A simulation approach for power calculation in large cohort studies based on multistate models

Realistic power calculations for large cohort studies and nested case control studies are essential for successfully answering important and complex research questions in epidemiology and clinical medicine. For this, we provide a methodical framework for general realistic power calculations via simulations that we put into practice by means of an R‐based template. We consider staggered recruitment and individual hazard rates, competing risks, interaction effects, and the misclassification of covariates. The study cohort is assembled with respect to given age‐, gender‐, and community distributions. Nested case‐control analyses with a varying number of controls enable comparisons of power with a full cohort analysis. Time‐to‐event generation under competing risks, including delayed study‐entry times, is realized on the basis of a six‐state Markov model. Incidence rates, prevalence of risk factors and prefixed hazard ratios allow for the assignment of age‐dependent transition rates given in the form of Cox models. These provide the basis for a central simulation‐algorithm, which is used for the generation of sample paths of the underlying time‐inhomogeneous Markov processes. With the inclusion of frailty terms into the Cox models the Markov property is specifically biased. An “individual Markov process given frailty” creates some unobserved heterogeneity between individuals. Different left‐truncation‐ and right‐censoring patterns call for the use of Cox models for data analysis. p‐values are recorded over repeated simulation runs to allow for the desired power calculations. For illustration, we consider scenarios with a “testing” character as well as realistic scenarios. This enables the validation of a correct implementation of theoretical concepts and concrete sample size recommendations against an actual epidemiological background, here given with possible substudy designs within the German National Cohort.     

Refined three-dimensional structures of two cyanobacterial C-phycocyanins at 2.1 and 2.5 A resolution. A common principle of phycobilin-protein interaction

The crystal structure of the light-harvesting protein-pigment complex C-phycocyanin (C-PC) from Mastigocladus laminosus (at 2.1 A resolution (1 A = 0.1 nm] has been refined by energy-restrained least-squares methods to a conventional R-factor of 21.7%. In the same way, the crystal structure of C-PC from Agmenellum quadruplicatum has been refined further (2.5 A, R = 18.4%); pyrrole rings C and D of the chromophore at position A84 have been corrected with respect to the previously reported structure. The two C-PC structures are very similar, 213 C alpha positions have a root-mean-square deviation of 0.49 A. Polar and ionic side-chain interactions are discussed in detail and the two subunits of C-PC from M. laminosus are compared to each other. All three chromophores are completely defined and their tetrapyrroles exhibit very similar geometry. The structure of a C-PC chromophore resembles a cleaved porphyrin which has been twisted roughly 180 degrees around the C-5-C-6 and C-14-C-15 bonds. Accordingly, the configuration/conformation of the chromophores is Z-anti, Z-syn, Z-anti (with the exception of the "configuration" of C-15 of chromophore B155, which is almost midway between Z and E). The three chromophores interact similarly with the protein. They arch around aspartate residues (A87, B87 and B39), and the nitrogens of pyrroles B and C are within hydrogen-bonding distance of one of the carboxylate oxygens. Most of the propionic side-chains of the chromophores form salt bridges with arginine and lysine residues. The updated relative chromophore distances and orientations confirm our conclusion that hexameric aggregates are probably the basic functional units, and that inter-hexameric energy transfer takes place preferentially via the central B84 chromophores.     

Electrolocation in the presence of jamming signals: behavior

Electrolocation behavior of Apteronotus leptorhynchus was studied by monitoring the animal's ability to maintain orientation to a variety of moving electrolocation targets. The primary goal of this study was to determine the relative effectiveness of various types of electrical 'jamming signals' in disrupting electrolocation performance. 1. Two measures of electrolocation performance were used: The latency between the electrolocation target motion and the fish's following response, and the minimum distance separating the fish from the target during the target movement sequence. Latency increased and minimum fish-target distance decreased as target size was decreased, and when large diameter ceramic targets were used as control stimuli the fish were less able to avoid, and frequently collided with, these 'electrically transparent' objects. 2. Four types of jamming signals were used in attempts to mask the electrosensory input used for electrolocation. Broad-band noise and sinusoidal signals, different in frequency by a few Hz from the animal's personal electric organ discharge (DF stimuli), were used to jam the tuberous electroreceptors. Five Hz and 50 Hz sinusoidal signals were used to jam the low-frequency or ampullary receptor system. Both the noise and the DF stimuli were effective in reducing electrolocation performance, and the threshold intensity for behavior deterioration was about three-fold lower for DF stimuli as compared to the noise. The rate of change of response deterioration as a function of increasing jamming intensity was, however, not different for these two types of stimuli. Neither the 50 Hz nor the 5 Hz jamming signals caused electrolocation deterioration when presented alone. However, 5 Hz jamming, when added to either the noise or DF jamming, did result in significant increments in response deterioration. This suggests that the ampullary receptors can provide supplementary information for electrolocation. 3. Electrolocation performance deterioration was also studied with various difference frequencies between an animal's EOD and the sinusoidal jamming stimulus. Increasing DF results in decreased electrolocation deterioration, but even at the highest DF frequencies used (128 Hz) significant response degradation was observed. 4. The apparent differences in the effectiveness of noise and DF stimulation in reducing electrolocation performance are largely accounted for by the differential effects of the tuberous electroreceptor filter characteristics on these two types of signals.     

