Effect of serum albumin supplementation on in vitro capacitation and fertilization of caprine oocytes

The aim of the investigation was to study the effect of purity and the type of serum albumin on in vitro fertilization (IVF) and cleavage rate of in vitro matured goat oocytes. Ovaries were collected from the local abattoir and transported within 4 h to the laboratory in warm saline (37 °C) containing 100 IU penicillin-G and 100 μg streptomycin sulfate per ml. A total of 2509 cumulus oocyte complexes (COCs) were collected from 1313 ovaries. Oocytes were matured in TCM-199 medium containing FSH (5 μg/ml), LH (5 μg/ml) and estradiol-17β (1 μg/ml), supplemented with 20% fetal bovine serum at 38.5 °C and 5% CO₂ in an incubator under humidified air for 27 h. After 27 h of in vitro maturation (IVM), oocytes were denuded, washed and randomly divided into 4 groups. Group 1 consisted of in vitro matured oocytes (n = 627) co-incubated with sperm in a 50 μl drop of TALP medium containing a 6 mg/ml crystalline bovine serum albumin (BSA) fraction V and 10 μg/ml heparin. Group 2 was comprised of in vitro matured oocytes (n = 470), co-incubated with sperm in a 50 μl drop of TALP medium containing 3 mg/ml crystalline BSA fraction V, 10% estrous goat serum and 10 μg/ml heparin. Group 3 was comprised of in vitro matured oocytes (n = 489) co-incubated with sperm in a 50 μl drop of TALP medium containing a 6 mg/ml fatty acid free BSA and 10 μg/ml heparin. Group 4 consisted of in vitro matured oocytes (n = 422) co-incubated with sperm in a 50 μl drop of TALP medium containing 20% estrous goat serum and 10 μg/ml heparin. After 18 h of co-incubation, the oocyte–sperm mixture was washed in the culture medium 15–20 times and cultured in 50 μl EDM. Cleavage of the in vitro fertilized oocytes were recorded 48 h post-insemination under an inverted phase contrast microscope. The average oocyte recovery rate/ovary and maturation rate was 1.91% and 80.03%, respectively. The cleavage rate in Group 1, Group 2, Group 3 and Group 4 was 1.59%, 8.93%, 11.86% and 35.30%, respectively. It could be concluded that the use of fatty acid free albumin resulted in a significantly higher (P < 0.05) cleavage rate, compared to unmodified albumin, and the supplementation of 20% estrous goat serum in the fertilization medium, significantly (P < 0.05) increased the cleavage rate of in vitro matured goat oocytes, compared to defatted albumin.     

Heterologous expression and characterisation of a biosynthetic thiolase from Clostridium butyricum DSM 10702

Biosynthetic thiolases (EC 2.3.1.9) are key enzymes in the branched catabolism of diverse clostridia as their activity and regulation influence the production of organic acids and solvents. In Clostridium butyricum, they are also involved in the production of hydrogen as a sustainable and environmentally benign energy source. In this study, the gene coding for thiolase from C. butyricum DSM 10702 was cloned by genome walking. It was found to consist of 1179 bp coding for a protein with 393 amino acids and a deduced molecular weight of 41.4 kDa. The enzyme was fused to an N-terminal his-tag, expressed in Escherichia coli, purified to near homogeneity and characterised for biochemical and kinetic properties. Gel filtration chromatography revealed that the catalytically active enzyme consists of a homotetramer. The enzyme showed a K_M of ～32 μM towards acetoacetyl-CoA and of ～21 μM towards CoASH at 30 °C and pH 8.0. Claisen condensation of acetyl-CoA by thiolase was analysed in a coupled enzyme assay, where β-hydroxybutyryl-CoA dehydrogenase was applied catalysing the subsequent NADH-dependant reduction of the formed condensation product acetoacetyl-CoA. For this purpose the latter enzyme was cloned from C. butyricum DSM 10702 and recombinantly expressed in E. coli. The K_M of thiolase towards acetyl-CoA was ～674 μM at 30 °C and pH 7.5. Acetyl-CoA condensation was inhibited even at micromolar concentrations of CoASH indicating that CoASH has an important regulatory function in vivo.     

