Biosynthesis and Molecular Genetics of Clavulanic Acid

The biosynthesis of clavulanic acid and related clavam metabolites is only now being elucidated. Understanding of this pathway has resulted from a combination of both biochemical studies of purified biosynthetic enzymes, and molecular genetic studies of the genes encoding these enzymes. Clavulanic acid biosynthesis has been most thoroughly investigated in Streptomyces clavuligerus where the biosynthetic gene cluster resides immediately adjacent to the cluster of cephamycin biosynthetic genes. A minimum of eight structural genes have been implicated in clavulanic acid biosynthesis, although more are probably involved. While details of the early and late steps of the pathway remain unclear, synthesis proceeds from arginine and pyruvate, as the most likely primary metabolic precursors, through the monocyclic -lactam intermediate, proclavaminic acid, to the bicyclic intermediate, clavaminic acid, which is a branch point leading either to clavulanic acid or the other clavams. Conversion of clavaminic acid to clavulanic acid requires side chain modfication as well as inversion of ring stereochemistry. This stereochemical change occurs coincident with acquisition of the -lactamase inhibitory activity which gives clavulanic acid its therapeutic and commercial importance. In contrast, the other clavam metabolites all arise from clavaminic acid with retention of configuration and lack -lactamase inhibitory activity.     

cDNA cloning and expression of a potato (Solanum tuberosum) invertase

A cDNA clone encoding an invertase isoenzyme has been isolated from a potato leaf cDNA library. The deduced amino acid sequence shows significant similarities to previously characterised invertases. The highest degree of overall similarity, including the signal peptide sequence, is to carrot cell wall invertase, suggesting that the potato gene encodes an apoplastic enzyme. Expression of the gene, as determined by RT-PCR, is detected in stem and leaf tissue, and at lower levels in tuber, but is absent from roots.     

Secondary structural changes in the intact and the disulfide bridges cleaved beta-lactoglobulin A and B in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate

The relative proportions of -helix, -sheet, and unordered form in -lactoglobulin A and B were examined in solutions of urea, guanidine, and sodium dodecyl sulfate (SDS). In the curve-fitting method of circular dichroism (CD) spectra, the reference spectra of the corresponding structures determined by Chen et al. (1974) were modified essentially according to the secondary structure of -lactoglobulin B predicted by Creamer et al. (1983), i.e., that the protein has 17% -helix and 41% -sheet. The two variants showed no appreciable difference in structural changes. The reduction of disulfide bridges in the proteins increased -sheet up to 48% but did not affect the -helical proportion. The -helical proportions of nonreduced -lactoglobulin A and B were not affected below 2 M guanidine or below 3 M urea, but those of the reduced proteins began to decrease in much lower concentrations of these denaturants. By contrast, the -helical proportions of the nonreduced and reduced proteins increased to 40–44% in SDS. The -sheet proportions of both nonreduced and reduced proteins, which remained unaffected even in 6 M guanidine and 9 M urea, decreased to 24–25% in SDS.     

Expression of one of the members of the Arabidopsis chaperonin 60β gene family is developmentally regulated and wound-repressible

To study the pattern of gene regulation of the plastid chaperonin 60 gene family a chimaeric gene was constructed fusing the 5-flanking region of the chaperonin 60 B3 gene to the -glucuronidase reporter gene. Histochemical and fluorometric analysis of the GUS activity present in transgenic plants harbouring this gene construct showed that the B3 promoter is expressed in leaves, stem, petioles and several flower tissues. The pattern of cell type-specific expression in stems and flowers was found to be developmentally regulated. Expression of the B3 promoter was found not to be heat-inducible, but highly repressed by wounding. The rapid decay in GUS activity upon wounding indicates that, at least under some physiological conditions, the gene product of this reporter gene is not as stable as has been previously thought.     

The wise use of wetlands: identifying development options for Ghana's coastal Ramsar sites

The Government of Ghana has recently declared five coastal wetlands to be Ramsar sites. This requires the wise use of wetlands of international importance for bird habitat, but does not preclude human habitation or ecologically friendly development. The sites are Muni-Pomadze, Densu Delta, Sakumo, Songor and Keta. An attempt was made to identify and assess a range of development options for initiation within the sites that would be compatible with the environmental concerns of Ramsar, while also being technically and economically viable and socially acceptable to the communities in the sites. The appraisal included developing a detailed understanding of the physical, biological, human and institutional resources of the areas, the land uses and prevailing livelihood systems of local people, and the pressures placed on the natural resources of the wetlands. Many of the developments nominated by stakeholders were rejected, but twenty topics passed the initial tests. These were considered in more detail using information collected for the study by national specialists. Most of the options were considered suitable for local level implementation by community groups or local entrepreneurs. Government assistance will be needed to ensure an enabling institutional framework for small-scale business development is provided.     

