Multidrug-resistant pathogenic Escherichia coli isolated from wild birds in a veterinary hospital.

Wild birds are carriers of Escherichia coli. However, little is known about their role as reservoirs for extra-intestinal pathogenic E. coli (ExPEC). In this work we investigated E. coli strains carrying virulence genes related to human and animal ExPEC isolated from free-living wild birds treated in a veterinary hospital. Multidrug resistance was found in 47.4% of the strains, but none of them were extended-spectrum beta-lactamase producers. Not only the virulence genes, but also the serogroups (e.g. O1 and O2) detected in the isolates of E. coli have already been implicated in human and bird diseases. The sequence types detected were also found in wild, companion and food animals, environmental and human clinical isolates in different countries. Furthermore, from the 19 isolates, 17 (89.5%) showed a degree of pathogenicity on an in vivo infection model. The isolates showed high heterogeneity by pulsed-field gel electrophoresis indicating that E. coli from these birds are clonally diverse. Overall, the results showed that wild birds can be reservoirs and/or vectors of highly pathogenic and multidrug-resistant E. coli that have the potential to cause disease in humans and poultry.

A Reliable Method for Quantifying Plasma Cell-Free DNA Using an Internal Standard Strategy: Evaluation in a Cohort of Non-Pregnant and Pregnant Women.

The use of plasma cell-free DNA (cfDNA) as a useful biomarker in obstetric clinical practice has been delayed due to the lack of reliable quantification protocols. We developed a protocol to quantify plasma cfDNA using an internal standard strategy to overcome difficulties posed by low levels and high fragmentation of cfDNA. cfDNA was isolated from plasma samples of non-pregnant (NP, n = 26) and pregnant (P, n = 26) women using a commercial kit and several elution volumes were evaluated. qPCR parameters were optimized for cfDNA quantification, and several quantities of a recombinant standard were evaluated as internal standard. Absolute quantification was performed using a standard curve and the quality of the complete method was evaluated. cfDNA was eluted in a 50-µl volume, actin-ß (ACTB) was selected as the target gene, and qPCR parameters were optimized. The ACTB standard was constructed and 1000 copies were selected as internal standard. The standard curve showed R2 = 0.993 and E = 109.7%, and the linear dynamic range was defined between 102 and 106 ACTB copies/tube. Repeatability and reproducibility in terms of CV were 19% and up to 49.5% for ACTB copies per milliliter of plasma, respectively. The range of cfDNA levels was 428-18,851 copies/mL in NP women and 4031-2,019,363 copies/mL in P women, showing significant differences between the groups. We recommend the application of internal standard strategy for a reliable plasma cfDNA quantification. This methodology holds great potential for a future application in the obstetric field.

Sorting of newly synthesized galactosphingolipids to the two surface domains of epithelial cells.

The high concentration of glycosphingolipids on the apical surface of epithelial cells may be generated by selective transport from their site of synthesis to the cell surface. Previously, we showed that canine kidney MDCK and human intestinal Caco-2 cells converted a ceramide carrying the short fluorescent fatty acid C6-NBD to glucosylceramide (GlcCer) and sphingomyelin (SM), and that GlcCer was preferentially transported to the apical surface as compared to SM. Here, we address the point that not all glycosphingolipid classes are apically enriched in epithelia. We show that a ceramide containing the 2-hydroxy fatty acid C6OH was preferentially converted by MDCK and Caco-2 cells to galactosylceramide (GalCer) and its derivatives galabiosylceramide (Ga2Cer) and sulfatide (SGalCer) as compared to SM and GlcCer--all endogenous lipid classes of these cells. Transport to the apical and basolateral cell surface was monitored by a BSA-depletion assay. In MDCK cells, GalCer reached the cell surface with two- to sixfold lower apical/basolateral polarity than GlcCer. Remarkably, in Caco-2 cells GalCer and GlcCer displayed the same apical/basolateral polarity, but it was sixfold lower for lipids with a C6OH chain than for C6-NBD lipids. Therefore, the sorting of a sphingolipid appears to depend on lipid structure and cell type. We propose that the different ratios of gluco- and galactosphingolipid synthesis in the various epithelial tissues govern lipid sorting in the membrane of the trans Golgi network by dictating the composition of the domains from where vesicles bud to the apical and basolateral cell surface.

Next generation sequencing characterizes the extent of HLA diversity in an Argentinian registry population.

