[The study of interaction of different forms of human recombinant anti-mullerian hormone with a chimeric analogue of the AMH type II]. / Issledovanie vzaimodeistviia razlichnykh form rekombinantnogo antimiullerova gormona cheloveka s khimernym analogom ego retseptora II tipa.

The homodimeric glycoprotein, anti-mullerian hormone (AMH), described over 70 years ago by A. Jost, is the least studied member of the transforming growth factor beta superfamily. Despite the antitumor activity of AMH discovered at the end of the last century, the creation of effective drugs based on AMH is hindered primarily by the lack of information on the mechanism of various AMH forms interaction with a specific type II receptor (MISRII). Previously, we have shown that not only the full-length activated hormone but also its C-terminal fragment (C-rAMH) could bind to MISRII. In this work, using the surface plasmon resonance technique, we compared the interaction of three forms of recombinant AMH (rAMH) with the MISRII analogue - the chimeric protein MISRII-Fc containing AMH type II receptor and a Fc-fragment of the human IgG1 heavy chain. Comparison of the binding of MISRII-Fc, immobilized on a chip with group specificity for human immunoglobulins, to C-rAMH, to intact rAMH (pro-rAMH), and to rAMH containing one uncleaved monomer (hc-rAMH), showed that the KD of the complexes increased: 1.7 nM, 88 nM and 110 nM, respectively. Thus, we have shown that C-terminal fragment of AMH has the maximum affinity for the recombinant MISRII analogue, which indicates the prospects for the development of drugs based on this hormone derivative.

[Mullerian inhibiting substance type II receptor as a potential target for antineoplastic therapy]. / Retseptor antimiullerova gormona II tipa kak potentsial'naia mishen' dlia protivoopukholevoi terapii.

The review considers properties of the type II anti-Mullerian hormone receptor (mullerian inhibiting substance receptor type II, MISRII), a transmembrane sensor with its own serine/threonine protein kinase activity, triggering apoptosis of the Mullerian ducts in mammalian embryogenesis and providing formation of the male type reproductive system. According to recent data, MISRII overexpression in the postnatal period is found in cells of a number of ovarian, mammary gland, and prostate tumors, and anti-Mullerian hormone (AMH) has a pro-apoptotic effect on MISRII-positive tumor cells. This fact makes MISRII a potential target for targeted anti-cancer therapy. Treatment based on targeting MISRII seems to be a much more effective alternative to the traditional one and will significantly reduce the drug dose. However, the mechanism of MISRII-AMH interaction is still poorly understood, so the development of new anticancer drugs is complicated. The review analyzes MISRII molecular structure and expression levels in various tissues and cell lines, as well as current understanding of the AMH binding mechanisms and data on the possibility of using MISRII as a target for the action of AMH-based antineoplastic drugs.

[The dynamics of human total and activated anti-mullerian hormone serum levels in different life periods.]

Here, changes in the serum level of total anti-mullerian hormone (AMH) and its activated form in children of both sexes and women with different reproductive status are investigated. This TGFß superfamily cytokine is known to provide the formation of the male-type reproductive system in mammalian embryogenesis, and regulate folliculogenesis, spermatogenesis and the balance of sex hormones after birth. The biologically active form of the hormone (aAMH) is formed as a result of limited proteolysis of the AMH molecule; it is not reliably known in which tissues and under the action of which enzyme it occurs. The serum level of aAMH seems to be a more informative clinical indicator than the content of total AMH (tAMH), but there are no ELISA systems at the world market that provide direct quantitative detection of aAMH. In this work, quantitative detection of total hormone (tAMH) and its biologically active form (aAMH) in serum was performed using specially developed enzyme immunoassay systems. We showed that in girls, the total serum AMH level, as well as the concentration ratio aAMH / tAMH, practically does not change with age, whereas in boys, there is not only a significant decrease in the total serum AMH level previously described in the literature (Pearson correlation coefficient R = - 0.86, p <0.001), but also in the ratios of the aAMH / tAMH level (R = -0.531, p <0.001). It was also found that in pregnant women, the amount of total AMH and the proportion of aAMH in serum was significantly higher (p <0.01 and p <0.001, respectively) than in the control group women. The obtained results are in good agreement with the available data on the total and activated AMH content in the blood serum of people of different sex and age and indicate a change in the ratio of aAMH / tAMH serum levels in pregnancy. These data may be important both for deepening the understanding of AMH biology and for interpreting the results obtained using AMH detection based diagnostics.

