BmGeminin2 interacts with BmRRS1 and regulates Bombyx mori cell proliferation.

Geminin is a master regulator of cell-cycle progression that ensures the timely onset of DNA replication and prevents re-replication in vertebrates and invertebrates. Previously, we identified two Geminin genes, BmGeminin1 and BmGeminn2, in the silkworm Bombyx mori, and we found that RNA interference of BmGeminin1 led to re-replication. However, the function of BmGeminin2 remains poorly understood. In this study, we found that knockdown of BmGeminin2 can improve cell proliferation, and upregulated G2/M-associated gene-cyclinB/CDK1 expression. Then, we performed yeast two-hybrid screening to identify interacting proteins. Our results yielded 23 interacting proteins, which are involved in DNA replication, chromosome stabilization, embryonic development, energy, defense, protein processing, or structural protein. Here, we focused on BmRRS1, a chromosome congression-related protein that is closely related to cell cycle G2/M progression. The interaction between BmGeminin2 and BmRRS1 was confirmed by immunofluorescence and immunoprecipitation. Analysis of its expression profile showed that BmRRS1 was related to BmGeminin2. In addition, BmGeminin2 overexpression downregulated the BmRRS1 transcript. Knockdown of BmGeminin2 led to upregulation of the BmRRS1 transcript. Furthermore, overexpression of BmRRS1 can upregulate G2/M-associated gene-cyclinB/CDK1 expression, and improved cell proliferation, consistent with the effects of BmGeminin2 knockout. In addition, BmRRS1 RNA interference can eliminate the impact of BmGem2 knockout on cell proliferation, the ratio of cell cycle stage and the expression of cyclinB/CDK1. These data suggested that the cell proliferation advantage of BmGeminin2 knockout was closely related to BmRRS1. Our findings provide insight into the functions of Geminin and the mechanisms underlying the regulation of the cell cycle in the silkworm.

A Novel Mathematical Model for Correcting the Physiologic Variance of Two-Dimensional Echocardiographic Measurements in Healthy Chinese Adults.

BACKGROUND: To facilitate differentiation between normal and abnormal values, it is necessary to correct echocardiographic measurements for physiologic variance induced by age, gender, and body size variables. METHODS: A total of 34 two-dimensional echocardiographic parameters were measured in 1,224 healthy Chinese adults with body mass index < 25.0 kg/m2. An optimized multivariate allometric model and scaling equations were first developed in 858 subjects (group A), and their reliability was then verified in the remaining 366 subjects (group B). The traditional single-variable isometric model in which parameters are linearly corrected by a single body size variable (height, weight, body mass index, or body surface area) was used for comparison. The success of correction was defined as the absence of significant correlations (r > 0.20, P < .05) between the corrected values and age or any body size variables, while maintaining high correlations (r > 0.80) between the corrected and uncorrected values. RESULTS: Before correction, all 34 parameters correlated significantly with one or more of the physiologic variables of age and body size and differed significantly between men and women on 29 parameters (85.3%) in both groups. The success rate of correction with the single-variable isometric model was only 11.0% (15 of 136 corrections due to four variable corrections used for each parameter), while use of the optimized multivariate allometric model successfully corrected all 34 parameters (100%) for physiologic variance induced by age and body size variables and eliminated the gender differences in 32 parameters (94.1%). A new set of reference values for corrected echocardiographic measurements independent of age, gender, and body size variables were established. CONCLUSIONS: The novel optimized multivariate allometric model developed in this study is superior to traditional the single-variable isometric model in the correction of echocardiographic parameters for physiologic effects of age, gender, and body size variables and thus should be encouraged in both scientific research and clinical practice.

Two Geminin homologs regulate DNA replication in silkworm, Bombyx mori.

