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Biomedical subjects

J T Thomas

Publications and source records attributed to J T Thomas.

At least 19 recordsLinked to original sources
PubMed2001

Cellular changes induced by low-risk human papillomavirus type 11 in keratinocytes that stably maintain viral episomes.

Infections by low-risk papillomavirus types, such as human papillomavirus (HPV) type 6 (HPV-6) and HPV-11, induce benign genital warts that rarely progress to malignancy. In contrast, lesions induced by high-risk HPV types have the potential to progress to cancer. Considerable information is available concerning the pathogenesis of high-risk HPV types, but little is known about the life cycle of low-risk HPV types. Although functionally distinct, both high- and low-risk virus types infect keratinocytes and induce virion production upon differentiation. This information suggests that they may share common mechanisms for regulating their productive life cycles. Using tissue culture methods developed to study high-risk HPV types, we examined the ability of HPV-11 to be stably maintained as episomes following transfection of normal human keratinocytes with cloned viral DNA. HPV-11 genomes were found to be maintained in keratinocytes for extended passages in cultures in 14 independent experiments involving transfection of cloned HPV-11 DNA. Interestingly, the HPV-11-positive cells exhibited an extended life span that averaged approximately twofold longer than that of control neomycin-transfected cells. In organotypic cultures, HPV-11-positive cells exhibited altered differentiation patterns, but the extent of disruption was less severe than that seen with high-risk HPV types. In addition, the amplification of HPV-11 DNA, as well as the induction of several viral messages, was observed following differentiation of transfected cells in semisolid media. To determine whether global changes in cellular gene expression induced by HPV-11 were similar to those observed with high-risk HPV-31 (Y. E. Chang and L. A. Laimins, J. Virol. 74:4174-4182, 2000), microarray analysis of 7,075 expressed sequences was performed. A spectrum of cellular genes different from that previously reported for HPV-31 was found to be activated or repressed by HPV-11. The expression of only a small set of genes was similarly altered by both high- and low-risk HPV types. This result suggests that different classes of HPVs have distinct effects on global cellular transcription patterns during infection. The methods described allow for a genetic analysis of HPV-11 in the context of its differentiation-dependent life cycle.

Cell Differentiation↗
PubMed2001

Early polyadenylation signals of human papillomavirus type 31 negatively regulate capsid gene expression.

The L1 and L2 capsid genes of human papillomavirus type 31 (HPV-31) are expressed upon keratinocyte differentiation from a promoter located in the E7 open reading frame (ORF) of the early region. Late transcripts must therefore pass through and ignore the early polyadenylation sequences to use the downstream late AAUAAA element located at the end of the L1 ORF. To identify sequences which modulate downstream capsid gene expression, a variety of substitution mutations were introduced into the early polyadenylation signal and studied first in the context of polycistronic luciferase reporter constructs. Removal of the G/U-rich cleavage stimulation factor (CstF) binding sites and the degenerate cleavage and polyadenylation specificity factor binding sites, UAUAUA, had minimal effect on downstream expression as defined by luciferase activities. This is in contrast to the deletion of the HPV-31 early AAUAAA element, which resulted in a dramatic increase in downstream expression. Additional sequences within the first 800 bp of the L2 ORF were also found to negatively regulate capsid expression in luciferase assays. To determine how these mutations influence gene expression in the context of the complete HPV-31 genome, recombinant genomes were constructed that contained a substitution in the AAUAAA sequence, an inserted strong CstF binding site, an inserted simian virus 40 (SV40) late poly(A) signal, or a substitution of the 5'-most 800 nucleotides of the L2 ORF. Reductions in both transient and stable replication were observed with the recombinant genomes containing the strong CstF site or the late SV40 signal, suggesting that alterations in the strength of the upstream poly(A) signal influence expression of viral replication factors. Similarly, disruption of the L2 ORF resulted in a significant reduction in genome replication and an inability to be maintained stably. In contrast, genomes containing a substitution of the AAUAAA sequence had increased levels of transient and stable replication. Quantitation of late transcripts following keratinocyte differentiation in methylcellulose also showed a reduction in downstream capsid gene expression in lines containing genomes with the strong CstF site or the late SV40 signal mutations, while a significant increase in expression was detected in the lines with genomes lacking the AAUAAA sequence. These studies demonstrate that capsid gene expression in HPV-31 requires an inefficient early poly(A) signal which is defined primarily by the AAUAAA element as well as a major negative regulatory element located within the L2 ORF.

