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The three yolk proteins of Drosophila melanogaster are synthesized in the fat body and ovarian follicle cells. A mutation in yolk protein 3, YP3S1, has been described in which the leader sequence is not cleaved from the protein. We describe here ultrastructural and molecular studies on the YP3S1 mutant and show that the mutant protein enters the secretory pathway and forms precipitates, often as electron dense material in excessive elaborations of the plasma membrane. Females homozygous for YP3S1 lay fewer eggs than wild type flies and these embryos are less viable. The abnormal ultrastructure of the yolk spheres observed suggests that whilst YP3 is not completely essential for viability, it is required for normal yolk sphere morphogenesis.

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A coplanar polychlorinated biphenyl (PCB) when eaten by test animals increased the rate of recombination in somatic cells, indicating a new mechanism of action for these compounds. Using the eye-mosaic test a high bioactivation strain of Drosophila that consumed 4,4'-dichlorobiphenyl (4,4'-DCB) manifested a genotoxicity rate that was three-fold greater than that in animals fed the solvent-spiked medium. This compound was not genotoxic in a suppressed bioactivation strain indicating that genotoxicity requires bioactivation of the compound. High bioactivation test strains made heterozygous for a paracentric inversion, a chromosomal rearrangement that suppresses homologous recombination, exhibited significantly reduced genotoxicity after treatment.

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Specific mutations in the yolk protein genes, yp1 and yp2, of Drosophila melanogaster cause the yolk proteins (YPs) they encode to precipitate, ultimately resulting in female sterility. YPs of the yp1 mutant fs(1)1163 are secreted normally but then precipitate as globules and occasionally as crystalline fibers in the subbasement membrane space of the fat body (Butterworth et al., 1991, J. Cell Biol. 112, 727-737). The present ultrastructural and immunological studies of the fat body of the yp2 mutant fs(1)K313 show that YP also precipitates as globules in the same tissue compartment. The globules are also incapable of passing into the hemolymph but they are morphologically distinct from those of fs(1)1163. Similar analyses were performed on developing oocytes in wild type and both mutant strains. YP-containing aggregates, ultrastructurally similar to those in the fat body of each respective mutant, were found in the space between the plasmalemma and the vitelline membrane and embedded within the membrane itself. The evidence suggests that the precipitates interfere with the correct assembly of the eggshell membranes, leading to the sterile phenotype. Immunogold studies demonstrate that newly synthesized YPs in the normal and mutant strains share secretory vesicles with putative, vitelline membrane proteins and that the translocation of follicle cell YP is not through the membrane along the interfollicular spaces but directly through the plasmalemma facing the oocyte. Further the YP precipitates in the mutants permit visualization of the polarity of exocytosis of YP from the follicle cells.

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Ultrastructural and genetic studies were carried out on the fat body of a female sterile mutant fs(1)1163 to ascertain why yolk protein 1 (YP1) is not secreted from this tissue. Earlier molecular studies demonstrated that (a) normally yolk protein is synthesized in the fat body, secreted into the hemolymph and taken up by the ovary, (b) the 1163 mutation causes a single amino acid substitution in YP1, and (c) females homozygous for the mutation, or heterozygous females raised at 29 degrees C, retain YP1 in the fat body. Ultrastructural analysis in this paper shows that the fat body of these females contains masses of electron-dense material deposited in the subbasement membrane space. This subbasement membrane material (SBMM), which occasionally has a crystalline-like, fibrous component, is found in females whose genotypes include at least one copy of the mutant 1163 gene. These strains include a deletion strain that is hemizygous for the 1163 gene and two strains that are transgenic for the mutant gene. Immunogold studies indicate that SBMM contains yolk protein. We propose that the mutant protein is secreted into the subbasement membrane space, but because of the amino acid substitution in YP1, the oligomers containing YP1 condense into SBMM, which cannot penetrate the basement membrane. The similarity of SBMM and deoxyhemoglobin S fibers is discussed.

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Using morphometric and cytochemical techniques we have described changes taking place in the fat body cells during three different stages of development. The cell number remains constant at about 2200 cells during larval life and then decreases gradually and continuously throughout metamorphosis and the first 3 days of the adult stage until no more cells can be observed. Cell size increases rapidly during the larval period and decreases steadily during metamorphosis and adult stage. The size of the nuclei increases during the larval instars and decreases during the pupal interval. The change in nuclear size is correlated with the amount of DNA present throughout development implying the nuclear DNA is synthesized during the larval period and degraded gradually during metamorphosis. The cell size changes are due in large part to accumulation or loss of reserve substances: lipid droplets, glycogen deposits and protein granules. During metamorphosis the amount of lipid decreases slightly whereas glycogen experiences two loss cycles. The protein granules in the form of lysosomes continue to increase in amount during the first day of metamorphosis because of a short period of massive autophagy. Then the lysosomes decrease in amount throughout the remainder of metamorphosis. The lysosomes stain positively for lipofuscin.

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The DNA content of fat body nuclei was measured cytophotometrically as a function of position along an anterior-posterior (A-P) axis of the tissue throughout larval development. There was a dramatic 55-fold increase in the average amount of DNA per nucleus during the first 3 days of this period, but there was no further increase during the final day. However, the rate of increase had regional specificity. The amount of DNA per nucleus correlated significantly with its position along the A-P axis of the tissue: nuclei in a posterior direction contain gradually increasing amounts of DNA. There was up to a seven-fold difference between the smallest anterior and largest posterior nucleus. In addition, for three of the ages studied there was a subgradient in the posterior region with a slope that was considerably steeper than that of the overall tissue gradient. The tissue has three characteristic morphological regions, anterior, medial, and posterior, which can be recognized early in development and which are maintained throughout the larval period. The distribution of nuclear DNA classes determined for cells in each region for the final 2 days of larval life became fixed before the final day of development. The significance of the DNA gradient in terms of a protein storage gradient is discussed.

