Cold Trichloroacetic Acid Research Articles

Structural characterization of exopolysaccharides obtained from Porphyridium cruentum exhausted culture medium

Microalgae produce and secrete large quantities of polysaccharides into the culture medium that is discarded when biomass is separated. The main objective of this study was to add value to the exhausted culture medium (ECM) of Porphyridium cruentum through the recovery of extracellular polysaccharides (EPS) by different extracting and purifying methods for the future biotechnological applications. The ECM was submitted to precipitation with cold absolute ethanol, using ultrasound, dialysis, and trichloroacetic acid (TCA) as purification methods. The purification provided a lower yield, with higher carbohydrate content. The TCA purified sample presented up to twice as much total carbohydrates as the non-purified samples, mainly composed of xylose, galactose, and glucose. Scanning Electron Microscopy (SEM) images showed the smooth structure of P. cruentum EPS, in which TCA post-treatment and dialysis allowed obtaining larger and purest particles, being a good candidate for film making. Microalgal polymeric by-products are a sustainable source to recover valuable compounds, and the purification treatment proved to be an important step to valorize this material.

Ion-pair hollow fiber liquid-phase microextraction combined with capillary electrophoresis for the determination of biogenic amines in rat tissues

Herein, the development of an ion-pair hollow fiber liquid-phase microextraction (IP-HF-LPME) procedure followed by capillary electrophoresis (CE) with indirect UV detection for the simultaneous extraction and determination of aliphatic biogenic amines including spermidine, spermine, and cadaverine in tissue samples of rat is described. In this study, tissue samples were firstly homogenized with a cold trichloroacetic acid solution then the developed method was applied to the supernatant resulting from tissue homogenization. Different CE separation and indirect UV detection conditions, as well as IP-HF-LPME extraction conditions were studied in detail and optimized. A 11 mmol L−1 imidazole solution containing 13 % (v/v) ethanol adjusted at pH 3.3 (by acetic acid) was the best running buffer for indirect UV detection of non-UV-absorbing amines. 1-Octanol in combination with salicylic acid, respectively as the membrane solvent and ion-pair reagent provided the highest extraction recovery for the studied amines. Method performances were established and good results of linearity, recovery, sensitivity, and repeatability were achieved for all the studied amines. Limits of detection were found to be between 2.8 and 4.5 ng mL−1 and limits of quantification were 15 ng mL−1 and the dynamic range was 15–2000 ng mL−1 for all three analytes.

A modified fluorimetric method for determination of hydrogen peroxide using homovanillic acid oxidation principle.

Hydrogen peroxide (H2O2) level in biological samples is used as an important index in various studies. Quantification of H2O2 level in tissue fractions in presence of H2O2 metabolizing enzymes may always provide an incorrect result. A modification is proposed for the spectrofluorimetric determination of H2O2 in homovanillic acid (HVA) oxidation method. The modification was included to precipitate biological samples with cold trichloroacetic acid (TCA, 5% w/v) followed by its neutralization with K2HPO4 before the fluorimetric estimation of H2O2 is performed. TCA was used to precipitate the protein portions contained in the tissue fractions. After employing the above modification, it was observed that H2O2 content in tissue samples was ≥2 fold higher than the content observed in unmodified method. Minimum 2 h incubation of samples in reaction mixture was required for completion of the reaction. The stability of the HVA dimer as reaction product was found to be >12 h. The method was validated by using known concentrations of H2O2 and catalase enzyme that quenches H2O2 as substrate. This method can be used efficiently to determine more accurate tissue H2O2 level without using internal standard and multiple samples can be processed at a time with additional low cost reagents such as TCA and K2HPO4.

Biochemical and Functional Characterization of the Klotho-VS Polymorphism Implicated in Aging and Disease Risk

Klotho (KL) is an age-regulating protein named after the Greek goddess who spins the thread of life. Mice deficient in KL are normal throughout development, but rapidly degenerate and display a variety of aging-associated abnormalities that eventually lead to decreased life expectancy. While multiple genetic association studies have identified KL polymorphisms linked with changes in disease risk, there is a paucity of concrete mechanistic data to explain how these amino acid substitutions alter KL protein function. The KLVS polymorphism is suggested to lead to changes in protein trafficking although the mechanism is unclear. Our studies have sought to further investigate the functional differences in the KLVS variant that result in increased risk of many age-related diseases. Our findings suggest that the F352V and C370S substitutions lead to alterations in processing as seen by differences in shedding and half-life. Their co-expression in KLVS results in a phenotype resembling wild-type, but despite this intragenic complementation there are still changes in homodimerization and interactions with FGFR1c. Taken together, these studies suggest that KLVS leads to altered homodimerization that indirectly leads to changes in processing and FGFR1c interactions. These findings help elucidate the functional differences that result from the VS polymorphism, which will help clarify how alterations in KL function can lead to human disease and affect cognition and lifespan.

