The mutation without children(rgl) causes ecdysteroid deficiency in third-instar larvae of Drosophila melanogaster.

Larvae homozygous for the recessive lethal allele without children(rgl) (woc(rgl)) fail to pupariate. Application of exogenous 20-hydroxyecdysone elicits puparium formation and pupation. Ecdysteroid titer measurements on mutant larvae show an endocrine deficiency in the brain-ring gland complex, which normally synthesizes ecdysone, resulting in a failure of the larvae to achieve a threshold whole body hormone titer necessary for molting. Ultrastructural investigation revealed extensive degeneration of the prothoracic cells of the ring gland in older larvae. The woc gene, located in polytene chromosomal region 97F, consists of 11 exons. A 6.8-kb transcript is expressed throughout development but is absent in the mutant woc(rgl) larvae. The woc gene encodes a protein of 187 kDa. Eight zinc fingers of the C2-C2 type point to a possible function as a transcription factor. The woc protein shows considerable homology to human proteins which have been implicated in both mental retardation and a leukemia/lymphoma syndrome.

An in vitro analysis of ecdysteroid-elicited cell death in the prothoracic gland of Manduca sexta.

The prothoracic glands of the tobacco hornworm, Manduca sexta, secrete the precursor of the insect molting hormone and normally undergo programmed cell death (PCD) during pupal-adult metamorphosis, between days 5 and 6 after pupation. This phenomenon can be elicited prematurely in vitro by the addition of 20-hydroxyecdysone (20E) to the gland cultures. To induce nuclear condensation in vitro in the glands from day-1 pupae, the effective dose range of 20E is 0.7-7 micrograms/ml and the minimum exposure period is 24 h. Prothoracic glands from different stages of pupal-adult development express different responsiveness to exogenous ecdysteroids. By utilizing terminal deoxynucleotidyl-transferase-mediated dUTP nick-end-labeling (TUNEL) and the apoptotic DNA laddering method together with transmission electron microscopy, it has been demonstrated that the ecdysteroid-induced cell death of the prothoracic glands occurs via not only apoptosis but also autophagy, i.e., the induced dying cells show both severe nuclear fragmentation and autophagic vacuole formation, characteristics typical of apoptotic and autophagic cell death. The composite data indicate that ecdysteroids regulate directly both apoptotic and autophagic mechanisms of PCD of the prothoracic glands.

Can the insect nervous system synthesize ecdysteroids?

The term "neurosteroid" refers to both classic and unique steroid molecules that are synthesized from cholesterol (C) by the central and peripheral nervous systems of higher vertebrates. Therein, they accumulate and modulate nervous activity by a variety of mechanisms other than the classic steroid receptor-mediated modulation of genomic activity, although such may also be involved. Since the insect nervous system expresses ecdysteroid receptors and responds both directly and developmentally to ecdysteroids, the possibility of ecdysteroidogenesis in the pupal and adult central and peripheral nervous system of Manduca sexta and the nervous system of Drosophila melanogaster larvae was investigated. The endogenous concentrations of the critical, dietary-derived delta 5,7-sterols ergosterol and 7-dehydrocholesterol (7dC) remained 10 to 20-fold higher in the Manduca pupal and adult nervous tissues than was found in the larval hemolymph at the cessation of feeding. In addition, it was determined that the Manduca pupal nervous system, but not that of the adult, could synthesize 3H/14C-7dC or 3H-7-dehydro-25-hydroxycholesterol (3H-7d25C) from 3H/14C-cholesterol (3H/14C-C) or the polar sterol substrate 3H-25-hydroxycholesterol (3H-25C), respectively. However, none of the nervous system samples from the two species and the several stages analyzed, a small window of neural development in these insects, were capable of incorporating any of the above tracer precursor sterols into a radiolabelled ecdysteroid, i.e. less than 0.0005%. Thus, the absence of neurosteroidogenesis by the insect nervous system stands in sharp contrast to previously described nervous system steroid hormone biosynthesis by the mammalian nervous system.

Juvenile hormone prevents the onset of programmed cell death in the prothoracic glands of Manduca sexta.

During normal pupal-adult development, programmed cell death of the prothoracic gland of Manduca sexta proceeds via apoptosis. By employing the DNA laddering technique, the earliest sign of DNA fragmentation in the cells comprising the gland occurred on Day 5 of pupal-adult development and DNA fragmentation peaked 1 day later. Since juvenile hormone (JH) is known to prevent adult development and since prothoracic gland degeneration occurs during the initial stages of adult development, we wished to determine the possible role of JH in prothoracic gland maintenance. JH was injected into pupae and the glands analyzed by DNA laddering and in situ TUNEL labeling. The administration of JH prevented apoptosis of the prothoracic gland even 11 days after JH injection into young pupae. The prothoracic glands remained intact and their ability to synthesize ecdysteroids was maintained at a fairly active level as ascertained by radioimmunoassay after in vitro incubation. The control glands had degenerated by this time and were almost devoid of ecdysteroidogenesis capability. Ultrastructural analysis confirmed that JH-treated prothoracic gland cells were rescued from the apoptotic sequence, i.e., nuclear condensation, cytoplasmic budding, and cell fragmentation. They exhibited a preserved smooth endoplasmic reticulum and intercellular channel system typical of active prothoracic gland cells. The composite data suggest that JH can both maintain and stimulate the prothoracic gland. Therefore, during normal pupal-adult metamorphosis the absence of JH is prerequisite for both the initiation and completion of prothoracic gland degeneration.

