Repulsive guidance molecule RGMa alters utilization of bone morphogenetic protein (BMP) type II receptors by BMP2 and BMP4

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily of multifunctional ligands that transduce their signals through type I and II serine/threonine kinase receptors and intracellular Smad proteins. Recently, we identified the glycosylphosphatidylinositol-anchored repulsive guidance molecules RGMa, DRAGON (RGMb), and hemojuvelin (RGMc) as coreceptors for BMP signaling (Babbit, J. L., Huang, F. W., Wrighting, D. W., Xia, Y., Sidis, Y., Samad, T. A., Campagna, J. A., Chung, R., Schneyer, A., Woolf, C. J., Andrews, N. C., and Lin, H. Y. (2006) Nat. Genet. 38, 531-539; Babbit, J. L., Zhang, Y., Samad, T. A., Xia, Y., Tang, J., Schneyer, A., Woolf, C. J., and Lin, H. Y. (2005) J. Biol. Chem. 280, 29820-29827; Samad, T. A., Rebbapragada, A., Bell, E., Zhang, Y., Sidis, Y., Jeong, S. J., Campagna, J. A., Perusini, S., Fabrizio, D. A., Schneyer, A. L., Lin, H. Y., Brivanlou, A. H., Attisano, L., and Woolf, C. J. (2005) J. Biol. Chem. 280, 14122-14129). However, the mechanism by which RGM family members enhance BMP signaling remains unknown. Here, we report that RGMa bound to radiolabeled BMP2 and BMP4 with Kd values of 2.4+/-0.2 and 1.4+/-0.1 nm, respectively. In KGN human ovarian granulosa cells and mouse pulmonary artery smooth muscle cells, BMP2 and BMP4 signaling required BMP receptor type II (BMPRII), but not activin receptor type IIA (ActRIIA) or ActRIIB, based on changes in BMP signaling by small interfering RNA inhibition of receptor expression. In contrast, cells transfected with RGMa utilized both BMPRII and ActRIIA for BMP2 or BMP4 signaling. Furthermore, in BmpRII-null pulmonary artery smooth muscle cells, BMP2 and BMP4 signaling was reduced by inhibition of endogenous RGMa expression, and RGMa-mediated BMP signaling required ActRIIA expression. These findings suggest that RGMa facilitates the use of ActRIIA by endogenous BMP2 and BMP4 ligands that otherwise prefer signaling via BMPRII and that increased utilization of ActRIIA leads to generation of an enhanced BMP signal.     

The folding transition state of protein L is extensive with nonnative interactions (and not small and polarized)

Progress in understanding protein folding relies heavily upon an interplay between experiment and theory. In particular, readily interpretable experimental data that can be meaningfully compared to simulations are required. According to standard mutational ? analysis, the transition state for Protein L contains only a single hairpin. However, we demonstrate here using ψ analysis with engineered metal ion binding sites that the transition state is extensive, containing the entire four-stranded β sheet. Underreporting of the structural content of the transition state by ? analysis also occurs for acyl phosphatase [Pandit, A. D., Jha, A., Freed, K. F. &; Sosnick, T. R., (2006). Small proteins fold through transition states with native-like topologies. J. Mol. Biol. 361, 755–770], ubiquitin [Sosnick, T. R., Dothager, R. S. &; Krantz, B. A., (2004). Differences in the folding transition state of ubiquitin indicated by ? and ψ analyses. Proc. Natl Acad. Sci. USA 101, 17377–17382] and BdpA [Baxa, M., Freed, K. F. &; Sosnick, T. R., (2008). Quantifying the structural requirements of the folding transition state of protein A and other systems. J. Mol. Biol. 381, 1362–1381]. The carboxy-terminal hairpin in the transition state of Protein L is found to be nonnative, a significant result that agrees with our Protein Data Bank-based backbone sampling and all-atom simulations. The nonnative character partially explains the failure of accepted experimental and native-centric computational approaches to adequately describe the transition state. Hence, caution is required even when an apparent agreement exists between experiment and theory, thus highlighting the importance of having alternative methods for characterizing transition states.     

