Antioxidant Capacity of Melatonin on Preimplantation Development of Fresh and Vitrified Rabbit Embryos: Morphological and Molecular Aspects

Embryo cryopreservation remains an important technique to enhance the reconstitution and distribution of animal populations with high genetic merit. One of the major detrimental factors to this technique is the damage caused by oxidative stress. Melatonin is widely known as an antioxidant with multi-faceted ways to counteract the oxidative stress. In this paper, we investigated the role of melatonin in protecting rabbit embryos during preimplantation development from the potential harmful effects of oxidative stress induced by in vitro culture or vitrification. Rabbit embryos at morula stages were cultured for 2 hr with 0 or 10⁻³ M melatonin (C or M groups). Embryos of each group were either transferred to fresh culture media (CF and MF groups) or vitrified/devitrified (CV and MV groups), then cultured in vitro for 48 hr until the blastocyst stage. The culture media were used to measure the activity of antioxidant enzymes: glutathione-s-transferase (GST) and superoxide dismutase (SOD), as well as the levels of two oxidative substrates: lipid peroxidation (LPO) and nitric oxide (NO). The blastocysts from each group were used to measure the expression of developmental-related genes (GJA1, POU5F1 and Nanog) and oxidative-stress-response-related genes (NFE2L2, SOD1 and GPX1). The data showed that melatonin promoted significantly (P<0.05) the blastocyst rate by 17% and 12% in MF and MV groups compared to their controls (CF and CV groups). The GST and SOD activity significantly increased by the treatment of melatonin in fresh or vitrified embryos, while the levels of LPO and NO decreased (P<0.05). Additionally, melatonin considerably stimulated the relative expression of GJA1, NFE2L2 and SOD1 genes in MF and MV embryos compared to CF group. Furthermore, melatonin significantly ameliorated the reduction of POU5F1 and GPX1 expression induced by vitrification. The results obtained from the current investigation provide new and clear molecular aspects regarding the mechanisms by which melatonin promotes development of both fresh and vitrified rabbit embryos.     

Pathogen-mediated modulation of host metabolism and trophic interactions in Spodoptera littoralis larvae

The success and sustainability of entomopathogens in insect control depend on their stress potential and ability to modulate certain physiological aspects of their insect hosts. In the present study, newly moulted fourth instars of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) were incubated for 24 h with the median lethal dose (LD₅₀) of Steinernema riobrave, Heterorhabditis bacteriophora, or Beauveria bassiana to determine their effects on certain metabolic activities and nutritional physiology compared to those of uninfected (control) larvae. Infection decreased total protein, lipid, and carbohydrate contents. Carbohydrase activity was found to be pathogen and enzyme dependent, with S. riobrave and H. bacteriophora infection increasing amylase, invertase, and trehalase activities. Beauveria bassiana infection decreased amylase and invertase activities but increased that of trehalase. Infection with the three pathogens also enhanced phosphatase activity. All pathogens reduced transaminase activity. Changes in the nutritional indices varied not only with the variation in the candidate entomopathogen and surviving instar but also with the variation in the age of the same instar, with a profound change in late last instars (3-day-old sixth instars). The link between the metabolic activities and nutritional indices was discussed. Overall, the entomopathogen-host interaction appears to be primarily nutritional. The pleiotropic effects of the median lethal infections on the insect pest might eventually help in the biological control potential of the entomopathogens tested.     

Mutations in the Beta Propeller WDR72 Cause Autosomal-Recessive Hypomaturation Amelogenesis Imperfecta

