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Biomedical subjects

G Ricci

Publications and source records attributed to G Ricci.

At least 55 records · Page 3Linked to original sources
PubMed2002

Genetic diversity and technological properties of Streptococcus thermophilus strains isolated from dairy products.

AIMS: To evaluate the genetic diversity and the technological properties of 44 strains of Streptococcus thermophilus isolated from dairy products. Methods METHODS AND RESULTS: Strains were analysed for some relevant technological properties, i.e. exopolysaccharide (EPS) production, growth kinetic in skim milk medium, urease activity and galactose fermentation. The EPS production, determined by evaluating the colour of the colonies grown in ruthenium red milk agar, was observed in 50% of the analysed strains. Urease activity, determined by colorimetric and conductimetric methods, showed that 91% of the isolates, all except four, could hydrolyse urea. A conductimetric approach was also used for the evaluation of the overall metabolic behaviour in milk of Strep. thermophilus strains and the differences observed allowed grouping of the strains in seven different clusters. A total of 11 strains were able to produce acid in presence of galactose. Genetic diversity of Streptococcus thermophilus strains, evaluated by Random Amplified Polymorphic DNA fingerprinting (RAPD) and amplified epsC-D restriction analysis, allowed the identification of 21 different genotypes. CONCLUSIONS: Comparison between the genotypic and phenotypic data highlights an interesting correlation between some important technological properties and well-defined genotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: The genetic and technological characterization carried out on several Strep. thermophilus strains of dairy origin should expand the knowledge on this important lactic acid bacteria species and lead to a simple, rapid, and reliable identification of strains on the basis of well-defined biotechnological properties.

Animals↗
PubMed2002

Apoptosis in human sperm: its correlation with semen quality and the presence of leukocytes.

BACKGROUND: Apoptosis plays an important role in regulating spermatogenesis. However, the biological significance of apoptosis in ejaculated sperm is not yet clear. This study set out to investigate how apoptosis correlates with semen quality and the presence of seminal leukocytes. METHODS: Fifty-seven semen samples from the male partners of infertile couples were classified as normal or abnormal according to World Health Organization guidelines. Flow cytometry was used to evaluate sperm populations and seminal leukocytes. Preliminary flow cytometry analysis using 6-carboxyfluoresceindiacetate (6-CFDA), which identifies live cells, and propidium iodide (PI), which stains only dead cells, was performed in order to pinpoint the sperm region accurately. Having thus gated the sperm population, bivariate Annexin V/PI analysis was then carried out in order to measure the percentage of apoptotic and necrotic sperm and the apoptotic index (the ratio between apoptotic:live sperm). Leukocytes were counted by the standard peroxidase test and by flow cytometry using monoclonal antibody (mAb) anti-CD45 or anti-CD53. RESULTS: No significant differences in the apoptotic index and the percentage of live and apoptotic sperm were detected between the subjects with normal and abnormal semen. A significant inverse correlation between sperm concentration and the apoptotic index was observed only in the normal sperm group. There was no correlation between the concentration of leukocytes, detected either by peroxidase or by mAb anti-CD45 or anti-CD53, either with the percentage of apoptotic sperm or with the apoptotic index. In contrast, the ratio between CD45 positive leukocytes and sperm showed a significant correlation with the apoptotic index. A weaker correlation was found when leukocytes were counted by peroxidase, while no correlation was observed using mAb anti-CD53. CONCLUSIONS: Sperm apoptosis did not seem to be correlated with semen quality. In the absence of genito-urinary infection, one of the main functions of seminal leukocytes is probably to provide for the removal of apoptotic sperm.

Adult↗
PubMed2002

[Postpartum perineal assessment].

BACKGROUND: To evaluate the incidence of urogenital and anorectal dysfunctions during puerperium, verify the correlation between obstetric perineal damage observed during labour and puerperal symptoms, test the efficacy of tests to evaluate perineal function in pelvic floor dysfunctions consequent to vaginal birth. METHODS: A total of 693 consecutive puerperae were recruited two months after birth. All completed a clinical and anamnestic questionnaire and underwent clinical urogynecological examination, digital test, vaginal manometry and uroflowmetric evaluation of the voluntary capacity to interrupt micturition. From a statistical point of view, persistent urinary incontinence was identified by graphic representation on frequency tables and predictive tests, and statistical "kappa" was used to evaluate the correlation between perineal function tests. RESULTS: Stress urinary incontinence (15.1%) represents the most widespread symptom, a postpartum perineal inspection provides a sufficiently accurate estimate of posterior damage, but does not identify those puerperae who will develop urinary incontinence. None of the tests used was able to predict persistant urinary incontinence. CONCLUSIONS: Postpartum perineal inspection seems to be effective in predicting anorectal dysfunctions, but is not so useful for urethro-vesical disorders. None of the diagnostic methods examined allowed a sufficiently accurate selection of those puerperae at risk.

