A role for antizyme inhibitor 2 in the biosynthesis and content of histamine and serotonin in mouse mast cells

Polyamines (putrescine, spermidine and spermine; PAs) are required for the survival of the majority of living cells. Antizymes and antizyme inhibitors are key regulatory proteins of PA levels by affecting ornithine decarboxylase, the rate-limiting biosynthetic enzyme, and PA uptake. In addition to PA, mast cells (MC) synthesize and store in their granules the biogenically active amines histamine (Hia) and serotonin (5-HT), which are of critical importance for their function. Previously, we have performed several studies in this cell type regarding the interplay between the metabolisms of PAs and Hia and 5-HT. Our results showed that PAs affect Hia synthesis during early stages of IL-3-induced bone marrow cell differentiation into bone marrow derived MCs (BMMCs) and demonstrated that PAs are present in MC secretory granules and are important for granule homeostasis, including Hia storage and 5-HT levels. A few years ago, a novel antizyme inhibitor (AZIN2) was described. In contrast to AZIN1, AZIN2 expression is restricted to a few tissues and cell types including brain, testis and MCs. In MCs, it was recently described that AZIN2 could act as a local regulator of PA biosynthesis in association with the 5-HT granule content and release. At present, our aim is to gain further insight into the role of AZIN2 in the biosynthesis, storage and release of both Hia and 5-HT. In this study, we have generated BMMCs from both wild-type and transgenic mice with severe Azin2 hypomorphism, and have analyzed the content of PAs, Hia and 5-HT, and some elements of their metabolisms. Both PAs and 5-HT levels were reduced in Azin2 hypomorphic BMMCs compared with wild-type controls, whereas the amount of Hia was increased. Accordingly, the level of tryptophan hydroxylase 1 (the key enzyme for 5-HT biosynthesis) was reduced and the amount of enzymatic activity of histidine decarboxylase (the enzyme responsible for histamine biosynthesis) was increased in Azin2 hypomorphic BMMCs. Taken together, our results show evidence that AZIN2 has an important role in the regulation of Hia and 5-HT biosynthesis and storage in MCs. Department of Molecular Biology and Biochemistry, and CIBER de Enfermedades Raras (CIBER-ER), Faculty of Sciences, University of Málaga, Málaga 29071, Spain. Corresponding author: I. Fajardo ([email protected]) This work was supported by SAF2011-26518 (MINECO, Spain) and P10-CVI-6585 and Bio-267 (Junta de Andalucia, Spain). CIBERER is an iniciative of Instituto de Salud Carlos III (Spain).Universidad de Málaga. Campus de Excelencia Internacional Andalucía-Tech. SAF2011-26518 (MINECO, Spain) and P10-CVI-6585 and Bio-267 (Junta de Andalucia, Spain. CIBERER is an iniciative of Instituto de Salud Carlos III (Spain)

A role for antizyme inhibitor 2 in the biosynthesis and content of serotonin and histamine in mouse mast cells

Polyamines (putrescine, spermidine and spermine; PAs) are essential for the majority of living cells. Antizymes and antizyme inhibitors are key regulatory proteins of PA levels by affecting ornithine decarboxylase and PA uptake. In addition to PAs, mast cells (MC) synthesize and store in their granules histamine (Hia) and serotonin (5-HT), which are critical for their function. Our previous studies have indicated a metabolic interplay among PAs, Hia and 5-HT in this cell type. For instance, we showed that PAs affect Hia synthesis during early stages of IL-3-induced bone marrow cell differentiation into bone marrow derived MCs (BMMCs) and demonstrated that PAs are present in MC secretory granules and are important for granule homeostasis, including Hia storage and 5-HT levels. A few years ago, a novel antizyme inhibitor (AZIN2) was described whose expression is restricted to a few tissues and cell types including brain, testis and MCs. In MCs, it was recently proposed that AZIN2 could act as a local regulator of PA biosynthesis in association with 5-HT-containing granules and with 5-HT release following MC activation. To gain insight into the role of AZIN2 in the biosynthesis and storage of 5-HT and also Hia, we have generated BMMCs from both wild-type and transgenic mice with severe Azin2 hypomorphism, and have analyzed the content of PAs, 5-HT and Hia, and some elements of their metabolisms. Spermine and 5-HT levels were reduced in Azin2 hypomorphic BMMCs compared with wild-type controls, whereas the amount of Hia was increased. Accordingly, the level of tryptophan hydroxylase 1 (the key enzyme for 5-HT biosynthesis) was reduced and the amount of enzymatic activity of histidine decarboxylase (the enzyme responsible for Hia biosynthesis) was increased in Azin2 hypomorphic BMMCs. Taken together, our results show evidence that AZIN2 has an important role in the regulation of 5-HT and Hia biosynthesis and storage in MCsUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This work was supported by SAF2011-26518 (MINECO, Spain) and P10-CVI-6585 and Bio-267 (Junta de Andalucia, Spain). CIBERER is an iniciative of Instituto de Salud Carlos III (Spain)

