Metabolism of N-ethyl-3-piperidyl benzilate in rats

The metabolic fate of N-ethyl-3-piperidyl benzilate (I) and its potential metabolites 3-piperidyl benzilate (II), N-ethyl-3-hydroxypiperidine (III), and 3-hydroxypiperidine (IV) was studied. Incubation of I with rat liver homogenates resulted in the formation of II and III. Only a trace of unchanged drug appeared in urine after intraperitoneal injection of I. Approximately 9% of the injected dose of I was excreted in urine as III and 2% in the form of metabolites that produced III after acid hydrolysis. After intraperitoneal injection of II in rats, 18% of the dose was excreted in urine as IV. Approximately 26% of the injected dose of III was present in urine as the unchanged drug, and 63% of the dose was excreted in the urine in the form of conjugates that produced III on acid hydrolysis. Urine of rats injected with IV contained approximately 50% of the injected dose as the unchanged drug and 50% of the dose in the form of a conjugate that produced IV on acid hydrolysis. The identity of the metabolites in extracts from urine was established by GLC-mass spectrometry. It is concluded that hydrolysis was one metabolic pathway for I and II. The major routes of elimination of these compounds are not yet known and may include excretion in feces or metabolic transformations resulting in the degradation of the piperidine ring.     

A prospective study of the outcome of anterior laxity of the knee after anterior cruciate ligament reconstruction with procedures using two different patellar tendon grafting methods

Oligonucleotides coding for linear epitopes of the fimbrial colonization factor antigen I (CFA/I) of enterotoxigenic Escherichia coli (ETEC) were cloned and expressed in a deleted form of the Salmonella muenchen flagellin fliC (H1-d) gene. Four synthetic oligonucleotide pairs coding for regions corresponding to amino acids 1 to 15 (region I), amino acids 11 to 25 (region II), amino acids 32 to 45 (region III) and amino acids 88 to 102 (region IV) were synthesized and cloned in the Salmonella flagellin-coding gene. All four hybrid flagellins were exported to the bacterial surface where they produced flagella, but only three constructs were fully motile. Sera recovered from mice immunized with intraperitoneal injections of purified flagella containing region II (FlaII) or region IV (FlaIV) showed high titres against dissociated solid-phase-bound CFA/I subunits. Hybrid flagellins containing region I (FlaI) or region III (FlaIII) elicited a weak immune response as measured in enzyme-linked immunosorbent assay (ELISA) with dissociated CFA/I subunits. None of the sera prepared with purified hybrid flagella were able to agglutinate or inhibit haemagglutination promoted by CFA/I-positive strains. Moreover, inhibition ELISA tests indicated that antisera directed against region I, II, III or IV cloned in flagellin were not able to recognize surface-exposed regions on the intact CFA/I fimbriae.     

Identification of hepatic metabolites of two highly carcinogenic polycyclic aza-aromatic compounds, 7,9-dimethylbenz[c]acridine and 7,10-dimethylbenz[c]acridine

The hepatic microsomal metabolites of the highly carcinogenic dimethylbenzacridines, 7,9-dimethylbenz[c]acridine (7,9-DMBAC), and 7,10-dimethylbenz[c]acridine (7,10-DMBAC) were obtained with preparations from 3-methylcholanthrene-pretreated rats. Metabolites were separated by reversed-phase HPLC and characterized using UV spectral data and chemical ionization-mass spectrometry after trimethylsilylation and GC. Comparisons with products formed in the presence of the epoxide hydrolase inhibitor, 1,1,1-trichloropropane 2,3-oxide and with those formed from the three synthetic alcohol derivatives of each parent compound, aided the assignment of firm or tentative structures to 16 products from 7,9-DMBAC found in 22 reversed-phase chromatographic peaks, and for 17 products of 7,10-DMBAC found in 19 chromatographic peaks. The more abundant metabolites were derived from oxidation of the methyl groups. Other metabolites were dihydrodiols, epoxides, phenols and secondary metabolites. The 9-methyl group prevented dihydrodiol formation at the 8,9-position from 7,9-DMBAC, and for each carcinogen, the 3,4-dihydrodiol was formed. As well, 3,4-dihydrodiols of methyl oxidized compounds were found.     

