Nanostructural alteration in bone quantified in terms of orientation distribution of mineral crystals: a possible tool for fracture risk assessment

There may be different causes of failures in bone; however, their origin generally lies at the lowest level of structural hierarchy, i.e., at the mineral-collagen composite. Any change in the nanostructure affects the affinity or bonding effectiveness between and within the phases at this level, and hence determines the overall strength and quality of bone. In this study, we propose a novel concept to assess change in the nanostructure and thereby change in the bonding status at this level by revealing change in the orientation distribution characteristics of mineral crystals. Using X-ray diffraction method, a parameter called Degree of Orientation (DO) has been quantified. The DO accounts for the azimuthal distribution of mineral crystals and represents their effective amount along any direction. Changes in the DOs in cortical bone samples from bovine femur with different preferential orientations of mineral crystals were estimated under external loads. Depending on the applied loads, change in the azimuthal distribution of the DOs and the degree of reversibility of the crystals was observed to vary. The characteristics of nanostructural change and thereby possible affect on the strength of bone was then predicted from the reversible or irreversible characteristics of distributed mineral crystals. Significant changes in the organization of mineral crystals were observed; however, variations in the applied stresses and elastic moduli were not evinced at the macroscale level. A novel concept to assess the alteration in nanostructure on the basis of mineral crystals orientation distribution has been proposed. The importance of nanoscale level information obtained noninvasively has been emphasized, which acts as a precise tool to estimate the strength and predict the possible fracture risks in bone.     

Pathological crystal imaging with single‐shot computational polarized light microscopy

Pathological crystal identification is routinely practiced in rheumatology for diagnosing arthritis disease such as gout, and relies on polarized light microscopy as the gold standard method used by medical professionals. Here, we present a single‐shot computational polarized light microscopy method that reconstructs the transmittance, retardance and slow‐axis orientation of a birefringent sample using a single image captured with a pixelated‐polarizer camera. This method is fast, simple‐to‐operate and compatible with all the existing standard microscopes without extensive or costly modifications. We demonstrated the success of our method by imaging three different types of crystals found in synovial fluid and reconstructed the birefringence information of these samples using a single image, without being affected by the orientation of individual crystals within the sample field‐of‐view. We believe this technique will provide improved sensitivity, specificity and speed, all at low cost, for clinical diagnosis of crystals found in synovial fluid and other bodily fluids.     

Dynamic measurement of pennation angle of gastrocnemius muscles during contractions based on ultrasound imaging

ABSTRACT: BACKGROUND: Muscle fascicle pennation angle (PA) is an important parameter related to musculoskeletal functions, and ultrasound imaging has been widely used for measuring PA, but manually and frame by frame in most cases. We have earlier reported an automatic method to estimate aponeurosis orientation based on Gabor transform and Revoting Hough Transform (RVHT). METHODS: In this paper, we proposed a method to estimate the overall orientation of muscle fascicles in a region of interest, in order to complete computing the orientation of the other side of the pennation angle, but the side found by RVHT. The measurements for orientations of both fascicles and aponeurosis were conducted in each frame of ultrasound images, and then the dynamic change of pennation angle during muscle contraction was obtained automatically. The method for fascicle orientation estimation was evaluated using synthetic images with different noise levels and later on 500 ultrasound images of human gastrocnemius muscles during isometric plantarflexion. RESULTS: The muscle fascicle orientations were also estimated manually by two operators. From the results it's found that the proposed automatic method demonstrated a comparable performance to the manual method. CONCLUSIONS: With the proposed methods, ultrasound measurement for muscle pennation angles can be more widely used for functional assessment of muscles.     

Energy distributions at the high-spin ferric sites in myoglobin crystals.

