Crystal structures of rare disaccharides, α-D-glucopyranosyl β-D-psicofuranoside, and α-D-galactopyranosyl β-D-psicofuranoside

The crystal structures of α-D-glucopyranosyl β-D-psicofuranoside and α-D-galactopyranosyl β-D-psicofuranoside were determined by a single-crystal X-ray diffraction analysis, refined to R(1)=0.0307 and 0.0438, respectively. Both disaccharides have a similar molecular structure, in which psicofuranose rings adopt an intermediate form between (4)E and (4)T(3). Unique molecular packing of the disaccharides was found in crystals, with the molecules forming a layered structure stacked along the y-axis.     

Combinatorial Pharmacogenetic Interactions of Bucindolol and β1, α2C Adrenergic Receptor Polymorphisms

Background

Pharmacogenetics involves complex interactions of gene products affecting pharmacodynamics and pharmacokinetics, but there is little information on the interaction of multiple genetic modifiers of drug response. Bucindolol is a β-blocker/sympatholytic agent whose efficacy is modulated by polymorphisms in the primary target (β₁ adrenergic receptor [AR] Arg389 Gly on cardiac myocytes) and a secondary target modifier (α_2C AR Ins [wild-type (Wt)] 322–325 deletion [Del] on cardiac adrenergic neurons). The major allele homozygotes and minor allele carriers of each polymorphism are respectively associated with efficacy enhancement and loss, creating the possibility for genotype combination interactions that can be measured by clinical trial methodology.

Methodology

In a 1,040 patient substudy of a bucindolol vs. placebo heart failure clinical trial, we tested the hypothesis that combinations of β₁389 and α_2C322–325 polymorphisms are additive for both efficacy enhancement and loss. Additionally, norepinephrine (NE) affinity for β₁389 AR variants was measured in human explanted left ventricles.

Principal Findings

The combination of β₁389 Arg+α_2C322–325 Wt major allele homozygotes (47% of the trial population) was non-additive for efficacy enhancement across six clinical endpoints, with an average efficacy increase of 1.70-fold vs. 2.32-fold in β₁389 Arg homozygotes+α_2C322–325 Del minor allele carriers. In contrast, the minor allele carrier combination (13% subset) exhibited additive efficacy loss. These disparate effects are likely due to the higher proportion (42% vs. 8.7%, P = 0.009) of high-affinity NE binding sites in β₁389 Arg vs. Gly ARs, which converts α_2CDel minor allele-associated NE lowering from a therapeutic liability to a benefit.

Conclusions

On combination, the two sets of AR polymorphisms 1) influenced bucindolol efficacy seemingly unpredictably but consistent with their pharmacologic interactions, and 2) identified subpopulations with enhanced (β₁389 Arg homozygotes), intermediate (β₁389 Gly carriers+α_2C322–325 Wt homozygotes), and no (β₁389 Gly carriers+α_2C322–325 Del carriers) efficacy.     

Ultrastructural localization of corticotropin, β-lipotropin,and αand β-endorphin in the porcine anterior pituitary

Summary The peroxidase-antiperoxidase immunocytochemical technique was used to identify the ACTH/endorphin cells in the porcine pituitary at the ultrastructural level and to determine the precise subcellular localization of the pro-ACTH/endorphin fragments. The cells display different aspects: 1) large, regular shapes with numerous and large secretory granules; 2) small, irregular and angular shapes with small granules aligned along the periphery of the cell; and 3) intermediate forms. The presence of and -endorphin not only in the same cells but also in the same secretory granules that contain ACTH and -LPH clearly indicates that both the precursor or its fragments and the abovementioned peptides are stored in the same granules and released simultaneously by the corticotropic cells. The presence of FSH in some corticotropic cells is also discussed.Abbreviations used in this Article ACTH corticotropin - -MSH -melanotropin (ACTH I–I3) - CLIP corticotropin-like intermediate lobe peptide (ACTH 18–39) - -LPH -lipotropin - -MSH -melanotropin (-LPH 41–58); -endorphin (-LPH 61–91); -endorphin (-LPH 61–76)     

