Case-inspired exploration of renin mutations in autosomal dominant tubulointerstitial kidney disease: not all paths lead to the endoplasmic reticulum.

BACKGROUND: Autosomal dominant tubulointerstitial kidney disease (ADTKD) results from mutations in various genes, including REN, UMOD, MUC1, and HNF1B. ADTKD due to REN mutations (ADTKD-REN) is often characterized as a proteinopathy that triggers the endoplasmic reticulum stress (ERS) cascade, potentially sharing similarities with ADTKD-UMOD and ADTKD-MUC1 at the cellular level. This study, inspired by a patient harboring a W17R mutation, investigates ERS activation by this mutation alongside two other renin variants, W10R and L381P. METHODS: We established stable cell lines expressing both wild-type and mutated renin forms (W17R, W10R, and L381P). Using luciferase reporter assays, RT-qPCR, and confocal microscopy, we evaluated ERS activation, determined the cellular localization of the renin variants, and characterized the mitochondrial network in the W17R line. RESULTS: The L381P line exhibited ERS activation, including transcriptional upregulation of MANF and CRELD2. No ERS activation was observed in the W17R line, while the W10R line exhibited intermediate characteristics. Notably, the W17R variant was misrouted to the mitochondria resulting in changes of the mitochondrial network organisation. CONCLUSIONS: ERS activation is not a universal response to different renin mutations in ADTKD-REN. The pathogenesis of the W17R mutation may involve mitochondrial dysfunction rather than the ER pathway, albeit further research is needed to substantiate this hypothesis fully. Testing CRELD2 and MANF as targeted therapy markers for a specific subgroup of ADTKD-REN patients is recommended. Additionally, fludrocortisone treatment has shown efficacy in stabilizing the renal function of our patient over a four-year period without significant side effects.

Overexpression of c-MYC Promoter Binding Protein-1 Enhances Proliferation and Glucose Metabolism of Melanoma Cells Lines.

BACKGROUND/AIM: c-MYC promoter binding protein (MBP-1) is a product of alternatively translated mRNA encoding alpha-enolase (ENO1). In contrast to ENO1, MBP-1 possesses no enzymatic activity but acts as a transcriptional repressor of c-MYC. Ectopic over-expression of MBP-1 in tumor cells was shown to reduce cell proliferation and tumorigenicity, thus making it an attractive target for anticancer strategies. This study aimed to assess the effects of MBP-1 over-expression on human cutaneous melanoma cell lines. MATERIALS AND METHODS: We overexpressed the full-length MBP-1 or its C-terminal truncated variant (MBP-1ΔC), in two human melanoma cell lines (A375, WM9) and assessed their subcellular localization. qPCR was then used to quantitate c-MYC transcription. Further, 5-ethynyl-2'-deoxyuridine incorporation assay was used to measure cell proliferation and a lactate assay was performed to measure the glycolysis rate of cells in normoxia and hypoxia. Finally, an in vitro wound-healing assay was performed to evaluate cell migration. RESULTS: The overexpressed MBP-1 variants predominantly localized in the cytoplasm and barely decreased c-MYC expression. Unexpectedly, the proliferation rate of MBP-1- transduced cells increased in comparison to controls, as did the rate of glucose metabolism in hypoxia. Furthermore, over-expression of MBP-1, but not MBP-1ΔC, led to a substantial decrease in the cell migration capacity of metastatic WM9 cells but not A375 cells from the primary tumor lesion. CONCLUSION: Misslocalization of over-expressed MBP-1 in the cytoplasm of two melanoma cell lines resulted in an unexpected tumor promoting activity by increasing cell proliferation and glycolysis rates in hypoxia.

Exposure to a 900 MHz electromagnetic field induces a response of the honey bee organism on the level of enzyme activity and the expression of stress-related genes.

