Anti-Müllerian hormone (AMH) levels in premenopausal breast cancer patients treated with taxane-based adjuvant chemotherapy - A translational research project of the SUCCESS A study.

BACKGROUND: Premenopausal women undergoing chemotherapy are at high risk for premature ovarian failure and its long-term consequences. Data on potential markers to evaluate ovarian reserve pre- and posttreatment are limited. Anti-Müllerian hormone (AMH) known for ovarian reserve in reproductive medicine could be a surrogate marker and was assessed in premenopausal breast cancer patients of the SUCCESS A study (EUDRA-CT no. 2005-000490-21). METHODS: We identified 170 premenopausal patients, age ≤ 40 years at trial entry, who received FEC-Doc as taxane-anthracylince based chemotherapy. Blood samples were taken at three time points: Before, four weeks after and two years after adjuvant chemotherapy. Serum AMH-levels were evaluated in a central laboratory by a quantitative immunoassay AMH Gen II ELISA (Beckman Coulter, Brea, USA). RESULTS: Median age was 36 years (21-40 years). Median serum AMH-level before chemotherapy was 1.37 ng/ml (range < 0.1-11.3 ng/ml). Four weeks after chemotherapy AMH-levels dropped in 98.6% of the patients to <0.1 ng/ml (range < 0.1-0.21 ng/ml). After two years, 73.3% (n = 101) showed no evidence of ovarian function recovery (AMH <0.1 ng/ml, range < 0.1-3.9 ng/ml). Permanent chemotherapy induced amenorrhea occurred only in 50.6% of the patients. CONCLUSIONS: In this analysis, premenopausal patients showed a high rate of ovarian impairment reflected by low AMH-levels after chemotherapy.

Developmental regulation of the A-type potassium-channel current in hippocampal neurons: role of the Kvbeta 1.1 subunit.

The rapidly inactivating A-type K+ current (IA) is prominent in hippocampal neurons; and the speed of its inactivation may regulate electrical excitability. The auxiliary K+ channel subunit Kvbeta 1.1 confers fast inactivation to Shaker-related channels and is postulated to affect IA. Whole-cell patch clamp recordings of rat hippocampal pyramidal neurons in primary culture showed a developmental decrease in the time constant of inactivation (tau(in)) of voltage-gated K+ currents: 17.9+/-1.5 ms in young neurons (5-7 days in vitro; n=53, mean+/-S.E.M.); 9.9+/-1.0 ms in mature neurons (12-15 days in vitro; n=72, mean+/-S.E.M., P<0.01). During the same developmental time, the level of Kvbeta 1.1 transcript increased more than two-fold in vitro and in vivo, determined by semi-quantitative reverse transcriptase-polymerase chain reaction for hippocampus. The hypothesis that up-regulation of Kvbeta 1.1 led to the changes in tau(in) was tested in vitro, using antisense knockdown. Kvbeta 1.1-specific antisense DNA was introduced with a modified herpes virus co-expressing enhanced green fluorescent protein and knockdown of Kvbeta 1.1 was verified by immunocytochemistry. Following transduction with the antisense virus, mature neurons reverted to tau(in) values characteristic of young neurons: 18.3+/-2.4 ms (n=20). The effect of antisense knockdown on electrical excitability was tested using current-clamp protocols to induce repetitive firing. Treatment with the antisense virus increased the interspike interval over a range of membrane depolarization (baseline membrane potential=-40 to +20 mV). This effect was most pronounced at -40 mV, where the ISI of the first pair of action potentials was nearly doubled. These data indicate that Kvbeta 1.1 contributes to the developmental control of IA in hippocampal neurons and that the magnitude of effect is sufficient to regulate electrical excitability. Viral-mediated antisense knockdown of Kvbeta 1.1 is capable of decreasing the electrical excitability of post-mitotic hippocampal neurons, suggesting this approach has applicability to gene therapy of neurological diseases associated with hyperexcitability.

Distinct mechanisms of block of Kv1.5 channels by tertiary and quaternary amine clofilium compounds.

The quaternary ammonium compound clofilium and its tertiary amine derivative LY97241 were used to analyze mechanisms of block in a voltage-gated potassium channel. Wild-type and mutant Kv1.5 channels expressed in Xenopus oocytes were recorded by two-electrode voltage clamp. Open-channel block to 20% of the control current amplitude was induced reversibly by 50 microM clofilium or 200 microM LY97241, and was seen as an acceleration of the macroscopic current decay. Although blockers remained present after application, channels recovered from block during each interpulse interval. The optimum voltage for recovery (-45 mV at pH 7.3) at the threshold for channel activation indicated that clofilium block and recovery occurred principally through the open channel state. In contrast, LY97241 appeared to exit from the closed state and the open state. In an acid-tolerant Kv1.5 mutant channel (H452Q), external pH was used to titrate LY97241. At low pH, which protonates the LY97241 amine group, recovery from block at hyperpolarized potentials was impaired in a manner similar to that seen with clofilium. Recovery from clofilium block was reduced at negative potentials independent of pH, an effect attributed to trapping of the permanently charged compound within the closed channels.

