Microbial Conversion of α-Ionone, α-Methylionone, and α-Isomethylionone

α-Ionone, α-methylionone, and α-isomethylionone were converted by Aspergillus niger JTS 191. The individual bioconversion products from α-ionone were isolated and identified by spectrometry and organic synthesis. The major products were cis-3-hydroxy-α-ionone, trans-3-hydroxy-α-ionone, and 3-oxo-α-ionone. 2,3-Dehydro-α-ionone, 3,4-dehydro-β-ionone, and 1-(6,6-dimethyl-2-methylene-3-cyclohexenyl)-buten-3-one were also identified. Analogous bioconversion products from α-methylionone and α-isomethylionone were also identified. From results of gas-liquid chromatographic analysis during the fermentation, we propose a metabolic pathway for α-ionones and elucidation of stereochemical features of the bioconversion.     

Selection of Astaxanthin-Overproducing Mutants of Phaffia rhodozyma with β-Ionone

β-Ionone, an end ring analog of β-carotene, inhibits astaxanthin production in the red yeast Phaffia rhodozyma. Astaxanthin-overproducing mutants of this yeast are easily spotted on β-ionone-containing yeast malt agar plates. β-Ionone appears to block astaxanthin synthesis at the β-carotene level.     

Stereo- and Regioselective Hydroxylation of α-Ionone by Streptomyces Strains

A total of 215 Streptomyces strains were screened for their capacity to regio- and stereoselectively hydroxylate β- and/or α-ionone to the respective 3-hydroxy derivatives. With β-ionone as the substrate, 15 strains showed little conversion to 4-hydroxy- and none showed conversion to the 3-hydroxy product as desired. Among these 15 Streptomyces strains, S. fradiae Tü 27, S. arenae Tü 495, S. griseus ATCC 13273, S. violaceoniger Tü 38, and S. antibioticus Tü 4 and Tü 46 converted α-ionone to 3-hydroxy-α-ionone with significantly higher hydroxylation activity compared to that of β-ionone. Hydroxylation of racemic α-ionone [(6R)-(−)/(6S)-(+)] resulted in the exclusive formation of only the two enantiomers (3R,6R)- and (3S,6S)-hydroxy-α-ionone. Thus, the enzymatic hydroxylation of α-ionone by the Streptomyces strains tested proceeds with both high regio- and stereoselectivity.     

Cross Talk between β1 and αV Integrins: β1 Affects β3 mRNA Stability

There is increasing evidence that a fine-tuned integrin cross talk can generate a high degree of specificity in cell adhesion, suggesting that spatially and temporally coordinated expression and activation of integrins are more important for regulated cell adhesive functions than the intrinsic specificity of individual receptors. However, little is known concerning the molecular mechanisms of integrin cross talk. With the use of beta(1)-null GD25 cells ectopically expressing the beta(1)A integrin subunit, we provide evidence for the existence of a cross talk between beta(1) and alpha(V) integrins that affects the ratio of alpha(V)beta(3) and alpha(V)beta(5) integrin cell surface levels. In particular, we demonstrate that a down-regulation of alpha(V)beta(3) and an up-regulation of alpha(V)beta(5) occur as a consequence of beta(1)A expression. Moreover, with the use of GD25 cells expressing the integrin isoforms beta(1)B and beta(1)D, as well as two beta(1) cytoplasmic domain deletion mutants lacking either the entire cytoplasmic domain (beta(1)TR) or only its "variable" region (beta(1)COM), we show that the effects of beta(1) over alpha(V) integrins take place irrespective of the type of beta(1) isoform, but require the presence of the "common" region of the beta(1) cytoplasmic domain. In an attempt to establish the regulatory mechanism(s) whereby beta(1) integrins exert their trans-acting functions, we have found that the down-regulation of alpha(V)beta(3) is due to a decreased beta(3) subunit mRNA stability, whereas the up-regulation of alpha(V)beta(5) is mainly due to translational or posttranslational events. These findings provide the first evidence for an integrin cross talk based on the regulation of mRNA stability.     

Purification of β-acetylglucosaminase and β-galactosidase from ram testis

1. The presence of beta-galactosidase (EC 3.2.1.23) in an acetic acid extract of ram testis is reported. Some properties of the crude enzyme preparation were studied. 2. The purification of beta-acetylglucosaminase (EC 3.2.1.30) and of beta-galactosidase from the ram-testis extract by ammonium sulphate precipitation and chromatography on a CM-cellulose column is described. 3. The final purifications of the separated enzymes achieved were for the beta-acetylglucosaminase 35 times and for the beta-galactosidase 99 times. 4. The possibility of using DEAE-cellulose and Sephadex G-200 to purify the enzymes was investigated.     

