Rapid molecular characterization of Hb Queens and Hb Siam: two variants easily misidentified as sickle Hb

OBJECTIVE: To establish a rapid differential diagnosis of hemoglobin (Hb) Queens and Hb Siam from other clinically relevant variants. DESIGN AND METHODS: Molecular and hematological features associated with two pregnant Thai women who were mistaken for Hb S were investigated. A simultaneous DNA diagnosis based on multiplex allele specific PCR approach was developed and tested with other common variants. RESULTS: Apart from mild anemia, the two subjects were generally healthy. DNA analysis identified that they were respectively carriers of Hb Siam [alpha15(A13)Gly-Arg] and Hb Queens [alpha34(B15)Leu-Arg]. A successful application of the multiplex allele specific PCR for differential diagnosis was demonstrated. CONCLUSION: Diagnosis of these clinically relevant hemoglobinopathies is problematic in the routine setting, and the method developed should prove useful in complementing routine Hb analysis for providing accurate diagnosis.     

Can adiponectin predict gestational diabetes?

The aim of the present study was to evaluate whether adiponectin is a predictive factor for gestational diabetes mellitus (GDM) and is appropriate as a screening test for GDM. Three-hundred and fifty-nine women with singleton pregnancy and indications for GDM screening according to criteria of the American College of Obstetricians and Gynecologists were enrolled in the study between July 5, 2004 and March 11, 2005. After confirming gestational age (GA) and number of fetuses by ultrasound, all women underwent a 1-h glucose challenge test with 50 g glucose load (50-g GCT) between 21 and 27 weeks of GA. Blood samples for determination of adiponectin levels were also obtained on the same day. Subsequently, between 24 and 28 weeks of GA, the women underwent an oral glucose tolerance test with 100 g glucose load (100-g OGTT). The diagnosis of GDM was established when two or more of the following criteria were fulfilled: (1) fasting glucose >95 mg/dl; (2) 1-h glucose >180 mg/dl; (3) 2-h glucose >155 mg/dl; (4) 3-h glucose >140 mg/dl. Sixty women were diagnosed with GDM, a prevalence of 16.7%. There was no difference in age between the GDM and non-GDM groups. Pre-pregnancy and sampling-day body mass index (BMI), increase in weight and all blood glucose levels were greater in women with GDM than in those without (p < 0.05). Adiponectin concentrations were significantly negatively correlated with GA and plasma glucose levels of the GCT and each OGTT. Using logistic regression analyses, adiponectin, but not age, pre-pregnancy BMI and increase in weight, was demonstrated as an independent predictive factor for GDM. The area under the receiver-operator characteristic curve of adiponectin was significantly lower than that of the GCT [0.63 (95% confidence interval (CI) 0.53-0.67) vs. 0.73 (95% CI 0.71-0.80), p < 0.001]. At a cut-off value of 140 mg/dl of the 50-g GCT, the sensitivity and specificity of the test were 90% and 61%, respectively. The 50-g GCT could identify GDM in 54 (90%) out of 60 women. On the other hand, at an arbitrary cut-off value of 10 microg/ml for adiponectin, sensitivity of 91% and specificity of 31% were achieved. If this cut-off value was used for ruling in or out pregnant women for the GDM screening, 27% of all women could be eliminated from needing to perform an OGTT, with five women (8.3%) misclassified. In conclusion, this study demonstrated that adiponectin was an independent predictor for GDM. As for GDM screening, adiponectin was not as strong a predictor as GCT. However, with advantage of being less cumbersome, adiponectin could be used to rule out pregnant women at low risk of GDM.     

Different AhR binding sites of diterpenoid ligands from Andrographis paniculata caused differential CYP1A1 induction in primary culture in mouse hepatocytes

Andrographis paniculata has been employed as a folklore remedy. Andrographolide (Andro), 14-deoxy-11,12-didehydroandrographolide (DHA), andrographiside (AS), and neoandrographolide (Neo), are major diterpenoids isolated from this plant. In the present study, influence of the four diterpenoids on CYP1A1 mRNA expression was investigated in primary cultured mouse hepatocytes. Additionally, binding of these compounds to aryl hydrocarbon receptor (AhR) was examined using molecular docking analysis to clarify mechanism of CYP1A1 induction. Andro and DHA induced CYP1A1 expression by itself, and co-treatment with a CYP1A1 inducer (BNF, beta-naphthoflavone) showed a synergistic increase of CYP1A1 expression. Andro demonstrated higher enhancing activity than DHA at every similar concentration. On the other hand, Neo suppressed BNF-induced CYP1A1 expression, but AS did not modify the induction. Results from molecular docking analysis of BNF and four diterpenoids on ligand binding domain of AhR were consistent with levels of CYP1A1 mRNA expressions. Furthermore, difference of binding sites of BNF in the presence of diterpenoids might affect the synergism or inhibition of CYP1A1 expression. These results suggest that use of A. paniculata as a health supplement should be concerned in term of herb-drugs interactions or risk of carcinogenesis, according to its ability to influence CYP1A1 expression.     

Impact of Andrographis paniculata crude extract on mouse hepatic cytochrome P450 enzymes

Modulatory influence of Andrographis paniculata crude extract on cytochrome P450 (CYP) enzymes was performed by administration of the crude extract of Andrographis paniculata to ICR male mice. Total hepatic P450 content was not significantly modified by either the aqueous or the alcoholic extracts of Andrographis paniculata. Assessment of hepatic microsomal P450 activities by alkoxyresorufin O-dealkylations noted that both the aqueous and alcoholic extracts of Andrographis paniculata significantly increased ethoxyresorufin O-dealkylase and pentoxyresorufin O-dealkylase activities, while those of methoxyresorufin O-dealkylase activities were not elevated. These results suggested that Andrographis paniculata might effectuate hepatic cytochrome P450 enzymes of which CYP1A1 and CYP2B are the responsive P450 isoforms.     

