Lipid Inclusions Research Articles

Different ecophysiological and structural strategies of toxic and non-toxic Microcystis aeruginosa (cyanobacteria) strains assessed under culture conditions

Microcystis aeruginosa causes severe problems in freshwater environments worldwide due to its capacity to form blooms and produce toxins. Natural populations contain a mixture of strains that vary in morphology, genotype and microcystin production. Beyond its toxic effect in animals, the possible role of microcystin in the metabolism of the producing cell is also a topic of interest. Recent studies pointed to a protective role against cellular oxidative stress. Since natural populations include microcystin producing and non-producing strains, the latter must rely on other ecophysiological traits to thrive. Here, we compared a toxic and a non-toxic strain of M. aeruginosa displaying different morphotypes and growth behaviors, aiming to explore the role of microcystin in an integrated way to the primary metabolism. The toxic strain formed large floating colonies (150–1850 μm) with densely aggregated cells, numerous gas vesicles and lipid inclusions and a typical irregular thylakoid arrangement. The non-toxic strain remained submerged, formed small colonies (< 100 μm) with a dense mucilage covering few sparse cells with rare gas vesicles and various types of thylakoids. It showed a higher abundance of carboxysomes, cyanophycin and polyphosphate granules and also greater amounts of pigments than the toxic strain. The antioxidant potential was higher for the toxic strain and it presented a higher expression of proteins related to photosynthesis, protein folding and cellular redox homeostasis. These traits of the toxic strain were compatible with growth under higher irradiance and microcystin can be a key factor to this adaptation. Growing submerged, the non-toxic strain showed preference for a lower light intensity and invested in intracellular reserves. This study illustrates different adaptive strategies of M. aeruginosa morphotypes that can be valuable in nature, enabling this species to widely exploit freshwater ecosystems.

Dissecting the membrane lipid binding properties and lipase activity of Mycobacteriumtuberculosis LipY domains.

The Mycobacteriumtuberculosis LipY protein, a prototype of the proline-glutamic acid (PE) family, exhibits a triacylglycerol (TAG) hydrolase activity that contributes to host cell lipid degradation and persistence of the bacilli. LipY is found either as a full-length intracytosolic form or as a mature extracellular form lacking the N-terminal PE domain. Even though the contribution of the extracellular form in TAG consumption has been partly elucidated, very little information is available regarding the potential interactions of either full-length LipY with the cytoplasmic membrane, or mature form LipY with the outer membrane. Herein, several LipY variants truncated in their N-terminal domain were produced and biochemically characterized in lipid-protein interaction assays, using the monomolecular film technique and FTIR. Comparison of the catalytic activities of these recombinant proteins showed that LipY∆149, corresponding to the extracellular form of LipY lacking the PE domain, is more active than the full-length protein. This confirms previous studies reporting that the PE domain negatively modulates the TAG hydrolase activity of LipY. Lipid-protein interaction studies indicate that the PE domain anchors LipY onto membrane lipids. Consistent with these findings, we show that LipY∆149 is loosely associated with the mycobacterial cell wall, and that this interaction is mediated by the sole lipase domain. Overall, our results bring new information regarding the molecular mechanisms by which LipY either binds and hydrolyses host cell lipids or degrades TAG, the major source of lipids within mycobacterial intracytosolic lipid inclusions.

Application of CNTF or FGF-2 increases the number of M2-like macrophages after optic nerve injury in adult Rana pipiens.

We have previously shown that a single application of the growth factors ciliary neurotrophic factor (CNTF) or fibroblast growth factor 2 (FGF-2) to the crushed optic nerve of the frog, Rana pipiens, increases the numbers and elongation rate of regenerating retinal ganglion cell axons. Here we investigate the effects of these factors on the numbers and types of macrophages that invade the regeneration zone. In control PBS-treated nerves, many macrophages are present 100 μm distal to the crush site at 1 week after injury; their numbers halve by 2 weeks. A single application of CNTF at the time of injury triples the numbers of macrophages at 1 week, with this increase compared to control being maintained at 2 weeks. Application of FGF-2 is equally effective at 1 week, but the macrophage numbers have fallen to control levels at 2 weeks. Immunostaining with a pan-macrophage marker, ED1, and a marker for M2-like macrophages, Arg-1, showed that the proportion of the putative M2 phenotype remained at approximately 80% with all treatments. Electron microscopy of the macrophages at 1 week shows strong phagocytic activity with all treatments, with many vacuoles containing axon fragments and membrane debris. At 2 weeks with PBS or FGF-2 treatment the remaining macrophages are less phagocytically active, containing mainly lipid inclusions. With CNTF treatment, at 2 weeks many of the more numerous macrophages are still phagocytosing axonal debris, although they also contain lipid inclusions. We conclude that the increase in macrophage influx seen after growth factor application is beneficial for the regenerating axons, probably due to more extensive removal of degenerating distal axons, but also perhaps to secretion of growth-promoting substances.

