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Lameness and lesions in the claws of 31 autumn‐calving Holstein Friesian dairy cows were recorded from before their first‐calving until their fifth lactation. The animals were managed by the same herdsman and housed adjacently in the same building in one of two herds grazed either on clover‐rich pastures (herd 1) or on conventional ryegrass (herd 2). All four hooves were examined routinely four times during each lactation, and claw lesions were scored for severity and drawn on hoof maps. Heel erosion and infectious skin conditions of the hoof were also recorded, and hoof conformation, hardness, and growth and wear were measured. The animals' locomotion was scored weekly throughout the winter housing period and any observed to be lame were examined to determine the cause. The development of lesions was modelled by using hierarchic smoothing splines. There was no significant effect of herd except on the prevalence of lameness in lactation 2 when the incidence of (inter)digital dermatitis was higher in herd 2. Lesion and locomotion scores were significantly higher by lactation 4 (P<0.05). There were significant effects (P<0.05) of weeks postcalving on lesion formation, claw conformation, and heel erosion.

Factorial experimental designs were employed to study the major factors governing the gelation of milkl κ-carrageenan mixtures. The relative effects of carrageenan concentration, pH, ionic strength, casein proteins, whey proteins and κ-casein genotype on the mechanical properties of the gels were studied using dynamic rheological techniques. Gel transition temperature was governed mainly by ionic strength and carrageenan concentration whilst gel strength was influenced by carrageenan concentration and pH. It was found that the relative importance of the milk proteins to gelation varied with carrageenan concentration. It appeared that at high levels of κ-carrageenan (i.e. 0.1% w/w) gelation was predominately the result of association of κ-carrageenan helices to produce self-supporting gels. Milk proteins appeared to have little effect at high levels of carrageenan. However, milk proteins interfered with gel formation at carrageenan concentrations lower than 0.018% w/w, thus increasing the amount of micellar casein by twofold inhibited gelation. Serum proteins also suppressed gelation, albeit at lower κ-carrageenan concentrations. These results suggested that, in the presence of milk proteins, gel formation involved mainly carrageenan—carrageenan cross-linkages and not carrageenan—casein or casein-casein linkages. Nevertheless, the possibility of a strong interaction between κ-carrageenan and κ-casein on the micelles which has to be satisfied first, leaving insufficient carrageenan to provide network forming capability, cannot be ruled out.

The vitronectin receptor (alpha v beta 3) is a member of the integrin superfamily that mediates cell attachment on arginine-glycine-aspartic acid (RGD)-containing adhesive proteins. A solid-phase microtiter assay was developed to investigate the binding properties of purified alpha v beta 3, using tritiated [3H]SK&F-107260 as the radiolabeled ligand. alpha v beta 3, purified from human platelets, human placenta, and chicken osteoclasts, bound [3H]SK&F-107260 saturably and specifically. Saturation binding studies using platelet alpha v beta 3 revealed a single class of high affinity binding sites, exhibiting a Kd of 1.44 nM and Bmax of 0.20 mol of [3H]SK&F-107260/mol of alpha v beta 3. [3H]SK&F-107260 binding was inhibited by a variety of RGD-containing peptides and by the snake venom protein echistatin, whereas an RGE-containing peptide and four nonpeptide fibrinogen receptor (alpha IIb beta 3) antagonists failed to do so. This study shows that alpha v beta 3 exhibits distinct ligand specificity from the structurally homologous fibrinogen receptor, alpha IIb beta 3. The relative potencies of the RGD-containing peptides in inhibiting [3H]SK&F-107260 binding to alpha v beta 3 were the same as their relative potencies in inhibiting biotinylated-fibrinogen binding to the receptor. alpha v beta 3 purified from chicken osteoclasts and human placenta bound [3H]SK&F-107260 with similar affinities and displayed the same pharmacological profile as the platelet vitronectin receptor. The alpha v beta 3 antagonists inhibited the attachment of MG63 human osteosarcoma cells or rat osteoclasts to recombinant rat osteopontin. The rank order of potency of the antagonists in the cell adhesion assays was similar to that of the receptor binding assay, suggesting that the purified alpha v beta 3-[3H]SK&F-107260 binding assay is a valid reflection of the ligand binding to alpha v beta 3 on cell systems.

