A general method for chimerization of monoclonal antibodies by inverse polymerase chain reaction which conserves authentic N-terminal sequences.

Chimerization of antibodies (Ab) by cloning the V (variable) regions encoding the light and heavy chains with degenerate oligodeoxyribonucleotide primers matching to framework region 1 and to the joining regions, leads to Ab with altered amino acids at the N-terminus compared to those of the parental Ab. This is due to N-terminal framework 1 sequences in the expression vectors [Larrick et al., Bio/Technology 7 (1989) 937-938; Le Boeuf et al., Gene (1989) 371-377; Orlandi et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837]. This might lead to Ab with altered affinity to the antigen due to interaction of framework sequences with complementarity determining regions. Moreover, some V regions may be refractory to cloning by this procedure. Here, we describe a method to circumvent these potential problems. The V regions for both chains of the Ab are cloned by inverse polymerase chain reaction (PCR) with primers matching the known constant region sequences of the Ab. After sequencing, PCR fragments corresponding to the V regions of both chains are inserted in-frame into appropriate expression vectors leading to Ab with unaltered N-terminal sequences after expression in mammalian cells. The procedure is illustrated with an Ab directed against the beta chain of the human interleukin-2 receptor.     

Micropipette suction for measuring piconewton forces of adhesion and tether formation from neutrophil membranes.

A new method for measuring piconewton-scale forces that employs micropipette suction is presented here. Spherical cells or beads are used directly as force transducers, and forces as small as 10-20 pN can be imposed. When the transducer is stationary in the pipette, the force is simply the product of the suction pressure and the cross-sectional area of the pipette minus a small correction for the narrow gap that exists between the transducer and the pipette wall. When the transducer is moving along the pipette, the force on it is corrected by a factor that is proportional to the ratio of its velocity relative to its drag-free velocity. With this technique, the minimum force required to form a membrane tether from neutrophils is determined (45 pN), and the length of the microvilli on the surface of neutrophils is inferred. The strength of this technique is in its simplicity and its ability to measure forces between cells without requiring a separate theory or a calibration against an external standard and without requiring the use of a solid surface.     

An improved Eschehchia coli-Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp. using electroporation

The genetic studies of metabolically diverse Rhodococcus spp. have been hampered by the lack of a system of introducing exogenous DNA. The authors improved an existing Escherichia coli-Rhodococcus shuttle vector (pMVS301) by removing much of the DNA not needed for replication and adding a multicloning site. This improved vector (pBS305) is 7·9 kb in length. Its ability to transform Rhodococcus was tested using electroporation parameters optimized for introduction of pMVS301 into Rhodococcus. Transformation efficiencies as high as 10⁵ cfu μg^-1 DNA were obtained although efficiencies varied depending on the Rhodococcus strain tested. The improved vector pBS305 offers great utility for genetic studies of Rhodococcus because its small size enables movement of large inserts of DNA into Rhodococcus , it has multicloning sites, contains a highly selective thiostrepton marker, and can be replicated in both E. coli and Rhodococcus.     

Five New Guaiane Sesquiterpenes from the Endophytic Fungus Xylaria sp. YM 311647 of Azadirachta indica

Five new guaiane sesquiterpenes, 1 – 5 , were isolated from the culture broth of the endophytic fungus Xylaria sp. YM 311647, isolated from Azadirachta indica A. Juss . The structures of these compounds were elucidated on the basis of spectroscopic analyses, and their inhibitory activities against five pathogenic fungi were evaluated. All guaiane sesquiterpenes showed moderate or weak antifungal activities in a broth microdilution assay.     

Nucleic acid binding activity of pns6 encoded by genome segment 6 of rice ragged stunt oryzavirus

Rice ragged stunt disease, caused by rice ragged stuntoryzavirus (RRSV), was first discovered in 1976–1977 inIndonesia and Philippines [1]. Subsequently the diseasewas found in most rice-growing countries in south-easternand far-eastern Asia [2] and may inflict heavy loss on thecrop. RRSV is the type species of the genus Oryzavirus in thefamily Reoviridae. The virus particle is icosahedral witha diameter of about 65–70 nm and the genome consistsof 10 double stranded RNA (dsRNA) segm…     

An eukaryotic translation initiation factor,AteIF5A‐2, affects cadmium accumulation and sensitivity in Arabidopsis

Cadmium (Cd) is one of the most toxic elements and can be accumulated in plants easily; meanwhile, eIF5A is a highly conserved protein in all eukaryotic organisms. The present work tried to investigate whether eIF5A is involved in Cd accumulation and sensitivity in Arabidopsis (Arabidopsis thaliana L.) by comparing the wild‐type Columbia‐0 (Col‐0) with a knockdown mutant of AteIF5A‐2, fbr12‐3 under Cd stress conditions. The results showed that the mutant fbr12‐3 accumulated more Cd in roots and shoots and had significantly lower chlorophyll content, shorter root length, and smaller biomass, suggesting that downregulation of AteIF5A‐2 makes the mutant more Cd sensitive. Real‐time polymerase chain reaction revealed that the expressions of metal transporters involved in Cd uptake and translocation including IRT1, ZIP1, AtNramp3, and AtHMA4 were significantly increased but the expressions of PCS1 and PCS2 related to Cd detoxification were decreased notably in fbr12‐3 compared with Col‐0. As a result, an increase in MDA and H₂O₂ content but decrease in root trolox, glutathione and proline content under Cd stress was observed, indicating that a severer oxidative stress occurs in the mutant. All these results demonstrated for the first time that AteIF5A influences Cd sensitivity by affecting Cd uptake, accumulation, and detoxification in Arabidopsis.