Four new β-lactones from the endophytic Streptomyces sp. T1B1

The detailed investigation of endophytic Streptomyces sp. T1B1 was performed during a search for new structural and active compounds. The strain T1B1 was isolated from the old bast tissue of Taxus yunnanensis and determined to be a member of Streptomyces, according to the 16S rRNA analysis. The extracts from the PDA solid fermentation media of Streptomyces sp. T1B1 were purified and four β-lactones were isolated. They were identified as 4α-(3,5-dihydroxy hexyl)-3α-methyl-2-oxetanone (1), 4α-(3-methyl-4-formyloxy-hexyl)-3α-methyl-2-oxetanone (2), 4α-(3,5-dihydroxy-heptyl)-3α-methyl-2-oxetanone (3) and 4α-(3-methyl-4-formyloxy-heptyl)-3α-methyl-2-oxetanone (4) on the basis of spectral data.     

The benefits of being β-crystallin heteromers: βB1-crystallin protects βA3-crystallin against aggregation during co-refolding

β-Crystallins are the major structural proteins in mammalian lens, and their stability is critical in maintaining the transparency and refraction index of the lens. Among the seven β-crystallins, βA3-crystallin and βB1-crystallin, an acidic and a basic β-crystallin, respectively, can form heteromers in vivo. However, the physiological roles of the heteromer have not been fully elucidated. In this research, we studied whether the basic β-crystallin facilitates the folding of acidic β-crystallin. Equilibrium folding studies revealed that the βA3-crystallin and βB1-crystallin homomers and the βA3/βB1-crystallin heteromer all undergo similar five-state folding pathways which include one dimeric and two monomeric intermediates. βA3-Crystallin was found to be the most unstable among the three proteins, and the transition curve of βA3/βB1-crystallin was close to that of βB1-crystallin. The dimeric intermediate may be a critical determinant in the aggregation process and thus is crucial to the lifelong stability of the β-crystallins. A comparison of the Gibbs free energy of the equilibrium folding suggested that the formation of heteromer contributed to the stabilization of the dimer interface. On the other hand, βA3-crystallin, the only protein whose refolding is challenged by serious aggregation, can be protected by βB1-crystallin in a dose-dependent manner during the kinetic co-refolding. However, the protection is not observed in the presence of the pre-existed well-folded βB1-crystallin. These findings suggested that the formation of β-crystallin heteromers not only stabilizes the unstable acidic β-crystallin but also protects them against aggregation during refolding from the stress-denatured states.     

NF-κB p65 represses β-catenin-activated transcription of cyclin D1

We have previously reported that obesity-induced diabetes developed in high-fat diet (HFD)-fed BDF1 mice. This is caused by insufficient insulin response to an excess glucose load. In this study, we have shown that the enhanced expression of retinaldehyde dehydrogenase 3 (Raldh3) causes functional disorders of pancreatic islets in diabetic mouse models. In the pancreatic islets of HFD-induced diabetic BDF1 mice and spontaneously diabetic C57BL/KsJ^db/db mice, gene expression analysis with oligonucleotide microarray revealed a significant increase in Raldh3 expression. Exposure to a culture medium containing a higher glucose concentration (25 mM) significantly increased Raldh3 expression in murine MIN6 and alphaTC1 clone 9 cells, which derived from the α and β-cells of pancreatic islets, respectively. Overexpression of Raldh3 reduced the insulin secretion in MIN6 cells, and surprisingly, increased the glucagon secretion in alphaTC1 clone 9 cells. Furthermore, the knockdown of Raldh3 expression with siRNA decreased the glucagon secretion in alphaTC1 clone 9 cells. Raldh3 catalyzes the conversion of 13-cis retinal to 13-cis retinoic acid and we revealed that 13-cis retinoic acid significantly reduces cell viability in MIN6 and alphaTC1 clone 9 cells, but not in cells of H4IIEC3, 3T3-L1, and COS-1 cell lines. These findings suggest that an increasing expression of Raldh3 deregulates the balanced mechanisms of insulin and glucagon secretion in the pancreatic islets and may induce β-cell dysfunction leading to the development of type 2 diabetes.     

