Biomedical subjects

M Fleischmann

Publications and source records attributed to M Fleischmann.

At least 19 recordsLinked to original sources

PubMed2006

Dynamics of the ion cyclotron resonance effect on amino acids adsorbed at the interfaces.

In this study we show a reproduction of the Zhadin experiment, which consists of the transient increase of the electrolytic current flow across an aqueous solution of L-arginine and L-glutamic acid induced by a proper low frequency alternating magnetic field superimposed to a static magnetic field of higher strength. We have identified the mechanisms that were at the origin of the so-far poor reproducibility of the above effect: the state of polarization of the electrode turned out to be a key parameter. The electrochemical investigation of the system shows that the observed phenomenon involves the transitory activation of the anode due to ion cyclotron frequency effect, followed again by anode passivation due to the adsorption of amino acid and its oxidation products. The likely occurrence of similar ion cyclotron resonance (ICR) phenomena at biological membranes, the implications on ion circulation in living matter, and the consequent biological impact of environmental magnetic fields are eventually discussed.

Adsorption↗

PubMed2002

On the "unreasonable" effects of ELF magnetic fields upon a system of ions.

A recent experiment on a physical, nonbiological system of ions at room temperature has proved that microscopic ion currents can be induced by applying simultaneously two parallel magnetic fields, one rather weak static field, (-->)B(0) and one much weaker alternating field, (-->) B(ac),[B(ac) approximately 10(-3) B(0)] whose frequency coincides with the cyclotron frequency v = qB(0)/2pim of the selected ion. As a result, ionic bursts lasting up to 20 s and with amplitude up to 10 nA arise. The much larger exchanges of energy induced by thermal agitation (the "kT-problem") appear to play no role whatsoever. We have analyzed this problem in the framework of coherent quantum electrodynamics, reaching the following conclusions: (a) as has been shown in previous articles, water molecules in the liquid and solute ions are involved in their ground state in coherent ordered configurations; (b) ions are able to move without collisions among themselves in the interstices between water coherence domains; (c) because of coherence, ions can follow classical orbits in the magnetic fields. A full quantitative understanding of the experiments is thus reached.

Electrochemistry↗

PubMed2000

Activation of protein kinase B/cAkt in hepatocytes is sufficient for the induction of expression of the gene encoding glucokinase.

Inhibitors of signalling pathways were used to dissect the mechanism of insulin action on expression of the gene encoding glucokinase in cultured rat hepatocytes. Wortmannin and LY 294002 completely prevented the insulin-induced increase in glucokinase mRNA seen in unhibited cells, indicating that the phosphoinositide 3-kinase module has a key role. A ligand inducible protein kinase B (PKB, also termed cAkt) fusion protein was expressed by using adenoviral transduction of hepatocytes in primary culture. The PKB activity of this protein was shown to be activated in transduced hepatocytes within 30 min of the addition of 4-hydroxytamoxifen and to stay high for 8 h, as a result of serine phosphorylation at position 473 of PKB. The increase in PKB activity was reflected in the hyperphosphorylation of phosphorylated, heat and acid stable regulated by insulin protein (PHAS-I; also termed 4E-BP1, for eukaryotic initiation factor 4E-binding protein 1), a protein involved in the regulation of translation initiation. These effects were comparable to the insulin-induced activation of endogenous PKB and phosphorylation of PHAS-I in non-transduced hepatocytes. The addition of tamoxifen to transduced hepatocytes resulted in an induction of glucokinase mRNA with kinetics and magnitude similar to those of insulin-induced mRNA accumulation. The effect of tamoxifen depended on stimulated PKB activity because it did not occur in hepatocytes that were transduced with a mutant PKB fusion protein that was refractory to activation with tamoxifen. These results establish that acute activation of PKB is sufficient to produce an insulin-like induction of glucokinase in isolated hepatocytes. Together with the inhibition by phosphoinositide 3-kinase inhibitors, they suggest that the activation of PKB might be critical in mediating the induction of glucokinase by insulin. In addition, experiments showed that PD98059 decreased by half the increase in glucokinase mRNA brought about by insulin, suggesting a contributory role of the mitogen-activated protein kinase cascade.

Adenoviridae↗

PubMed2000

Regulation of sterol regulatory-element binding protein 1 gene expression in liver: role of insulin and protein kinase B/cAkt.