Healthy dental enamel is the hardest and most highly mineralized human tissue. Though acellular, nonvital, and without capacity for turnover or repair, it can nevertheless last a lifetime. Amelogenesis imperfecta (AI) is a collective term for failure of normal enamel development, covering diverse clinical phenotypes that typically show Mendelian inheritance patterns. One subset, known as hypomaturation AI, is characterised by near-normal volumes of organic enamel matrix but with weak, creamy-brown opaque enamel that fails prematurely after tooth eruption. Mutations in genes critical to enamel matrix formation have been documented, but current understanding of other key events in enamel biomineralization is limited. We investigated autosomal-recessive hypomaturation AI in a consanguineous Pakistani family. A whole-genome SNP autozygosity screen identified a locus on chromosome 15q21.3. Sequencing candidate genes revealed a point mutation in the poorly characterized WDR72 gene. Screening of WDR72 in a panel of nine additional hypomaturation AI families revealed the same mutation in a second, apparently unrelated, Pakistani family and two further nonsense mutations in Omani families. Immunohistochemistry confirmed intracellular localization in maturation-stage ameloblasts. WDR72 function is unknown, but as a putative β propeller is expected to be a scaffold for protein-protein interactions. The nearest homolog, WDR7, is involved in vesicle mobilization and Ca²⁺-dependent exocytosis at synapses. Vesicle trafficking is important in maturation-stage ameloblasts with respect to secretion into immature enamel and removal of cleaved enamel matrix proteins via endocytosis. This raises the intriguing possibility that WDR72 is critical to ameloblast vesicle turnover during enamel maturation.     

Shedding of Vaccine Viruses with Increased Antigenic and Genetic Divergence after Vaccination of Newborns with Monovalent Type 1 Oral Poliovirus Vaccine

For the final stages in the eradication of poliovirus type 1 (P1), the World Health Organization advocates the selective use of monovalent type 1 oral poliovirus vaccine (mOPV1). To compare the immunogenicity of mOPV1 with that of trivalent OPV (tOPV) in infants, a study was performed in Egypt in 2005. Newborns were vaccinated with mOPV1 or tOPV immediately after birth and were challenged with mOPV1 after 1 month. Vaccination with mOPV1 at birth resulted in significantly higher seroconversion against P1 viruses and lower excretion of P1 viruses than vaccination with tOPV. Intratypic differentiation of the viruses shed by the newborns revealed the presence of remarkably high numbers of antigenically divergent (AD) P1 isolates, especially in the mOPV1 study group. The majority of these AD P1 isolates (71%) were mOPV1 challenge derived and were shed by newborns who did not seroconvert to P1 after the birth dose. Genetic characterization of the viruses revealed that amino acid 60 of the VP3 region was mutated in all AD P1 isolates. Isolates with substitution of residue 99 of the VP1 region had significantly higher numbers of nonsynonymous mutations in the VP1 region than isolates without this substitution and were preferentially shed in the mOPV1 study group. The widespread use of mOPV1 has proven to be a powerful tool for fighting poliovirus circulation in the remaining areas of endemicity. This study provides another justification for the need to achieve high vaccination coverage in order to prevent the circulation of AD strains.Polioviruses are the causative agents of human poliomyelitis and belong to the genus Enterovirus in the family Picornaviridae. The virus is transmitted primarily by the fecal-oral route and replicates in the human intestinal tract. The virus may also be transmitted through respiratory droplets and may replicate for a short period in the upper respiratory tract and tonsillar tissue. From either site of primary replication, the virus may invade the central nervous system and cause paralysis following infection and destruction of motor