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Biomedical subjects

J B Lawrence

Publications and source records attributed to J B Lawrence.

At least 37 records · Page 2Linked to original sources
PubMed1997

Identification of a nuclear matrix targeting signal in the leukemia and bone-related AML/CBF-alpha transcription factors.

Transcription factors of the AML (core binding factor-alpha/polyoma enhancer binding protein 2) class are key transactivators of tissue-specific genes of the hematopoietic and bone lineages. Alternative splicing of the AML-1 gene results in two major AML variants, AML-1 and AML-1B. We show here that the transcriptionally active AML-1B binds to the nuclear matrix, and the inactive AML-1 does not. The association of AML-1B with the nuclear matrix is independent of DNA binding and requires a nuclear matrix targeting signal (NMTS), a 31 amino acid segment near the C terminus that is distinct from nuclear localization signals. A similar NMTS is present in AML-2 and the bone-related AML-3 transcription factors. Fusion of the AML-1B NMTS to the heterologous GAL4-(1-147) protein directs GAL4 to the nuclear matrix. Thus, the NMTS is necessary and sufficient to target the transcriptionally active AML-1B to the nuclear matrix. The loss of the C-terminal domain of AML-1B is a frequent consequence of the leukemia-related t(8;21) and t(3;21) translocations. Our results suggest this loss may be functionally linked to the modified interrelationships between nuclear structure and gene expression characteristic of cancer cells.

Amino Acid Sequence↗
PubMed1997

Operant schedule transformations and human behavioral momentum.

Behavioral momentum, which can be defined as the persistence of behavior under altered environmental contingencies, is derived from Newtonian physics and operant psychology, and has relevance to behavior therapy in terms of shaping strong behaviors and ensuring effective relapse prevention strategies. The present study investigated whether changing operant schedule contingencies affects how humans respond to different stimuli when reinforcement density is systematically manipulated. Fifteen subjects participated in a computer study, in which each of two keys in a baseline condition was associated with the same schedule of reinforcement, multiple variable interval schedules, the only difference being that one reinforcer was ten times larger than the other. After six sessions, the contingency schedule changed to either an extinction condition, a variable time schedule, or a changed variable interval schedule to assess how 'subjects' responses persisted when reinforcement contingencies were systematically changed. Results of this study were found to be consistent with the general predictions of behavioral momentum. Subjects not only biased responding in favor of the more densely reinforcing key, but when contingencies changed, subjects showed continued biased responding. Implications for behavioral momentum for behavior modification and behavior therapy are discussed, and it is concluded that behavioral momentum has significant implications for designing new and comprehensive behavior change programs.

Behavior Therapy↗
PubMed1997

Evaluation of continuous infusion suramin in metastatic breast cancer: impact on plasma levels of insulin-like growth factors (IGFs) and IGF-binding proteins.

Suramin represents a new class of antitumor drugs that targets growth factor networks. In this Phase II trial, suramin was administered by continuous infusion to 10 patients with advanced breast cancer. The target level of 280 microgram/ml suramin was achieved in a median of 10 days; toxicities in this patient group were low. We monitored the insulin-like growth factor (IGF) network in these patients because of the previously defined growth-promoting role of the IGFs in breast cancer. Plasma levels of total IGF-I and total IGF-II showed variable responses to suramin with median decreases of 24 and 23%, respectively, for the 10 patients; for total IGF-I levels, this did not reach statistical significance. On the other hand, free IGF-I plasma levels were consistently and dramatically increased (over 250%) after suramin infusion. IGF-binding proteins (IGFBPs), modulators of IGF bioavailability, were also measured. Levels of IGFBP-3, the major carrier of IGFs in the circulation, were decreased 21% after suramin treatment when measured by immunoradiometric assay. However, the majority of the plasma IGFBP-3 remaining after suramin was not the intact high-affinity IGF-binding form but rather a 30-kDa fragment with markedly reduced affinity for IGF-I. IGFBP-3 protease activity was evident in the plasma of 3 of 10 patients after suramin. Measurements of plasma IGFBP-1, IGFBP-2, and IGFBP-4 revealed no significant changes in response to suramin. The dramatic increase in active free IGF-I seen after suramin raises concern and underscores the importance of measuring relevant biomarkers in clinical trials.

Adult↗
PubMed1996

Genomic analysis of a new mammalian distal-less gene: Dlx7.

