Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein.

We describe the construction of a plasmid (pCAT2AGUS) encoding a polyprotein in which a 19 amino acid sequence spanning the 2A region of the foot-and-mouth disease virus (FMDV) polyprotein was inserted between the reporter genes chloramphenicol acetyl transferase (CAT) and beta-glucuronidase (GUS) maintaining a single, long open reading frame. Analysis of translation reactions programmed by this construct showed that the inserted FMDV sequence functioned in a manner similar to that observed in FMDV polyprotein processing: the CAT2AGUS polyprotein underwent a cotranslational, apparently autoproteolytic, cleavage yielding CAT-2A and GUS. Analysis of translation products derived from a series of constructs in which sequences were progressively deleted from the N-terminal region of the FMDV 2A insertion showed that cleavage required a minimum of 13 residues. The FMDV 2A sequence therefore provides the opportunity to engineer either whole proteins or domains such that they are cleaved apart cotranslationally with high efficiency.     

Polymorphism analysis of the prion gene in BSE-affected and unaffected cattle

Polymerase chain reaction (PCR) primers designed to amplify the octapeptide repeat region of the bovine prion gene were used to test the association of genotypes with bovine spongiform encephalitis (BSE) in 56 BSE-affected and 177 unaffected animals. Three alleles (A, B, C) were detected as single-strand conformation polymorphisms (SSCPs) and two alleles (1,2 representing six or five copies of the octapeptide repeat respectively) were detected as amplified double-strand fragment length polymorphisms (AMFLPs). Observed genotypes of SSCPs and AMFLPs were analysed by x-square. The SSCP genotypes of nuclear family members of animals with BSE and BSE-affected animals were different (P < 0.001, P < 0.01) from unrelated animals of the same breed without BSE. No genotypic differences were found between the BSE-affected animals and their relatives (P > 0.469). No AMFLP genotypic differences were detected between BSE-affected animals, their relatives, unrelated animals of the same breed or animals of different breeds (P > 0.05). These data suggest that BSE-affected animals and their relatives are more likely to have the AA SSCP genotype than unrelated animals of the same breed or animals of different breeds.     

Vascular Development and Sap Flow in Apple Pedicels

Xylem and phloem tissues of the pedicel of apple fruit increasein cross-sectional area throughout development. The increasein phloem is similar in the two cultivars examined (Cox's OrangePippin and Royal Gala) and reflects a steadily increasing phloemsap flow to the fruit. The increase in xylem tissue is due toa proliferation of non-conducting, structural, components sinceclose examination reveals no increase in the number of vesselelements from just after flowering onwards. The greater number,and the larger diameter, of the vessels in Cox's explains theinitially higher xylem conductance found in this cultivar. In vitro measurements of xylem exudation reveal a decline duringthe growing season in the xylem conductance of both cultivarsand an increasing proportion of fruit (particularly in Cox's)in which the xylem comes to be totally non-conducting. Thisobservation is in line with previously reported measurementsof xylem sap flow in vivo. The straightforward techniques used in this study offer a feasiblealternative to more arduous methods of assessing xylem and phloemsap flows and their balance during growth.Copyright 1994, 1999Academic Press Apple, xylem, phloem, vascular development, sap flow, Malus domestica Borkh     

Follicular development during early pregnancy and the estrous cycle of the sow

The objective of this study was to monitor and compare follicle populations and follicular development in pregnant and nonpregnant sows from Day 3 to Day 20 after breeding. Twenty-four sows were paired within parity on the day of artificial insemination and were randomly allocated within pair for insemination with either killed (n=12) or live spermatozoa (n=12). All the sows were artificially inseminated with the pooled ejaculate of the same boar. From Day 3 through Day 20 post estrus, ovarian follicles were scanned daily by ultrasonography. Ultrasound images were recorded on videotape and were retrospectively analyzed. Follicles were mapped to indentify the existence of follicular waves. The follicles were then classified as small (< 3 mm), medium (3-5 mm), or large (>/=5 mm). Pregnancy diagnosis was performed on Day 21 by ultrasonography. Pregnant sows maintained a constant proportion of the follicle population in the small, medium and large follicle categories. However, in the nonpregnant sows, the proportion of follicles in the various size categories remained constant until Day 15. Thereafter, the proportion of small follicles decreased (P < 0.05) from Day 15 to 20, and the proportions of medium and large follicles increased (P < 0.05). The predictability of pregnancy status on Day 20 based on follicle populations in any of the 3 follicle categories was low. Moreover, there was no evidence of follicular waves during the estrous cycle or early pregnancy. In conclusion, the proportion of small follicles decreased while medium and large follicle increased from Day 15 through Day 20 of the estrous cycle, but not during a similar stage of pregnancy. This latter finding concurs with follicle recruitment from the pool of small follicles for ovulation following PGF2alpha secretion to induce luteolysis, which reduces progesterone concentrations and thereby allows for the stimulation of the pool of small follicles by gonadotropins.     