The air-blood barrier in the human lung

Summary The air-blood barrier was studied in replicas of freeze-fractured lung biopsies collected from healthy human subjects. Adjacent pneumocytes display a belt-like network composed of 3–7 superimposed ridges (fibrils) on the P face and complementary grooves on the E face, i.e., a structure corresponding to a tight junction. On the other hand, adjacent capillary endothelial cells show a continuous system of 2–4 membrane foldings. These appear mainly as smooth surfaced crests on the P face; on the E face furrows are seen, at the bottom of which a row of particles is situated. This arrangement suggests a leaky type of junction. Discontinuous occluding junctions are located in the pericytic venular segment of the alveolar vessels. The present findings are in agreement with previous physiological and ultrastructural tracer studies locating the main part of the diffusion barrier for small polar solutes and proteins in the alveolar epithelium. Communicating junctions are demonstrated between type I and type II pneumocytes, indicating intercellular cooperation between these cells of common embryonic origin, but which fulfill different functions in the adult. In the endothelium of the non-muscular alveolar vessels communicating junctions are lacking. Desmosomes occur in the epithelium between type I and type II pneumocytes; square arrays of particles characterize the plasma membrane of type I pneumocytes.A portion of this work was presented in partial fulfillment for the degree of Dr. med., Hannover Medical School     

A genetic component in human lysosomal enzyme excretion

Acid hydrolases are present in normal human urine in appreciable amounts. Their source appears to be lysosomes released by kidney proximal tubule epithelial cells. For a given lysosomal enzyme the total amount excreted is the product of two parameters, a general one describing the rate of lysosome secretion and a specific one describing the relative concentration of that enzyme in lysosomes. There is considerable population variation in both parameters. Studies of -glucuronidase, -galactosidase, -hexosaminidase, and -galactosidase in monozygotic and dizygotic twins show that an appreciable part of this variation is genetic in origin. This appears to be true for both total enzyme excretion and lysosome composition. Although it was not possible to test directly whether this is also true for the rate of lysosome secretion, the fact that the two former parameters are both heritable strongly suggests that the rate of lysosome excretion is also a heritable trait. Taken together with previous data, the results suggest polygenic control of these biochemical traits. It is particularly significant that -glucuronidase excretion in normal individuals is a heritable trait since the excretion of this enzyme has frequently been used as a measure of normal and pathological physiological changes.This study was supported by grants from the National Institutes of Health (GM-19521) and the Council for Tobacco Research—U.S.A., Inc. (1080). The work was done while the authors were in the Department of Molecular Biology, Roswell Park Memorial Institute, Buffalo, New York 14263.     

Neurohormonal regulation of calcium in the cell

Neurohormone C (NC) is a glycopeptide isolated from bovine hypothalamus, which inhibits Ca-calmodulin (CaM)-dependent cAMP and cGMP phosphodiesterase (PDE) and is a regulator of Ca in the cell. Distribution of [⁴⁵Ca]CaCl₂ in the mitochondria and reticulum (SR) of heart and brain mitochondria and changes of Ca-binding proteins in these organelles under NC influence have been studied in the myocardium before and after isoproterenol-induced necrosis. Intraperitoneal administration of 80–100 mU of PDE inhibitory activity of NC to rats did not cause any noticeable changes in the protein content of intracellular organelles, but altered the affinity of certain proteins to⁴⁵Ca²⁺. This property of NC was especially noticable after isoproterenol necrosis. Necrotic injury of the myocardium induced Ca²⁺ storage in the mitochondria and SR of brain, and decreased the Ca²⁺ concentration in myocardial mitochondria. NC injection to the animals with necrosis was followed by Ca²⁺ release from all the studied organelles.     

Survey and analysis of commercial cellulase preparations suitable for biomass conversion to ethanol

Commercial cellulase enzymes have been used in the food, detergent, and textile industries, and are potentially effective for processing biomass feedstocks. A survey was undertaken to identify major manufacturers/distributors of cellulases in the USA and to evaluate 13 representative commercial preparations for enzyme activity, protein concentration, and chemical composition. Samples were subjected to activity measurements using filter paper, carboxymethylcellulose, cellobiose, and p-nitrophenyl--d-glucopyranoside as substrates. To ascertain the microbial origin of the commercial preparations, Western blots utilizing monoclonal antibodies specific for Trichoderma reesei CBH I and Aspergillus niger -d-glucosidase were developed. Eleven of the cellulases tested were of T. reesei or T. viride origin and two were from A. niger.     

