The Leicester semi-automated olfactory threshold test--a psychophysical olfactory test for the 21st century.

OBJECTIVE: Develop a useful and cost-effective olfactometer for routine clinical use by providing a standardised threshold test for patients with olfactory disorders presenting in the ENT clinic. METHOD OF STUDY: A prospective study of olfactory thresholds in 48 healthy volunteers on 2 consecutive occasions, undergoing quantitative testing with an olfactometer. Further studies of 10 subjects performing 20 tests and 100 subjects performing a single test were performed. An olfactometer was designed to deliver a semi-automated threshold test for an odour. It contains 8 logarithmic dilutions of an odour along with a control valve operated by software from a laptop computer. Common potential variables for olfactory threshold testing were considered including peak inspiratory flow rate. The odours used were phenethyl alcohol (PEA) and eucalyptol (EUC). Subjects were asked to perform 2 tests within 1 month of each other and the mean threshold score for each was calculated to derive a test-retest score. MAIN RESULTS: Consistent olfactory thresholds for PEA were achieved with a mean concentration of 10-4. Test-retest reliability score (r(x)) for the olfactometer was r(x) = 0.78 (95% CI 0.67 to 0.89). PRINCIPAL CONCLUSIONS: The Leicester Olfactometer provides a simple and cost-effective method of reliably assessing olfactory thresholds in the outpatient clinic.

Does odour cross contamination alter olfactory thresholds in certain odours?

OBJECTIVES: To determine if 1) there is cross contamination between odours tested on thresholds achieved, 2) a delay period is necessary between testing different odours. METHODS: Thirty-five subjects underwent threshold testing with phenethyl alcohol (PEA), ethylmercaptan (MER), acetic acid (ACE), and eucalyptol (EUC) using serial logarithmic dilutions. On separate occasions subjects were exposed to high concentrations of PEA, ACE and EUC in random order for two minutes, and thresholds for all four odours re-tested. Pre- and post-high concentration odour thresholds were compared. RESULTS: Exposure to high concentrations of PEA, ACE and EUC does not alter olfactory thresholds by more than 10-2 for the other odours except in specific circumstances with ACE and EUV. CONCLUSIONS: There is limited cross contamination with ACE and EUC, which is avoided by specifying presentation order as: PEA, MER, ACE, EUC. Odours PEA, MER, ACE and EUC are recommended for olfactory testing.

The superosmic phenomenon.

BACKGROUND: Olfaction studies in the institution of Department of Otorhinolaryngology at Leicester Royal Infirmary have detected a previously unreported, variable phenomenon--'superosmia'--in which subjects' olfaction threshold concentrations are up to 100,000 smaller than the average value. OBJECTIVES: The aim of this report is to define and quantify this phenomenon. METHODS: Two hundred and thirty subjects, who had been screened for active nasal pathology (age range 20-60 years), underwent individual olfactory threshold tests for phenylethyl alcohol or eucalyptol, using a computer-driven olfactometer in a controlled laboratory setting. Some tests were single tests and others were repeated on a small cohort. RESULTS: Two per cent of subjects demonstrated the superosmic phenomenon on single testing, and 10 per cent demonstrated this phenomenon on variable occasions during repeated testing. The superosmic phenomenon was defined by: (1) confident olfactory perception of a threshold at least equal to if not greater than three threshold levels below the subject's average threshold; (2) repeated perception of the odour at this level for at least 10 responses (1:1024 probability of chance finding); and (3) (where time permitted) a sudden, rapid loss of superosmia. CONCLUSIONS: Superosmia is a distinct phenomenon, the stimulus or mechanism of which is currently the subject of further research. The enhancement of olfactory ability may be possible through activation of an accessory pathway or modulation of the existing olfactory apparatus.

Olfactory clearance: what time is needed in clinical practice?

