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Biomedical subjects

U Klein

Publications and source records attributed to U Klein.

At least 91 records · Page 5Linked to original sources
PubMed1994

Activity of the Chlamydomonas chloroplast rbcL gene promoter is enhanced by a remote sequence element.

The chloroplast gene rbcL encodes the large subunit of ribulose bisphosphate carboxylase. In Chlamydomonas reinhardtii, this gene is transcribed more actively than any other protein-encoding chloroplast gene studied to date. To delineate the rbcL gene promoter, chimeric reporter genes containing fragments of the 5' region of the rbcL gene fused to the coding sequence of the bacterial uidA gene, encoding beta-glucuronidase, were stably introduced into the chloroplast genome of Chlamydomonas by microprojectile bombardment. The relative transcription rates of endogenous and introduced genes were determined in transgenic cell lines in vivo. The basic rbcL promoter is located within the region of the gene extending from positions -18 to +63, taking position +1 as the site of initiation of transcription. A chimeric reporter gene containing only the basic promoter is transcribed only 1-15% as actively as the endogenous rbcL gene, depending on the conditions under which cells are grown and tested. However, a chimeric gene containing rbcL sequences extending to position +170 or beyond is transcribed at about the same rate as the endogenous gene. Deletion of the sequence between positions +170 and +126, well within the protein-encoding region, reduces the rate of transcription to that of reporter genes with the basic promoter alone.

Animals↗
PubMed1994

Variable region gene analysis of B cell subsets derived from a 4-year-old child: somatically mutated memory B cells accumulate in the peripheral blood already at young age.

Tonsillar germinal center and immunoglobulin M+ (IgM+)IgD+ B cells as well as peripheral blood (PB) CD5+ and CD5- (conventional) B cells from a 4-yr-old child were isolated and nucleotide sequences of expressed Ig heavy chain variable regions encoded by VH4 gene family members were determined from amplified cDNA. Whereas both tonsillar IgM+IgD+ cells and the majority of IgM-expressing CD5+ and CD5- PB B cells showed no or little somatic mutation, tonsillar germinal center (GC) B cells and IgG-expressing PB B cells carried a high load of somatic mutations in their V region genes. This suggests that somatically mutated memory B cells which have switched isotype accumulate in the PB already at young age. Their frequency seems to increase with age. On the other hand, the antibody repertoire of tonsillar IgM+IgD+ B cells and the majority of IgM-expressing PB B cells is determined by germline-encoded specificities and by generation of variability in the complementary determining region III through VH-DH-JH recombination. A fraction of IgM-bearing PB B cells carries somatically mutated V region genes and probably represents GC-derived B cells which have left the GC at an early stage of the GC reaction without undergoing isotype switching. 10 VH4 germline genes were found to be expressed. Three gene segments were overrepresented in the sequence collection (35 of 50 clones): VH4.21 (30%), V71-4 (20%), and 3D279D (20%). It appears that most potentially functional VH4 germline genes are expressed in peripheral B cells. Some members of this VH gene family are clearly overrepresented over others.

Amino Acid Sequence↗
PubMed1994

Reconstitution of the myometrial oxytocin receptor into proteoliposomes. Dependence of oxytocin binding on cholesterol.

The requirements for regaining high-affinity binding of the myometrial oxytocin receptor after detergent solubilization were investigated by reconstitution experiments. Large unilamellar liposomes were prepared by reverse-phase evaporation from different mixtures of phospholipids, cholesterol and cholesteryl hemisuccinate. In the presence of the oxytocin receptor solubilized from myometrial membranes from pregnant guinea pig uterus, liposomes were treated with 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate (Chapso) throughout the range of detergent concentrations that cause the transformation of lamellar structures to mixed micelles. Detergent removal was achieved using bio-beads SM-2 as adsorbent. The presence of cholesterol was a prerequisite for regaining high-affinity binding of [3H]oxytocin and 125I-oxytocin antagonist to reconstituted proteoliposomes. Binding of [3H]oxytocin but not of the antagonist was dependent on the presence of Mn2+ ions. Reconstitution after lectin chromatography and photoaffinity labeling of reconstituted vesicles resulted in the exclusive labeling of the oxytocin receptor with a molecular mass of 68-80 kDa.

