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L T Chow

Publications and source records attributed to L T Chow.

At least 127 records · Page 7Linked to original sources
PubMed1992

Control of human papillomavirus type 11 origin of replication by the E2 family of transcription regulatory proteins.

Replication of human papillomavirus type 11 (HPV-11) DNA requires the full-length viral E1 and E2 proteins (C.-M. Chiang, M. Ustav, A. Stenlund, T. F. Ho, T. R. Broker, and L. T. Chow, Proc. Natl. Acad. Sci. USA 89:5799-5803, 1992). Using transient transfection of subgenomic HPV DNA into hamster CHO and human 293 cells, we have localized an origin of replication (ori) to an 80-bp segment in the upstream regulatory region spanning nucleotide 1. It overlaps the E6 promoter region and contains a short A + T-rich segment and a sequence which is homologous to the binding site of the bovine papillomavirus type 1 (BPV-1) E1 protein in the BPV-1 ori. However, unlike the BPV-1 ori, for which half an E2-responsive sequence (E2-RS) or binding site suffices, an intact binding site is essential for the HPV-11 ori. Replication was more efficient when additional E2-RSs were present. The intact HPV-11 genome also replicated in both cell lines when supplied with E1 and E2 proteins. Expression vectors of transcription repressor proteins that lack the N-terminal domain essential for E2 transcriptional trans activation did not support replication in collaboration with the E1 expression vector. Rather, cotransfection with the repressor expression vectors inhibited ori replication by the E1 and E2 proteins. These results demonstrate the importance of the N-terminal domain of the E2 protein in DNA replication and indicate that the family of E2 proteins positively and negatively regulates both viral DNA replication and E6 promoter transcription.

Animals↗
PubMed1992

Solitary necrotic nodule of the liver: parasitic origin?

AIMS: To report further cases of solitary necrotic nodule of the liver and to study its nature. METHODS: Seven nodules were retrieved from 4000 necropsy and surgical liver specimens coming to light over the past five years. All of them satisfied the diagnostic criteria of solitary necrotic nodule: a solid lesion with a central necrotic core and a hyalinised fibrotic capsule containing elastic fibres. Their clinicopathological features were reviewed. RESULTS: The nodules were incidental findings at surgery or necropsy in four men and three women whose ages ranged from 48 to 79 years (mean 63.7 years). Four were found in the right lobe and three in the left. Six were subcapsular and only one deep in the parenchyma, with sizes ranging from 0.3-2.5 cm. Each of them was solitary, well demarcated, and round to oval with a firm, whitish rim and a core of yellowish white cheese-like to solid material. In addition to the basic architecture, there were a number of common and undescribed histological features: presence of varying numbers of small mural vessels with intimal fibrosis and obliteration, presence of cholesterol clefts and foamy cells among necrotic material, and sparsity of inflammatory cells. In the two cases where ghosts of degenerated cells and partially preserved liver reticulin pattern were noted, worms were identified, one being Clonorchis sinensis. CONCLUSIONS: The entity is believed to be a "burnt-out phase" of a variety of benign lesions. Parasitic infestation is another possible cause, and presence of ghosts of degenerate cells, partially preserved liver reticulin pattern, cholesterol clefts and foamy cells among necrotic material are auxiliary features pointing to such an aetiology. The variation in morphological fine details reflects both the lesion's diverse pathogenesis and the fact that it can be of varying duration.

Aged↗
PubMed1992

Recombinant retroviruses encoding human papillomavirus type 18 E6 and E7 genes stimulate proliferation and delay differentiation of human keratinocytes early after infection.

