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.     

The effects of chemical pollution on the bioturbation potential of estuarine intertidal mudflats

Bioturbation by benthic infauna has important implications for the fate of contaminants as well as for changes to the sediment structure, chemistry and transport characteristics. There is an extensive literature dealing with the influence of sedimentary variables on the structure and function of infaunal marine and estuarine organisms but less is known of the converse, the influence of biota on sedimentary structure. Although some work has been carried out regarding spatial and temporal patterns of bioturbation, little attention has been given to the effects of pollution. The paper gives a framework of animal sediment relationships in an intertidal environment and discusses the general role of macrofauna in structuring and modifying sedimentary features. A brief outline of the various techniques used for quantifying the degree of bioturbation is given and some of these techniques have then been used to demonstrate the effect of a petrochemical discharge on the bioturbation potential of intertidal communities in the Humber estuary, eastern England. These studies indicate an increase in bioturbation with increasing distance from the source of pollution, not only because of differences in abundance, animal size and depth of activity but also because of the difference in species composition between the communities. As a means of interpreting the responses, the species present have been broadly classified in terms of their feeding strategy and sediment modification potential. The paper concludes by discussing the potential impact, in terms of effect on sediment transport, of selectively removing the different guilds (by pollution). Received: 8 February 1999 / Received in revised form: 10 May 1999 / Accepted: 14 May 1999     

Molecular reaction mechanisms of proteins monitored by time-resolved FTIR-spectroscopy.

Time-resolved FTIR difference spectroscopy can provide a valuable insight into the molecular reaction mechanisms of proteins, especially membrane proteins. Isotopic labeling and site-directed mutagenesis allows an unequivocal assignment of IR absorption bands. Studies are presented which give insight into the proton pump mechanisms of proteins, especially bacteriorhodopsin. H-bonded network proton transfer via internal water molecules seems to be a general feature in proteins, also found in cytochrome c oxidase. Using caged GTP the intrinsic and GAP catalyzed GTPase activity of H-ras p21 is studied. Furthermore, protein folding reactions can be recorded with ns time-resolution.     

Ventilatory responses to specific CNS hypoxia in sleeping dogs.

Our study was concerned with the effect of brain hypoxia on cardiorespiratory control in the sleeping dog. Eleven unanesthetized dogs were studied; seven were prepared for vascular isolation and extracorporeal perfusion of the carotid body to assess the effects of systemic [and, therefore, central nervous system (CNS)] hypoxia (arterial PO(2) = 52, 45, and 38 Torr) in the presence of a normocapnic, normoxic, and normohydric carotid body during non-rapid eye movement sleep. A lack of ventilatory response to systemic boluses of sodium cyanide during carotid body perfusion demonstrated isolation of the perfused carotid body and lack of other significant peripheral chemosensitivity. Four additional dogs were carotid body denervated and exposed to whole body hypoxia for comparison. In the sleeping dog with an intact and perfused carotid body exposed to specific CNS hypoxia, we found the following. 1) CNS hypoxia for 5-25 min resulted in modest but significant hyperventilation and hypocapnia (minute ventilation increased 29 +/- 7% at arterial PO(2) = 38 Torr); carotid body-denervated dogs showed no ventilatory response to hypoxia. 2) The hyperventilation was caused by increased breathing frequency. 3) The hyperventilatory response developed rapidly (<30 s). 4) Most dogs maintained hyperventilation for up to 25 min of hypoxic exposure. 5) There were no significant changes in blood pressure or heart rate. We conclude that specific CNS hypoxia, in the presence of an intact carotid body maintained normoxic and normocapnic, does not depress and usually stimulates breathing during non-rapid eye movement sleep. The rapidity of the response suggests a chemoreflex meditated by hypoxia-sensitive respiratory-related neurons in the CNS.     

Baculoviruses Modulate a Proapoptotic DNA Damage Response To Promote Virus Multiplication

