Increased Phosphatidylserine on Blood Cells in Oral Squamous Cell Carcinoma.

The specific function of phosphatidylserine (PS) in the context of the development of a hypercoagulable state among individuals with oral squamous cell carcinoma (OSCC) is uncertain. The goal of this study was therefore to assess the exposure of PS on microparticles (MPs) as well as on endothelial and blood cells and to assess procoagulant activity (PCA) as a function of the stage of OSCC progression. We recruited patients with OSCC (n = 63) as well as healthy controls (n = 26) to participate in this study. PS exposure was then assessed via confocal microscopy and flow cytometry, revealing that patients with stage III/IV OSCC exhibited higher frequencies of PS-exposing blood cells, MPs, and serum-cultured endothelial cells (ECs) than did patients with stage I/II OSCC or healthy controls. When we conducted functional coagulation assays, we discovered that PS+ blood cells, MPs, and serum-cultured ECs from patients with stage III/IV OSCC mediated more rapid coagulation and more substantial production of FXa, thrombin, and fibrin as compared with controls. When samples were treated with the PS antagonist lactadherin, this resulted in an 80% disruption of PCA. Strikingly, when pre- and postoperative samples were compared from patients with stage III/IV OSCC undergoing resective surgery, PCA was significantly reduced in the postoperative samples. After stimulating ECs with inflammatory cytokines, we found by confocal microscopy that they expose PS on their cell membranes, thus generating FVa and FXa binding sites and mediating the formation of fibrin. Together our findings provide evidence that PS+ blood cells and MPs are important mediators of the development of a hypercoagulable and prothrombotic state among individuals afflicted by advanced-stage OSCC. As such, a PS blockade may be a viable therapeutic strategy for treating such patients.

Predominance of Cryptococcus neoformans var. grubii multilocus sequence type 5 and emergence of isolates with non-wild-type minimum inhibitory concentrations to fluconazole: a multi-centre study in China.

There are few data on the molecular epidemiology of cryptococcosis in China. Here we investigated the species distribution, molecular types and antifungal susceptibilities of 312 Cryptococcus neoformans species complex isolates from ten hospitals over 5 years. Isolates were identified by internal transcribed spacer (ITS) sequencing and by two matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems. Multilocus sequence typing (MLST) was used to verify species/variety and to designate molecular types. Susceptibility to six antifungal drugs was determined by the Sensititre YeastOne™ method. Cryptococcus neoformans was the predominant species (305/312 isolates (97.8%), all were ITS type 1, serotype A), of which 89.2% (272/305) were C. neoformans var. grubii MLST sequence type (ST) 5 and 6.2% (19/305) were ST31. Other C. neoformans var. grubii STs were rare but included six novel STs. Only two strains were C. neoformans var. neoformans (both serotype AD). Cryptococcus gattii was uncommon (n = 7, four ITS types) and comprised five MLST STs including one novel ST. For C. neoformans var. grubii, the proportion of isolates with non-wild-type MICs to fluconazole significantly rose in the fourth study year (from 0% (0/56 isolates) in the first year to 23.9% (17/71) in the fourth year), including five isolates with fluconazole MICs of ≥32 mg/L. The study has provided useful data on the species epidemiology and their genetic diversity and antifungal susceptibility. The proportional increase in isolates with non-wild-type MICs to fluconazole is noted.

Prevalence and genetic characterization of Toxoplasma gondii infection in bats in southern China.

