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R Kern

Publications and source records attributed to R Kern.

At least 73 records · Page 4Linked to original sources
PubMed1989

Dam methylated and hemimethylated oriC plasmids are replicated symmetrically; a novel and general test of replication symmetry.

Deoxyadenosine methylation (dam) of the numerous GATC sequences present in the Escherichia coli origin of chromosomal replication (oriC) has been shown to be important both in vivo and in vitro for efficient initiation of DNA synthesis. Recent in vivo data suggest that initiation is only inefficient when these sequences are hemimethylated. This raises the interesting possibility that initiation may be inefficient because it only takes place on one strand of the template, i.e., replication is asymmetric on hemimethylated DNA. We tested this possibility by a novel and rapid approach which relies on the specificities of the restriction endonucleases MboI, MboII and DpnI. Although we show that replication takes place equally well on both strands of methylated and hemimethylated oriC DNA templates, the method should be applicable to the analysis of replication symmetry on most DNA templates which contain methylated deoxyadenosine GATC sequences as part of MboII restriction sites.

Base Sequence↗
PubMed1989

A microscale procedure for isolating and sequencing the viroid RNA present in one gram of infected leaf tissue.

A microscale procedure for the isolation and purification of viroid RNA from one gram of viroid-infected leaf tissue and for its subsequent sequencing at the cDNA level is described using potato spindle tuber viroid (PSTV) as model system. Total nucleic acids are phenol-extracted and salt-fractionated with 2 M LiCl. The viroid-containing fraction is then subjected to bidirectional polyacrylamide gel electrophoresis. This removes all co-fractionated cellular RNAs from the circular viroid RNA which is finally recovered from the gel in pure form by isotachophoresis. Thus, from one gram of PSTV-infected tomato leaf tissue, about 100-250 ng of circular PSTV RNA can be obtained and used as template for several DNA primer-directed reverse transcription reactions. From the primer-extended overlapping cDNAs the entire sequence of the viroid progeny synthesized in an individual plant or plant leaf can thus be established by Maxam-Gilbert sequencing. This renders the procedure especially suited for the routine analysis of the in vivo fate of viroid mutants constructed in vitro.

Autoradiography↗
PubMed1989

Purification and characterization of a low-molecular-weight membrane protein with affinity for the Escherichia coli origin of replication.

A purification procedure was devised for a low-molecular-mass (about 10-kilodalton) membrane protein from Escherichia coli that was shown to bind specifically to the chromosomal replication origin region (oriC). Nitrocellulose membrane retention assays showed the binding site to be adjacent to the right boundary of the oriC minimal sequence. We determined the amino acid sequence of the N-terminal and C-terminal regions as well as the global amino acid composition of this membrane protein. Specific antibodies against the protein were produced and used to confirm the cell membrane location of the protein. These results demonstrate that this is a new membrane protein, different from the previously described B' protein, with specific binding activity for the oriC region. We propose that this protein be called membrane oriC-binding protein 2 (MOB2 protein).

Amino Acid Sequence↗
PubMed1987

Mechanism of leukotriene D4-induced bronchoconstriction in normal subject.

Leukotriene (LT) D4 is a potent constrictor of human airways and a putative mediator of asthma. However, its mechanism of action in man has not been established. In the present study we sought to determine the role of upper airway reflexes, cholinergic pathways, and cyclooxygenase products of arachidonic acid metabolism in mediating LTD4-induced bronchoconstriction in man. Six normal subjects underwent bronchoprovocation testing with LTD4 after pretreatment with either aerosolized phosphate-buffered saline, aerosolized atropine (1.5 mg), aerosolized lidocaine (80 to 160 mg), or oral indomethacin (50 mg three times daily for 10 doses). Specific airway conductance (SGaw), the flow rate at 30% of vital capacity from a partial forced expiratory maneuver (V30P), and the FEV1 were measured at each concentration of LTD4. We calculated the provocative concentration of LTD4 required to produce a 35% fall in SGaw (PC35SGaw) or a 30% fall in V30P (PC30V30P) and the slope of the LTD4 dose-response curve. Atropine increased baseline SGaw 49% (p less than 0.01) and V30P 43% (p less than 0.005). Atropine also increased the PC35SGaw and PC30V30P and increased the slope of the LTD4 dose-response curve. However, neither of these opposing effects was significant. Lidocaine had no effect on either baseline function or the airway response to LTD4. Indomethacin produced small decreases in baseline V30P (12%) and FEV1 (3%) (p less than 0.05 for both), but it too had no effect on the airway response to LTD4. These results indicate that the bronchoconstriction produced by aerosolized LTD4 in normal subjects is not mediated by cyclooxygenase products of arachidonic acid metabolism or irritant receptors in the upper airways. Although cholinergic pathways may play some role, the data suggest that bronchoconstriction results, at least in part, from a direct effect of LTD4 on airway smooth muscle.

