Inhibition of bovine adrenocortical cytochrome P-450scc by 3,3'-dimethoxybenzidine

The effect of 3,3'-dimethoxybenzidine ($\text{o}$-dianisidine) on the conversion of cholesterol to pregnenolone was investigated in a reconstituted side chain cleavage system using enzymes purified from bovine adrenal cortex; d-$\text{p}$-aminoglutethimide was also assayed under similar conditions for comparison. 3,3'-Dimethoxybenzidine was found to be a potent inhibitor of pregnenolone formation, causing 50% inhibition at a concentration of 1.5 μM when using 70 μM cholesterol — this dose is approximately one fourth that required of 3-methoxybenzidine and one twentieth that required of benzidine for equal inhibition. In the same system, d-$\text{p}$-aminoglutethimide exhibited an I₅₀ value of about 55 μM. No effects of 3,3'-dimetoxybenzidine on adrenodoxin reductase or adrenodoxin activities could be detected, and inhibition of side chain cleavage could be relieved by dilution suggesting that the inhibitor acts by reversibly binding to cytochrome P-450scc.     

Kinetic characterization of the ATPase cycle of the molecular chaperone Hsc66 from Escherichia coli

Hsc66 from Escherichia coli is a constitutively expressed hsp70 class molecular chaperone whose activity is coupled to ATP binding and hydrolysis. To better understand the mechanism and regulation of Hsc66, we investigated the kinetics of ATP hydrolysis and the interactions of Hsc66 with nucleotides. Steady-state experiments revealed that Hsc66 has a low affinity for ATP (K(m)(ATP) = 12.7 microM) compared with other hsp70 chaperones. The kinetics of nucleotide binding were determined by analyzing changes in the Hsc66 absorbance spectrum using stopped-flow methods at 23 degrees C. ATP binding results in a rapid, biphasic increase of Hsc66 absorbance at 280 nm; this is interpreted as arising from a two-step process in which ATP binding (k(a)(ATP) = 4.2 x 10(4) M(-1) s(-1), k(d)(ATP) = 1.1 s(-1)) is followed by a slow conformational change (k(conf) = 0. 1 s(-1)). Under single turnover conditions, the ATP-induced transition decays exponentially with a rate (k(decay) = 0.0013 s(-1)) similar to that observed in both steady-state and single turnover ATP hydrolysis experiments (k(hyd) = 0.0014 s(-1)). ADP binding to Hsc66 results in a monophasic transition in the absence (k(a)(ADP) = 7 x 10(5) M(-1) s(-1), k(d)(ADP) = 60 s(-1)) and presence of physiological levels of inorganic phosphate (k(a)(ADP(P(i)) = 0.28 x 10(5) M(-1) s(-1), k(d)(ADP(P(i)) = 9.1 s(-1)). These results indicate that ATP hydrolysis is the rate-limiting step under steady-state conditions and is >10(3)-fold slower than the rate of ADP/ATP exchange. Thus, in contrast to DnaK and eukaryotic forms of hsp70 that have been characterized to date, the R if T equilibrium balance for Hsc66 is shifted in favor of the low peptide affinity T state, and regulation of the reaction cycle is expected to occur at the ATP hydrolysis step rather than at nucleotide exchange.     

Rearing goat kids away from their dams 1. A survey to understand rearing methods

Despite an estimated global goat population of over one billion, little is known about methods being used to feed milk to artificially reared kids (reared away from their dams) and how kids are weaned from these systems. Quantifying and characterising current methods utilised on farms will enable future targeted research to investigate best practice methods for milk feeding and weaning of artificially reared kids. A recall-based survey that investigated on-farm kid-rearing practices (focusing on the milk feeding and weaning stages) was distributed via social media, and regional goat organisations across multiple countries. A total of 242 responses from 16 countries were collected and geographically grouped. Responses that could not be grouped sufficiently were removed (nine responses from eight countries). A total of 233 responses from eight countries (United States of America (USA) 72; United Kingdom (UK) 71; Australia 33; Canada 23; New Zealand 20; European Union (EU) 14), were analysed. Most farms (217; 93%) bred their own kids. The most common milk feeding method was bottle feeding, used on 135 farms (57.9%), followed by ad libitum feeding used by 72 (30.9%). A relationship between number of kids reared and feeding system was identified, χ²(3, N = 233) = 89.605, P < 0.001, with farms rearing > 100 kids more likely to feed milk ad libitum. A total of 170 farms (72.9%) were weaned based on a target age and 85 (36.4%) on a target weight, 53 (22.7%) used both and 45 (19.3%) neither. Target weaning ages and weights varied across countries; the median age was 84 days (interquartile range (IQR) 56–84), and the median weight was 16 kg (IQR 15–18). A difference was found between milk feeding systems for weaning method (Χ²(2, N = 232) = 63.797, P ≤ 0.001), with kids most likely to be abruptly weaned from ad libitum systems (or gradually weaned from bottle feeding). Abrupt weaning was used by 67 farms (28.8%), and gradual weaning was used by 165 (71.1%). Gradual weaning strategies included reducing milk quantity (150 farms; 93% of farms providing detail) and diluting milk (six farms; 4%). A total of 169 (72.5%) supplied enrichment that met the survey’s definition; items to climb on/hide in were most common, provided by 157 farms (92.8%). Findings suggest differing practices in smaller-scale bottle-fed versus larger-scale ad libitum milk systems, likely reflecting differing system needs. This highlights a requirement for welfare-focused research in kids reared artificially in order to identify and communicate best practices to ensure on-farm welfare is optimised within each system.     

