Identification of the lysine and histidine transporter family in Camellia sinensis and the characterizations in nitrogen utilization

In plants,the lysine and histidine transporter(LHT)family represent a class of proteins that mediate the uptake,translocation,and utilization of amino acids.The tea plant(Camellia sinensis)is a perennial evergreen with a relatively high level of amino acids.However,systematic identification and molecular characterization of the LHT gene family has rarely been reported in tea plants.In this study,22 CsLHTs were identified from the‘Shuchazao’genome and classified into two groups.The modeled three-dimensional structure and the conserved domains presented a high similarity among the LHTs proteins.Moreover,it was predicted that a few genes were conserved through the analysis of the physiochemical characters,structures and cis-elements in promoters.The expression patterns in tea plants revealed that CsLHT7 was mainly expressed in the roots,and CsLHT4 and CsLHT11 exhibited relatively high expression in both the roots and leaves.Moreover,the expression of all three genes could be induced by organic nitrogen.Additionally,heterogeneous expression of CsLHT4,CsLHT7 and CsLHT11 in Arabidopsis thaliana decreased the aerial parts biomass compared with that in WT plants while significantly increased the rosette biomass only for CsLHT11transgenic plants versus WT plants.Overall,our results provide fundamental information about CsLHTs and potential genes in N utilization for further analysis in tea plants.

基于金观音(Camellia sinensis)单体型基因组CsUGT等位基因的鉴定及表达

【目的】植物的尿苷二磷酸糖基转移酶(UGT)是催化次生代谢物糖基化的关键酶,UGT基因主要参与植物逆境胁迫的响应。对CsUGT等位基因进行鉴定与表达分析,为其在茶树抗性及乌龙茶加工中香气形成的作用提供理论参考。【方法】基于金观音(Camellia sinensis‘Jinguanyin’)单体型基因组,利用生物信息学方法分析,并通过实时荧光定量PCR技术分析CsUGT基因在低温(4℃)、干旱(10%PEG-6000溶液)和激素(100μmol·L^(−1)MeJA溶液)处理下0、3、6、12和24 h的表达,以及CsUGT基因在金观音乌龙茶加工过程中鲜叶、萎凋、一摇、二摇、三摇和杀青前的表达。【结果】生物信息学分析结果表明,从金观音单体型基因组中鉴定出了142对等位基因,多数定位于叶绿体中,并基于进化树将这些基因分为5个亚家族。CsUGT等位基因的结构较保守,相同基因的亲本等位基因的结构相似。转录组数据分析表明,发现多数CsUGT等位基因具有组织表达特性。实时荧光定量PCR检测发现,在MeJA处理下,CsUGT002、CsUGT047和CsUGT091的表达量在6 h达到峰值;在低温和干旱处理下,部分CsUGT基因的表达量分别在3或24 h时达到峰值。此外,在金观音乌龙茶加工过程中,CsUGT115的表达量显著上调,部分CsUGT基因在三摇时的表达量达到峰值。【结论】CsUGT蛋白多数为酸性和亲水性蛋白,并定位于叶绿体中;CsUGT等位基因结构较保守具有组织表达特性,部分CsUGT基因参与调控茶树的逆境胁迫及乌龙茶加工工艺中摇青时的香气合成。

Genetic analyses of ancient tea trees provide insights into the breeding history and dissemination of Chinese Assam tea(Camellia sinensis var.assamica)

Chinese Assam tea(Camellia sinensis var.assamica)is an important tea crop with a long history of cultivation in Yunnan,China.Despite its potential value as a genetic resource,its genetic diversity and domestication/breeding history remain unclear.To address this issue,we genotyped 469 ancient tea plant trees representing 26 C.sinensis var.assamica populations,plus two of its wild relatives(six and three populations of C.taliensis and C.crassicolumna,respectively)using 16 nuclear microsatellite loci.Results showed that Chinese Assam tea has a relatively high,but comparatively lower gene diversity(H_(S)=0.638)than the wild relative C.crassicolumna(H_S=0.658).Clustering in STRUCTURE indicated that Chinese Assam tea and its two wild relatives formed distinct genetic groups,with considerable interspecific introgression.The Chinese Assam tea accessions clustered into three gene pools,corresponding well with their geographic distribution.However,New Hybrids analysis indicated that 68.48%of ancient Chinese Assam tea plants from Xishuangbanna were genetic intermediates between the Puer and Lincang gene pools.In addition,10%of the ancient Chinese Assam tea individuals were found to be hybrids between Chinese Assam tea and C.taliensis.Our results suggest that Chinese Assam tea was domesticated separately in three gene pools(Puer,Lincang and Xishuangbanna)in the Mekong River valley and that the hybrids were subsequently selected during the domestication process.Although the domestication history of Chinese Assam tea in southwestern Yunnan remains complex,our results will help to identify valuable genetic resources that may be useful in future tea breeding programs.

