Identification of non-covalent structure in apocytochrome c by hydrogen exchange and mass spectrometry.

Apocytochrome c, the in vivo precursor to active cytochrome c, was analyzed by amide hydrogen exchange and mass spectrometry to search for fixed, non-covalent structure. The protein was incubated in H(2)O at pH 3.3 or 6.7 for various times, then exposed to D(2)O to initiate isotope labeling of unfolded regions. Following acid quenching of hydrogen exchange, the labeled apocytochrome c was digested with pepsin into fragments that were analyzed by directly coupled high-performance liquid chromatography/electrospray ionization mass spectrometry. The intermolecular distribution of deuterium and the deuterium levels in structurally distinctive populations were determined from the mass spectra of the peptic fragments. Spectra of peptic fragments derived from apocytochrome c incubated at pH 3.3 had single envelopes of isotope peaks with masses indicating that all of the amide hydrogens had been replaced with deuterium. These results showed that apocytochrome c at pH 3.3 offered little resistance to hydrogen exchange, indicating that it was unfolded with little fixed structure. However, mass spectra of peptic fragments including residues 81-94 of apocytochrome c incubated at pH 6.7 had two envelopes of isotope peaks, indicating that one population was unfolded and the other population was highly structured in this region. Mass spectra of peptic fragments including residues N-terminal to residue 81 indicated that this region of the protein remained unfolded with little fixed structure at pH 6.7.

Optimization of conditions for studies of protein unfolding by hydrogen exchange/mass spectrometry.

Understanding the forces driving protein folding and aggregation is an essential step in developing means for controlling these important processes. Amide hydrogen exchange, coupled with mass spectrometry, has become an important method for studying protein unfolding and refolding. To extend procedures developed to study unfolding of relatively soluble proteins to less soluble, aggregation-prone proteins requires special considerations. This publication describes a general strategy developed using yeast transaldolase, which aggregates easily under conditions required to study its unfolding. Results presented here show that reducing the protein concentration to the nanomolar range is essential for managing aggregation of transaldolase. In addition, the present results point to use of relatively high concentrations of denaturants and short incubation times to minimize aggregation. These results also show how amide hydrogen exchange, coupled with mass spectrometry, can be used to study soluble aggregates.

Telecytology of fine-needle aspiration biopsies of the pancreas: a study of well-differentiated adenocarcinoma and chronic pancreatitis with atypical epithelial repair changes.

Four experienced cytopathologists provided consultations using telecytology and routine microscopy. Twenty-seven fine-needle aspiration biopsies (FNABs) from patients with chronic pancreatitis with atypical epithelial repair changes (n = 9) and pancreatic low-grade adenocarcinomas (LG-AC) (n = 18) were studied. False-positive and false-negative diagnostic rates were 19.4% and 12.5% by microscopy and 11.1% and 2.8% by telecytology. Comparisons of agreements between the correct diagnoses and consultations rendered by the two methods and among the diagnoses rendered on the same cases by the two modalities yielded kappa coefficients ranging from 0.444-1.000. Telecytology yielded slightly better kappa coefficients than microscopy. This method, which to our knowledge has not been previously applied to pancreatic FNAB, provides a potentially useful consultative tool for the interpretation of these difficult specimens. The diagnosis of FNAB from patients with chronic pancreatitis and LG-AC is difficult even for experienced consultants, as underscored by the considerable intraobserver and interobserver variability encountered in this study.

Retinal ganglion cell and inner plexiform layer thickness measurements in regions of severe visual field sensitivity loss in patients with glaucoma.

PURPOSE: To better understand the effects of severe glaucoma on the thickness of the retinal ganglion cell (RGC) and inner plexiform (IP) layers measured with frequency-domain optical coherence tomography. METHODS: In experiment 1, macular cube scans were obtained in 11 patients with glaucoma and the thickness of both the RGC and IP layers were measured at locations corresponding to 3, 5, and 7° eccentricity. For patients, only locations with total deviation losses of -15 dB or worse on perimetry were included. In experiment 2, higher resolution, horizontal midline scans were obtained from 30 controls in order to obtain a precise measure of the thickness of the RGC and IP layers of the healthy retina. RESULTS: In regions of severe field loss (experiment 1), glaucoma decreased the thickness of both layers, leaving a residual layer. The residual thickness of the IP layer was larger than the residual thickness of the RGC layer. In healthy controls (experiment 2), the RGC layer was about 57% of the RGC+IP layer thickness at 3° as compared with only 36% at 10°, in agreement with a recent histological study. CONCLUSION: Glaucomatous optic neuropathy, with severe losses in visual field sensitivity, decreases the thickness of both the RGC and IP layers, but leaves a residual thickness of both. The IP layer contributes slightly more than the RGC to this residual, even just outside the center of the fovea where the RGC layer thickness exceeds the IP layer thickness in controls.

