New Multifunctional Agents for Potential Alzheimer's Disease Treatment Based on Tacrine Conjugates with 2-Arylhydrazinylidene-1,3-Diketones.

Alzheimer's disease (AD) is considered a modern epidemic because of its increasing prevalence worldwide and serious medico-social consequences, including the economic burden of treatment and patient care. The development of new effective therapeutic agents for AD is one of the most urgent and challenging tasks. To address this need, we used an aminoalkylene linker to combine the well-known anticholinesterase drug tacrine with antioxidant 2-tolylhydrazinylidene-1,3-diketones to create 3 groups of hybrid compounds as new multifunctional agents with the potential for AD treatment. Lead compounds of the new conjugates effectively inhibited acetylcholinesterase (AChE, IC50 0.24-0.34 µM) and butyrylcholinesterase (BChE, IC50 0.036-0.0745 µM), with weak inhibition of off-target carboxylesterase. Anti-AChE activity increased with elongation of the alkylene spacer, in agreement with molecular docking, which showed compounds binding to both the catalytic active site and peripheral anionic site (PAS) of AChE, consistent with mixed type reversible inhibition. PAS binding along with effective propidium displacement suggest the potential of the hybrids to block AChE-induced ß-amyloid aggregation, a disease-modifying effect. All of the conjugates demonstrated metal chelating ability for Cu2+, Fe2+, and Zn2+, as well as high antiradical activity in the ABTS test. Non-fluorinated hybrid compounds 6 and 7 also showed Fe3+ reducing activity in the FRAP test. Predicted ADMET and physicochemical properties of conjugates indicated good CNS bioavailability and safety parameters acceptable for potential lead compounds at the early stages of anti-AD drug development.

Effect of routine preoperative fasting on residual gastric volume and acid in patients undergoing myomectomy.

BACKGROUND: Preoperative fasting of patients aims to reduce the residual gastric volume (RGV). The magnitude of this reduction is yet to be ascertained in the Nigerian population. AIM: To compare the RGV and pH of patients fasted for 6-12 h with those allowed oral intake of fluid up to 2 h preoperatively. SUBJECTS AND METHODS: This randomized study involved 90 American Society of Anesthesiologists physical status I-II patients booked for abdominal myomectomy under general anesthesia. The patients were randomized into three groups. Preoperative fasting from midnight (Group F, n = 30) was fasted from midnight to the operation time. Carbohydrate-rich drink group (Group C, n = 30) received 800 mL of oral carbohydrate solution in the evening before surgery (22:00 h). An additional 400 mL was given 2 h before anesthesia. Placebo drink group (Group P, n = 30) received water in the same protocol as Group C. The Student's t-test was used to analyze RGV and pH postoperative satisfaction and postoperative nausea and vomiting (PONV) were compared on a visual analog scale. RESULTS: The RGV and pH were similar for all groups (P = 0.45 and 0.90, respectively). Antiemetic consumption and PONV scores were lower in Group C compared with Groups F and P (P = 0.01). Patients' in Group C had higher satisfaction (P < 0.001). CONCLUSION: Preoperative carbohydrate or water intake up to 2 h before surgery is safe with better satisfaction when compared to overnight fasting.

Influence of aging on ethanol-induced oxidative stress in digestive tract of rats.

Aging and ethanol induce oxidative stress due to increased prooxidant production and decreased antioxidative capacity. The aim was to investigate the influence of aging on oxidative stress in liver, stomach and pancreas in acute ethanol intoxication. Adult (3 months) and old (18 months) male Wistar rats were divided into the following groups: control (control group rats aged 3 months (C3) and control group rats aged 18 months (C18)) and ethanol-treated groups (ethanol-treated 3-month-old rats (E3) and ethanol-treated 18-month-old rats (E18)). Ethanol was administered in five doses of 2 g/kg at 12-h intervals by orogastric tube. Tissue samples were collected for the determination of oxidative stress parameters. Malondialdehyde (MDA) concentration was increased in all the experimental groups and investigated organs versus C3 group ( p < 0.01). The highest MDA level was observed in the stomach in E18 group when compared with C18 and E3 groups ( p < 0.01). Activity of total superoxide dismutase (SOD) and its isoenzymes (copper-/zinc-SOD and manganese-SOD) in E18 group was significantly decreased when compared with E3 and C18 groups ( p < 0.01). Nitrates and nitrites (NO x ) concentration was increased in stomach and pancreas for all the groups when compared with C3 group ( p < 0.01). Hepatic, gastric and pancreatic NO x level was significantly increased in E18 group when compared with E3 group ( p < 0.01). Moreover, level of NO x in liver and pancreas in E18 group was significantly increased when compared with C18 group ( p < 0.01). Aging potentiates ethanol-induced oxidative stress in liver, stomach and pancreas due to increased lipid peroxidation and nitrosative stress and decreased antioxidative tissue capacity.

