Search

The purpose of this study was to investigate age differences in remote pointing movements. The subjects were recruited from three age groups (ages 18-22 yr., 40-50 yr., and 60-70 yr., with 9 men and 9 women in each group). They were required to perform cursor-positioning tasks using a remote pointing device, in which the dependent measures were the time taken to reposition the cursor and the accuracy of subjects' movement trajectories. The movement time was further separated into two components, First Submovement duration and Adjustment Submovement duration. Analysis indicated that age groups showed reduced performance on remote pointing. Moreover, remote positioning movement for the young-adult group was mostly completed in their First Submovement phase, while the elderly subjects spent most of their movement time on the Adjustment Submovement phase. These results support the proposition that different age groups exhibit different kinds of movement patterns.

Splicing of mRNA precursors occurs in a massive structure known as the spliceosome and requires the function of several small nuclear RNAs (snRNAs). A number of studies have suggested potentially important roles for two snRNAs, U2 and U6, in splicing catalysis. These two RNAs interact extensively with each other, as well as with the pre-mRNA, and possible similarities with catalytic RNAs have been noted. An important feature of the U2-U6 complex is an intramolecular helix in U6, which forms in conjunction with activation of the spliceosome. Here we describe a detailed genetic analysis of residues that make up this helix in human U6 snRNA, using an in vivo assay in which splicing of a test pre-mRNA is dependent on exogenous U6 snRNA. Our results show that many, but not all, positions tested are sensitive to mutation. Unexpectedly, base pairing is fully compatible with function at all positions, and at many is both necessary and sufficient. For example, conversion of two noncanonical A-C pairs to G-C pairs did not affect splicing, nor did conversion of an A-G to C-G. Extension of the helix by a base pair was also tolerated, provided that base pairing was maintained. Most notable was the behavior of a bulged U (U74), which has been suggested previously to be of particular importance. Although U74 was sensitive to substitution or deletion, incorporation into the helix by insertion of an A across from it was without effect, even in the context of a second helix-stabilizing mutation. We discuss these results in terms of possible mechanisms by which U6 snRNA might function in splicing catalysis.

Recent studies have suggested that 3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione (beta-lapachone) inhibits DNA topoisomerase I by a mechanism distinct from that of camptothecin. To study the mechanism of action of beta-lapachone, a series of beta-lapachone and related naphthoquinones were synthesized, and their activity against drug-sensitive and -resistant cell lines and purified human DNA topoisomerases as evaluated. Consistent with the previous report, beta-lapachone does not induce topoisomerase I-mediated DNA breaks. However, beta-lapachone and related naphthoquinones, like menadione, induce protein-linked DNA breaks in the presence of purified human DNA topoisomerase IIalpha. Poisoning of topoisomerase IIalpha by beta-lapachone and related naphthoquinones is independent of ATP and involves the formation of reversible cleavable complexes. The structural similarity between menadione, a para-quinone, and beta-lapachone, an ortho-quinone, together with their similar activity in poisoning topoisomerase IIalpha, suggests a common mechanism of action involving chemical reactivity of these quinones. Indeed, both quinones form adducts with mercaptoethanol, and beta-lapachone is 10-fold more reactive. There is an apparent correlation between the rates of the adduct formation with thiols and of the topoisomerase II-poisoning activity of the aforementioned quinones. In preliminary studies, beta-lapachone and related naphthoquinones are found to be cytotoxic against a panel of drug-sensitive and drug-resistant tumor cell lines, including MDR1-overexpressing cell lines, camptothecin-resistant cell lines, and the atypical multidrug-resistant CEM/V-1 cell line.

Background: Pyrost bone substitute has been shown to be a promising orthopedic biomaterial. However, little is known about mechanisms that are responsible for the genesis and development of the bond between bone and the Pyrost bone substitute. The purpose of this study is to elucidate the in vitro cell behavior of osteoblasts on Pyrost bone substitute., Methods: By using primary culture of rat osteoblasts, the changes in cell morphology during adhesion and flattening onto the surface of Pyrost bone substitute were studied in vitro. At 1 hour, at 3 hours, and at days 1, 3, and 7 after layering, the cell behavior was observed with scanning electron microscope., Results: The processes of trypsinized osteoblast adhesion and spreading on Pyrost bone substitute consisted of 1) contact of rounded osteoblasts with the Pyrost substrate; 2) attachment of osteoblasts at point of contact; 3) centrifugal growth of filopodia; 4) flattening and spreading of the osteoblasts on the Pyrost substrate; 5) division and growth of osteoblasts; and 6) suspension of the osteoblasts across the pores by their processes., Conclusions: These results demonstrated that Pyrost can form a physico-chemical bond with osteoblasts. The Pyrost bone substitute not only supports osteoblasts attachment but also allows proliferation of the osteoblasts.

With advances in ceramics technology, calcium phosphate bioceramics have been applied as bone substitutes. The effects of implants on bony tissue have been investigated. The effects upon adjacent skeletal muscles have not been determined. The focus of this work is to elucidate the biological effects of various calcium phosphate bioceramics on skeletal muscles. Four different kinds of powder of calcium phosphate biomaterials including beta-tricalcium phosphate (beta-TCP), hydroxyapatite (HA), beta-dicalcium pyrophosphate (beta-DCP) and sintered beta-dicalcium pyrophosphate (SDCP), were tested by myoblast cell cultures. The results were analyzed by cell count, cell morphology and concentration of transforming growth factor beta 1 (TGF-beta 1) in culture medium. The cell population and TGF-beta 1 concentration of the control sample increased persistently as the time of culture increased. The changes in cell population and TGF-beta 1 concentration in culture medium of the beta-TCP and HA were quite low in the first 3 days of culture, then increased gradually toward the seventh day. The changes in cell population and TGF-beta 1 concentration in culture medium of the silica, beta-DCP, and SDCP were quite similar. They were lower during the first day of culture but increased and reached that of the control medium after 7 days' culture. Most cells on B-TCP and HA diminished in size with radially spread, long pseudopods. We conclude that HA and beta-TCP are thought to have an inhibitory effect on growth of the myoblasts. The HA and beta-TCP may interfere with the repair and regeneration of injured skeletal muscle after orthopedic surgery.