Next generation DNA sequencing is used to determine the HLA-A, -B, -C, -DRB1, and -DQB1 assignments of 1472 unrelated volunteers for the unrelated donor registry in Argentina. The analysis characterized all HLA exons and introns for class I alleles; at least exons 2, 3 for HLA-DRB1; and exons 2 to 6 for HLA-DQB1. Of the distinct alleles present, there are 330 class I and 98 class II. The majority (~98%) of the cumulative allele frequency at each locus is contributed by alleles that appear at a frequency of at least 1 in 1000. Fourteen (18.2%) of the 77 novel class I and II alleles carry nonsynonymous variation within their exons; 52 (75.4%) class I novel alleles carry only single, apparently random, nucleotide variation within their introns/untranslated regions. Alleles encoding protein variation not usually detected by typing focused only on the exons encoding the antigen recognition domain are 1.0% of the class I assignments and 7.3% of the class II assignments (predominantly DQB1*02:02:01, DQB1*03:19:01, and DRB1*14:54:01). Updates to the common and well documented list of alleles include 10 alleles previously thought to be uncommon but that are found at least 30 times. Five locus haplotypes estimated using the expectation-maximization algorithm as present 3 or more times total 187. While the known HLA diversity continues to increase, the conservation of known allele sequences is remarkable. Overall, the HLA diversity observed in the Argentinian population reflects its European and Native American ancestry.

[Hyperhomocysteinaemia and type 2 diabetes]. / Hyperhomocystéinémie et diabète de type 2.

OBJECTIVES: Hyperhomocysteinaemia is associated with cardiovascular events in nondiabetic individuals. The present study was aimed to explore the implication of hyperhomocysteinemia in development of cardiovascular events in patients with type 2 diabetes. DESIGN: A total of 185 patients with type 2 diabetes (115 women and 70 men, 30 to 93 years of age) have been included consecutively in the ambulatory unit at the Saint-Philibert Hospital. For each patient the concentration of homocysteine, cholesterol and triglyceride levels and HbA1c have been measured. In the studied population, 121 patients presented cardiovascular events (myocardial infarctus, peripheric arteriopathy, cerebrovascular accident). RESULTS: The patients with cardiovascular events were older, the concentration of homocysteine and creatinine were higher. The plasma homocysteine levels adjusted for age and creatinine levels were higher in patients with cardiovascular events than in patients without cardiovascular events (15.4 +/- 3.52 micromol/L and 13.13 +/- 2.26 micromol/L respectively; p = 2. 10(-5)). CONCLUSIONS: Hyperhomocysteinemia is an independant risk factor for cardiovascular events in type 2 diabetes, independent of age and renal function.

Oxidation of very-long-chain fatty acids in rat brain: cerotic acid is beta-oxidized exclusively in rat brain peroxisomes.

We studied the effect of sodium 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA), a potent inhibitor of mitochondrial carnitine palmitoyltransferase I, on fatty acid oxidation by rat brain cells. In cultured glial cells as well as in dissociated brain cells from adult rats palmitic acid (16:0) oxidation was inhibited by about 85% of control values when 25 microM POCA was added to the medium, whereas no inhibition of cerotic acid (26:0) oxidation was observed. Furthermore, omission of carnitine from the culture medium resulted in a 57.7% decrease in palmitic acid oxidation in cultured glial cells, whereas cerotic acid oxidation was not influenced. These results indicate that rat brain peroxisomes contribute only little (about 15%) to palmitic acid oxidation and provide conclusive evidence that cerotic acid is oxidized exclusively in rat brain peroxisomes.

Characterization of three arylsulfatases in semen: seminolipid sulfohydrolase activity is present in seminal plasma.

Sperm cells and seminal plasma of various mammals contain high levels of arylsulfatase. In the present study, we investigated the composition of soluble AS in these compartments of boar semen by analysing sperm cells and seminal plasma using anion-exchange chromatography. Seminal plasma contained both arylsulfatase B (2.4 units per ml), an enzyme which desulfates sulfoglycosaminoglycans and probably sulfoglycoproteins, and arylsulfatase A (10.2 units per ml), an enzyme which desulfates sulfogalactolipids. Sperm cells contained only arylsulfatase A, which differed biochemically from the extracellular arylsulfatase A of seminal plasma (2.6 units per ml). Both types of arylsulfatase A desulfate seminolipid, the natural sulfolipid substrate in sperm, as well as two brain sulfatides. The possible physiological consequences of the presence of extracellular arylsulfatases in seminal plasma for spermatozoa are discussed.

