Anthranilate synthase and chorismate mutase activities in transgenic tobacco plants overexpressing tryptophan decarboxylase fromCatharanthus roseus

TransgenicNicotiana tabacum L. Petit Havana SR1 F₁-plants expressing tryptophan decarboxylase cDNA (tdc) fromCatharanthus roseus (L.) G. Don under the control of the CaMV 35S promoter and terminator exhibited tryptophan decarboxylase (TDC) enzyme activity and accumulated tryptamine. The plants with the highest TDC activity contained 19 pkat per mg of protein. The influence of transgenic expression oftdc on the activities of anthranilate synthase (AS) and chorismate mutase (CM) were examined in 10 transgenic tobacco plants. The specific activities of these two chorismate-utilizing enzymes were not significantly affected by expression oftdc, despite their important functions as branch point enzymes in the shikimate pathway. The results indicate that the normal route of tryptophan biosynthesis in plants is sufficient to supply a considerable amount of this essential amino acid for the biosynthesis of secondary metabolites. Despite their increased tryptamine content, the growth and development of the transgenic tobacco plants expressingtdc appeared normal.     

Expressed sequence tags from roots and nodule primordia of Lotus japonicus infected with Mesorhizobium loti

Messenger RNA from young Lotus japonicus roots carrying root nodule primordia appearing after inoculation with Mesorhizobium loti bacteria were used to construct a cDNA expression library. Single-pass sequencing employing colony-polymerase chain reaction (PCR) and analysis of PCR products established a total of 2,397 new expressed sequence tags (ESTs). We have putatively identified 1,236 known and 484 hypothetical proteins coded by the corresponding mRNAs. The remaining cDNAs are unknown (316) or redundant overlapping cDNAs (361). We hope that this batch of ESTs will assist in the recognition of plant genes involved during development of nitrogen-fixing root nodules.     

Refinement of the three-dimensional solution structure of barley serine proteinase inhibitor 2 and comparison with the structures in crystals.

The three-dimensional structure of barley serine proteinase inhibitor, CI-2, has been determined using nuclear magnetic resonance spectroscopy. The present structure determination is a refinement of the structure previously determined by us, using in the present case stereo-specific assignments, and a virtually complete set of assignments of the two-dimensional nuclear Overhauser spectrum. The structure determination is based on the identification of more than 1300 nuclear Overhauser effects, of which 961 were used in the structure calculation as distance restraints, and on 94 dihedral angle restraints, of which 31 are for chi 1 angles in defined chiral centers. These have been used to calculate a series of 20 three-dimensional structures using a combination of distance geometry, simulated annealing and restrained molecular dynamics. Each of the 20 structures was in agreement within less than 0.5 A of each of the distance restraints and with all dihedral angle restraints. When compared to the geometric average structure of the 20 refined structures the root-mean-square differences for the backbone atoms were 0.8 (+/- 0.2) A and for all atoms were 1.6 (+/- 0.2) A. By comparison, the values obtained for the structures determined previously were 1.4 (+/- 0.2) A and 2.1 (+/- 0.1) A, respectively. The structures were also compared to the structure determined in the crystalline state by X-ray diffraction showing root-mean-square differences of 1.6 (+/- 0.2) A and 2.8 (+/- 0.2) A for the backbone and all atoms, respectively. Common features of the solution structure and the two crystal structures are the four-stranded beta-structure, composed of a pair of parallel strands, and three pairs of antiparallel beta-strands flanked on one side by a 12-residue alpha-helix and on the other side by a loop containing the serine proteinase binding site. The new analysis of the structure has revealed an additional pair of antiparallel beta-strands, consisting of residues 65 to 67 and 81 to 83, that was not seen in either of the crystal structures or the previous solution structure. Identification of this was based on nuclear magnetic resonance evidence for the hydrogen bond (67HN to 81CO) not reported previously. Also the presence of a bifurcated hydrogen bond involving Phe69 CO and HN atoms of Ala77 and Gln78 was observed in solution but not in crystals. Minor differences between the two structures were observed in the phi-angles of residues Met59 and Glu60 in the inhibitory site.     

Carboxylesterase 1 Gene Duplication and mRNA Expression in Adipose Tissue Are Linked to Obesity and Metabolic Function

Context and Aims

Carboxylesterase 1 (CES1) appears to play an important role in the control of the metabolism of triglycerides and cholesterol in adipocytes and other cell types including hepatocytes. Therefore, it is relevant to gain insights into the genetic versus non-genetic mechanisms involved in the control of CES1 mRNA expression. Here, we investigated CES1 mRNA expression level in adipose tissue and its association with measures of adiposity and metabolic function in a population of elderly twins. Furthermore, the heritability of CES1 mRNA expression level in adipose tissue and the effect of CES1 gene duplication were assessed.