Human papillomavirus (HPV) DNAs are detected in most genital dysplasias and cancers, suggesting that these viruses perturb epithelial growth and differentiation. The E6 and E7 genes of HPV type 18 induce immortality in keratinocytes cultured from genital tract epithelia, and the immortal cell lines display aberrant squamous differentiation. To examine whether the E6 and E7 proteins directly alter keratinocyte growth and differentiation, high-titer recombinant retroviruses were constructed for efficient transfer and expression of HPV-18 genes E6, E6* and E7 in cultures of normal human keratinocytes. Infection with retroviruses encoding E6 and E7 stimulated cell proliferation, reduced the requirement for bovine pituitary extract and induced immortality. E6 and E7 also delayed but did not prevent the onset of terminal squamous differentiation. The magnitude of effects on growth and differentiation of cultured cells was directly related to levels of E7 protein expression. Thus, expression of the HPV-18 E6 and E7 genes stimulates cell proliferation and delays differentiation of keratinocytes in vitro.

Blotting, Southern↗
PubMed1992

Epithelial-specific gene expression during differentiation of stratified primary human keratinocyte cultures.

Cultured epithelial cells are used to generate extensive patches of autologous skin equivalent for patients with burns or wounds and to investigate the growth and differentiation of epithelia in vitro. We have undertaken a comprehensive study of the morphological and molecular events that occur during culturing of human foreskin keratinocytes at the liquid-air interface on a dermal equivalent consisting of a collagen matrix containing fibroblasts. Using radioactively labeled RNA probes for mRNAs and monoclonal antibodies for proteins, we found that the expression of a comprehensive set of differentiation stage-specific genes was affected by the type of fibroblasts included in the matrix as well as by the age of the culture. The expression of these genes was not always coordinated and could not be predicted from the histological appearance of the stratified epithelium. Surprisingly, the mouse fibroblasts promoted epithelial differentiation much more closely resembling foreskin than did the homologous primary foreskin fibroblasts.

3T3 Cells↗
PubMed1992

Intraosseous lipoma. A clinicopathologic study of nine cases.

Intraosseous lipoma is one of the rarest benign primary tumors of the bone. The clinicopathologic features of nine cases of intraosseous lipoma, including one with multiple lesions of the lumbar spinous processes, were studied and analyzed. They represented 2.5% of all primary bone tumors seen in our institutions. They were subdivided into three groups according to their histologic appearances: Group 1, tumors consisting predominantly of viable fat cells; Group 2, tumors showing, in addition, areas of fat necrosis and calcification; and Group 3, lesions with extensive reactive ischemic bone formation and calcification, viable lipocytes and fat necrosis may or may not be visible. Since the tumor is located within the rigid confines of the bony trabeculae, the internal pressure created as it enlarges is believed to be most significant in the morphogenesis of the tumor. The recognition of this spectrum of morphologic features is important, since the secondary changes may predominate and overshadow the original lipomatous component, leading to the possibility of confusing it with other benign bone tumors, cysts, and bone infarction. A combined clinical, radiologic, and pathologic approach should be adopted in the diagnosis of this tumor.

Adult↗
PubMed1991

Complete structure of the human gene for 92-kDa type IV collagenase. Divergent regulation of expression for the 92- and 72-kilodalton enzyme genes in HT-1080 cells.