neurons. Three serologically different types of poliovirus can be distinguished (poliovirus type 1 [P1], P2, and P3), and only limited cross-protection exists between serotypes (35).In 1988, the World Health Assembly passed a resolution to eradicate wild poliovirus globally. A worldwide vaccination campaign with the trivalent oral poliovirus vaccine (tOPV) was launched by the World Health Organization (WHO). This vaccine contains the three attenuated poliovirus vaccine strains developed by Albert Sabin in the proportion of 10:1:6 for P1, P2, and P3, respectively. These OPV strains have been selected to replicate successfully in the human intestinal tract but not in the cells of the central nervous system. In addition to a strong humoral response, these strains generate strong intestinal immunity (12). Sabin type 1 is considered to be the most stable of the three attenuated poliovirus serotypes (19). This strain has 54 mutations compared to the parental Mahoney strain, of which 6 are primarily responsible for attenuation. Sabin type 2 has two major determinants of attenuation, and Sabin type 3 has three determinants of attenuation (11, 32). Upon replication in the human intestinal tract, the sites of attenuation can mutate, which results in reversion of the Sabin strains toward a parental neurovirulent phenotype. Also as a consequence of replication in the host, antibodies are produced that recognize the antigenic sites of the Sabin strains (42). This immunogenic pressure could favor the selection of antigenically divergent (AD) viruses with substituted residues in parts of these antigenic sites. AD Sabin viruses might circulate among a population for a long period and evolve into vaccine-derived polioviruses (VDPVs; with differences of >1% from the prototype Sabin viruses in the VP1 region) capable of causing outbreaks. These viruses might escape current diagnostic screening methods, and the risk for generation of these viruses should be reduced as much as possible (1, 9, 16).The tOPV vaccination campaigns have been very successful, since the number of countries with endemic wild poliovirus circulation decreased from >125 in 1988 to 4 in 2006, and wild type 2 poliovirus has likely been eradicated since 1999 (5). The tOPV vaccine, however, is known to be less immunogenic against type 1 and 3 polioviruses. After tOPV administration, the superior replicative capacity of the P2 vaccine strain interferes with effective replication of the other two serotype viruses in the human intestine (30). To eradicate wild P1 as well, vaccination with monovalent type 1 oral poliovirus vaccine (mOPV1) was introduced in the remaining countries where poliovirus is endemic, since this vaccine is more immunogenic for type 1 than the tOPV (4, 20).In 2005/2006, a clinical study was conducted in Egypt to compare the immunogenicity of mOPV1 with that of the tOPV in newborns (15). Newborns were vaccinated with mOPV1 or tOPV as soon as possible after birth and were challenged with mOPV1 4 weeks later. Vaccination with mOPV1 at birth resulted in a higher humoral and mucosal protection against P1 at day 28 than vaccination with tOPV at birth.In line with the recommendations of the WHO Polio Laboratory Network, we determined the antigenic characters of all the viruses shed by the newborns of the Egyptian study by using an intratypic differentiation (ITD) enzyme-linked immunosorbent assay (ELISA). The outcome of this analysis, an unexpectedly high percentage of AD isolates, prompted further investigation. To determine the possible presence of VDPVs and to gain insight into the genetic and antigenic evolution of the mOPV1 and tOPV isolates shed by the newborns in this study, we determined the sequences of the capsid regions of these isolates. We looked for correlates with antigenic change and rates of mutagenesis in the viruses and compared the evolution rates of the viruses shed by vaccinees of both study groups. We also linked the serological data collected during the study to the excretion of Sabin 1 isolates.     

