Decreased expression of the Slc31a1 gene and cytoplasmic relocalization of membrane CTR1 protein in renal epithelial cells : a potent protective mechanism against copper nephrotoxicity in a mouse model of Menkes disease

Kidneys play an especial role in copper redistribution in the organism. The epithelial cells of proximal tubules perform the functions of both copper uptake from the primary urine and release to the blood. These cells are equipped on their apical and basal membrane with copper transporters CTR1 and ATP7A. Mosaic mutant mice displaying a functional dysfunction of ATP7A are an established model of Menkes disease. These mice exhibit systemic copper deficiency despite renal copper overload, enhanced by copper therapy, which is indispensable for their life span extension. The aim of this study was to analyze the expression of Slc31a1 and Slc31a2 genes (encoding CTR1/CTR2 proteins) and the cellular localization of the CTR1 protein in suckling, young and adult mosaic mutants. Our results indicate that in the kidney of both intact and copper-injected 14-day-old mutants showing high renal copper content, CTR1 mRNA level is not up-regulated compared to wild-type mice given a copper injection. The expression of the Slc31a1 gene in 45-day-old mice is even reduced compared with intact wild-type animals. In suckling and young copper-injected mutants, the CTR1 protein is relocalized from the apical membrane to the cytoplasm of epithelial cells of proximal tubules, the process which prevents copper transport from the primary urine and, thus, protects cells against copper toxicity

Exacerbation of neonatal hemolysis and impaired renal iron handling in heme oxygenase 1-deficient mice

In most mammals, neonatal intravascular hemolysis is a benign and moderate disorder that usually does not lead to anemia. During the neonatal period, kidneys play a key role in detoxification and recirculation of iron species released from red blood cells (RBC) and filtered out by glomeruli to the primary urine. Activity of heme oxygenase 1 (HO1), a heme-degrading enzyme localized in epithelial cells of proximal tubules, seems to be of critical importance for both processes. We show that, in HO1 knockout mouse newborns, hemolysis was prolonged despite a transient state and exacerbated, which led to temporal deterioration of RBC status. In neonates lacking HO1, functioning of renal molecular machinery responsible for iron reabsorption from the primary urine (megalin/cubilin complex) and its transfer to the blood (ferroportin) was either shifted in time or impaired, respectively. Those abnormalities resulted in iron loss from the body (excreted in urine) and in iron retention in the renal epithelium. We postulate that, as a consequence of these abnormalities, a tight systemic iron balance of HO1 knockout neonates may be temporarily affected

Regulation of the switch from early to late bacteriophage lambda DNA replication

There are two modes of bacteriophage k DNA replication following infection of its host, Escherichia coli. Early after infection, replication occurs according to the theta (teta or circle-to-circle) mode, and is later switched to the sigma (sigma or rolling-circle) mode. It is not known how this switch, occurring at a specific time in the infection cycle, is regulated. Here it is demonstrated that in wildtype cells the replication starting from orik proceeds both bidirectionally and unidirectionally, whereas in bacteria devoid of a functional DnaA protein, replication from orik is predominantly unidirectional. The regulation of directionality of replication from orik is mediated by positive control of lambda pR promoter activity by DnaA, since the mode of replication of an artificial lambda replicon bearing the ptet promoter instead of pR was found to be independent of DnaA function. These findings and results of density-shift experiments suggest that in dnaA mutants infected with lambda, phage DNA replication proceeds predominantly according to the unidirectional teta mechanism and is switched early after infection to the sigma mode. It is proposed that in wild-type E. coli cells infected with lambda, phage DNA replication proceeds according to a bidirectional teta mechanism early after infection due to efficient transcriptional activation of ori lambda, stimulated by the host DnaA protein. After a few rounds of this type of replication, the resulting increased copy number of teta genomic DNA may cause a depletion of free DnaA protein because of its interaction with the multiple DnaA-binding sites in lambda DNA. It is proposed that this may lead to inefficient transcriptional activation of orik resulting in unidirectional teta replication followed by sigma type replication

Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe.

