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A tandem chain extension-homoenolate formation reaction is described. The treatment of beta-keto esters with the Furukawa reagent followed by exposure to a catalytic amount of trimethylsilyl chloride provides access to an ester homoenolate. Reaction: see text.
Multiple studies indicate that mRNA processing defects cause mRNAs to accumulate in discrete nuclear foci or dots, in mammalian cells as well as yeast. To investigate this phenomenon, we have studied a series of GAL reporter constructs integrated into the yeast genome adjacent to an array of TetR-GFP-bound TetO sites. mRNA within dots is predominantly post-transcriptional, and dots are adjacent to but distinct from their transcription site. These reporter genes also localize to the nuclear periphery upon gene induction, like their endogenous GAL counterparts. Surprisingly, this peripheral localization persists long after transcriptional shutoff, and there is a comparable persistence of the RNA in the dots. Moreover, dot disappearance and gene delocalization from the nuclear periphery occur with similar kinetics after transcriptional shutoff. Both kinetics depend in turn on reporter gene 3'-end formation signals. Our experiments indicate that gene association with the nuclear periphery does not require ongoing transcription and suggest that the mRNPs within dots may make a major contribution to the gene-nuclear periphery tether.
The order of relative reactivity of nitriles for the formation of Fe(II) complexes (2s) with 3-(1-alkyl(or aryl)methyl)-1-imino-2,4-bis(2-pyridylmethylimine)(L(2)s) from that with 2,4-bis(2-pyridylmethylimono)pentane (L1), trans-[FeL(1)(MeCN)(2)][ClO(4)](2).MeCN (1), and various nitriles has been determined based on the following order: C(6)F(5)CN > 3,4-difluorobenzonitrile > 4-fluorobenzonitrile > C(6)H(5)CN > C(6)H(5)CH(2)CN > C(2)H(5)CN > MeCN > Me(2)CHCN >>Me(3)CN. An iron(II) complex with L1 in a cis-configuration was prepared as the ternary complex [FeL(1)(bpy)][ClO(4)](2).1.5MeNO(2).0.5H(2)O, 3a (bpy = bipyridine). Compounds 2s and 3a undergo enantiomeric interconversion with an activation energy of ca. 60 kJ mol(-1).
Horse spleen and human spleen ferritins increase the formation of hydroxyl radicals (OH) at both pH 4.5 and pH 7.4 in reaction mixtures containing ascorbic acid and H2O2. The generation of OH is inhibited by the chelator desferrioxamine. Human spleen haemosiderin also accelerates OH generation in identical reaction mixtures, but is far less effective (on a unit iron basis) than ferritin under all reaction conditions. It is proposed that conversion of ferritin into haemosiderin in iron overload is biologically advantageous in that it decreases the ability of iron to promote oxygen-radical reactions.
In an attempt to define structural regions of the type I inositol 1, 4,5-trisphosphate [Ins(1,4,5)P3] receptor [Ins(1,4,5)P3R] involved in its intracellular targeting to the endoplasmic reticulum (ER), we have employed the use of green fluorescent protein (GFP) to monitor the localization of a truncated Ins(1,4,5)P3R mutant containing just the putative transmembrane spanning domain and the C-terminal cytoplasmic domain [amino acids 2216-2749; termed inositol trisphosphate receptor(ES)]. We expressed a chimeric GFP-Ins(1,4, 5)P3R(ES) fusion protein in Xenopus laevis oocytes, and used fluorescence confocal microscopy to monitor its intracellular localization. Fluorescence confocal microscopy data showed an intense fluorescence in the perinuclear region and in a reticular-network under the animal pole of the oocyte, consistent with the targeting of expressed GFP-Ins(1,4,5)P3R(ES) to perinuclear ER and ER under the animal pole. These findings are consistent with the intracellular localization of the endogenous Xenopus Ins(1,4, 5)P3R shown previously. Furthermore, electron microscopy data indicate that expressed GFP-Ins(1,4,5)P3R(ES) is in fact targeted to the ER. Sodium carbonate extraction of microsomal membranes and cross-linking experiments indicate that the expressed chimeric protein is in fact membrane anchored and able to form a homotetrameric complex. Our data provides evidence that Ins(1,4, 5)P3R(ES) constitutes the membrane spanning domain of the Ins(1,4, 5)P3R and is able to mediate homotetramer formation, without the need for the large N-terminal cytoplasmic domain. Furthermore, the localization of GFP-Ins(1,4,5)P3R(ES) on the ER indicates that an ER retention/targeting signal is contained within the transmembrane spanning domain of the inositol trisphosphate receptor.
