Identification of key enzyme genes involved in biosynthesis of steroidal saponins and analysis of biosynthesis pathway in Polygonatum cyrtonema / 中国中药杂志

Steroidal saponins, which are the characteristic and main active constituents of Polygonatum, exhibit a broad range of pharmacological functions, such as regulating blood sugar, preventing cardiovascular and cerebrovascular diseases and anti-tumor. In this study, we performed RNA sequencing(RNA-Seq) analysis for the flowers, leaves, roots, and rhizomes of Polygonatum cyrtonema using the BGISEQ-500 platform to understand the biosynthesis pathway of steroidal saponins and study their key enzyme genes. The assembly of transcripts for four tissues generated 129 989 unigenes, of which 88 958 were mapped to several public databases for functional annotation, 22 813 unigenes were assigned to 53 subcategories and 64 877 unigenes were annotated to 136 pathways in KEGG database. Furthermore, 502 unigenes involved in the biosynthesis pathway of steroidal saponins were identified, of which 97 unigenes encoding 12 key enzymes. Cycloartenol synthase, the first key enzyme in the pathway of phytosterol biosynthesis, showed conserved catalytic domain and substrate binding domain based on sequence analysis and homology modeling. Differentially expressed genes(DEGs) were identified in rhizomes as compared to other tissues(flowers, leaves or roots).The 2 437 unigenes annotated by KEGG showed rhizome-specific expression, of which 35 unigenes involved in the biosynthesis of steroidal saponins. Our results greatly extend the public transcriptome dataset of Polygonatum and provide valuable information for the identification of candidate genes involved in the biosynthesis of steroidal saponins and other important secondary metabolites.

Cloning and characterization of chalcone synthase and chalcone isomerase genes in Arisaema heterophyllum / 中国中药杂志

Chalcone synthase( CHS) and chalcone isomerase( CHI) are key enzymes in the biosynthesis pathway of flavonoids. In this study,unigenes for CHS and CHI were screened from the transcriptome database of Arisaema heterophyllum. The open reading frame( ORFs) of chalcone synthase( Ah CHS) and chalcone isomerase( Ah CHI) were cloned from the plant by RT-PCR. The physicochemical properties,expression and structure characteristics of the encoded proteins Ah CHS and Ah CHI were analyzed. The ORFs of Ah CHS and Ah CHI were 1 176,630 bp in length and encoded 392,209 amino acids,respectively. Ah CHS functioned as a symmetric homodimer. The N-terminal helix of one monomer entwined with the corresponding helix of another monomer. Each CHS monomer consisted of two structural domains. In particular,four conserved residues define the active site. The tertiary structure of Ah CHI revealed a novel open-faced β-sandwich fold. A large β-sheet( β4-β11) and a layer of α-helices( α1-α7) comprised the core structure. The residues spanning β4,β5,α4,and α6 in the three-dimensional structure were conserved among CHIs from different species. Notably,these structural elements formed the active site on the protein surface,and the topology of the active-site cleft defined the stereochemistry of the cyclization reaction. The homology comparison showed that Ah CHS had the highest similarity to the CHS of Anthurium andraeanum,while Ah CHI had the highest similarity to the CHI of Paeonia delavayi. This study provided the basis for the functional study of Ah CHS and Ah CHI and the further study on plant flavonoid biosynthesis pathway.

Value of cytopathology in endobronchial ultrasound-guided transbronchial needle aspiration / 中华病理学杂志

