Хроматоденситометрическое изучение листьев брусники обыкновенной (Vaccinium vitis-idaea L.)

VACCINIUMБРУСНИКАРАСТЕНИЙ ЛИСТЬЯРАСТЕНИЯ СТРУКТУРНЫЕ ЧАСТИ НАДЗЕМНЫЕАРБУТИНХРОМАТОГРАФИЯ ЖИДКОСТНАЯ /ИСПХРОМАТОДЕНСИТОМЕТРИЧЕСКИЙ СПОСОБХРОМАТОГРАФИЧЕСКАЯ ЗОНАХРОМАТОГРАММАРАСТЕНИЙ ЭКСТРАКТЫРЕАКТИВ ГИББСАЛИНЕЙНАЯ ЗАВИСИМОСТЬПроанализированы листья брусники обыкновенной, заготовленные в местах естественного произрастания в окрестностях г. Витебска Республики Беларусь. С помощью хроматоденситометрического способа изучались условия проведения хроматоденситометрического определения арбутина в листьях брусники. Подобраны система растворителей, концентрация реагента-проявителя (реактив Гиббса) для хроматоденситометрического способа определения арбутина в листьях брусники обыкновенной. Изучен характер зависимости цветометрических характеристик пятен арбутина от его количества в хроматографической зоне. Выявлена линейная зависимость цветометрических характеристик зон арбутина от его количества в хроматографической зоне. Было проведено сравнение хроматоденситометрического способа определения арбутина в листьях брусники с методом ВЭЖХ. Установлено, что результаты ВЭЖХ сопоставимы с результатами хроматоденситометрического способа определения арбутина в листьях брусники обыкновенной, что дает основание сделать вывод о достаточно высокой точности хроматоденситометрического способа и делает возможным его применение для качественного и количественного определения арбутина в листьях брусники. Изучались различные условия экстракции арбутина из листьев брусники обыкновенной. Построены графики зависимости полноты экстракции арбутина из листьев брусники обыкновенной при различных условиях экстракции. Подобраны оптимальные условия экстракции арбутина (степень измельченности листьев брусники –2 мм, время экстракции – 40 минут, экстрагент – вода).The leaves of common lingonberry harvested in the places of its natural growth in the vicinity of the city of Vitebsk of the Republic of Belarus have been analyzed using the chromatodensitometric method. The conditions of the chromatodensitometric method use for determining arbutin in the common lingonberry leaves have been studied. The solvent system, the concentration of the reagent- developer (Gibbs reagent) for the chromatodensitometric method used to determine arbutin in the common lingonberry leaves have been selected. The character of the dependence of the colorimetric characteristics of arbutin spots on its quantity in the chromatographic zone has been studied. A linear dependence of the colorimetric characteristics of arbutin zones on its quantity in the chromatographic zone has been revealed. The chromatodensitometric method of the determination of arbutin in the common lingonberry leaves has been compared to that of HPLC. It has been established that the HPLC results are comparable with those obtained by means of the chromatodensitometric method of determining arbutin in the common lingonberry leaves, which gives grounds to conclude that the chromatodensitometric method is rather accurate and may be used for the quantitative and qualitative determination of arbutin in the common lingonberry leaves. Various conditions for the extraction of arbutin from the leaves of common lingonberry have been studied. The graphs of the dependence of the completeness of the common lingonberry leaves arbutin extraction under various extraction conditions have been constructed. Optimal conditions for the extraction of arbutin have been selected (the degree of crushing the lingonberry leaves – 2 mm, extraction time – 40 minutes, the extractant – water)

