The Acute Oral Toxicity of Reduced Iron

The LD₅₀ ± S.E. of Reduced Iron, N.F. IX, given orally to albino rats was found to be 98.6 ± 26.7 g. per kg. body weight, or over a hundredfold that of iron given as ferrous sulfate. The clinical and pathological signs of toxicity were somewhat similar to those of ferrous sulfate given under the same conditions. The results indicate that reduced iron could stand reinvestigation in respect of its practical value as a therapeutic agent with probably very low toxicity.     

Purification and Characterization of NADH-Glutamate Synthase from Alfalfa Root Nodules

Glutamate synthase (GOGAT), a key enzyme in the pathway for the assimilation of symbiotically fixed dinitrogen (N₂) into amino acids in alfalfa (Medicago sativa L.) root nodules, was purified and used to produce high titer polyclonal antibodies. Purification resulted in a 208-fold increase in specific activity to 13 micromole per minute per milligram of protein and an activity yield of 37%. Further purification to near homogeneity was achieved by fast protein liquid chromatography, but with substantial loss of activity. Enzymic activity was highly labile, losing 3% per hour even when substrates, stabilizers, and reducing agents were included in buffers. However, activity could be partially stabilized for up to 1 month by storing GOGAT at −80°C in 50% glycerol. The subunit molecular weight of GOGAT was estimated at 200 ± 7 kilodaltons with a native molecular weight of 235 ± 16 kilodaltons, which suggested that GOGAT is a monomer of unusually high molecular weight. The pl was estimated to be 6.6. The K_m values for glutamine, α-ketoglutarate, and NADH were 466, 33, and 4.2 micromolar, respectively. Antibodies were produced to NADH-GOGAT. Specificity of the antibodies was shown by immunotitration of GOGAT activity. Alfalfa nodule NADH-GOGAT antibodies cross-reacted with polypeptides of a similar molecular weight in a number of legume species. Western blots probed with anti-GOGAT showed that the high GOGAT activity of nodules as compared to roots was associated with increased levels of GOGAT polypeptides. Nodule NADH-GOGAT appeared to be highly expressed in effective nodules and little if any in other organs.     

Using a Hazard Quotient to Evaluate Pesticide Residues Detected in Pollen Trapped from Honey Bees (Apis mellifera) in Connecticut

Analysis of pollen trapped from honey bees as they return to their hives provides a method of monitoring fluctuations in one route of pesticide exposure over location and time. We collected pollen from apiaries in five locations in Connecticut, including urban, rural, and mixed agricultural sites, for periods from two to five years. Pollen was analyzed for pesticide residues using a standard extraction method widely used for pesticides (QuEChERS) and liquid chromatography/mass spectrometric analysis. Sixty pesticides or metabolites were detected. Because the dose lethal to 50% of adult worker honey bees (LD₅₀) is the only toxicity parameter available for a wide range of pesticides, and among our pesticides there were contact LD₅₀ values ranging from 0.006 to >1000 μg per bee (range 166,000X), and even among insecticides LD₅₀ values ranged from 0.006 to 59.8 μg/bee (10,000X); therefore we propose that in studies of honey bee exposure to pesticides that concentrations be reported as Hazard Quotients as well as in standard concentrations such as parts per billion. We used both contact and oral LD₅₀ values to calculate Pollen Hazard Quotients (PHQ = concentration in ppb ÷ LD₅₀ as μg/bee) when both were available. In this study, pesticide Pollen Hazard Quotients ranged from over 75,000 to 0.01. The pesticides with the greatest Pollen Hazard Quotients at the maximum concentrations found in our study were (in descending order): phosmet, Imidacloprid, indoxacarb, chlorpyrifos, fipronil, thiamethoxam, azinphos-methyl, and fenthion, all with at least one Pollen Hazard Quotient (using contact or oral LD₅₀) over 500. At the maximum rate of pollen consumption by nurse bees, a Pollen Hazard Quotient of 500 would be approximately equivalent to consuming 0.5% of the LD₅₀ per day. We also present an example of a Nectar Hazard Quotient and the percentage of LD₅₀ per day at the maximum nectar consumption rate.     

