Network approach identifies Pacer as an autophagy protein involved in ALS pathogenesis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a multifactorial fatal motoneuron disease without a cure. Ten percent of ALS cases can be pointed to a clear genetic cause, while the remaining 90% is classified as sporadic. Our study was aimed to uncover new connections within the ALS network through a bioinformatic approach, by which we identified C13orf18, recently named Pacer, as a new component of the autophagic machinery and potentially involved in ALS pathogenesis. METHODS: Initially, we identified Pacer using a network-based bioinformatic analysis. Expression of Pacer was then investigated in vivo using spinal cord tissue from two ALS mouse models (SOD1G93A and TDP43A315T) and sporadic ALS patients. Mechanistic studies were performed in cell culture using the mouse motoneuron cell line NSC34. Loss of function of Pacer was achieved by knockdown using short-hairpin constructs. The effect of Pacer repression was investigated in the context of autophagy, SOD1 aggregation, and neuronal death. RESULTS: Using an unbiased network-based approach, we integrated all available ALS data to identify new functional interactions involved in ALS pathogenesis. We found that Pacer associates to an ALS-specific subnetwork composed of components of the autophagy pathway, one of the main cellular processes affected in the disease. Interestingly, we found that Pacer levels are significantly reduced in spinal cord tissue from sporadic ALS patients and in tissues from two ALS mouse models. In vitro, Pacer deficiency lead to impaired autophagy and accumulation of ALS-associated protein aggregates, which correlated with the induction of cell death. CONCLUSIONS: This study, therefore, identifies Pacer as a new regulator of proteostasis associated with ALS pathology.

Quantifying the dark data in museum fossil collections as palaeontology undergoes a second digital revolution.

Large-scale analysis of the fossil record requires aggregation of palaeontological data from individual fossil localities. Prior to computers, these synoptic datasets were compiled by hand, a laborious undertaking that took years of effort and forced palaeontologists to make difficult choices about what types of data to tabulate. The advent of desktop computers ushered in palaeontology's first digital revolution-online literature-based databases, such as the Paleobiology Database (PBDB). However, the published literature represents only a small proportion of the palaeontological data housed in museum collections. Although this issue has long been appreciated, the magnitude, and thus potential significance, of these so-called 'dark data' has been difficult to determine. Here, in the early phases of a second digital revolution in palaeontology--the digitization of museum collections-we provide an estimate of the magnitude of palaeontology's dark data. Digitization of our nine institutions' holdings of Cenozoic marine invertebrate collections from California, Oregon and Washington in the USA reveals that they represent 23 times the number of unique localities than are currently available in the PBDB. These data, and the vast quantity of similarly untapped dark data in other museum collections, will, when digitally mobilized, enhance palaeontologists' ability to make inferences about the patterns and processes of past evolutionary and ecological changes.

Ultrasound shear wave velocity in skeletal muscle: A reproducibility study.

PURPOSE: The purpose of the study was threefold: to assess the reliability of shear wave velocities (SWV) measurements in normal skeletal muscles; to evaluate intra- and inter-operator reproducibility of measurements for a specific site of the muscle and for the mean value in the whole muscle. MATERIALS AND METHODS: Two sets of measurements were performed at three weeks intervals of each other on 16 volunteers by two radiologists on medial gastrocnemius and tibialis anterior muscles. Each muscle was evaluated in 5 different sites, with three measurements for each site in the transverse and longitudinal planes. Reliability of SWV measurements was assessed by means of intraclass correlation coefficient (ICC). RESULTS: Reliability of the three independent SWV measurements was excellent, slightly better in the longitudinal plane. Inter/intra-operator reproducibility per site was fair to good in the longitudinal plane and poor to fair in the transverse plane. For global values of the whole muscle, ICC showed good agreement in the longitudinal plane and fair agreement in the transverse plane. CONCLUSION: Quantitative SWV measurements are reliable when performed in rigorous conditions. In conditions that mirror clinical practice, inter/intra-operator reproducibility is moderate, better for longitudinal compared to transverse plane.

Cutting edge: control of Mycobacterium tuberculosis infection by a subset of lung parenchyma-homing CD4 T cells.

