Congenital myasthenic syndrome associated with epidermolysis bullosa caused by homozygous mutations in PLEC1 and CHRNE.

Mutations in the plectin gene (PLEC1) cause epidermolysis bullosa simplex (EBS), which may associate with muscular dystrophy (EBS-MD) or pyloric atresia (EBS-PA). The association of EBS with congenital myasthenic syndrome (CMS) is also suspected to result from PLEC1 mutations. We report here a consanguineous patient with EBS and CMS for whom mutational analysis of PLEC1 revealed a homozygous 36 nucleotide insertion (1506_1507ins36) that results in a reduced expression of PLEC1 mRNA and plectin in the patient muscle. In addition, mutational analysis of CHRNE revealed a homozygous 1293insG, which is a well-known low-expressor receptor mutation. A skin biopsy revealed signs of EBS, and an anconeus muscle biopsy showed signs of a mild myopathy. Endplate studies showed fragmentation of endplates, postsynaptic simplification, and large collections of thread-like mitochondria. Amplitudes of miniature endplate potentials were diminished, but the endplate quantal content was actually increased. The complex phenotype presented here results from mutations in two separate genes. While the skin manifestations are because of the PLEC1 mutation, footprints of mutations in PLEC1 and CHRNE are present at the neuromuscular junction of the patient indicating that abnormalities in both genes contribute to the CMS phenotype.

In vitro biological effects of sodium titanate materials.

UNLABELLED: Monosodium titanate (MST) particles effectively bind specific metals and are therefore promising compounds for delivery or sequestration of metals in biological contexts. Yet, the biological properties of MST are largely unexplored. Our previous study showed that the cytotoxicity of these compounds was mild, but the nature of the dose response curves suggested that residual titanates in culture may have interfered with the assay. In the current study, we assessed the importance of these artifacts, and extended our previous results using fibroblasts for biological evaluation. We also assessed the biological response to a new type of titanate (referred to as amorphous peroxo-titanate or APT) that shows more promising metal binding properties than MST. METHODS: The degree of titanate-induced interference in the MTT (mitochondrial activity assay) was estimated by means of cell-free assays with and without a final centrifugation step to remove residual titanate particulate. Cytotoxic responses to titanates were assessed by measuring succinate dehydrogenase activity (by MTT) in THP1 monocytes or L929 fibroblasts after 24-72 h exposures. Monocytic activation by APT was assessed by TNFalpha secretion (ELISA) from monocytes with or without lipopolysaccharide (LPS) activation. RESULTS: We confirmed that residual titanate particulates may alter the SDH activity assay, but that this effect is eliminated by adding a final centrifugation step to the standard MTT procedure. Addition of MST or APT at concentrations up to 100 mg/L altered succinate dehydrogenase activity by < 25% in both monocytes and fibroblasts. Fibroblasts displayed time-dependent adaptation to the MST. APT did not trigger TNFalpha secretion or modulate LPS-induced TNFalpha secretion from monocytes. CONCLUSIONS: Although further in vitro and in vivo assessment is needed, MST and APT exhibit biological properties that are promising for their use as agents to sequester or deliver metals in biological systems.

Expression and purification of feline thyrotropin (fTSH): immunological detection and bioactivity of heterodimeric and yoked glycoproteins.

The goal of this study was to express and purify recombinant feline TSH as a possible immunoassay standard or pharmaceutical agent. Previously cloned feline common glycoprotein alpha (CGA) and beta subunits were ligated into the mammalian expression vector pEAK10. The feline CGA-FLAG and beta subunits were cloned separately into the pEAK10 expression vector, and transiently co-transfected into PEAK cells. Similarly, previously cloned and sequenced yoked (single chain) fTSH (yfTSH) and the CGA-FLAG sequences were ligated into the same vector, and stable cell lines selected by puromycin resistance. Expression levels of at least 1 microg/ml were achieved for both heterodimeric and yoked fTSH forms. The glycoproteins were purified in one step using anti-FLAG immunoaffinity column chromatography to high purity. The molecular weights of feline CGA-FLAG subunit, beta subunit and yfTSH were 20.4, 17, and 45 kDa, respectively. Both heterodimeric and yoked glycoproteins were recognized with approximately 40% detection by both a commercial canine TSH immunoassay and an in-house canine TSH ELISA. The yoked glycoprotein exhibited parallelism with the heterodimeric form in the in-house ELISA, supporting their possible use as immunoassay standards. In bioactivity assays, the heterodimeric and yoked forms of fTSH were 12.5 and 3.4% as potent as pituitary source bovine TSH at displacing (125)I-bTSH and 45 and 24% as potent in stimulating adenylate cyclase activity in human TSH receptor-expressing JP09 cells. However, in addition to reduced receptor binding affinity, the recombinant glycohormones produced a reduced maximal effect at maximal concentration (E(max)) suggesting the possibility of the recombinant glycohormone constructs acting as partial agonists at the human TSH receptor.