ADP-ribosylation of Translation Elongation Factor 2 by Diphtheria Toxin in Yeast Inhibits Translation and Cell Separation

Eukaryotic translation elongation factor 2 (eEF2) facilitates the movement of the peptidyl tRNA-mRNA complex from the A site of the ribosome to the P site during protein synthesis. ADP-ribosylation (ADP(R)) of eEF2 by bacterial toxins on a unique diphthamide residue inhibits its translocation activity, but the mechanism is unclear. We have employed a hormone-inducible diphtheria toxin (DT) expression system in Saccharomyces cerevisiae which allows for the rapid induction of ADP(R)-eEF2 to examine the effects of DT in vivo. ADP(R) of eEF2 resulted in a decrease in total protein synthesis consistent with a defect in translation elongation. Association of eEF2 with polyribosomes, however, was unchanged upon expression of DT. Upon prolonged exposure to DT, cells with an abnormal morphology and increased DNA content accumulated. This observation was specific to DT expression and was not observed when translation elongation was inhibited by other methods. Examination of these cells by electron microscopy indicated a defect in cell separation following mitosis. These results suggest that expression of proteins late in the cell cycle is particularly sensitive to inhibition by ADP(R)-eEF2.

Characterization of LipL as a Non-heme, Fe(II)-dependent α-Ketoglutarate:UMP Dioxygenase That Generates Uridine-5′-aldehyde during A-90289 Biosynthesis

Fe(II)- and α-ketoglutarate (α-KG)-dependent dioxygenases are a large and diverse superfamily of mononuclear, non-heme enzymes that perform a variety of oxidative transformations typically coupling oxidative decarboxylation of α-KG with hydroxylation of a prime substrate. The biosynthetic gene clusters for several nucleoside antibiotics that contain a modified uridine component, including the lipopeptidyl nucleoside A-90289 from Streptomyces sp. SANK 60405, have recently been reported, revealing a shared open reading frame with sequence similarity to proteins annotated as α-KG:taurine dioxygenases (TauD), a well characterized member of this dioxygenase superfamily. We now provide in vitro data to support the functional assignment of LipL, the putative TauD enzyme from the A-90289 gene cluster, as a non-heme, Fe(II)-dependent α-KG:UMP dioxygenase that produces uridine-5'-aldehyde to initiate the biosynthesis of the modified uridine component of A-90289. The activity of LipL is shown to be dependent on Fe(II), α-KG, and O(2), stimulated by ascorbic acid, and inhibited by several divalent metals. In the absence of the prime substrate UMP, LipL is able to catalyze oxidative decarboxylation of α-KG, although at a significantly reduced rate. The steady-state kinetic parameters using optimized conditions were determined to be K(m)(α-KG) = 7.5 μM, K(m)(UMP) = 14 μM, and k(cat) ≈ 80 min(-1). The discovery of this new activity not only sets the stage to explore the mechanism of LipL and related dioxygenases further but also has critical implications for delineating the biosynthetic pathway of several related nucleoside antibiotics.

