The Contribution of the Blood Glutamate Scavenging Activity of Pyruvate to its Neuroprotective Properties in a Rat Model of Closed Head Injury

The removal of excess glutamate from brain fluids after acute insults such as closed head injury (CHI) and stroke is expected to prevent excitotoxicity and the ensuing long lasting neurological deficits. Since blood glutamate scavenging accelerates the removal of excess glutamate from brain into blood and causes neuroprotection, we have evaluated here whether the neuroprotective properties of pyruvate could be partly accounted to its blood glutamate scavenging activity. The neurological outcome of rats after CHI improved significantly when treated with intravenous pyruvate (0.9 mmoles/100 g) but not with pyruvate administered together with glutamate. Pyruvate, at 5 μmole/100 g rat was neither protective not able to decrease blood glutamate but displayed the latter two properties when combined with 60 μg/100 g of glutamate-pyruvate transaminase. Since the neurological recovery from CHI was correlated with the decrease of blood glutamate levels, we conclude that pyruvate blood glutamate scavenging activity contributes to the spectrum of its neuroprotective mechanisms.     

NKp46 O-Glycan Sequences That Are Involved in the Interaction with Hemagglutinin Type 1 of Influenza Virus

Natural killer (NK) cells serve as a crucial first-line defense against tumors and virus-infected cells. We previously showed that lysis of influenza virus (IV)-infected cells is mediated by the interaction between the NK receptor, NKp46, and the IV hemagglutinin (HA) type 1 expressed by the infected cells. This interaction requires the presence of sialyl groups on the NKp46-T225 O-glycoforms. In the current study, we analyzed the O-glycan sequences that are imperative for the interaction between recombinant NKp46 (rNKp46) and IV H1N1 strains. We first showed that rNKp46 binding to IV H1N1 is not mediated by a glycoform unique to the Thr225 site. We then characterized the O-glycan sequences that mediate the interaction of rNKp46 and IV H1N1; we employed rNKp46s with dissimilar glycosylation patterns and IV H1N1 strains with different sialic acid α2,3 and α2,6 linkage preferences. The branched α2,3-sialylated O-glycoform Neu5NAcα2,3-Galβ1,4-GlcNAcβ1,6[Neu5NAcα2,3-Galβ1,3]GalNAc competently mediated the interaction of rNKp46 with IV H1N1, manifesting a preference for α2,3 linkage. In contrast, the linear α2,3-sialylated O-glycoform Neu5NAcα2,3-Galβ1,3-GalNAc was not correlated with enhanced interaction between rNKp46 and IV H1N1 or a preference for α2,3 linkage. The branched α2,3- and α2,6-sialylated O-glycoform Neu5NAcα2,3-Galβ1,3[Neu5NAcα2,6]GalNAc competently mediated the interaction of rNKp46 with IV H1N1, manifesting a preference for α2,6 linkage. Previous viral HA-binding-specificity studies were performed with glycopolymer conjugates, free synthetic sialyl oligosaccharides, and sialidase-treated cells. This study shed light on the O-glycan sequences involved in the interaction of glycoprotein and viral hemagglutinins and may help in the design of agents inhibitory to hemagglutinin for influenza treatment.Hemagglutinin (HA) is the receptor-binding and membrane fusion protein of influenza virus (IV), as well as the target for infectivity-neutralizing antibodies (27). Terminal sialic acids of glycoproteins and glycolipids are the cellular receptors for the IV HA (27). Two major linkages between sialic acid and the penultimate galactose residues of carbohydrate side chains are found in nature, Neu5NAcα(2,3)-Gal and Neu5NAcα(2,6)-Gal (27); different HAs have different recognition specificities for these linkages and the sugar backbone beneath (23, 26, 30). However, all of the HA-binding specificity studies were performed with glycopolymer conjugates, free synthetic sialyl oligosaccharides, and sialidase-treated cells (8, 10, 20, 25). This could be sufficient for the design of IV-inhibitory agents, and yet, it contributes only partially to the understanding of the interaction of IV HAs with glycoproteins and glycolipids. We aimed to further explore the exact glycoform sequences conjugated to a specific glycoprotein''s glycosylation site that is recognized by different IV strains.For this purpose, we took advantage of our findings on the interaction of natural cytotoxicity receptors (NCRs) and IV HAs (2, 3, 13, 18, 19, 22, 34). We showed that the NKp44 and NKp46 NCRs but not the NKp30 NCR interact with IV HAs. This interaction requires the sialylation of NKp44 and NKp46 oligosaccharides, and the binding of these NCRs to viral HA is required for the lysis of virus-infected cells by NK cells (3, 13, 18). NKp46 displays two putative O-linked glycosylation sites at Thr125 and Thr225 and one N-linked glycosylation site at Asn216. In order to determine the specific sugar-carrying residue that is important for the HA1 recognition, site-directed mutagenesis of the three residues was performed to carry the glycan modifications. Only when Thr225 was replaced was a sharp decrease in the enhanced binding to IV HA1 and IV H1N1-infected cells observed (2). Therefore, for the NKp46 receptor, the interaction with IV HA1 is restricted to Thr225, one of its three glycosylation sites (2).We already showed that producing recombinant NKp46 (rNKp46) in different cell lines resulted in dissimilar glycosylation patterns and had a strong effect on the binding to its ligands (11). Therefore, we analyzed the O-glycan patterns of rNKp46 produced from various cell lines and utilized the dissimilar glycosylation patterns to elucidate the NKp46 O-glycan sequences that mediate the interaction with IV H1N1 strains. To associate the results with the IV preference for sialic acid α2,3 and/or α2,6 linkages, we employed A/PR/8/34 (H1N1), A/NC/20/99 (H1N1), and A/Brisbane/59/2007 (H1N1) grown in either hen egg amnion or Madin-Darby canine kidney (MDCK) cells. Our results pointed to two branched O-glycan sequences that mediated the interaction of the NKp46 glycoprotein with IV H1N1 in correlation with the sialic acid linkage preference of the IV strain.     

