The amino acid sequences of two alpha chains of hemoglobins from Komodo dragon Varanus komodoensis and phylogenetic relationships of amniotes

To elucidate phylogenetic relationships among amniotes and the evolution of alpha globins, hemoglobins were analyzed from the Komodo dragon (Komodo monitor lizard) Varanus komodoensis, the world's largest extant lizard, inhabiting Komodo Islands, Indonesia. Four unique globin chains (alpha A, alpha D, beta B, and beta C) were isolated in an equal molar ratio by high performance liquid chromatography from the hemolysate. The amino acid sequences of two alpha chains were determined. The alpha D chain has a glutamine at E7 as does an alpha chain of a snake, Liophis miliaris, but the alpha A chain has a histidine at E7 like the majority of hemoglobins. Phylogenetic analyses of 19 globins including two alpha chains of Komodo dragon and ones from representative amniotes showed the following results: (1) The a chains of squamates (snakes and lizards), which have a glutamine at E7, are clustered with the embryonic alpha globin family, which typically includes the alpha D chain from birds; (2) birds form a sister group with other reptiles but not with mammals; (3) the genes for embryonic and adult types of alpha globins were possibly produced by duplication of the ancestral alpha gene before ancestral amniotes diverged, indicating that each of the present amniotes might carry descendants of the two types of alpha globin genes; (4) squamates first split off from the ancestor of other reptiles and birds.      

Inhibitory effect of bFGF on endochondral heterotopic ossification

Basic fibroblast growth factor (bFGF) is reported to stimulate repair of fracture and bony defects in in vivo animal studies. However, most studies performed in vitro demonstrate inhibitory effect of bFGF on cartilage and bone differentiation. To understand the discrepancy observed in in vivo and in vitro studies, we evaluated the effect of bFGF on chondro-osteogenesis initiated by bone matrix powder (MP). MP was implanted in the murine hamstring muscles with or without administration of bFGF. Injection of 1 microg of bFGF markedly reduced the size of heterotopic bone induced by MP, as detected by X-ray. Injection of 10 microg of bFGF completely inhibited ossification and only fibrous tissues were observed at the site of MP implantation. The expressions of alkaline phosphatase and osteocalcin mRNAs, markers for bone differentiation, were completely suppressed by 10 microg of bFGF. These results demonstrate the inhibitory effect of bFGF on endochondral ossification in vivo, implicating a precaution for its use in musculo-skeletal disorders.     

Interruption of signal transduction between G protein and PKC-ε underlies the impaired myocardial response to ischemic preconditioning in postinfarct remodeled hearts

We have recently shown that the protective mechanism of ischemic preconditioning (PC) is impaired in the myocardium that survived infarction and underwent postinfarct ventricular remodeling. In this study, we examined the hypothesis that failure of PC to activate PKC- underlies the refractoriness of the remodeling heart to PC. Circumflex coronary arteries were ligated in rabbits to induce infarction and subsequent ventricular remodeling, and only sham operations were performed in controls. Hearts were isolated before (i.e. 4 days later) or after (i.e. 2 weeks later) remodeling of the left ventricle and used for isolated buffer-perfused heart experiments. Myocardial infarction was induced in isolated hearts by 30 min global ischemia/2 h reperfusion, and its size was measured by tetrazolium staining. Using separate groups of hearts, tissue biopsies were taken before and after PC, and PKC translocation was assessed by Western blotting. Areas infarcted in vivo by coronary ligation (CL) were excluded from subsequent infarct size/PKC analyses. In the hearts 4 days after CL, PC with 2 cycles of 5 min ischemia/5 min reperfusion induced PKC- translocation from cytosol to particulate fractions and limited infarct size to 40% of control value. In the hearts remodeled 2 weeks after CL, PC failed to induce PKC- translocation and infarct size limitation. In this group, PKC activity and hemodynamic responses to adenosine were similar to those in sham-operated controls. When remodeling after CL was prevented by valsartan infusion (10 mg/kg/day), an angiotensin II type 1 (AT₁) receptor blocker, PC could induce both infarct limitation and PKC- translocation. The present results suggest that persistent activation of AT₁ receptors during remodeling disturbed the PC signaling between G proteins and PKC-, which underlies the refractoriness of the remodeled myocardium to PC.     

Use of tree seedlings for the phytoremediation of a municipal effluent used in dry areas of north-western India: Plant growth and nutrient uptake

