ACL reconstruction in children: a transphyseal technique.

The annual incidence of ACL tears is increasing steadily in pediatric patients. Chronic anterior instability causes meniscal lesions at a frequency that increases significantly with the injury-to-surgery interval. Conservative therapy, simple suturing, and isolated extra-articular tendon reconstruction are associated with high failure rates. Intra-articular arthroscopy-assisted tendon reconstruction is a good treatment method, although several different techniques have been described. We used a transphyseal technique with a hamstring tendon graft to treat 14 knees in 13 patients with a mean age of 13 years and 7 months. Mean injury-to-surgery interval was 6 months. Strict compliance with technical rules is required when using this technique. Bone tunnel diameter must not exceed 8 mm. Bone tunnels must be as vertical and central as possible. The fixation material must not bridge the physis (at the femur, cortical fixation; and at the tibia, fixation using a resorbable screw no longer than 25 mm combined with a staple). Meniscal lesions were present in half the knees and meniscal preservation considered mandatory. Conservative treatment of concomitant lesions was performed routinely. After a mean follow-up of 15 months, no recurrent tears or revision procedures for meniscectomy had been recorded. The IKDC grade was A or B in 93% of knees. The mean subjective IKDC score was 83.3 and the Lysholm score was in the excellent or good range in 93% of knees. Of the 14 knees, 2 exhibited signs suggesting femoral epiphysiodesis, with 4° of valgus deformity compared to the contra-lateral knee and no clinical consequences. Transphyseal reconstruction with open physes conducted in strict compliance with technical rules can be performed to control the instability and preserve the menisci. Nevertheless, this technique carries a risk of epiphysiodesis, chiefly at the femur.

Universal method for holographic grating recording: multimode deformable mirrors generating Clebsch-Zernike polynomials.

Recording methods for making aberration-corrected holographic gratings are greatly simplified by use of a plane multimode deformable mirror (MDM) upon one of the two recording beams. It is shown that MDM compensators easily provide the superposition of many interesting active optics modes, which we have named Clebsch-Zernike modes. When we apply only a uniform loading or no loading at all onto the rear side of the MDM clear aperture, the available Clebsch-Zernike modes are made to belong to a subclass of the Zernike modes that includes the three modes of the third-order aberration theory as well as a well-defined part of the Zernike higher-order modes. Such a recording method is considered to be universal, since it does not require the use of a sophisticated optical system such as a compensator. Active optics 12-arm MDM's in the vase form have been designed from the elasticity theory. The design of six-arm MDM's is currently carried out with theoretical results. As an example of the method, the recording of three holographic gratings of the Hubble Space Telescope Cosmic Origins Spectrograph has been investigated. Substantial improvements in image quality have been found by use of a six-arm MDM as recording compensator. The result is that aberrations of much higher order can simultaneously be corrected so that the residual blur images of the spectra occupy areas approximately 10 (direction of dispersion) x 3 (cross dispersion) = 30 times smaller--also in terms of pixel number--than those obtained by our American colleagues. Therefore the active optics recording method appears to provide substantial gains in resolving power and in sensitivity: (i) For all three gratings the spectral resolution would be increased by a factor of 10, and (ii), in addition, for the two higher dispersion gratings, the limiting magnitude on the sky appears to be increased by a magnitude of approximately 1-1.2.

A 1.5-Mb physical map of the hidrotic ectodermal dysplasia (Clouston syndrome) gene region on human chromosome 13q11.

The HED (hidrotic ectodermal dysplasia) or Clouston syndrome gene (named ED2) has been mapped to the pericentromeric region of chromosome 13 (13q11) to a 2.4-cM interval flanked by markers D13S1828 and D13S1830. We have developed a BAC/PAC-based contig map of this region. This contig, comprising 23 clones and spanning 1.5 Mb, was established by mapping of 27 BAC/PAC end-derived STSs, 11 known polymorphic markers, 2 previously mapped genes, and 14 ESTs. The genomic clone overlaps were confirmed by restriction fragment fingerprint analysis. This contig provides the basis for genomic sequencing and gene identification in the ED2 critical region. Of the 14 ESTs mapped to the contig, 6 show homology to human genes and 8 appear to be novel. Expression patterns of the genes/ESTs were tested by Northern blot and RT-PCR. Full characterization of some of these genes, as well as the novel ESTs, will be useful in assessing their involvement in the HED/Clouston syndrome.

