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Biomedical subjects

J P Halle

Publications and source records attributed to J P Halle.

14 recordsLinked to original sources
PubMed2001

The Ca2+-binding proteins S100A8 and S100A9 are encoded by novel injury-regulated genes.

To gain insight into the molecular mechanisms underlying cutaneous wound repair, we performed a large scale screen to identify novel injury-regulated genes. Here we show a strong up-regulation of the RNA and protein levels of the two Ca(2+)-binding proteins S100A8 and S100A9 in the hyperthickened epidermis of acute murine and human wounds and of human ulcers. Furthermore, both genes were expressed by inflammatory cells in the wound. The increased expression of S100A8 and S100A9 in wound keratinocytes is most likely related to the activated state of the keratinocytes and not secondary to the inflammation of the skin, since we also found up-regulation of S100A8 and S100A9 in the epidermis of activin-overexpressing mice, which develop a hyperproliferative and abnormally differentiated epidermis in the absence of inflammation. Furthermore, S100A8 and S100A9 expression was found to be associated with partially differentiated keratinocytes in vitro. Using confocal microscopy, both proteins were shown to be at least partially associated with the keratin cytoskeleton. In addition, cultured keratinocytes efficiently secreted the S100A8/A9 dimer. These results together with previously published data suggest that S100A8 and S100A9 are novel players in wound repair, where they might be involved in the reorganization of the keratin cytoskeleton in the wounded epidermis, in the chemoattraction of inflammatory cells, and/or in the defense against microorganisms.

Activins↗
PubMed2000

Humalog Mix25 offers better mealtime glycemic control in patients with type 1 or type 2 diabetes.

To compare the postprandial glucodynamics of Humalog Mix25, (Humalog Mix75/25 in the US; Mix25), to human insulin 30/70 (Humulin 70/30 in the US; 30/70) in patients with type 1 or type 2 diabetes. Ninety-three patients with type 1 diabetes and 84 patients with type 2 diabetes were evaluated in two separate but identical protocols using a randomized, multicenter, double-blind, crossover design. Patients consumed test meals 5 minutes after equal doses of Mix25 or 30/70. Plasma glucose was measured at baseline and 15 minute intervals for 4 hours after the meal. Two-hour postprandial glucose (2pp), 2-hour glucose excursion (2pp(ex) ), glucose versus time area under the curve 0 to 4 hours (AUC(0-4) ) and glucose excursion area under the curve 0 to 2 and 0 to 4 hours (AUCex(0-2), AUCex(0-4) ) were calculated. For the combined patient population, Mix25 resulted in significantly lower 2pp (12.45 +/- 3.59 vs. 13.47 +/- 3.62 mmol/L; p <0.001), AUC(0-4) (44.45 +/- 12.20 vs. 47.25 +/- 11.97 mmol x h/L; p <0.001), and glucose excursion parameters: 2pp(ex) (3.20 +/- 2.72 vs. 4.40 +/- 2.81 mmol/L; p <0.001), AUCex(0-2) (5.45 +/- 3.15 vs 6.60 +/- 3.13 mmol x h/L; p <0.001), and AUCex(0-4) (7.57 +/- 8.37 vs. 11.02 +/- 8.47 mmol x h/L; p <0.001) compared to 30/70. Further analysis of the treatment by type of diabetes indicated that Mix25 provided nearly identical glucose excursion responses in type 1 and type 2 diabetes up to 2 hours after the test meal, in contrast to 30/70. Pre-meal injection of Mix25 resulted in lower postprandial blood glucose levels compared to 30/70. The postprandial blood glucose response following Mix25 was similar in patients with either type 1 or type 2 diabetes.

Adult↗
PubMed1999

Conserved cAMP responsive element and core promoter complex are critical for specificity of the distal T-cell receptor beta chain enhancer for its native promoter.

The Vbeta 8.1 promoter is regulated by T-cell-specific and ubiquitous transcription factors, which bind immediately upstream of and inside the core promoter region. The various Vbeta promoters contain two conserved elements, a cAMP responsive element (CRE) located upstream of the core promoter and a basal initiator flanked by two regulatory motifs. Here we have studied the interplay between the distal enhancer and its native promoter. We show that the remote enhancer acts specifically through its native promoter. Specific enhancer-promoter interplay is mediated through the conserved regions of the Vbeta promoters. Importantly, the conserved CRE serves as a functional recognition element for the enhancer whereas it barely contributes to promoter activity. The other conserved regions surrounding the initiation site are critical for activators that bind at and function through the core promoter region and thereby regulate both promoter and enhancer activity. The enhancer is highly sensitive to E1A-12S, which represses both general and specific enhancer activities. Enhancer activity and promoter-enhancer specificity is, at least in part, mediated by the coactivators CBP/p300.

