Membrane lipid organization and nicotinic acetylcholine receptor function: A two-way physiological relationship.

Nicotinic acetylcholine receptors (nAChRs) are involved in a great range of physiological and pathological conditions. Since they are transmembrane proteins, they interact strongly with the lipids surrounding them. Thus, the plasma membrane composition and heterogeneity play an essential role for the correct nAChR function, on the one hand, and the nAChR influences its immediate lipid environment, on the other hand. The aim of this work was to investigate in more detail the role of the biophysical properties of the membrane in nAChR function and vice versa, focusing on the relationship between Chol and nAChRs. To this end, we worked with different model systems which were treated either with (i) more Chol, (ii) cholesteryl hemisuccinate, or (iii) the enzyme cholesterol oxidase to generate different membrane sterol conditions and in the absence and presence of γTM4 peptide as a representative model of the nAChR. Fluorescence measurements with crystal violet and patch-clamp recordings were used to study nAChR conformation and function, respectively. Using confocal microscopy of giant unilamellar vesicles we probed the membrane phase state/order and organization (coexistence of lipid domains) and lipid-nAChR interaction. Our results show a feedback relationship between membrane organization and nAChR function, i.e. whereas the presence of a model of nAChRs conditions membrane organization, changing its lipid microenvironment, membrane organization and composition perturb nAChRs function. We postulate that nAChRs have a gain of function in disordered membrane environments but a loss of function in ordered ones, and that Chol molecules at the outer leaflet in annular sites and at the inner leaflet in non-annular sites are related to nAChR gating and desensitization, respectively. Thus, depending on the membrane composition, organization, and/or order, the nAChR adopts different conformations and locates in distinct lipid domains and this has a direct effect on its function.

2DEP cytometry: distributed dielectrophoretic cytometry for live cell dielectric signature measurement on population level.

In this work, a novel force equilibrium method called distributed dielectrophoretic cytometry (2DEP cytometry) was developed. It uses a dielectrophoresis (DEP)-induced vertical translation of live cells in conjunction with particle image velocimetry (PIV) in order to measure probabilistic distribution of DEP forces acting on an entire cell population. The method is integrated in a microfluidic device. The bottom of the microfluidic channel is lined with an interdigitated electrode array. Cells passing through the micro-channel are acted on by sedimentation forces, while DEP forces either oppose sedimentation, support sedimentation, or neither, depending on the dielectric (DE) signatures of the cells. The heights at which cells stabilize correspond to their DE signature and are measured indirectly using PIV, which enables simultaneous and high-throughput collection of hundreds of single-cell responses in a single PIV frame. The system was validated using polystyrene micro-particles. Preliminary experimental data quantify the DE signatures of immortalized myelogenous leukemia cell lines K562 and KG1. We show DEP-induced cell translation along the parabolic velocity profile can be measured by PIV with sub-micron precision, enabling identification of individual cell DE signatures. DE signatures of the selected cell lines are distinguishable. Throughput of the method enables measurement of DE signatures at 10 different frequencies in almost real time.

CAG Repeat Number in the Androgen Receptor Gene and Prostate Cancer.

Prostate cancer (PC) is the second leading cause of cancer deaths in men. The effects of androgens on prostatic tissue are mediated by the androgen receptor (AR) gene. The 5' end of exon 1 of the AR gene includes a polymorphic CAG triplet repeat that numbers between 10 to 36 in the normal population. The length of the CAG repeats is inversely related to the transactivation function of the AR gene. There is controversy over association between short CAG repeat numbers in the AR gene and PC. This retrospective case-control study evaluates the possible effect of short CAG repeats on the AR gene in prostate cancer risk in Macedonian males. A total of 392 male subjects, 134 PC patients, 106 patients with benign prostatic hyperplasia (BPH) and 152 males from the general Macedonian population were enrolled in this study. The CAG repeat length was determined by fluorescent polymerase chain reaction (PCR) amplification of exon1 of the AR gene followed by capillary electrophoresis (CE) on a genetic analyzer. The mean repeat length in PC patients was 21.5 ± 2.65, in controls 22.28 ± 2.86 (p = 0.009) and in BPH patients 22.1 ± 2.52 (p = 0.038). Short CAG repeats (<19) were found in 21.64% of PC patients vs. 9.43% in BPH patients (p = 0.0154). We also found an association of low Gleason score (<7) with short CAG repeat (<19) in PC patients (p = 0.0306), and no association between the age at diagnosis of PC and BPH and CAG repeat length. These results suggest that reduced CAG repeat length may be associated with increased prostate cancer risk in Macedonian men.

