The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant.

Release of cytochrome c from mitochondria triggers activation of caspase proteases and death of a cell by apoptosis. However, the mechanism and kinetics of cytochrome c release remain unknown. Here we study this event by using green fluorescent protein (GFP)-tagged cytochrome c, and find that the release of cytochrome-c-GFP always precedes exposure of phosphatidylserine and the loss of plasma-membrane integrity - characteristics of apoptotic cells. Once initiated, the release of cytochrome- c-GFP continues until all of the protein is released from all mitochondria in individual cells, within about 5 minutes, regardless of the type or strength of stimulus or the time elapsed since the stimulus was applied. Temperatures ranging from 24 degrees C to 37 degrees C do not change the duration of release, and nor does the addition of caspase inhibitors. Further, we find that the electron-transport chain can maintain the mitochondrial transmembrane potential even after cytochrome c has been released.

Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process.

During apoptosis, cytochrome c is released into the cytosol as the outer membrane of mitochondria becomes permeable, and this acts to trigger caspase activation. The consequences of this release for mitochondrial metabolism are unclear. Using single-cell analysis, we found that when caspase activity is inhibited, mitochondrial outer membrane permeabilization causes a rapid depolarization of mitochondrial transmembrane potential, which recovers to original levels over the next 30-60 min and is then maintained. After outer membrane permeabilization, mitochondria can use cytoplasmic cytochrome c to maintain mitochondrial transmembrane potential and ATP production. Furthermore, both cytochrome c release and apoptosis proceed normally in cells in which mitochondria have been uncoupled. These studies demonstrate that cytochrome c release does not affect the integrity of the mitochondrial inner membrane and that, in the absence of caspase activation, mitochondrial functions can be maintained after the release of cytochrome c.

Cytochrome c is released in a single step during apoptosis.

Release of cytochrome c from mitochondria is a central event in apoptotic signaling. In this study, we utilized a cytochrome c fusion that binds fluorescent biarsenical ligands (cytochrome c-4CYS (cyt. c-4CYS)) as well as cytochrome c-green fluorescent protein (cyt. c-GFP) to measure its release from mitochondria in different cell types during apoptosis. In single cells, the kinetics of cyt. c-4CYS release was indistinguishable from that of cyt. c-GFP in apoptotic cells expressing both molecules. Lowering the temperature by 7 degrees C did not affect this corelease, but further separated cytochrome c release from the subsequent decrease in mitochondrial membrane potential (DeltaPsi(m)). Cyt. c-GFP rescued respiration in cells lacking endogenous cytochrome c, and the duration of cytochrome c release was approximately 5 min in a variety of cell types induced to die by various forms of cellular stress. In addition, we could observe no evidence of caspase-dependent amplification of cytochrome c release or changes in DeltaPsi(m) preceding the release of cyt. c-GFP. We conclude that there is a general mechanism responsible for cytochrome c release that proceeds in a single step that is independent of changes in DeltaPsi(m).

p53 triggers apoptosis in oncogene-expressing fibroblasts by the induction of Noxa and mitochondrial Bax translocation.

The mechanism of p53-dependent apoptosis is still only partly defined. Using early-passage embryonic fibroblasts (MEF) from wild-type (wt), p53(-/-) and bax(-/-) mice, we observe a p53-dependent translocation of Bax to the mitochondria and a release of mitochondrial Cytochrome c during stress-induced apoptosis. These events proceed independent of zVAD-inhibitable caspase activation, are not prevented by dominant negative FADD (DN-FADD), but are negatively regulated by Mdm-2. Bcl-x(L) expression prevents the release of mitochondrial Cytochrome c and apoptosis, but not Bax translocation. At a single-cell level, enforced expression of p53 is sufficient to induce Bax translocation and Cytochrome c release. Real-time RT-PCR analysis reveals a significant induction of RNA expression of Noxa and Bax in p53(+/+), but not in p53(-/-) MEF. Noxa protein expression becomes detectable prior to Bax translocation, and downregulation of endogenous Noxa by RNA interference protects wt MEF against p53-dependent apoptosis. Hence, in oncogene-expressing MEF p53 induces apoptosis by BH3 protein-dependent caspase activation.

Aspirin induces apoptosis through release of cytochrome c from mitochondria.

Nonsteroidal anti-inflammatory drugs (NSAID) reduce the risk for cancer, due to their antiproliferative and apoptosis-inducing effects. A critical pathway for apoptosis involves the release of cytochrome c from mitochondria, which then interacts with Apaf-1 to activate caspase proteases that orchestrate cell death. In this study we found that treatment of a human cancer cell line with aspirin induced caspase activation and the apoptotic cell morphology, which was blocked by the caspase inhibitor zVAD-fmk. Further analysis of the mechanism underlying this apoptotic event showed that aspirin induces translocation of Bax to the mitochondria and mitochondrial release of cytochrome into the cytosol. The release of cytochrome c from mitochondria was inhibited by overexpression of the antiapoptotic protein Bcl-2 and cells that lack Apaf-1 were resistant to aspirin-induced apoptosis. These data provide evidence that the release of cytochrome c is an important part of the apoptotic mechanism of aspirin.

