Cryo-EM structures of the plant plastid-encoded RNA polymerase.

Chloroplasts are green plastids in the cytoplasm of eukaryotic algae and plants responsible for photosynthesis. The plastid-encoded RNA polymerase (PEP) plays an essential role during chloroplast biogenesis from proplastids and functions as the predominant RNA polymerase in mature chloroplasts. The PEP-centered transcription apparatus comprises a bacterial-origin PEP core and more than a dozen eukaryotic-origin PEP-associated proteins (PAPs) encoded in the nucleus. Here, we determined the cryo-EM structures of Nicotiana tabacum (tobacco) PEP-PAP apoenzyme and PEP-PAP transcription elongation complexes at near-atomic resolutions. Our data show the PEP core adopts a typical fold as bacterial RNAP. Fifteen PAPs bind at the periphery of the PEP core, facilitate assembling the PEP-PAP supercomplex, protect the complex from oxidation damage, and likely couple gene transcription with RNA processing. Our results report the high-resolution architecture of the chloroplast transcription apparatus and provide the structural basis for the mechanistic and functional study of transcription regulation in chloroplasts.

Structural basis of exoribonuclease-mediated mRNA transcription termination.

Efficient termination is required for robust gene transcription. Eukaryotic organisms use a conserved exoribonuclease-mediated mechanism to terminate the mRNA transcription by RNA polymerase II (Pol II)1-5. Here we report two cryogenic electron microscopy structures of Saccharomyces cerevisiae Pol II pre-termination transcription complexes bound to the 5'-to-3' exoribonuclease Rat1 and its partner Rai1. Our structures show that Rat1 displaces the elongation factor Spt5 to dock at the Pol II stalk domain. Rat1 shields the RNA exit channel of Pol II, guides the nascent RNA towards its active centre and stacks three nucleotides at the 5' terminus of the nascent RNA. The structures further show that Rat1 rotates towards Pol II as it shortens RNA. Our results provide the structural mechanism for the Rat1-mediated termination of mRNA transcription by Pol II in yeast and the exoribonuclease-mediated termination of mRNA transcription in other eukaryotes.

A cryo-EM structure of KTF1-bound polymerase V transcription elongation complex.

De novo DNA methylation in plants relies on transcription of RNA polymerase V (Pol V) along with KTF1, which produce long non-coding RNAs for recruitment and assembly of the DNA methylation machinery. Here, we report a cryo-EM structure of the Pol V transcription elongation complex bound to KTF1. The structure reveals the conformation of the structural motifs in the active site of Pol V that accounts for its inferior RNA-extension ability. The structure also reveals structural features of Pol V that prevent it from interacting with the transcription factors of Pol II and Pol IV. The KOW5 domain of KTF1 binds near the RNA exit channel of Pol V providing a scaffold for the proposed recruitment of Argonaute proteins to initiate the assembly of the DNA methylation machinery. The structure provides insight into the Pol V transcription elongation process and the role of KTF1 during Pol V transcription-coupled DNA methylation.

Pol IV and RDR2: A two-RNA-polymerase machine that produces double-stranded RNA.

DNA methylation affects gene expression and maintains genome integrity. The DNA-dependent RNA polymerase IV (Pol IV), together with the RNA-dependent RNA polymerase RDR2, produces double-stranded small interfering RNA precursors essential for establishing and maintaining DNA methylation in plants. We determined the cryo­electron microscopy structures of the Pol IV­RDR2 holoenzyme and the backtracked transcription elongation complex. These structures reveal that Pol IV and RDR2 form a complex with their active sites connected by an interpolymerase channel, through which the Pol IV­generated transcript is handed over to the RDR2 active site after being backtracked, where it is used as the template for double-stranded RNA (dsRNA) synthesis. Our results describe a 'backtracking-triggered RNA channeling' mechanism underlying dsRNA synthesis and also shed light on the evolutionary trajectory of eukaryotic RNA polymerases.

