Long- and short-term freezing induce different types of injury in Arabidopsis thaliana leaf cells.

In nature, intact plant cells are subjected to freezing and can remain frozen for prolonged periods. We assayed the survival of Arabidopsis thaliana leaf cells following freezing and found that short- and long-term exposures produced different types of cellular injury. To identify the cause of these injuries, we examined the ultrastructure of the cell plasma membranes. Our results demonstrate that ultrastructural changes in the plasma membrane due to short-term freezing are associated with interbilayer events, including close apposition of the membranes. In both acclimated and non-acclimated leaf cells, these interbilayer events resulted in "fracture-jump lesions" in the plasma membrane. On the other hand, long-term freezing was associated with the development of extensive protein-free areas caused by the aggregation of intramembrane proteins with consequent vesiculation of the affected membrane regions; this effect was clearly different from the ultrastructural changes induced by interbilayer events. We also found that prolonged exposure of non-acclimated leaf cells to a concentrated electrolyte solution produced effects that were similar to those caused by long-term freezing, suggesting that the ultrastructural changes observed in the plasma membrane following long-term freezing are produced by exposure of the leaf cells to a concentrated electrolyte solution. This study illustrates multiple causes of freezing-induced injury in plant cells and may provide useful information regarding the functional role of the diverse changes that occur during cold acclimation.

Subchronic (13-week) oral toxicity study, preceded by an in utero exposure phase, with arachidonate-enriched triglyceride oil (SUNTGA40S) in rats.

Polyunsaturated fatty acids (PUFAs), such as arachidonic acid (ARA) and docosahexaenoic acid (DHA) are natural constituents found in human milk, fish oil or egg yolk. Until recently, infant formulas, though providing the essential fatty acid precursors for these PUFAs, did not contain preformed ARA or DHA. In this study the safety of SUNTGA40S as source of ARA, not only for use in infant formulas but also for nutritional products or food supplements, was evaluated in a subchronic study in Wistar rats, preceded by a 4-week pretreatment period of parental (F(0)) rats and exposure of the F(0) dams throughout mating, gestation and lactation. SUNTGA40S was administered at dietary levels of 0.5%, 1.5% and 5% (wt/wt) adjusted with corn oil to 5.76% added fat. An additional group received 3.65% (wt/wt) SUNTGA40S in conjunction with 2.11% (wt/wt) high DHA Tuna oil, providing an ARA:DHA ratio of 2.7:1. High-fat and low-fat controls received basal diet with or without 5.76% corn-oil supplement. The content, stability and homogeneous distribution of the test substances in the diet were confirmed under study conditions. The administration of SUNTGA40S, with or without DHA oil, did not affect health, growth, fertility or reproductive performance of the parental rats, nor pup characteristics (condition, weight gain, viability, number per litter or sex ratio). In the subchronic study with the offspring (F(1)) rats, no significant differences were found in condition, neurobehavioural observations, ophthalmoscopy, growth, urinalysis or macroscopic and microscopic findings between the test groups and the low-fat or the high-fat controls. In males of the 5% SUNTGA40S and the SUNTGA40S/DHA group, red blood cell counts, haemoglobin concentration and packed cell volume were lower and reticulocytes were slightly higher than in the high-fat and low-fat control groups. Cholesterol, triglycerides and phospholipids in plasma were lower than in the high-fat controls in both sexes in the 5% SUNTGA40S and the SUNTGA40S/DHA group and (for triglycerides only) in the 1.5% SUNTGA group. Due to the administration of extra dietary fat, food intake and prothrombin time (males only) were lower and alkaline phosphatase activity was higher in all the high-fat groups, including the corn-oil controls, as compared to the low-fat controls. The weight of the spleen was higher in males of the 5% SUNTGA40S and the SUNTGA40S/DHA group compared to both the low-fat and the high-fat controls. The effects noted in this study at high dose levels of SUNTGA40S are consistent with previously reported physiological responses to dietary intake of high PUFA containing oils. The present results provide evidence that SUNTGA40S is a safe source of arachidonic acid. Except during lactation when the intake in dams doubled, 5% Suntga40S in the diet was equivalent to an overall intake of approximately 3g/kg body weight/day in F(0) and F(1) animals.

Generation of a recombinant Sendai virus that is selectively activated and lyses human tumor cells expressing matrix metalloproteinases.

