[Development and validation of prognostic nomogram for malignant pleural mesothelioma].

Objective: To development the prognostic nomogram for malignant pleural mesothelioma (MPM). Methods: Two hundred and ten patients pathologically confirmed as MPM were enrolled in this retrospective study from 2007 to 2020 in the People's Hospital of Chuxiong Yi Autonomous Prefecture, the First and Third Affiliated Hospital of Kunming Medical University, and divided into training (n=112) and test (n=98) sets according to the admission time. The observation factors included demography, symptoms, history, clinical score and stage, blood cell and biochemistry, tumor markers, pathology and treatment. The Cox proportional risk model was used to analyze the prognostic factors of 112 patients in the training set. According to the results of multivariate Cox regression analysis, the prognostic prediction nomogram was established. C-Index and calibration curve were used to evaluate the model's discrimination and consistency in raining and test sets, respectively. Patients were stratified according to the median risk score of nomogram in the training set. Log rank test was performed to compare the survival differences between the high and low risk groups in the two sets. Results: The median overall survival (OS) of 210 MPM patients was 384 days (IQR=472 days), and the 6-month, 1-year, 2-year, and 3-year survival rates were 75.7%, 52.6%, 19.7%, and 13.0%, respectively. Cox multivariate regression analysis showed that residence (HR=2.127, 95% CI: 1.154-3.920), serum albumin (HR=1.583, 95% CI: 1.017-2.464), clinical stage (stage Ⅳ: HR=3.073, 95% CI: 1.366-6.910) and the chemotherapy (HR=0.476, 95% CI: 0.292-0.777) were independent prognostic factors for MPM patients. The C-index of the nomogram established based on the results of Cox multivariate regression analysis in the training and test sets were 0.662 and 0.613, respectively. Calibration curves for both the training and test sets showed moderate consistency between the predicted and actual survival probabilities of MPM patients at 6 months, 1 year, and 2 years. The low-risk group had better outcomes than the high-risk group in both training (P=0.001) and test (P=0.003) sets. Conclusion: The survival prediction nomogram established based on routine clinical indicators of MPM patients provides a reliable tool for prognostic prediction and risk stratification.

[Relationship between health literacy and health-related behaviors of enterprise employees].

Objective: To explore the relationship between the health literacy of employees and smoking, drinking, diet and exercise, and provide scientific basis for health education and targeted intervention. Methods: From February to July 2019, a cluster random sampling method was adopted to select enterprise employees in Shihezi City to conduct a questionnaire survey. Self-made questionnaires and "National Resident Health Literacy Monitoring Questionnaire" were used to assess the social demographics, health literacy, and Health-related behaviors were investigated, and 1053 valid questionnaires were collected. Logistic regression model was used to analyze the relationship between health literacy and the three dimensions with smoking, drinking, diet and exercise. Results: Total score of health literacy of enterprise employees is (42.06±11.03) points and the employee health literacy rate of Shihezi City in 2019 is 19.47%. Univariate analysis showed that non-smokers had higher health literacy and three-dimensional health literacy availability than smokers (P<0.01) ; fewer drinkers had higher health literacy availability than overdrinkers (P<0.05) ; balanced diet health literacy availability The health literacy availability rates in the three dimensions were higher than those in the unbalanced diet (P<0.01) . In the logistic regression analysis, healthy lifestyle and behavioral literacy were independently related to smoking behavior (OR=1.571, P<0.05) ; the presence of health literacy and the three dimensions of health literacy were not statistically related to alcohol consumption (P>0.05) ; there is a statistical correlation between health literacy, healthy lifestyles, behaviors, and health skills and regular exercise among employees (OR=1.829、2.503、1.395, P<0.05) ; employees with health literacy and three dimensions of health literacy are more likely to have a balanced diet (P<0.01) . Conclusion: There is a correlation between the health literacy of enterprise employees and diet and exercise. The improvement of health literacy level is an important way to interfere with unhealthy behaviors.

[Clinical characteristics and prognostic analyses of 87 patients with pulmonary mucoepidermoid carcinoma].