Fecal Incontinence↗
PubMed2001

Human glutathione transferase P1-1 and nitric oxide carriers; a new role for an old enzyme.

S-Nitrosoglutathione and the dinitrosyl-diglutathionyl iron complex are involved in the storage and transport of NO in biological systems. Their interactions with the human glutathione transferase P1-1 may reveal an additional physiological role for this enzyme. In the absence of GSH, S-nitrosoglutathione causes rapid and stable S-nitrosylation of both the Cys(47) and Cys(101) residues. Ion spray ionization-mass spectrometry ruled out the possibility of S-glutathionylation and confirms the occurrence of a poly-S-nitrosylation in GST P1-1. S-Nitrosylation of Cys(47) lowers the affinity 10-fold for GSH, but this negative effect is minimized by a half-site reactivity mechanism that protects one Cys(47)/dimer from nitrosylation. Thus, glutathione transferase P1-1, retaining most of its original activity, may act as a NO carrier protein when GSH depletion occurs in the cell. The dinitrosyl-diglutathionyl iron complex, which is formed by S-nitrosoglutathione decomposition in the presence of physiological concentrations of GSH and traces of ferrous ions, binds with extraordinary affinity to one active site of this dimeric enzyme (K(i) < 10(-12) m) and triggers negative cooperativity in the vacant subunit (K(i) = 10(-9) m). The complex bound to the enzyme is stable for hours, whereas in the free form and at low concentrations, its life time is only a few minutes. ESR and molecular modeling studies provide a reasonable explanation of this strong interaction, suggesting that Tyr(7) and enzyme-bound GSH could be involved in the coordination of the iron atom. All of the observed findings suggest that glutathione transferase P1-1, by means of an intersubunit communication, may act as a NO carrier under different cellular conditions while maintaining its well known detoxificating activity toward dangerous compounds.

Binding, Competitive↗
PubMed2001

Specific identification of Lactobacillus helveticus by PCR with pepC, pepN and htrA targeted primers.

Specific regions in three genes coding for aminopeptidases C and N, and a trypsin-like serine protease were selected as species-specific primer sequences for rapid and reliable identification of Lactobacillus helveticus strains. The PCR procedures carried out gave specific 524-, 726- and 918-bp amplificates, with DNA isolated from L. helveticus. No PCR product was generated for closely related bacteria. The amplification products were also screened for their species specificity in dot blot hybridization with representatives of the most closely related genera and species and a number of other bacterial species.

Aminopeptidases↗
PubMed2001

Decreased platelet glutamate uptake and genetic risk factors in patients with Parkinson's disease.

Genetic risk factors seem to play a role in sporadic Parkinson's disease (PD), maybe triggering oxidative stress and excitotoxicity within substantia nigra. However, genetic factors act at systemic level: reduced activity of mitochondrial enzymes and decreased glutamate uptake have been shown in platelets from PD patients. In this study we investigated glutamate uptake in platelets from 38 sporadic PD patients, 13 patients with parkinsonian syndromes and 28 controls and assessed polymorphisms of alpha-synuclein and ApoE genes. A 48% reduction of glutamate uptake p)<0.0001) was observed in PD patients which, with respect to control groups, correlated with the disease severity (r = -0.44, p < 0.05). Genetic studies of this population did not show differences between PD and controls, nor correlations with platelet glutamate uptake.

Apolipoproteins E↗
PubMed2001

A simple method for fallopian tube sperm perfusion using a blocking device in the treatment of unexplained infertility.

