A comparison of covalent immobilization and physical adsorption of a cellulase enzyme mixture.

This paper reports the first use of a linker-free covalent approach for immobilizing an enzyme mixture. Adsorption from a mixture is difficult to control due to varying kinetics of adsorption, variations in the degree of unfolding and competitive binding effects. We show that surface activation by plasma immersion ion implantation (PIII) produces a mildly hydrophilic surface that covalently couples to protein molecules and avoids these issues, allowing the attachment of a uniform monolayer from a cellulase enzyme mixture. Atomic force microscopy (AFM) showed that the surface layer of the physically adsorbed cellulase layer on the mildly hydrophobic surface (without PIII) consisted of aggregated enzymes that changed conformation with incubation time. The evolution observed is consistent with the existence of transient complexes previously postulated to explain the long time constants for competitive displacement effects in adsorption from enzyme mixtures. AFM indicated that the covalently coupled bound layer to the PIII-treated surface consisted of a stable monolayer without enzyme aggregates, and became a double layer at longer incubation times. Light scattering analysis showed no indication of aggregates in the solution at room temperature, which indicates that the surface without PIII-treatment induced enzyme aggregation. A model for the attachment process of a protein mixture that includes the adsorption kinetics for both surfaces is presented.

Effects of nutritional supplementation and resistance training on muscle strength in free living elders. Results of one year follow.

PURPOSE: To assess the effects of a one year nutritional supplementation and resistance training program on muscle strength and walking capacity in the elderly. MATERIAL AND METHODS: Elderly subjects from two outpatient clinics received a nutritional supplement, that provided 400 Kcal, 15 g/protein and 50% of vitamin DRVs per day. Half the subjects receiving and not receiving the supplement were randomly assigned to a resistance exercise training program with two sessions per week. Every six months, body composition using DEXA, limb muscle strength, maximal inspiratory and expiratory pressures and walking capacity were assessed. RESULTS: One hundred forty nine subjects were considered eligible and 101 (31 supplemented and trained, 28 supplemented, 16 trained and 26 without supplementation nor training) completed the year of follow up. Overall compliance with the supplement was 48 22 % and trained subjects attended 56 21% of programmed sessions. No changes in fat free mass were observed in any of the groups, but fat mass increased from 22.5 7.3 to 23.2 7.3 kg in all groups (p < 0.001). Upper and lower limb strength and walking capacity increased significantly in trained subjects whether supplemented or not. Maximal inspiratory pressure and right hand grip strength increased only in the supplemented and trained group. CONCLUSIONS: Resistance training improved muscle strength and walking capacity.

Biochemical analysis of ocular surface mucin abnormalities in dry eye: the canine model.

This study examines the canine model of keratoconjunctivitis sicca (KCS, 'dry eye') in order to establish the biochemical basis of altered ocular mucin secretion in this condition. It follows a previous examination of ocular mucins in the normal dog. Mucus was collected by suction from the ocular surface of dogs with KCS, and dispersed in guanidine hydrochloride containing a cocktail of protease inhibitors. Caesium chloride density gradient centrifugation was used to separate floating 'rafts' of cell membranes from gradients containing secreted mucins. Gradient fractions were collected into pools on the basis of differential staining by Periodic Acid Schiff, Wheat Germ Agglutinin, and antibodies to MUC5AC peptide. High molecular weight glycoproteins were purified from the pooled material by gel filtration chromatography. Membrane-associated glycoproteins were also derived from the membrane rafts using octyl glucoside extraction and/or reduction and alkylation. Secreted mucins and membrane extracts from KCS samples were compared to equivalent material obtained from normal eyes. Density gradient staining profiles for normal and KCS mucus were similar over the buoyant density range typical for secreted mucins, enabling the collection of identical pools of gradient fractions for direct comparison. The following differences were observed in KCS secreted mucins compared to normal samples: an increase in the proportion of mucin with low buoyant density; a decrease in mannose content detected with Concanavalin A lectin; an increase in N-acetylglucosamine structures detected with Lycopersicon esculentum lectin; increased migration and lack of evidence for distinct subunit structure on agarose gels. In membrane extracts, the main difference was the presence of T antigen (Gal beta 1-3GalNAc) in KCS. These results demonstrate alterations in the subunit linkage of mucins in KCS, and suggest that glycosylation, core protein expression and/or post-synthetic modification of ocular surface mucins may also be changed.

Tear protein profiles vs. clinical characteristics of untreated and cyclosporine-treated canine KCS.