[Acupoint catgut embedding improves dysmenorrhea by suppressing activation of NLRP3 inflammasomes in uterine tissues in primary dysmenorrhea rats].

OBJECTIVE: To observe the effect of acupoint catgut embedding on the expression of nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome, interleukin (IL)-1ß and IL-18 in the uterine tissue of primary dysmenorrhea (PD) rats, so as to explore its underlying mechanisms in improving PD. METHODS: Forty female SD rats were randomly divided into control, model, acupoint catgut embedding and medication groups (n＝10 in each group). The PD model was established by subcutaneous injection of Estradiol Benzoate (0.5 mg/rat on the 1st and 10thday, and 0.2 mg/rat from 2nd to 9thday) and Oxytocin (2 U/rat, i．p.). The catgut embedding was applied to bilateral "Sanyinjiao" (SP6) and "Guanyuan" (CV4) on the 1st and 5th day after modeling. Rats of the medication group were treated by intragastric perfusion of Fenbid (0.8 mL/rat, 125 mg/100 mL) once daily for 10 days. The body writhing times in 30 min were recorded. The histopathological changes of the uterine were observed by H．E. staining. Western blot was used to detect the expression of NLRP 3, caspase-1, IL-1ß and IL-18 in uterine tissues. RESULTS: The body writhing times were notably more in the model group than in the control group (P<0.01), and obviously fewer in both medication and catgut embedding groups than in the model group (P<0.05, P<0.01). After modeling, the rats' endometrium was extensively exfoliated and got swelling, the histopathological score and the expression levels of NLRP3, caspase-1, IL-1ß and IL-18 proteins in the uterus tissue were evidently increased in the model group relevant to the control group (P<0.01). Following the treatment, the degree of endometrial exfoliation and edema of the uterus tissue was lightened, the pathological score was significantly reduced (P<0.01), and the expression levels of caspase-1, IL-1ß and IL-18 protein in uterus tissue were markedly decreased in both acupoint catgut embedding and medication groups (P<0.01, P<0.05). The NLRP3 protein expression was significantly decreased in the acupoint catgut embedding group compared with that in the model group (P<0.01). The therapeutic effect of acupoint catgut embedding was significantly superior to that of medication in reducing writhing times and down-regulating expression of NLPR3 protein (P<0.01). No significant differences were found between catgut embedding and medication in histopathological score, and expression levels of caspase-1, IL-1ß and IL-18 proteins (P>0.05). CONCLUSION: The acupoint catgut embedding can significantly alleviate the symptoms and pathological damage in PD rats, which may be related to its effect in inhibiting the activation of NLRP3 inflammasome in the uterine tissue.

[Effects of exogenous ABA on translocation of photosynthate to fruit of Fuji apple during late stage of fruit rapid-swelling.] / å¤–æºè„±è½é ¸å¯¹å¯Œå£«è‹¹æžœæžœå®žè†¨å¤§åŽæœŸå ‰åˆäº§ç‰©å‘æžœå®žè¿è¾“çš„å½±å“.

To clarify the effects and underlying mechanism of ABA on sugar accumulation in apple fruits, 13C trace technique was used to examined the effects of different ABA levels (0, 50, 100 and 150 mg·L-1) and fluoridone (ABA biosynthesis inhibitor) on translocation of photosynthate to fruit during late stage of fruit rapid-swelling in five-year-old 'Yanfu3'/M26/Malus hupehensis Rehd. The results showed that the activities of related enzyme in sugar metabolism, the relative expression of sucrose transporter gene MdSUT1, MdSUT2.2 and sorbitol transporter gene MdSOT3 tended to increase first and then decrease with increasing ABA concentration, with a peak in 100 mg·L-1ABA treatment. Fluridone treatment significantly inhibited the enzymes activities of sugar metabolism and the relative expression of sugar transporters. The treatment of 100 mg·L-1ABA significantly reduced leaf 13C content, increased fruit 13C content and increased the transport rate of photosynthate from leaves to fruits compared with other treatments. Our results indicated that exogenous ABA enhanced sink strength of fruit and promoted the transportation of more photosynthate to fruits, which increased the soluble sugar content in fruits.