Connexin-43 Interactions with ZO-1 and α- and β-tubulin

Gap junctions are composed of connexins that form transmembrane channels between adjacent cells. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated in the regulation of Cx43 channel gating. Interestingly, channel-independent processes regulated by Cx43 have also been postulated. In our studies to elucidate the mechanism of Cx43 channel gating by growth factors and to explore additional functions of gap junctions, we have identified three interacting partners of the C-terminal tail of Cx43 (Cx43CT). (i) the c-Src tyrosine kinase, which phosphorylates Cx43CT and is involved in G protein-mediated inhibition of Cx43 gap junctional communication, (ii) the ZO-1 ‘scaffold’ protein, which might recruit signaling proteins into Cx43-based gap junctions. (iii) microtubules (consisting of α/β-tubulin dimers), which extend with their distal ends to Cx43-based gap junctions, suggesting that Cx43 gap junctions may play a novel role in regulating microtubule stability in contacted cells. Here we show that Cx43 binds α-tubulin equally well as β-tubulin. In addition, we show that the second, but not the first, PDZ domain of ZO-1 binds directly to Cx43, and we confirm that the very C-terminal isoleucine residue of Cx43 is critical for ZO-1 binding.     

1,3,5-Tri-O-acetyl-2-deoxy- α, β-D-ERYTHRO-Pentofuranose from 2-Deoxy-D-ERYTHRO-Pentose

Abstract

A convenient, high-yield synthesis of 1,3,5-tri-O-acetyl-2-deoxy-α. β-D-erythro-pentofuranose from 2-deoxy-D-erythro-pentose is described.     

Synthesis of methyl α- and β-maltotriosides and aryl β-maltotriosides

Reaction of β-maltotriose hendecaacetate with phosphorus pentachloride gave 2′,2″,3,3′,3″,4″,6,6′,6″,-nona-O-acetyl-(2)-O-trichloroacetyl-β-maltotriosyl chloride (2) which was isomerized into the corresponding α anomer (8). Selective ammonolysis of 2 and 8 afforded the 2-hydroxy derivatives 3 and 9, respectively; 3 was isomerized into the α anomer 9. Methanolysis of 2 and 3 in the presence of pyridine and silver nitrate and subsequent deacetylation gave methyl α-maltotrioside. Likewise, methanolysis and O-deacetylation of 9 gave methyl β-maltotrioside which was identical with the compound prepared by the Koenigs—Knorr reaction of 2,2′,2″,3,3′,3″,4″,6,6′,6″-deca-O-acetyl-α-maltotriosyl bromide (12) with methanol followed by O-deacetylation. Several substituted phenyl β-glycosides of maltotriose were also obtained by condensation of phenols with 12 in an alkaline medium. Alkaline degradation of the o-chlorophenyl β-glycoside decaacetate readily gave a high yield of 1,6-anhydro-β-maltotriose.     

Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

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.     

α1,3 Fucosyltransferase, α-L-fucosidase, α-D-galactosidase, β-D-galactosidase, and Lex glycoconjugates in developing rat brain

Fucosyltransferases (FTs) and various glycosidases that are involved in the biosynthesis or degradation of SSEA-1 (Lex) antigens and their precursors in the CNS are developmentally regulated. In forebrain and cerebellum with lactosamine (LacNAc) as acceptor the FT activity was maximal at P15–P22, but with the glycolipid substrate paragloboside (nLc4) the maximal activity in cerebellum was obtained at P10–P15. The FT activity, with these substrates, was insensitive to N-ethylmaleimide (NEM) and the glycolipid product had an α1,3 linkage (Fuc to GlcNAc) suggesting similarities of the investigated enzyme to the cloned human and rat FT IV. However, the observation of different patterns of FT activity in isoelectrofocused fractions (pH 3.5–10) with different types of acceptors, and the differential expression of Lex containing glycolipids and glycoproteins during development strongly suggest the presence of more than one type of FT during development. Data on developmental expression of the hydrolytic enzymes, α-L-fucosidase, β-D-galactosidase and α-D-galactosidase, which can potentially hydrolyse SSEA-1 or its precursors, support the notion that SSEA-1 expression is the result of a dynamic balance between the activity of transferases and hydrolases. © 1998 Rapid Science Ltd     