Comparison of autoradiography and DNA histograms in assessing S-phase cells from patients with myeloid leukemias.

The studies reported here were designed to determine whether DNA histogram analysis provided the same estimate for the percentage of cells in S-phase as does 3H-TdR labeling index (LI). For DNA histogram analysis high resolution flow cytometry was employed together with 3 different computer based methods for histogram analysis. When tissue culture cells were employed there was a good correlation (r = 0.89) between the estimates for the percentage of cells in S-phase by the LI and DNA histogram techniques. In contrast, when 1138 bone marrow or peripheral blood specimens obtained from leukemic patients were studied the correlation was poor. For example when 243 pretherapy bone marrow aspirate cells were studied by both methods the correlation was only 0.46. A direct comparison of the clinical relevance of the two methods for measuring S-phase cells when high dose cytosine arabinoside therapy was used to treat patients demonstrated that only LI measurements provided clinically useful information. In conclusion, DNA histogram analysis provides an inaccurate estimate of the percentage of cells in S-phase.

From neuronal inclusions to neurodegeneration: neuropathological investigation of a transgenic mouse model of Huntington's disease.

Huntington's disease (HD) is an inherited progressive neurodegenerative disease caused by the expansion of a polyglutamine repeat sequence within a novel protein. Recent work has shown that abnormal intranuclear inclusions of aggregated mutant protein within neurons is a characteristic feature shared by HD and several other diseases involving glutamine repeat expansion. This suggests that in each of the these disorders the affected nerve cells degenerate as a result of these abnormal inclusions. A transgenic mouse model of HD has been generated by introducing exon 1 of the HD gene containing a highly expanded CAG sequence into the mouse germline. These mice develop widespread neuronal intranuclear inclusions and neurodegeneration specifically within those areas of the brain known to degenerate in HD. We have investigated the sequence of pathological changes that occur after the formation of nuclear inclusions and that precede neuronal cell death in these cells. Although the relation between inclusion formation and neurodegeneration has recently been questioned, a full characterization of the pathways linking protein aggregation and cell death will resolve some of these controversies and will additionally provide new targets for potential therapies.

Follicular lesions of thyroid: a 5-year fine-needle aspiration experience.

BACKGROUND: Fine-needle aspiration (FNA) of the thyroid is of limited value in discriminating between nonneoplastic and neoplastic lesions in approximately 5-29% of patients. Indeterminate lesions are due primarily to the overlapping cytologic features found in follicular lesions. In this report, the authors describe their experience with FNA biopsy of the thyroid, concentrating on the analysis of those aspirates placed in the follicular lesion category. METHODS: A blinded, retrospective analysis of 92 patients who underwent FNA and were diagnosed with follicular lesions was performed by three of the authors (T.S.G., B.D.F., and M.O.) at a multihead microscope. A worksheet assessing a variety of cytologic and architectural features was filled out for each FNA patient. The reviewers then reached a consensus diagnosis. RESULTS: The reviewers agreed with the reported FNA diagnosis of follicular lesion in 63 of the 92 patients studied. No distinguishing cytologic features predictive of the histologic outcome were found in any of these 63 patients. Seven patients were judged by the reviewers to have insufficient cells for evaluation. In the remaining 22 patients, the reviewers' diagnoses were in agreement with the histologic diagnoses in 17 patients. CONCLUSIONS. The authors found that there is a gray area in the cytologic diagnosis of patients with thyroid lesions by FNA due to inherent similarities at the light microscopic level. However, increased specificity may be achieved by careful attention to cytologic features and morphologic detail. Skillful application of FNA techniques, with the recovery of an adequate sample, will further decrease both interpretive errors and the number of patients diagnosed with "follicular lesions."