In vivo anti-arthritic effect and repeated dose toxicity of standardized methanolic extracts of Buddleja cordata Kunth (Scrophulariaceae) wild plant leaves and cell culture

Ethnopharmacological relevanceBuddleja cordata Humb. Bonpl. & Kunth, known by the population as Tepozán blanco, is a shrub plant used in traditional herbal medicine in Mexico for the treatment of tumors, cancer, sores, skin burns, rheumatic pains and diseases related to inflammatory processes such as arthritis; authors adjudicate this etno-medicinal effect to the presence of secondary metabolites in the plant such as verbascoside, however due to its low concentration in recent years biotechnological tools are applied as cell culture to biosynthesize these pharmacological active metabolites in greater quantities.Aim of the study: Evaluate the possible toxic effect after a daily administration of MeOH extracts from wild plant leaves (Bc-Wp), and cell culture (Bc-Cc) of B. cordata for 28 days, and after their anti-edematous and antioxidant activities in vivo, as well their effect on the cytokines profile during experimental arthritis induced by complete Freund's adjuvant. Materials and methodsBoth extracts were evaluated in CD1 male mice first in a toxicity test of repeated dose administrations (1 g/kg) for 28 days, after which pharmacological activity of both extracts was measure during experimental induced arthritis where three doses were tested, at the end of the study edema formation, body weight gain and antioxidant activity were measure in edema and ganglionic tissues. Finally, dose that exerted the best protective effect (250 mg/kg) was evaluated to quantify its effect over ganglionic tissue concentration of lymphocytes T CD4+, and cytokines (IL-1β, TNF-α and IL-10), as well histological analysis in arthritic mice. ResultsBoth extracts (Bc-Wp and Bc-Cc) did not exert lethality, however body weight gain and food in-take were lower than in healthy mice administered with vehicles, also extract-treated animals showed a decrease in serum lipid concentration and only Bc-Wp extract treated animals decrease serum alkaline phosphatase after 28 daily administration compared to healthy un-treated mice. During experimental arthritis best inhibition effect over edema development was observed in those animals administered with both extracts at dose of 250 mg/kg (Bc-Wp and Bc-Cc) on day 28, compared to CFA un-treated mice. Also both extracts reduce oxidative damage over lipids and proteins at the same dose, in both ganglionic and edema tissue, as well antioxidant enzymatic response was reduced in both tissues compared to arthritic un-treated group. In ganglionic tissue of arthritic mice, CD4+ lymphocytes concentration was reduced by Bc-Wp and Bc-Cc treatment (250 mg/kg) respectively, as well IL-1β, and TNF-α levels. Only arthritic animals treated with Bc-Cc extract at 250 mg/kg generated a significant increase of IL-10 doubling the levels compared to CFA un-treated group. Histological analysis of popliteal ganglion showed that both extracts decrease the incidence of lytic lesions, lipid inclusions and leukocyte infiltration. ConclusionMethanolic extracts of wild Buddleja cordata and its cell cultures did not generated lethality after a daily administration for 28 days at 1 g/kg, but it was observed that both showed a lipid-lowering effect. Also at dose of 250 mg/kg both extracts exerted anti-edematous, protection against the oxidation of lipid and proteins, regulation on antioxidant enzymatic response, down-regulation on lymphocytes CD4+ producers of IL-1β and TNF-α, an increase in IL-10 levels, which caused a decrease in leukocyte infiltration in ganglionic tissue during experimental arthritis.