OBJECTIVE: To evaluate the efficacy of fallopian sperm perfusion (FSP) using a new method similar to the FAST system in comparison with standard intrauterine insemination (IUI) in patients with unexplained infertility. DESIGN: Prospective, randomized, controlled study. SETTING: Assisted conception service in a University Hospital. PATIENT(S): Women with unexplained infertility undergoing controlled ovarian hyperstimulation (COH). INTERVENTION(S): After hCG administration, patients were randomized to either standard IUI or FSP. The women received the same treatment in the first and all subsequent cycles. A maximum of three cycles was performed. Intrauterine insemination was performed using a standard method, and fallopian sperm perfusion was performed using a commercial device for hysterosalpingography and tubal hydropertubation. MAIN OUTCOME MEASURE(S): Clinical and ongoing pregnancy rates. RESULT(S): A total of 132 cycles was completed: 66 IUI cycles and 66 FSP cycles. In the IUI group, there were 5 ongoing pregnancies, giving a pregnancy rate of 7.6 per cycle and 15.6% per patient; in the FSP group, 14 ongoing pregnancies occurred, giving a pregnancy rate of 21.2% per cycle and 42.4% per patient. The prevalence of multiple pregnancies, miscarriages and ectopic pregnancies was similar in the two insemination groups. Fallopian sperm perfusion was easy to perform, and no case of sperm reflux was observed. The procedure was well tolerated and no complications were observed. The costs were comparable with standard IUI. CONCLUSION(S): In the treatment of couples with unexplained infertility, the method for fallopian sperm perfusion described yields higher pregnancy rates than IUI, with no significant increase in costs or complications. However, these results need to be confirmed in larger studies before replacing IUI with FSP as standard practice.

Adult↗
PubMed2001

Lack of association of the common TaqIB polymorphism in the cholesteryl ester transfer protein gene with angiographically assessed coronary atherosclerosis.

The anti-atherogenic effect of cholesteryl ester transfer protein (CETP) genetic variants associated with lowered enzyme activity is controversial. Moreover, in a few studies, this effect has been evaluated in the presence of a certain risk factor constellation. We addressed this issue in a case-control study, where 415 subjects with angiographically documented coronary artery disease (CAD +), 397 subjects without CAD (in 215, CAD was excluded by coronarography (CAD-)), and 188 healthy population controls, were screened for the CETP TaqIB polymorphism. The prevalence of the low-activity TaqIB2 allele was 0.396 in CAD+, and 0.428 and 0.416 in CAD- and population controls, respectively (p = 0.40). Its presence was significantly associated with increased high-density lipoprotein cholesterol (HDL-C) in population controls (1.40 +/- 0.40 mmol/l in B1B1, 1.52 +/- 0.39 mmol/l in B1B2 and 1.58 + 0.46 mmol/l in B2B2; p < 0.03 for trend), but not in the other groups. The CETP TaqIB polymorphism accounted for < 1% of the HDL-C variance in the whole cohort (p = 0.048). After adjustment for other risk factors, the CETP TaqIB2 allele was found not to be associated with significant changes in CAD risk independently of an assumed either dominant (odds ratio (OR) 0.97; 95% confidence interval (CI) 0.66-1.44; p = 0.89) or recessive effect (OR 0.68; 95% CI 0.42-1.12; p = 0.13). The CETP TaqIB polymorphism did not show a significant interaction with other risk factors in influencing CAD risk. Our findings do not support the hypothesis that a genetic variant resulting in lowered CETP activity is associated with reduced risk of coronary atherosclerosis.

Adult↗
PubMed2001

Efficacy of maintenance therapy with topical boric acid in comparison with oral itraconazole in the treatment of recurrent vulvovaginal candidiasis.

OBJECTIVE: Our purpose was to examine the efficacy of a topical long-term treatment with boric acid versus an oral long-term treatment (itraconazole) in the cure and prevention of recurrent vulvovaginal candidiasis. STUDY DESIGN: A prospective, nonrandomized study of patients affected by recurrent vulvovaginal candidiasis was undertaken. In 3 years we recruited 22 consecutive patients who underwent therapy with itraconazole (group 1) or boric acid (group 2). Women were followed up for 1 year, with clinic and microbiologic controls after 1, 3, 6, and 12 months after the first visit. RESULTS: During the treatment, the positive culture results (15.1% vs 12.1%) and the signs and symptoms (33.3% vs. 24.2%) were similar within the 2 groups, with no significant statistical difference. With the withdrawal, after 6 months relapses were common in the 2 groups (54.5%). CONCLUSIONS: Boric acid seems to be a valid and promising therapy both in the cure of the vaginal infection and in the prevention of relapses of recurrent vulvovaginal candidiasis, but its efficacy ends with the suspension of the therapy.

Adult↗
PubMed2001

Lipoprotein(a) changes during natural menstrual cycle and ovarian stimulation with recombinant and highly purified urinary FSH.

