Cell-free expression of visual arrestin. Truncation mutagenesis identifies multiple domains involved in rhodopsin interaction.

Visual arrestin plays an important role in regulating light responsiveness via its ability to specifically bind to the phosphorylated and light-activated form of rhodopsin. To further characterize rhodopsin/arrestin interactions we have utilized a rabbit reticulocyte lysate translation system to synthesize bovine visual arrestin. The translated arrestin (404 amino acids) was demonstrated to be fully functional in terms of its ability to specifically recognize and bind to phosphorylated light-activated rhodopsin (P-Rh*). Competitive binding studies revealed that the in vitro synthesized arrestin and purified bovine visual arrestin had comparable affinities for P-Rh*. In an effort to assess the functional role of different regions of the arrestin molecule, two truncated arrestin mutants were produced by cutting within the open reading frame of the bovine arrestin cDNA with selective restriction enzymes. In vitro translation of the transcribed truncated mRNAs resulted in the production of arrestins truncated from the carboxyl terminus. The ability of each of the mutant arrestins to bind to dark (Rh), light-activated (Rh*), dark phosphorylated (P-Rh), and light-activated phosphorylated rhodopsin were then compared. Arrestin lacking 39 carboxyl-terminal residues binds specifically not only to P-Rh* but also to Rh* and P-Rh. This suggests that the carboxyl-terminal domain of arrestin plays an important regulatory role in ensuring strict arrestin binding selectivity to P-Rh*. Arrestin that has only the first 191 amino-terminal residues predominately discriminates the phosphorylation state of the rhodopsin; however, it also retains some binding specificity for the activation state. These results suggest that the amino-terminal half of arrestin contains key rhodopsin recognition sites responsible for interaction with both the phosphorylated and light-activated forms of rhodopsin.


An element regulating adrenal-specific steroid 21-hydroxylase expression is located within the slp gene.

In this report we demonstrate that a transcriptional regulatory element for one gene lies within a second, seemingly unrelated gene. Specifically, the 3' portion of the murine sex-limited protein (slp) gene, located within the class III region of the major histocompatibility complex, contains an element that regulates expression of the linked steroid 21-hydroxylase gene. A 4.2-kilobase (kb) major histocompatibility complex region, located between -2.2 and -6.4 kb upstream of 21OH-A, is required for expression of a chloramphenicol acetyltransferase reporter gene in transgenic mice. Two short regions of DNA, located between -5.3 and -6.0 kb, stimulate chloramphenicol acetyltransferase expression in Y1 adrenocortical tumor cells, and both of these active regions lie within the slp gene. A 21-base pair sequence, which is required for activity of the most 3' region, does not contain any of over 100 previously identified transcriptional regulatory elements. This juxtaposition of structural and regulatory elements of otherwise unrelated genes suggests a mechanism by which the evolutionarily conserved genetic linkage of 21OH-A and slp (or the homologous complement component C4) might provide a selective advantage. Analogous genetic arrangements may explain other examples of conserved linkage of disparate genes.


Role of tryptophan 49 in the heparin cofactor activity of human antithrombin III.

To probe the functional role of tryptophan 49 in human antithrombin III, a mutant antithrombin, W49K, has been expressed in baby hamster kidney cells. The mutation reduces the affinity for heparin pentasaccharide by 1.8 kcal mol-1 but does not alter the heparin enhancement of the rate of factor Xa inhibition. 1H NMR spectra of W49K antithrombin show that the structure of the protein and the mode of heparin binding appear to be unaltered by the mutation, although tryptophan 49 is perturbed by heparin binding. 19F NMR spectra of 6-fluorotryptophan-substituted antithrombin show that tryptophan 49 is in a solvent-exposed environment. The heparin-induced fluorescence enhancement of W49K antithrombin is significantly different from that of wild-type antithrombin. Pentasaccharide induces only a 24% enhancement of antithrombin fluorescence, while high affinity heparin induces an enhancement of 40%. The results indicate that tryptophan 49 is probably a heparin contact residue but can be mutated without altering the remaining heparin-antithrombin interactions or the heparin-induced conformational change and resultant activation toward Factor Xa. Hydrophobic as well as charge interactions are thus probably involved in the specificity of the antithrombin-heparin pentasaccharide interaction. The lower fluorescence enhancements suggest that the heparin-induced 40% fluorescence enhancement used as the hallmark of activating heparin species is not the best indicator of the structural change in antithrombin that results in enhancement of the rate of proteinase inhibition.


Mammalian nitrobenzylthioinosine-sensitive nucleoside transport proteins. Immunological evidence that transporters differing in size and inhibitor specificity share sequence homology.

Polyclonal antibodies were raised against the nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporter of human erythrocyte membranes. On Western blots of these membranes they labeled the broad "band 4.5" region (average apparent M(r) 55,000), which contains both the nucleoside and glucose transport proteins. However, they did not recognize the glucose transporter when this was prepared free of nucleoside transporter by expression from a cDNA clone. Their specificity for the nucleoside transporter was confirmed by the ability to immunoadsorb NBMPR- but not cytochalasin B-binding sites from a detergent-solubilized mixture of band 4.5 proteins. Although a large proportion of the antibodies recognized extracellular epitopes, these appeared to be located primarily on the polypeptide moiety of the glycoprotein, as demonstrated by the ability of the antibodies strongly to label the deglycosylated transporter (apparent M(r) 45,000) on Western blots. The antibodies were species-cross-reactive, recognizing nucleoside transporters from pig and rabbit erythrocytes and from rat liver. The pig protein is similar to the human transporter in its inhibitor sensitivity but is considerably larger (apparent M(r) 57,000 after deglycosylation). In contrast, the rat protein is similar in size to the human transporter (apparent M(r) 45,000 after deglycosylation) but much less sensitive to the inhibitors dilazep and dipyridamole. These findings indicate that despite their differences in size and inhibitor specificity, the NBMPR-sensitive nucleoside transporters of these mammalian species are related in amino acid sequence.


Human plasma lipoproteins regulate apolipoprotein E secretion from a post-Golgi compartment.

The molecular regulation of apolipoprotein E (apoE) synthesis and secretion is incompletely understood. In this study, we have examined the effect of human low density lipoprotein (LDL) on apoE mRNA and protein levels in HepG2 and other eukaryotic cells. Exposing HepG2 cells to LDL for times up to 4 h resulted in an increase in 35S-labeled apoE accumulation in the medium by 2.2-fold, relative to serum free controls (n = 10, p < 0.001), with no changes in apoE mRNA levels. Similar observations have been made in JeG-3 cells and Chinese hamster ovary cells stably transfected with human apoE cDNA constructs. These results indicate that the LDL effect operates at a post-transcriptional level. In pulse-chase experiments, the LDL effect on apoE accumulation in the media was observed when it was added only during the chase even in the presence of cycloheximide, indicating that LDL is functioning at a post-translational level. The use of brefeldin A (BFA), an agent that impedes protein transport from the endoplasmic reticulum to the Golgi apparatus, suggests that the LDL effect occurs in a post-Golgi compartment. The addition of protease inhibitors could not duplicate the effects of LDL on the apoE accumulation in the medium. ApoA-I accumulation in the medium of HepG2 cells, but not albumin, was also significantly increased by 1.9-fold (n = 5, p < 0.001).


Guinea pigs possess a highly mutated gene for L-gulono-gamma-lactone oxidase, the key enzyme for L-ascorbic acid biosynthesis missing in this species.

Guinea pigs cannot synthesize L-ascorbic acid because of their deficiency in L-gulono-gamma-lactone oxidase, a key enzyme for the biosynthesis of this vitamin in higher animals. In this study we isolated the L-gulono-gamma-lactone oxidase gene of the rat and the homologue of this gene of the guinea pig by screening rat and guinea pig genomic DNA libraries in lambda phage vectors, respectively, using a rat L-gulono-gamma-lactone oxidase cDNA as a probe. Sequencing analysis showed that the amino acid sequence of the rat enzyme is encoded by 12 exons and that all the intron/exon boundaries follow the GT/AG rule. On the other hand, regions corresponding to exons I and V were not identified in the guinea pig L-gulono-gamma-lactone oxidase gene homologue. Other defects found in this gene homologue are a deletion of the nucleotide sequence corresponding to a 3' 84-base pair part of rat exon VI, a 2-base pair deletion in the remaining exon VI-related region, and nonconformance to the GT/AG rule at one of the putative intron/exon boundaries. Furthermore, a large number of mutations were found in the amino acid-coding regions of the guinea pig sequence; more than half of them lead to nonconservative amino acid changes, and there are three stop codons as well. Thus it is clear that the guinea pig homologue of the L-gulono-gamma-lactone oxidase gene exists as a pseudogene that randomly accumulated a large number of mutations without functional constraint since the gene ceased to be active during evolution. On the basis of the neutral theory of evolution, the date of the loss of L-gulono-gamma-lactone oxidase in the ancestors of the guinea pig was roughly calculated to be less than 20 million years ago.


A missense mutation of L-gulono-gamma-lactone oxidase causes the inability of scurvy-prone osteogenic disorder rats to synthesize L-ascorbic acid.

The osteogenic disorder Shionogi (ODS) rat is a mutant Wistar rat that is subject to scurvy, because it lacks L-gulono-gamma-lactone oxidase, a key enzyme in L-ascorbic acid biosynthesis. Sequencing of polymerase chain reaction-amplified cDNAs for mutant and normal rat L-gulono-gamma-lactone oxidases demonstrated that the mutant cDNA has a single base mutation from G to A at nucleotide 182, which mutation alters the 61st amino acid residue from Cys to Tyr. To test the effect of this mutation on the expression of L-gulono-gamma-lactone oxidase, we inserted a region of the cDNAs coding for normal and mutant L-gulono-gamma-lactone oxidases into an expression vector, pSVL, and transfected COS-1 cells with such vectors. The result indicated that the defined amino acid substitution does decrease both the amount of immunologically detectable protein and the level of enzyme activity to about one-tenth of their normal values, while it does not affect the amount of the mRNA produced in the transfected cells. This situation is similar to our previous observation that L-gulono-gamma-lactone oxidase is expressed in the liver of the ODS rat at a very low level irrespective of the presence of a normal amount of L-gulono-gamma-lactone oxidase-specific mRNA of a normal size (Nishikimi, M., Koshizaka, T., Kondo, K., and Yagi, K. (1989) Experientia (Basel) 45, 126-129). Thus it became clear that the Cys-->Tyr substitution is responsible for the L-gulono-gamma-lactone oxidase deficiency in the ODS rat.