There are many artificial sources of radiofrequency electromagnetic field (RF-EMF) in the environment, with a value between 100 MHz and 6 GHz. The most frequently used signal is with a frequency of around 900 MHz. The direction of these changes positively impacts the quality of life, enabling easy communication from almost anywhere in the world. All living organisms in the world feel the effects of the electromagnetic field on them. The observations regarding the influence of a RF-EMF on honey bees, describing the general impact of RF-EMF on the colony and/or behavior of individual bees, such as reduction in the number of individuals in colonies, extended homing flight duration, decrease in breeding efficiency, changes in flight direction (movement of bees toward the areas affected by RF-EMF), increase in the intensity and frequency of sounds characteristic for those announcing the impending danger. In this work, we describe the changes in the levels of some of the stress-related markers in honey bees exposed to varying intensities and duration of RF-EMF. One-day-old honeybee worker bees were used for the study. The bees were randomly assigned to 9 experimental groups which were exposed to the following 900 MHz EMF intensities: 12 V/m, 28 V/m, and 61 V/m for 15 min, 1 h and 3 h. The control group was not exposed to the RF-EMF. Each experimental group consisted of 10 cages in which were 100 bees. Then, hemolymph was collected from the bees, in which the activity was assessed AST, ALT, ALP, GGTP, and level of nonenzymatic antioxidants albumin, creatinine, uric acid, and urea. Bees were also collected for the analysis of rps5, ppo, hsp10, hsp70, hsp90, and vitellogenin gene expression. Our study shows that exposure to a 900 MHz electromagnetic field induces a response in the honey bees that can be detected in the level of enzyme activity and the expression of stress-related genes. The response is similar to the one previously described as a result of exposition to UVB irradiation and most likely cannot be attributed to increased temperature.

New insights into the criteria of functional heterozygosity of the Apis mellifera complementary sex determining gene-Discovery of a functional allele pair differing by a single amino acid.

The complementary sex determiner (csd) gene is responsible for controlling the sex-determination molecular switch in western honey bees (Apis mellifera): bees that are heterozygous for csd develop into females, whereas bees that are hemizygous or homozygous develop into males. The homozygous diploid males are destroyed at an early stage of their development. It has been proposed that the minimal number of amino acid differences between two csd alleles needed to fully determine femaleness is five and it has also been shown that smaller differences may result in forming an evolutionary intermediate that is not fully capable of female determination, but has increased fitness compared to the homozygous genotype. In this study, we have implemented a terminal restriction length polymorphism-based method of identifying and distinguishing paternal alleles in a given bee colony and assigning them to a particular maternal allele in order to gather information on large number of functional csd pairs and also to identify, to some extent, genotypes that are underrepresented or absent in bee colonies. The main finding of this study is the identification of a fully functional genotype consisting of csd alleles that differed from each other by a one amino acid position. The individuals carrying this genotype expressed only female-specific transcripts of feminizer and double-sex genes. By comparing the sequences differences between the csd pair identified in our study with those described earlier, we conclude that functional heterozygosity of the csd gene is dependent not only on the number of the amino acid differences but also on the sequence context and position of the change. The discovery of a functional allele pair differing by a single amino acid also implies that the generation of a new csd specificity may also occur during a single mutation step with no need for evolutionary intermediates accumulating further mutations.

Investigating the Role of Methylation in Silencing of VDR Gene Expression in Normal Cells during Hematopoiesis and in Their Leukemic Counterparts.

(1) Background: Vitamin D receptor (VDR) is present in multiple types of blood cells, and its ligand, 1,25-dihydroxyvitamin D (1,25D), is important for the proper functioning of the immune system. Activity of VDR is higher in hematopoietic stem and progenitor cells than in fully differentiated blood cells of mice and humans. In some human acute myeloid leukemia (AML) blasts, the expression of the VDR gene is also high. The mechanism of silencing the VDR gene expression during differentiation of blood cells has been addressed in this work. (2) Methods: The cells have been obtained using fluorescence activated sorting from murine tissues and from human umbilical cord blood (UCB). Then, the expression of the VDR gene and transcriptional activity of the VDR protein has been tested in real-time polymerase chain reaction (PCR). Eventually, the methylation of VDR promoter regions was tested using bisulfite sequencing. (3) Results: The CpG islands in VDR promoters were not methylated in the cells studied both in mice and in humans. The use of hypomethylating agents had no effect toward expression of human VDR transcripts, but it increased expression of the VDR-target gene, CYP24A1. (4) Conclusions: The expression of the VDR gene and transcriptional activity of the VDR protein varies at successive stages of hematopoietic differentiation in humans and mice, and in blasts from AML patients. The experiments presented in this case indicate that methylation of the promoter region of the VDR gene is not the major mechanism responsible for these differences.

