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.     

Monoclonal antibodies to neurospora adenylate cyclase

A monoclonal antibody against $\text{Neurospora}$ soluble adenylate cyclase was obtained. The antibody inhibits cyclase activities from several lower eucaryotic organisms but not activities associated to testicular cytosol or turkey erythrocyte membranes.     

Studies on activation and inhibition of cathepsin B from buffalo liver

Cathepsin B (EC 3.4.22.1) was purified from buffalo liver. The enzyme activity against-benzoyl-dl-arginine-naphthylamme (BANA) was substantially reduced by heat (above 37C) and by nondenaturing concentrations of urea (3 M) and guanidine hydrochloride (1 M). Cathepsin B was significantly activated by 1.5 mM EDTA alone. The activation of the enzyme was further enhanced in the presence of thiol compounds, e.g., cysteine thioglycolic acid, 2,3-dimercapto-1-propenol, and dithioerythritol (DTE). The minimum concentration of the thiol compound required for optimal activation of cathepsin B was found to be lowest (0.2 mM) for DTE. The BANA hydrolyzing activity of cathepsin B was substantially reduced by Cu²⁺ (20–200M) and Ca²⁺ (30–250 mM) as well as by thiol blocking reagents, e.g., iodoacetate, 5,5-dithiobis(2-nitro-benzoic acid) (DTNB), andp-hydroxymercuribenzoate (pHMB). The enzyme activity was completely abolished when the molar ratio of the reagent: cathepsin B was close to 1. The number of free sulfhydryl groups in cathepsin B was determined to be 2 by titration against DTNB and pHMB. Modification of one free thiol group of cathepsin B resulted in complete loss of BANA hydrolyzing activity.     

Hydrogen-ion titration curve of ovomucoid.

A systematic study of the H+ titration curve of purified ovomucoid was made at three temperatures (15, 25 and 35 degrees C) and three ionic strengths (0.05, 0.15 and 1.0). In all, 49 protons were dissociated reversibly in the pH range, 2.0-12.0. From the analysis of the results up to pH 12.0, the numbers of different dissociable groups per 28 300 g protein, together with their intrinsic pK values in parentheses were found tp be' 27 sode-chain carboxyl (pKint=4.0), four imidazole (pKint=6.5), one alpha-amino (pKint=7.5), 12 epsilon-amino (pKint=9.6), one guanidino (pKint=11.8) and one alpha-carboxyl group with abnormally low pK. The total number of basic nitrogens per mole of the protein was 22 so that four guanidino groups remained untitrated up to pH 12.0. Spectrophotometric titration showed that three out of five phenolic groups were titrated reversibly up to pH 11.9 with an intrinsic pK of 10.25; the remaining two groups became accessible only on protein denaturation. Viscosity results suggested absence of conformational change in the pH range 2.0-11.2. This explains the constancy of the pK values of carboxyl groups in the pH range 2.0-5.0. The empirical value of the electrostatic interaction factor, w, was 0.04, both in the carboxyl and phenolic regions.     

Objective Measurement of Daytime Napping,Cognitive Dysfunction and Subjective Sleepiness in Parkinson’s Disease

Introduction

Sleep-wake disturbances and concomitant cognitive dysfunction in Parkinson’s disease (PD) contribute significantly to morbidity in patients and their carers. Subjectively reported daytime sleep disturbance is observed in over half of all patients with PD and has been linked to executive cognitive dysfunction. The current study used daytime actigraphy, a novel objective measure of napping and related this to neuropsychological performance in a sample of PD patients and healthy, age and gender-matched controls. Furthermore this study aimed to identify patients with PD who may benefit from pharmacologic and behavioural intervention to improve these symptoms.

Methods

Eighty-five PD patients and 21 healthy, age-matched controls completed 14 days of wrist actigraphy within two weeks of neuropsychological testing. Objective napping measures were derived from actigraphy using a standardised protocol and subjective daytime sleepiness was recorded by the previously validated Epworth Sleepiness Scale.

Results

Patients with PD had a 225% increase in the mean nap time per day (minutes) as recorded by actigraphy compared to age matched controls (39.2 ± 35.2 vs. 11.5 ± 11.0 minutes respectively, p < 0.001). Significantly, differences in napping duration between patients, as recorded by actigraphy were not distinguished by their ratings on the subjective measurement of excessive daytime sleepiness. Finally, those patients with excessive daytime napping showed greater cognitive deficits in the domains of attention, semantic verbal fluency and processing speed.

Conclusion

This study confirms increased levels of napping in PD, a finding that is concordant with subjective reports. However, subjective self-report measures of excessive daytime sleepiness do not robustly identify excessive napping in PD. Fronto-subcortical cognitive dysfunction was observed in those patients who napped excessively. Furthermore, this study suggests that daytime actigraphy, a non-invasive and inexpensive objective measure of daytime sleep, can identify patients with PD who may benefit from pharmacologic and behavioural interventions to improve these symptoms.