Isolation and Characterization of a Mutant of Arabidopsis thaliana Resistant to alpha-Methyltryptophan

Mutants of Arabidopsis thaliana have been selected for resistance to growth inhibition at the seedling stage by α-methyltryptophan (aMT). One mutant, amt-1 has been characterized in detail. The appearance and growth rate of the mutant in the absence of the inhibitor are similar to wild type, both as plants and callus. However, mutant plant growth is unaffected by 25 micromolar aMT and mutant callus growth by 50 micromolar aMT, concentrations that completely inhibit the growth of wild-type plants and callus, respectively. Tryptophan levels in mutant and wild-type plants are 24.3 ± 2.7 and 4.7 ± 1.2 micrograms per gram fresh weight, respectively, and in the corresponding callus 64.0 ± 2.6 and 31.8 ± 8.4 micrograms per gram fresh weight, respectively. Anthranilate synthase (AS) activity levels in crude extracts from whole plants are 3.09 ± 0.54 nanomoles per milligram protein per hour in amt-1 and 1.32 ± 0.21 nanomoles per milligram protein per hour in wild-type plants. In crude extracts from callus, anthranilate synthase levels are 11.54 ± 2.05 nanomoles per milligram protein per hour and 7.74 ± 1.58 in amt-1 and wild type, respectively. Enzyme extracts are inhibited by l-tryptophan; the concentrations required for 50% inhibition (I₅₀) are 3.9 and 1.9 micromolar for amt-1 and for wild type, respectively. The mutation segregates as a single nuclear allele and shows incomplete dominance. The concomitant increases in both AS activity and its I₅₀ for tryptophan suggest that the mutation amt-1 either resides in one of the AS structural genes or causes increased expression of an AS isoform with an I₅₀ greater than the average for the entire extract.     

THE INFLUENCE OF ENVIRONMENTAL TEMPERATURE ON THE UTILIZATION OF FOOD ENERGY IN BABY CHICKS

1. An optimum of environmental temperature is to be expected for the utilization of food energy in warm blooded animals if their food intake is determined by their appetite. 2. Baby chicks were kept in groups of five chicks in a climatic cabinet at environmental temperatures of 21°, 27°, 32°, 38°, and 40°C. during the period of 6 to 15 days of age. The intake of qualitatively complete food was determined by their appetite. Food intake, excretion, and respiratory exchange were measured. Control chicks from the same hatch as the experimental groups were raised in a brooder and were given the same food as the experimental chicks. The basal metabolism of each experimental group was determined from 24 to 36 hours without food at the age of 16 days. 3. The daily rate of growth increased with decreasing environmental temperature from 2.74 gm. at 40°C. to 4.88 gm. at 21°C. This was 4.2 to 6.5 per cent of their body weight. 4. The amount of food consumed increased in proportion to the decrease in temperature. 5. The availability of the food, used for birds instead of the digestibility and defined as See PDF for Structure showed an optimum at 38°C. 6. The CO₂ production increased from 2.95 liters CO₂ per day per chick at 40°C. to 6.25 liters at 21°C. Per unit of the 3/4 power of the body weight, 23.0 liters CO₂ per kilo^3/4 was produced at 40°C. and 43.4 liters per kilo^3/4 at 21°C. The CO₂ production per unit of 3/4 power of the weight increased at an average rate of approximately 1 per cent per day increase in age. The R.Q. was, on the average, 1.04 during the day and 0.92 during the night. 7. The net energy is calculated on the basis of C and N balances. A maximum of 11.8 Cal. net energy per chick per day was found at 32°C. At 21°C. only 6.9 Cal. net per day per chick was produced and at 40°C. an average of 6.7 Cal. 8. The composition of the gained body substance changed according to the environmental temperature. The protein stored per gram increase in body weight varied from 0.217 to 0.266 gm. protein and seemed unrelated to the temperature. The amount of fat per gram gain in weight dropped from a maximum of 0.153 gm. at 32°C. to 0.012 gm. at 21°C. and an average of 0.107 gm. at 40°C. The energy content per gram of gain in weight had its maximum of 2.95 Cal. per gm. at 38°C. and its minimum of 1.41 Cal. per gm. at 21°C. at which temperature the largest amount of water (0.763 gm. per gm. increase in body weight) was stored. 9. The basal metabolism increased from an average of 60 Cal. per kilo^3/4 at an environmental temperature of 40°C. to 128 Cal. per kilo^3/4 at 21°C. No indication of a critical temperature was found. 10. The partial efficiency, i.e. the increase in net energy per unit of the corresponding increase in food energy, seemed dependent on the environmental temperature, reaching a maximum of 72 per cent of the available energy at 38°C. and decreasing to 57 per cent at 21°C. and to an average of 60 per cent at 40°C. 11. The total efficiency, i.e. the total net energy produced per unit of food energy taken in, was maximum (34 per cent of the available energy) at 32°C., dropped to 16 per cent at 21°C., and to an average of 29 per cent at 40°C.     