The effect of Coulomb repulsion on the space-time resolution limits for ultrafast electron diffraction.

The development of electron sources capable of temporal resolution on the order of 1 ps or less raises a number of questions associated with the estimation of the physical meaning and accuracy of the dynamic parameters based on the analysis of time-dependent scattering intensity. The use of low brightness ultrashort pulses with few electrons leads to the necessity for increasing the total exposure time and lengthening the time of data acquisition, with attendant problems with the limited sample. The sample restrictions can be mitigated by increasing the charge per pulse, i.e., by going to high brightness sources. Increasing in the number of electrons, however, is limited by the Coulomb repulsion between them, which leads on one hand to distortion of the diffraction pattern and on the other hand to an increase in the duration of the pulse. An analytical technique for estimating the deformation of the diffraction pattern caused by the Coulomb repulsion of the electrons in electron bunches with duration of less than 10 ps and the influence of this effect on the accuracy of determination of the interatomic distances is developed for the non-relativistic and relativistic regimes for electron energies.

[Immunity of a leguminous plant infected by nodular bacteria Rhizobium spp. F.]

Recent studies of the immune system of leguminous plants infected with nodular bacteria (rhizobia) are summarized. The possibility of blocking the invasion of rhizobia into plant organs not affected by the primary infection is discussed. The concept of local and systemic resistance of the leguminous plant to rhizobial infection is introduced. The Nod factors of rhizobia are considered, as well as the plant receptors that interact with these factors upon the formation of symbiosis of the plant and bacteria. The role of bacterial surface exopolysaccharides in the suppression of the protective system of the plants is discussed. The innate immunity of leguminous plant cells is assumed to affect the formation and functioning of the symbiosis of the plant and the bacteria.

[EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS IN ADULT RATS AFTER ADMINISTRATION OF INTERLEUKIN-1beta DURING DIFFERENT PERIODS OF EARLY LIFE].

Multiple sclerosis is a chronic disease of the CNS that affects people of working age, in which the targets of aggressive immune cells become the myelin and myeline producing cells, as well as neurons. It is assumed that a predisposition to MS is forming in childhood, due to common infections. In this paper the experimental allergic encephalomyelitis (EAE) was examined in rats administered IL-1beta at different periods of the early postnatal ontogenesis. EAE was induced in rats at the age of 3 months by single subcutaneous immunization with a homologous homogenate of spinal cord in complete Freund's adjuvant. The number of sick animals were evaluated, as well as the severity of the disease and its duration. It was shown that in rats after administration of IL-1beta on 1st and on 4th week of life EAE is more severe than corresponding control groups of rats. Discusses the damaging or protective effects of injections of IL-1beta during different periods of early postnatal ontogenesis, role of stress reactivity and communication with the "hygiene hypothesis".

[THE EFFECTS OF CHRO- NIC ADMINISTRATION OF MODERATE DOSE OF PROINFLAMMATORY CYTOKINE INTERLEUKIN-1ß ON RAT INVESTIGATIVE BEHAVIOR AND SPATIAL MEMORY].

The aim of this study was to discover the effects of chronic intraperitoneal administration of proinflammatory cytokine interleukin-1ß (IL-1ß) on rat investigative behavior and spatial memory. Rats were injected with a moderate pyrogenic dose of IL-1ß (0.5 mkg/kg) daily during 14 days (7 days before tests and 7 testing days). The behavior was examined in 23.5 hours after the previous injection of cytokine. The test battery included: "The open field" (within 3 consecutive days), "The exploration of novel objects", and "The Morris water maze". The animals treated with IL-1ß differed from the control animals in an essential decrease of locomotor activity, slight increase of anxiety and suppression of exploratory behavior. The impairment of spatial memory was not revealed.

