Pharmacophore-based identification and in vitro validation of apoptosis inducers as anticancer agents.

Ligand-based pharmacophore modelling and virtual screening along with in vitro screening were performed as a rational strategy for the identification of novel compounds as apoptosis inducers and anticancer agents from the chemical database. Known apoptosis inducers were selected from the literature for generation of pharmacophore models, which were subjected to validation using Receiver operating characteristic (ROC) and Günere-Henry (GH) scoring methods. Based on highest fitness score of 4680.61, ROC value of 0.872 and GH score of 0.758, pharmacophore model-2 was selected as the best model. Model-2 as 3D search query was searched against the IBS database to find novel compounds as hits. Three hits were selected with a QFIT value more than 82 for in vitro screening as apoptosis inducers and anticancer agents. In vitro anticancer activity was performed using resazurin cell variability assay, and apoptosis inducing activity was determined using caspase-3 activation and annexin-FITC assays. One of the retrieved hit, STOCK5S-44056 demonstrated IC50 value of 23.56 µM in cell variability assay, and had EC50 value of 26.95 µM in caspase-3 activation assay. STOCK5S-44056 also indicated late stage induction of apoptosis in annexin assay. The results of in vitro activity revealed that STOCK5S-44056 has a potential to become anticancer agents.

Identification of novel PfDHODH inhibitors as antimalarial agents via pharmacophore-based virtual screening followed by molecular docking and in vivo antimalarial activity.

Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) catalyses the fourth reaction of de novo pyrimidine biosynthesis in parasites, and represents an important target for the treatment of malaria. In this study, we describe pharmacophore-based virtual screening combined with docking study and biological evaluation as a rational strategy for identification of novel hits as antimalarial agents. Pharmacophore models were established from known PfDHODH inhibitors using the GALAHAD module with IC50 values ranging from 0.033 µM to 142 µM. The best pharmacophore model consisted of three hydrogen bond acceptor, one hydrogen bond donor and one hydrophobic features. The pharmacophore models were validated through receiver operating characteristic and Günere-Henry scoring methods. The best pharmacophore model as a 3D search query was searched against the IBS database. Several compounds with different structures (scaffolds) were retrieved as hit molecules. Among these compounds, those with a QFIT value of more than 81 were docked in the PfDHODH enzyme to further explore the binding modes of these compounds. In silico pharmacokinetic and toxicities were predicted for the best docked molecules. Finally, the identified hits were evaluated in vivo for their antimalarial activity in a parasite inhibition assay. The hits reported here showed good potential to become novel antimalarial agents.

Is the deficiency of vitamin B12 related to oxidative stress and neurotoxicity in Parkinson's patients?

This review deals with the results showing the relation between vitamin B(12) deficiency and neurotoxicity of homocysteine and nitrite (a metabolite of nitric oxide) in Parkinson's patients treated with levodopa (L-Dopa). We have already reported a linear relationship between the CSF levels of nitrite with glutamic acid and homocysteine suggesting that the production of nitrite is interrelated with the neurotoxic level of homocysteine. The levels of nitrite and homocysteine resulting in the deficiency of vitamin B(12) are some of the factors promoting degeneration in Parkinson's disease. This review emphasizes the importance of these parameters in designing suitable drug therapy for Parkinson disease. Additionally, there is evidence that increased homocysteine levels might accelerate dopaminergic cell death in Parkinson disease (PD), through neurotoxic effects. Furthermore, levodopa (L-Dopa) treatment of PD results in hyperhomocysteinemia as a consequence of L-Dopa methylation by catechol-O-methyltransferase (COMT). Therefore, higher dietary intakes of folate, vitamin B12, and vitamin B6 might decrease the risk of PD through decreasing plasma homocysteine.

Effect of carbimazole induced hypothyroidism and thyroxine replacement on the growth of the long bones in albino rats of different age groups.

