Stabilisation and partial purification of Triton X-100 solubilised trihydroxycoprostanoyl-CoA synthetase from rat liver

The surface characteristics of sixteen "monobloc" titanium-6% aluminum-4% vanadium (Ti6A14V) femoral components (two of the 6-Ti-28 type and 14 of the 6-Ti-32 type) retrieved after periods of 78-131 months following loosening of the femoral component, as well as two unimplanted controls, were studied. The femoral heads were examined by a combination of noncontact light profilometry, scanning electron microscopy, and energy-dispersive X-ray analysis. No consistent correlations were found between classical surface roughness parameters (average, root mean square, peak-to-valley roughness, and radius of curvature) and any clinical parameter studied (patient gender, weight, and height; primary diagnosis; implantation time; or calculated force applied on the hip joint). This extensive quantitative topographic analysis suggests that wear mechanisms in vivo are complex and that wear of titanium alloy femoral heads is partly attributed to a combination of an imperfect nature of the surface before implantation, removal of the oxide layer causing abrasion of the alloy, subsequent deformation of the bearing surface including polishing, and, to a very small degree, patient parameters.     

Maternal age and births in the 1980s

Projections from the US Bureau of the Census show that as the large "baby boom" cohort ages in the 1980s, the percentage of births to women 35 years or older will increase by 37%, while the percentage of births to teenaged women, the small post-baby-boom cohort, will decrease by 32%. Between 1980 and 1990, for women aged 35 to 44 years, fertility rates are projected to increase modestly, whereas for teenagers aged 15 to 19 years, fertility rates are projected to decrease modestly. Assuming that half of pregnant women aged 35 years or older request prenatal chromosomal diagnosis, an estimated 1.1 million pregnant women aged 35 years or older will request this service during the 1980s, increasing substantially the demand for it. Simultaneously, demand for prenatal care for teenagers will decrease, due to the decrease in births to teenagers.     

Citron rho-interacting kinase, a novel tissue-specific ser/thr kinase encompassing the Rho-Rac-binding protein Citron

We have identified a novel serine/threonine kinase belonging to the myotonic dystrophy kinase family. The kinase can be produced in at least two different isoforms: a approximately 240-kDa protein (Citron Rho-interacting kinase, CRIK), in which the kinase domain is followed by the sequence of Citron, a previously identified Rho/Rac binding protein; a approximately 54-kDa protein (CRIK-short kinase (SK)), which consists mostly of the kinase domain. CRIK and CRIK-SK proteins are capable of phosphorylating exogenous substrates as well as of autophosphorylation, when tested by in vitro kinase assays after expression into COS7 cells. CRIK kinase activity is increased severalfold by coexpression of costitutively active Rho, while active Rac has more limited effects. Kinase activity of endogenous CRIK is indicated by in vitro kinase assays after immunoprecipitation with antibodies recognizing the Citron moiety of the protein. When expressed in keratinocytes, full-length CRIK, but not CRIK-SK, localizes into corpuscular cytoplasmic structures and elicits recruitment of actin into these structures. The previously reported Rho-associated kinases ROCK I and II are ubiquitously expressed. In contrast, CRIK exhibits a restricted pattern of expression, suggesting that this kinase may fulfill a more specialized function in specific cell types.     

Cross-class inhibition of the cysteine proteinases cathepsins K, L, and S by the serpin squamous cell carcinoma antigen 1: a kinetic analysis

The human squamous cell carcinoma antigens (SCCA) 1 and 2 are tandemly arrayed genes that encode two high-molecular-weight serine proteinase inhibitors (serpins). Although these proteins are 92% identical, differences in their reactive site loops suggest that they inhibit different types of proteinases. Our previous studies show that SCCA2 inhibits chymotrypsin-like serine proteinases [Schick et al. (1997) J. Biol. Chem. 272, 1849-1855]. We now show that, unlike SCCA2, SCCA1 lacks inhibitory activity against any of the more common types of serine proteinases but is a potent cross-class inhibitor of the archetypal lysosomal cysteine proteinases cathepsins K, L, and S. Kinetic analysis revealed that SCCA1 interacted with cathepsins K, L, and S at 1:1 stoichiometry and with second-order rate constants >/= 1 x 10(5) M-1 s-1. These rate constants were comparable to those obtained with the prototypical physiological cysteine proteinase inhibitor, cystatin C. Also relative to cystatin C, SCCA1 was a more potent inhibitor of cathepsin K-mediated elastolytic activity by forming longer lived inhibitor-proteinase complexes. The t1/2 of SCCA1-cathepsin S complexes was >1155 min, whereas that of cystatin C-cathepsin complexes was 55 min. Cleavage between the Gly and Ser residues of the reactive site loop and detection of a stable SCCA1-cathepsin S complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that the serpin interacted with the cysteine proteinase in a manner similar to that observed for typical serpin-serine proteinase interactions. These data suggest that, contingent upon their reactive site loop sequences, mammalian serpins, in general, utilize their dynamic tertiary structure to trap proteinases from more than one mechanistic class and that SCCA1, in particular, may be involved in a novel inhibitory pathway aimed at regulating a powerful array of lysosomal cysteine proteinases.     

