A natural variant of Bacillus licheniformis alpha-amylase isolated from flour mill wastewaters sheds light on the origin of high thermostability

AIMS: Understanding the origin of high thermostability exhibited by the alpha-amylase produced by a natural strain of Bacillus licheniformis. METHODS AND RESULTS: The MSH320 alpha-amylase gene has been cloned from a native strain of B. licheniformis isolated from flour mill wastewaters in Kashan, central Iran, and its nucleotide sequence was determined (GenBank Accession Number AF438149). Whereas previously cloned B. licheniformisalpha-amylase (BLA) genes are nearly identical, the MSH320 gene coding sequence presents only 93% identity with the reference 'wild-type' BLA gene, most of the nucleotide changes leading to silent mutations. Amino acid substitutions occurred at 19 of the 483 residues of the matured protein, distributed all along the protein sequence. Nevertheless, the natural BLA variant presents thermoinactivation kinetics similar to that of the reference BLA. Protein modelling and structural predictions at the substitution sites suggest that half of the mutations may have a significant stabilizing or destabilizing effect on the protein structure. Compensatory mutations thus occurred in the natural variant in order to maintain thermostability to the level of the reference enzyme. CONCLUSIONS: The exceptional high thermostability of BLA, although produced by a nonthermophilic organism, is not fortuitous but subject to a selective pressure still at work in natural environments. SIGNIFICANCE AND IMPACT OF THE STUDY: BLA thermal performances are not naturally maximized and can be substantially improved by protein engineering.     

The effect of saffron, Crocus sativus stigma, extract and its constituents, safranal and crocin on sexual behaviors in normal male rats

In this study, the aphrodisiac activities of Crocus sativus stigma aqueous extract and its constituents, safranal and crocin, were evaluated in male rats. The aqueous extract (80, 160 and 320 mg/kg body wt.), crocin (100, 200 and 400 mg/kg body wt.), safranal (0.1, 0.2 and 0.4 ml/kg), sildenafil (60 mg/kg body wt., as a positive control) and saline were administered intraperitoneally to male rats. Mounting frequency (MF), intromission frequency (IF), erection frequency (EF), mount latency (ML), intromission latency (IL) and ejaculation latency (EL) were the factors evaluated during the sexual behavior study. Crocin, at all doses, and the extract, especially at doses 160 and 320 mg/kg body wt., increased MF, IF and EF behaviors and reduced EL, IL and ML parameters. Safranal did not show aphrodisiac effects. The present study reveals an aphrodisiac activity of saffron aqueous extract and its constituent crocin.     

Total synthesis,structural, and biological evaluation of stylissatin A and related analogs

The natural product cyclic peptide stylissatin A ( 1a ) was reported to inhibit nitric oxide production in LPS‐stimulated murine macrophage RAW 264.7 cells. In the current study, solid‐phase total synthesis of stylissatin A was performed by using a safety‐catch linker and yielded the peptide with a trans‐Phe⁷‐Pro⁶ linkage, whereas the natural product is the cis rotamer at this position as evidenced by a marked difference in NMR chemical shifts. In order to preclude the possibility of 1b being an epimer of the natural product, we repeated the synthesis using d ‐allo‐Ile in place of l ‐Ile and a different site for macrocyclization. The resulting product (d ‐allo‐Ile²)‐stylissatin A ( 1c ) was also found to have the trans‐Phe⁷‐Pro⁶ peptide conformations like rotamer 1b . Applying the second route to the synthesis of stylissatin A itself, we obtained stylissatin A natural rotamer 1a accompanied by rotamer 1b as the major product. Rotamers 1a , 1b , and the epimer 1c were separable by HPLC, and 1a was found to match the natural product in structure and biological activity. Six related analogs 2–7 of stylissatin A were synthesized on Wang resin and characterized by spectral analysis. The natural product ( 1a ), the rotamer ( 1b ), and (d ‐allo‐Ile²)‐stylissatin A ( 1c ) exhibited significant inhibition of NO^.. Further investigations were focused on 1b , which also inhibited proliferation of T‐cells and inflammatory cytokine IL‐2 production. The analogs 2–7 weakly inhibited NO^. production, but strongly inhibited IL‐2 cytokine production compared with synthetic peptide 1b . All analogs inhibited the proliferation of T‐cells, with analog 7 having the strongest effect. In the analogs, the Pro⁶ residue was replaced by Glu/Ala, and the SAR indicates that the nature of this residue plays a role in the biological function of these peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Production,optimization and purification of a novel extracellular protease from the moderately halophilic bacterium <Emphasis Type="Italic">Halobacillus karajensis</Emphasis>