OBJECTIVE: To determine olfactory adaptation and clearance times for healthy individuals, and to assess the effect of common variables upon these parameters. STUDY DESIGN AND SETTING: Fourteen healthy volunteers were recruited for a series of tests. Their initial olfactory threshold levels for phenethyl alcohol were determined. After olfactory exposure to a saturated solution of phenethyl alcohol (i.e. olfactory adaptation), the time taken for subjects to return to their initial olfactory threshold was then recorded (i.e. olfactory clearance). Visual analogue scale scores for subjective variables were also recorded. RESULTS: The 14 subjects performed 120 tests in total. Despite consistent linear trends within individuals, olfactory clearance times varied widely within and between individuals. The mean olfactory clearance time for phenethyl alcohol was 170 seconds (range 81-750). Univariate analysis showed a relationship between olfactory clearance times and age (p = 0.031), symptoms (p = 0.029) and mood (p = 0.048). CONCLUSIONS: When testing a person's sense of smell in a clinical setting, recent exposure to similar smells should be noted, and a period of 15 minutes needs to be allowed before retesting if using phenethyl alcohol. Other variables need not be controlled, but greater clearance time may be needed for older patients.

What is the short term effect of perfumes on olfactory thresholds?

OBJECTIVES: Body sprays and perfumes are commonly worn by patients attending ENT out-patients clinics. Their effect on performance in olfactory testing is unknown. The aim of this study was to determine whether olfactory thresholds are altered by the presence of such fragrances. MATERIALS AND METHODS: One hundred and sixty healthy volunteers, aged 18 to 65 years, underwent olfactory thresholds testing. Each was then exposed to one of four strong perfumes, applied in a facemask for two minutes, and the thresholds were retested. RESULTS AND ANALYSIS: All olfactory thresholds worsened after being exposed to the strong perfumes of Lynx and Impulse body sprays, with the strongest effect being on olfactory detection of phenylethyl alcohol (p<0.001). CONCLUSIONS: Strong perfumes can have a negative effect on olfactory thresholds. SIGNIFICANCE: Patients attending olfactory threshold testing need to be advised not to wear body sprays or perfumes.

Which variables matter in smell tests in the clinic?

BACKGROUND: Testing of olfactory thresholds in the clinic is becoming more common, with commercially produced tests now available. The effect of common potential variables in the clinic setting on these results is unclear. If many variables must be controlled, tests become more complex and a universally accepted olfactory test becomes less likely. OBJECTIVES: The aim of this study was to determine which potential variables the clinician needs to consider when testing olfaction in the out-patient clinic. METHODS: The study was conducted in a clinic setting at a university hospital, using 103 normal volunteers, comprising staff members and patients and relatives from the ENT clinic waiting room. The subjects recruited had no active rhinological complaints, were not taking any medications and were aged between 16 and 70 years. An olfactory threshold was established for each subject for the odour eucalyptol. Gender, smoking status, age, peak nasal inspiratory flow, ambient temperature and relative humidity were all recorded. RESULTS: For eucalyptol, the distribution of values for olfactory thresholds in the normal population lies around the concentration 10(-3) log vol/vol. There was no significant effect of smoking status, tester, ambient temperature or humidity on the thresholds obtained. CONCLUSIONS: The above variables do not have a significant effect on olfactory thresholds elicited in the clinic. The clinician therefore need not attempt to control these factors when testing olfaction in the out-patient setting. These findings bring the implementation of a universal, reliable and easily administered measurement of olfaction a step closer.

Which solvent for olfactory testing?

The physical properties of any carrier can deteriorate over time and thus alter the results in any olfactory test. The aim of this study was to evaluate clinically potential solvents as a clean odourless carrier for olfactory testing. Sweet almond oil, pure coconut oil, pure peach kernel oil, dipropylene glycol, monopropylene glycol, mineral oil and silicone oil were studied. The experimentation was conducted in two parts. First, an olfactory device was used to conduct air through the solvents on a weekly basis using a cohort of six volunteers to assess the perceived odour of each solvent at weekly intervals. Secondly a cross-reference test was performed using small bottled solutions of phenylethyl-alcohol and 1-butanol in 10-fold dilutions to compare any perceived difference in concentrations over a period of 8 weeks. We concluded that mineral oil is the most suitable carrier for the purpose of olfactory testing, possessing many desirable characteristics of an olfactory solvent, and that silicone oil may provide a suitable alternative for odorants with which it is miscible.

