Clinical presentations and skin denervation in amyloid neuropathy due to transthyretin Ala97Ser.

OBJECTIVE: Familial amyloid polyneuropathy (FAP) due to amyloidogenic transthyretin (TTR) is often associated with impairment of thermonociceptive functions. This study investigated skin innervation and its clinical significance in genetically defined FAP due to a hot-spot Ala97Ser TTR mutation (Ala97Ser). METHODS: Skin biopsies were performed on the distal leg of patients with Ala97Ser, and intraepidermal nerve fiber (IENF) densities were quantified. RESULTS: There were 19 unrelated patients with Ala97Ser manifesting a late-onset (59.47 +/- 5.70 years) generalized neuropathy with disabling motor, sensory, and autonomic symptoms. Against a background of a slowly progressive course, 7 patients (36.8%) exhibited additional rapid declines in neurologic deficits, which were associated with elevation of the protein content in the CSF (p < 0.001). The IENF density was markedly reduced in Ala97Ser patients compared to age- and gender-matched controls (0.99 +/- 1.11 vs 8.31 +/- 2.87 fibers/mm, p < 0.001). Skin denervation was present in all patients and was lower in patients with a higher disability grade (0.17 +/- 0.26 vs 1.37 +/- 1.16 fibers/mm, p = 0.003). Albuminocytologic dissociation in the CSF was observed in 14 patients (73.7%), and the IENF density was negatively correlated with the CSF protein concentration (p = 0.015). CONCLUSIONS: Skin denervation was common in Ala97Ser, and degeneration of cutaneous nerve terminals was correlated with the severity of clinical phenotypes and the level of CSF protein.

Application of the mixed venous blood concentration equation in desflurane anesthesia.

BACKGROUND: We have previously proposed an equation derived from Fick's law and Lin's concept of effective blood concentration (EBC) to calculate the mixed venous blood concentration (MVBC) of isoflurane. Desflurane has a lower blood/air partition coefficient than isoflurane and, as such, promotes a faster induction and recovery from anesthesia. In this study, we investigated the application of the MVBC equation to predict the MVBC of desflurane. METHODS: We maintained anesthesia with a fixed inspired concentration (CI) of desflurane (10%) during cardiac anesthesia in 11 patients. In order to measure the real concentrations of desflurane in mixed venous blood, pulmonary arterial blood samples were collected at different time points via a Swan-Ganz catheter for gas chromatographic-mass spectrometric determination. The relationship between the calculated concentrations and the actual blood sample concentrations of desflurane in mixed venous blood was investigated. Lin's EBC method was also used and the results were compared with those of MVBC. RESULTS: The calculations from our derived MVBC equation and the actual blood concentrations showed a similar kinetic pattern; the concentration levels were approximately the same and correlated well (r = 0.89) during anesthesia. However, the EBC method failed to accurately estimate the actual blood concentrations. CONCLUSIONS: The results demonstrate that our equation, but not the EBC method, may be useful for estimating pulmonary blood concentrations of desflurane. The clinical significance and the importance of the method merit further investigation.

Application of MVBC equation to predict mixed venous blood concentrations of sevoflurane in cardiac anaesthesia.

We have proposed an equation for estimating the real-time mixed venous blood concentration (MVBC) of isoflurane in cardiac anaesthesia. However, information related to the application of our method to sevoflurane is lacking. We studied 12 patients undergoing cardiac surgery and anaesthetised with sevoflurane. At different time points, pulmonary arterial blood samples were collected for gas chromatography-mass spectrometry (GC-MS) to determine the real mixed venous concentrations of sevoflurane. The inspired and expired concentrations of sevoflurane, measured by a gas monitor, were used for the MVBC calculations. Using Bland-Altman analyses, we found that the calculated MVBCs accurately represent the actual concentrations of sevoflurane in pulmonary arterial blood, as shown by a near-zero percentage bias with a 0.14% precision between the two concentrations. The results demonstrated that our equation could be a useful method for estimating the pulmonary blood concentration of sevoflurane.

A novel method for determining the blood/gas partition coefficients of inhalation anesthetics to calculate the percentage of loss at different temperatures.

