Subcellular Localization of Thioredoxin/Thioredoxin Reductase System-A Missing Link in Endoplasmic Reticulum Redox Balance.

The lumen of the endoplasmic reticulum (ER) is usually considered an oxidative environment; however, oxidized thiol-disulfides and reduced pyridine nucleotides occur there parallelly, indicating that the ER lumen lacks components which connect the two systems. Here, we investigated the luminal presence of the thioredoxin (Trx)/thioredoxin reductase (TrxR) proteins, capable of linking the protein thiol and pyridine nucleotide pools in different compartments. It was shown that specific activity of TrxR in the ER is undetectable, whereas higher activities were measured in the cytoplasm and mitochondria. None of the Trx/TrxR isoforms were expressed in the ER by Western blot analysis. Co-localization studies of various isoforms of Trx and TrxR with ER marker Grp94 by immunofluorescent analysis further confirmed their absence from the lumen. The probability of luminal localization of each isoform was also predicted to be very low by several in silico analysis tools. ER-targeted transient transfection of HeLa cells with Trx1 and TrxR1 significantly decreased cell viability and induced apoptotic cell death. In conclusion, the absence of this electron transfer chain may explain the uncoupling of the redox systems in the ER lumen, allowing parallel presence of a reduced pyridine nucleotide and a probably oxidized protein pool necessary for cellular viability.

More than one millennium (2nd-16th century CE) of the White Plague in the Carpathian Basin - New cases, expanding knowledge.

The causative agent of tuberculosis is still a widespread pathogen, which caused the death of ca. 1.6 million people globally in 2021. The paleopathological study of human remains revealed the antiquity of the disease and its continuous presence throughout the history of humankind. The Carpathian Basin has always been a biocultural melting pot, since it has seen several migrations over the centuries, and served as a location of admixture and interaction for numerous populations of different cultures. Thus, this geographical territory is ideal for the examination of the coevolutionary processes of hosts and their pathogens. We aimed to reveal the spatial and temporal distribution of tuberculosis cases excavated inside the borders of Hungary between the 2nd and 16th centuries CE. We established a comprehensive database by collecting 114 already published cases and introducing 39 new cases. The involved cases include those that have been confirmed by different molecular methods, as well as possible infections that were identified based on the presence of macromorphological and radiological alterations. The progress of future molecular and paleopathological studies can be facilitated by our dataset, as it presents spatial and temporal information concerning the spread of the disease in the Carpathian Basin, as well as the biological profile and detailed paleopathological description of lesions illustrated by photo- and radiographs.

Arterial Tortuosity Syndrome: An Ascorbate Compartmentalization Disorder?

Significance: Cardiovascular disorders are the most important cause of morbidity and mortality in the Western world. Monogenic developmental disorders of the heart and vessels are highly valuable to study the physiological and pathological processes in cardiovascular system homeostasis. The arterial tortuosity syndrome (ATS) is a rare, autosomal recessive connective tissue disorder showing lengthening, tortuosity, and stenosis of the large arteries, with a propensity for aneurysm formation. In histopathology, it associates with fragmentation and disorganization of elastic fibers in several tissues, including the arterial wall. ATS is caused by pathogenic variants in SLC2A10 encoding the facilitative glucose transporter (GLUT)10. Critical Issues: Although several hypotheses have been forwarded, the molecular mechanisms linking disrupted GLUT10 activity with arterial malformations are largely unknown. Recent Advances: The vascular and systemic manifestations and natural history of ATS patients have been largely delineated. GLUT10 was identified as an intracellular transporter of dehydroascorbic acid, which contributes to collagen and elastin cross-linking in the endoplasmic reticulum, redox homeostasis in the mitochondria, and global and gene-specific methylation/hydroxymethylation affecting epigenetic regulation in the nucleus. We revise here the current knowledge on ATS and the role of GLUT10 within the compartmentalization of ascorbate in physiological and diseased states. Future Directions: Centralization of clinical, treatment, and outcome data will enable better management for ATS patients. Establishment of representative animal disease models could facilitate the study of pathomechanisms underlying ATS. This might be relevant for other forms of vascular dysplasia, such as isolated aneurysm formation, hypertensive vasculopathy, and neovascularization. Antioxid. Redox Signal. 34, 875-889.

