Induction and repair of double-strand breaks in the replicating DNA of HeLa cells

The photochemical (lambda < 400 nm) decomposition of some monocyclic and polycyclic nitramines produces .NO2, which can be detected in the respective nitramine crystals at 77 K by EPR (electron paramagnetic resonance). In solutions of perdeutero-dimethylsulfoxide (DMSO-d6) the .NO2 produced by photolytic decomposition of dissolved nitramines can be spintrapped by the solvent to give a radical having the structure CD3-(SO2)-(NO.)-CD3. In this article, we examine this reaction for two nitramines: cyclotrimethylenetrinitramine (RDX) and hexanitrohexaazaisowurzitane (HNIW), which are energetic materials. The decay of the spin-adduct radical (I) follows first-order kinetics for both nitramines studied, having a rate constant (k) of congruent to 7.1 x 10(-4) s-1. The net growth in spin concentration of (1) measured from EPR spectra is fitted by a first-order rate equation taking into account the simultaneous competitive decay rate of spin adduct (I). Using the rate data and EPR spin concentration data, the ratio of free .NO2 produced per parent nitramine molecule is estimated as 1:1 for RDX and 4:1 for HNIW. Biological implications of trapping of .NO2 by dimethyl sulfoxide are discussed.     

Enhancement of recombinant gamma-aminobutyric acid type A receptor currents by chronic activation of cAMP-dependent protein kinase

alpha 1, beta 1, and gamma 2S gamma-aminobutyric acid (GABA) type A receptor (GABAR) subunit cDNAs were transiently expressed in derivative cell lines of mouse L929 fibroblasts, which possessed different levels of the catalytic subunit of cAMP-dependent protein kinase (PKA). These cell lines included L929 (intermediate levels of kinase), C alpha 12 (elevated levels of kinase), and RAB10 (low levels of kinase) cells. Pharmacological analysis of GABA-evoked whole-cell currents revealed that, compared with expression in L929 and RAB10 cells, expression of alpha 1 beta 1 gamma 2S GABARs in C alpha 12 cells produced a selective enhancement of single whole-cell current amplitudes. No other pharmacological properties (Hill slope, EC50, or diazepam sensitivity) of the expressed alpha 1 beta 1 gamma 2S GABARs were modified. The GABAR current enhancement in C alpha 12 cells was blocked by substitution of a beta 1 subunit mutated at the PKA consensus phosphorylation site, Ser409 [beta 1(S409A)], for the wild-type beta subunit. Interestingly, enhancement was specific for GABARs containing all three subunits, because it was not seen after expression of alpha 1 beta 1 or alpha 1 beta 1 (S409A) GABAR subunit combinations. Single-channel conductance and gating properties were not different for alpha 1 beta 1 gamma 2S or alpha 1 beta 1 (S409A) gamma 2S GABARs expressed in each cell line, suggesting that PKA did not enhance whole-cell currents by altering these properties of GABARs. These results suggested that unlike acute application of PKA, which has been shown to produce a decrease in GABAR current, chronic elevation of PKA activity can result in enhancement of GABAR currents. More importantly, this effect occurred only with GABARs composed of alpha 1 beta 1 gamma 2S subunits and not alpha 1 beta 1 subunits and was mediated by a single amino acid residue (Ser409) of the beta 1 subunit.     

Short-term modulation of the androgen milieu alters pulsatile, but not exercise- or growth hormone (GH)-releasing hormone-stimulated GH secretion in healthy men: impact of gonadal steroid and GH secretory changes on metabolic outcomes