DNA replication is rigorously controlled in cells to ensure that the genome duplicates exactly once per cell cycle. Geminin is a small nucleoprotein, which prevents DNA rereplication by directly binding to and inhibiting the DNA replication licensing factor, Cdt1. In this study, we have identified 2 Geminin genes, BmGeminin1 and BmGeminn2, in silkworm, Bombyx mori. These genes contain the Geminin conserved coiled-coil domain and are periodically localized in the nucleus during the S-G2 phase but are degraded at anaphase in mitosis. Both BmGeminin1 and BmGeminin2 are able to homodimerize and interact with BmCdt1 in cells. In addition, BmGeminin1 and BmGeminin2 can interact with each other. Overexpression of BmGeminin1 affects cell cycle progression: cell cycle is arrested in S phase, and RNA interference of BmGeminin1 leads to rereplication. In contrast, overexpression or knockdown of BmGeminin2 with RNAi did not significantly affect cell cycle, while more rereplication occurred when BmGeminin1 and BmGeminin2 together were knocked down in cells than when only BmGeminin1 was knocked down. These data suggest that both BmGeminin1 and BmGeminin2 are involved in the regulation of DNA replication. These findings provide insight into the function of Geminin and contribute to our understanding of the regulation mechanism of cell cycle in silkworm.

DIVERSITY OF VASCULAR REACTIVITY AND THE TREATMENT RESPONSE IN DIABETIC, HYPERTENSIVE, HYPERLIPIDEMIC, AND HEALTHY RATS SUBJECTED TO HEMORRHAGIC SHOCK.

The current diagnosis and treatment guidelines for severe trauma and shock are all for healthy population. Few studies focused on the pathophysiological features and treatments in metabolic diseases after severe trauma and shock. Vascular reactivity is significantly decreased after severe trauma and shock. Improving the vascular reactivity with arginine vasopressin (AVP) and phorbol-12 myristate-13-acetate (PMA) is beneficial to trauma and shock. Whether the cardiovascular function and treatment responses have the own features in hypertensive, diabetic, and hyperlipidemic patients after traumatic hemorrhagic shock is not known. Using hypertensive, diabetic, and hyperlipidemic and healthy rats, we compared the change patterns in cardiovascular function including vascular reactivity, tissue perfusion, and the hemodynamics after hemorrhagic shock and their responses to AVP, PMA, and common antishock agents including dopamine and norepinephrine. A same degree of hemorrhagic shock (40% hemorrhage or mean arterial pressure maintained at 40 mm Hg for 2 h) resulted in a more obvious decrease in vascular reactivity, hemodynamics, tissue perfusion, and mitochondrial function of liver and kidney in hypertensive, diabetic, and hyperlipidemic rats, and a more rapidly natural death than in healthy rats. The effectiveness of AVP and PMA in these diseased rats was lower than in healthy rats. The effective dosage of common antishock agents including norepinephrine, dopamine, and AVP in healthy rats was wider than that in these diseased rats. Among the antishock agents used in the current study, AVP had the best effect in improving animal survival and vascular reactivity both in healthy and in diseased rats. These findings suggest that hypertensive, diabetic, and hyperlipidemic rats have a worse vascular reactivity and organ function than the healthy rats after traumatic hemorrhagic shock, which result in the worse treatment responses and effects to vasoactive agents. Lower dose of AVP can be recommended as the first-line antishock agents for these diseased rats.

Effects of starvation and hormones on DNA synthesis in silk gland cells of the silkworm, Bombyx mori.

