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.

The biosynthesis of nitrogenase MoFe protein polypeptides in free-living cultures of Rhizobium japonicum.

The biosynthesis of the constituent polypeptides of nitrogenase component I (Rj 1) in free-living cultures of Rhizobium japonicum (strain 110) was investigated under different growth conditions. Cells were pulse-labelled and the proteins analysed by one and two-dimensional gel electrophoresis. The positions of the constituent Rj 1 polypeptides were identified by co-electrophoresis with purified Rj 1 isolated from bacteroids of soybean nodules, and by comparison with an immunoprecipitate from a culture induced for nitrogenase. The synthesis of the proteins preceded any detectable enzyme activity and increased with time, reaching a maximum after 3 days. At this time, between 6 and 8% of the total sodium dodecyl sulfate-soluble protein synthesized was Rj 1. Exposure to air led to a dramatic decrease in the rate of Rj 1 synthesis, with almost complete regression after 20 min. In the presence of KNO3, there was no nitrogenase activity, but the proteins were present in similar amounts (7%) as the control culture. When mannitol and glycerol were used as the sole carbon sources, the amount of Rj 1 synthesized was extremely low.

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.

Isolation and characterization of a Rhizobium loti gene required for effective nodulation of Lotus pedunculatus.

A Rhizobium loti gene required for effective invasion of the host Lotus pedunculatus has been identified by transposon Tn5 mutagenesis. Cosmids that complemented a previously isolated mutation (239) at this invasion (inv) locus were identified by in planta complementation and used to construct a physical map of the gene region. The insertion site of Tn5 in PN239 was mapped to a 7.5-kb EcoRI fragment, which complemented the mutation when subcloned into pLAFR1. Further Tn5 mutagenesis of the 7.5-kb fragment was carried out in Escherichia coli using bacteriophage lambda 467, and the mutations homogenotized into R. loti NZP2037. Three additional Fix- mutations were isolated, and these were found to map adjacent to the position of the original mutation in strain PN239. All the other Tn5 insertions isolated in the 7.5-kb fragment gave a Fix+ phenotype on L. pedunculatus. Electron microscopic examination of the L. pedunculatus nodules induced by the isolated Fix- mutants showed that bacteria were either blocked in release from the infection threads or were unable to undergo normal bacteroid development. The inv locus as defined by the Tn5 insertions was sequenced, and a single open-reading frame (ORF) of 576 bp, corresponding to a polypeptide of 21.3 kDa, was identified. The position and orientation of this ORF were consistent with those of the isolated Tn5 Fix- insertions.

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.

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 study of the effects of zimelidine on the pharmacokinetics and pharmacodynamics of temazepam in healthy volunteers.

In this double-blind two-period crossover study, ten healthy volunteers received either 200 mg zimelidine each morning for 5 days, or placebo on the same schedule. On day 5 they received 20 mg temazepam 2 h after zimelidine or placebo. A battery of psychometric tests and subjective measurements was carried out on days 4 and 5. Blood samples were collected on day 5 for pharmacokinetic analysis of temazepam. All the measures of psychomotor performance showed the effects of temazepam, as did two of the subjective measures, the "alert/drowsy" and "steady/dizzy" visual analogue scales. No effect of zimelidine alone on performance or subjective state was seen. Zimelidine showed no discernible interaction with the effects of temazepam as assessed by subjective reports, by psychomotor tests, or by pharmacokinetic analysis.

Effects of amitriptyline and zimelidine in combination with ethanol.

Six healthy male volunteers took part in this three-period crossover study. In each session, a dose of trial drug -- either placebo, zimelidine 200 mg, or amitriptyline 75 mg -- was given at 09.00 h. Ethanol (50 g) was taken orally at 1200 h. Blood samples were taken for measurement of drug and ethanol concentrations, and body sway and subjective sedation were determined. No differences in the pharmacokinetics of ethanol were seen between the three treatment sessions. Amitriptyline and ethanol showed marked sedative effects, and the results suggest that these two effects may be additive. The combination of amitriptyline and ethanol results in a particularly marked increase in body sway. No sedative nor alerting effect of zimelidine was seen, nor was any interaction between zimelidine and ethanol apparent.

Effects of remoxipride on measures of psychological performance in healthy volunteers.

