Chemoselective Oxidation of Thiols with Oxoammonium Cations.

The oxidation of various aryl and aliphatic thiols with the commercially available and environmentally benign reagent Bobbitt's salt (1) has been investigated. The reaction affords the corresponding disulfide products in good to excellent yields (71-99%) and can be accomplished in water, methanol, or acetonitrile solvent. Moreover, the process is highly chemoselective, tolerating traditionally oxidation-labile groups such as free amines and alcohols. Combined experimental and computational studies reveal that the oxidation takes place via a polar two-electron process with concomitant and unexpected deoxygenation of the oxoammonium cation through homolysis of the weak N-O bond, differing from prototypical radical-based thiol couplings. This unusual consumption of the oxidant has significant implications for the development of new nitroxide-based radical traps for probing S-centered radicals, the advancement of new electrochemical or catalytic processes involving nitroxide/oxoammonium salt redox couples, and applications to biological systems.

Novel Strategies to Improve Resident Selection by Improving Cultural Fit: AOA Critical Issues.

Residency selection is a perennial multifactorial process that differs considerably from the recruitment processes that other professional occupations enjoy. The 2016 meeting of the American Orthopaedic Association's Council of Orthopaedic Residency Directors highlighted a series of symposia that sought to present a novel manner of resident selection. Specifically, the presenters for each symposium were asked to do the following: present some general recruitment best practices in industries outside of medicine, present how branding of a program may translate into a better interview season, investigate evidence that the applicant pool to orthopaedic surgery may have changed and that residency program brands may have to reflect this, and assess our current evaluation techniques for talent identification and resident selection with respect to a specific department's appearance or brand. The meeting concluded with an understanding of the level to which programs can successfully create or adopt a brand and how this may go a long way in focusing the entire match process and allow emphasis to be placed on applicants who possess desired traits. The goal for this meeting was that attendees would leave with tangible practices and techniques that could be adopted at their home institutions.

Estimation of the impact of genital warts on health-related quality of life.

OBJECTIVES: One of the two new human papillomavirus (HPV) vaccines protects against HPV types 6 and 11, which cause over 95% of genital warts, in addition to protecting against HPV types 16 and 18. In anticipation of HPV vaccine implementation, the impact of genital warts on health-related quality of life (HRQoL) was measured to assess the potential benefits of the quadrivalent over the bivalent vaccine. METHODS: Genitourinary medicine clinic patients aged 18 years and older with a current diagnosis of genital warts were eligible; 81 consented and were interviewed by a member of the research team. A generic HRQoL questionnaire, the EQ-5D (comprising EQ-5D index and EQ visual analogue scale (VAS) scores) and a disease-specific HRQoL instrument, the CECA10, were administered. Previously established UK population norms were used as a control group for EQ-5D comparisons. RESULTS: Cases (with genital warts) had lower EQ VAS and EQ-5D index scores than controls. After adjusting for age a mean difference between cases and controls 30 years of age and under (n = 70) of 13.9 points (95% CI 9.9 to 17.6, p<0.001) for the EQ VAS and 0.039 points (95% CI 0.005 to 0.068, p = 0.02) on the EQ-5D index (also adjusted for sex) was observed. The difference between cases and controls for the EQ VAS was especially notable in young women. CONCLUSIONS: Genital warts are associated with a significant detriment to HRQoL. The potential added benefit of preventing most cases of genital warts by HPV vaccination should be considered in decisions about which HPV vaccine to implement in the United Kingdom.

Protein-protein interactions: lessons learned.

Protein-Protein (P-P) interactions play a pivotal role in determining cellular structure and in all cellular processes. The nature of P-P interactions is complex, and despite the large amount of research that has occurred in the field, is still poorly understood. Abnormal P-P interactions are particularly important because of their association with a variety of diseases, including cancer. This review examines P-P interactions with particular emphasis on the discovery of new anti-tumor drugs, including underlying physical forces that determine affinity and specificity and discusses classical and newer strategies used to discover inhibitors of P-P interactions, providing a number of recent cancer-related case studies and commentary.

