Role of convection in thin-layer electrodeposition.
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Journal Article↗
Biomedical subjects
A Kuhn
Publications and source records attributed to A Kuhn.
Major coat proteins of bacteriophage Pf3 and M13 as model systems for Sec-independent protein transport.
The membrane insertion of bacteriophage coat proteins occurs independent of the Sec-translocase of Escherichia coli. Detailed study of the Pf3 and M13 coat proteins has elucidated two fundamental mechanisms of how proteins invade the membrane, most likely by direct interaction with the lipid bilayer. The Sec-independent translocation of amino-terminal regions across the inner membrane is limited to a short length and a small number of charged residues. Protein regions that contain several charged residues are efficiently translocated across the membrane when these regions are flanked by two adjacent hydrophobic segments interacting synergistically. The relevance of these findings for the membrane insertion mechanism of multispanning membrane proteins is discussed.
Adenosine Triphosphatases↗
Solventogenic enzymes of Clostridium acetobutylicum: catalytic properties, genetic organization, and transcriptional regulation.
The enzymes acetoacetate decarboxylase and coenzyme A transferase catalyse acetone production from acetoacetyl-CoA in Clostridium acetobutylicum. The adc gene encoding the former enzyme is organized in a monocistronic operon, while the ctf genes form a common transcription unit with the gene (adhE) encoding a probable polyfunctional aldehyde/alcohol dehydrogenase. This genetic arrangement could reflect physiological requirements at the onset of solventogenesis. In addition to AdhE, two butanol dehydrogenase isozymes and a thiolase are involved in butanol synthesis. RNA analyses showed a sequential order of induction for the different butanol dehydrogenase genes, indicating an in vivo function of BdhI in low level butanol formation. The physiological roles of AdhE and BdhII most likely involve high level butanol formation, with AdhE being responsible for the onset of solventogenesis and BdhII ensuring continued butanol production. Addition of methyl viologen results in artificially induced butanol synthesis which seems to be mediated by a still unknown set of enzymes. Although the signal that triggers the shift to solventogenesis has not yet been elucidated, recent investigations suggest a possible function of DNA supercoiling as a transcriptional sensor of the respective environmental stimuli.
Base Sequence↗
Purification and partial characterization of an aldo-keto reductase from Saccharomyces cerevisiae.
A cytosolic aldo-keto reductase was purified from Saccharomyces cerevisiae ATCC 26602 to homogeneity by affinity chromatography, chromatofocusing, and hydroxylapatite chromatography. The relative molecular weights of the aldo-keto reductase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography were 36,800 and 35,000, respectively, indicating that the enzyme is monomeric. Amino acid composition and N-terminal sequence analysis revealed that the enzyme is closely related to the aldose reductases of xylose-fermenting yeasts and mammalian tissues. The enzyme was apparently immunologically unrelated to the aldose reductases of other xylose-fermenting yeasts. The aldo-keto reductase is NADPH specific and catalyzes the reduction of a variety of aldehydes. The best substrate for the enzyme is the aromatic aldehyde p-nitrobenzaldehyde (Km = 46 microM; kcat/Km = 52,100 s-1 M-1), whereas among the aldoses, DL-glyceraldehyde was the preferred substrate (Km = 1.44 mM; kcat/Km = 1,790 s-1 M-1). The enzyme failed to catalyze the reduction of menadione and p-benzoquinone, substrates for carbonyl reductase. The enzyme was inhibited only slightly by 2 mM sodium valproate and was activated by pyridoxal 5'-phosphate. The optimum pH of the enzyme is 5. These data indicate that the S. cerevisiae aldo-keto reductase is a monomeric NADPH-specific reductase with strong similarities to the aldose reductases.
Alcohol Oxidoreductases↗
Evidence for a loop-like insertion mechanism of pro-Omp A into the inner membrane of Escherichia coli.
We have studied the insertion of pro-OmpA into the Escherichia coli membrane in vivo using various mutants that have either alterations in the amino-terminal parts of the signal peptide or in the mature region that flanks the signal peptide. A pro-OmpA mutant with an amino terminal extension of 142 residues derived from ribulokinase (AraB) was analysed for its membrane insertion. The AraB portion, which includes a cluster of seven charged residues close to the signal sequence, did not interfere with the Sec components and allowed efficient export of OmpA. During translocation the AraB portion remained in the cytoplasm. Further mutants of OmpA were constructed in the carboxy-terminal region flanking the signal sequence. Pro-OmpA does not translocate across the membrane when a charge cluster, comprised of Lys-Arg-Arg-Glu-Arg, is introduced after positions 5, 11 or 15 of the mature region, but is translocated when the cluster is introduced after position 22. This defines a region of about 20 residues in the mature part of pro-OmpA that is crucial for membrane insertion. These results suggest that in the case of the Sec-dependent pro-OmpA, as with the Sec-independent M13 procoat, the precursor assumes a loop-like structure involving the signal peptide and the early part of the mature region, leaving the amino terminus of the signal peptide at the cytoplasmic face.
