Voluntary femoro-tibial subluxations: a benign differential diagnosis in the snapping knee of a child.

PURPOSE: Voluntary femoro-tibial subluxation is a rare entity predominantly found in pretoddlers. It presents as a dynamic phenomenon with uni- or bilateral audible snapping of the knee, often in a context of fatigue or irritation at the end of the day. The aim of the study was to observe the evolution and recovery in these patients. METHODS: Ten children were included. Other causes of dislocating joints and pathologies with snapping of the lateral meniscus were excluded from this study. Six-week immobilisation with a splint at 70° of knee flexion was primarily recommended to all patients. RESULTS: The mean age at onset of symptoms was 10 months. Forty percent of the patients presented with bilateral symptoms. Clinically, four patients were hyperlax. In all patients, subluxations could be reproduced passively by rotating the foot externally and advancing the internal tibial plateau anteriorly. Three of the patients were treated with a dorsal splint and experienced significantly less or cessation of symptoms. Two patients underwent surgery, one for a concomitant anterior cruciate ligament (ACL) rupture and meniscus tear that worsened the symptoms, another for concomitant patella dislocation and a meniscus tear. One patient's parents refused treatment and four patients experienced less symptoms at the time of consultation and were not immobilized. Except for the two patients undergoing surgeries, no sequelae were observed. In general, the symptoms got less frequent when the patient began to walk. CONCLUSION: Voluntary femoro-tibial subluxation in children is a rare and benign condition that often resolves spontaneously without sequelae. The risk of meniscus tear should, however, be considered if subluxations do not cease. LEVEL OF EVIDENCE: IV.

Recovery of arctic tundra from thermal erosion disturbance is constrained by nutrient accumulation: a modeling analysis.

Abstract. We calibrated the Multiple Element Limitation (MEL) model to Alaskan arctic tundra to simulate recovery of thermal erosion features (TEFs) caused by permafrost thaw and mass wasting. TEFs could significantly alter regional carbon (C) and nutrient budgets because permafrost soils contain large stocks of soil organic matter (SOM) and TEFs are expected to become more frequent as the climate warms. We simulated recovery following TEF stabilization and did not address initial, short-term losses of C and nutrients during TEF formation. To capture the variability among and within TEFs, we modeled a range of post-stabilization conditions by varying the initial size of SOM stocks and nutrient supply rates. Simulations indicate that nitrogen (N) losses after the TEF stabilizes are small, but phosphorus (P) losses continue. Vegetation biomass recovered 90% of its undisturbed C, N, and P stocks in 100 years using nutrients mineralized from SOM. Because of low litter inputs but continued decomposition, younger SOM continued to be lost for 10 years after the TEF began to recover, but recovered to about 84% of its undisturbed amount in 100 years. The older recalcitrant SOM in mineral soil continued to be lost throughout the 100-year simulation. Simulations suggest that biomass recovery depended on the amount of SOM remaining after disturbance. Recovery was initially limited by the photosynthetic capacity of vegetation but became co-limited by N and P once a plant canopy developed. Biomass and SOM recovery was enhanced by increasing nutrient supplies, but the magnitude, source, and controls on these supplies are poorly understood. Faster mineralization of nutrients from SOM (e.g., by warming) enhanced vegetation recovery but delayed recovery of SOM. Taken together, these results suggest that although vegetation and surface SOM on TEFs recovered quickly (25 and 100 years, respectively), the recovery of deep, mineral soil SOM took centuries and represented a major ecosystem C loss.

Recovery from disturbance requires resynchronization of ecosystem nutrient cycles.

