Effect of Nano red elemental selenium on GPx activity of broiler chick kidney cells<Emphasis Type="Italic">in vitro</Emphasis>

A new selenium source, Nano red elemental selenium (Nano-Se) was used to study the effect on the GPx activity of broiler chick kidney cells (BCKC)in vitro, Sodium selenite (Na₂SeO₃) and seleno-1-methionine (Se-Met) were used as the controls. The results showed that the effects of three kinds of Se forms on the GPx activity of BCKC were accordant (p>0.05) compared with each other at 0.01. 0.05 and 0.10 μmol/L Se concentrations treatments. In the range of 0.00–0.10 μmol/L Se concentrations, the GPx activity increased with elevation of Se concentrations in medium. For the three kinds of Se forms, the GPx activity reached the climax at 0.10 μmol/L Se concentration. At 0.20 and 0.30 μmol/L Se concentrations, the influnces of three kinds of Se forms were not accordant with one another. For Nano-Se, the GPx activity at 0.20 and 0.30 μmoi/L Se concentrations remained the same as that at 0.10 μmol/L Se concentration treatment. For Se-Met, the GPx activity at 0.20 μmol/L Se concentration treatment remained the same with 0.10 μmol/L treatment; the GPx activity at 0.30 μmol/L Se concentration treatment was declined significantly (p<0.05) compared with 0.10 or 0.20 μmol/L treatment. For Na₂SeO₃, the GPx activity falled gradually with Se concentration increasing from 0.10 μmol/L to 0.30 μmol/L, and at 0.30 μmol/L Se concentration treatment, the GPx activity was less than the original of BCKC. The results implicated, on the GPx activity of BCKCin vitro, the ranking of width range of the most suitable Se concentration for nutrition curve of the three Se formes is Nano-Se>Se-Met>Na₂SeO₃. Foundation item: Supported by the Key Item of the Science and Technology Bureau of Zhejiang Province (021122680) Biography: Xu Bao-hua (1966-), male, Associate professor, Ph. D. research direction: nanobiology and animal nutrition.     

Interaction between integrin αIIbβ3 and synthesized cyclic hexapeptide containing RGD

The RGD sequence generally exists in the extracellular matrix proteins and can be recognized by many integrin proteins. The binding ability of immobilized biotinylated cyclic hexapeptide [cyclo(-Arg-Gly-Asp-D-Phe-Lys-Gly-)] containing RGD to integrin α_αbβ₃ was tested by the methods of ELISA and SPR. Results showed that a spacer of 1.48–2.2 nm between the peptide and the biotin residue was long enough to send the RGD sequence into the binding center embccedded within α_IIbβ₃, and the equilibrium dissociation constant was 1.1 μm. The work provides an ideal model system for the research of cell adhesion on solid surfaces.     

Circular dichroism spectral studies on the recombinant human neuroglobin

Neuroglobin (NGB) is a newly discov- ered member of the hemoglobin superfamily that is primarily expressed in the brain of humans and other vertebrates. The effects of protein concentration, solvent, pH and temperature on the secondary structure of NGB were investigated by employing far UV circular dichroism (CD) spectroscopy. The results show that NGB exists mainly in α-helix form when its concentration is less than 10 μmol/L. However, its α-helix content decreases with the increase of con- centration in the range of 10―40 μmol/L and remains unchanged when the concentration is higher than 40 μmol/L, which suggest that NGBs form intermolecular disulfide bond and aggregate in higher concentration. The α-helix content of NGB in methanol and ethanol is a little higher than that in water, indicating a higher stability of NGB in these solvents. NGB loses its α-helical secondary structure in either acidic or alka- line solution to some extent. Although increased temperature destabilizes the α-helices of NGB, over 16% of α-helices can be kept at 110°C. Therefore, NGB is a protein with hyperthermal stability.     

Surface Dissociation Properties of Short Chain Carboxyl Mercaptan Self-Assembled Monolayers by Impedance Titration

Impedance titration was used to determine the surface dissociation characteristics of short-chain carboxyl mercaptan self-assembled monolayers （SAMs）. Based on the change of the cyclic voltammetric peak current and the charge-transfer resistance, which was related to pH value of the solution, the surface pKa of mercaptoacetic acid（MA）, 3-mercaptopropionic acid（MPA） and ω-mercaptohexanic acid（MHA） self-assembled membranes, with ionic strength being 0.1 mol/L, were determined to be 5.20, 4.80, 7.40, respectively. In addition, factors such as time needed for assembling, structure of monolayers and ionic strength, which effected the surface pKa, were studied as well. Such surface pKa shifts were sufficiently explained by interactions between interfacial molecules and hydrophobicity.     

