Isolation and characterization of a low phytic acid rice mutant reveals a mutation in the rice orthologue of maize MIK.

Using a forward genetics approach, we isolated two independent low phytic acid (lpa) rice mutants, N15-186 and N15-375. Both mutants are caused by single gene, recessive non-lethal mutations, which result in approximately 75% (N15-186) and 43% (N15-375) reductions in seed phytic acid (inositol hexakisphosphate). High-performance liquid chromatography and GC-MS analysis of seed extracts from N15-186 indicated that, in addition to phytic acid, inositol monophosphate was significantly reduced whereas inorganic phosphorus and myo-inositol were greatly increased when compared with wild-type. The changes observed in N15-186 resemble those previously described for the maize lpa3 mutant. Analysis of N15-375 revealed changes similar to those observed in previously characterized rice lpa1 mutants (i.e. significant reduction in phytic acid and corresponding increase in inorganic phosphorus with little or no change in inositol phosphate intermediates or myo-inositol). Further genetic analysis of the N15-186 mutant indicated that the mutation, designated lpa N15-186, was located in a region on chromosome 3 between the microsatellite markers RM15875 and RM15907. The rice orthologue of maize lpa3, which encodes a myo-inositol kinase, is in this interval. Sequence analysis of the N15-186 allele of this orthologue (Os03g52760) revealed a single base pair change (C/G to T/A) in the first exon of the gene, which results in a nonsense mutation. Our results indicate that lpa N15-186 is a mutant allele of the rice myo-inositol kinase (OsMIK) gene. Identification and characterization of lpa mutants, such as N15-186, will facilitate studies on the regulation of phytic acid biosynthesis and accumulation and help address questions concerning the contribution of the inositol lipid-dependent and independent biosynthetic pathways to the production of seed phytic acid.

A randomized controlled trial of an asthma clinical pathway for children in general practice.

AIMS: To evaluate the effect of the implementation of an asthma clinical pathway on asthma in children in general practice. METHODS: A randomized, controlled trial involving 270 general practitioners. One group of general practitioners implemented the asthma clinical pathway for children (intervention group) and the control group continued with their usual asthma medical care management. The main outcome measures were admissions to hospital for asthma and attendance at the Children's Emergency Department. Compliance with the guidelines was assessed by examining asthma drug prescriptions. RESULTS: Admissions to hospital for asthma dropped 40% in the intervention group, by 33% in the control group and by 22% in general practitioners not participating in the trial. The differences between the intervention and control and between the intervention and non-participating general practitioners were not statistically significant. The decrease in attendance at the Children's Emergency Department decreased by 25%, 30% and 19%, respectively, but this was not statistically significant. There was a significant decrease in prescriptions for oral relievers, dry powder relievers in the under 6s, mast cell stabilizers and methylxanthines in both control and intervention groups. However, only for oral relievers was there a significant difference between the intervention group and control, with the decrease larger in the intervention group (p < 0.001). CONCLUSIONS: Admissions to hospital for asthma decreased, as did attendance at the Children's Emergency Department. Prescriptions for asthma medication changed in the direction anticipated with compliance with the asthma clinical pathway. However, we found no evidence within the study that implementation of the asthma clinical pathway by general practitioners resulted in lower morbidity than those general practitioners who did not implement the pathway. Possible explanations are that these general practitioners were already providing care according to the recommendations of the pathway, or that there was contamination of the control group by the intervention, or that the guidelines, although based on currently accepted recommendations, are ineffective.

Raised dietary n-6 polyunsaturated fatty acid intake increases 2-series prostaglandin production during labour in the ewe.