Affinity Labels↗
PubMed1994

Enhanced selectivity of oxytocin antagonists containing sarcosine in position 7.

Neurohypophyseal hormone analogues containing sarcosine (Sar) in position 7 were prepared to design more potent and selective oxytocin antagonists. The three analogues (1-3) of [Sar7]arginine-vasopressin ([Sar7]AVP) and six analogues (4-9) of [Sar7]arginine-vasotocin ([Sar7]AVT) had a reduced affinity for antidiuretic V2 receptors. The [Sar7]AVP derivatives (1-3) were potent antiuterotonic (in vitro pA2 = 7.5-8.4, in vivo 6.6-7.1) and antipressor (pA2 = 7.2-8.0) agents. The [Sar7]AVT analogues (4-9) were more potent and selective uterotonic antagonists (in vitro pA2 = 7.9-8.6, in vivo 7.1-7.5); their antipressor potencies were reduced (pA2 = 6.4-7.7). The change of the antagonistic potencies was paralleled by a change in the receptor affinities. Among other antiuterotonic analogues, [Mca1, D-Phe2, Sar7]AVT (4, Mca = beta-mercapto- beta,beta-cyclopentamethyl-enepropionic acid) and [Mca1, D-Tyr(OEt)2,Sar7]AVT (6) were synthesized, two highly potent antiuterotonic compounds (in vitro pA2 = 8.3, in vivo 7.4 and 7.5, respectively) with reduced antipressor activity (pA2 = 6.4) and reduced binding affinity to V2 receptors (Kd = 421 and 35 nM, respectively) and no anti-antidiuretic effect. Another potent antiuterotonic analogue, [Mca1,D-Trp2,-Sar7]AVT (9, in vitro pA2 = 7.9, in vivo 7.5) has virtually no binding capability to V2 receptors (Kd approximately 0.3 mM). These analogues should lead to the design of even more potent and selective oxytocin antagonists.

Amino Acid Sequence↗
PubMed1994

[PCB concentration in hair, blood, tissues and secretions of chronically exposed dairy cows, fattening bulls and calves].

Fattening bulls (n = 8) and cows (n = 10) affected chronically with PCB were kept in barns on PCB-free feeding (silage, straw, concentrates). The cows were dried off. The kinetics of PCB-concentrations in hair, blood and feces were examined. Fattening bulls (mean 460 kg bw, german black and white and interbreeding german black and white and Simmental) with low (320 g) and medium (730 g) daily weight gain for a period of 5 months and 3 bulls with medium (640 g) weight gain within 18 months were examined. A drying off period of 3-5 months was recorded for the cows. The distribution of PCB in the carcass of some animals was registered in addition. The following results are to be emphasized: Reduction of PCB in the studied substrates in the fattening bulls depends on a high degree on daily weight gain. This is important in order to calculate biological half life times of PCB. By standardized weight gain of 10 kg in bulls, fed PCB-free diets for a 5 months period, a reduction of PCB-concentrations from 1 to 3% was found (hair and feces: 1%, blood 3%). Within a period of 18 months and with higher weights, in the examined substrates (blood, hair, feces) mean declines of the PCB-concentrations of 2% were found. On knowledge so far this value is an essential point to estimate biological half life times, because metabolic PCB-elimination is negligible.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Cloning, DNA sequence analysis and partial characterization of pepN, a lysyl aminopeptidase from Lactobacillus delbrückii ssp. lactis DSM7290.

