Interleukin 10 gene promoter polymorphisms in women with early-onset pre-eclampsia.

Pre-eclampsia is one of the most serious disorders of human pregnancy and T helper type 1 (Th1)/Th2 imbalance plays a major role in its aetiology. The Th2 cytokine, interleukin (IL)-10, plays a significant role in the maintenance of pregnancy. The present study is aimed at understanding the role of IL-10 promoter polymorphisms (-1082 G/A; -592 A/C and -819 C/T) and their haplotypes in early-onset pre-eclampsia. A total of 120 patients and an equal number of women with normal pregnancy, from Government Maternity Hospital, Petlaburz, Hyderabad, India, were considered for the present study. A standard amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) was carried out for genotyping followed by agarose gel electrophoresis. Appropriate statistical methods were applied to test for the significance of the results. It was found that the IL-10 -819 C allele (P = 0·003) and -592 A (P = 0·005) allele frequencies increased significantly in patients compared to controls. No significant difference was found with regard to -1082 promoter polymorphism. Haplotype analysis of the IL-10 single nucleotide polymorphisms (SNPs) revealed a significant association with ACC haplotype with a twofold increased risk in patients compared to controls. The frequencies of two common IL-10 haplotypes (GCC and ATA) did not show any significant difference. Further, the diplotype analysis revealed five genotypes: -1082A with -819C (P = 0·0016); -1082G with -819C (P = 0·0018); -819C with -592C (P = 0·001); -1082A with -592C (P = 0·032); and -1082G with -592C (P = 0·005) associated with the disease. These findings support the concept of contribution of IL-10 gene polymorphisms in the pathogenesis of early-onset pre-eclampsia.

In vitro growth characteristics of melanocytes obtained from adult normal and vitiligo subjects.

The in vitro growth characteristics of melanocytes obtained from uninvolved and perilesional skin of vitiligo vulgaris subjects have been investigated in comparison to those from healthy adult donors. Normal human melanocytes have been found to grow exponentially in the presence of 10(-11) M cholera toxin and 10 ng/ml of 12-O-tetradecanoylphorbol-13-acetate in routine tissue culture media. They could be trypsinized up to 3-4 passages. Melanocytes of the uninvolved skin of vitiligo subjects manifested a lag of 8-11 days for the onset of growth and they could not be passaged. Melanocytes obtained from both hypo- and hyper-pigmented perilesional skin failed to grow under these conditions. Only in a few cases where the perilesional skin was normally pigmented did the melanocytes manifest some growth after a lag of 15 days. The initial seeding capacity of the melanocytes from uninvolved and perilesional skin of vitiligo patients were, respectively, 50% and 25% of the normal individuals. Vitiligo lesions themselves gave rise to unidentified dendritic cells that survived for 10-15 days without manifesting any growth. Our results suggest that melanocytes of individuals with vitiligo are defective. This fact has to be taken into account in any theory on the etiology of vitiligo.

Demonstration of tyrosinase in the vitiligo skin of human beings by a sensitive fluorometric method as well as by 14C(U)-L-tyrosine incorporation into melanin.

Tyrosinase activity (Monophenol, dihydroxyphenylalanine: oxygen oxidoreductase EC 1.14.18.1) in vitiligo and normal epidermal homogenates of skin from human beings was measured by estimating beta 3,4-dihydroxyphenylalanine (dopa) by a highly sensitive fluorometric method described in this paper. The tyrosine activity in the vitiligo skin was about 4 to 37% of corresponding normal skin. The activity of tyrosinase in normal human skin from different individuals and from different regions of the body was in the range of 4 to 140 picomoles of beta 3,4-dihydroxyphenylalanine formed per min/mg protein of epidermal homogenate. The enzyme from vitiligo and normal skin was severely inhibited by substance(s) of low molecular weight. The enzyme exhibits a lag of about 4 hr in the absence of added beta 3,4-dihydroxyphenylalanine and 1 hr in presence of 5 microM dopa. Tyrosinase from the normal and vitiligo skin was inhibited by excess concentration of tyrosine. The homogenates from vitiligo skin could synthesize melanin from C14(U)-L-Tyrosine. The rate of tyrosine incorporation into melanin by the epidermal homogenates is increased by 3,4-dihydroxyphenylalanine (dopa) disproportionate to its effect on tyrosinase activity. Based on the data presented in this paper it is concluded that melanocytes are present in the vitiligo skin. A tentative hypothesis is put forward to explain the lack of melanin synthesis by the vitiligo skin under in vivo conditions, although melanocytes are present.