Interaction of PvALF and VP1 B3 domains with the β-phaseolin promoter

Caffeine (1,3,7-trimethylxanthine) is derived from xanthosine through three successive transfers of methyl groups and a single ribose removal in coffee plants. The methyl group transfer is catalyzed by N-zmethyltransferases, xanthosine methyltransferase (XMT), 7-methylxanthine methyltransferase (MXMT) and 3,7-dimethylxanthine methyltransferase (DXMT). We previously cloned three genes encoding each of these N-methyltransferases from coffee plants, and reconstituted the final sequence of the caffeine synthetic pathway in vitro. In the present study, we simultaneously expressed these coffee genes in tobacco plants (Nicotiana tabacum), using a multiple-gene transfer method, and confirmed successful caffeine production up to 5 μg g⁻¹ fresh weight in leaves of the resulting transgenic plants. Their effects on feeding behavior of tobacco cutworms (Spodoptera litura), which damage a wide range of crops, were then examined. Leaf disc choice test showed that caterpillars selectively fed on the wild-type control materials, or positively avoided the transgenic materials. The results suggest a novel approach to confer self-defense by producing caffeine in planta. A second generation of transgenic crops containing caffeine may save labor and agricultural costs and also mitigate the environmental load of pesticides in future.     

Computational analysis of pathogen-borne metallo β-lactamases reveals discriminating structural features between B1 types

Background

Disrupted-in-Schizophrenia 1 (DISC1) is considered to be a candidate susceptibility gene for psychiatric disorders, including schizophrenia, bipolar disorder, and major depression. A recent study reported that N-ethyl-N-nitrosourea (ENU)-induced mutations in exon 2 of the mouse Disc1 gene, which resulted in the amino acid exchange of Q31L and L100P, caused an increase in depression-like behavior in 31 L mutant mice and schizophrenia-like behavior in 100P mutant mice; thus, these are potential animal models of psychiatric disorders. However, remaining heterozygous mutations that possibly occur in flanking genes other than Disc1 itself might induce behavioral abnormalities in the mutant mice. Here, to confirm the effects of Disc1-Q31L and Disc1-L100P mutations on behavioral phenotypes and to investigate the behaviors of the mutant mice in more detail, the mutant lines were backcrossed to C57BL/6JJcl through an additional two generations and the behaviors were analyzed using a comprehensive behavioral test battery.

Results

Contrary to expectations, 31 L mutant mice showed no significant behavioral differences when compared with wild-type control mice in any of the behavioral tests, including the Porsolt forced swim and tail suspension tests, commonly used tests for depression-like behavior. Also, 100P mutant mice exhibited no differences in almost all of the behavioral tests, including the prepulse inhibition test for measuring sensorimotor gating, which is known to be impaired in schizophrenia patients; however, 100P mutant mice showed higher locomotor activity compared with wild-type control mice in the light/dark transition test.

Conclusions

Although these results are partially consistent with the previous study in that there was hyperactivity in 100P mutant mice, the vast majority of the results are inconsistent with those of the previous study; this discrepancy may be explained by differences in the genetic background of the mice, the laboratory environment, experimental protocols, and more. Further behavioral studies under various experimental conditions are necessary to determine whether these Disc1 mutant mouse lines are suitable animal models of schizophrenia and major depression.     

Epigenetic silencing of Na,K-ATPase β1 subunit gene ATP1B1 by methylation in clear cell renal cell carcinoma

The Na,K-ATPase or sodium pump carries out the coupled extrusion of Na⁺ and uptake of K⁺ across the plasma membranes of cells of most higher eukaryotes. We have shown earlier that Na,K-ATPase-β₁ (NaK-β) protein levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients' tumor samples. The mechanism(s) regulating the expression of NaK-β in tumor tissues has yet to be explored. We hypothesized that DNA methylation plays a role in silencing the NaK-β gene (ATP1B1) expression in kidney cancers. In this study, to the best of our knowledge we provide the first evidence that ATP1B1 is epigenetically silenced by promoter methylation in both renal cell carcinoma (RCC) patients’ tissues and cell lines. We also show that knockdown of the von Hippel-Lindau (VHL) tumor suppressor gene in RCC cell lines results in enhanced ATP1B1 promoter AT hypermethylation, which is accompanied by reduced expression of NaK-β. Furthermore, treatment with 5-Aza-2′-deoxycytidine rescued the expression of ATP1B1 mRNA as well as NaK-β protein in these cells. These data demonstrate that promoter hypermethylation is associated with reduced NaK-β expression, which might contribute to RCC initiation and/or disease progression.     

