G protein γ subunit modulates expression of plant-biomass-degrading enzyme genes and mycelial-development-related genes in Penicillium oxalicum.

Heterotrimeric-G-protein-mediated signaling pathways modulate the expression of the essential genes in many fundamental cellular processes in fungi at the transcription level. However, these processes remain unclear in Penicillium oxalicum. In this study, we generated knockout and knockout-complemented strains of gng-1 (POX07071) encoding the Gγ protein and found that GNG-1 modulated the expression of genes encoding plant-biomass-degrading enzymes (PBDEs) and sporulation-related activators. Interestingly, GNG-1 affected expression of the cxrB that encodes a known transcription factor required for the expression of major cellulase and xylanase genes. Constitutive overexpression of cxrB in ∆gng-1 circumvented the dependence of PBDE production on GNG-1. Further evidence indicated that CxrB indirectly regulated the transcription levels of key amylase genes by controlling the expression of the regulatory gene amyR. These data extended the diversity of Gγ protein functions and provided new insight into the signal transduction and regulation of PBDE gene expression in filamentous fungi. KEY POINTS: • GNG-1 modulates the expression of PBDE genes and sporulation-related genes. • GNG-1 controls expression of the key regulatory gene cxrB. • Overexpression of cxrB circumvents dependence of PBDE production on GNG-1.

A mitogen-activated protein kinase PoxMK1 mediates regulation of the production of plant-biomass-degrading enzymes, vegetative growth, and pigment biosynthesis in Penicillium oxalicum.

Mitogen-activated protein kinase (MAPK) cascades are broadly conserved and play essential roles in multiple cellular processes, including fungal development, pathogenicity, and secondary metabolism. Their function, however, also exhibits species and strain specificity. Penicillium oxalicum secretes plant-biomass-degrading enzymes (PBDEs) that contribute to the carbon cycle in the natural environment and to utilization of lignocellulose in industrial processes. However, knowledge of the MAPK pathway in P. oxalicum has been relatively limited. In this study, comparative transcriptomic analysis of P. oxalicum, cultured on different carbon sources, found ten putative kinase genes with significantly modified transcriptional levels. Six of these putative kinase genes were knocked out in the parental strain ∆PoxKu70, and deletion of the gene, Fus3/Kss1-like PoxMK1 (POX00158), resulted in the largest reduction (91.1%) in filter paper cellulase production. Further tests revealed that the mutant ∆PoxMK1 lost 37.1 to 92.2% of PBDE production, under both submerged- and solid-state fermentation conditions, compared with ∆PoxKu70. In addition, the mutant ∆PoxMK1 had reduced vegetative growth and increased pigment biosynthesis. Comparative transcriptomic analysis showed that PoxMK1 deletion from P. oxalicum downregulated the expression of major PBDE genes and known regulatory genes such as PoxClrB and PoxCxrB, whereas the transcription of pigment biosynthesis-related genes was upregulated. Comparative phosphoproteomic analysis revealed that PoxMK1 deletion considerably modified phosphorylation of key transcription- and signal transduction-associated proteins, including transcription factors Mcm1 and Atf1, RNA polymerase II subunits Rpb1 and Rpb9, MAPK-associated Hog1 and Ste7, and cyclin-dependent kinase Kin28. These findings provide novel insights into understanding signal transduction and regulation of PBDE gene expression in fungi.Key points• PoxMK1 is involved in expression of PBDE- and pigment synthesis-related genes.• PoxMK1 is required for vegetative growth of P. oxalicum.• PoxMK1 is involved in phosphorylation of key TFs, kinases, and RNA polymerase II.

Development and characterization of microsatellite markers in Prunus sibirica (Rosaceae).

