Soil formate regulates the fungal nitrous oxide emission pathway.

Fungal activity is a major driver in the global nitrogen cycle, and mounting evidence suggests that fungal denitrification activity contributes significantly to soil emissions of the greenhouse gas nitrous oxide (N(2)O). The metabolic pathway and oxygen requirement for fungal denitrification are different from those for bacterial denitrification. We hypothesized that the soil N(2)O emission from fungi is formate and O(2) dependent and that land use and landforms could influence the proportion of N(2)O coming from fungi. Using substrate-induced respiration inhibition under anaerobic and aerobic conditions in combination with (15)N gas analysis, we found that formate and hypoxia (versus anaerobiosis) were essential for the fungal reduction of (15)N-labeled nitrate to (15)N(2)O. As much as 65% of soil-emitted N(2)O was attributable to fungi; however, this was found only in soils from water-accumulating landforms. From these results, we hypothesize that plant root exudates could affect N(2)O production from fungi via the proposed formate-dependent pathway.

Multiple transcripts of a gene for a leucine-rich repeat receptor kinase from morning glory (Ipomoea nil) originate from different TATA boxes in a tissue-specific manner.

TATA boxes are the most common regulatory elements found in the promoters of eukaryotic genes because they are associated with basal transcription initiation by RNA polymerase II. Often only a single TATA element is found in a given promoter, and tissue-, stage- and/or stimulus-specific expression occurs because the TATA box is associated with other cis -acting elements that enhance or repress transcription. We used software tools for gene analysis to assist in locating potential TATA box(es) in an AT-rich region of the promoter of a gene, inrpk1, which codes for a leucine-rich receptor protein kinase in morning glory (Ipomoea nil). Through the use of RT-PCR and various combinations of forward primers bracketing most of the promoter region we were able to define the 5'-ends of transcripts in this region. The region was then targeted for analysis by RNA Ligase-Mediated-5' Rapid Amplification of cDNA Ends (RLM-5' RACE) to identify the transcript initiation site(s). Positioning of initiation sites with respect to TATA boxes identified by gene analysis tools allowed us to identify three operational TATA elements which regulate basal transcription from this gene. Two TATA boxes were responsible for all of the inrpk1 transcripts found in leaves and cotyledons, and about 25-30% of the transcripts in roots. A third TATA box was involved only in expression in roots and accounted for the remaining 50-70% of root transcripts. RNAs expressed from this element lack two potentially functional upstream AUG codons, and may be translated more efficiently than transcripts originating from the other TATA boxes.

High resolution measurement of translucent plastic wall thicknesses by computerized tomography and neural networks.

Today there is a great deal of interest in the field of plastics design. Several methods can be employed to create plastic products such as injection molding, compression and transfer molding, and blow molding. This paper is concerned with blow molding which is a procedure employed to create hollow plastic containers such as those used to contain liquids and solids in the wholesale and retail markets. An important aspect of blow molding is the measurement of the wall thickness of semi-liquid plastic before the molding procedure has been initiated. Minimization of waste is rapidly becoming a critical consideration within the plastics community due to the cost of raw polymers. Unfortunately, it is also an extremely difficult task to measure the thickness considering the high temperatures and elasticity of the polymers in question. This paper presents initial research on a non-invasive approach for wall thickness measurements of semi-liquid plastics through the utilization of computerized tomography and neural networks. The work described here is based on simulations and on modeling data obtained through experimental means. This technique can be extended to other fields of research as well, such as those related to the development of glass and ceramic products.

DNA amplification.

The polymerase chain reaction has become a mainstream tool for the molecular biologist. The sensitivity, efficiency, and speed of this method is unparalleled for the amplification and detection of exquisitely minute quantities of nucleic acids. Through repetitive cycles of heat denaturation of samples, followed by the base pairing of primers designed to identify one DNA sequence among the cellular heterogeneity, and finally synthesis of new DNA strands identical to the target, single molecules and individual genes can be detected and subsequently characterized. This method has revolutionized the study of gene organization, structure, and expression, not to mention offering newer, faster, and more economical means for the clinical detection infectious disease. That PCR has been fruitful is undisputed; however, the method is not without shortcomings. Among the major limitations of this method are the absolute requirement for well-designed primers, the super sensitivity of this method to biological contaminants from any of a variety of sources, and subtle, though very important, inter- and intra-laboratory variations in technique.

