Breakdown Intermediates Research Articles

Visible-light induced effective and sustainable remediation of nitro organics pollutants using Pd-doped ZnO nanocatalyst

Nitroaromatic compounds represent a class of highly toxic pollutants discharged into aquatic environments by various industrial activities, posing significant threats to ecological integrity and human health due to their persistent and hazardous nature. In this study, Pd-doped ZnO nanoparticles were investigated as a potential solution for the degradation of nitro organics, offering heightened photocatalytic efficacy and prolonged stability. The synthesis of Pd-doped ZnO NPs was achieved via the hydrothermal method, with subsequent analysis through XRD spectra and XPS confirming successful Pd doping within the ZnO matrix. Characterization through FESEM and HRTEM unveiled the heterogeneous morphologies of both undoped and Pd-doped ZnO nanoparticles. Additionally, UV–vis and PL spectroscopy provided insights into the optical properties, chemical bonding, and defect structures of the synthesized Pd-doped ZnO NPs. Pd doping induces a redshift in ZnO’s absorption spectra, reducing the bandgap from 3.12 to 2.94 eV as Pd concentration rises from 0 to 0.2 wt.%. The photocatalytic degradation, following pseudo-first-order kinetics, achieved 90% nitrobenzene abatement (200 µg/L, pH 7) under visible light within 320 min with a catalyst loading of 16 µg/mL. The photocatalytic efficacy of 0.08 wt% Pd-doped ZnO (k = 0.058 min⁻1) exhibited a 25-fold enhancement compared to bare ZnO (k = 3.1 × 10–4 min-1). Subsequent quenching and ESR experiments identified hydroxyl radicals (OH•) as the predominant active species in the degradation mechanism. Mass spectrometry analysis unveiled potential breakdown intermediates, illuminating a plausible degradation pathway. The investigated Pd-doped ZnO nanoparticles demonstrated reusability for up to five successive treatment cycles, offering a sustainable solution to nitro organics contamination challenges.

Host-microbe interactions rewire metabolism in a C. elegans model of leucine breakdown deficiency.

In humans, defects in leucine catabolism cause a variety of inborn errors in metabolism. Here, we use Caenorhabditis elegans to investigate the impact of mutations in mccc-1, an enzyme that functions in leucine breakdown. Through untargeted metabolomic and transcriptomic analyses we find extensive metabolic rewiring that helps to detoxify leucine breakdown intermediates via conversion into previously undescribed metabolites and to synthesize mevalonate, an essential metabolite. We also find that the leucine breakdown product 3,3-hydroxymethylbutyrate (HMB), commonly used as a human muscle-building supplement, is toxic to C. elegans and that bacteria modulate this toxicity. Unbiased genetic screens revealed interactions between the host and microbe, where components of bacterial pyrimidine biosynthesis mitigate HMB toxicity. Finally, upregulated ketone body metabolism genes in mccc-1 mutants provide an alternative route for biosynthesis of the mevalonate precursor 3-hydroxy-3-methylglutaryl-CoA. Our work demonstrates that a complex host-bacteria interplay rewires metabolism to allow host survival when leucine catabolism is perturbed.

Dietary fibre complexity and its influence on functional groups of the human gut microbiota

The aim of this review is to provide an overview of the complex interactions between dietary fibre and the resident microbial community in the human gut. The microbiota influences both health maintenance and disease development. In the large intestine, the microbiota plays a crucial role in the degradation of dietary carbohydrates that remain undigested in the upper gut (non-digestible carbohydrates or fibre). Dietary fibre contains a variety of different types of carbohydrates, and its breakdown is facilitated by many different microbial enzymes. Some microbes, termed generalists, are able to degrade a range of different carbohydrates, whereas others are more specialised. Furthermore, the physicochemical characteristics of dietary fibre, such as whether it enters the gut in soluble or insoluble form, also likely influence which microbes can degrade it. A complex nutritional network therefore exists comprising primary degraders able to attack complex fibre and cross feeders that benefit from fibre breakdown intermediates or fermentation products. This leads predominately to the generation of the short-chain fatty acids (SCFA) acetate, propionate and butyrate, which exert various effects on host physiology, including the supply of energy, influencing glucose and lipid metabolism and anti-carcinogenic and anti-inflammatory actions. In order to effectively modulate the gut microbiota through diet, there is a need to better understand the complex competitive and cooperative interactions between gut microbes in dietary fibre breakdown, as well as how gut environmental factors and the physicochemical state of fibre originating from different types of diets influence microbial metabolism and ecology in the gut.