Toxoplasma gondii can infect a wide variety of warm-blooded animals, including bats. Limited information on T. gondii infection in bats is available in China. The objective of the present study was to determine prevalence and genetic diversity of T. gondii infection in bats in southern China. A total of 608 bats representing 12 species, including 120 Aselliscus stoliczkanus, 59 Myotis chinensis, 11 Miniopterus schreibersii, 53 Rhinolophus affinis, 32 Rhinolophus pusillus, 81 Hipposideros armiger, 28 Hipposideros fulvus, 32 Cynopterus brachyotis, 14 Cynopterus sphinx, 45 Eonycteris spelaea, 109 Hipposideros larvatus, and 24 Taphozous melanopogon, were collected from Yunnan and Guangxi provinces, southern China. They were examined for the presence of T. gondii DNA by amplification of the B1 gene using a nested PCR, and the positive samples were genotyped at 11 genetic loci (SAG1, 5'- and 3'-SAG2, alternative SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) using multilocus polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technology. Fifty-nine (9.7%) of these bats were detected positive by PCR but only five of these positive DNA samples were completely typed at all loci; of which 4 samples, 2 from A. stoliczkanus, and 2 from H. larvatus, belonged to ToxoDB Genotype #10, and the other one from H. larvatus was identified as ToxoDB Genotype #9 (http://toxodb.org/toxo/). To our knowledge, this is the first report of molecular detection and genetic characterization of T. gondii infection in bats in China. The results show that these bats are potential reservoirs for T. gondii transmission, which may pose a threat to human health.

Effects of overexpression of endogenous phenylalanine ammonia-lyase (PALrs1) on accumulation of salidroside in Rhodiola sachalinensis.

Salidroside, a novel effective adaptogenic drug extracted from the medicinal plant Rhodiola sachalinensis A. Bor, can be derived from phenylalanine or tyrosine. Due to the scarcity of R. sachalinensis and its low yield of salidroside, there is great interest in enhancing production of salidroside by the plant. In this study, a cDNA clone encoding phenylalanine ammonia-lyase (PAL) was isolated from R. sachalinensis using rapid amplification of cDNA ends. The resulting cDNA was designated PALrs1. It is 2407-bp long and encodes 710 deduced amino acid residues. Southern blot analysis of genomic DNA indicated that the PAL gene family is composed of three to five genes in the R. sachalinensis genome. Northern blot analysis revealed that transcripts of PALrs1 were present in calli, leaves and stems, but expression in roots was very low. The PALrs1 under the 35S promoter with double-enhancer sequences from CaMV-Omega and TMV-Omega fragments was transferred into R. sachalinensis via Agrobacterium tumefaciens. PCR and PCR-Southern blot confirmed that the PALrs1 gene had been integrated into the genome of transgenic plants. Northern blot analysis revealed that the PALrs1 gene had been expressed at the transcriptional level. High-performance liquid chromatography indicated that overexpression of the PALrs1 gene resulted in a 3.3-fold increase in p-coumaric acid content, as expected. In contrast, levels of tyrosol and salidroside were 4.7-fold and 7.7-fold, respectively, lower in PALrs1 transgenic plants than in controls. Furthermore, overexpression of the PALrs1 gene resulted in a 2.6-fold decrease in tyrosine content. These data suggest that overexpression of the PALrs1 gene and accumulation of p-coumaric acid did not facilitate tyrosol biosynthesis; tyrosol, as a phenylethanoid derivative, is not derived from phenylalanine; and reduced availability of tyrosine most likely resulted in a large reduction in tyrosol biosynthesis and accumulation of salidroside.

Atemoyacin E, a bis-tetrahydrofuran annonaceous acetogenin from Annona atemoya seeds.

Atemoyacin E (I), a new adjacent bis-tetrahydrofuran annonaceous acetogenin was isolated and characterized from the seeds of Annona atemoya.

Enantioselective syntheses of monotetrahydrofuran Annonaceous acetogenins tonkinecin and annonacin starting from carbohydrates.

The total synthesis of two mono-THF acetogenins, tonkinecin (1) and annonacin (2), is reported in full detail. Terminal acetylene 3 prepared from D-glucono-delta-lactone and asymmetric dihydroxylation was employed as a common intermediate for both targets 1 and 2. Pd(0)-catalyzed coupling reaction of 3 with vinyl iodides 4 and 5, the chiral centers of which were taken from D-xylose and S-(-)-ethyl lactate, afforded enyne 26 and 27, respectively. Selective hydrogenation of 26 or 27 with diimide followed by removal of MOM ethers completed the synthesis of 1. A coupling reaction between the lithium derivative of 3 and epoxide 6 in the presence of boron trifluoride etherate gave 42. Both chiral centers in epoxide 6 were taken from L-ascorbic acid. Subsequent catalytic hydrogenation and MOM protection led to 43b. Introduction of the butenolide moiety by aldol condensation of protected S-lactal followed by cleavage of all MOM ethers completed the synthesis of 2.

