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Biomedical subjects

K F Jensen

Publications and source records attributed to K F Jensen.

At least 55 records · Page 3Linked to original sources
PubMed1994

Neonatal destruction of dopaminergic neurons.

Rats treated as neonates with 6-hydroxydopamine are proposed to model the dopamine deficiency associated with Lesch-Nyhan syndrome (LNS). To understand the neurobiological basis of specific behaviors in LNS, investigations were undertaken in these neonatally lesioned rats. Several new findings resulted from these studies. The first was that D1-dopamine receptors are essential for the action of D2-dopamine receptors, a phenomenon called "coupling" of receptor function. Another finding was that D1-dopamine receptors must be repeatedly stimulated before maximal behavioral sensitivity can be observed. This has been referred to as "priming" of D1-dopamine receptor responsiveness. This priming action by repeated administration of a D1-dopamine agonist was antagonized by NMDA antagonists indicating a potential role of glutamate in this sensitization. Ongoing work suggests that DARPP-32 is not involved in priming of D1-dopamine receptor responsiveness. However, we have observed an accumulation of GFAP in brain following repeated administration of a D1-dopamine agonist. In addition, immunoblots employing an antibody to phospho-DARPP-32 revealed a protein present in lesioned rats that was not present in control rats. Studies in these lesioned rats are expected to continue to contribute to our basic understanding of adaptive changes caused by lesioning of dopaminergic neurons during development.

Animals↗
PubMed1993

The rates of macromolecular chain elongation modulate the initiation frequencies for transcription and translation in Escherichia coli.

Here we show that most macromolecular biosynthesis reactions in growing bacteria are sub-saturated with substrate. The experiments should in part test predictions from a previously proposed model (Jensen & Pedersen 1990) which proposed a central role for the rates of the RNA and peptide chain elongation reactions in determining the concentration of initiation competent RNA polymerases and ribosomes and thereby the initiation frequencies for these reactions. We have shown that synthesis of ribosomal RNA and the concentration of ppGpp did not exhibit the normal inverse correlation under balanced growth conditions in batch cultures when the RNA chain elongation rate was limited by substrate supply. The RNA chain elongation rate for the polymerase transcribing lacZ mRNA was directly measured and found to be reduced by two-fold under conditions of high ppGpp levels. In the case of translation, we have shown that the peptide elongation rate varied at different types of codons and even among codons read by the same tRNA species. The faster translated codons probably have the highest cognate tRNA concentration and the highest affinity to the tRNA. Thus, the ribosome may operate close to saturation at some codons and be unsaturated at synonymous codons. Therefore, not only translation of the codons for the seven amino acids, whose biosynthesis is regulated by attenuation, but also a substantial fraction of the other translation reactions may be unsaturated. Recently, we have obtained results which indicate that also many ribosome binding sites are unsaturated with their substrate, i.e. with ribosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1993

The Escherichia coli K-12 "wild types" W3110 and MG1655 have an rph frameshift mutation that leads to pyrimidine starvation due to low pyrE expression levels.

The widely used and closely related Escherichia coli "wild types" W3110 and MG1655, as well as their common ancestor W1485, starve for pyrimidine in minimal medium because of a suboptimal content of orotate phosphoribosyltransferase, which is encoded by the pyrE gene. This conclusion was based on the findings that (i) the strains grew 10 to 15% more slowly in pyrimidine-free medium than in medium containing uracil; (ii) their levels of aspartate transcarbamylase were highly derepressed, as is characteristic for pyrimidine starvation conditions; and (iii) their levels of orotate phosphoribosyltransferase were low. After introduction of a plasmid carrying the rph-pyrE operon from strain HfrH, the growth rates were no longer stimulated by uracil and the levels of aspartate transcarbamylase were low and similar to the levels observed for other strains of E. coli K-12, E. coli B, and Salmonella typhimurium. To identify the mutation responsible for these phenotypes, the rph-pyrE operon of W3110 was cloned in pBR322 from Kohara bacteriophage lambda 2A6. DNA sequencing revealed that a GC base pair was missing near the end of the rph gene of W3110. This one-base-pair deletion results in a frame shift of translation over the last 15 codons and reduces the size of the rph gene product by 10 amino acid residues relative to the size of RNase PH of other E. coli strains, as confirmed by analysis of protein synthesis in minicells. The truncated protein lacks RNase PH activity, and the premature translation stop in the rph cistron explains the low levels of orotate phosphoribosyltransferase in W3110, since close coupling between transcription and translation is needed to support optimal levels of transcription past the intercistronic pyrE attenuator.

