Immune Proteasomes Research Articles

Activation of immune cell proteasomes in peripheral blood of smokers and COPD patients: implications for therapy.

BackgroundImmune cells contain a specialised type of proteasome, i.e. the immunoproteasome, which is required for intracellular protein degradation. Immunoproteasomes are key regulators of immune cell differentiation, inflammatory activation and autoimmunity. Immunoproteasome function in peripheral immune cells might be altered by smoking and in chronic obstructive pulmonary disease (COPD), thereby affecting immune cell responses.MethodsWe analysed the expression and activity of proteasome complexes in peripheral blood mononuclear cells (PBMCs) isolated from healthy male young smokers as well as from patients with severe COPD and compared them with matching controls.ResultsProteasome expression was upregulated in COPD patients as assessed by quantitative reverse transcriptase-PCR and mass spectrometry-based proteomic analysis. Proteasome activity was quantified using activity-based probes and native gel analysis. We observed distinct activation of immunoproteasomes in the peripheral blood cells of young male smokers and severely ill COPD patients. Native gel analysis and linear regression modelling confirmed robust activation and elevated assembly of 20S proteasomes, which correlated significantly with reduced lung function parameters in COPD patients. The immunoproteasome was distinctly activated in COPD patients upon inflammatory cytokine stimulation of PBMCs in vitro. Inhibition of the immunoproteasome reduced pro-inflammatory cytokine expression in COPD-derived blood immune cells.ConclusionsGiven the crucial role of chronic inflammatory signalling and the emerging involvement of autoimmune responses in COPD, therapeutic targeting of the immunoproteasome might represent a novel therapeutic concept for COPD.

Estrogen Receptors and Ubiquitin Proteasome System: Mutual Regulation.

This review provides information on the structure of estrogen receptors (ERs), their localization and functions in mammalian cells. Additionally, the structure of proteasomes and mechanisms of protein ubiquitination and cleavage are described. According to the modern concept, the ubiquitin proteasome system (UPS) is involved in the regulation of the activity of ERs in several ways. First, UPS performs the ubiquitination of ERs with a change in their functional activity. Second, UPS degrades ERs and their transcriptional regulators. Third, UPS affects the expression of ER genes. In addition, the opportunity of the regulation of proteasome functioning by ERs—in particular, the expression of immune proteasomes—is discussed. Understanding the complex mechanisms underlying the regulation of ERs and proteasomes has great prospects for the development of new therapeutic agents that can make a significant contribution to the treatment of diseases associated with the impaired function of these biomolecules.

Proteasome Expression in Ovarian Heterotopic Allografts of Wistar and August Rats under Induction of Donor-Specific Tolerance

The aim of this work is to investigate the dynamics for the ovarian tissue engraftment of inbred August rats transplanted to outbred Wistar rats and vice versa on the background of the induction of donor-specific tolerance and to identify the features of proteasome pools in the surviving grafts and tissue replacing the rejected grafts in animals of both groups. By day 14 after transplantation, a slight difference in graft engraftment was revealed in studied recipients: 87% in Wistar rats and 80% in August rats. At the same time, ovarian tissue allografts with well-preserved structure and significant vascularization represented 76% in Wistar rats and only 29% in August rats on day 37. The difference revealed in the remote period is apparently connected with the special condition of the central nervous system of August rats caused by the increased content of monoamines and heat-shock protein 70. The total proteasome level on day 37 was the same both in intact donor tissue and in the surviving grafts, as well as in the tissue replacing the rejected grafts, and did not depend on the donor–recipient differences. However, the increased expression of immune proteasomes was found in the surviving tissue and tissue replacing the rejected grafts compared to intact donor tissue. The surviving grafts were enriched with proteasomes with immune subunit LMP2, and the tissue replacing the rejected grafts was enriched with proteasomes with immune subunit LMP7 equally in August and Wistar rats. In addition, the surviving grafts in both groups of animals were characterized by equally high expression of proteasome activator РА28αβ compared to the replacement and intact donor tissues. Thus, transplant proteasomes containing immune subunits LMP2, and probably associated with the activator RA28αβ, serve as one of the key factors for engraftment regardless of the genetically determined differences of the recipients.

Changes in the Content of Mononuclear Liver Cells Expressing Immune Proteasomes after Transplantation of Ovarian Tissues Depending on Donor-Recipient Differences in Rats.

