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Introduction: Fetal heart rate monitoring during labor can aid healthcare professionals in identifying alterations in the heart rate pattern. However, discrepancies in guidelines and obstetrician expertise present challenges in interpreting fetal heart rate, including failure to acknowledge findings or misinterpretation. Artificial intelligence has the potential to support obstetricians in diagnosing abnormal fetal heart rates., Methods: Employ preprocessing techniques to mitigate the effects of missing signals and artifacts on the model, utilize data augmentation methods to address data imbalance. Introduce a multi-scale long short-term memory neural network trained with a variety of time-scale data for automatically classifying fetal heart rate. Carried out experimental on both single and multi-scale models., Results: The results indicate that multi-scale LSTM models outperform regular LSTM models in various performance metrics. Specifically, in the single models tested, the model with a sampling rate of 10 exhibited the highest classification accuracy. The model achieves an accuracy of 85.73%, a specificity of 85.32%, and a precision of 85.53% on CTU-UHB dataset. Furthermore, the area under the receiver operating curve of 0.918 suggests that our model demonstrates a high level of credibility., Discussion: Compared to previous research, our methodology exhibits superior performance across various evaluation metrics. By incorporating alternative sampling rates into the model, we observed improvements in all performance indicators, including ACC (85.73% vs. 83.28%), SP (85.32% vs. 82.47%), PR (85.53% vs. 82.84%), recall (86.13% vs. 84.09%), F1-score (85.79% vs. 83.42%), and AUC(0.9180 vs. 0.8667). The limitations of this research include the limited consideration of pregnant women's clinical characteristics and disregard the potential impact of varying gestational weeks., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Rao, Lu, Wu, Zhao, Lu and Li.)

Chemical constituents from the ethanol extract of Picrorhiza scrophulariiflora were isolated and purified by column chromatography. Their structures were identified by HR-MS, 1D and 2D-NMR, and their cytotoxicity was assessed by CCK-8 assay. Four compounds were isolated and identified as follows: 2β-D-glucosyloxy-3β,16α,20β-trihydroxy-9-methyl-19-norlanosterol-5,25-diene-22-one(1), 2β-D-glucosyloxy-3β,16α,20β-trihydroxy-9-methyl-19-norlanosta-5,24-diene-22-one(2), 25-acetoxy-2β-glucosyloxy-3β,16α,20β-trihydroxy-9-methyl-19-norlanosta-5-ene-22-one(3) and 25-acetoxy-2β-glucosyloxy-3β,16α,20β-trihydroxy-9-methyl-19-norlanosta-5,23-(E)-diene-22-one(4). Compound 1 represents a new cucurbitane glycoside. The half inhibitory concentrations of the 4 compounds exceeded 100 μmol·L~(-1) against four tumor cell lines, indicating no significant cytotoxicity.

Background: COVID-19 has caused more than 2.6 billion infections and several million deaths since its outbreak 2 years ago. We know very little about the long-term cellular immune responses and the kinetics of neutralizing antibodies (NAbs) to SARS-CoV-2 because it has emerged only recently in the human population., Methods: We collected blood samples from individuals who were from the first wave of the COVID-19 epidemic in Wuhan between December 30, 2019, and February 24, 2020. We analyzed NAbs to SARS-CoV-2 using pseudoviruses and IgG antibodies to SARS-CoV-2 spike (S) and nucleocapsid (N) protein using enzyme-linked immunosorbent assay in patients' sera and determined SARS-CoV-2-specific T-cell responses of patients with ELISpot assays., Results: We found that 91.9% (57/62) and 88.9% (40/45) of COVID-19 patients had NAbs against SARS-CoV-2 in a year (10-11 months) and one and a half years (17-18 months), respectively, after the onset of illness, indicating that NAbs against SARS-CoV-2 waned slowly and possibly persisted over a long period time. Over 80% of patients had IgG antibodies to SARS-CoV-2 S and N protein one and a half years after illness onset. Most patients also had robust memory T-cell responses against SARS-CoV-2 one and a half years after the illness. Among the patients, 95.6% (43/45) had an IFN-γ-secreting T-cell response and 93.8% (15/16) had an IL-2-secreting T-cell response. The T-cell responses to SARS-CoV-2 were positively correlated with antibodies (including neutralizing antibodies and IgG antibodies to S and N protein) in COVID-19 patients. Eighty percent (4/5) of neutralizing antibody-negative patients also had SARS-CoV-2-specific T-cell response. After long-term infection, protective immunity was independent of disease severity, sex, and age., Conclusions: We concluded that SARS-CoV-2 infection elicited a robust and persistent neutralizing antibody and memory T-cell response in COVID-19 patients, indicating that these sustained immune responses, among most SARS-CoV-2-infected people, may play a crucial role in protection against reinfection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yan, Liu, Li, Zhou, Li, Wang, Shen, Lu, Long, Xiao, Wang, Li, Han, Yu, Zhou, Lv and Yu.)

Background: Neoadjuvant therapy is associated with nodal downstaging and improved oncological outcomes in patients with lymph node (LN)-positive pancreatic cancer. This study aimed to develop and validate a nomogram to preoperatively predict LN-positive disease., Methods: A total of 558 patients with resected pancreatic cancer were randomly and equally divided into development and internal validation cohorts. Multivariate logistic regression analysis was used to construct the nomogram. Model performance was evaluated by discrimination, calibration, and clinical usefulness. An independent multicenter cohort consisting of 250 patients was used for external validation., Results: A four-marker signature was built consisting of carbohydrate antigen 19-9 (CA19-9), CA125, CA50, and CA242. A nomogram was constructed to predict LN metastasis using three predictors identified by multivariate analysis: risk score of the four-marker signature, computed tomography-reported LN status, and clinical tumor stage. The prediction model exhibited good discrimination ability, with C-indexes of 0.806, 0.742 and 0.763 for the development, internal validation, and external validation cohorts, respectively. The model also showed good calibration and clinical usefulness. A cut-off value (0.72) for the probability of LN metastasis was determined to separate low-risk and high-risk patients. Kaplan-Meier survival analysis revealed a good agreement of the survival curves between the nomogram-predicted status and the true LN status., Conclusions: This nomogram enables the identification of pancreatic cancer patients at high risk for LN positivity who may have more advanced disease and thus could potentially benefit from neoadjuvant therapy., (Copyright © 2021 First Affiliated Hospital, Zhejiang University School of Medicine in China. Published by Elsevier B.V. All rights reserved.)