Adult↗
PubMed1987

[A case of syphilitic uveitis simulating Harada disease].

On the basis of clinical findings and fluorescein angiography the authors diagnosed Harada's disease in a 30-year-old Caucasian woman. A drastic deterioration occurred under treatment with corticosteroids and immunosuppressive agents. Another cause of the uveitis was looked for and a reactive serology for lues was found in the blood and the cerebrospinal fluid. The severe inflammation was then treated successfully with high doses of penicillin, intravenously administered; visual acuity also improved as a result this treatment. The differential diagnosis of the two conditions is discussed.

Adult↗
PubMed1986

Characterization of the airway response to inhaled leukotriene D4 in normal subjects.

To better understand the role of leukotriene D4 (LTD4) in the pathogenesis of airway hyperreactivity, we administered aerosolized LTD4 on multiple occasions to 6 normal subjects and measured specific airway conductance (SGaw) by body plethysmography and the flow rate at 30% of vital capacity from partial forced expiratory maneuvers (V30P). Dose-response curves generated from standard bronchoprovocation tests revealed that LTD4 was 280 to 590 times more potent as a bronchoconstrictor than was methacholine. The airway response to single bronchoconstricting doses of LTD4 administered on separate days was reproducible for SGaw (r = 0.86, p less than 0.05) and V30P (r = 0.95, p less than 0.005). The response to 3 consecutive single-dose LTD4 challenges performed on the same day, allowing SGaw and V30P to return to greater than 90% of the control value between challenges, demonstrated tachyphylaxis: the response to the third dose was significantly less than the response to the first dose for SGaw (p less than 0.001) and for V30P (p less than 0.01). A single bronchoconstricting dose of LTD4, given before a standard methacholine dose-response challenge, lowered the concentration of methacholine needed to decrease V30P 30% (p less than 0.05) and SGaw 35% (p = 0.09). A single bronchoconstricting dose of methacholine preceding a standard methacholine challenge did not have this effect. LTD4 and methacholine demonstrated a cumulative dose-effect when progressively higher concentrations were inhaled. In summary, the airway response to LTD4 is reproducible, exhibits tachyphylaxis, and a cumulative dose-effect occurs. As well, LTD4 increases the responsiveness of normal airways to methacholine.

Adult↗
PubMed1985

[Drug therapy of glaucoma with adrenergic and anti-adrenergic agents].

This paper presents a survey for practitioners: The antagonism between the cholinergic and the adrenergic systems originally seemed clear and easy to understand. With the increasing differentiation of the peripheral adrenergic receptors alone into presynaptic alpha 2 and beta 2 and postsynaptic alpha 1, beta 1, and beta 2 subtypes, the current explanation of the antagonism as a simple process, the regulation and correction of aqueous humor dynamics, is increasingly being questioned. For example, the distribution of adrenergic receptors in the extraocular muscles is different in various species of mammals; both stimulation and blockage of beta 2-adrenoreceptors induce a fall in intraocular pressure. With the introduction of adrenergic and antiadrenergic agents into the treatment of the glaucomas, more and more questions arise concerning sympathomimetic action and interaction at subcellular level.

Adrenergic Agonists↗
PubMed1985

[Complications and after care in aphakic and pseudophakic patients].

The aim of every cataract operation is optical rehabilitation. With the greater social demands made today by aphakic and pseudophakic patients, the recognition and treatment of diseases with early or late onset after cataract extraction assumes greater importance. The early aims are rapid mobilization, complete absence of irritation, and normalization of pressure. Regular administration of corticosteroids, essential in the post-operative treatment of pseudophakia, may mask a bacterial or mycotic infection. The sole indication for removal of an implant is the uveitis-glaucoma-hyphema syndrome. The prevention and treatment of cystoid macular edema is discussed: whether or not the posterior lens capsule is intact seems to play a major role in the pathogenesis of this early or late complication in both aphakia and pseudophakia.

Adrenal Cortex Hormones↗
PubMed1985

Internally standardized determination of anipamil in human plasma by means of high-pressure liquid chromatography.

A high pressure liquid chromatographic method with internal analogue standardization for the determination of anipamil in plasma is described. The method comprises extraction from plasma diluted with citrate buffer pH 3 using n-heptane/isoamylalcohol (95/5, v/v), distribution between this organic phase and a methanol/citric acid mixture, and quantification by means of fluorescense detection after HPLC separation using a reverse phase. When using 1 ml plasma the lower limit of detection is 0.2 ng/ml. Under routine conditions the limit of determination is estimated at 1 ng/ml, with higher numbers of replicates and with a predetermined precision limit of 15% concentrations as low as 0.6 ng/ml can be determined reliably. The variation coefficients drop from about 10% in the low range to about 5% at 2 ng/ml or more. The determination of anipamil is not impaired by its N-nor-compound, which is to be expected as metabolite. Neither do other potential metabolites of anipamil with very similar chromatographic behaviour interfere with its quantification.

