Kinetics of β-N-methylamino-L-alanine (BMAA) and 2, 4-diaminobutyric acid (DAB) production by diatoms: the effect of nitrogen

The neurotoxins β-N-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB) are produced by cyanobacteria, diatoms and dinoflagellates and have been detected in seafood worldwide. Our present knowledge of their metabolism or biosynthesis is limited. In this study, the production of BMAA and DAB as a function of time was monitored in five strains representing four species of diatoms, i.e. Phaeodactylum tricornutum, Thalassiosira weissflogii, Thalassiosira pseudonana and Navicula pelliculosa, previously identified as BMAA and DAB producers. Subsequently, three strains were selected and exposed to three nitrogen treatments – starvation, control (the standard concentration in f/2 medium) and enrichment, because BMAA metabolism has been suggested to be closely associated with cellular nitrogen metabolism in both cyanobacteria and diatoms. Chlorophyll a and total protein concentrations were also determined. Our results indicate that BMAA and DAB production in diatoms is species- and strain-specific. However, production might also be affected by stress, particularly as related to nitrogen starvation and cell density. Furthermore, this study shows a significant correlation between the production of the two neurotoxins which might further suggest common steps in the metabolic pathways.     

Anti-Bacterial Activity of Recombinant Human β-Defensin-3 Secreted in the Milk of Transgenic Goats Produced by Somatic Cell Nuclear Transfer

The present study was conducted to determine whether recombinant human β-defensin-3 (rHBD3) in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (S. agalactiae) that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90–111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5–10.5, 21.8–23.0 and 17.3–18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05). The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×10³ and 95.4×10³ CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC) in infected glands reached up to 260.4×10⁵ and 622.2×10⁵ cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals.     

Peptide-specific Transfer of N-Acetylgalactosamine to O-Linked Glycans by the Glycosyltransferases β1,4-N-Acetylgalactosaminyl Transferase 3 (β4GalNAc-T3) and β4GalNAc-T4

N- and O-linked oligosaccharides on pro-opiomelanocortin both bear the unique terminal sequence SO(4)-4-GalNAcβ1,4GlcNAcβ. We previously demonstrated that protein-specific transfer of GalNAc to N-linked oligosaccharides on glycoprotein substrates is dependent on the presence of both an oligosaccharide acceptor and a peptide recognition motif consisting of a cluster of basic amino acids. We characterized how two β1,4-N-acetylgalactosaminyltransferases, β4GalNAc-T3 and β4GalNAc-T4, require the presence of both the peptide recognition motif and the N-linked oligosaccharide acceptors to transfer GalNAc in β1,4-linkage to GlcNAc in vivo and in vitro. We now show that β4GalNAc-T3 and β4GalNAc-T4 are able to utilize the same peptide motif to selectively add GalNAc to β1,6-linked GlcNAc in core 2 O-linked oligosaccharide structures to form Galβ1,3(GalNAcβ1,4GlcNAcβ1,6)GalNAcαSer/Thr. The β1,4-linked GalNAc can be further modified with 4-linked sulfate by either GalNAc-4-sulfotransferase 1 (GalNAc-4-ST1) (CHST8) or GalNAc-4-ST2 (CHST9) or with α2,6-linked N-acetylneuraminic acid by α2,6-sialyltransferase 1 (ST6Gal1), thus generating a family of unique GalNAcβ1,4GlcNAcβ (LacdiNAc)-containing structures on specific glycoproteins.     

Kinetics and mechanism of the oxidative addition of iodomethane to β-diketonatobis(triphenylphosphite)rhodium(I) complexes

The kinetics and mechanism of the oxidative addition of CH₃I to [Rh(β-diketone)(P(OPh)₃)₂] complexes was studied in acetone medium at various temperatures. The experimental rate law is R = k[Rh(β-diketone)(P(OPh₃)₂][CH₃I]. The order of the effect of the β-diketone on the reactivity of the complexes is acac >; BA >; DBM >; TFAA >; TFBA >; HFAA indicating that electronegative substituents of the β-diketone decrease the reactivity of the complexes towards oxidative addition reactions. The volume of activation for some of the reactions was determined in various solvents. The large negative values of the volume and entropy of the activation indicated a mechanism which occurs via a polar transition state.     

Are Female Starlings Able to Recognize the Scent of Their Offspring?

Background

Although there is growing evidence that birds may have individual chemical profiles that can function in several social contexts, offspring recognition based on olfactory cues has never been explored. This ability should be more likely evolved in colonial birds and/or species suffering brood parasitism, in which the risk of being engaged in costly misdirected parental care is high.