Impacts of municipal effluent (ME) irrigation on soil physicochemical properties and its remediation by tree species were assessed with a view to utilize this resource in growing woodlot, controlling land degradation and improving environmental quality in suburban areas. Acacia nilotica L. (babool), Dalbergia sissoo L. (sissoo) and Eucalyptus camaldulensis seedlings planted in July 1998 were irrigated with ME at ½ PET (T₂), 1 PET (T₃), 2 PET (T₄), and with canal water at 1 PET (T₅). The control was soil without seedlings irrigated with ME at 1 PET (T₁). Application of ME increased minerals concentration from T₂ to T₄ in both soil and seedling in June 1999 and 2000. The increase in soil pH, EC, SOC was by <2.00-fold and availability of potassium (K), copper (Cu), iron (Fe), manganese (Mn) and zinc (Zn) by >2-fold, NH₄–N by 10.44-fold and PO₄–P by 6.57-fold in T₄ than those in T₅ treatment in 2000. Available NH₄–N, PO₄–P, Mg and K were higher in 0–15 cm soil layer but continued irrigation and low soil carbon influenced leaching of NO₃–N, Na, Cu, Fe, Mn and Zn resulting their higher concentrations in 60–90 cm soil layer (P < 0.01). Lower soil nutrients in T₃ than in T₁ suggested soil amelioration by the planted seedlings and because of lowest concentration of most of the nutrients E. camaldlensis showed highest soil amelioration capacity. But nutrient utilization efficiency was highest in A. nilotica for K, Ca, Mg, Na, Fe, Cu and Zn, D. sissoo for N and P, and E. camaldulensis for Mn. Thus, planting tree seedlings, particularly E. camaldulensis, under ecological amelioration could in this way help in controlling land degradation and enhancing biomass and aesthetic benefits, although long-term application of effluent would lead to mineral/salt accumulation in soil and plants.     

Reduced transforming growth factor-beta signaling in cartilage of old mice: role in impaired repair capacity

Osteoarthritis (OA) is a common joint disease, mainly effecting the elderly population. The cause of OA seems to be an imbalance in catabolic and anabolic factors that develops with age. IL-1 is a catabolic factor known to induce cartilage damage, and transforming growth factor (TGF)-beta is an anabolic factor that can counteract many IL-1-induced effects. In old mice, we observed reduced responsiveness to TGF-beta-induced IL-1 counteraction. We investigated whether expression of TGF-beta and its signaling molecules altered with age. To mimic the TGF-beta deprived conditions in aged mice, we assessed the functional consequence of TGF-beta blocking. We isolated knee joints of mice aged 5 months or 2 years, half of which were exposed to IL-1 by intra-articular injection 24 h prior to knee joint isolation. Immunohistochemistry was performed, staining for TGF-beta1, -2 or -3, TGF-betaRI or -RII, Smad2, -3, -4, -6 and -7 and Smad-2P. The percentage of cells staining positive was determined in tibial cartilage. To mimic the lack of TGF-beta signaling in old mice, young mice were injected with IL-1 and after 2 days Ad-LAP (TGF-beta inhibitor) or a control virus were injected. Proteoglycan (PG) synthesis (³⁵S-sulfate incorporation) and PG content of the cartilage were determined. Our experiments revealed that TGF-beta2 and -3 expression decreased with age, as did the TGF-beta receptors. Although the number of cells positive for the Smad proteins was not altered, the number of cells expressing Smad2P strongly dropped in old mice. IL-1 did not alter the expression patterns. We mimicked the lack of TGF-beta signaling in old mice by TGF-beta inhibition with LAP. This resulted in a reduced level of PG synthesis and aggravation of PG depletion. The limited response of old mice to TGF-beta induced-IL-1 counteraction is not due to a diminished level of intracellular signaling molecules or an upregulation of intracellular inhibitors, but is likely due to an intrinsic absence of sufficient TGF-beta receptor expression. Blocking TGF-beta distorted the natural repair response after IL-1 injection. In conclusion, TGF-beta appears to play an important role in repair of cartilage and a lack of TGF-beta responsiveness in old mice might be at the root of OA development.     

Purification and characterization of fumarase from Corynebacterium glutamicum

Fumarase (EC 4.2.1.2) from Corynebacterium glutamicum (Brevibacterium flavum) ATCC 14067 was purified to homogeneity. Its amino-terminal sequence (residues 1 to 30) corresponded to the sequence (residues 6 to 35) of the deduced product of the fumarase gene of C. glutamicum (GenBank accession no. BAB98403). The molecular mass of the native enzyme was 200 kDa. The protein was a homotetramer, with a 50-kDa subunit molecular mass. The homotetrameric and stable properties indicated that the enzyme belongs to a family of Class II fumarase. Equilibrium constants (K(eq)) for the enzyme reaction were determined at pH 6.0, 7.0, and 8.0, resulting in K(eq)=6.4, 6.1, and 4.6 respectively in phosphate buffer and in 16, 19, and 17 in non-phosphate buffers. Among the amino acids and nucleotides tested, ATP inhibited the enzyme competitively, or in mixed-type, depending on the buffer. Substrate analogs, meso-tartrate, D-tartrate, and pyromellitate, inhibited the enzyme competitively, and D-malate in mixed-type.     