Preterm labour is the major cause of perinatal morbidity and mortality in humans. The incidence is around 10% and the causes are often unknown. Consumption of dietary n-6 polyunsaturated fatty acids (PUFAs) in western societies is increasing. These are metabolized to arachidonic acid, the precursor for 2-series prostaglandins (PGs), major signalling molecules during labour. This study investigated the effect of dietary supplementation with linoleic acid (LA, 18: 2, n-6) on parturition. Ewes were fed a control or LA-supplemented diet from 100 days gestation. Labour was induced using a standardized glucocorticoid challenge (dexamethasone, Dex) to the fetus, starting on day 139. Electromyographic (EMG) activity and fetal and maternal circulating PG concentrations were monitored. One third of LA-fed ewes delivered early (pre-Dex) although basal uterine EMG activity preceding Dex was higher in control ewes (P < 0.05). A steep increase in EMG activity occurred 18-38 h after the start of Dex infusion. Twice basal EMG activity (defined as established labour) occurred on average 7 h earlier in the LA-supplemented ewes (P < 0.05). The basal concentrations of maternal and fetal PGFM and fetal PGE(2) were approximately doubled in LA-supplemented ewes before the start of Dex infusion (P < 0.01). The rise in fetal PGE(2) and maternal oestradiol concentrations post-Dex occurred earlier in the LA-supplemented ewes. All PG measurements remained significantly higher in the LA-supplemented ewes during labour onset. This study suggests that consumption of a high LA diet in late pregnancy can enhance placental PG production and may thus increase the risk of preterm labour.

Hidden blood loss following hip and knee arthroplasty. Correct management of blood loss should take hidden loss into account.

Following total hip arthroplasty (THA) and total knee arthroplasty (TKR) only the 'visible' measured blood loss is usually known. This underestimates the 'true' total loss, as some loss is 'hidden'. Correct management of blood loss should take hidden loss into account. We studied 101 THAs and 101 TKAs (with re-infusion of drained blood). Following THA, the mean total loss was 1510 ml and the hidden loss 471 ml (26%). Following TKA, the mean total loss was 1498 ml. The hidden loss was 765 ml (49%). Obesity made no difference with either operation. THA involves a small hidden loss, the total loss being 1.3 times that measured. However, following TKA, there may be substantial hidden blood loss due to bleeding into the tissues and residual blood in the joint. The true total loss can be determined by doubling the measured loss.

Effect of nutritional state on Hsp60 levels in the rotifer Brachionus plicatilis following toxicant exposure.

The nutritional state of an organism can affect the results of toxicity testing. Here we exemplified this fact by examining the effect of nutritional deprivation on heat shock protein 60 (hsp60) production in the rotifer Brachionus plicatilis following exposure to two proven inducers of hsp60, a water-accommodated fraction of crude oil (WAF) and a dispersed oil preparation (DO). Both DO and WAF exposures of unfed rotifers resulted in significantly greater hsp60 levels than that of fed DO and WAF exposed rotifers at 8 h: 870 and 3100% of control, respectively. Results clearly demonstrate that a poor nutritional state potentiates stress protein induction upon exposure to water-soluble petroleum products. It is therefore critical to define the organismal nutritional status when reporting toxic responses.

The Everglades Nutrient Removal Project test cells: STA optimization--status of the research at the north site.

The Everglades is an oligotrophic ecosystem that is being adversely impacted by hydrologic changes and nutrient-rich runoff generated from urban and agricultural sources. The Stormwater Treatment Area (STA) Optimization Research and Monitoring program is mandated by the 1994 Everglades Forever Act and will assist the South Florida Water Management District in developing operational strategies that maximize performance of emergent macrophyte STAs. The primary objective of this research is to examine how hydrologic conditions may influence STA performance. The study was conducted in 0.2 ha, shallow, fully lined test cells located within the perimeter of the Everglades Nutrient Removal Project. Experiments were designed to examine the effect of increased and decreased hydraulic loading rate (HLR) on wetland performance and to determine, if possible, the HLR at which STA treatment fails to reduce outflow total phosphorus concentration to the interim target of 50 microg-P/L. To date, two HLR experiments have been completed at the north site. Preliminary data indicated at all HLRs tested that particulate phosphorus and dissolved organic phosphorus ratios remained virtually unchanged from inflow to outflow. The dissolved organic and particulate compounds within these test cells are extremely recalcitrant, and are not easily assimilated within the system. High HLRs may not result in detention times long enough to mineralize these forms into easily assimilated inorganic compounds, resulting in mean TP concentrations greater than 50 microg-P/L.