The complete structure of the human gene for 92-kDa type IV collagenase was determined. Two overlapping genomic clones spanning 26 kilobases (kb) of genomic DNA were shown to contain the entire 7.7-kb structural gene together with 15 and 3.5 kb of 5'-end and 3'-end flanking regions, respectively. The 92-kDa type IV collagenase gene contains 13 exons as does the 72-kDa type IV collagenase gene. All intron locations of the 92-kDa enzyme gene coincided with intron locations in the 72-kDa enzyme gene. Exons 5, 6, and 7 which were 174, 174, and 177 base pairs long, respectively, each encoded one complete internal repeat which resembles the collagen-binding domains of fibronectin. The sequence coding for a unique 48-residue segment in the 92-kDa type IV collagenase that has no counterpart in other metalloproteinases was not present in a separate exon, but was contained in exon 9 which also codes for sequences with homology to the other metalloproteinases. The initiation site for transcription was determined by primer extension analysis. Sequencing analysis of 599 base pairs of the 5'-end flanking region showed that the promoter does not have a TATA motif, but a TTAAA sequence at position -29 to -25. A CAAT motif was not observed but there was one GC box. Two putative 12-O-tetradecanoyl-phorbol-13-acetate (TPA) response elements, that might serve as binding sites for the transcription factor AP-1 and a consensus sequence of a transforming growth factor beta 1 (TGF-beta 1) inhibitory element were found in the promoter region. Gelatinase assay of enzyme secreted by cultured human fibrosarcoma cells (HT-1080) revealed only low levels of 92-kDa type IV collagenase activity, whereas considerable activity of the 72-kDa enzyme was present. Northern hybridization analysis confirmed these findings. Treatment of the HT-1080 cells with TPA resulted in induction of the secretion of 92-kDa type IV collagenase activity. This induction could not be significantly inhibited by concomitant incubation with TGF-beta 1. TPA and TGF-beta 1 did not markedly affect the activities of the 72-kDa enzyme. The activities of the secreted 92- and 72-kDa enzymes by HT-1080 cells correlated with the amounts of mRNA as estimated by Northern analyses.

Amino Acid Sequence↗
PubMed1991

Structure of the human laminin B2 chain gene reveals extensive divergence from the laminin B1 chain gene.

The exon-intron structure of the human laminin B2 chain gene was elucidated from genomic lambda phage clones spanning 2 kilobase pairs (kb) of the 5'-flanking region, 58 kb of the structural gene and 10 kb of the 3'-flanking region. The entire gene was shown to contain 28 exons. The promoter region has no TATA or CAAT boxes whereas it contains five GC boxes and three AP-2-like binding sites. Comparison with the promoter region of the mouse gene revealed six highly conserved sequences of 14 to 42 base pairs in length. Sequencing of the last exon of the gene showed that the 3'-untranslated region of the mRNA can be up to 2797 nucleotides with five AATAAA potential polyadenylation signals. The similarity of the human 3'-untranslated sequence with that of mouse was shown to be 68.8%. The exon-intron structure of the laminin B2 chain gene demonstrated extensive divergence from the human laminin B1 chain gene, which has 34 exons. Only three intron locations are conserved in these two genes. The overall exon profile of the laminin B2 chain gene correlates only marginally with the pattern of structural domains and internal cysteine-rich repeats in the laminin B2 polypeptide chain.

Amino Acid Sequence↗
PubMed1991

Characterization of the 3' half of the human type IV collagen alpha 5 gene that is affected in the Alport syndrome.

We have determined the exon-intron structure of the 3' half of the gene for the human type IV collagen alpha 5 chain that is affected in X-chromosome-linked Alport syndrome. Six overlapping lambda phage genomic clones containing exons 1-14 (as counted from the 3' end) and two additional overlapping genomic clones containing exons 16-19 spanned a total of 60 kb, 9.5 kb of which were the 3' flanking region. The exon-intron structure was elucidated by restriction enzyme mapping, nucleotide sequencing, and heteroduplex analyses. The sequences of all of the 19 most 3' exons and their flanking sequences were determined from the genomic clones, with the exception of exon 15, which was sequenced after amplification from genomic DNA with the polymerase chain reaction. The results show that the genes for the alpha 5(IV) and alpha 1(IV) chains have an almost identical exon size pattern in the 3' half. In contrast, there is not a clear conservation of intron sizes between the two genes, although both genes may have a similar total size. The current results have allowed the identification of three mutations in the alpha 5(IV) gene in three kindreds with Alport syndrome, and the gene structure and sequencing data presented should facilitate the analysis of other as yet unidentified mutations in this heterogeneous genetic disease.

Amino Acid Sequence↗
PubMed1991

An E1M--E2C fusion protein encoded by human papillomavirus type 11 is asequence-specific transcription repressor.

