Can recombinant tree shrew (Tupaia belangeri chinensis) chymosin coagulate cow (Bos taurus) milk?

Genetically engineered chymosin from the tree shrew (Tupaia belangeri chinensis) has been obtained and partially characterized for the first time. The target enzyme was produced in Escherichia coli, strain BL21(DE3). It was shown that tree shrew recombinant chymosin coagulates cow milk (Bos taurus). The total and specific milk-clotting activity of the obtained enzyme was 0.7–5.3 IMCU/mL and 8.8–16.6 IMCU/mg. The nonspecific proteolytic activity of tree shrew recombinant chymosin in relation to total bovine casein was 30 and 117% higher than that of recombinant chymosin of cow and of single-humped camel respectively. It was found that in comparison with most of the known genetically engineered chymosins, the tree shrew enzyme showed exceptionally low thermal stability. After heating at 45°C, the coagulation ability of tree shrew recombinant chymosin decreased by more than 40%, and at 50°C the enzyme lost more than 90% of the initial milk-clotting activity. The Michaelis constant (Km), enzyme turnover number (kcat), and catalytic efficiency (kcat/Km) for genetically engineered chymosin from the tree shrew were 6.3 ± 0.1 µM, 11 927 ± 3169 s–1 and 1968 ± 620 µM–1 s–1, respectively. Comparative analysis showed that the primary structure of the chymosin-sensitive site of cow kappa-casein and the supposed similar sequence of tree shrew kappa-casein differed by 75%. The ability of tree shrew recombinant chymosin to coagulate cow’s milk, along with a low thermal stability and high catalytic efficiency with respect to the substrate, imitating the chymosin-sensitive site of cow kappa-casein, suggests that this enzyme is of potential interest for cheese making

The effect of thioredoxin and prochymosin coexpression on the refolding of recombinant alpaca chymosin

The milk-clotting enzyme chymosin is a member of the group of aspartate proteinases. Chymosin is the main component of rennet traditionally obtained from the stomachs of dairy calves and widely used to coagulate milk in the production of various types of cheese. Another source of chymosin, which does not require the killing of animals, is based on recombinant DNA technology. Recombinant alpaca chymosin has a number of valuable technological properties that make it attractive for use in cheese-making as an alternative to recombinant bovine chymosin. The purpose of this work is to study the effect of coexpression of thioredoxin and prochymosin on the refolding of the recombinant zymogen and the activity of alpaca chymosin. To achieve this goal, on the basis of the pET32a plasmid, an expression vector was constructed containing the thioredoxin A gene fused to the N-terminal sequence of the marker enzyme zymogen, alpaca prochymosin. Using the constructed vector, pETTrxProChn, a strain-producer of the recombinant chimeric protein thioredoxin-prochymosin was obtained. The choice of prochymosin as a model protein is due to the ability of autocatalytic activation of this zymogen, in which the pro-fragment is removed, together with the thioredoxin sequence attached to it, with the formation of active chymosin. It is shown that Escherichia coli strain BL21 transformed with the pET-TrxProChn plasmid provides an efficient synthesis of the thioredoxin-prochymosin chimeric molecule. However, the chimeric protein accumulates in inclusion bodies in an insoluble form. Therefore, a renaturation procedure was used to obtain the active target enzyme. Fusion of thioredoxin capable of disulfide-reductase activity to the N-terminal sequence of prochymosin provides optimal conditions for zymogen refolding and increases the yield of recombinant alpaca chymosin immediately after activation and during long-term storage by 13 and 15 %, respectively. The inclusion of thioredoxin in the composition of the chimeric protein, apparently, contributes to the process of correct reduction of disulfide bonds in the prochymosin molecule, which is reflected in the dynamics of the increase in the milk-clotting activity of alpaca chymosin during long-term storage

Biochemical and technological properties of moose (<i>Alces alces</i>) recombinant chymosin