Bovine β-crystallin complementary DNA clones: Alternating proline/alanine sequence of βB1 subunit originates from a repetitive DNA sequence

A library of recombinant plasmids carrying complementary DNA sequences synthesized from bovine lens messenger RNAs was constructed. Clones coding for five different β-crystallin subunits: βB1, βB3, βBp, βs, βA3 (and βA1), were identified by means of hybridization selection, followed by one- and two-dimensional gel electrophoresis of the translational products. Under rather stringent conditions each of these clones hybridizes with its corresponding mRNA and does not show significant cross-hybridization with mRNAs coding for other β-crystallins, except in the case of the homologous βA3 and βA1-crystalline. The βA3 and βA1 subunits seem to be encoded by one mRNA using two different AUG codons as start position for translation. We have also determined the nucleotide sequence of a βB1-crystallin cDNA (pBLβB1) which enabled us to deduce the complete amino acid sequence of the protein. The βB1-crystallin, a characteristic component of the high molecular weight crystallin aggregate (β_H), is internally homologous both at DNA and protein level as has been reported for γ-and other β-crystallins. This is in agreement with the idea that these proteins had a common ancestral precursor gene that internally duplicated. The G + C content of the coding sequence of βB1 is very high: 67% overall and even 84.2% for the first 170 nucleotides, due to a remarkable non-random codon usage. A proline/ alanine repetition in the N-terminal domain of the protein is encoded by a repetitive “simple” DNA sequence.     

Molecular Cloning of the Dog β1 and β2 Adrenergic Receptors

Abstract

We report the isolation of the genes encoding the β1 and β2 adrenergic receptors from dog genomic DNA. Sequence analysis of both genes revealed intronless open reading frames of 473 and 415 amino acid residues, receptively. Heterologous expression of both receptors in CHO cells indicated that both receptors are functionally similar to the human homologs. Comparing the dog β1 and β2 adrenergic receptors, the β1 receptor appears to bind to G proteins more tightly than the β2 receptor. Heterologously expressed receptors provide a convenient system for evaluating novel receptor agonists and antagonists.     

Interaction of PRK1 Receptor-like Kinase with a Putative elF2B β-Subunit in Tobacco

PRK1, a receptor-like kinase that is expressed in pollen, pollen tubes, and ovaries, has been shown to play important roles in pollen development and embryo sac development in Petunia inflata. We have used the kinase domain of PRK1 as a bait in the yeast two-hybrid system to identify PRK1-interacting proteins. The screening resulted in isolation of a cDNA encoding a protein highly homologous to the human and yeast beta-subunit of translation initiation factor 2B (eIF2B-beta), which was designated NeIF2Bbeta. eIF2B is a guanine nucleotide exchange protein that functions in the regulation of translation in eukaryotic cells. Deletion mutants of NeIF2Bbeta were analyzed for their interaction with PRK1, and the results suggested that the N-terminal half of NeIF2Bbeta, especially the region between residue 103 and 235, is important for the interaction. This protein association was confirmed by in vitro binding assay of the recombinant NeIF2Bbeta and PRK1 proteins. Despite high sequence homology between NeIF2Bbeta and its yeast counterpart, the NeIF2Bbeta cDNA could not rescue the phenotype of the yeast mutant strain lacking the GCD7 gene encoding eIF2B-beta, when transferred into the mutant strain.     

Interaction of PRK1 Receptor-like Kinase with a Putative elF2B β-Subunit in Tobacco

PRK1, a receptor-like kinase that is expressed in pollen, pollen tubes, and ovaries, has been shown to play important roles in pollen development and embryo sac development in Petunia inflata. We have used the kinase domain of PRK1 as a bait in the yeast two-hybrid system to identify PRK1-interacting proteins. The screening resulted in isolation of a cDNA encoding a protein highly homologous to the human and yeast -subunit of translation initiation factor 2B (eIF2B-), which was designated NeIF2B. eIF2B is a guanine nucleotide exchange protein that functions in the regulation of translation in eukaryotic cells. Deletion mutants of NeIF2B were analyzed for their interaction with PRK1, and the results suggested that the N-terminal half of NeIF2B, especially the region between residue 103 and 235, is important for the interaction. This protein association was confirmed by in vitro binding assay of the recombinant NeIF2B and PRK1 proteins. Despite high sequence homology between NeIF2B and its yeast counterpart, the NeIF2B cDNA could not rescue the phenotype of the yeast mutant strain lacking the GCD7 gene encoding eIF2B-, when transferred into the mutant strain.     

