Showing papers by "University of Marburg published in 1993"
2,550 citations
TL;DR: Exendin-4 is an agonist and exendin-(9-39)-amide is a specific GLP-1 receptor antagonist, and both peptides stimulated the proinsulin gene expression at the level of transcription and reduced the effects of cAMP.
940 citations
TL;DR: It is reported here that regenerated tobacco plants containing stilbene synthase genes are more resistant to infection by Botrytis cinerea, the first report of increased disease resistance in transgenic plants based on an additional foreign phytoalexin.
Abstract: Although phytoalexins have long been inferred to be important in the defence of plants against fungal infection, there are few reports showing that they provide resistance to infection. Several plants, including grapevine, synthesize the stilbene-type phytoalexin resveratrol when attacked by pathogens. Stilbenes with fungicidal potential are formed in several unrelated plant species, such as peanut (Arachis hypogaea), grapevine (Vitis vinifera) and pine (Pinus sylvestris). Stilbene biosynthesis only specifically requires the presence of stilbene synthase. Furthermore, the precursor molecules for the formation of hydroxy-stilbenes are malonyl-CoA and p-coumaroyl-CoA, both present in plants. To investigate the potential of stilbene biosynthetic genes in a strategy of engineering pathogen resistance, we isolated stilbene synthase genes from grapevine, where they are expressed at a high level, and transferred them into tobacco. We report here that regenerated tobacco plants containing these genes are more resistant to infection by Botrytis cinerea. This is, to our knowledge, the first report of increased disease resistance in transgenic plants based on an additional foreign phytoalexin.
670 citations
TL;DR: In this review, the various genetic mechanisms leading to mosaicism and the resulting cutaneous patterns are considered and the concept of cutaneous mosaicism is important for gene mapping because here the authors have the opportunity to study two populations of cells differing only with regard to the mutation causing mosaicism.
Abstract: Background: The skin is especially suitable for the study of mosaicism. In this review, the various genetic mechanisms leading to mosaicism and the resulting cutaneous patterns are considered. Observations: Mosaicism may produce different cutaneous patterns such as the lines of Blaschko, the checkerboard pattern, the phylloid pattern, and a patchy pattern without midline separation. A unique lateralization pattern is observed in the CHILD syndrome. Two major genetic categories are functional mosaics resulting from X inactivation and genomic mosaics caused by autosomal mutations. Functional mosaicism may be caused by either male-lethal or nonlethal X-linked mutations. Similarly, autosomal mutations resulting in genomic mosaicism may be either lethal or nonlethal. Many mosaics are caused by loss of heterozygosity, and uncommonly this mechanism may give rise to twin spots such as vascular twin nevi. Some cutaneous mosaic phenotypes virtually always occur sporadically, but exceptionally may show a familial aggregation. This paradox may be explained by paradominant inheritance. Heterozygous individuals are, as a rule, unaffected, but they express the birthmark when allelic loss occurs during embryogenesis. Conclusions: The concept of cutaneous mosaicism is important for gene mapping because here we have the opportunity to study two populations of cells differing only with regard to the mutation causing mosaicism. Future research will probably show that a specific genetic anomaly, when present as a mosaic, always produces the same type of cutaneous pattern. ( Arch Dermatol. 1993;129:1460-1470)
475 citations
TL;DR: Ultrafast relaxation processes in poly(p-phenylenevinylene) and its oligomers are investigated using femtosecond luminescence spectroscopy and a quasi-instantaneous luminecence rise and the absence of luminescent near the excitation energy indicate very rapid vibronic relaxation.
Abstract: Ultrafast relaxation processes in poly(p-phenylenevinylene) and its oligomers are investigated using femtosecond luminescence spectroscopy. A quasi-instantaneous luminescence rise and the absence of luminescence near the excitation energy indicate very rapid vibronic relaxation. The subsequent transient redshift of the spectra is attributed to ultrafast energy relaxation of optical excitations within an inhomogeneously broadened density of states.
365 citations
TL;DR: MetalStent insertion for palliation of hilar malignancies does not only offer higher success rates and higher patency rates compared to plastic stent insertion, but is also cost-effective since patients require fewer re-interventions.