An international cohort study of autosomal dominant tubulointerstitial kidney disease due to REN mutations identifies distinct clinical subtypes.

There have been few clinical or scientific reports of autosomal dominant tubulointerstitial kidney disease due to REN mutations (ADTKD-REN), limiting characterization. To further study this, we formed an international cohort characterizing 111 individuals from 30 families with both clinical and laboratory findings. Sixty-nine individuals had a REN mutation in the signal peptide region (signal group), 27 in the prosegment (prosegment group), and 15 in the mature renin peptide (mature group). Signal group patients were most severely affected, presenting at a mean age of 19.7 years, with the prosegment group presenting at 22.4 years, and the mature group at 37 years. Anemia was present in childhood in 91% in the signal group, 69% prosegment, and none of the mature group. REN signal peptide mutations reduced hydrophobicity of the signal peptide, which is necessary for recognition and translocation across the endoplasmic reticulum, leading to aberrant delivery of preprorenin into the cytoplasm. REN mutations in the prosegment led to deposition of prorenin and renin in the endoplasmic reticulum-Golgi intermediate compartment and decreased prorenin secretion. Mutations in mature renin led to deposition of the mutant prorenin in the endoplasmic reticulum, similar to patients with ADTKD-UMOD, with a rate of progression to end stage kidney disease (63.6 years) that was significantly slower vs. the signal (53.1 years) and prosegment groups (50.8 years) (significant hazard ratio 0.367). Thus, clinical and laboratory studies revealed subtypes of ADTKD-REN that are pathophysiologically, diagnostically, and clinically distinct.

Lack of NWC protein (c11orf74 homolog) in murine spermatogenesis results in reduced sperm competitiveness and impaired ability to fertilize egg cells in vitro.

NWC is an uncharacterised protein containing three strongly conserved domains not found in any other known protein. Previously, we reported that the NWC protein is detected in cells in the germinal layer in murine testes (strain: C57BL/6), and its knockout results in no obvious phenotype. We determined the NWC expression pattern during spermatogenesis, and found this protein in spermatocytes and round spermatids, but not in epididymal sperm. Although NWC knockout males are fertile, we further characterised their reproductive potential employing non-standard mating that better simulates the natural conditions by including sperm competition. Such an approach revealed that the sperm of knockout males fail to successfully compete with control sperm. After analysing selected characteristics of the male reproductive system, we found that NWC knockout sperm had a reduced ability to fertilize cumulus-intact eggs during IVF. This is the first report describing a subtle phenotype of NWC knockout mice that could be detected under non-standard mating conditions. Our results indicate that NWC plays an important role in spermatogenesis and its deficiency results in the production of functionally impaired sperm.

The evolutionary conservation of the bidirectional activity of the NWC gene promoter in jawed vertebrates and the domestication of the RAG transposon.

The RAG-1 and RAG-2 genes form a recombinase complex that is indispensable for V(D)J recombination, which generates the diversity of immunoglobulins and T-cell receptors. It is widely accepted that the presence of RAGs in the genomes of jawed vertebrates and other lineages is a result of the horizontal transfer of a mobile genetic element. While a substantial amount of evidence has been gathered that clarifies the nature of the RAG transposon, far less attention has been paid to the genomic site of its integration in various host organisms. In all genomes of the jawed vertebrates that have been studied to date, the RAG genes are located in close proximity to the NWC gene. We have previously shown that the promoter of the murine NWC genes exhibits a bidirectional activity, which may have facilitated the integration and survival of the RAG transposon in the host genome. In this study, we characterise the promoters of the NWC homologues that are present in the representatives of other jawed vertebrates (H. sapiens, X. tropicalis and D. rerio). We show that the features that are characteristic for promoters as the hosts of a successful transposon integration (in terms of the arrangement, bidirectional and constitutive activity and the involvement of the Zfp143 transcription factor in the promoter regulation) are evolutionarily conserved, which indicates that the presence of RAG genes in jawed vertebrates is a direct result of a successful transposon integration into the NWC locus.

