Characterization of two soybean repetitive proline-rich proteins and a cognate cDNA from germinated axes

We have resolved and analyzed two proline-rich proteins isolated from the walls of soybean cells in culture. The proteins are similar in amino acid content, containing 20% proline, 20% hydroxyproline, 20% lysine, 16% valine, 10% tyrosine, and 10% glutamate. The proteins undergo a rearrangement or a limited cleavage in dilute NaOH, but are otherwise remarkably stable to a high concentration of alkali. We have cloned and sequenced a cDNA from soybean axes germinated for 31 hours (1A10-2) coding for a protein that closely corresponds in its amino acid content to that of the proline-rich proteins. The cDNA sequence predicts a decameric repeat of Pro-Pro-Val-Tyr-Lys-Pro-Pro-Val-Glu-Lys. Consequently, this class of proteins is referred to as repetitive proline-rich proteins, i.e., RPRP2 and RPRP3. We have also analyzed RNA gel blots with probes that discriminate between the new cDNA clone and a related cDNA previously reported [SbPRP1; Hong, Nagao, and Key (1987). J. Biol. Chem. 262, 8367-8376]. Messenger RNAs from young seedlings and from soybean suspension cultures correspond primarily to the new RPRP clone (1A10-2), whereas the predominant mRNA accumulating later in the roots corresponds to SbPRP1.     

Plants,microorganisms, and soil temperatures contribute to a decrease in methane fluxes on a drained Arctic floodplain

As surface temperatures are expected to rise in the future, ice‐rich permafrost may thaw, altering soil topography and hydrology and creating a mosaic of wet and dry soil surfaces in the Arctic. Arctic wetlands are large sources of CH₄, and investigating effects of soil hydrology on CH₄ fluxes is of great importance for predicting ecosystem feedback in response to climate change. In this study, we investigate how a decade‐long drying manipulation on an Arctic floodplain influences CH₄‐associated microorganisms, soil thermal regimes, and plant communities. Moreover, we examine how these drainage‐induced changes may then modify CH₄ fluxes in the growing and nongrowing seasons. This study shows that drainage substantially lowered the abundance of methanogens along with methanotrophic bacteria, which may have reduced CH₄ cycling. Soil temperatures of the drained areas were lower in deep, anoxic soil layers (below 30 cm), but higher in oxic topsoil layers (0–15 cm) compared to the control wet areas. This pattern of soil temperatures may have reduced the rates of methanogenesis while elevating those of CH₄ oxidation, thereby decreasing net CH₄ fluxes. The abundance of Eriophorum angustifolium, an aerenchymatous plant species, diminished significantly in the drained areas. Due to this decrease, a higher fraction of CH₄ was alternatively emitted to the atmosphere by diffusion, possibly increasing the potential for CH₄ oxidation and leading to a decrease in net CH₄ fluxes compared to a control site. Drainage lowered CH₄ fluxes by a factor of 20 during the growing season, with postdrainage changes in microbial communities, soil temperatures, and plant communities also contributing to this reduction. In contrast, we observed CH₄ emissions increased by 10% in the drained areas during the nongrowing season, although this difference was insignificant given the small magnitudes of fluxes. This study showed that long‐term drainage considerably reduced CH₄ fluxes through modified ecosystem properties.     

Persistent alterations in heart rate variability, baroreflex sensitivity, and anxiety-like behaviors during development of heart failure in the rat