Heterochromatin content and chiasma distribution in the megameric chromosomes of Stethophyma gracile and S. lineatum (Orthoptera: Acrididae)

Single populations of Stethophyma gracile and S. lineatum from Eastern Canada show substantial differences in the relative amount of heterochromatic and euchromatic material in the extensively heterochromatic (megameric) M₉ autosome. No differences in chromosome phenotype between these two species have been previously reported. A new polymorphism with respect to an interstitial, supernumerary H-block was also found in the S. lineatum population, and it appears to have some interchromosomal influence upon chiasma condition. These findings suggest that the North American Stethophyma forms may parallel the extensive and deme-specific heterochromatin variation found in the European populations of the Palaearctic S. grossum. Special attention was focused upon the structural organization of the proximal and distal H-segments on the M₉ during the early prophase of the first meiotic division in the male sex. These conspicuous, hetregions, prior to diplotene, are clearly interrupted and contain short, euchromatic sequences. This condition can readily account for the absence of chiasma localization and the distribution of chiasmata along the H-blocks at diplotene-diakinesis in the megameric element. This is the only member of the complement showing no evidence of pronounced proximal or proximal/distal chiasma localization at diplotene. Possible explanations for this interesting phenomenon are suggested.     

Modification of hemoglobin A with dimethyl adipimidate. Contribution of individual reacted subunits to changes in oxygen affinity.

The effect of dimethyl adipimidate, a bifunctional imidoester, on the oxygen affinity of hemoglobin A has been studied. Treatment of human oxyhemoglobin with 5 mM dimethyl adipimidate at pH 8.5, room temperature is accompanied by an increase in oxygen affinity in the presence and absence of 2,3-diphosphoglyceric acid. Circular dichroism measurements in the ultraviolet region indicate that dimethyl adipimidate-treated hemoglobin exhibits a reduced conformational change upon deoxygenation. In order to study the contribution of reacted individual subunits, alpha and beta subunits of dimethyl adipimidate-treated and untreated hemoglobin have been separated and reconstituted to form hybrid tetramers containing either the alpha-treated (alpha t beta c) or the beta-treated subunits (alpha c beta t). Electrophoresis on sodium dodecyl sulfate polyacrylamide gels of isolated alpha and beta globin subunits as well as hybrid tetramers from dimethyl adipimidate-treated hemoglobin reveals that 20% of the globin subunits are cross-linked. In the absence of 2,3-diphosphoglyceric acid, modification of alpha subunits increases the oxygen affinity and reduces the conformational change of the tetramer upon deoxygenation whereas modification of beta subunits has no effect. However, treatment of beta subunits decreases the effect of 2,3-diphosphoglyceric acid on the oxygen affinity of the hybrids and reduces the 2,3-diphosphoglyceric acid-induced spectral changes in oxyhemoglobin. Therefore the interaction of dimethyl adipimidate with both the alpha and beta subunits contributes to regulating the oxygen affinity of human hemoglobin.     

Culturing of stoneworts and submersed angiosperms with phosphate uptake exclusively from an artificial sediment

1. We aimed to demonstrate reproducible nutrition and growth of macrophytes in non‐axenic laboratory cultures preventing growth of phytoplankton and epiphytes. 2. Macrophyte shoot segments were planted in a mixture of commercial acid‐washed silica sand with crystalline tricalcium phosphate, and this artificial sediment was covered with a layer of pure silica sand. The liquid mineral media used did not contain phosphorus but were rich in all other nutrient elements. A CO₂ reservoir provided sustainable CO₂ supply to macrophyte cultures by gas diffusion through a polyethylene membrane. 3. Chara hispida, Chara tomentosa, Chara baltica, Elodea canadensis, Potamogeton pectinatus and Zanichellia palustris could be cultivated for long term without medium exchange and aeration. Microalgae growth was prevented by the absence of phosphate in the water column. Mobilisation of tricalcium phosphate and phosphate uptake by the rhizoids of C. hispida enabled sustainable rapid shoot growth and increased the concentration of inorganic phosphate in the shoot dry weight by five to six times in comparison with plants cultivated on pure silica sand. A significant growth support from tricalcium phosphate was also observed for E. canadensis, but the rate of phosphate uptake by the roots was not sufficient to maintain a storage pool of inorganic phosphate (P_i) in the growing shoots of this plant. 4. Membrane‐controlled CO₂ supply from a reservoir and artificial sediments like the one described provide attractive options for the laboratory culture of macrophytes.     