Activation of the Cellular Immune Response in Drosophila melanogaster Larvae

During the last 40 years, Drosophila melanogaster has become an invaluable tool in understanding innate immunity. The innate immune system of Drosophila consists of a humoral and a cellular component. While many details are known about the humoral immune system, our knowledge about the cellular immune system is comparatively small. Blood cells or hemocytes constitute the cellular immune system. Three blood types have been described for Drosophila larvae. Plasmatocytes are phagocytes with a plethora of functions. Crystal cells mediate melanization and contribute to wound healing. Plasmatocytes and crystal cells constitute the blood cell repertoire of a healthy larva, whereas lamellocytes are induced in a demand-adapted manner after infection with parasitoid wasp eggs. They are involved in the melanotic encapsulation response against parasites and form melanotic nodules that are also referred to as tumors. In my thesis, I focused on unraveling the mechanisms of how the immune system orchestrates the cellular immune response. In particular, I was interested in the hematopoiesis of lamellocytes. In Article I, we were able to show that ectopic expression of key components of a number of signaling pathways in blood cells induced the development of lamellocytes, led to a proliferative response of plasmatocytes, or to a combination of lamellocyte activation and plasmatocyte proliferation. In Article II, I combined newly developed fluorescent enhancer-reporter constructs specific for plasmatocytes and lamellocytes and developed a “dual reporter system” that was used in live microscopy of fly larvae. In addition, we established flow cytometry as a tool to count total blood cell numbers and to distinguish between different blood cell types. The “dual reporter system” enabled us to differentiate between six blood cell types and established proliferation as a central feature of the cellular immune response. The combination flow cytometry and live imaging increased our understanding of the tempo-spatial events leading to the cellular immune reaction. In Article III, I developed a genetic modifier screen to find genes involved in the hematopoiesis of lamellocytes. I took advantage of the gain-of-function phenotype of the Tl10b mutation characterized by an activated cellular immune system, which induced the formation blood cell tumors. We screened the right arm of chromosome 3 for enhancers and suppressors of this mutation and uncovered ird1. Finally in Article IV, we showed that the activity of the Toll signaling pathway in the fat body, the homolog of the liver, is necessary to activate the cellular immune system and induce lamellocyte hematopoiesis

Activation of the Cellular Immune Response in Drosophila melanogaster Larvae

During the last 40 years, Drosophila melanogaster has become an invaluable tool in understanding innate immunity. The innate immune system of Drosophila consists of a humoral and a cellular component. While many details are known about the humoral immune system, our knowledge about the cellular immune system is comparatively small. Blood cells or hemocytes constitute the cellular immune system. Three blood types have been described for Drosophila larvae. Plasmatocytes are phagocytes with a plethora of functions. Crystal cells mediate melanization and contribute to wound healing. Plasmatocytes and crystal cells constitute the blood cell repertoire of a healthy larva, whereas lamellocytes are induced in a demand-adapted manner after infection with parasitoid wasp eggs. They are involved in the melanotic encapsulation response against parasites and form melanotic nodules that are also referred to as tumors. In my thesis, I focused on unraveling the mechanisms of how the immune system orchestrates the cellular immune response. In particular, I was interested in the hematopoiesis of lamellocytes. In Article I, we were able to show that ectopic expression of key components of a number of signaling pathways in blood cells induced the development of lamellocytes, led to a proliferative response of plasmatocytes, or to a combination of lamellocyte activation and plasmatocyte proliferation. In Article II, I combined newly developed fluorescent enhancer-reporter constructs specific for plasmatocytes and lamellocytes and developed a “dual reporter system” that was used in live microscopy of fly larvae. In addition, we established flow cytometry as a tool to count total blood cell numbers and to distinguish between different blood cell types. The “dual reporter system” enabled us to differentiate between six blood cell types and established proliferation as a central feature of the cellular immune response. The combination flow cytometry and live imaging increased our understanding of the tempo-spatial events leading to the cellular immune reaction. In Article III, I developed a genetic modifier screen to find genes involved in the hematopoiesis of lamellocytes. I took advantage of the gain-of-function phenotype of the Tl10b mutation characterized by an activated cellular immune system, which induced the formation blood cell tumors. We screened the right arm of chromosome 3 for enhancers and suppressors of this mutation and uncovered ird1. Finally in Article IV, we showed that the activity of the Toll signaling pathway in the fat body, the homolog of the liver, is necessary to activate the cellular immune system and induce lamellocyte hematopoiesis

Edin expression in the fat body is required in the defense against parasitic wasps in Drosophila melanogaster