Sulfur assimilation byOxyrrhis marinafeeding on a35S‐DMSP‐labelled prey

A laboratory grazing experiment was conducted with the aim of quantifying the sulfur assimilation by a herbivore protist feeding on a dimethylsulfoniopropionate (DMSP)-containing phytoplankter. When supplied with dissolved (35)S-DMSP, cultures of an axenic strain of the diatom Thalassiosira pseudonana took up 60-95% of the added radioisotope and accumulated it untransformed in the cytoplasm. Radiolabelled diatom cells were offered as prey to the heterotrophic dinoflagellate Oxyrrhis marina. After 32 h in the dark, all the prey had been grazed and digested, leaving only radiolabelled O. marina in the grazing bottles and thus providing an estimate of the percentage of DMSP-sulfur retained by the predator. Subsequent precipitation with cold trichloroacetic acid (TCA) provided the fraction of retained DMSP-S that had been assimilated into the micrograzer macromolecules. In parallel incubations with predator and dissolved (35)S-DMSP only (no prey), O. marina (and their closely associated bacteria) took up the radiolabelled substrate osmotrophically to an activity of 0.04 dpm cell(-1) and assimilated it all into macromolecules. By correcting grazing (35)S-DMSP assimilation for osmotrophic (35)S-DMSP assimilation, and comparing it with the ingested radioisotope, the percentage of ingested DMSP-sulfur retained and assimilated by the predator was determined to be 32 +/- 4%. This is the first study that provides direct evidence that ingestion of a DMSP-containing prey supplies structural sulfur to a herbivore protist and that quantifies this assimilative supply at one-third of ingested DMSP.

A Disease-causing Point Mutation in Human Mitochondrial tRNAMet Results in tRNA Misfolding Leading to Defects in Translational Initiation and Elongation

The mitochondrial tRNA genes are hot spots for mutations that lead to human disease. A single point mutation (T4409C) in the gene for human mitochondrial tRNA(Met) (hmtRNA(Met)) has been found to cause mitochondrial myopathy. This mutation results in the replacement of U8 in hmtRNA(Met) with a C8. The hmtRNA(Met) serves both in translational initiation and elongation in human mitochondria making this tRNA of particular interest in mitochondrial protein synthesis. Here we show that the single 8U-->C mutation leads to a failure of the tRNA to respond conformationally to Mg(2+). This mutation results in a drastic disruption of the structure of the hmtRNA(Met), which significantly reduces its aminoacylation. The small fraction of hmtRNA(Met) that can be aminoacylated is not formylated by the mitochondrial Met-tRNA transformylase preventing its function in initiation, and it is unable to form a stable ternary complex with elongation factor EF-Tu preventing any participation in chain elongation. We have used structural probing and molecular reconstitution experiments to examine the structures formed by the normal and mutated tRNAs. In the presence of Mg(2+), the normal tRNA displays the structural features expected of a tRNA. However, even in the presence of Mg(2+), the mutated tRNA does not form the cloverleaf structure typical of tRNAs. Thus, we believe that this mutation has disrupted a critical Mg(2+)-binding site on the tRNA required for formation of the biologically active structure. This work establishes a foundation for understanding the physiological consequences of the numerous mitochondrial tRNA mutations that result in disease in humans.

Perturbation of the tRNA Tertiary Core Differentially Affects Specific Steps of the Elongation Cycle

The tRNA tertiary core region is important for both tRNA stability and activity in the translation elongation cycle. Here we report the effects of mutating each of two highly conserved base pairs in the tertiary core of Phe-tRNA(Phe), 18-55 and 19-56, on rate and equilibrium constants for specific steps of this cycle, beginning with formation of aminoacyl-tRNA.EF-Tu.GTP ternary complexs and culminating with translocation of A-site-bound peptidyl-tRNA into the P-site. We find that codon-dependent binding of aminoacyl-tRNA to the A/T-site and proofreading of near-cognate tRNA are sensitive to perturbation of either base pair; formation of the ternary complex and accommodation from the A/T to the A-site are sensitive to 18-55 perturbation only, and translocation of peptidyl-tRNA from the A- to P-site is insensitive to perturbation of either. These results underline the importance of the core region in promoting the efficiency and accuracy of translation, and they likely reflect different requirements for structural integrity of the core during specific steps of the elongation cycle.