To evaluate the effect of carbimazole induced hypothyroidism and thyroxine replacement, on the growth of long bones of albino rats of different age groups. Experimental albino rats were developed with carbimazole and carbimazole plus thyroxine for a period of six weeks. At the end of the experiment the animals were sacrificed, fixed and processed to demonstrate the bony and cartilaginous parts. The ulna and tibia of both sides were measured for intact bone length & diameter and the data compared. The reduction in length and circumference observed, at the end of experiment, in ulna was 10.89%, & 11.94% and in tibia it was 12.52%, 14.81% in carbimazole treated group respectively, while in carbimazole plus thyroxin treated group the reduction in length & circumference of ulna was 1.37% & 1.88% and in tibia it was 1.86% & 3.08% respectively. They were compared to their age matched controls. The reduction in length and circumference in ulna was 5.58% & 6.25% and 6.42% and 5.88% in tibia respectively among the carbimazole treated animals while in the carbimazole plus thyroxine treated animals the reduction was only 0.63% and 3.12% in ulna and 0.91% and 1.06% in tibia respectively. The results show that hypothyroidism and its replacement therapy affects the endochondral as well as periosteal bone growth and results in reduction in length as well as circumference of long bones.

Modified method of AgNOR staining for tissue and interpretation in histopathology.

This study was conducted in the department of Pathology King Edward Medical University, from June to December 2002 to introduce the new method of AgNOR staining and its interpretation to increase its reliability. A total of 60 brain specimens were stained with modified AgNOR technique. The diagnosis of malignancy was made on H & E staining. AgNOR counts, variation in size and dispersion of AgNOR dots in cells were graded and compared in malignant and non-malignant lesions. Modified method of AgNOR staining and interpretation was an easy, reliable and reproducible alternative to traditional AgNOR techniques for evaluating proliferation activity of cells in malignant and benign brain lesions. mAgNOR counts of different grades of astrocytoma (2.97+/-0.96, 3.97+/-0.43, 6.01+/-2.74 and 8.01+/-3.56) were significantly (P<0.01) greater when compared with counts of normal brain (0.40+/-0.01), and reactive gliosis (0.60+/-0.01). AgNOR size and dispersion were of higher grade in a significantly greater proportion of malignancy when compared with benign conditions (P<0.05). The AgNOR dots were brighter and more clear with modified staining when compared with previous studies. We conclude that modified AgNOR staining technique is simple, quick and reliable to evaluate cell proliferation by detecting AgNORs size and dispersion. In future, AgNOR size and dispersion should be considered rather than the count only. We recommend the use of morphometry for AgNOR size in future. We also recommend the use of modified AgNOR staining for obtaining sound and confidant results in routine paraffin sections.

Mutation of p53 in skin papilloma and tubular breast adenoma of albino mice.

OBJECTIVE: To determine the mutation of p53 in chemically induced carcinogenesis on albino mice in skin papilloma and tubular adenoma breast by immunohistochemistry. DESIGN: An experimental study. PLACE AND DURATION OF STUDY: The animal house of Postgraduate Medical Institute and Pathology Department of King Edward Medical College University, Lahore, for the duration of 20 weeks, from 15 February, 2004 to 15 July, 2004. SUBJECTS AND METHODS: Twenty five albino mice (male and female) were selected for a study on chemical carcinogenesis. These animals were divided into five groups (A-E), five animals in each. DMBA (Dimethylebenz[a] Anthracene) and TPA (Tetradecanoyl-phorbal-13-Acetic Acid) [chemical carcinogens] were given to produce the tumors and mutation of p53 expression was evaluated on the tumors appearing during this period of carcinogenesis. Squamous cell papillomas and tubular adenoma breast were selected for this study. RESULTS: All the papillomas showed faint reactivity for immunomarker p53, while tubular adenomas were nonreactive. CONCLUSION: The results of this study show that p-53 is a marker for premalignant lesions and helps in selecting patients for constant monitoring, upon the clinical verification of these results.

Impact of selenium, iron, copper and zinc in on/off Parkinson's patients on L-dopa therapy.