Objective: To evaluate the accuracy of the automated oscillometric upper-arm blood pressure (BP) monitor Globalcare GCE603 for home BP monitoring according to the International Protocol of the European Society of Hypertension (ESH) revision 2010., Methods: BP was sequentially measured in 33 adult participants and compared with a standard mercury sphygmomanometer. A total of 99 comparison pairs were obtained and analyzed according to the ESH international protocol., Results: For the 33 eligible participants, the device achieved 80/99, 98/99 and 99/99 measurements of absolute differences between device and observers within 5, 10 and 15 mmHg for systolic BP (SBP) and 87/99, 98/99 and 99/99 for diastolic BP (DBP), respectively. The average device-observer difference was 1.01 ± 4.11 mmHg for SBP and -0.69 ± 3.56 mmHg for DBP, respectively. The number of participants with two or three of the device-observer difference within 5 mmHg was 28 for SBP and 30 for DBP, and there was no subject with none of the device-observer difference within 5 mmHg., Conclusion: According to the validation results on the basis of the ESH international protocol revision 2010, the Globalcare GCE603 automated upper-arm BP monitor can be recommended for home measurement in general population.

Background: To explore the association of platelet activation markers, vitamin D, and antiplatelet drugs resistance in ischemic stroke patients., Methods: A total of 230 patients with ischemic stroke were enrolled in this study. Platelet aggregation, platelet activation marker (CD62p), and vitamin D were measured after 7-14 days of dual antiplatelet treatment (aspirin + clopidogrel). All individuals were divided into a drug resistance group and a drug sensitive group according to the platelet maximum aggregation rate induced by antagonist adenosine diphosphate or arachidonic acid., Results: In this study, the prevalence of aspirin resistance was low (1.2%), while the prevalence of clopidogrel resistance (CR) was 24.8%, so we focused on CR. The percentage of CD62p on activated platelet [(25.74 ± 4.61) versus (12.41 ± 3.93), P < .001] and the prevalence of hypertension [93.0% (53) versus 79.8% (138), P = .021] in CR group were significantly higher than those in clopidogrel sensitive (CS) group, while the vitamin D concentration [(8.96 ± 4.41) versus (13.9 ± 4.84) ng/mL, P = .003] in CR group was significantly lower compared with the CS group. No significant difference was found in soluble P-selectin between these 2 groups [(56.2 ± 16.13) versus (54.2 ± 14.87) ng/mL, P = .258], neither in calcium [(2.29 ± .12) versus (2.33 ± .13) mmol/L, P = .821]. Logistic regression analysis showed that hypertension (odds ratio [OR] = 5.348, 95% confidence intervals [CI] 1.184-23.350, P = .026), expression of platelet CD62p (OR = 1.095, 95% CI 1.052-1.201, P = .018) and vitamin D level (OR = .832, 95% CI .763-.934, P = .005) were associated with CR in ischemic stroke patients., Conclusions: CR in ischemic stroke patients is associated with several independent predictors, including increased platelet activation marker CD62p, decreased vitamin D level, and hypertension., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: Nasopharyngeal carcinoma is the most common head and neck tumor in Southern China and Southeast Asia, presenting high rates of local invasion and early distant metastasis. Abnormally expressed miR-99a has been discovered in many tumors, and it is involved in nasopharyngeal carcinoma as well. This study aims to explore the molecular mechanisms of miR-99a and mTOR in regulating nasopharyngeal carcinoma., Patients and Methods: MiR-99a expression in nasopharyngeal carcinoma cells was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was performed for accessing cell proliferative capacity. Dual-Luciferase reporter gene assay was employed to verify the combination between miR-99a and mTOR., Results: We found that miR-99a was downregulated while mTOR was upregulated in nasopharyngeal carcinoma cell lines CNE1 and SUNE1. Low expression of miR-99a or high expression of mTOR predicted poor prognosis of nasopharyngeal carcinoma. MiR-99a overexpression inhibited the proliferation of CNE1 and SUNE1 cells through targeting mTOR., Conclusions: We provided evidence that miR-99a inhibits NPC cell proliferative ability by inhibiting mTOR. The newly identified miR-99a/mTOR axis provides novel insight into the pathogenesis of NPC and represents a potential therapeutic target for NPC.

Nuclear hormone receptors (NRs), such as retinoic acid receptors (RARs), play critical roles in vertebrate development and homeostasis by regulating target gene transcription. Their activity is controlled by ligand-dependent release of corepressors and subsequent recruitment of coactivators, but how these individual receptor modes contribute to development are unknown. Here, we show that mice carrying targeted knockin mutations in the corepressor Silencing Mediator of Retinoid and Thyroid hormone receptor (SMRT) that specifically disable SMRT function in NR signaling (SMRT mRID ), display defects in cranial neural crest cell-derived structures and posterior homeotic transformations of axial vertebrae. SMRT mRID embryos show enhanced transcription of RAR targets including Hox loci, resulting in respecification of vertebral identities. Up-regulated histone acetylation and decreased H3K27 methylation are evident in the Hox loci whose somitic expression boundaries are rostrally shifted. Furthermore, enhanced recruitment of super elongation complex is evident in rapidly induced non-Pol II-paused targets in SMRT mRID embryonic stem cells. These results demonstrate that SMRT-dependent repression of RAR is critical to establish and maintain the somitic Hox code and segmental identity during fetal development via epigenetic marking of target loci., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Aim: To establish a severe acute cholangitis (SAC) model in mice., Methods: Cholecystic catheterization was performed under the condition of bile duct ligation (BDL). Trans-cholecystic injection of lipopolysaccharide (LPS) was defined as the SAC animal model. Sham operation group, intraperitoneal injection of LPS without BDL group, intraperitoneal injection of LPS with BDL group and trans-cholecystic injection of normal saline with BDL group were defined as control groups. The survival rates and tissue injuries in liver, lungs and kidney were evaluated., Results: Mice in the SAC group showed a time-dependent mortality and much more severe tissue injuries in liver, lungs and kidney, compared with other groups. However, relieving biliary obstruction could effectively reduce mortality and attenuate liver injury in the SAC mouse model., Conclusion: Trans-cholecystic injection of LPS under the condition of biliary obstruction could establish a repeatable and reversible mouse model of SAC., Competing Interests: Conflict-of-interest statement: The authors declare that there are no other conflicts of interest.

Enhanced expression of the ATP-binding cassette (ABC) transporter protein ABC sub-family G member 2 (ABCG2) in cancer stem cells (CSCs) plays a major role in chemotherapeutic drug efflux, which results in therapy failure and tumor relapse. In addition to downregulating apoptosis in CSCs, it has been reported that the transcriptional upregulation of the redox sensing factor Nrf2 is involved in the upregulation of ABCG2 expression and consequent chemoresistance. The current study investigated the presence of cancer stem-like side population (SP) cells from head and neck squamous cell carcinoma (HNSCC) samples, and evaluated the Nrf2 expression profile and multidrug resistance properties of HNSCC stem cells. Fluorescence-activated cell sorting was used for SP cells detection, while reverse transcription-polymerase chain reaction was used for the analysis of Nrf2 expression. The present study identified ~2.1% SP cells present in HNSCC specimens, which were positive for cluster of differentiation (CD)133 expression and displayed significantly elevated messenger RNA expression of Nrf2, compared with non-SP cells. These data suggest that the ABC transporter ABCG2 is highly upregulated in SP cells, and this results in multidrug resistance. In addition, these CD133 + cells underwent rapid proliferation and exhibited high self-renewal and tumorigenic properties. Taken together, the present findings suggest that elevated expression of Nrf2 mediated drug resistance in HNSCC CSCs, which may be one of the causative factors for cancer treatment failure. Therefore, novel anti-cancer drugs that downregulate the Nrf2 signaling pathway could effectively improve the treatment and survival rate of patients with HNSCC.

