Basement membrane-like substance in cytologic diagnosis in clear cell adenocarcinoma of the minor salivary gland of the palate. A case report

BACKGROUND: Clear cell adenocarcinoma (CCA) of the minor salivary gland accounts for < 1% of all tumors of the salivary gland. CASE: A 32-year-old woman with a history of papillary carcinoma of the thyroid 1 year earlier complained of pain on the left side of the neck. After a detailed examination, the patient underwent the resection of a tumor located at the palate. Imprint cytology of the tumor revealed cohesive tumor cells of uniform size containing an abundant clear cytoplasm and round nuclei with extra but fine granular chromatin and conspicuous nucleoli. A basement membrane-like substance (BMS) was stained in light green with Papanicolaou staining and was positive for laminin with immunohistochemical staining. Histopathologic analysis confirmed the trabecular or nest-like arrangement of the cells with the clear cytoplasm and BMS substance surrounded by tumor cells, which were positive for laminin and AE1 immunohistochemically. CONCLUSION: Although CCA of the palate is extremely rare, an accurate cytologic diagnosis can be made if the characteristic findings of CCA, including BMS, are imaged.     

Dynamics of Rhodobacter capsulatus [2FE-2S] ferredoxin VI and Aquifex aeolicus ferredoxin 5 via nuclear resonance vibrational spectroscopy (NRVS) and resonance Raman spectroscopy

We have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study the Fe(2)S(2)(Cys)(4) sites in oxidized and reduced [2Fe-2S] ferredoxins from Rhodobacter capsulatus (Rc FdVI) and Aquifex aeolicus (Aa Fd5). In the oxidized forms, nearly identical NRVS patterns are observed, with strong bands from Fe-S stretching modes peaking around 335 cm(-1), and additional features observed as high as the B(2u) mode at approximately 421 cm(-1). Both forms of Rc FdVI have also been investigated by resonance Raman (RR) spectroscopy. There is good correspondence between NRVS and Raman frequencies, but because of different selection rules, intensities vary dramatically between the two kinds of spectra. For example, the B(3u) mode at approximately 288 cm(-1), attributed to an asymmetric combination of the two FeS(4) breathing modes, is often the strongest resonance Raman feature. In contrast, it is nearly invisible in the NRVS, as there is almost no Fe motion in such FeS(4) breathing. NRVS and RR analysis of isotope shifts with (36)S-substituted into bridging S(2-) ions in Rc FdVI allowed quantitation of S(2-) motion in different normal modes. We observed the symmetric Fe-Fe stretching mode at approximately 190 cm(-1) in both NRVS and RR spectra. At still lower energies, the NRVS presents a complex envelope of bending, torsion, and protein modes, with a maximum at 78 cm(-1). The (57)Fe partial vibrational densities of states (PVDOS) were interpreted by normal-mode analysis with optimization of Urey-Bradley force fields. Progressively more complex D(2h) Fe(2)S(2)S'(4), C(2h) Fe(2)S(2)(SCC)(4), and C(1) Fe(2)S(2)(Cys)(4) models were optimized by comparison with the experimental spectra. After modification of the CHARMM22 all-atom force field by the addition of refined Fe-S force constants, a simulation employing the complete protein structure was used to reproduce the PVDOS, with better results in the low frequency protein mode region. This process was then repeated for analysis of data on the reduced FdVI. Finally, the degree of collectivity was used to quantitate the delocalization of the dynamic properties of the redox-active Fe site. The NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins.     

Functional requirement of CCN2 for intramembranous bone formation in embryonic mice

CCN2 is best known as a promoter of chondrocyte differentiation among the CCN family members, and Ccn2 null mutant mice display skeletal dysmorphisms. However, little is known concerning the roles of CCN2 during bone formation. We herein present a comparative analysis of wild-type and Ccn2 null mice to investigate the roles of CCN2 in bone development. Multiple histochemical methods were employed to analyze the effects of CCN2 deletion in vivo, and effects of CCN2 on the osteogenic response were evaluated with the isolated and cultured osteoblasts. As a result, we found a drastic reduction of the osteoblastic phenotype in Ccn2 null mutants. Importantly, addition of exogenous CCN2 promoted every step of osteoblast differentiation and rescued the attenuated activities of the Ccn2 null osteoblasts. These results suggest that CCN2 is required not only for the regulation of cartilage and subsequent events, but also for the normal intramembranous bone development.     

