PubMed Health⌕ Search

Biomedical subjects

R Kern

Publications and source records attributed to R Kern.

At least 55 records · Page 3Linked to original sources
PubMed1996

Co-ordination between membrane oriC sequestration factors and a chromosome partitioning protein, TolC (MukA).

oriC DNA in the hemimethylated (but not in the fully methylated) state reacts with an Escherichia coli K-12 outer membrane preparation. This reaction is drastically reduced when the membrane preparation of a seqA null mutant is used. An in vitro reconstitution of the activity was undertaken by adding a partially purified SeqA protein to a seqA mutant membrane without success. A possible reason for this failure might be a profound modification of the outer membrane of the seqA mutant (as revealed by the fact that membrane from the mutant sediments more slowly than that from the wild type during ultracentrifugation). There is also a reduction in the content of OmpF protein. Moreover, one of the minor outer membrane proteins involved in partitioning of newly synthesized chromosomes, the ToiC (MukA) protein, was also found to be downregulated in the seqA mutant. This is also true of the hobH mutant grown in a high-osmolarity medium. Mutants of both seqA and hobH stop dividing after hyperosmotic shock, forming filaments (as observed in dam mutants).

Bacterial Outer Membrane Proteins↗
PubMed1996

Differentiation of delayed kidney graft function with gadolinium-DTPA-enhanced magnetic resonance imaging and Doppler ultrasound.

RATIONALE AND OBJECTIVES: The authors differentiate acute tubular necrosis from transplant rejection in patients with delayed kidney graft function using gadolinium (Gd)-DTPA enhanced magnetic resonance (MR) imaging. METHODS: Twenty-four patients after renal transplantation (10 with normal graft function, 14 with delayed graft function) underwent conventional and Doppler sonography and MR imaging examination after bolus application of Gd-DTPA. Within a time period of 512 seconds, 39 single-slice MR images were obtained. Measurements of signal intensity in three regions of interests (cortex, medulla, renal pelvis) resulted in a graphic description of the dynamics of the contrast enhancement. The time between the start of the scan and the peaks of the curves was measured. RESULTS: In patients with normal graft function the curves reached the peaks between 39 and 55 seconds (cortex), 44 and 61 seconds (medulla), and between 161 and 318 seconds (renal pelvis). Six patients with acute tubular necrosis showed normal values for the curves 1 and 2 but markedly prolonged time for curve 3 (between 420 and 512 seconds). In all patients with histologically proven transplant rejection, the peaks of all curves were not reached before the ends of the scans. CONCLUSION: The authors' preliminary results suggest that MR imaging seems to be a sensitive, noninvasive diagnostic tool to differentiate acute tubular necrosis from transplant rejection in the critical early postoperative period.

Adult↗
PubMed1996

Ultrafast computed tomography and three-dimensional image processing of CT sialography in patients with parotid masses poorly defined by magnetic resonance imaging.

The purpose of this study was to determine the efficacy of ultrafast computed tomography (UF CT) in patients with parotid masses poorly defined by magnetic resonance imaging (MRI) and to evaluate the diagnostic potential of three-dimensional (3-D) UF CT sialography when compared with conventional CT sialograms. Thirteen patients with clinical suspicion of a parotid mass, in whom MRI was degraded by motion, underwent UF CT of the parotid region. Two radiologists independently assessed the CT and MR with respect to tumor localization, intraglandular tumor location, tumor margin characteristics, and infiltration of surrounding tissue. In 9 patients, CT sialography was performed using 3-D image processing. Anatomical details and pathologic findings were assessed by three readers using a numerical grad and compared with the findings derived from conventional CT sialography. Histopathologic specimens were obtained in all cases and correlated with the radiographic findings in a consensus manner following the blinded interpretations. UF CT and (suboptimal) MRI provided the same diagnostic information for the evaluation of tumor localization, and intraglandular location. UF CT was superior to MRI in the detection of tumor infiltration, and definition of tumor margins in 2 cases (15%), resulting in a substantial difference in treatment. Three-dimensional CT sialography offered significant improvement in demonstration of anatomic detail (2.5 +/- 0.2 vs 1.5 +/- 0.1, respectively) and pathologic findings (2.6 +/- 0.1 vs 1.3 +/- 0.2, respectively) when compared with conventional CT sialography. UF CT is a viable alternative in uncooperative patients with parotid masses. UF CT 3-D sialography has the potential to allow more precise pre-surgical planning and contributes to the diagnosis and therapy planning of parotid masses.