Dietary and Gut Microbiota Polyamines in Obesity- and Age-Related Diseases

The polyamines putrescine, spermidine, and spermine are widely distributed polycationic compounds essential for cellular functions. Intracellular polyamine pools are tightly regulated by a complex regulatory mechanism involving de novo biosynthesis, catabolism, and transport across the plasma membrane. In mammals, both the production of polyamines and their uptake from the extracellular space are controlled by a set of proteins named antizymes and antizyme inhibitors. Dysregulation of polyamine levels has been implicated in a variety of human pathologies, especially cancer. Additionally, decreases in the intracellular and circulating polyamine levels during aging have been reported. The differences in the polyamine content existing among tissues are mainly due to the endogenous polyamine metabolism. In addition, a part of the tissue polyamines has its origin in the diet or their production by the intestinal microbiome. Emerging evidence has suggested that exogenous polyamines (either orally administrated or synthetized by the gut microbiota) are able to induce longevity in mice, and that spermidine supplementation exerts cardioprotective effects in animal models. Furthermore, the administration of either spermidine or spermine has been shown to be effective for improving glucose homeostasis and insulin sensitivity and reducing adiposity and hepatic fat accumulation in diet-induced obesity mouse models. The exogenous addition of agmatine, a cationic molecule produced through arginine decarboxylation by bacteria and plants, also exerts significant effects on glucose metabolism in obese models, as well as cardioprotective effects. In this review, we will discuss some aspects of polyamine metabolism and transport, how diet can affect circulating and local polyamine levels, and how the modulation of either polyamine intake or polyamine production by gut microbiota can be used for potential therapeutic purposes

MGRN1 as a Phenotypic Determinant of Human Melanoma Cells and a Potential Biomarker

Mahogunin Ring Finger 1 (MGRN1), a ubiquitin ligase expressed in melanocytes, interacts with the α melanocyte-stimulating hormone receptor, a well-known melanoma susceptibility gene. Previous studies showed that MGRN1 modulates the phenotype of mouse melanocytes and melanoma cells, with effects on pigmentation, shape, and motility. Moreover, MGRN1 knockdown augmented the burden of DNA breaks in mouse cells, indicating that loss of MGRN1 promoted genomic instability. However, data concerning the roles of MGRN1 in human melanoma cells remain scarce. We analyzed MGRN1 knockdown in human melanoma cells. Transient MGRN1 depletion with siRNA or permanent knockdown in human melanoma cells by CRISPR/Cas9 caused an apparently MITF-independent switch to a more dendritic phenotype. Lack of MGRN1 also increased the fraction of human cells in the S phase of the cell cycle and the burden of DNA breaks but did not significantly impair proliferation. Moreover, in silico analysis of publicly available melanoma datasets and estimation of MGRN1 in a cohort of clinical specimens provided preliminary evidence that MGRN1 expression is higher in human melanomas than in normal skin or nevi and pointed to an inverse correlation of MGRN1 expression in human melanoma with patient survival, thus suggesting potential use of MGRN1 as a melanoma biomarker.This research was funded by grant SAF2018_RTI2018-094929-B-I00 financed by FEDER/Ministerio de Ciencia e Innovación—Agencia Estatal de Investigación (Spain) (to C.J.-C. and J.C.G.-B.), and by grant UPV/EHU GIU20/035 (to S.A and M.D.B.)