Phosphorylation of the Rex protein of human T-cell leukemia virus type I.

Rex protein, the posttranscriptional regulator of human T-cell leukemia virus type I (HTLV-I), is required for the control of viral structural protein expression and virus replication. Rex is a phosphoprotein found predominantly in the cell nucleolus, whose function is thought to be regulated by its nucleolar localization and phosphorylation. Therefore, we investigated the in vivo phosphorylation of Rex protein in more detail. Phosphorylation of Rex occurred in all HTLV-I-infected cell lines examined in vivo, primarily at serine residues and to a very small extent at threonine residues. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) led to significant but transient enhancement of the incorporation of [32P]orthophosphate into Rex protein. N-terminal truncation of Rex protein abolished TPA-dependent phosphorylation. Chymotryptic digestion of phosphorylated Rex yielded two phosphopeptides. In vivo phosphorylation sites were identified as serine residues 70 and 177 and threonine residue 174. Serine 70 was a TPA-dependent phosphorylation site within a regulatory domain. We have already shown that the protein kinase C inhibitor H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) specifically blocked accumulation of viral unspliced gag-pol mRNA. Therefore, the phosphorylation at serine 70 may be involved in the regulation of Rex function in response to extracellular stimuli.


Angiogenin is a cytotoxic, tRNA-specific ribonuclease in the RNase A superfamily.

Angiogenin is a 14.4-kDa human plasma protein with 65% homology to RNase A that retains the key active site residues and three of the four RNase A disulfide bonds. We demonstrate that recombinant angiogenin functions as a cytotoxic tRNA-specific RNase in cell-free lysates and when injected into Xenopus oocytes. Inhibition of protein synthesis by angiogenin correlates with degradation of endogenous oocyte tRNA. Exogenous, radiolabeled tRNA is also hydrolyzed by angiogenin, whereas oocyte rRNA and mRNA are not detectably degraded by angiogenin. Protein synthesis was restored to angiogenin-injected oocytes by injecting the RNase inhibitor RNasin plus total Xenopus or calf liver tRNAs, thereby demonstrating that the tRNA degradation induced by angiogenin was the sole cause of cytotoxicity. A similar tRNA-reversible inhibition of protein synthesis was seen in rabbit reticulocyte lysates. Angiogenin therefore appears to be a specific cellular tRNase, whereas five homologues in the RNase A superfamily lack angiogenin's specificity for tRNA. One of these homologues purified from human eosinophils, eosinophil-derived neurotoxin, nonspecifically degrades oocyte RNA similar to RNase A and is also cytotoxic at very low concentrations.


A fluid-structure interaction problem in biomechanics: prestressed vibrations of the eye by the finite element method.

The object of this work has been to develop a mechanical and numerical model of the eye submitted to vibrations, and in particular, to calculate the influence of intraocular pressure (IOP) on the eye resonance frequencies. Our mechanical model of the eye relies upon the theory of the mechanics of continuous media. The numerical model results from a model analysis of the vibrations of the eye using a finite element method (FEM) for discretization. The eye can be schematically represented as a prestressed shell, filled by an inviscid barotropic compressible fluid, which leads us to formulate and solve a problem of vibrations of a coupled fluid-structure system. The corneoscleral shell has been modeled as a thin and thick shell, taking into account material nonlinearities in the thick case. Numerical results obtained for the attached eye demonstrate a fair sensitivity of the resonance frequencies to the variations of the IOP; thus, founding the interest of the surveillance of the resonance frequency of the eye.


Description of the deformation of the left ventricle by a kinematic model.

A model of left ventricular (LV) kinematics is essential to identify the fundamental physiological modes of LV deformation during a complete cardiac cycle as observed from the motion of a finite number of markers embedded in the LV wall. Kinematics can be described by a number of modes of motion and deformation in succession. An obvious mode of LV deformation is the ejection of cavity volume while the wall thickens. In the more sophisticated model of LV kinematics developed here, seven time-dependent parameters were used to describe not only volume change but also torsion and shape changes throughout the cardiac cycle. Rigid-body motion required another six parameters. The kinematic model employed a deformation field that had no singularities within the myocardium, and all parameters describing the modes of deformation were dimensionless. Note that torsion, volume and symmetric shape changes all require the definition of a cardiac coordinate system, which has generally been related to the measured cardiac geometry by reference to approximate anatomical landmarks. However, in the present study the coordinate system was positioned objectively by a least-squares fit of the kinematic model to the measured motion of markers. Theoretically, at least five markers are needed to find a unique set of parameters.(ABSTRACT TRUNCATED AT 250 WORDS)


Dependence of local left ventricular wall mechanics on myocardial fiber orientation: a model study.

The dependence of local left ventricular (LV) mechanics on myocardial muscle fiber orientation was investigated using a finite element model. In the model we have considered anisotropy of the active and passive components of myocardial tissue, dependence of active stress on time, strain and strain rate, activation sequence of the LV wall and aortic afterload. Muscle fiber orientation in the LV wall is quantified by the helix fiber angle, defined as the angle between the muscle fiber direction and the local circumferential direction. In a first simulation, a transmural variation of the helix fiber angle from +60 degrees at the endocardium through 0 degrees in the midwall layers to -60 degrees at the epicardium was assumed. In this simulation, at the equatorial level maximum active muscle fiber stress was found to vary from about 110 kPa in the subendocardial layers through about 30 kPa in the midwall layers to about 40 kPa in the subepicardial layers. Next, in a series of simulations, muscle fiber orientation was iteratively adapted until the spatial distribution of active muscle fiber stress was fairly homogeneous. Using a transmural course of the helix fiber angle of +60 degrees at the endocardium, +15 degrees in the midwall layers and -60 degrees at the epicardium, at the equatorial level maximum active muscle fiber stress varied from 52 kPa to 55 kPa, indicating a remarkable reduction of the stress range. Moreover, the change of muscle fiber strain with time was more similar in different parts of the LV wall than in the first simulation. It is concluded that (1) the distribution of active muscle fiber stress and muscle fiber strain across the LV wall is very sensitive to the transmural distribution of the helix fiber angle and (2) a physiological transmural distribution of the helix fiber angle can be found, at which active muscle fiber stress and muscle fiber strain are distributed approximately homogeneously across the LV wall.


Finite element simulation of pulsatile flow through arterial stenosis.

The problem of blood flow through a stenosis is solved using the incompressible Navier-Stokes equations in a rigid circular tube presenting a partial occlusion. Calculations are based on a Galerkin finite element method. The time marching scheme employs a predictor-corrector technique using a variable time step. Results are obtained for steady and physiological pulsatile flows. Computational experiments analyse the effect of varying the degree of stenosis, the stricture length, the Reynolds number and Womersley number. The method gives results which agree well with previous computations for steady flows and experimental findings for steady and pulsatile flows.


Three-dimensional geometrical and mechanical modelling of the lumbar spine.

The main objective of this study is to design a three-dimensional geometrical and mechanical finite element model of the lumbar spine. The model's geometry is constructed using six parameters per vertebra. These parameters are digitized from two X-rays (anterio-posterior and lateral), thus yielding an individualized model which can be arrived at from the radiographs of a tested specimen. This procedure makes the model validation easier, as geometry is generally a factor of dispersion in experimental results. The geometrical reconstruction, in the form of a finite elements mesh, was effected for the whole lumbar spine. The global coherence of the model was verified.


The onset and progression of spinal injury: a demonstration of neutral zone sensitivity.

Spinal injuries are a great cost to society and the afflicted individuals. It is well known that most spinal injuries are not bony fractures but rather soft tissue lesions falling in the 'subfailure' region. For the clinical diagnosis of spinal injuries, abnormal motion patterns under physiological loads are considered an important factor. The purpose of the present study was to determine the onset and progression of spinal injury, and compare the sensitivity of three motion parameters: neutral zone (NZ), elastic zone (EZ), and range of motion (ROM). Spinal injury was defined as a significant increase in any of the three motion parameters. A repeatable high-speed flexion-compression load vector was applied individually to six porcine cervical spine specimens. Several impacts of increasing severity were applied to each specimen. After each impact, flexion-extension motion was measured. Neutral zone was the residual deformation from the neutral position to the position under zero load at the start of the final load cycle. Elastic zone was the displacement from zero load to the maximum load on the final load cycle. Range of motion was the sum of the neutral and elastic zones. The first significant increase in motion was determined by the neutral zone parameter with few observable anatomic lesions on the specimens. This was the onset of spinal injury. The next significant motion increase was also determined by the neutral zone parameter. After this motion increase, termed the progression of injury, ligament ruptures were observed in some specimens. It was concluded that the neutral zone was the most sensitive motion parameter in defining the onset and progression of spinal injury.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of changes in segmental values and timing of both torque and torque reversal in simulated throws.

An overarm throw in the sagittal plane was simulated using a three-segment model representing the upper arm, forearm and hand plus ball. Torque inputs at each joint were turned on at systematically varied times and maintained constant once initiated. All simulations began from identical initial conditions. The aim was to determine the sequence of onset of joint torques which gave the maximal range which the ball would travel and the maximal velocity of the ball irrespective of direction. Best throws proved to be sequential in that joint torques were turned on in a proximal to distal (P-D) temporal sequence. The P-D sequence was also demonstrated by time of peak joint angular velocities. The P-D sequence also proved to be best when segmental constants and joint torques were changed. As this sequence is a common feature of skilled throwing and striking, it is concluded that the linked segmental nature of the limb, irrespective of normal muscle characteristics, primarily predisposes the system to the use of a P-D sequence. The algebraic sign of the shoulder and elbow torques was reversed instantaneously to represent the use of antagonistic muscles. This led to increased output if performed late in the throw and in a P-D sequence. It is concluded that the use of antagonism leads to beneficial redistributions of angular velocity amongst limb segments.


