Energy-dispersive X-ray diffraction using an annular beam.

We demonstrate material phase identification by measuring polychromatic diffraction spots from samples at least 20 mm in diameter and up to 10 mm thick with an energy resolving point detector. Within our method an annular X-ray beam in the form of a conical shell is incident with its symmetry axis normal to an extended polycrystalline sample. The detector is configured to receive diffracted flux transmitted through the sample and is positioned on the symmetry axis of the annular beam. We present the experiment data from a range of different materials and demonstrate the acquisition of useful data with sub-second collection times of 0.5 s; equating to 0.15 mAs. Our technique should be highly relevant in fields that demand rapid analytical methods such as medicine, security screening and non-destructive testing.

Visualization of 3D osteon morphology by synchrotron radiation micro-CT.

Cortical bone histology has been the subject of scientific inquiry since the advent of the earliest microscopes. Histology - literally the study of tissue - is a field nearly synonymous with 2D thin sections. That said, progressive developments in high-resolution X-ray imaging are enabling 3D visualization to reach ever smaller structures. Micro-computed tomography (micro-CT), employing conventional X-ray sources, has become the gold standard for 3D analysis of trabecular bone and is capable of detecting the structure of vascular (osteonal) porosity in cortical bone. To date, however, direct 3D visualization of secondary osteons has eluded micro-CT based upon absorption-derived contrast. Synchrotron radiation micro-CT, through greater image quality, resolution and alternative contrast mechanisms (e.g. phase contrast), holds great potential for non-destructive 3D visualization of secondary osteons. Our objective was to demonstrate this potential and to discuss areas of bone research that can be advanced through the application of this approach. We imaged human mid-femoral cortical bone specimens derived from a 20-year-old male (Melbourne Femur Collection) at the Advanced Photon Source synchrotron (Chicago, IL, USA) using the 2BM beam line. A 60-mm distance between the target and the detector was employed to enhance visualization of internal structures through propagation phase contrast. Scan times were 1 h and images were acquired with 1.4-µm nominal isotropic resolution. Computer-aided manual segmentation and volumetric 3D rendering were employed to visualize secondary osteons and porous structures, respectively. Osteonal borders were evident via two contrast mechanisms. First, relatively new (hypomineralized) osteons were evident due to differences in X-ray attenuation relative to the surrounding bone. Second, osteon boundaries (cement lines) were delineated by phase contrast. Phase contrast also enabled the detection of soft tissue remnants within the vascular pores. The ability to discern osteon boundaries in conjunction with vascular and cellular porosity revealed a number of secondary osteon morphologies and provided a unique 3D perspective of the superimposition of secondary osteons on existing structures. Improvements in resolution and optimization of the propagation phase contrast promise to provide further improvements in structural detail in the future.

Relating age and micro-architecture with apparent-level elastic constants: a micro-finite element study of female cortical bone from the anterior femoral midshaft.

Homogenized elastic properties are often assumed for macro-finite element (FE) models used in orthopaedic biomechanics. The accuracy of material property assignments may have a strong effect on the ability of these models to make accurate predictions. For cortical bone, most macro-scale FE models assume isotropic elastic material behaviour and do not include variation of material properties due to bone micro-architecture. The first aim of the present study was to evaluate the variation of apparent-level (homogenized) orthotropic elastic constants of cortical bone with age and indices of bone micro-architecture. Considerable age-dependent differences in porosity were noted across the cortical thickness in previous research. The second aim of the study was to quantify the resulting differences in elastic constants between the periosteum and endosteum. Specimens were taken from the anterior femoral midshaft of 27 female donors (age 53.4 +/- 23.6 years) and micro-FE (gFE) analysis was used to derive orthotropic elastic constants. The variation of orthotropic elastic constants (Young's moduli, shear moduli, and Poisson's ratios) with various cortical bone micro-architectural indices was investigated. The ratio of canal volume to tissue volume, Ca.V/TV, analogous to porosity, was found to be the strongest predictor (r2(ave) = 0.958) of the elastic constants. Age was less predictive (r2(ave) = 0.385) than Ca.V/TV. Elastic anisotropy increased with increasing Ca.V/TV, leading to lower elastic moduli in the transverse, typically less frequently loaded, directions. Increased Ca.V/TV led to a more substantial reduction in elastic constants at the endosteal aspect than at the periosteal aspect. The results are expected to be most applicable in similar midshaft locations of long bones; specific analysis of other sites would be necessary to evaluate elastic properties elsewhere. It was concluded that Ca.V/TV was the most predictive of cortical bone elastic constants and that considerable periosteal-endosteal variations in these constants can develop with bone loss.

