DNA polymerase beta and DNA synthesis in Xenopus oocytes and in a nuclear extract.

The identities of the DNA polymerases required for conversion of single-strand (ss) M13 DNA to double-strand (ds) M13 DNA were examined both in injected Xenopus laevis oocytes and in an oocyte nuclear extract. Inhibitors and antibodies specific to DNA polymerases alpha and beta were used. In nuclear extracts, inhibition by the antibody to polymerase beta could be reversed by purified polymerase beta. The polymerase beta inhibitors, dideoxythymidine triphosphate (ddTTP) and dideoxycytidine triphosphate (ddCTP), also blocked DNA synthesis and indicated that polymerase beta is involved in the conversion of ssDNA to dsDNA. These results also may have particular significance for emerging evidence of an ssDNA replication mode in eukaryotic cells.

Arrested DNA replication in Xenopus and release by Escherichia coli mutagenesis proteins.

Xenopus oocytes and oocyte nuclear extracts repair ultraviolet photoproducts on double-stranded (ds) DNA and replicate single-stranded (ss) to ds DNA. M13 ss DNA molecules containing cyclobutane pyrimidine dimers were maintained but not replicated in Xenopus oocytes yet were replicated in progesterone-matured oocytes. The replication arrest functioned only in cis. The replication arrest was alleviated by injection into oocytes of messenger RNAs encoding the prokaryotic mutagenesis proteins UmuD'C or MucA'B. These results may help explain how cells stabilize repair or replication events on DNA with unrepairable lesions.

Rapid and apparently error-prone excision repair of nonreplicating UV-irradiated plasmids in Xenopus laevis oocytes.

Repair of UV-irradiated plasmid DNA microinjected into frog oocytes was measured by two techniques: transformation of repair-deficient (delta uvrB delta recA delta phr) bacteria, and removal of UV endonuclease-sensitive sites (ESS). Transformation efficiencies relative to unirradiated plasmids were used to estimate the number of lethal lesions; the latter were assumed to be Poisson distributed. These estimates were in good agreement with measurements of ESS. By both criteria, plasmid DNA was efficiently repaired, mostly during the first 2 h, when as many as 2 x 10(10) lethal lesions were removed per oocyte. This rate is about 10(6) times the average for removal of ESS from repair-proficient human cells. Repair was slower but still significant after 2 h, but some lethal lesions usually remained after overnight incubation. Most repair occurred in the absence of light, in marked contrast to differentiated frog cells, previously shown to possess photoreactivating but no excision repair activity. There was no increase in the resistance to DpnI restriction of plasmids (methylated in Escherichia coli at GATC sites) incubated in oocytes; this implies no increase in hemimethylated GATC sites, and hence no semiconservative DNA replication. Plasmid substrates capable of either intramolecular or intermolecular homologous recombination were not recombined, whether UV-irradiated or not. Repair of Lac+ plasmids was accompanied by a significant UV-dependent increase in the frequency of Lac- mutants, corresponding to a repair synthesis error frequency on the order of 10(-4) per nucleotide.

Directly photocrosslinked nucleotides joining transfer RNA to aminoacyl-tRNA synthetase in methionine and tyrosine systems.

We have used ultraviolet photocrosslinking and 32P post-labeling to help define the contact surface between transfer RNAs and aminoacyl-tRNA synthetases for the methionine and tyrosine systems. Photocrosslinking between tRNAs and synthetases is shown to occur only in cognate complexes. The increased sensitivity of our procedures reduces the amounts of interacting macromolecules and permits lower ultraviolet light doses, thereby minimizing radiation damage. These procedures have detected crosslinks only within the 3'-terminal RNase T1 fragments in yeast tRNAMeti and Escherichia coli tRNATyr2; and although the photoadducts were unstable, we have identified the crosslinked nucleotides. These crosslinks occur at positions C74 and A76 in yeast tRNAMeti and position U64 in E. coli tRNATyr1&2 (conventional tRNA numbering system of Gauss & Sprinzl, 1981). This work demonstrates that even labile photocrosslinks can be exploited for mapping crosslinked nucleotides.

Monte Carlo simulation of base and nucleotide excision repair of clustered DNA damage sites. I. Model properties and predicted trends.

DNA is constantly damaged through endogenous processes and by exogenous agents, such as ionizing radiation. Base excision repair (BER) and nucleotide excision repair (NER) help maintain the stability of the genome by removing many different types of DNA damage. We present a Monte Carlo excision repair (MCER) model that simulates key steps in the short-patch and long-patch BER pathways and the NER pathway. The repair of both single and clustered damages, except double-strand breaks (DSBs), is simulated in the MCER model. Output from the model includes estimates of the probability that a cluster is repaired correctly, the fraction of the clusters converted into DSBs through the action of excision repair enzymes, the fraction of the clusters repaired with mutations, and the expected number of repair cycles needed to completely remove a clustered damage site. The quantitative implications of alternative hypotheses regarding the postulated repair mechanisms are investigated through a series of parameter sensitivity studies. These sensitivity studies are also used to help define the putative repair characteristics of clustered damage sites other than DSBs.

