Roles of Mitogen-Activating Protein Kinase Kinase Kinase Kinase-3 (MAP4K3) in Preterm Skeletal Muscle Satellite Cell Myogenesis and Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation Regulation.

BACKGROUND Preterm skeletal muscle genesis is a paradigm for myogenesis. The role of mitogen-activating protein kinase kinase kinase kinase-3 (MAP4K3) in preterm skeletal muscle satellite cells myogenesis or its relationship to mammalian target of rapamycin complex 1 (mTORC1) activity have not been previously elaborated. MATERIAL AND METHODS Small interfering RNA (siRNA) interference technology was used to inhibit MAP4K3 expression. Leucine stimulation experiments were performed following MAP4K3-siRNA interference. The differentiation of primary preterm skeletal muscle satellite cells was observed after siRNA-MAP4K3 interference. Western blot analysis was used to determine the expression of MAP4K3, MyHC, MyoD, myogenin, p-mTOR, and p-S6K1. The immunofluorescence fusion index of MyHC and myogenin were detected. MAP4K3 effects on preterm rat satellite cells differentiation and its relationship to mTORC1 activity are reported. RESULTS MAP4K3 siRNA knockdown inhibited myotube formation and both MyoD and myogenin expression in primary preterm rat skeletal muscle satellite cells, but MAP4K3 siRNA had no effect on the activity of mTORC1. In primary preterm rat skeletal muscle satellite cells, MAP4K3 knockdown resulted in significantly weaker, but not entirely blunted, leucine-induced mTORC1 signaling. CONCLUSIONS MAP4K3 positively regulates preterm skeletal muscle satellite cell myogenesis, but may not regulate mTORC1 activity. MAP4K3 may play a role in mTORC1 full activation in response to leucine.

Epidermal growth factor receptor signalling mediates growth hormone-induced growth of chondrocytes from sex hormone-inhibited adolescent rats.

1. Growth hormone (GH) has been demonstrated to overcome the inappropriate deceleration of growth rate in children with central precocious puberty treated with gonadotropin-releasing hormone analogue (GnRHa). However, the underlying mechanisms remain largely unclear. In the present study, we investigated the potential involvement of the epidermal growth factor receptor (EGFR) pathway in the growth promotion by GH using in vitro cultured growth plate chondrocytes isolated from adolescent rats treated with GnRHa. 2. Chondrocytes were stimulated with GH in the presence or absence of the Janus tyrosine kinase (JAK) 2 inhibitor AG490 (1, 10 and 100 nmol/L), the EGFR kinase inhibitor AG1478 (0.1, 1 and 10 nmol/L), U0126 (an inhibitor of extracellular signal-regulated kinase (Erk) activation; 10 µmol/L) or a neutralizing antibody against epidermal growth factor (EGF Ab; 0.1, 1 and 10 µg/mL). The proliferation of chondrocytes was assessed by the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay and immunostaining for proliferating cell nuclear antigen (PCNA). Phosphorylation of Erk1/2 and EGFR was detected by western-blotting. Intracellular mRNA and extracellular protein levels of EGF were detected using reverse transcription-polymerase chain reaction and ELISA, respectively. 3. Growth hormone promoted the proliferation of chondrocytes, which was correlated with increased phosphorylation of Erk1/2 and EGFR and enhanced expression of EGF. Pretreatment with AG490, AG1478, U0126 or EGF Ab completely or partially inhibited the proliferation of chondrocytes and activation of Erk1/2 and EGFR. Pretreatment with AG490, AG1478, or U0126 partially inhibited the expression of EGF. 4. The findings indicate that GH promotes chondrocyte proliferation by activating EGFR signalling.

Metformin attenuates ventricular hypertrophy by activating the AMP-activated protein kinase-endothelial nitric oxide synthase pathway in rats.