Abstract: This prospective and randomized trial sought to compare large-bore plastic endoprostheses (14 French) and self-expanding metal stents (24 French) in the palliative treatment of obstructive jaundice due to biliary hilar malignancies. Twenty patients with Type II-IV (Bismuth classification) hilar obstruction were randomized to treatment with either plastic or metal stents. Both treatment groups were well matched with regard to all assessed clinical criteria before stenting. Stent placement was uniformly successful in the metal group and in 88.9% of the plastic group. Early stent failure ( 30 days), stent failure was observed in 50% of the plastic group and 18.2% of the metal stent group. All differences were not statistically significant. The number of re-interventions required to manage stent-related problems proved to be significantly higher in the plastic group (2.4 +/- 2.6) compared to the metal group (0.4 +/- 0.5). Hospitalization for treatment of stent complications was also significantly higher in the plastic treatment group. The costs calculated for stents and hospital stay for required re-interventions were therefore higher in the plastic stent group. In conclusion, metal stent insertion for palliation of hilar malignancies does not only offer higher success rates and higher patency rates compared to plastic stent insertion, but is also cost-effective since patients require fewer re-interventions.
316 citations
TL;DR: Elevated aldosterone levels play an important role in the adverse remodeling of the myocardium in arterial hypertension and the antifibrotic effects of spironolactone, if confirmed in human studies, may be a valuable strategy in treating hypertensive heart disease.
Abstract: Activation of the renin-angiotensin-aldosterone system in arterial hypertension can lead to remodeling of the myocardial collagen network, with progressive collagen accumulation in the cardiac interstitium. This reactive myocardial fibrosis, which is not secondary to myocyte necrosis, appears to be an important determinant of diastolic dysfunction and thus of pathologic hypertrophy. To examine the effects of the aldosterone antagonist spironolactone on myocardial fibrosis, we analyzed interstitial fibrosis in 7 different models of arterial hypertension in rats: 2 kidney, 1 clip model of renovascular hypertension (RHT); continuous subcutaneous aldosterone (0.75 micrograms/hr) infusion; RHT and aldosterone models treated with 20 mg/kg per day of subcutaneous spironolactone; uninephrectomized rats on a high sodium diet; and age- and sex-matched controls with or without spironolactone treatment. Systolic arterial pressure was comparably elevated in RHT and aldosterone models; it was modestly lowered but not normalized by 8 weeks of spironolactone treatment at the low doses used. Such treatment also failed to prevent left ventricular hypertrophy (LVH) in all experimental groups with hypertension. Spironolactone, however, was able to prevent myocardial fibrosis in RHT and aldosterone models of acquired arterial hypertension irrespective of the development of LVH and the presence of hypertension. These findings provide further evidence that elevated aldosterone levels play an important role in the adverse remodeling of the myocardium in arterial hypertension. The antifibrotic effects of spironolactone, if confirmed in human studies, may be a valuable strategy in treating hypertensive heart disease.
267 citations
TL;DR: High amplitude synchronized oscillation is recorded at the level of spike activity and local field potential from the primary visual cortex of an awake monkey and the dominant frequencies were considerably higher than those observed previously in cats and monkeys.
Abstract: IT has been proposed that synchronized oscillations play a key role in perceptual feature linking and sensory integration. This idea was supported by the discovery of strongly synchronized stimulus-specific oscillations in the visual cortex of anaesthetized cats. The ‘synchronization hypothesis’ was
255 citations
TL;DR: The results indicate that in human fetal tissues the paternal H19 allele is inactive and this work provides the first direct evidence for differential imprinting in the human and murine genome.
Abstract: In mouse, four genes have been found to undergo genomic imprinting resulting in differential expression of maternally and paternally inherited alleles. To determine whether the cognate genes are also subject to imprinting in humans, we have studied allele-specific expression patterns of insulin-like growth factor 2, IGF2-receptor and H19 in human fetal and adult tissues. In keeping with previous findings in mice, our results indicate that in human fetal tissues the paternal H19 alleles is inactive. IGF2 is monoallelically expressed in various tissues but surprisingly not in adult human liver. The human IGF2R gene, another classic example of imprinting in mice, was found to be expressed from both alleles. We provide the first direct evidence for differential imprinting in the human and murine genome.
236 citations
TL;DR: The present in vitro and in vivo data suggest that TGF-β1 has the capacity to diminish the deleterious consequences of an excitotoxic or ischemic insult.
