Methylated lysine in storage body protein of sheep with hereditary ceroid-lipofuscinosis.

Juvenile ceroid-lipofuscinosis (Batten disease) is a hereditary storage disease with an autosomal-recessive mode of transmission. This disorder has been identified in humans, dogs and sheep. It is characterized by massive accumulations of autofluorescent storage bodies in many tissues. This storage body accumulation is accompanied by functional decline and degeneration of the affected tissues, and ultimately results in premature death. The primary defect responsible for juvenile ceroid-lipofuscinosis has not been identified. Previous studies have indicated that the storage material is primarily protein. Why this protein accumulates in storage bodies remains to he determined. In affected humans, the storage body protein appears to be abnormally rich in a methylated derivative of lysine (epsilon-N-trimethyllysine). Studies were undertaken to determine whether the storage bodies from sheep with hereditary ceroid-lipofuscinosis were also characterized by the presence of this modified amino acid. Chromatographic and mass spectral analyses of hydrolysates of the storage body protein indicated a significant fraction of the lysine residues in this protein were present as the epsilon-N-trimethyl derivative. This modified amino acid was not detected in hydrolysates of protein from normal sheep tissues or from tissues of sheep with GM1 gangliosidosis, nor did it appear to be present in the storage body protein from a human subject with the late infantile form of ceroid-lipofuscinosis. Thus, it is apparently specific to the storage body protein that accumulates in the juvenile type of this disease. The abnormal presence of epsilon-N-trimethyllysine in proteins could interfere with their sorting or degradation within cells and thus cause them to accumulate in the storage bodies characteristic of the human juvenile and ovine ceroid-lipofuscinoses.

In Vivo Photomodification of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Holoenzyme by Ultraviolet-B Radiation (Formation of a 66-Kilodalton Variant of the Large Subunit).

Increased levels of solar ultraviolet (290-320 nm) (UV-B) radiation could have profound effects on plant proteins because the aromatic amino acids in proteins absorb strongly in this spectral region. We have investigated the effects of UV-B radiation on plant proteins and have observed a novel 66-kD protein. This product was formed in vivo when Brassica napus L. plants grown for 21 d in 65 [mu]mol m-2 s-1 photosynthetically active radiation were subsequently exposed to 65 [mu]mol m-2 s-1 photosynthetically active radiation plus UV-B radiation (1.5 [mu]mol m-2 s-1). The protein appeared after 4 h of UV-B irradiation and accumulated during the next 16 h in UV-B. The 66-kD protein cross-reacted with an antiserum against the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) holoenzyme. Analysis of soluble leaf proteins revealed that the 66-kD product had a number of isoforms corresponding closely to those of the large subunit of Rubisco (LSU). Partial proteolytic digests of the LSU and the 66-kD protein resulted in an equivalent pattern of protein fragments, leading to the conclusion that the 66-kD protein was a photomodified form of the LSU. A similar high molecular mass variant of Rubisco was observed in soluble protein extracts from leaves of tomato (Lycopersicon esculentum), tobacco (Nicotiana tabacum), and pea (Pisum sativum L.) plants treated in vivo with UV-B, suggesting that it might be a common product, at least among C3 plants. It is interesting that the 66-kD product appears to be generated after incorporation of the LSU into holoenzyme complexes. This conclusion was drawn from two lines of evidence. First, the LSU variant co-purified with holoenzyme complexes isolated by nondenaturing polyacrylamide gel electrophoresis. Second, a UV-B-specific 66-kD protein did not accumulate in a tobacco mutant that synthesizes the Rubisco subunits but does not assemble them into normal holoenzyme complexes.

Storage protein in hereditary ceroid-lipofuscinosis contains S-methylated methionine.