Nitrogen (N) and phosphorus (P) are tightly cycled in most terrestrial ecosystems, with plant uptake more than 10 times higher than the rate of supply from deposition and weathering. This near-total dependence on recycled nutrients and the stoichiometric constraints on resource use by plants and microbes mean that the two cycles have to be synchronized such that the ratio of N:P in plant uptake, litterfall, and net mineralization are nearly the same. Disturbance can disrupt this synchronization if there is a disproportionate loss of one nutrient relative to the other. We model the resynchronization of N and P cycles following harvest of a northern hardwood forest. In our simulations, nutrient loss in the harvest is small relative to postharvest losses. The low N:P ratio of harvest residue results in a preferential release of P and retention of N. The P release is in excess of plant requirements and P is lost from the active ecosystem cycle through secondary mineral formation and leaching early in succession. Because external P inputs are small, the resynchronization of the N and P cycles later in succession is achieved by a commensurate loss of N. Through succession, the ecosystem undergoes alternating periods of N limitation, then P limitation, and eventually co-limitation as the two cycles resynchronize. However, our simulations indicate that the overall rate and extent of recovery is limited by P unless a mechanism exists either to prevent the P loss early in succession (e.g., P sequestration not stoichiometrically constrained by N) or to increase the P supply to the ecosystem later in succession (e.g., biologically enhanced weathering). Our model provides a heuristic perspective from which to assess the resynchronization among tightly cycled nutrients and the effect of that resynchronization on recovery of ecosystems from disturbance.

The ETS family member Tel antagonizes the Fli-1 phenotype in hematopoietic cells.

The ETS family member Tel is rearranged in human leukemia of both myeloid and lymphoid origin while the ETS member Fli-1 is insertionally activated in Friend erythroleukemia in mice and is translocated to the EWS locus in Ewing's sarcoma. In previous studies we demonstrated that Tel binds to Fli-1 and blocks transactivation of megakaryocytic promoters by Fli-1. In this study we demonstrate that expression of Fli-1 in the leukemia cell line K562 induces a megakaryocytic phenotype and the expression of the platelet markers GPIX, GP1balpha, and GPIIb. Introduction of Tel blocked the megakaryocytic phenotype induced by Fli-1, suggesting a biological correlation to the biochemical interaction of Tel and Fli-1 reported previously.

Modelling carbon responses of tundra ecosystems to historical and projected climate: a comparison of a plot- and a global-scale ecosystem model to identify process-based uncertainties.

We are developing a process-based modelling approach to investigate how carbon (C) storage of tundra across the entire Arctic will respond to projected climate change. To implement the approach, the processes that are least understood, and thus have the most uncertainty, need to be identified and studied. In this paper, we identified a key uncertainty by comparing the responses of C storage in tussock tundra at one site between the simulations of two models - one a global-scale ecosystem model (Terrestrial Ecosystem Model, TEM) and one a plot-scale ecosystem model (General Ecosystem Model, GEM). The simulations spanned the historical period (1921-94) and the projected period (1995-2100). In the historical period, the model simulations of net primary production (NPP) differed in their sensitivity to variability in climate. However, the long-term changes in C storage were similar in both simulations, because the dynamics of heterotrophic respiration (RH ) were similar in both models. In contrast, the responses of C storage in the two model simulations diverged during the projected period. In the GEM simulation for this period, increases in RH tracked increases in NPP, whereas in the TEM simulation increases in RH lagged increases in NPP. We were able to make the long-term C dynamics of the two simulations agree by parameterizing TEM to the fast soil C pools of GEM. We concluded that the differences between the long-term C dynamics of the two simulations lay in modelling the role of the recalcitrant soil C. These differences, which reflect an incomplete understanding of soil processes, lead to quite different projections of the response of pan-Arctic C storage to global change. For example, the reference parameterization of TEM resulted in an estimate of cumulative C storage of 2032 g C m-2 for moist tundra north of 50°N, which was substantially higher than the 463 g C m-2 estimated for a parameterization of fast soil C dynamics. This uncertainty in the depiction of the role of recalcitrant soil C in long-term ecosystem C dynamics resulted from our incomplete understanding of controls over C and N transformations in Arctic soils. Mechanistic studies of these issues are needed to improve our ability to model the response of Arctic ecosystems to global change.