Aim: To investigate the feasibility of a dual-input two-compartment tracer kinetic model for evaluating tumorous microvascular properties in advanced hepatocellular carcinoma (HCC)., Methods: From January 2014 to April 2015, we prospectively measured and analyzed pharmacokinetic parameters [transfer constant (Ktrans), plasma flow (Fp), permeability surface area product (PS), efflux rate constant (kep), extravascular extracellular space volume ratio (ve), blood plasma volume ratio (vp), and hepatic perfusion index (HPI)] using dual-input two-compartment tracer kinetic models [a dual-input extended Tofts model and a dual-input 2-compartment exchange model (2CXM)] in 28 consecutive HCC patients. A well-known consensus that HCC is a hypervascular tumor supplied by the hepatic artery and the portal vein was used as a reference standard. A paired Student's t-test and a nonparametric paired Wilcoxon rank sum test were used to compare the equivalent pharmacokinetic parameters derived from the two models, and Pearson correlation analysis was also applied to observe the correlations among all equivalent parameters. The tumor size and pharmacokinetic parameters were tested by Pearson correlation analysis, while correlations among stage, tumor size and all pharmacokinetic parameters were assessed by Spearman correlation analysis., Results: The Fp value was greater than the PS value (FP = 1.07 mL/mL per minute, PS = 0.19 mL/mL per minute) in the dual-input 2CXM; HPI was 0.66 and 0.63 in the dual-input extended Tofts model and the dual-input 2CXM, respectively. There were no significant differences in the kep, vp, or HPI between the dual-input extended Tofts model and the dual-input 2CXM (P = 0.524, 0.569, and 0.622, respectively). All equivalent pharmacokinetic parameters, except for ve, were correlated in the two dual-input two-compartment pharmacokinetic models; both Fp and PS in the dual-input 2CXM were correlated with Ktrans derived from the dual-input extended Tofts model (P = 0.002, r = 0.566; P = 0.002, r = 0.570); kep, vp, and HPI between the two kinetic models were positively correlated (P = 0.001, r = 0.594; P = 0.0001, r = 0.686; P = 0.04, r = 0.391, respectively). In the dual input extended Tofts model, ve was significantly less than that in the dual input 2CXM (P = 0.004), and no significant correlation was seen between the two tracer kinetic models (P = 0.156, r = 0.276). Neither tumor size nor tumor stage was significantly correlated with any of the pharmacokinetic parameters obtained from the two models (P > 0.05)., Conclusion: A dual-input two-compartment pharmacokinetic model (a dual-input extended Tofts model and a dual-input 2CXM) can be used in assessing the microvascular physiopathological properties before the treatment of advanced HCC. The dual-input extended Tofts model may be more stable in measuring the ve; however, the dual-input 2CXM may be more detailed and accurate in measuring microvascular permeability.

Cases of gastric fistula secondary to drainage tube penetration have rarely been reported. The current study presents a case of gastric penetration caused by misplacement of a drainage tube after a splenectomy. The patient was admitted to the Department of Hepatobiliary Surgery, (Shaoxing People's Hospital, Shaoxing Hospital of Zhejiang University, Shaoxing, Zhejiang, China) for blunt abdominal trauma due to injuries sustained in an automobile accident. A ruptured spleen was found and successfully removed surgically. On post-operative day 7, the patient complained of slight discomfort and tenderness in the left upper quadrant of the abdomen. In addition, 500 ml of bile-colored fluid with small food particles was noted in the drainage tube. Barium X-ray revealed a gastric fistula in the upper gastrointestinal tract. Gastroscopy indicated infiltration of the drainage tube into the gastric cavity. No significant peritoneal effusion was observed, as revealed by abdominal ultrasound examination. These results confirmed the diagnosis of a gastric fistula secondary to perforation by the drainage tube. Following conservative treatment with antibiotics and total parenteral nutrition, the general condition of the patient improved significantly. The drainage tube was withdrawn progressively, as the amount of fluid being discharged was decreasing. Gastroenterography confirmed perforation closure and the tube was finally removed on post-operative day 44.

Objective: A large body of evidence suggests that stroke and depression are accompanied by activation of inflammatory pathways. Thus, the primary purpose of this study was to assess the high-sensitivity C-reactive protein (Hs-CRP) to the presence of post stroke depression (PSD)., Methods: Two hundred and twenty-six ischemic stroke patients admitted to the hospital within the first 24 hours after stroke onset were consecutively recruited and followed up for 6 months. Clinical information was collected. Serum Hs-CRP levels were measured at baseline. Based on the symptoms, diagnoses of depression were made in accordance with DSM-IV criteria for depression at 6-month after stroke., Results: At 6-month, ninety-five patients (42.0%) showed depression at 6 months after admission and in 69 patients (30.5%) this depression was classified as major. In the 69 patients with major depression, our results showed significantly higher Hs-CRP levels (1.54[IQR, 0.79-2.27]mg/dL vs. 0.43[IQR, 0.31-1.27]mg/dL, P<0.0001) at admission than patients without major depression. After adjusting for NIHSS on admission and all other recorded confounders, Hs-CRP still was an independent predicator of PSD with an adjusted OR of 1.339 (95% CI, 1.231-1.456; P<0.001). Further, in our study, we found that an increased risk of PSD was associated with serum Hs-CRP levels ≥0.85mg/dL (adjusted OR 7.830, 95% CI: 4.193-14.620) after adjusting for above recorded confounders., Conclusion: Elevated Hs-CRP serum levels at admission was found to be associated with depression 6-month after stroke, suggesting that these alterations might participate in the pathophysiology of depression symptoms in stroke patients., (Copyright © 2015 John Wiley & Sons, Ltd.)