We have isolated a putative, spliced E5 cDNA of human papillomavirus type 11 (HPV-11) by polymerase chain reaction amplification of cDNAs from an experimental condyloma. Using retrovirus-mediated gene transfer, we isolated two novel HPV-11 cDNAs, one of which had a splice linking nucleotides 1272 and 3377. This transcript also existed in experimental condylomata and in cervical carcinoma cells transfected with cloned genomic HPV-11 DNAs. The 5' end of the transcript in transfected cells originated upstream of the initiation codon of the E1 open reading frame (ORF). It could conceptually encode a fusion protein consisting of the amino-terminal 23% of the E1 ORF and the carboxy-terminal 40% of the E2 ORF. This E1M--E2C fusion protein contained both the DNA replication modulator domain E1M, as defined in the bovine papillomavirus system, and the DNA binding domain of the E2 protein, which regulates viral transcriptional activities. Indirect immunofluorescence with polyclonal antibodies raised against the bacterially expressed TrpE-HPV-11 E2 protein demonstrated nuclear localization of the E1M--E2C protein in cells transiently transfected with an expression plasmid. Immunoprecipitation revealed a specific protein with an apparent molecular weight of 42,000 in transfected cells. The chloramphenicol acetyltransferase assay established that the putative E1M--E2C protein was a potent transcriptional repressor of both E2-dependent and E2-independent HPV-11 enhancer/promoter activities. Northern (RNA) blot hybridization indicated the repression was on the transcriptional level. Mutational analysis suggested that the E1M--E2C protein is an E2-binding site-specific repressor. The fusion protein also repressed bovine papillomavirus type 1 (BPV-1) E2 protein-dependent BPV-1 enhancer activity. When constitutively expressed in mouse C127 cells, the E1M--E2C protein inhibited BPV-1 transformation and episomal DNA replication, consistent with a role in the modulation of replication.

Condylomata Acuminata↗
PubMed1990

Structure of the human laminin B1 chain gene.

The exon-intron structure of the human laminin B1 chain gene was determined from genomic clones that spanned 90 kilobase pairs (kb), including 80 kb of the structural gene, about 1 kb of the 5'-flanking region, and 9 kb of the 3'-flanking region. DNA sequencing and heteroduplex analyses demonstrated that the gene consists of 34 exons. The intron sizes vary from 92 base pairs to more than 15,000 base pairs. The clones did not completely contain introns 13 and 14 and, therefore, the exact size of the gene remains to be determined. The first exon encodes a 5'-untranslated region, with the ATG translation start codon being in exon 2. The promoter region does not contain TATA or CAAT boxes, but it has four GC boxes. Additionally, two GC boxes are located in the 5'-untranslated sequence. A glucocorticoid response element-like sequence and a sequence resembling the binding sequence for transcription factor AP-2 are present in the 5'-flanking region. Another potential AP-2 binding sequence is located in the 5'-untranslated sequence of exon 1. A TGACC motif present in the binding region for the retinoic acid response element of the mouse laminin B1 gene promoter is also present in the human counterpart. The overall exon pattern of the gene correlates only slightly with the highly conserved structural domains and internal repeats of the B1 polypeptide chain. Furthermore, the exon profile differs considerably from that of the laminin B2 gene. A HincII/HpaI restriction fragment length polymorphism was identified in exon 31.

Amino Acid Sequence↗
PubMed1990

Structure of the human type IV collagenase gene.

The structure of the gene for human 70-kDa type IV collagenase (gelatinase) was determined. Three overlapping genomic clones were isolated and shown to contain 0.4 kilobase (kb) of the 5'-flanking region, the 27-kb structural gene, and 4.5 kb of the 3'-flanking region. The gene has 13 exons that vary in length from 110 to 901 base pairs (bp) and 12 introns that range from 175 to 4350 bp. Alignment of intron locations demonstrated that introns 1-4 and 8-12 of the type IV collagenase gene coincide with intron locations in the interstitial collagenase and stromelysin genes, indicating a close structural relationship of these metalloproteinase genes. Exons 5-7 are each 174 bp in size, and each codes for one complete internal repeat that resembles the collagen-binding domains of fibronectin. The transcription initiation site was determined by primer extension and S1 nuclease analyses. Analysis of the 0.4-kb 5'-flanking region of the gene showed that, in contrast to the genes of interstitial collagenease and stromelysin, there is no TATA box or 12-O-tetradecanoylphorbol-13-acetate-responsive element present in the promoter region, whereas there are two GC boxes. There is no CAAT box, but a potential binding site (CCCCAGGC) for the transcription factor AP-2 is located in the first exon.