We have quantitated CSF and serum levels of Selenium, iron, copper and zinc by Atomic absorption spectrophotometer in 36 patients with parkinson's disease all on L-dopa therapy. Out of these 19 showed on or positive response to L-dopa where as 21 patients showed on and off response. These data were compared with 21 healthy controls. The results showed that serum levels of iron, copper and zinc remained unchanged where as in CSF, significant decrease in zinc was found in both on and on/off PD patients indicating the deficiency of zinc which continues in the worsening clinical condition of off patients. The level of copper remained unchanged in both on and on/off PD patients. Iron and selenium increase in CSF of both patients which is a clear evidence of relationship between increased iron and selenium level in brain which could be correlated with decrease in dopamine levels and oxidative stress in PD Patients.

Polyamines as cancer markers: applicable separation methods.

Spermine, spermidine, putrescine and cadaverine are aliphatic amines widely spread in the human body. Their concentrations together with their acetyl conjugates increase significantly in the biological fluids and the affected tissues of cancer patients. Their concentrations decrease with the improvement in the patient's condition on multiple therapy. Various chromatographic techniques are frequently used in monitoring concentrations of di- and polyamines in cancer. Among these techniques, thin-layer chromatography and liquid chromatography using pre- or postcolumn derivatization, separating on a reversed-phase or an ion-exchange column are the most commonly used. Besides, high-resolution capillary column gas chromatography (GC) is increasingly used over packed column GC, and in recent years, capillary zone electrophoresis has also gained some importance in polyamine determinations. The review examines the prospects and the limitations of polyamines as cancer markers using chromatographic and electrophoretic techniques.

Variation in cerebrospinal fluid levels of neuropeptide Y, cholecystokinin and substance P in patients with neurological disorders.

Neuropeptide Y, cholecystokinin (tetra- and octasulphated peptides) and substance P were measured in lumbar cerebrospinal fluid obtained from patients with various neurologic disorders such as Parkinson's disease, cerebrovascular disorders, multiple sclerosis, tuberculous meningitis and aseptic meningitis. These results are statistically compared with healthy results. The results accumulated showed that the data collected can provide the vital information necessary for designing drug therapy.

Non-Hodgkin's lymphoma as an unexpected diagnosis in a shoulder arthroplasty.

Shoulder arthroplasty (SA) is commonly performed in patients with rheumatoid arthritis (RA) who have been treated with long-term immunosuppressive medication. RA is associated with an increased risk of neoplasms of the immune system. A case of non-Hodgkin's lymphoma as an unexpected diagnosis after the routine pathologic examination of the soft tissues after SA was detected in a 54-year-old woman with long-standing RA and prolonged immunosuppressive therapy. Although this case does not support the cost-effectiveness of routine specimen evaluation during SA, we suggest that histological analysis of the surgical tissues is appropriate and should be performed in all patients who have been treated with prolonged immunosuppressive medication, especially RA patients as well as patients who have suspicious surgical findings.

The neurochemical markers in cerebrospinal fluid to differentiate between aseptic and tuberculous meningitis.

In this study, the use of neurochemical markers in patients with aseptic and tuberculous meningitis has been investigated. The cerebrospinal fluid levels of amino acids, nitrite (a metabolite of nitric oxide), vitamin B12 and homocysteine were quantitated in both groups of patients. Among the amino acids, aspartic acid and glutamic acid both excitatory amino acid, GABA, glycine and tryptophan were all significantly increased in both patient groups whereas decreased level of taurine and increased level of phenylalanine were only found in patients with tuberculous meningitis. The levels of nitrite and its precursor arginine were significantly higher in patients with tuberculous meningitis whereas unchanged levels were found in patients with aseptic meningitis. A significantly increased homocysteine level and a decreased level of vitamin B12 were found only in patients with tuberculous meningitis whereas unchanged levels were found in patients with aseptic meningitis. This indicates that patients with tuberculous meningitis are particularly prone to vitamin B12 deficiency resulting into increased level of HC, and involvement of free radical showing the importance of these biological markers for promoting the possibility for the design of therapeutic approach.

