Aequorin variants with improved bioluminescence properties.

The photoprotein aequorin has been widely used as a bioluminescent label in immunoassays, for the determination of calcium concentrations in vivo, and as a reporter in cellular imaging. It is composed of apoaequorin (189 amino acid residues), the imidazopyrazine chromophore coelenterazine and molecular oxygen. The emission characteristics of aequorin can be changed by rational design of the protein to introduce mutations in its structure, as well as by substituting different coelenterazine analogues to yield semi-synthetic aequorins. Variants of aequorin were created by mutating residues His16, Met19, Tyr82, Trp86, Trp108, Phe113 and Tyr132. Forty-two aequorin mutants were prepared and combined with 10 different coelenterazine analogues in a search for proteins with different emission wavelengths, altered decay kinetics and improved stability. This spectral tuning strategy resulted in semi-synthetic photoprotein mutants with significantly altered bioluminescent properties.

Posterior scleral thickness in perfusion-fixed normal and early-glaucoma monkey eyes.

PURPOSE: To characterize posterior scleral thickness in the normal monkey eye and to assess the effects of acute (15- to 80-minute) and short-term chronic (3- to 7-week) intraocular pressure (IOP) elevations. METHODS: Both eyes of four normal monkeys (both eyes normal) and four monkeys with early glaucoma (one eye normal and one eye with induced chronic elevation of IOP) were cannulated. In each monkey, IOP was set to 10 mm Hg in the normal eye and 30 or 45 mm Hg in the contralateral eye (normal or early glaucoma) for 15 to 80 minutes. All eight monkeys were perfusion fixed, yielding eight low IOP-normal eyes, four high IOP-normal eyes, and four high IOP-early glaucoma eyes. Posterior scleral thickness was measured histomorphometrically at 15 measurement points within each eye, and the data were grouped by region: foveal, midposterior, posterior-equatorial, and equatorial. RESULTS: Overall, posterior scleral thickness was significantly different in the various regions and among the treatment groups (P < 0.0001). In the low IOP-normal eyes, the posterior sclera was thickest in the foveal region (307 microm) and thinner in the midposterior (199 microm), posterior-equatorial (133 microm), and equatorial (179 microm) regions. In the high IOP-normal and high IOP-early glaucoma eyes, the posterior sclera was thinner both overall and within specific regions, compared with the low IOP-normal eyes. CONCLUSIONS: The posterior sclera in the perfusion-fixed normal monkey eye thins progressively from the fovea to the equator and is thinnest just posterior to the equator. Acute and short-term chronic IOP elevations cause regional thinning within the posterior sclera of some monkey eyes, which significantly increases stresses in the scleral wall.

Does the Stanford Health Assessment Questionnaire have potential as a monitoring tool for subjects with rheumatoid arthritis?

OBJECTIVE: To assist in the interpretation of the Stanford Health Assessment Questionnaire (HAQ) score changes for individual patients with rheumatoid arthritis (RA), by determining the minimum size of score change that can confidently be considered to reflect a significant change in disability from the patient's perspective. METHOD: HAQ score changes were calculated for 40 clinic patients with RA who had reported no change to health in general over two months. These were considered to reflect both inconsistencies in questionnaire completion and any true but minor changes not considered significant enough by the patients to represent a change to their health in general. HAQ score changes over one year were also calculated for 207 clinic patients with RA. RESULTS: The range within which 95% of score changes would be expected to lie in the absence of significant change was estimated as +/-0.48 points (+/-2SD of the score changes) and 80% within +/-0.31 points (+/-1.29SD). A chi(2) test showed no significant association between an HAQ score increase of >0.31 over one year and decline in health related to arthritis reported by the patient over the same period. CONCLUSION: As a general guideline, an HAQ score needs to change by 0.48 points or more for 95% confidence that it reflects significant change (0.31 for 80% confidence). Although the value of HAQ as a group outcome measure is well established, this study questions the usefulness of monitoring individual HAQ scores in a clinical setting.

Chlorocatechol detection based on a clc operon/reporter gene system.