Estrogen response element binding induces alterations in estrogen receptor-alpha conformation as revealed by susceptibility to partial proteolysis.

Genes whose expression is highly induced by estradiol (E(2)) contain multiple estrogen response elements (EREs) in their promoters. Previously we reported that estrogen receptor-alpha (ERalpha) binds cooperatively to and E(2) synergistically activates reporter gene expression from three or four tandem copies of a consensus ERE (EREc38). Here we evaluated how ERalpha binding to one, two, three or four tandem copies of EREc38 affects ERalpha conformation as detected by altered ERalpha trypsin digestion patterns in Western blots. E(2)- or 4hydroxytamoxifen (4-OHT)-occupied ERalpha bound to the pS2 ERE or to a single copy of EREc38 showed enhanced susceptibility to trypsin digestion compared to E(2)- or 4-OHT-ERalpha incubated with DNA lacking an ERE. ERalpha binding to multiple tandem copies of EREc38 further increased sensitivity to trypsin digestion. These results correlate with synergistic transcription and cooperativity of ERalpha binding to multiple tandem copies of EREc38. These observations suggest that EREc38 binding alters the overall conformation of ERalpha and that multiple tandem copies of EREc38 enhance these conformational changes. We hypothesize that ERE-induced alterations in ERalpha conformation modulate interaction with coregulatory proteins, resulting in synergistic transcriptional activation.

Identification of E2F-1/Cyclin A antagonists.

A simple method for the synthesis of a rationally designed (S,S)-[Pro-Leu]-spirolactam scaffold is described. This was expanded to a small biased library of compounds mimicking the 'ZRXL' motif in order to identify E2F-1/Cyclin A antagonists. The synthesized compounds were evaluated in an E2F-1/Cyclin A binding assay and moderately active analogues were identified. In addition, the critical roles of Phe, Leu, Lys, and Arg residues of the identified motif were determined.

A new Antennapedia-derived vector for intracellular delivery of exogenous compounds.

We describe the design, synthesis and cell translocation capacity of a peptide derived from the third alpha-helix of the homeodomain of Antennapedia. The new sequence appears to be an efficient and nontoxic means to deliver a covalently linked peptide cargo into cells.

Reversible G(1) arrest induced by inhibition of the epidermal growth factor receptor tyrosine kinase requires up-regulation of p27(KIP1) independent of MAPK activity.

We have used quinazoline inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase to study the link between EGFR signaling and G(1) to S traverse. Treatment of A431 and MDA-468 human tumor cells with 0.1-10 microM AG-1478 inhibited basal and ligand-stimulated EGFR phosphorylation without a decrease in receptor content, EGF-binding sites, or binding affinity. Incubation of A431 cells with 0.1-1 microM AG-1517 abrogated (125)I-EGF internalization. Both AG-1478 and AG-1517 markedly inhibited A431 and MDA-468 colony formation in soft agarose at concentrations between 0.01 and 1 microM. Daily injections of AG-1478 at 50 mg/kg delayed A431 tumor formation in athymic nude mice. A transient exposure of A431 cells to AG-1478 resulted in a dose-dependent up-regulation of the cyclin-dependent kinase inhibitor p27, down-regulation of cyclin D1 and of active MAPK, and hypophosphorylation of the retinoblastoma protein (Rb). These changes were temporally associated with recruitment of tumor cells in G(1) phase and a marked reduction of the proportion of cells in S phase. Upon removal of the kinase inhibitor, EGFR and Rb phosphorylation and the levels of cyclin D1 protein were quickly restored, but the cells did not reenter S phase until p27 protein levels were decreased. Phosphorothioate p27 oligonucleotides decreased p27 protein in A431 cells and abrogated the quinazoline-mediated G(1) arrest. Treatment of A431 cells with PD 098509, a synthetic inhibitor of MEK1, inhibited MAPK activity without inducing G(1) arrest or increasing the levels of p27. However, treatment with LY 294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited basal Akt activity, up-regulated p27, and recruited cells in G(1). These data suggest that p27 is required for the growth arrest that follows interruption of the EGFR kinase in receptor-overexpressing cells. In addition, the G(1) arrest and up-regulation of p27 resulting from EGFR blockade are not due to the interruption of MAPK, but to the interruption of constitutively active PI3K function.