Effect of CO<Subscript>2</Subscript> concentrations on the activity of photosynthetic CO<Subscript>2</Subscript> fixation and extracelluar carbonic anhydrase in the marine diatom <Emphasis Type="Italic">Skeletonema costatum</Emphasis>

The growth and activity of photosynthetic CO2 uptake and extracellular carbonic anhydrase (CAext) of the marine diatom Skeletonema costatum were investigated while cultured at different levels of CO2 in order to see its physio-logical response to different CO2 concentrations under either a low (30 靘ol·m-2·s-1) or high (210 靘ol·m-2·s-1) irradiance. The changes in CO2 concentrations (4—31 靘ol/L) affected the growth and net photosynthesis to a greater extent under the low than under the high light re-gime. CAext was detected in the cells grown at 4 mol/L CO2 but not at 31 and 12 靘ol/L CO2, with its activity being about 2.5-fold higher at the high than at the low irradiance. Photo- synthetic CO2 affinity (1/ K1/2(CO2)) of the cells de-creased with increased CO2 concentrations in culture. The cells cultured under the high-light show significantly higher photosynthetic CO2 affinity than those grown at the low-light level. It is concluded that the regulations of CAext activity and photosynthetic CO2 affinity are dependent not only on CO2 concentration but also on light availability, and that the de-velopment of higher CAext activity and CO2 affinity under higher light level could sufficiently support the photosyn-thetic demand for CO2 even at low level of CO2.     

Involvement of Oxidative Stress and Down-Regulation of Bcl-2 in Arachidonic Acid-Induced Apoptosis in HUVECs

0IntroductionAraacicdhi,diosn fiocu ancidd p(reAdAo)m i,naanntelsys eantt itahle p sonl-y2u npsoastiutiroante doff actetl-ylular phospholipids . Normal free AAconcentrationin humanblood ranges from5 .8μmol/Lto 49 .3μmol/L[1].It is re-leased mostlythroughactivation of phospholipase A2by physi-ological and pathological sti muli[2]. Free AAcan be metabo-lizedinto various eicosanoids via specific enzymes such as cy-clooxygenases ( COX) , lipoxygenase and cytochromesP-450[3]. During AA met…     

Bis(7)-Tacrine, a promising anti-Alzheimer's agent, attenuates glutamate-induced cell injury in primary cultured cerebrocortical neurons of rats

The effects of bis(7)-tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, on glutamate-induced cell injury were investigated in primary cerebral cortical neurons of rats. Exposure of cultured neurons (12 days after plating) to 0.5 mmol/L glutamate for 30 min resulted in significant cell damage. Pretreatment with bis(7)-tacrine (0.03–1.0 μmol/L) reduced the glutamate-induced neurotoxicity in a concentration dependent manner and the maximal response was seen at 1 μmol/L with approximately, 30% protection. A receptor binding assay showed that bis(7)-tacrine can completely displace MK-801 binding to rat cortical membrane with an IC₅₀ of 0.57 μmol/L. These findings suggest that bis(7)-tacrine can directly interact with N-methyl-D-aspartate receptor channel complex, which may contribute to the inhibitor's protective effects against glutamate-induced excitotoxicity. Thus, it is possible that anti-glutamate/anti-AChE synergism is responsible for potentially better Alzheimer's therapy of bis(7)-tacrine relative to tacrine. Biography: Zhang Bai-fang (1974-), female, Ph. D. candidate, research direction: Alzheimer's disease and ischemia     

Synergistic effect of trace elements and flavonoids from Epimedium koreanum Nakai on primary osteoblasts