[Diagnosis and treatment of Sturge-Weber syndrome associated with retinal detachment].

OBJECTIVE: To access the diagnosis and treatment of Sturge-Weber syndrome at late stage. METHODS: The male patient aged 65 visited the eye clinic with presenting symptom of visual loss of the left eye for 2 months. Ocular examination disclosed a facial hemangioma with the size of 2 cm x 3 cm on the nasal side of the left upper eyelid associated with engorged bulbar conjunctival and episcleral vessels in the upper nasal quadrant. Ophthalmoscopy revealed total detachment of the retina. Esotropia was measured as 10 degrees. Intraocular pressure was 24 mmHg. Electroretinogram (ERG) demonstrated an indistinguished pattern. Posterior trans-scleral and ciliary body cryocoagulation was applied followed by external release of the subretinal fluid. RESULTS: The retina became reattached with visual acuity of 0.3 and intraocular pressure was 15 mmHg of the left eye at 5 months postoperatively. Fluorescein angiographical findings were consistent with diffuse hemangioma of the choroid. Follow-up study for 6 years revealed that the retina remained attached with visual acuity of 0.8 and essentially normal electroretinogram. CONCLUSION: The small facial hemangioma was the clue for the diagnosis of Sturge-Weber syndrome and cryocoagulation was the procedure of choice for the treatment of retinal detachment with favorable visual outcome.

[Expression and characterization of two kinds of recombinant snake neurotoxins].

The cDNA encoding the precursor of cobrotoxin was cloned from the venom gland of the Chinese continental cobra (Naja naja atra) by RT-PCR. Its deduced amino acid sequence analysis showed that the mature protein was identical to that identified from the Taiwan cobra (Naja naja atra) by protein sequencing technique. The cDNA encoding the mature protein was then subcloned into the expression vector pMAL-P2. The gene of CM11, which was formed by ligation of the fragments of the synthetic oligonucleotides, was also cloned into the expression vector pMAL-P2. After induction of IPTG, both of the neurotoxins were overexpressed as soluble fusion proteins which were confirmed by SDS-PAGE and western blotting. The expressed fusion proteins was purified by sepharose 6B-amylose affinity chromatography and DEAE-sepharose FF chromatography. Both of the recombinant proteins achieved after digestion by factor Xa showed the in vivo toxicity.

Preparation and cDNA sequence analysis of two novel monoclonal antibodies against magaininII.

By using intrasplenic immunization and the conventional B lymphocyte hybridoma technique, we have established two novel hybridoma cell lines stably secreting specific monoclonal antibodies (MAbs) to magaininII, termed as 2D1 and 3F8, respectively. The two cell lines were then subjected to RNA extraction and the VH and VL segments were obtained by reverse transcription of RNA followed by polymerase chain reaction (RT-PCR) and characterized by nucleotide sequence analysis. The VH segments of 2D1 and 3F8 belong to the VH5 family and the VL segments of 2D1 and 3F8 belong to VK10 and VK1 groups, respectively. The two MAbs utilize different VL segments and have disparities in their HCDR3 regions, which may contribute to the different epitope recognition of the two antibodies.

The first total synthesis of annonacin, the most typical monotetrahydrofuran annonaceous acetogenins.

The first total synthesis of annonacin (1) was achieved by a highly convergent synthetic strategy. All the stereogenic centers were derived from three natural hydroxy acids respectively, except that those at C19 and C20 were produced from a Sharpless AD reaction.

The Linxian cataract studies. Two nutrition intervention trials.

