturation-promoting factor.

MPM-2 antigens, a discrete set of phosphoproteins that contain similar phosphoepitopes recognized by the monoclonal antibody MPM-2, are phosphorylated during M-phase induction. Our previous studies suggested that certain MPM-2 antigens are involved in the appearance of maturation-promoting factor (MPF) activity. Because the central mitotic regulator cdc2 kinase has been shown to exhibit MPF activity, we explored the possibility that certain MPM-2 antigens are regulators of cdc2 kinase. We found that MPM-2 binding of its antigens would inhibit the autoamplification of cdc2 kinase in Xenopus oocytes and interfere with cyclin-activation of cdc2 kinase in Xenopus interphase egg extract. Immunodepletion of MPM-2 antigens from cyclin-induced M-phase egg extract caused the inactivation of cdc2 kinase, which was accompanied by an inhibitory phosphorylation of p34cdc2 on Thr 14 and Tyr 15, indicating that at least one MPM-2 antigen is a positive regulator of p34cdc2 dephosphorylation. We then showed that cdc25 from M-phase arrested egg extract is an MPM-2 antigen. These results suggest that phosphorylation of the epitope recognized by MPM-2 may be a crucial event in the activation of cdc25 and that the kinase(s) that phosphorylates this MPM-2 epitope may be an important regulator of cdc2 kinase activation.

The G2 block induced by DNA damage: a caffeine-resistant component independent of Cdc25C, MPM-2 phosphorylation, and H1 kinase activity.

Treatment of cells with agents that cause DNA damage often results in a delay in G2. There is convincing evidence showing that inhibition of p34cdc2 kinase activation is involved in the DNA damage-induced G2 delay. In this study, we have demonstrated the existence of an additional pathway, independent of the p34cdc2 kinase activation pathway, that leads to a G2 arrest in etoposide-treated cells. Both the X-ray-induced and the etoposide-induced G2 arrest were associated with inhibition of the p34cdc2 H1 kinase activation pathway as judged by p34cdc2 H1 kinase activity and phosphorylation of cdc25C. Caffeine treatment restored these activities after either of the treatments. However, the etoposide-treated cells did not resume cycling, revealing the presence of an alternative pathway leading to a G2 arrest. To explore the possibility that this additional pathway involved phosphorylation of the MPM-2 epitope that is shared by a large family of mitotic phosphoproteins, we monitored the phosphorylation status of the MPM-2 epitope after DNA damage and after treatment with caffeine. Phosphorylation of the MPM-2 epitope was depressed in both X-ray and etoposide-treated cells, and the depression was reversed by caffeine in both cases. The results indicate that the pathway affecting MPM-2 epitope phosphorylation is involved in the G2 delay caused by DNA damage. However, it is not part of the caffeine-insensitive pathway leading to a G2 block seen in etoposide-treated cells.

Linking genomic reorganization to tumor initiation via the giant cell cycle.

To investigate the mechanisms underlying our recent paradoxical finding that mitotically incapacitated and genomically unstable polyploid giant cancer cells (PGCCs) are capable of tumor initiation, we labeled ovarian cancer cells with α-tubulin fused to green fluorescent protein, histone-2B fused to red fluorescent protein and FUCCI (fluorescent ubiquitination cell cycle indicator), and tracked the spatial and time-dependent change in spindle and chromosomal dynamics of PGCCs using live-cell fluorescence time-lapse recording. We found that single-dose (500 nm) treatment with paclitaxel paradoxically initiated endoreplication to form PGCCs after massive cell death. The resulting PGCCs continued self-renewal via endoreplication and further divided by nuclear budding or fragmentation; the small daughter nuclei then acquired cytoplasm, split off from the giant mother cells and acquired competency in mitosis. FUCCI showed that PGCCs divided via truncated endoreplication cell cycle (endocycle or endomitosis). Confocal microscopy showed that PGCCs had pronounced nuclear fragmentation and lacked expression of key mitotic proteins. PGCC-derived daughter cells were capable of long-term proliferation and acquired numerous new genome/chromosome alterations demonstrated by spectral karyotyping. These data prompt us to conceptualize a giant cell cycle composed of four distinct but overlapping phases, initiation, self-renewal, termination and stability. The giant cell cycle may represent a fundamental cellular mechanism to initiate genomic reorganization to generate new tumor-initiating cells in response to chemotherapy-induced stress and contributes to disease relapse.