Capsid↗
PubMed2001

A novel cell culture model of chondrocyte differentiation during mammalian endochondral ossification.

Endochondral ossification (EO) occurs in the growth plate where chondrocytes pass through discrete stages of proliferation, maturation, hypertrophy, and calcification. We have developed and characterized a novel bovine cell culture model of EO that mirrors these events and will facilitate in vitro studies on factors controlling chondrocyte differentiation. Chondrocytes derived from the epiphyses of long bones of fetal calves were treated with 5-azacytidine (aza-C) for 48 h. Cultures were maintained subsequently without aza-C and harvested at selected time points for analyses of growth and differentiation status. A chondrocytic phenotype associated with an extensive extracellular matrix rich in proteoglycans and collagen types II and VI was observed in aza-C-treated and -untreated cultures. aza-C-treated cultures were characterized by studying the expression of several markers of chondrocyte differentiation. Parathyroid hormone-related protein (PTHrP) and its receptor, both markers of maturation, were expressed at days 5-9. Type X collagen, which is restricted to the stage of hypertrophy, was expressed from day 11 onward. Hypertrophy was confirmed by a 14-fold increase in cell size by day 15 and an increased synthesis of alkaline phosphatase during the hypertrophic period (days 14-28). The addition of PTHrP to aza-C-treated cultures at day 14 led to the down-regulation of type X collagen by 6-fold, showing type X collagen expression is under the control of PTHrP as in vivo. These findings show that aza-C can induce fetal bovine epiphyseal chondrocytes to differentiate in culture in a manner consistent with that which occurs during the EO process in vivo.

Animals↗
PubMed1999

Human papillomavirus type 31 oncoproteins E6 and E7 are required for the maintenance of episomes during the viral life cycle in normal human keratinocytes.

The E6 and E7 oncoproteins of the high-risk human papillomavirus (HPV) types are able to immortalize human keratinocytes in vitro and likely contribute to the development of anogenital malignancies in vivo. The role of these oncoproteins in the productive viral life cycle, however, is not known. To begin to examine these possible roles, mutations in E6 were introduced in the context of the complete HPV 31 genome. Although transfected wild-type HPV 31 genomes, as well as genomes containing an E6 translation termination linker, an E6 frameshift mutation, and a point mutation in the p53 interacting domain were able to replicate in transient assays, only the wild-type genome was stably maintained as an episome. Interestingly, mutant genomes in either the E6 splice-donor site or splice-acceptor site were reduced in replication ability in transient assays; however, cotransfection of E1 and E2 expression vectors restored this function. In a similar fashion, genomes containing mutant HPV 31 E7 genes, including a translation termination mutant, two Rb-binding site mutants, a casein kinase II phosphorylation site mutant, and a transformation deficient mutant, were constructed. Although transient replication was similar to wild type in all of the E7 mutants, only the casein kinase II mutant had the ability to maintain high copies of episomal genomes. These findings suggest a role for E6 and E7 in the viral life cycle beyond their ability to extend the life span of infected cells.

Casein Kinase II↗
PubMed1999

Role of CDMP-1 in skeletal morphogenesis: promotion of mesenchymal cell recruitment and chondrocyte differentiation.

Cartilage provides the template for endochondral ossification and is crucial for determining the length and width of the skeleton. Transgenic mice with targeted expression of recombinant cartilage-derived morphogenetic protein-1 (CDMP-1), a member of the bone morphogenetic protein family, were created to investigate the role of CDMP-1 in skeletal formation. The mice exhibited chondrodysplasia with expanded cartilage, which consists of the enlarged hypertrophic zone and the reduced proliferating chondrocyte zone. Histologically, CDMP-1 increased the number of chondroprogenitor cells and accelerated chondrocyte differentiation to hypertrophy. Expression of CDMP-1 in the notochord inhibited vertebral body formation by blocking migration of sclerotome cells to the notochord. These results indicate that CDMP-1 antagonizes the ventralization signals from the notochord. Our study suggests a molecular mechanism by which CDMP-1 regulates the formation, growth, and differentiation of the skeletal elements.

