ULTRASTRUCTURAL ORGANIZATION OF NEURONS OF THE LATERAL PREOPTIC NUCLEUS OF THE HYPOTHALAMUS OF OLD RATS UNDER VARIOUS LIGHT CONDITIONS

Purpose – to study the changes in the ultrastructure of neurons of the lateral preopticnucleus (LPON) of the hypothalamus of old rats under various light conditions.Material and methods. The experiments were carried out on 36 old white male rats.The material was fixed in a 2.5% solution of glutaraldehyde, prepared on the basisof phosphate buffer with a pH of 7.2–7.4. Then, postfixation was performed in a 1%solution of osmium tetraoxide and dehydrated in propylene oxide, after which it waspoured into a mixture of epoxy resins. Ultrathin sections made on an ultramicrotomeLKB-3 were contrasted with uranium acetate and lead citrate according to the Reynoldsmethod and studied under electron microscope TEM - 125 K.Results. Studies of LPON neurons under the standard light mode revealed nuclei withuneven contours. The nucleoli are quite large. The neuroplasm contains well-developedtubules of the rough endoplasmic reticulum (ER) and small cisternae of the Golgi complex(GC). Mitochondria are rounded, small, with moderately pronounced cristae. Underconditions of round-the-clock darkness, we have found that the cell nuclei are rounded,less often determined by the nucleoli. In the neuroplasm there are locally dilated tubulesof the ER and cisternae of the GC, mitochondria with enlightened matrix and fragmentedcristae. Under conditions of round-the-clock illumination, the nuclei of the rounded formwith uneven contours of a nuclear membrane forming deep intussusception are revealed.Nucleoli were rarely identified. In the hyaloplasm, locally dilated tubules of the ERare identified. Mitochondria are small in size with an enlightened matrix and reducedcristae.Conclusions. The obtained results of submicroscopic examination of LPON neuronsof the hypothalamus of old rats revealed their relatively increased functional activityin the dark period. Under conditions of round-the-clock lighting, more pronouncedhypertrophic and initial destructive changes of the nuclei and organelles of the neuronsof the LPON of the hypothalamic were revealed, compared with the animals that wereunder the conditions of round-the-clock darkness. This is confirmed by the change in theultrastructure of nerve cells at 2 am the appearance of "dark" cells.

MORPHOGENESIS OF THE WALL OF GLANDULAR PART OF THE STOMACH IN CHICKENS DURING POSTNATAL PERIOD OF ONTOGENESIS

Knowledge about the morphological features of the structure and functions of the digestive organs provide the basis for rational and effective use of feed, prevention and treatment of gastrointestinal diseases in poultry. In this regard, the study of the morphogenesis of the digestive system in birds and the mechanisms of their regulation is of great importance. The object of the study was the glandular part of the stomach (proventriculus) in chickens of Shaver 579 strain. The material for macro- and microscopic examinations was selected from birds at age of 1, 30, 60, 90, 120, 150, 180, 210, 240, 270, and 300 days and 1, 2, and 3 years; it was fixed in a 10% neutral formalin solution and embedded into paraffin according to conventional methods. For submicroscopic examinations, the material was selected from hens of this strain at age of 180 days. The structure of epitheliocytes in the superficial epithelium and secretory cells of the deep glands was studied in ultrathin sections. Digital indicators of research results were statistically processed by a personal computer using Microsoft Excel program. The glandular stomach in chickens is a direct extension of the esophagus and has the form of a thick-walled tube, the wall of which is formed by mucous, muscular, and serous membranes. The superficial epithelium of the mucosa is represented by cylindrical epithelial cells that are located within the basal membrane. They are linked to each other by different types of contacts and have a well-defined polar differentiation. The lobules of the deep glands are formed by cells with well-developed synthesizing organelles and secretory granules. The morphogenesis of the glandular part of the stomach in chickens according to age aspect is manifested by changes in morphometric parameters of the thickness and the area of the membranes of its wall. The wall thickness increases (between the folds 4223.23 ± 189.25 and in the area of the folds 5561.32 ± 45.01 μm) unevenly in chickens up to age of 240 days. The most developed membrane of the wall of the glandular stomach is the mucosa. Its area increases (by 82.14 ± 0.56%) in chickens up to age of 180 days, and area of muscular and serous membranes decreases (by 15.54 ± 0.65 and 2.32 ± 0.33%, respectively). In older birds, the thickness of the wall and the area of the membranes of the glandular stomach do not change significantly.