Regulation of the megakaryocytic glycoprotein IX promoter by the oncogenic Ets transcription factor Fli-1.

Glycoprotein (GP) IX is a subunit of the von Willebrand receptor, GPIb-V-IX, which mediates adhesion of platelets to the subendothelium of damaged blood vessels. Previous characterization of the GPIX promoter identified a functional Ets site that, when disrupted, reduced promoter activity. However, the Ets protein(s) that regulated GPIX promoter expression was unknown. In this study, transient cotransfection of several GPIX promoter/reporter constructs into 293T kidney fibroblasts with a Fli-1 expression vector shows that the oncogenic protein Fli-1 can transactivate the GPIX promoter when an intact GPIX Ets site is present. In addition, Fli-1 binding of the GPIX Ets site was identified in antibody supershift experiments in nuclear extracts derived from hematopoietic human erythroleukemia cells. Comparative studies showed that Fli-1 was also able to transactivate the GPIbalpha and, to a lesser extent, the GPIIb promoter. Immunoblot analysis identified Fli-1 protein in lysates derived from platelets. In addition, expression of Fli-1 was identified immunohistochemically in megakaryocytes derived from CD34(+) cells treated with the megakaryocyte differentiation and proliferation factor, thrombopoietin. These results suggest that Fli-1 is likely to regulate lineage-specific genes during megakaryocytopoiesis.

ESR study of plasmatic membrane of the transplantable melanoma cells in relation to their biological properties.

Using the spin labelling method we studied changes in the structure and dynamics of molecular mobility in the plasmatic membrane accompanied by a spontaneous alteration of a melanotic melanoma line into an amelanotic form with a higher growth rate, changed antigenicity and immunogenicity. The calculated ratio of the low-field line (A) intensity to the central line (C) intensity of the spectrum showed statistically significant differences in the order of parameters in the plasmatic membranes of both forms of melanocytes. The significantly broader central line (deltaW0) in the spectra of labelled amelanotic melanoma cells than in the original cell line indicated changes in the membrane structure leading to a lowering of the degree of order in the phospholipid bilayer. It has been suggested that a progression of transplantable melanomas is accompanied by an increase in membrane fluidity and reduction in molecular mobility dynamics within it.

The ets family member Tel binds to the Fli-1 oncoprotein and inhibits its transcriptional activity.

The tel gene, recently shown to be translocated in a spectrum of acute and chronic human leukemias, belongs to the ets family of sequence-specific transcription factors. To determine the role of Tel in normal hematopoietic development, we used the tel gene as the bait in the yeast two-hybrid system to screen a hematopoietic stem cell library. Two partners were identified: Tel binds to itself, and Tel binds to the ets family member Fli-1. In vitro and in vivo assays confirmed these interactions. In transient transfection assays, Fli-1 transactivates megakaryocytic specific promoters, and Tel inhibits this effect of Fli-1. Transactivation studies using deletion mutants of Tel, and the Tel-AML-1 fusion protein, indicate that the helix-loop-helix domain of Tel only partially inhibits transactivation and that complete inhibition requires the full-length Tel molecule, including the DNA binding domain. The Tel and Fli-1 proteins are expressed early in hematopoiesis, and the inability of Tel fusion proteins such as Tel-AML-1 to counteract Fli-1 mediated transactivation may contribute to the malignant phenotype in human leukemias where this fusion protein is present.

Human NK cells demonstrate increased adherence and lysis of porcine endothelium following activation with IL-2.

