Covid-19 pandemic and clinical activity stop: oral health study in a group of children.

AIM: Public and private health services, which provide both preventive and health promotion interventions, were forced to suddenly stop their activities to limit the risk of infections during the pandemic emergency. Oral health administration, including that of children, was affected by these planned medical service closures, from both therapeutic and preventive perspectives. This study aims to analyse the consequences, at the oral cavity level, of failures to treat patients of childhood age, considering the impact of carious pathology on quality of life and incorrect eating and oral hygiene habits, which may occur in this age group. METHODS: This is a cross-sectional, single-center, observational study. One hundred patients from the Odontostomatological University Center (C.O.U.) of Perugia were randomly enrolled. CONCLUSION: Oral health status of the examined sample is satisfactory overall, considering the clinic's interruption of treatments with the resulting long period of no follow-up and the emotional and economic stress generated by the pandemic condition for both the young patients and their caregivers.

Delayed recovery from severe acute respiratory syndrome coronavirus-2 related anosmia predicts incomplete olfactory restoration.

OBJECTIVE: This study aimed to assess the olfactory recovery rates and patterns in a cohort of coronavirus disease 2019 positive patients, and to investigate the clinical predictors of poor long-term olfactory restoration. METHODS: An observational retrospective study was conducted on 146 patients between September 2020 and January 2021 at a tertiary referral hospital. Coronavirus disease 2019 positive patients with olfactory dysfunction were sent a modified version of the COVID-19 Anosmia Reporting Tool for Clinicians via e-mail. RESULTS: The difference in median recovery time between complete recovery and incomplete or no recovery was statistically significant. On multivariate analysis, the only significant factor associated with incomplete or no recovery was anosmia duration. CONCLUSION: After a mean time of 5.6 months from severe acute respiratory syndrome coronavirus-2 infection, persistent olfactory disorders were self-reported in 36.7 per cent of patients. Complete recovery was more likely to occur within 15 days. Given the high prevalence of coronavirus disease 2019, a large number of patients are expected to suffer from long-term olfactory morbidity.

Epigenetically silenced miR-34b/c as a novel faecal-based screening marker for colorectal cancer.

BACKGROUND: MicroRNAs are tiny non-coding small endogenous RNAs that regulate gene expression by translational repression, mRNA cleavage and mRNA inhibition. The aim of this study was to investigate the hypermethylation of miR-34b/c and miR-148a in colorectal cancer, and correlate this data to clinicopathological features. We also aimed to evaluate the hypermethylation of miR-34b/c in faeces specimens as a novel non-invasive faecal-DNA-based screening marker. METHODS: The 5-aza-2'-deoxycytidine treatment and methylation-specific PCR were carried out to detect the hypermethylation of miR-34b/c and miR-148a. RESULTS: The miR-34b/c hypermethylation was found in 97.5% (79 out of 82) of primary colorectal tumours, P=0.0110. In 75% (21 out of 28) of faecal specimens we found a hypermethylation of miR-34b/c while only in 16% (2 out of 12) of high-grade dysplasia. In addition, miR-148a was found to be hypermethylated in 65% (51 out of 78) of colorectal tumour tissues with no significant correlation to clinicopathological features. However, a trend with female gender and advanced age was found, P=0.083. We also observed a trend to lower survival rate in patients with miR-148a hypermethylation with 10-year survival probability: 48 vs 65%, P=0.561. CONCLUSIONS: These findings show that aberrant hypermethylation of miR-34b/c could be an ideal class of early screening marker, whereas miR-148a could serve as a disease progression follow-up marker.

Cortical metabolic changes and clinical outcome in normal pressure hydrocephalus after ventriculoperitoneal shunt: Our preliminary results. / Cambios metabólicos corticales y resultado clínico en la hidrocefalia normotensiva después de la derivación ventrículo-peritoneal: nuestros resultados preliminares.

