Excess NF-κB induces ectopic odontogenesis in embryonic incisor epithelium.

Nuclear factor kappa B (NF-κB) signaling plays critical roles in many physiological and pathological processes, including regulating organogenesis. Down-regulation of NF-κB signaling during development results in hypohidrotic ectodermal dysplasia. The roles of NF-κB signaling in tooth development, however, are not fully understood. We examined mice overexpressing IKKß, an essential component of the NF-κB pathway, under keratin 5 promoter (K5-Ikkß). K5-Ikkß mice showed supernumerary incisors whose formation was accompanied by up-regulation of canonical Wnt signaling. Apoptosis that is normally observed in wild-type incisor epithelium was reduced in K5-Ikkß mice. The supernumerary incisors in K5-Ikkß mice were found to phenocopy extra incisors in mice with mutations of Wnt inhibitor, Wise. Excess NF-κB activity thus induces an ectopic odontogenesis program that is usually suppressed under physiological conditions.

Treatment of severe congenital erythropoietic porphyria by bone marrow transplantation.

Congenital erythropoietic porphyria (CEP), which is the result of a deficiency of uroporphyrinogen (URO) III synthase activity, is the most disfiguring porphyria in humans. Various methods of treatment have been used to treat CEP with varying success, including erythrocyte transfusion, hydroxyurea, and splenectomy. The only treatment that corrects the enzymatic defect resulting in a cure is bone marrow/stem cell transplantation, which has been reported previously in only 5 patients worldwide. We describe the first patient with CEP who underwent successful bone marrow transplantation performed in the United States and review the therapeutic options in the management of this challenging type of porphyria.

Cloning, functional expression, and chromosomal localization of the human and mouse gp180-carboxypeptidase D-like enzyme.

We previously reported that a host cell glycoprotein, gp180, binds duck hepatitis B virus particles, and is encoded by a member of the carboxypeptidase gene family (Kuroki, K., Eng, F., Ishikawa, T., Turck, C., Harada, F., Ganem, D., 1995. gp180, a host cell glycoprotein that binds duck hepatitis B virus particles, is encoded by a member of the carboxypeptidase gene family. J. Biol. Chem. 270, 15022-15028). After that report, carboxypeptidase D (CPD) was subsequently purified from bovine pituitary and characterized as a novel carboxypeptidase E (CPE)-like enzyme, with many characteristics in common with duck gp180 (Song, L., Fricker, L.D., 1995. Purification and characterization of carboxypeptidase D, a novel carboxypeptidase E-like enzyme, from bovine pituitary. J. Biol. Chem. 270, 25007-25013). CPD is now supposed to play an important role in a secretory pathway. To clarify the function of gp180 further, we have isolated and analyzed human and mouse homologues of duck gp180. cDNA clones derived from human HepG2 cells and mouse livers have been isolated on the basis of homology to the duck gp180. The suggested open reading frames of the human and mouse cDNA encode 1380 and 1377 amino acid proteins, respectively and have three carboxypeptidase homologous domains (A, B, and C). Domains A and B have completely conserved the residues known to have the enzymatic activity of carboxypeptidase, but domain C in each cDNA does not. Northern blotting revealed a ubiquitous tissue distribution of human gp180 mRNA with several transcript species. Expression of human gp180 cDNA in transfected 293Tcells exhibited carboxypeptidase activity upon radiometric assay. The human and mouse homologues of duck gp180 have many characteristics in common with bovine CPD. Fluorescence in-situ hybridization reveals that the gene encoding human gp180 is located in region 17q11.2.

gp180, a host cell glycoprotein that binds duck hepatitis B virus particles, is encoded by a member of the carboxypeptidase gene family.

Duck hepatitis B virus particles bearing the L and S envelope proteins bind a cellular glycoprotein of 180 kDa (gp180) with high affinity and specificity. Binding is mediated by the pre-S region of the L protein and is blocked by neutralizing but not by non-neutralizing monoclonal antibodies to the virus. These and other properties have suggested that gp180 may be a component of the viral entry machinery. Here we report the purification of gp180 from duck liver and the isolation and characterization of cDNA encoding it. DNA sequence analysis of this cDNA indicates that gp180 is a novel member of the basic carboxypeptidase gene family.

Presence of env genes in members of the RTVL-H family of human endogenous retrovirus-like elements.