Malignant tumor cells often express matrix metalloproteinases (MMPs) at a high level to enable their dissemination and metastasis. Sendai virus (SeV), a nonsegmented negative strand RNA virus, spreads in the target tissues in vivo via cleavage activation of the viral fusion glycoprotein by a tissue-specific, trypsin-like enzyme. By deleting the viral matrix protein, we previously generated a recombinant SeV that does not bud to mature virions, but is highly fusogenic and spreads extensively from cell to cell in a trypsin-dependent manner. Here, we changed the tryptic cleavage site of the fusion glycoprotein of this virus to a site susceptible to MMPs. The resulting recombinant virus was no longer activated by trypsin but spread efficiently in cultured cells supplemented with MMP2 or MMP9 and in human tumor cell lines expressing these MMPs. Furthermore, the virus spread extensively in tumor cells xenotrasplanted to nude mice without disseminating to the surrounding normal cells, leading to the inhibition of the tumor growth in the mice. These results demonstrate the selective targeting and killing of human tumor cells by recombinant SeV technology and greatly advance the reemerging concept of oncolytic virotherapy, which currently appears to rely largely upon a natural preference of certain viruses for cancer cells.

Postischemic administration of Sendai virus vector carrying neurotrophic factor genes prevents delayed neuronal death in gerbils.

Sendai virus (SeV) vector-mediated gene delivery of glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) prevented the delayed neuronal death induced by transient global ischemia in gerbils, even when the vector was administered several hours after ischemia. Intraventricular administration of SeV vector directed high-level expression of the vector-encoded neurotrophic factor genes, which are potent candidates for the treatment of neurodegenerative diseases. After occlusion of the bilateral carotid arteries of gerbils, SeV vector carrying GDNF (SeV/GDNF), NGF (SeV/NGF), brain-derived neurotrophic factor (SeV/BDNF), insulin-like growth factor-1 (SeV/IGF-1) or vascular endothelial growth factor (SeV/VEGF) was injected into the lateral ventricle. Administration of SeV/GDNF, SeV/NGF or SeV/BDNF 30 min after the ischemic insult effectively prevented the delayed neuronal death of the hippocampal CA1 pyramidal neurons. Furthermore, the administration of SeV/GDNF or SeV/NGF as late as 4 or 6 h after the ischemic insult also prevented the death of these neurons. These results indicate that SeV vector-mediated gene transfer of neurotrophic factors has high therapeutic potency for preventing the delayed neuronal death induced by transient global ischemia, and provides an approach for gene therapy of stroke.

Preparation of carrier-free 7Be by ion-exchange following charged particle and photonuclear reactions.

Carrier-free 7Be has been produced by 7Li(p,n)7Be and 10B(gamma,p2n)7Be (as well as 11B(gamma,p3n)7Be) reactions using a cyclotron and electron linear accelerator, respectively. Radiochemical methods for purification of the carrier-free radioactive 7Be isotope from the irradiated lithium and boron compounds have been investigated. A simple separation scheme is proposed.

Monitoring of morphological development of the arachidonic-acid-producing filamentous microorganism Mortierella alpina.

Morphological parameters, such as hyphal growth rate, tip formation rate, tip extension rate and branch formation rate, of Mortierella alpina have been measured using a flow-through chamber under 25 different combinations of carbon and nitrogen concentrations. Morphological parameters were influenced not by C/N ratio but by carbon concentration in the medium. Specific rates of hyphal growth and tip formation both remained constant at a low carbon concentration of 5 g/l. Tip extension rate from one tip was 60 microm tip(-1) h(-1) at a carbon concentration below 15 g/l, and the branching formation rate was independent of carbon concentration. Tip extension rate was a function of specific hyphal growth rate, which in turn was linearly proportional to the specific tip formation rate, demonstrating that tip extension rate was exponentially proportional to the specific tip formation rate. Branch formation rate per hyphal element remained unchanged even at tip extension rates lower than 60 microm tip(-1) h(-1) and at specific hyphal growth rates lower than 0.83 h(-1), but decreased drastically at higher rates of tip extension and hyphal growth.

Sucrose incubation increases freezing tolerance of asparagus (Asparagus officinalis L.) embryogenic cell suspensions.