Hereditary ceroid-lipofuscinoses are neurodegenerative disorders characterized by the accumulation in numerous tissues of a storage material with lipofuscin-like fluorescence properties. Investigations were undertaken to determine the chemical nature of this storage material isolated from the cerebral cortex of human subjects with the late infantile form of the disease. The storage material was mainly protein that consisted of a mixture of polypeptides ranging in apparent molecular weight from 13 to 67 kDa. Protein-bound fluorophores apparently were largely responsible for the autofluorescence of the storage bodies. The disease-related storage body protein was rich in S-methylmethionine [(3-amino-3-carboxypropyl) dimethyl sulfonium ion], an amino acid that does not normally occur in animal proteins. Methylation of proteins to form this unusual charged amino acid may impair proteolytic degradation or other aspects of protein metabolism, and account for the accumulation of protein-filled inclusions in cells of individuals with ceroid-lipofuscinoses. Similar amino acid modifications that block proteolysis could be involved in age pigment accumulation.

Fatty acid pattern and cholesterol in skeletal muscle of men aged 22 to 73.

In order to examine the relationship between fatty acid distribution of skeletal muscle membranes and age, a needle biopsy was performed on the vastus lateralis muscle of 20 healthy, non-obese males, ranging in age from 22 to 73 years. The muscle sample was homogenized, centrifuged at 100,000 x g, and the resulting pellet was saponified and acidified. The fatty acids and cholesterol were removed by a single hexane extraction and analyzed by gas--liquid chromatography with flame ionization detection. All subjects regardless of age had no consistent differences in the fatty acid profiles and cholesterol composition in the tissue. Correlation coefficients indicated no significant relationship between the age of the individual and any of the analyzed lipids. The results of this study indicated that aging may not be reflected by gross changes in the composition of structural lipids in the cell.

Human spinal cord retains substantial structural mass in chronic stages after injury.

In chronic stages of human spinal cord injury, atrophy of the cord has been reported in regions both at and distant to the injury site. Local cord atrophy results from the direct effects of bony impact and ischemia, whereas distant atrophy results from anterograde (Wallerian) and retrograde axonal degeneration. However, the actual extent of degenerative changes in the chronically injured human spinal cord both at and remote from the injury site has rarely been reported, and has not been rigorously quantified to date. In the present study, we quantified the extent of spinal cord atrophy in 12 humans with chronic injury (2-34 years posttrauma) utilizing quantitative stereological assessment of spinal cord magnetic resonance images, and compared the results to uninjured human spinal cords. Focal cystic atrophy of the cord, characterized by signal attenuation on T1-weighted images, was regularly present at the actual site of impact injury and replaced a mean longitudinal area equaling less than one spinal cord segment in length (2.01 +/- 0.60 cm2, or a loss of 89.3 +/- 17.4% of the longitudinal area of one spinal cord segment). Spinal cord segments immediately rostral to the zone of cystic degeneration showed atrophy of only 19.4 +/- 7.5% of normal cord longitudinal area, and spinal cord segments immediately caudal to the zone of cystic degeneration showed atrophy of 16.5 +/- 4.1% of normal cord longitudinal area. Extensive spinal cord atrophy extending beyond the region of injury occurred in two of twelve cases (16.7%), and both were caused by late syrinx formation. Thus, spinal cord atrophy after trauma remains primarily restricted to the original site of injury. Experimental neural repair strategies should take into account the importance of "bridging" relatively short zones of cystic atrophy, then promoting axonal regeneration through potentially long segments of remaining cord parenchyma.

Effect of abdominal vibroacoustic stimulation on sound and acceleration levels at the head of the fetal sheep.