Functional Characterisation of Germinant Receptors in Clostridium botulinum and Clostridium sporogenes Presents Novel Insights into Spore Germination Systems

Clostridium botulinum is a dangerous pathogen that forms the highly potent botulinum toxin, which when ingested causes a deadly neuroparalytic disease. The closely related Clostridium sporogenes is occasionally pathogenic, frequently associated with food spoilage and regarded as the non-toxigenic equivalent of Group I C. botulinum. Both species form highly resistant spores that are ubiquitous in the environment and which, under favourable growth conditions germinate to produce vegetative cells. To improve the control of botulinum neurotoxin-forming clostridia, it is imperative to comprehend the mechanisms by which spores germinate. Germination is initiated following the recognition of small molecules (germinants) by a specific germinant receptor (GR) located in the spore inner membrane. The present study precisely defines clostridial GRs, germinants and co-germinants. Group I C. botulinum ATCC3502 contains two tricistronic and one pentacistronic GR operons, while C. sporogenes ATCC15579 has three tricistronic and one tetracistronic GR operons. Insertional knockout mutants, allied with characterisation of recombinant GRs shows for the first time that amino acid stimulated germination in C. botulinum requires two tri-cistronic encoded GRs which act in synergy and cannot function individually. Spore germination in C. sporogenes requires one tri-cistronic GR. Two other GRs form part of a complex involved in controlling the rate of amino-acid stimulated germination. The suitability of using C. sporogenes as a substitute for C. botulinum in germination studies and food challenge tests is discussed.     

Clostridium difficile survives minimal temperature recommended for cooking ground meats

We quantified the thermal inhibitory effect of 71 °C (recommended for cooking ground meats), and re-heating at 85 °C, on food- and food-animal-derived Clostridium difficile spores. All C. difficile strains tested (n = 20) survived 71 °C for 2 h, but 90% died within 10 min when re-heated at 85 °C. Current cooking recommendations would need revision to include C. difficile.     

Supplementary studies and molecular data on Henneguya cerebralis Pronin, 1972 (Myxozoa: Myxosporea), a parasite from Kosogol grayling Thymallus arcticus nigrescens in Mongolia

Henneguya cerebralis Pronin, 1972 (Myxozoa) was described from Kosogol graylings Thymallus arcticus nigrescens Dorogostaisky, 1923 in Lake Khovsgol (Mongolia) in 1972.H. cerebralis was redescribed using critical morphological features and 18S ribosomal DNA (rDNA) gene sequence. Parasite infects cranial cartilage of fish host. Plasmodia are white rounded or ovoid, by 0.1 to 2 mm in size, containing large quantities of spores. Spore body is ovoid or rounded, 11.18 ± 0.13 μm (range 9,71–12,56) in length and 9.06 ± 0.16 μm (range 7.22–10,06) in width with equal polar capsules (4.7 × 2.6 μm). The two caudal appendages have different lengths (one of them was shorter in 20%).Phylogenetic position inferred by 18S rDNA shows that H. cerebralis is closely related with H. zschokkei, H. nuesslini, H. salminicola and H. cartilaginis which are histozoic parasites of salmonid fish.     

Glomus drummondii and G. walkeri,two new species of arbuscular mycorrhizal fungi (Glomeromycota)