Genome-Wide Identification of ABCC Gene Subfamily Members and Functional Analysis of CsABCC11 in Camellia sinensis

The ATP-binding cassette(ABC)transporter is a gene superfamily in plants.ATP-binding cassette subfamily C(ABCC)protein is a multidrug resistance-associated(MRP)transporter.They play various roles in plant growth,development,and secondary metabolite transport.However,there are few studies on ABCC transporters in tea plants.In this study,genome-wide association study(GWAS)analysis of epigallocatechin gallate(EGCG)content in 108 strains of Kingbird revealed that CsABCCs may be involved in EGCG transport.We identified 25 CsABCC genes at the genomic level of the tea plant,their phylogenetic tree,gene structure,targeted miRNA and other bioinformatics were analyzed.The expression patterns of CsABCCs in eight different tissues and abiotic stress indicate that they have potential roles in regulating the growth,development,and defense of tea plants.The correlation analysis revealed that the expression of the CsABCC11 gene was closely related to the EGCG content in tea buds of 108 strains of the Kingbird,and the subcellular localization experiments in tobacco showed that CsABCC11 protein was localized on the plasma membrane.The virus-induced gene silencing(VIGS)strategy in tea plants further verified that CsABCC11 was involved in EGCG accumulation.Our study laid a foundation for studying the biological function of CsABCC and provided a new candidate molecular marker gene for further EGCG-related variety breeding,which will be of great interest to breeders.

Analysis of NPK in Camellia sinensis, Gliricidia sepium, and Musa acuminata Biomasses for Preparation of an Organic Fertilizers Formula for Young Tea Plants (Musa acuminata) and Studying of Their Nutrient Release Capacity in the Biodegradation Process

This study investigates the nitrogen (N), phosphorous (P), and potassium (K) contents in raw biomasses of Camellia sinensis, Gliricidia sepium, and Musa acuminata. Therein, the highest N and P content was seen in Camellia sinensis 116.80 ± 0.08 mg and 66.00 ± 0.14 mg respectively. The highest K content (106.80 ± 0.04 mg) was observed in Musa acuminata. Next, all three types of plant materials were allowed to decompose in water for 3 weeks, and a sample from each was analyzed for NPK after the 1st, 2nd, and 3rd week during decomposition. A significant increase in the release of N, P, and K by the Camellia sinensis to water (P Musa acuminate were not significantly changed (P > 0.05) over time. The ratio for N:P:K was calculated for raw biomass samples and decomposed samples to find the best fitting N:P:K ratio to apply to young tea plants as organic fertilizers. In addition to that, the microbial insight of these organic compounds was analyzed by observing how microbial population increased with decomposition by the enumeration of the total microbial count. A considerable increment in total microbial count was observed up to 3.28 × 106, 1.21 × 1010, 2.18 × 108, and 6.49 × 107 CFU/ml for Camellia sinensis, Gliricidia sepium, Musa accuminata (leaves), and Musa accuminata (trunk) respectively. The presence of phosphate solubilizing bacteria (PSB) and nitrogen solubilizing bacteria (NSB) throughout the decomposition period was confirmed by their growth on NBRIP and a modified nutrient medium that was specifically designed for the identification of ammonifiers respectively. Prepared fertilizer samples were applied to young tea plants that were grown in the Mawanella area in Sri Lanka (7°15'12.42"N 80°26'47.62"E) and according to the results, it is clear that fertilizer mixture 1 (N:P:K, 10:5:10, tea dust + Gliricidia + banana trunk) and fertilizer mixture 2 (N:P:K, 10:5:10, tea dust + Gliricidia + banana leaves) has the potential to increase the growth of young tea plants.

