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Biomedical subjects

H Werner

Publications and source records attributed to H Werner.

At least 19 recordsLinked to original sources
PubMed1992

RNA polymerase III catalysed transcription can be regulated in Saccharomyces cerevisiae by the bacterial tetracycline repressor-operator system.

We have investigated whether the RNA polymerase III-driven transcription of eukaryotic tRNA genes can be regulated by the prokaryotic tetracycline operator-repressor system. The bacterial tet operator (tetO) was inserted at two different positions (-7 and -46) upstream of a tRNA(Glu) (amber) suppressor gene. Both constructs are transcribed in Saccharomyces cerevisiae and yield functional tRNAs as scored by suppression of an amber nonsense mutation in the met8-1 allele. Controlled expression of Tet repressor was achieved by fusing the bacterial tetR gene to the yeast gal1 promoter. This leads to expression of Tet repressor in yeast on galactose--but not on glucose--containing media. Regulation of the su-tRNA gene with the tetO fragment inserted at position -7 has been demonstrated. Under conditions which allow tetR expression, cells exhibit a met- phenotype. This methionine auxotrophy can be conditionally reverted to prototrophy by adding tetracycline. However, a su-tRNA gene with the tetO fragment inserted at position -46 cannot be repressed. Our results demonstrate clearly that the bacterial repressor protein binds to its operator in the yeast genome. Formation of this complex in the vicinity of the pol III transcription initiation site reduces the level of su-tRNA at least 50-fold as concluded from quantitative primer extension analyses. This indicates for the first time that class III gene expression can be regulated by a DNA binding protein with its target site in the 5'-flanking region and that a prokaryotic repressor can confer regulation of a suitably engineered tRNA gene.

Alleles↗
PubMed1992

German multicentre study on the in vitro susceptibility of Bacteroides species. The German Bacteroides Study Group.

In 1990 the first German multicentre study on the in vitro susceptibility of Bacteroides species was completed. Employing a commercially prepared microbroth dilution assay, nine participating institutions evaluated approximately 100 consecutive isolates of Bacteroides species from relevant clinical specimens. A total of 911 strains (449 Bacteroides fragilis, 201 Bacteroides thetaiotaomicron, 79 Bacteroides ovatus, 78 Bacteroides vulgatus, 77 Bacteroides distasonis, 25 Bacteroides uniformis, 2 others) were tested. Most of the isolates came from surgical patients (72%); other sources included gynaecological patients (9%) and medical patients (5%). Seventy-eight percent of the anaerobes were found in mixed culture together with at least one aerobic organism (Escherichia coli 36%, streptococci 15%, or enterococci 13%), while in 22% of the cases, the anaerobes were the only bacteria grown from the specimens. The results showed that many of the strains were potent beta-lactamase producers (as judged by resistance to amoxicillin). However, all but one of the isolates demonstrated susceptibility in vitro when clavulanic acid was added to amoxicillin or ticarcillin. At the same time, 13% of the organisms were resistant to mezlocillin, 5% to cefoxitin and 4% to clindamycin. Three strains were reported resistant to imipenem and one strain to metronidazole.

Anti-Bacterial Agents↗
PubMed1992

Cellular pattern of type-I insulin-like growth factor receptor gene expression during maturation of the rat brain: comparison with insulin-like growth factors I and II.