Phytase dosing affects phytate degradation and Muc2 transporter gene expression in broiler starters

This study was conducted to determine effects of high phytase use on growth performance, amino acid (AA) digestibility, intestinal phytate breakdown, and nutrient transporter expression in starter broiler chickens. Male Ross 308 chicks were allocated to 24 pens, at 15 birds/pen and assigned to one of 4 dietary treatments. Treatments were: a control diet (PCa+) that contained adequate levels of calcium (Ca) and phosphorus (P) for growing broiler chicks; a reduced Ca and P diet (PCa–:–1.5 g P/kg and –1.6 g Ca/kg), and 2 additional diets in which phytase was supplemented in the PCa– diet at 1,500 (PCa-Phy1500) and 3,000 (PCa-Phy3000) FTU/kg feed. A common starter diet was fed from day 1 to 8. From day 8 to 22, birds were fed the 4 experimental diets. On day 22, birds were killed for sample collection. From day 8 to 15, average daily gain and average daily feed intake were not different across treatments (P < 0.05) but gain-to-feed ratio (G:F) was reduced (P < 0.006) in the PCa– treatment compared with other treatments. There were no further performance differences, but a tendency of phytase treatments improving the overall G:F (P = 0.079; day 8–22). Up to both the duodenum–jejunum and ileum, phytate, P, and Ca disappearance were increased (P < 0.05) in the PCa-Phy1500 and PCa-Phy3000 treatments compared with PCa– treatment. Phytase dose dependently increased myoinositol (MI) concentration in the digesta from both the duodenum–jejunum and ileum (P < 0.001). The highest concentration of MI was found in the PCa-Phy3000 treatment. Plasma MI concentration was increased by phytase supplementation (P < 0.001). Prececal disappearance of Cys was lower (P < 0.05) in the PCa– treatment than in PCa1and PCa-Phy3000 treatment. Expression of MUC2 in the duodenum–jejunum was higher (P < 0.05) in the PCa-Phy3000 treatment than in other treatments. Phytase-induced hydrolysis of phytate led to elevated digesta and plasma MI concentrations and reduced digesta concentrations of phytate breakdown intermediates

Biodegradation of polycyclic aromatic hydrocarbons by native Ganoderma sp. strains: identification of metabolites and proposed degradation pathways.

Since polycyclic aromatic hydrocarbons (PAHs) are mutagenic, teratogenic, and carcinogenic, they are of considerable environmental concern. A biotechnological approach to remove such compounds from polluted ecosystems could be based on the use of white-rot fungi (WRF). The potential of well-adapted indigenous Ganoderma strains to degrade PAHs remains underexplored. Seven native Ganoderma sp. strains with capacity to produce high levels of laccase enzymes and to degrade synthetic dyes were investigated for their degradation potential of PAHs. The crude enzymatic extracts produced by Ganoderma strains differentially degraded the PAHs assayed (naphthalene 34-73%, phenanthrene 9-67%, fluorene 11-64%). Ganoderma sp. UH-M was the most promising strain for the degradation of PAHs without the addition of redox mediators. The PAH oxidation performed by the extracellular enzymes produced more polar and soluble metabolites such as benzoic acid, catechol, phthalic and protocatechuic acids, allowing us to propose degradation pathways of these PAHs. This is the first study in which breakdown intermediates and degradation pathways of PAHs by a native strain of Ganoderma genus were determined. The treatment of PAHs with the biomass of this fungal strain enhanced the degradation of the three PAHs. The laccase enzymes played an important role in the degradation of these compounds; however, the role of peroxidases cannot be excluded. Ganoderma sp. UH-M is a promising candidate for the bioremediation of ecosystems polluted with PAHs.

