Preparation of carotenoid extracts and nanoemulsions from Lycium barbarum L. and their effects on growth of HT-29 colon cancer cells.

Lycium barbarum L., a traditional Chinese herb widely used in Asian countries, has been demonstrated to be protective against chronic diseases such as age-related macular degeneration. The objectives of this study were to determine the carotenoid content in L. barbarum by high-performance liquid chromatography-mass spectrometry, followed by preparation of a carotenoid nanoemulsion to evaluate the mechanism of inhibition on HT-29 colon cancer cells. The highest extraction yield of carotenoids was attained by employing a solvent system of hexane-ethanol-acetone (1:1:1, v/v/v). Nine carotenoids, including neoxanthin (4.47 µg g-1), all-trans-zeaxanthin and its cis-isomers (1666.3 µg g-1), all-trans-ß-cryptoxanthin (51.69 µg g-1), all-trans-ß-carotene and its cis-isomers (20.11 µg g-1), were separated within 45 min and quantified using a YMC C30 column and a gradient mobile phase of methanol-water (9:1, v/v) (A) and methylene chloride (B). A highly stable carotenoid nanoemulsion composed of CapryolTM 90, Transcutol®HP, Tween 80 and deionized water was prepared with a mean particle size of 15.1 nm. Characterization of zeaxanthin standard, blank nanoemulsion, carotenoid extract and carotenoid nanoemulsion by differential scanning calorimetry curves and Fourier transform infrared spectra revealed a good dispersion of zeaxanthin-dominated carotenoid extract with no significant chemical change after incorporation into nanoemulsion. The in vitro release kinetic study showed a higher release profile at pH 5.2 than at physiological pH 7.4, suggesting a rapid release of carotenoids in the acidic environment (pH 4.5-6.5) characteristic of tumors. Both the carotenoid nanoemulsion and the extract were effective at inhibiting growth of HT-29 colon cancer cells, with an IC50 of 4.5 and 4.9 µg ml-1, respectively. Also, both treatments could up-regulate p53 and p21 expression and down-regulate CDK2, CDK1, cyclin A and cyclin B expression and arrest the cell cycle at G2/M. The study may form a basis for further exploration of L. barbarum nanoemulsion in cancer treatment.

Nasotracheal intubation in patients with limited mouth opening: a comparison between fibreoptic intubation and the Trachway®.

In patients with limited mouth opening, traditional laryngoscopy and videolaryngoscopes are not useful when performing nasotracheal intubation. Eighty patients with limited mouth opening who required nasotracheal intubation were randomly assigned to either fibreoptic intubation (n = 40) or the Trachway(®) (n = 40). Using the modified nasal intubation difficulty scale, 22 (55%) patients who received fibreoptic intubation were categorised as no difficulty compared with 40 (100%) patients in the Trachway group (p < 0.001). Mean (SD) total intubation time was 71.8 (23.3) s in patients who received fibreoptic intubation compared with 35.4 (9.8) s in the Trachway group (p < 0.001). We conclude that the Trachway technique for nasotracheal intubation is quicker and easier compared with fibreoptic intubation in patients with limited mouth opening.

Xanthelasma is not associated with increased risk of carotid atherosclerosis in normolipidaemia.

OBJECTIVES: Extracranial carotid artery (ECCA) atherosclerosis is well known to be associated with cardiovascular diseases. This study aims to investigate the difference of ECCA atherosclerosis between patients with xanthelasma and control subjects in normolipidaemia. METHODS: Carotid atherosclerosis (CA) of 41 (8 males and 33 females) patients with xanthelasma and normolipidaemia, defined as levels of cholesterol below 6.21 mmol/l and triglyceride below 2.26 mmol/l, recruited from Department of Dermatology was compared with that of 85 age- and gender-matched control subjects. The extent and severity of CA were measured by high-resolution B-mode ultrasound and expressed as the mean intima-media thickness (IMT) of the common carotid artery (CCA) and ECCA plaque score. Mixed-effects model and multivariate logistic regression analyses were used to estimate the association between xanthelasma and CA. RESULTS: Patients with xanthelasma showed significantly higher levels of low-density lipoprotein cholesterol (LDL-C) levels and higher body mass index (BMI) compared with the control group. Mixed models identified age, male gender, smoking and subjects of hypertension with medication, but not the presence of xanthelasma, were associated with an increase of CCA IMT. Multivariate logistic regression analysis revealed subjects of male gender, and hypertension with medication, but not the presence of xanthelasma, associated with thicker IMT, defined as IMT >or= 75th percentile, or ECCA plaque score >or= 3. CONCLUSIONS: Normolipidaemia with xanthelasma is not significantly associated with CA, but did relate with adverse cardiovascular profiles, such as higher BMI, waist circumference and LDL-C levels.

