Inhibition of atrazine degradation by cyanazine and exogenous nitrogen in bacterial isolate M91-3

A variety of s-triazine herbicides and nitrogen fertilizers frequently occur as co-contaminants at pesticide manufacturing and distribution facilities. The degradation of atrazine and cyanazine by the bacterial isolate M91-3 was investigated in washed-cell suspensions and crude cellular extracts. Cyanazine competitively inhibited atrazine degradation. The maximum atrazine degradation rate (V _max) was 41 times higher and the half-saturation constant for the inhibitor (K _i) was 1.3 times higher in the crude cellular extract than in the washed-cell suspension, suggesting that cellular uptake influenced degradation of the s-triazines. Cultures that had received prior exposure to atrazine and simazine exhibited comparable atrazine degradation rates, while cells exposed to cyanazine, propazine, ametryne, cyanuric acid, 2-hydroxyatrazine, biuret, and urea exhibited a lack of atrazine-degradative activity. Growth in the presence of exogenous inorganic nitrogen inhibited subsequent atrazine-degradative activity in washed-cell suspensions, suggesting that regulation of s-triazine and nitrogen metabolism are linked in this bacterial isolate. These findings have significant implications for the environmental fate of s-triazines in agricultural settings since these herbicides are frequently applied to soils receiving N fertilizers. Furthermore, these results suggest that bioremediation of s-triazine-contaminated sites (common at pesticide distribution facilities in the cornbelt) may be inhibited by the presence of N fertilizers that occur as co-contaminants. Received: 3 March 1998 / Received revision: 24 September 1998 / Accepted: 11 October 1998     

Reference genes for quantitative reverse transcription-polymerase chain reaction expression studies in wild and cultivated peanut

Background

Molecular genetic studies on rare tumour entities, such as bone tumours, often require the use of decalcified, formalin-fixed, paraffin-embedded tissue (dFFPE) samples. Regardless of which decalcification procedure is used, this introduces a vast breakdown of DNA that precludes the possibility of further molecular genetic testing. We set out to establish a robust protocol that would overcome these intrinsic hurdles for bone tumour research.

Findings

The goal of our study was to establish a protocol, using a modified DNA isolation procedure and quality controls, to select decalcified samples suitable for array-CGH testing. Archival paraffin blocks were obtained from 9 different pathology departments throughout Europe, using different fixation, embedding and decalcification procedures, in order to preclude a bias for certain lab protocols. Isolated DNA samples were subjected to direct chemical labelling and enzymatic labelling systems and were hybridised on a high resolution oligonucleotide chip containing 44,000 reporter elements. Genomic alterations (gains and losses) were readily detected in most of the samples analysed. For example, both homozygous deletions of 0.6 Mb and high level of amplifications of 0.7 Mb were identified.

Conclusions

We established a robust protocol for molecular genetic testing of dFFPE derived DNA, irrespective of fixation, decalcification or sample type used. This approach may greatly facilitate further genetic testing on rare tumour entities where archival decalcified, formalin fixed samples are the only source.     

14C UPTAKE BY THE MARINE ANGIOSPERM PHYLLOSPADIX SCOULERP

Rhizomes and attached leaves of Phyllospadix scouleri Hook, were collected in the intertidal zone along the central California coast and exposed to a solution of NaH¹⁴CO₃ in seawater under controlled laboratory conditions. Over a 90-min period roots and rhizomes absorbed very little ¹⁴C compared to leaves. Translocation during that time was minor. Plants pretreated with the photosynthetic inhibitor DCMU showed no ¹⁴C uptake, indicating that under normal circumstances the carbon which is absorbed by leaves is fixed and accumulates as photosynthate. The rate of gross photosynthesis was about 13 mg CO₂ g dry wt⁻¹ hr⁻¹. Gross photosynthesis of wet leaves exposed to ¹⁴CO₂ in air was significantly less than leaves exposed to NaH¹⁴CO₃. The effect of a leaf-grazing limpet (Notoacmea paleacea) on leaf anatomy and ¹⁴C uptake is discussed.     