Cholecystokinin peptides in cerebrospinal fluid: a study in healthy male subjects.

The clinical reliability of measuring cholecystokinin (CCK) peptides in the cerebrospinal fluid (CSF) has not been fully elucidated. Therefore, we have assayed CCK-8S and CCK-4 in CSF obtained from 14 healthy male subjects, lumbar-punctured at the L4-5 level following a strictly standardised procedure. CSF concentrations of free CCK-8S and free CCK-4 were used as dependent variables while age, height, body weight, atmospheric pressure and some other factors served as independent variables. It was shown that the CCK-8S ratio between the second (7-12 ml) and first (0-6 ml) CSF fractions, correlated significantly with the atmosphere pressure at the time of puncture. Neither CCK-8S nor CCK-4 displayed concentration gradients in CSF. The CCK-4 levels, expressed as pmol l-1 in the total amount of CSF were found to be positively correlated with the neuraxis distance in the lying position and negatively with the neuraxis distance in the sitting position. Furthermore, CCK-4, expressed as pmol l-1 per min of tapping-time (pmol l-1 min-1), showed a negative correlation with storage time, presumably mirroring a proteolytic process. CCK-8S and CCK-4 intercorrelated positively independently of whether expressed as pmol l-1 or pmol l-1 min-1. In conclusion, the results of this exploratory study indicate that the neuraxis distance (in the sitting and lying positions) and storage-time have to be accounted for when interpreting data on CSF levels of CCK-4. Attention has to be paid to the potential influence of atmospheric pressure on the concentration ratio of CCK-8S.

Repetitive behaviors in schizophrenia: a single disturbance or discrete symptoms?

Schizophrenia patients often display multiple repetitive behaviors. We investigated relations among nine repetitive behaviors and evaluated the hypothesis that these behaviors are varied manifestations of a single underlying biobehavioral disturbance. Nine repetitive behaviors from the Elgin Behavioral Rating Scale were assessed in 400 schizophrenia patients residing at a state hospital. A majority of patients were smokers (76.3%) and very few had pica (3%). Several other repetitive behaviors showed substantial frequency. A principal components analysis revealed eight of nine behaviors shared at least 10% of their variance with a single, common component. However, a principal factor analysis suggested a five-factor model best represented the data. The five factors and items identifying them were: (1) 'oral consumption' behaviors-polydipsia and smoking; (2) 'Kluver-Bucy' behaviors-bulimia and hypersexuality; (3) 'movement' behaviors-mannerisms/postures and pacing; (4) 'bizarre use of objects'-bizarre grooming and hoarding; (5) 'Pica'. Associations among repetitive behaviors varied. Symptoms such as smoking and polydipsia appeared reliably related, and others such as pica appeared discrete and independent. Overall, the data did not support the 'single disturbance' hypothesis and suggested a multifactorial model is needed to characterize repetitive behavior disturbances in schizophrenia.

Nitric oxide synthase pathway may mediate human natural killer cell cytotoxicity.

The present study provides evidence that the human natural killer (NK) cell effector mechanism causing target cytolysis has a requirement for L-arginine. In a deficient medium (DM) containing only salts, buffer system and glucose, NK cell-mediated cytotoxicity was found to decrease by 70% as compared to that obtained in a complete medium (CM). However, adding L-arginine to such DM could restore the activity of NK cells to the normal level. Many other components of CM, such as serum, glutamine and vitamins did not improve NK cell-mediated killing in DM. When all amino acids except L-arginine were added to DM only a partial recovery of NK cell functional cytolysis was seen. L-arginine enhanced the NK cell activity in a dose-dependent manner. Additionally, the inhibitor of both inducible and constitutive nitric oxide synthase, N-monomethyl-L-arginine (L-NMMA) inhibited NK cytolytic activity in DM supplemented with L-arginine indicating participation of nitric oxide (NO). The results also show that the stimulatory effect of L-arginine on human NK cell-mediated cytotoxicity was accompanied by an increase in NO formation as determined by accumulation of nitrite and citrulline. L-NMMA gave a dose-dependent reduction in NO generation as well. The nitrite and citrulline production dose-dependently correlated with not only the concentration of L-arginine in the cultivation medium, but also the enhanced NK cell-mediated cytolysis. Taken together, these findings could define a L-arginine/NO-linked effector mechanism in human NK cells. Nitrite and citrulline were not formed when NK cell-mediated target cell killing took place in a L-arginine-free DM supplemented with additives. Thus, it appears as if human NK cells may cause target cell killing via both NO-dependent and -independent processes.

