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Biomedical subjects

A Kuhn

Publications and source records attributed to A Kuhn.

At least 91 records · Page 5Linked to original sources
PubMed1998

Sporulation and time course expression of sigma-factor homologous genes in Clostridium acetobutylicum.

The gene for the vegetative sigma factor A of Clostridium acetobutylicum was constitutively transcribed during growth and formed an operon together with dnaE. Sporulation-specific sigma factors E, G, and K were sequentially induced shortly before mature endospores could be detected. Maximal transcription in the course of spore formation was found to be in the order sigE-sigG-sigK, thus matching the pattern described for Bacillus subtilis. From primer extension experiments promoter structures could be deduced with high homology to the Bacillus consensus. Upstream of the spoIIGA-sigE operon a gene with significant similarity to ftsZ could be detected.

Clostridium↗
PubMed1998

[Experimental reconstruction of the sheep orbit with biodegradable implants].

In a complex animal model in sheep, polydioxanone (PDS) and polylactic membranes were used for the reconstruction of large orbital-wall defects. In a long-term experiment over 1 year, polylactic implants alone showed the best performance as compared with combinations involving autogenous bone grafts and titanium miniplate fixation. As soon as these polylactic implants are approved for human surgery, they will be used to solve the still challenging problem of anatomical reconstruction of large comminuted fractures of more than one orbital wall.

Animals↗
PubMed1998

Aberrant timing in epidermal expression of inducible nitric oxide synthase after UV irradiation in cutaneous lupus erythematosus.

Photosensitivity is a main criterion for the diagnosis of systemic lupus erythematosus (LE), and ultraviolet (UV) irradiation plays a key role in the pathogenesis of cutaneous LE. Patients with a tentative diagnosis of LE are routinely tested for skin lesion development after experimental UV irradiation, providing an ideal opportunity to evaluate early, preclinical events involved in the pathogenesis of LE. Several reports have shown expression of the cytokine-inducible nitric oxide synthase (iNOS) in autoimmune diseases. Therefore, we investigated the role of iNOS expression at mRNA and protein level in the pathogenesis of LE lesions. Skin biopsies from patients with different subtypes of LE were examined, and iNOS expression was found in six of 18 biopsies from cutaneous LE patients and two of three biopsies from systemic LE patients. In biopsies taken 4-20 d after UV irradiation, epidermal iNOS expression was seen in all patients (n = 10) after UVB and in four of 10 patients provoked by UVA. In healthy controls (n = 8) epidermal iNOS expression was detected 24 h after UV irradiation, persisting for another day before subsiding on day 3. In LE patients (n = 8) the exact reverse situation was seen: an iNOS-specific signal was undetectable in keratinocytes for 2 d after UV irradiation, but became positive on day 3 and persisted for up to 25 d in the evolving skin lesions. Our findings demonstrate a time-restricted, UV-induced iNOS expression in human skin; moreover, the results indicate that both the kinetics of iNOS induction as well as the time span of local iNOS expression may be critical to the development of cutaneous LE lesions.

Adult↗
PubMed1997

Negatively charged amino acid residues play an active role in orienting the Sec-independent Pf3 coat protein in the Escherichia coli inner membrane.

The coat protein of Pseudomonas aeruginosa phage Pf3 is transiently inserted into the bacterial inner membrane with a single transmembrane anchor sequence in the N(out)C(in) orientation. The N-terminal sequence immediately flanking the membrane anchor contains one negatively charged residue, whereas the C-terminal hydrophilic segment has two positively charged residues. To investigate how the orientation of this protein is achieved, the three flanking charged amino acid residues were altered. Membrane insertion was analyzed in vivo using the accessibility to externally added protease and in vitro by testing the insertion into inverted Escherichia coli membrane vesicles. In both systems, the orientation of the protein was completely reversed for the oppositely charged mutant coat protein (RD mutant). In addition, we show in vivo that the electrochemical membrane potential is necessary for the translocation of both the wild-type and the mutant Pf3 coat proteins, suggesting that membrane insertion is driven by electrophoretic forces.