1. Metformin is an activator of AMP-activated protein kinase (AMPK). Recent studies suggest that pharmacological activation of AMPK inhibits cardiac hypertrophy. In the present study, we examined whether long-term treatment with metformin could attenuate ventricular hypertrophy in a rat model. The potential involvement of nitric oxide (NO) in the effects of metformin was also investigated. 2. Ventricular hypertrophy was established in rats by transaortic constriction (TAC). Starting 1 week after the TAC procedure, rats were treated with metformin (300 mg/kg per day, p.o.), N(G)-nitro-L-arginine methyl ester (L-NAME; 50 mg/kg per day, p.o.) or both for 8 weeks prior to the assessment of haemodynamic function and cardiac hypertrophy. 3. Cultured cardiomyocytes were used to examine the effects of metformin on the AMPK-endothelial NO synthase (eNOS) pathway. Cells were exposed to angiotensin (Ang) II (10⁻6 mol/L) for 24 h under serum-free conditions in the presence or absence of metformin (10⁻³ mol/L), compound C (10⁻6 mol/L), L-NAME (10⁻6 mol/L) or their combination. The rate of incorporation of [³H]-leucine was determined, western blotting analyses of AMPK-eNOS, neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) were undertaken and the concentration of NO in culture media was determined. 4. Transaortic constriction resulted in significant haemodynamic dysfunction and ventricular hypertrophy. Myocardial fibrosis was also evident. Treatment with metformin improved haemodynamic function and significantly attenuated ventricular hypertrophy. Most of the effects of metformin were abolished by concomitant L-NAME treatment. L-NAME on its own had no effect on haemodynamic function and ventricular hypertrophy in TAC rats. 5. In cardiomyocytes, metformin inhibited AngII-induced protein synthesis, an effect that was suppressed by the AMPK inhibitor compound C or the eNOS inhibitor L-NAME. The improvement in cardiac structure and function following metformin treatment was associated with enhanced phosphorylation of AMPK and eNOS and increased NO production. 6. The findings of the present study indicate that long-term treatment with metformin could attenuate ventricular hypertrophy induced by pressure overload via activation of AMPK and a downstream signalling pathway involving eNOS-NO.

Fully automated simultaneous peripheral arteriovenous exchange transfusion not seen to aggravate brain function and the disorder of the internal environment in neonates with severe hyperbilirubinemia.

BACKGROUND: The acute changes in brain function in newborn infants undergoing ET remain unclear. This study aimed to determine whether fully automated simultaneous peripheral arteriovenous ET would influence the brain function. METHODS: A retrospective analysis was conducted on the clinical data of 39 neonates with hyperbilirubinemia who received ET. Seventeen patients were in the encephalopathy group, and the other 22 patients were in the non-encephalopathy group. Changes in amplitude-integrated electroencephalogram (aEEG) during ETs were analyzed, including background activities, sleep-wake cycling (SWC), and seizures. Before and after the ET, routine blood test parameters, electrolytes, blood glucose, and blood gas parameters were measured. RESULTS: After ETs, there were no significant changes in the levels of pH, PaO2, PaCO2, lactate, and red blood cells, while the levels of total bilirubin, indirect bilirubin, blood potassium, blood sodium, serum calcium, while blood cells, and platelets were significantly lower and the level of blood glucose was significantly higher compared to those before therapy. There was no significant difference in the changes of electroencephalographic activities during ETs, including background activities, SWC, and seizures. However, there were significant differences in suppressions on background activities, while there were no significant statistical differences in SWC or seizures between the 2 groups. CONCLUSION: Fully automated simultaneous peripheral arteriovenous ET is safe and efficient without significant influence on the disorder of the internal environment and electroencephalographic activities after ET in neonates. However, background activities are more significantly depressed in infants of bilirubin encephalopathy than infants of non-encephalopathy during ET.

[Stanozolol activates the cross-talk of estrogen receptor alpha-insulin-like growth factor-1 receptor-extracellular-signal regulated kinase 1/2 in the growth plate chondrocytes of estrogen-inhibited adolescent rats in vitro].

