[Results of using Spine Assist Mazor in surgical treatment of spine disorders].

In this paper, we describe the possibility of using a bone-mounted miniature robot based on the experience of different surgeries performed in 77 patients divided into four groups according to the general pathology (degenerative stenosis of the vertebral canal, fractures of vertebral bodies, spondylolisthesis, hemangiomas, and tumors). All the patients underwent surgical intervention using Spine Assist Mazor, such as stabilization using the Go-Lif system, transpedicular systems, vertebroplasty, and vertebral body biopsy. The new method and the technology of stabilizing surgeries using Spine Assist Mazor allow one to perform preoperative virtual planning based on CT images and to identify an ideal and safe trajectory of placing screws or needles for vertebroplasty or biopsy.

Identification of p53 as a sequence-specific DNA-binding protein.

The tumor-suppressor gene p53 is altered by missense mutation in numerous human malignancies. However, the biochemical properties of p53 and the effect of mutation on these properties are unclear. A human DNA sequence was identified that binds specifically to wild-type human p53 protein in vitro. As few as 33 base pairs were sufficient to confer specific binding. Certain guanines within this 33-base pair region were critical, as methylation of these guanines or their substitution with thymine-abrogated binding. Human p53 proteins containing either of two missense mutations commonly found in human tumors were unable to bind significantly to this sequence. These data suggest that a function of p53 may be mediated by its ability to bind to specific DNA sequences in the human genome, and that this activity is altered by mutations that occur in human tumors.

Robotic assisted spinal surgery--from concept to clinical practice.

After several years of product development, animal trials and human cadaver testing, the SpineAssist--a miniature bone-mounted robotic system--has recently entered clinical use. To the best of the authors' knowledge, this is the only available image-based mechanical guidance system that enables pedicle screw insertion with an overall accuracy in the range of 1 mm in both open and minimally invasive procedures. In this paper, we describe the development and clinical trial process that has brought the SpineAssist to its current state, with an emphasis on the various difficulties encountered along the way and the corresponding solutions. All aspects of product development are discussed, including mechanical design, CT-to-fluoroscopy image registration, and surgical techniques. Finally, we describe a series of preclinical trials with human cadavers, as well as clinical use, which verify the system's accuracy and efficacy.

Effect of protein tyrosine phosphatase 1B expression on transformation by the human neu oncogene.

Many oncogenes encode proteins with a tyrosine kinase activity that appears to be directly involved in the process of transformation. Because these kinases are themselves activated for transformation by tyrosine phosphorylation, proteins which remove phosphate from tyrosine residues, protein tyrosine phosphatases (also termed phosphotyrosine phosphatases and protein phosphotyrosyl phosphatases), are intuitive candidate transformation suppressors. The human PTP1B gene, previously cloned in our laboratory and encoding the low molecular weight protein tyrosine phosphatase PTPase 1B, was introduced into NIH 3T3 cells. Subsequent transformation of these PTPase 1B-expressing cells by an oncogenic form of the human neu gene was suppressed relative to control NIH 3T3 cells. This suppression of transformation was observed in assays for focus formation, anchorage-independent growth, and tumorigenicity. Tumorigenicity assays indicated a complex effect of PTPase 1B expression on transformation.

Depletion of the erbB-2 gene product p185 by benzoquinoid ansamycins.

Herbimycin A, a benzoquinoid ansamycin, is widely used as an inhibitor of tyrosine kinases. We have examined the effects of herbimycin A and several analogues on p185, the tyrosine kinase encoded by the erbB2 gene in human breast cancer cells. Exposure to 0.35 microM herbimycin A reduced tyrosine phosphorylation of p185 in SKBr3 cells by 80% after 2 h, and the p185 protein level was reduced by 90% after 6 h. The reduction of p185 resulted primarily from increased degradation of p185; cellular protein synthesis was reduced only 16% in SKBr3 cells treated with herbimycin A, RNA synthesis was inhibited only 10%, and erbB2 mRNA levels were unchanged. Examination of the major cellular glycoproteins indicated that most glycoproteins were unaffected under conditions that substantially depleted p185. Studies with cell lines transfected with erbB2 containing defined deletions indicated that susceptibility to the depletion of p185 by herbimycin and its analogues required the domain encoded by amino acids 751-971. The benzoquinoid ansamycins therefore initiate a process of specific degradation of tyrosine kinases by a mechanism that remains unknown.