Characterization of fat body cells at different developmental stages of Culex pipiens

The fat body, originates from mesoderm, has many metabolic functions which changes as the embryonic development of the insect progresses. It plays an important role in the intermediate metabolism and in the metabolism of proteins, lipids and carbohydrates. It has roles in synthesis, absorption and storage of nutrients from hemolymph. It is also responsible for the production of immunological system components, antibacterial compounds and blood clotting proteins. The most common type of fat body cells are trophocytes (the basic cells of the fat body) and oenocytes are found associated with the fat body. In this study, it is aimed at determining the cell types contained in the fat body of Culex pipiens at different developmental stages as well as identifying the molecules such as carbohydrate, protein and lipid contained in each of these cells. Knowing the regional distribution of the fat body cells and the concentration of its content at each developmental stage is important in understanding the process related to its physiology and it may help in fighting against the pest C. pipiens, which is a vector species for many contagious diseases observed in humans and other species.To achieve our goal, we have employed different histochemical techniques (fixatives and staining methods) for staining C. pipiens preparates of different developmental stages and analyzed the structure of the fat body, its distribution, its cell types and the macromolecular contents of the cells. We only observed trophocytes and oenocytes as fat body components in C. pipiens. The trophocytes had all the three macromolecules (lipids, proteins, carbohydrates) in the cytoplasm varying in concentration between the different regions and different stages. The oenocytes were observed below the integument as well as between the muscles in the larvae of Culex pipiens. They were present either as single cells or in clusters and also varied in size. Their cytoplasm was stained strongly for proteins when bromophenol blue staining was applied, but it was rather heterogeneous due to the lipid inclusions. On the contrary, oenocytes were not observed among the adult C. pipiens preparations.

Pathomorphological changes in parenchymatose organs of rabbits in case of chronic passalurosis

Pathomorphological changes, developing in case of pasalurosis chronic course in rabbits are described in the article. The research was held on ten rabbits of Large Grey breed, which were held on household farms of Poltava region. The diagnosing was complex: based on anamnesis data, clinical symptoms’ analysis, and the results of pathomorphological examination. The fragments of the liver, spleen, and kidneys were taken for histological analysis. While making histological analysis of the spleen it was revealed that lymphoid nodules were enlarged, some of them were of uniform structure, and others were in the state of hyperplasia. Non-uniformity of structure in addition to changing the size was registered in those nodules: there was less concentration of lymphoid cells in their central part and more concentration on the periphery. The situation was contrary in separate lymphoid nodules. The red pulp consisted of a large number of lymphoid cells and erythrocytes, the largest number of which were registered in the zones adjoining the places of lymphoid nodules’ location. Sometimes there were megakaryocytes in the field of vision. It was established, that blood filling of arteries was less than normal. In case of using some preparations, bilirubin inclusions were found at having big enlargements in red pulp. Grainy and fatty hepatocytes’ dystrophies, and also lymphohistiocytic interstitial hepatitis were also registered. At the same time, renal corpuscles were enlarged. Rather big spaces, filled with transparent substance containing vessel and cellular fragments (detritus), were revealed in the majority of them between glomeruli and the capsule.Capsule epithelial cells were destroyed in some places. Petechial hemorrhages were registered inside glomeruli. The destruction of separate podocytes and deep cells was noticed. The tubular epithelial cells were enlarged in size, and in some cases they were of cylinder, but not cubic shape; the spaces of such tubular were becoming correspondingly less. The epithelial cell cytoplasm of the tubular in some cases was not uniformand it was of cloudy-grey color: in other cases it had rather large colorless areas and, probably, looked like lipid inclusions (which were washed out from the cells as a result solvent impact during making histological preparations). Concentrations of eosinophilic masses (protein cylinders) sometimes containing certain amount of erythrocytes were often found in canaliculi spaces. Sometimes the destruction of epithelial cells was registered in the tubulеs, and their fragments were found in spaces. As a result of histological examination, the diffuse lymphohistiocytic infiltration in the renal interstitial tissue was found.

