An evaluation of the effect of altering nutrition and nutritional strategies in early lactation on reproductive performance and estrous behavior of high-yielding Holstein-Friesian dairy cows

Reproductive performance in the high-yielding dairy cow has severely decreased in the last 40 yr. The aim of this study was to compare the effectiveness of 4 nutritional strategies in improving the reproductive performance of high-yielding dairy cows. It was hypothesized that offering cows a high-starch ration in early lactation would enhance the onset of luteal activity, and that decreasing the severity of negative energy balance in the early postcalving period would improve reproductive parameters. Nutritional regimens aimed at improving fertility were applied to 96 Holstein-Friesian dairy animals. Upon calving, animals were allocated in a balanced manner to one of 4 dietary treatments. Primiparous animals were balanced according to live weight, body condition score and calving date. Multiparous animals were balanced according to parity, previous lactation milk yield, liveweight, body condition score and calving date. Treatment 1 was based on an industry best practice diet (control) to contain 170 g of crude protein/kg of dry matter. Treatment 2 was an individual cow feeding strategy, whereby the energy balance (EB) of individual animals was managed so as to achieve a predetermined target daily EB profile (+/- 10 MJ/d). Treatment 3 was a high-starch/high-fat combination treatment, whereby an insulinogenic (high-starch) diet was offered in early lactation to encourage cyclicity and followed by a lipogenic (low-starch, high-fat) diet to promote embryo development. Treatment 4 was a low-protein diet, containing 140 g of crude protein/kg of dry matter, supplemented with protected methionine at an inclusion level of 40 g per animal per day. The nutritional strategies implemented in this study had no statistically significant effects on cow fertility measures, which included the onset of luteal activity, conception rate, in-calf rate, and the incidence of atypical cycles. The individual cow feeding strategy improved EB in early lactation but had no benefit on conception rate to first insemination. However, conception rate to second insemination, 100-d pregnancy rate (from the commencement of breeding), and overall pregnancy rate tended to be higher in this group. The high-starch/high-fat treatment tended to decrease the proportion of delayed ovulations and increase the proportion of animals cycling by d 50 postcalving. Animals that failed to conceive to first insemination had a significantly longer luteal phase in the first cycle postpartum and a longer inter-ovulatory interval in the second cycle postpartum. With regards to estrous behavior, results indicate that as the size of the sexually active group increased, the intensity of estrus and the expression of mounting or attempting to mount another cow also increased. Furthermore, cows that became pregnant displayed more intense estrous behavior than cows that failed to become pregnant.

Immunovirological Correlates in Human Rabies Treated With Therapeutic Coma

A 37-year-old woman was admitted to hospital and over the next 5 days developed a progressive encephalitis. Nuchal skin biopsy, analyzed using a Rabies TaqMan(C) PCR, demonstrated rabies virus RNA. She had a history in keeping with exposure to rabies whilst in South Africa, but had not received pre- or post-exposure prophylaxis. She was treated with a therapeutic coma according to the

Isoprenylation of the G protein gamma subunit is both necessary and sufficient for beta gamma dimer-mediated stimulation of phospholipase C

We have previously shown that isoprenylation and/or additional pest-translational processing of the G protein gamma(1) subunit carboxyl terminus is required for beta(1) gamma(1) subunit stimulation of phospholipase C-beta(2) (PLC beta(2)) [Dietrich, A., Meister, M., Brazil, D., Camps, M., & Gierschik, P. (1994) Eur. J. Biochem. 219, 171-178]. To examine whether isoprenylation of the gamma(1) subunit alone is sufficient for beta(1) gamma(1)-mediated PLC beta(2) stimulation or whether any of the two subsequent modifications, proteolytic removal of the carboxyl-terminal tripeptide and/or carboxylmethylation, is required for this effect, nonisoprenylated recombinant beta(1) gamma(1) dimers were produced in baculovirus-infected insect cells, purified to near homogeneity, and then isoprenylated in vitro using purified recombinant protein farnesyltransferase. Analysis of the beta(1) gamma(1) dimer after in vitro farnesylation by reversed phase high-performance liquid chromatography followed by delayed extraction matrix-assisted laser desorption/ionization mass spectrometry confirmed that the gamma(1) subunit was carboxyl-terminally farnesylated but not proteolyzed and carboxylmethylated. Functional reconstitution of in vitro-farnesylated beta(1) gamma(1) dimers with a recombinant PLC beta(2) isozyme revealed that farnesylation rendered recombinant nonisoprenylated beta(1) gamma(1) dimers capable of stimulating PLC beta(2) and that the degree of this stimulation was only approximately 45% lower for in vitro-farnesylated beta(1) gamma(1) dimers than for fully modified native beta(1) gamma(1) purified from bovine retinal rod outer segments. Taken together, these results suggest that isoprenylation of the gamma subunit is both necessary and sufficient for beta gamma dimer-mediated stimulation of phospholipase C.