Evaluation of the value of p53 protein expression in the extra-capsular extension of prostate cancer.

INTRODUCTION AND OBJECTIVES: The objective of this study is to identify the nuclear expression of the p53 protein in prostate cancer and to determine its relationship with clinico-pathological variables. MATERIAL AND METHODS: The research included 83 patients, 43 of whom are patients with prostate cancer who underwent radical prostatectomy and a control group of 40 patients with benign hyperplasia of the prostate in whom a transurethral resection or a transvesical prostatectomy was undertaken. In all cases the nuclear expression of p53 protein was evaluated. A hystopatological evaluation of the tumour characteristics and the data of the local progression of the cancer were undertaken in the research group. RESULTS: The results show that the expression of the p53 protein does not have an important correlation with the preoperative PSA, but that it is in direct correlation with the malign potential of the cancer (Gleason score, Gleason sum, primary tumour) and with the features of the disease (metastatic lymph nodes, stage of the disease). CONCLUSION: p53 protein could be used as a valid biomarker in determining the malignant potential of the tumour and the prognosis of the disease. There is no practical use in predicting the extraprostatic extension.

Does prostate specific antigen density correlates with aggressiveness of the prostate cancer?

BACKGROUND: As already documented, a high prostate specific antigen in men with normal size of prostate gland is more likely to be associated with an aggressive cancer as compared to others with the same prostate specific antigen and a large gland size. In this retrospective study we tested the association between Prostate Specific Antigen Density (PSAD) and tumor aggressiveness in patients with clinically localized Prostate Cancer (PCa) surgically treated by radical prostatectomy. METHODS: We evaluated data from patients records in a cohort of 72 patients who underwent radical prostatectomy between January 2000 and June 2007. PSAD was calculated as ratio between the preoperative total prostatic specific antigen (PSA) in nanograms per milliliter with the prostate weight (PW) of prostatectomized specimen in grams or prostate volume measured with ultrasound (US). The patients were stratified into four PSAD categories: 0.1-0.15, 0.16- 0.20, 0.21-0.5 and greater than 0.51 ng/ml/gr. Parameters that were included into analysis were: PSA, measurement of the prostate volume by ultrasound (preoperatively) and prostate weight, pathological tumor stage, Gleason sum, Gleason grade, metastatic lymph nodes, seminal vesicle involvement and organ confine disease (postoperatively). Worsening of the clinicopathological properties was defined as aggressiveness. RESULTS: There was a significant correlation between US-PSAD and PW-PSAD (p<0.001). In US-PSAD categories the statistic tests found significant correlation with the primary tumor (R=0.303, p<0.01), metastatic lymph nodes (R=0.331, p<0.01), and the organ confine disease (R=0.296, p<0.05). The PW-PSAD categories correlated significantly with the pathologic findings from other parameters. Hence, a statistically significant correlation was found with Gleason sum (R=0.246, p<0.05), Gleason grade (R=0.234, p<0.05), primary tumor (R=0.285, p<0.05), metastatic lymph node (R=0.287, p<0.05) and organ confine disease (R=0.303, p<0.01). CONCLUSIONS: Prostate specific antigen density measurement is useful tool for the assessment of the degree of aggressiveness in clinically localized prostate cancer, and further investigation regarding its possible use as a prediction marker is justified.

[Intraoperative diagnosis of lymphatic metastases in colon cancer].

Colon carcinoma is leading cause of death in oncology. The precise staging in colon carcinoma by finding micrometastases is extremely difficult task using routine methods. By discovering and assessing the sentinel nodes we could determine more precisely the lymph status of the patient. The basic methods for intraoperative marking of sentinel nodes are based on using of dyes and radionuclides.

Urinary diversion: 30 years experience of a single centre in Republic of Macedonia.