Amino Acid Sequence↗
PubMed1997

[Comparative studies of the rate of restenosis after implantation of Palmaz-Schatz and AVE-Micro stents].

Late results of interventional procedures utilizing coronary stents are largely determined by the rate of restenosis. So far few data are available addressing the effect of stent design, implantation pressure and morphologic factors on this crucial variable. Therefore we analyzed the coronary angiograms obtained in 259 patients before, immediately after and at 3 to 6 months following stent implantation for obstructive coronary disease. A total of 196 AVE-Micro-Stents and 142 Palmaz-Schatz-Stents were implanted into 307 stenoses. In 126 stenoses there were implanted only Palmaz-Schatz-Stents, in 170 only AVE-Micro-Stents and in 11 stenoses there were implanted Palmaz-Schatz- as well as Micro-Stents. Restenosis was defined as an over 50% stenosis at follow up. No significant difference was detected with regard to global restenosis rate at an average of 4 months following implantation (Palmaz-Schatz 33%, Micro-Stent 27%). If results were analyzed according to implantation pressure however, there was a significantly lower restenosis rate for AVE-Micro-Stents implanted with > 10 atm (17%) as compared to < or = 10 atm (35%, p < 0.02) and as compared to Palmaz-Schatz-Stents (34%, p < 0.02), which were also implanted with high pressure over 10 atm. In addition to implantation pressure, vessel segment and morphology of stenosis proved to be important determinants of late results. In this series of patients the AVE-Micro-Stent compared favourably to the Palmaz-Schatz-Stent not only with respect to a significantly lower restenosis rate, when implanted with pressures > 10 atm, but also with regard to its superior flexibility and handling characteristics.

Aged↗
PubMed1997

The kdp system of Clostridium acetobutylicum: cloning, sequencing, and transcriptional regulation in response to potassium concentration.

The complete sequence of the kdp gene region of Clostridium acetobutylicum has been determined. This part of the chromosome comprises two small open reading frames (orfZ and orfY), putatively encoding hydrophobic peptides, and the genes kdpA, kdpB, kdpC, and kdpX, followed by an operon encoding a pair of sensor-effector regulatory proteins (KdpD and KdpE). Except for orfZ, orfY, and kdpX, all genes showed significant homology to the kdp genes of Escherichia coli, encoding a high-affinity potassium transport ATPase and its regulators. The complete genome sequence of Synechocystis sp. strain PCC 6803 and a recently published part of the Mycobacterium tuberculosis genome indicate the existence of a kdp system in these organisms as well, but all three systems comprise neither a second orf upstream of kdpA nor an additional kdpX gene. Expression of the clostridial kdp genes, including the unique kdpX gene, was found to be inducible by low potassium concentrations. A transcription start point could be mapped upstream of orfZ. A promoter upstream of kdpD was active only under noninducing conditions. Lowering the potassium content of the medium led to formation of a common transcript (orfZYkdpABCXDE), with a putative internal RNase E recognition site, which could be responsible for the instability of the common transcript. Except for the two small peptides, all gene products could be detected in in vitro transcription-translation experiments.

Adenosine Triphosphatases↗
PubMed1997

Vaccinia virus membrane proteins p8 and p16 are cotranslationally inserted into the rough endoplasmic reticulum and retained in the intermediate compartment.