Adenovirus E1A Proteins↗
PubMed1998

Accelerated proliferative senescence of rat embryo fibroblasts after stable transfection of multiple copies of the c-Myc DNA-binding sequence.

The protooncogene c-myc positively regulates cellular proliferation whereas it exhibits negative effects on both cellular senescence and differentiation. Ectopic overexpression of c-myc in transfection experiments or titration of the c-myc mRNA by antisense oligonucleotides has demonstrated that small changes of the concentration of cellular c-myc mRNA or protein levels can be crucial for these processes. In view of the role of c-Myc as a transcription factor, most of these effects may be mediated via its binding to specific DNA sequences. Here we studied the cellular reactions after manipulating the cellular concentration of c-Myc DNA-binding sites. Multiple copies of the c-Myc-binding sequence GACCACGTGGTC or, alternatively, the control sequence GACCAGCTGGTC that displays only a poor affinity for c-Myc were stably introduced into the genome of rat embryo fibroblasts. Transfection with the c-Myc-binding sequence yielded much lower clone numbers and sizes than transfection with the control sequence. After polyclonal selection and further subcultivation cells transfected with c-Myc-binding sequence exhibited a reduced growth rate and achieved less than two-thirds of the cumulative population doublings before becoming senescent and irreversibly growth arrested compared to the controls. Southern blot analysis demonstrated that 30 binding sequences on average were integrated into the cellular genome. Our results can be interpreted as competition of the ectopically introduced c-Myc-binding sequences with the functional genomic ones and assume that a fairly low number of the latter exist in the normal cellular genome. Hence, only a low copy number of introduced c-Myc-binding sequences is sufficient to cause signs of accelerated proliferative senescence.

Animals↗
PubMed1998

Involvement of negative cofactor NC2 in active repression by zinc finger-homeodomain transcription factor AREB6.

The transcription factor AREB6 contains a homeodomain flanked by two clusters of Krüppel type C2H2 zinc fingers. AREB6 binds to the E-box consensus sequence, CACCTGT, through either the N- or the C-terminal zinc finger cluster. To gain insights into the molecular mechanism by which AREB6 activates and represses gene expression, we analyzed the domain structure of AREB6 in the context of a heterologous DNA-binding domain by transient-transfection assays. The C-terminal region spanning amino acids 1011 to 1124 was identified as a conventional acidic activation domain. The region containing amino acids 754 to 901, which was identified as a repression domain, consists of 40% hydrophobic amino acids displaying no sequence similarities to other known repression domains. This region repressed transcription in vitro in a HeLa nuclear extract but not in reconstituted transcription systems consisting of transcription factor IID (TFIID), TFIIB, TFIIE, TFIIH/F, and RNA polymerase II. The addition of recombinant negative cofactor NC2 (NC2alpha/DRAP1 and NC2beta/Dr1) to the reconstituted transcription system restored the activity of the AREB6 repression domain. We further demonstrated interactions between the AREB6 repression domain and NC2alpha in yeast two-hybrid assay. Our findings suggest a mechanism of transcriptional repression that is mediated by the general cofactor NC2.

Animals↗
PubMed1997

A conserved tissue-specific structure at a human T-cell receptor beta-chain core promoter.

The T-cell receptor (TCR) beta-chain promoters have been characterized as nonstructured basal promoters that carry a single conserved ubiquitous cyclic AMP-responsive element. Our investigation of the human TCR beta gene uncovers a surprisingly complex and tissue-specific structure at the TCR Vbeta 8.1 promoter. The core of the promoter (positions -42 to +11) is recognized by the lymphoid cell-specific transcription factors Ets-1, LEF1, and AML1 as well as by CREB/ATF-1, as is demonstrated in gel shift and footprinting experiments. With the exception of LEF1, these factors activate transcription in T cells. Binding sites at the core region show little conservation with consensus sites. Nonetheless, CREB, Ets-1, and AML1 bind and activate cooperatively and very efficiently through the nonconsensus binding sites at the core promoter region. Moderate ubiquitous activation is further induced by CREB/ATF and Sp1 factors through proximal upstream elements. The tissue-specific core promoter structure is apparently conserved in other T-cell-specifically expressed genes such as the CD4 gene. Our observations suggest that both the enhancer and the promoter have a complex tissue-specific structure whose functional interplay potentiates T-cell-specific transcription.

