[Permeability disorders of the blood-brain barrier following intravascular contrast medium administration in kidney failure].
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Aged↗
Biomedical subjects
D Kern
Publications and source records attributed to D Kern.
Kinetics of 1,25-dihydroxyvitamin D metabolism in the aging rat.
To determine whether postmaturational aging influences the kinetics of 1,25-dihydroxyvitamin D [1,25(OH)2D] metabolism in the rat, we measured the metabolic clearance (MCR) and production (PR) rates of 1,25(OH)2D in 6-, 12-, 18-, and 24-mo-old Fischer 344 rats using the constant infusion method. Plasma calcium, phosphorus, and parathyroid hormone (PTH), urinary calcium and phosphorus, and glomerular filtration rate (GFR) were also measured. MCR and PR increased 57 and 91%, respectively (when expressed per rat), and 32 and 39%, respectively (when expressed per kg body wt), between 6 and 24 mo of age, with the greatest increase occurring between 18 and 24 mo. The plasma concentration of 1,25(OH)2D remained unchanged. Plasma PTH, when compared with 6-mo-old animals, was significantly elevated at 18 mo (147%) and even higher at 24 mo (240%). GFR (51Cr-labeled EDTA clearance) remained unchanged through 18 mo but tended to be reduced at 24 mo. Plasma phosphorus tended to decrease with age, whereas plasma calcium, urinary calcium, and urinary phosphorus did not differ among the age groups. These data indicate that both clearance and production of 1,25(OH)2D increase with postmaturational aging in the rat. They also suggest that the early age-related rise in plasma PTH is neither a consequence of low plasma calcium nor of renal insufficiency.
Aging↗
Are media just media? (Part III).
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Blood↗
An exploration of the variables involved when instituting a do-not-resuscitate order for patients undergoing bone marrow transplantation.
Written do-not-resuscitate (DNR) orders can facilitate the transition from aggressive care to supportive care in patients undergoing bone marrow transplantation who are critically ill. The DNR decision often is complicated by clinical and ethical problems. To assist nurses in analyzing these complex situations, a retrospective review was conducted on medical records of 40 patients who died on an eight-bed bone marrow transplant unit in which a formal DNR policy was in place. A review of specific variables for patients designated DNR and non-DNR showed that the groups did not vary in age, diagnosis, disease, or type of transplant. The non-DNR group developed life-threatening complications earlier in their transplant course, whereas multisystem failure was the common factor among the patients with DNR designation. The few ethical problems documented in the medical records did not appear to reflect the complexity of the DNR decision.
Adolescent↗
Isolation and characterization of active N-terminal truncated apo- and holoenzyme of mammalian liver tyrosine aminotransferase.
Limited proteolysis was used to probe the structure of the apo- and holoenzyme of rat liver tyrosine aminotransferase. Both were subjected to trypsinolysis and the major fragments were isolated and characterized. Trypsin cleaves the apoenzyme after residues Arg57, Lys64, and Lys71 and the holoenzyme after Arg37 and Lys38. The difference in the accessibility of the enzyme deprived or associated with pyridoxal 5'-phosphate reflects two distinct conformations. The activity, the affinity for the ligands and the thermostability of the purified truncated enzyme forms are similar to those of the native apo- and holoenzyme. A model for the domain structure of mammalian tyrosine aminotransferase and a mechanism for its rapid turnover are proposed.
Amino Acid Sequence↗
Expression of mammalian tyrosine aminotransferase in Saccharomyces cerevisiae and Escherichia coli. Purification to homogeneity and characterization of the enzyme overproduced in the bacteria.