We have cloned a new Dlx gene (Dlx7) from human and mouse that may represent the mammalian orthologue of the newt gene NvHBox-5. The homeodomains of these genes are highly similar to all other vertebrate Dlx genes, and regions of similarity also exist between mammalian Dlx7 and a subset of vertebrate Dlx genes downstream of the homeodomain. The sequence divergence between human and mouse Dlx7 in these regions is greater than that predicted from comparisons of other vertebrate Dlx genes, however, and there is little sequence similarity upstream of the homeodomain both between these two genes and with other Dlx genes. We present evidence for alternative splicing of mouse Dlx7 upstream of the homeodomain that may account for some of this divergence. We have mapped human DLX7 distal to the 5' end of the HOXB cluster at an estimated distance of between 1 and 2 Mb by FISH. Both the human and the mouse Dlx7 are shown to be closely linked to Dlx3 in a convergently transcribed orientation. These mapping results support the possibility that vertebrate distal-less genes have been duplicated in concert with the Hox clusters.

Amino Acid Sequence↗
PubMed1996

Structure and partial genomic sequence of the human E2F1 gene.

The E2F family of transcription factors appears to play a critical role in the transcription of certain genes required for cell cycle progression. E2F1, the first cloned member of this family, is regulated during the cell cycle at the mRNA level by changes in transcription of the E2F1 gene and at the protein level by complex formation with proteins such as the retinoblastoma gene product (pRB), cyclin A and DP1. E2F1 can override a pRB-induced G1/S block and can behave as an oncogene in certain cells. E2F1 was cloned and was found to contain seven exons. The dinucleotides at the 5' and 3' splice sites of intron 4 do not agree with consensus splice site sequences. Fluorescence in situ hybridization localized E2F1 to chromosome 20q11. Knowledge of the organization of E2F1 may facilitate identification of additional E2F family members, as well as detection of E2F1 abnormalities in human tumors.

Base Sequence↗
PubMed1996

Chromosomal assignment of human nuclear envelope protein genes LMNA, LMNB1, and LBR by fluorescence in situ hybridization.

We have used fluorescence in situ hybridization to establish precise chromosomal localizations for three human genes encoding four different nuclear envelope proteins. Lamin A/C (LMN1, HGMW-approved symbol LMNA) mapped to 1q21.2-q21.3, with a most probable gene assignment to 1q21.3; lamin B receptor (LBR) was localized to 1q42.1; and lamin B1 (LMNB1) was mapped to the interface of bands 5q23.3-q31.1. Assignments were determined by direct placement of signals relative to high-resolution DAPI or G-bands. Comparison of these results of band positions predicted from fractional length measurements to signal placement indicated that more accurate predictions are made using Francke idiograms and that measurement strategy avoids variance due to polymorphic chromosome segments.

Chromosome Banding↗
PubMed1996

XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure.

The XIST gene is implicated in X chromosome inactivation, yet the RNA contains no apparent open reading frame. An accumulation of XIST RNA is observed near its site of transcription, the inactive X chromosome (Xi). A series of molecular cytogenetic studies comparing properties of XIST RNA to other protein coding RNAs, support a critical distinction for XIST RNA; XIST does not concentrate at Xi simply because it is transcribed and processed there. Most notably, morphometric and 3-D analysis reveals that XIST RNA and Xi are coincident in 2- and 3-D space; hence, the XIST RNA essentially paints Xi. Several results indicate that the XIST RNA accumulation has two components, a minor one associated with transcription and processing, and a spliced major component, which stably associates with Xi. Upon transcriptional inhibition the major spliced component remains in the nucleus and often encircles the extra-prominent heterochromatic Barr body. The continually transcribed XIST gene and its polyadenylated RNA consistently localize to a nuclear region devoid of splicing factor/poly A RNA rich domains. XIST RNA remains with the nuclear matrix fraction after removal of chromosomal DNA. XIST RNA is released from its association with Xi during mitosis, but shows a unique highly particulate distribution. Collective results indicate that XIST RNA may be an architectural element of the interphase chromosome territory, possibly a component of nonchromatin nuclear structure that specifically associates with Xi. XIST RNA is a novel nuclear RNA which potentially provides a specific precedent for RNA involvement in nuclear structure and cis-limited gene regulation via higher-order chromatin packaging.