Th1 cells are critical for containment of Mycobacterium tuberculosis infection, but little else is known about the properties of protective CD4 T cell responses. In this study, we show that the pulmonary Th1 response against M. tuberculosis is composed of two populations that are either CXCR3(hi) and localize to lung parenchyma or are CX3CR1(hi)KLRG1(hi) and are retained within lung blood vasculature. M. tuberculosis-specific parenchymal CD4 T cells migrate rapidly back into the lung parenchyma upon adoptive transfer, whereas the intravascular effectors produce the highest levels of IFN-γ in vivo. Importantly, parenchymal T cells displayed greater control of infection compared with the intravascular counterparts upon transfer into susceptible T cell-deficient hosts. Thus, we identified a subset of naturally generated M. tuberculosis-specific CD4 T cells with enhanced protective capacity and showed that control of M. tuberculosis correlates with the ability of CD4 T cells to efficiently enter the lung parenchyma rather than produce high levels of IFN-γ.

Active multilayered capsules for in vivo bone formation.

Interest in the development of new sources of transplantable materials for the treatment of injury or disease has led to the convergence of tissue engineering with stem cell technology. Bone and joint disorders are expected to benefit from this new technology because of the low self-regenerating capacity of bone matrix secreting cells. Herein, the differentiation of stem cells to bone cells using active multilayered capsules is presented. The capsules are composed of poly-L-glutamic acid and poly-L-lysine with active growth factors embedded into the multilayered film. The bone induction from these active capsules incubated with embryonic stem cells was demonstrated in vitro. Herein, we report the unique demonstration of a multilayered capsule-based delivery system for inducing bone formation in vivo. This strategy is an alternative approach for in vivo bone formation. Strategies using simple chemistry to control complex biological processes would be particularly powerful, as they make production of therapeutic materials simpler and more easily controlled.

Lipid-based nanotherapeutics for siRNA delivery.

RNA interference (RNAi) is a specific gene-silencing mechanism triggered by small interfering RNA (siRNA). The application of RNAi in the clinic requires the development of safe and effective delivery systems. Inspired by progress with lipid-based systems in drug delivery, efforts have been dedicated to the development of liposomal siRNA delivery systems. Many of the lipid-based delivery vehicles self-assemble with siRNA through electrostatic interactions with charged amines, generating multi-lamellar lipoplexes with positively charged lipid bilayers separated from one another by sheets of negatively charged siRNA strands. Internalization of lipid-based siRNA delivery systems into cells typically occurs through endocytosis; accordingly, delivery requires materials that can facilitate endosomal escape. The size of the carrier is important as carriers <100 nm in diameter have been reported to have higher accumulation levels in tumours, hepatocytes and inflamed tissue, whereas larger particles tend to be taken up by Kupffer cells or other components of the reticuloendothelial system (RES). To reduce RES uptake and increase circulation time, carriers have been modified on the surface with hydrophilic materials, such as polyethyleneglycol. Herein, we review the molecular and structural parameters of lipid-based siRNA delivery systems.

A simple model for circadian timing by mammals.

Circadian timing is structured in such a way as to receive information from the external and internal environments, and its function is the timing organization of the physiological and behavioral processes in a circadian pattern. In mammals, the circadian timing system consists of a group of structures, which includes the suprachiasmatic nucleus (SCN), the intergeniculate leaflet and the pineal gland. Neuron groups working as a biological pacemaker are found in the SCN, forming a biological master clock. We present here a simple model for the circadian timing system of mammals, which is able to reproduce two fundamental characteristics of biological rhythms: the endogenous generation of pulses and synchronization with the light-dark cycle. In this model, the biological pacemaker of the SCN was modeled as a set of 1000 homogeneously distributed coupled oscillators with long-range coupling forming a spherical lattice. The characteristics of the oscillator set were defined taking into account the Kuramoto's oscillator dynamics, but we used a new method for estimating the equilibrium order parameter. Simultaneous activities of the excitatory and inhibitory synapses on the elements of the circadian timing circuit at each instant were modeled by specific equations for synaptic events. All simulation programs were written in Fortran 77, compiled and run on PC DOS computers. Our model exhibited responses in agreement with physiological patterns. The values of output frequency of the oscillator system (maximal value of 3.9 Hz) were of the order of magnitude of the firing frequencies recorded in suprachiasmatic neurons of rodents in vivo and in vitro (from 1.8 to 5.4 Hz).