BACKGROUND: For clinical xenotransplantation of pig organs to be successful, the significant barriers of host cellular immunity against the pig will need to be overcome. Natural killer (NK) cells appear to play an important role in rejection of discordant xenografts, and IL-2 activated human NK cells are cytotoxic to human endothelium. We addressed the role of IL-2 activated human and baboon NK cells in the primate immune response to pig xenoantigens in vivo and in vitro. METHODS: Human natural killer (NK) cells were isolated from pooled human peripheral mononuclear cells (PBMC) using density gradient centrifugation method. Phenotype of isolated cells was confirmed by flow cytometry. Purified NK cells were than tasted for spontaneous and IL-2 augmented adhesion and lysis of cultured pig aortic endothelial cells (PAEC) monolayers in a short-term 51 Cr-release assay. The effects of human macrophage derived cytokines TNF alpha and IL-1 on adhesion of IL-2 activated NK cells were studied. RESULTS: Human NK cells demonstrate prominent adherent and lytic activity against pig endothelium, which can be significantly augmented by IL-2. Macrophage derived cytokines can further augment the adherent properties of these NK cells. CONCLUSIONS: These result suggest that human NK cells are likely to play a significant role in rejection of pig-to-human xenografts.

Human leukocyte integrin CD18 promoter directs low levels of expression of a mutated human CD4 reporter gene in leukocytes of transgenic mice.

The human leukocyte integrin CD18 molecule exists on the leukocyte surface in heterodimeric complexes with individual CD11 subunits, which mediate important leukocyte adhesion reactions. The CD18 subunit is developmentally regulated with the highest levels present on mature leukocytes of all lineages. To identify the regulatory sequences responsible for the tissue- and stage-specific expression of the CD18 subunit, we used 3.5 kb of regulatory sequence upstream from the human CD18 gene transcription start site to drive expression of a modified human CD4 reporter gene in transgenic mice. Despite the inclusion of Sp1 and PU.1 sites in the construct, and the generation of founder lines possessing multiple copies of the transgene, the reporter gene was expressed in low levels in the leukocytes of the transgenic mice. These studies indicate that although PU.1 and Sp1 sites are required for CD18 promoter activity in vitro, additional regulatory regions appear to be required for high levels of copy number dependent expression in vivo.

Cloning of two cDNAs encoding calnexin-like and calreticulin-like proteins from maize (Zea mays) leaves: identification of potential calcium-binding domains.

Two cDNAs encoding calnexin (Cln)-like and calreticulin (Crl)-like proteins have been isolated by immunoscreening of a maize leaf cDNA library. In the deduced amino acid (aa) sequences, several regions that are conserved for Cln and Crl proteins from all sources have been identified. These regions can be classified into two distinct motifs which are repeated four times each in Cln and three times each in Crl sequences. One of these motifs, containing a highly acidic 17-aa sequence, has high homology to a Ca(2+)-binding domain previously characterized in both Cln and Crl from mammalian tissues. Motifs for retention in endoplasmic reticulum (Crl) and for an integral membrane-spanning sequence (Cln) have also been identified.

Increased sensitivity to MHC-nonrestricted lysis of BL6 melanoma cells by transfection with class I H-2Kb gene.

An H-2Kb- negative clone of BL6 melanoma (BL6-8) was transfected with neor, H-2Kb, or H-2IAk genes. In an 18-h cytotoxicity assay clones with high levels of H-2Kb Ag expression were found more sensitive to lysis by spleen cells of syngenic and allogeneic mice than H-2Kb low clones. NK cells were involved in the lysis of H-2Kb+ BL6 melanoma clones, with spleen cell cytotoxicity of mice increased after poly I:C stimulation or decreased after pretreatment with anti-asialo GM1 serum or NK1.1 mAb. Anti-TNF Ab were also able to reduce the cytotoxicity of normal spleen cells and completely abolished the cytotoxicity of the NK-depleted spleen cells suggesting involvement of NC cells in lysis of H-2Kb+ BL6 melanoma clones. Increase in sensitivity of H-2Kb+ BL6 cells to natural cell-mediated cytotoxicity was associated with the appearance of NK recognizable determinants as assessed by the cold target inhibition assay. All BL6 clones, irrespective of sensitivity to natural cell-mediated cytotoxicity, showed high sensitivity to lysis by LGL-derived granules. In contrast, all H-2Kb low BL6 clones were resistant and all H-2Kb highly positive clones were sensitive to lysis by TNF-alpha. When an H-2Kb highly positive clone was selected in vitro for resistance to TNF, it concomitantly showed increased resistance to cytotoxicity by spleen cells, confirming the importance of TNF in spleen cell cytotoxicity against H-2Kb+ melanoma cells. Taken together, the data indicate that class I H-2Kb but not class II H-2IAk gene product could increase the sensitivity of BL6 cells to lysis by NK and natural cytotoxic cells as well as TNF. We hypothesize that these effects could be due to pleiotropic effects of H-2Kb gene products on various biologic properties of BL6 melanoma cells some of which may be more directly involved in regulation of tumor cell sensitivity to lysis by NK and/or natural cytotoxic cells.