Effects of lactation on the regulation of hepatic metabolism in the rat and sheep: adrenergic receptors and cyclic AMP responses

The number and coupling efficiency of -adrenoceptors in liver membranes and intact hepatocytes of lactating and non-lactating female rats were compared to assess whether or not alterations in this signalling system could contribute towards the changed pattern of hepatic metabolism during lactation. In view of the different adaptations of hepatic metabolism to lactation in ruminants, the adrenergic receptor profile of sheep liver membranes was also determined. Post-receptor responses at two stages down-stream of cyclic AMP generation were also evaluated in rat hepatocytes in response to the -adrenergic agonist isoprenaline. No changes in the number or affinity of hepatic -adrenoceptors were found in sheep or rats when lactating and non-lactating individuals were compared. Sheep liver was found to have a much greater concentration of -adrenoceptors than rat liver, and a much higher ratio of :₁. The sensitivity and responsiveness of cyclic AMP generation in response to isoprenaline were similar in hepatocytes prepared from lactating and non-lactating rats, although the response to saturating concentrations of glucagon was diminished in hepatocytes from lactating rats. The activity ratio of cyclic AMP-dependent protein kinase (PK-A) also reacted similarly (in respect of both responsiveness and sensitivity) to isoprenaline in these two groups of hepatocytes. Contrastingly, the sensitivity of rat hepatocyte phosphorylase activity to -adrenergic stimulation was greatly diminished during lactation.     

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.     

Activity and stability of Escherichia coli β-galactosidase in cosolvent systems

The kinetic parameters of E.coli -galactosidase were not altered by the addition of 2-propanol or ethyl acetate (1.6% v/v). While ethylene glycol (1.6% v/v) doubled the values of both KM (0.29 mM) and kcat (1393 s–), tetraethyleneglycol-dimethylether (Tetraglyme,1.6% v/v) preserved KM, but decreased kcat. At 50°C all the cosolvents dramatically shortened the enzymatic half life, and so did Tetraglyme and 2-propanol at 28°C. At 28°C, both ethyl acetate and ethylene glycol stabilised the enzyme 9- and 6-fold respectively. This fact, together with the activation effect of ethylene glycol may lead to practical applications. © Rapid Science Ltd. 1998     

Tail-to-tail orientation of the Atlantic salmon alpha- and beta-globin genes

We report the cloning of a cDNA and two corresponding -globin genes of the Atlantic salmon (Salmo salar L.) as well as two genes for -globins. Nucleotide sequence analysis of the cDNA shows that the predicted -globin peptide comprises 148 amino acids with a calculated molecular mass of 16,127 Da and an overall amino acid similarity of 40–50% to higher vertebrates and 60–90% to fish sequences. The study of the genomic organization of - and -globin genes shows that, as is the case in Xenopus, the salmon genes are adjacent. Two sets of linked - and -globin genes were isolated and restriction-enzyme polymorphisms indicate that they belong to two distinct loci, possibly as a result of the salmon tetraploidy. In each locus the - and -globin genes are oriented 3 to 3 relative to each other with the RNA coding sequences located on opposite DNA strands. This is the first evidence for this type of arrangement found for globin genes. Moreover, while the linkage found in salmon and Xenopus supports the hypothesis of an initial tandem duplication of a globin ancestor gene, our results raise the question of the actual original orientation of the duplicated genes. Correspondence to: F. Gannon     

Copy Number Related Transgene Expression and Mosaic Somatic Expression in Hemizygous and Homozygous Transgenic Tilapia (Oreochromis Niloticus)

Three lines of transgenic tilapia (Oreochromisniloticus) fish were generated with a constructcontaining a lacZ reporter gene spliced to a 4.7kb 5 regulatory region of a carp beta actin gene. All these three lines contain different copy numbers oftransgenes and the levels of lacZ expressionwere found to be related to transgene copy number.Mosaic patterns of somatic lacZ expression wereobserved in these three lines which differed between linesbut were consistent within a line. We also observedthat expression of the reporter gene in homozygoustransgenic fish was approximately two-fold greater thanin the hemizygous transgenics. Analysis of expressionof the reporter gene on a tissue-to-tissue basisdemonstrated that lacZ expression of thereporter gene in stably transformed fish occured withvariable intensity in different organs and tissues andwas also sometimes variable in different cells of thesame tissue in G1and G2 generations of the transgenic lines.     