The purpose of this study was to re-evaluate the bone regeneration power and the in vitro biocompatibility of the Pyrost bone substitute. Twenty-four adult New Zealand White rabbits were used. Bony defect over both iliac crest and mid-diaphyseal portion of the ulna bone were created. Appropriate sized-block of Pyrost bone substitute were implanted. Four of the animals were killed at each postoperative month to evaluate its bone regeneration power by histologic study. The Pyrost bones were co-cultured with osteoblasts to evaluate its biocompatibility. The results showed that Pyrost bone substitute was quite stable and incorporated well with active bone regeneration. The Pyrost heal better at the iliac crest than at the ulnar defect. The Pyrost was compatible to the osteoblasts. Osteoblasts had successfully seeded and mitotically expanded on the porous surface of the Pyrost bone graft. The result showed that Pyrost bone obviously exerts an intense stimulus on osteo-regeneration in the presence of osteoblasts. We consider Pyrost to be an alternate to the conventional preserved allografts that is occasionally necessary.

Oligonucleotide directed triple helix formation allows the sequence-specific recognition of the major groove of double-helical DNA. Recently synthesized base analogs and backbones, such as N3'-->P5' phosphoramidates, allow stable triplexes to be formed under physiological conditions. However, it remains a challenge to design new oligomers that would extend the range of recognition sequences (which are still limited to oligopurine-rich tracts). Oligonucleotide directed triple helix formation could be used to control biological processes such as transcription and replication. Three-stranded structures formed during recombination processes have been further characterized.

Autogenous bone transfer is an important part of reconstructive plastic surgery. Presently available techniques have the disadvantages of limitation of available donor site, loss of donor tissue and the possibility of donor defect or deformity. In the present study, a vascularized bone graft was created and cultured in the groin area of the New Zealand rabbit. The cylindrical ceramic chambers, 15 mm in length, 6 mm in outer diameter and 3 mm in inner diameter, were prepared by the addition of sintered porous beta-Ca2P2O7 with 5 wt% Na4P2O7.10H2O. In the first group, the chambers impregnated with autogenous bone fragments and allogenous demineralized bone matrix with volume ratio 1:1 were cultured in the rabbit's groin area with saphenous vessels passing through. In the second group, the chambers were treated by the same procedures as the first group but without saphenous vessels passing through. In the third group, the chambers were not impregnated, and were cultured in the groin area with saphenous vessels. After 2, 4, 6, 8 and 12 wk of operation, the animals were killed with an overdose of intravenous pentobarbital. The viability of the osseous tissue in the chamber was evaluated by histological examination, microangiograms and fluorochrome incorporation for the three groups. The autogenous bone chips could survive and retain their osteogenic properties while packed into the sintered porous beta-Ca2P2O7 (with 5 wt% Na4P2O7.10H2O addition) ceramic chamber and implanted in the rabbit groin area up to 12wk. However, even at the longest time periods, considerable amounts of dead bone were present in the chambers. In addition, we observed bone resorption in the three groups up to 12 wk, which might be attributed to lack of physiological stress. There were significant differences in new bone formation and osseous cell viability among the three groups. The prevascularized vessels and autogenous bone chips were both necessary for the formation of new bone and osteogenic property in the chamber under these heterotopic circumstances. The biodegradable ceramic used in this study was gradually absorbed and dissolved in the physiological environment. However, the degradation debris of the ceramic caused no injury to the new bone formation. These findings support the concept of creating a preformed vascularized bone graft to reconstruct segmental bone defects.

We have examined the effects of benzopyridoindole derivatives on triple helices with antiparallel third strands. Absorption spectroscopy, footprinting, and gel retardation experiments demonstrate that a benzopyridoindole derivative (BePI) is able to induce formation of a triple helix with an antiparallel (G, T)-containing third strand, which does not form in the absence of this ligand. This triple-helical complex is very stable with a half-dissociation temperature as high as 51 degrees C, and its formation is pH independent. Antiparallel oligonucleotides containing thymine and guanine bind strongly to double-helical DNA under physiological conditions in the presence of only 0.5 microM BePI. Formation of a BePI-stabilized triple helix strongly inhibits cleavage of the target duplex by DNase I.

Oligonucleotide analogs with N3'-->P5' phosphoramidate linkages bind to the major groove of double-helical DNA at specific oligopurine.oligopyrimidine sequences. These triple-helical complexes are much more stable than those formed by oligonucleotides with natural phosphodiester linkages. Oligonucleotide phosphoramidates containing thymine and cytosine or thymine, cytosine, and guanine bind strongly to the polypurine tract of human immunodeficiency virus proviral DNA under physiological conditions. Site-specific cleavage by the Dra I restriction enzyme at the 5' end of the polypurine sequence was inhibited by triplex formation. A eukaryotic transcription assay was used to investigate the effect of oligophosphoramidate binding to the polypurine tract sequence on transcription of the type 1 human immunodeficiency virus nef gene under the control of a cytomegalovirus promoter. An efficient arrest of RNA polymerase II was observed at the specific triplex site at submicromolar concentrations.

Little is known about the morphological response of muscle after long term traction. The purpose of this study was to investigate the morphological changes of skeletal muscle during limb lengthening. After application of mini-extraskeletal fixator, the hindlimb of New Zealand white rabbit was osteotomized and then slowly lengthened at the rate of 1 mm/day up to a 20 mm gain in length. The muscles of hindlimbs were perfused and dissected. Morphological studies were performed at electron microscopic level. Transmission electron microscopy revealed foci of microtrauma at the myotendinous junction. The distance between the muscle fibers and tendon parenchyma increased, with numerous primitive mesenchyme-like cells interposed within this gap. The cytoplasmic space of these cells was devoid of myofibril formation at the ends of stretched fibers. Within the satellite near the myotendinous junction myofilament production was observed in various gradations of maturation. It is concluded that myofibrillogenesis with traction neogenesis of skeletal muscle during limb lengthening does exist and occurs mainly near the myotendinous junction. The myotendinous junction in mature skeletal muscle actively participated in the process of limb lengthening.

