Silent ovulation, based on walking activity and milk progesterone concentrations, in Holstein cows housed in a free-stall barn

The objectives of the current study were to determine the incidence of silent ovulation (based on walking activity and milk progesterone profiles), identify risk factors for silent ovulation, and investigate its impact on reproductive performance in high-yielding dairy cows in free-stall housing. Overall, 277 lactations in 161 Holstein Friesian cows from a commercial dairy herd in northern Japan were studied. Walking activity (measured with pedometers) >80% above the mean for the preceding 2 d was defined as estrus, whereas day of ovulation was estimated using milk progesterone concentrations. Ovulation not preceded by increased walking activity was considered silent ovulation; the incidence was 55.2%, 23.8%, 21.3%, and 10.5% at the first, second, third, and fourth ovulations postpartum, respectively. Moderate and high milk yield significantly increased the risk of silent ovulation at second (odds ratio [OR] = 2.7 and 1.2; P = 0.04) and third and/or fourth ovulations (OR = 6.7 and 12.9; P = 0.03). Based on survival analysis, silent ovulations at the first, second, third, and/or fourth ovulations were associated with 28% (hazard ratio [HR] = 0.72), 55% (HR = 0.45), and 47% (HR = 0.53) reductions in pregnancy rate, respectively, and 41% (HR = 0.59), 66% (HR = 0.34), and 65% (HR = 0.35) reductions in artificial insemination (AI) submission rate. Cows with at least one silent ovulation (with the exception of the first ovulation) had a longer interval from calving to first AI (72 vs. 54 d, P < 0.001) and to achievement of pregnancy (133 vs. 80 d, P < 0.001). In conclusion, approximately one third of the ovulations (based on milk progesterone concentrations) in Holstein cows within 90 d postpartum were silent. Silent ovulations at the second to fourth ovulations were associated with high milk yields and at all ovulations were associated with impaired reproductive performance.     

Two doses of prostaglandin improve pregnancy rates to timed-AI in a 5-day progesterone-based synchronization protocol in beef cows

The objective was to compare reproductive performance of Angus-cross beef cows synchronized with GnRH, a progesterone-based intravaginal insert (Controlled Internal Drug Release, CIDR) for 5-d, and one dose of either dinoprost (PGF) or cloprostenol (CLP, a PGF analogue) or two doses of PGF on the day of CIDR withdrawal. All cows (N = 830) at six locations received 100 μg of GnRH and a CIDR on Day 0. Within farm, cows were randomly allocated to receive 25 mg of PGF at the time of CIDR insert removal on Day 5 (1 × PGF; N = 277), two 25 mg doses of PGF, the first given on Day 5 at the time of CIDR removal and the second 7 h later (2 × PGF; N = 282), or 500 μg of CLP at the time of CIDR removal on Day 5 (1 × CLP; N = 271). All cows were given 100 μg of GnRH on Day 8 (72 h after CIDR removal) and concurrently inseminated (5-d CO-Synch + CIDR). Cows were fitted with a pressure-sensitive estrus detection device at the time of CIDR withdrawal. Timed-AI pregnancy rates were greater (P < 0.0001) in the 2 × PGF (69.0%) than the 1 × PGF (52.0%) and 1 × CLP (54.3%) treatments. However, breeding-season pregnancy rates were not different among treatments (87.0% for 1 × PGF, 92.9% for 2 × PGF and 87.5% for 1 × CLP; P > 0.1). In conclusion, cows that received two doses of PGF on the day of CIDR removal in a 5-d CO-Synch + CIDR synchronization protocol had excellent timed-AI pregnancy rates that were greater than in cows receiving a single treatment with either PGF or CLP.     

Comparison of accuracy of ultrasonography, progesterone, and pregnancy-associated glycoprotein tests for pregnancy diagnosis in semidomesticated reindeer