Depressed heart rate variability and mood are associated with increased mortality in patients with congestive heart failure (CHF). Here autonomic indexes were assessed 3 and 7 wk after left coronary artery ligation in telemetered rats, after which anxiety-like behaviors were assessed in an elevated plus maze. Low frequency (LF) and high frequency (HF) heart rate variability were reduced in CHF rats 3 wk after infarction (LF, 1.60 +/- 0.52 vs. 6.97 +/- 0.79 ms(2); and HF, 1.53 +/- 0.39 vs. 6.20 +/- 1.01 ms(2); P < 0.01). The number of sequences of interbeat intervals that correlated with arterial pressure was decreased in CHF rats at 3 and 7 wk (week 3, 26.60 +/- 10.85 vs. 59.75 +/- 11.4 sequences, P < 0.05; and week 7, 20.80 +/- 8.97 vs. 65.38 +/- 5.89 sequences, P < 0.01). Sequence gain was attenuated in CHF rats by 7 wk (1.34 +/- 0.06 vs. 2.70 +/- 0.29 ms/mmHg, P < 0.01). Coherence between interbeat interval and mean arterial blood pressure variability in the LF domain was reduced in CHF rats at 3 (0.12 +/- 0.03 vs. 0.26 +/- 0.05 k(2), P < 0.05) and 7 (0.16 +/- 0.02 vs. 0.31 +/- 0.05 k(2), P < 0.05) wk. CHF rats invariably entered the open arm of the elevated plus maze first and spent more time in the open arms (36.0 +/- 15% vs. 4.6 +/- 1.9%, P < 0.05). CHF rats also showed a tendency to jump head first off the apparatus, whereas controls did not. Together the data indicate that severe autonomic dysfunction is accompanied by escape-seeking behaviors in rats with verified CHF.     

VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia

Vascular endothelial growth factor (VEGF-A) is a major regulator of blood vessel formation and function. It controls several processes in endothelial cells, such as proliferation, survival, and migration, but it is not known how these are coordinately regulated to result in more complex morphogenetic events, such as tubular sprouting, fusion, and network formation. We show here that VEGF-A controls angiogenic sprouting in the early postnatal retina by guiding filopodial extension from specialized endothelial cells situated at the tips of the vascular sprouts. The tip cells respond to VEGF-A only by guided migration; the proliferative response to VEGF-A occurs in the sprout stalks. These two cellular responses are both mediated by agonistic activity of VEGF-A on VEGF receptor 2. Whereas tip cell migration depends on a gradient of VEGF-A, proliferation is regulated by its concentration. Thus, vessel patterning during retinal angiogenesis depends on the balance between two different qualities of the extracellular VEGF-A distribution, which regulate distinct cellular responses in defined populations of endothelial cells.     

Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.

The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway.     

Optimal control strategies and cost-effectiveness analysis of a malaria model

The aim of this paper is to investigate the effectiveness and cost-effectiveness of three malaria preventive measures (use of treated bednets, spray of insecticides and a possible treatment of infective humans that blocks transmission to mosquitoes). For this, we consider a mathematical model for the transmission dynamics of the disease that includes these measures. We first consider the constant control parameters’ case, we calculate the basic reproduction number and investigate the existence and stability of equilibria; the model is found to exhibit backward bifurcation. We then assess the relative impact of each of the constant control parameters measures by calculating the sensitivity index of the basic reproductive number to the model's parameters. In the time-dependent constant control case, we use Pontryagin's Maximum Principle to derive necessary conditions for the optimal control of the disease. We also calculate the Infection Averted Ratio (IAR) and the Incremental Cost-Effectiveness Ratio (ICER) to investigate the cost-effectiveness of all possible combinations of the three control measures. One of our findings is that the most cost-effective strategy for malaria control, is the combination of the spray of insecticides and treatment of infective individuals. This strategy requires a 100% effort in both treatment (for 20 days) and spray of insecticides (for 57 days). In practice, this will be extremely difficult, if not impossible to achieve. The second most cost-effective strategy which consists of a 100% use of treated bednets and 87% treatment of infective individuals for 42 and 100 days, respectively, is sustainable and therefore preferable.     