The loss by blood/gas (lambda) partition of inhalation anesthetics can be estimated by an equation for the percentage of loss. However, because lambdas of inhalation anesthetics at different temperatures have not been fully determined so far, the percentage of loss at varying temperature in various headspace volumes cannot be estimated. Therefore, a novel method was developed for the determination of inhalation anesthetic lambda, in this study. The method was precise, with a relative standard deviation of less than 5%. The average of lambda from seven distinct blood samples at 4 degrees C, 25 degrees C, and 37 degrees C were determined as 6.68, 2.04, and 1.32 of isoflurane; 3.47, 1.10, and 0.65 of sevoflurane; and 2.31, 0.75, and 0.46 of desflurane, respectively. In addition, increasing temperature was found to decrease lambda profoundly by a secondary order mechanism. Using the obtained value of lambda, the percentage of loss of isoflurane, sevoflurane, and desflurane were then predicted using a 5-mL vacuum tube as a collecting container for an example. In conclusion, a novel method was developed here for lambda determination, and lambdas of isoflurane, sevoflurane, and desflurane at various temperatures were given for estimating the loss resulting from liquid/gas partitioning.

Simultaneous determination of fluorinated inhalation anesthetics in blood by gas chromatography-mass spectrometry combined with a headspace autosampler.

Although the fluorinated inhalation anesthetics, including desflurane, sevoflurane, isoflurane, enflurane, and halothane are commonly used, fatal cases resulting from their abuse or misuse have been reported. To date, gas chromatography (GC) equipped with different kinds of detectors has been utilized to analyze inhalation anesthetics. However, none of them can detect desflurane reliably or analyze all five common anesthetics simultaneously. The purpose of the present work is to further modify the previously developed headspace (HS) GC-MS method for blood isoflurane determination to analyze and distinguish five common clinical inhalation anesthetics, simultaneously. The modified HS-GC-MS method adopts a 60 m x 0.25 mm I.D., 0.25 microm film thickness DB-5 capillary column along with an adequate GC temperature program, which gives the five inhalation anesthetics, including isoflurane and its isomer, enflurane, a high resolution. The method also takes both the volatility and the influence of the top space on the obtained concentration into consideration and therefore keeps the sample loss acceptable even for analyzing the highly volatile desflurane. Within a certain concentration range of the calibration standard (about 20-300 microg/ml), this method shows a good linearity with correlation coefficients greater than 0.999. In addition, both within- and between-run precision and accuracy results meet the validation requirements as well as the tested results of practical blood samples of desflurane. In summary, this is a reliable analytical method to simultaneously determine the concentration of five common inhalation anesthetics in blood. Such a method is very practical for both clinical and occupational monitoring, as well as for analytical toxicology.

Reliable gas chromatographic-mass spectrometric method combined with a headspace autosampler for isoflurane determination in blood.

Isoflurane is a nonflammable, liquid, volatile inhalation anesthetic administered by vaporizing. Although it is now commonly used, fatal cases resulting from its abuse or misuse have been reported. A combined system of a gas chromatograph-mass spectrometer and a headspace autosampler is therefore proposed for the detection of blood isoflurane. This analytic method showed sharp and well separated peaks, and revealed a good linear relationship (r=0.9994) with a function of y = 7.3768x - 0.0222 at concentrations between 18.7 and 299.2 microg/ml. The limits of detection and quantitation of this method were 1.2 and 4.7 microg/ml, respectively. The within- and between-run precision for spiked samples, assessed by the coefficient of variations, ranged from 1.7 to 10.0% and from 4.1 to 12.8%, respectively. The within- and between-run accuracy, assessed by errors from theoretical values, were 2.2-7.8% and 2.4-9.6%, respectively. In addition, practical sample analysis showed a good applicability, with a within-run precision rate of 5.6 to 7.7% and a between-run precision rate of 5.2-10.6%. In summary, the present work presents a valid alternative for blood isoflurane analysis.

DHEA inhibits cell growth and induces apoptosis in BV-2 cells and the effects are inversely associated with glucose concentration in the medium.