In cell extracts of Lactobacillus delbrückii ssp. lactis DSM7290 a peptidase with the ability to hydrolyse Phe-beta-naphthylamide (Phe-beta-NA) and His-beta-NA could be detected. Escherichia coli lacking the enzyme activity in an enzymic plate assay was used to screen high-copy-number and low-copy-number plasmid libraries of size-fractionated Lactobacillus DNA. Clones with the desired phenotype were detected, and the gene, designated pepN, was further subcloned and sequenced. A large open reading frame of 2529 nucleotides is predicted to encode a protein of 843 amino acids (95358 Da). Comparison of the pepN gene from Lb. delbrückii ssp. lactis DSM7290 indicates that it is homologous to genes of the family of Zn(2+)-metallohydrolases and PepN shows identity with the active centre Zn(2+)-binding motif of these enzymes. The substrate Lys-beta-NA is more effectively cleaved than Phe-beta-NA or His-beta-NA which were used for screening in E. coli. The cloned pepN gene was efficiently overexpressed in E. coli and subcloning of the gene in Lactobacillus casei resulted in a moderate overexpression of approximately 20-fold. The pepN gene product was purified from the pepN-deficient E. coli strain CM89, using the substrate Lys-p-nitroanilide (Lys-NH-Ph) in the assay procedure. In a four-step procedure including streptomycin sulfate precipitation, anion-exchange chromatography and gel filtration the peptidase was purified to electrophoretic homogeneity.

Amino Acid Sequence↗
PubMed1993

5' sequences are important positive and negative determinants of the longevity of Chlamydomonas chloroplast gene transcripts.

We have found that sequences in the 5' leader of the Chlamydomonas chloroplast rbcL gene, when fused 5' to foreign genes, destabilize transcripts of these chimeric genes in the chloroplast of transgenic Chlamydomonas but that 5' sequences of the rbcL structural gene prevent this destabilization. Transcripts of the chloroplast rbcL gene are about equally abundant at all times in Chlamydomonas reinhardtii growing on an alternating 12-h light/12-h dark cycle. However, Chlamydomonas chloroplast transformants, harboring chimeric genes containing the same rbcL promoter with 63 or 92 bp of the rbcL 5' leader sequence fused upstream of the Escherichia coli uidA (beta-glucuronidase, GUS) gene, accumulated GUS transcripts only in the dark. Transcripts disappeared rapidly upon illumination of the cells. The same phenomenon was exhibited by transcripts of chimeric genes in which the GUS gene coding sequence was replaced by other unrelated genes. The precipitous light-induced drop in GUS transcript abundance was found to be due to an approximately 16-fold increase in the rate of degradation of GUS transcripts in light rather than to a decrease in the rate of transcription of the GUS gene. Transcripts of a chimeric rbcL-GUS construct in which the leader sequence of the rbcL gene was replaced by 103 bp of the leader sequence of the atpB gene were stable in illuminated cells. The destabilizing effect of the rbcL 5' leader sequence was reversed by adding 257 bp of the 5' coding region of the rbcL gene. The results show that chloroplast transcript levels in illuminated Chlamydomonas cells--and perhaps in other cases--can be determined, at least to some extent, by sequences and interactions of sequences transcribed from the 5' ends of genes.

Animals↗
PubMed1993

Human IgM+IgD+ B cells, the major B cell subset in the peripheral blood, express V kappa genes with no or little somatic mutation throughout life.

Peripheral blood B cells of a 67-year-old person were separated into IgM+IgD+, IgM+IgD-, and IgM-IgD- subsets, and nucleotide sequences of expressed immunoglobulin light chain variable (V) regions encoded by V kappa 3 and V kappa 4 gene family members were determined from amplified cDNA. V region sequences from IgM+IgD+ cells (the major B cell population in the blood) showed no or little somatic mutation (0.3%), in contrast to V kappa sequences from IgM+IgD- and IgM-IgD- B cells (2.0% and 3.9%, respectively). This suggests that in the human like in the mouse, and independently of age, somatically mutated memory B cells accumulate in the compartment of IgM-IgD- cells, whereas the IgM+IgD+ subpopulation consists of cells whose antibody repertoire is mainly determined by V region gene rearrangements and N-region insertion, at the molecular level. The somatically mutated IgM+IgD- cells may represent early descendants of IgM+IgD+ cells recruited into the memory cell compartment.

Aged↗
PubMed1993

Functional in vivo analyses of the 3' flanking sequences of the Chlamydomonas chloroplast rbcL and psaB genes.