Noninvasive determination of ulnar stiffness from mechanical response--in vivo comparison of stiffness and bone mineral content in humans

An approach referred to as Mechanical Response Tissue Analysis (MRTA) has been developed for the noninvasive determination of mechanical properties of the constituents of the intact limb. Of specific interest in the present study is the bending stiffness of the ulna. The point mechanical impedance properties in the low frequency regime, between 60 and 1,600 Hz are used. The procedure requires a proper design of the probe for good contact of the skin at midshaft and proper support of the proximal and distal ends of the forearm to obtain an approximation to "simple support" of the ulna. A seven-parameter model for the mechanical response is then valid, which includes the first mode of anterior-posterior beam bending of the ulna, the damping and spring effect of the soft tissue between probe and bone, and the damping of musculature. A dynamic analyzer (HP3562A) provides in seconds the impedance curve and the pole-zero curve fit. The physical parameters are obtained from a closed-form solution in terms of the curve-fit parameters. The procedure is automated and is robust and analytically reliable at about the five percent level. Some 80 human subjects have been evaluated by this mechanical response system and by the Norland single photon absorptiometer, providing for the first time in vivo, a comparison of elastic bending stiffness (ulna) and bone mineral content (radius). Three functional parameters of potential clinical value are the cross-sectional bending stiffness EI, the axial load capability Pcr (Euler buckling load) and the bone "sufficiency" S, defined as the ratio of Pcr to body weight. The correlation between EI and bone mineral (r = 0.81) is only slightly less than previous in vitro results with both measurements on the same bone (r = 0.89). When sufficiency is taken into consideration, the correlation of Pcr and bone mineral content is improved (r = 0.89). An implication is that "quality" of bone is a factor which is not indicated by bone mineral content but which is indicated by stiffness. Bone mineral is necessary for proper stiffness but not sufficient. Therefore mechanical measurement should provide a new dimension to be used toward a better understanding of the factors related to bone health and disease.     

Extracellular lipid droplets promote hemozoin crystallization in the gut of the blood fluke Schistosoma mansoni

Hemozoin (Hz) is a heme crystal produced upon hemoglobin digestion as the main mechanism of heme disposal in several hematophagous organisms. Here, we show that, in the helminth Schistosoma mansoni, Hz formation occurs in extracellular lipid droplets (LDs). Transmission electron microscopy of adult worms revealed the presence of numerous electron-lucent round structures similar to LDs in gut lumen, where multicrystalline Hz assemblies were found associated to their surfaces. Female regurgitates promoted Hz formation in vitro in reactions partially inhibited by boiling. Fractionation of regurgitates showed that Hz crystallization activity was essentially concentrated on lower density fractions, which have small amounts of pre-formed Hz crystals, suggesting that hydrophilic-hydrophobic interfaces, and not Hz itself, play a key catalytic role in Hz formation in S. mansoni. Thus, these data demonstrate that LDs present in the gut lumen of S. mansoni support Hz formation possibly by allowing association of heme to the lipid-water interface of these structures.     

On the mechanisms involved in biological heme crystallization

Blood-feeding organisms digest hemoglobin, releasing large quantities of heme inside their digestive tracts. Free heme is very toxic, and these organisms have evolved several mechanisms to protect against its deleterious effects. One of these adaptations is the crystallization of heme into the dark-brown pigment hemozoin (Hz). Here we review the process of Hz formation, focusing on organisms other than Plasmodium that have contributed to a better understanding of heme crystallization. Hemozoin has been found in several distinct classes of organisms including protozoa, helminths and insects and Hz formation is the predominant form of heme detoxification. The available evidence indicates that amphiphilic structures such as phospholipid membranes and lipid droplets accompanied by specific proteins play a major role in heme crystallization. Because this process is specific to a number of blood-feeding organisms and absent in their hosts, Hz formation is an attractive target for the development of novel drugs to control illnesses associated with these hematophagous organisms.     

Expression,secretion and surface display of a human alkaline phosphatase by the ciliate <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Background  

Tetrahymena thermophila possesses many attributes that render it an attractive host for the expression of recombinant proteins. Surface proteins from the parasites Ichthyophthirius multifiliis and Plasmodium falciparum and avian influenza virus antigen H5N1 were displayed on the cell membrane of this ciliate. Furthermore, it has been demonstrated that T. thermophila is also able to produce a functional human DNase I. The present study investigates the heterologous expression of the functional human intestinal alkaline phosphatase (hiAP) using T. thermophila and thereby presents a powerful tool for the optimization of the ciliate-based expression system.