Viral and bacterial community responses to stimulated Fe(III)-bioreduction during simulated subsurface bioremediation

The delivery of fermentable substrate(s) to subsurface environments stimulates Fe(III)-bioreduction and achieves detoxification of organic/inorganic contaminants. Although, much research has been conducted on the microbiology of such engineered systems at lab and field scales, little attention has been given to the phage-host interactions and virus community dynamics in these environments. The objective was to determine the responses of soil bacterial communities and viral assemblages to stimulated anaerobic Fe(III)-bioreduction following electron donor (e.g. acetate) addition. Microbial communities, including viral assemblages, were investigated after 60 days of Fe(III)-bioreduction in laboratory-scale columns continuously fed with acetate-amended artificial groundwater. Viral abundances were greatest in the influent section and decreased along the flow path. Acetate availability was important in influencing bacterial diversity, microbial interactions and viral abundance and community composition. The impact of acetate addition was most evident in the influent section of the columns. The increased relative abundance of Fe(III)-reducing bacteria coincided with an increase in viral abundance in areas of the columns exhibiting the most Fe(III) reduction. The genetic composition of viruses in these column sections also differed from the control column and distal sections of acetate-treated columns suggesting viral communities responded to biostimulated Fe(III)-bioreduction.     

Quantification of angiogenesis in estrogen receptor-positive and negative breast carcinoma

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.

     

Spatiotemporal changes in Ha-ras p21 expression through the hepatocyte cell cycle during liver regeneration.

The protein product of the ras oncogene, Ha-ras (p21), is thought to be an important regulator of cell growth. The cytoplasmic relocalization of p21 in the cell during the cell cycle suggests a transient signaling role for this protein in association with its signal transduction function. Because of the importance of this role we examined spatial patterns in vivo of p21 expression at the protein and mRNA levels in hepatocytes during compensatory growth in rat liver following partial hepatectomy. A low level of p21 was immunolocalized on the cytoplasmic membrane of nonregenerating hepatocytes. The level of hepatic p21 increased significantly and without spatial restriction within the liver from 36 to 60 hr after partial hepatectomy (PH). p21 was localized in the cytoplasm of dividing hepatocytes and on the hepatic cytoplasmic membrane. The elevated p21 level decreased and was found mainly on hepatocyte plasma membranes by 96 hr after PH. Immunogold electron microscopy showed p21 localized over mitochondrial membranes and nuclei in nondividing regenerating hepatocytes. Approximately 50% of nonregenerating hepatocytes show nuclear localization of p21. This percentage changes with time following PH. The decrease in nuclear localization was accompanied with an increase in the low number of hepatocytes which demonstrated cytoplasmic localization in nondividing hepatocytes in regenerating liver. Flow cytometric analysis revealed a significant increase of p21 at 36 hr after PH which was 12 hr after the initial induction of ras mRNA. ras mRNA level increased 1.5-fold at 24 hr after PH and a maximum twofold induction was observed at 48 hr. Cell-cycle analysis of regenerating hepatocytes indicated a synchronized first peak of cell division 36-40 hr after PH. Dual parameter flow cytometry revealed that the level of p21 in hepatocytes in S phase and G2/M phase of the cell cycle was significantly higher than that in G0/G1 phase during regeneration. These findings suggest that p21 is important for the progression of regenerating hepatocytes to S phase and then to G2/M phase.     

Nutrition and fetal brain maturation : II. Impact of maternal starvation on changing levels of acetylcholinesterase and enolase in vitro

