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.     

STS markers linked to the Rf 1 fertility restorer gene of cotton

Marker-assisted selection (MAS) can accelerate the process of plant breeding, and sequence-tagged site (STS) markers are highly specific for regions of DNA being used for MAS. The objective of this research was to develop STS markers tightly linked with Rf₁, the fertility restoring gene for cytoplasmic male sterility (CMS) in cotton (Gossypium hirsutum L.). Bulked segregant analysis was employed to screen for Rf₁-linked RAPD markers in a backcross population. Four RAPD markers were identified, three of which co-segregated with Rf₁ (UBC147₁₄₀₀, UBC607₅₀₀, and UBC679₇₀₀). Another fragment, UBC169₈₀₀, co-segregated with the previously reported UBC169₇₀₀ in repulsion phase at a distance of 4.5 cM from Rf₁. A marker published by others (UBC659₁₅₀₀) mapped to 2.7 cM from Rf₁ and 1.8 cM from UBC169₈₀₀. Four sets of STS primer pairs were designed based on the RAPD fragment sequences. The primer pairs from the UBC147₁₄₀₀ and UBC607₅₀₀ fragments both amplified a single fragment specific to fertile plants. The UBC679₇₀₀ and UBC659₁₅₀₀ STS primer pairs each amplified one fragment specific to fertile plants and a monomorphic fragment. These four Rf₁-linked STS markers were also present in the Rf₁ donor species G. harknessii (D_2-2). The three primer pairs that produced co-segregating STS markers also amplified fragments from G. trilobum (D₈). However, the D₈ fragment amplified by the UBC147₁₄₀₀ STS primers was larger than that from D_2-2, and G. trilobum does not restore fertility to CMS-D2-2 lines. These STS markers will be useful in the development of restorer parental lines in cotton CMS breeding efforts.     

Lysine-spermine conjugates: hydrophobic polyamine amides as potent lipopolysaccharide sequestrants

Lipopolysaccharides (LPS), otherwise termed 'endotoxins', are outer-membrane constituents of Gram-negative bacteria. Lipopolysaccharides play a key role in the pathogenesis of 'Septic Shock', a major cause of mortality in the critically ill patient. Therapeutic options aimed at limiting downstream systemic inflammatory processes by targeting lipopolysaccharide do not exist at the present time. We have defined the pharmacophore necessary for small molecules to specifically bind and neutralize LPS and, using animal models of sepsis, have shown that the sequestration of circulatory LPS by small molecules is a therapeutically viable strategy. In this paper, the interactions of a focused library of lysine-spermine conjugates with lipopolysaccharide (LPS) have been characterized. Lysine-spermine conjugates with the epsilon-amino terminus of the lysinyl moiety derivatized with long-chain aliphatic hydrophobic substituents in acyl or alkyl linkage bind and neutralize bacterial lipopolysaccharides, and may be of use in the prevention or treatment of endotoxic shock states.     

Synthesis and activity of N-acyl azacyclic urea HIV-1 protease inhibitors with high potency against multiple drug resistant viral strains

As part of our efforts to identify potent HIV-1 protease inhibitors that are active against resistant viral strains, structural modification of the azacyclic urea (I) was undertaken by incorporating acyl groups as P₁′ ligands. The extensive SAR study has yielded a series of N-acyl azacyclic ureas (II), which are highly potent against both wild-type and multiple PI-resistant viral strains.     

Specific sensing between inositol epimers by a bis(boronate)

Bis(boronates) that utilize internal photoinduced electron transfer (PET) quenching mechanisms can specifically signal the binding of chiro-inositol without responding to its epimer, myo-inositol.     

The burgeoning molecular genetics of the Lyme disease spirochaete

Lyme disease is the most commonly reported vector-borne disease in North America and Europe, yet we know little about which components of the causative agent, Borrelia burgdorferi, are critical for infection or virulence. Molecular genetics has provided a powerful means by which to address these topics in other bacterial pathogens. Certain features of B. burgdorferi have hampered the development of an effective system of genetic analysis, but basic tools are now available and their application has begun to provide information about the identities and roles of key bacterial components in both the tick vector and the mammalian host. Increased genetic analysis of B. burgdorferi should advance our understanding of the infectious cycle and the pathogenesis of Lyme disease.     

A primer-dependent polymerase function of pseudomonas aeruginosa ATP-dependent DNA ligase (LigD)

Pseudomonas aeruginosa encodes two putative DNA ligases: a classical NAD(+)-dependent DNA ligase (LigA) plus an ATP-dependent DNA ligase (LigD). LigD exemplifies a family of bacterial proteins that consist of a ligase domain fused to flanking domains that resemble nucleases and/or polymerases. Here we purify LigD and show that it possesses an intrinsic polymerase function resident within an autonomous C-terminal polymerase domain, LigD-(533-840), that flanks an autonomous DNA ligase domain, LigD-(188-527). Native LigD and the polymerase domain are both monomeric proteins. The polymerase activity is manifest in three ways: (i) non-templated nucleotide addition to a blunt-ended duplex DNA primer; (ii) non-templated addition to a single-stranded DNA primer; and (iii) templated extension of a 5'-tailed duplex DNA primer-template. The divalent cation cofactor requirement for non-templated and templated polymerase activity is satisfied by manganese or cobalt. rNTPs are preferred over dNTPs as substrates for non-templated blunt-end addition, which typically entails the incorporation of only 1 or 2 nucleotides at the primer terminus. Templated dNMP addition to a 5'-tailed substrate is efficient with respect to dNTP utilization; the primer is elongated to the end of the template strand and is then further extended with a non-templated nucleotide. The polymerase activity is abolished by alanine substitution for two aspartates (Asp-669 and Asp-671) within the putative metal-binding site. We speculate that polymerase activity is relevant to LigD function in nonhomologous end-joining.     

The role of endothelin-1 in myocarditis and inflammatory cardiomyopathy: old lessons and new insights

Endothelin-1 has emerged as an important participant in the pathophysiology of a variety of cardiovascular diseases, where it may act on endocrine, paracrine and autocrine bases. Here we review its regulated biosynthesis, receptor-mediated signaling, and functional consequences in the heart, with particular emphasis on cardiac development and disease. Exploring published data employing molecular genetic mouse models of endothelin dysregulation, we highlight its heretofore underappreciated role as a pro-inflammatory cytokine. We also present novel micro-array data from one such mouse model, which implicate the specific downstream pathways that may mediate endothelin-1's effects.