Complement anaphylatoxin C3a is selectively protective against NMDA-induced neuronal cell death.

The anaphylatoxin C3a is a potent inflammatory polypeptide released at sites of complement activation. To test whether C3a might alter neuronal outcome following an ischemic insult, we determined the effects of purified human C3a on murine primary cortical cell cultures exposed to apoptotic or excitotoxic paradigms. C3a prevented neither serum deprivation-induced apoptotic neuronal death, nor AMPA/kainate-mediated excitotoxicity. However, in mixed cultures of neurons and astrocytes, C3a dose-dependently protected neurons against NMDA toxicity (47% neuroprotection using 100 nM C3a, p < 0.01, n = 12). The neuroprotective effect of C3a was observable only in the presence of astrocytes. These observations suggest that C3a is involved in excitotoxicity-mediated neuronal death through astrocyte stimulation and extend its role beyond immune functions.

C3A binds to the seven transmembrane anaphylatoxin receptor expressed by epithelial cells and triggers the production of IL-8.

The complement (C) plays an important role in many acute inflammatory processes. C3a is an inflammatory polypeptide named anaphylatoxin, generated during C activation and which acts through a specific receptor C3aR. In this study, we demonstrated that the epithelial cell line ECV 304 constitutively expressed C3aR (by flow cytometry and immunofluorescence) and that binding of purified C3a to epithelial cells resulted in a time- and dose-dependent upregulation of interleukin-8 (IL-8). Pre-treatment of ECV 304 with pertussis toxin inhibited IL-8 response induced by C3a, indicating that the action of C3a was mediated by a G protein coupled pathway.

The role of reactive oxygen species in membrane potential changes in macrophages and astrocytes.

Involvement of reactive oxygen species (ROS) in changes of the plasma membrane potential of mouse peritoneal macrophages and astrocytes (U118 cell line) under the action of different agents has been studied. Membrane potential was measured using the voltage-dependent fluorescent oxonol dye DiBAC4(3). Agonists which stimulate macrophages to release ROS (the fMLP peptide and platelet activating factor) caused prolonged hyperpolarization. Experiments with the fluorescent probe 2',7'-dichlorofluorescein diacetate have shown that astrocytes release ROS upon the action of C5a complement anaphylatoxin (but not C3a). The effect of C5a was accompanied with hyperpolarization of the astrocyte plasma membrane. Treatment of the cells with agents which do not induce ROS generation (C3a, lipopolysaccharide, interferon-gamma) depolarized the plasma membrane. Hyperpolarization of both cell types was significantly decreased in the presence of superoxide dismutase (but not catalase). Moreover, the O2- -generating system caused a marked hyperpolarization of both cell types. The data obtained suggest that O2- is involved in the macrophage and astrocyte hyperpolarization response.

Inhibition of IgE-mediated triggering of mast cells by complement-derived peptides interacting with the Fc epsilon RI.

Mucosal type mast cells, in contrast to the serosal type ones, do not respond to cationic agents, or to the complement-derived peptides C3a and C5a. Earlier we have found that while C3a does not activate the rat mucosal type mast cells (line RBL-2H3), it strongly inhibits the IgE-mediated triggering of these cells, by interfering with the Fc epsilon RI-initiated signaling pathway. In the present study we further investigated the mechanism of this process. It is shown, that C3a interacts with the beta-chain of the Fc epsilon RI complex. Binding of the complement peptide to the cells apparently causes a decrease in the proximity of the IgE-binding Fc epsilon RI. Investigating certain sequences of C3a we found that the inhibition is caused by the C-terminal sequences of the complement-peptide, ranging from positions 56 to 77 and also by a shorter sequence, ranging from positions 56 to 64. The inhibitory effect of these peptides was observed both in the case of RBL-2H3 cells and mouse bone marrow derived mast cells.