The freezing tolerance of asparagus (Asparagus officinalis L.) embryogenic cells, as determined by electrolyte leakage, was increased by the incubation of samples in medium containing 0.8 M sucrose. To elucidate the mechanism involved, we investigated the changes in soluble carbohydrates, cell ultrastructure and proteins accompanying the increase in freezing tolerance following incubation in sugar-rich medium. During sugar incubation, the intracellular sucrose content increased from 67 mol g-1FW to 429 mol g-1FW; it was also metabolized into fructose and glucose, as determined by high-performance liquid chromatography. Microscopy revealed that sugar incubation induced plasmolysis of embryogenic cells and drastic changes in cell ultrastructure with the appearance of rough endoplasmic reticulum (rER). Furthermore, immunoblotting analysis with anti-dehydrin antiserum revealed that a dehydrin-like protein appeared only when maximal freezing tolerance was induced by sugar incubation. These results suggest that freezing tolerance of asparagus embryogenic cells is increased by a complex mechanism involving notably changes in cell ultrastructure and accumulation of certain sugars and proteins during sugar incubation.

Loading process of sugars into cabbage petiole and asparagus shoot apex cells by incubation with hypertonic sugar solutions.

The freezing tolerance of cabbage petioles and asparagus shoot apexes was increased by preincubation with 0.8 M sugar solutions. In cabbage petioles with an initial freezing tolerance of -3 degrees C (temperature for 50% cell survival), as determined by both electrolyte leakage and fluorescein diacetate vital staining, the freezing tolerance was increased to -13 degrees C by incubation with sorbitol solutions for 3 h. In meristematic cells of asparagus shoot apexes with an initial freezing tolerance of -7.5 degrees C, as determined by fluorescein diacetate vital staining, the freezing tolerance was increased to -30 degrees C by incubation with 0.8 M sugar solutions for 3 h, although other cells in the shoot apexes were killed by higher freezing temperatures. During incubation of both cabbage petioles and asparagus shoot apexes with sugar solutions, sugars were intracellularly taken up by osmotically induced fluid-phase endocytotic vesicles, as indicated by comovement of Lucifer Yellows carbohydrazide (LYCH) observed with a confocal laser scanning microscope. The amounts of intracellularly taken up sugars increased concomitantly with the formation of endocytotic vesicles depending on the time of incubation in parallel with a gradual increase of freezing tolerance. However, the endocytotic vesicles and their contents were retained not only after prolonged incubation after maximum freezing tolerance had been achieved but also after recovery of these tissue cells to isotonic conditions or after freeze-thawing. These results suggest that although sugars are intracellularly taken up by endocytotic vesicles, they might be sequestered within vesicles, casting doubt on their protective role to the plasma membranes as a main site of freezing injury. The pretreatment with 1 mM p-chloromercuribenzenesulfonic acid (PCMBS), an inhibitor of sugar transport, reduced the amounts of intracellular sugar uptake without affecting the formation of endocytotic vesicles, suggesting that sugars were, at least partly, taken up by sugar transporters. In the pretreatment with PCMBS, the freezing tolerance of incubated tissues with sugar solutions was significantly reduced, although addition of PCMBS per se did not affect survival. These results suggest that sugars taken up by sugar transporters, rather than sugars taken up by endocytotic vesicles, are mainly responsible for the increased freezing tolerance of cabbage petioles and asparagus shoot apexes. Furthermore, we aimed to study the occurrence of fluid-phase endocytosis with LYCH in an isotonic condition. Our results indicated that uptake of LYCH by fluid-phase endocytotic vesicles was not detected microscopically in isotonic condition, although LYCH was spectrofluorimetrically taken up in isotonic condition. Spectrofluorimetric uptake of LYCH was inhibited by addition of probenecid, an anion transport inhibitor. These results suggest that in cabbage petioles and asparagus shoot apexes, LYCH is taken up by anion transport but not by fluid-phase endocytosis in isotonic condition, and uptake of LYCH by fluid-phase endocytosis is restricted to occur only in hypertonic condition.

Skeletal muscle regeneration after insulin-like growth factor I gene transfer by recombinant Sendai virus vector.

