Radiation-induced red cell damage: role of reactive oxygen species

BACKGROUND: Cellular blood components are irradiated to prevent graft- versus-host disease in transfusion recipients at risk for this syndrome. Because gamma radiation can result in the production of reactive oxygen species, the role of reactive oxygen species was investigated in radiation-induced red cell damage. STUDY DESIGN AND METHODS: Whole blood from normal donors was exposed to various doses of t-butyl hydroperoxide (0-1 mM) and/or to gamma-radiation (0-50 Gy). Oxidative damage was assessed by the extent of lipid peroxidation (measured by thiobarbituric acid-reactive substances [TBARS]) and hemoglobin oxidation. Fresh blood was divided into three parts-one initially irradiated and stored, another stored with portions irradiated weekly, and a third stored without irradiation. TBARS and hemoglobin oxidation were measured weekly. RESULTS: As expected, t- butyl hydroperoxide induced TBARS formation and hemoglobin oxidation in a dose-dependent fashion. The gamma-radiation not only increased hemoglobin oxidation and TBARS formation, but also enhanced the t-butyl hydroperoxide effect on red cells. Red cell storage increased TBARS generation and hemoglobin oxidation in a time-dependent fashion. When radiation was administered either initially or after weekly storage, TBARS production and hemoglobin oxidation were increased over that measured in unirradiated paired controls. CONCLUSION: Gamma radiation at clinically used doses increases lipid peroxidation and hemoglobin oxidation in human red cells. The effect of gamma-radiation is accentuated by blood storage and induces damage independent of time of storage.     

Plasma homocysteine levels and folate status in children with sickle cell anemia.

PURPOSE: A sensitive inverse relationship between plasma homocysteine concentration and folate status has been demonstrated. Although children with sickle cell anemia (SCA) are at potential risk for folate deficiency, plasma homocysteine levels have not been reported in such patients. Therefore, a study was designed to assess plasma homocysteine levels as a marker of folate status. DESIGN: Plasma homocysteine concentrations were measured in 120 children with SCA (102 in steady state and 18 during an acute complication) who had never received supplemental folic acid. Folate status was directly assessed in 34 of these patients. RESULTS: Plasma homocysteine levels in the patients with SCA and control subjects were similar. The mean value +/- 1 SD was 5.8+/-2.5 micromol/L (range, 1.6 to 14.1 micromol/L) in the patients with SCA and 6.1+/-2.7 micromol/L (range, 1.7 to 15.3 micromol/L) in 73 pediatric control subjects. In a subpopulation of the study group (34 children), simultaneous serum folate, red cell folate, and total homocysteine concentrations were also measured. Their serum folate and red cell folate concentrations were normal: 12.4+/-10.0 nmol/L (range, 1 to 42 nmol/L) and 604+/-374.7 nmol/L (range, 205 to 1741 nmol/L), respectively. There was no correlation of plasma homocysteine concentration with various clinical or laboratory measures or with red cell folate concentration. CONCLUSION: Folate stores in children with SCA not receiving folic acid supplements are adequate despite an underlying hemolytic anemia.     

Positional cloning of the gene for X-linked retinitis pigmentosa 3: homology with the guanine-nucleotide-exchange factor RCC1

The gene for retinitis pigmentosa 3 (RP3), the most frequent form of X- linked RP (XLRP), has been mapped previously to a chromosome interval of less than 1000 kbp between the DXS1110 marker and the OTC locus at Xp21.1-p11.4. Employing a novel technique, YAC Representation Hybridization (YRH)', we have recently identified a small XLRP associated microdeletion in this interval, as well as several putative exons including the 3' end of a gene that was truncated by the deletion. cDNA library screening and sequencing of a cosmid centromeric to the deletion has now enabled us to identify numerous additional exons and to detect several point mutations in patients with XLRP. The predicted gene product shows homology to RCC1, the guanine-nucleotide- exchange factor (GEF) of the Ras-like GTPase Ran. Our findings suggest that we have cloned the long-sought RP3 gene, and that it may encode the GEF of a retina-specific GTP-binding protein.      

大鼠视皮质胼胝体轴突的生后发育和形态──生物素结合的葡聚糖胺顺行示踪法研究

采用生物素结合的葡聚糖胺顺行示踪法研究了大鼠视皮质主要胼胝体投射区即１７／１８ａ交界区胼胝体轴突的生后发育和形态。在生后５天时，此交界区胼胝体轴突从白质向灰质Ⅰ层垂直生长，在灰质内仅有极少量的侧支抽芽。至生后１３天时，皮质Ⅰ层最先出现致密的由胼胝体轴突终支组成的终末丛。到生后１７天时，类似的终末丛也见于皮质Ⅱ／Ⅲ，Ⅴ和Ⅵ层，这种分布型式与成年大鼠者相似。以上结果表明，绝大部分胼胝体轴突首先生长到达Ⅰ层并先在Ⅰ层发出终支，然后再在其它皮质层发出侧支及终支，因而提示皮质Ⅰ层在胼胝体联系的生后发育中可能发挥重要作用。     