Despite its essential role in the (patho)physiology of several diseases, CB2R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB2R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB2R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB2R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB2R based drugs

The Localization of the ATP7B protein in Testis and Epididymis Male Mice

Homeostaza jest stanem organizmu najbardziej dla niego korzystnym. Stąd organizmy żywe wykształciły szereg mechanizmów regulujących stężenie różnych jonów, w tym jonów miedzi. Do mechanizmów tych należy między innymi działanie białka ATP7B oparte na wyrzucie miedzi z komórki, a także wbudowywaniu jonów tego pierwiastka w centra aktywne enzymu- ceruloplazminy. Miedź odgrywa ważną rolę w procesie spermatogenezy, jednakże nadmiar jonów tego mikroelementu może prowadzić do poważnych patologii.Jądra i najądrza są elementami męskiego układu rozrodczego i odpowiadają za proces spermatogenezy i steroidogenezy, a także za proces dojrzewania gamet męskich. Przez wzgląd na toksyczny wpływ nadmiaru miedzi na gamety męskie, homeostaza tego pierwiastka musi być w powyższych organach ściśle regulowana. W obecnej pracy zbadano lokalizację białka ATP7B w jądrach i najądrzach samców myszy przy użyciu reakcji immunohistochemicznej. Badanie przeprowadzono na materiale pochodzącym od samców myszy z dwóch grup wiekowych: 23 dni i 6 miesięcy. Białko ATP7B zlokalizowano w komórkach Leydiga i komórkach mioidalnych w jądrze samców 23-dniowych, natomiast w jądrze samców 6-miesięcznych białko to dodatkowo występuje w spermatydach wydłużonych. Z kolei w najądrzu powyższe białko zostało zlokalizowane w komórkach mioidalnych i komórkach głównych nabłonka.Homeostasis is the most advantageous state of the body. Thus organisms developed special mechanisms to regulate the concentration of different ions, including copper ions. One of these mechanisms is activity of ATP7B protein which is responsible for efflux of the copper from the cells and for the integration of copper ions into active sites of enzyme- ceruloplasmin. Also, copper plays a crucial role in spermatogenesis, but the excess of this trace element can lead to serious pathologies.Testes and epididymides are elements of male reproductive system and they are responsible for spermatogenesis, steroidogenesis and maturation of sperms. It is known that the excess of copper has toxic impact on the sperms, because of that homeostasis of this element has to be strictly regulated in above organs. In this work there was analysed the localization of the ATP7B protein in mouse testis and epididymides by the immunohistochemistry reaction. The study was performed on two age groups of mice: 23 days and 6 months. The ATP7B protein was localized in Leydig cells and peritubular myoid cells in the testis of 23-day-old males, whereas in the testis of 6-month-old males this protein also occurs in elongating spermatids. On the other hand, in epididymis ATP7B protein was localized in peritubular myoid cells and main cells

The analysis of genes and proteins expression which are connected with iron metabolism in neonatal period in mice liver with knockout of gene Hmox1