Identification of two S-containing metabolites of 1-allyl-3,5-diethyl-6-chlorouracil in rabbit urine (author's transl)

The synthesis of 2-14C-1-allyl-3,5-diethyl-6-chlorouracil (2-14C-Aclu; Acluracil) is described. After application in rabbits, 2-14C-Aclu is biotransformed in one S-free major metabolite I and two S-containing minor metabolites II and III, which are more polar than Aclu. The metabolites have been isolated and purified by thick layer-, column- and gaschromatography. With the help of 1N-NMR- and mass spectroscopy, metabolite II could be identified as 1-allyl-3-ethyl-5-(2-hydroxy ethyl)-6-methylmercaptouracil and metabolite III as 1-allyl-3,5-diethyl-6-methylmercaptouracil. The introduction of methylmercapto group (-SCH3) in metabolites II and III represents a new biochemical pathway which to the best of our knowledge has not been reported in the literature up to now.     

Comparison of different DNA fingerprinting techniques for molecular typing of Bartonella henselae isolates

Seventeen isolates of Bartonella henselae from the region of Freiburg, Germany, obtained from blood cultures of domestic cats, were examined for their genetic heterogeneity. On the basis of different DNA fingerprinting methods, including pulsed-field gel electrophoresis (PFGE), enterobacterial repetitive intergenic consensus (ERIC)-PCR, repetitive extragenic palindromic (REP) PCR, and arbitrarily primed (AP)-PCR, three different variants were identified among the isolates (variants I to III). Variant I included 6 strains, variant II included 10 strains, and variant III included only one strain. By all methods used, the isolates could be clearly distinguished from the type strain, Houston-1, which was designated variant IV. A previously published type-specific amplification of 16S rDNA differentiated two types of the B. henselae isolates (16S rRNA types 1 and 2). The majority of the isolates (16 of 17), including all variants I and II, were 16S rRNA type 2. Only one isolate (variant III) and the Houston-1 strain (variant IV) comprised the 16S rRNA type 1. Comparison of the 16S rDNA sequences from one representative strain from each of the three variants (I to III) confirmed the results obtained by 16S rRNA type-specific PCR. The sequences from variant I and variant II were identical, whereas the sequence of variant III differed in three positions. All methods applied in this study allowed subtyping of the isolates. PFGE and ERIC-PCR provided the highest discriminatory potential for subtyping B. henselae strains, whereas AP-PCR with the M13 primer showed a very clear differentiation between the four variants. Our results suggest that the genetic heterogeneity of B. henselae strains is high. The methods applied were found useful for typing B. henselae isolates, providing tools for epidemiological and clinical follow-up studies.     

Clinical presentation and prognostic factors in sodium monofluoroacetate intoxication

From 1970 to 1992 a total of 63 patients underwent operation for ampullary tumor: 40 pancreatoduodenectomies (PDs), 3 total PDs, 8 ampullectomies, and 12 bypass or exploratory laparotomies. The resectability rate was 68%. There were 9 benign tumors, 1 anaplastic tumor, and 53 adenocarcinomas. According to Martin's classification, there were 7 stage I, 11 stage II, 14 stage III, and 21 stage IV tumors. All patients with stage I, II, and III tumors underwent resection. Patients with stage IV tumors had either resection (n = 11) or bypass (n = 10). The mean duration of hospital stay was 20.6 days. Operative mortality was 12.7% for the whole series and 7.5% after PD (2.5% for the last 10 years). Overall survival was 40% at 5 years (85% for stage I, 65% for stage II, 44% for stage III, and 8% for stage IV). Survival was better for stages I, II, and III after PD than after ampullectomy. For stage IV patients survival was 70% after PD versus 20% after bypass at 1 year and 25% versus 0% after 2 years. In our opinion, PD should be proposed even for benign lesions because two of our patients had to undergo repeat operation (PD) 4 and 22 years later, respectively, for stage IV disease. PD is our choice for all tumors of the ampulla.     

Outcome and life prospects after surgical management of medically intractable epilepsy in patients under 18 years of age

A retrospective analysis of seizure outcome and quality of life assessment was done in 64 patients under 18 years of age with medically refractory epilepsy who underwent 64 primary and 16 repeat operative procedures in an attempt to control their epilepsy. At least 2 years' follow-up data were available for each patient. Operative procedures were 44 temporal lobe resections; 16 extratemporal resections; and 4 hemispherectomies. Effective control of previously intractable seizures was obtained in most patients: 55%, 11%, and 17% achieved Engel class I, II, and III status, respectively. Successful seizure control was thus obtained in 83%, while 17% (Engel class IV) failed to improve significantly after operation. Quality-of-life measures parallelled the improvements in seizures control, being highest in Engel I, outcome group and lowest in Engel IV outcome group. In appropriately selected pediatric and adolescent patients with medical refractory epilepsy, surgical management can offer a safe and effective adjunct to medication.     