Evaluation of two dental identification computer systems: DAVID and WinID3.

Human identification, by comparing dental characteristics, is considered to be one of the most reliable, accurate and rapid methods of resolving the identity of visually un-identifiable deceased persons. In recent decades computer programs have evolved to aid odontologists by suggesting records that have similar dental features. The aim of the present study was to compare two of those programs; Disaster And Victim IDentification (DAVID) and WinID3 in terms of effectiveness, accuracy and speed of data entry and to further compare them with the efficiency of the classical method of manually matching postmortem and antemortem dental records. An open disaster was simulated whereby 52 fragmented remains made of acrylic replicas and 77 provisional victims were represented on Interpol F2 postmortem and antemortem forms. The results assessed were the first seven possible matches made by each program. Manual matching of dental characteristics performed better than both programs (P<0.001) yielding 29 identifications. Eleven and six positive matches were the result of the DAVID and the WinID3 programs respectively (P=0.185). Data entry was quicker for WinID3. It was concluded that both programs are still not as accurate as the time-consuming manual matching method. The difference in performance between the DAVID and the WinID3 programs was attributed to the inclusion of more comparable dental characteristics, the inclusion of the type of dentition (deciduous or permanent) and the weighting of those characteristics by the DAVID program.

Lacunar-canalicular network in femoral cortical bone is reduced in aged women and is predominantly due to a loss of canalicular porosity.

The lacunar-canalicular network (LCN) of bone contains osteocytes and their dendritic extensions, which allow for intercellular communication, and are believed to serve as the mechanosensors that coordinate the processes of bone modeling and remodeling. Imbalances in remodeling, for example, are linked to bone disease, including fragility associated with aging. We have reported that there is a reduction in scale for one component of the LCN, osteocyte lacunar volume, across the human lifespan in females. In the present study, we explore the hypothesis that canalicular porosity also declines with age. To visualize the LCN and to determine how its components are altered with aging, we examined samples from young (age: 20-23 y; n = 5) and aged (age: 70-86 y; n = 6) healthy women donors utilizing a fluorescent labelling technique in combination with confocal laser scanning microscopy. A large cross-sectional area of cortical bone spanning the endosteal to periosteal surfaces from the anterior proximal femoral shaft was examined in order to account for potential trans-cortical variation in the LCN. Overall, we found that LCN areal fraction was reduced by 40.6% in the samples from aged women. This reduction was due, in part, to a reduction in lacunar density (21.4% decline in lacunae number per given area of bone), but much more so due to a 44.6% decline in canalicular areal fraction. While the areal fraction of larger vascular canals was higher in endosteal vs. periosteal regions for both age groups, no regional differences were observed in the areal fractions of the LCN and its components for either age group. Our data indicate that the LCN is diminished in aged women, and is largely due to a decline in the canalicular areal fraction, and that, unlike vascular canal porosity, this diminished LCN is uniform across the cortex.

New software for computer-assisted dental-data matching in Disaster Victim Identification and long-term missing persons investigations: "DAVID Web".

In 1997 an internally supported but unfunded pilot project at the Victorian Institute of Forensic Medicine (VIFM) Australia led to the development of a computer system which closely mimicked Interpol paperwork for the storage, later retrieval and tentative matching of the many AM and PM dental records that are often needed for rapid Disaster Victim Identification. The program was called "DAVID" (Disaster And Victim IDentification). It combined the skills of the VIFM Information Technology systems manager (VW), an experienced odontologist (JGC) and an expert database designer (JC); all current authors on this paper. Students did much of the writing of software to prescription from Monash University. The student group involved won an Australian Information Industry Award in recognition of the contribution the new software could have made to the DVI process. Unfortunately, the potential of the software was never realized because paradoxically the federal nature of Australia frequently thwarts uniformity of systems across the entire country. As a consequence, the final development of DAVID never took place. Given the recent problems encountered post-tsunami by the odontologists who were obliged to use the Plass Data system (Plass Data Software, Holbaek, Denmark) and with the impending risks imposed upon Victoria by the decision to host the Commonwealth Games in Melbourne during March 2006, funding was sought and obtained from the state government to update counter disaster preparedness at the VIFM. Some of these funds have been made available to upgrade and complete the DAVID project. In the wake of discussions between leading expert odontologists from around the world held in Geneva during July 2003 at the invitation of the International Committee of the Red Cross significant alterations to the initial design parameters of DAVID were proposed. This was part of broader discussions directed towards developing instruments which could be used by the ICRC's "The Missing" project that seeks to identify the victims of civil unrest and other atrocities. The most significant of these recommendations was that the next version of DAVID should be web-based allowing it to be used anywhere in the world and on any computer platform. The original intention that the software should be made available as freeware was strongly reiterated and endorsed. During 2005 these recommendations have been realized. This paper will describe the design parameters of the new software "DAVID web" and compare its features and performance with alternative packages.