Context-Dependent Pharmacology Exhibited by Negative Allosteric Modulators of Metabotropic Glutamate Receptor 7

Phenotypic studies of mice lacking metabotropic glutamate receptor subtype 7 (mGluR7) suggest that antagonists of this receptor may be promising for the treatment of central nervous system disorders such as anxiety and depression. Suzuki et al. (J Pharmacol Exp Ther 323: 147-156, 2007) recently reported the in vitro characterization of a novel mGluR7 antagonist called 6-(4-methoxyphenyl)-5-methyl-3-(4-pyridinyl)-isoxazolo[4,5-c]pyridin-4(5H)-one (MMPIP), which noncompetitively inhibited the activity of orthosteric and allosteric agonists at mGluR7. We describe that MMPIP acts as a noncompetitive antagonist in calcium mobilization assays in cells coexpressing mGluR7 and the promiscuous G protein G alpha(15). Assessment of the activity of a small library of MMPIP-derived compounds using this assay reveals that, despite similar potencies, compounds exhibit differences in negative co-operativity for agonist-mediated calcium mobilization. Examination of the inhibitory activity of MMPIP and analogs using endogenous G(i/o)-coupled assay readouts indicates that the pharmacology of these ligands seems to be context-dependent, and MMPIP exhibits differences in negative cooperativity in certain cellular backgrounds. Electrophysiological studies reveal that, in contrast to the orthosteric antagonist (2S)-2-amino-2-[(1S,2S)-2-carboxyclycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495), MMPIP is unable to block agonist-mediated responses at the Schaffer collateral-CA1 synapse, a location at which neurotransmission has been shown to be modulated by mGluR7 activity. Thus, MMPIP and related compounds differentially inhibit coupling of mGluR7 in different cellular backgrounds and may not antagonize the coupling of this receptor to native G(i/o) signaling pathways in all cellular contexts. The pharmacology of this compound represents a striking example of the potential for context-dependent blockade of receptor responses by negative allosteric modulators.

The Respiratory Syncytial Virus G Protein Conserved Domain Induces a Persistent and Protective Antibody Response in Rodents

Respiratory syncytial virus (RSV) is an important cause of severe upper and lower respiratory disease in infants and in the elderly. There are 2 main RSV subtypes A and B. A recombinant vaccine was designed based on the central domain of the RSV-A attachment G protein which we had previously named G2Na (aa130–230). Here we evaluated immunogenicity, persistence of antibody (Ab) response and protective efficacy induced in rodents by: (i) G2Na fused to DT (Diphtheria toxin) fragments in cotton rats. DT fusion did not potentiate neutralizing Ab responses against RSV-A or cross-reactivity to RSV-B. (ii) G2Nb (aa130–230 of the RSV-B G protein) either fused to, or admixed with G2Na. G2Nb did not induce RSV-B-reactive Ab responses. (iii) G2Na at low doses. Two injections of 3 µg G2Na in Alum were sufficient to induce protective immune responses in mouse lungs, preventing RSV-A and greatly reducing RSV-B infections. In cotton rats, G2Na-induced RSV-reactive Ab and protective immunity against RSV-A challenge that persisted for at least 24 weeks. (iv) injecting RSV primed mice with a single dose of G2Na/Alum or G2Na/PLGA [poly(D,L-lactide-co-glycolide]. Despite the presence of pre-existing RSV-specific Abs, these formulations effectively boosted anti-RSV Ab titres and increased Ab titres persisted for at least 21 weeks. Affinity maturation of these Abs increased from day 28 to day 148. These data indicate that G2Na has potential as a component of an RSV vaccine formulation.