OBJECTIVE: To determine whether the vitamin/mineral supplements used in two cancer intervention trials affected the risk of developing age-related cataracts. DESIGN: Two randomized, double-masked trials with a duration of 5 to 6 years and end-of-trial eye examinations. SETTING: Rural communes in Linxian, China. PARTICIPANTS: In trial 1, 2141 participants aged 45 to 74 years, and, in trial 2, 3249 participants aged 45 to 74 years. INTERVENTIONS: Multivitamin/mineral supplement or matching placebo in trial 1; factorial design to test the effect of four different vitamin/mineral combinations in trial 2 (retinol/zinc, riboflavin/niacin, ascorbic acid/molybdenum, and selenium/alpha-tocopherol/beta carotene). MAIN OUTCOME MEASURES: Prevalence of nuclear, cortical, and posterior subcapsular cataracts in treatment groups at end of trials. RESULTS: In the first trial, there was a statistically significant 36% reduction in the prevalence of nuclear cataract for persons aged 65 to 74 years who received the supplements. In the second trial, the prevalence of nuclear cataract was significantly lower in persons receiving riboflavin/niacin compared with persons not receiving these vitamins. Again, persons in the oldest group, 65 to 74 years, benefited the most (44% reduction in prevalence). No treatment effect was noted for cortical cataract in either trial. Although the number of posterior subcapsular cataracts was very small, there was a statistically significant deleterious effect of treatment with riboflavin/niacin. CONCLUSIONS: Findings from the two trials suggest that vitamin/mineral supplements may decrease the risk of nuclear cataract. Additional research is needed in less nutritionally deprived populations before these findings can be translated into general nutritional recommendations.

Profiling human leukocyte antigens in Vogt-Koyanagi-Harada syndrome.

Human leukocyte antigen typing was performed in 32 consecutive Chinese patients with Vogt-Koyanagi-Harada syndrome and 52 unrelated healthy Chinese individuals. Results indicated that HLA-DR4 was identified in 24 of the 32 patients with Vogt-Koyanagi-Harada syndrome (75.0%), but only in 12 (23.1%) of the 52 control subjects (P = .0003; relative risk, 10.0). Human leukocyte antigen-DQw7, also correlated with the disease, was identified in 19 (59.4%) patients, and in 19 control subjects (36.5%; P = .0230). The two-haplotype association detection demonstrated that HLA-DR4 and HLA-DQw7 were related through linkage disequilibrium, suggesting that the disease was primarily associated with only one of the antigens. The comparison between HLA-DR4-positive and HLA-DR4-negative patients with Vogt-Koyanagi-Harada syndrome in regard to clinical manifestations has shown that the HLA-DR4-positive group had a lower visual acuity at the first visit than did the HLA-DR4-negative group. However, both groups responded well to corticosteroid treatment. No other significant correlations between HLA-DR4 positivity and ocular features, including complications or systemic features, were found. Therefore, we concluded that the presence of HLA-DR4 may represent susceptibility to Vogt-Koyanagi-Harada syndrome, but may not represent specific tissue involvement or determine the prognosis. A decreased frequency of HLA-DQw1 in the patient group was also noticed. Further studies showed a higher percentage of HLA-DQw1 in HLA-DR4-positive control subjects than in the HLA-DR4-positive patients (P = .0308), which indicated that HLA-DQw1 was negatively associated with the disease. This protective effect from HLA-DQw1 was also studied.

Reduced absolute rate of myo-inositol biosynthesis of cultured bovine retinal capillary pericytes in high glucose.

De novo biosynthesis of myo-inositol (MI) by permeabilized cultured bovine retinal capillary pericytes (BRCP) and feline retinal pigment epithelial cells (FRPE), grown in different concentrations of glucose, were studied. After incubation with a physiological concentration of [14C]glucose 6-phosphate (G6P), the radioactive G6P derivatives were quantitated by a single HPLC column. Based on the determined specific activity of [14C]G6P, activities of inositol 1-phosphate synthase (MI synthase) were calculated. The activity of MI synthase was reduced 48% by growing BRCP in a high-glucose medium (20 mM) in comparison with that in the normal medium (glucose 5 mM). In contrast, the de novo MI biosynthesis by FRPE was not changed with increasing concentrations of glucose in the medium. As compared with MI uptake previously studied, the synthesized MI contributes a substantial proportion of cellular MI pool in BRCP. Therefore, in BRCP growing in high glucose the reduced MI biosynthesis aggravates the low MI content resulting from the inhibited MI uptake, and thus leads to altered inositol phospholipid metabolism.