Mechanical properties of the human lumbar anterior longitudinal ligament.

A new technique incorporating a motion analysis system and a materials testing machine was used to investigate regional differences in the tensile mechanical properties of the lumbar spine anterior longitudinal ligament (ALL). Bone-ALL-bone specimens were prepared from young human cadaveric motion segments with no disc or bony pathology. Each specimen was distracted until failure at a constant crosshead displacement rate of 2.5 mm s-1 (approximately 1.0% strain per second). Strains were evaluated from digitized video recordings of markers attached to the ALL at 12 sites along its length and width, including the ligament substance and insertions. The 'overall' strain in the ligament was calculated from the outermost pairs of markers along the ligament length. The average tensile strength, the 'overall' tensile modulus and the 'overall' strain of the ALL at failure were 27.4 MPa (S.D. 5.9), 759 MPa (S.D. 336) and 4.95% (S.D. 1.51), respectively. Large and significant variations in the strains were present along the width and length of the ALL. Peak substance strains were over twofold greater than peak strains at the ligament insertion sites, whereas across the ligament width, peak strains in the outer portion of the ligament were over 40% greater than in the central region. Failure consistently occurred in the ligament mid-substance and ultimate strains at the ligament failure site averaged 12.1% (S.D. 2.3). These results indicate that the strains are highly nonuniform in the normal ALL.


The effect of lower-limb anatomy on knee loads during seated cycling.

Overuse knee joint injuries are the primary injuries to cyclists. Overuse injuries have been intuitively linked to the anatomic structure of the foot because external loads are applied to the foot in cycling. Thus, the structure and function of the foot should dictate in part how the loads are transmitted to the knee joint. Therefore, it was hypothesized that patterns in knee loads are related to the anatomic structure of the foot. To test this hypothesis, peak knee loads (dependent variables) were related to anatomical variables (independent variables) through statistical analyses. This required first the detailed evaluation (i.e. measurement) of the anatomical structure of the foot and leg for 23 subjects. Next, three-dimensional knee joint loads were determined for a standardized riding condition. The results of the statistical analyses indicated that a group of cyclists with the most extreme inversion of the forefoot relative to the transverse plane developed significantly greater average posterior knee force and extensive knee moment. In addition, a number of anatomical variables significantly accounted for the variability in peak values of the posterior force, the extensive moment, the varus/valgus moment and the external axial moment. Based on these results, the hypothesis is accepted.


An explicit expression for the moment in multibody systems.

The equations of motion are formulated for a set of interacting rigid bodies. An explicit formulation could be derived for the total moment of the forces on such a system. This is applied to (1) the intersegmental moment in a multisegment rigid-body model, and (2) the interpretation of the 'centre of pressure' as defined in the studies of human posture.


Mechanical pulse wave propagation in gel, normal and oedematous tissues.

The velocity, attenuation and frequency content of the mechanical pulse wave propagation in gels of various water contents, in normal tissues from various sites and in oedematous tissues from different patients were investigated. The properties of the propagated pulse wave depend on the water content of the gel and the viscoelastic properties of the tissues. From the dependence of the pulse wave propagation velocity on elasticity, viscosity and density, information may be obtained concerning the effects of oedema on the mechanical properties of tissue.


SPACAR: a software subroutine package for simulation of the behavior of biomechanical systems.

Direct dynamics computer simulation is gaining importance as a research tool in the biomechanical study of complex human movements. Therefore, the need for general-purpose software packages with which the equations of motion can be derived automatically and solved numerically is growing. In this paper such a method is described: SPACAR. The method is compared to well-known commercially available software packages. On the basis of the results obtained on a test problem simulated with both SPACAR and DADS, it is concluded that both methods are accurate; DADS is much faster. The user-friendliness of SPACAR is less than that of DADS. However, SPACAR has two major advantages. First is the basic deformability of all elements, which allows handling of all kinds of problems within a unified framework; second is the full availability of the source code, which allows the experienced user to broaden the scope of possibilities to any extent.


Calibration characteristics of a video dimension analyser (VDA) system.

A comprehensive calibration and error analysis of a video dimension analyser (VDA) system was carried out to determine the effects of various parameters relevant to actual test conditions on the characteristics of the system. The parameters considered were: (1) varying distances or angles between the test specimen and the camera; (2) refractive effects of glass tank and physiologic solutions; and (3) dynamic response of the VDA system. The error analysis shows that, at low strain levels, the strain computed by the VDA is sensitive to camera placement and orientation as well as the media through which the object is observed. Very large errors can occur at small nominal strains, depending on the system parameters (e.g. 80% error at 1% nominal strain for one set of parameters). At higher strain levels, the sensitivity of the system is reduced, and the strain computed by the VDA system approaches the nominal strain (10% error at 10% nominal strain).


Ground reaction forces during termination of human gait.

This is the first published report of the ground reaction forces during gait termination. Two mechanisms appear to be used to stop walking: increased braking forces and decreased push-off force. There appears to be a short interval of time during the gait cycle in which a decision to take an additional step is to be made.


Step length and frequency effects on ground reaction forces during walking.

It is well established that the speed of walking or running significantly affects ground reaction force (GRF) characteristics. While it is sometimes assumed that the variations in step length (SL) and step frequency (SF) also affect GRF patterns, little documentation of this can be found in the literature. Ten young adults performed overground walking at 1.43 m s-1 across a force platform under five SL conditions: preferred SL and SLs that were longer and shorter than the preferred by 5 and 10% of greater trochanter height. The contact time, anteroposterior braking and propulsive force and impulse descriptors, and vertical impulse per step increased systematically as SL increased. In contrast, vertical peak forces and impulse per meter walked showed little change with SL manipulation. Despite the systematic effect of SL on several GRF descriptors, constraint of SL and SF in gait assessments is not recommended as this would prohibit the evaluation of representative gait kinematics and kinetics. Rather, these results suggest that researchers should report SL and SF data when comparing GRF characteristics between experimental groups or conditions, and should be alert to the association between SL/SF and GRF's when interpreting GRF trends.


Characterization of in vivo strain in the rat tibia during external application of a four-point bending load.

This paper describes a technique for characterizing strains and stresses induced in vivo in the rat tibia during application of an external four-point bending load. An external load was applied through the muscle and soft tissue with a four-point bending device, to induce strain in a 11 mm section of the right tibiae of ten adult female Sprague-Dawley rats. Induced strains were measured in vivo on the lateral surface of the tibia. Inter-rat difference, leg positioning and strain gage placement were evaluated as sources of variability of applied strains. Beam bending theory was used to predict externally induced in vivo strains. Finite element analysis was used to quantify the magnitude of shear stresses induced by this type of loading. There was a linear relationship between applied load and induced in vivo strains. In vivo strains induced by external loading were linearly correlated (R2 = 0.87) with the strains calculated using beam bending theory. The finite element analysis predicted shear stresses at less than 10% of the longitudinal stresses resulting from four-point bending. Strains predicted along the tibia by finite element analysis and beam bending theory were well-correlated. Inter-rat variability due to tibia size and shape difference was the most important source of variation in induced strain (CV = 21.6%). Leg positioning was less important (CV = 9.5%).


Biomechanics of the finger with anatomical restrictions--the significance for the exercising hand of the musician.

Exercise and teaching of musicians presupposes in the individual the constitutive ability to freely execute the finger movements required in the playing of the instrument. However, in the hand anatomical restrictions may exist that limit the mobility of the fingers and, thereby, the possibility to determine their movements voluntarily. In this article we investigate the kinematics of a monodigital system in which restrictions are present.


Coordination of the leg muscles in backlift and leglift.

Net joint moments are often used to quantify the loading of structures (e.g. the intervertebral disc at L5S1) during lifting. This quantification method is also used to evaluate the loading of the knee, for instance, to determine the effect of backlifting as opposed to leglifting. However, the true loading of the joint as derived from net joint moments can be obscured by a possible co-contraction of antagonists. To unravel the mechanisms that determine the net joint moments in the knee, the leglift was compared to the backlift. Although a completely different net knee moment curve was found when comparing the two lifting techniques, it appeared to be closely related to the ground reaction force vector and its orientation with respect to the joint centre of rotation (R > 0.995). This close relation was established by co-contraction of both flexors and extensors of the knee. Furthermore, a close relation appeared to exist between the joint moment difference between hip and knee and the activity difference between rectus femoris muscle and hamstring (R = 0.72 and 0.83 in leglift and backlift, respectively). The knee-ankle joint moment difference and the activity of the gastrocnemius showed a close relation as well (R = -0.89 and 0.96 in leglift and backlift, respectively). These relations can be interpreted as a mechanism to distribute net moments across joints. It is concluded that during lifting tasks the intermuscular coordination is aimed at coupling of joint moments, such that the ground reaction force points in a direction that provides balance during the movement. The use of net joint moments as direct indicators for joint loading (e.g. knee) seems, therefore, questionable.


A kinematic and strain gauge study of the reciprocal apparatus in the equine hind limb.

Hind limb kinematics were recorded in five horses at walk and trot using an opto-electronic CODA-3 system. Simultaneously, in vivo strain in the completely tendinous peroneus tertius muscle was registered by implanted mercury-in-silastic strain gauges. The origin-insertion length patterns of the peroneus tertius were calculated from raw kinematic data and from data corrected for the error caused by skin displacement, and compared with the directly measured strain. The strain patterns calculated from externally measured kinematic data appeared to be in accordance with the directly measured strain gauge data. However, a correction for skin displacement is an obligatory prerequisite to obtain reliable results. The amplitudes of strain did not exceed 3% and appeared to be of about the same magnitude at both walk and trot.


Relation between diameter and flow in major branches of the arch of the aorta.

