Beef production potential of Norwegian Red and Holstein-Friesian bulls slaughtered at two ages

There is a paucity of data on the beef production potential of Norwegian Red (NOR) compared with 'modern' Holstein-Friesian (HF) cattle. The present study used a total of 64 bulls in a 2 × 2 factorial design study encompassing two breeds (HF and NOR) and two slaughter ages (485; E, and 610; L, days). The mean initial age and live weight of the HF bulls were 179 (s.d. 47.1) days and 203 (s.d. 64.0) kg, while the corresponding data for the NOR bulls were 176 (s.d. 39.7) days and 185 (s.d. 63.6) kg, respectively. Bulls were offered a 50 : 50 mixture (dry matter (DM) basis) of grass silage and concentrates. No breed × slaughter group interactions were recorded for any parameters evaluated (P > 0.05). HF bulls had higher (P < 0.001) DM intake and poorer (P < 0.01) efficiency of conversion of food to carcass gain than NOR bulls. HF bulls tended (P = 0.07) to have a higher rate of live-weight gain and were heavier (P < 0.001) at slaughter than NOR bulls, though both carcass weight and rate of carcass gain did not differ between the breeds (P > 0.05). NOR bulls had higher (P < 0.001) dressing proportion and carcass conformation score than HF bulls, while breed of bull had no influence (P > 0.05) on carcass fat classification, depth of subcutaneous fat, marbling score or on the weight of fat in the internal depots. Daily food intakes did not differ (P > 0.05) across the two slaughter age groups, though efficiency of conversion of food to carcass gain was poorer (P < 0.05) in the L compared with E bulls. Rate of live-weight gain was lower (P < 0.01) for L bulls, although rate of carcass gain did not differ (P > 0.05) between the E and L bulls. Increasing age at slaughter increased (P < 0.01 or greater) dressing proportion, carcass fat class, depth of subcutaneous fat, marbling score and internal fat depots, but had no effect (P > 0.05) on the carcass conformation score. Instrumental measures of meat quality indicated that meat from NOR bulls was tougher (P < 0.01) than meat from HF bulls, while delaying slaughter increased (P < 0.001) a* and C*ab, and decreased (P < 0.01) h0, indicating improved redness. It is concluded that NOR bulls have higher food efficiency and produce more highly conformed carcasses than HF bulls, but HF bulls produce more tender meat.     

Sampling multiple life stages significantly increases estimates of marine biodiversity

Biodiversity assessments are critical for setting conservation priorities, understanding ecosystem function and establishing a baseline to monitor change. Surveys of marine biodiversity that rely almost entirely on sampling adult organisms underestimate diversity because they tend to be limited to habitat types and individuals that can be easily surveyed. Many marine animals have planktonic larvae that can be sampled from the water column at shallow depths. This life stage often is overlooked in surveys but can be used to relatively rapidly document diversity, especially for the many species that are rare or live cryptically as adults. Using DNA barcode data from samples of nemertean worms collected in three biogeographical regions—Northeastern Pacific, the Caribbean Sea and Eastern Tropical Pacific—we found that most species were collected as either benthic adults or planktonic larvae but seldom in both stages. Randomization tests show that this deficit of operational taxonomic units collected as both adults and larvae is extremely unlikely if larvae and adults were drawn from the same pool of species. This effect persists even in well-studied faunas. These results suggest that sampling planktonic larvae offers access to a different subset of species and thus significantly increases estimates of biodiversity compared to sampling adults alone. Spanish abstract is available in the electronic supplementary material.     

Determination of the functional epitopes of human interleukin-18-binding protein by site-directed mutagenesis

The human interleukin (IL)-18-binding protein (hIL-18BP) is a naturally occurring antagonist of IL-18, a proinflammatory cytokine that is related to IL-1beta and has an important role in defense against microbial invaders. As its name implies, the hIL-18BP binds to IL-18 with high affinity and prevents the interaction of IL-18 with its receptor. We genetically modified the C terminus of hIL-18BP by appending a 15-amino acid biotinylation recognition site and a six-histidine tag and then performed site-directed mutagenesis to determine the functional epitopes that mediate efficient binding to IL-18. The mutated IL-18BPs were secreted from mammalian cells, captured by metal affinity chromatography, biotinylated in situ, eluted, and immobilized on streptavidin-coated chips. Using surface plasmon resonance, we identified seven amino acids of hIL-18BP which, when changed individually to alanine, caused an 8-750-fold decrease in binding affinity, largely because of increased off-rates. These seven amino acids localized to the predicted beta-strand c and d of hIL-18BP immunoglobulin-like domain, and most had hydrophobic side chains. Just two amino acids, tyrosine 97 and phenylalanine 104, contributed approximately 50% of the binding free energy. Information obtained from these studies could contribute to the design of molecular antagonists of IL-18 for treatment of inflammatory diseases.     