OBJECTIVES: The aim of this report is to present our 30 years experience with various types of urinary diversions, in particular the Bricker and Studer techniques for the management of muscle invasive bladder cancer at our institution. Perioperative, early and late complications are also evaluated. MATERIAL AND METHODS: Between 1977 and 2007, 186 male and 15 female patients underwent combined radical cystectomy, pelvic lymphadenectomy and urinary diversion. In two subgroups of patients we evaluated the complications, divided as early and late, and subdivided as those related or unrelated to the neobladder. Mean follow up time was 28 months (range 12-60 months). RESULTS: Two main types of urinary diversion were performed: the ileal conduit diversion using a technique previously described by Bricker and the ileal neobladder diversion using a technique previously described by a Studer. The ages at surgery ranged from 40 to 82 years with a mean age of 60 years. Histopathologically, transitional cell carcinoma was the most common tumor cell type (93,7%), followed by difuse papilomatosis (5.5%) and adenocarcinoma (0.7%). The pathological tumor stage was pT1 (4.7%), pT2 (31.4%), pT3 (50.3 %) and pT4a (13,3%). Histological evidence of regional lymph node involvement was seen in 25% of the cases. From 52 patients from the Studer subgroup perioperative complications were found in 16 patients (30.7%). Specific early complications directly related to the neobladder occurred in 14 (26.9%) patients. Prolonged ileus in 2 patient (3.8%), ureteral leakage in 9 patients (17.3%), mucous buildup within the diversion in 3 patients (5.7%). Late complications occurred in 10 patients (19.2%): retention of the urine in 4 patients (7.6%) (stricture of the urethra-pouch anastomosis in one 1 patient) and to big reservoir in 3 patients. One patient (1.9%) developed prolonged metabolic acidosis. Stone formation was observed in one patient, two years postoperatively. Unilateral hydroureteronephrosis was observed in 2 patients whereas bilateral hydroureteronephrosis was observed in one patients at one year postoperatively. Perioperative and late complications were similar in the 32 patients from the Bricker subgroup. CONCLUSION: We show that our results with urinary diversion are promising in patients requiring radical cystoprostatectomy. The two methods preferred in our institution offer a sufficient protection of the upper urinary tract with a low complication rate, good voiding function and continence.

Effects of single and long-term theophylline treatment on the threshold of mechanical nociception: contribution of adenosine A1 and alpha2-adrenoceptors.

The effects of single and long-term treatment with theophylline as well as the influence of adenosine A1 receptor agonist cyclopentyladenosine (CPA) and a-adrenergic receptor antagonists prazosin and yohimbine were assessed in the paw pressure test in rats. Both single (37.5 and 75 mg/kg) and long-term (75 mg/kg/day, 14 days i.p.) theophylline treatments exerted antinociceptive effect by increasing the mechanical pain threshold. Single treatment of theophylline (75 mg/kg) antagonized the antinociceptive effect of CPA (0.1 mg/kg); CPA (0.1 mg/kg) abolished the theophylline-induced antinociception. Chronic treatment with theophylline did not change the antinociceptive effect of CPA, while CPA decreased the theophylline antinociception. Yohimbine (0.5 mg/kg), an a 2-adrenoceptor antagonist, diminished the antinociception of a single dose (75 mg/kg) of theophylline, whereas prazocin, an a 1-adrenoceptor antagonist, did not affect it. These results suggest that adenosine A1 and a 2-adrenoceptors take part in the antinociception induced by a single dose of theophylline. The antinociception induced by chronic theophylline treatment probably has a more complex mechanism in which the involvement of adenosine A1.

Radical retropubic prostatectomy for prostate carcinoma; first results of the Clinic of Urology in Skopje.