Implantation of ovarian tissue allografts in outbred Wistar rats and inbred August rats against the background of induction of donor-specific tolerance was accompanied by an increase in liver content of mononuclear cells expressing LMP2 immune of proteasome subunit by day 37 after transplantation in comparison with day 0. Graft rejection, on the contrary, was associated with a decrease in the number of LMP2+ cells in the liver of rats of both lines during this period. The difference in the content of these cells and the graft take rate were higher in Wistar rats. The number of mononuclear cells expressing LMP7 immune proteasome subunit in the liver did not change in rats of both lines by day 37 in comparison with day 0. Thus, the level of immune proteasomes with LMP2 subunit in mononuclear cells of the liver is related to fine mechanisms of regulation of immune responses and their shift toward graft take or rejection.

Changes in the Expression of Immune Proteasomes in the Liver after the Induction of Portal Tolerance Depending on Donor-Recipient Differences in Rats.

Induction of donor-specific tolerance in outbred Wistar rats (RT1u) and inbred August rats (RT1c) increased the expression of immune proteasome subunits in liver with a peak on day 7 after beginning of the induction. The increase in the level of immune subunits LMP2 and LMP7 was more pronounced in the liver of August rats in comparison with Wistar rats (by 2 and 6 times, respectively), which was associated with higher concentrations of monoamines in the CNS of August rats. After induction of donor-specific tolerance in August and Wistar rats, the immune subunits were in cells of sinusoidal lining and in cells located in sinusoid lumens. It can be suggested that immune proteasomes in these cells producing antigenic peptides for presentation to immunocompetent participate in the suppression of their activity and form the molecular basis for the development of donor-specific tolerance at very early stages of this process.

Detection of active proteasome structures in brain extracts: proteasome features of August rat brain with violations in monoamine metabolism.

The aim of this work was to detect changes in proteasome pools of brain parts of August rats with monoamine metabolism violations in comparison with that of control Wistar rats. To reveal active proteasome structures, a method of native electrophoresis for the analysis of crude tissue fractions was developed. By means of this method and following Western blotting, the most pronounced changes in reorganization of proteasome structures were detected in proteasome pool of the brain cortex of August rats. Main findings are the enhanced expression of immune proteasome subtypes containing proteolytic subunit LMP2 and activator PA28αβ as well as immune proteasome subtypes containing proteolytic subunit LMP7 and activator PA700 and simultaneously decreased expression of subtypes with subunit LMP2 and activator PA700 in the brain cortex of August rats compared to that of Wistar rats. These results were indirectly confirmed by SDS PAGE method followed by Western blotting, which showed the increased quantities of immune subunits and proteasome activators in the brain cortex of August rats compared to that of Wistar rats. Immune proteasomes were revealed by immunohistochemistry in neurons, but not in glial cells of August and Wistar rat cortex. The detected reorganization of proteasome pools is likely to be important for production of special peptides to provide the steady interaction between neurons and adaptation of central nervous system to conditions caused by monoamine metabolism deviations.

Native structure of rat liver immune proteasomes.

Native structure of active forms of rat liver immune proteasomes has been studied by two-dimensional electrophoresis method modified for analysis of unpurified protein fractions. The developed method allowed revealing the proteasome immune subunits LMP7 and LMP2 in 20S subparticles and in the structures bound to one or two PA28αβ activators, but not to the PA700 activator, which is involved in the hydrolysis of ubiquitinated proteins. The results obtained indicate the participation of the immune proteasomes in delicate regulatory mechanisms based on the production of biologically active peptides and exclude their participation in processes of crude degradation of "rotated" ubiquitinated proteins.