Amino Acid Sequence↗
PubMed1990

Identification of mutations in the COL4A5 collagen gene in Alport syndrome.

X-linked Alport syndrome is a hereditary glomerulonephritis in which progressive loss of kidney function is often accompanied by progressive loss of hearing. Ultrastructural defects in glomerular basement membranes (GBM) of Alport syndrome patients implicate an altered structural protein as the cause of nephritis. The product of COL4A5, the alpha 5(IV) collagen chain, is a specific component of GBM within the kidney, and the gene maps to the same X chromosomal region as does Alport syndrome. Three structural aberrations were found in COL4A5, in intragenic deletion, a Pst I site variant, and an uncharacterized abnormality, which appear to cause nephritis and deafness, with allele-specific severity, in three Alport syndrome kindreds in Utah.

Blotting, Southern↗
PubMed1990

Human papillomavirus type 1 produces redundant as well as polycistronic mRNAs in plantar warts.

Human papillomavirus type 1 (HPV-1) causes plantar warts. On the basis of previously mapped mRNAs and sequence homologies of HPV-1 to other papillomaviruses, we designed oligonucleotide primers and employed the polymerase chain reaction to recover HPV-1 cDNAs from plantar warts. Seven spliced RNA species were characterized, including three not previously detected, and the coding potentials of each were deduced. The most abundant viral mRNA encodes an E1i--E4 protein. One new species is predicted to encode the full-length E2 protein, and another can, theoretically, encode the E2-C or E1-M proteins, three products that regulate mRNA transcription and DNA replication. One RNA species originating from a novel HPV promoter in the upstream regulatory region has the potential to encode the minor capsid protein L2. A newly recognized E5a open reading frame (ORF) is contained in all mRNAs that are polyadenylated at the E-region poly(A) site and also in a putative L2 mRNA. Three distinct species, two of which are derived from the upstream regulatory region promoter, have the potential to encode the L1 protein; the third species also contains the entire coding region of the E1i--E4 protein 5' to the L1 ORF. Both the E1i--E4 mRNA and the potentially bicistronic L1 mRNA are derived from a promoter located in the E7 ORF. We uncovered no evidence of alternatively spliced mRNAs that could account for the multiple, abundant E4 proteins in plantar warts, suggesting that posttranslational modification is mainly responsible for the observed protein heterogeneity.

Base Sequence↗
PubMed1990

Infectious cycle of human papillomavirus type 11 in human foreskin xenografts in nude mice.

We have performed the first molecular analysis of a time course of infection by a papillomavirus. The Hershey isolate of the human papillomavirus type 11 was used to infect human foreskin tissues, which were then implanted under the renal capsules of nude mice. The xenografts were recovered every 2 weeks for 14 weeks, fixed in formalin, and embedded in paraffin. Four-micrometer serial sections were examined by light microscopy for morphological changes, by immunocytochemistry for virion antigen production, and by in situ hybridization with 3H-labeled RNA probes for viral DNA replication and expression of the major mRNA species. After a lag period, probes spanning the E4 and E5 open reading frames, which are present in all E region viral mRNAs, generated the first detectable signals at week 4. Signals of other E region probes were minimally detected at week 6. Between weeks 6 and 8, there was an abrupt change in the implant such that cellular proliferation, viral DNA replication, and E and L region mRNA transcription were robust and reached a plateau. By weeks 10 to 12, the experimental condylomata were morphologically and histologically indistinguishable from naturally occurring condylomata acuminata. These findings suggest that cellular hyperproliferation and the morphologic features of condylomata are direct results of viral genetic activities. Unlike other DNA viruses, the E region transcripts increased with cell age and cellular differentiation and persisted throughout the entire experiment. In particular, the mRNA encoding the E1iE4 and perhaps E5 proteins remained overwhelmingly abundant. In contrast, viral DNA replication, L region mRNA synthesis, and virion antigen production were restricted to the most differentiated, superficial cells.

