Kinetic disposition of an aqueous formulation of alphacypermethrin applied to the dorsal mid-line of sheep with long wool and its effect on lice

A group of 5 adult Merino sheep with fleeces about 70 mm long (7-months growth of wool) was treated with a topical formulation of the synthetic pyrethroid insecticide, alphacypermethrin, applied to the dorsal mid-line. Insecticide concentrations at the tip, middle and base of wool staples collected from meridians along the back, upper and lower flanks were measured at intervals from 1 to 98 days after treatment. Some movement of the alphacypermethrin from the back to the lower body occurred within 24h after treatment, but despite careful application of the insecticide there was wide variation in the concentration between and within meridians. The majority of the alphacypermethrin remained close to the dorsal mid-line and near the tip of the staple. There were significant differences in the concentration between the tip, middle and base segments of the staples in the back and lower flank meridians (P < 0.05). Despite exposure of the sheep to normal weathering, there was no significant difference in the concentration of alphacypermethrin between samples collected at day 1 or day 98 after treatment (P > 0.05). Numbers of pyrethroid-susceptible lice surviving exposure in vitro for 20 h differed significantly between samples collected at different times after treatment (P < 0.05). The numbers of lice surviving in samples collected within 28 days after treatment tended to be lower than in those collected from 28 to 98 days but, in some samples, regardless of time after treatment, lice survived for 20 h in wool taken from parts of the fleece that contained high concentrations of alphacypermethrin.     

Influx and efflux of inorganic carbon in Synechococcus UTEX625

The CO₂ and HCO₃^? fluxes in air-grown cells of Synechococcus UTEX 625 al pH 8-0 were measured during dark to light and light to dark transitions using a mass spectrometer and sampling of the reaction medium. The kinetic parameters for initial uptake of CO₂ and HCO₃^? were determined during the initial period of illumination. The development of the internal C_i pool was followed up to steady-state photosynthesis, which occurred when the size of the internal inorganic carbon pool remained apparently constant for a limited period. The experimental procedure confirmed that only CO₂ transport occurred with 100mmolm^?3 Na⁺ and that both CO₂ and HCO^?3 transport occurred with 25molm^?3 Na⁺. The K_1/2 values of initial CO₂ and HCO₃ uptake were 0.7 and 17.2 mmolm^?3respectively and agreed closely with the K_1/2 values of net CO₂ and HCO₃^? transport during steady-state photosynthesis, which were 0.66 and 17.1 mmolm^?3 respectively. Maximum rates of CO₂and HCO₃^? transport were 423 and 219mmolh^?1 g^?1 Chl. Maximum CO₂ efflux observed upon darkening was 118mmolh^?1 g^?1 Chl. A permeability coefficient of the cell for CO₂ of 3 × 10^?8 m s^?1 was determined from the dark CO₂ efflux assuming an internal pH of 7.2 in the dark. Following the initial CO₂ uptake in the light, the extracellular [CO₂] steadily declined when only CO₂ transport was allowed, but an increase in the extracellular [CO₂] when HCO₃^? transport was allowed to proceed suggested that an enhanced CO₂ efflux occurred as a result of the larger size of the intracellular C_i pool.     

A flash-photolysis study of the reactions of acaa 3-ttype cytochrome oxidase with dioxygen and carbon monoxide

The time course of absorbance changes following flash photolysis of the fully-reduced carboxycytochrome oxidase fromBacillus PS3 in the presence of O₂ has been followed at 445, 550, 605, and 830 nm, and the results have been compared with the corresponding changes in bovine cytochrome oxidase. The PS3 enzyme has a covalently bound cytochromec subunit and the fully-reduced species therefore accommodates five electrons instead of four as in the bovine enzyme. In the bovine enzyme, following CO dissociation, four phases were observed with time constants of about 10 s, 30 s, 100 s, and 1 ms at 445 nm. The initial, 10-s absorbance change at 445 nm is similar in the two enzymes. The subsequent phases involving hemea and Cu_A are not seen in the PS3 enzyme at 445 nm, because these redox centers are re-reduced by the covalently bound cytochromec, as indicated by absorbance changes at 550 nm. A reaction scheme consistent with the experimental observations is presented. In addition, internal electron-transfer reactions in the absence of O₂ were studied following flash-induced CO dissociation from the mixed-valence enzyme. Comparisons of the CO recombination rates in the mixed-valence and fully-reduced oxidases indicate that more electrons were transferred from hemea ₃ toa in PS3 oxidase compared to the bovine enzyme.     

