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PURPOSE Transitions in nursing education and professionalism that align with global nursing standards are elucidated as critical success factors in transforming health professionals and health care in Albania. Progressive educational and regulatory pathways throughout the 2000s (1999-2020) are emphasized for their impact on the Albanian health system, including the achievement of universal healthcare coverage. METHODS Data collected by the Ministry of Health and Sport and the Regulatory Authority for nursing and other healthcare professions in Albania were analyzed and outcomes explicated with regard to Albania's major health challenges. DISCUSSION AND CONCLUSIONS Three milestones affirmed nursing as a driving force in the Albanian healthcare system: (a) nurses constitute the largest health professional workforce via a nurse-patient ratio of 1:400 in contrast to a physician-patient ratio of 1:2,500; (b) nurses are frontline care providers via clinical leadership in the management of primary healthcare centers, which ensure universal healthcare coverage; and (c) nurses are first responders via their presence and compassionate caring in the primary healthcare centers, including making critical shifts in converting primary healthcare centers to urgent care centers as needed. CLINICAL RELEVANCE Nursing advancements have implicated quality care and professionalism in Albania across the health professions via three critical pathways: (a) health professional education at a university degree level for entry into practice (since 1999), which was prompted by and driven by nursing's quest to be a self-regulated profession (achieved in 2007); (b) healthcare global standards sparked by nursing's mandate toward professional autonomy, as achieved via regulatory procedures and policies; and (c) interprofessional healthcare initiatives that serve as collaborative platforms for innovative educational, clinical, and research projects.

One might think that after over 100 years of study we now know all there is to know about Hemoglobin and its function. However, the purpose of this review is to outline that this fascinating protein has still much to say in the field of biological modulation. Hence, we like to focus on a number of parallel functions of hemoglobin besides its basic function of oxygen transport. Among these we like to recall the following main functions: a) modulation of erythrocyte metabolism; b) Heme oxidation and erythrocytes senescence; c) resistance to malaria; d) molecular heat transducer e) Enzymatic activity; f) Hemorphins, carbon monoxide and nitric oxide.

Background: In this study, human neuroblastoma cells (IMR32) treated with Amyloid Beta Peptide (APβ), were used as model to evaluate the molecular basis of protective role of S100b, a neurotrophic factor and neuronal survival protein, highly expressed by reactive astrocytes close to amyloid deposition in the cortex of Alzheimer's patients. The aim of this work is to value the effect of S100b on ROS production in cells treated with Amyloid Beta Peptide and the subsequent influence on globin gene expression. Method: In this study we investigated the effect of S100b on ROS production and on globin gene expression in human neuroblastoma cells (IMR32) treated with Amyloid Beta Peptide (APβ). Results: Our results have shown that at nanomolar concentrations, S100b protects cells against AP mediated cytotoxicity and the protective mechanism could be related, almost in part, to the control of ROS production through an over expression of Myoglobin gene. Conclusion: In light of our results, we speculate that over-expression of the Myoglobin gene could be read as a possible attempt of the cell to increase the scavengers of reactive oxygen species (ROS).

[Image: see text] Autoreactive T cells specific to human collagen type II have a crucial role in the development of rheumatoid arthritis (RA) in the context of MHC class II allele HLA-DRB1-*04. The protein–protein interactions between the T cell receptor (TCR) and the type II collagen bound to the allele MHC of class II may thus represent the target for the development of new drugs against RA. In this study, a structure-based pharmacophore model for potential small molecule inhibitors was developed from protein–protein interface structure. The 3D model obtained was used for a virtual screening workflow, which resulted in three hits for experimental follow up. Three compounds have been identified that interfere with the TCR/collagenII-MHCII (K(i) values below 10 μM) and open up new possibilities in the treatment of RA.

Glycolysis and pentose phosphate pathway (PPP) in red blood cell (RBC) are modulated by the cell oxygenation state. This metabolic modulation is connected to variations in intracellular nicotinamide adenine dinucleotide phosphate-reduced form (NADPH) and adenosine triphosphate (ATP) levels as a function of the oxygenation state of the cell, and, consequently, it should have physiologic relevance. In the present study, we analysed the effects of amyloid beta peptide (1-42) (Abeta) on RBC metabolism and its relationship with the activity of protein kinase C (PKC). Our results showed that metabolic response to Abeta depended on the degree of cell oxygenation. In particular, under high O2 pressure, in Abeta-treated RBC, glucose metabolized through PPP approached that metabolized by RBC under low O2 pressure, differently to that observed in untreated cells. The effect of Abeta on RBC metabolism was paralleled by increase in PKC enzyme activity, but cytosolic Ca2+ concentration does not seem to be involved in this mechanism. Incubation of Abeta-treated RBC with a specific inhibitor of PKC partially restores PPP flux. A possible rationalization of the different metabolic behaviours shown by RBC following Abeta treatment is proposed. It takes into account the known post-translational modifications to cytoskeleton proteins induced by PKC. The reduction in PPP flux may lead to a weakened defence system of antioxidant reserve in RBC, becoming a source of reactive species, and, consequently, its typical, structural and functional features are lost. Therefore, oxidative stress may outflow from the RBC and trigger damage events in adjacent cells and tissue, thus contributing to vascular damage.

Widely confirmed reports were published on association between hyperhomocysteinemia, B vitamin deficiency, oxidative stress, and amyloid-beta in Alzheimer's disease (AD). Homocysteine, cysteine, cysteinylglycine and glutathione are metabolically interrelated thiols that may be potential indicators of health status and disease risk; they all participate in the metabolic pathway of homocysteine. Previous data obtained in one of our laboratories showed that B vitamin deficiency induced exacerbation of AD-like features in TgCRND8 AD mice; these effects were counteracted by S-adenosylmethionine (SAM) supplementation, through the modulation of DNA methylation and antioxidant pathways. Since the cellular response to oxidative stress typically involves alteration in thiols content, a rapid and sensitive HPLC method with fluorescence detection was here used to evaluate the effect of SAM and superoxide-dismutase (SOD) supplementation on thiols level in plasma, in TgCRND8 mice. The quantitative data obtained from HPLC analysis of mice plasma samples showed significant decrease of thiols level when the B vitamin deficient diet was supplemented with SAM + SOD and SOD alone, the latter showing the greatest effect. All these considerations point out the measurement of plasma thiols concentration as a powerful tool of relevance for all clinical purposes involving the evaluation of oxidative stress. The coupling of HPLC with fluorimetric detection, here used, provided a strong method sensitivity allowing thiols determination at very low levels.