BACKGROUND: Topically administered cyclosporine A (CsA) decreases ocular surface inflammation in canine keratoconjunctivitis sicca (KCS) and exerts lacrimomimetic effects. This study was performed to find correlations between clinical signs and tear protein levels in untreated and CsA-treated canine KCS. METHODS: Clinical profiles were scored in 16 KCS-affected dogs before and 6 weeks after commencing treatment with 0.2% topical CsA emulsion. Tear samples were also collected using polished micropipettes for specific protein assay by high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA). RESULTS: Tear levels of serum proteins correlated with conjunctival clinical signs. Levels of lacrimal gland proteins in tears correlated most often with corneal clinical signs. CONCLUSIONS: The inflammatory features of KCS appear to link conjunctival signs to serum proteins in tears, while corneal signs are linked to lacrimal gland proteins.

Chronic tenosynovitis of the tibialis posterior tendon and the use of tenography.

Tenosynovitis of the tibialis posterior tendon and the use of the tenogram is presented. The tenogram is used as a diagnostic aid in determining the etiology as well as the course of treatment of a symptomatic tibialis posterior tendon. Various treatments for tenosynovitis have also been described.

Threshold sensitivity and frequency specificity in auditory brainstem response audiometry.

Frequency-specific electric response audiometry can be performed on difficult to test young children if the child is sedated and proper choices are made of acoustic stimuli and recording parameters, although certain compromises are necessary. A very satisfactory sedative is secobarbital, administered intramuscularly in doses related to the weight of the child. As stimuli we recommend '2-1-2' tone bursts at 500, 1 000, 2 000, and 4 000 Hz: i.e., with a rise and fall of two periods and a plateau of one period of the modulated tone. A very robust and sensitive response that is not significantly modified by the sedation and is effective for all four frequencies is the P6-SN10 of the early brainstem sequence. To record this complex favorably requires a bandpass input filter of the Butterworth type with pass-band (at -3 dB) from 50 to 1 700 Hz and rejection rates of 24 dB/octave. With this combination, polarity of stimulus is unimportant and sweep time, rate of stimulation and number of responses averaged may be selected for convenience and simplicity. A routine that requires about an hour of testing time is described and the necessary correction factors are given for estimating a child's behavioral pure-tone thresholds. We believe that our threshold estimates are generally correct within 10 dB, and are sufficiently frequency-specific for proper selection of a hearing aid.

Fenfluramine treatment in infantile autism. Neurochemical, electrophysiological, and behavioral effects.

As part of a multicenter, collaborative project, response to fenfluramine was assessed in 10 autistic outpatients. After 4 months of treatment, blood serotonin concentrations decreased an average of 60 per cent and returned to pretreatment levels after 2 months on placebo. This reduction was accompanied by a decrease in certain behavioral symptoms, including motor activity, distractibility, and mood disturbances. Baseline evoked potential recordings indicated that autistic patients tended to have a larger amplitude of the P3 component to frequent tones as compared to age-matched controls. A tendency toward "normalization" of the P3 effect was observed during the medication trial and during the final placebo period. Treatment response was not related to initial serotonin levels, and no major clinical side effects were associated with fenfluramine.

Prospective evaluation of hearing impairment as a sequela of acute bacterial meningitis.

As part of a prospective study of acute bacterial meningitis in children, we studied for five years the hearing of 185 infants and children who had acute bacterial meningitis when they were more than one month of age. Nineteen (10.3 per cent) of the patients had persistent bilateral or unilateral sensorineural hearing loss. The incidence of hearing loss as determined by electric-response audiometry and conventional tests was 31 per cent with Streptococcus pneumoniae, 10.5 per cent with Neisseria meningitidis, and 6 per cent with Hemophilus influenzae infections. Transient conductive hearing impairment was found in 16 per cent of the sample, but in no case was there apparent improvement in a sensorineural deficit over time. The site of disease resulting in impaired hearing cannot be stated with certainty, but involvement of the inner ear or auditory nerve was suspected. The number of days of illness (symptoms) before hospitalization and institution of antibacterial treatment was not correlated with the development of sensorineural deafness.

Frequency selectivity and thresholds of brief stimuli suitable for electric response audiometry.