Methodology/Principal Findings

We performed a choice experiment to examine whether females of the spotless starling, Sturnus unicolor, a species that is colonial, and where a fraction of the population is exposed to intraspecific brood parasitism, can discriminate between the scent of their offspring and that of unrelated nestlings. We also explored whether the development of the uropygial gland secretion may play a role in such olfactory discrimination by performing the choice experiments to females rearing nestlings of two different ages, that is, without and with developed uropygial glands. Results showed that female starlings did not preferentially choose the scent of their offspring, independently of whether the gland of nestlings was developed or not.

Conclusions/Significance

Our results suggest that female starlings do not have or do not show the ability to distinguish their offspring based on olfaction, at least up to 12–14 days of nestling age. Further research is needed to examine whether odour-based discrimination may function when fledgling starlings leave the nest and the risk of costly misidentification is likely to increase.     

Is Socioeconomic Status of the Rearing Environment Causally Related to Obesity in the Offspring?

We attempt to elucidate whether there might be a causal connection between the socioeconomic status (SES) of the rearing environment and obesity in the offspring using data from two large-scale adoption studies: (1) The Copenhagen Adoption Study of Obesity (CASO), and (2) The Survey of Holt Adoptees and Their Families (HOLT). In CASO, the SES of both biological and adoptive parents was known, but all children were adopted. In HOLT, only the SES of the rearing parents was known, but the children could be either biological or adopted. After controlling for relevant covariates (e.g., adoptee age at measurement, adoptee age at transfer, adoptee sex) the raw (unstandardized) regression coefficients for adoptive and biological paternal SES on adoptee body mass index (BMI: kg/m²) in CASO were -.22 and -.23, respectively, both statistically significant (p = 0.01). Controlling for parental BMI (both adoptive and biological) reduced the coefficient for biological paternal SES by 44% (p = .034) and the coefficient for adoptive paternal SES by 1%. For HOLT, the regression coefficients for rearing parent SES were -.42 and -.25 for biological and adoptive children, respectively. Controlling for the average BMI of the rearing father and mother (i.e., mid-parental BMI) reduced the SES coefficient by 47% in their biological offspring (p≤.0001), and by 12% in their adoptive offspring (p = .09). Thus, despite the differing structures of the two adoption studies, both suggest that shared genetic diathesis and direct environmental transmission contribute about equally to the association between rearing SES and offspring BMI.     

Analysis of Proteins Binding to the ITAM Motif of the β-Subunit of the High-Affinity Receptor for IgE (FcεRI)

Aggregation of the multichain (α β γ₂) high-affinity IgE receptor (Fcε RI) initiates a signaling cascade that results in the release of allergic mediators. The cytoplasmic tails of the Fcε RI-β and -γ subunits contain immunoreceptor tyrosine-based activation motifs (ITAMs). Phosphorylation of the γ ITAM mediates activation of Syk kinase and is sufficient for triggering the responses induced by Fcε RI crosslinking. Phosphorylation of the β ITAM is insufficient to mediate cell activation. The rat β ITAM contains three tyrosines (Tyr²¹⁸, Tyr²²⁴, and Tyr²²⁸) with an intermediate noncanonical tyrosine. Synthetic peptides based on the ITAM of the Fcε RI-β subunit were used to investigate the role of each phosphotyrosine in the binding of signaling proteins to this motif. Among the proteins that bind to phosphorylated β ITAM are Syk, Grb2, Shc, SHIP, and SHP-1, and binding does not depend on previous cell activation. Nonphosphorylated peptides do not bind these proteins. Syk binding to β -peptides is dependent on the number and position of phosphotyrosines in the ITAM. Phosphorylation of Tyr²¹⁸ seems to be most important for Syk binding. Recruitment of Syk and other signaling proteins to the β -subunit might be important for its amplifier role.     

