Cell blocks from scraping of cytology smear: comparison with conventional cell block

OBJECTIVE: To compare cytomorphology preservation and immunohistochemistry results between conventional cell blocks (CCB) and cytoscrape cell blocks (SCB). STUDY DESIGN: Fine needle aspiration (FNAC) was done in 17 consecutive cases. Air-dried smears for May-Grünwald-Giemsa stain and wet-fixed smear for hematoxylin-eosin (H-E) stain were prepared. Simultaneously another pass was made in each case for preparation of material for CCB. One of the H-E-stained smears was spared for SCB. SCB was compared with CCB for cell morphology. Immunostaining was performed both cell blocks, as well as on FNA smears in 8 cases. Results were evaluated for intensity of staining and percentage of cells showing positivity. RESULTS: CCB and SCB sections showed adequate cellularity in all cases. Morphologic preservation was good in SCB sections. There was good architectural and nuclear preservation in all cases of SCB. Immunostaining results showed better and clear intensity of staining with little background in all cell block cases. CONCLUSION: SCB is a valuable technique in cell blocks from stained FNA smears. The cytomorphologic details are equally good in SCB and CCB. Additional panels of immunostaining can be done on SCB for better diagnosis and classification, particularly in cases in which repeat FNA is not possible.     

Todea from the Lower Cretaceous of western North America: implications for the phylogeny, systematics, and evolution of modern Osmundaceae

The first fossil evidence for the fern genus Todea has been recovered from the Lower Cretaceous of British Columbia, Canada, providing paleontological data to strengthen hypotheses regarding patterns of evolution and phylogeny within Osmundaceae. The fossil consists of a branching rhizome, adventitious roots, and leaf bases. The dictyoxylic stem has up to eight xylem bundles around a sclerenchymatous pith. Leaf traces diverge from cauline bundles in a typical osmundaceous pattern and leaf bases display a sheath of sclerenchyma around a C-shaped xylem trace with 2-8 protoxylem strands. Within the adaxial concavity of each leaf trace, a single sclerenchyma bundle becomes C-shaped as it enters the cortex. The sclerotic cortex is heterogeneous with an indistinct outer margin. The discovery of Todea tidwellii sp. nov. reveals that the genus Todea evolved by the Lower Cretaceous. A phylogenetic analysis combining morphological characters of living and extinct species with a previously published nucleotide sequence matrix confirms the taxonomic placement of T. tidwellii. Results also support the hypothesis that Osmunda s.l. represents a paraphyletic assemblage and that living species be segregated into two genera, Osmunda and Osmundastrum. Fossil evidence confirms that Osmundaceae originated in the Southern Hemisphere during the Permian, underwent rapid diversification, and species extended around the world during the Triassic. Crown group Osmundaceae originated by the Late Triassic, with living species appearing by the Late Cretaceous.     

Removal of detergents from protein digests for mass spectrometry analysis

Detergents are commonly used for the extraction of hydrophobic proteins and must be removed for sensitive detection of peptides by mass spectrometry. We demonstrate that ethyl acetate is able to extract octylglycoside from a protease digest without loss of peptides or interference with the peptide mass spectral profile. Ethyl acetate extraction was also found to reduce interference by sodium dodecyl sulfate, Nonidet P-40, or Triton X-100 in the mass spectrometry analysis.     