Functional Rac-1 and Nck signaling networks are required for FGF-2-induced DNA synthesis in MCF-7 cells.

The effects of Fibroblast Growth Factor-2 (FGF-2) on breast cancer cell DNA synthesis are controversial. To elucidate the mechanisms by which FGF-2 stimulates or inhibits DNA synthesis, we analysed FGF-2 signaling pathways in breast cancer MCF-7 and MCF-7 cells overexpressing Ha-Ras (MCF-7ras). We found that FGF-2-induction of DNA synthesis correlates with Ras transient activation, FRS-2 tyrosine phosphorylation and low level of expression of p66Shc. In addition, Nck-associated proteins are highly tyrosine phosphorylated and JNK reaches a higher level of activation when FGF-2 triggers DNA synthesis. Interestingly upon FGF-2 treatment, JNK activation and DNA synthesis are dependent on Rac-1 activity. These results confirm that in MCF-7 cells, induction of DNA synthesis by FGF-2 requires a transient activation of the Ras/MAPK cascade and demonstrates for the first time that intact Rac-1 and Nck signaling networks are required.

Glycosaminoglycans promote HARP/PTN dimerization.

Heparin affin regulatory peptide (HARP), also called pleiotrophin (PTN), is a secreted polypeptide which binds to heparin and plays a key role in cellular growth and differentiation. In order to assess the determinants potentially important to its biological activity, we tested the ability of HARP to oligomerize, a process involved in mitogenic activity of the heparin-binding fibroblast growth factor. Using dissuccinimidyl suberate cross-linking experiments and affinity chromatography, we report that human HARP forms noncovalent dimers. Dimerization is dependent on the presence of heparin or other sulfated glycosaminoglycans, as chlorate treatment of cells inhibits this process. In vitro, different glycosaminoglycans, such as dermatan sulfate and chondroitin sulfate-C, also induce a dimer assembly of HARP. The relevance of this process was supported by experiments demonstrating that HARP is secreted as a dimer in conditioned medium of NIH-3T3 cells that overexpressed this growth factor and is also associated to the cell surface or to the extracellular matrix.

Fibroblast growth factor receptors participate in the control of mitogen-activated protein kinase activity during nerve growth factor-induced neuronal differentiation of PC12 cells.

The current paradigm for the role of nerve growth factor (NGF) or FGF-2 in the differentiation of neuronal cells implies their binding to specific receptors and activation of kinase cascades leading to the expression of differentiation specific genes. We examined herein the hypothesis that FGF receptors (FGFRs) are involved in NGF-induced neuritogenesis of pheochromocytoma-derived PC12 cells. We demonstrate that in PC12 cells, FGFR expression and activity are modulated upon NGF treatment and that a dominant negative FGFR-2 reduces NGF-induced neuritogenesis. Moreover, FGF-2 expression is modulated by NGF, and FGF-2 is detected at the cell surface. Oligonucleotides that specifically inhibit FGF-2 binding to its receptors are able to significantly reduce NGF-induced neurite outgrowth. Finally, the duration of mitogen-activated protein kinase (MAPK) activity upon FGF or NGF stimulation is shortened in FGFR-2 dominant negative cells through inactivation of signaling from the receptor to the Ras/MAPK pathway. In conclusion, these results demonstrate that FGFR activation is involved in neuritogenesis induced by NGF where it contributes to a sustained MAPK activity in response to NGF.

Identification and characterization of an intracellular protein complex that binds fibroblast growth factor-2 in bovine brain.