Lower plasma adiponectin is correlated to higher alanine aminotransferase independent of metabolic factors and hepatitis B virus carrier status.

BACKGROUND: Adiponectin has been linked to the metabolic syndrome and coronary artery disease in recent years. The animal and human data also suggest that adiponectin may be beneficial for liver functions. The aim of this study was to investigate the correlation between plasma adiponectin level and liver function tests in adults with or without chronic hepatitis B virus (HBV) infection. METHODS: We analysed the blood levels of liver enzymes and adiponectin in 140 apparently healthy adults, including 21 HBV carriers. RESULTS: We found that the plasma adiponectin levels were inversely correlated to aspartate aminotransferase (r = -0.314, P = 0.000) and alanine aminotransferase (ALT) (r = -0.430, P = 0.000). Among the HBV carriers, the ALT correlated with the plasma adiponectin levels (r = -0.521, P = 0.015). In linear regression models adjusting for age, sex and the other metabolic variables, the ALT was independently related to the plasma adiponectin levels (beta = -0.371 +/- 0.134, P = 0.007), even in HBV carriers (beta = -1.143 +/- 0.482, P = 0.034). The ALT was also independently correlated to the plasma adiponectin levels (beta = 0.552 +/- 0.132, P < 0.001) with adjustment for age, sex and insulin-resistance index by homeostasis model assessment, even in HBV carriers (beta = -1.202 +/- 0.562, P = 0.048). The subjects with normal ALT had a significantly higher least square mean of plasma adiponectin than those with abnormal ALT (4.01 +/- 0.19 vs 3.30 +/- 0.30, P = 0.014) with adjustment for age, sex, homeostasis model assessment insulin resistance and HBV status. CONCLUSION: ALT was inversely related to adiponectin levels, independent of the metabolic factors and HBV status. Whether there is any potential prognostic and therapeutic value of adiponectin in human liver diseases remains to be investigated.

The effects of arecoline on the release of cytokines using cultured peripheral blood mononuclear cells from patients with oral mucous diseases.

In Taiwan there is a significant correlation between oral precancer diseases and oral cancer associated with the betel quid chewing habit. The carcinogenic components of betel quid are arecoline, arecaidine and safrole. However, it is unknown whether these substances influence the immune functions. This study investigated the effects of betel quid on the immune system using cultured peripheral blood mononuclear cells from patients with oral mucous diseases. In our experiment, mononuclear cells from 10 normal persons, 12 patients with precancer lesions, and 16 patients with squamous cell carcinoma were separated from blood samples and cultured. After stimulation by arecoline, the amounts of IL-2, TNF-alpha, TGF-beta and IFN-gamma secreted by mononuclear cells were measured using the ELISA method. The results showed that IL-2, TNF-alpha, and TGF-beta were significantly lower in mononuclear cells of normal persons as stimulated by arecoline. The TGF-beta amount in cells from oral submucous fibrosis patients with betel quid chewing habit (OSF-B) was lower than normal persons or patients who had long term betel quid chewing habit but were without oral mucosal diseases (N-B), and was also lower than the squamous cell carcinoma with betel quid chewing group (SCC-B). TNF-alpha was significantly lower in the squamous cell carcinoma with long term betel quid chewing group (SCC-B) than in normal persons. TNF-alpha was significantly higher in the squamous cell carcinoma without betel quid chewing group (SCC-N) than in normal persons and SCC-B groups. In addition, IFN-gamma was significantly lower in patients who had long term betel quid chewing but were without oral mucous lesions than the normal person and the OSF group. The results proved that betel quid influences cytokines production by mononuclear cells.

Ectopic expression of negative ARNT2 factor disrupts fish development.

ARNT factors are a cluster of bHLH-PAS factors that heterodimerize with other specific bHLH-PAS factors to mediate a wide range of biological responses. Previously, we obtained a truncated form of ARNT2-like factor, ARNT2A, from zebrafish, which encompasses the basic-helix-loop-helix and PAS A/B domains, but lacks a transactivation domain at its carboxyl end. Herein, we report another truncated ARNT2-like factor, ARNT2X, in zebrafish, which differs from ARNT2A at its N-terminal region. In cultured ZLE cells, transiently expressed ARNT2X and ARNT2A inhibited 2,3,7,8-TCDD-activated cyp1a1 transcription with different efficiencies. In the developing embryo, arnt2X mRNA was consistently expressed in the retinal and neural tube regions until the hatching stages, but it exhibited a more specific pattern at larval stages, including expression in the brain, eyes, hypothalamus, pharyngeal skeleton, heart, liver, pronephros duct, pectoral fin, and epithelial cells of the swim bladder. In contrast, arnt2A transcription diminished after hatching. Microinjecting a recombinant arnt2X-expression vector into fertilized eggs before cleavage stages caused severe defects in brain, eyes, pectoral fin, heart, and gut development. This suggests that the ARNT-mediated signal transduction pathways play important roles in fish tissue development.