Dehydroepiandrosterone (DHEA), a major steroid secreted by the adrenal gland which decreases with age after adolescence, is available as a nutritional supplement. DHEA is known to have antiproliferative effects but the mechanism is unclear. In this study using BV-2 cells, a murine microglial cell line, we investigated the effect of DHEA on cell viability and the interaction between DHEA and glucose concentrations in the medium. We showed that DHEA inhibited cell viability and G6PD activity in a dose-dependent manner and that the effect of DHEA on cell viability was inversely associated with glucose concentrations in the medium, i.e. lowered glucose strongly enhanced the inhibition of cell viability by DHEA. DHEA inhibited cell growth by causing cell cycle arrest primarily in the G0--G1 phase, and the effect was more pronounced at zero glucose (no glucose added, G0) than high glucose (4.5 mg/ml of the medium, G4.5). Glucose deprivation also enhanced apoptosis induced by DHEA. At G4.5, DHEA did not induce formation of DNA ladder until it reached 200 microM. However, at G0, 100 microM DHEA was able to induce apoptosis, as evidenced by the formation of DNA ladder, elevation of histone-associated DNA fragmentation and increase in cells positively stained with annexin V-FITC and annexin V-FITC/propidium iodide. The interactions between DHEA and glucose support the contention that DHEA exerts its antiproliferative effects through alteration of glucose metabolism, possibly by inhibition of G6PD activity leading to decreased supply of ribose-5-phosphate for synthesis of DNA and RNA. Although DHEA is only antiproliferative at pharmacological levels, our results indicate that its antiproliferative effect can be enhanced by limiting the supply of glucose such as by energy restriction. In addition, the present study shows that glucose concentration is an important factor to consider when studying the antiproliferative and toxicological effects of DHEA.

Evaluation of the quality of surgical gloves among four different manufactures.

A randomized trial was conducted to evaluate the quality of four different brands of surgical gloves in terms of the perforation rate, ventilation, fitness, allergic reaction, elasticity, thickness, powder, and satisfaction. Gloves of four different manufactures which were used by various medical centres were distributed to participants according to a computer-generated randomization table. A structured questionnaire was self-administered by volunteers immediately after the surgical procedure to gather the information from participants, including the demographic data, allergy history, length of use, and variables of quality measures. Two brands, A and D, were significantly inferior to the best manufacture among the four, B, in terms of the ventilation, elasticity, and thickness, odds ratios ranging from 6 to 24, p < 0.05. For the amount of corn starch powder and satisfaction, all three other brands were inferior to brand B, odds ratios ranging from 6 to 44, p < 0.05. Gloves worn longer than 2 hours had a slightly higher perforation rate post procedures (11.5% vs. 2.1%, p = 0.048). The rate of latex allergic reaction was not significantly different between surgeons (8.3%) and the others (6.7%). No difference of the allergic reaction rate was found between subjects with allergy history (7.7%) and those without the history (7.5%). The quality of surgical gloves differs from brand to brand. The government and institutions should take the responsibility of monitoring the quality of surgical gloves in order to provide a safer and more comfortable environment for the surgical personnel and patients.

Participation of Galaninergic Neurotransmission at the Paraventricular Hypothalamic Nucleus in the Suppression of Baroreceptor Reflex Response by Locus ceruleus in the Rat.

We evaluated the potential participation of galanin (GAL) at the paraventricular nucleus of hypothalamus (PVN) in the suppression of baroreceptor reflex (BRR) response by locus ceruleus (LC), using adult male Sprague-Dawley rats anesthetized with pentobarbital sodium. Microinjection of GAL (100 pmol) bilaterally into the PVN significantly depressed the BRR response. This suppressive effect was appreciably antagonized when GAL (100 pmol) and GAL antiserum (1:20) were coadministered into the bilateral PVN. Whereas bilateral microinjection of GAL antiserum into the PVN by itself elicited minimal effect, it nevertheless significantly attenuated the suppressive effect of either electrical or chemical activation of LC on the BRR response. Pretreatment with the same amount of normal rabbit serum (1:20), on the other hand, was ineffective. These results suggest that a galaninergic projection from the LC to PVN may participate in the suppression of BRR response by this dorsal pontine nucleus. Copyright 1997 S. Karger AG, Basel

A novel, multilayer structure of a helical peptide.