Here we describe the characterization of a novel human CC chemokine, tentatively named monocyte chemotactic protein (MCP-4). This chemokine was detected by random sequencing of expressed sequence tags in cDNA libraries. The full-length cDNA revealed an open reading frame for a 98-amino acid residue protein, and a sequence alignment with known CC chemokines showed high levels of similarity (59-62%) with MCP-1, MCP-3, and eotaxin. MCP-4 cDNA was cloned into Drosophila S2 cells, and the mature protein (residues 24-98) was purified from the conditioned medium. Recombinant MCP-4 induced a potent chemotactic response (EC50 = 2.88 +/- 0.15 nM) and a transient rise in cytosolic calcium concentration in fresh human peripheral blood monocytes but not in neutrophils. Binding studies in monocytes showed that MCP-4 and MCP-3 were very potent in displacing high affinity binding of 125I-MCP-1 (IC50 for MCP-4, MCP-3, and unlabeled MCP-1 of 2.1 +/- 1.4, 0.85-1.6, and 0.7 +/- 0.2 nM respectively), suggesting that all three chemokines interact with the CC chemokine receptor-2 (MCP-1 receptor). This was confirmed in binding studies with Chinese hamster ovary cells, stably transfected with the CC chemokine 2B receptor. Northern blot analysis in extracts of normal human tissues showed expression of mRNA for MCP-4 in small intestine, thymus, and colon, but the level of protein expression was too low to be detected in Western blot analysis. However, expression of MCP-4 protein was demonstrated by immunohistochemistry in human atherosclerotic lesion and found to be associated with endothelial cells and macrophages.

The site of action of a series of pyridinyl imidazole compounds that are selective inhibitors of p38 mitogen-activated protein kinase in vitro and block proinflammatory cytokine production in vivo has been determined. Using Edman sequencing, 125I-SB206718 was shown to cross-link to the nonphosphorylated Escherichia coli-expressed p38 kinase at Thr175, which is proximal to the ATP binding site. Titration calorimetric studies with E. coli-expressed p38 kinase showed that SB203580 bound with a stoichiometry of 1:1 and that binding was blocked by preincubation of p38 kinase with the ATP analogue, FSBA (5'-[p-(fluorosulfonyl)benzoyl]adenosine), which covalently modifies the ATP binding site. The intrinsic ATPase activity of the nonphosphorylated enzyme was inhibited by SB203580 with a Km of 9.6 mM. Kinetic studies of active, phosphorylated yeast-expressed p38 kinase using a peptide substrate showed that SB203580 was competitive with ATP with a Ki of 21 nM and that kinase inhibition correlated with binding and biological activity. Mutagenesis indicated that binding of 125I-SB206718 was dependent on the catalytic residues K53 and D168 in the ATP pocket. These findings indicate that the pyridinyl imidazoles act in vivo by inhibiting p38 kinase activity through competition with ATP and that their selectivity is probably determined by differences in nonconserved regions within or near the ATP binding pocket.

The in vitro activation of the recombinant purified human cathepsin K (EC 3.4.22.38) was examined by mutagenesis. Cathepsin K was expressed as a secreted proenzyme using baculovirus-infected Sf21 insect cells. Spontaneous in vitro activation of procathepsin K occurred at pH 4 and was catalyzed by exogenous mature cathepsin K. Three intermediates were identified as resulting from cleavages after Glu19, Ser98, and Glu110. The mature enzyme was composed of mixture of enzymes with N termini of Gly113, Arg114, and Ala115 with varying ratios depending on the preparation. Molecular weight determinations were consistent with the absence of carbohydrate in the mature protein, while electrospray mass spectroscopy indicated that six of the eight cysteine residues were in disulfide linkage, and that the protein had Met329 as the C-terminal residue. A mutant was constructed in which the active site Cys139 was changed to Ser. [Ser139,Ala163]Procathepsin K (containing mutation C139S,S163A) failed to spontaneously process and was only partially processed in the presence of 1% exogenous wild-type mature cathepsin K forming intermediates, which were identical to those observed in the activation of wild-type. [Ser139,Ala163]Procathepsin K could be fully processed to mature enzyme by including one equivalent of wild-type procathepsin K in the activation mixture. These results indicated that in vitro activation of the procathepsin K was an autocatalytic process.

