Predominant end-products of prophage Mu DNA transposition during the lytic cycle are replicon fusions

The structure of l-arabinose-binding protein (M_r 33, 100), an essential component of the osmotic shock-sensitive, high-affinity l-arabinose transport system in Escherichia coli, has been determined at 2.4 Å resolution. The phases were solved by the method of multiple isomorphous replacement, using four derivatives, p-chloromercuribenzenesulfonate and CdI₂ (data to 2.4 Å resolution), and p-chloromercurinitrophenol and (NH₄)₂PtCl₄ to 3.5 Å resolution. A final mean figure of merit of 0.65 was obtained for 9628 reflections.With the aid of the amino acid sequence determined by Hogg &; Hermodson (1977), a complete model of the protein molecule has been determined using initially an optical comparator. The entire model was subsequently examined in detail using a computer graphic system.The protein molecule is ellipsoidal (axial ratio of 2:1), and consists of two globular domains (designated P and Q). Each domain is made from two separate polypeptide chain segments. Despite the discontinuity in the folding, the arrangements of the secondary structure in the two domains are very similar. Both domains contain a six-stranded parallel β-sheet (with the exception of the sixth anti-parallel strand in the Q domain) flanked by two α-helices on either side. The packing topology is α/β. A C-terminal helix is shared by both domains.The two domains show significant conformational similarity but lack sequence homology. A comparison of the two domains revealed that of the 139 α-carbons in the P domain and 152 in the Q domain, 92 were found to be equivalent with a root-mean-square distance of 2.6 Å.The cleft formed by the packing of the two domains is predominantly lined with hydrophilic residues. The sugar-binding site is located in this cleft.     

Transposition studies of mini-Mu plasmids constructed from the chemically synthesized ends of bacteriophage Mu

We describe below the chemical synthesis of the right and left ends of bacteriophage Mu and characterize the activity of these synthetic ends in mini-Mu transposition. Mini-Mu plasmids were constructed which carry the synthetic Mu ends together with the Mu A and B genes under control of the bacteriophage λ p_L promoter. Derepression of p_L leads to a high frequency of mini-Mu transposition (5.6 × 10⁻²) which is dependent on the presence of the Mu ends and the Mu A and B proteins. Five deletion mutants in the Mu ends were tested in the mini-Mu transposition system and their effects on transposition are described.     

Blind trials evaluating in vitro infectivity of Cryptosporidium oocysts using cell culture immunofluorescence

An optimized cell culture immunofluorescence (IFA) procedure, using the HCT-8 cell line, was evaluated in blind trials to determine the sensitivity and reproducibility of measuring the infectivity of flow-cytometry-prepared inocula of Cryptosporidium parvum oocysts. In separate trials, suspensions consisting of between 0% and 100% viable oocysts were prepared at the US Environmental Protection Agency, shipped to the American Water Laboratory, and analyzed blindly by cell culture IFA. Data indicated the control (100% live) oocyst suspensions yielded statistically similar results to cell culture dose-response curve data developed previously at the American Water Laboratory. For test samples containing oocyst suspensions of unknown infectivity, cell culture IFA analyses indicated a high degree of correlation (r2 = 0.89; n = 26) with the values expected by the US Environmental Protection Agency. Cell culture infectivity correlates well with neonatal mouse infectivity assays, and these blind validation trials provide credibility for the cell culture IFA procedure as a cost-effective and expedient alternative to mouse infectivity assays for determining in vitro infectivity of C. parvum oocysts.     

Regulation of bacteriophage mu and mini-mu DNA replication in vivo

To study the regulation of bacteriophage Mu DNA's integrative-replication (transposition) during lytic growth in a cell containing both a Mu and a helper-dependent Mini-Mu (short, internally-deleted Mu genome), we placed "marker" genes (bla, lacZ) within either genome and then measured their encoded enzymes as indicators of the gene dosage. These results, corroborated using DNA-DNA hybridization, show that Mu and Mini-Mu DNA transposition is well regulated, requires both the Mu A and B gene products, and can be readily monitored by measuring beta-galactosidase and beta-lactamase expressed from the lacZ and bla genes, respectively.     

Effects of prophage Mu induction on expression of adjacent host genes

The extent of induction and approximate amount of DNA replication of a Mu prophage carrying a gene for ampicillin resistance can be monitored by assaying the level of -lactamase. The expression of thelacZ gene adjacent to either end of an induced Mu prophage remains virtually unaffected, until late in the Mu lytic cycle, while Mu DNA is replicating and transposing.     