Animals↗
PubMed1999

Intergenerational concepts of adolescent sexuality: implications for community-based reproductive health care with Haitian immigrants.

Intergenerational conflict occurs when immigrant parents hold different values from those of their more rapidly acculturating offspring. These conflicts frequently involve disparate views related to sex roles and reproduction. A community-based study of 19 immigrant Haitian parent-adolescent pairs in South Florida compared their attitudes and values about sexuality and reproduction. Data were obtained through focused, open-ended interviews. Content analysis procedures at the level of words and phrases facilitated the categorization of responses. Data revealed considerable differences between parents and adolescents about the sources and types of information learned about reproduction and contraception, when such information is learned, and expectations regarding premarital sexual intercourse. Both parents and adolescents lacked accurate biomedical information about contraception, placed responsibility for contraceptive use primarily on the female partner, considered reproduction a natural rather than a medical event, and believed parents have the major responsibility for educating children about reproduction and contraception. Implications for culturally-appropriate health care center on increasing the role of the public health nurse in health education, minimizing intergenerational and intercultural conflict, and engaging the Haitian immigrant community in the promotion of reproductive health.

Adolescent↗
PubMed1998

Expression pattern of two Frizzled-related genes, Frzb-1 and Sfrp-1, during mouse embryogenesis suggests a role for modulating action of Wnt family members.

Wnt proteins have been implicated in regulating growth and pattern formation in a variety of tissues during embryonic development. We previously identified Frzb-1, a gene which encodes a secreted protein with homology in the ligand binding domain to the Wnt receptor Frizzled, but lacking the domain encoding the putative seven transmembrane segments. Frzb-1 has recently been shown to bind to Wnt proteins in vitro, and to inhibit the activity of Xenopus Wnt-8 in vivo. We report now that mFrzb-1 and Wnt transcripts display both complementary and overlapping expression patterns at multiple sites throughout embryonic development. By Northern analysis, the expression of mFrzb-1 in the developing mouse embryo is greatest from 10.5 to 12.5 days postcoitum (dpc). In the early embryo, mFrzb-1 is expressed in the primitive streak, presomitic mesoderm, somites, and brain. Later, mFrzb-1 exhibits sharp boundaries of expression in the limb bud, branchial arches, facial mesenchyme, and in cartilaginous elements of the appendicular skeleton. We conclude from these experiments that Frzb-1 is expressed at a time and location to modulate the action of Wnt family members during development of the limbs and central nervous system.

Animals↗
PubMed1998

Human papillomavirus oncoproteins E6 and E7 independently abrogate the mitotic spindle checkpoint.

The E6 and E7 genes of the high-risk human papillomavirus (HPV) types encode oncoproteins, and both act by interfering with the activity of cellular tumor suppressor proteins. E7 proteins act by associating with members of the retinoblastoma family, while E6 increases the turnover of p53. p53 has been implicated as a regulator of both the G1/S cell cycle checkpoint and the mitotic spindle checkpoint. When fibroblasts from p53 knockout mice are treated with the spindle inhibitor nocodazole, a rereplication of DNA occurs without transit through mitosis. We investigated whether E6 or E7 could induce a similar loss of mitotic checkpoint activity in human keratinocytes. Recombinant retroviruses expressing high-risk E6 alone, E7 alone, and E6 in combination with E7 were used to infect normal human foreskin keratinocytes (HFKs). Established cell lines were treated with nocodazole, stained with propidium iodide, and analyzed for DNA content by flow cytometry. Cells infected with high-risk E6 were found to continue to replicate DNA and accumulated an octaploid (8N) population. Surprisingly, expression of E7 alone was also able to bypass this checkpoint. Cells expressing E7 alone exhibited increased levels of p53, while those expressing E6 had significantly reduced levels. The p53 present in the E7 cells was active, as increased levels of p21 were observed. This suggested that E7 bypassed the mitotic checkpoint by a p53-independent mechanism. The levels of MDM2, a cellular oncoprotein also implicated in control of the mitotic checkpoint, were significantly elevated in the E7 cells compared to the normal HFKs. In E6-expressing cells, the levels of MDM2 were undetectable. It is possible that abrogation of Rb function by E7 or increased expression of MDM2 contributes to the loss of mitotic spindle checkpoint control in the E7 cells. These findings suggest mechanisms by which both HPV oncoproteins contribute to genomic instability at the mitotic checkpoint.

