Biosynthesis of lactosamine in bovine mammary gland

Lactosamine (beta-D-Galp-(1-->4)-D-GlcN) was isolated from bovine milk sampled after intravenous infusion of glucosamine through the jugular vein of a lactating cow. The chemical structure was established by 2D NMR spectroscopy and electrospray ionisation mass spectrometry (ESIMS). Selected ion monitoring liquid chromatography-mass spectrometry (SIMLC-MS) of the perbenzoylated carbohydrate fraction showed the presence of the novel disaccharide in the milk sample after infusion, but not in the control bovine milk sample. The results showed the uptake of glucosamine in bovine mammary gland, and also indicated that a part of glucosamine was metabolised to the product lactosamine.     

Caspase-independent cell death and mitochondrial disruptions observed in the Apaf1-deficient cells

Apaf1 is a critical molecule in the mitochondria-dependent apoptotic pathway. Here we show that Apaf1-deficient embryonic fibroblasts died at a later phase of apoptotic induction, although these cells were resistant to various apoptotic stimulants at an early phase. Neither caspase 3 activation nor nuclear condensation was observed during this cell death of Apaf1-deficient cells. Electron microscopic examination revealed that death in response to apoptotic stimulation resembled necrosis in that nuclei were round and swollen with low electron density. Necrosis-like cell death was also observed in wild-type cells treated with z-VAD-fmk. Mitochondria were not only morphologically abnormal but functionally affected, since mitochondrial transmembrane potential (DeltaPsim) was lost even in cells with intact plasma membrane integrity. These mitochondrial alterations were also observed in the wild-type cells dying of apoptosis. Combined, these data suggest that cells without caspase activation, such as Apaf1-deficient cells or cells treated with caspase inhibitors, die of necrosis-like cell death with mitochondrial damage in response to "apoptotic stimulation."     

The analysis of heparin-protein interactions using evanescent wave biosensor with regioselectively desulfated heparins as the ligands

Evanescent wave biosensor has been recently employed as a powerful tool for analyses of macromolecular interactions. In the present study, evanescent wave biosensor analysis was developed to analyze the heparin-protein interaction using as ligands a series of heparin derivatives regioselectively desulfated by chemical methods, particularly to evaluate the effect of each sulfate group of heparin. The method for immobilizing heparin on the cuvette of the evanescent wave biosensor equipment was optimized to obtain the high response required for accurate measurement. The best result was achieved when the amino group introduced at the reducing end of heparin was coupled with carboxymethyl dextran on the surface of the cuvette using glycolchitosan as a multivalent linker. The established system appeared to describe well the interactions of heparin with such proteins as acidic and basic fibroblast growth factors and tissue factor pathway inhibitor.     

Molecular cloning of Na(+)-ATPase cDNA from a marine alga, Heterosigma akashiwo

We cloned novel Na(+)-ATPase (HANA) cDNA from marine alga Heterosigma akashiwo. The full-length HANA cDNA was 4467 bp long and coded for a 1330 amino acid protein with a molecular weight of 146,306. The deduced product exhibited around 40% identity in amino acids with Na(+)/K(+)-ATPase alpha-subunits. A hydrophilic sequence of 285 amino acid residues that showed no homology with any sequence listed in databases existed in the M7--M8 junction of HANA. This is the first report on the primary structure of putative Na(+)-transporting ATPase from plant cells.     

Brief exposure to low-pH stress causes irreversible damage to the growing root in Arabidopsis thaliana: pectin-Ca interaction may play an important role in proton rhizotoxicity

The viability of Arabidopsis thaliana (strain Landsberg) roots exposed to a low pH (4.5 or 4.7) solution that contained 100 microM CaCl(2) was examined by staining with fluorescein diacetate-propidium iodide. The elongation zone of growing roots lost viability within 1-2 h following exposure to low pH, but non-growing roots showed no damage under the same treatment. Low-pH damage in growing roots was irreversible after 1 h incubation at pH 4.5 as judged by regrowth in growing medium at pH 5.6. Growing lateral roots also lost viability in the same treatment, whereas non-growing lateral roots remained viable during and after the treatment. The low-pH damage was ameliorated by the simultaneous application of calcium, indicating the involvement of a calcium-requiring process in overcoming proton toxicity. At pH 5.0, growing roots required 25 microM of calcium to maintain elongation, and at pH 4.8 and pH 4.5 more than 250 microM and 750 microM, respectively. The low-pH damage was ameliorated by divalent cations in the order of Ba2+, approximately Sr2+>/=Ca2+>Mg2+. The monovalent cation K+ showed no ameliorative effect, but borate showed a strong ameliorative effect with Ca2+. These results indicate that the primary target of proton toxicity may be linked to a disturbance of the stability in the pectic polysaccharide network, where calcium plays a key role in plant roots.     

Differential susceptibility of heart, skin, and islet allografts to T cell-mediated rejection

Although it is widely accepted that there is a hierarchy in the susceptibility of different allografts to rejection, the mechanisms responsible are unknown. We show that the increased susceptibility of H-2K(b+) skin and islet allografts to rejection is not based on their ability to activate more H-2K(b)-specific T cells in vivo; heart allografts stimulate the activation and proliferation of many more H-2K(b)-specific T cells than either skin or islet allografts. Rejection of all three types of graft generate memory cells by 25 days posttransplant. These data provide evidence that neither tissue-specific Ags nor, surprisingly, the number of APCs carried in the graft dictate their susceptibility to T cell-mediated rejection and suggest that the graft microenvironment and size may play a more important role in determining the susceptibility of an allograft to rejection and resistance to tolerance induction.     

Radical scavenging activity of tea catechins and their related compounds

(-)-Epigallocatechin gallate was found to be the most effective scavenger among tea catechins for the superoxide anion, hydroxyl radical, and 1,1-diphenyl-3-picrylhydrazyl radical. Examination of the scavenging effects of tea catechins and their glucosides on superoxide anion showed that the presence of at least an ortho-dihydroxyl group in the B ring and a galloyl moiety at the 3 position was important in maintaining the effectiveness of the radical scavenging ability. Stoichiometric factors of tea catechins were estimated to be 2 for (+)-catechin and (-)-epicatechin, 5 for (-)-epigallocatechin, 7 for (-)-epicatechin gallate, and 10 for (-)-epigallocatechin gallate.     

cDNA sequence and expression of a cold-responsive gene in Citrus unshiu

A cDNA clone encoding a protein (CuCOR19), the sequence of which is similar to Poncirus COR19, of the dehydrin family was isolated from the epicarp of Citrus unshiu. The molecular mass of the predicted protein was 18,980 daltons. CuCOR19 was highly hydrophilic and contained three repeating elements including Lys-rich motifs. The gene expression in leaves increased by cold stress.     

Alpha4 protein as a common regulator of type 2A-related serine/threonine protein phosphatases

The catalytic activity of the C subunit of serine/threonine phosphatase 2A is regulated by the association with A (PR65) and B subunits. It has been reported that the alpha4 protein, a yeast homolog of the Tap42 protein, binds the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A-related protein phosphatases such as protein phosphatase 4 and protein phosphatase 6. In the present study, we showed that alpha4 binds these three phosphatases and the association of alpha4 reduces the activities of these phosphatases in vitro. In contrast, PR65 binds to the C subunit of serine/threonine phosphatase 2A but not to protein phosphatase 4 and protein phosphatase 6. These results suggest that the alpha4 protein is a common regulator of the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A-related protein phosphatases.