Chronic subdural cortical stimulation for phantom limb pain: report of a series of two cases.

Phantom limb pain is a complex, incompletely understood pain syndrome that is characterized by chronic painful paresthesias in a previous amputated body part. Limited treatment modalities exist that provide meaningful relief, including pharmacological treatments and spinal cord stimulation that are rarely successful for refractory cases. Here, we describe our two-patient cohort with recalcitrant upper extremity phantom limb pain treated with chronic subdural cortical stimulation. The patient with evidence of cortical reorganization and almost 60 years of debilitating phantom limb pain experienced sustained analgesic relief at a follow-up period of 6 months. The second patient became tolerant to the stimulation and his pain returned to baseline at a 1-month follow-up. Our unique case series report adds to the growing body of literature suggesting critical appraisal before widespread implementation of cortical stimulation for phantom limb pain can be considered.

DXA evaluation of femoral bone mineral density and cortical width in patients with prior total knee arthroplasty.

Periprosthetic fractures after total knee arthroplasty (TKA) have devastating consequences. Osteoporosis increases periprosthetic fracture risk, but distal femur bone mineral density (BMD) is not measured post-TKA. This study measured distal femur BMD and cortical width; both were lower in the TKA compared to the non-operated leg. BMD measurement reproducibility was good. Standardized DXA regions of interest are proposed. INTRODUCTION: Periprosthetic fractures following total knee arthroplasty (TKA) are not rare. We hypothesized that TKA is associated with low BMD, potentially increasing periprosthetic fracture risk. However, distal femur dual energy x-ray (DXA) measurement is virtually never performed after TKA due to lack of standardized approaches. Thus, this study's aims were to develop standard DXA femur regions of interest (ROIs), assess cortical width, and determine measurement reproducibility in TKA patients. METHODS: Thirty adults (15 M/15 F) age 59-80 years with unilateral, primary TKA within 2-5 years had femoral DXA scans performed in duplicate using a Lunar iDXA densitometer. In prior work, we established that femur BMD was lowest in the distal metaphysis and highest in mid-shaft. Thus, BMD and cortical width were measured at 15%, 25%, and 60% of the femur length measured from the distal notch. Femur BMD and cortical width were compared between limbs (TKA vs. non-operated side) by paired t test. RESULTS: BMD was 3.2-9.9% lower (p < 0.001) in the operated femur at all custom ROIs; substantial between individual differences existed with some up to 30% lower. Cortical width was lower (p < 0.05) at the 25% ROI on the TKA side. BMD reproducibility was excellent; CV 0.85-1.33%. CONCLUSIONS: Distal femur BMD can be reproducibly measured using DXA and is ~ 10% lower on the TKA leg. Similarly, medial and lateral cortices are thinner at the 25% ROI. These bone changes likely increase periprosthetic fracture risk. Further work to define and mitigate periprosthetic fracture risk after TKA is needed.

Surgery alters parameters of vitamin D status and other laboratory results.

Fracture liaison services often perform laboratory testing, but these results may be altered by surgery. In 40 hip arthroplasty patients, many laboratory parameters of bone health relevance were reduced by 8-22% on the first post-operative day. Laboratory results obtained in the immediate post-surgery interval do not reliably ascertain baseline status. INTRODUCTION: As secondary causes of osteoporosis are common, fracture liaison services often perform laboratory testing in the immediate post-fracture interval. We hypothesized that laboratory results obtained shortly after surgery may not accurately ascertain baseline status. If true, such alterations might confound subsequent fracture prevention efforts. METHODS: Patients undergoing elective total hip arthroplasty were studied as a surrogate for hip fracture patients. Blood and urine were obtained 2 weeks before surgery, before anesthetic induction, on post-operative day one, and 6 weeks after surgery. Serum total and free 25-hydroxyvitamin D (25(OH)D), vitamin D-binding protein (DBP), calcium, creatinine, albumin (Alb), alkaline phosphatase (ALP), plasma hemoglobin (Hgb) and urinary DBP/creatinine ratio (UDBP/Cr) were measured. RESULTS: Forty volunteers (28 women; 12 men) with mean age of 65.7 [8.7] years were studied. Laboratory results were stable from 2 weeks before to the day of surgery. On the first day after surgery, total 25(OH)D, DBP, calcium, creatinine, ALP, and Alb declined 8-22% (p < 0.0001); free 25(OH)D and Hgb declined by 8 and 15% (p < 0.01), respectively; and UDBP/Cr increased 32% (p < 0.01). Using a 25(OH)D <30 ng/mL threshold, vitamin D inadequacy prevalence increased from 38% before surgery to 68% the day after (p < 0.001). All laboratory values returned to baseline at 6 weeks after surgery. CONCLUSIONS: Laboratory values are reduced immediately following elective total hip arthroplasty. Testing at that time does not accurately ascertain baseline status and may lead to elevated estimates of vitamin D inadequacy, incorrect interventions, and misallocation of healthcare resources.