The impact of maternal starvation during Days 17-20 of gestation was examined in 20-day fetal rat brain tissue cultured for 6 days in MEM and 10% adult rat serum. Acetylcholinesterase (AChE) activities were consistently greater in fetal brain cell cultures from starved mothers. When fetal tissues from starved mothers were continuously exposed to 72-h fasted serum, AChE activities increased from 1.03 +/- 0.14 to 1.59 +/- 0.21 mumol/h/mg protein (P less than 0.001). In fetal tissues from fed mothers, lower AChE activities were increased from 0.78 +/- 0.09 to 1.04 +/- 0.07 mumol/h/mg protein (P less than 0.05) when 72-h fasted serum was used to replace the fed serum during incubation. When fetal brain cell cultures from fed mothers were exposed for 6 days to graded concentrations of fed serum (2.5-15%), the activities of AChE fell reciprocally from 1.34 +/- 0.10 to 0.82 +/- 0.12 mumol/h/mg protein (P less than 0.05). The levels of AChE activity in tissues exposed to fasted serum were consistently greater, but fell similarly from 1.62 +/- 0.10 to 0.97 +/- 14 mumol/h/mg protein (P less than 0.01), when serum concentrations were increased from 2.5 to 15%. AChE activities were 30% higher in tissues incubated with cycloheximide 10(-3) M (P less than 0.02). Unlike AChE, fetal brain enolase activities were unaffected by maternal starvation. In fetal brain cell cultures from fed mothers, enolase fell from 1.85 +/- 0.10 to 1.37 +/- 0.12 mumol/min/mg protein following exposure to fasted instead of fed serum (P less than 0.02). In fetal cultures from starved mothers, enolase activities were depressed similarly from 1.76 +/- 0.08 to 1.41 +/- 0.09 mumol/min/mg protein when fasted replaced fed serum (P less than 0.02). Thus, the fetal brain cell cultures appear to maintain enzymatic realignments imposed by maternal starvation for at least 6 days. In addition, serum from fasted animals has significant growth inhibiting properties manifested by heightened activities of AChE and lower activities of enolase.     

Sampling Natural Viral Communities from Soil for Culture-Independent Analyses

An essential first step in investigations of viruses in soil is the evaluation of viral recovery methods suitable for subsequent culture-independent analyses. In this study, four elution buffers (10% beef extract, 250 mM glycine buffer, 10 mM sodium pyrophosphate, and 1% potassium citrate) and three enumeration techniques (plaque assay, epifluorescence microscopy [EFM], and transmission electron microscopy [TEM]) were compared to determine the best method of extracting autochthonous bacteriophages from two Delaware agricultural soils. Beef extract and glycine buffer were the most effective in eluting viable phages inoculated into soils (up to 29% recovery); however, extraction efficiency varied significantly with phage strain. Potassium citrate eluted the highest numbers of virus-like particles from both soils based on enumerations by EFM (mean, 5.3 × 10⁸ g of dry soil⁻¹), but specific soil-eluant combinations posed significant problems to enumeration by EFM. Observations of virus-like particles under TEM gave confidence that the particles were, in fact, phages, but TEM enumerations yielded measurements of phage abundance (mean, 1.5×10⁸ g of dry soil⁻¹) that were about five times lower. Clearly, the measurement of phage abundance in soils varies with both the extraction and enumeration methodology; thus, it is important to assess multiple extraction and enumeration approaches prior to undertaking ecological studies of phages in a particular soil.     

PHYSIOLOGICAL CONTROL OF CHAMISE SHOOT GROWTH AFTER FIRE

Five controlled burns (ca. 2 ha each) were conducted in the Coast Range of northern California near Hopland, California, between November 18, 1975 and September 15, 1976 to determine the effect of shrub phenology on the sprouting response of chamise (Adenostoma fasciculatum H&A) following fire. The time of burn had little effect on the amount of shoot growth that occurred after fire, although the pattern of growth was altered. Shrubs burned on June 22 or August 2, 1976 grew continually until August 1977, while unburned shrubs or those burned at other times ceased seasonal growth during the first summer after fire. Neither the amount nor the pattern of shoot growth was influenced by the shrub water status before the fire occurred. The relationship between chamise shoot growth after fire, ¹⁴CO₂ fixation, xylem sap tension, and air temperature was also studied. The growth rate of regrowing shrubs was highly correlated with ¹⁴CO₂ fixation prior to the growth measurement. Water status (pre-dawn xylem sap tension) had a negative correlation with ¹⁴CO₂ fixation. The abundance of carbohydrate at the shoot apex is believed to influence the seasonal pattern and rate of chamise shoot growth following fire.