Growth defects of melanocytes in culture from vitiligo subjects are spontaneously corrected in vivo in repigmenting subjects and can be partially corrected by the addition of fibroblast-derived growth factors in vitro.

Melanocytes cultured from uninvolved skin of untreated vitiligo subjects have decreased initial seeding capacities, manifest a lag period for the onset of the growth phase, and cannot be passaged. In contrast, melanocytes obtained from uninvolved and perilesional skin of vitiligo subjects actively repigmenting under 8-methoxy psoralen plus sunlight (PUVA) therapy have higher initial seeding capacities, grow faster without a lag period, and can be passaged to more than 12 passages. Extracts of a fetal lung fibroblast cell line (PMR-GF) that promote the growth rates and passage capacities of melanocytes from normal adult donors have been found also to promote the growth rates and passage capacities of melanocytes from the uninvolved skin of vitiligo subjects. Extracts of a fetal lung fibroblast cell line (PMR-GF), however, did not have any further stimulatory effect on the growth of melanocytes obtained from repigmenting vitiligo subjects. Melanocytes cultured from normal and untreated vitiligo subjects grew individually dispersed in the absence of PMR-GF, but tended to grow in clusters in its presence. Melanocytes from the repigmenting vitiligo subjects, however, tended to grow in clusters even in the absence of PMR-GF. These results indicate that the defective in vitro growth characteristics of melanocytes from vitiligo subjects may be related to the pathogenesis of this disease. It is possible that growth factors may be involved in the process of repigmentation in vitiligo subjects.

Lag kinetics of tyrosinase: its physiological implications.

The various theories put forward to explain the characteristic lag kinetics of oxidation of L-tyrosine by tyrosinase a rate regulatory step in the biosynthesis of melanin are reviewed Examination of the evidence in the literature and from experiments in the author's laboratory indicate that one of the hypotheses, that is, competition of tyrosine and dopa for met-tyrosinase and the formation of a dead-end complex of met-enzyme with tyrosine as explanation for lag kinetics is not consistent with available information. The alternative hypothesis that tyrosinase is an allosteric enzyme with tyrosine having negative effector site on the enzyme and dopa competing for it as an explanation for lag kinetics of tyrosinase is not yet disproved. Irrespective of the actual explanation for the lag kinetics of tyrosinase, it is suggested that the highly conserved lag kinetics may serve a physiological function. It is suggested that this function is to keep the enzyme essentially inactive during its transport to the specific organelle, namely the melanosome, in which an acidic environment exists. Only at acidic pH is the enzyme able to catalyze the biosynthesis of melanin.

Etiology of vitiligo. A new hypothesis.

Serum from actively repigmenting human vitiligo subjects had maximum mitogenic effect on the growth of melanocytes in culture, followed by the serum from normal donors, and from untreated vitiligo subjects in that order. Based on these findings, a new hypothesis is suggested for the etiology of vitiligo.

Properties of phosphofructokinase from the mucosa of rat jejunum and their relation to the lack of Pasteur effect.