Diverse Regulation of Vitamin D Receptor Gene Expression by 1,25-Dihydroxyvitamin D and ATRA in Murine and Human Blood Cells at Early Stages of Their Differentiation.

Vitamin D receptor (VDR) is present in multiple blood cells, and the hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is essential for the proper functioning of the immune system. The role of retinoic acid receptor α (RARα) in hematopoiesis is very important, as the fusion of RARα gene with PML gene initiates acute promyelocytic leukemia where differentiation of the myeloid lineage is blocked, followed by an uncontrolled proliferation of leukemic blasts. RARα takes part in regulation of VDR transcription, and unliganded RARα acts as a transcriptional repressor to VDR gene in acute myeloid leukemia (AML) cells. This is why we decided to examine the effects of the combination of 1,25D and all-trans-retinoic acid (ATRA) on VDR gene expression in normal human and murine blood cells at various steps of their development. We tested the expression of VDR and regulation of this gene in response to 1,25D or ATRA, as well as transcriptional activities of nuclear receptors VDR and RARs in human and murine blood cells. We discovered that regulation of VDR expression in humans is different from in mice. In human blood cells at early stages of their differentiation ATRA, but not 1,25D, upregulates the expression of VDR. In contrast, in murine blood cells 1,25D, but not ATRA, upregulates the expression of VDR. VDR and RAR receptors are present and transcriptionally active in blood cells of both species, especially at early steps of blood development.

Uneven distribution of complementary sex determiner (csd) alleles in Apis mellifera population.

The complementary sex determiner (csd) gene determines the sex of the western honey bee (Apis mellifera L.). Bees that are heterozygous at the csd locus develop into females; whereas hemizygous bees develop into males. The co-occurrence of two identical csd alleles in a single diploid genome leads to the genetic death of the bee. Thus, the maintenance of csd diversity in the population is favoured. The number and distribution of csd alleles is particularly interesting in light of the recent decline in the honey bee population. In this study, we analysed the distribution of csd alleles in two Polish populations separated by about 100 km. We analysed the maternal alleles of 193 colonies and found 121 different alleles. We also analysed the distribution and frequency of the alleles, and found that they are distributed unevenly. We show that the methods that have been used so far to estimate the total worldwide number of csd alleles have significantly underestimated their diversity. We also show that the uneven distribution of csd alleles is caused by a large number of infrequent alleles, which most likely results from the fact that these alleles are generated very frequently.

Regulation of vitamin D receptor expression by retinoic acid receptor alpha in acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is the predominant acute leukemia among adults, characterized by an accumulation of malignant immature myeloid precursors. A very promising way to treat AML is differentiation therapy using either all-trans-retinoic acid (ATRA) or 1,25-dihydroxyvitamin D3 (1,25D), or the use of both these differentiation-inducing agents. However, the effect of combination treatment varies in different AML cell lines, and this is due to ATRA either down- or up-regulating transcription of vitamin D receptor (VDR) in the cells examined. The mechanism of transcriptional regulation of VDR in response to ATRA has not been fully elucidated. Here, we show that the retinoic acid receptor α (RARα) is responsible for regulating VDR transcription in AML cells. We have shown that a VDR transcriptional variant, originating in exon 1a, is regulated by RARα agonists in AML cells. Moreover, in cells with a high basal level of RARα protein, the VDR gene is transcriptionally repressed as long as RARα agonist is absent. In these cells down-regulation of the level of RARα leads to increased expression of VDR. We consider that our findings provide a mechanistic background to explain the different outcomes from treating AML cell lines with a combination of ATRA and 1,25D.

Search for the Function of NWC, Third Gene Within RAG Locus: Generation and Characterization of NWC-Deficient Mice.

NWC is a third gene within recombination activating gene (RAG) locus, which unlike RAG genes is ubiquitously expressed and encodes a unique protein containing three strongly evolutionarily conserved domains not found in any other known protein. To get insight into its function we identified several proteins co-immunoprecipitating with NWC protein and generated new NWC-deficient mice. Here, we present evidence that unlike many other ubiquitously expressed evolutionarily conserved proteins, functional inactivation of NWC does not cause any gross developmental, physiological or reproductive abnormalities and that under physiological conditions NWC may be involved in assembling and functioning of cilia, cell surface organelles found on nearly every eukaryotic cell.

