Comprehensive analysis of UGT1A1 polymorphisms through high-resolution melting analysis and DNA sequencing.

BACKGROUND: Uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1) is a key conjugating enzyme of bilirubin and the anti-tumor medication irinotecan. Comprehensive analysis of UGT1A1 gene polymorphisms may provide benefit by predicting pharmacokinetics and outcomes of treatment with irinotecan and certain antiviral medications. METHODS: A high-resolution melting (HRM) analysis was designed to characterize the UGT1A1 gene. Genomic DNA from 110 healthy subjects was extracted from peripheral blood samples. The promoter and 11 exons from UGT1A1 were screened by HRM, and all results were subsequently confirmed by direct DNA sequencing. RESULTS: HRM analysis readily identified UGT1A1 gene mutations. We identified 5 different known variants of UGT1A1 including c.211 G > A; G71R, c.686 C > A; P229Q, c.1091 C > T; c.-3279 T > G; and c.-3156 G > A in 110 normal Taiwanese individuals. We also identified 8 new sequence variants, namely, c.-3296 C > T; c.43 C > A; c.45 G > A; c.234 G > A; c.577 G > A; c.614 C > T; c.1011 T > C; and c.1352 C > T. Each UGT1A1 variant was easily identifiable by differences in curves plotted from HRM data. CONCLUSIONS: HRM analysis was rapid, accurate, and economical for screening UGT1A1 gene mutations.

Antimutagenic and antioxidant properties of milk-kefir and soymilk-kefir.

This study was aimed at evaluating the antimutagenic and antioxidant properties of milk-kefir and soymilk-kefir. Such antimutagenic activity was determined by means of the Salmonella mutagenicity assay, whereas the antioxidant properties of kefir were evaluated by assessing the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity, lipid peroxidation inhibition activity, ferrous ion chelating ability, reducing power, and antioxidative enzyme activity. Both milk-kefir and soymilk-kefir demonstrated significantly greater antimutagenic activity than milk and soymilk. Milk-kefir and soymilk-kefir also displayed significantly greater scavenging activity upon DPPH radicals, an inhibition effect upon linoleic acid peroxidation, and more substantial reducing power but displayed a reduced glutathione peroxidase activity than was the case for milk and soymilk. Milk and soymilk fermented by kefir grains did not alter the ferrous ion chelating ability and superoxide dismutase activity of the original materials. These findings have demonstrated that milk-kefir and soymilk-kefir possess significant antimutagenic and antioxidant activity and suggest that milk-kefir and soymilk-kefir may be considered among the more promising food components in terms of preventing mutagenic and oxidative damage.

Stevens-Johnson syndrome following cataract surgery.

A case of Stevens-Johnson syndrome in a healthy 58-year-old woman who underwent cataract surgery under topical anesthesia is reported. General skin erosions developed 2 hours after surgery. The patient's family doctor diagnosed that she was allergic to seafood. One month later, she underwent phacoemul-sification surgery in the other eye. After surgery, she developed Stevens-Johnson syndrome with general skin lesions, erythema nodosa, genital mucosa erosion, oral ulcers, gastritis, and conjunctiva edema. The symptoms subsided 2 weeks later after immunotherapy. Although nonpreserved anesthesia (2% lidocaine) has seldom been reported to cause allergic reactions via the intravenous or the intramuscular route, it is possible that an intracameral injection of 0.2% lidocaine during cataract surgery can induce Stevens-Johnson syndrome. Careful evaluation of the patient's history and proper treatment were recommended to prevent additional complications.

Increase EGFR Mutations Detection Rate in Lung Adenocarcinoma by Real-Time PCR Screening Followed by Direct Sequencing.

BACKGROUND: Recently, a number of small-molecule tyrosine kinase inhibitors (TKIs) have been developed to target the ATP-binding cleft of the epidermal growth factor receptor (EGFR). The presence of EGFR mutations in non-small cell lung cancer (NSCLC) correlates with the responsiveness to TKIs. Therefore, the identification of EGFR mutations before the administration of TKIs of NSCLC has become important. The aim of the present study was to investigate the occurrence of EGFR mutations in the southern Taiwanese population with NSCLC using a combination of real-time polymerase chain reaction (PCR) kit and direct sequencing. METHODS: In the present study, DNAs were extracted from 249 cases of formalin-fixed, paraffin-embedded NSCLC samples for clinical EGFR mutational analysis by real-time PCR kit and direct sequencing. RESULTS: The results showed that the frequency of EGFR mutations is 63% in the southern Taiwanese population. Most of the EGFR mutations are located at exons 19 and 21. In addition, we indicated that a combination of real-time PCR kit and direct sequencing increases the rate of mutation by 4%. Direct sequencing revealed 9 EGFR mutations including 6 reported EGFR mutations and 3 novel EGFR mutations. CONCLUSIONS: In the present study, we have demonstrated that a combination of real-time PCR kit and direct sequencing increases the detection rate of EGFR mutations. Therefore, our proposed EGFR mutation detection strategy could be applied in clinical settings. In addition, our results indicated the prevalence of EGFR mutational status in the southern Taiwanese population.