Novel Crizotinib-GnRH Conjugates Revealed the Significance of Lysosomal Trapping in GnRH-Based Drug Delivery Systems.

Several promising anti-cancer drug-GnRH (gonadotropin-releasing hormone) conjugates have been developed in the last two decades, although none of them have been approved for clinical use yet. Crizotinib is an effective multi-target kinase inhibitor, approved against anaplastic lymphoma kinase (ALK)- or ROS proto-oncogene 1 (ROS-1)-positive non-small cell lung carcinoma (NSCLC); however, its application is accompanied by serious side effects. In order to deliver crizotinib selectively into the tumor cells, we synthesized novel crizotinib analogues and conjugated them to a [d-Lys6]-GnRH-I targeting peptide. Our most prominent crizotinib-GnRH conjugates, the amide-bond-containing [d-Lys6(crizotinib*)]-GnRH-I and the ester-bond-containing [d-Lys6(MJ55*)]-GnRH-I, were able to bind to GnRH-receptor (GnRHR) and exert a potent c-Met kinase inhibitory effect. The efficacy of compounds was tested on the MET-amplified and GnRHR-expressing EBC-1 NSCLC cells. In vitro pharmacological profiling led to the conclusion that that crizotinib-GnRH conjugates are transported directly into lysosomes, where the membrane permeability of crizotinib is diminished. As a consequence of GnRHR-mediated endocytosis, GnRH-conjugated crizotinib bypasses its molecular targets-the ATP-binding site of RTKs- and is sequestered in the lysosomes. These results explained the lower efficacy of crizotinib-GnRH conjugates in EBC-1 cells, and led to the conclusion that drug escape from the lysosomes is a major challenge in the development of clinically relevant anti-cancer drug-GnRH conjugates.

Glucose Transport and Transporters in the Endomembranes.

Glucose is a basic nutrient in most of the creatures; its transport through biological membranes is an absolute requirement of life. This role is fulfilled by glucose transporters, mediating the transport of glucose by facilitated diffusion or by secondary active transport. GLUT (glucose transporter) or SLC2A (Solute carrier 2A) families represent the main glucose transporters in mammalian cells, originally described as plasma membrane transporters. Glucose transport through intracellular membranes has not been elucidated yet; however, glucose is formed in the lumen of various organelles. The glucose-6-phosphatase system catalyzing the last common step of gluconeogenesis and glycogenolysis generates glucose within the lumen of the endoplasmic reticulum. Posttranslational processing of the oligosaccharide moiety of glycoproteins also results in intraluminal glucose formation in the endoplasmic reticulum (ER) and Golgi. Autophagic degradation of polysaccharides, glycoproteins, and glycolipids leads to glucose accumulation in lysosomes. Despite the obvious necessity, the mechanism of glucose transport and the molecular nature of mediating proteins in the endomembranes have been hardly elucidated for the last few years. However, recent studies revealed the intracellular localization and functional features of some glucose transporters; the aim of the present paper was to summarize the collected knowledge.

Decreased Nuclear Ascorbate Accumulation Accompanied with Altered Genomic Methylation Pattern in Fibroblasts from Arterial Tortuosity Syndrome Patients.

Ascorbate requiring Fe2+/2-oxoglutarate-dependent dioxygenases located in the nucleoplasm have been shown to participate in epigenetic regulation of gene expression via histone and DNA demethylation. Transport of dehydroascorbic acid is impaired in the endomembranes of fibroblasts from arterial tortuosity syndrome (ATS) patients, due to the mutation in the gene coding for glucose transporter GLUT10. We hypothesized that altered nuclear ascorbate concentration might be present in ATS fibroblasts, affecting dioxygenase activity and DNA demethylation. Therefore, our aim was to characterize the subcellular distribution of vitamin C, the global and site-specific changes in 5-methylcytosine and 5-hydroxymethylcytosine levels, and the effect of ascorbate supplementation in control and ATS fibroblast cultures. Diminished nuclear accumulation of ascorbate was found in ATS fibroblasts upon ascorbate or dehydroascorbic acid addition. Analyzing DNA samples of cultured fibroblasts from controls and ATS patients, a lower global 5-hydroxymethylcytosine level was found in ATS fibroblasts, which could not be significantly modified by ascorbate addition. Investigation of the (hydroxy)methylation status of specific regions in six candidate genes related to ascorbate metabolism and function showed that ascorbate addition could stimulate hydroxymethylation and active DNA demethylation at the PPAR-γ gene region in control fibroblasts only. The altered DNA hydroxymethylation patterns in patient cells both at the global level and at specific gene regions accompanied with decreased nuclear accumulation of ascorbate suggests the epigenetic role of vitamin C in the pathomechanism of ATS. The present findings represent the first example for the role of vitamin C transport in epigenetic regulation suggesting that ATS is a compartmentalization disease.