Cooperative mechanisms in the activation dependence of the rate of force development in rabbit skinned skeletal muscle fibers

Regulation of contraction in skeletal muscle is a highly cooperative process involving Ca(2+) binding to troponin C (TnC) and strong binding of myosin cross-bridges to actin. To further investigate the role(s) of cooperation in activating the kinetics of cross-bridge cycling, we measured the Ca(2+) dependence of the rate constant of force redevelopment (k(tr)) in skinned single fibers in which cross-bridge and Ca(2+) binding were also perturbed. Ca(2+) sensitivity of tension, the steepness of the force-pCa relationship, and Ca(2+) dependence of k(tr) were measured in skinned fibers that were (1) treated with NEM-S1, a strong-binding, non-force-generating derivative of myosin subfragment 1, to promote cooperative strong binding of endogenous cross-bridges to actin; (2) subjected to partial extraction of TnC to disrupt the spread of activation along the thin filament; or (3) both, partial extraction of TnC and treatment with NEM-S1. The steepness of the force-pCa relationship was consistently reduced by treatment with NEM-S1, by partial extraction of TnC, or by a combination of TnC extraction and NEM-S1, indicating a decrease in the apparent cooperativity of activation. Partial extraction of TnC or NEM-S1 treatment accelerated the rate of force redevelopment at each submaximal force, but had no effect on kinetics of force development in maximally activated preparations. At low levels of Ca(2+), 3 microM NEM-S1 increased k(tr) to maximal values, and higher concentrations of NEM-S1 (6 or 10 microM) increased k(tr) to greater than maximal values. NEM-S1 also accelerated k(tr) at intermediate levels of activation, but to values that were submaximal. However, the combination of partial TnC extraction and 6 microM NEM-S1 increased k(tr) to virtually identical supramaximal values at all levels of activation, thus, completely eliminating the activation dependence of k(tr). These results show that k(tr) is not maximal in control fibers, even at saturating [Ca(2+)], and suggest that activation dependence of k(tr) is due to the combined activating effects of Ca(2+) binding to TnC and cross-bridge binding to actin.     

Immunogenicity and protective efficacy of oligomeric human immunodeficiency virus type 1 gp140

The biologically active form of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein is oligomeric. We previously described a soluble HIV-1 IIIB Env protein, gp140, with a stable oligomeric structure composed of uncleaved gp120 linked to the ectodomain of gp41 (P. L. Earl, C. C. Broder, D. Long, S. A. Lee, J. Peterson, S. Chakrabarti, R. W. Doms, and B. Moss, J. Virol. 68:3015-3026, 1994). Here we compared the antibody responses of rabbits to gp120 and gp140 that had been produced and purified in an identical manner. The gp140 antisera exhibited enhanced cross-reactivity with heterologous Env proteins as well as greater neutralization of HIV-1 compared to the gp120 antisera. To examine both immunogenicity and protective efficacy, we immunized rhesus macaques with oligomeric gp140. Strong neutralizing antibodies against a homologous virus and modest neutralization of heterologous laboratory-adapted isolates were elicited. No neutralization of primary isolates was observed. However, a substantial fraction of the neutralizing activity could not be blocked by a V3 loop peptide. After intravenous challenge with simian-HIV virus SHIV-HXB2, three of the four vaccinated macaques exhibited no evidence of virus replication.     

Visualization of intracellular movement of vaccinia virus virions containing a green fluorescent protein-B5R membrane protein chimera

We produced an infectious vaccinia virus that expressed the B5R envelope glycoprotein fused to the enhanced green fluorescent protein (GFP), allowing us to visualize intracellular virus movement in real time. Previous transfection studies indicated that fusion of GFP to the C-terminal cytoplasmic domain of B5R did not interfere with Golgi localization of the viral protein. To determine whether B5R-GFP was fully functional, we started with a B5R deletion mutant that made small plaques and inserted the B5R-GFP gene into the original B5R locus. The recombinant virus made normal-sized plaques and acquired the ability to form actin tails, indicating reversal of the mutant phenotype. Moreover, immunogold electron microscopy revealed that both intracellular enveloped virions (IEV) and extracellular enveloped virions contained B5R-GFP. By confocal microscopy of live infected cells, we visualized individual fluorescent particles, corresponding to IEV in size and shape, moving from a juxtanuclear location to the periphery of the cell, where they usually collected prior to association with actin tails. The fluorescent particles could be seen emanating from cells at the tips of microvilli. Using a digital camera attached to an inverted fluorescence microscope, we acquired images at 1 frame/s. At this resolution, IEV movement appeared saltatory; in some frames there was no net movement, whereas in others movement exceeded 2 microm/s. Further studies indicated that IEV movement was reversibly arrested by the microtubule-depolymerizing drug nocodazole. This result, together with the direction, speed, and saltatory motion of IEV, was consistent with a role for microtubules in intracellular transport of IEV.     

