Die sogenannten Indirekten Nieren-Clearance-Methoden

Zusammenfassung Vergleichsuntersuchungen in einem Untersuchungsgang zwischen der indirekten Inulin- und PAH-Clearance mit BilanzmessungBettge undSimon;Uhlmann) und direkter Inulin- und PAH-Clearance mit konstantem Plasmaspiegel ergaben nur bei der PAH-Clearance eine häufige Übereinstimmung (16 von 22 Fällen), während die indirekte Bestimmung derrenalen Inulin-Clearance wegen der Unmöglichkeit einer Bilanzmessung nicht anwendbar ist. Versuche über die Blutspiegelkinetik von Inulin und PAH ließen wiederum einen Abfall nach den Gesetzen einer Exponentialfunktion vermissen.Die Untersuchungen wurden mit Hilfe der Deutschen Forschungsgemeinschaft durchgeführt.     

Evidence for a direct relationship between mitochondrial genome organization and regeneration ability in hexaploid wheat somatic tissue cultures

Summary Embryogenic and non-embryogenic long-term callus cultures of hexaploid wheat exhibit differences in the organization of their mitochondrial genome. Embryogenic and non-embryogenic fractions of callus cultures initiated from immature embryos of the wheat cultivar Chinese Spring have been isolated and subsequently subcultured. DNA-DNA hybridization experiments using labelled cloned wheat mitochondrial DNA fragments have shown that the mitochondrial DNA organization of embryogenic subcultures derived from embryogenic parts of Chinese Spring calli is closely related to that of the initial Chinese Spring calli, while non-embryogenic subcultures derived from non-embryogenic fragments of Chinese Spring calli exhibit a mitochondrial DNA organization similar to that found in non-embryogenic calli derived from cultivar Aquila. In addition, somatic tissue cultures initiated from three other non-embryogenic wheat cultivars (Talent, Thésée and Capitole) display mitochondrial DNA arrangements similar to those found in cultivar Aquila. These results strongly suggest that, in wheat callus cultures, a particular mitochondrial genome organization is correlated with the ability of cultured cells to regenerate whole plants.Abbreviations mtDNA mitochondrial DNA - ctDNA chloroplast DNA - rRNA ribosomal RNA - kb kilobase pair - cv cultivar - 2,4-D 2,4-dichlorophenoxyacetic acid     

Molecular organisation of an A mating type factor of the basidiomycete fungus Coprinus cinereus

Summary The A3 and A3 genes, which together constitute the A42 mating type factor of Coprinus cinereus, were isolated from a cosmid genomic library by walking 50 kb, a map distance of 0.5 units, from the closely linked metabolic gene pab-1. Cosmid clones having A gene function were identified by transformation into compatible A6 (22) and A5 (11) host cells where either 3 or 3 was expected to elicit the A factor — regulated development of unfused clamp cells. DNAs were digested with various enzymes before transformation in order to identify the smallest fragments containing an active 3 or 3 gene. Two non-overlapping fragments were identified as containing the 3 and 3 genes respectively. Southern hybridisation analyses showed that these two cloned genes had no detectable sequence homology, and that there was little or no hybridisation to the and gene alleles that constitute the A5 and A6 factors. 3 and 3 were shown to be less than 2.0 kb apart and embedded in a DNA sequence extending over 9.0 kb which was unique to our A42 strain and may contain a third A factor gene.     

The role ofγδ T cells in the normal and disordered immune system

Summary A small population of T cells does not express the conventional T cell receptor characterized by the and polypeptide chains (TCR) but instead, two polypeptides termed and (TCR). This alternative receptor is able to recognize antigen. It appears early in T cell ontogeny, but its role in the thymus prior to the availability of TCR remains unclear. In selected sites such as skin or gut TCR predominates in mice which might suggest a role of T cells in the first line of defense against infection, T cells secrete lymphokines and display cytotoxic activity. However, their activation requirements may differ from what is known for T cells since MHC-nonrestricted and also CD4 and CD8 negative T cells have been described. Preferential activation by mycobacterial antigens possibly indicates a special repertoire of the T cells. In various diseases slightly increased numbers of T cells were found, but these preliminary studies have not yet provided evidence for a major pathogenetic role of T cells.List of abbreviations C constant region (immunoglobulin or TCR gene segment) - CD4 cluster of differentiation 4 (mainly on helper cells) - CD8 cluster of differentiation 8 (mainly on cytotoxic cells) - D diversity region (immunoglobulin or TCR gene segment) - DNA desoxyribonucleic acid - IL2 interleukin 2 - J joining region (immunoglobulin or TCR gene segment) - kD kiloDalton - MHC major histocompatibility complex - NK natural killer (cells) - RA rheumatoid arthritis - TCR T cell receptor - V variable region (immunoglobulin or TCR gene segment)     

