Prevalence and genotypes of alpha- and beta-thalassemia carriers in Hong Kong -- implications for population screening

BACKGROUND: The thalassemias are common in southern China. We determined the prevalence of heterozygous carriers of these genetic disorders in Hong Kong and assessed the feasibility of a community-based screening program. METHODS: An educational and screening program for the thalassemias was carried out in three high schools with a total of 2420 students. Seventy-five percent of the students agreed to undergo screening, which consisted of blood counts, hemoglobin electrophoresis, serum ferritin measurements, and DNA analyses. RESULTS: Of the 1800 blood samples tested, 150 (8.3 percent) had microcytosis (mean corpuscular volume, <80 microm3). Ninety students (5.0 percent) were carriers of alpha-thalassemia, of whom 81 (4.5 percent) were carriers of the Southeast Asian type of deletion, in which both alpha-globin genes on the same chromosome 16 are deleted. Sixty-one students (3.4 percent) were carriers of either beta-thalassemia or the mutation coding for hemoglobin E. Six students were carriers of both alpha- and beta-thalassemias. On the basis of these figures, the estimated numbers of pregnancies in Hong Kong in which the fetus is at risk for homozygous alpha-thalassemia and beta-thalassemia major or intermedia are 145 and 80 per year, respectively. In Hong Kong the actual numbers of women referred for prenatal diagnoses of these disorders are approximately 95 and 40 per year, respectively. CONCLUSIONS: Despite the availability of hospital-based screening and prenatal diagnosis for many years in Hong Kong, many women carrying fetuses at risk for thalassemia are not referred for genetic counseling. A community-based program of education, screening, and counseling is needed in Hong Kong and southern China.     

Comparison of the Ca2+ movement by activation of alpha1-adrenoceptor subtypes in HEK-293 cells

We studied the Ca2+ movement induced by activation of alpha1A-, alpha1B- and alpha1D-adrenoceptor subtypes in transfected HEK-293 cells with the fura-2 probe. All these alpha1-AR subtypes induced both Ca2+ release and Ca2+ entry. The effect on Ca2+ release in alpha1b transfected HEK-293 cells was bigger than that in alpha1a and alpha1d transfected HEK-293 cells, and the effects on Ca2+ entry were the same in alpha1a, alpha1b and alpha1d transfected HEK-293 cells. The Ca2+ entry was inhibited by 1 mM NiSO4, but not by nifedipine. Cyclopiazonic acid (CPA) produced a biphasic Ca2+ signal response in Ca2+ medium, and only induced a transient response in Ca2+-free medium. After depletion of CPA-sensitive Ca2+ pool by 10 microM CPA in Ca2+-free medium, 10 microM adrenaline (Adr) still transiently increased [Ca2+]i in three different alpha1-adrenoceptor subtype transfected HEK-293 cells. However, after depletion of adrenaline-sensitive Ca2+ pool by 10 microM Adr, CPA transiently elevated [Ca2+]i only in alpha1a and alpha1d transfected HEK-293 cells, not in alpha1b transfected HEK-293 cells. U73122, a phospholipase C (PLC) inhibitor, inhibited both Ca2+ release and Ca2+ entry induced by activation of alpha1A alpha1B and alpha1D subtypes in transfected HEK-293 cells. These results suggest that HEK-293 cell line contains two functionally separate intracellular Ca2+ pools, CPA-sensitive and Adr-sensitive pools. Activation of alpha1B-AR stimulates Ca2+ release from both CPA-sensitive and Adr-sensitive Ca2+ pools. Alpha1A and alpha1D subtypes induce Ca2+ release only from Adr-sensitive Ca2+ pool.     

Random versus selective membrane phospholipid oxidation in apoptosis: role of phosphatidylserine

The formation of reactive oxygen species has been associated with apoptosis. To assess the role of lipid peroxidation in apoptosis, we used 2,2'-azobis(2,4-dimethylisovaleronitrile) (AMVN) to generate peroxyl radicals within cellular membranes of HL-60 cells. cis-Parinaric acid (cis-PnA) metabolically integrated into phospholipids of HL-60 cells was used as a probe to assess the extent of lipid peroxidation within specific phospholipid classes. Within 2 h, AMVN (500 microM) randomly oxidized more than 85% of cis-PnA contained in all major classes of phospholipids. AMVN-induced lipid peroxidation was followed by apoptosis as determined by nuclear condensation, DNA fragmentation, and annexin V binding to externalized phosphatidylserine (PS). Fluorescamine derivatization of external aminophospholipids revealed that PS, but not phosphatidylethanolamine, was externalized. The vitamin E analogue, 6-hydroxy-2,2,5,7,8-pentamethylchromane (PMC), inhibited overall oxidation of cis-PnA in phospholipids by more than 85%. Not all phospholipids, however, were equally protected. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and sphingomyelin were nearly completely protected by PMC, while oxidation of PS was unaffected in whole living cells. The insensitivity of PS to PMC was not an intrinsic property because PMC protected all lipids equally during AMVN oxidation of liposomes prepared from cis-PnA-labeled cells. The potential role for PS oxidation in apoptosis was further suggested by the faithful execution of apoptosis following coexposure of cells to AMVN and PMC.     

The transducing pathway of Ca2+ influx during progesterone-initiated acrosome reaction of guinea pig sperm

Although idiopathic membranous nephropathy (IMN) is thought to represent a diffuse glomerulopathy, it was found that three of 31 children histologically diagnosed as IMN showed focal and segmental deposition of immunoglobulin G (IgG) and C3 on the glomerular capillary walls. The present study attempted to comparatively investigate clinical and pathological features of the diffuse group and the focal segmental group in 31 IMN children. Immunofluorescence study revealed that 28 of 31 IMN exhibited diffuse granular deposition of IgG along glomerular capillary walls. In contrast, focal and segmental deposition of IgG and C3 was noted in three children with IMN. In addition, focal and segmental electron-dense deposits were identified in these cases. In two children of the focal segmental group, immunofluorescent patterns of IgG deposition were unchanged even at the second biopsy. The focal segmental form of IMN tended to occur in younger children than diffuse IMN. However, other clinical parameters such as the range of proteinuria, hematuria, serum albumin and prognosis did not show any significant differences between both groups. Electrophoretic profiles of urinary proteins on sodium dodecylsulfate-polyacrylamide gel electrophoresis were not different between both groups. It is proposed that the focal segmental form of IMN may have a distinctive glomerulopathy from the typical form of IMN.     

Overexpression of human DNA topoisomerase II alpha by fusion to enhanced green fluorescent protein

DNA topoisomerase (topo) II alpha is a major target for many anticancer agents. However, progress towards understanding how these agents interact with this enzyme in human cells and how resistance to these agents arises is greatly impeded by difficulties in expressing this gene. Here, we report on achieving a high level of expression of a full-length human topo II alpha gene in human cells. We started with the topo II alpha cDNA driven by a strong cytomegalovirus promoter and transiently transfected HeLa cells. Although topo II alpha mRNA was consistently detected in transfected cells, no exogenous topo II alpha protein was detected. By contrast, when the same cDNA was fused to an enhanced green fluorescent protein (EGFP), we detected a high level of expression at both mRNA and protein levels. The exogenous topo II alpha was localized to cell nuclei as expected, indicating that the fusion protein is properly folded. Furthermore, overexpression of the EGFP-topo II alpha fusion protein increased the sensitivity of the transfected cells to teniposide, suggesting that it functions as the endogenous counterpart. Thus, in addition to being used as a gene tag, the GFP fusion approach may be generally applicable for expressing genes, such as topo II alpha, that are difficult to express by conventional methods.