Genetic influences in individual susceptibility to noise: a review.

Individual animals and humans show differing susceptibility to noise damage even under very carefully controlled exposure conditions. This difference in susceptibility may be related to unknown genetic components. Common experimental animals (rats, guinea pigs, chinchillas, cats) are outbred-their genomes contain an admixture of many genes. Many mouse strains have been inbred over many generations reducing individual variability, making them ideal candidates for studying the genetic modulation of individual susceptibility. Erway et al. (1993) demonstrated a recessive gene associated with early presbycusis in the C57BL/6J inbred mouse. A series of studies have shown that mice homozygous for Ahl allele are more sensitive to the damaging effects of noise. Recent work has shown that mice homozygous for Ahl are not only more sensitive to noise, but also are probably damaged in a different manner by noise than mice containing the wild-type gene (Davis et al., 2001). Recent work in Noben-Trauth's lab (Di Palma et al., 2001) has shown that the wild-type Ahl gene codes for a hair cell specific cadherin. Cadherins are calcium dependent proteins that hold cells together at adherins junctions to form tissues and organs. The cadherin of interest named otocadherin or CDH23, is localized to the stereocillia of the outer hair cells. Our working hypothesis, suggests that otocadherin may form the lateral links between stereocilia described by Pickles et al (1989). Reduction of, or missing otocadherin weakens the cell and may allow stereocilia to be more easily physically damaged by loud sounds and by aging.

Genetic basis for susceptibility to noise-induced hearing loss in mice.

The C57BL/6J (B6) and DBA/2J (D2) inbred strains of mice exhibit an age-related hearing loss (AHL) due to a recessive gene (Ahl) that maps to Chromosome 10. The Ahl gene is also implicated in the susceptibility to noise-induced hearing loss (NIHL). The B6 mice (Ahl/Ahl) are more susceptible to NIHL than the CBA/CaJ (CB) mice (+(Ahl)). The B6xD2.F(1) hybrid mice (Ahl/Ahl) are more susceptible to NIHL than the CBxB6.F(1) mice (+/Ahl) [Erway et al., 1996. Hear. Res. 93, 181-187]. These genetic effects implicate the Ahl gene as contributing to NIHL susceptibility. The present study demonstrates segregation for the putative Ahl gene and mapping of such a gene to Chromosome 10, consistent with other independent mapping of Ahl for AHL in 10 strains of mice [Johnson et al., 2000. Genomics 70, 171-180]. The present study was based on a conventional cross between two inbred strains, CBxB6.F(1) backcrossed to B6 with segregation for the putative +/Ahl:Ahl/Ahl. These backcross progeny were exposed to 110 dB SPL noise for 8 h. All of the progeny were tested for auditory evoked brainstem responses and analyzed for any significant permanent threshold shift of NIHL. Cluster analyses were used to distinguish the two putative genotypes, the least affected with NIHL (+/Ahl) and most affected with PTS (Ahl/Ahl). Approximately 1/2 of the backcross progeny exhibited PTS, particularly at 16 kHz. These mice were genotyped for two D10Mit markers. Quantitative trait loci analyses (log of the odds=15) indicated association of the genetic factor within a few centiMorgan of the best evidence for Ahl [Johnson et al., 2000. Genomics 70, 171-180]. All of the available evidence supports a role for the Ahl gene in both AHL and NIHL among these strains of mice.

Quantitative measure of genetic differences in susceptibility to noise-induced hearing loss in two strains of mice.

The CBA/CaJ (CB) and C57BL/6J (B6) inbred strains of mice were exposed for 1 h to noise intensities between 98 and 119 dB SPL. Previous studies indicated that the B6 mice exhibited permanent threshold shifts (PTS) after 1h exposure to 110 dB, whereas the CB mice did not exhibit any PTS. These differences in susceptibility to noise-induced hearing loss (NIHL) appear to be due to a gene for age-related hearing loss (AHL). The current study was designed to determine dose-response curves for NIHL over the ranges of intensities of noise that would characterize the B6 and CB inbred strains of mice. Because of the considerable differences in sensitivity to NIHL, the noise exposures for the two strains overlapped only at 110 and 113 dB. Nevertheless, the two strains exhibited two different dose-response curves, offset and with different slopes. We postulate that the B6 strain of mice exhibits a more linear increase for PTS from 98-113 dB, consistent with incremental effects on some metabolic physiological mechanism(s); the abrupt transition in NIHL between 113 and 116 dB for the CB mice is consistent with an ototraumatic structural injury.