Construction and use of facial archetypes in anthropology and syndrome diagnosis.

This paper describes the benefits of moving from recording simple Euclidian distances and angles between landmarks on the face to full three-dimensional visualisation and mapping using modern optical scanning techniques. Pilot experiments are reported on that strive to create facial archetypes which are accurately descriptive of various cohorts of people. Issues considered include variation amongst people of the same sex, age and population-of-origin. The study has discovered that very few people are needed to construct an "average" face, which is measurably indistinguishable from another average constructed using the faces of other people from within the group studied. This discovery has given the researchers confidence in the reliability of the archetypes which they have produced and this is important if such an analytical technique is to find application in discriminating between peoples on a population basis and in syndrome diagnosis.

A mechanostatistical approach to cortical bone remodelling: an equine model.

In this study, the development of a mechanostatistical model of three-dimensional cortical bone remodelling informed with in vivo equine data is presented. The equine model was chosen as it is highly translational to the human condition due to similar Haversian systems, availability of in vivo bone strain and biomarker data, and furthermore, equine models are recommended by the US Federal Drugs Administration for comparative joint research. The model was derived from micro-computed tomography imaged specimens taken from the equine third metacarpal bone, and the Frost-based 'mechanostat' was informed from both in vivo strain gauges and biomarkers to estimate bone growth rates. The model also described the well-known 'cutting cone' phenomena where Haversian canals tunnel and replace bone. In order to make this model useful in practice, a partial least squares regression (PLSR) surrogate model was derived based on training data from finite element simulations with different loads. The PLSR model was able to predict microstructure and homogenised Young's modulus with errors less than 2.2% and 0.6%, respectively.

Classification of fracture and non-fracture groups by analysis of coherent X-ray scatter.

Osteoporotic fractures present a significant social and economic burden, which is set to rise commensurately with the aging population. Greater understanding of the physicochemical differences between osteoporotic and normal conditions will facilitate the development of diagnostic technologies with increased performance and treatments with increased efficacy. Using coherent X-ray scattering we have evaluated a population of 108 ex vivo human bone samples comprised of non-fracture and fracture groups. Principal component fed linear discriminant analysis was used to develop a classification model to discern each condition resulting in a sensitivity and specificity of 93% and 91%, respectively. Evaluating the coherent X-ray scatter differences from each condition supports the hypothesis that a causal physicochemical change has occurred in the fracture group. This work is a critical step along the path towards developing an in vivo diagnostic tool for fracture risk prediction.

The relationship between porosity and specific surface in human cortical bone is subject specific.

A characteristic relationship for bone between bone volume fraction (BV/TV) and specific surface (BS/TV) has previously been proposed based on 2D histological measurements. This relationship has been suggested to be bone intrinsic, i.e., to not depend on bone type, bone site and health state. In these studies, only limited data comes from cortical bone. The aim of this paper was to investigate the relationship between BV/TV and BS/TV in human cortical bone using high-resolution micro-CT imaging and the correlations with subject-specific biometric data such as height, weight, age and sex. Images from femoral cortical bone samples of the Melbourne Femur Collection were obtained using synchrotron radiation micro-CT (SPring8, Japan). Sixteen bone samples from thirteen individuals were analysed in order to find bone volume fraction values ranging from 0.20 to 1. Finally, morphological models of the tissue microstructure were developed to help explain the relationship between BV/TV and BS/TV. Our experimental findings indicate that the BV/TV vs BS/TV relationship is subject specific rather than intrinsic. Sex and pore density were statistically correlated with the individual curves. However no correlation was found with body height, weight or age. Experimental cortical data points deviate from interpolating curves previously proposed in the literature. However, these curves are largely based on data points from trabecular bone samples. This finding challenges the universality of the curve: highly porous cortical bone is significantly different to trabecular bone of the same porosity. Finally, our morphological models suggest that changes in BV/TV within the same sample can be explained by an increase in pore area rather than in pore density. This is consistent with the proposed mechanisms of age-related endocortical bone loss. In addition, these morphological models highlight that the relationship between BV/TV and BS/TV is not linear at high BV/TV as suggested in the literature but is closer to a square root function.