Gonadal steroids are known to alter GH secretion as well as tissue metabolism. The present study was designed to examine the effects of short term (2- to 3-week) alterations in gonadal steroids on basal pulsatile (nonstimulated) and exercise- and GH-releasing hormone-stimulated GH secretion, urinary nitrogen excretion, and basal and exercise-stimulated oxygen consumption. Two protocols were conducted, which reflect a total of 18 separate studies. In the first paradigm, 5 healthy young men were each studied in a double blind, randomized manner during 3 different gonadal hormone manipulations, in which serum testosterone was varied from hypogonadal (induced by leuprolide) to eugonadal (saline injections) to high levels (testosterone enanthate, 3 mg/kg.week, i.m.). There was a washout period of 8 weeks between treatments. In the second protocol, 3 of the original subjects were studied after 2 weeks of treatment with stanozolol (0.1 mg/kg.day). Two to 3 weeks after the desired changes in serum testosterone, each subject was admitted to the General Clinical Research Center for study. The hypogonadal state (serum testosterone, 33 ng/dL) increased urinary nitrogen loss (by 34%; P < 0.005) and decreased basal metabolic rate (by 12%; P < 0.02) compared with the eugonadal state (testosterone, 796 ng/dL). High dose testosterone (1609 ng/dL) further decreased urinary nitrogen loss over the eugonadal state (by 16%; P < 0.05). Stanozolol yielded the lowest urinary nitrogen excretion of all (P < 0.03). Like urinary nitrogen, the basal metabolic rate showed the greatest change between the hypogonadal and eugonadal states (12%; P < 0.02), with a lesser change during high dose testosterone treatment (4%). Analogously, end-exercise oxygen consumption rose by 11% between the hypogonadal and eugonadal states (P < 0.05). Between the hypogonadal and eugonadal states, no significant changes in pulsatile (nonstimulated), exercise-stimulated, or GRF-stimulated GH secretion or serum insulin-like growth factor I concentrations were observed. Raising testosterone to supraphysiological levels increased pulsatile GH secretion by 62% over that with leuprolide and by 22% over that with saline (P < 0.05). High dose testosterone treatment also increased serum insulin-like growth factor I concentrations by 21% and 34% over those during the eugonadal and hypogonadal states, respectively (P < 0.01). Testosterone did not affect either exercise- or GRF-stimulated GH secretion. In protocol 2, stanozolol did not affect any parameter of GH secretion. To examine the interaction between GH secretion and testosterone on urinary nitrogen excretion and basal metabolic rate, a one-way analysis of covariance was undertaken. Statistical examination of GH production as the covariate and testosterone (by tertile) as the interactive factor demonstrated significant relationships between serum testosterone levels and either urinary nitrogen (P < 0.02) or basal metabolic rate (P < 0.01), but not GH secretion (P = NS). In summary, these results demonstrate that short term modulation of the androgen milieu affects metabolic outcome without necessitating changes in GH secretion. These results have significance for both normal physiology and for the treatment of hypogonadal GH-deficient patients.     

Effects of beta3-integrin blockade (c7E3) on the response to angioplasty and intra-arterial stenting in atherosclerotic nonhuman primates

