Plant Growth Promoting Characterization of Indigenous Azotobacteria Isolated from Soils in Iran

It has been well known that the bacteria of the genus Azotobacter, in addition to the beneficial N₂-fixing activity, are able to improve plant growth by a number of direct and indirect mechanisms. To identify this potential in indigenous azotobacteria, the efficiency of 17 isolates of Azotobacter from the rhizosphere of wheat and barley plants cultivated in salt- and/or drought-affected soils in Iran were evaluated for their ability to dissolve inorganic and organic phosphates, siderophore secretion, indole acetic acid (IAA) production; and protease, chitinase, and ACC deaminase (ACCD) activities. First, they were biochemically characterized and one isolate (strain) was identified by 16S rDNA sequencing. Eight isolates were designated as Azotobacter vinelandii and the remaining isolates were identified as A. chroococcum. All isolates hydrolyzed the organic and inorganic phosphate compounds and effectively produced IAA. Fifteen isolates produced siderophore, but only one isolate showed protease activity which is being reported for the first time in relation to Azotobacter. None of the 17 isolates was capable of producing ACCD or chitinase. However, polymerase chain reaction amplification of the ACCD coding genes, by the use of the gene-specific primers, indicated that not all contain the ACCD gene. The standard screening methods with slight modifications, especially in the case of ACCD assay, were applied. The results showed that the use of specific screening methods, modified according to bacterial nutritional requirements, are the efficient methods for precise evaluation of the plant growth promoting rhizobacteria activity.     

Effects of L-thyroxine on incorporation of (32)P into phospholipids of freshwater eels

The effects of L-thyroxine on phospholipid biosynthesis, via (32)P incorporation, were studied in gill, kidney, liver and muscle tissue of eels acclimatized at 11 degrees C. L-thyroxine treatment had no effect on tissue content of lipid, inorganic and organic acid-soluble phosphorus. Only an increase of the specific radioactivities of lipid, inorganic and organic acid-soluble phosphorus was observed in the muscle. Percentage distribution of (32)P among classes of phospholipid were significantly altered in liver and muscle, without change in phospholipid composition. A specific effect of L-thyroxine on (32)P incorporation into phosphatidic acid in muscle and liver has been shown. As expected by the higher specific radioactivity of muscle inorganic and organic acid-soluble phosphorus, the increased incorporation of (32)P into phosphatidic acid probably results from a higher specific radioactivity of muscle ATP phosphorus.     

Feasibility studies in spheronization and scale-up of ibuprofen microparticulates using the rotor disk fluid-bed technology

The aim of this study was to develop spheronized microparticulates as a drug delivery system using the 1-step closed rotor disk fluid-bed technology, and to scale up the batch spheronization process. Ibuprofen was used as the model drug and microcrystalline cellulose/sodium carboxymethyl cellulose hydrocolloid (Avicel(R) RC-581 or CL-611) was present as the diluent/binder. The mixture, in 1:1 ratio, was blended with and without 1% sodium lauryl sulfate (SLS) and spheronized with the rotor disk insert, using either water or hydroxypropylmethyl cellulose (HPMC) as binder. Fluid-bed machines (Vector/Freund Flo-Coater model) FLM-1 (with 9-inch rotor insert for 0.75 kg) and FLM-15 (with a 12-inch and 19-inch rotor inserts for 1 kg and 5, 10 kg, respectively) were used. The critical process parameters included inlet air temperature, rotor disk speed and configuration, air flow, and rate of binder application. The 1 kg batch containing SLS that was made with 12-inch smooth stainless steel or waffle teflon plates rotating at 500 rpm had desirable characteristics. The sphericity values were 0.88 and 0.91, with percent yield of 85.4 and 91.2 and drug content values of 94.47% and 91.44%, respectively. The spheroids showed good flow properties with respective rapid drug release (Q20 = 83.27 and 91.75). No difference was seen in the Avicel RC-581 and CL-611. Based on the 1 kg data, Avicel RC-581 and smooth stainless steel and waffle teflon plates (12 inch and 19 inch), the batch was scaled up to 5 and 10 kg. The scale-up parameters included rotor speed (124 -300 rpm) and spray rate (90-140 g/min). The scale-up batches had similar flow characteristics, release rate, and size distribution. The geometric mean diameter increased as batch size increased, and slightly bigger spheroids were obtained using the waffle teflon plate. Ibuprofen spheres with very good physical characteristics were developed using the rotor disk fluid-bed technology, a 1-step closed process that did not require additional unit processes.     

