Cancer Cell Analyses at the Single Cell-Level Using Electroactive Microwell Array Device

Circulating tumor cells (CTCs), shed from primary tumors and disseminated into peripheral blood, are playing a major role in metastasis. Even after isolation of CTCs from blood, the target cells are mixed with a population of other cell types. Here, we propose a new method for analyses of cell mixture at the single-cell level using a microfluidic device that contains arrayed electroactive microwells. Dielectrophoretic (DEP) force, induced by the electrodes patterned on the bottom surface of the microwells, allows efficient trapping and stable positioning of single cells for high-throughput biochemical analyses. We demonstrated that various on-chip analyses including immunostaining, viability/apoptosis assay and fluorescent in situ hybridization (FISH) at the single-cell level could be conducted just by applying specific reagents for each assay. Our simple method should greatly help discrimination and analysis of rare cancer cells among a population of blood cells.     

Analysis of human terminal deoxynucleotidyl transferase cDNA expressible in mammalian cells

Human Molt3 cDNA library was constructed using pcD vector system which permits the expression of cDNA inserts in mammalian cells. Nearly full-length human terminal deoxynucleotidyltransferase (TdT) cDNA was cloned using a fragment of bovine TdT cDNA as a probe. The human TdT cDNA contains an open reading frame of 1,557 bp coding for 519 amino acids, including 31 bp and 341 bp from 5' and 3' untranslated regions, respectively. The TdT cDNA was transfected into COS7 monkey fibroblasts directed the synthesis of enzymatically active protein of Mr 59,495. The cloned TdT cDNA hybridized with poly A+ RNAs of 2,100 b and 3,300 b from stable T-cell leukemia Molt3 and Molt4 cells.     

Regional assignment of the gene for diphtheria toxin sensitivity using subchromosomal fragments in microcell hybrids

Human x mouse microcell hybrids resistant to G418 were constructed between mouse hepatoma cells and human x mouse whole cell hybrids containing only intact human chromosome 5 and 22 with an integrated neo ^r-gene. Among these, microcell hybrid BG15 produced four subclones, BG15-4, BG15-6, BG15-7 and BG15-9, which contained variously sized complements of human chromosome 5. BG15-6 contained an intact human chromosome 5, BG15-7 a deleted human chromosome 5 (5pter-q22) and BG15-4 and BG15-9 a translocation between parts of human chromosome 5 (pter-qter? and pter-q23, respectively) and a mouse chromosome. Southern DNA blot analysis showed that the human dihydrofolate reductase (DHFR) gene was present in all four subclones, whereas the human homolog of the v-fms gene was present in BG15-4 and 15-6, but absent from BG15-7 and 15-9. BG15-4, 15-6 and 15-9 were sensitive to diphtheria toxin, and only BG15-7 was resistant to the toxin. We used these microcell hybrids to restrict further the regional location of the gene for diphtheria toxin sensitivity to the q23 region of human chromosome 5.     

Regional assignment of five genes on human chromosome 19

A human-mouse hybrid segregant HM76Dd40-6 with new characteristics was derived from the hybrid cell line HM76Dd containing human chromosome 19 as the only human chromosome. Three virus sensitivities located on human chromosome 19 (PVS, E11S and RDRC) were lost in HM76Dd40-6, while six other genes (C3, LDLR, EF2, GPI, PEPD and MANB) were retained. Cytogenetic analysis and in situ hybridization using human or mouse repeated sequences as probes showed that the region q13.1-qter of human chromosome 19 had been replaced by a fragment of mouse chromosome. Our results permit further regional assignment for the following five genes on human chromosome 19: GPI in the region cen-q12, MANB in p13.2-q12, E11S and RDRC in q13.1-qter, and EF2 in pter-q12.     

Role in translation of a triple tandemly repeated sequence in the 5''-untranslated region of human thymidylate synthase mRNA.

A triple tandem repeat (TTR) consisting of 90 nucleotides exists immediately upstream of the ATG initiator codon in human thymidylate synthase (TS) cDNA (pcHTS-1). To investigate the role of the TTR in the expression of the TS cDNA, we used pcHTS-1 to construct mutant cDNA clones in which part of the TTR was deleted or an additional element was inserted. The mutant cDNA plasmid was introduced into murine TS-negative mutant cells and the relative translation efficiencies of the mutant cDNAs were determined by measuring the transient expression of TS activity and the amount of TS mRNA transcribed. The translation efficiency in transient expression of the mutants was increased by deletions covering all the first two repeated elements, and the part of the third closest to the ATG initiator codon, but was not affected by deletions of only parts of the first two repeated elements at the 5' end. The translation efficiency was also not affected by insertion of an additional repeated element into the TTR. These results suggest that the first two repeated elements at the 5' end both have inhibitory effects on translation of the TS mRNA, probably due to the unique structural feature of this element.     