The acute psychomotor effects after oral doses of 30, 60 and 120 mg remoxipride, a new selective D2 receptor blocker, and placebo were investigated in a double-blind crossover study in 11 healthy male volunteers. Two out of the first three subjects given 120 mg remoxipride experienced marked akathisia, and therefore no subsequent subjects were given this dose. There were no other clearly drug-related adverse effects reported below 120 mg, although restlessness was reported at 60 mg. Remoxipride was associated with increases in error scores on a continuous attention task and on auditory vigilance, and with a reduction in critical flicker frequency, suggesting a decrease in arousal level. There were no significant changes in psychomotor measures such as choice reaction time, decision making time, or body sway. Subjective assessments using visual analogue scales showed a slight dose-related increase in drowsiness, while the calm-excited scale showed a small change in the excited direction with 30 mg only. The peak effects were at 4-6 h after drug intake, which was later than expected from previous pharmacokinetic data. These results indicate that remoxipride may have a slight depressant effect in the dose-range used. The pattern of changes is consistent with current theories on the role of dopamine in attention and arousal, and with the effects of other neuroleptics. It differs, however, from tranquilisers such as the benzodiazepines, which show a more global pattern of effects.

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.

Fracture properties of human enamel and dentin.

Fracture in enamel is anisotropic with respect ot the orientation of the enamel rods, with Wf for fracture parallel to the rods being 0.3 X 10(2) J/meter2 but on the order of 2.0 X 10(2) J/meter2 for fracture perpendicular to the rods. Fractographs of enamel showed that the enamel rods behaved as integral units during controlled fracture. A model was proposed to explain the fracture properties of enamel involving the assumption that the hydroxyapatite crystals did not fracture but that their orientation determined the fracture properties. Dentin was also anisotropic, with fracture parallel to the dentinal tubules being the strong direction - Wf was 5.5 X 10(2) J/meter2 for fracture parallel to the tubules, but 2.7 X 10(2) J/meter2 for fracture perpendicular to the tubules. Wf for dentin was of the same magnitude as the high strain rate Wf found for bone (2.0 X 10(2) J/meter2 [ref 3]). The data obtained in this investigation indicated that dentin and enamel are brittle substances and that forces occasionally imposed during mastication or bruxism probably are capable of initiating fracture when the normal anatomy of the tooth has been altered by cavity preparation. As consequence, the design of cavity preparations should include considerations to reduce stress concentrations and thus improve the practical fracture strength of tooth structure.

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.

Plethysmography during aorto-iliac surgery.

Cardiac output, arterial and central venous pressures and calf blood flow were used to monitor patients undergoing abdominal aortic surgery. Measurement of calf blood flow by means of venous occlusion plethysmography showed that measurable blood flow to the legs persisted after clamping of the aorta in patients with occlusive aortoiliac lesions and that the hyperaemic response following release of the clamp was reduced. Arterial hypotension after clamp release was of the same time course as the hyperaemia. Leg blood flow measurement was a valuable monitoring procedure.

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.

History of Mendelson's syndrome.

The history of Mendelson's Syndrome is briefly covered and the current problem of the condition in regard to prevention, amelioration and treatment is discussed.

An outbreak of ringworm in karakul sheep caused by a physiological variant of Trichophyton verrucosum Bodin.

An outbreak of ringworm in Karakul sheep occurred during 1973 among the flocks of at least 14 farmers in South West Africa and the northern Cape Province. Infections spread from one farm to another as a result of introducing infected stud rams. On one farm a few cattle, a goat and a horse developed lesions after being in contact with infected sheep. Some of the attendants handling infected sheep also contracted the disease. Strains recovered from infected animals resembled Trichophyton verrucosum morphologically, but when grown on laboratory media they did not require an exogenous source of vitamins, which was found to be indispensable for strains of this species studied previously.

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.

Black grain mycetoma (maduromycosis) in horses.

Black grain mycetoma occurred in two horses, one a cross-bred pony and the other a Thoroughbred racehorse. Lesions were limited to wart-like growths in one case and a deep-seated girth gall in the other. In both cases the granules in the affected tissues were similar in pigmentation and structure to those produced by Curvularia geniculata in dogs. Since all the specimens were received in formalin, the causative fungus could not be isolated. These are the first cases of maduromycosis in animals to be recorded in South Africa.

A fatal accident after epidural anesthesia for cesarean section.

It is a sobering fact that mishaps during the administration of anesthesia sometimes have a way of initiating problems which escalate step by step to a situation of total disaster for the patient. Inexperience and poor judgment often lie behind such tragedies and the following case report is presented not only as an illustration of this, but also as a useful lesson. The authors only became involved in this case after the anesthesiologist concerned made available his account of the clinical events together with the hospital records. For obvious reasons the anesthesiologist's name has not been given, but he has given his full permission to publish this case. A case report is presented in which a healthy 31-year-old a term para II, requested epidural anesthesia for an elective Cesarean section for pelvic disproportion. Repeated attempts to perform epidural anesthesia were necessary. Eventually she developed acute pulmonary edema and resuscitation was not successful. Both mother and fetus died.