The homeobox genes vox and vent are redundant repressors of dorsal fates in zebrafish.

Ventralizing transcriptional repressors in the Vox/Vent family have been proposed to be important regulators of dorsoventral patterning in the early embryo. While the zebrafish genes vox (vega1) and vent (vega2) both have ventralizing activity in overexpression assays, loss-of-function studies are needed to determine whether these genes have distinct or redundant functions in dorsoventral patterning and to provide critical tests of the proposed regulatory interactions among vox, vent and other genes that act to establish the dorsoventral axis. We show that vox and vent are redundant repressors of dorsal fates in zebrafish. Mutants that lack vox function have little or no dorsoventral patterning defect, and inactivation of either vox or vent by injection of antisense morpholino oligonucleotides has little or no effect on the embryo. In contrast, embryos that lack both vox and vent function have a dorsalized phenotype. Expression of dorsal mesodermal genes, including chordin, goosecoid and bozozok, is strongly expanded in embryos that lack vox and vent function, indicating that the redundant action of vox and vent is required to restrict dorsal genes to their appropriate territories. Our genetic analysis indicates that the dorsalizing transcription factor Bozozok promotes dorsal fates indirectly, by antagonizing the expression of vox and vent. In turn, vox and vent repress chordin expression, restricting its function as an antagonist of ventral fates to the dorsal side of the embryo. Our results support a model in which BMP signaling induces the expression of ventral genes, while vox and vent act redundantly to prevent the expression of chordin, goosecoid and other dorsal genes in the lateral and ventral mesendoderm.

Patterning the early zebrafish by the opposing actions of bozozok and vox/vent.

Fish and frog embryos are patterned along the dorsal-ventral axis during the gastrula stage by opposing gradients of Bmps and Bmp inhibitory proteins. Three transcriptional repressors with partially overlapping expression domains have been proposed to be important mediators of Bmp function in Xenopus. We find that two related factors are expressed in the early zebrafish embryo. Although these factors are considerably divergent from the related Xenopus genes, they are expressed in domains similar to those of their Xenopus relatives throughout embryogenesis. Both of the zebrafish genes, which we have named vox and vent, are potent ventralizing factors in both zebrafish and Xenopus embryos. Using mutants in the Bmp pathway, we find that there are Bmp-dependent and Bmp-independent domains of vox expression, whereas vent is mostly dependent upon Bmp signaling. We show that ectopic vox or vent negatively regulates expression of the early dorsal gene bozozok (boz) and that ectopic boz eliminates vox and vent expression. Moreover, the normal exclusion of vox and vent from the organizer region is lost in boz mutant embryos. Our results show that boz and vox/vent are mutually antagonistic and indicate that the early establishment of the size of the organizer domain is dependent on an interplay between these early expressed transcriptional repressors.

Regulation of dorsal gene expression in Xenopus by the ventralizing homeodomain gene Vox.

Patterning in the vertebrate embryo is controlled by an interplay between signals from the dorsal organizer and the ventrally expressed BMPs. Here we examine the function of Vox, a homeodomain-containing gene that is activated by the ventralizing signal BMP-4. Inhibition of BMP signaling using a dominant negative BMP receptor (DeltaBMPR) leads to the ectopic activation of dorsal genes in the ventral marginal zone, and this activation is prevented by co-injection of Vox. chordin is the most strongly activated of those genes that are up-regulated by DeltaBMPR and is the gene most strongly inhibited by Vox expression. We demonstrate that Vox acts as a transcriptional repressor, showing that the activity of native Vox is mimicked by a Vox-repressor fusion (VoxEnR) and that a Vox-activator fusion (VoxG4A) acts as an antimorph, causing the formation of a partial secondary axis when expressed on the ventral side of the embryo. Although Vox can ectopically activate BMP-4 expression in whole embryos, we see no activation of BMP-4 by VoxG4A, demonstrating that this activation is indirect. Using a hormone-inducible version of VoxG4A, we find that a critical time window for Vox function is during the late blastula period. Using this construct, we demonstrate that only a subset of dorsal genes is directly repressed by Vox, revealing that there are different modes of regulation for organizer genes. Since the major direct target for Vox repression is chordin, we propose that Vox acts in establishing a BMP-4 morphogen gradient by restricting the expression domain of chordin.