Improved analysis method of neuromuscular junction in Drosophila larvae by transmission electron microscopy

The Drosophila neuromuscular junction is an excellent model for neuroscience research. However, the distribution of neuromuscular junctions is very diffuse, and it is not easy to accurately locate during ultrathin sectioning, which seriously interferes with the ultrastructural analysis under electron microscopy that only has a small field of view. Here, we reported an efficient method for acquiring the ultrastructural picture of neuromuscular junctions in Drosophila larva under electron microscopy. The procedure was as follows: first, the larval sample of body wall muscle was placed between the metal mesh and was dehydrated with alcohol and infiltrated with epoxy resin to prevent the sample from curling or bending, after it was dissected and fixed into thin slices. Second, the sample was embedded in resin into a flat sheet to facilitate the positioning of the muscles. Third, carefully and gradually remove the excess resin and the cuticle of the larvae, cut off both ends of the special body segment, and trim the excess specific muscles according to the recommended ratio of trimming muscles, which would reduce the workload exponentially. At last, the trimmed sample were prepared into serial about 1000 ultrathin sections that was about total 80 microns thickness, and 30–40 sections were gathered into a grid to stain with lead citrate and uranyl acetate. This method could also be applied to the other small and thin samples such as the Drosophila embryo, ventral nerve cord and brain.

Establishment of a New Cell Line of Canine Mammary Tumor CMT-1026

Canine mammary tumors (CMTs) have histopathological, epidemiologic and clinical characteristics similar to those in humans and are known to be one of the best models for human breast cancer (HBC). This research aimed to describe a newly established canine cell line, CMT-1026. Tumor samples were collected from a female dog exhibiting clinical mammary neoplasm, and the adherent cells were cultured. Both the histology and immunohistochemistry (IHC) of tumor samples were estimated. Cell growth, ultrastructural, cytological and immunocytochemistry (ICC) features of CMT-1026 were examined. CMT-1026 cells were inoculated into 10 female BALB/c nude mice to evaluate oncogenicity and metastatic ability. Hematoxylin-eosin (H.E.) staining of the tumors revealed an epithelial morphology. Electron microscopy was used to detect histological and cytological of smears, and ultrathin sections showed that CMT-1026 cells were polygonal and characterized by atypia and high mitotic index in the tumor, with prominent nucleoli and multinucleated cells. IHC characterization of CMT-1026 indicated ER-, PR-, HER-2, p63+, CK5/6+, and α-SMA+ epithelial cells. ICC characterization of CMT-1026 showed high expression of Claudin-1, Delta-catenin, SOX-2, and KI-67. At 2 weeks after inoculation of the CMT-1026 cells, phyma was found in 100% of the mice. The xenograft cancers showed conservation of the original H.E. features of the female dog cancer. In conclusion, CMT-1026 may be a model of canine mammary cancer that can be used in research on the pathogenesis of both CMT and HBC.

Existence of giant mitochondria-containing sheet structures lacking cristae and matrix in the etiolated cotyledon of Arabidopsis thaliana

Mitochondria are essential organelles involved in the production and supply of energy in eukaryotic cells. Recently, the use of serial section scanning electron microscopy (S3EM) has allowed accurate three-dimensional (3D) reconstructed images of even complex organelle structures. Using this method, ultrathin sections of etiolated cotyledons were observed 4 days after germination of Arabidopsis thaliana in the dark, and giant mitochondria were found. To exclude the possibility of chemical fixation artifacts, this study confirmed the presence of giant mitochondria in high-pressure frozen samples. The 3D reconstructed giant mitochondria had a complex structure that included not only the elongated region but also the flattened shape of a disk. It contained the characteristic sheet structure, and the sheet lacked cristae and matrix but consisted of outer and inner membranes. Whether this phenomenon could be observed in living cells was investigated using the transformant with mitochondrial matrix expressing green fluorescent protein. Small globular mitochondria observed in light-treated samples were also represented in etiolated cotyledons. Although no giant mitochondria were observed in light-treated samples, they were found in the dark 3 days after germination and rapidly increased in number on the fourth day. Therefore, giant mitochondria were observed only in dark samples. These findings were supported by electron microscopy results.