Association of cell free mitochondrial DNA and caspase-1 expression with disease severity and ARTs efficacy in HIV infection

HIV infection is a global health concern. Current HIV-diagnostics provide information about the disease progression and efficacy of anti-retroviral therapies (ARVs), but this information is very limited and sometimes imprecise. Present study assessed the potential role of mononuclear cell (MNC) death, expression of caspases (1&3) and cell free mitochondrial DNA (CF mt-DNA) in HIV infected individuals. Apoptosis, cell-count, expression of caspases and CF mt-DNA were measured through flow cytometry and qPCR, respectively, in HIV infected individuals (n?=?120) divided in two groups i.e. ARVs-receiving (treated, n?=?87), ART-naïve (untreated, n?=?37) and healthy individuals (n?=?47). Data showed significant (p?<?0.0001) cell death in untreated individuals than treated and healthy individuals. CD4-positive T-cell percentage declined (p?<?0.0001) in untreated as compared to treated individuals. Caspase-1, an indicator of pyroptosis, and CF mt-DNA were also elevated in untreated HIV infected individuals. Untreated individuals when administered with ARVs showed improved CD4-positive T-cell percentage, lower caspase-1, CF mt-DNA and cell death. Data elucidated positive co-relation between cell death and CF mt-DNA in treated and untreated HIV infected individuals. While CD4-positive T-cell percentage was negatively correlated with caspase-1 expression and CF mt-DNA. Elevated levels of CF mt-DNA and caspase-1 in HIV infected individuals, positive correlation between cell death and CF mt-DNA, negative correlation of CD4-positive T-cell percentage with CF mt-DNA and caspase-1 expression clearly indicated the potential of CF mt-DNA and caspase-1 as a novel disease progression and ARTs effectiveness biomarkers in HIV.

     

Biodiversity characteristics of Teucrium polium species in Saudi Arabia

Teucrium (Lamiaceae) is a large and polymorphic genus distributed mainly in Europe, North Africa and in the temperate parts of Asia. In this study, the anatomical features of the leaf and stem of Teucrium polium are investigated. Teucrium has 19 taxa in Iran, which mainly grow in the Irano-Turanian region between 700 and 2000 m above sea level. T. polium belonging to sect. Polium, is a perennial herb growing on Lorestan province. The leaves clearly exhibit xeromorphy due to features such as thick cuticle layer, thick outer epidermal cell wall, high density of trichomes and thick palisade layer of the mesophyll. Anatomical studies on T. polium revealed that the stem shares the general characteristics of the Labiatae family. The aim of our approach was to study the morphological and taxonomical parameters for T. polium in Saudi Arabia. The results of this study showed that there was no influence of environment on the structure of stomata and trichomes as studying species with T.polium. In conclusion our study shows we have studied the geographical distribution of the species in Saudi Arabia and in the world.     

Application of Box-Behnken Design to Hybrid Electrokinetic-Adsorption Removal of Mercury from Contaminated Saline-Sodic Clay Soil

A hybrid electrokinetic-adsorption (HEKA) technique using uniform electric field and granular activated carbon (GAC) produced from date palm pits was investigated for the removal of mercury from natural saline-sodic clay heavily contaminated with heavy metals, phenol, and kerosene. Response surface methodology (RSM) was employed to model, optimize, and interpret the results obtained with the aid of Design Expert software. According to the Box-Behnken experimental design, 15 experiments were conducted each with residence time of three weeks. The effects of voltage gradient (0.2–1 V/cm), initial Hg concentration (mg/Kg), and polarity reversal interval (0-48 hours) on Hg removal efficiency and energy consumed for Hg removal were investigated. Respectively, the responses fitted reduced cubic (R² = 99.3%) and quadratic models (R² = 92.3%) with the overall relative contributions of the investigated parameters on the responses following the order: voltage gradient > initial Hg concentration > polarity reversal interval based on analysis of variance (ANOVA). The optimal conditions obtained with desirability of 90% aimed at maximizing Hg removal were 24 hours polarity reversal interval, 0.2 V/cm voltage gradient, and 100 mg/kg initial Hg concentration. This optimum operating condition yielded good removal of Hg (99.5%) at reduced energy consumption of 50.1kWh.m^?3mg^?1. Experimental validation of the models showed good prediction of Hg removal efficiency (0.0368% prediction error). The results presented herein suggest that HEKA technology could be utilized effectively for the removal of Hg from contaminated, low permeable soils under extreme soil and contamination conditions.     

Serum hormonal, electrolytes and trace element profiles in the rutting and non-rutting one-humped male camel (Camelus dromedarius)

The camel is known to be a seasonal breeder and, in the male camel, the breeding activity maximizes during the rutting period (winter and spring seasons). During the non-rutting period (summer and autumn), however, the breeding activity almost ceases completely. Serum steroid hormonal, electrolytes and trace element profiles were studied in 16 adult male camels aged 5-9 years, slaughtered during the rutting (n=8) and non-rutting periods (n=8). Moreover, testicular and epididymal tissue contents of electrolytes and trace elements were also studied during these two periods. Serum levels of testosterone, progesterone and T(4):T(3) ratio were higher in the rutting animals when compared to the non-rutting ones, while the reverse was true for serum T(3), T(4) and corticosterone levels (p<0.01). Among minerals, serum levels of sodium, calcium, copper and zinc were higher, while those of potassium were lower, in the rutting than non-rutting camels. In the testicular tissue, the levels of sodium, calcium, copper and iron were higher in the rutting group, while magnesium was higher in non-rutting one. In the epididymis, the tissue levels of chloride, iron and zinc were higher in the rutting than non-rutting period. These results demonstrate a clear evidence of the distribution of steroid hormones, electrolytes and trace elements forming and maintaining a certain level at two different physiological states in camels.