Thermally-induced structural and chemical alteration of organic-walled microfossils: an experimental approach to understanding fossil preservation in metasediments.

The identification and confirmation of bona fide Archean-Paleoproterozoic microfossils can prove to be a challenging task, further compounded by diagenetic and metamorphic histories. While structures of likely biological origin are not uncommon in Precambrian rocks, the search for early fossil life has been disproportionately focused on lesser thermally altered rocks, typically greenschist or lower-grade metamorphism. Recently, however, an increasing number of inferred micro- and macrofossils have been reported from higher-grade metasediments, prompting us to experimentally test and quantify the preservability of organic-walled microfossils over varying durations of controlled heating and under two differing redox conditions. Because of their relatively low-intensity natural thermal alteration, acritarchs from the Mesoproterozoic Ruyang Group were chosen as subjects for experimental heating at approximately 500°C, with durations ranging from 1 to 250 days and in both oxic (normal present day conditions) and anoxic conditions. Upon extraction, the opacity, reflectivity, color, microchemistry, and microstructures of the heated acritarchs were characterized using optic microscopy, scanning electron microscopy, Raman spectroscopy, and X-ray photoelectron spectroscopy. The results differ for acritarchs prepared under oxic vs. anoxic conditions, with the anoxic replicates surviving experimental heating longer and retaining biological morphologies better, despite an increasing degree of carbonization with continuous heating. Conversely, the oxic replicates show aggressive degradation. In conjunction with fossils from high-grade metasediments, our data illustrate the preservational potential of organic-walled microfossils subjected to metamorphism in reducing conditions, offer insights into the search for microfossils in metasediments, and help to elucidate the influence of time on the carbonization/graphitization processes during thermal alteration.

Occupational fatalities due to animal-related events.

OBJECTIVE: To better understand the extent of animal-related fatalities in the workplace. METHODS: This study utilized Census of Fatal Occupational Injuries files from the US Department of Labor for the years 1992-1997 to describe the events surrounding human workplace fatalities associated with animals. RESULTS: During the 6-year time period, 350 workplace deaths could be associated with an animal-related event. Cattle and horses were the animals primarily involved, and workers in the agricultural industry experienced the majority of events. Many deaths involved transportation events, either direct collision with the animal or highway crashes trying to avoid collision with an animal. Exotic animals, primarily elephants and tigers, were responsible for a few deaths. A small number of workers died of a zoonotic infection. CONCLUSIONS: We found that approximately 1% of workplace fatalities are associated with an animal-related event. Methods to decrease the frequency of an animal injury are suggested.

Pet dogs as sentinels for environmental contamination.

The presence of environmental contaminants in air, water and food may pose significant health risks to the exposed human population. However, problems associated with assessing chronic exposure to low doses of environmental chemicals, multiple exposure routes, diseases with long latency periods, and non-specific health outcomes make it difficult to conduct the appropriate human epidemiologic studies. It may be useful to complement human epidemiology with animal studies. Animals monitored or evaluated in situ for the appropriate suite of endpoints can provide information about both exposure levels and potential adverse health effects. Animals have served as sentinel indicators for health effects associated with a number of environmental exposures, including pesticides and asbestos. Pet dogs may be particularly valuable sentinels because they share the human environment. In addition, dogs respond to many toxic insults in ways analogous to humans, they have physiologically compressed life spans, and they are free from some important lifestyle risk factors for disease. An example of how pet dogs may be used as sentinels for potential human health hazards involves a study of the genotoxic effects resulting from exposure to a mixture of chemicals from nearby Superfund sites. We conducted a cross-sectional study of exposed dogs (living in the community with the Superfund sites) and controls (living in a nearby community). The pet owners completed a questionnaire, and we collected a blood sample from each dog. The blood samples were analyzed for standard clinical parameters and assays for possible genotoxic effects (peripheral blood lymphocyte micronucleus frequency and lymphocyte subtyping). Pet dogs living near the Superfund sites had a higher micronucleus frequency than control animals, suggesting that the dogs may have been exposed to environmental contaminants from these sites.

Sequence and analysis of chromosome 1 of the plant Arabidopsis thaliana.