Because the beta3-antagonist abciximab (c7E3 Fab) has significantly improved late outcomes after coronary angioplasty, the beta3 integrins have been implicated in the arterial response to injury. However, the mechanisms underlying this benefit are unknown. The observation that c7E3 binds beta3 integrins on vascular cells (alphavbeta3) with affinity equal to that for the platelet glycoprotein IIb/IIIa integrin has led to the hypothesis that c7E3 may act directly on the artery wall to prevent restenosis after angioplasty. To test this hypothesis, we studied the effects of c7E3 on structural changes within the artery wall after angioplasty or stent angioplasty in 23 male cynomolgus monkeys with established atherosclerosis. Animals were randomly assigned to receive either a bolus of c7E3 (0.4 mg/kg IV, n=11) followed by a 48-hour infusion (0. 2 microg. kg-1. min-1) or an equal volume of vehicle (n=12). Animals received weight-adjusted aspirin and heparin and then underwent unilateral iliac artery experimental angioplasty and subclavian artery stent angioplasty (Palmaz). Iliac artery lumen diameter (LD) was determined by angiography at baseline (LDPre), after angioplasty (LDPost), and 35 days later (LDDay35). Arteries were then fixed by perfusion and removed for analysis. Lumen, intima, media, and external elastic lamina (EEL) areas were measured in iliac artery cross sections. Values from each injured iliac artery were normalized to the contralateral uninjured iliac artery to control for interanimal variability in baseline artery size and atherosclerosis extent. Intimal area was also measured in subclavian stent cross sections. c7E3 blocked platelet aggregation and prolonged the bleeding time from 2.8+/-1.1 to 19.8+/-2.5 minutes, P<0.001. Experimental angioplasty increased LDPost an average of 28%, and the initial gain was similar in both groups (P=NS). Despite an anti-platelet effect, c7E3 did not inhibit iliac lumen narrowing (LDDay35-LDPost: c7E3, -0.69+/-0.17 versus vehicle, -0.99+/-.17 mm, P=0.35); intimal hyperplasia (neointima area: c7E3, 1.12+/-.28 versus vehicle, 1.22+/-.20 mm2, P=0.77); or decrease in artery wall size (EEL area [percent of uninjured control]: c7E3, 101+/-7% versus vehicle, 121+/-7%). Stent intimal hyperplasia was also unaltered by c7E3 treatment (neointimal area: c7E3, 1.09+/-0.16 versus vehicle, 1. 28+/-0.11 mm2, P=0.36). These results suggest that the benefits of c7E3 treatment in coronary angioplasty were not from inhibition of intimal hyperplasia or improved artery wall remodeling. Alternative mechanisms should be explored to explain improved late outcomes after angioplasty in patients treated with c7E3.     

Single dose pharmacokinetics of manidipine in hepatic impaired patients and healthy controls

The pharmacokinetics and safety of a single oral dose of 20 mg manidipine dihydrochloride have been studied in 8 patients with mild to moderate hepatic impairment (grade A or B in Child's classification, or score < or = 7 in Pugh's modification of Child's classification), and in 12 healthy subjects. They received one 20 mg manidipine dihydrochloride tablet with 100 ml of tap water after a standard breakfast. Manidipine was determined using HPLC with electrochemical detection from plasma samples taken up to 24 or 36 h after dosing. The medication was well tolerated. A trend toward higher Cmax, AUC, and MRT was observed in patients with a more severe hepatic impairment, as a consequence of reduction in the liver metabolic function. Patients with grade A hepatic impairment did not exhibit significantly altered pharmacokinetics with respect to healthy subjects, while grade B impairment patients had significantly higher AUC and MRT. Tmax values pointed to reduced absorption rate in patients compared to healthy subjects; the changes were more evident in grade B than grade A patients, although statistical significance was not reached. The reduction in absorption rate in grade B patients is probably related to their higher mean age, since this effect has been reported for manidipine. The pharmacokinetics of manidipine seem only modified in patients with a certain degree of hepatic impairment (at least Pugh grade 6 and Child grade B); therefore, adaptation of the dosing regimen does not seem to be generally recommendable, but should be modulated according to the liver status of the patient.     

Equating the MOS SF36 and the LSU HSI Physical Functioning Scales

This study equates the physical functioning subscales of the Medical Outcomes Study Short Form 36 (SF36) and the Louisiana State University Health Status Instruments (LSU HSI). Data from the SF36's 10-item physical functioning scale, the PF10, and the LSU HSI's 29-item Physical Functioning Scale (PFS), were fit to separate and mixed Rasch rating scale models. Data were provided by a convenience sample of 285 patients waiting for appointments in a public hospital general medicine clinic. Difficulty estimates for a subset of similar items from the two instruments were highly correlated (.95), indicating that the items from the two scales are working together to measure the same variable. The measures from the two equated instruments correlate .80 (.86 when disattenuated for error). Of the two instruments, the PFS's error is lower, model fit is better, and reliability coefficients are higher. Both instruments measure physical functioning, and can do so in a common unit of measurement. Conversion tables are provided for transforming raw scores from either instrument into the common metric.