Aberrant mobility phenomena of the DNA repair protein XPA.

The DNA repair protein XPA recognizes a wide variety of bulky lesions and interacts with several other proteins during nucleotide excision repair. We recently identified regions of intrinsic order and disorder in full length Xenopus XPA (xXPA) protein using an experimental approach that combined time-resolved trypsin proteolysis and electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry (MS). MS data were consistent with the interpretation that xXPA contains no post-translational modifications. Here we characterize the discrepancy between the calculated molecular weight (31 kDa) for xXPA and its apparent molecular weight on SDS-PAGE (multiple bands from approximately 40-45 kDa) and gel filtration chromatography ( approximately 92 kDa), as well as the consequences of DNA binding on its anomalous mobility. Iodoacetamide treatment of xXPA prior to SDS-PAGE yielded a single 42-kDa band, showing that covalent modification of Cys did not correct aberrant mobility. Determination of sulfhydryl content in xXPA with Ellman's reagent revealed that all nine Cys in active protein are reduced. Unexpectedly, structural constraints induced by intramolecular glutaraldehyde crosslinks in xXPA produced a approximately 32-kDa monomer in closer agreement with its calculated molecular weight. To investigate whether binding to DNA alters xXPA's anomalous migration, we used gel filtration chromatography. For the first time, we purified stable complexes of xXPA and DNA +/- cisplatin +/- mismatches. xXPA showed at least 10-fold higher affinity for cisplatin DNA +/- mismatches compared to undamaged DNA +/- mismatches. In all cases, DNA binding did not correct xXPA's anomalous migration. To test predictions that a Glu-rich region (EEEEAEE) and/or disordered N- and C-terminal domains were responsible for xXPA's aberrant mobility, the molecular weights of partial proteolytic fragments from approximately 5 to 25 kDa separated by reverse-phase HPLC and precisely determined by ESI-FTICR MS were correlated with their migration on SDS-PAGE. Every partial tryptic fragment analyzed within this size range exhibited 10%-50% larger molecular weights than expected. Thus, both the disordered domains and the Glu-rich region in xXPA are primarily responsible for the aberrant mobility phenomena.

Identification of intrinsic order and disorder in the DNA repair protein XPA.

The DNA-repair protein XPA is required to recognize a wide variety of bulky lesions during nucleotide excision repair. Independent NMR solution structures of a human XPA fragment comprising approximately 40% of the full-length protein, the minimal DNA-binding domain, revealed that one-third of this molecule was disordered. To better characterize structural features of full-length XPA, we performed time-resolved trypsin proteolysis on active recombinant Xenopus XPA (xXPA). The resulting proteolytic fragments were analyzed by electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance mass spectrometry and SDS-PAGE. The molecular weight of the full-length xXPA determined by mass spectrometry (30922.02 daltons) was consistent with that calculated from the sequence (30922.45 daltons). Moreover, the mass spectrometric data allowed the assignment of multiple xXPA fragments not resolvable by SDS-PAGE. The neural network program Predictor of Natural Disordered Regions (PONDR) applied to xXPA predicted extended disordered N- and C-terminal regions with an ordered internal core. This prediction agreed with our partial proteolysis results, thereby indicating that disorder in XPA shares sequence features with other well-characterized intrinsically unstructured proteins. Trypsin cleavages at 30 of the possible 48 sites were detected and no cleavage was observed in an internal region (Q85-I179) despite 14 possible cut sites. For the full-length xXPA, there was strong agreement among PONDR, partial proteolysis data, and the NMR structure for the corresponding XPA fragment.

Simulation of mechanisms of viral interference in influenza.

Biological interference among viral agents might have significant implications for disease prevention and therapy. Field data for influenza yield conflicting evidence concerning the independence of infection rates, or disease severity, for two co-circulating viruses. To examine the effects of several assumed modes of interference for influenza, simulations of a Monte Carlo micropopulation model of influenza epidemics have been performed. Model parameters were selected so that the simulated attack rates for each of two different viral strains matched actual field data. Rates of infection were compared for single agents and for two viruses with only behavioural interference. Other simulations included temporary immunity to the other virus for the duration of the infection, and/or reduced shedding of viral particles for dual infections. Simulated viral competition had little impact on epidemic severity, duration, or size distribution. Under the conditions studied, viral interference in natural populations would be difficult to infer from field observations of attack rates. Other simulations extended a partial immunity and/or reduced viral shedding during an infection with a second virus. These indicated that interference might be suggested by field data, but it could not be demonstrated conclusively. Still other simulations showed that for epidemics with much higher attack rates for both viruses, it would be relatively easy to demonstrate interference. However, in order to observe interference between influenza strains, it would be necessary to monitor on an almost daily basis, using a method of viral detection which would have to be both highly specific and also very sensitive.