β subunits determine the time course of desensitization in rat α3 neuronal nicotinic acetylcholine receptors

Standard two-electrode voltage-clamp techniques were used to investigate some of the pharmacological and functional properties of two types of rat neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes after pairwise injection of 34 or 32 mRNAs. Currents of several A amplitude were elicited by fast application of micromolar concentrations of either acetylcholine (ACh) or 1,1-dimethyl-4-piperazine (DMPP). The activation of either receptor type by DMPP showed cooperativity (Hill coefficient, n1.7) with a half-maximal activation concentration (EC₅₀) of 15–30 M. In 34 receptors, ACh displayed cooperativity (n=1.8) but was less efficacious than DMPP, yet its EC₅₀ was about equal to that of DMPP. Finally, in 32 receptors, ACh was much less efficacious and had a much lower EC₅₀. Desensitization induced by either DMPP or ACh was slow in 34 nicotinic ACh receptors but was rapid and extensive in 32 receptors, causing a significant proportion of the response to wane within the first few seconds of agonist application.     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Isolation and identification of the Aspergillus nidulans gdhA gene encoding NADP-linked glutamate dehydrogenase

Summary The Neurospora crassa am gene was used as a heterologous probe to identify clones from two independently constructed Aspergillus nidulans gene libraries. These clones have a common HindIII 1.85 kb fragment. This A. nidulans nucleotide stretch hybridises to a N. crassa 2.7 kb BamHI fragment of wild type DNA but not to a co-migrating fragment from the DNA of the N. crassa am132 deletion mutant. One A. nidulans clone was shown to complement the N. crasse am132 deletion strain. The N. crassa transformants show low levels (5%) of heterologous glutamate dehydrogenase activity. The A. nidulans gdhA gene was found to locate in N. crassa at both the homologous (i.e. am) site as well as non-nomologous sites. Partial nucleotide analysis of the fragment has revealed the 5 end of the locus and considerable homology with the N. crassa am gene. We concluded that we have cloned the A. nidulans gdhA gene.Abbreviations bp base pairs - kb 1,000 bp - GDH NADP-Iinked glutamate dehydrogenase - NADP nicotinamide adenine dinuc leotide phosphate - SDS sodium dodecyl sulfate - phage lambda - DTT dithiothreitol - SSC 0.15 M NaCl, 0.015 Na₃ citrate pH 7.0     

Molecular size-dependent leakage of intracellular molecules from frog skeletal muscle fibers permeabilized with β-escin

The permeability of -escin-treated cell membrane was characterized in terms of the permeant molecular size, by monitoring the leak of cytoplasmic molecules in frog skeletal muscle fibers. With a low concentration of -escin (5 M), most of the cellular ATP was lost within 30–40 min (as revealed by rigor force generation), whereas a fluorescence-labeled dextran injected into the cytoplasm (10 kDa) and cytoplasmic proteins (14–80 kDa) slowly leaked out of the cell. A high concentration of -escin (50–100 M) accelerated the leak of large molecules. Therefore, low concentrations of -escin may be used as a means of permeabilizing the cell membrane to relatively small molecules, while retaining a major fraction of the cellular macromolecules.Abbreviations MOPS 3-[N-morpholino]propanesulfonic acid - KMS potassium methanesulphonate - PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - EGTA ethylene glycol-bis( -amino-ethyl ether)N,N,N,N-tetraacetic acid - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Molecular and cytogenetic organization of the 5S ribosomal DNA array in chicken (Gallus gallus)