Woody-tissue respiration for Simarouba amara and Minquartia guianensis,two tropical wet forest trees with different growth habits

We measured CO₂ efflux from stems of two tropical wet forest trees, both found in the canopy, but with very different growth habits. The species were Simarouba amara, a fast-growing species associated with gaps in old-growth forest and abundant in secondary forest, and Minquartia guianensis, a slow-growing species tolerant of low-light conditions in old-growth forest. Per unit of bole surface, CO₂ efflux averaged 1.24 mol m^–2 s^–1 for Simarouba and 0.83 mol m^–2s^–1 for Minquartia. CO₂ efflux was highly correlated with annual wood production (r ²=0.65), but only weakly correlated with stem diameter (r ²=0.22). We also partitioned the CO₂ efflux into the functional components of construction and maintenance respiration. Construction respiration was estimated from annual stem dry matter production and maintenance respiration by subtracting construction respiration from the instantaneous CO₂ flux. Estimated maintenance respiration was linearly related to sapwood volume (39.6 mol m^–3s^–1 at 24.6° C, r ²=0.58), with no difference in the rate for the two species. Maintenance respiration per unit of sapwood volume for these tropical wet forest trees was roughly twice that of temperate conifers. A model combining construction and maintenance respiration estimated CO₂ very well for these species (r ²=0.85). For our sample, maintenance respiration was 54% of the total CO₂ efflux for Simarouba and 82% for Minquartia. For our sample, sapwood volume averaged 23% of stem volume when weighted by tree size, or 40% with no size weighting. Using these fractions, and a published estimate of aboveground dry-matter production, we estimate the annual cost of woody tissue respiration for primary forest at La Selva to be 220 or 350 g C m^–2 year^–1, depending on the assumed sapwood volume. These costs are estimated to be less than 13% of the gross production for the forest.     

Purification of Potato Leaf Plasma Membrane Protein pp34, a Protein Phosphorylated in Response to Oligogalacturonide Signals for Defense and Development

A potato (Solanum tuberosum L.) plasma membrane protein called pp34, the only known example of a plasma membrane protein that is phosphorylated specifically in response to defined Oligogalacturonide signals in plants, has been purified to apparent homogeneity. Polyclonal antibodies raised in rabbits against the purified pp34 protein immunoprecipitated a single thiophosphorylated protein species from potato plasma membranes, as analyzed by two-dimensional denaturing electrophoresis and fluorography. The pp34 antibodies also recognized a single protein in tomato (Lycopersicon esculentum L.) membranes that is thiophosphorylated in response to Oligogalacturonide elicitors, as demonstrated by western blotting and specific immunoprecipitation. These experiments confirm the identity of the tomato membrane protein as a pp34 homolog and establish the high monospecificity of the pp34 antibodies. This will permit further investigation of the role of protein phosphorylation in oligouronide signaling for defensive genes in potato and tomato plants.     