Insulin-like growth factors have a number of potent trophic effects on cultured neural tissue and most if not all of these effects appear to be mediated by the type-I insulin-like growth factor receptor. In order to establish the identity of cell types expressing this receptor in the rat central nervous system during development and maturity, we have used in situ hybridization to map sites of type-I insulin-like growth factor receptor mRNA synthesis in the developing and adult rat brain. In order to identify possible local sources of peptide ligands for this receptor, we have also mapped the sites of insulin-like growth factors I and II mRNA synthesis in parallel brain sections. From early development onward, there is a uniform and stable pattern of type-I insulin-like growth factor receptor gene expression in all neuroepithelial cell lineages, in which regional variations reflect primarily differences in cell density. In addition to this generalized pattern, during late postnatal development, high levels of type-I insulin-like growth factor receptor gene expression are found in specific sets of sensory and cerebellar projection neurons in conjunction with abundant insulin-like growth factor-I gene expression in these same neurons. While insulin-like growth factor-I expression is confined to the principal neurons in each system, receptor mRNA is also found in local interneurons. In the cerebral cortex and hippocampal formation, type-I insulin-like growth factor receptor mRNA and insulin-like growth factor-I are concentrated in different cell populations: receptor mRNA is abundant in pyramidal cells in Ammon's horn, in granule cells in the dentate gyrus, and in pyramidal cells in lamina VI of the cerebral cortex. Insulin-like growth factor-I mRNA is found in isolated medium- to large-sized cells which are rather irregularly distributed throughout the hippocampus and isocortex. In the hypothalamus, receptor mRNA is concentrated in the suprachiasmatic nucleus but is in low abundance elsewhere, including the median eminence, while insulin-like growth factor-I mRNA is not detected in this region at all. Type-I insulin-like growth factor receptor and insulin-like growth factor-II mRNAs are both abundant in choroid plexus, meninges and vascular sheaths from early development to maturity, but insulin-like growth factor-II mRNA is not detected in cells of neuroepithelial origin at any stage of development. This study provides evidence for two fundamentally different patterns of gene expression for the brain type-I insulin-like growth factor receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMed1992

In vitro fragmentation of gallstones with lithotriptors for use in laparoscopic cholecystotomy.

In preliminary in vitro experiments, five different lithotriptors for use in laparoscopic cholecystotomy were tested to determine the fragmentation time and rate of a defined group of gallstones. The Alexandrit laser and the ultrasonic lithotriptor were too ineffective to warrant further investigation. In phantom tests with pig gallbladders, the pulsed dye laser and the electrohydraulic lithotriptor had a fragmentation rate of 100%, but the rate of clearing of the gallbladder was 0% as stone debris always remained in the gallbladder. The clearing rate of the RotoLith mechanical lithotriptor was 84.6%. The RotoLith lithotriptor was the most effective device because the stones were ground into such small particles that they could easily be rinsed out of the gallbladder. As the RotoLith procedure does not need to be monitored optically, the opening of the gallbladder need not exceed 3 mm compared to 10 mm for the other lithotriptors. This makes closure of the gallbladder safer and it would seem feasible to perform the procedure using the RothoLith lithotriptor under local anaesthesia.

Animals↗
PubMed1992

Establishment of a system for conditional gene expression using an inducible tRNA suppressor gene.

We investigated the use of the prokaryotic tetracycline operator-repressor system as a regulatory device to control the expression of Dictyostelium discoideum tRNA genes. The tetO1 operator fragment was inserted at three different positions in front of a tRNA(Glu) (Am) suppressor gene from D. discoideum, and the tetracycline repressor gene was expressed under the control of a constitutive actin 6 promoter. The effectiveness of this approach was determined by monitoring the expression of a beta-galactosidase gene engineered to contain a stop codon that could be suppressed by the tRNA. When these constructs were introduced into Dictyostelium cells, the repressor bound to the operator in front of the tRNA gene and prevented expression of the suppressor tRNA. Addition of tetracycline (30 micrograms/ml) to the growth medium prevented repressor binding, allowed expression of the suppressor tRNA, and resulted in beta-galactosidase synthesis. The operator-repressor complex interfered with tRNA gene transcription when the operator was inserted immediately upstream (position +1 or -7) of the mature tRNA coding region. Expression of a tRNA gene carrying the operator at position -46 did not respond to repressor binding. This system could be used to control the synthesis of any protein, provided the gene contained a translational stop signal.

Animals↗
PubMed1992

Altered expression of insulin-like growth factor-I (IGF-I) and IGF receptor genes after unilateral nephrectomy in immature rats.