Clathrin-coated pits are the major ports of entry into the cell and are responsible for the internalization of a variety of biologically important macromolecules. These transport intermediates form as a result of the co-ordinated assembly of a number of cytosolic proteins on to the membrane which results in specific cargo recruitment. We have used a variety of approaches including permeabilized cell assays and light and electron microscopy to identify and characterize the proteins and enzymes involved in coated vesicle formation.
PC12 cells undergo apoptosis as well as necrosis following exposure to hypoxia. Following a 6-h hypoxic treatment, a time-dependent increase in intracellular ceramide level was observed with a concurrent decrease in sphingomyelin. It was also shown that the hypoxia-induced ceramide accumulation resulted from activation of neutral magnesium-dependent sphingomyelinase. Comparative kinetic analyses of the neutral sphingomyelinase in the cells under normoxia and hypoxia showed that hypoxia increased Vmax but did not affect Km of the enzyme. In PC12 cells overexpressing Bcl-2 which show strong resistance to hypoxia, sphingomyelin hydrolysis was decreased and activation of neutral sphingomyelinase was reduced. Addition of exogenous C2-ceramide induced cell death and activated caspase-3 as markedly as the hypoxia treatment. On the other hand, in PC12 cells overexpressing Bcl-2, significant decreases in cell death and inhibition of caspase-3 activation were observed after exogenous addition of C2-ceramide. The inhibitors of caspase-3 prevented cell death by either hypoxia or C2-ceramide. These results suggest that ceramide generated by activation of neutral magnesium-dependent sphingomyelinase mediates hypoxic cell death and that Bcl-2 has inhibitory effects on ceramide formation and caspase activation.
Beta2-microglobulin (beta2-m), a typical immunoglobulin domain made of seven beta-strands, is a major component of amyloid fibrils formed in dialysis-related amyloidosis. To understand the mechanism of amyloid fibril formation in the context of full-length protein, we prepared various mutants in which proline (Pro) was introduced to each of the seven beta-strands of beta2-m. The mutations affected the amyloidogenic potential of beta2-m to various degrees. In particular, the L23P, H51P, and V82P mutations significantly retarded fibril extension at pH 2.5. Among these, only L23P is included in the known "minimal" peptide sequence, which can form amyloid fibrils when isolated as a short peptide. This indicates that the residues in regions other than the minimal sequence, such as H51P and V82P, determine the amyloidogenic potential in the full-length protein. To further clarify the mutational effects, we measured their stability against guanidine hydrochloride of the native state at pH 8.0 and the amyloid fibrils at pH 2.5. The amyloidogenicity of mutants showed a significant correlation with the stability of the amyloid fibrils, and little correlation was observed with that of the native state. It has been proposed that the stability of the native state and the unfolding rate to the amyloidogenic precursor as well as the conformational preference of the denatured state determine the amyloidogenicity of the proteins. The present results reveal that, in addition, stability of the amyloid fibrils is a key factor determining the amyloidogenic potential of the proteins.
