Sonic hedgehog restricts adhesion and migration of neural crest cells independently of the Patched- Smoothened-Gli signaling pathway.

In the vertebrate embryo, neural cell types are organized spatially along the dorsoventral axis of the neural tube and differ by expression of cell-intrinsic determinants and by their adhesive and locomotory properties. Thus, dorsally, neural crest cells (NCC) show a strong propensity to disperse and migrate, whereas cells situated ventrally are highly cohesive and poorly motile. Members of the bone morphogenetic proteins have been shown to exert a dual role in the specification of dorsal neuroepithelial cells and in the dispersion of NCCs. To test whether Sonic hedgehog (Shh), another signaling molecule involved in the patterning of the ventral neural tube, might also contribute to the control of the adhesive and migratory potential of neuroepithelial cells, we analyzed the effect of ectopic Shh on NCC dispersion from neural tube explants cultured in vitro. The addition of Shh to the migration substrate of NCC caused inhibition of their dispersion. The effect of Shh on cell migration was reversible and was not accounted for by alterations of the specification, delamination, proliferation, and survival of NCCs but could be essentially attributed to a decreased cell-substrate adhesion mediated by integrins. In addition, Shh activity on cell migration was mediated by a specific N-terminal region of the molecule and was independent from the signaling cascade elicited by the Patched-Smoothened receptor and involving the Gli transcription factors. Our study therefore reveals an unanticipated role for Shh in regulating adhesion and migration of neuroepithelial cells that is discernable from its inductive, mitogenic, and trophic functions.

The morphogen Sonic hedgehog is an indirect angiogenic agent upregulating two families of angiogenic growth factors.

Sonic hedgehog (Shh) is a prototypical morphogen known to regulate epithelial/mesenchymal interactions during embryonic development. We found that the hedgehog-signaling pathway is present in adult cardiovascular tissues and can be activated in vivo. Shh was able to induce robust angiogenesis, characterized by distinct large-diameter vessels. Shh also augmented blood-flow recovery and limb salvage following operatively induced hind-limb ischemia in aged mice. In vitro, Shh had no effect on endothelial-cell migration or proliferation; instead, it induced expression of two families of angiogenic cytokines, including all three vascular endothelial growth factor-1 isoforms and angiopoietins-1 and -2 from interstitial mesenchymal cells. These findings reveal a novel role for Shh as an indirect angiogenic factor regulating expression of multiple angiogenic cytokines and indicate that Shh might have potential therapeutic use for ischemic disorders.

Sonic hedgehog induces the proliferation of primitive human hematopoietic cells via BMP regulation.

A pool of stem cells that arise from the mesoderm during embryogenesis initiates hematopoiesis. However, factors that regulate the expansion of blood stem cells are poorly understood. We show here that cytokine-induced proliferation of primitive human hematopoietic cells could be inhibited with antibodies to hedgehog (Hh). Conversely, Sonic hedgehog (Shh) treatment induced the expansion of pluripotent human hematopoietic repopulating cells detected in immunodeficient mice. Noggin, a specific inhibitor of bone morphogenetic protein 4 (BMP-4), was capable of inhibiting Shh-induced proliferation in a similar manner to anti-Hh; however, anti-Hh had no effect on BMP-4-induced proliferation. Our study shows that Shh functions as a regulator of primitive hematopoietic cells via mechanisms that are dependent on downstream BMP signals.

The reliability of cryoSEM for the observation and quantification of xylem embolisms and quantitative analysis of xylem sap in situ.

The reliability of cryoSEM for visualizing gas embolisms in xylem vessels of intact, functioning roots is examined and discussed. The possibility that these embolisms form as a result of freezing water columns under tension is discounted by a double-freeze experiment. Two regions of the same root, one frozen under tension, the other isolated from the tension by the first freeze, had the same percentage of embolisms, as did also long pieces of root frozen simultaneously along their length. The reliability of energy-dispersive X-ray analysis to measure xylem sap concentration in situ in frozen tissue was established by measurement of KCl standard solution frozen on stubs, and within xylem vessels. Solute heterogeneity within the vessels varied with freezing procedure; deep-freeze > LN2 > cryopliers > liquid ethane, but only the deep-freeze method gave unsatisfactory estimates of concentration for the standard solution. It is concluded that cryoanalytical SEM is useful for direct observation of gas and liquid-filled compartments, and for solute analyses at depth within intact plant organs.

Quantitative characterizations of speech rhythm: syllable-timing in Singapore English.