The orientation and temperature dependence (4.2-2.5 K) of electron paramagnetic resonance (EPR) power saturation and spin-lattice relaxation rate, and the orientation dependence of signal linewidth, were measured in single crystals of the aquo complex of ferric sperm whale skeletal muscle myoglobin. The spin-packet linewidth was found to be temperature independent and to vary by a factor of seven within the heme plane. An analysis is presented which enables one to arrive at (a) hyperfine component line-widths and, from the in-plane angular variation of the latter, at (b) the widths of distributions in energy differences between low-lying electronic levels and (c) the angular spread in the in-plane principal g-directions. The values of the energy level distributions in crystals obtained from the measurements and analysis reported here are compared with those obtained by a different method for the same protein complex in frozen solution. The spread in the rhombic energy splitting is significantly greater in solution than in the crystal.     

Thermal properties of water in myoglobin crystals and solutions at subzero temperatures.

The water of hydration in myoglobin crystals and solutions was studied at subzero temperatures by calorimetry and infrared spectroscopy (ir). For comparison we also investigated glycine, DL-alanine and DL-valine solutions. The hydration water remains amorphous at low temperatures. We find a broad glass transition between 180 and 270 K depending on the degree of hydration. The ice component shows a noncolligative melting point depression that is attributed to a finite conformational flexibility. The ir spectrum and the specific heat of water in myoglobin crystals was determined for the first time between 180 and 290 K. The glass transition in crystals is qualitatively similar to what is found in amorphous samples at the same water content. These data are compared with M?ssbauer experiments and dielectric relaxation of water in myoglobin crystals. The similar temperature dependencies suggest a cross correlation between structural fluctuations and the thermal motion of crystal water. A hydrogen bond network model is proposed to explain these features. The essential ingredients are cooperativity and a distribution of hydrogen-bonded clusters.     

Lateral packing of mineral crystals in bone collagen fibrils

Combined small-angle x-ray scattering and transmission electron microscopy studies of intramuscular fish bone (shad and herring) indicate that the lateral packing of nanoscale calcium-phosphate crystals in collagen fibrils can be represented by irregular stacks of platelet-shaped crystals, intercalated with organic layers of collagen molecules. The scattering intensity distribution in this system can be described by a modified Zernike-Prins model, taking preferred orientation effects into account. Using the model, the diffuse fan-shaped small-angle x-ray scattering intensity profile, dominating the equatorial region of the scattering pattern, could be quantitatively analyzed as a function of the degree of mineralization. The mineral platelets were found to be very thin (1.5 nm ∼ 2.0 nm), having a narrow thickness distribution. The thickness of the organic layers between adjacent mineral platelets within a stack is more broadly distributed with the average value varying from 6 nm to 10 nm, depending on the extent of mineralization. The two-dimensional analytical scheme also leads to quantitative information about the preferred orientation of mineral stacks and the average height of crystals along the crystallographic c axis.     

Characterization of acetylcholinesterase molecular forms in slow and fast muscle of rat

Multiple molecular forms of acetylcholinesterase (AChE EC 3.1.1.7) from fast and slow muscle of rat were examined by velocity sedimentation. The fast extensor digitorum longus muscle (EDL) hydrolyzed acetylcholine at a rate of 110 mumol/g wet weight/hr and possessed three molecular forms with apparent sedimentation coefficients of 4S, 10S, and 16S which contribute about 50, 35, and 15% of the AChE activity. The slow soleus muscle hydrolyzed acetylcholine at a rate of 55 mumol/g wet weight/hr and has a 4S, 10S, 12S, and 16S form which contribute 22, 18, 34, and 26% of AChE activity, respectively. A single band of AChE activity was observed when a 1M NaCl extract with CsCl (0.38 g/ml) was centrifuged to equilibrium. Peak AChE activity from EDL and SOL extracts were found at 1.29 g/ml. Resedimentation of peak activity from CsCl gradients resulted in all molecular forms previously found in both muscles. Addition of a protease inhibitor phenylmethylsulfonyl chloride did not change the pattern of distribution. The 4S form of both muscles was extracted with low ionic strength buffer while the 10S, 12S, and 16S forms required high ionic strength and detergent for efficient solubilization. All molecular forms of both muscles have an apparent Km of 2 x 10(-4) M, showed substrate inhibition, and were inhibited by BW284C51, a specific inhibitor of AChE. The difference between these muscles in regards to their AChE activity, as well as in the proportional distribution of molecular forms, may be correlated with sites of localization and differences in the contractile activity of these muscles.     

