Proteomic characterization of human multiple myeloma bone marrow extracellular matrix.

The extracellular matrix (ECM) is a major component of the tumor microenvironment, contributing to the regulation of cell survival, proliferation, differentiation and metastasis. In multiple myeloma (MM), interactions between MM cells and the bone marrow (BM) microenvironment, including the BM ECM, are critical to the pathogenesis of the disease and the development of drug resistance. Nevertheless, composition of the ECM in MM and its role in supporting MM pathogenesis has not been reported. We have applied a novel proteomic-based strategy and defined the BM ECM composition in patients with monoclonal gammopathy of undetermined significance (MGUS), newly diagnosed and relapsed MM compared with healthy donor-derived BM ECM. In this study, we show that the tumor ECM is remodeled at the mRNA and protein levels in MGUS and MM to allow development of a permissive microenvironment. We further demonstrate that two ECM-affiliated proteins, ANXA2 and LGALS1, are more abundant in MM and high expression is associated with a decreased overall survival. This study points to the importance of ECM remodeling in MM and provides a novel proteomic pipeline for interrogating the role of the ECM in cancers with BM tropism.

Lamellipodin promotes invasive 3D cancer cell migration via regulated interactions with Ena/VASP and SCAR/WAVE.

Cancer invasion is a hallmark of metastasis. The mesenchymal mode of cancer cell invasion is mediated by elongated membrane protrusions driven by the assembly of branched F-actin networks. How deregulation of actin regulators promotes cancer cell invasion is still enigmatic. We report that increased expression and membrane localization of the actin regulator Lamellipodin correlate with reduced metastasis-free survival and poor prognosis in breast cancer patients. In agreement, we find that Lamellipodin depletion reduced lung metastasis in an orthotopic mouse breast cancer model. Invasive 3D cancer cell migration as well as invadopodia formation and matrix degradation was impaired upon Lamellipodin depletion. Mechanistically, we show that Lamellipodin promotes invasive 3D cancer cell migration via both actin-elongating Ena/VASP proteins and the Scar/WAVE complex, which stimulates actin branching. In contrast, Lamellipodin interaction with Scar/WAVE but not with Ena/VASP is required for random 2D cell migration. We identified a phosphorylation-dependent mechanism that regulates selective recruitment of these effectors to Lamellipodin: Abl-mediated Lamellipodin phosphorylation promotes its association with both Scar/WAVE and Ena/VASP, whereas Src-dependent phosphorylation enhances binding to Scar/WAVE but not to Ena/VASP. Through these selective, regulated interactions Lamellipodin mediates directional sensing of epidermal growth factor (EGF) gradients and invasive 3D migration of breast cancer cells. Our findings imply that increased Lamellipodin levels enhance Ena/VASP and Scar/WAVE activities at the plasma membrane to promote 3D invasion and metastasis.

Quantifying heritable variation in fitness-related traits of wild, farmed and hybrid Atlantic salmon families in a wild river environment.

Farmed fish are typically genetically different from wild conspecifics. Escapees from fish farms may contribute one-way gene flow from farm to wild gene pools, which can depress population productivity, dilute local adaptations and disrupt coadapted gene complexes. Here, we reanalyse data from two experiments (McGinnity et al., 1997, 2003) where performance of Atlantic salmon (Salmo salar) progeny originating from experimental crosses between farm and wild parents (in three different cohorts) were measured in a natural stream under common garden conditions. Previous published analyses focussed on group-level differences but did not account for pedigree structure, as we do here using modern mixed-effect models. Offspring with one or two farm parents exhibited poorer survival in their first and second year of life compared with those with two wild parents and these group-level inferences were robust to excluding outlier families. Variation in performance among farm, hybrid and wild families was generally similar in magnitude. Farm offspring were generally larger at all life stages examined than wild offspring, but the differences were moderate (5-20%) and similar in magnitude in the wild versus hatchery environments. Quantitative genetic analyses conducted using a Bayesian framework revealed moderate heritability in juvenile fork length and mass and positive genetic correlations (>0.85) between these morphological traits. Our study confirms (using more rigorous statistical techniques) previous studies showing that offspring of wild fish invariably have higher fitness and contributes fresh insights into family-level variation in performance of farm, wild and hybrid Atlantic salmon families in the wild. It also adds to a small, but growing, number of studies that estimate key evolutionary parameters in wild salmonid populations. Such information is vital in modelling the impacts of introgression by escaped farm salmon.

