DNA microarray analysis of human gingival fibroblasts from healthy and inflammatory gingival tissues

In the inflammatory gingival tissues of patients with periodontitis, cytokines such as interleukin (IL)-1 alpha, IL-1 beta, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha have been detected. Gingival fibroblasts are the major constituents of gingival tissue. We recently demonstrated that lipopolysaccharide (LPS) from periodontopathic bacteria induces inflammatory reactions in various tissues via CD14 and/or Toll-like receptors (TLRs) in gingival tissues [Biochem. Biophys. Res. Commun. 273 (2000) 1161]. To confirm this, we examined the expression of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF-alpha, CD14, TLR2, and TLR4 in human gingival fibroblasts (HGFs) obtained from patients with healthy or inflammatory gingiva using DNA microarray analysis. We also studied the expression levels of these proteins by flow cytometric analysis (FACS). The expression levels of all eight genes in the HGFs of the Inflammatory group were significantly higher than those in the Healthy group on DNA microarray analysis. FACS revealed that the expression levels of all eight proteins on the HGFs of the Inflammatory group were higher than those on the Healthy group. Our data indicated that these eight proteins in HGFs are involved in inflammatory conditions in the gingiva, including periodontal disease. Our results suggested that these eight proteins, in turn, act directly or indirectly on the immune response by activating host cells involved in inflammatory processes.     

8-nitroguanine formation in the liver of hamsters infected with Opisthorchis viverrini

Nucleic acid damage by reactive nitrogen and oxygen species may contribute to the carcinogenesis associated with chronic infection and inflammation. We examined 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation and nitric oxide (NO) production in hamsters infected with Opisthorchis viverrini (OV). Formation of 8-nitroguanine was assessed immunohistochemically with an antibody specific for 8-nitroguanine. 8-nitroguanine formation was found mainly in the cytoplasm and slightly in the nucleus of inflammatory cells and epithelial lining of bile duct at inflammatory areas in the liver. 8-nitroguanine immunoreactivity reached the highest intensity on day 30. A time profile of 8-nitroguanine formation was closely associated with that of plasma nitrate/nitrite. HPLC with an electrochemical detector revealed that the amount of 8-oxodG in the liver reached the maximal level on day 21. The mechanisms of 8-oxodG and 8-nitroguanine formation via O2*- and NO production triggered by OV infection were discussed in relation to cholangiocarcinoma development.     

Molecular basis of spectral tuning in the newt short wavelength sensitive visual pigment

Previously we reported the sequence of the member of the short wavelength sensitive 2 (SWS2) family of vertebrate visual pigments from the retina of the Japanese common newt, Cynops pyrrhogaster[Takahashi, Y. et al. (2001) FEBS Lett. 501, 151-155]. Now we have expressed the apopigment and regenerated it with A1 retinal. Its absorption maximum, 474 nm, is greatly red shifted compared to other known SWS2 pigments (418-455 nm). To determine the amino acid residues that control its spectral tuning, we replaced the residues that were near the chromophore and which differed between the newt and the bullfrog (lambda(max) = 430 nm) wild-type SWS2 pigments: Pro91Ser, Ser94Ala, Ile122Met, Cys127Ser, Ser211Cys, Tyr261Phe, and Ala292Ser. Each of these site-directed mutants led to blue shifts of the newt pigment with five of them causing substantial shifts; their sum was about equal to the difference between the absorption maximum of the bullfrog and newt pigments, 44 nm. The 32 nm shift of the absorption maximum of the multiple seven-residue mutant to 442 nm is fairly close to that of the wild-type bullfrog pigment. Thus, the seven amino acid residues that we replaced are the major cause of the red shift of the newt SWS2 pigment's spectrum. Two of the residues, 91 and 94, have not previously been identified as wavelength regulating sites in visual pigments. One of these, 91, probably regulates color via a new mechanism: altering of a hydrogen bonding network that is connected via a water to the chromophore, in this case its counterion, Glu113.     