A simple model for circadian timing by mammals

Circadian timing is structured in such a way as to receive information from the external and internal environments, and its function is the timing organization of the physiological and behavioral processes in a circadian pattern. In mammals, the circadian timing system consists of a group of structures, which includes the suprachiasmatic nucleus (SCN), the intergeniculate leaflet and the pineal gland. Neuron groups working as a biological pacemaker are found in the SCN, forming a biological master clock. We present here a simple model for the circadian timing system of mammals, which is able to reproduce two fundamental characteristics of biological rhythms: the endogenous generation of pulses and synchronization with the light-dark cycle. In this model, the biological pacemaker of the SCN was modeled as a set of 1000 homogeneously distributed coupled oscillators with long-range coupling forming a spherical lattice. The characteristics of the oscillator set were defined taking into account the Kuramoto's oscillator dynamics, but we used a new method for estimating the equilibrium order parameter. Simultaneous activities of the excitatory and inhibitory synapses on the elements of the circadian timing circuit at each instant were modeled by specific equations for synaptic events. All simulation programs were written in Fortran 77, compiled and run on PC DOS computers. Our model exhibited responses in agreement with physiological patterns. The values of output frequency of the oscillator system (maximal value of 3.9 Hz) were of the order of magnitude of the firing frequencies recorded in suprachiasmatic neurons of rodents in vivo and in vitro (from 1.8 to 5.4 Hz).

Epigenetic regulation and nucleosome positioning in the human TATA-less IL-1 alpha promoter.

Interleukin-1 alpha (IL-1 alpha) is secreted by a variety of cell types and is a major player in immune and inflammatory processes. Genes involved in immunological processes are known to be strictly regulated; however, how epigenetic mechanisms contribute to this regulation in not understood. To gain insight into the epigenetic regulation of the human TATA-less IL-1A gene, we show that active and silent chromatin modifications characterize the regulatory regions of IL-1 alpha in expressing and non-expressing cells, respectively, and that the DNA methylation in the proximal promoter is associated with the expression status of the cells. Interestingly, although nucleosome depletion in active promoters is found in yeast and fly genes, now it has been reported in human promoters. We here show on the level of single DNA molecules that in expressing cells, a nucleosome is absent in about half of the proximal IL-1 alpha promoters. This observation might reflect a more subtle regulation of nucleosome positioning in TATA-less genes or human genes in general.

[Competence approach in teaching-learning process]. / Abordagem por competências no processo ensino-aprendizagem.

The work relates the experience of the teaching-learning process based on the competence approach. The detail of that teaching process reveals the authors concern with the few occurrence of studies about the practical development of the competences theme in the Educational Practice and, point out to the need of more studies production in that dimension.

Abordagem por competências no processo ensino-aprendizagem / Competence approach in teaching-learning process

O trabalho relata a experiência de um processo de ensino-aprendizagem baseado na abordagem por competências. O detalhamento desse processo de ensino revela a preocupação dos autores com a pouca ocorrência de estudos sobre o desenvolvimento prático do tema competências no âmbito das práticas educativas e, apontam para a necessidade de produção de mais estudos nessa dimensão.

Quenching of the intrinsic fluorescence of bovine serum albumin by chlorpromazine and hemin.

The binding of chlorpromazine (CPZ) and hemin to bovine serum albumin was studied by the fluorescence quenching technique. CPZ is a widely used anti-psychotic drug that interacts with blood components, influences bioavailability, and affects function of several biomolecules. Hemin is an important ferric residue of hemoglobin that binds within the hydrophobic region of albumin with high specificity. Quenching of the intrinsic fluorescence of bovine serum albumin (BSA) was observed by selectively exciting tryptophan residues at 290 nm. Emission spectra were recorded in the range from 300 to 450 nm for each quencher addition. Stern-Volmer graphs were plotted, and the quenching constant estimated for BSA solution titrated with hemin at 25 masculine C was 1.44 (+/- 0.05) x 10(5) M(-1). Results showed that bovine albumin tryptophans are not equally accessible to CPZ, in agreement with the idea that polar or charged quenchers have more affinity for amino acid residues on the outer wall of the protein. Hemin added to albumin solution at a molar ratio of 1:1 quenched about 25% of their fluorescence. The quenching effect of CPZ on albumin-hemin solution was stronger than on pure BSA. This increase can be the result of combined conformational changes in the structure of albumin caused firstly by hemin and then by CPZ. Our results suggest that the primary binding site for hemin on bovine albumin may be located asymmetrically between the two tryptophans along the sequence formed by subdomains IB and IIA, closer to tryptophan residue 212.