Requirements for the internalization of a murine monoclonal antibody directed against the HER-2/neu gene product c-erbB-2.

A murine monoclonal antibody, TA1, is directed against an epitope on the extracellular domain of the HER-2/neu (c-erbB-2) gene product. Requirements for TA1-induced internalization of c-erbB-2 have been studied using the SKBr3 human breast cancer cell line and several rat fibroblast cell lines that express either wild-type or mutant human c-erbB-2. Internalization of TA1 was monitored by assaying protease-resistant uptake of 125I-labeled TA1, by electron microscopy of gold-labeled TA1, and by inhibition of clonogenic growth of cells incubated with TA1 that had been conjugated with blocked ricin. Similar rates of internalization of TA1 were observed in SKBr3 and in rat fibroblasts that expressed human c-erbB-2. The route of endocytosis was the same as that observed with antibodies against other membrane receptors. Anti-c-erbB-2 and anti-transferrin receptor cointernalized through clathrin-coated pits, coated vesicles, endosomes, and multivesicular bodies. Products of mutant c-erbB-2 that lacked a portion of the tyrosine kinase domain or that lacked most of the cytoplasmic domain were endocytosed in the presence of TA1 as promptly as the wild-type c-erbB-2 product. Slightly more rapid internalization of TA1 was observed in rat cells that expressed c-erbB-2 with a single point mutation in the transmembrane domain. Taken together, our data suggest that neither the intracytoplasmic domain nor receptor phosphorylation is required for antibody-mediated endocytosis of c-erbB-2.

Six amino acids from the retroviral gene gag greatly enhance the transforming potential of the oncogene v-erb-B.

Avian erythroblastosis virus (AEV) induces both erythroblastosis and fibrosarcomas in susceptible birds and transforms the corresponding cells in culture. Neoplastic transformation by AEV is mediated principally by an oncogene known as v-erb-B. We have explored the means by which this gene is expressed from the genome of AEV and uncovered an important structural determinant for the potency of the oncogene. In order to define the boundaries of v-erb-B and the supplementary oncogene, v-erb-A, we sequenced all but a small portion of the genome of the ES4 strain of AEV. We then demonstrated that, during expression in infected cells, splicing fuses the first six amino acids of the retroviral gene gag to the body of the v-erb-B protein. In order to explore the impact of this fusion on the function of v-erb-B, we constructed vectors with Murine Leukemia Virus that express the oncogene either with or without the fusion to gag. Viruses generated from these two vectors differed greatly in their abilities to transform cells: fusion of v-erb-B with gag enhanced its transforming ability 50 to 100-fold as determined by focus transformation assays and growth in soft agar. Our data suggest that the difference in transforming ability is not due to alterations in transcription or translation but, rather, may result from changes in post-translational modification.

Transformation of murine myelomonocytic cells by myc: point mutations in v-myc contribute synergistically to transforming potential.

The v-myc oncogenes of chicken retroviruses (including MC29) bear point mutations relative to chicken c-myc. These mutations result in several amino acid differences in the encoded proteins. We have used recombinant murine retroviruses containing various myc alleles to analyse the myelomonocytic transforming potential of the myc oncogene. The myc alleles used were MC29 v-myc, chicken c-myc, chimeric genes combining 5' sections of v- or c-myc with 3' sections of c- or v-myc, and mouse c-myc. The same retroviral vector (based on the genome of Moloney leukemia virus) was used for each allele and the genes were translated from genomic message. By infecting the primary mouse tissues, bone marrow, peritoneal-derived macrophages and mixed embryonic tissue with the recombinant viruses, variation was found in the transforming efficacy of these alleles: v-myc was most effective, followed by the two chimeric genes, whereas c-myc (chicken or mouse) was least effective in eliciting myelomonocytic transformation. Viral gag sequences were not necessary for this transformation. In each case, the transformed monocytes were growth factor-dependent and non-immortal. However, v-myc transformed monocytes (though not monocytes transformed by other myc alleles) were able to progress to an immortal, growth factor-independent phenotype. Our results indicate that v-myc is far more effective than c-myc in eliciting myelomonocytic transformation; that this is due to combinatorial effects of 5' and 3' mutations in the v-myc gene; and that secondary events in addition to these mutations are required for transformation of myelomonocytic cells to an immortal, tumorigenic phenotype.