Amino Acid Sequence↗
PubMed1992

Overexpression and rapid purification of the orfE/rph gene product, RNase PH of Escherichia coli.

The pyrE gene, encoding the pyrimidine biosynthetic enzyme orotate phosphoribosyltransferase, is the promoter distal gene of the dicistronic orfE-pyrE operon. The promoter proximal orfE gene, whose transcription and translation is important for regulation of the pyrE attenuator, encodes a 238-amino acid residue protein which was recently identified as the phosphorolytic ribonuclease, RNase PH, that removes nucleotides from the 3' ends of tRNA precursors. In this paper we report the construction of a plasmid, which overexpresses the orfE and pyrE gene products substantially, as well as the purification of the OrfE protein by ammonium sulfate precipitation and chromatography on phosphocellulose. The highly purified protein catalyzes the phosphorolytic cleavage of poly(A) at a rate of 1.6 mumol/min/mg and the formation of CDP from tRNA-CCA-Cn and orthophosphate at a rate equal to 0.14 mumol/min/mg, as characteristic for RNase PH. OrfE/RNase PH contains helix-turn-helix motifs resembling those in DNA-binding proteins, and it binds nonspecifically to DNA. On SDS gels, OrfE/RNase PH migrates as two distinct protein bands. This heterogeneity might be caused by post-translational modification other than proteolysis, or may be an electrophoretic artifact. The native protein is composed of two or more subunits.

Amino Acid Sequence↗
PubMed1992

Attenuation in the rph-pyrE operon of Escherichia coli and processing of the dicistronic mRNA.

We have substituted on a plasmid the native promoter of the Escherichia coli rph-pyrE operon with an inducible transcription-initiation signal. The plasmid was used to study the mRNA chains derived from the operon at different intracellular concentrations of UTP and as a function of time following induction of transcription. The results showed that dicistronic rph-pyrE mRNA was formed when the UTP pool was low, and that a monocistronic rph mRNa was the major transcription product in high-UTP pools, thus supporting an UTP-controlled attenuation mechanism for regulation of pyrE gene expression. However, the dicistronic rph-pyrE transcript was rapidly processed into two monocistronic mRNA units, and a cleavage site was mapped near the attenuator in the intercistronic region, close to the site where transcription was terminated in high-UTP pools. Furthermore, the major 3' end of the pyrE mRNA was mapped near a palindromic structure of similarity to the family of repetitive extragenic palindromic sequences, 35 nucleotide residues after stop codon of the pryE gene.

Base Sequence↗
PubMed1992

Anomalous phosphorylated neurofilament aggregations in central and peripheral axons of hens treated with tri-ortho-cresyl phosphate (TOCP).

Previous biochemical studies demonstrated a dramatic increase in phosphorylation of cytoskeletal proteins that occurs early in organophosphorus ester-induced delayed neurotoxicity (OPIDN). In this report we present immunohistochemical evidence that there is anomalous aggregation of phosphorylated neurofilaments within central and peripheral axons following organophosphate exposure. The morphology, location, and time of appearance of these aggregations are consistent with the hypothesis that the aberrant phosphorylation of cytoskeletal elements is an antecedent to the focal axonal swelling and degeneration characteristic of OPIDN.

Animals↗
PubMed1992

Decreasing transcription elongation rate in Escherichia coli exposed to amino acid starvation.