OBJECTIVE: To measure the vibratory response of the fetal head and abdominal wall in sheep during vibroacoustic stimulation. METHODS: A piezoresistive accelerometer was attached to the skulls of seven sheep fetuses (128-134 days' gestational age), and a miniature hydrophone was attached to the skin overlying the fetal temporal bone. During fetal preparation and vibroacoustic stimulation procedures, ewes were anesthetized and supine. Vibroacoustic stimulation of the maternal abdomen was produced by each of two clinical devices that differed in spectral content, and an electric toothbrush. RESULTS: The approximate fundamental frequencies (f0) and first overtones (f1), as determined by both recordings of intrauterine sound pressure level and fetal head acceleration, were as follows: fetal acoustic stimulator, 75 and 150 Hz; electronic larynx, 150 and 300 Hz; and electric toothbrush, 25 and 50 Hz, respectively. At fundamental frequencies and first overtones, the ranges of fetal head accelerations (expressed in 1/12-octave bands) were as follows: fetal acoustic stimulator, 10-53 and 25-224 mm/sec2; electronic larynx, 10-53 and 18-114 mm/sec2; and electric toothbrush, 33-792 and 8-116 mm/sec2, respectively. Sound pressure levels exceeded 110 dB in all cases. High sound pressure levels in the uterus were proportional to fetal head vibration levels. CONCLUSION: Vibroacoustic stimulation of the surface of the abdomen of pregnant sheep is accompanied by both acoustic and vibratory exposure of the fetus.

Sound levels in the human uterus.

OBJECTIVE: We sought to determine the degree to which noises and voices are attenuated or enhanced as they pass into the uterus. METHODS: In eight parturients, a hydrophone in the uterus was used to measure sound pressure levels for externally generated one-third-octave band noises, male and female voices, and the subject's voice. RESULTS: Low-frequency sounds (0.125 kHz) generated outside the mother were enhanced by an average of 3.7 dB. There was a gradual increase in attenuation for increasing frequencies, with a maximum attenuation of 10.0 dB at 4.0 kHz. Sound attenuation was slightly less if the insonation was from in front of the woman rather than behind. Intrauterine sound levels of the mother's voice were enhanced by an average of 5.2 dB, whereas external male and female voices were attenuated by 2.1 and 3.2 dB, respectively. The effect of frequency on attenuation, the differences between front and back insonation, and the differences between speakers in attenuation were all statistically significant. CONCLUSIONS: The intrauterine environment is rich with externally generated sounds. This may imply fetal risk from maternal noise exposure and may aid in understanding fetal imprinting from prenatal exposure to voices.

Lectin-mediated drug delivery: influence of mucin on cytoadhesion of plant lectins in vitro.

As the mucous layer represents the first barrier to peroral lectin-mediated drug delivery, the influence of mucin on the cytoadhesive properties of lectins was studied in vitro by establishing a rapid and simple microplate format assay using pig gastric mucin (PGM) for coating the wells. The lectin-binding capacity of mucin followed the order WGA>>UEA-I>>LCA=STL>PNA>DBA. The PGM-binding of wheat germ agglutinin (WGA) was strongly dependent on pH being highest at pH 5.0. In comparison, PGM-binding of WGA was about 15% at gastric pH and 60-70% at intestinal pH. This points to unimpeded gastric transit of WGA-grafted formulations and favorable conditions within the intestine for binding to mucus coated enterocytes. Moreover the WGA-PGM interaction was concentration-dependent, specific and fully reversible. According to a competitive assay in the presence of Caco-2 monolayers, the PGM-binding of WGA was saturated and influenced by the lectin-concentration yielding 28% Caco-2 bound WGA (125 ng WGA/0.29 cm(2) monolayer) and 68% Caco-2 bound WGA (4 microg WGA/0.29 cm(2) monolayer), respectively. Following on from these results, lectins are expected to suffer at least partially from premature inactivation by shed off mucus like bioadhesives of the first generation, however initial but reversible mucus-binding of lectins offers partititioning to the cell membrane followed by uptake into the enterocyte.

Effect of high-intensity sound on local cerebral glucose utilization in fetal sheep.