Two new ectocarpic arbuscular mycorrhizal fungal species, Glomus drummondii and G. walkeri (Glomeromycota), found in maritime sand dunes of northern Poland and those adjacent to the Mediterranean Sea are described and illustrated. Mature spores of G. drummondii are pastel yellow to maize yellow, globose to subglobose, (58–)71(–85) μm diam, or ovoid, 50–80 × 63–98 μm. Their wall consists of three layers: an evanescent, hyaline, short-lived outermost layer, a laminate, smooth, pastel yellow to maize yellow middle layer, and a flexible, smooth, hyaline innermost layer. Spores of G. walkeri are white to pale yellow, globose to subglobose, (55–)81(–95) μm diam, or ovoid, 60–90 × 75–115 μm, and have a spore wall composed of three layers: a semi-permanent, hyaline outermost layer, a laminate, smooth, white to pale yellow middle layer, and a flexible, smooth, hyaline innermost layer. In Melzer's reagent, only the inner- and outermost layers stain reddish white to greyish rose in G. drummondii and G. walkeri, respectively. Both species form vesicular–arbuscular mycorrhizae in one-species cultures with Plantago lanceolata as the host plant. Phylogenetic analyses of the ITS and parts of the LSU of the nrDNA of spores placed both species in Glomus Group B sensu Schüßler et al. [Schüßler A, Schwarzott D, Walker C, 2001. A new fungal phylum, the Glomeromycota: phylogeny and evolution. Mycolological Research 105: 1413-1421.]     

Scolopendin 2, a cationic antimicrobial peptide from centipede,and its membrane-active mechanism

Scolopendin 2 is a 16-mer peptide (AGLQFPVGRIGRLLRK) derived from the centipede Scolopendra subspinipes mutilans. We observed that this peptide exhibited antimicrobial activity in a salt-dependent manner against various fungal and bacterial pathogens and showed no hemolytic effect in the range of 1.6 μM to 100 μM. Circular dichroism analysis showed that the peptide has an α-helical properties. Furthermore, we determined the mechanism(s) of action using flow cytometry and by investigating the release of intracellular potassium. The results showed that the peptide permeabilized the membranes of Escherichia coli O157 and Candida albicans, resulting in loss of intracellular potassium ions. Additionally, bis-(1,3-dibutylbarbituric acid) trimethine oxonol and 3,3′-dipropylthiacarbocyanine iodide assays showed that the peptide caused membrane depolarization. Using giant unilamellar vesicles encapsulating calcein and large unilamellar vesicles containing fluorescein isothiocyanate-dextran, which were similar in composition to typical E. coli O157 and C. albicans membranes, we demonstrated that scolopendin 2 disrupts membranes, resulting in a pore size between 4.8 nm and 5.0 nm. Thus, we have demonstrated that a cationic antimicrobial peptide, scolopendin 2, exerts its broad-spectrum antimicrobial effects by forming pores in the cell membrane.     

Microbial transformation of β-lapachone to its glycosides by Cunninghamella elegans ATCC 10028b

β-lapachone (1) has entered phases I and II clinical trials for the treatment of solid tumors and the therapeutic efficacy of β-lapachone is closely related to its metabolic process. In order to contribute to a better understanding of human metabolism of β-lapachone, Cunninghamella elegans ATCC 10028b was used as a microbial model of mammalian metabolism to biotransform β-lapachone and two new glycosylated derivatives were produced. The chemical structures were elucidated as 6-hydroxy-2,2-dimethyl-3,4-dihydro-2H-naphtho[1,2-b]pyran-5-O-β-d-glucopyranoside (2) and 5-hydroxy-2,2-dimethyl-3,4-dihydro-2H-naphtho[1,2-b]pyran-6-O-β-d-glucopyranoside (3) by ¹H NMR, ¹³C NMR, HMBC, HMQC, COSY and HRMS analyses. The major derivative (3) displayed a lower activity against breast cancer cell line SKBR-3 (IC₅₀ = 312.5 μM) than β-lapachone (IC₅₀ = 5.6 μM), but did not show cytotoxicity against normal fibroblasts cell line GM07492-A, whereas β-lapachone was highly toxic (IC₅₀ = 7.25 μM). These metabolites were reported here for the first time and are similar to those that occur in phase II of human metabolism     

Sex-specific light acclimation of Chara canescens (Charophyta)

