Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.Download video file.^{(173M, mp4)}     

Reliability of detecting rRNA sequences of Chlamydia trachomatis with fluorescence in situ hybridization without amplification

OBJECTIVE: To use fluorescence in situ hybridization (FISH) using ribosomal RNA (rRNA) oligonucleotide probes as the target nucleic acid for the detection of Chlamydia trachomatis. STUDY DESIGN: Suitable sequences selected from the rRNA sequence of C trachomatis were labeled with a fluorescent dye and used in FISH for detecting chlamydial inclusion bodies and/ or elementary bodies in paraformaldehyde-fixed urogenital swab samples. The sensitivity and specificity of the FISH assay were compared with those of the polymerase chain reaction (PCR) using plasmid primers. Positive known C trachomatis-infected McCoy cells were used as positive controls. Urogenital swab specimens that were C trachomatis negative on culture and PCR were used as negative controls. RESULT: Among the 128 samples included in the study, FISH was positive in 28 (21.8%) and PCR in 33 (25.7%). A significant correlation was found between the 2 detection methods. Results of PCR and FISH were consistent in 115 of the 128 samples (R = 0.89). Thirteen samples showed discordant results. Of these, 9 FISH negative samples were PCR positive and 4 FISH positive samples were PCR negative. CONCLUSION: FISH was a highly specific and fairly sensitive technique for detecting C trachomatis. Signal amplification techniques and use of different fluorophores may further increase the sensitivity of this technique.     

DsbD-catalyzed transport of electrons across the membrane of Escherichia coli

Dsb proteins catalyze folding and oxidation of polypeptides in the periplasm of Escherichia coli. DsbC reduces wrongly paired disulfides by transferring electrons from its catalytic dithiol motif (98)CGYC. Genetic evidence suggests that recycling of this motif requires at least three proteins, the cytoplasmic thioredoxin reductase (TrxB) and thioredoxin (TrxA) as well as the DsbD membrane protein. We demonstrate here that electrons are transferred directly from thioredoxin to DsbD and from DsbD to DsbC. Three cysteine pairs within DsbD undergo reversible disulfide rearrangements. Our results suggest a novel mechanism for electron transport across membranes whereby electrons are transferred sequentially from cysteine pairs arranged in a thioredoxin-like motif (CXXC) to a cognate reactive disulfide.     

The RNA components of Schizosaccharomyces pombe RNase P are essential for cell viability

The fission yeast Schizosaccharomyces pombe contains in the haploid genome one copy of the gene (designated rrkl) for the RNA components of RNase P. Gene disruption in diploid cells of one copy of rrkl resulted in a moderate reduction of the level of cellular RNase P activity. Haploidization by meiosis demonstrated that rrkl is required for cell growth. Thus, the RNA components of S. pombe RNase P are essential in vivo. This is similar to the situation in Escherichia coli.     

Morphological and ultrastructural changes in vegetative cells and heterocysts of Anabaena variabilis grown with fructose.

The morphology and ultrastructure of Anabaena variabilis grown in medium with and without 40 mM fructose were compared. Vegetative cells and young heterocysts in fructose-supplemented medium were significantly larger, were filled with glycogen granules, and had fewer thylakoids. Developing heterocysts contained large numbers of glycogen granules well into mature stages, and envelope formation was precocious. As heterocysts enlarged in fructose medium, their shape became more broadly oblong compared with the more rectangular heterocysts in fructose-free medium.     

The effects of dietary iodine on thyroid ultrastructure

This is a morphological study of changes in thyroid cells following iodine deficiency and iodine excess. Fifteen young male Sprague-Dawley rats were divided into three groups and fed one of the following diets for 6 weeks: low iodine (LID), normal iodine (NID) and high iodine (HID). Then the thyroid glands were removed and processed for light and electron microscopy. Thyroid tissue from the NID group was normal in appearance. The most outstanding feature of HID thyroids was the presence of numerous cells which contained irregularly shaped and stained lysosomes. These displaced other cell organelles and caused the apical cell surfaces to project into the follicle lumen. Thyroids from the LID group were three times heavier than the other two groups. Their follicles were very small, contained very little colloid. They were surrounded by dilated capillaries. Mitoses were frequent. Cells were columnar and contained abundant dilated endoplasmic reticulum, numerous apical vesicles, long microvilli and many mitochondria. Mitochondria were especially abundant in greatly infolded lateral and basal cell membranes. These findings show that there is a redistribution of organelles in thyroid cells in response to iodine deficiency and iodine excess which can be related to alterations in intracellular iodine metabolism.     

A new natural intergeneric cyprinid hybrid from the Jordan River drainage, with a key to the large barbine cyprinids of the southern Levant

In Ain al-Qunaiya. an isolated source within the Jordan River drainage basin of Jordan, several fishes were found to have characters intermediate between two cyprinid species. Barbus canis Valenciennes in Cuvier & Valenciennes. 1842 and Capoeta damascina (Valenciennes in Cuvier & Valenciennes. 1842). The hypothesis of their hybrid origin is advanced. A key to the large barbine cyprinids of the Jordan River system and their hybrids is given.