Lack of differential motor outcome with subthalamic nucleus region stimulation in Parkinson's disease.

Deep brain stimulation (DBS) has emerged as a viable therapy for Parkinson's disease (PD). The impact of subthalamic nucleus (STN) lead placement (lateral versus medial) on motor outcome, however, has not been systematically evaluated. Forty-eight patients with PD underwent STN-DBS surgery and were evaluated postoperatively for 48 weeks for motor improvement as measured by the Unified Parkinson's Disease Rating Scale (UPDRS) part III (standardized motor examination) and levodopa equivalent daily dose (LEDD). Postoperative MRI was used to identify the location of the active stimulating contact and motor outcome was analyzed. STN-DBS was associated with significant improvement in motor outcome as determined by a reduction in the UPDRS part III subscore from 34.44 ± 1.29 at baseline to 18.76 ± 1.06 at end visit (p<0.0001) and a reduction in LEDD from 1721 ± 152 mg/day at baseline to 1134 ± 119 mg/day at end visit (p=0.0024). Patients with stimulating contacts in the medial STN compared to the lateral STN did not demonstrate any significant differences in motor outcome (UPDRS, p=0.5811; LEDD, p=0.7341). No significant differences were found in motor outcome between patients with STN stimulation compared to stimulation of surrounding fiber tracts (p=0.80). No significant difference in stimulation voltage was noted with respect to lead location. Our study did not find a significant effect for the location of active contact and motor outcome neither within the subregions of the STN nor between the STN and surrounding fibers. Further research is needed to better understand the neurophysiological basis for these results.

Long-term stabilization of place cell remapping produced by a fearful experience.

Fear is an emotional response to danger that is highly conserved throughout evolution because it is critical for survival. Accordingly, episodic memory for fearful locations is widely studied using contextual fear conditioning, a hippocampus-dependent task (Kim and Fanselow, 1992; Phillips and LeDoux, 1992). The hippocampus has been implicated in episodic emotional memory and is thought to integrate emotional stimuli within a spatial framework. Physiological evidence supporting the role of the hippocampus in contextual fear indicates that pyramidal cells in this region, which fire in specific locations as an animal moves through an environment, shift their preferred firing locations shortly after the presentation of an aversive stimulus (Moita et al., 2004). However, the long-term physiological mechanisms through which emotional memories are encoded by the hippocampus are unknown. Here we show that during and directly after a fearful experience, new hippocampal representations are established and persist in the long term. We recorded from the same place cells in mouse hippocampal area CA1 over several days during predator odor contextual fear conditioning and found that a subset of cells changed their preferred firing locations in response to the fearful stimulus. Furthermore, the newly formed representations of the fearful context stabilized in the long term. Our results demonstrate that place cells respond to the presence of an aversive stimulus, modify their firing patterns during emotional learning, and stabilize a long-term spatial representation in response to a fearful encounter. The persistent nature of these representations may contribute to the enduring quality of emotional memories.

The nucleus accumbens as a potential target for central poststroke pain.