Differential sensitivity of voltage-gated potassium channels Kv1.5 and Kv1.2 to acidic pH and molecular identification of pH sensor.

Kv1.2 and Kv1.5 are two subtypes of voltage-gated potassium channels expressed in heart that are thought to contribute to phase 1 (ITO) and phase 3 (IK) components of cardiac action potential repolarization. Although the effect of decreased pH in prolonging cardiac action potentials is well documented, the molecular target of acidification has not previously been determined. We expressed Kv1.2 and Kv1.5 in Xenopus oocytes to study the effect of acidic and alkaline extracellular pH on channel function. Using two-electrode voltage clamp and cellattached patch clamp, we demonstrate that Kv1. 5 channels show enhanced C-type inactivation at acidic pH that is relevant to pathophysiological conditions. In contrast, homologous Kv1.2 channels are resistant to acidic pH. Both channel types are insensitive to alkaline pH. A histidine residue in the third extracellular loop of Kv1.5 (H452) accounts for the difference in pH sensitivity between the Kv1.5 and Kv1.2 channels. Mutation of histidine H452 to a glutamine residue in Kv1.5 yields a channel that no longer shows enhanced inactivation with acidification. These data provide insight into mechanisms subserving known pH effects on cellular signaling functions. Our results demonstrate that H452 in the third extracellular loop of Kv1.5 plays a role in C-type inactivation, thus expanding the known complement of protein regions that contribute to the slow K+ channel inactivation mechanism.

Residual stresses in the human aorta and their influences by growth and remodelling.

The aim of the present work is to demonstrate of the influence of the adventitia on distribution of residual stresses in the human aorta. The biomaterial increase in media as well as in adventitia in the course of the aorta's growth is formed by an intussusceptive mechanism of growth. In children's aortas, formative elastin lamellae are wavy with high amplitude perpendicular to the aortic surface. In adults' aortas the waves become smoother (stepwise) towards the adventitia. Also introduced is the growth function, which characterizes the biomaterial growth and the interference between the media and adventitial layers. It is possible to expect an extraordinary variability in the formation of the aortic wall tissue in the course of its growth as a result of metabolic and humoral influences and magnitude of the residual stresses in the course of growth.

Relationship of marital structure and interactions to opiate abuse relapse.

Several aspects of marital functioning were associated with subsequent relapse to opiate abuse in 17 married addicts. The addicts and spouses were evaluated in a task-oriented interview and rated using the Beavers Timberlawn Family Assessment instrument. The global health-pathology ratings on this instrument indicated that most couples had rigid patterns of interacting, rather than a chaotic lack of structure or a flexible, negotiated partnership. Within this range of rigid functioning, higher ratings were associated with longer times drug-free (up to 18 months with a mean of 7 months). On the seven subscales of the Beavers', five were significantly correlated with the time drug-free: effective and clear leadership, closeness between the spouses, a nonhostile mood, empathy, and efficient negotiation and problem solving. The subscales associated with drug abstinence were quite different for a group of seven single ex-addicts participating in the same outpatient program, but living with their parents. For these single ex-addicts three subscales were correlated with the time drug-free: parental reaction to separation strivings, the open expression of thoughts and feelings, and empathy. This difference in the subscales associated with abstinence for married versus single addicts suggested some specificity in the characteristics of family structure and interaction that may be related to drug abstinence.

The effect of multiple family therapy on addict family functioning: a pilot study.

Family therapy may help addicts remain drug abstinent by improving family functioning. In a outpatient pilot study eight addict families were evaluated before and after 16 weeks of multiple family therapy (MFT), while the addict was maintained on naltrexone, an opiate antagonist. The Beavers Timberlawn Family Assessment was used to rate videotapes on problem solving, family structure, individual autonomy, and affect. The 8 families showed significant improvement in global functioning, problem solving, structure, and autonomy, but not in affect. One addict relapsed during the 10 month follow up, and his was the only family that functioned worse at follow up. We concluded that MFT can help addict families progress from chaotic interactions to more stable family structures and from rigid to more flexible family functioning. This improvement in family functioning may be associated with ex-addicts remaining abstinent.