Staphylococcal β-Hemolysin I. Purification of β-Hemolysin

The purification of staphylococcal β-hemolysin was accomplished by the successive use of three protein fractionation methods. The first method employed was a double precipitation with the use of ammonium sulfate at 65% saturation. The second phase of purification used Sephadex G-100 column fractionation. The third phase utilized either carboxymethyl cellulose or diethylaminoethyl cellulose fractionation. The last two fractionation methods both resulted in the separation of a relatively high concentration of cationic hot-cold lysin and a low concentration of anionic hot-cold lysin. Because of the low concentration of the anionic component, its purity could not be assessed. However, the purity of the cationic component was demonstrated by immunodiffusion, microimmunoelectrophoresis, and by disc polyacrylamide gel electrophoresis. In addition, antisera against purified cationic β-hemolysin yielded one line of precipitate when tested against the original crude β-hemolysin. The purified cationic β-hemolysin was stable in the lyophilized state. Crude β-hemolysin was dermonecrotic, whereas purified cationic β-hemolysin was not dermonecrotic even after Mg⁺⁺ activation.     

Microbial 7α-Hydroxylation of 3-Ketobisnorcholenol

The transformation of 22-hydroxy-23,24-bisnorchol-4-en-3-one to 7α-22-dihydroxy-23,24-bisnorchol-4-en-3-one by Botryodiploida theobromae, Lasiodiplodia theobromae, and various Botryosphaeria strains is described. Factors affecting the reaction were incubation temperature, sonication of the substrate, and addition of 2,2′-dipyridyl, extra carbohydrate, and Amberlite XAD-7. The enzyme responsible for the reaction appeared to be very specific and was not characteristic of all members of the genera listed above.     

The α5β1 Integrin Mediates Elimination of Amyloid-β Peptide and Protects Against Apoptosis

The amyloid-β peptide (Aβ) can mediate cell attachment by binding to β1 integrins through an arg-his-asp sequence. We show here that the α5β1 integrin, a fibronectin receptor, is an efficient binder of Aβ, and mediates cell attachment to nonfibrillar Aβ. Cells engineered to express α5β1 internalized and degraded more added Aβ1-40 than did α5β1-negative control cells. Deposition of an insoluble Aβ1-40 matrix around the α5β1-expressing cells was reduced, and the cells showed less apoptosis than the control cells. Thus, the α5β1 integrin may protect against Aβ deposition and toxicity, which is a course of Alzheimer's disease lesions.     

Statistical-Mechanical Studies of the α⇄β Transformation in Keratin: IV. The Hill Model

The relationship between the two-dimensional Hill model and the David-Schor extension of the one-dimensional Zimm-Bragg model for the alpha right arrow over left arrow beta transformation in keratins is developed. On the basis of the assumptions of the David Schor model, it appears unlikely that the Hill model in its present form can give detailed agreement with the experimental tension-length isotherms.     

Statistical-Mechanical Studies of the α ⇄ β Transformation in Keratins: II. The Tension-Length Isotherms

In attempting to understand the yield region of the α β transformation in keratins (Astbury and Woods, 1933), we recently proposed a statistical-mechanical model (David and Schor, 1965) which generalized the work done by others on the helix random coil transformation (Zimm and Bragg, 1959; Gibbs and DiMarzio, 1959) (thermal denaturation) to the case of a polypeptide under external tension (Birstein, 1962). We wish now to report the comparison of the quantitative aspects of this model to the observed tension-length isotherms (in the yield region) of Cotswold wool.     

Haloalkane Dehalogenase LinB Is Responsible for β- and δ-Hexachlorocyclohexane Transformation in Sphingobium indicum B90A

Incubation of resting cells of Sphingobium indicum B90A, Sphingobium japonicum UT26, and Sphingobium francense Sp+ showed that they were able to transform β- and δ-hexachlorocyclohexane (β- and δ-HCH, respectively), the most recalcitrant hexachlorocyclohexane isomers, to pentachlorocyclohexanols, but only resting cells of strain B90A could further transform the pentachlorocyclohexanol intermediates to the corresponding tetrachlorocyclohexanediols. Moreover, experiments with resting cells of Escherichia coli expressing the LinB proteins of strains B90A, UT26, and Sp+ indicated that LinB was responsible for these transformations. Purified LinB proteins from all three strains also effected the formation of the respective pentachlorocyclohexanols. Although the three LinB enzymes differ only marginally with respect to amino acid sequence, they showed interesting differences with respect to substrate specificity. When LinB from strain B90A was incubated with β- and δ-HCH, the pentachlorocyclohexanol products were further transformed and eventually disappeared from the incubation mixtures. In contrast, the LinB proteins from strains UT26 and Sp+ could not catalyze transformation of the pentachlorocyclohexanols, and these products accumulated in the incubation mixture. A mutant of strain Sp+ lacking linA and linB did not degrade any of the HCH isomers, including β-HCH, and complementation of this mutant by linB from strain B90A restored the ability to degrade β- and δ-HCH.     