Triple helix formation requires a polypurine- polypyrimidine sequence in the target DNA. Recent works have shown that this constraint can be circumvented by using alternate strand triplex-forming oligonucleotides. We have previously demonstrated that (T,G)-containing triplex- forming oligonucleotides may adopt a parallel or an antiparallel orientation with respect to an oligopurine target, depending upon the sequence and, in particular, upon the number of 5'-GpT-3' and 5'-TpG-3' steps [Sun et al. (1991) C.R. Acad. Sci. Paris Ser III, 313, 585-590]. A single (T,G)-containing oligonucleotide can therefore interact with two oligopurine stretches which alternate on the two strands of the target DNA. The (T,G) switch oligonucleotide contains a 5'-part targeted to one of the oligopurine sequences in a parallel orientation followed by a 3'-part that adopts an antiparallel orientation with respect to the second oligopurine sequence. We show that a limitation to the stability of such a triplex may arise from the instability of the antiparallel part, composed of reverse-Hoogsteen C.GxG and T.AxT base triplets. Using DNase I footprinting and ultraviolet absorption experiments, we report that a benzo[e]pyridoindole derivative [(3-methoxy- 7H-8-methyl-11-[(3'-amino-propyl) amino] benzo[e]pyrido [4,3-b]indole (BePI)], a drug interacting more tightly with a triplex than with a duplex DNA, strongly stabilizes triplexes with reverse-Hoogsteen C.GxG and T.AxT triplets thus allowing a stabilization of the triplex-forming switch (T,G) oligonucleotide on alternating oligopurine- oligopyrimidine 5'-(Pu)14(Py)14-3' duplex sequences. These results lead to an extension of the range of oligonucleotide sequences for alternate strand recognition of duplex DNA.

Introduction: Based on molecular modeling studies, a model has been proposed for intercalation of triple-helix-specific ligands (benzopyridoindole (BPI) derivatives) into triple helices, in which the intercalating compounds interact mainly with the Hoogsteen-paired strands of the triple helix. We set out to test this model experimentally using DNA duplexes capable of forming parallel Hoogsteen base-paired structures., Results: We have investigated the possible formation of a parallel DNA structure involving Hoogsteen hydrogen bonds by thermal denaturation, FTIR spectroscopy and gel-shift experiments. We show that BPI derivatives bind to Hoogsteen base-paired duplexes and stabilize them. The compounds induce a reorganization from a non-perfectly matched antiparallel Watson- Crick duplex into a perfectly matched parallel Hoogsteen-paired duplex., Conclusions: These results suggest that preferential intercalation of BPI derivatives in triple helices is due to their ability to interact specifically with the Hoogsteen-paired bases. The results are consistent with a model proposed on the basis of molecular modeling studies using energy minimization, and they open a new field of investigations regarding the biological relevance of Hoogsteen base-pairing.

Glomus tumours of the hand are benign tumours that arise from the neuromyoarterial apparatus. The most common site for a glomus tumour is in the distal phalanx. They frequently display a classic triad of symptoms-pain, tenderness and temperature sensitivity. Ten treated cases examined with magnetic resonance imaging (MRI) preoperatively are described. All were female with a mean age of 43 years. All except 2 suffered the classic triad of symptoms. The mean duration from onset of symptoms to therapeutic intervention was 7.4 years. MRI depicted the tumours in excellent detail and aided diagnosis in the 4 patients without subungual blue discolouration. The lesions showed a low signal on the T1-weighted image and a high signal on the T2-weighted image. All except 3 were located in the subungual region. All patients underwent surgical excision with pathological confirmation. The postoperative course was smooth and there were no sequelae. High resolution MRI, providing a clear contrast between tumorous and normal tissues, has proved to be a useful non-invasive method for imaging glomus tumours in the hand.

To elucidate the effect of nerve stimulation on the mechanical property of muscle-tendon unit, the triceps surae muscle of New Zealand White rabbit was tested with material testing machine. After anaesthesia, the triceps surae muscle-tendon unit of rabbit was tested with cyclic loading during nerve stimulation. The differences between initial and residual force disappeared in the presence of low amplitude and low frequency nerve stimulation. Loading energy applied to the muscle-tendon unit did not change, but the unloading energy increased and energy loss decreased significantly. However, the residual force appeared again at the tetanic stimulation; the loading energy, unloading energy and energy loss all increased significantly.

The specific cleavage of a DNA triple helix by FeII.bleomycin (BLM) is demonstrated. Triplex-specific cleavage was observed on both strands of the 32-base pair (bp) duplex at the duplex-triplex junctions. Strand scission products and alkali labile lesions were both formed. The strongest BLM cleavage site was located at the 5'-duplex-triplex junction, which is also the preferred triplex binding site of intercalating agents [Collier, D. A., Mergny, J.-L., Thuong, N. T., & Hélène, C. (1991) Nucleic Acids Res. 19, 4219-4224]. The preference of BLM for the 5'-junction does not appear to derive from selective intercalative binding at this site. This is supported by the observation that phleomycin, which contains a thiazolinylthiazole moiety rather than a planar bithiazole ring system, exhibited the same selectivity of triplex cleavage as BLM. Cleavage of the triple helix by FeII.BLM was unaffected by concentrations of Mg2+ up to 5 mM, suggesting possible therapeutic applications of this novel DNA target. Molecular-modeling calculations of the triplex region suggested that dramatic variations in minor groove width and depth occur at the duplex-triplex junctions, particularly at the 5'-junction. Moreover, the minor groove at these sites was calculated to be somewhat shallower and wider than the minor groove of B-DNA. These results suggest that the preference of BLM for the duplex-triplex junctions derives from selective recognition of minor groove shape at these sites and thus reflects conformation-selective, rather than sequence-selective, DNA recognition by FeII.BLM.

A new concept is presented to design and synthesize modified oligonucleotides in order to extend the range of double-helical DNA sequences that can be recognized by oligonucleotides via triple helix formation. The DNA target is composed of adjacent oligopurine.oligopyrimidine domains where the oligopurine sequences alternate on the two DNA strands. Canonical (C,T)-motif triple helices are formed with each oligopurine.oligopyrimidine domain of the target sequence. The two third-strand oligonucleotides were joined together via an appropriate linker between the two terminal bases with either a 3'-3' or a 5'-5' polarity. Molecular modeling was used to predict the optimal length of the linker bridging two terminal bases. The interaction of DNA with such a modified oligonucleotide containing a C3'-3'U linkage was studied by thermal dissociation, footprinting, and gel retardation experiments. They provide experimental evidence that the oligonucleotide does form a switched triple helix on this extended DNA target sequence. The binding of the so-called "switch oligonucleotide" is enhanced as compared to the two unlinked parental oligonucleotides which form triple helices with each oligopurine.oligopyrimidine domain of the target sequence.