Recombinant chymosins (rСhns) of the cow and the camel are currently considered as standard milk coagulants for cheese-making. The search for a new type of milk-clotting enzymes that may exist in nature and can surpass the existing “cheese-making” standards is an urgent biotechnological task. Within this study, we for the first time constructed an expression vector allowing production of a recombinant analog of moose chymosin in the expression system of Escherichia coli (strain SHuffle express). We built a model of the spatial structure of moose chymosin and compared the topography of positive and negative surface charges with the correspondent structures of cow and camel chymosins. We found that the distribution of charges on the surface of moose chymosin has common features with that of cow and camel chymosins. However, the moose enzyme carries a unique positively charged patch, which is likely to affect its interaction with the substrate. Biochemical and technological properties of the moose rChn were studied. Commercial rСhns of cow and camel were used as comparison enzymes. In some technological parameters, the moose rChn proved to be superior to the reference enzymes. Сompared with the cow and camel rСhns, the moose chymosin specific activity is less dependent on the changes in CaCl2 concentration in the range of 1–5 mM and pH in the range of 6–7, which is an attractive technological property. The total proteolytic activity of the moose rСhn occupies an intermediate position between the rСhns of cow and camel. The combination of biochemical and technological properties of the moose rСhn argues for further study of this enzyme

К механизму активации восстановительных процессов в печени при использовании общей РНК клеток костного мозга

Objective: to study the cellular mechanisms of activation of regenerative processes in the liver when using total RNA (tRNA) of bone marrow cells (BMCs) based on an extended liver resection (ELR) model. Materials and methods. Male Wistar rats (n = 80) with ELR model (70%) were divided into 2 groups: group 1 (control group) had a single saline injection, while group 2 (experimental group) received a single tRNA injection at a 30 μg/100 g dose of animal weight. The biochemical parameters of liver function and weight were monitored over time. Also monitored were microstructural changes in hepatocytes 48 hours after ELR by examining mitotic activity, caspase-9 expression and morphometric parameters. Results. It was found that in group 2, in comparison to group 1, there was faster normalization of biochemical parameters (by 10–14 days), a higher mitotic index of hepatocytes (23.45‰ versus 5.37‰), and initially sharper decrease and then faster recovery of liver mass (by 10–12 days versus 18–20 days). Both groups showed almost total expression of caspase-9, including in mitotically splitting hepatocytes. Group 1 demonstrated decreased values of morphometric parameters of single and binuclear cells, decreased number of binucleated hepatocytes and increased total density of hepatocytes as compared to the intact liver. Intraperitoneal administration of tRNA increased morphometric parameters of mononuclear hepatocytes, did not affect their number, but increased the area of the nuclei of binuclear hepatocytes as compared to the control group. Conclusion. The proven capability of cell-bone marrow total RNA to simultaneously support apoptosis in liver cells after ELR and induce mitotic activity indicates that tRNA can switch activated apoptosis to cell proliferation at the early phase of the regenerative process. This effect may be due to the presence of regulatory RNA molecules in tRNA, including numerous non-coding RNAs.Цель – на модели обширной резекции печени (ОРП) изучить клеточные механизмы активации восстановительных процессов в печени при использовании общей РНК (оРНК) клеток костного мозга (ККМ). Материалы и методы. Крыс-самцов породы Вистар (n = 80) с моделью ОРП (70%) разделили на 2 группы: группа 1 – контроль с однократным введением физиологического раствора и группа 2 – опытная с однократным введением оРНК в дозе 30 мкг/100 г веса животного. Контролировали в динамике биохимические показатели функции и массу печени, а также микроструктурные изменения гепатоцитов через 48 часов после ОРП, исследуя митотическую активность, экспрессию каспазы 9 и морфометрические показатели. Результаты. Установлено, что в группе 2 по сравнению с группой 1 имеет место: более быстрая нормализация биохимических показателей (к 10–14-м суткам), более высокий митотический индекс гепатоцитов (23,45‰ против 5,37‰), первоначально более резкое снижение, а затем более быстрое восстановление массы печени (к 10–12-м суткам против 18–20-х суток). В группах 1 и 2 выявлена практически тотальная экспрессия каспазы 9, в том числе в митотически делящихся гепатоцитах. В группе 1 выявлено уменьшение значений морфометрических показателей одно- и двухъядерных клеток, уменьшение количества двухъядерных гепатоцитов и увеличение общей плотности гепатоцитов по сравнению с интактной печенью. Внутрибрюшинное введение оРНК приводило к увеличению значений морфометрических показателей одноядерных гепатоцитов, не влияло на их количество, но увеличивало площадь ядер двухъядерных гепатоцитов по сравнению с контролем. Заключение. Доказанное свойство оРНК из ККМ одновременно поддерживать в клетках печени после ОРП процессы апоптоза и индуцировать митотическую активность свидетельствует о том, что оРНК способна на ранней фазе регенерационного процесса переключать активировавшийся апоптоз на пролиферацию клеток. Обнаруженный эффект может быть обусловлен наличием в составе оРНК регуляторных молекул РНК, в том числе многочисленных белок-некодирующих РНК

