Genotypic and phenotypic characterization of rhizobia that nodulate snap bean (Phaseolus vulgaris L.) in Egyptian soils

Snap bean fields in 12 of the 25 governorates of Egypt were surveyed to determine the distribution and taxonomy of snap bean-nodulating rhizobia. Nodulation rates in the field were very low, indicating that Egyptian soils do not have sufficient numbers of snap bean-compatible Rhizobium spp. A total of 87 rhizobial isolates were assayed on the most commonly grown cultivars in order to identify the most effective strains. The five most effective isolates (R11, R13, R28, R49 and R52) were fast-growing and utilized a wide range of carbon and nitrogen sources. A phylogenetic assignment of these strains by analysis of the 16S ribosomal RNA gene suggested that all fell within the Rhizobium etli–Rhizobium leguminosarum group. Strains R11, R49 and R52 all clustered with other identified R. etli strains, while strains R13 and R28 were more distinct. The distinctness of R13 and R28 was supported by physiological characteristics, such as their ability to utilize citrate, erythritol, dulcitol and lactate. Strains R13 and R28 also yielded the highest plant nitrogen content of all isolates.The highly effective strains isolated in this study, in particular strains R13 and R28, are promising candidates for improving crop yields. The data also suggested that these two strains represented a novel sub-group within the R. etli–R. leguminosarum group. As snap bean is a crop of great economic value to Egypt, the identification of highly effective rhizobial strains adapted to Egyptian soils, such as strains R13 and R28, is of great interest.     

On-line casein micelle disruption for downstream purification of recombinant human myelin basic protein produced in the milk of transgenic cows

Downstream purification of a model recombinant protein (human myelin basic protein) from milk of transgenic cows is described. The recombinant protein was expressed as a His tagged fusion protein in the milk of transgenic cows and was found associated with the casein micellar phase. While difficulties in obtaining good recoveries were found when employing conventional micelle disruption procedures, direct capture using the cation exchanger SP Sepharose Big Beads? was found successful in the extraction of the recombinant protein. Early breakthrough suggested a slow release of the recombinant protein from the micelles and dictated micelle disruption in order to obtain good yields. A new approach for deconstruction of the calcium core of the casein micelles, employing the interaction between the micellar calcium and the active sites of the cation exchanger resin was developed. Milk samples were loaded to the column in aliquots with a column washing step after each aliquot. This sequential loading approach successfully liberated the recombinant protein from the micelles and was found superior to the conventional sample loading approach. It increased the recovery by more than 25%, reduced fouling due to milk components and improved the column hydrodynamic properties as compared to the conventional sample loading approach. Hardware and software modifications to the chromatography system were necessary in order to keep the whole process automated. A second purification step using a Ni²⁺ affinity column was used to isolate the recombinant protein at purity more than 90% and a recovery percentage of 78%.     

Ion transport mechanisms linked to bicarbonate secretion in the esophageal submucosal glands

The esophageal submucosal glands (SMG) secrete HCO(3)(-) and mucus into the esophageal lumen, where they contribute to acid clearance and epithelial protection. This study characterized the ion transport mechanisms linked to HCO(3)(-) secretion in SMG. We localized ion transporters using immunofluorescence, and we examined their expression by RT-PCR and in situ hybridization. We measured HCO(3)(-) secretion by using pH stat and the isolated perfused esophagus. Using double labeling with Na(+)-K(+)-ATPase as a marker, we localized Na(+)-coupled bicarbonate transporter (NBCe1) and Cl(-)-HCO(3)(-) exchanger (SLC4A2/AE2) to the basolateral membrane of duct cells. Expression of cystic fibrosis transmembrane regulator channel (CFTR) was confirmed by immunofluorescence, RT-PCR, and in situ hybridization. We identified anion exchanger SLC26A6 at the ducts' luminal membrane and Na(+)-K(+)-2Cl(-) (NKCC1) at the basolateral membrane of mucous and duct cells. pH stat experiments showed that elevations in cAMP induced by forskolin or IBMX increased HCO(3)(-) secretion. Genistein, an activator of CFTR, which does not increase intracellular cAMP, also stimulated HCO(3)(-) secretion, whereas glibenclamide, a Cl(-) channel blocker, and bumetanide, a Na(+)-K(+)-2Cl(-) blocker, decreased it. CFTR(inh)-172, a specific CFTR channel blocker, inhibited basal HCO(3)(-) secretion as well as stimulation of HCO(3)(-) secretion by IBMX. This is the first report on the presence of CFTR channels in the esophagus. The role of CFTR in manifestations of esophageal disease in cystic fibrosis patients remains to be determined.     