Rat liver tyrosine aminotransferase has been expressed in Saccharomyces cerevisiae and Escherichia coli. In yeast, the extent of production is 20-fold higher than that in rat liver after induction by dexamethasone, and reaches 250-fold higher in an E. coli strain carrying the T7 RNA polymerase transcription system. About 250 mg pure and homogeneous enzyme was obtained from 50 g transformed E. coli cells. Determination of Mr and pI, as well as analysis of N- and C-terminal amino acids, suggest that the isolated protein is native. The catalytic properties, similar to those of the enzyme from rat liver, confirm that it is fully active and that post-translational modifications in the mammalian cells are not essential for activity. Pyridoxal 5'-phosphate strongly protects the enzyme against thermal inactivation. After denaturation, 10 thiol groups, out of 16 in the polypeptide chain, react with 5,5'-dithiobis(2-nitrobenzoic acid) whereas only five or six are accessible under native conditions. Two thiols are rapidly modified with concomitant inactivation of the apoenzyme, but pyridoxal 5'-phosphate partially protects them in the holoenzyme. The results are interpreted in the light of the structure/function relationship in this enzyme.
Amino Acid Sequence↗
Cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae. Study of its functional organisation by deletion analysis.
Aspartyl-tRNA synthetase (AspRS) from yeast, a homodimer of 125 kDa, was shortened by several residues from the C- and N-termini, via site-directed mutagenesis, to examine the contribution of the removed peptides to the enzyme properties. This study showed that the N-terminal sequence up to amino acid 70 (which confers peculiar ionic properties to the protein) is dispensable for activity. Domains located beyond amino acid 70 appeared to have increasing catalytic importance; the removal of 80 or 90 residues affected the Km values for ATP and deletions of 101 or 140 amino acids profoundly modified the physiochemical properties of AspRS, and by consequence, its structural organisation (extraction of the mutated proteins out of the cells required the presence of SDS). On the C-terminal side, very limited modifications readily affected the enzyme properties. Deletion of as few as three residues increased the Km for ATP and reduced the aminoacylation kcat as well as the thermostability of the adenylate synthesis activity; the kcat of this step was impaired after deletion of two further residues. Finally, shortening the C-terminal decapeptide completely inactivated AspRS, whilst affecting neither its affinity for tRNAAsp nor its dimerisation capacity. These data reveal the role of the C-terminal decapeptide as a determinant in both reactions catalysed by AspRS. This peptide is involved in ATP binding, stabilising the functional conformation of the amino-acid-activating domain and probably maintaining the tRNA-acceptor end in a reactive position with regard to the activated amino acid.
Adenosine Triphosphate↗
Identification of structurally and functionally important histidine residues in cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae.
Cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae is an alpha 2 dimer (alpha, Mr 63,000), each alpha containing 12 histidines. The covalent incorporation of 6-7 mol of diethyl pyrocarbonate per monomer corresponded to complete enzyme inactivation. This inactivation was reversed by hydroxylamine hydrolysis which regenerates free histidine (and tyrosine) while leaving the carbethoxy group still attached to the epsilon-amino group of lysine. Three histidines, one tyrosine, and four lysines were the main targets of the reagent. Site-directed mutagenesis was also tried to replace each of these modified residues. Given the unstability of the carbethoxy-imidazole bond, the nine histidines that were not modified by diethyl pyrocarbonate were mutated too. For these experiments, the enzyme was expressed in Escherichia coli by using a vector bearing the structural gene in which the first 13 codons were replaced by the first 14 of the CII lambda gene. This substitution had no effect on the kinetic parameters. The combined results of chemical modification and site-directed mutagenesis show that one histidine seems to be part of the active site while two others play an important structural role. On the other hand, labeled lysines and tyrosine are nonessential residues. These results are discussed in light of two recent articles establishing the existence of a second family of aminoacyl-tRNA synthetases devoid of the HIGH and KMSKS consensus sequences and containing no Rossmann's domain in their three-dimensional structures.
Amino Acid Sequence↗
The three cysteine residues of cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae are not essential for its activity.
Cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae is a dimer made up of identical subunits (Mr 63,000) each of these containing three cysteines (residues 255, 512 and 519 in the amino acid sequence). Thiol-specific probes were used to label these cysteines and study the resulting effect of the modification on the kinetic parameters of both the ATP/PPi exchange and tRNA aminoacylation reactions. Using the classical techniques of protein chemistry it was shown that none of the three cysteines was labelled with iodoacetic acid, whilst N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoate) reacted with Cys512 and Cys255, respectively. Only the latter modification was accompanied by a decrease in the rates of both enzyme activities whilst the Km values for the various substrates remained unaffected. Site-directed mutagenesis was also used to replace each of the three cysteines by other residues, either individually or simultaneously. For these experiments the enzyme was expressed in Escherichia coli using an expression vector bearing the structural gene in which the first 13 codons were replaced by the first 14 of the CII lambda gene. The resulting substitution in the amino-terminal part of the expressed enzyme had no effect on the kinetic parameters, compared to those of the enzyme purified from S. cerevisiae. Taking into account the consequences of such substitutions, as well as those of chemical modifications on the two reactions catalysed by the enzyme. ATP/PPi exchange and tRNA aminoacylation, it could be concluded that none of these three cysteines plays any essential role in either substrate binding or catalysis.
Adenosine Triphosphate↗
Primary care internal medicine: a challenging career choice for the 1990s.
A career in primary care internal medicine can be challenging and rewarding, yet during the last decade fewer medical students have selected training in internal medicine. We wish to inform medical students, their advisors, and other physicians about the field of primary care internal medicine. We define the discipline, compare it with traditional internal medicine and with family practice, and describe features of strong primary care internal medicine training programs. We discuss common misconceptions and concerns about training programs and the career and give examples of career paths chosen by graduates of primary care programs. We encourage students to consider the option of primary care internal medicine when making career decisions and provide faculty advisors unfamiliar with primary care internal medicine training programs with a reference resource.
Career Choice↗
Stimulatory effect of ammonium sulfate at high concentrations on the aminoacylation of tRNA and tRNA-like molecules.
The influence of various salts on the aminoacylation of tRNA(Val) and the tRNA-like structure from turnip yellow mosaic virus RNA by yeast valyl-tRNA synthetase has been studied. As expected, increasing the concentration of salts inhibits the enzymatic reaction. However, in the presence of high concentration of ammonium sulfate, and only this salt, the inhibitory effect is suppressed. Under such conditions, the aminoacylation becomes comparable to that measured in the absence of salt. It was shown that ammonium sulfate affects both the catalytic rate of the reaction and the affinity between valyl-tRNA synthetase and the RNAs. Because the affinity between the partners in the complex is increased when the concentration of the salt is high, it is suggested that hydrophobic effects are involved in tRNA/synthetase interactions.
Amino Acyl-tRNA Synthetases↗
A control for fetal bovine serum.
The FBS Control is designed to assist cell culture scientists who have been frustrated with lack of consistency in FBS lots. Continued use will reduce the time and expense needed in classical lot selection methods.
Animals↗
The interior of a whole and unmodified biological object--the zymogen granule--viewed with a high-resolution X-ray microscope.
We report the ability of focused soft X-rays to visualize at spatial resolution well beyond that of the optical microscope (less than 100 nm) the interior of a small, whole biological object without fixation, staining, dehydration or sectioning. Quantitative estimation of its protein content with unique femtogram sensitivity is also reported. The present results represent a significant step towards the goals of natural imaging and chemical mapping of biological structures with soft X-rays.
Animals↗
Study of the arrangement of the functional domains along the yeast cytoplasmic aspartyl-tRNA synthetase.
Aspartyl-tRNA synthetase from yeast (AspRS) was screened for functional domains by measuring the effect of two types of amino acid mutations on its catalytic properties: (a) insertion of a dipeptide or a tetrapeptide along the polypeptide chain, (b) deletion of various lengths from the enzyme C-terminal. It was shown that insertion mutations significantly affect the kinetic properties of AspRS only when occurring in the second quarter of the molecule and the two centrally located mutations even inactivate the enzyme completely. Analysis of kinetic data strongly suggests that, in fact, all the observed activity modifications result from alteration of the activation reaction rate constant, kappa cat only. This led to the conclusion that the domain involved in aspartic acid activation should be located in the second quarter of the molecule. Furthermore, a deletion mutant with a modification of the last five amino acid residues was isolated. This mutant is fully active in the activation step, but has lost 80% of the wild-type aminoacylation activity. This involvement of the C-terminus in acylation implies that it has to be folded towards strategic regions of the enzyme, thus favouring conformations required for catalysis or maintaining the tRNA in a functional position.