Abstract: Transforming growth factor-β1 (TGF-β1) has been shown to be an injury-related peptide growth factor within the mammalian central nervous system. We tested whether TGF-β1 has the capacity to protect rat neocortical neurons against excitotoxic damage in vitro and mouse neocortex against ischemic injury in vivo. After 14 days in vitro, cultured neurons from rat cerebral cortex were exposed to 1 mM l-glutamate in serum-free culture medium. The cultures received TGF-β1 immediately after the addition of glutamate. Eighteen hours later, the cell viability of the cultures was determined using trypan blue exclusion. TGF-β1 (1–10 ng/ml) significantly reduced the excitotoxic neuronal damage in a concentration-dependent manner. In vivo, male NMRI mice were subjected to a permanent occlusion of the left middle cerebral artery by microbipolar electrocoagulation. After 48 h, the animals received a transcardiac injection of carbon black. The area of ischemia (devoid of carbon) was restricted to the neocortex and its size...
222 citations
TL;DR: The three-dimensional structure of CspB, the major cold-shock proteins of Escherichia coli and B. subtilis, is determined by nuclear magnetic resonance spectroscopy and resembles that of staphylococcal nuclease and the gene-5 single-stranded-DNA-binding protein.
Abstract: The cold-shock domain (CSD) is found in many eukaryotic transcriptional factors and is responsible for the specific binding to DNA of a cis-element called the Y-box. The same domain exists in the sequence of the Xenopus RNA-binding proteins FRG Y1 and FRG Y2 (refs 1, 3). The major cold-shock proteins of Escherichia coli (CS7.4) and B. subtilis (CspB) have sequences that are more than 40 per cent identical to the cold-shock domain. We present here the three-dimensional structure of CspB determined by nuclear magnetic resonance spectroscopy. The 67-residue protein consists of an antiparallel five-stranded beta-barrel with strands connected by turns and loops. The structure resembles that of staphylococcal nuclease and the gene-5 single-stranded-DNA-binding protein. A three-stranded beta-sheet, which contains the conserved RNA-binding motif RNP1 as well as a motif similar to RNP2 in two neighbouring antiparallel beta-strands, has basic and aromatic residues at its surface which could serve as a binding site for single-stranded DNA. CspB binds to single-stranded DNA in gel retardation experiments.
TL;DR: The results show that gabexate mesilate was not effective in preventing complications and mortality in acute pancreatitis, and the effect of high doses of the low molecular weight protease inhibitor gabExate Mesilate on mortality and complications associated with moderate and severe acute Pancreatitis.
Journal Article•
TL;DR: Data suggest that LHRH analogues can directly inhibit the in vitro proliferation of human ovarian cancer cells and that this effect might be mediated through the high affinity L HRH binding sites.
Abstract: Recently, specific binding sites for luteinizing hormone releasing hormone (LHRH) and its analogues have been demonstrated in biopsy samples of human epithelial ovarian cancer. Their biological significance remained obscure. In this study we ascertained whether such LHRH-binding sites are also present in the human epithelial ovarian cancer cell lines EFO-21 and EFO-27 and if they could mediate antiproliferative effects of LHRH analogues. Using [125I, D-Trp6]LHRH, a high affinity/low capacity binding site was detected in both lines: EFO-21 (Kd1 = 1.5 x 10(-9) M; binding capacity (Bmax1) = 4.9 fmol/10(6) cells) and EFO-27 (Kd1 = 1.7 x 10(-9) M; Bmax1 = 3 fmol/10(6) cells). In addition, a second class of low affinity/high capacity binding sites (EFO-21: Kd2 = 7.5 x 10(-6) M; Bmax2 = 24 pmol/10(6) cells; EFO-27: Kd2 = 4.3 x 10(-6) M; Bmax2 = 14.5 pmol/10(6) cells) was demonstrated. Specific binding of [125I, D-Trp6]LHRH was displaced with nearly equal efficiency by unlabeled [D-Trp6]LHRH, the LHRH-antagonists SB-75 and Hoe-013, and by native LHRH but not by unrelated peptides such as oxytocin and somatostatin. In the presence of 10(-5) M agonist [D-Trp6]LHRH, the proliferation of both cell lines was significantly reduced to 77% of controls after 24 h and to approx. 60% after 6 days. Lower concentrations (10(-9) M) of the agonist, significantly decreased the proliferation to 87.5% for EFO-21 and 86% for EFO-27 after 6 days. These antiproliferative effects were enhanced by increasing doses of [D-Trp6]LHRH and were maximal at 10(-5) M (EFO-21: 65.5% of control, EFO-27: 68% of control). Similar dose-dependent antiproliferative effects were obtained in EFO-21 line with the LHRH-antagonists SB-75 and Hoe-013, while these analogues had no effects on the proliferation of EFO-27 cells. SB-75 partly antagonized the antiproliferative effect of [D-Trp6]LHRH in a dose dependent way in the EFO-27 line. These data suggest that LHRH analogues can directly inhibit the in vitro proliferation of human ovarian cancer cells. This effect might be mediated through the high affinity LHRH binding sites.