Objective: To investigate the clinicopathological characteristics, therapy and prognosis of patients with pulmonary mucoepidermoid carcinoma. Methods: The clinical data of 87 patients diagnosed as pulmonary mucoepidermoid carcinoma at The First Affiliated Hospital of Zhengzhou University from April 2011 to May 2016 were retrospectively analyzed. The clinicopathological features were summarized and the prognoses were analyzed. Results: Among the 87 patients with lung mucoepidermoid carcinoma, 53 were male and 34 were female, the gender ratio between men and women was 1.56∶1.The ages of patients were from 16 to 79 years and the median age was 52.5 years. Seventeen cases were diagnosed as stage Ⅰ, 28 cases were stage Ⅱ, 26 cases were stage Ⅲ, 16 cases were stage Ⅳ.Thirty-six patients were examined by immunohistochemistry, of which 29 cases were cytokeratin (CK) 5/6 positive, 29 cases were CK7 positive, 10 cases were CK positive, 28 cases were p63 positive, 14 cases were p40 positive, 17 cases were thyroid transcription factor-1 (TTF-1) positive, 11 cases were NapsinA positive, 2 cases were epithelial membrane antigen (EMA) positive, 4 cases were CK8/18 positive, 3 cases were surfactant proteins A (SPA) positive, 1 case was CAM5.2 positive and 1 case was CK14 positive. Among the 87 patients, 34 cases were treated by operation alone, 23 cases were treated by operation combined with chemotherapy, 5 cases were treated by radiotherapy combined with chemotherapy, 14 cases were treated by chemotherapy alone, 2 cases were treated by particle implantation combined with chemotherapy, 2 cases were treated by local radiofrequency hyperthermia combined with chemotherapy, and 7 cases without special treatment.Five patients with brain metastasis were treated with cerebral radiotherapy combined with sequential chemotherapy. The 1-year, 2-year and 5-year survival rates of 87 patients were 90.7%, 81.6% and 46.3%, respectively. The median survival time was 60 months. The prognoses of patients with lung mucoepidermoid carcinoma were related with the clinical stage, smoking and operative therapy (all P<0.05). Conclusions: The age distribution of patients with pulmonary mucoepidermoid is a wide range, the incidence of male is higher than that of female. The diagnosis of pulmonary mucoepidermoid carcinoma mainly relies on the morphological diagnosis and the immunohistochemical detection is non-specific. The prognoses of patients with lung mucoepidermoid carcinoma are related with clinical stage, smoking and operative therapy. For patients who are inoperable and with single distant metastasis, local radiotherapy, other local treatment and chemotherapy can significantly improve their prognoses.

Application of a triblock copolymer additive modified polyvinylidene fluoride membrane for effective oil/water separation.

A hollow fibre membrane was fabricated by blending polyvinylidene fluoride (PVDF) with a triblock copolymer additive polymer that has both hydrophilic and oleophobic surface properties. The novel membrane was characterized and examined for oil/water separation under various system conditions, including different cross-flow rate, feed temperature, trans-membrane pressure, and its rejection and cleaning efficiency, etc. By applying the membrane into the filtration of synthesized oil/water emulsion, the membrane constantly achieved an oil rejection rate of above 99%, with a relatively constant permeate flux varied in the range of 68.9-59.0 l m-2 h-1. More importantly, the fouling of the used membrane can be easily removed by simple water flushing. The membrane also demonstrated a wide adaptability for different types of real oily wastewater, even at very high feed oil concentration (approx. 115 000 mg l-1 in terms of chemical oxygen demand (COM)). Hence, the novel triblock copolymer additive-modified PVDF membrane can have a great prospect in the continuing effort to expand the engineering application of polymeric membranes for oily wastewater treatment.

[Prenatal diagnosis of women with an adverse reproductive history using both traditional karyotyping and SNP-array].