Mineral Elements in Different Varieties of Camellia sinensis

To understand the mineral elements in different varieties of tea （ Camellia sinensis）, 17 mineral elements in eight tea varieties including Yunnan Dayezhong, No. 43 Longjing and No. 6 Zaobaijian, were measured. The results showed that nine elements, such as P, K, Ba, Mn, Cu, were significantly different among varieties, others did not. Black tea varieties usually contain high contents of Cu and K. As a conclusion, mineral elements should be extensively considered in breeding tea varieties.

茶Camellia sinensis L.对铝的吸收与累积研究

对广东省几个大型茶场中不同土壤背景值的 8个茶园 ,进行了土壤及茶树各部位的铝含量调查研究。地处我国南方的广东省茶园 ,其土壤中铝的含量都较高 ,全量在 6 0 0 0 0～ 15 0 0 0 0mg·kg- 1 之间 ,但有效态铝含量相对较低 ,仅为 30～ 40 0mg·kg- 1 之间 ,而且与土壤中全铝量大小无关 ,而与土壤酸碱度 (pH)呈极显著的负相关 ,相关系数r=- 0 9732。铝在茶树各部位含量大小依次为落叶 >成熟叶 >根 >枝条 >幼叶。茶树凋落叶铝含量最高 ,平均达 (112 71± 332 )mg·kg- 1 ,而用于加工成饮用茶的幼叶 (一芽两叶 )部分最低 ,平均只有 (6 39±2 32 )mg·kg- 1 ,同时期的相应茶场的茶叶制成品中 ,铝的含量亦接近这一水平 ,为 (978± 2 32 )mg·kg- 1 。土壤中的有机质、酸碱度 (pH)、总钾和有效钾、全铝和有效铝都对茶树各部位铝的累积产生较大影响 ,但只有土壤pH值对铝在茶树体内向幼叶部分的转移和累积影响达显著程度 ,幼叶铝含量与土壤pH之间的相关系数r =0 772 ,呈显著正相关 (P <0 0 5 )。

茶(Camellia sinensis L.)对铜的吸收与累积

对广东省5个大型茶场中不同土壤背景值的8个茶园进行了土壤及茶(CamelliasinensisL.)树各部位的铜含量分析调查。地处我国南方的广东省茶园,土壤中铜含量在2.39～53.05mg kg之间。有效态铜含量相对较低,仅在0.45～3.17mg kg之间。茶树中各部位铜含量大小依次为枝条>幼叶>根>成熟叶。茶树枝条和幼叶铜含量较高,平均分别达14.5和14.3mg kg,远高于成熟叶铜含量水平(9.7mg kg)。而同时期相应茶场的茶叶制成品铜含量则较幼叶(一芽两叶)铜的含量高。土壤中的总钾、有效磷和总铜等都对茶树各部位铜的累积产生较大影响,但只有土壤有效磷含量对铜在茶树体内向枝条部分的转移和累积影响达显著程度,枝条铜含量与土壤有效磷之间的相关系数r=-0.74,呈显著负相关(P<0.05)。

中亚热带东部茶树(Camellia sinensis)叶片光合及呼吸的研究

茶树（Ｃａｍｅｌｉａｓｉｎｅｎｓｉｓ）叶片光合速率（Ｐｎ）与光照强度（Ｌ）、气温（ｔ）多元相关呈极显著水平，其回归方程可作为光合速率估算的模型；日光合量与平均气温呈极显著正相关，呼吸速率与气温呈极显著正相关，其回归方程均可作为叶片日光合量、呼吸速率的估算模型．茶树的年净光合量、年呼吸量估算结果分别为３．４３×１０４ｍｇＣＯ２·ｄｍ－２·ａ－１和３．５３×１０４ｍｇＣＯ２·ｄｍ－２·ａ－１．

茶（Camellia sinensis Kuntze）离体培养体细胞胚胎发生的组织细胞学研究

以茶树叶片为外植体．以ＭＳ培养基附加４ｍｇ·Ｌ－１６－ＢＡ，２ｍｇ·Ｌ－１ＩＡＡ．３ｍｇ·Ｌ－１ＧＡ３和０．２—０．３％活性碳诱导出愈伤组织和胚状体，进一步形成小植株．切片观察表明．茶叶愈伤组织胚状体的发生，起源于愈伤组织表层及其内部的单个细胞和细胞团，胚状体发育顺序与合子胚大致相似．经球形胚、心形胚、鱼雷胚和子叶胚阶段．但发育过程中常有畸形胚出现．