The alpha-, beta-, and gamma-crystallins are the major structural proteins within the eye lens and are responsible for its exceptional stability and transparency. Under mildly denaturing conditions, all three types of bovine crystallin assemble into fibrillar structures in vitro. Characterization by transmission electron microscopy, dye binding assays, and x-ray fiber diffraction shows that these species have all of the characteristics of fibrils associated with the family of amyloid diseases. Moreover, the full-length proteins are incorporated into the fibrils, (i.e. no protein cleavage is required for these species to form), although for the gamma-crystallins some fragmentation occurs under the conditions employed in this study. Our findings indicate that the inherent stability of the beta-sheet supramolecular structure adopted by the crystallins in the eye lens and the chaperone ability of alpha-crystallin must be crucial for preventing fibril formation in vivo. The crystallins are very stable proteins but undergo extensive post-translational modification with age that leads to their destabilization. The ability of the crystallins to convert into fibrils under destabilizing conditions suggests that this process could contribute to the development of cataract with aging.
In the domestic chick, different versions of a particular learning experience are elaborated by right and left hemispheres (left: selection of cues allowing selection of appropriate response; right: elaboration of relatively complete and unselected record). During memory formation, further processing of the traces is associated with a series of brief points of trace reactivation, which recur with differing periodicity in association with the two hemispheres (16 min: left; 25 min: right). As a result there is a series of near coincidences, which allow interaction between the two traces. The first, at 48-50 min, is associated with such marked changes in what is available to recall that it has been identified as the onset of long-term memory. The period is shown here to begin with a left hemisphere event, which overlaps with a right hemisphere event, beginning at 50 min, in a way that helps to explain why the interaction that occurs at this time is predominantly one in which the left hemisphere accesses right hemisphere material. It is argued that the main change during hemispheric interaction is the establishment of linkages between different trace ''fragments''.
N-Hydroxy-2-acetylaminofluorene (N-OH-AAf) is metabolically converted into reactive N,O-esters which are capable of forming covalent adducts with DNA in rat liver in vivo. The effect of inhibiting one of the proposed pathways, N-O-sulfation, on DNA adduct formation was studied by using a specific sulfotransferase inhibitor, pentachlorophenol. Rats were pretreated with pentachlorophenol and, after 45 min, N-OH-AAF was administered. Four hours after dosing the animals were sacrificed and hepatic DNA was isolated. In DNA from control livers two acetylaminofluorene-and one aminofluorene-substituted deoxyguanosine adducts were found. The acetylaminofluorene derivatives, N-(deoxyguanosin-8-yl)-2--acetylaminofluorene and 3-(deoxyguanosin-N2-yl)-2acetylaminofluorene, accounted for 40% of the total binding in the hydrolyzed DNA. The aminofluorene adduct, N-(deoxyguanosin-8-yl)-2-aminofluorene, accounted for the remainder. In rats that were pretreated with pentacholorphenol, total DNA binding was decreased by 26%. The same three adducts were found, but the acetylaminofluorene adducts were now only 13% of the total, while the aminofluorene adduct accounted for 87%. The absolute amount of aminofluorene adduct was not altered as compared to control rats. These data demonstrate the involvement of N-O-sulfation in carcinogen-DNA binding and indicate that at least 70% of the acetylaminofluorene bound to deoxyguanosine in rat liver DNA, in vivo, is formed through N-O-sulfation of N-OH-AAF.
When recently arisen spontaneous petite mutants of Saccharomyces cerevisiae are crossed, respiratory competent diploids can be recovered. Such restored strains can be divided into two groups having sectored or unsectored colony morphology, the former being due to an elevated level of spontaneous petite mutation. On the basis of petite frequency, the sectored strains can be subdivided into those with a moderate frequency (5-16%) and those with a high frequency (greater than 60%) of petite formation. Each of the three categories of restored strains can be found on crossing two petites, suggesting either that the parental mutants contain a heterogeneous population of deleted mtDNAs at the time of mating or that different interactions can occur between the defective molecules. Restriction endonuclease analysis of mtDNA from restored strains that have a wild-type petite frequency showed that they had recovered a wild-type mtDNA fragmentation pattern. Conversely, all examined cultures from both categories of sectored strains contained aberrant mitochondrial genomes that were perpetuated without change over at least 200 generations. In addition, sectored colony siblings can have different aberrant mtDNAs. The finding that two sectored, restored strains from different crosses have identical but aberrant mtDNAs provides evidence for preferred deletion sites from the mitochondrial genome. Although it appears that mtDNAs from sectored strains invariably contain duplications, there is no apparent correlation between the size of the duplication and spontaneous petite frequency.