Amino Acid Sequence↗
Structural analysis of electroless deposits in the diffusion-limited regime.
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Journal Article↗
Synergistic insertion of two hydrophobic regions drives Sec-independent membrane protein assembly.
We have studied the membrane insertion of two proteins from the inner membrane of Escherichia coli, both with two transmembrane segments connected by a short periplasmic loop: the M13 procoat protein and a mutant "inverted" leader peptidase. Neither molecule depends on the Sec machinery for insertion. We show that the introduction of a charged residue in the second transmembrane segment completely blocks insertion of both proteins. In contrast, a Sec-dependent procoat mutant, where the periplasmic region has been lengthened, inserts into the membrane even in the presence of a charged residue in the second hydrophobic domain. In addition, a large deletion within the second transmembrane domain of the leader peptidase mutant allows membrane translocation, but only under conditions where the SecA protein is functional. Furthermore, we show that the first hydrophobic domain is required for insertion of the short periplasmic loop of the "inverted" leader peptidase. These results suggest that Sec-independent insertion occurs by a synergistic entry of the two neighboring hydrophobic domains into the lipid bilayer.
Bacterial Proteins↗
[Paradoxical cerebral embolism during fibrinolysis therapy in deep vein thrombosis and pulmonary embolism].
A 37-year-old woman with increasing dyspnoea over several months suddenly developed severe ortho- and tachypnoea as well as cyanosis of the lips and acrocyanosis. Pulmonary angiography revealed massive bilateral pulmonary emboli with a systolic pulmonary artery pressure of 75 mm Hg. Phlebography demonstrated a thrombotic occlusion of the deep veins of the left leg extending to the distal femoral vein. Thrombolysis treatment was started via an indwelling pulmonary artery catheter (500,000 IU urokinase and 10,000 IU heparin as bolus, then 1 mill. IU urokinase and 1,000 IU heparin per hour). After two hours an incomplete left-sided paresis occurred (involving ocular and facial muscles, dysarthria, left arm and left leg) and the thrombolytic infusion was stopped. But cerebral computed tomography (CT) did not demonstrate any intracerebral haemorrhage. The heparin infusion was restarted (partial thromboplastin time between 70 and 90 s). CT examinations during the next few days showed the development of an ischaemic infarction in the distribution of the right medial cerebral artery. Angiography demonstrated occlusion of the right internal carotid artery. The diagnosis of a paradoxical embolus was supported by easy cardiac catheter passage through a patent foramen ovale. Subsequent pulmonary angiography demonstrated a thrombus-free pulmonary arterial circulation with a normal pulmonary arterial pressure. There was gradual and extensive regression of the incomplete hemiparesis.
Adult↗
Functional differences between the two splice variants of the nucleolar transcription factor UBF: the second HMG box determines specificity of DNA binding and transcriptional activity.
The nucleolar transcription factor UBF consists of two proteins, UBF1 and UBF2, which originate by alternative splicing. Here we show that deletion of 37 amino acids within the second of five HMG box motifs in UBF2 is important for the dual role of UBF as transcriptional activator and antirepressor. UBF1 is a potent antirepressor and transcriptional activator, whereas the ability of UBF2 to counteract histone H1-mediated repression and to stimulate ribosomal gene transcription both in vivo and in vitro is at least one order of magnitude lower. The difference in transcriptional activity between UBF1 and UBF2 is due to their different binding to the ribosomal gene promoter and enhancer. Apparently, the presence of an intact HMG box2 modulates the sequence-specific binding of UBF to rDNA control elements. However, the interaction of UBF with rDNA does not entirely depend on sequence recognition. Both UBF isoforms bind efficiently to four-way junction DNA, indicating that they recognize defined DNA structures rather than specific sequences. The results demonstrate that the HMG boxes are functionally diverse and that HMG box2 plays an important role in specific binding of UBF to rDNA.
3T3 Cells↗
A dual role for phosphatidylglycerol in protein translocation across the Escherichia coli inner membrane.
