Arabidopsis ALF4 encodes a nuclear-localized protein required for lateral root formation

Lateral root formation, the primary way plants increase their root mass, displays developmental plasticity in response to environmental changes. The aberrant lateral root formation (alf)4-1 mutation blocks the initiation of lateral roots, thus greatly altering root system architecture. We have positionally cloned the ALF4 gene and have further characterized its phenotype. The encoded ALF4 protein is conserved among plants and has no similarities to proteins from other kingdoms. The gene is present in a single copy in Arabidopsis. Using translational reporters for ALF4 gene expression, we have determined that the ALF4 protein is nuclear localized and that the gene is expressed in most plant tissues; however, ALF4 expression and ALF4's subcellular location are not regulated by auxin. These findings taken together with further genetic and phenotypic characterization of the alf4-1 mutant suggest that ALF4 functions independent from auxin signaling and instead functions in maintaining the pericycle in the mitotically competent state needed for lateral root formation. Our results provide genetic evidence that the pericycle shares properties with meristems and that this tissue plays a central role in creating the developmental plasticity needed for root system development.     

Modulation of Rubisco activity in leaves of <Emphasis Type="Italic">Prosopis juliflora</Emphasis> in response to tropical conditions in north India

The success of P. juliflora, an evergreen woody species has been largely attributed to temperature acclimation and stomatal control of photosynthesis under wide range of environmental conditions prevalent in India. We studied the contribution of the enzyme ribulose-1,5 bisphosphate carboxylase/oxygenase (Rubisco) in diurnal and seasonal photosynthesis changes in P. juliflora. The changes observed in photosynthesis under natural conditions could be effected by the growth temperatures, which ranged from 10–30 °C in winter to 30–47 °C in summer. However, the Total Rubisco activity displayed a constant diurnal pattern and showed a maximum at 1200 in all seasons namely spring, summer, monsoon and winter irrespective of the changes in temperature. The Total Rubisco activity from two cohorts of leaves produced in spring and monsoon appeared to be down-regulated differentially at low PPFD during the evening. The in vivo and in vitro measurements of carboxylation efficiency of Rubisco showed wide variation during the day and were correlated with the photosynthesis rate. The light activation of Rubisco showed the acclimation to moderately high temperatures in different seasons except in summer. The exceptionally high temperatures (>45 °C) in summer, though not affecting Total activity, severely inhibited the light activation of Rubisco and also modulated the recovery process for the activation of Rubisco. Our studies suggest that the modulation of Rubisco driven by Rubisco activase and not Rubisco per se was crucial for the diurnal regulation of photosynthesis. NBRI Publication No.: 528     

Lack of adverse effects in pregnant/lactating female rats and their offspring following pre- and postnatal exposure to ELF magnetic fields

We have recently reported that exposure of pregnant rats to 60 Hz at field strengths up to 0.5 mT during the entire period of pregnancy did not induce any biologically significant effects on both pregnant dams and embryo-fetal development. The present study was carried out to investigate the potential effects of gestational and lactational MF exposure on pregnancy, delivery, and lactation of dams and growth, behavior, and mating performance of their offspring in rats. Timed-pregnant female Sprague-Dawley (SD) rats (24/group) received continuous exposure to 60 Hz magnetic field (MF) at field strengths of 0 (sham control), 5 microT, 83.3 microT, or 0.5 mT. Dams received MF or sham exposures for 21 h/day from gestational day 6 through lactational day 21. Experimentally generated MF was monitored continuously throughout the study. No exposure-related changes in clinical signs, body weight, food consumption, pregnancy length, and necropsy findings were observed in dams. Parameters of growth, behavior, and reproductive performance of offspring showed no changes related to MF exposure. There were no adverse effects on embryo-fetal development of F2 offspring from dams exposed to MF. In conclusion, exposure of pregnant SD rats to 60 Hz at field strengths up to 0.5 mT from gestational day 6 to lactational day 21 did not produce biologically significant effects in dams, F1 offspring, or F2 fetuses.     