We have found that the RTVL-H family of human endogenous retrovirus-like elements was highly expressed in lung squamous cell carcinoma tissue and several human cell lines. We determined the nucleotide sequences of several cDNA clones and found a unique sequence that was not present in the published "prototypic" RTVL-H sequence. An open reading frame in this region encoded an amino acid sequence homologous to the conserved sequence in the transmembrane envelope protein of various retroviruses. Two genomic clones that encoded this sequence were obtained and sequenced. One RTVL-H element containing the env gene was 8.7 kilobase pairs in length. The putative env gene, consisting of about 1800 base pairs, had two open reading frames interrupted by a termination codon. The amino acid sequence of this region showed significant homology with those of other retroviral envelope proteins and contained eight potential glycosylation sites. There were estimated to be about 100 copies of RTVL-H elements containing the env gene per haploid human genome.

Specific detection of c-erbB-2 mRNA expression in gastric cancers by the polymerase chain reaction following reverse transcription.

The expression of the c-erbB-2 mRNA in human embryonic lung fibroblasts, a gastric cancer cell line, mature placenta, and 25 gastric cancer tissues was examined by using the polymerase chain reaction following reverse transcription. This technique can be used to examine c-erbB-2 mRNA expression in a small endoscopic biopsy specimen before surgery.

Specific detection of metastasized human tumor cells in embryonic chicks by the polymerase chain reaction.

We have established a highly sensitive method for specific detection of metastasized human tumor cells in embryonic chicks using the polymerase chain reaction (PCR). The cells (HT-1080 or KMST-6) were inoculated into the chorioallantoic membrane vein of the chick embryo and DNA of each embryonic organ was extracted. Then, a human beta-globin-related sequence (576 bp) in the DNA from the embryonic liver and lung was specifically amplified and detected by gel electrophoresis and by a specific oligonucleotide probe. The amplified fragments from the liver DNA samples increased gradually from 2 h to 7 days after HT-1080 inoculation. On the other hand, with inoculation of non-tumorigenic human embryonal fibroblast KMST-6 cells, the DNA from the embryonic liver 7 days after inoculation did not show the PCR-amplified product. This detection technique can contribute significantly to the precise detection of microscopic metastasis.

A simple, general method for detecting retroviral RNAs expressed in cells.

A simple, efficient method has been developed for detecting retroviral RNAs expressed in cells. In this method, total RNAs or poly A+ RNAs extracted from various human cells are separated by electrophoresis and hybridized with synthetic oligonucleotides corresponding to the 3'-terminal 18 nucleotides of various tRNAs. Genomic and subgenomic RNAs of HTLV-I and HTLV-II in virus-infected cells and of xenotropic murine leukemia virus expressed in human lung cancer cells were easily detected with the tRNA(Pro)-derived oligonucleotide probe. This technique can be used to search for unidentified retroviruses expressed in human cancer cells and tissues.

Isolation of three kinds of human endogenous retrovirus-like sequences using tRNA(Pro) as a probe.

Three kinds of human endogenous retrovirus-like sequences (HuERS-P1, 2 and 3) were isolated from a HeLa cell genomic library using the 3'-half fragment of proline tRNA as a hybridization probe. These elements contained putative primer binding sites complementary to the 3'-terminus of proline tRNA and long terminal repeats (LTRs) characteristic of retrovirus provirus. The LTR sequence of HuERS-P1 consisted of about 690 nucleotides and contained a CAT box, a TATA box and a polyadenylation signal. A complete unit of an Alu family sequence was inserted into the 5'-LTR of one of the clones. HuERS-P2 also contained a TATA box and a polyadenylation signal in its LTR (about 840 nucleotides long), but the LTR sequence of this element was quite different from that of HuERS-P1. Although clone HuERS-P3 contained only the 5'-LTR region, this LTR sequence contained a CAT box, a TATA box and a poly-adenylation signal and was quite similar to the LTR sequence of the recently isolated human retrovirus-related sequence HuRRS-P (Kröger, B. and Horak, I. (1987) J. Virol., 61, 2071-2075). Human and simian DNAs contain 10 to 40 copies of these elements, but mouse DNA does not contain these elements.

Cloning of cDNAs for two beta-tubulin isotypes expressed in murine T cell lymphoma, L5178Y and analysis of their translation products.

