Chromosomal location and cloning of the gene (trmD) responsible for the synthesis of tRNA (m1G) methyltransferase in Escherichia coli K-12

Summary The trmD gene, which governs the formation of 1-methyl-guanosine (m¹G) in transfer ribonucleic acid (tRNA), has been located by phage P1 transduction at 56 min on the chromosomal map of Escherichia coli. Cotransduction to tyrA at 56 min is 80%. From the Clarke and Carbon collection a ColE1-tyrA ⁺ hybrid plasmid was isolated, which carried the trmD ⁺ gene and was shown to over-produce the tRNA (m¹G)methyltransferase. By subcloning restriction enzyme fragments in vitro, the trmD ⁺ gene was located to a 3.4 kb DNA fragment 6.5 kb clockwise from the tyrA ⁺ gene. The mutation trmD1, which renders the tRNA (m¹G) methyltransferase temperaturesensitive both in vivo and in vitro could be complemented by trmD ⁺ plasmids. These results suggest that the gene trmD ⁺ is the structural gene for the tRNA (m¹G)methyltransferase (EC 2.1.1.3.1).     

Scale down of recombinant protein production: a comparative study of scaling performance

A large bioreactor is heterogeneous with respect to concentration gradients of substrates fed to the reactor such as oxygen and growth limiting carbon source. Gradient formation will highly depend on the fluid dynamics and mass transfer capacity of the reactor, especially in the area in which the substrate is added. In this study, some production-scale (12 m³ bioreactor) conditions of a recombinant Escherichia coli process were imitated on a laboratory scale. From the large-scale cultivations, it was shown that locally high concentration of the limiting substrate fed to the process, in this case glucose, existed at the level of the feedpoint. The large-scale process was scaled down from: (i) mixing time experiments performed in the large-scale bioreactor in order to identify and describe the oscillating environment and (ii) identification of two distinct glucose concentration zones in the reactor. An important parameter obtained from mixing time experiments was the residence time in the feed zone of about 10 seconds. The size of the feed zone was estimated to 10%. Based on these observations the scale-down reactor with two compartments was designed. It was composed of one stirred tank reactor and an aerated plug flow reactor, in which the effect of oscillating glucose concentration on biomass yield and acetate formation was studied. Results from these experiments indicated that the lower biomass yield and higher acetate formation obtained on a large scale compared to homogeneous small-scale cultivations were not directly caused by the cell response to the glucose oscillation. This was concluded since no acetate was accumulated during scale-down experiments. An explanation for the differences in results between the two reactor scales may be a secondary effect of high glucose concentration resulting in an increased glucose metabolism causing an oxygen consumption rate locally exceeding the transfer rate. The results from pulse response experiments and glucose concentration measurements, at different locations in the reactor, showed a great consistency for the two feeding/pulse positions used in the large-scale bioreactor. Furthermore, measured periodicity from mixing data agrees well with expected circulation times for each impeller volume. Conclusions are drawn concerning the design of the scale-down reactor.     

Rural indebtedness in North‐Eastern Nepal part I: The ethnography and its implications*

This paper discusses ideas about ‘the nation’ and ‘the people’ expressed by residents of a low‐income district in the city of Salvador, capital of the state of Bahia, Brazil. The analysis relates positive and negative evaluations of national identity to a dialectic of hegemony and resistance. It focuses on gender and race in the local making or negating of nationalism, concluding that constitutive discourses and practices must be understood in relation to social inequality and the class structure.     

Sex-hormone-binding globulin and albumin concentrations in human cerebrospinal fluid

Polyacrylamide gel electrophoresis of plasma and concentrated cerebrospinal fluid (CSF) preincubated with tritium labelled 5 alpha-dihydrotestosterone (DHT) showed identical migration of the radioactivity, indicating the presence of sex-hormone-binding globulin (SHBG) in human CSF. The concentrations of SHBG (measured as the binding capacity) and albumin were measured in concentrated CSF (12 women and 1 man) and samples of plasma of some patients (9 women). SHBG could not be detected in 6 of the CSF samples, and the mean value of the determinable samples was 42.3 +/- 13.4 pmol/l. The mean +/- SE of the SHBG concentration in plasma was 90.8 +/- 8.9 nmol/l and the mean albumin concentrations in CSF and plasma were 3.4 +/- 0.6 mumol/l and 670 +/- 107 mumol/l respectively. The distribution ratio for SHBG over the blood-CSF barrier was 10 times higher than for albumin. It was concluded that the SHBG-binding in the CSF is negligible but that the albumin-binding may contribute to the CSF concentrations of testosterone and estradiol, which are 10-25% above the plasma unbound concentrations.     