Selection for Rate and Efficiency of Lean Gain in the Rat

Full-sib family selection for rate (WP) or efficiency (WP/F) of protein gain in rats from 3 to 9 weeks of age was applied for five generations. Three rats per litter were killed to estimate carcass protein. Standardized response/cumulative selection for WP was.19±.10 for WP,.28±.10 for 3- to 9-week gain,.28±.08 for 9-week weight,.16±.08 for litter size,.22±.12 for skinning loss and -.07±.09 for fraction of protein in the live weight. Response from selection for WP/F was.18±.16 for WP/F,.20±.11 for WP,.21±.11 for weight gain,.16±.11 for 3-week weight,.21±.10 for 9-week weight, but negligible for skinning loss or body protein. Response to WP/F selection was extremely variable among generations, associated with generation differences in weight and composition at 9 weeks. Estimates of heritability from offspring-midparent regression were.20±.12 for WP and.24±.08 for WP/F. Estimates of genotype-generation environment interaction were large for growth, feed intake and skinning loss. Maternal effects were large for weaning weight, fraction of body protein and WP. Sire component genetic correlations were 1.08±.13 for WP with total gain,.92±.08 for WP/F with gross efficiency and.29±.25 for WP with WP/F. A partitional calorimeter was used to evaluate heat production of rats. Lines differed in average heat loss but not in heat loss per unit actual or metabolic weight. Response to selection has been steady for WP but probably could be improved by selecting for WP/F at a constant weight rather than a constant age.     

Relationship between Thermal Transitions and Freezing Injury in Pea and Soybean Seeds

In an attempt to correlate freezable water with freezing injury, the thermal behavior of pea (Pisum sativum L.) and soybean (Glycine max L. Merr) seed parts at different moisture contents were compared with survival of the seeds when exposed to low temperatures. Thermal transitions between −150 and 10°C were studied using differential scanning calorimetry. In pea, reduction of germinability, after exposure of seeds to temperatures between − 18 and − 180°C, occurred at a constant moisture content (about 0.33 gram H₂O/gram dry weight) regardless of the temperature; this moisture level was above that at which freezable water was first detectable by differential scanning calorimetry (0.26 gram H₂O/gram dry weight). In contrast, damage to soybean seeds was observed at progressively lower moisture contents (from 0.33 to 0.20 gram H₂O/gram dry weight) when the temperature was decreased from −18°C to −50°C. At −18 and −30°C, moisture contents at which damage to soybean seeds was evident were above that at which freezable water was first detectable (0.23 gram H₂O/gram dry weight). However, at −50, −80, and −180°C, damage was evident even when freezable water was not detectable. The data suggest that, while the quantity of water is important in the expression of freezing injury, the presence of freezable water does not account for the damage.     