Physiological effects of platyhelminth RFamide peptides on muscle-strip preparations of Fasciola hepatica (Trematoda: Digenea)

The effects of each of the known platyhelminth neuropeptides were determined on muscle-strip preparations from the liver fluke, Fasciola hepatica. The activity of synthetic replicates of the C-terminal nonapeptide of neuropeptide F (NPF9, Moniezia expansa), and the FMRFamide-related peptides (FaRPs), GNFFRFamide, RYIRFamide, GYIRFamide and YIRFamide, were examined. Muscle-strip activity was recorded from 1 mm segments of muscle prepared from 28 to 32-day-old worms, using a photo-optic transducer system. None of the peptides (less than or equal to 10 mu M) altered baseline tension significantly; however, each of the peptides increased the amplitude and frequency of muscle contraction. The threshold for activity of each of the peptides examined was, respectively, 1 nM (RYIRFamide), 0.3 mu M (GYIRFamide and YIRFamide), and 10 mu M (GNFFRFamide and NPF9). All of the effects were reversible and repeatable, following wash-out. Muscle-strip integrity was tested following experimentation, using arecoline (10 mu M) and high-K+ bathing medium (90 mM K+).

Cell type-dependent uptake, localization, and cytotoxicity of 1.9 nm gold nanoparticles

Background: This follow-up study aims to determine the physical parameters which govern the differential radiosensitization capacity of two tumor cell lines and one immortalized normal cell line to 1.9 nm gold nanoparticles. In addition to comparing the uptake potential, localization, and cytotoxicity of 1.9 nm gold nanoparticles, the current study also draws on comparisons between nanoparticle size and total nanoparticle uptake based on previously published data.

Methods: We quantified gold nanoparticle uptake using atomic emission spectroscopy and imaged intracellular localization by transmission electron microscopy. Cell growth delay and clonogenic assays were used to determine cytotoxicity and radiosensitization potential, respectively. Mechanistic data were obtained by Western blot, flow cytometry, and assays for reactive oxygen species.

Results: Gold nanoparticle uptake was preferentially observed in tumor cells, resulting in an increased expression of cleaved caspase proteins and an accumulation of cells in sub G1 phase. Despite this, gold nanoparticle cytotoxicity remained low, with immortalized normal cells exhibiting an LD50 concentration approximately 14 times higher than tumor cells. The surviving fraction for gold nanoparticle-treated cells at 3 Gy compared with that of untreated control cells indicated a strong dependence on cell type in respect to radiosensitization potential.

Conclusion: Gold nanoparticles were most avidly endocytosed and localized within cytoplasmic vesicles during the first 6 hours of exposure. The lack of significant cytotoxicity in the absence of radiation, and the generation of gold nanoparticle-induced reactive oxygen species provide a potential mechanism for previously reported radiosensitization at megavoltage energies.

Characterisation of the Timing and Prevalence of Receptor Tyrosine Kinase Expression Changes in Esophageal Carcinogenesis

Despite being common in epithelial malignancies, the timing of receptor tyrosine kinase (RTK) up-regulation is poorly understood and therefore hampers the identification of the receptor to target for effective treatment. We aimed to determine if RTK expression changes were early events in carcinogenesis. Esophageal adenocarcinoma and its pre-invasive lesion, Barrett's esophagus, were used for immunohistochemical analysis of the RTK panel, EGFR, ErbB2, ErbB3, Met and FGFR2, by utilising a cohort of patients with invasive disease (n = 367) and two cohorts with pre-invasive disease, one cross-sectional (n = 110) and one longitudinal in time (n = 91). The results demonstrated that 51% of esophageal adenocarcinomas over-expressed at least one of the RTK panel; with 21% of these over-expressing multiple receptors. Up-regulation of RTK expression was an early event corresponding with low grade dysplasia development (25% in areas without dysplasia vs. 63% in low grade dysplasia, p

Unraveling the Structure and Function of G Protein-Coupled Receptors Through NMR Spectroscopy

G protein-coupled receptors (GPCRs) are a large superfamily of signaling proteins expressed on the plasma membrane. They are involved in a wide range of physiological processes and, therefore, are exploited as drug targets in a multitude of therapeutic areas. In this extent, knowledge of structural and functional properties of GPCRs may greatly facilitate rational design of modulator compounds. Solution and solid-state nuclear magnetic resonance (NMR) spectroscopy represents a powerful method to gather atomistic insights into protein structure and dynamics. In spite of the difficulties inherent the solution of the structure of membrane proteins through NMR, these methods have been successfully applied, sometimes in combination with molecular modeling, to the determination of the structure of GPCR fragments, the mapping of receptor-ligand interactions, and the study of the conformational changes associated with the activation of the receptors. In this review, we provide a summary of the NMR contributions to the study of the structure and function of GPCRs, also in light of the published crystal structures.