A Critical Evaluation of Interlaboratory Data on Total, Elemental, and Isotopic Carbon in the Carbonaceous Particle Reference Material, NIST SRM 1649a.

Because of increased interest in the marine and atmospheric sciences in elemental carbon (EC), or black carbon (BC) or soot carbon (SC), and because of the difficulties in analyzing or even defining this pervasive component of particulate carbon, it has become quite important to have appropriate reference materials for intercomparison and quality control. The NIST "urban dust" Standard Reference Material(®) SRM 1649a is useful in this respect, in part because it comprises a considerable array of inorganic and organic species, and because it exhibits a large degree of ((14)C) isotopic heterogeneity, with biomass carbon source contributions ranging from about 2 % (essentially fossil aliphatic fraction) to about 32 % (polar fraction). A primary purpose of this report is to provide documentation for the new isotopic and chemical particulate carbon data for the most recent (31 Jan. 2001) SRM 1649a Certificate of Analysis. Supporting this is a critical review of underlying international intercomparison data and methodologies, provided by 18 teams of analytical experts from 11 institutions. Key results of the intercomparison are: (1) a new, Certified Value for total carbon (TC) in SRM 1649a; (2) (14)C Reference Values for total carbon and a number of organic species, including for the first time 8 individual PAHs; and (3) elemental carbon (EC) Information Values derived from 13 analytical methods applied to this component. Results for elemental carbon, which comprised a special focus of the intercomparison, were quite diverse, reflecting the confounding of methodological-matrix artifacts, and methods that tended to probe more or less refractory regions of this universal, but ill-defined product of incomplete combustion. Availability of both chemical and (14)C speciation data for SRM 1649a holds great promise for improved analytical insight through comparative analysis (e.g., fossil/biomass partition in EC compared to PAH), and through application of the principle of isotopic mass balance.

Trioxane dimers have potent antimalarial, antiproliferative and antitumor activities in vitro.

A series of tetracyclic and tricyclic trioxane dimers has been prepared with ether and ester tethers of varying length and flexibility. Several of these trioxane dimers have been found to have potent and potentially therapeutically valuable antimalarial, antiproliferative, and antitumor activities in vitro.

Helicase-primase complex of herpes simplex virus type 1: a mutation in the UL52 subunit abolishes primase activity.

The UL52 gene product of herpes simplex virus type 1 (HSV-1) comprises one subunit of a 3-protein helicase-primase complex that is essential for replication of viral DNA. The functions of the individual subunits of the complex are not known with certainty, although it is clear that the UL8 subunit is not required for either helicase or primase activity. Examination of the predicted amino acid sequence of the UL5 gene reveals the existence of conserved helicase motifs; it seems likely, therefore, that UL5 is responsible for the helicase activity of the complex. We have undertaken mutational analysis of UL52 in an attempt to understand the functional contribution of this protein to the helicase-primase complex. Amino acid substitution mutations were introduced into five regions of the UL52 gene that are highly conserved among HSV-1 and the related herpesviruses equine herpesvirus 1, human cytomegalovirus, Epstein-Barr virus, and varicella-zoster virus. Of seven mutants analyzed by an in vivo replication assay, three mutants, in three different conserved regions of the protein, failed to support DNA replication. Within one of the conserved regions is a 6-amino-acid motif (IL)(VIM)(LF)DhD (where h is a hydrophobic residue), which is also conserved in mouse, yeast, and T7 primases. Mutagenesis of the first aspartate residue of the motif, located at position 628 of the UL52 protein, abolished the ability of the complex to support replication of an origin-containing plasmid in vivo and to synthesize oligoribonucleotide primers in vitro. The ATPase and helicase activities were unaffected, as was the ability of the mutant enzyme to support displacement synthesis on a preformed fork substrate. These results provide experimental support for the idea that UL52 is responsible for the primase activity of the HSV helicase-primase complex.