The time required for transcription of the lacZ gene in Escherichia coli was determined during exponential growth and under conditions, when the bacterium was exposed to partial isoleucine starvation. To do this, RNA was extracted from the cells at 10 s intervals following induction and quantified by Northern hybridization with probes complementary to either the beginning or the end of the lacZ mRNA. The time lag between inducer addition and the appearance of a hybridization signal at the 'late' probe represents the transit time for RNA polymerase on the lacZ gene, and this parameter and the known length of the transcribed sequence were used to calculate the lacZ mRNA chain growth-rate. The transcription elongation rate was c. 43 nucleotides s-1 during exponential growth and decreased abruptly to c. 20 nucleotides s-1 in a relA+ strain after the onset of isoleucine starvation, when massive concentrations of guanosine tetraphosphate (ppGpp) accumulated in the cells. The starvation condition did not affect initiation of transcription at the lac-promoter, but a substantial fraction of the initiated lacZ mRNA chains was never completed. For the rel+ strain the polarity was moderate, since c. 25% of the initiated lacZ mRNA' chains were continued into full-length mRNAs, but for the relA strain the polarity was so strong that no completed lacZ mRNA could be detected. The protein chain elongation rates decreased from 13 amino acids (aa) s-1 in the unperturbed growth phase to approximately 6 as s-1, when the cells starved for isoleucine. In combination, these results suggest that ppGpp plays a major role in maintaining the coupling between transcription and translation during the downshift by inhibiting mRNA chain elongation. The implications of this result for the control of stable RNA synthesis during the stringent response are discussed.

Amino Acids↗
PubMed1992

Comparison of the D1-dopamine agonists SKF-38393 and A-68930 in neonatal 6-hydroxydopamine-lesioned rats: behavioral effects and induction of c-fos-like immunoreactivity.

Administration of the selective D1-dopamine receptor agonist 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine (SKF-38393) to neonatal 6-hydroxydopamine-lesioned rats results in profound behavioral manifestations and induction of striatal c-fos-like immunoreactivity. The full D1-dopamine agonist I,[R,S]1-aminomethyl-3,4-dihydro-5,6-dihydroxy-3-phenyl-1H-2-benzopyran hydrochloride (A-68930), like SKF-38393, produced a dose-dependent, D1-selective increase in locomotor activity and striatal c-fos-like immunoreactivity. These responses were antagonized by a D1-dopamine antagonist, but not by a D2-dopamine antagonist. A-68930 produced locomotor activation at a lower dose than SKF-38393, but no dose of A-68930 was able to produce the magnitude of locomotor activation seen with SKF-38393. Both A-68930 and SKF-38393 induced similar stereotyped behaviors and possessed similar propensities to induce self-injurious behavior in neonatally lesioned rats; however, A-68930 was significantly more potent than SKF-38393 in inducing these behaviors. When either SKF-38393 or A-68930 were administered repeatedly at 2-week intervals, behavioral sensitization (priming) occurred. However, unlike SKF-38393, a high dose of A-68930 produced seizure activity and markedly desensitized D1-dopamine receptor activation for up to 3 days after administration. These results with A-68930 provide additional evidence that the specific behavioral and biochemical responses observed in neonatally lesioned rats after SKF-38393 administration are due to actions on D1-dopamine receptors, and indicate that A-68930 provides a new tool for investigating D1-dopamine receptor function.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1992

Enhanced expression of glial fibrillary acidic protein and the cupric silver degeneration reaction can be used as sensitive and early indicators of neurotoxicity.

The mammalian central nervous system is composed of a large and diverse array of its two principle cell types, neurons and glia. Given this extreme cellular heterogeneity, it is not surprising that targets of neurotoxic insult often are diverse and unpredictable. In devising strategies to assess neurotoxicity, one must overcome the obstacle posed by not knowing where to look for damage. Our approach to the detection and localization of sites of toxicant-induced damage has been to take advantage of two general responses to nervous system insult, astrogliosis and argyrophilia. Assays for GFAP, the principle intermediate filament protein of astrocytes, have been devised to quantify astrogliosis and a modified cupric silver degeneration stain has been employed to examine patterns of argyrophilia. Using these methods, we found that increases in GFAP and the occurrence of argyrophilia are consistent responses to neurotoxic insults. Moreover, increments in these two indicators reveal dose-, time- and region-dependent patterns of neurotoxicity at toxicant dosages below those that cause light microscopic evidence of cell loss or damage. The rapid onset of astrogliosis and argyrophilia following specific toxicant exposures suggests that the signals underlying these events are among the earliest that can be linked to the neurotoxic state.

