A. A. Ogienko

Basic Aspects of Ovarian Development in Drosophila melanogaster

Modern views of the development and structural organization of the female reproductive system in Drosophila melanogaster are reviewed. Special emphasis is placed on the generation and development of follicles in the germarium and the interactions of germline and somatic cells in the egg chamber. Detailed consideration is given to the main events that ensure and regulate the transport of mRNA, proteins, and organelles from nurse cells to the oocyte in the germarium and at later stages of egg chamber development.

Molecular and genetic description of a new hypomorphic mutation of Trithorax-like gene and analysis of its effect on Drosophila melanogaster oogenesis

The Trithorax-like (Trl) gene of Drosophila melanogaster encodes the multifunctional protein GAGA involved in many cellular processes. We have isolated and described a new hypomorphic mutation of The Trl gene—Trlen82. The mutation is the insertion of a 1.4 kb P-element into the 5′ untranslated region. Trl expression decreased in the ovaries of mutant flies by about 30%; however, it caused abnormalities. The Trlen82 mutation combined with the null allele of Trl caused female sterility: the females laid a few small eggs with abnormal shape. Many egg chambers demonstrated abnormalities in the Trlen82 mutants: the oocyte had a regular shape and intruded into the egg chamber region with nurse cells; the rapid transport of nurse cell cytoplasm into the oocyte was disturbed, which resulted in the “dumpless” phenotype of the chambers in mutants; follicular cells often did not completely cover the oocyte and concentrated on its posterior end; and the migration of centripetal cells was affected. We propose that the sterility of the Trlen82 females is due to the abnormal functioning of follicular cells resulting from low Trlexpression. This proposal is confirmed by normalizing the mutant phenotype of Trlen82 females after the transfection of Trl cDNA. Note that even an insignificant decrease in Trl expression in such females seriously affected the somatic cell functioning, while a significant decrease in its expression in strong hypomorphic mutants affected both somatic and germline cells in the egg chambers.

GAGA protein is essential for male germ cell development in Drosophila

The Drosophila Trithorax‐like (Trl) gene encodes a GAGA factor which regulates a number of developmentally important genes. In this study, we identify a new function for Drosophila GAGA factor in male germ cell development. Trl mutants carrying strong hypomorphic alleles display loss of primordial germ cells during their migration in embryogenesis and severe disruption in mitochondria structure during early spermatogenesis. The mutation resulted in small testes formation, a deficit of germ cells, abnormal mitochondrial morphogenesis, spermatocyte death through autophagy, and partial or complete male sterility. Pleiotropic mutation effects can be explained by the misexpression of GAGA factor target genes, the products of which are required for germ cell progression into mature sperm. genesis 52:738–751, 2014.

Hemozoin is a product of heme detoxification in the gut of the most medically important species of the family Opisthorchiidae.

Many species of trematodes such as Schistosoma spp., Fasciola hepatica and Echinostoma trivolvis are blood-feeding parasites. Nevertheless, there is no consensus on the feeding habits of the family Opisthorchiidae (Opisthorchis felineus, Opisthorchis viverrini and Clonorchis sinensis). Previously, histological studies of O. felineus and C. sinensis revealed some dark stained material in their gut lumen. In this study we conducted a comprehensive analysis of the gut contents of three members of the family Opisthorchiidae (O. felineus, O. viverrini and C. sinensis). Using transmission electron microscopy, we demonstrated for the first known time the presence of disintegrating blood cells in the gut of O. felineus as well as electron-dense crystals in the gut of O. felineus and C. sinensis. Electron energy loss spectroscopy revealed iron atoms in these crystals, and mass spectrometry of the purified pigment demonstrated the presence of heme. Fourier-transform infrared spectroscopy identified the signature peaks of the common iron-carboxylate bond characteristic in crystals isolated from O. felineus and C. sinensis. Scanning electron microscopy showed layered ovoid crystals of various sizes from 50 nm to 2 μm. Morphological, chemical and paramagnetic properties of these crystals were similar to those of hemozoin from Schistosoma mansoni. Crystal formation occurs on the surface of lipid droplets in O. felineus and C. sinensis guts. Our results suggest that the diet of O. felineus and C. sinensis includes blood. Detoxification of the free heme produced during the digestion proceeds via formation of insoluble crystals that contain iron and heme dimers, i.e. crystals of hemozoin. Furthermore, we believe that biocrystallisation of hemozoin takes place on the surface of the lipid droplets, similar to S. mansoni. Hemozoin was not detected in the closely related species O. viverrini.

Capping protein beta is required for actin cytoskeleton organisation and cell migration during Drosophila oogenesis.

Capping protein (CP) is a well‐characterised actin‐binding protein important for regulation of actin filament (AF) assembly. CP caps the barbed end of AFs, inhibiting the addition and loss of actin monomers. In Drosophila melanogaster, the gene encoding CP β‐subunit is named capping protein beta (cpb; see Hopmann et al. [1996] J Cell Biol 133: 1293–305). The cpb level is reduced in the Drosophila bristle actin cytoskeleton and becomes disorganised with abnormal morphology. A reduced level of the CP protein in ovary results in disruption of oocyte determination, and disturbance of nurse cell (NC) cortical integrity and dumping. We describe novel defects appearing in cpb mutants during oogenesis, in which cpb plays an important role in border and centripetal follicle cell migration, ring canal development and cytoplasmic AF formation. The number of long cytoplasmic AFs was dramatically reduced in cpb hypomorphs and abnormal actin aggregates was seen on the inner side of NC membranes. A hypothesis to explain the formation of abnormal short‐cut cytoplasmic AFs and actin aggregates in the cpb mutant NCs was proffered, along with a discussion of the reasons for ‘dumpless’ phenotype formation in the mutants.