The genome of the flowering plant Arabidopsis thaliana has five chromosomes. Here we report the sequence of the largest, chromosome 1, in two contigs of around 14.2 and 14.6 megabases. The contigs extend from the telomeres to the centromeric borders, regions rich in transposons, retrotransposons and repetitive elements such as the 180-base-pair repeat. The chromosome represents 25% of the genome and contains about 6,850 open reading frames, 236 transfer RNAs (tRNAs) and 12 small nuclear RNAs. There are two clusters of tRNA genes at different places on the chromosome. One consists of 27 tRNA(Pro) genes and the other contains 27 tandem repeats of tRNA(Tyr)-tRNA(Tyr)-tRNA(Ser) genes. Chromosome 1 contains about 300 gene families with clustered duplications. There are also many repeat elements, representing 8% of the sequence.

Attenuation of histamine-induced endothelial permeability responses after pacing-induced heart failure: role for endogenous catecholamines.

OBJECTIVE: After congestive heart failure (CHF), lung endothelial permeability responses to a number of perturbations, including acute barotrauma, angiotensin II, and thapsigargin are blunted. Our hypothesis was that similar attenuation of permeability responses occurs in peripheral vascular beds after CHF. We compared peripheral microvascular permeability responses to the autacoid histamine in control dogs and in dogs paced to heart failure (245 bpm for approximately 36 days). Since catecholamines attenuate autacoid-induced increases in microvascular permeability in skin and muscle in normal animals, we also tested whether the known elevation in catecholamines in CHF was involved in any downregulation of permeability responses in this group. METHODS: Control and paced dogs were anesthetized, intubated, and ventilated, and a hindpaw lymphatic cannulated. The reflection coefficient for total proteins (sigma) was measured at baseline and during one-hour, local intra-arterial histamine infusion. RESULTS: In controls, sigma fell from 0.83 +/- 0.02 to 0.73 +/- 0.04 after histamine (p < 0.05), while in the paced group sigma was no different from that at baseline (0.77 +/- 0.02). To test whether this difference was due to endogenous catecholamines, dogs were pretreated with propranolol (controls only) or the specific beta 2-antagonist ICI 118,551 prior to histamine infusion. After beta-blockade, histamine significantly reduced sigma in both control (0.83 +/- 0.01 to 0.55 +/- 0.05) and paced (0.83 +/- 0.01 to 0.57 +/- 0.07) groups (p < 0.05). CONCLUSION: We conclude that endogenous catecholamines, acting via beta 2-adrenergic receptors, attenuate the permeability response to histamine in pacing-induced heart failure.

The interaction between rac1 and its guanine nucleotide dissociation inhibitor (GDI), monitored by a single fluorescent coumarin attached to GDI.

The interaction of rac with guanine nucleotide dissociation inhibitor protein (rhoGDI) is described, using GDI fluorescently labeled on its single cysteine with N-[2-(1-maleimidyl)ethyl]-7-diethylaminocoumarin-3-carboxamide (MDCC). The labeled GDI shows a 70% decrease in fluorescence emission on binding geranylgeranylated rac1.GDP and has an affinity for rac1 within a factor of 2 of the unlabeled GDI. The labeled GDI was used to determine the kinetic mechanism of the interaction by measuring the association and dissociation in real time. The kinetics are interpreted in terms of a two-step mechanism: binding of rac to GDI and then a conformational change of the complex with an overall dissociation constant of 0.4 nM. The conformational change has a rate constant of 7.3 s-1 (pH 7.5, 30 degrees C), and the reverse has a rate constant of 1.4 x 10(-)3 s-1. To overcome difficulties inherent in using and manipulating lipid-modified rac, we also used a combination of unmodified rac1, expressed in Escherichia coli and produced with C-terminal truncation (thus lacking the cysteine that is the site of lipid attachment), and farnesylated C-terminal peptide. This combination can mimic geranylgeranylated rac1, producing a complex with the coumarin-labeled GDI, and was used to examine the relative importance of different regions of rac1 in interaction with GDI.

Mechanism of inorganic phosphate interaction with phosphate binding protein from Escherichia coli.

