Acute tolerance to ethanol using drug discrimination and open-field procedures in rats

This study examined the phenomenon of acute tolerance to ethanol (ETOH) using drug discrimination learning (DDL), and open-field (OF) procedures. In DDL, rats were trained to discriminate between ETOH (1.2 g/kg) and saline. Doses of ETOH lower (0.6 and 0.9 g/kg), or higher (1.8 and 2.4 g/kg) than the training dose were tested to examine possible influence of ETOH pretreatment doses on the expression of acute tolerance. To assess concentrations of ETOH in the organism, a rebreathed air procedure was used. Equal concentrations after different ETOH doses were achieved by postponing the tests until sufficient time had elapsed. Only doses of ETOH higher than the training dose produced acute tolerance in the DDL procedure. For the response-time data no acute tolerance was observed. In the OF experiment, the occurrence of acute tolerance was examined for different spontaneous behaviours in drug-naive animals. At equal ETOH concentrations, the group examined during the descending phase of intoxication (1.8 g/kg, 60 min post-injection), reared significantly more than the group tested during the ascending phase (1.5 g/kg, 10 min post-injection). Other OF behaviours did not differ significantly between the two time intervals. Thus, it is suggested that acute tolerance is seen both in ETOH naive and in ETOH pre-exposed rats. However, in DDL acute tolerance was observed only when doses higher than the training dose of ETOH were evaluated.     

Early neuromuscular customized training after surgery for lumbar disc herniation: a prospective controlled study

A prospective and controlled study of training after surgery for lumbar disc herniation (LDH). The objective was to determine the effect of early neuromuscular customized training after LDH surgery. No consensus exists on the type and timing of physical rehabilitation after LDH surgery. Patients aged 15–50 years, disc prolapse at L4–L5 or L5–S1. Before surgery, at 6 weeks, 4, and 12 months postoperatively, the following evaluations were performed: low back pain and leg pain estimated on a visual analog scale, disability according to the Roland–Morris questionnaire (RMQ) and disability rating index (DRI). Clinical examination, including the SLR test, was performed using a single blind method. Consumption of analgesics was registered. Twenty-five patients started neuromuscular customized training 2 weeks after surgery (early training group=ETG). Thirty-one patients formed a control group (CG) and started traditional training after 6 weeks. There was no significant difference in pain and disability between the two training groups before surgery. Median preoperative leg pain was 63 mm in ETG and 70 mm in the CG. Preoperative median disability according to RMQ was 14 in the ETG and 14.5 in the CG. Disability according to DRI (33/56 patients) was 5.3 in the ETG vs. 4.6 in the CG. At 6 weeks, 4 months, and 12 months, pain was significantly reduced in both groups, to the same extent. Disability scores were lower in the ETG at all follow-ups, and after 12 months, the difference was significant (RMQ P=.034, DRI P=.015). The results of the present study show early neuromuscular customized training to have a superior effect on disability, with a significant difference compared to traditional training at a follow-up 12 months after surgery. No adverse effects of the early training were seen. A prospective, randomized study with a larger patient sample is warranted to ultimately demonstrate that early training as described is beneficial for patients undergoing LDH surgery.     

Real-time PCR methods for independent quantitation of TTV and TLMV

There is considerable interest in the possible clinical effects of the human circoviruses TT virus (TTV) and TTV-like mini virus (TLMV). Most people appear to have at least one of these viruses replicating actively in their bodies, thus mere correlation of the presence of virus and disease states are probably less informative than a quantitative analysis of viraemia. Real-time PCR based methods, with either SYBR Green or TaqMan probe, designed to quantitate selectively TTV and TLMV are described. The suggested TaqMan-based protocols were suitable for quantitation of viruses in the range of 10(2)-10(9) copies/ml of sample; and proved, by sequencing of PCR products, to be specific for each of the two viruses.     

Complete Sequence of a 93.4-kb Contig from Chromosome 3 of Trypanosoma cruzi Containing a Strand-Switch Region

We have initiated large-scale sequencing of the third smallest chromosome of the CL Brener strain of Trypanosoma cruzi and we report here the complete sequence of a contig consisting of three cosmids. This contig covers 93.4 kb and has been found to contain 20–30 novel genes and several repeat elements, including a novel chromosome 3-specific 400-bp repeat sequence. The intergenic sequences were found to be rich in di- and trinucleotide repeats of varying lengths and also contained several known T. cruzi repeat elements. The sequence contains 29 open reading frames (ORFs) longer than 700 bp, the longest being 5157 bp, and a large number of shorter ORFs. Of the long ORFs, seven show homology to known genes in parasites and other organisms, whereas four ORFs were confirmed by sequencing of cDNA clones. Two shorter ORFs were confirmed by a database homology and a cDNA clone, respectively, and one RNA gene was identified. The identified genes include two copies of the gene for alanine-aminotransferase as well as genes for glucose-6-phosphate isomerase, protein kinases and phosphatases, and an ATP synthase subunit. An interesting feature of the sequence was that the genes appear to be organized in two long clusters containing multiple genes on the same strand. The two clusters are transcribed in opposite directions and they are separated by an ~20-kb long, relatively GC-rich sequence, that contains two large repetitive elements as well as a pseudogene for cruzipain and a gene for U2snRNA. It is likely that this strand switch region contains one or more regulatory and promoter regions. The reported sequence provides the first insight into the genome organization of T. cruzi and shows the potential of this approach for rapid identification of novel genes.     