Large scale in vitro experiment system for 2 GHz exposure

A beam formed radiofrequency (RF) exposure-incubator employing a horn antenna, a dielectric lens, and a culture case in an anechoic chamber is developed for large scale in vitro studies. The combination of an open type RF exposure source and a culture case through which RF is transmitted realizes a uniform electric field (+/-1.5 dB) in a 300 x 300 mm area that accommodates 49 35 mm diameter culture dishes. This large culture dish area enables simultaneous RF exposure of a large number of cells or various cell lines. The RF exposure source operates at 2142.5 MHz corresponding to the middle frequency of the downlink band of the International Mobile Telecommunication 2000 (IMT-2000) cellular system. The dielectric lens, which has a gain of 7 dB, focuses RF energy in the direction of the culture case and provides a uniform electric field. The culture case is sealed and connected to the main unit for environmental control, located outside the anechoic chamber, via ducts. The temperature at the center of the tray, which contains the culture dishes in the culture room, is maintained at 37.0 +/- 0.2 degrees C by air circulation. In addition, the appropriate CO2 density and humidity supplied to the culture case realizes stable long-term culture conditions. Specific absorption rate (SAR) dosimetry is performed using an electric field measurement technique and the Finite Difference Time Domain (FDTD) calculation method. The results indicate that the mean SAR of the culture fluid at the bottom of the 49 (7 x 7 array) culture dishes used in the in vitro experiments is 0.175 W/kg for an antenna input power of 1 W and the standard deviation of the SAR distribution is 59%. When only 25 culture dishes (5 x 5 array) are evaluated, the mean SAR is 0.139 W/kg for the same antenna input power and the standard deviation of the SAR distribution is 47%. The proliferation of the H4 cell line in 72 h in a pair of RF exposure-incubators reveals that the culture conditions are equivalent to those of a common CO2 incubator.     

Optimal conditions for hepatitis B core antigen production in shaked flask fermentation

The effects of various environmental factors such as pH (5, 6, 7, 8 and 9), temperature (30, 37 and 40°C) and rotational speed (150, 200 and 250 rpm) on the growth and the hepatitis B core antigen (HBcAg) production ofEscherichia coli W3110IQ were examined in the present study. The highest growth rate is achieved at PH 7, 37°C and at a rotational speed of 250 rpm which is 0.927 h⁻¹. The effect of pH on cell growth is more substantial compared to other parameters; it recorded a 123% different between the highest growth rate (0.927 h⁻¹) at pH 7 and lowest growth at pH 5. The highest protein yield is achieved at pH 9, rotational speed of 250 rpm and 40°C. The yield of protein at pH 7 is 154% higher compared to the lowest yield achieved at pH 5. There is about 28% different of the protein yield for theE. coli cultivated at 250 rpm compared to that at 150 rpm which has the lowest HBcAg yield. The yield of protein at 40°C is 38% higher compared to the lowest yield achieved, at 30°C.     

Modelling the effect of temperature and water activity on growth of Aspergillus niger strains and applications for food spoilage moulds

AIMS: To develop a model for the combined effect of water activity (a(w)) and temperature on growth of strains of Aspergillus niger, and comparison with data on food spoilage moulds in the literature. METHODS AND RESULTS: An extended combined model describing the growth of two strains of A. niger, as a function of temperature (25-30 degrees C) and a(w) (0.90-0.99) was developed. The growth rate (micro) was expressed as the increase in colony radial growth per unit of time. This extends the previous square root model showing the relationship between temperature and bacterial growth rate developed by Ratkowsky et al. (1983) and the parabolic relationship between the logarithm of the growth rate and a(w) developed by Gibson et al. (1994). A good correlation between the experimental data and the model predictions was obtained, with regression coefficients (r(2)) > 0.99. In addition, the use of this model allowed predictions of the cardinal a(w) levels: a(w(min)), and a(w(opt)). The estimation of the minimum a(w) levels (a(w(min))) was in accordance with data in the literature for similar and a range of other Aspergillus and related species, regardless of the solutes used for a(w) modification. The estimation of the optimal a(w) (a(w(opt))) and the optimal growth rate (micro(opt)) were in good agreement with the experimental results and data from the literature. CONCLUSIONS: This approach enables accurate prediction of the combined effects of environmental factors on growth of spoilage fungi for rapid prediction of cardinal limits using surface response curves. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach is a rapid method for predicting optimal and marginal conditions for growth of a wide range of spoilage micro-organisms in relation to interacting environmental conditions and will have applications for improving shelf-life of intermediate moisture foods.     