Bisexual populations of the charophyte Chara canescens (Desv. et Loisel. in Loisel., 1810) containing male and female individuals are rarely found. Two experiments were carried out to study whether male and female algae from the same site exhibit different physiological capacities, especially with respect to light acclimation.Algae from two different shore levels and from laboratory cultures acclimated to six irradiance conditions (35–500 μmol photons m⁻² s⁻¹) were compared. Field measurements showed that both female and male algae of C. canescens are able to acclimate to daily changes in solar irradiance. The quantum yield of Photosystem II (PSII) decreased with increasing irradiance in the morning and increased with decreasing irradiance in the afternoon. Growth experiments showed increasing growth rates from 35 μmol photons m⁻² s⁻¹ (∼7 mg FW) up to 500 μmol photons m⁻² s⁻¹ (∼27 mg FW) in female and male C. canescens. The irradiance saturation point for photosynthesis (E_k) was about 140 μmol m⁻² s⁻¹ for both sexes within the whole range of acclimation irradiances. The maximum photosynthesis rate at saturating irradiances (P_max) of male algae was highest at E_k, whereas P_max of female algae was highest at 500 μmol photons m⁻² s⁻¹. The photosynthetic efficiency in the light-limited range (α) increased in female C. canescens and decreased in male C. canescens. The ratio of the non-photochemical quenching parameter (NPQ) to the relative electron transport rates rETR_(MT) increased in both sexes with irradiance, but showed a steeper increase in male than in female algae. Pigment analysis showed similar acclimation pattern for male and female C. canescens. Chl a/Chl b ratios of both sexes were constant over the whole range of E_g, whereas Chl a/carotenoid ratios in male and female C. canescens decreased from 70 μmol photons m⁻² s⁻¹ upwards. Pigment analysis pointed out that the carotenes α-, β- and γ-carotene were more prominent in male than in female algae.Our results indicate that female C. canescens are more efficient in light acclimation than male algae from the same site. Nevertheless, further investigations of bisexual C. canescens populations resolving CO₂-uptake mechanisms and/or genetic differences are needed.     

Fine structure of Chloromyxum menticirrhi n. sp. (Myxozoa) infecting the urinary bladder of the marine teleost Menticirrhus americanus (Sciaenidae) in Southern Brazil

A myxosporidian was found in the urinary bladder of the teleost Menticirrhus americanus Linnaeus, 1758 (Sciaenidae) collected from the South Atlantic coast of Brazil. Polysporic amoeboid plasmodia containing sporoblasts, developing pansporoblasts and spores were free in the bladder lumen. The prevalence of infection was 17.64% (15/85). Unfixed spores were spherical to subspherical, on average 10.5 μm long, 9.8 μm wide and 10.1 μm thick (n=25), and fixed spores measured 10.1×9.5×9.7 μm. The two spore valves were of equal size and each possessed prominent sutural lines and about 41 (37–45) surface ridges aligned parallel with the suture line. These ridges gave transverse sections a cog-wheel-like outline. The spores contained four pyriform polar capsules of equal size (3.20×2.0 μm) (n=25) (fixed), each with a polar filament having 3–4 (rarely 5) coils. The binucleate sporoplasm was irregular in shape, with granular matrix and randomly distributed dense bodies. The shape and dimensions of the spore, as well as the number, position and arrangement of the surface ridges, polar capsules and polar filament indicate that this is a new species, herein designated Chloromyxum menticirrhi. The gill, liver, gall bladder and intestine of the host showed no abnormalities.     

Biomorphometric characteristics of different types of sensilla detected on the antenna of Helicoverpa armigera by scanning electron microscopy

