Sensitivity of Escherichia coli (MutT) and human (MTH1) 8-oxo-dGTPases to in vitro inhibition by the carcinogenic metals, nickel(II), copper(II), cobalt(II) and cadmium(II)

The toxicity of Ni(II), Co(II) and Cu(II) in animals, and that of Cd(II) in cultured cells, has been associated with generation of the promutagenic lesion 8-oxo-7,8-dihydroguanine (8-oxoguanine) in DNA, among other effects. One possible source of this base may be 8-oxo-7,8- dihydro-2'-deoxyguanosine-5'-triphosphate (8-oxo-dGTP), a product of oxidative damage to the nucleotide pool, from which it is incorporated into DNA. To promote such incorporation, the metals would have to inhibit specific cellular 8-oxo-dGTPases that eliminate 8-oxo-dGTP from the nucleotide pool. The present study was designed to test such inhibition in vitro on 8-oxo-dGTPases from two different species, the human MTH1 protein and Escherichia coli MutT protein. In the presence of Mg(II), the natural activator of 8-oxo-dGTPases, all four metals were found to inhibit both enzymes. For MTH1, the IC50 values (+/- SE; n = 3-4) were 17 +/- 2 microM for Cu(II), 30 +/- 8 microM for Cd(II), 376 +/- 71 microM for Co(II) and 801 +/- 97 microM for Ni(II). For MutT, they were 60 +/- 6 microM for Cd(II), 102 +/- 8 microM for Cu(II), 1461 +/- 96 microM for Ni(II) and 8788 +/- 1003 microM for Co(II). Thus, Cu(II) and Cd(II) emerged as much stronger inhibitors than Ni(II) and Co(II), and MTH1 appeared to be generally more sensitive to metal inhibition than MutT. Interestingly, in the absence of Mg(II), the activity of the enzymes could be restored by Co(II) to 73% of that with Mg(II) alone for MutT, and 34% for MTH1, the other metals being much less or non-effective. The difference in sensitivity to metal inhibition between the two enzymes may reflect the differences in the amino acid ligands, especially the cysteine ligand, outside their evolutionarily conserved Mg(II)-binding active sites, which might indicate predominantly non-competitive or uncompetitive mechanism of the inhibition. The overall results suggest that inhibition of 8-oxo- dGTPases may be involved in the mechanisms of induction of the 8- oxoguanine lesion in DNA by the metal ions studied, especially the non- redox-active Cd(II) cation.      

Localized muscular mucormycosis in a child with acute leukemia

Mucormycosis is a rare fungal infection of childhood, occurring mainly in patients with chronic illnesses such as diabetes and malignancies. The fungus seldom grows in culture and confirmation of the diagnosis depends on histologic examination of infected tissues. To date, the reported natural history of the disease has been rapid progression and a fatal outcome. Therefore, the importance of early diagnosis by tissue biopsy and early treatment with surgical debridement and systemic antifungal therapy cannot be overemphasized. The pulmonary system is the most common site for mucormycosis in patients with leukemia. We report what we believe to be the first successfully treated case of isolated muscular mucormycosis occurring in a child with biphenotypic acute leukemia. The diagnosis was made promptly by tissue examination at the time of surgical debridement. The patient was also given systemic amphotericin-B therapy.      

Modulation of ethylnitrosourea-induced toxicity and mutagenicity in human cells by O6-benzylguanine.

