Differences in the kinetic properties of thymidine kinase isoenzymes in unstimulated and phytohemagglutinin-stimulated human lymphocytes

Summary The two thymidine kinases, TK 1 and TK 2, found in phytohemagglutinin-stimulated human lymphocytes and the thymidine kinase, TK 2N, found in unstimulated human lymphocytes were purified and characterized. All three kinases had molecular weights between 70000 and 75000 which increased to 170000–200000 in the presence of 2 mM ATP.Studies on the kinetic properties of the enzymes with thymidine and ATP as the substrates and dTTP as the inhibitor showed clear differences between TK 1 and TK 2, but a close similarity between TK 2 and TK 2N. With thymidine as the variable substrate, TK 1 showed Michaelis-Menten kinetics, whereas TK 2 and TK 2N showed characteristic biphasic kinetics. With ATP as the variable substrate, all three enzymes showed positive cooperative kinetics, but TK 2 and TK 2N lost the cooperativity in the presence of dTTP. The results from inhibition studies showed, that dTTP was a cooperative inhibitor of TK 1 but a non-cooperative inhibitor of TK 2 and TK 2N.     

Fine structure of Merkel cells in lampreys

Summary The structure of Merkel cells occurring in the epidermis of adult and larval stages of Lampetra spp. is described; it is comparable to that reported from the gnathostome classes. The cells bear microvilli, grouped on the distal and proximal aspects, and are associated with sparsely branching and varicose nerve fibres. One branch of the neurite bears a spur-like process which indents the proximal side of the Merkel cell. Most of the specific Merkel granules are situated in the vicinity of this neurite projection; the cell membrane adjacent to the tip of the spur process bears structures resembling presynaptic densities. Occasionally, desmosome-like junctions are found between the neurite and the Merkel cell.The authors thank the Fresh Water Biological Association and the Department of Biological Sciences, University of Bath, for supplying the material, Dr. H. Fox for giving some prepared blocks of Lampetra planeri adults, Mr. B.L. Pirie for technical assistance, and the Science Research Council for support through grant GR/A/3740.6     

Characterization of Neisseria meningitidis capsular polysaccharides containing sialic acid by pyrolysis mass spectrometry

The group B, C, W-135, and Y capsular polysaccharides of Neisseria meningitidis which contain sialic acid were differentiated by Curie-point pyrolysis low-voltage mass spectrometry. A large series of partially purified group B polysaccharide preparations obtained from pathogenic as well as nonpathogenic strains were analyzed by the same technique. It was shown that the carbohydrate structure of these group B polysaccharides appears to be the same throughout the whole series. Slight immunogenicity of some of the group B polysaccharide preparations tested is probably due to protein impurities. Automated pyrolysis mass spectrometry coupled with multivariate analysis of the spectral data by computer turns out to be a rapid method of characterizing microgram samples of large series of polysaccharide preparations.     

Ceruloplasmin Polymorphism and Parentage Test in Hereford Cattle

In recent years parentage control by means of blood grouping tests (blood and protein systems) has been required for bulls to be registered in the Danish Hereford Herd Book. Because the Hereford breed shows less variation in the blood and protein systems, the probability of excluding an incorrectly stated bull (or cow) is estimated to be some 15 % lower in Hereford than in Danish dairy breeds (RDM, SDM and Jersey).     

Thymidine kinase isoenzymes in human acute monocytic leukemia

Summary Two thymidine kinase isoenzymes, TK 3 and TK 4, from mononuclear leucocytes from a patient with acute monocytic leukemia, were purified and characterized in regard to the molecular weights and kinetic properties.The molecular weights of TK 3 and TK 4 were 60 000 and 45 000, respectively. In the presence of 2 mM ATP, the molecular weight of TK 3 increased to 200 000, whereas the molecular weight of TK 4 was unchanged.Studies of the kinetic properties showed clear differences between TK 3 and TK 4. With thymidine as substrate, TK 3 showed biphasic kinetics with a K_m of 22 µM, and TK 4 showed Michaelis-Menten kinetics with a K_m of 0.33 µM With ATP as substrate, TK 3 showed Michaelis-Menten kinetics with a K_m of 100 µM, and TK 4 showed biphasic kinetics with a Km of 3.5 µM. With dTTP as inhibitor, TK 3 showed cooperative inhibition kinetics, and TK 4 showed non-cooperative competitive inhibition kinetics. The dTTP concentration at 50% inhibition was 75 µM for TK 3 but 380 µM for TK 4.Comparison of the molecular weights and the kinetic properties of TK 3 and TK 4 with the corresponding data previously obtained for TK 1 and TK 2 from normal human lymphocytes indicate the existence of four thymidine kinase isoenzymes in human leucocytes.     