Animals↗
PubMed1991

Pargyline and gamma-butyrolactone enhance tyrosine hydroxylase immunostaining of nigrostriatal axons.

Administration of pargyline or gamma-butyrolactone enhanced immunostaining of tyrosine hydroxylase immunoreactive axons in the striatum. The qualitative characteristics of the enhancement were compound dependent and the enhancement was not associated with an increase in the amount of tyrosine hydroxylase within the striatum. These results demonstrate the pharmacological enhancement of immunostaining for neurotransmitter synthetic enzymes in the absence of increased synthesis.

4-Butyrolactone↗
PubMed1991

Glutamate neurotoxicity in the developing rat cochlea: physiological and morphological approaches.

The neurotoxic effects of exogenous glutamate were studied in the rat cochlea. Glutamate-treated rats (4 g/kg/day i.p., postnatal days 2-9) exhibited electrophysiologically-measured elevations in high frequency thresholds usually associated with hair cell loss in the basal region of the cochlea. While surface preparations of the organ of Corti revealed no loss of hair cells, there was a dramatic and selective reduction of neurons in the basal, high frequency-related portion of the spiral ganglion. This sensitivity of developing spiral ganglion cells to the neurotoxicity of glutamate is consistent with the hypothesis that glutamate or a structurally related substance is a neurotransmitter at afferent synapses of cochlear hair cells.

Acoustic Stimulation↗
PubMed1991

Acquisition and loss of a neuronal Ca2+/calmodulin-dependent protein kinase during neuronal differentiation.

Calcium ions play a critical role in neural development. Insights into the ontogeny of Ca(2+)-signaling pathways were gained by investigating the developmental expression of granule cell-enriched Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr) in the cerebellum and hippocampus of the rat. Neurons of these brain regions displayed characteristic schedules by which they acquired and lost CaM kinase-Gr during differentiation. In the cerebellum, granule cells did not begin to express CaM kinase-Gr until after birth when they migrated into the granule cell layer, and this expression persisted in the adult. Purkinje cells expressed CaM kinase-Gr prenatally and lost this expression by postnatal day 14. In contrast, the granule and pyramidal cells of the hippocampus expressed the enzyme prenatally and in the adult. Moreover, CaM kinase-Gr was localized to the processes and nuclei of developing neurons. This subcellular localization together with the scheduled expression of CaM kinase-Gr can serve to regulate a developing neuron's sensitivity to Ca2+ at different subcellular levels.

Animals↗
PubMed1991

Nuclear and axonal localization of Ca2+/calmodulin-dependent protein kinase type Gr in rat cerebellar cortex.

The granule cell-enriched Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr) is a recently discovered neuron-specific enzyme. The kinase avidly phosphorylates synapsin I and contains a polyglutamate sequence, which suggests an association with chromatin as well. A possible role in synapsin I phosphorylation and in nuclear Ca2+ signaling was supported by immunochemical and ultrastructural examination of CaM kinase-Gr distribution. CaM kinase-Gr immunoreactivity was present in the molecular and granule cell layers of the rat cerebellum. This pattern corresponded to the occurrence of the enzyme in the granule cell axons and nuclei, respectively. Immunoblots confirmed these findings. Thus, CaM kinase-Gr may mediate and coordinate Ca2(+)-signaling within different subcellular compartments.

Animals↗
PubMed1991

Functional deficits produced by 3-methylindole-induced olfactory mucosal damage revealed by a simple olfactory learning task.