Cell Cycle↗
PubMed1996

X inactivation analysis and DNA methylation studies of the ubiquitin activating enzyme E1 and PCTAIRE-1 genes in human and mouse.

Previously reported data on the X inactivation status of the ubiquitin activating enzyme E1 (UBE1) gene have been contradictory, and the issue has remained unsettled. Here we present three lines of evidence that UBE1 is expressed from the inactive X chromosome and therefore escapes X inactivation. First, by RNA in situ hybridization, UBE1 RNA is detected from both the active and inactive X chromosomes in human female fibroblasts. Second, UBE1 is expressed in a large panel of somatic cell hybrids retaining inactive human X chromosomes, including two independent hybrids that did not require UBE1 expression for survival. And third, sites at the 5' end of UBE1 are unmethylated on both active and inactive X chromosomes, consistent with the gene escaping inactivation. In order to address whether other genes that escape inactivation map to the same region of the X chromosome, we have also examined the expression of genes mapping adjacent to UBE1. The gene for PCTAIRE-1 (PCTK1) maps within 5 kb of UBE1 and similarly escapes X inactivation by the somatic cell hybrid assay, whereas six other genes that are within 1 Mb of UBE1 in Xp11.23 are silenced on the inactive X chromosome. Comparative mapping studies of the homologous loci in mouse establish that Ube1-x and Pctk1 are also within close physical proximity on the murine X chromosome, and expression studies of the Pctk1 gene determine that, similar to Ube1-x, it is subject to X inactivation in mouse. Methylation of CpG residues at restriction sites at the 5' end of both genes on the murine inactive X chromosome is consistent with both genes being subject to X inactivation in mouse, in contrast to their expression status in humans.

Animals↗
PubMed1995

In situ hybridization to chromosomes stabilized in gel microdrops.

Conventional chromosome in situ hybridization procedures rely on fixation to glass slides followed by microscopic evaluation. This report describes the development of a microdrop in situ hybridization to chromosomes in suspension. Chromosomes encapsulated in gel microdrops (GMDs) composed of an agarose matrix withstood stringent hybridization and denaturation conditions. Because of the increased stability, hybridization to encapsulated chromosomes was detected by flow cytometry as well as conventional microscopy. Thus, the MISH method offers a means for chromosome hybridization without slides and may enable identification and isolation of chromosome using hybridization rather than nucleic acid binding dyes.

3T3 Cells↗
PubMed1995

Reliability of automated platelet counts: comparison with manual method and utility for prediction of clinical bleeding.

The 20 x 10(9)/L (20,000/microliters) threshold for prophylactic platelet transfusion may be unnecessarily high. The widespread use of this threshold may reflect lack of confidence in the reliability of low platelet counts. We evaluated the performance of automated platelet counts and their relation to clinical bleeding. First, we prepared serial blood dilutions with "target" platelet counts from 2 to 40 x 10(9)/L. For the 48 measurements on 2 x 10(9)/L "target" dilutions, values of 1 or 2 x 10(9)/L were obtained with the Sysmex NE-8000 analyzer (mean 1.44 x 10(9)/L; SD 0.31 x 10(9)/L). Similarly, for 5 x 10(9)/L "target" counts, automated counts were 3-6 x 10(9)/L (mean 4.42 x 10(9)/L; SD 0.18 x 10(9)/L). Similar results were observed with all other "target" levels, with coefficients of variation (CV) from 6.39% to 7.71% with 10-40 x 10(9)/L "target" values. Secondly, we compared triplicate automated and manual platelet counts on thrombocytopenic patients with platelet counts from 4-30 x 10(9)/L. The triplicate automated platelet counts differed by no more than 5 x 10(9)/L among themselves, whereas the manual counts varied by as much as 30 x 10(9)/L. Mean platelet counts: automated, 14.40 x 10(9)/L (CV 10.12%); manual, 16.48 x 10(9)/L (CV 30.39%) (P = 0.038 for counts; P < 0.001 for CV). Finally, we prospectively evaluated bleeding in thrombocytopenic patients (1,809 patient-days of observation). Univariate and multivariate logistic regression analysis revealed highly significant correlations between the automated platelet count and major and minor bleeding manifestations. Thus, automated platelet counts are highly reliable and accurately predict clinical bleeding. The use of automated analyzers should facilitate improved prophylactic platelet transfusion protocols.