P13: Features of proteasome system functioning in the tumor and microenvironment cells of breast cancer

Background The tumor microenvironment plays an important role in the progression of cancer and may be regulated by proteolysis, including the proteasomes. The proteasomes modify the biologically important molecules involved in the pathogenesis and progression of a variety of malignancies, including breast cancer (BC). The aim of this study was to investigate the features of the subunit composition of proteasomes in the tumor and its microenvironment of breast cancer. Material and Methods The material for investigation was samples of tumor tissue of invasive ductal breast cancer. The study was conducted to estimate the distribution of the total pool of proteasome, proteasomes activator PA700, immune proteasome forms containing LMP7 and/or LMP2 subunit in tumor cells and stromal component using immunofluorescence. Furthermore, there was evaluated the distribution of the proteasome in the tumor. The expression of immune proteasomes and proteasomes activators in the cells was studied by immunofluorescence labeling of the cells by antibodies to immune proteasome subunits and cell markers. Fluorescence was analyzed by using a fluorescence microscope DM RXA2 (“Leica”, Germany) and confocal microscope TCS SP (“Leica”, Germany). The specificity of the primary antibodies was confirmed by check samples, at which the reaction was carried out only with the second antibody. No cross reaction between the first and second antibodies was tested by incubation of each primary antibodies with the opposing second antibodies. In addition, there was carried out labeling the cell nuclei by reagent Hoechst 33,342. Results It was found that the tumor cells comprise immune proteasomes, also PA700 and PA28αb activators. Activator PA28αb and immune proteasomes are localized in the cytoplasm of tumor cells, whereas α1, 2, 3, 5, 6, 7 subunits and PA700 activator detected in the cytoplasm and in the nuclei of tumor cells. Availability of α1, 2, 3, 5, 6, 7 subunits in the nuclei of tumor cells shows the expression of constitutive proteasome subunits. Stromal cells are characterized by a high ratio of the α1, 2, 3, 5, 6, 7 subunits to the immune LMP2 subunit as compared to cells of invasive ductal carcinoma. This means that the pool of proteasome in tumor cells of invasive ductal cancer is enriched by the immune proteasomes as compared to stromal cells. Thus, samples of invasive ductal breast cancer contain predominantly tumor cells enriched by immune proteasomes, activators PA700 and PA28αb. The presence of proteasomes in stromal component indicates that the tumor microenvironment also has active processes of proteolysis, with involving proteasome system. Probably the processes occurring in the stromal component contribute to the output of the proteasome into the extracellular space, which is confirmed by other researchers about the existence of circulating proteasome pools and their further participation in dissemination of cancer. This work was supported by the Russian Foundation for Basic Research (Grant numbers 13-04-00169 ).

Features of immune proteasome expression in the development of rat central nervous system

Formation of the central nervous system in ontogeny and function in adult mammals are controlled by universal ubiquitin-proteasome proteolytic system. The aim of this work was to study the dynamics of expression of immune proteasomes in comparison with the dynamics of ChLA and CLA proteasome and expression of the transcription factor Zif268 in the structures of the brain (cortex, hippocampus, and brainstem) in embryonic (E19, E21 days of embryonic development) and early postnatal (P1, P3, P4, P5, P7, P15 days of post-natal development) development in rats. ChLA and CLA in clarified homogenates of rat brain structures were determined by hydrolysis of fluorogenic commercial oligopeptides Suc-LLVY-AMC and Z-LLG-AMC, respectively. In the cortex and hippocampus of the brain was observed upregulation of immune subunits LMP7 during the active formation of biochemical mediatory structure and efferent neuronal projections at the period P7-P15. In the cerebral cortex during this period ChLA and CLA also are increased. In all structures of the brain the LMP2 immune subunits content was significantly increased at the period P7-P15. Contents of proteolytic constitutive subunit β1 in all structures decreased by P4 compare to P1 levels and was increased on P15 relative to the P1 levels. However, the level of expression of proteolytic constitutive subunit β5 increased in cortex, hippocampus and brainstem from E21 and reached maximum values on P3, P5 and P1, respectively with a sharp decrease to P7 in all studied structures. In all structures expression of LM P2 immune subunits and β1 constitutive subunits increased simultaneously with LMP7 immune subunits and sharply on P15. Also shown a positive correlation of increased expression regulator PA28 and constitutive β5 subunits in the hippocampus during the period P3-P5 and in the brainstem at the period P1-P5. The peculiarity of the studied brain regions during P7-P15 of rat early development is a correlation of expression of immune subunits LMP2 and LMP7 proteasome and ChLA with the expression of the transcription factor Zif268. Probably immune proteasome plays an important role in the regulation of key biochemical processes in the early ontogenesis of the central nervous system and are necessary for the emergence and realization of synaptic plasticity in the brain structures studied in rats.