Syndecan-1 regulates BMP signaling and dorso-ventral patterning of the ectoderm during early Xenopus development

Extracellular regulation of growth factor signaling is a key event for embryonic patterning. Heparan sulfate proteoglycans (HSPG) are among the molecules that regulate this signaling during embryonic development. Here we study the function of syndecan1 (Syn1), a cell-surface HSPG expressed in the non-neural ectoderm during early development of Xenopus embryos. Overexpression of Xenopus Syn1 (xSyn1) mRNA is sufficient to reduce BMP signaling, induce chordin expression and rescue dorso-ventral patterning in ventralized embryos. Experiments using chordin morpholinos established that xSyn1 mRNA can inhibit BMP signaling in the absence of chordin. Knockdown of xSyn1 resulted in a reduction of BMP signaling and expansion of the neural plate with the concomitant reduction of the non-neural ectoderm. Overexpression of xSyn1 mRNA in xSyn1 morphant embryos resulted in a biphasic effect, with BMP being inhibited at high concentrations and activated at low concentrations of xSyn1. Interestingly, the function of xSyn1 on dorso-ventral patterning and BMP signaling is specific for this HSPG. In summary, we report that xSyn1 regulates dorso-ventral patterning of the ectoderm through modulation of BMP signaling.     

Fluorescent lipoprotein probe

The fluorescent cholesterol analog cholesta-5,7,9(11)triene-3-β-ol was used to label high-density and low-density lipoproteins in vivo (rabbit) and in vitro (human). Rabbit feeding experiments demonstrated that in vivo both the esterified and nonesterified forms of the fluorophore were incorporated by these particles. Using in vitro labeling techniques, it was possible to selectively incorporate either the free form of the fluorophore or both the free and the esterified forms depending upon the presence or absence of serum esterases during incubation. Subsequent to labeling, the thermotropic behavior of the low- and high-density lipoproteins was evaluated using temperature-dependent fluorescence intensity measurements. Purified low-density lipoprotein samples (human and rabbit) containing both forms of the fluorophore were observed to undergo a thermotropic transition between 27 and 32°C. However, this transition was not observed in low-density lipoprotein samples containing only the nonesterified form of the probe nor was it observed in any of the high-density lipoprotein samples, even those containing both forms of the fluorophore. These results provide further evidence that the previously reported thermotropic transition in low-density lipoprotein is due to a reordering of the low-density lipoprotein cholesterol ester core (Deckelbaum, R.J., Shipley, G. G., Small, D. M., Lees, R. S., and George, P. K. (1975) Science190, 392–394; Sears, B., Deckelbaum, R. J., Janiak, M. J., Shipley, G. G., and Small, D. M. (1976) Biochemistry15, 4151–4157; Chana, G. S., Sheppard, R. J., Mills, G. L., and Grant, E. H. (1980) Phys. Med. Biol.25, 427–432).     

BMP morphogen gradients in flies

Bone morphogenetic proteins (BMPs) act as morphogens to control patterning and growth in a variety of developing tissues in different species. How BMP morphogen gradients are established and interpreted in the target tissues has been extensively studied in Drosophila melanogaster. In Drosophila, Decapentaplegic (Dpp), a homologue of vertebrate BMP2/4, acts as a morphogen to control dorsal–ventral patterning of the early embryo and anterior–posterior patterning and growth of the wing imaginal disc. Despite intensive efforts over the last twenty years, how the Dpp morphogen gradient in the wing imaginal disc forms remains controversial, while gradient formation in the early embryo is well understood. In this review, we first focus on the current models of Dpp morphogen gradient formation in these two tissues, and then discuss new strategies using genome engineering and nanobodies to tackle open questions.     