This comparative study, focused on the anion kinetic of band 3 protein of pig erythrocytes, investigates in vitro the metabolic functionality of the red blood cells of this mammal which show particular characteristics of the cell membrane and significant s...

Molecules of the plant world are proving their effectiveness in countering, slowing down, and regressing many diseases. The resveratrol for its intrinsic properties related to its stilbene structure has been proven to be a universal panacea, especially for a wide range of neurodegenerative diseases. This paper evaluates (in vivo and in vitro) the various molecular targets of this peculiar polyphenol and its ability to effectively counter several neurodegenerative disorders such as Parkinson’s, Alzheimer’s, and Huntington’s diseases and amyotrophic lateral sclerosis. What emerges is that, in the deep heterogeneity of the pathologies evaluated, resveratrol through a convergence on the protein targets is able to give therapeutic responses in neuronal cells deeply diversified not only in morphological structure but especially in their function performed in the anatomical district to which they belong.

Methionine sulfoxide reductase A (MsrA) has been postulated to act as a catalytic antioxidant system involved in the protection of oxidative stress-induced cell injury. MsrA has recently turned attention in coupling with the neurodegenerative disorders and in particular with Alzheimer disease. In fact this neurodegenerative disorder depends to a deposit of beta amyloid a peptide with an oxidizable methionine in position 35 which is proved able to modulate the expression to MsrA in neuronal cells. Here, we firstly provided evidence that pretreatment with Resveratrol and Punicalagin (a potent antioxidant extracted from pomegranate), up-regulate the expression and enzymatic activity of MsrA in human neuroblastoma IMR-32 cells with beta amyloid peptides. This effect determines a lowering of oxidative potential of the cells as demonstrated by the ROS measurement and a protective effect on cellular availability. Therefore we hypothesize a possible prevent role for these molecules in Alzheimer and in other neurodegenerative diseases.

Palytoxin (PTX), a marine toxin, represents an increasing hazard for human health. Despite its high toxicity for biological systems, the mechanisms triggered by PTX, are not well understood. The high affinity of PTX for erythrocyte Na(+)/K(+)-ATPase pump is largely known, and it indicates PTX as a sensitive tool to characterize the signal transducer role for Na(+)/K(+)-ATPase pump. Previously, it has been reported that in red blood cells (RBC), probably via a signal transduction generated by the formation of a PTX-Na(+)/K(+)-ATPase complex, PTX alters band 3 functions and glucose metabolism. The present study addresses the question of which other signaling pathways are regulated by Na(+)/K(+)-ATPase in RBC. Here it has been evidenced that PTX following its interaction with Na(+)/K(+)-ATPase pump, alters RBC morphology and this event is correlated to decreases by 30% in nitrites and nitrates levels, known as markers of plasma membrane eNOS activity. Orthovanadate (OV), an antagonist of PTX binding to Na(+)/K(+)-ATPase pump, was able to reverse the effects elicited by PTX. Finally, current investigation firstly suggests that Na(+)/K(+)-ATPase pump, following its interaction with PTX, triggers a signal transduction involved in NO metabolism regulation.

This article reports the role of resveratrol in the erythrocyte as a result of its interaction with hemoglobin and describes the effect of this interaction on the metabolism, the redox state, and the release of ATP. The drug crosses the erythrocyte membrane and binds to hemoglobin, altering its modulation and the release of ATP. Our study correlates the variation of the phosphorylation balance induced by resveratrol with the change in the intracellular concentration of ATP and with the decrease in ATP release from red blood cell and the consequent paracrine alteration on the vascular epithelium. Molecular docking calculations indicate larger specificity of binding for oxy-hemoglobin that correlates well with the stabilization of the R-quaternary structure and with the functional modulation of resveratrol on the protein. Finally, we locate a putative binding site at the central cavity of hemoglobin and characterize its key interacting residues with the drug. Computational results support the assumption that resveratrol may act as a protector agent against oxidative protein damage by interacting with hemoglobin.

Backgrounds Recently, we have considered two new findings in genetics of 21-hydroxylase deficiency with great interested: the existence of rare RCCX trimodular haplotypes, where the CYP21A2 like-gene downstream of the TNXA gene carries from one to six pseudogene mutations, and population specific allelic frequencies of wild-type CYP21A2 loci in the CYP21A1P pseudogene. Both these events represent a further complication in CYP21A2 genetics. Therefore, the choice of the molecular protocol becomes a crucial point when genetic analysis is required. In this regard, we must consider that the literature is still lacking consistent data on the Italian population. For this reason, we report genetic results obtained on 375 healthy individuals of Italian origin. Methods Different genetic protocols were compared and novel molecular strategies were performed. Results In our group, only two known haplotypes were identified. In addition, specific allelic frequencies of CYP21A2 wild-type loci in the pseudogene have been established. Conclusions Based on our results, we can affirm that the employment of different molecular methods is necessary to ensure a correct CYP21A2 genotyping. In order to avoid mistakes both in patient diagnosis and/or in risk evaluation of the relatives, each case should be investigated in function of a careful clinical evaluation and the molecular test should be performed in specialized centres.

Amyloid beta (1-42) peptide is considered responsible for the formation of senile plaques that accumulate in the brain of patients with Alzheimer’s disease (AD). In the past years considerable attention has been focused on identifying new protective substances that prevent or almost retard the appearance of amyloid beta (1-42)-related neurotoxic effects. In this study, human neuroblastoma cells (IMR-32) was used as system model to evaluate the protective role of S100b, a neurotrophic factor and neuronal survival protein, that is highly expressed by reactive astrocytes in close vicinity of beta-amyloid deposits, against amyloid beta (1-42)-dependent toxicity. Our results show that at nanomolar concentrations, S100b protects cells against Aβmediated cytotoxicity, as assessed by MTS vitality test. The protective mechanism seems to be related to the effect on bcl-2 (an anti-apoptotic gene) expression, which is highly down-regulated by amyloid beta (1-42) treatment, while resulted more expressed in the presence of S100b. On the contrary, Bax, a proapoptotic gene, resulted down-regulated by the treatment with S100 compared with the results obtained in the presence of amyloid beta (1-42) peptide. However, at micromolar doses, S100b is toxic for IMR-32 cells and its toxicity adds to that of the Aβpeptide, suggesting that additional molecular mechanisms may be involved in theneurotoxic process.