Proteasomes on thyroid tissue allotransplantation under induction of donor specific tolerance in rats

The proteasomes in the liver of August rats (RT1C) were investigated 30 days after the allotransplantation of Wistar rat (RT1u) thyroid tissue under renal capsule with/without induction of donor specific tolerance by donor splenocyte intraportal administration. The level of the total proteasome pool, immune proteasomes containing the LMP2 and/or LMP7 subunits, proteasome 19S- and 11S-regulators was defined. The intact and sham-operated August rats were used as control groups. The level of all immune proteasome forms and 11S regulator increased while the level of the total proteasome pool and 19S regulator decreased in the liver of experimental animals compared to the control groups that indicated changes of liver functional state after transplantation. The 19S/11S ratio increased in the liver of non-tolerated rats compared to tolerated animals. In the liver of tolerated rats with survived transplants, the quantity of mononuclear cells, expressing the immune subunit LMP2, greatly increased in comparison with control and non-tolerated animals. Study of the survived transplants showed the increase of the ratio of LMP2/LMP7 immune subunits and 19S/11S regulators in them compared to the tissue replacing the rejected transplants. In the control intact thyroid tissue, the immune proteasomes were almost not revealed, while 19S/11S ratio was maximal. Thus, the development of the immune reaction or its suppression is accompanied by change of the balance between different proteasome forms. The immune subunit LMP7 and 11S regulator are connected with the response against donor tissue. On the contrary, the immune subunit LMP2 and 19S regulator are likely to be important for the immune tolerance development and survived tissue functioning. The low content of the immune proteasomes in the follicle cells was found by immunofluorescence assay. The formation of antigens for major histocompatibility complex class I molecules was impaired by low immune proteasome content that led to immunological tolerance to hormone-producing follicle cells.

Immune proteasomes in the development of the rat immune system

The dynamics of the expression of LMP7 and LMP2 proteasome subunits in embryonic and early postnatal development of rat spleen and liver is investigated in comparison with the dynamics of chymotrypsin-like and caspase-like proteasome activities and expression of MHC (major histocompatibility complex) class I molecules. The immune subunits LMP7 and LMP2 distribution in spleen and liver cells in the development process is also studied. A mutual for both organs tendency to the increase of the expression of both LMP7 subunit and LMP2 one on P21 (the 21st postnatal day) as compared to the embryonic period is discovered. However, the total proteasome level is shown to be constant. At definite development stages, the dynamics of immune subunits expression in the spleen and liver was different. In the spleen gradual enhancement of both immune subunits level being detected on P1, P18 and P21, in the liver gradual enhancement periods on E16 (the 16th embryonic day) and E18 changed to the stage of the shrink of immune subunits level on P5. This level did not reliably change till P18 and was augmented on P21. The alterations revealed were accompanied by chymotrypsin-like activity raise and caspase-like activity drop in spleen by P21 as compared with the embryonic period, which proves the enlargement of proteasome ability to form antigenic epitopes for MHC class I molecules. In the liver, both activities increased by P21 in comparison with the embryonic period. Such dynamics of caspase-like activity can be explained not only by the change of proteolytic constitutive and immune subunits, but also by additional regulatory mechanisms. Besides, it is discovered that the increment of immune subunits expression in the early spleen development is connected with the process of successive forming the white pulp by B- and T-lymphocytes enriched by immune subunits. In the liver, the growth of immune subunits level by P21 was accompanied by their expression expansion in hepatocytes, while their plunge by P5 may be related to the loss of liver function of a primary lymphoid organ of the immune system by this stage and disappearance of B-lymphocytes enriched by immune proteasomes in it. In the spleen and liver, MHC class I molecules were revealed at the periods of the raise of proteasome immune subunits level. On E21 , the liver was enriched by neuronal NO-synthase, its level decreased after birth and enhanced to P18. This fact indicates the possibility of the induction of the immune subunits LMP7 [character: see text] LMP2 expression in hepatocytes in signal way with neuronal NO-synthase participation. The results obtained prove that T-cell immune response with spleen participation as regards rat liver cells is possible starting with P19-P21 stage. First, at this period, white pulp T-area is formed in the spleen. Second, enhanced immune proteasomes and MHC class I molecules levels in hepatocytes can procure antigenic epitopes formation from foreign proteins and their delivery to cell surface for their subsequent presentation for cytotoxic T-lymphocytes.