British English and Singapore English are said to differ in rhythmic patterning. British English is commonly described as stress-timed, but Singapore English is claimed to be syllable-timed. In the present paper, we explore the acoustic nature of the suggested cross-varietal difference. In directly comparable samples from British English and Singapore English, two types of acoustic measurements were taken; we calculated a variability index reflecting changes in vowel length over utterances, and measurements reflecting vowel quality. Our findings provide acoustic data which support the hypothesized cross-varietal difference in rhythmic patterning; we show (1) that successive vowel durations are more nearly equal in Singapore English than in British English, and (2) that reduced vowels pattern more peripherally in the F1/F2 formant space in Singapore English than in British English. We complete the paper with a comparison of our vowel variability index with a set of acoustic measures for rhythm proposed by Ramus, Nespor, and Mehler (1999), which focus on variability in vocalic and intervocalic intervals. We conclude that our variability index is more successful in capturing rhythmic differences than Ramus et al. (1999)'s measures, and that an application of our index to Ramus et al.'s intervocalic measure may provide a further diagnostic of rhythmic class.

Characterization of antihuman IFNAR-1 monoclonal antibodies: epitope localization and functional analysis.

The type I interferon receptor (IFNAR) is composed of two subunits, IFNAR-1 and IFNAR-2, encoding transmembrane polypeptides. IFNAR-2 has a dominant role in ligand binding, but IFNAR-1 contributes to binding affinity and to differential ligand recognition. A panel of five monoclonal antibodies (mAb) to human IFNAR-1 (HuIFNAR-1) was produced and characterized. The reactivity of each mAb toward HuIFNAR-1 on native and transfected cells and in Western blot and ELISA formats was determined. In functional assays, one mAb, EA12, blocked IFN-a2 binding to human cells and interfered with Stat activation and antiviral activity. Epitopes for the mAb were localized to subdomains of the HuIFNAR-1 extracellular domain by differential reactivity of the mAb to a series of human/bovine IFNAR-1 chimeras. The antibody EA12 seems to require native HuIFNAR-1 for reactivity and does not map to a single subdomain, perhaps recognizing an epitope containing noncontiguous sequences in at least two subdomains. In contrast, the epitopes of the non-neutralizing mAb FB2, AA3, and GB8 mapped, respectively, to the first, second, and third subdomains of HuIFNAR-1. The mAb DB2 primarily maps to the fourth subdomain, although its reactivity may be affected by other determinants.

Bovine type I interferon receptor protein BoIFNAR-1 has high-affinity and broad specificity for human type I interferons.

The type I interferon receptor (IFNAR) is composed of two transmembrane polypeptides, IFNAR-1 and IFNAR-2. Human IFNAR-1 has low intrinsic affinity for IFNs, but enhances the affinity for IFNs of the complex over that of HuIFNAR-2 alone, and modulates the ligand specificity. Bovine cells respond to human alpha interferons. The bovine homologue of HuIFNAR-1, BoIFNAR-1, when expressed in heterologous cells, confers high-affinity binding and broad specificity for human type I IFNs. A soluble fusion protein of the ectodomain of BoIFNAR-1 and an immunoglobulin Fc domain was produced. In contrast to HuIFNAR-1, this protein competes strongly with human cells for IFN binding, and directly binds a wide spectrum of human type I IFNs, including diverse IFN-alphas, IFN-beta and IFN-omega, with moderate to high affinity. This accounts for much of the specificity for human IFNs possessed by bovine cells, with several exceptions. The BoIFNAR-1 ectodomain, in contrast to HuIFNAR-1, may be useful for studies of binary and ternary complexes with IFNs and IFNAR-2, and for purification, assay and biological neutralization protocols.

Daily embolism and refilling of xylem vessels in the roots of field-grown maize.