A CE-tandem MS method was optimised and validated for selective and specific determination of LVV- and VV-hemorphin-7 peptides in cerebrospinal fluid. These two small peptides originate from haemoglobin beta chains. They possess relevant biological activity and recently a potential biomarker role in posterior cranial fossa paediatric brain tumour disease was evidenced. The separation was optimised using formic acid as background electrolyte and a water/methanol mixture, containing 0.1% (v/v) formic acid, as sheath liquid. The two peptides, differing in only one amino acid of the sequence at the N-terminal side were baseline separated in less than 15 min. The method allowed a very reduced and rapid sample pretreatment and was successfully applied to hemorphins determination in patient samples without matrix interferences. The method successfully passed bioanalytical validation showing linearity, accuracy and precision data on cerebrospinal fluid matrix within the acceptable values. The analysis of cerebrospinal fluid of patients affected by different posterior cranial fossa tumour forms confirmed our previous findings showing the absence of hemorphins in the pre-surgical cerebrospinal fluid and their presence in the post-ones and controls. The present method saves costs and time due to capillary electrophoresis miniaturisation and to the absence of chromatographic column and gradient elution and allows numerous injections per sample starting from few microlitres of cerebrospinal fluid. (C) 2012 Elsevier B.V. All rights reserved.

Until few years ago, many studies of Alzheimer’s disease investigated the effects of this syndrome in the central nervous system. Only recently, the detection of amyloid beta peptide (Ab) in the blood has evidenced the necessity to extend studies on extraneuronal cells, particularly on erythrocytes. Ab is also present in brain capillaries, where it interacts with the erythrocytes, inducing several metabolic and functional alterations. Recently, functionally active endothelial type nitric oxide synthase (eNOS) was discovered in human erythrocytes. The goal of the present study was to evidence the effect of Ab on erythrocyte eNOS. We found that Ab following to 24-h exposure causes a decrease in the immune staining of erythrocyte eNOS. Concurrently, Ab alters erythrocyte cell morphology, decreases nitrites and nitrates levels, and affects membrane acetylcholinesterase activity. Propidium, an acetylcholinesterase inhibitor, was able to reverse the effects elicited by Ab. These events could contribute to the vascular alterations associated with Alzheimer’s disease disease. Copyright © 2012 John Wiley & Sons, Ltd. key words—amyloid beta peptide; erythrocytes; nitric oxide synthase; acetylcholinesterase; NO metabolites

In the present paper we have updated the G6PD mutations database, including all the last discovered G6PD genetic variants. We underline that the last database has been published by Vulliamy et al. [1] who analytically reported 140 G6PD mutations: along with Vulliamy's database, there are two main sites, such as http://202.120.189.88/mutdb/ and www.LOVD.nl/MR, where almost all G6PD mutations can be found. Compared to the previous mutation reports, in our paper we have included for each mutation some additional information, such as: the secondary structure and the enzyme 3D position involving by mutation, the creation or abolition of a restriction site (with the enzyme involved) and the conservation score associated with each amino acid position. The mutations reported in the present tab have been divided according to the gene's region involved (coding and non-coding) and mutations affecting the coding region in: single, multiple (at least with two bases involved) and deletion. We underline that for the listed mutations, reported in italic, literature doesn't provide all the biochemical or bio-molecular information or the research data. Finally, for the "old" mutations, we tried to verify features previously reported and, when subsequently modified, we updated the specific information using the latest literature data.

The aim of this research was to investigate the effect of caffeine on band 3 (the anion exchanger protein), haemoglobin function, caspase 3 activation and glucose-6-phosphate metabolism during the oxygenation–deoxygenation cycle in human red blood cells. A particular attention has been given to the antioxidant activity by using in vitro antioxidant models. Caffeine crosses the erythrocyte membrane and interacts with the two extreme conformational states of haemoglobin (the T and the R-state within the framework of the simple two states allosteric model) with different binding affinities. By promoting the high affinity state (R-state), the caffeine–haemoglobin interaction does enhance the pentose phosphate pathway. This is of benefit for red blood cells since it leads to an increase of NADPH availability. Moreover, caffeine effect on band 3, mediated by haemoglobin, results in an extreme increase of the anion exchange, particularly in oxygenated erythrocytes. This enhances the transport of the endogenously produced CO2 thereby avoiding the production of dangerous secondary radicals (carbonate and nitrogen dioxide) which are harmful to the cellular membrane. Furthermore caffeine destabilizes the haeme–protein interactions within the haemoglobin molecule and triggers the production of superoxide and met-haemoglobin. However this damaging effect is almost balanced by the surprising scavenger action of the alkaloid with respect to the hydroxyl radical. These experimental findings are supported by in silico docking and molecular dynamics studies and by what we may call the “caspase silence”; in fact, there is no evidence of any caspase 3 activity enhancement; this is likely due to the promotion of positive metabolic conditions which result in an increase of the cellular reducing power.

