Presence of the bacterial hemoglobin gene improves α-amylase production of a recombinantEscherichia coli strain

A recombinant plasmid (pMK57) was constructed by cloning theBacillus stearothermophilus α-amylase gene into pUC8; plasmid pMK79 was then derived from pMK57 by inserting the bacterial (Vitreoscilla) hemoglobin gene into the latter plasmid. Both pMK57 and pMK79 were transformed intoEscherichia coli strain JM103 to make strains MK57 and MK79, respectively. Both MK57 and MK79 produced α-amylase and MK79 produced hemoglobin. MK79 outgrew MK57 in shake flasks in LB medium, the advantage of the former appearing in late log phase. MK79 produced more α-amylase than MK57, on both per cell and per volume bases, in both mid and late log phases; the maximum advantage of MK79 (on a per volume basis) occurred in late log phase, at which time it produced 3.3 times as much α-amylase as MK57. The numbers of copies per cell of both pMK57 and pMK79 were significantly lower than that of pUC8.     

Effects of Pro-Tex® on the expression of Hsp70 gene and immune response parameters in the Persian sturgeon fingerlings,Acipenser persicus,infected with Aeromonas hydrophila

The induction of Hsps (heat shock protein) recognized as a promising approach to limiting disease and improving health in aquaculture. This investigation aimed to study the impacts of Pro-Tex®, an extract from the prickly pear cactus (Opuntia ficus indica), on the expression of Hsp70 gene and induction of immune response parameters in Acipenser persicus infected with Aeromonas hydrophila ATCC^®7966^TM. Fish were pretreated with 25, 50 and 100 mg/L of Pro-Tex and then injected in the intra-peritoneal cavity with A. hydrophila. The expression level of Hsp70 gene, lysozyme activity (LYZ) and complement C3 (C3), and immunoglobulin M (IgM) levels were assessed in liver, gill, and intestine on the days 3 and 7 post-infection. Tex-OE® increased expression of Hsp70 in a dose-dependent way in A. persicus, but this expression significantly reduced on the 7-days post-injection. The Hsp70 expression pattern was variable in each tissue, also, LYZ activity, C3, and IgM increased, depending on the concentration, and showed a decreasing trend in a time-dependent way. In conclusion, our data indicated that Pro-Tex as an Hsp70 inducer increases the resistance of sturgeon fry against fish pathogens by induction of different immunity factors.     

Copy number and loss of heterozygosity detected by SNP array of formalin-fixed tissues using whole-genome amplification

The requirement for large amounts of good quality DNA for whole-genome applications prohibits their use for small, laser capture micro-dissected (LCM), and/or rare clinical samples, which are also often formalin-fixed and paraffin-embedded (FFPE). Whole-genome amplification of DNA from these samples could, potentially, overcome these limitations. However, little is known about the artefacts introduced by amplification of FFPE-derived DNA with regard to genotyping, and subsequent copy number and loss of heterozygosity (LOH) analyses. Using a ligation adaptor amplification method, we present data from a total of 22 Affymetrix SNP 6.0 experiments, using matched paired amplified and non-amplified DNA from 10 LCM FFPE normal and dysplastic oral epithelial tissues, and an internal method control. An average of 76.5% of SNPs were called in both matched amplified and non-amplified DNA samples, and concordance was a promising 82.4%. Paired analysis for copy number, LOH, and both combined, showed that copy number changes were reduced in amplified DNA, but were 99.5% concordant when detected, amplifications were the changes most likely to be 'missed', only 30% of non-amplified LOH changes were identified in amplified pairs, and when copy number and LOH are combined ～50% of gene changes detected in the unamplified DNA were also detected in the amplified DNA and within these changes, 86.5% were concordant for both copy number and LOH status. However, there are also changes introduced as ～20% of changes in the amplified DNA are not detected in the non-amplified DNA. An integrative network biology approach revealed that changes in amplified DNA of dysplastic oral epithelium localize to topologically critical regions of the human protein-protein interaction network, suggesting their functional implication in the pathobiology of this disease. Taken together, our results support the use of amplification of FFPE-derived DNA, provided sufficient samples are used to increase power and compensate for increased error rates.     

Variation in Polyphenolic Profiles,Antioxidant and Antimicrobial Activity of Different Achillea Species as Natural Sources of Antiglycative Compounds

A comparative study was carried out on the methanolic extracts from six Achillea species and the examined polyphenols from these plants on the formation of advanced glycation end‐products (AGE) in vitro. A. pachycephala which was richer in flavonoids (15 mg quercetin/g W) and phenolics (111.10 mg tannic acid/g DW) with substantial antioxidant activity (IC₅₀ = 365.5 μg/ml) presented strong anti‐AGE properties. Chlorogenic acid, luteolin, quercetin and caffeic acid were identified as the major polyphenols in the extracts by HPLC. In general, polyphenolic content follows the order of A. pachycephalla > A. nobilis > A. filipendulina > A. santolina > A. aucheri > A. millefolium. Most extracts exhibited marked anti‐AGE ability in the bovine serum albumin (BSA)/methylglyoxal (MG) system, though A. pachycephala showed the highest potential. The formation of AGEs was assessed by monitoring the production of fluorescent products and circular dichroism (CD) spectroscopy. Diminution in free radical production (assessed by 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) assays) is discussed as potential mechanism for delay or reduced AGE. The results demonstrate the antiglycative, antioxidant and antimicrobial potential of Achillea species which can be attributed to polyphenols content and the effectiveness on generation of AGEs, thus Achillea species can be considered as natural sources for slowing down glycation related diseases.     