Lack of phenotypic and functional impairment in dendritic cells from chimpanzees chronically infected with hepatitis C virus

Dendritic cells (DCs), which are potent antigen-presenting cells (APCs), are used as adjuvants for the treatment of cancer and infectious diseases in human and nonhuman primates, with documented clinical efficacy. The hepatitis C virus (HCV)-chimpanzee model is the best available model for testing the immunotherapeutic effects of DCs in the setting of a chronic infection, as chimpanzees develop a persistent infection resembling that seen in humans. However, several reports have suggested that DCs derived from chronically infected individuals or nonhuman primates are functionally compromised. As a prelude to clinical studies, we evaluated whether functionally mature DCs could be generated in chimpanzee plasma by good manufacturing practice using CD14(+) mononuclear precursors from chronically infected chimpanzees. DCs generated in a medium with HCV-negative plasma and treated with a defined cocktail of cytokines or a CD40 ligand trimer matured fully, as measured by the induction of CD83 expression and the upregulation of costimulatory molecules. Furthermore, the expression of CCR7 was induced, suggesting an acquisition of migration capacity. Mature DCs were capable of stimulating allogeneic T cells, antigen-specific memory CD4(+) T cells, and HCV-specific CD8(+)-T-cell clones. In all cases, there was no evidence of HCV infection in DCs. Furthermore, these DCs maintained their phenotype and APC function after cryopreservation. Finally, no discernible differences were noted between DCs derived from HCV-infected and uninfected chimpanzees. In summary, precursor cells from HCV-infected chimpanzees are fully capable of differentiating into functional, mature DCs, which can now be reproducibly prepared for investigations of their immunotherapeutic potential in the setting of chronic HCV infection.     

Mosaic prophages with horizontally acquired genes account for the emergence and diversification of the globally disseminated M1T1 clone of Streptococcus pyogenes

The recrudescence of severe invasive group A streptococcal (GAS) diseases has been associated with relatively few strains, including the M1T1 subclone that has shown an unprecedented global spread and prevalence and high virulence in susceptible hosts. To understand its unusual epidemiology, we aimed to identify unique genomic features that differentiate it from the fully sequenced M1 SF370 strain. We constructed DNA microarrays from an M1T1 shotgun library and, using differential hybridization, we found that both M1 strains are 95% identical and that the 5% unique M1T1 clone sequences more closely resemble sequences found in the M3 strain, which is also associated with severe disease. Careful analysis of these unique sequences revealed three unique prophages that we named M1T1.X, M1T1.Y, and M1T1.Z. While M1T1.Y is similar to phage 370.3 of the M1-SF370 strain, M1T1.X and M1T1.Z are novel and encode the toxins SpeA2 and Sda1, respectively. The genomes of these prophages are highly mosaic, with different segments being related to distinct streptococcal phages, suggesting that GAS phages continue to exchange genetic material. Bioinformatic and phylogenetic analyses revealed a highly conserved open reading frame (ORF) adjacent to the toxins in 18 of the 21 toxin-carrying GAS prophages. We named this ORF paratox, determined its allelic distribution among different phages, and found linkage disequilibrium between particular paratox alleles and specific toxin genes, suggesting that they may move as a single cassette. Based on the conservation of paratox and other genes flanking the toxins, we propose a recombination-based model for toxin dissemination among prophages. We also provide evidence that a minor population of the M1T1 clonal isolates have exchanged their virulence module on phage M1T1.Y, replacing it with a different module identical to that found on a related M3 phage. Taken together, the data demonstrate that mosaicism of the GAS prophages has contributed to the emergence and diversification of the M1T1 subclone.     