Superelectrophilic amidine dications: dealkylation by triflate anion.

Superelectrophiles: Formamides were designed that when treated with triflic anhydride would be transformed into superelectrophilic amidine dications. These dications were so electrophilic that they underwent in situ dealkylation by the triflate anion (see scheme; Tf = trifluoromethanesulfonyl). DFT calculations were used to determine the mechanistic details of the dealkylation reaction.

The effects of omega 3 fatty acid supplementation on brain tissue oxidative status in aged wistar rats.

BACKGROUND: The omega 3 fatty acids play an important role in many physiological processes. Their effect is well documented in neurodegenerative diseases and inflammatory diseases. Also, aging as a biophysiological process could be influenced by eicosapentanoic acid (EPA) and docosahexanoic acid (DHA) components of fish oil. However there are not many studies showing the effect of PUFA (polyunsaturated FA) suplementation in eldery brain functions and the response to oxidative strees. The aim of this study was to investigate the effects of dietary omega-3 fatty acid supplementation on levels of lipid peroxidation and oxidant/antioxidant status of brain tissue in aged (24 months old) Wistar rats. METHODS: Animals were divided in two groups. Control group (n=8) was fed with standard laboratory food and received water ad libitum. Treated group (n=8) was also fed with standard laboratory food, water ad libitum and received fish oil capsules (EPA+DHA) for 6 weeks. Daily dose was 30mg EPA and 45mg DHA (capsules: 200mg EPA and 300mg DHA; in-house method). At the end of treatment animals were sacrificed and brains were collected and frozen on -80ºC. The levels of lipid peroxidation (malondialdehyde - MDA), activity of catalase (CAT) and activity of superoxide dismutase (SOD) were examined in cerebral cortex. Catalase activity was determined by measuring the decrease in absorbance (H2O2 degradation) at 240 nm for 3 min and expressed as U/mg protein. Total SOD (superoxide dismutase) activity was performed at room temperature according to the method of Misra and Fridovich. The extent of lipid peroxidation (LPO) was estimated as the concentration of thiobarbituric acid reactive product malondialdehyde (MDA) by using the method of Aruoma et al. The incorporation of fatty acids in cellular membranes was confirmed by gas chromatography. RESULTS: Our results showed that lipid peroxidation significantly decreased in treated animal group, where MDA concentration was 0.38±0.001 vs. 0.43±0.001 nM/ml (p<0.05) in control. However SOD activity increased significantly in treated animal group 1.57±0.24 vs. 4.12±0.15 U/gHb/L (p<0.01) in control. CAT activity decreased in treated group but not significantly. CONCLUSION: Incorporation of omega-3 fatty acids after their supplementation had beneficial effects on brain tissue. Omega-3 fatty acids increased activity of SOD and decreased lipid peroxidation. Changes in oxidative/antioxidative balance are a result of EPA and DHA effects on lipids and enzymes of antioxidative system.

Cardiovascular autonomic dysfunction in systemic lupus, rheumatoid arthritis, primary Sjögren syndrome and other autoimmune diseases.