Acetoacetate is a cholesterogenic precursor for myelinating rat brain and spinal cord. Incorporation of label from [3-14C]acetoacetate, [14C]glucose and 3H2O.

Rat pups, 3 weeks old, were injected i.p. with combinations of 3H2O and either [3-14C]acetoacetate or [14C]glucose. 3H/14C incorporation ratios were measured in lipid fractions of homogenates and myelin prepared from whole brain and spinal cord. Spinal cord synthesized at least twice as much fatty acids and 3-fold more sterols than whole brain. Both tissues used acetoacetate preferentially for sterol synthesis, whereas label from [14C]glucose was distributed between fatty acids and sterols in the same way as 3H from 3H2O. The relative contributions of acetoacetate to sterol synthesis in whole tissue and in the purified myelin fraction were about the same, both for the cerebrum and for the spinal cord.

Preferential utilization of ketone bodies for the synthesis of myelin cholesterol in vivo.

1. The distribution of radioactivity among lipid classes of myelin and other subcellular brain fractions of young rats (18-21 days) was determined after in vivo injection of (3-(14)C-labelled ketone bodies, [U-(14)C] glucose or [2-(14)C] glucose. 2. The incorporation ratios (sterol/fatty acids) were 0.67, 1.48, 0.25, 0.62 and 0.54 for whole brain, myelin, mitochondria, microsomes and synaptosomes, respectively, with (3-(14)C)-labelled ketone bodies as substrate and 0.37, 0.89, 0.19, 0.34 and 0.29 with [U-(14)C] glucose as substrate. These data show that, both in whole brain and in subcellular brain fractions, acetyl groups derived from ketone bodies are used for sterol synthesis to a large extent than acetyl groups originating from glucose. 3. The specific radioactivity of cholesterol is much higher in myelin than in whole brain or in the other brain fractions, particularly after administration of labelled ketone bodies as substrate. 4. The incorporation patterns of acetoacetate and D-3-hydroxybutyrate were very similar, indicating that both ketone bodies contribute acetyl groups for lipid synthesis via the same metabolic route. 5. Our data suggest that a direct metabolic path from ketone bodies towards cholesterol exists - possibly via acetoacetyl-CoA formation in the cytosol of brain cells - and that this process is most active in oligodendrocytes.

Effects of triiodothyronine on the synthesis of sulfolipids by oligodendrocyte-enriched glial cultures.

Glial cultures were obtained from the brains of 1-week-old rats and were grown in a chemically defined, serum-free medium. We investigated the development of oligodendrocytes in these cultures and the synthesis of sulfolipids in the presence and absence of triiodothyronine (T3) in the medium: (1) In the presence of T3, the incorporation of [35S]sulfate into sulfolipids exhibited a developmental profile which is comparable to that found in the developing brain in vivo. A sharp peak of sulfolipid synthesis was observed at day 5 in vitro, which is equivalent to day 12 after birth. As observed in vivo, the percentage of label incorporated into sulfogalactosyldiradylglycerols decreased with time in culture. (2) Addition of T3 to the medium stimulated sulfolipid synthesis by oligodendrocytes in a dose-related manner (optimal T3 concentration, 30 nM). The hormone also enhanced the rates of cholesterogenesis and lipogenesis but to a lesser extent than sulfolipid synthesis. (3) The temporary omission of T3 from the medium resulted in lower rates of sulfolipid synthesis that could not be restored by readdition of T3. This inhibitory effect was most pronounced if the hormone was omitted from the medium on days 2 and 3 in culture. (4) Omission of T3 also resulted in the development of fewer oligodendrocytes in the cultures. Our results show that T3 is essential for the development of oligodendrocytes in our neurone-free culture system. They also indicate that the stimulation of myelination by thyroid hormones can, at least partially, be explained as a direct effect of T3 on oligodendrocytes, independent of an effect of T3 on neuronal growth.

Cultured oligodendrocytes metabolize a fluorescent analogue of sulphatide; inhibition by monensin.