Macrophage population state and proliferative activity of spleen cells under liver regeneration conditions

Relevance. Currently, the participation of immune system cells in the regulation of reparative processes is attracting more and more attention of researchers. There is an anatomical connection between the liver and spleen by means of portal vein. Thus, cytokines and other biologically active substances can enter the liver from the spleen through the portal vein, as well as cells can migrate to the liver. However, the specific mechanisms of mutual influence of the mentioned organs, including in reparative processes, remain poorly studied. The aim of our work was to study the state of spleen monocyte-macrophage population after liver resection, as well as the proliferative activity of spleen cells during liver regeneration . Materials and Methods . The model of liver regeneration after 70 % resection in mouse was reproduced in this work. The animals were taken out of the experiment after 1, 3 and 7 days. The marker of cell proliferation Ki67 was immunohistochemically detected, the state of spleen monocyte-macrophage population was evaluated by markers CD68, CD115, CD206, F4/80 by methods of immunohistochemistry and flow cytometry. Results and Discussion . The liver regeneration had a pronounced effect on the cytoarchitectonics of the spleen. In 1 day after liver resection in the spleen there was observed a decrease in the share of Ki67+cells, according to the flow cytometry data there was a decrease in the number of CD115+cells, in 3 and 7 days there was a decrease in the number of F4/80+ macrophages. Conclusion . Liver resection causes changes in the state of cell populations of the spleen as well. First of all, to the decrease in the activity of proliferative processes in it, as well as to the changes in the state of the monocyte-macrophage system. A decrease in the content of CD115+ and F4/80+ cells in the spleen was found, which indirectly indicates the migration of monocytes/macrophages after liver resection, which can also influence the course of reparative processes in the liver

Migration, proliferation and cell death of regenerating liver macrophages in an experimental model

Relevance . Macrophages are the leading regulatory cell-lineage taking part in reparative processes in mammals, and the liver is no exception. The ratio of monocyte migration, proliferation and death of macrophages during liver regeneration requires further studies. The aim was to quantify the intensity of monocyte migration, cell proliferation and apoptosis of resident liver macrophages after its 70 % resection in a mouse model. Materials and Methods. We performed 70 % liver resection in sexually mature male BalbC mice. Cells of liver monocyte-macrophage system were obtained by magnetic sorting by marker F4/80. The immunophenotype of the isolated cells was further studied by cytofluorimetry, the level of proliferation and cell death, the content of cyclins and P53 was determined by western blot. Results and Discussion . It was found that after partial hepatectomy there is a marked migration of monocytes/macrophages positive for Ly6C and CD11b markers to the liver, the migration process starts already in the first day after the operation. On the same terms there is a rise in proliferative activity of macrophages, established by Ki67 marker, the peak of proliferation - 3 days after partial hepatectomy. A significant increase in the number of dying macrophages was found early after liver resection. Conclusion . The obtained data indicate that liver regeneration in mammals on the model in mice is accompanied by proliferation migration and cell death of macrophages. Taking into account the immunophenotype of macrophages, we can conclude that Ly6C+ blood monocytes migrate to the liver, and resident macrophages participate in proliferation. The obtained data confirm the universality of the course of reparative processes in mammals

Phenotypical and Functional Polymorphism of Liver Resident Macrophages

Liver diseases are one of the main causes of mortality. In this regard, the development of new ways of reparative processes stimulation is relevant. Macrophages play a leading role in the regulation of liver homeostasis in physiological conditions and in pathology. In this regard, the development of new liver treatment methods is impossible without taking into account this cell population. Resident macrophages of the liver, Kupffer cells, represent a unique cell population, first of all, due to their development. Most of the liver macrophages belong to the self-sustaining macrophage cell population, whose origin is not bone marrow. In addition, Kupffer cells are involved in such processes as regulation of hepatocyte proliferation and apoptosis, remodeling of the intercellular matrix, lipid metabolism, protective function, etc. Such a broad spectrum of liver macrophage functions indicates their high functional plasticity. The review summarizes recent data on the development, phenotypic and functional plasticity, and participation in the reparative processes of liver macrophages: resident macrophages (Kupffer cells) and bone marrow-derived macrophages

