The PIKK inhibitors caffeine and wortmannin had been extra to your finish processing reactions at concentrations previously shown to inhibit the kinase action of ATM . Each inhibitors had been capable of abolishing the protective effects of 0.2nM purified ATM and within the control nuclear extract within the presence of ATP. This was evident through the sharp decline in the intensity of full length goods . 3.6. ATM autophosphorylation just isn’t ample for end safety The dependency on ATP to repress degradation along with the inhibition of this repression by wortmannin or caffeine reflects the requirement for kinase exercise for DNA endprotection. This requirement could reflect a dependence on ATM autophosphorylation alone; or it could indicate the want for phosphorylation of the downstream substrate by ATM or by a different component on the strategy. Consequently, to examine no matter if an ATM autophosphorylation occasion was ample to confer protection to DNA ends not having the need to have for subsequent kinase pursuits, we incubated pre phosphorylated purified ATM having a duplex presenting a five AATTC overhang in an A T nuclear extract together with wortmannin or caffeine .
This was accomplished inside the presence with the phosphatase inhibitor fostriecin to make sure that ATM remained phosphorylated through the reaction. We made use of fostriecin at a concentration previously proven to inhibit ATM dephosphorylation Panobinostat solubility by PP2A . The addition of fostriecin had no result on finish protection by purified ATM or by a handle nuclear extract . Pre phosphorylated ATM was capable of repressing DNA enddegradation. Having said that, itwas not able to do so during the presence of either wortmannin or caffeine as reflected by a sharp decline in detectable full length products and a rise in intensities of shorter items . These information indicate that autophosphorylation of ATM is necessary but not sufficient and that downstream kinase pursuits are in all probability necessary to stop degradation of DNA ends. We ensured that ATM remained phosphorylated inside the extract through parallel monitoring of 32P labeled ATM incubated having a T nuclear extract, wortmannin, fostriecin and DNA duplex below standard fix reaction situations .
Non homologous finish joining is believed to become the key DNA DSB restore mechanism in mammalian cells all through G0, G1 and early S phase in the cell cycle. Proteins Veliparib selleck involved with the NHEJ pathway comprise the Ku70 Ku80 heterodimer, DNA PKcs, XRCC4, DNA Ligase IV and Artemis. Microhomology mediated NHEJ, then again, may possibly involve the MRN complicated . NHEJ deficient cells fail to restore up to 60 of induced DSBs . Then again, cells with ATM deficiencies, or maybe a T cells, show ranges of residual un repaired DSBs which are similar to people detected in controls or at most somewhat elevated .
Subsequent, we have been enthusiastic about learning no matter whether focusing on ATM by miR 100 could sensitize the cells to ionizing radiation induced killing mainly because ATM plays a vital position in promoting the HRR pathway , and AT cells while not the ATM function are incredibly sensitive to IR induced killing. 3.4. Targeting ATM by miR one hundred sensitizes the cells to IR induced killing To find out the effect of miR 100 on cell sensitivity to IR, we utilized the clonogenic assay. The outcomes showed that when miR one hundred were up expressed in M059K cells , the cells became even more sensitive to IR than the cells transfected using the empty vector , suggesting that miR 100 can be used as a tool to sensitize cells to IR. mTOR is also a target of miR one hundred , mTOR expression is reduced in M059J cells than in M059K cells, and upregulating miR a hundred in M059K cells resulted inside the down regulation of mTOR from the cells . To find out regardless if the low expression of mTOR by miR 100 in M059K also contributed to your effects of miR a hundred about the sensitization from the cells to IR, we examined the result of rapamycin, an mTor inhibitor, on cell radiosensitivity. The outcomes showed that when mTOR inside the cells was inhibited by rapamycin, the cells didn’t transform their sensitivity to IR .
According to these outcomes, we could conclude that mTOR won’t affect cell radiosensitivity and above expression of miR one hundred in the M059K TAK-875 selleckchem cells induced radiosensitivity just isn’t resulting from the lowexpression of mTOR. To confirm that the low expression of ATM induced from the in excess of expression of miR one hundred in M059K cells was the sole motive for the cell radiosensitization,weexamined the result of siRNA of ATM within the radiosensitivity of M059K cells because single miRNA could target multi genes and miR one hundred may target several other genes that also play a function in affecting the cell radiosensitivity. The outcomes showed that once the ATM level in M059K cells was down regulated from the siRNA , M059K cells became additional sensitive to IR induced killing , as well as sensitization level is much like that induced by miR 100 . These outcomes verify that up regulating miR a hundred in M059K cells induced radiosensitization, and it is the consequence from the low expression of ATM.