Anatomical features associated with preaxial duplication (pd): a recessive mutation in the rat

Preaxial polydactyly of the fore- and hindlimbs was found in Wistar-derived rats in 1978. Genetic analysis indicated that the polydactyly was due to the effects of an autosomal recessive gene (gene symbol; pd). Polydactylous homozygous rats had two or three pollices (six or seven digits) in the forelimbs and one to three preaxial extra digits (six to eight digits) in the hindlimbs. Skeletal examination revealed the presence of the extra carpal, metacarpal, and phalangeal bones that seemed to be complete or incomplete duplication of the navicular, greater multangular, first metacarpal, and phalanges of digit I in the forelimbs. In the hindlimbs, extra tarsal, metatarsal, and phalangeal bones were also observed preaxially. These extra elements seemed to be mirror-image duplications of the talus, navicular, second cuneiform, third cuneiform, cuboid, and metatarsals and phalanges of digits II-V with the absence of the first cuneiform, tibiale, first metatarsal, and phalanges of digit I. In addition, morphological changes were observed in the humerus, radius, and ulna in the forelimbs and femur, tibia, and fibula in the hindlimbs. Especially in the radius and tibia, thickening and bifurcation were found, indicating incomplete duplication of these bones. Based on these findings, the limb anomaly was classified as preaxial carpometacarpal/tarsometatarsal-type polydactyly with incomplete duplication of the radius and tibia. The mutant rats had other associated anomalies such as accessory spleens and cryptorchism. The males are sterile, whereas the females breed normally.     

Structure of human phenylethanolamine n-methyltransferase gene: existence of two types of mRNA with different transcription initiation sites

The chromosomal gene for human phenylethanolamine N-methyltransferase (PNMT; EC 2.1.1.28) was isolated from a human genomic library using a cloned human PNMT cDNA as a probe, and the nucleotide sequence was determined. PNMT is encoded in a single gene which consists of three exons. We observed newly the presence of minor PNMT mRNA (type B) besides the major mRNA (type A) as reported previously (Kaneda et al., J. Biol. Chem. 263, 7672–7677, 1988) by Northern hybridization. Type B mRNA carries an approximately 700 nucleotide-long untranslated region in the 5′ terminus. This suggests that two types of mRNA are produced from a single gene through the use of two alternative promoters. A TATA-like sequence locates 30 base pair upstream from the cap site of type A mRNA. Upstream of the cap site, there are several sequences resembling Spl binding sites and glucocorticoid responsive elements, with the latter also found in the first intron.     

Three-dimensional structure of endothelial cells in hepatic sinusoids of the rat as revealed by the Golgi method

Summary The three-dimensional structure of endothelial cells in the hepatic sinusoids of the rat was studied by application of light- and electron microscopy on Golgi-impregnated specimens. A number of endothelial cells could thus be individually delineated throughout the hepatic lobules. The cytoplasm, showing heavy silver deposits, consists of two distinct areas, a thick and thin portion. The thick portion, issuing from the region of the perikaryon, branches and tapers toward the cell periphery. The thin portion, occupying the remainder of the cytoplasm, consists largely of highly fenestrated sieve plates. Some intralobular variation can be noted; the thick portion of the endothelial cells is well developed in the periportal zone, while the cells in the centrilobular zone are relatively rich in thin portions. In addition, the area of distribution of an individual endothelial cell is larger in the centrilobular sinusoids than in the periportal zone. Some endothelial cells also possess unique cytoplasmic processes projecting into the intercellular space between hepatocytes and connecting the sinusoidal walls of neighboring sinusoids. These processes may anchor the endothelial cells to the hepatic plates.     

Expression of four types of human tyrosine hydroxylase in COS cells

Alternative splicing from a single gene produces four kinds of human tyrosine hydroxylase (types 1-4), which have structural diversity only in the N-terminal region. We attempted expression of the type 1-4 enzymes in COS cells and performed kinetic analyses. All had enzymatic activities. The Km values of the four types for L-tyrosine and 6-methyl-5,6,7,8-tetrahydropteridine were similar, although their relative homospecific activities were clearly different. The type 1 enzyme displayed the highest activity.