A sensitive and selective sensing system for chlorocatechols (3-chlorocatechol and 4-chlorocatechol) was developed based on Pseudomonas putida bacteria harboring the plasmid pSMM50R-B'. In this plasmid, the regulatory protein of the clc operon, ClcR, controls the expression of the reporter enzyme beta-galactosidase. When bacteria containing components of the clc operon are grown in the presence of chlorocatechols, ClcR activates the clcA promoter, which is located upstream from the beta-galactosidase gene. Thus, the concentration of chlorocatechols can be related to the production of beta-galactosidase in the bacteria. The concentration of beta-galactosidase expressed in the bacteria was determined by measuring the chemiluminescence signal emitted with the use of a 1,2-dioxetane substrate. ClcR has a high specificity for chlorocatechols and provides the sensing system with high selectivity. This was demonstrated by evaluating several structurally related organic compounds as potential interfering agents. Both 3-chlorocatechol and 4-chlorocatechol can be detected with this sensing system at concentrations as low as 8 x 10(-10) and 2 x 10(-9) M, respectively, using a 2-h induction period. In the case of 3-chlorocatechol, a highly selective sensing system was developed that can detect this species at concentrations as low as 6 x 10(-8) M after a 5-min induction period; the presence of 4-chlorocatechol at concentrations as high as 2 x 10(-4) M did not interfere with this system.

Altered entrainment and feedback loop function effected by a mutant period protein.

The period (per) and timeless (tim) genes encode interacting components of the circadian clock. Levels and phosphorylation states of both proteins cycle with a circadian rhythm, and the proteins drive cyclic expression of their RNAs through a feedback mechanism that is, at least in part, negative. We report here that a hypophosphorylated mutant PER protein, produced by creating a small internal deletion, displays increased stability and low-amplitude oscillations, consistent with previous reports that phosphorylation is required for protein turnover. In addition, this protein appears to be defective in feedback repression because it is associated with relatively high levels of RNA and high levels of TIM. Transgenic flies carrying the mutant PER protein display a temperature-dependent shortening of circadian period and are impaired in their response to light, particularly to pulses of light in the late night that normally advance the phase of the rhythm. Interestingly, per RNA is induced by light in these flies, most likely because of the removal of the light-sensitive TIM protein, thus implicating a more direct role for TIM in transcriptional inhibition. These data have relevance for mechanisms of feedback repression, and they also address existing models for the differential behavioral response to light at different times of the night.

A role for the proteasome in the light response of the timeless clock protein.

The cyclic expression of the period (PER) and timeless (TIM) proteins is critical for the molecular circadian feedback loop in Drosophila. The entrainment by light of the circadian clock is mediated by a reduction in TIM levels. To elucidate the mechanism of this process, the sensitivity of TIM regulation by light was tested in an in vitro assay with inhibitors of candidate proteolytic pathways. The data suggested that TIM is degraded through a ubiquitin-proteasome mechanism. In addition, in cultures from third-instar larvae, TIM degradation was blocked specifically by inhibitors of proteasome activity. Degradation appeared to be preceded by tyrosine phosphorylation. Finally, TIM was ubiquitinated in response to light in cultured cells.

Alterations of per RNA in noncoding regions affect periodicity of circadian behavioral rhythms.

Circadian rhythms in Drosophila depend on a molecular feedback loop that includes products of the period (per) and timeless (tim) genes. RNA and protein products of both genes cycle with a circadian period and the proteins feedback to inhibit expression of their own mRNAs. While cyclic expression of PER protein appears to be necessary for rhythmic behavior, the function of per RNA cycling is somewhat controversial. Rhythmic transcription accounts, in part, for cycling of per RNA, but it is clear now that posttranscriptional mechanisms also contribute to the cyclic expression of both per RNA and protein. As posttranscriptional mechanisms, such as mRNA stability and translation, are frequently mediated by 3' untranslated regions (UTR) of genes, the authors examined the role of this region of per in the regulation of circadian rhythms. Removal of most of per's 3' UTR had a small effect on the function of a per transgene. However, replacement of per's 3'UTR with corresponding sequences of the tubulin gene led to the rescue of behavioral rhythms in per01 flies with periods that were 3 h shorter than those generated by a wild-type per transgene. The hybrid RNA cycles, but the protein produced by it accumulates earlier in a day-night cycle than the PER protein produced by a control per transgene carrying its own 3'UTR, perhaps because the tubulin sequences counteract the effect of destabilizing elements in the per RNA at earlier points in the circadian cycle. These data indicate that the appropriate regulation of per RNA expression, effected by transcriptional as well as posttranscriptional mechanisms, is critical for the determination of circadian period.

Bacterial biosensors for monitoring toxic metals.

Biosensors utilize biological components to provide selectivity for monitoring compounds of environmental, clinical and industrial importance. A number of biosensors based on bacteria have recently been developed for monitoring toxic metals in the environment. The advantages and disadvantages of these types of biosensors are discussed.