There is a developmental difference in the initial phase of compensatory renal growth (CRG) following unilateral nephrectomy (UNX), in that CRG is GH-dependent in adult rats and GH-independent in immature rats. Furthermore, CRG in immature rats is associated with an increase in renal IGF-I mRNA, an effect not seen in adult rats. In this study we have examined the age-related differences in expression of the insulin-like growth factor-I (IGF-I) and IGF-II genes as well as in IGF-I and IGF-II receptors and membrane binding after UNX. Immature (22-24 days of age) and adult (4 months of age) male Wistar rats underwent a sham operation or left UNX and were killed 24 or 48 h later. Levels of mRNA for IGF-I and IGF-II and their receptors were determined in the left (control) and right (compensated) remnant kidneys using solution hybridization/RNase protection assays. Steady state levels of IGF-I mRNA as well as IGF-I receptor and IGF-II/mannose-6-phosphate receptor mRNAs were increased 3- to 4-fold in immature remnant kidneys, but not in adult kidneys. The findings related to IGF-I gene expression were confirmed by in situ hybridization to immature and adult kidney slices. The increase in IGF-I gene expression in the immature remnant kidneys was localized to the thick ascending limbs of the loops of Henle. Furthermore, in concert with the changes in mRNA levels, membrane binding studies showed significant increases in specific binding to IGF-I in cortical membranes and increases in specific binding to IGF-II in whole kidney membranes from immature, but not adult, rats. Thus, these findings demonstrate that the initial phase of CRG in the immature rat is associated with increased renal IGF-I gene expression as well as enhanced specific renal binding of IGF-I and IGF-II to plasma membranes and support the notion that this period of rapid renal growth in the immature UNX rat may involve the paracrine influence of the IGFs.

Animals↗
PubMed1992

Structural and functional analysis of the insulin-like growth factor I receptor gene promoter.

The insulin-like growth factor I receptor (IGF-I-R) gene is expressed in most body tissues. The levels of IGF-I-R mRNA, however, are regulated by a number of physiological conditions (development, differentiation, and hormonal milieu) as well as in certain pathological states (diabetes and tumors). To understand the molecular mechanisms which control the transcription of the IGF-I-R gene, we have cloned the promoter of the rat receptor gene and have characterized its activity by transient expression assays. Different fragments of the 5'-flanking region (subcloned upstream of a luciferase reporter gene) were transfected into buffalo rat liver 3A cells (a cell line with a low number of IGF-I binding sites) and Chinese hamster ovary cells (a cell line with a higher number of cell-surface receptors). In both cell lines, most of the promoter activity was located in the proximal 416 base pairs of 5'-flanking region. However, further dissection of this proximal fragment revealed a cell type-specific pattern of promoter activity. Thus, in buffalo rat liver 3A cells, subfragments of this region each contributed to total activity, suggesting that contiguous cis-elements can act together to activate transcription. In Chinese hamster ovary cells, on the other hand, subfragments of the proximal promoter region partially substituted for the proximal 416 base pairs of 5'-flanking region. Coexpression studies using an IGF-I-R promoter reporter construct together with an Sp1 expression vector (under the control of an ADH promoter) were performed in SL2 cells, a Drosophila cell line which lacks endogenous Sp1. The results obtained showed that Sp1 can trans-activate the IGF-I-R promoter in vivo. Transient transfection assays were complemented with gel-retardation assays and DNase I footprinting experiments, which showed that transcription factor Sp1 is potentially an important regulator of IGF-I-R gene expression.

Animals↗
PubMed1991

Insulin-like growth factor I (IGF-I) receptors and IGF-I action in oligodendrocytes from rat brains.