In the analysis of arterial branching the classical "cube law' has provided a working model for the relation between the diameter of a blood vessel and the flow which the vessel carries on a long-term basis. The law has shown good agreement with biological data, but questions remain regarding its applicability to all levels of the arterial tree. The present study tests the hypothesis that the cube law may not be valid in the first few generations of the arterial tree, where vessel capacitance and gross anatomy may play important roles. Biological data have shown some support for this hypothesis in the past but the heterogeneity characteristic of past data has not allowed a conclusive test so far. We present new data which have been obtained from the same location on the arterial tree and in sufficient number to make this test possible for the first time. Also, while past tests have been based primarily on correlation of the measured data with an assumed power law, we show here that this can be misleading. The present data allow a simpler test which does not involve correlation and which leads to more direct conclusions. For the vessels surveyed, the results show unequivocally that the relation between diameter and flow is governed by a 'square law' rather than the classical cube law. Coupled with past findings this suggests that the square law may apply at the first few levels of the arterial tree, while the cube law continues from there to perhaps the precapillary levels.


On the relationship between the microstructure of bone and its mechanical stiffness.

A recent study of bone structure shows that the plate-shaped carbonate apatite crystals in individual lamellae are arranged in layers across the lamellae, and that the orientation of these layers are different in alternate lamellae. Based on these findings, a new micromechanical model for the Young's modulus of bone is proposed, which accounts for the anisotropy and geometrical characteristics of the material. The model incorporates the platelet-like geometry of the basic reinforcing unit, the presence of alternating thin and thick lamellae, and the orientations of the crystal platelets in the lamellae. The thin and thick lamellae are modeled as orthotropic composite layers made up of thin rectangular apatite platelets within a collagen matrix, and classical orthotropic elasticity theory is used to calculate the Young's modulus of the lamellae. Bone is viewed as an assembly of such orthotropic lamellae bent into cylindrical structures, and having a constant, alternating angle between successive lamellae. The micromechanical model employs a modified rule-of-mixtures to account for the two types of lamellae. The model provides a curve similar to the published experimental data on the angular dependence of Young's modulus, including a local maximum at an angle between 0 and 90 degrees. A rigorous testing of the model awaits additional experimental data.


Changes in the microstructure of cultured porcine aortic endothelial cells in the early stage after applying a fluid-imposed shear stress.

Time course changes in the cell shape and in the patterns of microfilament distribution were analyzed quantitatively using cultured porcine aortic endothelial cell monolayers before and after a shear flow exposure. Geometrical parameters of the cell and of the microfilament were measured on fluorescent photomicrographs of the cells stained with rhodamine-phalloidin. After the shear flow exposure (20 dyn cm-2, 0-24 h), the endothelial cells on glass were elongated and oriented to the direction of the flow. Under the no-flow condition, F-actin filaments were mainly localized at the periphery of the cell, although some filaments were seen in the more central portion. The angles of the filaments were randomly distributed. After 3 h, the stress fiber-like structure of an F-actin bundle was formed in the central part of the cells, and these filaments were oriented to the direction of the flow. The degree of orientation increased as the time of exposure to shear stress became longer. This change in F-actin preceded cell elongation and orientation; these changes were statistically significant only after 6 h. After 24 h, peripheral filaments were again observed, and the fluorescence intensity of rhodamine-phalloidin-stained cells was enhanced. These findings suggest that the redistribution of F-actin filaments is one of the early cellular responses to the onset of shear stress and that it is one of the most important factors controlling cell elongation and orientation to the direction of the flow.


Force-length properties and functional demands of cat gastrocnemius, soleus and plantaris muscles.

The purpose of this study was to measure isometric force-length properties of cat soleus, gastrocnemius and plantaris muscle-tendon units, and to relate these properties to the functional demands of these muscles during everyday locomotor activities. Isometric force-length properties were determined using an in situ preparation, where forces were measured using buckle-type tendon transducers, and muscle-tendon unit lengths were quantified through ankle and knee joint configurations. Functional demands of the muscles were assessed using direct muscle force measurements in freely moving animals. Force-length properties and functional demands were determined for soleus, gastrocnemius and plantaris muscles simultaneously in each animal. The results suggest that isometric force-length properties of cat soleus, gastrocnemius and plantaris muscles, as well as the region of the force-length relation that is used during everyday locomotor tasks, match the functional demands.


Theoretical analysis of pressure pulse propagation in arterial vessels.

An original mathematical model of viscous fluid motion in a tapered and distensible tube is presented. The model equations are deduced by assuming a two-dimensional flow and taking into account the nonlinear terms in the fluid motion equations, as well as the nonlinear deformation of the tube wall. One distinctive feature of the model is the formal integration with respect to the radial coordinate of the Navier-Stokes equations by power series expansion. The consequent computational frame allows an easy, accurate evaluation of the effects produced by changing the values of all physical and geometrical tube parameters. The model is employed to study the propagation along an arterial vessel of a pressure pulse produced by a single flow pulse applied at the proximal vessel extremity. In particular, the effects of the natural taper angle of the arterial wall on pulse propagation are investigated. The simulation results show that tapering considerably influences wave attenuation but not wave velocity. The substantially different behavior of pulse propagation, depending upon whether it travels towards the distal extremity or in the opposite direction, is observed: natural tapering causes a continuous increase in the pulse amplitude as it moves towards the distal extremity; on the contrary, the reflected pulse, running in the opposite direction, is greatly damped. For a vessel with physical and geometrical properties similar to those of a canine femoral artery and 0.1 degree taper angle, the forward amplification is about 0.9 m-1 and the backward attenuation is 1.4 m-1, so that the overall tapering effect gives a remarkably damped pressure response. For a natural taper angle of 0.14 degrees the perturbation is almost extinct when the pulse wave returns to the proximal extremity.


Biomechanical properties of human lumbar spine ligaments.

Biomechanical properties of the six major lumbar spine ligaments were determined from 38 fresh human cadaveric subjects for direct incorporation into mathematical and finite element models. Anterior and posterior longitudinal ligaments, joint capsules, ligamentum flavum, interspinous, and supraspinous ligaments were evaluated. Using the results from in situ isolation tests, individual force-deflection responses from 132 samples were transformed with a normalization procedure into mean force-deflection properties to describe the nonlinear characteristics. Ligament responses based on the mechanical characteristics as well as anatomical considerations, were grouped into T12-L2, L2-L4, and L4-S1 levels maintaining individuality and nonlinearity. A total of 18 data curves are presented. Geometrical measurements of original length and cross-sectional area for these six major ligaments were determined using cryomicrotomy techniques. Derived parameters including failure stress and strain were computed using the strength and geometry information. These properties for the lumbar spinal ligaments which are based on identical definitions used in mechanical testing and geometrical assay will permit more realistic and consistent inputs for analytical models.


Evaluation of frontal radiographs of scoliotic spines--Part I. Measurement of position and orientation of vertebrae and assessment of clinical shape parameters.

A new method largely exploiting the shape information which may be obtained from frontal radiographs of scoliotic patients is presented. For a complete description of spinal deformity, six position parameters are needed for each vertebra. From a strictly mathematical point of view, none of them can be determined from a single standard radiograph. However, the four most important parameters can be measured if some reasonable assumptions are made. For a better interpretation, three of these parameters (lateral coordinate x, lateral tilt alpha and axial rotation rho) are plotted as a function of the fourth parameter, the longitudinal coordinate y. These functions may well be approximated by sinusoidal curves (or possibly by Fourier series). The data smoothing implied by this procedure improves the reliability of the data. The method has been tested with 478 radiographs of 113 patients (Cobb angles up to 52 degrees). The results are compared with scoliosis parameters which have been determined according to the conventional clinical rules. A particular advantage of approximation by a sinusoidal function lies in the direct relation of the curve parameters to common scoliosis parameters. Moreover, a mathematical analysis of the interrelations between different parameters--for example, between lateral deviation and axial rotation--is possible in this case.


A comparison between the inlet and outlet diameters of the normal aortic valve.

Two studies are described comparing the inlet and outlet diameters of the normal aortic valve. Both studies show the valve inlet to be smaller than the valve outlet. The first study is of measurements made on 12 casts of physiologically pressurised human aortic valves. The mean ratio between the diameter of the aortic ring and of the aorta just distal to the sinus ridge was 1.1, and the mean ratio between the diameter of the aortic ring and the maximum diameter of the valve leaflets was 1.18. The second study presents echocardiographic data from normal volunteers. The mean ratio between the diameter of the aortic ring and of the aorta just distal to the sinus ridge was 1.17. It is suggested that stents made to support the leaflets of prosthetic valves are made in conical or hyperbolic form, with the outlet being approximately 20% larger than the inlet.


Early and late loosening of the acetabular cup after low-friction arthroplasty.

Between 1971 and 1979, 680 low-friction arthroplasties of the hip were performed in 598 patients. The average duration of follow-up was twelve years and eight months. Sixty-one acetabular cups had loosening as seen on roentgenograms eighteen years postoperatively, resulting in a total cumulative probability of loosening of 19 per cent, according to survivorship analysis. In twenty-nine cups, the loosening appeared within ten years after the operation (early loosening) and in thirty-two, more than ten years after the operation (late loosening). Early loosening was associated with deficient structure of the bone of the acetabulum, a previous congenital dislocation of the hip, acetabular fracture, or acetabular protrusion in all instances (p < 0.01). Late loosening was associated with the depth of acetabular wear. Of the thirty-two cups that had more than two millimeters of wear, eighteen (56 per cent) had loosening on the roentgenograms (p < 0.001). In hips that had early loosening, migration was the most frequent finding, and its rate of progression was higher than in hips that had late loosening (p < 0.001). In late loosening, a complete bone-cement radiolucency of more than two millimeters was the most frequent finding. Clinical failure was seen in twenty-two (76 per cent) of the twenty-nine cups that loosened early and in nine (28 per cent) of the thirty-two cups that loosened late. The probability of extensive resorption of bone necessitates close observation of patients who have early loosening, while a reasonable period of observation is possible for those who have late loosening.