Cytochrome b5, Not Superoxide Anion Radical, Is a Major Reductant of Indoleamine 2,3-Dioxygenase in Human Cells

The heme protein indoleamine 2,3-dioxygenase (IDO) initiates oxidative metabolism of tryptophan along the kynurenine pathway, and this requires reductive activation of Fe(3+)-IDO. The current dogma is that superoxide anion radical (O(2)(*-)) is responsible for this activation, based largely on previous work employing purified rabbit IDO and rabbit enterocytes. We have re-investigated this role of O(2)(*-) using purified recombinant human IDO (rhIDO), rabbit enterocytes that constitutively express IDO, human endothelial cells, and monocyte-derived macrophages treated with interferon-gamma to induce IDO expression, and two cell lines transfected with the human IDO gene. Both potassium superoxide and O(2)(*-) generated by xanthine oxidase modestly activated rhIDO, in reactions that were prevented completely by superoxide dismutase (SOD). In contrast, SOD mimetics had no effect on IDO activity in enterocytes and interferon-gamma-treated human cells, despite significantly decreasing cellular O(2)(*-) Similarly, cellular IDO activity was unaffected by increasing SOD activity via co-expression of Cu,Zn-SOD or by increasing cellular O(2)(*-) via treatment of cells with menadione. Other reductants, such as tetrahydrobiopterin, ascorbate, and cytochrome P450 reductase, were ineffective in activating cellular IDO. However, recombinant human cytochrome b(5) plus cytochrome P450 reductase and NADPH reduced Fe(3+)-IDO to Fe(2+)-IDO and activated rhIDO in a reconstituted system, a reaction inhibited marginally by SOD. Additionally, short interfering RNA-mediated knockdown of microsomal cytochrome b(5) significantly decreased IDO activity in IDO-transfected cells. Together, our data show that cytochrome b(5) rather than O(2)(*-) plays a major role in the activation of IDO in human cells.

Cloning and Characterization of a Streptomyces Single Module Type Non-ribosomal Peptide Synthetase Catalyzing a Blue Pigment Synthesis

In the present study, we cloned a gene, designated bpsA, which encodes a single module type non-ribosomal peptide synthetase (NRPS) from a D-cycloserine (DCS)-producing Streptomyces lavendulae ATCC11924. A putative oxidation domain is significantly integrated into the adenylation domain of the NRPS, and the condensation domain is absent from the module. When S. lividans was transformed with a plasmid carrying bpsA, the transformed cells produced a blue pigment, suggesting that bpsA is responsible for the blue pigment synthesis. However, to produce the blue pigment in Escherichia coli, the existence of the 4'-phosphopantetheinyl transferase (PPTase) gene from Streptomyces was necessary, in addition to bpsA. The chemical structure of the pigment was determined as 5,5'-diamino-4,4'-dihydroxy-3,3'-diazadiphenoquinone-(2,2'), called indigoidine. The bpsA gene product, designated BPSA, was overproduced in an E. coli host-vector system and purified to homogeneity, demonstrating that the recombinant enzyme prefers L-Gln as a substrate. The in vitro experiment using L-Gln also showed that the blue pigment was formed by the purified BPSA only when the enzyme was phosphopantetheinylated by adding a Streptomyces PPTase purified from E. coli cells. Each site-directed mutagenesis experiment of Lys(598), Tyr(601), Ser(603), and Tyr(608), which are seen in the oxidation domain of BPSA, suggests that these residues are essential for the binding of FMN to the protein and the synthesis of the blue pigment.

15-Deoxyspergualin Primarily Targets the Trafficking of Apicoplast Proteins in Plasmodium falciparum

15-Deoxyspergualin, an immunosuppressant with tumoricidal and antimalarial properties, has been implicated in the inhibition of a diverse array of cellular processes including polyamine synthesis and protein synthesis. Endeavoring to identify the mechanism of antimalarial action of this molecule, we examined its effect on Plasmodium falciparum protein synthesis, polyamine biosynthesis, and transport. 15-Deoxyspergualin stalled protein synthesis in P. falciparum through Hsp70 sequestration and subsequent phosphorylation of the eukaryotic initiation factor eIF2alpha. However, protein synthesis inhibition as well as polyamine depletion were invoked only by high micromolar concentrations of 15-deoxyspergualin, in contrast to the submicromolar concentrations sufficient to inhibit parasite growth. Further investigations demonstrated that 15-deoxyspergualin in the malaria parasite primarily targets the hitherto underexplored process of trafficking of nucleus-encoded proteins to the apicoplast. Our finding that 15-deoxyspergualin kills the malaria parasite by interfering with targeting of nucleus-encoded proteins to the apicoplast not only exposes a chink in the armor of the malaria parasite, but also reveals new realms in our endeavors to study this intriguing biological process.

