Efficacy of an injection of dinoprost tromethamine when given subcutaneously on luteal regression in lactating Holstein cows

The objectives of these studies were to evaluate the efficacy of a PGF(2alpha) (PGF) analog given through different routes on causing luteal regression in lactating dairy cows. In Experiment 1, lactating Holstein cows (n=118) at random stages of lactation were blocked by parity and days in milk (DIM) and, within each block, randomly assigned to receive PGF as an intra-muscular (IM) injection in the semimembranous/semitendinous muscle (CON), subcutaneous (SC) injection in the cervical area (SCN), or SC injection in the ischio-rectal fossa (IRF). Blood was sampled at 0, 12, 24, 36, and 48 h after treatment for assessment of progesterone concentration. In Experiment 2, a total of 379 lactating Holstein cows, 46+/-7 DIM, were blocked by DIM and, within each block, randomly assigned to receive treatment similar to CON or IRF groups from Experiment 1. Blood was sampled 0 and 48 h after treatment for assessment of progesterone concentration. Cows were classified as experiencing luteal regression when progesterone concentration was <1.0 ng/mL or <40% of initial concentration (0 h=100%). In Experiment 1, there was no effect of route of PGF treatment on decline in progesterone concentration and on the proportion of cows experiencing luteal regression by 12, 24, 36, and 48 h after treatment. Similarly, in Experiment 2, route of treatment did not affect either the decline in progesterone concentration or the proportion of cows that had luteal regression by 48 h after treatment. Treatment of lactating dairy cows with 25mg of PGF given SC in the ischio-rectal fossa did not affect either the decline in progesterone concentration or the proportion of cows that experienced luteal regression by 12, 24, 36, and 48 h after PGF treatment.     

Differential N-glycosylation of a monoclonal antibody expressed in tobacco leaves with and without endoplasmic reticulum retention signal apparently induces similar in vivo stability in mice

Plant cells are able to perform most of the post-translational modifications that are required by recombinant proteins to achieve adequate bioactivity and pharmacokinetics. However, regarding N-glycosylation the processing of plant N-glycans in the Golgi apparatus displays major differences when compared with that of mammalian cells. These differences in N-glycosylation are expected to influence serum clearance rate of plant-derived monoclonal antibodies. The monoclonal antibody against the hepatitis B virus surface antigen expressed in Nicotiana tabacum leaves without KDEL endoplasmic reticulum (ER) retention signal (CB.Hep1(-)KDEL) and with a KDEL (Lys-Asp-Glu-Leu) fused to both IgG light and heavy chains (CB.Hep1(+)KDEL) were tested for in vivo stability in mice. Full characterization of N-glycosylation and aggregate formation in each monoclonal antibody batch was determined. The mouse counterpart (CB.Hep1) was used as control. Both (CB.Hep1(-)KDEL) and (CB.Hep1(+)KDEL) showed a faster initial clearance rate (first 24 h) compared with the analogous murine antibody while the terminal phase was similar in the three antibodies. Despite the differences between CB.Hep1(+)KDEL and CB.Hep1(-)KDEL N-glycans, the in vivo elimination in mice was indistinguishable from each other and higher than the murine monoclonal antibody. Molecular modelling confirmed that N-glycans linked to plantibodies were oriented away from the interdomain region, increasing the accessibility of the potential glycan epitopes by glycoprotein receptors that might be responsible for the difference in stability of these molecules.     

Purification, characterization, and cloning of a serine proteinase inhibitor from the ectoparasite Haematobia irritans irritans (Diptera: Muscidae)

The fly Haematobia irritans irritans is one of the most important ectoparasites in cattle production, due to its ability to suck large amounts of blood. This report describes the purification and characterization of a serine proteinase inhibitor (HiTI) present in H. i. irritans head and thorax extracts. The HiTI purified by affinity chromatography on trypsin-Sepharose has a molecular mass of 7029Da by MALDI-TOF mass spectrometry. HiTI inhibited bovine trypsin, human neutrophil elastase, and a trypsin-like enzyme purified from H. i. irritans abdomen with dissociation constants of 0.57, 1.30, and 0.20nM, respectively. The HiTI partial amino acid sequence allowed its classification into the BPTI-Kunitz-type family. An HiTI cDNA fragment was cloned in the pGEMT vector using RT-PCR. The translated amino acid sequence of HiTI cDNA confirmed a unique Kunitz-type-domain protein. Our results suggest that HiTI could control some endogenous enzyme, e.g., the H. i. irritans trypsin-like protein.     