Influence of chlorobenzoates on the utilisation of chlorobiphenyls and chlorobenzoate mixtures by chlorobiphenyl/chlorobenzoate-mineralising hybrid bacterial strains

Chlorobenzoates (CBA) arise as intermediates during the degradation of polychlorinated biphenyls (PCBs) and some chlorinated herbicides. Since PCBs were produced as complex mixtures, a range of mono-, di-, and possibly trichloro-substituted benzoates would be formed. Chlorobenzoate degradation has been proposed to be one of the rate-limiting steps in the overall PCB-degradation process. Three hybrid bacteria constructed to have the ability to completely mineralise 2-, 3-, or 4-monochlorobiphenyl respectively, have been studied to establish the range of mono- and diCBAs that can be utilised. The three strains were able to mineralise one or more of the following CBAs: 2-, 3-, and 4-monochlorobenzoate and 3,5-dichlorobenzoate. No utilisation of 2,3-, 2,5-, 2,6-, or 3,4-diCBA was observed, and only a low concentration (0.11 mM) of 2,4-diCBA was mineralised. When the strain with the widest substrate range (Burkholderia cepacia JHR22) was simultaneously supplied with two CBAs, one that it could utilise plus one that it was unable to utilise, inhibitory effects were observed. The utilisation of 2-CBA (2.5 mM) by this strain was inhibited by 2,3-CBA (200 M) and 3,4-CBA (50 M). Although 2,5-CBA and 2,6-CBA were not utilised as carbon sources by strain JHR22, they did not inhibit 2-CBA utilisation at the concentrations studied, whereas 2,4-CBA was co-metabolised with 2-CBA. The utilisation of 2-, 3-, and 4-chlorobiphenyl by strain JHR22 was also inhibited by the presence of 2,3- or 3,4-diCBA. We conclude that the effect of the formation of toxic intermediates is an important consideration when designing remediation strategies.Abbreviations PCB Polychlorinated biphenyl - CBA Chlorobenzoate     

Anchor-linked intermediates in peptide amide synthesis are caused by dimeric anchors on the solid supports

Cleavage and kinetic studies have been carried out using commercially obtained H-Tyr(tBu)-5-(4′-aminomethyl-3′,5′-dimethoxyphenoxy)valeric acid-TentaGelS (H-Tyr(tBu)-4-ADPV-TentaGelS) and H-Tyr (tBu)-4-ADPV-Ala-aminomethyl-resin (H-Tyr(tBu)-4-ADPV-AM-resin) prepared from commercially available resin and loaded with commercially available Fmoc-4-ADPV-OH amide anchor. Cleavage with pure trifluoroacetic acid (TFA) gave the intermediate H-Tyr-4-ADPV-NH₂, which was then degraded to H-Tyr-NH₂, and cleavage with TFA/dichloromethane (1:9) yielded H-Tyr-4-ADPV-NH₂ which could be isolated in preparative amounts. Cleavage reactions with ¹⁵N-labelled H-Ala-4-ADPV-[¹⁵N]-Gly-AM-resin yielded the intermediate H-Ala-4-ADPV-NH₂, which contained no ¹⁵N as demonstrated by ¹H-NMR. The analysis of the commercial Fmoc-4-ADPV-OH amide anchor showed the presence of Fmoc-4-ADPV-4-ADPV-OH as an impurity in high amounts. This dimeric anchor molecule is the cause of formation of the anchor-linked peptide intermediate obtained during the cleavage from the resin. The particularly high acid-lability of the amide bond between the two ADPV moieties was utilized to synthesize sidechain and C-terminally 4-ADPV protected pentagastrin on a double-anchor resin, and to cleave it using 5% trifluoroacetic acid in dichloromethane. This method may offer a new way for the synthesis of protected peptide amides with improved solubility to be used in fragment condensation.     