Beaufort trout MicroPlex: a high-throughput multiplex platform comprising 38 informative microsatellite loci for use in resident and anadromous (sea trout) brown trout Salmo trutta genetic studies.

A flexible panel consisting of 38 informative microsatellite markers for Salmo trutta is described. These markers were selected from a pool of over 150 candidate loci that can be readily amplified in four multiplex PCR groups but other permutations are also possible. The basic properties of each markers were assessed in six population samples from both the Burrishoole catchment, in the west of Ireland, and Lough Neagh, in Northern Ireland. A method to assess the relative utility of individual markers for the detection of population genetic structuring is also described. Given its flexibility, technical reliability and high degree of informativeness, the use of this panel of markers is advocated as a standard for S. trutta genetic studies.

Evidence that the biofungicide Serenade (Bacillus subtilis) suppresses clubroot on canola via antibiosis and induced host resistance.

This study investigated how the timing of application of the biofungicide Serenade (Bacillus subtilis QST713) or it components (product filtrate and bacterial cell suspension) influenced infection of canola by Plasmodiophora brassicae under controlled conditions. The biofungicide and its components were applied as a soil drench at 5% concentration (vol/vol or equivalent CFU) to a planting mix infested with P. brassicae at seeding or at transplanting 7 or 14 days after seeding (DAS) to target primary and secondary zoospores of P. brassicae. Quantitative polymerase chain reaction (qPCR) was used to assess root colonization by B. subtilis as well as P. brassicae. The biofungicide was consistently more effective than the individual components in reducing infection by P. brassicae. Two applications were more effective than one, with the biofungicide suppressing infection completely and the individual components reducing clubroot severity by 62 to 83%. The biofungicide also reduced genomic DNA of P. brassicae in canola roots by 26 to 99% at 7 and 14 DAS, and the qPCR results were strongly correlated with root hair infection (%) assessed at the same time (r = 0.84 to 0.95). qPCR was also used to quantify the transcript activity of nine host-defense-related genes in inoculated plants treated with Serenade at 14 DAS for potential induced resistance. Genes encoding the jasmonic acid (BnOPR2), ethylene (BnACO), and phenylpropanoid (BnOPCL and BnCCR) pathways were upregulated by 2.2- to 23-fold in plants treated with the biofungicide relative to control plants. This induced defense response was translocated to the foliage (determined based on the inhibition of infection by Leptosphaeria maculans). It is possible that antibiosis and induced resistance are involved in clubroot suppression by Serenade. Activity against the infection from both primary and secondary zoospores of P. brassicae may be required for maximum efficacy against clubroot.

Phylogeographic structure of brown trout Salmo trutta in Britain and Ireland: glacial refugia, postglacial colonization and origins of sympatric populations.

The phylogeographical structure of brown trout Salmo trutta in Britain and Ireland was studied using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of four mitochondrial DNA segments (16S/ND1, ND5/6, COXIII/ND5 and ND5/12S). Analysis of 3636 individuals from 83 sites-morphotypes revealed a total of 25 haplotypes. These haplotypes were nested in seven two-step clades. Although there was a clear geographical patterning to the occurrence of derived clades, admixture among ancestral clades was extensive throughout the studied area. A relevant feature of the data was that some populations contained mixtures of highly divergent clades. This type II phylogeographic pattern is uncommon in nature. Clade intermixing is likely to have taken place during earlier interglacials as well as since the Last Glacial Maximum. The anadromous life history of many S. trutta populations has probably also contributed to clade mixing. Based on the data presented here and published data, postglacial colonization of Britain and Ireland most likely involved S. trutta from at least five potential glacial refuges. Probable locations for such refugia were: south of England-western France, east of the Baltic Sea, western Ireland, Celtic Sea and North Sea. Ferox S. trutta, as defined by their longevity, late maturation and piscivory, exhibited a strong association with a particular clade indicating that they share a common ancestor. Current evidence indicates that the Lough Melvin gillaroo S. trutta and sonaghen S. trutta sympatric types diverged prior to colonization of Lough Melvin and, although limited gene flow has occurred since secondary contact, they have remained largely reproductively isolated due to inlet and outlet river spawning segregation. Gillaroo S. trutta may reflect descendents of a previously more widespread lineage that has declined due to habitat alterations particularly affecting outlet rivers. The mosaic-like distribution of mtDNA lineages means that conservation prioritization in Britain and Ireland should be based on the biological characteristics of local populations rather than solely on evolutionary lineages.