X-ray diffraction from bone employing annular and semi-annular beams.

There is a compelling need for accurate, low cost diagnostics to identify osteo-tissues that are associated with a high risk of fracture within an individual. To satisfy this requirement the quantification of bone characteristics such as 'bone quality' need to exceed that provided currently by densitometry. Bone mineral chemistry and microstructure can be determined from coherent x-ray scatter signatures of bone specimens. Therefore, if these signatures can be measured, in vivo, to an appropriate accuracy it should be possible by extending terms within a fracture risk model to improve fracture risk prediction.In this preliminary study we present an examination of a new x-ray diffraction technique that employs hollow annular and semi-annular beams to measure aspects of 'bone quality'. We present diffractograms obtained with our approach from ex vivo bone specimens at Mo Kα and W Kα energies. Primary data is parameterized to provide estimates of bone characteristics and to indicate the precision with which these can be determined.

The micro-architecture of human cancellous bone from fracture neck of femur patients in relation to the structural integrity and fracture toughness of the tissue.

Osteoporosis is clinically assessed from bone mineral density measurements using dual energy X-ray absorption (DXA). However, these measurements do not always provide an accurate fracture prediction, arguably because DXA does not grapple with 'bone quality', which is a combined result of microarchitecture, texture, bone tissue properties, past loading history, material chemistry and bone physiology in reaction to disease. Studies addressing bone quality are comparatively few if one considers the potential importance of this factor. They suffer due to low number of human osteoporotic specimens, use of animal proxies and/or the lack of differentiation between confounding parameters such as gender and state of diseased bone. The present study considers bone samples donated from patients (n = 37) who suffered a femoral neck fracture and in this very well defined cohort we have produced in previous work fracture toughness measurements (FT) which quantify its ability to resist crack growth which reflects directly the structural integrity of the cancellous bone tissue. We investigated correlations between BV/TV and other microarchitectural parameters; we examined effects that may suggest differences in bone remodelling between males and females and compared the relationships with the FT properties. The data crucially has shown that TbTh, TbSp, SMI and TbN may provide a proxy or surrogate for BV/TV. Correlations between FT critical stress intensity values and microarchitecture parameters (BV/TV, BS/TV, TbN, BS/BV and SMI) for osteoporotic cancellous tissue were observed and are for the first time reported in this study. Overall, this study has not only highlighted that the fracture model based upon BMD could potentially be improved with inclusion of other microarchitecture parameters, but has also given us clear clues as to which of them are more influential in this role.

Age and temperature related changes to the ultrastructure and composition of human bone mineral.

This X-ray diffraction (XRD) investigation of heat-treated human femoral bone showed that the main mineral phase of both unheated bone and bone heated to 600 degrees C resembled that of a poorly crystalline form of hydroxyapatite. The rod-shaped apatite crystals in unheated bone persisted in bone heated up to 400 degrees C. Recrystallization at approximately 600 degrees C, produced larger crystals, which either retained their original morphology or changed to tabular or equidimensional shapes. The size of the apatite crystals in unheated and heated bone specimens was dependent on both temperature and age. When heated above 600 degrees C the crystallinity of the bone mineral increased, and the XRD pattern more closely resembled that of hydroxyapatite. Partial decomposition of the hydroxyapatite phase to calcium oxide above 1000 degrees C, and beta-tricalcium phosphate, alpha-tricalcium phosphate, and calcium oxide phosphate between 1200 degrees C and 1400 degrees C, indicated that the original apatite phase was both calcium deficient and contained carbonate. The relative peak intensities of the thermal decomposition products were related to some extent to the age of the deceased person and reflected the compositional changes that occur during bone aging. Because the thermally induced changes to the composition and ultrastructure of bone mineral were influenced by the age of the individual, this investigation proposed that the heat treatment of bone tissue may offer an alternative way of studying bone aging.

An automated analysis of intracortical porosity in human femoral bone across age.