A SAW-based commutation signaling modem for broadband indoor wireless communication.

Commutation signaling is a bandwidth expanding modulation scheme that is robust to multipath induced intersymbol interference making it suitable for wireless digital communications. By using multipath diversity combining, commutation signaling exploits the time diversity that is inherent in a multipath propagation environment. This paper considers a surface acoustic wave (SAW) implementation of a commutation signaling modem for broadband indoor wireless communication. The modem employs differential encoding and a form of direct sequence spread spectrum modulation with the following specifications: data rate 40 Mb/sec, chip rate 200 MHz, and IF frequency 1 GHz. The differential coherent detector is a key element of a low cost, low complexity commutation signaling modem. A commutation signaling differential coherent detector has been implemented using SAW and RF integrated circuit (RF IC) technologies. The SAW devices have been fabricated on 1280-rotated Y-cut, X-propagating lithium niobate using approximately 1 micron line widths. RF IC technology Is used to implement the high-speed bilinear multipliers needed for differential coherent detection as well as the low-impedance buffers used to drive these multipliers.

Programmed cell death of the prothoracic glands of Manduca sexta during pupal-adult metamorphosis.

The degeneration of the prothoracic glands of Manduca sexta during pupal-adult metamorphosis was analyzed by both light and transmission electron microscopy, and correlated with a functional analysis of the ability of the prothoracic glands to synthesize ecdysteroids in vitro. Apoptosis of degenerating glandular cells was also examined by in situ indirect immunofluorescein detection of digoxigenin-labeled genomic DNA cleavage (the TUNEL method). The ability of the prothoracic gland to synthesize ecdysteroids reached a peak 4 days after pupation, decreased drastically on day 6 and then decreased further to the basal level 2 days later. Prothoracic gland degeneration was initiated on day 6 and was characterized by nuclear condensation, cytoplasmic budding, giant autophagic vacuoles, the disappearance of the smooth endoplasmic reticulum and intercellular channels, and the fragmentation of the cytoplasm into membrane-bound bodies. The cell debris of these degenerating cells was then engulfed by numerous phagocytic hemocytes. The results of the analysis of apoptosis by immunofluorescence detection are in complete accord with the histological, ultrastructural and radioimmunoassay data, suggesting that apoptosis is the basic mechanism for programmed cell death of the cells comprising these vital glands.

Differential incorporation of cholesterol and cholesterol derivatives into ecdysteroids by the larval ring glands and adult ovaries of Drosophila melanogaster: a putative explanation for the l(3)ecd1 mutation.

Studies in vitro revealed that intact ring glands of Drosophila melanogaster convert tritiated cholesterol (C) and 25-hydroxycholesterol (25C) via 7-dehydrocholesterol (7dC) and 7-dehydro-25-hydroxycholesterol (7d25C), respectively, to ecdysone (E) and 2-deoxyecdysone (2dE), while both intact and homogenized ovaries synthesize only 2dE from these precursors. Emulsified 7d25C was incorporated directly into ecdysteroids by these tissue preparations at a much greater rate than was 7d25C made in situ from 25C. To probe the basis of the biochemical defect in the ecdysteroid deficient conditional mutant ecdysoneless (ecd1), the differential incorporation into ecdysteroids of C (via 7dC), and particularly of 25C (via 7d25C), was measured relative to that observed after the incubation of 7d25C directly with both wild type and mutant tissues in vitro at 30 degrees C, the restrictive temperature. Both C and 25C were equally 7,8-dehydrogenated in situ to 7dC or 7d25C, respectively, by both wild type and mutant tissues at 30 degrees C. However, the rate of subsequent conversion of either of these delta 5,7-sterol intermediates synthesized in situ to ecdysteroids was reduced an average of 50% in the mutant tissues relative to the wild type. Yet, when emulsified 7d25C was incubated directly with either the wild type or mutant tissues at the restrictive temperature, the amplified rate of conversion of the freely available 7d25C to ecdysteroid by these tissues was identical. These data suggest that the defect in ecd1 tissue-mediated ecdysteroidogenesis does not involve a "hit" on any of the enzymes involved in either the 7,8-dehydrogenation of C or 25C or in the subsequent oxidation of 7d25C or 7dC to ecdysteroid. Rather, the mutation appears to affect the expression of a gene governing the translocation of delta 5,7-sterol intermediates from the subcellular compartment where they are synthesized and/or stored to the site of subsequent oxidation to ecdysteroid.

