Molecular biology of cannabinoid receptors

During the last decade, research on the molecular biology and genetics of cannabinoid receptors has led to a remarkable progress in understanding of the endogenous cannabinoid system, which functions in a plethora of physiological processes in the animal. At present, two types of cannabinoid receptors have been cloned from many vertebrates, and three endogenous ligands (the endocannabinoids arachidonoyl ethanolamide, 2-arachidonoyl glycerol and 2-arachidonoyl-glycerol ether) have been characterized. Cannabinoid receptor type 1 (CB(1)) is expressed predominantly in the central and peripheral nervous system, while cannabinoid receptor type 2 (CB(2)) is present almost exclusively in immune cells. Cannabinoid receptors have not yet been cloned from invertebrates, but binding proteins for endocannabinoids, endocannabinoids and metabolic enzyme activity have been described in a variety of invertebrates except for molting invertebrates such as Caenorhabditis elegans and Drosophila. In the central nervous system of mammals, there is strong evidence emerging that the CB(1) and its ligands comprise a neuromodulatory system functionally interacting with other neurotransmitter systems. Furthermore, the presynaptic localization of CB(1) together with the results obtained from electrophysiological experiments strengthen the notion that in cerebellum and hippocampus and possibly in other regions of the central nervous system, endocannabinoids may act as retrograde messengers to suppress neurotransmitter release at the presynaptic site. Many recent studies using genetically modified mouse lines which lack CB(1) and/or CB(2) finally could show the importance of cannabinoid receptors in animal physiology and will contribute to unravel the full complexity of the cannabinoid system.     

Novel promoter/transactivator configurations for macrolide- and streptogramin-responsive transgene expression in mammalian cells

Background

The recently developed heterologous macrolide‐ (E.REX system) and streptogramin‐ (PIP system) responsive gene regulation systems show significant differences in their regulation performance in diverse cell lines.

Methods

In order to provide optimal regulation modalities for a wide variety of mammalian cell lines, we have performed a detailed analysis of E.REX and PIP systems modified in (i) the transactivation domains of the antibiotic‐dependent transactivators, (ii) the type of minimal promoter used, and (iii) the spacing between the operator module and the minimal promoter.

Results

These novel E.REX and PIP regulation components showed not only dramatically improved regulation performance in some cell types, but also enabled their use in cell lines which had previously been inaccessible to regulated transgene expression.

Conclusions

Due to their modular set‐up the novel E.REX and PIP regulation systems presented here are most versatile and ready for future upgrades using different cell‐specific key regulation components. Copyright © 2002 John Wiley & Sons, Ltd.

     

Maxillofacial reconstruction with prefabricated osseous free flaps: a 3-year experience with 24 patients

Between January of 1998 and May of 2002, 25 prefabricated osseous free flaps (23 fibula and two iliac crest flaps) were transferred in 24 patients to repair maxillary (six flaps) or mandibular (eight flaps) defects after tumor resection, severe maxillary (four flaps) or mandibular (one flap) atrophy (Cawood VI), maxillary (one flap) or mandibular (three flaps) defects after gunshot injury, and maxillary (two flaps) defects after traffic accidents. Prefabrication included insertion of dental implants, positioned with a drilling template in a preplanned position, and split-thickness grafting. Drilling template construction was based on the prosthetic planning. The template determined the position of the implants and the site and angulation of osteotomies, if necessary. The mean delay between prefabrication and flap transfer was 6 weeks (range, 4 to 8 weeks). While the flap was harvested, a bar construction with overdentures was mounted onto the implants. The overdentures were used as an occlusal key for exact three-dimensional positioning of the graft within the defect. The bar construction also helped to stabilize the horseshoe shape of the graft. The follow-up period ranged from 2 months to 4 years (mean, 21 months), during which time two total and three partial flap losses occurred. One total loss was due to thrombosis of the flap veins during the delay period, whereas the other total loss was caused by spasm of the peroneal artery. Two partial losses were due to oversegmentation of the flaps with necrosis of the distal fragment, whereas one partial loss was caused by disruption of the vessel from the distal part. Of the 90 implants that were inserted into the prefabricated flaps during the study period, 10 were lost in conjunction with flap failure; of the remaining 80 implants, four were lost during the observation period, for a success rate of 95 percent. Flap prefabrication based on prosthetic planning offers a powerful tool for various reconstructive problems in the maxillofacial area. Although it involves a two-stage procedure, the time for complete rehabilitation is shorter than with conventional procedures.     

