M. Nicotra

STRUCTURAL FLEXIBILITY MODULATES THE ACTIVITY OF HUMAN GLUTATHIONE TRANSFERASE P1-1 : ROLE OF HELIX 2 FLEXIBILITY IN THE CATALYTIC MECHANISM

Presteady-state and steady-state kinetic studies performed on human glutathione transferase P1-1 (EC) with 1-chloro-2,4-dinitrobenzene as co-substrate indicate that the rate-determining step is a physical event that occurs after binding of the two substrates and before the σ-complex formation. It may be a structural transition involving the ternary complex. This event can be related to diffusion-controlled motions of protein portions as k°cat/kcat linearly increases by raising the relative viscosity of the solution. Similar viscosity dependence has been observed for KGSHm, while KCDNBm is independent. No change of the enzyme structure by viscosogen has been found by circular dichroism analysis. Thus, kcat and KGSHm seem to be related to the frequency and extent of enzyme structural motions modulated by viscosity. Interestingly, the reactivity of Cys-47 which can act as a probe for the flexibility of helix 2 is also modulated by viscosity. Its viscosity dependence parallels that observed for kcat and KGSHm, thereby suggesting a possible correlation between kcat, KGSHm, and diffusion-controlled motion of helix 2. The viscosity effect on the kinetic parameters of C47S and C47S/C101S mutants confirms the involvement of helix 2 motions in the modulation of KGSHm, whereas a similar role on kcat cannot be ascertained unequivocally. The flexibility of helix 2 modulates also the homotropic behavior of GSH in these mutants. Furthermore, fluorescence experiments support a structural motion of about 4 Å occurring between helix 2 and helix 4 when GSH binds to the G-site.

Molecular Cloning of hMena (ENAH) and Its Splice Variant hMena+11a: Epidermal Growth Factor Increases Their Expression and Stimulates hMena+11a Phosphorylation in Breast Cancer Cell Lines

hMena (ENAH), an actin regulatory protein involved in the control of cell motility and adhesion, is modulated during human breast carcinogenesis. In fact, whereas undetectable in normal mammary epithelium, hMena becomes overexpressed in high-risk benign lesions and primary and metastatic tumors. In vivo, hMena overexpression correlates with the HER-2(+)/ER(-)/Ki67(+) unfavorable prognostic phenotype. In vitro, neuregulin-1 up-regulates whereas Herceptin treatment down-modulates hMena expression, suggesting that it may couple tyrosine kinase receptor signaling to the actin cytoskeleton. Herein, we report the cloning of hMena and of a splice variant, hMena(+11a), which contains an additional exon corresponding to 21 amino acids located in the EVH2 domain, from a breast carcinoma cell line of epithelial phenotype. Whereas hMena overexpression consistently characterizes the transformed phenotype of tumor cells of different lineages, hMena(+11a) isoform is concomitantly present only in epithelial tumor cell lines. In breast cancer cell lines, epidermal growth factor (EGF) treatment promotes concomitant up-regulation of hMena and hMena(+11a), resulting in an increase of the fraction of phosphorylated hMena(+11a) isoform only. hMena(+11a) overexpression and phosphorylation leads to increased p42/44 mitogen-activated protein kinase (MAPK) activation and cell proliferation as evidenced in hMena(+11a)-transfected breast cancer cell lines. On the contrary, hMena knockdown induces reduction of p42/44 MAPK phosphorylation and of the proliferative response to EGF. The present data provide new insight into the relevance of actin cytoskeleton regulatory proteins and, in particular, of hMena isoforms in coupling multiple signaling pathways involved in breast cancer.

Transvaginal sonography of the yolk sac in normal and abnormal pregnancy

A cross‐sectional study comprising 117 consecutive first trimester singleton pregnancies was performed using transvaginal sonography (TVS) to evaluate size abnormalities of the secondary yolk sac (YS) vis‐à‐vis pregnancy outcome. In normal pregnancy outcome (NPO) the YS diameter showed an increase from the 5th to the 11th week, menstrual age, followed by a decrease and its disappearance after 12 weeks. A YS of abnormal size was statistically significant (p < 0.001) in spontaneous abortion (SA) versus NPO, with a sensitivity of 68.7%, a specificity of 99%, a positive predictive value of 91.6% and a negative predictive value of 95.2%. These preliminary results indicate that a measurement of the YS very early in gestation may be a useful marker of pregnancy outcome.