Automation↗
PubMed1995

U2 and U1 snRNA gene loci associate with coiled bodies.

The coiled bodies are nuclear structures rich in a variety of nuclear and nucleolar components including snRNAs. We have investigated the possibility that coiled bodies may associate with snRNA genes and report here that there is a high degree of association between U2 and U1 genes with a subset of coiled bodies. As investigated in human HeLa cells grown in monolayer culture, about 75% of nuclei had at least one U2 gene associated with a coiled body, and 45% had at least one U1 locus associated. In another suspension-grown HeLa cell strain, 92% of cells showed associated of one or more U2 genes with coiled bodies. In contrast to the U2 and U1 gene associations, a locus closely linked to the U2 gene cluster appeared associated with a coiled body only in 10% of cells. Associated snRNA gene signals were repeatedly positioned at the edge of the coiled body. Thus, this associated was highly nonrandom and spatially precise. Our analysis revealed a much higher frequency of association for closely spaced "doublet" U2 gene signals, with over 80% of paired signals associated as opposed to 35% for single U2 signals. This finding, coupled with the fact that not all genes were associated in all cells, suggested the possibility of a cell-cycle-dependent, possibly S-phase, association. However, an analysis of S- and non-S-phase cells using BrdU incorporation or cell synchronization did not indicate an increased level of association in S-phase. These and other results suggested that a substantial fraction of paired U2 signals represented association of U2 genes on homologous chromosomes rather than only replicated DNA. Furthermore, triple label analysis showed that in a significant fraction of cells U1 and U2 genes were both associated with the same coiled body. U1 and U2 genes were closely paired in approximately 20% of cells, over 60% of which were associated with a readily identifiable coiled body. This finding raises the possibility that multiple genes of a particular class may be in association with each coiled body. Thus, the coiled body may be a dynamic structure which transiently interacts with or is formed by one or more specific genetic loci, possibly carrying out some function related to their expression.

Alleles↗
PubMed1995

Compartmentalization of specific pre-mRNA metabolism: an emerging view.

It is increasingly recognized that the mammalian interphase nucleus contains a number of non-membranous compartments in which macromolecules associated with different nuclear functions concentrate. This review focuses on the function of a major compartment consisting of domains highly enriched in pre-mRNA splicing components and poly (A) RNA, commonly identified by the splicing factor, SC-35. RNA synthesis, as judged interdomain space. However, uridine labels several types of nuclear RNA, only a fraction of which is pre-mRNA, and such studies cannot address the question of whether specific genes are transcribed in specific places. Similarly, interpretations of transcriptional inhibition studies are compromised by the global impact that inhibition has on nuclear structure and function, and by conflicting results. Localization of specific protein coding genes or RNAs circumvents these limitations. For several sequences studied thus far, a non-random relationship to SC-35 domains has been observed, with most, but not all, active genes encoding intron-containing pre-mRNAs showing a very high degree of association. In some cases this was directly demonstrated to be the site of transcription and processing. Consistent with earlier uridine incorporation studies, we have found that transcription occurs at the outer edge of the SC-35 domain, likely corresponding to the border of ultrastructures termed interchromatin granule clusters. These preliminary glimpses into gene localization strongly argue for a sequence-specific spatial association of some transcriptionally active genes with SC-35 domains, and suggest an integrated functional organization of the genome with these nuclear compartments enriched in splicing factors and poly (A) RNA.

Animals↗
PubMed1995

Sex differences in platelet adherence to subendothelium: relationship to platelet function tests and hematologic variables.

Men have significantly more atherosclerotic disease than women. Platelet-mediated thrombosis plays a role in the initiation of myocardial infarction and stroke. Citrated whole blood from male and female donors was perfused through an annular system over everted human umbilical artery segments. Comparisons were made between platelet adherence and thrombus formation on subendothelium, platelet aggregation in citrated whole blood, hematologic variables, and the bleeding time. Platelet spreading and adherence were approximately 22% greater with male blood (P < 0.001), whereas thrombus formation on subendothelium and collagen- and arachidonic acid-induced platelet aggregation did not show sex-related differences. Platelet aggregation with adenosine diphosphate was greater in women, related to their lower hematocrit values. By contrast, in women hematocrit values showed a slight but significant positive correlation with platelet adherence on subendothelium. Fibrinogen was significantly correlated with collagen- and adenosine-diphosphate-induced platelet aggregation and with platelet adherence, spreading, and thrombus formation on subendothelium. The mean bleeding time was slightly longer in women than in men (P = 0.118). Platelet aggregation was not associated with the bleeding time except for collagen-induced platelet aggregation in males; the latter was significantly correlated with platelet adherence and spreading in both sexes, while arachidonic acid-induced platelet aggregation predicted platelet adherence and spreading in males. Male blood shows enhanced primary hemostatic activity; this may predispose men to atherosclerosis.

