Using a quality of life (QoL)-monitor: preliminary results of a randomized trial in Dutch patients with early breast cancer.

PURPOSE: The diagnosis and treatment of cancer negatively affect patients' physical, functional and psychological wellbeing. Patients' needs for care cannot be addressed unless they are recognized by healthcare providers (HCPs). The use of quality of life (QoL) assessments with feedback to HCPs might facilitate the identification and discussion of QoL-topics. METHODS: 113 patients with stage I-IIIB breast cancer treated with chemotherapy were included in this randomized controlled trial. Patients were randomly allocated to receive either usual care, or usual care with an intervention consisting of a QoL-monitor assessing QoL, distress and care needs before every chemotherapy cycle visit. Patients completed questionnaires regarding QoL, illness perceptions, self-efficacy, and satisfaction with communication. From the 2nd visit onwards, patients in the intervention arm and their HCPs received a copy of the QoL overview and results were shown in patients' medical files. Audio-recordings and patients' self-reports were used to investigate effects on communication, patient management and patient-wellbeing. A composite score for communication was calculated by summing the number of QoL-topics discussed during each consultation. RESULTS: Use of the QoL-monitor resulted in a higher communication score (0.7 topics increase per visit, p = 0.04), especially regarding the disease-specific and psychosocial issues (p < 0.01). There were no differences in patient management, QoL, illness perceptions or distress. Patients in the experimental arm (n = 60) had higher scores on satisfaction with communication (p < 0.05). CONCLUSIONS: Use of a QoL-monitor during chemotherapy in patients with early breast cancer might result in a more frequent discussion of QoL-topics, associated with high levels of patients' satisfaction.

Risk of End-Stage Renal Disease in HIV-Positive Potential Live Kidney Donors.

New federal regulations allow HIV-positive individuals to be live kidney donors; however, potential candidacy for donation is poorly understood given the increased risk of end-stage renal disease (ESRD) associated with HIV infection. To better understand this risk, we compared the incidence of ESRD among 41 968 HIV-positive participants of North America AIDS Cohort Collaboration on Research and Design followed for a median of 5 years with the incidence of ESRD among comparable HIV-negative participants of National Health and Nutrition Examination III followed for a median of 14 years. We used risk associations from multivariable Cox proportional hazards regression to derive cumulative incidence estimates for selected HIV-positive scenarios (no history of diabetes, hypertension, AIDS, or hepatitis C virus coinfection) and compared these estimates with those from similarly selected HIV-negative scenarios. For 40-year-old HIV-positive individuals with health characteristics that were similar to those of age-matched kidney donors, viral load <400 copies/mL, and CD4+ count ≥500 cells/µL, the 9-year cumulative incidence of ESRD was higher than that of their HIV-negative peers, yet still low: 2.5 versus 1.1 per 10 000 among white women, 3.0 versus 1.3 per 10 000 among white men, 13.2 versus 3.6 per 10 000 among black women, and 15.8 versus 4.4 per 10 000 among black men. HIV-positive individuals with no comorbidities and well-controlled disease may be considered low-risk kidney donor candidates.

Cross-cultural comparison of breast cancer patients' Quality of Life in the Netherlands and Japan.