The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) initiates apoptosis in diverse insects through events triggered by virus DNA (vDNA) replication. To define the proapoptotic pathway and its role in antivirus defense, we investigated the link between the host''s DNA damage response (DDR) and apoptosis. We report here that AcMNPV elicits a DDR in the model insect Drosophila melanogaster. Replication of vDNA activated DDR kinases, as evidenced by ATM-driven phosphorylation of the Drosophila histone H2AX homolog (H2Av), a critical regulator of the DDR. Ablation or inhibition of ATM repressed H2Av phosphorylation and blocked virus-induced apoptosis. The DDR kinase inhibitors caffeine and KU55933 also prevented virus-induced apoptosis in cells derived from the permissive AcMNPV host, Spodoptera frugiperda. This block occurred at a step upstream of virus-mediated depletion of the cellular inhibitor-of-apoptosis protein, an event that initiates apoptosis in Spodoptera and Drosophila. Thus, the DDR is a conserved, proapoptotic response to baculovirus infection. DDR inhibition also repressed vDNA replication and reduced virus yields 100,000-fold, demonstrating that the DDR contributes to virus production, despite its recognized antivirus role. In contrast to virus-induced phosphorylation of Drosophila H2Av, AcMNPV blocked phosphorylation of the Spodoptera H2AX homolog (SfH2AX). Remarkably, AcMNPV also suppressed SfH2AX phosphorylation following pharmacologically induced DNA damage. These findings indicate that AcMNPV alters canonical DDR signaling in permissive cells. We conclude that AcMNPV triggers a proapoptotic DDR that is subsequently modified, presumably to stimulate vDNA replication. Thus, manipulation of the DDR to facilitate multiplication is an evolutionarily conserved strategy among DNA viruses of insects and mammals.     

Micropropagation of Dendrocalamus asper {Schult. & Schult. F.} Backer ex k. Heyne): an exotic edible bamboo

Effect of season, media type, carbon source, growth regulators and transplanting media on micropropagation of Dendrocalamus asper, an important bamboo species, was examined. The season of explant collection played an important role in axillary bud sprouting and spring (February?CApril) was found to be the best period for explant collection. Among the different media MS was found to be the best for micropropagation. Maximum numbers (4.83/explant) of shoots were initiated in MS?+?15???M BAP. For shoot multiplication, MS medium supplemented with 10???M BAP and 75???M Adenine sulfate was used. BAP was superior to KIN for both explant establishment, as well as, shoot multiplication. Optimal rooting was achieved in shoots cultured on ? strength MS medium supplemented with 5???M each of IBA and NAA. Regenerated plantlets were acclimatized and hardened in green house using dune sand and vermi-compost (3:1) with 92.34% success and transferred to the field with 100% survival rate. In the field, plants supplied with FYM along with urea showed better growth and development. Macroproliferation, plant multiplication by separating the rooted tillers of well established in vitro raised plantlets after 5 to 6?months of growth in the green house could double the multiplication rate. More than 25000 in vitro raised plants were successfully transferred to the field and no morphological variations in growth were observed, thus proving the potential of tissue culture for raising large scale plantations of D. asper.     

Inhibition of Escherichia coli DNA polymerase-I by the anti-cancer drug cis-diaminedichloroplatinum(II): what roles do polymerases play in cis-platin-induced cytotoxicity?

Activities of Escherichia coli DNA polymerase-I were examined in the presence of the anti-tumor drug cis-diaminedichloroplatinum(II) and its inactive geometric isomer trans-diaminedichloroplatinum(II). The trans-isomer did not inhibit the enzyme activity. The anti-tumor drug, on the other hand, retarded the enzyme in its ability to extend the primer strand of DNA. Two alternative mechanisms of inhibition, covalent binding of cis-diaminedichloroplatinum(II) to the polymerase and to the template DNA, were explored. Selective preincubations of the platinum drug with the polymerase and DNA reveal that the inhibition is primarily due to covalent binding to the enzyme. The rates of inhibition were found to be first order in enzyme and zeroth order in platinum in the concentration range 0.05-3.0 mM. A mechanism that deals with the formation of an initial platinum-polymerase-I complex with a binding constant > 10(5) M(-1) followed by a further reaction to form an inhibitory complex is consistent with the kinetic data. The rate limiting first order rate constant for the formation of the inhibitory complex is comparable to that observed for the thiol coordination of peptides containing cysteine residues. Analyses of known structures and functions of catalytic domains of various polymerases point to the direction that the inhibition is perhaps due to the distortion of the DNA binding domain of the enzyme due to platinum coordination.     

Modulating effect of cerulein on benzodiazepine receptors

Subcutaneous administration of caerulein (100-500 micrograms/kg) significantly reduced the development of picrotoxin (8 mg/kg) seizures in male mice. The same doses of caerulein inhibited 3H-flunitrazepam binding in in vivo experiments. Proglumide, an antagonist of cholecystokinin receptors, in low dose (5 mg/kg) potentiated the effects of caerulein (100 micrograms/kg), whereas the administration of proglumide in high dose (25 mg/kg) reduced the action of caerulein on 3H-flunitrazepam binding and picrotoxin seizures. Caerulein (5-1000 nM) decreased 3H-flunitrazepam binding in in vitro experiments only after supplementation of the binding medium with 120 mM NaCl and 5mM KCl. The results suggest the possible interaction of caerulein with chloride ionophor. It seems probable that the direct interaction of caerulein with chloride ionophor in involved in the inhibitory effect of caerulein on picrotoxin seizures and 3H-flunitrazepam binding.