Growth and root nodule nitrogenase activity of Pisum sativum as influenced by transpiration

The influence of shoot transpiration on the rates of growth and nitrogen fixation was investigated in Pisum sativum L. cv. Rondo. In short term experiments, rates of transpiration and acetylene reduction of intact plants were measured simultaneously, using air-tight perspex vessels enclosing the basal part of the nodulated root. In long term experiments, accumulation of dry matter and reduced nitrogen in the plant were determined as well. Transpiration rate changed diurnally and was varied by manipulating the vapour saturation deficit or the flow rate of the air in the growth cabinet. The rate of acetylene reduction declined after subjecting intact plants to high transpiration rates. This decline was accompanied by a desiccation of the root nodules. Dry matter and reduced nitrogen accumulation were not affected by transpiration rate. At low transpiration rate reduced nitrogen content of the root nodules was higher than at high transpiration rate. However, in these nodules the rate of acetylene reduction was not significantly affected. It is concluded that the nitrogenase activity of pea root nodules is insensitive to changes in the flow rate and the organic N concentration of the xylem sap within a wide range of transpiration conditions under the applied growth conditions.     

Study of the carbohydrate part of yeast acid phosphatase

It has been found that the carbohydrate part of acid phosphatase from yeast Saccharomyces cerevisiae consists of 16 N-glycosidically linked carbohydrate chains containing from 14 to about 150 mannose units. The presence of very small amounts of O-glycosidically linked chains was indicated. Acetolysis studies pointed to a high similarity in the structure of acid phosphatase and mannan carbohydrate chains. A new method is described for cross-linking of acid phosphatase specifically via carbohydrate chains. The possibility to cross-link the enzyme subunits intramolecularly is in accordance with the suggestion that carbohydrate chains play a role in subunit associations.     

Allosteric antibody inhibition of human hepsin protease

Hepsin is a type II transmembrane serine protease that is expressed in several human tissues. Overexpression of hepsin has been found to correlate with tumour progression and metastasis, which is so far best studied for prostate cancer, where more than 90% of such tumours show this characteristic. To enable improved future patient treatment, we have developed a monoclonal humanized antibody that selectively inhibits human hepsin and does not inhibit other related proteases. We found that our antibody, hH35, potently inhibits hepsin enzymatic activity at nanomolar concentrations. Kinetic characterization revealed non-linear, slow, tight-binding inhibition. This correlates with the crystal structure we obtained for the human hepsin-hH35 antibody Fab fragment complex, which showed that the antibody binds hepsin around α3-helix, located far from the active centre. The unique allosteric mode of inhibition of hH35 is distinct from the recently described HGFA (hepatocyte growth factor activator) allosteric antibody inhibition. We further explain how a small change in the antibody design induces dramatic structural rearrangements in the hepsin antigen upon binding, leading to complete enzyme inactivation.     

Combined effects of soil moisture and nitrogen availability variations on grass productivity in African savannas

Savannas cover about 20% of the Earth’s land area and 50% of Africa. As an indispensable component of savanna, grasses play an important role in these ecosystems. A better understanding of grass productivity and its controlling factors in savanna ecosystems could therefore be a key to understand the functioning of savannas and predict savanna responses to future climatic changes. In this study, a stable isotope fertilization experiment was conducted to determine how factors limiting grass production in savannas differ across regional climate gradients. The study was conducted on the geomorphically homogenous Kalahari Transect (KT), which offers an ideal setting to study nutrient and vegetation dynamics independently of confounding soil effects. The results show that the grasses assimilated the added fertilizer at all the sites but they did not respond to nitrogen fertilization for both dry and wet years, and at both dry and wet ends of the Transect. Although prior studies have proposed a switch between water and nitrogen limitations between arid and mesic savannas, our results suggest that nitrogen availability may not limit grass productivity across the whole KT. Thus, although the traditional classifications as nutrient poor (broad-leaf) and nutrient rich (fine-leaf) savanna ecosystems may still be useful, it does not necessarily imply the existence of nitrogen limitation in the nutrient poor area; in fact, it is more likely that the herbaceous species found in the more humid sites (nutrient poor sites) are already adapted to lower nitrogen availability.     

A nonlinear calcification response to CO2-induced ocean acidification by the coral Oculina arbuscula

Anthropogenic elevation of atmospheric pCO₂ is predicted to cause the pH of surface seawater to decline by 0.3–0.4 units by 2100 AD, causing a 50% reduction in seawater [CO₃ ²⁻] and undersaturation with respect to aragonite in high-latitude surface waters. We investigated the impact of CO₂-induced ocean acidification on the temperate scleractinian coral Oculina arbuscula by rearing colonies for 60 days in experimental seawaters bubbled with air-CO₂ gas mixtures of 409, 606, 903, and 2,856 ppm pCO₂, yielding average aragonite saturation states (Ω_A) of 2.6, 2.3, 1.6, and 0.8. Measurement of calcification (via buoyant weighing) and linear extension (relative to a ¹³⁷Ba/¹³⁸Ba spike) revealed that skeletal accretion was only minimally impaired by reductions in Ω_A from 2.6 to 1.6, although major reductions were observed at 0.8 (undersaturation). Notably, the corals continued accreting new skeletal material even in undersaturated conditions, although at reduced rates. Correlation between rates of linear extension and calcification suggests that reduced calcification under Ω_A = 0.8 resulted from reduced aragonite accretion, rather than from localized dissolution. Accretion of pure aragonite under each Ω_A discounts the possibility that these corals will begin producing calcite, a less soluble form of CaCO₃, as the oceans acidify. The corals’ nonlinear response to reduced Ω_A and their ability to accrete new skeletal material in undersaturated conditions suggest that they strongly control the biomineralization process. However, our data suggest that a threshold seawater [CO₃ ²⁻] exists, below which calcification within this species (and possibly others) becomes impaired. Indeed, the strong negative response of O. arbuscula to Ω_A = 0.8 indicates that their response to future pCO₂-induced ocean acidification could be both abrupt and severe once the critical Ω_A is reached.     