Objective: Accumulating evidence has shown that microRNAs (miRNAs) are aberrantly expressd in many malignancies and crucial to tumorigenesis. Herein, we identified the role and mechanism of miR-206 in laryngeal squamous cell carcinoma (LSCC) growth., Patients and Methods: Quantitative real-time PCR was performed to detect the relative expression level of miR-206 in LSCC tissues. Crystal violet and flow cytometry were conducted to explore the effects of miR-206 on the proliferation and cell cycle of human LSCC cell line, respectively. The impact of miR-206 overexpression on putative target cyclinD2 were subsequently verified via Western blot. Tumor growth assay was performed to testify the effect of miR-206 on the tumor growth in vivo., Results: MiR-206 expression was frequently (p < 0.05) down-regulated in LSCC specimens. Overexpression of miR-206 in Hep-2 cell inhibited the proliferation by blocking the G1/S transition as well as suppressed the growth of xenograft tumors in mice, implying that miR-206 functions as a tumour suppressor in the progression of LSCC. Overexpression of miR-206 significantly decreased (p < 0.05) the protein level of cyclinD2, which has previously been identified as a direct targets of miR-206., Conclusions: Altogether, our results identify a crucial tumour suppressive role of miR-206 in LSCC growth, at least partly via up-regulation of cyclinD2 protein levels, and suggest that miR-206 might be a candidate prognostic predictor or an anticancer therapeutic target for LSCC patients.

Adenosquamous carcinoma rarely occurs in the pancreas, and is characterized by the presence of cellular components from both duct adenocarcinoma and squamous carcinoma. Here, we describe a rare case of pancreatic adenosquamous carcinoma with sarcomatous change. Immunohistochemistry showed that the sarcomatous lesion lost the epithelial marker and aberrantly expressed of acquired mesenchymal markers, which indicated that this special histological phenotype may be attributed to epithelial-mesenchymal transition. This case also indicated that a routine radical surgery without aggressive treatment strategies was still appropriate for adenosquamous carcinoma of the pancreas with sarcomatoid change.

Bile duct injuries (BDIs) are difficult to avoid absolutely when the biliary tract has a malformation, such as accessory hepatic duct. Here, we investigated the management strategies for BDI combined with accessory hepatic duct during laparoscopic cholecystectomy.

Growth and carcass traits are economically important quality characteristics of beef cattle and are complex quantitative traits that are controlled by multiple genes. In this study, 2 candidate genes, H-FABP (encoding the heart fatty acid-binding protein) and PSMC1 (encoding the proteasome 26S subunit of ATPase 1) were investigated in Qinchuan beef cattle of China. PCR-SSCP and DNA sequencing methods were used to detect mutations in the H-FABP and PSMC1 genes in Qinchuan cattle, and a T>C mutation in exon 1 of H-FABP and a T>C mutation in exon 9 of PSMC1 were identified. The association of these 2 single nucleotide polymorphisms with growth and carcass traits of Qinchuan cattle was analyzed. The T>C mutation in H-FABP was significantly associated with body length and dressing percentage (P < 0.05) and the T>C mutation in PSMC1 with body length and hip width (P < 0.05), indicating that both of the 2 mutations in H-FABP and PSMC1 had effects on growth and carcass traits in the Qinchuan beef cattle breed. Thus, the results of our study suggest that the H-FABP and PSMC1 gene polymorphisms could be used as genetic markers in marker-assisted selection for improving Qinchuan beef cattle.

Aim of the Study: The present study aims to explore the value of application of hepaticojejunostomy in surgical treatment of Bismuth-Corlette type III hepatic hilar cholangiocarcinoma., Material and Methods: The clinical data of hepaticojejunostomy in 6 patients with Bismuth-Corlette type III hepatic hilar cholangiocarcinoma from January 2008 to October 2011 were retrospectively analyzed. There were 5 males and 1 female, aged 45-69 years, on average 58 years old. There were 3 cases of Bismuth-Corlette type IIIa and 3 cases of Bismuth-Corlette type IIIb., Results: The 6 patients all successfully underwent hepaticojejunostomy, with operation time of 160-310 min, on average 210 ±28 min. One patient suffered a small amount of bile leakage (3-20 ml) and his drainage tube was taken out after being cured with conservative treatment for 18 days. Three patients showed postoperative mild increases of aspartate aminotransaminase (AST), alanine transaminase (ALT) and AKP, which were improved after drug treatments. There was no biliary stricture in the 6 patients after follow-up for 3-18 months., Conclusions: Hepaticojejunostomy was simple and flexible, with a good effect on biliary reconstruction of Bismuth-Corlette type III hepatic hilar cholangiocarcinoma. It solved the difficulties in multiple choledochojejunostomy, and with less complications; therefore, it is worth being popularized.

The basic helix-loop-helix transcription factor Twist1 is essential for normal limb development. Twist1(-/-) embryos die at midgestation. However, studies on early limb buds found that Twist1(-/-) mutant limb mesenchyme has an impaired response to FGF signaling from the apical ectodermal ridge, which disrupts the feedback loop between the mesenchyme and AER, and reduces and shifts anteriorly Shh expression in the zone of polarizing activity. We have combined Twist1 null, hypomorph and conditional alleles to generate a Twist1 allelic series that survives to birth. As Twist1 activity is reduced, limb skeletal defects progress from preaxial polydactyly to girdle reduction combined with hypoplasia, aplasia or mirror symmetry of all limb segments. With reduced Twist1 activity there is striking and progressive upregulation of ectopic Shh expression in the anterior of the limb, combined with an anterior shift in the posterior Shh domain, which is expressed at normal intensity, and loss of the posterior AER. Consequently limb outgrowth is initially impaired, before an ectopic anterior Shh domain expands the AER, promoting additional growth and repatterning. Reducing the dosage of FGF targets of the Etv gene family, which are known repressors of Shh expression in anterior limb mesenchyme, strongly enhances the anterior skeletal phenotype. Conversely this and other phenotypes are suppressed by reducing the dosage of the Twist1 antagonist Hand2. Our data support a model whereby multiple Twist1 activity thresholds contribute to early limb bud patterning, and suggest how particular combinations of skeletal defects result from differing amounts of Twist1 activity., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The actin depolymerizing factors (ADFs) play important roles in several cellular processes that require cytoskeletal rearrangements, such as cell migration, but little is known about the in vivo functions of ADFs in developmental events like branching morphogenesis. While the molecular control of ureteric bud (UB) branching during kidney development has been extensively studied, the detailed cellular events underlying this process remain poorly understood. To gain insight into the role of actin cytoskeletal dynamics during renal branching morphogenesis, we studied the functional requirements for the closely related ADFs cofilin1 (Cfl1) and destrin (Dstn) during mouse development. Either deletion of Cfl1 in UB epithelium or an inactivating mutation in Dstn has no effect on renal morphogenesis, but simultaneous lack of both genes arrests branching morphogenesis at an early stage, revealing considerable functional overlap between cofilin1 and destrin. Lack of Cfl1 and Dstn in the UB causes accumulation of filamentous actin, disruption of normal epithelial organization, and defects in cell migration. Animals with less severe combinations of mutant Cfl1 and Dstn alleles, which retain one wild-type Cfl1 or Dstn allele, display abnormalities including ureter duplication, renal hypoplasia, and abnormal kidney shape. The results indicate that ADF activity, provided by either cofilin1 or destrin, is essential in UB epithelial cells for normal growth and branching., Competing Interests: The authors have declared that no competing interests exist.