Analysis of weakly guiding cladded acoustic fibers of hexagonal crystal symmetry.

An approximate analysis of the weakly guiding cladded acoustic fiber consisting of a core and infinite thick cladding both with hexagonal crystal symmetry is presented. The crystalline Z axis is parallel to the fiber axis. Weak guidance conditions require that the stiffness constants of the core are slightly less than those of the cladding, and the density of the core and cladding are almost the same. Approximate dispersion and cutoff equations are derived for all pure guided modes. Dispersion curves of several lower-order guided torsional, radial-axial and flexural modes are evaluated using both exact and approximate formulas and they are in good agreement. A simple approximate dispersion equation for leaky (longitudinal-type) modes is also obtained.

Analysis of cladded acoustic fibers of hexagonal crystal symmetry.

A cladded acoustic fiber consisting of a core and an infinite thick cladding both having a hexagonal crystal symmetry is analyzed. The crystalline Z axes of both core and cladding are parallel to the fiber axis. Dispersion equations and field distributions for all modes are derived, and previously reported results for the isotropic case are retrieved. Numerical results for a few lowest-order pure guided torsional, radial-axial and flexural modes are shown. Material considerations are discussed.

An ultrastructural analysis of the ecdysoneless (l(3)ecd1ts) ring gland during the third larval instar of Drosophila melanogaster.

In the late third larval instar of Drosophila melanogaster, the prothoracic gland, an endocrine portion of the ring gland, synthesizes ecdysteroids at an accelerated rate. The resultant ecdysteroid titer peak initiates the events associated with metamorphosis. The normal prothoracic gland displays several ultrastructural features at this developmental stage that reflect increased steroidogenic activity, including extensive infoldings of the plasma membrane (membrane invaginations) and an increase in both the concentration of smooth endoplasmic reticulum (SER) (or transitional ER) and elongated mitochondria. By contrast, the prothoracic glands of larvae homozygous for a conditional larval lethal mutation, l(3)ecd1ts, not only fail to produce ecdysteroids at normal levels at the restrictive temperature (29 degrees C), but also acquire abnormal morphological features that reflect the disruptive effects of the mutation. These abnormalities include an accumulation of lipid droplets presumed to contain sterol precursors of ecdysteroids, a disappearance of SER and a drastic reduction of membrane invaginations in the peripheral area of the cell. These morphological defects are observed in prothoracic glands dissected from larvae transferred from 18 degrees C to 29 degrees C approximately 24 h before observation and also within 4 h of an in vitro transfer to 29 degrees C following dissection from wandering third instar larvae reared at 18 degrees C. No ultrastructural abnormalities were noted in the corpus allatum portion of mutant ring glands. These observations further indicate the direct involvement of the ecd gene product in ecdysteroid synthesis and suggest a role for the gene in the proper transport of precursors to the site where they can be utilized in ecdysteroid biosynthesis.

Metamorphosis of the corpus allatum and degeneration of the prothoracic glands during the larval-pupal-adult transformation of Drosophila melanogaster: a cytophysiological analysis of the ring gland.

The degeneration of the prothoracic glands of Drosophila melanogaster during pupal-adult metamorphosis was analyzed by light microscopy, scanning, and transmission electron microscopy. The ultrastructural observations were correlated with the ability of the ring gland to synthesize ecdysteroids in vitro. The ring gland is prominent during larval life and is identifiable until just before adult eclosion but undergoes dramatic changes in location, shape, size, ultrastructure, and function during pupal-adult development. Prothoracic gland degeneration is characterized by: a gradual decrease in its ability to synthesize ecdysteroids; a decreasing quantity of smooth endoplasmic reticulum (SER) and mitochondria; the absence of intercellular channels; cytoplasmic fragmentation; and the separation of the prothoracic gland from the corpus allatum and corpus cardiacum. An ultrastructural analysis of the corpus allatum during larval-pupal-adult metamorphosis and adult life was also correlated with function, i.e., juvenile hormone biosynthesis, using a radiochemical assay of ring glands and adult corpora allata in vitro. A relatively high concentration of SER, mitochondria, and mitochondrion-scalariform junction complexes are typical features of an active corpus allatum cell. The migration of the corpus allatum from the ring gland to its position as a separate gland in the adult fly was studied in detail. The capacity of the corpus allatum to synthesize juvenile hormone is at its peak in the ring gland of the early wandering third instar larva, whereas the corpus allatum of 2-day-old female adults displayed the greatest synthetic activity during adult life. The physiological significance of the alterations in gland activity is discussed.

An analysis of thin-rod flexural acoustic wave gravimetric sensors immersed in liquid.

A perturbation analysis of acoustic wave propagation properties for a stiff thin rod surrounded by a liquid is presented. A study is made of the velocity sensitivity of the lowest flexural acoustic mode, F(11), along a thin rod due to mass changes in the rod and variations in the surrounding liquid density. It is shown that the presence of the liquid decreases the velocity of the F(11 ) mode and also the mass sensitivity.