A proof-of-concept study comparing tinnitus and neural connectivity changes following multisensory perceptual training with and without a low-dose of fluoxetine.

Background. This proof-of-concept study investigated a method of multisensory perceptual training for tinnitus, and whether a short, low-dose administration of fluoxetine enhanced training effects and changed neural connectivity.Methods. A double-blind, randomized placebo controlled design with 20 participants (17 male, 3 female, mean age = 57.1 years) involved 30 min daily computer-based, multisensory training (matching visual, auditory and tactile stimuli to perception of tinnitus) for 20 days, and random allocation to take 20 mg fluoxetine or placebo daily. Behavioral measures of tinnitus and correlations between pairs of a priori regions of interest (ROIs), obtained using resting-state functional magnetic resonance imaging (rs-fMRI), were performed before and after the training.Results. Significant changes in ratings of tinnitus loudness, annoyance, and problem were observed with training. No statistically significant changes in Tinnitus Functional Index, Tinnitus Handicap Inventory or Depression Anxiety Stress Scales were found with training. Fluoxetine did not alter any of the behavioural outcomes of training compared to placebo. Significant changes in connectivity between ROIs were identified with training; sensory and attention neural network ROI changes correlated with significant tinnitus rating changes. Rs-fMRI results suggested that the direction of functional connectivity changes between auditory and non-auditory networks, with training and fluoxetine, were opposite to the direction of those changes with multisensory training and placebo.Conclusions. Improvements in tinnitus measures were correlated with changes in sensory and attention networks. The results provide preliminary evidence for changes in rs-fMRI accompanying a multisensory training method in persons with tinnitus.

Molecular mechanism of spectral tuning in sensory rhodopsin II.

Sensory rhodopsin II (SRII) is unique among the archaeal rhodopsins in having an absorption maximum near 500 nm, blue shifted roughly 70 nm from the other pigments. In addition, SRII displays vibronic structure in the lambda(max) absorption band, whereas the other pigments display fully broadened band maxima. The molecular origins responsible for both photophysical properties are examined here with reference to the 2.4 A crystal structure of sensory rhodopsin II (NpSRII) from Natronobacterium pharaonis. We use semiempirical molecular orbital theory (MOZYME) to optimize the chromophore within the chromophore binding site, and MNDO-PSDCI molecular orbital theory to calculate the spectroscopic properties. The entire first shell of the chromophore binding site is included in the MNDO-PSDCI SCF calculation, and full single and double configuration interaction is included for the chromophore pi-system. Through a comparison of corresponding calculations on the 1.55 A crystal structure of bacteriorhodopsin (bR), we identify the principal molecular mechanisms, and residues, responsible for the spectral blue shift in NpSRII. We conclude that the major source of the blue shift is associated with the significantly different positions of Arg-72 (Arg-82 in bR) in the two proteins. In NpSRII, this side chain has moved away from the chromophore Schiff base nitrogen and closer to the beta-ionylidene ring. This shift in position transfers this positively charged residue from a region of chromophore destabilization in bR to a region of chromophore stabilization in NpSRII, and is responsible for roughly half of the blue shift. Other important contributors include Asp-201, Thr-204, Tyr-174, Trp-76, and W402, the water molecule hydrogen bonded to the Schiff base proton. The W402 contribution, however, is a secondary effect that can be traced to the transposition of Arg-72. Indeed, secondary interactions among the residues contribute significantly to the properties of the binding site. We attribute the increased vibronic structure in NpSRII to the loss of Arg-72 dynamic inhomogeneity, and an increase in the intensity of the second excited (1)A(g)(-) -like state, which now appears as a separate feature within the lambda(max) band profile. The strongly allowed (1)B(u)(+)-like state and the higher-energy (1)A(g)(-) -like state are highly mixed in NpSRII, and the latter state borrows intensity from the former to achieve an observable oscillator strength.

Evidence for a perturbation of arginine-82 in the bacteriorhodopsin photocycle from time-resolved infrared spectra.

Arginine-82 (R82) of bacteriorhodopsin (bR) has long been recognized as an important residue due to its absolute conservation in the archaeal rhodopsins and the effects of R82 mutations on the photocycle and proton release. However, the nature of interactions between R82 and other residues of the protein has remained difficult to decipher. Recent NMR studies showed that the two terminal nitrogens of R82 experience a highly perturbed asymmetric environment during the M state trapped at cryogenic temperatures [Petkova et al. (1999) Biochemistry 38, 1562-1572]. Although previous low-temperature FT-IR spectra of wild-type and mutant bR samples have demonstrated effects of R82 on vibrations of other amino acid side chains, no bands in these spectra were assignable to vibrations of R82 itself. We have now measured time-resolved FT-IR difference spectra of bR intermediates in the wild-type and R82A proteins, as well as in samples of the R82C mutant with and without thioethylguanidinium attached via a disulfide linkage at the unique cysteine site. Several bands in the bR --> M difference spectrum are attributable to guanidino group vibrations of R82, based on their shift upon isotope substitution of the thioethylguanidinium attached to R82C and on their disappearance in the R82A spectrum. The frequencies and intensities of these IR bands support the NMR-based conclusion that there is a significant perturbation of R82 during the bR photocycle. However, the unusually low frequencies attributable to R82 guandino group vibrations in M, approximately 1640 and approximately 1545 cm(-)(1), would require a reexamination of a previously discarded hypothesis, namely, that the perturbation of R82 involves a change in its ionization state.