Neuronal protection from cerebral ischemia by synthetic fibronectin peptides to leukocyte adhesion molecules

Leukocytes play an important role in the development of ischemia/reperfusion injury. Recent work in our laboratory has demonstrated that a mixture of synthetic fibronectin peptides to leukocyte adhesion molecules reduces ischemic brain damage after transient focal cerebral ischemia. The purpose of this study was to evaluate the efficacy of the individual peptides on leukocyte accumulation, infarct size, and neurological outcome in rats subjected to 1 h of cerebral ischemia and 48 h of reperfusion. Thirty-five animals were divided into five groups: transient ischemia without treatment (Group I), treatment with arginyl-glycyl-aspartic acid (RGD) peptide (Group II), connecting segment (CS)-1 peptide (Group III), fibronectin (FN)-C/H-V peptide (Group IV), and scrambled FN-C/H-V peptide (Group V). Groups III and IV showed a significant decrease in the degree of leukocyte infiltration in the lesion and in the infarct size (p < 0.05) when compared to Groups I, II, and V. The neurological grade of Groups III and IV was significantly better than in Groups I, II, and V at 48 h after reperfusion (p < 0.01). Thus, in addition to demonstrating the potential efficacy of synthetic peptides as therapeutic agents for ischemia-reperfusion, these results also offer new insights into the mechanisms of leukocyte arrest and recruitment in ischemia/reperfusion injury.     

Donor treatment with the lazeroid U74389G reduces ischemia-reperfusion injury in a rat lung transplant model

BACKGROUND: Antioxidant treatment with lazeroids has proven beneficial for the amelioration of reperfusion injury in experimental lung transplantation. This study compares the effect of donor versus recipient treatment on immediate postoperative graft function. METHODS: A model of acute double-lung transplantation in rats was used to assess graft function. Transplanted controls after 2 (group I) and 16 hours of ischemia (group II) were compared to a recipient (group III; 16-hour ischemia) and a donor treatment group (group IV; 16-hour ischemia) using the lazeroid U74389G (6 mg/kg). Serial assessment of alveolar-arterial oxygen difference, dynamic lung compliance, airway and pulmonary vascular resistance was obtained during a 2-hour reperfusion period. Final analysis included survival, weight gain, and histologic examination. RESULTS: Graft function was significantly better after 2 hours of ischemia than in any of the three 16-hour ischemia groups (II, III, IV). After 16 hours of ischemia, donor treatment provided superior graft function with respect to dynamic lung compliance, airway resistance, and alveolar-arterial oxygen difference when compared with groups II and III. The pulmonary vascular resistance was significantly higher in group III when compared with groups II and IV. Graft weight increase reflecting edema was highest in groups III (104%) and II (98%). CONCLUSIONS: After prolonged ischemia only donor treatment with the lazeroid U74389G was able to significantly reduce ischemia-reperfusion-related graft dysfunction.     

The use of the radioimmunoassay in the characterization of antibodies to basement membrane collagen

This study describes the use of the radioimmunoassay for the characterization of antibodies to basement membrane (type IV) collagen from bovine anterior lens capsule. The immunogen was extracted from calf anterior lens capsules by limited pepsin digestion and injected into rabbits. The antisera were characterized using gel diffusion, haemagglutination and the radioimmunoassay in which 125I-labelled types I, II, III, and IV bovine collagen were employed. In the direct radioimmunoassay there was no reaction with either native or denatured types I, II or III bovine collagen, whereas there were high titres towards both native and denatured type IV bovine collagen. Radioimmune inhibition studies using unlabelled types I, II, III and IV bovine collagen, collagenase digested and repepsinized type IV collagen showed that there was marked inhibition by either native, denatured or repepsinized type IV collagen, and slight inhibition by native type I collagen; native type II and type III, denatured types I, II and III, and collagenase digested type IV collagen had no inhibitory effects.     