PURPOSE: Cultural differences are hypothesized to influence patients' Quality of Life (QoL) reports. However, there is a lack of empirical cross-cultural studies comparing QoL of patients with cancer. This study aims to compare QoL of women with breast cancer in the Netherlands and Japan, and to investigate the association of QoL with sociodemographic, clinical, and psychological variables (illness perceptions). METHODS: Dutch (n = 116) and Japanese (n = 148) women with early breast cancer undergoing chemotherapy completed the EORTC QLQ-C30 and Brief Illness Perception Questionnaire immediately before their second cycle of chemotherapy. RESULTS: Dutch women reported poorer Physical, Role, Emotional, and Cognitive functioning than Japanese women. Additionally, illness perceptions were significantly different in Japan and the Netherlands, but these did not vary across treatment type. In Japan, QoL of women receiving AC-chemotherapy was better than that of women receiving FEC-chemotherapy, whereas in the Netherlands, QoL did not vary as a function of chemotherapy. Illness perceptions about symptom severity, adverse consequences, and emotional representations were negatively related to most domains of patients' QoL in both countries. Adding illness perceptions as covariates to the ANOVA analyses rendered the effects of country and treatment type on QoL non-significant. CONCLUSIONS: Comparing Dutch and Japanese women with early breast cancer revealed important differences in treatment modalities and illness perceptions which both appear to influence QoL. Perceptions about cancer have been found to vary across cultures, and our study suggests that these perceptions should be considered when performing cross-cultural studies focusing on patient-reported outcomes.

Improvements of complex post-traumatic stress disorder symptoms during a multimodal psychodynamic inpatient rehabilitation treatment - results of an observational single-centre pilot study.

Background: Complex post-traumatic stress disorder (CPTSD) describes chronic disturbances in self-organization (i.e. affect dysregulation; negative self-concept; severe difficulties in relationships) which are frequently observed in survivors of prolonged, repeated or multiple traumatic stressors. So far, evidence of psychodynamic treatment approaches for CPTSD is scarce.Methods: In this single-centre observational pilot study, symptom change during a 6-week psychodynamic inpatient treatment in a multimodal psychosomatic rehabilitation centre was evaluated using repeated measures analyses of variance (ANOVAs). Patients completed questionnaires on PTSD and CPTSD symptoms (ITQ), anxiety, depression and somatization (BSI-18), functional impairment (WHODAS) and epistemic trust, mistrust and credulity (ETMCQ) before (T1) and at the end of treatment (T2). A hierarchical linear regression analysis was calculated to identify factors associated with improved CPTSD symptoms.Results: A total of n = 50 patients with CPTSD were included in the study, of whom n = 40 (80%) completed treatment. Patients reported a significant reduction of CPTSD symptoms during treatment with a large effect size (-3.9 points; p < .001; η2 = .36), as well as a significant reduction of psychological distress (p < .001; η2 = .55) and functional impairment (p < .001; η2 = .59). At the end of treatment, 41.0% of patients no longer fulfilled the diagnostic criteria for CPTSD. Changes in epistemic stance included improved epistemic trust (ß = -.34, p = .026) and decreased epistemic credulity (ß = .37, p = .017), which together with lower age (ß = .43, p = .012) and lower depression levels at baseline (ß = .35, p = .054) were significantly associated with baseline adjusted mean change of CPTSD symptoms during therapy and explained 48% of its variance.Discussion: In our study, patients reported a significant reduction of CPTSD symptoms and comorbid symptoms during a multimodal psychodynamic inpatient rehabilitation treatment. Improved epistemic trust may facilitate the establishment of a trusting therapeutic relationship, thus fostering an environment of openness for knowledge transfer (i.e. social learning) and the exploration of diverse viewpoints and perspectives in the therapeutic process.

Complex post-traumatic stress disorder (CPTSD) is a condition often found in individuals who have experienced severe trauma, such as childhood abuse or torture.A study involving 50 patients with CPTSD showed significant improvements in symptoms and overall quality of life after undergoing a 6-week integrative multimodal psychodynamic inpatient rehabilitation treatment.The study also highlighted that improvement in epistemic trust could be a potential mechanism of change contributing to the positive therapeutic outcomes.

[Neuropeptide effects on the trigeminal system: pathophysiology and clinical significance for migraine]. / Neuropeptidwirkungen im trigeminalen System: Pathophysiologie und klinische Bedeutung bei der Migräne.