Cortical bone trajectory for lumbar pedicle screws.

BACKGROUND CONTEXT: Achieving solid implant fixation to osteoporotic bone presents a clinical challenge. New techniques and devices are being designed to increase screw-bone purchase of pedicle screws in the lumbar spine via a novel cortical bone trajectory that may improve holding screw strength and minimize loosening. Preliminary clinical evidence suggests that this new trajectory provides screw interference that is equivalent to the more traditionally directed trajectory for lumbar pedicle screws. However, a biomechanical study has not been performed to substantiate the early clinical results. PURPOSE: Evaluate the mechanical competence of lumbar pedicle screws using a more medial-to-lateral path (ie, "cortical bone trajectory") than the traditionally used path. STUDY DESIGN: Human cadaveric biomechanical study. METHODS: Each vertebral level (L1-L5) was dual-energy X-ray absorptiometry (DXA) scanned and had two pedicle screws inserted. On one side, the traditional medially directed trajectory was drilled and tapped. On the contralateral side, the newly proposed cortical bone trajectory was drilled and tapped. After qCT scanning, screws were inserted into their respective trajectories and pullout and toggle testing ensued. In uniaxial pullout, the pedicle screw was withdrawn vertically from the constrained bone until failure occurred. The contralateral side was tested in the same manner. In screw toggle testing, the vertebral body was rigidly constrained and a longitudinal rod was attached to each screw head. The rod was grasped using a hydraulic grip and a quasi-static, upward displacement was implemented until construct failure. The contralateral pedicle screw was tested in the same manner. Yield pullout (N) and stiffness (N/mm) as well as failure moment (N-m) were compared and bone mineral content and bone density data were correlated with the yield pullout force. RESULTS: New cortical trajectory screws demonstrated a 30% increase in uniaxial yield pullout load relative to the traditional pedicle screws (p=0.080), although mixed loading demonstrated equivalency between the two trajectories. No significant difference in construct stiffness was noted between the two screw trajectories in either biomechanical test or were differences in failure moments (p=0.354). Pedicle screw fixation did not appear to depend on bone quality (DXA) yet positive correlations were demonstrated between trajectory and bone density scans (qCT) and pullout force for both pedicle screws. CONCLUSIONS: The current study demonstrated that the new cortical trajectory and screw design have equivalent pullout and toggle characteristics compared with the traditional trajectory pedicle screw, thus confirming preliminary clinical evidence. The 30% increase in failure load of the cortical trajectory screw in uniaxial pullout and its juxtaposition to higher quality bone justify its use in patients with poor trabecular bone quality.

Cell-matrix adhesion in vascular development.

Vascular development requires correct interactions among endothelial cells, pericytes and surrounding cells. These interactions involve many cell adhesion interactions, including cell-matrix interactions both with basement membranes and with surrounding extracellular matrices. Investigations of the contributions of these various interactions in vascular development and angiogenesis have been rather uneven and incomplete over the past 10-15 years. There has been considerable concentration on a few receptors, matrix proteins and proteolytic fragments with the goal of finding means to control angiogenesis. Many other potential contributors have received much less attention. Even for those molecules that have been subject to intensive investigation, our knowledge is incomplete. This review will survey the spectrum of extracellular matrix (ECM) proteins and cell-matrix adhesion receptors (particularly integrins) that are likely to contribute to angiogenesis and discuss what is known and not known about the roles of each of them.

Beta 3 integrins regulate lymphocyte migration and cytokine responses in heart transplant rejection.

