The sialidase superfamily and its spread by horizontal gene transfer

Sialidases (neuraminidases, EC 3.2.1.18) belong to a class of glycosyl hydrolases that release terminal N-acylneuraminate (slalic acid) residues from glycoproteins, glycolipids, and polysaccharides. These enzymes are common in animals of the deuterostomate lineage (Echinodermata through Mammalia) and also in diverse microorganisms that mostly exist as animal commensals or pathogens. Sialidases, and their sialyl substrates, appear to be absent from plants and most other metazoans. Even among bacteria, sialidase is found irregularly so that related species or even strains of one species differ in this property. This unusual phylogenetic distribution makes sialidases interesting for evolutionary studies. The biochemical diversity among bacterial sialidases does not indicate close relationships. However, at the molecular level, homologies are detectable, supporting the hypothesis of a common sialidase origin and thus of a sialidase super family. Some findings indicate that sialidase genes were recently transferred via phages among bacteria. The proposal of a sialidase origin in higher animals is suggested by the presence of apparently homologous enzymes in this kingdom, supporting the idea that some microbes may have acquired the genetic information during association with their animal hosts.     

Influence of indole carbinols and growth hormone on the metabolism of 4-androstenedione by rat liver microsomes

The effect of indole-3-carbinol (IC), an anticarcinogen present in cruciferous vegetables, to alter the metabolism of 4-androstenedione (AD) by female rat liver microsomes was investigated and compared to that of its main gastric conversion product, diindolylmethane (DIM) as well as other specific cytochrome P450 inducers. DIM was a more potent inducer of the hydroxylase which converts androsterone to its 6β-hydroxylated derivative 3,6β-dihydroxy-5-androstan-17-one (A) than IC after either oral or intraperitoneal administration and was also a better in vitro inhibitor. Isosafrole (ISF), which like IC and DIM, induces CYP1A2 as well as gestodene, were powerful inhibitors of the in vitro reaction. Naringenin produced only a weak inhibitory effect while 3-methylcholanthrene was inactive. SKF-525A, a prototypic hydroxylase inhibitor, or 17β-N,N-diethylcarbamoyl-4-methyl-4-aza-5-androst-1-ene-3-one which inhibits steroid 5-reductase, also decreased the formation of A from AD by liver microsomes. The infusion of human growth hormone by osmotic minipump, which feminizes hepatic steroid metabolism, increased the ability of male rat liver microsomes to convert AD to A and to respond to induction by IC. The identity of A, the main polar derivative of AD, induced by IC, DIM and ISF, was tentatively assigned by a combination of GC-MS and results from metabolic studies with intermediates in the pathway leading to its formation. It is proposed that the protective role of indole carbinols against mammary carcinoma due to decreased formation of 16-hydroxyestrone from estrone may be further enhanced by the diminished availability of AD for aromatization to estrone.     

Real-time monitoring of biomass during Escherichia coli high-cell-density cultivations by in-line photon density wave spectroscopy

An efficient monitoring and control strategy is the basis for a reliable production process. Conventional optical density (OD) measurements involve superpositions of light absorption and scattering, and the results are only given in arbitrary units. In contrast, photon density wave (PDW) spectroscopy is a dilution-free method that allows independent quantification of both effects with defined units. For the first time, PDW spectroscopy was evaluated as a novel optical process analytical technology tool for real-time monitoring of biomass formation in Escherichia coli high-cell-density fed-batch cultivations. Inline PDW measurements were compared to a commercially available inline turbidity probe and with offline measurements of OD and cell dry weight (CDW). An accurate correlation of the reduced PDW scattering coefficient µ_s′ with CDW was observed in the range of 5–69 g L⁻¹ (R² = 0.98). The growth rates calculated based on µ_s′ were comparable to the rates determined with all reference methods. Furthermore, quantification of the reduced PDW scattering coefficient µ_s′ as a function of the absorption coefficient µ_a allowed direct detection of unintended process trends caused by overfeeding and subsequent acetate accumulation. Inline PDW spectroscopy can contribute to more robust bioprocess monitoring and consequently improved process performance.     