Neuropeptides, such as calcitonin gene-related peptide (CGRP), substance P and vasoactive intestinal polypeptide (VIP) are considered important mediators in primary headaches. Increased concentrations of CGRP have been found in jugular venous plasma during attacks of migraine and, concomitant with VIP elevation, during cluster headache. Substance P and CGRP are produced from subsets of trigeminal afferents whereas VIP derives from parasympathetic efferents. Release of these neuropeptides in the meninges causes arterial vasodilatation, mast cell degranulation and plasma extravasation in animal experiments. Particularly CGRP seems to be important, as receptor antagonists have recently been shown to have a therapeutic effect on migraine. Animal models have confirmed the role of CGRP in meningeal nociception. The activity of spinal trigeminal neurons is a sensitive integrative measure of trigeminal activity and CGRP released from central terminals of trigeminal afferents in the spinal trigeminal nucleus has been shown to facilitate nociceptive transmission, most likely by a presynaptic action. The proposed CGRP functions are supported by the distribution of CGRP receptor components localized in the rat cranial dura mater, the trigeminal ganglion and the spinal trigeminal nucleus. The currently available data indicate multiple sites of CGRP action in trigeminal nociception and the pathogenesis of migraine but central CGRP receptors are probably the essential targets in the treatment of migraine using CGRP receptor antagonists.

Calcitonin gene-related peptide receptor inhibition reduces neuronal activity induced by prolonged increase in nitric oxide in the rat spinal trigeminal nucleus.

Infusion of nitric oxide (NO) donors is known to induce delayed attacks of migraine and cluster headache or aggravate tension-type headaches in patients suffering from these primary headaches. Previously we have reported that infusion of NO donors in the rat causes delayed neuronal activity in the spinal trigeminal nucleus, which parallels the above clinical observations. Suggesting that endogenous NO production is involved in the generation of primary headaches, we used this animal model of meningeal nociception to determine whether a prolonged increase in NO levels causes an increase in neuronal activity. In anaesthetized rats spinal trigeminal neurons with afferent input from the exposed dura were recorded. Continuous intravenous infusion of the NO donors sodium nitroprusside (25 microg/kg/h) or glycerol trinitrate (250 microg/kg/h) for 2 h induced a persisting increase in neuronal activity but no change in systemic blood pressure. In this activated trigeminal system the calcitonin gene-related peptide (CGRP) receptor antagonist BIBN4096BS (900 microg/kg) was infused. Spinal trigeminal activity was significantly reduced within minutes and to a similar extent as previously reported in animals not treated with NO. Slow continuous NO infusion may be a model of the active headache phase, and inhibition of CGRP receptors can reverse the induced neuronal activity.

Increase in NADPH-diaphorase-positive and neuronal NO synthase immunoreactive neurons in the rat spinal trigeminal nucleus following infusion of a NO donor--evidence for a feed-forward process in NO production involved in trigeminal nociception.

Nitric oxide (NO) donors, which cause delayed headaches in migraineurs, have been shown to activate central trigeminal neurons with meningeal afferent input in animal experiments. Previous reports indicate that this response may be due to up-regulation of NO-producing cells in the trigeminal brainstem. To investigate this phenomenon further, we determined nitric oxide synthase (NOS)-containing neurons in the rat spinal trigeminal nucleus (STN), the projection site of nociceptive trigeminal afferents, following infusion of the NO donor sodium nitroprusside (SNP). Barbiturate anaesthetized rats were infused intravenously with SNP (50 microg/kg) or vehicle for 20 min or 2 h, and after periods of 3-8 h fixed by perfusion. Cryostat sections of the medulla oblongata containing the caudal STN were histochemically processed for detection of nicotineamide adenine dinucleotide phosphate (NADPH)-diaphorase or immunohistochemically stained for NOS isoforms and examined by light and fluorescence microscopy. The number of neurons positive for these markers was determined. Various forms of neurons positive for NADPH-diaphorase or immunoreactive to neuronal NOS (nNOS) were found in superficial and deep laminae of the STN caudalis and around the central canal. Neurons were not immunopositive for endothelial (eNOS) or inducible (iNOS) NOS isoforms. The number of NADPH-diaphorase-positive neurons increased time dependently after SNP infusion by a factor of more than two. Likewise, the number of nNOS-immunopositive neurons was increased after SNP compared with vehicle infusion. Around the central canal the number of NADPH-diaphorase-positive neurons was slightly increased and the number of nNOS+ neurons not changed after SNP treatment. NO donors increase the number of neurons that produce NO in the STN, possibly by induction of nNOS expression. Increased NO production may facilitate neurotransmitter release and promote nociceptive transmission in the STN. This mechanism may explain the delayed increase in neuronal activity and headache after infusion of NO donors.