Crystal structure determination of an acidic neurotoxin (BmK M8) from scorpion Buthm martensii Karsch at 0.25nm resolution

The crystal structure of an acidic neurotoxin, BmK M8, from Chinese scorpion Buthus martensii Karsch was determined at 0.25 nm resolution. The X-ray diffraction data of BmK M8 crystals at 0.25nm resolution were collected on a Siemens area detector. Using molecular replacement method with a basic scorpion toxin AaH II in a search model, the cross-rotation function, PC-refinement and translation function were calculated by X-PLOR program package. The correct orientation and position of BmK M8 molecule in crystal were determined in a resolution range of 1.5 - 0.35nm, The oystallographic refinement was further performed by stereo-chemical restrict least-square technique, followed by simulated annealing, slow-cooling protocols. The final crystallographic R-factor at 0.8-0.25 nm is 0.171. The standard deviations of bond length and bond angle from ideality are 0.001 7nm and 2.24° , respectively. The final model of BmK M8 structure is composed of a dense core of secondary structure elements by a stretch of α-     

Capillary tortuosity in skeletal muscles of mammals depends on muscle contraction

Capillary orientation (anisotropy) was compared in hindlimb muscles of mammals of different size and/or different aerobic capacity (dog, goat, pony, and calf). All muscles were fixed by vascular perfusion at sarcomere lengths ranging from 1.5 to 2.7 micron. The ratios of capillary counts per fiber cross-sectional area on two sets of sections (0 and 90 degrees) to the muscle fiber axis were used to estimate capillary anisotropy and the coefficient c(K,0) relating 1) capillary counts on transverse sections (a commonly used parameter to assess muscle capillarity) and 2) capillary length per volume of fiber (i.e., capillary length density). Capillary orientation parallel to the muscle fiber axis decreased substantially with muscle fiber shortening. In muscles fixed at sarcomere lengths of 2.69 microns (dog vastus intermedius) and 1.52 microns (dog gastrocnemius), capillary tortuosity and branching added 7 and 64%, respectively, to capillary length density. The data obtained in this study are highly consistent with the previously demonstrated relationship between capillary anisotropy and sarcomere length in extended vs. contracted rat muscles, by use of the same method. Capillary anisotropy in mammalian locomotory muscles is curvilinearly related to sarcomere length. No systematic difference was found in capillary tortuosity with either body size, athletic ability, or aerobic capacity. Capillary tortuosity is a consequence of fiber shortening rather than an indicator of the O2 requirements of the tissue.     

Understanding site-specific residual strain and architecture in bovine cortical bone

Living bone is considered as adaptive material to the mechanical functions, which continually undergoes change in its histological arrangement with respect to external prolonged loading. Such remodeling phenomena within bone depend on the degree of stimuli caused by the mechanical loading being experienced, and therefore, are specific to the sites. In the attempts of understanding strain adaptive phenomena within bones, different theoretical models have been proposed. Also, the existing literatures mostly follow the measurement of surface strains using strain gauges to experimentally quantify the strains experienced in the functional environment. In this work, we propose a novel idea of understanding site-specific functional adaptation to the prolonged load in bone on the basis of inherited residual strains and structural organization. We quantified the residual strains and amount of apatite crystals distribution, i.e., the degree of orientation, using X-ray diffraction procedures. The sites of naturally existing hole in bone, called foramen, are considered from bovine femur and metacarpal samples. Significant values of residual strains are found to exist in the specimens. Trends of residual strains noted in the specimens are mostly consistent with the degree of orientation of the crystallites. These features explain the response behavior of bone to the mechanical loading history near the foramen sites. Preferential orientation of crystals mapped around a femoral foramen specimen showed furnished tailored arrangement of the crystals around the hole. Effect of external loading at the femoral foramen site is also explained by the tensile loading experiment.     

