Thermostabilization of Bacillus amyloliquefaciens alpha-amylase by chemical cross-linking

Chemical cross-linking of a mesophilic alpha-amylase from Bacillus amyloliquefaciens (BAA) was carried out. Intra-molecular cross-links between lysine residues upon treatment of the enzyme with ethylene glycol bis(succinic acid N-hydroxy succinimide ester) resulted in enhancement of thermostability as indicated by irreversible thermoinactivation experiments. Enhancement of thermostability coincided with a dramatic protection against aggregation, combined with a decrease in surface hydrophobicity. Deamidation, another important mechanism of irreversible thermoinactivation, was also diminished upon modification. While no significant changes in the kinetic parameters are evident, rigidification of the protein structure is suggested by circular dichroism (CD) and fluorescence studies.     

Embryotrophic effects of ethylenediaminetetraacetic acid and hemoglobin on in vitro porcine embryos development

This study investigated the embryotrophic effects of ethylenediaminetetraacetic acid (EDTA) and hemoglobin (Hb) on porcine preimplantation embryo development. Porcine embryos produced by in vitro maturation/fertilization were cultured for 6 days in modified North Carolina State University-23 medium (mNCSU-23) supplemented with EDTA and/or Hb. In Exp. 1, culturing porcine zygotes with 100 microM EDTA significantly increased cleavage frequencies (85.3%) at 48 h post insemination and the number of inner cell mass (ICM) (9.6+/-5.5) compared to the control (7.0+/-2.8). However, 100 microM EDTA did not improve blastocyst formation compared to 0, 1 or 10 microM EDTA. In Exp. 2, in vitro fertilized oocytes were cultured with 0, 1 or 10 microg/ml Hb. Culturing with Hb did not promote porcine embryo development, but significantly increased the cell numbers of blastocysts in 1 microg/ml Hb compared to 0 or 10 microg/ml Hb. In Exp. 3, culturing embryos with 100 microM EDTA+1 microg/ml Hb significantly improved frequencies of cleavage, blastocyst formation, and total cell numbers in blastocysts compared to the control. Moreover, 100 microM EDTA, 1 microg/ml Hb and their combination reduced reactive oxygen species (ROS) accumulation and decreased the incidence of apoptosis. In conclusion, the present study clearly demonstrated that the combining treatment of EDTA and Hb improved IVF porcine embryo development.     

Strengthening medical ethics by strategic planning in the Islamic Republic of Iran

To bring attention to medical ethics and to enhance the quality of health care in Iran, the Ministry of Health and Medical Education has introduced a strategic plan for medical ethics at a national level. This plan was developed through the organization and running of workshops in which experts addressed important areas related to medical ethics. They analysed strengths and weaknesses, opportunities and threats, and outlined a vision, a mission and specific goals and essential activities surrounding medical ethics. The current strategic plan has six main goals that will be reviewed in this paper. Some major activities that were carried out in recent years, and some future plans, will be also reviewed.     

Cytokine single nucleotide polymorphisms in Iranian patients with pulmonary tuberculosis

BACKGROUND: Several genes coding for different cytokines may affect host susceptibility to tuberculosis. METHODS: In the present study, the allele and genotype frequencies of a number polymorphic genes coding for cytokines or cytokine receptors were investigated in Iranian patients with pulmonary tuberculosis (PTB). RESULTS: From the IL-1 cluster, a positive, significant difference was found at position -889, where the T/T genotype was over represented in PTB patients (p = 0.01); a positive, significant increase was found in the IL1R PstI 1970 C/C genotype, where the C allele was over represented in the PTB patients (p = 0.01). A significant negative association at codon 10 TGF-beta, T allele, was shown in our patients and the C allele and C/C genotype were over represented in the PTB patients (P<0.005). For TNF-alpha at position -238, we found a negative association for the G/A genotype and a positive association for the G/G genotype (p = 0.0009). Significant negative associations at position -590 IL-4, T allele and the T/T genotype were shown in our patients (p = 0.0007); also, the C allele and T/C genotype were significantly increased in our patients (P<0.05). With IL-6 at -174, G/G increased and G/C decreased significantly in the patients (P<0.005). CONCLUSION: Pro-inflammatory cytokines such as TNF-alpha and TGF-beta seem to be decreased, and IL-6 increased in PTB patients.     