The production of a protease was investigated under conditions of high salinity by the moderately halophilic bacterium Halobacillus karajensis strain MA-2 in a basal medium containing peptone, beef extract, maltose and NaCl when the culture reached the stationary growth phase. Effect of various temperatures, initial pH, salt and different nutrient sources on protease production revealed that the maximum secretion occurred at 34°C, pH 8.0–8.5, and in the presence of gelatin. Replacement of NaCl by various concentrations of sodium nitrate in the basal medium also increased the protease production. The secreted protease was purified 24-fold with 68% recovery by a simple approach including a combination of acetone precipitation and Q-Sepharose ion exchange chromatography. The enzyme revealed a monomeric structure with a relative molecular mass of 36 kDa by running on SDS-PAGE. Maximum caseinolytic activity of the enzyme was observed at 50°C, pH 9.0 and 0.5 M NaCl, although at higher salinities (up to 3 M) activity still remained. The maximum enzyme activity was obtained at a broad pH range of 8.0–10.0, with 55 and 50% activity remaining at pH 6 and 11, respectively. Moreover, the enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), Pefabloc SC and EDTA; indicating that it probably belongs to the subclass of serine metalloproteases. These findings suggest that the protease secreted by Halobacillus karajensis has a potential for biotechnological applications from its haloalkaline properties point of view.     

Loss of heterozygosity on chromosome 5 in Iranian esophageal cancer patients

There is a high incidence of esophageal squamous cell carcinoma (ESCC) in Iran. Non-functionality of some tumor suppressor genes has been reported in esophageal cancer. Loss of heterozygosity on chromosome 5 has also been reported in esophageal carcinomas. We assessed loss of heterozygosity along a region of the long arm of chromosome 5 (5q), from 5q23.1 to 5q23.2, by PCR amplifying DNA fragments of tumor tissues from patients with ESCC and their corresponding normal samples. The PCR products were electrophoresed on 6% non-denaturing polyacrylamide gels, and band intensity was shown by silver staining. Of 40 patients with ESCC, 27, 25 and 36% of informative cases showed allelic losses at microsatellite markers D5S1384, D5S1478 and D5S1505, respectively. Two of the 40 patients studied had microsatellite instability at marker D5S1384. Based on the fact that loss of heterozygosity with more than 22% incidence for a specific marker cannot be regarded as a random event, we add support to previous reports concerning the presence of tumor suppressor genes in this chromosome region and that they affect esophageal cancer development. According to the data in NCBI UniSTS, the PCR product size of human DNA with primers of the D5S1505 marker ranges from 243 to 275 bp, containing about 20 repeats of the TAGA tetranucleotide, while the amplicon size of one allele of one of our cases was 207 bp, with about 10 repeats of the TAGA tetranucleotide, which would be the shortest sequence reported so far.     

Effect of thymoquinone and Nigella sativa seeds oil on lipid peroxidation level during global cerebral ischemia-reperfusion injury in rat hippocampus

