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In this study, we set out to establish whether fludarabine could enhance the DNA interstrand crosslinking capacity of SJG-136 in primary human chronic lymphocytic leukaemia (CLL) cells and thereby offer a rationale for its clinical use in combination with SJG-136. SJG-136 rapidly induced DNA crosslinking in primary CLL cells which was concentration-dependent. Further, the level of crosslinking correlated with sensitivity to SJG-136-induced apoptosis (P=0.001) and higher levels of crosslinking were induced by the combination of SJG-136 and fludarabine (P=0.002). All of the samples tested (n=40) demonstrated synergy between SJG-136 and fludarabine (mean combination index (CI)=0.54+/-0.2) and this was even retained in samples derived from patients with fludarabine resistance (mean CI=0.62+/-0.3). Transcription of the excision repair enzyme, ERCC1, was consistently increased (20/20) in response to SJG-136 (P0.0001). In contrast, fludarabine suppressed ERCC1 transcription (P=0.04) and inhibited SJG-136-induced ERCC1 transcription when used in combination (P=0.001). Importantly, the ability of fludarabine to suppress ERCC1 transcription correlated with the degree of synergy observed between SJG-136 and fludarabine (r(2)=0.28; P=0.017) offering a mechanistic rationale for the synergistic interaction. The data presented here provides a clear indication that this combination of drugs may have clinical utility as salvage therapy in drug-resistant CLL.

AP50 is a subunit of the assembly polypeptide (AP) subclass AP-2 from bovine brain coated vesicles. It can be phosphorylated in vivo and in vitro on a threonine residue by means of the AP50 kinase activity associated with AP. We have undertaken an analysis of the amino acid sequence around the AP50 phosphorylation site. After phosphorylation in vitro of AP50 followed by tryptic cleavage, only one radioactive peptide was isolated following Mono-Q ion-exchange f.p.l.c. and reverse-phase h.p.l.c. The amino acid sequence of this peptide: Glu146-Glu-Gln-Ser-Gln-Ile-Thr-Ser-Gln-Val-Thr*-Gly-Gly-Ile-Gly-Tr p-Arg162, displayed two threonine residues. Analysis of the yield and radioactivity of the product from automated Edman degradation indicated that only Thr-156 was phosphorylated, reflecting the presence of a single phosphorylation site in AP50. AP phosphorylated the corresponding synthetic peptide on the same threonyl residue. We demonstrated that AP50 was a phosphorylation substrate unable to autophosphorylate. The enzyme involved in the AP50 phosphorylation was shown to be associated with AP-1 and with a soluble protein complex co-purified with APs but resolved from the latter by hydroxyapatite-column exclusion chromatography. This AP50 kinase activity corresponded to a 280 kDa protein complex according to gel-filtration data.

The in vitro and in vivo effects of S 16118 [p-guanidobenzoyl-[Hyp3, Thi5,D-Tic7,Oic8]bradykinin (BK)], a new BK receptor antagonist, were studied. S 16118 inhibited the contraction produced by BK in the rabbit jugular vein, but was ineffective in the rabbit aorta, indicating the BK B2 receptor specificity of the compound. In isolated organs from various species including humans, S 16118 was a potent antagonist (Ki, pA2 or pKB value from 9.58-7.37). The effect of S 16118 was specific as it did not show any affinity for a number of other receptors or channels and did not possess residual agonistic properties in most of the tissues studied. Furthermore, S 16118 is a poor secretagogue agent either in the rat or human mast cells and is resistant to degradation with an in vitro half-life in blood from different species, including humans, of more than 24 hr. In vivo, in the rabbit, i.v. injection of S 16118 inhibited the hypotension induced by BK up to 4 hr after administration. In the guinea pig, it was also effective in inhibiting the bronchoconstriction induced by BK, although when administered i.v. it had a shorter duration than in the rabbit. However, in the same species, when aerosolized, S 16118 was effective and long-acting against BK-induced bronchoconstriction. Changes in permeability induced by BK injection in the guinea pig trachea and bronchus, and by BK superfusion in the hamster cheek pouch, were abolished by i.v. pretreatment with S 16118.(ABSTRACT TRUNCATED AT 250 WORDS)

Hyaluronic acid-binding region and trypsin-link protein were prepared from bovine nasal cartilage proteoglycan complex after trypsin digestion. Binary complexes were reformed between trypsin-link protein and hyaluronic acid-binding region or hyaluronate. Upon trypsin treatment of these complexes, two fragments deriving from trypsin-link protein were characterized. One of them, of 20 kDa, corresponds in fact to a 140-amino acid long fragment and bears the glycosylated site of trypsin-link protein; it appears to be involved in proteoglycan/link protein interaction. The other, of 22 kDa, corresponds to the 200 C-terminal amino acids of trypsin-link protein; it appears to be involved in the binding of link protein with hyaluronic acid. A structural model of bovine trypsin-like protein depicting two distinct domains involved in hyaluronate and proteoglycan subunit interactions is proposed.

Hyaluronic acid-binding region and trypsin-link protein were prepared from bovine nasal cartilage proteoglycan complex after trypsin digestion. Binary complexes were reformed between trypsin-link protein and hyaluronic acid-binding region or hyaluronate. Upon trypsin treatment of these complexes, two fragments deriving from trypsin-link protein were characterized. One of them, of 20 kDa, corresponds in fact to a 140-amino acid long fragment and bears the glycosylated site of trypsin-link protein; it appears to be involved in proteoglycan/link protein interaction. The other, of 22 kDa, corresponds to the 200 C-terminal amino acids of trypsin-link protein; it appears to be involved in the binding of link protein with hyaluronic acid. A structural model of bovine trypsin-like protein depicting two distinct domains involved in hyaluronate and proteoglycan subunit interactions is proposed.