Monoacylglycerols Activate TRPV1 – A Link between Phospholipase C and TRPV1

Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H₁ receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H₁ receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous “entourage” compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations. Furthermore, intracerebroventricular injection of JZL184 produced TRPV1-dependent antinociception in the mouse formalin test. Our results show that intact 2-arachidonoylglycerol and 1-arachidonoylglycerol are endogenous TRPV1 activators, contributing to phospholipase C-dependent TRPV1 channel activation and TRPV1-mediated antinociceptive signaling in the brain.     

Identification and design of a novel series of MGAT2 inhibitors

[Acyl CoA]monoacylglycerol acyltransferase 2 (MGAT2) is of interest as a target for therapeutic treatment of diabetes, obesity and other diseases which together constitute the metabolic syndrome. In this Letter we report our discovery and optimisation of a novel series of MGAT2 inhibitors. The development of the SAR of the series and a detailed discussion around some key parameters monitored and addressed during the lead generation phase will be given. The in vivo results from an oral lipid tolerance test (OLTT) using the MGAT2 inhibitor (S)-10, shows a significant reduction (68% inhibition relative to na?ve, p <0.01) in plasma triacylglycerol (TAG) concentration.     

Altered sensitivity of eukaryotic elongation factor 2 for trypsin after phosphorylation and ribosomal binding

We have used variations in the trypsin sensitivity of eukaryotic protein synthesis elongation factor 2 (eEF-2) to probe for structural alterations induced by phosphorylation, ribosomal binding, or guanosine nucleotides. We could not detect any nucleotide-related effect on the tryptic cleavage rate of Arg66. However, eEF-2 was protected from trypsin after ribosomal binding. Also, phosphorylation of eEF-2 led to a protection of Arg66. This indicates that phosphorylation leads to a structural rearrangement that could explain the reduced affinity of the phosphorylated factor for ribosomes (Carlberg, U., Nilsson, A., and Nyg?rd, O. (1990) Eur. J. Biochem. 191, 639-645). Cleavage of Arg66 led to a complete loss of the ability of the factor to be phosphorylated. Furthermore, ribosome-bound eEF-2 was found to be inaccessible for phosphorylation. Based on these findings and previously published data, we suggest that the region around the sites of phosphorylation and trypsin cleavage is vitally important for the factor function and ribosomal binding.     

Genetic evidence that the putative receptor binding domain of apolipoprotein B (residues 3130 to 3630) is not the only region of the protein involved in interaction with the low density lipoprotein receptor

We have searched for sequence differences in the region of the apolipoprotein B (apo B) gene encoding amino acids 3130-3630 in eight individuals with reduced affinity of low density lipoprotein (LDL) for the normal LDL-receptor. All individuals were hypercholesterolaemic and were selected either on the basis of reduced fractional catabolic rate (FCR) of autologous LDL or substantially reduced binding of their LDL to normal LDL-receptors determined by an in vitro cell growth assay using the U937 macrophage-like cell line. Segments of the apo B gene were amplified by the polymerase chain reaction. Using a combination of cloning and sequencing the amplified fragment, together with chemical cleavage mismatch analysis, no sequence differences were identified in this region of the gene. We therefore conclude that variation outside the region of the apo B gene that codes for amino acids 3130-3630 must be responsible for the reduced LDL clearance in these patients.     

Plasmodium falciparum: identification and localization of a knob protein antigen expressed by a cDNA clone

Differential screening of cDNA libraries constructed from knobby and predominantly knobless Plasmodium falciparum isolates, identified the sequence SD17. Chromosome blotting experiments have shown that this sequence, which is located on chromosome 2 of most isolates, was deleted in the cloned parasite line E12 of the FCQ27/PNG isolate. Here we show that erythrocytes infected with the SD17-containing cloned line D10 have typical knob structures on their surfaces, whereas those infected with the line E12 lack knobs. An expression clone was constructed from SD17 and used to affinity purify antibodies from the sera of individuals living in areas of Papua New Guinea where malaria is endemic. The antibodies reacted in immunoblotting experiments with a single polypeptide that varied in Mr from 85,000 to 105,000 among different isolates. The antigen was not expressed in the knobless clone E12. Postembedding immunoelectron microscopy showed localization of the antigen over the knobs of FC27 and two other isolates, largely on the cytoplasmic side. We conclude that the parasite antigen corresponding to clone SD17 is a knob protein.