Embolisms in the vessels of maize axile roots of different types were observed directly after rapid freezing of intact, functioning roots in the field, by cryo-scanning electron microscopy. Quantification of the degree of embolization in each root was made by counting empty and full vessels of both the late and early metaxylem (LMX & EMX), and expressed as percent embolized vessels of the LMX, and %EMX poles containing embolized vessels. Contents of the connecting xylem (CX) at branch root junctions, and of xylem in branch roots were observed also, but not systematically quantified. Records of % embolized vessels were made from dawn to dusk on summer days in Ottawa under moderate irradiance, and in Canberra under high irradiance. Measurements in Canberra were supported by estimates of irradiance, of stomatal conductance, and of chamber balance pressure of bagged and unbagged leaves. Soon after sunrise embolisms appeared in all types of vessel, at balance pressures c. 300-400 kPa, and increased rapidly with increasing irradiance. During the middle of the day % embolized vessels reached a maximum (LMX ≈70% in Ottawa, and ≈80% in Canberra). At all times the EMX vessels were less embolized. The midday maximum was brief in Ottawa, and % embolized vessels fell to a low value during the afternoon. In Canberra the maximum was prolonged into late afternoon. By dusk nearly all vessels were once again filled with sap. The balance pressures measured during vessel refilling in Canberra ranged from 500 kPa to 1200 kPa. At all times of the day sap was seen entering some embolized vessels. Almost all were refilling by mid- to late-afternoon. Such refilling was especially frequent at junctions of branch roots with the axile roots. X-ray microanalysis of the sap entering the vessels, and of the liquid filling or partly filling vessels, showed the concentration of mineral solutes present in the sap was below the threshold of detection (≈12 mM). These results are discussed in relation to current opinions about embolisms and vessel refilling.

Osteopontin N-terminal domain contains a cryptic adhesive sequence recognized by alpha9beta1 integrin.

Osteopontin is an adhesive glycoprotein implicated in numerous diseases associated with inflammation and remodeling. There are several structural domains in osteopontin that are of particular interest. The RGD motif is a cell attachment sequence shown to be critical for cell adhesion through alphav-containing integrins. In close proximity to the RGD domain is the thrombin cleavage site. Previous observations suggest that thrombin cleavage of osteopontin occurs in vivo and may be physiologically important. To study the functional significance of osteopontin cleavage by thrombin, we made glutathione S-transferase-osteopontin fusion proteins. These proteins contain either the N- or C-terminal domains expected to be formed following thrombin cleavage at the Arg169-Ser170 peptide bond. We compared these osteopontin fragments with native osteopontin in their ability to support adhesion of several different cell lines and identified the receptors mediating these interactions. Our data show that the N-terminal osteopontin fragment, which contains the RGD domain, supports adhesion of a melanoma cell line that is unable to bind native osteopontin. This suggests that osteopontin adhesive interactions may be regulated by thrombin cleavage. We also demonstrate that osteopontin contains a cryptic binding activity, which can be recognized by a novel osteopontin receptor. This receptor has been identified as the alpha9beta1 integrin.

Configuration of the interferon-alpha/beta receptor complex determines the context of the biological response.

Constituents of the Type 1 interferon (IFN) receptor (IFNABR) identified to date include the alpha and beta transmembrane subunits and the associated intracellular kinases, Jak 1 and Tyk 2. In this report, we demonstrate that a human cell type that expresses both subunits of IFNABR, together with Jak 1 and Tyk 2, exhibits a limited binding capacity for and is only partially sensitive to the effects of IFN-alpha/beta, despite adequate levels of the cytoplasmic transcription factors Stat1, Stat2, and Stat3. Specifically, a low affinity interaction between IFN-alpha/beta and cell surface receptors results in ISGF3 (Stat1:2) activation and an antiviral response, yet no IFN-inducible growth inhibition. Using a panel of murine cells that are variably configured with respect to the human IFNABR-alpha/beta subunits, we provide evidence that an additional component(s) encoded on human chromosome 21 is required to confer high affinity binding and IFN-inducible growth inhibition to cells that express the alpha and beta subunits of the IFNABR. The data indicate that transcriptional activation that leads to an antiviral response is mediated by IFN-alpha/beta activation of IFNABR-alpha and IFNABR-beta in the context of a low affinity interaction, yet a high affinity interaction is necessary for signal transducing events that mediate growth inhibition. We provide evidence that the extent of ISGF3 activation correlates directly with the magnitude of an antiviral but not a growth inhibitory response.

Human type I interferon receptor, IFNAR, is a heavily glycosylated 120-130 kD membrane protein.