Identification of the binding site on cytochrome c1 for cytochrome c.

The reagent 1-ethyl-3-(3-[14C]trimethylaminopropyl)carbodiimide (ETC) was used to identify specific carboxyl groups on the cytochrome bc1 complex (ubiquinol-cytochrome c reductase, EC 1.10.2.2) involved in binding cytochrome c. Treatment of the cytochrome bc1 complex with 2 mM ETC led to inhibition of the electron transfer activity with cytochrome c. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both the cytochrome c1 heme peptide and the Mr = 9175 "hinge" peptide were radiolabeled by ETC. In addition, a new band appeared at a position consistent with a 1:1 cross-linked cytochrome c1-hinge peptide species. Treatment of a 1:1 cytochrome bc1-cytochrome c complex with ETC led to the same inhibition of electron transfer activity observed with the uncomplexed cytochrome bc1, but to decreased radiolabeling of the cytochrome c1 heme peptide. Two new cross-linked species corresponding to cytochrome c-hinge peptide and cytochrome c-cytochrome c1 were formed in place of the cytochrome c1-hinge peptide species. In order to identify the specific carboxyl groups labeled by ETC, a purified cytochrome c1 preparation containing both the heme peptide and the hinge peptide was dimethylated at all the lysines to prevent internal cross-linking. The methylated cytochrome c1 preparation was treated with ETC and digested with trypsin and chymotrypsin, and the resulting peptides were separated by high pressure liquid chromatography. ETC was found to label the cytochrome c1 peptides 63-81, 121-128, and 153-179 and the hinge peptides 1-17 and 48-65. All of these peptides are highly acidic and contain one or more regions of adjacent carboxyl groups. The only peptide consistently protected from labeling by cytochrome c binding was 63-81, demonstrating that the carboxyl groups at residues 66, 67, 76, and 77 are involved in binding cytochrome c. These residues are relatively close to the heme-binding cysteine residues 37 and 40 and indicate a possible site for electron transfer from cytochrome c1 to cytochrome c.

Microcalorimetric studies of the interactions between cytochromes c and c1 and of their interactions with phospholipids.

Thermotropic properties of purified cytochrome c1 and cytochrome c have been studied by differential scanning calorimetry under various conditions. Both cytochromes exhibit a single endothermodenaturation peak in the differential scanning calorimetric thermogram. Thermodenaturation temperatures are ionic strength, pH, and redox state dependent. The ferrocytochromes are more stable toward thermodenaturation than the ferricytochromes. The enthalpy changes of thermodenaturation of ferro- and ferricytochrome c1 are markedly dependent on the ionic strength of the solution. The effect of the ionic strength of solution on the enthalpy change of thermodenaturation of cytochrome c is rather insignificant. The formation of a complex between cytochromes c and c1 at lower ionic strength causes a significant destabilization of the former and a slight stabilization of the latter. The destabilization of cytochrome c upon mixing with cytochrome c1 was also observed at high ionic strength, under which conditions no stable complex was detected by physical separation. This suggests formation of a transient complex between these two cytochromes. When cytochrome c was complexed with phospholipids, no change in the thermodenaturation temperature was observed, but a great increase in the enthalpy change of thermodenaturation resulted.

Psychosis and depression arising in the midlife period. A comparison.

Two groups of hospitalized psychotic patients were compared to a hospitalized nonpsychotic group composed mainly of major unipolar depressives. In the main, demographic and historical dimensions did not differ. Interpersonal and social role development favored the nonpsychotic group, but only employment history and maternal function reached significance. All groups were judged to be under substantial stress in association with the index hospitalization. Loss of a supportive relationship was the most frequent stress category for all groups but was relatively more frequent in the nonpsychotic (depressive) group. Medical stress was significantly higher in the psychotic groups.

Medical condition, adherence to treatment regimens, and family functioning. Their interactions in patients receiving long-term dialysis treatment.

Twenty-three medically stable patients receiving long-term dialysis treatment and their families were studied to investigate the relationship between medical condition, adherence to treatment, and patterns of family interaction. We found significant correlations between ratings of overall family functioning and overall medical condition, and a near-significant relationship between ratings of adherence to treatment and overall family functioning. In addition, specific family variables that related either to medical condition or to adherence were identified. Our findings suggest that family assessment can be used for early identification of patients at risk for poor adherence to treatment or poor medical progress. Furthermore, it may be possible to improve medical condition and adherence by working with the family in specific areas of family functioning found to be related to medical condition or adherence.