A method of isolating and analysing drugs from cancer cells for preclinical research.

The presented paper describes a new isolation method of recovery and analysis of selected drugs developed for preclinical research. The method uses the RP-HPLC technique (in a single chromatographic separation) and serves the recovery and analysis of selected drugs from neoplastic cells. It enables the determination of cytostatics statins, fibrates, and pioglitazone. Chromatographic separations of the tested compounds were carried out on a Gemini-NX 5 µ C18 (4.6 × 150 mm i.d.) column, in a gradient system with a mobile phase consisting of ACN (0.1% TFA) and water (0.1% TFA) at ambient temperature. The separations were carried out at a flow of 1 ml/min and UV detection of 220 nm. The inter-day and intra-day precision and accuracy of the method were determined. Extending the extraction time at reduced temperature resulted in a significant increase in the recovery of the pharmaceuticals in comparison with traditional extraction methods. The presence of the tested pharmaceuticals at defined retention times was confirmed by mass spectrometry. A recovery procedure for the tested compounds from biological material (medium, cell pellets) was developed at a level ranging between 93 and 99%. The utility of the new HPLC method has been confirmed in drug absorption studies as screening tests for the analysis of the new therapeutic compositions on melanoma cell lines.

Application of the ETD/PTR reactions in top-down proteomics as a faster alternative to bottom-up nanoLC-MS/MS protein identification.

Our goal was to compare two popular analytical techniques used nowadays in proteomic investigations for proteins/peptides sequencing and identification, a widely used nanoLC-MS/MS approach applied in the bottom-up proteomics and electron transfer dissociation/proton transfer reaction fragmentation preferably used when top-down strategy is applied. Comparison was carried out with the aid of the ESI-quadrupole ion-trap instrument using the following criteria: total time of analysis including sample preparation, sequence coverage, Mascot scoring, capability to detect modifications, quality of the results as a function of protein molecular weight and sample consumption.

Modulation of neuropeptide FF (NPFF) receptors influences the expression of amphetamine-induced conditioned place preference and amphetamine withdrawal anxiety-like behavior in rats.

Many data indicate that endogenous opioid system is involved in amphetamine-induced behavior. Neuropeptide FF (NPFF) possesses opioid-modulating properties. The aim of the present study was to determine whether pharmacological modulation of NPFF receptors modify the expression of amphetamine-induced conditioned place preference (CPP) and amphetamine withdrawal anxiety-like behavior, both processes relevant to drug addiction/abuse. Intracerebroventricular (i.c.v.) injection of NPFF (5, 10, and 20 nmol) inhibited the expression of amphetamine CPP at the doses of 10 and 20 nmol. RF9, the NPFF receptors antagonist, reversed inhibitory effect of NPFF (20 nmol, i.c.v.) at the doses of 10 and 20 nmol and did not show any effect in amphetamine- and saline conditioned rats. Anxiety-like effect of amphetamine withdrawal was measured 24h after the last (14 days) amphetamine (2.5mg/kg, i.p.) treatment in the elevated plus-maze test. Amphetamine withdrawal decreased the percent of time spent by rats in the open arms and the percent of open arms entries. RF9 (5, 10, and 20 nmol, i.c.v.) significantly reversed these anxiety-like effects of amphetamine withdrawal and elevated the percent of time spent by rats in open arms at doses of 5 and 10 nmol, and the percent of open arms entries in all doses used. NPFF (20 nmol) pretreatment inhibited the effect of RF9 (10 nmol). Our results indicated that stimulation or inhibition of NPFF receptors decrease the expression of amphetamine CPP and amphetamine withdrawal anxiety, respectively. These findings may have implications for a better understanding of the processes involved in amphetamine dependence.

Strain rate and tissue tracking imaging in quantitation of left ventricular systolic function in endurance and strength athletes.

