Comparison of the enzymatic behavior of high molecular weight and free lysyl-tRNA synthetase from rat liver: kinetic analysis of lysylation of tRNA

Lysyl-tRNA synthetase occurs in the high molecular weight form in rat liver. The high molecular weight lysyl-tRNA synthetase has been previously demonstrated to exist as multienzyme complexes of aminoacyl-tRNA synthetases. The multienzyme complexes can be dissociated by hydrophobic interaction chromatography and yield fully active, free lysyl-tRNA synthetase. The free form is found to be twice as active as the complexed form in lysylation. Bisubstrate and product inhibition kinetics of lysylation are systematically carried out for highly purified free lysyl-tRNA synthetase and the 18 S synthetase complex. Surprisingly, the two enzyme forms exhibit distinctly different kinetic patterns in bisubstrate and product inhibition kinetics under identical conditions. The 18 S synthetase complex shows kinetic patterns consistent with an ordered bi uni uni bi ping pong mechanism, while the results of free lysyl-tRNA synthetase do not. We conclude that structural organization of lysyl-tRNA synthetase beyond quaternary structure of proteins may alter the enzyme behavior.     

Synthesis of diadenosine 5',5' -P1,P4-tetraphosphate by lysyl-tRNA synthetase and a multienzyme complex of aminoacyl-tRNA synthetases from rat liver

An 18 S multienzyme complex of aminoacyl-tRNA synthetases is found to be active in the synthesis of diadenosine-5',5'-P1,P4-tetraphosphate (AppppA). Most of the activity is attributed to lysyl-tRNA synthetase in the complex. Free lysyl-tRNA synthetase dissociated from the synthetase complex is about 6-fold more active than the complex in AppppA synthesis, while their apparent Michaelis constants for ATP and lysine are similar. AMP, which reportedly activates AppppA synthesis (Hilderman, R.H. (1983) Biochemistry 22, 4353-4357), has no effect on AppppA synthesis. The higher activity of free Lys-tRNA synthetase is in part due to the higher stimulation of AppppA synthesis by Zn2+. These results suggest that association of aminoacyl-tRNA synthetases may affect AppppA synthesis.     

New branched Porolithon species (Corallinales,Rhodophyta) from the Great Barrier Reef,Coral Sea,and Lord Howe Island

Porolithon is one of the most ecologically important genera of tropical and subtropical crustose (non-geniculate) coralline algae growing abundantly along the shallow margins of coral reefs and functioning to cement reef frameworks. Thalli of branched, fruticose Porolithon specimens from the Indo-Pacific Ocean traditionally have been called P. gardineri, while massive, columnar forms have been called P. craspedium. Sequence comparisons of the rbcL gene both from type specimens of P. gardineri and P. craspedium and from field-collected specimens demonstrate that neither species is present in east Australia and instead resolve into four unique genetic lineages. Porolithon howensis sp. nov. forms columnar protuberances and loosely attached margins and occurs predominantly at Lord Howe Island; P. lobulatum sp. nov. has fruticose to clavate forms and free margins that are lobed and occurs in the Coral Sea and on the Great Barrier Reef (GBR); P. parvulum sp. nov. has short (<2 cm), unbranched protuberances and attached margins and is restricted to the central and southern GBR; and P. pinnaculum sp. nov. has a mountain-like, columnar morphology and occurs on oceanic Coral Sea reefs. A rbcL gene sequence of the isotype of P. castellum demonstrates it is a different species from other columnar species. In addition to the diagnostic rbcL and psbA marker sequences, the four new species may be distinguished by a combination of features including thallus growth form, margin shape (attached or unattached), and medullary system (coaxial or plumose). Porolithon species, because of their ecological importance and sensitivity to ocean acidification, need urgent documentation of their taxonomic diversity.     

