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1.
The aim of this study was to analyze the growth response of HeLa cells over a prolonged period of time to a single exposure of physiological and supraphysiological concentrations of 4-hydroxynonenal (HNE), a peroxidation product of omega-6-polyunsaturated fatty acids. Furthermore, the growth modulating effect of serum factors, particularly albumin, on the growth pattern was examined. The effects of HNE on the growth rate and viability of the cells, as well as on the incorporation of labelled amino acids were monitored daily over a period of four days. Fetal calf serum not only had a growth stimualting effect but also modulated the action of HNE. In neither respect was albumin able to substitute for serum indicating that the influence of serum was not exerted via an albumin–HNE conjugate. HNE had a clear dose-dependent effect and a distinction could be made between a supraphysiological concentration (100 μM), which was primarily cytotoxic and a physiological range (below 10 μM) which showed growth modulatory effects. These effects consisted of a transient inhibition in the initial phase of the cell growth, which under optimal conditions (in presence of serum) was followed by a period of increased proliferation, compared to untreated control cultures, until confluence was attained. It is suggested that HNE is not only a toxic product of lipid peroxidation, but a physiological growth regulating factor as well.  相似文献   
2.
Hepatic lipid metabolism is controlled by integrated metabolic pathways. Excess accumulation of hepatic TG is a hallmark of nonalcoholic fatty liver disease, which is associated with obesity and insulin resistance. Here, we show that KH-type splicing regulatory protein (KSRP) ablation reduces hepatic TG levels and diet-induced hepatosteatosis. Expression of period 2 (Per2) is increased during the dark period, and circadian oscillations of several core clock genes are altered with a delayed phase in Ksrp−/− livers. Diurnal expression of some lipid metabolism genes is also disturbed with reduced expression of genes involved in de novo lipogenesis. Using primary hepatocytes, we demonstrate that KSRP promotes decay of Per2 mRNA through an RNA-protein interaction and show that increased Per2 expression is responsible for the phase delay in cycling of several clock genes in the absence of KSRP. Similar to Ksrp−/− livers, both expression of lipogenic genes and intracellular TG levels are also reduced in Ksrp−/− hepatocytes due to increased Per2 expression. Using heterologous mRNA reporters, we show that the AU-rich element-containing 3′ untranslated region of Per2 is responsible for KSRP-dependent mRNA decay. These findings implicate that KSRP is an important regulator of circadian expression of lipid metabolism genes in the liver likely through controlling Per2 mRNA stability.  相似文献   
3.
Aminoacyl-tRNA synthetases catalyze ATP-dependent covalent coupling of cognate amino acids and tRNAs for ribosomal protein synthesis. Escherichia coli isoleucyl-tRNA synthetase (IleRS) exploits both the tRNA-dependent pre- and post-transfer editing pathways to minimize errors in translation. However, the molecular mechanisms by which tRNAIle organizes the synthetic site to enhance pre-transfer editing, an idiosyncratic feature of IleRS, remains elusive. Here we show that tRNAIle affects both the synthetic and editing reactions localized within the IleRS synthetic site. In a complex with cognate tRNA, IleRS exhibits a 10-fold faster aminoacyl-AMP hydrolysis and a 10-fold drop in amino acid affinity relative to the free enzyme. Remarkably, the specificity against non-cognate valine was not improved by the presence of tRNA in either of these processes. Instead, amino acid specificity is determined by the protein component per se, whereas the tRNA promotes catalytic performance of the synthetic site, bringing about less error-prone and kinetically optimized isoleucyl-tRNAIle synthesis under cellular conditions. Finally, the extent to which tRNAIle modulates activation and pre-transfer editing is independent of the intactness of its 3′-end. This finding decouples aminoacylation and pre-transfer editing within the IleRS synthetic site and further demonstrates that the A76 hydroxyl groups participate in post-transfer editing only. The data are consistent with a model whereby the 3′-end of the tRNA remains free to sample different positions within the IleRS·tRNA complex, whereas the fine-tuning of the synthetic site is attained via conformational rearrangement of the enzyme through the interactions with the remaining parts of the tRNA body.  相似文献   
4.