Quenching of the intrinsic fluorescence of bovine serum albumin by chlorpromazine and hemin

The binding of chlorpromazine (CPZ) and hemin to bovine serum albumin was studied by the fluorescence quenching technique. CPZ is a widely used anti-psychotic drug that interacts with blood components, influences bioavailability, and affects function of several biomolecules. Hemin is an important ferric residue of hemoglobin that binds within the hydrophobic region of albumin with high specificity. Quenching of the intrinsic fluorescence of bovine serum albumin (BSA) was observed by selectively exciting tryptophan residues at 290 nm. Emission spectra were recorded in the range from 300 to 450 nm for each quencher addition. Stern-Volmer graphs were plotted, and the quenching constant estimated for BSA solution titrated with hemin at 25ºC was 1.44 (± 0.05) x 10(5) M-1. Results showed that bovine albumin tryptophans are not equally accessible to CPZ, in agreement with the idea that polar or charged quenchers have more affinity for amino acid residues on the outer wall of the protein. Hemin added to albumin solution at a molar ratio of 1:1 quenched about 25 percent of their fluorescence. The quenching effect of CPZ on albumin-hemin solution was stronger than on pure BSA. This increase can be the result of combined conformational changes in the structure of albumin caused firstly by hemin and then by CPZ. Our results suggest that the primary binding site for hemin on bovine albumin may be located asymmetrically between the two tryptophans along the sequence formed by subdomains IB and IIA, closer to tryptophan residue 212.

Morphometrical and stereological analysis of the superior cervical ganglion of rattus norvegicus submitted to chronic treatment with cortisol

The present work investigates the corticoid action on the growth of the superior cervical ganglion of the rat and describes the cortisol effect during early stages of development. The study was based on morphometric and stereological analysis of the perikarya. Eight rats were treated intraperitoneally with cortisol (1mg/Kg/day) during 36 days. Treatment was initiated in the 8th day after birth and was withdrawn one day before the sacrifice. There was a significant difference (úP0,05) for the neural mean diameter between the control group (16.78 ± 1.11mm) and treated animals (15.84 ± 0.99mm). The decrease of perikarya neuronal diameter was also demonstrated by stereological methods. Morphometrical findings may suggest alterations in superior cervical ganglion neuronal activity in rats treated for long term with cortisol.

New synthetic approaches to polycyclic aromatic hydrocarbons and their carcinogenic oxidized metabolites: derivatives of benzo[s]picene, benzo[rst]pentaphene, and dibenzo[b,def]chrysene.

A new synthetic approach to polycyclic aromatic compounds is described that entails in the key steps double Suzuki coupling of PAH bisboronic acid derivatives with o-bromoaryl aldehydes to furnish aryl dialdehydes that are converted to larger polycyclic aromatic ring systems by either (a) conversion to diolefins by Wittig reaction followed by photocyclization or (b) reductive cyclization with triflic acid and 1,3-propanediol. This synthetic method provides convenient access to as many as three different polycyclic aromatic ring systems from a single Suzuki coupled intermediate. It was utilized to synthesize substituted derivatives of benzo[s]picene, benzo[rst]pentaphene, dibenzo[b,def]chrysene, and 13,14-dihydro-benz[g]indeno[2,1-a]fluorene, as well as the putative carcinogenic bisdihydrodiol metabolites of benzo[s]picene, benzo[rst]pentaphene, and dibenzo[b,def]chrysene.

Erratic oscillatory characteristics of plasma insulin concentrations in patients with insulinoma: mechanism for unpredictable hypoglycemia.

Patients with insulin-producing tumors may have hypoglycemic symptoms at unpredictable times. This study evaluated whether plasma insulin oscillations, known to occur in normal individuals but not explored in patients with insulinomas, could be an underlying mechanism for such events. Nine normal subjects and five patients with proven insulinomas were studied in the fasting state. Serial sampling of arterialized blood over 80-100 min, at 2- or 3-min intervals was performed. In normal subjects, mean plasma glucose and insulin concentrations were 5.3 +/- 0.1 mmol/L and 58 +/- 9 pmol/L, respectively. Regular, low-amplitude plasma insulin oscillations were observed, with a period of 10-17 min. The subjects with insulinomas had lower mean plasma glucose and higher insulin concentrations than controls, 3.6 +/- 0.3 mmol/L (P = 0.01) and 150 +/- 42 pmol/L (P = 0.01), respectively. They also had insulin oscillations that appeared unstable as a result of variability in duration and amplitude compared with controls. The insulin pulses were irregular, and interpeak intervals varied between 4-54 min in different subjects; in some subjects, the amplitude was also variable, with sudden spontaneous pulses as high as 565 pmol/L, with an associated glucose decrement. We conclude that large spontaneous bursts of insulin secretion occur in patients with insulinomas as part of an erratic pattern of oscillatory insulin secretion, and these can account for unpredictable occurrences of hypoglycemia.