Amino Acid Sequence↗
Non-essential role of lysine residues for the catalytic activities of aspartyl-tRNA synthetase and comparison with other aminoacyl-tRNA synthetases.
Essential lysine residues were sought in the catalytic site of baker's yeast aspartyl-tRNA synthetase (an alpha 2 dimer of Mr 125,000) using affinity labeling methods and periodate-oxidized adenosine, ATP, and tRNA(Asp). It is shown that the number of periodate-oxidized derivatives which can be bound to the synthetase via Schiff's base formation with epsilon-NH2 groups of lysine residues exceeds the stoichiometry of specific substrate binding. Furthermore, it is found that the enzymatic activities are not completely abolished, even for high incorporation levels of the modified substrates. The tRNA(Asp) aminoacylation reaction is more sensitive to labeling than is the ATP-PPi exchange one; for enzyme preparations modified with oxidized adenosine or ATP this activity remains unaltered. These results demonstrate the absence of a specific lysine residue directly involved in the catalytic activities of yeast aspartyl-tRNA synthetase. Comparative labeling experiments with oxidized ATP were run with several other aminoacyl-tRNA synthetases. Residual ATP-PPi exchange and tRNA aminoacylation activities measured in each case on the modified synthetases reveal different behaviors of these enzymes when compared to that of aspartyl-tRNA synthetase. When tested under identical experimental conditions, pure isoleucyl-, methionyl-, threonyl- and valyl-tRNA synthetases from E. coli can be completely inactivated for their catalytic activities; for E. coli alanyl-tRNA synthetase only the tRNA charging activity is affected, whereas yeast valyl-tRNA synthetase is only partly inactivated. The structural significance of these experiments and the occurrence of essential lysine residues in aminoacyl-tRNA synthetases are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine↗
Requirements for T cell recognition and elimination of retrovirally-transformed cells.
An adoptive therapy model has been utilized to examine the requirements for T cells to promote eradication of a disseminated, retrovirus-induced, syngeneic leukemia. Complete tumor elimination required that the transferred T cells proliferate in the host and mediate an anti-tumor effect for more than 30 days. Non-cytolytic L3T4+ T helper (Th) cells were capable of eliminating disseminated tumor without the participation of Lyt-2+ cytotoxic T cells (Tc). Purified or cloned Lyt-2+ T cells were also effective in therapy, but required the concurrent administration of either L3T4+ Th or interleukin 2 (IL-2) for optimal efficacy. L3T4+ Th appear to function via secretion of lymphokines that activate macrophages to a cytotoxic state. Lyt-2+ Tc, in addition to direct cytotoxicity, may mediate tumor eradication in part by secretion of lymphokines that activate in vivo tumoricidal macrophages. These studies suggested that the reported efficacy of individual T cell subsets in therapy of particular tumors might not reflect resistance or susceptibility to a cytotoxic effector mechanism, but rather the efficiency with which a T cell subset is activated by the tumor and/or recognizes the tumor antigen. Methods were developed to independently assess the activation and proliferation requirements of each subset. L3T4+ Th required that macrophages degrade tumor antigens in lysosomes and present the antigens in the context of class II molecules, and produced IL-2 and IL-4 as endogenous growth factors. By contrast, Lyt-2+ T cells recognized the tumor directly, required macrophages only to produce IL-1 for activation, and produced IL-2 but not IL-4 as an endogenous growth factor. The ability of T cell subsets to recognize the distinct retroviral tumor antigens expressed on FBL leukemia was assessed using cell lines or recombinant vaccinia viruses transfected with selected retroviral genes. Highly selective antigen recognition was detected, with Lyt-2+ Tc cells recognizing products of gag but not envelope genes, and L3T4+ Th recognizing envelope but not gag products. The results suggest that even complex unique tumor antigens may elicit only limited host T cell responses.
Animals↗
Occupational and post-traumatic arthritis.
Secondary osteoarthritis (OA) may result from a number of causes including trauma or may be related to a specific occupation or activity. In patients who present with atypical OA one should consider the possibility of such an etiology. This article discusses post-traumatic osteoarthritis and specific forms of occupational OA.
Arthritis↗