OBJECTIVES: The aim of this retrospective study is to present our experience and results in the management of prostate carcinoma, with radical retropubic prostatectomy, for a period of seven years. MATERIAL AND METHODS: From December 1997 to April 2005, 61 radical retropubic prostatectomies for prostate carcinoma were performed at the Clinic of Urology in Skopje. Mean age of the treated patients was 66.4 years. Mean serum PSA level was 32.75 ng/ml. None of the patients had distant or bone metastases. Mean operative time was 160 minutes and from 2 to 4 units of blood were transfused intra and postoperatively. Mean follow up time was 39 months. RESULTS: In all of 61 patients, the RRP was performed for adenocarcinoma of the prostate. The pathological findings postoperatively showed the following pTNM grade: pT2a in 8, pT2b in 10, pT3a in 10, pT3b in 27and pT4 in 6 patients. Positive lymph nodes were found in 14 cases. Intraoperative complications occurred in 6 patients. Early postoperative complications were seen in 12 patients. Urine leakage was seen in 2 patients, incontinence (day and night) in 8 and pulmonary embolia in 2 patients. Late postoperative complications occurred in 11 patients. Stenosis of the vesicourethral anasthomosis was seen in 3 patients and incontinence (during the night only) in 8 patients. The rate of potency was not evaluated but in the last 30 cases we insisted on preservation of the neurovascular bundles in the cases that it was possible. CONCLUSION: Radical retropubic prostatectomy is the method of choice and the golden standard for treatment of organ confined prostate carcinoma in patients with long life expectancy, no neither local nor distant metastases and good overall status. With this technique complication rates are minimal, the cure rate is very big and the patients have high quality of life. The experience of the surgeon is very important since the learning curve is crucial for diminishing operative time, postoperative complications and blood transfusions.

The effects of peptide and nonpeptide antagonists of angiotensin II receptors on the noradrenaline uptake of different brain structures in rats with angiotensin II-induced increase of water intake.

Angiotensin II (ANG II) significantly increased noradrenaline (NA) uptake by cortical, hypothalamic and hippocampal synaptosomes thus activating noradrenergic neurotransmission. ANG II did not affect NA uptake by striatal synaptosomes. The interaction between AT1 receptors and noradrenergic neurons and the involvement of brain noradrenergic neurotransmitter system in ANG II-induced drinking in rats is suggested by the increase of NA uptake in hypothalamus and frontal cortex which are rich in AT1 receptors and are of importance for drinking behavior. The ANG II-receptor antagonists losartan, EXP 3174, sarmesin and saralasin decreased NA uptake in all brain regions studied as compared to the uptake in the same brain regions of ANG II-injected animals thus antagonising the effect of ANG II. There is no relationship between the inhibition of ANG II-induced water intake and the changes of NA uptake under the effect of the ANG II-receptor antagonists.

The effects of sarmesin, an Angiotensin II analogue on seizure susceptibility, memory retention and nociception.

The present research studies the effects of sarmesin [Sar(1)Tyr(OMe)(4)] Angiotensin II (ANG II), an analogue of ANG II, on the seizure susceptibility, memory activity and nociception. It was found that this octapeptide, administered i.c.v., dose-dependently decreased the seizure intensity (pentylenetetrazol (PTZ) generalized seizure model and PTZ kindling) and augmented PTZ seizure threshold in mice. Sarmesin impaired the memory upon re-testing of rats 24 h later in the passive avoidance test. It decreased the pain threshold in a paw pressure nociceptive assay in rats. ANG II exerted pronociceptive effect as well. Taken together, these results reveal sarmesin as a behaviorally active peptide in the studied experimental animal models.

The effects of peptide and nonpeptide antagonists of angiotensin II receptors on the level of brain biogenic monoamines in rats with angiotensin II-induced increase of water intake.

The effects of peptide and nonpeptide angiotensin II (Ang II)-receptor antagonists (losartan, EXP-3174, saralasin and sarmesin) on the levels of the biogenic monoamines dopamine, noradrenaline and serotonin in the frontal cortex, striatum, hypothalamus and hippocampus of rats with Ang II-induced water intake were investigated. Ang II administered i.c.v. at a dose inducing drinking behavior in rats significantly changed the levels of biogenic monoamines. The latter were also significantly affected by the Ang II-receptor antagonists, as in most cases the drugs antagonized the effect of Ang II. Most pronounced were the effects of Ang II and Ang II-receptor antagonists on the dopamine levels. These levels were reduced to zero after Ang II in all brain structures studied. The drugs tested increased the dopamine levels, restoring their values to the values in vehicle-injected rats. Ang II-receptor antagonists exerted mosaic effects on noradrenaline and 5-HT (serotonin) levels depending on both--the type of biogenic monoamine and the brain structure. There was no relationship between the inhibition of Ang II-induced water intake and the changes in the levels of brain biogenic monoamines under the effect of the Ang II-receptor antagonists. These antagonists may play a role in the modulation of brain monoaminergic neurotransmitter systems.