Esterase Autodisplay: Enzyme Engineering and Whole-Cell Activity Determination in Microplates with pH Sensors

Among the GDSL family of serine esterases/lipases is a group of bacterial enzymes that posses C-terminal extensions involved in outer membrane anchoring or translocation. ApeE from Salmonella enterica serovar Typhimurium, a member of this group, has been expressed in Escherichia coli and was resistant to protease digestion when the protease was added to whole cells, indicating a periplasmic localization. The five consensus blocks conserved within all GDSL esterases were identified in ApeE by multiple sequence alignment and separated from the C-terminal extension. The DNA sequence spanning the four invariant residues Ser, Gly, Asn, and His, and hence representing the catalytic domains of ApeE, was amplified by PCR and fused in frame to the transport domains of the autodisplay system. The resulting artificial esterase, called EsjA, was overexpressed in the cell envelope of E. coli and was shown to be active by the use of α-naphthyl acetate (α-NA) as a substrate in an in-gel activity stain after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Surface exposure of EsjA was indicated by its accessibility to protease added to whole cells. The esterase activity of whole cells displaying EsjA was determined by a pH agar assay and by the use of microplates with integrated pH-dependent optical sensors. α-NA, α-naphthyl butyrate, and α-naphthyl caproate were used as substrates, and it turned out that the substrate preferences of artificial EsjA were altered in comparison to original ApeE. Our results indicate that autodisplay of esterase in combination with pH sensor microplates can provide a new platform technology for the screening of tailor-made hydrolase activities.     

Methionine sulfoxides on PrPSc: a prion-specific covalent signature

Prion diseases are fatal neurodegenerative disorders believed to be transmitted by PrP (Sc), an aberrant form of the membrane protein PrP (C). In the absence of an established form-specific covalent difference, the infectious properties of PrP (Sc) were uniquely ascribed to the self-perpetuation properties of its aberrant fold. Previous sequencing of the PrP chain isolated from PrP(27-30) showed the oxidation of some methionine residues; however, at that time, these findings were ascribed to experimental limitations. Using the unique recognition properties of alphaPrP mAb IPC2, protein chemistry, and state of the art mass spectrometry, we now show that while a large fraction of the methionine residues in brain PrP (Sc) are present as methionine sulfoxides this modification could not be found on brain PrP (C) as well as on its recombinant models. In particular, the pattern of oxidation of M213 with respect to the glycosylation at N181 of PrP (Sc) differs both within and between species, adding another diversity factor to the structure of PrP (Sc) molecules. Our results pave the way for the production of prion-specific reagents in the form of antibodies against oxidized PrP chains which can serve in the development of both diagnostic and therapeutic strategies. In addition, we hypothesize that the accumulation of PrP (Sc) and thereafter the pathogenesis of prion disease may result from the poor degradation of oxidized aberrantly folded PrP.     

Saccharomyces cerevisiae Pra1p/Yip3p interacts with Yip1p and Rab proteins.

The regulation of membrane traffic involves the Rab family of Ras-related GTPases, of which there are a total of 11 members in the yeast Saccharomyces cerevisiae. Previous work has identified PRA1 as a dual prenylated Rab GTPase and VAMP2 interacting protein [Martinic et al. (1999) J. Biol. Chem. 272, 26991-26998]. In this study we demonstrate that the yeast counterpart of PRA1 interacts with Rab proteins and with Yip1p, a membrane protein of unknown function that has been reported to interact specifically with the Rab proteins Ypt1p and Ypt31p. Yeast Pra1p/Yip3p is a factor capable of biochemical interaction with a panel of different Rab proteins and does not show in vitro specificity for any particular Rab. The interactions between Pra1p/Yip3p and Rab proteins are dependent on the presence of the Rab protein C-terminal cysteines and require C-terminal prenylation.     

One-electron reduction potentials of dietary carotenoid radical cations in aqueous micellar environments

The one-electron reduction potentials of the radical cations of five dietary carotenoids (β-carotene, canthaxanthin, zeaxanthin, astaxanthin and lycopene) in aqueous micellar environments have been obtained from a pulse radiolysis study of electron transfer between the carotenoids and tryptophan radical cations as a function of pH, and lie in the range of 980–1060 mV. These values are consistent with our observation that the carotenoid radical cations oxidise tyrosine and cysteine. The decays of the carotenoid radical cations in the absence of added reactants suggest a distribution of exponential lifetimes. The radicals persist for up to about 1 s, depending on the medium.