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R Lehner

Publications and source records attributed to R Lehner.

67 records · Page 4Linked to original sources
PubMed1993

Purification of an acyl-CoA hydrolase from rat intestinal microsomes. A candidate acyl-enzyme intermediate in glycerolipid acylation.

We have purified to apparent homogeneity an acyl-CoA hydrolase activity from rat intestinal villus cell microsomes by heparin and anion exchange and affinity chromatography. The purified 54-kDa acyl-CoA hydrolase along with several microsomal proteins form a covalent acyl-protein bond upon incubation with an activated fatty acid (acyl-CoA). The acyl moiety of the acylated acyl-CoA hydrolase is stable to denaturation and extraction with organic solvents, but is displaced by neutral hydroxylamine or mercaptoethanol, indicating a labile high energy (thio)ester linkage. The enzyme activity is inhibited by thiol-directed reagents and activated by the presence of dithiothreitol suggesting the presence of a cysteine residue(s) at or near the active site. Common serine-esterase inhibitors (NaF, phenylmethylsulfonyl fluoride) and activators (Mg2+, Ca2+) had no effect on the hydrolase activity. The enzyme hydrolyzed (transferred to water) 14-20 carbon acyl-CoA with similar efficiencies and did not utilize glycerophospholipids or mono- and diacylglycerols as potential acyl donors/acceptors. Phospholipids and mono- and diradylglycerols at concentrations below 100 microM or polyclonal antibodies raised against the purified hydrolase did not inhibit the enzyme activity. However, the acyl-CoA hydrolase activity could be immunoprecipitated from solubilized microsomes or purified enzyme preparations with corresponding decrease of the hydrolase activity in the supernatant of the immunoprecipitate. Immunoblotting studies show cross-reactivity with a protein of an identical molecular mass in other rat or human tissues. It is concluded that the microsomal acyl-CoA hydrolase deserves consideration as a candidate acyl-enzyme intermediate in glycerolipid synthesis when associated with appropriate acyltransferases.

Acylation↗
PubMed1993

Triacylglycerol synthesis by an sn-1,2(2,3)-diacylglycerol transacylase from rat intestinal microsomes.

A membrane-associated sn-1,2(2,3)-diacylglycerol transacylase activity was purified 550-fold from the microsomes of rat intestinal villus cells. The enzyme was solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and was isolated and purified by sequential chromatography on hydroxylapatite, an anion exchanger, Affi-Gel heparin and Cibacron blue A-agarose. SDS-polyacrylamide gel electrophoresis revealed two major closely migrating polypeptides of apparent molecular mass of 50 and 52 kDa. Incubation of the isolated enzyme with sn-1,2(2,3)dioleoyl-[2-3H]glycerol yielded trioleoyl[2-3H]glycerol and 2-oleoyl[2-3H]glycerol. The synthesis of triacylglycerols was independent of acyl-CoA, as indicated by the absence of incorporation of radioactivity from [1-14C]oleoyl-CoA into mono-, di-, or triacylglycerols under these conditions. The transacylase did not hydrolyze triacylglycerols readily and was not affected by the acyltransferase inhibitors, N-ethylmaleimide and 4,4'-diisothiocyanostilbene-2,2'-disulfonate, while the lipase/hydrolase inhibitors NaF, phenylmethylsulfonyl fluoride, and diethyl p-nitrophenyl phosphate caused partial inactivation. The enzyme was specific toward 1(3)-positions of rac 1,2-diacylglycerols but did not utilize the -1,3-diacylglycerols or other neutral lipid esters effectively and was not capable of removing fatty acids from phosphatidylcholine or of transfer of fatty acids from X (unspecified enantiomer)-1,2-diacylglycerol to lysophosphatidylcholine. Since the diacylglycerol transacylation did not lead to accumulation of either sn-1,2- or sn-2,3-diacylglycerols during incubation with the racemates, it is concluded that both sn-1,2- and sn-2,3-s are utilized at similar rates.

Acyltransferases↗
PubMed1993

Stereospecificity of monoacylglycerol and diacylglycerol acyltransferases from rat intestine as determined by chiral phase high-performance liquid chromatography.

