Production of fertile transgenic wheat plants by laser micropuncture.

A modified, non-damaging, protocol for the production of fertile transgenic wheat (Triticum aestivum L. cultivar Giza 164) plants by laser micropuncture was developed. The new homemade setup secures the transformation of as many as 60 immature embryo-derived calli (10000 cells each) in less than one hour using a UV excimer laser with two dimensional translation stages, a suitable computer program and a proper optical system. Five-day-old calli were irradiated by a focused laser microbeam to puncture momentarily made self-healing holes ( approximately 0.5 microm) in the cell wall and membrane to allow uptake of the exogenous DNA. The plant expression vector pAB6 containing bar gene as a selectable marker for the herbicide bialaphos resistance and GUS (uidA) gene as a reporter gene was used for transformation. No selection pressure was conducted during the four-week callus induction period. Induced calli were transferred to a modified MS medium with 1 mg l(-1) bialaphos for regeneration, followed by selection on 2 mg l(-1) bialaphos for rooting. Three regenerated putative transgenic events were evaluated for the integration and stable expression of both genes and results indicated that this modified procedure of laser-mediated transformation can be successfully used in transforming wheat.     

Correlation ofLegionella pneumophila virulence with the presence of a plasmid

Legionella pneumophila is the causative agent of Legionellosis in man and considered an opportunistic intracellular Gram-negative bacterium that preferentially infects macrophages. The presence of a plasmid in these organisms was determined in cultures of the bacteria grown in vitro. A correlation was observed between the growth of virulent strains of theLegionella in murine macrophages and growth on standard buffered charcoal yeast extract agar supplemented with 0.1% -ketoglutarate (BCYE) agar medium rich in cysteine and widely used for growth of the bacteria in vitro. In contrast, the avirulent isolates of these strains grew well on supplemented Mueller-Hinton (SMH) agar utilized for differentiating virulent from avirulentLegionella. However, one virulent strain ofLegionella (the Iowa strain) was found to grow moderately well on the SMH agar. In addition, test strains ofLegionella that infect in vitro human monocytes were found to grow moderately well on the BCYE- agar, but did not grow on the SMH agar. Examination of these strains for plasmid DNA expression showed that extra chromosomal DNA-containing molecules were present in theL. pneumophila strains characterized as virulent for in vitro growth in macrophages. However, the avirulent strains that replicated in the human monocytes readily but only poorly in the permissive murine macrophages did not show evidence of similar plasmid DNA expression.     

Bioprocess development to produce a hyperthermostable S-methyl-5′-thioadenosine phosphorylase in Escherichia coli

Nucleoside phosphorylases are important biocatalysts for the chemo-enzymatic synthesis of nucleosides and their analogs which are, among others, used for the treatment of viral infections or cancer. S-methyl-5′-thioadenosine phosphorylases (MTAP) are a group of nucleoside phosphorylases and the thermostable MTAP of Aeropyrum pernix (ApMTAP) was described to accept a wide range of modified nucleosides as substrates. Therefore, it is an interesting biocatalyst for the synthesis of nucleoside analogs for industrial and therapeutic applications. To date, thermostable nucleoside phosphorylases were produced in shake flask cultivations using complex media. The drawback of this approach is low volumetric protein yields which hamper the wide-spread application of the thermostable nucleoside phosphorylases in large scale. High cell density (HCD) cultivations allow the production of recombinant proteins with high volumetric yields, as final optical densities >100 can be achieved. Therefore, in this study, we developed a suitable protocol for HCD cultivations of ApMTAP. Initially, optimum expression conditions were determined in 24-well plates using a fed-batch medium. Subsequently, HCD cultivations were performed using E. coli BL21-Gold cells, by employing a glucose-limited fed-batch strategy. Comparing different growth rates in stirred-tank bioreactors, cultivations revealed that growth at maximum growth rates until induction resulted in the highest yields of ApMTAP. On a 500-mL scale, final cell dry weights of 87.1–90.1 g L⁻¹ were observed together with an overproduction of ApMTAP in a 1.9%–3.8% ratio of total protein. Compared to initially applied shake flask cultivations with terrific broth (TB) medium the volumetric yield increased by a factor of 136. After the purification of ApMTAP via heat treatment and affinity chromatography, a purity of more than 90% was determined. Activity testing revealed specific activities in the range of 0.21 ± 0.11 (low growth rate) to 3.99 ± 1.02 U mg⁻¹ (growth at maximum growth rate). Hence, growth at maximum growth rate led to both an increased expression of the target protein and an increased specific enzyme activity. This study paves the way towards the application of thermostable nucleoside phosphorylases in industrial applications due to an improved heterologous expression in Escherichia coli.     