The Harris-Galante porous-coated acetabular component with screw fixation. Radiographic analysis of eighty-three primary hip replacements at a minimum of five years.

The results of eighty-three consecutive primary total hip arthroplasties in which a Harris-Galante porous-coated acetabular component had been used were reviewed after a minimum of five years. In all patients, the stated diameter of the acetabular component (the diameter printed on the packaging for the implant) used was equal to the stated diameter of the reamer (the diameter printed on the reamer) that had been used last in the preparation of the acetabulum. As there was little or no press-fit stability, stability was obtained initially with multiple transfixing screws. No component was revised because of loosening, and none were radiographically loose at an average of sixty-eight months and a maximum of seven years after the operation. There was no evidence of disruption of the titanium porous mesh, and no screw had bent or broken. Two sockets, however, had been revised because of failure of the liner-locking mechanism as well as disassociation of the polyethylene liner from the titanium-alloy shell. Lysis of bone occurred in only one patient, around one screw. Areas of non-contact (gaps) between the porous mesh at the periphery of the acetabular component and the bone were seen on the immediate postoperative radiographs of nearly half of the patients. New areas of radiolucency, which had not been seen immediately postoperatively, were identified at two years in forty-nine hips. These radiolucent lines were never wider than one millimeter and were most frequently located in zone 3 and, less frequently, in zone 1. At the time of the most recent follow-up evaluation, a progressive radiolucent line was identified around twenty-two components and a discontinuous radiolucent line was present in all three zones around eleven components. No continuous radiolucent line was identified at the mesh-bone interface of any component. These results are superior to our results with cemented acetabular components after a similar period of follow-up. A longer period of follow-up is needed before the importance of these thin radiolucent lines can be determined, but experience with cemented acetabular components indicates that progressive or extensive radiolucent lines, or both, may represent resorption of bone at the porous mesh-bone interface and this can lead to loosening of the component. Our data suggest that the technique used for implantation may be important not only for the initial fixation and ingrowth of bone, but also for the long-term durability of the fixation of a porous-coated acetabular component.(ABSTRACT TRUNCATED AT 400 WORDS)


Prognostic factors in congenital dislocation of the hip treated with closed reduction. The importance of arthrographic evaluation.

We reviewed the clinical records, arthrograms, and roentgenograms of sixty-one children who had seventy-two congenitally dislocated hips in an effort to identify factors that can be used to predict the outcome of treatment. Only patients who had been followed clinically and roentgenographically for a minimum of two years after the initial closed reduction were included in the study. The mean age at the time of closed reduction was thirteen months (range, three to forty-one months). The mean age at the time of the most recent follow-up was six years (range, two to fifteen years). The mean duration of follow-up after the initial closed reduction was five years (range, two to thirteen years). There was no statistical difference between the good, fair, and poor-result groups with regard to sex, the age at the initial reduction, the traction station, the side of involvement, the initial acetabular index, the initial grade of displacement, the effect of adductor tenotomy, and several of the arthrographic measurements. The medialization ratio (the percentage of the horizontal radius of the cartilaginous femoral head that lay medial to the Perkins line), measured at the time of the reduction, was significantly different (p < 0.04) between the hips for which the result was good or fair and those for which the result was poor. The medialization ratio averaged 75 per cent in the hips for which the result was good, 66 per cent in those for which the result was fair, and 57 per cent in those for which the result was poor. Limbus shapes 5 through 8 were associated with avascular necrosis (p < 0.05) and a poor result (p < 0.03).


An intraosseous device for studies of bone-healing. The effect of transforming growth-factor beta.

A novel implantable device, the analytic bone implant, was used in order to establish a model for studies of bone-healing and the evaluation of factors that augment the process, such as transforming growth-factor beta (TGF-beta). This device was implanted into the tibiae of four baboons. After healing, bone was removed from the center chamber. Recombinant human TGF beta-1 was then delivered to the core of the device. After twenty-two days of healing, the device was disassembled and the newly formed bone was removed from the core of the implant for histomorphometric analysis. An analysis of the bone revealed a substantial effect of TGF-beta on osteoblastic activity and proliferation compared with that seen in control and placebo groups. However, despite increased osteoblastic activity, trabecular bone volumes at twenty-two days were equivalent among the groups. The number of osteoclasts and the erosion of the surface were also increased, although not significantly so. Substantial endochondral formation of bone was seen in the supraperiosteal tissues directly over the implants that contained TGF-beta but not over the implants in the control and placebo groups. These data demonstrate the utility of this bone-implant model for studies of bone-healing with minimally invasive methods. In addition, use of the device provided the first in vivo data on the effects of TGF-beta at an intermediate (twenty-two-day) time-point in the healing process in a non-human primate.


Treatment of open fractures of the tibial shaft with the use of interlocking nailing without reaming.

Fifty open fractures of the tibial shaft that were treated with débridement and interlocking nailing without reaming were followed for an average of twelve months. Most of the fractures were the result of high-energy trauma, and 68 per cent of the fracture wounds were grade III. Forty-eight (96 per cent) of the fifty fractures united at an average of seven months; there were no malunions. There were four infections (8 per cent), all at the sites of grade-III fractures. Locking screws broke in five tibiae (10 per cent), but the breakage did not result in a loss of reduction. Three nails broke, two at the sites of ununited fractures and one at the site of a healed fracture. These results are comparable with, or better than, those obtained with other forms of fixation, including immobilization with a cast, unlocked intramedullary nailing, and external fixation.


Continued growth of the proximal part of the tibia after prosthetic reconstruction of the skeletally immature knee. Estimation of the minimum growth force in vivo in humans.

We studied five skeletally immature patients who had a cemented endoprosthetic replacement involving the proximal part of the tibia because of a malignant tumor. In each patient, the cement-column fractured, allowing additional physeal growth. With plain radiographs and scanograms, we determined the cross-sectional areas of the physes, the cement-mantle, and the tibial component. Using the known tensile strength of polymethylmethacrylate cement, we then calculated the minimum force that the growth plates must have overcome to fracture the cement. This averaged 584 newtons per square centimeter. This observation of continued tibial growth after partial physeal ablation with a cemented prosthesis in skeletally immature patients presented a unique opportunity to estimate the force generated in the human physis during growth.


Palsy of the deep peroneal nerve after proximal tibial osteotomy. An anatomical study.

Iatrogenic, isolated weakness or paralysis of the extensor hallucis longus muscle is a common complication in patients who have had a proximal tibial and fibular osteotomy. To investigate why this complication occurs, we dissected the deep peroneal nerve and neighboring structures, such as the tibia and fibula and the muscles of the leg, in twenty-nine specimens from cadavera, paying special attention to the motor branches supplying the extensor hallucis longus. Of forty-six motor nerves that were identified, eight entered the muscle from the lateral side in an area seventy to 150 millimeters distal to the fibular head; all of them ran close to the fibular periosteum. We suggest that, in some patients, the nerve supply to the extensor hallucis longus is at high risk for injury during a tibial osteotomy because of the proximity of the bone to the motor branches.


Lateral meniscal variant with absence of the posterior coronary ligament.

We reviewed the cases of 3468 patients who had had arthroscopy of the knee between January 1976 and December 1988. Twenty-six patients (0.8 per cent) had a partial or a complete discoid lateral meniscus, and seven (0.2 per cent) had the Wrisberg-variant-type lateral meniscus. Of the seven patients, six had operative stabilization of the meniscus and one had a partial lateral meniscectomy because of an irreparable complex tear. Subjective, objective, and radiographic evaluations were performed on the patients who had had stabilization of the meniscus. According to the scale of Tegner and Lysholm, the result was excellent in four patients, good in one, and fair in one. None of the six patients had a tear of the sutured meniscus after an average follow-up of thirty-two months (range, twenty-four to forty months). Two patients had progressive symptoms attributable to osteoarthrosis, but the changes in the articular cartilage had been present at the time of the index procedure. None of the seven Wrisberg-variant-type menisci demonstrated a true discoid shape of the meniscus. We therefore classified this lesion as a lateral meniscal variant with absence of the posterior coronary ligament.


Revision of ankle arthrodesis with external fixation for non-union.

We evaluated the cases of twenty-six patients (twenty-six ankles) who had had revision of an ankle arthrodesis with external fixation for a nonunion, to determine the reasons for the failure of the previous arthrodesis. Eighteen patients had had supplemental bone-grafting in addition to the external fixation. The failure of the previous arthrodesis was related to inadequate fixation technique in seven patients and to technical problems in two patients; in the other seventeen patients at least one risk factor was identified. We also determined the functional results of the revision operation with external fixation for all patients. The average duration of follow-up was five years (range, two to ten years) in the twenty-two patients who did not have a reoperation for a persistent nonunion. The results were excellent in eleven patients, good in five, fair in four, and poor in six. The over-all rate of union was twenty (77 per cent) of twenty-six, comparable with that after primary arthrodesis; however, supplemental bone-grafting is usually necessary. In the current series, rigid fixation, precise apposition of bone and alignment of the foot, and early treatment of perioperative infection gave satisfactory results.


Assessment of the posterior malleolus as a restraint to posterior subluxation of the ankle.

We assessed the function of the posterior malleolus, the anterior tibiofibular ligament, and the fibula with regard to posterior stability of the talus in ten ankles of cadavera. Posteriorly directed loads of as much as 200 newtons were applied. Two groups of ankles were tested; in the first group, three ankles in which the ligamentous and osseous structures were intact were tested after transection of the posterior capsule and after removal of 10, 20, 30, and 40 per cent of the articular surface of the distal end of the tibia from the posterolateral corner. In the second group, seven ankles were tested in the same sequence, but the anterior tibiofibular ligament and the fibula were transected before sectioning of the articular surface. Compared with the results for the intact ankle, the experiments on the first group demonstrated less than one millimeter of additional posterior translation of the talus after removal of as much as 40 per cent of the articular surface. In the second group, in which the anterior tibiofibular ligament and the fibula had been transected, significant posterior translation of the talus (more than three millimeters) occurred after removal of 30 per cent of the articular surface (p < 0.01). This represented a 160 per cent increase in translation compared with that in the intact ankle.


