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 production of hematopoietic growth factors by endothelial accessory cells

Monocytes are known to produce both hematopoietic growth factors and other factors, monokines, which do not directly stimulate hematopoiesis. Monokines such as interleukin-1 (IL-1) and tumor necrosis factor (TNF) may indirectly stimulate mesenchymal cells to produce hematopoietic growth factors. The identity of all the factors produced by monocytes or mesenchymal cells has not been established because of overlapping activities on biologic assay. The purpose of this study was to identify the individual growth factors produced by endothelial cells before and after stimulation with various monokines. We prepared conditioned media and extracted RNA from endothelial cells before and after stimulation with monokines. The results show that immortalized endothelial cells produce maximal detectable amounts of granulocyte-macrophage colony-stimulating factor (GM-CSF) constitutively. In contrast, GM-CSF production by primary endothelial cells requires induction with either IL-1 or TNF.     

Biosynthesis of glyantrypine by Aspergillus clavatus

The biosynthesis of glyantrypine from radiolabelled amino acid precursors has been shown experimentally to involve anthranilic acid, tryptophan and glycine. Low values for percentage incorporation of radiolabel into glyantrypine were partly influenced by a complex array of other novel alkaloids shown by the radiolabelling experiments to be related to glyantrypine. Interpretation of radiolabel incorporation from [14C-carboxyl]-anthranilic acid into microbial metabolites seen to contain an anthranilyl moiety in various biosynthetic arrangements is discussed. The possibility of diversion of anthranilic acid from the kynurenine pathway to glyantrypine biosynthesis is recognised.     

Interactions between Males of the Lizard Liolaemus tenuis: Roles of Familiarity and Memory

For territorial organisms, recognition of familiar individuals can reduce the frequency and intensity of aggressive encounters (‘dear enemy’ phenomenon), stabilize social systems, and reduce the cost of territory maintenance. Here, we investigated the behavioural events displayed during contests between familiar and unfamiliar individuals in the lizard Liolaemus tenuis (Liolaemidae), a species in which males are territorial. The behaviours recorded were attack, warning, evasion, and submission, and the latencies to the first aggressive (attack or warning) behaviour. Additionally, we assessed the ability of individuals to remember a familiar conspecific after a period without social interaction. Individual males reduced and delayed aggressive behaviour directed towards socially familiar individuals compared with unfamiliar ones. These results suggest that males distinguished between familiar and unfamiliar conspecific males and are in agreement with the ‘dear enemy’ phenomenon. Other behaviours were similar in the contests between familiar and unfamiliar individuals. Recognition of familiar conspecifics was lost after 20 d without social interactions. This may be relevant for interactions with floater males or with neighbours that lose their territory and subsequently attempt to fight for their ex‐neighbour's territory.     

Inhibition of hexokinase activity by a fructose 2,6-bisphosphate-dependent cytosolic protein from liver

Mammalian and yeast hexokinases were found to be reversibly inhibited by fructose 2,6-bisphosphate, an effect requiring the presence of a cytosolic protein factor. Experimental evidence suggests that this factor (inhibitor) is a regulatory protein, the interactions of which with hexokinases are modulated by fructose 2,6-bisphosphate. The Vmax of hexokinase D was decreased, and no changes on other kinetic parameters were observed. The inhibitor was present in fresh liver cytosol filtered through Sephadex G-25 and was partially isolated by negative absorption on DEAE-cellulose followed by ammonium sulfate fractionation. The inhibitor was also present in brain and kidney, but not in muscle. A molecular mass of 200,000 was determined by gel filtration. The inhibition was dependent on the concentrations of both the inhibitory protein and fructose 2,6-bisphosphate. No delay in fructose 2,6-bisphosphate inhibition was observed. Several other hexose phosphates were tested and were not effective. In the presence of amounts of inhibitor sufficient to produce complete inhibition of hexokinase D, the concentration of fructose 2,6-bisphosphate required to produce 50% inhibition was about 0.5 microM. The inhibitor was unstable and was stabilized by the presence of fructose 2,6-bisphosphate.     