Although deep brain stimulation (DBS) has been found to be efficacious for some chronic pain syndromes, its usefulness in patients with central poststroke pain (CPSP) has been disappointing. The most common DBS targets for pain are the periventricular gray region (PVG) and the ventralis caudalis of the thalamus. Despite the limited success of DBS for CPSP, few alternative targets have been explored. The nucleus accumbens (NAC), a limbic structure within the ventral striatum that is involved in reward and pain processing, has emerged as an effective target for psychiatric disease. There is also evidence that it may be an effective target for pain. We describe a 72-year-old woman with a large right hemisphere infarct who subsequently experienced refractory left hemibody pain. She underwent placement of 3 electrodes in the right PVG, ventralis caudalis of the thalamus, and NAC. Individual stimulation of the NAC and PVG provided substantial improvement in pain rating. The patient underwent implantation of permanent electrodes in both targets, and combined stimulation has provided sustained pain relief at nearly 1 year after the procedure. These results suggest that the NAC may be an effective DBS target for CPSP.

Clostridium difficile infection in the community: a zoonotic disease?

Clostridium difficile infections (CDIs) are traditionally seen in elderly and hospitalized patients who have used antibiotic therapy. In the community, CDIs requiring a visit to a general practitioner are increasingly occurring among young and relatively healthy individuals without known predisposing factors. C. difficile is also found as a commensal or pathogen in the intestinal tracts of most mammals, and various birds and reptiles. In the environment, including soil and water, C. difficile may be ubiquitous; however, this is based on limited evidence. Food products such as (processed) meat, fish and vegetables can also contain C. difficile, but studies conducted in Europe report lower prevalence rates than in North America. Absolute counts of toxigenic C. difficile in the environment and food are low, however the exact infectious dose is unknown. To date, direct transmission of C. difficile from animals, food or the environment to humans has not been proven, although similar PCR ribotypes are found. We therefore believe that the overall epidemiology of human CDI is not driven by amplification in animals or other sources. As no outbreaks of CDI have been reported among humans in the community, host factors that increase vulnerability to CDI might be of more importance than increased exposure to C. difficile. Conversely, emerging C. difficile ribotype 078 is found in high numbers in piglets, calves, and their immediate environment. Although there is no direct evidence proving transmission to humans, circumstantial evidence points towards a zoonotic potential of this type. In future emerging PCR ribotypes, zoonotic potential needs to be considered.

Spatially complex distribution of dissolved manganese in a fjord as revealed by high-resolution in situ sensing using the autonomous underwater vehicle Autosub.

Loch Etive is a fjordic system on the west coast of Scotland. The deep waters of the upper basin are periodically isolated, and during these periods oxygen is lost through benthic respiration and concentrations of dissolved manganese increase. In April 2000 the autonomous underwater vehicle (AUV) Autosub was fitted with an in situ dissolved manganese analyzer and was used to study the spatial variability of this element together with oxygen, salinity, and temperature throughout the basin. Six along-loch transects were completed at either constant height above the seafloor or at constant depth below the surface. The ca. 4000 in situ 10-s-average dissolved Mn (Mnd) data points obtained provide a new quasi-synoptic and highly detailed view of the distribution of manganese in this fjordic environment not possible using conventional (water bottle) sampling. There is substantial variability in concentrations (<25 to >600 nM) and distributions of Mnd. Surface waters are characteristically low in Mnd reflecting mixing of riverine and marine end-member waters, both of which are low in Mnd. The deeper waters are enriched in Mnd, and as the water column always contains some oxygen, this must reflect primarily benthic inputs of reduced dissolved Mn. However, this enrichment of Mnd is spatially very variable, presumably as a result of variability in release of Mn coupled with mixing of water in the loch and removal processes. This work demonstrates how AUVs coupled with chemical sensors can reveal substantial small-scale variability of distributions of chemical species in coastal environments that would not be resolved by conventional sampling approaches. Such information is essential if we are to improve our understanding of the nature and significance of the underlying processes leading to this variability.