Ago-Allosteric Modulation and Other Types of Allostery in Dimeric 7TM Receptors

Conventionally, an allosteric modulator is neutral in respect of efficacy and binds to a receptor site distant from the orthosteric site of the endogenous agonist. However, recently compounds being ago-allosteric modulators have been described i.e., compounds acting both as agonists on their own and as enhancers for the endogenous agonists in both increasing agonist potency and providing additive efficacy—superagonism. The additive efficacy can also be observed with agonists, which are neutral or even negative modulators of the potency of the endogenous ligand. Based on the prevailing dimeric concept for 7TM receptors, it is proposed that the ago-allosteric modulators bind in the orthosteric binding site, but–importantly–in the “other” or allosteric protomer of the dimer. Hereby, they can act both as additive co-agonists, and through intermolecular cooperative effects between the protomers, they may influence the potency of the endogenous agonist. It is of interest that at least some endogenous agonists can only occupy one protomer of a dimeric 7TM receptor complex at a time and thereby they leave the orthosteric binding site in the allosteric protomer free, potentially for binding of exogenous, allosteric modulators. If the allosteric modulator is an agonist, it is an ago-allosteric modulator; if it is neutral, it is a classical enhancer. Molecular mapping in hetero-dimeric class-C receptors, where the endogenous agonist clearly binds only in one protomer, supports the notion that allosteric modulators can act through binding in the “other” protomer. It is suggested that for the in vivo, clinical setting a positive ago-allosteric modulator should be the preferred agonist drug.     

Cholesterol accumulation caused by low density lipoprotein receptor deficiency or a cholesterol-rich diet results in ectopic bone formation during experimental osteoarthritis

Introduction

Osteoarthritis (OA) is associated with the metabolic syndrome, however the underlying mechanisms remain unclear. We investigated whether low density lipoprotein (LDL) accumulation leads to increased LDL uptake by synovial macrophages and affects synovial activation, cartilage destruction and enthesophyte/osteophyte formation during experimental OA in mice.

Methods

LDL receptor deficient (LDLr^−/−) mice and wild type (WT) controls received a cholesterol-rich or control diet for 120 days. Experimental OA was induced by intra-articular injection of collagenase twelve weeks after start of the diet. OA knee joints and synovial wash-outs were analyzed for OA-related changes. Murine bone marrow derived macrophages were stimulated with oxidized LDL (oxLDL), whereupon growth factor presence and gene expression were analyzed.

Results

A cholesterol-rich diet increased apolipoprotein B (ApoB) accumulation in synovial macrophages. Although increased LDL levels did not enhance thickening of the synovial lining, S100A8 expression within macrophages was increased in WT mice after receiving a cholesterol-rich diet, reflecting an elevated activation status. Both a cholesterol-rich diet and LDLr deficiency had no effect on cartilage damage; in contrast, ectopic bone formation was increased within joint ligaments (fold increase 6.7 and 6.1, respectively). Moreover, increased osteophyte size was found at the margins of the tibial plateau (4.4 fold increase after a cholesterol-rich diet and 5.3 fold increase in LDLr^−/− mice). Synovial wash-outs of LDLr^−/− mice and supernatants of macrophages stimulated with oxLDL led to increased transforming growth factor-beta (TGF-β) signaling compared to controls.

Conclusions

LDL accumulation within synovial lining cells leads to increased activation of synovium and osteophyte formation in experimental OA. OxLDL uptake by macrophages activates growth factors of the TGF-superfamily.     

Aerobic Fitness in Children and Young Adults with Primary Ciliary Dyskinesia

Background

Although aerobic fitness is regarded as an overall prognostic measure of morbidity and mortality, its evaluation in the chronic progressive sinopulmonary disease primary ciliary dyskinesia (PCD) has been infrequently and inconsistently reported. Here we assessed peak oxygen uptake (VO_2peak) in a large well-characterized cohort of PCD patients, and explored whether VO_2peak was associated with parameters of pulmonary function, self-reported physical limitations, and physical activity level.

Methods

VO_2peak, spirometry, diffusing capacity, whole-body plethysmography, and nitrogen multiple breath inert gas washout (N₂ MBW) were assessed in a cross-sectional, single-occasion study of clinically stable children and young adults with PCD. We used a questionnaire including self-reported physical limitations in everyday life or in vigorous activities, and estimation of weekly hours of strenuous physical activity. VO_2peak in PCD patients was compared with that in matched, healthy control subjects and a national reference.

Results

Forty-four PCD patients aged 6–29 years exhibited reduced VO_2peak compared to healthy controls (P<0.001) and the national reference. VO_2peak was abnormal (z-score <–1.96) in 34% of PCD patients. Spirometric values, RV/TLC, and indices of N₂ MBW were significantly abnormal, but VO_2peak only correlated with FEV₁ and DL_CO/V_A. VO_2peak correlated with complaints of moderate or significant limitations in vigorous activities (P = 0.0001), exhibited by 39% of PCD patients.

Conclusion

One-third of PCD patients exhibited substantially lower aerobic fitness than healthy subjects. Aerobic fitness correlated with FEV₁, DL_CO/V_A and self-reported complaints of limitations in vigorous physical activity. These findings are most likely explained by PCD pulmonary disease and its impact on pulmonary function and physical ability. Considering fitness as an important outcome and including regular strenuous physical activity in PCD treatment would probably altogether increase pulmonary clearance, lung function, aerobic fitness, and quality of life, and prevent lifestyle-related diseases.