Transformation of early erythroid precursor cells (BFU-E) by a recombinant murine retrovirus containing v-erb-B.

Avian erythroblastosis virus (AEV) is a replication-defective retrovirus that transforms erythroid and fibroblast cells in vitro and in vivo. The transforming ability of AEV is due primarily to the oncogene v-erb-B. A recombinant murine retrovirus has been constructed by inserting a chimeric gag-v-erb-B gene into a Moloney murine leukemia virus based vector. This retrovirus was used to examine v-erb-B-induced transformation of murine hematopoietic cells. Infection of murine primary fetal liver, adult bone marrow or adult spleen cells with the recombinant virus generated large hemoglobinized erythroid colonies in the absence of exogenous growth factors. Generation of such colonies usually requires the presence of erythropoietin (Epo) and interleukin-3 (IL-3). These growth-factor independent colonies were shown to be derived from early (BFU-E) and not late (CFU-E) erythroid progenitor cells, and the effect was not attributable to growth factors elicited by the virus-producing cell lines. In order to confirm that the recombinant virus was responsible for this transformation of BFU-E to growth factor independence, bone marrow cells from post 5-fluorouracil treated mice were infected and used to repopulate lethally-irradiated mice. Growth factor-independent BFU-E were obtained in up to 30% of day-13 spleen colonies and it was shown by DNA analysis that cells from these colonies contained integrated provirus. Our results indicate that v-erb-B transforms early erythroid progenitors to growth factor independent growth and subsequent differentiation to erythrocytes -a process that normally requires Epo plus either IL-3 or granulocyte-macrophage colony stimulating factor (GM-CSF).

Structure of the Spec1 gene encoding a major calcium-binding protein in the embryonic ectoderm of the sea urchin, Strongylocentrotus purpuratus.

We have identified and characterized the structure of the Spec1 gene in the sea urchin Strongylocentrotus purpuratus. In earlier studies we demonstrated that a small family of messenger RNAs, termed Spec mRNAs for S. purpuratus ectodermal mRNAs, begins to accumulate 20 hours after fertilization in ectoderm cells of the sea urchin embryo. The Spec mRNAs code for a group of low molecular weight proteins belonging to the troponin C superfamily. Spec1 transcripts, the predominant mRNAs of the family, are heterogeneous in their 3' untranslated sequences but code for a single protein, recently shown to be a calcium-binding protein. Spec complementary DNA clones were used to isolate genomic clones from two lambda libraries. These genomic clones comprise a 41 kb (kb = 10(3) bases or base-pairs) region of the S. purpuratus genome and contain a Spec1 gene closely linked to another Spec gene, Spec2c. The Spec1 gene is 10.3 kb in length and contains six exons. The genomic clones containing the Spec1 gene can be placed into two groups based on restriction fragment length differences and differences in hybridization strengths using probes derived from Spec1 3' untranslated regions. Evidence that these groups probably correspond to two alleles of the Spec1 gene was obtained by probing genomic DNA blots of sperm DNA from different individuals with 3' untranslated sequences of Spec1 complementary DNA clones. These blots show that two of the Spec1 mRNAs we have characterized, and probably a third, are alleles of the Spec1 gene. Thus, there appears to be a single polymorphic Spec1 gene in the sea urchin genome. We used S1 protection and primer extension procedures to map the 5' end of the Spec1 gene. Results from these experiments indicate that the initiation of transcription of the Spec1 mRNA begins at an A residue 220 bases from the 3' end of the first exon. Adding support to this claim, cannonical T-A-T-A and C-A-A-T sequences, indicative of many eukaryotic promoters, are found 23 bases and 60 bases upstream from this site, respectively. Analysis of sequences within a few kb of the Spec1 gene show that there are five members of a repetitive sequence family near the gene, three upstream and two downstream. The 5' leader sequence of another Spec mRNA, Spec2a, also contains a member of this repeat family.(ABSTRACT TRUNCATED AT 400 WORDS)

A p53 growth arrest protects fibroblasts from anticancer agents.