Structural basis for multimeric heme complexation through a specific protein-heme interaction: the case of the third neat domain of IsdH from Staphylococcus aureus

To elucidate the heme acquisition system in pathogenic bacteria, we investigated the heme-binding properties of the third NEAT domain of IsdH (IsdH-NEAT3), a receptor for heme located on the surfaces of pathogenic bacterial cells, by using x-ray crystallography, isothermal titration calorimetry, examination of absorbance spectra, mutation analysis, size-exclusion chromatography, and analytical ultracentrifugation. We found the following: 1) IsdH-NEAT3 can bind with multiple heme molecules by two modes; 2) heme was bound at the surface of IsdH-NEAT3; 3) candidate residues proposed from the crystal structure were not essential for binding with heme; and 4) IsdH-NEAT3 was associated into a multimeric heme complex by the addition of excess heme. From these observations, we propose a heme-binding mechanism for IsdH-NEAT3 that involves multimerization and discuss the biological importance of this mechanism.     

A helical string of alternately connected three-helix bundles for the cell wall-associated adhesion protein Ebh from Staphylococcus aureus

The 1.1 MDa cell-wall-associated adhesion protein of staphylococci, Ebh, consists of several distinct regions, including a large central region with 52 imperfect repeats of 126 amino acid residues. We investigated the structure of this giant molecule by X-ray crystallography, circular dichroism (CD) spectrometry, and small-angle X-ray scattering (SAXS). The crystal structure of two repeats showed that each repeat consists of two distinct three-helix bundles, and two such repeats are connected along the long axis, resulting in a rod-like structure that is 120 A in length. CD and SAXS analyses of the samples with longer repeats suggested that each repeat has an identical structure, and that such repeats are connected tandemly to form a rod-like structure in solution, the length of which increased proportionately with the number of repeating units. On the basis of these results, it was proposed that Ebh is a 320 nm rod-like molecule with some plasticity at module junctions.     

Aluminum concentrations in human deciduous enamel and dentin related to dental caries

The aluminum (Al) concentrations in the enamel and dentin of 314 human deciduous teeth were determined in order to examine the relationship between Al and dental caries. The sample teeth were divided into three groups: the sound tooth group, carious tooth group and filled tooth group. The teeth of the carious tooth group were further classified into three groups depending on the stage of caries. The Al content was determined using graphite furnace atomic absorption spectrometry. In both the enamel and dentin, the Al concentrations were unaffected by sex, but did depend on tooth type. In enamel, the Al concentration was significantly higher in the sound tooth group (42.8 +/- 37.3 microg/g) than in the three carious groups (20.7 +/- 17.1-24.9 +/- 22.0 microg/g) and the filled tooth group (27.3 +/- 25.5 microg/g). As for dentin, the Al concentration was also significantly higher in the sound tooth group (36.2 +/- 35.1 microg/g) than in the three carious groups (15.1 +/- 13.3-24.5 +/- 23.4 microg/g) and the filled tooth group (17.2 +/- 20.6 microg/g). Even when analyzing incisors alone, the Al concentrations were significantly higher in the sound tooth group than in the other groups, for both enamel and dentin. Furthermore, the Al levels in carious enamel and dentin did not decrease with the advance of caries. These findings indicated that the deciduous teeth containing higher Al concentrations on average had less caries than the teeth with lower Al concentrations, and suggest that Al acts as a possible cariostatic agent by itself.     