Association of hereditary cataracts in strain 13/N guinea-pigs with mutation of the gene for zeta-crystallin.

Congenital nuclear cataracts transmitted by an autosomal dominant gene are present in a line of strain 13/N guinea-pigs. Studies on the lens proteins from these animals demonstrate changes in both the composition and structure of the crystallins relative to normal controls. The most prominent difference is in the zeta-crystallin, a taxon-specific crystallin which has been shown to be related to the alcohol dehydrogenases. In animals homozygous for the cataract phenotype the normal zeta-crystallin polypeptide is absent from the lens. Quantitation is difficult in the cataractous lenses from heterozygotes because of protein changes secondary to opacification: however in liver and kidney which have catalytic levels of the protein, the concentrations are approximately half that present in tissue from normal control animals. These findings suggest that in the cataractous animals a mutation has occurred in the gene for zeta-crystallin. In addition, a novel protein which is very similar to zeta-crystallin is synthesized only in the lenses of animals with cataract. This protein appears to be the product of the mutant gene for zeta-crystallin. These data support the hypothesis that this hereditary congenital cataract results from a specific mutation in the zeta-crystallin gene.

Intramural pericyte degeneration in early diabetic retinopathy study in vitro.

An experimental model of diseased pericytes was established by using cultured bovine retinal capillary pericytes in high--glucose medium. The high glucose stimulated polyol pathway, reduced cellular myo-inositol content and disturbed inositol phospholipid metabolism which resulted in a decrease in inositol trisphosphate (IP3) level. The correlation of suppressed IP3 levels with reduced DNA synthesis was evident. These findings suggested the biochemical mechanism by which retinal pericytes degenerate in high glucose. To supplement myo--inositol and/or an aldose reductase inhibitor to the high--glucose medium largely reversed the suppressed IP3 level and the decreased DNA synthesis. Therefore, these two manipulations may be considered as in vitro therapy to treat sick pericytes induced by high glucose.

DNA-synthesis regulation and correlation with inositol trisphosphate levels in cultured bovine retinal capillary pericytes.

Inositol phosphate (IP), inositol bisphosphate (IP2) and inositol trisphosphate (IP3) in cultured bovine retinal capillary pericytes (BRCP) were quantitated by an ion-pair reverse-phase HPLC. BRCP were grown in media with standard (5 mM) or high (30 mM) glucose, and were either labeled with myo-[2-3H]inositol (20 microCi ml-1) for 60 hr or with dual isotopes (20 microCi ml-1 myo-[2-3H]inositol and 2 microCi ml-1 [14C]glycerol) for 8 hr. In parallel, BRCP in different glucose-media were incubated with 1 microCi ml-1 [3H]thymidine for 4 hr. High glucose significantly suppressed the accumulation of [3H]label in IP, IP2 and IP3, and specifically reduced the incorporation of [14C]glycerol into inositol phospholipids, but not that of neutral lipids and other types of phospholipids. The reduced IP3 level correlated with the decrease in the incorporation of [3H]thymidine into DNA. Both the reduced IP3 formation and DNA synthesis which were induced by high glucose were significantly reversed by adding either myo-inositol or AL1576, an aldose reductase inhibitor (ARI). However, the addition of neither myo-inositol nor ARI stimulated IP3 formation and/or DNA synthesis when BRCP were grown in the standard medium (5 mM glucose). These findings indicate that myo-inositol metabolism and the polyol pathway affect inositol phospholipid-mediated pericyte division in vitro only under the high-glucose condition. These data are compatible with the hypothesis that altered inositol phospholipid metabolism accounts for the loss of pericytes in diabetic retinopathy.

Age-related cataract in the Tibet Eye Study.