Changes in proteasome pool in human papillary thyroid carcinoma development

Abstract Searching the antitumor drug targets among proteasomes, “ubiquitous” enzyme systems, may provide a new impulse to the antitumor drug discovery. In this study, changes in the proteasome pool in the development of human papillary thyroid carcinoma were determined. Proteasome activities were evaluated by hydrolysis of commercial fluorogenic peptides. Changes in the expression of the total proteasome pool, proteasome 19S activator and proteolytic constitutive subunits X(β5), Y(β1) and immune subunits LMP7 (β5i) and LMP2 (β1i) were investigated by Western blotting. The distribution of the proteasome subunits in thyroid gland cells was detected by immunohistochemistry. It was shown that the chymotrypsin- and caspase-like activities as well as the expression of the total proteasome pool, proteasome 19S activator and immune subunits increased gradually in the tumors at the T2N0M0 and T3N0M0 stages in comparison with the control tissues. Among the structures studied, the expression of the 19S activator and immune proteasomes, which contain the LMP2 (β1i) subunit, was enhanced to the largest degree in tumor cells. The data obtained may be implicated in a new therapeutic strategy. Taking into consideration the antitumor function of the immune proteasomes, we advance the 19S activator as the target for the development of a novel antitumor therapy.

Pattern of MHC class I and immune proteasome expression in Walker 256 tumor during growth and regression in Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis

Dynamics of the expression of MHC class I, immune proteasomes and proteasome regulators 19S, PA28, total proteasome pool and proteasome chymotrypsin-like activity in Walker 256 tumor after implantation into Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis was studied. The tumor growth and regression in Brattleboro rats were accompanied by changes in the proteasome subunit level unlike the tumor growth in WAG rats with normal expression of arginine-vasopressin gene. In the tumor implanted into Brattleboro rats the immune proteasome level was maximal between days 14 and 17, when the tumor underwent regression. Conversely, the expression of proteasome regulators tended to decrease during this period. Immune proteasomes are known to produce antigen epitopes for MHC class I to be presented to CD8+ T lymphocytes. Enhanced expression of immune proteasomes coincided with the recovery of MHC class I expression, suggesting the efficient presentation of tumor antigens in Brattleboro rats.

Ontogenesis of rat immune system: Proteasome expression in different cell populations of the developing thymus

Immune proteasomes in thymus are involved in processing of self-antigens, which are presented by MHC class I molecules for rejection of autoreactive thymocytes in adults and probably in perinatal rats. The distribution of immune proteasome subunits LMP7 and LMP2 in thymic cells have been investigated during rat perinatal ontogenesis. Double immunofluorescent labeling revealed LMP7 and LMP2 in thymic epithelial and dendritic cells, as well as in CD68 positive cells – macrophages, monocytes – at all developmental stages. LMP2 and LMP7 were also detected by flow cytometry in almost all thymic CD90 lymphocytes through pre- and postnatal ontogenesis. Our results demonstrate that the immune proteasomes are expressed in all types of thymic antigen presenting cells during perinatal ontogenesis, suggesting the establishment of the negative selection in the thymus at the end of fetal life. The observation of the immune proteasome expression in T lymphocytes suggests their role in thymocyte differentiation besides antigen processing in thymus.

Multiple Forms of Proteasomes and their Role in Tumor Fate

Mammalian and human cells contain multiple forms of proteasomes different in their structure and functions. 26S-proteasome pool regulates most cellular processes through ATP- and ubiquitin-dependent hydrolysis of proteins participating in these processes. This function is possible due to 19S-subparticle capable to recognize ubiquitinated proteins, unfold and direct them into the proteolytic chamber. 20S-proteasome pool is capable to degrade some damaged and foreign proteins in an ATP- and ubiquitin-independent manner. Among both proteasome pools, the immune proteasomes effectively produce antigen epitopes for MHC class I molecules and play a crucial role in the antitumor immunity. Excluding the immune proteasomes from their cells, numerous tumors avoid the immune system. On the contrary, tumor cells enhance the expression of the housekeeping proteasomes and 19S-subparticle. At this time, bortezomib, a patented proteasome inhibitor, is used as an anticancer therapy. This drug induces the cell cycle arrest and apoptosis of dividing tumor cells. However, bortezomib causes, firstly, deviations in immune functions, secondly, the increase of the housekeeping proteasome expression by a feedback mechanism. Taking into account the recent patents, we consider prospects of the development of new drugs directed to the regulation of the expression of the immune and housekeeping proteasomes and 19S-subparticle in tumor cells.