Base Sequence↗
PubMed1997

Copy number, epigenetic state and expression of the rRNA genes in young and senescent rat embryo fibroblasts.

The recent cloning of the gene that causes the premature aging in Werner syndrome patients has evoked speculations that deficits in expression of the ribosomal RNA genes could be related to cellular aging in general. Here we compare the state of the rRNA genes and the rRNA metabolism in young and senescent (aged) rat embryo fibroblasts (REF). Southern blot analysis revealed that the copy number and the methylation state of the genes did not change significantly with increasing cumulative population doublings (CPD) of the culture. Hence, young (low numbers of CPD) and senescent REF (high numbers of CPD), respectively, have the same repertoire of rDNA units that can be transcribed. The rRNA synthesis in these cells was analyzed by incorporation of labeled uridine at conditions allowing the measurement of absolute rather than relative rRNA synthesis rates. We revealed that the cell density dependence of the rRNA synthesis diminishes in senescent cells. Exponentially growing young REF exhibited an rRNA synthesis of 16 amol uridine incorporation per minute and cell. The rRNA synthesis decreased 10-fold in quiescent cells at saturating cell densities. Exponentially growing REF near the end of their replicative lifespan exhibited a 2-fold lower rRNA synthesis rate compared to young cells. However, in senescent REF the rRNA synthesis rate decreased only 2-fold with increasing cell densities resulting in a 3-fold higher rRNA synthesis rate compared to young cells at saturating cell densities. These data could be confirmed by calculating the rRNA synthesis rates from the rRNA content, the rRNA half-life, and the proliferation rate of the cells. Hence, senescent REF exhibited a higher rRNA synthesis rate when compared to young cells at similar growth rates resulting in the generally observed higher rRNA content (and cell size) of senescent cells. We conclude that cellular senescence of REF is not accompanied by rRNA expression deficiencies.

Animals↗
PubMed1995

Activation of transcription by recombinant upstream stimulatory factor 1 is mediated by a novel positive cofactor.

The transcription factor USF1 belongs to the family of basic helix-loop-helix proteins that are involved in the regulation of various important cellular processes. Here we characterized the factors involved in the activation of transcription by upstream stimulatory factor 1 (USF1) in a reconstituted class II gene transcription system. Activation of transcription by both wild type USF1 and a GAL-USF (amino acids 1-94 of the yeast activator protein GAL4 fused to amino acids 17-196 of USF) fusion protein required the presence of at least one positive cofactor. A novel positive cofactor (PC5) that functions specifically through the activation domain of USF1 was partially purified and biochemicaly distinguished from previously described positive cofactors. The mechanism by which PC5 mediates activation of transcription through USF1 was investigated in order-of-addition experiments. PC5 had to be present during binding of transcription factor (TF) IID to the TATA box to observe transcriptional activation. However, this event alone did not result in transcriptional activation, which also required the presence of the activator and of PC5 after binding of TFIID. Hence, PC5 may enter transcription during binding of TFIID to function in concert with the activator during subsequent steps in transcription.

DNA-Binding Proteins↗
PubMed1995

Changes of the methylation pattern of the c-myc gene during in vitro aging of IMR90 human embryonic fibroblasts.

DNA modification by cytosine methylation has received considerable interest in the context of mammalian cell differentiation but is discussed controversially with respect to cellular aging. As the expression of c-myc affects strongly cellular aging and terminal differentiation, we have analysed the sequence-specific methylation pattern of the c-myc gene during proliferative aging in vitro of human embryonic fibroblasts. In this study, both, 5-methylcytidine sensitive restriction enzymes as well as genomic sequencing were used. The overall methylation pattern was found essentially stable during proliferative aging. However, specific hypermethylation of exon II during aging was observed. Furthermore, one specific cytidine located in the consensus sequence of the DNA binding factor PEBP2 was found completely methylated during most of the course of proliferative aging of the cells but became demethylated as the cells reached the end of their proliferative life span. Our results indicate the importance of establishing the sequence-specific changes of the methylation pattern of the genome during in vitro aging.