The aim of the study was to compare transrectal ultrasound with progesterone (P4) and pregnancy-associated glycoproteins (PAGs) as pregnancy detection methods for semidomesticated reindeer (Rangifer tarandus tarandus) in field conditions. Female reindeer (n = 195) were scanned transrectally by a 7.5-MHz linear array transducer, and blood was sampled either in December 2005 (n = 33), December 2006 (n = 92), or January 2007 (n = 70) during early or mid gestation. Plasma levels of P4 and PAGs were assessed by radioimmunoassay (RIA). Based on calving records, the sensitivity, specificity, predictive values, and the overall accuracy of the three tests were calculated. The overall calving rate calculated from the calving records was 86.2%. The overall accuracy of transrectal ultrasound was 99.5%. The sensitivity and specificity of transrectal ultrasound were 99.4% and 100%, respectively. In the plasma P4 test, the threshold level of 5.0 nmol/L gave the highest overall accuracy (94.9%). The sensitivity of the P4 test decreased from 96.4% to 81.5%, when the threshold level increased from 5.0 nmol/L to 8.0 nmol/L, while the specificity remained at 85.2% over the range of these cutoff values. The overall accuracy of the plasma PAG test decreased from 96.4% to 64.1% when the plasma PAG threshold level increased from 0.5 ng/mL to 3.5 ng/mL, whereas sensitivity decreased from 99.4% to 58.3%. Specificity increased from 77.8% to 100% when the plasma PAG threshold level reached 3.0 ng/mL. Transrectal ultrasound showed higher diagnostic values than those of plasma P4-RIA and PAG-RIA in diagnosing pregnancy of reindeer, with the advantage that diagnoses can be made in real time in field conditions.     

Infrared technology for estrus detection and as a predictor of time of ovulation in dairy cows in a pasture-based system

The development and application of an algorithm to assess the ability of an infrared thermography (IRT) device to predict cows in estrus and about to ovulate was investigated. Twenty cows were synchronized using a controlled internal drug release and PGF2α. Vulval and muzzle temperatures were measured every 12 hours from controlled internal drug release insertion to 32 hours after PGF2α treatment and then every 4 hours until ovulation occurred or until 128 hours after PGF2α treatment (whichever occurred first). Thermal images obtained with a FLIR T620 series infrared camera were analyzed using ThermaCAM Researcher Professional 2.9 software. Cows were also monitored for behavioral signs of estrus and color changes of an Estrotect applied to the tail head of each cow 36 hours after PGF2α treatment. Algorithms were developed by adjusting body surface temperature of individual animals for ambient temperature and humidity during each observation period, and were expressed as a deviation from the baseline temperature. Of the 20 cows enrolled in this study, 12 (60%) ovulated. An IRT estrus alert was defined using different thresholds (D = 1 °C, 1.25 °C, and 1.5 °C). Sensitivity and specificity to predict estrus depended upon the chosen threshold level. At a threshold D = 1 °C, the highest sensitivity (92%; n = 11) and the lowest specificity (29%) and positive predictive value (64%) were observed. Conversely, D = 1.5 °C resulted in sensitivity of 75%, specificity of 57%, and positive predictive value of 69%. The mean ± standard deviation intervals between onset and the end of IRT estrus alert to ovulation were 30.7 ± 8.2 and 13.3 ± 7.7 hours, respectively. Ovulation occurred 24 to 47 hours after the onset of the IRT estrus alert for eight out of the 11 ovulated cows (73%). Although the sensitivity of the IRT alert was greater than visual observation (67%) and Estrotect activation (67%), the specificity and positive predictive value were lower than these two aids (i.e., the IRT overpredicted the incidence of ovulation). Results presented indicate that IRT shows some potential as an estrus detection aid; however, further studies investigating the potential to improve the specificity and capturing data throughout entire 21-day reproductive cycles would be worthwhile.     

Field evaluation of the MM3-SERO ELISA for detection of anti-Fasciola IgG antibodies in milk samples from individual cows and bulk milk tanks

We carried out a field evaluation of the MM3-SERO ELISA for the diagnosis of Fasciola hepatica infection, by analysing serum and milk samples from individual cows and samples from bulk milk tanks. The diagnostic performance of the assay was assessed with serum samples from all 257 cows in eight fluke-free herds, and 240 cows with natural fasciolosis (diagnosed in vivo and/or post-mortem). Assay performance for individual milk samples was determined by analysis of paired serum and milk samples from 947 lactating cows from 33 F. hepatica-infected farms. The diagnostic usefulness of the assay for bulk tank milk was evaluated by analysis of bulk milk from infected (33) and non-infected (35) farms. For serum samples, the sensitivity, specificity and diagnostic accuracy of the assay were respectively 99.2% (95% CI: 97.0%–99.9%), 100% (95% CI: 98.6%–100%) and 0.997 (95% CI: 0.987–1.000). The only two infected animals in which serum antibodies were not detected had very low parasitic burdens (with only 2 and 3 flukes observed). The performance of the MM3 SERO ELISA for individual milk samples was similar to that for serum samples, and the stepwise linear regression revealed a strong correlation between the results for the milk samples and the serum samples (R² = 0.84; p < 0.001). The agreement between results obtained with pairs of serum and milk samples was very high: there was matching classification in 96% (910/947) of paired samples (kappa = 0.92; p < 0.001). Individual milk samples may therefore be used, instead of serum samples, in the MM3-SERO ELISA, for reliable detection of seropositive cows. Testing bulk tank milk samples enabled detection of infected herds, even when the within-herd prevalence of infection was as low as 12%. We conclude that the MM3-SERO ELISA is a sensitive and highly specific test for serodiagnosis of bovine fasciolosis, and can be used with individual samples of either serum or milk. Use of the assay with bulk milk samples enables estimation of the within-herd prevalence of infection.     