Interaction of angiotensin II and III with adenosine A(1) receptor-related drugs in passive avoidance conditioning in rats.

The present study examines the functional interaction between angiotensins (ANG II and III) and adenosine A(1) receptor-related drugs on passive avoidance (step-through) conditioning in rats. ANG II and III were administered intracerebroventricularly (i.c.v.) while N(6)-cyclohexyladenosine (CHA) and theophylline-intraperitoneally (i.p.), immediately after the training trial. ANG II (0.1,0.5,1 microg) induced dose-dependent (inverted-U) increase of the retention while ANG III in the same doses decreased it upon re-testing of rats 24 h and 7 days later. The selective adenosine A(1) receptor agonist CHA (0.1 mg/kg) attenuated memory-enhancing effect of ANG II (0.1 microg) 24 h but not 7 days after the training session. Conversely, CHA had opposite i.e. facilitating effect on ANG III (0.1 microg) response upon re-testing 24 h and 7 days later. The pretreatment with ANG III attenuated the retention-improving effect exerted by the non-specific adenosine A(1)/A(2) receptor antagonist theophylline (75 mg/kg) 24 h and 7 days after the training trial. Taken together, the results show a mutual interaction of the drugs belonging to the adenosine and angiotensin modulatory systems in memory consolidation of rats.

Effects of angiotensin III and angiotensin IV on pentylenetetrazol seizure susceptibility (threshold and kindling): interaction with adenosine A(1) receptors.

The effects of angiotensin (ANG) III and ANG IV on pentylenetetrazol (PTZ) seizure susceptibility--threshold and kindling in mice--as well as the influence of adenosine A(1) receptor agents (agonist and antagonist) on these effects were studied. It was found that ANG III and ANG IV increased dose-dependently the PTZ seizure threshold and decreased the seizure intensity in PTZ kindled mice. Cyclohexyladenosine (CHA), an adenosine A(1) receptor agonist, potentiated the effects of ANG III and ANG IV on the seizure threshold and kindling, whereas DPCPX (an A(1) receptor antagonist) reversed peptide-induced effects on the PTZ kindling. Taken together, ANG III and ANG IV decrease the PTZ seizure susceptibility. We could suggest that these effects are realized in part through interaction with adenosine A(1) receptors.

Interaction between angiotensin IV and adenosine A(1) receptor related drugs in passive avoidance conditioning in rats.

The functional interaction between ANG (3--8) (ANG IV) and adenosine A(1) receptor related drugs in passive avoidance (step-through) task in rats was studied in Wistar rats. ANG IV exerted dose-dependent (inverted-U) improvement of the retention while sarilesin (an angiotensin II analog) impaired this effect. Co-administration of theophylline and ANG IV, both in ineffective doses, enhanced the retention. The selective adenosine A(1) receptor agonist cyclopentyladenosine (CPA) attenuated ANG IV-induced memory enhancement.

Pharmacotherapy of ascariasis.

Ascaris lumbricoides, the most frequent human intestinal nematode, is the causative agent of ascariasis, with an estimated worldwide prevalence of over one billion people, especially in moist tropical and subtropical regions, but also in cooler climates. Although characterised with low morbidity and mortality rates, the global prevalence of ascariasis still results in approximately 20,000 deaths annually, primarily as a consequence of intestinal obstruction. In humans, transmission usually occurs by hand-to-mouth route by way of contaminated agricultural products and food, or from dirty hands. Three phases of ascariasis may be present, namely, the pulmonary, intestinal and the complications stage. Although generally asymptomatic, heavy infestation may cause serious pulmonary disease, or partial or complete obstruction of biliary or intestinal tracts. Anthelminthic chemotherapy is required to eradicate the parasites and prevent potentially serious complications. Mebendazole, albendazole and pyrantel pamoate are the most widely used agents to treat ascariasis. Preventive chemotherapy delivered to communities in endemic regions may serve as an affordable and cost-effective strategy to reduce the prevalence and morbidity in endemic regions. Under unusual circumstances, Ascaris suum, the cause of helminthic infection in pigs, may also cause disease in humans.