Dystroglycan (DG) is a cell surface receptor which is composed of two subunits that interact noncovalently, namely alpha- and beta-DG. In skeletal muscle, DG is the central component of the dystrophin-glycoprotein complex (DGC) that anchors the actin cytoskeleton to the extracellular matrix. To date only the three-dimensional structure of the N-terminal region of alpha-DG has been solved by X-ray crystallography. To expand such a structural analysis, a theoretical molecular model of the murine alpha-DG C-terminal region was built based on folding recognition/threading techniques. Although there is no a significant (

Palytoxin (PTX) is classified as one of the most powerful marine biotoxins (of high molecular weight and no protein origin) because it is able to interact strongly with important cellular structures influencing their function in different biological processes. This study of the effects of PTX on red blood cells (RBC) extends the knowledge about its toxicity, which concerns not only the well-known action on Na(+)/K(+)-ATPase but also band 3 protein (B3 or AE1), the role of which is essential for anion transport and for the structure, function, and metabolic integrity of the erythrocyte. The effects of PTX on RBC can be summarized as follows: it alters the anionic flux and seriously compromises not only CO(2) transport but also the metabolic modulation centered on the oxy-deoxy cycle of hemoglobin; it stabilizes the plasma membrane by preventing lipid peroxidation; and its effect does not lead to activation of caspases 3 and 8. From what is reported in steps 2 and 3, and on the basis of the results obtained on hemolysis, methemoglobin levels, and phosphatase activity, an increase of the reducing power of the erythrocytes (RBC) in the presence of PTX clearly emerges. The results have enabled us to outline some metabolic adaptations induced in the RBC by PTX.

Glucose-6-phosphate dehydrogenase (G6PD), an X-linked hereditary deficiency, is the most common of all clinically significant enzyme defects. While many drugs are responsible for haemolytic anaemia in G6PD-deficient patients, acetaminophen's imputability is still under debate, although an overdose of this drug can provoke acute haemolytic events. We report a case of a Philipino child carrying the G6PD-Vanua Lava mutation with acute haemolytic crisis related to infection in progress and acetaminophen's administration. Fever and concomitant infection, through an increment of erythrocyte glutathione depletion, sensitized the infant to the haemolytic event. In this condition, acetaminophen (or paracetamol [PCM]) was capable of inducing a haemolytic crisis in our G6PD-deficient patient although administered under standard conditions. PCM seems to have induced the haemolytic event, probably by the alteration of its catabolism due to dehydration and fever. The enzymatic G6PD instability associated to the presence of the G6PD-Vanua Lava mutation could have led to an increment of red blood cells' sensitivity to lysis; hence, it is possible that PCM toxicity may also be due to the presence of this particular mutation. Finally, we propose a new biochemical classification of this G6PD variant.

Objective: Aim of the present study was to evaluate the cytotoxicity of Real Seal 1 compared to other commercially available endodontic filling materials: Real Seal (SybronEndo, Orange, CA, USA) and Thermafil (Tulsa Dental, Tulsa, OK, USA). Material and Methods: Periodontal ligament cells from healthy patients were cultured. The eluate of Real Seal 1 TM (RS1), Real Seal (RS) and Thermafil (TF) samples was used for the cells viability tests, both diluted (50%) or undiluted (100%). Incubation of the specimens was performed in culture medium for 24 h, 48 h and 72 h at 37 °C under sterile conditions. The cellular mortality was evaluated by MTT test. Results were statistically analysed and the statistical significance was set at p< 0.05. Results: None of the studied materials showed toxic effects during the period of observation (0 -72 h) when compared to the control group. Only RS induced a very modest increase in cell mortality (about 3% at both concentrations used, during the first 24 hours), when increasing the incubation time, however, only the lower concentration continued to show modest toxicity. Conclusions: Results of the present study showed that all tested materials did not exhibit cytotoxic effects when compared to the control group.

Cardiovascular disease (CVD) is the leading cause of death in Western societies and accounts for up to a third of all deaths worldwide. In comparison to the Northern European or other Western countries, the Mediterranean area has lower rates of mortality from cardiovascular diseases and cancer, and this is attributed, at least in part, to the so-called Mediterranean diet, which is rich in plantderived bioactive phytochemicals. Identification of the active constituents of the Mediterranean diet is therefore crucial to the formulation of appropriate dietary guidelines. Lycopene is a natural carotenoid found in tomato, an essential component of the Mediterranean diet, which, although belonging to the carotenoid family, does not have pro-vitamin A activity but many other biochemical functions as an antioxidant scavenger, hypolipaemic agent, inhibitor of pro-inflammatory and pro-thrombotic factors, thus potentially of benefit in CVD. In particular, the review intends to conduct a systematic analysis of the literature (epidemiological studies and interventional trials) in order to critically evaluate the association between lycopene (or tomato products) supplementation and cardiovascular diseases and/or cardiovascular disease risk factors progression, and to prepare provision of evidence-based guidelines for patients and clinicians. Several reports have appeared in support of the role of lycopene in the prevention of CVD, mostly based on epidemiological studies showing a dose-response relationship between lycopene and CVD. A less clear and more complex picture emerges from the interventional trials, where several works have reported conflicting results. Although many aspects of lycopene in vivo metabolism, functions and clinical indications remain to be clarified, supplementation of low doses of lycopene has been already suggested as a preventive measure for contrasting and ameliorating many aspects of CVD.

The dissection of phylogenetic and environmental components in biological evolution is one of the main themes of general biology. Here we propose an approach to this theme relying upon the comparison between a phylogenetic oriented metrics spanning the hemoglobin beta chains of different fishes and a more physiologically oriented metrics defining the same sequences in terms of the dynamical features of their hydrophobic distributions. By analyzing the set of sequences more similar to the Gadus morhua (Atlantic cod) hemoglobin beta chain, we were able to give a proof of concept of the possibility to discriminate the phylogenetic and environmental (evolutive convergence) components by the comparative analysis of the Clustal W (phylogenetics first) and Recurrence Quantification Analysis (physiology first) metrics in which the sequences were embedded. The use of a molecular system like hemoglobin playing a crucial role in fishes adaptation to environmental cues allowed us to span different levels of biological variability by means of the same paradigm. Starting from the reconstruction of the general taxonomy of vertebrate groups we went down to the exploitation of the peculiar role played by Met55Val and Lys62Ala polymorphisms in the beta1 hemoglobin chain of the Atlantic cod able to influence the geographical distribution of its various stocks.

