hSSB1 (human single strand DNA binding protein 1) has been shown to participate in homologous recombination (HR)-dependent repair of DNA double strand breaks (DSBs) and ATM-mediated checkpoint pathways. Here we present evidence that hSSB2, a homolog of hSSB1, plays a role similar to hSSB1 in DNA damage response pathways. This was evidenced by findings that hSSB2-depleted cells resemble hSSB1-depleted cells in hypersensitivity to DNA damaging reagents, reduced efficiency in HR-dependent repair of DSBs, and defective ATM-dependent phosphorylation. Notably, hSSB1 and hSSB2 form separate complexes with two identical proteins, INTS3 and hSSBIP1 (C9ORF80). Cells depleted of INTS3 and hSSBIP1 also exhibited hypersensitivity to DNA damage reagents, chromosomal instability, and reduced ATM-dependent phosphorylation. hSSBIP1 was rapidly recruited to laser-induced DSBs, a feature also similar to that reported for hSSB1. Depletion of INTS3 decreased the stability of hSSB1 and hSSBIP1, suggesting that INTS3 may provide a scaffold to allow proper assembly of the hSSB complexes. Thus, our data demonstrate that hSSB1 and hSSB2 form two separate complexes with similar structures, and both are required for efficient HR-dependent repair of DSBs and ATM-dependent signaling pathways.

DNA interstrand cross-links have important biological consequences and are useful biotechnology tools. Phenylselenyl substituted derivatives of thymidine ( 1) and 5-methyl-2'-deoxycytidine ( 5) produce interstrand cross-links in duplex DNA when oxidized by NaIO 4. The mechanism involves a [2,3]-sigmatropic rearrangement of the respective selenoxides to the corresponding methide type intermediates, which ultimately produce the interstrand cross-links. Determination of the rate constants for the selenoxide rearrangements indicates that the rate-determining step for cross-linking is after methide formation. Cross-linking by the thymidine derivative in duplex DNA shows a modest kinetic preference when flanked by pyrimidines as opposed to purines. In contrast, the rate constant for cross-link formation from 5 opposite dG in duplex DNA is strongly dependent upon the flanking sequence and, in general, is at least an order of magnitude slower than that for 1 in an otherwise identical sequence. Introduction of mispairs at the base pairs flanking 5 or substitution of the opposing dG by dI significantly increases the rate constant and yield for cross-linking, indicating that stronger hydrogen bonding between the methide derived from it and dG compared to dA and the respective electrophile derived from 1 limits reaction by increasing the barrier to rotation into the required syn-conformation. Incorporation of 1 or 5 in triplex forming oligonucleotides (TFOs) that utilize Hoogsteen base pairing also yields interstrand cross-links. The dC derivative produces ICLs approximately 10x faster than the thymidine derivative when incorporated at the 5'-termini of the TFOs and higher yields when incorporated at internal sites. The slower, less efficient ICL formation emanating from 1 is attributed to reaction at N1-dA, which requires local melting of the duplex. In contrast, 5 produces cross-links by reacting with N7-dG. The cross-linking reactions of 1 and 5 illustrate the versatility and utility of these molecules as mechanistic probes and tools for biotechnology.

Novel mycophenolic adenine dinucleotide (MAD) analogues have been prepared as potential inhibitors of inosine monophosphate dehydrogenase (IMPDH). MAD analogues resemble nicotinamide adenine dinucleotide binding at the cofactor binding domain of IMPDH; however, they cannot participate in hydride transfer and therefore inhibit the enzyme. The methylenebis(phosphonate) analogues C2-MAD and C4-MAD were obtained by coupling 2',3'-O-isopropylideneadenosine 5'-methylenebis(phosphonate)(22) with mycophenolic alcohols 20 and 21 in the presence of diisopropylcarbodiimide followed by deprotection. C2-MAD was also prepared by coupling of mycophenolic methylenebis(phosphonate) derivative 30 with 2',3'-O-isopropylideneadenosine. Compound 30 was conveniently synthesized by the treatment of benzyl-protected mycophenolic alcohol 27 with a commercially available methylenebis(phosphonic dichloride) under Yoshikawa's reaction conditions. C2-MAD and C4-MAD were found to inhibit the growth of K562 cells (IC(50)= 0.7 microM and IC(50)= 0.1 microM, respectively) as potently as mycophenolic acid (IC(50)= 0.3 microM). In addition, C2-MAD and C4-MAD triggered vigorous differentiation of K562 cells an order of magnitude more potently than tiazofurin, and MAD analogues were resistant to glucuronidation in vitro. These results show that C2-MAD and C4-MAD may be of therapeutic interest in the treatment of human leukemias.