Early Diagnosed and Treated Glutaric Acidemia Type 1 Female Presenting with Subependymal Nodules in Adulthood.

Glutaric acidemia type 1 (GA-1, OMIM no. 231670) is an autosomal recessive disorder caused by the deficiency of glutaryl-CoA dehydrogenase (GCDH). The subsequent accumulation of the amino acids lysine, hydroxylysine, and tryptophan and their breakdown intermediates can be neurotoxic and particularly cause injury to the basal ganglia.Roughly 1 of 100,000 infants is affected with GA-1, and a common feature at birth is macrocephaly. Stress, such as in febrile illnesses, can precipitate encephalopathic crises in children generally less than 2years with variable recovery. Many infants develop dystonia with complex movement disorders and subtle cognitive and fine motor deficits. Common neuroradiologic findings include hypoplasia of temporal and frontal lobes, striatal lesions, white matter changes, and subdural effusions.There are three previous reports of subependymal nodules found on neuroimaging in GA-1 patients who were diagnosed as adults and untreated for GA-1. We present a unique case of an adult female who was diagnosed at age 2months and managed prior to any metabolic decompensation. Her initial diagnosis was made based on biochemical and enzymatic analysis, and then later confirmed with genetic sequencing. She started experiencing frequent headaches at age 12years. Neuroimaging in adulthood revealed common features seen in GA-1 in addition to the finding of subependymal nodules.This case may provide some insight into the natural progression of the disease despite early treatment. Though subependymal nodules are typically seen in tuberous sclerosis, the significance of these lesions in GA-1 is not well understood. Disease courses of more early diagnosed and treated patients with GA-1 need to be documented.

Unravelling glutathione conjugate catabolism in Saccharomyces cerevisiae: the role of glutathione/dipeptide transporters and vacuolar function in the release of volatile sulfur compounds 3-mercaptohexan-1-ol and 4-mercapto-4-methylpentan-2-one.

Sulfur-containing aroma compounds are key contributors to the flavour of a diverse range of foods and beverages, such as wine. The tropical fruit characters of Sauvignon Blanc wines are attributed to the presence of the aromatic thiols 3-mercaptohexan-1-ol (3-MH), its acetate ester 3-mercaptohexyl acetate (3-MHA), and 4-mercapto-4-methylpentan-2-one (4-MMP). These aromatic thiols are not detectable in grape juice to any significant extent but are released by yeast during alcoholic fermentation. While the processes involved in the release of 3-MH and 4-MMP from their cysteinylated precursors have been studied extensively, degradation pathways for glutathione S-conjugates (GSH-3-MH and GSH-4-MMP) have not. In this study, a candidate gene approach was taken, focusing on genes known to play a role in glutathione and glutathione-S-conjugate turnover in Saccharomyces cerevisiae. Our results confirm the role of Opt1p as the major transporter responsible for uptake of GSH-3-MH and GSH-4-MMP, and identify vacuolar Ecm38p as a key determinant of 3-MH release from GSH-3-MH. ECM38 was unimportant, on the other hand, for release of 4-MMP, and abolition of vacuolar biogenesis caused an increase in the amount of 4-MMP released. The alternative cytosolic glutathione degradation pathway was not involved in release of either thiol from their glutathionylated precursors. Finally, cycling of GSH-3-MH and/or its breakdown intermediates between the cytosol and the vacuole or extracellular space was implicated in modulation of 3-MH formation. Together, these results provide new targets for development of yeast strains that optimize release of these potent volatile sulfur compounds, and further our understanding of the processes involved in glutathione-S-conjugate turnover.