Reversible inhibitors of the cell cycle such as the TGF-betas have been exploited to protect dividing cells from exposure to anticancer drugs and radiation. Here, rat embryo fibroblast (REF) lines expressing different p53 mutations were used to test whether the p53 growth arrest could also chemoprotect cells from high doses of anticancer drugs. Whereas the doubling times of the different REF lines at 37 degrees C were similar, cells bearing temperature-sensitive mutations (mouse 135V or human 143A) were growth arrested at 31 degrees C. Temperature-dependent p53 activity was associated with increased levels of MDM2 and p21/WAF1, and the induction of an integrated p53-responsive luciferase gene. The REF lines exhibited similar sensitivities to common anticancer drugs when grown at 37 degrees C. However, when exposed to the same agents following transient incubation at 31 degrees C, the p53-arrested cells exhibited a marked survival advantage as shown by colony-forming assays. Chemoprotection was not universal, in that colony formation was not enhanced significantly after treatment with cisplatin or 5-fluorouracil, two drugs which can cause cellular damage throughout the cell cycle. Like other negative growth regulators, an activated p53 checkpoint may mediate the survival of cells exposed to drugs that target DNA synthesis or mitosis.

A four-amino-acid insertion in the ligand-binding domain inactivates hRXRbeta and renders dominant negative activity.

Retinoid X receptors (RXRs) are members of the steroid and thyroid hormone receptor superfamily of hormone-dependent transcription factors that mediate the pleiotropic effect of retinoids. Here, we report the initial characterization of an isoform of hRXR beta, termed hRXR beta3, which was previously identified as an H-2RIIBP isoform (Epplen and Epplen, 1992). The hRXR beta3 isoform cotains an in-frame insertion of four amino acids (SLSR) in the ligand binding domain at codon 419. The isoform is generated by alternate use of a 3' splice acceptor site and was detectable by reverse transcription polymerase chain reaction (RT-PCR) in all human tumor cell lines and mouse tissues examined. Chimeric receptors, in which the ligand-binding domain of hRXR alpha was substituted by the corresponding domain from hRXR beta3, were used to investigate the consequences of the SLSR insertion on the transactivation and DNA-binding functions of the chimeric receptor. Co-transfection assays revealed that a chimera RXR alpha/beta3 receptor failed to transactivate the RXR-specific CRBPII promoter, whereas the identical chimera lacking the SLSR insertion was active. The RXR alpha/beta3 receptor exhibited dominant negative activity against active retinoid X and retinoic acid receptors on retinoid-responsive promoters. Moreover, the RXR alpha/beta3 protein failed to interact physically with the retinoic acid receptor (RAR) to form heterodimers as detected by physical association assays, and failed to bind DNA containing an RAR-responsive element. Therefore, this suggests that the SLSR insertion in the ligand-binding domain of the RXR alpha/beta3 receptor is responsible for the altered behavior of the chimera. Our findings raise the possibility that RXR alpha/beta3, and perhaps hRXR beta3 isoform, function by titrating a limiting adaptor molecule that is involved in mediating retinoid function.

Cellular processing of (125)I- and (111)in-labeled epidermal growth factor (EGF) bound to cultured A431 tumor cells.

Low molecular weight of epidermal growth factor (EGF) enables better intratumoral penetration in comparison with larger targeting proteins, but the cellular retention of EGF-associated radioactivity is poor for directly iodinated EGF. An attempt was made to improve intracellular retention by the use of metal-diethylenetriaminepentaacetic acid or nonphenolic linker (N-succinimidyl-para-iodobenzoate) as labeling agents. The use of nonphenolic linker did not improve retention of the radioactivity in A431 carcinoma cell line. The use of the radiometal label provided an appreciable prolongation of radioactivity residence inside the cell.