Análisis de la expresión y función del inhibidor de antizimas 2 (AZIN2) en ratones transgénicos : estudio de nuevos ortólogos y parálogos de AZIN2

BACKGROUND AND AIMS Polyamines are ubiquitous organic cations that play multiple essential roles in mammalian physiology. Ornithine decarboxylase (ODC) is the rate limiting enzyme in polyamine biosynthesis and it is regulated by antizymes, a family of small proteins whose synthesis is stimulated by high polyamine levels. AZs bind to ODC inhibiting its enzymatic activity and target it for degradation by the 26S proteasome. AZs are themselves regulated by another ODC-related family named antizyme inhibitors (AZINs), consisting of two members: AZIN1 and AZIN2. These proteins lack enzymatic activity but they bind to AZs with higher affinity than ODC, counteracting the effects of antizymes on ODC. AZIN1 is a ubiquitous protein that participates in the regulation of polyamine metabolism and cell growth and its disruption in mice causes death soon after birth. AZIN2 is mainly expressed in brain and testis, followed by other secretory tissues and cells. Although the physiological role of AZIN2 is mostly unknown, some data suggests that it is involved in cell growth, vesicular trafficking, secretion and spermiogenesis. In order to study the expression pattern of AZIN2 and its physiological role in mouse, AZIN2 KO mice were generated by the insertion of a beta geo cassette, resulting in a fused protein formed by the N-terminus extreme of AZIN2 and the reporter enzyme β-galactosidase. On the other hand, advances in genome sequencing revealed the existence of many orthologous of Azin2, and a new paralogue of Azin2 in mouse, named Gm853, whose study by transient transfected cells is a main aim of the present work. RESULTS AND CONCLUSIONS 1. Expression of the reporter gene lacZ in tissues of mice lacking Azin2. AZIN2 KO generated mice provide qualitative and quantitative information about the expression and function of AZIN2. Hystochemical and biochemical analysis of the reporter β-galactosidase activity in different tissues of AZIN2 KO mice confirmed significant expression of AZIN2 in epidydymis, adrenal glands, pancreas, heart, lung and eyes, in addition to testis and brain, pointing to secretory regions. However, none of the KO tissues examined showed significantly variation in polyamine content. Finally, the microarray analysis of gene differential expression between WT and KO mice did not show common changes, except the marked reduction of Azin2 expression. 2. The role of AZIN2 in male reproductive system. Hystological sections from testis and epididymis of AZIN2 KO and WT mice showed that AZIN2 was mainly expressed in haploid cells but also in Leydig cells, suggesting that AZIN2 is required not only for the synthesis but also for the function of mature sperm cells. Besides, the sperm motility test revealed that most AZIN2 KO sperm cells were not able to move across the test medium. However, this deficiency in motor skills did not have major consequences in male fertility, as AZIN2 KO mice demonstrated to be as fertile as their WT counterparts. Finally, the deprivation of AZIN2 in male reproductive system was also associated to decreased biosynthesis and secretion of testosterone. 3. Expression and function of AZIN2 in other secretory cells and tissues. Phenotypic analysis of AZIN2 KO mice. The role of AZIN2 in murine physiology was analysed in other secretory cells and tissues apart from testis by comparing the gain or the loss of functions in AZIN2 KO mice. In brain, AZIN2 prevails in the cerebellum and hippocampus areas and its disruption in these areas correlates with a deficient motor function in mice. The expression of AZIN2 in adrenal glands and pancreas was restricted to the adrenal medulla and to Langerhans islets, respectively. In other tissues such as heart, eyes, lung and kidney, the expression of AZIN2 was also restricted to specific types of cells. In pancreas, the absence of AZIN2 generates a slight hyperglycemia associated to impaired synthesis and secretion of insulin from β cells. 