Self-organization of core Golgi material is independent of COPII-mediated endoplasmic reticulum export.

The Golgi is a highly organized and dynamic organelle that receives and distributes material from and to the endoplasmic reticulum (ER) and the endocytic pathway. One open question about Golgi organization is whether it is solely based on ER-to-Golgi transport. Here, we analyzed the kinetics of Golgi breakdown in the absence of COPII-dependent ER export with high temporal and spatial resolution using quantitative fluorescence microscopy. We found that Golgi breakdown occurred in two phases. While Golgi enzymes continuously redistributed to the ER, we consistently observed extensive Golgi fragmentation at the beginning of the breakdown, followed by microtubule-dependent formation of a Golgi remnant structure (phase 1). Further Golgi disintegration occurred less uniformly (phase 2). Remarkably, cisternal Golgi morphology was lost early in phase 1 and Golgi fragments instead corresponded to variably sized vesicle clusters. These breakdown intermediates were devoid of COPI-dependent recycling material, but contained typical 'core' Golgi components. Furthermore, Golgi breakdown intermediates were able to disassemble and reassemble following cell division, indicating that they retained important regulatory capabilities. Taken together, these findings support the view that Golgi self-organization exists independently of ER-to-Golgi transport.

Evaluation of CV2025 ω-transaminase for the bioconversion of lignin breakdown products into value-added chemicals: synthesis of vanillylamine from vanillin

Lignin is an essential component of the cell wall of various plants and represents an abundant and renewable natural resource. Both thermo-chemical and biological pre-treatment can be applied to break down the phenylpropanoid polymer subunits present in lignin. These liberate a range of phenolic compounds which represent potential substrates for bioconversion by ω-transaminases. In this work, the CV2025 ω-transaminase (ω-TAm) from Chromobacterium violaceum DSM30191, heterologously expressed in E. coli, was explored for selective amination of lignin breakdown intermediates into value-added products. Eight potential ω-TAm substrates were initially screened using (S)-α-methylbenzylamine (MBA) as the amino donor. Vanillin was identified as the best potential substrate which is converted into vanillylamine; an intermediate in the preparation of pelargonic acid vanillylamide used as a hyperemia inducing active substance in wound dressings. At low vanillin and MBA concentrations (< 10 mM) and with an excess of the amine donor (1:4 mol/mol) 100% w/w conversion of vanillin into vanillylamine was observed within 25 min. At vanillin concentrations above 10 mM, substrate inhibition was observed decreasing the rate and yield of the bioconversion. High concentrations of the reaction product (vanillylamine) and by-product (acetophenone) also limited the conversion due to increased backward reaction rate and inhibition. Vanillylamine synthesis could be carried out by both whole cell and clarified lysate forms of the CV2025 ω-TAm while fed-batch bioconversions (feeding low concentrations of both vanillin and MBA) could help overcome substrate inhibition and double the final product concentrations obtained. These results demonstrate the potential for bioconversion of lignin breakdown products into value-added chemicals but illustrate the need for enzymes with improved substrate range and implementation of techniques to overcome product inhibition and equilibrium constraints.

Arabidopsis STAY-GREEN2 Is a Negative Regulator of Chlorophyll Degradation during Leaf Senescence