Efficacy of decreasing the dose of GnRH used in a protocol for synchronization of ovulation and timed AI in lactating dairy cows

To determine the efficacy of reducing the dosage of GnRH used in a protocol for synchronization of ovulation and timed AI, primiparous and multiparous lactating Holstein cows (n=237) were randomly assigned to 1 of 2 treatment groups. Ovulation was synchronized for cows in the first group using intramuscular injections of GnRH and PGF₂ as follows: Day 0, 100 μg GnRH; Day 7, 25 mg PGF₂; Day 9, 100 μg GnRH. Ovulation was synchronized in the second group of cows using the same injection schedule and dosage of PGF₂ but only 50 μg GnRH per injection. All cows underwent a timed AI at 12 to 18 h after the second GnRH injection. The proportion of cows ovulating in response to the second GnRH injection (synchronization rate) and pregnancy status at 28 and 56 d post AI were determined using transrectal ultrasonography. The synchronization rate, double-ovulation rate, conception rate at 28 and 56 d post AI, and pregnancy loss from 28 to 56 d post AI did not differ statistically between treatment groups. For all cows, synchronization rate was 84.0%, and double-ovulation rate was 14.1%. Conception rates calculated using all cows receiving synchronization of ovulation were 41.1% at 28 d and 34.4% at 56 d post AI. Conception rates calculated for only synchronized cows were 47.6% at 28 d and 40.1% at 56 d post AI. For all cows, pregnancy loss from 28 to 56 d post AI was 13.5%, with an attrition rate of 0.5% per day. Estimated savings in hormone costs using 50 rather than 100 μg GnRH per injection for synchronizing ovulation were $6.40 per cow and $20.27 per pregnancy. Thus, decreasing the dosage of GnRH used for synchronization of ovulation and timed AI in lactating dairy cows reduces synchronization costs per cow and per pregnancy without compromising the efficacy of the synchronization protocol.     

Co-construction of a method for evaluating the intrinsic quality of bovine milk in relation to its fate

There are time-tested assessments for the environmental and economic aspects of sustainability. Its societal aspect has mainly been approached through the assessment of animal welfare. However, the intrinsic quality of milk is seldom taken into account. We developed a participatory construction method for the overall assessment of intrinsic milk quality in its different dimensions (sensory, technological, nutritional and health), according to the fate of the raw milk. Two assessment models were developed, for semi-skimmed standardized ultra-high temperature (UHT) milk and for pressed uncooked non-standardized raw milk cheese. They were constructed by a participatory approach involving experts in the dairy sector with the aim to obtain a diagnostic tool that could be used in the field to help farmers to manage the quality of their milk (by prioritizing improvements on major problems). They were shaped from prerequisite specifications (limited costs and time of application, desire to obtain a transparent tool with all the steps kept visible) and current technical and scientific knowledge. They were based on indicators obtained from raw bulk tank milk analyses (30 for UHT milk and 50 for cheese assessments), which were then aggregated into criteria, principles, dimensions and overall intrinsic quality at farm level. The assessment models had parts in common, for example, same four dimensions, common indicators for health and nutritional dimensions. They also had process-specific features: units chosen, criteria, indicators and weightings in relation to the final product specifications. For instance, sensory and technological dimensions are more complex and preponderant in the cheese assessment (three principles for cheese vs one for UHT milk in both dimensions). Another example is the lack of microbial pathogens (as potential health risk for consumer) in the UHT milk assessment because of pasteurization. The assessment models then underwent a sensitivity analysis and an application in 30 farms in indoor and grazing periods to finally obtain overall UHT milk and cheese quality scores at a 1-year level. The tool was found to be applicable at farm level. However, we observed low overall quality scores with a narrow dispersion, characteristic of a severe evaluation. Even so, the assessment models showed up seasonal differences of the UHT milk and cheese quality at both overall and dimensional levels. In the light of new scientific knowledge and future quality objectives, these are adaptable to other dairy products allowing for their specific features.     