Type I interferons (IFNs) bind and signal through cell surface receptors that share at least one common component. One candidate for such a component is the interferon-alpha receptor (IFNAR). Genetic studies have shown that the IFNAR gene product is required for response to many type I interferons. However, these studies also suggest that the IFNAR protein interacts with an additional receptor component(s) to form functionally complete type I IFN receptors. Although these genetic studies have contributed significantly to understanding the type I IFN receptors. Although these genetic studies have contributed significantly to understanding the type I IFN receptors, little biochemical characterization of IFNAR and its function has been reported. To facilitate biochemical studies of the IFNAR gene product, a monoclonal antibody, GB8, recognizing the extracellular domain of IFNAR was prepared. The epitope for GB8 maps to the second extracellular domain of IFNAR between amino acids 278 and 293. GB8 identifies IFNAR in western blots of cell membranes as a broad band with molecular mass ranging from 100 to 150 kD in membranes from CHO cells overexpressing the human IFNAR gene to 136-150 kD in Daudi cell membranes. Such variations in the mean value and the range of molecular mass between IFNAR in different cell lines suggest differences in glycosylation. The majority of glycosylation is N-linked, although there may also be a small amount O-linked oligosaccharide. Deglycosylation of IFNAR in Daudi cell membranes results in a 70 kD IFNAR species, indicating that nearly half of the apparent molecular mass of Daudi cell IFNAR is contributed by carbohydrate moieties.

Purification and characterization of human erythrocyte phosphatidylinositol 4-kinase. Phosphatidylinositol 4-kinase and phosphatidylinositol 3-monophosphate 4-kinase are distinct enzymes.

PtdIns 4-kinase has been purified 83,000-fold from human erythrocyte membranes. The major protein detected by SDS/PAGE is of molecular mass 56 kDa, and enzymic activity can be renatured from this band of the gel. The characteristics of this enzyme are similar to other type II PtdIns kinases previously described: PtdIns presented in Triton X-100 micelles is preferred as a substrate over PtdIns vesicles, the enzyme possesses a relatively low Km for ATP (20 microM), and adenosine is an effective inhibitor. A monoclonal antibody raised against bovine brain type II PtdIns 4-kinase is an effective inhibitor of the purified enzyme. PtdIns(4,5)P2 inhibits by approx. 50% when added in equimolar amounts with PtdIns; PtdIns4P has little effect on activity. A PtdIns3P 4-kinase activity has also been detected in erythrocyte lysates. Approximately two-thirds of this activity is in the cytosolic fraction and one-third in the membrane fraction. No PtdIns3P 4-kinase activity could be detected in the purified type II PtdIns 4-kinase preparation, nor could this activity be detected in a bovine brain type III PtdIns 4-kinase preparation. The monoclonal antibody that inhibits the type II PtdIns 4-kinase does not affect the PtdIns3P 4-kinase activity in the membrane fraction. The cytosolic PtdIns3P 4-kinase can be efficiently recovered from a 60%-satd.-(NH4)2SO4 precipitate that is virtually free of PtdIns 4-kinase activity. We conclude that PtdIns3P 4-kinase is a new enzyme distinct from previously characterized PtdIns 4-kinases, and that this enzyme prefers PtdIns3P over PtdIns as a substrate.

Transformation-defective mutants of polyomavirus middle T antigen associate with phosphatidylinositol 3-kinase (PI 3-kinase) but are unable to maintain wild-type levels of PI 3-kinase products in intact cells.

Middle T antigen (MT) of polyomavirus causes transformation by associating with a number of cellular proteins. The association with and activation of two such proteins, phosphatidylinositol 3-kinase (PI 3-kinase) and pp60c-src, appears to be necessary for transformation by MT. The tyrosine kinase activity of MT-associated pp60c-src is significantly increased when assayed in vitro, and levels of phosphotyrosine-containing proteins are elevated in vivo. Similarly, levels of the PI 3-kinase products phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] and phosphatiylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] are constitutively elevated in MT-transformed cells. However, the formation of a complete MT/cellular protein complex and the activation of tyrosine kinase are not sufficient to cause transformation, since the transformation-defective mutants 248m and dl1015 associate with all wild-type MT-associated proteins, including PI 3-kinase and pp60c-src, and neither mutant appears to be defective in MT-associated tyrosine kinase activity. Studies presented here compared (i) the amount of PI 3-kinase activity associated with the MT complex and (ii) levels of [3H]inositol incorporation into PI 3-kinase products in cells expressing mutant or wild-type MT. The results show that dl1015 is defective in both assays, whereas 248m is defective only for incorporation of [3H]inositol into PI(3,4,5)P2 and PI(3,4)P3. These findings identify a biochemical defect in the 248m mutant and corroborate previous results correlating transformation and elevated levels of PI 3-kinase products in vivo. In addition, they indicate that PI 3-kinase product levels are affected by factors other than simply the amount of PI 3-kinase activity associated with the MT complex.

A completely transformation-defective point mutant of polyomavirus middle T antigen which retains full associated phosphatidylinositol kinase activity.