Neurological manifestations are known to occur in patients with autoimmune diseases, often subclinically, but autonomic nervous system (ANS) involvement has rarely been studied, and studies have shown conflicting results. We performed cardiovascular ANS assessment in 125 patients with autoimmune diseases in this case-control study, including 54 patients with systemic lupus erythematosus (SLE), 39 with rheumatoid arthritis (RA), 20 with primary Sjbgren syndrome (pSS), eight patients with polymyalgia rheumatica (PR), four patients with scleroderma (Ssc) and 35 healthy control subjects. The control group was formed to approximately match the mean age of SLE, RA and pSS patients; controls did not differ significantly by gender from the autoimmune pations. All patients with were in stable condition. Autonomic nervous system dysfunction was diagnosed by applying cardiovascular reflex tests according to Ewing, and was considered to exist if at least two tests were positive. Vagal dysfunction was established by applying three tests: Valsalva manoeuvre, deep breathing test, and heart rate response to standing. Sympathetic dysfunction was examined by applying two tests: blood pressure response to standing and handgrip test. In all cardiovascular reflex tests, frequencies of abnormal results were significantly higher among the patients than among the controls (P < 0.05). The difference between the autoimmune patients and the controls was particularly significant in sympathetic and parasympathetic tests, with P < 0.0001. No correlation was found between disease duration, clinical manifestations, cardiovascular risk factors and diseases activity on the one hand, and ANS dysfunction on the other hand. Cardiovascular autonomic dysfunction was revealed in the majority of autoimmune patients.

Ganglioside a/b ratio in different rat brain regions following chronic diazepam treatment.

Gangliosides are sialic acid-containing glycosphingolipids that play a variety of important functions in neurons. The main purpose of this study was to determine the a/b ratio of gangliosides in different rat brain regions (cerebral cortex, hypothalamus, caudate nucleus, hippocampus, thalamus and cerebellum) after prolonged diazepam treatment. Male Wistar rats were maintained on a nutritionally adequate diet and diazepam was administered in a dose of 10 mg/kg day for 180 days. Total gangliosides were extracted according to Harth and the total ganglioside-NeuAc content was determined by Svennerholm's resorcinol method, modified by Miettinen and Takki-Luukkainen. The a/b ratio remained unchanged in rat hypothalamus, thalamus and cerebellum. It was slightly decreased in the caudate nucleus and hippocampus, but this was not statistically significant. A drastic decrease ( p<0.01) in ganglioside content, compared to control animals, was found in rat cerebral cortex. Ganglioside a/b profile did not change significantly in most of the brain regions (except in cerebral cortex), which suggests that adaptive changes occurred upon prolonged exposure to diazepam, in order to maintain the physiological ratio of ganglioside a- and b-series in distinct brain areas.

Ganglioside content and composition in rat cerebellum after prolonged diazepam treatment.

The main purpose of this study was to determine the content and composition of cerebellar gangliosides after prolonged diazepam treatment and their possible recovery after diazepam withdrawal. Male Wistar rats were administered diazepam in a dose of 10 mg/kg/day in drinking water for 3, 5 or 6 months. A additional group of rats had a one-month recovery period after five months of diazepam treatment. Control animals were age-matched and pair-fed. At the end of the experiment, the animals were sacrificed and the total cerebellar contents of ganglioside-NeuAc as well as its content in particular ganglioside fractions were estimated. After three months of diazepam consumption, no changes of ganglioside-NeuAc in investigated fractions (G(Q1b), G(T1b), G(D1b), G(D1a), G(M1), G(M2), and G(M3)) were observed. Five months of diazepam treatment caused a significant decrease in the total amount of gangliosides, which was evident in most of the investigated fractions, with the exception of the monosialoganglioside G(M2). Six months of treatment induced a generalized decrease in all the investigated ganglioside fractions. The diazepam-induced ganglioside reduction found after five months of treatment was also present after a one-month recovery period. The only fraction, which recovered and reached its control value, was monosialoganglioside G(M3).

[Gamma aminobutyric acid--its function, disorders and their sequelae]. / Gama-aminobuterna kiselina--funkcija, poremecaji i posledice.