p38α MAPK Is Required for Tooth Morphogenesis and Enamel Secretion

An improved understanding of the molecular pathways that drive tooth morphogenesis and enamel secretion is needed to generate teeth from organ cultures for therapeutic implantation or to determine the pathogenesis of primary disorders of dentition (Abdollah, S., Macias-Silva, M., Tsukazaki, T., Hayashi, H., Attisano, L., and Wrana, J. L. (1997) J. Biol. Chem. 272, 27678–27685). Here we present a novel ectodermal dysplasia phenotype associated with conditional deletion of p38α MAPK in ectodermal appendages using K14-cre mice (p38α^K14 mice). These mice display impaired patterning of dental cusps and a profound defect in the production and biomechanical strength of dental enamel because of defects in ameloblast differentiation and activity. In the absence of p38α, expression of amelogenin and β₄-integrin in ameloblasts and p21 in the enamel knot was significantly reduced. Mice lacking the MAP2K MKK6, but not mice lacking MAP2K MKK3, also show the enamel defects, implying that MKK6 functions as an upstream kinase of p38α in ectodermal appendages. Lastly, stimulation with BMP2/7 in both explant culture and an ameloblast cell line confirm that p38α functions downstream of BMPs in this context. Thus, BMP-induced activation of the p38α MAPK pathway is critical for the morphogenesis of tooth cusps and the secretion of dental enamel.     

Spray technique for applying stripmix (cellulose acetate) to tlc plates for tritiated organic compounds

A modification of the recent rolling technique of Redgwell, R. J., Turner, N A., and Bieleski, R. L. [(1974) J. Chromatogr.88, 25–31] for stripping thin layers from chromatographic plates for radioactive measurements has been developed. Use of this rolling technique in the case of tritiated benzo[α]pyrene and metabolites yields erratic results. By the simple expedient of spraying (instead of rolling) of the Stripmix, highly reproducible data were obtained.     

Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis

Bone morphogenetic protein (BMP) family members, including BMP2, BMP4, and BMP7, are expressed throughout limb development. BMPs have been implicated in early limb patterning as well as in the process of skeletogenesis. However, due to complications associated with early embryonic lethality, particularly for Bmp2 and Bmp4, and with functional redundancy among BMP molecules, it has been difficult to decipher the specific roles of these BMP molecules during different stages of limb development. To circumvent these issues, we have constructed a series of mouse strains lacking one or more of these BMPs, using conditional alleles in the case of Bmp2 and Bmp4 to remove them specifically from the limb bud mesenchyme. Contrary to earlier suggestions, our results indicate that BMPs neither act as secondary signals downstream of Sonic Hedghog (SHH) in patterning the anteroposterior axis nor as signals from the interdigital mesenchyme in specifying digit identity. We do find that a threshold level of BMP signaling is required for the onset of chondrogenesis, and hence some chondrogenic condensations fail to form in limbs deficient in both BMP2 and BMP4. However, in the condensations that do form, subsequent chondrogenic differentiation proceeds normally even in the absence of BMP2 and BMP7 or BMP2 and BMP4. In contrast, we find that the loss of both BMP2 and BMP4 results in a severe impairment of osteogenesis.     

Metabolism of phosphatidylglycerol and bis(monoacylglycero)-phosphate in macrophage subcellular fractions

Bis(monoacylglycero)phosphate (BMP) is synthesized from exogenous phosphatidylglycerol (PG) by macrophages (Cochran, F. R., Roddick, V. L., Connor, J. R., Thornburg, J. T., and Waite, M. (1987) J. Immunol. 138, 1877-1883). Previous work from our laboratory showed that arachidonic acid in BMP was released by the macrophages upon challenge of the cells with PMA (Cochran, F. R., Connor, J. R., Roddick, V. L., and Waite, M. (1985) Biochem. Biophys. Res. Commun. 130, 800-806). Here we extend those studies using a model cultured cell line of macrophages, RAW 264.7. When PG labeled with 32P- and [3H]glycerol in both moieties was added to the culture medium, 32P/[3H]BMP was synthesized in a time-dependent manner. Fractionation of cell homogenates on a discontinuous sucrose gradient in which the light membranes were floated from dense sucrose showed an enrichment of [3H]BMP in light membrane fractions. The precursor [3H]PG was also found in the light fractions but, relative to the [3H]BMP, was more abundant in the denser membrane fractions. The appearance of [3H]PG and [3H]BMP in the light membrane fraction was time-dependent which suggested that the initial uptake and metabolism of [3H]PG was into the denser membranes. Incubation of the light membranes under conditions that are optimal for the lysosomal phospholipase A1 led to significant metabolism of [3H]PG. Both degradation of [3H]PG to water-soluble compounds and its conversion to acylphosphatidylglycerol occurred while no lyso-PG was detected. On the other hand, little BMP was found to be degraded. From these studies we postulate that in lysosomes acylphosphatidylglycerol is a precursor of BMP and that the previously reported turnover of arachidonic acid by BMP may occur via transacylation rather than hydrolysis.     