Evaluation of a rapid ELISA technique for detection of circulating antigens of Toxoplasma gondii

To evaluate a modified rapid ELISA method for detecting CAg during Toxoplasma gondii infection, we analyzed the specificity and sensitivity of the ELISA method by using experimental Toxoplasma infection in rabbits and also tested this method in human samples including 5428 serum, 548 cerebrospinal fluid and two breast milk samples. We prepared PcAb, and used it for rapid one-step sandwich ELISA testing in which an incubation time in the regular ELISA procedure was omitted. This method detected CAg at the concentration of 31.2 ng/mL, and no cross-reaction was found with antigens of protozoa (Cryptosporidium parvum, Plasmodium falciparum), trematode (Schistosoma japonicum, Paragonimus sp.) and nematode (Brugia malayi, Ancylostoma duodenale, Ascaris lumbricoides and Trichinella spiralis). CAg was detected in rabbit serum 3 days after infection, and optical density values reached a peak 9-13 days after infection, then declined gradually. Among human serum samples, the positive rate of CAg was 2.11% in cerebral paralysis patients, whereas it was 0.22% or 0.71% in patients without neurological symptoms or in uncomplicated pregnant women. The difference among these three groups was statistically significant (P < 0.05). The positive rate of cerebrospinal fluid samples from cerebral paralysis patients was 10.58%. There is a statistically significant difference between the positive rates of meat-packing workers and blood donors (P < 0.01). In the retrospective analysis, CAg was detected in accordance with the onset of clinical symptoms, suggesting that CAg could reflect the clinical course in humans. Together with these results, CAg detected in the modified rapid sandwich ELISA could be a sensitive marker for acute and active infection of T. gondii.     

Incorporation of ultrasonic cell disintegration into a membrane bioreactor for zero sludge production

For the prevention of excess sludge production from a membrane bioreactor (MBR), an ultrasonic cell disintegration process was incorporated. The results of this study showed that excess sludge production could be prevented using an ultrasound hybrid (MBR-US) system at an organic loading of around 0.91 kg BOD₅/m³ per day. Under the same organic loading rate, the mixed liquor suspended solid (MLSS) of MBR-US system was maintained at 7000–8000 mg/l while the MLSS of a conventional MBR increased from 7000 to 13,700 mg/l during the experimental period. While sludge production was completely prevented, the effluent quality of the MBR-US system slightly deteriorated. The additional organic loading caused by disintegrated sludge return was considered to be a reason. With sonication the volume of the average particle size of the sludge in the aeration tank decreased from 132 to 95 μm. In the MBR-US system, around 25–30% of total phosphorus removal was achieved without sludge removal from the aeration tank.     

Effects of ovine CRF injections into the dorsomedial, dorsolateral and lateral columns of the periaqueductal gray: a functional role for the dorsomedial column

Corticotropin-releasing factor (CRF) and its receptor subtypes have been implicated in the regulation of endocrine, behavioral and autonomic responses to stress, fear and anxiety. Ovine CRF (oCRF) is a nonspecific CRF receptor agonist that produces anxiogenic-like effects when injected locally into the dorsal aspects of the periaqueductal gray (PAG). This structure is subdivided into four distinct longitudinal columns but their exact functional role is not fully understood. The purpose of the present study was to characterize the effects of oCRF (0.25, 0.5 and 1 microg/0.2 microL) injections into the dorsomedial (dmPAG), dorsolateral (dlPAG) and lateral (lPAG) columns of the PAG using an analysis of the exploratory behavior of rats in the elevated plus-maze (EPM) test. The results showed that microinjections of oCRF intra-dmPAG reduced entries and time spent in the open arms and decreased end-arm exploration and head-dipping. In contrast, oCRF intra-dlPAG or lPAG did not affect the exploratory behavior of the animals in the EPM. These findings point to a columnar specificity for the oCRF effects in the PAG, that is, it increased spatial avoidance measures of the EPM test only in the dmPAG. The proaversive effects of oCRF in the dmPAG gain further relevance when combined with previous immunohistochemical studies showing that CRF-containing projections from the periventricular hypothalamic system arch dorsomedially to the PAG, which could function as an important relay station in the midbrain tectum for avoidance behaviors.     