Crystal structure of the ββα-Me type II restriction endonuclease Hpy99I with target DNA

The ββα-Me restriction endonuclease (REase) Hpy99I recognizes the CGWCG target sequence and cleaves it with unusual stagger (five nucleotide 5′-recessed ends). Here we present the crystal structure of the specific complex of the dimeric enzyme with DNA. The Hpy99I protomer consists of an antiparallel β-barrel and two β4α2 repeats. Each repeat coordinates a structural zinc ion with four cysteine thiolates in two CXXC motifs. The ββα-Me region of the second β4α2 repeat holds the catalytic metal ion (or its sodium surrogate) via Asp148 and Asn165 and activates a water molecule with the general base His149. In the specific complex, Hpy99I forms a ring-like structure around the DNA that contacts DNA bases on the major and minor groove sides via the first and second β4α2 repeats, respectively. Hpy99I interacts with the central base pair of the recognition sequence only on the minor groove side, where A:T resembles T:A and G:C is similar to C:G. The Hpy99I–DNA co-crystal structure provides the first detailed illustration of the ββα-Me site in REases and complements structural information on the use of this active site motif in other groups of endonucleases such as homing endonucleases (e.g. I-PpoI) and Holliday junction resolvases (e.g. T4 endonuclease VII).     

The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

Mammalian adenoviruses (AdVs) comprise more than ~350 types including over 100 human (HAdVs) and just three mouse AdVs (MAdVs). While most HAdVs initiate infection by high affinity/avidity binding of their fiber knob (FK) protein to either coxsackievirus AdV receptor (CAR), CD46 or desmoglein (DSG)-2, MAdV-1 (M1) infection requires arginine-glycine-aspartate (RGD) binding integrins. To identify the receptors mediating MAdV infection we generated five novel reporter viruses for MAdV-1/-2/-3 (M1, M2, M3) transducing permissive murine (m) CMT-93 cells, but not B16 mouse melanoma cells expressing mCAR, human (h) CD46 or hDSG-2. Recombinant M1 or M3 FKs cross-blocked M1 and M3 but not M2 infections. Profiling of murine and human cells expressing RGD-binding integrins suggested that αvβ6 and αvβ8 heterodimers are associated with M1 and M3 infections. Ectopic expression of mβ6 in B16 cells strongly enhanced M1 and M3 binding, infection, and progeny production comparable with mαvβ6-positive CMT-93 cells, whereas mβ8 expressing cells were more permissive to M1 than M3. Anti-integrin antibodies potently blocked M1 and M3 binding and infection of CMT-93 cells and hαvβ8-positive M000216 cells. Soluble integrin αvβ6, and synthetic peptides containing the RGDLXXL sequence derived from FK-M1, FK-M3 and foot and mouth disease virus coat protein strongly interfered with M1/M3 infections, in agreement with high affinity interactions of FK-M1/FK-M3 with αvβ6/αvβ8, determined by surface plasmon resonance measurements. Molecular docking simulations of ternary complexes revealed a bent conformation of RGDLXXL-containing FK-M3 peptides on the subunit interface of αvβ6/β8, where the distal leucine residue dips into a hydrophobic pocket of β6/8, the arginine residue ionically engages αv aspartate215, and the aspartate residue coordinates a divalent cation in αvβ6/β8. Together, the RGDLXXL-bearing FKs are part of an essential mechanism for M1/M3 infection engaging murine and human αvβ6/8 integrins. These integrins are highly conserved in other mammals, and may favour cross-species virus transmission.     

Staphylococcal β-Hemolysin II. Phospholipase C Activity of Purified β-Hemolysin

Sheep erythrocyte ghosts released water-soluble organic phosphorus when treated with purified beta-hemolysin. Phospholipid analysis demonstrated that sphingomyelin accounted for 53% of the phospholipids present in sheep erythrocytes. Purified beta-hemolysin showed phospholipase C activity when purified ox brain or sheep erythrocyte sphingomyelin was used as substrate. Such studies have also revealed that the disappearance of sphingomyelin from the reaction mixture was accompanied by a comparable increase in the concentration of phosphoryl choline. Thin-layer chromatography of phospholipids, extracted from sheep erythrocytes which had been exposed to beta-hemolysin, demonstrated that sphingomyelin was rapidly degraded. Activators of beta-hemolysin, such as Mg(++), enhanced the release of organic phosphorus from erythrocyte ghosts and from sphingomyelin. Inhibitors of beta-hemolysin, such as ethylenediaminetetraacetic acid, p-chloromercuribenzoate, and iodoacetamide, also inhibited the release of organic phosphorus from erythrocyte ghosts and from sphingomyelin. These studies strongly suggested that beta-hemolysin enzymatically degraded the sphingomyelin of the erythrocyte membrane. Such degradation probably resulted in the eventual lysis of the erythrocyte.     