INTRODUCTION: Our objective was to evaluate the cortical metabolic changes and clinical outcome in patients affected by idiopathic normal pressure hydrocephalus (iNPH) after a placement of ventriculoperitoneal (VP) shunt. MATERIALS AND METHODS: 10 patients affected by suspected iNPH underwent a CSF hydrodynamics evaluation based on a lumbar infusion test (LIT). The main selection criterion for surgery was based on intracranial elasticity (IE)>0.30. All subjects with an IE>0.30 underwent a PET scan with 18 fluorodeoxiglucose (18F-FDG) at baseline (PET1) and 1 month after surgery (PET2). Furthermore, the same patients were submitted to clinical evaluation before and 1 month after surgery through neuropsychological tests and gait analysis. RESULTS: An overall number of 20 18F-FDG PET scans were performed in all the enrolled patients. As compared to PET1, PET2 showed an increase in glucose consumption in the left frontal and left parietal lobe in PET2 as compared to PET1 (P<.001). All the enrolled patients presented a significant increase in neuropsychological scores (i.e Frontal Assessment Battery and Montreal Cognitive Assessment) and have clinically improved at gait analysis. A significant correlation was found between the increase of cortical glucose consumption in the left parietal area and the cognitive improvement as detectable by neuropsychological assessment. CONCLUSIONS: Improvement in 18F FDG PET glucose metabolism could be considered a useful imaging marker for the assessment of iNPH response to VP shunting.

Melanoma-associated markers expression in blood: MUC-18 is associated with advanced stages in melanoma patients.

BACKGROUND: Multimarker reverse transcriptase-polymerase chain reaction (RT-PCR) was originally reported to reveal melanoma-associated mRNAs (MAMs) in melanoma cells but not in the peripheral blood of healthy individuals. OBJECTIVES: To evaluate the expression of MAMs in the peripheral blood of melanoma patients at different American Joint Committee on Cancer (AJCC) stages, and to correlate their presence with early and/or advanced stages of the disease. MATERIALS AND METHODS: One hundred blood samples of melanoma patients (AJCC I-IV) were analysed using multimarker RT-PCR to assess the co-expression of Tyr-OH, MART-1, MAGE-3, MUC-18/MCAM and p97. Patients were stratified into two disease categories: early and advanced stages. The former includes in situ and melanoma stages AJCC I-II, the latter AJCC III-IV. chi(2) and Fisher's exact tests were used to statistically evaluate the association between each MAM and disease categories. The recognized significant associations were subsequently resubmitted to univariate logistic regression. Furthermore, sensitivity and specificity were established. RESULTS: At least one MAM could be detected in 24% of our series. Tyr-OH was the most common marker (14%), followed by MUC-18 (12%), MART-1 (5%), MAGE-3 (4%) and p97 (3%). No significant association among Tyr-OH, MART-1, MAGE-3, p97 and disease stages were evidenced. Only MUC-18 was statistically associated (P < 0.009) with advanced stages alone or co-expressed with other MAMs. According to logistic regression univariate analysis, MUC-18 increases the probability (odds ratio: 33) being in advanced stages and the incidence of recurrences (95% CI 2.9-374). CONCLUSIONS: MUC-18 RT-PCR assay could be proposed as an adjunctive molecular method in the management of melanoma patients and is useful in the monitoring of study protocols.

The usefulness of the prognostic inflammatory and nutritional index (PINI) in a haemodialysis population.

BACKGROUND AND AIM: Protein-Energy Wasting and inflammation are the principal risk factors of haemodialysis complications. We evaluated the reliability of a simple and non expensive test, the Prognostic Inflammatory and Nutritional Index (PINI), for regular screening of maintenance haemodialysis (MHD) patients in order to detect early onset of inflammation and malnutrition. METHODS AND RESULTS: 121 adult patients on maintenance dialysis were followed up for 32 months and screened every 6 months for PINI, calculated as alpha1-Acid Glycoprotein (alpha1-AG)xC-Reactive Protein (CRP)/AlbuminxTransthyretin. PINI score < or =1 was considered normal. Patients were stratified according to their PINI score: 86 patients (71.66%) had a normal score, whereas 35 (28.33%) had PINI > or = 1. The latter also had higher CRP levels, despite no clinical evidence of inflammation at the time of enrolment. Survival in patients with normal PINI was similar to patients with normal CRP, while in patients with abnormal PINI it was significantly lower than in patients with low serum albumin (p<0.05) or elevated CRP (p<0.05). After follow-up, all surviving MHD patients with PINI > or = 1 had at least one cardiovascular event vs 2.5% of patients with PINI > or = 1. CONCLUSION: The assessment of PINI can reliably identify MHD patients at higher risk of mortality and morbidity even in the absence of overt Malnutrition-Inflammation Complex Syndrome (MICS). This simple test appears to be more sensitive and specific of the single components, and not expensive, so that it could be routinely used to identify patients with sub-clinical inflammation and/or malnutrition.

Stromelysin gene promoter polymorphism and common carotid geometry in diabetic subjects.