Infantile facioscapulohumeral muscular dystrophy revisited: Expansion of clinical phenotypes in patients with a very short EcoRI fragment.

Contrary to the classical form, infantile facioscapulohumeral muscular dystrophy (FSHD) usually denotes a severe phenotype and is frequently associated with extramuscular involvements. To elucidate the genotype-phenotype correlation in this severe subgroup, we identified a cohort of nine patients with infantile FSHD who also carried a very short (10-13kb) EcoRI fragment. Their current age ranged from 8 to 33 years and age of onset ranged from 0.4 to 5 years. One patient even manifested his first FSHD-related symptoms at as early as 5 months of age, including inability to smile, poor response to call, and infantile spasms. To date, four patients were wheelchair-bound and six patients had asymmetric weakness. Sensorineural hearing loss and abnormal fundoscopic findings were observed in eight and all of patients respectively. Three with the smallest EcoRI fragments (10-11kb, with normal length being 50-300kb) had mental retardation. Two of these had epilepsy. Cardiac arrhythmias were found in five patients. Restrictive ventilatory defects were observed in seven patients, with one progressing to chronic respiratory failure. Two had swallowing difficulties; one of these required gastrostomy. We identified several rarely reported phenotypes in infantile FSHD, including cardiac arrhythmia, respiratory insufficiency, and swallowing difficulties. There seems to be a correlation between the severity of phenotype and the very short EcoRI fragment in the chromosome 4q35 region. We conclude that the high frequency of multi-organ involvements in this severe FSHD variant suggests the need for an early and multidisciplinary intervention.

Development of a high-resolution melting method for the screening of Wilson disease-related ATP7B gene mutations.

BACKGROUND: Wilson disease is an autosomal recessive inherited disorder of copper metabolism. The condition is characterized by excessive deposition of copper in many organs and tissues. The major physiologic aberration is excessive absorption of copper from the small intestine and impaired biliary copper excretion. The genetic defect is located at copper-transporting adenosine triphosphatase (ATPase) gene (ATP7B). METHODS: A high-resolution melting analysis (HRM) was designed to characterize the ATP7B hotspot mutations. Genomic DNA was extracted from peripheral blood samples from 14 patients and 50 normal controls. The 21 exons of ATP7B were screened by HRM analysis. Our methodology was confirmed by direct DNA sequencing. RESULTS: We have confirmed the 10 different hotspot mutations and 7 polymorphisms in the ATP7B gene, and also identified 1 newly-identified sequence variant (p.A476T) and 1 novel SNP (p.L776L) in 50 normal Taiwanese individuals. We estimate that the carrier frequency of WD in the Taiwanese population as probably 0.03. CONCLUSIONS: HRM analysis is accepted as a rapid, accurate and low-cost method to screen ATP7B gene mutations.

Detection of the JAK2 V617F missense mutation by high resolution melting analysis and its validation.

BACKGROUND: Janus kinase 2 (JAK2) is a tyrosine kinase involved in the cytokine signaling of several growth factors such as erythropoietin and thrombopoietin in normal and neoplastic cells. The G to T exchange at nucleotide 1849 in exon 14 of the JAK2 gene leads to a substitution of valine with phenylalanine at the amino acid position 617 (V617F) of the JAK2 protein. Currently, the occurrence of the JAK2 V617F mutation is well recognized in chronic myeloproliferative disorders (MPDs). METHODS: We identified JAK2 V617F missense mutation in patients with MPD by high resolution melting (HRM) analysis. HRM analysis is a new gene scan tool that quickly performs the PCR and identifies sequences alterations without requiring post-PCR treatment. This study included 7 PV patients (41.1%), 6 ET patients (35.3%), and 4 myelofibrosis patients (23.5%). Additionally, our methodology was compared with amplification refractory mutation system (ARMS) assay. RESULTS: Up to 5% of the JAK2 V617F mutation was successfully detected in patients with MPD using HRM analysis. Eleven out of 17 patients (64.7%) were positive for the presence of JAK2 V617F mutation. The prevalence of mutation in the different subtypes of MPDs was 85.7% in PV (6 of 7 patients), 66.7% in ET (4 of 6) and 5.9% in myelofibrosis (1 of 4). The results proved 100% comparable to those obtained by ARMS assay. CONCLUSIONS: The HRM analysis is a rapid and effective technique for the detection of JAK2 V617F missense mutation.