Long-term results after Russe bone-grafting: the effect of malunion of the scaphoid.

Twenty-five patients had Russe anterior corticocancellous bone-grafting between 1973 and 1984 for twenty-six symptomatic established non-unions of the scaphoid. The mean duration of follow-up was eleven years (range, seven to eighteen years). Twenty-one (81 per cent) of the twenty-six scaphoid bones united. We developed two rating scales to evaluate the results of the operation. One scale, based on objective findings, included the radiographic appearance of the wrist, the range of motion, and strength; the other scale, based on subjective findings, comprised function, pain, perception of a decrease in performance because of limitation of motion or strength, and satisfaction. These scales were used to compare the objective and subjective results in patients who had a malunion of the scaphoid in which the lateral intrascaphoid angle was more than 45 degrees convex dorsally between the proximal and distal poles (a so-called flexion or humpback deformity, which results in extension of the proximal fragment of the scaphoid at the radiocarpal joint) with the results in patients who had no such deformity. The lateral intrascaphoid angle was more than 45 degrees in thirteen (50 per cent) of the twenty-six wrists. Although the difference in the objective results between the wrists that had a malunion and those that did not have a malunion was highly significant (p = 0.001), there was no significant difference in the subjective results between the two groups, including satisfaction of the patient (p = 0.39). Twenty-three patients (92 per cent) returned to full-time employment and twenty-two (88 per cent), to sports activities. Twenty-three patients (92 per cent) reported that they had pronounced relief of pain and that the procedure had improved their quality of life. The presence of this deformity of the scaphoid after bone-grafting for a symptomatic non-union was not predictive of a poor long-term subjective outcome.


Anterior capsulotomy and continuous passive motion in the treatment of post-traumatic flexion contracture of the elbow. A prospective study.

Thirty-three patients who had a post-traumatic flexion contracture of the elbow were managed consecutively with anterior capsulotomy without tenotomy of the biceps tendon or myotomy of the brachialis muscle. The first fifteen patients (Group I) did not receive continuous passive motion postoperatively. Preoperative active extension for Group I was to an average of 48 degrees short of full extension, which improved to 19 degrees at a mean follow-up time of forty-five months. Subsequently, eighteen patients (Group II) received continuous passive motion postoperatively for a mean of six weeks. Preoperative active extension for Group II was to an average of 55 degrees short of full extension, which improved to 23 degrees at a mean duration of follow-up of thirty-five months. The mean preoperative arc of motion for Group I was 69 degrees, which improved to 94 degrees postoperatively. The mean preoperative arc of motion for Group II was 48 degrees, which improved to 95 degrees postoperatively. Five patients in Group I and six patients in Group II had severe preoperative heterotopic ossification. There was no correlation, however, between preoperative heterotopic ossification and the amount that extension of the elbow improved postoperatively. There was no postoperative increase in heterotopic ossification. Four patients in Group I and six patients in Group II had severe post-traumatic osteoarthrosis preoperatively. Anterior capsulotomy is an effective treatment of post-traumatic flexion contracture of the elbow. Although the postoperative use of continuous passive motion did not significantly improve mean active extension, it did improve active flexion and the total arc of motion.


Ligamentous reconstruction for posterolateral rotatory instability of the elbow.

Eleven consecutively seen patients who had posterolateral rotatory instability of the elbow joint were managed operatively. The radial collateral-ligament complex was advanced and imbricated in three of them. In seven patients, the ulnar band of the radial collateral ligament (the lateral ulnar collateral ligament) was reconstructed with the palmaris longus tendon and in two of the seven, the reconstruction was augmented with a prosthetic ligament. The ligament was reconstructed with the lateral one-third of the triceps fascia in the remaining patient. Stability was obtained in ten patients, and seven patients had an excellent functional result. There was one failure in one of the patients in whom the ulnar band of the radial collateral ligament had been reconstructed with the palmaris longus tendon and augmented with a prosthetic ligament.


The fate of traumatic anterior dislocation of the shoulder in children.

The cases of twenty-one patients who had open physes and were treated for radiographic evidence of traumatic anterior dislocation of the shoulder at either the Adelaide Childrens Hospital or The Hospital for Sick Children, Toronto, during a fifteen-year period, were reviewed. All twenty-one patients had had one or more recurrent dislocations. Treatment, which included immobilization in a sling and swathe for as long as six weeks, had no effect on the rate of recurrence.


False-negative biopsy for testicular intraepithelial neoplasia.

A routine biopsy of the contralateral testis obtained during orchiectomy for embryonal carcinoma in a 26-year-old patient was negative for testicular intraepithelial neoplasia (TIN; carcinoma in situ of the testis). However, a rebiopsy that was taken because of unexplained elevation of alpha-fetoprotein 15 months later proved to be positive for TIN. Six previously reported cases of false-negative testicular biopsies obtained during a search for TIN are reviewed. In the light of several thousands of biopsies performed world-wide to date, the number of false-negative biopsies is probably very low. Although TIN is obviously not randomly dispersed throughout the testis in all patients, a routine biopsy of the contralateral testicle in patients with testis cancer remains a valuable tool for early detection of bilateral testicular tumors.-cal distribution of TIN in testes removed for this lesion. Their results suggested that after puberty TIN is usually randomly dispersed throughout the testicle. Support for this concept was recently given by Mumperow et al. (1992). These authors examined tumor-bearing testes and they did not find differences in the presence of TIN in biopsies taken from a location close to the tumor and taken from a location distant from the tumor. Thus, one single biopsy is regard to be representative for the entire testis and one biopsy taken after puberty is also assumed to be reliable for predicting whether the testis will ever develop cancer (Berthelsen and Skakkebaek 1981 a). Conversely, if the biopsy is negative for TIN, a future tumor manifestation in the testicle examined is not expected according to this theory (Skakkebaek et al. 1987). Taken together, the concept of TIN would constitute an ideal avenue for the early detection of testis cancer in high-risk populations with the biopsy being a safe means of discriminating between individuals who will or who will not develop testis cancer.


Radiation sensitivities in various anticancer-drug-resistant human lung cancer cell lines and mechanism of radiation cross-resistance in a cisplatin-resistant cell line.

To determine whether there exists cross-resistance between anticancer drugs and radiation, six drug-resistant human lung cancer cell lines and their parental cell lines were examined for radiosensitivity using a growth-inhibition assay. Only one cisplatin-resistant cell line, PC-9/CDDP, showed cross-resistance to radiation. The other three cisplatin-resistant cell lines (PC-7/CDDP, PC-4/CDDP, and H69/CDDP), an etoposide-resistant cell line (H69/VP) and a camptothecin-resistant cell line (PC-7/CPT) did not show cross-resistance to radiation. To analyze the mechanism of radiation resistance in PC-9/CDDP cells, the formation and repair of radiation-induced DNA single-strand breaks (ssb) and double-strand breaks (dsb) were examined by alkaline elution and neutral elution respectively. Although the formation of DNA ssb and repair of both DNA ssb and DNA dsb were the same for both cell lines, the formation of DNA dsb in PC-9/CDDP cells was significantly less than those in PC-9 cells. Measurement of intracellular glutathione content in all of the cell lines revealed that only PC-9/CDDP cells had a significant increase of glutathione content compared to the parental cells. Buthionine sulfoximine treatment of PC-9/CDDP cells caused an increase of DNA dsb to the same levels as in PC-9 cells after irradiation and caused a complete radiosensitization. These results indicate that cross-resistance to radiation in drug-resistant cells in a rare phenomenon, and increased glutathione content may play a crucial role in the emergence of cross-resistance to radiation in the drug-resistant cells.


Establishment and characterization of a new human oestradiol- and progesterone-receptor-positive mammary carcinoma serially transplantable in nude mice.

A human mammary carcinoma originating from a postmenopausal patient was successfully transplanted into nude mice. According to the adopted criteria the tumour proved to be oestradiol- and progesterone-receptor-positive. Histological studies of the patient tumour revealed a ductal invasive mammary carcinoma with 80% tubular growth pattern. Following transplantation the adenoid structures decreased to 30%; the mitosis rate and grade of malignancy increased. Treatment of the nude mice with 20 micrograms oestradiol benzoate/mouse caused a loss of the oestradiol receptor of the mammary carcinoma. The mammary carcinoma 3366 can be used for testing of antineoplastic substances, antihormones and for studies in regard to down-regulation or blocking of hormone receptors and possible consequences for therapies.


Toxicity associated with the formation and clearance of immune complexes between antitumour monoclonal antibodies and syngeneic anti-idiotypic antibodies in mice.

We have previously shown that Balb/c mice immunised against syngeneic monoclonal antibodies (mAbs), so that they have anti-idiotypic responses against those mAbs, will clear the mAbs from the circulation as a result of immune complex formation. We report here that with three separate mAbs, this clearance of complexes can result in toxicity to the animals. This was particularly severe with one mAb (NCRC-2), in some cases leading to death. It was not seen with Fab/c or Fab fragments of NCRC-2. This toxic response could be passively transferred with serum, indicating that it was due to formation of immune complexes or to their subsequent clearance. Treatment of mice with cobra venom factor, to reduce complement levels, reduced clearance of complexes but had no effect on toxicity. The anti-histamine pyrilamine did not reduce toxicity. This is one of only a few situations in which an anti-(mouse antibody) response has been reported to be potentially dangerous, and is particularly remarkable since it occurs as a result of such a limited anti-antibody response.


Suppression of human nasopharyngeal carcinoma cell growth in nude mice by the wild-type p53 gene.