Several trace elements, particularly, manganese （Mn） and zinc （Zn）, are essential in bone metabolism as cofactors for specific enzymes. It has been reported that there exists the relationship between osteoporosis and trace element-deficiency and the efficacy of Ca, Mn and Zn supplementation on spinal bone mineral density in postmenopausal women. Traditional Chinese medicines （TCM）, such as Herba epimedii, were proved to be effective for prevention of osteoprosis in vivo; however, the efficacy of the main constituents and/or crude extract was not ideal in vitro, which suggested that they may work in another way. The purpose of the present study was to examine whether the combination of icariin and total flavonoids （TF） from Herba epimedii with mineral elements, which were abundant in Herba epimedii, would have a more beneficial effect on the viability and differentiation of primary osteoblasts than either agent alone, and to analyze the dada for a possible synergistic, additive or antagonistic effect. The combinations of 10 μmol/L Zn, Ca and Mn with icariin and total flavonoids greatly improved the cell viability and meanwhile dramatically enhanced the alkaline phosphatase activity as compared to each agent alone. On the other hand, an increased cell growth inhibition was also observed by combining 0.1 μmol/L, 1 pmol/L Zn with 10μmol/L icariin, and 10 μmol/L Mn with 0.06 μg/mL total flavonoids. Meanwhile a decreased alkaline phosphatase activity was also found in several icariin-Zn/Mn and total flavonoids-Zn/Ca/Mn combinations. These results suggested that mineral elements （Zn, Ca, Mn） greatly enhanced the efficacy of icariin and total flavonoids from Herba epimedii on the viability and differentiation of primary osteoblasts by certain combinations.     

Characterization of γ-Fe2O3 nanoparticles prepared by transfor- mation of α-FeOOH

γ-Fe2O3 nanoparticles were successfully synthesized by a chemically induced transformation of α-FeOOH.In this method,the precursor(α-FeOOH)was prepared by chemical precipitation,and then treated with a mixed FeCl2/NaOH solution to produce the nanoparticles.X-ray diffraction indicated that when the precursor was treated with FeCl2(0.22 mol/L)and NaOH(0.19 mol/L),pure γ-Fe2O3 nanoparticles were obtained.However,when the concentration of FeCl2 was<0.22 mol/L or the concentration of NaOH was<0.19 mol/L,α-FeOOH and γ-Fe2O3 phases co-existed in the nanoparticles.Transmission electron microscopy observations showed that in the samples with co-existing phases,the nanoparticles did not have identical morphologies.The pure γ-Fe2O3 nanoparticles were polygonal rather than spherical.The volume ratio of α-FeOOH and γ-Fe2O3 was estimated for the two-phase samples from magnetization data obtained from a vibrating sample magnetometer.This chemically induced transformation is novel,and could provide an effective route for the synthesis of other metal oxide nanocrystallites.     

Effect of Cl- on photosynthetic bicarbonate uptake in two cyanobacteria Microcystis aeruginosa and Synechocystis PCC6803

Photosynthetic inorganic carbon utilization was investigated in two cyanobacteria Microcystis aeruginosa and Synechocystis PCC6803 grown in standing culture. Photosynthetic rates for the two algae reached about 10 times the theoretical CO₂ supply rate at low dissolved inorganic carbon (DIC) of 100 μmol/L, and the rates were unaffected by the addition of 20 mmol/L Na⁺, indicating that the two algae possessed Na+-independent HCO^-₃ utilization for photosynthesis under low DIC. Their photo- synthetic rates at low DIC were inhibited by higher Cl^- and the degrees of inhibition were increased with the rise of CI^- concentration, and in the presence of Diphenylamine-2-carboxylate (DPC), a reported Cl^- channel inhibitor, the rates decreased by 74%－82%, implying that putative DPC-sensitive Cl^- channels participate in Na+-independent HCO₃^- uptake for photosynthesis. The experiment of intracellular 14C-DIC accumulation for photosynthesis showed that internal DIC pools decreased by about 80% with 200 μmol/L DPC and by 64%－70% with 100 mmol/L Cl^-. The experiment of chlorophyll a fluorescence quenching showed that initial rates and extents of fluorescence quenching obviously decreased with 200 μmol/L DPC or 100 mmol/L Cl^-. The two experiments gave further evidence that putative DPC-sen- sitive Cl^- channels participate in Na+-independent HCO^-₃ uptake for photosynthesis in the two algae grown in standing culture.     