Bromine oxidation of methyl α- and β-pyranosides of D-galactose,D-glucose,and D-mannose

Methyl α- and β-pyranosides of D-galactose, D-glucose, and D-mannose have been oxidized with bromine in aqueous solution at various pH values. The resulting keto glycosides were converted into their more-stable O-methyloxime derivatives which were characterized by spectroscopy and chromatography. Oxidation at a ring carbon atom where the hydrogen is axial is hindered by bulky substituents in syn (i.e., a 1,3) diaxial relationship. Thus, the aglycon group in the α anomers protects position 3, the axial HO-4 in galactopyranosides protects position 2, and the axial HO-2 in mannopyranosides protects position 4 from oxidation.     

Cell adhesion to anosmin via α5β1, α4β1, and α9β1 integrins

Anosmin is an extracellular matrix protein, and genetic defects in anosmin result in human Kallmann syndrome. It functions in neural crest formation, cell adhesion, and neuronal migration. Anosmin consists of multiple domains, and it has been reported to bind heparan sulfate, FGF receptor, and UPA. In this study, we establish cell adhesion/spreading assays for anosmin and use them for antibody inhibition analyses to search for an integrin adhesion receptor. We find that α5β1, α4β1, and α9β1 integrins are needed for effective adhesive receptor function in cell adhesion and cell spreading on anosmin; adhesion is inhibited by both RGD and α4β1 CS1-based peptides. This identification of anosmin-integrin adhesion receptors should facilitate studies of anosmin function in cell and developmental biology.     

Attempt at Affinity Labeling of α- and β- Amylases by α- and β-d-Glucopyranosides and α- and β-Maltooligosaccharides with 2,3-Epoxypropyl Residue as Aglycone: Specific Inactivation of β-Amylases

Among 2,3-epoxypropyl α-d-glucopyranoside and 2,3-epoxypropyl α-maltooligosaccharides and the β-anomers, 2,3-epoxypropyl α-d-glucopyranoside (α-EPG) strongly inactivated the β-amylases [EC 3.2.1.2] of sweet potato, barley, and Bacillus, cereus, in addition to soybean β amylase [J. Biochem., 99, 1631 (1986)]. However, none of the compounds used inactivated any α-amylases [EC 3.2.1.1] of porcine pancreas, Aspergillus oryzae, or Bacillus amyloliquefaciens. Irreversible incorporation of ¹⁴C-labeled α-EPG into β-amylases was stoichiometric, i.e., one α-EPG per active site of the enzyme was bound, and the inactivations were almost complete. The results suggest that α-EPG is an affinity labeling reagent selective for β-amylase. Slow inactivations by the other compounds were also observed, depending on the difference of source of β amylase.     

Investigation of the Relationship between the Lectin Activity of Azospirilla and Their α-Glucosidase, β-Glucosidase,and β-Galactosidase

The activities of -glucosidase, -glucosidase, and -galactosidase were studied during the isolation and purification of lectins from Azospirillum brasilenseSp7 and Azospirillum lipoferum59b cells. These enzymatic activities were revealed in crude extracts of surface proteins, protein fraction precipitated with ammonium sulfate or ethanol–acetone mixture, and protein fraction obtained by gel filtration on Sephadex G-75. The distribution of the enzymes between different protein fractions varied for the azospirilla studied. The cofunction of the A. brasilenseSp7 lectin and -galactosidase on the cell surface is assumed. A strong interaction between the A. lipoferum59b lectin and glucosidases was revealed. The lectin from A. lipoferum59b may possess saccharolytic activity.     

Studies on the Interaction between αs1- and β-Caseins

The interaction of α_s1-casein with β-, dephosphorylated β-,γ- and R-caseins was studied. It was proved by the sedimentation velocity experiments that α_s1-casein formed a complex with each of these components at 25±C in the presence of 3 mm CaCl₂.

In the presence of 10 mm CaCl₂, β- and dephosphorylated β-casein prevented the precipitation of α_s1-casein and gave micelle-like turbid solutions. However, γ- and R-caseins, fragments of β-casein, did not stabilize α_s1-casein. It was concluded from these results that α-casein interacted with α_s1-casein through its hydropholic region corresponding to R-casein and that hydrophilic region of β-casein was responsible for the stabilization of α_s1-casein.     