Large porous particles for respiratory drug delivery. Glycine-based formulations

Abstract Large porous particles are becoming increasingly popular as carriers for pulmonary drug delivery with both local and systemic applications. These particles have high geometric diameters (5–30 &mgr;m) but low bulk density (˜ 0.1 g/cm3 or less) such that the aerodynamic diameter remains low (1–5 &mgr;m). In this study salbutamol and budesonide serve as model inhalable drugs with poor water solubility. A novel method is proposed for the production of dry powder inhaler formulations with enhanced aerosol performance (e.g. for salbutamol‐glycine formulation the fine particle fraction (FPF ≤ 4.7 &mgr;m) value is 67.0 ± 1.3%) from substances that are poorly soluble in water. To overcome the problems related to extremely poor aqueous solubility of the APIs, not individual solvents are used for spray freeze‐drying of API solutions, but organic‐water mixtures, which can form clathrate hydrates at low temperatures and release APIs or their complexes as fine powders, which form large porous particles after the clathrates are removed by sublimation. Zwitterionic glycine has been used as an additive to API directly in solutions prior to spray freeze‐drying, in order to prevent aggregation of powders, to enhance their dispersibility and improve air‐flow properties. The clathrate‐forming spray freeze‐drying process in the multi‐component system was optimized using low‐temperature powder X‐ray diffraction and thermal analysis. Graphical abstract Figure. No Caption available.

[Generation and analysis of novel mutations of the trithorax-like gene in Drosophila melanogaster].

The Trithorax-like (Trl) gene of Drosophila melanogaster encodes the multifunctional GAGA factor. The expression of Trl is known to depend on numerous factors, such as the organ, the tissue, the ontogenetic stage, and the ambient temperature. Apparently, this expression is controlled by a complex system of regulatory elements, which so far has been scarcely studied. Our preliminary results indicate that the second intron of the Trl gene bears functionally significant elements. To test this assumption, we generated 23 novel alleles of the gene via P-induced male recombination and analyzed them cytogenetically. Of these mutations, 13 (recessive lethals) are deletions, disrupting the coding gene region. Ten mutations (seven deletions and three duplications) remove parts of the second Trl intron only. Some of these mutant stocks exhibit lower viability at different temperatures. These results suggest that the second intron region harbors functionally significant elements. The deletion mapping results verified the localization of the Trl gene in the 70F1-2 region.

Analysis of a novel hypomorphic mutation in Trithorax-like gene affecting Drosophila melanogaster oogenesis

We generated and characterized a new hypomorphic mutation of Drosophila melanogaster Trithorax-like (Trl) gene named Trl362. The Trl362 homozygous females are sterile and lay a small number of eggs; most embryos die at the early developmental stages. The transcriptional Trl level of adult Trl362 females was markedly lowered. Little or no GAGA protein, encoded by Trl, was detected in the nurse cell nuclei. The ovaries of Trl362 females showed impairments, such considerable changes in the structure of both ovarioles and individual egg chambers. We believe that the observed ovarian defects in Trl362 mutants are mostly due to a decreased amount of GAGA protein in the germline cells. An increase of GAGA-519 protein caused by introduction of hsp83:GAGA-519 transgene against Trl362 background rescued partially the female fertility. It may well be that a decrease of GAGA protein in Trl362 germline cells leads to a defective expression of the genes regulated by transcription factor GAGA, whose products are essential for normal Drosphila oogenesis.

A new method for obtaining fine powders of paracetamol for compression without excipients

Paracetamol (N(phydroxyphenyl)acetamide) is a widely used nonnarcotic analgesic having also anti inflammatory and antipyretic action. Several crystalline polymorphs of paracetamol are known. One of these (monoclinic form I) is thermodynamically stable and is readily prepared but cannot be compressed to tablets without excipients (fillers). Another polymorph (orthorhombic form II) can be readily compressed to tablets without excipients [1, 2] and is better soluble but its formation as a pure phase is not reproducible, and, what is worse, it is metastable and is spontaneously converted to the monoclinic form on storage [1]. The idea of obtaining compressible forms of paracetamol that would be stable on storage attracts considerable attention of the scientific community and pharmaceutical companies. For solving this problem, it has been proposed to use, instead of pure paracetamol, its mixtures with polyvinylpyrrolidone [3], carbohydrates [4], chitosan and sodium alginate [5] or mixed crystals based on oxalic acid, naphthalene and other compounds [6] or the inclusion compounds with hydroxypropylβcyclodextrin [7].

Role of GAGA factor in drosophila primordial germ cell migration and gonad development

The GAGA protein of drosophila is a factor involved in epigenetic transcription regulation of a large gene group controlling developmental processes. In this paper, the role of GAGA factor in germ cell migration is demonstrated as well as its effect on the gonad development in drosophila embryogenesis. Mutations in the Trl gene, encoding GAGA factor, prematurely induces the active migration program and relocation of the primordial cells inward the embryo before the beginning of gastrulation. The germ cells that prematurely separated from the main group migrate ectopically, lose orientation, and stay out of gonad development. Expression pattern of the Trl gene suggests its activity in epithelial cells of the embryonic blastoderm, part of which contact primordial cells. Thus, GAGA factor influences migration of these cells in an indirect manner via their somatic environment.