Oct-6: a regulator of keratinocyte gene expression in stratified squamous epithelia.

POU domain proteins have been implicated as regulators of differentiation and development, particularly in early embryogenesis and in neural morphogenesis. Given that neural and epidermal lineages originate from a common precursor (ectodermal) cell, we explored the possibility that POU proteins are involved in epidermal differentiation. Using reverse transcription-PCR and degenerate oligonucleotides, we generated several POU domain cDNAs from cultured human epidermal mRNAs. One of these encoded a sequence identical to the rodent Tst-1/SCIP/Oct-6 POU domain. Subsequently, we isolated a cDNA encoding a 45.3-kDa protein with 98% sequence identity to rat Tst-1/SCIP and 94% identity to mouse Oct-6. This protein bound specifically to the canonical octamer motif, warranting its designation as human Oct-6. By RNase protection assays, by PCR, and by immunoblot analysis, Oct-6 was expressed in cultured epidermal keratinocytes. By in situ hybridization, Oct-6 mRNA was detected not only in epidermis but also a variety of other stratified squamous epithelia and with greater signals than testis, the tissue in which this POU protein was originally discovered. Moreover, Oct-6 exerted a marked and specific negative influence on expression of the K5 and K14 genes, abundantly expressed in most dividing stratified squamous epithelial cells and downregulated as cells commit to terminally differentiate. The repressive effect was complex, but it was not observed with Oct-1, nor was it seen with a truncated Oct-6 missing the POU domain. Taken together, our studies suggest that Oct-6 may play an important role in controlling gene expression in stratified squamous epithelia, including epidermis.

[Incidence of tuberculosis in the hunting tribe E-Lun-Chun in Northeast China]. / Inzidenz der Tuberkulose beim Jagdstamm E-Lun-Chun (Orotschenen) in Nordostchina.

The E-Lun-Chun, or Ortochen, a national minority in North East China, consisted of 2,262 members in 1953. In the mid-17th century a Russian invasion had displaced them from their original home in southward direction over the Amur river towards the Sin An Lin mountain range. While coming into closer contact with neighbouring peoples, tuberculosis and other infectious diseases spread rapidly among the members of the tribe. Under the Japanese occupation (1932-1945) the incidence of pulmonary tuberculosis was estimated at about 30% of the inhabitants. After initiation of modern measures to fight disease (case finding, drug treatment, BCG vaccination) the incidence dropped from 12.8% in 1954 to 6.5% in 1985, representing a reduction by 49.2% in 31 years. In 1963 a tuberculosis hospital was set up exclusively for E-Lung-Chun and indigenous medical personnel was suitably trained. However, the expected success of drug therapy was hampered by the tribe's abuse of alcohol and their unsteady life as hunters. To achieve a more stable state of health among the E-Lun-Chun, information work and measures to fight tuberculosis should be intensified in future.

Characterization of genomic clones specifying rabbit alpha- and beta-ventricular myosin heavy chains.

We have isolated gene sequences coding for the alpha- and beta-myosin heavy chains (HC) of rabbit ventricular muscle. A rabbit genomic library was screened with previously characterized cDNA clones specifying part of the light meromyosin and the entire subfragment 2 portion of alpha- and beta-myosin HCs, as well as with a clone containing the 3' nontranslated sequences of the alpha-myosin HC mRNA. Seven strongly hybridizing clones were analyzed in detail. One genomic clone encoded all of the 3' nontranslated sequences of an alpha-cDNA clone and, therefore, contained the 3' end of the alpha-myosin HC gene. Electron microscopic heteroduplex analysis and DNA sequence analysis showed that this clone overlapped a second genomic clone providing more than 25 kilobase pairs of the alpha-myosin HC gene. The exons within this region corresponded to approximately equal to 85% of the mRNA and were separated by at least 28 introns. A clone for the beta-myosin HC gene was also identified by Southern blot hybridization, by heteroduplex mapping, and by comparing the DNA sequence of a subfragment 2 exon to sequences of the alpha- and beta-cDNA clones. The introns of the alpha- and beta-myosin HC genes were in the same position but showed marked variation in length. These results conclusively showed that the alpha- and beta-myosin HCs are products of separate genes.

Cloned mRNA sequences for two types of embryonic myosin heavy chains from chick skeletal muscle. II. Expression during development using S1 nuclease mapping.