In summary, our data, to the very best of our practical knowledge, demonstrate for the 1st time that ATM stands out as the target of miR a hundred, and indicate that more than expression of miR 100 is mainly accountable for your low expression of ATM in M059J cells. These information AMN-107 also show that miR 100 targeting ATM could sensitize the cells to IR induced killing. Furthermore, according to these results, we could identify miRNAs that target DNA restore genes to sensitize tumor cells to radiotherapy or chemotherapy and therefore enhance cancer treatment method . Whenever a cell encounters a problem this kind of as DNA injury and inhibiting of DNA replication, various self defense mechanisms are induced to resolve the challenge.
Right after centrifugation at , g for min, the pellet was handled with nuclear extraction reagent with vortexing for sec each and every min for a total of min. Following centrifequencing. SATB particular siRNA sequences were synthesized in line with people as reported by Han et al. and inserted into the pGCsi H Neo GFP siNEGative vector , which coexpresses GFP to permit identification of transfection efficiency. The SATB shRNA sequence was: SATB shRNA ? GTCCACCTTGTCTTCTCTC ?. The non exact shRNA sequence was: management shRNA ? ACGTGACACGTTCGGAGAA ? . All constructs were confirmed by sequencing. Transient transfection and luciferase assays Jurkat cells were transfected with g luciferase reporter plasmids plus ng pRL vectors making use of an electroporator at F and V in a . cm cuvette at a concentration of cells L in RPMI medium containing FBS. Each and every electroporation was plated right into a mm diameter tissue culture dish and incubated for h. Forty eight h immediately after transfection, cells had been washed with PBS and lysed using passive lysis buffer, and L of cell extract was assayed for firefly and Renilla luciferase activity working with Dual Luciferase? reporter Assay System kit according to the manufacturer?s directions.
Western blotting analysis Entire cell extracts have been ready from cells transiently transfected with SATB RNAi plasmids or manage plasmids employing lysis buffer containing mmol L Tris . NP and . SDS that has a cocktail of protease inhibitors. Total Pazopanib protein was boiled for min in loading buffer, chilled on ice after which separated on sodium dodecyl sulfate polyacrylamide gels. Subsequent to transfer onto PVDF membranes , non particular protein interactions have been blocked by incubation in nonfat dry milk in TST buffer at C for h. Membranes were then incubated at C overnight with polyclonal anti SATB or anti actin monoclonal antibody in fresh blocking buffer. Horseradish peroxide conjugated secondary antibody was additional for h at room temperature. The blot was created with ECL reagent . Prestained markers were utilised as internal molecular bodyweight standards. RNA isolation and RT RCR Complete RNA was isolated with Trizol reagent in line with the producer?s protocol.
RNA integrity was assessed by visualizing the ribosomal bands on the agarose gel assessed. Finally, cDNA was synthesized from total RNA working with AMV Reverse Transcriptase in accordance with the producer?s instructions , and oligo was used because the primer. The reactions have been incubated at C for min and after that Tivantinib selleck stored at C before use. The authentic time PCR disorders have been C for min, and C for min followed by cycles of denaturation at C for sec, and annealing at C for min. Statistical evaluation Outcomes were expressed as imply SD. Data were analyzed utilizing Student?s t check. Statistical analysis was performed with statistical evaluation software program SPSS P . was deemed to have statistically major difference.
Serum amounts of endostatin and VEGF in individuals and management topics had been determined making use of the quantitative sandwich enzyme immunoassay technique according to the producer?s instructions. The immunoassay approach involved trapping both endostatin or VEGF current in serum concerning two distinct antibodies with one particular antibody staying enzymatically linked for colorimetric detection. Serum samples had been diluted as critical. The optical density was measured at nm with correction wavelength set at nm. All serum samples were analyzed in triplicates for precision. The intraassay and interassay coefficient of variation for endostatin and VEGF ranged amongst . and and concerning . and respectively. The values of endostatin and VEGF in serum are expressed as geometric indicate normal deviation ng mL and pg mL, respectively. RNA isolation, complementary DNA synthesis, primer style, and polymerase chain response . Skin tissue samples have been homogenized employing Polytron in chilled homogenization tubes containing TRIzol at , rpm for bursts of s just about every.