Selective killing of transformed cells by cyclin/cyclin-dependent kinase 2 antagonists.

Recent studies identified a short peptide motif that serves as a docking site for cyclin/cyclin-dependent kinase (cdk) 2 complexes. Peptides containing this motif block the phosphorylation of substrates by cyclin A/cdk2 or cyclin E/cdk2. Here we report that cell membrane-permeable forms of such peptides preferentially induced transformed cells to undergo apoptosis relative to nontransformed cells. Deregulation of E2F family transcription factors is a common event during transformation and was sufficient to sensitize cells to the cyclin/cdk2 inhibitory peptides. These results suggest that deregulation of E2F and inhibition of cdk2 are synthetically lethal and provide a rationale for the development of cdk2 antagonists as antineoplastic agents.

Unliganded epidermal growth factor receptor dimerization induced by direct interaction of quinazolines with the ATP binding site.

Receptor dimerization is critical for signaling by the epidermal growth factor receptor (EGFR) tyrosine kinase. This occurs after binding of the receptor's extracellular domain by ligand or bivalent antibodies. The role of other receptor domains in dimerization is less clear, and there are no examples of dimers induced by direct perturbation of the EGFR kinase domain. Submicromolar concentrations of AG-1478 and AG-1517, quinazolines specific for inhibition of the EGFR kinase, induced reversible receptor dimerization in vitro and in intact A431 cells. Consistent with the inhibitory effect of quinazolines on receptor kinase activity, the dimers formed lacked a detectable Tyr(P) signal. Quinazoline-induced EGFR dimerization was abrogated in vitro by ATP and the ATP analog adenyl-5'-yl imidodiphosphate. Receptors with a single-point mutation in the ATP binding site as well as wild-type EGFR with a covalent modification of the ATP site failed to dimerize in response to AG-1478 and AG-1517. These data suggest that EGFR dimerization can be induced by the interaction of quinazolines at the ATP site in the absence of receptor ligand binding. In SKBR-3 cells, the quinazolines induced the formation of inactive EGFR/ErbB-2 heterodimers, potentially sequestering ErbB-2 from interacting with other coreceptors of the ErbB family. Structural studies of the quinazoline interaction with the EGFR tyrosine kinase domain should allow for an analysis of receptor-specific chemical features required for binding to the ATP site and disruption of signaling, a strategy that can be perhaps applied to other tumor cell receptor systems.

Blockade of transforming growth factor-beta signaling does not abrogate antiestrogen-induced growth inhibition of human breast carcinoma cells.

We have studied the role of autocrine transforming growth factor-beta (TGF-beta) signaling on antiestrogen-mediated growth inhibition of hormone-dependent T47D and MCF-7 human breast carcinoma cells. Tamoxifen treatment increased the secretion of TGF-beta activity into serum-free cell medium and the cellular content of affinity cross-linked type I and III TGF-beta receptors in both cell lines. Anti-pan-TGF-beta antibodies did not block anti-estrogen-induced recruitment in G1 and inhibition of anchorage-dependent and -independent growth of both cell lines. Early passage MCF-7 cells, which exhibit detectable type II TGF-beta receptors at the cell surface and exquisite sensitivity to exogenous TGF-beta1, were transfected with a tetracycline-controllable dominant-negative TGF-betaRII (DeltaRII) construct. Although the TGF-beta1 response was blocked by removal of tetracycline in MCF-7/DeltaRII cells, tamoxifen-mediated suppression of Rb phosphorylation, recruitment in G1, and inhibition of cell proliferation were identical in the presence and absence of tetracycline. TGF-beta1 treatment up-regulated the Cdk inhibitor p21 and induced its association with Cdk2 in MCF-7 cells; these responses were blocked by the DeltaRII transgene product. In MCF-7 cells with a functional TGF-beta signaling pathway, tamoxifen did not up-regulate p21 nor did it induce association of p21 with Cdk2, suggesting alternative mechanisms for antiestrogen-mediated cytostasis. Finally, transfection of late-passage, TGF-beta1 unresponsive MCF-7 cells with high levels of TGF-betaRII restored TGF-beta1-induced growth inhibition but did not enhance tamoxifen response in culture. Taken together these data strongly argue against any role for TGF-beta signaling on tamoxifen-mediated growth inhibition of hormone-dependent breast cancer cells.