Methodology

A total of 295 monozygotic and dizygotic twin subjects (62–83 years) with (n = 48) or without (n = 247) type 2 diabetes mellitus were enrolled in the study. They were subjected to a standard oral glucose tolerance test and excision of abdominal subcutaneous fat biopsies during the fasting state. Levels of CES1 mRNA and copy number of the gene were assessed by quantitative PCR.

Results

CES1 mRNA expression level in adipose tissue was positively associated with body-mass index (P<0.001), homeostasis model assessment-insulin resistance (P = 0.003) and level of fasting glucose (P = 0.002), insulin (P = 0.006), and triglycerides (P = 0.003). The heritability for the expression of CES1 mRNA in adipose tissue was high. CES1 gene duplication was positively associated with insulin sensitivity (P = 0.05) as well as glucose tolerance (P = 0.03) and negatively associated with homeostasis model assessment-insulin resistance (P = 0.02). Duplication of CES1 was not linked to mRNA level of this gene (P = 0.63).

Conclusion

CES1 mRNA in adipose tissue appears to be under strong genetic control and was associated with measures of metabolic function raising the possibility of a potential role of this enzyme in the development of type 2 diabetes mellitus. Further studies are needed to understand the potential effect of CES1 gene duplication on adipocyte and whole-body metabolic functions.     

Coexpression of Notch3 and Rgs5 in the pericyte-vascular smooth muscle cell axis in response to pulp injury

Recent studies have shown that the pulp of human teeth contains a population of cells with stem cell properties and it has been suggested that these cells originate from pericytes. Molecules of the Notch signaling pathway regulate stem cell fate specification, while Rgs5 represents an excellent marker for pericytes. Pathological conditions such as dental trauma and carious lesion stimulate pulp stem cells to elaborate reparative dentin. Previous studies have shown that genes involved in the Notch pathway are activated in response to pulp injury in rodent and humans. To demonstrate the importance of pericytes as a source of stem cells during dental repair, we have studied Rgs5 and Notch3 mRNA expression by in situ hybridization in developing, adult intact and injured rodent teeth. Furthermore, we have examined the distribution of Notch3 protein in carious and injured human teeth using immunohistochemistry. Overlapping expression patterns of Rgs5 and Notch3 were observed during rodent tooth development as well as immediately after injury. Both genes were expressed in vascular structures during development and in perivascular and single capillary cells of injured teeth. However, the expression patterns of Rgs5 and Notch3 were different during tooth repair, with relatively extensive Rgs5 expression along the pericyte-vascular smooth muscle cell axis in central pulp arterioles. These results show co-expression of Rgs5 and Notch3 in pericytes of developing and injured teeth and furthermore indicate the importance of vascular-derived stem cells during pulp healing.     

Single cell studies and simulation of cell-cell interactions using oscillating glycolysis in yeast cells

The observation of oscillations in the concentrations of NADH and other intermediates in glycolysis in dense yeast cell suspensions is generally believed to be the result of synchronization of such oscillations between individual cells. The synchrony is believed to be a property of cell density and the question is: does metabolism in each individual yeast cell continue to oscillate, but out of phase, in the absence of synchronization? Here we have used high-sensitivity fluorescence microscopy to measure NADH in single isolated yeast cells under conditions where we observe oscillations of glycolysis in dense cell suspensions. However, we have not been able to detect intracellular oscillations in NADH in these isolated cells, which cannot synchronize their metabolism with other cells. However, addition of acetaldehyde to a single cell as pulses with a frequency similar to the oscillations in dense cell suspensions will induce oscillations in that cell. Ethanol, another product of glycolysis, which has been proposed as a synchronizing agent of glycolysis in cells, was not able to induce oscillations when added as pulses. The experiments support the notion that the intracellular oscillations are associated with the cell density of the yeast cell suspension and mediated by acetaldehyde and perhaps also other substances.     

A luminescent oxygen channeling immunoassay for the determination of insulin in human plasma