[Effects of split combined application of organic-inorganic fertilizers on plant growth, 15N absorption, utilization and loss of Gala apple tree]. / æœ‰æœºæ— æœºè‚¥åˆ†æ¬¡é æ–½å¯¹å˜Žå•¦è‹¹æžœç”Ÿé•¿ã€15N-å°¿ç´ å¸æ”¶åˆ©ç”¨åŠæŸå¤±çš„å½±å“.

Fifteen-year-old 'Gala'/M. hupehensis Rehd. trees and 15N trace technique were used to explore the effects of split combined application of organic-inorganic fertilizers on plant growth, 15N absorption, utilization and loss. The main results were as follows: compared to control, combined application of organic-inorganic fertilizers significantly increased the root-shoot ratio, chlorophyll content, total nitrogen content of leaves and mean fruit mass. The effects of split combined application of organic-inorganic fertilizers treatment were more obvious than one time combined application. Combined application of organic-inorganic fertilizers treatment improved the capacity of 15N derived from fertilizer (Ndff) of different organs, with the effects of split combined application of organic-inorganic fertilizers treatment being more significant. The Ndff value of fruits in different treatments were the highest, followed by leaves, biennial branches, fine roots, large roots and perennial branches, and lowest in trunks. Total N content of plant and 15N-urea utilization rate of the split combined application of organic-inorganic fertilizers treatment were 395.39 g and 28.4% respectively, which were obviously higher than the treatments of one time combined application (342.77 g and 21.1%) and no organic fertilizer application (296.41 g and 14.6%), while 15N loss rate was 51.3%, which was obviously lower than the treatments of one time combined application (57.5%) and no organic fertilizer application (60.6%).

Pathological Changes in APP/PS-1 Transgenic Mouse Models of Alzheimer's Disease Treated with Ganoderma Lucidum Preparation.

Objective To explore the efficacy of ganoderma lucidum preparation(Ling Zhi) in treating APP/PS-1 transgenic mouse models of Alzheimer's disease(AD).Methods APP/PS-1 transgenic mice of 4 months were randomly divided into model group,ganoderma lucidum treatment groups,including high [2250 mg/(kg·d)] and middle [750 mg/(kg·d)] dose groups,i.e.LZ-H and LZ-M groups,and the positive control group(treated with donepezil hydrochloride [2 mg/(kg·d)]).In addition,C57BL/6J wild mice were selected as normal group.The animals were administered for 4 months.Histopathological examinations including hematoxylin-eosin(HE) staining,immunohistochemistry,special staining,and electron microscopy were applied,and then the pathological morphology and structures in different groups were compared. Results The senile plaques and neurofibrillar tangles in the cerebrum and cerebellum were dissolved or disappeared in LZ-H and LZ-M groups.Decrease of amyloid angiopathy was found in LZ-H and LZ-M groups.The immature neurons appeared more in hippocampus and dentate nucleus of LZ-H and LZ-M groups than those in AD model and donepezil hydrochloride groups(hippcampus:F=1.738,P=0.016;dentate nucleus:F=1.924,P=0.026),and these immature neurons differentiated to be neurons.More Purkinje cells loss occurred in AD model mice than that in LZ-H and LZ-M groups(F=9.46,P=0.007;F=9.46,P=0.010).The LZ-H and LZ-M groups had more new neuron stem cells grown up in cerebellum.Electromicroscopic examination showed the hippocampal neurons in LZ-H and LZ-M group were integrated,the nuclear membrane was intact,and the mitochondria in the cytoplasm,endoplasmic reticulum,Golgi bodies,microtubules,and synapses were also complete.The microglial cell showed no abnormality.No toxicity appeared in the pathological specimens of mice treated with ganoderma lucidum preparation.Conclusion The ganoderma lucidum preparation can dissolve and decline or dismiss the senile plaques and neurofibrillar tangles in the brain of AD mice and also reduce the amyloid angiopathy.