Effects of cadmium, 17β-estradiol and their interaction in the male Chinese loach （Misgurnus anguillicaudatus）

To assess the interaction effect between cadmium (Cd) and 17-estradiol (E2),male Chinese loaches (Misgurnus anguillicaudatus) were exposed to E2 (1 g/L) and Cd (50 or 500 g/L) alone and in combination using a semi-static waterborne exposure system.The effects of E2 on the accumulation and distribution of Cd,as well as the effects of Cd on vitellogenin (Vtg) synthesis induced by E2,were investigated.Cd mainly accumulated in the kidneys,liver,intestines,and gills,with little amounts in muscles,bones,and gonads.Co-exposure with E2 did not change the main targets for Cd.E2 could induce Vtg synthesis in male Chinese loaches,and co-exposure with 50 or 500 g/L Cd could inhibit the Vtg induced by 1 g/L E2.Compared with the results reported in the literature,it can be concluded that factors such as fish species,Cd dosage,and manner of exposure might make contributions to the interaction between Cd and E2.Our results also suggested that male Chinese loaches are susceptible to Cd,and can be recommended as a potential sentinel species to study the ecotoxicology of heavy metals.     

Synergistic defensive mechanism of phytochelatins and antioxidative enzymes in <Emphasis Type="Italic">Brassica chinensis</Emphasis> L. against Cd stress

Brassica chinensis L. was chosen and exposed to different concentrations of Cd exposure to evaluate its Cd-accumulating capacity and its potential cellular defensive mechanisms. Cd accumulation in the shoots and roots of B. chinensis was up to 1348.3±461.8 and 3761.0±795.0 mg per killogram of dry weight, respectively, under 200 μmol/L of Cd exposure. Increasing Cd accumulation in the plant was accompanied by rapid accumulation of phytochelatins （PCs）, and the sequestration of Cd by PCs provided a primary cellular mechanism for Cd detoxification and tolerance of B. chinensis. Furthermore, malondialdehyde formation, hydrogen peroxide content and antioxidative enzyme activities such as superoxide dismutase, catalase, guaiacol peroxidase and ascorbate peroxidase were observed in the shoots of Cd-stressed B. chinensis. Increasing enzyme activities in response to concentrations of 5 to 50 μmol/L Cd showed an efficient defense against oxidative stress, suggesting that the antioxidative system was a secondary defensive mechanism. These resulted in reduced free Cd damage and enhanced Cd accumulation and tolerance. Glutathione plays a pivotal role in these two detoxification pathways. In general, these results suggested that PCs and the antioxidative system are synergistic in combatting Cd-induced oxidative stress and that they play important roles in Cd detoxification of B. chinensis, and also give a deep understanding of the natural defensive mechanisms in plants under heavy metal stress.     

Coral reef ecosystems in the South China Sea as a source of atmospheric CO<Subscript>2</Subscript> in summer

Field measurements of air-sea CO2 exchange in three coral reef areas of the South China Sea (i.e. the Yongshu Reef atoll of the Nansha Islands, southern South China Sea (SCS); Yongxing Island of Xisha Islands, north-central SCS; and Luhuitou Fringing Reef in Sanya of Hainan Island, northern SCS) during the summers of 2008 and 2009 revealed that both air and surface seawater partial pressures of CO2 (pCO2) showed regular diurnal cycles. Minimum values occurred in the evening and maximum values in the morning. Air pCO2 in each of the three study areas showed small diurnal variations, while large diurnal variations were ob-served in seawater pCO2. The diurnal variation amplitude of seawater pCO2 was ~70 μmol mol–1 at the Yongshu Reef lagoon, 420–619 μmol mol–1 on the Yongxing Island reef flat, and 264–579 μmol mol–1 on the reef flat of the Luhuitou Fringing Reef, and 324–492 μmol mol–1 in an adjacent area just outside of this fringing reef. With respect to spatial relations, there were large differences in air-sea CO2 flux across the South China Sea (e.g. ~0.4 mmol CO2 m–2 d–1 at Yongshu Reef, ~4.7 mmol CO2 m–2 d–1 at Yongxing Island, and ~9.8 mmol CO2 m–2 d–1 at Luhuitou Fringing Reef). However, these positive values suggest that coral reef ecosystems of the SCS may be a net source of CO2 to the atmosphere. Additional analyses indicated that diurnal variations of surface seawater pCO2 in the shallow water reef flat are controlled mainly by biological metabolic processes, while those of deeper water lagoons and outer reef areas are regulated by both biological metabolism and hydrodynamic factors. Unlike the open ocean, inorganic metabolism plays a significant role in influencing seawater pCO2 variations in coral reef ecosystems.     