Using chiral phase high-performance liquid chromatography of diacylglycerols, we have redetermined the ratios of 1,2-/2,3-diacyl-sn-glycerols resulting from acylation of 2-monoacylglycerols by membrane bound and solubilized triacylglycerol synthetase of rat intestinal mucosa. With 2-oleoyl[-3H]glycerol as the acyl acceptor and oleoyl-CoA as the acyl donor, 97-98% of the diacylglycerol product was 1,2(2,3)-dioleoyl-sn-glycerol, 90% of which was the sn-1,2- and 10% the sn-2,3-enantiomer. The remaining diacylglycerol (less than 3%) was the sn-1,3-isomer. The overall yield of acylation products was 70%, of which 60% were diacylglycerols and 40% triacylglycerols. With 2-oleylglycerol ether as the acyl acceptor and [1-14C]oleoyl-CoA as the acyl donor, 90% of the diradylglycerol was 1-oleoyl-2-oleyl-sn-glycerol and 10% was the 2-oleyl-3-oleoyl-sn-glycerol. The diradylglycerols made up 96% and the triradylglycerols 4% of the radioactive product. With 1-palmitoyl-sn-glycerol as the acyl acceptor and [1-14C]oleoyl-CoA as the acyl donor, the predominant reaction product was 1-palmitoyl-3-oleoyl-sn-glycerol. The 3-palmitoyl-sn-glycerol was not a suitable acyl acceptor. Both 1,2- and 2,3-diacyl-sn-glycerols were substrates for diacylglycerol acyltransferase as neither isomer was favored when 1,2-dioleoyl-rac-[2-3H]glycerol was used as the acyl acceptor. There was a marked decrease in the acylation of the 1(3)-oleoyl-2-oleyl-sn-glycerol to the 1,3-dioleoyl-2-oleyl-sn-glycerol. It is concluded that neither monoacylglycerol nor diacylglycerol acyltransferase exhibit absolute stereospecificity for acylglycerols as fatty acid acceptors.

Acyltransferases↗
PubMed1992

Utilization of 2-monoacylglycerols for phosphatidylcholine biosynthesis in the intestine.

Conventional preparations of intestinal microsomes were observed to incorporate acetone-solubilized 2-oleoyl-[2-3H]glycerol into dioleoylglycerophosphocholine in the presence of oleoyl CoA and CDP-choline. The apparent Km values for CDP-choline utilization were 77 +/- 10 microM in rat and 72 +/- 5 microM in hamster. The incorporation ratio of glycerol into triacylglycerols and phosphatidylcholines was 4.5:1 and 25:1 in the rat and hamster, respectively. Endogenous diacylglycerols generated by phospholipase C treatment of microsomes readily equilibrated with the diacylglycerols arising via the monoacylglycerol pathway as indicated by a dilution of the radioactivity in the triacylglycerol and phosphatidylcholine synthesized from radioactive 2-monooleoylglycerol. These results suggest an alternative pathway for glycerophospholipid formation in the intestinal mucosa during possible inhibition of the phosphatidic acid pathway by dietary 2-monoacylglycerols. It is concluded that exogenously added monoacylglycerol can serve as a precursor for microsomal diacyl- and triacylglycerol as well as phosphatidylcholine. The inability to demonstrate comparable monoacylglycerol utilization in earlier experiments in attributed to the inhibition of choline phosphotransferase by the detergents used to solubilize the acylglycerols.

Animals↗
PubMed1991

[Differentiation antigens on normal bone marrow cells and following culture with growth factors in patients with acute myeloid leukemia (AML)].

In an attempt to detect residual leukemic cells during complete remission (CR), we used 4 monoclonal antibodies detecting markers at different stages of myeloid maturation. Bone marrow cells of AML patients at diagnosis/relapse or in CR were compared with normal controls at day 0 and 7, after incubation with colony stimulating factors, by the alkaline phosphatase/antialkaline phosphatase method. In AML patients at diagnosis/relapse, the expression of the early differentiation markers (CD34, HLA DR) was significantly increased and that of the late marker CD15 significantly decreased at day 0. After day 7 liquid cultures, the markers HLA DR and CD13 were significantly increased and CD15 significantly decreased. During CR a significant increase in day 7 liquid cultures of the markers HLA DR and CD13 was found compared to normal controls. These results may reflect the proliferation in culture of residual leukemic cells in CR patients.

Adolescent↗
PubMed1991

Comparative sequence analysis of human cytomegalovirus strains.

Three regions of DNA from five low-passage clinical isolates of human cytomegalovirus were amplified by polymerase chain reaction. The DNA sequences as well as the predicted amino acid sequences were compared with those of the laboratory strains AD169 and Towne. The genomic regions consisted of (i) three regions from the major glycoprotein (gp58/116, unique long [UL]55), (ii) three regions from the integral membrane protein (IMP, UL100), and (iii) a region from the major immediate-early 1 and 2 (IE-1/2) enhancer/promoter. Homologies ranged from 75.8 to 100.0% on the nucleotide level and from 47 to 100% on the amino acid level. The following two patterns were observed. (i) There are regions with a high degree of conservation with few scattered point mutations (mainly in the IE-1/2 enhancer/promoter and in the IMP gene). (ii) There are clusters of highly variable regions (parts of the gp58/116 gene and of the IMP gene). Within the areas of high variability, the strains could be classified into a limited number of subtypes.

Amino Acid Sequence↗
PubMed1989

Identification and characterization of a human cytomegalovirus gene coding for a membrane protein that is conserved among human herpesviruses.

A rabbit antiserum was raised against envelope material from purified human cytomegalovirus strain AD169. The serum recognized polypeptides 200, 170, 160, 75, 58, and 45 kilodaltons in size. It was used to screen a cDNA library constructed from poly(A)+ RNA from human cytomegalovirus-infected cells in the expression vector lambda gt11. A recombinant bacteriophage expressing cytomegalovirus-specific sequences was identified, and the corresponding gene was mapped to the HindIII R fragment. The gene is transcribed into a late 1.5-kilobase RNA. The nucleotide sequence of the coding region was determined. Computer analysis of the gene product revealed a polypeptide containing multiple potential membrane-spanning domains, representing a type of protein not identified in the envelope of herpesviruses before. The protein shows homology on the amino acid level to hypothetical proteins from reading frames BBRF3 of Epstein-Barr virus, UL10 of herpes simplex virus type 1, and ORF50 of varicella-zoster virus. By using an antiserum raised against procaryote-expressed parts of the cytomegalovirus membrane protein, a 45-kilodalton structural component of the virus was identified as the gene product.