Integrin alpha v beta 3 is important for cell survival, signaling and migration, particularly during angiogenesis and tumorigenesis, where it has been proposed as a therapeutic target. alpha v beta 3 is up-regulated following transplantation and beta 3 polymorphisms are associated with increased acute kidney rejection, suggesting that alpha v beta 3 may also play a role in transplant rejection. Here, using a model of allogeneic heart transplantation, we show that allograft survival is prolonged in beta 3 integrin-deficient (beta 3(-/-)) mice. This is associated with Th2-type immune responses and reduced T-cell infiltration into grafts and T cells from beta 3(-/-) mice show impaired adhesion and migration, consistent with a role for alpha v beta 3 in transmigration. These studies provide evidence that targeting beta 3 integrins impairs recruitment of effector cells and alters cytokine production, so prolonging graft survival. We also show that low doses of blocking antibodies against leukocyte function associated antigen-1 (LFA-1)/alpha L beta 2 and very late antigen-4 (VLA-4)/alpha 4 beta 1, when combined with deletion of beta 3, lead to long-term survival of allografts with no evidence of chronic rejection. Hence we provide strong mechanistic evidence supporting previous genetic studies, demonstrate the involvement of beta 3 integrins in both acute and chronic rejection and identify beta 3 as a new target for immunosuppressive therapy.

Fibrinogen and von Willebrand factor-independent platelet aggregation in vitro and in vivo.

BACKGROUND: Fibrinogen (Fg) has been considered essential for platelet aggregation. However, we recently demonstrated formation of occlusive thrombi in Fg-deficient mice and in mice doubly deficient for Fg and von Willebrand factor (Fg/VWF(-/-)). METHODS AND RESULTS: Here we studied Fg/VWF-independent platelet aggregation in vitro and found no aggregation in citrated platelet-rich plasma of Fg/VWF(-/-) mice. Surprisingly, in Fg/VWF(-/-) plasma without anticoagulant, adenosine diphosphate induced robust aggregation of Fg/VWF(-/-) platelets but not of beta(3)-integrin-deficient (beta(3) (-/-)) platelets. In addition, beta(3) (-/-) platelets did not significantly incorporate into thrombi in Fg/VWF(-/-) mice. This Fg/VWF-independent aggregation was blocked by thrombin inhibitors (heparin, hirudin, PPACK), and thrombin or thrombin receptor activation peptide (AYPGKF-NH(2)) induced aggregation of gel-filtered Fg/VWF(-/-) platelets in 1 mm Ca(2+) PIPES buffer. Notably, aggregation in PIPES buffer was only 50-60% of that observed in Fg/VWF(-/-) plasma. Consistent with the requirement for thrombin in vitro, hirudin completely inhibited thrombus formation in Fg/VWF(-/-) mice. These data define a novel pathway of platelet aggregation independent of both Fg and VWF. Although this pathway was not detected in the presence of anticoagulants, it was observed under physiological conditions in vivo and in the presence of Ca(2+)in vitro. CONCLUSIONS: beta(3) integrin, thrombin, and Ca(2+) play critical roles in this Fg/VWF-independent aggregation, and both plasma and platelet granule proteins contribute to this process.

Expression and localisation of alphav integrins in human odontoblasts.

Integrin alphabeta heterodimers mediate adhesion to the extracellular matrix and at cell-cell contacts and initiate intracellular signalling cascades in response to a variety of inductive factors. Apart from the expression of alphavbeta3 that we have previously reported, little is known about the expression of integrins in odontoblasts. Here, we investigated the expression of alphav-binding beta integrin subunits in healthy human dental pulp in vivo and in odontoblasts differentiated in vitro. Reverse transcription/polymerase chain reaction analysis revealed the expression of alphav, beta1, beta5 and beta8 integrin mRNA, but not beta6, in whole pulp cells. Flow cytometry showed that the alphav and beta1 subunits were the most intensely expressed. Immunohistochemistry demonstrated that the beta1 subunit was localised in newly differentiated odontoblasts in the root and in mature odontoblasts in the crown, including their intradentinal cell processes. The alphav chain was predominantly expressed by mature odontoblasts and alphavbeta5 was only observed in mature odontoblasts. In vitro differentiated odontoblasts expressed genes for alphav, beta1 and beta5, but not for beta6 and beta8. A comparison of integrin profiles between cultured pulp cells and in vitro differentiated odontoblasts revealed that odontoblast maturation was characterised by a significant increase in the expression of alphav and beta1 subunits and alphavbeta5 integrin. The beta8 subunit was detected in nerve cells only. Histological analysis of teeth from alphav knockout mice showed no obvious structural modification in the odontoblast layer. Thus, human mature odontoblasts express alphavbeta3, alphavbeta5 and perhaps alphavbeta1 integrins, with the possible presence of alpha-beta1 pairs. The roles that these molecules play in the exchange of information throughout the odontoblast layer remain to be determined.