[Effectiveness of Ganoderma lucidum preparation in treating simian acquired immune deficiency syndrome].

OBJECTIVE: To explore the effectiveness of the traditional Chinese herbal medicine Lingzhi (Ganoderma lucidum) preparation in treating simian acquired immune deficiency syndrome (SAIDS). METHODS: Five female adult Chinese rhesus monkeys were inoculated rectally with SIVmac239, and were all diagnosed as SAIDS by laboratory and clinical examinations 17 months later. Of these 5 monkeys, 3 (#393, #374, and #381; treatment group) were orally administered with Ganoderma lucidum (2 spores powder capsules plus 2 spores oil capsules on a daily basis), and the remaining other two monkeys (#348 and #361) served as control and did not receive treatment. RESULTS: Animal #393 (treatment group), #361 (control group) and #348 (control group) died of SAIDS (opportunity infection) 3.5 months, 6 months, and 11 months later, respectively. Two animals (#374 and #381) survived. The necropsy revealed depletion and/or exhaustion of their lymphoid tissue. In the monkey #374, the peripheral CD4(+) T lymphocyte increased by 30% in the 6(th) month compared with the baseline level and then fluctuated. The plasma viral load gradually fell and reached about 1 log(10) in the treatment group, but remained stable in the control group. As shown by pathological examinations, the lymph node and spleen of monkeys #374 (treatment group) and #381 (treatment group) showed rehabilitation and reconstruction in the lymphatic tissue, thymus, nerve tissue of gyrus hippocampi, pituitary gland, pineal body, thyroid gland, adrenal gland, and ovary. In the control group, however, animals experienced depletion of lymph nodes, atrophy of spleen, disappearance of thymus, and other disorders in endocrine organs. CONCLUSION: Ganoderma lucidum preparation may have certain protective effect on the immune system, nervous system, and endocrine system of monkeys with SAIDS.

Beneficial effect of HHI-I on cerebral microcirculation, blood-brain barrier in rats and anti-hypoxic activity in mice.

OBJECTIVE: To investigate the effect of HHI-I (I) on the cerebral microcirculation, the blood-brain barrier permeability in rats and anti-hypoxic activity in mice. METHODS: (1) The blood microcirculation of the brain in rats was investigated by laser Doppler flowmetry with the probes laid on the cerebral pia mater or inserted into the brain parenchyma. (2) The protective action of HHI-I against the brain microcirculation disturbance induced by intravenous injection of high-molecular dextran (10%, 9 mL/kg) was observed. (3) The protective effect of HHI-I against lethal hypoxia in mice was observed with a hypoxic chamber containing 5% oxygen. (4) The disruption of the blood-brain barrier (BBB) permeability in rats was caused by phenylephrine-induced hypertension, and the effect of intravenous injection of HHI-I on the BBB permeability was determined using Evans blue as the marker. RESULTS: HHI-I could increase the blood flow of the cerebral microcirculation in rats and possess some protective effects on the brain microcirculatory disturbance. Besides, HHI-I could decrease the brain edema occurring in the process of lethal hypoxia in mice. While increasing the blood flow of brain, HHI-I could lower the BBB permeability in rats. CONCLUSION: HHI-I has several beneficial effects on the cerebral microcirculation, blood-brain barrier in rats and anti-hypoxic activity in mice.

[Comparison of intravenous and intrarectal SIVmac239 infections in rhesus monkeys of Chinese origin].