Insulin stimulates the transcription of the sterol regulatory- element binding protein (SREBP) 1/ADD1 gene in liver. Hepatocytes in primary culture were used to delineate the insulin signalling pathway for induction of SREBP1 gene expression. The inhibitors of phosphoinositide 3-kinase (PI 3-kinase), wortmannin and LY 294002, abolished the insulin-dependent increase in SREBP1 mRNA, whereas the inhibitor of the mitogen- activated protein kinase cascade, PD 98059, was without effect. To investigate the role of protein kinase B (PKB)/cAkt downstream of PI 3-kinase, hepatocytes were transduced with an adenovirus encoding a PKB--oestrogen receptor fusion protein. The PKB activity of this recombinant protein was rapidly activated in hepatocytes challenged with 4-hydroxytamoxifen (OHT), as was endogenous PKB in hepatocytes challenged with insulin. The addition of OHT to transduced hepatocytes resulted in accumulation of SREBP1 mRNA, with a time-course and magnitude similar to the effect of insulin in non-transduced cells. The level of SREBP1 mRNA was not increased by OHT in hepatocytes expressing a mutant form of the recombinant protein whose PKB activity was not activated by OHT. Thus acute activation of PKB is sufficient to induce SREBP1 mRNA accumulation in primary hepatocytes, and might be the major signalling event by which insulin induces SREBP1 gene expression in the liver.

Adenoviridae↗

PubMed2000

Rapid and sensitive detection of enhanced green fluorescent protein expression in paraffin sections by confocal laser scanning microscopy.

Various forms of green fluorescent protein (GFP) have become important reporters of gene transfer and expression after transfection or infection of cells in cell culture. Frequently, molecular biological assays (Northern blots, PCR) are applied to detect reporter gene expression in target organs. However, these methods are not suitable for evaluation of tissue- or cell-specific expression which would be of great interest especially in case of using tissue-specific promoters. Therefore, organs of transgenic mice with the enhanced green fluorescent protein (EGFP) gene under control of the cytomegalovirus (CMV) promoter were processed for histology by formaldehyde fixation and embedding in paraffin. Sections were deparaffinized, mounted and evaluated for fluorescence in a confocal laser scanning microscope. This method combines the advantages of direct exploitation of tissue sections without further staining procedures with evaluable tissue-, cell-, and even subcellular-specific distribution patterns of EGFP expression in tissues. Results obtained by direct evaluation of EGFP fluorescence in paraffin sections were confirmed by immunohistochemical staining with anti-EGFP. In the present report, we demonstrate that application of confocal microscopy on routinely processed histological preparations is very suitable for determining gene transfer efficiency and promotor activities.

Animals↗

PubMed1999

Human nerve growth factor beta (hNGF-beta): mammary gland specific expression and production in transgenic rabbits.

Transgenic rabbits carrying gene constructs encoding human nerve growth factor beta (hNGF-beta) cDNA were generated. Expression of hNGF-beta mRNA was restricted to the mammary gland of lactating rabbits. Western Blot analysis revealed a polypeptide of 13.2 kDa in the milk of transgenic animals. hNGF-beta was purified from the milk by a two-step chromatographic procedure. Electrospray mass spectroscopy analysis of purified hNGF-beta depicted a molecular weight of 13,261 Da per subunit. The biological activity of the hNGF-beta was tested using PC12W2 cells and cultures of dorsal root ganglion neurons from chicken embryos. Crude defatted milk from transgenic animals and purified hNGF-beta demonstrated full biological activity when compared to commercial recombinant hNGF-beta.

Animals↗

PubMed1999

The 2.3 kb smooth muscle myosin heavy chain promoter directs gene expression into the vascular system of transgenic mice and rabbits.

BACKGROUND: Smooth muscle cells (SMC) are a preferential target for gene therapeutic approaches in atherosclerosis and restenosis. However, the undesirable expression of putative therapeutic genes in tissues other than the vascular wall is a considerable safety limitation for clinical trials, thus requiring the identification of a smooth-muscle-specific promoter sequence. Since the 2.3 kb rabbit Smooth Muscle Myosin Heavy Chain (SMHC) promoter was shown to be transcriptionally active in primary vascular but not visceral or other non-SMC in vitro, this fragment was chosen for in vivo analysis. METHODS AND RESULTS: Transgenic mice and rabbits were established expressing a luciferase reporter gene under control of the 2.3 kb rabbit SMHC promoter. In contrast to the endogenous expression pattern of the SMHC gene both species revealed light emission predominantly in the arterial system including coronary arteries. Low activities were measured in large veins and the gastrointestinal system. In situ hybridization of murine embryos using a luciferase riboprobe confirmed reporter gene expression in large arteries with no detectable mRNA in the viscera. Unlike adult animals, ectopic luciferase activities were found in ventricular myocardium during murine development ceasing 1 week post partum. CONCLUSIONS: In two animal species, the 2.3 kb SMHC promoter appeared to be effective in discriminating between the pathways regulating vascular and visceral smooth muscle gene expression. The vascular-specific expression profile of the 2.3 kb SMHC promoter suggests that the 2.3 kb SMHC promoter contains the regulatory elements necessary for selective gene targeting into vascular SMC of large arteries including coronary arteries in vivo.