Buprenorphine and naloxone combinations: the effects of three dose ratios in morphine-stabilized, opiate-dependent volunteers.

Sublingual buprenorphine is a promising new treatment for opiate dependence, but its opioid agonist effects pose a risk for parenteral abuse. A formulation combining buprenorphine with the opiate antagonist naloxone could discourage such abuse. The effects of three intravenous (IV) buprenorphine and naloxone combinations on agonist effects and withdrawal signs and symptoms were examined in 12 opiate-dependent subjects. Following stabilization on a daily dose of 60 mg morphine intramuscularly, subjects were challenged with IV doses of buprenorphine alone (2 mg) or in combination with naloxone in ratios of 2:1, 4:1, and 8:1 (1, 0.5, or 0.25 mg naloxone), morphine alone (15 mg) or placebo. Buprenorphine alone did not precipitate withdrawal and had agonist effects similar to morphine. A naloxone dose-dependent increase in opiate withdrawal signs and symptoms and a decrease in opioid agonist effects occurred after all drug combinations. Buprenorphine with naloxone in ratios of 2:1 and 4:1 produced moderate to high increases in global opiate withdrawal, bad drug effect, and sickness. These dose ratios also decreased the pleasurable effects and estimated street value of buprenorphine, thereby suggesting a low abuse liability. The dose ratio of 8:1 produced only mild withdrawal symptoms. Dose combinations at 2:1 and 4:1 ratios may be useful in treating opiate dependence.

Kinematics of posterior cruciate ligament-retaining and -sacrificing mobile bearing total knee arthroplasties. An in vitro comparison of the New Jersey LCS meniscal bearing and rotating platform prostheses.

The posterior cruciate ligament (PCL)-retaining, meniscal bearing and the PCL-sacrificing rotating platform designs of the LCS prosthesis (DePuy, Warsaw, IN) were designed to minimally constrain knee kinematics while minimizing bone-cement-prosthesis interface stresses and polyethylene wear. The kinematics and stability of the knee following arthroplasty with these devices rely on adequate tensioning of the remaining soft tissues by management of the flexion/extension gaps at the time of surgery. In this in vitro study, the knee kinematics of the function of the quadriceps mechanism for 8 cadaveric knees were measured quantitatively before and after implantation of these 2 prosthesis designs. Following implantation of the PCL-retaining, meniscal bearing prosthesis, anterior translations during anterior drawer testing were significantly greater (P < .05) than those seen in the intact knee. Implantation of the PCL-retaining, meniscal bearing prosthesis resulted in an increase in the extension gap of 2 mm. Quadriceps force needed to achieve full extension was increased by 30% over that needed in the intact knee. The PCL-sacrificing, rotating platform prosthesis constrained anterior translation such that nearly normal anterior knee stability was reestablished; however, the extension gap was increased by 4 mm and the quadriceps force needed to achieve full extension was 50% greater than that needed in the intact knee. Attempts to achieve joint stability by increases in the thickness of the tibial component to widen the flexion/extension gaps results in compromises of quadriceps efficiency, particularly in the absence of a functioning PCL, as demonstrated in this in vitro model. Patients receiving the PCL-sacrificing prosthesis may experience difficulty in those activities requiring quadriceps power near full extension, such as rising from a chair or ascending or descending stairs.

Spatial regulation of floating head expression in the developing notochord.