Oligodendrocyte progenitor cells were prepared by mechanical dissociation of 1-day-old rat brain cultures. These cells undergo proliferation and differentiation into oligodendrocytes as demonstrated by the expression of proliferation and differentiation-related specific antigens. We have used this unique culture system to characterize insulin-like growth factor I (IGF-I) receptors and their action in the central nervous system (CNS). 125I-IGF-I specifically binds to these cultures with high affinity. Competition-inhibition data suggest that IGF-I is most potent in competing for 125I-IGF-I binding, followed by IGF-II and insulin. Scatchard analyses of the binding data indicate a curvilinear plot with a Kd for high affinity of 0.2 nM, and a Bmax of 247 fmol/mg, and a Kd for low affinity of 3.2 nM and Bmax of 1213 fmol/mg protein. Covalent cross-linking followed by SDS-PAGE analysis demonstrated a radioactive band of Mr 135,000 which corresponds to the alpha subunit of the IGF-I receptor. Solution hybridization/RNase protection assay produced a single protected band corresponding to IGF-I receptor messenger RNA, further confirming the presence of these receptors. Incubation of progenitor cells with IGF-I resulted in a time- and concentration-dependent increase in [3H]thymidine incorporation and cell numbers. This effect appears to be mediated by IGF-I receptors since IGF-II and insulin were proportionately less potent. In addition to its effect on proliferation, IGF-I also increased the number of 4E7- and GC-antigen positive cells. These observations indicate that oligodendrocytes in primary culture express specific IGF-I receptors and that the interaction of IGF-I with these receptors results in the proliferation as well as differentiation of oligodendrocytes.

Animals↗
PubMed1991

Comparison of three methods for measuring respiratory mechanics in ventilated children.

Continuous measurements of airway pressure, gas flow, and tidal volume were made in 22 mechanically ventilated children, both during steady state conditions and following airway occlusion at end-inflation. For each child, three methods of analyzing the stored data were used to generate values of respiratory system compliance and resistance: 1) end-inspiratory hold technique (Bone: Respir Care 28:597, 1983; Rossi et al. Am Rev Respir Dis 131:672, 1985); 2) constant flow technique (Rossi et al. J Appl Physiol 58:1849, 1985; Suratt et al. J Appl Physiol 49:1116, 1980); and 3) multiple linear regression (Roy et al. Comput Biomed Res 7:21, 1974; Bhutani et al. Pediatr Pulmonol 4:150, 1988). In the absence of an accepted standard, we used the inspiratory hold technique as a reference. All methods gave comparable values for respiratory mechanics over a wide clinical range. However, multiple linear regression was the most convenient of the three: it can be automated and continuously displayed, there is no subjective input, values are taken through the respiratory cycle, and it is completely noninvasive. We also found that respiratory system resistance was largely a measure of endotracheal tube resistance and that respiratory compliance is a more sensitive monitor of lung function in intubated children.

Airway Resistance↗
PubMed1991

Pilot study for the development of a monitoring device for ventilated children.

Airway pressure and air flow were measured at the endotracheal tube in 13 children on a variety of ventilators. These signals were stored for analysis on a computer. Further data sets were obtained after 24 hours or following major interventions. Air flow rate was integrated to give volume. Pulmonary resistance and elastance were obtained by multiple linear regression. Pressure-volume, pressure-flow and flow-volume loops were plotted. "Closed" pressure-volume and pressure-flow loops (by subtraction of the resistive or elastic pressure components, respectively) were also displayed, giving compliance and resistance loops. The loops from the initial data set were taken as the baseline, and loops from later data sets were superimposed to provide visual comparisons. Change in clinical status was reflected by the change in slope of compliance and resistance loops. A 30% change in compliance or resistance was easily observed. There was minimal interference with patient care. This pilot study demonstrates that changes in respiratory mechanics can be displayed safely and easily in ventilated patients using resistance and compliance loops. Further work is necessary to confirm the usefulness of real time of these displays.

Child↗
PubMed1991

Positive selection for Dictyostelium discoideum mutants lacking UMP synthase activity based on resistance to 5-fluoroorotic acid.