Tinnitus is the perception of sound in the absence of an apparent acoustic stimulus. More than 35 million Americans experience tinnitus, with 2-3 million severely debilitated by this distressing symptom. The prevalence increases with age and there is a high incidence associated with both noise-induced and age-related hearing loss. Although there are several theories regarding the pathophysiology of tinnitus, the precise mechanism remains to be elucidated. The most compelling of these is the hypothesis that tinnitus occurs as a result of spontaneous and aberrant neural activity at any level along the auditory axis, even after cochlear nerve transection or labyrinthine ablation.There are numerous aetiologies associated with tinnitus. Tinnitus, in clinical practice, is characterised as either objective or subjective. The distinction is relevant in terms of both aetiology and treatment. Despite a large number of therapeutic interventions and studies claiming success in treating tinnitus, a cure remains elusive. However, there are several potential treatment options that offer patients varying degrees of symptomatic improvement and enhanced quality of life.It is imperative to formulate a rational and systematic approach in evaluating an older adult with tinnitus. An individualised treatment regimen and the creation of a strong therapeutic relationship are the hallmarks of successful management of the patient with tinnitus.

OBJECTIVES: The study goals were to evaluate the safety and effectiveness of continuous gentamicin therapy (CGT) in the treatment of Meniere's disease and to evaluate the effect of flow rate. STUDY DESIGN: In a retrospective study, physicians who were known to have used CGT in the treatment of Meniere's disease were asked to report patient information on a standardized data collection form. RESULTS: In patients treated with low-flow CGT, vertigo was eliminated or substantially controlled in 90%, tinnitus was improved in 69%, pressure was improved in 77%, hearing was worse in 23%, and vestibular function was ablated in only 14% of patients. CONCLUSION: On the basis of an average 10-month follow-up period, of the methods currently available to treat Meniere's patients with gentamicin, low-flow CGT seems to provide an excellent combination of vertigo control and tinnitus and pressure improvement, with a relatively low risk to hearing and vestibular function. SIGNIFICANCE: The clinical use of low-flow CGT appears to be justified based on these data.

OBJECTIVES/HYPOTHESIS:: The purpose of the study was to determine the effect of electrical stimulation of the auditory cortex in patients with tinnitus. STUDY DESIGN:: Nonrandomized clinical trial. METHODS:: Two patients with debilitating tinnitus refractory to conventional therapies were treated. Patients were evaluated with validated questionnaires and psychoacoustic measures to determine the frequency and pitch of their tinnitus. Tones at these frequencies were then presented to the first patient (RP) under magnetoencephalography (MEG) and functional magnetic resonance imaging (fMRI) to determine the tonotopic map for these frequencies in Heschl's gyrus. These tonotopic sites were targeted for implant with a quadripolar electrode. In the second patient (MV), only the fMRI tonotopic map was performed. These fMRI results detected an area of increased activity, which was selected as the site for the implanted bipolar electrode. RESULTS:: Patient RP (bilateral tinnitus for 2 years) has experienced a sustained reduction to near elimination of tinnitus with intracerebral implanted electrodes, whereas patient MV (unilateral tinnitus for 7 years) had an unsustained reduction in her tinnitus. CONCLUSION:: These findings suggest that the perception and annoyance of tinnitus may be modulated or reduced through electrical stimulation of the auditory cortex. These unsustained effects for patient MV may have been influenced by the longstanding nature of her tinnitus or by another reason as yet undetermined.