Minimally modified LDL is an oxidized LDL enriched with oxidized phosphatidylcholines

The oxidative modification of low-density lipoprotein (LDL) is involved in atherogenesis. Among a variety of modified LDLs mentioned in the literature, so-called minimally modified LDL (MM-LDL) was reported to have pro-atherogenic properties despite minimal changes in its oxidative measures. After treatment of LDL with 1 micro M FeSO(4) at 4 degrees C for 96 h, the resulting MM-LDL showed a slight increase in thiobarbituric acid-reactive substances (TBARS) and little association with macrophages. On the other hand, heavily oxidized LDL, which was prepared by copper-induced oxidation of LDL at 37 degrees C, showed a sharp increase in TBARS and strong association with macrophages. By introducing a fluorometric procedure to detect aldehyde-containing phosphatidylcholines (aldehyde-PCs), we examined the amounts of aldehyde-PCs in modified LDL preparations. Aldehyde-PCs increased to 23.4 pmol/ microg protein in MM-LDL, which was more than four-fold higher than in the heavily oxidized LDL. We conclude that MM-LDL is a unique type of oxidized LDL enriched with aldehyde-PCs.     

Site-directed mutagenesis of benzalacetone synthase. The role of the Phe215 in plant type III polyketide synthases

Benzalacetone synthase (BAS) and chalcone synthase (CHS) are plant-specific type III polyketide synthases (PKSs) that share approximately 70% amino acid sequence identity. BAS catalyzes a one-step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce a diketide benzalacetone, whereas CHS performs sequential condensations with three malonyl-CoA to generate a tetraketide chalcone. A homology model suggested that BAS has the same overall fold as CHS with cavity volume almost as large as that of CHS. One of the most characteristic features is that Rheum palmatum BAS lacks active site Phe-215; the residues 214LF conserved in type III PKSs are uniquely replaced by IL. Our observation that the BAS I214L/L215F mutant exhibited chalcone-forming activity in a pH-dependent manner supported a hypothesis that the absence of Phe-215 in BAS accounts for the interruption of the polyketide chain elongation at the diketide stage. On the other hand, Phe-215 mutants of Scutellaria baicalensis CHS (L214I/F215L, F215W, F215Y, F215S, F215A, F215H, and F215C) afforded increased levels of truncated products; however, none of them generated benzalacetone. These results confirmed the critical role of Phe-215 in the polyketide formation reactions and provided structural basis for understanding the structure-function relationship of the plant type III PKSs.     

Analysis of the molecular species of hydrogenase in the cells of an obligately chemolithoautotrophic, marine hydrogen-oxidizing bacterium, Hydrogenovibrio marinus

Hydrogenovibrio marinus was suggested to have only membrane-bound hydrogenase (MBH). The change of cultivation pO2 did not affect the molecular species of hydrogenase expressed. We propose the MBH is grouped in class I [NiFe] MBH according to the subunit composition, size (Mw 38,000 and Mw 74,000 subunits) and N-terminal sequences of the subunits, and arrangement of the structural genes. Ni-requirement for the autotrophic growth on H2 also suggested the MBH is the Ni-containing type. Southern hybridization analysis using a part of the MBH gene showed a possibility of the presence of two highly homologous MBHs which were not separated by SDS-PAGE.     

High-cut characteristics of the baroreflex neural arc preserve baroreflex gain against pulsatile pressure

A transfer function from baroreceptor pressure input to sympathetic nerve activity (SNA) shows derivative characteristics in the frequency range below 0.8 Hz in rabbits. These derivative characteristics contribute to a quick and stable arterial pressure (AP) regulation. However, if the derivative characteristics hold up to heart rate frequency, the pulsatile pressure input will yield a markedly augmented SNA signal. Such a signal would saturate the baroreflex signal transduction, thereby disabling the baroreflex regulation of AP. We hypothesized that the transfer gain at heart rate frequency would be much smaller than that predicted from extrapolating the derivative characteristics. In anesthetized rabbits (n = 6), we estimated the neural arc transfer function in the frequency range up to 10 Hz. The transfer gain was lost at a rate of -20 dB/decade when the input frequency exceeded 0.8 Hz. A numerical simulation indicated that the high-cut characteristics above 0.8 Hz were effective to attenuate the pulsatile signal and preserve the open-loop gain when the baroreflex dynamic range was finite.     

