An efficient chemical approach to bispecific antibodies and antibodies of high valency

Irreversible chemical programming of monoclonal aldolase antibody (mAb) 38C2 has been accomplished with β-lactam equipped mono- and bifunctional targeting modules, including a cyclic-RGD peptide linked to either the peptide (d-Lys⁶)-LHRH or another cyclic RGD unit and a small-molecule integrin inhibitor SCS-873 conjugated to (d-Lys⁶)LHRH. We also prepared monofunctional targeting modules containing either cyclic RGD or (d-Lys⁶)-LHRH peptides. Binding of the chemically programmed antibodies to integrin receptors α(v)β(3) and α(v)β(5) and to the luteinizing hormone releasing hormone receptor were evaluated. The bifunctional and bivalent c-RGD/LHRH and SCS-783/LHRH, the monofunctional and tetravalent c-RGD/c-RGD, and the monofunctional bivalent c-RGD chemically programmed antibodies bound specifically to the isolated integrin receptor proteins as well as to integrins expressed on human melanoma M-21 cells. c-RGD/LHRH, SCS-783/LHRH, and LHRH chemically programmed antibodies bound specifically to the LHRH receptors expressed on human ovarian cancer cells. This approach provides an efficient, versatile, and economically viable route to high-valency therapeutic antibodies that target defined combinations of specific receptors. Additionally, this approach should be applicable to chemically programmed vaccines.     

Basic carboxyl groups of hemoglobin S: Influence of oxy-deoxy conformation on the chemical reactivity of Glu-43(β)

The -carboxyl groups of Glu-43() and Glu-22() of hemoglobin-S (HbS), two intermolecular contact residues of deoxy protein, are activated by carbodiimide atp H 6.0. The selectivity of the modification by the two nucleophiles, glycine ethyl ester (GEE) and glucosamine, is distinct. Influence ofN-hydroxysulfosuccinimide, a reagent that rescues carbodiimide-activated carboxyl (O-acyl isourea) as sulfo-NHS ester, on the overall selectivity and efficiency of the coupling of Glu-22() and Glu-43() with nucleophiles has been investigated. Sulfo-NHS increases the extent of coupling of nucleophiles to HbS. The rescuing efficiency of sulfo-NHS(increase in modification) with GEE and galactosamine as nucleophiles is 2.0 and 2.8, respectively. In the presence of sulfo-NHS, the extent of modification of a carboxyl group is a direct reflection of the extent to which it is activated (i.e., the protonation state of the carboxyl group). The modification reaction exhibits very high selectivity for Glu-43() with GEE and galactosamine (GA) in the presence of sulfo-NHS. From the studies of the kinetics of amidation of oxy-HbS at its Glu-43() (i.e., chemical reactivity) as a function of thepH in the region of 5.5–7.5, the apparentpKa of its -carboxyl group has been calculated to be 6.35. Deoxygenation of HbS, nearly doubles the chemical reactivity of Glu-43() of HbS atpH 7.0. It is suggested that the increased hydrophobicity of the microenvironment of Glu-43(), which occurs on deoxygenation of the protein, is reflected as the increased chemical reactivity of the -carboxyl group and could be one of the crucial preludes to the polymerization process.     

Platelet count on slow induction to high altitude

Platelet counts were estimated at sea level in 50 lowlanders. They were divided at random in two groups (A and B) of 25 each. Group A went up by train/road transport to 3658 m, while group B reached the same height after 8 days of acclimatisation enroute. Platelet counts were estimated serially in both groups at high altitude. Symptoms of high altitude exposure were also recorded. No significant change in the counts was noted in either group and none became Symptomatic. All were brought back to sea level by air and deinduction studies carried out on days 1 and 4 of return. The importance of these findings in the light of our existing knowledge is discussed.     

In vitro characterization of salt stress effects and the selection of salt tolerant plants in rice (Oryza sativa L.)

Summary The response of plant cells to salt stress was studied on embryo derived calli of rice (Oryza sativa L.) in order to identify cellular phenotypes associated with the stress. The feasability of selecting salt tolerant callus and its subsequent regeneration to plants was also studied. Callus was grown on agar-solidified media containing 0%, 1% and 2% (w/v) NaCl for 24 days. Parameters such as fresh weight, dry weight, soluble protein and proline content were measured. The callus growth decreased markedly with increasing NaCl concentration in the medium. The proline content was enhanced several fold in salt stressed calli. A prolonged exposure of callus to the salt environment led to discolouration and arrested growth in the majority of the calli and only a small number of callus cells maintained healthy and stable growth. These variants were subcultured every three weeks for a period of four months onto medium containing 1% NaCl to identify tolerant lines. At the end of the third cell passage, the tolerant calli were transferred to regeneration medium to regenerate plants. The regeneration frequency in the salt-selected lines was enhanced when compared to unselected lines.     