Objective: To explore the occurrence of fetal chromosomal abnormalities among pregnant women with an adverse reproductive history using traditional karyotyping and single nucleotide polymorphism microarray (SNP-array) technology. Methods: Totally 94 in 2 163 (4.35%) cases of singleton pregnant women with an adverse reproductive history were performed amniocentesis in Jinhua Maternal and Child Health Care Hospital from June 2015 to June 2017. Traditional karyotyping and SNP-array were employed simultaneously for prenatal diagnosis, and the detection rates of the two methods were compared. Results: All of the 94 specimens were successfully analyzed, 11 cases were found with chromosomal anomaly, the overall detection rate was 11.7%(11/94). Seven (7.4%,7/94) abnormalities cases were detected by karyotyping, and 7(7.4%) by SNP-array. The karyotyping results of trisomy 21, and 45,X and the deletion of chromosome 13 were consistent with SNP-array. Only 3 (3.2%, 3/94) microdeletion/duplications (the sizes of duplications and deletions were between 422.4-1 708.4 kb) and 1 (1/4) loss of heterozygosity were detected by SNP-array, but were missed by karyotyping. Furthermore, 2 cases' copy number variation were found pathogenic gene related, while the other 2 were considered benign or variant of uncertain significance. Four cases (4/7) of abnormalities were detected by karyotyping, while confirmed balanced translocation and inversion by SNP-array. All patients were informed and chosen to continue the pregnancy. Conclusions: The rate of abnormal fetal chromosomes in pregnant women with an adverse reproductive history is still high. SNP-array is a new molecular genetic technique, and combined with use of traditional karyotyping, it could improve the detection rate of fetal chromosomal abnormalities and reduce abortion rate, thus providing a basis for genetic counseling and prenatal diagnosis.

Aging and contribution of MyD88 and TRIF to expression of TLR pathway-associated genes following stimulation with Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Periodontal disease is a highly complex chronic inflammatory disease of the oral cavity. Multiple factors influence periodontal disease, including socio-economic status, genetics and age; however, inflammation elicited by the presence of specific bacteria in the subgingival space is thought to drive the majority of soft- and hard-tissue destruction. Porphyromonas gingivalis is closely associated with periodontal disease. Toll-like receptors (TLRs) and their intracellular signaling pathways play roles in the host response to P. gingivalis. The focus of the current study was to use microarray analysis to define the contributions of the TLR adaptor molecules myeloid differentiation factor 88 (MyD88) and Toll/interleukin-1 receptor domain-containing adaptor inducing interferon-beta (TRIF), and aging, on the expression of TLR pathway-associated mRNAs in response to P. gingivalis. MATERIAL AND METHODS: Bone marrow-derived macrophages (BMØ) from wild-type (Wt), MyD88 knockout (MyD88-KO) and Trif(Lps2) [i.e. containing a point mutation in the lipopolysaccharide 2 (Lps2) gene rendering the Toll/interleukin (IL)-1 receptor domain-containing adaptor inducing interferon-beta (TRIF) protein nonfunctional] mice, at 2-and 12-mo of age, were cultured with P. gingivalis. Expression of genes in BMØ cultured with P. gingivalis was determined in comparison with expression of genes in BMØ cultured in medium only. RESULTS: Using, as criteria, a twofold increase or decrease in mRNA expression, differential expression of 32 genes was observed when Wt BMØ from 2-mo-old mice were cultured with P. gingivalis compared with the medium-only control. When compared with 2-mo-old Wt mice, 21 and 12 genes were differentially expressed (p < 0.05) as a result of the mutations in MyD88 or TRIF, respectively. The expression of five genes was significantly (p < 0.05) reduced in Wt BMØ from 12-mo-old mice compared with those from 2-mo-old mice following culture with P. gingivalis. Age also influenced the expression of genes in MyD88-KO and Trif(Lps2) mice challenged with P. gingivalis. CONCLUSIONS: Our results indicate that P. gingivalis induces differential expression of TLR pathway-associated genes, and both MyD88 and TRIF play roles in the expression of these genes. Age also played a role in the expression of TLR-associated genes following stimulation of BMØ with P. gingivalis.

Role of the Fyn kinase in calcium release during fertilization of the sea urchin egg.