Issues related to smoking cessation among substance abusers.

Initial studies have found that stop-smoking treatments for newly recovering substance abusers have been neither harmful to sobriety nor effective in achieving smoking cessation. The development of more effective stop-smoking treatments for this population could be aided by delineating their particular smoking-related characteristics. This article describes the biopsychosocial characteristics of newly recovering substance abusers that are relevant to smoking cessation, and suggests that there are notable differences between abusers and nonabusers that may contribute to abusers' greater difficulty in quitting smoking. It also recommends changes in existing treatment protocols where applicable and identifies key areas for future research.

Synthesis and evaluation of potential N pi and N sigma metal chelation sites within the beta-hydroxy-L-histidine subunit of bleomycin A2: functional characterization of imidazole N pi metal complexation.

The synthesis and evaluation of 4 and 5, fully functionalized deglycobleomycin A2 (2) analogues incorporating an oxazole and a pyrrole in place of the beta-hydroxy-L-histidine imidazole, are detailed. The oxazole agent is only capable of Npi metal complexation through a form related to the N1-H imidazole tautomer of bleomycin A2 (1) while the pyrrole agent may potentially mimic the Nsigma metal complexation capabilities of the imidazole N3-H tautomer. Metal complexes (Fe-II, Fe-III) of 4 and 5 were found to cleave duplex DNA in the presence of O2 (Fe-II) or H2O2 (Fe-III). The oxazole agent 4 which is incapable of Nsigma metal chelation was found to behave analogous to, albeit slightly less effectively than, deglycobleomycin A2 resulting in the characteristic 5'-GC/5'-GT sequence selective cleavage of duplex DNA directly confirming that imidazole/oxazole Npi metal chelation is sufficient for functional reactivity. Importantly, the effective substitution of the oxazole O-1 for the histidine N-1 further illustrates that this group does not require deprotonation upon metal complexation, oxygen activation, or the ensuing oxidation reactions, that the functional bleomycin A2 tautomer is the imidazole N'-H tautomer, and that the imidazole N'-H functionality is not contributing to the polynucleotide recognition through H-bonding to the phosphate backbone or nucleotide bases. In contrast, the pyrrole agent 5 which is incapable of Npi metal chelation, but possesses the capabilities of functioning as a Nsigma metal donor was also found to cleave duplex DNA, but does so in a nonsequence selective fashion with a significantly reduced efficiency and a diminished double to single strand cleavage ratio both only slightly above that of background iron itself. These observations are analogous to those made with 3 which lacks the imidazole altogether and further support the observations that Nsigma coordination, not Npi coordination, of the imidazole is required for the functional activity of bleomycin A2.

Cigarette smoking in alcohol and cocaine abusers.

This study compared the cigarette smoking of substance abusers whose primary substance of abuse was cocaine (cocaine group: n = 18) or alcohol (alcohol group: n = 23). Cigarette smoking and smoking topography was assessed daily (via self-report and single cigarette topography assessments) at baseline and following a switch to a cigarette brand with 30% lower nicotine. The alcohol and cocaine groups did not differ at baseline on cigarettes smoked per day, cigarette nicotine, smoking topography, or the Fagerstrom Tolerance Questionnaire. However, the cocaine group exhibited marked increases in compensatory smoking relative to the alcohol group following the 30% reduction in cigarette nicotine, as evidenced by decreases in the average time interval between each puff, p < .05, increases in the total amount of time spent puffing, p < .05, and increases in estimated total amount of time spent puffing per day, p < .05. These findings provide initial data that cocaine and alcohol abusers may titrate nicotine differently and suggest that cocaine abusers may require additional or modified smoking cessation treatments.

