A synthetic peptide homologous to functional domain of human IL-10 down-regulates expression of MHC class I and Transporter associated with Antigen Processing 1/2 in human melanoma cells

Tumor cells treated with IL-10 were shown to have decreased, but peptide-inducible expression of MHC class I, decreased sensitivity to MHC class I-restricted CTL, and increased NK sensitivity. These findings could be explained, at least partially, by a down-regulation of TAP1/TAP2 expression. In this study, IT9302, a nanomeric peptide (AYMTMKIRN), homologous to the C-terminal of the human IL-10 sequence, was demonstrated to mimic these previously described IL-10 effects on MHC class I-related molecules and functions. We observed a dose-dependent down-regulation of MHC class I at the cell surface of melanoma cells after 24-h treatment with IT9302. The IL-10 homologue peptide also caused a dose-dependent inhibition of the IFN-gamma-mediated surface induction of MHC class I in a melanoma cell line. We demonstrated, using Western blot and flow cytometry, that IT9302 inhibits the expression of TAP1 and TAP2 proteins, but not MHC class I H chain or low molecular protein molecules. Finally, peptide-treated melanoma cells were shown to be more sensitive to lysis by NK cells in a dose-dependent way. Taken together, these results demonstrate that a small synthetic peptide derived from IL-10 can mimic the Ag presentation-related effects mediated by this cytokine in human melanomas and increase tumor sensitivity to NK cells, which can be relevant in the designing of future strategies for cancer immune therapy.     

Fine needle aspiration cytology of dedifferentiated acinic cell carcinoma of the parotid gland: a case report

BACKGROUND: Dedifferentiation of acinic cell carcinoma (ACC) to undifferentiated carcinoma occurs rarely and entails a poor prognosis. Most cases of dedifferentiation occur as recurrences of a previously excised ACC. More rarely the neoplasm presents with areas of well-differentiated ACC coexisting with dedifferentiated ones. CASE: An 85-year-old man presented with 2 nodular areas in the right parotid area. Fine needle aspiration of both nodules was performed. In both cases cytology revealed a double epithelial component in similar proportions. The first one corresponded to cohesive groups of small to intermediate-sized, polygonal cells with round, monomorphic nuclei. They were distributed in small and larger, branching groups with acinic morphology. Intermixed with this population, irregular groups of larger, pleomorphic cells with irregular nuclei, prominent nucleoli and scarce cytoplasm were present. In addition, smears showed an abundant lymphoid background. A cytologic diagnosis of "salivary carcinoma with coexisting areas of acinic cell differentiation and high grade, undifferentiated carcinoma" was given. Histopathology revealed a well-differentiated ACC with areas of high grade undifferentiated carcinoma (dedifferentiated ACC). CONCLUSION: The current case expands the cytomorphologic spectrum of ACC. Cytology may permit the preoperative recognition of dedifferentiation, allowing a more sound therapeutic approach.     

Cytologic features of pheochromocytoma and retroperitoneal paraganglioma: a morphologic and immunohistochemical study of 13 cases

OBJECTIVE: To review the cytologic features and potential pitfalls of pheochromocytoma and retroperitoneal paraganglioma and to evaluate complications of the aspiration procedure and the diagnostic utility of immunocytochemistry. STUDY DESIGN: We reviewed 15 cytologic specimens from 12 patients with 13 tumors (1 bilateral case). Ten were adrenal (pheochromocytomas) and 3 extraadrenal paragangliomas. Eleven specimens were from fine needle aspiration (FNA) procedures that were performed in collaboration with radiologists using 23-25-gauge needles. In 3 patients the cytologic material was obtained during intraoperative diagnosis. Immunocytochemistry was performed on alcohol-fixed smears. RESULTS: Two aspirates were hypocellular, while the remainder were cellular. Cells were distributed singly or formed discohesive groups. When present, cytoplasm was abundant and ill defined. Most cells had an eccentric nucleus and plasmacytoid morphology. Nuclear pleomorphism, binucleation and multinucletaion, naked nuclei and intranuclear preudoinclusions were common findings. In 2 cases a lipid background was seen focally. Evident cytoplasmic immunoexpression of synaptophysin or chromogranin was detected in the 10 cases analyzed. One patient developed a hypertensive episode during the FNA procedure. It was controlled medically without complications. CONCLUSION: When adequate cytologic material is present, the recognition of pheochromocytoma and extraadrenal paraganglioma is possible. Together with morphology, immunocytochemical studies allow a specific preoperative diagnosis. Scarce material can be a source of diagnostic errors. FNA of pheochromocytomas is not necessarily contraindicated. When analytic data are not diagnostic, FNA may follow. Aspiration must be performed in an area equipped with the therapeutic tools necessary to control a pheochromocytoma crisis.     