Adult↗
PubMed1996

Internally standardized simultaneous assay of gallopamil enantiomers in human plasma by means of high performance liquid chromatography.

A high performance liquid chromatographic method with internal analogue standardisation for the simultaneous assay of the gallopamil enantiomers in human plasma after administration of racemic gallopamil is described. The method comprises extraction from alkalinized plasma into an n-hexane/2-propanol phase (95:5), back extraction into 1 N hydrochloric acid and extraction into n-hexane/ 2-propanol (95:5) after addition of 5 N sodium hydroxide. Separation of the optical antipodes was achieved by the use of a chiral phase (Daicel Chiralcel OD-H) and quantification by means of fluorescence detection. A limit of detection of about 0.2 ng/ml was determined for both enantiomers. Under routine conditions the limit of determination (variation coefficient < 20%) was about 0.5 ng/ml for (R)- and (S)-gallopamil. The method is not impaired by the known metabolites. The suitability of the method for clinical pharmacology studies is demonstrated with samples obtained from healthy subjects. First results of kinetic studies in humans showed that the (R)-enantiomer dominates after oral administration of racemic gallopamil.

1-Propanol↗
PubMed1994

Parental strand recognition of the DNA replication origin by the outer membrane in Escherichia coli.

The outer membrane of Escherichia coli binds the origin of DNA replication (oriC) only when it is hemimethylated. We report here the results of a footprinting analysis with the outer membrane which demonstrate that its interaction with oriC occurs mainly at the left moiety of the minimal oriC, where 10 out of 11 Dam methylation sites are concentrated. Two regions, flanking the Integration Host Factor (IHF) sites, are preferentially recognized at the minimum membrane concentration at which oriC plasmid replication is inhibited in vitro. We have identified the putative proteins involved in hemimethylated oriC binding and cloned one of the corresponding genes (hobH). The purified LacZ-HobH fusion protein specifically binds oriC DNA at the same preferential sites as the membrane. A mutant of the hobH gene reveals partial asynchronous initiation of DNA replication.

Amino Acid Sequence↗
PubMed1994

Magnetic resonance assessment of age-related development of the sphenoid sinus.

Magnetic resonance images of the sphenoid sinus in 401 patients under 15 years old were reviewed to establish normal age-related standards. T1-weighted sagittal and T2-weighted axial scans were evaluated for bone marrow conversion, development of pneumatization, spatial enlargement and septation of the sphenoid sinus. The sphenoid sinus had a uniformly low signal intensity (red bone marrow) on T1-weighted images in all children less than 4 months old. Signal intensity changes from hypointense to hyperintense (bone marrow conversion) started at the age of 4 months. Onset of pneumatization was observed in 12% of the patients at age 13-15 months. By age 43-48 months, 85% of the patients showed pneumatization of the anterior part of the sphenoid bone. Pneumatization was complete in all patients older than 10 years. Enlargement of the sinus showed a characteristic profile in each dimension. Median septation was observed irregularly with age, with a maximum of 77%. Septum variants were noticed between 4.5% and 20%. Because paediatric sinus disease is a challenging problem in children, these results may be useful as baseline standards of normal age-related development of the sphenoid sinus during childhood.

Adolescent↗
PubMed1994

Internally standardized simultaneous assay of propafenone and 5-hydroxypropafenone enantiomers in human plasma by means of high performance liquid chromatography.

A high performance liquid chromatographic method with internal analog standardization for the quantification of propafenone and 5-hydroxypropafenone enantiomers in human plasma is described. The method comprises extraction from alkalinized plasma into a toluene/diethylether phase (9:1), separation of the optical antipodes on a silica gel column using N-tert-Boc-(L)-proline as the chiral additive to the mobile phase and UV detection. A detection limit of 10 pmol/assay was established for the propafenone enantiomers, whereas for the optical antipodes of 5-hydroxypropafenone the limit was around 50 pmol/assay. Under routine conditions the limit of quantification (variation coefficient 15%) for the propafenone enantiomers is about 13 pmol/assay and for the 5-hydroxypropafenone enantiomers it is 66 pmol/assay. The determination method for the enantiomers is not impaired by the other metabolites. The suitability of the method for human pharmacology studies is demonstrated with samples obtained from healthy subjects.