The effects of external noise on fetal sheep cerebral glucose utilization were determined with the [14C]deoxyglucose method. Seventeen animals were prepared at 130 days gestation with catheters and electrodes for assessing fetal behavioral state. Five to 7 days later, 7 animals were studied under normal laboratory sound conditions (65-70 dB), 5 animals were exposed to 105-120 dB broadband noise levels produced by two earphones applied to the abdomen of the ewe, and 5 fetuses were stimulated with an electronic artificial larynx (EAL), positioned on the abdomen directly over the fetal head. There were no significant differences between local cerebral glucose utilization in controls and earphone ewes, and no obvious alteration in behavioral states. However, there were marked, significant differences in glucose utilization along the central auditory pathway during EAL stimulation. These autoradiographs revealed isofrequency-like bands in medial geniculate body and irregular darkening of cortex of the temporal lobe. Total time spent in clearly defined high and low voltage electrocortical activity did not change during EAL stimulation.

Cocaine alters behavioral states in fetal sheep.

Intrauterine cocaine exposure causes subtle neurologic abnormalities in human newborn infants; however, the mechanism for these abnormalities is not known. To investigate whether cocaine alters fetal behavioral state, the electrocortical, electro-ocular and neck muscle electrical activity was monitored in 7 chronically instrumented fetal sheep before and during both saline and cocaine HCl infusions directly to the fetus. Saline infusion to the fetus had no effect on the percentage of time spent in rapid eye movement sleep compared to no infusion (37.5 +/- 11.6% vs 46.3 +/- 4.6%, mean +/- SD, P greater than 0.1). Cocaine infusion directly to the fetus had no effect on fetal arterial pO2, but did increase mean arterial pressure from 53.6 +/- 15 mmHg to 61.0 +/- 21 mmHg (P less than 0.01). In addition, during cocaine infusion, the percentage of time spent in rapid eye movement sleep dropped to 3.9 +/- 5.1% (P less than 0.0001) and the average duration of rapid eye movement epochs decreased from 10.1 +/- 3.0 min precocaine infusion to 1.9 +/- 2.6 min during cocaine infusion (P less than 0.02). The influence of cocaine was noted in a frequency analysis of the electrocorticogram. The amplitude of the energy centered at 1 Hz during cocaine infusion (73.8 +/- 4.0 dB) was greater than the amplitude during rapid eye movement sleep (65.5 +/- 4.7 dB) and less than the amplitude during non-rapid eye movement periods (79.9 +/- 4.5 dB) (P = 0.01). Cocaine appears to alter fetal behavioral state directly and this may play a role in the abnormal behavior in newborn infants exposed to cocaine in utero.

Effect of cocaine on electrocortical activity in fetal sheep.

The effect of cocaine on the behavioral state of six fetal sheep was studied during gestational ages between 128-135 days. Two to eight days after surgery, fetuses received either a continuous 60 min intravenous infusion of cocaine HCl (33.4 mg) or saline. The infusions were preceded and followed by control periods of 102 min. Cocaine induced a disruption in fetal behavioral state cyclicity and a decrease in the amount of time spent in rapid eye movement sleep (P < 0.01) and non-rapid eye movement sleep (P < 0.05) during the infusion, but not during the recovery period. Spectral amplitude of the electrocortical activity at all three cortical locations increased within most one-third octave bands between 0.8-4 Hz and decreased within most bands between 16-25 Hz (P < 0.05) compared to controls. There were no differences in spectral amplitude between pre- and post-cocaine periods at any location over the 25 frequency bands studied (P > 0.6) except for one frequency band centered at 12.5 Hz. The effects of a one hour cocaine infusion on fetal cortical electrical activity are diffuse, but short-lived, and occur independently of changes in fetal oxygenation.

Fetal hearing: characterization of the stimulus and response.

Before sounds originating outside the abdomen of pregnant women can reach the inner ear of the fetus, they must first pass through the tissues and fluids surrounding the fetal head. Low-frequency sound energy easily penetrates to the fetal head, less than 5 dB attenuation for frequencies below 500 Hz, whereas higher frequencies are attenuated by up to 20 to 30 dB. The sound energy in amniotic fluid stimulates fetal hearing through a bone conduction route rather than through the external and middle ear systems. During passage through the bones of the skull, sound energy is slightly diminished for frequencies less than 250 Hz (10 to 20 dB), yet significantly reduced for frequencies from 500 to 2,000 Hz (40 to 50 dB). Thus, the fetus in utero can easily detect low-frequency sound energy (< 500 Hz) produced at levels that are comfortably loud for its mother, but probably cannot detect acoustic energy at frequencies higher than 500 Hz.