A cryptic deletion of 2q35 including part of the PAX3 gene detected by breakpoint mapping in a child with autism and a de novo 2;8 translocation.

We report a de novo, apparently balanced (2;8)(q35;q21.2) translocation in a boy with developmental delay and autism. Cross species (colour) paint (Rx) and SKY FISH, forward and reverse chromosome painting, and FISH with subtelomeric probes were used to examine the patient's karyotype, but further rearrangements were not detected. FISH with region specific clones mapping near 2q35 and 8q21.2 breakpoints and STS mapping performed on the isolated derivative chromosomes were used to refine the location of the breakpoints further. A cryptic deletion of between 4.23 and 4.41 Mb in extent and involving at least 13 complete genes or transcription units was found at the breakpoint on 2q35. The deletion includes the promoter and 5' untranslated region of the paired box 3 (PAX3) gene. The child has very mild dystopia canthorum which may be associated with the PAX3 haploinsufficiency. The 8q21.2 breakpoint is within MMP16 which encodes matrix metalloproteinase 16. We postulate that the cryptic deletion and rearrangement are responsible for the patient's phenotype and that a gene (or genes) responsible for autism lies at 2q35 or 8q21.2. The results present a step towards identifying genes predisposing to autism.

Use of flow-sorted canine chromosomes in the assignment of canine linkage, radiation hybrid, and syntenic groups to chromosomes: refinement and verification of the comparative chromosome map for dog and human.

The mapping of the canine genome has recently been accelerated by the availability of chromosome-specific reagents and publication of radiation hybrid (RH), genetic linkage, and dog/human comparative maps, but the assignment of mapping groups to chromosomes is incomplete. To assign published radiation hybrid, linkage, and "syntenic" groups to chromosomes, individual markers found within each group have been amplified from canine and vulpine flow-sorted, chromosome-specific DNAs as templates. Here a further 102 type I genetic markers (previously mapped in human) and 21 further type II markers are assigned to canine chromosomes using marker-specific PCR. We have assigned all linkage, RH, and syntenic groups in the two most recently published canine genome maps to chromosomes. This demonstrates directly that there is at least one published mapping group for each of the 38 canine autosomes and thus that the coverage of the canine chromosome map is approaching completion. The dog/human comparative map is one of the most complex so far described, with 90 separate segments of chromosomal homology previously seen in dog-on-human cross-species chromosome-painting studies. The total of 142 type I markers now placed on canine chromosomes using this method of marker mapping has allowed us to confirm the placement of the great majority (83) of the 90 homologous segments. The positions of the remaining homologous segments were confirmed in new cross-species chromosome-painting experiments (dog-on-human, fox-on-human).

Reciprocal chromosome painting illuminates the history of genome evolution of the domestic cat, dog and human.

Domestic cats and dogs are important companion animals and model animals in biomedical research. The cat has a highly conserved karyotype, closely resembling the ancestral karyotype of mammals, while the dog has one of the most extensively rearranged mammalian karyotypes investigated so far. We have constructed the first detailed comparative chromosome map of the domestic dog and cat by reciprocal chromosome painting. Dog paints specific for the 38 autosomes and the X chromosomes delineated 68 conserved chromosomal segments in the cat, while reverse painting of cat probes onto red fox and dog chromosomes revealed 65 conserved segments. Most conserved segments on cat chromosomes also show a high degree of conservation in G-banding patterns compared with their canine counterparts. At least 47 chromosomal fissions (breaks), 25 fusions and one inversion are needed to convert the cat karyotype to that of the dog, confirming that extensive chromosome rearrangements differentiate the karyotypes of the cat and dog. Comparative analysis of the distribution patterns of conserved segments defined by dog paints on cat and human chromosomes has refined the human/cat comparative genome map and, most importantly, has revealed 15 cryptic inversions in seven large chromosomal regions of conserved synteny between humans and cats.

Cemented rotating-platform total knee replacement. A nine to twelve-year follow-up study.