The Zinc Finger Protein Ynr046w Is Plurifunctional and a Component of the eRF1 Methyltransferase in Yeast

Protein release factor eRF1 in Saccharomyces cerevisiae, in complex with eRF3 and GTP, is methylated on a functionally crucial Gln residue by the S-adenosylmethionine-dependent methyltransferase Ydr140w. Here we show that eRF1 methylation, in addition to these previously characterized components, requires a 15-kDa zinc-binding protein, Ynr046w. Co-expression in Escherichia coli of Ynr046w and Ydr140w allows the latter to be recovered in soluble form rather than as inclusion bodies, and the two proteins co-purify on nickel-nitrilotriacetic acid chromatography when Ydr140w alone carries a His tag. The crystal structure of Ynr046w has been determined to 1.7 A resolution. It comprises a zinc-binding domain built from both the N- and C-terminal sequences and an inserted domain, absent from bacterial and archaeal orthologs of the protein, composed of three alpha-helices. The active methyltransferase is the heterodimer Ydr140w.Ynr046w, but when alone, both in solution and in crystals, Ynr046w appears to be a homodimer. The Ynr046w eRF1 methyltransferase subunit is shared by the tRNA methyltransferase Trm11p and probably by two other enzymes containing a Rossman fold.

Physiological Regulation of Phospholipid Methylation Alters Plasma Homocysteine in Mice

Biological methylation reactions and homocysteine (Hcy) metabolism are intimately linked. In previous work, we have shown that phosphatidylethanolamine N-methyltransferase, an enzyme that methylates phosphatidylethanolamine to form phosphatidylcholine, plays a significant role in the regulation of plasma Hcy levels through an effect on methylation demand (Noga, A. A., Stead, L. M., Zhao, Y., Brosnan, M. E., Brosnan, J. T., and Vance, D. E. (2003) J. Biol. Chem. 278, 5952-5955). We have further investigated methylation demand and Hcy metabolism in liver-specific CTP:phosphocholine cytidylyltransferase-alpha (CTalpha) knockout mice, since flux through the phosphatidylethanolamine N-methyltransferase pathway is increased 2-fold to meet hepatic demand for phosphatidylcholine. Our data show that plasma Hcy is elevated by 20-40% in mice lacking hepatic CTalpha. CTalpha-deficient hepatocytes secrete 40% more Hcy into the medium than do control hepatocytes. Liver activity of betaine:homocysteine methyltransferase and methionine adenosyltransferase are elevated in the knockout mice as a mechanism for maintaining normal hepatic S-adenosylmethionine and S-adenosylhomocysteine levels. These data suggest that phospholipid methylation in the liver is a major consumer of AdoMet and a significant source of plasma Hcy.

Mitotic Kinesin Inhibitors Induce Mitotic Arrest and Cell Death in Taxol-resistant and -sensitive Cancer Cells

Taxanes are powerful chemotherapy agents that target the microtubule cytoskeleton, leading to mitotic arrest and cell death; however, their clinical efficacy has been hampered due to the development of drug resistance. Therefore, other proteins involved in spindle assembly are being examined as potential targets for anticancer therapy. The mitotic kinesin, Eg5 is critical for proper spindle assembly; as such, inhibition of Eg5 leads to mitotic arrest making it a potential anticancer target. We wanted to validate Eg5 as a therapeutic target and determine if Eg5 inhibitors retain activity in Taxol-resistant cells. Using affinity chromatography we first show that the compound HR22C16 is an Eg5 inhibitor and does not interact with other microtubule motor proteins tested. Furthermore, HR22C16 along with its analogs, inhibit cell survival in both Taxol-sensitive and -resistant ovarian cancer cells with at least 15-fold greater efficacy than monastrol, the first generation Eg5 inhibitor. Further analysis with HR22C16-A1, the most potent HR22C16 analog, showed that it retains efficacy in PgP-overexpressing cells, suggesting that it is not a PgP substrate. We further show that HR22C16-A1 induces cell death following mitotic arrest via the intrinsic apoptotic pathway. Interestingly, the combination of HR22C16-A1 with Taxol results in an antagonistic antiproliferative and antimitotic effect, possibly due to the abrogation of Taxol-induced mitotic spindles by HR22C16-A1. Taken together, our results show that Eg5 inhibitors have promising anticancer activity and can be potentially used to overcome Taxol resistance in the clinical setting.