Roles of individual domains of annexin I in its vesicle binding and vesicle aggregation: a comprehensive mutagenesis study

To understand the mechanism by which annexin I induces membrane aggregation, a comprehensive mutagenesis of all six Ca2+-binding sites was performed. When the cap residues of type II Ca2+-binding sites were systematically mutated to Ala, a type II site in domain II was shown to be essential for Ca2+-dependent vesicle binding of annexin I. Domain II was not, however, directly involved in vesicle aggregation. Instead, type II sites in domains III and IV, respectively, and type III sites in domains I and IV were involved in vesicle aggregation. When all type II sites were deactivated, three type III sites provided residual vesicle binding and aggregating activities. Their contributions to these activities in the presence of type II sites were, however, relatively insignificant. To further investigate the role of each domain harboring a type II site, a set of mutants containing only a specific type II site(s) were generated and their activities measured. These measurements again underscored the importance of domain II in vesicle binding of annexin I and the involvement of domains III and IV in vesicle aggregation. The roles of individual domains in vesicle binding and aggregation can be accounted for by the conformational change of membrane-bound annexin I involving modular rotation of domains (I/IV) following the initial membrane adsorption of domains (II/III). In conjunction with mutagenesis studies on other annexins, these results show that individual domains of annexins, although structurally homologous, have distinct functions and that different annexins might interact with membranes via different domains.     

5-aryl-2,3-dihydro-5H-imidazo[2,1-a]isoindol-5-ols. A novel class of anorectic agents

A series of 5-aryl-2,3-dihydro-5H-imidazo[2,1-a]isoindol-5-ols (IV), prepared by the LiA1H4 reduction of the corresponding 9b-aryl-1,2,3,9b-tetrahydro-5H-imidazo[2,1-a]isoindol-5-ones (II), was evaluated for suppression of food consumption in rats. One member of this series, 5-p-chlorophenyl-2,3-dihydro-5H-imidazo[2,1-a]isoindol-5-ol (6, mazindol), was evaluated in squirrel and capuchin monkeys and found to have anorexic activity approximately equal to d-amphetamine.     

Isolation and identification of a new cinnamate ester from liaoxi propolis

A new cinnamate ester drivitive (II) and three flavonoids (I, III, IV) were isolated from Liaoxi propolis. Their chemical structures were established as benzyl caffeate (II), 7-O-methylchrysin (I), genkwanin (III) and rhamnazin (IV) by spectral analysis. II is a new natural compound; I, III and IV were found from the propolis for the first time.     

Metabolism of isbufylline in humans. Isolation, identification, and synthesis of plasma and urine metabolites

Isbufylline metabolism after oral administration to humans was studied. The main metabolites detected by the HPLC method, in plasma, were 1-methyl-7-(2-hydroxy-2-methyl-propyl) xanthine (I), 1,3-dimethyl-7-(2-hydroxy-2-methyl-propyl) xanthine (II), and 1-methyl-7-(2-methyl-propyl) xanthine (III). The main metabolites detected in urine were 1-methyl-7-(2-hydroxy-2-methyl-propyl) xanthine (I), 1,3-dimethyl-7-(2-carboxy-propyl) xanthine (IV), and 1,3-dimethyl-7-(2-hydroxymethyl-propyl) xanthine glucuronic acid (V)-Gluc. They were isolated by HPLC, identified by GC/MS, HPLC/MS, or HPLC/MS/MS, and finally synthesized. Recovery of these metabolites, along with the absence of unmetabolized isbufylline in the urine, indicated biotransformation and renal excretion as the main routes of isbufylline elimination in humans. HPLC quantitation of the characterized urine metabolites revealed that 49% of the drug was eliminated as (I), 9% as (V)-Gluc, and 5% as (IV).     

High-performance liquid chromatography study of stereospecific microsomal enzymes catalysing the reduction of a potential cytostatic drug, oracin. Interspecies comparison

One of the main metabolites of oracin (I) ?6-[2-(2-hydroxyethyl)aminoethyl]-5,11-dioxo-5,6-dihydro-11H-indeno[1,2- c] isoquinoline?, a potential cytostatic drug, is 11-dihydrooracin (II) ?(+),(-)-6-[2-(2-hydroxyethyl)aminoethyl]-5-oxo-11-hydroxy-5,6-dihydro-1 1H- indeno[1,2-c]isoquinoline?, a metabolite formed by the reduction of oracin's pro-chiral centre on C 11. This metabolite has been found in all laboratory species in vitro and in vivo and it constitutes the main metabolite in man. The stereospecificity of reducing enzymes participating in the oracin biotransformation pathway was investigated using microsomal preparations from standard laboratory animals. Enzyme stereospecificity has been defined as preferential formation by the enzyme of the (+) or (-) stereoisomer of II. Significant interspecies differences were observed in the stereospecificity of the respective biotransformation enzymes. HPLC quantitative determinations of both enantiomers were performed using a Chiralcel OD-R column as chiral stationary phase with excellent resolution and stability.     

