Augmentation of murine natural killer cell and antibody dependent cellular cytotoxicity activities by phyllanthus emblica, a new immunomodulator

When administered orally, Phyllanthus emblica, an excellent source of vitamin C (ascorbate), has been found to enhance natural killer (NK) cell activity and antibody dependent cellular cytotoxicity (ADCC) in syngeneic BALB/c mice, bearing Dalton's lymphoma ascites (DLA) tumor. P. emblica elicited a 2-fold increase in splenic NK cell activity on day 3 post tumor inoculation. Enhanced activity was highly significant on days 3, 5, 7 and 9 after tumor inoculation with respect to the untreated tumor bearing control. A significant enhancement in ADCC was documented on days 3, 7, 9, 11 and 13 in drug treated mice as compared to the control. An increase in life span (ILS) of 35% was recorded in tumor bearing mice treated with P. emblica. This increased survival was completely abrogated when NK cell and killer (K) cell activities were depleted either by cyclophosphamide or anti-asialo-GM, antibody treatment. These results indicate: (a) an absolute requirement for a functional NK cell or K cell population in order that P. emblica can exert its effect on tumor bearing animals, and (b) the antitumor activity of P. emblica is mediated primarily through the ability of the drug to augment natural cell mediated cytotoxicity.

Cytochrome c oxidase is preferentially synthesized in the rough endoplasmic reticulum--mitochondrion complex in rat liver

The specific activity and content of cytochrome oxidase in the rough endoplasmic reticulum--mitochondrion complex are higher than in the mitochondrial fraction. Radiolabelling studies with the use of hepatocytes and isolated microsomal and rough endoplasmic reticulum--mitochondrion fractions, followed by immunoprecipitation with anti-(cytochrome oxidase) antibody, reveal that the nuclear-coded cytoplasmic subunits of cytochrome oxidase are preferentially synthesized in the latter fraction. The results have a bearing on the mechanism of transport of these subunits into mitochondria.

Biosynthesis of cytoplasmic subunits of cytochrome C oxidase in rat liver

The biosynthesis of the cytoplasmic subunits of cytochrome oxidase from rat liver has been studied in vitro by translating liver poly (A)-containing RNA in the wheat germ cell-free system and immunoprecipitating the products with anti-cytochrome oxidase antibody. Analysis of the labelled immunoprecipitate on SDS-gels does not reveal the presence of a polyprotein precursor. On the other hand discrete products which are either slightly bigger or closely similar to the mature subunits present in purified cytochrome oxidase have been detected.

Acetylation of Transition Protein 2 (TP2) by KAT3B (p300) Alters Its DNA Condensation Property and Interaction with Putative Histone Chaperone NPM3

The hallmark of mammalian spermiogenesis is the dramatic chromatin remodeling process wherein the nucleosomal histones are replaced by the transition proteins TP1, TP2, and TP4. Subsequently these transition proteins are replaced by the protamines P1 and P2. Hyperacetylation of histone H4 is linked to their replacement by transition proteins. Here we report that TP2 is acetylated in vivo as detected by anti-acetylated lysine antibody and mass spectrometric analysis. Further, recombinant TP2 is acetylated in vitro by acetyltransferase KAT3B (p300) more efficiently than by KAT2B (PCAF). In vivo p300 was demonstrated to acetylate TP2. p300 acetylates TP2 in its C-terminal domain, which is highly basic in nature and possesses chromatin-condensing properties. Mass spectrometric analysis showed that p300 acetylates four lysine residues in the C-terminal domain of TP2. Acetylation of TP2 by p300 leads to significant reduction in its DNA condensation property as studied by circular dichroism and atomic force microscopy analysis. TP2 also interacts with a putative histone chaperone, NPM3, wherein expression is elevated in haploid spermatids.Interestingly, acetylation of TP2 impedes its interaction with NPM3. Thus, acetylation of TP2 adds a new dimension to its role in the dynamic reorganization of chromatin during mammalian spermiogenesis.

Structural basis for the function of anti-idiotypic antibody in immune memory

We had earlier proposed a hypothesis to explain the mechanism of perpetuation of immunological memory based on the operation of idiotypic network in the complete absence of antigen. Experimental evidences were provided for memory maintenance through anti-idiotypic antibody (Ab2) carrying the internal image of the antigen. In the present work, we describe a structural basis for such memory perpetuation by molecular modeling and structural analysis studies. A three-dimensional model of Ab2 was generated and the structure of the antigenic site on the hemagglutinin protein H of Rinderpest virus was modeled using the structural template of hemagglutinin protein of Measles virus. Our results show that a large portion of heavy chain containing the CDR regions of Ab2 resembles the domain of the hemagglutinin housing the epitope regions. The similarity demonstrates that an internal image of the H antigen is formed in Ab2, which provides a structural basis for functional mimicry demonstrated earlier. This work brings out the importance of the structural similarity between a domain of hemagglutinin protein to that of its corresponding Ab2. It provides evidence that Ab2 is indeed capable of functioning as surrogate antigen and provides support to earlier proposed relay hypothesis which has provided a mechanism for the maintenance of immunological memory.

