Concomitant Information in Competing Risk Analysis

A competing risk model, accommodating both Type I censoring and random withdrawals, is expanded to incorporate concomitant information by allowing the parameters of the underlying distributions to be a linear function of two covariates. The model is developed for two competing risks, one following a Weibull distribution and the other a Rayleigh distribution, and random withdrawals following a Weibull distribution. A method is developed for testing the equality of the coefficients for a given covariate for each of the competing risks using MLE'.     

The cyclophilin multigene family of peptidyl-prolyl isomerases. Characterization of three separate human isoforms.

Cyclophilin (CyP), a major cytosolic protein possessing peptidyl-prolyl cis-trans isomerase activity, has been implicated as the specific receptor of the immunosuppressive drug cyclosporin A (CsA). To identify other potential CsA receptors related to CyP, two human cDNA libraries were screened under low stringency conditions using human CyP cDNA (encoding hCyP1) as a probe. Two cDNAs were identified which encode distinct proteins related to human hCyP1. These two novel proteins, designated hCyP2 and hCyP3, share 65 and 76% amino acid sequence homology with hCyP1, respectively. Both hCyP2 and hCyP3 contain NH2-terminal hydrophobic extensions of 32 and 42 amino acids, respectively. Protein-specific antibodies revealed the predominant association of hCyP2 and hCyP3 with membranes and subcellular organelles, which suggests that the amino-terminal leader sequences of the two CyP isoforms may act as signal peptides. In contrast to the results with hCyP1, Southern blot analysis indicated that both hCyP2 and hCyP3 gene sequences are represented infrequently in the human genome. Northern and Western blot analysis showed that the distribution of mRNA and proteins of the three hCyPs in differing tissues and cell types was similar. Each hCyP protein was expressed in Escherichia coli, purified, and shown to be an active peptidyl-prolyl isomerase. Substrate specificity was examined with 11 synthetic peptides (Suc-Xaa-Yaa-Pro-Phe-4-nitroanilide), and inhibition of the peptidyl-prolyl isomerase activities associated with hCyP1, hCyP2, and hCyP3 was studied with CsA, MeAla6-CsA and MeBm2t1-CsA. From both equilibrium considerations and the results of kinetic characterizations it is proposed that of these three CyP proteins, hCyP1 is the most likely intracellular target for CsA.     

Reaction profile of the last step in cytochrome P-450 catalysis revealed by studies of model complexes

 A series of oxoiron(IV) porphyrin cation radical complexes was investigated as compound I analogs of cytochrome P-450. Both the spectroscopic features and the reactivities of the complexes in oxygen atom transfer to olefins were examined as a function of only one variable, the axial ligand trans to the oxoiron(IV) bond. The results disclosed two important kinetic steps – electron transfer from olefin to oxoiron(IV) and intramolecular electron transfer from metal to porphyrin radical – which are affected differently by the axial ligands. The large kinetic barrier of the latter step in the reaction of olefins with the perchlorato-bound oxoiron(IV) porphyrin cation radical complex enabled the trapping of a reaction intermediate in which the metal, but not the porphyrin radical, is reduced. The first electron transfer step is probably followed by σ-bond formation, which readily accounts for formation of isomerized organic products at low temperatures. It is finally postulated that part of the enhanced oxygenation activities of cytochrome P-450 monooxygenases and chloroperoxidases is due to a lowering of the energy barrier for the second electron transfer step via participation of their redox-active cysteinate ligand. Received: 16 January 1997 / Accepted: 24 May 1997     

Cyclic AMP response to recombinant human relaxin by cultured human endometrial cells--a specific and high throughput in vitro bioassay.

A specific and high throughput 96-well format bioassay for recombinant human relaxin (rhRLX) has been developed using human endometrial cells (NHE cells). rhRLX caused a time- and dose-dependent stimulation of cyclic AMP (cAMP) with 1/2 maximal activity of 3.56 +/- 0.65 ng/ml (n = 30). The range of the standard curve was 0.39 to 25 ng/ml with interplate precision of 17 and 22% CV for high and low controls respectively. The cAMP response requires forskolin and 3-isobutyl-1-methylxanthine, and is enhanced by prostaglandin E2 and F2 alpha. The NHE cells do not respond to A or B chains of rhRLX, or a whole array of hormones. Preincubation of rhRLX with specific monoclonal antibody completely abolished the cAMP response. This bioassay has been used to determine the biological activity of several manufactured lots of recombinant human relaxin.     

Preparation of Agglutinating Antisera and Fluorescent-Antibody Conjugates Against Pasteurella tularensis in Equines

The serological response in burros and horses to the viable LVS strain of Pasteurella tularensis was studied. High-titered agglutinating antisera and fluorescent-antibody conjugates were obtained in both groups of animals. Maximum titers were obtained in horses 14 to 21 days after the start of vaccination and in burros 21 to 28 days after the start of vaccination. The use of Woodhour's adjuvants or booster inoculations did not result in increased titers.     

The human ASM (adult skeletal muscle) gene: expression and chromosomal assignment to 11p15

A rat adult skeletal muscle probe (Asm15) originated from a rhabdomyosarcoma was used to isolate the human homologous sequence from a placenta cDNA library. Among several positive clones the longest EcoRI-EcoRI insert (ASM1) obtained was 1875 bp long with 72% homology with rat Asm15 cDNA sequence. Important variations of ASM1 RNA level were observed in different adult skeletal muscles. Expression of a 29kD ASM1 protein was demonstrated in human adult skeletal muscle lysates using an antiserum (PB1579) raised against the C terminal region of the rat Asm15 protein. The human ASM gene was assigned by somatic cell analysis with human (ASM1) and rat (Asm15) probes to chromosome 11, and by in situ hybridization with the human probe to 11p15, a chromosome region involved in human embryonal rhabdomyosarcomas. Except for the presence of a HindII restriction site, the results obtained for the restriction map and the sequence of ASM1 cDNA (data not shown) exhibited extensive homology with the human H19 DNA sequence which have been mapped with a mouse probe also in 11p15. This suggests that ASM/Asm and H19 may represent the same sequence (in this hypothesis the presence of the supplementary HindII site in our ASM1 probe is explained by polymorphic variability). However it was reported that human and mouse H19 mRNA did not encode for a protein but acted as an RNA molecule whereas in our present study ASM protein was detected in human adult skeletal muscle. This could be explained by important regulation of ASM protein expression during development and cell differentiation. However we cannot exclude for the different species studied (mouse, rat, and man) the hypothesis that H19 and ASM/Asm mRNA may represent two distinct messengers from the same gene or even from duplicated genes.