To elucidate the strain effects of dynamic cyclic loading on the skeletal muscle-tendon unit, the muscle-tendon unit of New Zealand white rabbits were tested with different strains and strain rates under ketamine general anaesthesia. The results indicated that the dynamic responses of muscular tissues were both strain and strain-rate dependent with appreciably non-linear characteristic. The muscle-tendon unit behaved viscoelastically below 12% strain; but when stretching more than 16% strain it began to behave plastically. The loading energies and unloading energy are more dependent on strain than on strainrate. RELEVANCE: This study offers firm evidence that plasticity may be one of the important biomechanical properties of the skeletal muscle. The findings in this report imply that the expression of skeletal muscle mechanical behaviour may not be limited to phenomena represented by a composition of elastic and viscous elements. The elongation of skeletal muscle under sustained stretching might be explained by the plastic property. Elucidation of the existence of viscoplasticity in skeletal muscle gives a good reference point to determine the functional capabilities and limitation of muscular tissue.

The ultimate goal of implantation of biomaterials in the skeleton is to reach full integration of the non-living implant with the living bone. The biomaterial can be used much as a bone graft, resorbing or dissolving as bone growth occurs, and the end result is a new remoulded bone. Calcium pyrophosphate, Ca2P2O7, is one of the intermediate products of bone mineralization. beta-Dicalcium pyrophosphate (beta-DCP) doped with certain amounts of Na4P2O7.10H2O was prepared as the developed material. Na4P2O7.10H2O was used as a liquid-phase additive to improve the sintering process and promote physiological bioresorbability. Compressive strength and four-point bending strength were measured by the Bionix test system 858. The mechanical strength of the sintered beta-DCP increased with the addition of Na4P2O7.10H2O up to 5 wt%, but thereafter decreased. The microstructure and crystal structure were analysed by the techniques of SEM, EPMA, TEM and XRD. The relationship between the mechanical strength of the sintered bioceramics and the Na4P2O7.10H2O dopant was examined in terms of the presence of NaCa(PO3)3, grain growth and abnormal grain coalescence while the dopant increased. Preliminary in vivo evaluation was studied by rabbit femur condyle implantation. There was no inflammation or any toxic sign during the experimental period. The histological section of intraosseous implantation revealed that the new bone deposited directly on the surface of the material in the fourth week after operation. The implant gradually decreased in volume and was replaced by the surrounding regenerated bone in the rabbit condyle in vivo environment. The results led us to conclude that the developed material has great potential as a biodegradable bone substitute.

The hindlimb of New Zealand white rabbit was osteotomized and then slowly lengthened at a rate of 1 mm/day until a 2.0-cm gain in length was reached. The triceps surae muscle-tendon unit was then tested either with or without 2 months fixation for bony consolidation. After limb lengthening, the strain at peak load was decreased and the axial rigidity was increased significantly; but other biomechanical parameters such as peak load, maximal deformation at peak load, stiffness, and energy absorption before peak load did not show any significant difference. We conclude that healthy triceps surae muscle has a great potential to be lengthened despite the changes in intrinsic structural properties of the muscle. The change in the biomechanical properties occurred during the time of distraction and was not affected by the time of bony consolidation. RELEVANCE: As noted in this study, many biomechanical parameters of the triceps surae muscle were not affected after prolonged distraction. This fact suggested that the healthy muscle-tendon unit is not a main causative factor of joint contracture after limb lengthening. However, there were intrinsic changes in the structural properties of the triceps surae muscle. Equinus contracture after limb lengthening may be caused by the aggravated intrinsic structural changes of the injured or denervated scarred muscular tissue.

Splicing of mRNA precursors requires a complex and dynamic set of RNA-RNA base-pairing interactions in which the U2 and U6 snRNAs play central roles. Using a genetic suppression assay, we refine and extend a U2-U6 snRNA structure that may comprise the catalytic center of the spliceosome. We first show that a critical U2-U6 helix proven in yeast, helix Ia, is also essential for mammalian splicing. Mutations in the adjacent helix Ib, however, cannot be similarly suppressed, and relevant residues in both U2 and U6 are shown to participate in intramolecular, rather than intermolecular, base-pairing. We next demonstrate the requirement for a novel U2-U6 helix, helix III, which involves bases extending 3' from the branch site recognition sequence in U2 and 5' from an evolutionarily invariant sequence in U6 implicated previously in 5' splice site recognition. This configuration suggests that helix III may help juxtapose the pre-mRNA 5' splice site and branch site. We provide evidence for this by demonstrating that a branch site mutation can be suppressed by a mutation in the 5' splice site, provided that compensatory changes are made in the appropriate bases in U2 and U6. Our results provide new insights into how U2 and U6 snRNAs interact with each other and with the pre-mRNA to initiate the first catalytic step in splicing.

Molecular modeling of complexes between the octanucleotide d(CGATATCG)2 and either a monocationic or biscationic distamycin-ellipticine hybrid molecule predicted that the extra positive charge on the latter conjugate ligand should ensure tight fitting into the minor groove of the duplex without affecting intercalation of the ellipticine chromophore. To test this prediction, we have synthesized a biscationic compound Distel (2+) and investigated its interaction with DNA using various optical and gel electrophoresis techniques. Viscosity, fluorescence lifetime, and circular and linear dichroism measurements bear out the validity of the calculations and show that Distel (2+) does indeed come to lie with its distamycin moiety in the minor groove of DNA and its ellipticine ring intercalated nearby. Linear dichroism experiments with a range of polynucleotides indicate that, unlike its monocationic homologue, the biscationic ligand engages in bidentate binding to AT sequences but not to GC sequences. Footprinting studies employing DNase I and methidiumpropyl-EDTA.FeII as DNA cleaving agents reveal that the biscationic hybrid is notably selective for AT-rich sequences in DNA. The concentrations required to detect a clear footprint at AT sites with Distel (2+) are 4- to 10-fold lower than those required to produce comparable DNase I footprints with distamycin alone. Also, in accord with the energy-minimized model of the hybrid-oligonucleotide complex, chemical probing experiments using diethyl pyrocarbonate and osmium tetroxide reveal that the hybrid causes significant distortion of the DNA helix, explicable in terms of bending of the duplex toward the minor groove, which greatly enhances the reactivity toward probes in the major groove of the DNA. The experimental results help to identify the determinant factors, predominantly steric and electrostatic interactions, which shape the DNA-binding reaction. Thus, molecular modeling has correctly predicted the DNA-binding properties of a doubly charged ligand and shown that appending an auxiliary basic group onto the distamycin moiety was the right way to proceed in order to convert a nonspecific conjugate into a highly specific DNA reader.

