MDM2 inhibition improves cisplatin-induced renal injury in mice via inactivation of Notch/hes1 signaling pathway.

OBJECTIVE: To explore the potential function of MDM2-mediated Notch/hes1 signaling pathway in cisplatin-induced renal injury. METHODS: The acute renal injury models of mice after intraperitoneal injection of cisplatin in vivo, and the apoptotic models of human renal tubular epithelial (HK-2) cells induced by cisplatin in vitro, were conducted respectively. The renal function-related parameters were measured. The renal tissue pathological changes and apoptosis were observed by PAS staining and TUNEL staining, respectively. Cell viability and apoptosis were detected by MTT and flow cytometry. Notch/hes1 pathway-related proteins were tested by Western blotting. RESULTS: After mice injected by cisplatin, the levels of Cr, BUN, urine cystatin C, urine NGAL and urine ACR were increased and GFR was decreased with the elevation of renal tubular injury scores, the upregulation of the expressions of MDM2, N1ICD, Hes1 and Cleaved caspase-3, as well as the enhancement of cell apoptosis accompanying decreased ratio of Bcl-2/Bax. However, these cisplatin-induced renal injuries of mice have been improved by MDM2 inhibition. Besides, the declined viability, increased cytotoxicity, and enhanced apoptosis were observed in cisplatin-induced HK-2 cells, with the activated Notch/hes1 pathway. Notably, the phenomenon was alleviated in cisplatin-induced HK-2 cells transfected with MDM2 shRNA, but was severer in those co-treated with AdMDM2. Moreover, Notch1 siRNA can reverse the injury of AdMDM2 on HK-2 cells. CONCLUSION: Inhibiting MDM2 could reduce cell apoptosis through blocking Notch/hes1 signaling pathway, thus alleviating the acute renal injury caused by cisplatin.

MicroRNA-210 promotes spinal cord injury recovery by inhibiting inflammation via the JAK-STAT pathway.

OBJECTIVE: To investigate the effect of microRNA-210 on the spinal cord injury (SCI) and its underlying mechanism. MATERIALS AND METHODS: The mouse SCI model was established. Mice were randomly assigned into 4 groups, namely the sham operation group (sham group), surgery group (SCI group), surgery+NC group (SCI+NC group) and surgery+microRNA-210 overexpression group (SCI+microRNA-210 mimics group). The mRNA levels of microRNA-210 and the key genes in the JAK-STAT pathway of the four groups were detected by Real-Time Polymerase Chain Reaction (RT-PCR) at different time points. Protein levels of JAK2 and STAT3 in mice of the four groups were detected by Western blot. To investigate the role of microRNA-210 in SCI recovery, changes in the motor function of mice were detected. RESULTS: Grip strengths of right and left forelimbs in mice from the sham group were temporarily decreased at the early stage after surgery, which were gradually recovered to the preoperative levels on the 3rd postoperative day. However, mice in SCI group were unable to complete the grip strength determination at the early stage after surgery. Mice in SCI group were capable of grasping on the 7th postoperative day. Besides, grip strengths of mice in SCI group were remarkably lower than those of sham group until the end-point (on the 50th day). Furthermore, mRNA levels of microRNA-210 in mice of SCI group were decreased in a time-dependent manner (p<0.05). Higher grip strengths were observed in mice of SCI+microRNA-210 mimics group in comparison with those of SCI group and SCI+NC group (p<0.05). In addition, Western blot showed that protein levels of JAK2 and STAT3 in mice of SCI group were increased in a time-dependent manner (p<0.05). Moreover, protein levels of JAK2, STAT3, and MCP-1 in mice of SCI+NC group were remarkably higher than those in the sham group and SCI+microRNA-210 mimics group (p<0.05). CONCLUSIONS: MicroRNA-210 is down-regulated in SCI mice. Grip strengths of SCI mice can be recovered after microRNA-210 overexpression via inhibiting inflammatory response by the JAK-STAT pathway.

MiR-137 attenuates spinal cord injury by modulating NEUROD4 through reducing inflammation and oxidative stress.