Protein tyrosine kinase activity has been implicated as part of the signaling mechanism leading to the sperm-induced calcium transient following fertilization. In the present study, we have tested the role of the Fyn kinase in triggering the calcium transient by microinjecting domain-specific fusion proteins encoding regions of Fyn sequence as inhibitors of Fyn function in vivo. A fusion protein encoding the SH2 domain of Fyn caused an increase in the latent period between sperm-egg fusion and the beginning of the calcium transient and reduced the amplitude of the calcium signal. A fusion protein encoding the U + SH3 domains also caused a small increase in the latent period. Microscopic examination revealed that a large percentage of eggs injected with the U+SH3 or SH2 domains became polyspermic as a result of the delayed block to polyspermy. Affinity experiments demonstrated that the U+SH3 and SH2 domains of Fyn were capable of forming a stable complex with phospholipase Cgamma from the sea urchin egg. The results suggest that the Fyn kinase participates in the signaling events leading up to the calcium transient and may directly regulate phospholipase Cgamma activity at fertilization.

A specific inhibitor of p34(cdc2)/cyclin B suppresses fertilization-induced calcium oscillations in mouse eggs.

Fertilization-induced Ca(2+) oscillations in mouse eggs cease at the time of pronuclear formation when maturation-promoting factor (MPF) is inactivated, but the Ca(2+) oscillations are ceaseless if eggs are arrested at metaphase by colcemid, which maintains the activity of MPF. To determine the possible role of MPF in regulation of cytoplasmic Ca(2+) excitability, roscovitine, a specific inhibitor of p34(cdc2)/cyclin B kinase, was used to inactivate MPF, and its effect on fertilization-induced Ca(2+) oscillations was investigated. Our results showed that roscovitine at >/= 50 microM suppressed fertilization-induced Ca(2+) oscillations in normal and colcemid-treated metaphase II (MII) eggs after the first 1-2 Ca(2+) spikes. Roscovitine inhibition of fertilization-induced Ca(2+) oscillations could be reversed by extensive washing of the eggs. Histone H1 kinase activity in colcemid-treated MII eggs was similarly inhibited by roscovitine, which suggested that the cessation of fertilization-induced Ca(2+) oscillations is due to the inactivation of MPF. Thimerosal-induced Ca(2+) oscillations in Ca(2+)-, Mg(2+)-free medium was also suppressed by roscovitine, suggesting a general inhibitory effect of roscovitine on Ca(2+) oscillations. The inhibition may be achieved by disruption of Ca(2+) release and refilling of the calcium store. Thapsigargin, an inhibitor of the endoplasmic reticulum Ca-ATPase, induced significantly less Ca(2+) release in roscovitine-treated eggs than in the non-drug-treated eggs. Taken together, our results suggest that MPF plays an important role in regulation of the cytoplasmic Ca(2+) excitability in mouse eggs.

Protein tyrosine kinase-dependent release of intracellular calcium in the sea urchin egg.

The aminoguanide, methylglyoxal bis(guanylhydrazone) (MGBG), was shown to stimulate phosphorylation of RR-SRC, a synthetic protein tyrosine kinase (PTK) substrate, and different levels of tyrosyl phosphorylation of endogenous proteins in a sea urchin egg membrane-cortex preparation. Stimulating protein tyrosine kinase activity in the sea urchin egg stimulated intracellular Ca2+ release, because microinjection of 1-5 mM of MGBG into unfertilized eggs triggered a transient rise in intracellular Ca2+ activity ([Ca2+]i) after a brief latent period. Pretreating eggs with PTK-specific inhibitors, genistein or tyrphostin B42, significantly inhibited the MGBG-induced rise in [Ca2+]i. Methylglyoxal bis(guanylhydrazone) stimulation of PTK activities in the unfertilized sea urchin egg appeared to trigger Ca2+ release through phospholipase C (PLC)-dependent inositol 1,4,5-trisphosphate (InsP3) production. The MGBG-induced Ca2+ response could be suppressed in eggs preloaded with the InsP3 receptor antagonist, heparin, and was reduced in eggs pretreated with U73122, a PLC inhibitor. However, the response was unchanged in eggs treated with nicotinamide, an inhibitor of ADP-ribosyl cyclase, or nifedipine, an inhibitor of nicotinic acid adenine dinucleotide phosphate activity. These results suggest that MGBG may be useful as a chemical agonist of PTK in sea urchin eggs and allow direct testing of the PTK requirement for the transient rise in [Ca2+]i in sea urchin eggs during fertilization. Although genistein was observed to significantly delay the onset, the sperm-induced Ca2+ response in PTK inhibitor-loaded eggs otherwise appeared normal. Therefore, it was concluded that sea urchin eggs contain a PTK-dependent pathway that can mediate intracellular Ca2+ release, but PTK activity does not appear to be required for the fertilization response.