Classifying glycerol dehydratase by its functional residues and purifying selection in its evolution

Glycerol dehydratase (GD) catalyses glycerol reductive conversion to 3-hydroxypropanaldehyde (3-HPA), this being the first step required for the microbial conversion of glycerol to 1, 3 -propanodiol. GD has been functionally characterised to date and two main groups have been determined, one of them being vitamin B(12)-dependent and the other B(12)-independent. GD evolutionary history has been described and an exhaustive analysis made for detecting the functional residues responsible for type I divergence. GD phylogenetic tree topology was seen to be statistically robust and the data indicated strong purifying selection operating on the GD proteins within it. Two clades were indentified, one for vitamin B(12)-dependent and the other for B(12)- independent classes. The ancient hot-pot residues responsible for protein divergency for each clade were also identified. The basic evolutionary biology for GD proteins has been described, thereby opening the way forward for developing rational mutagenesis studies.     

Relaxase DNA Binding and Cleavage Are Two Distinguishable Steps in Conjugative DNA Processing That Involve Different Sequence Elements of the nic Site

TrwC, the relaxase of plasmid R388, catalyzes a series of concerted DNA cleavage and strand transfer reactions on a specific site (nic) of its origin of transfer (oriT). nic contains the cleavage site and an adjacent inverted repeat (IR₂). Mutation analysis in the nic region indicated that recognition of the IR₂ proximal arm and the nucleotides located between IR₂ and the cleavage site were essential for supercoiled DNA processing, as judged either by in vitro nic cleavage or by mobilization of a plasmid containing oriT. Formation of the IR₂ cruciform and recognition of the distal IR₂ arm and loop were not necessary for these reactions to take place. On the other hand, IR₂ was not involved in TrwC single-stranded DNA processing in vitro. For single-stranded DNA nic cleavage, TrwC recognized a sequence embracing six nucleotides upstream of the cleavage site and two nucleotides downstream. This suggests that TrwC DNA binding and cleavage are two distinguishable steps in conjugative DNA processing and that different sequence elements are recognized by TrwC in each step. IR₂-proximal arm recognition was crucial for the initial supercoiled DNA binding. Subsequent recognition of the adjacent single-stranded DNA binding site was required to position the cleavage site in the active center of the protein so that the nic cleavage reaction could take place.     

Selective spatiotemporal induction of matrix metalloproteinase-2 and matrix metalloproteinase-9 transcription after myocardial infarction

Myocardial remodeling after myocardial infarction (MI) is associated with increased levels of the matrix metalloproteinases (MMPs). Levels of two MMP species, MMP-2 and MMP-9, are increased after MI, and transgenic deletion of these MMPs attenuates post-MI left ventricular (LV) remodeling. This study characterized the spatiotemporal patterns of gene promoter induction for MMP-2 and MMP-9 after MI. MI was induced in transgenic mice in which the MMP-2 or MMP-9 promoter sequence was fused to the beta-galactosidase reporter, and reporter level was assayed up to 28 days after MI. Myocardial localization with respect to cellular sources of MMP-2 and MMP-9 promoter induction was examined. After MI, LV diameter increased by 70% (P < 0.05), consistent with LV remodeling. beta-Galactosidase staining in MMP-2 reporter mice was increased by 1 day after MI and increased further to 64 +/- 6% of LV epicardial area by 7 days after MI (P < 0.05). MMP-2 promoter activation occurred in fibroblasts and myofibroblasts in the MI region. In MMP-9 reporter mice, promoter induction was detected after 3 days and peaked at 7 days after MI (53 +/- 6%, P < 0.05) and was colocalized with inflammatory cells at the peri-infarct region. Although MMP-2 promoter activation was similarly distributed in the MI and border regions, activation of the MMP-9 promoter was highest at the border between the MI and remote regions. These unique findings visually demonstrated that activation of the MMP-2 and MMP-9 gene promoters occurs in a distinct spatial relation with reference to the MI region and changes in a characteristic time-dependent manner after MI.     

Developmental regulation of the activation of signaling components leading to translation initiation in skeletal muscle of neonatal pigs

The rapid growth of neonates is driven by high rates of skeletal muscle protein synthesis. This high rate of protein synthesis, which is induced by feeding, declines with development. Overnight-fasted 7- and 26-day-old pigs either remained fasted or were refed, and the abundance and phosphorylation of growth factor- and nutrient-induced signaling components that regulate mRNA translation initiation were measured in skeletal muscle and liver. In muscle, but not liver, the activation of inhibitors of protein synthesis, phosphatase and tensin homolog deleted on chromosome 10, protein phosphatase 2A, and tuberous sclerosis complex 1/2 increased with age. Serine/threonine phosphorylation of the insulin receptor and insulin receptor substrate-1, which downregulates insulin signaling, and the activation of AMP-activated protein kinase, an inhibitor of protein synthesis, were unaffected by age and feeding in muscle and liver. Activation of positive regulators of protein synthesis, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), and eIF4E-binding protein-1 (4E-BP1) decreased with age in muscle but not liver. Feeding enhanced mTOR, S6K1, and 4E-BP1 activation in muscle, and this response decreased with age. In liver, activation of S6K1 and 4E-BP1, but not mTOR, was increased by feeding but was unaffected by age. Raptor abundance and the association between raptor and mTOR were greater in 7- than in 26-day-old pigs. The results suggest that the developmental decline in skeletal muscle protein synthesis is due in part to developmental regulation of the activation of growth factor and nutrient-signaling components.