BACKGROUND: Although the LCS (low contact stress) rotating-platform mobile-bearing knee replacement has been used extensively, there have been few intermediate or long-term clinical and radiographic follow-up studies evaluating the device. The purpose of this study was to report the nine to twelve-year results of a consecutive series of patients who had a primary total knee replacement performed with this device. METHODS: Between November 1985 and November 1988, the senior author (R. C. J.) performed 119 consecutive total knee arthroplasties in eighty-six patients with LCS rotating-platform femoral and tibial components and a Townley all-polyethylene dome patellar component. All components were fixed with cement. The average age of the patients at the time of the operation was seventy years (range, thirty-seven to eighty-eight years). Fifty-two patients (seventy-six knees) were female, and thirty-four patients (forty-three knees) were male. The patients were evaluated with clinical knee ratings and radiographic analysis nine to twelve years following the knee replacement. RESULTS: At the time of the nine to twelve-year follow-up, sixty-four patients (eighty-six knees) were alive, eighteen patients (twenty-eight knees) had died, and four patients (five knees) had been lost to follow-up. Of the 114 knees in the eighty-two patients for whom the final outcome was known, none required a reoperation and none had a dislocation of the mobile-bearing prosthesis. For the forty-five patients (sixty-six knees) who returned for final clinical and radiographic follow-up examinations at nine to twelve years, the average clinical and functional Knee Society ratings were 30 points (range, 2 to 70 points) and 44 points (range, 0 to 80 points) preoperatively and 90 points (range, 63 to 102 points) and 75 points (range, 30 to 100 points) at the final follow-up evaluation. The average Hospital for Special Surgery knee rating was 57 points (range, 28 to 80 points) preoperatively and 84 points (range, 59 to 97 points) at the final follow-up evaluation. The average active range of knee flexion was from 0 degrees (range, 0 to 10 degrees) to 102 degrees (range, 15 to 120 degrees) at the final follow-up evaluation. Seven of the sixty-six knees were painful anteriorly. There was no periprosthetic osteolysis and no evidence of loosening on follow-up radiographs. CONCLUSIONS: After nine to twelve years of follow-up, the cemented LCS rotating-platform knee replacement was found to be performing well, with durable clinical and radiographic results.

Unicompartmental knee replacement. A minimum 15 year followup study.

One hundred forty Marmor cemented unicompartmental knee replacements were inserted in 103 patients between 1975 and 1982. Fifty-two patients were women and 51 were men. One hundred twenty-five were medial compartment knee replacements and 15 were lateral knee replacements. At minimum 15 year followup 34 patients with 48 knee replacements were living; only four patients with four knee replacements were lost to followup. Average preoperative and final followup Hospital for Special Surgery knee scores were 57 and 82 points, respectively for the knees of living patients. Average preoperative and final followup Knee Society clinical and Knee Society functional scores were 31 and 42, and 85 and 71 points, respectively. For all knees, 10.2% (14 knees) were revised [4.4% (six knees) for tibial loosening, 5.1% (seven knees) for disease progression, and .7% (one knee) for pain]. For patients living 15 years, 12.5% (six knees) were revised [2.1% (one knee) for tibial loosening, and 10.4% (five knees) for disease progression]. Revision for failure of fixation of these unicompartmental replacements was comparable with that reported for fixed bearing total knee replacement. Disease progression (46%; 62 of 136 knees) and tibial subsidence with wear (10.4%; 15 of 136 knees, five of which required revision) were the major long term problems in this group of patients.

The integration of canine genetic maps with the canine karyotype using specific gene amplification of chromosome-specific DNA.

We have used a rapid approach to place markers that are already represented in current genetic maps onto individual chromosomes in species for which chromosome paints exist. PCR-based techniques are used to look for the presence of individual marker genes within each chromosome-specific DNA pool. The presence of a given marker within a DNA pool allows assignment of the complete radiation hybrid group, or linkage group from which the marker is drawn, to an individual chromosome. We have used this method with a new set of canine chromosome paints (Yang et al., 1999). In this way, we have assigned 39 of 44 published RH or syntenic RH groups to canine chromosomes, together with 33 of 40 canine linkage groups in a recently published map (Neff et al., 1999).