Organic matter digestibility of condensed tannin-containing Lotus corniculatus and its prediction in vitro using cellulase/hemicellulase enzymes

Three digestion experiments involving cryptorchid weaned lambs were conducted for 14-day periods in the spring, summer and autumn to determine the in vivo digestibility of dry matter (DMD), gross energy (GE), organic matter (OMD), digestible organic matter in the dry matter (DOMD) and estimated metabolisable energy (ME) concentration of Lotus corniculatus L. (birdsfoot trefoil; cv. Grasslands Goldie) at different stages of maturity. The experiments were carried out indoors at Massey University's Riverside farm, in the Wairarapa, North Island, New Zealand. In vivo digestibility samples were then used as standards to investigate if the enzymic in vitro system of [Roughan, P.G., Holland, R., 1977. Predicting in vitro digestiblities of herbages by exhaustive enzymic hydrolysis of cell walls. J. Sci. Food Agric. 28, 1057–1064] could predict OMD and DOMD of condensed tannin (CT)-containing L. corniculatus.Digestibility of L. corniculatus declined as it matured, but the rate of decline was much less than that which occurs for perennial ryegrass (Lolium perenne)/white clover (Trifolium repens) pasture. It was concluded that the in vitro enzymic system of [Roughan, P.G., Holland, R., 1977. Predicting in vitro digestiblities of herbages by exhaustive enzymic hydrolysis of cell walls. J. Sci. Food Agric. 28, 1057–1064] can be used to predict OMD and DOMD of L. corniculatus with a high degree of accuracy, provided a standard curve involving in vivo data generated with L. corniculatus is used. Using a standard curve with in vivo data from perennial ryegrass/white clover pasture led to bias which increased at lower OMD values. Reasons for the differences between L. corniculatus and pasture standard curves are discussed, including possible effects of bound CT.     

Analysis of the high- and low-spin Soret bands of horse-heart metmyoglobin complexes

Interest in the thermodynamics of the iron-binding site in hemoproteins has increased in recent years due to refinements in x-ray crystallographic studies of hemoproteins [see Deathage, J. F., Lee, R. S., Anderson, C. M. & Moffat, K. (1976) J. Mol. Biol. 104 , 687–706; Heidner, E. J., Ladner, R. C. & Perutz, M. F. (1976) J. Mol. Biol. 104 , 707–722; Deathage, J. F., Lee, R. S. & Moffat, K. (1976) J. Mol. Biol. 104 , 723–728; Ladner, R. C., Heidner, E. J. & Perutz, M. F. (1976) J. Mol. Biol. 114 , 385–414; Fermi, G. & Perutz, M. F. (1977) J. Mol. Biol. 114 , 421–431; Takano, T. (1977) J. Mol. Biol. 110 , 537–568 and 569–589], the synthesis and x-ray analysis of model heme compounds [see Scheidt, W. R. (1977) Acc. Chem. Res. 10 , 339–345; Kastner, M. E., Scheidt, W. R., Mashino, T. & Reed, C. A. (1978) J. Am. Chem. Soc. 100 , 666–667; Mashiko, T., Kastner, M. E., Spartalian, K., Scheidt, W. R. & Reed, C. A. (1978) J. Am. Chem. Soc. 100 , 6354–6362; Hill, H. A. O., Skite, P. P., Buchler, J. W., Luchr, H., Tonn, M., Gregson, A. K. & Pellizer, G. (1979) Chem. Commun. 4 , 151–152; and Scheidt, W. R., Cohen, I. A. & Kastner, M. E. (1979) Biochemistry 18 , 3546–3556], and the numerous data on heme–protein interactions that account for the differences observed in ligand binding between the various species of animals. Numerous probes have been used and provide information about the structure and thermodynamics of the binding site, but no single probe can provide the complete picture [see Iizuka, T. & Yonetani, T. (1970) Adv. Biophys. 1 , 157–182; Smith, D. W. & Williams, R. J. P. (1970) Struct. Bond. 7 , 1–45; and Spiro, T. G. (1975) Biochim. Biophys. Acta 416 , 169–189].     