The 5S ribosomal (r) RNA genes encode a small (120-bp) highly-conserved component of the large ribosomal subunit. The objective of the present research was to study the molecular and cytogenetic organization of the chicken 5S rDNA. A predominant 2.2-kb gene (5S) consisting of a coding and intergenic spacer (IGS) region was identified in ten research and commercial populations. A variant gene repeat of 0.6kb (5S) was observed in some of the populations. Genetic linkage analysis and cytogenetic localization by fluorescence in-situ hybridization assigned the 5S rDNA to chromosome 9. The 5S rDNA array was determined to be 80.2±7.0kb upon electrophoretic sizing following EcoRV digestion. Sequence analysis of 5S IGS regions revealed considerable conservation between chicken subspecies (98.4% identity) as well as homology with vertebrate Pol III promoter and regulatory sequence motifs. Minor intraindividual sequence variation within 1000bp of IGS was observed in four cloned Red Jungle Fowl (Gallus gallus gallus) 5S repeats (95.5% identity in this region). Sequence comparisons between IGS regions of 5S and 5S genes indicated two short continuous (>20bp) and many short non-continuous homologous regions as well as other conserved features such as promoter and termination motifs.     

Die in vitro-Empfindlichkeit von Trichomonas vaginalis gegen Metronidazol

Zusammenfassung Die in vitro-Empfindlichkeit von 105 Trichomonas vaginalis-Stämmen gegenüber Metronidazol lag zwischen 0,0625 und 8,0/ml (Verdünnung 116000000 bis 1125000). 58% der Stämme wurden bis 0,5, 77% bis 1,0, 90% bis 2,0 und 99% bis 4,0/ml total gehemmt. Nur bei einem Stamm konnte eine Hemmung erst durch 8,0/ml beobachtet werden. Die Anwesenheit von Escherichia coli oder das gleichzeitige Wachstum von Candida albicans bzw. von Hefen anderer Art im Nährmedium hat keinen Einfluß auf den Ausfall des Empfindlichkeitstests. An Hand von Vergleichsuntersuchungen wird bei der Bestimmung der Grenzkonzentration die Notwendigkeit des Anlegens von Subkulturen herausgestellt. Zwischen klinischem Heilerfolg und in vitro-Empfindlichkeit kann vorerst ein Zusammenhang nicht gefunden werden. In bisher noch nicht abgeschlossenen Versuchen war in vitro eine Gewöhnung von Trichomonas vaginalis an Metronidazol nicht möglich.     

Glucocorticoids modulate TGF-beta production

TGF- is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGB- isoforms affect TGF- production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFL-1) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TFG-1, -2, and -3. TGF- mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TGF-2 and -3 stimulated TGF-1 production significantly (p < 0.01 relative to control). TGF-1 stimulated TGF-2 production (p < 0.01 relative to control). TGF-3 was undetectable. Glucocorticoids significantly inhibited TGF-1 and -2 production and reduced expression of the upregulated TGF-1 and -2 mRNA induced by exogenous TGF-1, -2 or -3 (p < 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF--stimulated cells, it was reduced by glucocorticoids. Thus, TGF- isoforms may stimulate production of various TGF- isoforms in the lung. Glucocorticoids then may block TGF- production by modulating mRNA levels and c-Jun.     

The use of electrophoretic karyotypes in the classification of yeasts: Kluyveromyces marxianus and K. lactis

Summary The relationship between varieties marxianus and lactis of Kluyveromyces marxianus was investigated. Strains of these varieties readily form hybrids, but their classification in one species has recently been contested. Enzyme patterns and the GC contents of their DNAs differed significantly. Moreover, DNA-DNA reassociation was less than 15%. Since the generally accepted definition of a species is based on gene exchange, we used two approaches for directly detecting genetic recombination in crosses between representatives of both species. First, multiple marked strains were crossed, and the offspring from the resulting hybrids analyzed. Secondly, the fate of individual chromosomes in identical crosses was followed by comparing the karyotypes of the parent strains, the hybrids formed between them, and the descendants of these hybrids. Karyotypes were obtained by orthogonal-field-altemation gel electrophoresis (OFAGE). Gene exchange was not detected with either method. We therefore concluded that the formation of hybrids, even when they produced viable offspring, was not sufficient to include var. lactis and var. marxianus in one species.     