A morphometric study on H. armigera antenna showed four styles of sensilla, i.e., styloconica, chaetica, coeloconica, and trichodea, and their numbers were estimated. Sensilla trichodea detect inter and intraspecific communication signals and was the most numerous. They were divided into three types: type I, the longest, with a length of 34.04 ± 3.16 μm and about 2.16 to 2.42 μm in diameter at its base; 2) type II, intermediate, with a length of 22.58 ± 0.77 μm and basal diameter of 1.8–2.52 μm; 3) type III, the shortest sensilla trichodea, with a length of 7.62 ± 0.4 μm and a range in diameter similar to that of type II. The length of the female sensilla trichodea was longer than that of the male. The total number of sensilla trichodea was estimated to be 7520 on the antenna of the female, and 6831 on the male antenna. The lengths of the sensilla trichodea type I and type III were significantly different on male (t = 4.6881, P = 0.0034) and female antenna (t = 18.9852, P = 0.0001). An estimation of the predicted surface area of the most numerous type I on sampled segments between the 12th and 20th segments from a female of H. armigera showed a surface area of 5 × 10³ μm² and a sensillar density of 38 sensilla/10³ μm². The fraction of sensilla-occupied surface area was 0.4 μm².     

Heat activation and germination–promotion of Bacillus subtilis spores by infrared irradiation

The influence of infrared (IR) radiation on the viability and heat-activation of Bacillus subtilis spores, suspended in phosphate-buffered saline, was investigated. Two types of IR heaters with different spectral distributions were used. Near-infrared (NIR) and far-infrared (FIR) heaters with main wavelengths of approximately 1 μm and 3–6 μm, respectively, were utilized. Although both irradiation treatments decreased the number of B. subtilis colonies at a bulk temperature of approximately 75 °C, the mode of action was clearly different. In the case of the NIR heater, the number of colony-counts decreased gradually. In contrast, use of the FIR heater resulted in heat activation of the spores during the early stage of irradiation at a low bulk temperature (40–60 °C) over several minutes, followed by a decrease in the number of colonies. Consequently, FIR irradiation inactivated 90% of B. subtilis spores more effectively as compared to NIR irradiation for 20 min with a suspension volume of 20 ml and irradiation energy of 7.57 kW m^?2. Spore exposure to FIR irradiation accelerated their germination rate in nutrient broth; however, this was not true for treatment with the NIR heater. The absorption IR spectrum of B. subtilis spores indicated that FIR radiation was absorbed easily by the spore cell components and might activate the bioactive substances involved in germination. Even at the same irradiation energy, the influence of infrared radiation on spore germination was dependent on the IR spectral distribution. Bacterial spores undergoing germination lose their resistance to stressors, such as heat, chemicals and ultraviolet rays. FIR heating promotes heat activation and germination, thereby producing vegetative cells that are more susceptible to other killing methods, enabling the killing of bacterial spores at lower stress without product damage.     

Antimicrobial properties of two novel peptides derived from Theobroma cacao osmotin

The osmotin proteins of several plants display antifungal activity, which can play an important role in plant defense against diseases. Thus, this protein can be useful as a source for biotechnological strategies aiming to combat fungal diseases. In this work, we analyzed the antifungal activity of a cacao osmotin-like protein (TcOsm1) and of two osmotin-derived synthetic peptides with antimicrobial features, differing by five amino acids residues at the N-terminus. Antimicrobial tests showed that TcOsm1 expressed in Escherichia coli inhibits the growth of Moniliophthora perniciosa mycelium and Pichia pastoris X-33 in vitro. The TcOsm1-derived peptides, named Osm-pepA (H-RRLDRGGVWNLNVNPGTTGARVWARTK-NH₂), located at R23-K49, and Osm-pepB (H-GGVWNLNVNPGTTGARVWARTK-NH₂), located at G28-K49, inhibited growth of yeasts (Saccharomyces cerevisiae S288C and Pichia pastoris X-33) and spore germination of the phytopathogenic fungi Fusarium f. sp. glycines and Colletotrichum gossypi. Osm-pepA was more efficient than Osm-pepB for S. cerevisiae (MIC = 40 μM and MIC = 127 μM, respectively), as well as for P. pastoris (MIC = 20 μM and MIC = 127 μM, respectively). Furthermore, the peptides presented a biphasic performance, promoting S. cerevisiae growth in doses around 5 μM and inhibiting it at higher doses. The structural model for these peptides showed that the five amino acids residues, RRLDR at Osm-pepA N-terminus, significantly affect the tertiary structure, indicating that this structure is important for the peptide antimicrobial potency. This is the first report of development of antimicrobial peptides from T. cacao. Taken together, the results indicate that the cacao osmotin and its derived peptides, herein studied, are good candidates for developing biotechnological tools aiming to control phytopathogenic fungi.     