Effect of iron deficiency on heterocyst differentiation and physiology of the filamentous cyanobacterium<Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC 7120

The effect of iron deficiency on heterocyst differentiation and some physiological properties of the filamentous cyanobacteriumAnabaena sp. PCC 7120 was investigated. Under moderate iron limitation conditions, achieved by addition of iron chelator 2, 2′-Dipyridyl (<80 μmol/L) led to delayed heterocyst differentiation, no heterocyst differentiation was observed under severe iron limitation conditions, when the concentration of 2,2′-Dipyridyl in the medium was more than 100 μmol/L. it seemed that there are certain iron-regulated genes or operons whose function is to control heterocyst development. In addition, iron deficiency impaired the growth. Low iron cells had a decrease in the quantities of pigment content (chlorophyll and phycocyanin content), the whole cellin vivo absorbance spectra confirmed the decrease, the protein electrophoretic profiles revealed that iron-deficient cells had less protein bands, with the increase of 2,2′-Dipyridyl, the protein bands was more and more less. And differently, iron deficiency also caused an increase of ROS (Reactive Oxygen Species) and SOD activity, it suggests that iron deficiency led to oxidative stress, which generally occured under high-iron conditions. Foundation item: Supported by the National Natural Sciences Foundation of China (30070154), the Frontier Science Projects Programme of the Institute of Hydrobiology, the Chinese Academy of Sciences (220316), State Key Project on Cyanobacterial Bloom Control in Lake Danchi (K9905-35-01) Biography: Xu Wenllang (1974-), male, Ph. D. research direction: molecular genetics of eyanobacteria.     

Pinacidil,a K<Subscript>atp</Subscript> channel opener,identified as a novel agonist for TRPA1

The transient receptor potential Ankyrin 1(TRPA1) cation channel is activated by various pungent and irritant compounds,and it also mediates the perception of noxious cold.Identification of different agonists for this channel is important for understanding its activation mechanism.Therefore,a screen for novel TRPA1 agonists was performed using an agonist-induced calcium influx assay.Out of 90 compounds screened,pinacidil was identified as a novel agonist for this channel.Pinacidil is a known opener of the K atp channel,for which it has an EC50 value of 1-3 μmol/L.In comparison,the EC50 value of pinacidil for TRPA1 is relatively high(260 μmol/L).Recombinant HEK-TRPA1 cells did not respond to P1075,another K atp channel opener,suggesting that the effect of pinacidil on TRPA1 was highly specific.Further studies revealed that the agonist activity of pinacidil could be blocked by the TRP channel inhibitors,ruthenium red and HC-030031.Using glutathione(GSH) and site-specific mutagenesis,we demonstrated that pinacidil could activate TRPA1 by covalent modification of the critical amino acids C619,C639 and C663 in the N-terminus of TRPA1.     

Inhibition of bromophenols against PTP1B and anti-hyperglycemic effect of <Emphasis Type="Italic">Rhodomela confervoides</Emphasis> extract in diabetic rats

Protein tyrosine phosphatase 1B （PTPIB） plays an important role as a negative regulator In insulin signaling pathways. PTPIB is an effective target for the treatment of type 2 diabetes mellitus. Four bromophenol derivatives from red algae Rhodomela confervoides, 2,2＇,3,3＇-tetrabromo-4,4＇,5,5＇-tetrahydroxydiphenyl methane （1）, 3-bormo-4,5-bis（2,3-dibromo-4,5-dihydroxybenzyl） pyrocatechol （2）, bis（2,3-dibromo-4,5-dihydroxybenzyl） ether （3） and 2,2＇,3-tribromo-3＇,4,4＇,5-tetrahydroxy-6＇-ethyloxymethyldiphenylmethane （4） showed significant inhibitory activity against PTPIB （IC50 were 2.4, 1.7, 1.5 and 0.84 μmol/L, respectively） as potential therapeutical agents for the treatment of type 2 diabetes mellitus. The anti-hyperglycemic effects of the ethanol extracts from If. confervoides on streptozotocin-induced diabetes （STZ-diabetes） in male Wistsr rats fed with high fat diet were investigated. The STZ-diabetic rats treated with medium-dose and high-dose alga extracts showed remarkable reductions in fasting blood glucose （FBG） as compared with the STZ-diabetic control. The results indicate that the in vivo anti-hyperglycemic activity of the R. confervoides extracts can be partially attributed to the inhibitory actions against PTPIB of the bromophenol derivatives and that may be of clinical Importance in improving the management of type 2 diabetes mellitus.     

