IV. Determination of aromatic L-amino acid decarboxylase in serum of various animals by high-performance liquid chromatography with electrochemical detection

This paper describes the distribution of aromatic L-amino acid decarboxylase (AADC), using both L-DOPA and L-5-hydroxytryptophan (L-5-HTP) as substrates, in serum of various animals. The ratio of the activities of the enzyme towards both substrates was also determined in the same serum. AADC activity was discovered in serum using our new and highly sensitive assay for AADC activity by high-performance liquid chromatography (HPLC) with electrochemical detection (ED) with L-DOPA and L-5-HTP as substrates. It was found that among the species used, guinea pig serum had the highest activity, and we made a systematic study on guinea pig serum AADC.     

Topology-informed strategies for the overexpression and purification of membrane proteins

Membrane proteins represent a significant fraction of all genomes and play key roles in many aspects of biology, but their structural analysis has been hampered by difficulties in large-scale production and crystallisation. To overcome the first of these hurdles, we present here a systematic approach for expression and affinity-tagging which takes into account transmembrane topology. Using a set of bacterial transporters with known topologies, we tested the efficacy of a panel of conventional and Gateway? recombinational cloning vectors designed for protein expression under the control of the tac promoter, and for the addition of differing N- and C-terminal affinity tags. For transporters in which both termini are cytoplasmic, C-terminal oligohistidine tagging by recombinational cloning typically yielded functional protein at levels equivalent to or greater than those achieved by conventional cloning. In contrast, it was not effective for examples of the substantial minority of proteins that have one or both termini located on the periplasmic side of the membrane, possibly because of impairment of membrane insertion by the tag and/or att-site-encoded sequences. However, fusion either of an oligohistidine tag to cytoplasmic (but not periplasmic) termini, or of a Strep-tag II peptide to periplasmic termini using conventional cloning vectors did not interfere with membrane insertion, enabling high-level expression of such proteins. In conjunction with use of a C-terminal Lumio? fluorescence tag, which we found to be compatible with both periplasmic and cytoplasmic locations, these findings offer a system for strategic planning of construct design for high throughput expression of membrane proteins for structural genomics projects.     

Comparative study of the gastrocnemius and soleus muscles of the Egyptian desert rodent Gerbillus pyramidum and the albino rat.

The hind limb muscles of the Egyptian desert rodent Gerbillus pyramidum and the albino rat were studied macroscopically and microscopically, using ordinary and histochemical techniques. The results revealed a relatively long hind limb and a relatively long tendon of the gastrocnemius group in G. pyramidum as compared with the albino rat. The dark fibres (red) were dominant in the muscles of the former animal and its spindles similarly contained more dense intrafusal fibres than the spindles of the albino rat. The correlation between these findings and the frequency and power of movement of the hind limbs in G. pyramidum was discussed.     

Structure and Antibacterial Activity of Brominated Diterpenes from the Red Alga Sphaerococcus coronopifolius

Four new tetracyclic brominated diterpenes, 1 – 4 , were isolated from the organic extract of Sphaerococcus coronopifolius, collected from the rocky coasts of Corfu Island. The structures of the new natural products, as well as their relative configurations, were elucidated on the basis of extensive spectral analyses, including 2D‐NMR experiments. The isolated metabolites were evaluated for their antibacterial activity against a panel of bacteria including multidrug‐resistant (MDR) and methicillin‐resistant Staphylococcus aureus (MRSA) with MIC values in the range of 16–128 μg/ml.     