X-ray diffraction analysis at 1.5 A resolution has confirmed the helical conformation of a de novo designed 18-residue peptide. However, the crystal structure reveals the formation of continuous molecular layers of parallel-packed amphiphilic helices as a result of much more extensive helix-helix interactions than predicted. The crystal packing arrangement, by virtue of distinct antiparallel packing interactions, segregates the polar and apolar surfaces of the helices into discrete and well-defined interfacial regions. An extensive "ridges-into-grooves" interdigitation characterizes the hydrophobic interface, whereas an extensive network of salt bridges and hydrogen bonds dominates the corresponding hydrophilic interface.

Reversion of v-H-ras-transformed NIH 3T3 cells by apigenin through inhibiting mitogen activated protein kinase and its downstream oncogenes.

Apigenin, a plant flavonoid, induced the reversion of transformed phenotypes of v-H-ras-transformed NIH 3T3 cells at a quite low concentration of 12.5 microM. In the present study, we have examined the components of this Ras-mediated signaling transduction to determine whether they were involved in the apigenin-induced reversion process. Interestingly, the consitutively activated mitogen activated protein kinase (MAPK) in the ras transformant was inhibited significantly and rapidly by 25 microM apigenin within 30 min, and this reduction continued for more than 4 h. Corroborating these observations, expression of the downstream oncogenes c-jun and c-fos was also dramatically reduced during the first 4 h of treatment. We found that the levels of ras protein and mRNA were not affected by 24 h of treatment with apigenin. These findings indicate that apigenin-induced reversion of v-H-ras-transformed NIH 3T3 cells may occur by inhibiting MAPK activity and its downstream oncogenes rather than by affecting the expression of the ras gene.

Reversion of the transformed phenotypes of v-H-ras NIH3T3 cells by flavonoids through attenuating the content of phosphotyrosine.

Fifteen flavonoids were employed to examine their effects on the morphological changes, foci formation in soft agar and cellular growth in v-H-ras-transformed NIH3T3 cells. The data presented here demonstrated that only three specific flavonoids--apigenin, kaempferol, and genistein--exhibited the reverting effect on the transformed phenotypes of ras-3T3 cells. For example, treatment with 25 microM of these flavonoids could effectively reverse the transformed morphology of ras-3T3 cells into flatter cells with contact inhibition. Colony formation in soft agar was decreased to 0.11%, 0.15%, and 0.35% by 25 microM apigenin, kaempferol, and genistein, respectively, as compared with control (0.92%). It was also found that the proliferation of ras-3T3 cells was significantly inhibited by these compounds in a dose-dependent manner. Finally, two biochemical parameters, the content of phosphotyrosine and cAMP, were examined to see whether affected by these compounds. The results showed the phosphotyrosine content in ras-3T3 cells was dramatically decreased by apigenin and kaempferol, but that was slightly reduced by genistein. By contrast, these three flavonoids all failed to significantly alter the level of cAMP within this transformant. Based on these observations, we suggest that some specific flavonoids are capable of reverting the transforming properties of v-H-ras transformed cells. The possible mechanism of this reversion is not mediated by activating the protein kinase A or its associated pathways, but rather inhibiting tyrosine kinases, subsequently leading to the blockage of p21ras-mediated signal transduction circuitry.

Effects of signalling transduction modulators on the transformed phenotypes in v-H-ras-transformed NIH 3T3 cells.