RNA extraction was carried out according to the producer?s protocol. The RNA pellet was dissolved in RNase cost-free water and stored at C right up until subsequent examination. Complete RNA samples have been quantified and purity checked utilizing NanoDrop ND UV Vis spectrophotometer . The integrity of total RNA was assessed employing Vandetanib the RNA Nano Lab Chip kit with Agilent Bioanalyzer System . Very first strand cDNA was synthesized from total RNA samples with ProtoScript M MuLV Initially Strand cDNA Synthesis Kit applying anchored oligo dTprimer in line with the producer?s instructions. The cDNA was diluted with nuclease free of charge sterile water and stored at C until subsequent utilization. The 2nd strand synthesis of b actin, endostatin collagen XVIII, and VEGF had been carried out on the gradient Thermocycler with PCR response combine comprising mL to begin with strand cDNA, mmol L primers, and red dye PCR Master Combine . The PCR amplification was performed on the following circumstances: initial denaturation at C for min, followed by cycles of denaturation at C for s, annealing at Ta C for s, extension at C for s, followed by ultimate extension C for min.
The annealing temperatures for that primer combinations were optimized at C significantly less than the melting temperatures with the forward and reverse primer pair . The PCR cycle number was optimized at cycles right after preliminary PCR reactions have been carried out at and cycles with respect for the significantly less expressed gene namely, collagen XVIII. The amplified PCR items have been fractionated on the agarose gel and visualized by ethidium bromide staining. The images have been obtained by using GelDoc Salbutamol XR and bands were quantified by using Quantity A single software package .
Similarly, TLE expression was not persistently regulated by Wnt expression or perhaps catenin knockdown in T L preadipocytes . These information suggest that Wnt, Wnta or perhaps Wntb very likely prevents adipogenesis independently of effects upon TLE mRNA expression. Discussion Wnt and additionally Wnta since regulators of MSC fate Even though Wnt ligands have been identified in animals , few of the have been researched within the context of MSC fate. In addition to Wntb, ectopic Wnt and recombinant Wnta each suppress adipogenesis in vitro , and Wnta happens to be recorded to suppress adipogenesis . Conversely, some other studies report stimulation of adipogenesis by Wnta, and as by Wnt and also Wntb . Nishizuka mais aussi al. also reported suppression of Wnt mRNA during the course of adipogenesis; however, they failed to study whether Wnt controls adipogenesis . Similarly, Wnta happens to be proposed as a good endogenous inhibitor of brown adipogenesis , however it has not been empirically demonstrated. Therefore, the present learn is the very first to display which Wnt and Wnta regulate fate of mesenchymal precursors.
Disruption of Wnt catenin signaling promotes spontaneous adipogenesis in vitro , supporting the notion that endogenous Wnt ligands inhibit adipogenesis. Wntb has long Pazopanib selleck been touted as the endogenous inhibitory Wnt; however, no published studies have conclusively demonstrated this. Although knockdown of pro adipogenic Wnt or Wnta impairs adipogenesis , to our knowledge no previous studies have used stable Wnt knockdown to investigate endogenous anti adipogenic Wnts. Our attempts to knock down Wnt, Wnta or Wnta individually were complicated by technical difficulties in detecting Wnt knockdown in ST cells. The robust knockdown of catenin protein suggests that our Wnt knockdowns may be more apparent if assessed at the protein level, because the almost total knockdown of catenin protein is far greater than the knockdown detected for catenin mRNA . Unfortunately, lack of reliable antibodies againstWnt,Wnta orWntb undermined our attempts to detect these proteins .
Nevertheless, our Wnt knockdown cells consistently display decreased catenin protein, enhanced adipogenesis and impaired osteoblastogenesis, suggesting functional Wnt knockdown in each of these cell lines. Another observation from our shWnt expressing cell lines is that, in all cases, Wnt knockdown is associated with decreased expression of other Wnts. This indicates potential positive feedback between Wnts, consistent with our previous finding that Wnt Ostarine structure stimulates expression of Wnt and Wnta in preadipocytes . Although the mechanisms underpinning such cross regulation remain unclear, catenin is unlikely to be involved because knockdown of catenin does not affect endogenous expression of Wnt, Wnta or Wntb .