茶(Camellia sinensis)蜂花粉及其蜂粮的营养成分研究

测定中国茶 (Camellia sinensis)的新鲜蜂花粉、5 d、1 0 d、1 5 d茶蜂粮中水解氨基酸的含量 ,茶蜂粮中平均含量为2 3 .3 6% ,比新鲜茶蜂花粉高 0 .2 6% ;新鲜茶蜂花粉和混合 (5～ 1 0 d)茶蜂粮样品中游离氨基酸含量分别为 1 0 3 7.46mg/1 0 0 g和 80 1 .75 mg/1 0 0 g。混合 (5～ 1 0 d)茶蜂粮和新鲜茶蜂花粉样品中维生素 A、B1 、B2 、C、D、E的含量 (mg/1 0 0 g)分别是0 .1 9、0 .1 5、1 .1 5、0 .3 1、0 .0 1 5 6、1 0 .2和 0 .79、0 .0 9、2 .74、1 .2 0、0 .0 1 5 8、6.6。新鲜茶蜂花粉和混合茶蜂粮样品中软脂酸、硬脂酸、亚油酸、亚麻酸的含量分别为 1 .3 %、0 .2 %、0 .1 6%、2 .5 %和 1 .1 %、1 .0 %、0 .3 %、1 .0 %。1 d茶蜂粮中的过氧化氢酶、淀粉酶的活力比新鲜茶蜂花粉高 ,而过氧化物歧化酶的活力则比新鲜茶蜂花粉略低 ;随着酿制时间的延长 ,茶蜂粮中过氧化氢酶、过氧化物歧化酶的活力下降 。

Study on the Transferability of Camellia sinensis EST-SSRs in Theaceae Plant

[Objective] The research aimed to study the transferability of Camellia sinensis EST-SSRs in Theaceae plant.[Method] Seven pairs of EST-SSRs primers which derived from Camellia sinensis EST sequence were used to amplify the nineteen materials of Theaceae plant.[Result] Five pairs in the seven pairs of EST-SSRs primers could effectively amplify the nineteen tested varieties,and the transferability rate was 71.43%.The amplification rate of Camellia retiacalate FengShanCha,Camellia japouica CaiXia and Camellia retiacalate JuBan was the highest.The amplification rate of Camellia synaptica Sealy and Adinandra sagonica var.wallichiana（oc）Ming was the lowest.Moreover,four pairs in the five pairs of primers which could effectively amplify showed the rich polymorphism whose difference was obvious in the tested materials.[Conclusion] SSR primers which were developed from Camellia sinensis genome had the higher transferability in the different genus and species of Theaceae plant,could be used in the comparative genome research and analysis mark research of Theaceae plant.

茶(Camellia sinensis)花粉及其蜂粮中花粉粒的显微结构

采用光学显微镜和扫描电镜观察茶蜂花粉及其不同酿制时间的蜂粮中花粉粒的外部形态和萌发沟,未发现花粉壁和花粉粒结构被破坏的迹象.通过体外模拟消化试验,采用光学显微镜和透射电镜观察,发现经模拟消化的蜂粮中花粉粒内含物外吐的数量比蜂花粉多,而且随着酿制时间的延长,其内含物外吐的花粉粒所占的比例增加.结果提示,同种粉源的蜂粮比蜂花粉更易于被消化.

外源24-表油菜素内酯诱导茶树(Camellia sinensisL.)耐热性的生理机制

以茶树品种龙井43为试验材料,研究外源24-表油菜素内酯(EBR)处理对高温胁迫下茶树叶片光合特性、叶绿素荧光参数、丙二醛(MDA)含量和抗氧化酶活性的影响。研究发现:叶面喷施EBR能显著提高高温胁迫下茶树叶片的净光合速率(Pn),气孔导度(Gs),Rubisco最大羧化速率(V_(c,max))、Rubisco的最大再生速率(J_(max))和PSⅡ最大光化学效率(F_v/F_m);同时外源EBR处理后,高温胁迫下茶树叶片中的抗坏血酸过氧化物酶(APX)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)和过氧化物酶(POD)活性显著提高,而MDA含量则显著下降。外源EBR主要通过解除非气孔因素的限制、促进光合碳反应的进行来缓解高温胁迫对茶树叶片光合作用的抑制;同时EBR能够有效利用多种抗氧化途径以清除其细胞内的自由基和活性氧,最终缓解高温胁迫对茶树的伤害。