U73122 blocked the cGMP-induced calcium release in sea urchin eggs.

U73122, a phospholipase C inhibitor, dose dependently blocks the cGMP-induced Ca2+ release in sea urchin eggs and homogenates. U73122 inhibition was prevented by cotreatment with dithiothreitol (DTT), but DTT is ineffective when eggs or homogenates were pretreated with U73122. U73122 action is different from the other sulfhydryl reagents, thimerosal and N-ethylmaleimide, which cause Ca2+ release in egg homogenates at high concentration, but at lower concentration have no significant effect on cGMP-induced Ca2+ release. Histone, a reported NAD glycohydrolase (NADase) activator, was found to induce Ca2+ release in egg homogenates via the same pathway as the cGMP response, since histone-induced Ca2+ release is blocked by Rp-8-pCPT-cGMPS, a cGMP-dependent protein kinase (PKG) inhibitor, and nicotinamide, a NADase inhibitor. Histone-induced Ca2+ release is similarly blocked by U73122. The aminosteroid U73122 does not inhibit cADPR-induced Ca2+ release, which is significantly reduced by PKG inhibitors. Furthermore, U73122 has no significant effect on phorbol 12-myristate 13-acetate induced-cytoplasmic alkalinization in intact eggs, which depends on protein kinase C activity. These results suggest that U73122 does not act as a general serine-threonine protein kinase inhibitor, and the aminosteroid inhibition of the cGMP-induced Ca2+ release may interfere with ADP ribosyl cyclase activity.

The calcium transient in sea urchin eggs during fertilization requires the production of inositol 1,4,5-trisphosphate.

The production of inositol 1,4,5-trisphosphate (InsP3) has been reported to mediate the transient rise in intracellular Ca2+ activity ([Ca2+]i) in sea urchin eggs during fertilization. However, direct evidence of an absolute requirement for generation of InsP3 during fertilization is still lacking. We investigated this question by blocking the InsP3 synthesizing enzyme phospholipase C (PLC) during fertilization with U73122, an aminosteroid. U73122 inhibited the sperm-induced Ca2+ release in a dose-dependent manner, although above 15 microM U73122 eggs showed an elevated resting [Ca2+]i and a lower fertilization rate. The inhibition of Ca2+ transient by U73122 was not due to a failure of fertilization, since incorporated sperm nuclei were evident in eggs used to measure the Ca2+ response. U73122 also prevented the accompanying rise in intracellular pH (pHi), which is mediated by the activation of the Na+-H+ antiporter. The antiporter is regulated through activation of protein kinase C by 1,2-diacylglycerol, which is the other hydrolytic product of phosphatidylinositol 4,5-bisphosphate by PLC. Further evidence of the specificity of U73122 action was inhibition of the increase in InsP3 mass during the first 2 min of fertilization. In addition, U73122 inhibited the GTPgammaS-induced Ca2+ release and pHi rise in unfertilized eggs. These results suggested that the transient rise in Ca2+ in sea urchin during fertilization requires the production of InsP3.

The cyclic GMP-mediated calcium release pathway in sea urchin eggs is not required for the rise in calcium during fertilization.

The mechanisms required for cGMP-induced Ca2+ release in the sea urchin egg were investigated using both egg homogenates and intact eggs. The postulated pathway of cGMP-dependent protein kinase (PKG) activation of ADP-ribosyl cyclase for production of cADPR to activate the ryanodine receptor Ca2+ channel was tested with a variety of activators (cGMP analogs and cIMP) and inhibitors (Rp-8-pCPT-cGMPS, 3-aminopyridine NAD, nicotinamide, and spermine). Our observations are consistent with Ca2+ release by cGMP in the egg being dependent on an isoform of PKG that is distinct from the mammalian enzyme. PKG activity in the sea urchin egg was activated by cIMP, but was insensitive to cGMP analogs, which are potent activators of mammalian isoenzymes. Surprisingly, it appears the activation of the cGMP-dependent Ca2+ release pathway was unnecessary during fertilization. Inhibitors of either PKG or ADP-ribosyl cyclase activities did not prevent the transient rise in intracellular Ca2+ activity in heparin-loaded eggs during fertilization. These results suggest the synthesis of cADPR during fertilization is not necessary for regulating the Ca2+ event.