Less than one from the carotid blood movement was distributed on the contralateral half with the head and under 0.01 within the microspheres was detected within the heart and kidneys. three.two. Result of 5 HT for the distribution of prevalent carotid artery blood movement The effects of intracarotid infusion of five HT on haemodynamics as well as distribution within the ipsilateral popular carotid artery blood flow had been quite equivalent to individuals reported earlier . The amine brought on a 46 7 decrease in ipsilateral carotid blood movement and, since mean arterial stress was nearly unaffected, a equivalent boost from the ipsilateral carotid resistance . The decrease in complete carotid movement was substantially lower than that in its arteriovenous anastomotic fraction as the ipsilateral extracerebral fraction elevated . The tissues which contributed most to this boost had been the skin as well as ears whilst muscle movement was doubled . The component of carotid blood distributed to brain was not impacted by five HT. The improvements in movement and resistance on the contralateral side were negligible. The haemodynamic variables measured 20 min after the end of your 5 HT infusion had been comparable to individuals beneath control problems.
three.three. Hemodynamic results of ketanserin and Wal 1307 The results of ketanserin and Wal 1307 within the heart charge, indicate arterial blood strain and carotid movement and resistance are shown in table 2. Neither drug purchase Entinostat selleckchem brought about any appreciable transform in these cardiovascular variables, except to get a slight lessen in arterial pressure. The responses from the haemodynamic parameters to one g kgnoradrenaline had been not markedly altered immediately after ketanserin or Wal 1307 in any of your animals . three. four. Effects of ketanserin within the five HT induced redistribution of total widespread carotid artery blood flow Ketanserin itself didn’t bring about any adjust while in the distribution of carotid blood flow. Even so, the drug totally eradicated the reduction with the complete carotid blood flow elicited by five HT. The response with the arteriovenous anastomotic movement was somewhat but substantially diminished and that in the extracerebral flow was enhanced .
Tissue blood movement to a variety zafirlukast of structures, particularly on the skin and ears, increased immediately after 5 HT . The colour of each skin and ear of the ipsilateral side modified to vivid red. Baseline values have been reached 20 rain following the amine infusion had been stopped. Whilst ketanserin itself didn’t cause any change in the tissue blood flows, the drug enhanced the vasodilatory response to 5 HT while in the skin and ears. The colour improvements in the skin and ears were also present all through the 2nd infusion of 5 HT. 3.five. Results of Wal 1307 within the 5 HT mduced redistribution of total normal carotid blood movement The results of Wal 1307 over the carotid blood movement and its redistribution by five HT are proven in figs. four and 5.
Similarly, distinctive responses of five HT, receptors in the course of differentiation and ontogeny are already demonstrated . This suggests that alternate splice variants or subunit substitution may well take place below some conditions. ELECTROPHYSIOLOGY OF five HT, RECEPTORS Derkach et al. have been the very first to report that the 5 HT, receptor can be a ligand gated ion channel from their studies making use of single recent recordings from outdoors out patches of guinea pig submucosal plexus neurons. Far more current electrophysiological scientific studies in cultured cell lines have also proven that 5 HT creates transient electrical responses that could be blocked by selective 5,HT, receptor antagonists . The relative permeability to K? and Na? appears to fall into two principal groups in different research, one near a I? of 0.4 and 1 near a P,IP of one.0, even though rather extensively ranging values are actually obtained in comparable preparations and by various investigators. Whilst cell culture problems or recording procedures could possibly be no less than partly accountable to the discordant data, the existence of distinct five HT, receptor subtypes might also be postulated . Massive organic cations also can pass with the five HT, receptor channel , but divalent cations this kind of as Ca and Mg are relatively excluded . This may possibly be as a result of permeability within the receptor to Ca2 , activation of voltage dependent Ca ch annels as a consequence of five HT, receptormediated depolarization, or release Ponatinib selleckchem of stored intracellular Ca pools . Measurement of Hill coefficient values significantly higher than one signifies co operativity, requiring the binding of two 5 HT molecules for the receptor in advance of channel opening can take place . It really is unclear as to if binding of 5 HT towards the five HT, receptor internet site accountable for channel opening is additionally accountable for the speedy, reversible, desensitization observed in many scientific studies a different blog possessing been advised by some studies . Throughout continuous exposure to five HT, the very low conductance channel appeared to desensitize, whereas the open probability in the high conductance channel doesn’t adjust . In other preparations, remarkably smaller conductance values of 0.6 to 4 pS are actually obtained . In a comparable examine, activation of 5 HT, receptors with two methyl 5 HT stimulated the production of cGMP in NG108 15 cells , with this effect currently being blocked by unique five HT, receptor antagonists such as ondansetron . It looks that coupling of five HT, receptors with second messenger systems effected via Gproteins won’t take place considering the fact that pertussis toxin MG-132 selleckchem will not alter 5 HT s mediated responses and this kind of responses proceed to happen in nucleotide 100 % free bathing solutions following numerous hrs . In addition, intracellular dialysis with G protein activators, this kind of as CsF, AlF and GTPyS, will not alter responses to 5 HT, receptor activation .