Temperature adaptation of glutathione S-transferase P1-1. A case for homotropic regulation of substrate binding.

Human glutathione S-transferase P1–1 (GST P1–1) is a homodimeric enzyme expressed in several organs as well as in the upper layers of epidermis, playing a role against carcinogenic and toxic compounds. A sophisticated mechanism of temperature adaptation has been developed by this enzyme. In fact, above 35u2009°C, glutathione (GSH) binding to GST P1–1 displays positive cooperativity, whereas negative cooperativity occurs below 25u2009°C. This binding mechanism minimizes changes of GSH affinity for GST P1–1 because of temperature fluctuation. This is a likely advantage for epithelial skin cells, which are naturally exposed to temperature variation and, incidentally, to carcinogenic compounds, always needing efficient detoxifying systems. As a whole, GST P1–1 represents the first enzyme which displays a temperature-dependent homotropic regulation of substrate (e.g. GSH) binding.

Adenosine Deaminase and Human Reproduction: A Comparative Study of Fertile Women and Women with Recurrent Spontaneous Abortion

PROBLEM: We have investigated the possible role of adenosine deaminase (ADA) genetic polymorphism in human fertility through a comparative study of couples with recurrent spontaneous abortion (RSA) and healthy puerperae.

The Genetics of Signal Transduction and the Feto-Maternal Relationship. A Study of Cytosolic Low Molecular Weight Phosphotyrosine Phosphatase

Intracellular kinases mediate positive signalling from surface receptors by phosphorylating critical target proteins whereas phosphatases inhibit this process. Differential phosphatase activity at the feto-maternal interface could determine the appropriate relative growth and development on each side of the placenta. The highly polymorphic cytosolic low molecular weight phosphotyrosine-phosphatase (ACP1-cLMWPTPase) has been studied in 170 women who had at least two consecutive spontaneous abortions along with their husbands and in 352 normal puerperae along with their newborn babies. Symmetry analysis of joint wife/husband and mother/infant distribution suggests that when ACP1 activity is lower in the mother than in either her aborted fetus or her child, the probability of abortion is higher and the survival to term is lower as compared to pairs in which the ACP1 activity is higher in the mother than in her fetus. Further analysis has shown that the effect is due to S isoform: i.e. a high mother/fetus S isoform ratio favours intrauterine survival. Analysis of gestational duration and birth weight suggests that a high ACP1 maternal activity coupled with a low or moderate fetal activity favour fetal growth and developmental maturation. The present data indicate that maternal-fetal genetic differences in signal transduction could contribute significantly to variability of intrauterine developmental parameters and to pathological manifestation of pregnancy.

Interaction between ABO blood groups and ADA genetic polymorphism during intrauterine life

A total of 203 couples with unexplained habitual abortions and 364 consecutive normal puerperae along with their live-born babies were studied. The analysis of wife-husband joint ABO blood group distribution in couples with habitual abortion showed an excess of A incompatible mating type and a defect of B incompatible type as compared with expected proportions assuming random mating. The joint wife-husband ABO blood group distribution was further analysed in relation to the adenosine deaminase (ADA) genotype. A defect of O-A and A-O couples when the wife carries the ADA*1/*1 genotype and the husband carries the ADA*2 allele, and a defect of O-O and A-A when the wife carries the ADA*2 allele were observed. In the sample of normal puerperae, analysis of the joint mother-newborn ABO distribution in relation to the ADA genotype showed a pattern similar to that observed in couples with habitual abortion, i.e. there is a defect of O-A and A-O when the mother carries the ADA*1/*1 genotype and the newborn carries the ADA*2 allele and a defect of O-O and A-A types when the mother carries the ADA*2 allele. Altogether the data suggest an early loss of O-A and A-O zygotes when they carry the ADA*2 allele and an early loss of O-O and A-A zygotes when the mother carries the ADA*2 allele resulting in a deficit of these zygotic classes among both spontaneously aborted fetuses and live-born infants. The pattern of association observed in the mother-fetus type O-A (incompatible according to conventional terminology) appears similar to that observed for the reciprocal A-O type (compatible according to conventional terminolgy). Therefore strictly conventional immunological mechanisms cannot explain the whole pattern of associations. Cell to cell interactions involving ABO antigens may have an important role at implantation: ADA, through the control of local adenosine concentration, could modulate these interactions influencing the probability of successful implantation.