An overnight insulin infusion algorithm provides morning normoglycemia and can be used to predict insulin requirements in noninsulin-dependent diabetes mellitus.

Initial insulin requirements in noninsulin-dependent diabetes mellitus (NIDDM) are difficult to estimate because of individual variability in insulin sensitivity and secretion. We evaluated a simple, nurse-managed algorithm for overnight delivery of insulin, for its ability to provide morning near-normoglycemia and as a means to predict initial insulin requirements in NIDDM. Twenty-seven patients with poorly controlled NIDDM were studied on 30 occasions. A 12-h iv insulin infusion was begun at 2000 h, and bedside blood glucose concentrations were measured at hourly intervals. The rate of insulin infusion was adjusted according to blood glucose levels. We estimated the preprandial insulin dose requirement for the following day in 16 patients based on overnight insulin requirements to maintain normoglycemia. Preprandial insulin doses were adjusted for prevailing blood glucose concentrations. At 2000 h, the mean (+/-SEM) blood glucose concentration was 265.7 +/- 10.8; at 0300 h, it was 122.8 +/- 3.4; and at 0700 h, it was 123.8 +/- 5.1 mg/dL. On the next day, mean blood glucose levels (before and 2 h after a meal) were: breakfast, 102.5 +/- 5.9 and 177.3 +/- 19.2; lunch, 138.9 +/- 15.5 and 136.3 +/- 11.4; dinner, 105.7 +/- 7.2 and 178.1 +/- 15.7 mg/dL. There was no significant difference between mean calculated and administered total insulin dosage the next day (84.2 +/- 7.0 vs. 78.2 +/- 8.2 U). Thus, a weight-based algorithm for iv insulin infusion induced near-normoglycemia in NIDDM and successfully predicted the insulin dose requirement. We conclude that initiating insulin therapy in NIDDM patients can be achieved rapidly and efficiently based on a nurse-managed overnight insulin infusion.

Analysis of 7-methylbenz[a]anthracene-DNA adducts formed in SENCAR mouse epidermis by 32P-postlabeling.

The present study has analysed the DNA adducts formed in SENCAR mouse epidermis following topical application of 7-methylbenz[a]anthracene (7-MBA). Mice were treated with 400 nmol of 7-MBA, which represents an initiating dose of this hydrocarbon for SENCAR mice. DNA adducts were analysed 24 h after topical application of the hydrocarbon by 32P-postlabeling coupled with either HPLC analysis or an improved TLC procedure giving better resolution of DNA adducts through the use of a D6 solvent [isopropanol:4N NH4OH (1:1)] following D5. Twenty-four hours after topical application of 400 nmol 7-MBA, the level of total covalent binding was 0.37 +/- 0.07 pmol/mg DNA as determined by 32P-postlabeling. This level of binding correlated well with the relative tumor initiating activity of this hydrocarbon compared to 7,12-dimethylbenz[a]anthracene (6.4 +/- 0.01 pmol/mg DNA) and dibenz[a,j]anthracene (0.03 +/- 0.01 pmol/mg DNA). Analysis of the 32P-labeled 3',5'-diphosphodeoxyribonucleosides by HPLC and TLC revealed the presence of deoxyguanosine (dGuo) and deoxyadenosine (dAdo) adducts formed from both the anti- and syn-bay-region diol-epoxides of 7-MBA (anti- and syn-7-MBADEs). The major DNA adduct derived from 7-MBA in mouse epidermis was tentatively identified as (+) anti-7-MBADE-trans-N2-dGuo. In addition, a minor dGuo adduct derived from the bay-region syn-diol-epoxide of 7-MBA was detected as well as a minor dAdo adduct from this diol-epoxide. Another minor dAdo adduct was also detectably present which arose from either the anti- or syn-diol epoxide. Furthermore, several unidentified DNA adducts were present in both HPLC and TLC chromatograms of DNA samples from 7-MBA-treated mice. These results are discussed in terms of the role of specific 7-MBA-DNA adducts in tumor initiation by this hydrocarbon.

The effect of fluoro substituents on reactivity of 7-methylbenz[a]anthracene diol epoxides.