Auditory evoked potentials are nearly all on-effects and the 'effective stimuli' for them are necessarily brief. Their frequency specificity is therefore limited, especially for the brainstem responses, because of the well-known trade-off between duration and frequency specificity. Brainstem responses are of special interest because they are unchanged in the sleep-like sedation that is required for difficult-to-test children. The middle-latency responses do not meet this requirement. Two patterns of tone burst that are appropriate and promising for the slow cortical potentials and for brainstem potentials, respectively, have rise and fall times of 2 periods of the modulated tone and plateaus of 10 (or 7) periods and 1 period, respectively. Their behavioral thresholds are nearly insensitive to difference in repetition rate between 4 and 40 stimuli/s. Their peak equivalent SPL threshold values at 500, 1 000, 2 000 and 4 000 Hz have been determined for 16 otologically normal ears. Using these reference levels, audiograms have been obtained for subjects with impaired hearing. The audiograms for 'flat' hearing losses do not differ significantly from the corresponding conventional pure-tone audiograms. The slopes for steep high-frequency hearing losses are underestimated, however, particularly with the brief (2-1-2) pattern. Nevertheless, the 2-1-2 pattern appears to be close to the best possible compromise.

A slow brain stem response for low-frequency audiometry.

Proper choices of stimuli and of brain stem electric responses allow us to estimate peripheral auditory thresholds at 500, 1 000, 2 000 and 4 000 Hz with an accuracy of about +/- 10 dB. With the help of sedation (secobarbital), such audiograms may be obtained from each ear of a child of any age in a single session. Tone pips (filtered clicks) or very brief tone bursts give a frequency selectivity that is clinically adequate. The rise time must be adjusted to the center frequency. A rise time of two periods with a plateau from zero to one period gives a good compromise between frequency specificity and a synchronous neural discharge. The best threshold indicator for tone pips of 2 000 Hz or higher (or unfiltered clicks) is P6 (Jewett V). At 60 dB nHL its latency is 6.0-7.0 ms (for children of 1 year or older), but near threshold it is 8.0-9.5 ms. An input pass-band of 140-3 000 Hz is appropriate. The best threshold indicator at 500 or 1 000 Hz is a nearly neglected slower wave with a scalp-negative crest at about 10 ms following a 60-dB click. Latency is 15 ms following a 500-Hz tone pip at 15 dB SL. We call this wave "slow negative (ten)" or SN10. To see it well a wider input pass-band such as 40-3 000 Hz is needed. SN10 is usually obscured by P6 or by frequency-following response at stimulus levels above 35 dB SL. The details are given of a clinical routine that allows the determination (+/- 10 dB) of 8 threshold endpoints within about 80 min. Several precautions and limitations are discussed, and also the origin of the SN10 wave.

Brain stem electric response audiometry (BSERA).

Brain stem electric responses, recorded with external electrodes on vertex and ear lobes, are excellent for audiometry of young children. The vertex-positive wave with latency of 6 to 9 msec resembles closely the action potential of the auditory nerve, with the same high-intensity short-latency component and low-intensity long-latency component. Thresholds are reliable with filtered clicks at 1 000 Hz and higher. Practical advantages and theoretical limitations are summarized.

The audiometric utility of brain stem responses to low-frequency sounds.

The human scalp-recorded vertex-positive brain stem response to a click or high-frequency tone pip is an excellent audiometric indicator. Its latency of 6-9 ms is practically independent of the polarity of the stimulus but is inversely related to intensity. With a 4,800- or 2,400-Hz tone pip (filtered click) its threshold of detectability is usually at or below 10 dB SL. With a 500-Hz tone pip, with rise and fall of 2-3 ms, the response at 30 dBSL is low in amplitude, rounded in wave form, and has a latency of about 10 ms. When the polarity of the stimulus is reversed, the latency shifts by 1 ms. At and above 40 dB, this late response is obsured by a larger and earlier response. High-pass (1,500 Hz) masking noise does not affect the low-level response but the earlier high-level response is reduced in amplitude and delayed by about 1 ms. The large early response seems to be initiated by stimulation of the basal turn of the cochlea by the low-frequency transient. The frequency-following response (FFR) to a 500-Hz tone burst with a 2-ms rise time has a threshold at about 40 dB SL. Its relatively short latency is appropriate to the basal turn. A later low-amplitude apically generated response can sometimes be detected, either at a lower stimulus level or in the presence of high-pass masking noise. The usual FFR often has complex wave forms and some individuals show only an onset response, even at 70 dB SL. It is almost impossible to edentify with certainty the first individual waves of FFR as they relate to the individual waves of the tone burst and as they change amplitude with intensity. The audiometric usefulness of the high-threshold responses to 500 Hz that are initiated in the basal turn is doubtful. The low-threshold responses initiated in the apical turn are so difficult to identify with certainty that they are not likely to be of clinical value unless high-pass masking noise can be used to clarify them.