Animals↗
PubMed1997

Disruption of human limb morphogenesis by a dominant negative mutation in CDMP1.

Chondrodysplasia Grebe type (CGT) is an autosomal recessive disorder characterized by severe limb shortening and dysmorphogenesis. We have identified a causative point mutation in the gene encoding the bone morphogenetic protein (BMP)-like molecule, cartilage-derived morphogenetic protein-1 (CDMP-1). The mutation substitutes a tyrosine for the first of seven highly conserved cysteine residues in the mature active domain of the protein. We demonstrate that the mutation results in a protein that is not secreted and is inactive in vitro. It produces a dominant negative effect by preventing the secretion of other, related BMP family members. We present evidence that this may occur through the formation of heterodimers. The mutation and its proposed mechanism of action provide the first human genetic indication that composite expression patterns of different BMPs dictate limb and digit morphogenesis.

Amino Acid Sequence↗
PubMed1996

A reversible defect in alpha-beta T cell receptor assembly.

A human leukemia cell line, SUP-T13, can gain and lose TCR/CD3 expression at rates incompatible with spontaneous mutation. In this study, we determined (i) the generality of this phenomenon among other T cell lines, (ii) the specificity of this phenomenon to the TCR/CD3 complex, and (iii) the molecular mechanism of TCR/CD3 loss in the SUP-T13 cell line at a biochemical level. We show that two other T cell lines can undergo gain and loss of TCR/CD3 expression at similar rates. However, class I MHC molecules do not switch expression on and off, demonstrating that such switching is not an artifact of the analysis. To determine the mechanism for loss of surface TCR/CD3 expression, pulse-chase labeling and immunoprecipitation were performed on SUP-T13 TCR/CD3 negative cells. These analyses revealed that TCR alpha proteins are produced, but do not covalently associate with TCR beta-CD3 proteins in the negative cells. Thus, these variants represent a novel level of posttranslational regulation of TCR/CD3 expression, namely, the disulfide linkage of alpha and beta TCR chains.

Glycosylation↗
PubMed1996

A human chondrodysplasia due to a mutation in a TGF-beta superfamily member.

The TGF-beta superfamily comprises a number of functionally diverse growth factors/signalling molecules (1) which elicit their response upon binding to serine-threonine kinase receptors (2). We recently reported the isolation and characterization of two new members of the family, designated cartilage-derived morphogenetic protein (CDMP) 1 and 2 (ref. 3) which are closely related to the sub-family of bone morphogenetic proteins. CDMP-1 is predominantly expressed at sites of skeletal morphogenesis (3), and we now show that a mutation in hCDMP-1 is associated with a recessive human chondrodysplasia (acromesomelic chondrodysplasia, Hunter-Thompson type (4,5)). The disorder, characterized by skeletal abnormalities restricted to the limbs andlimb joints, is phenotypically similar to murine brachypodism (bp) which is due to mutations in growth/differentiation factor-5 (Gdf-5) (6), the mouse homologue of hCDMP-1. Affected individuals are homozygous for a 22-bp (tandem-duplication) frameshift mutation in the mature region of CDMP-1. The resulting phenotype provides direct evidence for the involvement of CDMP-1 in human skeletal development and represents the first human disorder attributable to a mutation in a TGF-beta superfamily member.

Adolescent↗
PubMed1996

The expression of types X and VI collagen and fibrillin in rat mandibular condylar cartilage. Response to mastication forces.