The matrix of human cortical bone is arranged around a network of vascular spaces (hereafter referred to as "pores"). Our aim was to investigate age-related differences in human cortical porosity (total pore area divided by cortical bone area), pore size and number, and surface to volume ratios, while adjusting for sex, height, and weight. Ninety-six specimens of entire transverse sections of human femoral diaphysis, from subjects aged 21-92 years, were examined. We used our established automated image acquisition and analysis system which measures pores from entire sections of multiple specimens of bone. Over 400,000 pores were recorded. Results showed a greater porosity in older bone (p < 0.01) but marked variation in porosity for any given age. The cohort median, of the specimen medians, of pore cross-sectional area was 2050 microns 2. Older specimens did not have more pores than younger specimens but had a greater proportion of larger pores (p < 0.05) and greater intraspecimen variation in pore size (p < 0.001). The pore surface to bone matrix volume ratio was a median 2.3 mm2/mm3. This varied more than 4-fold between individuals but did not relate to age. No simple relationships were found between any of the measured parameters and either sex, height, or weight, even after adjustment for age. We conclude that the greater porosity in older specimens is due to greater pore size rather than a larger number of pores. Age, however, explains little of the inter-individual variation in the parameters studied.

Effect of pretreatment with sodium phenobarbital on the toxicity of soman in mice.

Pretreatment with sodium phenobarbital induces hepatic microsomal enzymes which are responsible for the metabolic breakdown of a large number of endogenous and exogenous chemical compounds. A previous study [K. P. DuBois and F. K. Kinoshita, Proc. Soc. exp. Biol. Med. 129, 699 (1968)] reported that phenobarbital pretreatment reduced the toxicity of various organophosphorus anticholinesterases; however, the exact mechanism for the increased detoxification was not investigated. In this study, the effect of phenobarbital pretreatment on the toxicity of soman was investigated. Male mice were injected daily for 4 days with sodium phenobarbital (100 mg/kg, i.p.) and used in the various experiments 24 hr after the last injection. Phenobarbital pretreatment produced a significant increase in liver weight and decreased the sodium pentobarbital (75 mg/kg, i.p.) induced sleep-time to 41 min compared to 141 min in controls. The lethality of soman was reduced following phenobarbital pretreatment. In control mice, the soman 24 hr LD50 values (microgram/kg) were 130, 393 and 42 following s.c., i.p. and i.v. administration, respectively, whereas in phenobarbital-pretreated mice the soman 24 hr LD50 values (microgram/kg) were 261, 746 and 63 following s.c., i.p. and i.v. administration respectively. Acetylcholinesterase activity was increased in the plasma (90%) but not in brain or diaphragm following phenobarbital pretreatment. Liver somanase activity was not affected. Liver aliesterase and serum aliesterase were both increased significantly following phenobarbital pretreatment. An increase in the amount of nonspecific binding sites for soman (esterases in liver and plasma) and not an increase in the metabolism of soman in vivo probably accounts for the protection afforded by phenobarbital pretreatment in mice.

HI-6: reactivation of central and peripheral acetylcholinesterase following inhibition by soman, sarin and tabun in vivo in the rat.

HI-6, ([[[(4-aminocarbonyl)pyridino]methoxy]methyl]-2-] (hydroxyimino)methyl]-pyridinium dichloride), is an oxime which, when combined with atropine, is an extremely effective therapy against organophosphate poisoning. It was found that, following soman (287 micrograms/kg) poisoning, HI-6 reactivated acetylcholinesterase in the diaphragm and intercostal muscles but not in the brain. At a lower dose of soman (110 micrograms/kg), HI-6 reactivated sarin-inhibited acetylcholinesterase in the brain and in the respiratory musculature but did not reactivate tabun-inhibited acetylcholinesterase. It was also found that soman produced a differential inhibition of diaphragm and intercostal muscle acetylcholinesterase in vivo, whereas the in vitro I50 for soman was the same in both areas. HI-6 was capable of reactivating soman-inhibited acetylcholinesterase when administered up to 30 min post-soman, indicating that the rate of aging of the soman-acetylcholinesterase complex is slower than previously reported. The above results suggest that, in severe soman poisoning, the primary lesion occurs in peripheral acetylcholinesterase in the respiratory musculature (specifically the diaphragm).

Importance of aliesterase as a detoxification mechanism for soman (Pinacolyl methylphosphonofluoridate) in mice.