Silk gland cells of silkworm larvae undergo multiple cycles of endomitosis for the synthesis of silk proteins during the spinning phase. In this paper, we analyzed the endomitotic DNA synthesis of silk gland cells during larval development, and found that it was a periodic fluctuation, increasing during the vigorous feeding phase and being gradually inhibited in the next molting phase. That means it might be activated by a self-regulating process after molting. The expression levels of cyclin E, cdt1 and pcna were consistent with these developmental changes. Moreover, we further examined whether these changes in endomitotic DNA synthesis resulted from feeding or hormonal stimulation. The results showed that DNA synthesis could be inhibited by starvation and re-activated by re-feeding, and therefore appears to be dependent on nutrition. DNA synthesis was suppressed by in vivo treatment with 20-hydroxyecdysone (20E). However, there was no effect on DNA synthesis by in vitro 20E treatment or by either in vivo or in vitro juvenile hormone treatment. The levels of Akt and 4E-BP phosphorylation in the silk glands were also reduced by starvation and in vivo treatment with 20E. These results indicate that the activation of endomitotic DNA synthesis during the intermolt stages is related to feeding and DNA synthesis is inhibited indirectly by 20E.

Mitochondrial Apoptotic Pathway Is Activated by H2O2-Mediated Oxidative Stress in BmN-SWU1 Cells from Bombyx mori Ovary.

Apoptosis is a known regulator of morphogenetic events. In mammals, the critical role of oxidative stress-induced apoptosis has been well-studied; however, in insects the role of oxidative stress in apoptosis is not clear. In a previous study, we showed that apoptosis-related genes are present in the silkworm Bombyx mori, an important lepidopteran insect model. In this study, we evaluated the effect of H2O2-induced oxidative stress on apoptosis, reactive oxygen species (ROS) levels, mitochondrial response, cytochrome c release and apoptosis-related gene expression in the BmN-SWU1 cell line from B. mori ovaries. Our results showed that BmN-SWU1 cells exposed to H2O2 showed cell protuberances, cytoplasmic condensation, apoptotic bodies, DNA ladder formation and caspase activities indicating apoptosis. H2O2-induced apoptosis also increased intracellular ROS level, changed mitochondrial distribution, reduced mitochondrial membrane potential and increased the release of cytochrome c from mitochondria. Furthermore, western blot analysis revealed a significant increase in p53 and cytochrome c expression, and a decrease in Bcl-2 expression compared to the controls. Moreover, quantitative real-time PCR (qRT-PCR) showed an increase in the transcript levels of BmICE, Bmapaf-1 and BmEndoG by 439.5%, 423.9% and 42.2%, respectively, after treatment with 1 µM H2O2 for 24 h. However, the transcript levels of Bmbuffy declined by 41.4% after 24 h of exposure to 1 µM H2O2. These results show that H2O2 treatment induced apoptosis in BmN-SWU1 cells via the mitochondrial apoptotic pathway. Further, it appears that oxidative stress induced by H2O2 activates both caspase-dependent and caspase-independent mitochondrial apoptotic pathways in silkworm cells. Taken together, these findings improve our knowledge of apoptosis in silkworm and the apoptotic pathways in insects.

Diabetes and hyperlipidemia induce dysfunction of VSMCs: contribution of the metabolic inflammation/miRNA pathway.

Vascular endothelial cell injury is considered to be the major factor inducing vascular complications in metabolic diseases and plays an important role in other organ damage. With diabetic and hyperlipidemic rats and cultured VSMCs, the present study was aimed at investigating whether the early damage of VSMCs during metabolic diseases plays a critical role in vascular dysfunction and the underlying mechanisms and would be a promising treatment target. With diabetic and hyperlipidemic rats and cultured VSMCs, the changes and relationships of vascular relaxation and contractile function to the vital organ damage and the underlying mechanisms were investigated; meanwhile, the protective and preventive effects of lowering blood lipid and glucose and inhibition of diabetes and hyperlipidemia-induced vascular hyperreactivity were observed. Diabetic and hyperlipidemic rats presented hyperreactivity in vascular contractile response in the early stages. Hyperglycemia and hyperlipidemia directly affected the contractile function of VSMCs. Early application of fasudil, a specific antagonist of Rho kinase, significantly alleviated diabetes and hyperlipidemia-induced organ damage by inhibiting vascular hyperreactivity. Diabetes and hyperlipidemia-induced inflammatory response could upregulate the expression of connexins and Rho kinase by selective downregulation of the expression of miR-10a, miR-139b, miR-206, and miR-222. These findings suggest that hyperglucose and lipid may directly impair VSMCs and induce vascular hyperreactivity in the early stages. Metabolic inflammation-induced changes in the miRNA-connexin/Rho kinase regulatory pathway are the main mechanism for vascular hyperreactivity and organ damage. Measures inhibiting vascular hyperreactivity are promising for the prevention of organ damage induced by metabolic diseases.