CRISPR/Cas9-mediated knockout of Abcd1 and Abcd2 genes in BV-2 cells: novel microglial models for X-linked Adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD), the most frequent peroxisomal disorder, is associated with mutation in the ABCD1 gene which encodes a peroxisomal ATP-binding cassette transporter for very long-chain fatty acids (VLCFA). The biochemical hallmark of the disease is the accumulation of VLCFA. Peroxisomal defect in microglia being now considered a priming event in the pathology, we have therefore generated murine microglial cells mutated in the Abcd1 gene and its closest homolog, the Abcd2 gene. Using CRISPR/Cas9 gene editing strategy, we obtained 3 cell clones with a single or double deficiency. As expected, only the combined absence of ABCD1 and ABCD2 proteins resulted in the accumulation of VLCFA. Ultrastructural analysis by electron microscopy revealed in the double mutant cells the presence of lipid inclusions similar to those observed in brain macrophages of patients. These observations are likely related to the increased level of cholesterol and the accumulation of neutral lipids that we noticed in mutant cells. A preliminary characterization of the impact of peroxisomal defects on the expression of key microglial genes such as Trem2 suggests profound changes in microglial functions related to inflammation and phagocytosis. The expression levels of presumed modifier genes have also been found modified in mutant cells, making these novel cell lines relevant for use as in vitro models to better understand the physiopathogenesis of X-ALD and to discover new therapeutic targets.

Genome Sequencing and Comparative Transcriptomics Provide a Holistic View of 4-Nitrophenol Degradation and Concurrent Fatty Acid Catabolism by Rhodococcus sp. Strain BUPNP1.

Rhodococcus sp.strain BUPNP1 can utilize the priority environmental pollutant 4-nitrophenol (4-NP) as its sole source of carbon and energy. In this study, genome and transcriptome sequencing were used to gain mechanistic insights into 4-NP degradation. The draft BUPNP1 genome is 5.56 Mbp and encodes 4,963 proteins, which are significantly enriched in hypothetical proteins compared to other Rhodococcus sp. A novel 4-NP catabolic 43 gene cluster “nph” was identified that encodes all the genes required for the conversion of 4-NP into acetyl-CoA and succinate, via 4-nitrocatechol. The cluster also encodes pathways for the catabolism of other diverse aromatic compounds. Comparisons between BUPN1 growing on either 4-NP or glucose resulted in significant changes in the expression of many nph cluster genes, and, during 4-NP growth, a loss of lipid inclusions. Moreover, fatty acid degradation/synthesis genes were found within the nph cluster, suggesting fatty acids may be concurrently catabolised with 4-NP. A holistic model for the action of the nph gene cluster is proposed which incorporates genetic architecture, uptake and metabolism of aromatic compounds, enzymatic activities and transcriptional regulation. The model provides testable hypotheses for further biochemical investigations into the genes of the nph cluster, for potential exploitation in bioremediation.

Karyometric and electron microscopic studies of dark and light cells of the adrenal cortex in mammals

The purpose of this study is a comparative study of dark and light cells of cortical parenchyma of the adrenal glands in mammals using light and electron microscopy. Material and methods. Cariometric and electron microscopic studies of the adrenal glands of 9 mammalian species have been carried out: Vulpes vulpes (fox), Vulpes lagopus (arctic fox), Canis familiaris (dog), Мartes zibellina (sable), Mustela vison (mink), Enhydra lutris (sea ape); Castor fiber (river beaver); Callorhinus ursinus (fur seal); Ovis aries (sheep). Serial paraffin sections were stained with hematoxylin and eosin, iron hematoxylin, Heidenheim azan, the PAS-reaction was used, the color of the trichrome-PAS and the tetrachromium-PAS. RNA was detected by the reaction of Brashe and gallocyanin by Einarsson. For the detection of lipids, frozen sections were stained with Sudan III + IV, Black Sudan. The number of dark and light cells in the cortex of the adrenal mink in different seasons was determined. On electron micrographs, the amount of lipid droplets and mitochondria in the fascicle of the adrenal cortex was counted. Results. In the cortex of the adrenal glands of mammals, dark and light cells are present. Dark cells, as far as accumulation of sudanophilic substances are converted into light cells, which with further accumulation of lipids undergo destructive changes. Also found are the dark dying cells of the adrenal cortex, characterized by densification of the cytoplasm, pycnosis of nuclei, accumulation of yellow-brown pigment. Dark cells have larger nuclei, give more intense reaction to RNA and ketosteroids and are more active secretory cells compared to light adrenocorticocytes. Electron microscopy in dark cells reveals a large number of mitochondria and a significant amount of lipid droplets are present. Light cells contain many lipid inclusions and few mitochondria.

Human cellular mitochondrial remodelling is governed by miR-2909 RNomics.