We scrutinized the applicability and efficacy of Sendai virus (SeV) vectors expressing either LacZ or human insulin-like growth factor-I (hIGF-I) in gene transfer into skeletal muscle. Seven days after the intramuscular injection of LacZ/SeV X-gal labeled myofibers were demonstrated in rat anterior tibialis muscle with/without bupivacaine treatment and the transgene expression persisted up to 1 month after injection. Recombinant hIGF-I was detected as a major protein species in culture supernatants of a neonatal rat myoblast cell line L6 and thus induced the cells to undergo myogenetic differentiation. The introduction of hIGF-I/SeV into the muscle showed a significant increase in regenerating and split myofibers which were indicative of hypertrophy, and also an increase in the total number of myofibers, in comparison to that seen in the LacZ/SeV-treated control muscle. These results demonstrate that SeV achieves high-level transgene expression in skeletal muscle, and that hIGF-I gene transfer using SeV vector may therefore have great potential in the treatment of neuromuscular disorders.

Mycelial pellet intrastructure and visualization of mycelia and intracellular lipid in a culture of Mortierella alpina.

The intrastructure of mycelial pellets of Mortierella alpina, which accumulate fatty acids in mycelia, was visualized following labeling with fluorescein isothiocyanate (FITC) and Nile red using fluorescence microscopy. The pellet was an ellipse shape, but its intrastructure was shaped as a doughnut with a cave inside. Using three-dimensional image analysis, it was shown that the lipid was produced on the edge of the pellet, which corresponded to the area where the mycelial density was high. The cavity ratio of the pellet section was determined on the basis of the FITC fluorescence intensity, and in the early culture stage remained at 0.2 in a 10-kl fermentor culture, but finally increased to 0.35. Mycelial pellet volume paralleled the cavity ratio. Application of the technique used here allows analysis of the intrastructure of fungal pellets and new types of fungal biological study.

Cold acclimation-induced WAP27 localized in endoplasmic reticulum in cortical parenchyma cells of mulberry tree was homologous to group 3 late-embryogenesis abundant proteins.

We have shown that two 27-kD proteins, designated as WAP27A and WAP27B, were abundantly accumulated in endoplasmic reticulum-enriched fractions isolated from cortical parenchyma cells of mulberry tree (Morus bombycis Koidz.) during winter (N. Ukaji, C. Kuwabara, D. Takezawa, K. Arakawa, S. Yoshida, S. Fujikawa [1999] Plant Physiol 120: 480--489). In the present study, cDNA clones encoding WAP27A and WAP27B were isolated and characterized. The deduced amino acid sequences of WAP27A and WAP27B cDNAs had 12 repeats of an 11-mer amino acid motif that was the common feature of group 3 late-embryogenesis-abundant proteins. Under field conditions, transcripts of WAP27 genes were initially detected in mid-October, reached maximum level from mid-November to mid-December, and then gradually decreased. The transcript levels of WAP27 genes in cortical parenchyma cells harvested in October was drastically induced by cold treatment within a few days, whereas those in cortical parenchyma cells harvested in August were low even by cold treatment for 3 weeks. Immunocytochemical analysis by electron microscopy confirmed that WAP27 was localized specifically in vesicular-form ER and also localized in dehydration-induced multiplex lamellae-form ER. The role of WAP27 in the ER is discussed in relation to acquisition of freezing tolerance of cortical parenchyma cells in mulberry tree during winter.

Efficient High-Pulse-Energy Green-Beam Generation by Intracavity Frequency Doubling of a Quasi-Continuous-Wave Laser-Diode-Pumped Nd:YAG Laser.

A 50-mJ green beam was generated at a 1-kHz repetition rate by intracavity frequency doubling of a quasi-cw laser-diode-pumped Nd:YAG laser. The green laser was used for 12-mJ fourth-harmonic beam generation with a CsLiB(6)O(10) crystal.

28% electrical-efficiency operation of a diode-side-pumped Nd:YAG rod laser.

We propose a highly efficient quasi-cw Nd:YAG rod laser with a novel side-pumping design that uses microlens-free diode stacks. We demonstrate 320-W output power with 28% electrical-to-optical efficiency, which is, to our knowledge, the highest efficiency reported for diode-pumped solid-state lasers.

Effect of consumed carbon to nitrogen ratio of mycelial morphology and arachidonic acid production in cultures of Mortierella alpina.

