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We identified five cDNA clones of the Six gene family which are expressed in retina. They are Six2, Six3ct and Six3[~ (which are derived from alternative splicing forms), Six5, and AREC3/Six4. All of these Six family genes possess extensive sequence similarity among each other in the so-homologous region (Six domain and homeodomain) but differ greatly in structure in some other regions. The amino acid sequence similarity of the so-homologous region to the previously identified AREC31Six4 is 70.1% for Six2, 57.3% for Six3ct and Six3[~, and 70.3% for Six5. The expression of these genes was observed in inner and outer nuclear layer, ganglion cell layer, and pigment epithelium of mouse retina by in situ hybridization. The so-homologous region of each Six family protein has specific DNA binding activity. Six5 and Six2 bind to the same sequence as does AREC31Six4, while Six3 does not. These observations suggest that some of the Six family genes can regulate the same target genes.cloned. The roles of these Six family genes in retina formation are discussed. Materials and methods Screening of the cDNA library and DNA sequencingThe mouse retina eDNA library from 1-month-old BALB/c mice was kindly supplied by Dr. Ananda Swaroop [7]. A SalI-DraI (-17 2486) fragment of AREC3 eDNA (pSVSPORTMI8) [3] was labeled with [32P]dCTP with a Megaprime labeling kit (Amersham). About 1.2x 10 6 plaques were screened and we obtained 13 positive clones. They were grouped into 4 based on restriction mapping and partial nucleotide sequencing. The overlapping clones were sequenced and we obtained four species of Six family cDNAs. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank nucleotide sequence databases with the following accession numbers: D83147 (Six2), D83144 (Six3cq, D83145 (Six313) and D83146 (Six5).

Pattern recognition receptors, which include the toll-like receptors (TLRs), are considered to play an important role in the response against lipopolysaccharide (LPS). In this study, we performed a reverse transcriptase/polymerase chain reaction (RT-PCR) study, Western analysis, immunohistochemical staining, and RT-PCR-amplified in situ hybridization of TLR2 and TLR4 in the case of LPS-induced lung injury. The expression of TLR2 and TLR4 increased in the lung rapidly after LPS inhalation and peaked at 24 h, followed by a gradual decrease. TLR2 and TLR4 expression was observed on the bronchial epithelium and tissue macrophages. In the early hours after inhalation of fluorescein-isothiocyanate (FITC)-labeled LPS, LPS was detected mainly on the bronchial epithelium and on a few of tissue macrophages. One day after inhalation, the LPS signals disappeared in the lungs of the mice, except for a few alveolar macrophages. The expression of TLR2, TLR4, and CD14 was coincident with the signals of FITC-labeled LPS. Instillation of liposome-encapsulated dichloromethylene diphosphonate induced a significant decrease in alveolar macrophages. In the macrophage-depleted mice, however, expression of TLR2 and TLR4 mRNA or protein was slightly suppressed in the lung after LPS inhalation. These data suggest that the bronchial epithelium and macrophages play crucial roles in LPS-induced lung injury through TLR2 and TLR4.

AAV vectors can efficiently transduce rat VSMC in vitro. AAV-mediated ex vivo gene transfer into the normal aorta resulted in efficient gene transfer into endothelial and adventitial cells but not into medial VSMC. These findings suggest that AAV-based vectors are promising for use in cardiovascular gene therapy.

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We describe an improved copper ferrocyanide-based method for cytochemical detection of glucose-6-phosphate dehydrogenase (G6PD), which was used to localize the enzyme within the ultrastructure of rat hepatocytes and adrenocortical cells. With this method, glutaraldehyde fixation and the addition of exogenous electron carriers (for example, phenazine methosulfate) to the cytochemical reaction medium were essential. Copper ferrocyanide reaction product showing the distribution of G6PD was readily recognized at the light microscopic level as Hatchett's brown staining and at the electron microscopic level as electron-dense deposits. Within stained regions, enzyme cytochemical G6PD activity was found to be associated with ribosome-like structures. Because G6PD is a soluble, cytosolic enzyme, its displacement or extraction may occur during conventional fixation. We, therefore, combined a rapid-freezing technique with G6PD enzyme cytochemistry. The resultant rapid-freezing enzyme cytochemistry enabled us to show the subcellular distribution of G6PD in a more life-like state; the localization of G6PD in rapidly frozen cells was in substantial agreement with that in conventionally fixed cells.

Photodynamic diagnosis/therapy (PDD/PDT) are novel modalities for the diagnosis and treatment of cancer. The photosensitizer protoporphyrin IX is metabolized from 5-aminolevulinic acid (5-ALA) intracellularly, and PDD/PDT using 5-ALA have been approved in dermatologic malignancies and gliomas. However, the molecular mechanism that defines the efficacy of PDD/PDT is unknown. In this study, we analyzed the functions of ATP-binding cassette (ABC) transporters in PDD using 5-ALA. Most of the human gastrointestinal cancer line cells examined showed a homogenous staining pattern with 5-ALA, except for the pancreatic cancer line PANC-1, which showed heterogeneous staining. To analyze this heterogeneous staining pattern, single cell clones were established from PANC-1 cells and the expression of ABC transporters was assessed. Among the ABC transporter genes examined, ABCG2 showed an inverse correlation with the rate of 5-ALA-positive staining. PANC-1 clone #2 cells showed the highest level of ABCG2 expression and the lowest level of 5-ALA staining, with only a 0.6% positive rate. Knockdown of the ABCG2 gene by small interfering RNAs increased the positive rate of 5-ALA staining in PANC-1 wild-type and clone cells. Interestingly, PANC-1 clone #2 cells showed the high sphere-forming ability and tumor-formation ability, indicating that the cells contained high numbers of cancer stem cells (CSCs). Knockdown or inhibition of ABCG2 increased the rate of 5-ALA staining, but did not decrease sphere-forming ability. These results indicate that gastrointestinal cancer cell lines expressing high levels of ABCG2 are enriched with CSCs and show low rates of 5-ALA staining, but 5-ALA staining rates can be improved by inhibition of ABCG2.

In the present investigation the ultrastructural site of ferricyanide reduction by reductases in niitochondria in tissues (heart and kidney) of the normal adult rat was studied by the copper ferrocyanide method developed recently in our laboratory. Substrates used were sodium succinate, cytochrome c, dihydronicotinamide-adenine dinucleotide (NADH2), sodium DL-j3-hydroxybutyrate, phenazine methosulfate and ubiquinone. All reductases tested were positive in mitochondria and the activities of the succinate-ferri-. cyanide reductase, the cytochrome c-ferricyanide reductase, the NADHrferricye.nlde reductase and $.hydroxybutyrate-.ferricyanide reductase were most evident. An ambiguous trace of activity was observed when phenazine methosulfate and ubiquinone were used as substrate. It was also observed that not all of the mitochondria revealed the reductase activity and the existence of functional heterogeneity among mitochondria was postulated. In enzymatically positive mitochondria, the reaction product, copper ferrocyanide, was found both in the mitochondrial membranes and in spaces