TL;DR: Antiproliferative effects are enhanced by increasing the doses of triptorelin and were maximal in this series of experiments at 10(-5) mol/L, the proliferation in the HEC-1A line being 62 +/- 1% and in the Ishikawa line 52 +/- 2% of controls, respectively.
Abstract: Although specific binding sites for LH-releasing hormone (LHRH) and its analogs have been demonstrated in biopsy samples of human endometrial cancer, their biological significance remains obscure. In this study we evaluated whether binding sites for LHRH are also present in the human endometrial cancer cell lines HEC-1A and Ishikawa and if such sites could mediate antiproliferative effects of LHRH analogs. Using [125I,D-Trp6]LHRH as a ligand, a high affinity/low capacity binding site was detected in both lines: HEC-1A line, dissociation constant (Kd)1 = 5.7 x 10(-9) mol/L, binding capacity (Bmax)1 = 78 fmol/10(6) cells; Ishikawa line, Kd1 = 4.2 x 10(-9) mol/L, Bmax1 = 29 fmol/10(6) cells. In addition, a second class of low affinity/high capacity binding sites for LHRH was demonstrated (HEC-1A line, Kd2 = 1.4 x 10(-6) mol/L, Bmax2 = 21 pmol/10(6) cells; Ishikawa, Kd2 = 4 x 10(-6) mol/L, Bmax2 = 32 pmol/10(6) cells). In the presence of 10(-5) mol/L agonist [D-Trp6]LHRH (triptorelin), the proliferation of HE...
TL;DR: In this paper, a general class of asymptotically distribution-free tests of a linear hypothesis in the linear regression model is proposed, which are based on regression rank scores.
Abstract: We propose a general class of asymptotically distribution-free tests of a linear hypothesis in the linear regression model. The tests are based on regression rank scores, recently introduced by Gut...
TL;DR: ABA triblock copolymers were synthesized by bulk polymerization, using aluminium triisopro-poxide as catalyst as mentioned in this paper, and they show striking differences in their physicochemical properties compared to their homopolymers.
TL;DR: It is proposed that the RNA structure of a given snRNP particle determines at least in part whether the particle's m3G cap is required for nuclear transport or can be dispensed with.
Abstract: We have investigated the nuclear transport of U1 and U5 snRNPs by microinjection studies in oocytes from Xenopus laevis using snRNP particles prepared by reconstitution in vitro. Competition studies with snRNPs showed that the Sm core domain of U1 snRNPs contains a nuclear location signal that acts independently of the m3G cap. The transport of U1 snRNP can be blocked by saturation with competitor U1 snRNPs or by U5 snRNPs, which indicates that the signals on the respective Sm core domains interact with the same transport receptors. Further, by using a minimal U1 snRNP particle reconstituted in vitro and containing only the Sm core RNP domain and lacking stem-loops I to III of U1 RNA, we show that this is targeted actively to the nucleus, in spite of the absence of the m3G cap. This indicates that under certain conditions the NLS in the Sm core domain not only is an essential, but may also be a sufficient condition for nuclear targeting. We propose that the RNA structure of a given snRNP particle determines at least in part whether the particle's m3G cap is required for nuclear transport or can be dispensed with.
TL;DR: In Bacillus species, starvation leads to the activation of a number of processes that affect the ability to survive during periods of nutritional stress, which include the development of genetic competence, sporulation, the synthesis of degradative enzymes, motility, and antibiotic production.
Abstract: In Bacillus species, starvation leads to the activation of a number of processes that affect the ability to survive during periods of nutritional stress. Activities that are induced include the development of genetic competence, sporulation, the synthesis of degradative enzymes, motility, and antibiotic production. The genes that function in these processes are activated during the transition from exponential to stationary phase and are controlled by mechanisms that operate primarily at the level of transcription initiation. One class of genes functions in the synthesis of special metabolites such as the peptide antibiotics tyrocidine and gramicidin S as well as the cyclic lipopeptide surfactin. These genes include the grs and tyc operons in Bacillus brevis, which encode gramicidin S synthetase and tyrocidine synthetase, respectively, and the srfA operon of Bacillus subtilis which encodes the enzymes of the surfactin synthetase complex. Peptide antibiotic biosynthesis genes are regulated by factors as diverse as the early sporulation gene product Spo0A, the transition-state regulator AbrB, and gene products (ComA, ComP, and ComQ) required for the initiation of the competence developmental pathway.