Academia as partner in organizational change.

Partnerships are valuable strategies for promoting organizational change. Collaboration between academia and service can provide rapid access to new clinical service delivery mechanisms and real-world laboratories for implementing and testing novel approaches to care delivery. Academic-service partnerships also provide opportunities for work force development. One example of a partnership is described in this article. Details of a Nursing Care Management Institute illustrate principles of good practice for community-campus partnerships.

Trends in hearing protector usage in American manufacturing from 1972 to 1989.

This study investigated the trends in hearing protector use in United States manufacturing industries. Using data from the National Institute for Occupational Safety and Health-sponsored National Occupational Hazard Survey (1972), the National Occupational Exposure Survey (1983), and the Occupational Safety and Health Administration-sponsored National Survey of Personal Protective Equipment Usage (1989), estimates were made of numbers of workers using hearing protection in various industries. Unique to this study is discussion of the impact of enactment of hearing conservation regulations during the same time frame as the two earlier surveys. In general, higher percentages of workers utilized hearing protection in 1989 than in 1972. Increased hearing protection use over time was also found when size of facility (number of employees) was taken into account. Differences in the use of hearing protection over the period 1972-1989 varied in individual industries, ranging from less than 10 to more than 30%.

Cumulative effects of prevention education on substance use among 11th grade students in Ohio.

This article examines participation in school-based prevention activities from a statewide sample of 11th grade students in Ohio. About 42% of subjects indicated they had never been involved in a prevention education activity. Differences existed in mean number of activities by both gender and White/nonWhite status. Popular prevention education activities included participation in D.A.R.E. (Drug Abuse Resistance Education) at elementary, junior high, and senior high levels, "Just Say No Clubs"; Quest; and Red Ribbon Week. Only a small proportion of youth participated exclusively in any one of these activities. An association existed between student participation in prevention education and level of drug involvement. Students in each activity had lower mean scores for drug involvement when compared to students who had never participated in a prevention education activity. Also, the lowest mean scores occurred among students who had participated in multiple prevention activities.

The role of the chinchilla pinna and ear canal in electrophysiological measures of hearing thresholds.

Measurements of the acoustic transfer function (ATF) of the pinnae of 8 chinchillas were compared with the auditory-evoked potential (AEP) thresholds of 16 chinchillas measured in free field and with insert earphones. The ATF was measured in anesthetized chinchillas in a far-field condition in a semi-anechoic room using a logarithmic frequency sweep from 100 Hz to 20 kHz. Probe microphone measurements were collected with the probe opening at the tympanic membrane and in the same approximate position with the chinchilla removed from the sound field. For each animal's acoustic transfer function, the average of five in-the-ear and three free-field measurements were determined. The ATF exhibited a 5-dB passive gain at about 1 kHz and a broad resonance between 2.5 and 6 kHz of about a 10-dB gain. AEP thresholds were obtained from monaural chronically implanted chinchillas at 0.5, 1, 2, 4, and 8 kHz using first free-field and then insert earphone stimuli. The free-field sound pressure was measured with a microphone in the approximate position of the chinchilla's head. The earphone sound pressures were measured with a probe microphone positioned near the tympanic membrane. The free-field AEP hearing thresholds exhibited +10-dB gain at 4 kHz compared to the insert earphone AEP thresholds. The agreement between the ATF and AEP derived transfer function suggested that the threshold differences at 4 kHz between the two testing configurations can be accounted for by the pinna and ear canal gain.

Genetics of age-related hearing loss in mice. III. Susceptibility of inbred and F1 hybrid strains to noise-induced hearing loss.