Immune proteasomes in the developing rat spleen

Changes in the structure of the rat spleen and the distribution of immune proteasomes in it during early postnatal development have been studied using double immunofluorescent staining of tissue sections with antibodies to the LMP7 immune proteasome subunit and to specific markers of T and B lymphocytes. It has been shown that the white pulp on postnatal day 5 is not yet colonized by lymphocytes and contains a smaller amount of immune proteasomes than the red pulp. At this stage, T and B lymphocytes concentrate mainly in the red pulp. On day 8, B lymphocytes occupy the marginal zone, while T lymphocytes aggregate into dense strands close to the white pulp. By day 18, T lymphocytes form periarteriolar sheaths in the white pulp, and the contents of immune proteasomes in the red and white pulp become equally high. An increase in the total content of immune proteasomes in the spleen on the third postnatal week was revealed in our previous study by Western blotting. In addition to T and B lymphocytes, immune proteasomes have also been revealed in other spleen cell types, probably in macrophages and reticular cells of the white pulp. Thus, the postnatal development of the spleen is associated with an increase in the contents of immune proteasomes in it.

Immune proteasomes in the developing rat thymus

The age dynamics of the content of the immune proteasome subunits LMP2 and LMP7 in rat thymus during prenatal and early postnatal ontogeny was studied. The LMP2 and LMP7 immune subunits were detected by Western blotting already by the 18th day of embryonic development, their amount increased to the 21st day to the level characteristic of the postnatal state. Double immunofluorescent labeling showed that in the thymus tissue the largest amount of LMP2 and LMP7 is localized in epithelial cells, whereas the level of their expression in thymocytes is lower. The results suggest that the establishment in thymus of selection processes, which depend on activity of immune proteasomes, can take place already in prenatal ontogeny. Analysis of age dynamics of the natural apoptosis level in thymocytes also favors this supposition. The presence of immune proteasomes in thymocytes during perinatal ontogeny suggests that, besides the antigen presentation, immunoproteasomes may possess other important functions.

Formation of immune proteasomes and development of immune system in ontogenesis of mammals

Current concepts of the structure of immune proteasomes and their role in immune response have been considered. The main attention has been paid to the formation of immune proteasomes in secondary lymphoid and nonlymphoid organs during ontogenesis of mammals. The causes of ineffective formation of immune system in early postnatal development have been discussed.

Peculiarities of proteasome pool formation in rat spleen and liver during postnatal development

Changes in the specific activity and amounts of 26S and 20S proteasome pools in rat spleen and liver during postnatal development and appearance in them of immune subunits were studied. Two decreases in chymotrypsin-like activity of the proteasome pools were recorded during the first three weeks after birth. The activity minimum fell on the 11th and 19th days, and the first decrease was more prolonged and pronounced than the second. The decrease in the specific activity of the 26S proteasome pools was associated with a reduction of their quantity. The 20S proteasome pools displayed no such decreases. Noticeable quantities of immune subunits LMP7 and LMP2 were revealed by Western blotting in the spleen on the 7th day and on the 19th day in the liver, concurrently with the beginning of the decrease in the proteasome activity. It was concluded that during the first three weeks of postnatal development the proteasome pools in rat spleen and liver were replaced twice, and in the spleen (a lymphoid organ) a qualitatively new pool containing immune subunits appeared nearly two weeks earlier than in the liver (a non-lymphoid organ). The appearance of immune proteasomes in different organs and tissues during some weeks after birth seems to explain the immune system inefficiency during embryogenesis and early postnatal development.

Immune proteasomes and immunity

A lot of facts that require understanding have been accumulated since immune proteasomes were discovered and their relationship with the immune response was established. For example, why are immune proteasomes present in all studied mammalian organs and tissues, including nonlymphoid tissues? What is responsible for differences in the ratio of immune to constitutive proteasomes in different organs? Are the functions of immune proteasomes related to the T-cell immune response alone, as was shown initially, or not? Are immune proteasomes formed simultaneously in different organs during ontogenesis? An attempt is made in this review to answer these and other related questions.