TL;DR: In situ hybridization of mitochondrial DNA has been performed on extraocular muscles of humans over 70 years of age to show whether mutated mtDNA with the so called common deletion of 4,977 basepairs at position 8,482–13,460 of mtDNA accumulates in the cytochrome c oxidase-deficient fibres.
Abstract: Previous studies have revealed an increase of cytochrome c oxidase-deficient fibres/cells in the skeletal and heart muscle of humans during ageing. The enzyme defect is due to a lack of both mitochondrial and nuclear coded enzyme subunits. In the present investigation in situ hybridization of mitochondrial DNA (mtDNA) has been performed on extraocular muscles of humans over 70 years of age to show whether mutated mtDNA with the so called common deletion of 4,977 basepairs at position 8,482–13,460 of mtDNA accumulates in the cytochrome c oxidase-deficient fibres. The cytochrome c oxidase-deficient fibres revealed different hybridization patterns: a normal hybridization signal with three different mtDNA probes, a reduced or lacking signal with all three probes indicating depletion of mtDNA and a selective hybridization defect with the probe recognizing the “common deletion” region of mtDNA as evidence of mtDNA deletion. The results suggest that during ageing defects of cytochrome c oxidase are associated with different molecular alterations of mtDNA. Deletion and depletion of mtDNA are not the only nor probably the leading mechanisms responsible for the loss of respiratory chain capacity during ageing. The normal hybridization signal in most of the cytochrome c oxidase-deficient fibres and the loss of mitochondrial and nuclear protein subunits indicate the involvement of other, especially nuclear factors.
TL;DR: The concept of the taxonomic order Mononegavirales for all nonsegmented negative-strand RNA viruses and the classification of Marburg virus, Ebola virus, and Reston virus in the family Filoviridae are supported.
Abstract: The family Filoviridae contains extremely pathogenic human viruses causing a fulminating, febrile hemorrhagic disease. Filoviruses are enveloped, filamentous particles with a nonsegmented negative-strand RNA genome showing the gene arrangement 3′-NP-VP35-VP40-GP-VP30-VP24-L-5′. Genes are flanked by highly conserved transcriptional signals and are generally separated by variable intergenic regions. They are transcribed into monocistronic polyadenylated messenger RNAs which contain relatively long 5′ and 3′ untranslated regions. Seven structural proteins are encoded by the genome of which four form the helical nucleocapsid (NP-VP35-VP30-L), two are membrane-associated (VP40-VP24), and one is a transmembrane glycoprotein (GP). Comparison of filovirus genomes with those of other nonsegmented negative-strand RNA viruses suggest comparable mechanisms of transcription and replication and a common evolutionary lineage for all these viruses. Sequence analyses of single genes, however, showed that filoviruses are more closely related to paramyxoviruses, particularly human respiratory syncytial virus. These data support the concept of the taxonomic order Mononegavirales for all nonsegmented negative-strand RNA viruses and the classification of Marburg virus, Ebola virus, and Reston virus in the family Filoviridae, separate from the families Paramyxoviridae and Rhabdoviridae.
TL;DR: The role of three N-linked glycans which are conserved among various hemagglutinin (HA) subtypes of influenza A viruses was investigated by eliminating the conserved glycosylation (cg) sites at asparagine residues 12,cg1, 28, and 478 residues by site-directed mutagenesis.