Gama-Aminobutyric acid (GABA) is an important neurotransmitter that mediates inhibition in the central nervous system. Approximately 30-50% of all synapses are defined as GABA-ergic. GABA is a neurotransmitter in cortical and hippocampal interneurones. GABA-RECEPTORS: Till today, three receptor subtypes have been known: GABAA, GABAB and GABAC, which are pharmacologically different. GABAA receptor is postsynaptic and localized in central and peripheral sympathetic neurones. Its agonist is muscimol and is antagonized by bicucculline. GABAB is a presynaptic receptor of vegetative and central nerve terminals. Its agonist is baclofen. The main difference between these two subtypes is that the first one acts directly on Cl ionphore, while GABAB activity is mediated by Gi protein. GABAC receptors are the integral part of the membrane, which stabilise the resting potential of the cell by increasing conductivity for Cl. Their most effective agonist is TACA. GABA ACTIVITY ON SYNAPSES: GABA is the most powerful inhibitory neurotransmitter in CNS. Synaptic inhibition decreases cell's ability to communicate with other cells and it is realised by various inhibitory mechanism of GABA, such as preventing of stimuluss generation, dendritic inhibition and dendro-dendritic inhibition. GABA AND NEUROENDOCRINE REGULATION: Besides physiological significance in maintaining regular excitation and inhibition balance. GABA plays an important role in neuroendocrine regulation of the following hormones: LH, FSH, PRL, STH, SS, ACTH, TSH, TRH, MSH, VP and OX. GABA IN NEUROLOGICAL DISEASES: Increasing or decreasing of GABA-ergic tone, due to different reasons, may lead to numerous neurodegenerative disorders (epilepsy, hepatic encephalopathy, Huntington's chorea, spinocerebellar degeneration, dementia and psychosis).

Phospholipid content and fatty acid composition in the rat heart after chronic diazepam treatment.

The effects of chronic diazepam treatment (10 mg/kg/day for 180 days) on the fractional distribution and fatty acid composition of heart phospholipids were studied in male Wistar rats. It was found that diazepam treatment increased the content of phosphatidylcholine and cardiolipin in the heart and slightly increased its phosphatidylcholine fraction. There were no significant changes in fatty acid composition after diazepam treatment in heart phospholipids, with the exception of significant decrease of 20:3n-6 and 20:5n-3 fatty acids. Our findings suggest that diazepam, probably through peripheral benzodiazepine binding sites, altered the content of heart cardiolipin and caused changes in the flux of oxidative phosphorylation in the heart.

Factors that influence biochemical failure after radiation therapy for stage T1c prostate cancer.

More patients are now being diagnosed with nonpalpable prostate cancer after a needle biopsy is performed for an elevated prostate-specific antigen (PSA) level (stage T1c). The purpose of this study was to identify prognostic factors that are associated with biochemical failure after definitive external beam radiation therapy. This study included 75 patients with the diagnosis of T1c prostate cancer who were referred to four radiation oncology centers in the West Chicago area from 1992 to 1995. All patients were treated with megavoltage external beam radiotherapy to doses between 66 and 70 Gy. Biochemical failure was defined as three consecutive rising PSA values of at least 10% of the prior reading in posttreatment serial measurements. The mean age of the patients was 72 years. The mean follow-up was 1.7 years (range, 1-3 1/2 years). Of the 75 patients, 72 (96%) are clinically with no evidence of disease, three of the 75 are alive with disease, and 60 (80%) remain biochemically free of disease (bNED). The significant factors for bNED status were an initial PSA level of <15 ng/ml (p = 0.0001), achievement of a posttreatment nadir PSA level of < or = 1.5 ng/ml (p = 0.0001), and a Gleason score of <6 (p = 0.034). Multisextant involvement with tumor or bilobar disease was not significant. On multivariate analysis, an initial PSA level of <15 ng/ml (p = 0.0001), Gleason score of <6 (p = 0.02), and nadir PSA level of < or = 1.5 ng/ml (p = 0.03) were significant predictors of bNED survival. Men with T1c prostate cancer comprise a heterogeneous group. Patients with a high PSA level (>15 ng/ml) and high Gleason score (>6) are at increased risk for biochemical failure. Failure to achieve a posttreatment nadir PSA level of < or = 1.5 ng/ml is a predictor of ultimate biochemical failure.

Molecular studies on a new genetic locus linked to the common nodulation genes in Bradyrhizobium japonicum.