Inside the presence of atropine , neither granisetron nor SB 204070 affected the contractions to substance P or neurokinin A , demonstrating the lack of interference of those five HT receptor antagonists together with the tachykinin receptors concerned.The current research exposed some interesting variations involving the pathway to contraction upon both five HT 3 or five HT four receptor selective stimulation. Provided the lack of impact of hexamethonium, 5 HT three receptors seem to be situated principally on motor neurons as they never seem to rely upon interneurons which synapse while in the myenteric ganglia. Tetrodotoxin was in general ineffective to inhibit the five HT 3 receptor mediated responses, while there was some inhibition at one concentration of two methyl 5 HT along with a tendency to inhibit 5 HT induced 5 HT three receptor mediated contractions during the presence of atropine. The concentration of tetrodotoxin was sufficient nevertheless, as tetrodotoxin in the very same concentration did abolish the five HT 4 receptor mediated contractions. Action possible propagation along axons is Na mediated and will therefore be blocked from the concentration of tetrodotoxin applied. Nonetheless, tetrodotoxin insensitive action potentials are actually observed in the minor subset of myenteric neurons .
Its also conceivable that tetrodotoxin insensitive release takes place as a consequence of stimulation of receptors localized over the nerve endings . Without a doubt, within the presence of tetrodotoxin, atropine was nevertheless capable to strongly inhibit the remaining contraction upon five HT three receptor stimulation, which demonstrates SB 271046 selleck chemicals that tetrodotoxin insensitive acetylcholine release is usually elicited . It’s consequently proposed that the 5 HT three receptor mediated contraction is largely thanks to stimulation of 5 HT three receptors that happen to be localized on the nerve endings from the motor neurons , whilst also a minor population of five HT three receptors might be localized proximal towards the nerve endings. The five HT 4 receptor mediated contraction looks to involve interneurons as well as motor neurons, like a partial block attributable to hexamethonium was seen. Tetrodotoxin thoroughly prevented the five HT four receptor mediated effects. This suggests that, on the motor neurons, five HT 4 receptors have to be localized on the soma .
With respect for the five HT four receptors on the interneurons, it cannot be absolutely concluded irrespective of whether they are existing around the nerve endings or to the soma. Electrophysiological experiments with myenteric neurons from the guinea pig ileum and stomach have uncovered 3 forms of response Taxifolin upon 5 HT application: quick depolarization and a decrease in membrane resistance attributable to five HT three receptor stimulation, slow depolarization and a rise in membrane resistance due to putative five HT w receptor stimulation and hyperpolarization with concomitant decrease in membrane resistance because of 5 HT1A receptor activation . No effects on input resistance or membrane prospective have been observed that appeared for being mediated by 5 HT 4 receptors.
All results confirm that emodin protected the liver from injury and fibrogenesis caused by CCl4 in the rat model. Chronic liver injury may lead to development of fibrosis, a process in which HSC play a major role. As a result of liver injury, HSC, which in the healthy organ store vitamin A, undergo a process of activation that is mediated by the concerted action of resident hepatic cell types such as Kupffer cells, liver endothelial cells, and hepatocytes. The phenotype of activated HSC is characterized by smooth muscle actin expression . SMA expression in the liver tissues is an indicator of hepatic stellate cell activation, which is recognized as being critical in liver fibrogenesis. Thus inhibition of the accumulation of activated HSCs is an important therapeutic strategy . Our results showed the levels of SMA in rat liver tissues increased significantly after CCl4 administration for 12 wk. Emodin reduced SMA expression at mRNA and protein levels. Inflammation is commonly associated with hepatic fibrogenesis during chronic liver diseases . CCl4 is metabolized in the liver by cytochrome P450 into the free radical CCl3 .