The zebrafish homeobox gene floating head (flh) is essential for notochord development and is one of the earliest genes to be expressed in notochord precursors. To understand how flh is regulated during notochord development, we compared the wild-type flh expression pattern to that in embryos mutant for flh and no tail (ntl), the zebrafish homologue of Brachyury. In the early gastrula, the pattern of flh expression is not affected in either flh or ntl mutants, implying that the initial establishment of a gastrula notochord domain is independent of the function of these genes. However, flh RNA is expressed at lower levels in flh mutants suggesting that flh positively regulates its own expression. During gastrulation, flh mutants show an abrupt loss of flh expression in cells which have involuted and entered the hypoblast, while the rest of the expression pattern appears normal, thus flh+ function is specifically required to maintain flh expression in hypoblast cells. The anterior-most part of the notochord rudiment differentially maintains flh expression in both wild types and flh mutant embryos, suggesting that there is unique regulation of flh in this region of the developing notochord. In ntl mutants the spatial pattern of flh expression is altered as early as the late gastrula stage, becoming broad and diffuse. We hypothesize that ntl+ is required for the proper convergence movements of flh-expressing cells.

Buprenorphine and naloxone interactions in opiate-dependent volunteers.

OBJECTIVE: Sublingual buprenorphine appears useful in the treatment of opiate dependence. A combination sublingual dose of buprenorphine and naloxone could have less potential for parenteral use by opiate-dependent individuals. To estimate the abuse potential of a combination formulation, we assessed the parenteral effects of a buprenorphine and naloxone combination in untreated heroin addicts. METHODS: Eight healthy, opiate-dependent daily users of heroin were given, under double-blind conditions on four separate occasions, either (1) 2 mg buprenorphine, (2) 2 mg naloxone, (3) 2 mg buprenorphine and 2 mg naloxone combined, or (4) placebo as a single intravenous infusion during a 30-second interval. Opiate agonist and antagonist physiologic and subjective effects were measured. Data were analyzed by analysis of variance. RESULTS: Buprenorphine increased opiate intoxication and relieved withdrawal. The buprenorphine and naloxone combination precipitated opiate withdrawal and was unpleasant and dysphoric in all subjects. Fifty percent of the subjects were unable to distinguish between naloxone alone and the combined medications during the first hour of testing. CONCLUSIONS: The buprenorphine and naloxone combination has a low abuse potential in opiate-dependent daily heroin users.

Specification of cell fates at the dorsal margin of the zebrafish gastrula.

Using fate mapping techniques, we have analyzed development of cells of the dorsal marginal region in wild-type and mutant zebrafish. We define a domain in the early gastrula that is located just at the margin and centered on the dorsal midline, in which most cells generate clones that develop exclusively as notochord. The borders of the notochord domain are sharp at the level of single cells, and coincide almost exactly with the border of the expression domain of the homeobox gene floating head (flh; zebrafish homologue of Xnot), a gene essential for notochord development. In flh mutants, cells in the notochord domain generate clones of muscle cells. In contrast, notochord domain cells form mesenchyme in embryos mutant for no tail (ntl; zebrafish homologue of Brachyury). A minority of cells in the notochord domain in wild-type embryos develop as unrestricted mesoderm, invariably located in the tail, suggesting that early gastrula expression of flh does not restrict cellular potential to the notochord fate. The unrestricted tail mesodermal fate is also expressed by the forerunner cells, a cluster of cells located outside the blastoderm, adjacent to the notochord domain. We show that cells can leave the dorsal blastoderm to join the forerunners, suggesting that relocation between fate map domains might respecify notochord domain cells to the tail mesodermal fate. An intermediate fate of the forerunners is to form the epithelial lining of Kupffer's vesicle, a transient structure of the teleost tailbud. The forerunners appear to generate the entire structure of Kupffer's vesicle, which also develops in most flh mutants. Although forerunner cells are present in ntl mutants, Kupffer's vesicle never appears, which is correlated with the later severe disruption of tail development.

A homeobox gene essential for zebrafish notochord development.

The notochord is a midline mesodermal structure with an essential patterning function in all vertebrate embryos. Zebrafish floating head (flh) mutants lack a notochord, but develop with prechordal plate and other mesodermal derivatives, indicating that flh functions specifically in notochord development. We show that floating head is the zebrafish homologue of Xnot, a homeobox gene expressed in the amphibian organizer and notochord. We propose that flh regulates notochord precursor cell fate.