OBJECTIVE: The purpose of this study was to determine the association between bilomas and pseudoaneurysm complications after severe hepatic injury. METHODS: Angiography was performed in patients with American Association for the Surgery of Trauma grade > or = III hepatic injury on contrast-enhanced computed tomographic scanning. When contrast extravasation was observed, transarterial embolization (TAE) was performed. After TAE, technetium-99m pyridoxyl-5-methyl-tryptophan cholescintigraphy was performed to detect the coexistence of bilomas. Follow-up angiography was performed when a biloma was detected. Eighty consecutive patients underwent angiography; after angiography, five patients died. The remaining 75 patients who underwent cholescintigraphy were included in this study. RESULTS: All 11 patients who had bilomas had angiographic evidence of contrast extravasation. The biloma frequency was higher in patients with grades IV and V injuries than in those with grade III injury (p = 0.024). Follow-up angiography revealed pseudoaneurysms in 7 of these 11 patients. All six patients in whom only gelatin sponge pledget injection was used to embolize had pseudoaneurysms. Among them, two patients had computed tomographic evidence of massive intra-abdominal fluid collection. In contrast, only one of five patients who received the combination of gelatin sponge pledget injection and stainless steel coils to permanently embolize injured arteries had a pseudoaneurysm. In this patient, the pseudoaneurysm was found in the peripheral part of the collateral vessels. All patients with pseudoaneurysms underwent repeat TAE and were discharged from the hospital uneventfully. CONCLUSION: In patients with high-grade hepatic injury and arterial bleeding who developed biloma, use of a gelatin sponge, an absorbable embolic material, is associated with a risk of pseudoaneurysm formation. Permanent arterial embolization using stainless steel coils is indicated to decrease this risk.
Megamitochondria were induced within 36-40 hours in mouse hepatocytes by injecting cuprizone into the peritoneal cavity. Induction of megamitochondria was dependent upon the amount and the time intervals of the injection of cuprizone: 200 mg of cuprizone/kg of body weight-injected every 12 hours or 400 mg of cuprizone/kg of body weight-injected every 24 hours. When the latter amount of the noxious reagent was administered to the animal every 12 hours, fatty changes of the liver was observed. Involvement of the fusion phenomenon in the mechanism of megamitochondrial formation is discussed in the light of turnover rates for various components of the mitochondrion.
The role of zone 3 hepatocytes in bile formation was determined when they were selectively destroyed by 3.8 mmol/kg b.w. of bromobenzene injected i.p. for 48 h, as compared to appropriate controls. Bromobenzene treatment resulted in 29 +/- 4.4% hepatic lobule necrosis localized in the zone 3 hepatocytes. Although bile flow and bile salt-independent flow were not affected, this treatment was associated with a significant reduction in bile salt, and phospholipid secretion. The bile salt pool and bile salt synthesis were also significantly decreased. These results suggest that necrosis of zone 3 hepatocytes induced by bromobenzene reduced bile acid synthesis which decreased bile salt pool and affected bile salt and phospholipid secretion rates. However, necrosis of zone 3 hepatocytes did not affect bile flow or the bile salt-independent flow, suggesting that hepatocytes of zones 1 and 2 maintained the normal bile salt-independent flow when zone 3 hepatocytes were damaged.
