Generation of antiserum to specific epitopes

The ability to prevent disease by immunization with subunit vaccines that incorporate specific epitopes was demonstrated by DiMarchi et al. (1), who used a synthetic peptide to protect cattle against foot-and-mouth disease. However, generation of antibody to peptide antigens is often difficult owing to the small molecular mass and limited chemical complexity. We tested the hypothesis that recombinant DNA and synthetic peptide techniques would make it possible to stimulate vigorous immune responses to specific epitopes of an outer membrane protein ofNeisseria gonorrhoeae. The MtrC AP1 sequence from the invariant mtrC gonococcal lipoprotein was genetically fused to maltose binding protein. The resultant fusion protein was used as the primary immunogen to stimulate MtrC AP1-specific antiserum. To enhance antibody production specific to MtrC AP1, boosting immunizations were performed with synthetic MtrC AP1 sequence contained in a multiple antigenic peptide system immunogen. The MtrC AP1-specific antiserum strongly recognized the MtrC protein on Western blots and appeared to bind native MtrC proteinin situ. The generation of antibody in this fashion provides the technology to produce antibody to defined epitopes of any protein, including those found in the gonococcal outer membrane. The ability of those antibodies to inhibit bacterial growth or to activate complement protein can then be tested.     

Sequence specific antibodies to a deoxyribodinucleotide

Antibodies were elicited in rabbits to the dinucleotide, dpTpA, conjugated to a carrier protein. Among a few deoxyribomono- and oligonucleotides tested for binding to the antibodies by nitrocellulose tilter binding assay, only dpTpA and dpTpA containing oligonucleotides showed binding. The inhibition of the binding of 3H-dpTpA to the antibodies by various nonradioactive mono- and oligonucleotides showed that the antibodies were sequence specific and recognized the whole molecule of dpTpA. dpTpA specific antibodies were purified by a two step affinity chromatography. By competition studies, it was found that the purified antibodies bound denatured DNA at dpTpA sequences. The antibodies did not bind RNA.     

Structural features specific to plant metallothioneins

The metallothionein (MT) superfamily combines a large variety of small cysteine-rich proteins from nearly all phyla of life that have the ability to coordinate various transition metal ions, including Zn^II, Cd^II, and Cu^I. The members of the plant MT family are characterized by great sequence diversity, requiring further subdivision into four subfamilies. Very peculiar and not well understood is the presence of rather long cysteine-free amino acid linkers between the cysteine-rich regions. In light of the distinct differences in sequence to MTs from other families, it seems obvious to assume that these differences will also be manifested on the structural level. This was already impressively demonstrated with the elucidation of the three-dimensional structure of the wheat E_c-1 MT, which revealed two metal cluster arrangements previously unprecedented for any MT. However, as this structure is so far the only one available for the plant MT family, other sources of information are in high demand. In this review the focus is thus set on any structural features known, deduced, or assumed for the plant MT proteins. This includes the determination of secondary structural elements by circular dichroism, IR, and Raman spectroscopy, the analysis of the influence of the long linker regions, and the evaluation of the spatial arrangement of the sequence separated cysteine-rich regions with the aid of, e.g., limited proteolytic digestion. In addition, special attention is paid to the contents of divalent metal ions as the metal ion to cysteine ratios are important for predicting and understanding possible metal–thiolate cluster structures.     