<Emphasis Type="SmallCaps">l</Emphasis>-Methioninase Production by Filamentous Fungi: I-Screening and Optimization Under Submerged Conditions

Findings show 21 fungal isolates belonging to eight genera recovered from Egyptian soils that have the potential to attack l-methionine under submerged conditions. Aspergillus flavipes had the most methioninolytic activity, giving the highest yield of l-methioninase (10.78 U/mg protein), rate of methionine uptake (93.0%), and growth rate (5.0 g/l), followed by Scopulariopsis brevicaulis and A. carneus. The maximum l-methioninase productivity (11.60 U/mg protein) by A. flavipes was observed using l-methionine (0.8%) as an enzyme-inductive agent and glucose (1%) as a co-dissimilated carbon source. A significant reduction in l-methioninase biosynthesis by A. flavipes was detected using carbon-free medium, suggesting the lack of ability to use l-methionine as a carbon and nitrogen source. Potassium dihydrogen phosphate (0.25%), the best source of phosphorus, favors enzyme biosynthesis and enhances the level of methionine uptake by A. flavipes. The maximum l-methioninase productivity (12.58 U/mg protein) and substrate uptake (95.6%) were measured at an initial pH of 7.0.     

Pharmacokinetics, immunogenicity and anticancer efficiency of Aspergillus flavipesl-methioninase

Methionine starvation can powerfully modulate DNA methylation, cell cycle transition, polyamines and antioxidant synthesis of tumor cells, in contrary to normal ones. Aspergillus flavipesl-methioninase was previously characterized by our studies, displaying affordable biochemical properties comparing to Pseudomonas putida enzyme (ONCASE). Thus, the objective of current study was to evaluate the catalytic properties of Af-METase in New Zealand rabbits, exploring its antitumor efficacy. In vivo, Af-METase (40.8U/ml) have T(1/2) 19.8h, elimination constant 0.088U/h and apparent volume distribution 85U/ml. Also, Af-METase has two maxima one at A(280nm) (apo-enzyme) and at A(420nm) (internal Schiff base of PLP), unlike control plasma (without enzyme). The two peaks of absorption spectra were detected maximally at 15min then the absorbance at 420nm was subsequently decreased with circulation time, due to dissociation of the co-enzyme. The A(280/420) ratio was increased from 1.69 to 5.81 with circulation time from 15 to 30h. Rabbits plasma methionine was depleted from 18.7μM (control) to 8.8μM after 1h of enzyme injection and completely omitted after 2h till 19h, assuming the sustainability of negligible levels of methionine (<2μM) in plasma of rabbits, for about 17h. Upon infusion of PLP, the T(1/2) of Af-METase was significantly prolonged by 3.2 fold, assuming the fully reconstitution of the enzyme. The holo-AfMETase still retained its co-enzyme, completely, till 33h of PLP infusion. From spectral studies, the internal aldimine linkage of apo-Af-METase was constructed upon PLP infusion, with fully catalytic structure after less than 4h of its infusion, the A(280/420) ratio being not relatively changed till 45h. After 25 days of last enzyme dose, the titer of IgG was increase by about 1.66 fold comparing to control (without enzyme). However, IgM was not detected along the tested challenge points. In vitro, plasma anti-Af-METase neutralizing antibodies (NAb) were assessed, with no significant reduction on activity of Af-METase by Nab. All the hematological parameters were in normal range, otherwise, the RBCs titer and platelet level was slightly increased, after 25 days of Af-METase injection, comparing to control. There is no obvious negative effect on chemistry of liver, kidney, glucose, lipids, and other electrolytes. Additionally, the anticancer activity of Af-METase was evaluated against five types of human cancer cell lines, in vitro. The enzyme showed a powerful activity against prostate (PC3), liver (HEPG2) and breast (MCF7) cancers, with IC(50) 0.001U/ml, 0.26U/ml and 0.37U/ml, respectively.     

Synthesis, biological evaluation and molecular modeling study of pyrazole and pyrazoline derivatives as selective COX-2 inhibitors and anti-inflammatory agents. Part 2

New pyrazole and pyrazoline derivatives have been synthesized and their ability to inhibit ovine COX-1/COX-2 isozymes was evaluated using in vitro cyclooxygenase (COX) inhibition assay. Among the tested compounds, N-((5-(4-chlorophenyl)-1-phenyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)methylene)-3,5-bis(trifluoromethyl)aniline 8d exhibit optimal COX-2 inhibitory potency (IC(50)=0.26 lM) and selectivity (SI)=>192.3] comparable with reference drug celecoxib (IC(50) value of 0.28 lM and selectivity index of 178.57). Moreover, the anti-inflammatory activity of selected compounds, which are the most selective COX-2 inhibitors in the COX inhibition assay, was investigated in vivo using carrageenan-induced rat paw edema model. Molecular modeling was conducted to study the ability of the active compounds to bind into the active site of COX-2 which revealed a similar binding mode to SC-558, a selective COX-2 inhibitor.     