Simulation studies of influenza epidemics: assessment of parameter estimation and sensitivity.

The influenza simulation model of Elveback et al is used to evaluate the accuracy of the maximum likelihood procedure of Longini et al for estimating the secondary attack rate in households. The sample population from the Tecumseh Respiratory Illness Study is mapped into the simulation model and simulations are carried out over a range of parameter values and conditions, some of which were derived from influenza seasons in Tecumseh and from the Seattle Flu Study for the years 1975-1980. The estimation procedure is found to be quite robust for parameter values preset within appropriate limits for influenza. However, a significant difference is found between the preset and estimated household contact parameter for epidemics of medium and high intensity when the preset value is zero. Incremental increases in the household contact parameter are shown to produce marked increases in the overall infection attack rate demonstrating that household spread is an important link in maintaining infection in other mixing groups such as schools, preschool groups and neighbourhood clusters of households.

Economic impact of a computer-based centralized organization in a clinical laboratory.

The Department of Laboratory Medicine, University of Washington, has explored various management of organizational approaches over the past seven years. The Centralized Processing Area supported by a computerized information system has evolved from these efforts. Despite a decrease in U.W. hospital usage since 1968-69, laboratory volume has steadily increased. The cost of these services is shown in relation both to three successive management systems and to the impact of inflation on laboratory costs. Chemistry and hematology costs per procedure, including administration and computer costs, are compared with microbiology cost per procedure, where the management system was not used. There is growing acceptance of the intangible benefits of computerization in the laboratory. Economic benefits are often presumed but have not been proven. Favorable economic effects of the Centralized Processing Area together with the computerized information system are demonstrated.

Investigation of statistical decision rules for sequential hematologic laboratory tests.

A statistical data processing program for identifying patients who are likely to have abnormalities on various hematologic laboratory tests is described. The prediction of abnormal levels of serum vitamin B12, serum folate, transferrin saturation, and reticulocyte counts is based on a statistical analysis of the patient's age, sex, and routine blood cell measurements. The program was developed using data from normal-value studies and data from patients who had these laboratory abnormalities. The sensitivity and specificity of the program were evaluated in a controlled prospective study of about 5,000 ambulatory adult patients. The program's predictions also were compared with the laboratory tests requested by the patients' attending physicians. The program was most sensitive for predicting low serum vitamin B12 (74%) and low transferrin saturation (78%). In the prospective evaluation, the predictive values varied from 11% for predicting low serum folate to 65% for predicting low transferrin saturation.

An integrated confocal and magnetic resonance microscope for cellular research.

Complementary data acquired with different microscopy techniques provide a basis for establishing a more comprehensive understanding of health and disease at a cellular level, particularly when data acquired with different methodologies can be correlated in both time and space. In this Communication, a brief description of a novel instrument capable of simultaneously performing confocal optical and magnetic resonance microscopy is presented, and the first combined images of live Xenopus laevis oocytes are shown. Also, the potential benefits of combined microscopy are discussed, and it is shown that the a priori knowledge of the high-resolution optical images can be used to enhance the boundary resolution and contrast of the MR images.

Iontophoretic delivery of morphine for postoperative analgesia.

Iontophoresis is a method of transdermal administration of ionized drugs in which electrically charged molecules are propelled through the skin by an external electrical field. This was a prospective, randomized, single-blind study to determine the effectiveness of iontophoretically delivered morphine HCl for the control of postoperative pain. Thirty-eight patients who underwent total knee or hip replacement completed this clinical trial. Informed consent was obtained before surgery and patients were instructed on the use of a patient-controlled analgesia (PCA) device. Postoperatively, pain in the recovery room was initially controlled with IV meperidine, and thereafter with PCA therapy using meperidine, 2 mg/cc, with a dose of 10 mg IV and a lock-out period of 15 min. The dose was adjusted as necessary and the lock-out period remained the same. The number of patient requests and the dose (mg) administered was recorded hourly. On the morning following surgery, iontophoresis devices were attached for 6 hr to patients who received either morphine HCl or lactated ringers solution. During this period and for 12 hr following completion of iontophoresis, PCA analgesia remained available to patients. Venous blood samples for determination of morphine levels were obtained every 30 min during iontophoresis, then every 60 min for 2 hr following iontophoresis. Of the 38 patients, 17 received iontophoresed morphine, and 21 received iontophoresed lactated ringers. The morphine group utilized the PCA device more than the control group during the baseline period. However, following the institution of iontophoresis and continuing up to 12 hr following completion of iontophoresis, the morphine group used significantly less PCA meperidine to maintain analgesia than the control group (p = 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Diffusion of n-butyl-p-aminobenzoate (BAB), lidocaine and bupivacaine through the human dura-arachnoid mater in vitro.