PREMISE OF THE STUDY: Microsatellite loci were developed for Prunus sibirica to investigate genetic diversity, population genetic structure, and marker-assisted selection of late-blooming cultivars in the breeding of P. sibirica. • METHODS AND RESULTS: Using a magnetic bead enrichment strategy, 19 primer pairs were developed and characterized across 40 individuals from three P. sibirica wild populations and six individuals of P. armeniaca. The number of alleles per locus varied from three to 11 and the observed and expected heterozygosities ranged from 0.063 to 0.917 and 0.295 to 0.876, respectively, in the three P. sibirica wild populations. All primer pairs could be successfully amplified in six individuals of P. armeniaca. • CONCLUSIONS: These microsatellite primer pairs should be useful for population genetics, germplasm identification, and marker-assisted selection in the breeding of P. sibirica and related species.

Epigallocatechin-3-gallate inhibits angiotensin II and interleukin-6-induced C-reactive protein production in macrophages.

BACKGROUND: Consumption of green tea has been associated with health benefits against multiple diseases including cardiovascular diseases. However, the action mechanisms of green tea and its major ingredient epigallocatechin-3-gallate (EGCG) against cardiovascular diseases are still unclear. Emerging evidence has suggested a common role for C-reactive protein (CRP) in the pathogenesis of inflammation and atherosclerosis. Therefore, the effect of EGCG on angiotensin II (Ang II)- and interleukin-6 (IL-6)-induced CRP production in U937 macrophages and the possible mechanisms were observed. METHODS: U937 macrophages were cultured, and Ang II and IL-6 were used as stimulants for generation of CRP. U937 macrophages were preincubated with EGCG at 1, 3, 10 µM for 1 h prior to the stimulation. mRNA expression and protein level were determined by RT-PCR and ELISA, respectively. ROS production was observed by a fluorescence microscope. RESULTS: Pretreatment of macrophages with EGCG prior to the stimulation concentration-dependently inhibited Ang II- and IL-6-induced expression of CRP both in protein and mRNA levels. Meanwhile, EGCG reduced Ang II- and IL-6-stimulated generation of ROS in macrophages. CONCLUSION: EGCG is able to inhibit Ang II- and IL-6-stimulated CRP expression in macrophages to produce an anti-inflammation by interfering with ROS generation. The finding is helpful to update understanding of anti-atherosclerotic effects of EGCG.

Rosiglitazone inhibits angiotensin II-induced C-reactive protein production in human aortic endothelial cells through regulating AT(1)-ROS-MAPK signal pathway.

OBJECTIVE: Atherosclerosis is an inflammatory disease. As an inflammatory molecule, C-reactive protein (CRP) plays a direct role in atherogenesis. Our previous study confirmed that angiotensin II (Ang II) is capable of inducing CRP generation in human aortic endothelial cells (HAECs). The present study observed the effect of rosiglitazone on Ang II-induced CRP expression in HAECs and molecular mechanisms. METHODS: HAECs were cultured, and Ang II (10(-6) M) was used as a stimulant for the generation of CRP and reactive oxygen species (ROS). HAECs were preincubated with rosiglitazone at 1, 10, 100 µM for 18 h prior to the stimulation. mRNA and protein expressions were identified by reverse transcription polymerase chain reaction and Western blot, respectively. ROS production was observed by a fluorescence microscope. RESULTS: Pretreatment of HAECs with rosiglitazone prior to Ang II stimulation markedly downregulated Ang II-induced mRNA and protein expressions of CRP (maximal inhibition of 55.2 and 99.1 %, P < 0.001 vs. Ang II alone) and AT(1) (maximal inhibition of 66.4 and 90.5 %, P < 0.001 vs. Ang II alone) in a concentration-dependent manner, inhibited Ang II-stimulated ROS production (P < 0.01 vs. Ang II alone), and attenuated Ang II-induced phosphorylation of ERK1/2 and JNK (P < 0.001 vs. Ang II alone). Meanwhile, AT(1) receptor blocker losartan also reduced Ang II-stimulated ROS generation in HAECs (P < 0.001 vs. Ang II alone). CONCLUSIONS: Rosiglitazone at the concentrations used in the present experiment is able to inhibit Ang II-induced CRP generation in HAECs by regulating AT(1)-ROS-MAPK signal pathway. These results strengthen our understanding of the anti-inflammatory and anti-atherosclerotic effects of rosiglitazone.