Possible roles of untranslated sequences at the 3' ends of chloroplast genes, which include inverted repeat elements, were investigated in Chlamydomonas reinhardtii in vivo. Chlamydomonas chloroplast rbcL or psaB 3' flanking regions were coupled in various arrangements 3' to a chimeric gene consisting of a Chlamydomonas chloroplast atpB promoter sequence fused 5' to the Escherichia coli uidA (GUS) structural gene. These genes were introduced into the Chlamydomonas chloroplast genome at the same location by homologous recombination following microprojectile bombardment. Transformants harboring chimeric GUS genes fused to rbcL or psaB gene 3' inverted repeat sequences in their normal forward orientations accumulated GUS transcripts of a single size, whereas GUS transcripts of heterogenous sizes accumulated in transformants harboring the same gene lacking an inverted repeat sequence at its 3' end. Thus, the 3' flanking regions of the rbcL and psaB genes can define the location of the 3' terminus of a transcript in vivo. In chloroplast transformants harboring chimeric GUS genes fused to multiple inverted repeat sequences in their normal forward orientations, only GUS transcripts accumulated that were terminated by the first inverted repeat sequence. The latter data suggest that the 3' ends of these RNAs are the products of either transcription termination or endonucleolytic cleavage. Analyses of GUS transcripts in transformants harboring GUS genes terminated by rbcL or psaB gene 3' flanking regions in reversed orientations indicate that transcript 3' end formation in vivo requires nucleotide sequences located outside the inverted repeat elements. Inasmuch as decay rates of GUS transcripts were found to be independent of the presence of a 3' inverted repeat sequence, RNA stabilization does not appear to be a major in vivo function of these elements in the Chlamydomonas chloroplast transcripts studied.

Animals↗
PubMed1993

Light-regulated and endogenous fluctuations of chloroplast transcript levels in Chlamydomonas. Regulation by transcription and RNA degradation.

Changes in the relative sizes of pools of transcripts of organelle genes during plastid development are common in flowering plants, but technical difficulties have prevented direct determinations of the effects of changes in rates of transcription and degradation on such fluctuations. It has been possible to follow both rates in Chlamydomonas reinhardtii. In synchronous or asynchronous cultures of cells grown in 12 h light/12 h dark periods, sizes of pools of transcripts of the chloroplast genes atpA, atpB, tufA, and psaB fluctuate. Differences in chloroplast transcript abundances in light/dark cycles were found to be cell cycle-independent but controlled by either an endogenous rhythm (atpA, atpB, and tufA) or by light (psaB). In vivo labeling experiments showed that transcriptional regulation and light/dark-regulated degradation both contribute, in gene-specific manners, to the level of transcripts of individual C. reinhardtii chloroplast genes in cells grown in alternating light/dark cycles.

Animals↗
PubMed1993

[Connector for double-lumen tubes in thoracic surgery interventions].

The junction between double-lumen endotracheal tube (DLT) and anaesthetic circuit is of crucial importance to modern anaesthesiological management in thoracic surgery. We present a connector which allows for all the essential procedures, such as clinical control of DLT position, use of fiberoptic bronchoscope (FOB) to control or correct DLT position, application of differential lung ventilation patterns or simple suction manoeuvres by easy handling, minimal interference, and avoidance of disconnection or clamping.

Bronchoscopes↗
PubMed1992

THE INSECT V-ATPase, A PLASMA MEMBRANE PROTON PUMP ENERGIZING SECONDARY ACTIVE TRANSPORT: IMMUNOLOGICAL EVIDENCE FOR THE OCCURRENCE OF A V-ATPase IN INSECT ION-TRANSPORTING EPITHELIA.