It has been suggested that oligodendrocytes can actively phagocytose myelin debris during active myelination or after injury and experimental demyelination. Therefore, we have used a fluorescent analogue (N-lissamine rhodaminyl-(12-aminododecanoyl) cerebroside 3-sulphate) to study the metabolic fate of sulphatide, a galactosphingolipid that is highly enriched in myelin membranes. The fluorescent sulphatide was incorporated in small unilamellar vesicles and administered to cultured oligodendrocytes. The association of the lipid probe to the cells in culture was saturable in time and with the concentration of the probe. The processes of association, internalization and subcellular distribution were followed by confocal scanning laser microscopy and appeared to be very rapid. Within 20 min a marked perinuclear staining was seen. After prolonged incubation the fluorescence distributed gradually over the cytoplasm and into cellular branches along structures suggestive of cytoskeletal elements. Lipid analysis demonstrated that ceramide was the major metabolite present in the cells but galactosylceramide, sphingomyelin and free fatty acid were also detected. In the culture medium only free fatty acid and sphingomyelin were found. Monensin did not affect the cellular association and internalization of the fluorescent sulphatide but markedly reduced its conversion to metabolic products. These results indicate that exogenous sulphatide is targeted to the Golgi apparatus prior to its lysosomal degradation.

Acetoacetate and glucose as substrates for lipid synthesis by rat brain oligodendrocytes and astrocytes in serum-free culture.

We have compared glucose and acetoacetate as precursors for lipogenesis and cholesterogenesis by oligodendrocytes and astrocytes, using mixed glial cultures enriched in oligodendrocytes. In order to differentiate between metabolic processes in oligodendrocytes and those in astrocytes, the other major cell type present in the mixed culture, we carried out parallel incubations with cultures from which the oligodendrocytes had been removed by treatment with anti-galactocerebroside serum and guinea-pig complement. The following results were obtained: 1. Both oligodendrocytes and astrocytes in culture actively utilize acetoacetate as a precursor for lipogenesis and cholesterogenesis. 2. In both cell types, the incorporation of acetoacetate into fatty acids and cholesterol exceeds that of glucose by a factor of 5-10 when the precursors are present at concentrations of 1 mM and higher. 3. Glucose stimulates acetoacetate incorporation into fatty acids and cholesterol, whereas acetoacetate reduces the entry of glucose into these lipids. This suggests that glucose is necessary for NADPH generation, but that otherwise the two precursors contribute to the same acetyl-CoA pool. 4. Both with acetoacetate and with glucose as precursor, oligodendrocytes are more active in cholesterol synthesis than astrocytes. 5. Using incorporation of 3H2O as an indicator for total lipid synthesis, we estimated that acetoacetate contributes one third of the acetyl groups and glucose one twentieth when saturating concentrations of both substrates are present.

Developmental profiles of arylsulfatases A and B in rat cerebral cortex and spinal cord.

Arylsulfatases A (EC 3.1.6.1) and B (EC 3.1.6.12) are lysosomal enzymes that can remove sulfate groups from sulfatides and sulfo-glycosaminoglycans, respectively. The activities of these enzymes in cerebral cortex and in spinal cord of developing rat pups were measured. The tissues were homogenized and the arylsulfatases A and B in the soluble fraction were separated from each other by anion exchange chromatography on DE-52 cellulose. Subsequently, the enzyme activities were assayed with p-nitrocatechol sulfate as substrate at 37 degrees C and pH 5.6. We observed a developmental profile of arylsulfatase A, similar to that previously reported for cerebroside sulfatase (EC 3.1.6.8; (Van der Pal et al. (1990) Biochim. Biophys. Acta 1043, 91-96]. The activity of arylsulfatase A increased gradually during development, whereas arylsulfatase B rose more steeply, peaked around day 15 and declined thereafter. As a consequence the ratio between B and A forms of arylsulfatase dropped from about 4 in 1-week-old pups to 2.2 (cortex) and 0.7 (cord) in 7-week-old rat pups.

A rapid procedure for the preparation of oligodendrocyte-enriched cultures from rat spinal cord.

Spinal cords and cerebra from 7-day-old rat pups were compared as tissue sources for the isolation of oligodendrocytes and for studies on the development of these cells in culture. After 1 day in culture the serum-containing medium was replaced by a chemically-defined medium, which contained a cocktail of hormones that stimulated oligodendrocyte development. The cultures were characterized with various immunocytochemical markers; monoclonal A2B5 for bipotential glial progenitor cells, anti-galactocerebroside (GC) serum for oligodendrocytes, and anti-glial fibrillary acidic protein (GFAP) serum for astrocytes. The number of positive cells was counted and expressed as a percentage of total cells. At 1 day in culture the cell cultures from spinal cord contained 30% GC+ cells, increasing to 90% after 7 days in culture. In cultures derived from cerebra the percentage of GC+ cells was always lower than in cultures from spinal cord. In cerebral cultures GFAP+ cells increased from 15% at 1 day in culture to 30% at 7 days in culture, whereas it remained low in spinal cord cultures. The activity of oligodendroglial marker enzyme 2',3'-cyclic-nucleotide 3'-phosphodiesterase was followed during development in culture. The specific activity increased rapidly in both types of culture but was more than threefold higher in cultures derived from spinal cord. This procedure yields, within one week and without subculture, primary glial cultures from rat spinal cord, that are highly enriched in oligodendrocytes (greater than or equal to 90%; 3.10(5) oligodendrocytes per rat pup).