Effects of chemical speciation in growth media on the toxicity of mercury(II).

The toxicity of metals, including mercury, is expressed differently in different media, and the addition of soluble organics to the growth medium can have a significant impact on bioassay results. Although the effect of medium composition on metal toxicity is generally attributed to its effect on metal speciation (i.e., the chemical form in which the metal occurs), the importance of individual metal-ligand species remains largely unclear. Here, we report the results of a study that investigated, both experimentally and from a modeling perspective, the effects of complex soluble organic supplements on the acute toxicity (i.e., 50% inhibitory concentration [IC50]) of mercury to a Pseudomonas fluorescens isolate in chemically well-defined synthetic growth media (M-IIX). The media consisted of a basal inorganic salts medium supplemented with glycerol (0.1%, vol/vol) and a variety of common protein hydrolysates (0.1%, vol/vol), i.e., Difco beef extract (X = B), Casamino Acids (X = C), peptone (X = P), soytone (X = S), tryptone (X = T), and yeast extract (X = Y). These were analyzed to obtain cation, anion, and amino acid profiles and the results were used to compute the aqueous speciation of Hg(II) in the media. Respirometric bioassays were performed and IC50s were calculated. Medium components varied significantly in their effects on the acute toxicity of Hg(II) to the P. fluorescens isolate. IC50s ranged from 1.48 to 14.54 micrograms of Hg ml-1, and the acute toxicity of Hg(II) in the different media decreased in the order M-IIC >> M-IIP > M-IIB >> M-IIT > M-IIS >>> M-IIY.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of C-myc and C-Ha-ras oncogene expression by cell shape.

The influence of cell shape on the expression of proto-oncogenes was examined in normal and malignant human cells that varied in their sensitivities to contact-inhibition of proliferation. Cells were constrained into varying degrees of roundness by plating onto culture surfaces coated with different concentrations of poly(2-hydroxyethyl methacrylate) (poly[HEMA]) and assayed for proliferation capacity and levels of c-myc, c-ras, c-fos, and c-fes mRNAs. Proliferation of contact-inhibited normal CUA-1 fibroblasts and the variant HT-IFNr cells was highly coupled to cell shape. As these cells became more rounded, a critical degree of roundness was reached at which proliferation ceased. In contrast, proliferation of non-contact-inhibited malignant HT-1080 cells was independent of cell shape. Northern analysis revealed that expression of c-myc and c-ras was highly sensitive to cell shape in the normal CUA-1 cells but not in the malignant HT-1080 or variant HT-IFNr cells. Levels of c-myc and c-ras mRNAs declined to nearly undetectable levels in CUA-1 cells at degrees of roundness that correlated with loss of proliferative ability. Expression of c-fos and c-fes oncogenes were independent of cell shape in all cells tested. Quantification of transcription rates by the nuclear run-off assay showed that shape modulation of c-myc and c-ras oncogene expression occurred at the transcriptional level. These data suggest that changes in cell shape can modulate expression of certain oncogenes and that these changes correlate with the cell's ability to proliferate. Moreover, inability to regulate c-myc and c-ras oncogene expression is associated with loss of shape-dependent growth controls and contact inhibition but that loss of this regulation alone is not sufficient to release cells from contact-inhibited controls.

Biotoxicity of mercury as influenced by mercury(II) speciation.