Endosomal Proteolysis of Internalized Insulin at the C-terminal Region of the B Chain by Cathepsin D

The endosomal compartment of hepatic parenchymal cells contains an acidic endopeptidase, endosomal acidic insulinase, which hydrolyzes internalized insulin and generates the major primary end product A(1--21)-B(1--24) insulin resulting from a major cleavage at residues Phe(B24)-Phe(B25). This study addresses the nature of the relevant endopeptidase activity in rat liver that is responsible for most receptor-mediated insulin degradation in vivo. The endosomal activity was shown to be aspartic acid protease cathepsin D (CD), based on biochemical similarities to purified CD in 1) the rate and site of substrate cleavage, 2) pH optimum, 3) sensitivity to pepstatin A, and 4) binding to pepstatin A-agarose. The identity of the protease was immunologically confirmed by removal of greater than 90% of the insulin-degrading activity associated with an endosomal lysate using polyclonal antibodies to CD. Moreover, the elution profile of the endosomal acidic insulinase activity on a gel-filtration TSK-GEL G3000 SW(XL) high performance liquid chromatography column corresponded exactly with the elution profile of the immunoreactive 45-kDa mature form of endosomal CD. Using nondenaturating immunoprecipitation and immunoblotting procedures, other endosomal aspartic acid proteases such as cathepsin E and beta-site amyloid precursor protein-cleaving enzyme (BACE) were ruled out as candidate enzymes for the endosomal degradation of internalized insulin. Immunofluorescence studies showed a largely vesicular staining pattern for internalized insulin in rat hepatocytes that colocalized partially with CD. In vivo pepstatin A treatment was without any observable effect on the insulin receptor content of endosomes but augmented the phosphotyrosine content of the endosomal insulin receptor after insulin injection. These results suggest that CD is the endosomal acidic insulinase activity which catalyzes the rate-limiting step of the in vivo cleavage at the Phe(B24)-Phe(B25) bond, generating the inactive A(1--21)-B(1--24) insulin intermediate.

Circadian change of adenylate cyclase activity in rabbit ciliary processes

Purpose. To characterize in vivo changes of adenylate cyclase activity in rabbit ciliary processes during the circadian cycle. Methods. After removal of vitreous, lens, retina and choroid from freshly enucleated rabbit eyes, the anterior segment and attached sclera was chilled in cold buffer containing the non-selective phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Then ciliary processes were excised and homogenized in ice cold trichloro-acetic acid (TCA) 2.5 min after IBMX treatment. Increased cyclic AMP in response to PDE inhibition was measured in ciliary processes at six times during the circadian cycle, after preganglionic section of the cervical sympathetic trunk (decentralization, DX), and after topical instillation of hydroxyamphetamine, timolol, brimonidine, rauwolscine or a soluble derivative of forskolin. Results. The increase of rabbit ciliary process cyclic AMP levels in response to PDE inhibition with IBMX was enhanced at night. Much of the enhanced nocturnal increase persisted in constant dark and was blunted by DX. Topical instillation of hydroxyamphetamine enhanced the increase during the day; whereas, timolol, a ß-adrenergic antagonist, or brimonidine, an a 2 -adrenergic agonist, blunted the IBMX induced increase. Neither instillation of rauwolscine, an a 2 -adrenergic antagonist, nor the soluble forskolin derivative enhanced the increase of cyclic AMP after IBMX. Conclusions. The technique reported here can be used to estimate ciliary process adenylate cyclase activity in vivo. There is a circadian rhythm of adenylate cyclase activity in rabbit ciliary processes that is driven in part by ocular sympathetic input and stimulation of ß-adrenergic receptors.