In vitro selection of RNA molecules that displace cocaine from the membrane-bound nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (AChR) controls signal transmission between cells in the nervous system. Abused drugs such as cocaine inhibit this receptor. Transient kinetic investigations indicate that inhibitors decrease the channel-opening equilibrium constant [Hess, G. P. & Grewer, C. (1998) Methods Enzymol. 291, 443-473]. Can compounds be found that compete with inhibitors for their binding site but do not change the channel-opening equilibrium? The systematic evolution of RNA ligands by exponential enrichment methodology and the AChR in Torpedo californica electroplax membranes were used to find RNAs that can displace inhibitors from the receptor. The selection of RNA ligands was carried out in two consecutive steps: (i) a gel-shift selection of high-affinity ligands bound to the AChR in the electroplax membrane, and (ii) subsequent use of nitrocellulose filters to which both the membrane-bound receptor and RNAs bind strongly, but from which the desired RNA can be displaced from the receptor by a high-affinity AChR inhibitor, phencyclidine. After nine selection rounds, two classes of RNA molecules that bind to the AChR with nanomolar affinities were isolated and sequenced. Both classes of RNA molecules are displaced by phencyclidine and cocaine from their binding site on the AChR. Class I molecules are potent inhibitors of AChR activity in BC3H1 muscle cells, as determined by using the whole-cell current-recording technique. Class II molecules, although competing with AChR inhibitors, do not affect receptor activity in this assay; such compounds or derivatives may be useful for alleviating the toxicity experienced by millions of addicts.     

Sixty adrenal masses of large dimensions: hormonal and morphologic evaluation

OBJECTIVES: To evaluate the nature and function of adrenal masses of large dimensions (macrotumors). METHODS: Sixty consecutive patients (31 women, 29 men, age range 15 to 84 years) with adrenal masses 4.0 cm in diameter or larger (range 4.0 to 15.0 cm) underwent morphologic study by computed tomography (CT); the majority also underwent 131-I-6beta-norcholesterol (131I-NC) or 131I-MIBG scintigraphy. Basal evaluation of glucocorticoids, mineralcorticoids, and catecholamines was performed in all patients, and in 38 cases determination of androgens was also made. In addition, on the basis of various clinical suspicions, a dynamic hormonal study was performed. RESULTS: Macrotumors were benign in 78.3% of cases and included pheochromocytomas (n = 17), nonfunctioning cortical adenomas (n = 12), and cortisol-secreting tumors (n = 7, Cushing's syndrome). Malignant forms were 21.7% of the total, including pheochromocytomas (n = 3), cortical carcinomas (n = 6), and metastases (n = 4). On CT, malignant masses were larger (8.4+/-0.9 cm) than benign ones (5.7+/-0.3 cm) (P < 0.0001) and the mass size was strictly related to malignancy (P < 0.03). CT did not offer other diagnostic criteria for malignancy, except irregular margins and regional lymph node enlargement, which were more frequently (P < 0.0001) found in malignant forms. 131I-MIBG scintigraphy showed tracer uptake in all pheochromocytomas, both benign and malignant. By contrast, on 131I-NC scintigraphy, cortical malignancies never accumulated the radiotracer, whereas uptake was observed in all cases of solid cortical benign adenomas. Patients with cortical carcinomas showed plasma sex steroids above the normal range, pheochromocytomas were asymptomatic in 15% of cases, and almost half of the patients with Cushing's syndrome did not show clinical features of the disease (pre-Cushing's syndrome). CONCLUSIONS: Adrenal macrotumors frequently show endocrine activity and the medulla seems to be involved more than the cortex. Pheochromocytomas and cortisol-secreting adenomas are sometimes asymptomatic. Malignancy is often found in macromasses and, at least for the cortical forms, size of the tumor on CT, 131I-NC uptake on scintigraphy, and determination of levels of sex steroids seem to be useful criteria for predicting the nature of the mass.