Studies on a N-acetyl-β-d-glucosaminidase produced by Fusarium oxysporum F3 grown in solid-state fermentation

Fusarium oxysporum F3 produced N-acetyl-β-d-glucosaminidase when grown on wheat bran and chitin as carbon sources in solid-state fermentation. The initial moisture content and pH of growth medium were 65% and 6.0, respectively, and the enzyme yield 23.6 U g⁻¹ carbon source. Two isozymes of N-acetyl-β-d-glucosaminidase, called N-acetyl-β-d-glucosaminidases I and II, were isolated from the culture filtrate of F. oxysporum F3. The filtrate was subjected to ammonium sulphate fractionation followed by anion exchange, gel filtration, hydrophobic interaction and cation exchange chromatography. The optimum pH of isozymes I and II was 5.0 and 6.0, respectively, whereas maximum activity of both isozymes was obtained at 40 °C. The K_m of isozymes I and II was 49.6 and 48.6 μM and the V_max 1.24 and 0.26 μmol mg⁻¹ min⁻¹, respectively, on p-nitrophenyl N-acetyl-β-d-glucosaminide as substrate. The molecular mass of isozymes I and II was calculated to be 67 kDa by SDS–PAGE.     

Synthesis and carbonic anhydrase inhibitory properties of sulfamides structurally related to dopamine

A series of novel sulfamides incorporating the dopamine scaffold were synthesized. Reaction of amines and tert-butyl-alcohol/benzyl alcohol in the presence of chlorosulfonyl isocyanate (CSI) afforded sulfamoyl carbamates, which were converted to the title compounds by treatment with trifluoroacetic acid or by palladium-catalyzed hydrogenolysis. Inhibition of six α-carbonic anhydrases (CAs, EC 4.2.1.1), that is, CA I, CA II, CA VA, CA IX, CA XII and CA XIV, and two β-CAs from Candida glabrata (CgCA) and Mycobacterium tuberculosis (Rv3588) with these sulfamides was investigated. All CA isozymes were inhibited in the low micromolar to nanomolar range by the dopamine sulfamide analogues. K_is were in the range of 0.061–1.822 μM for CA I, 1.47–2.94 nM for CA II, 2.25–3.34 μM for CA VA, 0.041–0.37 μM for CA IX, 0.021–1.52 μM for CA XII, 0.007–0.219 μM for CA XIV, 0.35–5.31 μM for CgCA and 0.465–4.29 μM for Rv3588. The synthesized sulfamides may lead to inhibitors targeting medicinally relevant CA isoforms with potential applications as antiepileptic, antiobesity antitumor agents or anti-infective.     

Embryo production and progesterone profiles in ewes superovulated with different hormonal treatments