Whole-genome comparison of leucine-rich repeat extensins in Arabidopsis and rice. A conserved family of cell wall proteins form a vegetative and a reproductive clade

We have searched the Arabidopsis and rice (Oryza sativa) genomes for homologs of LRX1, an Arabidopsis gene encoding a novel type of cell wall protein containing a leucine-rich repeat (LRR) and an extensin domain. Eleven and eight LRX (LRR/EXTENSIN) genes have been identified in these two plant species, respectively. The LRX gene family encodes proteins characterized by a short N-terminal domain, a domain with 10 LRRs, a cysteine-rich motif, and a variable C-terminal extensin-like domain. Phylogenetic analysis performed on the conserved domains indicates the existence of two major clades of LRX proteins that arose before the eudicot/monocot divergence and then diversified independently in each lineage. In Arabidopsis, gene expression studies by northern hybridization and promoter::uidA fusions showed that the two phylogenetic clades represent a specialization into "reproductive" and "vegetative" LRXs. The four Arabidopsis genes of the "reproductive" clade are specifically expressed in pollen, whereas the seven "vegetative" genes are predominantly expressed in various sporophytic tissues. This separation into two expression classes is also supported by previous studies on maize (Zea mays) and tomato (Lycopersicon esculentum) LRX homologs and by information on available rice ESTs. The strong conservation of the amino acids responsible for the putative recognition specificity of the LRR domain throughout the family suggests that the LRX proteins interact with similar ligands.     

Redox labelled avidin for enzyme sensor architectures

Conjugates of avidin with ferrocene and with microperoxidase 8 have been used as electrochemically active molecular building blocks. Assemblies of the conjugates with biotinylated glucose oxidase or lactate oxidase on gold electrodes were tested as enzyme sensors for glucose and lactate. The electrochemical detection is based either on ferrocene-mediated oxidation of the substrate in oxygen-free solution, or on microperoxidase-catalysed reduction of H2O2 which is enzymatically produced from the substrate and molecular oxygen. Glucose and lactate were detectable with both detection principles in concentrations down to 1 or 0.1 mM, respectively. The molecular architecture concept allows quick adaptation of the sensors to other analytes, and it provides a platform for arrays of sensors with different selectivity.     

Reduction in cross-sucking in calves by the use of a modified automatic teat feeder

Cross-sucking may be a problem in group-housed calves fed by automatic teat feeders. In the present study, the behaviour of calves fed by a conventional feeder with an open feeding stall (n=15 calves) was compared with the behaviour of calves fed by a modified feeder, closing in the rear after the calf has entered (n=14 calves). It was found that the calves fed by the feeder with the closed stall stayed longer in the feeding stall following milk ingestion (P<0.001), showed longer bouts of non-nutritive sucking directed to the teat after milk ingestion (P<0.005) and performed less cross-sucking in the first 15min after milk ingestion (P<0.001) than calves fed by the feeder with the open stall. The design of the feeder had, however, no significant influence on the incidence of cross-sucking performed without close temporal association with milk ingestion. As the modification in the feeding stall reduces cross-sucking and is not costly, it should be feasible to introduce it in practice.     

Biodegradation of N-Methylmorpholine-N-oxide

N-Methylmorpholine-N-oxide (NMMO) is capable of dissolving cellulose without any further addition of chemicals. The solution can be used to produce cellulosic staple fibres by pressing it through spinning jets into an aqueous spinning bath. Because of results from conventional biodegradation tests using non-adapted activated sludge, the solvent is generally considered being persistent. The object of the described work was to show, whether and how activated sludge can be adapted to N-methylmorpholine-N-oxide and whether it is possible to purify NMMO-containing wastewaters in conventional wastewater treatment plants. The experiments showed that the sludge can be adapted within about 15–20 days. Adapted sludge can degrade the substance itself and its most important metabolites to concentrations below their detection levels and retain this ability even during limited periods without solvent being present in the wastewater. The main requirement for a successful adaptation is a high sludge age. The degradation takes place in several steps. First, NMMO is reduced to N-methylmorpholine. The next step is a demethylation of N-methylmorpholine to morpholine. This step is crucial for the adaptation process. Once morpholine has been formed, the adaptation proceeds very quickly until none of the substances in question can be detected any longer. So the next step must be the cleavage of the morpholine ring structure.     

HMQC and HSQC experiments with water flip-back optimized for large proteins

An HMQC experiment is proposed, dubbed FHMQC, where water flip-back is achieved by a single water-selective pulse preceding the basic HMQC pulse sequence. The scheme is demonstrated with a ¹⁵N, ¹H-HMQC spectrum of uniformly ¹⁵N/²H-labelled S. aureus DNA gyrase B with a molecular weight of 45 kDa for the unlabelled protein. The sensitivity of the experiment is improved compared to that of an FHSQC spectrum. It is further shown that the original FHSQC experiment can be shortened by the use of bipolar gradients. Relaxation times of different ¹⁵N magnetizations and coherences were measured. The new FHMQC scheme is implemented in 3D NOESY-¹⁵N-HMQC and 3D¹⁵ N-HMQC-NOESY-¹⁵N-HMQC pulse sequences which are demonstrated with a 24 kDa fragment of uniformly ¹⁵N/¹³C/²H-labelled S. aureus DNA gyrase B.