This prospective, randomized, controlled study compared the effects of recombinant human FSH (r-hFSH) and highly purified urinary FSH (u-hFSH HP) on lipoprotein(a) [Lp(a)] concentrations in women undergoing ovarian stimulation. Fifty infertile women were randomly allocated into two equally sized treatment groups (n = 25 per group). Thirty normal ovulation women were recruited as controls. The infertile women received u-hFSH or r-hFSH 150 IU/day starting on cycle day 2. From cycle day 6 the dose was adjusted according to ovarian response. Human chorionic gonadotrophin 10,000 IU was administered once there was at least one follicle > or =18 mm in diameter. The luteal phase was supported with progesterone 50 mg/day for at least 15 days. Repeated measurements of Lp(a) concentrations were performed during both stimulated and natural cycles. A significant increase in luteal phase Lp(a) concentrations was detected in the stimulated cycles, whereas no significant changes in serum Lp(a) concentrations were observed during natural cycles. There were no significant differences between the urinary and recombinant FSH effects on serum Lp(a). The luteal Lp(a) increase was transitory because after 1 month Lp(a) concentrations returned to baseline values if pregnancy failed to occur; in pregnant women persistent increased Lp(a) concentrations were found at the 8th week. The percentage changes in serum Lp(a) were positively correlated with the luteal progesterone increase (r = 0.40, P < 0.05), but not with follicular or luteal oestradiol increase. The women with low baseline Lp(a) (< or =5 mg/dl) had a greater increase of the Lp(a) concentrations at midluteal phase than women with baseline Lp(a) >5 mg/dl. In conclusion, the recombinant or urinary hFSH administration does not directly influence Lp(a) concentrations. The luteal Lp(a) increase in stimulated cycles is not related to gonadotrophin treatment per se, but appears to be related to the high luteal progesterone concentrations, physiologically or pharmacologically determined. Our results also suggest that the sensitivity to the progesterone changes could be related to apolipoprotein(a) phenotype.

Adult↗
PubMed2001

"Linear" and "derived" otoacoustic emissions in newborns: a comparative study.

OBJECTIVE: To investigate the effects of a specific aspect of the acquisition procedure, the averaging technique, on the evaluation of click-evoked otoacoustic emissions (CEOAEs) in newborns. DESIGN: CEOAEs were recorded by an Otodynamic ILO88 system from 89 full-term newborns at the third day after delivery. For each ear and in the same test session, CEOAEs were evoked by 75 to 85 dB pSPL acoustic clicks and averaged according to two different modes: the "linear" (classic average) and the "derived" mode, which allows the cancellation of linear behaving components (such as acoustic artifacts). All examined ears had a normal auditory function as assessed by conventional ABR between the ages of 2 and 4 mo. CEOAEs obtained by both averaging techniques were compared on the basis of several quantitative parameters: the waveform similarity; the levels of signal and noise and the inter-test reproducibility of the broadband response and of four different frequency bands centered at 1.6, 2.4, 3.2, and 4 kHz; the amplitude as a function of time; the test time. To eliminate the contribution of the stimulus artifact, linear CEOAEs were windowed 6 to 20 msec, whereas derived emissions were windowed using the default ILO88 window (2.5 to 20 msec). Additionally, CEOAEs were classified as "pass" or "fail" accordingly to screening criteria used in the daily clinical practice. RESULTS: Linear and derived emissions had very similar wave shapes and no time shifts during the first 12.5 msec. On the contrary, clear differences in the waveforms and time shifts were observed at longer latencies. The use of both averaging techniques resulted in identical CEOAE levels for both the broadband response and for the first two tested frequencies. For the last two frequencies, emission levels were lower when averaged with the linear technique owing to the use of the time window 6 to 20 msec, which reduces the amplitudes of high-frequency components. The residual noise in derived traces is 6 dB higher than that from linear traces. Also, derived CEOAEs had a lower inter-test reproducibility in both the broadband compound emission and in the four frequency bands examined here. The greatest difference in reproducibility was observed at the lowest band (1.2 to 2 kHz). Scoring of emissions was influenced by the averaging technique: 14% CEOAEs obtained with linear averaging and scored as passes were classified as fails when averaged with the derived mode. Moreover, if a CEOAE was scored as pass when using the derived technique, it also was scored as pass when using linear averaging. The increased number of false positives most likely was due to the higher noise floor/lower signal to noise ratio (SNR) of CEOAEs obtained with the derived technique. CONCLUSIONS: In the tested newborns and at the levels of stimulation used in this study, the emissions obtained with the derived technique were noisier than those obtained with the linear technique, this being intrinsically due to the type of averaging. Therefore, screening criteria based on the evaluation of the SNR (or similar parameters) could be influenced by the type of averaging used during the acquisition.