The mechanism of Pi interaction with phosphate binding protein of Escherichia coli has been investigated using the A197C mutant protein labeled with a coumarin fluorophore (MDCC-PBP), which gives a fluorescence change on binding Pi. A pure preparation of MDCC-PBP was obtained, in which the only significant inhomogeneity is the presence of equal amounts of two diastereoisomers due to the chiral center formed on reaction of the cysteine with the maleimide. These diastereoisomers could not be separated, but Pi binding data suggest that they differ in affinity and fluorescence change. When Pi binds to MDCC-PBP, the fluorescence quantum yield increases 8-fold and the fluorescence intensity at 465 nm increases 13-fold. The kinetics of Pi binding show saturation of the rate at high Pi concentrations, and this together with other information suggests a two-step mechanism with the fluorescence change after binding, concomitant with a conformational change of the protein that closes the cleft containing the Pi binding site. Cleft closure has a rate constant of 317 s-1 (pH 7.0, 5 degrees C), and opening has a rate constant of 4.5 s-1. The fluorescence increase is likely to arise from a change in the hydrophobic environment during this closure as the steady state fluorescence emission (lambdamax and intensity) on Pi binding is mimicked by the addition of ethanol to aqueous solutions of an MDCC-thiol adduct. Fluorescence lifetimes in the absence and presence of Pi were 0.3 and 2.4 ns, respectively, consistent with the change in quantum yield. The rotational correlation time of the coumarin increases only 2-fold from 15 to 26 ns on binding Pi as measured by time-resolved polarization, consistent with the main rotation being determined by the protein even in the open conformation, but with greater local motion. Circular dichroism of the coumarin induced by the protein is weak in the absence of Pi and increases strongly upon saturation by Pi. These data are also consistent with an open to closed conformational model.

Purine nucleoside phosphorylase: its use in a spectroscopic assay for inorganic phosphate and for removing inorganic phosphate with the aid of phosphodeoxyribomutase.

The kinetics of the phosphorolysis of 7-methylated guanosine analogues catalyzed by purine nucleoside phosphorylase has been analyzed to understand the use of this system as a "Pi mop" to remove Pi from solutions and as a spectroscopic assay for Pi at micromolar concentrations. An expression system was developed for the phosphorylase from Escherichia coli: this protein (subunit molecular mass 26 kDa) and one from a commercial source (29 kDa) were used in this study. Rates of >50 s-1 were obtained for the phosphorolysis at 30 degrees C, so that when the phosphorylase is coupled to the phosphatase being studied, rates of Pi release from the phosphatase can be measured close to this rate. The kinetic mechanism appears to obey the Michaelis-Menten model in the steady state with the bond cleavage rate limiting. Slow hydrolysis of ribose-1-phosphate to Pi catalyzed by the phosphorylase limits the efficiency of the Pi mop. To overcome this, phosphodeoxyribomutase was used to catalyze the conversion of ribose-1-phosphate to ribose-5-phosphate, enabling the Pi mop to remove large amounts of Pi quantitatively. Acyclovir diphosphate provides a simple method to switch off the Pi mop as it is a tight inhibitor (Kd 12 nM) of purine nucleoside phosphorylase.

Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase.

A probe has been developed that can rapidly measure micromolar concentrations of inorganic phosphate (Pi), in particular to follow the release of Pi in real time from enzymes such as phosphatases. Its application is described to investigate the mechanism of actomyosin subfragment 1 ATPase. The probe uses the A197C mutant of Escherichia coli phosphate binding protein (PBP), generated by oligonucleotide-directed mutagenesis. A new fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC), was attached to the single cysteine to produce the reporter molecule that was purified free of unlabeled protein and unattached MDCC. The labeled protein has an excitation maximum at 425 nm and emission maximum at 474 nm in the absence of Pi, shifting to 464 nm with a 5.2-fold increase in fluorescence (lambda max/lambda max) when complexed with Pi at pH 7.0, low ionic strength, 22 degrees C. The fluorescence increase is not much altered by change to pH 8 or by increase in ionic strength to 1 M. Pi binds tightly (Kd approximately 0.1 microM) and rapidly (1.36 x 10(8) M-1 s-1) and the dissociation rate constant is 21 s-1, at pH 7.0, low ionic strength, 22 degrees C. A variety of phosphate esters were tested to investigate the specificity of the MDCC-PBP and none gave a significant fluorescence increase at 100 microM or higher concentration. ATP weakly inhibited the Pi-induced fluorescence change, indicating that it binds at least 3000-fold weaker than Pi. Because Pi is a widespread contaminant, the probe is used in conjunction with a "Pi mop", consisting of 7-methylguanosine and purine nucleoside phosphorylase, to remove free Pi from solutions by its conversion to ribose 1-phosphate. Because the equilibrium constant of this reaction is > 100, free Pi can be reduced below 0.1 microM. The probe was used to measure the rate of Pi release during a single turnover of ATP hydrolysis with actomyosin subfragment 1 from rabbit skeletal muscle, to determine to what extent Pi release contributes to the rate limitation of this ATPase. Using a stopped-flow apparatus, a small lag prior to rapid Pi release was detected at pH 7.0, low ionic strength, between 5 and 22 degrees C at both high and low [ATP]. For measurements of a single turnover at low [ATP], the observed rate increased with [actin], showing saturation with a Km with respect to actin of 26 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Substitutional and insertional RNA editing of the cytochrome c oxidase subunit 1 mRNA of Physarum polycephalum.