Hyperostosis frontalis interna in ancient populations from the Carpathian Basin - A possible relationship between lifestyle and risk of development.

OBJECTIVE: The prevalence of hyperostosis frontalis interna (HFI) was examined in different periods of the Carpathian Basin from 4900 BCE to 17th century AD. The study seeks to evaluate temporal changes in HFI and the possible impact of lifestyle on it. MATERIALS: The studied material consisted of 4668 crania from Hungary and Serbia. METHODS: The crania were analyzed employing macroscopic and endoscopic examination. RESULTS: In historic periods, sex and age played a pivotal role in HFI development. Among predominantly pastoralist populations of the 5th-8th and 10th centuries, prevalence of HFI was considerably higher than in the medieval populations of the 9th-17th centuries. CONCLUSIONS: In addition to age and sex, other factors could be implicated in HFI development. The physiological effects of the pastoralist lifestyle and diet on insulin regulation could explain the increased risk of developing HFI in the 5th-8th and 10th-century populations. SIGNIFICANCE: The study provides the first comprehensive dataset of HFI from different archaeological periods from the Carpathian Basin. It has implications for lifestyle and risk of HFI development in past populations. LIMITATIONS: The archaeological periods are not equally represented. SUGGESTIONS FOR FURTHER RESEARCH: In order to better understand the etiology of HFI, lifestyle factors can be used to elucidate the risk of developing HFI in ancient populations.

GLUT10-Lacking in Arterial Tortuosity Syndrome-Is Localized to the Endoplasmic Reticulum of Human Fibroblasts.

GLUT10 belongs to a family of transporters that catalyze the uptake of sugars/polyols by facilitated diffusion. Loss-of-function mutations in the SLC2A10 gene encoding GLUT10 are responsible for arterial tortuosity syndrome (ATS). Since subcellular distribution of the transporter is dubious, we aimed to clarify the localization of GLUT10. In silico GLUT10 localization prediction suggested its presence in the endoplasmic reticulum (ER). Immunoblotting showed the presence of GLUT10 protein in the microsomal, but not in mitochondrial fractions of human fibroblasts and liver tissue. An even cytosolic distribution with an intense perinuclear decoration of GLUT10 was demonstrated by immunofluorescence in human fibroblasts, whilst mitochondrial markers revealed a fully different decoration pattern. GLUT10 decoration was fully absent in fibroblasts from three ATS patients. Expression of exogenous, tagged GLUT10 in fibroblasts from an ATS patient revealed a strict co-localization with the ER marker protein disulfide isomerase (PDI). The results demonstrate that GLUT10 is present in the ER.

[Angiography of the ocular fundus without dye: Optical coherence tomography based angiography in exsudative age-related macular degeneration]. / Szemfenéki érfestés festék nélkül: Az optikai koherencia tomográfia alapú angiográfia exsudativ típusú idoskori maculadegenerációban.