AIM: Stromelysin (MMP3), through its action on collagen and other matrix metalloproteinases, influences arterial wall remodeling. In healthy subjects, the 5A/6A polymorphism located in the promoter of the MMP3 gene is associated with common carotid remodeling, 6A/6A subjects having increased arterial diameter, wall thickness (intima-media thickness, IMT) and decreased wall shear stress (WSS). In the present study, we have investigated the influence of the 5A/6A polymorphism on common carotid remodeling in subjects with diabetes mellitus. METHODS: Diabetic subjects (N.=136) and age-matched healthy male controls (N.=101) have been studied. Common carotid diameter, IMT and flow velocity have been measured by echo-Doppler. Blood viscosity has been measured by a cone/plate viscometer. WSS has been calculated. RESULTS: Diabetic patients had increased common carotid diameter, IMT, and decreased flow velocity and WSS (all P<0.05), compared with controls. In controls, subjects homozygous for the 6A allele had increased diameter, IMT and decreased WSS. In diabetics, no difference was observed in vascular parameters among the three genotypes. CONCLUSION: The 5A/6A polymorphism of the MMP3 gene influences arterial remodeling of the common carotid artery in healthy subjects, but not in patients with diabetes mellitus. Therefore, the significance of the 5A/6A polymorphism as a marker of risk in this high cardiovascular risk population seems to be somehow blunted.

Missense mutations in the TGM2 gene encoding transglutaminase 2 are found in patients with early-onset type 2 diabetes. Mutation in brief no. 982. Online.

Transglutaminase 2 (TG2 or TGM2) is a multi-functional enzyme which catalyzes transamidation reactions or acts as a G-protein in intracellular signalling. Tgm2-/- Mice lacking TG2 activity are glucose intolerant and show impairment of insulin secretion, suggesting an important physiological role for TG2 in the pancreatic beta cell. We have previously described a TGM2 heterozygous missense mutation ((c.998A>G, p.N333S) in a 14 year-old patient with insulin-treated diabetes and in his diabetic father. The aim of this study was to further investigate the role of TG2 in early-onset type 2 diabetes. We analysed the TGM2 gene in 205 patients with clinically defined Maturity Onset Diabetes of the Young (MODY) or early-onset type 2 diabetes. We found two novel heterozygous mutations (c.989T>G, p.M330R; c.992T>A, p.I331N), which were not detected in 300 normoglycemic controls. All mutations were in residues which are located close to the catalytic site and impaired transamidating activity in vitro. Gene expression of TGM family genes and localization of TG2 in normal human pancreas indicated that TG2 is the only transglutaminase significantly expressed in human pancreatic islet cells. We conclude that reduced TG2 activity can contribute to disorders of glucose metabolism possibly via an impairment of insulin secretion.

[Feasibility study of biological monitoring of chemical agents by means of evaluation of the effects on their in vitro effects on the complement system]. / Studio di fattibilità del monitoraggio biologico di agenti chimici mediante valutazione degli effetti su sistemi multienzimatici del plasma coinvolti nella risposta immune.

Immunological methods for the study of the plasma complement system have been standardized in order to be good and reproducible indicators of some biological effects of the substances under study in in vitro experiments. The substances tested were not capable of interfering within 10 times the possible hypothetical plasma concentration reached in vivo with the function of the different reagents used in the study of complement. Five substances (Skin-ACGIH) have been studied for their effects on the complement system in vitro; four of them could be fully studied (allylic alcohol, cyclohexanone, phenol, dimethylacetamide). After this deep insight we can conclude that: 1. These substances are capable of interfering with the immune response through their complement activating capacity 2. These substances, throughout complement activation, can induce inflammation and reduction of important defensive functions that are complement mediated. 3. The results obtained encourage to study the complement system and especially CH50 in workers exposed to the selected substances in order to verify the possibility to enclose this test in the medical surveillance program.

Long-term usability and bio-integration of polyimide-based intra-neural stimulating electrodes.