Improved Glycaemia Correlates with Liver Fat Reduction in Obese,Type 2 Diabetes,Patients Given Glucagon-Like Peptide-1 (GLP-1) Receptor Agonists

Glucagon-like peptide-1 receptor agonists (GLP-1 RA) are effective for obese patients with type 2 diabetes mellitus (T2DM) because they concomitantly target obesity and dysglycaemia. Considering the high prevalence of non-alcoholic fatty liver disease (NAFLD) in patients with T2DM, we determined the impact of 6 months’ GLP-1 RA therapy on intrahepatic lipid (IHL) in obese, T2DM patients with hepatic steatosis, and evaluated the inter-relationship between changes in IHL with those in glycosylated haemoglobin (HbA₁c), body weight, and volume of abdominal visceral and subcutaneous adipose tissue (VAT and SAT). We prospectively studied 25 (12 male) patients, age 50±10 years, BMI 38.4±5.6 kg/m² (mean ± SD) with baseline IHL of 28.2% (16.5 to 43.1%) and HbA₁c of 9.6% (7.9 to 10.7%) (median and interquartile range). Patients treated with metformin and sulphonylureas/DPP-IV inhibitors were given 6 months GLP-1 RA (exenatide, n = 19; liraglutide, n = 6). IHL was quantified by liver proton magnetic resonance spectroscopy (¹H MRS) and VAT and SAT by whole body magnetic resonance imaging (MRI). Treatment was associated with mean weight loss of 5.0 kg (95% CI 3.5,6.5 kg), mean HbA_1c reduction of 1·6% (17 mmol/mol) (0·8,2·4%) and a 42% relative reduction in IHL (−59.3, −16.5%). The relative reduction in IHL correlated with that in HbA₁c (ρ = 0.49; p = 0.01) but was not significantly correlated with that in total body weight, VAT or SAT. The greatest IHL reduction occurred in individuals with highest pre-treatment levels. Mechanistic studies are needed to determine potential direct effects of GLP-1 RA on human liver lipid metabolism.     

Water Content during Abscisic Acid Induced Freezing Tolerance in Bromegrass Cells

Changes in water content and dry weight were determined in control cells and those induced to cold harden in response to abscisic acid (ABA) treatment (7.5 × 10⁻⁵ molar). Bromegrass (Bromus inermis Leyss cv Manchar) cells grown in suspension culture at room temperature (23°C) for 7 days acclimated to −28°C (LT₅₀) when treated with ABA, or to −5°C when untreated. ABA significantly reduced cell growth rates at 5 and 7 days after treatment. Growth reduction was due to a decrease in cell number rather than cell size. When the cell water content was expressed as percent water (percent H₂O) or as grams water per gram dry weight (gram H₂O/gram dry weight [g DW]), the water content of hardy, ABA-treated cells decreased from 85% to 77% or from 6.4 to 3.3 g H₂O/g DW in 7 days. Control cell water content remained static at approximately 87% and 7.5 g H₂O/g DW. However, cell water content, expressed as milligrams water per million cells (milligram H₂O/10⁶ cells), did not differ in ABA-treated or control cells. The dry matter content of ABA-treated cells, expressed as milligram DW/10⁶ cells increased to 3.3 milligram/10⁶ cells in 7 days, whereas the dry weight of the control cells remained between 1.4 to 2.1 milligrams/10⁶ cells. The osmotic potential of ABA-treated cells decreased by the fifth day while that of control cells increased significantly and then decreased by day 7. Elevated osmotic potentials were not associated with increased ion uptake. In contrast to much published literature, these results suggest that cell water content does not decrease in ABA-treated cells during the induction of freezing tolerance, rather the dry matter mass per cell increased. Cell water content may be more accurately expressed as a function of cell number when accompanying changes to dry cell matter occur.     

Effect of Some Variables on theIn VivoDetermination of Scorpion and Viper Venom Toxicities

