Mitochondrial role in hair cell survival after injury.

The role of mitochondrial biogenesis in hair cell survival after injury was evaluated by inhibiting mitochondrial protein synthesis with chloramphenicol and then studying the effects on hair cell survival after exposure to two different types of ototoxins, gentamicin and acoustic trauma. Seven- to 10-day-old chicks were treated with either a single injection of gentamicin (250 mg/kg) or noise (1500 Hz at 120 dB sound pressure level for 14 hours). A subset of the gentamicin- and noise-treated animals also received chloramphenicol (1200 mg/kg during a 24-hour period) through a subcutaneous osmotic pump. A control group received chloramphenicol alone (1200 mg/kg during a 24-hour period). All animals were sacrificed after 5 days, and their basilar papillae (cochleas) were prepared for scanning electron microscopy. Hair cell loss was quantified with stereologic techniques. Animals treated with chloramphenicol alone did not have any evidence of hair cell loss. Gentamicin-treated animals had characteristic hair cell loss beginning at the basal tip and tapering out along the inferior edge more distally. The addition of chloramphenicol to gentamicin treatment significantly increased hair cell loss by 30%, extending the area of hair cell loss into the superior hair cell region at the distal boundary of the lesion. Pure-tone noise exposure characteristically produced hair cell loss along the inferior edge and occasionally included hair cells along the most superior edge. Addition of chloramphenicol to noise exposure significantly increased hair cell loss by 80%, with extension of the lesion across the full width of the sensory epithelium and basally. These results demonstrate that mitochondrial biogenesis is involved in cellular responses to injury. They suggest that mitochondrial function may regulate the probability of survival after metabolic challenges to hair cell integrity.

Head and neck manifestations of Maffucci's syndrome: chondrosarcoma of the nasal septum.

The possibility of a dyschondroplastic syndrome should be considered in any patient presenting with a cartilagenous tumor in the head and neck. Classification of the patient as Maffucci's syndrome or Ollier's disease increases the need for vigilant observation for the development of sarcomatous lesions and can help differentiate between low-grade chondrosarcoma and atypical enchondroma.

Increased deafferentation-induced cell death in chick brainstem auditory neurons following blockade of mitochondrial protein synthesis with chloramphenicol.

Second-order auditory neurons in nucleus magnocellularis (NM) of the chick brainstem undergo a series of rapid metabolic changes following unilateral cochlea removal, culminating in the death of 25% of NM neurons. Within hours of cochlea removal, ipsilateral NM neurons show marked increases in histochemical staining for the mitochondrial enzymes succinate dehydrogenase and cytochrome oxidase (CO). We have shown previously in an ultrastructural study that these increases in oxidative capacity are mediated in part by a rapid increase in mitochondrial volume within deafferented neurons. In neurons that are destined to die as a result of deafferentation, mitochondria are smaller, stain poorly for CO, and often contain vacuoles associated with oxidative dysfunction. Our present set of experiments is designed to test the hypothesis that increases in oxidative metabolism are necessary for NM neuronal survival following removal of afferent input. We used chloramphenicol (CAP), a mitochondrial protein synthesis inhibitor, to block the characteristic increase in CO activity and mitochondrial proliferation following cochlea removal. We then studied the effects of CAP on NM neuronal survival following deafferentation. When CAP was administered continuously at 600 mg/kg/d for 5 d following cochlea removal, deafferentation-induced neuronal death in NM was significantly increased from 22% to 36%. Higher-dose (1200 mg/kg/d) pulses of CAP were administered for the first 6, 12, or 24 hr following cochlea removal. After 5 d survival, greater increases in neuronal cell death were found in animals treated for the first 12 or 24 hr (65% neuronal death). CAP administration for the first 6 hr had no significant effect on neuronal survival. The effects of CAP on neuronal cell death in NM are not likely to be due to systemic effects of the drug, but instead to a specific change in mitochondrial function. Our results suggest that enhanced oxidative enzyme function plays an important role in the survival of NM neurons during the first 24 hr after deafferentation. The nature of the signal(s) eliciting mitochondrial enhancement and the means by which it influences NM survival are not known.

