Reversal of endothelial dysfunction by nicotinamide mononucleotide via extracellular conversion to nicotinamide riboside

Nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) are effective substrates for NAD synthesis, which may act as vasoprotective agents. Here, we characterize the effects of NMN and NR on endothelial inflammation and dysfunction and test the involvement of CD73 in these effects. Materials and methods: The effect of NMN and NR on IL1β- or TNFα-induced endothelial inflammation (ICAM1 and vWF expression), intracellular NAD concentration and NAD-related enzyme expression (NAMPT, CD38, CD73), were studied in HAECs. The effect of NMN and NR on angiotensin II-induced impairment of endotheliumdependent vasodilation was analyzed in murine aortic rings. The involvement of CD73 in NMN and NR effects was tested using CD73 inhibitor-AOPCP, or CD73$^{-/-}$ mice. Results: 24 h-incubation with NMN and NR induced anti-inflammatory effects in HAEC stimulated by IL1β or TNFα, as evidenced by a reduction in ICAM1 and vWF expression. Effects of exogenous NMN but not NR was abrogated in the presence of AOPCP, that efficiently inhibited extracellular endothelial conversion of NMN to NR, without a significant effect on the metabolism of NMN to NA. Surprisingly, intracellular NAD concentration increased in HAEC stimulated by IL1β or TNFα and this effect was associated with upregulation of NAMPT and CD73, whereas changes in CD38 expression were less pronounced. NMN and NR further increased NAD in IL1β- stimulated HAECs and AOPCP diminished NMN-induced increase in NAD, without an effect on NR-induced response. In ex vivo aortic rings stimulated with angiotensin II for 24 h, NO-dependent vasorelaxation induced by acetylcholine was impaired. NMN and NR, both prevented Ang II-induced endothelial dysfunction in the aorta. In aortic rings taken from CD73$^{-/-}$ mice NMN effect was lost, whereas NR effect was preserved. Conclusion: NMN and NR modulate intracellular NAD content in endothelium, inhibit endothelial inflammation and improve NO-dependent function by CD73-dependent and independent pathways, respectively. Extracellular conversion of NMN to NR by CD73 localized in the luminal surface of endothelial cells represent important vasoprotective mechanisms to maintain intracellular NAD

National experience with adenosine deaminase deficiency related SCID in Polish children

IntroductionDeficiency of adenosine deaminase (ADA) manifests as severe combined immunodeficiency (SCID), caused by accumulation of toxic purine degradation by-products. Untreated patients develop immune and non-immune symptoms with fatal clinical course. According to ESID and EBMT recommendations enzyme replacement therapy (ERT) should be implemented as soon as possible to stabilize the patient’s general condition, normalize transaminases, treat pulmonary proteinosis, bone dysplasia, and protect from neurological damage. Hematopoietic stem cell transplantation (HSCT) from a matched related donor (MRD) is a treatment of choice. In absence of such donor, gene therapy (GT) should be considered. HSCT from a matched unrelated donor (MUD) and haploidentical hematopoietic stem cell transplantation (hHSCT) are associated with worse prognosis.Material and methodsWe retrospectively evaluated the clinical course and results of biochemical, immunological and genetic tests of 7 patients diagnosed in Poland with ADA deficiency since 2010 to 2022.ResultsAll patients demonstrated lymphopenia affecting of T, B and NK cells. Diagnosis was made on the basis of ADA activity in red blood cells and/or genetic testing. Patients manifested with various non-immunological symptoms including: lung proteinosis, skeletal dysplasia, liver dysfunction, atypical hemolytic-uremic syndrome, and psychomotor development disorders. Five patients underwent successful HSCT: 3 patients from matched unrelated donor, 2 from matched sibling donor, and 1 haploidentical from a parental donor. In 4 patients HSCT was preceded by enzyme therapy (lasting from 2 to 5 months). One patient with multiple organ failure died shortly after admission, before the diagnosis was confirmed. None of the patients had undergone gene therapy.ConclusionsIt is important to diagnose ADA SCID as early as possible, before irreversible multi-organ failure occurs. In Poland HSCT are performed according to international immunological societies recommendations, while ERT and GT are less accessible. Implementation of Newborn Screening (NBS) for SCID in Poland could enable recognition of SCID, including ADA-SCID

Metabolity nikotynamidu i ich rola w fizjologii i patologii człowieka

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Hydrophobicity of protein determinants influences the recognition of substrates by EDEM1 and EDEM2 in human cells