OBJECTIVE: To investigate the effects and the mechanisms of stanozolol (ST) on the proliferation, maturation and differentiation of in vitro cultured growth plate chondrocyte isolated from gonadotropin releasing hormone analogue (GnRHa)-treated adolescent rats, to study if ST mediates the proliferation of chondrocytes via the estrogen receptor alpha (ERalpha), androgen receptor (AR) and/or insulin-like growth factor-1 receptor (IGF-1R) and interactions of the two receptor and IGF-1R receptor signaling pathway, to investigate the mechanism of the biological effects in ST promoting bone growth/maturity at molecular level. METHOD: The rats were weaned at the end of 3 weeks and intramuscular injection of triptorelin of GnRHa preparations, qow x 2 was started. The rats were sacrificed at the end of 7 weeks, and then the tibiae growth plates were taken out with sterile procedure. The chondrocytes were obtained by two-time enzyme digestion method, and the experiments were carried out with the primary chondrocytes. Immunohistochemical staining of proliferating cell nuclear antigen (PCNA) and Western blot analysis were applied. RESULT: The results of PCNA demonstrated that stanozolol enhanced the proliferation of the chondrocytes, time-course studies showed that the proliferation were maximally stimulated by stanozolol after 2 days of incubation and decreased again after longer periods of incubation. The expression of p-ERalpha, p-IGF-1R and p-extracellular-signal regulated kinase 1/2 (ERK1/2) increased with the incubation period of ST treatment, and reached the peak value at a certain time, and then gradually decreased. The expression of p-ERalpha, p-IGF-1R and p-ERK1/2 increased with the elevation of ST concentration, and reached the peak value at 10(-9) - 10(-8) mol/L, then gradually decreased. ST induced-p-ERalpha expression was partially blocked by ERalpha and mitogen-activated protein kinase kinase inhibitors. ST induced-p-IGF-1R expression was partially blocked by ERalpha and IGF-1R inhibitors. ST induced-p-ERK1/2 expression was partially blocked by mitogen-activated protein kinase kinase and IGF-1R inhibitors. CONCLUSION: As an androgen derivation, ST exerts its biological effects of promoting proliferation of the long bone growth plate chondrocytes via activating the classic ERalpha receptor pathway and mitogen-activated protein kinase pathway, and at the same time, by activation of IGF-1R. Both IGF-1R and ERalpha can promote "cross-talk" of two systems' receptor signal through mitogen-activated protein kinase signal pathway.

[Clinical investigation on the treatment of HCMV hepatitis in children].

OBJECTIVE: To investigate the effective therapeutic method of human cytomegalovirus (HCMV) hepatitis in children. METHODS: Twenty-five children with HCMV hepatitis were randomly assigned to a treated group (n=13) or a control group (n=12). Both groups were treated with prednisone, glucurone, luminal and Xiaoyanlidanpian. But the treated group was given ganciclovir (GCV) + intravenous immunoglobulin (IVIG) in addition. Each infant of the two groups was checked for blood routine, liver function and HCMV copy numbers on admission and before discharge. They were seen at the third, sixth and ninth month after discharge. On each visit blood specimens were collected for HCMV copy numbers (fluorescence quantitative PCR, FQ-PCR). RESULTS: The viral load of the treated group decreased significantly. A significant difference in viral copy numbers was found between the two groups on admission, discharge, and third, sixth and ninth month after discharge (P less than 0.001). The number of HCMV DNA copy fell to 10(3) copies/ml on discharge while that of the control group fell to the same level after the third month. The differences between the two groups in the length of hospitalization, time of initial jaundice disappearance and complete disappearance were statistically significant (P less than 0.05). The need for transfusion in the treated group was significantly less than that in the control group (chi-square=4.012, P less than 0.05). CONCLUSION: Combination of GCV with a high dosage of IVIG to treat HCMV active infection could decrease viral load remarkably; The duration of disease, severity of symptoms, degree of anemia and the need for blood transfusion were reduced. No adverse effects related to the combination of GCV with IVIG therapy were observed.

Clinical applications of real-time PCR for diagnosis and treatment of human cytomegalovirus infection in children.

There are many methods of detecting human cytomegalovirus (HCMV) infection. So far, the quantitative polymerase chain reaction (PCR) has been very useful not only in aiding in the diagnosis of HCMV but also in determining the severity and predicting HCMV infection. However, it is time-consuming and labor intensive. Real-time PCR (RT-PCR) is an exception, for it allows rapid quantification of HCMV DNA load. Our group used this method for detecting and monitoring HCMV and compared it with the diagnostic criterion recommended by the Pediatric Branch of Chinese Medical Association, in 45 children suspected of having HCMV infection. The response to two types of antiviral treatment on HCMV DNA load was also monitored in HCMV hepatitis cases. RT-PCR was positive in 30 cases while the diagnostic criterion, which includes enzyme-linked immunosorbent assay (ELISA) and/or conventional PCR, was positive in 32 cases. The decrease in the HCMV DNA load was achieved earlier in the modified treatment group compared with the conventional treatment group. A 10(3) copies/ml of HCMV DNA load of is a useful cut-off value in predicting patients who will have symptoms of the disease. RT-PCR can be used not only in detecting HCMV but also in monitoring response to antiviral treatment and risk of having symptoms of the disease.

[Validation study on the criteria for clinical classification of small for gestational age infants].

