Monitoring oil spill bioremediation using marsh foraminifera as indicators.

A controlled experiment was conducted in June 2000 to identify the environmental impacts of weathered crude oil on an Atlantic coastal salt marsh to help evaluate in situ biological remediation techniques for restoring the environment. Foraminifera, marsh microfossils known to be sensitive to a range of environmental stress factors, were used to monitor the effects of the residual oil and the experimental treatments. Results show that the foraminifera responded quickly to the oil and that the oil had a statistically significant, negative impact, as demonstrated by a dramatic increase in deformities in the tests of Miliammina fusca, compared to specimens from the non-oiled control plots. The results clearly show that foraminifera can be excellent indicators of oil pollution using only the percent of deformed tests. The advantages that foraminifera provide are the ease of sampling, processing and examination, with the added benefit that these organisms leave a fossil record.

An NMDA receptor ER retention signal regulated by phosphorylation and alternative splicing.

Formation of mature excitatory synapses requires the assembly and delivery of NMDA receptors to the neuronal plasma membrane. A key step in the trafficking of NMDA receptors to synapses is the exit of newly assembled receptors from the endoplasmic reticulum (ER). Here we report the identification of an RXR-type ER retention/retrieval motif in the C-terminal tail of the NMDA receptor subunit NR1 that regulates receptor surface expression in heterologous cells and in neurons. In addition, we show that PKC phosphorylation and an alternatively spliced consensus type I PDZ-binding domain suppress ER retention. These results demonstrate a novel quality control function for alternatively spliced C-terminal domains of NR1 and implicate both phosphorylation and potential PDZ-mediated interactions in the trafficking of NMDA receptors through early stages of the secretory pathway.

EAF1 regulates vegetative-phase change and flowering time in Arabidopsis.

We have identified a new locus that regulates vegetative phase change and flowering time in Arabidopsis. An early-flowering mutant, eaf1 (early flowering 1) was isolated and characterized. eaf1 plants flowered earlier than the wild type under either short-day or long-day conditions, and showed a reduction in the juvenile and adult vegetative phases. When grown under short-day conditions, eaf1 plants were slightly pale green and had elongated petioles, phenotypes that are observed in mutants altered in either phytochrome or the gibberellin (GA) response. eaf1 seed showed increased resistance to the GA biosynthesis inhibitor paclobutrazol, suggesting that GA metabolism and/or response had been altered. Comparison of eaf1 to other early-flowering mutants revealed that eaf1 shifts to the adult phase early and flowers early, similarly to the phyB (phytochrome B) and spy (spindly) mutants. eaf1 maps to chromosome 2, but defines a locus distinct from phyB, clf (curly leaf), and elf3 (early-flowering 3). These results demonstrate that eaf1 defines a new locus involved in an autonomous pathway and may affect GA regulation of flowering.

The requirement for exopolysaccharide precedes the requirement for flavolan-binding polysaccharide in nodulation of Leucaena leucocephala by Rhizobium loti.

Rhizobium loti strain PN4115 (NZP2213 str-1) ineffectively nodulates Leucaena leucocephala, i.e., strain PN4115 induces nodulation (Nod+) and is able to invade these nodules (Inv+), but fails to fix nitrogen (Fix-). Strain PN4115 does not synthesize a flavolan-binding polysaccharide (FBP), which is synthesized by the fully effective (Nod+Inv+Fix+) R. loti strain PN184 (NZP2037 str-1). The FBP may offer protection from prodelphinidin-rich flavolans synthesized by Lc. leucocephala. In this work, we show that exopolysaccharide (EPS)-negative mutants derived from strain PN4115 have a more severe ineffective phenotype (Nod+Inv-Fix-) on Lc. leucocephala than strain PN4115. This suggests that EPS from strain PN4115 is functional during invasion of Lc. leucocephala and that the requirement for EPS precedes the requirement for FBP.

Novel and complex chromosomal arrangement of Rhizobium loti nodulation genes.