Ectopic Pax2 expression in chick ventral optic cup phenocopies loss of Pax2 expression

Pax2 is essential for the development of the urogenital system, neural tube, otic vesicle, optic cup and optic tract [Dressler, G.R., Deutsch, U., et al., 1990. PAX2, a new murine paired-box-containing gene and its expression in the developing excretory system. Development 109 (4), 787-795; Nornes, H.O., Dressler, G.R., et al., 1990. Spatially and temporally restricted expression of Pax2 during murine neurogenesis. Development 109 (4), 797-809; Eccles, M.R., Wallis, L.J., et al., 1992. Expression of the PAX2 gene in human fetal kidney and Wilms’ tumor. Cell Growth Differ 3 (5), 279-289]. Within the visual system, a loss-of-function leads to lack of choroid fissure closure (known as a coloboma), a loss of optic nerve astrocytes, and anomalous axonal pathfinding at the optic chiasm [Favor, J., Sandulache, R., et al., 1996. The mouse Pax2(1Neu) mutation is identical to a human PAX2 mutation in a family with renal-coloboma syndrome and results in developmental defects of the brain, ear, eye, and kidney. Proc. Natl. Acad. Sci. U. S. A. 93 (24), 13870-13875; Torres, M., Gomez-Pardo, E., et al., 1996. Pax2 contributes to inner ear patterning and optic nerve trajectory. Development 122 (11), 3381-3391]. This study is directed at determining the effects of ectopic Pax2 expression in the chick ventral optic cup past the normal developmental period when Pax2 is found. In ovo electroporation of Pax2 into the chick ventral optic cup results in the formation of colobomas, a condition typically associated with a loss of Pax2 expression. While the overexpression of Pax2 appears to phenocopy a loss of Pax2, the mechanism of the failure of choroid fissure closure is associated with a cell fate switch from ventral retina and retinal pigmented epithelium (RPE) to an astrocyte fate. Further, ectopic expression of Pax2 in RPE appears to have non-cell autonomous effects on adjacent RPE, creating an ectopic neural retina in place of the RPE.     

Left-right function of dmrt2 genes is not conserved between zebrafish and mouse

Background

Members of the Dmrt family, generally associated with sex determination, were shown to be involved in several other functions during embryonic development. Dmrt2 has been studied in the context of zebrafish development where, due to a duplication event, two paralog genes dmrt2a and dmrt2b are present. Both zebrafish dmrt2a/terra and dmrt2b are important to regulate left-right patterning in the lateral plate mesoderm. In addition, dmrt2a/terra is necessary for symmetric somite formation while dmrt2b regulates somite differentiation impacting on slow muscle development. One dmrt2 gene is also expressed in the mouse embryo, where it is necessary for somite differentiation but with an impact on axial skeleton development. However, nothing was known about its role during left-right patterning in the lateral plate mesoderm or in the symmetric synchronization of somite formation.

Methodology/Principal Findings

Using a dmrt2 mutant mouse line, we show that this gene is not involved in symmetric somite formation and does not regulate the laterality pathway that controls left-right asymmetric organ positioning. We reveal that dmrt2a/terra is present in the zebrafish laterality organ, the Kupffer''s vesicle, while its homologue is excluded from the mouse equivalent structure, the node. On the basis of evolutionary sub-functionalization and neo-functionalization theories we discuss this absence of functional conservation.