After application of a modified Orthofix mini-extraskeletal fixator, the hind limb of New Zealand White rabbits was osteotomized and then slowly lengthened at a rate of 1 mm/day. After a 20 mm gain in length, the net weight and the length of muscular and tendinous portions were measured and histological examination was carried out in triceps surae muscles. Quantitative analysis showed a significant increase in the gained length of the muscular portion (28.05% to 30.65%). Histological studies of these lengthened muscles showed a generalized increase in cellularity with scanty inflammatory cell infiltration near the myotendinous junction. The increased cellularity is due to the presence of muscle precursor cells characterized by large, oval and pale-stained vesicular nuclei and two prominent nucleoli. The nuclei of these precursor cells were larger and more numerous near the myotendinous junction, and gradually changed into a flattened and more condensed form at a distance from the junction. Occasionally, chains of centrally-located nuclei of primitive myoblasts were also visible. It is concluded that traction neogenesis of the skeletal muscle during limb lengthening does exist and occurs mainly near the myotendinous junction.

Extrastriate visual areas on the banks of the middle suprasylvian sulcus were inactivated by cooling to assess the behavioral contribution of this cortical region to the extraction of a stationary figure from a moving mask. Cooling blocked figure-ground separation when the mask was moving but had no influence when the mask was static. This difference provides strong evidence that the areas bounding the middle suprasylvian sulcus contribute to the neural separation of stationary from moving visual stimuli.

The purpose of this study was to obtain quantitative measures of the influence of callosal input to cells at the area 17/18 border region where transcallosal axons terminate most densely. Single-cell recordings were performed at the area 17/18 border region of the right hemisphere, while gamma-aminobutyric acid (GABA) or its antagonist, bicuculline, were applied to the transcallosal projecting regions of the left hemisphere to either block or overactivate the cells which projected to the neurons at the recording site. The results showed that visually evoked responses of the cells at the area 17/18 border were affected by administration of GABA or bicuculline to the contralateral hemisphere. Blockade of transcallosal input by application of GABA in the left hemisphere diminished the visually evoked responses of 51% of the neurons in the right hemisphere, and led to an increase in response magnitude for 17% of the neurons. Disinhibition of transcallosal input by application of bicuculline increased the evoked activity of 40% of the neurons and diminished the response magnitude of 20% of the neurons in the right hemisphere. GABA and bicuculline failed to show antagonistic effects on some cells. Thirty-two percent of the cells were affected by only one type of drug administration, and 13% of the cells showed either an increase or a decrease in responses after both GABA and then bicuculline administration. This study demonstrated complex interactions between neurons connected by the transcallosal pathway. A model of the transcallosal circuitry was proposed to explain the results.

In order to further characterize the binding of the hybrid molecule NetOPC [bis(pyrrolecarboxamide)-oxazolopyridocarbazole conjugate] to double-stranded DNA, we have performed a molecular modeling study to investigate the binding modes of the complexes possibly formed between NetOPC and synthetic polynucleotides poly [(dA-dT)]2, poly [(dA).d(T)], and poly [d(G-C)]2 and interpreted the results in the light of the experimentally determined binding parameters. In agreement with experimental data, the modeling study suggests that whatever was the binding mode of the complex formed, the complexation energy is markedly lower (thus favorable) for AT-containing polynucleotides than for poly d[(G-C)]2. With both poly [d(A).d(T)] and poly [d(A-T)]2, the most energetically favored complex has netropsin and OPC moieties bound simultaneously in the minor groove of DNA. The second favored complex exhibits the bimodal binding, i.e., intercalation of OPC and minor groove binding of the netropsin moiety. For both types of complex, the energy of complex formation is slightly lower with poly [d(A).d(T)]. The binding site sizes of the modeled complexes are about seven and four base pairs to the full groove and bimodal binding, respectively.

To study the material properties of the skeletal muscle-tendion unit, the peroneus longus muscle of a rabbit, rubber and plastic materials were tested with on MTS machine. After anesthesia, the peroneus longus muscle of a rabbit was dissected out and then stretched to failure by an MTS Bionix 858 machine. The load-deformation curves of the muscle and non-biological materials were analyzed and compared. The results showed that the load-deformation curve of a muscle-tendon unit can be separated into 4 different portions: first, the viscoelastic portion, which corresponds to the physiological range of motion; second, the portion of microfailure; third, the portion of macrofailure; and fourth, the portion of rupture and separation of a muscle-tendon unit. The cause of these behaviors are explained by the presence of actinmyosin cross bridges. Elucidation of the existence of microfailure of skeletal muscle gives a good reference point to determine the functional capabilities and limitation of muscular tissue. This will be useful in determining the beneficial effect of stretch programs in sports and rehabilitation and preventing the potential impact of tissue injury.

The triceps surae muscle-tendon units of rabbits were tested in vivo by a passive extension technique using an MTS Bionix 858 machine. The effect of nerve function on the failure mechanism was evaluated. The sites of failure were always at the muscular portion near the musculotendinous (MT) junction. It always occurred within 0.1 to 1.0 mm of the distal MT junction of the soleus muscle and within 5 to 10 mm of the distal MT junction in the lateral head of the gastrocnemius muscle. In the medial head of the gastrocnemius muscle, it always occurred within 5 to 10 mm of the proximal MT junction. The loss of nerve function significantly reduced the peak force and the absorption of energy before the point of peak force, but it produced no significant changes in the failure length and the total energy absorption. The functioning nerve might have a protective mechanism which works on the muscle-tendon unit by increasing the peak force and the energy requirement before the muscle is injured.

Tritiated proline and leucine were injected into areas 17 and 18 of intact cats and into the medial bank of the lateral suprasylvian (LS) cortex of intact cats and cats from which areas 17 and 18 had been removed on postnatal day 1 (P1), P28, or in adulthood (A). The density of label transported to the superior colliculus was quantified using image-analysis equipment. The results from the intact cats confirmed previous reports that areas 17 and 18 project most heavily to stratum zonale (SZ) and stratum griseum superficiale (SGS) and LS cortex projects most heavily to stratum opticum (SO) of the superior colliculus. However, in cats with lesions of areas 17 and 18, the projections from LS cortex showed an age-dependent reorganization. LS projections to SGS and SZ were enhanced following ablation of areas 17 and 18 on P1, and projections to SGS were enhanced following an ablation on P28. The pattern of LS-collicular projection following ablations incurred in adulthood was indistinguishable from the pattern presented by intact cats. This study demonstrates that the LS corticocollicular projection expands in SGS and possibly substitutes for inputs eliminated by the removal of areas 17 and 18 from the immature brain. This enhanced pathway may contribute to compensatory neuronal changes and to spared behaviors that accompany damage of immature cortex.