Objective: To investigate the association between distribution of protein gene product (PGP) 9.5-immunoactive nerve fibers in peritoneal endometriotic lesions and disease-associated pain symptoms., Methods: Thirty two peritoneal endometriotic lesions from patients with endometriosis (16 cases with pain and 16 cases without pain) and matched with 20 peritoneal tissues from patients with uterine leiomyoma without endometriosis were stained immunohistochemically for PGP9.5-immunoactive nerve fibers., Results: The positive rate and density of PGP9.5-immunoreactive nerve fibers in peritoneal endometriotic leision were 62% (10/16) and (3.8+/-1.7)/mm2 in endometriosis patients with pain, which were significantly higher than 19% (3/16) and (1.7+/-0.5)/mm2 in endometriosis patients without pain (P<0.05) and 25% (5/20) and (1.3+/-0.6)/mm2 in peritoneal tissues in women without endometriosis (P<0.05). However, no differences were found between endometriosis patients without pain and women without endometriosis (P>0.05). Moreover, the density of PGP9.5-immunoreactive nerve fibers in peritoneal lesions in endometriosis patients with pain was positively correlated with the severity of pain (r=0.855, P<0.05). In addition, the density of PGP9.5-immunoreactive nerve fibers in peritoneal lesions was statistically higher in endometriosis patients with chronic pelvic pain and (or) dysmenorrhea than those in endometriosis patients with other type of pain (P<0.05), which was not associated with active lesion, site and staging (P>0.05)., Conclusion: It suggested that PGP9.5-immunoreactive nerve fibers might confer the mechanism of pelvic pain with endometriosis.

Glial cell line-derived neurotrophic factor signaling through the Ret receptor tyrosine kinase is crucial for ureteric bud branching morphogenesis during kidney development, yet few of the downstream genes are known. Here we show that the ETS transcription factors Etv4 and Etv5 are positively regulated by Ret signaling in the ureteric bud tips. Mice lacking both Etv4 alleles and one Etv5 allele show either renal agenesis or severe hypodysplasia, whereas kidney development fails completely in double homozygotes. We identified several genes whose expression in the ureteric bud depends on Etv4 and Etv5, including Cxcr4, Myb, Met and Mmp14. Thus, Etv4 and Etv5 are key components of a gene network downstream of Ret that promotes and controls renal branching morphogenesis.

Objective: To investigate nerve fibers distribution in endometrium of adenomyosis and their relationship with dysmenorrhea., Methods: Endometrial tissue was sampled from 74 hysterectomy specimens including 32 cases with adenomyosis and 42 cases with uterine fibroids. Two-step Envision immunohistochemical staining was used to detect distribution of nerve fibers in endometrium. Highly specific polyclonal rabbit anti-protein gene product 9.5 (PGP9.5) and monoclonal mouse anti-neurofilament protein (NF) were used to demonstrate both myelinated and unmyelinated nerve fibers in endometrium in women with adenomyosis and uterine fibroids., Results: The positive rate of PGP9.5 immunoreactive nerve fibers in the functional layer of endometrium of pain patients were with 64% (14/22) in adenomyosis and 67% (10/15) in uterine fibroids. And their density were 0.6 (0 - 9.4)/mm(2) and 0.6 (0 - 6.0)/mm(2) without reaching statistical difference (P > 0.05). No expression of NF could be detected in the functional layer of endometrium of adenomyosis and uterine fibroids. There were no PGP9.5 immunoreactive nerve fibers in the functional layer of endometrium in non-pain women with adenomyosis and uterine fibroids. Moreover, No NF immunoreactive nerve fibers in the functional layer of endometrium were shown in non-pain patients with adenomyosis and uterine fibroids. PGP9.5 immunoreactive nerve fibers and the nerve density in the basal layer of endometrium were 64% (14/22), 1.1 (0 - 12.0)/mm(2) in pain adenomyosis and 50% (5/10), 0.6 (0 - 3.0)/mm(2) in non-pain adenomyosis. NF immunoreactive nerve fibers and the density in the basal layer of endometrium were 23% (5/22), (0 - 0.6)/mm(2) in pain adenomyosis and 20% (2/10), (0 - 1.0)/mm(2) in non-pain adenomyosis. PGP9.5 immunoreactive nerve fibers and the nerve density in the basal layer of endometrium were 80% (12/15) and 1.6 (0 - 10.0)/mm(2) in pain fibroids and 44% (12/27), 0 (0 - 5.0)/mm(2) in non-pain fibroids. NF immunoreactive nerve fibers and the nerve density in the basal layer of endometrium were 40% (6/15), 0 (0 - 0.4)/mm(2) in pain fibroids and 15% (4/27), 0 (0 - 1.0)/mm(2) in non-pain fibroids. There was no statistical different PGP9.5 and NF immunoreactive nerve fibers distribution in basal layer of endometrium between pain adenomyosis and pain fibroids or between non-pain adenomyosis and non-pain fibroids (all P > 0.05). However, PGP9.5 immunoreactive nerve fibers density in basal layer of endometrium was higher in pain adenomyosis and fibroids when compared with non-pain adenomyosis and fibroids (P < 0.05)., Conclusions: PGP9.5 immunoreactive nerve fibers might confer the occurrence of pelvic pain, however, NF immunoreactive nerve fibers may not involved in the pathogenesis of pain.

Intestinal malrotation is a congenital anomaly of intestinal rotation and fixation that generally becomes symptomatic during the neonatal period. However, it rarely occurs in adulthood, and synchronous colon cancer is even rarer. This report presents the case of a 45-year old man who suffered from intestinal malrotation associated with colon cancer.