4. Structural and functional properties of Xenopus Azin2. Studies about ODC in Xenopus reveal the existence of two homologous proteins named XODC1 and XODC2, with different spatial and temporary patterns of expression. XODC2 appears on gene databases as XAZIN2, thus, it is considered an orthologous of mAZIN2 and hAZIN2. Unlike mAZIN2, the orthologous of AZIN2 in Xenopus laevis (xlAZIN2) is degraded by AZ1 and possesses decarboxylase activity of ornithine and lysine, showing higher affinity for lysine than for ornithine. The comparative analysis of xlAZIN2 with mODC and mAZIN2 revealed additional similarities with mODC, based on the cytosolic subcellular localization and the ability to form aggregates such as homotetramers. Similarly to mODC, xlAZIN2 stimulates agmatine uptake, decreases that of putrescine and spermidine and presents a short half-life, lower than 30 minutes. 5. Study of mouse Gm853, a new paralogue of Odc and Azin2. Comparative analysis of protein sequences from ODC paralogues revealed GM853 as a new homologue protein of ODC and AZINs in mouse, presenting all conserved residues for enzymatic activity. Experiments with transfected cells demonstrated that GM853 is not an antizyme inhibitor as it does not prevent the interaction between ODC and AZ1. Otherwise, it is a very stable protein mainly located in the cytosol and it is able to form homodimers and homotetramers but no heterodimers with other paralogues. Besides, GM853 presents enzymatic activity, being the first decarboxylase for L-Leucine described in mammals. The expression of GM853 in mice is restricted to renal tissue with higher representation in male kidneys, related to increased levels of testosterone. The product of Leucine decarboxylation is the isopenthylamine and it is rapidly excreted with urine.   ANTECEDENTES Y OBJETIVOS Las poliaminas son cationes orgánicos presentes en todas las células con funciones esenciales en la fisiología de todos los mamíferos. Ornitina descarboxilasa (ODC) es la enzima limitante en la biosíntesis de poliaminas y está regulada por los antizimas (AZs), una familia de pequeñas proteínas cuya síntesis es estimulada por las propias poliaminas. Los AZs se unen a ODC inhibiendo su actividad enzimática y la conducen al proteasoma 26S para su degradación. Los AZs son a su vez regulados por otra familia de proteínas relacionadas con ODC, denominadas inhibidores de antizimas (AZINs), formada por dos miembros: AZIN1 y AZIN2. Estas proteínas carecen de actividad enzimática pero se unen a los AZs con más afinidad que ODC, contrarrestando los efectos de los antizimas sobre ODC. AZIN1 es una proteína ubicua que participa en la regulación del metabolismo de poliaminas y el crecimiento celular y su abatimiento en ratones causa la muerte poco después de nacer. AZIN2 se expresa fundamentalmente en cerebro y testículos, además de otros tejidos y células secretoras. Aunque el papel fisiológico de AZIN2 no se conoce con certeza, algunos experimentos sugieren que está implicado en el crecimiento celular, el tráfico vesicular, secreción y espermatogénesis. Con el fin de estudiar el patrón de expresión de AZIN2 y su implicación fisiológica en ratón, se generaron ratones KO de AZIN2 mediante la inserción de un cassette beta geo, que dio lugar a una proteína recombinante formada por el extremo N-terminal de AZIN2 y la enzima reportera β-galactosidasa. Por otro lado, los avances en el ámbito de la secuenciación de genomas, han revelado la existencia de un gran número de ortólogos de Azin2, así como un nuevo parálogo de Azin2 en ratón, denominado Gm853, cuyo estudio mediante células transfectadas también es objeto de esta tesis doctoral. RESULTADOS Y CONCLUSIONES 1. Expresión del gen reportero lacZ en tejidos de ratones con abatimiento de Azin2. El modelo de ratón transgénico con abatimiento del gen Azin2 generado, permitía el estudio de la expresión y posible función de la proteína AZIN2. El análisis histoquímico y bioquímico de la actividad β-galactosidasa en tejidos de ratones KO de AZIN2 confirmó la expresión de AZIN2 en epidídimo, glándula adrenal, páncreas, corazón, pulmón y ojos, aparte de testículo y cerebro, apuntando a células secretoras. Sin embargo, en ninguno de los tejidos estudiados en ratones deficientes en AZIN2 se observó reducción significativa de los niveles tisulares de poliaminas. Finalmente, el estudio comparativo de la expresión de genes entre ratones WT y KO mediante microarrays, no mostró cambios comunes en todos ellos salvo la marcada reducción de la expresión de Azin2. 