The Release of CO<Subscript>2</Subscript> from Riverwaters – the Contribution of Excess CO<Subscript>2</Subscript> from Groundwater

The dissolved CO ₂ concentration of stream waters is an important component of the terrestrial carbon cycle and an important pathway for release of CO₂ to the atmosphere. This study uses data from the UK's largest groundwater monitoring network to estimate the importance of groundwater in contributing excess dissolved CO₂ to the atmosphere. The study shows that:

Structure of the “Peroxy-Y Base” from Liver tRNA<Superscript>Phe</Superscript>

WE wish to report that reconstituted sperm whale myoglobin prepared by the method of Breslow¹ (except that pH 2 was found sufficient to remove all the haem) (I) crystallizes² in a different habit from those prepared by the method of Rossi-Fanelli et al.³ (II) using haemin of Sigma lot 77B-0220 and our own ⁵⁷Fe photoporphyrin preparation and the native myoglobin (III). Although all three form type A³ monoclinic prisms, the best developed plane is [001] for II and III, it is [100] for I. There seems to be great interest in reconstituted haemoproteins^4,5, so it is important that crystallization habit may be a sensitive test for subtle changes in protein structures.     

Transient releases of acetaldehyde from tree leaves − products of a pyruvate overflow mechanism?

Emissions of acetaldehyde from tree leaves were investigated by proton‐transfer‐reaction mass spectrometry (PTR‐MS), a technique that allows simultaneous monitoring of different leaf volatiles, and confirmed by derivatization and high‐performance liquid chromatography analysis. Bursts of acetaldehyde were released by sycamore, aspen, cottonwood and maple leaves following light–dark transitions; isoprene emission served as a measure of chloroplastic processes. Acetaldehyde bursts were not accompanied by ethanol, but exposure of leaves to inhibitors of pyruvate transport or respiration, or anoxia, led to much larger releases of acetaldehyde, accompanied by ethanol under anoxic conditions. These same leaves have an oxidative pathway for ethanol present in the transpiration stream, resulting in acetaldehyde emissions that are inhibited in vivo by 4‐methylpyrazole, an alcohol dehydrogenase (Adh) inhibitor. Labelling of leaf volatiles with ¹³CO₂ suggested that the pools of cytosolic pyruvate, the proposed precursor of acetaldehyde bursts, were derived from both recent photosynthesis and cytosolic carbon sources. We hypothesize that releases of acetaldehyde during light–dark transitions result from a pyruvate overflow mechanism controlled by cytosolic pyruvate levels and pyruvate decarboxylase activity. These results suggest that leaves of woody plants contribute reactive acetaldehyde to the atmosphere under different conditions: (1) metabolic states that promote the accumulation of cytosolic pyruvate, triggering the pyruvate decarboxylase reaction; and (2) leaf ethanol oxidation resulting from ethanol transported from anoxic tissues.     

Ability of Vasconcellea × heilbornii lipase to catalyse the synthesis of alkyl esters from vegetable oils

Lipase-catalysed synthesis of alkyl esters is regarded as a potential alternative to chemical catalysis. Owing to its availability as a waste material from the babaco fruit production, its strong lipolytic activity and its natural immobilization, the dried latex of Vasconcellea × heilbornii appears as a good candidate to produce alkyl esters. The ability and performance of this lipase to catalyse the alcoholysis of sunflower oil with various primary alcohols was evaluated in a solvent-free system. A linear correlation between the final reaction rate and the alcohol polarity was established. For methanolysis, the influence of substrates ratio on final conversion rate was studied at different temperatures. At 30 °C, the lipase was inactivated by shaking in a mixture containing more than 0.5 molar equivalents of methanol; the minimum methanol concentration for enzyme deactivation increased with temperature. Moreover, for a 0.5:1 methanol/TAG molar ratio, conversion rates of 73, 66 and 55% were obtained at 30, 40 and 55 °C respectively, showing that the increase of temperature diminished the final methanolysis conversion rate. These facts were associated to the miscibility of methanol in oil and to the thermodynamic state of the medium. To overcome the inactivation of the lipase by methanol, alcoholysis was carried out by fractionated addition of methanol. In those conditions, Vasconcellea × heilbornii latex could catalyse the conversion of 70% of sunflower TAGs in methyl esters at 30 °C.     