Schistosoma mansoni genome project: an update

A schistosome genome project was initiated by the World Health Organization in 1994 with the notion that the best prospects for identifying new targets for drugs, vaccines, and diagnostic development lie in schistosome gene discovery, development of chromosome maps, whole genome sequencing and genome analysis. Schistosoma mansoni has a haploid genome of 270 Mb contained on 8 pairs of chromosomes. It is estimated that the S. mansoni genome contains between 15000 and 25000 genes. There are approximately 16689 ESTs obtained from diverse libraries representing different developmental stages of S. mansoni, deposited in the NCBI EST database. More than half of the deposited sequences correspond to genes of unknown function. Approximately 40-50% of the sequences form unique clusters, suggesting that approximately 20-25% of the total schistosome genes have been discovered. Efforts to develop low resolution chromosome maps are in progress. There is a genome sequencing program underway that will provide 3X sequence coverage of the S. mansoni genome that will result in approximately 95% gene discovery. The genomics era has provided the resources to usher in the era of functional genomics that will involve microarrays to focus on specific metabolic pathways, proteomics to identify relevant proteins and protein-protein interactions to understand critical parasite pathways. Functional genomics is expected to accelerate the development of control and treatment strategies for schistosomiasis.     

Role of hyaluronidase inhibitors in the neutralization of toxicity of Egyptian horned viper Cerastes cerastes venom

Hyaluronidase “venom spreading factor” is a common component of snake venoms and indirectly potentiates venom toxicity. It may cause permanent local tissue destruction at the bite site/systemic collapse of the envenomated victim. The present study was performed to assess the benefits of inhibiting the hyaluronidase activity of Egyptian horned viper, Cerastes cerastes (Cc). The aqueous extracts of some medicinal plants were screened for their inhibitory effect on hyaluronidase activity of Cc venom. The results revealed that the Rosmarinus officinalis (Ro) extract is the most potent hyaluronidase inhibitor among the tested extracts. The Ro extract is more potent inhibitory effect on the hyaluronidase activity than the prepared rabbit monoclonal antiserum of previously purified hyaluronidase enzyme from Cc venom (anti-CcHaseII). In addition, the Ro extract is efficiently inhibited the activity of hemorrhagic toxin previously purified from Cc venom, and it also neutralized the edema inducing activity of the Cc venom in vivo. Furthermore, the Ro extract markedly increased the survival time of experimental mice injected with lethal dose of Cc venom up to 7 h in compared to mice injected with venom alone or with venom/anti-CcHaseII (15 ± 5, 75 ± 4 min), respectively. Our findings imply the significance of plant-derived hyaluronidase inhibitor in the neutralization of local effects of Cc venom and retardation of death time. Therefore, it may use as a therapeutic value in complementary snakebite therapy.     

Implications of life-history transitions on the population genetic structure of the toxigenic marine dinoflagellate Alexandrium tamarense

Genotypic or phenotypic markers for characterization of natural populations of marine microalgae have typically addressed questions regarding differentiation among populations, usually with reference to a single or few clonal isolates. Based upon a large number of contemporaneous isolates from the same geographical population of the toxigenic species Alexandrium tamarense from the North Sea, we uncovered significant genetic substructure and low but significant multilocus linkage disequilibrium (LD) within the planktonic population. Between the alternative molecular genotyping approaches, only amplified fragment length polymorphism (AFLP) revealed cryptic genetic population substructure by Bayesian clustering, whereas microsatellite markers failed to yield concordant patterns. Both markers, however, gave evidence for genetic differentiation of population subgroups as defined by AFLP. A considerable portion of multilocus LD could be attributed to population subdivision. The remaining LD within population subgroups is interpreted as an indicator of frequency shifts of clonal lineages during vegetative growth of planktonic populations. Phenotypic characters such as cellular content and composition of neurotoxins associated with paralytic shellfish poisoning (PSP) and allelochemical properties may contribute to intra- or inter-annual differentiation of planktonic populations, if clonal lineages that express these characters are selectively favoured. Nevertheless, significant phenotypic differentiation for these characters among the genetically differentiated subgroups was only detected for PSP toxin content in two of the four population subgroups. By integrating the analysis of phenotypic and genotypic characteristics, we developed a conceptual population genetic model to explain the importance of life-cycle dynamics and transitions in the evolutionary ecology of these dinoflagellates.