Utrzymanie przez organizm prawidłowej gospodarki żelazowej jest istotne zarówno dla funkcjonowania komórek, jak i całego ustroju. Jest ono niezbędne w trakcie całego życia postnatalnego, a szczególnie w okresie noworodkowym ze względu na zachodzącą wówczas wzmożoną hemolizę. Organizmy żywe wykształciły określone mechanizmy pozwalające na utrzymanie homeostazy żelaza. Organem, który odgrywa kluczową rolę w metabolizmie tego pierwiastka, jest wątroba. Stanowi ona miejsce magazynowania żelaza, odpowiedzialna jest także za wytwarzanie komórek krwi w okresie życia prenatalnego i noworodkowego ssaków. W regulację metabolizmu żelaza zaangażowany jest szereg białek, do których zaliczane są między innymi: oksygenaza hemowa 1 (HO-1) oraz ferroportyna 1 (FPN1). HO-1 umożliwia uwolnienie jonu żelazawego z hemu podczas degradacji hemoglobiny. FPN1 z kolei odpowiada za wyrzut jonów żelaza z komórek do osocza. W poniższej pracy zbadano ekspresję genów (Hmox1 i Slc40a1) i białek (odpowiednio: HO-1 i FPN1) w obrębie wątroby noworodków myszy z nokautem genu Hmox1. Badanie to przeprowadzono w organach pochodzących od myszy 3-, 5-, 7-, 9- i 11-dniowych. Z wykorzystaniem reakcji RT-PCR i Real-Time PCR wykazano różnice w ekspresji genu Slc40a1 między osobnikami dzikimi a nokautami oraz między poszczególnymi grupami wiekowymi. U osobników z nokautem ekspresja tego genu była niższa niż u osobników dzikich. Z zastosowaniem metody immunohistochemicznej wykazano obecność białka FPN1 w komórkach Browicza-Kupffera u 5-dniowych myszy dzikich i nokautów. U osobników z nokautem ekspresja tego białka była niższa niż u osobników dzikich. Wykorzystanie barwienia hematoksylina-eozyna umożliwiło wykazanie obecności oraz różnic w liczbie wysp erytroblastycznych między osobnikami dzikimi a nokautami oraz między poszczególnymi grupami wiekowymi. Wyspy te były obecne u wszystkich grup wiekowych osobników dzikich i nokautów, jednak zdecydowanie dłużej utrzymały się w znacznej liczbie w wątrobie osobników z nokautem. Barwienie błękitem pruskim wykazało obecność i różnice w liczbie depozytów żelaza niehemowego między osobnikami dzikimi a nokautami oraz między poszczególnymi grupami wiekowymi.Maintenance of the proper iron balance by an organism is crucial in both cells and whole body functioning. It is essential during whole postnatal life, however, it is especially crucial in the neonatal period because of intensified haemolysis. Living organisms have developed specified mechanisms which enable maintenance of iron homeostasis. An organ which plays a key role in iron metabolism is the liver. It storages iron reserves and it is responsible for mammalian blood cells production in the prenatal and neonatal period. In the iron metabolism regulation is involved a wide array of proteins. Among them, heme oxygenase 1 (HO-1) and ferroportin 1 (FPN1) are known to be important in the iron metabolism regulation at the cellular level. HO-1 is involved in ferrous ions release during haemoglobin degradation. FPN1, in turn, is responsible for iron ions export from the cells. In this work was analysed the genes (Hmox1 and Slc40a1) and proteins (HO-1 and FPN1, respectively) expression in the livers of neonatal mice with knockout of gene Hmox1. The study was performed on five age groups of mice: 3-, 5-, 7-, 9-, 11-day-old. Due to the RT-PCR and Real-Time reaction, there were shown differences in Slc40a1 gene expression between wild type individuals and knockout individuals and between respective age groups. The analysis has demonstrated that Slc40a1 gene expression was lower with knockout mice than with wild type mice. Due to the immunohistochemistry method, there was shown the presence of FPN1 protein in Browicz-Kupffer cells with 5-day-old wild type and knockout mice. The expression of this protein was lower with knockout mice than with wild type mice. Thanks to the hematoxylin and eosin stain method there were shown the presence and the differences in a number of erythroblastic islands between wild type individuals and knockout individuals and between respective age groups. These islands occurred in knockout and wild type mice livers with all of the age groups, however, erythroblastic islands persisted with knockout mice longer in greater number than with wild type mice. Due to the Prussian blue staining, there were shown the presence and the differences in a number of “non-heme” iron deposits between wild type individuals and knockout individuals and between respective age groups

Molecular Regulation of Copper Homeostasis in the Male Gonad during the Process of Spermatogenesis

Owing to its redox properties, copper is a cofactor of enzymes that catalyze reactions in fundamental metabolic processes. However, copper–oxygen interaction, which is a source of toxic oxygen radicals generated by the Fenton reaction, makes copper a doubled-edged-sword in an oxygen environment. Among the microelements influencing male fertility, copper plays a special role because both copper deficiency and overload in the gonads worsen spermatozoa quality and disturb reproductive function in mammals. Male gametes are produced during spermatogenesis, a multi-step process that consumes large amounts of oxygen. Germ cells containing a high amount of unsaturated fatty acids in their membranes are particularly vulnerable to excess copper-mediated oxidative stress. In addition, an appropriate copper level is necessary to initiate meiosis in premeiotic germ cells. The balance between essential and toxic copper concentrations in germ cells at different stages of spermatogenesis and in Sertoli cells that support their development is handled by a network of copper importers, chaperones, recipient proteins, and exporters. Here, we describe coordinated regulation/functioning of copper-binding proteins expressed in germ and Sertoli cells with special emphasis on copper transporters, copper transporting ATPases, and SOD1, a copper-dependent antioxidant enzyme. These and other proteins assure copper bioavailability in germ cells and protection against copper toxicity