Inhibition of BmNPV replication in silkworm cells using inducible and regulated artificial microRNA precursors targeting the essential viral gene lef-11.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major silkworm pathogen, causing substantial economic losses to the sericulture industry annually. We demonstrate a novel anti-BmNPV system expressing mature artificial microRNAs (amiRNAs) targeting the viral lef-11 gene. The mature amiRNAs inhibited the lef-11 gene in silkworm BmN-SWU1 cells. Antiviral assays demonstrated that mature amiRNAs silenced the gene and inhibited BmNPV proliferation efficiently. As constitutive overexpression of mature amiRNAs may induce acute cellular toxicity, we further developed a novel virus-induced amiRNA expression system. The amiRNA cassette is regulated by a baculovirus-induced fusion promoter. This baculovirus-induced RNA interference system is strictly regulated by virus infection, which functions in a negative feedback loop to activate the expression of mature amiRNAs against lef-11 and subsequently control inhibition of BmNPV replication. Our study advances the use of a regulatable amiRNA cassette as a safe and effective tool for research of basic insect biology and antiviral application.

DNA replication events during larval silk gland development in the silkworm, Bombyx mori.

The silk gland is an important organ in silkworm as it synthesizes silk proteins and is critical to spinning. The genomic DNA content of silk gland cells dramatically increases 200-400 thousand times for the larval life span through the process of endomitosis. Using in vitro culture, DNA synthesis was measured using BrdU labeling during the larval molt and intermolt periods. We found that the cell cycle of endomitosis was activated during the intermolt and was inhibited during the molt phase. The anterior silk gland, middle silk gland, and posterior silk gland cells asynchronously exit the endomitotic cycle after day 6 in 5th instar larvae, which correlated with the reduced expression of the cell cycle-related cdt1, pcna, cyclin E, cdk2 and cdk1 mRNAs in the wandering phase. Additional starvation had no effect on the initiation of silk gland DNA synthesis of the freshly ecdysed larvae.

A septal angle measured on computed tomographic pulmonary angiography can noninvasively estimate pulmonary vascular resistance in patients with chronic thromboembolic pulmonary hypertension.

PURPOSE: To explore the correlation between a septal angle measured on computed tomographic pulmonary angiography (CTPA) and pulmonary vascular resistance (PVR) determined by right heart catheterization in patients with chronic thromboembolic pulmonary hypertension (CTEPH). MATERIALS AND METHODS: Eighty-six patients with CTEPH (54 men, mean age: 53.08±12.43 y) were retrospectively reviewed, and 86 sex-matched and age-matched individuals without pulmonary artery hypertension and pulmonary embolism were used as the control group. All patients with CTEPH underwent CTPA before right heart catheterization. Septal angle was measured on transverse CTPA images as the angle between the interventricular septum and the line joining the midpoint of the sternum to the thoracic vertebral spinous process. Hemodynamic PVR was calculated on the basis of the data from right heart catheterization. RESULTS: Septal angle was 65.27±12.24 degrees and 39.43±9.79 degrees in the CTEPH group and control group, respectively, which was statistically significant (t=18.139, P=0.000). The septal angle correlated positively with PVR (r=0.629, P=0.000). By stepwise linear regression analysis, the septal angle was shown to be the only variable (r=0.578) that was independently associated with PVR levels, leading to the following equation: PVR=21.591×septal angle-374.641. By receiver operating characteristic analysis, septal angle ≥67.55 degrees had a sensitivity of 71.6% and a specificity of 73.4% for predicting PVR≥1000 dyne s/cm with an area under the curve of 0.739±0.055, which was higher than the area under the curve of right ventricular area/left ventricular area (0.627±0.061) and that of transverse diameter of right ventricle/transverse diameter of left ventricle (0.612±0.060). CONCLUSION: The septal angle is a useful tool for estimating PVR in patients with CTEPH.