A mutational and structural analysis of Rhizobium loti nodulation genes in strains NZP2037 and NZP2213 was carried out. Unlike the case with other Rhizobium strains examined to date, nodB was found on an operon separate from nodACIJ. Sequence analysis of the nodACIJ and nodB operon regions confirm that R. loti common nod genes have a gene organization different from that of other Rhizobium spp. At least 4 copies of nodD-like sequences were identified in R. loti. The complete nucleotide sequence of one of these, nodD3, was determined. A new host-specific nod gene, nolL, was identified adjacent to nodD3. NolL shares homology with NodX and other O-acetyl transferases. Mutational analysis of the nod regions of strains NZP2037 and NZP2213 showed that nodD3, nodI, nodJ, and nolL were all essential for R. loti strains to effectively nodulate the extended host Lotus pedunculatus, but were not necessary for effective nodulation of the less restrictive host, Lotus corniculatus. Both nodD3 and nolL were essential for R. loti strains to nodulate Leucaena leucocephala.

An in vitro comparison of fluid leakage after dural puncture with Atraucan, Sprotte, Whitacre, and Quincke needles.

BACKGROUND AND OBJECTIVES: The study was designed to evaluate the influence of needle size and design on the rate of leakage following dural puncture. METHODS: An in vitro model and fresh human lumbar dura were used to examine the rate of fluid leakage after puncture with Sprotte (24-gauge and 26-gauge), Atraucan (24-gauge and 26-gauge), Quincke (26-gauge and 29-gauge), and Whitacre (22-gauge and 25-gauge) needles. RESULTS: The study confirmed that finer-gauge needles tend to produce less leakage and that traditional Quincke pattern bevels result in greater leakage than pencil-point designs of the same diameter. CONCLUSIONS: The comparably low leakage rate produced by the Atraucan, a new needle with a terminal opening, suggests that this needle is worthy of further clinical evaluation.

The dawn of a new era.

This paper reviews the scientific events culminating in the fluoridation of communal water supplies. Dental and medical studies completed by 1942 had established the safety and benefits of exposure to drinking water naturally containing fluoride. Researchers and public health workers concluded that it was possible to test the hypothesis that the dental benefits attained where fluoride levels around 1 ppm occurred naturally in drinking water could be safely replicated in low-fluoride areas by raising the level to this optimal concentration. Grand Rapids became the first test site and by the time the demonstration ended in 1959, around 40 million people in about 2,000 communities already were drinking water with fluoride levels that had been adjusted to optimal. The success of fluoridation brought the dawn of the era of caries control and created great opportunities for research and public health.

Nodulating strains of Rhizobium loti arise through chromosomal symbiotic gene transfer in the environment.

Rhizobia were isolated from nodules off a stand of Lotus corniculatus established with a single inoculant strain, ICMP3153, 7 years earlier in an area devoid of naturalized Rhizobium loti. The isolates showed diversity in growth rate, Spe I fingerprint of genomic DNA, and hybridization pattern to genomic DNA probes. The 19% of isolates that grew at the same rate as strain ICMP3153 were the only isolates that had the same fingerprint as strain ICMP3153. Sequencing of part of the 16S rRNA gene of several diverse isolates confirmed that they were not derived from the inoculant strain. Nevertheless, all non-ICMP3153 strains gave EcoRI and Spe I hybridization patterns identical to ICMP3153 when hybridized to nodulation gene cosmids. Hybridization of digests generated by the very rare cutting enzyme Swa I revealed that the symbiotic DNA region (at least 105 kb) was chromosomally integrated in the strains. The results suggest that the diverse strains arose by transfer of chromosomal symbiotic genes from ICMP3153 to nonsymbiotic rhizobia in the environment.

Serious complications associated with epidural/spinal blockade in obstetrics: a two-year prospective study.