The effect of temperature, humidity and peak inspiratory nasal flow on olfactory thresholds.

Temperature, humidity and nasal peak inspiratory flow rate (PIFR) are potential variables in the quantitative measurement of olfactory thresholds in the clinic. To date, these variables have not been properly evaluated with respect to olfactory perception, and therefore the aim of this study was to determine their effect on the thresholds. These variables were measured on 10 occasions in 10 subjects over a 10-week period. The results obtained were then subjected to statistical analysis using a linear mixed-effect model. This demonstrated that olfactory thresholds are sufficiently independent of room temperature, peak humidity and nasal PIFR in a routine outpatient clinic environment in normal subjects, with no evidence of any statistically significant influence by these variables.

Evolution of functional diversity in the cupin superfamily.

The cupin superfamily of proteins is among the most functionally diverse of any described to date. It was named on the basis of the conserved beta-barrel fold ('cupa' is the Latin term for a small barrel), and comprises both enzymatic and non-enzymatic members, which have either one or two cupin domains. Within the conserved tertiary structure, the variety of biochemical function is provided by minor variation of the residues in the active site and the identity of the bound metal ion. This review discusses the advantages of this particular scaffold and provides an evolutionary analysis of 18 different subclasses within the cupin superfamily.

Germin is a manganese containing homohexamer with oxalate oxidase and superoxide dismutase activities.

Germin is a hydrogen peroxide generating oxalate oxidase with extreme thermal stability; it is involved in the defense against biotic and abiotic stress in plants. The structure, determined at 1.6 A resolution, comprises beta-jellyroll monomers locked into a homohexamer (a trimer of dimers), with extensive surface burial accounting for its remarkable stability. The germin dimer is structurally equivalent to the monomer of the 7S seed storage proteins (vicilins), indicating evolution from a common ancestral protein. A single manganese ion is bound per germin monomer by ligands similar to those of manganese superoxide dismutase (MnSOD). Germin is also shown to have SOD activity and we propose that the defense against extracellular superoxide radicals is an important additional role for germin and related proteins.

Crystallization and preliminary X-ray analysis of the auxin receptor ABP1.

Auxin-binding protein (ABP1) is an important receptor for the plant hormone auxin that is involved with many growth and developmental responses in plants. The maize ABP1 gene has been expressed in insect cells, purified and crystallized. Type II crystals are monoclinic, with two glycosylated homodimers in the asymmetric unit, and diffract to 1.9 A using synchrotron radiation.

Mutagenesis and kinetic studies of a plant cysteine proteinase with an unusual arrangement of acidic amino acids in and around the active site.

We report the cloning, overexpression, kinetic analysis, and modeling of the tertiary structure of an unusual plant cysteine proteinase. Ananain (EC 3.4.22.31), from Ananas comosus (pineapple) is distinguished from all other cysteine proteinases in the papain superfamily by having a unique combination of acidic amino acids. As well as lacking the acidic residue immediately preceding the active site histidine (position 158 in papain), it also lacks the extensive surface network of acidic residues that were postulated to compensate for the loss of charge at position 158 in mammalian cathepsins. Ananain has the fewest acidic residues, so far reported, of any plant cysteine proteinase, but two of the carboxyl residues (E50 and E35) postulated to have an enabling role in catalysis, the so-called "electrostatic switch", remain conserved. Comparisons of the kinetics of recombinant wild-type ananain with E50A and E35A mutants proves that these charged groups are not essential for catalysis. Hence this research does not confirm the presence of an electrostatic switch in this cysteine proteinase, and the role of acidic residues in the enhancement of catalytic competence in these enzymes is discussed in light of this new evidence.