Characterization of hypervariable locus-specific probes derived from a (CAC)5/(GTG)5 multilocus fingerprint in various Eurasian populations

Summary Population genetic studies were performed using oligonucleotide probes (Hz1103, Hz4103, and Hz4201) that recognize three hypervariable loci (D11S859, D9S128 and D22S265) in the human genome. DNA from 17 Eurasian population samples including 37 monozygotic twin pairs were digested with HinfI and hybridized with Hz4103. Allele frequency distribution profiles and high degrees of heterozygosity were similar in each ethnic group. Among 804 unrelated individuals tested, we detected one case of mosaicism caused by a somatic recombination event in a monozygotic twin. In addition, samples of DNA from three ethnic groups (Germans, Assamese Hindus and Thais) and from German and Thai families were restricted with MboI and probed with Hz1103, Hz4103, and Hz4201. The results showed considerable degrees of heterozygosity and locus-specific allele distribution profiles, rather than interpopulation differences. Among 262 meioses (12 three-generation families with a total of 131 children) analyzed, a single recombination event was observed following hybridization with the DNA probe Hz4201.     

Phosphofructokinase is responsible for the fructose 2,6-bisphosphate inhibition of hexokinase in tissue extracts

Mammalian and yeast hexokinases were reported to be reversibly inhibited by fructose 2,6-bisphosphate in the presence of cytosolic proteins (H. Niemeyer, C. Cerpa, and E. Rabajille (1987) Arch. Biochem. Biophys. 257, 17-26). Reinvestigation of this finding using a radioassay with [14C]glucose as substrate showed no effect of fructose 2,6-bisphosphate on hexokinase activity of rat liver cytosols. Detailed reexamination of the spectrophotometric assay resulted in the observation that the fructose 2,6-bisphosphate-dependent inhibition was a function of the cytosolic phosphoglucose isomerase and phosphofructokinase activities compared to the amount of glucose-6-phosphate dehydrogenase used as auxiliary enzyme. The diminution or loss of the fructose 2,6-bisphosphate-dependent inhibition produced in aged cytosols was restored by addition of crystalline muscle phosphofructokinase, as well as by decreasing the amount of glucose-6-phosphate dehydrogenase in the assay. When phosphoglucose isomerase, phosphofructokinase, and hexokinase activities were separated by DEAE-chromatography of liver cytosol, no fructose 2,6-bisphosphate-dependent inhibition of hexokinase was found in any single fraction of the chromatogram. However, combination of fractions containing both phosphoglucose isomerase and phosphofructokinase displayed the fructose 2,6-bisphosphate-dependent inhibition on either endogenous hexokinase or added yeast hexokinase. From these results we conclude that the activation of phosphofructokinase elicited by fructose 2,6-bisphosphate is responsible for the hexokinase inhibition observed in the coupled spectrophotometric assay.     

Functional properties of palmaris longus muscles of rhesus monkeys transplanted as index finger flexors

This experiment with skeletal muscle autografts in monkeys was designed to retest previous findings that transplanted skeletal muscle can regenerate to a functional degree in primates without predenervation and to test a new hypothesis that increased functional demands on regenerated muscle grafts in monkeys may result in improved functional capacity of the grafts. Rhesus monkey index flexors were replaced with free palmaris longus muscle autografts with microneural anastomoses between the graft motor nerve and the severed profundus motor nerve. One monkey was taught selective index flexion before grafting and continued with this program after grafting to test the effect of training on the graft. Mature grafts were evaluated for in vivo contractile properties and by histology and histochemistry and were compared with a group of normal Rhesus palmaris longus muscles. The results reconfirm the capacity of nonpredenervated monkey skeletal muscle grafts to regenerate and to achieve some contractile ability and suggest that training of free muscle grafts may enhance recovery of their functional and structural properties.