The influence of the consumed carbon to nitrogen (C/N) ratio on arachidonic acid (AA) production and mycelial morphology was investigated in cultures of Mortierella alpina using shake flasks and a fermentor. The consumed C/N ratio was varied from 5 to 32 under the condition that the total initial amount of carbon and nitrogen sources was 50 g/l. Cellular yield increased markedly at C/N ratios below 7; carbon utilization was switched from cellular growth to lipid biosynthesis in the C/N ratio range of 7-15; lipid biosynthesis was most active when the C/N ratio was in the range of 15-32. However, for C/N ratios higher than 15, the mycelial concentration decreased due to nitrogen limitation but the lipid yield still increased. In the presence of excess nitrogen, the biomass concentration depended on the amount of the nitrogen source, but the AA yield was inversely related to this. On the other hand, in the presence of excess carbon, the fatty acid concentration increased with carbon source concentration but the AA concentration remained constant. From the viewpoint of AA production, the optimum C/N ratio was in the range of 15 to 20 with a balance between the amounts of carbon and nitrogen sources. When an enriched medium was used at a fixed C/N ratio of 20, the cellular and AA concentrations were shown to be proportional to the total concentrations of carbon and nitrogen sources in both flasks and the fermentor. The whole pellet size and width of pellet annular regions did not change with increasing C/N ratio for C/N ratios below 20 in the flask cultures. However, when the C/N ratio was higher than 20, these sizes increased in proportion to the C/N ratio.

Vestibular neuropathy accompanying auditory and peripheral neuropathies.

OBJECTIVE: To define the incidence of measurable vestibular disorders in patients with auditory and peripheral neuropathies. DESIGN: Descriptive study of the case features of auditory neuropathy in 14 patients, 8 of whom had concomitant peripheral neuropathies. SETTING: University referral center. PATIENTS: Fourteen patients aged from 10 to 75 years and diagnosed as having auditory neuropathy, 8 of whom had concomitant peripheral neuropathies. MAIN OUTCOME MEASURES: Incidence of abnormal vestibular caloric test results and the relationship of such incidence to clinical variables including the ages of the subjects, the presence of a concomitant peripheral neuropathy, vestibular symptoms, and audiological findings. RESULTS: Abnormal vestibular caloric test results occurred in 9 of the 14 patients. These 9 patients were on average older (35.6 years) than patients with normal caloric responses (17.8 years). Seven of the 9 patients with abnormal caloric responses had concomitant peripheral neuropathies compared with only 1 of the 5 patients with normal caloric responses. None of the 14 patients experienced symptoms of vestibular disorder. CONCLUSIONS: Asymptomatic vestibular disorders are common in patients with auditory neuropathy when a peripheral neuropathy is also present. The reason for the abnormal vestibular test results is likely a neuropathy of the vestibular nerves. Arch Otolaryngol Head Neck Surg. 2000;126:1453-1456

Cryo-scanning electron microscopic study on freezing behavior of xylem ray parenchyma cells in hardwood species

