Quantification of supplemental enzymes in animal feedingstuffs by radial enzyme diffusion

Methods are described which facilitate quantification of supplemental cellulase, protease and -amylase when added to animal feedingstuffs at normal industrial inclusion levels. The methods entail extraction of the enzymes from the feedingstuffs by agitation in buffer followed by quantification of extract activity using radial diffusion techniques. A linear relationship between the diameter of the zone of hydrolyzed substrate and the log of the enzyme activity applied is observed over a broad activity range. Assay of a feedingstuff supplemented with 1 kg t^–1 cellulase, protease and -amylase yielded net supplemental activity recoveries of 104±11.7%, 91.3±6.74% and 126±29.5%, respectively. A similar assay method did not prove sufficiently sensitive to facilitate detection of xylanase at typical in-feed inclusion levels. The levels of endogenous cellulase, protease and -amylase activity detected in the unsupplemented feedingstuffs were equivalent to 6.4±0.47%, 6.6±0.82% and 29.0±14.1%, respectively, of a 1 kg t^–1 supplement. The methods are technically straightforward and will facilitate determination of enzyme stabilities during processes such as high-temperature pelleting of feedingstuffs, as well as allowing more rigorous quality control related to enzyme-supplemented animal feedingstuffs.     

Development of solution phase hybridisation PCR-ELISA for the detection and quantification of Enterococcus faecalis and Pediococcus pentosaceus in Nurmi-type cultures

Nurmi-type cultures (NTCs), derived from the fermentation of caecal contents of specifically pathogen-free (SPF) birds, have been used successfully to control salmonella colonisation in chicks. These cultures are undefined in nature and, consequently, it is difficult to obtain approval from regulatory agencies for their use as direct fed microbials (DFMs) for poultry. Progress towards the generation of effective defined probiotics requires further knowledge of the composition of these cultures. As such, species-specific, culture-independent quantification methodologies need to be developed to elucidate the concentration of specific bacterial constituents of NTCs. Quantification of specific bacterial species in such ill-defined complex cultures using conventional culturing methods is inaccurate due to low levels of sensitivity and reproducibility, in addition to slow turnaround times. Furthermore, these methods lack selectivity due to the nature of the accompanying microflora. This study describes the development of a rapid, sensitive, reliable, reproducible, and species-specific culture-independent, solution phase hybridisation PCR-ELISA procedure for the detection and quantification of Enterococcus faecalis and Pediococcus pentosaceus in NTCs. In this technique, biotin-labelled primers were designed to amplify a species-specific fragment of a marker gene of known copy number, in both species. Resulting amplicons were hybridised with a dinitrophenol (DNP)-labelled oligonucleotide probe in solution and were subsequently captured on a streptavidin-coated microtitre plate. The degree of binding was determined by the addition of IgG (anti-DNP)-horseradish peroxidase conjugate, which was subsequently visualised using a chromogenic substrate, tetramethylbenzidine. This novel quantitative method was capable of detecting E. faecalis and P. pentosaceus at levels as low as 5 CFU per PCR reaction.     

Caspase-mediated specific cleavage of BubR1 is a determinant of mitotic progression

The fidelity of chromosomal duplication is monitored by cell cycle checkpoints operational during mitosis. One such cell cycle delay is invoked by microtubule-targeting agents such as nocodazole or paclitaxel (Taxol) and is mediated by mitotic checkpoint proteins that include BubR1. Relatively little is known about the regulation of expression and stability of BubR1 (or other checkpoint proteins) and how these factors dictate the durability of the cell cycle delay. We report here that treatment of HeLa cells with spindle-disrupting agents resulted in caspase activation and precipitated the cleavage of BubR1. This mechanism ultimately leads to reduced levels of full-length protein, which are accompanied by abrogation of the mitotic block; the checkpoint abrogation is substantially accelerated by inhibition of de novo protein synthesis. In contrast, inhibition of caspase activity blocked BubR1 degradation and prolonged mitosis. To confirm a direct link between caspase activity and BubR1 protein expression, we identified by site-directed mutagenesis the specific caspase cleavage sites cleaved after exposure to paclitaxel. Surprisingly, BubR1 has two sites of cleavage: primarily at Asp607/Asp610 and secondarily at Asp576/Asp579. BubR1 mutated at both locations (BubR1Delta579Delta610) was resistant to paclitaxel-induced degradation. Expression of BubR1Delta579Delta610 augmented the mitotic delay induced by spindle disruption in transfected cells as well as in clones engineered to inducibly express the mutant protein upon exposure to doxycycline and ultimately led to increased aneuploidy. Underscoring the importance of these caspase cleavage sites, both tetrapeptide motifs are identified in the amino acid sequences of human, mouse, chicken, and Xenopus BubR1. These results are potentially the first to link the control of the stability of a key mitotic checkpoint protein to caspase activation, a regulatory pathway that may be involved in killing defective cells and that has been evolutionarily conserved.     

