A novel bHLH-PAS factor with close sequence similarity to hypoxia-inducible factor 1␣ regulates the VEGF expression and is potentially involved in lung and vascular development (vasculogenesis͞tubular Medicine, Ann Arbor, MI, February 14, 1997 (received for review December 26, 1996 ABSTRACTWe have isolated and characterized a cDNA for a novel Per-Arnt͞AhR-Sim basic helix-loop-helix (bHLH-PAS) factor that interacts with the Ah receptor nuclear translocator (Arnt), and its predicted amino acid sequence exhibits significant similarity to the hypoxia-inducible factor 1␣ (HIF1␣) and Drosophila trachealess (dTrh) gene product. The HIF1␣-like factor (HLF) encoded by the isolated cDNA bound the hypoxiaresponse element (HRE) found in enhancers of genes for erythropoietin, vascular endothelial growth factor (VEGF), and various glycolytic enzymes, and activated transcription of a reporter gene harboring the HRE. Although transcription-activating properties of HLF were very similar to those reported for HIF1␣, their expression patterns were quite different between the two factors; HLF mRNA was most abundantly expressed in lung, followed by heart, liver, and other various organs under normoxic conditions, whereas HIF1␣ mRNA was ubiquitously expressed at much lower levels. In lung development around parturition, HLF mRNA expression was markedly enhanced, whereas that of HIF1␣ mRNA remained apparently unchanged at a much lower level. Moreover, HLF mRNA expression was closely correlated with that of VEGF mRNA. Whole mount in situ hybridization experiments demonstrated that HLF mRNA was expressed in vascular endothelial cells at the middle stages (9.5 and 10.5 days postcoitus) of mouse embryo development, where HIF1␣ mRNA was almost undetectable. The high expression level of HLF mRNA in the O 2 delivery system of developing embryos and adult organs suggests that in a normoxic state, HLF regulates gene expression of VEGF, various glycolytic enzymes, and others driven by the HRE sequence, and may be involved in development of blood vessels and the tubular system of lung.

Background: Vertebrates have numerous lateral asymmetries in the position of their organs, but the molecular basis for the determination of left-right (L-R) asymmetries remains largely unknown. TGFbrelated genes such as lefty and nodal are L-R asymmetrically expressed in developing mouse embryos, and may be involved in L-R determination.

From a cDNA library of mouse skeletal muscle, we have isolated mouse Sim1 (mSim1) cDNA encoding a polypeptide of 765 amino acids with striking amino acid identity in basic helix-loop-helix (89% identity) and PAS (89% identity) domains to previously identified mSim2, although the carboxy-terminal third of the molecule did not show any similarity to mSim2 or Drosophila Sim (dSim). Whole-mount in situ hybridization showed restricted and characteristic expression patterns of the two mSim mRNAs in various tissues and organs during embryogenesis, such as those for the somite, the nephrogenic cord, and the mesencephalon (for mSim1) and those for the diencephalon, branchial arches, and limbs (for mSim2). From sequence similarity and chromosomal localization, it is concluded that mSim2 is an ortholog of hSIM2, which is proposed to be a candidate gene responsible for Down's syndrome. The sites of mSim2 expression showed an overlap with the affected regions of the syndrome, further strengthening involvement of mSim2 in Down's syndrome.

Fragments of the spacer region of genes for rat 5S ribosomal RNA (rDNA), which are tandemly repeated, were amplified by PCR with primers specific to the two ends of the coding region for 5S rRNA. Two amplified fragments of ∼1.6 kb were subcloned and sequenced. The spacer sequences showed a high degree of sequence identity to each other (99.2%) but substantial divergence from those of analogous mouse clones. The homologous regions in the mouse clones were interrupted by the duplication or deletion of small segments of DNA. A 12-mer, 5-GGCTCTTGGGGC-3’, thought to be responsible for efficient transcription, was located from position -33 to position -22 in the rat 5S clones. The genes were mapped by fluorescence in situ hybridization with cloned fragments of rat 5S rDNA as probe. The genes were localized exclusively in a single telomeric region of chromosome 19.