Radiation Resistance and Injury of Yersinia enterocolitica

The D values of Yersinia enterocolitica strains IP134, IP107, and WA, irradiated at 25°C in Trypticase soy broth, ranged from 9.7 to 11.8 krad. When irradiated in ground beef at 25 and −30°C, the D value of strain IP107 was 19.5 and 38.8 krad, respectively. Cells suspended in Trypticase soy broth were more sensitive to storage at −20°C than those mixed in ground beef. The percentages of inactivation and of injury (inability to form colonies in the presence of 3.0% NaCl) of cells stored in ground beef for 10 days at −20°C were 70 and 23%, respectively. Prior irradiation did not alter the cell's sensitivity to storage at −20°C, nor did storage at −20°C alter the cell's resistance to irradiation at 25°C. Added NaCl concentrations of up to 4.0% in Trypticase soy agar (TSA) (which contains 0.5% NaCl) had little effect on colony formation at 36°C of unirradiated Y. enterocolitica. With added 4.0% NaCl, 79% of the cells formed colonies at 36°C; with 5.0% NaCl added, no colonies were formed. Although 2.5% NaCl added to ground beef did not sensitize Y. enterocolitica cells to irradiation, when added to TSA it reduced the number of apparent radiation survivors. Cells uninjured by irradiation formed colonies on TSA when incubated at either 36 or 5°C. More survivors of an exposure to 60 krad were capable of recovery and forming colonies on TSA when incubated at 36°C for 1 day than at 5°C for 14 days. This difference in count was considered a manifestation of injury to certain survivors of irradiation.     

Selective Synthesis and Structure of 6-Arylvinyl-2- and 4-Glucosyl-1,2,4-triazines of Expected Interesting Biological Activity

Abstract

The 6-β-arylvinyl-2- and 4-glucosyl-3-thioxo-2,3-dihydro-1,2,4-triazin-5(4H)-ones were synthesized with high selectivity using the recently developed amino protecting group strategy. The structure of these new glucosides was established chemically and spectroscopically. Also, some interesting chemical transformations and rearrangements were observed.     

Acetylcholinesterase mediated susceptibility of soldiers and workers of formosan subterranean termite (Isoptera: Rhinotermitidae) to chlorpyrifos

Target site studies were undertaken to examine the difference in susceptibility of Formosan subterranean termite, Coptotermes formosanus Shiraki, workers and soldiers to chlorpyrifos. Workers exhibited significantly greater acetylcholinesterase activity per insect than soldiers (118.63 +/- 48.51 versus 47.98 +/- 22.59 mOD/min/insect equivalent). Likewise, enzyme activity (mean +/- SD) per milligram of protein was greater in workers than soldiers (440.30 +/- 267.43 versus 311.53 +/- 149.83 mOD/min/mg protein). The enzyme of soldiers was more sensitive to the acetylcholinesterase (AChE) inhibitors eserine and chlorpyrifos-oxon than that of workers. The I50s of chlorpyrifos-oxon were 2.66 and 4.59 nM for soldiers and workers, respectively, whereas the I50s of eserine were 16.56 and 25.41 nM for soldiers and workers, respectively. The amount of protein was significantly higher in workers than in soldiers with mean values of 0.270 +/- 0.102 and 0.154 +/- 0.054 mg/insect equivalent, respectively. We suggest that the differential response of workers and soldiers to chlorpyrifos may be due to the difference in AChE sensitivity to inhibition and the amount of protein between them.     