Stimulus change dis-habituates operant responding supported by water reinforcers

The present experiment examined whether habituation contributes to within-session decreases in operant responding for water reinforcers. The experiment asked if this responding can be dishabituated, a fundamental property of habituated behavior. During baseline, rats’ lever pressing was reinforced by water on a variable interval 15-s schedule. During experimental conditions, rats responded on the same schedule and a new stimulus was introduced for 5 min at 15, 30 or 45 min into the 60-min session. The new stimulus was extinction, continuous reinforcement or flashing lights in different conditions. Rate of responding primarily decreased within the session during baseline. Introducing a new stimulus sometimes suppressed (extinction, continuous reinforcement) and sometimes increased (flashing lights) responding while it was in effect. The new stimulus increased responding after it ended and before it was presented in the session. The results are incompatible with the idea that non-habituation satiety factors (e.g., cellular hydration and blood volume) contributed to within-session changes in responding. These satiety factors should increase with increases in consumption, decrease with decreases in consumption and remain constant with constant consumption of water. Nevertheless, all stimulus changes increased operant responding for water. These results support the idea that habituation contributes to within-session decreases in responding for water reinforcers.     

An AFLP-based survey of genetic diversity among accessions of sea oats (Uniola paniculata, Poaceae) from the southeastern Atlantic and Gulf coast states of the United States

Uniola paniculata, commonly known as sea oats, is a C₄ perennial grass capable of stabilizing sand dunes. It is most abundant along the Gulf of Mexico and southeastern Atlantic coastal regions of the United States. The species exhibits low seed set and low rates of germination and seedling emergence, and so extensive clonal reproduction is achieved through production of rhizomes, which may contribute to a decline in genetic diversity. To date, there has been no systematic assessment of genetic variability and population structure in naturally occurring stands in the USA. This study was conducted to assess the genetic relationship and diversity among nineteen U. paniculata accessions representing eight states: Texas, Louisiana, Mississippi, Alabama, Florida, South Carolina, North Carolina, and Virginia, using amplified fragment length polymorphism (AFLP). Twelve AFLP EcoRI+MseI primer combinations generated a wide range of polymorphisms (42–81%) with a mean of 59%. Overall, the sea oats plants exhibited a low range of genetic similarity. Florida accessions, FL-33 and FL-39, were most genetically diverse and the accessions from both Carolinas and Virginia (NC-1, NC-11, SC-15, and VA-53) harbored less genetic variability. Cluster analysis using the UPGMA approach separated U. paniculata plants into four major clusters which were also confirmed by principal coordinate analysis (PCO). Further examination of the different components of genetic variation by analysis of molecular variance (AMOVA) indicated the largest proportion of variability at the state level (47.8%) followed by the variation due to the differences among the genotypes within an accession (34.4%), and the differences among the accessions within a state (17.8%). The relationship between genetic diversity and geographic source of sea oats populations of the United States as revealed through this comprehensive study will be helpful to resource managers and commercial nurseries in identifying suitable plant materials for restoration of new areas without compromising the adaptation and genetic diversity.     

Towards normalization of soybean somatic embryo maturation

Soybean (Glycine max L. Merrill) somatic embryos have been useful for assaying seed-specific traits prior to plant recovery. Such traits could be assessed more accurately if somatic embryos more closely mimicked seed development. Amino acid supplements, carbon source, and abscisic acid and basal salt formulations were tested in an effort to modify existing soybean embryogenesis histodifferentiation/maturation media to further normalize the development of soybean somatic embryos. The resultant liquid medium, referred to as soybean histodifferentiation and maturation medium (SHaM), consists of FNL basal salts, 3% sucrose, 3% sorbitol, filter-sterilized 30 mM glutamine and 1 mM methionine. SHaM-derived somatic embryos are more similar to seed in terms of protein and fatty acid/lipid composition, and conversion ability, than somatic embryos obtained from traditional soybean histodifferentiation and maturation media.     