An adequate assessment of scorpion and snake venom LD₅₀is an important step for accurate evaluation of antivenom sera potencies and the optimization of serotherapy. The LD₅₀variation of Tunisian scorpion (Androctonus australis garzonii: Aag andButhus occitanus tunetanus: Bot) venoms with body weight, sex and strain (Swiss or C57Bl/6) of mice used, the route of venom injection, the venom-milking procedures (manually or electrically) and the venom batches have been studied over a 7-year period (1990–1996). Aag venom is 3–4 times more toxic than Bot venom. However for both venoms, the LD₅₀determined in C57Bl/6 mice, in small body weight animal or by intraperitoneal route were respectively significantly lower than those determined in Swiss mice, in high body weight or by subcutaneous route. Significant LD₅₀variations (25–50%) were also seen from one electrically prepared batch to another. A good correlation (r=0·982) was observed between the concentrations of the crude venom toxic fraction determined by ELISA and LD₅₀values when assessedin vivo.The LD₅₀variation of Tunisian viper (Cerastes cerastes: Cc andVipera lebetina: VI) venoms with the strain (Swiss or BALB/c), sex and body weight of mice used, the season and the year of venom milking were also investigated over a 3-year period (1990–1992). No significant LD₅₀variations were observed with the mouse strain, the sex or the season of venom milking. However, LD₅₀varies significantly with the year of the venom collection and the body weight of mice used. Furthermore, SDS–PAGE analysis shows annual variation for VI venom composition where no such variations were observed for Cc venom. These results stress the need either for the standardization of the venom LD₅₀evaluation or the venom quality used for the development of an efficient antivenom.     

Photosynthetic and respiratory rates of two psychrophilic diatoms

The photosynthetic rates in two psychrophilic diatoms, Chaetoceros sp. strain K3-10 and Nitzschia sp. K3-3 for cells grown at 0°C were 8 to 10 microliters O₂ evolved per milligram dry weight per hour, and 10-fold higher, about 80 for cells grown at 10°C. The respiration rates followed the same pattern, with a value of around 1 microliter dark uptake per milligram dry weight per hour for both organisms grown at 0°C, and 6 to 10 for cells grown at 10°C. When cells grown at 0°C were immediately shifted to 10°C or cells grown at 10°C were shifted to 0°C, the respiratory rates quickly adapted to values characteristic of cells grown at the shift temperature. On the other hand, the light-saturated rate of O₂ evolution showed much less immediate adaptation, especially on the up shift, 0° to 10°C. The chlorophyll a content of 0°C grown cells was about 0.5% of dry weight, in 10°C grown cells 1.3% (strain K3-10) and 2.2% (strain K3-3). In addition to a diminished chlorophyll a content in 0°C grown cells, there seemed proportionally (by absorbance and calculation) less c to a than in 10°C grown cells. The relative fluorescence excitation spectra of 680-nm emission also showed a lower contribution by both chlorophyll c and fucoxanthin in 0°C grown cells of Chaetoceros sp. strain K3-10 as compared to 10°C grown cells. The data at hand suggest that in psychrophilic diatoms continuously growing at 0°C there may be problems associated with synthesis of an effective accessory pigment system, and as a working hypothesis it is suggested this is related to restriction of synthesis of one or several accessory pigment proteins.     

Differential effect of chromium compounds on the immune response of the African mouth breeder Oreochromis mossambicus (Peters)

The objective of this study was to investigate the effect of trivalent (chromic chloride) and hexavalent (potassium dichromate) forms of chromium in the African mouth breeder Oreochromis mossambicus (Peters), with reference to the humoral immune response and lymphoid cells/organs. The 96 h LD₅₀for hexavalent and trivalent chromium was found to be 75 and 1000 μg fish⁻¹, respectively. Groups of fishes were injected intraperitoneally with 10, 1, 0·1 and 0·01% LD₅₀hexavalent and trivalent forms of chromium and subsequently immunised with bovine serum albumin (5 mg in 0·2 ml physiological saline). Both forms of chromium suppressed the antibody response, with hexavalent chromium being more suppressive than trivalent chromium. Reduction in spleen weight, splenocyte number and the percentage of blood lymphocytes was observed following administration of both forms of chromium. The possible immunological mechanisms behind the differential suppression of the antibody response and the reduction in spleen weight, splenocyte and lymphocyte counts are discussed.     