Validity of force platform measures for stance stability under varying sensory conditions.

Maintaining stance stability under varying sensory environment is an essential function in the elderly and among patients. Testing sensory organization ability of standing balance, the Sensory Organization Test (SOT), has become a standard procedure in many clinical and laboratory settings. The stance stability can be quantified by two forceplate measures in the SOT: the equilibrium score (ES) and the sway area (SA). This study compares the validity of the ES and the SA in detecting gender, trial, and sensory effects on stability in twenty (ten male, ten female) healthy young adults. Subjects were tested under six sensory conditions: eyes open (EO), eyes closed (EC), sway-referenced vision (Vs), sway-referenced support surface (Ss), eyes closed sway-referenced support surface (ECSs), and sway-referenced visual surround and support surface (VsSs). A visual surround and/or the support surface were tilted proportionately to the subject's spontaneous sway in the sway-referenced conditions. Three trials, 20-second for each trial, were repeated for each sensory condition. Above results demonstrated that the Pearson correlation coefficients between the ES and the SA were all highly significant (p < .0001) except for the first trial of the EO condition. The consistencies in which the two measures discriminated among sensory conditions were tested by the Kendall's coefficient of concordance. The Kendall's coefficient for the ES (W = .843) and the SA (W = .866) were high and similar. Separate ANOVA procedure for the ES and SA revealed that both measures satisfactorily detected a significant sensory condition and trial effects and insignificant gender effect. We can conclude that ES and SA are valid measures of stance stability during the SOT. Our results confirm that healthy young adults have a poorer postural stability when the visual and somatosensory inputs are simultaneously altered. Moreover, the learning effect is observed during repeated trials within test conditions.

Ca2+ release triggered by nicotinate adenine dinucleotide phosphate in intact sea urchin eggs.

Nicotinate adenine dinucleotide phosphate (NAADP) was recently identified [Lee and Aarhus (1995) J. Biol. Chem. 270, 2152-2157; Chini, Beers and Dousa (1995) J. Biol. Chem. 270, 3116-3223] as a potent Ca(2+)-releasing agent in sea urchin egg homogenates. NAADP triggered Ca2+ release by a mechanism that was distinct from inositol 1,4,5-trisphosphate (InsP3)- and cyclic ADP-ribose (cADPR)-induced Ca2+ release. When NAADP was microinjected into intact sea urchin eggs it induced a dose-dependent increase in cytoplasmic free Ca2+ which was independent of the extracellular [Ca2+]. The Ca2+ waves elicited by microinjections of NAADP originated at the site of injection and swept across the cytosol. As previously found in sea urchin egg homogenates, NAADP-induced Ca2+ release in intact eggs was not blocked by heparin or by prior desensitization to InsP3 or cADPR. Thio-NADP, a specific inhibitor of the NAADP-induced Ca2+ release in sea urchin homogenates [Chini, Beers and Dousa (1995) J. Biol. Chem. 270, 3116-3223] blocked NAADP (but not InsP3 or cADPR) injection-induced Ca2+ release in intact sea urchin eggs. Finally, fertilization of sea urchin eggs abrogated subsequent NAADP-induced Ca2+ release, suggesting that the NAADP-sensitive Ca2+ pool may participate in the fertilization response. This study demonstrates that NAADP acts as a selective Ca(2+)-releasing agonist in intact cells.

Thiophosphorylated RCM-lysozyme, an active site-directed protein tyrosine phosphatase inhibitor, inhibits G2/M transition during mitotic cell cycle and uncouples MPF activation from G2/M transition.