Manganese-histidine cluster as the functional center of the water oxidation complex in photosynthesis

The recent model of Kambara and Govindjee for water oxidation [Kambara T. and Govindjee (1985) Proc. Natl. Acad. Sci. U.S.A., 82:6119–6123] has been extended in this paper by examining all the data in order to identify the most likely candidate for the redox-active ligand (RAL), suggested to operate between the water oxidizing complex (WOC) and Z, the electron donor to the reaction center P680. We have concluded that a very suitable candidate for RAL is the imidazole moiety of a histidine residue. The electrochemical data available on imidazole derivatives play heavily in this identification of RAL. Thus, we suggest that histidine might play the role of an electron mediator between the WOC and Z. A model of S-states in terms of their plausible chemical identity is presented here.Abbreviations J electronic spin of ion - P680 reaction center chlorophyll - RAL Redox active ligand - S_n state of the oxygen-evolving system - WOC water oxidation complex - Z electron donor to P680 Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement     

Mobilization of cloned luciferase genes into Vibrio harveyi luminescence mutants

A recombinant plasmid which carried a 5 kb fragment of Vibrio harveyi DNA containing the luxA and luxB genes was mobilized from Escherichia coli into luminescence-deficient mutants of V. harveyi. The cloned genes complemented a temperature sensitive luciferase mutation, but failed to complement lesions in two different aldehyde deficient mutants. Expression of the cloned genes was not subject to autoinduction in either E. coli or in V. harveyi.     

Adhesive apparatus in certain Indian hill stream fishes

The results of a detailed study on the adhesive apparatus of Indian hill stream cyprinid and sisorid fishes are presented. The structure of the mental suctorial disc in the genus Garra is described. Histological study of the mental region of Crossocheilus and Psilorhynchus has also been made and compared with the mental disc of Garra . The muscles of the disc which control its movements in bringing a partial vacuum during adhesion have been investigated. The complexity of the geniohyoideus muscles in Garra is interesting. Variations in the degree of development and histological modifications of the mental disc are exhibited by the fishes of the genus Garra .
A thoracic adhesive apparatus in the form of longitudinal or transverse ridges and grooves is present in the sisorids, Laguvia, Glyptothorax and Pseudecheneis except Gagata . A close study has revealed that the adhesive apparatus of the catfishes works mainly on the principle of friction. The variations in shape, size and location of the adhesive apparatus in various species are correlated to their respective habitats.
Structural variation in the adhesive apparatus of Pseudecheneis is maximum in comparison to that in Laguvia and Glyptothorax . It is discovered that the modifications are associated with the presence of multi-spinous layers, formation of caps by the basal or holdfast cells and with the transformation of loose areolar tissue into thick collagenous dermis. The presence of a pad of adipose tissue in the region of the adhesive apparatus has been reported and its physiological significance as a source of stored food suggested.
The anterior part of the protractor ischii muscle is modified to control the action of the adhesive apparatus. In Pseudecheneis , besides the m. protractor ischii, the ventral part of m. mesioventral is also associated with the adhesive apparatus.     

Biosynthesisin vitro of neolactotetraosylceramide by a galactosyltransferase from mouse T-lymphoma: purification and kinetic studies; synthesis of neolacto and polylactosamine core