Base Sequence↗
PubMed1994

Proliferative and metabolic capacity of rat embryo fibroblasts immortalized by c-myc depends on cellular age at oncogenic transfection.

It is well known that secondary rat embryo fibroblasts are immortalized and transformed with respect to requirements of growth factors by transfection with an overexpressed c-myc protooncogene. On the other hand, c-myc expression of nontransformed cells was shown to be independent of cellular age in vitro. In order to elucidate further the role of the c-myc protooncogene in the process of aging of rat embryo fibroblasts, we have transfected these cells at low (< or = 2) and at high (> or = 16) cumulative population doublings with SV40-promoter/enhancer-driven murine c-myc. These cells transformed young or aged, as well as their nontransformed, young and aged controls, were characterized with respect to their expression of c-myc at the mRNA and the protein level. Furthermore, we have investigated in detail their cell density-dependent growth, dependence of cell proliferation on stimulation by combinations of growth factors, and dependence of entry into cell cycle on cell size. In addition, we have measured rates of synthesis and degradation of cellular RNA and protein. The main result was that cells transformed at old age cannot be distinguished from nontransformed old cells by any of the characteristics investigated except by their immortalization. Thus, the cell lines overexpressing c-myc are immortalized and fixed in the proliferative/metabolic state achieved at the time of transfection. It is concluded that the intracellular effects of c-myc depend on the epigenetic status of the cells.

Animals↗
PubMed1993

Genomic sequencing by ligation mediated polymerase chain reaction using direct blotting and non-radioactive detection.

Genomic sequencing has become an important tool for analyzing uncloned cellular DNA with regard to the methylation status of cytidines as well as to DNA-protein interactions within cells. The hybridization step of the genomic sequencing procedure requires a very high sensitivity, rendering the method fairly difficult. Using a modified ligation mediated polymerase chain reaction procedure (LMPCR) and a sensitive non-radioactive detection method, we have developed a procedure avoiding the high amounts of radioactivity formerly needed for detection of chemically cleaved genomic DNA. The detection limit of our method of genomic sequencing is less than 1 microgram mammalian DNA, which is much better than the detection limit of the original genomic sequencing method and comparable with the detection limit of radioactive detection after the LMPCR procedure. In addition to the advantages of the non-radioactive detection technique we simplified the blotting step of the genomic sequencing procedure by using the direct blotting electrophoresis method. The method was applied to a region 5' to the human c-myc promoter of HeLa cells and was able to verify the sequence obtained by other authors and to specify the methylation status of five CpG-pairs within this sequence.

Base Sequence↗
PubMed1992

Production of alginate beads by emulsification/internal gelation. I. Methodology.

Small diameter alginate beads (microspheres) were formed via internal gelation of alginate solution emulsified within vegetable oil. Gelation was initiated by addition of an oil-soluble acid thereby reducing the pH of the alginate solution and releasing soluble Ca2+ from the citrate complex. Smooth, spherical, micron-sized beads were formed. The mean diameter ranged from 200 to 1000 microns, controlled by the reactor impeller design and rotational speed. The technique has potential for large-scale and continuous applications in immobilization.

Alginates↗
PubMed1985

Effect of plasma, serum and platelets from diabetics on DNA synthesis in cultured vascular smooth muscle cells.

Abnormalities in the regulation of proliferation of vascular smooth muscle are believed to be involved in the development of atherosclerosis. This study addresses the question of whether altered levels or activity of circulating factors in diabetes may influence the growth of vascular smooth muscle cells and fibroblasts. Plasma prepared from a group of patients with insulin-dependent diabetes mellitus was less capable of stimulating DNA synthesis in cultured vascular smooth muscle cells and human lung fibroblasts than plasma from control subjects. In contrast a platelet lysate prepared from the same patients caused significantly greater DNA synthesis than did a platelet lysate prepared from the controls. Thus both increased and decreased growth promoting activity exist in diabetes. The end result of these abnormalities may depend on the sensitivity of the target organ and platelet function, but may be related to the increased risk of atherosclerosis amongst the diabetic population.

Animals↗