Cress and potato soluble epoxide hydrolases: purification, biochemical characterization, and comparison to mammalian enzymes.

Affinity chromatographic methods were developed for the one-step purification to homogeneity of recombinant soluble epoxide hydrolases (sEHs) from cress and potato. The enzymes are monomeric, with masses of 36 and 39 kDa and pI values of 4.5 and 5.0, respectively. In spite of a large difference in sequence, the two plant enzymes have properties of inhibition and substrate selectivity which differ only slightly from mammalian sEHs. Whereas mammalian sEHs are highly selective for trans- versus cis-substituted stilbene oxide and 1,3-diphenylpropene oxide (DPPO), plant sEHs exhibit far greater selectivity for trans- versus cis-stilbene oxide, but little to no selectivity for DPPO isomers. The isolation of a covalently linked plant sEH-substrate complex indicated that the plant and mammalian sEHs have a similar mechanism of action. We hypothesize an in vivo role for plant sEH in cutin biosynthesis, based on relatively high plant sEH activity on epoxystearate to form a cutin precursor, 9,10-dihydroxystearate. Plant sEHs display a high thermal stability relative to mammalian sEHs. This stability and their high enantioselectivity for a single substrate suggest that their potential as biocatalysts for the preparation of enantiopure epoxides should be evaluated.

Laser therapy of spider leg veins: clinical evaluation of a new long pulsed alexandrite laser.

BACKGROUND: The response of spider leg veins to laser or intense pulsed light therapy has generally been characterized by varying degrees of success and frequently inconsistent clinical response rates. OBJECTIVE: The purpose of this study was to examine the effectiveness of the 755 nm long pulsed infrared alexandrite (LPA) laser for the treatment of leg telangiectasias. METHODS: This study was constructed in four phases. Phase I examined 28 patients with variable sized telangiectasias using 5 treatment parameters (15 J/cm2 x 1 pulse, 20 J/cm2 x 1 pulse, 20 J/cm2 x 2 pulses, 20 J/cm2 x 3 pulses, or 30 J/cm2 x 1 pulse). Each patient received 3 treatments at 4 week intervals with the LPA. Patient diaries were obtained to examine the effects of the treatments. Subjective grading was performed at each follow-up visit by the investigators. Blinded objective grading was performed at the conclusion of the study by trained observers. Phase II examined the effects of these treatment parameters on varying vessel diameters. Vessels were grouped into small (<0.4 mm), intermediate (0.4-1.0 mm), and large (1.0-3.0 mm) subsets. Phase III examined the effects of a combination of LPA treatment followed by 23.4% hypertonic saline sclerotherapy. Subjective and blinded objective grading was used to determine improvement after a single treatment with the LPA at 20 J/cm2, single pulsed with a pulse duration of 5 or 10 msec followed by treatment with 23.4% hypertonic saline injected 3, 7, 14, or 28 days after laser therapy. Phase IV involved biopsies after LPA treatment alone at time intervals of immediately posttreatment and 5 and 21 days posttreatment. RESULTS: These evaluations revealed that the optimal treatment parameters for LPA therapy alone appeared to be 20 J/cm2, double pulsed at a repetition rate of one Hz. After 3 treatments at 4 week intervals, subjective grading indicated a 63% reduction in leg telangiectasias. Medium diameter vessels responded best with small vessel diameters responding poorly, if at all. The addition of 23.4% hypertonic saline sclerotherapy performed 3 to 7 days after laser therapy (LPA at 20 J/cm2, single pulsed with a pulse duration of 5 msec) produced 87% reduction in leg telangiectasias. Biopsies after LPA treatment revealed vessel wall endothelial cell necrosis at 5 days with fibrosis occurring at 3 weeks. The optimal clinical "window" for sclerotherapy seems to coincide with the period of endothelial cell necrosis. CONCLUSION: LPA therapy is most effective for leg telangiectasias 0.4-3.0 mm in diameter. This LPA technique is significantly improved with the addition of sclerotherapy.