AIM: The aim of this study was to assess the impact of long-term physical training on left ventricular longitudinal contraction by strain rate analysis and tissue tracking imaging. METHODS AND RESULTS: The study population comprised 17 male elite endurance and 15 male elite strength athletes and 12 male control subjects of similar age. Tissue Doppler imaging was recorded in the apical views and used for analysis of the longitudinal systolic myocardial velocity, annular diastolic velocities, strain rate and tissue tracking. Left ventricular mass index was significantly increased in both endurance athletes (209+/-40 g/m(2)) and strength athletes (138+/-38 g/m(2)) compared with normal subjects (96+/-20 g/m(2), P<0.001). Tissue tracking score index and mean strain rate of the 16 segments were significantly increased in strength athletes (7.9+/-1.1 mm and -1.4+/-0.3 s(-1), respectively) compared with endurance athletes (7.5+/-0.9 mm and -1.0+/-0.4 s(-1), P<0.01 for both) and normal subjects (7.4+/-1.0 mm and -1.0+/-0.3 s(-1), P<0.01 for both). CONCLUSION: Despite significant left ventricular hypertrophy and extensive training in elite athletes, we found normal longitudinal left ventricular systolic function, and in strength athletes performing isometric exercise even increased function.

Morphinome--proteome of the nervous system after morphine treatment.

Proteome is a natural consequence of the post-genome era when the HUGO project (Human Genome Organization) has almost been completed. Here, a specifically aimed proteome in drug dependence--morphinome, is described, including tasks, strategies and pitfalls of the methodology.

Dynorphin A inhibits nociceptin-converting enzyme from the rat spinal cord.

Cysteine proteinase found in the spinal cord of rat, called nociceptin-converting enzyme (NCE), is competitively inhibited by dynorphin A and its fragment des-[Tyr(1)]-DYN A. This proteinase converts orphanin FQ/nociceptin (OFQ/N) to two major fragments: OFQ/N(1-11) and further OFQ/N(1-6) with analgesic properties. Dynorphin A at the concentration of 10 microM increases K(M) from 15.0 to 55.9 microM. The calculated K(i) for this interaction was estimated at 3.7 microM. This observation may suggest an interaction between opioid and nociceptive systems which may be affected by the balance between opioid and antiopioid systems. This balance between particular OFQ/N sequences that are derived from the same precursor and regulated by proteinases may play an important role in pain. Interestingly, dynorphin B does not reveal a similar action on the NCE.

C-Terminal glycine is crucial for hyperalgesic activity of nociceptin/orphanin FQ-(1-6).

A C-terminal analog of the hexapeptide orphanin FQ/nociceptin-(1-6), [Ala(6)]-orphanin FQ/nociceptin-(1-6), and a pentapeptide orphanin FQ/nociceptin-(1-5) were tested in vivo for their analgesic/hyperalgesic activity in the hot-plate test with rats. Replacement of the C-terminal glycine by L-alanine (Phe-Gly-Gly-Phe-Thr-Ala) in orphanin FQ/nociceptin-(1-6) abolished the hyperalgesic potency of native orphanin FQ/nociceptin-(1-6) (Phe-Gly-Gly-Phe-Thr-Gly), but analgesic activity was retained and was diminished by naloxone. Removal of the C-terminal amino acid (glycine or alanine) from orphanin FQ/nociceptin-(1-6) caused a significant loss of analgesic activity. It is anticipated that glycine plays a crucial role in the biphasic activity of orphanin FQ/nociceptin-(1-6). This may suggest the existence of a mechanism for terminating the biological action of orphanin FQ/nociceptin.

Determination of nociceptin-orphanin FQ metabolites by capillary LC-MS.

Nociceptin-orphanin FQ (OFQ/N) is a newly discovered peptide involved in pain transmission. The method is described to identify metabolic pathway of this neuropeptide in the spinal cord of rats using capillary size-exclusion liquid chromatography coupled to electrospray ionization mass spectrometry. The applied technique is rapid and selective, and allows for simultaneous measurement and quantitation of several fragments in the incubation mixture.

Orphanin FQ/nociceptin inhibits morphine withdrawal.