Kinematic in-vitro comparison between the normal knee and two techniques for reconstruction of the anterior cruciate ligament.

The influence of anterior cruciate ligament sectioning and the results of its reconstruction on joint kinematics were studied using eight cadaver knees and a computerized system for analysis of three-dimensional motions. A Dacron(®) prosthesis was used in a transcondylar isometric drill-guide-determined route, and in an over-the-top route. After sectioning of the anterior cruciate ligament, the tibial motion increased in several passive laxity tests including anterior/posterior translation as well as varus/valgus and internal/external rotation. Increased valgus rotation was also seen during a simulated active extension manoeuvre. The effect of anterior cruciate ligament reconstruction was seen only on anterior/posterior translation, where the drill-guide technique restored, and the over-the-top technique partially restored, normal sagittal play at 90° of knee flexion. At 30° of knee flexion both methods reduced, but did not normalize, anterior/posterior translation. An adverse effect of both techniques, more prominent with the drill-guide technique which involved an extensive notchplasty, was abnormal external tibia[ rotation in 90° of knee flexion with the tibia in a gravity-determined position. Both techniques resulted in less than normal external rotation in the screw-home mechanism.

In vitro comparison of over-the-top and through-the-condyle anterior cruciate ligament reconstructions.

Knee kinematics, during standard knee examination maneuvers, were measured on 15 fresh cadaveric knees in their normal state and after isolated sectioning of the anterior cruciate ligament using a six degree of freedom electrogoniometer. Proximal iliotibial band autografts were used to perform two anterior cruciate ligament reconstructions on the cadaveric knees: an over-the-top reconstruction and a through-the-condyle procedure. Both of these reconstructions reduced the abnormal anterior translation of the anterior cruciate ligament-deficient knee seen in Lachman testing. However, neither reconstruction restored the normal anterior translation. In addition, other motions remained uncorrected: 1) the internal rotation due to internal torque at full extension; 2) coupled anterior translation during internal rotation at full extension; and 3) coupled medial translation with anterior translation in Lachman testing. There were no statistical differences noted in the joint kinematics created by either reconstruction.

The gene encoding rat insulinlike growth factor-binding protein 1 is rapidly and highly induced in regenerating liver.

The liver is an epithelioid organ that can regenerate following partial hepatectomy. Although it is composed mainly of hepatocytes, it has a complex, multicellular architecture, implying that intercellular communications must exist during regeneration. As in other mitogen-stimulated cells, immediate-early growth response genes induced in the absence of prior protein synthesis are likely to play an important regulatory role in the regenerative process. Through differential screening of regenerating liver cDNA libraries, we found that one of the most highly expressed immediate-early genes in liver regeneration encodes the rat homolog of the low-molecular-weight insulinlike growth factor (IGF)-binding protein (IGFBP-1). This protein has been implicated in enhancing the mitogenic effect of IGF on tissues. IGFBP-1 gene induction is transcriptionally mediated and specific to regenerating liver, as the gene is not expressed in mitogen-stimulated fibroblasts. IGFBP-1 expression has been shown to increase under low-insulin conditions such as diabetes, and the complex regulation of expression is indicated by our finding that insulin treatment of H35 rat hepatoma cells, which induces proliferation, also causes a rapid decrease in transcription and expression of the IGFBP-1 gene. Of note, IGFBP-1 mRNA is abundant in fetal rat liver, implying that it participates in normal liver growth and development. Although regenerating liver cells continue to produce IGF-I, we did not detect IGF-I receptor mRNA during the first 24 h after hepatectomy. However, some IGFBPs may act to enhance the activity of IGF-I independently of IGF-I receptors. Thus, IGF-1 and IGFBPs may interact with hepatocytes or nonparenchymal liver cells, through either IGF-I or novel receptors. In this way, IGFBP-I and IGF-I could act in a paracrine and/or autocrine fashion in maintaining normal liver architecture during regeneration.

