Evolution and function of the sucrose-phosphate synthase gene families in wheat and other grasses

Suc-phosphate synthase (SPS) is a key regulatory enzyme in the pathway of Suc biosynthesis and has been linked to quantitative trait loci controlling plant growth and yield. In dicotyledonous plants there are three SPS gene families: A, B, and C. Here we report the finding of five families of SPS genes in wheat (Triticum aestivum) and other monocotyledonous plants from the family Poaceae (grasses). Three of these form separate subfamilies within the previously described A, B, and C gene families, but the other two form a novel and distinctive D family, which on present evidence is only found in the Poaceae. The D-type SPS proteins lack the phosphorylation sites associated with 14-3-3 protein binding and osmotic stress activation, and the linker region between the N-terminal catalytic glucosyltransferase domain and the C-terminal Suc-phosphatase-like domain is 80 to 90 amino acid residues shorter than in the A, B, or C types. The D family appears to have arisen after the divergence of mono- and dicotyledonous plants, with a later duplication event resulting in the two D-type subfamilies. Each of the SPS gene families in wheat showed different, but overlapping, spatial and temporal expression patterns, and in most organs at least two different SPS genes are expressed. Analysis of expressed sequence tags indicated similar expression patterns to wheat for each SPS gene family in barley (Hordeum vulgare) but not in more distantly related grasses. We identified an expressed sequence tag from rice (Oryza sativa) that appears to be derived from an endogenous antisense SPS gene, and this might account for the apparently low level of expression of the related OsSPS11 sense gene, adding to the already extensive list of mechanisms for regulating the activity of SPS in plants.     

Regulation of sulfate uptake and expression of sulfate transporter genes in Brassica oleracea as affected by atmospheric H(2)S and pedospheric sulfate nutrition

Demand-driven signaling will contribute to regulation of sulfur acquisition and distribution within the plant. To investigate the regulatory mechanisms pedospheric sulfate and atmospheric H(2)S supply were manipulated in Brassica oleracea. Sulfate deprivation of B. oleracea seedlings induced a rapid increase of the sulfate uptake capacity by the roots, accompanied by an increased expression of genes encoding specific sulfate transporters in roots and other plant parts. More prolonged sulfate deprivation resulted in an altered shoot-root partitioning of biomass in favor of the root. B. oleracea was able to utilize atmospheric H(2)S as S-source; however, root proliferation and increased sulfate transporter expression occurred as in S-deficient plants. It was evident that in B. oleracea there was a poor shoot to root signaling for the regulation of sulfate uptake and expression of the sulfate transporters. cDNAs corresponding to 12 different sulfate transporter genes representing the complete gene family were isolated from Brassica napus and B. oleracea species. The sequence analysis classified the Brassica sulfate transporter genes into four different groups. The expression of the different sulfate transporters showed a complex pattern of tissue specificity and regulation by sulfur nutritional status. The sulfate transporter genes of Groups 1, 2, and 4 were induced or up-regulated under sulfate deprivation, although the expression of Group 3 sulfate transporters was not affected by the sulfate status. The significance of sulfate, thiols, and O-acetylserine as possible signal compounds in the regulation of the sulfate uptake and expression of the transporter genes is evaluated.     

Current density threshold for the stimulation of neurons in the motor cortex area

The aim of this study was to determine a current density threshold for exciting the motor cortex area of the brain. The current density threshold for excitation of nerve fibres (20 microm in diameter) found in the literature is approximately 1 A/m(2) at frequencies lower than 1 kHz. In consideration of a safety factor of 100, the International Commission on Non-Ionizing Radiation Protection (ICNIRP) recommends to restrict the exposure to 0.01 A/m(2). The electromagnetic stimulation of neurons in the motor cortex is used in the clinical diagnosis of nerve lesions and neuropathy by means of magnetic or electrical transcranial stimulation. Combining medical data from clinical studies and technical specifications of the Magstim Model 200 stimulator, we were able to compute the current density threshold for the excitation of the human motor cortex by means of the finite element method (FEM). A 3D-CAD head model was built on the basis of magnetic resonance imaging (MRI) slices and segmented into four anatomical structures (scalp, skull, brain, and ventricular system) with different conductivities. A current density threshold for the stimulation of the motor cortex area of the upper limbs of 6 and 2.5 A/m(2) at 2.44 kHz and 50 Hz, respectively, was calculated. As these values lie above the recommended ICNIRP values by two orders of magnitude there is no need for lower safety standards with regard to stimulation of the brain.     

Stress- and mitogen-induced phosphorylation of the small heat shock protein Hsp25 by MAPKAP kinase 2 is not essential for chaperone properties and cellular thermoresistance.