Animals↗

PubMed1999

Stable long-term germ-line transmission of transgene integration sites in mice.

Transgenic mice provide a valuable tool in all fields of basic and applied biological and medical research. In this study, we describe the fate of integrated transgenes in the mammalian host genome over a large number of generations. The stability of the germ-line transmission of integrated tyrosinase transgene copies was monitored up to generation F20 in a large number of individuals from seven transgenic mouse lines. Phenotypic and molecular genetic analysis of the offspring both within the different lines and in cross-breeding experiments revealed the high stability of the transgene integration sites in mice. Only very few individuals were affected by a transgene copy loss. These results indicate that, once homozygous transgenic lines are established, breeding programs can be continued to a high number of generations without further stringent molecular genetic analysis.

Animals↗

PubMed1999

Identification of cis-acting RNA leader elements required for chloroplast psbD gene expression in Chlamydomonas.

The psbD mRNA of Chlamydomonas reinhardtii is one of the most abundant chloroplast transcripts and encodes the photosystem II reaction center polypeptide D2. This RNA exists in two forms with 5' untranslated regions of 74 and 47 nucleotides. The shorter form, which is associated with polysomes, is likely to result from processing of the larger RNA. Using site-directed mutagenesis and biolistic transformation, we have identified two major RNA stability determinants within the first 12 nucleotides at the 5' end and near position -30 relative to the AUG initiation codon of psbD. Insertion of a polyguanosine tract at position -60 did not appreciably interfere with translation of psbD mRNA. The same poly(G) insertion in the nac2-26 mutant, which is known to be deficient in psbD mRNA accumulation, stabilized the psbD RNA. However, the shorter psbD RNA did not accumulate, and the other psbD RNAs were not translated. Two other elements were found to affect translation but not RNA stability. The first comprises a highly U-rich sequence (positions -20 to -15), and the second, called PRB1 (positions -14 to -11), is complementary to the 3' end of the 16S rRNA. Changing the PRB1 sequence from GGAG to AAAG had no detectable effect on psbD mRNA translation. However, changing this sequence to CCUC led to a fourfold diminished rate of D2 synthesis and accumulation. When the psbD initiation codon was changed to AUA or AUU, D2 synthesis was no longer detected, and psbD RNA accumulated to wild-type levels. The singular organization of the psbD 5' untranslated region could play an important role in the control of initiation of psbD mRNA translation.

5' Untranslated Regions↗

PubMed1998

Cardiac specific expression of the green fluorescent protein during early murine embryonic development.

We demonstrate the establishment of transgenic mice, where the expression of the green fluorescent protein (GFP) is under control of the human cardiac alpha-actin promoter. These mice display cardiac specific GFP expression already during early embryonic development. Prominent GFP fluorescence was observed at the earliest stage of the murine heart anlage (E8). Cardiomyocytes of different developmental stages proved GFP positive, but the intensity varied between cells. We further show that contractions of single GFP positive cardiomyocytes can be monitored within the intact embryo. At later stages of embryonic development, the skeletal musculature was also GFP positive, in line with the known expression pattern of cardiac alpha-actin. The tissue specific labeling of organs is a powerful new tool for embryological as well as functional investigations in vivo.

Actins↗

PubMed1997

The expression of the C. elegans labial-like Hox gene ceh-13 during early embryogenesis relies on cell fate and on anteroposterior cell polarity.

Clusters of homeobox-containing HOM-C/hox genes determine the morphology of animal body plans and body parts and are thought to mediate positional information. Here, we describe the onset of embryonic expression of ceh-13, the Caenorhabditis elegans orthologue of the Drosophila labial gene, which is the earliest gene of the C. elegans Hox gene cluster to be activated in C. elegans development. At the beginning of gastrulation, ceh-13 is asymmetrically expressed in posterior daughters of anteroposterior divisions, first in the posterior daughter of the intestinal precursor cell E and then in all posterior daughters of the AB descendants ABxxx. In this paper, we present evidence that supports position-independent activation of ceh-13 during early C. elegans embryogenesis, which integrates cell fate determinants and cell polarity cues. Our findings imply that mechanisms other than cell-extrinsic anteroposterior positional signals play an important role in the activation and regulation of the C. elegans Hox gene ceh-13.