Microinjection of thiophosphotyrosylated RCM-lysozyme (TRCML), a potent and specific inhibitor of protein tyrosine phosphatases (PTPs) into sea urchin (Lytechinus pictus) eggs prior to fertilization inhibited the first zygotic cell division in a concentration-dependent fashion. Microinjection of TRCML at varying times after fertilization indicated that at least one site of action is late in the first cell cycle near the G2/M boundary. In order to further study the mechanism for the TRCML effect, a cell-free cell cycling system prepared from electrically activated Xenopus eggs was used. The addition of TRCML to cycling extracts delayed the entrance and progression of extracts through mitosis, as indicated by the inhibition of chromatin condensation, nuclear envelope breakdown, M-phase-promoting factor (MPF) inactivation, and cyclin degradation. Surprisingly, TRCML did not inhibit MPF activation. These results suggest that (1) the target(s) of TRCML lies in late G2- or early M-phase before the onset of metaphase, (2) TRCML uncouples MPF activation from progression through M-phase, and (3) there is a potential involvement of a novel PTP(s) in the control of the cell cycle which may act either downstream of the MPF activation or alternatively in an additional but essential mitotic pathway that is parallel to the MPF activation pathway.

Synergistic calcium release in the sea urchin egg by ryanodine and cyclic ADP ribose.

A transient rise in cytoplasmic Ca2+ activity in the sea urchin egg occurs during fertilization due to release from an intracellular store. Two intracellular receptor Ca2+ channels for inositol 1,4,5-trisphosphate (IP3) and ryanodine have been identified by physiological and immunological techniques. While IP3 is the endogenous messenger for the IP3 receptor, a corresponding physiological messenger for the ryanodine receptor is unknown. A variety of recent experimental evidences suggest that cyclic ADP ribose (cADPR) may be a possible candidate. In this study using both egg homogenates and intact eggs, we show that subthreshold concentrations of cADPR and ryanodine can act synergistically to potentiate Ca2+ release. Addition of 10-20 nM cADPR, which causes little net increase in Ca2+, generally enhances the action of subthreshold concentrations of ryanodine. Similarly the addition of 60-80 microM ryanodine causes a slight transient increase but potentiates maximal Ca2+ increase by a subsequent subthreshold addition of cADPR. While the target of Ca2+ release by ryanodine and cADPR may be the ryanodine receptor, their actions appear to be different and more complex than simply opening the release mechanism. There are significant differences in the kinetics of release by the two agonists. In addition we used a poorly metabolized analog of IP3 and an inhibitor of endoplasmic reticulum Ca2+ ATPase activity, to show that the unfertilized egg contains a rapidly filled Ca2+ store, which is commonly released by both IP3-mediated and ryanodine-mediated release mechanisms.

Sources of calcium in sea urchin eggs during the fertilization response.

Shortly after sperm-egg interaction the sea urchin egg is traversed by a Ca2+ wave, which is necessary for metabolic activation of the quiescent cell. Several sources including influx across the egg plasma membrane and release from intracellular stores may contribute to the rise in cytoplasmic Ca2+ activity. Inositol 1,4,5-trisphosphate (InsP3), cyclic ADP ribose (cADPR), and ryanodine have been reported to induce intracellular Ca2+ release. We used confocal laser scanning microscopy to image the Ca2+ transient during fertilization and parthenogenetic activation by microinjection of Ca2+ release agonists. A near instantaneous rise in Ca2+ localized to the egg cortex occurred near the time of sperm-egg binding, followed by a distinctive delay before the onset of the Ca2+ wave. Since the rise in cortical Ca2+ activity was absent when Ca2+ influx was prevented, it appeared that this change in Ca2+ activity was due to the opening of membrane Ca2+ channels. Blocking the influx did not alter the onset of the Ca2+ wave. The Ca2+ wave during the fertilization response seemed to require Ca2+ release mediated by InsP3-, cADPR-, and ryanodine-sensitive mechanisms. Parthenogenetic activation by microinjection of these three agents had different spatiotemporal patterns of Ca2+ release. Most significantly the injection of either InsP3 or cADPR, but not ryanodine, induced an enhanced pronucleus-associated Ca2+ release, which was similar to the Ca2+ response during fertilization.