Molecular Basis for Protein-specific Transfer of N-Acetylgalactosamine to N-Linked Glycans by the Glycosyltransferases β1,4-N-Acetylgalactosaminyl Transferase 3 (β4GalNAc-T3) and β4GalNAc-T4

Two closely related β1,4-N-acetylgalactosaminyltransferases, β4GalNAc-T3 and β4GalNAc-T4, are thought to account for the protein-specific addition of β1,4-linked GalNAc to Asn-linked oligosaccharides on a number of glycoproteins including the glycoprotein hormone luteinizing hormone and carbonic anhydrase-6 (CA6). We have utilized soluble, secreted forms of β4GalNAc-T3 and β4GalNAc-T4 to define the basis for protein-specific GalNAc transfer in vitro to chimeric substrates consisting of Gaussia luciferase followed by a glycoprotein substrate. Transfer of GalNAc by β4GalNAc-T3 and β4GalNAc-T4 to terminal GlcNAc is divalent cation-dependent. Transfer of GalNAc to glycoprotein acceptors that contain a peptide recognition determinant is maximal between 0.5 and 1.0 mm MnCl(2); however, transfer is increasingly inhibited by concentrations of MnCl(2) above 1 mm and by anion concentrations above 15 mm. In contrast, transfer of GalNAc to the simple sugar acceptor N-acetylglucosamine-β-p-nitrophenol (GlcNAcβ-pNP) is not inhibited by concentrations of MnCl(2) or anions that would inhibit transfer to glycoprotein acceptors by >90%. This finding indicates that interaction with the peptide recognition determinant in the substrate is sensitive to the anion concentration. β4GalNAc-T3 and β4GalNAc-T4 have similar but distinct specificities, resulting in a 42-fold difference in the IC(50) for transfer of GalNAc to chimeric glycoprotein substrates by agalacto human chorionic gonadotropin, comprising 29 nm for β4GalNAc-T3 and 1.2 μm for β4GalNAc-T4. Our in vitro analysis indicates that enzymatic recognition of the peptide determinant and the oligosaccharide acceptor are independent events.     

The β(1a) subunit of the skeletal DHPR binds to skeletal RyR1 and activates the channel via its 35-residue C-terminal tail

Although it has been suggested that the C-terminal tail of the β(1a) subunit of the skeletal dihyropyridine receptor (DHPR) may contribute to voltage-activated Ca(2+) release in skeletal muscle by interacting with the skeletal ryanodine receptor (RyR1), a direct functional interaction between the two proteins has not been demonstrated previously. Such an interaction is reported here. A peptide with the sequence of the C-terminal 35 residues of β(1a) bound to RyR1 in affinity chromatography. The full-length β(1a) subunit and the C-terminal peptide increased [(3)H]ryanodine binding and RyR1 channel activity with an AC(50) of 450-600 pM under optimal conditions. The effect of the peptide was dependent on cytoplasmic Ca(2+), ATP, and Mg(2+) concentrations. There was no effect of the peptide when channel activity was very low as a result of Mg(2+) inhibition or addition of 100 nM Ca(2+) (without ATP). Maximum increases were seen with 1-10 μM Ca(2+), in the absence of Mg(2+) inhibition. A control peptide with the C-terminal 35 residues in a scrambled sequence did not bind to RyR1 or alter [(3)H]ryanodine binding or channel activity. This high-affinity in vitro functional interaction between the C-terminal 35 residues of the DHPR β(1a) subunit and RyR1 may support an in vivo function of β(1a) during voltage-activated Ca(2+) release.     

Mapping of the Antibody-Binding Regions on the HN-Domain (Residues 449–859) of Botulinum Neurotoxin A with Antitoxin Antibodies from Four Host Species. Full Profile of the Continuous Antigenic Regions of the H-Chain of Botulinum Neurotoxin A

Previously, we mapped the antibody (Ab) and T-cell recognition regions on the HC domain (residues 855-1296) of the 848-residue heavy (H) chain of botulinum neurotoxin A (BoNT/A). We have mapped here the HN-domain (residues 449-859) regions that bind protective anti-BoNT/A Abs raised in four different species. We synthesized, purified, and characterized 29 19-residue peptides that spanned the entire HN and overlapped consecutively by 5 residues, and also region L218-231 around the L-chain's substrate-binding site. Human, horse, mouse, and chicken anti-BoNT/A Abs did not bind to the L-peptide but recognized similar HN regions within peptides 519-537/533-551/547-565/561-579 (with slight left- or right-shifts), 743-761, 785-803, and 813-831/827-845 overlap. Recognition of other peptides that bound lower Ab levels showed similarities and also some differences. Peptide 463-481, strongly immunodominant with horse antisera, did not bind human, mouse, and chicken Abs. However, peptide 449-467 bound Abs in these three antisera, and the region may have shifted to the right (peptide 463-481) with horse Abs. The overlap 659-677/673-691 reacted strongly with human Abs whereas with mouse and chicken antisera, only peptide 673-691 showed low reactivity. Horse antisera had no detectable Ab binding to region(s) 659-691. The Ab-recognition regions on the H chain occupy surface locations in BoNT/A three-dimensional structure, but the great part of the surface is not immunogenic. Regions recognized by the protective antisera of the four different species are prime candidates for inclusion in synthetic vaccine designs.     