2. The role of AZIN2 in male reproductive system. El análisis histoquímico de secciones de testículos y epidídimos WT y KO de AZIN2 mostró que AZIN2 se expresa fundamentalmente en células haploides, pero también en las células de Leydig, sugiriendo su participación no solo enla síntesis sino también en la maduración de los espermatozoides. Además, el test de movilidad espermática mostró que la mayoría de los espermatozoides KO de AZIN2 no eran capaces de desplazarse en el medio de ensayo. Sin embargo, esta deficiencia en la motilidad no tuvo consecuencias en la fertilidad de los ratones machos, demostrando ser tan fértiles como los WT. Finalmente, el abatimiento de AZIN2 en el aparato reproductor masculino se asoció a una disminución en la biosíntesis y la secreción de testosterona. 3. Expresión y función de AZIN2 en otros tejidos y células secretoras. Análisis fenotípico de los ratones KO de AZIN2. El papel de AZIN2 en la fisiología murina en otros tejidos que expresan AZIN2 se analizó comparando los cambios sobre la función en ratones KO de AZIN. En el cerebro de ratón, AZIN2 predomina en el cerebelo y en el hipocampo y su disrrupción en estas áreas está relacionada con un una función motora deficiente. La expression de AZIN2 en la glándula adrenal y el páncreas se limitó a la médula adrenal y a los islotes de Langerhans, respectivamente. En otros tejidos como corazón, ojos, pulmón y riñón, la expression de AZIN2 se concentró en tipos de células específicas. En páncreas, la ausencia de AZIN2 genera una ligera hiperglucemia porque afecta a la síntesis y secreción de insulina por las células β. 4. Propiedades estructurales y funcionales de Azin2 de Xenopus. El estudio de ODC en Xenopus reveló la existencia de dos proteínas homólogas denominadas XODC1 y XODC2, con diferente patró de expresión espacial y temporal. XODC2 aparece en las bancos de genes como XAZIN2, siendo considerado un ortólogo de mAZIN2 y hAZIN2. Al contrario que AZIN2 de ratón, su ortólogo en Xenopus (xlAZIN2) es degradado por AZ1 y presenta actividad descarboxilasa de ornitina y lisina, con una afinidad superior por lisina que por ornitina. El análisis comparativo de xlAZIN2 con mODC y mAZIN2 mostró una mayor semejanza con mODC, basándose en su localización subcelular citosólica y en la capacidad de ambas proteínas para formar oligómeros. Además, al igual que mODC, xlAZIN2 presenta una vida media muy corta (inferior a 30 minutos) y estimula la captación de agmatina en detrimento de la de putrescina y espermidina. 5. Estudio de Gm853, un nuevo parálogo de Odc y Azin2 en ratón. El análisis comparativo de la secuencia proteica de los distintos parálogos de ODC reveló la existencia de GM853, una nueva proteína homóloga de ODC y AZINs en ratón, que conserva todos los residuos relacionados con la actividad catalítica. Estudios con células transfectadas demostraron que GM853 no es un inhibidor de antizimas, ya que no evita la interacción entre ODC y AZ1. En cambio, es una proteína estable localizada fundamentalmente en el citosol y es capaz de formar homodímeros y homotetrámeros pero no heterodímeros con otros parálogos. GM853 presenta actividad enzimática, siendo la primera proteína descrita en mamíferos con actividad descarboxilasa de leucina. En ratón, GM853 se encuentra en el riñón y su expresión depende de los niveles de testosterona, predominando en el riñón de ratones machos. El producto de la descarboxilación de la leucina es la isopentilamina, una amina que es rápidamente eliminada a través de la orina