Objective: To investigate the metabolism of lipid, calcium and phosphorus in women with endometriosis., Methods: Clinical data of 223 patients with endometriosis and 200 patients without endometriosis were retrospectively analyzed. Electrochemiluminoimmunoassay was used to detect the levels of serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T), estradiol (E2) and progesterone (P), and photoelectric colorimetry was used to determine the concentrations of serum triglyceride (TG), total cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein (HDL), calcium and phosphorus in the patients with and without endometriosis., Results: The levels of serum P were 2.0 nmol/L and 1.8 nmol/L in endometriosis patients with age < or = 35 years old (case group A) and > 35 years old (case group B), and 1.6 nmol/L and 1.2 nmol/L in patients without endometriosis at age < or = 35 years old (control group A) and > 35 years old (control group B), respectively. A significant difference was found between case group A and control group A, and between case group B and control group B. The levels of serum LH was significantly higher in case group B (7.2 U/L) than that in control group B (6.1 U/L), however, there was no significant difference between case group A (7.0 U/L) and control group A (6.5 U/L). Also no significant difference in serum FSH, T and E2 levels was found between case group A (respectively, 6.2 U/L, 1.1 nmol/L and 269 pmol/L) and control group A (respectively, 6.3 U/L, 1.1 nmol/L and 264 pmol/L), also between case group B (respectively, 6.6 U/L, 1.0 nmol/L and 345 pmol/L) and control group B (6.6 IU/L, 0.9 nmol/L and 279 pmol/L). The concentrations of serum TG in case group A and B (0.71 mmol/L and 0.72 mmol/L) were significantly lower than in control group A and B (0.92 mmol/L and 1.08 mmol/L), respectively. The concentrations of serum LDL in case group A and B [(2.2 +/- 0.5) mmol/L and (2.4 +/- 0.6) mmol/L]were also significantly lower than in control group A and B [(2.4 +/- 0.7) mmol/L and (2.62 +/- 0.63) mmol/L], respectively. However, the concentrations of serum HDL in case group A and B [(1.62 +/- 0.31) mmol/L and (1.53 +/- 0.32) mmol/L] were significantly higher than in control group A and B [(1.48 +/- 0.21) mmol/L and (1.37 +/- 0.22) mmol/L] , respectively. In addition, the concentrations of serum TC were not significantly different between case group A and control group A [(4.2 +/- 0.7) mmol/L and (4.29 +/- 0.71) mmol/L], and between case group B and control group B [(4.4 +/- 0.8) mmol/L and (4.5 +/- 0.7) mmol/L]. The levels of serum phosphorus in case group A and B [(1.01 +/- 0.22) mmol/L and (0.89 +/- 0.18 mmol/L] were significantly lower than in control group A and B [1.23 +/- 0.24 mmol/L and (1.10 +/- 0.13) mmol/L], respectively. But the levels of serum calcium had no significant difference between case group A and control group A [(2.33 +/- 0.23) mmol/L and (2.41 +/- 0.12) mmol/L], and between case group B and control group B [(2.40 +/- 0.28) mmol/L and (2.42 +/- 0.20) mmol/L]., Conclusion: The abnormal metabolism of lipid and phosphorus, and the higher levels of serum P may playing a role in the pathogenesis of endometriosis.

Objective: To explore the clinicopathologic characteristics, treatment and prognosis of differentiated thyroid carcinoma (DTC) in adolescents., Methods: The data of 46 patients with DTC under the age of 18 years were retrospectively reviewed., Results: Twenty patients were misdiagnosed in this group (43.5%). All patients received operation, including 39 unilateral neck dissection and 6 bilateral neck dissection, followed by postoperative thyrotropin suppressive therapy. There were 42 cases of papillary carcinoma (91.3%) and 4 cases of follicular carcinoma (8.7%). Cervical lymph node metastasis was found in 39 cases (84.8%). In the follow-up period of 1 to 25 years (mean 10 years), no death of thyroid carcinoma occurred., Conclusions: The most common DTC in adolescents is papillary carcinoma with better prognosis regardless of the higher incidence of cervical lymph node metastasis. The optimal extent of primary thyroidectomy and neck dissection followed by postoperative thyrotropin suppressive therapy in adolescents with DTC may improve the quality of life and decrease the incidence of complications.

The purposes of the present research are (i) to establish the karyotype of Neursopora crassa using visualization of kinetochores in the synaptonemal complex (SC) spreads, (ii) to assign each chromosome to a linkage group, and (iii) to examine chromosome pairing and recombination nodules in quadrivalents. Two strains containing reciprocal translocations were used: T(I;II)4637, which involves linkage groups I and II, and alcoy, which contains 3 independent translocations involving I and II, IV and V, and III and VI. Visualization of kinetochores in the spreads requires the use of freshly prepared fixatives. Kinetochore locations and arm ratios were documented in all 7 N. crassa chromosomes. This new information, based on kinetochore position, arm ratios, chromosome length, and quadrivalent analyses, enabled unequivocal confirmation of chromosome assignments to genetic linkage groups. Chromosome pairing in a translocation quadrivalent starts at the 4 terminal regions, and proceeds right up to the translocation break point. Recombination nodules are found in all 4 arms of quadrivalents. The ability to identify a specific chromosome to a genetic linkage group together with the ability to visualize recombination nodules and their locations will allow future cytological analysis of recombination events.

Among many white-cap mutants of Coprinus cinereus, four distinct classes have been identified cytologically. Mutants of one class progress through meiosis normally but fail to sporulate; the defect is post-meiotic and it triggers apoptosis in the tetrad stage. Mutants of the other three classes have defects in meiotic prophase and these are: (1) those that assemble synaptonemal complexes (SCs) normally; (2) those that assemble axial elements (AEs) but not SCs; and (3) those that assemble neither AEs nor SCs even though the chromosomes are condensed and also paired. All three meiotic mutant classes arrest at meiotic metaphase I and the arrest triggers meiosis-specific apoptosis showing characteristic chromatin condensation, DNA fragmentation as shown by the TUNEL assay, cytoplasmic shrinkage, and finally total DNA degradation. Apoptosis is very cell-type specific; it occurs only in the basidia while the neighboring somatic cells are perfectly healthy and the mushroom continues to develop and mature with very few basidiospores produced. The meiotic apoptosis in C. cinereus is under strict cell cycle control rather than at any time after defect; apoptosis is triggered only after entry to meiotic metaphase. It is intriguing to note that C. cinereus has two checkpoints for arrest and entry to apoptosis: one is meiotic at the metaphase I spindle checkpoint regardless of the time of defects, and one is post-meiotic at the tetrad stage. This is in striking contrast to multiple checkpoint arrests and entries to meiotic apoptosis found in the mouse.

Meiosis progression in Coprinus cinereus is controlled by light/dark cycles. Light is essential to propel basidia into karyogamy and light intensity determines the timing of meiotic events. The higher the light intensities, the faster the fruiting bodies enter karyogamy. The critical period when light has this influence is between 16 and 6 h before karyogamy. The control is highly stage specific. A 3-h dark period is essential for a Java dikaryon and the Japanese A(mut)B(mut) homokaryon to enter meiotic metaphase; without it the fruit body is permanently arrested at diffused diplotene. This arrest is light intensity-dependent (>20 hlx) and temperature-dependent (e.g., 27 degrees C). The placement of the dark period is very stage specific; it has no effect when placed before karyogamy stage. A dikaryon of London origin is light blind and able to complete meiosis under continuous high light regime. Fruiting bodies arrested under a continuous high light can be rescued by a 3-h dark treatment, but there is always an 8-h lag time to enter meiotic metaphase. It is possible that the dark effect signals cellular processes leading to division events. Cytological studies of arrested fruiting bodies showed that chromosomes are normal in meiotic prophase through pachytene and diplotene, but are unable to undergo chromosome condensation. Genetic crosses between a monokaryon of Java stock J6;5.4 and a monokaryon BL55 or H5 of London stock showed that light-blindness is dominant, and is controlled by a single Mendelian gene.