Innervation of holothurian body wall muscle: inhibitory effects and localization of 5-HT

We investigated innervation to body wall muscles as well as distribution of 5-HT (serotonin) and its effects on longitudinal muscles of body wall (LMBW) of the sea cucumber Apostichopus japonicus. With serial sections we found neural branches and fibers extending from hyponeural part of radial nerve towards LMBW and circular muscles of body wall. With the aqueous aldehyde (Faglu) method yellow fluorescence indicating indolamines was observed in LMBW and in the mesentery connecting LMBW to the body wall. With indirect immunohistochemistry 5-HT-like immunoreactivity was observed in LMBW and in mesentery. These results strongly suggested that both LMBW and mesentery contained 5-HT. The effects of monoamine neurotransmitters were studied in LMBW. Putative neurotransmitters tested were 5-HT, adrenaline, noradrenaline, dopamine, and DOPA at the concentration of 10(-6) M. The application of 5-HT caused no contraction or relaxation, but it inhibited the contraction induced by 10(-6)-10(-5) M acetylcholine (ACh). Catecholamines were ineffective by themselves and had no effects on the contraction induced by ACh. The present histological, histochemical, and pharmacological studies strongly suggested that holothurian LMBW was innervated by inhibitory serotonergic neurons of the hyponeural nervous system.     

Proteins of Bacillus thuringiensis delta-endotoxin crystals]

Pure crystals (at least 99% purification) of sigma-endotoxin were isolated from Bac. thuringiensis var. galleriae. The complete dissolution of crystals might be achieved by the increase of pH up to 12 and higher or by a combined action of S = S-reducing and denaturing agents. Electrophoresis of the solubilized crystal proteins in 5% polyacrylamide gels containing 0,1% sodium dodecyl sulfate and 8 M urea reveals two major bands corresponding to molecular weights of 120000--140000 (65%) and 65000 (8-10%), and some minor components whose molecular weights varied from 65000 to 340000. Urea (3--8 M) causes to partial dissolution of the crystals; the component with molecular weight of 65000 is mainly found in the solution (component A). In dithioerythritol extracts at pH 9 the major component of the crystal is the protein with molecular weight 120000--140000 (component B). The crystals, alkali-soluble components and proteins isolated from crystals by selective extraction (3--8 M urea or 0.01 M dithioerythrytol, pH 9) were found toxic for the larvae of Galleria mellonella.     

十七种一变种美登木叶片解剖与分类的关系

我们从国内外收集美登木属17种1变种,从叶片外部形状及内部结构进行比较,,发现在美登木属17种1变种美登木叶片组织中存在两种晶体(簇晶和方晶),可把美登木属分成三大类,一类具簇晶,二类具方晶,三类具簇晶和方晶。然后根据晶体的大小及其在组织中的分布部位,又将本属中的种分开。另外我们从美登木叶片解剖发现,成熟叶主脉横切面维管束的形状常是固定的,它不受外界环境的影响,也不受地理位置的影响,如滇南美登木分别采自云南省景洪和耿马不同地区不同生境,成熟叶横切面上维管束形状是相同的。成熟叶横切面上维管束的形状较稳定。所以用细胞内的晶体类型和维管束的形状把美登术属不同种进行分类是有意义的。     

Studies of the diffuse x-ray scattering from contracting frog skeletal muscles.