Bioprocess development to produce a hyperthermostable S-methyl-5′-thioadenosine phosphorylase in Escherichia coli

Nucleoside phosphorylases are important biocatalysts for the chemo-enzymatic synthesis of nucleosides and their analogs which are, among others, used for the treatment of viral infections or cancer. S-methyl-5′-thioadenosine phosphorylases (MTAP) are a group of nucleoside phosphorylases and the thermostable MTAP of Aeropyrum pernix (ApMTAP) was described to accept a wide range of modified nucleosides as substrates. Therefore, it is an interesting biocatalyst for the synthesis of nucleoside analogs for industrial and therapeutic applications. To date, thermostable nucleoside phosphorylases were produced in shake flask cultivations using complex media. The drawback of this approach is low volumetric protein yields which hamper the wide-spread application of the thermostable nucleoside phosphorylases in large scale. High cell density (HCD) cultivations allow the production of recombinant proteins with high volumetric yields, as final optical densities >100 can be achieved. Therefore, in this study, we developed a suitable protocol for HCD cultivations of ApMTAP. Initially, optimum expression conditions were determined in 24-well plates using a fed-batch medium. Subsequently, HCD cultivations were performed using E. coli BL21-Gold cells, by employing a glucose-limited fed-batch strategy. Comparing different growth rates in stirred-tank bioreactors, cultivations revealed that growth at maximum growth rates until induction resulted in the highest yields of ApMTAP. On a 500-mL scale, final cell dry weights of 87.1–90.1 g L⁻¹ were observed together with an overproduction of ApMTAP in a 1.9%–3.8% ratio of total protein. Compared to initially applied shake flask cultivations with terrific broth (TB) medium the volumetric yield increased by a factor of 136. After the purification of ApMTAP via heat treatment and affinity chromatography, a purity of more than 90% was determined. Activity testing revealed specific activities in the range of 0.21 ± 0.11 (low growth rate) to 3.99 ± 1.02 U mg⁻¹ (growth at maximum growth rate). Hence, growth at maximum growth rate led to both an increased expression of the target protein and an increased specific enzyme activity. This study paves the way towards the application of thermostable nucleoside phosphorylases in industrial applications due to an improved heterologous expression in Escherichia coli.     

Induction of the human growth hormone gene placed under human hsp70 promoter control in mouse cells: A quantitative indicator of metal toxicity

An in vitro test method for general metal toxicity screening was designed, based on the cellular response to stress. The expression of a transfected human growth hormone gene sequence driven by the human heat-shock protein 70 promoter in NIH/3T3 cells was used as marker of noxious contact with metal compounds. Out of a series of31 metals, 17 were competentfor inducing this stress response system. According to the effective concentration and to the intensity of the response, three different clusters of positive compounds emerged and were ranked as strong, intermediate strength and weak inducers. These results correlated well with data from other in vivo and in vitro metal toxicity studies, including LD₅₀ in mice. Apparently the positivelnegative compounds also fitted well with data from genotoxicity and carcinogenesis studies on metal salts.Abbreviations hGH human growth hormone - hsp70 70 kDa heat-shock protein     

Effects of temperature on metabolic rate and lower dissolved oxygen tolerance of juvenile speckled peacock bass Cichla temensis

The speckled peacock bass Cichla temensis is a popular sport and food fish that generates substantial angling tourism and utilitarian harvest within its range. Its popularity and value make this species important for management and a potential aquaculture candidate for both fisheries enhancement and food fish production. However, little is known of optimal physiochemical conditions in natural habitats, which also are important for the development of hatchery protocols for handling, spawning and grow-out. Speckled peacock bass have been documented to have high sensitivity to extreme temperatures, but the metabolic underpinnings have not been evaluated. In this study, the effects of temperature (25, 30 and 35°C) on the standard metabolic rate (SMR) and lower dissolved oxygen tolerance (LDOT) of juvenile speckled peacock bass (mean ± standard error total length 153 ± 2 mm and wet weight 39.09 ± 1.37 g) were evaluated using intermittent respirometers after an acclimation period of 2 weeks. Speckled peacock bass had the highest SMR at 35°C (345.56 ± 19.89 mgO₂ kg⁻¹ h⁻¹), followed by 30°C (208.16 ± 12.45 mgO₂ kg⁻¹ h⁻¹) and 25°C (144.09 ± 10.43 mgO₂ kg⁻¹ h⁻¹). Correspondingly, the Q₁₀, or rate of increase in aerobic metabolic rate (MO₂) relative to 10°C, for 30–35°C was also greater (2.76) than from 25 to 30°C (2.08). Similarly, speckled peacock bass were the most sensitive to hypoxia at the warmest temperature, with an LDOT at pO₂ of 90 mmHg (4.13 mg l⁻¹) at 35°C compared to pO₂ values of 45 mmHg (2.22 mg l⁻¹) and 30 mmHg (1.61 mg l⁻¹) at 30 and 25°C, respectively. These results indicate that speckled peacock bass are sensitive to temperatures near 35°C, therefore we recommend managing and rearing this species at 25–30°C.     