Fruiting body formation in the basidiomycete Coprinus cinereus is a developmental process that occurs as a response of the mycelium to external stimuli. First, localized, highly branched hyphal structures (knots) are formed as a reaction to nutritional depletion. Hyphal-knot formation is repressed by light; however, light signals are essential for the development of the hyphal knot into an embryonic fruiting body (primordium) as well as karyogamy, meiosis and fruiting body maturation. The role of the different environmental signals in the initial phases of fruiting body development was analysed. It was observed that two fungal galectins, Cgl1 and Cgl2, are differentially regulated during fruiting body formation. cgl2 expression initiated in early stages of fruiting body development (hyphal knot formation) and was maintained until maturation of the fruiting body, whereas cgl1 was specifically expressed in primordia and mature fruiting bodies. Immunofluorescence and immuno-electron microscopy studies detected galectins within specific fruiting body tissues. They localized in the extracellular matrix and the cell wall but also in membrane-bound bodies in the cytoplasm. Heterologous expression of Cgl2 in Saccharomyces cerevisiae indicated that secretion of this protein occurred independently of the classical secretory pathway.

Galectins are members of a genetically related family of beta-galactoside-binding lectins. At least eight distinct mammalian galectins have been identified. More distantly related, but still conserving amino acid residues critical for carbohydrate-binding, are galectins in chicken, eel, frog, nematode, and sponge. Here we report that galectins are also expressed in a species of fungus, the inky cap mushroom, Coprinus cinereus. Two dimeric galectins are expressed during fruiting body formation which are 83% identical to each other in amino acid sequence and conserve all key residues shared by members of the galectin family. Unlike most galectins, these have no N-terminal post-translational modification and no cysteine residues. We expressed one of these as a recombinant protein and studied its carbohydrate-binding specificity using a novel nonradioactive assay. Binding specificity has been well studied for a number of other galectins, and like many of these, the recombinant C. cinereus galectin shows particular affinity for blood group A structures. These results demonstrate not only that the galectin gene family is evolutionarily much older than previously realized but also that fine specificity for complex saccharide structures has been conserved. Such conservation implies that galectins evolved to perform very basic cellular functions, presumably by interaction with glycoconjugates bearing complex lactoside carbohydrates resembling blood group A.

A protocol was developed to spread the synaptonemal complex (SC) of the fungus Neurospora crassa. It involves direct mechanical breakage of meiotic cells before spreading. This technique makes it possible to examine the SC of the same nucleus with both light and electron microscopy. This protocol is potentially applicable for other Pyrenomycetes. The SCs were examined at zygotene, pachytene and diplotene. The central elements and the recombination nodules (RN) were well revealed by silver staining. Ten to 13 RNs were counted at pachytene. The total genomic SC length varied with the stage. This whole mount electron microscopy of the SC is particularly useful for studying chromosomal rearrangements.

The naturally synchronous meiosis of the fungus, Coprinus cinereus, provides an ideal system for the investigation of differential gene expression in relation to meiosis and fruiting body development. We have cloned a cDNA from the fruiting body of C. cinereus encoding the 12-kDa subunit of a meiotic endonuclease (mENase). The identification of the 12-kDa subunit cDNA clone was achieved by the mENase antiserum against a lambda gt11 cDNA expression library. It was confirmed by a direct match of the amino acid (aa) sequence obtained from purified 12-kDa polypeptide with the nucleotide sequence. Northern blot analysis using the cDNA clone as a probe showed that the mENase-encoding gene (MenA) for the 12-kDa subunit was expressed mainly in fruiting bodies and at a very low level in the asexual vegetative mycelium. In addition, it was differentially expressed in the early meiotic stages. The MenA transcript was most abundant in fruiting body primordia prior to the premeiotic S-phase; it remained high from karyogamy to early pachytene, declined drastically by late pachytene and diplotene, and was undetectable by sterigma stage. Western blot analysis showed that the mENase protein was produced at a very low level in mycelium; it was produced in great quantity during the early meiotic stages and decreased to a low level at the end of meiosis.

An endo-exonuclease has been identified and partially purified from the basidiocarp tissues of the basidiomycete Coprinus cinereus, which include synchronous meiosis at karyogamy-pachytene stages. Its peak activity appears during the meiotic prophase. The Coprinus endo-exonuclease has a single-strand specific endonuclease activity that converts the supercoiled DNA to relaxed DNA. The endonucleolytic cleavage of single-strand DNA generates 3'-phosphomonoester termini. It is also a single-strand-specific exonuclease and it hydrolyzes linear DNA in a 3' to 5' direction, but is unable to hydrolyze single-strand DNA having a 3'-phosphomonoester terminus. It requires Mg2+ with an optimal concentration of 25 mM. It has an optimal pH of 8.3, a peak enzyme activity at 50 degrees C, and it contains a single 43-kilodalton polypeptide. Coprinus meiotic endo-exonuclease may be involved in the substrate preparation for meiotic recombination.

A functional recombination assay involving the tetracycline mutant plasmids, pUW1 and pUW4, was used to assess (i) the nature of the DNA substrates needed and (ii) the involvement of Coprinus endonuclease in preparing substrate, for the RecA-directed recombination process. A gapped circular plasmid and a linear or a nicked circular plasmid are efficient substrate combinations in this system to achieve a 160-fold increase in the in vitro recombination frequency over the control levels. The Coprinus endonuclease obtained from early meiotic prophase can produce such substrates. The recombination frequency obtained with the combination of gapped pUW1 plasmids initially relaxed by the Coprinus endonuclease and linear pUW4 plasmids produced by the site-specific BamHI digest is 10-fold lower than that obtained when both substrates are digested by BamHI. The results suggest that the Coprinus endonuclease creates random nicks on plasmid DNA. Glyoxal gel electrophoretic analysis was used to confirm this random nicking activity of Coprinus endonuclease.

The kinetics of incorporation of 32P into DNA have unequivocally shown that the premeiotic S-phase in Coprinus lagopus occurs before the onset of karyogamy. It takes 8 h under the control conditions (25 degrees C with a 16 h light-8 h dark regime) but only 6 h under the arrest-release conditions. An important discovery in this study is that the initiation of premeiotic DNA replication is subject to an arrest by restrictive conditions (35 degrees C under a continuous light regime) whereas that of the mitotic replication is not. Once initiated, meiotic DNA replication can continue even under the restrictive conditions. Incorporation of 32P into DNA at pachytene is quite extensive. These replications are considered to be repair replications.

We find three interesting characteristics of the genes encoding the ribosomal RNA (rRNA) in the basidiomycete Coprinus cinereus. First, there are only 60 to 90 copies of the genes, fewer than in other fungi. Second, the genes are organized in an unusual arrangement. The 5S rRNA genes are located in the repeat unit which encodes the other rRNAs and all four rRNAs are transcribed in the same direction. Third, meiotic recombination is inhibited within the ribosomal DNA.