Adrenergic beta-Antagonists↗
PubMed1993

In vitro formation of a photoreversible adduct of phycocyanobilin and tobacco apophytochrome B.

The light-stable tobacco phytochrome apoprotein (PHYB) expressed in yeast can be assembled with phycocyanobilin to give a photoreversible adduct. The spectral properties of the reconstituted PHYB-phycocyanobilin species were determined by absorbacen and difference absorbance spectroscopies. The holoprotein exhibits absorbance maxima at 408 nm and 712 nm for the far-red-light-absorbing (Pfr) form and 356 nm and 658 nm for the red-light-absorbing (Pr) form. The ligation of the chromophores to the dimeric PHYB apoprotein resulted in a PHYB-phycocyanobilin adduct with the spectral properties of the Pr form. Kinetic analyses of the in vitro reconstitution for PHYB apoprotein under saturating concentrations of phycocyanobilin revealed a pseudo first-order rate constant of 2.8 x 10(-2)s.-1. The similarity with the reported rate constant for the reconstitution of light-labile phytochrome (PHYA) from oat [Li, L. & Lagarias, J.C. (1992) Phytochrome assembly, J. Biol. Chem. 267, 19,204-19,210] suggests that the mechanisms of chromophore attachment are probably very similar for PHYA and PHYB.

Apoproteins↗
PubMed1993

Calcium/calmodulin-dependent and -independent phytochrome signal transduction pathways.

Phytochrome is a well-characterized plant photoreceptor, able to modulate many morphological, physiological, and biochemical events through as yet undefined mechanisms. By developing single-cell assays to visualize phytochrome responses, we have studied the effects of microinjecting putative signaling intermediates into phytochrome-deficient tomato cells. We demonstrate that phytochrome phototransduction initially involves the activation of one or more G proteins that are coupled to at least two different pathways; one pathway requires calcium and activated calmodulin and can stimulate expression of a photoregulated cab-GUS reporter gene together with the synthesis and assembly of some, but not all, of the photosynthetic complexes. The other pathway, controlling anthocyanin biosynthesis, does not require calcium. Furthermore, our results reveal that phytochrome signaling is cell autonomous and is not likely to require any light-activated steps downstream of the G protein.

ATP Synthetase Complexes↗
PubMed1992

Inhibition of DNA synthesis at the hemimethylated pBR322 origin of replication by a cell membrane fraction.

The replication of both ColE1-type plasmids and plasmids bearing the origin of replication of the Escherichia coli chromosome (oriC) has been shown to be inhibited by hemimethylation of adenine residues within GATC sequences. In the case of oriC plasmids, this inhibition was previously shown to be mediated by the specific affinity of the hemimethylated origin DNA for an outer cell membrane fraction. Here, we suggest that a similar mechanism is operating in the case of the ColE1-like plasmid pBR322 as (i) a hemimethylated DNA fragment carrying the promoter for the RNA which primes DNA synthesis (RNAII) is specifically bound by the same membrane fraction and, (ii) the addition of the membrane fraction to a soluble assay of pBR322 replication results in preferential inhibition of initiation on the hemimethylated template. We suggest that membrane sequestration of hemimethylated origin DNA and/or associated replication genes following replication may be a common element restricting DNA replication to precise moments in the cell cycle.

Base Sequence↗
PubMed1992

Internally standardized simultaneous assay of verapamil and N-demethyl-verapamil enantiomers in human plasma by means of high performance liquid chromatography.

A high performance liquid chromatographic method with internal analog standardization for the simultaneous assay of the verapamil and N-demethyl-verapamil enantiomers in human plasma after administration of racemic verapamil is described. The method comprises extraction from alkalinized plasma into an n-hexane/2-propanol phase (95:5), separation of the optical antipodes on a chiral phase (Daicel OJ) and quantification by means of fluorescence detection. A detection limit of about 6 pmol was determined for all four enantiomers. Under routine conditions the limit of quantification (variation coefficient < 10%) was about 8 pmol for the (R)- and (S)-enantiomers. The determination method for verapamil and N-demethyl-verapamil enantiomers is not impaired by their known metabolites. The suitability of the method for clinical pharmacology studies is demonstrated with samples obtained from healthy subjects. First results of comparative kinetics studies in humans and various animal species show marked species-dependency with prevailing (R)-enantiomer levels in humans and dogs, and the opposite in rats.