In the preceding report (Ladner, R.D., McNulty, D.E., Carr, S.A., Roberts, G.D., and Caradonna, S.J. (1996) J. Biol. Chem. 271, 7745-7751), we identified two distinct isoforms of dUTPase in human cells. These isoforms are individually targeted to the nucleus (DUT-N) and mitochondria (DUT-M). The proteins are nearly identical, differing only in a short region of their amino termini. Despite the structural differences between these proteins, they retain identical affinities for dUTP (preceding article). In previous work, this laboratory demonstrated that dUTPase is posttranslationally phosphorylated on serine residue(s) (Lirette, R., and Caradonna, S. (1990) J. Cell. Biochem. 43, 339-353). To extend this work and determine if both isoforms of dUTPase are phosphorylated, a more in depth analysis of dUTPase phosphorylation was undertaken. [32P]Orthophosphate-labeled dUTPase was purified from HeLa cells, revealing that only the nuclear form of dUTPase is phosphorylated. Electrospray tandem mass spectrometry was used to identify the phosphorylation site as Ser-11 in the amino-terminal tryptic peptide PCSEETPAIpSPSKR (the NH2-terminal Met is removed in the mature protein). Mutation of Ser-11 by replacement with Ala blocks phosphorylation of dUTPase in vivo. Analysis of the wild type and Ser-11 --> Ala mutant indicates that phosphorylation does not regulate the enzymatic activity of the DUT-N protein in vitro. Additionally, experiments with the Ser-11 --> Ala mutant indicate that phosphorylation does not appear to play a role in subunit association of the nuclear form of dUTPase. The amino acid context of this phosphorylation site corresponds to the consensus target sequence for the cyclin-dependent protein kinase p34(cdc2). Recombinant DUT-N was specifically phosphorylated on Ser-11 in vitro with immunoprecipitated p34(cdc2). Together, these data suggest that the nuclear form of dUTPase may be a target for cyclin-dependent kinase phosphorylation in vivo.

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase; EC 3.6.1.23) was purified from HeLa cells by immunoaffinity chromatography. Based on SDS-polyacrylamide gel electrophoresis, two distinct forms of dUTPase were evident in the purified preparation. These proteins were further characterized by a combination of NH2-terminal protein sequencing, mass spectrometry, and mass spectrometry-based protein sequencing. These analyses indicate that the two forms of dUTPase are largely identical, differing only in a short region of their amino-terminal sequences. Despite the structural difference, both forms of dUTPase exhibited identical binding characteristics for dUTP. Each form of dUTPase has a distinct cellular localization. Cellular fractionation and isopycnic density centrifugation indicate that the lower molecular weight form of dUTPase (DUT-N) is associated with the nucleus, while the higher molecular weight species (DUT-M) fractionates with the mitochondria. The DUT-N isoform is approximately 30-fold more abundant in HeLa cells than DUT-M as determined by densitometry. The NH2-terminal protein sequence of both DUT-N and DUT-M did not match previous reports of the predicted amino-terminal sequence for human dUTPase (McIntosh, E.M., Ager, D.D., Gadsden, M.H., and Haynes, R.H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 8020-8024; Strahler, J.R., Zhu X., Hora, N., Wang, Y.K., Andrews, P.C., Roseman, N.A., Neel, J.V., Turka, L., and Hanash, S.M. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4991-4995). A cDNA corresponding to the DUT-N isoform was isolated utilizing an oligonucleotide probe based on the determined NH2-terminal sequence. The cDNA contains a 164-amino acid open reading frame, encoding a protein of Mr 17,748. The DUT-N cDNA sequence matches the previously cloned cDNAs with the exception of a few discrepancies in the 5' end. Our data indicate a 69-base pair addition to the 5' end of the previously reported open reading frame.

Human cathepsin K is a recently identified protein with high primary sequence homology to members of the papain cysteine protease superfamily including cathepsins S, L, and B and is selectively expressed in osteoclasts (Drake, F.H., Dodds, R., James I., Connor J., Debouck, C., Richardson, S., Lee, E., Rieman, D., Barthlow, R., Hastings, G., and Gowen, M. (1996) J. Biol., Chem. 271, 12511-12516). To characterize its catalytic properties, cathepsin K has been expressed in baculovirus-infected SF21 cells and the soluble recombinant protein isolated from growth media was purified. Purified protein includes an inhibitory pro-leader sequence common to this family of protease. Conditions for enzyme activation upon removal of the pro-sequence have been identified. Fluorogenic peptides have been identified as substrates for mature cathepsin K. In addition, two protein components of bone matrix, collagen and osteonectin, have been shown to be substrates of the activated protease. Cathepsin K is inhibited by E-64 and leupeptin, but not for by pepstatin, EDTA, phenylmethylsulfonyl fluoride, or phenanthroline, consistent with its classification within the cysteine protease class. Leupeptin has been characterized as a slow binding inhibitor of cathepsin K (kobs/[I] = 273,000 m(-1).s(-1)). Cathepsin K may represent the elusive protease implicated in degradation of protein matrix during bone resorption and represents a novel molecular target in treatment of disease states associated with excessive bone loss such as osteoporosis.