Expression of recombinant microfilarial chitinase and analysis of domain function.

A family of chitinase isozymes was previously characterized from the microfilariae of Brugia malayi and Brugia pahangi. The expression of these enzymes correlates with the onset of microfilarial infectivity for the mosquito vector. To study the role of chitinase activity in filarial transmission, the p70 chitinase from Brugia malayi was cloned and expressed in two forms: a full-length product of approximately 62 kDa and a truncated product of 43 kDa containing only the N-terminal catalytic domain. Two epitopes defined by monoclonal antibodies were preserved only in the full-length recombinant enzyme. It was found that deletion of the cysteine-rich C-terminal domain increased the yield of the recombinant expression product, and did not affect the K(m) for di- or trisaccharide substrates. However, affinity for high molecular weight chitin was specific to the full-length molecule, and is apparently mediated by the cysteine-rich domain, suggesting a role for this part of the protein in targeting the secreted enzyme to its substrate.

A single cell analysis of apoptosis. Ordering the apoptotic phenotype.

Microcinematography was applied to the analysis of the kinetics of apoptotic cell death. Apoptosis was found to be a process that proceeds in different cells at different times after an initial stress, and therefore kinetic studies of apoptotic events in bulk cultures can be problematic. Using single cell analysis we found that stronger apoptotic stimuli induce an earlier onset of apoptosis, but that there is no relationship between time of onset and duration of the apoptotic process. That is, cells that initiate apoptosis shortly after induction do not proceed more rapidly through the process. This suggests an all-or-non-mechanism that is supported by some models of the biochemical pathways of apoptosis.

Rise of medical specialization and organizations affecting otolaryngology.

As we enter the third millennium, there are in the United States 24 medical specialties recognized by the American Board of Medical Specialties. The majority of the members of each of these specialties have their education, training, and knowledge "certified" by an examining board unique to their specialty. One hundred years ago virtually none of the foregoing existed. At the turn of the 20th century, nearly all physicians practiced all of medicine. How and why did this evolution occur and what controls evolved to contain this? The goal of this presentation is to review the rise of medical specialties and the board examination system and describe some of the many organizations, often known by acronyms, which deal with this now complex architecture.

Closure of pharyngocutaneous fistulae with the rhomboid flap.

This technique has been used successfully in 15 cases of pharyngocutaneous or antrocutaneous fistulae. Only 1 case failed which later closed with conservative management.

Pseudomonas aeruginosa in chronic maxillary sinusitis.

Pseudomonas aeruginosa (Ps. Au.) infection of the maxillary sinus has been reported as an incidental finding on routine antrostomy; however, it has also been noted in several studies as the significant organism in the etiology of chronic sinusitis. Four case reports of culture verified Ps. Au. maxillary sinusitis are presented. The therapeutic modality used in two of the cases was a Caldwell-Luc operation and in two, an intranasal antrostomy. In all cases, multiple irrigations through the surgically created nasoantral windows were done postoperatively, as was the instillation of gentamicin ophthalmic drops intranasally. In all four cases the infection cleared with this combined surgical and medical therapy.

Advanced laryngeal cancer. Relevance of pathologic stage to survival and therapy.

Sixty-seven laryngectomies performed for stage III and stage IV laryngeal carcinoma were reviewed. Stage III disease was managed by surgery alone. Treatment of stage IV disease was divided equally between surgery only and surgery plus radiotherapy. Five-year survival rates by clinical stage were 73% for stage III and 39% for stage IV. Clinical underestimation of disease occurred in 25% of stage III lesions. Unrecognized cartilage invasion and nodal disease occurred with equal frequency. Survival rates computed on the basis of pathologic staging were 91% for stage III and 41% for stage IV. Patients with stage IV disease who were treated with surgery alone had a 28% survival rate, while those receiving both radiotherapy and surgery had a 56% survival rate. In our opinion, surgical pathologic staging more accurately predicts survival than does clinical staging. Surgery alone appears to be adequate therapy for pathologic stage III laryngeal cancer. Addition of radiotherapy significantly improves survival in stage IV disease.

Blindness secondary to injections in the nose, mouth, and face: cause and prevention.

The potential hazard of blindness from injections in the face, nose, and mouth and the mechanisms by which this complication takes place are discussed. Precautionary measures, which we originally recommended for nasal turbinate injections, appear to be applicable for all injections in this area. We encourage their usage.

Head pain.

Head pain can be a challenging, difficult, and interesting diagnostic problem. A detailed history and carefully performed and directed physical and laboratory examination will aid in the complex differential diagnosis--which includes structural disease of the eye, ear, nose, throat, teeth; trauma; central and peripheral nervous system infections and tumors; and various neuropathies of unknown cause. Systemic abnormalities such as hypertension are a frequent cause, as are emotional upset and tension. Other isolated and extremely rare causes, such as the anatomic abnormality of an elongated styloid process, cannot be discussed in a presentation of this length, but are included in the bibliography.