INTRODUCTION: Epidural administration of a suspension of n-butyl-p-aminobenzoate (BAB) to humans resulted in a selective, ultra-long lasting sensory block without motor function impairment. The absence of motor block was attributed to 'spatial' confinement of active concentrations of dissolved BAB within the epidural space. This study was designed to investigate the diffusion of BAB through the human dura-arachnoid membrane in vitro relative to lidocaine and bupivacaine and to quantify the influence of the composition of the suspension formulation on this flux. MATERIALS AND METHODS: Human dura-arachnoid specimens were mounted between the donor and the receiver compartment of a diffusion cell. Five concentrations of BAB, lidocaine and bupivacaine in phosphate-buffered saline, pH 7.4, were added to the donor compartment and the increase of the concentration of the agent in time in the receiver compartment was measured by automated UV-spectrometry. Fluxes and permeabilities were calculated. The influence of pH, polysorbate 80 (PS 80) and polyethylene glycol 3350 (PEG 3350) on the flux of BAB in solution and in suspension formulations were analyzed. RESULTS: The flux of both lidocaine and bupivacaine at pH 4 was considerably smaller than at pH 7.4. Permeabilities decreased in the order bupivacaine>lidocaine&z.Gt;BAB and at the level T12>T1. PS 80 at concentrations exceeding 0.025 mg/ml and PEG 3350 decreased the flux of BAB from BAB-solutions. Used in the preparation of the suspension, PS 80 and PEG 3350 did significantly reduce the permeability. DISCUSSION: The results of this study are consistent with the hypothesis that the selective action of epidurally applied BAB suspension can be attributed to the spatial confinement of active BAB-concentrations within the epidural space. Additives used in the preparation of the aqueous suspension formulation may substantially influence the local pharmacokinetics and by that the pharmacodynamic effects.

Pancreatitis induced by mesalamine.

Sulphasalazine is an active agent in the treatment of chronic inflammatory bowel disease, but there are a number of well-known side effects, including pancreatitis. The newer 5-ASA agents are thought to be equally effective but less toxic. Here we describe a patient who developed a pancreatitis due to mesalamine; this was confirmed at rechallenge.

Viral encephalitis: imaging with SPECT.

SPECT brain imaging performed in two patients with presumed herpes encephalitis demonstrated greater sensitivity and more precise localization than either planar brain imaging or CT scanning.

Effects of sampling location on data sensitivity in a three compartment model.

A computer simulation of a three compartment model was developed to examine the effect of sampling location on the accuracy of estimates of system parameters. Results indicate that the best estimates are obtained when all three compartments are sampled. Sampling from only one compartment tends to result in the poorest estimates.

LINKERS: a simulation programming system for generating populations with genetic structure.

Significant features are described of a Monte Carlo simulation system, LINKERS, that generates nuclear family data conforming to one locus and two loci genetic models. Software engineering techniques help to produce a user friendly environment for studies of genetic ascertainment. LINKERS' 39 code modules are maintained and optimized using VAXset. Within a VAX/VMS workstation environment LINKERS generates a useful population of 10,000 families in 1 min. An example is presented to illustrate the use of LINKERS. Generated data were analysed using the segregation program POINTER. Likelihood ratio tests showed the effects of multiple alleles, gene frequency and incorrect specification of disease prevalence.

Simulation of stochastic micropopulation models--I. The SUMMERS simulation shell.

A generic, abstract model and the simulation shell based on it, both called SUMMERS, are used as a framework for the implementation of stochastic micropopulation models; in these, each individual is followed separately while moving through a sequence of states. The shell supports groups of interacting members, individual characteristics and multiple simultaneous activities. Stochastic decisions may be made using Monte Carlo rules. Keywords control the simulations and the reports generated. A sensitivity analysis utility allows assessment of the dependency of outcomes on model features. Extensive use has been made of software engineering techniques. Specializations of SUMMERS are described in subsequent papers.

Simulation of stochastic micropopulation models--III. COGNET: an artificial neural network for visual recognition.

COGNET, based on a neural network first described by Fukushima, demonstrates the relationship between connectionist and other micropopulation models. Its success and physiological orientation led to an implementation using the SUMMERS simulation shell. After self-supervised learning, COGNET uses forward and backward propagation of signals to recognize partial and noisy patterns, and to reconstruct the originals. Stochastic features include variable thresholds for neuronal firing and occasional cell death. The successful implementation of COGNET demonstrates the generality of the concepts embodied in SUMMERS, which in turn promotes the reusability of software and facilitates the extension of computational models in biomedical research. COGNET itself forms a framework for building other physiologically oriented neural network models.