Infection of nonhuman primates with recombinant human metapneumovirus lacking the SH, G, or M2-2 protein categorizes each as a nonessential accessory protein and identifies vaccine candidates

Recombinant human metapneumovirus (HMPV) in which the SH, G, or M2 gene or open reading frame was deleted by reverse genetics was evaluated for replication and vaccine efficacy following topical administration to the respiratory tract of African green monkeys, a permissive primate host. Replication of the deltaSH virus was only marginally less efficient than that of wild-type HMPV, whereas the deltaG and deltaM2-2 viruses were reduced sixfold and 160-fold in the upper respiratory tract and 3,200-fold and 4,000-fold in the lower respiratory tract, respectively. Even with the highly attenuated mutants, there was unequivocal HMPV replication at each anatomical site in each animal. Thus, none of these three proteins is essential for HMPV replication in a primate host, although G and M2-2 increased the efficiency of replication. Each gene-deletion virus was highly immunogenic and protective against wild-type HMPV challenge. The deltaG and deltaM2-2 viruses are promising vaccine candidates that are based on independent mechanisms of attenuation and are appropriate for clinical evaluation.     

Phylogenetic congruence of <Emphasis Type="Italic">Sarcocystis neurona</Emphasis> Dubey et al., 1991 (Apicomplexa: Sarcocystidae) in the United States based on sequence analysis and restriction fragment length polymorphism (RFLP)

The objectives of the present study were to assess the genetic diversity, phylogeny and phylogeographical relationships of available Sarcocystis neurona isolates from different localities in the United States. All 13 Sarcocystis isolates from different hosts were subjected to polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analyses using two published DNA markers (25/396 and 33/54). The 334 bp sequence of the 25/396 marker of these isolates and Besnoitia darlingi, B. bennetti, Toxoplasma gondii and Neospora caninum were sequenced and compared. Phylogenetic analysis was performed using neighbour-joining (NJ), maximum parsimony (MP) and minimum evolution (ME) methods based on the sequences of the 25/396 marker of the 13 Sarcocystis isolates obtained in this study and sequences of 10 related isolates from GenBank. Phylogenetic trees revealed a close relatedness among S. neurona isolates in the US (nucleotide sequence diversity <5.0%). US isolates formed a monophyletic group and appeared more closely related to each other than to the South American isolates, which formed a separate lineage. NJ and ME trees with Kimura 2-parameter model separated S. neurona into two separate groups: a northern US group and a Southern US group. These findings suggest a correlation between grouping of the isolates and geographical segregation and were consistent with a genetic bottleneck hypothesis during opossum colonisation of North America. These data do not support either the view of S. neurona as a single super-species or its division into multiple subspecies.     

Voltage-gated proton channels help regulate pHi in rat alveolar epithelium

Voltage-gated proton channels are expressed highly in rat alveolar epithelial cells. Here we investigated whether these channels contribute to pH regulation. The intracellular pH (pH(i)) was monitored using BCECF in cultured alveolar epithelial cell monolayers and found to be 7.13 in nominally HCO(3)(-)-free solutions [at external pH (pH(o)) 7.4]. Cells were acid-loaded by the NH(4)(+) prepulse technique, and the recovery was observed. Under conditions designed to eliminate the contribution of other transporters that alter pH, addition of 10 microM ZnCl(2), a proton channel inhibitor, slowed recovery about twofold. In addition, the pH(i) minimum was lower, and the time to nadir was increased. Slowing of recovery by ZnCl(2) was observed at pH(o) 7.4 and pH(o) 8.0 and in normal and high-K(+) Ringer solutions. The observed rate of Zn(2+)-sensitive pH(i) recovery required activation of a small fraction of the available proton conductance. We conclude that proton channels contribute to pH(i) recovery after an acid load in rat alveolar epithelial cells. Addition of ZnCl(2) had no effect on pH(i) in unchallenged cells, consistent with the expectation that proton channels are not open in resting cells. After inhibition of all known pH regulators, slow pH(i) recovery persisted, suggesting the existence of a yet-undefined acid extrusion mechanism in these cells.     

Refolding, purification, and crystallization of apical membrane antigen 1 from Plasmodium falciparum

Extracellular domains of malaria antigens almost invariably contain disulphide linkages but lack N- and O-linked glycosylation. The best practical approach to generating recombinant extracellular Plasmodium proteins is not established and the problems encountered when using a bacterial expression/refolding approach are discussed in detail. Limited proteolysis experiments were used to identify a relatively non-flexible core region of the Plasmodium falciparum protein apical membrane antigen 1 (AMA1), and refolding/purification was used to generate two fragments of AMA1. Several chromatographically distinct AMA1 variants were identified that are presumably differentially refolded proteins. One of these AMA1 preparations proved to be crystallizable and generated two crystal forms that diffracted X-rays to 2 A resolution.