Stunned myocardium following prolonged cardiopulmonary bypass: effect of warm versus cold cardioplegia in the canine model.

"Stunned myocardium" is defined as the prolonged but transient postischemic contractile dysfunction of viable myocardium that has been salvaged by reperfusion. This phenomenon, although first characterized in the experimental canine model of coronary artery occlusion/reperfusion, also occurs following transient global ischemia. Moreover, despite the superb cardioprotection conferred by administration of cold cardioplegia during aortic cross-clamping, stunned myocardium is a well-recognized sequela of prolonged cardiopulmonary bypass. Using the anesthetized open chest dog, we tested the concept that continuous retrograde infusion of warm blood cardioplegia would effectively prevent ischemia during prolonged aortic cross-clamping and thereby preclude the development of stunned myocardium following bypass. Thirteen dogs were placed on cardiopulmonary bypass and randomized to receive: (1) continuous retrograde administration of warm blood cardioplegia (n = 8); or (2) intermittent retrograde cold blood cardioplegia (n = 5) during a 3-hour cross-clamp period. Left ventricular (LV) systolic function (i.e., area LV ejection fraction and posterior LV free wall thickening assessed by two-dimensional echocardiography) and hemodynamic parameters were monitored at baseline and at 1 and 2 hours postbypass and, at the end of the protocol, transmural myocardial biopsies were obtained for electron microscopic analysis. All dogs in both treatment groups showed electron microscopic evidence of mild and reversible morphological injury indicative of stunned myocardium, with no difference between dogs that received warm versus cold cardioplegia. Direct comparison of LV function between the two groups was confounded by a profound decrease in afterload in dogs that received cold cardioplegia. However, incorporation of systemic vascular resistance as a covariate revealed that LV function following bypass was modestly depressed at approximately 85% of baseline values, and that continuous administration of warm cardioplegia did not prevent this hypokinesis. Thus, in our canine model: (1) morphological injury and LV dysfunction induced by 3 hours of aortic cross-clamping is subtle; and (2) continuous retrograde infusion of warm blood cardioplegia during the cross-clamp period failed to preclude myocardial stunning following prolonged cardiopulmonary bypass.

Mutagenesis by apurinic sites in normal and ataxia telangiectasia human lymphoblastoid cells.

We used a shuttle vector based on the Epstein-Barr virus origin of plasmid replication (oriP) to determine the types of mutations induced by depurination in human cells. Plasmid DNA was incubated at pH 2 at 40 degrees C for various times to induce up to 20 apurinic (AP) sites per 9.7-kb plasmid and electroporated into lymphoblastoid cells derived from either a normal individual or an ataxia telangiectasia patient. After replication of the vector in the human cells, plasmid DNA was isolated and analyzed for mutations induced in the plasmid-encoded herpes simplex virus type 1-thymidine kinase gene. Both the frequencies and types of mutations induced by depurination were essentially identical for normal and ataxia telangiectasia cells. The mutant frequency at 20 AP sites/plasmid was 10-fold to 13-fold greater than that observed for untreated DNA. Deletion and frameshift events accounted for 46-55% of the mutants induced by depurination. The induced deletions were relatively small (median size, 100-150 bp) and characterized by short (1-5 bp) regions of sequence homology at the endpoints. These mutations and the frameshifts, a majority of which occurred in runs of identical nucleotides, are consistent with a model involving AP-site-induced template dislocation during DNA synthesis. A broad spectrum of base-substitution mutations, which accounted for 19-36% of the induced mutants, was observed. The apparent preference for insertion opposite AP sites in human cells was G (43-55%) greater than A approximately C (18-21%) greater than T (9-14%). Our results in human cells contrast markedly with those published previously for the mutational specificity of AP sites in Escherichia coli, in which a large majority of the mutants resulted from insertion of an A opposite the abasic site.

Reduction to homozygosity is the predominant spontaneous mutational event in cultured human lymphoblastoid cells.