Types X and VI collagen and fibrillin were localized by in situ hybridization and immunohistochemical methods in the mandibular condyles of rats, and the response of these molecules to post-weaning diets of soft food, ordinary pellets, or hardened pellets was studied. Type X collagen was synthesized, particularly in conditions of soft food consistency, by cells in the perichondrium-periosteum and in the bone and by cells at the erosion front between cartilage and bone. Type X collagen synthesis diminished under higher compression forces due to mastication and with increasing age. Type VI collagen and fibrillin were synthesized by cells in the perichondrium-periosteum and by chondrocytes and by stromal osteoblasts and were not modified by higher mechanical forces. In contrast to previous findings in the growth plate of long bones, type X collagen in the mandibular condyle was not synthesized by hypertrophic chondrocytes but was associated with cells of the osteoblastic rather than the chondroblastic phenotype.

Animals↗
PubMed1995

Sequence comparison of three mammalian type-X collagen promoters and preliminary functional analysis of the human promoter.

The mechanism(s) controlling the specific expression of the type-X collagen (COL10A1)-encoding gene in the growth plate of developing long bones is not known. In preparation for identifying and characterizing the 5'-regulatory sequences and transcription factors which control mammalian Col10a1 gene expression, we have isolated and sequenced the first exon and 5' flanking promoter regions of bovine Col10a1. Sequence comparisons, including those previously published for mouse Col10a1, highlighted a number of conserved domains within the promoter and upstream elements. Reporter cat gene (encoding chloramphenicol acetyltransferase, CAT) constructs containing 5'-regulatory sequences of human COL10a1 (hCOL10a1) were transfected into primary cultures of foetal bovine growth plate chondrocytes producing COL10A1 and non-producing epiphyseal cartilage chondrocytes. Constructs containing up to 900 bp of promoter sequence exhibited low levels of CAT production in expressing cells and non-expressing cells. Addition of a further 1.5 kb of upstream sequence resulted in a dramatic increase in CAT production in expressing cells only. The results demonstrate the presence of enhancer-like elements between 900 bp and 2.4 kb upstream of the transcription start point(s) of hCOL10a1, which is distinctly different from that reported for the chick.

Animals↗
PubMed1995

Feeding and weaning practices of Cuban and Haitian immigrant mothers.

The decline of breast feeding among immigrant mothers is of concern to transcultural nurses and other health care professionals. A descriptive survey of 30 Cuban and 30 Haitian immigrant mothers in South Florida explored their feeding and weaning beliefs and practices. Findings revealed that social, economic, and political factors in their country of origin and in South Florida affected their traditional health-culture beliefs and practices related to breast and bottle feeding and weaning, initial fluid intake, introduction of supplemental foods, and administration of vitamins, minerals, and medications. Implications for transcultural nursing care are presented.

Adult↗
PubMed1994

Cartilage-derived morphogenetic proteins. New members of the transforming growth factor-beta superfamily predominantly expressed in long bones during human embryonic development.

Partially purified extracts from newborn calf articular cartilage were found to induce cartilage and bone when subcutaneously implanted in rats. This activity showed characteristics of bone morphogenetic proteins (BMPs). Degenerate oligonucleotide primer sets derived from the highly conserved carboxyl-terminal region of the BMP family were designed and used in reverse transcription-polymerase chain reactions with poly(A)+ RNA from articular cartilage as template to determine which BMPs are produced by chondrocytes. Two novel members of the transforming growth factor-beta (TGF-beta) superfamily were identified and designated cartilage-derived morphogenetic protein-1 (CDMP-1) and -2 (CDMP-2). Their carboxyl-terminal TGF-beta domains are 82% identical, thus defining a novel subfamily most closely related to BMP-5, BMP-6, and osteogenic protein-1. Northern analyses showed that both genes are predominantly expressed in cartilaginous tissues. In situ hybridization and immunostaining of sections from human embryos showed that CDMP-1 was predominantly found at the stage of precartilaginous mesenchymal condensation and throughout the cartilaginous cores of the developing long bones, whereas CDMP-2 expression was restricted to the hypertrophic chondrocytes of ossifying long bone centers. Neither gene was detectable in the axial skeleton during human embryonic development. The cartilage-specific localization pattern of these novel TGF-beta superfamily members, which contrasts with the more ubiquitous presence of other BMP family members, suggests a potential role for these proteins in chondrocyte differentiation and growth of long bones.

Amino Acid Sequence↗