An immunogenetic and molecular basis for differences in outcomes of invasive group A streptococcal infections

The role of host genetic factors in conferring predisposition or protection in infectious diseases has become evident. Infection with group A streptococci causes a wide spectrum of disease ranging from pharyngitis to streptococcal toxic shock syndrome. The release of inflammatory cytokines triggered by streptococcal superantigens has a pivotal role in invasive streptococcal disease. However, individuals infected with the same strain can develop very different manifestations. We report here that the immunogenetics of the host influence the outcome of invasive streptococcal infection, and demonstrate the underlying mechanism for these genetic associations. Specific human leukocyte antigen class II haplotypes conferred strong protection from severe systemic disease, whereas others increased the risk of severe disease. Patients with the DRB1*1501/DQB1*0602 haplotype mounted significantly reduced responses and were less likely to develop severe systemic disease (P < 0.0001). We propose that human leukocyte antigen class II allelic variation contributes to differences in severity of invasive streptococcal infections through their ability to regulate cytokine responses triggered by streptococcal superantigens.     

Incorporation of ω6 polyunsaturated fatty acids into phospholipids of the crab Carcinus maenas

The incorporation in vivo of ¹⁴C-18:2 ω6 and ³H-20:4 ω6 fatty acids in phospholipids isolated from gills, hepatopancreas and hemolymph of the crab Carcinus maenas was analysed. PC was the most heavily labelled phospholipid from these ω6-unsaturated fatty acids and appeared to play an important part in the phospholipids metabolism in Crustaceans. The pathway of fatty acids synthesis in phospholipids of C. maenas seems to be similar to those described for mammals. It is at the level of tissue Pl of C. maenas that the renewal of the 20:4 ω6 fatty acid is the most important. It is suggested that the rapid reorganization of phospholipid molecular species composition in the crab is checked by deacylation—reacylation cycle.     

Fluorescence spectral properties of troponin C mutant F22W with one-, two-, and three-photon excitation.

We report the first measurements of protein fluorescence with three-photon excitation, using a mutant of troponin C (TnC) that contains a single tryptophan residue F22W. From the emission intensity dependence on laser power we determine that TnC F22W displays one-, two-, and three-photon excitation at 285, 570, and 855 nm, respectively. The emission spectra and intensity decays are identical for one-, two-, or three-photon excitation. The steady-state and time 0 anisotropies are distinct for each mode of excitation, but the correlation times were the same, suggesting that three-photon excitation of proteins can be accomplished without significant effects of the locally intense illumination. The excitation anisotropy spectrum from 830 to 900 nm displays only negative values, suggesting dominant excitation via the 1Lb state of tryptophan from 830 to 900 nm.     

Differential signal requirements in T-cell activation by mitogen and superantigen

The requirement for co-stimulatory molecules in T-cell stimulation by mitogens and superantigens in the absence of antigen-presenting cells (APC) was investigated. Phytohemagglutinin (PHA) induced interleukin (IL)-2 receptor (IL-2R) expression on purified T-cells, but proliferation occurred only when exogenous IL-2 was added. In contrast, the proliferative response to a pepsin-extracted type 5 M-protein from Streptococcus pyogenes (pep M5), a recently identified superantigen, required signals provided by phorbol 12-myristate 13-acetate (PMA), IL-1 and IL-6. pep M5 alone did not induce IL-2R expression; however, when combined with PMA, IL-1 and IL-6, IL-2R was expressed. Differences were also observed in the response of the leukemic T-cell line, Jurkat, to PHA and pep M5. Soluble PHA, but not pep M5, induced IL-2 production by these cells in the presence of PMA. Cross-linking by its specific antibody or adsorption of pep M5 to microtiter plates was required to activate Jurkat cells. Both PHA and pep M5 induced Ca²⁺ mobilization in Jurkat cells; however, only PHA induced a rise in intracellular Ca²⁺ in purified T-cells, whereas pep M5 was unable to induce this activity unless IL-1, IL-6 and PMA were added. Our data provide biochemical evidence that mitogenic and superantigenic stimulation of T-cells is different.