Structural basis for the function of anti-idiotypic antibody in immune memory

We had earlier proposed a hypothesis to explain the mechanism of perpetuation of immunological memory based on the operation of idiotypic network in the complete absence of antigen. Experimental evidences were provided for memory maintenance through anti-idiotypic antibody (Ab(2)) carrying the internal image of the antigen. In the present work, we describe a structural basis for such memory perpetuation by molecular modeling and structural analysis studies. A three-dimensional model of Ab(2) was generated and the structure of the antigenic site on the hemagglutinin protein H of Rinderpest virus was modeled using the structural template of hemagglutinin protein of Measles virus. Our results show that a large portion of heavy chain containing the CDR regions of Ab(2) resembles the domain of the hemagglutinin housing the epitope regions. The similarity demonstrates that an internal image of the H antigen is formed in Ab(2), which provides a structural basis for functional mimicry demonstrated earlier. This work brings out the importance of the structural similarity between a domain of hemagglutinin protein to that of its corresponding Ab(2). It provides evidence that Ab(2) is indeed capable of functioning as surrogate antigen and provides support to earlier proposed relay hypothesis which has provided a mechanism for the maintenance of immunological memory.

Protein A: nature's universal anti-antibody

Staphylococcal protein A specifically interacts with immunogobulins. This fact is being used in various disciplines of biology and some of the unique properties of protein A and their applications are summarized in this review.

Immunologic studies on nucleic acids and their components II. Reversible inhibition of anti-nucleoside antibodies in aqueous pyridine and its application in antibody

The effect of aqueous pyridine on a hapten—antihapten system was investigated by the quantitative precipitin reaction and by the membrane filtration method. It was found that dilute solutions of pyridine inhibited the reaction between isopentenyladenosine and its antiserum. Other solvents examined were less effective. The effect of pyridine was reversible at concentrations where complete inhibition occurred, thus indicating its use for the dissociation of antigen—antibody complexes. The inhibitory effect of pyridine was exploited in a single-step purification method for anti—isopentenyladenosine and antideoxy-adenylate antibodies. In addition, generally applicable methods for linking nucleosides and nucleotides to aminoethyl-Sepharose are described.

Immunologic studies on nucleic acids and their components II. Reversible inhibition of anti-nucleoside antibodies in aqueous pyridine and its application in antibody purification

The effect of aqueous pyridine on a hapten—antihapten system was investigated by the quantitative precipitin reaction and by the membrane filtration method. It was found that dilute solutions of pyridine inhibited the reaction between isopentenyladenosine and its antiserum. Other solvents examined were less effective. The effect of pyridine was reversible at concentrations where complete inhibition occurred, thus indicating its use for the dissociation of antigen—antibody complexes. The inhibitory effect of pyridine was exploited in a single-step purification method for anti—isopentenyladenosine and antideoxy-adenylate antibodies. In addition, generally applicable methods for linking nucleosides and nucleotides to aminoethyl-Sepharose are described.

Protein A: nature's universal anti-antibody

Staphylococcal protein A specifically interacts with immunogobulins. This fact is being used in various disciplines of biology and some of the unique properties of protein A and their applications are summarized in this review.

A CD8(+) T cell clone specific for antigen also recognizes peptidomimics present in anti-idiotypic antibody: Implications for T cell memory

The relay hypothesis [R. Nayak, S. Mitra-Kaushik, M.S. Shaila, Perpetuation of immunological memory: a relay hypothesis, Immunology 102 (2001) 387-395] was earlier proposed to explain perpetuation of immunological memory without requiring long lived memory cells or persisting antigen. This hypothesis envisaged cycles of interaction and proliferation of complementary idiotypic B cells (Burnet cells) and anti-idiotypic B cells (Jerne cells) as the primary reason for perpetuation of immunological memory. The presence of pepti-domimics of antigen in anti-idiotypic antibody and their presentation to antigen specific T cells was postulated to be primary reason for perpetuation of T cell memory. Using a viral hemagglutinin as a model, in this work, we demonstrate the presence of peptidomimics in the variable region of ail anti-idiotypic antibody capable of functionally mimicking the antigen derived peptides. A CD8(+) CTL clone was generated against the hemagglutinin protein which specifically responds to either peptidomimic synthesizing cells or peptidomimic pulsed antigen presenting cells. Thus, it appears reasonable that a population of activated antigen specific T cells is maintained in the body by presentation of peptidomimic through Jerne cells and other antigen presenting cells long after immunization. (C) 2007 Elsevier Inc. All rights reserved.