The involvement of phosphatidylglycerol in the SecA-independent translocation of M13 procoat in Escherichia coli was demonstrated. Processing of procoat to mature coat protein was retarded when the level of phosphatidylglycerol was reduced. In vitro translocation experiments using inner membrane vesicles isolated from a strain with inducible synthesis of phosphatidylglycerol, showed that translocation of procoat and of a SecA-dependent procoat analog was proportional to the content of phosphatidylglycerol. Moreover, introduction of phosphatidylglycerol by means of a lipid transfer method into phosphatidylglycerol-depleted inner membrane vesicles, efficiently restored procoat translocation. The phosphatidylglycerol dependence in both the SecA-dependent and -independent translocation pathway indicates that phosphatidylglycerol plays a dual role in translocation. We suggest that besides membrane binding of SecA this lipid has a direct interaction with the M13 procoat in translocation across the inner membrane.
Adenosine Triphosphatases↗
Pf3 coat protein forms voltage-gated ion channels in planar lipid bilayers.
The coat protein of bacteriophage Pf3 forms discrete and stable ion channels of uniform size in planar bilayers of asolectin. Its primary sequence suggests a channel formed by a bundle of transmembrane helices. Since the apparent transmembrane region only consists of strongly hydrophobic residues, it represents a new class of channel-forming proteins. The channel activity is strongly voltage-dependent. The single-channel conductance of 60 pS (at 100 mV) in 0.2 M NaCl is slightly voltage-dependent, indicating conformational changes of the pore upon variation of the transmembrane electric field. The channel is unselective which suggests that the pore is of aqueous character. For the observed conductance, a channel diameter of 3.6 A is consistent with a tetrameric alpha-helix bundle, as calculated from a barrel-stave model. A pronounced dependence of the gating kinetics with increasing voltage arises from two opposing effects: an increase in the number of open channel structures, and a simultaneous, more than 3-fold decrease in the channel lifetime. Thus, a maximum activity is reached around 100 mV, a range which corresponds well with physiological membrane potentials. The channels activate only upon application of a positive voltage on the side of the membrane to which the protein had been added. The slow relaxation of the mean current upon application of sudden voltage jumps indicates a strong activation barrier in the channel gating process, which may result from the membrane translocation of the charged residues of the peptide ends. A channel-mediated import mechanism is suggested for the bacterial infection by phage DNA.
Amino Acid Sequence↗
In vitro comparison of various types of sodium perborate used for intracoronal bleaching of discolored teeth.
Fifty-eight extracted incisors were artificially stained to compare the efficacy of sodium perborate preparations used for intracoronal bleaching. All teeth were bleached for a 6-day period and the bleaching pastes replaced at days 1 and 3 (walking bleach technique). Sodium perborate-monohydrate, trihydrate, or tetrahydrate in conjunction with 30% H2O2 as well as tetrahydrate, either mixed with H2O or prepared as an experimental gel, were placed intracoronally at a level of 1-mm below the vestibular cementoenamel junction. Standardized slides were used to evaluate the color changes during bleaching. Success rates between 46 and 77% could be achieved, but no significant differences in final bleaching results between any of the sodium perborate types were observed. The use of the experimental gel resulted in comparable esthetic results (54%), although the portion of tetrahydrate in the gel was lower than that of the other preparations. In contrast to general recommendations that bleaching agents be left for 3 to 7 days in the access cavity before replacement, shorter bleaching intervals did not seem to affect the success.
Borates↗
Spatiotemporal morphological transitions in thin-layer electrodeposition: The Hecker effect.
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Journal Article↗
Multiple cytochrome P450 isozymes in murine skin: induction of P450 1A, 2B, 2E, and 3A by dexamethasone.
Cytochrome P450s (P450s) are a supergene family of enzymes responsible for the metabolism of a wide range of endogenous and foreign compounds. P450 isozymes possess overlapping substrate specificity. Systemic administration of dexamethasone, a widely used topical agent in dermatologic practice, to animals is known to result in the induction of multiple P450 isozymes in liver. In this study the effect of topical application of dexamethasone to mice on P450-dependent monooxygenase activities, expression of P450 isozymes, and P450 mRNA levels in skin was assessed. The treatment of mice with dexamethasone resulted in significant induction of 7-ethoxyresorufin O-deethylase (2.3 times), 7-pentoxyresorufin O-depentylase (19.2 times), para-nitrophenol hydroxylase (7.5 times), and erythromycin N-demethylase (2.2 times) activities; the monooxygenases catalyzed preferentially by P450 isozymes 1A1, 2B1, 2E1, and 3A, respectively. Immunoblot analysis of cutaneous microsomes, employing antibodies directed against purified P450s 1A1/2, 2B1/2, 2E1, and 3A, showed that dexamethasone treatment results in an increased immunoreactivity (1.8-13.9 times). In immunohistochemical staining of skin with antibody against P4502B1/2, topical application of dexamethasone resulted in an increased reactivity towards microsomal protein in the suprabasal layer of the epidermis and with the cells of the hair follicles. Whereas constitutive expression of mRNAs for CYP1A1 and CYP2E1 was evident in murine skin, any change in the levels of these mRNAs following treatment with dexamethasone was not apparent. The results of our study indicate that the application of dexamethasone to murine skin results in the induction of several families of P450 isozymes, suggesting that murine skin contains multiple inducible P450 isozymes capable of participating in the metabolism of a wide range of xenobiotics and endogenous compounds.