Polymorphisms within the Novel Type 2 Diabetes Risk Locus MTNR1B Determine β-Cell Function

Background

Very recently, a novel type 2 diabetes risk gene, i.e., MTNR1B, was identified and reported to affect fasting glycemia. Using our thoroughly phenotyped cohort of subjects at an increased risk for type 2 diabetes, we assessed the association of common genetic variation within the MTNR1B locus with obesity and prediabetes traits, namely impaired insulin secretion and insulin resistance.

Methodology/Principal Findings

We genotyped 1,578 non-diabetic subjects, metabolically characterized by oral glucose tolerance test, for five tagging single nucleotide polymorphisms (SNPs) covering 100% of common genetic variation (minor allele frequency >0.05) within the MTNR1B locus (rs10830962, rs4753426, rs12804291, rs10830963, rs3781638). In a subgroup (N = 513), insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp, and in a further subgroup (N = 301), glucose-stimulated insulin secretion was determined by intravenous glucose tolerance test. After appropriate adjustment for confounding variables and Bonferroni correction for multiple comparisons, none of the tagging SNPs was reliably associated with measures of adiposity. SNPs rs10830962, rs4753426, and rs10830963 were significantly associated with higher fasting plasma glucose concentrations (p<0.0001) and reduced OGTT- and IVGTT-induced insulin release (p≤0.0007 and p≤0.01, respectively). By contrast, SNP rs3781638 displayed significant association with lower fasting plasma glucose levels and increased OGTT-induced insulin release (p<0.0001 and p≤0.0002, respectively). Moreover, SNP rs3781638 revealed significant association with elevated fasting- and OGTT-derived insulin sensitivity (p≤0.0021). None of the MTNR1B tagging SNPs altered proinsulin-to-insulin conversion.

Conclusions/Significance

In conclusion, common genetic variation within MTNR1B determines glucose-stimulated insulin secretion and plasma glucose concentrations. Their impact on β-cell function might represent the prevailing pathomechanism how MTNR1B variants increase the type 2 diabetes risk.     

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with β-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins^1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of β-arrestins³. β-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization ^4-6.Leukotriene B₄ (LTB₄) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB₄ receptor 1 (BLT1; a high affinity receptor) and LTB₄ receptor 2 (BLT2; a low affinity receptor)^7-9. The LTB₄-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis^10-17. The current paper describes the methodologies developed to monitor LTB₄-induced leukocyte migration and the interactions of BLT1 with β-arrestin and , receptor translocation in live cells using microscopy imaging techniques^18-19.Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described ^20-21. These cells were tested in live cell imaging methods to demonstrate LTB₄ induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and β-arrestin1 tagged with green fluorescent protein (β-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines^18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.Download video file.^{(88M, mov)}     

Disulfide cross-links in the interaction of a cataract-linked αA-crystallin mutant with βB1-crystallin

A number of αA-crystallin mutants are associated with hereditary cataract including cysteine substitution at arginine 49. We report the formation of affinity-driven disulfide bonds in the interaction of αA-R49C with βB1-crystallin. To mimic cysteine thiolation in the lens, βB1-crystallin was modified by a bimane probe through a disulfide linkage. Our data suggest a mechanism whereby a transient disulfide bond occurs between αA- and βB1-crystallin followed by a disulfide exchange with cysteine 49 of a neighboring αA-crystallin subunit. This is the first investigation of disulfide bonds in the confine of the chaperone/substrate complex where reaction rates are favored by orders of magnitude. Covalent protein cross-links are a hallmark of age-related cataract and may be a factor in its inherited form.     