The cellular immune response against parasitoid wasps in Drosophila involves the activation, mobilization, proliferation and differentiation of different blood cell types. Here, we have assessed the role of Edin (elevated during infection) in the immune response against the parasitoid wasp Leptopilina boulardi in Drosophila melanogaster larvae. The expression of edin was induced within hours after a wasp infection in larval fat bodies. Using tissue-specific RNAi, we show that Edin is an important determinant of the encapsulation response. Although edin expression in the fat body was required for the larvae to mount a normal encapsulation response, it was dispensable in hemocytes. Edin expression in the fat body was not required for lamellocyte differentiation, but it was needed for the increase in plasmatocyte numbers and for the release of sessile hemocytes into the hemolymph. We conclude that edin expression in the fat body affects the outcome of a wasp infection by regulating the increase of plasmatocyte numbers and the mobilization of sessile hemocytes in Drosophila larvae

Transdifferentiation and Proliferation in Two Distinct Hemocyte Lineages in Drosophila melanogaster Larvae after Wasp Infection

Cellular immune responses require the generation and recruitment of diverse blood cell types that recognize and kill pathogens. In Drosophila melanogaster larvae, immune-inducible lamellocytes participate in recognizing and killing parasitoid wasp eggs. However, the sequence of events required for lamellocyte generation remains controversial. To study the cellular immune system, we developed a flow cytometry approach using in vivo reporters for lamellocytes as well as for plasmatocytes, the main hemocyte type in healthy larvae. We found that two different blood cell lineages, the plasmatocyte and lamellocyte lineages, contribute to the generation of lamellocytes in a demand-adapted hematopoietic process. Plasmatocytes transdifferentiate into lamellocyte-like cells in situ directly on the wasp egg. In parallel, a novel population of infection-induced cells, which we named lamelloblasts, appears in the circulation. Lamelloblasts proliferate vigorously and develop into the major class of circulating lamellocytes. Our data indicate that lamellocyte differentiation upon wasp parasitism is a plastic and dynamic process. Flow cytometry with in vivo hemocyte reporters can be used to study this phenomenon in detail

Control of Drosophila blood cell activation via toll signaling in the fat body

The Toll signaling pathway, first discovered in Drosophila, has a well-established role in immune responses in insects as well as in mammals. In Drosophila, the Toll-dependent induction of antimicrobial peptide production has been intensely studied as a model for innate immune responses in general. Besides this humoral immune response, Toll signaling is also known to activate blood cells in a reaction that is similar to the cellular immune response to parasite infections, but the mechanisms of this response are poorly understood. Here we have studied this response in detail, and found that Toll signaling in several different tissues can activate a cellular immune defense, and that this response does not require Toll signaling in the blood cells themselves. Like in the humoral immune response, we show that Toll signaling in the fat body (analogous to the liver in vertebrates) is of major importance in the Toll-dependent activation of blood cells. However, this Toll-dependent mechanism of blood cell activation contributes very little to the immune response against the parasitoid wasp, Leptopilina boulardi, probably because the wasp is able to suppress Toll induction. Other redundant pathways may be more important in the defense against this pathogen

Genetic Screen in Drosophila Larvae Links ird1 Function to Toll Signaling in the Fat Body and Hemocyte Motility.

To understand how Toll signaling controls the activation of a cellular immune response in Drosophila blood cells (hemocytes), we carried out a genetic modifier screen, looking for deletions that suppress or enhance the mobilization of sessile hemocytes by the gain-of-function mutation Toll10b (Tl10b). Here we describe the results from chromosome arm 3R, where five regions strongly suppressed this phenotype. We identified the specific genes immune response deficient 1 (ird1), headcase (hdc) and possibly Rab23 as suppressors, and we studied the role of ird1 in more detail. An ird1 null mutant and a mutant that truncates the N-terminal kinase domain of the encoded Ird1 protein affected the Tl10b phenotype, unlike mutations that affect the C-terminal part of the protein. The ird1 null mutant suppressed mobilization of sessile hemocytes, but enhanced other Tl10b hemocyte phenotypes, like the formation of melanotic nodules and the increased number of circulating hemocytes. ird1 mutants also had blood cell phenotypes on their own. They lacked crystal cells and showed aberrant formation of lamellocytes. ird1 mutant plasmatocytes had a reduced ability to spread on an artificial substrate by forming protrusions, which may explain why they did not go into circulation in response to Toll signaling. The effect of the ird1 mutation depended mainly on ird1 expression in hemocytes, but ird1-dependent effects in other tissues may contribute. Specifically, the Toll receptor was translocated from the cell membrane to intracellular vesicles in the fat body of the ird1 mutant, and Toll signaling was activated in that tissue, partially explaining the Tl10b-like phenotype. As ird1 is otherwise known to control vesicular transport, we conclude that the vesicular transport system may be of particular importance during an immune response