The fibroblast growth factor (FGF) family is composed of polypeptides with sequence identity which signal through transmembrane tyrosine kinase receptors. We report here the purification from bovine brain microsomes of an FGF-2-binding complex composed of three proteins of apparent molecular masses 150 kDa, 79 kDa and 46 kDa. Only the 150 kDa and 79 kDa proteins bound FGF-2 in cross-linking and ligand-blotting experiments. Binding of FGF-2 to p79 is enhanced in the presence of calcium. Peptide sequences allowed the identification of p150 and the cloning of the cDNAs encoding p79 and p46. The deduced amino acid sequence of p79 reveals high similarity to those of gastrin-binding protein and mitochondrial enoyl-CoA hydratase/hydroxyacyl-CoA dehydrogenase. p46 is similar to mitochondrial ketoacyl-CoA thiolase. Stable transfection of FR3T3 rat fibroblast cells with p79 cDNA analysed by electron microscopy following immunolabelling of ultra-thin cryosections revealed a localization of p79 in the secretory pathway, mainly in the endoplasmic reticulum and the Golgi region, where it is specifically associated with the molecular chaperone calnexin. In vivo a protein similar to the Golgi protein MG-160 forms a complex with FGF-2 and p79.

Myofibroblastoma of breast--an appraisal of cytoskeletal phenotypes.

Myofibroblastoma of the breast is a recently described entity. Since its first description in 1987, less than 50 cases have been reported. We present the first (reported) myofibroblastoma to be detected as a non-palpable mass on a routine screening mammogram and emphasize the importance of not mis-diagnosing this rare cellular lesion as malignant on frozen section. Review of the literature demonstrates changes in the clinical presentation of myofibroblastomas. Once considered more common in men than in women, myofibroblastomas are now being reported with increasing frequency in women. The age at presentation is a decade earlier, and not surprizingly, the size of the earlier detected lesion is smaller. Recently four different cytoskeletal phenotypes (V, VA, VAD and VD) of myofibroblastomas have been described, depending upon the vimentin (V), actin (A), and desmin (D) immunoreactivity. Whereas vimentin reactivity is universal, actin and desmin immunoreactivity is variable, desmin being more frequently positive than actin. As more is known about the clinical behavior of myofibroblastomas, their rate of recurrence and malignant potential, if any, the relationship of the cytoskeletal content to prognosis may become clearer. Currently, complete immunohistochemical analysis and electron microscopic examination of this interesting breast lesion is recommended. List of abbreviations-Vimentin (V), actin (A), and desmin (D), vimentin and actin (VA), vimentin and desmin (VD), vimentin, actin and desmin (VAD).

Recording method for obtaining high-resolution holographic gratings through use of multimode deformable plane mirrors.

We propose a new way of recording in which a spherical blank and a deformable mirror are used to obtain high-resolution holographic gratings. The reflection of one of the two laser recording waves upon this mirror provides the deformations necessary to image correction to as much as seventh-order aberrations inclusively.

Illustration of the use of multimode deformable plane mirrors to record high-resolution concave gratings: results for the Cosmic Origins Spectrograph gratings of the Hubble Space Telescope.

To illustrate the efficiency of using a deformable plane mirror to record holographic gratings, we have computed the three gratings for the Cosmic Origins Spectrograph. Their working conditions are severe, since they have to correct the residual spherical aberration of the Hubble Space Telescope. Nevertheless, all images obtained are largely diffraction limited with regard to the resolution.

Production and purification of active FGF2 via recombinant fusion protein.

Basic fibroblast growth factor (FGF2) is involved in both cell proliferation and differentiation processes. Heparin may interfere in the stability and biological activities of FGFs. However, it is difficult to obtain FGF preparation without traces of heparin since heparin affinity chromatographies are routinely used to prepare this growth factor. We have therefore devised a means of production of active recombinant FGF2 devoid of heparin traces. The bovine FGF2 gene was inserted into the pMAL-c prokaryotic expression vector and the recombinant protein was synthesised as a fusion product between the maltose binding protein (MBP) and FGF2. Purification of the FGF2 fusion protein was performed by an amylose affinity chromatography. Yields were similar to those obtained by using a traditional heparin affinity column purification procedure. The fusion protein (MBP-FGF2) and the cleaved-off FGF2 were tested for some of their biological properties and compared to recombinant FGF2 purified by heparin affinity chromatography. Mitogenic activity on Chinese hamster lung fibroblasts (CCL39) and neurite outgrowth on pheochromocytoma culture cells (PC12) were used as biological assays. The cleaved-off FGF2 was as active as commercially available recombinant FGF2 (ED50 at 0.16 and 0.04 nM respectively). However MBP-FGF2 was less active (ED50 at 0.9 nM) in both tests.