Rapid increase in mitochondrial volume in nucleus magnocellularis neurons following cochlea removal.

Second-order auditory neurons in nucleus magnocellularis (NM) of the chick brainstem undergo a series of rapid metabolic changes following unilateral cochlea removal, culminating in the death of 25% of NM neurons. Within hours of cochlea removal, ipsilateral NM neurons show marked increases in histochemical staining for the mitochondrial enzymes succinate dehydrogenase and cytochrome oxidase. We investigated corresponding ultrastructural changes in NM neurons by preparing animals undergoing unilateral cochlea removal for transmission electron microscopy. We quantified changes in NM mitochondrial volume by stereological methods and qualitatively compared mitochondrial morphology between NM neurons destined to survive and those destined to die after cochlea removal. Within hours of cochlea removal, ipsilateral NM neurons show striking increases in mitochondrial volume (84% at 6 hours and 236% at 12 hours after cochlea removal compared to unoperated, control animals). At 2 week survival times, ipsilateral NM neurons contain fewer mitochondria than contralateral neurons. Surprisingly, anesthesia alone causes short-term increases in NM mitochondrial volume. Animals anesthetized with pentobarbital and ketamine and sacrificed 6 or 12 hours later showed a 45% increase in mitochondrial volume compared to previously unanesthetized animals. NM neurons destined to die within days of cochlea removal can be identified within several hours after deafferentation by the appearance of their ribosomes. We observed qualitative differences in mitochondrial morphology in dying neurons. Mitochondria in neurons destined to die consistently showed mitochondrial swelling and vacuolization indicative of metabolic dysfunction. Similar mitochondrial changes have been reported when mitochondria take up excess calcium. Ultrastructural changes in NM after cochlea removal display features of both programmed and pathological cell death, in which increased intracellular calcium is thought to play a role.

The sequence of squash NADH:nitrate reductase and its relationship to the sequences of other flavoprotein oxidoreductases. A family of flavoprotein pyridine nucleotide cytochrome reductases.

Nucleotide sequences were determined for cDNA clones for squash NADH:nitrate oxidoreductase (EC 1.6.6.1), which is one of the most completely characterized forms of this higher plant enzyme. An open reading frame of 2754 nucleotides began at the first ATG. The deduced amino acid sequence contains 918 residues, with a predicted Mr = 103,376. The amino acid sequence is very similar to sequences deduced for other higher plant nitrate reductases. The squash sequence has significant similarity to the amino acid sequences of sulfite oxidase, cytochrome b5, and NADH:cytochrome b5 reductase. Alignment of these sequences with that of squash defines domains of nitrate reductase that appear to bind its 3 prosthetic groups (molybdopterin, heme-iron, and FAD). The amino acid sequence of the FAD domain of squash nitrate reductase was aligned with FAD domain sequences of other NADH:nitrate reductases, NADH:cytochrome b5 reductases, NADPH:nitrate reductases, ferredoxin:NADP+ reductases, NADPH:cytochrome P-450 reductases, NADPH:sulfite reductase flavoproteins, and Bacillus megaterium cytochrome P-450BM-3. In this multiple alignment, 14 amino acid residues are invariant, which suggests these proteins are members of a family of flavoenzymes. Secondary structure elements of the structural model of spinach ferredoxin:NADP+ reductase were used to predict the secondary structure of squash nitrate reductase and the other related flavoenzymes in this family. We suggest that this family of flavoenzymes, nearly all of which reduce a hemoprotein, be called "flavoprotein pyridine nucleotide cytochrome reductases."

Cytochrome oxidase response to cochlea removal in chicken auditory brainstem neurons.