Deviation of eyes and head in acute cerebral stroke

Background  

It is a well-known phenomenon that some patients with acute left or right hemisphere stroke show a deviation of the eyes (Prévost's sign) and head to one side. Here we investigated whether both right- and left-sided brain lesions may cause this deviation. Moreover, we studied the relationship between this phenomenon and spatial neglect. In contrast to previous studies, we determined not only the discrete presence or absence of eye deviation with the naked eye through clinical inspection, but actually measured the extent of horizontal eye-in-head and head-on-trunk deviation. In further contrast, measurements were performed early after stroke onset (1.5 days on average).     

The calpastatin-derived calpain inhibitor CP1B reduces mRNA expression of matrix metalloproteinase-2 and -9 and invasion by leukemic THP-1 cells

The ubiquitous proteases mu- and m-calpain are Ca(2+)-dependent cysteine endopeptidases. Besides involvement in a variety of physio(patho)logical processes, recent studies suggest a pivotal role of calpains in differentiation of hematopoietic cells and tumor cell invasion. However, the precise actions of calpains and their endogenous inhibitor, calpastatin, in these processes are only partially understood. Here we have studied the role of the calpain/calpastatin system in the invasion of leukemic cells under basal and differentiation-stimulating conditions. To further differentiate the human leukaemic cell line THP-1 (monocytic), the cells were treated for 24 hours with the differentiation-stimulating reagents phorbol 12-myristate 13-acetate (PMA) and dimethyl sulfoxide (DMSO). Macrophage- and granulocyte-like differentiation was confirmed by induction of vimentin expression as well as by microscopic and fluorescence-assisted cytometric analysis. Extracellular matrix (ECM) invasion of both the basal and differentiation-stimulated cells in a Matrigel assay was inhibited by pre-incubation of the cells with the specific calpain inhibitor CP1B for 24 hours. Inhibition of invasiveness correlated with decreased mRNA expression and secretion of the matrix metalloproteinases MMP-2 and MMP-9. In contrast, addition of CP1B only during the invasion process did neither influence transmigration nor MMP release. This is the first report showing that the calpain/calpastatin system mediates MMP-mRNA expression of the leukemic THP-1 cells and as a consequence their invasiveness.     

Recombinant domains of mouse nidogen-1 and their binding to basement membrane proteins and monoclonal antibodies.

The basement membrane protein, nidogen-1, was previously shown to consist of three globular domains, G1 to G3, and two connecting segments. Nidogen-1 is a major mediator in the formation of ternary complexes with laminins, collagen IV, perlecan and fibulins. In the present study, we have produced recombinant proteins of these predicted domains in mammalian cells and used these proteins for crystallographic and binding epitope analyses. These fragments included G1, G2, the rod domain and a slightly larger G3 structure; all were obtained in good yields and were shown to be properly folded using electron microscopy. Surface plasmon resonance assays demonstrated high affinity binding (Kd = 3-9 nM) of domain G2 for collagen IV, perlecan domain IV-1 and fibulin-2, and a more moderate Kd for fibulin-1C. Domain G3 contained high affinity binding sites for the laminin gamma1 chain and collagen IV (Kd = 1 nM) and weaker binding sites for fibulin-1C and fibulin-2. A moderate binding affinity was also observed between domain G1 and fibulin-2, while no activity could be detected for the nidogen rod domain. Together, these data indicate the potential of nidogen-1 for multiple interactions within basement membranes. A similar binding repertoire was also identified for seven rat monoclonal antibodies that bound with Kd = 2-30 nM to either G1, G1-G2, G2, the rod domain or G3. Three of the antibodies showed strongly reduced binding to G2 and G3 after complex formation with either a perlecan domain or laminin-1.     

Stimulation of ATPase activity in barley (Hordeum vulgare) root plasma membranes after treatment with triacontanol and calmodulin

Triacontanol (TRIA) treatment of plasma membrane-enriched vesicles from barley ( Hordeum vulgare L., cv. Conquest) roots resulted in stimulation of membrane-associated, divalent cation-dependent ATPase activity (EC 3.6.1.3). The stimulation at physiologically active concentrations of TRIA (10⁻¹¹–10⁻⁹ M ) occurred only when the vesicles were treated with TRIA in the presence of calmodulin. Octacosanol, the C₂₈-analogue of TRIA, had no effect on divalent cation-dependent ATPase activity. Consistent with in vivo studies, simultaneous treatment of vesicles with weight equivalents of TRIA and octacosanol reduced the stimulation of ATPase activity. The effect of calmodulin on the stimulation of ATPase activity was diminished by calmidazolium, a specific inhibitor of calmodulin. Circular dichroism studies did not show a change in the α-helix content of calmodulin in the presence of TRIA. TRIA also had no apparent effect on soluble calcium-calmodulin 3',5'-cyclic nucleotide phosphodiesterase activity. Removal of excess TRIA from the medium after treatment still resulted in stimulation of divalent cation-dependent ATPase activity in the presence of calmodulin was comparable to treated vesicles from which excess TRIA had not been removed. These data further support the contention that TRIA affects membrane structure and function.