Transforming growth factor-beta 3 mediated modulation of cell cycling and attenuation of 5-fluorouracil induced oral mucositis.

Mucositis is a common, dose-limiting complication in patients receiving cancer chemotherapy, which derives from damage to the epithelial cell layer. We have shown that transforming growth factor-beta 3 (TGF-beta 3) negatively regulates epithelial cell proliferation and reduces the incidence of oral mucositis. Here, we report the findings of a large study examining the effects of TGF-beta 3 administration in a hamster model on oral epithelial cell cycling in vivo, on oral mucositis, on weight retention and on survival. Topical application of TGF-beta 3 to the buccal mucosa significantly reduced basal cell proliferation, as measured by proliferating cell nuclear antigen (PCNA) immunohistochemistry and DNA ploidy. Administration of topical TGF-beta 3 prior to chemotherapy with 5-fluorouracil (5-FU) significantly reduced the severity of mucositis with respect to time, reduced chemotherapy-associated weight loss and increased survival.

[Percutaneous trephine for vertebral biopsy].

We performed percutaneous vertebral biopsy on 18 patients (March 1978 to March 1992) using a Craig trephine needle under fluoroscopic control, a more reliable and safer procedure than open vertebral body biopsy. Material for histopathologic examination was obtained in 17 of the 18 patients and there were no complications. Possible complications involve mostly in the cervical and dorsal spine. They can be prevented by using very precise and delicate technique under reliable fluoroscopic control in the operating room.

Energization of glucose transport by Pseudomonas fluorescens.

We have measured the capacity of Pseudomonas fluorescens to transport the glucose analog 2-deoxy-d-glucose and the amino acids l-alanine and alpha-aminoisobutyric acid under conditions in which the cells could generate (i) both a membrane proton motive force and high-energy phosphate compounds, (ii) a proton motive force but not high-energy phosphate compounds, and (iii) neither a proton motive force nor high-energy phosphate compounds. This was done by depleting cells of adenosine triphosphate stores by treatment with sodium arsenate and then suspending them in a phosphate-free medium, where they could generate a proton motive force but not phosphate bond energy, or in a phosphate-containing medium, where they could generate both a proton motive force and phosphate bond energy. Inclusion of the proton-conducting ionophore carbonyl cyanide-m-chlorophenyl hydrazone under either condition precluded the generation of both a proton motive force and phosphate bond energy. The amino acids l-alanine and alpha-aminoisobutyric acid were transported independently of phosphate bond energy and required only a proton motive force. 2-Deoxy-d-glucose was transported only under conditions in which phosphate bond energy could be generated. These results are consistent with the findings of others that Pseudomonas aeruginosa produces an inducible shock-sensitive glucose-binding protein and conform to the generalization that binding protein-associated transport systems are energized by phosphate bond energy.

Transforming growth factor-B3 protects murine small intestinal crypt stem cells and animal survival after irradiation, possibly by reducing stem-cell cycling.

Damage to the normal replacing tissues of the body, specifically the gastro-intestinal tract, limits the treatment and hence, cure rate of cancer patients. Here, we investigate the possibility that the sensitivity of the gastro-intestinal tract can be manipulated by transforming growth factor beta3 (TGF-beta3), making it more resistant to radiation in a murine model. The effects of TGF-beta3 were assessed using the crypt microcolony assay, a test of crypt stem-cell functional competence, in animal survival studies examining diarrhoea severity, labelling index and crypt size. Prior treatment with TGF-beta3 can result in a 3- to 4-fold increase (protection factor, PF) in surviving crypts, whilst longer exposure can raise the PF to almost 12. Protection of intestinal clonogenic stem cells results in marked protection of survival with a corresponding reduction in the duration and level of diarrhoea and ultimate restoration of normal histology in surviving mice. Inhibition of proliferation can be demonstrated when sufficient TGF-beta3 exposure is studied. Crypt size is also reduced. In conclusion, TGF-beta3 protects small intestinal clonogenic stem cells from radiation damage, reducing diarrhoea and animal mortality. The mode of action is believed to be specific inhibition of stem-cell proliferation.