OBJECTIVE: To explore the role of microRNA (miR) 137 in spinal cord injury and its mechanism. MATERIALS AND METHODS: The model of spinal cord injury in mice was established to detect the recovery differences of grip strength in upper and lower limbs of mice. The expressions of miR-137 and neuronal differentiation 4 (NEUROD4) were detected at the same time. The inflammation level and the oxidative stress response after spinal cord injury were subsequently detected after overexpression of miR-137. Target genes of miR-137 were identified by bioinformatics. Finally, dual-luciferase reporter gene assay was used to identify the target genes of miR-137. RESULTS: By establishing the model of spinal cord injury in mice, the strength of upper and lower limbs recovered after 7 days of injury in mice. The expression of miR-137 in spinal cord injury was found to decrease in a time-dependent manner by quantitative Real-time polymerase chain reaction (qRT-PCR), while the expression of NEUROD4 gradually increased. Inflammation indicators and oxidative stress level were found to be significantly higher after spinal cord injury. However, the inflammation level and oxidative stress were significantly reduced after transfection of miR-137. Finally, we predicted the target gene of miR-137 through bioinformatics website and found that NEUROD4 was a potential target gene of miR-137. Using dual luciferase reporter assays, we found that NEUROD4 bound to miR-137. After overexpression of miR-137, the expression of NEUROD4 was significantly reduced. Overexpression of NEUROD4 could promote spinal cord injury inflammation and oxidative stress. After intracellular transfection of NEUROD4 and miR-137 at the same time, the inflammation level and oxidative stress of spinal cord injury decreased significantly. CONCLUSIONS: These results suggested that miR-137 promoted the recovery of spinal cord injury by degrading NEUROD4 to relieve the spinal cord inflammation and the progression of oxidative stress, thus promoting the recovery of spinal cord injury.

MicroRNA-125b promotes the regeneration and repair of spinal cord injury through regulation of JAK/STAT pathway.

OBJECTIVE: Spinal cord injury (SCI) is a severe trauma to the central nervous system. Long non-coding RNAs have been reported to play essential roles in spinal cord injury. This study mainly explored the role of micro-125 in the regulation of spinal cord injury by regulating STAT3. MATERIALS AND METHODS: The stable mouse model of cervical spinal cord contusion was established by Infinite Horizon spinal cord striker, and the model mice' motor function was analyzed. Bioinformatics databases were used to screen the target mRNAs of micro-125b. qRT-PCR was performed to detect the expression of micro-125b and its target genes in injury area of mice' spinal cord. Western Blot and ELISA were introduced to detect the expression of inflammation and apoptosis-related proteins in each group. The recovery status of spinal cord after SCI was assessed by motor function scores and axon counts of mice in each group. RESULTS: Micro-125b appeared to be significantly down-regulated over-time after SCI. JAK1 and STAT1, two important neuregulin proteins, were predicted to be the target genes of micro-125b, and overexpression of micro-125b induced the decrease of phosphorylated JAK1 and STAT1. Enhanced micro-125b expression also allowed axons from the injury area of spinal cord to extend into the outer periphery of the damaged area, thus improving the motor function of the injured rats. Besides, overexpression of micro-125b demonstrated significant neuronal protective effects by reducing apoptosis and inflammatory responses in neurons. CONCLUSIONS: Our data revealed that micro-125b was down-regulated in injured spinal cord, and overexpression of micro-125b promoted the repair and regeneration following spinal cord injury through the regulation of the JAK/STAT pathway.

The influence of different movements on ambulatory blood pressure in hypertensive subacute stroke patients.

OBJECTIVE: This study evaluated variation in blood pressure (BP) in hypertensive subacute stroke patients performing eight different types of active movement, and variations in BP over time. METHODS: The study included 35 subacute stroke patients (60 - 74 years old) and 15 age-matched healthy volunteers. Ambulatory systolic and diastolic BP was measured over 4 consecutive days, before and during active movement. RESULTS: The greatest effect of the different active movements in stroke patients was on mean systolic BP variability (BPV). There was a significant difference in systolic and diastolic BPV between stroke patients at different time-points and compared with healthy volunteers. Systolic BPV during shifting from the ward to the rehabilitation centre was significantly higher than for all other active movements. Mean systolic BPVs during the sessions on the first and second days were significantly higher than for the sessions on the third and fourth days in stroke patients and compared with healthy volunteers. CONCLUSIONS: Systolic BP was found to be increased in hypertensive subacute stroke patients during their first and/or second attempts at performing active movements. Therapists should consider the BP of hypertensive subacute stroke patients during these first two attempts, especially for activities involving the patient moving from the ward to the rehabilitation centre.