BackgroundThere exists a general recognition of the fact that mitochondrial remodelling as a result of aerobic glycolysis ensures human somatic cells to revert to a more primitive-form exhibiting stem-like phenotype. The present study is an attempt to demonstrate that miR-2909 RNomics within human peripheral blood mononuclear cells (PBMCs) has the inherent capacity to re-program these cells to exhibit mitochondrial remodelling paralleled by aerobic glycolysis together with intracellular lipid inclusions. Such re-programmed PBMCs also expressed genes having ability to sustain their de-differentiation state and survival.Material and methodsHuman PBMCs were programed to ectopically express miR-2909. Expression levels of genes including glucose transporter-1 (Glut-1), hexokinase (HK), hypoxia inducia factor-1 (HIF-1α), c-Myc, p53,mechanistic target of rapamycin complex (mTORC1), polycombcomplex protein (Bmi-1), Notch,Nanog,Tie-2, Oct-4,CD59, p53, CD34, B-cell lymphoma-2 (Bcl2),sterol regulatory element-binding protein2 (SREBP2), peroxisome proliferator-activated receptor gamma (PPARγ) nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (Tfam), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) within miR-2909 expression vector transfected human PBMCs as well as PBMCs transfected with control vector containing scrambled sequence after 48h post-transfection using RT-qPCR and cellular ultrastructural features induced by miR-2909 ectopic expression were observed using transmission electron microscopy and morphometric analysis of an electron micrograph.ResultsEctopic expression of miR-2909 within human PBMCs resulted in their reprogramming into stem-like phenotype indicated by mitochondrial globular shaped coupled with cristae-poor morphology. Nuclear to cytoplasmic ratio (N/C), quantification of ATP levels, GSSG/GSH ratio, mitochondrial cytochrome c oxidase activity, secreted lactate concentrations, activity of antioxidant enzymes, levels of esterified cholesterol and triglycerides and flow-cytometric detection of apoptosis confirmed the compromised nature of mitochondrial function induced by ectopic miR-2909 expression in human PBMCs.ConclusionBased upon these results we propose that AATF gene-encoded miR-2909 may act as an epigenetic switch for cellular aerobic-glycolysis to ensure de-differentiation.

Heparin-Binding Hemagglutinin Adhesin (HBHA) Is Involved in Intracytosolic Lipid Inclusions Formation in Mycobacteria.

The heparin-binding hemagglutinin adhesin (HBHA) is an important virulence factor of Mycobacterium tuberculosis. It is a surface-displayed protein that serves as an adhesin for non-phagocytic cells and is involved in extra-pulmonary dissemination of the tubercle bacillus. It is also an important latency antigen useful for the diagnosis of latently M. tuberculosis-infected individuals. Using fluorescence time-lapse microscopy on mycobacteria that produce HBHA-green fluorescent protein chimera, we show here that HBHA can be found at two different locations and dynamically alternates between the mycobacterial surface and the interior of the cell, where it participates in the formation of intracytosolic lipid inclusions (ILI). Compared to HBHA-producing mycobacteria, HBHA-deficient mutants contain significantly lower amounts of ILI when grown in vitro or within macrophages, and the sizes of their ILI are significantly smaller. Lipid-binding assays indicate that HBHA is able to specifically bind to phosphatidylinositol and in particular to 4,5 di-phosphorylated phosphatidylinositol, but not to neutral lipids, the main constituents of ILI. HBHA derivatives lacking the C-terminal methylated, lysine-rich repeat region fail to bind to these lipids and these derivatives also fail to complement the phenotype of HBHA-deficient mutants. These studies indicate that HBHA is a moonlighting protein that serves several functions depending on its location. When surface exposed, HBHA serves as an adhesin, and when intracellularly localized, it participates in the generation of ILI, possibly as a cargo to transport phospholipids from the plasma membrane to the ILI in the process of being formed.