Integration of physicochemical procedures for studying mercury(II) speciation with microbiological procedures for studying the effects of mercury on bacterial growth allows evaluation of ionic factors (e.g., pH and ligand species and concentration) which affect biotoxicity. A Pseudomonas fluorescens strain capable of methylating inorganic Hg(II) was isolated from sediment samples collected at Buffalo Pound Lake in Saskatchewan, Canada. The effect of pH and ligand species on the toxic response (i.e., 50% inhibitory concentration [IC50]) of the P. fluorescens isolated to mercury were determined and related to the aqueous speciation of Hg(II). It was determined that the toxicities of different mercury salts were influenced by the nature of the co-ion. At a given pH level, mercuric acetate and mercuric nitrate yielded essentially the same IC50s; mercuric chloride, on the other hand, always produced lower IC50s. For each Hg salt, toxicity was greatest at pH 6.0 and decreased significantly (P = 0.05) at pH 7.0. Increasing the pH to 8.0 had no effect on the toxicity of mercuric acetate or mercuric nitrate but significantly (P = 0.05) reduced the toxicity of mercuric chloride. The aqueous speciation of Hg(II) in the synthetic growth medium M-IIY (a minimal salts medium amended to contain 0.1% yeast extract and 0.1% glycerol) was calculated by using the computer program GEOCHEM-PC with a modified data base. Results of the speciation calculations indicated that complexes of Hg(II) with histidine [Hg(H-HIS)HIS+ and Hg(H-HIS)2(2+)], chloride (HgCl+, HgCl2(0), HgClOH0, and HgCl3-), phosphate (HgHPO4(0), ammonia (HgNH3(2+), glycine [Hg(GLY)+], alanine [Hg(ALA)+], and hydroxyl ion (HgOH+) were the Hg species primarily responsible for toxicity in the M-IIY medium. The toxicity of mercuric nitrate at pH 8.0 was unaffected by the addition of citrate, enhanced by the addition of chloride, and reduced by the addition of cysteine. In the chloride-amended system, HgCl+, HgCl2(0), and HgClOH0 were the species primarily responsible for observed increases in toxicity. In the cysteine-amended system, formation of Hg(CYS)2(2-) was responsible for detoxification effects that were observed. The formation of Hg-citrate complexes was insignificant and had no effect on Hg toxicity.

Construction and evaluation of a potassium-selective tube-mounted membrane electrode.

A simple procedure for the rapid construction of inexpensive potassium-selective electrodes with valinomycin-based PVC membranes is described. Potassium-selective membranes were formed on the end of Parafilm- or Tygon-covered glass tubes by dipping the tubes into a mixture of PVC, valinomycin, and dioctyl sebacate dissolved in tetrahydrofuran. Small internal Ag/AgCl reference electrodes were made with silver wire and placed inside the tubes with AgCl-saturated potassium chloride solution. This procedure yields tube-mounted membrane electrodes that perform as well as commercially available potassium-selective electrodes in terms of their response characteristics and practical applications with soil extracts. Moreover, it facilitates the evaluation of membranes with different compositions, for making ion-selective electrodes.

Serotonin metabolism and platelet monoamine oxidase activity in patients with medullary carcinoma of the thyroid and pheochromocytoma.

Occasional patients with medullary carcinoma of the thyroid (Multiple Endocrine Neoplasia Type II [MEN II]) are reported to have excessive serotonin (5-HT) production from the MCT; almost all patients with metastatic MCT have elevations in plasma concentration of the amine oxidase, histaminase. The elevated 5-HT production is thought ot contribute to the troublesome diarrhea experienced by patients with MEN II. We compared the urinary excretion of 5-hydroxyindoleacetic acid (5-HIAA), the principle metabolite of 5-HT, of 33 patients with MCT with the urinary excretion of 5-HIAA in 33 control subjects. Six of the 33 MCT patients (18%) had severe diarrhea. The 5-HIAA excretion of the MCT patients did not differ from that of normal subjects. We also compared the platelet monoamine oxidase (MAO) activity of 27 MCT patients and 27 control subjects. The platelet MAO activity of the two groups did not differ. The 5-HT content and MAO activity of 6 of the MCTs was similar to normal thyroid tissue. The MAO activity of two follicular adenomas of the thyroid was greater than the MAO activity of MCTs. In contrast to the uniform elevation of plasma histaminase in patients with MCT, the platelet MAO activity is not altered and the majority of MCTs do not produce excessive amounts of 5-HT.

Plasma norepinephrine response to standing in patients with pheochromocytoma or medullary carcinoma of the thyroid.