Solid-phase fragment condensation for synthesis of peptides from the immunodominant sequence of β<Subscript>1</Subscript>-adrenoreceptor

The P26 peptide corresponding to the 197–222 sequence of the second extracellular loop of the β₁-adrenoreceptor (β₁-AR) was synthesized by solid-phase fragment condensation on the Wang polymer. Pentapeptide fragments were prepared on the 2-chlorotrityl resin. The racemization degree of the C-terminal alanine residue of the pentapeptide was experimentally evaluated for the synthetic H-Glu-Ser-Asp-Glu-Ala-Arg-OH hexapeptide β₁-АR-(202–207) which was prepared by the 5 + 1 fragment condensation with the use of various condensing agents. A content of the diastereoisomeric peptide in the products of the fragment condensation was determined by HPLC on a reversed phase. The D-alanine-containing hexapeptide was specially synthesized and used for a comparison. The minimum racemization degree of the C-terminal alanine residue was observed if complex F was applied to the synthesis of the hexapeptide.     

Dietary histories of herbivorous loricariid catfishes: evidence from δ<Superscript>13</Superscript>C values of otoliths

The ecology of many Neotropical fishes is difficult or often impossible to study during rainy seasons. Thus, ecological studies of tropical fishes are usually performed on fish captured only during dry seasons. Because otoliths preserve a record of life history, this study evaluated the utility of otolith stable isotope values for the investigation of trophic ecology of Neotropical fishes (specifically herbivorous loricariid catfish) throughout their lives. Because plant dietary materials have δ¹³C values that are determined by their photosynthetic pathways, metabolism and environmental conditions, different plants may impart different isotope values on fish otoliths that reflect consumption of these plants. The δ¹³C_(otolith) values of xylophagous Panaque nigrolineatus captured in the field were significantly lower than those of algivorous Hypostomus regani from a nearby region. A laboratory experiment wherein Hypostomus sp. had δ¹³C_(otolith) values that reflected the δ¹³C values of their plant diet and additional evidence indicate that δ¹³C_(otolith) values in loricariid catfish otoliths can record dietary history.     

Metabolism of Uridine 5′-Diphosphate-Glucose in Golgi Vesicles from Pea Stems

Uridine 5′-diphosphate-glucose (UDP-Glc) is transported into the lumen of the Golgi cisternae, where is used for polysaccharide biosynthesis. When Golgi vesicles were incubated with UDP-[³H]Glc, [³H]Glc was rapidly transferred to endogenous acceptors and UDP-Glc was undetectable in Golgi vesicles. This result indicated that a uridine-containing nucleotide was rapidly formed in the Golgi vesicles. Since little is known about the fate of the nucleotide derived from UDP-Glc, we analyzed the metabolism of the nucleotide moiety of UDP-Glc by incubating Golgi vesicles with [α-³²P]UDP-Glc, [β-³²P]UDP-Glc, and [³H]UDP-Glc and identifying the resulting products. After incubation of Golgi vesicles with these radiolabeled substrates we could detect only uridine 5′-monophosphate (UMP) and inorganic phosphate (Pi). UDP could not be detected, suggesting a rapid hydrolysis of UDP by the Golgi UDPase. The by-products of UDP hydrolysis, UMP and Pi, did not accumulate in the lumen, indicating that they were able to exit the Golgi lumen. The exit of UMP was stimulated by UDP-Glc, suggesting the presence of a putative UDP-Glc/UMP antiporter in the Golgi membrane. However, the exit of Pi was not stimulated by UDP-Glc, suggesting that the exit of Pi occurs via an independent membrane transporter.     

Temporal variation of δ<Superscript>13</Superscript>C of larch leaves from a montane boreal forest in Mongolia