By using a random mutagenesis procedure combined with a recombinant retrovirus vector, mutants of polyomavirus middle T antigen (MTAg) were generated. Three new MTAg mutants with various degrees of transformation competence were more thoroughly characterized. All of the mutants produced a stable MTAg, as assessed by metabolic labeling or immunoblotting, and each mutant possessed wild-type levels of associated tyrosine kinase activity and associated phosphatidylinositol-3 (PI-3) kinase activity. One of these mutants, with a substitution of leucine for proline at amino acid 248 of MTAg (248m) was completely transformation defective, as measured in a focus-forming assay. Furthermore, the pattern of phosphorylation of 248m in vivo was identical to that of wild-type MTAg, and the kinetics of association of MTAg with an 85-kilodalton protein, the putative PI kinase, was not altered. Similarly, the pattern of PI derivatives obtained in an in vitro kinase assay was not altered by the substitution at amino acid 248. Since the single base pair mutation at amino acid 248 resulted in an MTAg that was completely transformation defective despite possessing wild-type levels of kinase activities, this suggests that neither tyrosine kinase nor PI-3 kinase activity nor the combination of both are sufficient for transformation by MTAg.

Characterization and purification of membrane-associated phosphatidylinositol-4-phosphate kinase from human red blood cells.

The membrane-bound form of phosphatidylinositol-4-phosphate (PtdInsP) kinase was purified 4,300-fold from human red blood cells to a specific activity of 117 nmol min-1 mg-1. Although this enzyme copurified with red blood cell membranes, it was solubilized by high salt extraction in the absence of detergent indicating that it is a peripheral membrane protein. The major protein seen in the most purified preparation migrated at 53,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major PtdInsP kinase activity in this preparation was also coincident with this 53,000-dalton band upon renaturation of activity from SDS-PAGE. To test further whether the 53,000-dalton protein contained PtdInsP kinase activity, antibodies were prepared against the gel-purified 53,000-dalton protein. This antiserum was able to precipitate both the 53,000-dalton peptide and PtdInsP kinase activity from red blood cell membranes. The apparent size of the native enzyme in the most purified preparation was determined to be 150,000 +/- 25,000 daltons by gel filtration. This PtdInsP kinase activity was at least 100-fold more active in phosphorylating PtdInsP than phosphatidylinositol and was easily separated from the red cell membrane phosphatidylinositol kinase by salt extraction. Analysis of the reaction product, phosphatidylinositol 4,5-bisphosphate, indicates that the enzyme phosphorylates phosphatidylinositol 4-phosphate specifically at the 5'-hydroxyl of the inositol ring. The apparent Km for ATP was 2 microM, and the concentrations of Mg2+ and Mn2+ giving half-maximal activity were 2 and 0.2 mM, respectively. Mg2+ supported 3-fold higher activity than Mn2+ at optimal concentrations. The enzymatic activity was inhibited by its product, phosphatidylinositol 4,5-bisphosphate and enhanced by phosphatidylserine.

Sequence of the junction in adenovirus 2-SV40 hybrids: examples of illegitimate recombination.

The nucleotide sequences of six Ad2-SV40 junctions from three Ad2-SV40 hybrid viruses (Ad2++HEY, Ad2++LEY and Ad2+D1) were determined. Comparison of parental adenovirus 2 and SV40 DNA sequences with the sequence at the Ad2-SV40 junctions revealed that 5 out of 6 junctions are abrupt transitions from Ad2 to SV40 DNA, and in one case (Ad2++LEY, right junction) there is an additional nucleotide at the junction, which cannot be ascribed to either DNA. Ad2++HEY and Ad2+D1 right junctions are identical and Ad2++LEY and Ad2+ND4 left junctions are identical, a result that strongly suggests these Ad2-SV40 hybrids arose by recombination between the linear Ad2 DNA and circular SV40 DNA, followed by recombination between Ad2 DNA and SV40 DNA present in the Ad2-SV40 hybrid DNA. The unambiguous transition of Ad2 DNA into SV40 DNA at the junction sites is an example of recombination events which have apparently occurred without any homology at the recombination site.

Scanning electron microscopy: low-magnification pictures of uncoated zoological specimens.

Good low-magnification (x 5 to x 500) scanning electron microscope pictures of dry, uncoated zoological specimens may be obtained with a low accelerating voltage (1.5 to 3 kilovolts) in conjunction with a short exposure to the scanning beam.