Effects of the alpha subunit on imidacloprid sensitivity of recombinant nicotinic acetylcholine receptors.

1. Imidacloprid is a new insecticide with selective toxicity for insects over vertebrates. Recombinant (alpha4beta2) chicken neuronal nicotinic acetylcholine receptors (AChRs) and a hybrid nicotinic AChR formed by co-expression of a Drosophila melanogaster neuronal alpha subunit (SAD) with the chicken beta2 subunit were heterologously expressed in Xenopus oocytes by nuclear injection of cDNAs. The agonist actions of imidacloprid and other nicotinic AChR ligands ((+)-epibatidine, (-)-nicotine and acetylcholine) were compared on both recombinant nicotinic AChRs by use of two-electrode, voltage-clamp electrophysiology. 2. Imidacloprid alone of the 4 agonists behaved as a partial agonist on the alpha4beta2 receptor; (+)-epibatidine, (-)-nicotine and acetylcholine were all full, or near full, agonists. Imidacloprid was also a partial agonist of the hybrid Drosophila SAD chicken beta2 receptor, as was (-)-nicotine, whereas (+)-epibatidine and acetylcholine were full agonists. 3. The EC50 of imidacloprid was decreased by replacing the chicken alpha4 subunit with the Drosophila SAD alpha subunit. This alpha subunit substitution also resulted in an increase in the EC50 for (+)-epibatidine, (-)-nicotine and acetylcholine. Thus, the Drosophila (SAD) alpha subunit contributes to the greater apparent affinity of imidacloprid for recombinant insect/vertebrate nicotinic AChRs. 4. Imidacloprid acted as a weak antagonist of ACh-mediated responses mediated by SADbeta2 hybrid receptors and as a weak potentiator of ACh responses mediated by alpha4beta2 receptors. This suggests that imidacloprid has complex effects upon these recombinant receptors, determined at least in part by the alpha subunit.

Caenorhabditis elegans levamisole resistance genes lev-1, unc-29, and unc-38 encode functional nicotinic acetylcholine receptor subunits.

We show that three of the eleven genes of the nematode Caenorhabditis elegans that mediate resistance to the nematocide levamisole and to other cholinergic agonists encode nicotinic acetylcholine receptor (nAChR) subunits. unc-38 encodes an alpha subunit while lev-1 and unc-29 encode non-alpha subunits. The nematode nAChR subunits show conservation of many mammalian nAChR sequence features, implying an ancient evolutionary origin of nAChR proteins. Expression in Xenopus oocytes of combinations of these subunits that include the unc-38 alpha subunit results in levamisole-induced currents that are suppressed by the nAChR antagonists mecamylamine, neosurugatoxin, and d-tubocurarine but not alpha-bungarotoxin. The mutant phenotypes reveal that unc-38 and unc-29 subunits are necessary for nAChR function, whereas the lev-1 subunit is not. An UNC-29-GFP fusion shows that UNC-29 is expressed in body and head muscles. Two dominant mutations of lev-1 result in a single amino acid substitution or addition in or near transmembrane domain 2, a region important to ion channel conductance and desensitization. The identification of viable nAChR mutants in C. elegans provides an advantageous system in which receptor expression and synaptic targeting can be manipulated and studied in vivo.

ACR-3, a Caenorhabditis elegans nicotinic acetylcholine receptor subunit. Molecular cloning and functional expression.