Accelerated re-epithelialization in beta3-integrin-deficient- mice is associated with enhanced TGF-beta1 signaling.

The upregulation of TGF-beta1 and integrin expression during wound healing has implicated these molecules in this process, but their precise regulation and roles remain unclear. Here we report that, notably, mice lacking beta(3)-integrins show enhanced wound healing with re-epithelialization complete several days earlier than in wild-type mice. We show that this effect is the result of an increase in TGF-beta1 and enhanced dermal fibroblast infiltration into wounds of beta(3)-null mice. Specifically, beta(3)-integrin deficiency is associated with elevated TGF-beta receptor I and receptor II expression, reduced Smad3 levels, sustained Smad2 and Smad4 nuclear localization and enhanced TGF-beta1-mediated dermal fibroblast migration. These data indicate that alpha(v)beta(3)-integrin can suppress TGF-beta1-mediated signaling, thereby controlling the rate of wound healing, and highlight a new mechanism for TGF-beta1 regulation by beta(3)-integrins.

Thrombospondin-1 gene expression affects survival and tumor spectrum of p53-deficient mice.

In vitro and in vivo data indicate that thrombospondin-1 (TSP1) inhibits tumor progression in several ways including direct effects on cellular growth and apoptosis in the stromal compartment. To evaluate the importance of TSP1 for the progression of naturally arising tumors in vivo, we have crossed TSP1-deficient mice with p53-deficient mice. In p53-null mice, the absence of TSP1 decreases survival from 160 +/- 52 days to 149 +/- 42 days. A log-rank test comparing survival curves for these two populations yields a two-sided P value of 0.0272. For mice that are heterozygous for the p53-null allele, survival is 500 +/- 103 days in the presence of TSP1 expression, and 426 +/- 125 days in its absence (P = 0.0058). Whereas TSP1 expression did not cause a measurable change in the incidence of the majority of tumor types, a statistically significant (P < or = 0.05) decrease in the incidence of osteosarcomas is observed in the absence of TSP1. To determine more directly if host TSP1 inhibits tumor growth, B16F10 melanoma and F9 testicular teratocarcinoma cells have been implanted in C57BL/6J and 129Sv TSP1-null mice, respectively. The B16F10 tumors grow approximately twice as fast in the TSP1-null background and exhibit an increase in vascular density, a decrease in the rate of tumor cell apoptosis, and an increase in the rate of tumor cell proliferation. Increased tumor growth is also observed in the absence of TSP1 on the 129Sv genetic background. These data indicate that endogenous host TSP1 functions as a modifier or landscaper gene to suppress tumor growth.

Thrombospondin-1 suppresses spontaneous tumor growth and inhibits activation of matrix metalloproteinase-9 and mobilization of vascular endothelial growth factor.

Growth of tumors and metastasis are processes known to require neovascularization. To ascertain the participation of the endogenous angiogenic inhibitor thrombospondin-1 (TSP1) in tumor progression, we generated mammary tumor-prone mice that either lack, or specifically overexpress, TSP1 in the mammary gland. Tumor burden and vasculature were significantly increased in TSP1-deficient animals, and capillaries within the tumor appeared distended and sinusoidal. In contrast, TSP1 overexpressors showed delayed tumor growth or lacked frank tumor development (20% of animals); tumor capillaries showed reduced diameter and were less frequent. Interestingly, absence of TSP1 resulted in increased association of vascular endothelial growth factor (VEGF) with its receptor VEGFR2 and higher levels of active matrix metalloproteinase-9 (MMP9), a molecule previously shown to facilitate both angiogenesis and tumor invasion. In vitro, enzymatic activation of proMMP9 was suppressed by TSP1. Together these results argue for a protective role of endogenous inhibitors of angiogenesis in tumor growth and implicate TSP1 in the in vivo regulation of metalloproteinase-9 activation and VEGF signaling.