Knock down of <i>edin</i> in the fat body decreases the encapsulation and killing ability of <i>Drosophila</i> larvae.

<p><b>(A)</b> The encapsulation response of two different <i>edin</i> RNAi lines (<i>edin</i>^<i>14289</i> and <i>edin</i>^{<i>109528</i>}) was analyzed 27-29h after a wasp infection. The <i>C564</i>-<i>GAL4</i> (<i>C564>)</i>, <i>Fb-GAL4</i> (<i>Fb></i>) and <i>Hml</i>^<i>Δ</i>;<i>He-GAL4</i> (<i>HH></i>) drivers were used to drive the expression of the RNAi constructs. <i>w</i>^<i>1118</i> (<i>w</i>) was used as control. Data were pooled from one to eight individual experiments, as depicted on each column, each experiment with at least 50 analyzed individual infected larvae. <b>(B)</b> The ability of <i>Drosophila</i> larvae to kill wasp eggs was assessed with two different <i>edin</i> RNAi lines (<i>edin</i>^<i>14289</i> and <i>edin</i>^{<i>109528</i>}) 48-50h after infection. The <i>C564</i>-<i>GAL4</i> (<i>C564></i>) and <i>Fb-GAL4</i> (<i>Fb></i>) drivers were used to drive the expression of the RNAi constructs. <i>w</i>^<i>1118</i> (<i>w</i>) was used as control. Data are pooled from three to sixteen independent experiments, as indicated on each column, and at least 50 infected larvae were scored per experiment. Error bars in A and B show standard deviations. Knocking down the expression of <i>edin</i> in several tissues including the fat body or in the fat body alone caused a significant decrease in the encapsulation activity and killing response of <i>Drosophila</i> larvae compared to controls, whereas knocking down <i>edin</i> in hemocytes had no effect.</p

<i>Edin</i> expression is induced upon a wasp infection.

<p><b>(A)</b> Wasp infection causes a 6.7-fold increase in <i>edin</i> expression in 2^nd instar <i>Canton S</i> larvae. Data are pooled from two independent experiments, n = 2 for each experiment, where one sample represents 10 larvae. <b>(B)</b><i>Edin</i> expression is induced in the fat bodies of <i>Canton S</i> larvae 24 hours post infection. The data are pooled from four independent experiments, and each experiment consisted of two samples, where one sample represents 8–10 larval fat bodies.</p

Quantification of hemocytes in <i>edin</i> RNAi larvae after a wasp infection.

<p><b>(A-B)</b> Hemocytes of infected larvae were bled 48–50 hours post-infection and visualized with the <i>eaterGFP</i> (green) and <i>msnCherry</i> (red) reporters. Uninfected controls contained only GFP-positive cells that corresponded to plasmatocytes (green). (<b>A’ and B’</b>) <i>msnCherry</i> expression was detected in the infected samples and this included lamellocytes (asterisks) and cells that express both <i>eaterGFP</i> and <i>msnCherry</i> indicating that they were undergoing lamellocyte transition. Lamellocytes were present also in the infected <i>edin</i> RNAi larvae suggesting that <i>edin</i> expression is not necessary for lamellocyte differentiation. Scale bars are 10 μm <b>(C-E)</b> Flow cytometry was carried out to quantify the amount of hemocytes in the unchallenged and the wasp infected <i>edin</i> RNAi larvae. (C = control, inf = infected)</p