Differential thermal analysis (DTA) has indicated that xylem ray parenchyma cells (XRPCs) of hardwood species adapt to freezing of apoplastic water either by deep supercooling or by extracellular freezing, depending upon the species. DTA studies indicated that moderately cold hardy hardwood species exhibiting deep supercooling in the XRPCs were limited in latitudinal distribution within the -40 degrees C isotherm, while very hardy hardwood species exhibiting extracellular freezing could distribute in colder areas beyond the -40 degrees C isotherm. Predictions based on the results of DTA, however, indicate that XRPCs exhibiting extracellular freezing may appear not only in very hardy woody species native to cold areas beyond the -40 degrees C isotherm but also in less hardy hardwood species native to tropical and subtropical zones as well as in a small number of moderately hardy hardwood species native to warm temperate zones. Cryo-scanning electron microscopic (cryo-SEM) studies on the freezing behavior of XRPCs have revealed some errors in DTA. These errors are originated mainly due to the overlap between exotherms produced by freezing of water in apoplastic spaces (high temperature exotherms, HTEs) and exotherms produced by freezing of intracellular water of XRPCs by breakdown of deep supercooling (low temperature exotherms, LTEs), as well as to the shortage of LTEs produced by intracellular freezing of XRPCs. In addition, DTA results are significantly affected by cooling rates employed. Further, cryo-SEM observations, which revealed the true freezing behavior of XRPCs, changed the previous knowledge of freezing behavior of XRPCs that had been obtained by freeze-substitution and transmission electron microscopic studies. Cryo-SEM results, in association with results obtained from DTA that were reconfirmed or changed by observation using a cryo-SEM, revealed a clear tendency of the freezing behavior of XRPCs in hardwood species to change with changes in the temperature in the growing conditions, including both latitudinal and seasonal temperature changes. In latitudinal temperature changes, XRPCs in less hardy hardwood species native to tropical and subtropical zones exhibited deep supercooling to -10 degrees C, XRPCs in moderately hardy hardwood species native to temperate zones exhibited a gradual increase in the supercooling ability to -40 degrees C from warm toward cool temperate zones, and XRPCs in very hardy hardwood species native to boreal forests exhibited extracellular freezing via an intermediate form of freezing behavior between deep supercooling and extracellular freezing. In seasonal temperature changes, XRPCs in hardwood species native to temperate zones changed their supercooling ability from a relatively low degree in summer to a high degree in winter. XRPCs in hardwood species native to boreal forests changed their freezing behavior from deep supercooling to -10 degrees C in summer to extracellular freezing in winter. These results indicate that the freezing behavior of XRPCs in hardwood species tends to shift gradually from supercooling of -10 degrees C, to a gradual increase in the deep supercooling ability to -40 degrees C or less, and finally to extracellular freezing as a result of cold acclimation in response to both latitudinal and seasonal temperature changes. It is thought that these temperature-dependent changes in the freezing behavior of XRPCs in hardwood species are mainly controlled by changes in cell wall properties, although no distinct changes were detected by electron microscopic observations in cell wall organization between hardwood species or between seasons. Evidence of temperature-dependent changes in the freezing behavior of XRPCs in hardwood species provided by the results of studies using a cryo-SEM has indicated the need for further investigation to clarify cold acclimation-induced cell wall changes at the sub-electron microscopic level in order to understand the mechanisms of freezing adaptation.

Scopoletin uptake from culture medium and accumulation in the vacuoles after conversion to scopolin in 2,4-D-treated tobacco cells.

Tobacco (Nicotiana tabacum L. Bright Yellow) T-13 cell line has the ability to produce scopoletin endogenously and release some of it into the culture medium. We investigated the mechanism of scopoletin uptake following treatment of a tobacco culture with 2,4-dichlorophenoxyacetic acid (2,4-D). Addition of [14C]-labeled scopoletin showed that scopoletin was taken up by 2,4-D-treated cells and converted to scopolin, a 7-O-glucoside of scopoletin. This uptake of scopoletin began 6 h after 2,4-D addition to the cells. Experiments using several inhibitors showed that this uptake was energy-dependent. The phenomenon of 2,4-D-stimulated uptake was observed only for 7-hydroxycoumarins, such as scopoletin, umbelliferone and esculetin. To further investigate the site for scopoletin accumulation, we separated the vacuoles from T-13 cells and quantified the coumarin contents in this fraction. Most of the scopoletin in the vacuoles was present as glucoconjugate, scopolin. Moreover, glucosylation activity was absent from isolated vacuoles and, therefore, is likely to be located in the cytosol. Therefore, we can state that 2,4-D treatment of tobacco cells stimulated scopoletin uptake. The scopoletin was converted into scopolin in the cytoplasm, and then transferred into the vacuoles.

External and middle ear effects on infant hearing screening test results.

This study investigated the relationship between external and middle ear factors and hearing screening results by automated auditory brain stem response (ABR) and transient-evoked otoacoustic emissions (EOAEs). The ears of 200 healthy newborns aged 5 to 48 hours underwent screening by ABR and EOAE, followed by otoscopic examination. The pass rates for ABR and EOAE were 91% and 58.5%, respectively. On otoscopic examination, 28% (112/400) ears had occluding vernix obscuring the view of the tympanic membrane. Cleaning of vernix was successfully performed in all but 2 ears that had occluding vernix. Cleaning of vernix significantly increased the pass rates of all 400 ears for ABR and EOAE to 96% and 69%. Decreased tympanic membrane mobility was found in 22.7% (90/396) of ears that were evaluated otoscopically. Decreased tympanic membrane mobility had a significant effect on EOAE screening; only 33.4% of ears passed EOAE testing. Decreased tympanic membrane mobility did not significantly affect pass rates for ABR screening; 95% of these ears passed the automated ABR screen. Implications for newborn hearing screening are discussed.