Chlorophyll (Chl) degradation causes leaf yellowing during senescence or under stress conditions. For Chl breakdown, STAY-GREEN1 (SGR1) interacts with Chl catabolic enzymes (CCEs) and light-harvesting complex II (LHCII) at the thylakoid membrane, possibly to allow metabolic channeling of potentially phototoxic Chl breakdown intermediates. Among these Chl catabolic components, SGR1 acts as a key regulator of leaf yellowing. In addition to SGR1 (At4g22920), the Arabidopsis thaliana genome contains an additional homolog, SGR2 (At4g11910), whose biological function remains elusive. Under senescence-inducing conditions, SGR2 expression is highly up-regulated, similarly to SGR1 expression. Here we show that SGR2 function counteracts SGR1 activity in leaf Chl degradation; SGR2-overexpressing plants stayed green and the sgr2-1 knockout mutant exhibited early leaf yellowing under age-, dark-, and stress-induced senescence conditions. Like SGR1, SGR2 interacted with LHCII but, in contrast to SGR1, SGR2 interactions with CCEs were very limited. Furthermore, SGR1 and SGR2 formed homo- or heterodimers, strongly suggesting a role for SGR2 in negatively regulating Chl degradation by possibly interfering with the proposed CCE-recruiting function of SGR1. Our data indicate an antagonistic evolution of the functions of SGR1 and SGR2 in Arabidopsis to balance Chl catabolism in chloroplasts with the dismantling and remobilizing of other cellular components in senescing leaf cells.

Stay-Green Not Always Stays Green

Senescence is a highly regulated recycling process in which nutrients are translocated from deteriorating leaves to newly developing leaves, flowers, seeds, or roots (Lim et al., 2007; Gregersen et al., 2013). Chloroplast dismantling, a major event during senescence, involves the degradation of light-harvesting chlorophyll a/b-binding proteins (LHCPs) and the release of free chlorophyll (Chl) which is catabolized to phototoxic, cell-damaging Chl breakdown intermediates.

Genesis of antibiotic resistance II: monitoring of antibiotics and their metabolic breakdown products in the Rio Grande River water by United States Geological Survey (986.2)

An analysis of the data on the detection and concentration of antibiotics, its metabolic breakdown intermediates in Site number: 08450900, Site Name: Rio Grande bl Amistad Dam nr Del Rio, TX of USGS: &lt;http://waterdata.usgs.gov/nwis&gt;: has been undertaken. We have obtained the Water‐Data Report TX‐2005 08450900 USGS Rio Grande below Amistad Dam near Del Rio, TX; Rio Grande River Basin; LOCATION.‐‐Lat 29°25′30″, long 101°02′27″ referenced to North American Datum of 1927, Val Verde County, Hydrologic Unit 13080001, and 2.2 mi downstream from Amistad Dam. Data analysis was performed on WATER‐QUALITY RECORDS CHEMICAL DATA: July 1968 to Aug. 01, 2005 (discontinued); BIOCHEMICAL DATA: Oct. 1995 to Aug. 01, 2005 (discontinued); PESTICIDE DATA: Oct. 1995 to Aug. 01, 2005 (discontinued); SEDIMENT DATA: Oct. 1995 to Aug. 01, 2005 (discontinued). The available data indicates that no antibiotics or its metabolic breakdown products are monitored in the Rio Grande Water. However, the following indirect metabolic derivatives were detected: Disulfoton; Simazine; Terbacil; Terbu‐fos. At present efforts are underway to obtain the water data from 2005 to present from THE USGS for the detection of antibiotics and its metabolic derivatives. The detailed database search on sifter phrases and data analysis are to be presented in Experimental Biology 2014.Grant Funding Source: Professional Development Funds from SWTJC‐Eagle Pass 78852

Abstract B156: Regulation of tumor metabolism by the investigational proteasome inhibitor ixazomib in KRAS wild-type and KRAS mutant xenograft tumors.