Background: Mouse lefty1 and lefty2 genes are expressed on the left side of developing embryos and are required for left-right determination. Here we have studied expression and transcriptional regulatory mechanisms of human LEFTY genes.

Tympanic membrane perforation causes a sound conduction disturbance, and the size of this conduction disturbance is proportional to the perforation area. However, precise evaluation of perforation size is difficult, and there are few detailed reports addressing this issue. Furthermore, such evaluation becomes more difficult for irregularly shaped perforations. This study conducted a quantitative evaluation of tympanic membrane perforations, using image analysis equipment.A significant correlation was found between the degree of sound conduction disturbance and the perforation area; this correlation was greater at low frequencies following a traumatic perforation. The conductive disturbance associated with chronic otitis media was significantly greater at low frequencies. Circular perforations caused only minor conduction disturbance. Perforations in the anteroinferior quadrant were associated with greater conduction disturbance. Traumatic spindle-shaped perforations and malleolar perforations were associated with greater conduction disturbance.

Labyrinthine fistula is one of the most common complications of chronic otitis media associated with cholesteatoma. The optimal management of labyrinthine fistula, however, remains controversial. Between 1995 and 2005, labyrinthine fistulae were detected in 31 (6 per cent) patients in our institution. The canal wall down technique was used in 27 (87 per cent) patients. The cholesteatoma matrix was completely removed in the first stage in all patients. Bone dust and/or temporalis fascia was inserted to seal the fistula in 29 (94 per cent) patients. A post-operative hearing test was undertaken in 27 patients; seven (26 per cent) patients showed improved hearing, 17 (63 per cent) showed no change and three (11 per cent) showed a deterioration. The study findings indicate that there are various treatment strategies available for cholesteatoma, and that the treatment choice should be based on such criteria as auditory and vestibular function, the surgeon's ability and experience, and the location and size of the fistula.

To identify the novel receptor tyrosine kinases (RTKs) critical to the proliferation of hematopoietic stem cells, we performed polymerase chain reaction-based cloning from highly purified murine hematopoietic stem cells. Lineage marker-negative, c-KIT-positive, and Ly6A/E- or Sca- 1-positive (Lin-c-KIT+Sca-1+) cells were sorted by a fluorescence- activated cell sorter. Two sets of degenerate oligonucleotide primers were directed to the conserved sequences of the catalytic domain, and were used to amplify cDNAs that encode protein tyrosine kinases (PTKs). One hundred cDNA clones were sequenced and 8 RTKs were identified, as well as 12 non-RTKs and 2 serine/threonine kinases. Sixteen cDNAs were identical to the known kinase genes (PKC beta, JAK-1, JAK-2, TYK-2, HCK, FGR, FYN, BLK, c-FES, FER, c-ABL, c-KIT, FLK-1, FLK-2, IGF1R, and ECK). Six novel cDNA sequences (stk series) were identified. However, three of them turned out to be BPK, RYK, and TEK. The remaining three showed high homology to S6 kinase II, JAK-2, and v-SEA/c-MET, respectively. Characterization of full-length cDNA sequence of the v- SEA/cMET-related gene showed that this was a novel RTK gene and we named this gene STK (stem cell-derived tyrosine kinase). We identified two distinct forms of STK cDNA; the short one encoded a putative truncated protein that lacked most of the extracellular domain. STK was expressed at various stages of hematopoietic cells, including stem cells, but we could not detect any apparent expression in other adult tissues. The expression of the truncated form of mRNA was more predominant than that of the complete form. STK was assigned by fluorescent in situ hybridization to the R-positive F1 band of chromosome 9, the same region to which hepatic growth factor-like protein has been assigned. Characterization of these PTKs, including STK, will be helpful to elucidate the molecular mechanism of the growth regulation of hematopoietic stem cells.