ORFA, an actively transcribed genetic locus linked to the common nodulation genes in Bradyrhizobium japonicum USDA110, was sequenced and analysed. The expression of ORFA is neither dependent on the regulatory proteins NifA, NtrC, NtrB and NodD1 nor on either copy of sigma 54, RpoN1 and RpoN2. The transcriptional start site of ORFA was determined and found to overlap the oppositely transcribed nodZ gene by 224 nucleotides. An appropriately located -10 sequence identical to the consensus proposed for rhizobia and a homologous -35 region were identified upstream of the transcriptional start site. ORFA showed no significant homologies to known sequences in gene databases, and its mutational inactivation had no effect on the nodulation of five legume species. Nevertheless, ORFA seems to be conserved among bradyrhizobia, since an ORFA probe hybridised to total DNA extracted from other Bradyrhizobium strains.

Cloning of the rpoD analog from Rhizobium etli: sigA of R. etli is growth phase regulated.

Rhizobium bacteria fix atmospheric nitrogen during symbiosis with legume plants only after bacterial division is arrested. The role of the major vegetative sigma factor, SigA, utilized by Rhizobium bacteria during symbiosis is unknown. By using PCR technology, a portion of the sigA gene corresponding to domain II was directly amplified from Rhizobium etli total DNA by using two primers designed in accordance with the published sequence of sigA from Agrobacterium tumefaciens. The amplified fragment was cloned and used as a hybridization probe for cloning of the R. etli sigA gene. Sequencing data revealed an open reading frame of 2,055 bp showing extensive similarity to various vegetative sigma factors. The 5' end of the sigA transcript was determined and revealed a long, seemingly untranslated region of 170 nucleotides. Quantitative analysis of the sigA transcript by RNase protection and by primer extension assays indicated its down-regulation during entry into the stationary phase. On the basis of the structures of various vegetative sigma factors and considering previous information on heterologous expression, we speculate on the function of domain I of vegetative sigma factors.

Liver, plasma and erythrocyte phospholipid content after chronic diazepam treatment in the rat.

Male Wistar rats were maintained on a nutritionally adequate diet and diazepam was administered in a dose of 10 mg/kg/day. Control animals were pair-fed an adequate diet. Feeding was continued for 180 days, and the effects on the liver, plasma and erythrocyte phospholipid content were studied. It was found that the contents of sphingophospholipids and phosphatidylinositol + phosphatidylserine were significantly reduced in the erythrocytes of diazepam-treated rats. There was a significantly increased content of phosphatidylcholine in the liver an erythrocytes after 180 days of diazepam treatment. Such treatment did not cause statistically significant changes in the plasma of diazepam-treated rats. These investigations are in agreement with the hypothesis that extended or chronic use of drugs such as diazepam may alter membrane-dependent processes.

[Gangliosides as neurobiochemical markers]. / Gangliozidi kao neurobiohemijski obelezivaci.

Gangliosides are primary brain membrane lipids which, due to their localization, present specific markers of certain nerve cells. Their structure and content is tightly connected with cytogenesis and histogenesis of brain in mammals. Their biologic function, at the surface of the neurons' membrane, is important for bioelectric neuronal activity and synaptic transmission. Apart from that, gangliosides are important as receptors of bacterial toxins. Effects that gangliosides may have on dendrites' and axons' growth have been studies and it can be concluded that they have a significant impact on recovery of ischemic brain lesions. They are also important therapeutic and diagnostic markers of certain malignant tumors. The most valuable results are expected in future by examination of gangliosides metabolism in neurologic diseases by application of cell cultures.

Comparison of treatment plans for irradiating adenocarcinoma of the prostate.