Monitoring the Interaction between β2-Microglobulin and the Molecular Chaperone αB-crystallin by NMR and Mass Spectrometry: αB-CRYSTALLIN DISSOCIATES β2-MICROGLOBULIN OLIGOMERS*

The interaction at neutral pH between wild-type and a variant form (R3A) of the amyloid fibril-forming protein β₂-microglobulin (β2m) and the molecular chaperone αB-crystallin was investigated by thioflavin T fluorescence, NMR spectroscopy, and mass spectrometry. Fibril formation of R3Aβ2m was potently prevented by αB-crystallin. αB-crystallin also prevented the unfolding and nonfibrillar aggregation of R3Aβ2m. From analysis of the NMR spectra collected at various R3Aβ2m to αB-crystallin molar subunit ratios, it is concluded that the structured β-sheet core and the apical loops of R3Aβ2m interact in a nonspecific manner with the αB-crystallin. Complementary information was derived from NMR diffusion coefficient measurements of wild-type β2m at a 100-fold concentration excess with respect to αB-crystallin. Mass spectrometry acquired in the native state showed that the onset of wild-type β2m oligomerization was effectively reduced by αB-crystallin. Furthermore, and most importantly, αB-crystallin reversibly dissociated β2m oligomers formed spontaneously in aged samples. These results, coupled with our previous studies, highlight the potent effectiveness of αB-crystallin in preventing β2m aggregation at the various stages of its aggregation pathway. Our findings are highly relevant to the emerging view that molecular chaperone action is intimately involved in the prevention of in vivo amyloid fibril formation.     

A study of genetic polymorphisms of milk β-lactoglobulin, αS1-casein, β-casein,and κ-casein in five dairy breeds

Gene frequencies of the milk -lactoglobulin, _S1-casein, -casein, and -casein loci have been estimated from 1663 cows of five dairy breeds. Departure from Hardy-Weinberg equilibrium was found only in the -casein system in Jerseys. However, chance alone could have accounted for this single significant finding. Results of pairwise comparisons among the five breeds of allele frequencies at these milk protein loci indicate that of the 40 possible tests, only six comparisons are not significant at the 5% probability level. It would appear that these breeds are characterizable in terms of the gene frequencies of these milk protein loci. Nonindependent assortment of genotypes among these milk protein loci was also studied. The closely linked casein loci were not independent in almost all the breeds where tests could be carried out. The only exception was between the _S1-casein and -casein loci in Holsteins. -Lactoglobulin was independent of the casein loci in all breeds except Brown Swiss, where it was found to be significantly associated with -casein. Close linkage is proposed as an important factor for maintaining the observed milk protein polymorphisms.This paper represents a portion of a doctoral dissertation submitted by the first author as partial fulfillment of the requirements for the degree of Doctor of Philosophy at the University of Massachusetts at Amherst.     

Cytosolic carboxypeptidase 1 is involved in processing α- and β-tubulin

The Purkinje cell degeneration (pcd) mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1). This study tested two proposed functions of CCP1: degradation of intracellular peptides and processing of tubulin. Overexpression (2-3-fold) or knockdown (80-90%) of CCP1 in human embryonic kidney 293T cells (HEK293T) did not affect the levels of most intracellular peptides but altered the levels of α-tubulin lacking two C-terminal amino acids (delta2-tubulin) ≥ 5-fold, suggesting that tubulin processing is the primary function of CCP1, not peptide degradation. Purified CCP1 produced delta2-tubulin from purified porcine brain α-tubulin or polymerized HEK293T microtubules. In addition, CCP1 removed Glu residues from the polyglutamyl side chains of porcine brain α- and β-tubulin and also generated a form of α-tubulin with two C-terminal Glu residues removed (delta3-tubulin). Consistent with this, pcd mouse brain showed hyperglutamylation of both α- and β-tubulin. The hyperglutamylation of α- and β-tubulin and subsequent death of Purkinje cells in pcd mice was counteracted by the knock-out of the gene encoding tubulin tyrosine ligase-like-1, indicating that this enzyme hyperglutamylates α- and β-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from β- as well as α-tubulin in vitro and in vivo.