Interaction of oddball probability and primary task type on P300 in the dual-task paradigm

Using a dual-task paradigm with an oddball secondary task, P300 amplitude and latency were studied as a function of factorially manipulated oddball probability (low = .22, high = .44) and primary task type. In addition to a Baselinecondition (oddball task only), three primary tasks were used: (1) Pure Sensory;watching a movie; (2) Pure Motor (manipulating a flashlight); and (3) Sensory/Motor(using the flashlight to trace the outlines of characters in a movie). The findings included the usual significant effects of probability on amplitude. There was also a significant effect of task type on amplitude, and a significant interaction of oddball probability with task type. In the low but not high probability condition, a pure Sensory task depressed P300 amplitude. In both probability conditions, the Sensory/motortask depressed P300 amplitude. Only task type had a significant effect on P300 latency. The results confirm the ability of other labs (using Sensory/motor primary tasks) to demonstrate P300 depression at high oddball probability, in view of the difficulty in our lab of achieving P300 depression with pure sensory tasks and high oddball probabilities. The results are discussed in terms of partial overlap of processing resource pools. A preliminary report of these data was presented at the 1990 meetings of the Society for Psychophysiological Research.     

The amino-terminal one-third of pseudorabies virus glycoprotein gIII contains a functional attachment domain, but this domain is not required for the efficient penetration of Vero cells.

We have examined the attachment and penetration phenotypes of several glycoprotein gIII mutants of pseudorabies virus (PRV) and have identified the first one-third of gIII as a region that mediates efficient virus attachment to PK15 and Vero cells. This portion of gIII, amino acids 25 through 157 of the wild-type sequence, appeared to support attachment by binding to heparinlike molecules on cell surfaces. Virions containing the first one-third of gIII were sensitive to heparin competition and showed greatly reduced infectivity on cells treated with heparinase. PRV virions lacking the first one-third of the mature glycoprotein exhibited only residual binding to cells if challenged by vigorous washing with phosphate-buffered saline at 2 h postinfection at 4 degrees C. This residual binding was resistant to heparin competition, and strains lacking the first one-third of gIII were able to infect cells treated with heparinase as effectively as untreated cells. When we determined the penetration phenotypes for each strain, we found that gIII-mediated virus attachment was necessary for timely penetration of PK15 cells but remarkably was not required for efficient virus penetration of Vero cells. Moreover, wild-type PRV was actually prohibited from rapid penetration of Vero cells by a gIII-heparan sulfate interaction. Our results indicate that initial virus binding to heparan sulfate via glycoprotein gIII is not required for efficient PRV infection of all cell types and may in fact be detrimental in some instances.     

Interaction between Aluminum Toxicity and Calcium Uptake at the Root Apex in Near-Isogenic Lines of Wheat (Triticum aestivum L.) Differing in Aluminum Tolerance

Aluminum (Al) is toxic to plants at pH < 5.0 and can begin to inhibit root growth within 3 h in solution experiments. The mechanism by which this occurs is unclear. Disruption of calcium (Ca) uptake by Al has long been considered a possible cause of toxicity, and recent work with wheat (Triticum aestivum L. Thell) has demonstrated that Ca uptake at the root apex in an Al-sensitive cultivar (Scout 66) was inhibited more than in a tolerant cultivar (Atlas 66) (J.W. Huang, J.E. Shaff, D.L. Grunes, L.V. Kochian [1992] Plant Physiol 98: 230-237). We investigated this interaction further in wheat by measuring root growth and Ca uptake in three separate pairs of near-isogenic lines within which plants exhibit differential sensitivity to Al. The vibrating calcium-selective microelectrode technique was used to estimate net Ca uptake at the root apex of 6-d-old seedlings. Following the addition of 20 or 50 [mu]M AlCl3, exchange of Ca for Al in the root apoplasm caused a net Ca efflux from the root for up to 10 min. After 40 min of exposure to 50 [mu]M Al, cell wall exchange had ceased, and Ca uptake in the Al-sensitive plants of the near-isogenic lines was inhibited, whereas in the tolerant plants it was either unaffected or stimulated. This provides a general correlation between the inhibition of growth by Al and the reduction in Ca influx and adds some support to the hypothesis that a Ca/Al interaction may be involved in the primary mechanism of Al toxicity in roots. In some treatments, however, Al was able to inhibit root growth significantly without affecting net Ca influx. This suggests that the correlation between inhibition of Ca uptake and the reduction in root growth may not be a mechanistic association. The inhibition of Ca uptake by Al is discussed, and we speculate about possible mechanisms of tolerance.