The effect of dihydrofolate reductase-mediated gene amplification on the expression of transfected immunoglobulin genes

Murine/human chimeric gamma 1 and K Ig genes were cloned adjacent to the gene coding for methotrexate-resistant dihydrofolate reductase. These constructs were introduced into myeloma cells, and lines containing stably integrated genes were selected. The integrated Ig genes were then amplified by selection of the cells in increasing concentrations of methotrexate. The extent of gene amplification, mRNA accumulation, and production of Ig was studied in transfectomas containing introduced light chain genes, heavy chain genes, or both. When the light chain gene was introduced alone, it was expressed at low levels, but after selection with methotrexate, light chain expression was increased as much as 63-fold. In contrast, the transfected heavy chain genes were highly expressed, but production of the corresponding protein was increased a maximum of only fourfold by methotrexate treatment. Cellular toxicity of unassembled heavy chain monomer was not observed, even at amounts equivalent to 2% of total cellular protein. Cointroduction of the heavy and light chain constructs with subsequent amplification resulted in as much as 25-fold increase in secretion of intact antibody relative to unamplified cells. The results demonstrate that amplification of Ig genes can induce transfectomas to secrete antibody at nearly the rate of hybridomas.     

Aldimine to ketoamine isomerization (Amadori rearrangement) potential at the individual nonenzymic glycation sites of hemoglobin a: Preferential inhibition of glycation by nucleophiles at sites of low isomerization potential

The relative roles of the two structural aspects of nonenzymic glycation sites of hemoglobin A, namely the ease with which the amino groups could form the aldimine adducts and the propensity of the microenvironments of the respective aldimines to facilitate the Amadori rearrangement, in dictating the site selectivity of nonenzymic glycation with aldotriose has been investigated. The chemical reactivity of the amino groups of hemoglobin A forin vitro reductive glycation with aldotriose is distinct from that in the nonreductive mode. The reactivity of amino groups of hemoglobin A toward reductive glycation (i.e., propensity for aldimine formation) decreases in the order Val-1(), Val-1(), Lys-66(), Lys-61(), and Lys-16(). The overall reactivity of hemoglobin A toward nonreductive glycation decreased in the order Lys-16(), Val-1(), Lys-66(), Lys-82(), Lys-61(), and Val-1(). Since the aldimine is the common intermediate for both the reductive and nonreductive modification, the differential selectivity of protein for the two modes of glycation is clearly a reflection of the propensity of the microenvironments of nonenzymic glycation sites to facilitate the isomerization reaction (i.e., Amadori rearrangement). A semiquantitative estimate of this propensity of the microenvironment of the nonenzymic glycation sites has been obtained by comparing the nonreductive (nonenzymic) and reductive modification at individual glycation sites. The microenvironment of Lys-16() is very efficient in facilitating the rearrangement and the relative efficiency decreases in the order Lys-16(), Lys-82(), Lys-66(), Lys-61(), Val-1(), and Val-1(). The propensity of the microenvironment of Lys-16() to facilitate the Amadori rearrangement of the aldimine is about three orders of magnitude higher than that of Val-1() and is about 50 times higher than that of Val-1(). The extent of nonenzymic glycation at the individual sites is modulated by various factors, such as thepH, concentration of aldotriose, and the concentration of the protein. The nucleophiles—such as tris, glycine ethyl ester, and amino guanidine—inhibit the glycation by trapping the aldotriose. The nonenzymic glycation inhibitory power of nucleophile is directly related to its propensity to form aldimine. Thus, the extent of inhibition of nonenzymic glycation at a given site by a nucleophile directly reflects the relative role ofpK _a of the site in dictating the glycation at that site. The nonenzymic glycation of an amino group of a protein is an additive/synergestic consequence of the propensity of the site to form aldimine adducts on one hand, and the propensity of its microenvironment to facilitate the isomerization of the aldimines to ketoamines on the other. The isomerization potential of microenvironment plays the dominant role in dictating the site specificity of the nonenzymic glycation of proteins.     