Animals↗
PubMed1989

Differentiation-linked human papillomavirus types 6 and 11 transcription in genital condylomata revealed by in situ hybridization with message-specific RNA probes.

Human papillomaviruses (HPVs) infect specific human epithelial tissues. Because viral propagation in cultured cells has not been achieved, studies of HPV genetic activities have been difficult and rely largely on analyses of patient specimens by conventional biochemical methods. HPV type 6 and type 11 infections often result in genital warts (condylomata acuminata). Structural mapping of RNAs from such warts reveals that they use alternative promoters, splice sites, and polyadenylation sites to produce complex families of overlapping mRNAs that span multiple open reading frames. Based on the mRNA structures, we have developed message-specific subgenomic clones of HPV-6 and HPV-11 in pGEM vectors. Tritium-labeled, single-stranded RNA probes were synthesized in vitro and applied to serial thin sections of patient specimens for in situ hybridization. Our results reveal the qualitative and quantitative transcription patterns of different viral messages in relationship to one another, to viral DNA replication, and to cellular differentiation. The viral "E region" is transcribed before the onset of vegetative DNA replication and continues to be expressed in increasing amounts in the maturing epithelium. Even in mature epithelia, E region messengers are far more abundant than "L region" mRNAs. The L region messages encoding capsid proteins are truly late in that they appear concomitant with or after the onset of vegetative viral DNA replication and are only present in the superficial strata of the epithelium, which contain the oldest and most differentiated keratinocytes. Abundant intron material derived from processing E region transcripts accumulates in the nuclei. Strictly nuclear signals from the L region transcripts in the midepithelium suggest that regulation of their expression is at the level of transcription elongation.

Condylomata Acuminata↗
PubMed1989

Characterization of cDNAs of spliced HPV-11 E2 mRNA and other HPV mRNAs recovered via retrovirus-mediated gene transfer.

Human papillomaviruses (HPVs) are associated with hyperproliferations of cutaneous or mucosal epithelium. These viruses cannot be propagated in any cell culture system. Because cloning cDNA copies of HPV mRNAs recovered from human lesions has met with only very limited success, the characterization of HPV mRNAs has been problematic. Using the Moloney murine leukemia virus vector system (C.L. Cepko, B.E. Roberts, and R.C. Mulligan, 1984, Cell 37, 1053-1062), we have recovered cDNAs of spliced E2 mRNAs of human papillomavirus type 11 and additional mRNAs of type 11 and type 18 and determined the utilization of open reading frames (ORFs) in the DNA sequences. The recovery of cDNA copies of messages with splice sites identical to those previously described strongly suggests that the newly characterized splice donors and acceptors are also authentic. The HPV-11 E2 cDNA contains the intact E6 and E7 ORFs and the beginning of the E1 ORF in the first exon, which is then spliced from nt 847 to the second exon at nt 2622, 100 nucleotides upstream from the initiation codon for the E2 ORF. The initiation codon in the E1 ORF is followed by four additional in-frame AUG triplets and an in-frame termination codon positioned 30 nucleotides upstream from the initiation codon for the E2 protein. The authenticity of this putative E2 cDNA was shown by its ability to provide enhancer transactivating activity in chloramphenicol acetyltransferase (CAT) assays in several cell lines. A mutation in the genomic DNA at this splice acceptor site eliminates its activity, demonstrating that the splice is essential for the expression of the E2 protein. We conclude that the translation of the HPV-11 E2 protein requires internal initiation.

Base Sequence↗