Reduction of simian-human immunodeficiency virus 89.6P viremia in rhesus monkeys by recombinant modified vaccinia virus Ankara vaccination

Since cytotoxic T lymphocytes (CTLs) are critical for controlling human immunodeficiency virus type 1 (HIV-1) replication in infected individuals, candidate HIV-1 vaccines should elicit virus-specific CTL responses. In this report, we study the immune responses elicited in rhesus monkeys by a recombinant poxvirus vaccine and the degree of protection afforded against a pathogenic simian-human immunodeficiency virus SHIV-89.6P challenge. Immunization with recombinant modified vaccinia virus Ankara (MVA) vectors expressing SIVmac239 gag-pol and HIV-1 89.6 env elicited potent Gag-specific CTL responses but no detectable SHIV-specific neutralizing antibody (NAb) responses. Following intravenous SHIV-89.6P challenge, sham-vaccinated monkeys developed low-frequency CTL responses, low-titer NAb responses, rapid loss of CD4+ T lymphocytes, high-setpoint viral RNA levels, and significant clinical disease progression and death in half of the animals by day 168 postchallenge. In contrast, the recombinant MVA-vaccinated monkeys demonstrated high-frequency secondary CTL responses, high-titer secondary SHIV-89.6-specific NAb responses, rapid emergence of SHIV-89.6P-specific NAb responses, partial preservation of CD4+ T lymphocytes, reduced setpoint viral RNA levels, and no evidence of clinical disease or mortality by day 168 postchallenge. There was a statistically significant correlation between levels of vaccine-elicited CTL responses prior to challenge and the control of viremia following challenge. These results demonstrate that immune responses elicited by live recombinant vectors, although unable to provide sterilizing immunity, can control viremia and prevent disease progression following a highly pathogenic AIDS virus challenge.     

PBXL-1: a new fluorochrome applied to detection of proteins on membranes

An easy, sensitive and direct fluorescent immunodetection method for proteins is described using the new fluorochrome PBXL-1 imaged with the FMBIO II Laser Scanning Imaging System. PBXL-1 is derived from a protein supra-molecular complex that contains a large number of chromophores. This complex, the phycobilisome, is extracted from a red alga then chemically stabilized to allow its use in specific binding assays. PBXL-1 was cross-linked to goat anti-rabbit IgG or streptavidin with heterobifunctional cross-linkers. The detection limit of PBXL-1 was determined by applying it on nitrocellulose membranes then imaging the membrane using an ytterbium aluminum garnet (YAG) laser. Evaluation of PBXL-1 sensitivity in a specific binding assay was tested on streptavidin/biotin and an antibody system. PBXL-1 provides high sensitivity in direct fluorescent applications due to a physical amplification of signal (i.e., a large number of fluorophores per binding event). PBXL-1 provides a linear response over two orders of magnitude while providing sub-amol sensitivity, indicating broad applicability for detection of a variety of targets. To our knowledge, this is the most sensitive direct fluorescent detection method available.     

Characterization of a novel GDP-mannose:Serine-protein mannose-1-phosphotransferase from Leishmania mexicana

Protozoan parasites of the genus Leishmania secrete a number of glycoproteins and mucin-like proteoglycans that appear to be important parasite virulence factors. We have previously proposed that the polypeptide backbones of these molecules are extensively modified with a complex array of phosphoglycan chains that are linked to Ser/Thr-rich domains via a common Manalpha1-PO4-Ser linkage (Ilg, T., Overath, P., Ferguson, M. A. J., Rutherford, T., Campbell, D. G., and McConville, M. J. (1994) J. Biol. Chem. 269, 24073-24081). In this study, we show that Leishmania mexicana promastigotes contain a peptide-specific mannose-1-phosphotransferase (pep-MPT) activity that adds Manalpha1-P to serine residues in a range of defined peptides. The presence and location of the Manalpha1-PO4-Ser linkage in these peptides were determined by electrospray ionization mass spectrometry and chemical and enzymatic treatments. The pep-MPT activity was solubilized in non-ionic detergents, was dependent on Mn2+, utilized GDP-Man as the mannose donor, and was expressed in all developmental stages of the parasite. The pep-MPT activity was maximal against peptides containing Ser/Thr-rich domains of the endogenous acceptors and, based on competition assays with oligosaccharide acceptors, was distinct from other leishmanial MPTs involved in the initiation and elongation of lipid-linked phosphoglycan chains. In subcellular fractionation experiments, pep-MPT was resolved from the endoplasmic reticulum marker BiP, but had an overlapping distribution with the cis-Golgi marker Rab1. Although Man-PO4 residues in the mature secreted glycoproteins are extensively modified with mannose oligosaccharides and phosphoglycan chains, similar modifications were not added to peptide-linked Man-PO4 residues in the in vitro assays. Similarly, Man-PO4 residues on endogenous polypeptide acceptors were also poorly extended, although the elongating enzymes were still active, suggesting that the pep-MPT activity and elongating enzymes may be present in separate subcellular compartments.