FAST-Modelfree: a program for rapid automated analysis of solution NMR spin-relaxation data

Herein we describe the program FAST-Modelfree for the fully automated, high throughput analysis of NMR spin-relaxation data. This program interfaces with the program Modelfree 4.1 and provides an intuitive graphical user interface for configuration as well as complete standalone operation during the model selection and rotational diffusion parameter optimization processes. FAST-Modelfree is also capable of iteratively assigning models to each spin and optimizing the parameters that describe the diffusion tensor. Tests with the protein Ribonuclease A indicate that using this iterative approach even poor initial estimates of the diffusion tensor parameters will converge to the optimal value within a few iterations. In addition to improving the quality of the final fit, this represents a substantial timesaving compared to manual data analysis and minimizes the chance of human error. It is anticipated that this program will be particularly useful for the analysis and comparison of data collected under different conditions such as multiple temperatures and the presence and absence of ligands. Further, this program is intended to establish a more uniform protocol for NMR spin-relaxation data analysis, facilitating the comparison of results both between and within research laboratories. Results obtained with FAST-Modelfree are compared with previous literature results for the proteins Ribonuclease H, E. coli glutaredoxin-1 and the Ca²⁺-binding protein S100B. These proteins represent data sets collected at both single and multiple static magnetic fields and which required analysis with both isotropic and axially symmetric rotational diffusion tensors. In all cases results obtained with FAST-Modelfree compared favorably with the original literature results.     

Reproductive consequences of a reciprocal chromosomal translocation in two Duroc boars used to provide semen for artificial insemination

Cytogenetic analysis of 58 boars at an artificial insemination (AI) centre revealed the presence of a reciprocal chromosome translocation, rcp(1;11)(q−;p+), in two Duroc boars. Pedigree analysis of these two boars suggested familial transmission of the chromosome rearrangement. The reproductive consequences of this translocation were determined in a herd of sows that had received semen doses from these and other boars. All sows underwent multiple AI, with different groups established retrospectively depending on the percentage of semen doses provided by the carrier boars ([number of carrier boar doses/total number doses provided] x 100): 0%, 25%, 50%, 75%, 100%. The fertility rates (percentage of successful multiple AIs/total multiple AIs) recorded for multiple AI including semen doses from the carrier boars were not significantly different from those recorded when all semen doses were supplied by normal-karyotype boars. A reduction in litter size of 29.38% was observed, however, in litters sired by one of the carrier boars when its participation in multiple AI was 100%. The number of live-born piglets per litter gradually decreased (P < 0.05) as the percentage participation in multiple AI (25, 50, or 75%) of the carrier boar increased. In addition, both carrier boars sired some piglets with signs of cleft palate and complex malformations of the front legs; these died soon after birth. In conclusion, the boars carrying the translocation rcp(1;11)(q−;p+) showed reduced reproductive performance.     

Evaluation of a standardized F1 capsular antigen capture ELISA test kit for the rapid diagnosis of plague

Rapid detection of soluble F1 capsular antigen in serum, bubo fluid or urine of patients proved to be a valuable tool in the presumptive diagnosis of plague. We evaluated a F1 capsular antigen capture ELISA resembling a commercially available test kit. The minimal detectable concentration was 4 ng/ml. The specificity was 100% when investigating 47 sera from healthy Malagasy subjects and 98.4% when 365 sera from German blood donors were studied. Sensitivity was determined on sera (n=11) and buboes (n=18) from bacteriologically confirmed Malagasy plague patients. Sensitivity was 90.1% for serum and 100% for buboes. A standardized F1 capsular antigen capture ELISA test kit might be well suited for the early detection of plague particularly in non-endemic areas where clinical microbiological laboratories have only limited access to alternative techniques for rapid identification of Yersinia pestis.     