Fluorophores at the N Terminus of Nascent Chloramphenicol Acetyltransferase Peptides Affect Translation and Movement through the Ribosome

Structurally different fluorescent probes were covalently attached to methionyl-tRNA(f) and tested for their incorporation into nascent peptides and full-length protein using an Escherichia coli cell-free coupled transcription/translation system. Bovine rhodanese and bacterial chloramphenicol acetyltransferase (CAT) were synthesized using derivatives of cascade yellow, eosin, pyrene, or coumarin attached to [(35)S]Met-tRNA(f). All of the probes tested were incorporated into polypeptides, although less efficiently when compared with formyl-methionine. Eosin, the largest of the fluorophores used with estimated dimensions of 20 x 11 A, caused the largest reduction in product formed. The rate of initiation was reduced with the fluorophore-Met-tRNA(f) compared with fMet-tRNA(f) with pyrene having the least and eosin the biggest effect. Analysis of the nascent polypeptides showed that the modifications at the N terminus affected the rate at which nascent CAT peptides were elongated causing accumulation of peptides of about 4 kDa, possibly by steric hindrance inside the tunnel within the 50 S ribosomal subunit. Fluorescence measurements indicate that the probe at the N terminus of nascent pyrene-CAT peptides is in a relatively hydrophilic environment. This finding is in agreement with recent data showing cross-linking of the N terminus of nascent peptides to nucleotides of the 23 S ribosomal RNA.

Influence of Site and Fertiliser Addition on Nutrient Cycling in Eucalyptus globulus Plantations in Gippsland, South-eastern Australia. I. Foliage and Litter Quality

The productivity of Eucalyptus plantations on many sites in south-eastern Australia is limited by nitrogen and phosphorus supply. Therefore, after canopy closure, nutrient return and decomposition are key processes maintaining productivity. To gain a better understanding of the effects of site and fertilisers on these processes, foliage and litter quality in E. globulus (Labill.) plantations in Gippsland, south-eastern Australia were characterised on three sites covering a range of soil types, inherent soil fertility and fertiliser treatments. Foliage and litter quality were estimated by sequential extraction of labile forms of N, P and C with cold, then hot, trichloroacetic acid (TCA). Selected treatments were sampled in N × P factorial fertiliser trials of 6-year-old trees where nutrients were added up to 2 years of age. Foliage and litter were categorised as recent or old depending on sampling position. Site significantly influenced concentrations of total and labile N and P (P < 0.0001) in foliage and litter. Phosphorus fertiliser increased total P concentrations in old foliage at two sites, with the greatest absolute and relative increases at the least fertile site (Glencoe). Inorganic P extracted by cold (4°C) TCA accounted for 30-55% of total leaf and litter P and was the fraction most responsive to P fertiliser addition. Total N concentration and N fractions in foliage and litter were not influenced by N fertiliser addition. Inorganic N extracted by cold and hot (90°C) TCA accounted for less than 2% of total N and was not significantly different among fertiliser treatments. Both sugar and phenol concentrations in foliage and litter varied significantly between sites, with the least fertile site showing significantly higher concentrations of phenols in recent litter. Sugars and phenols extracted in cold TCA decreased from old foliage to litter at all sites and were not influenced by N and P fertiliser addition. The results show that additions of 200 kg ha-1 of P cause perturbations in P cycle that are bigger in magnitude and are sustained for longer periods of time compared to changes in N cycle with 400 kg ha-1 of N additions.

Glycosomes — the organelles of glycogen metabolism

This article reviews the data concerning the electron microscopical interpretation of glycogen. It demonstrates that glycogen in the cell is associated with the enzymes involved in its metabolism and that the glycogen-protein complex forms morphologically distinct cell organelles called glycosomes. Glycogen can be visualized in the electron microscope (EM) by histochemical procedures, or by negative staining, but it does not react with heavy metals such as uranium and lead. The protein component of glycosomes, stainable by heavy metals, appears in EM as 20–30 nm granules. While biochemical findings have long indicated the association of glycogen and protein in the cell, morphological interpretation traditionally defined the protein component of glycosomes as particles of glycogen. Accordingly, the term α or β particles, introduced to define particles of glycogen, became subsequently applied to the protein component visible in sections stained by heavy metals. The history of microscopic research reveals the conditions which led to such interpretation. Morphological analysis of the reaction of glycosomes to the acids shows that glycosomes deposited free in the cytosol (lyoglycosomes) are acid labile, whereas the others (desmoglycosomes), intimately associated with different cellular structures, are acid-resistant. These 2 groups correspond to lyo- and desmoglycogen distinguished in early biochemical studies on the basis of their different resistance to the cold trichloroacetic acid. The theory of glycosomes provides a new paradigm which clarifies numerous unexplained data in the microscopic literature on glycogen, and opens a vast field for the research on the cellular metabolism of glycogen, with the use of modern molecular and cellular biology techniques.