Distribution of interstitial telomere-like repeats and their adjacent sequences in a dioecious plant, <Emphasis Type="Italic">Silene latifolia</Emphasis>

The dioecious plant Silene latifolia has large, heteromorphic X and Y sex chromosomes that are thought to be derived from rearrangements of autosomes. To reveal the origin of the sex chromosomes in S. latifolia, we isolated and characterized telomere-homologous sequences from intra-chromosomal regions (interstitial telomere-like repeats; ITRs) and ITR-adjacent sequences (IASs). Nine genomic DNA fragments with degenerate 84- to 175-bp ITRs were isolated from a genomic library and total genome of male plants. Comparing the nucleotide sequences, the IASs of the nine ITRs were classified into seven elements (IAS-a, IAS-b, IAS-c, IAS-d, IAS-e, IAS-f, and IAS-g) by sequence similarity. The ITRs were grouped into two classes (class-I and -II ITRs) according to the classification of IASs. The class-I ITRs were sub-grouped into three subclasses (subclasses-IA, -IB, and -IC ITRs) based on the arrangement of IAS elements. By contrast, the class-II ITR was located between two different IASs (IAS-f and IAS-g). Genomic Southern analyses showed that both the male and female genomes contained six (IAS-f) to 153 (IAS-d) copies of each IAS per haploid genome. Fluorescence in situ hybridization analyses showed that one IAS element, IAS-d, was distributed in the interstitial and proximal regions of the sex chromosomes of S. latifolia. The distribution of IAS-d is important evidence for past telomere-mediated chromosome rearrangements during the evolution of the sex chromosomes of S. latifolia.     

Imprint cytology of epithelioid angiomyolipoma in a patient with tuberous sclerosis. A case report

BACKGROUND: Angiomyolipoma composed predominantly of epithelioid cells has been referred to as epithelioid angiomyolipoma. As this subtype shows considerable cellular atypia, it may be erroneously diagnosed as malignant epithelioid tumor, such as renal cell carcinoma and hepatocellular carcinoma. So far, only one report describing the cytologic findings of epithelioid angiomyolipoma has been documented, and epithelioid angiomyolipoma occurring in the peritoneal cavity has not been reported. CASE: Eleven years after resection of a renal epithelioid angiomyolipoma in a 34-year-old male with tuberous sclerosis, a tumor appeared in the peritoneal cavity and three masses in the liver. The intraoperative smears imprinted from part of the peritoneal mass revealed many large, atypical cells. The well-preserved atypical cells showed abundant, round to polyhedral, granular cytoplasm. Bizarre, giant nuclei with hyperchromasia and huge nucleoli were occasionally seen. Intranuclear cytoplasmic inclusions and mitotic figures were occasionally observed. As the epithelioid cells were markedly pleomorphic, we could not rule out hepatocellular carcinoma, cytologically and histologically, in the intraoperative consultation. In permanent sections the tumor was composed predominantly of epithelioid cells showing an alveolar pattern or sheetlike arrangement. Mitotic counts were zero to one per 10 high-power fields. Immunohistochemically, the epithelioid tumor cells were positive for vimentin, alpha-smooth muscle actin and HMB-45, consistent with epithelioid angiomyolipoma. MIB-1-labeling index was 1.6%. CONCLUSION: When one sees atypical epithelioid tumor cells in a tuberous sclerosis patient during an intraoperative consultation, one must consider epithelioid angiomyolipoma.