A family of acetylcholine-gated chloride channel subunits in Caenorhabditis elegans

The genome of the nematode Caenorhabditis elegans encodes a surprisingly large and diverse superfamily of genes encoding Cys loop ligand-gated ion channels. Here we report the first cloning, expression, and pharmacological characterization of members of a family of anion-selective acetylcholine receptor subunits. Two subunits, ACC-1 and ACC-2, form homomeric channels for which acetylcholine and arecoline, but not nicotine, are efficient agonists. These channels are blocked by d-tubocurarine but not by alpha-bungarotoxin. We provide evidence that two additional subunits, ACC-3 and ACC-4, interact with ACC-1 and ACC-2. The acetylcholine-binding domain of these channels appears to have diverged substantially from the acetylcholine-binding domain of nicotinic receptors.     

Activity and function in lung injuries due to sulphur mustard

Aim. Pulmonary complications are known to occur in over half the patients exposed to sulphur mustard. Many studies have focused on the clinical complications, often ignoring the pathogenesis of sulphur mustard. Also, the reasons for the variable severity of lung injuries caused by sulphur mustard are unclear. Hence, the current study was performed to evaluate the correlation between superoxide dismutase (SOD) and catalase (CAT) activity and pulmonary function in patients exposed to sulphur mustard. Methods. Our study was a comparative cross-sectional survey. Two hundred and fifty incident survivors were selected from the Sardasht population who were exposed to sulphur mustard in 1987. A control group from non-exposed civilians was also selected. We used a pulmonary function test, and SOD and CAT activity was measured in these groups. Results. The mean SOD activity in the healthy control group (70.5±10.8 U ml^?1) was higher than in the moderate-to-severe group (67.0±6.1 U ml^?1) (p <0.001, one-tail ANOVA, least significant difference (LSD) post hoc). The mean activity in the mild group (72.5±6.9 U ml^?1) was no higher than in the healthy control group (70.5±10.8 U ml^?1) (p=0.095 one-tail ANOVA, LSD post hoc). The mean CAT activity in the healthy control group (4.9±1.5 U ml^?1) was lower than in the moderate-to-severe group (8.0±1.8 U ml^?1) (p <0.001, one-tail ANOVA, LSD post hoc) and in the mild group (7.5±1.5 U ml^?1) (p=0.012 one-tail ANOVA, LSD post hoc). Conclusion. According to our findings, it is reasonable to hypothesize that re-establishment of the activation–inactivation or oxidant–antioxidant balance in favour of the activation and antioxidant balances would be useful as a therapeutic strategy to suppress pathological mechanisms underlying lung injuries.     

The proteomes of human parotid and submandibular/sublingual gland salivas collected as the ductal secretions

Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.     

Lecithin soybean phospholipid nano-transfersomes as potential carriers for transdermal delivery of the human growth hormone

Pharmaceutical molecules such as peptides and proteins are usually injected into the body. Numerous efforts have been made to find new noninvasive ways to administer these peptides. In this study, highly flexible vesicles (transfersomes [TFs]) were designed as a new modern transdermal drug delivery system for systemic drug administration through the skin, which had also been evaluated in vitro. In this study, two growth hormone-loaded TF formulations were prepared, using soybean lecithin and two different surfactants; F₁_sodium deoxycholate and F ₂_sodium lauryl sulfate. Thereafter, the amount of skin penetration by the two formulas was assessed using the Franz diffusion cell system. TF formulations were evaluated for size, zeta potential and in vitro skin penetration across the rat skin. Results indicated that vesicle formulations were stable for 4 weeks and their mean sizes were 241.33 ± 17 and 171 ± 12.12 nm in the F ₁ and F ₂ formulation, respectively. After application to rat skin, transport of the human growth hormone (hGH) released from the TF formulations was found to be higher than that of the hGH alone. Maximum amounts of transdermal hormone delivery were estimated to be 489.54 ± 8.301 and 248.46 ± 4.019 ng·cm−2 , for F ₁ and F ₂, respectively. The results demonstrate the capability of the TF-containing growth hormone in transdermal delivery and superiority of the F ₁ to F ₂ TFs.     

Heat-Induced Production of Human Growth Hormone by High Cell Density Cultivation of Recombinant Escherichia Coli

The temperature-induced, over-expression of the human growth hormone gene in a recombinant E. coli during high cell density cultivation is reported. Human growth hormone (hGH) production and stability were tested under different heat shock conditions. Cell densities were 25 and 60 g l(-1) in a pH-stat fed-batch mode in defined and complex medium, respectively, and the fermentation time was decreased from 41 to 32 h. hGH was produced at 2 g l(-1) in complex medium. By using glycerol as main carbon source in the complex medium with exponential feeding, cell density and hGH production were increased to 100 g l(-1) and 2.7 g l(-1), respectively.