Changes in cytochrome oxidase (CO) activity were studied in the chick brainstem auditory nuclei, n. magnocellularis (NM) and n. laminaris (NL), following unilateral cochlea removal. Chickens aged 10 days or 56 weeks underwent unilateral cochlea removal. Following survival periods of 30 minutes to 14 days for the 10-day-old birds and 6 hours or 14 days for the 56-week-old birds, the animals were perfused with paraformaldehyde/glutaraldehyde fixative. Cryostat sections of the brainstem were then prepared for CO histochemistry. Microdensitometry was used to quantify the difference in CO staining in NM and NL ipsilateral and contralateral to the cochlea removal. Since the cochlea projects to the ipsilateral NM, the contralateral NM was used as a within-animal control. In normal chickens, NM cell bodies and the cell bodies and dendrites of NL neurons stain darkly for CO in both young and adult birds. In 10-day-old birds, there is no significant change in CO staining in NM from 30 minutes to 3 hours after cochlea removal. Then, a rapid biphasic change in CO staining was found in the ipsilateral NM. An increase in staining was observed 6 to 24 hours postoperatively, followed by a decrease in CO staining at 3- to 14-day survival times. In the 56-week-old birds, no increases in CO staining were observed 6 hours after cochlea removal, but a decrease in CO staining was found 14 days postoperatively. In NL, no changes were observed until 3 days (10-day-old birds) or 14 days (56-week-old birds) after cochlea removal. Then a decrease in CO staining was observed in the dendritic and glial/fiber regions of NL containing axons from the deafferented NM. Thus it appears that afferent input has a regulatory effect on the oxidative metabolism of neurons in the chicken auditory brainstem nuclei, an effect that differs with the age of the animal at the time of afferent manipulation.

High-level expression in Escherichia coli of the catalytically active flavin domain of corn leaf NADH:nitrate reductase and its comparison to human NADH:cytochrome B5 reductase.

Higher plant nitrate reductase can be divided into three functional domains representing its prosthetic groups: 1) flavin; 2) cytochrome b; and 3) Mo-pterin. The flavin domain has been synthesized by heterologous expression in Escherichia coli using a fragment of a corn leaf NADH:nitrate reductase cDNA clone, Zmnr1, which we had previously isolated and sequenced. A Xho2-BamH1 fragment was cut from Zmnr1, containing the sequence for the flavin domain, and ligated in the BamH1 site of expression vector pET3c. When this construct was expressed in E. coli, a 30 kD polypeptide was found to be newly synthesized. The flavin domain was purified to homogeneity using blue Sepharose and shown to have a molecular weight of 30 kD. The recombinant flavin domain has a ferricyanide reductase specific activity of 1000 mumols NADH oxidized/min/mg protein and a visible spectrum virtually identical to that of human NADH:cytochrome b5 reductase.

Monoclonal antibody-based immunoaffinity chromatography for purifying corn and squash NADH: nitrate reductases. Evidence for an interchain disulfide bond in nitrate reductase.

NADH: nitrate reductase (EC 1.6.6.1) (NR) is present in small amounts in plant tissues and its polypeptide in inherently labile. Consequently, NR is difficult to purify. We have generated 20 monoclonal antibodies (McAb) for corn and squash NR and selected two for use in immunoaffinity chromatography. Squash McAb CM 15(11) and corn McAb ZM 2(69)9, which both bind corn and squash NR, were covalently coupled to Sepharose and used for purification of NR with elution of the purified enzyme by a pH 11 buffer. Although this procedure yielded highly purified NR, its activity was diminished by the pH 11 treatment. When corn leaf crude extract was applied to McAb CM 15(11)-Sepharose, NR bound and could be eluted in homogeneous form by its substrate, NADH. Corn leaf NR prepared by substrate elution retained a high level of NADH: NR activity. Immunoaffinity-purified corn and squash NR were shown to have an interchain disulfide bond as well as a reactive thiol group. These results are discussed in relation to the recently obtained sequences of NR clones and suggestions made for site-directed mutagenesis experiments to aid in identifying the cysteine residues of NR associated with these features of the enzyme.