The superovulatory response and embryo yield following hormonal treatments of Merino ewes during late spring and their estrous cycle were evaluated. Ewes (n=17) were treated with progestagen-impregnated sponges and assigned to Group I (800 IU PMSG plus 11.5 mg FSH-p); Group II (1200 IU PMSG); Group III (1600 IU PMSG). Ewes were naturally mated and followed by laparotomy 6 days later. After laparotomy, ewes were injected with a prostaglandin analogue (PGF) and serum samples were obtained prior to surgery and then for 25 days to measure progesterone (P4) by radioimmunoassay. There were no differences among groups neither for estrous incidence (Group I: 83.3%; Group II: 83.3%; Group III: 100%), nor for the time interval to estrous onset (Group I: 26.4±2.4 h; Group II: 28.8±2.9 h; Group III: 24.0±3.8 h). Group I had more corpora lutea than Group II (14.2±1.2 and 6.2±0.8; P<0.05), and Group III was intermediate (11.0±3.0). There was a low incidence of persistent follicles in all treatments (Group I: 0.5±0.5; Group II: 0.6±0.4; Group III: 1.8±1.2). Number of collected ova were 9.0±2.6, 3.8±0.6 and 6.5±0.9 for Groups I, II and III, respectively. Significant differences in number of ova were detected between Groups I and II. Unfertilized ova did not differ among groups (Group I: 3.5±1.0; Group II: 2.8±0.8; Group III: 5.2±1.4; P>0.05). Embryos and high viability embryos were higher (P<0.05) in Group I (5.2±1.9 and 4.8±2.0) than in Group II (1.0±0.5 and 1.0±0.5) or Group III (1.2±0.6 and 1.0±0.5). Total plasma progesterone (P4) and P4 per corpus luteum before PGF administration did not vary (P>0.05) among groups (Group I: 71.0±14.7 and 4.9±0.7 nmol/l; Group II: 50.6±13.3 and 7.9±1.6 nmol/l; Group III: 90.4±42.6 and 6.8±1.8 nmol/l). There was a significant and positive correlation between P4 before PGF administration and number of corpora lutea (r=0.76). No significant differences were detected among groups for: interval PGF to P4 <3.18 nmol/l (Group I: 2.7±0.3 days; Group II: 1.8±0.6 days; Group III: 2.2±0.5 days), cycle length (Group I: 18.3±1.4 days; Group II: 17.9±0.5 days; Group III: 16.8±0.9 days), duration of P4 levels <3.18 nmol/l (Group I: 11.3±1.9 days; Group II: 7.1±1.0 days; Group III: 7.2±2.4 days), duration of P4 levels ≥3.18 nmol/l (Group I: 7.0±1.3 days; Group II: 10.8±0.8 days; Group III: 9.5±1.7 days) and peak of P4 (Group I: 7.4±0.4 nmol/l; Group II: 10.8±1.6 nmol/l; Group III: 9.2±1.9 nmol/l). It was concluded that PMSG–FSH-p treatment was more efficient than PMSG alone for superovulation and embryo production in ewes while P4 profiles were similar among groups.     

Purification and partial characterization of a 36-kDa chitinase from Bacillus thuringiensis subsp. colmeri,and its biocontrol potential

Chitinase A (ChiA) produced by Bacillus thuringiensis subsp. colmeri 15A3 (Bt. 15A3) was expressed in Escherichia coli XL-Blue. The ChiA was purified using Sephadex G-200 and its molecular mass was estimated to be 36 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Detection of chitinase activity on SDS-PAGE after protein renaturation indicated that the molecular mass of the protein band expressing chitinase activity was approximately 72 kDa. This suggests that the dimeric form of ChiA is the enzymatically active form when glycol chitin is used as a substrate. ChiA has optimal activity at 50 °C and retains most of its activity between 20 and 60 °C. The optimum pH for ChiA activity is pH 5.0, and the enzyme is active between pH 4.0 and 8.0. The enzyme activity was significantly inhibited by Ag⁺ and Zn²⁺. ChiA significantly inhibited the spore germination of four species of fungi. The median inhibitory concentrations (IC₅₀) of ChiA on the spore germination of Penicillium glaucum and Sclerotinia fuckelian were 11.27 and 10.57 μg/ml, respectively. In surface contamination bioassays, the crude ChiA protein (12.6 mU) reduced the LC₅₀ (50% lethal concentration) of the crystal protein of Bt. 15A3 against the larvae of Spodoptera exigua and Helicoverpa armigera.     