Abstract The investigational proteasome inhibitor ixazomib (MLN2238) inhibits cell growth in a broad panel of solid tumor and hematological cell lines when tested in vitro. In contrast, antitumor activity in xenograft-bearing mice is model-dependent, with some solid tumor models showing no response to ixazomib treatment. A survey of 15 NSCLC and 6 colon xenograft models showed a striking relationship between the degree of antitumor activity of ixazomib and KRAS genotype. Tumors with wild-type (WT) KRAS showed higher sensitivity to ixazomib compared to the tumors harboring a KRAS activating mutation. These xenografts included cell-line-derived subcutaneous xenografts as well as patient derived primary tumors. To confirm that KRAS mutation impacts response to ixazomib, we used SW48 isogenic colon cancer cell lines, in which a KRAS-G13D mutation was introduced into KRAS-WT SW48 cells to generate stable SW48-KRAS-G13D cells. Although in vitro sensitivity to ixazomib was similar between the paired cell lines, in vivo antitumor activity was observed in only SW48 KRAS WT tumors, but not SW48-KRAS-G13D tumors. These data confirm the association of KRAS status and response to ixazomib. Since KRAS activating mutations are known to be associated with metabolic reprogramming, we performed metabolite profiling using in vivo SW48 isogenic tumor pairs treated with or without ixazomib. As expected, prior to treatment there were significant baseline differences between SW48 WT and SW48-KRAS-G13D tumor metabolite profiles in categories such as glutathione levels and glycogen breakdown intermediates, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. After a single dose of ixazomib, significant metabolic regulation was noted in categories including free amino acid pools, and polyamine levels, and some of these changes were more pronounced in the SW48 KRAS WT tumors as compared to the KRAS-G13D tumors. Our data also showed activation of GCN2 mediated signaling following ixazomib treatment, possibly as a result of amino acid deprivation in the tumors. The results suggest additional non-clinical avenues to explore mechanisms of sensitivity and resistance to proteasome inhibitors in solid tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B156. Citation Format: Nibedita Chattopadhyay, Allison Berger, James Garnsey, Hugues Bernard, Erik Koenig, Eric Lightcap, Ben Amidon. Regulation of tumor metabolism by the investigational proteasome inhibitor ixazomib in KRAS wild-type and KRAS mutant xenograft tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B156.

7-Hydroxymethyl chlorophyll a reductase functions in metabolic channeling of chlorophyll breakdown intermediates during leaf senescence

During natural or dark-induced senescence, chlorophyll degradation causes leaf yellowing. Recent evidence indicates that chlorophyll catabolic enzymes (CCEs) interact with the photosynthetic apparatus; for example, five CCEs (NYC1, NOL, PPH, PAO and RCCR) interact with LHCII. STAY-GREEN (SGR) and CCEs interact with one another in senescing chloroplasts; this interaction may allow metabolic channeling of potentially phototoxic chlorophyll breakdown intermediates. 7-Hydroxymethyl chlorophyll a reductase (HCAR) also acts as a CCE, but HCAR functions during leaf senescence remain unclear. Here we show that in Arabidopsis, HCAR-overexpressing plants exhibited accelerated leaf yellowing and, conversely, hcar mutants stayed green during dark-induced senescence. Moreover, HCAR interacted with LHCII in in vivo pull-down assays, and with SGR, NYC1, NOL and RCCR in yeast two-hybrid assays, indicating that HCAR is a component of the proposed SGR-CCE-LHCII complex, which acts in chlorophyll breakdown. Notably, HCAR and NOL are expressed throughout leaf development and are drastically down-regulated during dark-induced senescence, in contrast with SGR, NYC1, PPH and PAO, which are up-regulated during dark-induced senescence. Moreover, HCAR and NOL are highly up-regulated during greening of etiolated seedlings, strongly suggesting a major role for NOL and HCAR in the chlorophyll cycle during vegetative stages, possibly in chlorophyll turnover.

STAY-GREEN and chlorophyll catabolic enzymes interact at light-harvesting complex II for chlorophyll detoxification during leaf senescence in Arabidopsis.