Vibration of the abdominal segment in pregnant sheep.

Mechanical vibration of the abdominal wall results in a frequency-related distribution of intra-abdominal sound pressure levels. A greater attenuation of applied signals of equal dynamic force occurs as frequency increases. A broad resonance peak exists between 6 and 18 Hz. Transducers fixed to the fetal head show clear increases in acceleration levels during stimulation of the abdominal surface with the artificial electronic larynx. Sine-wave stimulation results in a frequency-dependent increase in vibration levels of the abdominal wall of 4% to 140% of the input levels. At the fetal head, a broad peak in response was noted between 6 and 12 Hz, but the overall levels never exceeded 4% of the input level.

Gas-liquid chromatography-mass spectrometry of hydroxy fatty acids as their methyl esters tert.-butyldimethylsilyl ethers.

tert.-Butyldimethylsilyl ethers of secondary hydroxy fatty acid methyl esters (tBDMS-O-FAMEs) produce stable derivatives amenable to gas-liquid chromatography (GLC) and mass spectrometry (MS). Derivatives produce prominent molecular mass minus 57 [M-57]+ fragment ions and unique marker fragment ions indicating the location of the secondary hydroxyl groups along the aliphatic chain from the omega-2 carbon to carbon numbers 5 from the carboxylic terminus, in addition to yielding information regarding carbon chain length, and degree of unsaturation. The tBDMS-derivatives of C-2, C-3 hydroxy fatty acids and the unique GLC-MS data of gamma- and delta-lactones are also presented. Though several tBDMS-O-FAMEs with centrally located hydroxyl groups were not chromatographically resolved, the combination of GLC retention times and monitoring of key diagnostic fragment ions of each tBDMS-derivative, when applied to mixtures containing all hydroxy isomers of palmitic through arachidic acid methyl esters, and to several monounsaturated, monohydroxylated fatty acid methyl esters, allowed for their unambiguous identification. Coupled with derivative stability, permitting their purification and concentration, this method was applied to the identification of trace lipids isolated from bovine skim milk which contained a complex mixture of hydroxy fatty acids of which 19 were newly identified.

Temporary threshold shifts induced by low-pass and high-pass filtered noises in fetal sheep in utero.

Auditory brainstem responses (ABRs) were obtained from nine late gestational age fetal sheep in utero before and after a 16-h exposure to low-pass (cut-off frequency 1.0 kHz) and high-pass (cut-off frequency 1.0 kHz) noises (approximately 120 dB sound pressure level, recorded in air). Bone-conduction ABRs were elicited by broadband clicks and 0.5, 1.0 and 2.0 kHz tone bursts. Following low-pass noise exposure, ABR thresholds and wave IV latencies increased significantly for 0.5 and 1.0 kHz tone bursts. The high-pass noise exposure produced significant shifts in ABR thresholds and wave IV latencies only for the 1.0 kHz tone bursts. These findings confirm previous reports of low-frequency sound transmission into the fetal inner ear.

Noise induced hearing loss in fetal sheep.

The auditory brainstem response (ABR) was recorded in utero from chronically instrumented fetal sheep prior to and following exposure of pregnant ewes to intense broadband noise (120 dB SPL for 16 h). ABRs were elicited by clicks and tone bursts (0.5, 1, 2, and 4 kHz) delivered through a bone oscillator secured to the fetal skull. Latency-intensity functions for most of the four vertex-positive waves (labelled I-IV) were prolonged and ABR thresholds were temporarily elevated by an average of 8 dB following the noise exposure. Results show that exogenous sounds can penetrate the uterus and result in alterations of the fetal ABR.

Effect of stress on cochlear glucocorticoid protein: acoustic stress.