Abstract: The role of three N-linked glycans which are conserved among various hemagglutinin (HA) subtypes of influenza A viruses was investigated by eliminating the conserved glycosylation (cg) sites at asparagine residues 12 (cg1), 28 (cg2), and 478 (cg3) by site-directed mutagenesis. An additional mutant was constructed by eliminating the cg3 site and introducing a novel site 4 amino acids away, at position 482. Expression of the altered HA proteins in eukaryotic cells by a panel of recombinant vaccinia viruses revealed that rates and efficiency of intracellular transport of HA are dependent upon both the number of conserved N-linked oligosaccharides and their respective positions on the polypeptide backbone. Glycosylation at two of the three sites was sufficient for maintenance of transport of the HA protein. Conserved glycosylation at either the cg1 or cg2 site alone also promoted efficient transport of HA. However, the rates of transport of these mutants were significantly reduced compared with the wild-type protein or single-site mutants of HA. The transport of HA proteins lacking all three conserved sites or both amino-terminally located sites was temperature sensitive, implying that a polypeptide folding step had been affected. Analysis of trimer assembly by these mutants indicated that the presence of a single oligosaccharide in the stem domain of the HA molecule plays an important role in preventing aggregation of molecules in the endoplasmic reticulum, possibly by maintaining the hydrophilic properties of this domain. The conformational change observed after loss of all three conserved oligosaccharides also resulted in exposure of a normally mannose-rich oligosaccharide at the tip of the large stem helix that allowed its conversion to a complex type of structure. Evidence was also obtained suggesting that carbohydrate-carbohydrate interactions between neighboring oligosaccharides at positions 12 and 28 influence the accessibility of the cg2 oligosaccharide for processing enzymes. We also showed that terminal glycosylation of the cg3 oligosaccharide is site specific, since shifting of this site 4 amino acids away, to position 482, yielded an oligosaccharide that was arrested in the mannose-rich form. In conclusion, carbohydrates at conserved positions not only act synergistically by promoting and stabilizing a conformation compatible with transport, they also enhance trimerization and/or folding rates of the HA protein.
TL;DR: GLP-1 might represent another neuropeptide that acts as neurotransmitter of the peptidergic, nonadrenergic-noncholinergic nervous system that innervates the airways, according to slide-mount autoradiography of fresh frozen lung tissue sections.
Abstract: Recent data revealed the existence of specific receptors for glucagon-like peptide-1(7–36)amide (GLP-1) on rat lung membranes. Utilizing slide-mount autoradiography of fresh frozen lung tissue sect...
TL;DR: The results obtained by pulse-chase experiments, cell fractionation and immunostaining techniques strongly suggest that cyclin D1 is a labile protein, which is located in the nucleus, which fundamentally differs from "classical" cyclins, such as the mitotic cyclin B, whose expression is clearly cell cycle-dependent.
Abstract: We show that the cyclin D1 gene is regulated by a variety of growth factors in human diploid fibroblasts (WI38). Expression of cyclin D1 mRNA is low in quiescent WI-38 cells and reaches a maximum around 10 hours after serum stimulation, i.e. approximately 8 hours prior to the onset of DNA synthesis. A cyclin D1-specific antiserum raised against a bacterially expressed fusion protein detected a 39 kDa polypeptide in WI-38 cells. In agreement with the RNA expression data, cyclin D1 protein synthesis is also serum-inducible, reaching a maxi mum around 9 hours post-stimulation. The results obtained by pulse-chase experiments, cell fractionation and immunostaining techniques strongly suggest that cyclin D1 is a labile protein ( tg ≈ 38 min), which is located in the nucleus. Cyclin D1 is directly induced by growth factors, i.e. in the presence of cycloheximide, and its expression does not significantly fluctuate during the cell cycle in synchronized cells. Cyclin D1 therefore fun damentally differs from “classical” cyclins, such as the mitotic cyclin B, whose expression is clearly cell cycledependent. Cyclin D1 may rather establish a direct link between growth control mechanisms and the cell cycle. Interestingly, cyclin D1 expression is stimulated by the protein kinase C activator TPA, but suppressed by dibutyryl-cAMP and the adenylate cyclase inducer forskolin, pointing to multiple regulatory pathways controlling cyclin D1 expression. SUMMARY
TL;DR: The data show that the state of phosphorylation of the Ul-specific 70K protein is critical for its participation in a pre-catalytic step of the splicing reaction, which is vital for the successful splicing of pre-mRNA.