The free radical attacks hepatocytes and causes necrosis of parenchymal cells, which promotes inflammatory responses in the liver . Results in this study indicated that emodin suppressed inflammation caused by CCl4, which might lead to the protection of the liver from injury. It is now widely accepted that the pro inflammatory cytokine TGF 1 is a major cytokine in the regulation of the production, degradation, and accumulation of ECM , and it screening compounds has been suggested that overexpression of TGF 1 for a prolonged period of time after tissue damage may induce a fibroproliferative response and deposition of ECM, resulting in fibrosis in vital organs . Many studies have detected the presence of TGF 1, in the form of either protein or message, in the fibrotic tissues of animal models or human samples . Partial inhibition of the accumulation of ECM using either anti TGF 1 serum or a TGF 1 binding protein has been reported in fibrosis models . Our results showed that TGF 1 mRNA levels and serum TGF 1 protein levels in normal rat were low.
After TH-302 dissolve solubility injection of CCl4 for 12 wk, mRNA and protein levels of TGF 1 increased significantly. Emodin down regulated mRNA levels of TGF 1 expression in liver tissue. Furthermore, serum TGF 1 levels in the model rats were also significantly down regulated by emodin treatment in a manner similar to hepatic fibrosis attenuation. These findings imply that emodin might attenuate hepatic fibrosis through down regulation of TGF 1 expression in vivo. Smad4 is well known to function as one of the downstream effectors of TGF 1, and it mediates TGF 1 induced collagen synthesis .
But the expression of most SM clusters and their concomitant goods continue to be veiled. Two approaches for activating otherwise silent clusters had been recently described. A single system, utilizing the awareness that numerous SM clusters have a pathway distinct transcription component, fused an inducible promoter to a cluster transcription component top on the production of hybrid polyketide nonribosomal peptide metabolites, the cytotoxic aspyridones A and B four. A second technique, dependant on genomic mining of microarrays generated from mutants in the global regulator of secondary metabolic process LaeA5, six, seven, led on the identification with the anti tumor compound terrequinone A eight. Efforts to uncover the regulatory function of LaeA uncovered that some subtelomeric SM clusters were positioned in heterochromatic regions on the genome the place suppression was relieved by deletion of the essential histone deacetylase9. The importance of histone modifications in SM clusters was even more reflected within the initiation and spread of histone H4 acetylation concurrent with transcriptional activation of your subtelomeric A.
parasiticus aflatoxin gene cluster10. A consideration of your accruing proof linking chromatin modifications with Olaparib molecular weight SM cluster regulation led us to examine the hypothesis that additional chromatin modifying proteins have been critical in SM cluster regulation. Specifically, we examined a member of the COMPASS complicated for doable regulatory roles in SM silencing. The COMPASS complicated is really a conserved eukaryotic transcriptional effector the two facilitating and repressing chromatin mediated processes via methylation of lysine 4 of histone three 11, twelve. Although H3K4me2 and H3K4me3 are located predominantly on active loci twelve, the COMPASS complicated also regulates homothallic mating silencing, ribosomal DNA silencing, telomere length, and subtelomeric gene expression in yeast 13 15. A essential member of your COMPASS complicated may be the SPRY domain protein designated Bre2 in Saccharomyces cerevisiae11. Examination from the A.
nidulans genome exposed a putative ortholog, here named CclA. Extracts of cclA deletants , deficient in H3K4 di and trimethylation , presented an altered chemical landscape as depicted by thin layer chromatography . Preceding operate has proven the main SM created by A. nidulans may be the polyketide sterigmatocystin Rosiglitazone . To cut back ST and ST precursor backgrounds, stcJ encoding a fatty acid synthase essential for ST production16, was also deleted, generating a double stcJ , cclA mutant. HPLC profiles of stcJ showed the production of two regarded metabolites of a. nidulans austinol and dehydroaustinol along with the absence of ST . Evaluation from the stcJ , cclA double mutant yielded not less than six additional aromatic compounds.