We have examined the expression of two embryonic myosin HC mRNAs using two cDNA clones (110 and 251) which we have previously constructed from RNA isolated from 14-day-old embryonic chick skeletal muscle. Sequence divergence in the 3' nontranslated regions enabled us to analyze the differential expression of the mRNAs corresponding to the two clones using the S1 nuclease mapping procedure. Clone 251 mRNA is expressed primarily in embryonic fast muscle, where its transcripts appear to be the predominant species. This mRNA is minimally expressed in the posthatching period, but it is not detected in adult leg and breast muscle. Messenger RNA for clone 110 is also primarily expressed in embryonic fast muscle. However, in the posthatching and adult stages of development, it continues to be expressed at a low level in leg muscle but not in breast muscle. The differential expression of these mRNAs during development strongly indicates that they correspond to two different genes coding for embryonic myosin HCs. Other myosin HC mRNAs which were partially homologous to the clone 110 or 251 mRNAs were also identified by S1 nuclease mapping. Using the probes from these two clones, a minimum of four other developmentally expressed forms were detected. Two of these correspond to "neonatal" myosin HCs, while the other two code for different adult myosin HCs present in leg and in breast muscle, respectively. The results therefore suggest a much greater diversity of myosin HC mRNAs expressed during development than previously reported.

Molecular cloning of mRNA sequences for cardiac alpha- and beta-form myosin heavy chains: expression in ventricles of normal, hypothyroid, and thyrotoxic rabbits.

We have isolated cDNA clones from thyrotoxic (pMHC alpha) and normal (pMHC beta) adult rabbit hearts. Restriction map analysis and DNA sequence analyses show that, although there is strong homology between overlapping regions of the two clones, they are distinctly different. The two clones exhibited 78-83% homology between the derived amino acid sequences and those determined by direct amino acid sequence analysis of rabbit fast skeletal muscle myosin heavy chains. The clones specify a segment of the myosin heavy chain corresponding to subfragment 2 and the COOH-terminal portions of subfragment 1. Nuclease S1 mapping was used to compare transcription of the two clones with expression of the alpha and beta forms of myosin heavy chains in the ventricles of thyrotoxic, hypothyroid (propylthiouracil-treated), and normal rabbits. Thyrotoxic ventricles contained only pMHC alpha transcripts whereas hypothyroid ventricles contained exclusively pMHC beta transcripts. These data correlate well with the presence of alpha- and beta-form myosin heavy chains. In the normal young adult rabbit, pMHC beta transcripts predominate, agreeing with the known beta form/alpha form ratio of 4:1. We therefore conclude that pMHC alpha and pMHC beta contain sequences of the alpha- and beta-form myosin heavy chain genes, respectively.

Molecular cloning of two fast myosin heavy chain cDNAs from chicken embryo skeletal muscle.

Recombinant DNA clones containing sequences for two different types of myosin heavy chain (HC) genes from chicken embryonic skeletal muscle were constructed and analyzed. Specificity of the clones for myosin HC was demonstrated by hybrid-arrested translation, by hybridization to a 7.0-kb mRNA, and by comparison of DNA sequences with known amino acid sequences of rabbit skeletal muscle myosin HC. Restriction enzyme and electron-microscopic heteroduplex analysis showed the presence of two distinct but homologous cDNA sequences. Hybrid melting curves indicated that both types of sequences represent fast myosin HC sequences.

Restriction endonuclease analysis of mitochondrial DNA from grande and genetically characterized cytoplasmic petite clones of Saccharomyces cerevisiae.

Digestion of grande mitochondrial DNA (mtDNA) BY EcoRI restriction endonuclease gives rise to nine fragments with a total molecular weight of 51.8 x 10(6). HindIII digestion yields six fragments with a similar total molecular weight. Specific restriction fragments can be detected despite the fact that yeast mtDNA consists of a heterogeneous distribution of randomly broken molecules. Digestion patterns of 10 genetically characterized petite clones containing various combinations of five antiobiotic resistance markers indicate that the petite mtDNA predominantly represents deletion of the grande genome. The petite mtDNAs contained up to seven EcoRI restriction fragments which comigrate with grande restriction fragments, and at least one fragment that did not correspond to any in the grande. Some strains contained multiple fragments with mobility different from that of grande; these fragments were usually present in less than molar concentrations. The genetic markers were associated with individual sets of restriction fragments. However, several internal inconsistencies prevent the construction of a definitive genetic fragment map. These anomalies, together with the digestion patterns, provide strong evidence that, in addition to single contiguous deletion, other changes such as multiple deletion and heterogeneity of mtDNA populations are present in some of the petite mtDNAs.