Nitrogen (N) and phosphorus (P), either individually or in combination, have been demonstrated to limit biomass production in terrestrial ecosystems. Field studies have been extensively synthesized to assess global patterns of N impacts on terrestrial ecosystem processes. However, to our knowledge, no synthesis has been done so far to reveal global patterns of P impacts on terrestrial ecosystems, especially under different nitrogen (N) levels. Here, we conducted a meta‐analysis of impacts of P addition, either alone or with N addition, on aboveground (AGB) and belowground biomass production (BGB), plant and soil P concentrations, and N : P ratio in terrestrial ecosystems. Overall, our meta‐analysis quantitatively confirmed existing notions: (i) colimitation of N and P on biomass production and (ii) more P limitation in tropical forest than other ecosystems. More importantly, our analysis revealed new findings: (i) P limitation on biomass production was aggravated by N enrichment and (ii) plant P concentration was a better indicator of P limitation than soil P availability. Specifically, P addition increased AGB and BGB by 34% and 13%, respectively. The effect size of P addition on biomass production was larger in tropical forest than grassland, wetland, and tundra and varied with P fertilizer forms, P addition rates, or experimental durations. The P‐induced increase in biomass production and plant P concentration was larger under elevated than ambient N. Our findings suggest that the global limitation of P on biomass production will become severer under increasing N fertilizer and deposition in the future.  相似文献   
5.
Studies were conducted to determine if in vivo exposure to dinitrotoluenes (DNT), which is associated with circulatory disorders of atherosclerotic etiology in humans, is associated with alterations of vascular smooth muscle cells (SMC) consistent with the atherogenic process. Sprague-Dawley rats (150-180 g) were injected IP for 5 days/week for 8 weeks with 2,4- or 2,6-DNT (0.5, 5, or 10 mg/kg) or medium chain triglyceride (MCT) oil. Histopathologic evaluation of aortae from animals exposed to either isomer showed dysplasia and rearrangement of SMC at all doses tested. Reduced 3H-thymidine incorporation was observed in primary cultures of aortic SMC from DNT-exposed animals relative to vehicle controls. This inhibitory response was maintained for up to two passages in culture after which a significant increase in thymidine incorporation was observed. Exposure of SMC from naive animals to DNT in vitro (1–100 µM) did not alter the extent of thymidine incorporation in cycling or growth-arrested cultures. In contrast, exposure to 2,4- or 2,6-diaminotoluene (DAT) (1–100 µM), carcinogens which share toxic metabolic intermediates in common with DNT, inhibited replicative DNA synthesis and stimulated unscheduled DNA synthesis in cycling and growth-arrested cultures of SMC, respectively. Our results suggest that modulation of DNA synthesis in aortic SMC by DNT metabolites generated in vivo contribute to the development of vascular lesions.Abbreviation DAT diaminotuluene - tDNT technical grade dinitrotoluene - DNT dinitrotoluenes - HU hydroxyurea - IP intraperitoneal - LDH lactate dehydrogenase - MCT oil medium chain triglyceride - NPTC non-protein thiol content - RDS replicative DNA synthesis - SEM standard error of the mean - SMC smooth muscle cells - UDS unscheduled DNA synthesis  相似文献   
6.
A series of novel 11-O-aralkylcarbamoyl-3-O-descladinosylclarithromycin derivatives were designed, synthesized and evaluated for their in vitro antibacterial activity. The results showed that the majority of the target compounds displayed potent activity against erythromycin-susceptible S. pyogenes, erythromycin-resistant S. pneumoniae A22072 expressing the mef gene and S. pneumoniae AB11 expressing the mef and erm genes. Besides, most of the target compounds exhibited moderate activity against erythromycin-susceptible S. aureus ATCC25923 and B. subtilis ATCC9372. In particular, compounds 11a, 11b, 11c, 11e, 11f and 11h were found to exert favorable antibacterial activity against erythromycin-susceptible S. pyogenes with the MIC values of 0.015–0.125?μg/mL. Furthermore, compounds 10e, 11a, 11b and 11c showed superior activity against erythromycin-resistant S. pneumoniae A22072 with the MIC values of 0.25–0.5?μg/mL. Additionally, compound 11c was the most effective against all the erythromycin-resistant S. pneumoniae strains (A22072, B1 and AB11), exhibiting 8-, 8- and 32-fold more potent activity than clarithromycin, respectively.  相似文献   
7.
Chlorophyll is synthesized from activated glutamate in the tetrapyrrole biosynthesis pathway through at least 20 different enzymatic reactions. Among these, the MgProto monomethylester (MgProtoME) cyclase catalyzes the formation of a fifth isocyclic ring to tetrapyrroles to form protochlorophyllide. The enzyme consists of two proteins. The CHL27 protein is proposed to be the catalytic component, while LCAA/YCF54 likely acts as a scaffolding factor. In comparison to other reactions of chlorophyll biosynthesis, this enzymatic step lacks clear elucidation and it is hardly understood, how electrons are delivered for the NADPH‐dependent cyclization reaction. The present study intends to elucidate more precisely the role of LCAA/YCF54. Transgenic Arabidopsis lines with inactivated and overexpressed YCF54 reveal the mutual stability of YCF54 and CHL27. Among the YCF54‐interacting proteins, the plastidal ferredoxin‐NADPH reductase (FNR) was identified. We showed in N. tabacum and A. thaliana that a deficit of FNR1 or YCF54 caused MgProtoME accumulation, the substrate of the cyclase, and destabilization of the cyclase subunits. It is proposed that FNR serves as a potential donor for electrons required in the cyclase reaction and connects chlorophyll synthesis with photosynthetic activity.  相似文献   
8.