OBJECTIVE: To investigate the biological and clinical features of Chinese rhesus monkeys after intravenous (IV) and intrarectal (IR) challenge with SIVmac239 in rhesus monkeys of Chinese origin, and compare the differences between the routes of infection. METHODS: Rhesus monkeys of Chinese origin were inoculated with SIVmac239 either by IV (n = 19) or IR (n = 6) routes. Simian immunodeficiency virus (SIV)-specific antibody titer, CD4 + T cell counting, plasma SIV load, lymph node pathology, and clinical manifestations were compared between these two groups 232 or 168 days after challenging. RESULTS: All SIVmac239-inoculated animals became seropositive for SIV-specific antibodies. SIV-specific IgM was detected in IV groups as from day 10 but was not detected in IR for all the time points. Although SIV-specific IgG was detected as from day 30 in both groups, the IgG titers were ten-fold higher in IV group than in IR group after day 168. CD4 + T-cell counting decreased progressively in IV group but remained stable in IR group over time. Plasma SIV RNA loads peaked in all animals between day 10 and day 14 (10(7) copies/ml), then declined to "setpoint" (10(3) - 10(6) copies/ml) about 2 months later. Most inoculated animals manifested lymphadenopathy. Two animals in IV group and one in IR group died of simian AIDS between day 150 and day 210, as evidenced by the autopsies showing the depletion of lymph tissues, Pneumocystis carinii pneumonia and other opportunity infections. Conclusion IV or IR inoculation of SIVmac239 in Chinese rhesus monkeys will result in chronic SIV infection with a similar clinical feature of natural HIV infection, which provides an excellent experimental animal model for AIDS.

[Autoantibodies and autoimmunity in simian immunodeficiency virus-infected monkeys].

OBJECTIVE: To study the relationship between simian acquired immunodeficiency syndromn (SAIDS) and autoimmunity in simian immunodeficiency virus (SIV)-infected monkeys. METHODS: Indirect immunofluorescence assays were performed to detect plasma or serum autoantibodies in SIV-infected monkeys. The heart, liver, spleen, lung, kidney, and lymph node of BALB/c mice, a strain of endothelial cell ECV304, and granulocytes were used as target antigens. These results were compared with HE stained slides of SIV-infected monkeys. RESULTS: The levels of various autoantibodies, including anti-lymphocyte autoantibodies, anti-endothelial cell autoantibodies, and anti-granulocyte antibodies, increased after SIV infection in monkeys. Moreover, pathological examinations showed injuries in the lymphoid tissue and vascular pathological changes in cerebral cortex, submucosa of gastrointestinal tract, interstitial capillaries of myocardium, nephron of the kidney, and sinusoid cell of liver. CONCLUSION: The increased autoantibodies and the pathological changes of tissues and organs confirm the existence of autoimmunity in SIV-infected monkeys.

Protective effect of YHI and HHI-I against experimental acute pancreatitis in rabbits.

AIM:To observe the protective effect of combined i.v. administraction of Yuanhu injection (YHI) and Huoxuehuayu injection-I (HHI-I) against acute pancreatitis (AP) in rabbits.METHODS:Sever acute pancreatitis (SAP) was induced by retrograde infusion of artificial bile juice into biliary-pancreatic duct, and treated with YHI and HHI-I intravenously. The protective effect was judged by the survival time and rate, serum amylase, serum interleukin-6, pancreatic microcirculation and pathological alteration.RESULTS:Combined use of YHI and HHI-I could markedly increase the rabbits' 5-day survival rate after AP (83.3% in the treatment group and 33.3% in control). The serum amylase value (x-± s) decreased to 1596.6U/L± 760.50U/L in the 5th day from the high level (6320.83U/L± 2614.12U/L) in the 1st day after AP in the treatment group, while in the control group the amylase activity in the 5th day was 2095.0U/L± 1081.87U/L, being significantly different from that before AP (837.17U/L± 189.12U/L). YHI and HHI-I also obviously improved the pancreatic microcirculation and lowered the serum interleukin-6 level, one of the indices of severe pancreatitis. Pathological examination indicated all the changes typical for AP in YHI and HHI-I treatment group were milder than those in the control.CONCLUSION:YHI and HHI-I used in combination might have protective effect against acute pancreatitis in rabbits.