AgNOR quantification in the diagnosis of follicular pattern thyroid lesions

OBJECTIVE: To evaluate the usefulness of silver staining of nucleolar organizer regions (AgNORs) in the preoperative diagnosis of follicular lesions in the thyroid with computer-aided morphometric analysis of the silver dots. STUDY DESIGN: Forty-eight cytologic smears of the thyroid were divided into 3 groups according to the results of postoperative histopathologic examination: hyperplastic nodules in nodular goiter (NG) (20), follicular thyroid adenoma (FTA) (20) and follicular thyroid carcinoma (FTC) (8). They were silver stained. The slides were analyzed with a computerized system for image analysis. Nearly 20 variables describing AgNORs were calculated (related to the area of the dots, number of dots and intranuclear localization of the dots). RESULTS: Only assessment of the total area of AgNORs in the nucleus allowed distinguishing between malignant and benign lesions. It was possible to determine the cutoff value of the total area of AgNORs in the nucleus (3.00 microns 2), limiting FTC from other lesions (observed ranges: NG, 1.64-2.87 microns 2; FTA, 1.81-2.85 microns 2; FTC, 3.01-3.97 microns 2). Evaluation of the mean number of AgNORs per nucleus did not improve the diagnosis of malignancy. CONCLUSION: Computer-aided morphometric analysis of silver dots may be useful in the preoperative diagnosis of thyroid lesions.     

O-N intramolecular acyl migration strategy in water-soluble prodrugs of taxoids

We synthesized a highly water-soluble canadensol prodrug 6 that formed canadensol 3 by a simple pH-dependent chemical mechanism via the O–N intramolecular acyl migration of the isobutyryl group. This prodrug, a 2′-O-isobutyryl isoform of 3, has no additional functional auxiliaries released during the conversion to 3. This is a significant advantage in toxicology and medical economics, since the potential side effects of reported water-soluble auxiliaries and the use of detergent for solubilization can be avoided. The solubility of 6 was 2.26 mg mL⁻¹ and only the parent drug 3 was released under physiological conditions (pH=7.4) while, in acidic medium, the release of 3 slowed until migration was completely obstructed at pH=2. In further consideration of this strategy, we elucidated the use of an ‘O–N acyl-like’ migration reaction of the Boc group in the design of a docetaxel prodrug. Both O–N migration and undesired hydrolysis of the Boc group occurred under physiological conditions, although no oxazolidinone formation was observed, suggesting the limitation of our water-soluble prodrug strategy to docetaxel.     

Structural analysis of N-glycans from human neutrophil azurocidin

N-glycans of human neutrophil azurocidin, enzymatic inactive homolog of serine proteinase playing important and multifunctional roles in antimicrobial defense, endotoxin binding, monocyte, and T-cell activation, were isolated by hydrazinolysis and fluorescence labeled. An ion-exchange chromatography on GlycoSep C column separated neutral, mono-, and disialylated glycans. The glycans from each group were separated subsequently on GlycoSep N and GlycoSep H columns. Sequential exoglycosidase treatment and HPLC mapping allowed determining 21 different glycan structures, majority of them being neutral (79.8%), the rest-mono- (13.1%) and disialylated (1.2%).     

Stem cell biology: a never ending quest for understanding

Stem cells (SC) research is an important part of biotechnology that could lead to the development of new therapeutic strategies. A lot of effort has been put to understand biology of the stem cells and to find genes and subsequently proteins that are responsible for their proliferation, self-renewal and differentiation. Different cytokines and growth factors has been used to expand stem cells, but no combination of these factors was identified that could effectively expand the most primitive stem cells. Recently, however, genes and receptors responsible for SC proliferation and differentiation have been described. Ligands for these receptors or these genes themselves are being already used for ex vivo expansion of stem cells and the first data are very promising. New markers, such as CXCR4 and CD133, have been discovered and shown to be present on surface of hematopoietic stem cells. The same markers were recently also found to be expressed on neuronal-, hepatic- or skeletal muscle-stem cells. By employing these markers several laboratories are trying to isolate stem cells for potential clinical use. New characteristics of stem cells such as transdifferentiation and cell fusion have been described. Our team has identified a population of tissue committed stem cells (TCSC). These cells are present in a bone marrow and in other tissues and they can differentiate into several cell types including cardiac, neural and liver cells.     

Effects of N-acylethanolamines on mitochondrial energetics and permeability transition

Effects of N-acylethanolamines (NAEs): N-arachidonoylethanolamine (anandamide), N-oleoylethanolamine and N-palmitoylethanolamine, on energy coupling and permeability of rat heart mitochondria were investigated. In nominally Ca2+-free media, these compounds exerted a weak protonophoric effect manifested by dissipation of the transmembrane potential and stimulation of resting state respiration. The strongest action was exhibited by N-arachidonoylethanolamine, followed by N-oleoylethanolamine, whereas N-palmitoylethanolamine was almost inactive. These protonophoric effects were resistant to cyclosporin A (CsA) and were much weaker than those of corresponding nonesterified fatty acids. In uncoupled mitochondria N-arachidonoylethanolamine and N-oleoylethanolamine partly inhibited mitochondrial respiration with glutamate and succinate but not with tetramethyl-p-phenylenediamine (TMPD) plus ascorbate as respiratory substrates. In mitochondria preloaded with small amounts of Ca2+, NAEs produced a much stronger dissipation of the membrane potential and a release of accumulated calcium, both effects being inhibited by CsA, indicative for opening of the mitochondrial permeability transition pore (PTP). Again, the potency of this action was N-arachidonoylethanolamine>N-oleoylethanolamine>N-palmitoylethanolamine. However, in spite of making the matrix space accessible to external [14C]sucrose, N-arachidonoylethanolamine and N-oleoylethanolamine resulted in only a limited swelling of mitochondria and diminished the rate of swelling produced by high Ca2+ load.     