Interactions between dietary calcium and caffeine consumption on calcium metabolism in hypertensive humans.

Abnormal calcium metabolism has been implicated in human hypertension. Caffeine consumption may contribute to hypertension since it increases urinary calcium excretion. Nineteen hypertensive subjects (HTN) and nineteen age and gender matched normotensive controls (NTC) who habitually consumed at least 175 mg caffeine daily were studied before and after abstinence from all caffeine (CAF) consumption for 2 weeks. Caffeine abstinence (CAF-) increased fasting serum ultrafiltrable calcium in HTN and NTC, but not serum total calcium. Parathyroid hormone (PTH) levels decreased after CAF abstinence in 14 of 18 HTN subjects, including all seven subjects consuming less than 700 mg calcium daily. Three day dietary calcium intakes and 72 h urinary excretion of calcium were not different between CAF+ and CAF- or between HTN and NTC. A morning caffeine dose of 6 mg/kg lean body mass increased urinary Ca/creatinine ratios similarly for 2 h after beverage consumption in both HTN and NTC. Caffeine consumption stresses calcium metabolism in hypertensive individuals, especially those consuming less than 700 mg calcium daily.

Interactions between dietary caffeine and calcium on calcium and bone metabolism in older women.

OBJECTIVE: Our purpose was to determine the effect of 2 weeks of caffeine abstinence on calcium (Ca) and bone metabolism in women habitually consuming caffeine and either low or moderate amounts of Ca. METHODS: Participants were 25 women, aged 39-76 years (mean 65 years, median 57 years) habitually consuming at least 200 mg caffeine daily. Three days of dietary records and 24-hour urine collections were made immediately prior to collection of fasting blood and 1-hour urine in a metabolic unit. Women were classified as low Ca consumers (414-584 mg daily) or moderate Ca consumers (662-1357 mg daily) based on 6 days of diet records. RESULTS: Women in the low Ca group had higher levels of serum ultrafiltrable Ca (UFCa) after caffeine abstinence (1.40 mmol/L CAF+ vs 1.52 mmol/L CAF-, p < 0.01), while there were no differences between experimental periods for UFCa in the moderate Ca group (1.35 mmol/L CAF+ vs 1.38 mmol/L CAF-, ns). Women in the low Ca group also had lower serum bone isoenzyme alkaline phosphatase levels after caffeine abstinence (9.3 U/L CAF+ vs 8.8 U/L CAF-, p < 0.05), while no significant changes were seen in bone alkaline phosphatase in the moderate Ca consumers (8.7 U/L CAF+ vs 8.9 U/L CAF-, ns). Fasting total serum Ca, urinary hydroxyproline/creatinine, and Ca/creatinine ratios were unchanged in both dietary groups. Three-day mean 24-hour urinary Ca excretion decreased after caffeine abstinence in the moderate Ca group only. There were no differences in dietary intakes of Ca from dairy products between CAF+ and CAF- in either the low or moderate Ca groups. CONCLUSION: Abstinence from moderate caffeine intake (mean 5.8 mg/kg lean body mass, 383 mg/day caffeine) raises ultrafiltrable Ca and decreases bone alkaline phosphatase in older women consuming < 600 mg Ca daily.

The hemodynamic effect of dietary calcium supplementation on rat renovascular hypertension.

Mechanisms which could be responsible for the hypotensive effects of increasing calcium intake from 1.2 to 2.5% of diet were examined in two kidney, one clip renovascular hypertension (2K, 1C RHV) in male Sprague-Dawley rats. Plasma renin activity was elevated similarly in NCA and HCA rats. No evidence for volume or phosphate depletion was found in HCA animals. Cardiac output was not different. Decreased sensitivity was demonstrated to moderate doses of norepinephrine in HCA rats, while vascular reactivity to exogenously administered angiotensin II over the range of doses used was not different between diets. The antihypertensive effect of supplemental calcium was associated with a 39% reduction in systemic vascular resistance. Therefore, dietary calcium supplementation lowers blood pressure in 2-K, 1C RVH primarily due to a systemic vasodilatation and reduced vascular reactivity to moderate doses of norepinephrine.