In vitro Activation of Protein Kinase C by β-N-oxalyl-l-α, β-diaminopropionic acid,the Lathyrus sativus Neurotoxin

-N-oxalyl-l-,-diaminopropionic acid (l-ODAP) toxicity has been associated with lathyrism; a spastic paraparesis caused by excessive dietary intake of the pulse Lathyrus sativus. We investigated the effect of Lathyrus neurotoxin l-ODAP on protein kinase C (PKC) activity under in vitro conditions. l-ODAP activated phosphorylation activity of purified chick brain PKC. Both lysine-rich (histone III-S) and arginine-rich (protamine sulfate) substrate phosphorylation was enhanced in the presence of l-ODAP. The activation is concentration dependent, and maximal activation is observed at 100 M concentration. Protamine sulfate phosphorylation was enhanced by 47%, whereas histone III-S phosphorylation was enhanced by 50% over PS/PDBu/Ca²⁺ dependent activity. The nontoxic d-isomer (d-ODAP) did not affect both histone III-S and protamine sulfate phosphorylation activity. These results indicate that l-ODAP taken up by neuronal cells could also contribute to PKC activation and so be associated with toxicity.     

Binding of agonists and antagonists to the porcine adipose tissue β-adrenergic receptor(s)

1. Affinities of agonists for porcine adipose tissue β-adrenergic receptors, determined by competitive ligand binding with ³H-dihydroalprenolol to crude adipose tissue membranes in vitro, varied from 50 times > to 25 times < than isoproterenol. Affinities for antagonists varied from 8 times > to 1000 times < propranolol.2. Receptor affinity was not related to the ability to stimulate or inhibit lipolysis, or to the agonist or antagonist purported receptor subtype specificity.3. Modeling of ligand-binding data indicated more than one binding site for several ligands. The assignment of β-adrenergic subtypes to the individual binding sites was unclear because this would depend on the individual ligands used to establish binding sites.     

A multivalent approach to the discovery of long-acting β(2)-adrenoceptor agonists for the treatment of asthma and COPD

A multivalent approach was applied to the design of long-acting inhaled β(2)-adrenoceptor agonists. A series of dimeric arylethanolamines based on the short acting β(2)-adrenoceptor agonist albuterol were prepared, varying the nature and length of the linker between the basic nitrogens. None of the C(2)-symmetric dimers demonstrated increased potency, however dimer 5j, derived from 4-phenethylamine, was found to have increased binding potency in vitro relative to the parent monomer. Optimization of this structure led to the identification of 22 (milveterol) which demonstrates high potency in vitro and long duration of action in a guinea pig model of bronchoprotection.     

Statins Promote the Degradation of Extracellular Amyloid β-Peptide by Microglia via Stimulation of Exosome-associated Insulin-degrading Enzyme (IDE) Secretion

Epidemiological studies indicate that intake of statins decrease the risk of developing Alzheimer disease. Cellular and in vivo studies suggested that statins might decrease the generation of the amyloid β-peptide (Aβ) from the β-amyloid precursor protein. Here, we show that statins potently stimulate the degradation of extracellular Aβ by microglia. The statin-dependent clearance of extracellular Aβ is mainly exerted by insulin-degrading enzyme (IDE) that is secreted in a nonconventional pathway in association with exosomes. Stimulated IDE secretion and Aβ degradation were also observed in blood of mice upon peripheral treatment with lovastatin. Importantly, increased IDE secretion upon lovastatin treatment was dependent on protein isoprenylation and up-regulation of exosome secretion by fusion of multivesicular bodies with the plasma membrane. These data demonstrate a novel pathway for the nonconventional secretion of IDE via exosomes. The modulation of this pathway could provide a new strategy to enhance the extracellular clearance of Aβ.     

Rapid detection of single nucleotide deletions: application to the β 6 (-A) mutation of the β-globin gene and to cystic fibrosis

The formation of heteroduplexes from the amplified products of homologous alleles has been shown to be useful in the identification of heterozygotes carrying deletion or insertion mutations. Here, we describe an improved procedure that allows the detection of single base pair (bp) deletions on nondenaturing polyacrylamide gels. Carriers for a common Mediterranean -thalassemic mutation, 6 (-A), could be easily detected by use of this method, as could carriers of a 1-bp deletion in the cystic fibrosis gene.