Fast growing greater amberjack post-larvae require a high energy-high protein weaning diet

Larvae and early juveniles of fast-growing fish species show tremendous growth potential, leading to higher requirements for protein, high-unsaturated fatty acids, and other nutrients. Several commercial weaning diets with relatively high success have been developed for low/moderate-growing species; however, additional challenges are outlined to meet growth potential and energy requirements of such fast-growing species. The objective of the present study was to evaluate two commercial microdiets for marine fish, one having simultaneously a very high protein and high lipid contents (HIGH), and another (MOD) with a high protein and moderate lipid content, in a growth performance trial with greater amberjack post-larvae from 33 to 78 days after hatching. Moreover, histological preparations of liver, anterior and posterior intestine were assessed for hepatic and intestinal lipid inclusions quantification and gut epithelial brush height measurement. Activities of the digestive enzymes: pepsin, trypsin, chymotrypsin, lipase and amylase were also analyzed. Post-larvae fed HIGH microdiet exhibited higher final weight and lower feed conversion ratio that those fed on MOD microdiet. Liver displayed a higher level of lipid inclusions for the MOD diet than for HIGH diet. Moreover, enterocytes of posterior intestine presented a much higher level of supranuclear vacuoles for the HIGH diet compared to MOD diet. The lower Trypsin/Chymotrypsin ratio observed at the end of the experiment in larvae fed on MOD diet may indicate a deficiency in protein of this diet. Together, these results support that larvae of greater amberjack, and likely other fast-growing marine fish species, require high protein–high lipid microdiets. The use of microdiets developed targeting slower growing marine species may lead to sub-optimal performances in fast-growing larvae.

Delineating the Physiological Roles of the PE and Catalytic Domains of LipY in Lipid Consumption in Mycobacterium-Infected Foamy Macrophages.

Within tuberculous granulomas, a subpopulation of Mycobacterium tuberculosis resides inside foamy macrophages (FM) that contain abundant cytoplasmic lipid bodies (LB) filled with triacylglycerol (TAG). Upon fusion of LB with M. tuberculosis-containing phagosomes, TAG is hydrolyzed and reprocessed by the bacteria into their own lipids, which accumulate as intracytosolic lipid inclusions (ILI). This phenomenon is driven by many mycobacterial lipases, among which LipY participates in the hydrolysis of host and bacterial TAG. However, the functional contribution of LipY's PE domain to TAG hydrolysis remains unclear. Here, enzymatic studies were performed to compare the lipolytic activities of recombinant LipY and its truncated variant lacking the N-terminal PE domain, LipY(ΔPE). Complementarily, an FM model was used where bone marrow-derived mouse macrophages were infected with M. bovis BCG strains either overexpressing LipY or LipY(ΔPE) or carrying a lipY deletion mutation prior to being exposed to TAG-rich very-low-density lipoprotein (VLDL). Results indicate that truncation of the PE domain correlates with increased TAG hydrolase activity. Quantitative electron microscopy analyses showed that (i) in the presence of lipase inhibitors, large ILI (ILI+3) were not formed because of an absence of LB due to inhibition of VLDL-TAG hydrolysis or inhibition of LB-neutral lipid hydrolysis by mycobacterial lipases, (ii) ILI+3 profiles in the strain overexpressing LipY(ΔPE) were reduced, and (iii) the number of ILI+3 profiles in the ΔlipY mutant was reduced by 50%. Overall, these results delineate the role of LipY and its PE domain in host and mycobacterial lipid consumption and show that additional mycobacterial lipases take part in these processes.

Autophagy in Hepatocytes during Distant Tumor Growth.

Structural changes in the liver of CBA mice were studied during the development of experimental hepatocarcinoma-29 inoculated into the hip. A decrease in the volume density of hepatocyte cytoplasm, mitochondria, endoplasmic reticulum, and lipid inclusions and an increase in the volume density of lysosomal structures during tumor growth were observed. All stages of intracellular autophagy were recorded by the method of electron microscopy. These stages included the appearance of autophagosomes, autophagolysosomes, and secondary lysosomes in the hepatocyte cytoplasm. Fragments of cytoplasm, glycogen rosettes, mitochondria, and fragments of endoplasmic reticulum with ribosomes were found in autophagosomes. The obtained data indicate the development of non-selective autophagy in the liver during distant tumor growth in aimed at the maintenance of intracellular homeostasis in hepatocytes and energy and trophic homeostasis of organism.

Morphology of the Ovaries in Condition of Inhalation Intoxication with Dust-Saline Aerosols of the Aral Sea in Female White Rats.