The use of two-dimensional gel electrophoresis has identified the gene products A14L (p16) and A13L (p8) as abundant membrane proteins of the first infectious form of vaccinia virus, the intracellular mature virus (IMV; O. N. Jensen, T. Houthaeve, A. Shevchenko, S. Cudmore, T. Ashford, M. Mann, G. Griffiths, J. Krijnse Locker, J. Virol. 70:7485-7497, 1996). In this study, these two proteins were characterized in detail. In infected cells, both proteins localize not only to the viral membranes but also to tubular-cisternal membranes of the intermediate compartment, defined by the use of antibodies to either rab1A or p21, which colocalize with rab1A (J. Krijnse Locker, S. Schleich, D. Rodriguez, B. Goud, E. J. Snijder, and G. Griffiths, J. Biol. Chem. 271:14950-14958, 1996). Both proteins appear to reach this destination via cotranslational insertion into the rough endoplasmic reticulum, as shown by in vitro translation and translocation experiments. Whereas p16 probably spans the membrane twice, p8 is inserted into the membrane by means of its single NH2-terminal hydrophobic domain, adopting a topology which leaves the C terminus exposed to the cytoplasm. Combined immunocytochemical and biochemical data show that p16 is a member of the inner of the two IMV membrane layers, whereas p8 localizes to both the inner and the outer membrane. These findings are discussed with respect to our model of IMV membrane assembly.

Blotting, Western↗
PubMed1997

[Effect of integrated distance studs on cement thickness of implanted PE-acetabulum cups--experimental in vitro studies for improved cementing technique].

Variations in cement thickness between PE cups and acetabular bone are biomechanically unfavourable, and may contribute to prosthetic loosening. Integrated distance studs should in the first instance, prevent local thinning of the cement. In an experimental study we compared the thickness of the cement mantel of implanted PE cups without such studs. In the case of cups with studs, significantly fewer areas of thin cement (0-1 mm) were found in comparison with cups with no studs. "Bald" areas of cement (0-0.2 mm) were virtually prevented by the studs. We are of the opinion that PE cups with integrated studs on the surface ensure a uniform cement thickness around the cup, as well as decreasing the risk of "bald" areas in the cement, thus reducing loosening.

Acetabulum↗
PubMed1996

Thermodynamics of the membrane insertion process of the M13 procoat protein, a lipid bilayer traversing protein containing a leader sequence.

For the first time, the standard free energy change, delta Gzero, of a membrane-inserting protein with a leader sequence has been determined experimentally, using M13 procoat protein as an example. The partition coefficient for the distribution of the procoat protein between the aqueous phase and the membrane phase of preformed lipid vesicles yielded a value of gamma = 6.5 x 10(5) M-1, corresponding to a delta Gzero of -10.4 kcal/mol, based on measurements of the fluorescence energy transfer between the intrinsic tryptophan of the protein and a suitably labeled lipid membrane of POPC. For comparison, the partition coefficient of the M13 coat protein between the aqueous and the POPC lipid bilayer phase was determined to be distinctly lower: gamma = 1 x 10(5) M-1 (delta Gzero = -9.3 kcal/mol). Proteinase K digestion experiments have been performed, showing that 20% of the procoat protein bound to lipid vesicles spontaneously integrate in a transbilayer form, whereas 80% remain inserted in the interfacial membrane region. By taking together these results, an upper limit for the free energy change of the transmembrane insertion of procoat protein was estimated to be -14.8 kcal/mol. In order to distinguish further the contribution arising from insertion of the procoat protein into the membrane interfacial region from that due to transmembrane insertion, the partition coefficient of the mutant procoat protein OM30R [which contains a positively charged amino acid in its mature hydrophobic segment (exchange of a Val to an Arg residue at position 30)] was determined, yielding gamma = 0.3 x 10(5) M-1 (delta Gzero = -8.6 kcal/mol). Previously reported in vivo experiments have shown that the OM30R mutant protein is not translocated across Escherichia coli membranes but only binds to the inner surface. The results presented here indicate that although the insertion of the procoat protein into the interfacial region of the lipid bilayer contributes the major part to delta Gzero, it is the final energy gain of the interaction of the hydrophobic portions of the folded pre-protein with the lipid chains which drives the transmembrane insertion of the M13 procoat protein. Neither the leader sequence nor the mature coat protein alone yields this free energy gain. For the different proteins investigated here, spontaneous membrane insertion occurs only for fluid lipid bilayers, but not for membranes in the crystalline lipid phase. Furthermore, by using lipid bilayers with negative membrane surface charges, it was shown that both procoat and coat proteins are electrostatically attracted to the surface of the lipid membrane, though only to a small extent, with apparent partition coefficients of the same order of magnitude as for the phosphatidylcholine lipid membrane.