We determined the effect of a lying/standing test on 22 patients with medullary carcinoma of the thyroid (MCT), seven patients with pheochromocytoma (six of whom also had MCT), six healthy first-degree family members of patients with MCT, and nine normal subjects who did not have a family history of MCT. The purpose of the study was to determine if the patients with MCT had an altered noradrenergic response to standing and to determine if this test would be useful in screening MCT patients for the presence of pheochromocytoma. All of the patients with MCT, as well as all of the healthy family members, had normal urinary excretion of catecholamines and their metabolites. Plasma norepinephrine (NE) concentrations, plasma cortisol levels, and blood pressure (BP) were determined during the lying/standing test. The plasma NE concentration showed the expected increase with a change in posture; the plasma cortisol concentration did not change. There was no significant difference in the plasma NE, plasma cortisol, and BP response in the four study groups. Only one of the seven patients with pheochromocytoma had a hypertensive episode in response to standing. We conclude that there is probably a normal noradrenergic response to standing in patients with MCT, and the standing/lying test, as performed in this study, is of limited value in screening for the presence of pheochromocytoma.

Alterations in plasma norepinephrine concentration during surgical resection of pheochromocytoma.

Using a sensitive and specific radioenzymatic assay, the plasma norepinephrine (NE) concentration was measured in seven patients with pheochromocytoma, one patient with bilateral adrenal medullary hyperplasia, one patient with a retroperitoneal paraganglioma, and two patients undergoing bilateral adrenalectomies for palliation of metastatic breast carcinoma. Surgical manipulation of the pheochromocytomas resulted in striking increases in plasma NE concentration with concomitant increases in blood pressure. There were either small changes or no changes in the patients' plasma NE and blood pressure during resection of the normal adrenal glands, the adrenal glands with medullary hyperplasia, or the retroperitoneal paraganglioma. Plasma dopamine-beta-hydroxylase (DBH) was measured in one patient with pheochromocytomas and the patient with medullary hyperplasia. There was no change in plasma DBH in either patient, supporting the concept that exocytosis is not the primary mechanism for catecholamine secretion from pheochromocytomas. It was also noted that enflurane is an excellent general anesthetic for the resection of pheochromocytomas, and that sodium nitroprusside (rather than phentolamine) may be the agent of choice for the management of the hypertensive episodes that occur during surgical manipulation of pheochromocytomas.

Plasma and tumor dopamine-beta-hydroxylase activity in patients with familial pheochromocytomas.

The majority of patients with sporadic pheochromocytomas (pheos) have been noted to have normal plasma dopamine-beta-hydroxylase (DBH) activity. We determined the activity of DBH in the plasma of 8 patients with pheos, secondary to multiple endocrine neoplasia Type 2 (MEN II) (medullary carcinoma of the thyroid [MCT], pheochromocytoma(s), and parathyroid hyperplasia). We also determined the activity of DBH in the pheos of six of the patients. Three of the eight patients (38%) had elevated plasma DBH in the preoperative period. After surgical resection of their pheos, the plasma DBH of two of the subjects, in whom it could be measured, returned to normal. Although two of the other patients had bilateral pheos containing large amounts of DBH and norepinephrine (NE), their plasma DBH was normal in the preoperative and postoperative periods. Despite elevated urinary vanillylmandelic acid (VMA) excretion in all eight patients, their homovanillic acid (HVA) excretion was normal. VMA reflects NE plus epinephrine (E) excretion, while HVA reflects dopamine (DA) excretion. We conclude that the majority of patients with pheos, associated with MEN II, have normal plasma DBH activity.

Medullary thyroid carcinoma: relationship of method of diagnosis to pathologic staging.