A bacteriophage-associated glycanase cleaving beta-pyranosidic linkages of 3-deoxy-D-manno-2-octulosonic acid (KDO).

A bacteriophage growing on Escherichia coli K13, K20, and K23 strains carries a glycanase that catalyzes the hydrolytic cleavage of the beta-ketopyranosidic linkages of 3-deoxy-D-manno-2-octulosonic acid (KDO) in the respective capsular polysaccharides. The main cleavage product of the K23 polysaccharide has been identified by 1H- and 13C-n.m.r. spectroscopy as beta beta Ribfl----7 beta KDOp2----3-beta Ribfl----7KDO. Cleavage of polysaccharides containing alpha-pyranosidic, or 5-substituted beta-pyranosidic KDO is not catalyzed by the enzyme.

Degradation of Citrobacter O-Serogroup Ci23Vi+ murein with Vi phages.

Dialysable products resulting from the digestion of the Citrobacter O-Serogroup Ci23Vi+ murein with Vi phages show a decrease of their reducing power/muropeptides. It is accompanied with an increase of free amino groups amount/muropeptide when compared to the one obtained after the lysozyme treatment. It leads to a suggestion, that Vi phage particles possess two enzyme activities: "lysozyme-like" and deacetylase which might be a transacetylase.

Substrate specificity of two bacteriophage-associated endo-N-acetylneuraminidases.

For Escherichia coli Bos12 (O16:K92:H-), a bacteriophage (phi 92) has been isolated which carries a depolymerase active on the K92 capsular polysaccharide. As seen under the electron microscope, phi 92 belongs to Bradley's morphology group A and is different from the phage phi 1.2 previously described (Kwiatkowski et al., J. Virol. 43:697-704, 1982), which grows on E. coli K235 (O1:K1:H-), depolymerizes colominic acid, and belongs to morphology group C. The specificity of the phi 1.2- and phi 92-associated endo-N-acetylneuraminidases has been studied with respect to the following substrates (all alkali treated, and where NeuNAc represents N-acetylneuraminic acid): (i) [-alpha-NeuNAc-(2 leads to 8)-]n (colominic acid), (ii) [-alpha-NeuNAc-(2 leads to 8)-alpha-NeuNAc-(2 leads to 9)-]n (E. coli K92 polysaccharide), and (iii) [-alpha-NeuNAc-(2 leads to 9)-]n (Neisseria meningitidis type C capsular polysaccharide). The increase in periodate consumption of these glycans upon incubation with purified phi 1.2 or phi 92 particles was measured, and the split products obtained from all substrates after exhaustive degradation were analyzed by gel chromatography. It was found that the Neisseria polysaccharide is not appreciably affected by either virus enzyme and that phi 1.2 only depolymerizes a small fraction of the K92 glycan. Colominic acid, however, is completely degraded by both agents, phi 92 yielding smaller fragments (one to six NeuNAc residues) than phi 1.2 (two to seven). Phage phi 92 additionally depolymerizes the K92 glycan, essentially to oligosaccharides of two, four, and six residues. The size distribution of these K92 oligosaccharides indicates that the phi 92 enzyme predominantly cleaves the alpha(2 leads to 8) linkages in this polymer.