[Placebo therapy. Analysis of extent and expectations in a tertiary referral center]. / Placebotherapie. Analyse von Umfang und Erwartung in einer Klinik der Maximalversorgung.

BACKGROUND: The dimensions of orally administered pharmacological placebos in routine clinical practice and the attitude of the clinical staff towards placebos are widely unknown. The aim of this report was to examine the frequency, indications and the intentions of placebo use at the Medical University of Hannover (MHH). METHODS: This study was performed as an anonymous cross-sectional written survey at the MHH. Quantitative data on placebo requests registered by the dispensary were obtained in advance. RESULTS: A total of 74% of respondents reported using placebos in clinical practice, including 53% of physicians and 88% of the nursing staff. Pain (76%) and insomnia (59%) were the most frequently reported reasons for administering placebos. Placebos were considered to be highly effective by 28.5% of physicians and 63.8% of the nursing staff. CONCLUSION: The effective use of pharmacological placebos appears to be an established component of the therapeutic options of a tertiary referral center. The placebo effect seems to contain remarkable potential. While the use of pharmacological placebos is ethically problematic within the clinical context, the improvement of caregiver-patient interactions and the utilization of positive suggestion could serve as an ideal adjunct to active therapy regimes.

Black TiO2 nanotubes: Efficient electrodes for triggering electric field-induced stimulation of stem cell growth.

TiO2 nanostructures represent a key platform for biomedical applications, due to the combination of biocompatibility and high surface area. Especially TiO2 nanotube layers have been widely investigated due to controllable nanotopographic effects as well as for electrodes in electrostimulation experiments. In the present work we produce Ar/H2-reduced 'black' TiO2 nanotube arrays with a strongly enhanced electrical conductivity and explore their interaction with mesenchymal stem cells when used as electrodes to apply electric fields (EF) across the cells. While we observe no significant change in cell adhesion and their focal contact formation on these high conductivity nanotubes, we do observe a rapid stem cell response when EF is engaged using the 'black' TiO2 nanotube arrays as electrodes. Compared to as-formed nanotube arrays, a faster stem cell growth was observed and a lower EF intensity caused an intracellular calcium level elevation. Our results indicate that the increased conductivity in TiO2 nanotubes significantly enhances the early stem cell response to minimal electric field stimuli. STATEMENT OF SIGNIFICANCE: The use of TiO2 nanostructures in biomedical applications is widely investigated, especially considering the nanostructured surface influence on the biomaterial-cell interactions. We have previously shown that an applied electric field (EF) on stem cells grown on TiO2 nanotubes leads to synergistic osteogenic stimulation in the absence of biochemical bone-inducing supplements. Here we report that black (i.e. highly conductive nanotubes obtained by reduction treatments) TiO2 nanotubes enable short-time EF effects on stem cells: we observe a faster stem cell growth and a significantly enhanced early stem cell response to minimal EF stimuli. The application of such nanostructures under electric field is promising for therapeutic interventions for bone regeneration and tissue engineering approaches.

Participation and mental well-being of mothers of home-living patients with spinal muscular atrophy.