Galactosylceramide sulfotransferase, arylsulfatase A and cerebroside sulfatase activity in different regions of developing rat brain.

The in vivo metabolism of sulfatides was studied in spinal cord and cerebral cortex of developing rat pups. Developmental changes in the rate of sulfolipid synthesis were measured after the intraperitoneal injection of 35SO4(2-). We also measured the accumulation of sulfatides, as well as the profiles of cerebroside sulfotransferase, cerebroside sulfatase and arylsulfatase A in both brain regions as a function of postnatal development. The accumulation of sulfatides was higher in spinal cord than in cerebral cortex. In addition, sulfatide metabolism was more active in spinal cord. In both brain regions, the developmental pattern of 35SO4(2-) incorporation into sulfolipids was closely correlated to the activities of cerebroside sulfotransferase and of arylsulfatase A. The activity of these enzymes was initially low, increased during the period of active myelination and declined thereafter. However, the activity of cerebroside sulfatase, measured with its physiological substrate, [35S]sulfatide, increased during development and did not decline. An explanation for the difference between the developmental profiles of the arylsulfatase A and cerebroside sulfatase reactions (which are supposed to be catalysed by the same enzyme) is proposed.

Antisepsys and profilatic antimicrobial therapy in prevention of surgical site infection of horses / [Antissepsia e terapia antimicrobiana profilática no controle infeccioso do foco cirúrgico em equinos

The efficacy of an antisepsis protocol comprising chlorhexidine gluconate and ethyl alcohol in combination with prophylactic antimicrobial therapy in controlling surgical site infection in horses was studied. To that purpose, seven mixed breed horses received potassium penicillin and gentamicin at least 30 minutes prior to surgery. The surgical site was scrubbed with chlorhexidine gluconate and rinsed with ethyl alcohol. Samples were collected at four time points: (A) - before and (B) - immediately following shaving of the hair coat, (C) - at the end of antisepsis procedures, and (D) - at the end of the surgical procedure. Duration of surgery was recorded. Samples were cultured in three different culture mediums: Mitis Salivarus (Streptococcus sp.), Staphylococcus 110 (Staphylococcus sp.), and Mac Conkey (Enterobacteria). A high level of bacterial growth was observed in all culture mediums at (A) and (B), with no bacterial growth in (C). Staphylococcus sp. growth was observed in (D) in a single patient whose surgical procedure lasted for 120 minutes. Shaving of the hair coat reduced microbial flora on the surface of the skin. Antisepsis in combination with prophylactic antimicrobial therapy was effective in controlling surgical site infection in elective procedures with an average duration of 90 minutes.(AU)

Objetivou-se averiguar a eficácia do protocolo de antissepsia com clorexidina degermante e álcool etílico hidratado 70%, em associação com terapia antimicrobiana profilática, no controle microbiano do foco cirúrgico de equinos submetidos a procedimentos cirúrgicos. Foram utilizados 07 cavalos adultos de raças variadas, onde ambos receberam o mesmo tratamento (terapia antimicrobiana profilática e antissepsia com clorexidina degermante 2% e álcool etílico hidratado 70%), coletando-se amostras em quatro tempos distintos [(A - antes da tricotomia), (B - imediatamente após tricotomia), (C - ao término da antissepsia), (D - ao término do procedimento cirúrgico)]. O tempo de cada procedimento cirúrgico foi contabilizado. Foram utilizados três meios de cultura diferentes, cada um com especificidade para um tipo de crescimento bacteriano. Constatou-se alta incidência de crescimento bacteriano nos três meios utilizados nos tempos de coleta A e B. Para o tempo C, não foi observado crescimento bacteriano. No tempo D averiguou-se crescimento bacteriano do tipo Staphylococcus sp. em um único paciente, cujo tempo cirúrgico foi de 120 minutos de duração. Desta forma, a tricotomia reduziu a carga microbiana na superfície da pele. A antissepsia associada à terapia antimicrobiana profilática mostrou-se eficaz no controle microbiano do foco cirúrgico em procedimentos eletivos, com duração média de 90 minutos.(AU)

Acetoacetate: a major substrate for the synthesis of cholesterol and fatty acids by isolated rat hepatocytes.