Administration, Topical↗
Immunohistochemical demonstration of myoepithelial cells in sweat gland carcinomas.
Although myoepithelial cells are detectable in many benign sweat gland tumours, little is known about their role in sweat gland carcinomas. To specifically demonstrate myoepithelial cells, paraffin sections from 46 sweat gland carcinomas were stained, using a standard avidin-biotin-peroxidase complex method, with the monoclonal alpha-smooth muscle actin antibody 1A4. Myoepithelial cells were not found in adenoid cystic eccrine carcinoma (n = 2), malignant nodular hidradenoma (n = 2), porocarcinoma (n = 4), extramammary Paget's disease (n = 12), sclerosing sweat duct carcinoma (n = 4) or in adenosquamous-mucoepidermoid carcinoma (n = 1). In contrast, myoepithelial cells were demonstrated in two of eight apocrine adenocarcinomas, one of six mucinous eccrine carcinomas and two of seven eccrine adenocarcinomas. In all these tumours myoepithelial differentiation was found in peripheral cells of solid tumour islands, or in basal cells of tubular structures. However, in most areas of the tumours, myoepithelial layers were discontinuous. Cells in the centre of solid tumour nodules, and luminal cells of tubular structures, were negative for alpha-smooth muscle actin. In analogy to breast tumours, in which malignancy and invasiveness correlate with scattered or absent myoepithelial cells, we suggest that disrupted myoepithelial layers in sweat gland carcinomas may be interpreted as a loss of the invasion barrier.
Actins↗
Radicular penetration of hydrogen peroxide during intra-coronal bleaching with various forms of sodium perborate.
The development of external cervical root resorption following internal bleaching of discoloured pulpless teeth is associated with the use of hydrogen peroxide. The aim of the study was to determine radicular penetration of hydrogen peroxide following intracoronal bleaching with various forms of sodium perborate. 63 extracted human incisors were root filled and stained artificially. Standardized cementum defects were created on the mesial and distal aspects of the root directly below the cemento-enamel junction (CEJ). Using the walking bleach technique all teeth were bleached for a 6-day period, with replacement of the bleaching paste after days 1 and 3. Sodium perborate monohydrate (MH), trihydrate (TRH) or tetrahydrate (TH) was mixed with H2O2 or H2O and subsequently placed intracoronally 1 mm below the labial CEJ. The teeth were divided into six groups: I. MH + H2O2(30%) (n = 12); II. TRH + H2O2(30%) (n = 12); III. TH + H2O2(30%) (n = 12); IV. TH + H2O (n = 12); V. TH + H2O, gel (n = 12); VI. no bleaching paste (n = 3). At baseline and at days 1, 3 and 6 the amount of H2O2 taken up from the surrounding medium of each root was indirectly recorded and calculated as p.p.m. Almost all teeth of the experimental groups showed leakage of hydrogen peroxide compared to those of the control group. The radicular penetration of hydrogen peroxide was significantly higher in teeth of groups I and III than in those of groups IV and V (P < or = 0.001). In conclusion, the amount of hydrogen peroxide leakage depends, among other factors, on the form of sodium perborate used.(ABSTRACT TRUNCATED AT 250 WORDS)
Borates↗
Plasmid Transfer into the Homoacetogen Acetobacterium woodii by Electroporation and Conjugation.
Shuttle vectors (pMS3 and pMS4) which replicated in Escherichia coli and in gram-positive Acetobacterium woodii were constructed by ligating the replication origin of plasmid pAMbeta1 with the E. coli cloning vector pUC19 and the tetM gene of streptococcal transposon Tn916. Electrotransformation of A. woodii was achieved at frequencies of 4.5 x 10 transformants per mug of plasmid DNA. For conjugal plasmid transfer, the mobilizable shuttle vector pKV12 was constructed by cloning the tetM gene into pAT187. Mating of E. coli containing pKV12 with A. woodii resulted in transfer frequencies of 3 x 10 to 7 x 10 per donor or recipient.
Journal Article↗
[The removable partial denture. A report on the annual congress of the Prosthodontics Society of Switzerland (ZPGS) of 3/4 September 1993 in Biel].
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Dental Materials↗