The influence of orphanin FQ/nociceptin (OFQ/N) on the morphine-withdrawal symptom was investigated. Withdrawal syndrome was induced in the morphine-dependent rats by an intraperitoneal (i.p.) injection of 2 mg/kg naloxone hydrochloride--an opioid receptors antagonist. Wet-dog shakes were used as a measure of the abstinence syndrome. Intraventricular injections of OFQ/N (5-20 microg/animal) caused significant inhibition of the withdrawal signs at doses between 15-20 microg, in the morphine-dependent rats. OFQ/N alone did not change behavior of the morphine-dependent animals. The obtained results indicate that OFQ/N can inhibit the morphine withdrawal symptoms induced by naloxone.

Metabolic fate of nociceptin/orphanin FQ in the rat spinal cord and biological activity of its released fragment.

Metabolism of orphanin FQ/nociceptin (OFQ/N) was studied in the spinal cord of rats. The heptadecapeptide was efficiently cleaved by a neutral serine endopeptidase, thus releasing the major metabolite, OFQ/N(1-11), further truncated to the final product, OFQ/N(1-6). Biologic activity of this latter fragment was tested in vivo, after intracerebroventricular and intrathecal injections. Hexapeptide exhibited a bi-phasic effect, causing antinociception up to 10 min after injection, followed by a hyperalgesia. The analgesic effect was blocked by naloxone and hyperalgesia was inhibited by NMDA--and NMDA/glycine site antagonists. The results indicate that shorter nociceptin fragments still possess their biologic activity though possibly acting via receptors other than ORL1.

Biotransformation of nociceptin/orphanin FQ by enzyme activity from morphine-naive and morphine-treated cell cultures.

The biotransformation of nociceptin/orphanin FQ (NOFQ) by enzyme activity isolated from U1690 human lung carcinoma and SH-SY5Y human neuroblastoma cell lines, and from rat brain cortex cells in primary culture was investigated. The identification and quantification of the cleavage products were performed using electrospray ionization mass spectrometry linked to size-exclusion chromatography. The effect of chronic morphine treatment of the cells (5 days) on NOFQ biotransformation was also studied. It was found that major products generated from NOFQ were the amino-terminal peptides N1-9 and N1-13. The pattern of NOFQ biotransformation was quite similar for all three cell cultures. However, different proportions of the formed peptides were noted. The cleavage was inhibited by EDTA, PMSF, Hg2+, Cu2+ and Zn2+. Dynorphin A2-13 inhibited NOFQ cleavage in a manner suggesting competition of the two peptides for the same enzyme. Chronic morphine treatment of the cell cultures resulted in a substantial increase in the enzyme activity, leading to higher levels of the major fragments and accumulation of N1-12 and the shorter peptides N1-5, N1-6. Since the effect of morphine treatment of the cells was blocked by naloxone, it is likely that it was receptor specific. Taken together, the findings suggest that a metallosensitive endopeptidase, the activity of which is increased by chronic morphine treatment of the cells, is responsible for the biotransformation of NOFQ with fragments N1-9 and N1-13 being the major products.

Magnetic resonance imaging evaluation of capsulolabral tears after traumatic primary anterior shoulder dislocation. A prospective comparison with arthroscopy of 25 cases.

The purpose of our study was to evaluate the use of static magnetic resonance imaging (MRI) as a preoperative diagnostic tool in young patients with a traumatic primary anterior shoulder dislocation. Twenty-five patients who had acute primary traumatic anterior shoulder dislocation were examined with MRI and arthroscopy. The patients (18 male and 7 female) were between 16 and 39 years old (mean age, 27 years). They had no previous shoulder dislocations. The dislocations were confirmed radiographically. Examination with MRI and arthroscopy was performed within 10 days after the trauma. The MRI evaluation was performed before the arthroscopic examination, and the images were interpreted by an experienced magnetic resonance radiologist. No information from the MRI examination was available to the orthopedic surgeons before arthroscopy. The standard of reference for comparison was arthroscopy. Subacute MRI evaluation identified 15 labral tears, 12 Hill-Sachs lesions, 1 total rotator cuff lesion, 1 partial joint side rotator cuff lesion, and 1 partial rupture of the biceps tendon. Arthroscopic examination revealed 22 labral tears, 15 Hill-Sachs lesions, 1 total rotator cuff lesion, 1 partial joint side rotator cuff tear, 1 partial rupture of the biceps tendon, and 1 osseous Bankart lesion. Anterior capsulolabral tears and Hill-Sachs lesions appeared with a high incidence after acute anterior primary shoulder dislocation. Conventional MRI was only moderately reliable in the preoperative evaluation of labral tears and Hill-Sachs lesions, and it failed to give an accurate, differentiated preoperative diagnosis of the capsulolabral lesions.