Hedgerows as an environment for forest plants: a comparative case study of five species

Many areas in Europe are dominated by agricultural land use, and as a consequence, many typical forest plant species suffer from habitat loss and fragmentation. Hedgerows, one of the common elements of rural landscapes, have been considered as potential refuges for these species. The main objective of this study was to examine whether forests and hedgerows differ in environmental conditions, and whether important life-history attributes of the populations differ between the two habitat types. We selected five species commonly found in the region in both forests and hedgerows (Adoxa moschatellina, Anemone nemorosa, Circaea lutetiana, Polygonatum multiflorum and Stellaria holostea), and sampled data on 10 populations of each species in each habitat type, including measurements of light and various soil factors. Hedgerows had higher relative light availability and tended to have higher soil nutrient contents and lower soil water values than forests. The comparison of plant performance values between habitat types did not show consistent patterns across species. Anemone and Polygonatum performed equally well in hedgerows and forests, whereas Stellaria appeared to have a higher fitness in hedgerows. In contrast, Circaea showed a higher reproduction under forest conditions. For Adoxa, the results were somewhat contradictory: whereas the reproductive output of this species was higher in forests, population density was higher in hedgerows. The abiotic factors most closely related to the performance values were relative light and soil water availability. The majority of plant performance values did not differ between hedgerows and forests. We therefore conclude that the tested forest species are capable of growing also in hedgerows and will survive equally well in forest and its “surrogate” habitat.     

Incorporation of the gene for a cell-cell channel protein into transformed cells leads to normalization of growth

Summary Incorporation of the gene for connexin 43, a cell-cell channel protein of gap junction, into the genome of communication-deficient transformed mouse 10T1/2 cells restored junctional communication and inhibited growth. Growth was slowed, saturation density reduced and focus formation suppressed, and these effects were contingent on overexpression of the exogenous gene and the consequent enhancement of communication. In coculture with normal cells the growth of the connexin overexpressors was completely arrested, as these cells established strong communication with the normal ones. Thus, in culture by themselves or in coculture, the connexin overexpressor cells grew like normal cells. These results demonstrate that the cell-cell channel is instrumental in growth control; they are the expected behavior if the channel transmits cytoplasmic growth-regulatory signals.     

Incorporation of DMSO and dextran-40 into a gelatin/alginate hydrogel for controlled assembled cell cryopreservation

A new cell cryopreservation strategy for cell-assembling constructs was proposed. With this strategy, different concentrations of dimethysulfoxide (DMSO) and dextran-40 were directly incorporated into the cell/gelatin/alginate systems, prototyped according to a predesigned structure, cryopreserved at −80 °C for 10 days and followed a thawing process at 17 °C. The rheological properties, bonding water contents and melting points of the gelatin/alginate hydrogel systems were changed with the addition of different amounts of DMSO. The microscopy analysis, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrasodium bromide (MTT) and hematoxylin and eosin (HE) staining indicated that the cell numbers were progressively in a selected DMSO concentration range. With DMSO 5% (v/v) alone, the metabolic rate in the construct attained (81.3 ± 5.7)%. A synergistic effect was achieved with the combination of the DMSO/gelatin/alginate and dextran-40/gelatin/alginate hydrogel systems. These results indicated that the inclusion of DMSO and dextran-40 in the hydrogel could effectively enhance the cell preservation effects. This cryopreservation strategy holds the ability to be widely used in organ manufacturing techniques.     

Incorporation of cis-parinaric acid,a fluorescent fatty acid,into synaptosomal phospholipids by an acyl-CoA acyltransferase

The cis-isomer of parinaric acid, a naturally occurring C-18 polyene fatty acid, was incubated with brain subcellular fractions and the polarization of fluorescence increased in a time dependent manner. Greatest increases occurred in synaptosomal and microsomal membranes. This increase in polarization of fluorescence was found with the cis, but not the trans, isomer of parinaric acid and required Mg²⁺ or Ca²⁺ and was stimulated by coenzyme A and ATP. Synaptosomes were incubated with cis-parinaric acid and lipids were extracted and examined by high performance liquid chromatography. The highest incorporations of cis-parinaric acid were found in phosphatidylcholine (71%) and phosphatidylethanolamine (20%) while only traces were found in phosphatidylserine and phosphatidylinositol. [³H]Oleic acid was also incorporated into membrane phospholipids and unlabeled oleic acid blocked incorporation of cis-parinaric acid. It is proposed that cis-parinaric acid, like fatty acids normally found in brain, is incorporated into membrane phospholipids by an acyl-CoA acyltransferase. The presence of this enzyme in nervous tissue may make it possible to easily introduce fluorescent fatty acid probes into membrane phospholipids and to thereby facilitate study of membrane-mediated processes.     