Several signalling transduction modulators were used to examine their effects on the morphological changes, foci formation in soft agar and cellular growth in v-H-ras-transformed NIH 3T3 cells. The results from this study showed that specific tyrosine kinase inhibitors (genistein and tyrphostin 23) and cyclic AMP-elevating agents (forskolin and 3-isobutyl-1-methyl-xanthine) could effectively induce differential flat phenotype of v-H-ras transformant at micromolar concentrations. At the same dose range, both signalling modulators also caused a significant suppression of anchorage-independent and cellular growth in the same transformant. By contrast, compound inhibitors such as protein kinase C (staurosporin and H-7), phospholipase A2 (aristolochic acid), phospholipase C (neomycin sulfate) and cyclooxygenase (indomethacin) all did not alter the cellular morphology or foci formation in soft agar, although PKC inhibitors exhibited a slight inhibition on the cellular growth. Based on these observations, we propose that the alterations of protein kinase A or tyrosine kinase-associated signal pathways is necessary and the original cause of the transformation event, but that increase of the activities of protein kinase C, phospholipase C, phospholipase A2 or cyclooxygenase probably is an indirect result of the v-H-ras-mediated transformation.

Fluorescence studies of rat cellular retinol binding protein II produced in Escherichia coli: an analysis of four tryptophan substitution mutants.

Rat intestinal cellular retinol binding protein II (CRBP II) is an abundant 134-residue protein that binds all-trans-retinol which contains 4 tryptophans in positions 9, 89, 107, and 110. Our ability to express CRBP II in Escherichia coli and to construct individual tryptophan substitution mutants by site-directed mutagenesis has provided a useful model system for studying the fluorescence of a multi-tryptophan protein. Each of the four mutant proteins binds all-trans-retinol with high affinity, although their affinities are less than that of the wild-type protein. Steady-state and time-resolved fluorescence analyses of these proteins indicate that W107 is at the hydrophobic binding site, W110 is in a polar environment, and the remaining two tryptophans are in a hydrophobic environment. Time-resolved fluorescence study indicates that excited-state energy transfer occurs from the hydrophobic tryptophans to W110. The Stern-Volmer analysis with acrylamide of these proteins reveals that static quenching occurs in the W9F mutant protein while others do not. The fluorescence of rat intestinal fatty acid binding protein (I-FABP), a related protein of known X-ray structure, was also studied for comparison. The results of these findings, coupled with those derived from NMR studies and molecular graphics, suggest that CRBP II undergoes minor structural changes in all of the mutant proteins. Since these effects may be cumulative on the protein structure and function, any conclusions derived from higher mutants in this family of proteins must be treated with caution.

Quantitative SPECT for indium-111-labeled antibodies in the livers of beagle dogs.

Results are presented for SPECT computations of liver volumes and 111In-labeled antibody activities in the livers of eight normal beagle dogs. Administered activities ranged from 1 to 2 mCi. SPECT studies were acquired 1 day postinjection using a rotating gamma camera system with elliptical orbits in a 360-degree rotation (128 views, 15 sec/view, 64 x 64 matrices). Uniformity-corrected images were reconstructed by use of the circular harmonic transform algorithm with computer software developed in-house. Liver volumes and activities were computed from transverse slices, 1 pixel (6.25 mm) in thickness. Comparison of SPECT and autopsy data demonstrated that absolute values of percent differences between measured and computed liver volumes ranged from 1.0% to 7.2%. Absolute values of percent differences between autopsy data and computed 111In activities in the liver ranged from 2.3% to 7.5%. These results suggest that quantitative SPECT has the potential of becoming an important tool in clinical trials for determining activities and localization volumes of radiolabeled antibodies directly from radionuclide images.

Validation of the circular harmonic transform (CHT) algorithm for quantitative SPECT.

The purpose of this study was to validate the use of the circular harmonic transform (CHT) algorithm for quantitative single-photon emission computed tomography (SPECT) with isotopes technetium-99m (99mTc) and indium-111 (111In) under clinically relevant conditions. Phantom studies were the principal tools used. Volumes of fillable organs within a tissue-equivalent anthropomorphic phantom were determined over a wide range (145-1960 ml) to within 6% by using a thresholding technique. Additionally, phantom studies with nonuniform activity distributions were made. These included a background of activity and hot as well as cold lesions. The hot lesion was computed to within 12% (111In) and 7.7% (99mTc), and contrast in the cold lesion was approximately 70% for both isotopes. The CHT algorithm incorporates the energy-distance relation (EDR) which minimizes the degrading effects of attenuation, scatter, collimator blur and poor statistics.