Proximal spinal muscular atrophy (SMA) causes severe physical limitations but also has a major impact on the lives of parents. The aim of this study was to investigate participation and mental well-being (burden, emotional distress and satisfaction with participation) of parents of home-living patients with SMA. Caregiver burden was assessed with the Caregiver Strain Index, emotional distress with the Hospital Anxiety and Depression Scale and satisfaction with participation with the Utrecht Scale for Evaluation of Rehabilitation-Participation. Because the majority of parents were mothers of home-living SMA patients (76%), further analyses were restricted to mothers. Seventy-seven percent of mothers of patients with SMA had paid work. A substantial proportion of mothers (76%) perceived high caregiver burden. Burden, emotional distress and satisfaction with participation were comparable between mothers of children and mothers of adults with SMA. Caregivers' participation in leisure activities was significantly related to their perceived level of caregiver burden, emotional distress and satisfaction with participation. Mothers engaging in more social and leisure activities reported lower emotional distress and caregiver burden. Considering the high level of burden attention should be paid to mental well-being of primary caregivers of patients with SMA. Caregivers should be motivated to keep participating in social/leisure activities.

Excitatory nicotinic and desensitizing muscarinic (M2) effects on C-nociceptors in isolated rat skin.

The actions of different cholinergic agonists and antagonists were investigated on nociceptive afferents using the rat skin-saphenous nerve preparation, in vitro. Nicotine was able to weakly excite C-nociceptors and to induce a mild sensitization to heat stimulation (in 77% of tested fibers) in a dose-dependent manner (10(-)6 to 10(-)5 m), but it caused no alteration in mechanical responsiveness tested with von Frey hairs. Muscarine did not induce a significant nociceptor excitation, but almost all fibers exhibited a marked desensitization to mechanical and heat stimuli in a dose-dependent manner (from 10(-)6 to 10(-)4 m). The muscarinic effects could be prevented by the general muscarinic antagonist scopolamine (10(-)5 m), by the M3 antagonist 1,1-dimethyl-4-diphenylacetoxypiperidium oxide (10(-)6 m) co-applied with the M2 antagonist gallamine (10(-)5 m), and by gallamine alone. As positive control we used the relatively M2-selective agonist arecaidine (10(-)6 to 10(-)5 m), obtaining a similar desensitizing effect as with muscarine. Finally, we performed an immunocytochemical study that demonstrated the presence of M2 but not M3 receptors in thin epidermal nerve fibers of the rat hairy skin. Altogether, these data demonstrate opposite effects of nicotinic and muscarinic receptor stimulation on cutaneous nociceptors. M2 receptor-mediated depression of nociceptive responsiveness may convey a therapeutic, i.e., analgesic or antinociceptive, potential.

Drug-binding and other physicochemical properties of a large tryptic and a large peptic fragment of human serum albumin.

The diazepam-binding behaviour of a large tryptic and a large peptic fragment of human serum albumin has been studied by circular dichroism and equilibrium dialysis in order to locate the primary diazepam-binding site on the albumin molecule. The analytical set-up of the FPLC was used to find the optimum experimental conditions for isolating the fragments. Conventional columns with a 100-fold higher loading capacity than the analytical FPLC columns were used to isolate large amounts of the fragments. The isolation procedure for the tryptic fragment (45 kDa, domains two and three of the albumin structure) is described in this paper. The description of the isolation procedure for the peptic fragment (46 kDa, domains one and two of the albumin structure) is published elsewhere (Bos, O.J.M., Fischer, M.J.E., Wilting, J. and Janssen, L.H.M. (1988) J. Chromatogr. 424, 13-21). The induced ellipticity of the diazepam-fragment complexes as well as the affinity of diazepam to the fragments turned out to be pH dependent. This pH dependence occurs in the region of the neutral to base transition of the albumin molecule. Difference CD-spectra of the proteins showed that the tryptic fragment and albumin have similar diazepam-binding properties, whereas the peptic fragment has different diazepam-binding properties. This result is in line with our equilibrium dialysis experiments which showed that the affinity of diazepam to the tryptic fragment and to albumin is of the same order of magnitude, whereas the affinity of diazepam to the peptic fragment is several orders of magnitude lower. On the basis of these results, it can be concluded that the tryptic fragment contains the primary diazepam-binding site and the peptic fragment one or more secondary diazepam-binding sites. This means that at least the main part of the primary diazepam-binding site is located in domain three of the albumin structure.