The present study has examined potential mechanisms for the influence of F-substituents on the biologic activity of methylbenz[a]anthracenes. DNA adducts derived from reaction of the racemic bay-region anti-diol epoxides of 7-methylbenz[a]anthracene, and its 9- and 10- fluoro derivatives, with calf thymus DNA in vitro were partially characterized. All three hydrocarbon diol epoxides produced similar DNA adduct profiles upon reaction with calf thymus DNA in vitro that were composed of two deoxyganosine and two deoxyadenosine adducts (tentatively identified as trans addition products). The extent of covalent binding to calf thymus DNA, as estimated by 32P-postlabeling, was similar for all three diol epoxides. The reactivity of the unsubstituted and 10-F-substituted diol epoxide was further assessed by measuring overall pseudo-first-order rate constants for hydrolysis in water or 0.1 M Tris-HCl buffer, pH 7.0, and in the presence or absence of native or denatured DNA. The rate constant for hydrolysis of 7-methylbenz[a]anthracene diol epoxide in the absence of DNA was similar to that of 10-F-7-methylbenz[a]anthracene diol epoxide (t1/2 = 138 min vs 115 min in water, respectively, and 93 vs 83 min in 0.1 M Tris-HCl buffer, respectively). In addition, the presence of DNA accelerated hydrolysis rates to similar extents for both diol expoxides. The skin tumor-initiating activities of the 9- and 10-F-substituted 3,4-diols of 7-methyl-, 12-methyl-, and 7,12-dimethylbenz[a]anthracene were determined in SENCAR mice. The presence of F-substituents in the 9- or 10- position did not enhance or in some cases reduced the tumor-initiating activity of the 3,4-diols of these hydrocarbons. Collectively, these results, as well as previous results from our laboratory, suggest that the influence of a F-substituent at position 10 of the benz[a]anthracene nucleus is not due to increased or altered reactivity of the bay-region diol epoxide but rather likely on the initial formation of the 3,4-diol.

Targeted A --> T and G --> T mutations induced by site-specific deoxyadenosine and deoxyguanosine adducts, respectively, from the (+)-anti-diol epoxide of dibenz[a,j]anthracene in M13mp7L2.

The studies described in this report directly examined the mutagenicity in Escherichia coli of both a deoxyadenosine (dAdo) and a deoxyguanosine (dGuo) adduct derived from (+)-anti-dibenz[a,j]-anthracene-3,4-diol 1,2-epoxide [(+)anti-DB[a,j]A-DE] that were site-specifically placed in a single-stranded M13mp7L2 replication vector. An 11-base oligonucleotide (5'-CTC ACG CTT CT-3') containing either a single (+)anti-DB[a,j]A-DE--trans-N2-dGuo or (+)anti-DB[a,j]A-DE--trans-N6dAdo adduct was successfully incorporated into single-stranded M13mp7L2 plasmid via ligation. In vitro studies using E. coli DNA polymerase I (Klenow fragment)indicated that both adducts were effective blocks for polymerase action. E. coli strains JM103 and JM103 uvrA6 were subsequently transformed with control (unadducted) and adduct-containing M13mp7L2 constructs followed by analysis of progeny DNA. In both JM103 and JM103 uvrA6 cells, plaque yields were markedly reduced with adduct containing vectors compared to control vectors. Activation of the inducible bacterial DNA repair system (SOS) by UV light only slightly increased the number of plaques recovered from either bacterial strain transformed with adduct-containing vectors. Targeted mutations were obtained with both adduct-containing vectors in both bacterial strains, whereas no mutations were detected in plaques recovered from control M13mp7L2 vectors. In JM103 cells, (+)anti-DB[a,j]A-DE--N6-dAdo induced exclusively A --> t transversions and (+)anti-DB[a,j]A-DE--N2-dGuo induced exclusively G --> T transversions. In JM103 uvrA6 cells, similar targeted transversion mutations were also obtained except that a few C deletions (i.e., aprroximately 10% of the mutations) were detected immediately 3' to the dAdo adduct. While mutagenesis was SOS dependent in JM103 cells [<0.15% (-SOS) vs approximately 1.3% (+SOS)], it appeared to be SOS independent in JM103 uvrA6 cells (approximately 1-2% in the presence or absence of SOS induction). It is argued that adduct-induced G --> T mutations can be rationalized by either misinformational or noninformational mechanisms. In contrast, A --> T mutations are unlikely to arise via a misinformational pathway, which provides the strongest support to date that bulky DNA adducts can induce mutations via a noninformational pathway.