This paper reports the temporal variation (2002–2004) in foliar δ¹³C values, which are indicative of long-term integrated photosynthetic and water use characteristics, of Siberian larch (Larix sibirica Ledeb.) trees in a montane forest at Mongonmorit, NE Mongolia. At the stand, the δ¹³C value for understory shaded leaves was more negative by 2‰ on average than that for sunlit leaves sampled concurrently from open and sun-exposed environments in a forest gap. The δ¹³C value of both sunlit and shaded leaves showed pronounced intra- but relatively small inter-seasonal variations. The δ¹³C value was more positive for juvenile than mature leaves. We conjecture that juvenile leaves may derive carbon reserves in woody tissues (e.g., stems). Regardless of leaf habitats, the δ¹³C value was also affected by insect herbivores occurred in mid summer of 2003, being more negative in newly emerging leaves from the twigs after defoliation than in non-defoliated mature leaves. This pattern seems to contrast with that for the juvenile leaves in the early growing season. We surmise that the newly emerging leaves used stored organic carbon that was depleted due to fractionation during remobilization and translocation for leaf regrowth. There was also intra- and inter-seasonal variation in the foliar N concentrations and C:N ratios. A good positive (negative) correlation between the foliar δ¹³C values and N concentrations (C:N ratios) was also observed for both sunlit and shaded leaves, suggesting that the relationship between water and nitrogen use is a crucial factor affecting the plant carbon–water relationship in this mid latitude forest with a cold semiarid climate. Our isotopic data demonstrate that the larches in NE Mongolia exhibits relatively higher water use efficiency with a distinct within-season variability.     

Ocular Disposition of ∆<Superscript>8</Superscript>-Tetrahydrocannabinol from Various Topical Ophthalmic Formulations

The purposes of this project are to enhance the trans-membrane penetration of Δ⁸-Tetrahydrocannabinol (Δ⁸-THC) and to study the effect of various lipid based systems in delivering the compound, non-invasively, to anterior and posterior ocular chambers. Solid lipid nanoparticles (SLNs), fast gelling films were manufactured using high pressure homogenization and melt cast techniques, respectively. The formulations were characterized for drug content, entrapment efficiency, particle size and subsequently evaluated in vitro for trans-corneal permeation. In vivo, the drug disposition was tested via topical administration in albino rabbits. The eye globes were enucleated at the end of experiment and tissues were analyzed for drug content. All formulations showed favorable physicochemical characteristics in terms of particle size, entrapment efficiency, and drug content. In vitro, the formulations exhibited a transcorneal flux that depended on the formulation’s drug load. An increase in drug load from 0.1 to 0.75% resulted in 12- to16-folds increase in permeation. In vivo, the film was able to deliver THC to all the tissues with high accumulations in cornea and sclera. The SLNs showed a greater ability in delivering THC to all the tissues, at a significantly lower drug load, due to their colloidal size range, which in turn enhanced corneal epithelial membrane penetration. The topical formulations evaluated in the present study were able to successfully deliver Δ⁸-THC in therapeutically meaningful concentrations (EC₅₀ values for CB₁: 6 nM and CB₂: 0.4 nM) to all ocular tissues except the vitreous humor, with pronounced tissue penetration achieved using SLNs as a Δ⁸-THC delivery vehicle.     

Structure of the Mn<Subscript>4</Subscript>–Ca cluster as derived from X-ray diffraction

The catalytic centre for light-induced water oxidation in photosystem II (PSII) is a multinuclear metal cluster containing four manganese and one calcium cations. Knowing the structure of this biological catalyst is of utmost importance for unravelling the mechanism of water oxidation in photosynthesis. In this review we describe the current state of the X-ray structure determination at 3.0 A resolution of the water oxidation complex (WOC) of PSII. The arrangement of metal cations in the cluster, their coordination and protein surroundings are discussed with regard to spectroscopic and mutagenesis studies. Limitations of the presently available structural data are pointed out and possible perspectives for the future are outlined, including the combination of X-ray diffraction and X-ray spectroscopy on single crystals.     

Structure and Expression of a Dhurrinase (β-Glucosidase) from Sorghum

Sorghum (Sorghum bicolor L. Moench) has two isozymes of the cyanogenic β-glucosidase dhurrinase: dhurrinase-1 (Dhr1) and dhurrinase-2 (Dhr2). A nearly full-length cDNA encoding dhurrinase was isolated from 4-d-old etiolated seedlings and sequenced. The cDNA has a 1695-nucleotide-long open reading frame, which codes for a 565-amino acid-long precursor and a 514-amino acid-long mature protein, respectively. Deduced amino acid sequence of the sorghum Dhr showed 70% identity with two maize (Zea mays) β-glucosidase isozymes. Southern-blot data suggested that β-glu-cosidase is encoded by a small multigene family in sorghum. Northern-blot data indicated that the mRNA corresponding to the cloned Dhr cDNA is present at high levels in the node and upper half of the mesocotyl in etiolated seedlings but at low levels in the root—only in the zone of elongation and the tip region. Light-grown seedling parts had lower levels of Dhr mRNA than those of etiolated seedlings. Immunoblot analysis performed using maize-anti-β-glucosidase sera detected two distinct dhurrinases (57 and 62 kD) in sorghum. The distribution of Dhr activity in different plant parts supports the mRNA and immunoreactive protein data, suggesting that the cloned cDNA corresponds to the Dhr1 (57 kD) isozyme and that the dhr1 gene shows organ-specific expression.     