Generating compatible translation initiation regions for heterologous gene expression in Escherichia coli by exhaustive periShine-Dalgarno mutagenesis. Human glutathione reductase cDNA as a model.

Adaptation of eucaryotic cDNA to heterologous expression was studied by mutating the translation initiation (TI) region upstream (mTI) and downstream (MTI) of the start codon. In the mTI subregion the 8 bases flanking the invariant Shine-Dalgarno motif GG-AG were mutagenized exhaustively, while the MTI subregion was subjected to random silent mutations at the wobble positions. The quality of a given TI sequence was judged on the basis of expressed enzyme activity. Low-yield and high-yield mutants of both TI subregions were selected and recombined systematically. The analysis of these double cartridges gave the following results: 1. As a rule, an unfavourable MTI subregion can be compensated for by mutations in the mTI subregion and vice versa. 2. The compatibility between mTI and MTI subregion is explainable at least in part by a low interaction tendency; a delta G(o)'-value of -10.7 kcal/mol appears to be a physical threshold for heterologous cDNA expression. 3. On the basis of periShine-Dalgarno mutations, the expression yield for different cDNA sequences could be increased by 1 to 2 orders of magnitude. One of these sequences encoded delta(1-15)human glutathione reductase, a mutant lacking the flexible N-terminal extension of the protein. In conclusion, to study and overcome TI region-based expression problems it is worthwhile to start out with a versatile vector containing exhaustive mutations in the periShine-Dalgarno sequences; as a rule the coding MTI subregion can be kept unchanged.     

Lymphocyte ecto-5'-nucleotidase activity in infancy: increasing activity in peripheral blood B cells precedes their ability to synthesize IgG in vitro

Ecto-5'-nucleotidase activity was measured in peripheral blood lymphocytes isolated from serial specimens from nine healthy full-term infants and two premature infants at 0, 2, 4, and 6 mo of age. The postnatal nadir in activity was 7.1 +/- 2.0 nmol/hr/10(6) cells, which is the same as the activity in cord blood lymphocytes (7.0 +/- 2 nmol/hr/10(6) cells). The activity rose twofold to 13.2 +/- 3.8 nmol/hr/10(6) cells at 6 mo of age (p less than 0.001, paired t-test), which is similar to the activity in adult peripheral blood lymphocytes (14.1 +/- 6.3 nmol/hr/10(6) cells). This increased activity in total lymphocytes reflects increased activity in the B cell population. B cell ecto-5'-nucleotidase activity in two infants at 12 to 13 mo of age was 19.3 and 25.2 nmol/hr/10(6) cells, values that are four-to fivefold higher than for cord blood B cells (5.6 +/- 2.8 nmol/hr/10(6) cells) and within the normal range for adult B cells (27.9 +/- 12 nmol/hr/10(6) cells). In spite of a greatly expanded peripheral blood B cell population, studies of immunoglobulin biosynthesis in vitro demonstrated that infant peripheral blood B cells are functionally immature with no synthesis of IgG in response to Epstein Barr virus. Thus, the increase in peripheral blood B lymphocyte ecto-5'-nucleotidase activity in infants precedes their acquisition of a capacity for IgG synthesis in vitro. Data from a hypogammaglobulinemic infant revealed a persistently low ecto-5'-nucleotidase activity over a 10-mo period until at 14 mo of age the activity was 8.8 nmol/hr/10(6) cells in total lymphocytes and 13.0 nmol/hr/10(6) cells in B cells, which correlated with in vivo and in vitro evidence of delayed B cell maturation. Thus, ecto-5'-nucleotidase activity may be a useful cell surface marker in studies of human postnatal B cell maturation.     