Phosphoglucomutase genetic polymorphism of newborns

An association of the phosphoglucomutase locus 1 (PGM1) genetic polymorphism with repeated spontaneous abortion (RSA), with intrauterine development in both normal and diabetic pregnancies, and with fertility has been reported in previous studies. In view of the evolutionary interest and of a possible clinical relevance of PGM1 selection during intrauterine life, this study considers healthy puerperae, consecutive newborns, and couples with RSA as well as two alleles (PGM1*1 and PGM1*2). The joint maternal–neonatal PGM1 distribution in a sample from an Italian rural population is significantly different from that expected assuming Hardy–Weinberg conditions for equilibrium. Deviation is dependent on maternal age and parity. The joint mother–newborn PGM1 genotype distribution is significantly associated with a positive history of previous spontaneous miscarriage, suggesting that the presence of the PGM1*2 allele in the father predisposes to spontaneous abortion. This hypothesis is also supported by the observation that in couples with RSA, the delivery of a live born infant within 5 years from the first episode of miscarriage is negatively associated with the presence of a PGM1*2 allele in the husband. Altogether these observations suggest the hypothesis of PGM1 maternal selection at the reproductive level involving a differential role of PGM1*1 and PGM1*2 alleles of paternal origin. Am. J. Hum. Biol. 13:9–14, 2001.

Phosphotyrosine-protein-phosphatases and human reproduction: an association between low molecular weight acid phosphatase (ACP1) and spontaneous abortion.

ACP1 (low molecular weight acid phosphatase) genetic polymorphism has been studied in 173 women with a history of two or more consecutive spontaneous abortions and in 1508 control subjects, including 482 normal pregnant women. The proportion of carriers of ACP1*C allele (*A/ *C, *B/*C) in women with a history of repeated spontaneous abortion is lower than in normal pregnant women and other control groups. Women with repeated spontaneous abortion show a specific decrease of ACP1 S isoform concentration as compared to normal pregnant women. The other component of ACP1 activity, the F isoform, does not show a significant difference between the two groups. The data suggest that women with ACP1 genotypes showing a high concentration of S isoform are relatively protected against spontaneous abortion. Preliminary analysis of a sample of 352 normal puerperae along with their newborn babies supports this hypothesis.

HLA and Complement Factors Alleles Sharing in Italian Couples with Recurrent Spontaneous Abortions

Recurrent Spontaneous Abortion (RSA) is postulated to be due to several factors including immunogenetic mechanisms. Many studies have been conducted on the effect of the MHC region in the reproductive phenomena suggesting an immunological or genetic involvement in RSA. We studied couples with 3 or more abortions among a larger group of couples in which female partners were anti-cardiolipin antibodies negative, resulting in a population of 43 couples typed for HLA-A, B, C, DR, DQ. In 16 of these 43 couples, complement factors C4A, C4B, and Bf were typed. The data shows a statistically significant increase of C4B*Q0 in RSA patients (N = 32) compared with the control population (N = 44) (pc = .00147) and also a statistically significant increase of C4B*Q0 sharing in aborting couples (43.75%) against the expected sharing rate in the control population (1.86%) (p < .001). Frequency increase of C4B*Q0 allele in aborting population leads to the hypothesis that an imbalance of complement factors expression and activity can have detrimental effects on implantation and embryo survival. Additionally, the significant sharing rate of C4B*Q0 in couples with RSA could indicate the existence of a gene in linked to this allele predisposing to RSA and acting in a recessive manner if present in double copies in the fetus.