The Drosophila disembodied gene controls late embryonic morphogenesis and codes for a cytochrome P450 enzyme that regulates embryonic ecdysone levels

Ecdysteroids regulate a wide variety of cellular processes during arthropod development, yet little is known about the genes involved in the biosynthesis of these hormones. Previous studies have suggested that production of 20-hydroxyecdysone in Drosophila and other arthropods involves a series of cytochrome P450 catalyzed hydroxylations of cholesterol. In this report, we show that the disembodied (dib) locus of Drosophila codes for a P450-like sequence. In addition, we find that dib mutant embryos have very low titers of ecdysone and 20-hydroxyecdysone (20E) and fail to express IMP-E1 and L1, two 20E-inducible genes, in certain tissues of the embryo. In situ hybridization studies reveal that dib is expressed in a complex pattern in the early embryo, which eventually gives way to restricted expression in the prothoracic portion of the ring gland. In larval and adult tissues, dib expression is observed in the prothoracic gland and follicle cells of the ovaries respectively, two tissues known to synthesize ecdysteroids. Phenotypic analysis reveals that dib mutant embryos produce little or no cuticle and exhibit severe defects in many late morphogenetic processes such as head involution, dorsal closure and gut development. In addition, we examined the phenotypes of several other mutants that produce defective embryonic cuticles. Like dib, mutations in the spook (spo) locus result in low embryonic ecdysteroid titers, severe late embryonic morphological defects, and a failure to induce IMP-E1. From these data, we conclude that dib and spo likely code for essential components in the ecdysone biosynthetic pathway and that ecdysteroids regulate many late embryonic morphogenetic processes such as cell movement and cuticle deposition.     

Genetic diversity of Agrobacterium vitis as determined by DNA fingerprints of the 5'-end of the 23S rRNA gene and Random Amplified Polymorphic DNA

Sixty-nine strains of Agrobacterium vitis , the causal agent of grape crown gall, originating from different geographical regions of the USA and Europe, were characterized by fingerprint analysis of the 5'-end of the 23S rRNA gene and by Random Amplified Polymorphic DNA (RAPD) markers. For 5'-end 23S fingerprinting, amplicons were digested with Taq I, Rsa I, Ava I, Cfo I and Alu I. For RAPD analysis, three 10-mer primers were used to generate PCR products. There was a high degree of correlation between strain groupings generated by the two methods. However, more diversity was identified when groupings were based on RAPDs. For example, 28 of 29 strains having nopaline type Ti plasmids generated identical 5'-end 23S patterns but formed two distinct RAPD groups that separated strains originating from the USA and Hungary. Similarly by RAPDs, one cluster of strains carrying vitopine-type Ti plasmids could be separated into those originating in the USA and Europe. The composition of strain groups generated by 5'-end 23S and RAPDs were highly correlated with a previous fingerprint analysis of the intergenic spacer region (located between the 16S and 23S rRNA genes) and with RFLP analysis for characterizing Ti plasmids. These findings show that among Ag. vitis strains there is a high level of correlation between two regions of the rRNA operon, total genomic DNA (as determined by RAPDs) and the type of Ti plasmid they carry.     

Effects of suppression of bone turnover on cortical and trabecular load sharing in the canine vertebral body

The relative biomechanical effects of antiresorptive treatment on cortical thickness vs. trabecular bone microarchitecture in the spine are not well understood. To address this, T-10 vertebral bodies were analyzed from skeletally mature female beagle dogs that had been treated with oral saline (n=8 control) or a high dose of oral risedronate (0.5 mg/kg/day, n=9 RIS-suppressed) for 1 year. Two linearly elastic finite element models (36-μm voxel size) were generated for each vertebral body—a whole-vertebra model and a trabecular-compartment model—and subjected to uniform compressive loading. Tissue-level material properties were kept constant to isolate the effects of changes in microstructure alone. Suppression of bone turnover resulted in increased stiffness of the whole vertebra (20.9%, p=0.02) and the trabecular compartment (26.0%, p=0.01), while the computed stiffness of the cortical shell (difference between whole-vertebra and trabecular-compartment stiffnesses, 11.7%, p=0.15) was statistically unaltered. Regression analyses indicated subtle but significant changes in the relative structural roles of the cortical shell and the trabecular compartment. Despite higher average cortical shell thickness in RIS-suppressed vertebrae (23.1%, p=0.002), the maximum load taken by the shell for a given value of shell mass fraction was lower (p=0.005) for the RIS-suppressed group. Taken together, our results suggest that—in this canine model—the overall changes in the compressive stiffness of the vertebral body due to suppression of bone turnover were attributable more to the changes in the trabecular compartment than in the cortical shell. Such biomechanical studies provide an unique insight into higher-scale effects such as the biomechanical responses of the whole vertebra.     