A CE ion trap tandem MS method was optimised for the analysis of arginine, monomethyl- and (symmetric and asymmetric) dimethylarginines in human plasma after a very reduced sample pretreatment step involving a simple protein precipitation with ACN. Several parameters affecting the analytes MS ionization and the capillary electrophoretic separation were carefully studied and optimised. The complete separation of arginine, monomethylarginine and symmetric and asymmetric dimethylarginine was obtained in formic acid BGE in short analysis time with high specificity due to MS(2) detection of specific analytes fragments. In order to achieve the detection sensitivity suitable for the analysis of asymmetric and symmetric dimethylarginine in human plasma, the field amplified sample injection was applied. Due to stacking effects, this methodology allowed to operate a consistent on-line preconcentration of the analytes before running the electrophoresis. The method was validated for linearity, repeatability, recovery and accuracy and applied to the quantitative analysis of arginine, monomethyl- and dimethylarginines in human plasma of healthy subjects.

Background: 5,10-methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of homocysteine metabolism. Hyperhomocysteinemia, frequently associated with cardiovascular disease, congenital defects, pregnancy complications, and cancer, can derive from MTHFR impaired activity, depending on some common mutations such as C677T and A1298C. In this study we evaluated the genotype and the allele frequencies of the polymorphisms C677T and A1298C of MTHFR in 104 newborns from central-southern Italy. Materials and Methods: C677T and A1298C polymorphisms were analyzed by polymerase chain reaction (PCR-RFLP). Results: The 677TT and 1298CC genotypes showed a frequency of 25.0% and 12.5% respectively; the frequency of the mutated alleles was 50.5% for 677T and 32.7% for 1298C. The analysis of the genotype combination showed a significant negative linkage disequilibrium. Conclusions: The results showed interesting new data concerning the higher prevalence of the 1298CC genotype; furthermore, the frequency of the 677TT genotype could induce consideration of a nutrition policy based on folic acid supplementation.

Background More than 90% of all cases of congenital adrenal hyperplasia (CAH) result from steroid 21-hydroxylase gene (CYP21A2) mutations. Most of these mutations result from intergenic recombinations between CYP21A2 and closely linked CYP21A1P pseudogene. Rare mutations not generated by gene conversion account for 5-10% of 21-hydroxylase deficiency alleles. Objective Functional analysis of two novel CYP21A2 missense mutations (p.R224W and p.D407N) was performed. Design Our study was composed of two Italian patients suffering from a very mild form of nonclassic CAH (NC-CAH). To assay the enzymatic activity of mutants, the in vitro analysis was performed in transiently transfected COS-1 cells. Results The residual activities obtained for p.R224W and p.D407N mutants allow their classification as NC-CAH mutations. These results correlate with the rate of severity of the patients' disease. Conclusions In this paper, we report two novel CYP21A2 mutations in two Italian individuals affected by 21-hydroxylase deficiency. Based on the functional in vitro analysis we can classify these mutations as NC-CAH variants.

The interaction between a-dystroglycan (alpha-DG) and beta-dystroglycan (beta-DG), the two constituent subunits of the adhesion complex dystroglycan, is crucial in maintaining the integrity of the dystrophin-glycoprotein complex. The importance of the alpha-beta interface can be seen in the skeletal muscle of humans affected by severe conditions, such as Duchenne muscular dystrophy, where the alpha-beta interaction can be secondarily weakened or completely lost, causing sarcolemmal instability and muscular necrosis. The reciprocal binding epitopes of the two subunits reside within the C-terminus of alpha-DG and the ectodomain of beta-DG. As no ultimate structural data are yet available on the alpha-beta interface, site-directed mutagenesis was used to identify which specific amino acids are involved in the interaction. A previous alanine-scanning analysis of the recombinant beta-DG ectodomain allowed the identification of two phenylalanines important for alpha-DG binding, namely F692 and F718. In this article, similar experiments performed on the alpha-DG C-terminal domain pinpointed two residues, G563 and P565, as possible binding counterparts of the two beta-DG phenylalanines. In 293-Ebna cells, the introduction of alanine residues instead of F692, F718, G563 and P565 prevented the cleavage of the DG precursor that liberates alpha- and beta-DG, generating a pre-DG of about 160 kDa. This uncleaved pre-DG tetramutant is properly targeted at the cell membrane, is partially glycosylated and still binds laminin in pull-down assays. These data reinforce the notion that DG processing and its membrane targeting are two independent processes, and shed new light on the molecular mechanism that drives the maturation of the DG precursor.

Structural analysis of the hemoglobin (Hb) system of Delphinus delphis revealed a high globin multiplicity: HPLC-electrospray ionization-mass spectrometry (ESI-MS) analysis evidenced three major beta (beta1 16,022 Da, beta2 16,036 Da, beta3 16,036 Da, labeled according to their progressive elution times) and two major alpha globins (alpha1 15,345 Da, alpha2 15,329 Da). ESI-tandem mass and nucleotide sequence analyses showed that beta2 globin differs from beta1 for the substitution Val126 --Leu, while beta3 globin differs from beta2 for the isobaric substitution Lys65 --Gln. The alpha2 globin differs from the alpha1 for the substitution Ser15 --Ala. Anion-exchange chromatography allowed the separation of two Hb fractions and HPLC-ESI-MS analysis revealed that the fraction with higher pI (HbI) contained beta1, beta2 and both the alpha globins, and the fraction with lower pI (HbII) contained beta3 and both the alpha globins. Both D. delphis Hb fractions displayed a lower intrinsic oxygen affinity, a decreased effect of 2,3-BPG and a reduced cooperativity with respect to human HbA(0), with HbII showing the more pronounced differences. With respect to HbA(0), either the substitution Probeta5 --Gly or the Probeta5 --Ala is present in all the cetacean beta globins sequenced so far, and it has been hypothesized that position 5 of beta globins may have a role in the interaction with 2,3-BPG. Regarding the particularly lowered cooperativity of HbII, it is interesting to observe that the variant human HbA, characterized by the substitution Lysbeta65 --Gln (HbJ-Cairo) has a decreased cooperativity with respect to HbA(0).

Glucose-6-phosphate dehydrogenase (G6PD), a X-linked hereditary deficiency, is one of most common clinically significant enzyme defects. Despite its largely known role in acute and life-threatening haemolytic crises, G6PD deficiency may be also associated with neonatal jaundice that, when severe and untreated, may lead to the potential of bilirubin encephalopathy. A prolonged neonatal jaundice was found to be associated with a rare G6PD mutation (c.383T>G; p.L128R), the latter simply annotated in literature database. In this article, we clinically and phenotipically describe a case of an Italian neonate carrying the c.383T>G G6PD mutation. Finally, we named this variant "G6PD Salerno."

Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most common defect of red blood cells. Although some different laboratory techniques or methods are employed for the biochemical screening, a strict relationship between biochemists, clinicians, and molecular biologists is necessary for a definitive diagnosis. This article represents an overview on the current laboratory tests finalized to the screening or to the definitive diagnosis of G6PD-deficiency, underlying the problems regarding the biochemical and molecular identification of heterozygote females other than those regarding the standardization of the clinical and laboratory diagnostic procedures. Finally, this review is aimed to give a flow-chart for the complete diagnostic approach of G6PD-deficiency.

Capillary electrophoresis (CE) coupled to tandem mass spectrometry was applied to the chiral separation of baclofen using sulfobutylether-β-cyclodextrin chiral selector in partial filling counter current mode. On-line UV detection was simultaneously used. Method optimization was performed by studying the effect of cyclodextrin and BGE concentration as well as sheath liquid composition on analyte migration time and enantiomeric resolution. The cyclodextrin showed stereoselective complexation towards baclofen enantiomers, allowing chiral resolution at low concentration. The CE capillary protrusion from the ESI needle relevantly affected the chiral resolution and the analyte migration time. Complete enantiomeric separation was obtained by using 0.25 M formic acid BGE containing 1.75 mM of chiral selector and water/methanol (30:70, v/v) 3% formic acid as sheath liquid. The method exhibited a LOD of 0.1 μg/mL (racemic concentration) in MS3 product ion scan mode of detection and was applied to the analysis of racemic baclofen in pharmaceutical formulations.

Arthropod hemocyanins transport and store oxygen and are composed of six subunits, or multiples thereof depending on the species. Palinurus gilchristi hemocyanin is found only as 1 x 6-mers, as normally occurs in spiny lobsters. An alkaline pH and removal of calcium ions induce a wholly reversible dissociation into monomers. The oxygen-binding properties of 1 x 6-meric hemocyanin from P. gilchristi were investigated with respect to pH and modulating effect exerted by calcium, lactate and urate. The oxygen affinity was highly affected by pH in the presence of calcium ions, while in its absence the Bohr coefficient became 60% lower. The protein is insensitive to lactate, but affected by urate which markedly increased hemocyanin-oxygen affinity, acting as the physiological major positive effector. Calcium ions decrease oxygen affinity at low concentration range (0-1 mM), while as concentration becomes higher than 100 mM, the oxygen affinity increases, indicating the presence of two independent types of calcium-binding sites with high and low affinity, respectively. The previous hypothesis, that the presence of high-affinity binding sites in addition to low affinity ones could be a characteristic feature of Palinuran hemocyanins, has been tested by analyzing, with respect to calcium-hemocyanin interaction, three other species belonging to Palinura.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most well-known human genetic defects, identified in more than 400 million individuals in the world. To date, no commercial kits are available for the mutation screening of this disease. Seventy G6PD-deficient Italian individuals admitted to the Laboratory of Clinical Molecular Biology of Hospital "Agostino Gemelli" of Rome were screened for the most frequent Italian mutations, by means of allele-specific PCR, followed by restriction fragment length electrophoresis. The present study compares two techniques for the identification of restriction patterns: agarose gel electrophoresis versus Experion system. When the first screening was negative, the entire G6PD gene was sequenced using the ABI 3100 Avant Instrumentation. The G6PD variants identified and their frequencies were the following: G6PD Mediterranean (75.7%), G6PD Seattle (7.1%), G6PD A(-) 202 + 376 (7.1%), and G6PD Cassano (2.8%). In addition, we identified by direct sequencing two new mutations, namely Buenos Aires and Rignano. With the Experion method, the size band determination was more accurate than that obtained by gel electrophoresis. The Experion system resulted as a valid, easy, and reproducible diagnostic method for the screening of G6PD mutation as compared with the agarose electrophoretic analysis.

The recent development of compounds that induce cell differentiation in various types of cancer cells has enabled the molecular mechanisms governing this kind of induced cancer regression to be investigated. Moreover, this approach to investigating the pathophysiology of neoplasia represents a promising experimental model for proteomic analysis of cancer cells. Modulating neoplastic cell differentiation grade may reveal cytodifferentiation-related protein expression changes, and doing so in vitro has the advantage of less biological variation. Hence, this analysis brings attention to molecular targets of the so-called differentiating factors (i.e., retinoids, hybrid polar compounds, tyrosine kinase inhibitors, etc.) as well as proteins that are frequently associated with differentiation/dedifferentiation processes. The in vitro study of these proteins and of their pathogenetic roles in cancer may ultimately result in the discovery of cancer biomarkers with diagnostic, prognostic, and therapeutic applications.

Resveratrol is widely known as an antioxidant and anti-inflammatory molecule. The present study first reports the effects of trans-δ-viniferin (TVN), a dimer of resveratrol, on human erythrocytes. The antioxidant activity of TVN was tested using in vitro model systems such as hydroxy radical scavenging, DPPH and lipid peroxidation. In addition we have examined the influence of the 15R,22R- and 15S,22S-enantiomers (abbreviated R,R-TVN, and S,S-TVN, respectively) on anion transport, ATP release, caspase 3 activation. Given that hemoglobin (Hb) redox reactions are the major source of RBC oxidative stress, we also explored the effects of TVN on hemoglobin function. TVN showed moderate antioxidant properties and good protective activity from hemoglobin oxidation. Potential binding sites of R,R-TVN and S,S-TVN with oxy- and deoxy-Hb were also investigated through an extensive in silico docking approach and molecular dynamics calculations. The whole molecular modeling studies indicate that binding of R,R-TVN and S,S-TVN to Hb lacks of specific ligand-target interactions. This is the first report on the biological activity of the individual enantiomers of a resveratrol-related dimer.