CBDP (2-/O-cresyl/4H:1:2-benzodioxaphosphorin-2-oxide) pretreatment produced a dramatic increase in the toxicity of soman in mice following the subcutaneous (s.c.) or intraperitoneal (i.p.) route of administration. This increase in soman toxicity was very highly correlated with inhibition of plasma aliesterase activity. Other enzymes (e.g. liver aliesterase and plasma cholinesterase) were inhibited by CBDP pretreatment; however, they did not appear to play a significant role in the potentiation of soman toxicity by CBDP. Liver aliesterase was not inhibited by doses of CBDP which produced significant increases in soman toxicity. Similarly, doses of Iso-OMPA, a selective inhibitor of pseudocholinesterase, which completely inhibited plasma cholinesterase, had no effect on soman toxicity. Pyridostigmine pretreatment which inhibited brain, diaphragm and plasma acetylcholinesterase 27, 57 and 60%, respectively, while not inhibiting plasma aliesterase, did not affect soman toxicity. The results of this study demonstrate that, in mice, plasma aliesterase is an extremely important detoxification route for soman.

Soman and sarin inhibition of molecular forms of acetylcholinesterase in mice. Time course of recovery and reactivation by the oxime HI-6.

The in vivo sensitivity of the molecular forms of the enzyme acetylcholinesterase to inhibition by either soman or sarin, reactivation by HI-6 and the time course of recovery following inhibition by soman were investigated in mice. Administration of HI-6 (50 mg/kg, i.p.) immediately after soman (100 micrograms/kg, s.c.) or sarin (150 micrograms/kg, s.c.) resulted in an apparent selective reactivation of the 10S and 16S molecular forms of acetylcholinesterase and no reactivation of the 4S form of diaphragm acetylcholinesterase. The apparent selectivity of the reactivation of the molecular forms of the acetylcholinesterase was probably due to the fact that the 10S and 16S forms of acetylcholinesterase are located primarily extracellularly and the 4S form intracellularly. The HI-6 was restricted primarily to the extracellular compartment due to its quaternary, hydrophilic nature. If the administration of HI-6 was delayed until 60 min following soman (100 micrograms/kg, s.c.) injection, no reactivation of any of the molecular forms of acetylcholinesterase could be found in the diaphragm. The soman-inhibited acetylcholinesterase had probably aged and, thus, was not susceptible to reactivation by HI-6. The time course of recovery of the molecular forms in the diaphragm occurred rather quickly with the smaller 4S and 10S forms recovering to control levels faster than the larger 16S form. It took between 8 and 16 days for the 16S form to recover to normal. In the brain, hypothalamic acetylcholinesterase molecular forms such as the 4S recovered faster than the 10S form which had not recovered to control 16 days after soman administration; the 16S form of acetylcholinesterase was not detected in the brain.

Low blood-brain barrier permeability to azidothymidine (AZT), 3TC, and thymidine in the rat.

The blood-brain barrier (BBB) permeability to [3H]-azidodeoxythymidine (AZT), deoxythiacytidine (3TC), and thymidine was studied using both an intravenous injection/external organ (IV/EO) method and an internal carotid artery perfusion (ICAP) technique in parallel with [14C]-sucrose as a plasma volume marker. The brain volumes of distribution (VD) of the three compounds approximated that of sucrose with either method. Although the lipid solubility of AZT, as determined by the 1-octanol/buffer partition coefficient (P), was 16-fold higher than that of thymidine, the BBB permeability-surface area (PS) products were almost identical, consistent with preferential efflux of AZT from brain to blood.

Pharmacological actions of HS-6, an oxime, on the neuromuscular junction.

The effect of HS-6 [1-(2-hydroxyiminomethylpyridinium)-1-(3-carboxamidopyridinium)-dimethyl ether] on neuromuscular (NM) transmission was investigated using chick biventer cervicis (CBC), rat diaphragm (RD) and guinea pig ileum longitudinal muscle strip (GPLS) preparations. In the CBC preparation HS-6 did not cause a contraction, whereas it produced a dose-related contraction of the GPLS preparation. HS-6 produced a hemicholinium-like NM blockade in the CBC and RD preparations. This was supported by the fact that HS-6 inhibited choline uptake in erythrocytes. HS-6 inhibited the contractions of various nicotinic agonists in the CBC preparation and augmented the acetycholine contractions. The latter was due to AChE inhibition produced by HS-6.