Deciphering codon usage patterns and evolutionary forces in chloroplast genes of Camellia sinensis var. assamica and Camellia sinensis var. sinensis in comparison to Camellia pubicosta

Codon usage bias(CUB) is a unique property of genome which refers to non-random usage of synonymous codons in coding sequences. The present study makes an attempt to find out the pattern of CUB in chloroplast(cp) genes among three tea species, i.e., Camellia sinensis var. assamica(Assam tea), Camellia sinensis var. sinensis(Chinese tea) and Camellia pubicosta(wild tea species) as no work on CUB was reported earlier. To understand the patterns of codon usage among the cp genes of three tea groups, we used bioinformatic tools to investigate the protein coding sequences of cp genes. In our present study, the mean nucleobase T was the highest whereas C was the lowest in all the three tea groups. The overall AT content was more than GC content, i.e., genes were AT rich. The scaled chi-square(SCS) value indicated that the CUB of cp genes was low. The codon CGT(Arg) was over-represented in C. sinensis var. sinensis whereas GGA(Pro) was over-represented in C. pubicosta species. Heatmap study revealed that most of the GC ending codons showed positive correlations between codon usage and GC3 while AT ending codons exhibited negative correlations. From neutrality plot analysis, it was evident that natural selection had played a major role, while mutation pressure exerted a minor effect in the CUB of cp genes in three tea groups. Highly significant(P<0.01) positive correlation was found between SCS and synonymous codon usage order(SCUO) of cp genes which suggested that high expression of cp genes was associated with high degree of CUB.

Computational Identification of Conserved microRNAs and Their Targets in Tea (Camellia sinensis)

MicroRNAs (miRNAs) are a class of ~22 nucleotides long non coding RNA molecules which play an important role in gene regulation at the post transcriptional level. The conserved nature of miRNAs provides the basis of new miRNA identification through homology search. In an attempt to identify new conserved miRNAs in tea, previously known plant miRNAs were used for searching their homolog in a tea Expressed Sequence Tags and full length nucleotide sequence database. The sequences showing homolog no more than four mismatches were predicted for their fold back structures and passed through a series of filtration criteria, finally led us to identify 13 conserved miRNAs in tea belonging to 9 miRNA families. A total of 37 potential target genes in Arabidopsis were identified subsequently for 7 miRNA families based on their sequence complementarity which encode transcription factors (8%), enzymes (30%) and transporters (14%) as well as other proteins involved in physiological and metabolic processes (48%). Overall, our findings will accelerate the way for further researches of miRNAs and their functions in tea.

Comparative Effect of Ca, K, Mn and B on Post-Drought Stress Recovery in Tea [<i>Camellia sinensis</i>(L.) O <i>Kuntze</i>]

Tea (Camellia sinensis) is one of the most important economic crops. Being perennial in nature, tea plant often experiences natural drought, which affects its growth and productivity. The present investigation was undertaken to understand the mechanism of post-drought stress recovery on rehydration and the effect of nutrients in the recovery process of the selected clones of Camellia sinensis L. (TV-1, TV-20, TV-29 & TV-30). The results demonstrated that decrease in relative water content (RWC), dry mass of leaf and antioxidants like-ascorbate and glutathione in all the tested clones, as a result of imposed water stress, which caused damage was not permanent. Increase in phenolic content with decrease in O2-, H2O2 and lipid peroxidation was indication of the recovery of stress induced oxidative damage following the post stress rehydration. Further, the post drought recovery was enhanced by foliar spray of K, Ca, Mn & B. Differential activities of enzymes like SOD, CAT, POX, GR and PPO in response to foliar spray of nutrients in rehydrated plant improved the recovery process. The present study reveals that the tested nutrients (K, Ca, Mn & B) showed some positive response in influencing growth and antioxidative responses during post drought recovery process, where K and Ca showed comparatively better effect in improving post drought recovery potential in tea plant.