Pravastatin inhibits C-reactive protein generation induced by fibrinogen, fibrin and FDP in isolated rat vascular smooth muscle cells.

OBJECTIVE: The available evidence indicates that C-reactive protein (CRP) participates directly in atherosclerosis formation as an inflammatory molecule. Our previous investigation suggested that fibrinogen, fibrin and fibrinogen degradation products (FDP) produce a pro-inflammatory effect on vascular smooth muscle cells (VSMCs) through inducing CRP generation. In the present study, we observed the effect of pravastatin on CRP generation induced by fibrinogen, fibrin and FDP in rat VSMCs. METHODS: VSMCs from Sprague-Dawley rats were cultured. Fibrinogen, fibrin and FDP were used as stimulants for CRP generation. VSMCs were preincubated with pravastatin at 10, 30, 100 µM for 30 min prior to stimulation. CRP mRNA expression was studied by reverse transcription polymerase chain reaction (RT-PCR). CRP levels in the supernatant of VSMCs were measured by enzyme-linked immunosorbent assay (ELISA). CRP expression in VSMCs was examined with immunocytochemical staining. RESULTS: ELISA analysis showed that the pravastatin concentration-dependently reduced fibrinogen-, fibrin- and FDP-stimulated generation of CRP in VSMCs, with maximal inhibition of 56.6, 55.7 and 62.3%, respectively. Immunocytochemical staining and RT-PCR revealed that pravastatin inhibited protein and mRNA expression of CRP in VSMCs significantly. CONCLUSIONS: Pravastatin at the concentrations used in the present experiment has ability to relieve vascular inflammation and to restrain atherosclerotic processes via inhibiting the CRP production induced by fibrinogen, fibrin and FDP in VSMCs, which helps explain the beneficial effects of pravastatin on atherosclerosis.

Effects of Panax notoginseng saponins on proliferation and apoptosis of vascular smooth muscle cells.

AIM OF THE STUDY: Atherosclerosis is a common cardiovascular disease, and linked with the development of many cardiovascular complications, such as myocardial ischemia and stroke. Although pathogenesis of atherosclerosis is not completely elucidated, increasing evidence has demonstrated that abnormal proliferation of vascular smooth muscle cells (VSMCs) plays an important role in formation of atherosclerosis. Previous studies showed that saponins from Panax notoginseng (PNS) possess anti-atherosclerotic properties. However, the mechanism of PNS against atherosclerosis is not well understood. Therefore, the present study observed the effects of PNS on proliferation and apoptosis of VSMCs. MATERIALS AND METHODS: Rat VSMCs were cultured, and platelet-derived growth factor (PDGF) was used to stimulate cell proliferation. The viability of VSMCs was assessed with the MTT method. VSMCs apoptosis was detected by flow cytometry. Expressions of apoptosis related protein p53, Bax, caspase-3 and Bcl-2 were determined using Western blot. RESULTS: Pretreatment of the cells with PNS (200, 400, 800 µg/mL) significantly inhibited proliferation of PDGF-stimulated VSMCs, and induced apoptosis of the proliferated VSMCs in a concentration-dependent way. Western blot analysis showed that PNS upregulated expressions of pro-apoptotic protein p53, Bax and caspase-3, downregulated expression of anti-apoptotic protein Bcl-2, and enlarged Bax/Bcl-2 ratio in the proliferated VSMCs induced by PDGF. CONCLUSIONS: This study demonstrates that PNS both inhibits VSMCs proliferation and induces VSMCs apoptosis through upregulating p53, Bax, caspase-3 expressions and downregulating Bcl-2 expression, which constitute the pharmacological basis of its anti-atherosclerotic action.

[Mechanism of Dahuang Zhechong pill against atherosclerosis induced by balloon angioplasty in rabbits].