Purpose: The squamous cell carcinoma (SCC) antigen is still considered the most accurate serologic tumor marker in cervical carcinoma. We assessed the contribution of the SCC assay to the detection of recurrences in patients treated with radiotherapy. Methods and Materials: The pattern of recurrence and follow-up data were prospectively recorded for 135 patients. Of the 135 patients, 103 (76.3%) had primary cervical carcinoma and 32 (23.7%) had already experienced disease recurrence that had been successfully treated with surgery (n = 2), surgery plus radiotherapy (n = 2), radiotherapy (n = 5), or concomitant chemoradiotherapy (n = 23). The follow-up evaluations (chest X-ray, abdominopelvic magnetic resonance imaging, gynecologic examination with colposcopy, Papanicolaou smear, and SCC assay) were performed at 6-month intervals; the evaluation was done earlier if recurrent disease was suspected. The median follow-up time was 29 months (range, 6-131). The SCC serum levels were assayed, and a cost analysis was done. Results: A total of 481 SCC determinations were performed. Of the 135 patients, 43 (31.8%) experienced disease recurrence. The SCC levels were higher in those with recurrent disease than in the disease-free patients. Elevation of SCC was documented in 34 (79.1% sensitivity) of 43 recurrences before symptoms appeared. Of the 38 patients with serum SCC elevation, 34 developed a recurrence (positive predictive value, 89.5%). Of the 97 patients with negative SCC serum levels, 88 had negative findings at the clinicoradiologic evaluation (negative predictive value, 90.7%). A simplified approach (SCC plus gynecologic examination) was evaluated. Compared with the complete follow-up program, the rate of missed recurrence was 2.2%. The total projected cost per patient for 5 years of follow-up for the simplified procedure was approximately 12.2-fold lower than the standard approach. Conclusions: Our results have shown that a simplified diagnostic approach, including the SCC assay and gynecologic examination, can detect a high rate of recurrence from cervical cancer, with a very favorable cost-effective profile. © 2007 Elsevier Inc. All rights reserved.

Objectives: To analyze a possible association between glutathione-S-transferase T1 (GSTM1) and/or glutathione-S-transferase M1 (GSTM1) polymorphisms and chronic or aggressive forms of periodontitis in a Caucasian ethnic group. Design and methods: Sixty-nine chronic, 14 aggressive periodontitis and 61 controls, deeply analyzed from a clinical point of view, were studied for their GSTM1 and GSTT1 polymorphisms by allelic specific PCR techniques. As a second control group, 64 blood donors were also included. Results: A significant association was found between GSTM1-null genotype and both chronic and aggressive periodontitis. The aggressive forms were associated with the double null GSTM1 and GSTT1 combination. These results were independent of the patients' age, gender, hygienic habits and smoke (evaluated as tobacco smoking yes/no, cigarettes/day and pack years) as confirmed by multivariate logistic regression analysis. Conclusions: The GSTM1-null variant is statistically associated with the two forms of periodontitis, while the aggressive one also presents a second null variant: GSTT1.

Hemolysate from white stork displayed a single hemoglobin component, thus resulting into two bands and two globin peaks in dissociating PAGE and reversed phase-HPLC, respectively. Stripped hemoglobin showed an oxygen affinity higher than that of human HbA, a small Bohr effect, and a cooperative oxygen binding. A small decrease of oxygen affinity, of the same extent in all the pH range examined, was observed by addition of chloride, thus indicating an unusual chloride-independent Bohr effect (DeltalogP50/Deltalog pH=-0.24). Saturating amounts of inositol hexakisphosphate, largely decreased hemoglobin-oxygen affinity (DeltalogP(50)=1.17 at pH 7.0), and increased the extent of its Bohr effect (DeltalogP50/DeltalogpH=-0.45). The phosphate binding curve allowed to measure a very high overall binding constant (K=1.18 x 10(5) M(-1)). The effect of temperature on the oxygen affinity was measured, and the enthalpy change of oxygenation resulted almost independent on pH. Structural-functional relationships are discussed by considering some amino acid residues situated at alpha1/beta1 and alpha1/beta2 interfaces, such as alpha38 and alpha89 positions. The presence of only one hemoglobin component, a rare event among birds, and its functional properties have been related to the physiological oxygen requirements of this soaring migrant bird and to its technique of flight during migration.

Histatin 1 (His-1) derivatives showing serial mass increases of 80.0 +/- 0.1 Da were detected in human saliva by HPLC-ESI-MS. The same derivatives were also found in granules of submandibular glands and secretions of submandibular/sublingual glands, but not in granules and secretions of parotid glands. Only one phosphate group was present in His-1 and its derivatives, since treatment with alkaline phosphatase provided an 80.0 Da mass decrease. His-1 derivatives were almost completely transformed into His-1 by treatment with 1 M HCl at 100 degrees C, suggesting the presence of O-sulfotyrosine, which is more labile than phospho-Tyr to acidic hydrolysis. CE-MS analysis of pronase extensive digestion of derivatives confirmed the presence of sulfotyrosine. Derivatives were digested by trypsin, proteinase K, and protease V-8 and analyzed by different MS strategies. The results allowed locating sulfation on the last four tyrosines (Tyr 27, 30, 34, and 36). This study is the first report of the gland-specific sulfation of a salivary phosphopeptide in vivo.

A capillary electrophoresis apparatus was used as sampler for flow injection analysis (FIA) in tandem mass spectrometry of l -carnitine and its acetyl- and propionyl-metabolites in human plasma. The capillary electrophoresis instrument was coupled to the ion trap mass spectrometer by an electrospray ionization coaxial sheath liquid interface. The electrophoresis capillary introduced the sample directly into the source by applying a prolonged sample injection. The use of the capillary electrophoresis apparatus miniaturised the FIA procedure, substantially reducing the quantities of solvents and samples used, and allowed rapid automated sequential analyses. The method was optimised and validated using a dialyzed human plasma matrix. The plasma samples were analysed after a simple, rapid deproteinisation procedure with acetonitrile and diluted 70 times before direct injection into the mass spectrometer for product ion scan MS/MS analysis in positive ionisation. The total analysis time was 5 min, including capillary preconditioning and acquisition time (3 min). The method was sensitive, allowing the determination of l -, l -acetyl- and l -propionyl-carnitines at 140, 14 and 3.6 nM concentrations (injected values) corresponding to lower limit of quantitation values in plasma of 10, 1 and 0.25 μM, respectively. The method was processed for full validation and applied to the analysis of l -carnitine and its short chain derivatives in human plasma samples.