Methyl methacrylate levels in unwashed salvage blood following unilateral total knee arthroplasty.

To evaluate the safety of autologous reinfusion of drain blood in total knee arthroplasty (TKA), eight patients were prospectively evaluated to quantify levels of methyl methacrylate (MMA) monomer in systemic blood, and in their drain blood after unilateral cemented TKA. The systemic blood was analyzed before and after reinfusion of the drain blood. The drain blood was analyzed before reinfusion, and both before and after filtration through a 40-microm filter. A separate study was performed on 10 patients to assess the effect of blood, time, and filtration on MMA levels. Levels of MMA monomer in salvage blood were low enough to allow safe reinfusion. Systemic blood showed no evidence of MMA monomer either before reinfusion of salvage blood or at 5 minutes after reinfusion. Elimination of MMA is dependent on the time that MMA is exposed to blood and is independent of filtration.

Body contouring: a preliminary report on the use of the silhouette device for treating cellulite.

Cellulite is a common condition affecting 85% of postadolescent women. Recent advances in aesthetic techniques have initiated a new pursuit into understanding the cause and treatment of this condition. A recently introduced "roller massage therapy" device, Silhouette, was selected for evaluation in this study. Three patients were enrolled into this private practice, medical school-affiliated, prospective pilot project. The study objective was to identify whether the Silhouette device could generate reproducible reductions in the appearance of cellulite or alter body appearance, specifically in the abdominal, buttock, and thigh regions. A secondary goal was to evaluate the effectiveness of various measuring devices used to quantify cellulite regression. These included serial body weights, percent body fat, relative fat distribution, specific anatomic measurements, diagnostic ultrasound, and serial morphed photographic analysis and laboratory data. Initial results showed that 16 biweekly treatments produced minimal changes in body weight or percent body fat. On average, thigh circumferences increased by 1.7 mm and 8.7 mm in the right and left proximal thighs and decreased by 25 mm and 22 mm in the right and left distal thighs, respectively. Diagnostic ultrasound scans showed trends that may provide further insight into a possible mechanism of action. Relative fat distribution values taken from four selected sites proved the most significant finding with selected treatment sites improving on average from 0.8 to 2.1 units (mean 1.5 units/site). This technique provided safe yet modest improvement in the appearance of cellulite. The mechanism of this improvement remains unknown. Further research is needed to determine the mechanism of this improvement, its longevity, optimum treatment parameters, and whether maintenance therapy is needed.

The clinical advantages of autologous transfusion. A randomized, controlled study after knee replacement.

We have carried out a randomised, controlled trial on 70 patients having unilateral total knee replacement in which transfusion was either with homologous bank blood or by reinfusion of unwashed blood salvaged after operation. No complications or adverse effects were observed from reinfusion. The need for bank blood was reduced by 86% in the reinfusion group but, more importantly, the number of infective episodes was significantly less when the use of bank blood was avoided. The mean length of stay in hospital was also reduced by more than two days.

Crystallization and preliminary X-ray diffraction studies of the influenza C virus glycoprotein.

Influenza C virus contains a single surface glycoprotein in its lipid envelope which is the hemagglutinin-esterase-fusion glycoprotein (HEF). HEF binds cell-surface receptors, is a receptor-destroying enzyme (a 9-O-acetylesterase), and mediates the fusion of virus and host cell membranes. A bromelain-released soluble form of HEF has been crystallized. Two different tetragonal forms have been identified from crystals with the same morphology [P(1(3))22, a = b = 154.5, c = 414.4 A, and P4(1(3))2(1)2, a = b = 217.4, c = 421.4 A]. Both crystal forms share a common packing scheme. Synchrotron data collection and flash cooling of crystals have been used for high-resolution data collection.

Ciliary neurotrophic factors enhances peripheral nerve regeneration.