1. The properties of phosphofructokinase after its slight purification from the mucosa of rat jejunum were studied. 2. The enzyme is inhibited by almost 100% by an excess of ATP (1.6mm), with 0.2mm-fructose 6-phosphate. AMP, ADP, P(i) and NH(4) (+) at 0.2, 0.76, 1.0 and 2mm respectively do not individually prevent the inhibition of phosphofructokinase activity by 1.6mm-ATP with 0.2mm-fructose 6-phosphate to any great extent, but all of them together completely prevent the inhibition of phosphofructokinase by ATP. 3. One of the effects of high concentrations of ATP on the enzyme was to increase enormously the apparent K(m) value for the other substrate fructose 6-phosphate, and this increase is largely counteracted by the presence of AMP, ADP, P(i) and NH(4) (+). At low concentrations of ATP the above effectors individually decrease the concentration of fructose 6-phosphate required for half-maximum velocity and when present together they decrease it further, in a more than additive way. 4. When fructose 6-phosphate is present at a saturating concentration (5mm), 0.3mm-NH(4) (+) increases the maximum velocity of the reaction 3.3-fold; with 0.5mm-fructose 6-phosphate, 4.5mm-NH(4) (+) is required for maximum effect. The other effectors do not change the maximum reaction velocity. 5. The results presented here suggest that NH(4) (+), AMP, ADP and P(i) synergistically decrease the inhibition of phosphofructokinase activity at high concentrations of ATP by decreasing the concentration of fructose 6-phosphate required for half-maximum velocity. Such synergism among the effectors and an observed, low ;energy charge' [(ATP+(1/2)ADP)/(AMP+ADP+ATP)] in conjunction with the possibility of a relatively high NH(4) (+) and fructose 6-phosphate concentration in this tissue, may keep the mucosal phosphofructokinase active and uninhibited by ATP under aerobic conditions, thus explaining the high rate of aerobic glycolysis and the lack of Pasteur effect in this tissue.

Melanosomal proteins promote melanin polymerization.

In melanocytes, enzymes involved in the generation of melanin monomers are present and active in coated vesicles which are known to be acidic. Melanin polymerization however, occurs only in melanosomes. In vitro, it is not possible to generate melanin at the acidic pH of melanosomes using 3,4-dihydroxyphenylalanine (DOPA) and tyrosinase alone whereas melanin readily forms at higher pH with these reagents. Dimerization and elongation of the melanin polymer is known to require deprotonation. We have hypothesized that the amino acid side chains of melanosomal proteins act as proton acceptors to initiate polymerization and that the protonated basic groups serve to attract the negatively charged oligomers thus aiding polymerization and binding to proteins. We show that basic model proteins and basic premelanosomal proteins promote polymerization at an acidic pH and that positively charged surfaces allow binding of the growing melanin polymer. With progressive polymerization and exhaustion of the proton abstracting ability of melanosomal proteins, melanosomal pH drops further, which, we argue, is an additional controlling step that limits tyrosinase activity and melanin polymerization.

pH-dependent interconvertible forms of mushroom tyrosinase with different kinetic properties.

The lag in cresolase activity and inhibition by excess tyrosine of mushroom tyrosinase which was observed when assayed at pH 6.8 was found to be absent when assayed at pH 5.0. The absence of lag and inhibition by excess tyrosine of tyrosinase at pH 5.0 were brought about only after the enzyme was kept at pH 5.0, at 0-4 degrees C, for 1.5 h. The enzyme kept at pH 5.0 for 1.5-3 h at 0-4 degrees C when brought back to pH 6.8, acquires lag and inhibition by excess tyrosine when its activity was measured at pH 6.8. The pH-dependent changes in the kinetic properties of the mushroom tyrosinase are similar to the pH-dependent changes in the kinetic properties of tyrosinase from B-16 murine melanoma and human skin, and thus appear to be a general property of tyrosinase from diverse sources.

Citrate activates tyrosinase from B-16 murine melanoma and human skin.