Endo-N-acetylneuraminidase associated with bacteriophage particles.

A bacteriophage (phi 1.2) has been isolated for Escherichia coli K235 (O1:K1:H-). phi 1.2 is specific for the host capsular polysaccharide (colominic acid). The phage forms plaques with acapsular halos and thus carries a glycanase activity for colominic acid, a homopolymer of alpha (2 leads to 8)-linked N-acetylneuraminic acid (NeuNAc) residues. Upon incubation with purified phi 1.2 particles, a solution of K1 polysaccharide loses viscosity and consumes increasing amounts of periodate. Also, by gel filtration, the production of colominic oligosaccharides (down to a size of two to three NeuNAc residues) can be demonstrated. No NeuNAc monomers, however, are formed. The capsules of E. coli strains with the K92 antigen, which consists of NeuNAc residues linked by alternating alpha (2 leads to 8) and alpha (2 leads to 9) bonds, are also depolymerized by the phi 1.2 enzyme. Under the electron microscope, phage phi 1.2 is seen to belong to Bradley's morphology group C (D. E. Bradley, Bacteriol. Rev. 31:230-314, 1967); it has an isometric head, carrying a baseplate with six spikes. By analogy to other virus particles with host capsule depolymerase activity, it is probable that the phi 1.2 endo-N-acetylneuraminidase activity is associated with these spikes.

The murein of citrobacter O-serogroup Ci23 Vi+.

The isolation and analysis of the Citrobacter O-serogroup Ci23Vi+ murein are described. The murein consists of alanine, glutamic acid, diaminopimelic acid (occurring in the molar ratio 1.5 : 1: 0.9), N-acetylmuramic acid and N-acetylglucosamine. Dialysable products resulting from the digestion of the Citrobacter O-serogroup Ci23Vi+ murein with egg white lysozyme resemble closely those obtained from the E. coli B murein.

On the deacetylase activity of Vi bacteriophage III particles.

1. Using the complete phage particles as an enzyme, O-acetyl (1 leads to 4)-alpha-D-galacturonan (acetylated pectic acid) as a substrate, and gas-liquid-chromatography for the determination of the acid liberated, the virus-catalysed deacetylation of the polymer was studied. The activity was found to be stable up to about 50 degrees C, and from pH 4.5 to 9, with an optimum at pH 7.8; it was not affected by EDTA, or by 1,10-phenanthroline. The initial reaction velocity (at 37 degrees C) exhibited a simple hyperbolical dependence on the substrate concentration, with Km = 10.5 mM for O-acetyl (independent of virus concentration), and Vmax = 15 nmoles/min and 10(10) plaque forming units. The reaction was, however, rapidly inhibited by a partially deacetylated product (but neither by acetate, nor by pectic acid itself). 2. Using the natural substrate, acetylated (1 leads to 4)-2 amino-2-deoxy-alpha-D-galacturonan (Vi polysaccharide, Vi antigen), and a variety of structural analogues, the following conclusions about the substrate specificity of the Vi phage III deacetylase (acetyl-alpha-1,4-galacturonan acylhydrolase) were reached: (a) acetylated galacturonan is as good a substrate as acetylated aminogalacturonan; (b) of the two substrate diastereomers, acetylated alpha-L-guluronan (also 1 ax leads to 4 ax-linked units, but with axial acetyl residues at C-3), and beta-D-mannuronan (1 eq leads to 4 eq-linkages, and axial acetyl groups at C-2), only the former was acted upon, possibly indicating a specificity for the conformation of the polymer rather than for the configuration of the single residues; (c) all acyl analogues tested, O-monofluoroacetyl, O-propionyl, and O-butyryl galacturonan, were inert, showing a high degree of specificity for O-acetyl; (d) the oligomers, acetylated tri- and digalacturonic acid, as well as methyl-alpha-D-galacturonide, were still deacetylated, although more slowly, demonstrating tolerance of the enzyme of substrate size.