Ikaros and RAG-2-mediated antisense transcription are responsible for lymphocyte-specific inactivation of NWC promoter.

Recombination activating gene-2 (RAG-2) and NWC are strongly evolutionarily conserved overlapping genes which are convergently transcribed. In non-lymphoid cells the NWC promoter is active whereas in lymphocytes it is inactive due to the DNA methylation. Analysing the mechanism responsible for lymphocyte-specific methylation and inactivation of NWC promoter we found that Ikaros, a lymphocyte-specific transcription factor, acts as a repressor of NWC promoter--thus identifying a new Ikaros target--but is insufficient for inducing its methylation which depends on the antisense transcription driven by RAG-2 promoter. Possible implications of these observations for understanding evolutionary mechanisms leading to lymphocyte specific expression of RAG genes are discussed.

Bidirectional activity of the NWC promoter is responsible for RAG-2 transcription in non-lymphoid cells.

The recombination-activating genes (RAG-1 and RAG-2) encode a V(D)J recombinase responsible for rearrangements of antigen-receptor genes during T and B cell development, and RAG expression is known to correlate strictly with the process of rearrangement. In contrast to RAG-1, the expression of RAG-2 was not previously detected during any other stage of lymphopoiesis or in any other normal tissue. Here we report that the CpG island-associated promoter of the NWC gene (the third evolutionarily conserved gene in the RAG locus), which is located in the second intron of RAG-2, has bidirectional activity and is responsible for the detectable transcription of RAG-2 in some non-lymphoid tissues. We also identify evolutionarily conserved promoter fragments responsible for this bidirectional activity, and show that it is activated by transcription factor ZFP143. The possible implications of our findings are briefly discussed.

Complexity of transcriptional regulation within the Rag locus: identification of a second Nwc promoter region within the Rag2 intron.

Nwc represents a mysterious third evolutionarily conserved gene within the Rag locus. Here, we analyzed the phenotype of Nwc(tmpro1) mice, in which the Rag2 intragenic region containing the previously identified promoter responsible for initiating transcription of Nwc in all cells except lymphocytes was deleted by homologous recombination. Despite strong nonlymphocyte-specific inhibition of Nwc transcription which runs through the regulatory region of Rag genes, their expression remained suppressed, and no developmental, morphological, anatomical, functional, physiological, or cellular defects in Nwc(tmpro1) mice could be observed. However, careful analysis of the Rag2 intergenic region uncovered a second evolutionarily conserved Nwc promoter region from which a previously unknown Nwc transcript can be generated in nonlymphocytes of Nwc(tmpro1) and normal mice. The above results reveal an unexpected additional complexity of transcriptional regulation within the Rag/Nwc locus and show that strong inhibition of Nwc transcription in nonlymphoid cells is well tolerated. Complete inactivation of Nwc is necessary to get insight into its function at transcriptional and posttranscriptional levels.

Peripheral Thy1+ lymphocytes rearranging TCR-gammadelta genes in LAT-deficient mice.

Linker for activation of T cells (LAT) is an adaptor molecule indispensable for development of alphabeta and gammadelta T lymphocytes. Surprisingly, using a new model of LAT-deficient mice we found that despite arrested thymic development, a discrete population of cells with active Lat promoter, expressing Thy1 molecules, accumulated in peripheral lymphoid organs of homozygous (Lat(Inv/Inv)) mutant mice. By measuring frequencies of TCR gene rearrangements in conjunction with a panel of cell surface Ag, we dissected two subsets of these Thy1(+) cells. Thy1(dull) cells expressed markers of NK lymphocytes and contained low frequency of TCR-gamma gene rearrangements without detectable TCR-delta rearrangements. Thy1(high) cells resembled immature CD44(+)CD25(+) thymocytes and contained high frequency of non-productive TCR-gamma and TCR-delta rearrangements, indicating that cells displaying molecular signatures of commitment toward gammadelta T-cell lineage can develop and populate lymphoid tissues of LAT-deficient mice. Phenotypically similar Thy1(high) cells were also found in lymph nodes of lymphocyte-deficient (Rag2(-/-)) mice but not in T lymphocyte proficient, heterozygous Lat(+/Inv) mice suggesting that Thy1(high) cells of LAT-deficient mice identified in this study accumulate in peripheral lymphoid organs as a result of congenital lymphopenia.