The Tibet Eye Study was designed to estimate the prevalence of age-related cataract in Duilong-Deqing County, west of Lhasa, China (altitude, 4000 m). Previous reports have suggested an unusually high prevalence of age-related cataract in Tibet. A two-stage probability sample of persons aged 20 years or older from the 35 townships of the county targeted 2884 persons for inclusion in the study; 2665 (92.4%) were examined. Age-related cataract was diagnosed when (1) visual acuity was worse than 6/12 (20/40) because of nuclear or cortical (including posterior subcapsular) opacities, or (2) aphakia associated with a history of age-related cataract was present in either eye. The prevalence of age-related cataract among persons aged 20 to 39 years was 0.2%; among persons 40 years old or older, the prevalence was 11.8%. Cortical cataracts were by far the most common type of cataract diagnosed. Age- and sex-adjusted prevalence in Tibet was 60% higher than the prevalence in a similar, previously conducted study of 6951 person in Shunyi County, northeast of Beijing (altitude, 50 m). A second, independent slit-lamp classification of lens status was conducted in the Tibet Eye Study using standard photographs previously described. Age-specific cataract prevalence was similar with the two examination techniques. Results from the Tibet Eye Study support previous suggestions of a high prevalence of age-related cataract in Tibet.

Attenuation of phosphoinositidase activity and phosphatidylinositol bisphosphate level of bovine retinal capillary pericytes in high glucose.

Both phosphoinositidase (PIase) and individual species of inositol phospholipid (IPL) of bovine retinal capillary pericytes (BRCP) were quantitatively determined. When glucose in growth medium was increased from 5- to 15- or 30 mM, PIase activity was attenuated to 82% or 55%, respectively. In contrast, when glucose (5-, 15-, 30 mM) was added to an enzyme extract from cells grown in the standard growth medium (5 mM glucose, 0.04 mM myo-inositol) the PIase activity was not changed, indicating that the reduced PIase activity was not due to the direct effect of glucose. When IPLs from BRCP were analysed by HPLC and TLC, we observed reduction of the total and newly formed IPLs including the substrate of PIase. Phosphatidylinositol bisphosphate (PIP2). Reduced levels of IPLs were associated with a decrease in myo-inositol and an increase in sorbitol. The changes in IPL metabolism were reversed by adding either free myo-inositol or AL1576, an aldose reductase inhibitor (ARI), to the high-glucose medium. However, the addition of myo-inositol to the growth medium with a standard concentration of glucose only caused a marked increase in phosphatidylinositol, but not in PIP or PIP2, while the supplement of AL1576 in the standard medium did not cause any changes in IPL formation. These findings suggest that the alteration in IPL metabolism in BRCP may be related to insufficient myo-inositol or activated sorbitol pathway under high-glucose conditions. Further explanation of the role of the altered hydrolysis of PIP2 triggered by PIase may provide clues to understanding of the mechanism of decreased pericyte viability in the presence of high glucose concentrations.

Reduction of inositol triphosphate in retinal microvessels by glucose and restimulation by myo-inositol.

The formation of inositol phospholipids (IPLs) and inositol phosphate esters (IPEs) in response to glucose was studied in isolated retinal microvessels from porcine eyes. Retinal microvessels incubated from 60 hr with myo-[3H]inositol were sequentially extracted to obtain IPLs and IPEs. [3H]Inositol-labelled IPLs were deacylated to produce the corresponding glycero derivatives. Both deacylation products and water-soluble IPEs were monitored by anion-exchange chromatography. In the presence of high glucose (30 mM) the labelling in inositol triphosphate (IP3) was reduced to 77% and was restimulated by adding myo-inositol (final concentration 0.4 mM) to 158% of the control under physiological conditions of glucose (5 mM) and myo-inositol (0.04 mM). With a fixed glucose concentration (5 mM), IPE accumulation was observed with increasing concentrations of exogenous myo-inositol. Under physiological conditions (glucose 5 mM, myo-inositol 0.04 mM) the distribution (percentage) of radioactivity in phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-phosphate (PIP2) was 63:19:18. The myo-inositol concentration dependence of IPL formation was also demonstrated. A decrease in IP3 in response to high glucose without changing PIP2 but with a reduction in PI indicated that PI may act as a reservoir to replace a possible loss of PIP2. These findings suggest that availability of myo-inositol by retinal microvessels may be essential to maintain the normal signal transduction and cell proliferation associated with IPL turnover under high glucose concentration.