Metabolic activation of unsaturated derivatives of valproic acid. Identification of novel glutathione adducts formed through coenzyme A-dependent and -independent processes

The ability of 2-n-propyl-4-pentenoic acid (Δ⁴-VPA) and 2-n-propyl-2(E)-pentenoic acid ([E]-Δ²-VPA), two unsaturated metabolites of valproic acid (VPA), to form reactive intermediates, deplete hepatic glutathione (GSH) and cause accumulation of liver triglycerides was investigated in the rat. With the aid of ionspray liquid chromatography-tandem mass spectrometry (LC-MS/MS), three GSH adducts were detected in the bile of Δ⁴-VPA-treated animals and were identified as 4-hydroxy-5-glutathion-S-yl-VPA-γ-lactone, 5-glutathion-S-yl-(E)-Δ³-VPA and 3-oxo-5-glutathion-S-yl-VPA. A fourth conjugate was identified tentatively as 4-glutathion-S-yl-5-hydroxy-VPA. Quantitative analysis of the corresponding N-acetylcysteine (NAC) conjugates in urine indicated that metabolism of Δ⁴-VPA via the GSH-dependent pathways accounted for approximately 20% of an acute dose (100 mg kg⁻¹ i.p.). In contrast, when rats were given an equivalent dose of (E)-Δ²-VPA, only one GSH adduct (5-glutathion-S-yl-(E)-Δ³-VPA) was detected at low concentrations in bile. In vitro experiments with rat liver mitochondria demonstrated that Δ⁴-VPA undergoes coenzyme A- and ATP-dependent metabolic activation in this organelle via the β-oxidation pathway to intermediates which bind covalently to proteins. When liver homogenates and hepatic mitochondria from rats injected with Δ⁴-VPA, (E)-Δ²-VPA or VPA were analyzed for GSH content, it was found that only Δ⁴-VPA depleted GSH pools significantly. Treatment of rats with Δ⁴-VPA and (to a lesser extent) VPA led to an accumulation of liver triglycerides, whereas (E)-Δ²-VPA had no measurable effect. It is concluded that Δ⁴-VPA undergoes metabolic activation by both microsomal cytochrome P-450-dependent and mitochondrial coenzyme A-dependent processes, and that the resulting electrophilic intermediates, which are trapped in part by GSH, may mediate the hepatotoxic effects of this compound. In contrast, (E)-Δ²-VPA is not transformed to any appreciable extent to reactive metabolites, which thus accounts for the apparent lack of hepatotoxicity of this positional isomer in the rat.     

Kinetics of leucrose formation from sucrose by dextransucrase

Leucrose formation from sucrose and fructose by dextransucrase is of practical interest. It has been investigated at different experimental conditions, including the influence of temperature on reaction rate and selectivity. Under appropriate conditions high product yield can be obtained. Furthermore, a model is presented that allows interpretation of the experimental data.     

Native disulfide bonds greatly accelerate secondary structure formation in the folding of lysozyme.

To assess the respective roles of local and long-range interactions during protein folding, the influence of the native disulfide bonds on the early formation of secondary structure was investigated using continuous-flow circular dichroism. Within the first 4 ms of folding, lysozyme with intact disulfide bonds already had a far-UV CD spectrum reflecting large amounts of secondary structure. Conversely, reduced lysozyme remained essentially unfolded at this early folding time. Thus, native disulfide bonds not only stabilize the cfinal conformation of lysozyme but also provide, in early folding intermediates, the necessary stabilization that favors the formation of secondary structure.     

Thermodynamics of staphylococcal nuclease denaturation. II. The A-state.

Staphylococcal nuclease, at low pH and in the presence of high salt concentrations, has previously been proposed to exist in a partially folded or molten globule form called the "A-state" (Fink et al., 1993, Protein Sci 2:1155-1160). We have found that the A-state of nuclease at pH 2.1 in the presence of moderate to high salt concentrations and at low temperature exists in a substantially folded form structurally more similar to a native state. The A-state has the far-UV circular dichroism spectra characteristic of the native protein, which indicates that it has a large degree of secondary structure. Upon heating, the A-state denatures with a sigmoidal change in far-UV ellipticity and an observable peak in a differential scanning calorimeter trace, indicating that it is thermodynamically distinct from the denatured state. Three different mutations in a residue normally buried in the protein's core stabilize or destabilize the A-state in the same way as they affect the denaturation of the native state. The A-state must, therefore, contain at least some tertiary packing of side chains. Unlike the native state, which shows cold denaturation at low temperatures, the A-state is most stable at temperatures below 0 degrees C.