It has been previously reported that Nigella sativa oil (NSO) and thymoquinone (TQ), active constituent of N. sativa seeds oil, may prevent oxidative injury in various models. Therefore, we considered the possible effect of TQ and NSO on lipid peroxidation level following cerebral ischemia-reperfusion injury (IRI) in rat hippocampus. Male NMRI rats were divided into nine groups, namely, sham, control, ischemia and ischemia treated with NSO or TQ. TQ (2.5, 5 and 10 mg/kg), NSO (0.048, 0.192 and 0.384 mg/kg), phenytoin (50 mg/kg, as positive control) and saline (10 ml/kg, as negative control) were injected intraperitoneally immediately after reperfusion and the administration was continued every 24h for 72 h after induction of ischemia. The transient global cerebral ischemia was induced using four-vessel-occlusion method for 20 min. Lipid peroxidation level in hippocampus portion was measured as malondialdehyde (MDA) based on its reaction with thiobarbituric acid (TBA) following ischemic insult. The transient global cerebral ischemia induced a significant increase in TBA reactive substances (TBARS) level (p<0.001), in comparison with sham-operated animal. Pretreatment with TQ and NSO were resulted a significant decrease in MDA level as compared with ischemic group (66.9+/-1.5 vs. 297+/-2.5 nmol/g tissue for TQ, 10 mg/kg; p<0.001 and 153.5+/-1.3 nmol/g tissue for NSO, 0.384 mg/kg; p<0.001). Using a reversed-phase HPLC system, the amount of TQ in NSO was also quantified and was 0.58% w/w. These results suggest that TQ and NSO may have protective effects on lipid peroxidation process during IRI in rat hippocampus.     

Toxicity of Carum copticum essential oil‐loaded nanogel against Sitophilus granarius and Tribolium confusum

Myristic acid–chitosan (MA‐chitosan) nanogels were synthesized with the technique of self‐assembly and loaded with Carum copticum (L.) essential oil. Scanning electron microscopy (SEM) study indicated nanogels with smooth surfaces and near spherical appearance, and according to transmission electron microscopy (TEM) image, core–shell stricter of nanogels was reported. Subsequently, fumigant toxicity of C. copticum oil and oil‐loaded nanogels was assessed against Sitophilus granarius (L.) and Tribolium confusum Jacquelin du Val. Adults were exposed to different concentrations of the oil and oil‐loaded nanogels at 27 ± 1°C and 55 ± 5% r.h. in continuous darkness. The mortality was counted after 3, 6, 10, 24 and 48 h of exposure. Mortality increased with increasing concentration levels. Fumigant toxicity of oil‐loaded nanogels was significantly more than the oil even after 48 h. According to the findings, oil‐loaded nanogels were 8.9‐ and 3.7‐fold more toxic than the oil against S. granarius and T. confusum, respectively. The persistence of oil and oil‐loaded nanogels was also investigated over period of time. Oil‐loaded nanogels lost the insecticidal effectiveness after 21 days post‐application for S. granarius and 33 days in the case of T. confusum, while the efficacy of oil decreased in the early days of application. Chemical composition of C. copticum essential oil was measured, and six components were identified. Thymol, ρ‐cymene and γ‐terpinolene were the major components of the oil. The essential oil‐loaded nanogels may have potential to be used as an alternative to synthetic insecticides for stored products protection. However, more research is necessary to improve the findings.     

Efficacy of Iranian diatomaceous earth deposits against Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae)

Laboratory bioassays were carried out to evaluate the insecticidal efficacy of two Iranian deposits of diatomaceous earths (DEs) and a commercial formulation, SilicoSec®, against adult confused flour beetle (Tribolium confusum Jacquelin du Val). The Iranian DEs were dried and sieved to get particle sizes of 0–149, 74–149, 0–74 μm, and 0–37 μm. First, DE samples were applied at the four concentration levels of 500, 1000, 1500 and 2000 ppm and each concentration was replicated four times. Tests were carried out at 27 ± 1 °C and 55 ± 5% r.h. in continuous darkness. The beetle mortality was counted at 2, 7, and 14 days after exposure. Moreover, another experiment was conducted to estimate the LC₅₀ values of the DEs. For the first experiment, adult beetle mortality exceeded 51% when exposed for 2 d to 2000 ppm of SilicoSec®. Complete mortality was recorded at each concentration of SilicoSec® at an exposure longer than 7 days except for 500 ppm; while mortalities of T. confusum at 2000 ppm of Maragheh and Mamaghan samples with the 0–149 μm particle size were 40.62% and 85.41%, respectively. Mortality of T. confusum was influenced by concentration and time of exposure to the DEs. SilicoSec® was the most effective DE followed by Mamaghan samples. The Maragheh samples were the least effective. In addition, in most cases fractions with smaller particles were more effective than larger ones. More experiments are necessary to process natural DEs and make them commercially exploitable.