Developmental and genotypic differences in the response of pea stem segments to auxin

The objective of this investigation was to examine the response to exogenous auxin (indole-3-acetic acid; IAA)of stem segments at two developmental stages. The standard auxin response of excised stem segments and intact plants consists of an initial growth response and a prolonged growth response. We found that this biphasic response does not occur in internodes at very early stages. Stem segments of light grown pea of various genotypes were cut when the fourth internode was at 6–13% of full expansion (early-expansion) or at 18–25% of full expansion (mid-expansion). Length measurements of excised segments were made after 48 hours of incubation on buffer with or without auxin. An angular position transducer linked to a computerized data collection system provided high-resolution measurement of growth of stacks of segments incubated in buffer over 20 hours. Early-expansion segments of all genotypes deviated from the standard auxin response, while mid-expansion segments responded in a manner consistent with previous reports. Early-expansion segments of tall, light-grown plants were unique in showing an auxin-induced inhibition of growth. The auxin-induced inhibition correlated with high endogenous auxin content, as determined by HPLC and GC/MS, across genotypes and between early-expansion and mid-expansion segments of tall plants. Measurement of ethylene evolved from stem segments in response to auxin, and treatment of segments with the ethylene action inhibitor, norbornadiene, showed the inhibition to be mediated in part by heightened ethylene sensitivity. Growth of early-expansion segments of dwarf and severe dwarf plants was stimulated by exogenous auxin, but the growth rate increase was delayed compared to that in mid-expansion segments. This is the first time that such a growth response, termed the delayed growth response has been emonstrated. It is concluded that developmental stage and endogenous hormone content affect tissue response to exogenous auxin.     

Drought signal transduction in plants

Water deficit is one of the most common environmental limitations of crop productivity by affecting growth through alterations in metabolism and gene expression. The mechanisms involved in drought perception and signal transduction pathways are poorly understood. The participation of the plant hormone abscisic acid (ABA) has been well established. ABA levels increase when there are changes in the environment that result in cellular dehydration. Different approaches have been taken to understanding the molecular responses to desiccation and how ABA regulates gene expression. Recent efforts have identified particular topics of importance in the dissection of the signal transduction pathway which are summarized as follows: physiological approaches: identification of signalling molecules. Genetic approaches: the use of mutants, and Molecular approaches: promoter analysis.     

Algorithm independent properties of RNA secondary structure predictions

Algorithms predicting RNA secondary structures based on different folding criteria – minimum free energies (mfe), kinetic folding (kin), maximum matching (mm) – and different parameter sets are studied systematically. Two base pairing alphabets were used: the binary GC and the natural four-letter AUGC alphabet. Computed structures and free energies depend strongly on both the algorithm and the parameter set. Statistical properties, such as mean number of base pairs, mean numbers of stacks, mean loop sizes, etc., are much less sensitive to the choice of parameter set and even of algorithm. Some features of RNA secondary structures, such as structure correlation functions, shape space covering and neutral networks, seem to depend only on the base pairing logic (GC or AUGC alphabet). Received: 16 May 1996 / Accepted: 10 July 1996     

Isozyme analysis of intercontinental disjuncts within Styrax (Styracaceae): implications for the Madrean —Tethyan hypothesis

Various hypotheses have been put forward to explain the presence of sclerophyllous plant disjuncts between western North America and the Mediterranean region. The Madrean–Tethyan hypothesis postulates that the two regions were floristically connected in the Early to Middle Tertiary by way of a low-latitude migration route. Others deny the possibility of such a route, and instead postulate convergence to xerophytic conditions from more widespread mesophytic ancestors, or suggest long-distance dispersal scenarios. One example of a “Madrean–Tethyan link” between the two regions is composed of four species within the genus Styrax: S. officinalis subsp. officinalis from the Mediterranean region, S. officinalis subsp. redivivus and subsp. fulvescens from California, and three closely related species in Texas and northeastern Mexico (S. texanus, S. platanifolius, and S. youngiae). This group was examined with isozymes to assess whether patterns of genetic variation are consistent with those predicted by the Madrean–Tethyan hypothesis. Ten populations from California, six from the Mediterranean region, and three from Texas were sampled. Pairwise comparisons revealed mean genetic identity (I) estimates of 0.581 between Mediterranean and California populations, 0.470 between Mediterranean and Texas populations, and 0.640 between California and Texas populations. Two populations of a species thought by many to be the closest relative of S. officinalis on morphological grounds (S. jaliscanus) exhibited low I (0.299–0.321) relative to all other group comparisons. Intercontinentally disjunct populations of S. officinalis possessed an I value that warrants species status for the Californian and Mediterranean groups. Divergence time estimates between Madrean and Tethyan Styrax range from 5.0 to 13.8 Mya, too recent to be consistent with the Madrean–Tethyan hypothesis. However, alternative explanations for this disjunction are suboptimal in that they require the invocation of either long-distance dispersal, which appears unlikely in this group, or extinction. Nonetheless, the evidence presented here and in other recent studies casts substantial doubt on the Madrean–Tethyan hypothesis as a general explanation for the presence of Madrean and Tethyan taxa similar in overall appearance. More plants with Madrean–Tethyan distributions must be sampled before definitive conclusions regarding this aspect of Madrean and Tethyan vegetation can be reached.     