Three beta-tubulin isoforms, M beta Ib, M beta Ia and M beta II, were detected by isoelectric focusing gel electrophoresis (IEF) in extracts of cultured cells of a mouse T cell lymphoma, L5178Y. To investigate the origin(s) of the isoforms, we have isolated cDNA clones encoding beta-tubulin from a cDNA library prepared from poly (A)+ RNA of L5178Y cells. Seventeen cDNA clones carrying the entire coding sequences of beta-tubulin were isolated and classified into two distinct isotypes, represented by two clones designated pMT27 and pMT49, according to the results of restriction mapping. On the basis of the nucleotide sequences of the two cDNAs, pMT27 and pMT49 were identified as mouse beta-tubulin isotypes 3 and 5, respectively. By using in vitro translation products of hybrid-selected mRNAs and of the SP6 in vitro transcripts of the cDNAs, polypeptides encoded by the two cDNA clones were analyzed by IEF. We found that pMT27 and pMT49 encode M beta Ib and M beta Ia, respectively. In addition, M beta II was detected in translation products of mRNA specifically hybridized to pMT49, but not in those of the in vitro transcript of pMT49 DNA. These results suggest that M beta II is the translation product of mRNA whose 3'-untranslated region is highly homologous to that of pMT49.

Two mammalian heat shock proteins, HSP90 and HSP100, are actin-binding proteins.

Two high molecular weight heat shock proteins, HSP90 (Mr, 90,000) and HSP100 (Mr, 100,000), were separately purified from extracts of cultured cells of a mouse lymphoma cell line, L5178Y. Both of the HSPs exist in homodimeric form under physiological conditions. Their physicochemical properties are quite similar to each other. Each of the purified HSPs was shown to coprecipitate with rabbit skeletal muscle actin under actin-polymerizing conditions. Both HSP90 and HSP100 increased the low-shear viscosity of filamentous actin solutions in a dose-dependent manner, which suggests that these HSPs cross-link actin filaments. Although some molecular properties and the effects described above on actin solution of HSP90 and HSP100 resemble those of alpha-actinin, the HSPs were distinguished from alpha-actinin by various means, including visualization of molecular shapes by electron microscopy with the aid of the low-angle rotary shadowing technique. Immunofluorescence staining by specific antisera against HSP90 revealed that HSP90 was localized in ruffling membranes in addition to the cytoplasmic space.

Occurrence of two beta-tubulin isoforms with different polymerizing abilities in L5178Y cells.

Mouse lymphoma L5178Y cells express at least two isoforms of beta-tubulin, designated M beta I and M beta II, as revealed by isoelectrofocusing, whereas two independently isolated normal T-cell clones, 3D10 and K23, express only M beta I. M beta II-tubulin is more acidic (pI, 5.10) than M beta I-tubulin (pI, 5.15). L5178Y cells were disrupted under the microtubule-stabilizing conditions, followed by centrifugation to separate fractions containing polymerized and unpolymerized tubulin. We found that a proportion of M beta II to total beta-tubulins is larger in the fraction containing unpolymerized tubulin than in that containing polymerized tubulin. In addition, when tubulin was purified from extracts of L5178Y cells by repeated cycles of polymerization-depolymerization, the M beta II-tubulin isoform was gradually lost during the successive purification steps. The low recovery of M beta II-tubulin was observed, irrespective of the presence or absence of MAPs, and even in the presence of an excess amount of essentially polymerizable porcine brain tubulin. These results indicate that M beta II-tubulin is less able to polymerize than is M beta I-tubulin, both in vivo and in vitro.

New U1 RNA species found in Friend SFFV (spleen focus forming virus)-transformed mouse cells.

The U1 RNA species in 10 mouse cell lines were examined by two-dimensional polyacrylamide gel electrophoresis. Seven cell lines that were not infected by Friend spleen focus forming virus gave only one (I) or two (I and II) U1 RNA-containing spots. However, two Friend cell lines (FVTCT and Friend 745a cells) gave three spots (I, II, and III) and another Friend cell line, K-1 cells, gave four spots (I, II, III, and IV). As a result of further separation and fingerprinting analysis of each spot, FVTCT and Friend 745a cells were found to contain U1a-1, U1b-1, -2, and -6 RNAs whereas K-1 cells were found to contain several U1 RNAs, which we call U1a-1 and -2, U1b-4, -5, and -6 RNAs. We determined the sequences of these seven U1 RNAs and found that mouse U1 RNAs had two basic sequences (U1a and -b). The nucleotide sequence of U1a-1 RNA was identical to that of rat U1a RNA, while U1a-2 RNA was one base different from U1a-1 RNA. Relative to U1a-1 RNA all of the U1b RNAs had five base substitutions and one additional base and were under-methylated in the center. U1b-6 RNA contained two base substitutions and one base addition in the 3'-terminal portion of U1b-1 RNA. U1b-2, -4, and -5 RNAs, which were observed only in Friend cells, each had an additional base substitution in the 5'-half of U1b-1 RNA.