Expression of cytokines by human astrocytomas following stimulation by C3a and C5a anaphylatoxins: specific increase in interleukin-6 mRNA expression.

C3a and C5a anaphylatoxins are two proinflammatory peptides generated during complement activation that act through distinct Gi protein-coupled receptors named C3aR and C5aR, respectively. We have demonstrated previously that human astrocytes expressed C3aR and C5aR constitutively and were able to produce a functional complement. In this study, we examined the effect of an anaphylatoxin stimulation on cytokine expression by human astrocyte cell lines. Interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha, and transforming growth factor-beta mRNA expression was studied by quantitative RT-PCR. Whereas IL-1beta, tumor necrosis factor-alpha, and transforming growth factor-beta mRNA levels remained unchanged, stimulation of astrocytoma cells (T98G, CB193, U118MG) by C3a, C5a, and peptidic C3aR and C5aR agonists induced an increase in the IL-6 mRNA level. The amount of IL-6 was markedly increased at 3 and 6 h and returned to the basal level at 9 h of stimulation. This response was specific, because pretreatment of cells with pertussis toxin or with polyclonal anti-C3aR or anti-C5aR antibodies completely blocked the IL-6 mRNA increase. The IL-6 response was also investigated at the protein level, but IL-6 protein was detected neither in cell lysates nor in supernatants of stimulated cells. The anaphylatoxin-mediated transcriptional activation of IL-6 gene suggests that C3a and C5a could play a role in priming glial cells during the inflammatory process in the brain.

Expression of a functional anaphylatoxin C3a receptor by astrocytes.

Human astrocyte cell lines reportedly contain a specific receptor for the complement anaphylatoxin C3a based on ligand-binding studies, functional responses, and RNA analysis by RT-PCR. Uptake of 125I-C3a by astrocytes was specific and reversible. Scatchard analysis indicated the presence of two classes of binding sites. High-affinity binding sites were abundantly expressed (20,000-80,000 sites per cell) with an estimated K(D) of 1-2 nM. Low-affinity binding sites with a K(D) of 209 nM were largely expressed (n > or = 4 x 10(6) sites per cell) and probably did not reflect a receptor-mediated binding, but rather an ionic interaction between C3a and the membrane. Analysis of astrocyte mRNA by RT-PCR with three different sets of primers covering 60% of the C3a receptor (C3aR) mRNA sequence indicated that glial C3aR was identical to the leukocytic one. Western blot analysis using a specific anti-C3aR evidenced a C3aR with a molecular mass of 60,000 Da. C3a and a superagonist peptide, E7, induced a transient increase of intracellular [Ca2+] in primary culture of astrocytes. Treatment of the ligands by carboxypeptidase B to eliminate the C-terminus Arg considerably decreased the [Ca2+] response. Moreover, flow cytometry experiments demonstrated the expression of C3aR on normal rat astrocyte membrane. This report brings new insight for the role of the complement system in the brain inflammation response.

Characterization of rat C5a anaphylatoxin receptor (C5aR): cloning of rat C5aR cDNA and study of C5aR expression by rat astrocytes.

Complement system activation within the central nervous system (CNS) is involved in demyelinating and neurodegenerative disorders, but the role of complement in the pathogenic process or in the repair remains unclear. Besides the direct lytic effects of complement on target cells (oligodendrocytes or neurons), complement can exert other functions through interaction of complement fragments with specific receptors. The C5a anaphylatoxin, an inflammatory peptide which is formed during complement activation, might play a role in the CNS pathogenesis, and activation and recruitment of glial cells by binding to its receptor (C5aR) on CNS cells. Using degenerate primers corresponding to homologous regions between human and mouse C5aR cDNAs, we have cloned a rat C5aR cDNA probe from rat monocytes RNAs after RT-PCR experiment. The rat C5aR probe isolated by this procedure allowed us to clone the rat C5aR cDNA-coding sequence using a library screening cloning strategy. This probe was also used to study the expression of the C5aR mRNA in the rat CNS. Northern blotting and RT-PCR experiments demonstrated the constitutive expression of C5aR mRNA in brain, spleen, kidney and lung. This transcript was also observed in primary culture of rat astrocytes. Microfluorimetry experiments demonstrated that C5aR expressed by astrocytes in culture is functional since the addition of C5a induced a dose-dependent increase of intracellular calcium concentration. The expression of the C5aR by astrocytes suggests new roles for the C5a anaphylatoxin in reactive astrogliosis to CNS injuries.