Conclusions/Significance

Our results show that the role of dmrt2 gene is not conserved during zebrafish and mouse embryonic development.     

Evidence for an anisotropic magnetic interaction between the (bacteriopheophytin) intermediary acceptor and the first quinone acceptor in bacterial photosynthesis

We have investigated electron spin polarization effects occurring in protonated and perdeuterated reaction centers of Rhodospirillum rubrum with electron spin resonance at 9 and 35 GHz (X- and Q-band). As for Rhodopseudomonas sphaeroides strains 2.4.1 and R-26 (Gast, P. and Hoff, A.J. (1979) Biochim. Biophys. Acta 548, 520–535; Gast, P., Mushlin, R.A. and Hoff, A.J. (1982) J. Phys. Chem. 86, 2886–2891), electron spin polarization effects of the prereduced first quinone acceptor Q^?_A in R. rubrum are strongly nonuniform. This nonuniformity is due to an anisotropic magnetic coupling between the intermediary bacteriopheophytin acceptor (I^?) and Q^?_A. It is argued that the anisotropy is too strong to arise solely from an anisotropy in the exchange interaction between I^? and Q^?_A and that dipolar contributions to the magnetic coupling between I^? and Q^?_A are important. The anisotropy in the magnetic coupling for reaction centers of Rps. sphaeroides strains 2.4.1 and R-26 is different from that of R. rubrum wild type. The combination of the 4-fold higher resolution at Q-band and the line narrowing upon deuteration has enabled us to obtain the principal g values and two hyperfine interaction constants of the reduced first quinone acceptor Q^?_A. The principal g values are g_x = 2.0067, g_y = 2.0056 and g_z = 2.0024; the hyperfine constant of the CH₂ group at position 1 is 1.6 G and that of the CH₃ group at position 2 is 2.1 G. These values are close to those found for ubisemiquinone in vitro (Okamura, M.Y., Debus, R.J., Isaacson, R.A. and Feher, G. (1980) Fed. Proc. 39, 1802; Hales, B.J. (1975) J. Am. Chem. Soc. 97, 5993–5997).     

zen and the art of phenotypic maintenance: Canalization of embryonic dorsal-ventral patterning in Drosophila

We recently uncovered a novel genetic mechanism that generates the phenotypic uniformity, or canalization, of BMP signaling and cell fate specification during patterning of the dorsal-ventral (D/V) axis in D. melanogaster embryos. We went on to show that other wild-type Drosophila species lack this canalizing genetic circuitry and, consequently, have non-robust D/V patterning. In this review, we propose molecular mechanisms that may give rise to stereotyped BMP signaling, and we identify an additional species that could have decanalized D/V patterning. Extension of these analyses could in turn help explain why canalization is not a universal necessity for species survival.     

Pea-derived vaccines demonstrate high immunogenicity and protection in rabbits against rabbit haemorrhagic disease virus

Vaccines against rabbit haemorrhagic disease virus (RHDV) are commercially produced in experimentally infected rabbits. A genetically engineered and manufactured version of the major structural protein of RHDV (VP60) is considered to be an alternative approach for vaccine production. Plants have the potential to become an excellent recombinant production system, but the low expression level and insufficient immunogenic potency of plant‐derived VP60 still hamper its practical use. In this study, we analysed the expression of a novel multimeric VP60‐based antigen in four different plant species, including Nicotiana tabacum L., Solanum tuberosum L., Brassica napus L. and Pisum sativum L. Significant differences were detected in the expression patterns of the novel fusion antigen cholera toxin B subunit (CTB)::VP60 (ctbvp60_SEKDEL) at the mRNA and protein levels. Pentameric CTB::VP60 molecules were only detected in N. tabacum and P. sativum, and displayed equal levels of CTB, at approximately 0.01% of total soluble protein (TSP), and traces of detectable VP60. However, strong enhancement of the CTB protein content via self‐fertilization was only observed in P. sativum, where it reached up to 0.7% of TSP. In rabbits, a strong decrease in the protective vaccine dose required from 48–400 µg potato‐derived VP60 [ Castanon, S., Marin, M.S., Martin‐Alonso, J.M., Boga, J.A., Casais, R., Humara, J.M., Ordas, R.J. and Parra, F. (1999) Immunization with potato plants expressing VP60 protein protects against rabbit hemorrhagic disease virus. J. Virol. 73 , 4452–4455; Castanon, S., Martin‐Alonso, J.M., Marin, M.S., Boga, J.A., Alonso, P., Parra, F. and Ordas, R.J. (2002) The effect of the promoter on expression of VP60 gene from rabbit hemorrhagic disease virus in potato plants. Plant Sci. 162 , 87–95] to 0.56–0.28 µg antigenic VP60 (measured with VP60 enzyme‐linked immunosorbent assay) of crude CTB::VP60 pea extracts was demonstrated. Rabbits immunized with pea‐derived CTB::VP60 showed anti‐VP60‐specific antibodies, similar to RikaVacc^®‐immunized rabbits, and survived RHDV challenge.     