OBJECTIVE: To study the mechanism of Dahuang Zhechong pill (DHZCP) against atherosclerosis induced by balloon angioplasty in rabbits. METHODS: Atherosclerosis model was established by the combination of balloon angioplasty-induced endothelial injury and high cholesterol feeding in rabbit. Male New Zealand rabbits were divided into six groups randomly: normal control, sham, model, positive control and two doses of DHZCP-treated groups. Rabbits in DHZCP-treated groups were intragastrically administered 0.9 and 1.8 g/kg DHZCP for 60 days respectively,and rabbits in positive control group were given 0.5 g/kg Danshen. MDA, NO levels and SOD activity in serum, and MPO activity in the vascular wall were determined with spectrophotometry. Expressions of proliferating cell nuclear antigen (PCNA) and BCL-2 in the vascular wall were detected by SP immuohistochemical technique. RESULTS: Compared with the model group, DHZCP significantly reduced serum MDA level and MPO activity in the vascular wall, increased serum NO level and SOD activity,and inhibited PCNA and BCL-2 expressions in the vascular wall. CONCLUSION: DHZCP inhibits the formation and development of atherosclerosis through anti-oxidative action, protecting endothelium from injury,inhibiting proliferation and promoting apoptosis of vascular smooth muscle cells.

[Study on in vitro release and transdermal behaviors of mumps cataplasm of complex prescription].

OBJECTIVE: To investigate the in vitro release and transdermal behaviors of mumps cataplasm of complex prescription. METHODS: The improved Franz difficusion was used and the concentration of drugs was detected. DCS was determined by HPLC and flavanoid contents were determined by UV. RESULTS: The accumulation skin permeation percentage of DCS and flavanoid was 32% and 21%, respectively, the in vitro release percentage of DCS and flavanoid was 47% and 42%, respectively. CONCLUSION: The transdermal behavior of mumps cataplasm of complex prescription is an zero-order kinetics progress. The in vitro release behavior is in accordance with the Higuchi equation.

Over-expression of the apple spermidine synthase gene in pear confers multiple abiotic stress tolerance by altering polyamine titers.

An apple spermidine synthase (SPDS) gene (MdSPDS1) was verified to encode a functional protein by the complementation of the spe3 yeast mutant, which lacks the SPDS gene. To justify our hypothesis that apple SPDS is involved in abiotic stress responses and to obtain transgenic fruit trees tolerant to abiotic stresses as well, MdSPDS1-over-expressing transgenic European pear (Pyrus communis L. 'Ballad') plants were created by Agrobacterium-mediated transformation. A total of 21 transgenic lines showing various spermidine (Spd) titers and MdSPDS1 expression levels were obtained. Selected lines were exposed to salt (150 mM NaCl), osmosis (300 mM mannitol), and heavy metal (500 microM CuSO4) stresses for evaluating their stress tolerances. Transgenic line no. 32, which was revealed to have the highest Spd accumulation and expression level of MdSPDS1, showed the strongest tolerance to these stresses. When growth increments, electrolyte leakage (EL), and values of thiobarbituric acid reactive substances (TBARS) were monitored, line no. 32 showed the lowest growth inhibition and the least increase in EL or TBARS under stress conditions. Spd titers in wild-type and transgenic lines showed diverse changes upon stresses, and these changes were not consistent with the changes in MdSPDS1 expressions. Moreover, there were no differences in the sodium concentration in the shoots between the wild type and line no. 32, whereas the copper concentration was higher in the wild type than in line no. 32. Although the mechanism(s) underlying the involvement of polyamines in stress responses is not known, these results suggest that the over-expression of the SPDS gene substantially increased the tolerance to multiple stresses by altering the polyamine titers in pear. Thus, MdSPDS1-over-expressing transgenic pear plants could be used to improve desert land and/or to repair polluted environments.

Suppression subtractive hybridization identifies genes induced in response to UV-B irradiation in apple skin: isolation of a putative UDP-glucose 4-epimerase.