During leaf senescence, plants degrade chlorophyll to colorless linear tetrapyrroles that are stored in the vacuole of senescing cells. The early steps of chlorophyll breakdown occur in plastids. To date, five chlorophyll catabolic enzymes (CCEs), NONYELLOW COLORING1 (NYC1), NYC1-LIKE, pheophytinase, pheophorbide a oxygenase (PAO), and red chlorophyll catabolite reductase, have been identified; these enzymes catalyze the stepwise degradation of chlorophyll to a fluorescent intermediate, pFCC, which is then exported from the plastid. In addition, STAY-GREEN (SGR), Mendel's green cotyledon gene encoding a chloroplast protein, is required for the initiation of chlorophyll breakdown in plastids. Senescence-induced SGR binds to light-harvesting complex II (LHCII), but its exact role remains elusive. Here, we show that all five CCEs also specifically interact with LHCII. In addition, SGR and CCEs interact directly or indirectly with each other at LHCII, and SGR is essential for recruiting CCEs in senescing chloroplasts. PAO, which had been attributed to the inner envelope, is found to localize in the thylakoid membrane. These data indicate a predominant role for the SGR-CCE-LHCII protein interaction in the breakdown of LHCII-located chlorophyll, likely to allow metabolic channeling of phototoxic chlorophyll breakdown intermediates upstream of nontoxic pFCC.

Pheophytin pheophorbide hydrolase (pheophytinase) is involved in chlorophyll breakdown during leaf senescence in Arabidopsis.

During leaf senescence, chlorophyll is removed from thylakoid membranes and converted in a multistep pathway to colorless breakdown products that are stored in vacuoles. Dephytylation, an early step of this pathway, increases water solubility of the breakdown products. It is widely accepted that chlorophyll is converted into pheophorbide via chlorophyllide. However, chlorophyllase, which converts chlorophyll to chlorophyllide, was found not to be essential for dephytylation in Arabidopsis thaliana. Here, we identify pheophytinase (PPH), a chloroplast-located and senescence-induced hydrolase widely distributed in algae and land plants. In vitro, Arabidopsis PPH specifically dephytylates the Mg-free chlorophyll pigment, pheophytin (phein), yielding pheophorbide. An Arabidopsis mutant deficient in PPH (pph-1) is unable to degrade chlorophyll during senescence and therefore exhibits a stay-green phenotype. Furthermore, pph-1 accumulates phein during senescence. Therefore, PPH is an important component of the chlorophyll breakdown machinery of senescent leaves, and we propose that the sequence of early chlorophyll catabolic reactions be revised. Removal of Mg most likely precedes dephytylation, resulting in the following order of early breakdown intermediates: chlorophyll --> pheophytin --> pheophorbide. Chlorophyllide, the last precursor of chlorophyll biosynthesis, is most likely not an intermediate of breakdown. Thus, chlorophyll anabolic and catabolic reactions are metabolically separated.

CHLOROPHYLL DEGRADATION DURING SENESCENCE

The catabolic pathway of chlorophyll (Chl) during senescence and fruit ripening leads to the accumulation of colorless breakdown products (NCCs). This review updates an earlier review on Chl breakdown published here in 1999 ( 69 ). It summarizes recent advances in the biochemical reactions of the pathway and describes the characterization of new NCCs and their formation inside the vacuole. Furthermore, I focus on the recent molecular identification of three chl catabolic enzymes, chlorophyllase, pheophorbide a oxygenase (PAO), and red Chl catabolite reductase (RCCR). The analysis of Chl catabolic mutants demonstrates the importance of Chl breakdown for plant development and survival. Mutants defective in PAO or RCCR develop a lesion mimic phenotype, due to the accumulation of breakdown intermediates. Thus, Chl breakdown is a prerequisite to detoxify the potentially phototoxic pigment within the vacuoles in order to permit the remobilization of nitrogen from Chl-binding proteins to proceed during senescence.

Proteolysis of bovine beta-lactoglobulin during thermal treatment in subdenaturing conditions highlights some structural features of the temperature-modified protein and yields fragments with low immunoreactivity.