Influences of sea level changes and volcanic eruptions on Holocene vegetation in Tonga

Here, we investigate Mid- to Late-Holocene vegetation changes in low-lying coastal areas in Tonga and how changing sea levels and recurrent volcanic eruptions have influenced vegetation dynamics on four islands of the Tongan archipelago (South Pacific). To investigate past vegetation and environmental change at Ngofe Marsh (‘Uta Vava’u), we examined palynomorphs (pollen and spores), charcoal (fire), and sediment characteristics (volcanic activity) from a 6.7-m-long sediment core. Radiocarbon dating indicated the sediments were deposited over the last 7700 years. We integrated the Ngofe Marsh data with similar previously published data from Avai’o’vuna Swamp on Pangaimotu Island, Lotofoa Swamp on Foa Island, and Finemui Swamp on Ha’afeva Island. Plant taxa were categorized as littoral, mangrove, rainforest, successional/ disturbance, and wetland groups, and linear models were used to examine relationships between vegetation, relative sea level change, and volcanic eruptions (tephra). We found that relative sea level change has impacted vegetation on three of the four islands investigated. Volcanic eruptions were not identified as a driver of vegetation change. Rainforest decline does not appear to be driven by sea level changes or volcanic eruptions. From all sites analyzed, vegetation at Finemui Swamp was most sensitive to changes in relative sea level. While vegetation on low-lying Pacific islands is sensitive to changing sea levels, island characteristics, such as area and elevation, are also likely to be important factors that mediate specific island responses to drivers of change.     

Interaction between the black yeast Aureobasidium pullulans and the gall midge Lasioptera ephedricola in gall formation on the desert shrub Ephedra trifurca

Galls produced by the cecidomynd Lastoptera ephedncola on Ephedia trifurca always have a black ring associated with them while galls produced by the congener L ephedrae never do Black ring material after microscopic examination and culture proved to be Aureobasidium pullulans In addition to lacking black ring material neither L ephedrae galls nor healthy stems consistently yielded Aureobasidium on culture Gall and larva size measurements indicated that continued larval presence is not necessary for gall development, suggesting fungus initiated gall formation However inoculation of healthy stems with Aureobasidium caused lesions hut not galls The mycelium m galls did not appear grazed and neither larvae nor pupae contained Aureobasidium propagules suggesting that larvae do not feed directly on fungi These data also suggest that there is no trans-pupal passage of fungus from larvae or pupae to adults Newly emerged females do not carry fungal propagules suggesting that thcy are not inoculated upon exiting the gall Gall position leaf culture and stem culture data suggest that the fungus is picked up from leaves prior to oviposition     

HPLC separation of the enantiomers of nicotine and nicotine-like compounds

A sensitive and reproducible HPLC method utilizing a commercially available chiral α₁-acid glycoprotein (AGP) phase has been developed to separate and quantify the enantiomers of nicotine. The method is suitable for routine use as indicated by column life. The quantification of (R/S:0.05/99.95)-nicotine or (R/S:99/1)-nicotine was possible. In addition, the separation or at least partial separation of the enantiomers of nornicotine and nornicotine-derived compounds was achieved. © 1993 Wiley-Liss, Inc.     

A 4D HCC(CO)NNH experiment for the correlation of aliphatic side-chain and backbone resonances in 13C/15N-labelled proteins

Summary We recently proposed a novel four-dimensional (4D) NMR strategy for the assignment of backbone nuclei in spectra of ¹³C/¹⁵N-labelled proteins (Boucher et al. (1992) J. Am. Chem. Soc., 114, 2262–2264 and J. Biomol. NMR, 2, 631–637). In this paper we extend this approach with a new constant time 4D HCC(CO)NNH experiment that also correlates the chemical shifts of the aliphatic sidechain (¹H and ¹³C) and backbone (¹H, ¹³C and ¹⁵N) nuclei. It separates the sidechain resonances, which may heavily overlap in spectra of proteins with large numbers of similar residues, according to the backbone nitrogen and amide proton chemical shifts. When used in conjunction with a 4D HCANNH or HNCAHA experiment it allows, in principle, complete assignment of aliphatic sidechain and backbone resonances with just two 4D NMR experiments.