[Reduction of traumatic primary anterior shoulder dislocation under local analgesia]. / Reponering af traumatisk primaer anterior skulderluksation i lokal analgesi.

The aim of the present study was to evaluate the value of local versus intravenous anaesthesia in the reduction of acute shoulder dislocations. Patients with a primary traumatic dislocation of the shoulder were randomized to either local lidocaine or intravenous anaesthesia with pethidine/diazepam. The local method was performed with 20 ml of 1% lidocaine. The intravenous method was performed with pethidine/diazepam injected intravenously. The patients were observed for any complication during and after the procedure and the used methods were evaluated using a Visual Analogue Scale (VAS). In the period from November 1991 to September 1993 81 patients were admitted to our departments and 68 patients were included. Average age was 48 years (range 15-79) with 29 men and 39 women. Thirty-five patients were randomized to intravenous anaesthesia, 33 had a successful reduction and two failed. Thirty-three patients received local anaesthesia, 32 succeeded and one failed. Ten patients treated with the intravenous method had respiratory depression and six required antidote. No systemic or local side effects and no neuro-vascular injuries were recorded with the use of lidocaine. We did not observe any superficial or deep infection in the lidocaine group. There was no statistical difference between the average VAS value in the two groups. Local anaesthesia used to reduce acute primary anterior dislocation of the shoulder is a simple, safe and well-accepted method with significantly fewer respiratory complications.

Reduction of traumatic secondary shoulder dislocations with lidocaine.

The aim of the present study was to evaluate the value of local anaesthesia versus the commonly used intravenous pethidine/diazepam in the reduction of acute secondary shoulder dislocations. Patients with a traumatic secondary dislocation of the shoulder were randomized to either locally injected lidocaine or intravenously injected pethidine/diazepam. The local method was performed with 20 ml of 1% lidocaine. The patients were observed for any complication during and after the procedure, and the methods used were evaluated with a visual analogue scale (VAS). From November 1991 to September 1993, 62 patients were admitted to our departments of whom 52 were included in the study. Average age was 47 years (range 18-89 years) with 24 men and 28 women. Twenty-six patients were randomized to pethidine/diazepam; 22 had a successful reduction, and 4 were failures. Twenty-six patients received lidocaine, of whom 18 were successful and 8 not. Three patients treated with the intravenous method suffered respiratory depression, and one required an antidote. No systemic or local side-effects, no neurovascular damage and no early or late superficial or deep infection were recorded in the lidocaine group. There was no statistical difference between the average VAS value in the two groups. Lidocaine used to reduce acute secondary dislocations of the shoulder is a simple and safe method. It is as effective as the standard intravenous method and is well accepted by patients.

Intraarticular findings in the chronically painful shoulder. A study of 32 posttraumatic cases.

32 consecutive patients suffering from chronic shoulder pain for more than 6 months after a single, nondislocating shoulder trauma were examined clinically and by special radiographs, dynamic sonography, MRI and arthroscopy. Typical complaints were pain during loading, especially during over the head activities. Symptoms of a "dead arm" and instability were also present. Patients with previous dislocations, traumas or radiographic signs of degenerative shoulder lesions were excluded. The patients had a decreased active range of motion and positive signs of apprehension and impingement, but only 4 had clinical signs of shoulder instability. Diagnostic evaluation identified labral tears, partial and total rotator cuff lesions with subacromial impingement and tendinitis of the biceps tendon. Surgery was performed in 24 patients, using capsulolabral and rotator cuff reconstruction, arthroscopic labral resection and open subacromial decompression. In conclusion, patients with chronic posttraumatic shoulder pain have intraarticular injuries, especially tears of the glenoid labrum. History, clinical findings, radiography and sonography are seldom diagnostic. MRI is valuable, particularly for identification of labral pathology, but arthroscopy appears necessary for a preoperative assessment.