Ephrin-B1 localizes at the slit diaphragm of the glomerular podocyte.

Ephs and ephrins are a family of membrane-bound proteins that function as receptor-ligand pairs. Members of the Eph-ephrin-B family have recently been reported to regulate the paracellular permeability of epithelial cells. In this study, we analyzed the expression and the function of ephrin-B1 in glomeruli. Using immunofluorescence (IF), we found that ephrin-B1 was expressed along the glomerular capillary loop. Immunoelectron microscopy revealed that ephrin-B1 expression was restricted at the slit diaphragm. Dual labeled IF showed ephrin-B1 colocalized with the slit diaphragm proteins nephrin and CD2-associated protein. Ephrin-B1 colocalized with nephrin at the late capillary loop stage of kidney development. Additionally, injection of rats with a nephritogenic anti-nephrin antibody (ANA) reduced ephrin-B1 expression. When podocytes were cultured in vitro, they extruded processes that co-stained for ephrin-B1 and for CD2-associated protein. When these podocytes were treated in culture with small interfering RNA for ephrin-B1, CD2-associated protein was reduced in the processes, with a remaining faint perinuclear staining. We suggest that ephrin-B1 has a role in maintaining barrier function at the slit diaphragm.

Changes in immunoelectron microscopic localization of cathepsin D in muscle induced by conditioning or high-pressure treatment.

Changes in the localization of cathepsin D in postmortem and pressurized rabbit muscles were investigated by immunoelectron microscopy. The anti-cathepsin D monoclonal antibody strongly labeled a large vesicle in a subsarcolemmal part of the cell , which strongly suggests that this is the primary lysosome. The liberation of the cathepsin D entrapped in the lysosomes in the muscle prepared immediately after death proceeded with the progress of the conditioning. The release of almost all cathepsin D from the lysosomes and its absorption on the myofibrils were observed in the muscle conditioned for 14 days. The accumulation of lysosomes having various volumes and shapes accompanied with the disruption of myofibrillar structure was also observed. The liberation of cathepsin D from the lysosomes can be attributed to the modification of membranes permeability of the lysosomes during conditioning. When the muscle was pressurized at 100 MPa, the modification of the round shape of the lysosome was observed. With the increase of the pressure applied to the muscle, the release of cathepsin D from the lysosome due to the disruption of membrane was accelerated, and absorption of the released cathepsin D on the myofibrils was observed. From the results obtained, it was clear that the changes in the localization of cathepsin D accompanied with the modification of lysosomes induced by the brief exposure to high pressure were drastic in comparison with that in the muscle during conditioning.

Meat allergy: investigation of potential allergenic proteins in beef.

The potential allergenic proteins in beef were investigated. The sera of ten beef-allergic patients suffering from atopic dermatitis and having a positive RAST score to beef, aged 3-18 years, were obtained from Yoshida Hospital in Japan, and five non-allergic individuals were subjected to this study. The sera of the ten patients reacted strongly to a beef extract, but not to pork and chicken extracts by both ELISA and immunoblotting. The sera of the five control subjects did not react to any of these meat extracts. Three bands having molecular masses of approximately 200 kDa, approximately 67 kDa and approximately 60 kDa were observed by immunoblotting after SDS-PAGE. Two fractions of the beef extract from a Sephadex-gel (G-200) filtration column strongly reacted with the sera of the beef-allergic patients by ELISA and immunoblotting: one fraction had the approximately 67 kDa component and the other had the approximately 200 kDa and approximately 60 kDa components. One of them (approximately 67 kDa) was confirmed to be bovine serum albumin (BSA) by an analysis of the N-terminal amino acid sequence. We could not identify the others by sequencing, but the approximately 200 kDa and approximately 60 kDa components were presumed to be glycoproteins. Bovine gamma (BGG:globulin M.W. approximately 160 kDa) is a glycoprotein and has several subunits. The beef-allergic patients showed strong reactivity to the approximately 200 kDa and approximately 60 kDa components of pure BGG by immunoblotting. Inhibition-ELISA showed that pure BGG preparations strongly inhibited the binding of sera from the beef-allergic patients to the beef extract. These results suggest that the approximately 200 kDa, approximately 67 kDa and approximately 60 kDa components in the beef extract had strong allergenicity: approximately 67 kDa was BSA, and approximately 200 kDa and approximately 60 kDa were presumably aggregated BGG and it's heavy chain, respectively.