Polyamine biosynthesis in Xenopus laevis: the xlAZIN2/xlODC2 gene encodes a lysine/ornithine decarboxylase.

Ornithine decarboxylase (ODC) is a key enzyme in the biosynthesis of polyamines, organic cations that are implicated in many cellular processes. The enzyme is regulated at the post-translational level by an unusual system that includes antizymes (AZs) and antizyme inhibitors (AZINs). Most studies on this complex regulatory mechanism have been focused on human and rodent cells, showing that AZINs (AZIN1 and AZIN2) are homologues of ODC but devoid of enzymatic activity. Little is known about Xenopus ODC and its paralogues, in spite of the relevance of Xenopus as a model organism for biomedical research. We have used the information existing in different genomic databases to compare the functional properties of the amphibian ODC1, AZIN1 and AZIN2/ODC2, by means of transient transfection experiments of HEK293T cells. Whereas the properties of xlODC1 and xlAZIN1 were similar to those reported for their mammalian orthologues, the former catalyzing the decarboxylation of L-ornithine preferentially to that of L-lysine, xlAZIN2/xlODC2 showed important differences with respect to human and mouse AZIN2. xlAZIN2 did not behave as an antizyme inhibitor, but it rather acts as an authentic decarboxylase forming cadaverine, due to its higher affinity to L-lysine than to L-ornithine as substrate; so, in accordance with this, it should be named as lysine decarboxylase (LDC) or lysine/ornithine decarboxylase (LODC). In addition, AZ1 stimulated the degradation of xlAZIN2 by the proteasome, but the removal of the 21 amino acid C-terminal tail, with a sequence quite different to that of mouse or human ODC, made the protein resistant to degradation. Collectively, our results indicate that in Xenopus there is only one antizyme inhibitor (xlAZIN1) and two decarboxylases, xlODC1 and xlLDC, with clear preferences for L-ornithine and L-lysine, respectively

New insights of polyamine metabolism in testicular physiology: A role of ornithine decarboxylase antizyme inhibitor 2 (AZIN2) in the modulation of testosterone levels and sperm motility.

The specific role of polyamines in the testis physiology is not fully understood. Antizymes (OAZs) and antizyme inhibitors (AZINs) are modulators of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis and polyamine uptake. Although the three known OAZs are expressed in the testis, only OAZ3 is testis specific and has been proven to have an essential role in male fertility. Regarding the two existing AZINs, AZIN2 is the most abundantly expressed member in this gonad. Whereas previous studies suggested that AZIN2 might participate in mouse spermatogenesis, immunohistological analysis of human testicular sections revealed that AZIN2 is also detected in the steroidogenic Leydig cells but not in the germinal epithelium. In the present study, we found a close ontogenic similarity in the mRNA levels of OAZs and AZINs between mice and rats, but an opposite expression pattern of ODC activity. Further analysis of AZIN2 and OAZ3 in the testis of mice with different alterations in spermatogenesis and fertility, induced either genetically or pharmacologically, corroborated that both AZIN2 and OAZ3 are mainly expressed in the haploid germinal cells. Finally, by using transgenic mice with a truncated Azin2 gene fused to the bacterial lacZ gene, we studied the expression of Azin2 in testes, epididymides and spermatozoa. AZIN2 was detected in spermatids and spermatozoa, as well as in Leydig cells, and in epithelial epidydimal cells. Azin2 knock-out male mice were fertile; however, they showed marked decreases in testicular putrescine and plasma and testicular testosterone levels, and a dramatic reduction in the sperm motility. These results suggest an important role for AZIN2 in testicular cells by modulating polyamine concentrations, testosterone synthesis and sperm function. Overall, our data corroborate the relevance of polyamine regulation in testis functions, where both AZIN2 and OAZ3 play fundamental roles

The organization of large high-efficiency web-applications development

Problēma, kas ir izskatīta šajā bakalaura darbā, sākumā var izlikties pārāk plaša – tik, ka tikai vien šī darba ietvaros tiks izskatīti problemātikas atslēgas momenti. Bet tomēr autors nolēma izvēlēties tematu saistītu ar tīmekļa tehnoloģijām tieši tāpēc, ka nesen viņam pašam nācās patstāvīgi nodarboties ar līdzīgo uzdevumu risināšanu. Noderīgas zināšanas bija iegūtas, studējot Latvijas Universitātes Fizikas un matemātikas fakultātes Datorikas nodaļā, un patstāvīgi strādājot šajā jomā ārpus tās. Darbā izskatītie jautājumi skar plāšu spektru no serveru un izstrādes vides uzstādīšanas līdz darbam ar repozitorijiem, no specifikāciju sagatavošanas un uzdevumu sadalījuma komandā līdz ievāru izvēlei un apmācībām darbam ar tiem, no augsta līmeņa kešdarbu un akseleratoru pielietojuma organizācijās līdz pareizi uzbūvētajai klasteru sistēmai. Autora piedāvātie risinājumi un to aspekti, var kļūt par praktiskas informācijas bagāto avotu, kurš noderēs pie sarežģīto un augstražīgo tīmekļu ātra un kvalitatīva izstrādes procesa.The problem, described in this bachelor work at first may seem too ambitious - so that in this paper addresses only the key points related issues. Nevertheless, the author decided to choose this topic because recently he himself had to deal with its own solution. In this useful knowledge acquired while studying at the Department of Computer Science Faculty of Physics and Mathematics, University of Latvia, and independent work in this area - from installation of servers and development environment to work with the repository, from writing the specifications and the assignment in the team to choosing and training frameworks work with them, from organizing high-level caching and application accelerator to properly constructed cluster system. A spectrum and solutions proposed by the author, can be a useful practical tool in organizing a rapid and qualitative development and production of complex web systems