Animals↗

PubMed1996

Allele-specific suppression of a Saccharomyces cerevisiae prp20 mutation by overexpression of a nuclear serine/threonine protein kinase.

The yeast PRP20 protein is homologous to the RCC1 protein of higher eukaryotes and is required for mRNA export and maintenance of nuclear structure. RCC1/PRP20 act as guanine nucleotide exchange factors for the nuclear Ras-like Ran/GSP1 proteins. In a search for prp20-10 allele-specific high-copy-number suppressors, the KSP1 locus, encoding a serine/threonine protein kinase was isolated. Ksp1p is a nuclear protein that is not essential for vegetative growth of yeast. Inactivation of the kinase activity by a mutation affecting the catalytic center of the Ksp1p eliminated the suppressing activity. Based on the isolation of a protein kinase as a high-copy-number suppressor, the phosphorylation of Prp20p was examined. In vivo labeling experiments showed that Prp20p is a phosphoprotein; however, deletion of the KSP1 kinase did not affect Prp20p phosphorylation.

Alleles↗

PubMed1996

Long-term interferon-alpha therapy induces autoantibodies against epidermis.

BACKGROUND: While interferon (IFN) is known for its immunoregulatory properties, it has also been shown to induce autoimmune disorders such as lupus erythematosus, hypothyroidism, antiphospholipid syndrome and, recently, bullous pemphigus-like eruptions. OBJECTIVE: The purpose of this study was to determine the percentage of antibodies against epidermis induced by IFN-alpha therapy, as detected by indirect immunofluorescence and Western blotting (WB). METHOD: We have studied the sera of 47 patients treated with low doses of IFN-alpha 2a for malignant melanoma or cutaneous T cell lymphoma for a period of 12 months. These sera were tested by standard IIF and WB. RESULTS: 32% of patient sera were positive after 6 months of treatment. Antibodies against epidermis were most often of the pemphigus type as confirmed by WB. Two types of labeling were noted for anti-basement membrane antibodies: basal cytoplasmic pattern in 4 sera and along the dermal side of the basal keratinocytes in 2 sera. CONCLUSION: This study brings biological arguments to confirm the direct role of IFN-alpha in the development of pemphigus-like eruption, and emphasizes the need for clinical follow-up of patients treated with IFN-alpha over a long period of time.

Adult↗

PubMed1994

Site-directed mutagenesis of the yeast PRP20/SRM1 gene reveals distinct activity domains in the protein product.

Prp20/Srm1, a homolog of the mammalian protein RCC1 in Saccharomyces cerevisiae, binds to double-stranded DNA (dsDNA) through a multicomponent complex in vitro. This dsDNA-binding capability of the Prp20 complex has been shown to be cell-cycle dependent; affinity for dsDNA is lost during DNA replication. By analyzing a number of temperature sensitive (ts) prp20 alleles produced in vivo and in vitro, as well as site-directed mutations in highly conserved positions in the imperfect repeats that make up the protein, we have determined a relationship between the residues at these positions, cell viability, and the dsDNA-binding abilities of the Prp20 complex. These data reveal that the essential residues for Prp20 function are located mainly in the second and the third repeats at the amino-terminus and the last two repeats, the seventh and eighth, at the carboxyl-terminus of Prp20. Carboxyl-terminal mutations in Prp20 differ from amino-terminal mutations in showing loss of dsDNA binding: their conditional lethal phenotype and the loss of dsDNA binding affinity are both suppressible by overproduction of Gsp1, a GTP-binding constituent of the Prp20 complex, homologous to the mammalian protein TC4/Ran. Although wild-type Prp20 does not bind to dsDNA on its own, two mutations in conserved residues were found that caused the isolated protein to bind dsDNA. These data imply that, in situ, the other components of the Prp20 complex regulate the conformation of Prp20 and thus its affinity for dsDNA. Gsp1 not only influences the dsDNA-binding ability of Prp20 but it also regulates other essential function(s) of the Prp20 complex. Overproduction of Gsp1 also suppresses the lethality of two conditional mutations in the penultimate carboxyl-terminal repeat of Prp20, even though these mutations do not eliminate the dsDNA binding activity of the Prp20 complex. Other site-directed mutants reveal that internal and carboxyl-terminal regions of Prp20 that lack homology to RCC1 are dispensable for dsDNA binding and growth.

Alleles↗

PubMed1994

Presence of Epstein-Barr virus in cutaneous lesions of mycosis fungoides and Sézary syndrome.