Determination of branched beta-cyclodextrin-prostaglandin complexes using electrospray ionization mass spectrometry

Mass spectral measurements by electrospray ionization mass spectrometry (ESI-MS) detected the ions of beta-cyclodextrin (betaCD) or branched betaCDs (glucosyl-, galactosyl-, mannosyl- and maltosyl-betaCD)-prostaglandins (PGs: PGA(2), PGD(2), PGE(1), PGE(2), PGF(2alpha) and PGJ(2)) complexes, i.e., betaCD-PG complexes, with a host:guest ratio of 1:1 in the negative ion mode. This is the first study to report the ions of branched betaCD-PG complexes using ESI-MS. The inclusion complexes were determined by a flow injection analysis using acetonitrile/water. We could confirm by this method the presence of a betaCD-PGE(2) complex with a host:guest ratio of 1:1 in a solution-dissolved pharmaceutical formulation consisting of betaCD-PGE(2) (Prostarmon E tablet).     

A Simple Detection Method for Low-Affinity Membrane Protein Interactions by Baculoviral Display

Background

Membrane protein interactions play an important role in cell-to-cell recognition in various biological activities such as in the immune or neural system. Nevertheless, there has remained the major obstacle of expression of the membrane proteins in their active form. Recently, we and other investigators found that functional membrane proteins express on baculovirus particles (budded virus, BV). In this study, we applied this BV display system to detect interaction between membrane proteins important for cell-to-cell interaction in immune system.

Methodology/Principal Findings

We infected Sf9 cells with recombinant baculovirus encoding the T cell membrane protein CD2 or its ligand CD58 and recovered the BV. We detected specific interaction between CD2-displaying BV and CD58-displaying BV by an enzyme-linked immunosorbent assay (ELISA). Using this system, we also detected specific interaction between two other membrane receptor-ligand pairs, CD40-CD40 ligand (CD40L), and glucocorticoid-induced TNFR family-related protein (GITR)-GITR ligand (GITRL). Furthermore, we observed specific binding of BV displaying CD58, CD40L, or GITRL to cells naturally expressing their respective receptors by flowcytometric analysis using anti-baculoviral gp64 antibody. Finally we isolated CD2 cDNA from a cDNA expression library by magnetic separation using CD58-displayng BV and anti-gp64 antibody.

Conclusions

We found the BV display system worked effectively in the detection of the interaction of membrane proteins. Since various membrane proteins and their oligomeric complexes can be displayed on BV in the native form, this BV display system should prove highly useful in the search for natural ligands or to develop screening systems for therapeutic antibodies and/or compounds.     

Identification of prostaglandin F-producing cells in the liver

Prostaglandin (PG) F synthase forms PGF_2α and 9α, 11β-PGF₂ from PGH₂ and PGD₂, respectively. PGH₂ is synthesized from arachidonic acid by cyclooxygenase (COX) and then metabolized to various PGs and thromboxane by specific enzymes. PGD₂ is synthesized from PGH₂ by PGD synthase. To identify PGF₂-producing cells in the rat liver, the occurrence and localization of PGF synthase and COX were studied with immunochemical and immunohistochemical techniques using anti-liver-type PGF synthase and anti-COX antibodies. In Western blot analyses, positive bands of liver-type PGF synthase and constitutive COX-1 were observed at positions approximately 37 kDa and 70–72 kDa, respectively. However, inducible COX-2 was not detected. In the immunohistochemical study, PGF synthase was present in the cytoplasm of the sinusoidal endothelial cells. COX-1 was present on the membranes of the nucleus and endoplasmic reticulum of the endothelial cells and Kupffer cells. Double immunostaining for PGF synthase and COX-1 showed that both enzymes were present in the same endothelial cells. These results suggest that the main site of PGF₂ synthesis in the liver is the sinusoidal endothelial cell. Accepted: 12 October 1999     

Comparison of recombinant protein expression in a baculovirus system in insect cells (Sf9) and silkworm

Using a hybrid baculovirus system, we compared the expression of 45 recombinant proteins from six categories using two models: silkworm (larvae and pupae) and an Sf9 cell line. A total of 45 proteins were successfully expressed; preparation of hybrid baculovirus was unsuccessful for one protein, and two proteins were not expressed. A similar pattern of expression was seen in both silkworm and Sf9 cells, with double and multiple bands found in immunoblotting of the precipitate of both hosts. Degraded proteins were seen only in the silkworm system (particularly in the larvae). Production was more efficient in silkworms; a single silkworm produced about 70 times more protein than 10(6) Sf9 cells in 2 ml of culture medium.