Dopamine D1 or D2 antagonists enhance inhibition of consummatory ingestive behavior by CCK-8.

A dopamine D1 (SKF-38393, 1 mg)- or D2 (LY-171555, 0.1 mg)-receptor agonist inhibited intake of an intraorally infused solution of sucrose by male rats, a test of consummatory ingestive behavior. Treatment with a D1 (SCH-39166, 0.1 mg) or D2 (raclopride, 0.6 mg) antagonist reversed inhibition by the respective agonist but enhanced the inhibitory effect of cholecystokinin octapeptide (CCK-8; 1.8 micrograms). It was not possible to demonstrate specific effects of D1 and D2 agonists on intake of pellets, a test that does not discriminate consummatory ingestive behavior from appetitive ingestive behavior, i.e., behavior used to obtain food. The results demonstrate specific involvement of dopamine D1 and D2 receptors in inhibition of consummatory ingestive behavior.

Increased cerebrospinal fluid concentration of nitrite in Parkinson's disease.

The concentration of nitrite, a metabolite of nitric oxide (NO), was increased in the cerebrospinal fluid (CSF) of untreated patients with Parkinson's disease and in patients treated with L-DOPA in comparison with a group of patients without dopaminergic dysfunction. There was no difference in the concentration of L-arginine (ARG), a precursor of NO, between the groups. There was a highly significant, linear relationship between the concentration of nitrite and ARG in the CSF suggesting that the production of NO is dependent on the availability of ARG. The results support the possibility that production of NO is increased in the brain in Parkinson's disease.

Inhibition of serotonin synthesis attenuates inhibition of ingestive behavior by CCK-8.

Ingestive behavior was activated in male rats by intraoral infusion of a 1-M solution of sucrose. Injection of cholecystokinin octapeptide (CCK-8; 1.6 or 5.0 micrograms) inhibited ingestion of the sucrose solution and increased the concentration of 5-hydroxytryptamine (5-HT) in the paraventricular hypothalamic nuclei. The inhibitory effect of the low, but not the high, dose of CCK-8 was attenuated by depleting 5-HT in the brain with p-chlorophenylalanine (PCPA; 100 mg/kg for 3 days). Treatment with 5-hydroxytryptophan (20 mg/kg) increased the concentration of 5-HT in the brain of rats pretreated with either NaCl or PCPA and enhanced the inhibitory effect of CCK-8 on ingestive behavior in the PCPA-, but not NaCl-, treated rats. 5-HT may play a role in the mechanism of action of CCK-8 but additional factors must be involved.

Diet-independent suppression of ingestive behavior by cholecystokinin octapeptide and amino acids.

Male rats consumed much more of an intraorally administered mixed protein, fat and carbohydrate solution than of a carbohydrate solution. Injection of cholecystokinin octapeptide (CCK-8, 0.6-5.0 microgram) suppressed intake of both solutions, but the CCK-A receptor antagonist L-364, 718 (20-40 micrograms) facilitated only carbohydrate intake. Blood levels of CCK-8 were higher after intake of the carbohydrate than the mixed solution. Blood levels of isoleucine, leucine, lysine, threonine, valine, and tryptophan increased only after intake of the mixed solution. Injection of these amino acids suppressed intake of both solutions. Blood levels of amino acids were also less after the seventh than after the first session ingesting the mixed solution. Treatment with CCK-8 or amino acids inhibits intake of any diet, but when secreted endogenously, these signals may terminate the meal in a diet-dependent manner.