Stimulation of peripheral nerves has transiently restored lost sensation and has the potential to alleviate motor deficits. However, incomplete characterization of the long-term usability and bio-integration of intra-neural implants has restricted their use for clinical applications. Here, we conducted a longitudinal assessment of the selectivity, stability, functionality, and biocompatibility of polyimide-based intra-neural implants that were inserted in the sciatic nerve of twenty-three healthy adult rats for up to six months. We found that the stimulation threshold and impedance of the electrodes increased moderately during the first four weeks after implantation, and then remained stable over the following five months. The time course of these adaptations correlated with the progressive development of a fibrotic capsule around the implants. The selectivity of the electrodes enabled the preferential recruitment of extensor and flexor muscles of the ankle. Despite the foreign body reaction, this selectivity remained stable over time. These functional properties supported the development of control algorithms that modulated the forces produced by ankle extensor and flexor muscles with high precision. The comprehensive characterization of the implant encapsulation revealed hyper-cellularity, increased microvascular density, Wallerian degeneration, and infiltration of macrophages within the endoneurial space early after implantation. Over time, the amount of macrophages markedly decreased, and a layer of multinucleated giant cells surrounded by a capsule of fibrotic tissue developed around the implant, causing an enlargement of the diameter of the nerve. However, the density of nerve fibers above and below the inserted implant remained unaffected. Upon removal of the implant, we did not detect alteration of skilled leg movements and only observed mild tissue reaction. Our study characterized the interplay between the development of foreign body responses and changes in the electrical properties of actively used intra-neural electrodes, highlighting functional stability of polyimide-based implants over more than six months. These results are essential for refining and validating these implants and open a realistic pathway for long-term clinical applications in humans.

C-Met/miR-130b axis as novel mechanism and biomarker for castration resistance state acquisition.

Although a significant subset of prostate tumors remain indolent during the entire life, the advanced forms are still one of the leading cause of cancer-related death. There are not reliable markers distinguishing indolent from aggressive forms. Here we highlighted a new molecular circuitry involving microRNA and coding genes promoting cancer progression and castration resistance. Our preclinical and clinical data demonstrated that c-Met activation increases miR-130b levels, inhibits androgen receptor expression, promotes cancer spreading and resistance to hormone ablation therapy. The relevance of these findings was confirmed on patients' samples and by in silico analysis on an independent patient cohort from Taylor's platform. Data suggest c-Met/miR-130b axis as a new prognostic marker for patients' risk assessment and as an indicator of therapy resistance. Our results propose new biomarkers for therapy decision-making in all phases of the pathology. Data may help identify high-risk patients to be treated with adjuvant therapy together with alternative cure for castration-resistant forms while facilitating the identification of possible patients candidates for anti-Met therapy. In addition, we demonstrated that it is possible to evaluate Met/miR-130b axis expression in exosomes isolated from peripheral blood of surgery candidates and advanced patients offering a new non-invasive tool for active surveillance and therapy monitoring.

Divergent phenotype of two siblings human leukocyte antigen identical, affected by nonclassical and classical congenital adrenal hyperplasia caused by 21-hydroxylase deficiency.

CONTEXT: Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders most often caused by enzyme 21-hydroxylase deficiency. Most mutations causing enzymatic deficiency are generated by recombinations between the active gene CYP21 and the pseudogene CYP21P. Only 1-2% of affected alleles result from spontaneous mutations. The phenotype of CAH varies greatly, usually classified as classical or nonclassical, depending on variable degree in 21-hydroxylase activity. Here we report a divergent phenotype of two human leukocyte antigen identical siblings, affected by nonclassical and classical CAH caused by 21-hydroxylase deficiency due to different genotype. PATIENTS AND METHODS: Using direct sequencing method and Southern blot, we studied two children (one male and one female), affected, respectively, by nonclassical and classical CAH and their parents. RESULTS: The mother was heterozygous for the Q318X mutation, and the father was heterozygous for the V281L mutation. The brother was a compound heterozygote for the mutations V281L and Q318X, whereas the proband was compound heterozygote for the Q318X mutation and a large conversion. The two children are human leukocyte antigen identical (A*02;B*14;DRB1*01/A*33;B*14;DRB1*03). CONCLUSIONS: Different phenotype of the proband is the result of compound heterozygosity for the maternal mutation Q318X and a de novo large conversion.

Interaction between 1,4-thiazine derivatives and D-amino-acid oxidase.

Aminoethylcysteine-ketimine (2H-1,4-thiazine-5,6-dihydro-3-carboxylic acid) strongly inhibits D-amino-acid oxidase (D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3). The inhibition is purely competitive (Ki = 3.3 X 10(-7) M). Aminoethylcysteine-ketimine modifies the visible spectrum of the enzyme: the absorption maxima of bound FAD shift from 375-455 nm to 385-445 nm with a definite shoulder at 465 nm; the appearance of a large absorption band centered at 750 nm may be due to a charge-transfer complex formation. The dissociation constant for the aminoethylcysteine-ketimine-enzyme complex, calculated by a photometric procedure (4 X 10(-7) M), is in good agreement with kinetic data. The dicarboxylic analogue of this inhibitor (lanthionine-ketimine) is ineffective in D-amino-acid oxidase inhibition and does not produce any spectral modification of the enzyme. These results confirm structural requirements for D-amino-acid oxidase inhibitor reported by other researchers. Ketimine reduced forms (thiomorpholine-2-carboxylic acid and thiomorpholine-2,6-dicarboxylic acid) are chemically synthesized and checked as D-amino-acid oxidase substrates: only thiomorpholine-2-carboxylic acid is oxidized to aminoethylcysteine-ketimine (Km = 2 X 10(-4) M).