Synthesis of New Bis(pyrazolo[1,5-a]pyrimidines) Linked to Different Spacers as Potential MurB Inhibitors

An efficient protocol was adopted to efficiently prepare three new series of bis(pyrazolo[1,5-a]pyrimidines) linked to different spacers. The new bis(pyrazolo[1,5-a]pyrimidines) were prepared in 80–90 % yields by reacting the respective bis(enaminones) and 4-(4-substituted benzyl)-1H-pyrazole-3,5-diamines in pyridine at reflux temperature for 5–7 h. The new products showed a wide spectrum of antibacterial activity against six different bacterial strains. In general, propane- and butane-linked bis(pyrazolo[1,5-a]pyrimidines), which are attached to 3-(4-methyl- or 4-methoxybenzyl) units, had the best antibacterial activity with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values up to 2.5 and 5.1 μM, respectively. Additionally, the previous products demonstrated promising MurB inhibitory activity with IC₅₀ values up to 7.2 μM.     

Evaluation of the weed flora of Egypt from Predynastic to Graeco-Roman times

Macrofossils of weeds retrieved from archaeological sediments in Egypt are discussed in terms of their presence, preservation and representation significance. The study reveals 112 field weeds from 61 archaeological sites dating from Predynastic times (4500 B.C.) up to the Graeco-Roman period (A.D. 395). Most of the remains were preserved by desiccation. The 112 listed species include 24 taxa from Predynastic Hierakonpolis (3800–3500 B.C.) identified for the first time. This study is based on a selection of 97 species from the entire list. Interpretation of field weed finds from the archaeological contexts is discussed. The highest number of species, 63, is recorded from the Pharaonic period. The Predynastic era is represented by 46 species and the Graeco-Roman period by 34. The intensive archaeological excavation of Pharaonic settlements may explain the rich flora of that period compared with the two others. Floristic analysis shows that 57 species were introduced in association with crops from the Middle East and 40 may belong to the native vegetation of the Nile valley.     

Antimicrobial and immunomodulating activities of hesperidin and ellagic acid against diarrheic Aeromonas hydrophila in a murine model

Aims

The present study is designed to evaluate the in vitro and in vivo bactericidal and immunomodulating activities of hesperidin (HES) and ellagic acid (EA) against Aeromonas hydrophila. A hydrophila, an uncommon human pathogen, can cause invasive infections in immunocompromised individuals and common clinical presentations in acute gastrointestinal illness, soft-tissue infections and sepsis. The antimicrobial activities of medicinal plants against A. hydrophila have received only cursory attention.

Methods

We examined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values in vitro. Moreover, the effects of HES and EA against bacterial colonization were studied in vivo. Also, humoral immune response was tested against A. hydrophila-LPS or A. hydrophila-ECP antigen preparations and the intestinal histopathological alterations were studied.

Results

Data revealed that the treatments with HES and EA each had antimicrobial activities against A. hydrophila. Both HES and EA treatments significantly increased anti-LPS IgM levels and reduced anti-LPS and anti-ECP IgA levels to their normal values in comparison to the infected group, which recorded significantly elevated levels two week post-infection. In conclusion, the present data suggest that HES and EA have antimicrobial and immunomodulating activities against murine A. hydrophila infections.

Significant

These data warrant clinical studies to delineate HES and EA roles in human infectious diseases.     

Pollen morphology and ultrastructure of Marathrum schiedeanum (Podostemaceae)

The Podostemaceae, or river-weeds, comprise 46 genera and 270 species of dicots and are the largest family of strictly aquatic angiosperms. Despite the large size, specialized habitats, and enigmatic morphology of the family, relatively little is known about the palynology of Podostemaceae. In the current paper, pollen morphology and ultrastructure of Marathrum schiedeanum are described. Pollen grains are relatively small, spheroidal, and tricolpate to spiraperturate. The exine has a microechinate ornament, a tectate-granular sexine and a relatively thick nexine in non-apertural regions, and a semitectate sexine and thinner nexine in apertural regions. Although aperture variation occurs in the family, this is the first report of the spiral aperture type in Podostemaceae. The spiraperturate condition appears to be derived in river-weeds, as does the granular pollen wall, which represents a reduction of the typical columellae found in eudicots.     

Re-evaluation of a neglected layer in pollen wall development with comments on its evolution

This study focuses on one particular layer of the pollen wall, which develops below the endexine in the free microspore stage and prior to the initiation of the intine. This membranous-granular layer (MGL) has been described by different terms in the literature and has often been interpreted either as part of the endexine, or the intine. During ontogeny, however, the granular material shows a development that is clearly distinct, both in timing and mode of formation, from the endexine as well as the intine. Its chemical composition is also characteristic; the MGL resists acetolysis. Our ontogenetic observations from four dicot and one monocot species are used to illustrate the systematically widespread occurrence of this wall layer, its ultrastructure and histochemistry, and its comparable nature throughout angiosperms.