The effects of graft tensioning on the laxity and kinematics of the anterior cruciate ligament reconstructed knee.

An in vitro study of eight cadaveric knees was conducted to investigate the effect of initial graft tension on the laxity and full three-dimensional kinematics of the anterior cruciate ligament reconstructed knee. A parallel strand, prototype, expanded polytetrafluoroethylene graft (W. L. Gore and Associates, Flagstaff, AZ, U.S.A.) was used. The graft was placed in the over-the-top position with initial tensions of 18, 36, 54, 72, and 90 N applied with the knee in full extension or at 30 degrees of flexion. The motion of the tibia relative to the femur was measured by a 6 degrees-of-freedom spatial linkage, and the applied forces and moments, the quadriceps force, and the graft tension were measured by load cells. Near normal anterior laxity in the Lachman test was restored with all the tested initial graft tensions. However, over constraint, posterior, lateral, and external tibial subluxation, and abnormalities in joint stiffness developed as the initial graft tension increased. Graft tension-related posterior tibial subluxation resulted in an increase in quadriceps force needed to achieve full extension.

The immediate-early growth response in regenerating liver and insulin-stimulated H-35 cells: comparison with serum-stimulated 3T3 cells and identification of 41 novel immediate-early genes.

Liver regeneration provides a unique system for analysis of mitogenesis in intact, fully developed animals. Cellular immediate-early genes likely play an important role in cell cycle regulation and have been extensively studied in mitogen-stimulated fibroblasts lymphocytes but not in liver. We have begun to characterize the immediate-early growth response genes of mitogen-stimulated liver cells, specifically, regenerating liver and insulin-stimulated Reuber H-35 hepatoma cells, and to address differences in growth response between different cell types. Through subtraction and differential screening of cDNA libraries from regenerating liver and insulin-treated H-35 cells, we have extensively characterized 341 differentially expressed clones and identified 52 immediate-early genes. These genes have been partially sequenced and subjected to Northern (RNA) blot analysis, and 41 appear to be novel. Surprisingly, two-thirds of these genes are also expressed in BALB/c 3T3 cells, but only 10 were identified in previous studies of 3T3 cells, and of these, 6 include well-known genes like jun and fos, and only 4 are novel. Approximately one-third of the immediate-early genes identified in mitogen-stimulated liver cells or serum-stimulated NIH 3T3 cells are expressed in a tissue-specific fashion, indicating that cell type-specific regulation of the proliferative response occurs during the immediate-early period. Our findings indicate that the immediate-early response is unusually complex for the first step in a regulatory cascade, suggesting that multiple pathways must be activated. The abundance of immediate-early genes and the highly varied pattern of their expression in different cell types suggest that the tissue specificity of the proliferative response arises from the particular set of these genes expressed in a given tissue.

Immediate-early gene expression differs between regenerating liver, insulin-stimulated H-35 cells, and mitogen-stimulated Balb/c 3T3 cells. Liver-specific induction patterns of gene 33, phosphoenolpyruvate carboxykinase, and the jun, fos, and egr families.