Landscape-scale variation in the seed banks of floodplain wetlands with contrasting hydrology in China

1. At a local scale, the species composition, diversity and spatial variation of wetland plant communities are determined primarily by spatial and temporal heterogeneity in their environments. Less is known about variation at a landscape‐level. The floodplain of the Changjiang (Yangtze) River in China includes hydrologically connected, subtropical wetlands with different hydrological characteristics. 2. We examined seed‐bank species composition and richness in marshes of two contrasting hydrological types: permanent marshes, fed by local runoff, and lakeshore marshes more closely connected to the regulated river. Lakeshore marshes are flooded annually to depth of approximately 1 m and during flooding they support an alternate, aquatic vegetation type. The soil seed bank in March was a comparative estimator of species diversity. At the beginning of the growing season it included seeds from both phases of alternating vegetation types associated with the annual hydrological cycle. 3. A regional pool of 101 species was detected in the seed banks of six wetlands associated with the river and its tributaries: 56 occurred in permanent marshes and 59 in lakeshore marshes, with only 15 common to both. Species rarefaction curves indicated that more species occurred in permanent than lakeshore marshes at equal numbers of individuals sampled. However, the more heterogeneous lakeshore seed banks were estimated (Chao 2) to have greater total species richness (81) than permanent marsh (60). 4. Analysis using Sørensen's coefficient of similarity and DCA ordination revealed complex variation, with much greater differences between hydrological types than within them, irrespective of geographical distance. The types also differed significantly in the composition of four functional groups of species. 5. Despite the potential for dispersal of propagules via the annually pulsing river system (hydrochory), at a regional and landscape scale, diversity is maintained largely by large‐scale temporal hydrological heterogeneity and smaller scale spatial and topographic heterogeneity.     

Functional characterization of a three-component regulatory system involved in quorum sensing-based regulation of peptide antibiotic production in Carnobacterium maltaromaticum

Background  

Quorum sensing is a form of cell-to-cell communication that allows bacteria to control a wide range of physiological processes in a population density-dependent manner. Production of peptide antibiotics is one of the processes regulated by quorum sensing in several species of Gram-positive bacteria, including strains of Carnobacterium maltaromaticum. This bacterium and its peptide antibiotics are of interest due to their potential applications in food preservation. The molecular bases of the quorum sensing phenomenon controlling peptide antibiotic production in C. maltaromaticum remain poorly understood. The present study was aimed at gaining a deeper insight into the molecular mechanism involved in quorum sensing-mediated regulation of peptide antibiotic (bacteriocin) production by C. maltaromaticum. We report the functional analyses of the CS (autoinducer)-CbnK (histidine protein kinase)-CbnR (response regulator) three-component regulatory system and the three regulated promoters involved in peptide antibiotic production in C. maltaromaticum LV17B.     

Floral synorganization and its influence on mechanical isolation and autogamy in Marantaceae

The flowers of Marantaceae (～ 550 species) exhibit a highly derived pollination mechanism within Zingiberales, with a rapid and irreversible style movement based on a close synorganization of different floral parts. Given the complexity of the structure, we assume that little variation is possible if functionality is to be maintained. To test this, we investigated how much floral diversity exists in the clade and whether this diversity potentially influences the breeding system and placement of pollen on the pollinator. Flowers of 66 species covering the five major phylogenetic clades of the family were analysed. All species are similar in their basic flower construction: the fleshy staminode forms the tunnel‐shaped roof of the flower and narrows the tube with stiff swellings, and the hooded staminode holds the style under tension and narrows the flower entrance with its trigger appendage. Despite morphological diversity of the pollination apparatus, functionality is maintained by coordinated variation of the fleshy and hooded staminodes. Autogamy is usually avoided by herkogamy. However, in a few exceptions, subtle morphological changes alter the breeding system from allogamy to autogamy. Variable floral proportions allow for differential pollen deposition potentially causing mechanical isolation between sister taxa. This study clearly illustrates that structural variation is not only present in the highly synorganized flowers of Marantaceae, but that it also creates potentially new options for evolutionary diversification. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 168 , 300–322.     

Immunolocalization of MMP-19 in the human intervertebral disc: implications for disc aging and degeneration

Matrix metalloproteinases (MMPs) degrade components of the extracellular matrix of the disc, but the presence of MMP-19 has not been explored. In other tissues, MMP-19 is known to act in proteolysis of the insulin-like growth factor (IGF) binding protein-3, thereby exposing this protein to make it available to influence cell behavior. MMP-19 also has been shown to inhibit capillary-like formation and thus play a role in the avascular nature of the disc. Using immunohistochemistry, normal discs from six subjects aged newborn through 10 years and 20 disc specimens from control donors or surgical patients aged 15-76 (mean age 40.2 years) were examined for immunolocalization of MMP-19; six Thompson grade I discs, five Thompson grade II, eight Thompson grade III, five Thompson grade IV, and one Thompson grade V discs were analyzed. The results indicate that in discs from young subjects, MMP-19 was uniformly localized in the outer annulus. In discs from adult donors and surgical patients, outer and inner annulus cells only occasionally showed MMP-19 localization. The greatest expression of MMP-19 was observed in young discs, and little expression was seen in older or degenerating discs. Because MMP-19 has been shown to regulate IGF-mediated proliferation in other tissues, its decline in the aging/degenerating disc may contribute to the age-related decrease in disc cell numbers.