A prospective study of complications associated with epidural and spinal blockade in obstetrics was carried out during the years 1990 and 1991. 79 obstetric units in the United Kingdom took part. These units had a total of 467 491 deliveries during the two years. 108 133 mothers received epidural blockade and 14856 received spinal blockade. 22% of all mothers received epidural analgesia in labour, and 50% of caesarean sections were performed under either epidural or spinal anaesthesia. 128 complications (not including post dural puncture headache) were reported. Of these, 46 were neuropathies involving a single spinal or peripheral nerve. 26 unexpectedly high blockades were encountered. Backache was reported in 21 mothers and urinary retention in 8. Cardiac arrest occurred twice. Although resuscitation restored normal cardiac function in both cases, one patient was decerebrate and died some days later. Postmortem examination revealed evidence of amniotic fluid embolus. 20 reports were classified as 'miscellaneous' and presented individual clinical diagnoses. The overall complication rate (excluding post dural puncture headache) was approximately 1 per thousand.

A mutualistic fungal symbiont of perennial ryegrass contains two different pyr4 genes, both expressing orotidine-5'-monophosphate decarboxylase.

A fragment of the Claviceps purpurea pyr4 gene, encoding orotidine-5'-monophosphate decarboxylase (OMP decarboxylase), was used to screen a genomic library from an isolate of a fungus, Acremonium sp. (designated Lp1), which grows as an endophyte in perennial ryegrass (Lolium perenne). Three positive clones, lambda MC11, lambda MC12 and lambda MC14, were isolated. Two of these clones, lambda MC12 and lambda MC14, were overlapping clones from the same locus, while lambda MC11 was from a different locus. Fragments of these clones which hybridised with C. purpurea pyr4 were sequenced and found to have similarity with pyr4 from other Pyrenomycete fungi. The pyr4 gene from lambda MC12 and lambda MC14 was designated pyr4-1 and that from lambda MC11 was designated pyr4-2. The predicted ORFs of the two genes were highly conserved, with 97.5% identity at the nucleotide level, the 5' non-coding sequences were the least conserved with 88.5% identity and the 3' non-coding sequences had 93.0% identity. RT-PCR analysis of total RNA from Lp1 demonstrated that transcripts from the two genes were present at similar levels, and hybridisation of pyr4-1 to Northern blots of total RNA from Lp1 showed that full-length transcripts were being produced. Genomic fragments containing pyr4 were transformed into a strain of Aspergillus nidulans which has a mutation in pyrG (encoding OMP decarboxylase). Both pyr4-1 and pyr4-2 complemented the pyrG mutation in A. nidulans, indicating that both encode functional OMP decarboxylases. It has been proposed [Schardl et al., Genetics 136 (1994) 1307-1317] that the two pyr4 in Lp1 arose by interspecific hybridisation, most likely between the ryegrass choke pathogen, Epichloë typhina, and another endophyte from perennial ryegrass, Acremonium lolii. Analysis by PCR amplification and direct sequencing of the variable 5' non-coding regions of pyr4, from possible ancestors to Lp1 supports this hypothesis. Comparisons of these sequences to the 5' non-coding sequences from pyr4-1 and pyr4-2 demonstrated that E. typhina and A. lolii were the most likely ancestors of the two pyr4 found in Lp1.

A dose-response study of the effects of inhaled nitrous oxide on psychological performance and mood.

In this five-period randomised double-blind crossover study, 12 healthy volunteers inhaled mixtures of nitrous oxide at concentrations of 0% (placebo); 5%, 10%; 20% and 40% in oxygen. Each concentration was inhaled for about 1 h, each period being on a separate day. The effects of nitrous oxide were measured using a comprehensive battery of performance tests including measures of attention, psychomotor function, memory and cognition. Mood was assessed with visual analogue scales. All tests except critical flicker fusion showed substantial effects at the highest does (40%). No measure showed evidence of change at the lowest concentration (5%). Several measures showed significant impairment at 10%, viz: digit-symbol substitution, choice reaction time (latency and total), tapping, and continuous attention. Subjects felt dizzy and muzzy on nitrous oxide, but no significant effect was seen on the Alert-Drowsy VAS. The dose-response profiles of the various tests showed substantial differences. Thus tapping was virtually linear, while choice reaction motor time and body sway showed steeply accelerating impairment with increasing dose. These results indicate that comparisons of profiles of drug-induced change must take into account the variable effects of dose before interpretations in terms of specific drug effects can be made.