FGFs and their receptors, in vitro and in vivo studies: new FGF receptor in the brain, FGF-1 in muscle, and the use of functional analogues of low-affinity heparin-binding growth factor receptors in tissue repair.

Several heparin-binding growth factors (HBGFs) are thought to play a key role in the natural processes of tissue homeostasis, regeneration or repair. The HBGFs are active upon release from neighbouring inflammatory or circulating cells, as well as upon release from heparan sulfate proteoglycosaminoglycans that are associated with the extracellular matrix (ECM). To better understand the physiological role of these HBGFs, we have focused our effort on studying a subset of HBGFs, namely FGF-1 and FGF-2 and their receptors. We present the purification and characterisation of a new form of heparin-binding FGF receptor from adult bovine brain (Perderiset et al., 1992). This receptor has now been purified to homogeneity. Ligand blot and cross-linking experiments performed with labeled FGF-1 or FGF-2 revealed 80-kd and 130-kd bands. Preliminary sequence information indicates that receptor is different from the receptors, FGFR-1 to -4, but it may be related the cysteine-rich-FGF receptor (CFR). We have previously shown that FGF-1, but not FGF-2, is specifically expressed in myoblastic satellite cells during the proliferating phase preceding myoblast alignment and fusion. We have now transfected primary cultures of rat myoblastic satellite cells with FGF-1 cDNA and expressed this growth factor constitutively. The transfected cells were no longer able to form myotubes. Transfection with antisense FGF-1 induced myotube formation suggesting that endogenous expression of FGF-1 is associated with myoblastic cell differentiation. Numerous studies have concluded that the ECM represents a natural reservoir for various HBGFs.(ABSTRACT TRUNCATED AT 250 WORDS)

In situ grafting made easy. Modification of a technique.

In situ bypass grafting has come of age following a period of disenchantment. It has become, in some centers, the operation of choice for bypasses to the tibial and peroneal vessels. Two fundamentally different techniques for this procedure have evolved. The first, following the method of Hall, uses a retrograde valvulotome passed from the distal saphenous vein to the proximal vein; on withdrawal, it severs the valves. A second approach, that of Leather, uses microscissors and a right-angle valvulotome, along with a disposable valve stripper, to disrupt the valves. Proponents exist for both approaches. In 1983 we reported our protocol for in situ grafting using the Hall technique. A number of modifications have since been made to expedite the technique. We describe and provide illustrations of these modifications.

Imaging extreme ultraviolet spectrometer employing a single toroidal diffraction grating: the initial evaluation.

A high-efficiency extreme ultraviolet (EUV) imaging spectrometer has been constructed and tested. The spectrometer employs a concave toroidal grating illuminated at normal incidence in a Rowland circle mounting and has only one reflecting surface. The toroidal grating has been fabricated by a new technique employing an elastically deformable submaster grating which is replicated in a spherical form and then mechanically distorted to produce the desired aspect ratio of the toroidal surface for stigmatic imaging over the selected wavelength range. The fixed toroidal grating used in the spectrometer is then replicated from this surface. Photographic tests and initial photoelectric tests with a 2-D pulse-counting detector system have verified the image quality of the toroidal grating at wavelengths near 600 A. The results of these initial tests are described in detail, and the basic designs of two instruments which could employ the imaging spectrometer for astrophysical investigations in space are briefly described, namely, a high-resolution EUV spectroheliometer for studies of the solar chromosphere, transition region, and corona and an EUV spectroscopic telescope for studies of nonsolar objects.