The authors describe a homogeneous, sensitive, and rapid bead-based sandwich immunoassay with a broad analytical range for quantifying insulin in human plasma. The assay was performed as a 2-step reaction by incubating the sample with a mixture of biotinylated anti-insulin antibody and beads covalently coated with anti-insulin antibody for 1 h. This was followed by incubation with beads covalently coated with streptavidin for 30 min. After the incubation steps, light generated from a chemiluminescent reaction within the beads was quantitated. The assay was run in 384-well plates with a sample volume of 5 microL. The analytical range extended from 1 to 10,000 pM. Intra-assay precision (% coefficient of variation) ranged from 1.9% to 3.8% for various insulin concentrations. Interassay precision ranged from 4.6% to 7.3%. Assay detection limit was 0.3 pM. There was no interference from moderate hemolysis (with hemoglobin up to 375 mg/dL), bilirubin (up to at least 50 mg/dL), triglyceride (up to at least 1000 mg/dL), biotin (up to at least 7.7 ng/mL), or ascorbic acid (up to 100 mg/dL). However, gross hemolysis did affect the assay. Comparable results were obtained for plasma (ethylenediamine tetra-acetic acid, citrate, and heparin treated) and serum. The correlation with enzyme-linked immunosorbent assay (ELISA) was good (y = 1.25x + 1.19, R(2) = 0.98). This method is convenient and represents an alternative to ELISA.     

High inter-individual variation in the gestation length of the hedgehog tenrec, Echinops telfairi (Afrotheria)

The gestation length (GL) of Tenrecs (Tenrecinae, Afrotheria) is still uncertain. This lack of knowledge also applies to the lesser hedgehog tenrec, Echinops telfairi, the species most commonly bred and maintained in captivity. The animals used in this study were held under controlled conditions (light, temperature and humidity). In order to determine the GL, groups of female tenrecs were subjected to various mating procedures followed by isolation periods of different lengths. A total of n=249 pregnancies were analysed and the number of offspring per litter was 3.29+/-0.09. The length of gestation could be determined in n=199 pregnancies and a mean GL of 67.53+/-0.36 days was calculated. Initial attempts with isolation periods of less than 16 days did not allow to accurately define the GL. Experiments with longer isolation periods and females subjected to only one mating procedure (n=10) revealed a variation in the GLs of 57-79 days. However, in one female a GL of only 50 days was also observed indicating an even greater range in GL variation. There was a statistically significant tendency for shorter GLs in the animals that conceived later in the mating season, but no statistical evidence was found that age, parity or litter size played an essential role in determining the GL. In conclusion, an unexpected high variability in gestation length in E. telfairi was demonstrated although the study animals were kept under controlled environmental conditions. The factors and mechanisms regulating this high intra-species variability in gestation length need further investigations.     

CAF01 potentiates immune responses and efficacy of an inactivated influenza vaccine in ferrets

Trivalent inactivated vaccines (TIV) against influenza are given to 350 million people every year. Most of these are non-adjuvanted vaccines whose immunogenicity and protective efficacy are considered suboptimal. Commercially available non-adjuvanted TIV are known to elicit mainly a humoral immune response, whereas the induction of cell-mediated immune responses is negligible. Recently, a cationic liposomal adjuvant (dimethyldioctadecylammonium/trehalose 6,6'-dibehenate, CAF01) was developed. CAF01 has proven to enhance both humoral and cell-mediated immune responses to a number of different experimental vaccine candidates. In this study, we compared the immune responses in ferrets to a commercially available TIV with the responses to the same vaccine mixed with the CAF01 adjuvant. Two recently circulating H1N1 viruses were used as challenge to test the vaccine efficacy. CAF01 improved the immunogenicity of the vaccine, with increased influenza-specific IgA and IgG levels. Additionally, CAF01 promoted cellular-mediated immunity as indicated by interferon-gamma expressing lymphocytes, measured by flow cytometry. CAF01 also enhanced the protection conferred by the vaccine by reducing the viral load measured in nasal washes by RT-PCR. Finally, CAF01 allowed for dose-reduction and led to higher levels of protection compared to TIV adjuvanted with a squalene emulsion. The data obtained in this human-relevant challenge model supports the potential of CAF01 in future influenza vaccines.     

Lysine 219 participates in NADPH specificity in a flavin-containing monooxygenase from Saccharomyces cerevisiae

The flavin-containing monooxygenase from Saccharomyces cerevisiae (yFMO) uses NADPH and O(2) to oxidize thiol containing substrates such as GSH and thereby generates the oxidizing potential for the ER. The enzyme uses NADPH 12 times more efficiently than NADH. Amino acid sequence analysis suggests that Lys 219 and/or Lys 227 may act as counterions to the 2' phosphate of NADPH and to help determine the preference for pyridine nucleotides. Site directed mutations show that Lys 219 makes the greater contribution to cosubstrate recognition. Conversion of Lys 219 to Ala reduces NADPH dependent activity 90-fold, but has no effect on NADH-dependent activity. Conversion of Lys 227 to Ala reduces NADPH-dependent activity fivefold and NADH-dependent activity threefold. Dissociation constants for NADP(+) to oxidized yFMO were measured spectroscopically. K(d) is 12 microM for the wild-type enzyme and 243 microM for the K219A mutant, consistent with the role of Lys 219 in pyridine nucleotide binding.