The preparation, characterisation, and DNA adduct profile of 2-amino-2-methyl-3-butanoneoximedichloroplatinum(II), a platinum(II) complex designed to bind to GpA sequences of DNA

The complex, 2-amino-2-methyl-3-butanoneoximedichloroplatinum(II), [Pt(ambo)Cl2], was chosen because of its potential to bind to GpA sequences of duplex DNA. Crystals of [Pt(ambo)Cl2] are monoclinic, space group, P2(1)/n, a = 6.799(4), b = 17.642(5), c = 8.193(2) A, beta = 102.10(3) degrees, Z = 4, R = 0.033 (1864 F). The binding of [Pt(ambo)Cl2] to salmon-sperm DNA was studied using enzymatic digestion and HPLC analysis. [Pt(ambo)Cl2] was found to form fewer GpG and ApG intrastrand adducts and more monofunctional adducts than [Pt(en)Cl2]. Binding to GpA sequences could not be established, but [Pt(ambo)Cl2] forms substantially more adducts with adenine than does [Pt(en)Cl2].     

Type V collagen regulates the assembly of collagen fibrils in cultures of bovine vascular smooth muscle cells

Vascular smooth muscle cells (SMCs), the major cellular constituent of the medial layer of an artery, synthesize the majority of connective tissue proteins, including fibrillar collagen types I, III, and V/XI. Proper collagen synthesis and deposition, which are important for the integrity of the arterial wall, require the antioxidant vitamin C. Vitamin C serves as cofactor for the enzymes prolyl and lysyl hydroxylase, which are responsible for the proper hydroxylation of collagen. Here, the role of type V collagen in the assembly of collagen fibrils in the extracellular matrix (ECM) of cultured vascular SMCs was investigated. Treatment of SMCs with vitamin C resulted in a dramatic induction in the levels of the cell‐layer associated pepsin‐resistant type V collagen, whereas only a minor induction in the levels of types I and III collagen was detected. Of note, the deposition of type V collagen was accompanied by the formation of striated collagen fibrils in the ECM. Immunohistochemistry demonstrated that type V collagen, but not type I collagen, became masked as collagen fibrils matured. Furthermore, the relative ratio of type V to type I collagen decreased as the ECM matured as a function of days in culture, and this decrease was accompanied by an increase in the diameter of collagen fibrils. Together these results suggest that the masking of type V collagen is caused by its internalization on continuous deposition of type I collagen on the exterior of the fibril. Furthermore, they suggest that type V collagen acts as framework for the initial assembly of collagen molecules into heterotypic fibrils, regulating the diameter and architecture of these fibrils. J. Cell. Biochem. 80:146–155, 2000. © 2000 Wiley‐Liss, Inc.     

Summer bird abundance and distribution on set-aside fields on intensive arable farms in England

The abundance and distribution of breeding birds were compared on paired fields comprising one set-aside field and one crop field, on 11 intensive arable farms in eastern and western England. A single observer made four visits to all set-aside and crop fields between April and July and recorded all birds seen during standardised counts and transects.
Rotational and non-rotational set-aside supported higher densities and more species of birds than fields of wheat, brassicas, root crops and seed rye. These differences in density, between set-aside and crops, were evident across a suit of species including waders, gamebirds, pigeons and passerines and were particularly marked on rotational set-aside. The majority of species recorded in fields away from boundaries would have been feeding rather than nesting there and higher bird densities on set-aside compared with adjacent arable crops probably reflects greater food abundance in the former.
The study is the first to demonstrate a clear habitat preference for set-aside by a wide range of bird species throughout the breeding season and it allows a number of recommendations to be made concerning the development of cost-effective agri-environment measures. The fact that the majority of birds utilised the outer 5 m, or in some cases 20 m, margin of the field suggests that many of the benefits of whole field set-aside may be derived from marginal strips. A slight, but not significant preference for rotational over non-rotational set-aside suggest such margins should be managed to maintain a patchy, relatively diverse sward of arable plants. A key feature of set-aside is the scale at which it has been incorporated into the arable landscape. Agri-enviromental schemes will only provide similar national benefits if they are implemented on a wide scale in such as way as to promote high uptake by farmers.