Monoclonal antibodies specific to plant tubulin

Summary Tubulin was isolated from mung bean seedling by a combination of affinity (ethyl N-phenylcarbamate-Sepharose 4 B) and ion exchange (DEAE-Sephacel) chromatography. Using SDS-PAGE together with blotting with subunit-specific antitubulins, mung bean tubulin has been shown to consist of two -tubulin subunits, MBT₂ and MBT₃, of which MBT₃ is a minor component, and one -tubulin, MBT₁.Monoclonal antibodies were produced by fusing mouse myeloma cells and spleen cells from a Balb/c mouse immunized with mung bean tubulin. Antibody producing cell lines were identified by an ELISA assay and immunofluorescence microscopy and subsequently cloned by limiting dilution.The properties of monoclonal antibody (K₄E₇G₃) were examined by Western blot analysis and indirect immunofluorescence studies. K₄E₇G₃ reacts with MBT₂ and MBT₃ -tubulin subunits of mung bean tubulin, but not with MBT₁ -tubulin nor with the - and -subunits of sheep brain tubulin. Peptide fragments transferred onto nitrocellulose papers were treated with K₄E₇G₃ and with other monoclonal antibodies that are known to be specific to the -subunit of yeast tubulin and - or -subunit of mammalian brain tubulin. MBT₂ and MBT₃ are shown to be similar but not identical and are quite different from MBT₁ and the -subunit of sheep brain tubulin. K₄E₇G₃ reacts with peptide fragments in MBT₂ and MBT₃ that are not found in digests of brain tubulin, and that are either not reactive or only weakly reactive to the antibodies to yeast and brain -tubulin. It is concluded that K₄E₇G₃ and another monoclonal antibody, K₂D₇B₈, which has similar properties, are relatively specific for plant -tubulin.In indirect immunofluorescence studies on a wide range of plant cells, the epitopes recognised by these monoclonal antibodies are shown to be present in all types of microtubule array that were investigated. The spindle, preprophase band, phragmoplast and interphase microtubules were clearly observed in onion and mung bean root tip cells. Reactions with spindle microtubules ofFunaria spore mother cells and with the blepharoplast and flagella microtubules of fern spermatozoa are also seen. However, studies using several animal cell lines have shown that K₄E₇G₃ and K₂D₇B₈ do not give positive immunofluorescent localization of animal microtubules, correlating with the inability of K₄E₇G₃ to react with brain tubulin subunits on Western blot analysis.     

Idiotype-restricted antibody response to specific immune complexes

In this report, we compared the immunogenicity of specific antigen/antibody complexes with that of free antigen. The complexes were prepared in antigen excess using the TEPC-15 myeloma protein and a phosphorylcholine-containing polysaccharide antigen (PnC), and the PnC-specific antibody response was measured using a hemolytic plaque assay 5 days after immunization. The results showed that the complexes were as immunogenic as the free antigen; however, the PnC-specific antibody response induced with the complexes was completely dominated by one particular idiotope (defined by plaque inhibition with the AB1-2 monoclonal antibody). On the other hand, the response of mice immunized with free antigen (PnC) was dominated to a lesser degree by the AB1-2 idiotope, and there was a great degree of variability in idiotope expression among individual mice. The results suggest that immunization with antigen/antibody complexes restricts the response to the expression of idiotopes that are present in the immune complex.     

Drosophila strain specific response to cisplatin neurotoxicity

ABSTRACT

Drosophila melanogaster has recently been developed as a simple, in vivo, genetic model of chemotherapy-induced peripheral neuropathy. Flies treated with the chemotherapy agent cisplatin display both a neurodegenerative phenotype and cell death in rapidly dividing follicles, mimicking the cell specific responses seen in humans. Cisplatin induces climbing deficiencies and loss of fertility in a dose dependent manner. Drosophila sensitivity to cisplatin in both cell types is affected by genetic background. We show that mutation or RNAi-based knockdown of genes known to be associated with CIPN incidence in humans affect sensitivity of flies to CIPN. Drosophila is a promising model with which to study the effect of genetics on sensitivity to CIPN.     

Antibodies specific to a deoxyribodinucleotide sequence.

Antibodies were raised in rabbits against the bovine serum albumin conjugate of dpApT. Analysis by double diffusion in agar gel and quantitative precipitation test showed the presence of antibodies specific to the hapten in the antisera. Quantitative data on the specificity of the antibodies were obtained by studying the inhibition of the binding of 3H-dpApT to the antisera by various nonradioactive mono- and oligonucleotides, using a nitrocellulose membrane binding assay. The antibodies were found to be highly specific for the dinucleotide sequence dpApT. The antibodies were able to bind to synthetic oligonucleotides containing the sequence dpApT and to denatured calf thymus DNA.     