Use of PCR/DNA probes to identify circumsporozoite genotype of Plasmodium vivax in China.

The paper reports the result of identifying cirumsporozoite (CS) genotype of Plasmodium vivax by using PCR/DNA probe labeled with biotin. The sensitivity of this method to detect patient blood samples was 0.2 parasite/microl and also with high specific to P. vivax. CS genes from 52 blood samples collected from patients with P. vivax in Hainan and Yunnan Provinces were amplified by PCR and 49 were positive by gel-e electrophoresis analysis, positive rate was 94%. Then the amplified CS genes further were probed with special oligoprobes (PV210 and PV247) that hybridized with the predominant CS repeat region and the variant CS repeat region. The results showed 46 (88.5%) PV210 positive and 6 (11.5%) PV247 positive; 2 hybridized with both probes. The variant genotype was present only in samples from Yunnan Province. The above results showed that the PCR/DNA probe labeled with biotin was highly sensitive and specific to P. vivax and found a CS variant genotype of P. vivax in Yunnan Province of China.

Novel microbial hydroxylation of 13-ethyl-17 beta-hydroxy-18,19-dinor-17 alpha-pregn-4-en-20-yn-3-one.

The microbial transformation of the dl and the d-enantiomer of 13-ethyl-17 beta-hydroxy-18,19-dinor-17 alpha-pregn-4-en-20-yn-3-one (1) were investigated. Poor yields and poor resolutions were usually obtained for the hydroxylation reactions. Transformation of 1 by Cunninghamella blakesleeana gave 6 beta-, 7 beta-, 10 beta-, 15 alpha-hydroxy derivatives 4, 5, 6, 7, and 6 beta,10 beta-dihydroxy derivative 8; transformation of 1 by Cunninghamella echinulata afforded 5, 6, and 8. Biotransformation of dl-1 by Cunninghamella species usually gave 10 beta-hydroxy product with the low enanitomeric excess or as the racemic form. However, C. echinulata was able to efficiently differentiate the two enantiomers of 1 in the course of 6 beta,10 beta-dihydroxylation reactions. The d-enantiomer of the dl-1 was the better substrate for this type hydroxylation. The 7 beta and 15 alpha-hydroxylations of 1 by microbial cultures was unusual for 19-nor type steroids, and these hydroxylation reactions were presumably due to the presence of 17 alpha-ethynyl group.

Microbial hydroxylation of 13-ethyl-17 beta-hydroxy-18,19-dinor-17 alpha-pregn-4-en-20-yn-3-one.

The microbial transformation of the racemic mixture of 13-ethyl-17 beta-hydroxy-18,19-dinor-17 alpha-pregn-4-en-20-yn-3-one (1) was investigated. Rhizopus nigricans (AS 3.2050), R. arrhizus (AS 3.4523), Aspergillus niger (AS 3.2744), A. ochraceus (AS 3.1408), and Curvularia lunata (NRRL 4381) transformed 1 into its 10 beta-hydroxy derivative (2) as a major metabolite. Biotransformation of 1 by Aspergillus ochraceus AS 3.1408 afforded 7 beta-hydroxy derivative (3) as the only product.

[Synthesis of gona-3,5-dienes and studies on their activity for interruption of early pregnancy].

A series of gona-3,5-dienes were synthesized from norgestrel, norethisterone 17 alpha-hydroxyprogesterone and ethisterone via reduction of alpha, beta-unsaturated 3-ketones to allylic alcohols and dehydration with concomitant migration of the double bond. The structures of these gona-3,5-dienes were verified by MS and 1H-NMR spectral data. Pharmacological results showed that compound IVb2 in this series gave rise to significant interruption of early pregnancy in mice. The mechanism of the early pregnancy terminating effect of this compound was discussed.