UV-absorption spectrophotometry and molecular modeling have been used to study the influence of the chemical nature of sugars (ribose or deoxyribose) on triple helix stability. For the Pyrimidine.purine* Pyrimidine motif, all eight combinations were tested with each of the three strands composed of either DNA or RNA. The chemical nature of sugars has a dramatic influence on triple helix stability. For each double helix composition, a more stable triple helix was formed when the third strand was RNA rather than DNA. No stable triple helix was detected when the polypurine sequence was made of RNA with a third strand made of DNA. Energy minimization studies using the JUMNA program suggested that interactions between the 2'-hydroxyl group of the third strand and the phosphates of the polypurine strand play an important role in determining the relative stabilities of triple-helical structures in which the polypyrimidine third strand is oriented parallel to the polypurine sequence. These interactions are not allowed when the third strand adopts an antiparallel orientation with respect to the target polypurine sequence, as observed when the third strand contains G and A or G and T/U. We show by footprinting and gel retardation experiments that an oligoribonucleotide containing G and A or G and U fails to bind double helical DNA, while the corresponding DNA oligomers form stable triple-helical complexes.

Seventy-one consecutive patients were treated with a modified Steindler flexorplasty from 1970 to 1987. The mechanisms of injury and etiologies included 45 motor vehicle accidents, 13 birth injuries, and 13 cases of poliomyelitis. Additional operative procedures included shoulder fusion (45 patients), tendon transfer (20 patients), and wrist tenodesis (3). Follow-up averaged 8.2 years. The outcome was excellent in 32%, good in 47%, fair in 13%, and poor in 8%. Postoperatively, the mean arc of active elbow flexion was 114 degrees; the average elbow extension loss, 28 degrees; the mean active pronation, 74 degrees; and supination, 30 degrees. Wire breakage was found in two cases. Additional tendon transfer of flexor carpi ulnaris to extensor carpi radialis brevis improved the outcomes in the patients without active supination. The modified Steindler flexorplasty provided predictable functional improvement in carefully selected patients with paralyzed upper extremities.

A benzo[e]pyridoindole derivative, 3-methoxy-7H-8-methyl-11-[(3'-amino)propylamino] -benzo[e]pyrido[4,3-b]indole (BePI), and its interactions with double and triple-helical DNA have been investigated by a variety of fluorescence, spectrophotometric, hydrodynamic and molecular modeling techniques. Binding to DNA stabilizes the doubly charged (+2) form of BePI, increasing the apparent pKa of the 10-NH proton by approximately 1 pH unit. Binding to DNA also quenches the fluorescence of BePI, with a greater extent of quenching upon binding triplex relative to duplex DNA. BePI preferentially binds (and stabilizes) triple-helical relative to double-helical DNA. This preferential binding is not restricted to triplexes containing solely T x A.T base triplets. In addition, BePI preferentially stabilizes the poly(dA).poly(dT) relative to the poly[d(A-T)].poly[d(A-T)] duplex. Viscosity studies demonstrate that, upon binding, BePI induces the unwinding of negative supercoils in the pBR322 plasmid, and increases the relative contour lengths of double and triple-helical polydeoxynucleotides. Fluorescence studies reveal that energy transfer occurs from polynucleotide bases to bound BePI molecules in both BePI/duplex and BePI/triplex complexes. In a BePI/triplex complex, an average of 4.8 bases appear to transfer excitation energy totally to a bound BePI molecule, while in various BePI/duplex complexes an average of only 2.5 bases appear to do so, indicating that energy transfer is more efficient in the former complex. Measurements of fluorescence quenching indicate that BePI is protected from quenching by acrylamide when bound to either double or triple-helical polynucleotides. The viscosity and fluorescence behavior of BePI are fully consistent with the conclusion that BePI intercalates into both double and triple-helical DNA. Molecular modeling studies suggest that stronger stacking interactions between intercalated BePI and adjacent bases in BePI/triplex relative to BePI/duplex complexes may account for the enhanced thermal stability of the former complex.

Recent experimental studies of the structure of triple helices show that their conformation in solution differs from the A-like structure derived from diffraction data on triple helix fibers by Arnott and co-workers. Here we show by means of molecular modeling that a family of triple helix structures may exist with similar conformational energies, but with a variety of sugar puckers. The characteristics of these putative triple helices are analyzed for three different base sequences: (T.AxT)n, (C.GxC+)n, and alternating (C.GxC+/T.AxT)n. In the case of (C.GxC+)n triple helix, infrared and Raman spectra have been obtained and clearly reveal the existence of both N- and S-type sugars in solution. The molecular mechanics calculations allow us to propose a stereochemically reasonable model for this triple helix, in good agreement with the vibrational spectroscopy results.

Skeletal tuberculosis (TB) can present with a broad spectrum of variable clinical manifestations which may cause diagnostic difficulties. To further elucidate these possible diagnostic pitfalls, 14 cases of bacteriologically or histologically proven skeletal TB, which were initially diagnosed otherwise, are reviewed. In five cases with lumbar or midthoracic spinal involvement, concomitant traumatic history and degenerative radiological changes were the main reasons for not suspecting the possibility of skeletal TB. A differential diagnosis between TB and metastatic lesions can be difficult in the very early stage when X-ray images usually have a low contrast and are not sharp. In nine cases with bone and joint involvement, a long-lasting discharging sinus led the clinician to an initial impression of septic arthritis; sometimes concomitant gouty arthritis can mask the clinical manifestations of skeletal TB. In five cases, minor erosive lesions over the peripheral margin of the involved bone were the earliest findings on the plain X-ray films and were overlooked. To facilitate the early diagnosis of skeletal TB and to avoid the possibility of complications in the management of patients, medical practitioners should be familiar with the usual forms of the condition and the wide variety of clinical manifestations.