cDNA cloning, sequence analysis and molecular modeling of a new peptide from the scorpion Buthotus saulcyi venom

In this study, the cDNA of a new peptide from the venom of the scorpion, Buthotus saulcyi, was cloned and sequenced. It codes for a 64 residues peptide (Bsaul1) which shares high sequence similarity with depressant insect toxins of scorpions. The differences between them mainly appear in the loop1 which connects the beta-strand1 to the alpha-helix and seems to be functionally important in long chain scorpion neurotoxins. This loop is three amino acids longer in Bsaul1 compared to other depressant toxins. A comparative amino acid sequence analysis done on Bsaul1 and some of alpha-, beta-, excitatory and depressant toxins of scorpions showed that Bsaul1 contains all the residues which are highly conserved among long chain scorpion neurotoxins. Structural model of Bsaul1 was generated using Ts1 (a beta-toxin that competes with the depressant insect toxins for binding to Na(+) channels) as template. According to the molecular model of Bsaul1, the folding of the polypeptide chain is being composed of an anti-parallel three-stranded beta-sheet and a stretch of alpha- helix, tightly bound by a set of four disulfide bridges. A striking similarity in the spatial arrangement of some critical residues was shown by superposition of the backbone conformation of Bsaul1 and Ts1.     

Conception rates and serum progesterone concentration in dairy cattle administered gonadotropin releasing hormone 5 days after artificial insemination

The objectives of this study were to determine the effect of administration of exogenous GnRH 5days after artificial insemination (AI) on ovarian structures, serum progesterone concentration, and conception rates in lactating dairy cows. In experiment 1, 23 Holstein cows were synchronized using the Ovsynch protocol. Five days after AI (day 0) cows were assigned randomly to receive either saline (saline; n=11) or 100microg GnRH (GnRH; n=12). To examine ovarian structures, ultrasonography was performed on day 1 and every other day beginning on day 5 until day 13. On days 5 and 13 blood samples were obtained to measure serum progesterone concentrations. All cows in the GnRH-treated group developed an accessory corpus luteum (CL), whereas cows in the saline group did not. Mean serum progesterone concentrations did not differ between GnRH and saline groups on day 5 (1.64+/-0.46ng/ml versus 2.04+/-0.48ng/ml). On day 13 serum progesterone concentrations were greater (P<0.05) in the GnRH group compared with saline (5.22+/-0.46ng/ml versus 3.36+/-0.48ng/ml). In experiment 2, 542 lactating cows, at two different commercial dairies, were used to test the effect of administering GnRH 5 days after AI on conception rates. Cows were synchronized and detected for estrus according to tail chalk removal. Cows detected in estrus received AI within 1h after detection of estrus. Five days after AI, cows were assigned randomly to receive either GnRH (n=266) or saline (n=276). Pregnancy status was determined by palpation per rectum of uterine contents approximately 40 days after AI. There was no effect of farm on conception rate. There was no effect of treatment as conception rates did not differ between GnRH and saline groups (26.7% GnRH versus 24.3% saline). Regardless of treatment, days in milk, parity, milk yield, and number of services had no effect on the odds ratio of pregnancy. In summary, the results of this study indicated that GnRH administered 5 days after AI increased serum progesterone by developing an accessory CL but did not improve conception rates in dairy cattle.     

Refining and unifying the upper limits of the least limiting water range using soil and plant properties

The least limiting water range (LLWR) was introduced as an integrated soil water content indicator, measuring the impact of mechanical impedance, oxygen and water availability on water uptake and crop growth. However, a rigorous definition of the upper limit of the LLWR using plant physiological and soil physical concepts was not given. We introduce in this study an upper limit of the LLWR, based on soil physical and plant physiological properties. We further evaluate the sensitivity of this boundary to different soil and crop variables, and compare the sensitivity of the upper limit of the LLWR to previous definitions of soil water content at field capacity. The current study confirms that the upper limit of the LLWR can be predicted from knowledge of the soil moisture characteristic curve, plant root depth and oxygen consumption rate. The sensitivity analysis shows further that the upper limit of the LLWR approaches the volumetric soil water content at saturation when the oxygen consumption rate by plants becomes less than 2 µmol m^?3 s^?1. When plants are susceptible to aeration (e.g. potato and avocado), there is a big difference between the upper limit of the LLWR and the soil water content at field capacity, in particular for sandy soils. Results also show that the soil water content at aeration porosity corresponding to 10% cannot be considered as an appropriate upper limit of LLWR because it does not appropriately reflect the crop water requirements. Similar poor results are obtained when considering the soil water content at matric potential ?0.033 MPa or when defining the soil water content at field capacity based on drainage flux rate. It is observed that the upper limit of the LLWR is higher than either soil water content at ?0.033 MPa matric potential or soil water content at field capacity as based on drainage flux rate, especially in sandy soils.     