Expression of recessive mutant phenotypes can occur by a number of different mechanisms. Inactivation of the wild-type allele by base-substitution mutations, frameshift mutations or small deletions occurs at both hemizygous and heterozygous cellular loci, while other events, such as chromosome level rearrangements, may not be detected at hemizygous loci because of inviability of the resulting mutants. In order to assess the relative contribution of each type of mutational event, we isolated a human lymphoblastoid cell line that is heterozygous at the adenine phosphoribosyltransferase (aprt) locus. The mutation rate for the expression of the mutant phenotype (aprt(+/-)----aprt-/-) was 1.3 x 10(-5)/cell/generation. Molecular analysis of the DNA from 26 mutant clones revealed that 19% had undergone deletion of the entire wild-type allele. The aprt heterozygote carries a mutation in the coding sequence of the gene that results in the loss of a restriction site. Analysis of aprt-/- mutants for this restriction fragment length difference revealed that 23% of the mutants contained point mutations or small (less than 100 bp) deletions. The remainder of the mutants (58%) resulted from reduction to homozygosity of the mutant allele. We suggest that, as in tumor cells in vivo, reduction to homozygosity is a major mechanism for the expression of recessive mutant phenotypes in cultured human cells.

Molecular analysis of mutations induced in human cells by N-ethyl-N-nitrosourea.

Mutational activation of cellular proto-oncogenes is an important event in the pathogenesis of chemically induced tumors. We have used the ori P-tk shuttle vector, pHET, to analyze the types of DNA sequence changes induced after treating mammalian cells with the carcinogen N-ethyl-N-nitrosourea (ENU). This shuttle vector contains the putative replication origin of the Epstein-Barr virus (EBV) and is stably maintained as a plasmid in EBV-transformed human lymphoblastoid cells. Populations of plasmid-bearing cells were treated with ENU, and plasmid DNA was isolated approximately 7-8 population doublings after treatment for analysis of mutations induced at the herpes simplex virus type 1 thymidine kinase (HSV-tk) target gene. After ENU treatment, frequencies of four of the six possible base substitution mutations significantly increased. Transition mutations were the most common sequence change: 48% of the 46 mutants sequenced were GC----AT transitions and 17% were AT----GC transitions. In addition, the number of AT----TA (20%) and AT----CG (9%) transversion mutations significantly increased after ENU treatment. Based on the comparison of mutations induced by ENU in human cells with the types of base pair changes previously reported for other alkylating agents, we propose that the O2-ethylthymine adduct may be a significant premutagenic lesion in mammalian cells, capable of resulting in AT base pair transversion mutations. Studies from other laboratories have demonstrated the importance of AT----TA transversion mutations in the activation of cellular proto-oncogenes by ENU.

Chemically induced mutagenesis in a shuttle vector with a low-background mutant frequency.

We have developed a recombinant DNA shuttle vector that permits the molecular analysis of mutations induced in human cells by chemical or physical mutagens. The vector is able to replicate as a plasmid in Escherichia coli and in Epstein-Barr virus (EBV)-transformed human lymphoblastoid cell lines and contains the herpes simplex virus type 1 thymidine kinase gene (HSV tk) as the target for mutagenesis studies. After introduction of the vector into an EBV-transformed lymphoblastoid cell line (LCL-721) by electroporation, approximately equal to 2% of the transfected cells expressed the vector-encoded gene for hygromycin resistance. Plasmid DNA isolated from cells immediately after selection for hygromycin resistance (10 population doublings posttransfection) contained mutations in the HSV tk gene at a frequency of 6 X 10(-5). Treatment of plasmid-bearing LCL-721 cells with N-ethyl-N-nitrosourea resulted in a dose-dependent increase of up to 15-fold in the frequency of mutations in the HSV tk gene. The dose-response for the induction of mutations in the plasmid-encoded gene closely paralleled that for the induction of mutations in the cellular gene for hypoxanthine (guanine) phosphoribosyltransferase.