Mechanism of spectrin degradation induced by phenylhydrazine in intact human erythrocytes.

The exposure of human erythrocytes to phenylhydrazine results in the degradation of both monomers of spectrin, a major cytoskeleton membrane protein. The degradative process, characterized by a loss of spectrin without the appearance of high-molecular-weight products, either under reducing conditions or not, is almost complete in 10 min when a 5% erythrocyte suspension is treated with 1 mM phenylhydrazine. Under these conditions, we found a loss of 62.3 and 48.5% for the alpha and beta monomer, respectively. A similar degradative extent was obtained when the membrane ghost plus cellular free extracts, were dialyzed, and the membrane ghost plus hemoglobin was exposed to 1 mM phenylhydrazine for 10 min. The presence of different proteinase inhibitors and effectors, such as EDTA, diethylenetriaminepentaacetic acid, EGTA, leupeptin, aprotinin, phenylmethylsulfonyl fluoride, pepstatin, Ca2+ and ATP plus Mg2+, in the membrane ghost plus cellular free extract system (undialyzed) did not affect the degree of the spectrin-degradative process induced by phenylhydrazine. In addition, a purified spectrin tetramer preparation exposed to 1 mM phenylhydrazine in the presence of hemoglobin was degraded to an extent comparable to that with intact cells. Our data suggest that the initial degradative step of spectrin induced by phenylhydrazine in intact erythrocytes may be ascribed more to a direct oxidative breakdown, probably involving main-chain cleavage and side-chain cleavage processes, than to an eventual proteolytic system.

Effects of L-carnitine and its acetate and propionate esters on the molecular dynamics of human erythrocyte membrane.

EPR and fluorescence probes were used in this study to define the effects of L-carnitine and its short-chain esters, acetyl-L-carnitine and propionyl-L-carnitine, on the natural fluidity gradient and molecular packing of phospholipid headgroups of erythrocyte membrane in intact cells. Purified erythrocyte suspensions, labeled with different stearic acid derivatives containing a stable doxyl radical ring at the C-5, C-7, C-12 and C-16, were incubated with 0.5-5 mM L-carnitine and its esters for 60 min at 37 degrees C and washed twice with an isosmotic buffer. A decrease in the order parameter, calculated from the EPR spectra of the 5-doxylstearic acid derivative, was observed at all the concentrations of propionyl-L-carnitine and the extent of the decrease was dose and temperature dependent. An increase of the chain length between the doxyl ring and the carboxylic group of the spin label, resulted in a much lower efficacy of propionyl-L-carnitine in decreasing the order parameter. Acetyl-L-carnitine also showed a significant effect of decreasing the molecular order but only at the lower temperatures of red cells labeled with 5-doxyl and treated with the highest concentration of the drug. L-Carnitine did not modify the molecular dynamics at all the temperatures and concentrations used in this study. L-Carnitine and its short-chain derivatives did not alter significantly membrane fluidity of deeper regions of the erythrocyte membrane, measured by means of the excimer/monomer fluorescence intensity ratio of pyrene incorporated into the membrane of intact erythrocytes. However, these compounds were all capable of loosening the molecular packing of the polar head of erythrocyte membrane phospholipids evaluated by the membrane binding fluorescence properties of merocyanine-540. The binding of the fluorescent probe decreased in the order propionyl-L-carnitine > acetyl-L-carnitine > L-carnitine. Our findings suggest that this category of compounds affect the molecular dynamics of a membrane bilayer region close to the glycerol backbone of phospholipids, which might be relevant for the expression of membrane functions.

Purification and characterization of a novel glutathione transferase from Serratia marcescens.