Background EDEM1 and EDEM2 are crucial regulators of the endoplasmic reticulum (ER)-associated degradation (ERAD) that extracts misfolded glycoproteins from the calnexin chaperone system. The degradation of ERAD substrates involves mannose trimming of N-linked glycans; however the precise mechanism of substrate recognition and sorting to the ERAD pathway is still poorly understood. It has previously been demonstrated that EDEM1 and EDEM2 binding does not require the trimming of substrate glycans or even ERAD substrate glycosylation, thus suggesting that both chaperones probably recognize misfolded regions of aberrant proteins. Results In this work, we focused on the substrate recognition by EDEM1 and EDEM2, asking whether hydrophobicity of protein determinants might be important for these interactions in human cells. In the study we used ricin, a protein toxin that utilizes the ERAD pathway in its retrotranslocation from the ER to the cytosol, and a model misfolded protein, the pancreatic isoform of human β-secretase, BACE457. Mutations in the hydrophobic regions of these proteins allowed us to obtain mutated forms with increased and decreased hydrophobicity. Conclusions Our data provide the first evidence that recognition of ERAD substrates by EDEM1 and EDEM2 might be determined by a sufficiently high hydrophobicity of protein determinants. Moreover, EDEM proteins can bind hydrophobic transmembrane regions of misfolded ERAD substrates. These data contribute to the general understanding of the regulation of ERAD in mammalian cells

Plasma Nucleotide Dynamics during Exercise and Recovery in Highly Trained Athletes and Recreationally Active Individuals

Circulating plasma ATP is able to regulate local skeletal muscle blood flow and 02 delivery causing considerable vasodilatation during exercise. We hypothesized that sport specialization and specific long-term training stimuli have an impact on venous plasma [ATP] and other nucleotides concentration. Four athletic groups consisting of sprinters (n=11; age range 21–30 yr), endurance-trained athletes (n=16; age range 18–31 yr), futsal players (n=14; age range 18–30 yr), and recreationally active individuals (n=12; age range 22–33 yr) were studied. Venous blood samples were collected at rest, during an incremental treadmill test, and during recovery. Baseline [ATP] was 759±80 nmol·l−1 in competitive athletes and 680±73 nmol·l−1 in controls and increased during exercise by ~61% in competitive athletes and by ~31% in recreationally active participants. We demonstrated a rapid increase in plasma [ATP] at exercise intensities of 83–87% of VO2max in competitive athletes and 94% in controls. Concentrations reported after 30 minutes of recovery were distinct from those obtained preexercise in competitive athletes (P<0.001) but not in controls (P=0.61). We found a correlation between total-body skeletal muscle mass and resting and maximal plasma [ATP] in competitive athletes (r=0.81 and r=0.75, respectively). In conclusion, sport specialization is significantly related to plasma [ATP] at rest, during exercise, and during maximal effort. Intensified exercise-induced plasma [ATP] increases may contribute to more effective vessel dilatation during exercise in highly trained athletes than in recreational runners. The most rapid increase in ATP concentration was associated with the respiratory compensation point. No differences between groups of competitive athletes were observed during the recovery period suggesting a similar pattern of response after exercise. Total-body skeletal muscle mass is indirectly related to plasma [ATP] in highly trained athletes

Two-year follow-up of Sanfilippo Disease patients treated with a genistein-rich isoflavone extract: Assessment of effects on cognitive functions and general status of patients

Mucopolysaccharidoses (MPS) are inherited metabolic disorders caused by deficiencies in enzymes involved in degradation of glycosaminoglycans. MPS type III (Sanfilippo disease) is clinically characterized mainly by progressive and severe behavioral disturbances and cognitive dysfunction. Recent 1-year experimental treatment of 10 patients with a genistein (4’, 5, 7-trihydroxyisoflavone)-rich extract resulted in improvement of tested parameters, including cognitive and behavioral functions.Eight pediatric patients with Sanfilippo disease were enrolled into the study. The modified version of the Brief Assessment Examination was used to assess cognitive functions. Moreover, 18 different parameters concerning changes in conditions of patients were assessed by their parents.During the first year of the treatment, an improvement of cognitive functions in 7 patients and stabilization in 1 patient were assessed, while after the third year (2-year follow-up) further improvement was observed in 2 patients, stabilization in 3 patients and some deterioration in 3 patients. Monitoring of general and behavioral symptoms revealed improvement in all patients after the first year of the treatment, further improvement in 5 patients, and deterioration in 3 patients during the next 2 years. We conclude that the treatment of Sanfilippo patients with a genistein-rich soy isoflavone extract(called gene expression-targeted isoflavone therapy[GET IT]) may be effective in either inhibition (in some patients) or slowing down (in other patients) of behavioral and cognitive problems over a longer period. An increased dose of genistein may improve the efficacy of the treatment