A 1 year follow-up investigation of salivary acidic proline-rich proteins (aPRPs) in preterm and at-term newborns using HPLC-ESI-IT-MS showed that (i) this class of proteins is constitutive rather than inducible, as it is still found in the oral cavity of preterm newborns from 180 days of postconception age (PCA); (ii) the expression of PRH-2 locus anticipates that of PRH-1, since Db isoforms are expressed some months after the PRP-1 and PRP-2 isoforms. The evaluation of the relative abundances of the different aPRPs isoforms and derivatives (differently phosphorylated and cleaved) as a function of PCA showed that (iii) the proteolytic enzymes generating truncated isoforms are also constitutive because they are fully active since 180 days of PCA; (iv) the kinase involved in aPRP phosphorylation is not fully mature in preterm newborns, but its activity increases with PCA, synchronizing with that of at-term newborns and reaching the adult levels at about 500-600 days of PCA, in concomitance with the beginning of deciduous dentition.

Objective: To determine the type of mutation in a patient with clinical diagnosis of suspected APRT deficiency. Design and methods: A 51-year-old male patient, with a clinical history of two prior episodes of renal colic with urinary stone excretion (reported as uric acid stones in the first episode and as calcium oxalate stones in the second), was admitted to the hospital with severe non-oliguric renal failure (1.06 mmol/L serum creatinine), severe hyponatremia (114 mmol/L Na+), metabolic acidosis (14 mmol/L HCO3−) and uricemia in the normal range. Abnormalities at renal scan and persistency of severe renal failure required to start haemodialysis. Results of renal biopsy prompted us to undertake a biochemical and molecular biological evaluation of the patient for suspected adenine phosphoribosyltransferase (APRT) deficiency. Results: HPLC analysis of serum and urine, for determining purine derivative profile, showed the pathological presence of adenine in both biological fluids (3.57 μmol/L and 7.11 μmol/mmol creatinine in serum and urine, respectively; not detectable in both fluids in healthy controls). APRT assay in a sample of patient hemolysate showed no detectable activity of the enzyme (25.56 ± 9.55 U/L red blood cells in control healthy subjects). Molecular biological analysis of the amplified APRT gene revealed that the patient harboured in exon 3 a homozygous 254 bp deletion–8 bp insertion, previously described only once in a compound heterozygote. Analysis of the patient family showed that heterozygotes for this APRT gene mutation, in spite of a 69% lower APRT enzymatic activity than that of healthy subjects, had no detectable adenine concentrations in both serum and urine. Conclusions: Results of the first patient harbouring the homozygous 254 bp deletion–8 bp insertion of the APRT gene strongly indicated that definitive diagnosis of APRT deficiency (often under or misdiagnosed) would require a combined clinical, biochemical and molecular biological evaluation.

Antineoplastic therapy with anthracyclines like doxorubicin (DOX) and daunorubicin (DNR) is limited by the possible development of a dose-related cardiomyopathy. Secondary alcohol metabolites like doxorubicinol (DOXol) and daunorubicinol (DNRol), formed by cytoplasmic two-electron reductases, have been implicated as potential mediators of anthracycline-induced cardiomyopathy. In the present study, we characterized the effects of 12 chalcones on the formation of anthracycline secondary alcohol metabolites by rabbit or human heart cytosol and compared them with those of quercetin and other flavonoids. Both chalcones and flavonoids inhibited DOXol or DNRol formation in isolated rabbit heart cytosol. Structure--activity relationships showed that inhibition by chalcones was determined primarily by the position of hydroxyl groups in their phenolic A and B rings. In particular, the presence of a hydroxyl group at C-4' in the A ring was an important determinant of the inhibitory activity of chalcones. Among chalcones, 2',4',2-trihydroxychalcone exhibited the highest inhibition of both DOXol and DRNol formation, but it proved less efficient than quercetin. Different results were obtained with isolated human heart cytosol: in the latter, 2',4',2-trihydroxychalcone and other hydroxychalcones inhibited both DOXol and DNRol formation, whereas quercetin and other flavonoids inhibited DNRol formation but failed to inhibit or slightly stimulated DOXol formation. These results identify chalcones as versatile inhibitors of the cytoplasmic reductases that convert anthracyclines to cardiotoxic secondary alcohol metabolites.

The amyloid β-peptide (AβP) is the major protein component of brain senile plaques in Alzheimer's disease. The redox state of methionine-35 residue plays a critical role in peptide neurotoxic actions. We used the fragment 31–35 of AβP [AβP(31–35)], containing a single methionine-35 residue (Met-35), to investigate the relationship between the oxidative state of Met-35 and neurotoxic and pro-apoptotic actions induced by the peptide; in rat cerebellar granule cells (CGC), we compared the effects of AβP(31–35), in which the Met-35 is present in the reduced state, with those of a modified peptide with oxidized Met-35 [AβP(31–35)Met-35OX], as well as an AβP-derivative with Met-35 substituted by norleucine [AβP(31–35)Nle-35]. AβP(31–35) induced a time-dependent decrease in cell viability. AβP(31–35)Met-35OX was significantly less potent, but still induced a significant decrease in cell viability compared to control. No toxic effects were observed after treatment with AβP(31–35)Nle-35. AβP(31–35) induced a 2-fold increase in bax mRNA levels after 4 h, whereas AβP(31–35)Met-35OX raised bax mRNA levels by 41% and AβP(31–35)Nle-35 had no effect. Finally, AβP(31–35) caused a 43% increase in caspase-3 activity after 24 h; AβP(31–35)Met-35OX caused only a 18% increase, and AβP(31–35)Nle-35 had no effect. These findings suggest that AβP(31–35)-induced neurodegeneration in CGC is mediated by a selective early increase in bax mRNA levels followed by delayed caspase-3 activation; the redox state of the single Met-35 residue is crucial in the occurrence and extent of the above phenomena.