Synthesis of β-1, 3-Glucan and β-1, 3 and β-1, 4-Mixed Glucan from UDP-α-d-Glucose

A particulate enzyme preparation from Phaseolus aureus (mung bean) seedlings catalyzed the synthesis of a water insoluble β-1,3-glucan from UDP-α-d-glucose (UDPG) at high concentrations (0.4~20 mm) and an alkaline insoluble β-1,3 and β-1,4-mixed glucan from UDPG at a low concentration (8.5 µm).

Furthermore, the two kinds of β-glucan synthetases which were investigated with two reaction systems at high and low concentrations of UDPG had different properties in optimal pH, stability of enzyme activity, and metallic ion requirement.     

A novel transglycosylating β-galactosidase from Enterobacter cloacae B5

A strain of Enterobacter cloacae B5 producing β-galactosidase with transglycosylation activity was isolated from the soil. Its freeze-thawed cells synthesized galacto-oligosaccharides with a high yield of 55% from 275 g/L lactose at 50 °C for 12 h. A novel β-galactosidase capable of glycosyl transfer was purified from this strain. It was a homotetramer with molecular mass of about 442 kDa. The optimal pH and temperature for hydrolysis activity on o-nitrophenyl-β-d-galactopyranoside (oNPGal) were 6.5–10.5 and 35 °C, respectively. The enzyme showed a wide range of acceptor specificity for transglycosylation and catalyzed glycosyl transfer from oNPGal to various chemicals such as galactose, glucose, fructose, arabinose, mannose, sorbose, rhamnose, xylose, cellobiose, sucrose, trehalose, melibiose, inositol, mannitol, sorbitol and salicin, resulting in novel saccharide yields ranging from 0.8% to 23.5%. A gene encoding the enzyme was cloned and the recombinant enzyme from Escherichia coli had similar transglycosylation activity to the natural enzyme.     

Lysosomal-associated Transmembrane Protein 4B (LAPTM4B) Decreases Transforming Growth Factor β1 (TGF-β1) Production in Human Regulatory T Cells

Production of active TGF-β1 is one mechanism by which human regulatory T cells (Tregs) suppress immune responses. This production is regulated by glycoprotein A repetitions predominant (GARP), a transmembrane protein present on stimulated Tregs but not on other T lymphocytes (Th and CTLs). GARP forms disulfide bonds with proTGF-β1, favors its cleavage into latent inactive TGF-β1, induces the secretion and surface presentation of GARP·latent TGF-β1 complexes, and is required for activation of the cytokine in Tregs. We explored whether additional Treg-specific protein(s) associated with GARP·TGF-β1 complexes regulate TGF-β1 production in Tregs. We searched for such proteins by yeast two-hybrid assay, using GARP as a bait to screen a human Treg cDNA library. We identified lysosomal-associated transmembrane protein 4B (LAPTM4B), which interacts with GARP in mammalian cells and is expressed at higher levels in Tregs than in Th cells. LAPTM4B decreases cleavage of proTGF-β1, secretion of soluble latent TGF-β1, and surface presentation of GARP·TGF-β1 complexes by Tregs but does not contribute to TGF-β1 activation. Therefore, LAPTM4B binds to GARP and is a negative regulator of TGF-β1 production in human Tregs. It may play a role in the control of immune responses by decreasing Treg immunosuppression.     

Crystal structure of human β-galactosidase: structural basis of Gm1 gangliosidosis and morquio B diseases

G(M1) gangliosidosis and Morquio B are autosomal recessive lysosomal storage diseases associated with a neurodegenerative disorder or dwarfism and skeletal abnormalities, respectively. These diseases are caused by deficiencies in the lysosomal enzyme β-d-galactosidase (β-Gal), which lead to accumulations of the β-Gal substrates, G(M1) ganglioside, and keratan sulfate. β-Gal is an exoglycosidase that catalyzes the hydrolysis of terminal β-linked galactose residues. This study shows the crystal structures of human β-Gal in complex with its catalytic product galactose or with its inhibitor 1-deoxygalactonojirimycin. Human β-Gal is composed of a catalytic TIM barrel domain followed by β-domain 1 and β-domain 2. To gain structural insight into the molecular defects of β-Gal in the above diseases, the disease-causing mutations were mapped onto the three-dimensional structure. Finally, the possible causes of the diseases are discussed.