Biochemical and immunological responses of hairy cell leukemia patients to interferonβ

Summary Ten hairy-cell leukemia patients were treated with interferon (IFN-) at a dose rate of 2 × 10⁶ IU/m² × 5 days for 4 weeks (induction therapy) and, thereafter, at the same dose three times a week for 11 months (maintenance therapy). The effect of this treatment on serum neopterin, 2-microglobulin, (2–5)oligoadenylate [(2–5)A _n ] levels, intracellular (2–5)A _n values and human Mx protein synthesis was analysed. There were significant rises in serum neopterin and (2–5)A _n levels during both induction and maintenance, whereas 2-microglobulin levels rose only during induction. Rises in intracellular (2–5)A _n were documented mainly during induction, but they were not significantly higher than pretherapy values. IFN provoked an increase in human Mx protein synthesis over the entire induction — maintenance period, but was only significantly higher than baseline during induction. All markers proved useful for monitoring the effects of IFN dose schedules, but were not predictive of clinical outcome. Natural killer activity and IFN production, which were initially defective, followed a different trend from that of the other factors studied, in that increases were documented only late in the course of therapy when the disease was already in remission.     

The use of norbornene derivatives in the synthesis of conformationally constrained peptides and pseudo-peptides

The desymmetrisation of endo-norborn-5-ene-2,3-dicarboxylic anhydride by proline esters has been used to prepare conformationally constrained pseudo-peptides with two peptide chains parallel to one another. A Curtius rearrangement on the desymmetrisation adduct produced the corresponding isocyanate which was used to prepare both a peptide incorporating an endo-2-amino-3-carboxy-norborn-5-ene unit, and a pseudo-peptide with two peptide chains parallel to one another but offset by the presence of a urea unit. The conformational analysis of the resulting peptides was carried out, and the norbornene unit was found to induce the formation of -turns and parallel -sheets.     

Interactions between esterified whey proteins (alpha-lactalbumin and beta-lactoglobulin) and DNA studied by differential spectroscopy

Spectroscopic study of interactions between esterified whey proteins and nucleic acids, at neutral pH, showed positive differential spectra over a range of wavelength between 210 and 340 nm. In contrast, native forms of whey proteins added to DNA did not produce any differential spectra. The positive difference in UV absorption was observed after addition of amounts of proteins as low as 138 molar ratio (MR) of protein/DNA, indicating high sensitivity of the applied method to detect interactions between basic proteins and DNA. UV-absorption differences increased with MR of added whey protein up to saturation. The saturation points were reached at relatively lower MR in the case of methylated forms of the esterified protein as compared to its ethylated form. Saturation of nucleic acid (2996 bp long) was achieved using 850 and 1100 MR of methylated -lactoglobulin and of methylated -lactalbumin, respectively. Saturation with ethylated forms of the proteins was reached at MR of 3160 and 2750. Lysozyme, a native basic protein, showed a behavior similar to what was observed in the case of methylated forms of the dairy proteins studied. However, in the case of lysozyme, saturation was achieved at relatively lower MR (700). Methylated -casein failed to give positive spectra at pH 7 in the presence of DNA. It interacted with DNA only when the pH of the medium was lowered to 6.5, below its pI. Generally, amounts of proteins needed to saturate nucleic acid were much higher than those needed to neutralize it only electrostatically, demonstrating the presence on DNA of protein-binding sites other than the negative charges on the sugar-phosphate DNA backbones. Addition of 0.1% SDS to the medium suppressed totally all spectral differences between 210–340 nm. The presence of 5 M urea in the medium reduced only the spectral differences between 210–340 nm, pointing to the role played by hydrophobic interactions. Peptic hydrolysates of esterified and native proteins or their cationic fractions (pH > 7) produced negative differential spectra when mixed with DNA. The negative differences in UV absorption spectra were the most important in the case of peptic hydrolysates of methylated derivatives of whey proteins.     

Enzymic synthesis and hydrolytic resolution of alkyl β-d-glucopyranosides

Extracellular -glucosidases from Aspergillus oryzae (two strains), Fusarium oxysporum, Aspergillus terreus and Talaromyces flavus were either induced by cellobiose or 6-deoxyglucose, or produced without induction, i.e., either on casein acid hydrolyzate or on the Sabouraud medium. They were tested to catalyze a stereoselective synthesis of the cis- and trans-isomers of 2-(4-methoxybenzyl)-1-cyclohexyl -d-glucopyranosides with either d-glucose or phenyl -d-glucopyranoside as sources of the glucosyl unit. The enzymes also hydrolyzed (resolved) diastereoisomeric mixtures of alkyl -d-glucopyranosides, which were prepared by the Koenigs–Knorr method from the respective cis- and trans-isomers of 2-(4-methoxybenzyl)-1-cyclohexanol.