The effect of oral omeprazole on the disposition of lignocaine.

The effects of the gastric antisecretory drug, omeprazole, on the disposition of lignocaine were studied in 10 healthy male volunteers in a double-blind, randomised, placebo-controlled, two-period crossover trial. Omeprazole 40 mg or placebo was taken daily for one week before administration of lignocaine 1 mg.kg-1 (3.7 mumol.kg-1) given intravenously over 10 min. Venous concentrations of lignocaine and its metabolite monoethylglycinexylidine were measured in plasma with reversed phase liquid chromatography. The mean (95% CI) areas under the curve at infinity for lignocaine after pretreatment with omeprazole or placebo were 6.67 (4.90-8.45) mumol.h.l-1 and 6.14 (5.05-7.23) mumol.h.l-1, respectively (p = 0.44). The respective areas for monoethylglycinexylidine were 1.85 (1.25-2.45) mumol.h.l-1 and 1.79 (1.44-2.14) mumol.h.l-1 (p = 0.78). Similarly, omeprazole had no significant effect on the half-lives of lignocaine or methylglycinexylidine.

Origin of a fungal symbiont of perennial ryegrass by interspecific hybridization of a mutualist with the ryegrass choke pathogen, Epichloë typhina.

Seed-borne fungal symbionts (endophytes) provide many cool-season grass species with biological protection from biotic and abiotic stresses. The endophytes are asexual, whereas closely related sexual species of genus Epichloë (Clavicipitales) cause grass choke disease. Perennial ryegrass (Lolium perenne) is a host of two endophyte taxa, LpTG-1 (L. perenne endophyte taxonomic grouping one = Acremonium lolii) and LpTG-2, as well as the choke pathogen, Epichloë typhina (represented by isolate E8). Relationships among these fungi and other Epichloë species were investigated by analysis of gene sequences, DNA polymorphisms and allozymes. The results indicate that LpTG-2 is a heteroploid derived from an interspecific hybrid. The LpTG-2 isolates had two copies each of nine out of ten genes analyzed (the exception being the rRNA gene locus), and the profiles for seven of these were composites of those from E. typhina E8 and A. lolii isolate Lp5. Molecular phylogenetic analysis grouped the two beta-tubulin genes of LpTG-2 into separate clades. One (tub2-1) was related to that of E. typhina E8, and the other (tub2-2) to that of A. lolii. The mitochondrial DNA profile of LpTG-2 was similar to that of A. lolii, but its rRNA gene sequence grouped it with E. typhina E8. A proposed model for the evolution of LpTG-2 involves infection of a L. perenne-A. lolii symbiotum by E. typhina, followed by hybridization of the two fungi. Such interspecific hybridization may be a common and important mechanism for genetic variation in Epichloë endophytes.

Effects of ethanol on psychomotor performance under steady-state conditions.

Ethanol was administered to eight male volunteers using an oral loading dose followed by repeated small oral doses to achieve approximate steady-state drug concentrations in a double-blind placebo controlled cross over design. Ethanol or placebo were administered over a 5-h period in two sessions at least 7 days apart. The effects of ethanol were assessed using a short battery of psychomotor tests and visual analogue scales which was administered repeatedly during the steady-state period, and a long battery administered once before and once during the steady-state period. The concentrations of ethanol in plasma and breath were determined at 20-min intervals. Mean plasma concentrations of 94 mg/100 ml were obtained. Ethanol produced a clear impairment to psychomotor performance, with a 41% increase in body sway, a 61% increase in errors on a maze task, a 6.5% reduction in digit-symbol substitution and an 8% slowing in tapping. Subjective feelings of drunkenness and sedation were noted. No measure showed evidence for acute tolerance, as assessed by comparison of the slopes fitted to the performance measures in the short battery.

Effects of chlormethiazole on psychological performance under conditions of constant plasma concentrations.