The galactosyltransferase, GalT-4, which catalyses the biosynthesisin vitro of neolactotetraosylceramide, nLcOse4Cer (Gal1-4GleNAc1-3Gal1-4Glc-Cer) from lactotriaosylceramide, LcOse3Cer (GlcNAc1-3Gal1-4Glc-Cer), and UDP-galactose has been purified 107 500-fold from a mineral oil induced mouse T-lyphoma P-1798, using affinity columns. The purified enzyme is partially stabilized in the presence of phospholipid liposomes. Two closely migrating protein bands of apparent molecular weights 56 kDa and 63 kDa were observed after sodium dodecyl sulfate polyacrylamide gel electrophoresis of highly purified mouse GalT-4. These two protein bands, when subjected to limited proteolysis, resulted in three peptides with identical mobilities indicating amino acid sequence identity between the proteins. Both protein bands from P-1798 gave a positive immunostain when tested with polyclonal antibody against bovine lactose synthetase (UDP-Gal:Glc 4-galactosyltransferase) following Western blot analysis on nitrocellulose paper. The enzyme has a pH optimum between 6.5 and 7.0 and like all other galactosyltransferases, GalT-4 has absolute requirements for divalent cation (Mn²⁺). TheK _m values for the substrate LcOse3Cer and donor UDP-galactose are 110 and 250 µm, respectively. Substrate competition studies with LcOse3Cer and either asialo-agalacto-1-acid glycoprotein orN-acetylglucosamine revealed that these reactions might be catalysed by the same protein. The only other glycolipid which showed acceptor activity toward the purified GalT-4 was iLcOse5Cer (GlcNAc1-1-3Gal1-4Lc3), the precursor for polylactosamine antigens. However, competition studies with these two active substrates using the most purified enzyme fraction, revealed that these two reactions might be catalysed by two different proteins since the experimental values were closer to the theoretical values calculated for two enzymes. Interestingly however, it seems that the GalT-4 from P-1798 has an absolute requirement for anN-acetylglucosamine residue in the substrate since the lyso-derivative (GlcNH₂1-3Gal1-4Glc-sphingosine) of the acceptor glycolipid LcOse3Cer is completely inactive as substrate while theK _m andV _max of the reacetylated substrate (GlcNac1-3Gal1-4Glc-acetylsphingosine) was comparable with LcOse3Cer. Autoradiography of the radioactive product formed by purified P-1798 GalT-4 confirmed the presence of nLcOse4Cer, as the product cochromatographed with authentic glycolipid. The monoclonal antibody IB-2, specific for nLcOse4Cer, also produced a positive immunostained band on TLC as well as giving a positive ELISA when tested with radioactive product obtained using a highly purified enzyme from mouse P-1798 T-lymphoma.Abbreviations EDTA ethylenediamine tetraacetate - ME -mercaptoethanol - PEG polyethylene glycol - PBS phosphate buffered saline - Suc sucrose - Mn²⁺ manganese - Gal galactose - GlcNAc N-acetylglucosamine - UDP-Gal Uridine diphosphate galactose - Ab antibody - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - ECB embryonic chicken brain - Cer ceramide - nLc4 or NlcOse4Cer Gal1-4GleNac1-3Gal1-4Glc-Cer, neoLactotetraosylceramide - Lc3 or LcOse3Cer GlcNac1-3Gal1-4Glc-Cer, lactotriaosylceramide - iLc5 iLcOse5Cer, GlcNAc1-3nLcOse4Cer - nLc6 nLcOse6Cer, Gal1-4iLcOse5Cer - SA^–Gal^–₁AGP asialo-agalacto1-acid glycoprotein - TLC thin layer chromatography     

Inhibition by 2,4-D of somatic embryogenesis in carrot as explored by its reversal by difluoromethylornithine

The development of somatic embryos is, in many plants, inhibited by 2,4-dichlorophenoxyacetic acid (2,4-D) and other auxins. The finding that difluoromethylornithine (DFMO) can counteract this inhibition has been used to test some of the hypotheses for the mechanism of inhibition.
Inhibition of somatic embryogenesis in carrot ( Daucus carota L.) by exogenous ethylene (from ethephon), antioxidants (ascorbic acid and glutathione), ethanol/acetaldehyde and abscisic acid was not counteracted by DFMO, indicating that the inhibitory effect of 2,4-D is not manifest through the formation of these compounds. Embryogenesis was abolished by micromolar concentrations of the polar auxin transport inhibitors 2, 3, 5-triiodobenzoic acid (TIBA), N-1-naphthylphthalamic acid (NPA) and 9-hydroxyfluorene-9-carboxylic acid (HFCA). This inhibition was counteracted to a considerable extent by DFMO. Inhibition by relatively high concentrations of the antiauxin 2-( p -chlorophenoxy)-isobutyric acid (CPIB), which does not affect polar auxin transport, was in contrast not counteracted by DFMO. These findings indicate that exogenous auxins may inhibit embryogenesis by interfering with the ability of postglobular embryos to set up internal auxin gradients necessary for polarized growth.