Medullary thyroid carcinoma (MTC) develops in virtually all patients affected with multiple endocrine neoplasia type II (MEN II), a disease inherited as an autosomal dominant trait. The thyroid tumor cells secrete calcitonin (CT) and the detection of elevated plasma levels (>300 pg/ml) of this hormone in MEN II kindred members strongly suggests the presence of MTC even though it may not be evident clinically. Intravenously administered calcium ion (Ca(++)) and pentagastrin (Pg) are potent CT secretagogues which are of particular value in establishing the early diagnosis of MTC. In evaluating seven kindreds with MEN II, we detected 90 patients with MTC. Depending on the method of diagnosis, they could be divided into three categories: Group 1; patients with no clinical evidence of MTC whose undetectable basal plasma calcitonin levels became elevated following intravenous Ca(++) or Pg, Group II; patients with no clinical evidence of MTC who had elevated basal plasma CT levels, and Group III; patients with clinically evident MTC. At the time of diagnosis of MTC, the patients in Group I were younger (20.5 +/- 1.9 years) than the patients in Group II (32.5 +/- 4.7 years, p < 0.005) and Group III (34.3 +/- 2.0, p < 0.00005). The incidence of residual MTC, as indicated by an elevated plasma CT level following provocative testing postoperatively, was less frequent in patients diagnosed biochemically ([6/34]; Group I, 4/26 and Group II, 2/8) than in those diagnosed clinically (Group III, 15/26, p < 0.002). Furthermore, regional nodes were involved less often in patients diagnosed biochemically ([5/28]; Group I, 2/22 and Group II, 3/6) than in those diagnosed clinically (Group III, 15/24, p < 0.02). Distant metastases were only evident in Group III patients. Patients with MEN II who had the diagnosis of MTC established biochemically rather than clinically, had a more favorable pathological stage of disease at the time of thyroidectomy. This was especially true if the biochemical diagnosis had been by provocative testing.

The detection of elevated plasma levels of carcinoembryonic antigen in patients with suspected or established medullary thyroid carcinoma.

Plasma levels of carcinoembryonic antigen (CEA) and calcitonin (CT) were measured in 35 normal control subjects and in 37 patients with suspected or established medullary thyroid carcinoma (MTC). None of the normal control subjects had elevated basal plasma levels of either CEA (greater than 5 ng/ml) or CT (greater than 0.25 ng/ml). However, of the 37 patients with suspected or established MTC, 23 (62%) had elevated basal plasma levels of CEA (range 9.8--7,000 ng/ml) and 27 (73%) had elevated basal plasma CT values (range 0.30--500 ng/ml). Generally, patients with clinically apparent MTC, either primary or metastatic, had higher plasma CEA and CT levels than those with occult disease. A positive correlation was found (r = 0.785, p less than 0.01) when comparing basal plasma CEA and stimulated plasma CT levels in 20 patients. A marked increase above the basal plasma level of CT but not CEA was detected in each of six MTC patients following intravenous calcium or pentagastrin. These data demonstrate that basal plasma levels of CEA and CT were increased in a large percentage of patients with MTC. Plasma calcitonin levels unlike plasma CEA levels were more often elevated in patients with occult disease and were increased above basal following the intravenous administration of either calcium gluconate or pentagastrin.

Provocative agents and the diagnosis of medullary carcinoma of the thyroid gland.

Twenty-six patients with known or suspected medullary thyroid carcinoma (MTC) and 21 normal control subjects were tested intravenously on four separate days with calcium gluconate (CG), 2 mg Ca(++)/kg/1 min.; pentagastrin (P), 0.5 ug/kg/ 5 sec.; calcium chloride (CC), 3 mg Ca(++)/kg/10 min.; and a combination of calcium gluconate and pentagastrin (CG + P). Calcitonin (CT) levels were determined by radioimmunoassay on plasma collected before and immediately following each test infusion. In none of the 21 control subjects was there a clear increase in CT above 200 pg/ml following any of the four provocative tests. Conversely, in all 26 patients with known or suspected MTC, plasma CT levels were markedly increased (>300 pg/ml) following the combined infusion of CG + P. The peak CT response was greater with CG + P than with a) CG alone (22 of 24 patients, p < 0.002), b) P alone (25 of 26 patients, p < 0.002), or c) CC alone (17 of 17 patients, p < 0.002). Of 12 MTC patients with undetectable basal calcitonin levels, all had peak responses greater than 300 pg/ml following CG + P, whereas such responses occurred less often following CG alone (8 of 12) or P alone (8 of 12). The results demonstrate that the combined administration of pentagastrin and calcium gluconate constitutes a more effective and reliable stimulus for CT secretion from MTC cells than the use of either agent alone, and appears the most useful single screening test for the detection of occult MTC.