Simultaneous determination of lopinavir, saquinavir and ritonavirin in human plasma using liquid chromatography – ion trap mass spectrometry

Background: Lopinavir, saquinavir, and ritonavir are viral protease inhibitors (PIs) developed for and widely used in the therapy of human immunodeficiency virus (HIV)-related disease. These compounds are also active in vitro against the pathogens causing tuberculosis, malaria and coronavirus infections. PIs have been regarded as a platform for the design of inhibitors targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-encoded proteases. This study aimed to develop a liquid chromatography/mass spectrometry (LC/MS) procedure for accurate simultaneous determination of concentrations of these three PIs in the plasma. Methods: Samples of human plasma were protein precipitated with 0.3 M zinc sulfate in a water/methanol solution (30:70, v/v). The extracts were analyzed with reversed-phase chromatography coupled with the electrospray ionization (ESI) source of the ion trap mass detector operating in mEass spectrometry (MS) and tandem mass spectrometry (MS/MS) modes. Results: Calibration curves demonstrated good linearity from 0.01 to 10 µg/mL and acceptable reproducibilities and recoveries. Conclusions: The described procedure proves that a very basic ion-trap LC/MS system could be applied for selective, rapid, and precise determination of antiviral protease inhibitors

Genetic mobile elements in plants and other organisms

In this review the types of mobile genetic elements in prokaryotes and eukaryotes are presented. There is also information about their molecular characteristics, mechanisms ofmoving, evolution and their influence on genome structure and gene activity in organisms of plants, insects and humans. Transposable elements are abundant in genomes of lower and higher organisms. Mobile genetic elements are divided into two main groups: transposons and retrotransposons. The transposons, or "jumping genes" are fragments of DNA capable of moving, from a plasmid to another plasmid (or chromosome) in prokaryotes, and from one part of a chromosome to another (or to another chromosome) in eukaryotes. Transposons become transposed directly fromDNA to DNA eg. the Ac element of maize and the P element of Drosophila are similar to bacterial transposons. Retrotransposons accomplish transposition via an RNA intermediate that is reverse transcribed before integration into a new location within the host genome. They are ubiquitous in eukaryotes and constitute amajor portion of the nuclear genome in humans, animals and plants. They are dispersed as interspersed repetitive sequences through out the genome. Retrotransposons can be divided into two sub-groups: viral retrotransposons eg. Ty (yeast), copia (fruit fly), Bs1 (maize), LINEs (mammals), Cin4 (maize) and non-viral retrotransposons which comprise SINEs and processed pseudogenes. The properties of the genetic mobile elements have been exploited as genetic tools for plant genome analysis

The Effect of Training on Erythrocyte Energy Status and Plasma Purine Metabolites in Athletes

This study aimed to assess the changes in red blood cell (RBC) energy status and plasma purine metabolites concentration over a one-year training cycle in endurance-trained (EN; n = 11, 20&ndash;26 years), and sprint-trained (SP; n = 11, 20&ndash;30 years) competitive athletes in comparison to recreationally-trained individuals (RE; n = 11, 20&ndash;26 years). Somatic, physiological, and biochemical variables were measured in four training phases differing in exercise load profile: transition, general, specific, and competition. Significantly highest values of RBC adenylate energy charge (AEC; p &le; 0.001), ATP-to-ADP and ADP-to-AMP ratios (p &le; 0.05), and plasma levels of adenosine (Ado; p &le; 0.05) were noted in the competition phase in the EN and SP, but not in the RE group. Significantly lowest plasma levels of adenosine diphosphate (ADP; p &le; 0.05), adenosine monophosphate (AMP; p &le; 0.001), inosine (Ino; p &le; 0.001), and hypoxanthine (Hx; p &le; 0.001) accompanied by higher erythrocyte hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity (p &le; 0.001), were observed in the competition phase in both athletic groups. No significant alterations were found in the erythrocyte concentration of guanine nucleotides in any group. In conclusion, periodized training of competitive athletes&rsquo; results in a favorable adaptation of RBC metabolism. The observed changes cover improved RBC energy status (increased AEC and ATP/ADP ratio) and reduced purine loss with more efficient erythrocyte purine pool recovery (increased HGPRT activity and plasma levels of Ado; decreased Hx and Ino concentration)