Isolation, characterization, and cDNA sequencing of alpha-1-antiproteinase-like protein from rainbow trout seminal plasma

Seminal plasma of teleost fish contains serine proteinase inhibitors related to those present in blood. These inhibitors can be bound to Q-Sepharose and sequentially eluted with a NaCl gradient. In the present study, using a two-step procedure, we purified (73-fold to homogeneity) and characterized the inhibitor eluted as the second fraction of antitrypsin activity (inhibitor II) from Q-Sepharose. The molecular weight of this inhibitor was estimated to be 56 kDa with an isoelectric point of 5.4. It effectively inhibited trypsin and chymotrypsin but was less effective against elastase. It formed SDS-stable complexes with cod and bovine trypsin. Inhibitor II appeared to be a glycoprotein. Carbohydrate content was determined to be 16%. N-terminal Edman sequencing allowed identification of the first 30 N-terminal amino acids HDGDHAGHTEDHHHHLHHIAGEAHPQHSHG and 25 amino acids within the reactive loop IMPMSLPDTIMLNRPFLLFILEDST. The N-terminal sequence did not match any known sequence, however, the sequence within the reactive loop was significantly similar to carp and mammalian alpha1-antiproteinases. Both sequences were used to construct primers and obtain a cDNA sequence from liver. The mRNA coding the protein is 1675 nt in length including a single open reading frame of 1281 nt that encodes 426 amino acid residues. Analysis of this sequence indicated the presence of putative conserved serpin domains and confirmed the similarity to carp alpha1-antiproteinase and mammalian alpha1-antiproteinase. Our results indicate that inhibitor II belongs to the serpin superfamily and is similar to alpha1-antiproteinase.     

Domain swapping in N-truncated human cystatin C

Human cystatin C (HCC) inhibits papain-like cysteine proteases by a binding epitope composed of two beta-hairpin loops and the N-terminal segment. HCC is found in all body fluids and is present at a particularly high level in the cerebrospinal fluid. Oligomerization of HCC leads to amyloid deposits in brain arteries at advanced age but this pathological process is greatly accelerated with a naturally occurring Leu68Gln variant, resulting in fatal amyloidosis in early adult life. When proteins are extracted from human cystatin C amyloid deposits, an N-terminally truncated cystatin C (THCC) is found, lacking the first ten amino acid residues of the native sequence. It has been shown that the cerebrospinal fluid may cause this N-terminal truncation, possibly because of disintegration of the leucocytes normally present in this fluid, and the release of leucocyte proteolytic enzymes. HCC is the first disease-causing amyloidogenic protein for which oligomerization via 3D domain swapping has been observed. The aggregates arise in the crystallization buffer and have the form of 2-fold symmetric dimers in which a long alpha-helix of one molecule, flanked by two adjacent beta-strands, has replaced an identical domain of the other molecule, and vice versa. Consistent with a conformational change at one of the beta-hairpin loops of the binding epitope, the dimers (and also any other oligomers, including amyloid aggregates) are inactive as papain inhibitors. Here, we report the structure of N-truncated HCC, the dominant form of cystatin C in amyloid deposits. Although the protein crystallized under conditions that are drastically different from those for the full-length protein, the structure reveals dimerization by the same act of domain swapping. However, the new crystal structure is composed of four independent HCC dimers, none of which has the exact 2-fold symmetry of the full-length dimer. While the four dimers have the same overall topology, the exact relation between the individual domains shows a variability that reflects the flexibility at the dimer-specific open interface, which in the case of 3D domain-swapped HCC consists of beta-interactions between the open hinge loops and results in an unusually long intermolecular beta-sheet. The dimers are engaged in further quaternary interactions resulting in spherical, closed octameric assemblies that are identical to that present in the crystal of the full-length protein. The octamers interact via hydrophobic patches formed on the surface of the domain-swapped dimers as well as by extending the dimer beta-sheet through intermolecular contacts.     

A family of auxin-conjugate hydrolases that contributes to free indole-3-acetic acid levels during Arabidopsis germination

Auxins are hormones important for numerous processes throughout plant growth and development. Plants use several mechanisms to regulate levels of the auxin indole-3-acetic acid (IAA), including the formation and hydrolysis of amide-linked conjugates that act as storage or inactivation forms of the hormone. Certain members of an Arabidopsis amidohydrolase family hydrolyze these conjugates to free IAA in vitro. We examined amidohydrolase gene expression using northern and promoter-beta-glucuronidase analyses and found overlapping but distinct patterns of expression. To examine the in vivo importance of auxin-conjugate hydrolysis, we generated a triple hydrolase mutant, ilr1 iar3 ill2, which is deficient in three of these hydrolases. We compared root and hypocotyl growth of the single, double, and triple hydrolase mutants on IAA-Ala, IAA-Leu, and IAA-Phe. The hydrolase mutant phenotypic profiles on different conjugates reveal the in vivo activities and relative importance of ILR1, IAR3, and ILL2 in IAA-conjugate hydrolysis. In addition to defective responses to exogenous conjugates, ilr1 iar3 ill2 roots are slightly less responsive to exogenous IAA. The triple mutant also has a shorter hypocotyl and fewer lateral roots than wild type on unsupplemented medium. As suggested by the mutant phenotypes, ilr1 iar3 ill2 imbibed seeds and seedlings have lower IAA levels than wild type and accumulate IAA-Ala and IAA-Leu, conjugates that are substrates of the absent hydrolases. These results indicate that amidohydrolases contribute free IAA to the auxin pool during germination in Arabidopsis.