The amide functional group is ubiquitous in nature and one of the most important motifs in pharmaceuticals, agrochemicals, and other valuable products. While coupling amides and carboxylic acids is a trivial synthetic transformation, it often requires protective group manipulation, along with stoichiometric quantities of expensive and deleterious coupling reagents. Nature has evolved a range of enzymes to construct amide bonds, the vast majority of which utilize adenosine triphosphate to activate the carboxylic acid substrate for amine coupling. Despite the fact that these enzymes operate under mild conditions, as well as possessing chemoselectivity and regioselectivity that obviates the need for protecting groups, their synthetic potential has been largely unexplored. In this review, we discuss recent research into the discovery, characterization, and development of amide bond forming enzymes, with an emphasis on stand-alone ligase enzymes that can generate amides directly from simple carboxylic acid and amine substrates.  相似文献   
9.
Human DNA polymerases (pols) η and ι are Y-family DNA polymerase paralogs that facilitate translesion synthesis past damaged DNA. Both polη and polι can be monoubiquitinated in vivo. Polη has been shown to be ubiquitinated at one primary site. When this site is unavailable, three nearby lysines may become ubiquitinated. In contrast, mass spectrometry analysis of monoubiquitinated polι revealed that it is ubiquitinated at over 27 unique sites. Many of these sites are localized in different functional domains of the protein, including the catalytic polymerase domain, the proliferating cell nuclear antigen-interacting region, the Rev1-interacting region, and its ubiquitin binding motifs UBM1 and UBM2. Polι monoubiquitination remains unchanged after cells are exposed to DNA-damaging agents such as UV light (generating UV photoproducts), ethyl methanesulfonate (generating alkylation damage), mitomycin C (generating interstrand cross-links), or potassium bromate (generating direct oxidative DNA damage). However, when exposed to naphthoquinones, such as menadione and plumbagin, which cause indirect oxidative damage through mitochondrial dysfunction, polι becomes transiently polyubiquitinated via Lys11- and Lys48-linked chains of ubiquitin and subsequently targeted for degradation. Polyubiquitination does not occur as a direct result of the perturbation of the redox cycle as no polyubiquitination was observed after treatment with rotenone or antimycin A, which both inhibit mitochondrial electron transport. Interestingly, polyubiquitination was observed after the inhibition of the lysine acetyltransferase KATB3/p300. We hypothesize that the formation of polyubiquitination chains attached to polι occurs via the interplay between lysine acetylation and ubiquitination of ubiquitin itself at Lys11 and Lys48 rather than oxidative damage per se.  相似文献   
10.
    
Filamentous fungi are capable of secreting relatively large amounts of heterologous recombinant proteins. Recombinant human glycoproteins expressed in this system, however, carry only carbohydrates of the oligomannose type limiting their potential use in humans. One approach to the problem is genetic engineering of the fungal host to permit production of complex and hybrid N-glycans. UDP-GlcNAc:3-d-mannoside -1,2-N-acetylglucosaminyltransferase I (GnT I) is essential for the conversion of oligomannose to hybrid and complex N-glycans in higher eukaryotic cells. Since GnT I is not produced by fungi, we have introduced into the genome ofAspergillus nidulans the gene encoding full-length rabbit GnT I and demonstrated the expression of GnT I enzyme activity at levels appreciably higher than occurs in most mammalian tissues. All the GnT I activity in theAspergillus transformants remains intracellular suggesting that the rabbit trans-membrane sequence may be capable of targeting GnT I to the fungal Golgi apparatus.Abbreviations CM complete medium - Gal-T UDP-Gal:GlcNAc -1,4-galactosyltransferase (EC 2.4.1.38/90) - GnT I UDP-GlcNAc:3-d-mannoside -1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101) - HPLC high performance liquid chromatography - M3-octyl Man1-6[Man1-3]Man-octyl - PAGE polyacrylamide gel electrophoresis - MES 2-(N-morpholino)ethane sulfonate - PCR polymerase chain reaction - PEG polyethylene glycol - PMSF phenyl methyl sulfonyl fluoride - SDS sodium dodecyl sulfate - SSC (1×) 0.15m NaCl/0.015m sodium citrate (pH 7.0) - STC 1.2m sorbitol, 100mm Tris-HCl, pH 7.4, and 10mm CaCl2 - STET 0.1m NaCl, 10mm Tris-HCl, pH 8.0, 1mm EDTA, pH 8.0, 5% Triton-X-100 Deceased. This paper is dedicated to the memory of Lorne S. Reid.  相似文献   
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