Influence of muscle fibre type and pedal rate on the V̇O2-work rate slope during ramp exercise

We hypothesised that the ratio between the increase in oxygen uptake and the increase in work rate (O₂/WR) during ramp cycle exercise would be significantly related to the percentage type II muscle fibres at work rates above the gas exchange threshold (GET) where type II fibres are presumed to be active. We further hypothesised that ramp exercise at higher pedal rates, which would be expected to increase the proportional contribution of type II fibres to the total power delivered, would increase the O₂/WR slope at work rates above the GET. Fourteen healthy subjects [four female; mean (SD): age 25 (3) years, body mass 74.3 (15.1) kg] performed a ramp exercise test to exhaustion (25 W min^–1) at a pedal rate of 75 rev min^–1, and consented to a muscle biopsy of the vastus lateralis. Eleven of the subjects also performed two further ramp tests at pedal rates of 35 and 115 rev min^–1. The O₂/WR slope for exercise <GET (S ₁) was significantly correlated with O₂ peak in ml kg^–1 min^–1 (r=0.60; P<0.05), whereas the O₂/WR slope for exercise >GET (S ₂) was significantly correlated to percentage type II fibres (r=0.54; P=0.05). The ratio between the O₂/WR slopes for exercise above and below the GET (S ₂/S ₁) was significantly greater at the pedal rate of 115 rev min^–1 [1.22 (0.09)] compared to pedal rates of 35 rev min^–1 [0.96 (0.02)] and 75 rev min^–1 [1.09 (0.05), (P<0.05)]. The greater increase in S ₂ relative to S ₁ in subjects (1) with a high percentage type II fibres, and (2) at a high pedal rate, suggests that a greater recruitment of type II fibres contributes in some manner to the xs O₂ observed during ramp exercise.     

Transformation of Saccharomyces cerevisiae with plasmids containing fragments of yeast 2 μ DNA and a suppressor tRNA gene

Hybrid plasmids have been constructed containing segments of the yeast plasmid 2 DNA, the yeast ochre-suppressing SUP4.0 gene and the bacterial plasmid pBR322. Yeast transformation is detected with a host containing multiple ochre auxotrophic mutations. The transformed SUP4.0 gene is active and can promote growth in the absence of all the requirements. Plasmids containing different fragments of 2 DNA all appear to be active in high frequency transformation of yeast containing 2 DNA, except those containing the HindlII-D fragment. The transforming plasmids undergo recombination with the indigenous 2 DNA. Integration of the transforming plasmid into the host chromosome has been detected by hybridization of restriction enzyme cleaved DNA with labelled pBR322. The plasmids contain restriction enzyme sites which can be used for cloning other genes into yeast.Abbreviations kb kilobase pair - 2 the yeast plasmid of 6.2 kb size     

Inhibition of lung colonization of mouse colon 26 adenocarcinoma by recombinant mouse interferon β through a modification of platelet function

Recombinant murine interferon (MuIFN-) given i.v. efficiently inhibited both pulmonary arrest and formation of lung colonies of NL-17, a highly metastatic variant of mouse colon adenocarcinoma 26. NL-17 was rather resistant to MuIFN- in vitro and was highly resistant to natural killer cells of mice even though they were treated in vivo with MuIFN-. Platelets isolated from MuIFN--treated mice showed reduced aggregating activity induced by NL-17. Since lung colonization by NL-17 is influenced by platelet aggregation, the inhibition of colonization by MuIFN- could be partly mediated through modification of platelet function in vivo. The effect of MuIFN- on platelet function and its subsequent inhibition of lung colony formation give new insights into the action of recombinant MuIFN-.     

Identification and characterization of four new GCD genes in Saccharomyces cerevisiae

Summary Mutant strains, resistant against the amino acid analogues 5-methyltryptophan, 5-fluorotryptophan and canavanine were isolated, starting with a trp2 leaky auxotrophic strain. Of 10 such strains, only four turned out to be of the general control derepressed (gcd) mutant type. Three other isolates were shown to be defective in the general amino acid permease system, while the remaining three strains displayed low spore viability and were not further investigated. Complementation tests amongst the four new gcd-mutant strains, including strain RH558 gcd2-1 isolated earlier, yielded five complementation groups: GCD2, GCD3, GCD4, GCD5, and GCD6. All mutant strains showed a dual phenotype, which was not separable by wild type backcrosses: constitutive derepression and slow growth. Epistatis of all gcd mutations over gcn1-1, gcn2-1 and gcn3-1 was found with respect to both phenotypes, except for gcd5-1, which was lethal in these combinations. On the other hand gcn4-101 was found to be epistatic over all gcd mutations, but only with respect to the constitutive derepression phenotype, and not to slow growth; again the combination with gcd5-1 was lethal. Mutation gcd2-1 was mapped on chromosome VII, 50 cM from leu1 and 22 cM from ade6. A new model is discussed, in which GCD-genes are involved in the amino acid uptake into the vacuoles.     