Suppression subtractive hybridization (SSH) successfully identified 11 cDNAs in apple skin with highly induced expression as a result of ultraviolet (UV)-B irradiation. Apart from three putative flavonoid biosynthetic genes, chalcone synthase (CHS; A5C), flavanone-3-hydroxylase (F3H; B5F), and flavonol synthase (FLS; D1F), five clones (A1H, A10E, B11G, D5F, and D11H) were induced by low temperature (17 degrees C) as well, which is also known to induce anthocyanin accumulation in apple skin. Moreover, four clones (A1H, A10E, B11G, and D11H), showing higher expression levels in the skin, accumulated higher anthocyanin concentrations than their counterparts. Of the four clones, only A10E, a putative UDP-glucose 4-epimerase (UGE), was deemed to play an important role in anthocyanin accumulation in apple skin based on the facts that: (i) its transcription level was higher in the deep red cultivar, 'Jonathan', than in the pale red cultivar, 'Tsugaru'; and (ii) it could reversibly catalyse UDP-glucose to UDP-galactose, and the latter molecule is a major sugar donor for cyanidin-glycoside in apple. Therefore, the full-length cDNA of A10E was isolated by rapid amplification of cDNA ends (RACE) and designated as MdUGE1. Further analysis demonstrated that UGE enzymatic activity was positively correlated with anthocyanin accumulation in apple skin. Thus, MdUGE1 isolated by SSH could play an important role in anthocyanin biosynthesis in apple skin in concert with other flavonoid biosynthetic genes.

Polyamine biosynthesis of apple callus under salt stress: importance of the arginine decarboxylase pathway in stress response.

To clarify the involvement of the arginine decarboxylase (ADC) pathway in the salt stress response, the polyamine titre, putrescine biosynthetic gene expression, and enzyme activities were investigated in apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.] in vitro callus under salt stress, during recovery after stress, and when ADC was inhibited by D-arginine, an inhibitor of ADC. Salt stress (200 mM NaCl) caused an increase in thiobarbituric acid-reactive substances (TBARS) and electrolyte leakage (EL) of the callus, which was accompanied by an increase in free putrescine content, during 7 d of treatment. Conjugated putrescine was also increased, but this increase was limited to the early stage of salt stress. Accumulation of putrescine was in accordance with induction of ADC activity and expression of the apple ADC gene (MdADC). When callus that had been treated with 200 mM NaCl was transferred to fresh medium with (successive stress) or without (recovery) NaCl, TBARS and EL were significantly reduced in the recovery treatment, indicating promotion of formation of new callus cells, compared with the successive stress treatment. Meanwhile, MdADC expression and ADC activity were also decreased in the callus undergoing recovery, whereas those of the callus under successive stress were increased. Ornithine decarboxylase (ODC) activity showed a pattern opposite to that of ADC in these conditions. D-Arginine treatment led to more serious growth impairment than no treatment under salt stress. In addition, accumulation of putrescine, induction of MdADC, and activation of ADC in D-arginine-treated callus were not comparable with those of the untreated callus. Exogenous addition of putrescine could alleviate salt stress in terms of fresh weight increase and EL. All of these findings indicated that the ADC pathway was tightly involved in the salt stress response. Accumulation of putrescine under salt stress, the possible physiological role of putrescine in alleviating stress damage, and involvement of MdADC and ADC in response to salt stress are discussed.

Phylogenetic relationships among citrus and its relatives as revealed by SSR markers.

Phylogenetic relationships among 29 accessions belonging to Citrus, Poncirus, Fortunella, Microcitrus, Eremocitrus, Atlantia and Severinia were investigated using SSR markers. Seven SSR primers generated 114 polymorphic alleles, with an average of 16.3 alleles per primer. Cluster analysis via neighbour-joining method showed that Microcitrus was close to Citrus; Poncirus was distant from Citrus, which suggested that Poncirus could not be derived from Citrus. High frequency of the homozygous SSR locus supported the species status of Fumin trifoliate orange. Separation of neither Papeda and Citrus nor Archicitrus and Metacitrus was well resolved. The present work confirmed citron, pummelo and mandarin as basic species of cultivated citrus since they could be placed into three distinct clusters.