Heat Stress Impedes Development and Lowers Fecundity of the Brown Planthopper Nilaparvata lugens (St?l)

This study investigated the effects of sub-lethal high temperatures on the development and reproduction of the brown plant hopper Nilaparvata lugens (Stål). When first instar nymphs were exposed at their ULT₅₀ (41.8°C) mean development time to adult was increased in both males and females, from 15.2±0.3 and 18.2±0.3 days respectively in the control to 18.7±0.2 and 19±0.2 days in the treated insects. These differences in development arising from heat stress experienced in the first instar nymph did not persist into the adult stage (adult longevity of 23.5±1.1 and 24.4±1.1 days for treated males and females compared with 25.7±1.0 and 20.6±1.1 days in the control groups), although untreated males lived longer than untreated females. Total mean longevity was increased from 38.8±0.1 to 43.4±1.0 days in treated females, but male longevity was not affected (40.9±0.9 and 42.2±1.1 days respectively). When male and female first instar nymphs were exposed at their ULT₅₀ of 41.8°C and allowed to mate on reaching adult, mean fecundity was reduced from 403.8±13.7 to 128.0±16.6 eggs per female in the treated insects. Following exposure of adult insects at their equivalent ULT₅₀ (42.5°C), the three mating combinations of treated male x treated female, treated male x untreated female, and untreated male x treated female produced 169.3±14.7, 249.6±21.3 and 233.4±17.2 eggs per female respectively, all significantly lower than the control. Exposure of nymphs and adults at their respective ULT₅₀ temperatures also significantly extended the time required for their progeny to complete egg development for all mating combinations compared with control. Overall, sub-lethal heat stress inhibited nymphal development, lowered fecundity and extended egg development time.     

Comparison of dosimetric variation between prostate IMRT and VMAT due to patient's weight loss: Patient and phantom study

Aim

This study compared the dosimetric impact between prostate IMRT and VMAT due to patient''s weight loss.

Background

Dosimetric variation due to change of patient''s body contour is difficult to predict in prostate IMRT and VMAT, since a large number of small and irregular segmental fields is used in the delivery.

Materials and methods

Five patients with prostate volumes ranging from 32.0 to 86.5 cm³ and a heterogeneous pelvis phantom were used for prostate IMRT and VMAT plans using the same set of dose–volume constraints. Doses in IMRT and VMAT plans were recalculated with the patient''s and phantom''s body contour reduced by 0.5–2 cm to mimic size reduction. Dose coverage/criteria of the PTV and CTV and critical organs (rectum, bladder and femoral heads) were compared between IMRT and VMAT.

Results

In IMRT plans, increases of the D99% for the PTV and CTV were equal to 4.0 ± 0.1% per cm of reduced depth, which were higher than those in VMAT plans (2.7 ± 0.24% per cm). Moreover, increases of the D30% of the rectum and bladder per reduced depth in IMRT plans (4.0 ± 0.2% per cm and 3.5 ± 0.5% per cm) were higher than those of VMAT (2.2 ± 0.2% per cm and 2.0 ± 0.6% per cm). This was also true for the increase of the D5% for the right femoral head in a patient or phantom with size reduction due to weight loss.

Conclusions

VMAT would be preferred to IMRT in prostate radiotherapy, when a patient has potential to suffer from weight loss during the treatment.     

Persistence of Viruses in Desert Soils Amended with Anaerobically Digested Sewage Sludge

Pima County, Ariz., is currently investigating the potential benefits of land application of sewage sludge. To assess risks associated with the presence of pathogenic enteric viruses present in the sludge, laboratory studies were conducted to measure the inactivation rate (k = log₁₀ reduction per day) of poliovirus type 1 and bacteriophages MS2 and PRD-1 in two sludge-amended desert agricultural soils (Brazito Sandy Loam and Pima Clay Loam). Under constant moisture (approximately -0.05 × 10⁵ Pa for both soils) and temperatures of 15, 27, and 40°C, the main factors controlling the inactivation of these viruses were soil temperature and texture. As the temperature increased from 15 to 40°C, the inactivation rate increased significantly for poliovirus and MS2, whereas, for PRD-1, a significant increase in the inactivation rate was observed only at 40°C. Clay loam soils afforded more protection to all three viruses than sandy soils. At 15°C, the inactivation rate for MS2 ranged from 0.366 to 0.394 log₁₀ reduction per day in clay loam and sandy loam soils, respectively. At 27°C, this rate increased to 0.629 log₁₀ reduction per day in clay loam soil and to 0.652 in sandy loam soil. A similar trend was observed for poliovirus at 15°C (k = 0.064 log₁₀ reduction per day, clay loam; k = 0.095 log₁₀ reduction per day, sandy loam) and 27°C (k = 0.133 log₁₀ reduction per day, clay loam; k = 0.154 log₁₀ reduction per day, sandy loam). Neither MS2 nor poliovirus was recovered after 24 h at 40°C. No reduction of PRD-1 was observed after 28 days at 15°C and after 16 days at 27°C. At 40°C, the inactivation rates were 0.208 log₁₀ reduction per day in amended clay loam soil and 0.282 log₁₀ reduction per day in sandy loam soil. Evaporation to less than 5% soil moisture completely inactivated all three viruses within 7 days at 15°C, within 3 days at 27°C, and within 2 days at 40°C regardless of soil type. This suggests that a combination of high soil temperature and rapid loss of soil moisture will significantly reduce risks caused by viruses in sludge.     