Identification and characterization of baxepsilon, a novel bax variant missing the BH2 and the transmembrane domains.

The Bax gene is a member of the Bcl2 family that functions to regulate the programmed cell death process. A number of Bax isoforms have been previously identified: alpha, beta, gamma, delta, and omega. Here we report the identification and characterization of an additional Bax variant, termed Baxepsilon. The newly identified Bax variant contains a 97-base insertion generated by alternative splicing which includes a previously unidentified exon between exons 4 and 5. The insertion causes the production of a truncated Bax protein, termed Baxepsilon, which encodes a protein of 164 residues with a calculated molecular weight of 18 kDa. The last 69 amino acids of Baxalpha that encompass the BH2 and the TM domains are missing in Baxepsilon. The Baxepsilon protein, when expressed as a GST fusion protein, associated efficiently with Baxalpha, Baxepsilon, Bcl2, and Bcl-xL. In addition, Baxepsilon was active in inducing apoptosis when tested in a transient transfection assay. Furthermore, the presence of antiapoptotic genes including Bcl2, Bcl-xL, and baculovirus p35 abrogated Baxepsilon and Baxalpha function. Although the newly identified Bax variant was detectable by RT-PCR in several normal mouse tissues, the role of this variant in controlling programmed cell death is currently unknown.

Oral mucosal permeability and stability of transforming growth factor beta-3 in vitro.

PURPOSE: To investigate the permeability and localization of topically applied 125I-TGF-beta3 in porcine floor-of-mouth mucosa as a function of concentration and exposure. METHODS: The 125I-TGF-beta3 diluted in three different vehicles was applied to the tissue samples mounted in perfusion cells maintained at 37 degrees C. Flux and Kp values were calculated from the perfusate collected over a 24 hour period. The quantity of 125I-TGF-beta3 present in the tissue was determined by horizontal sectioning and subsequent counting. The stability of 125I-TGF-beta3 in saliva and in the tissue was analyzed by SDS polyacrylamide gradient gel electrophoresis. RESULTS: 125I-TGF-beta3 was relatively stable in saliva and in the epithelium; approximately 50% of the total counts in the deeper epithelium were resident in the 25kDa TGF-beta3 homodimer. A steady-state flux was reached approximately 6 hours post application and Kp value was 4.0+/-0.6 x 10(-6) (mean +/- sem). Penetration of 125I-TGF-beta3 to the basal cell layer was concentration dependent but reached nanomolar concentrations even after extensive surface rinsing, representing over one-thousand fold the IC50 for epithelial cell cycle arrest. CONCLUSIONS: The data suggest that topical application of TGF-beta3 to the oral mucosa in an appropriate vehicle can provide effective therapeutic delivery to the tissue.

Localization of a family of MRNAS in a single cell type and its precursors in sea urchin embryos.

Spec 1 mRNAs increase 100-fold in abundance per embryo during early sea urchin development. Previous studies indicated an enrichment of this mRNA in ectoderm fractions of gastrulae and plutei. We have determined the precise localization of this mRNA by in situ hybridization techniques. In pluteus larvae, the mRNA is highly restricted to a set of morphologically uniform ectoderm cells in the dorsal part of the embryo. The mRNA is not detectable in other regions of ectoderm or in endoderm and mesoderm. The pattern of localization is already established at the gastrula stage, before these cells are distinguishable by morphological criteria. This pattern of distribution of Spec 1 mRNA is distinct from that of bulk poly(A)+ mRNA. Measurements of the amount of Spec 1 mRNA per embryo and the number of cells containing this RNA indicate that there are about 500 Spec 1 mRNA molecules per cell at the pluteus stage and probably twice as many at the gastrula stage. These results indicate that the sensitivity of the in situ hybridization method allows detection of sequences that comprise approximately equal to 0.05% of the embryo mRNA nucleotides.