Ribonuclease activity of the “myofibrillary” fraction of normal and autolyzed muscle tissue

The separation in a sucrose gradient of the myofibrillary fraction of normal and autolyzed muscle tissue gave 4 components. During post-mortem destruction of the tissue there was observed a slight decrease of the myofibrillary fraction yield and also certain changes in the distribution of protein between different components. Under the selected conditions RNase activity was found in all 4 components. During the course of autolysis enzymatic activity increased in the whole myofibrillary fraction, as well as in the lysosomal-mitochondrial components of myofibrils.Research Laboratory, Ministry of Health of the USSR, Moscow. Translated from Buylleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 5, pp. 533–537, May, 1978.     

Transformation of ψ − Saccharomyces cerevisiae to ψ + with DNA co-purified with 3 μm circles

Summary DNA enriched for supercoiled plasmids prepared from the 3 m plasmid-enriched, [ ⁺], [2 m°] strain 6-1G-P188 and from the [2 m⁺] [⁺] strain LL20 can be used to transform a ^– recipient strain to ⁺. Fractionation of the former preparation by electrophoresis showed that the 3 Mm plasmid band contained the transforming activity.     

Female song preference and theperiod gene inDrosophila

Mutations at theperiod (per) locus inDrosophila melanogaster alter rhythmic components of the male courtship song. We have examined the mating speed of females homozygous for mutantper alleles when presented with artificial mutant songs. Mutant females retain a preference for wild-type over mutant songs, thus male song and female preference are probably under separate genetic control. In contrast,per-mutant females from an established laboratory stock which had been maintained for nearly two decades appear to have an enhanced response to the corresponding mutant song in that they no longer discriminate against mutant song. These results are discussed in terms of the genetic coupling and coevolution theories of complementarity between male and female components of communication systems.     

Effects of inhibitors and ion substitutions on oscillations of cell membrane potential in cells expressing the RAS oncogene

Previous studies revealed that in NIH fibroblasts expressing the ras oncogene but not in other NIH fibroblasts, bradykinin leads to sustained, calcium dependent oscillations of cell membrane potential by repetitive activation of calcium-sensitive K⁺ channels. The present study has been performed to test for ion and inhibitor sensitivity of these oscillations. Both, Lys-bradykinin (kallidin) and bradykinin, but not any shorter peptide tested, maintained the oscillations. The oscillations are abolished in the presence of the K⁺ channel blocker barium (10 nmol/l). The amplitude but not the frequency of the oscillations is dependent on the extracellular potassium concentration. The oscillations are not dependent on the presence of extracellular sodium, bicarbonate or chloride. The oscillations are abolished in the absence of extracellular calcium and their frequency is significantly decreased at reduced extracellular calcium (to 0.2 mmol/l). The oscillations are not inhibited by acute administration of ouabain (0.1 mmol/l), by dimethylamiloride (100 mol/l), furosemide (1 mmol/l) and hydrochlorothiazide (100 mol/l), by cobalt (100 mol/l), zinc (100 mol/l), gadolinium (100 mol/l), verapamil (10 mol/l) and diltiazem (10 mol/l), but are abolished in the presence of 100 mol/l lanthanum, 1 mmol/l cadmium, 10 mol/l nifedipine, 25 mol/l SK & F 96365 and 200 mol/l TMB-8. Stimulation of calcium entry by 10 mol/l ionomycin is frequently followed by oscillations of cell membrane potential even in the absence of bradykinin. In conclusion, in cells expressing the ras oncogene bradykinin leads to sustained activation of calcium channels at the cell membrane, which cause oscillations of the cell membrane potential by triggering intracellular calcium release.