Soil water availability for plants as quantified by conventional available water, least limiting water range and integral water capacity

There are different approaches to define the soil available water (SAW) for plants. The objectives of this study are to evaluate the SAW values of 12 arable soils from Hamadan province (western Iran) calculated by plant available water (PAW), least limiting water range (LLWR) and integral water capacity (IWC) approaches and to explore their relations with Dexter’s index of soil physical quality (i.e., S-value). Soil water retention and mechanical resistance were determined on the intact samples which were taken from the 5–10 cm layer. For calculation of LLWR and IWC, the van Genuchten-Mualem model was fitted to the observed soil water retention data. Two matric suctions (h) of 100 and 330 cm were used for the field capacity (FC). There were significant differences (P?<?0.01) between the SAW values calculated by PAW₁₀₀, PAW₃₃₀, LLWR₁₀₀, LLWR₃₃₀ and IWC. The highest (i.e., 0.210 cm³ cm^?3) and the lowest (i.e., 0.129 cm³ cm^?3) means of SAW were calculated for the IWC and LLWR₃₃₀, respectively. The upper limit of LLWR₃₃₀ for all of the soils was h of 330 cm, and that of LLWR₁₀₀ (except for one soil that was air-filled porosity of 0.1 cm³ cm^?3) was h of 100 cm. The lower limit of LLWR₃₃₀ and LLWR₁₀₀ for five soils was h of 15,000 cm and for seven soils was mechanical resistance of 2 MPa. The IWC values were smaller than those of LLWR₁₀₀ for two soils, equal to those of LLWR₁₀₀ for three soils and greater than those of LLWR₁₀₀ for the rest. There is, therefore, a tendency to predict more SAW using the IWC approach than with the LLWR approach. This is due to the chosen critical soil limits and gradual changes of soil limitations vs. water content in the IWC calculation procedure. Significant relationships of SAW with bulk density or relative bulk density were found but not with the clay and organic matter contents. Linear relations between IWC and LLWR₁₀₀ or LLWR₃₃₀ were found as: IWC?=??0.0514 + 1.4438LLWR₁₀₀, R ²?=?0.83; and IWC?=??0.0405 + 2.0465LLWR₃₃₀, R ²?=?0.84, respectively (both significant at P?<?0.01). Significant relationships were obtained between the SAW values and S indicating the suitability of the index S to explain the availability of soil water for plants even when complicated approaches like IWC are considered. Overall, the results demonstrate the importance of the choice of the approach to be used and its critical limits in the estimation of the soil available water to plants.     

An epitopic approach to designing and characterization of a multiple antigenic polypeptide against Botulinum neurotoxins A and E

Botulinum neurotoxin (BoNT) serotypes A, B and E are most frequently associated with human botulism. Recombinant vaccines against BoNTs are usually based on one or more domain(s) of the toxin molecule. In this study, we investigate a new-designed multiple antigenic polypeptide for serotypes A and E (MAP/AE), containing two linear epitopes from each serotype. A synthetic gene was used to express the recombinant MAP/AE, in E. coli. Anti-MAP/AE antibodies were produced by injecting the purified MAP/AE to Balb/C mice. The interactivity of these antibodies and BoNT/A and E has been shown by ELISA. High titers of anti-MAP/AE antibodies were detected in mice sera. The anti-MAP/AE antibody titer is clearly detectable even at 25,600 dilution level. The anti-MAP/AE antibodies bound to both BoNT/A and BoNT/E holotoxin molecules. Neutralization ability of the antibodies for both toxin serotypes was determined, by an inhibitory ELISA assay. Results are suggestive of the feasibility of this epitope targeting strategy to develop novel multivalent recombinant vaccines against BoNTs.