BACKGROUND:After some clinical studies of the reproductive health of the female population of the Aral Sea region, we concluded that it is necessary to confirm by experiment the theory of the direct influence of dust-saline aerosols of the Aral Sea on the reproductive system of female individuals.AIM:The purpose of this work is to study the effect of dust-saline aerosols of the Aral Sea on the folliculogenesis process in rats at inhalation intoxication.METHODS:Inhalation in rats was carried out for 30 days for 4 hours a day for 5 days a week in special inoculating cylindrical chambers with the extra-chamber placement of animals in individual boxes and dynamic aerosol delivery. Morphological changes were assessed using electron microscopy.RESULTS:Ultrastructural changes in the ovaries of the experimental study group were characterised by the pathology of all structures of the cortical substance of the organ. The accumulation of lutein pigment, the utilisation of lipid inclusions and the destruction of the complete cell reflected the pathology of estrogen production-an important factor in the postovulatory phase of the ovarian cycle.CONCLUSIONS:Taking into account the proven effect of dust-saline aerosols on the production of estrogen, the violation of the postovulatory phase of the ovarian cycle, we trace the mechanism of folliculogenesis disturbance. This confirms the data of our previous studies on primary and secondary infertility in women living in the Aral Sea region and the necessity of creation and development of preventive measures for the inhabitants of the region.

Tyrphostin AG17 inhibits adipocyte differentiation in vivo and in vitro

BackgroundExcessive subcutaneous adiposity in obesity is associated to positive white adipocyte tissue (WAT) differentiation (adipogenesis) and WAT expandability. Here, we hypothesized that supplementation with the insulin inhibitor and mitochondrial uncoupler, Tyrphostin (T-AG17), in vitro and in vivo inhibits adipogenesis and adipocyte hypertrophy.MethodsWe used a 3T3-L1 proadipocyte cell line to identify the potential effect of T-AG17 on adipocyte differentiation and fat accumulation in vitro. We evaluated the safety of T-AG17 and its effects on physiological and molecular metabolic parameters including hormonal profile, glucose levels, adipogenesis and adipocyte hypertrophy in a diet-induced obesity model using C57BL/6 mice.ResultsWe found that T-AG17 is effective in preventing adipogenesis and lipid synthesis in the 3T3-L1 cell line, as evidenced by a significant decrease in oil red staining (p < 0.05). In obese C57BL/6 mice, oral administration of T-AG17 (0.175 mg/kg for 2 weeks) lead to decreased fat accumulation and WAT hypertrophy. Further, T-AG17 induced adipocyte apoptosis by activating caspase-3. In the hepatocytes of obese mice, T-AG17 promoted an increase in the size of lipid inclusions, which was accompanied by glycogen accumulation. T-AG17 did not alter serum biochemistry, including glucose, insulin, leptin, free fatty acids, creatinine, and aspartate aminotransferase.ConclusionT-AG17 promotes adipocyte apoptosis in vivo and is an effective modulator of adipocyte differentiation and WAT hypertrophy in vitro and in vivo. Therefore, T-AG17 may be useful as a pharmacological obesity treatment.

Lopinavir, an HIV-1 peptidase inhibitor, induces alteration on the lipid metabolism of Leishmania amazonensis promastigotes.

The anti-leishmania effects of HIV peptidase inhibitors (PIs) have been widely reported; however, the biochemical target and mode of action are still a matter of controversy in Leishmania parasites. Considering the possibility that HIV-PIs induce lipid accumulation in Leishmania amazonensis, we analysed the effects of lopinavir on the lipid metabolism of L. amazonensis promastigotes. To this end, parasites were treated with lopinavir at different concentrations and analysed by fluorescence microscopy and spectrofluorimetry, using a fluorescent lipophilic marker. Then, the cellular ultrastructure of treated and control parasites was analysed by transmission electron microscopy (TEM), and the lipid composition was investigated by thin-layer chromatography (TLC). Finally, the sterol content was assayed by gas chromatography-mass spectrometry (GC/MS). TEM analysis revealed an increased number of lipid inclusions in lopinavir-treated cells, which was accompanied by an increase in the lipophilic content, in a dose-dependent manner. TLC and GC-MS analysis revealed a marked increase of cholesterol-esters and cholesterol. In conclusion, lopinavir-induced lipid accumulation and affected lipid composition in L. amazonensis in a concentration-response manner. These data contribute to a better understanding of the possible mechanisms of action of this HIV-PI in L. amazonensis promastigotes. The concerted action of lopinavir on this and other cellular processes, such as the direct inhibition of an aspartyl peptidase, may be responsible for the arrested development of the parasite.

