Standardization of amniotic fluid leptin levels and utility in maternal overweight and fetal undergrowth.

OBJECTIVE: Leptin is an adipokine that regulates energy homeostasis. The objective of this study was to establish a gestational age-specific standard for amniotic fluid leptin (AFL) levels and examine the relationship between AFL, maternal overweight and fetal growth restriction. STUDY DESIGN: Amniotic fluid was obtained at mid-gestation from singleton gravidas, and leptin was quantified using enzyme-linked immunosorbent assay. Amniotic fluid samples from 321 term pregnancies were analyzed. Clinical data, including fetal ultrasound measurements and maternal and infant characteristics, were available for a subset of patients (n=45). RESULTS: The median interquartile range AFL level was significantly higher at 14 weeks' gestation (2133 pg ml(-1) (1703 to 4347)) than after 33 weeks' gestation (519 pg ml(-1) (380 to 761), P trend<0.0001), an average difference of 102 pg ml(-1) per week. AFL levels were positively correlated with maternal pre-pregnancy body mass index (BMI) (r=0.36, P=0.03) adjusting for gestational age at measurement, but were not associated with fetal growth. CONCLUSIONS: AFL levels are higher at mid-gestation than at late gestation, and are associated with maternal pre-pregnancy BMI.

Juvenile hormone esterase (JHE) from Tenebrio molitor: full-length cDNA sequence, in vitro expression, and characterization of the recombinant protein.

Juvenile hormone regulates the development and reproduction in a variety of insects. Juvenile hormone esterase (JHE) is a selective enzyme, which hydrolyzes the methyl ester of JH and alters its activity. In Tenebrio molitor, JHE has been previously purified from pupae and a partial cDNA was amplified by RT-PCR using fat body mRNA. The previous report indicated that several forms of the JHE protein were present in pupal homogenate. In this study, we report the full-length cDNA, which was obtained by RACE methods. The deduced protein sequence corresponds to peptides from two proteins of different molecular weights in the previous study. The coding region of the full-length cDNA was subcloned into the AcMNPV genome and high levels of expression of the JHE enzyme from the viral p10 promoter were demonstrated in cell culture. The majority of JHE is secreted from the cells as a soluble enzyme. The recombinant JHE enzyme was biochemically characterized. The recombinant protein appears by PAGE analysis as a monomer of approximately the same MW (66000) and pI (4.9) as was expected from the deduced amino acid sequence of the cDNA.

In vitro expression and biochemical characterization of juvenile hormone esterase from Manduca sexta.

Juvenile hormone esterase (JHE) is a selective enzyme that hydrolyzes the methyl ester of juvenile hormone. This enzyme plays an important role in the regulation of metamorphosis in caterpillars, and is implicated in additional roles in development and reproduction in this and other orders of insect. The full length coding region of the JHE cDNA from Manduca sexta was subcloned into the baculovirus AcMNPV genome under the control of the p10 promoter. The recombinant virus demonstrated the expression of high levels of JHE activity when infected into Hi5 cells from Trichoplusia ni. The recombinant protein was partially purified by anion exchange chromatography and its biochemical characterization showed similar features to the wild type protein. The recombinant JHE has an estimated MW of 66500 Da. Some heterogeneity with the enzyme was observed when analyzed by isoelectric focusing, although the peak of JHE activity was observed at pI=6.0. It is highly sensitive to trifluoroketone inhibitors and certain phosphoramidothiolates, while relatively insensitive to other common esterase inhibitors. Incubating the enzyme with various organic solvents and detergents showed that the enzyme is activated at lower concentrations of solvents/detergents and remains significantly active even at high concentrations. The high tolerance of organic solvents may make this JHE enzyme useful in future applications as a synthetic catalyst.

Recombinant baculoviruses for insect control.