Fine-tuning by strigolactones of root response to low phosphate

Strigolactones are plant hormones that regulate the development of different plant parts. In the shoot,they regulate axillary bud outgrowth and in the root,root architecture and root-hair length and density. Strigolactones are also involved with communication in the rhizosphere,including enhancement of hyphal branching of arbuscular mycorrhizal fungi. Here we present the role and activity of strigolactones under conditions of phosphate deprivation.Under these conditions,their levels of biosynthesis and exudation increase,leading to changes in shoot and root development. At least for the latter,these changes are likely to be associated with alterations in auxin transport and sensitivity. On the other hand,strigolactones may positively affect plant–mycorrhiza interactions and thereby promote phosphate acquisition by the plant. Strigolactones may be a way for plants to fine-tune their growth pattern under phosphate deprivation.     

Investigation of Phycobilisome Subunit Interaction Interfaces by Coupled Cross-linking and Mass Spectrometry

The phycobilisome (PBS) is an extremely large light-harvesting complex, common in cyanobacteria and red algae, composed of rods and core substructures. These substructures are assembled from chromophore-bearing phycocyanin and allophycocyanin subunits, nonpigmented linker proteins and in some cases additional subunits. To date, despite the determination of crystal structures of isolated PBS components, critical questions regarding the interaction and energy flow between rods and core are still unresolved. Additionally, the arrangement of minor PBS components located inside the core cylinders is unknown. Different models of the general architecture of the PBS have been proposed, based on low resolution images from electron microscopy or high resolution crystal structures of isolated components. This work presents a model of the assembly of the rods onto the core arrangement and for the positions of inner core components, based on cross-linking and mass spectrometry analysis of isolated, functional intact Thermosynechococcus vulcanus PBS, as well as functional cross-linked adducts. The experimental results were utilized to predict potential docking interactions of different protein pairs. Combining modeling and cross-linking results, we identify specific interactions within the PBS subcomponents that enable us to suggest possible functional interactions between the chromophores of the rods and the core and improve our understanding of the assembly, structure, and function of PBS.     

TH1/TH2 cytokine secretion of first degree relatives of T1DM patients

Th1/Th2 cytokine imbalance has been demonstrated in Type 1 diabetes (T1DM) patients. We characterized the peak levels, secretory pattern and total cytokine production of the Th1 cytokines (IL-2 and IFN gamma) and Th2 cytokines (IL-4 and IL-10), by stimulated peripheral blood mononuclear cells of twenty six first-degree relatives of T1DM patients, and eleven matched controls. At enrollment, first degree relatives demonstrated a significant increase in peak and overall secretion of IL-2; P<0.01 and P<0.005 respectively and IL-4 cytokine; P<0.05 and P<0.01 respectively, as compared to normal controls. Their mean IFN gamma secretion increased significantly, P<0.05, after one year while their higher IL-2 and IL-4 secretion remained unchanged. Ab-negative and Ab-positive relatives demonstrated a similar cytokine secretion pattern. Four relatives all Ab positive, developed diabetes: Peak IL-4 levels were low in three and markedly decreased within one year in one of these relatives, while peak IL-2 and IFN gamma levels were elevated in all of them. These data demonstrate that secretion of both Th1 and Th2 cytokines is increased in first-degree relatives of T1DM patients independently of their diabetes-associated autoantibodies. The presence of low IL-4 and elevated IL-2 and IFN gamma levels in autoAb positive relatives is associated with progression to overt disease.     