Chromatography, High Pressure Liquid↗
PubMed1984

[Pseudophakia--correction of unilateral cataract following penetrating eye injuries in children].

Between 1972 and 1980 extracapsular cataract surgery was performed in 24 children, followed by implantation of an iris clip lens, after primary reconstruction of the anterior segment. Regular follow-ups were carried out at 3-month intervals. Discission of a secondary cataract was necessary in 19 cases. Visual acuity of 0.5 or more was achieved by 19 children. An orthophoria or exophoria was also found in 19 children with positive depth perception after a minimum of 2 and a maximum of 9.5 years' observation.

Adolescent↗
PubMed1984

Use of Healon for corneal trephination in penetrating keratoplasty.

We report the use of Healon to assist in the performance of penetrating keratoplasty. Filling the recipient's anterior chamber with Healon prior to trephination permits complete penetration of the cornea with less chance of damaging underlying structures.

Anterior Chamber↗
PubMed1983

Regulation by glucocorticoids of rat-liver phenylalanine hydroxylase in vivo.

Phenylalanine hydroxylase, a liver-associated enzyme, is induced markedly by glucocorticoids in two permanent rat-hepatoma cell lines. In order to gain evidence that this phenomenon also occurs in vivo, we examined the effect of adrenalectomy and/or hormone supplementation on the levels of phenylalanine hydroxylase in the livers of adult rats: glucocorticoid administration increases, and adrenal ablation reduces, the activity of the hepatic enzyme, and the diminution occurring in the latter instance is entirely prevented by concurrent hormone replacement. These results thus corroborate earlier findings from a single experiment and are consistent with the hypothesis that adrenal corticosteroid hormones participate in modulating phenylalanine-hydroxylase levels within the diploid hepatocyte.

Adrenalectomy↗
PubMed1983

Regulation of expression of genes for enzymes of the mammalian urea cycle in permanent cell-culture lines of hepatic and non-hepatic origin.

We present here the results of investigations conducted by ourselves and others on the regulation of the expression of genes encoding the enzymes of the mammalian urea cycle as manifest in cultured cells of both hepatic and extrahepatic origin. Upon consideration of the recently discovered discrete non-hepatic arginase genetic locus in man and our consequent hypothesis that the form of arginase thus transcribed in such extrahepatic cells functions principally in providing ornithine for protein anabolism and polyamine biosynthesis, rather than in detoxifying ammonia through urea formation, we have chosen instead to study permanent cell lines that are derived from liver and continue to perform a variety of hepatic functions in culture as experimental models for probing the molecular mechanisms underlying the control of ureagenesis within the mature liver cell. Of two such arginase-positive rat-hepatoma lines, we have characterized extensively in one (H4-II-E-C3) the mode of action of glucocorticoids in augmenting the cellular levels of this enzyme as well as of argininosuccinate synthetase. To this end, we have recently demonstrated that these stimulations are both mediated by binding of the hormones to classical cytoplasmic steroid receptors in a specific and saturable fashion and have thus concluded that the H4-II-E-C3 line will provide a suitable cell culture system for subsequent more detailed experiments from which the information garnered will continue to be relevant to the ureagenic pathway as modulated in the differentiated hepatocyte in vivo.

Animals↗
PubMed1982

Regulation of glucocorticoids of arginase and argininosuccinate synthetase in cultured rat hepatoma cells.

We have examined and characterized the regulation by glucocorticoids of the levels of arginase and argininosuccinate synthetase in two rat hepatoma cell lines (H4-II-E-C3 and MH1C1). Hydrocortisone elevates the activity of both enzymes in a time- and dose-dependent fashion. This effect was blunted markedly by small amounts of ethanol (0.1 to 0.9% [v/v]) and blocked substantially by a high molar excess of the "anti-inducer" steroid fluoxymesterone. The other "optimal" inducers dexamethasone and corticosterone were as effective as hydrocortisone in elevating the levels of these enzymes at saturating concentrations. Inhibition of these stimulations by cycloheximide indicated that ongoing cellular protein synthesis was required for both effects, and the admixture of extracts from fully stimulated and basal cells gave no evidence for the existence of direct inhibitors or activators of either enzyme. The results corroborate findings from earlier whole-animal studies and provide evidence for the following conclusions. (i) This stimulation by hydrocortisone of urea-cycle enzymes in the cultured hepatoma cells is mediated by a classical glucocorticoid mechanism involving initial binding to specific cytoplasmic steroid receptors and the eventual accumulation of new enzyme molecules. (ii) These cell lines thus constitute valid experimental models for use in further detailed studies on the molecular mechanism(s) through which glucocorticoids and intermediary metabolites effect a selective modulation of arginase and argininosuccinate-synthetase gene expression in the differentiated mammalian liver.

Animals↗