[Arthroscopy of post-traumatic shoulder pain]. / Artroskopi ved posttraumatiske skuldersmerter.

Thirty-two consecutive patients suffering chronic shoulder pain for more than six months after a non-dislocating shoulder trauma were examined clinically and with arthroscopy of the shoulder. The trauma was blunt or simple distortion of the shoulder. The patients complained of pain during loading, especially during over the head activities. Symptoms of dead arm and feeling of instability with a popping sensation inside the shoulder were also present. Patients with previous dislocations, trauma or radiographic signs of degenerative shoulder lesions were excluded. Clinically, 21 patients had decreased range of motion, 19 patients had a positive anterior apprehension sign and four of these had signs of shoulder instability. Nine patients had signs of impingement. The suspected preoperative diagnoses included a tear of the rotator cuff (17), a tear of the labrum (12), tendinitis of the biceps tendon (2) and periarthrosis of the shoulder (1). Arthroscopic findings consisted of 22 labral tears, six partial and three total rotator cuff lesions and three cases of synovitis of the rotator cuff with signs of subacromial impingement. Four patients had tendinitis of the biceps tendon. One patient had a lesion of the greater tubercle. Only two shoulders were found to be unstable under anaesthesia. In conclusion, patients with posttraumatic chronic shoulder pain after a non-dislocating trauma of the shoulder should be evaluated with arthroscopy in order to diagnose possible intraarticular lesions.

Reduction of traumatic, primary anterior shoulder dislocations with local anesthesia.

This study evaluates the use of local anesthesia in the reduction of acute shoulder dislocations. Patients with a primary traumatic dislocation of the shoulder were randomly assigned to receive either local anesthesia or intravenous anesthesia. The patients were observed for any complication during and after the procedure, and the methods used were evaluated with a visual analog scale. In the period from November 1991 to September 1993, 81 patients were admitted to our departments, and 68 patients were included in the study. Average age was 48 years (range 15 to 79 years); 29 men and 39 women were studied. Thirty-five patients were randomly assigned to receive intravenous anesthesia; 33 had a successful reduction, and two had a failed reduction. Thirty-three patients received local anesthesia; 32 had a successful reduction, and one had a failed reduction. Ten patients treated with the intravenous method had respiratory depression, and six required an antidote. No systemic or local side effects and no neurovasculor injuries were recorded. We did not observe any superficial or deep infection in the local anesthetic group. No statistical difference was found between the average visual analog value scale in the two groups. Local anesthesia to reduce acute primary anterior dislocation of the shoulder is a simple and safe method.

Effect of local prostaglandin E2 on fracture callus in rabbits.

We investigated the effect of local infusion with prostaglandin E2 (PGE2) in doses of 0.0003 to 4.0 mg/hour per kg body weight for 6 weeks on a plated unilateral osteotomy in rabbits. PGE2 caused a dose-dependent stimulation of callus formation. Total bone mineral content increased, although the mineral content per volume of the callus was reduced. In another experiment, PGE2 was infused either in the first half or in the second half of the healing period. No effect of PGE2 infusion could be observed in the first half of the 6-week healing period, whereas PGE2 infusion during the second half caused callus stimulation.

Glenohumeral movement patterns after puncture of the joint capsule: An experimental study.

In on experimental series comprising 22 shoulder specimens obtained at autopsy, we investigated the influence of an intact capsule on glenohumeral stability. Puncture of the capsule resulted in significant glenohumeral translation in unloaded and loaded specimens during shoulder abduction. A maximum of 16.6 mm of distal translation was observed at 20° of abduction. Concomitant with this translation the humerus spontaneously rotated externally, with a maximum rotation of 15.8° at 50° of abduction. After venting the capsule, anterior and posterior translation and external rotation were increased significantly. Maximum total increase in anteroposterior translation was 14 mm at 30° of abduction. The external rotation was increased up to 7.1° at 40° of abduction. These findings indicate that studies evaluating glenohumeral instability are compromised unless the translations resulting from capsular venting ore corrected. Evaluation of shoulder stability should be performed before violation of the intraarticular pressure mechanisms.