Kinetic evaluation of the oxidation of phenothiazine derivatives by methemoglobin and horseradish peroxidase in the presence of hydrogen peroxide. Implications for the reaction mechanisms.

The oxidation of ten 2-substituted 10-(3-(dimethylamino)propyl) phenothiazines (PHs) by methemoglobin (metHb) and horseradish peroxidase (HRP) in the presence of H2O2 was kinetically analysed based on an enzymic-chemical second-order reaction with substrate regeneration: PHs are oxidized enzymatically to their radical cations (PH+) which subsequently, in a second order reaction, react further to parent compound and PH-sulfoxide (PHSO). The enzymic reaction rate can be obtained from the accumulation curves of both radical cation formation and sulfoxide formation. In the case of chlorpromazine and promazine both methods gave similar reaction rates. The rate constant of PH+. decay could also be determined from the radical concentrations of their radicals. The rate constant of reaction of PHs with HRP compound II was also analysed. The logarithm of this rate constant correlated well with the Hammett sigma para and the Swain and Lupton F and R substituent constants, whereas no correlation with hydrophobic and steric parameters was found. This indicates that the interaction of PH with the porphyrin ring, which is the active site of HRP, is predominantly under electronic control. In the case of catalysis by hemoglobin (Hb), the formation of the reactive Hb form, ferry1Hb with a protein radical, appeared to be rate limiting in the oxidation of PHs by metHb-H2O2. Differences in the conversion rates of various PHs can be explained by a competition between their electron transfer reaction to the protein radical and the denaturation reaction(s) involving the protein radical. Our results confirm our earlier observation that the mechanism of oxidation by metHb-H2O2 differs from that of the classical peroxidases. In the former case, electron transfer from PH occurs most likely to a tyrosine residue on the globin part, whilst in the latter case electron transfer to the porphyrin moiety takes place.

Exploring computational lead optimisation with affinity constants obtained by surface plasmon resonance for the interaction of PorA epitope peptides with antibody against Neisseria meningitidis.

LUDI is a program used for de novo structure-based design of ligands and can predict binding of ligands quantitatively using a scoring function. Here we evaluate LUDI in a lead optimisation study with ligands for the antibody MN12H2, that has been raised against outer membrane protein PorA epitope P1.16 of Neisseria meningitidis. The ligands were synthetic peptides that are derived from the smallest binding epitope (182)DTNNN(186). LUDI's fragment building rules are used for the proposal of new peptide-ligands for MN12H2 and were focused on replacements of Asp(186) in the epitope. Accordingly, a series of peptides was synthesised with isosteric mutations. The interaction of the peptides with MN12H2 was analysed with a surface plasmon resonance competition assay yielding equilibrium binding constants in solution (K(S)). The binding affinity seems to be largely determined by entropy, and the side chain of Asn(186) is sensitive for charge, inversion, hydrophobicity and size. Head-to-tail cyclisation of the peptide in a nine-amino-acid ring gives little reduction in affinity. It is concluded that the scoring function of LUDI does not help in optimisation of the peptide lead for MN12H2 binding. Other more elaborate molecular mechanics calculations show similar results. This implies that our current knowledge of molecular recognition is insufficient for explaining a case of peptide-protein binding, where the design process requires subtle changes in structure (from lead finding to lead optimisation).

Ramipril prevents impaired endothelium-dependent relaxation in arteries from rabbits fed an atherogenic diet.