Immediate-early genes, whose expression increases independent of de novo protein synthesis during the transition from quiescence to proliferation, are postulated to play important regulatory roles in the growth response. The complement of immediate-early genes expressed must depend on the milieu of preexisting transcription factors in the quiescent cell as well as the type of mitogenic stimulation and, thus, may differ between cell types. We have begun characterizing the immediate-early response in regenerating liver and insulin-stimulated Reuber H-35 hepatoma cells in comparison with previously published results from mitogen-stimulated Balb/c 3T3 fibroblasts. The proliferating H-35 and regenerating liver cells maintain their similarity to quiescent liver as demonstrated by their continued production of the liver-specific albumin, CCAAT/enhancer binding protein, and phosphoenolpyruvate carboxykinase messenger RNAs (mRNA). Surprisingly, the phosphoenolpyruvate carboxykinase gene, which undergoes down-regulation in insulin-treated H-35 cells, was cloned by differential screening of a subtraction-enriched regenerating liver cDNA library and is an immediate-early gene in regenerating liver. H-35 cells treated with either insulin or phorbol 12-myristate 13-acetate express elevated levels of the jun genes, and phorbol 12-myristate 13-acetate pretreatment fails to abolish the insulin response, indicating that it does not depend on protein kinase C. jun family gene expression in regenerating liver differs from that in mitogen-treated fibroblasts in that the time course of expression of c-jun and junB is prolonged, and junD mRNA levels distinctly increase. Additionally, although c-fos and egr-1 mRNAs are expressed at elevated levels in stimulated liver cells, fos-B, fra-1, and egr-2 are not, which suggests that factors in addition to the serum response factor participate in the regulation of immediate-early gene induction. Interestingly, gene 33, which was cloned from a regenerating liver cDNA library by differential screening and lacks a recognizable serum response element, functions as an immediate-early gene in regenerating liver and in mitogen-treated H-35 and Balb/c 3T3 cells. These results suggest that gene 33 participates in the transition from quiescence to proliferation in many mitogen-treated cells in addition to its previously reported involvement in hormone responses. Overall, the results presented here suggest that the immediate-early response varies considerably between regenerating liver and mitogen-stimulated fibroblasts and could involve multiple, preexisting, tissue-specific, transcription-activating proteins.

A technique for quantitative assessment of three-dimensional motion with applications to human joints.

A technique is developed for quantitative measurement of general three-dimensional motion, and this technique is applied to the kinematics of anatomical joints. The spatial locations of three orthogonal points representing coordinate frames on each member of the joint are measured during motion of the joint by photo encoders of a three-dimensional mechanical pointer. Kinematic calculations are used to derive, from the experimentally collected data, the six orthogonal components of the motion of one member relative to the other. The accuracy of this technique is presented. Applications to the knee and ankle are discussed.

Anterolateral approach to the hip for bone graft epiphysiodesis in the treatment of slipped capital femoral epiphysis.

In an effort to accommodate the needs of the majority of orthopedic surgeons who might encounter cases of slipped capital femoral epiphysis, an operative approach familiar to everyday practice was sought. Thirty-two cases of slipped capital femoral epiphysis were treated by bone graft epiphysiodesis using an anterolateral approach in contrast to the more traditionally used iliofemoral (Smith-Peterson) approach. Experience has shown this to be a better approach based on reduced operating time, less blood loss, avoidance of damage to the lateral femoral cutaneous nerve, improved wound healing, and for the operating surgeon, familiarity with an exposure utilized in total hip replacement.

A 30-year experience with bone graft epiphysiodesis in the treatment of slipped capital femoral epiphysis.

A retrospective review was carried out of all cases treated by bone graft epiphysiodesis for slipped capital femoral epiphysis at Children's Hospital Medical Center of Akron between the years 1950 and 1980. Only those cases followed for a minimum of 1 year following treatment were included in the review and evaluated as to their status of avascular necrosis, acute cartilage necrosis, or other complications attendant to the surgical approach. One hundred seventy-six patients representing 207 hips underwent bone graft epiphysiodesis over this 30-year period. One hundred fifty-nine patients representing 185 hips were followed a minimum of 1 year and were included in this study. There were 25 cases of acute slipping representing 26 hips and 134 patients representing 159 hips involved with the chronic slipping process. In the acute group of patients representing 26 hips, there were two cases of avascular necrosis and one case of acute cartilage necrosis. In the chronic group of 159 hips, there were four cases of reslipping, one due to graft resorption, one due to placement of the graft short the growth plate, and two that went on to acute slipping. All cases were salvaged with good results. There was one case of avascular necrosis in the chronic group; not a single case of acute cartilage necrosis was encountered. Owing to the rapid nature of growth plate closure following bone graft epiphysiodesis, the avoidance of hardware removal or pin penetration, and the extremely low incidence of overall complications, we suggest that bone graft epiphysiodesis is at least as good as multiple pin fixation in the treatment of slipped capital femoral epiphysis.