Glutathione transferase mu 2 protects glioblastoma cells against aminochrome toxicity by preventing autophagy and lysosome dysfunction

U373MG cells constitutively express glutathione S-transferase mu 2 (GSTM2) and exhibit ³H-dopamine uptake, which is inhibited by 2 µM of nomifensine and 15 µM of estradiol. We generated a stable cell line (U373MGsiGST6) expressing an siRNA against GSTM2 that resulted in low GSTM2 expression (26% of wild-type U373MG cells). A significant increase in cell death was observed when U373MGsiGST6 cells were incubated with 50 µM purified aminochrome (18-fold increase) compared with wild-type cells. The incubation of U373MGsiGST6 cells with 75 µM aminochrome resulted in the formation of autophagic vacuoles containing undigested cellular components, as determined using transmission electron microscopy. A significant increase in autophagosomes was determined by measuring endogenous LC3-II, a significant decrease in cell death was observed in the presence of bafilomycin A₁, and a significant increase in cell death was observed in the presence of trehalose. A significant increase in LAMP2 immunostaining was observed, a significant decrease in bright red fluorescence of lysosomes with acridine orange was observed, and bafilomycin A₁ pretreatment reduced the loss of lysosome acidity. A significant increase in cell death was observed in the presence of lysosomal protease inhibitors. Aggregation of TUBA/α-tubulin (tubulin, α) and SQSTM1 protein accumulation were also observed. Moreover, a significant increase in the number of lipids droplets was observed compared with U373MG cells with normal expression of GSTM2. These results support the notion that GSTM2 is a protective enzyme against aminochrome toxicity in astrocytes and that aminochrome cell death in U373MGsiGST6 cells involves autophagic-lysosomal dysfunction.     

Hp1404, a New Antimicrobial Peptide from the Scorpion Heterometrus petersii

Antimicrobial peptides have attracted much interest as a novel class of antibiotics against a variety of microbes including antibiotics resistant strains. In this study, a new cationic antimicrobial peptide Hp1404 was identified from the scorpion Heterometrus petersii, which is an amphipathic α-helical peptide and has a specific inhibitory activity against gram-positive bacteria including methicillin-resistant Staphylococcus aureus. Hp1404 can penetrate the membrane of S. aureus at low concentration, and disrupts the cellular membrane directly at super high concentration. S. aureus does not develop drug resistance after multiple treatments with Hp1404 at sub MIC concentration, which is possibly associated with the antibacterial mechanism of the peptide. In addition, Hp1404 has low toxicity to both mammalian cells (HC₅₀  =  226.6 µg/mL and CC₅₀ > 100 µg/mL) and balb-c mice (Non-toxicity at 80 mg/Kg by intraperitoneal injection and LD₅₀  =  89.8 mg/Kg by intravenous injection). Interestingly, Hp1404 can improve the survival rate of the MRSA infected balb-c mice in the peritonitis model. Taken together, Hp1404 may have potential applications as an antibacterial agent.     