Identification and characterization of the complement C5a anaphylatoxin receptor on human astrocytes.

The C fragment C5a exerts its important physiologic and pathologic effects through interaction with a specific C5a receptor (C5aR) which is highly expressed on polymorphonuclear leukocytes and some other leukocytes. The presence of this receptor on epithelia and endothelia has recently been documented, raising the possibility that these other cells might also respond to locally generated C5a. C has been implicated in several brain disorders, notably demyelination and neurodegeneration, and cells within brain can synthesize a complete C system. It is thus of interest to examine the mechanisms by which C damages or activates brain cells. To this end we have examined the expression on human fetal astrocytes and astrocyte-derived cell lines of receptors for C fragments. We here report that human astrocytes and cell lines express a receptor for C5a (48 to 72 x 10(3) copies/cell), which is indistinguishable at the protein or mRNA level from that in leukocytes. The astrocyte C5aR was recognized by five different specific Abs, which revealed by Western blotting a protein of 40 to 45 kDa in primary human astrocytes and astrocyte cell lines. Expression was confirmed by RT-PCR using multiple primers. Neither inflammatory cytokines nor PMA caused up-regulation of the receptor on astrocytes. The receptor was functional in that addition of C5a (1 nM to 100 nM) or, at high doses (100 nM), C5adesArg, triggered a calcium transient in astrocytes. We propose that C5aR expression on astrocytes plays an important role in control of inflammation in brain and may be a central component of C-mediated brain injury.

Expression of the complement classical pathway by human glioma in culture. A model for complement expression by nerve cells.

In this paper we demonstrate the synthesis of the components of the classical complement pathway, namely C1q, C1r, C1s, C1-Inh, C2, C4, and C5, by human glioma cell lines (U118MG, T193, and T98G). All these components were structurally, antigenically, and functionally similar to their serum counterparts as determined by biosynthetic labeling experiments, Western blot analysis, and hemolytic assays. Northern blot analysis of mRNA demonstrated that, for each of these components, their specific mRNA had the same size as the equivalent mRNA from hepatic tissue. We could not detect the synthesis of C4bp by these cell lines, and the secretion of C1q was only detected after stimulation by interferon-gamma. All these syntheses were up-regulated by interferon-gamma and tumor necrosis factor. Interleukin-1 beta only increased C2 expression and reproducibly down-regulated C5 secretion when used at high doses. Glioma cell lines appear to be an efficient and convenient model for the analysis of complement expression in human astrocytic cells.

Expression of complement components of the alternative pathway by glioma cell lines.

Glioma cell lines express proteins of the complement alternative pathway, namely C3, factor B, factor H, and factor I. Secretion of these proteins was shown by a sensitive and specific ELISA. C3 and factor H were rapidly secreted by glioma cell line CB193 and reached a concentration of 140 ng/ml/10(6) cells after 72 h of culture. Factor B and factor I were secreted at a lower rate and reached concentrations of 25 and 15 ng/ml/10(6) cells, respectively. Western blot and immunoprecipitation experiments showed that secreted proteins were identical to the corresponding plasma proteins. For factor H, besides the well known 150-kDa species, an additional polypeptide of 45 kDa with factor H immunoreactivity was observed. This species corresponded to the N-terminal truncated form found in plasma. In preliminary experiments, we observed control of these syntheses by cytokines. IL-1 beta significantly increased C3 secretion, with no effect on factor H. Secretion of factor H was enhanced by IFN-gamma. These results show that a glioma cell line could be a useful tool to study complement biosynthesis by glial cells in humans.