Amino Acid Sequence↗
PubMed1996

Modelling of a photoacoustic probe designed for medical applications.

A model is presented describing the thermoelastic photoacoustic interaction in a layered medium within a transparent fluid, where a polymer transducer is used for the detection of ultrasonic pulses. By taking the optical absorption coefficient and finite layer thickness into account, the amplitude and shape of photoacoustic transients are calculated for both forward and backward travelling directions. Additionally, photoacoustic transient interaction with the PVDF transducer has been characterised using a discrete-time algorithm for the transducer response. Good agreement with experimental waveforms are demonstrated, so that this may form the basis of system characterisation when miniature laser-ultrasound probes are used in various applications.

Absorption↗
PubMed1996

[Development of a resorbable implant: experimental reconstruction of the orbits with polylactate membranes. Animal model and preliminary results].

In a complex animal model in five sheep, PDS or polylactic biodegradable membranes were used for reconstruction of large orbital defects in combination with autogenous bone grafts and stable internal fixation with titanium miniplates and screws. Because of anatomical similarities, animal model and human clinical findings are comparable. After eight weeks, in four sheep all reconstructions were separated from the maxillary and frontal sinus by a continuous mucosal layer. The bone grafts were integrated into the bony frame of the orbit. Foreign body reactions were milder with polylactic membranes compared to PDS, probably due to the slow degradation of polylactide. These preliminary results will lead to a further study with a larger series to compare the two biodegradable materials and to prepare the way for clinical use of polylactic membranes in orbital reconstruction.

Animals↗
PubMed1995

Activation of mammalian ribosomal gene transcription requires phosphorylation of the nucleolar transcription factor UBF.

The nucleolar factor UBF is phosphorylated by casein kinase II (CKII) at serine residues within the C-terminal acidic domain which is required for transcription activation. To investigate the biological significance of UBF modification, we have compared the trans-activating properties of cellular UBF and recombinant UBF expressed in Escherichia coli. Using a variety of assays we demonstrate that unphosphorylated UBF is transcriptionally inactive and has to be phosphorylated at multiple sites to stimulate transcription. Examination of cDNA mutants in which the serine residues within the C-terminal domain were altered by site-directed mutagenesis demonstrates that CKII-mediated phosphorylations of UBF contribute to, but are not sufficient for, transcriptional activation. Besides CKII, other cellular protein kinases phosphorylate UBF at distinct sites in a growth-dependent manner. The marked differences in the tryptic peptide maps of UBF from growing and serum-starved cells suggest that alterations in the degree of UBF phosphorylation may modulate rRNA synthetic activity in response to extracellular signals.

3T3 Cells↗
PubMed1995

The translocation of negatively charged residues across the membrane is driven by the electrochemical potential: evidence for an electrophoresis-like membrane transfer mechanism.

The role of the membrane electrochemical potential in the translocation of acidic and basic residues across the membrane was investigated with the M13 procoat protein, which has a short periplasmic loop, and leader peptidase, which has an extended periplasmically located N-terminal tail. For both proteins we find that the membrane potential promotes membrane transfer only when negatively charged residues are present within the translocated domain. When these residues are substituted by uncharged amino acids, the proteins insert into the membrane independently of the potential. In contrast, when a positively charged residue is present within the N-terminal tail of leader peptidase, the potential impedes translocation of the tail domain. However, an impediment was not observed in the case of the procoat protein, where positively charged residues in the central loop are translocated even in the presence of the membrane potential. Intriguingly, several of the negatively charged procoat proteins required the SecA and SecY proteins for optimal translocation. The studies reported here provide insights into the role of the potential in membrane protein assembly and suggest that electrophoresis can play an important role in controlling membrane topology.