A simplified screening strategy for thalassaemia and haemoglobin E in rural communities in south-east Asia

OBJECTIVE: To evaluate a simple screening strategy for thalassaemia and haemoglobin (Hb) E in a prevention and control programme for thalassaemia in rural communities with limited resources. METHODS: Blood samples from 301 Thai-Khmer participants were screened for thalassaemia and Hb E using a combined modified one-tube osmotic fragility (OF) test and a modified dichlorophenolindophenol (DCIP) precipitation test. Results were evaluated with standard haematological analyses including erythrocyte indices, Hb typing and quantification and polymerase chain reaction (PCR) analysis of alpha-globin and beta-globin genes. FINDINGS: Participants were divided into four groups according to the results of the combined tests. Altogether, 104 of 301 participants (34.6%) had negative results on both tests; 48 (15.9%) were positive on the OF test but not the DCIP test; 40 (13.3%) were negative on the OF test but positive on DCIP test; and 109 (36.2%) were positive on both tests. No carrier of clinically significant forms of thalassaemia (alpha(o)-thalassaemia, beta-thalassaemia) or Hb E was found among the group that had negative results for both tests. All participants with Hb E had positive DCIP tests. Carriers of alpha+-thalassaemia or Hb Constant Spring could generate either positive or negative OF test results but they all had negative DCIP tests. Using both tests as a preliminary screening for the three important groups of carriers gave a sensitivity of 100% and a specificity of 69.8%. The positive predictive value of the combined test was 77.2%. The negative predictive value was 100%. Further evaluation of the screening system by local staff at three community hospitals found a sensitivity of 98.1-100% and a specificity of 65.4-88.4% with positive predictive values of 75.0-86.9% and negative predictive values of 98.1-100%. CONCLUSION: A combined test using OF and DCIP could be used as an effective preliminary screening alternative to an electronic blood cell count for identifying carriers with alpha(o)-thalassaemia, beta-thalassaemia and Hb E. The strategy should prove useful for population screening in prevention and control programmes in rural communities in south-east Asia where laboratory facilities and economic resources are limited.     

Mutation analysis of the GNE gene in distal myopathy with rimmed vacuoles (DMRV) patients in Thailand

Distal myopathy with rimmed vacuoles (DMRV) is an early-adult-onset, distal myopathy caused by a mutation of the UDP-N-acetylglucosamine 2 epimerase/N-acetylmannosamine kinase (GNE) gene. We herein report four Thai patients with DMRV who carried compound heterozygous mutations of the GNE gene including three novel (p.G89R, p.P511T, and p.I656N) and two known mutations (p.A524V and p.V696M). All patients shared p.V696M in one allele. Our study demonstrates the mutation spectrum of the GNE gene in Thai patients with DMRV.     

Atypical hemoglobin H disease in a Thai patient resulting from a combination of α-thalassemia 1 and hemoglobin Constant Spring with hemoglobin J Bangkok heterozygosity

A case of hemoglobin H disease in combination with hemoglobin Constant Spring and a beta-globin chain variant is reported in a 3-yr-old Thai girl. On routine cellulose acetate electrophoresis, one abnormal band in addition to the hemoglobins A, A2, H, Bart's and Constant Spring was detected. The amount of this abnormal band movement towards more anodic to the hemoglobin A was 35.7%. DNA analysis of the alpha-globin gene cluster by polymerase chain reaction (PCR) revealed a combination of defects caused by the SEA-type alpha-thalassemia 1 and the alpha-Constant Spring gene. Analysis of beta-globin gene by PCR and DNA sequencing also detected the heterozygosity for the GGC-GAC mutation at codon 56, leading to a substitution of aspartic acid for glycine resulting in the hemoglobin J Bangkok. The hematologic data of this unusual case of hemoglobin H disease are presented and compared with two compound heterozygotes for hemoglobin J Bangkok and alpha-thalassemia 1 found in the patient's father and grandfather. A simple DNA assay based on an allele-specific PCR for rapid diagnosis of the hemoglobin J Bangkok is also described.     

Methanol extract of the ethnopharmaceutical remedy Smilax spinosa exhibits anti-neoplastic activity

Plants have been the source of several effective drugs for the treatment of cancer and over 60% of anticancer drugs originate from natural sources. Therefore, extracts of the rhizome of Smilax spinosa, an ethnomedicinal plant from Guatemala which is used for the treatment of inflammatory conditions, were investigated regarding their anti-neoplastic activities. By using several solvents the methanol extract was by far the most potent against HL60 cell proliferation (50% inhibition at 60 μg/ml). Furthermore, fractionation of this extract yielded fraction F2, which exhibited enforced pro-apoptotic activity, and activated CYP1A1. Proteins that are relevant for cell cycle progression and apoptosis, as well as proto-oncogenes were investigated by western blotting. This revealed that the methanol extract increased the levels of p21 and this may have caused cell cycle attenuation. The derivative fraction F2 induced apoptosis through the intrinsic pathway, which correlated with the inhibition of Stat3 phosphorylation and concomitant induction of caspase 9, then caspase 8 and caspase 3. In summary, the methanol extract and the derivative fraction F2 of S. spinosa showed anti-neoplastic effects in HL-60 cells and CYP1A1 activation in estrogen receptor-positive MCF-7 breast cancer cells but not in estrogen-negative MDA-MB231 breast cancer cells. Based on our data Smilax spinosa may be a promising source for novel anticancer agents.