A novel method for thermodynamic study on binding of copper ion with Alzheimer’s amyliod β peptide

The interaction of Cu²⁺ with the first 16 residues of the Alzheimer's amyliod β peptide, Aβ (1-16), was studied by employing isothermal titration calorimetry at pH 7.2 and 37℃ in aqueous solution. The Gholamreza Rezaei Behbehani (GRB) solvation model was used to reproduce the enthalpies of Cu²⁺+ Aβ(1-16) interaction over the whole Cu²⁺ concentrations. The binding parameters recovered from the solvation model were attributed to the structural change of Aβ (1-16) due to the metal ion interaction. It was found that there is a set of two identical and non interacting binding sites for Cu²⁺ ions. The molar enthalpy of binding is ΔH=27.895 kJ/mol. The association binding constants are 1.895 μm^-1 and 1.891 μm^-1 for the first and second binding sites respectively.     

The foxtail millet Si69 gene is a Wali7 (wheat aluminum-induced protein 7) homologue and may function in aluminum tolerance

We isolated a clone, named Si69, from a foxtail millet immature seed cDNA library. The protein encoded by Si69 contains a conserved Wali7 (wheat aluminum induced protein 7) domain and shares high-level homology with aluminum-induced proteins from other species including rice and Arabidopsis. The Si69 gene presents as a single locus in foxtail millet genome and is globally expressed in all tissues examined. Its expression is up-regulated by aluminum. The sequence feature and expression pattern suggest that the Si69 gene is involved in aluminum tolerance or detoxification. To confirm its biological functions, Si69 controlled by the CaMV35S promoter was introduced into Arabidopsis. Transgenic plants did not show any visible morphological changes compared to wild-type plants under normal growth conditions. However, when treated with 20 or 50 μmol/L Aluminum (Al), the root apices of wild-type plants were heavily stained by hematoxylin, whereas those of Si69 transgenic plants were not stained when treated with 20 μmol/L Al and slightly stained when treated with 50 μmol/L Al. Scanning electron microscopy (SEM) results further demonstrated that the damage of the root apices was severer in wild-type plants than in transgenic plants. Inhibition of root growth and accumulation of malondialdehyde (MDA), an indicator of lipid peroxidation, were lower in transgenic plants than in wild-type plants. The results show that overexpression of Si69 may increase Al tolerance in transgenic plants, indicating that a series of Wali7-containing genes may play similar roles in Al tolerance/detoxification.     

Effect of cadmium and herbicides on the growth,chlorophyll and soluble sugar content in rice seedlings

To investigate the combined effects of Cd^2＋ （ 0,10,100,500 μmol/L） , acetochlor （AC） （0,1.6,4.0,8.0μmol/L）, and bensulfuronmethyl （BSM） （ 0,0.16,0.40,0.80 μmol/L） on the biomass,leaf soluble sugar, total chlorophyll （chl） content, chl a/b ratio, and Cd content in roots and shoots in the rice seedlings, pot culture experiments were conducted with rice （Oryza sativa L. ） cultivar Jinyou 402. The results showed that the Cd and AC combined treatment （Cd＋ AC treatment） significantly inhibited the growth of the roots and shoots. The root dry weight/shoot dry weight （RDW/SDW） ratio, total chlorophyll content and chl a/b ratio decreased by 41%, 50%, 56% , respectively, in comparison with the control, as well as the leaf soluble sugar content, plant dry weight /plant fresh weight （PDW/PFW） ratio increased by 284% and 44% , respectively. Cd content in the roots and shoots had a decreasing tendency with the increasing concentration of AC under the Cd＋AC treatment. The Cd and BSM combined treatment （Cd＋BSM treatment） had a similar result to the Cd＋AC treatment. The results suggest that the toxicity of Cd to rice seedlings has been enhanced due to its combination with herbicides.