Chromatography, High Pressure Liquid↗
PubMed1992

The cucumber long hypocotyl mutant lacks a light-stable PHYB-like phytochrome.

A novel cDNA sequence homologous to a phytochrome B (phyB) gene that was isolated in a library from tobacco tissue has been used in an Escherichia coli expression system to raise anti-phytochrome B (anti-PHYB) polypeptide-specific monoclonal antibodies. The specificity of these antibodies has been tested by cross-reactivity against purified pea light-labile type 1 and light-stable type 2 phytochromes, with some antibodies reacting with the type 2 and none with the type 1 phytochromes. One such antibody, monoclonal mAT1, has been employed to analyze the phytochrome molecular species present in a photomorphogenic long hypocotyl (lh) mutant of cucumber. The results indicated that the mutant contains wild-type levels of the light-labile type 1 phytochrome polypeptide (PHYA), which has an apparent molecular mass of approximately 120 kD, but shows less than 1% (detection limit) of a light-stable polypeptide recognized by mAT1 in wild-type seedlings. This protein, not detectable in the lh mutant, has the properties of light-stable type 2 phytochrome, has an apparent molecular mass of 116 to 117 kD, and remains at constant levels under continuous low-fluence-rate red light. Therefore, we conclude that the lh mutant lacks at least one type 2 phytochrome-like polypeptide, most probably a phyB gene product. The correlation between the lack of this protein and the deficiency or absence of physiological responses to a light-stable phytochrome species in this mutant helps to identify the physiological roles played by the products of different subfamilies within the phytochrome gene family.

Antibodies, Monoclonal↗
PubMed1991

Waste treatment integration in space.

The circumstances and criteria for space-based waste treatment bioregenerative life-support systems differ in many ways from those needed in terrestrial applications. In fact, the term "waste" may not even be appropriate in the context of nearly closed, cycling, ecosystems such as those under consideration. Because of these constraints there is a need for innovative approaches to the problem of "materials recycling". Hybrid physico-chemico-biological systems offer advantages over both strictly physico-chemico or biological approaches that would be beneficial to material recycling. To effectively emulate terrestrial cycling, the use of various microbial consortia ("assemblies of interdependent microbes") should be seriously considered for the biological components of such systems. This paper will examine the use of consortia in the context of a hybrid-system for materials recycling in space.

Anaerobiosis↗
PubMed1990

The E. coli cell surface specifically prevents the initiation of DNA replication at oriC on hemimethylated DNA templates.

A particular outer membrane fraction previously defined as possessing specific affinity for the hemimethylated form of the origin of replication of the E. coli chromosome (oriC) is shown to inhibit the initiation of DNA synthesis at this site on hemimethylated DNA templates in vitro. The replication of fully methylated or unmethylated DNA templates is not affected. Also, no inhibition is observed if initiation takes place at random sites on the hemimethylated template. The key inactivation step appears to be membrane inhibition of DnaA initiator protein binding to oriC. Remethylation of the membrane-bound hemimethylated DNA results in reactivation. Our results demonstrate direct involvement of the membrane in the control of DNA replication. We propose that association/dissociation of the origin from the cell membrane is one of the control elements governing interinitiation times in E. coli.

Bacterial Proteins↗
PubMed1989

dam methylation and the initiation of DNA replication on oriC plasmids.

Plasmid DNA containing the replication origin of the Escherichia coli chromosome (oriC) has been shown to be inefficient as a template for DNA synthesis in vitro when isolated from dam mutants. Here, we extend this study to hemimethylated oriC plasmids and to replication in dam-3 mutant enzyme extracts. The results show that: (1) hemimethylated oriC plasmids replicate with the same low efficiency as nonmethylated DNA; (2) DNA synthesis starts at oriC regardless of the methylated state of the template; (3) replication in dam-3 enzyme extracts is inefficient because this strain is deficient in DnaA protein; and (4) consistent with this observation, the copy number of the oriC plasmid pFH271 is reduced in the dam-3 mutant. However, we have found that low DnaA protein levels in dam-3 mutants are not sufficient to explain the reduced transformation efficiency of oriC plasmids. We suggest that there must exist in vivo inhibitory factors not present or present in low quantities in vitro which specifically recognize the hemimethylated or nonmethylated forms of the oriC region.

Bacterial Proteins↗