Effects of tRNALeu1 overproduction in Escherichia coli

Strains of Escherichia coli have been produced which express very high levels of the tRNA^leu₁ isoacceptor. This was accomplished by transforming cells with plasmids containing the leuV operon which encodes three copies of the tRNA^Leu₁ gene. Most transformants grew very slowly and exhibited a 15-fold increase in cellular concentrations of tRNA^Leu₁ As a result, total cellular tRNA concentration was approximately doubled and 56% of the total was tRNA^Leu₁. We examined a number of parameters which might be expected to be affected by imbalances in tRNA concentration: in vivo tRNA charging levels, misreading, ribosome step time, and tRNA modification. Surprisingly, no increase in intracellular ppGpp levels was detected even though only about 40% of total leucyl tRNA was found to be charged in vivo. Gross ribosomal misreading was not detected, and it was shown that ribosomal step times were reduced between two- and threefold. Analyses of leucyl tRNA isolated from these slow-growing strains showed that at least 90% of the detectable tRNA^Leu₁ was hypomodified as judged by altered mobility on RPC-5 reverse-phase columns, and by specific modification assays using tRNA(m¹G)-methyltransferase and pseudo-uridylate synthetase. Analysis of fast-growing revertants demonstrated that tRNA concentration per se may not explain growth inhibition because selected revertants which grew at wild-type growth rates displayed levels of tRNA comparable to that of control strains bearing the leuV operon. A synthetic tRNA^Leu₁ operon under the control of the T7 promoter was prepared which, when induced, produced six- to sevenfold increases in tRNA^Leu₁ levels. This level of tRNA^Leu₁ titrated the modification system as judged by RPC-5 column chromatography. Overall, our results suggest that hypomodified tRNA may explain, in part, the observed effects on growth, and that the protein-synthesizing system can tolerate an enormous increase in the concentration of a single tRNA.     

Venous versus arterialised venous blood for assessment of blood glucose levels during glucose clamping: comparison in healthy men.

The aim of this study was to investigate the influence of the arteriovenous (A-V) gradient in blood glucose concentrations at low and high insulin levels on the determination of glucose requirements during glucose clamping in 9 healthy, insulin sensitive, male volunteers. In a random order two clamps were performed, once using arterialised venous blood (A Clamp, mean pO2 = 11.5 +/- 0.36 kPa, 86 +/- 2.7 mmHg), and once using venous blood (V clamp, mean pO2 = 7.9 +/- 0.21 kPa, 59 +/- 1.6 mmHg). Insulin levels were maintained at 48 +/- 2.4 mU/l from 0-180 min and at 1054 +/- 114 mU/l from 180-360 min. Elevation of insulin levels caused a significant rise of the A-V gradient: from 0.3 +/- 0.1 to 0.5 +/- 0.1 mmol/l (p < 0.05) and from 0.2 +/- 0.1 to 0.3 +/- 0.1 mmol/l (p < 0.05) during the A and V clamps, respectively. Despite these A-V glucose gradients no significant differences were found for the glucose requirements during the last 30 min of each period of insulin infusion between the A and V clamps: 43.70 +/- 3.4 vs 44.8 +/- 2.8 mumol.kg-1.min-1 during the low insulin level and 77.3 +/- 5.0 vs 76.2 +/- 3.4 mumol.kg-1.min-1 during the high insulin level. We conclude that the A-V glucose gradient, even at high insulin levels, does not influence the assessment of glucose requirements to a measurable extent, allowing the use of the simpler technique of taking venous rather than arterialised venous blood for the measurements of glucose levels during glucose clamping.     

Effects of salt stress on nitrogenase activity and growth of four legumes

The effects of salt stress on nitrogenase (N2-ase) activity, growth and nitrogen content ofVicia faba (L.),Medicago sativa (L.) Merrill,Glycine max andVigna sinensis (L.) were investigated. Four levels of salinity were applied and salt treatments were imposed on inoculated and N-fertilized plants.M. sativa tolerated mild levels of salinity but higher salt concentrations depressed N₂-ase activity of this species. The other three legumes were considerably affected by salt treatments, and N₂-ase activity was significantly reduced by salinity. Vicia faba, carrying elongate nodules, could restore a partial N₂-ase activity upon recovery from salt stress whereasG. max andV. sinesis, both with spherical nodules, could not regain significant activity when salinity was removed. Salt stress retarded growth of both inoculated and N-fertilized plants. The nitrogen content of both treatments was also affected by salinity and the effect was more severe for inoculated than N-fertilized plants.     

Studies on a strain of Fusarium solani (Mart.) Sacc. Isolated from a case of mycotic keratitis

A strain of Fusarium solani (Mart.) Sacc. (IMI-216517), isolated from a patient of mycotic keratitis, produced experimental keratomycosis in albino rabbit cornea and survived in internal tissues of albino mice for varying periods. Alantolactone, isolated from the plant — Inula racemosa Hook. f. exhibited marked in vitro fungistatic activity against this strain of F. solani at 100–200 g/ml concentrations. The strain was less sensitive to amphotericin B and showed more acid than alkaline proteinase and phosphatase activities.Communication No. 2526.     