Hypocholesterolaemic effects of milk-kefir and soyamilk-kefir in cholesterol-fed hamsters.

This study aimed to evaluate the hypocholesterolaemic property of milk-kefir and soyamilk-kefir. Male hamsters were fed on a cholesterol-free or cholesterol-enriched diet containing 10 % skimmed milk, milk-kefir, soyamilk or soyamilk-kefir for a period of 8 weeks. The soyamilk, milk-kefir and soyamilk-kefir diets all tended towards a lowering of serum triacylglycerol and total cholesterol concentrations, and a reduction of cholesterol accumulation in the liver, the decrease in serum cholesterol concentration being mainly in the non-HDL fraction. The soyamilk-kefir diet led to a significant increase in the faecal excretion of neutral sterols and bile acids compared with the other two diets. The soyamilk-kefir diet also elicited a significant decrease in the serum ratio of non-HDL-cholesterol to HDL-cholesterol, compared with the control, than was the case for the other diets. These findings demonstrate that soyamilk-kefir may be considered to be among the more promising food components in terms of preventing CVD through its hypocholesterolaemic action.

Change in Quality of Chinese-Style Sausage Inoculated with Lactic Acid Bacteria during Storage at 3°C and 25°C †.

To develop the utilization of lactic acid bacteria in Chinese-style sausage, Lactobacillus plantarum and a commercial culture (DS-66) were used as a starter cultures for manufacturing Chinese-style sausage. The products were vacuum packaged and stored at 3 or 25°C. The results showed that the pH value and residual sodium nitrite of the sausage significantly declined as storage time increased (P< 0.05). However, the thiobarbituric acid (TBA) and volatile base nitrogen (VBN) values gradually increased as storage progressed. Lactic acid bacteria propagation increased rapidly during the initial storage time and then decreased slightly after 4 weeks of storage. The sausage inoculated with one of the starter cultures had a lower pH value and could suppress the TBA and VBN values, dissipating residual sodium nitrite, and could inhibit spoilage bacteria during the storage time.

Antitumor activity of milk kefir and soy milk kefir in tumor-bearing mice.

The effects of oral administration of milk and soy milk kefirs on tumor growth in tumor-bearing mice and the mucosal immunoglobulin A response in mice were studied. Oral administration of milk and soy milk kefirs to mice inoculated with sarcoma 180 tumor cells resulted in 64.8% and 70.9% inhibition of tumor growth, respectively, compared with controls. In addition, oral administration of the two kefir types induced apoptotic tumor cell lysis. Total immunoglobulin A levels for tissue extracts from the wall of the small intestine were also significantly higher for mice fed a milk kefir or a soy milk kefir regimen for 30 days. These results suggest that milk and soy milk kefirs may be considered among the more promising food components in terms of cancer prevention and enhancement of mucosal resistance to gastrointestinal infection.

Production of recombinant porcine lactoferrin exhibiting antibacterial activity in methylotrophic yeast, Pichia pastoris.

Lactoferrin is a metal-binding glycoprotein exhibiting multifunctional immunoregulation of antibacterial, antioxidant, anti-endotoxin and antiviral activities. Uptake of porcine lactoferrin (PLF) has been shown to enhance resistance to diarrhea and anemia in neonatal piglets. In this study, the methylotrophic yeast, Pichia pastoris, was used to express a recombinant PLF (rPLF) gene from swine mammary gland. A synthetic secretion cassette was constructed using the inducible promoter of the alcohol oxidase-1 gene (AOX1) and the yeast alpha-mating factor signal peptide. After electroporation and Zeocin selection, several clones expressed high levels of rPLF protein which constitutes more than 30% of the total protein. A time-course study showed that rPLF mRNA transcripts are stably expressed during 120 h of culture induction. rPLF was exported into the culture supernatant at approximately 87 mg/l and a large portion of rPLF was accumulated in the cell cytoplasm at approximately 760 mg/l after 72 h of methanol induction. Recombinant PLF protein was purified via a heparin column using a fast protein liquid chromatography system. The glycosylation of P. pastoris-derived rPLF was analyzed and similar patterns to milk PLF were observed. Pepsin hydrolysate of rPLF displayed high bactericidal activity against Escherichia coli ATCC 25922 under scanning electron microscopy observation and minimal inhibitory concentration and minimal bactericidal concentration tests. Our results suggested that the methylotrophic yeast-inducible system is suitable for large-scale production of active antibacterial rPLF glycoprotein.