Synthesis of key analogs of bleomycin A2 that permit a systematic evaluation of the linker region: identification of an exceptionally prominent role for the L-threonine substituent.

The synthesis of a full series of analogs 2b-k of deglycobleomycin A2 (2a) containing systematic variations in the linker domain of bleomycin A2 (1) is described. The agents 2b-k, which are not accessible through structural modification of 1 or 2a, constitute key substructure analogs incorporating deep-seated structural modifications in the linker domain capable of delineating the contribution of the individual backbone substituents to the DNA cleavage efficiency, characteristic DNA cleavage selectivity, and double strand to single strand DNA cleavage ratio. The comparative examination of the DNA cleavage properties of the Fe(II) and Fe(III) complexes of 2a-k upon activation by O2-thiol or H2O2, respectively, revealed several characteristic features and trends. First, none of the substituents affect the characteristic 5'-GC, 5'-GT > 5'-GA DNA cleavage selectivity of bleomycin A2. In contrast, an exceptionally prominent role for the L-threonine substituent and an important role for the C4-methyl substituent of the (2S,3S,4R)-4-amino-3-hydroxy-2-methylpentanoic acid subunit were observed on the DNA cleavage efficiency of the agents. Similarly, the L-threonine substituent was found to substantially increase the ratio of double strand to single strand DNA cleavage events (2-3 times). In a w794 DNA cleavage assay, shortening the linker region by two carbons resulted in an exceptionally large reduction in DNA cleavage efficiency (125 times) and provided an agent that was only 1.3 times more effective than Fe(III) indicating that this deep-seated modification essentially destroys the DNA cleavage capabilities of the agent. The L-threonine substituent contributes in an exceptional manner, and its removal resulted in a 25 times reduction in DNA cleavage efficiency. A substantial contribution was observed for the C4-methyl group on the 4-aminobutanoic acid subunit and its removal resulted in a 7 times reduction in DNA cleavage efficiency. Little effect for the C3-hydroxyl and C2-methyl substituents on the 4- aminobutanoic acid subunit was observed (0-2.5 times) and even their inversion of stereochemistry had little impact on DNA cleavage efficiency or selectivity. Notably, the magnitude of the previously unappreciated L-threonine substituent contribution to the DNA cleavage efficiency and on the ratio of double to single strand DNA cleavage events is the largest effect observed to date including the well recognized disaccharide potentiation (6 times) of the DNA cleavage properties. Consequently, the past role and relative importance of the L-threonine subunit and substituent has been underestimated. Moreover, the cumulative effect of the two important linker chain substituents clearly illustrate that the functional role of this domain is much more important than its simply serving as a linker.

The metabolic cost of backpack and shoulder load carriage.

Eleven healthy male volunteer soldiers (mean [SD] age 24.0 [2.8] years, stature 174.1 [5.2] cm, body weight 73.2 [10.8] kg, body fat 14.2 [5.0]% and maximal oxygen uptake 4.1 [0.4] 1 min-1) walked at 4.8 km h-1 on a motor driven treadmill for 5 min at each of three gradients (0, 2.5 and 5%) whilst carrying a two-part 26 kg load either on each shoulder or strapped to a backpack frame. The load was made up of two cylinders, one weighing 18.4 kg and the other weighing 7.6 kg. For all treadmill gradients the mean (SD) backpacking heart rates and oxygen uptakes (0% gradient, 122 [10] beats min-1, 1.51 [0.11] 1 min-1; 2.5% gradient, 135 [10] beats min-1, 1.81 [0.13] 1 min-1; 5% gradient, 155 [7] beats min-1, 2.21 [0.11] 1 min-1) were significantly (p less than 0.001) lower than for shoulder load carriage (0% gradient, 130 [9] beats min-1, 1.70 [0.12] 1 min-1, 2.5% gradient, 147 [8] beats min-1; 2.01 [0.10] 1 min-1; 5% gradient 164 [9] beats min-1, 2.39 [0.11] 1 min-1). The relative oxygen cost of backpacking was 4.3-4.7% VO2 max lower than for shoulder load carriage. It is concluded that the metabolic cost of backpacking an asymmetric two part 26 kg load was significantly less than for shoulder load carriage when walking at 4.8 km h-1 on a treadmill over gradients of 0-5%.(ABSTRACT TRUNCATED AT 250 WORDS)