Adult↗
PubMed1995

Whole-blood platelet aggregation predicts in vitro and in vivo primary hemostatic function in the elderly.

Increased platelet aggregation is associated with higher coronary artery disease mortality. Enhanced platelet aggregation in platelet-rich plasma has also been described in the elderly. To define age-related changes in primary hemostasis, we studied 37 elderly and 31 young blood donors. There were no significant age-related differences in whole-blood platelet aggregation, platelet adherence and thrombus formation on human umbilical artery segments, or bleeding time. Plasma fibrinogen was significantly higher in elderly men and women, whereas activated factor VII was elevated only in elderly women. Collagen-induced platelet aggregation was significantly correlated with platelet adherence to the subendothelium in elderly (r = .488, P = .002) but not in young donors. Accordingly, collagen-induced platelet aggregation showed a significant inverse correlation with bleeding time only in the elderly (r = -.401, P = .014). Arachidonic acid-induced platelet aggregation was significantly associated with platelet adherence to the subendothelium (r = .658, P = .003) and bleeding time (r = -.540, P = .021) only in elderly men. In young donors, ADP-induced platelet aggregation was significantly correlated with platelet adherence to the thrombogenic adventitial surface (r = .395, P = .031); in the elderly this association only approached significance (r = .315, P = .058). Whole-blood platelet aggregation in response to collagen and arachidonic acid may be more useful in predicting primary hemostatic function in the elderly than in the young. Furthermore, in the elderly, the correlation between platelet aggregation in whole blood and platelet-arterial wall interactions in vitro and in vivo may contribute to the ability of this test to predict coronary risk.

Adenosine Diphosphate↗
PubMed1994

E2F-4, a new member of the E2F transcription factor family, interacts with p107.

The E2F family of transcription factors has been implicated in the regulation of cell proliferation, and E2F-binding sites are present in the promoters of several growth-regulating genes. E2F family members are functionally regulated, in part, by complex formation with one or more members of the nuclear pocket protein family, RB, p107, and p130. Pocket protein regulation of E2F likely contributes to normal cellular growth control. While the three cloned species of E2F, E2F-1, E2F-2, and E2F-3, are known to be targets of RB interaction, no E2F species has yet been shown to be a specific p107 or p130 target. Here, we describe the cloning of a new member of the E2F family, E2F-4, which forms heterodimers with a member(s) of the DP family and, unlike some family members, is present throughout the cell cycle and appears to be a differentially phosphorylated p107-binding partner. p107 binding not only can be linked to the regulation of E2F-4 transcriptional activity, but also to suppression of the ability of E2F-4 to transform an immortalized rodent cell line.

Adenovirus E2 Proteins↗
PubMed1994

Molecular cloning and functional analysis of the adenovirus E1A-associated 300-kD protein (p300) reveals a protein with properties of a transcriptional adaptor.

The growth-controlling functions of the adenovirus E1A oncoprotein depend on its ability ot interact with a set of cellular proteins. Among these are the retinoblastoma protein, p107, p130, and p300. We have isolated a cDNA encoding full-length human p300 and mapped the chromosomal location of the gene to chromosome 22q13. p300 contains three cysteine- and histidine-rich regions of which the most carboxy-terminal region interacts specifically with E1A. In its center, p300 contains a bromodomain, a hallmark of certain transcriptional coactivators. We have examined the ability of p300 to overcome the repressive effect of E1A on the SV40 enhancer. We show that p300 molecules lacking an intact E1A-binding site can bypass E1A repression and restore to a significant extent the activity of the SV40 enhancer, even in the presence of high levels of E1A protein. These results imply that p300 may function as a transcriptional adaptor protein for certain complex transcriptional regulatory elements.

Adenovirus E1A Proteins↗