Feeding of Sagitta enflata and vertical distribution of chaetognaths in relation to low oxygen concentrations

Zooplankton samples were collected in Mejillones Bay, northernChile (23°00'15'S, 70°26'43'W ). Sampling was conductedat 4 h intervals, for 24 h during three seasons, austral spring(October 2000), summer ( January 2001) and winter (August 2001)at three different strata (0–25, 25–50 and 50–100m). Five species of chaetognaths were collected. Sagitta enflatawas the most abundant species, representing up to 65% of allchaetognaths in total numbers, followed by Sagitta bierii, makingup 34% of the total abundance of chaetognaths. S. enflata wasdistributed mainly above the Oxygen Minimum Zone, while S. bieriiremained below this zone. Feeding rates were relatively constantwithin the upper layer (0–25 m depth), for each samplingdate, averaging 1.2 prey S. enflata day^–1, and decreasingwith depth. Gut content analyses demonstrated that predationwas principally focused on small copepods (<1500 µm),with greatest feeding activity occurring at night. The dailypredation impact on the total standing stock of small copepodsvaried seasonally between 6% in spring and 0.4% in winter. Thispercentage may represent a negligible impact on the entire copepodcommunity, but it is relevant at the species or genus level,since S. enflata removed more than 20% of the standing stockof Centropages brachiatus and Corycaeus sp. Thus, during someperiods of the year, chaetognaths may strongly influence theabundance and size distribution of copepods in coastal upwellingecosystems.     

Improved pulmonary artery buffering function during phenylephrine-induced pulmonary hypertension

The goal of this study was to determine the in vivo pulmonary arterial buffering function (BF) during acute and moderate pulmonary hypertension achieved by phenylephrine-induced smooth muscle activation.Pulmonary pressure (Konigsberg P7) and diameter (sonomicrometry) were measured in nine anesthetized sheep. Transit pulmonary arterial hypertension was induced by mechanical occlusion of the pulmonary artery (HP) and by phenylephrine infusion (5 g/kg/min) (PHE). A viscoelastic Kelvin-Voigt model was used. By increasing the values of the viscous modulus, the pressure-diameter hysteresis area was reduced to a minimum in order to obtain the purely elastic pressure-diameter relationship. The elastic index (E) was calculated as the first derivative of the exponential model of the purely elastic pressure-diameter relationship at the mean pressure point.Systolic, diastolic, mean and pulse pressures were similar during HP and PHE, but significantly higher with regard to control steady state. In HP, E and arterial diameter (both its minimum and maximum values) increased significantly. In contrast, when pulmonary hypertension was induced by VSM activation, E was maintained concomitantly with pulmonary artery vasoconstriction.Pulmonary hypertension produced by occlusion of the pulmonary artery increases elasticity. Smooth muscle activation may offset the deleterious effect of pulmonary hypertension on arterial wall elasticity by reducing E and impeding arterial dilatation and collagen recruitment, maintaining BF during pulmonary hypertension.     

Impact of aging on myocardial metabolic response to dobutamine

In humans, under resting conditions there is an age-related decrease in myocardial fatty acid utilization (MFAU) and oxidation (MFAO) and a relative increase in myocardial glucose utilization (MGU). The impact of age on an individual's myocardial metabolic response to catecholamines is not well defined. Sixteen younger (mean age, 26 +/- 5 yr) and 14 older (mean age, 69 +/- 4 yr) volunteers underwent positron emission tomography to measure myocardial blood flow, myocardial oxygen consumption (M.VO2), MFAU, MFAO, and MGU both under resting conditions and during dobutamine infusion. In response to dobutamine administration, the rate-pressure product, myocardial blood flow, and M.VO2 measurements increased by similar amounts in both groups. No age-related differences were noted in the responses of plasma insulin, glucose, fatty acid, or lactate levels to dobutamine. With dobutamine infusion, MFAU and MFAO increased by a similar extent in both younger and older volunteers (age/dobutamine interactions, P = 0.62 and 0.75, respectively). In contrast, MGU increased with dobutamine administration in the younger (from 149 +/- 71 to 209 +/- 78 nmol.g(-1).min(-1); P = 0.04) but not in the older (from 235 +/- 147 to 176 +/- 84 nmol.g(-1).min(-1); P = 0.23; age/dobutamine interaction, P = 0.03) group. With dobutamine infusion, hearts in both younger and older volunteers responded by increasing their MFAU and MFAO values. Whereas younger hearts also responded with an increase in MGU, older hearts did not. Although the clinical significance of these findings awaits further study, these results may partially explain the impaired contractile reserve and the increased incidence of cardiovascular disease in older individuals.     