We have examined the contributions of O6-alkylguanine-DNA alkyl-transferase (AGT) and nucleotide excision repair to the protection of human cells from the toxic and mutagenic effects of ethylnitrosourea. Three human lymphoblastoid cell lines were used: one which possesses both of these DNA repair pathways; one derived from a xeroderma pigmentosum complementation group A patient, which expresses AGT but is deficient in nucleotide excision repair; and a third which does not express AGT but is capable of excision repair. The level of active AGT in the cells was further modulated with the use of the AGT inhibitor, O6-benzylguanine. These cells were exposed to ethylnitrosourea in both the presence and absence of O6-benzylguanine, and population survival, growth, and mutagenesis at the hypoxanthine-guanine phosphoribosyl-transferase locus were measured. The results for all three measurements indicated that the lack of either AGT or nucleotide excision repair significantly impairs the ability of human cells to withstand DNA ethylation damage. Furthermore, the inhibition of AGT in xeroderma pigmentosum group A cells did not increase toxicity or mutagenicity, suggesting that AGT and nucleotide excision repair cooperate in the removal of DNA ethyl adducts. Related studies in our laboratory have shown that AGT and nucleotide excision repair are both necessary for the efficient removal of O6-ethyldeoxyguanosine.     

Factor VIII-von Willebrand factor requires calcium for facilitation of platelet adherence

The role of divalent cations in platelet adherence to deendothelialized human arteries in flowing blood was investigated in an annular perfusion chamber. Spreading of platelets on the subendothelium was impaired below 30 microM of free Ca2+ ions (Ca2+). When Ca2+ was replaced by Mg2+, adherence was unchanged in perfusates without exogenous factor VIII-von Willebrand factor (FVIII-vWF), but the ability of FVIII-vWF to support platelet adherence was lost. Binding of FVIII-vWF to the vessel wall was independent of divalent cations, but bound FVIII-vWF was only able to mediate adherence after exposure to Ca2+. Pretreatment of FVIII-vWF with the calcium chelator EGTA (10 mM) resulted in loss of the ability to facilitate platelet adherence, while the ristocetin cofactor activity remained intact. Full restoration of the ability to mediate platelet adherence could only be obtained by prolonged dialysis against Ca2+ in the millimolar range. These data indicate that divalent cations have at least two separate roles to play in supporting platelet adherence: (1) platelet spreading on the subendothelium requires Ca2+ or Mg2+; (2) FVIII-vWF should be exposed to Ca2+ to obtain its optimal biologic activity in supporting platelet adherence.     

Maternal and Neonatal Factors Adversely Affecting Breastfeeding in the Perinatal Period

Background

Despite its known advantages, breastfeeding rates are low world over. Large number of factors affect breastfeeding. This study was designed to detect maternal and neonatal factors that adversely affect breastfeeding in the perinatal period.

Methods

A prospective, single-blinded study was conducted on randomly chosen mother-infant pairs in the maternity ward of a tertiary care service hospital. Only full term singletons born by normal vaginal delivery were studied. The B.R.E.A.S.T observation score and time spent by the infant at the mother''s breast were primary outcome variables. Maternal age, gravida, para status and education level were recorded. Birth weight, sex, gestation age of the infant and time interval from birth to observation were also recorded. Initial univariate analysis followed by multivariate analysis was performed using SPSS ver 7.5 software.

Results

A total of 54 mother-infant pairs formed the study group; 19(35.2%) were primigravidas. Primigravidas status of the mother led to significantly lower scores (p<0.04; 95% CI 0.10 to 3.62) as did maternal age < 26 years (p<0.04; 95% CI 0.2. to 3.46) on univariate analysis. Low birth weight (<2500 g) was the only neonatal factor that significantly lowered breastfeeding scores (p<0.02;95%Cl 0.56 to 6.31). On multivariate analysis only primigravida status was significantly associated with lower scores (p<0.02). The alpha value of the study was 5% and the power was 74%. Time spent by infant on breast was not significantly different between primigravida and non-primigravida mothers.

Conclusion

Primigravida status adversely affects breastfeeding scores; therefore counseling and support should be focused on this group. Extra care should also be taken to ensure adequate breastfeeding by younger mothers and in those with low birth weight infants. Larger studies with long-term follow up will be able to identify other factors and dertermine the effects of focused counseling and support in the perinatal period upon long-term breastfeeding rates.Key Words: Breastfeeding, Primigravida, Counseling