Bovine beta-lactoglobulin was hydrolyzed with trypsin or chymotrypsin in the course of heat treatment at 55, 60 and 65 degrees C at neutral pH. At these temperatures beta-lactoglobulin undergoes significant but reversible structural changes. In the conditions used in the present study, beta-lactoglobulin was virtually insensitive to proteolysis by either enzyme at room temperature, but underwent extensive proteolysis when either protease was present during the heat treatment. High-temperature proteolysis occurs in a progressive manner. Mass spectrometry analysis of some large-sized breakdown intermediates formed in the early steps of hydrolysis indicated that both enzymes effectively hydrolyzed some regions of beta-lactoglobulin that were transiently exposed during the physical treatments and that were not accessible in the native protein. The immunochemical properties of the products of beta-lactoglobulin hydrolysis were assessed by using various beta-lactoglobulin-specific antibodies, and most epitopic sites were no longer present after attack of the partially unfolded protein by the two proteases.

Interactions of CCCH Zinc Finger Proteins with mRNA: TRISTETRAPROLIN-MEDIATED AU-RICH ELEMENT-DEPENDENT mRNA DEGRADATION CAN OCCUR IN THE ABSENCE OF A POLY(A) TAIL

The CCCH family of tandem zinc finger proteins has recently been shown to promote the turnover of certain mRNAs containing class II AU-rich elements (AREs). In the case of one member of this family, tristetraprolin (TTP), absence of the protein in knockout mice leads to stabilization of two mRNAs containing AREs of this type, those encoding tumor necrosis factor alpha (TNFalpha) and granulocyte-macrophage colony-stimulating factor. To begin to decipher the mechanism by which these zinc finger proteins stimulate the breakdown of this class of mRNAs, we co-transfected TTP and its related CCCH proteins into 293 cells with vectors encoding full-length TNFalpha, granulocyte-macrophage colony-stimulating factor, and interleukin-3 mRNAs. Co-expression of the CCCH proteins caused the rapid turnover of these ARE-containing mRNAs and also promoted the accumulation of stable breakdown intermediates that were truncated at the 3'-end of the mRNA, even further 5' than the 5'-end of the poly(A) tail. To determine whether an intact poly(A) tail was necessary for TTP to promote this type of mRNA degradation, we inserted the TNFalpha ARE into a nonpolyadenylated histone mRNA and also attached a histone 3'-end-processing sequence to the 3'-end of nonpolyadenylated interleukin-3 and TNFalpha mRNAs. In all three cases, TTP stimulated the turnover of the ARE-containing mRNAs, despite the demonstrated absence of a poly(A) tail. These studies indicate that members of this class of CCCH proteins can promote class II ARE-containing mRNA turnover even in the absence of a poly(A) tail, suggesting that the processive removal of the poly(A) tail may not be required for this type of CCCH protein-stimulated mRNA turnover.

Biomineralisation of azo dyes and their breakdown products in anaerobic-aerobic hybrid and UASB reactors

Batch toxicity and biodegradability of two azo dyes (Siriusgelb and Siriuslichtbraun) has been investigated. It was found that the former azo dye was significantly less toxic to methanogenic sludge than the latter one (IC50 are equal to 3.55 and 0.41 g COD/l, respectively). Neither of the azo dyes was biodegradable under aerobic conditions but both dyes were readily decolourised and slowly mineralised in anaerobic environments. In order to optimise the treatment strategy, the anaerobic and aerobic phases were combined into one single unit called the anaerobic-aerobic hybrid reactor. The performance of this innovative reactor was tested with a synthetic wastewater containing Siriusgelb and ethanol at 30°C and 56% removal of azo dye COD was achieved at volumetric load of 0.3 g azo dye COD/l/day. The effluent COD content could be attributed to the presence of non-biodegradable autooxidation products of Siriusgelb breakdown intermediates. A continuous biomineralisation of 2-aminobenzoic acid (2-ABA) – intermediate of the anaerobic decomposition of Siriuslichtbraun – was studied in a UASB reactor at 30°C. A high (&amp;gt;90%) removal of 2-ABA was achieved under volumetric loads of 1.5 g 2-ABA COD/l/day. However, a further increase in volumetric load led to a decrease in 2-ABA removal, probably due to low attachment ability of the bacteria responsible for primary decomposition of 2-ABA.