Wild-type and mutant human p53 genes were transfected into the nasopharyngeal carcinoma (NPC) cell line CNE-3. Tumorigenicity in nude mice showed that the tumor resulting from the cells transfected with the wild-type p53 gene grew more slowly and was smaller than that from the cells transfected with mutant p53 gene and that from control CNE-3 cells. In contrast, the tumor from the cells transfected with the mutant p53 gene grew faster than that produced by cells transfected with the wild-type p53 gene and that produced by control CNE-3 cells. The results demonstrate that the wild-type p53 gene could inhibit the NPC cell growth in nude mice and the mutant p53 gene could enhance the NPC cell growth in nude mice. The p53 gene may also play an important role in the pathogenesis of NPC.


Argyrophilic nucleolar organizer regions in human adrenocortical neoplasms.

Argyrophilic nucleolar organizer regions (AgNOR) in human adrenocortical neoplasms, including five carcinomas and ten adenomas, were studied using a semi-automatic image analyzer. Both the number and total area of AgNOR per nucleus in the carcinomas were found to be statistically greater than in adenomas and control tissues. However, there were no statistically significant differences in total AgNOR area per nuclear area or in the mean area of individual AgNOR dots. The AgNOR of neoplastic and normal cells were of four morphological types: type 1 had a few dots at the periphery of the nucleus, type 2 a few dots at the center, type 3 a large round dot along with several small ones at the center, and type 4 numerous diffusely distributed polymorphic dots. Most type 3 and 4 cells were found in carcinoma cases. Type 1 cells decreased in proportion to the severity of biological malignancy. It follows from these findings that careful observation of AgNOR should facilitate the distinction of malignant from benign adrenocortical neoplasms.


Phase 2 trial of chronic low-dose oral etoposide as salvage therapy of platinum-refractory ovarian cancer.

Eighteen previously treated patients with advanced ovarian cancer were entered into a phase 2 trial of chronic low-dose oral etoposide (50 mg/day for 20 days, repeated every 28 days) to determine the activity of this therapeutic strategy in organoplatinum-refractory disease. The treatment program was generally well tolerated, with mild neutropenia the most common side-effect encountered. One patient (6%; 95% confidence interval = 0-17%) achieved a partial response, which lasted for 11 months. Three additional patients (17%), who failed to meet the criteria of a partial response, demonstrated objective evidence of antineoplastic activity. Chronic low-dose oral etoposide administration is associated with definite, although modest, activity in platinum-refractory ovarian cancer.


Calcitonin therapy of aneurysmal bone cysts.

Seven aneurysmal bone cysts (ABC) were treated with the hormone calcitonin. Six of the cysts, which were hypovascular responded well to the calcitonin administered directly into the cyst. Ossification and rebuilding of the ABC occurred after some months in every case. One hypervascularized ABC, however, failed to respond either to embolo-therapy or to the calcitonin hormone treatment. The authors recommend calcitonin administration as a useful non-invasive method for the treatment of hypovascular ABC.


The cytoplasmic tail of the mannose 6-phosphate/insulin-like growth factor-II receptor has two signals for lysosomal enzyme sorting in the Golgi.

The mannose 6-phosphate/insulin-like growth factor-II (Man-6-P/IGF-II) receptor is known to cycle between the Golgi, endosomes, and the plasma membrane. In the Golgi the receptor binds newly synthesized lysosomal enzymes and transports them directly to an endosomal (prelysosomal) compartment without traversing the plasma membrane. Deletion of the carboxyl-terminal Leu-Leu-His-Val residues of the 163 amino acid cytoplasmic tail of the bovine Man-6-P/IGF-II receptor partially impaired this function, resulting in the diversion of a portion of the receptor-ligand complexes to the cell surface, where they were endocytosed. The same phenotype was observed when 134 residues of the cytoplasmic tail were deleted from the carboxyl terminus. Disruption of the Tyr24-Lys-Tyr-Ser-Lys-Val29 plasma membrane internalization signal alone had little effect on Golgi sorting, but when combined with either deletion resulted in a complete loss of this function. The mutant receptors retained the ability to recycle to the Golgi and bind cathepsin D. These results indicate that the cytoplasmic tail of the Man-6-P/IGF-II receptor contains two signals that contribute to Golgi sorting, presumably by interacting with the Golgi clathrin-coated pit adaptor proteins. The Leu-Leu-containing sequence represents a novel motif for mediating interaction with Golgi adaptor proteins.


Effects of brefeldin A on endocytosis, transcytosis and transport to the Golgi complex in polarized MDCK cells.

We have studied the effects of brefeldin A (BFA) on endocytosis and intracellular traffic in polarized MDCK cells by using the galactose-binding protein toxin ricin as a membrane marker and HRP as a marker of fluid phase transport. We found that BFA treatment rapidly increased apical endocytosis of both ricin and HRP, whereas basolateral endocytosis was unaffected, as was endocytosis of HRP in the poorly polarized carcinoma cell lines HEp-2 and T47D. Tubular endosomes were induced by BFA both apically and basolaterally in some MDCK cells, comparable with those seen in HEp-2 and T47D cells. In addition, in MDCK cells, BFA induced formation of small (< 300 nm) vesicles, labeled both after apical and basolateral uptake of HRP, as well as some very large (> 700 nm) vacuoles, which were only labeled when HRP was present in the apical medium. In contrast, neither in MDCK nor in HEp-2 or T47D cells, did BFA have any effect on lysosomal morphology. Moreover, transcytosis in the basolateral-apical direction was stimulated both for HRP and ricin. Other vesicular transport routes were less affected or unaffected by BFA treatment. Two closely related structural analogues of BFA (B16 and B21), unable to produce the changes in Golgi and endosomal morphology seen after BFA treatment in a number of different cell lines, were also unable to mimic the effects of BFA on MDCK cells.


Primary central nervous system lymphomas--an update.

Primary CNS lymphomas (PCNSL), until recently representing about 1% of all brain tumors, show dramatically increased incidence both in high-risk groups (immunocompromised, AIDS) and in the general population. They are extranodal diffuse non-Hodgkin's lymphomas, the morphology and classification of which are identical to those of systemic lymphomas, although PCNSL show different biological behavior and diagnosis according to the New Working Formulation and updated Kiel classification may be difficult. The majority are large B cell variants of high-grade malignancy; low-grade subtypes and T cell lymphomas are rare. Sixty per cent occur in the supratentorial space (hemispheres, periventricular) and 12% in the posterior fossa; 30% are multiple (50%-70% in AIDS). PCNSL show a male preponderance with a peak incidence in the 5th-7th decade (3rd-4th in AIDS). The duration of diffuse or focal clinical symptoms averages 1-2 months. Computed tomography and magnetic resonance imaging scans show single or multiple or diffuse, often typical lesions. Diagnosis is achieved by evaluation of stereotactic biopsy material or cerebrospinal fluid cytology using immunocytological markers. Current therapy in immunocompetent patients, radiation plus corticosteroids and pre- or postradiation polychemotherapy, shows response rates of 85% with a median survival of 17-44 months, a prognosis similar to that for glioblastoma. Meningeal PCNSL is treated with intrathecal methotrexate or cytosine arabinoside. Transliquoral seeding of PCNSL is frequent, distant metastases occurring in 6%-8%. Therapy of AIDS-related PCNSL makes use of radiation and corticosteroids, and rarely of chemotherapy. The pathogenesis of PCNSL is unknown, but Epstein-Barr virus may be a contributory factor.


The morphology but not the function of endosomes and lysosomes is altered by brefeldin A.

Brefeldin A (BFA) induces the formation of an extensively fused network of membranes derived from the trans-Golgi network (TGN) and early endosomes (EE). We describe in detail here the unaffected passage of endocytosed material through the fused TGN/EE compartments to lysosomes in BFA-treated cells. We also confirmed that BFA caused the formation of tubular lysosomes, although the kinetics and extent of tubulation varied greatly between different cell types. The BFA-induced tubular lysosomes were often seen to form simple networks. Formation of tubular lysosomes was microtubule-mediated and energy-dependent; interestingly, however, maintenance of the tubulated lysosomes only required microtubules and was insensitive to energy poisons. Upon removal of BFA, the tubular lysosomes rapidly recovered in an energy-dependent process. In most cell types examined, the extensive TGN/EE network is ephemeral, eventually collapsing into a compact cluster of tubulo-vesicular membranes in a process that precedes the formation of tubular lysosomes. However, in primary bovine testicular cells, the BFA-induced TGN/EE network was remarkably stable (for > 12 h). During this time, the TGN/EE network coexisted with tubular lysosomes, however, the two compartments remained completely separate. These results show that BFA has multiple, profound effects on the morphology of various compartments of the endosome-lysosome system. In spite of these changes, endocytic traffic can continue through the altered compartments suggesting that transport occurs through noncoated vesicles or through vesicles that are insensitive to BFA.


Aminopeptidase I of Saccharomyces cerevisiae is localized to the vacuole independent of the secretory pathway.

The Saccharomyces cerevisiae APE1 gene product, aminopeptidase I (API), is a soluble hydrolase that has been shown to be localized to the vacuole. API lacks a standard signal sequence and contains an unusual amino-terminal propeptide. We have examined the biosynthesis of API in order to elucidate the mechanism of its delivery to the vacuole. API is synthesized as an inactive precursor that is matured in a PEP4-dependent manner. The half-time for processing is approximately 45 min. The API precursor remains in the cytoplasm after synthesis and does not enter the secretory pathway. The precursor does not receive glycosyl modifications, and removal of its propeptide occurs in a sec-independent manner. Neither the precursor nor mature form of API are secreted into the extracellular fraction in vps mutants or upon overproduction, two additional characteristics of soluble vacuolar proteins that transit through the secretory pathway. Overproduction of API results in both an increase in the half-time of processing and the stable accumulation of precursor protein. These results suggest that API enters the vacuole by a posttranslational process not used by most previously studied resident vacuolar proteins and will be a useful model protein to analyze this alternative mechanism of vacuolar localization.


Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction.