INTRODUCTION: Vascular endothelial growth factor antibody therapy is an established treatment of exsudative age-related macular degeneration. AIM: The morphologic characterisation of the macular microvasculature after longstanding treatment. METHOD: Forty-eight patients (34 women and 14 men; age, 74.4 ± 8.0 years) were enrolled in the study. During follow-up time (53.8 ± 31.0 months), 7.6 ± 4.9 injections were administered in 56 eyes. Optical coherence tomography angiographic examination was performed with AngioVue (Optovue Inc. Fremont, CA, USA). RESULTS: Distortion of the superficial retinal plexus and foveal avascular zone enlargement were noted in 5/56 eyes, deep retinal plexus defect was detected in 9/56 cases. Destruction of the choriocapillaries and the former neovascularisation could be found in 4 different patterns: 1. pigment epithelium and choriocapillary atrophy, 2. submacular scar, 3. active leaking choroidal neovascularisation, 4. intraretinal cysts. CONCLUSION: Optical coherence tomography angiography is a novel non-invasive method, which enables the follow up of macular degeneration. Orv. Hetil., 2016, 157(42), 1683-1690.

Glucose transporter type 10-lacking in arterial tortuosity syndrome-facilitates dehydroascorbic acid transport.

Loss-of-function mutations in the gene encoding GLUT10 are responsible for arterial tortuosity syndrome (ATS), a rare connective tissue disorder. In this study GLUT10-mediated dehydroascorbic acid (DAA) transport was investigated, supposing its involvement in the pathomechanism. GLUT10 protein produced by in vitro translation and incorporated into liposomes efficiently transported DAA. Silencing of GLUT10 decreased DAA transport in immortalized human fibroblasts whose plasma membrane was selectively permeabilized. Similarly, the transport of DAA through endomembranes was markedly reduced in fibroblasts from ATS patients. Re-expression of GLUT10 in patients' fibroblasts restored DAA transport activity. The present results demonstrate that GLUT10 is a DAA transporter and DAA transport is diminished in the endomembranes of fibroblasts from ATS patients.

Generation of amphidiploids from hybrids of wheat and related species from the genera Aegilops, Secale, Thinopyrum, and Triticum as a source of genetic variation for wheat improvement.

We aim to improve diversity of domesticated wheat by transferring genetic variation for important target traits from related wild and cultivated grass species. The present study describes the development of F1 hybrids between wheat and related species from the genera Aegilops, Secale, Thinopyrum, and Triticum and production of new amphidiploids. Amphidiploid lines were produced from 20 different distant relatives. Both colchicine and caffeine were successfully used to double the chromosome numbers. The genomic constitution of the newly formed amphidiploids derived from seven distant relatives was determined using genomic in situ hybridization (GISH). Altogether, 42 different plants were analysed, 19 using multicolour GISH separating the chromosomes from the A, B, and D genomes of wheat, as well as the distant relative, and 23 using single colour GISH. Restructuring of the allopolyploid genome, both chromosome losses and aneuploidy, was detected in all the genomes contained by the amphidiploids. From the observed chromosome numbers there is an indication that in amphidiploids the B genome of wheat suffers chromosome losses less frequently than the other wheat genomes. Phenotyping to realize the full potential of the wheat-related grass germplasm is underway, linking the analyzed genotypes to agronomically important target traits.

Subcellular compartmentation of ascorbate and its variation in disease states.

Beyond its general role as antioxidant, specific functions of ascorbate are compartmentalized within the eukaryotic cell. The list of organelle-specific functions of ascorbate has been recently expanded with the epigenetic role exerted as a cofactor for DNA and histone demethylases in the nucleus. Compartmentation necessitates the transport through intracellular membranes; members of the GLUT family and sodium-vitamin C cotransporters mediate the permeation of dehydroascorbic acid and ascorbate, respectively. Recent observations show that increased consumption and/or hindered entrance of ascorbate in/to a compartment results in pathological alterations partially resembling to scurvy, thus diseases of ascorbate compartmentation can exist. The review focuses on the reactions and transporters that can modulate ascorbate concentration and redox state in three compartments: endoplasmic reticulum, mitochondria and nucleus. By introducing the relevant experimental and clinical findings we make an attempt to coin the term of ascorbate compartmentation disease.

Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat.

(1,3;1,4)-beta-d-Glucan (beta-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative beta-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total beta-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable beta-glucan was also reduced by about 50% in the transgenic lines, and the M(r) distribution of the fraction was decreased from an average of 79 to 85 x 10(4) g/mol in the controls and 36 to 57 x 10(4) g/mol in the transgenics. Immunolocalization of beta-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a beta-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.