Physicochemical basis for the rapid time-action of LysB28ProB29-insulin: dissociation of a protein-ligand complex.

The rate-limiting step for the absorption of insulin solutions after subcutaneous injection is considered to be the dissociation of self-associated hexamers to monomers. To accelerate this absorption process, insulin analogues have been designed that possess full biological activity and yet have greatly diminished tendencies to self-associate. Sedimentation velocity and static light scattering results show that the presence of zinc and phenolic ligands (m-cresol and/or phenol) cause one such insulin analogue, LysB28ProB29-human insulin (LysPro), to associate into a hexameric complex. Most importantly, this ligand-bound hexamer retains its rapid-acting pharmacokinetics and pharmacodynamics. The dissociation of the stabilized hexameric analogue has been studied in vitro using static light scattering as well as in vivo using a female pig pharmacodynamic model. Retention of rapid time-action is hypothesized to be due to altered subunit packing within the hexamer. Evidence for modified monomer-monomer interactions has been observed in the X-ray crystal structure of a zinc LysPro hexamer (Ciszak E et al., 1995, Structure 3:615-622). The solution state behavior of LysPro, reported here, has been interpreted with respect to the crystal structure results. In addition, the phenolic ligand binding differences between LysPro and insulin have been compared using isothermal titrating calorimetry and visible absorption spectroscopy of cobalt-containing hexamers. These studies establish that rapid-acting insulin analogues of this type can be stabilized in solution via the formation of hexamer complexes with altered dissociation properties.     

Development of a capture/enrichment sandwich ELISA for the rapid detection of enteropathogenic and enterohaemorrhagic Escherichia coli O26 strains

AIMS: To improve the sensitivity of a monoclonal antibody (MAb 2F3) based enteropathogenic Escherichia coli (EPEC)/enterohaemorrhagic E. coli (EHEC) serogroup O26-specific sandwich ELISA (sELISA), using a capture/enrichment format of the assay. METHODS AND RESULTS: The sELISA utilized an EPEC/EHEC O26-specific MAb 2F3 as the capture reagent and an E. coli serogroup O26 lipopolysaccharide-specific polyclonal antibody in the development stage. Wells containing faeces test samples from bovine enteritis cases and agar colony sweep cultures from human diarrhoea cases, after a 2-h capture stage, were washed and enrichment of the captured cells was encouraged by addition of tryptone soya broth. After overnight incubation, the contents of each well were transferred to sterile wells and the sELISA completed. Any sELISA positive samples were then subcultured onto blood agar to recover and further characterize the positive cultures. The assay had a sensitivity of 10(3) CFU ml(-1). ELISA positive samples consisted of 21 (4.8%) of the 442 bovine and 19 (3.7%) of the 519 human samples tested, and ELISA positive EPEC/EHEC O26 strains were isolated from 11 and three of these samples respectively. CONCLUSION: The capture/enrichment method improved the sensitivity of a MAb-based sELISA for the detection of EPEC/EHEC O26 strains, and also contributed to an improved isolation rate of the organism from field samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of a specific MAb in a capture/enrichment format of the sELISA, provides a prospectively suitable screening method for the detection of pathogenic bacteria from mixed culture samples.     

Incorporation of biodegradable nanoparticles into human airway epithelium cells-in vitro study of the suitability as a vehicle for drug or gene delivery in pulmonary diseases

PURPOSE: Nanoparticles are able to enhance drug or DNA stability for purposes of optimised deposition to targeted tissues. Surface modifications can mediate drug targeting. The suitability of nanoparticles synthesised out of porcine gelatin, human serum albumin, and polyalkylcyanoacrylate as drug and gene carriers for pulmonary application was investigated in vitro on primary airway epithelium cells and the cell line 16HBE14o-. METHODS: The uptake of nanoparticles into these cells was examined by confocal laser scan microscopy (CLSM) and flow cytometry (FACS). Further the cytotoxicity of nanoparticles was evaluated by an LDH-release-test and the inflammatory potential of the nanoparticles was assessed by measuring IL-8 release. RESULTS: CLSM and FACS experiments showed that the nanoparticles were incorporated into bronchial epithelial cells provoking little or no cytotoxicity and no inflammation as measured by IL-8 release. CONCLUSIONS: Based on their low cytotoxicity and the missing inflammatory potential in combination with an efficient uptake in human bronchial epithelial cells, protein-based nanoparticles are suitable drug and gene carriers for pulmonary application.