Levels of glucocorticoid (GR) receptor protein were determined by a quantitative enzyme-linked immunosorbent assay (ELISA) technique in inner ear tissue of rats exposed daily to 85 dB SPL white noise for 4 hours on 3 consecutive days. GR levels in spiral ligament and organ of Corti tissues were detected using a monoclonal antibody to the GR receptor, BuGR2. A non-significant 13% decrease in GR levels of spiral ligament tissues was observed in the noise exposed animals relative to untreated animals. A statistically significant decrease of 27% in GR protein levels was seen in the organ of Corti region (P < 0.03), however. There was a concomitant increase of serum corticosterone levels (P < 0.03) in noise exposed animals as opposed to those of controls. These results indicate a tissue specific response of GR receptor to acoustic stress. Inner ear GR protein therefore may be a useful marker in determining the effect of stress on the inner ear. Finally, such data may be applicable to support the hypothesis that stress is an etiological agent in Ménière's disease.

Electrochemical potentials and potassium concentration profiles recorded from perilymph, endolymph and associated inner ear tissues in adrenalectomized rats.

This study evaluated the electrochemical potentials and potassium concentration (Ck+) profiles in the perilymph, endolymph, marginal cells, and spiral ligament of adrenalectomized rats in which endogenous corticosteroids had been removed. Electrochemical potentials recorded at the four cochlear sites were not affected by adrenalectomy (ADX). Ck+ was greater in the endolymph of the ADX animals as compared to control animals. Additionally, there was an increase of Ck+ in the marginal cells, perilymph, and spiral ligament tissues of the ADX animals as compared to control animals, although the observed increases were not statistically significant. In a previous study (Ma et al., 1995a), it was found that potassium levels in the blood plasma of ADX animals were higher than those identified in normal rats; thus, ADX may have a systemic effect on Ck+ that is detectable in both tissues and fluids within the cochlea. Even though Ck+ was elevated within the cochlea in the ADX model, the functional response of the inner ear, as assessed electrophysiologically, was not altered.

Combined effects of adrenalectomy and noise exposure on compound action potentials, endocochlear potentials and endolymphatic potassium concentrations.

The effects of removal of endogenous corticosteroids via bilateral adrenalectomy in combination with noise exposure (30 min at 100 dB) were determined by recording compound action potential (CAP) and endocochlear potentials (EP), and by measuring potassium concentrations (K+e) within the endolymph. Thirty-eight Long-Evans rats were divided into groups according to experimental treatments: adrenalectomy (ADX) or non-ADX and noise exposure or non-noise exposure. CAP thresholds, EP and K+e values were subjected to repeated-measures analysis of variance with group and time as factors classifying the measurements. Noise exposure resulted in significant elevations of CAP thresholds in both the ADX and non-ADX animals, but had no effect on either EP or endolymphatic K+e. Recovery was noted during all post-exposure measurement periods and was significantly faster for ADX animals. EP and K+e did not change during or after noise exposure. ADX animals showed a non-significant reduction of EP and a statistically significant increase of K+e during all measurement periods as compared to non-ADX animals.

Correlative evidence of hypertension and altered cochlear microhomeostasis: electrophysiological changes in the spontaneously hypertensive rat.

The spontaneously hypertensive rat model has been used to show that hypertension is an important pathophysiological risk factor in age-related hearing loss. In the present study, compound action potential (CAP), electrochemical potential (ECP), and potassium concentration (CK+) measurements were taken from the cochlea of genetically predisposed, spontaneously hypertensive rats (SHR) and from normotensive Wistar-Kyoto (WKY) rats. In the SHR model, as the duration of hypertension increased with the animal's age (from 3 to 8 months), CAP thresholds increased, ECP increased in marginal cells only, and CK+ increased in both endolymph and marginal cells. Collectively, the data suggest that ionic alternations of cellular potentials are involved in hearing changes in the hypertensive state. Ultimately, such data may assist in understanding hearing loss in individuals who are diagnosed with hypertension.