Photoactivated disinfection (PAD) of dental root canal system – An ex-vivo study

AimTo investigate the efficacy of photo activated disinfection (PAD) in reducing colony-forming unit (CFU) counts of Enterococcus faecalis (E. faecalis) in infected dental root canals. The study compared the efficacy of PAD with conventional endodontic treatment (CET) and also a combination of CET along with PAD.Material and Methods53 maxillary incisors were taken for the study. Teeth were divided into 3 groups, CET (Group I) (n = 11), PAD (Group II) (n = 21), and a combination of CET and PAD (Group III) which consisted of (n = 21) samples, Group II and Group III were further divided into 2 subgroups, Group IIa, IIb and Group IIIa, IIIb. Strains of E. faecalis were inoculated in all the root canals. CET group samples were treated by chemo-mechanical preparation (CMP) alone, PAD samples were treated with laser alone at 2 different exposure time (4 min and 2 min). In the combination treatment, samples were treated initially by CET and then by PAD for a time period of 4 min and 2 min. Contents of the root canal were aspirated, diluted and plated in Tryptone Soya Broth (TSB) and plates were incubated for 24 h to observe the bacterial regrowth.ResultsShowed PAD used along with CMP reduced the bacterial load of E. faecalis by 99.5% at 4 min and 98.89% at 2 min.ConclusionPAD may be an adjunctive procedure to kill residual bacteria in the dental root canal systems after standard endodontic root canal preparation.     

Immunological characterization of diphtheria toxin recovered from Corynebacterium pseudotuberculosis

Diphtheria toxin (DT) is a potent toxin produced by the so-called diphtheria group which includes Corynebacterium diphtheriae (C. diphtheriae), Corynebacterium ulcerans (C. ulcerans), and Corynebacterium pseudotuberculosis (C. pseudotuberculosis). The present investigation is aimed to study in detail the production of DT by C. pseudotuberculosis. Twenty isolates were obtained from sheep diseased with caseous lymphadenitis (CLA) and twenty-six isolates were obtained from 26 buffaloes diseased with oedematous skin disease (OSD). All isolates were identified by standard microbiological and DT production was assayed serologically by modified Elek test and immunoblotting. All sheep isolates were nitrate negative, failed to hydrolyze starch and could not produce DT, while all buffalo isolates (biotype II) revealed positive results and a specific band of 62 kDa, specific to DT, was resulted in all concentrated cell fractions (CF), but was absent from non-toxigenic biotype I isolates. At the same time, another band of 31 kDa specific to the PLD gene was obtained with all isolates of biotype I and II. Moreover, all isolates showed positive synergistic hemolytic activity and antagonistic hemolysis with β-hemolytic Staphylococci. The obtained results also indicated that C. pseudotuberculosis could be classified into two strains; non-toxigenic biotype I strain, which failed to produce DT as well as being negative to nitrate and starch hydrolysis, and toxigenic biotype II strain, which can reduce nitrate, hydrolyze starch as well as produce DT.     

Hybrid fluorescent conjugates of COX-2 inhibitors: Search for a COX-2 isozyme imaging cancer biomarker

The observation that the cyclooxygenase-2 (COX-2) isozyme is over-expressed in multiple types of cancer, relative to that in adjacent non-cancerous tissue, prompted this investigation to prepare a group of hybrid fluorescent conjugates wherein the COX inhibitors ibuprofen, (S)-naproxen, acetyl salicylic acid, a chlororofecoxib analog and celecoxib were coupled via a linker group to an acridone, dansyl or rhodamine B fluorophore. Within this group of compounds, the ibuprofen-acridone conjugate (10) showed potent and selective COX-2 inhibition (COX-2 IC₅₀ = 0.67 μM; SI = 110.6), but its fluorescence emission (λ_em = 417, 440 nm) was not suitable for fluorescent imaging of cancer cells that over-express the COX-2 isozyme. In comparison, the celecoxib-dansyl conjugate (25) showed a slightly lower COX-2 potency and selectivity (COX-2 IC₅₀ = 1.1 μM; SI > 90) than the conjugate 10, and it possesses a better fluorescence emission (λ_em = 500 nm). Ultimately, a celecoxib-rhodamine B conjugate (28) that exhibited moderate COX-2 potency and selectivity (COX-2 IC₅₀ = 3.9 μM; SI > 25) having the best fluorescence emission (λ_em = 580 nm) emerged as the most promising biomarker for fluorescence imaging using a colon cancer cell line that over-expresses the COX-2 isozyme.