A system for the formation of apparently wall-free protoplasts from exponential-phase cells of Streptococcus faecalis ATCC 9790 in the absence of added lytic enzymes was developed. Exponential-phase cells suspended in 0.04 M ammonium acetate, pH 6.7, 1 mM magnesium acetate, and 0.5 M sucrose become osmotically fragile within 1 to 1.5 h due to the action of the native, autolytic enzyme on the cell wall peptidoglycan. However, maximal cell wall loss occurred much more slowly, being complete only after 3 to 6 h. Under these conditions, the autolytically formed protoplasts (autoplasts) remained intact for prolonged periods (up to 24 h) with less than 5% of their deoxyribonucleic acid, ribonucleic acid, and protein lost during the first 6 h. During dissolution of the cell wall, release of autolytic enzyme to the supernatant fluid began after 60% of the wall was lost. The addition of trypsin to the incubation mixture increased the rate of attainment of osmotic fragility and cell wall loss two- to threefold, apparently due to the activation of the latent form of the autolysin. Electron microscopy was used to confirm cell wall loss and the presence of intact protoplasts at the end of the incubation periods.
The nitrogen assimilation control protein (NAC) from Klebsiella pneumoniae is a very versatile regulatory protein. NAC activates transcription of operons such as hut (histidine utilization) and ure (urea utilization), whose products generate ammonia. NAC also represses the transcription of genes such as gdhA, whose products use ammonia. NAC exerts a weak repression at gdhA by competing with the binding of a lysine-sensitive activator. NAC also strongly represses transcription of gdhA (about 20-fold) by binding to two separated sites, suggesting a model involving DNA looping. We have identified negative control mutants that are unable to exert this strong repression of gdhA expression but still activate hut and ure expression normally. Some of these negative control mutants (e.g., NAC(86ter) and NAC(132ter)) delete the C-terminal domain, thought to be required for tetramerization. Other negative control mutants (e.g., NAC(L111K) and NAC(L125R)) alter single amino acids involved in tetramerization. In this work we used gel filtration to show that NAC(86ter) and NAC(L111K) are dimers in solution, even at high concentration (NAC(WT) is a tetramer). Moreover, using a combination of DNase I footprints and gel mobility shifts assays, we showed that when NAC(WT) binds to two adjacent sites on a DNA fragment, NAC(WT) binds as a tetramer that bends the DNA fragment significantly. NAC(L111K) binds to such a fragment as two independent dimers without inducing the strong bend. Thus, NAC(L111K) is a dimer in solution or when bound to DNA. NAC(L111K) (typical of the negative control mutants) is wild type for every other property tested: (i) it activates transcription at hut and ure; (ii) it competes with the lysine-sensitive activator for binding at gdhA; (iii) it binds to the same sites at the hut, ure, nac, and gdhA promoters as NAC(WT); (iv) the relative affinity of NAC(L111K) for these sites follows the same order as NAC(WT) (ure > gdhA > nac > hut); (v) it induces the same slight bend as dimers of NAC(WT); and (vi) its DNase I footprints at these sites are indistinguishable from those of NAC(WT) (except for features ascribed to tetramer formation). The only two phenotypes we know for negative control mutants of NAC are their inability to tetramerize and their inability to cause the strong repression of gdhA. Thus, we propose that in order for NAC(WT) to exert the strong repression, it must form a tetramer that bridges the two sites at gdhA (similar to other DNA looping models) and that the negative control mutants of NAC, which fail to tetramerize, cannot form this loop and thus fail to exert the strong repression at gdhA.
Phenocopy maps are constructed from the spatial pattern of the phenocopy spots using a distance function defined on the frequencies with which two compartments transform separately and jointly. The temporal changes in distances between different compartments in turn enable us to make inferences concerning the diffusion-like processes involved in prepattern formation of the metathorax. A model is presented which accounts for the data in terms of the propagation of two wavefronts in sequence and in approximately orthogonal directions. Sensitivity to transformation by ether is limited in time to the interval between the passing of the two wavefronts. Changes caused by 13 successive generations of ether treatment can be interpreted in terms of alterations in the timing and rates of wave propagation. A comparison of phenocopy and fate maps suggests that the prepatterning processes affect primarily the blastoderm cytoplasm or surface membrane.