Baculoviruses are double-stranded DNA viruses which are highly selective for several insect groups. They are valuable natural control agents, but their utility in many agricultural applications has been limited by their slow speed of kill and narrow host specificity. Baculoviruses have been genetically modified to express foreign genes under powerful promoters in order to accelerate their speed of kill. In our and other laboratories, the expression of genes coding for insect juvenile hormone esterases and various peptide neurotoxins has resulted in recombinant baculoviruses with promise as biological insecticides. These viruses are efficacious in the laboratory, greenhouse and field and dramatically reduce damage caused by insect feeding. The recombinant viruses synergize and are synergized by classical pesticides such as pyrethroids. Since they are highly selective for pest insects, they can be used without disrupting biological control. Because the recombinant virus produces fewer progeny in infected larvae than the wild-type virus, they are rapidly out-competed in the ecosystem. The viruses can be used effectively with crops expressing endotoxins of Bacillus thuringiensis. They can be produced industrially but also by village industries, indicating that they have the potential to deliver sustainable pest control in developing countries. It remains to be seen, however, whether the current generation of recombinant baculoviruses will be competitive with the new generation of synthetic chemical pesticides. Current research clearly indicates, though, that the use of biological vectors of genes for insect control will find a place in agriculture. Baculoviruses will also prove valuable in testing the potential utility of proteins and peptides for insect control.

Purification of juvenile hormone esterase and molecular cloning of the cDNA from Manduca sexta.

Juvenile hormone esterase (JHE) is a highly specific enzyme important for regulating the onset of metamorphosis in lepidopteran insects. After affinity chromatography of the hemolymph proteins of Manduca sexta, the pure JHE protein was digested with Lys-C and the resultant peptides were purified by microbore HPLC. Two peptides were selected for sequencing. Based upon these amino acid sequences, degenerate RT-PCR was performed in order to amplify a partial cDNA sequence from mRNA from the fat body of M. sexta. A 1512bp partial cDNA was generated and found to be highly homologous to the JHE from Heliothis virescens. 5' and 3' RACE were performed to obtain the full length cDNA sequence. The cDNA has a total length of 2220bp, with a 1749bp coding region. The deduced protein sequence contains 573 amino acids.

Isolation of juvenile hormone esterase and its partial cDNA clone from the beetle, Tenebrio molitor.

Juvenile hormone esterase (JHE) plays an essential role in insect development. It is partially responsible for the clearance of juvenile hormone (JH) which regulates various aspects of insect development and reproduction. Because of its role in regulating JH titer, this enzyme has been targeted for development of biologically-based insecticides. JHE was partially purified from the beetle, Tenebrio molitor, using a transition state analog as the affinity ligand. Two forms of JHE were characterized by activity analysis, isoelectric focusing, two-dimensional SDS-PAGE and N-terminal sequence analysis. The esterase is associated with two proteins of sizes 71 and 150 kDa, both of which are active on JH III. A partial cDNA clone for the enzyme was isolated based on the sequence of N-terminal and internal peptides. Its sequence indicates that JHE from T. molitor and Heliothis virescens may have a common origin.

Isolation and characterization of juvenile hormone esterase from hemolymph of Lymantria dispar by affinity- and by anion-exchange chromatography.

Juvenile hormone esterase (JHE), which catalyzes the hydrolysis of juvenile hormone, was isolated from the hemolymph of 5(th) instars of Lymantria dispar by two different procedures. One procedure was based on affinity chromatography and the other on anion-exchange chromatography. The material from both purifications showed bands of approximately 50 kDa when analyzed by SDS-PAGE. Isoelectric focusing (IEF) gels in combination with enzyme activity assays indicated two isoelectric forms with the same pI values (pH 5.1. and 5.3) from affinity purification and from anion-exchange chromatography. Amino acid sequencing of several internal peptides from the 50 kDa band following affinity purification and alignment of these sequences with JHEs from previously purified lepidopteran species (Heliothis virescens, Manduca sexta) showed high homology of these enzymes. The isolated JHE, at least in the stage of insect used, was different from the enzyme reported earlier [Valaitis, A.P., 1991. Characterization of hemolymph juvenile hormone esterase from Lymantria dispar. Insect Biochemistry 21, 583-595] to hydrolyze JH in the hemolymph of gypsy moth, based on molecular weight and amino acid sequence.