Phenotype and function of engrafted maternal T cells in patients with severe combined immunodeficiency

Engrafted maternal T cells from two patients with severe combined immunodeficiency (SCID) and graft-vs-host disease (GVHD) were characterized for surface phenotype, function, and ecto-5'-nucleotidase (ecto-5'-NT) activity. The majority of engrafted T cells from both patients were T6-, T3+, and Ia+; the ratio of T4+:T8+ cells varied from 0.89 to 3.1 for Patient 1 and was 0.17 for Patient 2. The sum of T4+ + T8+ cells was greater than the number of T3+ cells, and approximately one-third of the patients' T cells were T3-. Two-color immunofluorescent staining showed that one-third of the T cells from Patient 1 had a novel cell surface phenotype (T6-, T3-, T4+, T8+) that was not previously described. T cells from Patient 1 failed to proliferate in response to allogeneic cells or specific antigen and provided little help for PWM-driven Ig synthesis in vitro. However, they did suppress Ig synthesis in vitro and proliferate in response to PHA and Con A; thus they appeared to be more mature than the T cells of Patient 2 and of most previously reported patients with SCID and maternal T cell grafts. Both patients lacked detectable lymphocyte ecto-5'-NT activity, suggesting that either the ecto-5'-NT activity of maternal T cells is lost after engraftment or that a specific subset(s) of ecto-5'-NT-negative maternal T cells predominates in infants with SCID and GVHD. Thus, in vitro T cell function and the proportions of T cells bearing T4 and T8 may vary in SCID patients with maternal T cell grafts. However, the presence of the Ia antigen and the absence of ecto-5'-NT activity may be consistent features of activated maternal T cells responsible for GVHD.     

Guanethidine and Pupillary Reaction

Local application of guanethidine to the eye results in miosis. The sympathicolytic action of guanethidine on the pupil was proved by the consistent appearance of a Horner''s syndrome after instillation of a 10% solution into the conjunctival sac. Lack of cocaine mydriasis and unimpaired adrenaline mydriasis after guanethidine application are further evidence of this mode of action. Guanethidine is the first drug that can be consistently relied upon to produce miosis by inhibiting sympathetic impulses to the intraocular pupillary muscles; it also inhibits sympathetic impulses to Horner''s muscle of the upper lid. It is a reliable sympathicolytic agent for testing the reaction of abnormal pupils.     

Degradation of poly(3-hydroxyoctanoic acid) [P(3HO)] by bacteria: purification and properties of a P(3HO) depolymerase from Pseudomonas fluorescens GK13.

Twenty-five gram-negative bacteria and one gram-positive bacterium capable of growing on poly(3-hydroxyoctanoic acid) [P(3HO)] as the sole source of carbon and energy were isolated from various soils, lake water, and activated sludge. Most of the isolates degraded only P(3HO) and copolymers of medium-chain-length (MCL) hydroxyalkanoic acids (HA). Except for the gram-positive strain, which was able to hydrolyze P(3HO) and poly(3-hydroxybutyric acid) [P(3HB)], no isolate was able to degrade polymers of short-chain-length HA, such as P(3HB) or poly(3-hydroxyvalerate) [P(3HV)]. All strains utilized a large variety of monomeric substrates for growth. All gram-negative strains, but not the gram-positive strain, accumulated poly(hydroxyalkanoic acids) (PHA), consisting of MCL HA, if they were cultivated under accumulation conditions. One strain, which was identified as Pseudomonas fluorescens GK13 (biovar V), was selected and the extracellular P(3HO) depolymerase of this strain was purified from the culture medium of P(3HO)-grown cells by chromatography with Octyl-Sepharose CL4B and by gel filtration with Superose 12. The relative molecular weights of the native and sodium dodecyl sulfate-treated enzymes were 48,000 and 25,000, respectively. The purified enzyme hydrolyzed P(3HO), copolymers of MCL HA, and para-nitrophenyl esters of fatty acids. P(3HB), P(3HV), and characteristic substrates for lipases, such as Tween 80 or triolein, were not hydrolyzed. The P(3HO) depolymerase of P. fluorescens GK13 was insensitive to phenylmethylsulfonyl fluoride and dithioerythritol, unlike other PHA depolymerases. The dimeric ester of 3-hydroxyoctanoic acid was identified as the main product of enzymatic hydrolysis of P(3HO).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of a glutathione S-conjugate with glutathione reductase. Kinetic and X-ray crystallographic studies

S-Conjugates of glutathione influence the glutathione/glutathione disulfide (GSH/GSSG) status of hepatocytes in at least two ways, namely by inhibition of GSSG transport into the bile [Akerboom et al. (1982) FEBS Lett. 140, 73-76] and by inhibition of the enzyme GSSG reductase (EC 1.6.4.2). The interaction of GSSG reductase with a well-studied conjugate, namely S-(2,4-dinitrophenyl)-glutathione and its electrophilic precursor 1-chloro-2,4-dinitrobenzene are described. For short exposures both compounds are reversible inhibitors of the enzyme, the Ki values being 30 microM and 22 microM respectively. After prolonged incubation, 1-chloro-2,4-dinitrobenzene blocks GSSG reductase irreversibly, which emphasizes the need for rapid conjugate formation in situ. As shown by X-ray crystallography the major binding site of S-(2,4-dinitrophenyl)-glutathione in GSSG reductase overlaps the binding site of the substrate, glutathione disulfide. However, the glutathione moiety of the conjugate does not bind in the same manner as either of the glutathiones in the disulfide.