19F nuclear magnetic resonance studies of 6-fluorotryptophan-substituted rat cellular retinol binding protein II produced in Escherichia coli. An analysis of four tryptophan substitution mutants and their interactions with all-trans-retinol.

Rat cellular retinol binding protein (CRBP II) is a 134-amino acid intracellular protein synthesized in the polarized absorptive cells of the intestine. We have previously used 19F nuclear magnetic resonance (NMR) spectroscopy to survey the structural effects of ligand binding on the apoprotein. For these studies, all 4 Trp residues of rat CRBP II were efficiently labeled with 6-fluorotryptophan (6-F-Trp) by inducing its expression in a tryptophan auxotroph of Escherichia coli. Resonances corresponding to 2 of its Trp residues underwent large downfield shifts upon binding of all-trans-retinol and retinal, while resonances corresponding to the other 2 Trp residues underwent only minor perturbations in chemical shifts. To identify which Trp residues undergo changes in their environment upon ligand binding, we have constructed four CRBP II mutants where Trp9, Trp89, Trp107, or Trp110 have been replaced by another hydrophobic amino acid. By comparing the 19F NMR spectrum of each 6-F-Trp-labeled mutant with that of wild type 6-F-Trp CRBP II, we demonstrate that the 19F resonance corresponding to Trp107 undergoes the largest change in chemical shift upon ligand binding (2.0 ppm downfield). This is consistent with the position of this residue predicted from molecular modeling studies. The 19F resonance corresponding to Trp9 also undergoes a downfield change in chemical shift of 0.5 ppm associated with retinol binding even though it is predicted to be removed from the ligand binding site. By contrast, the resonances assigned to Trp89 and Trp110 undergo only minor perturbations in chemical shifts. These results have allowed us to identify residue-specific probes for evaluating the interactions of all-trans-retinol (and other retinoids) with this intracellular binding protein.

Nuclear magnetic resonance studies of 6-fluorotryptophan-substituted rat cellular retinol-binding protein II produced in Escherichia coli. Analysis of the apoprotein and the holoprotein containing bound all-trans-retinol and all-trans-retinal.

Rat cellular retinol-binding protein II (CRBP II) is a 15.6-kDa intestinal protein which binds all-trans-retinol and all-trans-retinal but not all-trans-retinoic acid. We have previously analyzed the interaction of Escherichia coli-derived rat apoCRBP II with several retinoids using fluorescence spectroscopic techniques. Interpretation of these experiments is complicated, because the protein has 4 tryptophan residues. To further investigate ligand-protein interactions, we have utilized 19F nuclear magnetic resonance (NMR) spectroscopy of CRBP II labeled at its 4 tryptophan residues with 6-fluorotryptophan. Efficient incorporation of 6-fluorotryptophan (93%) was achieved by growing a tryptophan auxotroph of E. coli harboring a prokaryotic expression vector with a full-length rat CRBP II cDNA on defined medium supplemented with the analog. Comparison of the 19F NMR spectra of 6-fluorotryptophan-substituted CRBP II with and without bound all-trans-retinol revealed that resonances corresponding to 2 tryptophan residues (designated WA and WB) undergo large downfield changes in chemical shifts (2.0 and 0.5 ppm, respectively) associated with ligand binding. In contrast, 19F resonances corresponding to two other tryptophan residues (WC and WD) undergo only minor perturbations in chemical shifts. The 19F NMR spectra of 6-fluorotryptophan-substituted CRBP II complexed with all-trans-retinal and all-trans-retinol were very similar, suggesting that the interactions of these two ligands with the protein are similar. Molecular model building, based on the crystalline structures of two homologous proteins was used to predict the positions of the 4 tryptophan residues of CRBP II and to make tentative resonance assignments. The fact that ligand binding produced residue-specific changes in the chemical shifts of resonances in CRBP II suggests that NMR analysis of isotopically labeled retinoid-binding proteins expressed in E. coli will provide an alternate, albeit it complementary, approach to fluorescence spectroscopy for examining the structural consequences of their association with ligand.