Mechanism of lymphocyte-specific inactivation of RAG-2 intragenic promoter of NWC: implications for epigenetic control of RAG locus.

NWC, third evolutionarily conserved gene within RAG locus is transcribed at high level in all cells except mature T and B lymphocytes and their RAG negative progenitors. It is so, because in lymphocytes expression of NWC is regulated by RAG-1 promoter, while in other cells it is controlled by RAG-2 intragenic promoter which in T and B lymphocytes is silent. Here we show that lymphocyte-specific inactivation of NWC promoter is caused by CpG island hypermethylation accompanied by site-specific blocking of chromatin accessibility, which in contrast to RAG promoters, is not accompanied by expected posttranslational modifications of histone H3. These results indicate that accessibility of NWC promoter and RAG promoters to trans-acting factors is regulated by different epigenetic mechanisms. The implications of our findings for understanding mechanisms regulating transcription within RAG/NWC locus in different cells are discussed and the model of epigenetic control of this locus is proposed.

Monte Carlo simulations of the age structure of the human population.

In this mini review, we present assumptions and some results of Monte Carlo simulations based on the Penna model, which support the mutation accumulation theory of aging. This microscopic model has been exploited for the quantitative description of many biological phenomena connected with the population evolution. We show how the results of simulations could describe the changes of mortality trajectories of the human populations during the last 150 years, and how the method could be used for predicting the human age distribution in the future. The main assumption of the model is that genes are expressed chronologically one after another in the same order in all individuals during their life span. Different forces of selection pressure exerted on genes expressed at different periods of life generate characteristic gradient of defective genes accumulated in the germline cells. The genes expressed after the minimum reproduction age are under weaker selection pressure, and the fraction of defects among them is higher than among the genes expressed before the minimum reproduction age. This gradient of defects generates a gradient of mortality for the part of the population in the reproduction age following the exponential Gompertz law. The limitations of the model and some biological interpretations of its parameters are also discussed.

Characterization of a membrane-linked Ser/Thr protein kinase in Bacillus subtilis, implicated in developmental processes.

PrkC was shown to be a eukaryotic-like (Hanks-type) protein kinase from Bacillus subtilis with a structural organization similar to that of the eukaryotic sensor Ser/Thr or Tyr kinases (e.g. the TGF beta or PDGF receptors). The molecule consists of a catalytic domain located in the cytoplasm, joined by a single transmembrane-spanning region (TMD) to a large extracellular domain. Using a genetic reporter system, involving the cI repressor of lambda, evidence was obtained indicating that PrkC forms a dimer, involving both the TMD and the external domain in dimerization. The purified catalytic domain of PrkC was shown to autophosphorylate and to phosphorylate an external target, MBP, in both cases on threonine. These two functions require the completely conserved K40 residue in subdomain II, which is essential for enzymatic activity. Importantly, both the mutant deleted for prkC and a K40R mutant exhibit decreased efficiency of sporulation and a significant reduction in biofilm formation, demonstrating that the catalytic activity of PrkC is necessary for these two developmental processes. In addition, we showed that the product of prpC, a PPM phosphatase encoded by the adjacent gene, co-transcribed with prkC, is also required for normal biofilm and spore formation.

How many protein-coding genes are there in the Saccharomyces cerevisiae genome?

We have compared the results of estimations of the total number of protein-coding genes in the Saccharomyces cerevisiae genome, which have been obtained by many laboratories since the yeast genome sequence was published in 1996. We propose that there are 5300-5400 genes in the genome. This makes the first estimation of the number of intronless ORFs longer than 100 codons, based on the features of the set of genes with phenotypes known in 1997 to be correct. This estimation assumed that the set of the first 2300 genes with known phenotypes was representative for the whole set of protein-coding genes in the genome. The same method used in this paper for the approximation of the total number of protein-coding sequences among more than 40 000 ORFs longer than 20 codons gives a result that is only slightly higher. This suggests that there are still some non-coding ORFs in the databases and a few dozen small ORFs, not yet annotated, which probably code for proteins.