Rhizobium population dynamics in the pea rhizosphere of rhizobial inoculant strain applied in different formulations.

The effect of inoculant formulation on the population dynamics of rhizobia in the pea rhizosphere was investigated using a streptomycin-resistant mutant of Rhizobium leguminosarum bv. viceae NITRAGIN128C56G (128C56G strR). The isolate was formulated into liquid, peat powder, and granular peat carriers, and was tested on pea at field sites near Saskatoon, Saskatchewan, and Beaverlodge, Alberta, in 1996 and 1997. The liquid and peat powder formulations were applied to seed while the granular inoculant was applied to soil. In three out of four site years, population dynamics were similar among formulations: an initial decline or lag period lasting 2-5 days followed by an increase to approximately 10(5) colony-forming units (CFU)/seedling by 14-28 days after planting (DAP) and, where sampled, a continuing increase from 10(7) to 10(8) CFU/plant at 63 DAP. In these same site years, nodule number (not determined at Beaverlodge in 1997) and nodule occupancy at 60 days were not significantly different among formulations. In contrast, soil populations of 128C56G strR from the liquid formulation declined to near zero by 28 DAP at Beaverlodge in 1996, when soil moisture was excessive in spring because of high rainfall. Populations increased in this treatment after this time, but remained significantly lower than the populations of the other two formulations throughout the sampling period. Pea seed yields were not significantly different among treatments in either year at Beaverlodge, but were significantly higher with granular inoculant than the noninoculated control in Saskatoon. Within inoculated treatments at Saskatoon, there were no significant differences in grain yield.

Functional comparison of the alpha3A and alpha3B cytoplasmic domain variants of the chicken alpha3 integrin subunit.

Integrin alpha3beta1 can be alternatively spliced to generate alpha3A and alpha3B cytoplasmic domain variants that are conserved among vertebrates. To identify distinct functions of these variants, we transfected cells with intact alpha3 integrins or chimeric receptors. alpha3Abeta1 and alpha3Bbeta1 each localized to focal contacts in keratinocytes on an extracellular matrix rich in laminin-5, to which both are known to bind with high affinity. However, alpha3B accumulated intracellularly in keratinocytes on collagen, suggesting that laminin binding may stabilize alpha3Bbeta1 surface expression. Neither alpha3 cytoplasmic domain affected recruitment of chimeric alpha5 integrins to fibronectin-induced focal contacts, and either substituted for the alpha5 cytoplasmic domain in alpha5beta1-mediated cell migration. However, the alpha5/alpha3B chimera localized to cell-cell borders in MDCK or CHO cells to a lesser extent than did the alpha5/alpha3A chimera. To determine whether the alpha3 cytoplasmic domains conferred distinct localization to a nonintegrin protein, we transfected cells with interleukin-2 receptor (IL-2R) chimeras containing the alpha3 cytoplasmic domains. The IL-2R/alpha3A chimera was expressed efficiently on the cell surface, while the IL-2R/alpha3B chimera accumulated intracellularly. Our findings suggest that the alpha3B cytoplasmic domain harbors a retention signal that is regulated in an intact integrin and can alter cell surface expression and distribution of alpha3beta1.

Targeted mutation of TNF receptor I rescues the RelA-deficient mouse and reveals a critical role for NF-kappa B in leukocyte recruitment.