Abstract: The U1 small nuclear ribonucleoprotein (snRNP) particle is one of the Sm class of snRNPs essential for splicing of precursor messenger RNA. Mammalian U1 snRNP contains a 165-nucleotide long RNA molecule and at least 11 proteins: the U1-specific 70K proteins A and C, and the common U snRNP proteins (B', B, D1, D2, D3, E, F and G). One of the functions of U1 snRNP is recognition of the 5' splice site, an event that requires both U1 RNA and U1 proteins. The 70K protein is the only heavily phosphorylated U1 protein in the cell. Isolated U1 snRNPs are associated with a kinase activity that selectively phosphorylates the 70K protein in vitro in a reaction requiring ATP. Here we investigate the role of phosphorylation of the 70K protein in the splicing of pre-mRNA. The 70K protein on U1 snRNPs was phosphorylated in vitro with either ATP, or with ATP-gamma S, which gave a thiophosphorylated product that was resistant to dephosphorylation by phosphatases. When HeLa nuclear splicing extracts that had been depleted of endogenous U1 snRNPs were complemented with U1 snRNPs possessing normal phosphorylated 70K protein, mature spliceosomes were generated and the splicing activity of the extracts was fully restored. By contrast, if thiophosphorylated U1 snRNPs were used instead, splicing was completely inhibited, although formation of the mature spliceosome was unaffected. Our data show that the state of phosphorylation of the U1-specific 70K protein is critical for its participation in a pre-catalytic step of the splicing reaction.
TL;DR: It is shown that theSF3a subunits correspond to three of the 17S U2 snRNP-specific polypeptides, which suggests a function for SF3a and SF3b in the incorporation of U2snRNP into the spliceosome.
Abstract: In the assembly of a prespliceosome, U2 small nuclear ribonucleoprotein (snRNP) functions in pre-messenger RNA (mRNA) splicing together with splicing factors (SFs) 3a, SF3b, and several other proteins. The 17S but not the 12S form of U2 snRNP is active in splicing-complex formation. Here it is shown that the SF3a subunits correspond to three of the 17S U2 snRNP-specific polypeptides. SF3a interacts with U2 snRNP in the presence of SF3b to generate a structure similar to 17S U2 snRNP, which suggests a function for SF3a and SF3b in the incorporation of U2 snRNP into the spliceosome. Furthermore, the 60-kilodalton subunit of SF3a is related to the yeast splicing protein PRP9.
TL;DR: The A‐to‐G transition mutation in the tRNALys gene of mitochondrial DNA, characteristic for the maternally inherited MERRF syndrome, has been identified by point mutation‐specific polymerase chain reaction in extraocular muscle from 11 of 16 healthy people of different ages.
TL;DR: It is reported that the abundance of transcripts from two other cell cycle genes, cdk2 and cdk4, thought to act during G1-->S progression, is significantly diminished in senescent cells of the diploid human fibroblast line WI-38, and this deregulation may be due to the absence of a regulatory loop that would normally control their expression.
Abstract: The state of cellular senescence is characterised by an irreversible arrest in the G1 phase of the cell cycle. It has previously been shown that three cell cycle genes, cyclin A, cyclin B and cdc2, are not expressed in senescent human fibroblasts. All three gene products have functions after S-phase entry, so that their suppression cannot explain the irreversible G1 arrest. Here, we report that the abundance of transcripts from two other cell cycle genes, cdk2 and cdk4, thought to act during G1—>S progression, is significantly diminished in senescent cells of the diploid human fibroblast line WI-38. Surprisingly, two other cyclins, D1 and E, behave in a completely different way, in that their expression is elevated in senescent cells, especially under conditions of serum starvation. Both the synthesis and the steady-state level of cyclin D1 protein were also found to be markedly higher in senescent cells (3- to 6-fold). Cyclins D1 and E are thus the first genes shown to be overexpressed or deregulated in senescent cells. It is tempting to speculate that this deregulation may be due to the absence, in senescent cells, of a regulatory loop that would normally control their expression. This is supported by our finding that cyclin E-associated kinase activity in senescent cells is reduced approx. 14-fold. Our data also suggest that the deregulated expression of cyclin D1 and E is not sufficient to drive senescent cells into DNA replication.
TL;DR: Results obtained identify TGFβ and TNFα as mediators stimulating key events in liver fibrogenesis (i.e. FSC proliferation, FSC transdifferentiation into MFBIC, and fibronectin synthesis).