Barley oxalate oxidase is a hexameric protein related to seed storage proteins: evidence from X-ray crystallography.

The oxalate oxidase enzyme expressed in barley roots is a thermostable, protease-resistant enzyme that generates H2O2. It has great medical importance because of its use to assay plasma and urinary oxalate, and it has also been used to generate transgenic, pathogen-resistant crops. This protein has now been purified and three types of crystals grown. X-ray analysis shows that the symmetry present in these crystals is consistent with a hexameric arrangement of subunits, probably a trimer of dimers. This structure may be similar to that found in the related seed storage proteins.

The enhancing of a cysteine proteinase activity at acidic pH by protein engineering, the role of glutamic 50 in the enzyme mechanism of caricain.

Carica papaya produces four cysteine proteinases. Calculations show that the Cys25, His159 essential ion pair is fully ionised at pH 2.99, where activity cannot be detected, but apparently an additional ionisation with a pKa of 4 is essential for activity (an electrostatic switch). Caricain (EC 3.4.22.30) wt and D158E genetic backgrounds were used to study the contribution of E50A to activity. E50 or E135 are candidates for the switch, E50A would be expected to reduce activity. However, activity increased at pH 5.0 in both backgrounds and at the pH optimum in D158E E50A but decreased slightly in the wt background. This challenges the hypothesis of an electrostatic switch.

A new glucose oxidase from Aspergillus niger: characterization and regulation studies of enzyme and gene.

A new glucose oxidase from Aspergillus niger was isolated and characterized. The enzyme showed different kinetic and stability characteristics when compared to a commercially available batch of A. niger glucose oxidase. The gene encoding the new glucose oxidase was isolated and DNA sequence analysis of the coding region showed 80% identity to the sequence of a glucose oxidase gene previously published. However, the similarity of the non-coding sequences up- and downstream of the open reading frame was much less, showing only 66% and 50% identity respectively. Despite the low degree of similarity between the promotor region of the new gene and the previously published one, the new glucose oxidase was likewise induced by calcium carbonate. In addition, we showed that this induction occurred on the transcriptional level. Observations concerning the effect of gluconolactone and the levels of glucose-6 phosphate isomerase upon calcium carbonate induction suggested that the enhancement of glucose oxidase biosynthesis by calcium carbonate was accompanied by a metabolic shift from glycolysis to the pentose phosphate pathway.

Rapid kinetic studies and structural determination of a cysteine proteinase mutant imply that residue 158 in caricain has a major effect upon the ability of the active site histidine to protonate a dipyridyl probe.

Cysteine proteinases are endopeptidases whose catalytic activity depends upon the nucleophilicity of the active site cysteine thiol group. An ion pair forms with an active site histidine. The presence in some cysteine proteinases of an aspartic acid close to the ion pair has been used as evidence of a "catalytic triad" as found in the serine proteinases. In these enzymes, the correct alignment of serine, histidine, and aspartate residues controls catalysis. However, the absence of the homologous aspartate residue in the mammalian cysteine proteinases cathepsins B and H argues against this pivotal role for aspartic acid. Instead, an Asn, physically close to the histidine in cysteine proteinases, has been proposed as a member of the catalytic triad. Protein engineering is being used to investigate these questions. In this study, the Asp158Glu mutant of the plant cysteine proteinase caricain was analyzed by stopped-flow rapid kinetics. The probe that was used was 2,2'-dipyridyl disulfide (2 PDS), and the profile of k versus pH gave results more closely allied to a small molecule active site model than the normal profile with cysteine proteinases. Multiple pKa's identified in the profile are as follows: pK1 = 3.4 (Cys 25), pK2 = 3.6, pK3 = 7.0, and pK4 = 8.6 (His 158). The structure of the enzyme with the bound inhibitor E64 was solved (R factor of 19.3%). Although the distance between the imadazolium and the surrounding charged amino acids is only slightly changed in the mutant, the reduced steady state activity and narrower pH range can be related to changes in the hydrogen-bonding capacity of the imadazolium.