For determination of the physiological role and mechanism of vacuolar proteolysis in the yeast Saccharomyces cerevisiae, mutant cells lacking proteinase A, B, and carboxypeptidase Y were transferred from a nutrient medium to a synthetic medium devoid of various nutrients and morphological changes of their vacuoles were investigated. After incubation for 1 h in nutrient-deficient media, a few spherical bodies appeared in the vacuoles and moved actively by Brownian movement. These bodies gradually increased in number and after 3 h they filled the vacuoles almost completely. During their accumulation, the volume of the vacuolar compartment also increased. Electron microscopic examination showed that these bodies were surrounded by a unit membrane which appeared thinner than any other intracellular membrane. The contents of the bodies were morphologically indistinguishable from the cytosol; these bodies contained cytoplasmic ribosomes, RER, mitochondria, lipid granules and glycogen granules, and the density of the cytoplasmic ribosomes in the bodies was almost the same as that of ribosomes in the cytosol. The diameter of the bodies ranged from 400 to 900 nm. Vacuoles that had accumulated these bodies were prepared by a modification of the method of Ohsumi and Anraku (Ohsumi, Y., and Y. Anraku. 1981. J. Biol. Chem. 256:2079-2082). The isolated vacuoles contained ribosomes and showed latent activity of the cytosolic enzyme glucose-6-phosphate dehydrogenase. These results suggest that these bodies sequestered the cytosol in the vacuoles. We named these spherical bodies "autophagic bodies." Accumulation of autophagic bodies in the vacuoles was induced not only by nitrogen starvation, but also by depletion of nutrients such as carbon and single amino acids that caused cessation of the cell cycle. Genetic analysis revealed that the accumulation of autophagic bodies in the vacuoles was the result of lack of the PRB1 product proteinase B, and disruption of the PRB1 gene confirmed this result. In the presence of PMSF, wild-type cells accumulated autophagic bodies in the vacuoles under nutrient-deficient conditions in the same manner as did multiple protease-deficient mutants or cells with a disrupted PRB1 gene. As the autophagic bodies disappeared rapidly after removal of PMSF from cultures of normal cells, they must be an intermediate in the normal autophagic process. This is the first report that nutrient-deficient conditions induce extensive autophagic degradation of cytosolic components in the vacuoles of yeast cells.


Immunocytochemical localization of alpha-protein kinase C in rat pancreatic beta-cells during glucose-induced insulin secretion.

To investigate the role of protein kinase C (PKC) in the regulation of insulin secretion, we visualized changes in the intracellular localization of alpha-PKC in fixed beta-cells from both isolated rat pancreatic islets and the pancreas of awake unstressed rats during glucose-induced insulin secretion. Isolated, perifused rat islets were fixed in 4% paraformaldehyde, detergent permeabilized, and labeled with a mAb specific for alpha-PKC. The labeling was visualized by confocal immunofluorescent microscopy. In isolated rat pancreatic islets perifused with 2.75 mM glucose, alpha-PKC immunostaining was primarily cytoplasmic in distribution throughout the beta-cells. In islets stimulated with 20 mM glucose, there was a significant redistribution of alpha-PKC to the cell periphery. This glucose-induced redistribution was abolished when either mannoheptulose, an inhibitor of glucose metabolism, or nitrendipine, an inhibitor of calcium influx, were added to the perifusate. We also examined changes in the intracellular distribution of alpha-PKC in the beta-cells of awake, unstressed rats that were given an intravenous infusion of glucose. Immunocytochemical analysis of pancreatic sections from these rats demonstrated a glucose-induced translocation of alpha-PKC to the cell periphery of the beta-cells. These results demonstrate that the metabolism of glucose can induce the redistribution of alpha-PKC to the cell periphery of beta-cells, both in isolated islets and in the intact animal, and suggest that alpha-PKC plays a role in mediating glucose-induced insulin secretion.


Structural changes and lateral redistribution of photosystem II during donor side photoinhibition of thylakoids.

The structural and topological stability of thylakoid components under photoinhibitory conditions (4,500 microE.m-2.s-1 white light) was studied on Mn depleted thylakoids isolated from spinach leaves. After various exposures to photoinhibitory light, the chlorophyll-protein complexes of both photosystems I and II were separated by sucrose gradient centrifugation and analysed by Western blotting, using a set of polyclonals raised against various apoproteins of the photosynthetic apparatus. A series of events occurring during donor side photoinhibition are described for photosystem II, including: (a) lowering of the oligomerization state of the photosystem II core; (b) cleavage of 32-kD protein D1 at specific sites; (c) dissociation of chlorophyll-protein CP43 from the photosystem II core; and (d) migration of damaged photosystem II components from the grana to the stroma lamellae. A tentative scheme for the succession of these events is illustrated. Some effects of photoinhibition on photosystem I are also reported involving dissociation of antenna chlorophyll-proteins LHCI from the photosystem I reaction center.


The cytoskeleton and the cellular traffic of the progesterone receptor.

Previous studies on glucocorticoid receptors have suggested the existence of interactions between the receptor and microtubule or actin networks. It was hypothesized that such interactions may contribute to the guidance of steroid hormone receptors towards the nucleus. We used a permanent L cell line expressing the delta 638-642 progesterone receptor. This mutant has all the characteristics of the wild type receptor except that the deletion of five amino acids inactivates the constitutive karyophilic signal. Consequently, the receptor is cytoplasmic in the absence of hormone but is shifted into the nucleus when administration of hormone activates the second karyophilic signal. Optical microscopy and confocal laser microscopy were used in intact cells or in cells depleted of soluble elements by permeabilization with detergents. By immunofluorescence, the receptor was found to be mainly concentrated in the perinuclear area. A small fraction of progesterone receptor (PR) persisted in this region after Triton X100 treatment. These observations suggested that the receptor could interact with some insoluble constituent(s) of the cytoplasm. However, careful colocalization studies showed that this heterogenous distribution was not due to interactions with microtubule, microfilament, or intermediate filament networks. Functional involvement of these networks in the translocation of the receptor into the nucleus was studied after cell treatment with cytoskeletal drugs such as nocodazole, demecolcine and cytochalasin. None of these compounds prevented or even delayed the hormone-dependent transfer of delta 638-642 PR into the nucleus. Similar conclusions were reached with the wild type receptor expressed by transfection in Cos-7 cells. PR was shifted from the nucleus into the cytoplasm by administration of energy-depleting drugs. After disruption of the various cytoskeletal networks normal nuclear reaccumulation of the receptor was observed when these drugs were removed. The results thus suggest that the progesterone receptor is not colocalized with the main cytoskeletal components. Disruption of the cytoskeletal networks does not prevent its nuclear translocation. Thus, karyophilic signals and interactions with the nuclear pore seem to be the primary determinants of the cellular traffic of the progesterone receptor.


The subcellular distribution of chromosome 6-encoded dystrophin-related protein in the brain.

Chromosome 6-encoded dystrophin-related-protein (DRP) shows significant structural similarities to dystrophin at the carboxyl terminus, though the two proteins are encoded on different chromosomes. Both DRP and dystrophin are expressed in muscle and brain and show some similarity in their subcellular localization. For example, in skeletal muscle both are expressed at neuromuscular and myotendinous junctions. However, while dystrophin is absent or severely reduced in Duchenne/Becker muscular dystrophy, DRP continues to be expressed. Within the brain, dystrophin is enriched at the postsynaptic regions of specific subsets of neurons, while the distribution of DRP is yet to be described. In this study we demonstrate a distinct though highly specific pattern of distribution of DRP in the brain. DRP is enriched in the choroid plexus, pia mater, intracerebral vasculature, and ependymal lining. Within the parenchyma proper, DRP is located at the inner plasma face of astrocytic foot processes at the abluminal aspect of the blood-brain barrier. The distribution of DRP is conserved across a large evolutionary distance, from mammals to elasmobranchs, suggesting that DRP may play a role in the maintenance of regional specializations in the brain.


Comparison of actin and cell surface dynamics in motile fibroblasts.

We have investigated the dynamic behavior of actin in fibroblast lamellipodia using photoactivation of fluorescence. Activated regions of caged resorufin (CR)-labeled actin in lamellipodia of IMR 90 and MC7 3T3 fibroblasts were observed to move centripetally over time. Thus in these cells, actin filaments move centripetally relative to the substrate. Rates were characteristic for each cell type; 0.66 +/- 0.27 microns/min in IMR 90 and 0.36 +/- 0.16 microns/min in MC7 3T3 cells. In neither case was there any correlation between the rate of actin movement and the rate of lamellipodial protrusion. The half-life of the activated CR-actin filaments was approximately 1 min in IMR 90 lamellipodia, and approximately 3 min in MC7 3T3 lamellipodia. Thus continuous filament turnover accompanies centripetal movement. In both cell types, the length of time required for a section of the actin meshwork to traverse the lamellipodium was several times longer than the filament half-life. The dynamic behavior of the dorsal surface of the cell was also observed by tracking lectin-coated beads on the surface and phase-dense features within lamellipodia of MC7 3T3 cells. The movement of these dorsal features occurred at rates approximately three times faster than the rate of movement of the underlying bulk actin cytoskeleton, even when measured in the same individual cells. Thus the transport of these dorsal features must occur by some mechanism other than simple attachment to the moving bulk actin cytoskeleton.


Astral microtubules are not required for anaphase B in Saccharomyces cerevisiae.

tub2-401 is a cold-sensitive allele of TUB2, the sole gene encoding beta-tubulin in the yeast, Saccharomyces cerevisiae. At 18 degrees C, tub2-401 cells are able to assemble spindle microtubules but lack astral microtubules. Under these conditions, movement of the spindle to the bud neck is blocked. However, spindle elongation and chromosome separation are unimpeded and occur entirely within the mother cell. Subsequent cytokinesis produces one cell with two nuclei and one cell without a nucleus. The anucleate daughter can not bud. The binucleate daughter proceeds through another cell cycle to produce a cell with four nuclei and another anucleate cell. With additional time in the cold, the number of nuclei in the nucleated cells continues to increase and the percentage of anucleate cells in the population rises. The results indicate that astral microtubules are needed to position the spindle in the bud neck but are not required for spindle elongation at anaphase B. In addition, cell cycle progression does not depend on the location or orientation of the spindle.