Production of fructooligosaccharides by crude enzyme preparations of β-fructofuranosidase from <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

Fructooligosaccharides (FOS) were produced from sucrose by using crude enzyme preparations of β-fructofuranosidases (FFases) obtained from sucrose-cultured cells of Aureobasidium pullulans DSM 2404. When the preparation mainly consisted of FFase I, that has high transfructosylating activity, the FOS yield was 62%. When the reaction was carried out with additional commercial glucose isomerase (GI) at an activity ratio of FFase and GI of 1:2, the maximum FOS yield reached 69%. This value was higher than those obtained previously using other Aureobasidium spp. (53–59%).     

Plasmodium falciparum Variability and Immune Evasion Proceed from Antigenicity of Consensus Sequences from DBL6ε; Generalization to All DBL from VAR2CSA

We studied all consensus sequences within the four least ‘variable blocks’ (VB) present in the DBL6ε domain of VAR2CSA, the protein involved in the adhesion of infected red blood cells by Plasmodium falciparum that causes the Pregnancy-Associated Malaria (PAM). Characterising consensus sequences with respect to recognition of antibodies and percentage of responders among pregnant women living in areas where P. falciparum is endemic allows the identification of the most antigenic sequences within each VB. When combining these consensus sequences among four serotypes from VB1 or VB5, the most often recognized ones are expected to induce pan-reactive antibodies recognizing VAR2CSA from all plasmodial strains. These sequences are of main interest in the design of an immunogenic molecule. Using a similar approach than for DBL6ε, we studied the five other DBL and the CIDRpam from VAR2CSA, and again identified VB segments with highly conserved consensus sequences. In addition, we identified consensus sequences in other var genes expressed by non-PAM parasites. This finding paves the way for vaccine design against other pathologies caused by P. falciparum.     

Antimicrobial effects of α-defensins from leukocytes of the hamadryas baboon <Emphasis Type="Italic">Papio hamadryas</Emphasis>

To understand the emergence and evolutionary selection of the efficient mechanisms of innate immunity it is necessary to accumulate knowledge about the structural and functional properties of antimicrobial peptides in different animal species. The cationic antimicrobial peptides, α-defensins, were isolated from leukocytic extracts of the lower narrow-nosed monkey, hamadryas baboon Papio hamadryas, using ultrafiltration, preparative electrophoresis and reverse-phase highperformance liquid chromatography. Analysis of the antimicrobial properties of α-defensins showed that they display a wide spectrum of antimicrobial activity, comparable with that of human α-defensin HNP1, and exert bactericidal and fungicidal effects at micromolar concentrations. A study of the influence of different medium conditions on antimicrobial activity of α-defensins revealed that a higher ionic strength or the presence of blood serum leads to a marked decrease in antimicrobial activity of α-defensins, while pH has no appreciable effect on it. We found that hamadryas baboon α-defensins are able to increase the permeability of the outer and inner membranes of E. coli, suggests that the bacterial membrane is one of the major targets of the antimicrobial effects of these peptides. The revealed differences in antimicrobial activity of α-defensins may result from their structural heterogeneity, which reflects different pathways of evolution of α-defensins in primates and underlies the selectivity of their antimicrobial effect.     