Circulating Lipids Are Associated with Alcoholic Liver Cirrhosis and Represent Potential Biomarkers for Risk Assessment

Liver disease is the greatest cause of death related to alcohol and a major public health problem. While excessive alcohol intake results in hepatosteatosis in most individuals, this can progress in some to more severe forms of liver disease including fibrosis and cirrhosis. An ongoing challenge in the management of alcoholic liver disease is the identification of liver injury early in the disease process such that intervention strategies can prevent serious long term outcomes. Given that excessive alcohol consumption results in dysregulation of lipid metabolism we applied lipid profiling technology to characterise and compare serum lipid profiles from excessive chronic drinkers with no liver disease to those with advanced alcoholic cirrhosis. In a cohort of 59 excessive drinkers (31 with liver cirrhosis and 28 with no evidence of liver disease) we used electrospray ionisation tandem mass spectrometry to measure over 300 individual lipid species in serum, including species of the major phospholipid, sphingolipid, glycerolipid and sterol classes. Six of the 25 lipid classes and subclasses were significantly associated with alcoholic liver cirrhosis; these included dihexosylceramide, trihexosylceramide, alkylphosphatidylcholine, lysoalkylphosphatidylcholine, phosphatidylinositol and free cholesterol. Multivariate classification models created with only clinical characteristics gave an optimal model with an AUC of 0.847 and an accuracy of 79.7%. The addition of lipid measurements to the clinical characteristics resulted in models of improved performance with an AUC of 0.892 and accuracy of 81.8%. The gain in AUC and accuracy of the combined models highlight the potential of serum lipids as markers of liver injury in alcoholic liver disease.     

The Impact of Tissue Morphology,Cross-Section and Turgor Pressure on the Mechanical Properties of the Leaf Petiole in Plants

This paper describes a study of petiole structural morphology in which tissue materials, cross-sectional geometry, layer-architecture and hydrostatic condition are variables that affect the overall structural properties of the organ. Philodendron melinonii is selected as a model species for characterizing the mechanical properties of the petiole. The shape of the petiole is modeled through the polar parameterization of the Lame's curves, i.e. Gielis formulation. A multiscale model of bending stiffness is proposed to capture the impact of changing the constituent tissues and the cross-sectional geometry. Stiffness and density of different tissues are used to plot the domain bounded by the limiting curve of the respective tissue material. Shape parameters and the respective tissue properties are used to generate structural efficiency maps displaying property domains within which fall all possible combinations of tissues that are shaped into a certain geometry during growth. The turgor pressure is also taken into account to show how the domain of the effective material properties changes with water content. Finite element analysis besides experimental data is used to validate the theoretical results. The maps may offer a source of inspiration for biomimetic design, as they help to gain insight into the efficiency of biological beams described by different tissues properties, geometry and turgidity.     

Gene cloning, sequencing and enzymatic properties of glutamate synthase from the hyperthermophilic archaeon Pyrococcus sp. KOD1

The gltA gene encoding a glutamate synthase (GOGAT) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned as a 6.6?kb HindIII-BamHI fragment. Sequence analysis indicates that gltA encodes a 481- amino acid protein (53?269?Da). The deduced amino acid sequence of KOD1-GltA includes conserved regions that are found in the small subunits of bacterial GOGAT: two cysteine clusters, an adenylate-binding consensus sequence and an FAD-binding consensus sequence. However, no sequences homologous to the large subunit of bacterial GOGAT were found in the upstream or downstream regions. In order to examine whether GltA alone can act as a functional GOGAT, GltA was overexpressed in Escherichia coli BL21 (DE3) cells using an expression plasmid. GltA was purified to homogeneity and shown to be functional as a homotetramer of approximately 205?kDa, which is equivalent to the molecular weight of the native GOGAT from KOD1, thus indicating that KOD1-GOGAT is the smallest known active GOGAT. GltA is capable of both glutamine-dependent and ammonia-dependent synthesis of glutamate. Synthesis of glutamate by KOD1-GltA required NADPH, indicating that this enzyme is an NADPH-GOGAT (EC 1.4.1.13). The optimum pH for both activities was 6.5. However, GltA exhibited different optimum temperatures for activity depending on the reaction assayed (glutamine-dependent reaction, 80°?C; ammonia-dependent reaction, 90°?C).