Activation of regenerative processes in the liver when using cell-bone marrow total RNA

Objective: to study the cellular mechanisms of activation of regenerative processes in the liver when using total RNA (tRNA) of bone marrow cells (BMCs) based on an extended liver resection (ELR) model. Materials and methods. Male Wistar rats (n = 80) with ELR model (70%) were divided into 2 groups: group 1 (control group) had a single saline injection, while group 2 (experimental group) received a single tRNA injection at a 30 μg/100 g dose of animal weight. The biochemical parameters of liver function and weight were monitored over time. Also monitored were microstructural changes in hepatocytes 48 hours after ELR by examining mitotic activity, caspase-9 expression and morphometric parameters. Results. It was found that in group 2, in comparison to group 1, there was faster normalization of biochemical parameters (by 10–14 days), a higher mitotic index of hepatocytes (23.45‰ versus 5.37‰), and initially sharper decrease and then faster recovery of liver mass (by 10–12 days versus 18–20 days). Both groups showed almost total expression of caspase-9, including in mitotically splitting hepatocytes. Group 1 demonstrated decreased values of morphometric parameters of single and binuclear cells, decreased number of binucleated hepatocytes and increased total density of hepatocytes as compared to the intact liver. Intraperitoneal administration of tRNA increased morphometric parameters of mononuclear hepatocytes, did not affect their number, but increased the area of the nuclei of binuclear hepatocytes as compared to the control group. Conclusion. The proven capability of cell-bone marrow total RNA to simultaneously support apoptosis in liver cells after ELR and induce mitotic activity indicates that tRNA can switch activated apoptosis to cell proliferation at the early phase of the regenerative process. This effect may be due to the presence of regulatory RNA molecules in tRNA, including numerous non-coding RNAs

Multipotent stromal cells stimulate liver regeneration by influencing the macrophage polarization in rat

AIM: To investigate the influence of the umbilical cordderived multipotent stromal cells (MSCs) on recovery of the liver after the subtotal resection, that is, removal of 80% of the organ mass, a renowned model of the small-for-size liver remnant syndrome. METHODS: The MSCs were obtained from the intervascular tissue of umbilical cords, dissected from rat fetuses, by the explant culture technique. The vital labeling of MSCs with PKH26 was carried out on the 3rd passage. The subtotal resection was performed on male Sprague-Dawley rats. The experimental group animals received a transplant 106 MSCs infused into the spleen. Hepatocyte proliferation was assessed by counting of either mitotic figures or Ki67-positive cells in microscopic images. MSC differentiation was assessed with antibodies to hepatocyte-specific marker cytokeratin 18 (CK18), cholangiocyte-specific protein CK19, smooth muscle cell-specific protein α-SMA, the endothelial cell marker CD31, or the active fibroblast marker FAPα. Total macrophages of the liver were selectively stained in cryosections incubated with anti-CD68 antibodies (1:100, Abcam), while the M2a and M2c macrophage populations were selectively stained with anti-CD206 antibodies. Expression of interleukin and growth factor genes was evaluated with PCR-RT. RESULTS: Intrasplenic allogeneic transplantation of the umbilical cord-derived multipotent stromal cells stimulates reparative processes within the residual liver tissue after subtotal resection (removal of 80% of the organ mass), as indicated by increased rates of hepatocyte proliferation and accelerated organ mass recovery. These effects may result from paracrine influence of the transplanted cells on the resident macrophage population of the liver. The transplantation favors polarization of macrophages to M2 phenotype (the M2-polarized macrophages specifically express CD206; they are known to suppress inflammation and support tissue repair). No differentiation of the transplanted cells into any of the liver cell types have been observed in the study. CONCLUSION: We found no direct evidence for the paracrine effect of MSCs on liver regeneration after the subtotal liver resection in rats. However, the paracrine mechanism of the therapeutic activity of transplanted MSC is indirectly indicated by a decrease in the total number of CD68 + macrophages and an increase in the proportion of M2 pro-repair macrophages in the regenerating liver as compared to animals in which the transplantation was only mimicked. © The Author(s) 2018