Linkage mapping of powdery mildew and greenbug resistance genes on recombinant 1RS from 'Amigo' and 'Kavkaz' wheat-rye translocations of chromosome 1RS.1AL.

Cultivated rye (Secale cereale L., 2n = 2x = 14, RR) is an important source of genes for insect and disease resistance in wheat (Triticum aestivum L., 2n = 6x = 42). Rye chromosome arm 1RS of S. cereale 'Kavkaz' originally found as a 1BL.1RS translocation, carries genes for disease resistance (e.g., Lr26, Sr31, Yr9, and Pm8), while 1RS of the S. cereale 'Amigo' translocation (1RSA) carries a single resistance gene for greenbug (Schizaphis graminum Rondani) biotypes B and C and also carries additional disease-resistance genes. The purpose of this research was to identify individual plants that were recombinant in the homologous region of.1AL.1RSV and 1AL.1RSA using both molecular and phenotypic markers. Secale cereale 'Nekota' (1AL.1RSA) and S. cereale 'Pavon 76' (1AL.1RSV) were mated and the F1 was backcrossed to 'Nekota' (1AL.1AS) to generate eighty BC1F2:3 families (i.e., ('Nekota' 1AL.1RSA x 'Pavon 76' 1AL.1RSV) x 'Nekota' 1AL.1AS). These families were genotyped using the secalin-gliadin grain storage protein banding pattern generated with polyacrylamide gel electrophoresis to discriminate 1AL.1AS/1AL.1RS heterozygotes from the 1AL.1RSA+V and 1AL.1AS homozygotes. Segregation of the secalin locus and PCR markers based on the R173 family of rye specific repeated DNA sequences demonstrated the presence of recombinant 1AL.1RSA+V families. Powdery mildew (Blumeria graminis) and greenbug resistance genes on the recombinant 1RSA+V arm were mapped in relation to the Sec-1 locus, 2 additional protein bands, 3 SSRs, and 13 RFLP markers. The resultant linkage map of 1RS spanned 82.4 cM with marker order and spacing showing reasonable agreement with previous maps of 1RS. Fifteen markers lie within a region of 29.7 cM next to the centromere, yet corresponded to just 36% of the overall map length. The map position of the RFLP marker probe mwg68 was 10.9 cM distal to the Sec-1 locus and 7.8 cM proximal to the powdery mildew resistance locus. The greenbug resistance gene was located 2.7 cM proximal to the Sec-1 locus.     

Cardiac Specific Expression of Threonine 5 to Alanine Mutant Sarcolipin Results in Structural Remodeling and Diastolic Dysfunction

The functional importance of threonine 5 (T5) in modulating the activity of sarcolipin (SLN), a key regulator of sarco/endoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA) pump was studied using a transgenic mouse model with cardiac specific expression of threonine 5 to alanine mutant SLN (SLNT5A). In these transgenic mice, the SLNT5A protein replaces the endogenous SLN in atria, while maintaining the total SLN content. The cardiac specific expression of SLNT5A results in severe cardiac structural remodeling accompanied by bi-atrial enlargement. Biochemical analyses reveal a selective downregulation of SR Ca²⁺ handling proteins and a reduced SR Ca²⁺ uptake both in atria and in the ventricles. Optical mapping analysis shows slower action potential propagation in the transgenic mice atria. Doppler echocardiography and hemodynamic measurements demonstrate a reduced atrial contractility and an impaired diastolic function. Together, these findings suggest that threonine 5 plays an important role in modulating SLN function in the heart. Furthermore, our studies suggest that alteration in SLN function can cause abnormal Ca²⁺ handling and subsequent cardiac remodeling and dysfunction.