Evidence is presented that isolated, intact rat hepatocytes can synthesize fatty acids and cholesterol from acetoacetate. The quantitative importance of these processes is evaluated by measuring total rates of fatty acid and cholesterol synthesis by incorporation of 3H from 3H2O. The contribution of acetoacetate varies from 14-54% and from 21-75% for de novo synthesized fatty acids and cholesterol, respectively, depending on the physiological condition of the donor rat. The relative contribution of acetoacetate to cholesterol synthesis is 1.4-2.3-times greater than to fatty acid synthesis.

Uptake and metabolism of fluorescent ceramide analogs by rat oligodendrocytes in culture.

We studied the metabolism of sphingolipids by oligodendrocytes derived from rat spinal cord by providing lipid vesicles with either N-lissamine-rhodaminyl-ceramide (LRh-Cer) or N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-ceramide (NBD-Cer) to the cells cultured in a chemically-defined medium. With both probes the major fluorescent product turned out to be sphingomyelin (SM). Most of LRh-SM was not cell-associated but recovered from the culture medium, probably due to back-exchange to the lipid vesicles. The accumulation of LRh-SM, both in the cells and in the medium, was inhibited in the presence of monensin or brefeldin A, whereas the production of NBD-SM was much less affected by these Golgi perturbing drugs. With LRh-Cer as substrate, LRh-labelled fatty acid (FA), galactosyl- and sulfogalactosyl-ceramides (GalCer and SGalCer) were also formed. NBD-Cer, however, was metabolized to glucosylceramide (GlcCer) and GalCer but not to SGalCer or NBD-FA. These data demonstrate that chemical modifications of ceramide alter its metabolism in oligodendrocytes and that the metabolites of LRh-Cer reflect the glycolipid composition of myelin more closely than those of NBD-Cer.

Sphingomyelin is synthesized at the plasma membrane of oligodendrocytes and by purified myelin membranes: a study with fluorescent- and radio-labelled ceramide analogues.

In most cell types sphingomyelin is synthesized predominantly in the cis-medial compartments of the Golgi stacks whereas the contribution of the plasma membrane is much lower. The aim of this study was to assess the contribution of both compartments to the synthesis of sphingomyelin in myelinating cells. Therefore, oligodendrocytes from rat spinal cord were incubated in culture with fluorescently- or radiolabelled ceramides, and the effects of a block in the vesicular flow (monensin, brefeldin A, low temperature) on surface synthesis of sphingomyelin were evaluated. The results indicate that approximately 50% of the sphingomyelin synthase is present at the plasma and myelin membranes of oligodendrocytes.

QSAR analyses of the substituted indanone and benzylpiperidine rings of a series of indanone-benzylpiperidine inhibitors of acetylcholinesterase.

QSAR analyses have been performed on the substituted indanone and benzylpiperidine ring substructures of a set of acetylcholinesterase, AChE, inhibitors of which 1-benzyl-4-[(5,6-dimethoxy-1-oxoindan-2-yl)methyl]piperidine hydrochloride is a potent in vitro and ex vivo inhibitor. The method of molecular decomposition-recomposition was used to define the sets of molecular substructures and corresponding in vitro inhibition databases. A QSAR involving the magnitude of the dipole moment, the highest occupied molecular orbital (HOMO) energy, and a specific pi-orbital wave function coefficient of the substituted indanone ring substructure was constructed and found to be significant. The absence of any molecular-shape or bulk term in the QSAR, coupled with some of the relatively large substituents used to construct the QSAR, suggests considerable space is available around the indanone ring during the inhibition process. A set of QSARs were constructed and evaluated for substituents on the aromatic ring of the benzylpiperidine substructure. The most significant QSAR involves a representation of molecular shape, the largest principal moment of inertia, and the HOMO of the substituted aromatic ring. It appears that upon binding the receptor "wall" is closely fit around the benzyl ring, especially near the para position. Overall, the QSAR analysis suggests inhibition potency can be better enhanced by substitution on the indanone ring, as compared to the aromatic sites of the benzylpiperidine ring. Moreover, inhibition potency can be rapidly diminished, presumably through steric interactions with the receptor surface of AChE, by substitution of moderate to large groups on the benzyl ring, particularly at the para position.