Citrate stimulates cresolase activity of tyrosinase from B-16 murine melanoma and human skin. Maximal stimulation by citrate was obtained at 2 mM, and stimulation was decreased at higher concentrations. Citrate stimulates tyrosinase not only from mammalian sources but also from mushroom. The stimulation was not due to reversal of inhibition of enzyme activity by excess tyrosine. On rapid decrease in pH of the enzyme solution from 6.8 to 5.0-5.2, the enzyme is no longer inhibited by excess tyrosine even when its activity was assayed at pH 6.8. Citrate also stimulates this form of enzyme. However, the stimulation is more at acidic pH than at pH 6.8. At higher concentrations of citrate the stimulatory effect decreases at both pH 5.0 and pH 6.8. Inhibition of this enzyme occurs at higher concentrations (22 mM) at pH 6.8. The physiological role of stimulation of cresolase activity of tyrosinase by citrate is yet to be unravelled.

pH-dependent interconvertible allosteric forms of murine melanoma tyrosinase. Physiological implications.

Murine melanoma melanosomal tyrosinase, solubilised at pH 6.8 and 1% Igepal, exhibits a lag in cresolase activity which increases with increasing concentration of tyrosine. The enzyme, solubilised at pH 5.0 and assayed at pH 5.0, does not exhibit lag even at inhibitory concentrations of tyrosine while the same enzyme when assayed at pH 6.8 exhibits characteristic lag. When the enzyme was solubilised from a melanosomal fraction with detergent/water without any buffer, significant linear activity for 2 h was seen at an inhibitory concentration of tyrosine, indicating for the first time the presence of a form of tyrosinase without lag and inhibition by excess tyrosine. Exposure of the enzyme solubilised in buffer/detergent at pH 6.8 to rapid decrease in pH to 5.0 or 4.7 makes the enzyme remain irreversibly in the form without characteristic lag, even at an inhibitory concentration of tyrosine and at pH 6.8. These results may be interpreted as follows. The enzyme at pH 6.8 exists in the E form with an allosteric site for tyrosine. Decrease of the pH of the enzyme solution from 6.8 to 5.0 or 4.7 by dialysis results in the reversible protonation of the enzyme, which no longer binds tyrosine at its allosteric site and consequently inhibition by excess tyrosine and lag were not observed at acidic pH. However, if the enzyme was rapidly brought to pH 5.0 from 6.8 it remains irreversibly in the protonated form even at pH 6.8. Ascorbic acid acts as an effective reductant for the hydroxylation of tyrosine by tyrosinase, while 3,4-dihydroxyphenylalanine is both an effective reductant and counteracts the inhibition by tyrosine at pH 6.8.

pH-dependent interconversion of two forms of tyrosinase in human skin.

1. We have shown that the characteristic lag in cresolase activity of human skin tyrosinase at inhibitory concentration of tyrosine was absent at all pH values studied, i.e. pH 5.2, 5.7, 6.2 and 6.8, if the enzyme solubilized at low pH was used as the source of enzyme, but the same enzyme when dialysed against buffers of various pH values showed linear activity only at pH 5.2 and was not inhibited by excess tyrosine, whereas at higher pH values it exhibited a lag and inhibition by excess tyrosine. 2. However, the enzyme solubilized in buffer/detergent, pH 6.8, when dialysed against buffer of the same pH showed linear activity at pH 5.2 and non-linear activity at pH 6.8. 3. The water/detergent-solubilized enzyme from human skin melanosomes showed linear activity even at inhibitory concentrations of tyrosine at pH 5.2 and 6.8 up to 2 h, but acceleration of rate was observed after 2 h for the enzyme measured at pH 6.8. 4. After dialysis of the water/detergent-solubilized enzyme against double-glass-distilled water, it still exhibits linear activity at inhibitory concentration of tyrosines at pH 6.8 for the first 2 h, but the same enzyme when dialysed against 0.02 M-sodium phosphate buffer, pH 6.8, exhibits negligible activity up to 1/2 h, in contrast with considerable activity before dialysis during the same interval of time, but without any loss of activity at later intervals of incubation time. 5. On the basis of these results, it is concluded that the enzyme exists in at least two interconvertible forms, one without lag and inhibition by excess tyrosine and the other with lag and inhibition by excess tyrosine. These two forms are interconvertible only by gradual change in pH over a period of hours.