Methods for assessing functional consequences of olfactory mucosal damage were examined in rats exposed to 3-methylindole (3-MI). Treatment with 3-MI (400 mg/kg) induced severe degeneration of olfactory sensory epithelium followed by regeneration, fibrous adhesions, and osseous remodeling of the nasal passages. At 100 mg/kg, there was mild Bowman's gland hypertrophy while the sensory epithelium remained intact. Rats receiving 3-MI demonstrated a treatment-related deficit in acquiring an olfactory learning task which was not due to altered cognitive abilities, as determined by subsequent testing in a step-through passive avoidance task. The results confirm the conclusion that alterations in functional indices resulted from 3-MI-induced anosmia and demonstrate the utility of simple learning tasks in assessing functional capacity following olfactory epithelial damage in rats.

Animals↗
PubMed1991

Role of transcription pausing in the control of the pyrE attenuator in Escherichia coli.

Expression of the Escherichia coli pyrE gene is regulated by transcription attenuation in the intercistronic orfE-pyrE region and modulated by the distance between the transcribing RNA polymerase and the leading ribosome as a function of the supply of UTP and GTP. In this communication we show that pyrE expression is hyper-repressed in vivo following addition of uracil in strains carrying the nusAcs10 mutation. This phenotype, previously seen in rpsL1204 strains whose ribosomes are pseudodependent on streptomycin and work at suboptimal elongation rate, indicates that RNA polymerase escapes from the ribosomes in the pyrE attenuator region in the nusA mutant. In vitro transcription studies revealed that the build-up of the full-length attenuated orfE transcript occurred more slowly in the presence of the NusA protein than in its absence. Moreover, the NusA protein enhanced several transcription pauses through the orfE gene. These effects were more pronounced when low concentrations of either UTP or GTP were used than at low concentrations of either CTP or ATP. The results indicate that the NusA protein is required for proper regulation of pyrE gene expression and is involved, together with the NTP pools, in maintaining the coupling between transcription and translation in the pyrE attenuator region by inhibiting RNA chain elongation.

Bacterial Proteins↗
PubMed1991

An unusual correlation between ppGpp pool size and rate of ribosome synthesis during partial pyrimidine starvation of Escherichia coli.

Escherichia coli was exposed to partial pyrimidine starvation by feeding a pyrBI strain orotate as the only pyrimidine source. Subsequently, differential rates of synthesis of rRNA and of a few ribosome-associated proteins as well as the pool sizes of nucleoside triphosphates and ppGpp were measured. As the orotate concentration in the medium was reduced, the growth rate decreased and the pools of pyrimidine nucleotides, particularly UTP, declined. We did not observe the normal inverse relation between concentration of ppGpp and growth rate; rather, we observed that the ppGpp pool was low at slow growth rates. Upshifts in growth rate were made by adding uracil to a culture growing slowly on orotate. Downshifts could be provoked by adding aspartate plus glutamate to a culture growing at a high concentration of orotate. Following the upshift, both the rates of synthesis of the ribosomal components and the pool of ppGpp increased rapidly, while they all decreased after the downshift. These results are discussed in relation to the role of ppGpp in the growth rate control and the stringent response.

Escherichia coli↗
PubMed1990

Influence of two growing seasons of experimental ozone fumigation on photosynthetic characteristics of white oak seedlings.

White oak (Quercus alba L.) seedlings were exposed to charcoal-filtered air or to above-ambient ozone concentrations for 19-20 weeks during each of two growing seasons in continuously stirred tank reactors in greenhouses. Ozone treatments were 0.15 ppm (300 microg m(-3)) for 8 h day(-1), 3 days week(-1) in 1988, and continuous 15% above ambient in 1989. The seedlings were grown in forest soil watered twice weekly with simulated rain of pH 5.2. Responses of net photosynthesis to photosynthetically active radiation and intercellular CO(2) concentration were measured three times each year. There were no significant differences in light-saturated net photosynthesis or stomatal conductance, dark respiration, quantum or carboxylation efficiencies, and light or CO(2) compensation points on any date between control and ozone-exposed seedlings.

Journal Article↗