Endothelium-dependent relaxation in arteries is attenuated in clinical and experimental atherosclerosis. This study investigates the endothelial preservation properties of the angiotensin converting enzyme inhibitor, ramipril, by assessing its ability to restore endothelium-dependent responsiveness in blood vessels from rabbits fed an atherogenic diet (0.25% cholesterol; 3% coconut oil; 12 weeks). Seven rabbits fed the atherogenic diet received ramipril (3 mg/kg mixed into their food daily) and 6 rabbits were maintained on the atherogenic diet alone. Control rabbits (n = 6) were fed a standard diet and did not receive ramipril. At the end of the dietary intervention, the rabbits were killed and blood was collected for measurement of the lipid profile. The thoracic aorta was isolated and half was frozen for pathologic review while the other half was cut into rings and placed in a muscle bath for measurement of isometric force development. Dose response curves to phenylephrine (10(-9) to 10(-5) M) and angiotensin II (10(-10) to 3 x 10(-7) M) were completed. There was a minimal decrease in responsiveness to phenylephrine in vessels from rabbits eating the atherogenic diet compared with controls and no significant differences in the response to angiotensin II for any of the vessels. Following contraction by phenylephrine, acetylcholine (10(-9) to 10(-5) M) and nitroglycerin (10(-10) to 10(-5) M) dose response curves were completed. Relaxation to acetylcholine in aortic rings from control rabbits was observed, although in arteries from atherogenic rabbits relaxation was attenuated. This effect was prevented in the atherogenic rabbits fed ramipril. Responsiveness to the endothelium-independent vasodilator, nitroglycerin, was similar in arteries from the three rabbit groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics and safety of transcorneal iontophoresis of tobramycin in the rabbit.

Transcorneal iontophoresis of tobramycin in normal eyes of New Zealand white rabbits was compared to an eye cup control and application of fortified topical drops. Iontophoresis was performed with 25 mg/ml of tobramycin at 0.8 mAmps for 10 or 5 min. The eye cup with 25 mg/ml of tobramycin was placed on the eye for 10 min without current. Topical fortified drops (13.6 mg/ml) were applied every half hour for 4 hr. Epithelium, stroma, and aqueous humor were assayed separately at 1, 4, 8 and 16 hr after treatment. The eyes were examined using the slit-lamp biomicroscope before and immediately after the treatment, and prior to sacrifice. Two eyes were examined by light and scanning electron microscopy 5 and 10 min after iontophoresis. Iontophoresis yielded significantly higher tobramycin concentrations than the eye cup or fortified topical drops at 1 hr and 4 hr after treatment (P = 0.001). In all treated eyes, iontophoresis resulted in epithelial edema and mucous discharge, which resolved by 24 hr after the treatment. Histologically there was evidence of epithelial disruption in the superficial layer after 5 min of iontophoresis and in all layers of the epithelium after 10 min of iontophoresis. Iontophoresis is a relatively safe, effective method to deliver medications to ocular tissues and may be useful alone or as an adjunct to current modes of antimicrobial chemotherapy.

Mechanism by which warfarin binds to human serum albumin. Stopped-flow kinetic experiments with two large fragments of albumin.

In order to obtain information about the kinetics of the process by which warfarin binds to human serum albumin at a molecular level, we performed stopped-flow kinetic experiments on albumin and on a large peptic fragment (residues 1-387) and a large tryptic fragment (residues 198-585) of albumin. From these experiments it can be concluded that the first interaction between warfarin and the proteins is almost certainly diffusion-controlled and is dependent on the net charges of the reactants. The next step in the binding process involves the formation of an activated warfarin-protein complex, whereafter the final complex is formed. The warfarin-albumin complex forms more slowly than the warfarin-fragment complexes, because the formation is sterically hindered by the albumin structure. We think it very unlikely that albumin has an oblate ellipsoid structure; it is much more likely to have a U-shaped structure, where the domains make contact with each other. If this hypothesis is correct, then this indicates that the domains do not act independently of each other. The formation of the activated warfarin-albumin complex is further influenced by the conformational state of the albumin molecule, i.e. the N-B transition. The possible role of this N-B transition in albumin-mediated transport of drugs through cellular membranes is discussed.