Urinary albumin excretion is correlated to fibrinogen levels and protein S activity in patients with type 1 diabetes mellitus without overt diabetic nephropathy.

The aim of the study was to test the hypothesis that in diabetic patients without overt nephropathy there may be a correlation between the activity of natural anticoagulant proteins and glomerular dysfunction. Assays for functional activity of proteins S and C, measurements of urinary albumin excretion, lipid parameters and haemoglobin A1c were performed in 91 patients with type 1 diabetes mellitus and 85 patients with type 2. Patients with type 1 diabetes and microalbuminuria had significantly higher mean age (44.1 +/- 10.9 vs. 37.9 +/- 12.7 years; p<0.05), fibrinogen level (3.75 +/- 1.0 vs. 3.21 +/- 0.8 g/l; p<0.01), protein S activity (92.3 +/- 17.6 vs. 84.5 +/- 15.5%; p<0.05) and higher prevalence of retinopathy (p<0.01) and macrovascular disease (p<0.01) than those with normoalbuminuria. Albumin excretion was significantly correlated to age (r=0.25, p<0.05), fibrinogen level (r=0.39, p<0.01), protein S activity (r=0.27; p<0.05), total cholesterol (r=0.23; p<0.05), apoprotein B (r=0.22; p<0.05), retinopathy (r=0.33; p<0.01) and macrovascular disease (r=0.33; p<0.01). Patients with type 2 diabetes mellitus and microalbuminuria had significantly higher apoprotein B levels (1.17 +/- 0.3 vs. 1.06 +/- 1.2 mg/dl; p<0.05) than those with normoalbuminuria, and apoprotein B was significantly correlated to albumin excretion (r=0.22; p<0.05). In a multivariate model of type 1 diabetes mellitus with fibrinogen, protein S and C activity, cholesterol, triglycerides, haemoglobin A1c, retinopathy, and macrovascular disease as independent parameters (r=0.53; p<0.003), there was significant independent correlation of fibrinogen (beta=0.28; p<0.01), protein S activity (beta=0.27; p<0.05) and retinopathy (beta=0.21; p<0.01) with albumin excretion. We conclude that in type 1 diabetes, relative elevation of fibrinogen level and protein S activity appear in the early stages of development of diabetic nephropathy, and may be related to the pathogenesis of diabetic kidney disease.

Diurnal blood pressure pattern may predict the increase of urinary albumin excretion in normotensive normoalbuminuric type 1 diabetes mellitus patients.

To characterise the relationship between diurnal blood pressure and the subsequent increase of urinary albumin excretion (UAE) in normotensive normoalbuminuric type 1 diabetic patients, ambulatory blood pressure monitoring (ABPM) was performed in 53 patients, who were then followed for 5 years. Albumin excretion rate changed from 12.4 (8.9-17.2) to 29.3 (15.2-47.0) mg/day. Macroalbuminuria developed in 2 (3.8%), microalbuminuria in 22 (41.5%) patients, 29 (54.7%) remained normoalbuminuric. Night-time diastolic blood pressure was significantly higher (64.3+/-6.5 vs. 60.9+/-5.5 mmHg, P<0.05), diastolic diurnal index significantly lower (15.5+/-9.7 vs. 22.3+/-6.2%, P<0.01) in patients who later progressed to micro- or macroalbuminuria. Diastolic diurnal index (r=-0.40; P<0.01) and nocturnal diastolic pressure (r=0.35; P<0.01) were correlated to the change in albumin excretion. In a multivariate analysis model with the change of albumin excretion as dependent, and means and diurnal indices of systolic and diastolic blood pressure, baseline UAE, cholesterol, triglycerides, HbA1c and retinopathy as independent parameters (r=0.68; P=0.001), diurnal index for diastolic blood pressure (beta=-0.30; r=0.013), baseline HbA1c (beta=0.32; P=0.010) and retinopathy (beta=0.44; P=0.001) were significant independent correlates. We conclude that the relative increase of nocturnal blood pressure is associated with the subsequent increase of albuminuria, which in turn is predictive of overt diabetic nephropathy.