Identification of a replicon from pCC3, a cryptic plasmid from <Emphasis Type="Italic">Leuconostoc citreum</Emphasis> C4 derived from kimchi,and development of a new host–vector system

Analysis of the structural properties of pCC3, a cryptic plasmid from Leuconostoc citreum C4 isolated from kimchi, determined its length as 3,338 bp and revealed three open reading frames (ORFs): ORF1–ORF3. ORF3 showed high homology with a replication initiation protein of the theta-type plasmid pTXL1. The fragment encompassing ORF3 and its upstream sequences (nt 1,299–1,634) was found to contain a functional plasmid replicon. A new shuttle vector, pUCC3E1, was constructed based on pCC3. Using Southern hybridization analysis, no single-stranded DNA intermediate was detected from Leu. citreum harboring pUCC3E1, which indicates that pCC3 replicated via the theta mechanism. The pUCC3E1 could be replicated in E. coli TG1 (5.8 × 10⁴ CFU/μg DNA) and the developed cloning hosts, Leu. citreum C16 (2.1 × 10² CFU/μg DNA) and Leu. citreum GJ7 (8.0 × 10¹ CFU/μg DNA). pUCC3E1 was stably maintained in Leu. citreum C16 (for 100 generations, ca. 94.2%) in the absence of erythromycin (5 μg/ml).     

25 years of recombinant proteins from reactor-grown cells - Where do we go from here?

The purpose of this review is to describe the current status and to highlight several emerging trends in the manufacture of recombinant therapeutic proteins in cultivated mammalian cells, focusing on Chinese hamster ovary cells as the major production host. Over the past 25 years, specific and volumetric productivities for recombinant cell lines have increased about 20-fold as the result of improvements in media and bioprocess design. Future yield increases are expected to come from further developments in gene delivery and genetic selection for more efficient recovery of high-producing cell lines and in high-throughput cultivation systems to simplify medium design and bioprocess development. Other emerging trends in protein manufacturing that are discussed include the use of disposal bioreactors and transient gene expression. We specifically highlight current research in our own laboratories.     

Characterization of two β-decarboxylating dehydrogenases from <Emphasis Type="Italic">Sulfolobus acidocaldarius</Emphasis>

Sulfolobus acidocaldarius, a hyperthermoacidophilic archaeon, possesses two β-decarboxylating dehydrogenase genes, saci_0600 and saci_2375, in its genome, which suggests that it uses these enzymes for three similar reactions in lysine biosynthesis through 2-aminoadipate, leucine biosynthesis, and the tricarboxylic acid cycle. To elucidate their roles, these two genes were expressed in Escherichia coli in the present study and their gene products were characterized. Saci_0600 recognized 3-isopropylmalate as a substrate, but exhibited slight and no activity for homoisocitrate and isocitrate, respectively. Saci_2375 exhibited distinct and similar activities for isocitrate and homoisocitrate, but no detectable activity for 3-isopropylmalate. These results suggest that Saci_0600 is a 3-isopropylmalate dehydrogenase for leucine biosynthesis and Saci_2375 is a dual function enzyme serving as isocitrate-homoisocitrate dehydrogenase. The crystal structure of Saci_0600 was determined as a closed-form complex that binds 3-isopropylmalate and Mg²⁺, thereby revealing the structural basis for the extreme thermostability and novel-type recognition of the 3-isopropyl moiety of the substrate.     

Carbenicillin as Inhibitor of β-Lactamase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

CARBENICILLIN was produced as a new, semi-synthetic penicillin with antibacterial activity against Pseudomonas aeruginosa and some other microorganisms¹, but this compound was known to be destroyed by staphylococcal penicillinase². Newsom et al.³ described the substrate profile of a constitutive β-lactamase from one strain of Pseudomonas aeruginosa and reported the hydrolysis of carbenicillin at a rate higher than benzylpenicillin. When compared with the inducible enzyme described by Sabath et al.⁴, it differed both in the substrate profile and the ability to hydrolyse carbenicillin. Lack of activity of the inducible enzyme on carbenicillin was also reported by Garber and Friedman⁵ when studying eight strains of Pseudomonas aeruginosa. Sykes and Richmond⁶ were able to identify three types of β-lactamases among fifty-six strains of Pseudomonas aeruginosa according to induci-bility, substrate profile and activity on carbenicillin. Type I (Sabath et al.⁴) was inducible, highly active on cephaloridine and showed no activity on carbenicillin. Types II (Sykes and Richmond⁷) and III (Newsom et al.³) were constitutive and inactivated carbenicillin at different rates. Only the constitutive enzymes conferred resistance towards carbenicillin. We have investigated the activity on carbenicillin of β-lactamases from strains of Pseudomonas aeruginosa isolated from clinical specimens. Activity on benzylpenicillin and cephaloridine was also studied.