Regulation of the Drosophila protein timeless suggests a mechanism for resetting the circadian clock by light.

Circadian behavioral rhythms in Drosophila depend on the appropriate regulation of at least two genes, period (per) and timeless (tim). Previous studies demonstrated that levels of PER and TIM RNA cycle with the same phase and that the PER and TIM proteins interact directly. Here we show the cyclic expression of TIM protein in adult heads and report that it lags behind peak levels of TIM RNA by several hours. We alsoshow that nuclear expression of TIM depends upon the expression of PER protein. Finally, we report that the expression of TIM, but not PER, is rapidly reduced by light, suggesting that TIM mediates light-induced resetting of the circadian clock. Since both PER and TIM RNA are unaffected by light treatment, the effects of light on TIM appear to be posttranscriptional.

Rhythmic expression of timeless: a basis for promoting circadian cycles in period gene autoregulation.

The clock gene timeless (tim) is required for circadian rhythmicity in Drosophila. The accumulation of tim RNA followed a circadian rhythm, and the phase and period of the tim RNA rhythm were indistinguishable from those that have been reported for per. The tim RNA oscillations were found to be dependent on the presence of PER and TIM proteins, which demonstrates feedback control of tim by a mechanism previously shown to regulate per expression. The cyclic expression of tim appears to dictate the timing of PER protein accumulation and nuclear localization, suggesting that tim promotes circadian rhythms of per and tim transcription by restricting per RNA and PER protein accumulation to separate times of day.

The long-term use of D-penicillamine for treating rheumatoid arthritis: is continuous therapy necessary?

Patients with RA often fail to comply with their therapy. We investigated the extent of non-compliance with D-penicillamine therapy and also whether patients needed continuous treatment after they had shown a therapeutic response. We developed a simple urinary test, measuring cysteine-penicillamine mixed disulphide by a high performance liquid chromatography technique, as an indicator of compliance. Using this method we evaluated compliance in 59 consecutive RA patients attending rheumatology outpatients for monitoring of D-penicillamine therapy. Evidence of poor compliance was shown in 39%. There was no relationship between poor compliance and disease activity. A possible explanation for this paradox emerged from a therapeutic study of the efficacy of intermittent treatment in patients in partial clinical remission on long-term D-penicillamine. In 14 randomly selected patients the daily dose of D-penicillamine was reduced in frequency over 6 months from 250-750 mg daily to the same dose taken 1 week out of every 4. The patients were compared to matched controls who remained on continuous therapy. After 30 months both the intermittent and continuous therapy groups had similar clinical and laboratory scores for disease activity. Our results suggest that many patients may not comply with their prescribed D-penicillamine regime, but such variation in compliance may not be clinically important since intermittent treatment may sustain response in the longer term.

Pharmacologically distinct sodium-dependent L-[3H]glutamate transport processes in rat brain.

The transport of L-[3H]glutamate into crude synaptosomal membrane fractions prepared from cerebellum, brainstem, hippocampus, cortex, striatum, and midbrain was characterized. In all brain regions, greater than 95% of the accumulation of radiolabel was sodium-dependent and the concentration-dependence was consistent with a single high affinity site. Dihydrokainate and L-alpha-aminoadipate were region specific inhibitors of uptake; this inhibition was consistent with a competitive mechanism. In the forebrain regions examined, dihydrokainate inhibited transport with IC50s of approx. 100 microM (range from 80 to 170 microM). Transport in cerebellum was essentially dihydrokainate-insensitive L-alpha-Aminoadipate inhibited transport in forebrain regions with IC50s of approx. 700 microM (range from 590 to 800 microM) and inhibited transport in cerebellum with an IC50 of 40 microM. The inhibition data obtained with forebrain and cerebellar tissues were consistent with nearly homogeneous (greater than 80%) populations of non-interacting sites. Inhibition data obtained with tissue prepared from brainstem were best fit to a mixture of the two sites (35-50% of the type observed in cerebellum). Other previously identified uptake inhibitors, including DL-threo-hydroxyaspartate, L-aspartate-beta-hydroxamate, beta-glutamate, and L-cysteine sulfinate were not selective for the two types of transport. These data demonstrate that there are two pharmacologically distinct sodium-dependent high affinity transport systems with heterogeneous regional distributions.

Evidence for the site of lambda insertion in the ilv gene cluster of Escherichia coli K-12.

The experiments reported herein provide evidence that the secondary site of lambda is in the ilvC instead of the ilvA gene.