POTENTIAL RAPID BIOASSAY FOR ALIMET® USING A METHIONINE ESCHERICHIA COLI AUXOTROPH

A potential rapid bioassay for methionine hydroxy analog (MHA) feed additive (ALIMET®) was examined using a methionine auxotroph E. coli strain. Bacterial cells were grown in minimal media containing a concentration range of 0 to 26.8 μM of either L-methionine or MHA as ALIMET®. Increasing either methionine or MHA concentration increased the growth rate of the methionine auxotroph. The estimated substrate affinities for methionine compared to MHA were not significantly different (P > 0.13) and the maximum growth rate estimates were also similar (P > 0.34). Methionine and MHA standard curves yielded linear responses (R²= 0.96) to increasing concentrations of the respective substrate. Based on these results it appears that the E. coli methionine auxotroph would have potential utility for further development of a rapid bioassay of ALIMET®.     

Effect of cooling rate,cryoprotectant and holding time at different transfer temperatures on the survival of cryopreserved cell suspension culture (Puccinellia distans (L.) Parl.)

Reflexed saltmarsh-grass suspension cultures produced by seed callus were frozen to the liquid nitrogen temperature. Cooling rates, cryoprotectants and holding times were taken as a function of transfer temperatures. The highest survival of cells (45%) was found at a freezing rate of 1°C min^-1, without cryoprotectant treatments. The cryoprotectants (proline, dimethyl sulphoxide, glycerol), used at different concentrations and transfer temperatures, increased the survival rate. The maximum value was 78% at 12.5% (w/v) of proline with –30°C transfer temperature. Considerable improvement of viability (from 0% to 95%) among the 12.5 and 15.0% (v/v) dimethyl sulphoxide cryopreserved cells was achieved by holding them at – 20°C for 10–30 min before plunging into the liquid nitrogen. A 20 min holding time at 15.0% (v/v) glycerol level and – 30°C transfer temperature significantly enhanced the viability of the explants from 42% to 92%. Plants were successfully regenerated from cells cryopreserved with proline (w/v) and dimethyl sulfoxide (v/v) levels of 12.5 and 15.0%, respectively.     

A karyological study of Asphodelus L. (Asphodelaceae) from the Western Mediterranean

The following aspects of Asphodelus karyology are analysed: base number, polyploidy, chromosome size, chromosome morphology, satellited chromosomes, structural heteromorphism, karyotype asymmetry and karyotype evolution. The base number 0 ×= 14 is common to all species except for A. refractus , which has the derived ×= 13. Three ploidy levels occur, often in the same species; diploid, tetraploid and hexaploid, with 2n = 28, 56 and 84. Chromosomes are generally small to medium-small, with the occasional presence of medium-large chromosomes. The most frequent chromosome types are metacentric of type m and submetacentric. Metacentric chromosomes of type M occur only in sections. Verineopsis, Verinea and Plagiasphodelus ; subtelocentric chromosomes occur only in sections Asphodelus and Plagiasphodelus. There is a wide variability in relation to the number of satellited chromosomes, relative to ploidy level. There are usually two to four in diploids, four to eight in tetraploids and usually six, exceptionally up to 12, in the hexaploid. Satellites are present on the shortest arm, exceptionally on the longest arm. There is a high degree of structural heteromorphism in practically all the species which affects satellited and non satellited chromosomes. Karyotype asymmetry is generally of type 2B. Inter-and intra-chromosomal differences are estimated by the A1 and A2 indexes. Both indices vary in the karyotype evolution of the genus, with a decrease of A1 and an increase of A2. The role of polyploidy, hybridization, asymmetry and decrease of chromosome size in the evolution of Asphodelus is discussed.     

Melanism and coat colour polymorphism in the Egyptian Wolf Canis lupaster Hemprich & Ehrenberg (Carnivora: Canidae) from Egypt

The Egyptian Wolf Canis lupaster was recently rediscovered as a distinct species on the basis of both morphologic and molecular genetic evidence. Phenotypical variability, including coat colour of this species across its vast, ecologically diverse range is yet to be investigated. In this paper, we present the first record of melanistic individuals of this species and compare their morphological characters and mitochondrial cytochrome b DNA sequences with those of typically coloured Canis lupaster and other closely related canids to verify their identity. We also study pelage polymorphism in a population of this species in the Egyptian Nile Valley and the Nile Delta and define its different colour variants. The typical colour, as well as the rare, very light and reddish coat colours are described. We discuss the possibility that the observed coat colour polymorphism is the result of hybridisation with the domestic dog and their potential adaptive significance.     

Sexual dimorphism and interpopulation differences in lizard hind limb length: locomotor performance or chemical signalling?