Differences between observed and predicted energy costs at rest and during exercise in three subsistence-level populations

Estimates of daily energy expenditure are important for many areas of research in human ecology and adaptability. The most common technique for estimating human energy expenditure under field conditions, the factorial method, generally relies on activity-specific energy costs derived from published sources, based largely on North American and European subjects. There is concern that such data may not be appropriate for non-Western populations because of differences in metabolic costs. The present study addresses this concern by comparing measured vs. predicted energy costs at rest and during sub-maximal exercise in 83 subjects (52 males, 31 females) from three subsistence-level populations (Siberian herders and high-land and coastal Ecuadorian farmers). Energy costs at rest (i.e., lying, sitting and standing) and while performing a standard stepping exercise did not significantly differ among the three groups. However, resting energy costs were significantly elevated over predicted levels (+16% in men, +11% in women), whereas exercising costs were comparable to predicted values (?6% in men, +3% in women). Elevations in resting energy needs appear to reflect responses to thermal stress. These results indicate that temperature adjustments of resting energy costs are critical for accurately predicting daily energy needs among traditionally living populations. o 1996 Wiley-Liss, Inc.     

A Quantitative Microtiter Plate Nuclease Assay Based on Ethidium/DNA Fluorescence

A microtiter plate assay was developed to quantitate the nuclease activity of the extracellularSerratia marcescensendonuclease under different buffer conditions. Substrate cleavage was followed as decrease in ethidium/DNA fluorescence using a uv-transilluminator and a video documentation system. Time courses of DNA cleavage were recorded and cleavage rates determined very precisely within a factor of 1.2. The assay has a linear dynamic range covering three orders of magnitude of nuclease activity and can be carried out very quickly within a few minutes. It can also be used with RNA as substrate. With appropriate modifications it should be possible to adapt this assay for other enzymatic reactions which are accompanied by changes in absorbance or fluorescence.     

Low multiplicity infection of insect cells with a recombinant baculovirus: The cell yield concept

In vitro infection of insect cells with baculoviruses is increasingly considered a viable means for the production of biopesticides, recombinant veterinary vaccines, and other recombinant products. Batch fermentation processes traditionally employ intermediate to high multiplicities of infection necessitating two parallel scale-up processes-one for cells and one for virus. In this study, we consider the use of multiplicities of infection as low as 0.0001 plaque-forming units per cell, a virus level low enough to enable infection of even large reactors (e.g., 10 m(3)) directly from a frozen stock. Using low multiplicities in the Sf9/beta-gal-AcNPV system, recombinant protein titers comparable with the maximum titer observed in high multiplicity infections were achieved. Cultures yielding the maximum titer were characterized by reaching a maximum cell density between 3 and 4 x 10(9) cell L(-1). This optimal cell yield did not depend on the multiplicity of infection, supporting the existing view that batch cultures are limited by availability of substrate. Up to a certain cell density, product titer will increase almost linearly with availability of biocatalyst, that is, cells. Beyond this point any further cell formation comes at the expense of final product titer. Low multiplicity infections were found not to cause any significant dispersion of the protein production process. Hence, product stability is not a major issue of concern using low multiplicities of infection. The sensitivity to initial conditions and disturbances, however, remains an issue of concern for the commercial use of low multiplicity infections. (c) 1996 John Wiley & Sons, Inc.     

Biological activity and metabolic clearance of recombinant human follicle stimulating hormone produced in Sp2/0 myeloma cells

Human follicle stimulating hormone is a pituitary glycoprotein that is essential for the maintenance of ovarian follicle development and testicular spermatogenesis. Like other members of the glycoprotein hormone family, it contains a common a subunit and a hormone specific subunit. Each subunit contains two glycosylation sites. The specific structures of the oligosaccharides of human follicle stimulating hormone have been shown to influence both thein vitro andin vivo bioactivity. Since the carbohydrate structure of a protein reflects the glycosylation apparatus of the host cells in which the protein is expressed, we examined the isoform profiles,in vitro bioactivity and metabolic clearance of a preparation of purified recombinant human follicle stimulating hormone derived from a stable, transfected Sp2/0 myeloma cell line, and pituitary human follicle stimulating hormone. Isoelectric focussing and chromatofocussing studies of human follicle stimulating hormone preparations both showed a more basic isoform profile for the recombinant human follicle stimulating hormone compared to that of pituitary human follicle stimulating hormone. The recombinant human follicle stimulating hormone had a significantly higher radioreceptor activity compared to that of pituitary human follicle stimulating hormone, consistent with a greaterin vitro potency. Pharmacokinetic studies in rats indicated a similar terminal half life (124 min) to that of the pituitary human follicle stimulating hormone (119 min). Preliminary carbohydrate analysis showed recombinant human follicle stimulating hormone to contain high mannose and/or hybrid type, in addition to complex type carbohydrate chains, terminating with both2,3 and2,6 linked sialic acids. These results demonstrate that recombinant human follicle stimulating hormone made in the Sp2/0 myeloma cells is sialylated, has a more basic isoform profile, and has a greaterin vitro biological potency compared to those of the pituitary human follicle stimulating hormone.