The molecular cloning and functional co-expression of a novel nicotinic acetylcholine receptor (nAChR) non-alpha subunit gene, acr-3, is described. Previously we determined the sequence and demonstrated the functional co-expression of acr-2, a nAChR non-alpha subunit gene from Caenorhabditis elegans. Analysis of the acr-2 genomic DNA revealed the existence of another potential nAChR subunit gene, acr-3, in the same orientation, only 281 bp downstream of acr-2. A cDNA containing the entire acr-3 coding sequence was isolated by RT-PCR and sequenced. The predicted protein contains the conserved features typical of nAChR non-alpha subunits and most closely resembles other invertebrate nAChR non-alpha polypeptides. Unusually, the highly conserved glycine residue (equivalent to residue 240 in the Torpedo alpha subunit) upstream of transmembrane domain 2 (m2) is replaced by a serine residue in ACR-3. When acr-3 cDNA was injected alone into Xenopus oocytes no levamisole-gated channel activity was observed. However when co-expressed with a C. elegans alpha subunit (UNC-38), ACR-3 contributed to the formation of levamisole-gated channels. The response of this hetero-oligomer to levamisole (100 microM) was reduced by the nAChR antagonists mecamylamine (1 microM) and d-tubocurarine (10 microM).

pH-dependent actions of THIP and ZAPA on an ionotropic Drosophila melanogaster GABA receptor.

The actions of THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) and ZAPA (Z-3-[(aminoiminomethyl)thio]prop-2-enoic acid) were tested on an ionotropic homo-oligomeric GABA receptor of Drosophila melanogaster. The amplitude of currents activated by THIP and ZAPA declined rapidly during agonist application and a rebound response was observed on washout. By correcting the pH shift induced by these acid salts, responses more typical of GABA agonists were seen. Less striking pH-dependence was observed in the case of GABA responses.

Analysis of osteoblast mineral deposits on orthopaedic/dental implant metals.

Neonatal rat calvarial osteoblasts were cultured on Ti-6Al-4V, Co-Cr-Mo alloy, 316L stainless steel and polystyrene (reference substrate) in the presence of ascorbic acid and 10 mM beta-glycerophosphate for 16, 17, 18, 19, 20, 21, 24 and 28 d. Scanning electron microscopy examination revealed that osteoblasts cultured on these orthopaedic/dental implant metals synthesized and deposited an extracellular matrix containing collagenous and non-collagenous components, as well as mineral nodules of various morphologies. Energy dispersive spectrometry revealed that the mineral deposits consisted of three distinct chemical compositions: calcium phosphate, calcium-sulphur-phosphorus, and calcium only. Backscattered electron imaging demonstrated that both the calcium phosphate and calcium-only deposits were electron dense, while the calcium-sulphur-phosphorus deposits were electron translucent. X-ray diffraction analysis indicated that the bulk of the osteoblast mineral deposits was amorphous hydroxyapatite; in addition, electron diffraction analysis revealed small regions of crystalline hydroxyapatite.

Wild-type and insecticide-resistant homo-oligomeric GABA receptors of Drosophila melanogaster stably expressed in a Drosophila cell line.

RDL is an ionotropic GABA receptor subunit, a product of the Rdl gene, originally identified in the Maryland strain of Drosophila melanogaster. Here, we report the generation of a Drosophila melanogaster cell line (S2-RDLA302S) stably expressing a mutated, dieldrin-resistant (A302S) form of RDL. The properties of this dieldrin-resistant, homo-oligomeric receptor have been compared with those of the stably expressed, wild-type form (S2-RDL). Using these stable lines, a striking reduction in sensitivity to both picrotoxinin and dieldrin was observed for responses to GABA of S2-RDLA302S compared to S2-RDL. To determine if these stable insect cell lines generate results similar to those obtained by transient expression in Xenopus laevis oocytes, we have examined the actions of two widely used convulsants, EBOB and TBPS, and a recently developed convulsant BIDN, on RDL-mediated GABA responses in the two expression systems. In both oocytes and S2 cells, the three convulsants suppressed the amplitude of responses to GABA. Thus, in accord with earlier work on agonist and allosteric sites, the S2-RDL cell line is found to yield similar pharmacological results to those obtained in transient expression studies. Stable cell lines are now available expressing susceptible and resistant forms of an ionotropic receptor by GABAergic insecticides.