We are developing triple helix forming oligonucleotides (TFOs) for gene targeting. Previously, we synthesized bioactive TFOs containing 2'-O-methylribose (2'-OMe) and 2'-O-aminoethylribose (2'-AE) residues. Active TFOs contained four contiguous 2'-AE residues and formed triplexes with high thermal stability and rapid association kinetics. In an effort to further improve bioactivity, we synthesized three series of TFOs containing the 2'-AE patch and additional ribose modifications distributed throughout the remainder of the oligonucleotide. These were either additional 2'-AE residues, the conformationally locked BNA/LNA ribose with a 2'-O,4'-C-methylene bridge, or the 2'-O,4'-C-ethylene analogue (ENA). The additionally modified TFOs formed triplexes with greater thermal stability than the reference TFO, and some had improved association kinetics. However, the most active TFOs in the biochemical and biophysical assays were the least active in the bioassay. We measured the thermal stability of triplexes formed by the TFOs in each series on duplex targets containing a change in sequence at a single position. The Tm value of the variant sequence triplexes increased as the number of all additional modifications increased. A simple explanation for the failure of the improved TFOs in the bioassay was that the increased affinity for nonspecific targets lowered the effective nuclear concentration. Enhancement of TFO bioactivity will require chemical modifications that improve interaction with the specific targets while retaining selectivity against mismatched sequences.

Triple helix forming oligonucleotides (TFOs) may have utility as gene targeting reagents for "in situ" gene therapy of genetic disorders. Triplex formation is challenged by negative charge repulsion between third strand and duplex phosphates, and destabilizing positive charge repulsion between adjacent protonated cytosines within pyrimidine motif third strands. Here we describe the synthesis of TFOs designed to target a site in the human beta-globin gene, which is the locus for mutations that underlie the beta-globinopathies, including sickle cell anemia. The target is an uninterrupted polypurine:polypyrimidine sequence, containing four adjacent cytosines, next to a psoralen cross-link site. Pyrimidine motif TFOs that contained four adjacent cytosines or 5-methylcytosines did not form stable triplexes at physiological pH, despite the introduction of otherwise stabilizing base and sugar analogues. We synthesized a series of pso-TFOs containing 2'-O-methyl (OMe) and 2'-O-aminoethoxy substitutions (AE), as well as 8-oxo-adenine (A(8)) and 2'-O-methylpseudoisocytidine (P) as neutral cytosine replacements. Thermal stability measurements indicated that TFOs with A(8) did not meet criteria established in previous work. However, TFOs with P did form triplexes with appropriate T(m)() and k(ON) values. A pso-TFO with AE and P residues was sufficiently active to permit the determination of targeting in living cells by direct measurement of cross-link formation at the target site. Our results validate the modification format described in our previous studies and indicate that P substitutions are an effective solution to the problem of targeting genomic sequences containing adjacent cytosines.

Alternate DNA structures other than double-stranded B-form DNA can potentially impede cellular processes such as transcription and replication. The DNA triplex helix and G4 tetraplex structures that form by Hoogsteen hydrogen bonding are two examples of alternate DNA structures that can be a source of genomic instability. In this study, we have examined the ability of human replication protein A (RPA), a single-stranded DNA binding protein that is implicated in all facets of DNA metabolism, to destabilize DNA triplexes and tetraplexes. Biochemical studies demonstrate that RPA efficiently melts an intermolecular DNA triple helix consisting of a pyrimidine motif third strand annealed to a 4 kb duplex DNA fragment at protein concentrations equimolar to the triplex substrate. Heterologous single-stranded DNA binding proteins ( Escherichia coli SSB, T4 gene 32) melt the triplex substrate very poorly or not at all, suggesting that the triplex destabilizing effect of RPA is specific. In contrast to the robust activity on DNA triplexes, RPA does not melt intermolecular G4 tetraplex structures. Cellular assays demonstrated increased triplex DNA content when RPA is transiently repressed, suggesting that RPA melting of triple helical structures is physiologically important. On the basis of our results, we suggest that the abundance of RPA known to exist in vivo is likely to be a strong deterrent to the stability of triplexes that can potentially form from human genomic DNA sequences.

The advent of microarray technology, coupled with the availability of mouse cDNA collections derived specifically from preimplantation embryos, helps to provide global gene expression profiles for the earliest stages of development. However, to determine the functions of the large numbers of genes of interest, massive systematic functional assays such as gene 'knockdown' experiments are required. As a first step, the relative suppression of blastocyst formation by differentially-modified antisense oligonucleotides to E-cadherin was assayed. The injection of 2'-methoxyethoxy (2'-MOE)-modified oligonucleotides blocked the formation of blastocysts in two-thirds of embryos, whereas the injection of either control missense 2'-MOE-oligonucleotides, or oligonucleotides with a Morpholino modification, had no significant effect on embryonic development. Thus, the 2'-MOE-modified antisense oligonucleotides are candidates for effective examination of roles of large numbers of genes during early embryological development.