Amino Acid Sequence↗
PubMed1989

Recurrent angioedema associated with hypogonadism or anti-androgen therapy.

Two male patients with hypogonadism and four female patients who received an anti-androgen as contraceptive (cyproteronacetate) and who had recurrent angioedema are described. In one male patient, augmentation of the plasma androgen level resulted in disappearance of symptoms. In the four female patients, recurrent angioedema and urticaria developed after initiation of the anti-androgen treatment. Cessation of cyproteronacetate and a change to another contraceptive resulted in complete resolution of the previously frequent angioedematous attacks. The women are still symptom free after more than 60 patient's months. These cases suggest that an androgen deficit due to either hypogonadism or to anti-androgen treatment may be another cause of angioedema. One of the two male patients was untreated and presented with 40% normal value of C1-INH. Androgen therapy normalized C1-INH concentration in this male patient. Functional C1-INH in the same patient, studied before and after the beginning of androgen therapy, clearly increased when assessed by inhibition of amidolytic activity of C1-esterase. The other male patient with hypogonadism had already been under androgen treatment for 4 years and had C1-INH levels in the normal range. In the female patients, complement profiles were normal before and after cessation of anti-androgen contraception; however, the C1-INH plasma levels were higher after cessation of anti-androgen anticonception. These results indicate an effect of androgen deficit on the level of C1-INH in circulating plasma but do not prove a role of C1-INH in angioedema associated with diminished androgen plasma levels.

Adult↗
PubMed1988

[Action of various growth factors on bone marrow cultures in acute myeloid leukemia (ALM)].

In an attempt to detect residual leukemic cells during remission, we examined differences in the stimulatory capacity of colony stimulating factors (CSF) added to agar cultures of AML patients at diagnosis/relapse (group I) and in remission (II), or of normal controls (III). Supernatant from the human bladder carcinoma cell line 5637 and human recombinant granulocyte-macrophage-CSF were compared with supernatant from human placenta as standard. Group I showed wide patient-to-patient variations. The stimulatory capacity of the 3 CSF produced significant differences within groups II and III, but not between those groups. In conclusion, our comparative investigations do not reveal residual leukemic cells in the marrow of AML patients in remission.

Adolescent↗
PubMed1980

Cytotoxicity of pivalylphenylephrine and pivalic acid to corneal endothelium.

We examined the effects of topically applied pivalylphenylephrine (PPE) and pivalic acid (PA) on the corneal endothelium of rabbits and the direct effects of PPE and PA on monolayer cultures of bovine corneal endothelium. The PPE-treated corneas without epithelium significantly increased in thickness, whereas no change in thickness was observed in corneas with epithelium intact. The PA did not alter the thickness of corneas with or without epithelium. Although 0.001% PE had no noticeable effect in two hours, 0.01% PPE caused breakdown of intercellular junctions in cultured cells in five minutes. Higher concentrations of PPE caused the cells to detach from the culture dishes within 30 minutes of treatment. Only 1% PA caused cell elongation and loss of intercellular contact after 60 to 90 minutes of exposure; lower concentrations did not effect cultured cells.

Administration, Topical↗
PubMedDate not supplied

A novel microperfusion system for the long-term local supply of drugs to the inner ear: implantation and function in the rat model.

Local therapy is practiced for middle and inner ear diseases but is usually restricted to cases of ear drum perforation or repeated invasive intratympanic drug application. Perfusion of drugs on the round window or through the scalae of animals using a pump system suggests that the chronic local drug treatment might also be feasible in humans. However, drug delivery systems that are currently on the market involve repeated reimplantation if they are to be used for long-term drug supply. A bone-anchored, totally implantable micro-drug delivery system (MDS) for patient-controlled drug supply has been developed [Lehner et al., 1997]. In this study, we show the first successful long-term in vivo test of the MDS micro-pump in rats. The process of implantation and first functional tests will be described. The biomaterial used to manufacture the delivery system did not cause any inflammation reaction in any of the 9 animals successfully implanted. After activation of the micro-pump, the drug reservoir and port was found to be fluid-tight. Bolus applications of tetrodotoxin (TTX) to the round window induced a transient decrease of evoked brainstem responses. In 2 animals which carried the MDS for more than 8 months the proper functioning of the pumping device was examined in a 2-3 week interval over a 3 month period. The MDS can be autoclaved even after long-term implantation and can then be reused for subsequent implantations. Designed for life-long implantation in humans, the demonstration of an effective long-term drug supply to the inner ear using the MDS provides an encouraging first step towards future long-term drug treatment of the inner ear in humans.

Administration, Topical↗