Statistical evaluation of ways to analyze nonideal systems by sedimentation equilibrim

Three equations describing sedimentation equilibrium are examined and tested for their ability to analyze data. The testing procedure using simulated data is similar to that described previously (Holladay, L. A., and Sophianopoulos, A. J. (1972) J. Biol. Chem.247, 427–439) and used with another equation. The equations examined here are found to be of much less statistical reliability and of a more restricted range of application than the previously examined equation. The equation described previously, (Holladay, L. A., and Sophianopoulos, A. J. (1972) J. Biol. Chem.247, 427–439) is also used here to examine the conditions necessary to detect isodesmic systems of more than four components. The self-association of lysozyme reported previously (Sophianopoulos, A. J., and Van Holde, K. E. (1964) J. Biol. Chem.239, 2516–2524) is reexamined at pH 8.2, 0.15 ionic strength, and 13°C. The tentative conclusion is that the system is mainly a monomer-dimer, with a small, uncertain amount of tetramer possibly present. Under the above conditions the second virial coefficient, B, is estimated to lie in the range 0–4.4 × 10^?6 mole·dl·g^?2, the dimerization constant. K₂₁, lies in the range 2.3–2.7 × 10^?3m, and the tetramerdimer constant, K₄₂, is in the range 1.5–15 × 10^?3m.     

Tracking the impact of drought on functionally different woody plants in a Mediterranean scrubland ecosystem

Climate warming is predicted to amplify drought stress. Thus, it is important to understand how coexisting plant species respond to severe droughts. Here we study how seven Mediterranean woody plant species with different evolutionary history and functional characteristics (Pinus halepensis Mill., Juniperus phoenicea L., Pistacia lentiscus L., Rhamnus lycioides L., Rosmarinus officinalis L., Genista scorpius (L.) DC., and Globularia alypum L.) responded to a severe winter drought during 2011–2012 in Spain. The study site is located in the Valcuerna valley, Monegros desert, northeastern Spain. We evaluated how the drought affected the annual growth-ring formation of the species by using dendrochronology and quantified the intensity of drought-induced defoliation and mortality and compared it between species and groups of species with different evolutionary history. Radial growth of all species was strongly reduced by the 2012 drought. The pre-Mediterranean species (P. halepensis, J. phoenicea, P. lentiscus and R. lycioides) reduced growth more than the Mediterranean species (R. officinalis, G. scorpius and G. alypum). Defoliation was significantly higher in pre-Mediterranean than in Mediterranean species. When species were analyzed separately we found that P. halepensis was the species with the highest growth reduction but J. phoenicea was defoliated more severely and showed higher mortality rates as a consequence of drought. In the case of the Mediterranean shrubs, drought-induced mortality was only noticeable in R. officinalis. Drought impacted growth of all species but this did not induce mortality in all of them. Growth reduction was dependent on evolutionary history. However, functional characteristics of the species such as leaf stomatal regulation and root architecture may be more important than evolutionary history on explaining drought-induced mortality. Indeed, species with shallow root systems such as J. phoenicea and R. officinalis were the most adversely affected by the drought.