Using x-rays from synchrotron radiation, we studied diffuse scattering, sometimes together with the myosin layer lines. With an area detector, sartorius muscles and a time resolution of 150 ms, earlier results from semitendinosus muscles contracting isometrically at 6 degrees C (Lowy, J., and F. R. Poulsen. 1987. J. Mol. Biol. 194:595-600) were confirmed and extended. Evidence from intensity changes both in the diffuse scattering and in the myosin layer lines showed that the majority of the heads become disordered at peak tetanic tension. With a linear detector and a time resolution of 5 ms, it was found that during tension rise the intensity increase of the diffuse scattering (which amounted maximally to 12% recorded near the meridian) runs approximately 20 ms ahead of the mechanical change, comparing half-completion times. This suggests that an appreciable number of heads change orientation before peak tension is reached. In quick release experiments the diffuse scattering intensity showed very little change. Recorded near the meridian during rapid shortening, however, it decreased progressively with a half-time of approximately 40 ms. This change amounted to approximately 35% of that observed during the initial tension rise. We interpret this to indicate that during rapid shortening a certain number of heads assume an orientation characteristic of the relaxed state. Viewed in the context of the behavior of the first myosin layer line and the (1, 1) equatorial reflection in similar experiments (Huxley, H. E., M. Kress, A. R. Faruqi, and R. M. Simmons. 1988.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural and cytochemical observation of calcium ion localization in the black-beetle pleural muscles

The authors examined the infrastructure and distribution of calcium ions in the plexural muscles of a black-beetle (Blatta orientalis L.) after being fixed in a solution of osmiumtetroxide buffered with a cacodylate buffer (pH = 7.4) and according to the method of Carasso-Favard (1966). The infrastructure of these muscles differs from the muscles of other insects, first of all, in the amount and distribution of the sarcoplasmic net (SR) and mitochondria and also in the amount and topography of the location of lead precipitations which mark the calcium ions. The authors ascertained an intensive and permanent positive reaction to the presence of calcium in the mitochondria and sarcotubular systems of the sarcoplasmic reticulum, and in the intrafibrillar spaces and in the Z and M lines of the sarcomeres calcium concentrations are not detectable. The authors checked the results by using the method of Carasso-Favard (1966).     

Immunohistochemical localization of pyruvate kinase isoenzymes in chicken tissues.

A method for the localization of pyruvate kinase isoenzymes type L, M2 and M1 in tissue sections is described. Mono-specific antibodies directed against isoenzymes of pyruvate kinase from chicken and the peroxidase antiperoxidase method were used. The following preferential localizations of the isoenzymes in chicken tissues were observed: Pyruvate kinase M1 was found in skeletal muscle. The white muscle fibers were more intensely stained than the red. Some dark muscles (e.g., anterior latissimus dorsi) and the heart muscle showed no reaction with antiserum against pyruvate kinase M1. Pyruvate kinase type L was found in the hepatocytes and in kidney cortex. Pyruvate kinase type M2 was seen in the distal tubules of kidney, in hepatocytes and sinusoidal cells in liver, in lung, adipose tissue, and in the spleen mainly in the bursa dependent areas. Pyruvate kinase type M2 was detected in high concentrations in the granulation tissue of regenerating liver after partial hepatectomy. Liver sections of a hen bearing a pancreatic tumor showed an unusually high content of pyruvate kinase type M2 in some hepatocytes, which were each clustered to spots in the liver parenchyma. Thus, contrary to previous reports, the tissue distribution of isoenzymes in chicken is similar to that of other vertebrates.     

Muscle activity and muscle agrin regulate the organization of cytoskeletal proteins and attached acetylcholine receptor (AchR) aggregates in skeletal muscle fibers.