Chemoattractant-induced NADPH oxidase activity in human monocytes is terminated without any association of receptor-ligand complex to cytoskeleton

When the chemotactic peptide formylmethionyl-leucyl-phenylalanine binds to its cell surface receptor, a transmembrane signal is generated that activates the superoxide-producing NADPH oxidase of human phagocytes. Comparing monocytes and neutrophils with regard to the production of superoxide anion induced by the peptide, we found a similar time-course for both types of cells. In neutrophils, ligand binding induced a conversion of the receptor to a high-affinity form, a change suggested to be due to an association of the receptor-ligand complex to the Triton X-100-insoluble cytoskeleton. This event has been hypothesized to terminate the signal that activates the NADPH oxidase and thereby results in cessation of the cellular production of superoxide anion. Neutrophils preincubated with the cytoskeleton-disrupting drug cytochalasin B showed an increased and prolonged superoxide anion production after activation with the peptide, thus indicating that the cytoskeleton is involved in terminating this response. Formylmethionyl-leucyl-phenylalanine was also found to induce polymerization of actin in monocytes; however, cytochalasin B had no effect on the peptide-induced generation of superoxide anion in these cells. Furthermore, also in monocytes, ligand binding induced a conversion of the receptor to a high-affinity form; however, the receptor-ligand complex did not coisolate with the Triton X-100-insoluble cytoskeleton. These results indicate that, in monocytes, the NADPH oxidase activating pathway is terminated without any association of the receptor-ligand complex to the Triton X-100-insoluble cytoskeleton.     

The regulatory role of FSH in steroid formation by luteinized human granulosa cells in culture

Granulosa cells were aspirated from human pre-ovulatory folliclesfollowing a combined clomiphene-gonadotrophin stimulation inan in-vitro fertilization (IVF) programme. The cells were culturedfor 8 days in medium M199 containing 10% bovine fetal calf serumunder 5% CO₂ in air. Pure human FSH and human LH were addedalone or in combination to the culture in various concentrationsand the progesterone (P) and oestradiol-17rnals.org/math/beta.gif" ALT="{beta}" BORDER="0"> (E₂) levels in themedium were measured every second day by a conventional RIAtechnique. In the presence of FSH or LH the formation of P increased2- to 3-fold with the pronounced effect after 4 to 6 days inculture. Addition of testosterone (T) (3 ? 10^–7 M) tothe culture medium affected neither basal nor gonadotrophinstimulated P formation. In this system, only minute amountsof E₂ were formed and neither FSH nor LH stimulated its formation.When the medium was fortified with T, basal E₂ formation increased50- to 100-fold. FSH further stimulated this conversion significantlyafter 6 and 8 days of culture, while LH had no significant influence.     

Manipulations in Maternal Environment Reverse Periodontitis in Genetically Predisposed Rats

The predisposition to develop periodontitis is partly genetically determined in humans as well as in animals. Here we demonstrate, however, that early manipulations in the maternal environment of an animal (rat) model of periodontitis can fully reverse the genetic predisposition to develop periodontitis at adult age.     

Characterization of the major secreted zinc metalloprotease- dependent glycerophospholipid:cholesterol acyltransferase, PlaC, of Legionella pneumophila

Legionella pneumophila, an intracellular pathogen causing a severe pneumonia, possesses distinct lipolytic activities which have not been completely assigned to specific enzymes so far. We cloned and characterized a gene, plaC, encoding a protein with high homology to PlaA, the major secreted lysophospholipase A of L. pneumophila and to other hydrolytic enzymes belonging to the GDSL family. Here we show that L. pneumophila plaC mutants possessed reduced phospholipase A and lysophospholipase A activities and lacked glycerophospholipid:cholesterol acyltransferase activity in their culture supernatants. The mutants' reduced phospholipase A and acyltransferase activities were complemented by reintroduction of an intact copy of plaC. Additionally, plaC conferred increased lysophospholipase A and glycerophospholipid:cholesterol acytransferase activities to recombinant Escherichia coli. Furthermore, PlaC was shown to be another candidate exported by the L. pneumophila type II secretion system and was activated by a factor present in the bacterial culture supernatant dependent on the zinc metalloprotease. Finally, the role of plaC in intracellular infection of Acanthamoeba castellanii and U937 macrophages with L. pneumophila was assessed, and plaC was found to be dispensable. Thus, L. pneumophila possesses another secreted lipolytic enzyme, a protein with acyltransferase, phospholipase A, and lysophospholipase A activities. This enzyme is distinguished from the previously characterized phospholipases A and lysophospholipases A by its capacity not only to cleave fatty acids from lipids but to transfer them to cholesterol. Cholesterol is an important compound of eukaryotic membranes, and an acyltransferase might be a tool for host cell modification to fit the needs of the bacterium.