Chemotherapy of enterobiasis (oxyuriasis).

Enterobius vermicularis (syn. Oxyurus vermicularis), also known as pinworm or seatworm, is the causative agent of human enterobiasis (oxyuriasis). The disease is more prevalent in temperate regions and is facilitated by factors such as overcrowding in schools and family groupings, as well as inadequate personal and community hygiene. Although the infection is more likely to occur in lower socioeconomic groups, enterobiasis has been reported to affect virtually every level of the general population and especially children. In the great majority of cases, enterobiasis is asymptomatic. One common symptom is intense pruritus ani that in some patients can lead to insomnia, restlessness and irritability. Scratching may cause skin irritation, and in more serious cases, eczematous dermatitis, haemorrhage or secondary bacterial infections. Ectopic migration of E. vermicularis often results in pinworm infestation of the female genital tract often causing granulomas of the uterus, ovary and the fallopian tubes and pelvic peritoneum. Anthelmintic therapies for enterobiasis are successful and include mebendazole, albendazole and pyrantel pamoate. Mass medication of affected groups reduced symptoms rapidly, progressively and in a cost-effective way.

(S)-Zopiclone Sepracor.

(S)-Zopiclone, a cyclopyrrolone sharing activity with benzodiazepines in the CNS, is a short-acting sedative being developed by Sepracorfor the potential treatment of sleeping disorders. In August 2000, the company had completed phase II trials of (S)-zopiclone for insomnia [380377]; by September 2000, patient enrollmentfor phase III studies for insomnia was completed and the trial initiation was planned for October 2000 [381361]. Merrill Lynch expects US filing to take place in the second half of 2001 [383230]. Sepracor was granted US-05786357 in August 1998 covering methods and compositions of (S)-zopiclone in the treatment of sleeping disorders [342938]. In August 2000, Merrill Lynch predicted (S)-zopiclone sales of US $30 million in 2002, rising to US $150 million in 2004 [383230].

Necatoriasis: treatment and developmental therapeutics.

Two hookworm parasites, Necator americanus and Ancylostoma duodenale, infect approximately one billion people worldwide. These hookworms are one of the leading causes of iron-deficiency anaemia especially in children, resulting directly from intestinal capillary blood loss following the feeding activities of fourth-stage (L(4)) larva and adult worms. If ignored, human hookworm infections can retard growth and the intellectual development of children. Another clinical manifestation often associated with hookworm infections is cutaneous larva migrans (CLM). It is a well recognised, usually self-limiting condition caused by the infectious larvae of nematodes, especially Ancylostoma spp. CLM is characterised by skin eruption and represents a clinical description rather than a definitive diagnosis. Of the hookworm parasites, the dog and cat worm A. braziliense and A. caninum are the most common nematodes causing CLM, although many other species have also been implicated. The major subject of this review article will be discussion of the evolution of therapies and treatment of human necatoriasis and the development of experimental infections with N. americanus. Difference in the clinical efficacy of mebendazole and albendazole will be discussed along with drug resistance of N. americanus.

Lateralized learning and memory effects of angiotensin II microinjected into the rat CA1 hippocampal area.

The effects of angiotensin II (ANG II) microinjected unilaterally (left or right) and bilaterally (left and right) at a dose of 0.5 microg (0.5 nmol) into the CA1 hippocampal area of male Sprague Dowley rats on learning and memory (shuttle box) were studied. Bilateral microinjections of ANG II improved learning, i.e. increased the number of avoidances during the two training days as compared to the respective controls microinjected with saline. ANG II facilitated learning and memory, especially when microinjected into the left CA1 hippocampal area as compared to the respective controls microinjected with saline. Left-side microinjection of ANG II increased the number of avoidances on the first and second training day as compared to the right-side microinjection of ANG II. These findings suggest asymmetric effects of ANG II on cognitive processes in hippocampus.