The term RNA editing encompasses two types of specific alterations in the coding potential of RNA molecules: base substitution and the insertion (or deletion) of nucleotides. Such changes in RNA sequence can have profound effects on gene expression, and, indeed, most genes in the mitochondria of plants, trypanosomatids, and Physarum appear to require editing for their expression. We describe here the first instance of the utilization of both types of RNA editing in the processing of a single mRNA, that of the mitochondrially encoded cytochrome oxidase subunit I of the acellular slime mold, Physarum polycephalum. Editing of this mRNA includes the insertion of cytidine, guanosine, and uridine residues, as well as the apparent conversion of cytidines to uridines. No edited version of this gene was detected in Physarum DNA, and amino acid alignments suggest that both types of RNA editing are required to produce a functional protein.

Interaction of GTPase-activating protein with p21ras, measured using a continuous assay for inorganic phosphate release.

The mechanism of GTPase-activating protein (GAP) activation of p21ras GTP hydrolysis has been investigated by measuring the kinetics of release of Pi during the hydrolysis. The measurement uses a continuous spectroscopic assay for Pi, based on a guanosine analogue, 2-amino-6-mercapto-7-methylpurine ribonucleoside, as substrate for purine nucleoside phosphorylase [Webb, M.R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4884-4887]. This phosphorolysis gives an absorbance increase at 360 nm, so that when the reaction is coupled to GTP hydrolysis, the change in absorbance gives the total amount of Pi released from the p21ras. The rate of the absorbance increase gives the GTPase activity. This provides a non-radioactive method of determining p21ras concentration and GAP activity. It was used to determine the interaction of GAP with wild-type p21ras and two mutants (Leu-61/Ser-186 and Asp-12), all in the GTP (or guanosine 5'-[ beta gamma-imido]triphosphate) form. The Leu-61/Ser-186 mutant binds 10-fold tighter than does the wild-type protein. The Asp-12 mutant binds to GAP with the same affinity as the wild-type protein. A novel GTPase activity was characterized whereby the EDTA-induced nucleotide release and GAP-activated cleavage of bound GTP leads to steady-state turnover of GTP hydrolysis. An assay for GAP is described based on this activity.

Nitrogenase of Klebsiella pneumoniae. Reversibility of the reductant-independent MgATP-cleavage reaction is shown by MgADP-catalysed phosphate/water oxygen exchange.

The steady-state kinetics of reductant-independent ATP hydrolysis by Klebsiella pneumoniae nitrogenase at 23 degrees C at pH 7.4 were determined as a function of component protein ratio (optimal at an oxidized Fe protein/MoFe protein ratio of 3:1) and MgATP concentration (Km 400 microM). Competitive inhibition was observed for MgADP (Ki 145 microM), [beta gamma-methylene]ATP (Mgp[CH2]ppA) (Ki 115 microM), [beta gamma-monofluoromethylene]ATP (Mgp[CHF]ppA) (Ki 53 microM) and [beta gamma-difluoromethylene]ATP (Mgp[CF2]ppA) (Ki 160 microM). The tighter binding of MgADP to free oxidized Fe protein (KD less than 10 microM) than to the oxidized Fe protein-MoFe protein complex (Ki 145 microM) is proposed as the driving force that induces rate-limiting protein dissociation in the catalytic cycle of nitrogenase. The reversible nature of the reductant-independent MgATP-cleavage reaction was demonstrated by an MgADP-induced enhancement of the rate of the phosphate/water oxygen exchange reaction with 18O-labelled phosphate ion. This enhancement, like the reductant-independent ATPase reaction, only occurred with the complex formed by oxidized Fe protein and MoFe protein and not with the individual proteins. The results are discussed in terms of the mechanism of ATP hydrolysis by nitrogenase and other systems involving protein-protein interactions.

Kinetics of ATP release and Pi binding during the ATPase cycle of lethocerus flight muscle fibres, using phosphate-water oxygen exchange.