In vivo hormonal environment leads to differential susceptibility of the corpus luteum to apoptosis in vitro

We evaluated the involvement of the in vivo hormonal environment on the ability of the rat corpus luteum (CL) to undergo apoptosis. Gel electrophoretic DNA fragmentation analysis revealed no apoptosis in CL isolated either the 2 last days of pregnancy (Days 21 and 22) or throughout the 4 days following parturition, suggesting that the number of cells undergoing apoptosis at the same time is not sufficient to allow for visualization of DNA breakdown. In contrast, CL incubated in serum-free medium underwent significant apoptosis, as evaluated by chromatin condensation and DNA fragmentation, regardless of their developmental stage in pregnancy. However, CL obtained on Day 7 of pregnancy and on Day 4 postpartum demonstrated higher sensitivity to apoptosis in vitro, but lactation reduced significantly the capacity of the CL to undergo apoptosis when maintained in culture. These data suggest that the exposure of the CL to different hormonal environments throughout pregnancy and after parturition is responsible for the differential susceptibility to apoptosis observed in vitro. We have previously shown that progesterone is a direct factor for survival of the CL. Prolactin stimulates luteal progesterone production; therefore, we examined whether prolactin prevents apoptosis in luteal cells independently of its stimulatory action on progesterone production. We used a luteal cell line (GG-CL) that expresses the prolactin receptor but does not produce progesterone. These cells undergo apoptosis under conditions of serum starvation, and addition of prolactin to the culture medium significantly reduced DNA fragmentation. These results indicate that the extent of luteal cell death induced by incubation of CL under serum-free conditions depends on the hormonal environment to which this endocrine gland is exposed in vivo. These results also indicate an important role for lactation in preventing apoptosis, which is further supported by the antiapoptotic activity of prolactin observed in luteal cells.     

Determination of the triacylglycerol composition of coffee beans by reverse-phase high-performance liquid chromatography

Reverse-phase HPLC with refractive index and light scattering detectors in isocratic and gradient elution modes, respectively, was applied for the separation of the major triacylglycerols (TAG) in coffee lipids. Twelve TAG species could be identified and determined using a linear gradient of acetonitrile in dichloromethane: dichloroethane. The quantitative evaluation was based on the relative area percentages derived directly from a data-station. The procedure was applied to determine the TAG composition of three types of coffee beans harvested in two coffee producing areas in Brazil and dried by two commonly used procedures. No significant differences in the TAG compositions due to the type, origin and drying procedure were found.     

TIP,a T-cell factor identified using high-throughput screening increases survival in a graft-versus-host disease model

A coordinated effort combining bioinformatic tools with high-throughput cell-based screening assays was implemented to identify novel factors involved in T-cell biology. We generated a unique library of cDNAs encoding predicted secreted and transmembrane domain-containing proteins generated by analyzing the Human Genome Sciences cDNA database with a combination of two algorithms that predict signal peptides. Supernatants from mammalian cells transiently transfected with this library were incubated with primary T cells and T-cell lines in several high-throughput assays. Here we describe the discovery of a T cell factor, TIP (T cell immunomodulatory protein), which does not show any homology to proteins with known function. Treatment of primary human and murine T cells with TIP in vitro resulted in the secretion of IFN-gamma, TNF-alpha, and IL-10, whereas in vivo TIP had a protective effect in a mouse acute graft-versus-host disease (GVHD) model. Therefore, combining functional genomics with high-throughput cell-based screening is a valuable and efficient approach to identifying immunomodulatory activities for novel proteins.