BACKGROUND: Adjunctive measures to enhance nerve repair have focused on a variety of trophic factors that alter the physiologic response to nerve injury through Schwann cell-axonal interactions. OBJECTIVE: To evaluate the effects of two trophic factors, ciliary neurotrophic factor and nerve growth factor, on axonal response to injury. DESIGN: A prospective, randomized, blinded animal study with a placebo control using lactated Ringer's solution. INTERVENTION: Rat sciatic nerves were transected and repaired as a model of injury following which experimental factors were delivered in vivo through an implantable osmotic pump. OUTCOME MEASURES: Functional nerve recovery, muscle mass, and gene expression in the three experimental groups were evaluated. RESULTS: The ciliary neurotrophic factor group (n=6) showed a higher sciatic functional recovery (P=.003) and preservation of affected muscle mass (P=.03) compared with the nerve growth factor (n=8) and control (n=8) groups. Molecular analysis of injured nerves showed no difference in expression of ciliary neurotrophic factor, myelin basic protein, or low-affinity neurotrophin receptor messenger RNA among the three groups. CONCLUSION: These data suggest that ciliary neurotrophic factor may serve as an important neurocytokine for axonal regrowth during peripheral nerve regeneration.

An experimental test of "the mozart effect": does listening to his music improve spatial ability?

This experiment was designed as a test of the 1993 findings of Rauscher, Shaw, and Ky who reported a positive effect of listening to classical music on spatial reasoning. Present results do not demonstrate the "Mozart effect." In our study, 114 students were pretested on items from the Raven's Progressive Matrices--Advanced Form, then instructed to listen to either 8 min. of Mozart's music, relaxation instructions, or silence. Then subjects were posttested on an equivalent set of Raven's items. The subjects were also asked to provide information about their musical background and preferences. All instructions and treatments were audiotaped and played to individual subjects through earphones in the university language laboratory, ensuring standardization of procedures. Subjects in all 3 treatment groups showed a practice effect, but this improvement in Raven's scores was not dependent on the type of treatment received. There were no differences in Raven's scores among groups before or after treatment so our results do not confirm the prior ones. There was no evidence that the brief music had a different effect on subsequent problem solving according to listeners' musical background and training.

Infection of soybean and pea nodules by Rhizobium spp. purine auxotrophs in the presence of 5-aminoimidazole-4-carboxamide riboside.

Purine auxotrophs of various Rhizobium species are symbiotically defective, usually unable to initiate or complete the infection process. Earlier studies demonstrated that, in the Rhizobium etli-bean symbiosis, infection by purine auxotrophs is partially restored by supplementation of the plant medium with 5-amino-imidazole-4-carboxamide (AICA) riboside, the unphosphorylated form of the purine biosynthetic intermediate AICAR. The addition of purine to the root environment does not have this effect. In this study, purine auxotrophs of Rhizobium fredii HH303 and Rhizobium leguminosarum 128C56 (bv. viciae) were examined. Nutritional and genetic characterization indicated that each mutant was blocked in purine biosynthesis prior to the production of AICAR. R. fredii HH303 and R. leguminosarum 128C56 appeared to be deficient in AICA riboside transport and/or conversion into AICAR, and the auxotrophs derived from them grew very poorly with AICA riboside as a purine source. All of the auxotrophs elicited poorly developed, uninfected nodules on their appropriate hosts. On peas, addition of AICA riboside or purine to the root environment led to enhanced nodulation; however, infection threads were observed only in the presence of AICA riboside. On soybeans, only AICA riboside was effective in enhancing nodulation and promoting infection. Although AICA riboside supplementation of the auxotrophs led to infection thread development on both hosts, the numbers of bacteria recovered from the nodules were still 2 or more orders of magnitude lower than in fully developed nodules populated by wild-type bacteria. The ability to AICA riboside to promote infection by purine auxotrophs, despite serving as a very poor purine source for these strains, supports the hypothesis that AICAR plays a role in infection other than merely promoting bacterial growth.