Extinction of prolactin gene expression in somatic cell hybrids is correlated with the repression of the pituitary-specific trans-activator GHF-1/Pit-1.

The tissue-specific expression of the PRL and GH genes is dependent on the presence of a pituitary-specific trans-activator, GHF-1/Pit-1. Previous studies indicate that somatic cell hybridization of rat pituitary GH3 cells with LB82 mouse fibroblasts frequently results in the extinction of GH and PRL expression. The extinction of the GH gene occurs at the transcriptional level, and is accompanied by repression of GHF-1/Pit-1 synthesis. To elucidate the mechanism of PRL extinction we further characterized these same somatic cell hybrid lines as well as the parental GH3 and LB82 cells. The pattern of PRL extinction and reexpression paralleled that of GH in the three hybrid lines that were examined. Two of these lines extinguished both GH and PRL synthesis, while a third displayed reexpression of both genes, apparently due to the loss of mouse chromosomal material. These studies revealed that the extinction of PRL expression in these hybrid lines occurs at the level of mRNA accumulation and is strongly correlated with the loss of GHF-1/Pit-1 mRNA and protein synthesis. These data suggest that in pituitary x fibroblast hybrids repression of the trans-activator GHF-1/Pit-1 is a primary mechanism for the extinction of PRL and GH gene expression.

Comparison of dentofacial morphology in monozygotic twin pairs with their siblings using LISREL.

Lateral cephalometric radiographs from 33 families of monozygotic twins and their siblings were digitized. Seven dentofacial parameters were measured: 1 to SN, 1 to NA, 1 to NA(mm), 1 to 1, 1 to Mandibular Plane Angle, 1 to NB(mm) and 1 to APg(mm). Data from monozygotic twin pairs and siblings were analyzed using LISREL to determine variance components of heredity, common environment and random environment. Five of the parameters measured showed heredity as the primary factor of variance, while two, 1 to NB(mm) and 1 to APg(mm) showed a common environment as having the greatest effect.

Comparison of craniofacial morphology in monozygotic twins with their siblings.

Lateral cephalometric radiographs from 33 families of monozygotic twins and their siblings were digitized. Eight skeletal parameters were measured: FA to SN, SNA, SNB, ANB, angle of convexity, mandibular plane to SN angle, Y axis and pogonion to NB distance. Monozygotic twin pairs and their siblings showed a normal distribution pattern for all the skeletal parameters, except the pogonion to NB distance measurement. There was found to be a statistically significant correlation between age and facial angle and ANB. A familial aggregation effect was found for SNA, ANB and angle of convexity. Some genetic influence was found for FA, mandibular plane angle and Y axis, while the Pg to NB distance showed neither genetic influence, nor a familial aggregation.

Plasma and erythrocyte electrolytes in affective disorders.

Plasma and erythrocyte sodium (Na+), total and free (ultrafiltrable) plasma magnesium (Mg2+) as well as erythrocyte magnesium were measured in patients with affective disorders and in healthy control subjects. Depressed and manic patients had higher total plasma Mg2+ than did hospitalized healthy control subjects, but concentrations of ultrafiltrable Mg2+ did not differ. Although erythrocyte Mg2+ was significantly elevated in the depressed subjects in comparison with that found in the non-hospitalized healthy controls, this difference was not seen between the depressives and the hospitalized healthy controls. Depressed, manic or healthy control subjects did not differ with respect to either plasma or erythrocyte.