In innervated skeletal muscle fibers, dystrophin and beta-dystroglycan form rib-like structures (costameres) that appear as predominantly transverse stripes over Z and M lines. Here, we show that the orientation of these stripes becomes longitudinal in denervated muscles and transverse again in denervated electrically stimulated muscles. Skeletal muscle fibers express nonneural (muscle) agrin whose function is not well understood. In this work, a single application of > or = 10 nM purified recombinant muscle agrin into denervated muscles preserved the transverse orientation of costameric proteins that is typical for innervated muscles, as did a single application of > or = 1 microM neural agrin. At lower concentration, neural agrin induced acetylcholine receptor aggregates, which colocalized with longitudinally oriented beta-dystroglycan, dystrophin, utrophin, syntrophin, rapsyn, and beta 2-laminin in denervated unstimulated fibers and with the same but transversely oriented proteins in innervated or denervated stimulated fibers. The results indicate that costameres are plastic structures whose organization depends on electrical muscle activity and/or muscle agrin.     

Homology of the muscles within the uropatagium membrane in Leschenault’s rousette (Rousettus leschenaultii)

Pteropodidae possess unique muscles in the uropatagium called Musculus uropatagialis and M. depressor ossis styliformis. The homology of these muscles is important for the phylogenetic analysis of bats because the wing membrane is a characteristic feature for them. Here, I discuss the homology of M. uropatagialis and M. depressor ossis styliformis in Rousettus leschenaultii by tracing their innervations. I found that the dominant nerve for the M. uropatagialis contains the components of the sciatic nerve associated with the dominant nerve of the flexor muscles of the thigh. This result shows that M. uropatagialis is homologous to the flexor muscles of the thigh. The dominant nerve of M. depressor ossis styliformis is the lateral plantar nerve derived from the tibial nerve. Thus, this innervation pattern proposes the hypothesis that M. depressor ossis styliformis is homologous to one of the muscles of the foot sole.     

Respiratory responses to microinjections of gastrin-releasing peptide into the solitary tract nucleus

In acute experiments on urethane-anesthetized rats, the respiratory effects ofmicroinjections of 10(-5), 10(-8) and 10(-10) M gastrin-releasing peptide (GRP) into the solitary tract nucleus were investigated. It was found that microinjections of the neuropeptide induced an increase in tidal volume, amplitude of diaphragm and external intercostal muscles firing activity and in expiratory duration. The most obvious respiratory responses observed when 10(-8) M GRP was used, while 10(-10) M GRP appeared to be sub-threshold and didn't alter the breathing pattern and activity of inspiratory muscles. In some experiments, where the blood pressure and the heart rate was monitored alone with breathing pattern, these parameters did not change after GRP microinjections into the solitary tract nucleus. The obtained data together with particularities of the distribution of GRP receptors in the brainstem suggest the possibility of GRP involvement into the respiratory control mechanisms at the level of solitary tract nucleus.     

Occlusal force and craniofacial biomechanics during growth in rhesus monkeys

The masticatory muscles in 132 anesthetized male and female rhesus monkeys ranging in age from juvenile to adult were unilaterally stimulated. Muscle forces and speeds were measured with a bite force transducer positioned at the incisors, premolars, and molars during twitch and tetanic contractions. Lateral cephalographs of all animals were used to estimate the orientation and mechanical advantage of the masticatory muscles. Results showed that maximal occlusal forces increased at a greater rate than body weight during growth. However, maximal occlusal forces increased isometrically relative to mandibular length. Mean forces at the incisors ranged from 70.3 newtons (n) in juveniles up to 139.9 n in adult males. Forces at the molars were 2-2.5 times greater than at the incisors. Time-to-peak tension decreased with increasing body size from 44.1 msec in juveniles to 37.4 msec in adult females to 31.0 msec in adult males. Regression analysis showed that adult males have faster muscles than adult females or juveniles even when corrected for body size. Temporalis and masseter orientation was found to change little throughout growth. The mechanical advantage of the masseter and temporalis muscles for producing occlusal forces on the distal molars improved between juveniles and adults, which is contrary to findings of Oyen et al. (Growth 43:174-187, 1979). Among adults, females had a greater mechanical advantage of the masseter muscles than males.