Intestinal and hepatic expression of BNIP3 in necrotizing enterocolitis: regulation by nitric oxide and peroxynitrite

Necrotizing enterocolitis (NEC) is characterized by the upregulation of proinflammatory proteins, nitrosative stress, and increased enterocyte apoptosis. We examined the expression and regulation of the Bcl-2/adenovirus EIB 19-kDa-interacting protein 3 (BNIP3), a pro-apoptotic gene regulated by nitric oxide (NO) in hepatocytes, in NEC. Newborn rats subjected to hypoxia and fed a conventional formula by gavage (FFH) developed NEC and demonstrated elevated expression of BNIP3 mRNA and protein in mucosal scrapings of the ileal samples and in the liver. In contrast, control rats [breast-fed (BF) without hypoxia] did not develop NEC or elevated BNIP3 expression in these tissues. BNIP3 expression paralleled the histological manifestation of NEC. Supplementation of the formula with L-Nomega-(1-iminoethyl)lysine, an inducible NO synthase inhibitor, reduced BNIP3 expression in FFH animals to the levels found in BF animals. Both hypoxia and peroxynitrite upregulated BNIP3 protein expression in human intestinal cells. Finally, ileal samples obtained from infants undergoing surgical resection for acute NEC demonstrated higher levels of BNIP3 protein. Because hypoxia and formation of reactive nitrogen species may promote gut barrier failure, we propose that upregulation of the cell death-related protein BNIP3 is one possible mechanism associated with enterocyte cell death observed in the intestine with NEC.     

Induction and the Turing-Child field in development

The central problem in biological development is the understanding of epigenesis. The dominant theory of development in the last 80 years that also purports to explain epigenesis is induction theory. It suggests that development is driven by sequential inductions where each "induction" (in one sense of the word induction) is effected by the action of an inducing part of the embryo on a responding part of the embryo. The theory stems from Spemann and Mangold (W.Roux' Arch.f.Entw.d.Organis.u.mikrosk.Anat.100 (1924) 599) who transplanted a tissue from the dorsal blastopore lip of Triturus into the ventral ectoderm of another gastrula and thus initiated and "induced" (in another sense of the word induction) gastrulation and embryogenesis in the ventral side of the host that became a double embryo (siamese twins). We explain this induction, i.e. the formation of the double embryo, according to the Child theory and the Turing-Gierer-Meinhardt theory when it is also assumed that cAMP and ATP are the Turing activator and inhibitor, respectively. Spemann and Mangold (W.Roux' Arch.f.Entw.d.Organis.u.mikrosk.Anat.100 (1924) 599) also suggested that the ingressing mesoderm induces the overlying ectoderm to form the neural plate and neural tube. This 'neural induction', the 'primary embryonic induction', became the cornerstone of induction theory, i.e. of the sequential induction concept referred to above. But we argue that the metabolic gradients that precede and accompany neurulation, as obtained by Child, also for Triturus, arise through a Turing self-organization if it is assumed that cAMP and ATP are the Turing morphogens, and these gradients are the cause and primary event of neurulation. Thus there is no need to invoke the 'neural induction'. It is argued that fundamental events such as gastrulation and also organ formation are caused by the Turing-Child field and not by sequential induction. Similar principles, such as bud formation caused by a radial metabolic pattern that transforms to a longitudinal pattern, govern the formation, for example, of the mouth and the gut. The formation and localization of bottle cells is explained according to the Child-Turing field and modern biochemistry. The chemical metabolic pre-pattern precedes, and causes, morphogenesis and differentiation as envisaged by Turing. The Spemann and Mangold (W.Roux' Arch.f.Entw.d.Organis.u.mikrosk.Anat.100 (1924) 599) transplantation experiment when performed on a sea urchin duplicates not only the phenotype but also the metabolic (reduction) pattern. These experimental results, by Horstadius, predicted by Child, follow from the Turing-Gierer-Meinhardt theory if it is assumed that cAMP and ATP are the Turing morphogens. If the transplantation is performed not onto the whole sea urchin but onto only a part of it, that manifests only a part of the metabolic pattern, then from the part a phenotypic whole underlain by a normal and a whole metabolic pattern can be rescued. These experimental results of Horstadius follow from Turing theory if cAMP and ATP are the Turing morphogens. Understanding how to transform a part into a whole can be valuable in regenerative medicine. Unspecific induction of a secondary amphibian embryo is similar to the induction of posterior structures at the anterior pole of an insect, and the "double abdomen" (and Kalthoff's experimental results) of the midge Smittia resulting from UV irradiation of the anterior pole, can be explained by Meinhardt theory of unspecific induction if ATP is the Turing morphogen. When not working on regeneration, Child investigated intact living organisms and his observation method was not disruptive to normal development, whereas workers in induction theory work with pieces and in general disrupt normal development. We conclude that the Turing-Child field causes all development and explains epigenesis. Sequential induction does not explain epigenesis and does not exist in normal development. But induction in the sense of a transplantation leading to double embryo or rescuing a whole phenotype from a part is of interest.