Crystal structure of a caricain D158E mutant in complex with E-64.

The structure of the D158E mutant of caricain (previously known as papaya protease omega) in complex with E-64 has been determined at 2.0 A resolution (overall R factor 19.3%). The structure reveals that the substituted glutamate makes the same pattern of hydrogen bonds as the aspartate in native caricain. This was not anticipated since in the native structure there is insufficient room to accommodate the glutamate side chain. The glutamate is accommodated in the mutant by a local expansion of the structure demonstrating that small structural changes are responsible for the change in activity.

Peripheral chemoresponses of infants measured by a minimally invasive method utilizing two-breath alternations in Fl,02.

The aim of this study was to develop a minimally invasive and reliable method for measuring peripheral chemoresponsiveness to oxygen in infants, and to establish baseline data from normal infants at 12 weeks of age. Two-breath alternations in fractional inspired oxygen (FI,O2), switching between 0.42 to 0.00 were given for 2 min periods via a face mask (held close to the face but without contact) to 18 healthy infants during quiet sleep. End-tidal oxygen concentrations alternated between 21 and 11%. Instantaneous minute ventilation (V'E) and its components tidal volume (VT), respiratory frequency (fR) inspiratory and expiratory times (tI and tE), inspiratory flow (VT/tT), and inspiratory duty cycle (tI/ttot) were measured by respiratory inductance plethysmography. Two-breath alternations in each of the ventilatory components were matched with the corresponding alternating end-tidal oxygen record and compared with contiguous pre- and post-test data obtained in control periods of air breathing. Alternations in all ventilatory components except fR changed significantly during FI,O2 alternations; VT 26%, tE-8%, VT/tI 18%, tI/ttot 11% and V'E 28% of baseline values. Within and between infant variances are reported for the individual components of ventilation. Differences among infants were best detected by alternations in V'E; within infant variance 76, between infant variance 171. We conclude that the test described is a safe, reliable and relatively easily applied method of measuring peripheral chemoresponsiveness, which is suitable for clinical application in infancy.

The effect of sleep deprivation on sleep states, breathing events, peripheral chemoresponsiveness and arousal propensity in healthy 3 month old infants.

We wished to investigate the effects of sleep deprivation on sleep, arousal propensity, respiratory events and peripheral chemoresponses in healthy infants, since these effects might be relevant to mechanisms concerned with some cases of sudden infant death syndrome. Paired observations were made overnight during natural sleep and following sleep deprivation, in a randomized fashion, in 15 healthy infants aged 78 (7) days (mean (SD)). Polysomnograms were recorded and sleep was scored using Anders' criteria. Respiratory events were categorized into central, mixed and obstructive apnoeas. Peripheral chemoresponses were measured during quiet sleep from the respiratory response to two-breath alternations in fractional inspiratory oxygen (F1, O2) (0.42 and 0.00). Arousal propensity was determined from awakening and arousal thresholds to graded photic and auditory stimuli during quiet sleep, and from spontaneous awakenings and limb movements. Compared with natural sleep, following sleep deprivation infants maintained a greater proportion of quiet sleep (39 vs 44%). There was no measurable change in arousal propensity. During quiet sleep, obstructed breathing events tended to be more common after sleep deprivation (0.1 vs 0 events.h-1) and the expiratory time during baseline breathing increased significantly (1.27 vs 1.58 s) although the decrease in respiratory rate was not significant (32 vs 30 breaths.min-1). Peripheral chemoresponses altered significantly, alternations in tidal volume/inspiratory time (VT/tI) as a measure of inspiratory drive increased after sleep deprivation (9 vs 21%). In conclusion, following short-term sleep deprivation in infancy, respiratory control alters, peripheral chemoresponsiveness increases in magnitude and the timing of baseline breathing alters, without any detectable alteration in arousal propensity. This state may be associated with an increased vulnerability to obstructive respiratory events.