It has been suggested that prolonged antigenic stimulation contributes to the development of epidermotropic cutaneous T cell lymphoma (CTCL), mycosis fungoides and Sézary syndrome, characterized by a cutaneous infiltration of proliferating helper T cells. Since Epstein-Barr virus (EBV) antibodies were increased in CTCL sera, we investigated a possible etiologic role for EBV in epidermotropic CTCL by looking for the EBV genome in 25 cutaneous biopsies of mycosis fungoides or Sézary syndrome and 12 reactional inflammatory skin lesions. The use of a non-isotopic in situ hybridization procedure based on the detection of Epstein-Barr encoded RNAs with biotinylated oligonucleotide probes (EBER) revealed 32% of the lesions with CTCL to be positive for EBV (3 in dermis, 3 in epidermis, 2 both in dermis and epidermis), as compared to no detection of the EBV genome in the reactional inflammatory skin lesions. Moreover, a combined hybridization (EBER probe) and immunochemistry technique (anti-CD3 or anti-Kil monoclonal antibody) permitted the identification of EBV in T cells of dermis and in keratinocytes, respectively. The identification of EBV in epidermotropic CTCL suggests that this virus could play a role in the development of these CTCLs, either as an etiological agent or more probably as a chronic activating agent. Indeed, the infection of keratinocytes by EBV could activate them and so induce the production of in situ cytokines (IL1a, IL6, TNFa) playing a role in the development of tumoral infiltrate.

Adult↗

PubMed1993

Nuclear PRP20 protein is required for mRNA export.

The yeast PRP20 protein is highly homologous in structure and function to the RCC1 protein of higher eukaryotes. The RCC1 protein is involved in the regulation of the onset of mitosis, whereas the PRP20 protein was shown to be required for accurate and efficient mRNA metabolism. The first observable phenotype in mutant prp20 cells when shifted from permissive to non-permissive temperature is a loss of nuclear PRP20 protein. Concomitantly, an accumulation of poly(A)+ RNA in the nucleus is observed. The temperature-sensitive RCC1 allele in the mutant hamster cell line tsBN2 leads to a similar accumulation of mRNA in the nucleus.

Alleles↗

PubMed1993

In vitro induction of basal keratinocyte MY7 antigen expression in cutaneous T-cell lymphoma is associated with response to interferon-alfa therapy.

BACKGROUND: Normal basal cell keratinocytes express an antigen recognized by monoclonal antibody MY7. This expression is lost in cutaneous T-cell lymphoma (CTCL) and may be reinduced under interferon-alfa-2a therapy. We investigated whether similar modulation of MY7 antigen could be obtained in vitro and determined the relationship between in vitro modulation and clinical response. SUBJECTS: We studied MY7 expression by basal cell keratinocytes in reconstituted skin specimens from 10 patients with CTCL and from skin specimens from five control patients and determined its modulation by interferon-alfa-2a and interleukin 1 using the indirect immunofluorescence technique. Concurrently, clinical examination and in vivo immunologic study on cutaneous biopsy specimens were carried out for these 10 patients before and while receiving interferon-alfa-2a therapy. RESULTS: In vitro studies showed that in five control specimens MY7 expression by basal cells was constant without modulation by interferon-alfa-2a or interleukin 1. Two of 10 CTCL specimens spontaneously expressed MY7 antigen while an additional five did so after incubation with interferon-alfa-2a and the last three never did. CONCLUSION: The three patients with CTCL for whom no MY7 expression was observed in reconstituted skin studies were "poor responders" to interferon-alfa-2a therapy. The five patients with CTCL for whom in vitro MY7 expression was induced by interferon-alfa-2a were responders. For the last two patients, results were variable. Thus, in vitro MY7 antigen is expressed in normal basal cell keratinocytes, absent in CTCL basal cell keratinocytes, but can be induced by interferon-alfa-2a. Moreover this in vitro modulation appears to be possibly correlated with interferon-alfa efficacy in vivo.

Antigens, CD↗

PubMed1993

[Blue rubber bleb nevus].

Blue Rubber Bleb Naevus (BRBN) is characterized by the presence of bluish haemangiomas on the skin and in the digestive tract. We report a new familial case of this rare entity. Analysis of this female patient's pedigree confirmed that BRBN is transmitted as an autosomal dominant trait. In this particular case gastrointestinal haemangiomas appeared over a 4-year period. Although all treatments of this disease have proved disappointing on the skin as well as in the digestive tract, we insist on the need for a regular and prolonged surveillance.

Adult↗