Copper tolerance in Frankia sp. strain EuI1c involves surface binding and copper transport

Several Frankia strains have been shown to be copper-tolerant. The mechanism of their copper tolerance was investigated for Frankia sp. strain EuI1c. Copper binding was shown by binding studies. Unusual globular structures were observed on the surface of the bacterium. These globular structures were composed of aggregates containing many relatively smaller “leaf-like” structures. Scanning electron microscopy with energy-dispersive X-ray (SEM-EDAX) analysis of these structures indicated elevated copper and phosphate levels compared to the control cells. Fourier transform infrared spectroscopy (FTIR) analysis indicated an increase in extracellular phosphate on the cell surface of copper-stressed cells. Bioinformatics’ analysis of the Frankia sp. strain EuI1c genome revealed five potential cop genes: copA, copZ, copC, copCD, and copD. Experiments with Frankia sp. strain EuI1c using qRT-PCR indicated an increase in messenger RNA (mRNA) levels of the five cop genes upon Cu²⁺ stress. After 5 days of Cu²⁺ stress, the copA, copZ, copC, copCD, and copD mRNA levels increased 25-, 8-, 18-, 18-, and 25-fold, respectively. The protein profile of Cu²⁺-stressed Frankia sp. strain EuI1c cells revealed the upregulation of a 36.7 kDa protein that was identified as FraEuI1c_1092 (sulfate-binding periplasmic transport protein). Homologues of this gene were only present in the genomes of the Cu²⁺-resistant Frankia strains (EuI1c, DC12, and CN3). These data indicate that copper tolerance by Frankia sp. strain EuI1c involved the binding of copper to the cell surface and transport proteins.     

Kinetic FP-TDI assay for SNP allele frequency determination

Strategies for identifying genetic risk factors in complex diseases by association studies require the comparison of allele frequencies of numerous SNPs between affected and control populations. Theoretically, hundreds of thousands of SNP markers across the genome will have to be genotyped in these studies. Genotyping SNPs one sample at a time is extremely costly and time consuming. To streamline whole genome association studies, some have proposed to screen SNPs by pooling the DNA samples initially for allele frequency determination and perform individual genotyping only when there is a significant discrepancy in allele frequencies between the affected and control populations. Here we describe a new method for determining the allele frequency of SNPs in pooled DNA samples using a two-color primer extension assay with real-time monitoring of fluorescence polarization (named kinetic FP-TDI assay). By comparing the ratio of the rate of incorporation of the two allele-specific dye-terminators, one can calculate the relative amounts of each allele in the pooled sample. The accuracy of allele frequency determination with pooled samples is within 3.3 +/- 0.8% of that determined by genotyping individual samples that make up the pool.     

Some biochemical aspects of the protective effect of strontium chloride on γ-irradiated rats

The effect of treatment with SrCl₂ (10 mg 100 g^–) on rats 15 min prior to whole body -irradiation (7.5 Gy) was studied. The hazardous effects of irradiation were greatly corrected in the treated group. The hyperglycemic effect and liver glycogen accumulation in the untreated group decreased to normal level. The enzymatic activities of serum alkaline phosphatase, alanine and aspartate aminotransferases, and lactate dehydrogenase were greatly affected, showing insignificant changes in the treated group of animals. Life span calculated on 50% survival was also significantly elongated by 36.3%. These results show the potentiality of SrCl₂ as a radioprotective agent. A proposed mechanism is discussed.     