Location and characterization of the warfarin binding site of human serum albumin. A comparative study of two large fragments.

The warfarin binding behaviour of a large tryptic fragment (residues 198-585 which comprise domains two and three) and of a large peptic fragment (residues 1-387 which comprise domains one and two) of human serum albumin has been studied by circular dichroism and equilibrium dialysis in order to locate and characterize the primary warfarin binding site. The induced ellipticity of the warfarin-peptic fragment complex turned out to be pH dependent. This pH dependence occurs in the region of the neutral-to-base transition of the albumin molecule. The induced ellipticity of the warfarin-tryptic fragment complex is pH independent. Difference CD-spectra showed that the peptic fragment and albumin have similar warfarin binding properties whereas the tryptic fragment has deviant warfarin binding properties. The equilibrium dialysis experiments showed that the affinity of warfarin to the peptic fragment and to albumin is practically the same whereas the affinity of warfarin to the tryptic fragment is a factor 2-8 lower than the affinity of warfarin to albumin. Our results indicate that the main part of the primary warfarin binding site is located in domain two of the albumin structure and that domain one plays an important role in the N-B transition of albumin.

Steroid binding to human serum albumin and fragments thereof. Role of protein conformation and fatty acid content.

The binding properties of the steroids testosterone and pregnenolone to human serum albumin (HSA) and derived fragments of albumin have been investigated by means of equilibrium dialysis and circular dichroism. The 46 kDa peptic fragment (P46) of HSA comprises domains one and two of the HSA structure, whereas the other fragment, the 45 kDa tryptic fragment (T45) is composed of domains two and three. A comparison of the binding behaviour of the steroid ligands to HSA and its fragments showed that the single primary testosterone binding site in all probability is located in the second domain of the HSA molecule. For pregnenolone it was found that at least two primary binding sites are present, also located in domain two. Both steroids show pH dependent binding profiles in the case of HSA and the P46 fragment. The binding of the steroids to the T45 fragment seems to be pH independent. The same phenomenon was observed with the circular dichroism experiments, indicating a link between the steroid binding properties and the structural behaviour of the proteins. In fact, the binding properties of the steroids can be assigned to the neutral-to-base (N-B) transition. The possible role of fatty acids as modulators in the transport processes of steroids in the body is discussed.

Effect of albumin on adenylate cyclase receptor-related signal transduction of human peripheral blood mononuclear cells.

In the present study we investigated in vitro the effect of human serum albumin (HSA) on receptor-stimulated cAMP production in isolated human peripheral blood mononuclear cells (PBMC). The cAMP production is strongly correlated with the pH of the medium during long incubations with albumin. Adenylate cyclase is stimulated by receptor agonists like histamine, forskolin, prostaglandin E2 and the beta-adrenergic agonist isoprenaline, in the presence or absence of HSA. This protein, at concentrations above 0.1%, dose-dependently inhibits both basal and agonist-stimulated cAMP levels in PBMC. In the presence of 0.5% HSA a significant reduction of 30-60% (cell batch dependent) is induced, a reduction which is not incubation time dependent. Washing the cells after a period of incubation with 2% HSA does not reverse the HSA-induced cAMP inhibition. Oleic acid-evoked conformational changes in HSA were not capable of influencing the inhibition processes of HSA on the isoprenaline-stimulated cAMP production. Structure-controlled interactions between HSA and membrane or adenylate cyclase are therefore unlikely. Bovine serum albumin and chicken albumin had different effects upon the agonist-stimulated cAMP production as compared with HSA. At this moment no explanation for this behavior can be provided. The findings indicate that albumin may inhibit non-specifically cAMP production in PBMC and possibly influences membrane-controlled processes.