Histological organization of intestinal villi in the crocodilian caiman yacare (Daudin, 1802) during dietary lipid absorption

Intestinal villi of Caiman yacare form longitudinal folds instead of the finger-like projections of most birds and mammals. Moreover, they lack Crypts of Lieberkuhn and the lamina epithelialis organization is dynamic, changing from pseudostratified to simple columnar epithelium after feeding. Because of these differences, we sought to verify whether intestinal villi of the crocodilian Caiman yacare are functionally compartmentalized along their length similarly to the finger-like villi that harbors Crypt of Lieberkuhn. For this, Caiman yacare were force-fed soybean oil, the intestinal mucosa was harvested and analyzed under light microscopy after lipid staining or immunohistochemistry for the proliferative marker PCNA. Functional compartmentalization was assessed by evaluating differences in lipid absorption along intestinal villi base-to-tip axis, by localizing the proliferative enterocytes and by verifying whether such cells were capable of absorbing lipids. Histological morphometric analyses of the extent of enterocyte hypertrophy caused by lipid inclusions and the contribution of such inclusions to histological remodeling from pseudostratified to simple columnar epithelium were also evaluated. Although lacking Crypts of Lieberkuhn, enterocytes present at villi base were PCNA positive and devoid of the great amount of lipid inclusions observed in the other intestinal villi domains, in a similar pattern to finger-like villi. Enterocytes doubled their volume because of lipid inclusions, and in spite of such enterocyte hypertrophy, lamina epithelialis continued to be pseudostratified within lateral sides, whereas villi tip were organized in a simple columnar epithelium.

In vivo anti-arthritic and antioxidant effects from the standardized ethanolic extract of Moussonia deppeana

Moussonia deppeana (Schltdl. & Cham.) Klotzsch ex Hanst., Gesneriaceae, known as tlachichinole, is a Mexican medicinal plant used for treatment of chronic inflammation-related diseases such as arthritis. In this paper, the main metabolite verbascoside was quantified in ethanolic extract; anti-arthritic and antioxidant activities were also evaluated in Complete Freund's Adjuvant induced arthritis in mice, with complete hematological evaluation, and oxidative stress measure in edema and ganglionic tissues on day 28. In popliteal ganglion, CD4+ lymphocytes and tumor necrosis factor alpha concentration were measured in addition to histological analysis. Ethanolic extract contained 79.2mg of verbascoside/g extract, and this extract at 450mg/kg generated an inhibition of 24% over paw edema development and increased body weight gain on final day. For hematological parameters, same dose decreased total leukocytes and lymphocytes, as well as decreased oxidation rate over biomolecules in edema and ganglionic tissues, and increased antioxidant enzyme activity. In ganglionic tissue, CD4+ lymphocytes and tumor necrosis factor alpha level showed no differences at any tested dose compared to complete Freund's adjuvant untreated group. Histological analysis of popliteal ganglion revealed moderate reduction of follicular hyperplasia, leukocyte infiltration and lipid inclusions at 450mg/kg dose. Ethanolic extract of M. deppeana possesses anti-edematous activity associated to a moderate reduction in follicular hyperplasia, with immune-modulatory and antioxidant effects during experimental arthritis in mice.

Intracellular Reorganization of Cardiomyocytes in Dyslipidemic Cardiomyopathies.

The study examined the myocardial ultrastructural alterations in rats maintained on various atherogenic diets. It revealed the complex ultrastructural alterations of cardiomyocytes and endotheliocytes (including the lytic and destructive changes of the intracellular organelles, upregulation of the autophagocytosis in the cardiomyocytes, and necrobiosis with apoptosis of endotheliocytes) reflecting the cytopathic features of circulating cholesterol and lipoproteins, whose elevation determined the intensity of destructive processes. The revealed peculiarities in the changes of lipid inclusions (their osmiophilic transformation) in cardiomyocytes can be provoked by entry of cholesterol into the cells and its further metabolic modifications. During moderate dyslipidemia, the cardiomyocytes demonstrated the ultrastructural signs of induction of intracellular regeneration (marked with the clusters of polysomes in the intermyofibrillar and subsarcolemmal spaces with appearance of neogenic myofilaments) and upregulated pinocytotic activity. In all cases, up-regulated autophagocytosis in cardiomyocytes was accompanied by accumulation of myelin- and vacuole-like structures in the intercellular spaces and capillary lumens paralleled with appearance of activated forms of macrophages and fibroblasts in the myocardium.