Characterization of a Rous sarcoma virus mutant defective in packaging its own genomic RNA: biochemical properties of mutant TK15 and mutant-induced transformants.

The accompanying paper (S. Kawai and T. Koyama , J. Virol. 51:147-153, 1984) describes the isolation and biological properties of a mutant, TK15 , derived from a Rous sarcoma virus mutant, tsNY68 . The cis-acting defect of the mutant is analyzed biochemically in this paper. TK15 virions released from virus-producing 15c (+) cells were deficient in viral genomic 39S RNA, although comparable amounts of viral RNAs were transcribed in 15c (+) and tsNY68 -infected cells. Analysis of provirus DNA occurring in 15c (+) cells suggested that the mutant genome had a deletion of ca. 250 bases near the 5' end of the genome somewhere between the primer binding site and the 5' end of the gag-coding region. These findings indicate that at least part of the sequence lost in the TK15 genome is indispensable for packaging viral genomic RNA into virions. TK15 induces nonvirus -producing 15c (-) transformants at high frequency. Southern blot analysis of DNAs from those 15c (-) clone cells revealed that TK15 -derived proviruses contained various extents of internal deletions. Many 15c (-) clones had a provirus carrying only the src gene with long terminal repeat sequences at both ends. The mechanism for the segregation of 15c (-) cells is discussed.

Nucleotide sequence of nuclear 5.4 S RNA of mouse cells.

The nucleotide sequence of nuclear 5.4 S RNA, a new species of small nuclear RNA (snRNA) of mouse cells, was determined. The 5.4 S RNA consists of 138 nucleotide residues containing 1 mol each of 2,2,7- trimethylguanosine (m3(2,2,7) G), 2'-O-methyladenosine (Am), 2'-O-methyluridine (Um) and pseudouridine as modified nucleosides. This RNA has a cap structure, m3(2,2,7) ++GpppAm -, at its 5'-terminus and sequences complementary to the terminal consensus sequences of introns. The sequence complementary to the 5'-splice junction, A-U-C-C-psi-U-A-C-C-U-G, is very similar to the 5'-terminal sequence of U1 RNA.

Minor serine tRNA containing anticodon NCA (C4 RNA) from human and mouse cells.

The nucleotide sequence of C4 RNA, one of the "4.5S RNAs" of HeLa cells, was determined. This RNA consists of 90 nucleotides containing C-C-A at its 3'-terminus. The sequence can be drawn to form a clover-leaf structure with several unusual features and with anticodon NCA. The short term labeled molecule contains triphosphates at its 5'-terminus, whereas the mature molecule contains only monophosphate. Therefore, there must be no precursor nucleotide at the 5'-end in the primary transcript of this tRNA. Mouse cells also contain C4 RNA. Only one base exchange was observed in the extra arm in human and mouse C4 RNAs. The molecule purified from mouse liver showed serine acceptor ability.

Biological and structural differences between tRNAVal species isolated from rat ascites hepatoma cells and normal rat liver.

On RPC-5 column chromatography, the main valine acceptor activity of tRNA (tRNA2Val) from rat ascites hepatoma cells was eluted later than that of normal rat liver tRNA (tRNA1Val). The tRNA2Val was aminoacylated by E. coli amino-acyl-tRNA synthetase, while tRNA1Val from normal rat liver was not. Rat fetal liver tRNAVal was also aminoacylated by E. coli aminoacyl-tRNA synthetase. tRNA1Val (rat liver) and tRNA2Val (ascites hepatoma) were each purified to a homogeneous state by RPC-5 column chromatography and two-dimensional polyacrylamide gel electrophoresis, and their sequences were determined by post-labeling techniques. Ascites hepatoma tRNA2Val differed from rat liver tRNA1Val in that Gm18, C32 and an unknown modified nucleoside, N34, in the latter tRNA were mostly replaced by G, Cm, and inosine, respectively. In addition, 3'-terminal adenosine was not present in tRNA1Val (normal rat liver), but was in tRNA2Val (ascites hepatoma). Other modifications and the primary structures of the two tRNAValS were found to be the same. Thus it was concluded that the new iso-acceptor species of tRNA Val in ascites hepatoma cells is due to a change of post-transcriptional modification, not to a change of tRNA transcription. The unique feature of the change of post-transcriptional modification in tRNA2Val (ascites hepatoma) is that both hypo- and hyper-modification take place simultaneously in the tRNA molecule depending the locations of nucleotide residues.