Active electrogenic K+ transport in insects serves as the energy source for secretion or absorption in gastrointestinal epithelia or for the receptor current in sensory epithelia. In the larval midgut of the tobacco hornworm Manduca sexta, a vacuolar-type proton pump (V-ATPase) and a K+/nH+ antiport represent the functional elements of the potassium pump. Several immunological findings support the hypothesis that active K+ transport in other insect epithelia may also be energized by a V-ATPase. In immunoblots, crude homogenates of sensilla-rich antennae and Malpighian tubules of M. sexta cross reacted with an immune serum directed to the purified plasma membrane V-ATPase from the midgut; the M. sexta midgut V-ATPase cross reacted with polyclonal antibodies to endomembrane V-ATPases from xenic origin. In immunocytochemical investigations of larvae of M. sexta and adults of Antheraea pernyi, monoclonal antibodies to defined subunits of the purified midgut V-ATPase or polyclonal antibodies to xenic endomembrane V-ATPase labelled the sites of active K+ transport: the goblet cell apical membrane in the midgut, the brush border of Malpighian tubules and the apical projections of the auxiliary cells in antennal sensilla. The functional mechanism of a primary H+-pumping V-ATPase and a secondary H+-dependent K+ transport postulated for K+-transporting insect epithelia may be further applicable to active Na+ or Cl- transport and would provide a unifying concept for all ouabain-insensitive electrogenic ion transport in insects. The findings from the midgut investigations, however, are the first instance in which a V-ATPase provides an alternative to the Na+/K+-ATPase in energizing secondary active transport in animal plasma membranes.

Journal Article↗
PubMed1992

Two types of chloroplast gene promoters in Chlamydomonas reinhardtii.

Structures of the promoters of Chlamydomonas reinhardtii plastid atpB and 16S rRNA-encoding genes were analyzed in vivo. Chimeric constructs, containing the Chlamydomonas chloroplast atpB or 16S rRNA-encoding gene promoter coupled to the Escherichia coli uidA (beta-glucuronidase, GUS) reporter gene and bordered by C. reinhardtii chloroplast sequences, were stably introduced into the chloroplast of Chlamydomonas by microprojectile bombardment. Activity of the promoters in the chloroplast of GUS gene-positive transformants was assayed by measuring the abundance of GUS transcripts and determining the relative rates of GUS transcription in vivo. Deletion analyses of the 16S rRNA gene and atpB promoter fragments showed that the two promoters differ structurally. The 16S rRNA gene promoter resembles the bacterial sigma 70 type with typical -10 and -35 elements. The atpB promoter, on the other hand, lacks a conserved motif in the -35 region but contains, in the -10 region, a characteristic octameric palindrome (TATAATAT) that is conserved in the promoter sequences of some other C. reinhardtii chloroplast genes. For maximum activity, the atpB promoter requires sequences of approximately 22 base pairs upstream and approximately 60 base pairs downstream of the transcription start site.

Animals↗
PubMed1992

Antibodies to mammalian and plant V-ATPases cross react with the V-ATPase of insect cation-transporting plasma membranes.

In immunobiochemical blots, polyclonal antibodies against subunits of plant and mammalian vacuolar-type ATPases (V-ATPases) cross-react strongly with corresponding subunits of larval Manduca sexta midgut plasma membrane V-ATPase. Thus, rabbit antiserum against Kalanchoe daigremontiana tonoplast V-ATPase holoenzyme cross-reacts with the 67, 56, 40, 28 and 20 kDa subunits of midgut V-ATPase separated by SDS-PAGE. Antisera against bovine chromaffin granule 72 and 39 kDa V-ATPase subunits cross-react with the corresponding 67 and 43 kDa subunits of midgut V-ATPase. Antisera against the 57 kDa subunit of both beet root and oat root V-ATPase cross-react strongly with the midgut 56 kDa V-ATPase subunit. In immunocytochemical light micrographs, antiserum against the beet root 57 kDa V-ATPase subunit labels the goblet cell apical membrane of both posterior and anterior midgut in freeze-substituted and fixed sections. The plant antiserum also labels the apical brush-border plasma membrane of Malpighian tubules. The ability of antibodies against plant V-ATPase to label these insect membranes suggests a high sequence homology between V-ATPases from plants and insects. Both of the antibody-labelled insect membranes transport K+ and both membranes possess F1-like particles, portasomes, on their cytoplasmic surfaces. This immunolabelling by xenic V-ATPase antisera of two insect cation-transporting membranes suggests that the portasomes on these membranes may be V-ATPase particles, similar to those reported on V-ATPase-containing vacuolar membranes from various sources.

Adenosine Triphosphatases↗
PubMed1992

Measurement of potential visual acuity in 343 patients with cataracts. A prospective clinical study.