Acoustic Stimulation↗
PubMed2001

Expression of cytokines, inducible nitric oxide synthase, and matrix metalloproteinases in pouchitis: effects of probiotic treatment.

OBJECTIVE: The efficacy of probiotic organisms in the treatment of pouchitis has been reported. In the present study, we evaluated the tissue levels of pro- and anti-inflammatory cytokines, nitric oxide synthase, and matrix metalloproteinases in control and inflamed pouches before and after antibiotic and probiotic treatment of patients with acute pouchitis. METHODS: Pouch biopsy samples were obtained from seven patients with pouchitis before and after antibiotic and probiotic treatment. Tissue samples from five patients with normal pouches were used as controls. Cytokines were determined by ELISA, matrix metalloproteinase activity was evaluated by zymograms, and nitric oxide synthase activity was determined by measuring arginine to citrulline conversion. RESULTS: Tissue levels of tumor necrosis factor a increased (p < 0.01) in pouchitis relative to uninflamed pouches and reduced after antibiotic and probiotic treatment. Also, interferon y and interleukin 1alpha (IL-1alpha) augmented in pouchitis, but their increase did not reach statistical significance. The latter, however, were lower (p < 0.05) after treatment with the antibiotics and probiotics. Tissue levels of IL-4 and IL-10 were unchanged in inflamed pouches and unaffected by antibiotic treatment. However, IL-10 increased (p < 0.05) after probiotic treatment. Moreover, inflamed pouches had higher levels of inducible nitric oxide synthase and gelatinase activities, which decreased after treatment. CONCLUSIONS: The ability of antibiotic and probiotic treatments to increase tissue levels of IL-10, at a higher level than those observed in control pouches, and to decrease, to levels present in control pouches, proinflammatory cytokine, inducible nitric oxide synthase, and matrix metalloproteinase activity may suggest a mechanism of action to explain the efficacy of this therapeutic regime in pouchitis.

Acute Disease↗
PubMed2001

Pregnancy in hyperprolactinemic infertile women treated with vaginal bromocriptine: report of two cases and review of the literature.

Vaginal bromocriptine has proven safe and effective in treating hyperprolactinemic women. However, there has been no long-term clinical assessment regarding the influence of daily vaginal bromocriptine administration on the ability to conceive. This article presents two cases of successful pregnancy resulting from this alternative treatment. An infertile woman with an empty sella and hyperprolactinemia was treated with vaginal bromocriptine because of intolerance to oral administration. Prolactin levels were quickly normalized and no side effects occurred. Repeated postcoital tests during treatment proved normal. Twelve months later, the patient conceived. The therapy was discontinued during pregnancy, without complications. Although bromocriptine treatment was not resumed after delivery, postpartum prolactin levels were lower than before treatment and magnetic resonance imaging revealed an unchanged empty sella. Another patient with infertility and pituitary microadenoma with intolerance to oral dopaminergic agonists received the same treatment. Prolactin quickly fell to within the normal range. Vaginal bromocriptine was well tolerated and postcoital test results were not impaired. Tumor regression occurred and 10 months later the patient conceived. Despite bromocriptine withdrawal, no significant complications occurred during pregnancy. It can therefore be concluded that a couple's fertility does not appear to be significantly affected by the persistent local presence of bromocriptine.

Adenoma↗
PubMed2001

Expression and functional role of hepatocyte growth factor receptor (C-MET) during postnatal rat testis development.

The met protooncogene encodes the hepatocyte growth factor receptor (HGFR, c-met). C-met, a tyrosine kinase receptor protein, is widely expressed in different cell types including the male reproductive tract. As we recently demonstrated, both c-met messenger RNA and protein are expressed in prebuberal rat testis. The aim of this work was to detect the expression of c-met during postnatal testis development and to study its functional role. Our findings show that in total rat testis c-met is expressed during postnatal life until the sexual maturation of the animals. To evaluate the receptor expression in the different cell types in the testis, homogeneous cell populations of Sertoli and peritubular myoid cells were isolated from the seminiferous tubules of 10- and 35-day-old animals. c-met gene is expressed in myoid cells at the ages considered and its expression decreases with increasing age. By contrast, in Sertoli cells c-met expression is first detectable at 25 days of life and its expression increases with the increasing age being well evident at 35 days of age. C-met protein was detected by immunocytochemistry and its expression correlates with gene expression. The receptor is functionally active because HGF administration induces morphological changes in myoid cells and in c-met-expressing Sertoli cells. As a consequence of HGF addition, Sertoli cells cultured on reconstituted basement membrane reorganize into cord-like structures that resemble testicular seminiferous cords. The data here reported demonstrate for the first time that in Sertoli cells c-met expression is developmentally regulated being present and functionally active in postpuberal Sertoli cells. Given that c-met expression persists in myoid cells during postnatal testis development and that in Sertoli cells its expression correlates over time with germ cell differentiation and lumen formation, we conclude that the c-met/HGF system is involved in testis development and function.