Chlormethiazole was administered intravenously to six healthy volunteers (four male, two female, aged 20-33 years) using a loading dose followed by a maintenance infusion lasting ∼ 90 min. Doses were individually calculated from previous pharmacokinetic investigations in these subjects to produce a target steady-state plasma concentration of 1.5 µg ml(-1). Effects of chlormethiazole were determined using a short battery consisting of digit-symbol substitution, body sway and visual analogue scales, which was performed repeatedly before, during and after the active infusion. A more comprehensive battery of performance tests was performed once before and once during the active infusion. The mean plasma concentration of chlormethiazole obtained was 1.33 µg ml(-1). This produced marked sedation, with subjects scoring themselves as much more drowsy on chlormethiazole than on placebo, and global impairment to performance. An analysis of the slopes of scores on the performance tests in the short battery showed no evidence of a diminution of the effects of chlormethiazole over the infusion period. The same was true of the majority of the visual analogue scales, but two scales, rating eye symptoms and nose symptoms, did decline over the period of the infusion. Recovery was rapid, subjects returning to approximately baseline levels of performance within 30 min of discontinuation of the infusion. These results suggest that acute tolerance to the CNS effects of chlormethiazole does not occur over this time scale, but is found for peripheral effects such as eye and nose symptoms.

Atraucan: a new needle for spinal anesthesia.

BACKGROUND AND OBJECTIVES: Atraucan 26-gauge spinal needles have a tip designed to make a small linear cut (as opposed to a V-shaped cut) in the dura mater. The cut is shorter than the outside diameter of the needle and is dilated as the needle passes through the dura. The needle is used with a 20-gauge introducer. In vitro, it causes less leakage of cerebrospinal fluid than Quincke 26-gauge or Sprotte 24-gauge needles. This study was designed to test the ease of use and any damage caused to the needle tip during lumbar dural puncture. METHODS: This was a multicenter trial (six centers in five countries) involving 362 patients undergoing spinal anesthesia. A detailed questionnaire was filled in for every patient by the anesthesiologist. All the needles were returned to the factory and examined microscopically for damage. RESULTS: Lumbar dural puncture was successful in all but one patient. Spinal anesthesia was satisfactory for the planned surgery in 97%. Microscopy of the needle tips showed only a minor degree (0.01-0.19 mm) of bending in 14%, and none of the tips had a "hook." Postdural puncture headache (PDPH) occurred in nine patients (2.5%), all but one of whom (a 15-year-old male) were females under 55 years of age. CONCLUSIONS: The Atraucan needle is easy to use and has a high success rate in identifying the subarachnoid space. Lumbar dural puncture causes minimal damage to the tip. The incidence of PDPH is low, but a larger comparative study needs to be performed.

Surrogate transformation of perennial ryegrass, Lolium perenne, using genetically modified Acremonium endophyte.

Conditions have been developed for transforming protoplasts of the perennial ryegrass endophyte Acremonium strain 187BB. Unlike most other ryegrass endophytes, this strain does not produce the lolitrem B neurotoxin and is therefore suitable as a host for surrogate introduction of foreign genes into grasses. Transformation frequencies of 700-800 transformants/micrograms DNA were obtained for both linear and circular forms of pAN7-1, a hygromycin (hph) resistant plasmid. Up to 80% of the linear transformants were stable on further culturing but only 25% of the circular transformants retained hygromycin resistance. Integration of pAN7-1 into the genome was confirmed by Southern blotting and probing of genomic digests of transformant DNA. Both single and tandemly repeated copies of the plasmid were found in the genome and both the number and sites of integration varied among the transformants. At least 13 chromosomes were identified in 187BB using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Probing of Southern blots of these gels confirmed that pAN7-1 had integrated into different chromosomes. The beta-glucuronidase (GUS) gene, uidA, was also introduced into 187BB by co-transformation of pNOM-2 with pAN7-1. GUS activity was detected by growing the transformants on plates containing 5-bromo-4-chloro-3-indolyl beta-D-glucuronic acid and by enzyme assays of mycelial extracts. Several hph- and uidA-containing transformants were reintroduced into ryegrass seedlings and expression of GUS visualized in vivo, demonstrating that 187BB can be used as a surrogate host to introduce foreign genes into perennial ryegrass.(ABSTRACT TRUNCATED AT 250 WORDS)