In vivo regulation of phenylalanine hydroxylase in the genetic mutant hph-1 mouse model

The hph-1 mouse has low liver activity of GTP cyclohydrolase 1, the rate limiting enzyme in the biosynthesis of tetrahydrobiopterin (BH₄). BH₄ is the cofactor for phenylalanine hydroxylase (PAH) and in the early stages of life the hph-1 mouse is hyperphenylalaninemic. At approximately 15 days after birth the blood phenylalanine levels normalize. During this period the animals provide an in vivo model which can be used to study the regulatory effects of phenylalanine on PAH, and for related pediatric metabolic disease in humans; from birth to youth. We therefore, examined; liver PAH activity using BH₄ and 6-methyltetrahydropterin (6MPH₄) as cofactor; PAH total enzyme concentration by Western blotting using the PH8 antibody, and PAH state of phosphorylation using the PH7 antibody from 4 to 18 days after birth. The findings were compared to the wild type animals that are not hyperphenylalaninemic during this period. PAH (6MPH₄) activity and total protein (PH8 antibody) rose steadily in the hph-1 mice. In control mice, both activity and total protein fluctuated. The degree of phosphorylation of PAH in the mutants and the state of activation (as measured by the 6MPH₄/BH₄ activity ratio) increased as phenylalanine levels rose, and decreased when they fell. Similar patterns were not seen in the control animals. These studies provide in vivo evidence that phenylalanine concentration regulates the activity of PAH in the hph-1 mouse and that this acts via a mechanism that includes phosphorylation of the PAH molecule. The kinetic values (K_m and V_max) for mouse PAH are also reported.     

Optimization of a method for deactivation of platelet-activating factor:acetylhydrolase in serum for use in in-vitro fertilization culture media

Embryos produced by in-vitro fertilization (IVF) may produce less platelet-activating factor (PAF) than is optimal for development. It was previously shown that supplementation of culture media with PAF results in a significant increase in pregnancy rate. Human embryos are often cultured in media supplemented with serum containing the enzyme PAF:acetylhydrolase (PAF:AH; EC 3.1.1.47), which hydrolyses PAF to its inactive form, lyso-PAF. Thus, effective supplementation of media with PAF requires inactivation of this enzyme. In this study we examine the efficacy of the methods of PAF:AH deactivation used for PAF supplementation of IVF culture medium. When the effectiveness of a commonly used acid treatment protocol (pH 3.0 at room temperature for 5 min) was examined, it was found that it was not completely effective for the majority of sera. When synthetic PAF was added to 18 serum samples which had been acid treated, five had 90-100% of the original PAF remaining after 24 h (showing that the acid treatment was effective), eight had from 10-90% of the original PAF remaining after 24 h, and five samples had 0-10%. The extent to which PAF:AH was susceptible to deactivation was not associated with the activity in the serum prior to treatment, the serum oestradiol concentration, or the cause of infertility. The period of acidification and the incubation temperature were assessed to develop a new acid-treatment protocol (20 min acid treatment at 37 degrees C) which was able to deactivate PAF:AH effectively in all sera (53/53) examined. A trial was performed to assess the effect of acid treatment of serum for 5 min at room temperature compared with the new protocol (20 min at 37 degrees C) on IVF outcome, following PAF supplementation of IVF culture medium. Oocyte recovery, fertilization and embryo development rates were equivalent for both groups and approximately equal numbers of embryos were transferred or cryopreserved. Pregnancy rates were not significantly different (14.6 versus 20.0%) for the two treatments, with a trend towards a higher pregnancy rate with the new acid- treatment protocol. The results show that this new procedure for acid treatment of serum in combination with PAF supplementation does not have detrimental effects on embryos and their pregnancy outcome and is therefore suitable for use in IVF.      

Hydrosalpinges adversely affect markers of endometrial receptivity

While in-vitro fertilization (IVF) was initially developed in women with tubal factor infertility, recent clinical studies have suggested that the presence of hydrosalpinges lowers implantation and pregnancy rates. We postulated that these hydrosalpinges cause impaired endometrial receptivity. A total of 103 women with hydrosalpinges were prospectively evaluated, and compared with 55 infertile and 44 fertile controls. All women had endometrial biopsies during the window of implantation, analysed by conventional histological criteria, and also stained for three integrin markers of endometrial receptivity (alpha1beta1, alpha4beta1 and alpha vbeta3). Women with hydrosalpinges (cases) expressed significantly less of the alpha vbeta3 integrin compared with controls. There was no difference in expression of alpha1beta1 or alpha4beta1 among groups. A significantly greater number of cases had out of phase histology and missing alpha vbeta3 (type I defects) and absent integrin expression despite normal histological maturation (type II) defects, compared with controls. Of 20 women with impaired endometrial receptivity who were also biopsied after hydrosalpinx surgery, 70% demonstrated increased alpha vbeta3 expression. Seventy-seven percent of type I and 57% of type II defects were corrected postoperatively. Using markers of endometrial receptivity, this study demonstrates that inflammatory hydrosalpinges have an adverse effect on endometrial receptivity, which in some cases may be overcome by surgical treatment of the hydrosalpinx.