Atypicalities in Perceptual Adaptation in Autism Do Not Extend to Perceptual Causality

A recent study showed that adaptation to causal events (collisions) in adults caused subsequent events to be less likely perceived as causal. In this study, we examined if a similar negative adaptation effect for perceptual causality occurs in children, both typically developing and with autism. Previous studies have reported diminished adaptation for face identity, facial configuration and gaze direction in children with autism. To test whether diminished adaptive coding extends beyond high-level social stimuli (such as faces) and could be a general property of autistic perception, we developed a child-friendly paradigm for adaptation of perceptual causality. We compared the performance of 22 children with autism with 22 typically developing children, individually matched on age and ability (IQ scores). We found significant and equally robust adaptation aftereffects for perceptual causality in both groups. There were also no differences between the two groups in their attention, as revealed by reaction times and accuracy in a change-detection task. These findings suggest that adaptation to perceptual causality in autism is largely similar to typical development and, further, that diminished adaptive coding might not be a general characteristic of autism at low levels of the perceptual hierarchy, constraining existing theories of adaptation in autism.     

The role of phospholipase A activity in rat liver microsomal lipid peroxidation

The involvement of phospholipase(s) A in lipid peroxidation of rat liver microsomes was investigated by: (a) determining the effects of phospholipase A inhibitors (p-bromophenylacyl bromide, chlorpromazine, mepacrine) on the accumulation of thiobarbituric acid reactivity or on levels of oxidized phospholipids in response to selected oxidative stimuli and (b) measurement of phospholipase A activities in response to these agents. Lipid peroxidation in response to various peroxidation systems was inhibited completely by exposure of microsomes to p-bromophenylacyl bromide (250 microM). The effectiveness of p-bromophenylacyl bromide was dependent on the presence of glutathione (200 microM) in preincubation mixtures. Chlorpromazine (100 microM) and mepacrine (100 microM) also effectively inhibited peroxidation, and their potency was independent of glutathione. The accumulation of oxidized phospholipids in response to the potent peroxidation stimulus alloxan/ferrous ion was similarly inhibited by p-bromophenylacyl bromide, although the level of oxidized phospholipid in response to the initiator ADP/ferrous ion was not affected. Microsomal phospholipase A1 activity, assessed using a liposomal substrate, was substantially enhanced by promoters of lipid peroxidation. Phospholipase A2 activity was not detected using a liposomal substrate but was evident using radiolabeled microsomes as endogenous substrate and was enhanced by oxidative stimuli. We conclude that phospholipase A activity may play an integral role in the microsomal lipid peroxidation mechanism. Based on this study, we hypothesize a role for phospholipases in facilitating propagation reactions.     

The binding of adenosine to polyuridylic acid in which uracil residues are chemically altered.

The binding of adenosine-¹⁴C to polyuridylic acid (poly(U)) and several modified poly(U)s has been studied by equilibrium dialysis. The poly(U) was modified by addition of appropriate reagents across the 5,6-double bond of the uracil ring to form the photohydrate, photodimer, dihydrouracil, the HOBr addition product and the HSO₃^? addition product. Modification of the uracil rings decreases the amount of adenosine which can be bound to the poly(U); the decrease in binding is a function of the fraction of uracil rings which have been changed. Using the expression S = S₀(1 ? αr)² to relate the fraction of uracil rings modified (r) to the number of binding “sites” remaining (S), it is found that α is about 1 for all the modifications except photodimer where it is about 2. These observations are taken to mean that the loss of binding capacity of the poly(U) resulting from modifications of the uracil ring is caused by loss of planarity of the uracil rings caused by the modifications, and consequent loss of double helix structure, but that for all modifications except photodimer there is no disruption of the poly(U) double helix on either side of the leison. There does appear to be local melting on either side of the photodimer lesion. The sigmoidal binding isotherms (A_b versus C_a) of modified and unmodified poly(U) can be approximated closely by the following equation: ((1)) (1) where A_b = bound A, C_a = free A, n = minimum number of adjacent A′s in complex, S = concentration of sites on poly(U), and K₁ = (K_m)^1/m for all m ≥ n. The stacking energy of adenosine (w) can be calculated accurately using the following equation, where dθ/d ln C_a is obtained from Eq. (1). ((2)) (2) For unmodified poly(U), w is ?2.0 kcal/mole and ΔG° (?;RT ln K₁) is ?3.2 kcal/mole. The difference (?1.2 kcal/mole) is attributed to hydrogen bonding. Heavily photohydrated poly(U) does not bind guanosine or guanosine-5′-phosphate.     

How do trabeculae affect the calculation of structural properties of bone?

One difficulty that arises in an analysis of the cross-sectional properties of bone is whether to include cancellous bone in the analysis. The purpose of this paper is to determine how different amounts of cancellous bone affect the measurement of structural properties of bone cross-sections. Thirty-two tibial and femoral cross-sections were chosen at random from a series of cross-sectioned nonhuman primate bones. Geometrical properties were calculated for the cross-sections, and torsional and bending stress analyses were performed. The results suggest that the effect of including cancellous bone in the analysis is closely related to the amount of bone, where it lies within the cross-section, and the type of analysis performed. Including cancellous bone in calculations of structural properties of bone cross-sections may cause the strength and stiffness of the bone to be exaggerated.