<p><b>OBJECTIVE</b>To evaluate the role of cytopathology in endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) for lung tumor diagnosis and staging.</p><p><b>METHODS</b>Two-hundred consecutive cases of lung tumor with EBUS-TBNA performed during the period from April, 2009 to September, 2010 in Shanghai Cancer Hospital were retrospectively reviewed. The cytologic diagnoses were categorized as non-diagnostic, negative, suspicious and malignant. When available, cell block preparation and immunohistochemistry were performed. On the 22 positive cases diagnosed by on-site evaluation, epidermal growth factor receptor (EGFR) mutation study was carried out.</p><p><b>RESULTS</b>In the 200 cases of cytology specimens, 122 cases (69.3%) were diagnosed as malignant, 42 cases (23.9%) as benign and 12 cases (6.8%) as suspicious for malignancy. The non-diagnostic rate was 12.0% (24/200). Amongst the 200 cases studied, 140 cases (70.0%) had histologic correlation available (via core biopsy, mediastinoscopic biopsy or surgical excision). The sensitivity and specificity of EBUS-TBNA cytologic diagnoses were 94.4% and 100%, when using histopathologic findings and clinical follow-up data as gold standard. The cell block preparation and immunohistochemistry were useful in subtyping and diagnosis of extrathoracic malignancy. EGFR mutations were detected in 8 cytology samples (36.4%).</p><p><b>CONCLUSIONS</b>EBUS-TBNA is a sensitive and specific tool for diagnosis and staging of lung cancer. The cytology samples can be used for further ancillary investigations including cell block preparation, immunohistochemistry and molecular studies.</p>

Effect of magnesium isoglycyrrhizinate on PLA2 during liver tissue injury following limb ischemia/reperfusion in rats / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effects of magnesium isoglycyrrhizinate (MI) on the changes of phospholipase A2 (PLA2) induced during liver tissue injury following limb ischemia/reperfusion (I/R) in rats.</p><p><b>METHOD</b>Twenty-four healthy male Sprague-Dawley rats weighing (230+/-30) g were randomly divided into three groups (n = 8 each) as follows: control (Group C: anesthetization without any ischemia); I/R injury (Group I/R: 4 h ischemia induced by rubber band ligation of the left hind limb around the roots of the hind limb, followed by 6 h of reperfusion, with 1 mL normal saline given via tail vein prior to reperfusion); MI-treated group (Group MI: underwent ischemia and reperfusion, with 1 mL MI (30 mg/kg) infused prior to reperfusion). Levels of TNFa and PLA2 in plasma and liver tissue were measured by enzyme-linked immunosorbent assay (ELISA). Levels of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase (CK), myeloperoxidase (MPO), and malondialdehyde (MDA), and activities of MPO and MDA in liver tissue were measured by colorimetry. Ultrastructural changes of liver tissue were observed by electron microscopy.</p><p><b>RESULTS</b>The MI group had significantly lower PLA2 and TNFa in liver homogenates and serum than the I/R group (both P less than 0.05). Serum ALT, AST, LDH, and CK were significantly lower in the MI group than in the I/R group (all P less than 0.05), as were the levels of MPO and MDA in liver homogenates and serum (all P less than 0.05). The I/R group showed significantly more liver tissue damage, which appeared to be attenuated in the MI group.</p><p><b>CONCLUSION</b>MI treatment can inhibit the I/R-induced TNFa, PLA2, and MDA in plasma and liver tissue, as well as decrease the I/R-induced MPO activity in rats. Thus, MI may have protective effects against liver tissue injury following limb ischemia/reperfusion.</p>

A study on pathological changes of closed diffuse brain injury in rats with H.E., esterification-silver stain and albumin immunohistochemical stain / 法医学杂志

OBJECTIVE@#To observed the pathological changes of closed diffuse brain injury in the rats died immediately and 15 min to 5 days after the injury.@*METHODS@#H.E. staind and esterification-silver stain were applied to investigate the closed diffuse brain injury.@*RESULTS@#In rats died immediately after the concussive injury, a number of shrunken neurons(type I change), distended neurons(type II change) and wave-like nerve fibers were identified in the brain tissue, especially in brain-stem. At 2 h and 8 h after injury, brain edema and axonal swelling appeared clearly in the cortex and white matter, especially in brain-stem. At post-traumatic 8 h and 24 h, the axonal retraction balls began to appear. The amount of neurons undergoing type I and II changes and constraction balls increased along with the survivor time. After 4 days and 5 days, brain edema alleviated, but the retraction balls and axonal swelling still existed. With Esterifica-tion silver stain, the above changes of neurons and nerve fibers were more obvious. With ABC stain, the distribution of albumin(Al) was extended from the perio-vascular area to diffuse distribution. Al positive staining were more obvious in injuried neurons and nerve fibers.@*CONCLUSION@#The distribution of the concussive damage in the brain are coup, contra-coup and centripental.