A finite element model of stress-mediated vascular adaptation: application to abdominal aortic aneurysms

Despite rapid expansion of our knowledge of vascular adaptation, developing patient-specific models of diseased arteries is still an open problem. In this study, we extend existing finite element models of stress-mediated growth and remodelling of arteries to incorporate a medical image-based geometry of a healthy aorta and, then, simulate abdominal aortic aneurysm. Degradation of elastin initiates a local dilatation of the aorta while stress-mediated turnover of collagen and smooth muscle compensates the loss of elastin. Stress distributions and expansion rates during the aneurysm growth are studied for multiple spatial distribution functions of elastin degradation and kinetic parameters. Temporal variations of the degradation function are also investigated with either direct time-dependent degradation or stretch-induced degradation as possible biochemical and biomechanical mechanisms for elastin degradation. The results show that this computational model has the capability to capture the complexities of aneurysm progression due to variations of geometry, extent of damage and stress-mediated turnover as a step towards patient-specific modelling.     

Distributional Learning of Appearance

Opportunities for associationist learning of word meaning, where a word is heard or read contemperaneously with information being available on its meaning, are considered too infrequent to account for the rate of language acquisition in children. It has been suggested that additional learning could occur in a distributional mode, where information is gleaned from the distributional statistics (word co-occurrence etc.) of natural language. Such statistics are relevant to meaning because of the Distributional Principle that ‘words of similar meaning tend to occur in similar contexts’. Computational systems, such as Latent Semantic Analysis, have substantiated the viability of distributional learning of word meaning, by showing that semantic similarities between words can be accurately estimated from analysis of the distributional statistics of a natural language corpus. We consider whether appearance similarities can also be learnt in a distributional mode. As grounds for such a mode we advance the Appearance Hypothesis that ‘words with referents of similar appearance tend to occur in similar contexts’. We assess the viability of such learning by looking at the performance of a computer system that interpolates, on the basis of distributional and appearance similarity, from words that it has been explicitly taught the appearance of, in order to identify and name objects that it has not been taught about. Our experiment tests with a set of 660 simple concrete noun words. Appearance information on words is modelled using sets of images of examples of the word. Distributional similarity is computed from a standard natural language corpus. Our computation results support the viability of distributional learning of appearance.     

Immuno-Regulatory Function of Indoleamine 2,3 Dioxygenase through Modulation of Innate Immune Responses

Successful long-term treatment of type-1 diabetes mainly relies on replacement of β-cells via islet transplantation. Donor shortage is one of the main obstacles preventing transplantation from becoming the treatment of choice. Although animal organs could be an alternative source for transplantation, common immunosuppressive treatments demonstrate low efficacy in preventing xenorejection. Immunoprotective effects of indoleamine 2,3-dioxygenase (IDO) on T-cell mediated allorejection has been extensively studied. Our studies revealed that IDO expression by fibroblasts, induced apoptosis in T-cells while not affecting non-immune cell survival/function. Since macrophages play a pivotal role in xenograft rejection, herein we investigated the effect of IDO-induced tryptophan deficiency/kynurenine accumulation on macrophage function/survival. Moreover, we evaluated the local immunosuppressive effect of IDO on islet-xenograft protection. Our results indicated that IDO expression by bystander fibroblasts significantly reduced the viability of primary macrophages via apoptosis induction. Treatment of peritoneal macrophages by IDO-expressing fibroblast conditioned medium significantly reduced their proinflammatory activity through inhibition of iNOS expression. To determine whether IDO-induced tryptophan starvation or kynurenine accumulation is responsible for macrophage apoptosis and inhibition of their proinflammatory activity, Raw264.7 cell viability and proinflammatory responses were evaluated in tryptophan deficient medium or in the presence of kynurenine. Tryptophan deficiency, but not kynurenine accumulation, reduced Raw264.7 cell viability and suppressed their proinflammatory activity. Next a three-dimensional islet-xenograft was engineered by embedding rat islets within either control or IDO–expressing fibroblast-populated collagen matrix. Islets morphology and immune cell infiltration were then studied in the xenografts transplanted into the C57BL/6 mouse renal sub-capsular space. Local IDO significantly decreased the number of infiltrating macrophages (11±1.47 vs. 70.5±7.57 cells/HPF), T-cells (8.75±1.03 vs. 75.75±5.72 cells/HPF) and iNOS expression in IDO-expressing xenografts versus controls. Islet morphology remained intact in IDO-expressing grafts and islets were strongly stained for insulin/glucagon compared to control. These findings support the immunosuppressive role of IDO on macrophage-mediated xeno-rejection.