In 343 patients with cataracts (the largest consecutive series in which this subject has been addressed) and 63 patients with clear optic media, who served as the control group, potential visual acuity was measured with the Guyton/Minkowski Potential Acuity Meter (PAM). The control group included 32 cases of macular degeneration, 22 cases of glaucoma, and 9 cases of amblyopia. The cataract group comprised 46 patients with macular degeneration, 46 with glaucoma, and 8 with amblyopia; 5-7 days postoperatively, corrected visual acuity was measured in this group. Because of dense cataracts in 59 patients, no PAM value could be obtained. The visual acuity of 70.4% of the patients, in whom complete data were obtained, was within one Snellen line of the predicted value or better (21%). In general, postoperative predictions were accurate in patients with moderate cataracts. The results were not affected by glaucoma or amblyopia. In 17.5% of patients with macular degeneration, false-positive results were obtained. The control group, in which distance visual acuity was compared with the PAM values, rendered comparable results, with 86.9% of the PAM predictions being correct.

Adult↗
PubMed1992

[PCB concentrations in hair, blood, tissues and secretions of chronically-exposed dairy cows, fattening bulls and calves. 1. Course and relation of some polychlorinated biphenyls in hair, milk and tissues in exposed nursing cows and conventionally raised dairy cows].

20 cows contaminated chronically with PCB were kept as nurse cows or in a tie-stall. The contamination in different substrates (hair, milk, blood, feces) was examined for a period up to 8 months. The distribution of PCB in the carcass of some animals was registered in addition. The system of keeping nurse cows turned out to be useful in order to overcome a contamination situation. Drying off gravid animals in the first step helps solving problems of handling contaminated milk. With low contaminated animals a short dry off period is sufficient, so that the milk--after calving--is marketable (report II, in preparation). In case of nurse cows with two calves post partum a continuous decline of PCB-concentrations in the milk can be observed. With biological half life periods of 2-4 months, milk with PCB 153-concentrations of 0.15-0.20 mg/kg fat will be marketable within this period. Cows milked by milking-machines show a decline of PCB in the milk similar to nurse cows. Two animals had a limited increase p.p., probably caused by mobilization of fat in the high lactation stage. Over longer periods, the biological half-life-periods were comparable to nursing cows. Hair is easy to sample and store; it is useful as a diagnostic substrate beside the milk in order to estimate the degree of contamination in individual animals. The ratio of PCB-concentrations between perirenal fat and hair is 1:0.95 (s = +/-0.192); the ratio of PCB-concentrations between perirenal fat and milk is 1:0.77 (s = +/- 0.122).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

[Long-term results of Mustard operation in transposition of the great arteries. Angiographic and nuclear medicine study of ventricular function].

The fate of the right ventricle as systemic ventricle after atrial repair of complete transposition of the great arteries has not been clearly elucidated. In order to assess the long-term results of the Mustard operation in patients with complete transposition of the great arteries we present the clinical data of 23 patients who had been operated in the years 1974 and 1975. Twenty of these patients had simple complete transposition of the great arteries with intact ventricular septum, two had an additional small ventricular septal defect and one an additional left ventricular outflow tract obstruction with a 40 mm Hg systolic pressure gradient. The Mustard operation had been performed at a mean age of 2.2 (1 to 3.7) years. Seventeen of the 23 patients underwent a postoperative hemodynamic study with angiocardiography 1.1 (1 to 1.8) years following surgery. At that time the right ventricular ejection fraction, which had been calculated from biplane angiographic right ventricular volume measurements in twelve patients was 62 (52 to 68) %. However the right ventricle was dilated and the mean enddiastolic volume was 132 (108 to 192) % of normal. In twelve of the 23 patients right ventricular function was reassessed 12.6 (11 to 15.3) years after surgery by Technetium-99m-scintigraphy at rest and in ten of those after exercise with a workload of 2 watt/kg. The mean ejection fraction was 51 (38 to 66) % at rest and 52 (40 to 80) % during exercise. Only three patients had a normal response to exercise, which was defined as an increase of ejection fraction with exercise of more than 5%.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiocardiography↗