Pre-competition anxiety and performance in female high school swimmers: a test of optimal function theory.

This investigation attempted to test Hanin's theory of optimal function in 15 female high school swimmers. Each swimmer completed the State Anxiety Inventory (STAI) and the Body Awareness Scale (BAS) under the following conditions: I) baseline; II) retrospective recall of "best", "usual", and "worst" performances; III) 24 h prior to an easy and difficult dual meet with instructions to respond as to "how you think you will feel 1 h before the meet"; and IV) 1 h before the meets utilizing the standard ("right now") instructional set. Success was determined in each meet by two methods: 1) objective ratings based on comparisons with the average of times from previous meets; and 2) subjective ratings of performance made by the coach. In the difficult meet significant (P less than 0.01) increases in precompetition anxiety and body awareness occurred, and the correlations between predicted and actual pre-competition were .95 (P less than 0.001) for both anxiety and body awareness. In the difficult meet swimmers subjectively classified as successful were more accurate (P less than 0.05) in predicting precompetition anxiety, and possessed pre-competition anxiety values that were closer to their recall of optimal pre-competition anxiety compared to the unsuccessful swimmers. No comparisons based on the objective classifications were significant, nor were any of the comparisons in the easy meet. The present findings support Hanin's optimal function theory.

Effects of dietary caffeine on renal handling of minerals in adult women.

Thirty-seven women, aged 31-78 years, on two separate mornings consumed a decaffeinated beverage to which 6 mg caffeine/kg lean body mass or no caffeine were added. Total urine output of water, calcium, magnesium, sodium, chloride, potassium and creatinine increased in the two hours following caffeine ingestion when compared to the control beverage. Increased urinary mineral (mg)/urinary creatinine (g) ratios were seen for calcium (120 to 200), magnesium (70 to 110), sodium (3,800 to 6,200) and chloride (9,200 to 14,800), following the caffeinated beverage. Creatinine clearance did not change significantly. The percent reabsorption of calcium (98.6% to 97.5%, p less than .001) and magnesium (97.0% to 94.2%, p less than .0001) decreased significantly during the post-caffeine period. The calcium and magnesium filtered loads did not differ significantly between the caffeine and no caffeine beverages. Therefore, caffeine-induced urinary loss of calcium and magnesium is largely attributable to a reduction in calcium and magnesium renal reabsorption, although the physiological mechanism and tubular segment affected remain to be established.

Measurement of drug displacement of protein-bound bilirubin using gel filtration.

Gel filtration was used to measure drug interaction with protein-bound bilirubin in 0.5 ml samples of Gunn rat serum, human serum and fraction V human serum albumin. Using sulfadimethoxine as a prototype differences in displacement were found in all 3 sera. The differences between human and rat serum were related to the binding characteristics of sulfadimethoxine whereas the differences between human serum and fraction V human serum albumin were attributed to displacement of bilirubin from albumin to other proteins in serum. Gel filtration permitted the use of small samples with bilirubin-albumin ratios less than 1.0 and provided data that were used for analysis of drug displacement of bilirubin using principles of drug-receptor theory. Ten of 14 drugs found to alter serum bilirubin concentrations in icteric Gunn rats had measurable effects on protein binding of bilirubin.

Effects of bilirubin on glucuronidation of p-nitrophenol and morphine.

Addition of bilirubin in vitro to hepatic microsomes or 9,333 g supernatant activated UDP glucuronyltransferase activity (UDPGT) for p-nitrophenol of livers from Sprague-Dawley rats, Wistar rats, icteric Gunn rats and nonicteric Gunn rats. However, injection of bilirubin at subtoxic doses using a variety of regimens failed to produce detectable activation of UDPGT in intact or bile duct-ligated rats. Pretreatment with intraperitoneal injections of bilirubin increased the rate of morphine glucuronide excretion in both Sprague-Dawley and icteric Gunn rats. Sulfadimethoxine did not affect morphin e excretion in Gunn rats. The data suggest that activation of UDPGT is of little, if any, significance in hyperbilirubinemia although the basis for increased excretion of morphine following bilirubin pretreatment has not been established.

Plasma disappearance of sulfobromophthalein or indocyanine green in unconjugated hyperbilirubinemia.

Unconjugated hyperbilirubinemia as occurs in Gunn rats did not affect the plasma disappearance of sulfobromophthalein (BSP) or indocyanine green (ICG). The simultaneous injection of bilirubin and ICG decreased the plasma disappearance of ICG apparently by decreasing hepatic uptake. The data suggest that although bilirubin, ICG and BSP bind to the intracellular hepatic transport protein ligandin one cannot achieve conditions in intact rats to demonstrate effects of competitive interaction with ligandin that affect plasma kinetics of these substances.