NF-kappaB binding sites are present in the promoter regions of many acute phase and inflammatory response genes, suggesting that NF-kappaB plays an important role in the initiation of innate immune responses. However, targeted mutations of the various NF-kappaB family members have yet to identify members responsible for this critical role. RelA-deficient mice die on embryonic day 15 from TNF-alpha-induced liver degeneration. To investigate the importance of RelA in innate immunity, we genetically suppressed this embryonic lethality by breeding the RelA deficiency onto a TNFR type 1 (TNFR1)-deficient background. TNFR1/RelA-deficient mice were born healthy, but were susceptible to bacterial infections and bacteremia and died within a few weeks after birth. Hemopoiesis was intact in TNFR1/RelA-deficient newborns, but neutrophil emigration to alveoli during LPS-induced pneumonia was severely reduced relative to that in wild-type or TNFR1-deficient mice. In contrast, radiation chimeras reconstituted with RelA or TNFR1/RelA-deficient hemopoietic cells were healthy and demonstrated no defect in neutrophil emigration during LPS-induced pneumonia. Analysis of RNA harvested from the lungs of mice 4 h after LPS insufflation revealed that the induction of several genes important for neutrophil recruitment to the lung was significantly reduced in TNFR1/RelA-deficient mice relative to that in wild-type or TNFR1-deficient mice. These results suggest that TNFR1-independent activation of RelA is essential in cells of nonhemopoietic origin during the initiation of an innate immune response.

Reduced blood vessel formation and tumor growth in alpha5-integrin-negative teratocarcinomas and embryoid bodies.

Embryonic stem (ES) cells-wild-type, heterozygous, or null for alpha5-integrin-were injected ectopically into syngeneic mice to develop teratocarcinomas. alpha5-null-derived teratocarcinomas were significantly smaller than the wild-type or alpha5 heterozygous tumors. Histological analysis revealed the presence of tissues derived from all three germ layers, in all tumors. However, alpha5-null teratocarcinomas displayed less undifferentiated tissue than did the controls. Decreased proliferation and increased apoptosis were observed in the undifferentiated areas of the alpha5-null teratocarcinomas. The expression of extracellular matrix proteins, fibronectin and tenascin-C, and the basement membrane components, laminin, entactin/nidogen, and collagen IV, was similar in the different tumors, although the deposition of these molecules was more disorganized in alpha5-null teratocarcinomas. The absence of alpha5-integrin in the various tissues of the alpha5-null tumors was confirmed by immunohistochemistry. Many vessels, but not all, stained positively for alpha5-integrin, showing that they were host derived. Analysis of the area occupied by vessels revealed, on average, an 8-fold decrease in alpha5-null teratocarcinomas compared with control tumors. Staining for smooth muscle alpha-actin showed that pericytes and smooth muscle cells were recruited around the vessels in all tumors, suggesting similar vessel differentiation. Deposition of EIIIA and EIIIB and fibronectin around the vessels was observed in all tumors. The fact that some, although few, alpha5-integrin-negative vessels existed in alpha5-null tumors indicated that alpha5-/- ES cells could differentiate into endothelial cells. Endothelial cell differentiation and vessel formation were analyzed also in vitro. alpha5-null ES cells were differentiated into embryoid bodies, although they were delayed in growth and attachment. Differentiation into endothelial cells was achieved, but the organization into a complex vasculature was delayed compared with controls. We conclude that alpha5beta1-integrin plays a significant role in vessel formation both in ES cell cultures and in teratocarcinomas. Reduced vascularization likely contributed to the reduced proliferation and increased apoptosis observed in alpha5-null teratocarcinomas.

Cytokinin production by plant growth promoting rhizobacteria and selected mutants.

One of the proposed mechanisms by which rhizobacteria enhance plant growth is through the production of plant growth regulators. Five plant growth promoting rhizobacterial (PGPR) strains produced the cytokinin dihydrozeatin riboside (DHZR) in pure culture. Cytokinin production by Pseudomonas fluorescens G20-18, a rifampicin-resistant mutant (RIF), and two TnphoA-derived mutants (CNT1, CNT2), with reduced capacity to synthesize cytokinins, was further characterized in pure culture using immunoassay and thin layer chromatography. G20-18 produced higher amounts of three cytokinins, isopentenyl adenosine (IPA), trans-zeatin ribose (ZR), and DHZR than the three mutants during stationary phase. IPA was the major metabolite produced, but the proportion of ZR and DHZR accumulated by CNT1 and CNT2 increased with time. No differences were observed between strain G20-18 and the mutants in the amounts of indole acetic acid synthesized, nor were gibberellins detected in supernatants of any of the strains. Addition of 10(-5) M adenine increased cytokinin production in 96- and 168-h cultures of strain G20-18 by approximately 67%. G20-18 and the mutants CNT1 and CNT2 may be useful for determination of the role of cytokinin production in plant growth promotion by PGPR.