Rate constants have been obtained using oxygen isotope exchange techniques for steps controlling ATP release and Pi binding in the ATPase cycle of insect flight muscle fibres from the giant waterbug Lethecerus. The new exchange data for Pi binding and ATP release are compatible with a model developed previously in which only the rate constants controlling Pi and ATP release change during fibre activation. Phosphate-water oxygen exchange occurs into ATP remaining after partial hydrolysis by chemically skinned fibres in (18O) water. For fully activated fibres, the results are compatible with a single set of rate constants controlling this exchange and give a rate constant for ATP release of 1 s-1 (21 degrees C, pH 7.0 I = 120 mM). Oxygen exchange also occurs between (18O4)Pi in the medium and water during ATP hydrolysis. There is a strong correlation between the measured rate constant of exchange and the value of keat for the ATPase activity at different levels of activation. For fibres fully activated by oscillation or strain, the rate constant for Pi binding to an actomyosin. ADP state is greater than 960 M-1 s-1.

Maintenance of DNA methylation level in SV40-infected human fibroblasts during their in vitro limited proliferative life span.

Methylation level as expressed by the molar ratio of 5-methylcytosine content to the combined content of cytosine and 5-methylcytosine was determined by HPLC and uv adsorption of cellular DNA extracted from SV40-infected and pretransformed MRC-5 human diploid fibroblasts (HDFs) during their limited in vitro life span. The level decreased slightly during early passages, and then was maintained within a certain range in the subsequent pretransformed stage of serial passages. When HDFs were treated with 5-aza-2'-deoxycytidine (5-aza-CdR) at an effective concentration shortly after the SV40 infection, the level decreased and then increased or was maintained again within a certain range in the subsequent pretransformed state. The proliferative life span potential of SV40-infected HDFs was not significantly decreased by the 5-aza-CdR treatment. These results are in contrast to the established observations for uninfected HDFs, that methylation level decreases during serial passages, and that, after treatment with 5-aza-CdR, the level, as well as the proliferative life span, is decreased in comparison to untreated populations. These results show that SV40-infected pretransformed HDFs are in an intermediate state between normal finite growth and an established permanent line, in that they retain limited in vitro cell proliferation, while acquiring the ability to maintain methylation levels.

Creatinine excretion rates for evaluation of kidney function in children.

A protein load protocol for evaluation of kidney function was tested in normal children and pediatric renal patients. An overnight, timed urine collection was used for calculation of the baseline creatinine clearance and creatinine excretion rate. One hour following ingestion of a standardized protein meal (baked chicken), a 2-3 h urine collection was begun. The post-protein meal changes in creatinine clearance showed considerable variation in both the normal children and those with renal disorders. In contrast, the rate of excretion of creatinine was consistently increased in the normal children following the protein meal (73.4 +/- 18%; range 48.2%-122.4%). Of 33 renal patients, 14 showed less than a 48% increase in creatinine excretion rate, even though 9 of these children had baseline creatinine clearances within the normal range. These 9 patients have evidence of less than normal quantities of functioning renal tissue. Serial studies over a year on 2 children who presented with acute renal failure showed a progressive increase in creatinine clearance with scant increases in creatinine excretion rate. These studies provide indirect evidence that a less than normal enhancement of the rate of creatinine excretion following a protein load reflects the presence of adaptive glomerular hyperfiltration and hyperperfusion.

Nonaccidental poisoning in childhood.

A boy aged 7 years 10 months was admitted to hospital on several occasions in an unconscius state with twitching and apnoeic episodes. Initial investigations failed to show a specific cause. During his time in hospital he had recurrent episodes of loss of consciousness and, on the last occasion, hypotension and ventricular tachycardia. A diagnosis of imipramine poisoning was established by the presence of imipramine in stomach washings and blood. The drug was being given to the child, both at home and in hospital, by his mother. The possibility of nonaccidental poisoning must be considered if there is no obvious cause for a child's illness. In this case the mother responded to psychiatric treatment.

Meiotic analysis of a pericentric inversion, inv(7) (p22q32), in the father of a child with a duplication-deletion of chromosome 7.

In a family in which a large pericentric inversion of chromosome 7 is segregating, two of the four progeny of inversion heterozygotes show severe psychomotor retardation and have the karyotype 46,XX,rec(7),dup q,inv(7)(p22q32), derived from crossing-over within the inversion. Meiotic analysis in one of the heterozygotes revealed no evidence of inversion loops in well-spread pachytene cells. In approximately 20% of cells in diakinesis, the presumptive bivalent 7 had only one chiasma. Two alternatives to the reversed loop mode of meiotic pairing of inversions are proposed. Review of the literature supports the view that "small" pericentric inversions have a much better genetic prognosis than "large" pericentric inversions.