THE ANTICANCER EFFECT AND ANTI－DNA TOPOISOMERASE II EFFECT OF EXTRACTS OF CAMELLIA PTILOPHYLLA CHANG AND CAMELLIA SINENSIS

The cytotoxic effect of extract of camellia ptilophyllachang(ECPC) and extract of camellia sinensis(ECS) onHeLa cell line, poorly differentiated nasopharyngealcarcinoma cell line(CNE2) and gastric cancer cell line(MGC-803 ) in vitro was studied using MIT assay method.The results showed that ECPC and ECS possessed significantcytotoxic effect on above three cell lines. The anticancer testin mice showed that ECPC had marked inhibitory effectagainst Ehrlich solid carcinoma(ESC) with inhibition ratesof 17. 8 48. 3% and with inhibition rates of 28. 3-54. 5% against reticular cell sarcoma(L2), and that ECShad inhibition rates of 31 . 5 -49. 4 % against ESC and 35. 8- 50% against L2. These two extracts had only marginalinhibitory effect against sarcoma- 180. The unknottingactivity of DNA topoisomerase II was inhibited completelyby ECPC and ECS at the concentration of 50 μg/ mlsuggesting that DNA topoisomerase II might be a targetenzyme of these two extracts.

Cold Stress-induced Glucosyltransferase CsUGT78A15 is Involved in the Formation of Eugenol Glucoside in Camellia sinensis

Eugenol is a natural phenolic compound known for its health-promoting properties and its ability to add a floral scent to tea plants.Plant eugenol glycosides have been identified and shown to make important contributions to fruit floral quality.However,the details of their biosynthesis and metabolism in tea plants are still unknown.Here,eugenol glucoside was unambiguously identified as a native metabolite in the tea plant,and its biosynthesis was shown to be induced by low temperature treatment.Through the analysis of UGTs induced by low temperature,the glycosyltransferase CsUGT78A15 was identified in tea,and its encoded protein was shown to catalyze the glucosylation of eugenol.Vmax/Km ratios showed that eugenol was the most suitable substrate for CsUGT78A15.Sugar donor preference analysis showed that CsUGT78A15 had a higher selectivity for glucose,followed by galactose and glucuronic acid.The expression of CsUGT78A15was correlatedwith the accumulation of eugenol glucoside in different tissues and genotypes of tea.Down-regulation of CsUGT78A15 led to a decreased eugenol glucoside content under cold stress,indicating that CsUGT78A15 plays an important role in the biosynthesis of eugenol glucoside under cold stress.The identification of eugenol glucoside in the tea plant and the discovery of a cold stress-induced eugenol glucosyltransferase in tea provide the foundation for the improvement of tea flavor under cold stress and the biotechnological production of eugenol glucoside.

Anti-inflammatory properties of oolong tea(Camellia sinensis)ethanol extract and epigallocatechin gallate in LPS-induced RAW 264.7 cells

Objective: To evaluate the anti-inflammatory activity of oolong tea ethanol extract(OTEE) and epigallocatechin gallate(EGCG) on lipopolysaccharide-induced murine macrophage cell line(RAW 264.7).Methods: A cytotoxic assay using MTS tetrazolium was conducted to find a nontoxic level of OTEE and EGCG toward RAW 264.7 cells. Interleukins(IL-6, IL-1 b), tumor necrosis factor-a(TNF-a), and cyclooxigenase-2(COX-2) levels were measured by ELISA, and nitric oxide(NO) levels measured by a nitrate/nitrite colorimetric assay to determine the inhibition activity of OTEE and EGCG.Results: Lipopolysaccharide induction increases NO, COX-2, IL-6, IL-1 b, and TNF-a levels compared with the untreated cell(negative control). The positive control,lipopolysaccharide-induced RAW 264.7 without treatments showed the highest level of all pro-inflammatory cytokines and modulators tested in this study. The positive control was used as standard to obtain OTEE and EGCG inhibition activity toward NO, COX-2,IL-6, IL-1 b, and TNF-a. OTEE had a higher inhibition activity toward NO, COX-2, IL-6,and IL-1 b than EGCG; the reverse was seen for TNF-a. However, both OTEE and EGCG suppressed production of NO, COX-2, IL-6, IL-1 b, and TNF-a.Conclusions: OTEE and EGCG have the potential for use as anti-inflammatory drugs,which is shown by their ability to reduce the production of NO, COX-2, IL-6, IL-1 b, and TNF-a in active macrophages.