Screening for mutations by enzyme mismatch cleavage with T4 endonuclease VII

BACKGROUND: The analytical pattern of ascitic fluid in peritoneal tuberculosis is frequently similar to that found in other causes of ascites. The diagnostic value of the ascitic fluid pH and lactate in cases of tuberculous peritonitis has not yet been established. METHODS: Ascitic fluid pH, lactate, total proteins, cell count, lactate dehydrogenase, glucose, and their blood-ascitic gradients were determined in 10 patients with tuberculous peritonitis (group I). These results were compared with those obtained from 40 patients with cirrhotic sterile ascites (group II), 16 patients with spontaneous bacterial peritonitis (group III), and 18 patients with malignant ascites (group IV). RESULTS: A decreased pH and an elevated lactate level in ascitic fluid were found in patients in group I in comparison with those in group II (p < 0.001). No significant differences were found between group I and groups III and IV. The arterial blood-ascitic fluid pH gradient was more than 0.10 (p < 0.001), and the ascitic fluid-serum lactate gradient was greater than 15 mg/dl (p < 0.001) in group I when compared with group II. No significant differences were found between group I and groups III and IV. CONCLUSIONS: Ascitic fluid pH and lactate are useful markers in differentiating tuberculous peritonitis from cirrhotic sterile ascites. However, these variables lack specificity, as they are also decreased and increased, respectively, in cases of malignant ascites and spontaneous bacterial peritonitis.     

Development of spatial orientation in infancy.

The ability of infants at 6, 11, and 16 mo to keep track of their relationship to a place in space was assessed in 4 experiments with 72 Ss. Ss were trained to expect an event to occur to their right or left; they were then moved so that their view of the space was reversed. The direction in which they turned in anticipation of the event indicated whether they were coding the location egocentrically or objectively. In Exp I, a longitudinal study of 24 infants, significant shift with age from egocentric responding at 6 and 11 mo to objective responding at 16 mo was revealed, a change that data in Exp II indicated was not simply due to previous experience with the experimental space. In Exps III and IV, manipulation of the type of experience Ss had during training failed to decrease egocentric responding at 6 and 11 mo. The overall pattern indicated that a landmark had its greatest impact at 11 mo. The data support Piaget's theory that in coding location, young infants rely on past accommodations to an object rather than its relationship to other objects or the larger space. (8 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Long-term detection and identification of metandienone and stanozolol abuse in athletes by gas chromatography-high-resolution mass spectrometry

The misuse of anabolic androgenic steroids (AAS) in human sports is controlled by gas chromatography-mass spectrometric analysis of urine specimens obtained from athletes. The analysis is improved with modern high-resolution mass spectrometry (HRMS). The detection and identification of metabolites of stanozolol (I) [3'-hydroxystanozolol (II) and 4 beta-hydroxystanozolol (III)] and metandienone (IV) I17 beta-methyl-5 beta-androst-1-ene-3 alpha,17 alpha-diol (V) and 18-nor-17,17-dimethyl-5 beta-androsta-1,13-dien-3 alpha-ol (VI)] with GC-HRMS at 3000 resolution yielded a large increase in the number of positive specimens. A total of 116 anabolic steroid positives were found in this laboratory in 1995 via GC-MS and GC-HRMS screening of 6700 human urine specimens collected at national and international sporting events and at out-of-competition testing. Of the 116 positive cases, 41 were detected using conventional (quadrupole) GC-MS screening. The other 75 positives were identified via GC-HRMS screening. To confirm the HRMS screening result, the urine sample was reanalyzed using a specific sample workup procedure to selectively isolate the metabolites of the identified substance. II and III were selectively isolated via immunoaffinity chromatography (IAC) using an antibody which was prepared for methyltestosterone and shows high cross reactivity to II and III. V and VI were isolated using high-performance liquid chromatography (HPLC) fractionation.