An efficient method to produce specific anti-actin

Summary The production of affinity column purified (specific) anti-actin is described. With the immunization scheme employed, all rabbits produced precipitating antibodies over several months, so that 30 mg specific anti-actin per rabbit could be isolated in 6 months. The antibodies against native and detergent denatured smooth muscle actin are characterized by immunodiffusion tests, staining of the I-band of isolated myofibrils and stress fibers in tissue culture cells, using indirect immunofluorescence.     

Antibodies specific to two deoxyribotrinucleotide sequences.

Antibodies to the deoxyribotrinucleotides dpApTpA and dpApApT were prepared by injecting the bovine serum albumin conjugates of the respective haptens in rabbits. The specificities of the antibodies were determined by estimating the inhibition of the binding of the tritiated haptens to the immunoglobulins by various nonradioactive mono- and oligonucleotides, using nitrocellulose membrane binding assay. Anti-dpApTpA and anti-dpApApT antisera were found to contain antibodies which were highly specific to the respective hapten sequence.     

Strain group specific and virus specific hypersensitive reactions to infection with potyviruses in potato cultivars

When 12 potato cultivars were inoculated with isolates (one each) of potato virus Y (PVY) ordinary (Y^o), C (Y^c) and tobacco veinal necrosis (Yⁿ) strain groups, potato virus A (PVA) and potato virus V (PVV), none of them responded hypersensitively to Yⁿ. However, with Y^o, Y^c, PVA and PW specific hypersensitive reactions developed depending on isolate-cultivar combination which were all independent of each other. When field isolates of PVY thought to be Y^oor Y^cwere inoculated to the same 12 cultivars, two did not fit into either strain group giving hypersensitive reactions in only two cultivars instead of seven with Y^oor eight with Y^c. These two isolates may represent a previously unreported PVY strain group (Y^z). When Y^owas graft-inoculated to seedlings of the cross Desiree × Maris Piper (hypersensitive × non-hypersensitive for Y^o), the segregation ratio obtained for non-hypersensitive:hypersensitive reactions was close to 1:1 suggesting that a single dominant gene (Ny_tbr) determining Y^ospecific hypersensitivity may be present in cv. Desiree (simplex condition). In tests using PVV and Desiree × Maris Piper (non-hypersensitive × hypersensitive for PVV) seedlings, the segregation ratio obtained was close to 1:5 indicating that a single dominant gene (Nv) determining PVV specific hypersensitivity may be present in cv. Maris Piper (duplex condition). Cultivars Corine, Pirola and clone G5457(4) which each carry one of the extreme resistance genes (Ry) from Solanum stoloniferum were graft-inoculated with Yⁿ, Y^o, Y^c, PVV and PVA. G5457(4) gave a strong localised hypersensitive reaction in all instances, while cv. Pirola did so with all except PVA to which it was immune. In cv. Corine a severe localised hypersensitive reaction developed with PVA, generalised hypersensitivity with PVV but an immune response with the three PVY strain groups. Large-scale grafting of Yⁿto plants of cvs Corine and Pirola gave no evidence of selection of a strain which overcomes Ry genes.     

Differences in hydration coupled to specific and nonspecific competitive binding and to specific DNA Binding of the restriction endonuclease BamHI

Using the osmotic stress technique together with a self-cleavage assay we measure directly differences in sequestered water between specific and nonspecific DNA-BamHI complexes as well as the numbers of water molecules released coupled to specific complex formation. The difference between specific and nonspecific binding free energy of the BamHI scales linearly with solute osmolal concentration for seven neutral solutes used to set water activity. The observed osmotic dependence indicates that the nonspecific DNA-BamHI complex sequesters some 120-150 more water molecules than the specific complex. The weak sensitivity of the difference in number of waters to the solute identity suggests that these waters are sterically inaccessible to solutes. This result is in close agreement with differences in the structures determined by x-ray crystallography. We demonstrate additionally that when the same solutes that were used in competition experiments are used to probe changes accompanying the binding of free BamHI to its specific DNA sequence, the measured number of water molecules released in the binding process is strikingly solute-dependent (with up to 10-fold difference between solutes). This result is expected for reactions resulting in a large change in a surface exposed area.