Small heat shock proteins (sHsps) show a very rapid stress- and mitogen-dependent phosphorylation by MAPKAP kinase 2. Based on this observation, phosphorylation of sHsps was thought to play a key role in mediating thermoresistance immediately after heat shock, before the increased synthesis of heat shock proteins becomes relevant. We have analysed the phosphorylation dependence of the chaperone and thermoresistance-mediating properties of the small heat shock protein Hsp25. Surprisingly, overexpression of Hsp25 mutants, which are not phosphorylated in the transfected cells, confers the same thermoresistant phenotype as overexpression of wild type Hsp25, which is either mono- or bis-phosphorylated at serine residues 15 and 86 within the cells. Furthermore, in vitro phosphorylated Hsp25 shows the same oligomerization properties and the same chaperone activity as the nonphosphorylated protein. No differences between phosphorylated and nonphosphorylated Hsp25 are detected in preventing thermal aggregation of unfolding proteins and assisting refolding of denatured proteins. The results suggest that chaperone properties of the small heat shock proteins contribute to the increased cellular thermoresistance in a phosphorylation-independent manner.     

Prevalence of HBsAg and HBsAg sub-types in the Canadian blood-donor population

After 8 years of screening all blood donations in Canada for HBsAg, first by CIEP and later by RIA, the prevalence of HBsAg in the regular panel of "repeat" donors has been reduced from 267/10(5) to 39/10(5). Marked geographic variations exist, but the available data do not indicate whether the high prevalence of HBsAg in young adults, particularly males, may be a factor. The ad : ay subtype ratio across Canada is 2.0, but noticeable geographic differences are present, varying from 3.5 in Quebec to 0.5 in the Atlantic Provinces.     

An unstructured C-terminal region of the Hsp90 co-chaperone p23 is important for its chaperone function.

p23 is a co-chaperone of the heat shock protein Hsp90. p23 binds to Hsp90 in its ATP-bound state and, on its own, interacts specifically with non-native proteins. In our attempt to correlate these functions to specific regions of p23 we have identified an unstructured region in p23 that maps to the C-terminal part of the protein sequence. This unstructured region is dispensible for interaction of p23 with Hsp90, since truncated p23 can still form complexes with Hsp90. In contrast, however, truncation of the C-terminal 30 amino acid residues of p23 affects the ability of p23 to bind non-native proteins and to prevent their non-specific aggregation. The isolated C-terminal region itself is not able to act as a chaperone nor is it possible to complement truncated p23 by addition of this peptide. These results imply that the binding site for Hsp90 is contained in the folded domain of p23 and that for efficient interaction of p23 with non-native proteins both the folded domain and the C-terminal unstructured region are required.     

Lysis of prostate carcinoma cells by trifunctional bispecific antibodies (alpha EpCAM x alpha CD3).

Bispecific monoclonal antibodies (bsAbs) are a promising immunotherapeutic option for treatment of cancer, especially in situations of minimal residual disease. The combination of an anti-CD3 and anti-tumor-associated antigen antibody redirects cytotoxic T-lymphocytes towards malignant cells. Using a trifunctional bispecific antibody against EpCAM x CD3, that additionally activates Fc gamma R(+) accessory cells via its Fc region, we investigated the interaction between three EpCAM(+) prostate carcinoma cell lines and peripheral blood mononuclear cells (PBMCs) of healthy donors and patients with prostate carcinoma (PC). Visualization was performed by double immunocytochemical methods and computerized sequential video microscopy. Tumor cells and PBMCs supplemented with alpha EpCAM x alpha CD3 in 16-well chamber slides resulted in lysis of tumor cells within 1--3 days without any differences between patient and healthy donor PBMCs. The characteristic necrotic way of tumor cell killing (rounding, swelling, disrupting) could be observed in computerized sequences of video frames. Simultaneously, we could not reveal any form of apoptotic signal using three different apoptotic markers (TUNEL, M30 cyto death, anti-active caspase 3). Within the first 48 hr we observed typical PBMC cluster formation with increasing cell proliferation. PBMCs surrounding the tumor cells were not dominated by CD4(+), CD8(+), or CD14(+) cells. Lymphocytes with pore-forming perforin proteins concentrated towards the tumor target cells. Our combination of double immunocytochemical and computerized video microscopic techniques may serve as an important improvement of validity of cell-cell interaction experiments using in vitro models. (J Histochem Cytochem 49:911-917, 2001)     

Plant sulphate transporters: co-ordination of uptake, intracellular and long-distance transport

Proton/sulphate co-transport in the plasma membrane of root cells is the first step for the uptake of sulphate from the environment by plants. Further intracellular, cell-to-cell and long-distance transport must fulfil the requirements for sulphate assimilation and source/sink demands within the plant. A gene family of sulphate transporters, which may be subdivided into five groups, has been identified with examples from many different plant species. For at least two groups, proton/sulphate co-transport activity has been confirmed. It appears that each group represents sulphate transporters with distinct kinetic properties, patterns of expression, and cell/tissue specificity related to specific roles in the uptake and allocation of sulphate. High-affinity sulphate uptake and low-affinity vascular transport, as well as vacuolar efflux, are controlled by the nutritional status of the plant. Most notably there is an apparent increase in capacity for cellular sulphate uptake and vacuolar efflux when sulphur supply is limiting. Within the groups, the individual sulphate transporters may be further subdivided by differences in temporal, cellular and tissue expression. Many of the transporters are regulated by the nutritional status of the individual tissues, to optimize sulphate movement within and between sink and source organs.