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The number of genomic replications of four separate tissues was determined in Drosophila melanogaster by summing the total number of nuclei and the maximum amount of DNA per nucleus in each tissue. It was found that for a given tissue the total number of nuclei was generally inversely proportional to the maximum amount of DNA per nucleus. In addition the sum of the number of divisions giving rise to the nuclei and the number of DNA replications occurring in the nucleus in each tissue never exceeded 20.

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A morphological and cytometric analysis of the adult fat body cells and oenocytes was made on sections of abdomens from immature, mature and senescent Drosophila melanogaster of both sexes. There are about 18,000 fat body cells in abdomens of female and mature male flies. Immature and senescent males have about 12,000 and 15,000 cells, respectively. The size of the cells is almost the same for immature flies of both sexes and increases about six-fold to approximately 2600 micron2, so that mature flies of both sexes have equivalent amounts of fat body tissue. The proportions of lipid, glycogen, and background cytoplasm of fat body cells also remain relatively constant throughout adult life, but dense, proteinaceous granules are observed in cells of senescent flies. The amounts of cellular components change dramatically due to change of cell size with age; the amount of lipid shows the greatest sexual difference with about 2x more in the females at all stages studied. The oenocytes number about 6,000 in the abdomens of all but immature male flies, which have approximately 4,000. Although the cells of both sexes triple in size to about 700 micron 2, the oenocytes of males reach maximum size earlier than those of females. The major features of oenocytes appear to be dense background cytoplasm, putative lipid droplets found only in mature flies, and pigmented granules first seen in the cells of mature flies which accumulate with age to 33% of the cytoplasm. The number of cells and their anticipated capacity for protein synthesis is discussed in relation to the production of yolk protein precursors.

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Progressive changes in the ultrastructure of the larval fat body of Drosophila melanogaster were studied during the third instar. In addition to electron microscopy, light microscopy and morphometric stereology were employed to evaluate the tissue at five 12-hr intervals: 48, 60, 72, 84, and 96 hr after hatching from the egg. Lipid and glycogen were found stored throughout the instar, whereas protein is stored in the form of cytoplasmic granules mainly during the final 24 hr. The cells increased in cross-sectional area, and there was a concomitant increase in the relative amounts of these substances. Based on morphological characteristics there were three types of protein granules which we called dense granules (D), heterogeneous granules (H), and autophagic vacuoles. The morphology, size range, time of appearance, and changes in frequency of these granules suggested that the H type arose from D granules, and that the autophagic vacuoles were derived from D and H types. Morphological evidence indicated D granules have the unusual characteristic of forming in the intercellular space before entering the cytoplasm.

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A nonspecific carboxylesterase (esterase 6) of Drosophila melanogaster shows greater activity in adult males than in females and is highly concentrated in the anterior ejaculatory duct of the reproductive tract of the male. Esterase 6 is depleted in males by copulation and is transferred to females early during copulation as a component of the seminal fluid. That esterase 6 may be involved in a system controlling the timing of remating is suggested by differences in the activity of this enzyme in a strain of Drosophila selected for a decrease in time to remating and by differences in the timing of remating in females initially inseminated by males lacking or having active esterase 6.

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Using radioimmunoassay, the levels of plasma cortisol of 23 patients with multiple sclerosis and 10 control subjects were determined at three or more times over a one-year period. The status of the disease was ascertained by neurological examination at the same time. Twelve of the patients were considered stable because they showed no changes in their disease status during the year. The remaining 11 patients were designated active since each had one or more relapses. The levels of cortisol of the control and stable groups remained relatively constant during the one-year period, but the levels of the active group fluctuated significantly more than either of the other two groups. Furthermore the active patients who had more than one episode had the greatest fluctuation in their cortisol levels. The fluctuations could indicate some endocrinological aberration in patients with active multiple sclerosis or may be secondary to the influence of the disease.

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Using transplantation techniques it was shown that immature and supposedly mature stages of fat body from the larvae ofDrosophila melanogaster do not lyse rapidly in the lytic, internal environment of the young adult. In the younger tissue the protein granules (probably lysosomes) were just beginning to form, whereas in the older tissue the protein granules were at a maximum level. In both cases the implanted tissues became steadily smaller independently of the environment. The decrease in implant size was interreted to mean that the cells were lysing, since the average cell size did not change, and since many cells appear cytologically degenerate. However, the estimated rate of cell loss was much slower than in the case where the cells pass through metamorphosis. Some of the immature cells produced relatively high amounts of protein granules independently of the environment. Although the protein granules are at a maximum amount in both stages, it would appear that additional development must be required for the cells to become susceptible to the lytic environment of the young adult.

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A lipid found exclusively in the ejaculatory bulb of adult male Drosophila melanogaster has been identified as cis-vaccenyl acetate. Identification is based on, spectral comparisons with a synthetic sample.

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A thin-layer chromatographic analysis of Drosophila melanogaster revealed a lipid to be found almost exclusively in the adult male. The compound or class of compounds having an R(F) close to that of methyl oleate is present in substantial amounts and is located predominantly in the ejaculatory bulb. It appears from genetic studies that the formulation of the lipid is not mediated by the Y chromosome.

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