Temperature-sensitive random insulin granule diffusion is a prerequisite for recruiting granules for release

Glucose-evoked insulin secretion exhibits a biphasic time course and is associated with accelerated intracellular granule movement. We combined live confocal imaging of EGFP-labelled insulin granules with capacitance measurements of exocytosis in clonal INS-1 cells to explore the relation between distinct random and directed modes of insulin granule movement, as well as exocytotic capacity. Reducing the temperature from 34 degrees C to 24 degrees C caused a dramatic 81% drop in the frequency of directed events, but reduced directed velocities by a mere 25%. The much stronger temperature sensitivity of the frequency of directed events (estimated energy of activation approximately 135 kJ/mol) than that of the granule velocities (approximately 22 kJ/mol) suggests that cooling-induced suppression of insulin granule movement is attributable to factors other than reduced motor protein adenosine 5'-triphosphatase activity. Indeed, cooling suppresses random granule diffusion by approximately 50%. In the single cell, the number of directed events depends on the extent of granule diffusion. Finally, single-cell exocytosis exhibits a biphasic pattern corresponding to that observed in vivo, and only the component reflecting 2nd phase insulin secretion is affected by cooling. We conclude that random diffusive movement is a prerequisite for directed insulin granule transport and for the recruitment of insulin granules released during 2nd phase insulin secretion.     

Challenges in constructing statistically based structure-activity relationship models for developmental toxicity

Regulatory agencies are increasingly called upon to review large numbers of environmental contaminants that have not been characterized for their potential to pose a health risk. Additionally, there is special interest in protecting potentially sensitive subpopulations and identifying developmental toxicants that may be present in the environment. Thus, there is an urgent need for efficient methods to screen large numbers of chemicals for their potential to pose a developmental hazard. One potential screening method involves the use of statistically based structure-activity relationship (SAR) tools to predict activity of untested chemicals. Such systems rely on statistical analyses to discern relationships between structure and activity for a training set of substances. Predictions can then be made for an untested substance as long as its structural features are encompassed by chemicals of the training set. In theory, such systems could assist regulatory agencies in their screening efforts; however, to date, there has been little independent evaluation of these tools for this use. To contribute to such an evaluation, the International Life Sciences Institute Risk Science Institute (ILSI RSI) convened a Working Group to examine methodology used to construct statistically based SAR systems for developmental toxicity. This document reports on the deliberations of the Working Group, which concluded that an improved process is needed for utilizing developmental toxicity data in the construction of statistically based SAR models. The process must be objective, reproducible, rational and transparent. Moreover, it must be informed by the expertise of developmental toxicologists and biologists and must be subject to peer review.     

Effects of protein deficient diets on the developmental toxicity of inorganic arsenic in mice

BACKGROUND: Inorganic arsenic, when given by injection to pregnant laboratory animals (mice, rats, hamsters), has been shown to induce malformations. Arsenic methylation may be a detoxification step, and diets deficient in protein are a poor source of methyl donors and may possibly result in impaired arsenic methylation. Human health effects from chronic arsenic exposure have been reported mainly in populations with low socioeconomic status. Individuals in such populations are likely to suffer from malnutrition, which can compromise embryonic/fetal development and diminish arsenic methylating capacity. We sought to determine if dietary protein deficiency affects the developmental toxicity of inorganic arsenic. METHODS: Mated females were randomly assigned to one of 12 treatment groups. Experimental groups received either AsIII or AsV i.p. on Gestation Day 8 (GD 8, plug=GD 0) and were maintained on a 5%, 10%, or 20% protein custom mixed diet from GD 1 until sacrifice. Controls received the custom diets alone, were given AsIII or AsV i.p. on GD 8 with Teklad LM-485 rodent diet, or were fed the LM-485 diet alone. Test females were sacrificed on GD 17, and their litters were examined for mortality and developmental defects. RESULTS: Arsenic plus dietary protein deficiency decreased maternal weight gain and increased the incidences of exencephaly, ablepharia, and skeletal defects, such as malformed vertebral centra, fused ribs, and abnormal sternebrae (bipartite, rudimentary, or unossified). CONCLUSIONS: These results demonstrate that dietary protein deficiency enhances the developmental toxicity of inorganic arsenic, possibly by impairment of arsenic methylation.