In the cellular slime mould Dictyostelium discoideum the two enzymatic activities of the pyrimidine pathway, orotidine-5'-phosphate decarboxylase (EC 4.1.1.23; OMPdecase) and orotate phosphoribosyl transferase (EC 2.4.2.10; OPRTase), are encoded by a single gene (DdPYR5-6). As in higher eukaryotes the bifunctional enzyme is referred to as UMP synthase. Here we present a method that allows efficient generation and selection of mutants lacking UMP synthase. D. discoideum cells are transformed with either of two different types of plasmids. One plasmid type contains no sequences homologous to the UMP synthase gene whereas the other type contains at least parts of this gene. UMP synthase- mutants, which were positively selected for in the presence of 5-fluoroorotic acid (5-FOA), were obtained with both plasmids. However, mutation rates were at least one order of magnitude higher if plasmids containing various portions of the UMP synthase gene were used as opposed to plasmids that lack any homology to the UMP synthase locus. Several mutant strains were extensively characterized. These strains lack OMPdecase activity and exhibit in addition to 5-FOA resistance a ura- phenotype. All mutants carry UMP synthase loci with deletions of various extents but integration of transforming plasmids was not detected. This efficient generation of 5-FOA resistance is part of a proposed complex selection scheme which allows multiple rounds of transformation of D. discoideum.

Blotting, Southern↗
PubMed1991

Developmental regulation of insulin-like growth factor-I-stimulated glucose transporter in rat brain astrocytes.

Astrocytic glial cells from 1- and 21-day-old rat brains were established in primary culture to study the expression of insulin-like growth factor-I (IGF-I) receptors and IGF-I-stimulated glucose transporter (Glut-1). Astrocytes from both age groups expressed specific high affinity IGF-I receptors, whose relative affinities for IGF-I, IGF-II, and insulin were comparable. However, the total number of binding sites and IGF-I receptor mRNA levels were 148% and 240% higher in astrocytes from 21-day-old compared with 1-day-old brains. IGF-I caused a dose-dependent stimulation of [3H]2-deoxy-D-glucose [( 3H]dGlc) uptake in astrocytes from 1-day-old brains. This was associated with increases in Glut-1 protein and mRNA levels. In contrast, astrocytes from 21-day-old brains exhibited a 58% decrease in the binding capacity and a 77% decrease in the steady state levels of Glut-1 protein and its mRNA. In addition, IGF-I failed to stimulate the Glut-1 system in these cells. This lack of IGF-I effect is not due to an alteration inherent to the Glut-1 system, since 12-O-tetradecanoyl-phorbol-13-acetate stimulated [3H]dGlc uptake and Glut-1 protein and its mRNA levels. These observations suggest that changes in basal and IGF-I-stimulated Glut-1 system in brain astrocytes may be developmentally regulated.

Animals↗
PubMed1991

Insulin-like growth factor receptor gene expression in the rat ovary: divergent regulation of distinct receptor species.

The intraovarian insulin-like growth factor (IGF) system constitutes a triad composed of ligands, receptors, and binding proteins. Although conventional radioligand receptor assays have documented the presence of specific receptors for insulin and insulin-like peptides in some rat somatic ovarian cell types, the exact cellular localization and hormonal regulation of the receptors in question remain matters of inquiry. To reevaluate the very presence, cellular localization, and hormonal regulation of the IGF receptor gene family in the rat ovary, solution hybridization/RNase protection assays were used wherein ovarian total RNA (20 micrograms) from immature (21-23 days old) rats was hybridized with 32P-labeled type I IGF receptor, type II IGF/mannose-6-phosphate receptor, and insulin receptor riboprobes. Single protected fragments 261 (type I IGF receptor), 500 (type II IGF/mannose-6-phosphate receptor), and 478 (insulin receptor) bases long were evident in whole ovary, granulosa, and theca-interstitial cells. Hypophysectomy of immature rats led to significant (P less than 0.05) albeit variable decrements in the relative (densitometrically quantified) ovarian abundance of transcripts corresponding to the type I IGF (but not insulin or type II IGF/mannose-6-phosphate) receptor. Treatment of immature hypophysectomized rats with FSH (10 micrograms/rat.day x 2.5 days) resulted in a significant (P less than 0.05) increase (4-fold) in transcripts corresponding to the type I IGF receptor in both whole ovarian material and freshly isolated granulosa cells. Similar (3.7-fold) increments (P less than 0.05) were noted after treatment with a diethylstilbestrol-containing sc silastic implant applied for a total of 5 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