A bifunctional molecule in which an ellipticine chromophore is attached to a distamycin residue via a diaminopropyl tether has been designed and synthesized in the expectation of creating a hybrid molecule capable of bidentate binding to DNA by both intercalation and minor-groove interactions. The strength and mode of binding to DNA of this conjugate have been studied by means of circular and linear dichroism as well as by stopped-flow kinetics and measurements of reactivity toward a chemical probe. The results converge to reveal that the ellipticine moiety of the hybrid largely dominates the binding reaction with DNA. In the presence of chromatin, the hybrid molecule binds preferentially to the internucleosomal DNA, a preference dictated by its intercalating chromophore. Theoretical computations were performed on the comparative complexation energies of distamycin, the ellipticine derivative, and the hybrid ligand with a B-representative octanucleotide, d(GCATATGC)2. The best binding configuration of the ellipticine derivative locates its aminoalkyl side chain in the minor groove where distamycin is also present. The molecular modeling analysis fully supports the involvement of a bimodal binding process for the hybrid and reveals that the binding of the conjugate to DNA favors a pronounced bending toward the minor groove. This effect is attributed to intercalation of the ellipticine chromophore. An interesting link is established between the DEPC reactivity experiments and the theoretical computations, suggesting that DEPC can be used as a probe for drug-induced DNA bending. On the basis of these results, we propose the design of a new hybrid ligand bearing an additional positively-charged amidine side chain to confer higher DNA-binding affinity.

We propose a classification of DNA structures formed from 1 to 4 strands, based only on relative strand directions, base to strand orientation and base pairing geometries. This classification and its associated notation enable all nucleic acids to be grouped into structural families and bring to light possible structures which have not yet been observed experimentally. It also helps in understanding transitions between families and can assist in the design of multistrand structures.

Concentrations of extractable and total phosphorus in leaves, stem, root and nodules of 12-week-old seedlings of two provenances of Acacia mangium Willd. were analyzed to identify the fraction of phosphorus and the plant part most suitable for predicting the phosphorus nutritional status of the seedlings.For both provenances, concentrations of extractable phosphorus were more sensitive to changes in soil phosphorus status and varied less among different plant parts than concentrations of total phosphorus. Concentrations of extractable phosphorus in the youngest fully expanded leaf (Leaf 3 from the apex) and the next two older leaves correlated closely with seedling dry mass and may be used to assess the phosphorus nutritional status of Acacia mangium seedlings.

The aims of the present theoretical study of the conformations of [alpha]-oligodeoxynucleotides forming triple helices with DNA duplexes are to understand the structural and energetic factors involved in [alpha]-triple helix formation by means of energy minimization, and to explain the experimentally observed dependence of strand orientation on the nucleotide sequence. It is found that the energetically preferred orientation of the [alpha]-oligonucleotide with respect to the homopurine strand depends on the sequence of the homopurine.homopyrimidine tracts. This is a consequence of the structural heteromorphism of base triplets in the intrinsically more stable reverse Hoogsteen hydrogen bonding configuration. Practical rules are proposed for determining the orientation of the nuclease-resistant [alpha]-oligodeoxynucleotide strand which will form the most stable triple helix.

Early soft tissue reconstruction is recommended in treating open grade III fractures. Yet, there are still controversies regarding the way to handle bony defects when the soft tissue reconstruction is completed. This is a retrospective study to evaluate various methods of treating bony defects after soft tissue reconstruction in open fractures. From May 1985 to June 1988, we experienced 16 cases of open grade III fractures of the extremities with bony defects which received either local treatment with flap transfers (five cases) or free flap transfers (11 cases). The bony defects ranged from 3 cm to 10 cm in length. Cancellous bone grafting was performed in nine cases, and two of them received internal fixation at the same time. Seven cases received vascularized bone grafts (six fibulas and one iliac bone). Our results showed that bone grafting, with either a conventional cancellous bone graft or a vascularized bone graft, is useful in treating bony defects after soft tissue reconstruction. Internal fixation of the fractures after removing an extraskeletal fixator has the disadvantages of soft tissue breakdown and of possible infection. Early posterolateral bone grafting was beneficial if a cancellous bone graft was used on the tibia. Double free tissue transfers were quite useful, and we did not lose any free tissue transfers in our series. Vascularized bone grafts should be considered in cases where the osseous gap is greater than 6 cm, or where the recipient bed is bad.

Pyrimidine oligoribonucleotides bind to the major groove of double-helical DNA at homopurine.homopyrimidine sequences. They recognize Watson-Crick base pairs by forming T.A x U and C.G x C base triplets via Hoogsteen hydrogen bonding. The stability of these triple helices is much higher than that of triple helices formed by oligodeoxyribonucleotides as shown by an increase of the temperature at which half-dissociation of the third strand occurs. When the 2'-hydroxyl group of ribose moieties is replaced by 2'-O-methyl substituent, triple helix stability is further increased.

The contribution of different factors in the interaction of ethidium intercalated into various sequences of a triple helix, or in the region of the junction between the double- and triple-stranded DNA has been studied by energy minimization. It is found that in the total energy of the ethidium- triple helix complexes, a particular electrostatic contribution emerges due to the presence of protonated cytosines in the triple helix. This parameters is determinant in the sequence-specificity of ethidium binding to the triple helix. The preferred intercalation sites of ethidium in the triple helix are proposed. The interaction of ethidium at the triplex-duplex junction, and its effects are also discussed. This study is aimed at searching for new drugs specific for the triple helix, or for the triplex-duplex junctions.

Visual response properties of single neurons in the superior colliculus of golden hamsters could be altered by iontophoretically applied gamma-aminobutyric acid (GABA) and its antagonist, bicuculline (Bicu). GABA decreased the responses of the superficial cells to stationary flashing stimuli, while Bicu increased the responses and suppressed the inhibition exerted by the surround. The number of spikes evoked by a moving bar/spot decreased after applying GABA in 76.6% of the cells (n = 60) and increased in 90.0% (n = 60) of the cells after Bicu. Similar effects on the spontaneous activities were observed. In addition, 65.0% of the 60 cells recorded have enlarged movement receptive fields after application of Bicu. GABA and Bicu have some effects on the orientation selectivity of the collicular cells too.

By means of molecular modelling, electrostatic interactions are shown to play an important role in the sequence-dependent structure of triple helices formed by a homopyrimidine oligonucleotide bound to a homopurine. homopyrimidine sequence on DNA. This is caused by the presence of positive charges due to the protonation of cytosines in the Hoogsteen-bonded strand, required in order to form C.GxC+ triplets. Energetic and conformational characteristics of triple helices with different sequences are analyzed and discussed. The effects of duplex mismatches on the triple helix stability are investigated via thermal dissociation using UV absorption.