Field and Laboratory Studies on Nile Phytoplankton in Egypt. I. Some Physical and Chemical Characteristics

Monthly variations of some physical and chemical characteristics of Nile waters at Assiut (375 km south from Cairo, Egypt) during the period from September 1980 to September 1982 were followed and discussed. As expected, the maximum temperature was recorded in summer and the minimum in winter. The pH values of Nile waters were recorded to be in the vicinity of 8.0. The highest oxygen concentrations were recorded in the summer months, mainly due to the relatively high photosynthetic activity. The total soluble salts and the total alkalinity exhibited almost identical trends. Nitrate, phosphate, silicate and chloride concentrations showed no major regular trends. The cation abundances by weight were as follows: Ca²⁺ > Na⁺ > Mg²⁺ > K⁺.     

ftsW is an essential cell-division gene in Escherichia coli

In the absence of exogenous promoters, plasmid-mediated complementation of the temperature-sensitive ftsW201 allele requires the presence of the full coding sequence of ftsW plus upstream DNA encompassing the C-terminus of mraY and the full coding sequence of murD . We used molecular and genetic techniques to introduce an insertional inactivation into the chromosomal copy of ftsW , in the presence of the plasmid-borne wild-type ftsW gene under the control of P_BAD. In the absence of arabinose, the ftsW -null strain is not viable, and a shift from arabinose- to glucose-containing liquid medium resulted in a block in division, followed by cell lysis. Immunofluorescence microscopy revealed that in ftsW -null filaments, the FtsZ ring is absent in 50–60% of filaments, whilst between one and three Z-rings per filament can be detected in the remainder of the population, with the majority of these containing only one Z-ring per filament. We also demonstrated that the expression of only ftsWS (the smaller of two ftsW open reading frames) from P_BAD is sufficient for complementation of the ftsW -null allele. We conclude that FtsW is an essential cell-division protein in Escherichia coli , and that it plays a role in the stabilization of the FtsZ ring during cell division.     

Genetic polymorphism of two genes associated with carcass trait in Egyptian buffaloes

Leptin and μ-calpain have been considered as two candidate genes for carcass performance and meat quality traits in the farm animals. The micromolar calcium-activated neutral protease (CAPN1) gene encodes μ-calpain that degrades myofibril proteins under the postmortem conditions which appears to be the primary enzyme in the postmortem tenderization process. Leptin is the hormone product of the obese (LEP) gene. The role of leptin as a lipostatic signal regulating whole-body energy metabolism makes it one of the best physiological markers of body weight, food intake, reproduction and immune system functions.Genomic DNA extracted from 100 healthy buffaloes was amplified using primers that were designed from the cattle CAPN1 and LEP gene sequences. The amplified fragments of CAPN1 obtained from all tested buffalo DNA at 670-bp were digested with FokI endonuclease. The result showed that all tested buffaloes are genotyped as CC for CAPN1. For LEP gene, the amplified fragments obtained from all tested buffalo DNA at 400-bp were digested with Sau3AI endonuclease. All buffalo animals investigated in the present study are genotyped as AA for LEP gene.     

The diagnostic efficacy of circulating miRNAs in monitoring the early development of colitis-induced colorectal cancer

Early detection of colorectal cancer and monitoring the progress in colon carcinogenesis stages is essential to reduce mortality. Therefore, there is continuous search for noninvasive biomarkers with high stability and good sensitivity and specificity. miRNAs have attracted attention as promising biomarkers as they are stably expressed in circulation. The aim of our study is to evaluate the aberrant expression of circulating miRNAs during the stepwise progress of colitis-associated colon cancer. This was accomplished through assessing the expression levels of five miRNAs (miR-141, miR-15b, miR-17-3p, miR-21, and miR-29a) in serum and their corresponding tissue samples through the different cycles of colorectal carcinogenesis cascade using the azoxymethane/dextran sulfate sodium murine model. We also compared the diagnostic performance of these selected miRNAs with the conventional tumor biomarkers CEA and CA 19-9. The results of our study revealed that the expression levels of those miRNAs were dynamically changing in accordance with the tumor development state. Moreover, their aberrant expression in serum was statistically correlated with that in tissue. Our data also revealed that serum miR-15b, miR-21, and miR-29a showed the best performance in terms of diagnostic power. Our findings highlight the efficiency of these circulating miRNAs not only for early diagnostics purposes, but also for monitoring progress in the colorectal carcinogenesis process, and therefore encouraging integrating these noninvasive biomarkers into the clinical diagnostic settings beside the traditional diagnostic markers for accurate screening of the early progress of colon carcinogenesis.