Two forms of DNA polymerase have been studied in the basidiomycete Coprinus. DNA polymerase from basidiocarp tissues at zygotene-pachytene stage has been purified 3,500-fold and defined as DNA polymerase b by virtue of its insensitivity to N-ethylmaleimide and by its low molecular weight (76,000). This enzyme has optimal activity at pH 7.0 to 7.5, at 200 mM KCl, and at 25 degrees C incubation temperature. It can use polycytidylic acid-oligo(dG)12-18 as template primer in addition to homodeoxypolymers. The DNA polymerase a is mainly produced in the exponentially growing mycelium. It is sensitive to N-ethylmaleimide and has a temperature optimum at 35 degrees C. At the premeiotic S phase, activities from both polymerase a and polymerase b are found in cell-free extracts. The b enzyme is the only DNA polymerase produced during meiotic prophase. Its assayable activity exhibits two peaks, one at premeiotic S stage and one at pachytene. It is possible that DNA polymerase b is responsible for pachytene repairs involved in recombination.

We have taken advantage of the synchronous meiotic process in the basidiomycete Coprinus cinereus to develop a simple and rapid method to selectively stain meiotic chromosomes and nucleoli in this fungus without prior removal of the cell wall. Electron microscopic examination of these silver-stained chromosomes indicated that the lateral elements of the synaptonemal complexes were prominently stained, and terminal attachment plaques were apparent. We found that a translocation quadrivalent could be recognized easily in the light microscope using these methods. The procedures appear suitable for the characterization of chromosome rearrangements in this small genome, and should facilitate cytogenetic analysis in this fungus.

Repair synthesis of DNA per se at pachytene is not needed for commitment to meiotic recombination although it is a necessary event to follow. Recombination frequency is governed by the rate of nicking and the time in which unrepaired nicks are allowed to match and crossover. Cold treatment at pachytene prevented repair synthesis, hence open nicks were accumulated to match and crossover, and a 3-fold increase in recombination resulted. The kinetics of cold temperature effect followed a quadratic function as shown by a computer simulation which agreed with our experimental data (Lu, 1974b). High temperature did not change the rate of repair synthesis. It did cause an increased nicking which led to a twofold increase in recombination and which entailed a higher rate of recovery repair synthesis.

Inhibition of protein synthesis by cycloheximide on processes in meiosis was used to probe the cellular program for the formation and dissolution of the synaptonemal complex (SC) in the synchronous meiotic system of Coprinus cinereus. The pathway for the synthesis and assembly of the synaptonemal complex is proposed to be as follows: (1) synthesis and assembly of lateral components on the chromosomes; (2) synthesis and assembly of the central components in the nucleolus; (3) the lateral components of the homologous chromosomes are brought together to pair when the homologous pairing occurs at zygotene; (4) the transport of the central components from the nucleolus to join the paired lateral components and thus complete the synaptonemal complex. Continued protein synthesis is required for all steps. Step (1) is nearly complete 2 h after the onset of karyogamy, because continued assembly is possible in the presence of cycloheximide. The transition point for step (2) is 4 h after the onset of karyogamy, as inhibition at this point results in accumulation of central components in the nucleolar dense body. The paired lateral components of step (3) are deprived of the central component. The transition point for step (4) is 5 h after the onset of karyogamy, for inhibition at this point no longer prevents transport. Two steps are proposed for dissociation and dissolution of the SC at the end of pachytene. Protein synthesis is required for the dissolution of SC. Inhibition at this point causes accumulation of polycomplexes. Mutations in various organisms from the literature relating to the SC support the validity of the proposed pathway.

An endonuclease was purified from the cap tissues of basidiocarp of Coprinus cinereus collected at early meiotic prophase. It has an optimal activity at pH 7.0 and 37 degrees C. It is a cationic enzyme with a molecular mass of 22 kDa by gel filtration, and contains a 12-kDa and a 14-kDa peptide as revealed by SDS gel electrophoresis and Western blot analysis. An antiserum was produced in rabbits against the purified Coprinus endonuclease. The specificity of this antiserum was demonstrated in a dot-blot analysis and, more critically, in an immunoinhibition of endonuclease activity. The Coprinus endonuclease requires Mg2+ and/or Ca2+ as co-factors. Ca2+ is more efficient than Mg2+ while the effect of combining both co-factors is the highest. The Coprinus endonuclease has a substrate preference for single-strand and supercoiled DNA. It gives only single-strand nicks on supercoiled DNA at low enzyme concentration and limited time of incubation. At high enzyme concentration and/or long incubation time, double-strand fragmentation occurred. As is discussed, this endonuclease is believed to be involved in the early phase of meiotic recombination.

The time-course study of meiosis in the fungus Coprinus cinereus (C. lagopus) by electron microscopy reveals that two monoglobular spindle pole bodies (SPB's) of prekaryogamy nuclei come together during karyogamy and are fused. The fusion SPB of postkaryogamy nucleus persists through zygotene and pachytene as evidenced by the presence of axial components and synaptonemal complexes. At early diplotene, the SPB divides. The divided SPB takes on a diglobular form, which grows in size to form two daughter SPB's. These separate and move to opposite poles at metaphase I.

At the restrictive conditions (35 degrees under continuous light) Coprinus lagopus is unable to initiate premeiotic S phase which takes place normally within 8-10 h of karyogamy. A shift-up to the restrictive conditions causes an arrest of the basidiocarps at this critical stage. A prolonged arrest causes a reversal to mitosis (Lu 1974b). Incubation of basidiocarps at the restrictive conditions before this critical stage causes no increase in recombination frequency (R.F.) in the loci studied. An arrest of 4 h at the critical stage still causes no R.F. increase, but 12-13 h and 18-19 h arrests cause increases of 50% and 90% over the controls, respectively. Thus R.F. can be increased even before the cells are fully committed to meiosis.-A 3-h heat treatment at the beginning of S phase (or 8 h before karyogamy) also causes some (30%) increase in R.F. while the same treatment at late S phase (or 3 h before karyogamy) causes a substantial (164%) increase in R.F. over the controls. A 3-h heat treatment before S phase causes no increase in R.F.-Pachytene is also responsive to temperature treatments (Lu 1969). The maximum R.f. increase is 100% by heat and 220% by cold treatment. The shortest time that can cause the maximum increase in recombination by high temperature is 3 h and that by cold treatment is 7 h. These durations are correlated with the length of the pachytene stage under the treatment conditions. The kinetic data show that the increase in R.F. caused by high and low temperatures follows two-hit kinetics and their rate of increase is almost identical. The higher increase in R.F. by low temperature can be attributed to the increased duration of pachytene and therefore R.F. is a function of time. The longer the homologous chromosomes are held together, the higher the recombination frequency.