The specificity of a homopyrimidine oligonucleotide binding to a homopurine-homopyrimidine sequence on double-stranded DNA was investigated by both molecular modeling and thermal dissociation experiments. The presence of a single mismatched triplet at the center of the triplex was shown to destabilize the triple helix, leading to a lower melting temperature and a less favorable energy of interaction. A terminal mismatch was less destabilizing than a central mismatch. The extent of destabilization was shown to be dependent on the nature of the mismatch. Both single base-pair substitution and deletion in the duplex DNA target were investigated. When a homopurine stretch was interrupted by one thymine, guanine was the least destabilizing base on the third strand. However, G in the third strand did not discriminate between a C.G and an A.T base pair. If the stretch of purines was interrupted by a cytosine, the presence of pyrimidines (C or T) in the third strand yielded a less destabilizing effect than purines. This study shows that oligonucleotides forming triple helices can discriminate between duplex DNA sequences that differ by one base pair. It provides a basis for the choice of antigene oligonucleotide sequences targeted to selected sequences on duplex DNA.

The hypocrellin B (HB)-sensitized photodamage on Na(+)-K+ ATPase and sodium permeability of human erythrocytes by means of NMR and biochemical techniques was studied in this paper. The decrease of the enzyme activity and increase of intracellular sodium concentration were usually observed simultaneously. The evidences suggested that the integrality of membrane phospholipid played an important role in maintaining the physiological sodium content of erythrocytes. The loss of the enzyme activity was a sensitive index compared with the increase of intracellular Na+ concentration during the photosensitization. From the comparison tests among HB, HA, protoporphyrin and bilirubin, we found that HB had more ability to increasing intracellular Na+ concentration than the other photosensitization even though the photodamage on the enzyme activity caused by HB, HA, and protoporphyrin were nearly the same. Besides the photoinactivation of Na(+)-K+ ATPase induced by HB and light, the enzyme was also inactivated in the medium containing HB in absence of light. The active oxygen radicals generated though HB mediated redox-cycling might be involved in the dark inactivation of the enzyme.

Base-pair sequences in double-stranded DNA can be recognized by homopyrimidine oligonucleotides that bind to the major groove at homopurine.homopyrimidine sequences thereby forming a local triple helix. To make oligodeoxynucleotides resistant to nucleases, we replaced the natural (beta) anomers of the nucleotide units by the synthetic (alpha) anomers. The 11-mer alpha oligodeoxynucleotide 5'-d(TCTCCTCCTTT)-3' binds to the major groove of DNA in an antiparallel orientation with respect to the homopurine strand, whereas a beta oligonucleotide adopts a parallel orientation. When an intercalating agent was attached to the 3' end of the alpha oligodeoxynucleotide, a strong stabilization of the triple helix was observed. A 16-base-pair homopurine.homopyrimidine sequence of human immunodeficiency virus proviral DNA was chosen as a target for a 16-mer homopyrimidine alpha oligodeoxynucleotide. A restriction enzyme that cleaves DNA at the junction of the homopurine.homopyrimidine sequence was inhibited by triple-helix formation. The 16-mer alpha oligodeoxynucleotide substituted by an intercalating agent was approximately 20 times more efficient than the unsubstituted oligomer. Nuclease-resistant alpha oligodeoxynucleotides offer additional possibilities to control gene expression at the DNA level.

Oligodeoxynucleotides containing G and T can bind to homopurine.homopyrimidine sequences on double-stranded DNA by forming C.G x G and T.A x T base triplets. The orientation of the third strand in such triple helices depends on the number of GpT and TpG steps. Therefore a single oligonucleotide can be designed to bind to two consecutive homopurine.homopyrimidine sequences where the two homopurine stretches alternate on the two strands of DNA. The oligonucleotide switches from one homopurine strand to the other at the junction between the two sequences. This result shows that it is possible to extend the range of DNA sequences that can be recognized by a single oligonucleotide.

From January 1984 to March 1988, 210 tibial shaft fractures in 207 patients treated by intramedullary nails, and by plate and screws and extraskeletal fixation were studied. There were 166 men and 41 women. Of the fractures, 95 (42.2%) of the 210 tibial shaft fractures were closed fractures and 115 (54.8%) were open fractures [Grade I: 44 (21.0%), Grade II: 39 (18.6%), and Grade III: 32 (15.2%)]. The incidence of associated injury was 44.3% and it was highest in the group with extraskeletal fixation (64.7%). Intramedullary nailing was performed in the majority of these fractures (127 out of 210) and yielded the best results in this series. Kuntscher's nailing resulted in nonunion in 3 cases (2.8%), superficial infections in 3 cases (2.8%) and deep infections in 4 cases (3.8%). Interlocking nailing resulted in nonunion in 1 case (4.8%), superficial infection in 1 case (4.8%) and deep infections in 3 cases (14.3%). Plate and screws fixation was performed primarily for metaphyseal fractures (71.4%) and resulted in nonunion in 4 cases (8.1%), superficial infection in 1 case (2.0%) and deep infection in 5 (10.2%). In the group treated with extraskeletal fixation, inasmuch as there were more cases of compromised soft tissue (open fracture, 97.1%), a higher incidence of associated major injuries (64.7%) and more cases of comminution of the bone (67.6%), the final results were less satisfactory. Nonunion was noted in 26.5% of the cases, superficial infection in 2.9% of the cases, and deep infection in 32.4% of the cases. From our study, we conclude that in appropriately selected patients, surgical fixation of tibial shaft fractures can produce a good functional recovery with an acceptable low rate of complications.

With the application of more refined examinations, angiodysplasia is now being recognized as a common cause of bleeding in patients suffering from lower gastrointestinal bleeding. The diagnosis can be made by angiography or occasionally by colonofiberscopy. Angiography allows one to make this diagnosis even after bleeding has ceased. The lesions may not be clinically evident at the time of surgery, and the diagnosis must be made preoperatively to allow for proper treatment. A right hemicolectomy is the treatment of choice since these lesions are frequently multiple and seldom, if ever, occur distal to the proximal transverse colon. The pathophysiology, diagnosis, management and salient features of these lesions are described. The management of 5 patients is discussed.