Adenosine Triphosphatases↗
PubMed1995

DNA-dependent protein kinase: a potent inhibitor of transcription by RNA polymerase I.

DNA-dependent protein kinase (DNA-PK) comprises a catalytic subunit of approximately 350 kD (p350) and a DNA-binding component termed Ku. Although DNA-PK can phosphorylate many transcription factors, no function for this enzyme in transcription has been reported thus far. Here, we show that DNA-PK strongly represses transcription by RNA polymerase I (Pol I). Transcriptional repression by DNA-PK requires ATP hydrolysis, and DNA-PK must be colocalized on the same DNA molecule as the Pol I transcription machinery. Consistent with DNA-PK requiring DNA ends for activity, transcriptional inhibition only occurs effectively on linearized templates. Mechanistic studies including single-round transcriptions, abortive initiation assays, and factor-independent transcription on a tailed template demonstrate that DNA-PK inhibits initiation (i.e., the formation of the first phosphodiester bonds) but does not affect transcription elongation. Repression of transcription involves phosphorylation of the transcription initiation complex, and rescue experiments reveal that the inactivated factor remains bound to the promoter and thus prevents initiation complex formation. We discuss the possible relevance of these findings in regard to the control of rRNA synthesis in vivo.

Adenosine Triphosphate↗
PubMed1995

Hemodynamic and antiischemic effects of intravenous elgodipine, a new dihydropyridine calcium channel blocker, in patients with chronic stable angina.

Elgodipine is a new second-generation dihydropyridine calcium antagonist. Its hemodynamic and antiischemic properties were evaluated in a single-blind, placebo-controlled trial in 22 males with chronic stable angina. Coronary artery disease was angiographically confirmed. Measurements were performed with a Swan-Ganz thermodilution catheter during a 30-minute period of rest and before the end of a 4-minute bicycle exercise test at maximum individual workload, both with placebo (IV infusion of 5 ml saline over 30 minutes) and elgodipine (10 micrograms/kg/2 min bolus IV, then IV infusion of 1 micrograms/kg/min for 28 minutes. Elgodipine caused very similar hemodynamic changes at rest and during exercise. Its major hemodynamic modification was the marked decrease in systemic vascular resistance, which was accompanied by an increase in cardiac index and stroke volume. Mean arterial blood pressure was slightly reduced, whereas the opposite small increase in heart rate meant that the double product remained unchanged. Contrary to resting conditions, pulmonary capillary wedge pressure, pulmonary artery pressures, pulmonary vascular resistance, and mean right atrial pressure remained normal or increased to a lesser extent during exercise after elgodipine. After elgodipine ischemic ST depression during exercise was diminished, and 11 of 16 assessable patients remained free from angina pectoris. We conclude that elgodipine is an efficacious antianginal drug. Its major mechanism of action is lowering of systemic vascular resistance. Thus elgodipine improves systolic cardiac function in patients with chronic stable angina and may delay the onset of ischemic diastolic dysfunction during exercise, as indicated by a normal left ventricular end-diastolic pressure (LVEDP). The data also suggest an improvement in coronary blood flow during exercise.

Adult↗
PubMed1995

Directionality in protein translocation across membranes: the N-tail phenomenon.

Protein translocation normally starts from an N-terminal signal peptide and proceeds in an N-to-C-terminal direction. However, in certain integral membrane proteins an N-terminal tail is translocated even though it is not preceded by a signal peptide. In eukaryotic cells this process involves the normal Sec-machinery. In contrast, recent studies in Escherichia coli show that translocation of such N-terminal tails occurs by a mechanism that does not appear to involve the Sec proteins and is most efficient for short tails lacking positively charged residues. These novel observations suggest that the Sec-machinery has an inherent N-to-C-terminal directionality and cannot work 'in reverse'.

Journal Article↗