Affinity Modulation of Platelet Integrin αIIbβ3 by β3-Endonexin, a Selective Binding Partner of the β3 Integrin Cytoplasmic Tail

Platelet agonists increase the affinity state of integrin α_IIbβ₃, a prerequisite for fibrinogen binding and platelet aggregation. This process may be triggered by a regulatory molecule(s) that binds to the integrin cytoplasmic tails, causing a structural change in the receptor. β₃-Endonexin is a novel 111–amino acid protein that binds selectively to the β₃ tail. Since β₃-endonexin is present in platelets, we asked whether it can affect α_IIbβ₃ function. When β₃-endonexin was fused to green fluorescent protein (GFP) and transfected into CHO cells, it was found in both the cytoplasm and the nucleus and could be detected on Western blots of cell lysates. PAC1, a fibrinogen-mimetic mAb, was used to monitor α_IIbβ₃ affinity state in transfected cells by flow cytometry. Cells transfected with GFP and α_IIbβ₃ bound little or no PAC1. However, those transfected with GFP/β₃-endonexin and α_IIbβ₃ bound PAC1 specifically in an energy-dependent fashion, and they underwent fibrinogen-dependent aggregation. GFP/β₃-endonexin did not affect levels of surface expression of α_IIbβ₃ nor did it modulate the affinity of an α_IIbβ₃ mutant that is defective in binding to β₃-endonexin. Affinity modulation of α_IIbβ₃ by GFP/β₃-endonexin was inhibited by coexpression of either a monomeric β₃ cytoplasmic tail chimera or an activated form of H-Ras. These results demonstrate that β₃-endonexin can modulate the affinity state of α_IIbβ₃ in a manner that is structurally specific and subject to metabolic regulation. By analogy, the adhesive function of platelets may be regulated by such protein–protein interactions at the level of the cytoplasmic tails of α_IIbβ₃.     

The stereospecific biosynthesis of plant sterols and α- and β-amyrin

1. A preparation of pea seedlings has been obtained that will incorporate [2-(14)C]mevalonate into squalene, alpha- and beta-amyrin and the phytosterols. 2. The (14)C/(3)H ratio in alpha- and beta-amyrin biosynthesized in the presence of [2-(14)C,4R-(3)H]-mevalonate is the same as in the starting material and in squalene; this gives experimental support to the mechanism for the cyclization of squalene proposed by Ruzicka for the formation of these pentacyclic triterpenoids. 3. The (14)C/(3)H ratio for beta-sitosterol was 5:3, the same as that in cholesterol in liver. 4. As the absence of (3)H from C-3 in beta-sitosterol was demonstrated (3)H must be present in the side chain and thus the H at C-24 is not lost during alkylation of the side chain; it probably migrates to C-25.     

Integrin Cross Talk: Activation of Lymphocyte Function-associated Antigen-1 on Human T Cells Alters α4β1- and α5β1-mediated Function

A regulated order of adhesion events directs leukocytes from the vascular compartment into injured tissues in response to inflammatory stimuli. We show that on human T cells, the interaction of the β2 integrin leucocyte function–associated antigen-1 (LFA-1) with its ligand intercellular adhesion molecule-1 (ICAM-1) will decrease adhesion mediated by α4β1 and, to a lesser extent, α5β1. Similar inhibition is also seen when T cells are exposed to mAb 24, which stabilizes LFA-1 in an active state after triggering integrin function through divalent cation Mg²⁺, PdBu, or T cell receptor/ CD3 complex (TCR/CD3) cross-linking. Such cross talk decreases α4β1 integrin–mediated binding of T cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In contrast, ligand occupancy or prolonged activation of β1 integrin has no effect on LFA-1 adhesion to ICAM-1. We also show that T cell migration across fibronectin, unlike adhesion, is mediated solely by α5β1, and is increased when the α4β1-mediated component of fibronectin adhesion is decreased either by cross talk or the use of α4-blocking mAb. The ability of mAb 24 Fab′ fragments to induce cross talk without cross-linking LFA-1 suggests signal transduction through the active integrin. These data provide the first direct evidence for cross talk between LFA-1 and β1 integrins on T cells. Together, these findings imply that activation of LFA-1 on the extravasating T cell will decrease the binding to VCAM-1 while enhancing the subsequent migration on fibronectin. This sequence of events provides a further level of complexity to the coordination of T cell integrins, whose sequential but overlapping roles are essential for transmigration.