In a clinic with multiple megavoltage beams (Co-60, 6 MV, 10 MV, 18 MV) available, the optimal dose distributions depends on the radiation technique, patient size, and beam energy. To determine optimal dose distributions for external beam irradiation for patients with localized adenocarcinoma of the prostate, we investigated the dependence of beam energy, treatment technique, and patient size. Various radiation isodose plans were prepared at the central plane of the treatment field using computerized tomography scans of the pelvis for small, medium, and large patients. The dose uniformity in the target volume, the dose received by sensitive tissues such as bladder, rectum, and femoral heads, and the irradiated volume of these normal tissues were used as the criteria for comparing the plans. In all cases, the target volume was within the 95% isodose line with good dose uniformity (+/- 5%, of the prescribed dose). The volume of the rectum and bladder enclosed within the 90% isodose curve depended on the choice of the beam energy and the treatment technique for different patient sizes. For the small size patient the optimal dose distribution was achieved by using 10 MV box and arc or oblique boost. For the medium size patient, using 18 MV X-rays with box technique and oblique boost gave the optimal treatment plan. For the large size patient, using 18 MV or 10 MV X-rays box technique with arc boost were both acceptable.

nodZ, a unique host-specific nodulation gene, is involved in the fucosylation of the lipooligosaccharide nodulation signal of Bradyrhizobium japonicum.

The nodulation genes of rhizobia are regulated by the nodD gene product in response to host-produced flavonoids and appear to encode enzymes involved in the production of a lipo-chitose signal molecule required for infection and nodule formation. We have identified the nodZ gene of Bradyrhizobium japonicum, whose product is required for the addition of a 2-O-methylfucose residue to the terminal reducing N-acetylglucosamine of the nodulation signal. This substitution is essential for the biological activity of this molecule. Mutations in nodZ result in defective nodulation of siratro. Surprisingly, although nodZ clearly codes for nodulation function, it is not regulated by NodD and, indeed, shows elevated expression in planta. Therefore, nodZ represents a unique nodulation gene that is not under the control of NodD and yet is essential for the synthesis of an active nodulation signal.

Beam attenuators and the risk of unrecognized large-fraction irradiation of critical tissues.

The use of radiation beam attenuators led to radiation injury of the spinal cord in one patient and of the peripheral nerve in another due to unsuspected large-fraction irradiation. The anatomic distribution of radiation dose was reconstructed in the sagittal plane for the patient who developed radiation myelopathy and in the axial plane for the patient who developed peripheral neuropathy. The actual dose delivered to the injured structure in each patient was taken from the dose distribution and recorded along with the time, number of fractions, and dose per fraction. The patient who developed radiation myelopathy received a total of 46.5 Gy in twenty-three 2.1 Gy fractions in 31 days to the upper cervical spinal cord where the thickness of the neck was less than the central axis thickness due to cervical lordosis and absence of a posterior compensating filter. The patient who developed peripheral neuropathy received 55 Gy in twenty-five 2.2 Gy fractions in 50 days to the femoral nerve using bolus over the groins and an anterior one-half value layer Cerrobend pelvic block to bias the dose anteriorly. Compensating filters and other beam attenuators should be used with caution because they may result in unsuspected large-fraction irradiation and total doses of radiation that exceed the tolerance of critical structures.

nolMNO genes of Bradyrhizobium japonicum are co-transcribed with nodYABCSUIJ, and nolO is involved in the synthesis of the lipo-oligosaccharide nodulation signals.

A host-inducible lacZ fusion was mapped down-stream of the nodYABCSUIJ operon in Bradyrhizobium japonicum strain USDA110. Sequencing of this region identified three novel genes, nolMNO. RNA dot blot analysis showed that nolO transcription is nodD1-dependent and that a polar mutation in nodS, located 5 kilobases upstream of nolO, blocks the transcription of nolO. Coupled with the host-inducible nature of nolO expression, these results indicate that nolMNO are part of a 9-kilobase operon, nodYABCSUIJnolMNO. The lipo-oligosaccharide nodulation signals produced by strains SL67 (nolO-) and SL65(nolNO-) were purified, and their chemical structures were determined. In addition to the wild-type signal molecules, both mutants produced modified compounds that are not produced by the parent strain USDA110. The most prevalent difference observed was the absence of the 2-O-methylfucosyl residue from the mutant structures. In addition, metabolites were found in which the N-acetylglucosamine residue at the reducing end was glycosidically linked to glycerol. These alterations in the profiles of nodulation signals produced by strains SL67 and SL65 were accompanied by reduced nodulation efficiency on all hosts tested.