A new ELISA plate based microtiter well assay for mycobacterial topoisomerase I for the direct screening of enzyme inhibitory monoclonal antibody supernatants

Antigen specific monoclonal antibodies present in crude hybridoma supernatants are normally screened by ELISA on plates coated with the relevant antigen. Screening for inhibitory monoclonals to enzymes would require the evaluation of purified antibodies or antibody containing supernatants for their inhibition of enzyme activity in a separate assay. However, screening for inhibitory antibodies against DNA transacting enzymes such as topoisomerase I (topo I) cannot be done using hybridoma supernatants due to the presence of nucleases in tissue culture media containing foetal calf serum which degrade the DNA substrates upon addition. We have developed a simple and rapid screening procedure for the identification of clones that secrete inhibitory antibodies against mycobacterial topo I using 96 well ELISA microtiter plates. The principle of the method is the selective capture of monoclonal antibodies from crude hybridoma supernatants by topo I that is tethered to the plate through the use of plate-bound polyclonal anti-topo I antibodies. This step allows the nucleases present in the medium to be washed off leaving the inhibitor bound to the tethered enzyme. The inhibitory activity of the captured antibody is assessed by performing an in situ DNA relaxation assay by the addition of supercoiled DNA substrate directly to the microtiter well followed by the analysis of the reaction products by agarose gel electrophoresis. The validity of this method was confirmed by purification of the identified inhibitory antibody and its evaluation in a DNA relaxation assay. Elimination of all enzyme-inhibitory constituents of the culture medium from the well in which the inhibitory antibody is bound to the tethered enzyme may make this method broadly applicable to enzymes such as DNA gyrases, restriction enzymes and other DNA transaction enzymes. Further, the method is simple and avoids the need of prior antibody purification for testing its inhibitory activity. (C) 2010 Elsevier B.V. All rights reserved.

Detection of IgG, IgA, IgM and IgE Antibodies in Invasive Amoebiasis in Endemic Areas

Entamoeba histolytica-specific serum IgG, IgA, IgM and IgE antibodies were assayed in cases of amoebiasis in an endemic area. Patient groups consisted of amoebic liver abscess (n=18), pre-abscess hepatic amoebiasis (n=22) and amoebic colitis (n=30). Control subjects comprised 26 asymptomatic cyst passers, 13 giardiasis cases, 20 typhoid patients and 24 non-amoebic individuals. Serum IgG was assayed by ELISA, using a monoclonal anti IgG β- galactosidase (IgG β-gal) conjugate, a polyclonal avidin biotin horse radish peroxidase (AB-HRP), and a polyclonal anti IgG horse radish peroxidase (IgG HRP) conjugate. IgA and IgM were assayed by the β-gal ELISA and IgE by AB-HRP. Diagnostically significant IgG and IgA while lower IgM and IgE antibody levels were seen in extraintestinal cases. About 40% of suspected pre-abscess hepatic amoebiasis cases were confirmed by antibody estimation. All isotype levels in most dysentery cases were in the range of the controls.

Baker's yeast expressing the Japanese encephalitis virus envelope protein on its cell surface: induction of an antigen-specific but non-neutralizing antibody response

Live recombinant Saccharomyces cerevisiae yeast expressing the envelope antigen of Japanese encephalitis virus (JEV) on the outer mannoprotein layer of the cell wall were examined for their ability to induce antigen-specific antibody responses in mice. When used as a modelantigen, parenteral immunization of mice with surface-expressing GFP yeast induced a strong anti-GFP antibody response in the absence of adjuvants. This antigen delivery approach was then used for a more stringent system, such as the envelope protein of JEV, which is a neurotropic virus requiring neutralizing antibodies for protection.Although 70% of cells were detected to express the total envelope protein on the surface by antibodies raised to the bacterially expressed protein, polyclonal anti-JEV antibodies failed to react with them. In marked contrast, yeast expressing the envelope fragments 238-398, 373-399 and 373-500 in front of a Gly-Ser linker were detected by anti-JEV antibodies as well as a monoclonal antibody but not by antibodies raised to the bacterially expressed protein. Immunization of mice with these surface-expressing recombinants resulted in a strong antibody response. However, the antibodies failed to neutralize the virus, although the fragments were selected based on neutralizing determinants.