Some humans and mice are genetically predisposed to age-related hearing loss (AHL), and others are variously susceptible to noise-induced hearing loss (NIHL). The inbred C57BL/6J (B6) mice exhibit AHL at an early age, whereas the inbred CBA/CaJ (CB) mice do not. The B6 mice are much more susceptible to NIHL than are the CB mice (Shone et al., 1991; Li, 1992a). The B6 mice possess an Ahl gene which maps to chromosome 10 (Erway et al., 1995). This study was designed, using these two inbred strains plus two F1 hybrid strains of mice, to begin to test the hypothesis that the Ahl genotypes may influence the susceptibility to NIHL. These strains of mice (with putative genotypes) are: inbred CB (+/+) and B6 (Ahl/Ahl); hybrid CBB6F1 (+/Ahl) and B6D2F1 (Ahl/Ahl; D2 represents inbred DBA/2J). Twenty-four mice of each of these four strains were exposed to noise (110 dB for 0, 1 or 2 h) and tested for auditory-evoked brainstem response (ABR) thresholds. The CB and CBB6F1 strains of mice did not differ significantly from each other, exhibiting mostly temporary threshold shifts. The B6 and B6D2F1 strains of mice did not differ significantly from each other, but did exhibit permanent threshold shifts. These results support the hypothesis that genetic predisposition to AHL may be revealed at a younger age by NIHL. This suggests that it may be possible to use the NIHL to distinguish segregating genotypes (+/Ahl vs. Ahl/Ahl) among backcross progeny and thereby to identify and map single genes for AHL.

Hearing loss in the chinchilla from impact and continuous noise exposure.

The relative hazard posed to the peripheral auditory system by impact/impulse and continuous noise of the same power spectrum was determined. Impact noise was generated by striking a nail with a hammer and was digitally recorded. The acoustical power spectrum of the impact was determined and pink noise was filtered to produce a continuous noise stimulus with the same acoustic power spectrum. Pre-exposure auditory evoked response (AER) thresholds were obtained at 1, 2, 4, and 8 kHz on 16 adult chinchillas. The pool of animals was divided into two equal groups based upon pre-exposure AER thresholds. One group was exposed to impact noise and the other group to the filtered pink noise. Exposures were 4 h/day for 5 days. Thirty days following the exposure, auditory evoked response thresholds were remeasured. Changes in auditory sensitivity were determined by subtracting the pre-exposure thresholds from the post-exposure thresholds. Hearing threshold shifts of the impact noise group were significantly greater (p less than 0.0001) than the hearing threshold shifts of the continuous noise group. These data indicate a need to more closely examine the parameters and effects of impact noise. There may be a need to develop expanded damage-risk criteria for occupational exposure to impulse/impact noise.

Analysis of a hearing conservation program data base: factors other than workplace noise.

The hearing conservation records of a large, multiple-facility printing company were obtained. The records were analyzed for factors associated with Standard Threshold Shift. These factors included hearing levels, employee age and sex, occupational and nonoccupational noise exposure histories, and medical history. The analysis revealed that statistically significant factors associated with Standard Threshold Shift were from medical and nonoccupational noise exposure histories, and not occupational noise exposure.

Dissociation of maximum concentration of kanamycin in plasma and perilymph from ototoxic effect.

Kanamycin was administered in total daily doses of 0 (vehicle), 100, 200 or 300 mg/kg to different groups of guinea pigs for two weeks. These total daily doses were administered according to three different dosing schedules, either as a single injection given once a day, divided into two equal doses and given twice a day, or divided into four equal doses and administered four times a day. It was found that the magnitude of the ototoxicity resulting from kanamycin administration was related to the total daily dose alone and not the dosing schedule. This lack of relationship between the dosing schedule and the magnitude of the ototoxicity due to kanamycin is the reverse of that reported for the nephrotoxicity resulting from gentamicin, tobramycin and netilmicin.

The ototoxic interaction of viomycin, capreomycin and polymyxin B with ethacrynic acid.

The ototoxic interaction between the aminoglycoside antibiotics (streptomycin, kanamycin, etc.) and the loop-inhibiting diuretics (ethacrynic acid, furosemide and bumetanide) has been well documented. This interaction causes extensive destruction of the hair cells of the cochlea. Brummett et al. (1974) demonstrated that this interaction did not occur with the non-loop-inhibiting diuretics and kanamycin. The present study was undertaken to determine if antibiotics other than the aminoglycosides could produce the ototoxic interaction when combined with a loop-inhibiting diuretic. Three antibiotics-viomycin, capreomycin, and polymyxin B- when given with ethacrynic acid were found to produce cochlear hair cell damage that was similar to that produced by aminoglycoside antibiotics administered with ethacrynic acid. Therefore, the interaction appears to be specific to the loop-inhibiting diuretics but not specific for the aminoglycoside antibiotics.