Abstract: Transforming growth factor-β (TGFβ1) and tumor necrosis factor alpha (TNFα) stimulate the trans-differentiation of fat-storing cells (FSC) in the rat liver into highly active and “synthetic” myofibroblast-like cells (MFBIC). This activation has been documented by differential-interference contrast and light microscopy using morphologic criteria (a reduction in the number and size of fat droplets, cell flattening and the development of long cytoplasmic extensions), by the loss of retinyl-palmitate (measured by HPLC) and by the enhanced expression of iso-α smooth muscle actin (demonstrated by immunofluorescence microscopy). Furthermore, while cell growth measured by the cell count and DNA content is slightly inhibited by TGFβ1 (0.81 of the control), the combination of TGFβ1 with TNFα stimulates cell proliferation to 1.44 times of the control. In addition the combination of TGFβ and TNFα potentiated the stimulatory effect on fibronectin synthesis (TGFβ alone: 1.4 times control; TNFα alone: 2.2 times control; TGFβ plus TNFα: 4.7 times control). The total protein synthesis was not altered by TGFβ or TNFα. In summary the results obtained identify TGFβ and TNFα as mediators stimulating key events in liver fibrogenesis (i.e. FSC proliferation, FSC transdifferentiation into MFBIC, and fibronectin synthesis).
TL;DR: Results show that Ta and food availability are effective in modifying both seasonal timing and extent of photoperiodically controlled adaptations, and indicates that Djungarian hamsters are capable of flexible physiological responses towards unpredictable climatic changes in the environment.
Abstract: We investigated the influence of ambient temperature (Ta) and food availability on seasonal timing and extent of physiological responses to short photoperiod (SP), in particular daily torpor, in Djungarian hamsters (Phodopus sungorus). Exposure of hamsters to cold temperature (Ta = 5 degrees C), relative to warm Ta (23 degrees C), resulted in: 1) a significant advance (P < 0.05) of the first occurrence of torpor among cold-exposed hamsters (days 52-97 vs. days 83-99 in SP); 2) a higher (P < 0.01) incidence of torpor (48% vs. 20% torpid animals/day); 3) a higher (P < 0.05) degree of molt into the winter pelt; and 4) an accelerated reduction of body weights (P < 0.001). However, within SP/cold-Ta exposed groups, individual hamsters clearly showed different tendencies for torpor (torpor on 0-95% of days observed). Therefore, we evaluated the effects of small changes in Ta on torpor frequency and extension by subjecting the same SP-adapted individuals to varying temperatures. Lowering of Ta from 15 degrees C to 10 degrees C and 5 degrees C caused significant (P < 0.05) increases in the incidence of torpor (20%, 33%, and 40%, respectively) and lower minimal body temperatures during hypothermia (P < 0.05). When the same animals were subjected to 24-48 h lasting periods of food restriction (60% of the ad libitum intake), torpor frequency further increased 1.8- to 2.6-fold at all Tas. These results show that Ta and food availability are effective in modifying both seasonal timing and extent of photoperiodically controlled adaptations. This integration of multiple environmental cues, combined with a pronounced within-species variability of winter adjustments, indicates that Djungarian hamsters are capable of flexible physiological responses towards unpredictable climatic changes in the environment.
TL;DR: It is found that backcross of E mu-Pim-1 transgenics into lpr/lpr mice results in strong acceleration of lymphoproliferation and dramatic enlargement of lymph nodes, and first insight into the functional basis for the oncogenic activity of PIM-1 is provided.
Abstract: Transgenic mice expressing the Pim-1 kinase are predisposed to develop T-cell lymphomas with a long latency period of about 7-9 months. However, the exact functional basis of the oncogenic activity of Pim-1 remains obscure. C57BL/6 mice homozygous for the lpr mutation develop a well-described lymphoproliferative syndrome at about 26-30 weeks of age. This syndrome is characterized mainly by the accumulation of abnormal T cells in lymph nodes because of the lack of Fas receptor-induced apoptosis. We find that backcross of E mu-Pim-1 transgenics (mice with a transgene that carries the mouse Pim-1 gene under the transcriptional control of the immunoglobulin heavy chain gene enhancer E mu) into lpr/lpr mice results in strong acceleration of lymphoproliferation and dramatic enlargement of lymph nodes. In addition, we show here that cultured lymph node cells from E mu-Pim-1 lpr/lpr mice are rescued from rapid apoptosis that normally occurs in nontransgenic lpr cells in vitro. We also present evidence that CD4+/CD8+ double-positive thymocytes from lpr/lpr mice are sensitive to dexamethasone-induced apoptosis, although lpr/lpr mice lack the Fas receptor. In contrast, E mu-Pim-1 lpr/lpr animals show considerable protection from dexamethasone-induced apoptosis. These results show that Pim-1 can strongly accelerate lymphoproliferation through inhibition of apoptosis and thereby provide first insight into the functional basis for the oncogenic activity of Pim-1.