Plant uptake is one of the major pathways by which sludge-borne potentially toxic metals enter the food chain. This study examined the accumulation of Cd, Cu, Ni, Pb and Zn in wheat, carrots and spinach grown on soils from 13 sites previously amended with sewage sludge. Winter wheat, carrots and spinach were grown consecutively under field like conditions. The results showed that plant availability of heavy metals differed widely among the crop species. The accumulation of Cd, Ni and Zn in the plants showed the greatest increases compared to their background levels. The Cu and Pb accumulation in the plants grown on sludge-amended soils showed only small increases compared to those grown on uncontaminated soils. Multiple regression analysis of various soil properties showed that the surest way to control the accumulation of metals in food plants is by controlling their concentrations in the soils. Furthermore, soils with a non-acidic pH and a clayey texture tended to achieve better control of metal accumulation in food plants compared to those with an acidic reaction and a coarse texture. Metal concentrations in the plants generally correlated well with those extracted from soils in 0·005 M DTPA, 0·05 M EDTA-(Na)2, 1 M NH4NO3 and 0·05 M CaCl2. The EDTA, however, proved to be a more reliable and consistent test in predicting the accumulation of metals in the plants. The results also showed that liming soils to pH 7 effectively reduced the metal contents in carrots and spinach, but liming to pH 6·5 had little effect on metal concentrations in wheat grain. © 1997 SCI.

An interlaboratory comparison on cheese making trials was conducted to examine the differences between model cheeses manufactured in pilot plants at six European laboratories. The experimental design (within each laboratory) for the model cheese was an unreplicated 23full factorial design in one block of eight cheese vats. The three factors were the pasteurization of skimmed milk, microfiltration of skimmed milk and pasteurization of cream. Although the manufacture, sampling and analyses were generally standardized, the gross composition and sensorial properties of the cheeses differed greatly across the laboratories. Good statistical design and analysis of the experimental data aided in minimizing the effect caused by the technical difficulties experienced by some of the laboratories during the cheese making trials. Despite the problems encountered, all six laboratories noted similar effects between model cheeses that were produced from milk in which the initial load of indigenous flora was reduced by either pasteurization or microfiltration. The cheeses produced from milk in which the raw milk flora had been reduced had lower concentrations of D-lactate and a less intense aroma and odour. With few exceptions the individual laboratories observed similar effects, although these were less significant when compared with the overall effect.

Two observables are called complementary if preparing a physical object in an eigenstate of one of them yields a completely random result in a measurement of the other. We investigate small sets of complementary observables that cannot be extended by yet another complementary observable. We construct explicit examples of unextendible sets up to dimension 16 and conjecture certain small sets to be unextendible in higher dimensions. Our constructions provide three complementary measurements, only one observable away from the ultimate minimum of two. Almost all our examples in finite dimensions are useful for discriminating pure states from some mixed states, and they help to shed light on the complex topology of the Bloch space of higher-dimensional quantum systems. [ABSTRACT FROM AUTHOR]

Thermally removing amine molecules that serve as chemical templates for vanadium oxide nanotubes is demonstrated to significantly improve the performance when tested as a cathode material in Li-ion battery cells. Capacity fading issues associated with blocked intercalation sites on the (010) faces of layered vanadium oxide that form the nanotubes are prevented. Thermal treatment of the nanotubes up to 600°C is shown to cause a specific conversion from nanotubes to polycrystalline nanorods and removal of the organic template. The conversion process was monitored by thermogravimetric analysis, X-ray diffraction, transmission electron microscopy and infra-red spectroscopy. In a potential window of 4.0–1.2 V drawing 30 μA (C/30), the nanorods show improved specific capacities of ∼280 mAh g−1with a modest 6% capacity fade compared to ∼8 mAh g−1with 62% capacity fade for the VONTs. The improvements in specific capacity and cycling performance are due to the successful removal of amine molecules and conversion to nanorods containing nanoscale crystals. The cathode material also demonstrated enhanced energy densities (∼700 W h kg−1) compared to composites of the same overall weight, without conductive carbon additives or polymeric binders.

The fungus Verticillium lecanii (V.1.) has been developed as a pesticide at two research centres, Tate & Lyle (T & L) and Glasshouse Crops Research Institute (GCRI) and staff at both locations have been screened for atopic status and sensitization to V.1 The majority of staff at T & L had full medical examination, respiratory testing, chest X-ray and full blood profile. In total 141 staff were skin tested for allergy: eight showed positive tests to V.l., four at GCRI and four at T & L; seven of these were allergic and one non-allergic, non atopic. Sensitization was not well correlated with heavy exposure. None were aware of symptoms handling V.l. and all responses were less than histamine control. Testing with two isolates of V.1, Ve2 and Ve6, revealed that the major allergens are in Ve2. The 85 T & L staff completing medical tests recorded revealed no abnormalities. All X-rays were normal. The normal variability of respiratory function was not altered by exposure. Immunoglobulins varied with allergic state but did not differ significantly between exposed and unexposed groups. There was no toxic effect on blood parameters following exposure to V.l.