[Relationship between spinal ventricular septal angle by computer tomographic pulmonary angiography and right cardiac functions, N-terminal brain natriuretic peptide in chronic thromboembolic pulmonary hypertension].

OBJECTIVE: To explore the relationship of spinal ventricular septal angle (SVSA) measured by computer tomographic pulmonary angiography (CTPA) and right cardiac functions, N-terminal brain natriuretic peptide (NT-proBNP) in the patients with chronic thromboembolic pulmonary hypertension (CTEPH). METHODS: Forty-four CTEPH patients, 26 males and 18 females aged (52 ± 12) years old on average, at our hospital from January 2008 to January 2010 were retrospectively reviewed. SVSA and such pulmonary artery obstruction indices as Qanadli and Mastora indices were evaluated by two independent radiologists. The parameters of right heart functions were evaluated by echocardiography and right-heart catheterization. The level of NT-proBNP was measured by enzyme linked immunosorbent assay (ELISA). RESULTS: SVSA was (63 ± 11)° in CTEPH and (40 ± 7)° in the control group. The differences were significant (t = 12.320, P = 0.000). SVSA had a moderately positive correlation with the level of NT-proBNP (r = 0.704, P = 0.000). A positive correlation existed between SVSA and right atrium transverse diameter (r = 0.381, P = 0.002), right atrium long axis diameter (r = 0.437, P = 0.000) and right ventricular transverse diameter (r = 0.449, P = 0.000) on echocardiography. But there was no correlation between SVSA and right ventricular ejection fraction (r = -0.175, P = 0.365, n = 24). Also there was a negative correlation between SVSA and cardiac output (r = -0.337, P = 0.025), cardiac index (r = -0.351, P = 0.020), right cardiac work (r = -0.307, P = 0.043) and right ventricular stroke work (r = -0.384, P = 0.010). CONCLUSION: Spinal ventricular septal angle measured on CTPA may serve as a better predictor for evaluating the level of NT-proBNP and right cardiac functions in CTEPH.

[Observation on therapeutic effect of digital acupoint pressure for treatment of the nerve root type of cervical spondylosis].

OBJECTIVE: To observe the therapeutic effect of digital acupoint pressure for treatment of the nerve root type of cervical spondylosis. METHODS: Four hundred cases were randomly divided into a digital acupoint pressure group (DAP group) and a medicine group, 200 cases in each group. Acupoints of Quepen (ST 12), Jianjing (GB 21) and Tianzong (ST 11) etc. were used for digital pressing in the DAP group; and Chinese herb medicine of Gentongping was routinely taken in the medicine group. After three treatment courses, the symptoms of pain and numbness, the signs of pressure measurement by compression of head, brachial plexus drawer test and arm myodynamia, as well as the total cumulative scores of daily living capability, were compared. RESULTS: After treatment, the total cumulative scores of numbness, pressure measurement by compression of head, brachial plexus drawer test, arm myodynamia and daily living capability in both groups were obviously better than those of before treatment (all P<0.01); but there was a significant difference on the total cumulative score of the symptoms and signs between the two groups. The cured rate of 78.0% and total effective rate of 99.0% in the DAP group were better than those of 61.0% and 87.0% in the medicine group, respectively (both P<0.01). CONCLUSION: Digital acupoint pressure plays an active role in improving the symptoms and signs on patients with nerve root type of cervical spondylosis, which is better than Chinese herb medicine of Gentongping.