Antizyme inhibitor 2 hypomorphic mice. New patterns of expression in pancreas and adrenal glands suggest a role in secretory processes.

The intracellular levels of polyamines, polycations implicated in proliferation, differentiation and cell survival, are regulated by controlling their biosynthesis, catabolism and transport. Antizymes and antizyme inhibitors are key regulatory proteins of polyamine levels by affecting ornithine decarboxylase, the rate-limiting biosynthetic enzyme, and polyamine uptake. We recently described the molecular function of a novel antizyme inhibitor (AZIN2). However, the physiological function of AZIN2 in mammals is mostly unknown. To gain insight on the tissue expression profile of AZIN2 and to find its possible physiological role, we have generated, transgenic mice with severe Azin2 hypomorphism. This mouse model expresses transgenic bacterial β-D-galactosidase as a reporter gene, under the control of the Azin2 endogenous promoter, what allows a very sensitive and specific detection of the expression of the gene in the different tissues of transgenic mice. The biochemical and histochemical analyses of β-D-galactosidase together with the quantification of Azin2 mRNA levels, corroborated that AZIN2 is mainly expressed in testis and brain, and showed for the first time that AZIN2 is also expressed in the adrenal glands and pancreas. In these tissues, AZIN2 was not expressed in all type of cells, but rather in specific type of cells. Thus, AZIN2 was mainly found in the haploid germinal cells of the testis and in different brain regions such as hippocampus and cerebellum, particularly in specific type of neurons. In the adrenal glands and pancreas, the expression was restricted to the adrenal medulla and to the Langerhans islets, respectively. Interestingly, plasma insulin levels were significantly reduced in the transgenic mice. These results support the idea that AZIN2 may have a role in the modulation of reproductory and secretory functions and that this mouse model might be an interesting tool for the progress of our understanding on the role of AZIN2 and polyamines in specific mammalian cells

Antizyme Inhibitor 2-Deficient Mice Exhibit Altered Brain Polyamine Levels and Reduced Locomotor Activity

Background: Alterations in the neural polyamine system are known to be associated with different brain pathological conditions. In addition, the regulation of enzymes involved in polyamine metabolism such as ornithine decarboxylase (ODC), antizymes (AZs), and antizyme inhibitors (AZINs) is critical during brain development. However, while most studies focus on ODC and AZs, less is known about AZIN expression and function in the brain. Thus, our aim was to analyze the expression pattern of AZIN2 during postnatal development, its brain distribution, and its possible implication in phenotypical alterations. Methods: The expression pattern of Azin2 and other genes related to polyamine metabolism was analyzed by RT-qPCR. β-D-galactosidase staining was used to determine the anatomical distribution of AZIN2 in a Azin2 knockout model containing the βGeo marker. Brain polyamine content was determined by HPLC. The Rota-Rod and Pole functional tests were used to evaluate motor skills in Azin2-lacking mice. Results: Our results showed that expression of genes codifying for AZs and AZINs showed a similar increasing pattern over time that coincided with a decrease in ODC activity and putrescine levels. The analysis of AZIN2 distribution demonstrated that it is strongly expressed in the cerebellum and distributed along the neuron body and dendrites. The ablation of Azin2 showed a decrease in putrescine levels and is related to reduced motor skills. Conclusions: Our study revealed that AZIN2 expression in the brain is particularly limited to the cerebellum. In addition, the ablation of Azin2 leads to a reduction in putrescine that relates to alterations in motor function, suggesting the role of AZIN2 in the functioning of dopaminergic neurons