Endogenous Gibberellin-Like Substances in Somatic Embryos of Grape (Vitis vinifera x Vitis rupestris) in Relation to Embryogenesis and the Chilling Requirement for Subsequent Development of Mature Embryos

Endogenous gibberellin (GA)-like substances were examined in suspension cultures of somatic embryos of a hybrid grape (Vitis vinifera × Vitis rupestris) during embryogenesis, and in mature embryos chilled at 4°C, and subsequently incubated at 26°C with and without abscisic acid (ABA). The extract was separated into a nonpolar fraction (would contain GA-precursors); a fraction that would contain free GAs; and a highly H₂O-soluble fraction (would contain GA glucosyl conjugates and very polar free GAs). Quantitation after SiO₂ partition chromatography was accomplished by microdrop and immersion dwarf rice bioassays. As embryogenesis developed, the free and highly H₂O-soluble GA-like substances, expressed on a dry weight basis, decreased (however, they increased on a per embryo basis). Chilling at 4°C for 1 week greatly increased activity of free GA-like substances (per g dry weight and per embryo), it then declined over the next three weeks of chilling. Activity (per g dry weight and per embryo) in the H₂O-soluble fraction declined throughout chilling. Activity in the GA-precursor fraction, however, increased steadily with chilling (per g dry weight and per embryo). Incubation at 26°C after chilling enhanced activity in the free GA and H₂O-soluble fractions (per g dry weight and per embryo), but activity in the GA-precursor fraction dropped dramatically. Incubation at 26°C with (±) ABA after chilling prevented germination and maintained high activity for GA precursors and less polar free GAs and low activity in the polar free GA and H₂O-soluble fractions.

Kaurene and kaurenoic acid were characterized in the GA-precursor fraction of chilled embryos by gas-liquid chromatography-mass spectrometry (GLC-MS). The existence of GA₄ and GA₉ in ABA-treated, chilled embryos was also confirmed by GLC-MS.

     

Effect of Temperature on Composting of Sewage Sludge

The effect of temperature on the composting reaction of sewage sludge was investigated at 50, 60, and 70°C. The total amount of CO₂ evolved and the final conversion of volatile matter were maximum at 60°C., suggesting that the optimal temperature for composting was around 60°C. The specific CO₂ evolution rate (moles of CO₂ evolved per hour per viable cell) was maximum at 70°C. The isolated thermophilic bacterium which was dominant at 60°C but did not grow at 70°C showed that the rate of O₂ consumption measured on the agar plate at 70°C was four times higher than that at 60°C. This showed that the energy yielded from catabolism is rather uncoupled with the anabolism at 70°C in the metabolism of microorganisms indigenous in the compost. A higher respiratory quotient was observed at 70°C than at any other temperature.     

Risk factors for premature death in middle aged men

The causes of premature death and the associated risk factors were analysed in a cohort of 7935 middle aged men participating in a preventive population programme in Malmö. They were screened when aged 46-48 and then followed up for 3½-8 years. Two hundred and eighteen died, of whom 181 (83%) underwent necropsy. Three major causes of death were established: cancer in 61 (28%), deaths related to consumption of alcohol in 55 (25%), and coronary heart disease in 50 (23%).Distinctly different patterns of risk factors were found to be associated with each of the three main causes of premature death. In death due to coronary heart disease smoking (p=0·0062), serum cholesterol concentration (p=0·00014), serum triglyceride concentration (p=0·00013), systolic blood pressure (p=0·000012), and diastolic blood pressure (p=0·0021) were the strongest single determinants but diastolic blood pressure ceased to be a predictive factor in a multivariate analysis whereas all the other variables could be combined in a highly predictive logistic model. In death related to consumption of alcohol equal or even stronger associations were found for serum γ glutamyltransferase activity (p<0·0001), points scored in a questionnaire screening for alcoholism (p<0·0001), and, inversely, serum cholesterol (p=0·0046) and serum creatinine (p<0·0001) concentrations both when applied independently and when combined in a logistic model. In death due to cancer significant associations were found for serum urate concentration (p=0·023) and, inversely, serum cholesterol concentration (p=0·056-0·031).Malignant diseases and diseases related to consumption of alcohol were at least as prominent as cardiovascular disorders in causing premature death in the cohort of men studied. All three types of conditions are potentially avoidable and seem to be associated with significant and distinctive patterns of risk factors. These patterns should be used, as blood pressure and serum lipid concentrations already are, to predict the risk of premature death and indicate preventive measures.