Structural and Biochemical Aspects Related to Resistance and Susceptibility of Rubber Tree Clones to Anthracnose

The aim of the present study was to evaluate structural and biochemical aspects related to the interaction of resistant (RRIM 937, IAC 502 and 507) and susceptible (RRIM 600) rubber tree clones with C. tamarillo. For such analysis, ultrathin sections of the leaf limb were embedded in historesin and differently stained to verify structural alterations and presence of starch grains, arginine, lipids, tannins and lignins. The total proteins and activity of the enzymes peroxidase and (PAL) were quantified. Stomatal density was also analyzed under a scanning electron microscope. Data indicated alterations in the cell content of resistant clones inoculated with the pathogen, as well as greater lignin and lipid accumulation in these samples. For tannins, there was no difference between inoculated and non-inoculated clones. Arginine was found at greater quantities in IAC 502 and 507. Starch grains were not detected in any of the analyzed samples. Protein level and stomatal density were lower in resistant clones. Peroxidase activity was more expressive in resistant clones. PAL activity, there was no significant difference between clones. The lignin and lipids, total protein, peroxidase activity and stomatal density may be related to the resistance of rubber tree clones to anthracnose.

STUDY OF THE COMPOSITION OF ESSENTIAL OIL ACHILLEA NOBILIS L. AND ITS ANTIBACTERIAL INFLU-ENCE ON CHANGE OF STAPHYLOCOCCUS AUREUS CELL ULTRASTRUCTURE

The component composition of the Achillea. nobilis essential oil, which grows in Azerbaijan, was studied for the first time by the gas-chromato-mass spectrometry method. In the essential oil, 35 components are identified, of which the predominant are artemisia ketone (23.706%), thujone 22.400%), 2-bornanone (6,367%), eudesm-7(11)-en-4-ol (6.321%), eucalyptol (4.507%), cubenol (3.317%), lavandulol (2.975%), β-thujone (2.933%), β-eudesmol (2.702%), methyl hinokiate (2.108%), terpinen-4-ol (1.715%), 1,2-longidione (1.313%), limonene-6-ol, pivalate (1.188%), neryl (S)-2-methylbutanoate (1.120%), caryophyllene oxide (1.014%). The effect of A. nobilis essential oil on the ultrastructural organization of bacteria (S. aureus) was studied using transmission electron microscopy. It was revealed that under the influence of essential oil, a violation of the plasma permeability of the plasma membrane is observed, accompanied by diffusion of the fine-grained osmiophilic precipitate of the destroyed cytoplasmic structures, which leads to the loss of the layered structure of the wall elements and to a significant compaction of the matrix in comparison with the control preparations. The death of bacterial cells was observed with complete destruction of the integrity of all structures that make up their wall. Along with this, on ultrathin sections, focal accumulations of dead bacterial cells are found, surrounded by structurally changed elements of their walls. The remains of destructively altered fragments of CP are found on various parts of unseparated bacterial cells. All of the above shows that the essential oil of A. nobilis has a pronounced antibacterial activity.

Characterization of Antennal Sensilla and Immunolocalization of Odorant-Binding Proteins on Spotted Alfalfa Aphid, Therioaphis trifolii (Monell)

The spotted alfalfa aphid [Therioaphis trifolii (Monell), Homoptera, Drepanosiphidae] is a well-known destructive pest that can significantly reduce alfalfa yields. Herein, the morphology of antennal sensilla of T. trifolii has been examined by using scanning electron microscopy and the ultrastructure of sensilla stellate and placoidea was described by transmission electron microscopy. Stellate sensilla, placoid sensilla, and coeloconic sensilla were found on the 6th segment, and a single sensillum placoidea was located on the 5th segment. Placoid sensilla were also present on the 3rd antennal segment of alate and apterous aphids, and the number was similar between two morphs. Two types of trichoid sensilla and coeloconic sensilla were found on the antennae, respectively. The results of ultrastructure showed that stellate sensilla are innervated by three neurons, while placoid sensilla present three groups of neurons, equipped with 2–3 dendrites in each neuron group. Immunocytochemical localization of odorant-binding proteins (OBPs) was performed on ultrathin sections of sensilla stellate and placoidea, and we observed that the antiserum against OBP6 intensively labeled all placoid sensilla from both primary and secondary rhinaria. OBP7 and OBP8 could also be detected in placoid sensilla, but less strongly than OBP6. In addition, OBP6, OBP7, and OBP8 were densely labeled in stellate sensilla, suggesting OBP6, OBP7, and OBP8 may sense alarm pheromone germacrene A in T. trifolii.