Four forms of glutathione transferase were resolved from the cytosol of Serratia marcescens CIP 6755 by GSH-affinity chromatography followed by isoelectric focusing. The major isoenzyme, named Sm-GST-7.3, is composed of two subunits each with a molecular mass of 22 kDa and has an isoelectric point at pH 7.3. Sm-GST-7.3, appears to be distinct from Pm-GST-6.0, previously characterized from Proteus mirabilis AF 2924 as indicated by its substrate specificity, immunological reactivity, subunit molecular mass as well as by its N-terminal amino acid sequence. None of the antisera raised against a number of human, rat and mouse GSTs cross-reacted with Sm-GST-7.3 indicating major structural differences between them and bacterial GST. This is further supported by the fact that the N-terminal sequence of Sm-GST-7.3 also differs significantly from the known sequences of mammalian GSTs of alpha, mu and pi classes. In addition, comparison with the known N-terminal amino acid sequences of helminth, plant and insect GSTs demonstrate that the latter enzymes are distantly related (less than 25% identity) to the Sm-GST-7.3. Immunoblotting experiments performed with antisera raised against Sm-GST-7.3 indicate that a GST immunologically identical to Sm-GST-7.3 is present in a number of other bacterial strains. All together the results obtained suggest that Sm-GST-7.3 is distinct from any known GST, including microbial and mammalian GSTs.

Investigation of the active site of human placenta glutathione transferase pi by means of a spin-labelled glutathione analogue.

A spin-labelled analogue of glutathione (sl-glutathione) has been used in order to characterize the active site of human placenta glutathione transferase pi. The sl-glutathione shows a competitive inhibition towards glutathione (Ki = 14 microM). Binding of sl-glutathione to the enzyme, followed by electron paramagnetic resonance spectroscopy, gives a Kd of 3 microM and two identical binding sites for dimeric unit. Inhibition of the enzyme, by modification of the Cys-47 residue, completely prevents the binding of sl-glutathione. The same results are obtained by monitoring the binding of glutathione by means of fluorescence spectroscopy. It is concluded that integrity of the thiolate of Cys-47 is necessary to maintain an active conformation of the enzyme able to efficiently bind glutathione into the active site.

Tryptophan environments in glutathione transferase of human placenta from temperature-dependent phosphorescence studies.

An investigation of the tryptophan emission properties of glutathione transferase from human placenta was conducted in order to characterize the environments of the two aromatic residues. The low-temperature phosphorescence spectra and temperature dependence of the phosphorescence quantum yield of the tryptophan residues revealed a difference in the chemical nature and dynamical structure of the surrounding protein matrix. Thus, one tryptophan residue seems to be deeply embedded within the polypeptide in a rigid weakly polar environment, characteristic of a beta-type secondary structure. The other is located in a more polar site, probably near the surface, in a rather flexible region of the macromolecule. At high temperature, the heterogeneity in the triplet lifetime of the internal residue attests to the presence of multiple conformers which are not in rapid equilibrium in the phosphorescence time scale. The anisotropy of the phosphorescence emission of glutathione transferase indicates that no energy transfer occurs between the two residues, and measurement of the rotational correlation time yields an hydrodynamic volume which is in good agreement with the molecular weight reported in the literature for the dimer.

Chemical modification of human placental glutathione transferase by pyridoxal 5'-phosphate.

Incubation of GST pi from human placenta with 8 mM PLP resulted in a rapid loss of activity during the first 10 min, concomitant with a Schiff base formation. This inactivation was probably due to the formation of a reversible adduct between PLP and the enzyme. After sodium borohydride treatment this adduct was reduced and stabilized. Stoichiometry and peptide isolation studies showed that three lysine residues were modified during reaction of GST and PLP. Protection of the enzyme against inactivation was achieved in the presence of 4 mM GSH suggesting that at least one lysyl residue is associated with the substrate binding site. Peptide mapping by digesting the enzyme with trypsin revealed that lysine shielded by GSH is Lys-127. Our results suggest that this residue may play an important role in enzymatic activity.

A study on the in vitro interaction between tyrosinase and glutathione S-transferase.

The actions of glutathione S-transferase and tyrosinase on the in vitro production of glutathionyl-3,4-dihydroxyphenylalanine and the dopachrome level in the presence of GSH and L-3,4-dihydroxyphenylalanine were studied. No clear evidence of complementarity between tyrosinase and glutathione S-transferase was observed; on the contrary, in the presence of glutathione S-transferase the glutathionyl-3,4-dihydroxyphenylalanine yield was lower than with tyrosinase only, as measured by HPLC. It is concluded that the spontaneous conjugation of GSH with dopaquinone should probably be high enough to scavenge the toxic quinone and to produce precursors for phaeomelanogenesis.