Animals↗
PubMed2001

Maxillary sinus elevation: the effect of macrolacerations and microlacerations of the sinus membrane as determined by endoscopy.

The authors evaluated the health of the maxillary sinus in a group of 18 patients who had undergone sinus lift surgery for implantation purposes, using computed tomography and endoscopic imaging as a means of diagnosis. The study was performed after a mean interval of 31 months. All sinus lift operations were carried out using the lateral flap technique. The authors evaluated graft characteristics and intraoperative and postoperative complications and compared the results with computed tomography and endoscopic images. Endoscopy of a maxillary sinus that underwent microlaceration of the mucosa during sinus lifting showed a situation compatible with healthy conditions. In cases complicated by macrolaceration, the endoscopic picture was typical of sinusitis, even where clinical conditions of this type were not present. In the absence of intraoperative complications, the endoscopic appearance of sinus mucosa appears to improve when autologous graft material is used.

Adult↗
PubMed2000

Valine 10 may act as a driver for product release from the active site of human glutathione transferase P1-1.

We have probed the electrophilic binding site (H-site) of human glutathione transferase P1-1 through mutagenesis of two valines, Val 10 and Val 35, into glycine and alanine, respectively. These two residues were previously shown to be the only conformationally variable residues in the H-site and hence may play important roles in cosubstrate recognition and/or product dissociation. Both of these mutant enzymes have been expressed in Escherichia coli and purified and their kinetic properties characterized. The results demonstrate that Val35Ala behaves similarly to wild-type, whereas Val10Gly exhibits a strong decrease of k(cat) and k(cat)/K(m) (cosub) toward two selected cosubstrates: ethacrynic acid and 1-chloro-2,4-dinitrobenzene. Pre-steady-state kinetic analysis of the GSH conjugation with ethacrynic acid shows that both wild-type and Val10Gly mutant enzymes exhibit the same rate-limiting step: the dissociation of product. However, in the Val10Gly mutant there is an increased energetic barrier which renders the dissociation of product more difficult. Similar results are found for the Val10Gly mutant with 1-chloro-2,4-dinitrobenzene as cosubstrate. With this latter cosubstrate, Val 10 also exerts a positive role in the conformational transitions of the ternary complex before the chemical event. Crystallographic analysis of the Val10Gly mutant in complex with the inhibitor S-hexyl-GSH suggests that Val 10 optimally orientates products, thus promoting their exit from the active site.

Alanine↗
PubMed2000

Human glutathione transferase T2-2 discloses some evolutionary strategies for optimization of the catalytic activity of glutathione transferases.

Steady state, pre-steady state kinetic experiments, and site-directed mutagenesis have been used to dissect the catalytic mechanism of human glutathione transferase T2-2 with 1-menaphthyl sulfate as co-substrate. This enzyme is close to the ancestral precursor of the more recently evolved glutathione transferases belonging to Alpha, Pi, and Mu classes. The enzyme displays a random kinetic mechanism with very low k(cat) and k(cat)/K(m)((GSH)) values and with a rate-limiting step identified as the product release. The chemical step, which is fast and causes product accumulation before the steady state catalysis, strictly depends on the deprotonation of the bound GSH. Replacement of Arg-107 with Ala dramatically affects the fast phase, indicating that this residue is crucial both in the activation and orientation of GSH in the ternary complex. All pre-steady state and steady state kinetic data were convincingly fit to a kinetic mechanism that reflects a quite primordial catalytic efficiency of this enzyme. It involves two slowly interconverting or not interconverting enzyme populations (or active sites of the dimeric enzyme) both able to bind and activate GSH and strongly inhibited by the product. Only one population or subunit is catalytically competent. The proposed mechanism accounts for the apparent half-site behavior of this enzyme and for the apparent negative cooperativity observed under steady state conditions. These findings also suggest some evolutionary strategies in the glutathione transferase family that have been adopted for the optimization of the catalytic activity, which are mainly based on an increased flexibility of critical protein segments and on an optimal orientation of the substrate.

Arginine↗