OBJECTIVE: To study the validity of criteria currently used in China for the classification of symmetric small for gestational age infants (SGA) as compared with its definition. METHODS: This study included 417 inpatients diagnosed as SGA in authors' hospital from January 1998 to June 2002. Symmetric SGA was diagnosed by the following three criteria: (1) the Ponderal Index (PI), (2) the crown-heel length-to-head circumference ratio (BL/HC) issued in Chin J Pediatr (1988;26:164 - 165), as well as (3) the SGA definition. The definition criterion was considered as the "gold standard". The sensitivity, specificity, false positive and negative values, positive and negative predictive values, exact agreement ratio, diagnosis index, and Cohen's Kappa value were used to evaluate the validity and agreement of the methods of PI and BL/HC. Receiver Operating Characteristic (ROC) analysis was used to evaluate the validity of the diagnosis. RESULTS: Of 417 SGA infants, 376 (90.17%), 376 (90.17%) and 187 (44.84%) subjects were diagnosed as symmetric type with PI, BL/HC and the definition criteria, respectively. (2) The agreement rate and Kappa value between PI and BL/HC was 80.82% and -0.093 (SEM 0.026), respectively. And the agreement rates between PI or BL/HC and the definition criterion were 49.88% and 50.84%, respectively. As compared with the definition criterion, the PI and BL/HC methods had sensitivities of 91.8% - 96.4%, specificities of 9.3% - 25.9%, positive predictive values of 45.8% - 51.1%, negative predictive values of 72.7% - 82.8%, diagnosis indices of 4.9% - 17.7% and Kappa values of 0.070 - 0.167. (3) The areas under the ROC curves in full-term and preterm infants by PI method were 0.635 (95% CI, 0.573 - 0.697) and 0.698 (95% CI, 0.622 - 0.725), respectively. PI cutoffs at 2.47 in full-term SGA, at 2.43 in preterm SGA, and BL/HC cutoff at 1.43 produced the maximum diagnosis indices that were 24.7%, 39.6% and 33.7%, respectively. When the PI at 2.50 (full-term), PI at 2.31 (preterm) and BL/HC values at 1.46, the sensitivity closed mostly to the specificity. The sensitivities and specificities in full-term and preterm infants were 59.4% and 59.3%, 65.3% and 65.5%, and 66.3% and 65.5%, respectively. CONCLUSION: In the classification of SGA, the results showed a poor agreement between PI or BL/HC and the definition criterion. The results suggested that the current cutoffs of PI and BL/HC might not be appropriate for the diagnosis of symmetric SGA. Low AUC suggested that PI and BL/HC could not give a valid diagnosis at any cutoffs.

[Effect of human cytomegalovirus on hematopoietic system].

OBJECTIVE: To investigate the mechanism and the suppression effect of human cytomegalovirus (HCMV) on hematopoietic system. METHODS: Semi-solid culture system was used to observe the effect of HCMV AD169 strain on colony forming unit granulocyte/macrophage (CFU-GM), CFU-erythroid (CFU-E), CFU-multipotent (CFU-Mix) and CFU-megakaryocyte (CFU-MK) growth. The techniques of in situ polymerase chain reaction (IS-PCR) and polymerase chain reaction (PCR) were used to demonstrate the existence of HCMV DNA in the colony cells of cultured CFU-GM, CFU-Mix, CFU-MK and CFU-E, respectively. The immediate early antigen (IEA) mRNA in CFU-MK and late antigen (LA) mRNA in CFU-E were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). HCMV early protein P52 was detected with immunohistochemical technique. RESULTS: HCMV AD169 suppressed the differentiation and proliferation of CFU-GM, CFU-E, CFU-Mix and CFU-MK in vitro significantly (P < 0.05). The suppression was dose-dependent. HCMV DNA was successfully detected in CFU-GM, CFU-Mix, CFU-MK colony cells from viral infection groups by IS-PCR, and was detected in CFU-E by PCR, while it was negative in blank control or mock control groups. CFU-MK colony cells expressed HCMV IEA mRNA with the size of 340 bp in virus infection groups of 10(3) plague forming unit (PFU), 10(4) PFU and 10(5) PFU, respectively. The HCMV LA mRNA was detected by RT-PCR and was 263 bp long in positive control group of HCMV-infected human embryonic fibroblasts. The expression of HCMV LA mRNA in CFU-E was negative. The early protein P52 of HCMV in 10(4) PFU group was also identified by immunohistochemical staining. CONCLUSION: HCMV AD169 strains inhibited the differentiation and proliferation of CFU-GM, CFU-E, CFU-Mix and CFU-MK by the infection of the hematopoietic progenitors. HCMV might cause the suppression of hematopoiesis by direct infection, which is thought to be one of the reasons of HCMV infection associated with thrombocytopenia, neutropenia and anemia.