Intraspecific variation in morphology has often been related to fitness differences through its effects on performance. In lizards, variation in hind limb length can be shaped by natural selection for increased locomotor performance, sexual selection on the number or size of femoral pores involved in chemical signalling, or both. Here, we analyse the selective forces involved in sexual dimorphism and differences in hind limb length between two populations of Psammodromus algirus living at different elevation. Males were more robust and had longer hind limbs and limb segments than females, and low‐elevation lizards had longer limbs than high‐elevation lizards. However, differences in locomotor performance were small and non‐significant, making natural selection for faster runs an unlikely explanation for the observed pattern. On the other hand, males had more femoral pores than females, and lizards had more pores at lower elevation, although the difference was significant only for males (which invest more in chemical signalling). In males, the number of pores, which remains constant along a lizard's life, was not correlated with hind limb length. However, femur length was positively correlated with mean pore size, allowing low‐elevation males to have larger than expected pores, which could increase the effectiveness with which they spread their signals in a dry and warm habitat where chemicals become volatile rapidly. Also, saturation of the sexual coloration of the head was higher for low‐elevation males, suggesting that sexual selection pressures may be more intense. Overall, our results indicate that sexual selection plays a significant role in shaping intraspecific variation in hind limb length. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104 , 318–329.     

Premalignant Variations in Extracellular Matrix Composition in Chemically Induced Hepatocellular Carcinoma in Rats

Chemical composition of extracellular matrix (ECM) plays a pivotal role in cellular and tissue development, regeneration, and differentiation. It also plays a key role in pathogenesis of hepatocellular carcinoma (HCC). This study explored premalignant changes in the liver tissue content of collagen (as hydroxyproline, HP), total glycosaminoglycans (TGAGs), free glucosamine (FGA), total sialic acid (TSA), lysosomal membrane integrity variations (calculated as total and free cathepsin D activities), and liver histology. Serum alfa-fetoprotein (AFP) level was used as an early marker for HCC in two groups of Wistar rats. One group of rats served as control and was provided normal saline orally. The other group was provided trichloroacetic acid (TCA) as 0.5 g/kg/day for five consecutive days by oral gavage. Animals were killed before tumor development. The treatment revealed dysplastic changes in addition to microsteatosis (fatty changes). Both sinusoids and the portal vein among dysplastic cells were dilated and congested. These dysplastic foci are believed to be premalignant and may be precancerous lesions. The following things were observed: a highly significant increase in serum AFP (as a key marker for HCC), a significant decrease in HP and TSA, a significant increase in FGA, nonsignificant decrease in TGAGs, significant up-regulation of free cathepsin D, nonsignificant decrease in total cathepsin D activities, and destabilization of lysosomal membrane integrity. Down-regulation of HP, TSA, and TGAGs seems to be a prerequisite for cancer development. This might be stimulated by up-regulation of free cathepsin D activity. Perhaps tissue fibrosis is not a condition for developing HCC because collagen was significantly depressed. Up-regulated FGA could be assumed to be a defense mechanism against TCA-induced proteolysis of membrane proteins because it is frequently reported to be of value in cancer chemotherapy. Studied ECM perturbations can be assumed as preliminary changes during chemical hepatocarcinogenesis at the tissue level. Prospective studies on blood levels of cathepsins, TGAGs, and individual ECM variables such as TSA, FGA, and Hp in patients at risk for HCC, performed in parallel with assessments of AFP, may provide a cost-effective way to find new links between tissue changes and circulation that would permit early prediction of disease. It may also provide a way to monitor HCC and compensate for the missed peak AFP values.     

Flow-through immunofiltration assay system for rapid detection of E. coli O157:H7

A flow-through amperometric immunofiltration assay system based on disposable porous filter-membranes for rapid detection of Escherichia coli O157:H7 has been developed. The analytical system utilizes flow-through, immunofiltration and enzyme immunoassay techniques in conjunction with an amperometric sensor. The parameters affecting the immunoassay such as selection of appropriate filter membranes, membrane pore size, antibody binding capacity and the concentrations of immunoreagents were investigated and optimized. Non-specific adsorption of the enzyme conjugate was investigated and minimized. A sandwich scheme of immunoassay was employed and the immunofiltration system allows to specifically and directly detect E. coli cells with a lower detection limit of 100 cells/ml. The working range is from 100 to 600 cells/ml with an overall analysis time of 30 min. No pre-enrichment was needed. This immunosensor can be easily adapted for assay of other microorganisms and may be a basis for a new class of highly sensitive bioanalytical devices for rapid quantitative detection of bacteria.     

Genetic markers between Biomphalaria glabrata snails susceptible and resistant to Schistosoma mansoni infection

The analysis of the genetic variability related to susceptibility to Schistosoma mansoni infection in the vector of the genus Biomphalaria is important in terms of a better understanding of the epidemiology of schistosomiasis itself, the possible pathological implications of this interaction in vertebrate hosts, and the formulation of new strategies and approaches for disease control. In the present study, the genetic variability of B. glabrata strains found to be resistant or susceptible to S. mansoni infection was investigated using DNA amplification by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). The amplification products were analyzed on 8% polyacrylamide gel and stained with silver. We selected 10 primers, since they have previously been useful to detect polymorphism among B. glabrata and/or B. tenagophila. The results showed polymorphisms with 5 primers. Polymorphic bands observed only in the susceptible strain. The RAPD-PCR methodology represents an adequate approach for the analysis of genetic polymorphisms. The understanding of the genetic polymorphisms associated to resistance may contribute to the future identification of genomic sequences related to the resistance/susceptibility of Biomphalaria to the larval forms of S. mansoni and to the development of new strategies for the control of schistosomiasis.